IMMUNOSEROLOGY

A collection of transes on Immunoserology

Bachelor of Science
in Medical Technology

Christine Joy R. Timbang | 0963-467-6983

 NATURE OF THE
IMMUNE SYSTEM

Christine Joy R. Timbang | 0963-467-6983

Christine Joy R. Timbang | 0963-467-6983
IMMUNOSEROLOGY 1.

Introduction to Immunity and the Immune System

OUTLINE NOTE
I. Introduction
A. Definitions Attenuation
II. Immunity and Immunization • make a pathogen less virulent
A. Immunity • takes place through heat, aging, or chemical means
B. History • remains the basis for many of the immunizations that
III. Innate Versus Adaptive Immunity are used today
A. History
B. Branches of Immunity
IV. Cells of the Innate Immune System INNATE VERSUS ADAPTIVE IMMUNITY
A. Leukocytes in Peripheral Blood A. History
B. Tissue Cells late 1800s
V. Cells of the Adaptive Immune System
• Elie Metchnikoff
A. Lymphocyte
o a Russian scientist, observed under a microscope
VI. Organs of the Immune System
that foreign objects introduced into transparent
A. Primary lymphoid organs
starfish larvae became surrounded by motile
B. Secondary lymphoid organs
amoeboid-like cells that attempted to destroy the
penetrating objects → phagocytosis
INTRODUCTION § based on the action of these scavenger
A. Definitions cells and was a natural, or innate, host
• Immunology defense
o study of a host’s reactions when foreign substances
are introduced into the body. Other
• Antigens • Emil von Behring
o foreign substances that induce a host response o demonstrated that diphtheria and tetanus toxins,
o all around us in nature and they vary from which are produced by specific microorganisms as
substances such as pollen that may make us sneeze they grow, could be neutralized by the noncellular
to serious bacterial pathogens portion of the blood of animals previously exposed
to the microorganisms
IMMUNITY AND IMMUNIZATION § leading to the theory of humoral immunity
A. Immunity
• condition of being resistant to infection 1903
• Almroth Wright
B. History o English physician
1500s o linked the two theories by showing that the
• Chinese inhaled powder made from smallpox scabs in immune response involved both cellular and
order to produce protection against this dreaded disease humoral elements
• Hypothesis: If a healthy individual was exposed as a child o observed that certain humoral, or circulating,
or young adult the effects of the disease would be factors called opsonins acted to coat bacteria so
minimized that they became more susceptible to ingestion by
phagocytic cells
late 1700s § These serum factors include specific
proteins known as antibodies, as well as
• Edward Jenner
other factors called acute-phase reactants
o English country doctor
that increase nonspecifically in any
o successfully prevent infection with smallpox by
infection.
injecting a more harmless substance— cowpox—
from a diseaseaffecting cows • Antibodies - serum proteins
produced by certain lymphocytes
1800s when exposed to a foreign
substance; react specifically with
• Louis Pasteur that foreign substance.
o father of immunology
o observed by chance that older bacterial cultures B. Branches of Immunity
would not cause disease in chickens 1. Innate or natural immunity
• Subsequent injections of more virulent organisms had no
• individual’s ability to resist infection by means of normally
effect on the birds that had been previously exposed to
present body functions
the older cultures. In this manner, the first attenuated
• considered nonadaptive or nonspecific and are the same
vaccine was discovered; this event can be considered the
for all pathogens or foreign substances to which one is
birth of immunology
exposed.

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• No prior exposure is required and the response lacks
memory and specificity.
• Many of these mechanisms are subject to influence by
such factors as nutrition, age, fatigue, stress, and genetic
determinants.

2. Adaptive immunity
• type of resistance that is characterized by specificity for
each individual pathogen, or microbial agent, and the
ability to remember a prior exposure. 1. Neutrophils
• Memory and specificity result in an increased response to
that pathogen upon repeated exposure, something that • polymorphonuclear neutrophilic (PMN) leukocyte
does not occur in innate immunity. • represents approximately 50% to 75% of the total
peripheral WBCs in adults
• often called segmented neutrophils, or “segs.”
CELLS OF THE INNATE IMMUNE SYSTEM
• Margination occurs to allow neutrophils to move from the
A. Leukocytes in Peripheral Blood
circulating blood to the tissues through a process known
• White blood cells (WBCs) or leukocytes as diapedesis, or movement through blood vessel walls.
o play a key role in both innate and adaptive
• They are attracted to a specific area by chemotactic
immunity
factors.
o Chemotaxins are chemical messengers that cause
cells to migrate in a particular direction.

2. Eosinophils
• 1% and 3% of the circulating WBCs in a nonallergic
person
• number increases in an allergic reaction or in response to
certain parasitic infections
• capable of phagocytosis but are much less efficient than
neutrophils because they are present in smaller numbers
and they lack digestive enzymes
• regulation of the immune response, including regulation of
mast cell function

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3. Basophils
• least numerous of WBCs found in peripheral blood
• function in inducing and maintaining allergic reactions
o Histamine contracts smooth muscle and heparin is
an anticoagulant
• have a short life span of only a few hours in the
bloodstream; they are then pulled out and destroyed by
macrophages in the spleen

3. Dendritic Cells
• covered with long membranous extensions that make
them resemble nerve cell dendrites
• After capturing an antigen in the tissue by phagocytosis or
endocytosis, dendritic cells present the antigen to T
lymphocytes to initiate the adaptive immune response in a
similar way as macrophages.
• considered the most effective APC in the body, as well as
the most potent phagocytic cell
4. Monocytes
• largest cells in the peripheral blood CELLS OF THE ADAPTIVE IMMUNE SYSTEM
• stay in peripheral blood for up to 30 hours → migrate to A. Lymphocyte
the tissues and become known as macrophages • only white blood cells (WBCs) or leukocytes in the
adaptive immune system
• nuclear chromatin is dense and tends to stain a deep blue
• arise from an HSC and then are further differentiated in
the primary lymphoid organs, namely the bone marrow
and the thymus
• Three major populations:
o T cells
o B cells
o natural killer (NK) cells

NOTE

clusters of differentiation (CD)

• scientists set up the Human Leukocyte Differentiation
Antigens Workshops to relate research findings.
• 15 Panels of antibodies from different laboratories were
B. Tissue Cells used for analysis and antibodies reacting similarly with
1. Macrophages standard cell lines → CD
• arise from monocytes
• macrophages contain no peroxidase (unlike monocytes)
• play an important role in initiating and regulating both Surface Markers on T, B, and NK Cells
innate and adaptive immune responses Antigen MOL WT Cell Type Function
• Killing activity is enhanced when macrophages become (KD)
“activated” by contact with microorganisms or with CD3 20–28 Thymocytes, Found on all
chemical messengers called cytokines, which are T cells T cells;
released by T lymphocytes during the immune response. associated
with T-cell
2. Mast Cells antigen
• larger than basophils with a small round nucleus and receptor
more granules CD4 55 T helper cells, Identifies T
• have a long-life span of between 9 and 18 months monocytes, helper cells;
macrophages also found
• play a role in allergic reactions, but they can also function
on most T
as antigen-presenting cells (APCs)

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 regulatory 2. T Cells
cells • named because they differentiate in the thymus
CD8 60–76 Thymocyte Identifies • Lymphocyte precursors called thymocytes enter the
subsets, cytotoxic T thymus from the bone marrow through the bloodstream.
cytotoxic T cells • As they mature, the T cells express unique surface
cells markers that allow them to recognize foreign antigens
CD16 50–80 Macrophages, Low affinity bound to cell membrane proteins called MHC molecules.
NK cells, Fc receptor • conducts the process “cell-mediated immunity”
neutrophils for antibody; • produce cytokines that contribute to immunity by
mediates stimulating B cells to produce antibodies, assisting in
phagocytosis killing tumor cells or infected target cells, and helping to
CD19 >120 B cells, Part of B-cell regulate both the innate and adaptive immune response
follicular coreceptor; • Main subtypes
dendritic cells regulates B- o helper, cytolytic, and regulatory T cells
cell develop- § identified by the presence of the CD3 marker
ment and on their cell surface, and either CD4, or CD8
activation • CD4 receptor - either helper or
CD21 145 B cells, Receptor for regulatory cells
follicular complement • CD8-positive (CD8+) population -
dendritic cells component cytotoxic T cells
C3d; part of o CD4+ to CD8+ cells - approximately 2:1 in
B-cell peripheral blood
coreceptor
with CD19 3. Natural Killer (NK) Cells
CD 56 175–220 NK cells, Not known • this small percentage of lymphocytes do not express the
subsets of T markers of either T cells or B cells
cells
• have the ability to kill target cells without prior exposure to
them
• do not require the thymus for development but appear to
mature in the bone marrow itself
• no surface markers that are unique to NK cells, but they
express a specific combination of antigens that can be
used for identification
o CD16
§ receptor for the nonspecific end of antibodies
§ Because of the presence of CD16, NK cells
are able to make contact with and then lyse
any cell coated with antibodies
o CD56
• also capable of recognizing any foreign cell and represent
the first line of defense against virally infected cells and
tumor cells
• play an important role as a transitional cell bridging the
innate and the adaptive immune response against
pathogens

ORGANS OF THE IMMUNE SYSTEM

1. B Cells A. Primary lymphoid organs
• derived from a lymphoid precursor that differentiates to • where maturation of B lymphocytes and T lymphocytes
become either a T cell, B cell, or NK cell depending on takes place
exposure to different cytokines
• remain in the environment provided by bone marrow 1. Bone Marrow
stromal cells • considered one of the largest tissues in the body and it
• can be recognized by the presence of membrane-bound fills the core of all long flat bones
antibodies of two types: (1) immunoglobulin M (IgM) and • main source of hematopoietic stem cells
(2) immunoglobulin (IgD)
• surface proteins 2. Thymus
o CD19 • small, flat, bilobed organ found in the thorax, or chest
o CD21 cavity, right below the thyroid gland and overlying the
o class II major histocompatibility complex (MHC) heart
molecules • reaches a weight of 30 to 40 g by puberty and then
• received their name because they were originally found to gradually shrinks in size
mature in birds in an organ called the bursa of Fabricius, • Each lobe of the thymus is divided into smaller lobules
which is similar to the appendix in humans filled with epithelial cells that play a central role in the
differentiation process.

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• Maturation of T cells takes place over a 3-week period as • large discriminating filter as it removes old and damaged
cells filter through the thymic cortex to the medulla. cells and foreign antigens from the blood
• receives a blood volume of approximately 350 mL/minute

Main Types
a. Red pulp
• makes up more than one-half of the total volume and its
function is to destroy old red blood cells (RBCs)
• Blood flows from the arterioles into the red pulp and then
exits by way of the splenic vein.

b. White pulp
• approximately 20% of the total weight of the spleen and
contains the lymphoid tissue, which is arranged around
arterioles in a periarteriolar lymphoid sheath (PALS)

B. Secondary lymphoid organs

• provide a location where contact with foreign antigens
• Lymphocytes in these organs travel through the tissue
and return to the bloodstream by way of the thoracic duct.
o The thoracic duct is the largest lymphatic vessel in
the body.
o It collects most of the body’s lymph fluid and
empties it into the left subclavian vein.
• T lymphocytes - effector cells that serve a regulatory
• B lymphocytes - produce antibodies
• Lymphopoiesis, or multiplication of lymphocytes, occurs in
the secondary lymphoid tissue and is strictly dependent
on antigenic stimulation. 2. Lymph Nodes
o Formation of lymphocytes in the bone marrow, • central collecting points for lymph fluid from adjacent
however, is antigen-independent, meaning that tissues
lymphocytes are constantly being produced without • Lymph fluid
the presence of specific antigens. • filtrate of the blood and arises from passage of water and
low-molecular-weight solutes out of blood vessel walls
1. Spleen and into the interstitial spaces between cells
• largest secondary lymphoid organ • flows slowly through spaces called sinuses, which are
• located in the upper-left quadrant of the abdomen just lined with macrophages, creating an ideal location where
below the diaphragm and is surrounded by a thin phagocytosis can take place
connective tissue capsule

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• located along lymphatic ducts and are especially c. cutaneous-associated lymphoid tissue or CALT
numerous near joints and where the arms and legs join • monocytes, macrophages, and dendritic cells
the body
• Secondary follicles Comparison of T, B, and NK Cells
o consist of antigen-stimulated proliferating B cells T Cells B Cells NK Cells
o germinal center - interior; where transformation of Develop in the Develop in the Develop in the
the B cells takes place thymus bone marrow bone marrow
• T lymphocytes Found in lymph Found in bone Found in spleen,
o mainly localized in the paracortex (region nodes, marrow, spleen, liver
between the follicles and the medulla) thoracic duct lymph nodes, 5–15% of
o in close proximity to APCs called interdigitating fluid 10–15% of circulating
cells 60–80% of circulating lymphocyte
• medulla - less densely populated than the cortex but circulating lymphocyte pool pool in blood
contains some T cells (in addition to B cells), lymphocyte pool in blood
macrophages, and numerous plasma cells in blood
• If contact with an antigen takes place, lymphocyte traffic Adaptive Adaptive Innate immunity:
shuts down. Lymphocytes able to respond to a particular immunity: end immunity: end lysis of virally
antigen proliferate in the node. products of product of infected cells and
• Accumulation of lymphocytes and other cells causes the activation activation is tumor cells;
lymph nodes to become enlarged, a condition known as are cytokines antibody production of
lymphadenopathy. cytokines
Antigens include Antigens include Antigens include
CD2, CD3, CD4, CD19, CD20, CD16, CD56
or CD8 CD21 surface
antibody

Primary And Secondary Lymphoid Organs

Lymphoid Organs Function
Organ Category Involved
Primary Bone marrow Produces
hematopoietic
stem cells;
maturation of B
and NK cells
Thymus Maturation of T
cells
Secondary Spleen Filters blood
Lymph nodes Places where
Mucosal contact between
associated T cells,
lymphoid tissue antigens, and B
(MALT) cells occur
Cutaneous-
associated
Structure of a lymph node. lymphoid tissue
A lymph node is surrounded by a tough outer capsule. Right (CALT)
underneath is the subcapsular sinus, where lymph fluid drains from
afferent lymphatic vessels. The outer cortex contains collections of B
cells in primary follicles. When stimulated by antigens, secondary Cells of the Immune System
follicles are formed. T cells are found in the paracortical area. Fluid Cell Type Where Function
drains slowly through sinusoids to the medullary region and out the Found
efferent lymphatic vessel to the thoracic duct. Neutrophil 50–75% of First
circulating responder
3. Other Secondary Organs WBCs, to infection,
a. Mucosal-associated tissue known as MALT also in phagocytosi
• found in the gastrointestinal, respiratory, and urogenital tissue
tracts
• represent some of the main ports of entry for foreign Eosinophil 1–3% of Kill
antigens, and thus, numerous macrophages and circulating parasites,
lymphocytes are localized here WBCs neutralize
basophil
b. Skin and mast
• considered the largest organ in the body and the cell
• epidermis contains a number of intraepidermal products,
lymphocytes regulate
• Most of these are T cells, which are uniquely positioned to mast cells
combat any antigens that enter through the skin

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Basophil < 1% of Induce and Review Questions
circulating maintain 1. Which of the following can be attributed to Pasteur?
WBCs allergic a. Discovery of opsonins
reactions, b. Observation of phagocytosis
stimulate c. First attenuated vaccines
production d. Theory of humoral immunity
of IgE 2. Which WBC is capable of further differentiation in
Mast cell Found in Antigen tissues?
skin, presentation a. Neutrophil
connective to T and B b. Eosinophil
tissue, cells; c. Basophil
mucosal enhanceme d. Monocyte
epithelium nt and 3. The cells that Metchnikoff first observed are associated
suppression with which phenomenon?
of the a. Innate immunity
adaptive b. Adaptive immunity
immune c. Humoral immunity
response d. Specific immunity
Monocyte 4–10% of Phagocytosi Where are all undifferentiated lymphocytes made?
circulating s; migrate to a. Bone marrow
WBCs tissues to b. Spleen
become c. Thymus
macrophage d. Lymph nodes
s 3. Which of the following statements is true of NK cells?
Macropha In lungs, Phagocytosi a. They rely upon memory for antigen recognition.
ge liver, brain, s; kill b. They have the same CD groups as B cells.
bone, intracellular c. They are found mainly in lymph nodes.
connective parasites; d. They kill target cells without prior exposure to
tissue, tumoricidal them.
other activity; 4. Which cell is the most potent phagocytic cell in the
tissue antigen tissue?
presentation a. Neutrophil
to T and B b. Dendritic cell
cells c. Eosinophil
Dendritic In skin, Most potent d. Basophil
cell mucous phagocytic 5. The ability of an individual to resist infection bymeans of
membrane cell; most normally present body functions is called
s, effective e. innate immunity.
heart, at antigen f. humoral immunity.
lungs, presentation g. adaptive immunity.
liver, h. cross-immunity.
kidney, 6. A cell characterized by a nucleus with two to five lobes,
other a diameter of 10 to 15 μm, and a large number of
tissue neutral staining granules is identified as a(n)
a. eosinophil.
Lymphocyt 20–40% of Subtypes b. monocyte.
e circulating are T cells, c. basophil.
WBCs; B cells, and d. neutrophil.
also found NK cells; T 7. Which of the following is a primary lymphoid organ?
in lymph cells a. Lymph node
nodes, produce b. Spleen
spleen, cytokines, B c. Thymus
other cells d. MALT
secondary produce 8. What type of cells would be found in a primary follicle?
lymphoid antibody in a. Unstimulated B cells
organs adaptive b. Germinal centers
immune c. Plasma cells
response, d. Memory cells
and NK 9. Which of the following is a distinguishing feature of B
cells are cells?
involved in a. Act as helper cells
innate b. Presence of surface antibody
immunity c. Able to kill target cells without prior exposure
d. Active in phagocytosis

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10. Where do lymphocytes mainly come in contact with
antigens?
a. Secondary lymphoid organs
b. Bloodstream
c. Bone marrow
d. Thymus
11. Which of the following is found on the T cell subset
known as helpers?
a. CD19
b. CD4
c. CD8
d. CD56
12. Which of the following statements best characterizes
adaptive immunity?
a. Relies on normally present body functions
b. Response is similar for each exposure
c. Specificity for each individual pathogen
d. Involves only cellular immunity
13. The main function of T cells in the immune response is
to
a. produce cytokines that regulate both innate
and adaptive immunity.
b. produce antibodies.
c. participate actively in phagocytosis.
d. respond to target cells without prior exposure.
14. Which of the following is a part of humoral immunity?
a. Cells involved in phagocytosis
b. Neutralization of toxins by serum
c. Macrophages and mast cells in the tissue
d. T and B cells in lymph nodes
15. Immunity can be defined as
a. the study of medicines used to treat diseases.
b. a specific population at risk for a disease.
c. the condition of being resistant to disease.
d. the study of the noncellular portion of the
blood.
16. A blood cell that has reddish staining granules and is
able to kill large parasites describes
a. basophils.
b. monocytes.
c. neutrophils.
d. eosinophils.
17. Which of the following statements best describes a
lymph node?
a. It is considered a primary lymphoid organ.
b. It removes old RBCs.
c. It collects fluid from the tissues.
d. It is where B cells mature.
18. Antigenic groups identified by different sets of antibodies
reacting in a similar manner to certain standard cell lines
best describes
a. cytokines.
b. clusters of differentiation (CD).
c. neutrophilic granules.
d. opsonins.

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IMMUNOSEROLOGY 2.

Nature of Antigens and the Major Histocompatibility

Complex
OUTLINE C. Dose
I. Introduction • significant quantity of an immunogen must be present in
A. Definitions order for an adaptive immune response to take place
II. Factors Influencing the Immune Response • very large amounts can result in T- and B-cell tolerance
A. Age
B. Overall health D. Route of inoculation
C. Dose • how we are exposed to them and where they get into our
D. Route of inoculation bodies determines the actual amount of immunogen
E. Genetic capacity needed to generate an immune response
III. Traits of Immunogens o intravenous (into a vein)
A. Immunogenicity o intradermal (into the skin)
IV. Epitopes o subcutaneous (beneath the skin)
A. Introduction o oral contact
B. Types • route where the immunogen enters the body also
V. Haptens determines which cell populations will be involved in the
VI. Adjuvants response
VII. Relationship of Antigens to The Host
A. Categories of Antigens E. Genetic capacity
VIII. Major Histocompatibility Complex • this predisposition is linked to the MHC and to the
A. Introduction receptors generated during T- and B-lymphocyte
B. Genes Coding for MHC Molecules (HLA development
Antigens) • the MHC is a system of genes that code for cell-surface
C. Structure of Class I and II MHC Molecules molecules that play an important role in antigen
D. Role of Class I and II Molecules in the recognition
Immune Response
TRAITS OF IMMUNOGENS
INTRODUCTION A. Immunogenicity
A. Definitions • ability of an immunogen to stimulate a host response
Immunogens
• trigger the immune response of lymphocytes Depends on
• macromolecules capable of triggering an adaptive 1. Macromolecular size
immune response by inducing the formation of antibodies • at least 10,000 to be recognized by the immune system
or sensitized T cells in an immunocompetent host • most active immunogens typically have a molecular
• specifically react with such antibodies or sensitized T cells weight of over 100,000 daltons
• greater the molecular weight, the more potent the
Antigen molecule is as an immunogen
• substance that reacts with an antibody or sensitized T
cells but may not be able to evoke an immune response in 2. Foreignness
the first place • able to distinguish between self and nonself
o nonself are immunogenic
NOTE • the more distant taxonomically the source of the
immunogen is from the host, the more successful it is as a
All immunogens are antigens but not all antigens are stimulus
immunogens.
3. Chemical composition and molecular complexity
• proteins and polysaccharides
FACTORS INFLUENCING THE IMMUNE RESPONSE o most effective immunogens
A. Age o powerful immunogens because they are made up of
• older individuals are more likely to have a decreased a variety of units of amino acids
response to antigenic stimulation o particular sequential arrangement of amino acids,
• neonates do not fully respond to immunogens because the primary structure, determines the secondary
their immune systems are not completely developed structure, which is the relative orientation of amino
acids within the chain
B. Overall health • Carbohydrates
• plays a role because individuals who are malnourished, o less immunogenic than protein because they are
fatigued, or stressed are less likely to mount a successful smaller than proteins
immune response o have a limited number of sugars available to create
their structures

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 HAPTENS
4. The ability to be processed and presented with MHC • too small to be recognized, but if they are combined with
molecules larger molecules they are then able to stimulate a
• involves enzymatic digestion to create small peptides or response
pieces that can be complexed to MHC molecules to • nonimmunogenic materials that, when combined with a
present to responsive lymphocytes carrier, create new antigenic determinants
• by themselves haptens are antigens but not immunogens
EPITOPES • precipitation or agglutination reactions will not occur
A. Introduction because a hapten has a single determinant site and
• the small part of a 10,000 dalton immunogen is actually cannot form the cross-links with more than one antibody
recognized in the immune response molecule that are necessary for precipitation or
• key portion of the immunogen is known as the agglutination
determinant site or epitope • may be artificially joined to carrier molecules in a
• molecular shapes or configurations that are recognized by laboratory setting or this may occur naturally within a host
B or T cells o e.g. Poison ivy (Rhus radicans) contains
o Epitopes recognized by B cells may differ from chemical substances called catechols, which are
those recognized by T cells. haptens. Once in contact with the skin, these can
§ Anything that is capable of cross-linking couple with tissue proteins to form the
surface immunoglobulin molecules is able immunogens that give rise to contact dermatitis.
to trigger B-cell activation. The immunogen
does not necessarily have to be degraded
first.
§ For T cells to be able to recognize an
immunogen it must first be degraded into
small peptides by an antigen presenting cell
(APC). Then the peptides form a complex
with MHC proteins and are carried to the
surface of the APC.

B. Types
1. Sequential or linear epitopes
• e.g. amino acids following one another on a single chain

2. Conformational epitope
• results from the folding of one chain or multiple chains,
bringing certain amino acids from different segments of a
linear sequence or sequences into close proximity with
each other so they can be recognized together

ADJUVANTS
• power of immunogens to generate an immune response
can be increased through the use of adjuvants
• substance administered with an immunogen that
increases the immune response in order to provide
immunity to a particular disease
• work by targeting APCs, which are key to the adaptive
immune response
• protect immunogens from degradation and allow a longer
response time that attracts a large number of immune
system cells to the injection site, which helps to boost the
strength of the response
• Aluminum salts - only ones currently approved for clinical
use

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 RELATIONSHIP OF ANTIGENS TO THE HOST Nomenclature
A. Categories of Antigens • notation HLA DRB1*1301
1. Autoantigens o indicates the actual gene involved in coding for the
• antigens that belong to the host β chain of an HLA DR1 antigen is the number 13
• do not evoke an immune response under normal o Specific Subtype: 01
circumstances
• if an immune response does occur to autoantigens → Three categories or classes of genes coding for MHC
autoimmune disease molecules
1. Class I genes
2. Alloantigens • found at three different locations or loci, termed A, B, and
• from other members of the host’s species and are capable C
of eliciting an immune response • there is only one gene coding for each particular molecule
• important to consider in tissue transplantation and in • involved in antigen recognition (influence the repertoire of
blood transfusions antigens to which T cells can respond)

3. Heteroantigens 2. Class II genes

• situated in the D region
• from other species, such as other animals, plants, or
microorganisms • several different loci: DR, DQ, and DP
• have one gene that codes for the α chain and one or more
4. Heterophile antigens genes that code for the β chain
• involved in antigen recognition (influence the repertoire of
• heteroantigens that exist in unrelated plants or animals
antigens to which T cells can respond)
but are either identical or closely related in structure so
that antibody to one will cross-react with antigen of the
3. Class III genes
other
• lies between the class I and class II regions on
• e.g. human blood group A and B antigens, which are
chromosome 6
related to bacterial polysaccharides
• code for the C4A, C4B, C2, and B complement proteins,
as well as cytokines such as tumor necrosis factor (TNF)
MAJOR HISTOCOMPATIBILITY COMPLEX • secreted proteins that have an immune function, but they
A. Introduction are not expressed on cell surfaces
Human leukocyte antigens (HLA) • have a completely different structure
• group of molecules where genetic capability to mount an
immune response is linked to
• by French scientist Dausset
• now known as MHC molecules because they determine
whether transplanted tissue is histocompatible and thus
accepted or recognized as foreign and rejected
• found on all nucleated cells in the body and they play a
pivotal role in the development of both humoral and
cellular immunity
• can function as antigens when transplanted from one
individual to another
• Main function: bring antigen in the body to the surface of The major histocompatibility complex.
cells for recognition by T cells Location of the class I, II, and III genes on chromosome 6. Class I
consists of loci A, B, and C, whereas class II has at least three loci:
B. Genes Coding for MHC Molecules (HLA Antigens) DR, DQ, and DP.
• MHC system
o most polymorphic system found in humans Alleles
§ because there are so many possible alleles • alternate forms of a gene that code for slightly different
at each location. varieties of the same product
• MHC genes • At each of these loci, or locations, there is the possibilityof
o code for proteins that play a pivotal role in immune multiple alleles.
recognition • One individual inherits two copies of chromosome 6; thus,
o it is thought that this polymorphism is essential to there is a possibility of two different alleles for each gene
our survival because it allows for an immune on the chromosome unless that person is homozygous
response to diverse immunogens (has the same alleles) at a given location.
• Genes coding for the MHC molecules in humans are o These genes are described as codominant,
found on the short arm of chromosome 6 meaning that all alleles that an individual inherits
code for products that are expressed on cells.
Clinical Significance
• uniqueness of the HLA antigens creates a major problem Haplotype
in matching organ donors to recipients because these • Because the MHC genes are closely linked, they are
antigens are highly immunogenic inherited together as this package
• One haplotype is inherited from each parent

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C. Structure of Class I and II MHC Molecules Structure of Class II MHC Molecules
Structure of Class I MHC Molecules • found on the APCs (include B lymphocytes, monocytes,
• expressed on all nucleated cells macrophages, dendritic cells, and thymic epithelium)
• differ in the level of expression • Dendritic cells
o highest on lymphocytes and myeloid cells and low • most effective APCs
or undetected on liver hepatocytes, neural cells, • have the highest levels of class II molecules on their
muscle cells, and sperm surface
§ This may explain why HLA matching is not
done in the case of liver transplants. Major class II molecules
• glycoprotein dimer made up of two noncovalently linked • consist of two noncovalently bound polypeptide chains
polypeptide chains that are encoded by separate genes in the MHC complex
• called heterodimers because they contain two different
Chains chains
1. α chain o DP - shortest supply
• has a molecular weight of 44,000 daltons o DQ
• folded into three domain o DR - expressed at the highest level because it
o α1 accounts for about one-half of all the class II
o α2 molecules on a particular cell
o α3 § DRβ gene - most highly
§ inserted into the cell membrane via a polymorphic; close to 2000
transmembrane segment that is hydrophobic different alleles
§ consist of about 90 amino acids each
• X-ray crystallographic studies: α1 and α2 domains each Chains
form an alpha helix and that these serve as the walls of a 1. α chain
deep groove at the top of the molecule • molecular weight of 34,000,
o functions as the peptide-binding site in antigen • anchored to the cell membrane
recognition
• α1 and α2 regions - where most of the polymorphism 2. β chain
resides • molecular weight of 29,000
• α3 region - reacts with CD8 on cytotoxic T cells • anchored to the cell membrane

2. β2–macroglobulin • Each chain has two domains and the α1 and β1 domains
• lighter chain come together to form the peptide-binding site, similar to
• has a molecular weight of 12,000 the one found on class I molecules
• encoded by a single gene on chromosome 15 that is not • Both ends of the peptide-binding cleft are open and thus
polymorphic allow class II molecules to capture longer peptides than
• every class I molecule contains the same β2– class I molecules
microglobulin • α1 domains and the β2 domains - highly conserved in a
• does not penetrate the cell membrane, but it is essential similar manner to the class I molecules
for proper folding of the α chain
Nonclassical class II genes
Nonclassical class I antigens • DM - helps to load peptides onto class II molecules
• designated E, F, and G • DN - not known
• not expressed on cell surfaces and do not function in • DO - modulates antigen binding
antigen recognition but may play other roles in the o products of these genes play a regulatory role in
immune response (except G) antigen processing
• G antigens - expressed on fetal trophoblast cells during
the first trimester of pregnancy, where they come in direct
contact with maternal tissue

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D. Role of Class I and II Molecules in the Immune • Once cleaved, the peptides must then be pumped from
Response the cytoplasm to the lumen of the endoplasmic reticulum
• Main role: antigen presentation by specialized transporter proteins
o process by which degraded peptides within cells
are transported to the plasma membrane where T transporters associated with antigen processing (TAP1 and
cells can then recognize them. T cells can only TAP2)
“see” and respond to antigens when they are • proteins responsible for the adenosine triphosphate
combined with MHC molecules dependent transport of peptides suitable for binding to
• Class I molecules class I molecules
o mainly present peptides synthesized within the cell • most efficient at transporting peptides that are between 8
to CD8 (cytotoxic) T cell to 16 amino acids in size
o watchdogs of viral, tumor, and certain parasitic • Tapasin - brings the TAP transporters into close proximity
antigens that are synthesized within the cell, to the newly formed MHC molecules and mediates
• Class II molecules interaction with them so that peptides can be loaded onto
o present exogenous antigen to CD4 (helper) T cells the class I molecules
o help to mount an immune response to bacterial • Once the α chain has bound the peptide, the class I MHC
infections or other pathogens found outside cells peptide complex is rapidly transported to the cell surface

NOTE

• The difference in functioning of the two molecules is

tied to the mechanisms by which processed antigen is
transported to the surface.
• Both types of molecules, however, must be capable of
presenting an enormous array of different antigenic
peptides to T cells.
• The chemistry of the MHC antigens controls what sorts
of peptides fit in the binding pockets.

Class I MHC-Peptide Interaction

• Endogenous pathway of antigen presentation
o Class I molecules are synthesized in the rough
endoplasmic reticulum and for a time they remain
anchored in the endoplasmic reticulum membrane.
o It is here that these molecules bind peptides.
o Because antigens that bind to class I proteins are
actually synthesized in the same cell as the class I
molecules
o binding of the newly synthesized proteins →
stabilize the association of the α chain of class I
with the β2–macroglobulin

Calnexin
• 88-kd molecule is membrane-bound in the endoplasmic
reticulum
• keeps the α chain in a partially folded state while it awaits
• As few as 10 to 100 identical antigen class I MHC
binding to β2–macroglobulin
complexes can induce a cytotoxic response.
ERp57 • Display of hundreds of class I molecules complexed to
antigen allows CD8+ T cells to continuously check cell
• binds to this complex also
surfaces for the presence of nonself antigen. If it
• released and two other chaperone molecules—calreticulin
recognizes an antigen as being foreign, the CD8+ T cell
and tapasin—associate with the complex and help to
produces cytokines that cause lysis of the entire cell.
stabilize it for peptide binding

Proteasomes
• proteases that carries out digestion of these intracellular
proteins
• reside in large cytoplasmic complexes
• packets of enzymes formed into a cylindrical shape
through which peptides pass and are cleaved.
• Peptides must be unfolded before entering the cylindrical
chamber of the proteasome; they are then cleaved into
the proper size for delivery to class I molecules.

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Class II MHC–Peptide Interaction • If binding occurs with a TCR on a CD4+ T cell, the T
• Exogenous pathway of antigen presentation helper (Th) cell recruits and triggers a B-cell response,
o means that antigen is taken into the cell from the resulting in antibody formation.
outside by means of either phagocytosis or
endocytosis, processes by which cells ingest
extracellular molecules by enclosing them in a small
portion of the plasma membrane

Invariant chain (Ii)

• where class II molecules associate with when synthesized
in the endoplasmic reticulum
• Because the open structure of class II molecules would
permit binding of segments of endogenous peptides within
the ER, Ii serves to protect the binding site.
• 31-kd protein that is made in excess so that enough is
available to bind with all class II molecules shortly after
they are synthesized
• may be responsible for helping to bring α and β chains
together in the ER lumen and then moving them out
through the Golgi complex to the endocytic vesicles where
digested antigen is found

• Once transported to an endosomal compartment, class II

molecules encounter peptides derived from endocytosed,
exogenous proteins.
• The invariant chain is gradually degraded by a protease,
leaving just a small fragment called class II invariant chain
peptide (CLIP) attached to the peptide-binding cleft.
• CLIP is then exchanged for exogenous peptides.
• Selective binding of peptides is favored by the low pH of
the endosomal compartment.
• HLA-DM molecules - help to mediate the reaction by
removing the CLIP fragment and helping to load peptides
into the binding groove.
• Hydrogen bonding - takes place along the length of the
captured peptide
o In contrast to class I molecule (bond at the amino
and carboxy-terminal ends)

E. Clinical Significance of MHC

• Testing for MHC antigens has typically been carried out
before tissue transplant procedures because both class I
and class II molecules can induce a response that leads
to graft rejection.
• MHC antigens also appear to play a role in the
development of autoimmune diseases.
• Inheritance of certain HLA antigens appears to predispose
a person to certain autoimmune diseases.
o inheritance of HLA B27 - ankylosing spondylitis →
progressive chronic inflammatory disorder
affecting the vertebrae of the spine
• On the other hand, the presence of a particular MHC
protein may confer additional protection
o HLA B8 - increased resistance to HIV infection
• On the cell surface, class II molecules are responsible for
forming a trimolecular complex that occurs between
antigen, class II molecule, and an appropriate TCR.

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 A Comparison of Class I and Class II MHC Molecules 8. Which of the following is true of class II MHC (HLA)
Class I MHC Class II MHC antigens?
Molecules Molecules a. They are found on B cells and macrophages.
Cellular All nucleated B cells, b. They are found on all nucleated cells.
Distribution cells monocytes, c. They all originate at one locus.
macrophages, d. They are coded for on chromosome 9.
dendritic cells, 9. Class II MHC molecules are recognized by which of the
thymic epithelial following?
cells a. CD4+ T cells
Structure One α chain and An α chain and a b. CD8+ T cells
β2–microglobulin β chain c. Natural killer cells
Classes A, B, C DP, DQ, DR d. Neutrophils
Size of Peptides 8 to 11 amino 13 to 18 amino 10. Which of the following best describes the role of TAP?
Bound acids acids a. They bind to class II molecules to help block the
Nature of Closed at both Open at both antigen-binding site.
b. They bind to class I proteins in proteasomes.
Peptide ends ends
c. They transport peptides into the lumen of the
Binding Cleft
endoplasmic reticulum.
Interaction with Presents Presents
d. They help cleave peptides for transport to
T Cells endogenous exogenous
endosomes.
antigen to CD8+ antigen to CD4+
11. What is the purpose of the invariant chain in antigen
T cells T cells
processing associated with class II MHC molecules?
a. Helps transport peptides to the binding site
Review Questions b. Blocks binding of endogenous peptides
1. All of the following are characteristics of an effective c. Binds to CD8+ T cells
immunogen except d. Cleaves peptides into the proper size for binding
a. internal complexity. 12. An individual is recovering from a bacterial infection and
b. large molecular weight. tests positive for antibodies to a protein normallyfound in
c. the presence of numerous epitopes. the cytoplasm of this bacterium. Which of the following
d. found on host cells. statements is true of this situation?
2. Which of the following best describes a hapten? a. Class I molecules have presented bacterial
a. Cannot react with antibody antigen to CD8+ T cells.
b. Antigenic only when coupled to a carrier b. Class I molecules have presented bacterial
c. Has multiple determinant sites antigen to CD4+ T cells.
d. A large chemically complex molecule c. Class II molecules have presented bacterial
3. Which would be the most effective immunogen? antigen to CD4+ T cells.
a. Protein with a molecular weight of 200,000 d. B cells have recognized bacterial antigen
b. Nylon polymer with a molecular weight of without help from T cells.
250,000 13. In relation to a human, alloantigens would need to be
c. Polysaccharide with a molecular weight of considered in which of the following events?
220,000 a. Transplantation of a kidney from one individual
d. Protein with a molecular weight of 175,000 to another
4. Which of the following individuals would likely respond b. Vaccination with the polysaccharide coat of a
most strongly to a bacterial infection? bacterial cell
a. An adult who is 75 years of age c. Oral administration of a live but heat-killed virus
b. A malnourished 40-year-old particle
c. A weightlifter who is 35 years old d. Grafting skin from one area of the body to
d. A newborn baby another
5. Which best describes an epitope? 14. Which is characteristic of class I MHC molecules?
a. A peptide that must be at least 10,000 MW a. Consists of one α and one β chain
b. An area of an immunogen recognized only by T b. Binds peptides made within the cell
cells c. Able to bind whole proteins
c. A segment of sequential amino acids only d. Coded for by DR, DP, and DQ genes
d. A key portion of the immunogen 15. Class I MHC antigens E and G serve which function?
6. Adjuvants act by which of the following methods? a. Enhance the response by macrophages
a. Protects antigen from being degraded b. Transport antigen for recognition by CD4+ T
b. Facilitates rapid escape from the tissues cells
c. Limits the area of the immune response c. Bind to A, B, and C antigens to protect the
d. Decreases number of APCs binding site
7. A heterophile antigen is one that d. Protect fetal tissue from destruction by NK cells
a. is a self-antigen. 16. Which best explains the difference between
b. exists in unrelated plants or animals. immunogens and antigens?
c. has been used previously to stimulate antibody a. Only antigens are large enough to be
response. recognized by T cells.
d. is from the same species but is different from b. Only immunogens can react with antibody.
the host. c. Only immunogens can trigger an immune
response.

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 d. Only antigens are recognized as foreign.
17. When a child inherits one set of six HLA genes together
from one parent, this is called a(n)
a. genotype.
b. haplotype.
c. phenotype.
d. allotype.
18. HLA molecules A, B, and C belong to which MHC class?
a. Class I
b. Class II
c. Class III
d. Class IV

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IMMUNOSEROLOGY 3.

Innate Immunity
OUTLINE B. Skin
I. Introduction • epidermis - outer layer of the skin; contains several layers
A. Definitions of tightly packed epithelial cells
II. External Defense System o cells are coated with a protein called keratin
A. Introduction § making the skin impermeable to most
B. Skin infectious agents
C. Mucosal membrane surfaces • outer skin layer - renewed every few days to keep it intact
III. Internal Defense System • dermis - a thicker layer just underneath the epidermis;
A. Introduction composed of connective tissue with blood vessels, hair
B. Pathogen Recognition Receptors follicles, sebaceous glands, sweat glands, and white
C. Acute-Phase Reactants blood cells (WBCs) including macrophages, dendritic
D. Inflammation cells, and mast cells
E. Phagocytosis • serve as a major structural barrier
F. Action of Natural Killer Cells • presence of several secretions on it discourages the
growth of microorganisms
o Lactic acid in sweat and fatty acids from
INTRODUCTION
sebaceous glands - maintain the skin at a pH of
A. Definitions
approximately 5.6
Innate immunity
• psoriasin
• consists of the defenses against infection that are ready o produced by the human skin cells
for immediate action when a host is attacked by a o a small protein that has antibacterial effects,
pathogen especially against gram-negative organisms such
as Escherichia coli
Two Parts
1. External defense system C. Mucosal membrane surfaces
• consists of anatomical barriers designed to keep Respiratory tract
microorganisms from entering the body
• mucous secretions block the adherence of bacteria to
• if these defenses are overcome → internal defense epithelial cells
system is triggered within minutes and clears invaders as
• contain small proteins called surfactants that are
quickly as possible
produced by the epithelial cells and bind to
microorganisms to help move pathogens out
2. Internal defenses
• motion of the cilia that line the nasopharyngeal passages
• include cellular responses that recognize specific
clears away almost 90% of the deposited material
molecular components of pathogens
• simple acts of coughing and sneezing also help to move
pathogens out of the respiratory tract
EXTERNAL DEFENSE SYSTEM
A. Introduction Urinary tract
• composed of physical, chemical, and biological barriers • flushing action of urine, plus its slight acidity, helps to
function together to prevent most infectious agents from remove many potential pathogens from the genitourinary
entering the body tract
• Lactic acid production - keeps the vagina at a pH of about
5

Digestive tract
• stomach’s hydrochloric acid keeps the pH as low as 1

NOTE

Lysozyme
• an enzyme found in many bodily secretions such as
tears and saliva
• attacks the cell walls of microorganisms, especially
those that are gram-positive

Colicins
• produced by resident microorganisms
• a type of protein that binds to the negatively charged
surface of certain bacteria and kills them by penetrating
the membrane
The external defense system.

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 INTERNAL DEFENSE SYSTEM The 10 Toll-Like Receptors
A. Introduction Receptor Substance Target
• composed of both cells and soluble factors that have Recognized Microorganism
specific and essential functions TLR Receptors Found on Cell Surfaces
• Phagocytic cells - engulf and destroy most of the foreign TLR1 Lipopeptides Mycobacteria
cells or particles that enter the body; most important TLR2 Peptidoglycan, Gram-positive
function of the internal defense system. lipoproteins, zymosan bacteria, myco-
• Phagocytosis - enhanced by specific receptors on cells bacteria, yeasts
that capture invaders through identification of unique TLR4 Lipopolysaccharide, Gram-negative
microbial substances. fusion proteins, bacteria, RSV fungi
• Acute-phase reactants - soluble factors that act by several mannan
different methods to either facilitate contact between TLR5 Flagellin Bacteria with
microbes and phagocytic cells or mop up and recycle flagellae
important proteins after the process of phagocytosis has TLR6 Lipopeptides, Mycobacteria, gram-
taken place lipoteichoic acid, positive bacteria,
zymosan yeasts
B. Pathogen Recognition Receptors TLR Receptors Found in Endosomal Compartments
Macrophages and dendritic cells TLR3 Double-stranded RNA viruses
• 10% and 15% of the total cellular population in the tissues RNA
most important cells involved in pathogen recognition TLR7 Single-stranded RNA RNA viruses
• able to distinguish pathogens from normally present TLR8 Single-stranded RNA RNA viruses
molecules in the body by means of receptors known as TLR9 Double-stranded DNA viruses,
pathogen recognition receptors (PRRs) DNA bacterial DNA
TLR10 Unknown Unknown
Pathogen recognition receptors (PRRs)
• encoded by the host’s genomic DNA and act as sensors
for extracellular infection
• play a pivotal role as a second line of defense if
microorganisms penetrate the external barriers
• Once these receptors bind to a pathogen, phagocytic cells
become activated and are better able to engulf and
eliminate any microorganisms
• able to distinguish self from nonself by recognizing
substances, known as pathogen-associated molecular
patterns (PAMPs), that are only found in microorganisms
o e.g. peptidoglycan in gram-positive bacteria,
lipoproteins in gram-negative bacteria, zymosan
in yeast, and flagellin in bacteria with flagellae

1. Toll-like receptor (TLR)

• toll
o a protein originally discovered in the fruit fly
Drosophila, plays an important role in antifungal
immunity in the adult fly
o highest concentration of these TLRs occurs on
monocytes, macrophages, and neutrophils
§ TLR1, TLR2, TLR4, TLR5, and TLR6 -
found on cell surfaces
§ TLR3, TLR7, TLR8, and TLR9 - found in the
endosomal compartment of a cell
• TLR2 - recognizes teichoic acid
and peptidoglycan found in gram-
positive bacteria
• TLR4 recognizes
lipopolysaccharide, which is found
in gram-negative bacteria
• TLR5 - recognizes bacterial
flagellin
• membrane-spanning glycoproteins that share a common
structural element called leucine-rich repeats (LRRs) 2. C-type lectin receptor (CLR)
• Once TLRs bind to their particular substances, host • other family of receptors that activate innate immune
immune responses are rapidly activated by production of responses
cytokines and chemokines. • plasma membrane receptors found on monocytes,
macrophages, dendritic cells, neutrophils, B cells, and T-
cell subsets
• bind to mannan and β-glucans found in fungal cell walls

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• produce cytokines and chemokines to eliminate microbes Reference values
• Normal: 5 to 8 μg/mL
3. retinoic acid-inducible gene-I-like receptors (RLRs)
• recognizes RNA from RNA viruses in the cytoplasm of 3. Complememt
infected cells and induces inflammatory cytokines and • A series of serum proteins involved in mediation of
type I interferons inflammation but also involved in
o opsonization (coating of microorganism)
4. nucleotide-binding oligomerization domain receptors o chemotaxis (migration of WBC)
(NOD) o cell lysis (main function)
• bind peptidoglycans found in bacterial cell walls and also • Nine proteins
help to protect against intracellular protozoan parasites o activated by classical cascade (bound antibodies in
• Mutations in NOD receptors → Crohn’s disease a sequence)

C. Acute-Phase Reactants 4. Alpha1-Antitrypsin (AAT)

• soluble factors • major component of the alpha band when serum is
• normal serum constituents that increase rapidly because electrophoresed
of infection, injury, or trauma to the tissues • plasma inhibitor of proteases released from leukocytes,
especially elastase
1. C-Reactive Protein • When the lungs do not have enough alpha1-antitrypsin,
• member of the family known as the pentraxins, all of elastase is free to destroy lung tissue.
which are proteins with five subunits o As a result, the lungs lose some of their ability to
• CRP acts somewhat like an antibody, as it is capable of expand and contract (elasticity) → emphysema →
opsonization, agglutination, precipitation, and activation of makes breathing difficult.
complement by the classical pathway. • collagen for lungs
• Main substrate: phosphocholine (common constituent of • Elastase
microbial membranes) o protease inhibitor
• most widely used indicator of acute inflammation o an enzyme secreted by neutrophils during
• risk marker for cardiovascular disease inflammation that can degrade elastin and collagen
• CRP is easily destroyed by heating serum to 56°C for 30 • AT deficiency can result in premature emphysema,
minutes especially in individuals who smoke or who have frequent
exposure to noxious chemicals
Levels • Homozygous inheritance of this particular gene may lead
• Increases rapidly within 4 to 6 hours of infection or injury. to development of cirrhosis, hepatitis, or hepatoma in
• Peak value within 48 hours. early childhood.
• Returns to normal rapidly once condition subsides. • also react with any serine protease, such as proteases
• Elevated levels are found in conditions such as bacterial generated by the triggering of the complement cascade or
infections, rheumatic fever, viral infections, malignant fibrinolysis.
diseases, tuberculosis, and after a heart attack
Levels
Reference values • Increases during acute inflammation.
• <1 mg/L - associated with a low risk for cardiovascular
disease 5. Haptoglobin
• 1 to 3 mg/L - associated with an average risk • an alpha2-globulin
• >3 mg/L - associated with a high risk • Binds irreversibly to free hemoglobin to protect kidneys
• Normal (adults): 0.47 to 1.34 mg/L from damage and prevent loss of iron by urinary
• Mean: 0.87 mg/L excretion.
• hemoglobin complex removed by RES, mainly spleen.
2. Serum Amyloid A (SAA) • Used to monitor hemolysis
• Other major protein secreted during the acute phase of
inflammation. Levels
• apolipoprotein that is synthesized in the liver • A two- to ten-fold increase in haptoglobin can be seen
• Has several roles, including: following inflammation, stress, or tissue necrosis
o Removes cholesterol from cholesterol-filled • Early in the inflammatory response: levels may drop
macrophages at site of injury – clean up. (because of intravascular hemolysis, consequently
o Recruitment of immune cells to inflammatory sites, masking the protein’s behavior as an acute-phase
and (can recruit other more cells) reactant)
o Thought to play a role in cholesterol metabolism
• has a high affinity for HDL cholesterol Reference values
• transported by HDL to the site of infection • Normal plasma concentrations: 40 to 290 mg/dL

Levels 6. Fibrinogen
• Peak between 24 to 48 hours after an acute infection • a coagulation factor integral to clot formation which serves
• Increased because of chronic inflammation, as a barrier to prevent spread of microorganisms further in
atherosclerosis, and cancer the body.

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• thought to play a key role in the inflammatory response D. Inflammation
and development of rheumatoid arthritis. • body’s overall reaction to injury or invasion by an
• most abundant coagulation factor infectious agent
• makes blood more viscous and serves to promote • plays a role in initiating, amplifying, or sustaining the
aggregation of red blood cells (RBCs) and platelets reaction and a delicate balance must be maintained for
the process to be speedily resolved
Levels
• Increase with tissue inflammation or tissue destruction. Four cardinal signs or clinical symptoms of inflammation are
1. redness (erythema)
Reference values 2. swelling (edema)
• Normal levels: 200 to 400 mg/dL 3. heat
4. pain
7. Ceruloplasmin
• converts the toxic ferrous ion (Fe2+) to the nontoxic ferric Major events that occur rapidly after tissue injury are
form (Fe3+) 1. Increased blood supply to the affected area. Dilation of
• principal copper transporting protein in plasma, plays a the blood vessels caused by the release of chemical
role in iron metabolism and histamine regulation. mediators such as histamine from injured mast cells
• Stimulates the immune system to fight infections, repair brings additional blood flow to the affected area,
injured tissues and promote healing. resulting in redness and heat.
• Copper deposits in the liver, brain, kidneys, and the eyes. 2. Increased capillary permeability caused by contraction
• The deposits of copper cause tissue damage, necrosis of the endothelial cells lining the vessels. The increased
(death of the tissues), and scarring, which causes permeability of the vessels allows fluids in the plasma to
decreased functioning of the organs affected. leak into the tissues, resulting in the swelling and pain
• Liver failure and damage to the central nervous system associated with inflammation.
(brain, spinal cord) are the most predominant, and the 3. Migration of WBCs, mainly neutrophils, from the
most dangerous, effects of the disorder. capillaries to the surrounding tissue in a process called
diapedesis. As the endothelial cells of the vessels
Levels contract, neutrophils move through the endothelial cells
of the vessel and out into the tissues. Soluble mediators,
• Depletion found in Wilson’s disease, causes the body to
which include acute-phase reactants, chemokines, and
absorb and retain excessive amounts of copper.
cytokines, act as chemoattractants to initiate and control
the response. Neutrophils are mobilized within 30 to 60
Reference values
minutes after the injury and their emigration may last 24
• Normal: 20 to 40 mg/dL
to 48 hours.
4. Migration of macrophages to the injured area. Migration
8. Mannose Binding Protein (MPB)
of macrophages and dendritic cells from surrounding
• Main function: cell lysis tissue occurs several hours later and peaks at 16 to 48
• also called mannosebinding lectin hours.
• trimer that acts as an opsonin, which is calcium- 5. Acute-phase reactants stimulate phagocytosis of
dependent microorganisms. Macrophages, neutrophils, and
• It is able to recognize foreign carbohydrates such as dendritic cells all attempt to clear the area through
mannose and several other sugars found primarily on phagocytosis; in most cases, the healing process is
bacteria, some yeasts, viruses, and several parasites. completed with a return of normal tissue structure

Characteristics of Acute-Phase Reactants

Protein Respo Normal Increas Function
nse Concen e
time tration
(hr) (mg/dl)
C-reactive Opsonixation,
protein 6-10 0.5 1000x complement
activation
Serum Removal of
24 3.0 1000x
amyloid A cholesterol
Alpha1- 200- Protease inhibitor
24 2-5x
antitrypsin 400
Fibrinoge 110- Clot formation
24 2-5x
n 400
Haptoglob Binds
24 40-200 2-10x
in hemoglobin
Cerulopla Binds copper and
48-72 20-40 2x
smin oxidizes iron
Complem Opsonization,
48-72 60-140 2x
ent C3 lysis
Mannose- Complement
0.15-
binding ? ? activation
1.0
protein

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E. Phagocytosis
Once the WBCs are attracted to the area, the actual process
of phagocytosis consists of seven main steps
1. Physical contact between the WBC and the foreign cell

• Chemotaxis - cells are attracted to the site of inflammation

by chemical substances such as soluble bacterial factors
or acute-phase reactants including complement
components and CRP.
• This binding process is enhanced by opsonins
o term derived from the Greek word meaning “to
prepare for eating.”
o serum proteins that attach to a foreign cell or
pathogen and help prepare it for phagocytosis.
o may act by neutralizing the surface charge on the
foreign particle, making it easier for the cells to
approach one another

2. Outflowing of the cytoplasm to surround the

microorganism

3. Formation of a phagosome
• After attachment to a foreign cell or pathogen has
occurred,
• the cell membrane invaginates and pseudopodia
(outflowing Two processes of elimination of pathogens:
• of cytoplasm) surround the pathogen.
1. Oxygen-dependent pathway
• The pseudopodia fuse to completely enclose the
• an increase in oxygen consumption, known as the
pathogen, forming a structure known as a phagosome.
oxidative burst, occurs within the cell as the pseudopodia
enclose the particle within a vacuole.
4. Fusion with lysosomal granules with the phagosome
• This mechanism generates considerable energy via
• The granules contain lysozyme, myeloperoxidase, and
oxidative metabolism
other proteolytic enzymes.
• The contents of the granules are released into the 2. Oxygen-independent pathway
phagolysosome and digestion occurs.
• Any undigested material is excreted from the cells by F. Action of Natural Killer Cells
exocytosis. • represent the first line of defense against cells that are
• Heavily opsonized particles are taken up in as little as 20 virally infected, cells infected with other intracellular
seconds and killing is almost immediate. pathogens, and tumor cells
• have the ability to recognize any damaged cell and to
5. Formation of the phagolysosome with release of eliminate such target cells without prior exposure to them
lysosomal contents
• lack specificity in their response
• stimulated by exposure to cytokines such as interleukin-
6. Digestion of microorganisms by hydrolytic enzymes
12, interferon-α, and interferon-β
7. Release of debris to the outside by exocytosis • peaks in about 3 days
o well before antibody production or a cytotoxic T-cell
response

Mechanism of Cytotoxicity
• The inhibitory signal is based on recognition of class I
major histocompatibility complex (MHC) proteins, which
are expressed on all healthy cells.
• If NK cells react with class I MHC proteins, then inhibition
of natural killing occurs.
o Examples of this type of inhibitory receptor
include killer cell immunoglobulin-like receptors
(KIRs)37 and CD94/NKG2A receptors, both of
which bind class I MHC molecules.
• Diseased and cancerous cells tend to lose their ability to
produce MHC proteins. NK cells are thus triggered by a
lack of MHC antigens, sometimes referred to as
recognition of “missing self.”
• This lack of inhibition appears to be combined with an
activating signal switched on by the presence of proteins

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 produced by cells under stress, namely those cells that • Any target cell coated with IgG can be bound and
are infected or cancerous. destroyed. This method is not unique to NK cells, as
o Examples of activating receptors that bind stress monocytes, macrophages, and neutrophils also exhibit
proteins are CD16 and NKG2D. such a receptor and act in a similar manner.
• If an inhibitory signal is not received when binding to • Nonetheless, the overall importance of NK cells as a
activating receptors occurs, then NK cells release defense mechanism is demonstrated by the fact that
substances called perforins and granzymes. patients who lack these cells have recurring, serious viral
o These substances are released into the space infections and an increased incidence of tumors.
between the NK cell and the target cell.
§ Perforins - proteins that form channels Mechanisms of Innate Immunity
(pores) in the target cell membrane. Type of Example Function
§ Granzymes - packets of enzymes that Defense
may enter through the channels and External Skin and mucous Biological barriers
mediate cell lysis. membranes
Lactic acid Keeps down growth of
microorganisms
Cilia Move pathogens out of
respiratory tract
Stomach acid Low pH keeps
pathogens from growing
Urine Flushes out pathogens
from the body

Lysozyme Attacks cell walls of

pathogens

Normal flora Compete with

pathogens
Produce antimicrobial
peptides
Internal Cells Participate in
phagocytosis
NK cells destroy target
cells using granzymes
and perforins
Pathogen Help phagocytic cells
recognition recognize pathogens
receptors
(e.g., Toll-like
receptors)
Acute-phase Recruit WBCs for
reactants phagocytosis
Coat pathogens to
enhance phagocytosis
Mop up debris
•
Review Questions
1. The term for enhancement of phagocytosis by coating of
foreign particles with serum proteins is
a. opsonization.
b. agglutination.
c. solubilization.
d. chemotaxis.
2. Which of the following plays an important role as an
external defense mechanism?
a. Phagocytosis
b. C-reactive protein
c. Lysozyme
d. Complement
Antibody-Dependent Cell Cytotoxicity (ADCC)
3. The process of inflammation is characterized by all of
• They recognize and lyse antibody-coated cells through a the following except
process called antibody-dependent cell cytotoxicity a. increased blood supply to the area.
(ADCC). b. migration of WBCs.
• Binding occurs through the CD16 receptor for the Fc c. decreased capillary permeability.
portion of immunoglobulin G (IgG). d. appearance of acute-phase reactants.
4. Skin, lactic acid secretions, stomach acidity, and

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G. the motion of cilia represent which type of immunity? 14. How does innate immunity differ from adaptive
a. Innate immunity?
b. Cross a. Innate immunity requires prior exposure to a
c. Adaptive pathogen.
d. Auto b. Innate immunity depends upon normally
5. The structure formed by the fusion of engulfed material present body functions.
and enzymatic granules within the phagocytic cell is c. Innate immunity develops later than adaptive
called a immunity.
a. phagosome. d. Innate immunity is more specific than adaptive
b. lysosome. immunity.
c. vacuole. 15. A 40-year-old male who is a smoker develops
d. phagolysosome symptoms of premature emphysema. The symptoms
6. The presence of human microbiota (normal flora) acts may be caused by a deficiency of which of the following
as a defense mechanism by which of the following acute-phase reactants?
methods? a. Haptoglobin
a. Maintaining an acid environment b. Alpha1-antitrypsin
b. Competing with potential pathogens c. Fibrinogen
c. Keeping phagocytes in the area d. Ceruloplasmin
d. Coating mucosal surfaces 16. Which statement best describes NK cells?
7. Measurement of CRP levels can be used for all of the a. Their response against pathogens is very
following except specific.
a. monitoring drug therapy with anti-inflammatory b. They only react when an abundance of MHC
agents. antigens is present.
b. tracking the progress of an organ transplant. c. They react when both an inhibitory and
c. diagnosis of a specific bacterial infection. activating signal is triggered.
d. determining active phases of rheumatoid d. They are able to kill target cells without
arthritis. previous exposure to them.
8. Pathogen recognition receptors act by
a. recognizing molecules common to both host
cells and pathogens.
b. recognizing molecules that are unique to
pathogens.
c. helping to spread infection because they are
found on pathogens.
d. all recognizing the same pathogens.
9. Which of the following are characteristics of acute-phase
reactants?
a. Rapid increase following infection
b. Enhancement of phagocytosis
c. Nonspecific indicators of inflammation
d. All of the above
10. Which is the most significant agent formed in the
phagolysosome for the elimination of microorganisms?
a. Proteolytic enzymes
b. Hydrogen ions
c. Hypochlorite ions
d. Superoxides
11. Which acute-phase reactant helps to prevent formation
of peroxides and free radicals that may damage
tissues?
a. Haptoglobin
b. Fibrinogen
c. Ceruloplasmin
d. Serum amyloid A
12. Which statement best describes Toll-like receptors
(TLRs)?
a. They protect adult flies from infection.
b. They are found on all host cells.
c. They only play a role in adaptive immunity.
d. They enhance phagocytosis.
13. The action of CRP can be distinguished from that of an
antibody because
a. CRP acts before the antibody appears.
b. only the antibody triggers the complement
cascade.
c. binding of the antibody is calcium-dependent.
d. only CRP acts as an opsonin.

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Christine Joy R. Timbang | 0963-467-6983
IMMUNOSEROLOGY 4.

Adaptive Immunity
OUTLINE • Migration occurs in waves and is driven by chemical
I. Introduction messengers called chemokines.
A. Definitions o Chemokines are a large family of cytokines that
II. T-Cell Differentiation have the ability to recruit specific cells to a
A. Introduction particular site.
B. Double-Negative Stage • Once in the thymus, precursors that are committed to
C. Double-Positive Stage becoming T cells are known as thymocytes.
D. Mature T Cells • As thymocytes travel through the thymus, there is an
III. Stages of B-Cell Differentiation orderly rearrangement of the genes coding for the antigen
A. Pro-B Cells receptor.
B. Pre-B Cells o At the same time, distinct surface markers appear
C. Immature B Cells during specific stages of development.
D. Mature B Cells • Maturation is an elaborate process that takes place over a
E. Plasma Cells 3-week period as cells filter through the cortex to the
IV. The Role of T Cells in The Adaptive Immune medulla.
Response • Thymic stromal cells include epithelial cells,
A. Introduction macrophages, fibroblasts, and dendritic cells, all of which
B. Action of T helper Cells play a role in T-cell development.5 Interaction with stromal
C. Action of Cytotoxic T Cells cells under the influence of cytokines, especially
V. The Role of B Cells in The Adaptive Immune interleukin-7 (IL-7), is critical for growth and differentiation.
Response • A positive and negative selection process occurs as
A. Response to T-Dependent Antigens maturation takes place because an estimated 97% of the
VI. Laboratory Identification of Lymphocytes cortical cells die intrathymically before becoming mature T
A. Introduction cells.
B. Cell flow cytometry
C. Point-of-care testing

INTRODUCTION
A. Definitions
Innate immunity
• type of resistance characterized by:
o Specificity for each individual pathogen or
microbial agent
o The ability to remember a prior exposure
o An increased response to that pathogen upon
repeated exposure
• more specific and longer lasting

Lymphocyte
• key cell involved in the adaptive immune response
• Types
o T cells - mature in the thymus and serve a
regulatory role by providing help to B cells in
responding to antigens as well as by killing virally
infected target cells
B. Double-Negative Stage
o B cells - mature in the bone marrow and
differentiate into plasma cells that produce • double-negative (DN) thymocytes
antibodies o early thymocytes that lack CD4 and CD8 markers
o actively proliferate in the outer cortex under the
influence of IL-7
T-CELL DIFFERENTIATION • Rearrangement of the genes that code for the antigen
A. Introduction receptor known as the T-cell receptor (TCR) begins at this
• T cells stage
o 60% to 80% of circulating lymphocytes o This random gene rearrangement is what builds in
o become differentiated in the thymus the diversity that allows T cells to respond to the
• Lymphocyte precursors enter the thymus from the bone myriads of different antigens that the body might
marrow. encounter in a lifetime.
• Within the lobules of the thymus are two main zones, the • Two specific chains of TCR
outer cortex and the inner medulla. o (1) alpha (α) and (2) beta (β) chains
o Early precursors enter the thymus at the cortico- § both contain variable regions that recognize
medullary junction and migrate to the outer cortex. specific antigens

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 § coded for by the selection of certain gene
segments and deletion of others in a random
fashion
• appearance of the β chain → triggers
the thymocyte to become CD4–positive
(CD4+) and CD8–positive (CD8+)
§ occur with six other chains that are common
to all T cells
• six chains of the nonspecific CD3
portion of the complex assist in
signaling when an antigen binds to the
T cells
o delta-epsilon (δ–ε)
o gamma-epsilon (γ–ε)
o tau-tau (ζ–ζ)
• CD3/TCR complex - sum of third eight chains
• pre-TRC receptor - combination of the β chain with the
rest of CD3
• allelic exclusion - selection of an allele on one
chromosome only

T-cell maturation in the thymus.

T-lymphocyte precursors (TP) enter the thymus at the
corticomedullary junction. They migrate upward in the cortex and
begin development of the T-cell receptor. A small percentage of
precursors develop gamma-delta chains, whereas the majority
develop alpha-beta chains and become double-positive (DP) (both
CD4 and CD8 are present). Positive and negative selection takes
place through the CD3/T-cell receptor for antigen. If positively
selected, the T cell becomes single-positive (SP); that is, either CD4+
or CD8+. Further interactions with macrophages or dendritic cells take
place to weed out any T cells able to respond to self-antigen.
Surviving CD4+ and CD8+ cells exit the thymus to the peripheral
blood.

C. Double-Positive Stage
• second stage
• double-positive (DP) thymocytes
o thymocytes express both CD4 and CD8 antigens
• Young DP thymocytes begin to rearrange the genes
coding for the α chain.
• When the CD3-αβ receptor complex (TCR) is complete
and expressed on the cell surface, a positive selection
process takes place that allows only DP cells with
functional TCR receptors to survive.
o T cells must recognize foreign antigen in
association with class I or class II MHC molecules.
o When thymocytes bind to self-MHC antigens in the
cortex by means of the newly formed TCR
receptors, an enzyme cascade involving a group of
enzymes called kinases is activated.
§ Enzyme activity causes changes in cell
shape and motility that lead to increased
cell survival.3 The selection of thymocytes
that will only interact with the MHC antigens

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 found on host cells is known as MHC D. Mature T Cells
restriction. • Survivors of selection exhibit only one type of marker,
o Any thymocytes that have either a very low or a either CD4 or CD8.
very high affinity for self-MHC antigens die by
apoptosis. CD4+ T cells
§ This weeding out is important, because • recognize antigen along with class II MHC protein
functioning T cells must be able to • bearing the CD4 receptor are T helper (Th) cells
recognize foreign antigen along with MHC • two-thirds of peripheral T cells
molecules.
• A second selection process, known as negative selection, T helper (Th) Subsets
takes place among the surviving DP T cells. 1. Th1 cells
o This second selection process takes place in the • produce interferon gamma (IFN-γ), interleukin-2 (IL-2),
corticomedullary region and the medulla of the and tumor necrosis factor-β (TNF-β
thymus as medullary epithelial cells express a wide o protect cells against intracellular pathogens by
variety of self-antigens activating cytotoxic lymphocytes and macrophages
o Strong reactions with self-peptides other than MHC
antigens triggers apoptosis. The process of 2. Th2 cells
elimination of clones of T cells that would be • produce IL-4, IL-5, IL-6, IL-9, IL-10, and IL-13
capable of an autoimmune response is called clonal
• help B cells produce antibodies
deletion.
• against extracellular pathogens and to generally regulate
B cell activity

3. Th9 cells
• produce interleukin-9 (IL-9)
• have a proinflammatory effect
• play a role at epithelial surfaces by warding off fungi and
extracellular bacteria
• in the process, they stimulate growth of hematopoietic
cells, especially mast cells; as such, they may promote
autoimmune inflammation

4. Th17 cells
• produce interleukin-17 (IL-17) and interleukin-22 (IL-22)
o increase inflammation and joint destruction
• associated with autoimmune diseases such as
rheumatoid arthritis, multiple sclerosis, and inflammatory
bowel disease

T regulatory (Treg) cells

Positive selection of thymocytes in the cortex. • additional T-cell subpopulation
Double-positive (CD4+ and CD8+) thymocytes interact with thymic • possess the CD4 antigen as well as CD25
epithelial cells. If very strong bonding occurs, cells are eliminated by • play an important role in suppressing the immune
apoptosis. If very weak or no bonding occurs, cells are also response to self-antigens
eliminated.
• inhibit proliferation of other T-cell populations by secreting
inhibitory cytokines and the response is antigen-specific

CD8+ T cells
• interact with antigen and class I MHC proteins
• consists of cytotoxic T (Tc) cells
• one-third of peripheral T cells

Negative selection of thymocytes in the medulla.

When self-antigen is presented by a macrophage, dendritic cell, or
thymic epithelial cell to a thymocyte, if the T-cell receptor (TCR) binds,
the thymocyte is eliminated by apoptosis.

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 Several transcription or growth factors are necessary to
differentiate common lymphoid precursors into pro-B cells:
• E2A
• EBF (early B-cell factor)
• interferon regulatory factor (IFR8)
• paired box protein 5 (PAX5)
• interleukin-7 (IL-7)

• During this maturation process, the first step in the pro-B

phase is the rearrangement of genes that code for the
heavy and light chains of an antibody molecule.
• Although portions of each chain are identical for every
antibody molecule, it is the so-called variable regions that
make each antibody molecule specific for a certain
antigen or group of antigens.
• Rearrangement of the DNA by cutting out certain regions
is similar to the process that occurs in T cells, where
antigen specificity is built into the α and β chains of the
TCR for antigen.
• Heavy chains of antibody molecules are coded for on
chromosome 14 and light chains are coded for on
chromosomes 2 and 22.
• Gene rearrangement of the DNA that codes for antibody
production occurs in a strict developmental sequence.
• Rearrangement of genes on chromosome 14, which code
for the heavy-chain part of the antibody molecule, takes
place first in a random fashion.
• C-Kit
o a receptor on the pro-B cell
o interacts with a cell surface molecule called stem
cell factor found on stromal cells.
§ This interaction triggers the activation
process.
T helper (Th) subsets. • The DNA is cleaved randomly at certain possible
Depending upon the type of pathogen encountered, antigen- recombination sites and the enzyme terminal
presenting cells (APCs) secrete a specific combination of polarizing deoxyribonucleotidyl transferase (TdT) helps to join the
cytokines that direct naïve CD4+ Th cells to further differentiate into
pieces back together by incorporating additional
one of five subsets: Th1, Th2, Treg, Th9, or Th17. These specialized
T cells release different types of cytokines to coordinate an nucleotides in the joining areas.
appropriate immune response against the pathogen. • Differentiation of pro-B cells into pre-B cells occurs upon
successful rearrangement of heavy-chain genes on one of
STAGES OF B-CELL DIFFERENTIATION the number 14 chromosomes.
o If rearrangement of genes on the first chromosome
A. Pro-B Cells
14 is not successful, then rearrangement of genes
• Surface markers: CD19, CD24, CD45R, CD43, C-kit
on the second chromosome 14 occurs.
o If neither rearrangement is successful,
B cells
development of the cell is halted.
• derived from a hematopoietic stem cell that develops into • Only pro-B cells that successfully rearrange one set of
an early lymphocyte progenitor in the bone marrow heavy-chain genes go on to become pre-B cells.
• remain and mature in the bone marrow itself
• B-cell precursors go through a developmental process of B. Pre-B Cells
three phases: • Surface markers: SAME, no CD43 and C-kit
o Development of mature immunocompetent B cells • µ chains
o Activation of B cells by antigen o first heavy chains synthesized
o Differentiation of activated B cells into plasma o belong to the class of immunoglobulins called
cells, which produce antibodies immunoglobulin M (IgM)
o accumulate in the cytoplasm
Antigen-independent phase § may also express µ chains on the cell
• first phase of B-cell development in the bone marrow surface, accompanied by an unusual light
• results in mature B cells that have not yet been exposed chain molecule called a surrogate light chain
to antigen • consist of two short polypeptide
• This phase can be divided according to formation of chains along with two shorter
• several distinct subpopulations: chains, Ig-α and Ig-β (signal-
o pro-B cells (progenitor B cells) transducing subunits)
o pre-B cells (precursor B cells) • Signaling through the pre-B receptors formed stimulates a
o immature B cells burst of clonal expansion.
o mature B cells

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• If, however, gene rearrangement does not work, then B- • Nucleus: eccentric or oval with heavily clumped chromatin
cell development is halted and cells are destroyed by that stains darkly
apoptosis. • abundant endoplasmic reticulum and a clear well-defined
Golgi zone are present in the cytoplasm
C. Immature B Cells • most fully differentiated lymphocyte
• Surface markers: SAME, with CD40 and MHC complex • Main function: antibody production
• CD21 • not normally found in the blood
o acts as a receptor for a breakdown product of the o located in germinal centers in the peripheral
complement component C3, known as C3d lymphoid organs or they reside in the bone
o enhances the likelihood of contact between B cells marrow.
and antigens because antigens frequently become • In the bone marrow, plasma cells can survive in niches
coated with complement fragments during the surrounded by stromal cells.
immune response • Stromal cells provide chemical stimulation by cytokines,
• distinguished by the appearance of complete IgM which allow plasma cells to be long-lived and continually
antibody molecules on the cell surface produce antibodies.
• indicates that rearrangement of the genetic sequence
coding for light chains on either chromosome 2 or 22 has
taken place by this time
• Once surface immunoglobulins appear, µ chains are no
longer detectable in the cytoplasm.
• At this stage, there is evidence that self-antigens give a
negative signal to immature B cells.
• Immature B cells that tightly bind self-antigens through
cross-linking of surface IgM molecules receive a signal to
halt development, resulting in arrested maturation and cell
death.
• The elimination of B cells that bear self-reactive receptors
is known as central tolerance.
• Immature B cells that survive this selection process leave
the bone marrow and proceed to the spleen, where they
become mature B cells.

D. Mature B Cells
• In the spleen, immature B cells develop into mature cells
known as either:
o marginal zone B cells
§ remain in the spleen in order to respond
quickly to any blood-borne pathogens they
may come into contact with B-cell development in the bone marrow.
o follicular B cells Selected markers are shown for the various stages in the
§ migrate to lymph nodes and other secondary differentiation of B cells. Stages up to the formation of mature B cells
organs occur in the bone marrow. (A) Pro-B cell. (B) Pre-B cell. (C) Immature
B cell. (D) Mature B cell.
§ constantly recirculating throughout the
secondary lymphoid organs
• Immunoglobulins: IgM and IgD
o Both IgM and IgD have the same specificity for a
particular antigen or group of antigens.
o These surface immunoglobulins provide the primary
activating signal to B cells when contact with
antigen takes place.
o IgD - not required for B-cell function, but it may
prolong the life span of mature B cells in the
periphery.
• If, however, a B cell is stimulated by antigen, it undergoes
• transformation to a blast stage that eventually forms
memory cells and antibody-secreting plasma cells.
o This process is known as the antigen-dependent
phase of B-cell development.
• The production of antibodies by plasma cells is called
humoral immunity.

E. Plasma Cells B-cell activation in peripheral lymph nodes.

B cells capture specific antigen by means of immunoglobulin
• spherical or ellipsoidal cells receptors. The activity of cytokines produced by Th cells produces
• 10 and 20 µm transformation of naïve B cells into antibody-producing plasma cells
• characterized by the presence of abundant cytoplasmic and memory cells.
immunoglobulin and little to no surface immunoglobulin

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 • Two major ways of killing target cells:
o release the contents of granules that damage the
cell; or
o they bind to the host cell and, using intracellular
signaling, induce apoptosis.
§ In either case, the target cell is induced to
undergo apoptosis, usually within 30 minutes
of contact
• Granules within cytotoxic T cells contain two different
types of toxins: granzymes and perforins.
o granzymes - class of enzymes called serine
o proteases perforins - pore-forming proteins that
insert themselves into the target cell membrane

THE ROLE OF T CELLS IN THE ADAPTIVE IMMUNE

RESPONSE
A. Introduction
• Interaction between T cells and antigen-presenting cells
(APCs) - initiating event in the adaptive immune response
• T cells circulate continuously through the blood-stream,
lymph nodes, and secondary lymphoid tissue searching
for antigen
• Each naïve or unstimulated T cell circulates from the
lymph nodes to the blood and back again within 12 to 24
hours.

B. Action of T helper Cells

• CD4+ T cell encounters an antigen along with a class II
MHC molecule and binds by using its antigen receptor
o first activating signal to induce transformation of a
T cell
• second signal: binding of CD28 on the T cell with CD80 Activation of Th cells.
and CD86 found on APCs Exposure to antigen presented by macrophages causes production of
• T memory cells CD25 receptors for interleukin-2 (IL-2). IL-2 causes sensitized CD4+
o arise early in the course of an immune response T cells to secrete cytokines, resulting in CD4+ effector cells that have
o may arise independently from effector cells or they various functions. Some CD4+ cells secrete interleukins that recruit
may arise as soon as the original T cell is macrophages and neutrophils, whereas others activate CD8+ T cells
stimulated. to increase cytotoxicity against virally infected cells. Activated Th
cells also enhance antibody production by B cells.
o have a higher affinity for antigen than unstimulated
T cells and are capable of immediate cytokine
production when they reencounter the initiating
antigen
o able to proliferate sooner than naïve T cells,
express a broader array of cytokines, and appear
to persist for years.

C. Action of Cytotoxic T Cells

• leave the secondary lymphoid tissue and circulate to sites
of infection → bind and kill infected cells by triggering
apoptosis
• Because all nucleated host cells express class I antigens
on their surfaces, cytotoxic T cells act as a primary
defense against intracellular pathogens such as viruses,
as well as other altered host cells such as tumor cells that
exhibit new antigens.
• The cytotoxic T cells bind to altered host cells by using the
TCR receptor for antigen and by CD8, which recognizes
class I MHC molecules
• Naïve CD8+ T cells require 5 to 8 days after antigen
activation to differentiate into cytotoxic lymphocytes.

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 • Within the follicles, however, B cells undergo further
differentiation under the influence of follicular Th cells.
The CD40 on B cells must interact with CD40L on Th cells
in order for germinal center formation to occur. Here, both
plasma cells and memory cells are formed.

THE ROLE OF B CELLS IN THE ADAPTIVE IMMUNE

RESPONSE
A. Response to T-Dependent Antigens
• B cells require two signals to be activated
o First signal: occurs when antigen binds to
membrane immunoglobulin receptors on the B-cell
surface and cross links them.
§ Cross-linking leads to activation of
intracellular signaling pathways that allow
stimulated B cells to interact with T cells.
o Second signal: provided by Th cells themselves,
which bind to the B cell both through its antigen
receptor and through CD40 on the B cell and
CD40L on the activated Th cell.
§ The bound T cell then delivers cytokines
and other signals to fully activate the B cell.

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 o receptors on a B cell to cross-link them and induce
proliferation and antibody production
o produce IgM only because the induction of
memory cells does not occur to any great extent
• e.g. plant lectins, polymerized proteins with repeating
molecular patterns, and lipopolysaccharides found in
bacterial cell walls

LABORATORY IDENTIFICATION OF LYMPHOCYTES

A. Introduction
• immunodeficiency diseases such as X-linked
hypogammaglobulinemia → B cells are frequently absent
• severe combined immunodeficiency disease (SCID) →
both T and B cells are either absent or present in very low
numbers
• Because the human immunodeficiency virus (HIV) infects
and progressively kills CD4+ T cells, assays for CD4+ T
cells are useful in evaluating the stage of infection

B. Cell flow cytometry

• gold standard for testing
• automated system for identifying cells based on the
scattering of light as cells in a stream of fluid flow in single
file by a laser beam.
• able to segregate lymphocytes into subsets using a
technique that relies on labeled monoclonal antibodies
against specific surface antigens

C. Point-of-care testing
• using either fluorescent or antibody-labeled beads
• measure the CD4 count and report it as a percentage of
the total T-cell count

Comparison of T and B Cells

T Cells B Cells
Develop in the thymus Develop in the bone
marrow
Found in blood (60–80% of Found in bone marrow,
circulating lymphocytes), spleen, lymph nodes
T- and B-cell cooperation in the immune response. thoracic duct fluid, lymph
CD4+ T cells recognize exogenous antigen on a macrophage along nodes
with class II MHC. Binding between CD28 and B7 enhances Identified by rosette Identified by surface
interaction between the cells. Th cells go through clonal expansion formation with SRBCs immunoglobulin
and produce cytokines, including interleukin-2 (IL-2). B cells capable End products of activation End product of activation is
of responding to the same antigen present antigen to Th cells through are cytokines antibody
the class II MHC receptor. The TCR binds antigen and CD4 binds to
class II MHC. CD40L bind to CD40, enhancing the reaction. Cytokine
Antigens include CD2, Antigens include CD19,
production by the T cell causes B cells to proliferate and produce CD3, CD4, CD8 CD20, CD21, CD40, class
plasma cells, which secrete antibody. II MHC
Located in paracortical Located in cortical region of
Memory cells region of lymph nodes lymph nodes
• progeny of B cells that have been exposed to antigen *SRBC = Sheep red blood cells
• characterized by a long life span and a rapid response to
Review Questions
• second exposure to the triggering antigen
1. Which MHC molecule is necessary for antigen
• similar in appearance to unstimulated B cells, but they
recognition by CD4+ T cells?
remain in an activated state for months or years, ready to
a. Class I
respond to the initial antigen.
b. Class II
• CD27 - marker to identify memory cells because they are
c. Class III
similar in appearance to mature B cells. d. No MHC molecule is necessary.
2. Which would be characteristic of a T-independent
B. Response to T-Independent Antigens
antigen?
• T-independent antigens a. The IgG antibody is produced exclusively.
o able to elicit antibody formation in the absence of b. A large number of memory cells are produced.
T cells c. Antigens bind only one receptor on B cells.
o able to interact with multiple immunoglobulin d. It consists of a limited number of repeating
determinants.

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3. Humoral immunity refers to which of the following? a. It consists of IgM and IgD molecules.
a. Production of antibody by plasma cells b. It is the same for all T cells.
b. Production of cytokines by T cells c. It is present in the double-negative stage.
c. Elimination of virally infected cells by cytotoxic d. Alpha and beta chains are unique for each
cells antigen.
d. Downregulation of the immune response 15. A cell flow cytometry pattern belonging to a 3-year-old
4. Where does antigen-independent maturation of B patient showed the following: normal CD4+ T-cell count,
lymphocytes take place? normal CD19+ B-cell count, low CD8+ T-cell count.
a. Bone marrow Which type of immunity would be affected?
b. Thymus a. Production of antibody
c. Spleen b. Formation of plasma cells
d. Lymph nodes c. Elimination of virally infected cells
5. In the thymus, positive selection of immature T cells is d. Downregulation of the immune response
based upon recognition of which of the following? 16. Which of the following is a unique characteristic of
a. Self-antigens adaptive immunity?
b. Stress proteins a. Ability to fight infection
c. MHC antigens b. Ability to remember a prior exposure to a
d. µ chains pathogen
6. Which of these are found on a mature B cell? c. A similar response to all pathogens encountered
a. IgG and IgD d. Process of phagocytosis to destroy a pathogen
b. IgM and IgD 17. Clonal deletion of T cells as they mature is important in
c. Alpha and beta chains which of the following processes?
d. CD3 a. Elimination of autoimmune responses
7. How do cytotoxic T cells kill target cells? b. Positive selection of CD3/TCR receptors
a. They produce antibodies that bind to the cell. c. Allelic exclusion of chromosomes
b. They engulf the cell by phagocytosis. d. Elimination of cells unable to bind to MHC
c. They stop protein synthesis in the target cell. antigens
d. They produce granzymes that stimulate 18. Where do germinal centers occur?
apoptosis. a. In the thymus
8. Which of the following can be attributed to antigen- b. In the bone marrow
stimulated T cells? c. In peripheral blood
a. Humoral response d. In lymph nodes
b. Plasma cells
c. Cytokines
d. Antibody
9. Which is a distinguishing feature of a pre-B cell?
a. µ chains in the cytoplasm
b. Complete IgM on the surface
c. Presence of CD21 antigen
d. Presence of CD25 antigen
10. When does genetic rearrangement for coding of
antibody light chains take place during B-cell
development?
a. Before the pre-B cell stage
b. As the cell becomes an immature B cell
c. Not until the cell becomes a mature B cell
d. When the B cell becomes a plasma cell
11. Which of the following antigens are found on the T-cell
subset known as helper/inducers?
a. CD3
b. CD4
c. CD8
d. CD11
12. Where does the major portion of antibody production
occur?
a. Peripheral blood
b. Bone marrow
c. Thymus
d. Lymph nodes
13. Which of the following would represent a double-
negative thymocyte?
a. CD2–CD3+CD4–CD8+
b. CD2–CD3–CD4+CD8–
c. CD2+CD3+CD4–CD8–
d. CD2–CD3–CD4+CD8–
14. Which of the following best describes the T-cell receptor
for antigen?

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Christine Joy R. Timbang | 0963-467-6983
IMMUNOSEROLOGY 5.

Antibody Structure and Function

OUTLINE TETRAPEPTIDE STRUCTURE OF
I. Introduction IMMUNOGLOBULINS
A. Antibody A. Introduction
II. Tetrapeptide Structure of Immunoglobulins • All immunoglobulin molecules are made up of a basic four
A. Introduction chain tetrapeptide unit
B. Rodney Porter o two large chains called heavy or H chains
C. Alfred Nisonoff § 3.5 S fraction (work by Gerald Edelman)
III. The Nature of Light Chains § molecular weight of approximately 50,000
IV. Heavy-Chain Sequencing o two smaller chains called light or L chains
V. Hinge Region § 2.2 S fraction
VI. Three-Dimensional Structure of Antibodies § molecular weight of 22,000,
A. Introduction • Each chain has a single variable region and one or more
B. Immunoglobulin G (IgG) constant regions.
C. Immunoglobulin M (IgM) • variable region
D. Immunoglobulin A (IgA) o unique to each specific antibody
E. Immunoglobulin D (IgD) o held together by noncovalent forces and disulfide
F. Immunoglobulin E (IgE) interchain bridges
VII. Theories To Explain Antibody Diversity • intact IgG molecules - had a sedimentation coefficient of 7
A. Ehrlich’s Side-Chain Theory S
B. Clonal Selection Hypothesis
VIII. Genes Coding for Immunoglobulins B. Rodney Porter
A. Introduction
• based on the use of the proteolytic enzyme papain
B. Rearrangement of Heavy-Chain Genes
• Carboxymethyl cellulose ion exchange chromatography
C. Light Chain Rearrangement
separated this material into two types of fragments, one of
IX. Monoclonal Antibody
which spontaneously crystallized at 4°C.
A. Introduction
o This fragment, known as the Fc fragment (for
B. Hybridomas
“fragment crystallizable”), had no antigen-binding
C. Clinical Applications
ability and is now known to represent the carboxy-
terminal halves of two H chains that are held
INTRODUCTION together by S–S bonding.
A. Antibody o The Fc fragment is important in effector functions
• also known as an immunoglobulin of immunoglobulin molecules, which include
• glycoproteins found in the serum portion of the blood opsonization and complement fixation.
• constitute approximately 20% of plasma proteins in o Fab fragments
healthy individuals § remaining two identical fragments
• composed of: § were found to have antigen-binding
o 86% to 98% polypeptide capacity
o 2% to 14% carbohydrate § Each Fab fragment thus consists of one L
• slowest moving proteins and appear primarily in the chain and one-half of an H chain held
gamma (γ) band together by disulfide bonding
• main humoral element of the adaptive immune response
• play an essential role in antigen recognition and in C. Alfred Nisonoff
biological activities related to the immune response such • used pepsin to obtain additional evidence for the structure
as opsonization and complement activation of immunoglobulins
• This proteolytic enzyme was found to cleave IgG at the
Five major classes on the basis of a part of the molecule carboxy-terminal side of the interchain disulfide bonds,
called the heavy chain: yielding one single fragment with a molecular weight of
1. IgG - heavy chain γ 100,000 d and all the antigen-binding ability, known as
2. IgM - heavy chain μ F(ab')2
3. IgA - heavy chain α • FC'
4. IgD - heavy chain δ o additional fragment called
5. IgE - heavy chain ε o was similar to FC except that it disintegrated into
• each type of immunoglobulin is made up of a number of several smaller pieces
regions called domains
o consists of approximately 110 amino acids each \ Join works → each L chain was bonded to an H
chain by means of an S–S bond and the H chains
were joined to each other by one or more S–S
bonds

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 o Heating to 80°C, they redissolve.
• Two main types of L chains
o kappa (κ) chains
o lambda (λ) chains
• Each contained between 200 and 220 amino acids
• from position number 111 onward
• it was discovered that each type had essentially the same
sequence
• This region was called the constant region and the amino-
terminal end was called the variable region.
• The difference between the κ and λ chains lies in the
amino acid substitutions at a few locations along the
chain.
• There are no functional differences between the two
types. Both κ and λ L chains are found in all five classes
of immunoglobulins, but only one type is present in a
given molecule.

HEAVY-CHAIN SEQUENCING
• demonstrates the presence of domains similar to those in
the L chains—that is, variable and constant regions.
• The first approximately 110 amino acids at the amino-
terminal end constitute the variable domain
• The remaining amino acids can typically be divided up
into three or more constant regions with very similar
sequences, designated
o CH1
o CH2
o CH3
• Each of these represents an isotype, a unique amino acid
sequence that is common to all immunoglobulin
molecules of a given class in a given species.
• Minor variations of these sequences that are present in
some individuals but not others are known as allotypes
o Allotypes occur in the four IgG subclasses, in one
IgA subclass, and in the κ L chain.
o These genetic markers are found in the constant
region and are inherited in simple Mendelian
fashion.
o Some of the best-known examples of allotypes are
variations of the γ chain known as G1m3 and
G1m17.
• The variable portions of each chain are unique to a
specific antibody molecule, and they constitute what is
known as the idiotype of the molecule.
• The amino-terminal ends of both L and H chains contain
these regions, which are essential to the formation of the
antigen-binding site. Together they serve as the antigen-
recognition unit.

THE NATURE OF LIGHT CHAINS

• Bence Jones proteins
o found in the urine of patients with multiple myeloma
o When heated to 60°C, they precipitate from urine

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 HINGE REGION
• segment of H chain located between the CH1 and CH2
regions
• has a high content of proline and hydrophobic residues
• high proline content allows for flexibility
• ability to bend lets the two antigen-binding sites operate
independently and engage in an angular motion relative to
each other and to the FC stem
o Gamma, delta, and alpha chains - all have a hinge
region
o Mu and epsilon chains - do not
• All types of immunoglobulins contain a carbohydrate
portion, which is localized between the CH2 domains of
the two H chains.
o Functions of the carbohydrate include
§ increasing the solubility of immunoglobulin
§ providing protection against degradation
§ enhancing functional activity of the FC
domains

THREE-DIMENSIONAL STRUCTURE OF ANTIBODIES

A. Introduction
• folded into compact globular subunits based on the
formation of balloon-shaped loops at each of the domains
• Intrachain disulfide - bonds stabilize these globular
regions
• Within each of these regions or domains, the polypeptide
chain is folded back and forth on itself to form what is
called a β-pleated sheet.
• The folded domains of the H chains line up with those of
the L chains to produce a cylindrical structure called an
immunoglobulin fold.
• Antigen is captured within the fold by binding to a small
number of amino acids at strategic locations on each
chain known as hypervariable regions. B. Immunoglobulin G (IgG)
o Three small hypervariable regions consisting of • predominant immunoglobulin in humans
approximately 30 amino acid residues are found
• has the longest half-life of any immunoglobulin class
within the variable regions of both H and L chains.
o Half-life: approximately 23 days
§ Each of these regions, called
• differ mainly in the number and position of the disulfide
complementarity-determining regions
bridges between the γ chains
(CDRs), is between 9 and 12 residues
long. • high diffusion coefficient that allows it to enter
extravascular spaces more readily than other
• They occur as loops in the folds of
immunoglobulin types
the variable regions of both L and H
chains. • Agglutination and precipitation reactions take place invitro
o The antigen-binding site is • better at precipitation reactions than at agglutination
actually determined by the because precipitation involves small soluble particles,
apposition of the six which are more easily brought together by the relatively
hypervariable loops, three small IgG molecule
from each chain.
Subclasses
1. IgG1
• 66%
• second most efficient at binding complement
• induced in response to protein antigens
• particularly good at initiating phagocytosis, because they
bind most strongly to FC receptors

2. IgG2
• 23%
• have shorter hinge segments
• poor mediators of complement activation
• All subclasses have the ability to cross the placenta
except IgG2.
• associated with polysaccharide antigens

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3. IgG3 • a glycoprotein made in plasma cells that contains several
• 7% cysteine residues
• has the largest hinge region and the largest number of • serve as linkage points for disulfide bonds between two
interchain disulfide bonds adjacent monomers
• most efficient at binding complement • One J chain is present per pentamer
• induced in response to protein antigens
• particularly good at initiating phagocytosis, because they Primary response
bind most strongly to FC receptors • predominantly IgM
• characterized by a long lag phase, a slow increase in
4. IgG4 antibody, and a short-lived response
• 4%
• have shorter hinge segments Secondary response
• poor mediators of complement activation • mainly IgG
• associated with polysaccharide antigens • or anamnestic response
• distinguished by a shortened lag period, a much more
\ All subclasses are able to participate in the rapid rise in antibody, and higher serum levels for a longer
secondary immune response, an enhanced and period of time.
quicker response to antigen, although their • result of the larger number of antigen-specific memory T
appearance depends on the triggering antigen. and B cells generated during the primary response

Major functions of IgG Functions of IgM

• Providing immunity for the newborn because IgG is the • complement fixation
only antibody that can cross the placenta • agglutination
• Fixing complement • opsonization
• Coating antigen for enhanced phagocytosis (opsonization) • toxin neutralization
• Neutralizing toxins and viruses
• Participating in agglutination and precipitation reactions

C. Immunoglobulin M (IgM)
• known as a macroglobulin because it has a sedimentation
rate of 19 S
• molecular weight of approximately 900,000
• Half-life: 6 days
D. Immunoglobulin A (IgA)
• If IgM is treated with mercaptoethanol → dissociates into
• appears as a monomer with a molecular weight of
• five 7 S units, each having a molecular weight of 190,000 approximately 160,000
and a four-chain structure that resembles IgG
• has a sedimentation coefficient of 7 S
o pentamer form - found in serum
• migrates between the β and γ regions on electrophoresis
o monomer form - occurs on the surface of B cells
• Major role: anti-inflammatory agent
• assumes a starlike shape with 10 functional binding sites
• IgA dimer - consists of two monomers held together by a J
• found mainly in the intravascular pool and not in other
chain
body fluids or tissues
• Secretory IgA - synthesized in plasma cells found mainly
• cannot cross the placenta
in mucosal-associated lymphoid tissue and is released in
• primary response antibody
dimeric form.
• first to appear after antigenic stimulation and the first to
• not capable of fixing complement by the classical pathway
appear in the maturing infant
• synthesized only as long as antigen remains present Subclasses
because there are no memory cells for IgM
1. IgA1
o Thus, IgM can be used to diagnose an acute
infection, as its presence indicates a primary • 13 amino acids of 22 are located in the hinge region
exposure to antigen • deleted in IgA2
• most efficient of all immunoglobulins at triggering the • mainly found in serum
classical complement pathway
2. IgA2
J or joining chain • 13 amino acids of 22 are deleted in IgA2
• joins the five monomeric units

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• more resistant to some bacterial proteinases that are able • second type of immunoglobulin to appear
to cleave IgA1 • Those cells bearing only IgM receptors appear incapable
• predominant form in secretions at mucosal surfaces of an IgG response, whereas those with both IgM and IgD
• found as a dimer along the respiratory, urogenital, and receptors are capable of responding to T-cell help and
intestinal mucosa; it also appears in breast milk, switching to synthesis of IgG, IgA, or IgE.5
colostrum, saliva, tears, and sweat o Thus, IgD may play a role in regulating B-cell
maturation and differentiation.
secretory component (SC) • more susceptible to proteolysis than other
• later attached to the FC region around the hinge portion of immunoglobulins (because of its unusually long hinge
the α chains region)
• This protein, consisting of five immunoglobulin-like • does not appear to serve a protective function because it
domains, is derived from epithelial cells found in close does not bind complement, it does not bind to neutrophils
proximity to the plasma cells. or macrophages, and it does not cross the placenta
• Once binding takes place, IgA and SC precursor are
taken inside the cell and then released to the opposite F. Immunoglobulin E (IgE)
surface by a process known as transcytosis. • very low concentration in serum and the fact that it has
the ability to activate mast cells and basophils
Functions of IgA • least abundant immunoglobulin in the serum
• plays an important role in neutralizing toxins produced by • molecular weight of 190,000
microorganisms and helps to prevent bacterial and viral • 8 S molecule
adherence to mucosal surfaces • has a carbohydrate content of 12%
• H chain is composed of around 550 amino acids that are
distributed over one variable and four constant domains
• A single disulfide bond joins each ε chain to an L chain
and two disulfide bonds link the H chains to one another.
• most heat-labile of all immunoglobulins
o heating to 56°C for between 30 minutes and 3
hours results in conformational changes and loss
of ability to bind to target cells
• does not participate in typical immunoglobulin reactions
such as complement fixation, agglutination, or
opsonization
• incapable of crossing the placenta
• high-affinity FC ε RI receptors: basophils, Langerhans
cells, eosinophils, and tissue mast cells by means of
specific surface proteins (after synthesis it attaches to
these)
• appears to be a nuisance antibody; however, it may serve
a protective role by triggering an acute inflammatory
reaction that recruits neutrophils and eosinophils to the
area to help destroy invading antigens that have
penetrated IgA defenses

Action of IgE on mast cells.

(A) IgE binds to specific ε receptors on mast cells. (B) When antigen
E. Immunoglobulin D (IgD) bridges two nearby IgE molecules, the membrane is disturbed and
• found in a patient with multiple myeloma degranulation results. Chemical mediators are released.
• extremely scarce in the serum, representing less than
0.001% of total immunoglobulins THEORIES TO EXPLAIN ANTIBODY DIVERSITY
• synthesized at a low level A. Ehrlich’s Side-Chain Theory
• Half-life: 1 to 3 days • certain cells had specific surface receptors for antigen that
• molecular weight of approximately 180,000 were present before contact with antigen occurred.
• migrates as a fast γ protein • Once antigen was introduced, it would select the cell with
• found on the surface of immunocompetent but the proper receptors, combination would take place, and
unstimulated B lymphocytes then receptors would break off and enter the circulation as
antibody molecules.

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• New receptors would form in place of those broken off, region. When RNA synthesis occurs, one constant region is attached
after which this process could be repeated. to the VDJ combination. μ H chains are made first, but the cell retains
its capacity to produce immunoglobulin of another class.
• Although this represented a rather simplistic explanation
for antibody synthesis, two key premises emerged: first,
C. Light Chain Rearrangement
the lock-and-key concept of the fit of antibody for antigen,
and second, the idea that an antigen selected cells with • exhibit a similar genetic rearrangement, except they lack a
the built in capacity to respond to it. D region
• Although this theory did not explain the kinetics of the
immune response or the idea of immunologic memory, it
laid the foundation for further hypotheses.

B. Clonal Selection Hypothesis

• Niels Jerne and Macfarlane Burnet
• Key premise: individual lymphocytes are genetically
preprogrammed to produce one type of immunoglobulin
and that a specific antigen finds or selects those particular
cells capable of responding to it, causing them to
proliferate.
• Drawback: If separate genes were present to code for.
antibody to every possible antigen, an overwhelming
amount of DNA would be needed.
o Resolution by Dreyer and Bennett: Constant and
variable portions of immunoglobulin chains are
actually coded for by separate genes

GENES CODING FOR IMMUNOGLOBULINS

A. Introduction
• H chain genes - located on chromosome 14
• κ chain genes - chromosome 2
• λ chain genes - chromosome 22
• Rearrangement involves a cutting and splicing process
that gets rid of much of the intervening DNA, resulting in a
functional gene that codes for a specific antibody. Assembly and expression of the κ L chain locus.
A DNA rearrangement fuses one V segment to one J segment. The
B. Rearrangement of Heavy-Chain Genes VJ segment is then transcribed along with a unique C region to form
• The genes that code for the variable region are divided mature κ mRNA. Unarranged J segments are removed during RNA
into three groups splicing
o VH - at least 39 VH (variable) genes
o D - 27 functional D (diversity) genes MONOCLONAL ANTIBODY
o J - 6 J (joining) genes A. Introduction
• set of genes (C) • Monoclonal antibodies are derived from a single parent
o codes for the constant region antibody-producing cell that has reproduced many times,
o includes one gene for each H chain isotype thus forming a clone.
o located in the following order: Cμ, Cδ, Cγ3, Cγ1, • Every cell in the clone is just like every other cell; the
Cα1, Cγ2, Cγ4, Cε, and Cα2 antibody produced by each cell is exactly the same as
that of every other cell.
• This differs from the normal response to an antigen,
which is heterogeneous, because even a purified antigen
has multiple epitopes that stimulate a variety of B cells.

B. Hybridomas
• hybridoma - fusion of an activated B cell with a myeloma
cell
• Myeloma cells has a deficiency of the enzyme
hypoxanthine guanine phosphoribosyltransferase
(HGPRT) that makes it incapable of synthesizing
nucleotides from hypoxanthine and thymidine, which are
needed for DNA synthesis.
• The fact that these myeloma cells cannot make their own
DNA means that they will die out unless they are fused to
a plasma cell that has the enzymes necessary to
synthesize DNA.

Coding for immunoglobulin H chains.

Four separate regions on chromosome 14 code for H chains. DJ
regions are spliced first, and then this segmentis joined to a variable

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Hybridoma Production The Five Classes of Immunoglobulins
IgG IgM IgA IgD IgE
Most Primary Monomer Present Binds to
abundan response and dimer on B mast
t in antibody cells cells
serum
Able to Pentame Protects Role in Triggers
cross r with 10 mucosal B-cell allergic
placenta antibody- surfaces activatio respons
combinin n e
g sites
Increase Indicates Has Identifie Role in
s with acute secretory s mature respons
second infection compone B cells e to
exposur nt parasite
e s

Properties of Immunoglobulins
IgG IgM IgA IgD IgE
Molecular 150,0 900,0 160,0 180,0 190,0
weight 00 00 00 00 00
Sedimentat 7 S 19 S 7S 7S 8S
ion
coefficient
Serum 23 6 5 1-3 2-3
half-life
(days)
H chain γ μ α δ ε
H chain γ1, - α1, α2 - -
subclasses γ2,
γ3, γ4
Constant 3 4 3 3 4
domains (H
chain)
Compleme Yes Yes No No No
nt fixation

Review Questions
1. Which of the following is characteristic of variable
domains of immunoglobulins?
a. They occur on both the H and L chains.
b. They represent the complement-binding site.
Formation of a hybridoma in monoclonal antibody production. c. They are at the carboxy-terminal ends of the
A mouse is immunized and spleen cells are removed. These cells are molecules.
fused with nonsecreting myeloma cells and then plated in a restrictive d. They are found only on H chains.
medium. Only the hybridoma cells will grow in this medium where 2. All of the following are true of IgM except that it
they synthesize and secrete a monoclonal immunoglobulin specific a. can cross the placenta.
for a single determinant on an antigen.
b. fixes complement.
C. Clinical Applications c. has a J chain.
d. is a primary response antibody.
• Monoclonal antibodies were initially used for in vitro
3. How does the structure of IgE differ from that of IgG?
diagnostic testing.
a. IgG has a secretory component and IgE does
• e.g. pregnancy testing, which uses antibody specific for not.
the β chain of human chorionic gonadotropin, thereby b. IgE has one more constant region than IgG.
eliminating many false-positive reactions.
c. IgG has more antigen-binding sites than IgE.
• As therapeutic agents d. IgG has more light chains than IgE.
• Treatment for cancer 4. How many antigen-binding sites does a typical IgM
• Treatment for autoimmune disease molecule have?
a. 2

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 b. 4 16. Which of the following can be attributed to the clonal
c. 6 selection hypothesis of antibody formation?
d. 10 a. Plasma cells make generalized antibody.
5. Bence Jones proteins are identical to which of the b. B cells are preprogrammed for specific antibody
following? synthesis.
a. H chains c. Proteins can alter their shape to conform to
b. L chains antigen.
c. IgM molecules d. Cell receptors break off and become circulating
d. IgG molecules antibody.
6. A Fab fragment consists of 17. All of the following are true of IgE except that it
a. two H chains. a. fails to fix complement.
b. two L chains. b. is heat stable.
c. one L chain and one-half of an H chain. c. attaches to tissue mast cells.
d. one L chain and an entire H chain. d. is found in the serum of allergic persons.
7. Which antibody best protects mucosal surfaces? 18. Which best describes coding for immunoglobulin
a. IgA molecules?
b. IgG a. All genes are located on the same chromosome.
c. IgD b. L chain rearrangement occurs before H chain
d. IgM rearrangement.
8. Which of the following pairs represents two different c. Four different regions are involved in coding of
immunoglobulin allotypes? H chains.
a. IgM and IgG d. Lambda rearrangement occurs before kappa
b. IgM1 and IgM2 rearrangement.
c. Anti-human IgM and anti-human IgG 19. What is the purpose of HAT medium in the preparation
d. IgG1m3 and IgG1m17 of monoclonal antibody?
9. The structure of a typical immunoglobulin consists of a. Fusion of the two cell types
which of the following? b. Restricting the growth of myeloma cells
a. 2L and 2H chains c. Restricting the growth of spleen cells
b. 4L and 2H chains d. Restricting antibody production to the IgM class
c. 4L and 4H chains 20. Papain digestion of an IgG molecule results in which of
d. 2L and 4 H chains the following?
10. Which of the following are L chains of antibody a. 2 Fab' and 1 Fc' fragment
molecules? b. F(ab')2 and 1 Fc' fragment
a. Kappa c. 2 Fab and 2 Fc fragments
b. Gamma d. 2 Fab and 1 Fc fragment
c. Mu 21. Which antibody provides protection to the growing fetus
d. Alpha because it is able to cross the placenta?
11. If the results of serum protein electrophoresis show a a. IgG
significant decrease in the gamma band, which of the b. IgA
following is a likely possibility? c. IgM
a. Normal response to active infection d. IgD
b. Multiple myeloma 22. Which best characterizes the secondary response?
c. Immunodeficiency disorder a. Equal amounts of IgM and IgG are produced.
d. Monoclonal gammopathy b. There is an increase in IgM only.
12. The subclasses of IgG differ mainly in c. There is a large increase in IgG but not IgM.
a. the type of L chain. d. The lag phase is the same as in the primary
b. the arrangement of disulfide bonds. response.
c. the ability to act as opsonins.
d. molecular weight.
13. Which best describes the role of the secretory
component of IgA?
a. A transport mechanism across endothelial cells
b. A means of joining two IgA monomers together
c. An aid to trapping antigen
d. Enhancement of complement fixation by the
classical pathway
14. Which represents the main function of IgD?
a. Protection of the mucous membranes
b. Removal of antigens by complement fixation
c. Enhancing proliferation of B cells
d. Destruction of parasitic worms
15. Which antibody is best at agglutination and complement
fixation?
a. IgA
b. IgG
c. IgD
d. IgM

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IMMUNOSEROLOGY 6.

Cytokines
OUTLINE
I. Introduction To Cytokines
A. Cytokines
II. Cytokines In the Innate Immune Response
A. Introduction
B. List
III. Cytokines In the Adaptive Immune Response
A. Introduction
B. List Naming
C. Cytokines Associated with T Regulatory Cells • Initially, cytokines were named based on their activities
IV. Th17 Cytokines in Innate And Adaptive Immune and types of cells from which they were first isolated.
Responses o e.g cytokines released from lymphocytes were
V. Hematopoietic Growth Factors called lymphokines, cytokines released from
A. Introduction monocytes and macrophages were called
B. Colony stimulating factors (CSFs) monokines, and cytokines secreted by leukocytes
VI. Cytokine And Anticytokine Therapies that mainly act on other leukocytes were called
VII. Clinical Assays for Cytokines interleukins.
A. ELISpot assay • Now, are grouped into families
B. Others o tumor necrosis factor (TNF)
o interferon (IFN)
INTRODUCTION TO CYTOKINES o chemokine,
A. Cytokines o transforming growth factor (TGF)
• chemical messengers that regulate the immune system, o colony stimulating factor (CSF)
orchestrating both innate immunity and the adaptive o interleukins (IL)
response to infection.
• small proteins produced by several different types of cells Functions
that influence the hematopoietic and immune systems 1. Pleiotropy - single cytokine can have many different
through activation of cell-bound receptor actions
• are induced in response to the binding of stimuli, such as 2. Redundancy - when different cytokines activate some of
bacterial lipopolysaccharides, flagellin, or other bacterial the same pathways and genes
products, to specific cell receptors or through the 3. Synergistic - if the effects complement and enhance
recognition of foreign antigens by host lymphocytes each other
4. Antagonism - one cytokine may counteract the action of
Effects of cytokines in vivo another cytokine
5. Cascade induction - cytokine secreted by a specific type
• regulation of growth, differentiation, and gene expression
of cell can activate target cells to produce additional
by many different cell types, including leukocytes
cytokines
• These effects are achieved through both
o autocrine stimulation - affecting the same cell that
secreted paracrine activities - affecting a target cell
in close proximity
o endocrine activities - systemic

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(A) Cytokine characteristics of synergy, antagonism, and cascade CYTOKINES IN THE INNATE IMMUNE RESPONSE
induction. Synergy: Neither cytokine A nor B induce a strong response
A. Introduction
individually; however, when combined, the net response is much
greater than the sum of the individual responses. Antagonism: • responsible for many of the physical symptoms attributed
individually, cytokine C induces a strong response and cytokine A to inflammation, such as fever, swelling, pain, and cellular
induces a weak (but positive) response. When combined, cytokine A infiltrates into damaged tissues
diminishes the action of cytokine C. In this illustration, the
concentrations of cytokines B and C are held constant, whereas B. List
cytokine A increases along the horizontal axis. (B) Synergistic and 1. Interleukin-1 (IL-1)
antagonistic interactions may be the result of cytokine A (1) altering
2. Tumor Necrosis Factors
the expression or function of the receptor for cytokine X, (2) altering
the activity of a key enzyme in the signaling pathway for cytokine X, 3. Interleukin-6
or (3) altering the stability and/or translation of the mRNA induced by 4. Chemokines
cytokine X. (C) Cytokines can induce release of other cytokines in an 5. Transforming Growth Factor-β
amplifying cascade. For example, following intravenous injection of a 6. Interferon-α and Interferon-β
bacterial toxin, TNF-α is first released by monocytes and
macrophages. Both the toxin and TNF-α induce subsequent release (see provided table)
of IL-1β. Then all three induce IL-6 release from a wide variety of cell
types.

Hypercytokinemia
• a condition commonly referred to as cytokine storm →
shock, multiorgan failure, or even death, thus contributing
to pathogenesis
• Select pathogens have been known to induce cytokine
storms.

Examples of Major Proinflammatory

and Anti-Inflammatory Cytokines
Major Pro-inflammatory Major Anti-Inflammatory
Cytokines Cytokines
TNF-α TGF-β
IL-1 IL-10
IL-6 IL-13
IFN-γ IL-35

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CYTOKINES IN THE ADAPTIVE IMMUNE RESPONSE C. Cytokines Associated With T Regulatory Cells
A. Introduction • induced Tregs (iTregs)
• cytokines involved in the adaptive immune response are o an additional population of Tregs
mainly secreted by T cells, especially Th cells, and affect o can develop from mature T cells in the periphery
T- and B-cell function more directly • TGF-β
• Once the T-cell receptor (TCR) captures antigen, clonal o induces expression of Foxp3 (a transcription factor
expansion of those particular CD4+ Th cells occurs. that causes Treg cells to suppress the activity of
Differentiation into Th1, Th2, or Treg cell lineages is other T cells
influenced by the spectrum of cytokines expressed in the o Tregs are also responsible for inducing IL-10 and
initial response. TGF-β expression in adaptive T regulatory 1 (Tr1)
cells in the peripheral circulation
o Tr1 cells
o CD4+ T cells that are induced from antigen-
activated naïve T cells in the presence of IL-10
o exert their suppressive activities on both Th1 and
Th2 cells by producing more IL-10, TGF-β, or IL-35
• IL-35
o immunosuppressive effects on Th1, Th2, and Th17
cells while promoting growth of Tregs.

Th17 CYTOKINES IN INNATE AND ADAPTIVE

IMMUNE RESPONSES
• secretes the IL-17 family of cytokines and plays critical
roles in both innate and adaptive immune responses
• TGF-β and IL-6 - key cytokines that differentiate T cells to
maintain them as Th17 cells
• IL-17 family
o potent proinflammatory cytokines and induce
expression of TNF-α, IL-1β, and IL-6 in epithelial,
endothelial, keratinocyte, fibroblast, and
macrophage cells
o produce proinflammatory mediators from myeloid
and synovial fibroblasts and perpetuate the
inflammatory process in RA
• Th17 cells
o play an important role in host defense against
bacterial and fungal infections at mucosal surfaces
o invade the infected area and secrete IL-17
cytokines necessary for continuous recruitment of
neutrophils
• IL-17A and IL-17F - induce epithelial cells, endothelial
cells, and fibroblasts to produce CXC ligand 8 (CXCL-8),
which is crucial for recruitment of neutrophils to the site of
inflammation
o ↑ Th17 cells and IL-17A = asthmatic and allergic
patients
o ↑ IL-17A and IL-17F = been associated with more
severe asthma.
• IL-17A - directly induces IgE production by B cells
• Interestingly, removal of Th17 cells from peripheral blood
mononuclear cells of allergic asthma patients has led to
decreased levels of IgE.

HEMATOPOIETIC GROWTH FACTORS

B. List A. Introduction
Th1 Cytokines • A number of cytokines produced during innate and
1. Interferon-γ (IFN-γ) adaptive immune responses stimulate the proliferation
2. Interleukin-2 (IL-2) and differentiation of bone marrow progenitor cells.

Th2 Cytokines B. Colony stimulating factors (CSFs)

1. Interleukin-4 (IL-4) • primary mediators of hematopoiesis
2. Interleukin-10 (IL-10) • stimulate the formation of colonies of cells in the bone
marrow
(see provided table)

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1. IL-3
• a multilineage CSF that induces bone marrow stem cells
to form T and B cells
• acts on bone marrow stem cells to begin the
differentiation cycle
• the activity of IL-3 alone drives the stem cells into the
lymphocyte differentiation pathway
• IL-3 in conjunction with GM-CSF drives the development
of basophils and mast cells,
• addition of IL-5 to IL-3 and GM-CSF drives the cells to
develop into eosinophils

2. erythropoietin (EPO)
• regulates RBC production in the bone marrow but is
primarily produced in the kidneys
• EPO-α
o form licensed for clinical use by the FDA
o prescribed to improve RBC counts for individuals
with anemia and for those with cancer who have
undergone radiation and chemotherapy
• improves oxygenation of the tissues and eventually
switches off EPO production
• Normal serum values: 5 to 28 U/L

3. granulocyte colony stimulating factor (G-CSF)

• If G-CSF is activated, the cells become neutrophils
• enhances the function of mature neutrophils and affects
the survival, proliferation, and differentiation of all cell
types in the neutrophil lineage
• decreases IFN-gamma production, increases IL-4 Influence of colony stimulating factors on growth and differentiation
production in T cells, and mobilizes multipotential stem of blood cells.
cells from the bone marrow Growth of hematopoietic stem cells (HSC) requires stem cell factor
(SCF) with differentiation determined by IL-7 or thrombopoietin (TPO).
4. macrophage colony stimulating factor (M-CSF) Growth of common myeloid progenitors (CMP) depends upon IL-3.
• if activated, cells become macrophages Differentiation is driven by granulocyte-macrophage colony
stimulating factor (GM-CSF) or erythropoietin (EPO). Common
• also increases phagocytosis, chemotaxis, and additional granulocyte/monocyte precursors (GMP) differentiate into
cytokine production in monocytes and macrophages granulocytes in response to granulocyte-CSF. Further specificity is
provided by IL-3 or IL-5. Macrophage-CSF (M-CSF) promotes
5. granulocyte-macrophage colony stimulating factor (GM- development of monocytes. CLP = common lymphoid progenitor, EB
CSF) = erythroblast
• acts to drive differentiation toward other WBC types
CYTOKINE AND ANTICYTOKINE THERAPIES
• murine monoclonal antibodies - able to function only for
short periods of time before the host mounted an immune
response against them
• Recombinant DNA techniques have allowed for the
production of humanized monoclonal antibodies that are
much less immunogenic and that function as cytokine
antagonists.
o e.g. infliximab (Remicade) - chimeric antibody
containing human constant regions and murine
antigen-specific arms that bind human TNF-α
• development of a class of hybrid proteins containing
cytokine receptor binding sites attached to
immunoglobulin constant regions
o e.g. Etanercept (Enbrel) - consists of the
extracellular domains of the type 2 TNF receptor
fused to the heavy-chain constant region of IgG1;
fusion protein can bind TNF-α and block its activity

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 CLINICAL ASSAYS FOR CYTOKINES
A. ELISpot assay
• employs the enzyme-linked immunosorbent assay
(ELISA) technique on in vitro-activated peripheral WBCs
• either a monoclonal or polyclonal antibody specific for the
chosen cytokine is precoated onto a microplate.
• Antigen-stimulated, mitogen-stimulated (positive control),
or saline-stimulated (negative control) WBCs are pipetted
into the wells and the microplate is placed into a
humidified CO2 incubator at 37°C for a specified period of
time.
• During the incubation period, the immobilized antibody in
the immediate vicinity of the secreting cells binds the
secreted cytokine.
• After any cells and unbound substances are washed
away, a biotinylated polyclonal antibody specific for the
chosen cytokine is added to the wells.
• Following a wash to remove any unbound biotinylated
antibody, alkaline-phosphatase conjugated to streptavidin
is added.
• Unbound enzyme is subsequently washed away and a
substrate solution is added.
• A colored precipitate forms and appears as spots at the
sites of cytokine localization, with each spot representing
an individual cytokine-secreting cell.
• The spots can be counted with a stereomicroscope or with
an automated ELISpot reader.

B. Others
• Multiplexed ELISAs and microbead assays
o can detect several cytokines in serum at a time
• One drawback of protein-based technologies is the short
half-life of certain cytokines.
o Resolution: looking at RNA expression in cells
using reverse transcription polymerase chain
reaction (PCR).
• The PCR product is made using a fluorescent-labeled
primer and can be hybridized to either solid-phase or
liquid microarrays.

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 Review Questions a. Blast formation
1. The ability of a single cytokine to alter the expression of b. T-cell proliferation
several genes is called c. Measurement of leukocyte chemotaxis
a. redundancy. d. ELISA testing
b. pleiotropy. 10. Selective destruction of Th cells by the human
c. autocrine stimulation. immunodeficiency virus contributes to immune
d. endocrine effect. suppression by which means?
2. Which of the following effects can be attributed to IL-1? a. Decrease in IL-1
a. Mediation of the innate immune response b. Decrease in IL-2
b. Differentiation of stem cells c. Decrease in IL-8
c. Halted growth of virally infected cells d. Decrease in IL-10
d. Stimulation of mast cells 11. Why might a colony stimulating factor be given to a
3. Which of the following precursors are target cells for IL- cancer patient?
3? a. Stimulate activity of NK cells
a. Myeloid precursors b. Increase production of certain types of
b. Lymphoid precursors leukocytes
c. Erythroid precursors c. Decrease the production of TNF
d. All of the above d. Increase production of mast cells
3. A lack of IL-4 may result in which of the following 12. Which of the following would result from a lack of TNF?
effects? a. Decreased ability to fight gram-negative
a. Inability to fight off viral infections bacterialinfections
b. Increased risk of tumors b. Increased expression of class II MHC
c. Lack of IgM molecules
d. Decreased eosinophil count c. Decreased survival of cancer cells
4. Which of the following cytokines is also known as the T- d. Increased risk of septic shock
cell growth factor? 13. Which cytokine acts to promote differentiation of T cells
a. IFN-γ to the Th1 subclass?
b. IL-12 a. IL-4
c. IL-2 b. IFN-α
d. IL-10 c. IL-12
5. Which of the following represents an autocrine effect of d. IL-10
IL-2? 14. What is the major function of T regulatory cells?
a. Increased IL-2 receptor expression by the Th a. Suppression of the immune response by
cell producing it producing TNF
b. Macrophages signaled to the area of antigen b. Suppression of the immune response by
stimulation inducing IL-10
c. Proliferation of antigen-stimulated B cells c. Proliferation of the immune response by
d. Increased synthesis of acute-phase proteins producing IL-2
throughout the body d. Proliferation of the immune response by
6. IFN-α and IFN-β differ in which way from IFN-gamma? inducing IL-4
a. IFN-α and IFN-β are called immune interferons, 15. Th17 cells affect the innate immune response by
and IFN-gamma is not. inducing production of which cytokines?
b. IFN-α and IFN-β primarily activate a. IFN-γ and IL-2
macrophages, whereas IFN-gamma halts viral b. IL-4 and IL-10
activity. c. IL-2 and IL-4
c. IFN-α and IFN-β are made primarily by d. TNF-α and IL-6
activated T cells, whereas IFN-gamma is made
by fibroblasts.
d. IFN-α and IFN-β inhibit cell proliferation,
whereas IFN-gamma stimulates antigen
presentation by class II MHC molecules.
7. A patient in septic shock caused by a gram-negative
bacterial infection exhibits the following symptoms: high
fever, very low blood pressure, and disseminated
intravascular coagulation. Which cytokine is the most
likely contributor to these symptoms?
a. IL-2
b. TNF
c. IL-12
d. IL-7
8. IL-10 acts as an antagonist to what cytokine?
a. IL-4
b. TNF-α
c. IFN-gamma
d. TGF-β
9. Which would be the best assay to measure a specific
cytokine?

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IMMUNOSEROLOGY 6.

Cytokines

CYTOKINES OF THE INNATE IMMUNE SYSTEM

CYTOKINE RECEPTOR SOURCE TARGET FUNCTION DISEASE ASSOCIATION
1. Interleukin-1 (IL-1) IL1RI and Macrophages, Macrophages, • Inflammatory; promotes activation, • ↑ = inflammatory bone
• IL-1α, IL-1β, and IL-1RA (IL-1 IL1R-AcP monocytes and thymocytes, costimulation, and secretion of • resorption; gout;
receptor antagonist) many others CNS, others cytokines and other acute- phase promotes Th17 response
• may be induced by the presence of proteins; pyrogenic
microbial pathogens, bacterial • acts as an endogenous pyrogen
lipopolysaccharides, or other (induces fever) in the acute-phase
cytokines response through its actions on the
hypothalamus
IL-1α • induces the production of vascular
• remains within the cells that cell-adhesion molecules as well as
produce it and is rarely found chemokines and IL-6
outside these cells • induces the production of CSFs in
• can be released after cell death the bone marrow, thereby increasing
and can help attract inflammatory the available number of phagocytic
cells to areas where cells and cells that can respond to the
tissues are being killed or damaged damaged tissues

IL-1β
• responsible for most of the
systemic activity attributed to IL-1,
including fever, activation of
phagocytes, and production of
acute-phase proteins

IL-1RA
• also produced by monocytes and
macrophages
• best characterized cytokine
inhibitor
• acts as an antagonist to IL-1 by
blocking the IL-1 receptor, which
helps to regulate the physiological
response to IL-1 and turn off the
response when no longer needed
2. Tumor Necrosis Factors Murine: TNFR, p55; Macrophages, Neutrophils, • Inflammatory; promotes activation • ↓ = disregulated
• first isolated from lymphocytes and TNFR, p75 Human: monocytes, T macrophages, and production of acute-phase • fever; increased
macrophages and were so named TNFR, p60; TNFR, p80 cells, others monocytes, proteins susceptibility to bacterial
because they induced lysis in endothelial • activate T cells through its ability to infection; enhanced
tumor cells cells induce expression of class II MHC

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• An inflammatory cytokine produced TNFR1 (TNF receptor Trigger: presence molecules, vascular adhesion • resistance to LPS-
by macrophages/ monocytes 1) of molecules, and chemokines in a induced septic shock
during acute inflammation and is • constitutively lipopolysaccharide, manner similar to IL-1 • ↑ = exacerbation of
responsible for a diverse range of expressed on most which is found in arthritis and colitis
signaling events within cells, tissues and binds gram-negative • large amounts of TNF
leading to necrosis or apoptosis. soluble TNF-α bacteria secreted in response to
• primary mediator of gram-negative bacterial
TNF-α TNF-α signal infections, causing a
• most prominent member of the transduction in most decrease in blood
TNF superfamily cell types pressure, reduced tissue
• activity is at least partially regulated perfusion, and
by soluble forms of both TNF TNFR2 disseminated
receptors. • Usually expressed in intravascular coagulation
• These receptors act to bind excess epithelial cells and → uncontrolled bleeding
TNF-α and, combined with the cells of the immune
short half-life of the soluble form, system and is
serve to limit the cytokine’s activated by the
signaling activity. membrane-bound
• present in rheumatoid synovial form of TNF-α.
fluids and synovial membranes of
patients with rheumatoid arthritis
(RA) (as well as IL-1)
• central mediator of pathological
processes in RA and other
inflammatory illnesses such as
• Crohn’s disease, ulcerative colitis,
and juvenile arthritis
3. Interleukin-6 (IL-6) IL-6Rα Thymic stromal B cells, T • Homeostasis, differentiation, and • ↓ = severe combined
• A cytokine featuring pleiotropic • the IL-6-specific cells, bone cells, survival immune deficiency
activity receptor marrow, and thymocytes • stimulates B cells to proliferate and (SCID)
• single protein produced by both spleen differentiate into plasma cells and • chronic psychosocial
lymphoid and non-lymphoid cell gp130 induces CD4+ T cells to produce stress → increases the
types • the common Trigger: IL-1 greater quantities of both pro- and risk of atherosclerotic
• part of the cytokine cascade • signal-transducing anti-inflammatory cytokine cardiovascular disease
released in response to receptor subunit
lipopolysaccharide and plays an used by several
important role in acute-phase cytokines
reactions
• • Binding of IL-6 to the
IL 6Rα induces
dimerization of
gp130 with the α-
subunit (Fig. 6–4).

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4. Chemokines Trigger: TNF-α • initiation and development of • HIV uses the chemokine
• family of cytokines that enhance and IL-6 inflammatory responses in receptors CXCR4 and
motility and promote migration of numerous disease processes CCR5 as co-receptors
many types of white blood cells • facilitate the extravasation of for infection of CD4+ T
(WBCs) toward the chemokine leukocytes into the tissues lymphocytes and
source via a process known as • Leukocytes rolling on capillary macrophages
chemotaxis endothelial cells activate their • CCR5-δ32 polymorphism
chemokine receptors in the is a 32 bp deletion in the
Classified into four families based on presence of chemokines. CCR5 gene and is the
the position of N-terminal cysteine • Integrins, or cell adhesion molecules most important of the
residues on leukocytes, are then activated, host resistance factors
• 1st group: the alpha, or CXC, leading to firm adhesion to the • Homozygous individuals
chemokines—contains a single endothelial cells. are protected from HIV
amino acid between the first and infection, whereas
second cysteines heterozygous persons
• 2nd group: the beta, or CC, exhibit longer periods
chemokines—has adjacent between HIV infection
cysteine residues. and AIDS development.
• 3rd group: the C chemokines—
lacks one of the cysteines.
• 4th group: CX3C, has three amino
acids between the cysteines.
5. Transforming growth factor-β IL2Rγ T cells, DCs, All leukocyte • Regulatory; inhibits growth and ↓ = increased susceptibility
(TGF-β) macrophages, population activation; Treg maintenance; to autoimmune disorders
• composed of three isoforms: TGF- others synergizes with IL-6 ↑ = Fibrotic diseases
β1, TGF-β2, and TGF-β3 • induces antiproliferative activity in a
• originally growth factor in tumor wide variety of cell types
cells • primarily a regulator of cell growth,
differentiation, apoptosis, migration,
and the inflammatory response.
Thus, it acts as a control to help
downregulate the inflammatory
response when no longer needed.
• both an activator and an inhibitor of
proliferation, depending on the
developmental stage of the affected
cells
• regulates the expression of CD8 in
CD4–CD8– thymocytes and acts as
an autocrine inhibitory factor for
immature thymocytes
• inhibits the activation of
macrophages and the growth of
many different somatic cell types
and functions as an anti-

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 inflammatory factor for mature T
cells
• blocks the production of IL-12 and
strongly inhibits the induction of IFN-
gamma
• production of TGF-β by T helper 2
(Th2) cells is now recognized as an
important factor in the establishment
of oral tolerance to bacteria normally
found in the mouth
• inhibits proliferation and may
function as an autocrine regulator to
limit the expansion of activated cell
6. Interferon-α and Interferon-β IFNɑR1, IFNɑR2 Macrophages, NK cells, • Promotes resistance to viral • ↓ = impaired antiviral
(IFN) fibroblasts, many others pathogens responses
• originally so named because they plasmacytoid DCs, • promotes increased expression of • IFN-β - efficacious in
interfere with viral replication others MHC class I treating multiple sclerosis
• type I • limit the infection to one relatively • IFN-α - used to treat
• induce production of proteins and dendritic cells small area of the body hepatitis C and Kaposi’s
pathways that directly interfere with • activates natural killer (NK) cells and sarcoma,
viral replication and cell division; enhances the expression of class I • as well as certain
antiviral protein MHC proteins, thus increasing the leukemias and
recognition and killing of virus- lymphomas
infected cells

CYTOKINES OF THE ADAPTIVE IMMUNE SYSTEM

CYTOKINE RECEPTOR SOURCE TARGET FUNCTION DISEASE ASSOCIATION
Th1 Cytokines
IL-12
• produced by dendritic cells in damaged tissues in response to certain stimuli such as mycobacteria, intracellular bacteria, and viruses
• also produced by macrophages and B cells
• has multiple effects on both T cells and NK cells
• binds to its receptor on naïve T cells and causes the expression of a new set of genes, including those that determine maturation into the Th1 lineage.
• Activation of Th1 cells induces high-level expression of IFN-gamma.22 IL-12 also increases the cytolytic ability of NK cells; therefore, it serves as an important link between the innate and adaptive
immune responses by enhancing defenses against intracellular pathogens.
1. Interferon-γ (IFN-γ) IFNγR1 Th1 cells, NK cells, CD8 Macrophages, NK cells, • Promotes activation of • ↓ = susceptibility to intracellular
• principal molecule IFNγR2 T cells T cells, others APCs and cell mediated pathogens
produced by Th1 cells, immunity; increased MHC • ↑ expression of class I and II MHC
and it affects the RNA class II expression molecules on antigen-presenting cells
expression • stimulation of antigen • (APCs) = ↑ the likelihood of antigen
• most potent activator presentation by class I capture and involvement of additional
of macrophages and MHC and class II MHC lymphocytes
boosts their tumoricidal molecules
activity → creating • regulation and activation of
CD4+ Th1 cells, CD8+

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 activated or “super” cytotoxic lymphocytes, and
macrophages NK cells
2. Interleukin-2 (IL-2) IL2Rɑ, IL2Rβ, Th1 cells T, B, NK, cells and Proliferation; enhancement of • ↓ = lymphoproliferative disease and
• Th1 cells also secrete and IL2Rγ macrophages cytotoxicity, IFNγ secretion, susceptibility to auto immune disease;
IL-2 in addition to IFN- and antibody production reduced Treg development.
γ β and γ drives the growth and • ↑ = Reduced Th17 development
• also known as the T- • subunits differentiation of both T and B
cell growth factor increase the cells and induces lytic activity
• IL-2 and IFN-γ - induce affinity of the in NK cells
the development of receptor for activate proliferation of Th2
Th1 cells → induces IL-2 and are cells and helps to generate
macrophage activation • responsible IgG1- and IgE-producing cells
and delayed type for most of
hypersensitivity. T the signal
• h1 cells stimulate the transduction
production of IgG1 and through the
IgG3 opsonizing and receptor
complement fixing
antibodies by antigen- γ subunits
activated B cells. • X-linked
severe
combined
immu -
nodeficiency
syndrome
and lack
functional T
and B cells
Th2 Cytokines
• primarily responsible for antibody-mediated immunity
1. Interleukin-4 (IL-4) IL5Rɑ and Th2 cells Eosinophils, B cells • Proliferation and activation; ↓ = eosinophil and B1 cell deficiency.
• One of the key IL3Rβ hallmark of Th2 effector ↑ = Allergic asthma
cytokines regulating cells
Th2 immune activities • induces production of
and helps drive MHC-I, IL-4, IL-5, IL-13,
antibody responses in and the costimulatory
a variety of diseases molecules CD80 and
• IL-4 activity on naïve T CD86
cells turns on the • stimulates the production
genes that generate of IgG2a and IgE and,
Th2 cells and turns off along with IL-5, drives the
the genes that promote differentiation and
Th1 cells. activation of eosinophils in
both allergic immune
responses and response to
parasitic infections

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 • IL-13 is a cytokine with
many of the same
properties as IL-4; both
cytokines induce worm
expulsion and favor IgE-
class switching.
• IL-13, however, differs
from IL-4 because it also
plays an anti-inflammatory
role by inhibiting activation
and cytokine secretion by
monocytes.
2. Interleukin-10 (IL- IL10R1 and monocytes, macrophages, CD8+ T cells, and Th2 • inhibits antigen • ↓ = Immune pathology due to
10) IL10R2 CD4+ T cells presentation by uncontrolled inflammation.
• Has anti-inflammatory macrophages and dendritic • ↑=
and suppressive cells • Inhibits sterile immunity to some
effects on Th1 cells • inhibition of IFN-gamma pathogens.
• Has primarily inhibitory production via the
effects on the immune suppression of IL-12
system synthesis by accessory
cells and the promotion of
a Th2 cytokine pattern
• antagonist to IFN-
gamma—it is a
downregulator of the
immune response

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IMMUNOSEROLOGY 7.

Complement System
OUTLINE o recognizes cellular debris such as apoptotic cells
I. Introduction and immune complexes, tagging them for removal
A. Complement by innate immune cells
II. Pathways of the Complement System • chronic activation → inflammation and tissue damage to
A. Classical Pathway the host
B. Alternative Pathway • deficiencies → increased susceptibility to infection or the
C. Lectin Pathway accumulation of immune complexes → autoimmune
III. The Classical Pathway disorders
A. Introduction o proteins - act as controls or regulators of the system
B. The Recognition Unit
C. The Activation Unit PATHWAYS OF THE COMPLEMENT SYSTEM
D. The Membrane Attack Complex (MAC) A. Classical Pathway
IV. The Lectin Pathway • involves nine proteins
A. Introduction • trigger: antigen-antibody combination
V. The Alternative Pathway
A. Introduction B. Alternative Pathway
B. Summary • discovered by Pillemer and colleagues
VI. System Controls • antibody-independent
A. Introduction • plays a major role as a natural defense system
B. Regulation of the Classical and Lectin • originally called the properdin system because the protein
Pathways properdin was thought to be the main initiator of this
C. Regulation of the Alternative Pathways pathway
D. Regulation of The Terminal Components
• (now) properdin’s - stabilize a key enzyme complex
VII. Complement Receptors and Their Biological Roles formed along the pathway and that the other forms of
A. CR2 activation are more prominent
B. CR3
C. CR4
C. Lectin Pathway
D. Collectin Receptors
• most ancient of the three
VIII. Biological Manifestations of Complement Activation
A. Introduction • antibody-independent
B. Anaphylatoxins and Chemotaxins • mannose- (or mannan-) binding lectin (MBL)
C. Opsonization o prototypic constituent
IX. Complement And Disease States o adheres to mannose found mainly in the cell walls
X. Complement Deficiencies or outer coating of bacteria, viruses, yeast, and
A. Major Pathway Components protozoa
B. Regulatory Factor Components
XI. Laboratory Detection of Complement Abnormalities MUST KNOW
A. Introduction
B. Immunologic Assays of Individual Site of Synthetization
Components • Liver
C. Assays for the Classical Pathway o where most plasma complement proteins are
D. Alternative Pathway Assays synthesized
XII. Interpretation Of Laboratory Findings • except C1 components
• Intestinal epithelial cells and Factor D
INTRODUCTION o where C1 components are produced
A. Complement
• Monocytes and macrophages
• complex series of more than 30 proteins that play a major
o additional sources of early complement
part in amplifying the inflammatory response to destroy
components
and clear foreign antigens
o including C1, C2, C3, and C4
• these soluble and cell-bound proteins interact in a very
o inactive precursors, or zymogens
specific way and have powerful abilities o converted to active enzymes in a very precise
• can lyse foreign cells, opsonize and tag the invaders for order
clearance, and direct the adaptive immune system to the
site of infection
• if activated → proinflammatory in its ability to increase
Proteins of the Complement System
vascular permeability, recruit monocytes and neutrophils Serum Molecular Concen Function
to the area of antigen concentration, and trigger secretion Protein Weight tration
of immunoregulatory molecules that amplify the immune (KD) (μg/mL)
response Classical Pathway
• important link between innate and adaptive immunity C1q 410 50 Binds to Fc region of
• “housekeeping roles”: IgM and IgG

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 C1r 85 50 Activates C1s 3. Membrane attack complex (MAC)
C1s 85 50 Cleaves C4 and C2 • C5 to C9
C4 205 300– Part of C3 convertase • completes the lysis of foreign particles
600 (C4b)
C2 102 25 Binds to C4b—forms
C3 convertase
C3 190 1,200 Key intermediate in
all pathways
C5 190 80 Initiates membrane
attack complex
C6 110 45 Binds to C5b in MAC
C7 100 90 Binds to C5bC6 in
MAC
C8 150 55 Starts pore formation
on membrane
C9 70 60 Polymerizes to cause
cell lysis
Alternative Pathway
Factor B 93 200 Binds to C3b to form
C3 convertase
Factor D 24 2 Cleaves Factor B
Properdin 55 15–25 Stabilizes C3bBb–C3
convertase
MBL Pathway
MBL 200–600 0.0002 Binds to mannose
–10
MASP-1 93 1.5–12 Unknown
MASP-2 76 Unkno Cleaves C4 and C2
wn

THE CLASSICAL PATHWAY

A. Introduction
• main antibody-directed mechanism for triggering
complement activation
• not all immunoglobulins are able to activate this pathway
o can activate: IgM, IgG1, IgG2, and IgG3
§ IgM - the most efficient of the activating
immunoglobulins because it has multiple
binding sites
• only one molecule attached to two
adjacent antigenic determinants to
initiate the cascade
• Some epitopes (Rh group) are too
far apart on the cell for this to B. The Recognition Unit
occur → unable to fix complement The Recognition Unit
• IgG3 - most efficient in the IgG group (followed by IgG1 C1 • 1st complement component to bind
and IgG2) • 740, 000 d
• Consists of three subunits:
Substances that can bind complement directly to initiate o C1q
classical cascade o C1r
• C-reactive protein (CRP) o C1s
• several viruses • require the presence of calcium to
• mycoplasmas maintain structure
• some protozoa • Complex: 1 C1q subunit and 2 C1r and 2
• certain gram-negative bacteria (such as Escherichia coli) C1s subunits
o C1q - part that binds to antibody
Three Main Stages of Complement Activation molecules
o C1r and C1s subunits - generate
1. Recognition unit
enzyme activity to begin the
• C1
cascade
C1q • 410,000 d
2. Activation unit
• once C1 is activated → C4, C2, and C3 • six strands that form six globular heads
with a collagen-like tail portion (bouquet of
• of the classical pathway (and the lectin pathway)

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 tulips with six blossoms extending C. The Activation Unit
outward) The Activation Unit
• As long as calcium is present in the serum, First amplification
C1r and C1s remain associated with C1q. • for every C1 attached approximately 30 molecules of
• “recognizes” the fragment crystallizable C4 are split and attached
(Fc) region of two adjacent antibody C1 cleaves C4 C4
molecules • 2nd most abundant complement
o but at least two of the globular protein (600 μg/mL)
heads of C1q must be bound to • 3 polypeptide chains
initiate the classical pathway • 198, 000 d
• each stalks is composed of three Split off of C4a & C4a
homologous polypeptide chains—A, B, C4b • 77 amino acid fragments
and C—that form a triple helix. • anaphylatoxin
Alanine residues
- interrupt the triple helix, make it C4b
semiflexible joint • thioester-containing active site
- C1r and C1s intertwined with C1q.
• must bind to protein or
Calcium
carbohydrate within few seconds
- present in the serum hence C1r and
• or it will react with water molecules
C1s remain associated with C1q.
to form iC4b (rapidly degraded)
(C1r, • S-shaped structure
• binds mainly to antigen in clusters
C1s) • unequal sizes of domains that are within a 40-nm radius of
complex • wraps itself around the arms of C1q C1
• serine protease proenzymes Combination of C2
• aka zymogens C2 & C4b in the • single-chain glycoprotein
presence of • 102,000 d
Recognition Unit Stage magnesium • closely associated with the gene
• As binding of C1q occurs, both are ions for Factor B (alternative pathway)
converted into active enzymes on chromosome 6 in the major
• Autoactivation of C1r results from a histocompatibility complex (MHC)
conformational change that takes place as
C1q is bound. ⨯ Mg ions - C4b in fluid phase
o Activated C1r → cleaves a thioester
✓ Mg ions - C4b attached to antigen
bond on C1s → activates it
C1s cleaved C2 C2a
§ Activated C1r - extremely
to form C2a & • 70,000d
specific; only known
C2b
substrate is C1s
C2b
• C1s - limited specificity; only substrates
being C4 and C2 • 34,000 d
o Activated C1s → the recognition
stage ends • C2 must be within a 60 nm radius
of bound C1s
Sequence of Binding • short life of these reactive species
• C1s-C1r-C1r-C1s serves as a mechanism of control,
keeping the reaction localized
Combination of C4b2a (C3 convertase)
C4b & C2a • sum of C4b + C2a
• also known as C3 convertase
• written as C4b2a to indicate that
the complex is an active enzyme
• not very stable
• Half-life: 15s to 3min
o C3 must be bound quickly
o If binding does occur → C3
is cleaved into two parts
(C3a and C3b)
Second amplification
• about 200 molecules are split for every molecule of
C4b2a
Cleaving of C3 to C3
C3a & C3b • major and central constituent of
the complement system
• 190, 000 d
• plasma concentration: 1 mg/mL to
1.5 mg/mL

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 • serves as pivotal point for all three
pathways
• consists of two polypeptide chains:
alpha (α) and beta (β)
o α chain - contains a highly
reactive thioester group
• When C3a is removed (by
cleavage of a single bond in the α
chain) the thioester is exposed

C3a
• Anaphylatoxin

C3b
• when thioester is exposed → C3b
is capable of binding to hydroxyl
groups on carbohydrates and
proteins in the immediate vicinity
• Half-life: 60 microseconds if not
bound to antigen
o only a small percentage of
cleaved C3 molecules bind to
antigen
§ most are hydrolyzed by
water molecules and
decay in the fluid phase
• a powerful opsonin
• required for the formation of the
MAC

\ cleavage of C3 to C3b - represents

the most significant step in the
entire process of complement
activation
Combination of C5 convertase
C4b2a & C3 • Sum of C4b2a & C3
• must occur in 40nm of C4b2a

\ cleaving of C5 with deposition of

C5b at another site on the cell
membrane - constitutes the
beginning of the MAC
D. The Membrane Attack Complex (MAC)
The Membrane Attack Complex (MAC)
Cleavage of C5 C5
by C5 • consists of two polypeptide chains
convertase (α and β) linked by disulfide bond
• 190, 000 d

C5 convertase
• C4b2b3b
• Splits off C5a
Split off of C5a & C5a
C5b • 74 amino acid piece
• an anaphylatoxin and chemotaxin
• released into circulation

C5b
• attaches to the cell membrane →
beginning of MAC
• extremely labile (must bind with
another complement protein [C6])

\ splitting of C5 and the cleavage

of C3 - represent the most
significant biological

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 consequences of the
complement system as explained
in the section on biological
manifestations of complement
activation
Subsequent C6 & C7
binding of C6 • 110, 000 d each
(once bound to • same physical & chemical
C5), C7, C8 & properties
C9
C8
• no enzymatic • 150, 000 d
activity • made up of three dissimilar chains
• present in joined by disulfide bonds
smaller
amounts in C9
serum • single polypeptide chain
• 70, 000 d
• Carboxy-terminal end: hydrophobic
(serves to anchor the MAC within
the target membrane)
• Amino-terminal end: hydrophilic
• if present → speeds up lysis

C6 binds to C5b • To stabilize C5b

C5b-C6 complex attaches to cell membrane
C7 binds to C5b- • Forming a tri-molecular complex
C6 complex
C8 bind to C7 • exposes a hydrophobic region
• interacts with the cell membrane to
form a small hole in the membrane
• C8 is inserted into the lipid bilayer
Note
• Lysis can be observed before C9 binds to C5-C8
complex
o binding of C8 causes a loss of potassium from
the cell
o leakage of amino acids and ribonucleotides
• Pore formed is small
o Lysing erythrocytes but not nucleated cells.
o That’s why C9 must bind to C8 THE LECTIN PATHWAY
A. Introduction
\ C5-C8 have an initial cell lysis but only lyse the • represents another means of activating complement
erythrocytes • activated by recognition of surface moieties that are found
\ C5-C9 capable of lysing all type of cells, nucleated or on pathogens
unnucleated • provides an additional link between the innate and
C9 binds to C5- • 1 to 18 mol of C9 can bind to one acquired immune response because it involves
C8 complex C8 nonspecific recognition of carbohydrates that are common
• Polymerized C9 forms a thin, constituents of microbial cell walls and that are distinct
hollow cylinder (transmembrane from those found on human cell surfaces
channel) • structurally similar to those of the classical
• 1 MAC unit has a functional size of o share the components C4 and C2
100A o once C4 and C2 are cleaved, the rest of the
C5b-9 or MAC pathway is identical to the classical pathway
• the complex of C5b-C6-C7-C8 and C9 • plays an important role as a defense mechanism in
• If the complex is soluble in circulation → known as infancy, during the interval between the loss of maternal
sC5b-9 antibody and the acquisition of a full-fledged antibody
• level measurement is an indicator of the amount of response to pathogens
terminal pathway activation that is occurring
• presents a pore of 70 to 100Å (allows ions to pass in C1q
and out of the membrane) • filled by three classes of recognition molecules in the
lectin pathway
o lectins
o ficolins
o CL-K1

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• structure is similar to that of C1q because they are all The Alternative Pathway
classed as collectins Bypass C1,C4 &
C2
mannose-binding, or mannan-binding, lectin (MBL) Activation of C3 C3
• a key lectin • Native C3 is not stable in plasma
• binds to mannose or related sugars in a calcium- • sometimes called iC3b
dependent manner to initiate this pathway • Water is able to hydrolyze a
• these sugars are found in glycoproteins or carbohydrates thioester bond, thus spontaneously
of a wide variety of microorganisms such as bacteria, activating a small number of these
yeasts, viruses, and some parasites molecules formed b spontaneous
• considered as acute phase protein because it is produced hydrolysis, from activation, or from
in the liver and is normally present in the serum but the classical or lectin pathways
increases during an initial inflammatory response • acts as the seed of activation of
the alternative pathway
MBL-associated serine proteases (MASPs) C3b binds to Factor B
• serine proteases Factor B • 93, 000 d
• plays the role of C1r and C1s in the lectin pathway • 200 μg/mL in serum
• identified as MASP-1, MASP-2, and MASP-3 • analogous to that of C2 in the
classical pathway because it forms
B. Summary • an integral part of a C3 convertase

iC3b
• More susceptible to cleavage by
serine protease
\ Once bound to C3b, Factor B
can be cleaved by Factor D
Cleavage of Factor D
Factor D with • plasma protein
bound Factor B • goes through a conformational
change when it binds to factor B
• a serine protease
• 24, 000 d
• 2 μg/mL (lowest of all the
complement proteins)
• high specific because it is the only
substrate of bound factor B
• When bound to factor B, catalytic
THE ALTERNATIVE PATHWAY site on factor D opens, making it
A. Introduction an open active enzyme
• Pillemer and his associates
• originally named for the protein properdin Split off of Factor Ba
o a constituent of normal serum with a concentration B to Ba & Bb • 33,000
of approximately 5 to 15 μg/mL
o initiate activation Bb
o stabilize the C3 convertase formed from activation • 60,000
of other factors
• remains attached to C3b, forming
• can be activated on its own the initial C3 convertase of the
• functions mainly as an amplification loop for activation alternative pathway
started from the classical or lectin pathways • rapidly inactivated unless it
• Factor B and Factor D becomes bound to a site on one of
o serum proteins the triggering cellular antigens
• Trigger: bacterial cell walls, especially those containing Formation of C3bBb
lipopolysaccharide, fungal cell walls, yeast, viruses, virally C3bBb • capable of cleaving additional C3
infected cells, tumor cell lines, and some parasites, into C3a and C3b (more efficient)
especially trypanosomes
• capable of cleaving C5
• serve as sites for binding the complex C3bBb, one of the
• also known as C3 convertase of
end products of this pathway
alternative pathway
conversion of C3
• All C3 present in plasma would be
• first step in this pathway rapidly converted by this method
were it not for the fact that the
enzyme C3bBb is extremely
unstable unless properdin binds to
the complex.

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 • Binding of properdin increases the
half-life of C3bBb from 90 seconds
to several minutes.

C3b
• attaches to cellular surfaces and
acts as a binding site for more
Factor B, resulting in an
amplification loop

C3bBb3bP
• C3b produced remains bound to
the C3 convertase, the enzyme is
altered to form this complement
• high affinity for C5 and exhibits C5
convertase activity
Formation of When C3b produced remains bound
C3bBb3bP in the C3 convertase
Cleavage of C3bBb3bP with C5 B. Summary
Split off of C5a & C5b
Subsequent binding of C6, C7, C8 & C9

Convergence of the classical, alternative, and lectin pathways.

The binding of C1qrs to two antibody molecules activates the classical
pathway, whereas the alternative pathway is started by hydrolysis of
C3. The lectin pathway is triggered by binding of MBP to mannose on
bacterial cell walls. MASP-1, MASP-2, and MASP-3 bind to form an

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activated C1-like complex. MASP-2 cleaves C2 and C4 and proceeds o serine protease that inactivates C3b and C4b when
like the classical pathway. Factor B and Factor D operate in the bound to one of these regulators
alternative pathway. Although C3 convertase is formed differently in
each pathway, C3 is a key component in each one. The C5
a. Soluble C4b-binding protein (C4BP)
convertase in the alternative pathway consists of C3bBb3bP. In the
classical and lectin pathways, C5 convertase is made up of C4b2a3b. • 520,000 d
After C5 is cleaved, the pathway is common to all. • combines with either fluid-phase or bound C4b, so C4b
cannot bind C2 and is made available for degradation by
SYSTEM CONTROLS Factor I
A. Introduction • If C4BP attaches to cell-bound C4b, it can dissociate it
• Activation of complement could cause tissue damage and from C4b2a complexes, causing the cessation of the
have devastating systemic effects if it were allowed to classical pathway
proceed uncontrolled.
• plasma proteins - act as system regulators b. Complement receptor type 1 (CR1)
• specific receptors on certain cells - also exert a controlling • aka CD35
influence on the activation process • large polymorphic glycoprotein
• Because activation of C3 is the pivotal step in all • 165,000 to 280,000
pathways, the majority of the control proteins are aimed at • It binds C3b and C4b but has the greatest affinity for C3b
halting accumulation of C3b. • found mainly on peripheral blood cells
Plasma Complement Regulators • once bound to CR1, both C4b and C3b can then be
Serum Molecular Concentration Function degraded by Factor I
Protein Weight (mg/mL) • Main function: act as a receptor on platelets and red blood
(KD) cells → mediate transport of C3b-coated immune
C1 inhibitor 105 240 Dissociates complexes to the liver and spleen → strip the immune
(C1-INH) C1r and C1s complexes from the red blood cells, process the
from C1q complexes, and return the red blood cells intact
Factor I 88 35 Cleaves C3b • immune adherence - ability of cells to bind complement-
and C4b coated particles
Factor H 150 300–450 Cofactor with
I to c. Membrane cofactor protein (MCP)
inactivate • aka CD46
C3b; • 50,000 to 70,000 d
prevents • found on all epithelial and endothelial cells except
binding of B erythrocytes
to C3b • most efficient cofactor for Factor I-mediated cleavage of
C4-binding 520 250 Acts as a C3b
protein cofactor with • cofactor for cleavage of C4b (not as effective as C4BP)
(C4BP) I to • helps to control the alternative pathway, since binding of
inactivate factor B to C3b is inhibited
C4b
S protein 84 500 Prevents d. Decay accelerating factor (DAF)
(vitronectin) attachment • aka CD55
of the C5b67 • membrane glycoprotein
complex to • 70,000 d
cell • third main receptor
membranes
• found on peripheral blood cells, on endothelial cells and
fibroblasts ,and on numerous types of epithelial cells
B. Regulation of the Classical and Lectin Pathways • capable of dissociating both classical and alternative
1. C1 inhibitor (C1-INH) pathway C3 convertases
• Glycoprotein • bind to both C3b and C4b in a similar manner to CR1
• 105,000 • does not prevent initial binding of either C2 or factor B to
• produced from liver & some monocyte the cell
• inhibits activation at the first stages of both the classical • rapidly dissociate both from their binding sites →
and lectin pathways preventing the assembly of an active C3 convertase
• Inactivate C1 by binding to the active sites of C1r and C1s • carboxy-terminal portion: covalently attached to a
o Clr and Cls become instantly and irreversibly glycophospholipid anchor that is inserted into the outer
dissociated from C1q layer of the membrane lipid bilayer
o C1q remains bound to antibody byt all enzymatic o allows DAF mobility within the membrane so it can
activity ceases reach C3 convertase sites
• The activation stops in the recognition • presence of DAF on host cells protects them from
• also activates MASP-2 binding to the MBL-MASP bystander lysis
complex → halts lectin pathway • one of the main mechanisms used in discrimination of self
from nonself
Further formation of C3 convertase in the classical and • does not permanently modify C3b or C4b
lectin pathways is inhibited by four main regulators: • capable of reforming elsewhere as active convertases
• all of these act with factor I

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C. Regulation of the Alternative Pathways
1. Factor H
• principal soluble regulator of the alternative pathway
• 160,000 d
• acts by binding to C3b, preventing the binding of Factor B
• In fluid phase
o C3b > factor B
o has a hundredfold greater affinity for Factor H than
for Factor B
• on cell surfaces
o C3b < factor B
• accelerates the dissociation of the C3bBb complex on cell
surfaces
• when factor H binds to C3bBb, Bb becomes displaced →
C3 convertase activity is curtailed in plasma and on cell
surfaces
• acts as a cofactor that allows factor I to break down C3b
• only those molecules with tightly bound Factor H acquire
high-affinity binding sites for Factor I
• When factor I binds, a conformational change takes place
and allows it to cleave C3b
• On cellular surfaces, C3b is cleaved to:
o C3f
§ small piece that is released into the plasma
o iC3b
§ remains attached but is no longer an active
enzyme.
§ Further broken down to C3c and C3dg by
factor I in conjunction with another cofactor,
the CR1 receptor
Complement controls.
CR1 receptor acts as a cofactor in the inactivation of C3b. Factor I
cleaves C3b to form C3dg and C3c. C3dg is not an effective opsonin
and is not capable of further participation in the complement cascade.

D. Regulation of The Terminal Components

1. S Protein
• soluble control protein
• aka vitronectin
• interacts with the C5b67 complex as it forms in the fluid
phase and prevents it from binding to cell membranes
• binding of C8 and C9 still proceeds, but polymerization of
C9 does not occur

2. Membrane inhibitor of reactive lysis (MIRL)

• aka CD59
• cell membranes of all circulating blood cells

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• bind to C8 and prevent insertion of C9 into host cell
membranes

COMPLEMENT RECEPTORS AND THEIR

BIOLOGICAL ROLES
A. CR2
• aka CD21
• mainly on B lymphocytes and follicular dendritic cells
• lost when conversion to plasma cells occurs
• ligands include: degradation products of C3b (such as
C3dg, C3d,and iC3b)
• Epstein-Barr virus gains entry to B cells by binding to this
receptor
• B-cell coreceptor for antigen
• with CD19, it binds complement-coated antigen
• cross-links it to membrane immunoglobulin to activate B
cells
• immune complexes are more effective at enhancing B-cell
differentiation production of memory cells than is antigen
by itself

B. CR3
• aka CD11b/CD18
• found on monocytes, macrophages, neutrophils, and NK
cells Receptors On Cell Membranes For Complement
• binds particles opsonized with iC3b, especially in Components
alternative pathway Receptor Ligand Cell Type Function
• mediates phagocytosis of particles coated w/ complement CR1 (CD35) C3b, RBCs, Cofactor for
fragments iC3b, neutrophils, Factor I;
C4b monocytes, mediates
• trigger surface adhesion and increased activity of
macrophages, transport of
phagocytic cells
eosinophils, B immune
and T cells, complexes
C. CR4
follicular
• aka CD11c / CD18 receptor
dendritic cells
• binds iC3b fragments in a calcium-dependent fashion on
CR2 (CD21) C3dg, B cells, B-cell co-
neutrophils, monocytes, tissue macrophages, activated
C3d, follicular receptor for
cells, dendritic cells, NK cells, and activated B cells
iC3b dendritic cells, antigen
epithelial with CD19
D. Collectin Receptors
cells
• receptors specific for Clq
CR3 iC3b, Monocytes, Adhesion
• on neutrophils, monocytes, macrophages, B cells, (CD11b/CD18 C3d, macrophages, and incr-
platelets, and endothelial cells C3b neutrophils, eased
• bind the collagen portion of Clq NK cells activity of
• enhance the binding of Clq to Fc receptors phagocytic
cells
CR4 iC3b, Monocytes, Adhesion
(CD11c/CD18) C3b macrophages, and
neutrophils, increased
NK cells, activity of
activated T phagocytic
and B cells, cells
dendritic cells
DAF (CD55) C3b, RBCs, Dissociates
C4b neutrophils, C2b or Bb
platelets, from
monocytes, binding
endothelial sites, thus
cells, preventing
fibroblasts, T formation
cells, of C3
B cells, convertase
epithelial cells
MIRL (CD59) C8 RBCs, Prevents
neutrophils, insertion of
C9

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 platelets, into cell C. Opsonization
monocytes, membrane • C4b, C3b, iC3b, and C3dg
endothelial • accumulate on cell membranes as complement activation
cells, proceeds, bind to specific receptors on erythrocytes,
epithelial cells neutrophils, monocytes, and macrophages
MCP (CD46) C3b, Neutrophils, Cofactor for • facilitates phagocytosis and clearance of foreign
C4b monocytes, Factor I substances or cellular debris, which is one of the key
macrophages, cleavage of functions of the complement system
platelets, T C3b and • attachment of C3 products to an antigen has been found
cells, B cells, C4b to enhance the B-cell response
endothelial
cells COMPLEMENT AND DISEASE STATES
• Complement can be harmful if
BIOLOGICAL MANIFESTATIONS OF COMPLEMENT o It is activated systemically on a large scale as in
ACTIVATION gram-negative septicemia
A. Introduction § ↑ C3a and C5a - in the case of septicemia
• Activation of complement is a very effective means of caused by a gram-negative organism →
amplifying the inflammatory response to destroy and clear leading to neutrophil aggregation and clotting
foreign antigens. The cycle does not always have to o It is activated by tissue necrosis such as myocardial
proceed to lysis for this to be accomplished; hence, some infarction
of the initiating proteins are much more plentiful than § cause complement activation and deposition
proteins that form the MAC. of MACs on cell surfaces
• Complement proteins also serve as a means of linking § Receptors for C3a and C5a have been found
innate and adaptive immunity. in coronary plaques, indicating that
• Recent work demonstrates that complement is necessary complement components may increase the
for maintaining immunologic memory. damage to heart tissue
• Effector molecules generated earlier in the cascade play a o Lysis of red blood cells occurs
major role in all these areas. § Hemolytic diseases such as cold
• Such molecules can be classified into three main autoimmune hemolytic anemia are
categories: anaphylatoxins, chemotaxins, and opsonins. characterized by the presence of an
autoantibody that binds at low temperatures.
B. Anaphylatoxins and Chemotaxins When these cells warm up, complement
• small peptide that causes increased vascular fixation results in lysis.
permeability, contraction of smooth muscle, and release
of histamine from basophils and mast cells COMPLEMENT DEFICIENCIES
A. Major Pathway Components
C3a and C5a lack of C2
• formed as cleavage products from larger complement • the most common deficiency
components • C2-deficient persons are often reported to have
• attach to specific receptors on neutrophils, basophils, decreases in Factor B also
mast cells, eosinophils, smooth muscle cells, and vascular
endothelium lack of C3
• rapidly inactivated by an enzyme in the plasma called • most serious deficiency
carboxypeptidase N to localize and control their effects Deficiencies of Complement Components
Deficient Associated Disease
C3a Component
• attaches to the C3a receptor (C3aR) C1 (q, r, or s) Lupuslike syndrome; recurrent
• cleaved in seconds infections
C2 Lupuslike syndrome; recurrent
C5a infections; atherosclerosis
• most potent C3 Severe recurrent infections;
• at least 200 times more powerful than C3a glomerulonephritis
• attaches to the C5a receptor (C5aR) C4 Lupuslike syndrome
• causes neutrophils to release hydrolytic enzymes, oxygen C5–C8 Neisseria infections
radicals, and prostaglandins, which aid in the destruction C9 No known disease association
of foreign antigens C1-INH Hereditary angioedema
• serves as a chemotaxin for neutrophils, basophils, DAF Paroxysmal nocturnal hemoglobinuria
eosinophils, mast cells, monocytes, and dendritic cells MIRL Paroxysmal nocturnal hemoglobinuria
• Binding of C5a to monocytes causes them to undergo an Factor H or Recurrent pyogenic infections
oxidative burst that includes increased production of Factor I
hydrolytic enzymes, neutrophil chemotactic factor, MBL Pneumococcal diseases, sepsis,
platelet-activating factors, interleukin-1 (IL-1), and toxic Neisseria infections
oxygen metabolites.
Properdin Neisseria infections
o IL-1 - protein that enhances T-cell activation
MASP-2 Pneumococcal diseases
• conversion of C5a occurs more slowly

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 B. Immunologic Assays of Individual Components
B. Regulatory Factor Components • most frequently used to measure individual components
Paroxysmal nocturnal hemoglobinuria (PNH) include radial immunodiffusion (RID) and nephelometry
• disease caused by a missing or defective regulatory • C3 and C4 levels - routinely measured in most clinical
component laboratories by nephelometry or by turbidity
• have RBCs that are deficient in DAF measurements that are often automated
• subject to lysis by means of the bystander effect once the • Nephelometry
complement system has been triggered • measures concentration according to the amount of light
• When C3b is deposited on erythrocytes through activation scattered by a solution containing a reagent antibody and
of either pathway, the result is complement-mediated a measured patient sample
intravascular and extravascular hemolysis, resulting in a • Generally, the more antigen–antibody complexes that are
chronic hemolytic anemia. present, the more a beam of light will scatter as it passes
• DAF deficiency is associated with a lack of CD59 (MIRL) through the solution.
• has the same glycophospholipid anchor • Enzyme-linked immunosorbent assay (ELISA) methods -
available for the measurement of the inhibitor C1-INH.
Hereditary angioedema (HAE)
• involves recurrent attacks of swelling that affect the
extremities, the skin, the gastrointestinal tract, and other
mucosal surfaces
• disease is caused by a deficiency or lack of C1-INH
• C1-INH - a serpin (serine protease inhibitor) that controls
many of the serine proteases on contact
• Types
o Type I
§ characterized by a decrease in the C1-INH
protein
o Type II
§ has normal levels of C1-INH, but the function
is decreased

Hemolytic uremic syndrome (HUS)

• most common cause of renal failure in children and is
characterized by hemolytic anemia, low platelet count,
and acute renal failure
• primary cause of HUS is a Shiga toxin related to infection
that is associated with acute diarrhea
• atypical form of HUS (aHUS)
• occurs because of complement dysregulation caused by
genetic polymorphisms
C. Assays for the Classical Pathway
• has a less acute onset and may not be associated with
diarrhea • measure lysis, the end point of complement activation

C3 glomerulopathies (C3G) Hemolytic titration (CH50) assay

• diseases involving the glomeruli of the kidneys • most commonly used for this purpose
• only C3 that is found in the deposits on the kidney • measures the amount of patient serum required to lyse
• C3 nephritic factors (C3NeF) 50% of a standardized concentration of antibody-
sensitized sheep erythrocytes
o autoantibodies
o binds the C3-convertase from the alternative • titer is expressed in CH50 units, which is the reciprocal of
pathway, C3bBb, holding it together and making it the dilution that is able to lyse 50% of the sensitized cells.
impervious to the normal control mechanisms • The 50% point is used because this is when the change in
o leads to uncontrolled cleavage of C3 with lytic activity per unit change in complement is at maximum
concomitant uncontrolled deposition of C3 on the
kidneys Radial hemolysis in agarose plates
• Rabbit RBCs that have been sensitized with antibody are
LABORATORY DETECTION OF COMPLEMENT implanted in agarose and patient serum is added to wells
punched in the gel.
ABNORMALITIES
• Lysis appears as a clear zone around each well; if
A. Introduction complement standards are run, the size of the zone can
• Techniques to determine complement abnormalities be related to complement concentration.
generally fall into two categories:
o measurement of components as antigens in serum Solid-phase IgM attached to the walls of microtiter plates
and
o measurement of functional activity • Anti-human antibody to C9 conjugated to alkaline
phosphatase is the indicator of complement activation.

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• When a substrate is added, if any C9 is present and the INTERPRETATION OF LABORATORY FINDINGS
antibody conjugate has attached, a color change will be • Blood should be collected in a clot tube with no serum
evident. separator.
• The tube should be spun down and the serum should be
frozen or placed on dry ice if it is not tested within 1 to 2
hours.
• If a specimen has been inadequately refrigerated, been
subjected to multiple freeze–thaws, or been in prolonged
storage, the results may be invalid and the test needs to
be repeated with a fresh specimen.
• If the CH50 is low but the AH50 is normal, the
components unique to the classical pathway should be
investigated.
• If the CH50 is normal but the AH50 is low, the alternative
pathway components need to be investigated.
• If both the CH50 and AH50 are low, suspicion should be
on the components of the terminal pathway because that
pathway is shared by both the classical and alternative
pathways.

Diagnosis of Complement Abnormalities

Impaired Classical Lectin Alternative
Function or Pathway Pathway Pathway
Deficiency
C1q, C1r, C1s Low Normal Normal
C4, C2 Low Low Normal
MBL, MASP2 Normal Low Normal
B, D, P Normal Normal Low
C3, C5, C6, Low Low Low
C7, C8, C9
C1-INH Low Low Low
Factor H and I Low Low Low
Improperly Low Low Low
handled
sera
Adapted from Seelen MA, et al. An enzyme-linked immunosorbent
assay-based method for functional analysis of the three pathways of
the complement system. In: Detrick B, Hamilton RG, and Folds JD,
eds. Manual of Molecular and Clinical Laboratory Immunology. 7th
ed. Washington, DC: ASM Press; 2006:124

Review Questions
1. The classical complement pathway is activated by
a. most viruses.
b. antigen–antibody complexes.
c. fungal cell walls.
d. mannose in bacterial cell walls.
2. Which of the following is characteristic of complement
components?
a. Normally present in serum
b. Mainly synthesized by B cells
c. Present as active enzymes
d. Heat stable
3. All of the following are true of the recognition unit except
D. Alternative Pathway Assays a. it consists of C1q, C1r, and C1s.
AH50 b. the subunits require calcium for binding together.
• same manner as the CH50 c. binding occurs at the FC region of antibody
• except magnesium chloride and ethylene glycol molecules.
tetraacetic acid (EGTA) are added to the buffer and d. C1q becomes an active esterase.
calcium is left out 4. Which of the following is referred to as C3 convertase?
• buffer chelates calcium, which blocks classical pathway a. C1qrs
activation b. C3bD
• rabbit RBCs - used as the indicator because they provide c. C3bBb
an ideal surface for alternative pathway activation d. C4b5a

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5. Mannose-binding protein in the lectin pathway is most 15. Which of the following would be most effective in
similar to which classical pathway component? preventing bystander lysis of RBCs?
a. C3 a. C1-INH
b. C1rs b. Factor B
c. C1q c. DAF
d. C4 d. Factor H
6. Which of the following describes the role of properdin in 16. A decreased CH50 level and a normal AH50 level
the alternative pathway? indicate which deficiency?
a. Stabilization of C3/C5 convertase a. Decrease in components in the lectin pathway
b. Conversion of B to Bb only
c. Inhibition of C3 convertase formation b. Decrease in components in the alternative
d. Binding and cleavage of Factor B pathway only
7. Which best characterizes the membrane attack complex c. Decrease in components of both classical and
(MAC)? alternative pathways
a. Each pathway uses different factors to form it. d. Decrease in components of the classical pathway
b. C5 through C9 are not added in any particular only
order. 17. Which best describes the role of an anaphylatoxin?
c. One MAC unit is sufficient to lyse any type of cell. a. Coats cells to increase phagocytosis
d. C9 polymerizes to form the transmembrane b. Attracts WBCs to the area of antigen
channel. concentration
8. All of the following represent functions of the c. Increases production of interleukin-1
complement system except d. Increases permeability of blood vessels
a. decreased clearance of antigen–antibody 18. Which best describes the role of Factor H?
complexes. a. Acts with DAF to break down C3b
b. lysis of foreign cells. b. Prevents binding of Factor B to C3b
c. increase in vascular permeability. c. Binds to the C5C6C7 complex
d. migration of neutrophils to the tissues. d. Binds to C1q to shut down the classical pathway
9. Which of the following is true of the amplification loop in 19. A lack of C1-INH might result in which of the following
complement activation? conditions?
a. It is only found in the alternative pathway. a. Paroxysmal nocturnal hemoglobinuria
b. The membrane attack unit is amplified. b. Hemolytic uremic syndrome
c. C3b is the product that is increased. c. Hereditary angioedema
d. Increasing amounts of C1qrs are produced. d. Increased bacterial infections
10. Factor H acts by competing with which of the following 20. Which would be most effective in measuring an
for the same binding site? individual complement component?
a. Factor B a. CH50 assay
b. Factor D b. Radial immunodiffusion
c. C3B c. AH50 assay
d. Factor I d. Lytic assay with liposome
11. A lack of CR1 receptors on RBCs would result in which
of the following?
a. Decreased binding of C3b to RBCs
b. Decreased clearance of immune complexes by
the spleen
c. Decreased breakdown of C1qrs
d. Decreased binding of Factor H
12. Which best describes the role of CR2 on cell
membranes?
a. Binds C1qrs to inactivate it
b. Acts as co-receptor on B cells for antigen
c. Increases clearance of immune complexes
d. Binds particles opsonized with C3b
13. Which of the following best characterizes hemolytic
uremic syndrome?
a. It is a rare cause of renal failure in children.
b. It can be associated with deficiencies in Factor H.
c. The major cause is lack of DAF on RBCs.
d. It is associated with antibody-to-C3 convertase.
14. The CH50 test measures which of the following?
a. Patient serum required to lyse 50% of sensitized
sheep RBCs
b. Functioning of both the classical and alternative
pathways
c. Genetic deficiencies of any of the complement
components
d. Functioning of the lectin pathway only

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BASIC IMMUNOLOGIC
PROCEDURES

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IMMUNOSEROLOGY 8.

Safety and Quality Management

OUTLINE microscopy, moderate complexity, and high-
I. Summary complexity testing
o TJC - provides accreditation and certification of
health-care organizations
SUMMARY o CAP - provides laboratory accreditation and
• Transmission of biological hazards that are encountered provision of proficiency testing
when testing patient specimens requires a chain of o CLSI - develops written standards and guidelines
infection, which consists of an infectious agent, reservoir, for sample collection, handling and processing,
portal of exit, mode of transmission, portal of entry, and a and laboratory testing and reporting
susceptible host. • The 12 quality essentials provide the management
• Hand hygiene and wearing PPE are essential actions to documentation needed to demonstrate quality work.
prevent transmission of infectious organisms. Standard Quality indicators are developed to monitor each phase of
precautions should be followed at all times. testing.
• Specimens, except urine, and contaminated supplies • The Lean system utilizes the “6S” tools (sort, straighten,
must be disposed of in a biohazard container. scrub, safety, standardize, and sustain) to enhance
• All sharps, including needles and holders, must be efficiency and proficiency.
disposed of in puncture-proof containers. Recapping of • The goal of the statistical Six Sigma method is to reduce
needles is prohibited. • variables and decrease errors to a level of 3.4 defects per
• The Occupational Exposure to Bloodborne Pathogens 1 million opportunities.
Standards are a means of providing protection from
accidental exposure to bloodborne pathogens through the Review Questions
use of engineering controls, work practice controls, and 1. A technologist who observes a red rash on her hands
use of PPE. after removing her gloves
• When transporting biological specimens, Department of a. should apply antimicrobial lotion to the hands.
Transportation and International Air Transit Association b. may be washing the hands too frequently.
regulations must be followed. They include placing c. may have developed a latex allergy.
specimens in screw cap containers, wrapping them in d. should not create friction when washing the
absorbent material, and placing them in a sturdy leakproof hands.
container. 2. In the chain of infection, a contaminated work area
• Follow specific directions when mixing chemicals and would serve as which of the following?
always add acid to water, rather than water to acid. a. Reservoir
• When chemical contact with the skin or eyes occurs, b. Means of transmission
immediately flush the area with water for 15 minutes. c. Portal of entry
• A SDS and a Chemical Hygiene Plan must be available to d. Portal of exit
employees. Dispose of chemicals per EPA guidelines. 3. The only biological waste that does not have to be
• Dispose of radioactive material following NRC guidelines. discarded in a container with a biohazard symbol is
• Be observant for frayed cords, overloaded circuits, and a. urine.
improperly grounded equipment. Avoid working with b. serum.
electrical equipment when you, or the equipment, is wet. c. feces.
• Follow routine safety protocols and maintain a clean, d. serum tubes.
organized work area to avoid physical hazards. 4. Patient specimens transported by the Department of
• The acronym RACE outlines the steps to follow when a Transportation must be labeled as a
fire is discovered: (R) rescue anyone in danger, (A) a. diagnostic specimen.
activate the fire alarm, (C) contain the fire, (E) extinguish b. clinical specimen.
the fire if possible or evacuate, closing the door. c. biological specimen, category b.
• Quality management is the overall process of d. laboratory specimen.
guaranteeing quality throughout the entire testing system. 5. A technician places tightly capped noninfectious serum
tubes in a rack and places the rack and the specimen
• Quality control involves performing individual procedures
data in a labeled leakproof metal courier box. Is there
using acceptable standards and control material at
anything wrong with this scenario?
various medically significant levels.
a. Yes, DOT requirements are not met.
• Documentation includes a procedure manual, policies to
b. No, the tubes are placed in a rack.
control and monitor procedure variables, and records of
c. Yes, absorbent material is missing.
competency assessment and continuing education.
d. No, the box contains the specimen data.
• Preexamination variables occur before sample testing. 6. The Occupational Exposure to Bloodborne Pathogens
Examination variables occur during the specimen testing. Standard developed by OSHA requires employers to
Postexamination variables occur during interpretation and provide all of the following except
reporting of test results. a. hepatitis B immunization.
• Agencies regulating the laboratory include: b. safety training.
o CLIA - provides requirements for persons c. hepatitis C immunization.
performing waived, provider-performed d. laundry facilities for nondisposable lab coats.

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7. An employee who receives an accidental needlestick b. Increased laboratory productivity
should immediately c. Improved patient outcomes
a. apply sodium hypochlorite to the area. d. Reproducible test results
b. notify a supervisor. 19. Would a control sample that has accidentally become
c. receive HIV prophylaxis. diluted produce a trend or a shift in the Levey-Jennings
d. receive a hepatitis B booster shot. plot?
8. The first thing to do when acid is spilled on the skin is to a. Trend
a. notify a supervisor. b. Shift
b. neutralize the area with a base.
c. apply burn ointment. Fill in the Blank
d. flush the area with water. 20. Indicate whether each of the following would be
9. When combining acid and water, considered a (1) preexamination, (2) examination, or (3)
a. acid is added to water. postexamination variable by placing the appropriate
b. water is added to acid. number in the space.
c. water is slowly added to acid. ____ Reagent expiration date
d. both solutions are combined simultaneously. ____ Rejection of a hemolyzed specimen
10. To determine the chemical characteristics of sodium ____ Construction of a Levey-Jennings chart
azide, an employee would consult the ____ Telephoning a critical result to the nurse
a. Chemical Hygiene Plan. ____ Calibrating the centrifuge
b. Merck manual. ____ Pipetting the diluent
c. SDS.
d. NRC guidelines.
11. A technician who is pregnant should avoid working with
a. organic chemicals.
b. radioisotopes.
c. HIV-positive serum.
d. needles and lancets.
12. Which of the following laboratory regulatory agencies
classifies laboratory tests by their complexity?
a. OSHA
b. CAP
c. TJC
d. CMS
13. Which of the following organizations publishes
guidelines that are considered the standard of care for
laboratory procedures?
a. CLIA
b. CLSI
c. TJC
d. CAP
14. Quality managment refers to
a. performance of two levels of testing controls.
b. reliable control results.
c. increased productivity.
d. quality of specimens and patient care.
15. When external quality control is run, what information
must be documented?
a. The lot number
b. Expiration date of the control
c. The test results
d. All of the above
16. What steps are taken when the results of the quality
control testing are outside of the stated confidence
limits?
a. Check the expiration date of the control
material
b. Run a new control
c. Open a new control bottle
d. All of the above
17. When a new bottle of QC material is opened, what
information is placed on the label?
a. The time the bottle was opened
b. The supervisor’s initials
c. The lot number
d. The date and the laboratory worker’s initials
18. What is the primary goal of TQM?
a. Precise test results

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IMMUNOSEROLOGY 9.

Principles of Serological Testing

OUTLINE • enables the user to dispense the exact measure of liquid
I. Introduction with a small drop left behind
A. Definitions • usually labeled TD (to deliver)
II. Blood Specimen Preparation and Measuring • have an oval bulb in the center and a tapered dispensing
A. Blood Specimen Collection and Preparation end
B. Measurement • used with some sort of suctioning device such as a rubber
III. Dilutions bulb
A. Introduction o bulb is squeezed to draw the measured amount of
B. Simple Dilutions liquidinto the pipette
C. Compound Dilutions • Excess fluid is wiped off the outside of the pipette and
IV. Test Parameters then the pipette is held vertically with the tip against the
A. Sensitivity and Specificity surface of a container
B. Positive and Negative Predictive Value • The suction is released and the liquid is allowed to flow by
gravity into the container.
INTRODUCTION
A. Definitions
Serology
• study of the fluid components in the blood, especially
antibodies.

Serum
• the liquid portion of the blood minus the coagulation
factors most frequently encountered specimen in
immunologic testing

BLOOD SPECIMEN PREPARATION AND

MEASURING 2. Graduated pipettes
A. Blood Specimen Collection and Preparation • have markings that allow for varying amounts of liquid to
• collected aseptically by venipuncture into a clean, dry, be measured
sterile tube • has marks all along its length
• hemolysis → false-positive test • labeled TC (to contain)
• allowed to clot at room temperature or at 4°C, depending • If they are filled to the end, the last drop of liquid must be
upon the protocol for the specific procedure forced out using a pipetting bulb or other device to deliver
• centrifuged an accurate volume.
• serum
o should be promptly separated into another tube a. Serological pipette
without transferring any cellular elements • marks go all the way down to the tip
o Fresh serum that has not been inactivated by • blowout pipette - have a frosted band around the opening
heating - usually recommended for testing
§ complement must be inactivated because
it interferes with test results
• Serum is heated to 56°C for 30
minutes to destroy any
complement present
• may be stored between 2°C and 8°C for up to 72 hours
• additional delay: frozen at –20°C or below

B. Measurement
Pipettes
• commonly used to measure either serum for testing or
liquid for making reagents and dilutions
• calibrated to transfer or deliver specific volumes as
marked on their surfaces

Categories of Pipettes
1. Volumetric pipettes
• marked and calibrated to deliver only one volume of the
specified liquid

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• Generally, a measuring pipette is held in a vertical
position
• when drawing up a liquid.
• The bottom of the meniscus should be level with the
calibration line on the pipette.
DILUTIONS
Micropipettes A. Introduction
• deliver volumes in the microliter (μL) range and can be • For many serology tests, it is the serum that is
used when very small volumes are needed concentrated; it may be necessary to dilute it with saline in
• mechanical pipettes that draw up and then release a order for a visible reaction to occur.
certain volume by depressing a plunger • If the relative proportions of antigen and antibody present
• one-time use disposable tip is used for each different are not similar, the reaction cannot be detected.
specimen • When too much antibody is present, an end point may not
• easier to use than pipettes with safety bulbs, as well as be reached. In this case, the serum that contains antibody
more accurate must be diluted.

B. Simple Dilutions
Dilution
• consist of two entities:
o solute - material being diluted
o diluent - medium making up the rest of the solution
§ relationship between these two is a ratio
that can also be expressed as a fraction
• e.g. 1:20 is 1/20 (implies 1 part of solute and 19 parts of
diluent)

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• Sometimes it is necessary to dilute a concentrate to a To give the final volume, we add the 50 mL of concentrate to the 950
specific percentage. mL of water to get 1,000 mL total.
o A percentage is simply a different way of
expressing a dilution. 4. A volume of 500 mL of a 10% solution of acetic acid is
o e.g. 10% solution can also be expressed as 1/10 needed. How much glacial acetic acid is needed and
how much diluent should be used?

1/Dilution = Amount of Solute/Total Volume

1/10 = x/500 mL

Examples Cross-multiplying and solving for x gives us 50 mL of glacial acetic

acid needed.
1. 2 mL of a 1:20 dilution is needed to run a specific Then the amount of diluent is the total volume minus the
serological test. How much serum and how much diluent concentrate, or 500 mL – 50 mL = 450 mL of diluent to make up the
are needed to make this dilution? 10% solution.

C. Compound Dilutions
1/20 = x/2 mL • a process of very large dilution
• more accurate and less costly to do this in several steps
20 - total number of parts in the solution rather than all at once
2 mL - total volume desired • dilution occurs in several stages
o e.g. if a 1:500 dilution is necessary, it would take
= 1 x x 49.9 mL of diluent to accomplish this in one step
20 2 with 0.1 mL of serum
• If only a small amount of solution is needed to run the
= 0.1 mL test, this is wasteful; furthermore, inaccuracy may occur if
the solution is not properly mixed. Therefore, it is helpful
Amount of diluent: to make several smaller dilutions
2.0 mL - 0 .1 mL
= 1.9 mL of diluent Steps to calculate a compound dilution problem
First step: Plan the number and sizes of simple dilutions
Checking: necessary to reach the desired end point
0.1 mL/(1.9 mL diluent + 0.1 mL serum) = 1/20
1:500 dilution can be achieved by making a 1:5 dilution of
2. A 1:5 dilution of patient serum is necessary to run a the original serum, a 1:10 dilution from the first dilution, and
serological test. There is 0.1 mL of serum that can be another 1:10 dilution. This can be shown as follows:
used. What amount of diluent is necessary to make this
dilution using all of the serum? Serum:
1:5 dilution 1:10 dilution 1:10 dilution
Equation: 0.1 mL serum 0.1 mL of 0.1 mL of
1:5 dilution 1:10 dilution
1/Dilution – 1 = Amount of Solute/Amount of Diluent 0.4 mL diluent 0.9 mL diluent 0.9 mL diluent

1/4 = 0.1 mL/x Multiplying 5 × 10 × 10 equals 500, or the total dilution.

Each of the simple dilutions is calculated individually by
x = 0.4 mL of diluent doing mental arithmetic or by using the formula given for
simple dilutions. In this example, the 1:500 dilution was
Final volume is obtained by adding 0.1 mL of solute to the 0.4 mL of made using very little diluent in a series of test tubes, rather
diluent. than having to use a larger volume in a flask. The volumes
Dividing the volume of the solute by the total volume of 0.5 mL yields were kept small enough so that mixing could take place
the desired 1:5 ratio.
easily. The final volume of 1.0 mL is all that is necessary to
3. Instructions that come with a buffer indicate that it must perform a test.
be mixed with 19 parts of water for use in a serological
test. The volume of the buffer concentrate is 50 mL. Serial dilution
How would we find out the amount of water to add and • If, in each step of the dilution, the dilution factor is exactly
what would be the final dilution factor? the same
• often used to obtain a titer (indicator of an antibody’s
Equation: strength)
• series of test tubes is set up with exactly the same
1/Dilution – 1 = 50 mL/ Amount of Diluent amount of diluent in each
• most common serial dilution is a doubling dilution in which
1/19 = 50/x x = 50 × 19 the amount of serum is cut in half with each dilution

x = 950 mL of diluent

First, multiply 50 mL by the 19 parts to give us 950 mL, which is the

amount of water that needs to be added to the buffer concentrate.

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 Sensitivity (%) = True Positives × 100
True Positives and False Negatives

Specificity
• proportion of people who do not have the disease or
condition and who have a negative test

Specificity (%) = True Negatives × 100

True Negatives and False Positives

Examples
1. A certain new laboratory test was used with a particular
population. The results were as follows: 200 patients
were tested and there were 160 true positives, 20 true
negatives, 8 false positives, and 12 false negatives.

Sensitivity (%) = 160/(160 + 12) × 100

Examples = 93%
1. Six test tubes can be set up with 0.2 mL of diluent in
each. If 0.2 mL of serum is added to the first tube, this Specificity (%) = 20/(20 + 8) × 100
becomes a 1:2 dilution. = 71.4%

0.2 mL Serum/(0.2 mL Serum + 0.2 mL Diluent) = Answer: Highly sensitive, not very specific
0.2 mL/0.4 mL = ½
B. Positive and Negative Predictive Value
Positive predictive value
When 0.2 mL of the 1:2 dilution is added to 0.2 mL of diluent, • probability that a person with a positive screening test
a 1:4 dilution is obtained. The final dilution is obtained by actually has the disease
counting the number of tubes and setting up a multiplication
series in which the original dilution factor is raised to a power
equal to the number of tubes. In this example, if the first tube Positive Predictive Value (%) = True Positives × 100
contains a 1:2 dilution, the dilution in tube number six is True Positives + False Positives

1/2 ⨯ 1/2 ⨯ 1/2 ⨯ 1/2 ⨯ 1/2 ⨯ 1/2 = 1/64 Negative predictive value
• probability that a person with a negative screening test
If, in this instance, an end point was reached at tube number does not have the disease
five, the actual titer would be 1:32. To avoid confusion, this is
customarily written as the reciprocal of the dilution—that is,
32. The titer is the last tube in which a positive reaction is Negative Predictive Value (%) = True Negatives × 100
visible. The tubes are read all the way to the end of the True Negatives + False Negatives
dilution; then, after finding the first negative tube, the positive
tube before it is reported out as the titer.
Review Questions
Serial dilutions do not always have to be doubling dilutions. 1. If serum is not tested immediately, how should it be
Consider the following set of test tube dilutions: treated?
a. It can be left at room temperature for 24 hours.
1:5→1:25→1:125→1:625→1:3125 b. It can be stored in the refrigerator for up to 72
hours.
For each successive tube, the dilution is increased by a c. It can be stored in the refrigerator for up to 48
factor of 5, so this would indeed be considered a serial hours.
dilution. d. It needs to be frozen immediately.
2. A 1:750 dilution of serum is needed to perform a
serological test. Which of the following series of
TEST PARAMETERS dilutions would be correct to use in this situation?
A. Sensitivity and Specificity a. 1:5, 1:15, 1:10
Sensitivity b. 1:5, 1:10, 1:5
• proportion of people who have a specific disease or c. 1:15, 1:10, 1:3
condition and who have a positive test d. 1:15, 1:3, 1:5
• indicates how small an amount can be measured and still 3. How much diluent needs to be added to 0.2 mL of
produce a positive test result serum to make a 1:20 dilution?
a. 19.8 mL
b. 4.0 mL

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 c. 3.8 mL 14. Which of the following would be the correct way to make
d. 10.0 mL a 5% solution of hydrochloric acid from concentrated
4. If glacial acetic acid needs to be diluted with water to hydrochloric acid?
make a 10% solution, what does the glacial acetic acid a. 0.5 mL of acid and 9.5 mL of water
represent? b. 0.5 mL of acid and 95 mL of water
a. Solute c. 0.1 mL of acid and 9.9 mL of water
b. Diluent d. 0.1 mL of acid and 4.9 mL of water
c. Titer 15. What is the final dilution of serum obtained from the
d. Serial dilution following serial dilutions: 1:4, 1:4, 1:4, 1:4, 1:4, 1:4?
5. A pipette that has markings all the way down to its tip is a. 1:24
called a b. 1:256
a. volumetric pipette. c. 1:1,024
b. serial pipette. d. 1:4,096
c. graduated pipette. 16. A new laboratory assay gave the following results:
d. micropipette. number of patients tested = 100; number of true
6. A serological test requires 5 mL of a 1:50 dilution. How positives = 54, number of true negatives = 42; number
much serum is required to make this dilution? of false positives = 2; number of false negatives = 2.
a. 0.5 mL What is the specificity of this assay in whole numbers?
b. 0.01 mL a. 75%
c. 1.0 mL b. 85%
d. 0.1 mL c. 95%
7. If 0.02 mL of serum is diluted with 0.08 mL of diluent, d. 98%
what dilution of serum does this represent? 17. What is the sensitivity of the assay in Question 16?
a. 1:4 a. 84%
b. 1:5 b. 90%
c. 1:10 c. 92%
d. 1:20 d. 96%
8. A tube containing a 1:40 dilution is accidently dropped. 18. A screening test gave the following results: number of
A 1:2 dilution of the specimen is still available. A volume patients tested = 150; number of true positives = 50;
of 4 mL is needed to run the test. How much of the 1:2 number of true negatives = 85; number of false
dilution is needed to remake 4 mL of a 1:40 dilution? positives = 5; number of false negatives = 10. What is
a. 0.2 mL the positive predictive value rounded off to a whole
b. 0.4 mL number for a patient whose test is positive?
c. 0.5 mL a. 91%
d. 1.0 mL b. 83%
9. If 0.4 mL of serum is mixed with 15.6 mL of diluent, what c. 89%
dilution of serum does this represent? d. 56%
a. 1:4
b. 1:40
c. 2:70
d. 1:80
10. How much diluent needs to be added to 0.1 mL of
serum to make a 1:15 dilution?
a. 1.4 mL
b. 1.5 mL
c. 5.0 mL
d. 15 mL
11. Which of the following choices would be considered a
serial dilution?
a. 1:5, 1:15, 1:20
b. 1:2, 1:10, 1:25
c. 1:15, 1:30, 1:40
d. 1:5, 1:15, 1:45
12. The following dilutions were set up to titer an antibody.
The following results were obtained: 1:4 +, 1:8 +, 1:16 +,
1:32 +, 1:64 –. How should the titer be reported out?
a. 4
b. 16
c. 32
d. 64
13. If a serological test is positive for an individual who does
not have a particular disease, the result was caused by
a problem with
a. sensitivity.
b. specificity.
c. accuracy.
d. poor pipetting.

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IMMUNOSEROLOGY 10.

Precipitation and Agglutination Reactions

OUTLINE • cross-reactivity - reacting with antigens resembling the
I. Introduction original antigen that induced antibody production
A. Definitions • The more the cross-reacting antigen resembles the
II. Antigen-Antibody Binding original antigen, the stronger the bond will be between the
A. Affinity antigen and the binding site.
B. Avidity • When the affinity is higher, the assay reaction is more
C. Law of Mass Action sensitive because more antigen–antibody complexes will
III. Precipitation Curve be formed and visualized more easily.
A. Zone of Equivalence
B. Prozone and Postzone
IV. Measurement of Precipitation by Light Scattering
A. Turbidimetry
B. Nephelometry
V. Passive Immunodiffusion Techniques
A. Introduction
B. Radial Immunodiffusion
C. Ouchterlony Double Diffusion
VI. Electrophoretic Techniques
A. Electrophoresis
B. Immunoelectrophoresis
C. Immunofixation electrophoresis
VII. Comparison of Precipitation Techniques
VIII. Principles of Agglutination Reactions
A. Agglutination
B. Immunoglobulins and Agglutination
IX. Types of Agglutination Reactions
A. Direct Agglutination
B. Passive or Indirect Agglutination B. Avidity
C. Reverse Passive Agglutination
• overall strength of antigen–antibody binding and is the
D. Agglutination Inhibition
sum of the affinities of all the individual antibody–antigen
X. Instrumentation
combining sites
A. Particle-counting immunoassay (PACIA)
• refers to the strength with which a multivalent antibody
binds a multivalent antigen and is a measure of the overall
INTRODUCTION stability of an antigen–antibody complex
A. Definitions • A high avidity can compensate for a low affinity.
Precipitation • The more bonds that form between antigen and antibody,
• combining soluble antigen with soluble antibody to the higher the avidity is.
produce insoluble complexes that are visible • IgM, for instance, has a higher avidity than IgG because
• first noted in 1897 by Kraus IgM has the potential to bind 10 different antigens

Agglutination
• process by which particulate antigens such as cells
aggregate to form larger complexes when a specific
antibody is present

NOTE

Precipitation and agglutination are considered

unlabeled assays because a marker label is not needed to
detect the reaction.

ANTIGEN-ANTIBODY BINDING
A. Affinity
• initial force of attraction that exists between a single Fab
site on an antibody molecule and a single epitope or
determinant site on the corresponding antigen
• held together by rather weak bonds occurring
• short distance of approximately 1⨯10–7 mm

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C. Law of Mass Action B. Prozone and Postzone
• states that free reactants are in equilibrium with bound Prozone phenomenon
reactants • in the case of antibody excess
• The equilibrium constant K represents the difference in • antigen combines with only one or two antibody molecules
the rates of the forward and reverse reactions according and no cross-linkages are formed
to the following equation: • only one site on an antibody molecule is used and many
free antibody molecules remain in solution
• false-negative reaction may take place because of high
K = [AgAb]/[Ab][Ag] antibody concentration
o Resolution: diluting out antibody and performing the
[AgAb] = concentration of the antigen–antibody complex (mol/L) test again may produce a positive result
[Ab] = concentration of free antibody (mol/L)
[Ag] = concentration of free antigen (mol/L) Postzone phenomenon
• where there is antigen excess
• The value of K depends on the strength of binding
• small aggregates are surrounded by excess antigen
between antibody and antigen. As the strength of binding,
• no lattice network is formed
or avidity, increases, the tendency of the antigen–antibody
complexes to dissociate decreases, which increases the • every available antibody site is bound to a single antigen
value of K. and no cross-links are formed
• When the value of K is higher, the amount of antigen– o Resolution: test is repeated with an additional
antibody complex is larger and the assay reaction is more patient specimen taken about a week later
visible or easily detectable.
• The ideal conditions in the clinical laboratory would be to MEASUREMENT OF PRECIPITATION BY LIGHT
have an antibody with a high affinity, or initial force of SCATTERING
attraction, and a high avidity, or strength of binding. A. Turbidimetry
• The higher the values are for both of these and the more • a measure of the turbidity or cloudiness of a solution
antigen–antibody complexes that are formed, the more • A detection device is placed in direct line with an incident
sensitive the test. light, collecting the light after it has passed through the
solution.
PRECIPITATION CURVE o This device measures the reduction in light
A. Zone of Equivalence intensity caused by reflection, absorption, or
• number of multivalent sites of antigen and antibody are scatter.
approximately equal • The amount of scatter is proportional to the size, shape,
• precipitation is the result of random, reversible reactions and concentration of molecules present in solution.
whereby each antibody binds to more than one antigen • It is recorded in absorbance units, a measure of the ratio
and vice versa, forming a stable network or lattice of incident light to that of transmitted light.
• lattice hypothesis • The measurements are made using a spectrophotometer
o formulated by Marrack or an automated clinical chemistry analyzer.
o based on the assumptions that each antibody
molecule must have at least two binding sites and B. Nephelometry
the antigen must be multivalent. • measures the light that is scattered at a particular angle
o As they combine, this arrangement results in a from the incident beam as it passes through a suspension
multimolecular lattice that increases in size until it • The amount of light scattered is an index of the solution’s
precipitates out of solution concentration.
• When the same amount of soluble antigen is added to • If a solution has excess antibody, adding increasing
increasing dilutions of antibody, the amount of amounts of antigen results in an increase in antigen–
precipitation increases up to the zone of equivalence. antibody complexes and thus an increase in light
• When the amount of antigen overwhelms the number of scattering.
antibody-combining sites present, precipitation begins to • used to detect either antigen or antibody, but typically it is
decline because fewer lattice networks are formed. run with antibody as the reagent and patient antigen as
the unknown

Rate nephelometry
• measurement of serum proteins
• rate of scattering increase is measured immediately after
the reagent antibody is added
• Quantification of immunoglobulins such as IgG, IgA, IgM,
and IgE, as well as kappa and lambda light chains

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 C. Ouchterlony Double Diffusion
• both antigen and antibody diffuse independently through a
semisolid medium in two dimensions, horizontally and
PASSIVE IMMUNODIFFUSION TECHNIQUES vertically
A. Introduction • Incubation period: 12 to 48 hours
Agarose • precipitin lines form where the moving front of antigen
meets that of antibody and the point of equivalence is
• a purified high-molecular-weight complex polysaccharide
reached
derived from seaweed
• The density of the lines reflects the amount of immune
• for precipitation of antigen–antibody complexes that can
complex formed.
also be determined in a support medium such as a gel
• used to identify fungal antigens such as Aspergillus,
• When antigen and antibody diffuse toward one another in
Blastomyces, Coccidioides, and Candida.
a gel matrix, visible lines of precipitation will form
• Several patterns are possible:
• The rate of diffusion is affected by the size of the particles,
o Fusion of the lines at their junction to form an arc
the temperature, the gel viscosity, and the amount of
represents serological identity or the presence of a
hydration.
common epitope,
o a pattern of crossed lines demonstrates two
B. Radial Immunodiffusion
separate reactions and indicates that the compared
• single-diffusion technique
antigens share no common epitopes, and
• antibody is uniformly distributed in the support gel and o fusion of two lines with a spur indicates partial
antigen is applied to a well cut into the gel identity
End-point method
• developed by Mancini
• one technique for the measurement of radial
immunodiffusion
• antigen is allowed to diffuse to completion; when
equivalence is reached, there is no further change in the
ring diameter
• allotted time of 24 to 72 hours
• square of the diameter is then directly proportional to the
concentration of the antigen

Kinetic or Fahey method

• uses ring diameter readings taken at about 19 hours
before equivalence is reached
• diameter is then proportional to the log of the
concentration and a graph is plotted using semi-log paper

Sources of error include

• overfilling or underfilling the wells
• nicking the side of the wells when filling
• spilling sample outside the wells
• improper incubation time and temperature
• incorrect measurement

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 ELECTROPHORETIC TECHNIQUES
A. Electrophoresis
• separates molecules according to differences in their
electric charge when they are placed in an electric field.
• A direct current is forced through the gel, causing antigen,
antibody, or both to migrate.
• As diffusion takes place, distinct precipitin bands are COMPARISON OF PRECIPITATION TECHNIQUES
formed. Comparison of Precipitation Techniques
Technique Application Sensitiv Principle
B. Immunoelectrophoresis
ity (μG
• a double-diffusion technique that incorporates AB/ML)
electrophoresis to enhance results.
Nephelometr Immunoglobul 1–10 Light that is
• Typically, the source of antigen is serum, which is y ins, scattered at
electrophoresed to separate out the main proteins. complement, an angle is
• A trough is then cut in the gel parallel to the line of C-reactive measured,
separation. protein, indicating
• Antiserum is placed in the trough and the gel is incubated other serum the amount
for 18 to 24 hours. proteins of antigen
• Double diffusion occurs at right angles to the or antibody
electrophoretic separation and precipitin lines develop present.
where specific antigen–antibody combination takes place. Radial Immunoglobul 10–50 Antigen
• Replaced by immunofixation electrophoresis immunodiffus ins, diffuses out
ion complement into gel that
C. Immunofixation electrophoresis is infused
• similar to immunoelectrophoresis except that after with
electrophoresis takes place, antiserum is applied directly antibody.
to the gel’s surface rather than placed in a trough. Measureme
• Agarose or cellulose acetate is used nt of the
• Less than 1 hour radius
• Typically, patient serum is applied to six lanes of the gel; indicates
after electrophoresis, five lanes are overlaid with one each concentratio
of the following antibodies: antibody to gamma, alpha, or n of antigen.
mu heavy chains and to kappa or lambda light chains. Ouchterlony Complex 20–200 Both antigen
o sixth lane - overlaid with antibody to all serum double antigens such and
proteins; reference lane diffusion as antibody
fungal diffuse out
Interpretation of Results antigens from wells in
• Hypogammaglobulinemias - characterized by low a gel. Lines
antibody production, will exhibit faintly staining bands of
• Polyclonal hypergammaglobulinemias (overproduction of precipitate
antibody) - show darkly staining bands in the gamma formed
region. indicate the
• Monoclonal antibody - such as is found in certain relationship
malignancies of the immune system, will result in dark and of antigens.
narrow bands in specific lanes Immuno- Differentiation 20–200 Electrophor
electrophore of serum esis of
sis proteins serum
followed by
diffusion of
antibody
from wells.

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 Immunofixati Over- or Variable Electrophor • often requires the use of enhancement techniques that
on underproducti esis of vary physicochemical conditions such as the ionic
electrophore on of antibody serum strength of the solution, the pH, and the temperature
sis followed by • agglutinate best at 30°C to 37°C
Direct • Antibodies to other human blood groups usually belong to
application the IgG class; reactions involving these must be run at
of antibody 37°C
to the gel. • requires the use of a second antibody, antihuman
immunoglobulin (Coombs reagent), to visualize a reaction
PRINCIPLES OF AGGLUTINATION REACTIONS o Coombs reagent - attach to the Fc portion of IgG
A. Agglutination and help to bridge the gap between RBCs so a
• visible aggregation of particles caused by combination visible agglutination reaction will occur
with specific antibody
• a two-step process that results in the formation of a stable
lattice network
• Agglutinins - antibodies that produce such reactions

Process
1. Sensitization
• involves antigen–antibody combination through single
antigenic determinants on the particle and is rapid and
reversible
• affected by the nature of the antigens on the agglutinating
particles.
• If epitopes are sparse or if they are obscured by other
surface molecules, they are less likely to interact with
antibody

2. Lattice formation
• represents the stabilization of antigen–antibody
complexes with the binding together of multiple antigenic
determinants
• red blood cells (RBCs) and bacterial cells - have a slight
negative surface charge; because like charges tend to
repel one another, it is sometimes difficult to bring such
cells together into a lattice formation

TYPES OF AGGLUTINATION REACTIONS

A. Direct Agglutination
• occurs when antigens are found naturally on a particle
• e.g. known bacterial antigens used to test for the
presence of unknown antibodies in the patient
• patient serum is diluted into a series of tubes or wells on a
slide and reacted with bacterial antigens specific for the
suspected disease
B. Immunoglobulins and Agglutination
IgM Widal test
• potential valence of 10 is over 700 times more efficient in • a rapid screening test used to help determine the
agglutination possibility of typhoid fever.
• strong agglutinins because of their larger size • A significant finding is a fourfold increase in antibody titer
• react best at temperatures between 4°C and 27°C over time when paired dilutions of serum samples are
tested with any of these antigens.
• naturally occurring antibodies - react against the ABO
blood groups belong to the IgM class, these reactions are
Hemagglutination
best run at room temperature
• If an agglutination reaction involves RBCs
IgG • Patient RBCs mixed with antisera of the IgM type can be
• valence of 2 used to determine the presence or absence of the A and
• often cannot bridge the distance between particles B antigens; this reaction is usually performed at room
temperature without the need for any enhancement
because their small size and restricted flexibility at the
hinge region may prohibit multivalent binding techniques

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• A titer that yields semiquantitative results can be C. Reverse Passive Agglutination
performed in test tubes or microtiter plates by making • antibody rather than antigen is attached to a carrier
serial dilutions of the antibody. particle
• The reciprocal of the last dilution still exhibiting a visible • antibody must still be reactive and is joined in such a
reaction is the titer, indicating the antibody’s strength. manner that the active sites are facing outward
• Positive: Cells that are spread across the well’s bottom, • often used to detect microbial antigens
usually in a jagged pattern with an irregular edge. Test • Principle: Latex particles coated with antibody are reacted
tubes also can be centrifuged and then shaken to see if with a patient sample containing the suspected antigen.
the cell button can be evenly resuspended. • The use of monoclonal antibodies has greatly cut down on
• Negative: If it is resuspended with no visible clumping, cross-reactivity, but there is still the possibility of
then the result is negative. interference or nonspecific agglutination.

C. Agglutination Inhibition
• based on competition between particulate and soluble
Grading of agglutination reactions: A. tube method.
If tubes are centrifuged and shaken to resuspend the button, reactions
antigens for limited antibody-combining sites
can be graded from negative to 4+, depending on the size of clumps o Positive: lack of agglutination
observed. B. Rapid slide method. o Negative: Agglutination
• involves haptens that are complexed to proteins
B. Passive or Indirect Agglutination • If the patient sample has no free hapten, the reagent
• employs particles that are coated with antigens not antibody is able to combine with the carrier particles and
normally found on their surfaces produce a visible agglutination.

Latex agglutination tests Hemagglutination inhibition

• it is found that IgG was naturally adsorbed to the surface • dilutions of patient serum are incubated with a viral
of polystyrene latex particles preparation.
• used to detect rheumatoid factor, antibodies to Group A • Then RBCs that the virus is known to agglutinate are
Streptococcus antigens, and antibodies to viruses such as added to the mixture.
rotavirus, cytomegalovirus, rubella, and varicella-zoster • If antibody is present, it will attach to the viral particles and
prevent agglutination

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• So a lack of or reduction in agglutination indicates the Particles indicating the
presence of patient antibody. compete with presence of
patient antigens patient antigen.
for a limited
number of
antibody sites.
Hemagglutination Red blood cells Lack of
inhibition spontaneously agglutination is a
agglutinate positive test,
if viral particles indicating the
are present. presence of
patient antibody.

Review Questions
1. In a precipitation reaction, how can the ideal antibody be
characterized?
a. Low affinity and low avidity
b. High affinity and low avidity
c. High affinity and high avidity
d. Low affinity and high avidity
Agglutination inhibition. 2. Precipitation differs from agglutination in which way?
Reagent antibody is added to the patient sample. If patient antigen is a. Precipitation can only be measured by an
present, antigen–antibody combination results. When antigen-coated automated instrument.
latex particles are added, no agglutination occurs, which is a positive
b. Precipitation occurs with univalent antigen,
test. If no patient antigen is there, the reagent antibody combines with
latex particles and agglutination results, which is a negative test. whereas agglutination requires multivalent
antigen.
INSTRUMENTATION c. Precipitation does not readily occur because few
antibodies can form aggregates with antigen.
A. Particle-counting immunoassay (PACIA)
d. Precipitation involves a soluble antigen,
• involves measuring the number of residual whereas agglutination involves a particulate
nonagglutinating particles in a specimen antigen.
• These particles are counted by means of a laser beam in 3. When soluble antigens diffuse in a gel that contains
an optical particle counter similar to the one that is antibody, in which zone does optimum
designed to count blood cells. precipitationoccur?
• Nephelometric methods are used to measure forward light a. Prozone
scatter. b. Zone of equivalence
• Latex particles are coated with whole antibody molecules c. Postzone
or with F(ab')2 fragments. Use of the latter reduces d. Prezone
interference and nonspecific agglutination. 4. Which of the following statements apply to rate
• If antigen is present, complexes will form and will be nephelometry?
screened out by the counter because of their large size. a. Readings are taken before equivalence is
• rate assay - rate at which the number of unagglutinated reached.
particles decrease b. It is more sensitive than turbidity.
• end-point assay - total number of unagglutinated particles c. Measurements are time dependent.
left at the end d. All of the above.
5. Which of the following is characteristic of the end-point
Comparison of Agglutination Reactions method of RID?
Type of Principle Results a. Readings are taken before equivalence.
Reaction b. Concentration is directly in proportion to the
Direct Antigen is Agglutination square of the diameter.
agglutination naturally found indicates the c. The diameter is plotted against the log of the
on a particle. presence of concentration.
patient antibody. d. It is primarily a qualitative rather than a
Indirect (passive) Particles coated Agglutination quantitative method.
agglutination with antigens not indicates the 6. In which zone might an antibody-screening test be false
normally found presence of negative?
on their surfaces. patient antibody. a. Prozone
b. Zone of equivalence
Reverse passive Particles are Agglutination c. Postzone
coated with indicates the d. None of the above
reagent presence of 7. How does measurement of turbidity differ from
antibody. patient antibody. nephelometry?
Agglutination Haptens Lack of a. Turbidity measures the increase in light after it
inhibition attached to agglutination is a passes through a solution.
carrier particles. positive test, b. Nephelometry measures light that is scattered at
an angle.
c. Turbidity deals with univalent antigens only.

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 d. Nephelometry is not affected by large particles 17. Reactions involving IgG may need to be enhanced for
falling out of solution. which reason?
8. Which of the following refers to the force of attraction a. It is only active at 25°C.
between an antibody and a single antigenic b. It may be too small to produce lattice formation.
determinant? c. It has only one antigen-binding site.
a. Affinity d. It is only able to produce visible precipitation
b. Avidity reactions.
c. Van der Waals attraction 18. For which of the following tests is a lack of agglutination
d. Covalence a positive reaction?
9. Immunofixation electrophoresis differs from a. Hemagglutination
immunoelectrophoresis in which way? b. Passive agglutination
a. Electrophoresis takes place after diffusion has c. Reverse passive agglutination
occurred in immunofixation electrophoresis. d. Agglutination inhibition
b. Better separation of proteins with the same 19. Typing of RBCs with reagent antiserum represents
electrophoretic mobilities is obtained in which type of reaction?
immunoelectrophoresis. a. Direct hemagglutination
c. In immunofixation electrophoresis, antibody is b. Passive hemagglutination
directly applied to the gel instead of being c. Hemagglutination inhibition
placed in a trough. d. Reverse passive hemagglutination
d. Immunoelectrophoresis is a much faster 20. In a particle-counting immunoassay using reagent
procedure. antibody attached to latex particles, if the particle count
10. If crossed lines result in an Ouchterlony in solution is very low, what does this mean about the
immunodiffusion reaction with antigens 1 and 2, what presence of patient antigen?
does this indicate? a. The patient has no antigen present.
a. Antigens 1 and 2 are identical. b. The patient has a very small amount of antigen.
b. Antigen 2 is simpler than antigen 1. c. The patient has a large amount of antigen
c. Antigen 2 is more complex than antigen 1. present.
d. The two antigens are unrelated. d. The test is invalid.
11. Which technique represents a single-diffusion reaction?
a. Radial immunodiffusion
b. Ouchterlony diffusion
c. Immunoelectrophoresis
d. Immunofixation electrophoresis
12. Which best describes the law of mass action?
a. Once antigen–antibody binding takes place, it is
irreversible.
b. The equilibrium constant depends only on the
forward reaction.
c. The equilibrium constant is related to strength of
antigen antibody binding.
d. If an antibody has a high avidity, it will dissociate
from antigen easily.
13. Agglutination of dyed bacterial cells represents which
type of reaction?
a. Direct agglutination
b. Passive agglutination
c. Reverse passive agglutination
d. Agglutination inhibition
14. If a single IgM molecule can bind many more antigens
than a molecule of IgG, which of the following is higher?
a. Affinity
b. Initial force of attraction
c. Avidity
d. Initial sensitization
15. Agglutination inhibition could best be used for which of
the following types of antigens?
a. Large cellular antigens such as erythrocytes
b. Soluble haptens
c. Bacterial cells
d. Coated latex particles
16. Which of the following correctly describes reverse
passive agglutination?
a. It is a negative test.
b. It can be used to detect autoantibodies.
c. It is used for identification of antigens.
d. It is used to detect sensitization of red blood
cells.

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IMMUNOSEROLOGY 11.

Labeled Immunoassays
OUTLINE This ratio can be illustrated by the following equation:
I. Introduction
A. Definitions 6Ag* + 2Ag + 4Ab → 3Ag*Ab + 1AgAb + 3Ag* + 1Ag
II. Format For Labeled Assays
A. Competitive Immunoassays In this example, labeled and unlabeled antigens occur in a
B. Noncompetitive Immunoassays 3:1 ratio.
III. Heterogenous Vs Homogenous Assays
A. Principle In this example, labeled and unlabeled antigens occur in a
B. Heterogeneous enzyme immunoassays 3:1 ratio. Binding to a limited number of antibody sites will
C. Homogeneous enzyme immunoassays take place in the same ratio. Thus, on the right side of the
IV. Radioimmunoassay (RIA) equation, three of the four binding sites are occupied by
V. Enzyme Immunoassays labeled antigen, whereas one site is filled by unlabeled
A. Introduction antigen. As the amount of patient antigen increases, fewer
B. Heterogeneous Enzyme Immunoassays binding sites will be occupied by labeled antigen, as
C. Homogeneous Enzyme Immunoassays demonstrated by the next equation:
D. Rapid Immunoassays
VI. Fluorescent Immunoassays
A. Introduction 6Ag* + 18Ag + 4Ab →1Ag*Ab + 3AgAb + 5Ag*+ 15Ag
B. Direct Immunofluorescent Assays
C. Indirect Immunofluorescent Assays In this case, the ratio of labeled to unlabeled antigen is 1:3.
D. Fluorescence Polarization Immunoassays
(FPIA) Binding to antibody sites takes place in the same ratio
VII. Chemiluminescent Immunoassays and the amount of bound label is greatly decreased in
E. Chemiluminescence comparison to the first equation.
F. Electrochemiluminescence immunoassay • A standard curve using known amounts of unlabeled
antigen can be used to extrapolate the concentration of
INTRODUCTION the unknown patient antigen.
A. Definitions • The detection limits of competitive assays are largely
Labeled immunoassays determined by the affinity of the antibody.
• designed for antigens and antibodies that may be small in
size or present in very low concentrations
• presence of such antigens or antibodies is determined
indirectly by using a labeled reactant to detect whether or
not specific binding has taken place

Analyte
• substance to be measured
• typically a protein
o e.g. bacterial antigens, hormones, drugs, tumor
markers, specific immunoglobulins, and many
other substances
• bound to molecules that react specifically with them
(typically a antibody)
• One reactant, either the antigen or the antibody, is labeled
with a marker so that the amount of binding can be
monitored.

FORMAT FOR LABELED ASSAYS

A. Competitive Immunoassays
• all the reactants are mixed together simultaneously
• labeled antigen competes with unlabeled patient antigen
for a limited number of antibody-binding sites
• the amount of bound label is inversely proportional to the
concentration of the labeled antigen, which means that
the more labeled antigen that is detected, the less there is
of patient antigen.

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B. Noncompetitive Immunoassays
• aka capture antibody
• first passively absorbed to a solid phase such as
microtiter plates, nitrocellulose membranes, or plastic
beads
• Excess antibody is present so that any patient antigen
present can be captured. Unknown patient antigen is then
allowed to react with and be captured by the solid-phase
antibody.
• After washing to remove unbound antigen, a second
antibody with a label is added to the reaction.
• The amount of label measured is directly proportional to
the amount of patient antigen.
• This type of assay is more sensitive than competitive
immunoassays.

HETEROGENOUS VS HOMOGENOUS ASSAYS

A. Principle
• Categorized according to whether or not it is necessary to
separate the bound reactants from the free ones

B. Heterogeneous enzyme immunoassays

• require a step to physically separate free from bound
analyte.
• Antigen or antibody is attached by physical adsorption;
when specific binding takes place, complexes remain
attached to the solid phase.
o This step provides a simple way to separate bound
and free reactants.
• The sample is then thoroughly washed and the remaining
activity is determined.

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C. Homogeneous enzyme immunoassays RADIOIMMUNOASSAY (RIA)
• do not need a separation step • first type of immunoassay developed
• The activity of the label attached to the antigen is • pioneered by Yalow and Berson
diminished when binding of antibody and antigen occurs. • used to determine the level of insulin–anti-insulin
• Typically, homogeneous assays involve an enzyme label, complexes in diabetic patients
chosen so that the enzyme is inactivated when bound to • uses a radioactive substance as a label
an antibody. • Radioactive elements
• This type of assay is simpler to perform because there is o have nuclei that decay spontaneously, emitting
no washing step. matter and energy.
• The sample containing patient antigen is incubated with o easily incorporated into protein molecules and emits
the labeled antigen and the antibody; the amount of gamma radiation, which is detected by a gamma
activity then can be measured directly. counter
• less sensitive than heterogeneous assays • extremely sensitive and precise technique for determining
trace amounts of analytes that are small in size
• Disadvantage: health hazard involved in working with
radioactive substances.

ENZYME IMMUNOASSAYS
A. Introduction
• using enzymes as labels
• can either be used
o qualitatively - to determine the presence of an
antigen or antibody
o quantitatively - to determine the actual
concentration of an analyte in an unknown
specimen
• typically chosen according to the number of substrate
molecules converted per molecule of enzyme, ease and
speed of detection, and stability
• colorimetric reactions include: horseradish peroxidase,
alkaline phosphatase, and β-D-galactosidase.
o Alkaline phosphatase and horseradish peroxidase -
highest turnover (conversion of substrate) rates,
high sensitivity, and are easy to detect, so they are
most often used in such assays

B. Heterogeneous Enzyme Immunoassays

Competitive Enzyme Immunoassays
• based on the principles of RIA
• Enzyme-labeled antigen competes with unlabeled patient
antigen for a limited number of binding sites on antibody
molecules that are attached to a solid phase.
• After carefully washing to remove any nonspecifically
bound antigen, enzyme activity is determined.
• Enzyme activity is inversely proportional to the
concentration of the test substance.
• used for measuring small antigens that are relatively pure,
such as drugs and hormones

Noncompetitive Enzyme Immunoassays

• have a higher sensitivity than their competitive
counterparts

enzyme-linked immunosorbent assays (ELISA)

• most noncompetitive indirect immunoassays
• enzyme-labeled reagent does not participate in the initial
antigen–antibody binding reaction, only binds after the
initial antigen–antibody reaction has taken place
• most frequently used immunoassays in the clinical
laboratory because of its sensitivity, specificity, simplicity,
and low cost
• Process
o Antigen is typically bound to solid phases:
microtiter plates, nitrocellulose membranes, and
magnetic latex or plastic beads

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 o When antigen is bound to solid phase, patient • e.g. enzyme-multiplied immunoassay technique (EMIT)
serum with unknown antibody is added and given • Principle: change in enzyme activity as specific antigen
time to react. antibody combination occurs
o After a wash step, an enzyme-labeled antiglobulin, • Reagent antigen is labeled with an enzyme tag. When
or secondary antibody, is added. antibody binds to specific determinant sites on the
o This second antibody reacts with any patient antigen, the active site on the enzyme is blocked,
antibody that is bound to solid phase. resulting in a measurable loss of activity.2 Free analyte
o If no patient antibody is bound to the solid phase, (antigen) competes with enzyme-labeled analyte for a
the second labeled antibody will not be bound. limited number of antibody-binding sites, so this is a
o After a second wash step, the enzyme substrate is competitive assay. Enzyme activity is directly in proportion
added. The amount of color, fluorescence, or to the concentration of patient antigen or hapten present
luminescence is measured using a detection in the test solution.
device and is compared with the amount of • The sensitivity of homogeneous assays is determined by
product according to a standard curve. the following:
o The amount of color, fluorescence, or o detectability of enzymatic activity;
luminescence detected is directly proportional to o change in that activity when antibody binds to
the amount of antibody in the specimen. antigen;
• used to measure antibody production to infectious agents o strength of the antibody’s binding; and
that are difficult to isolate in the laboratory and for o susceptibility of the assay to interference from
autoantibody testing endogenous enzyme activity, cross-reacting
• useful as a screening tool for detecting antibody to HIV, antigens, or enzyme inhibitors.
hepatitis B, and hepatitis C
• best employed where quantitation is not necessary and is Limitations
easily applied to point-of-care and home testing • Only certain enzymes are inhibited in this manner
• Enzymatic activity may be altered by steric exclusion of
Capture Assays the substrate or there may also be changes in the
• aka sandwich assays conformation structure of the enzyme, especially in the
• If antibody, rather than antigen, is bound to the solid region of the active site
phase o Malate dehydrogenase and glucose-6-phosphate
• Antigens captured in these assays must have multiple dehydrogenase - frequently used in this assay
epitopes.
• Excess antibody attached to solid phase is allowed to Disadvantages
combine with the test sample to capture any antigen • Some specimens may contain natural inhibitors
present. • Size of the enzyme label may be a limiting factor in the
• Process design of some assays.
o After an appropriate incubation period, enzyme- • Nonspecific protein binding
labeled antibody is added.
§ This second antibody recognizes a D. Rapid Immunoassays
different epitope or binding site than the • membrane based, easy to perform, and give reproducible
solid-phase antibody and completes the results
“sandwich.” • designed as single-use, disposable assays in a plastic
o Depending upon the particular enzyme used, cartridge
either a colored or chemiluminescent reaction • membrane is usually made of microporous nylon, which
product is detected
• is able to easily immobilize proteins
• Enzymatic activity is directly proportional to the amount of
• Either antigen or antibody can be coupled to the
antigen in the test sample.
membrane; the reaction is then read by looking for the
• best suited to antigens that have multiple determinants, presence of a colored reaction product.
such as antibodies, cytokines, proteins, tumor markers,
and microorganisms, especially viruses Immunochromatography
• subject to the hook effect
• another type of rapid assay
o an unexpected fall in the amount of measured
analyte when an extremely high concentration is • combines all the previously mentioned steps into one
present1. • Process:
o occurs in antigen excess, where the majority of o Analyte is applied at one end of the strip and
binding sites are filled and the remainder of patient migrates toward the distal end where there is an
antigen has no place to bind. absorbent pad to maintain a constant capillary flow
§ If this condition is suspected, serum rate.
dilutions must be made and then retested. o The labeling and detection zones are set between
the two ends.
C. Homogeneous Enzyme Immunoassays o As the sample is loaded, it reconstitutes the
• less sensitive than heterogeneous assays, but they are labeled antigen or antibody and the two form a
rapid, simple to perform, and adapt easily to automation complex that migrates toward the detection zone.
o An antigen or antibody immobilized in the
• No washing or separation steps are necessary
detection zone captures the immune complex and
• used the determination of low-molecular-weight analytes
forms a colored line for a positive test, which may
such as hormones, therapeutic drugs, and drugs of abuse
be in the form of a plus sign.
in both serum and urine

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 • The presence of a specific antigen is determined by the
appearance of localized color against a dark background.

B. Direct Immunofluorescent Assays

• antibody that is conjugated with a fluorescent tag is added
directly to unknown antigen that is fixed to a microscope
slide.
• Process:
o After incubation and a wash step, the slide is read
using a fluorescence microscope.
o Antigens are typically visualized as bright apple
green or orange-yellow objects against a dark
background.
• e.g. detected: Legionella pneumophila and Chlamydia
trachomatis.
• best suited to antigen detection in tissue or body fluids

C. Indirect Immunofluorescent Assays

• more commonly used
• Process:
o patient serum is incubated with a known antigen
attached to a solid phase.
o The slide is then washed and an anti-human
Immunochromatographic assay.
immunoglobulin containing a fluorescent tag is
(A) Patient sample is added to a cassette containing antibody labeled
with colloidal gold. (B) Sample combines with antibody and is moved added.
along by capillary flow. (C) Monoclonal antibody to the analyte o This immunoglobulin combines with the first
captures the patient antigen attached to gold-labeled antibody. (D) antibody to form a sandwich, which localizes the
Control line has antibody that captures colloidal gold-labeled fluorescence.
antibody. o In this manner, one antibody conjugate can be
used for many different types of reactions,
FLUORESCENT IMMUNOASSAYS eliminating the need for numerous purified, labeled
A. Introduction reagent antibodies.
• Albert Coons - demonstrated that antibodies could be • result in increased staining because multiple molecules
labeled with molecules that fluoresce can bind to each primary molecule, thus making this a
• fluorophores or fluorochromes more sensitive technique
o fluorescent compounds • useful in antibody identification and have been the
o can absorb energy from an incident light source and standard for detecting treponemal and antinuclear
convert that energy into light of a longer wavelength antibodies, as well as antibodies to a number of viruses
and lower energy as the excited electrons return to
the ground state
o typically organic molecules with a ring structure;
each has a characteristic optimum absorption range
• fluorescent probe
o should exhibit high intensity, which can be
distinguished easily from background fluorescence
o should also be stable
o fluorescein and rhodamine
§ two compounds used most often
§ usually in the form of isothiocyanates,
because these can be readily coupled with
antigen or antibody
• Fluorescein - absorbs maximally at 490
to 495 nm and emits a green color at
520 nm; has a high intensity, good
photostability, and a high quantum
yield.
• Tetramethyl-rhodamine - absorbs at
550 nm and emits red light at 585 nm
o Other compounds used: phycobiliprotein, europium
(β-naphthyl trifluoroacetone), and lucifer yellow VS.

Immunofluorescent assay
• Antibodies used to identify such antigens are highly
specific; when bound to antigen in the tissue, the
fluorescent probe attached to the antibody is detected
under ultraviolet light using a fluorescent microscope.

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 CHEMILUMINESCENT IMMUNOASSAYS
D. Fluorescence Polarization Immunoassays (FPIA) A. Chemiluminescence
• based on the change in polarization of fluorescent light • emission of light caused by a chemical reaction, typically
emitted from a labeled molecule when it is bound by an oxidation reaction, producing an excited molecule that
antibody decays back to its original ground state.
• Incident light directed at the specimen is polarized with a • A large number of molecules are capable of
lens or prism so that the waves are aligned in one plane. chemiluminescence
• If a molecule is small and rotates quickly enough, the • Some of the most common substances: luminol,
emitted light is unpolarized after it is excited by polarized acridinium esters, ruthenium derivatives, and nitrophenyl
light. oxalates.
• If, however, the labeled molecule is bound to antibody, the o e.g. when acridinium esters are oxidized by
molecule is unable to tumble as rapidly and it emits an hydrogen peroxide under alkaline conditions, they
increased amount of polarized light. emit a quick burst or flash of light. The light remains
• Thus, the degree of polarized light reflects the amount of for a longer time with luminol.
labeled analyte that is bound. • can be used for heterogeneous and homogeneous assays
because labels can be attached to either antigen or
antibody
• have an excellent sensitivity; the reagents are stable and
relatively nontoxic

B. Electrochemiluminescence immunoassay
• uses electrochemical compounds that generate light when
an oxidation-reduction reaction occurs.
• Ruthenium
o one of the common chemical substances used as
an indicator
o can be conjugated with antibody and applied to
sandwich type assays.
o undergoes an electrochemiluminescent reaction
with another chemical substance, tripropylamine
(TPA), at the surface of an electrode.
o When the ruthenium is oxidized and then returned
to its reduced state through interaction with TPA, it
gives off light that can be measured by a
photomultiplier tube.
• Magnetic beads are often used as solid phase to capture
the labeled antibody.

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 Labeled Immunoassays migrates form on the test
Type of Assay Principle Results through the strip where patient
Competitive Patient antigen Inverse ratio: The strip. Labeled antigen or antibody
competes with more patient antigen or is captured.
labeled antigen antigen is present, antibody binds
for limited the less the label and is captured
antibody- detected. by a second
binding sites. reagent in the
Noncompetitive Excess solid- All patient antibody detection zone.
or phase antigen is allowed to bind.
indirect ELISA binds patient Amount of label is Review Questions
antibody and a directly proportional 1. Which of the following statements accurately describes
second labeled to the amount of competitive binding assays?
antibody is patient antibody a. Excess binding sites for the analyte are
added. present. provided.
Capture or Excess solid- All patient antigen b. Labeled and unlabeled analyte are present in
sandwich phase antibody is allowed to bind. equal amounts.
binds patient Amount of label is c. The concentration of patient analyte is
antigen and a directly proportional inversely proportional to bound label.
second labeled to the amount of d. All the patient analyte is bound in the reaction.
antibody is patient antigen 2. How do heterogeneous assays differ from
added. present. homogeneous assays?
Homogeneous Patient antigen No separation step. a. Heterogeneous assays require a separation
and enzyme- Antibody in step.
labeled antigen solution. b. Heterogeneous assays are easier to perform
react with Inverse ratio than homogeneous assays.
reagent between patient c. The concentration of patient analyte is
antibody in antigen indirectly proportional to bound label in
solution. and amount of heterogeneous assays.
Enzyme label is label detected. d. Homogeneous assays are more sensitive
inactivated than heterogeneous ones.
when reagent 3. In the following equation, what is the ratio of bound
antigen binds to radioactive antigen (Ag*) to bound patient antigen (Ag)?
antibody. 12Ag* + 4Ag + 4Ab → :___Ag*
Direct Patient antigen If fluorescence is Ab + ___AgAb + Ag* +___Ag
fluorescent is attached to a detected, patient a. 1:4
slide. Specific antigen is present b. 1:3
fluorescent- and the test is c. 3:1
labeled positive. d. 8:4
antibody is 4. Which of the following responses characterizes a
added. capture or sandwich enzyme assay?
Indirect Reagent If fluorescence is a. Less sensitive than competitive enzyme
fluorescent antigen is detected, patient assays
attached to a antibody is present b. Requires two wash steps
slide. Patient and the test is c. Best for small antigens with a single
antibody is positive. determinant
allowed to d. A limited number of antibody sites on solid
react. A second phase
fluorescent- 5. Which of the following is an advantage of enzyme
labeled immunoassay over RIA?
antibody is a. Decrease in hazardous waste
added. b. Shorter shelf life of kit
Fluorescent Fluorescent- When patient c. Natural inhibitors do not affect results
polarization labeled antigen antigen binds, less d. Needs to be read manually
competes with reagent 6. Which of the following is characteristic of direct
patient antigen antigen binds and fluorescent assays?
for a limited less polarization a. The anti-immunoglobulin has the fluorescent
number of will be detected. tag.
soluble Inverse ratio b. Antibody is attached to a solid phase.
antibody- between patient c. Microbial antigens can be rapidly identified by
binding sites. antigen and this method.
amount of d. The amount of color is in inverse proportion to
polarization. the amount of antigen present.
Immuno- Patient sample If test is positive, a
chromatographic is added to a line or plus sign will
test strip and

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7. Which of the following is true of fluorescence 16. In a heterogeneous enzyme immunoassay, if the patient
polarization immunoassay? sample produces more color than the highest positive
a. Both antigen and antibody are labeled. control, what action should be taken?
b. Large molecules polarize more light than a. Report the results out as determined.
smaller molecules. b. Dilute the patient sample.
c. When binding occurs, there is quenching of the c. Repeat the assay using one-half the volume of
fluorescent tag. the patient sample.
d. The amount of fluorescence is directly d. Report the results as falsely positive.
proportional to concentration of the analyte.
8. A fluorescent substance is best described as one in
which
a. light energy is absorbed and converted to a
longer wavelength.
b. the emitted wavelength can be seen under
normal white light.
c. there is a long time between the absorption
and emission of light.
d. it spontaneously decays and emits light.
9. In a noncompetitive enzyme immunoassay, if a
negative control shows the presence of color, which of
the following might be a possible explanation?
a. No reagent was added.
b. Washing steps were incomplete.
c. The enzyme was inactivated.
d. No substrate was present.
10. Which of the following best characterizes
chemiluminescent assays?
a. Only the antigen can be labeled.
b. Tests can be read manually.
c. These are only homogeneous assays.
d. A chemical is oxidized to produce light.
11. Immunofluorescent assays may be difficult to interpret
for which reason?
a. Autofluorescence of substances in serum
b. Nonspecific binding to serum proteins
c. Subjectivity in reading results
d. Any of the above
12. Which statement best describes flow-through
immunoassays?
a. Results are quantitative.
b. They are designed for point-of-care testing.
c. Reagents must be added separately.
d. They are difficult to interpret.
13. Which of the following is characteristic of an indirect
enzyme immunoassay?
a. The first antibody has the enzyme label.
b. All reagents are added together.
c. Color is directly proportional to the amount of
patient antigen present.
d. Enzyme specificity is not essential.
14. In a homogeneous enzyme immunoassay, which best
describes the enzyme?
a. Enzyme activity is altered when binding to
antibody occurs.
b. The enzyme label is on the antibody.
c. Enzyme activity is directly proportional to the
amount of patient antigen present.
d. Most enzymes can be used in this type of
assay.
15. In an indirect immunofluorescent assay, what would be
the outcome of an improper wash after the antibody-
enzyme conjugate is added?
a. Results will be falsely decreased.
b. Results will be falsely increased.
c. Results will be unaffected.
d. It would be difficult to determine the effect.

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IMMUNOSEROLOGY 12.

Flow Cytometry and Laboratory Automation

OUTLINE • side scatter (SSC) or right angle light scatter (SSC) -
I. Cell Flow Cytometry indicative of granularity or the intracellular complexity of
A. Introduction the cell
B. Instrumentation
C. Data Acquisition and Analysis Extrinsic parameters
D. Sample Preparation • require the addition of a fluorescent probe for their
E. Clinical Applications detection
II. Immunoassay Automation • Fluorescent-labeled antibodies bound to the cell can be
A. Introduction detected by the laser.
B. Validation
\ combination of data allows for characterization of cells
according to size, granularity, DNA and RNA content,
CELL FLOW CYTOMETRY
antigens, total protein, and cell receptors
A. Introduction
• an automated system in which single cells (or beads) in a
fluid suspension are analyzed in terms of their intrinsic
light-scattering characteristics
• cells are simultaneously evaluated for their extrinsic
properties using fluorescent-labeled antibodies or probes
• fluorochrome
o or fluorescent molecule
o absorbs light across a spectrum of wavelengths
and emits light of lower energy across a spectrum
of longer wavelengths
o each fluorochrome has a distinctive spectral
pattern of absorption (excitation) and emission
• frequently used in leukemia and lymphoma diagnostics
• used in functional assays for chronic granulomatous
disease (CGD) and leukocyte adhesion deficiency, fetal
red blood cell (RBC) and F-cell identification in maternal
blood (replacing the Kleihauer-Betke assay), and
identification of paroxysmal nocturnal hemoglobinuria
(PNH)

B. Instrumentation
Fluidics
• For cellular parameters to be accurately measured in the
flow cytometer, it is crucial that cells pass through the
laser one cell at a time. Flow cytometry.
• Cells are processed into a suspension; the cytometer Components of a laser-based flow cytometer include the fluidics
draws up the cell suspension and injects the sample system for cell transportation, a laser for cell illumination,
inside a carrier stream of isotonic saline (sheath fluid) to photodetectors for signal detection, and a computer-based
form a laminar flow. management system. Both forward and 90 degree LS are measured,
• The sample stream is constrained by the carrier stream indicating cell size and type.
and is thus hydrodynamically focused so that the cells
pass single file through the intersection of the laser light
source.
• Each time a cell passes in front of a laser beam, light is
scattered and the interruption of the laser signal is
recorded.

Laser Light Source

• Solid-state diode lasers - typically used as light sources.
• The wavelength of monochromatic light emitted by a laser
- dictates which fluorochromes can be used in an assay.
• Not all fluorochromes can be used with all lasers because
each fluorochrome has distinct spectral characteristics.

Two specific angles measured or intrinsic parameters

• forward-angle light scatter (FSC) - indicator of size

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Optics and Photodetectors NOTE
• detected by
o photodiodes for FSC Detailed phenotypic analysis
o photomultiplier tubes for fluorescence • can determine the lineage and clonality, as well as the
• specificity of each photomultiplier tube for a given band degree of differentiation and activation of a specific cell
length of wavelengths is achieved through the population
arrangement of a series of optical filters that are designed • useful for differential diagnosis or clarification of closely
to maximize collection of light derived from a specific related lymphoproliferative disorders.
fluorochrome while minimizing collection of light from
other fluorochromes used to stain the cells
• When fluorescent light from fluorescently tagged
antibodies bound to cell surfaces reaches the
photomultiplier tubes, it creates an electrical current that is
converted into a voltage pulse.
• The voltage pulse is then converted (using various
methods, depending on the manufacturer) into a digital
signal.
• The digital signals are proportional to the intensity of light
detected.

C. Data Acquisition and Analysis

Single-parameter histogram
• first level of representation
• plots a chosen parameter (generally fluorescence) on the
x axis versus the number of events on the y axis
• operator can then set a marker to differentiate between
cells that have low levels of fluorescence (negative) from
cells that have high levels of fluorescence (positive) for a
particular fluorochrome-labeled antibody

Dual-parameter dot plot

• each dot represents an individual cell or event
• Two parameters, one on each axis, are plotted against
each other.
• operator can draw a “gate” around a population of interest
and analyze various parameters (extrinsic and intrinsic) of
the cells contained within the gated region
• gate
o allows the operator to screen out debris and
isolate subpopulations of cells of interest
o set of filtering rules for analyzing a very large
database
• Disadvantage: greater potential for added error
associated with using two distinct methods to derive the
absolute count

Analysis
• The operator divides the dot plot into four quadrants,
separating the positives from the negatives in each axis.
o Quadrant 1 - consists of cells that are positive for
fluorescence on the y axis and negative for
fluorescence on the x axis.
o Quadrant 2 - consists of cells that are positive for
fluorescence on both the x and y axes.
o Quadrant 3 - consists of cells that are negative
for fluorescence on both the x and y axes.
o Quadrant 4 - consists of cells that are positive for
fluorescence on the x axis and negative for
fluorescence on the y axis.
• The computer will then calculate the percentage of cells in
each quadrant based on the total number of events
counted (typically 10,000 to 20,000 events per sample).

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 D. Sample Preparation
• Commonly used: whole blood, bone marrow, and fluid
aspirates

Whole blood
• should be collected into ethylenediaminetetraacetic acid
(EDTA),
o the anticoagulant of choice for samples processed
within 30 hours of collection
• Heparin
o used for whole blood and bone marrow and can
provide improved stability in samples over 24
hours old.
• Blood should be stored at room temperature (20°C to
25°C) before processing and should be well mixed before
being pipetted into staining tubes.
• Hemolyzed or clotted specimens should be rejected.
Peripheral blood, bone marrow, and other samples with
large numbers of RBCs require erythrocyte removal to
allow for efficient analysis of WBCs.

density gradient centrifugation with Ficoll-Hypaque

• used to generate a cell suspension enriched for
lymphocytes or lymphoblasts
• results in selective loss of some cell populations and is
time consuming
• replaced by erythrocyte lysis techniques, both commercial
and noncommercial

Tissue specimens
• collected and transported in tissue culture medium (RPMI
1640) at either room temperature (if analysis is imminent)
or 4°C (if analysis will be delayed).
• disaggregated to form a single cell suspension, either by
mechanical dissociation or enzymatic digestion.
• Mechanical disaggregation, or “teasing,” is preferred and
is accomplished by the use of a scalpel and forceps, a
needle and syringe, or wire mesh screen.
• Antibodies are then added to the resulting cellular
preparation and processed for analysis.
• The antibodies used are typically monoclonal, each with a
different fluorescent tag.

Gating strategy to analyze lymphocyte subsets in a sample of whole E. Clinical Applications

blood. Routine applications of flow cytometry
Whole blood is incubated with fluorescent-labeled antibodies specific • immunophenotyping of peripheral blood lymphocytes
for CD3, CD4, CD8, and HLA-DR. The sample is washed, RBCs are • enumeration of CD34+ stem cells in peripheral blood and
lysed, and the sample is analyzed on the flow cytometer. To analyze
using gating strategies, the sample is first plotted on FSC versus SSC.
bone marrow for use in stem cell transplantation
(A) A gate, or region, is drawn around the lymphocyte population. (B) • immunophenotypic characterization of acute leukemias
On the subsequent plots of fluorescent markers, only the lymphocyte • non-Hodgkin’s lymphomas
population is analyzed. The dot plot is divided into four quadrants to • other lymphoproliferative disorders
isolate positive from negative populations. The computer calculates
the percentage of positive cells in each quadrant. The three flow
contour plots are analyzing two different cell surface markers. In the
Surface Markers on Leukocytes
first dot plot, quadrant 2 (upper right) identifies CD4+, CD3+ T helper Antigen Cell Type Function
cell lymphocytes. Quadrant 3 (lower left) identifies B lymphocyte and CD2 Thymocytes, T Involved in T-cell
NK cells. Quadrant 4 (lower right) identifies CD3+ CD4– T-cell cells, NK cells activation
lymphocytes. In the second dot plot, quadrant 1 (upper left) identifies CD3 Thymocytes, T Associated with T-cell
low intensity CD8+ CD3– NK cells. Quadrant 2 identifies CD3+ CD8+ cells antigen receptor; role in
T-cytotoxic lymphocytes. Quadrant 3 identifies any lymphocyte that is
TCR signal
not a CD8+, CD3+, or B-cell lymphocyte. Quadrant 4 identifies CD3+
CD8–T helper cell lymphocytes. In the third contour plot, quadrant 1 transduction
identifies CD38+ CD3– B cells, quadrant 2 identifies CD38+ CD3+ CD4 T helper cells, Co-receptor for class II
activated T cells, quadrant 3 identifies CD38– CD3– cells, and monocytes, MHC; receptor for HIV
quadrant 4 identifies CD3+ CD38– T cells, not activated. macrophages

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CD5 Mature T cells, Positive or negative CD38 Plasma cell Plasma cells,
thymocytes, modulation of T- and B- thymocytes, NK
subset of cell receptor signaling lymphocytes, early B,
B cells (B1) monocytes, multiple
CD7 T cells, Regulates peripheral T- myelomas, ALL, acute
thymocytes, NK cell and NK cell myeloblastic leukemia
cells, pre-B cytokine CD44 Most leukocytes Adhesion molecule
cells production mediating homing to
peripheral lymphoid
CD8 Thymocyte Co-receptor for class I organs
subsets, MHC CD45 All hemato- Tyrosine phosphatase,
cytotoxic T cells poietic cells augments signaling
CD10 B- and T-cell Protease; marker for CD45R Different forms Essential in T- and B-
precursors, pre-B CALLA on all hema- cell antigen-stimulated
bone marrow topoietic cells activation
stromal cells CD56 NK cells, Not known
CD11b Myeloid and NK αM subunit of integrin subsets of T
cells CR3, binds cells
complement CD57 NK cell sub, T-cell sub,
component iC3b small cell lung
CD13 Myelomonocytic Zinc metalloproteinase carcinoma
cells CD94 NK cells, Subunit of NKG2-A
CD14 Monocytic cells Lipopolysaccharide subsets of T complex involved in
receptor cells inhibition of NK cell
CD15 Neutrophils, Terminal trisaccharide cytotoxicity
eosinophils, expressed on CD103 Intraepithelial HCL, adult T-cell
monocytes glycolipids lymphocytes leukemia
CD138 Plasma cell, pre-B
CD16 Macrophages, Low affinity FC (weak)
NK cells, receptor, mediates HLA-DR B lymphocytes, Lack of expression
neutrophils phagocytosis activated T diagnostic of M3
and ADCC lymphocytes, myeloid
CD19 B cells, follicular Part of B-cell co- monocytes leukemia, T-cell
dendritic cells receptor, signal activation
transduction molecule FCM7 B-cell subset, Expressed in mantle
that regulates B-cell mantle cell lymphoma
development and lymphoma
activation Kappa B cells Light chain part of
CD20 B cells, T-cell Binding activates chains antibody molecule on B
subsets signaling pathways, cells
regulates B-cell Lambda B cells Light chain part of
activation chains antibody molecule on B
CD21 B cells, follicular Receptor for cells
dendritic cells, complement ADCC = antibody-dependent cell cytotoxicity; ALL= acute
subset of component C3d; part of lymphocytic leukemia; AML= acute myeloid leukemia; CALLA =
immature B-cell co-receptor with common acute lymphoblastic leukemia antigen; CLL= chronic
thymocytes CD19 lymphocytic leukemia; FC = fragment crystallizable; GM-CSF =
granulocyte-macrophage colony-stimulating factor; HCL = hairy cell
CD22 B cells B, B-ALL (surface and
leukemia; HIV = human immunodeficiency virus; MHC = major
cytoplasmic), B-CLL, histocompatibility class; NK = natural killer; PLL= prolymphocytic
HCL, PLL leukemia; TCR = CD3-αβreceptor complex.
CD23 B cells, Regulation of IgE
monocytes, synthesis; triggers Common Markers Used for Lymphoproliferative and
follicular release of IL-1, Myeloproliferative Studies in Clinical Flow Cytometry
dendritic cells IL-6, and GM-CSF from Antibodies Paired Interpretation
monocytes Chronic lymphocytic leukemia (CLL) and
CD25 Activated T Receptor for IL-2 prolymphocytic leukemia
cells, B cells, FMC7 with CD23 FMC7 negative and CD23
monocytes positive in CLL, CD23 negative
CD33 Myeloid cell Monocytes, in mantle cell lymphoma and
macrophages, FMC7 is positive
granulocytes (weak), CD5 with CD20 When a cell is both CD5
myeloid-Pro, myeloid positive and CD20 positive:
leukemia, AML characteristic of CLL and well-
CD34 Hematopoietic Hematopoietic-Pre, differentiated lymphocytic
progenitor cell endothelial cells, lymphoma (WDLL), as well as
immature leukemias mantle cell lymphoma

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CD19 with kappa CD19 positive with only one Myeloma plasmacytoid leukemia or lymphoma
CD19 with lambda light chain (kappa or lambda) is CD3 with HLA-DR CD3 negative; most are HLA-
expressed in low intensity; DR negative, although some
occasionally light chains are not early plasmacytoid cells may be
detected DR positive
CD45 with CD14 CD45 fluorescence is brightly CD5 with CD20 CD5 and CD20 negative
(Antiglycophorin added expressed and CD14 negative CD19 with kappa CD19 and surface Ig negative;
to bone marrows) Used to determine erythroid CD19 with lambda occasionally surface Ig is
component to the specimen positive; cytoplasmic Ig positive
Hairy cell leukemia (HCL) CD45 with CD38 CD45 negative or low intensity;
CD3 with CD23 CD3 negative and CD23 CD38 is high-intensity positive
negative CD40 with CD56 Usually CD40 positive; CD56
CD11c with CD22 Brightly expressed CD11c and has been reported to be
CD22, unlike CLL positive on myeloma cells but
CD20 with CD5 CD20 positive and CD5 negative on normal plasma
negative (occasionally weak cells
expression of CD5) CD10 CD10 positivity indicates poor
CD19 with kappa CD19 positive with one prognosis
monoclonal light chain CD138 Syndecan-1 positive in mature
expressed plasma cells
CD103 with CD25 CD103 is highly specific for CD45 with CD14 CD14 negative
HCL, and CD25 is usually T-cell acute lymphoblastic leukemia (ALL)
expressed; hairy cell variants CD1a with CD3 CD1a positivity associated with
may be negative for these two longer disease-free survival in
markers adult T-cell ALL; CD3 is
CD21 with HLA-DR CD21 negative and DR positive negative in 99% (exception is
CD45 with CD14 CD45 is brightly expressed and mature medullary thymocyte T-
(Antiglycophorin added CD14 negative cell ALL)
to bone marrows) To determine erythroid CD2 with CD25 CD2 variably expressed and
component to the specimen, CD25 negative
CD10 (Calla) is weakly CD38 with CD7 CD38 and CD7 are positive
expressed in 26% of cases CD4 with CD8 CD4 and CD8 variably
B cell Acute lymphocytic leukemia (ALL) expressed depending on
CD3 with HLA-DR CD3 (T-cell receptor) negative maturity; dual expression is
and DR positive common
CD5 with CD20 CD5 negative and CD20 CD5 with CD20 CD5 positive except for
variably positive (low intensity prothymocyte stage T-cell ALL,
or negative on precursor B-cell CD20 negative
ALL, positive on more mature CD45 with CD14 CD45 positive and CD14
B-cell ALL) negative
CD19 with kappa CD19 positive (stem cell is HLA-DR with CD34 DR positive T-cell ALL
CD19 with lambda negative) and surface Ig associated with a worse
negative (a mature B-cell ALL prognosis; CD34 in pediatric
may have surface patients associated with CNS
immunoglobulin) involvement and poor prognosis
CD34 with CD38 CD34 positive ALL correlates and predicts myeloid
with good prognosis in pediatric expression
patients and poor prognosis in TdT with CD10 TdT is positive; CD10 positive
adults; CD38 is positive from T-cell ALL associated with
stem cell to pre-B prolonged disease-free survival
TdT with CD10 TdT and CD10 (Calla) positive CD19 and kappa Negative
in common ALL and pre-B ALL; CD19 with lambda Negative
CD10 positivity is associated (Antiglycophorin added To determine erythroid
with favorable complete to bone marrows) component to the specimen
treatment Post-thymic T-cell leukemia or lymphoma
response and disease-free CD1a with CD3 CD1a negative, CD3 positive;
survival; CD10 is usually high note: peripheral T-cell
intensity lymphomas lack 1 or more pan-
TdT with CD33 CD33 negative; however, very T cell antigen (CD3, CD2, CD5,
early B-ALL may be positive or CD7) 75% of the time
CD45 with CD14 CD45 dimly expressed and Peripheral T-cell leukemia
(Antiglycophorin added CD14 negative CD2 with CD25 CD2 positive; CD25 positive in
to bone marrows) To determine erythroid adult T-cell leukemias and
component to the specimen some peripheral T-cell
lymphomas

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Adult T-cell leukemia CD19 with kappa CD19 occasionally expressed
CD5 with CD7 CD5 positive; CD7 positive on some primitive AML
except in adult T-cell leukemia CD19 with lambda Surface Ig negative
CD4 with CD8 Variable expression CD34 with HLA-DR Poorly differentiated AML often
CD19 with kappa Negative expresses CD34; high-intensity
CD19 with lambda Negative CD34 has worse prognosis;
CD45 with CD14 CD45 positive and CD14 CD34 coexpressed with HLA-
negative DR has worse prognosis; CD34
TdT with CD10 Negative coexpressed with CD has
(Antiglycophorin added To determine erythroid worse
to bone marrows) component to the specimen prognosis than CD34 alone;
Tγ proliferative disease (NK-like T-cell leukemia) NK- lack of HLA-DR indicates either
like T-cell lymphoma NK cell leukemia APL or very immature AML
CD2 with CD57 NK-like T-cell lymphoma tends CD10 with TdT CD10 is present on neutrophils
to be CD56 positive, CD57 (Antiglycophorin added To determine erythroid CD10 is
nega-tive and is usually to bone marrows) present on neutrophils
clinically aggressive; NK cell component present in the
leukemias tend to be CD56 or specimen
CD16 positive, CD57 negative MPO with CD117 Myeloperoxidase (MPO) is
and are usually clinically found on fairly mature AMLs,
aggressive; Tγ proliferative whereas CD117 is a myeloid
disease is CD56 negative,CD57 blast marker
positive and exhibits a chronic
indolent course; CD2 is IMMUNOASSAY AUTOMATION
usually positive for all, but there A. Introduction
are variants • Using a solid support for separating free and bound
CD3 with CD56, CD16 Surface CD3 is positive in Tγ analytes, these instruments have made it possible to
proliferative disease and NK- automate heterogeneous immunoassays even for low-
like T-cell lymphoma; CD3 is level peptides such as peptide hormones.
negative in NK cell leukemia;
for Two main types of immunoassay analyzers
CD56 and CD57, see above 1. Batch analyzers
CD11c with CD11b Usually positive • examine multiple samples and provide access to the test
CD4 with CD8 Usually CD8 positive, CD4 samples for the formation of subsequent reaction
negative; however, dual mixtures. permit only one type of analysis at a timestat
staining and CD4 positivity has samples cannot
been reported • be loaded randomly and there cannot be multiple
CD19 Negative analyses on any one sample
CD45 with CD14 CD45 positive and CD14
negative 2. Random access analyzers
Acute myelogenous leukemia (AML) • designed in a modular system that could be configured to
CD11c with CD11b CD11c positive on mature measure numerous analytes from multiple samples
myeloid cells; CD11b positive • many test samples can be analyzed and a number of
on myelomonocytic cells, different tests can be performed on any one sample
eosinophilic myelocytes,
eosinophils, and neutrophils; Automatic sampling
differentiated AML usually
• can be accomplished by several different methods:
expresses mature markers
• peristaltic pumps (older technology)
CD13 with CD15 Poorly differentiated AML
usually lacks CD15, CD11c, • positive-liquid displacement pipettes (newer technology)
CD11b, but positive for CD13
B. Validation
CD33 with TdT CD33 in the absence of CD34,
HLA-DR, or CD13 suggests • Clinical Laboratory Improvement Amendment (CLIA)
immature acute basophilic or o regulations for verifying the manufacturer’s
mast cell leukemia; TdT is often performance specifications
expressed in low intensity in • CLIA '88
poorly differentiated AML o specifies that the standard deviation and
CD14 with CD64 CD14 on early promonocytes to coefficient of variation should be calculated from
mature monocytes; high 10 to 20 day-to-day quality control results.
expression of CD14 and CD11b • Accuracy - refers to the test’s ability to actually measure
predicts poor outcome; CD64 what it claims to measure
on immature and mature • Precision - refers to the ability to consistently reproduce
monocytes the same result on repeated testing of the same sample
CD3 with CD7 CD3 negative; some immature • Analytic sensitivity - lowest measureable amount of an
AML expresses CD7 analyte

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• Analytic specificity - assay’s ability to generate a negative 8. The various signals generated by cells intersecting with
result when the analyte is not present a flow cytometry laser are captured by
• Reportable range - range of values that will generate a a. bandwidth waves.
positive result for the specimens assayed by the test b. wave channels.
procedure c. photomultiplier tubes.
• Reference interval - range of values found in healthy d. flow cells.
individuals who do not have the condition that is detected 9. Analysis of flow cytometer data of cells can be filtered in
by the assay, which is used to define the expected value many ways by using a method of
of a negative test. a. “gating” in a dot plot.
b. banding of a histogram.
c. single-parameter histogram monitoring.
Review Questions d. automatic sampling.
1. Flow cytometry characterizes cells on the basis of which 10. A newer flow cytometry technology that has the potential
of the following? to detect over 500 analytes from one sample of blood is
a. Forward and 90-degree side scatter of an called a/an
interrupted beam of light a. RBC fragmentation assay.
b. Front-angle scatter only of an interrupted light b. Dihydrorhodamine 123.
beam c. sucrose test.
c. Absorbance of light by different types of cells d. cytometric bead array.
d. Transmittance of light by cells in solution 11. Many flow cytometry laboratories now use the CD45
2. Forward-angle light scatter is an indicator of cell marker in combination with SSC in differentiating
a. granularity. various populations of WBCs to replace which of the
b. density. following combinations?
c. size. a. CD4 + SSC
d. number. b. CD4 + FSC
3. What is the single most important requirement for c. FSC + SSC
samples to be analyzed on a flow cytometer? d. FSC + CD45
a. Whole blood is collected into a serum-separator 12. Which cell surface marker is present on cells seen in
tube. hairy cell leukemia?
b. Cells must be in a single-cell suspension. a. CD138
c. Samples must be fixed in formaldehyde before b. CD33
processing. c. CD103
d. Blood must be kept refrigerated while d. CD34
processing. 13. CD45 is a pan-leukocyte marker expressed on WBCs in
4. Which statement represents the best explanation for a varying levels or amounts of expression, based on
flow cytometer’s ability to detect several cell surface a. size of a cell.
markers at the same time? b. granularity of a cell.
a. The forward scatter can separate out cells on c. maturity and lineage of a cell.
the basis of complexity. d. malignancy of a cell.
b. One detector can be used to detect many 14. Which of the following statements best describes a
different wavelengths. single-parameter histogram?
c. For each marker, a specific fluorochrome– a. Each event is represented by a dot.
antibody combination is used. b. Data is distributed in four quadrants.
d. Intrinsic parameters are separated out on the c. Positive and negative events are plotted on the
basis of the amount of side scatter. x and y axis.
5. Which of the following cell surface markers would be d. A chosen parameter is plotted versus the
present on a population of T helper (Th) cells? number of events.
a. CD3 and CD4 15. How many fluorochromes (colors) are current clinical
b. CD3 and CD8 flow cytometers capable of detecting?
c. CD3 only a. 2
d. CD4 only b. 6
6. If an analyzer consistently indicates a positive test when c. 8
the analyte in question is not present, this represents a d. 10
problem with 16. Which type of analyzer allows one to measure multiple
a. sensitivity. analytes from numerous samples, loaded at any time?
b. specificity. a. Batch analyzer
c. reportable range. b. Random access analyzer
d. precision. c. Front-end loaded analyzer
7. All of the following are clinical applications for flow d. Sequential access analyzer
cytometry except 17. Operational considerations when selecting automated
a. fetal hemoglobin. analyzers for your laboratory include all of the following
b. immunophenotyping of lymphocyte except
subpopulations. a. reagent stability.
c. HIV viral load analysis. b. test menu.
d. enumeration of stem cells in a peripheral blood c. STAT capability.
mononuclear cell product. d. purchase cost.

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18. Analyzers use different methods for mixing, including
magnetic stirring, rotation paddles, forceful dispensing,
and vigorous lateral shaking. Whichever method used, it
is imperative that
a. reagents always be kept refrigerated.
b. there is no splashing or carry-over between
samples.
c. samples are kept at room temperature.
d. multiple methods are not used simultaneously.
19. All of the following are benefits of automation except
a. greater accuracy.
b. increased turnaround time.
c. savings on controls.
d. less disposal of outdated reagents.
20. If an analyzer gets different results each time the same
sample is tested, what type of problem does this
represent?
a. Sensitivity
b. Specificity
c. Accuracy
d. Precision

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IMMUNE DISORDERS

Christine Joy R. Timbang | 0963-467-6983

Christine Joy R. Timbang | 0963-467-6983
IMMUNOSEROLOGY 13.

Hypersensitivity
OUTLINE • This can lead to cell damage by complement-mediated
I. Introduction lysis or other mechanisms, dysfunction of the cell by
A. Definitions blocking the binding of a ligand to a surface receptor, or
B. P. G. H. Gell and R. R. A. Coombs overstimulation of a cell’s function.
Classification System • classified as immediate hypersensitivity reactions
II. Type I Hypersensitivity because symptoms develop within a few minutes to a few
A. Introduction hours after exposure to the antigen
B. Immunologic Mechanism
C. Genetic and Environmental Influences on 3. Type III hypersensitivity reactions
Type I Hypersensitivity • referred to as complex-mediated hypersensitivity.
D. Clinical Manifestations of Type I • In this process, IgG or IgM antibodies react with soluble
Hypersensitivity antigens to form small complexes that precipitate in the
E. Treatment of Type I Hypersensitivity tissues and activate complement.
F. Testing for Type I Hypersensitivity • Recruitment of neutrophils to the site results in an
III. Type II Hypersensitivity inflammatory response that causes injury to the tissues.
A. Introduction • classified as immediate hypersensitivity reactions
B. Clinical Examples of Type II Hypersensitivity because symptoms develop within a few minutes to a few
C. Testing for Type II Hypersensitivity hours after exposure to the antigen
IV. Type III Hypersensitivity • classified as immediate hypersensitivity reactions
A. Introduction because symptoms develop within a few minutes to a few
B. Arthus Reaction hours after exposure to the antigen
C. Serum Sickness
D. Autoimmune Diseases and Other Causes of 4. Type IV hypersensitivity reactions
Type III Hypersensitivity • differs from the other three types because sensitized T
E. Testing for Type III Hypersensitivity cells, rather than antibody, are responsible for the
V. Type IV Hypersensitivity symptoms that develop.
A. Introduction
• This cell-mediated hypersensitivity involves the release of
B. Contact Dermatitis
cytokines that induce inflammation and tissue damage
C. Hypersensitivity Pneumonitis
• referred to as delayed hypersensitivity because its
D. Skin Testing for Delayed Hypersensitivity
manifestations are not seen until 24 to 48 hours after
contact with the antigen
INTRODUCTION
A. Definitions
Hypersensitivity
• antigen can persist, and the immune response can cause
damage to the host
• defined as an exaggerated response to a typically
harmless antigen that results in injury to the tissue,
disease, or even death

B. P. G. H. Gell and R. R. A. Coombs Classification

System
1. Type I hypersensitivity reactions
• also known as anaphylactic hypersensitivity
• exposure to an antigen induces production of specific
immunoglobulin E (IgE) antibody, which binds to receptors
on mast cells and basophils.
• Subsequent attachment of the antigen to adjacent cell-
bound
• IgE results in degranulation with release of chemical
mediators that produce characteristic allergy symptoms.
• classified as immediate hypersensitivity reactions
because symptoms develop within a few minutes to a few
hours after exposure to the antigen

2. Type II hypersensitivity reactions

• also known as antibody-mediated cytotoxic
hypersensitivity
• Immunoglobulin G (IgG) or immunoglobulin M (IgM)
antibodies react with antigens on the surface of host cells

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 TYPE I HYPERSENSITIVITY o Subsequent binding of allergen to IgE-sensitized
A. Introduction mast cells and basophils triggers degranulation
• commonly thought of as allergies with release of inflammatory mediators.
• allergens
o antigens that trigger type I hypersensitivity Activation Phase
o e.g. peanuts, eggs, and pollen • Adjacent cell-bound IgE molecules are cross-linked by a
• Distinguishing feature: short time lag, usually minutes, bivalent or multivalent antigen, causing aggregation of the
between exposure to allergen and the onset of clinical surface FcεRI receptors
symptoms • This action, in turn, initiates complex intracellular signaling
• serum factor events involving multiple phosphorylation reactions, an
o first clue about the cause of type I hypersensitivity influx of calcium, and secretion of cytokines.
o provided by Carl Wilhelm Prausnitz and Heinz • The increase in intracellular calcium triggers rapid
Küstner degranulation of the mast cells and basophils, which
o patients who exhibit allergic hypersensitivity release chemical mediators that have been previously
reactions produce a large amount of IgE antibody in made and stored in the granules.
response to a small concentration of allergen • histamine
o comprises approximately 10% of the total weight of
Passive cutaneous anaphylaxis the granules in mast cells
• In their historic experiment, serum from Küstner, who was o most abundant preformed mediator
allergic to fish, was injected into Prausnitz. o preformed substances are referred to as primary
• A later exposure to fish antigen at the same site resulted mediators
in redness and swelling. § e.g. heparin, eosinophil chemotactic factor of
anaphylaxis (ECF-A), neutrophil chemotactic
• occurs when serum is transferred from an allergic
factor, and proteases
individual to a non-allergic individual, and the second
§ esponsible for the early-phase symptoms
individual is challenged at a later time with the specific
seen in allergic reactions, which occur within
allergen
30 to 60 minutes after exposure to the
Atopy allergen
• a term derived from the Greek word atopos (“out of Late Phase
place”)
• In addition to immediate release of preformed mediators,
• refers to an inherited tendency to develop classic allergic
mast cells and basophils are triggered to synthesize other
responses to naturally occurring inhaled or ingested
reactants from the breakdown of phospholipids in the cell
allergens
membrane.
B. Immunologic Mechanism • Newly formed or secondary mediators include
o platelet-activating factor (PAF)
• Key immunologic components in Type I hypersensitivity:
o prostaglandin (PG) D2
IgE antibody, mast cells, basophils, and eosinophils
o leukotrienes (LT) B4, C4,D4, and E4
• The response begins when a susceptible individual is
o cytokines
exposed to an allergen and produces specific IgE
§ more potent than the primary mediators
antibody.
and are responsible for a late-phase
• IL-4 and IL-13 (among others) allergic reaction that can be seen in some
o these cytokines are responsible for the final individuals 6 to 8 hours after exposure to
differentiation that occurs in B cells the antigen
o initiates the transcription of the gene that codes for § release additional mediators that prolong
the epsilon-heavy chain of immunoglobulin the hyperactivity and may lead to tissue
molecules belonging to the IgE class damage
Sensitization Phase
• IgE antibody attaches to high-affinity receptors called
FcεRI, which bind the fragment crystallizable (Fc) region
of the epsilon-heavy chain
• Langerhans and dendritic cells internalize and process
allergens from the environment and transport the allergen-
MHC class II complex to local lymphoid tissue where
synthesis of IgE occurs.
• Binding of IgE to cell membranes increases the half-life of
the antibody from 2 or 3 days to at least 10 days.
• Once bound, IgE serves as an antigen receptor on mast
cells and basophils.
o Mast cells - principal effector cells of immediate
hypersensitivity
o Basophils respond to chemotactic stimuli during
inflammation and accumulate in the tissues,
where they can persist for a few days.

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 D. Clinical Manifestations of Type I Hypersensitivity
Anaphylaxis
• clinical manifestations caused by release of inflammatory
mediators from mast cells and basophils vary from a
localized skin reaction to a severe systemic response
o Ingestion of an allergen → gastrointestinal
Mediators of Type I Hypersensitivity
symptoms
Mediator Actions
o Injection into the bloodstream → systemic
Primary Histamine Smooth muscle response
(Preformed) contraction,
• most severe type of allergic response because it is an
vasodilation,
acute reaction that simultaneously involves multiple
increased vascular
organs
permeability
• means “without protection”
Heparin Smooth muscle
• triggered by glycoproteins or large polypeptides
contraction,
vasodilation, • severity of the reaction depends on the number of
increased vascular previous exposures to the antigen
permeability • because multiple exposures result in additional
Eosinophil Chemotactic for accumulation of IgE on the surface of the mast cells and
chemotactic eosinophils basophils
factor of
anaphylaxis Rhinitis
(ECF-A) • most common form of atopy, or allergy
Neutrophil Chemotactic for • Symptoms: paroxysmal sneezing; rhinorrhea, or runny
chemotactic neutrophils nose; nasal congestion; and itching of the nose and eyes
factor of • hay fever - seasonal allergic rhinitis, triggered by tree and
anaphylaxis grass pollens in the air during the spring in temperate
(NCF-A) climates
Proteases (e.g., Convert C3 to C3b,
tryptase, stimulate mucus Asthma
chymase) production, activate • derived from the Greek word for “panting” or
cytokines “breathlessness”
Secondary Prostaglandin Vasodilation, • caused by inhalation of small particles such as pollen,
(Newly PGD2 increased vascular dust, or fumes that reach the lower respiratory tract
Synthesized) permeability • defined clinically as recurrent airflow obstruction that
Leukotriene Chemotactic for leads to intermittent sneezing, breathlessness, and,
LTB4 neutrophils, occasionally, a cough with sputum production
eosinophils • airflow obstruction is caused by bronchial smooth muscle
Leukotrienes Increased vascular contraction, mucosal edema, and heavy mucus secretion
LTC4, LTD4, permeability,
LTE4 bronchoconstriction, Food allergies
mucus secretion • Symptoms: cramping, vomiting, and diarrhea
Platelet Platelet aggregation • spread of antigen through the bloodstream may cause
activating factor hives and angioedema on the skin, asthma, rhinitis, or
(PAF) anaphylaxis
Cytokines IL-1, Increase inflammatory
IL-3, IL-4, IL-5, cells in area, and Acute urticaria or eczema
IL-6, IL-9, IL-13, increase IgE • local inflammation of the skin, or dermatitis
IL-14, production
IL-16, TNF-α,
a. Urticaria, or hives
GM-CS
• appear within minutes after exposure to the allergen and
are characterized by severe itching, erythema (redness)
C. Genetic and Environmental Influences on Type I • caused by local vasodilation, leakage of fluid into the
Hypersensitivity surrounding area, and a spreading area of redness
• The development of IgE responses and allergy appears to around the center of the lesion
depend on complex interactions between genetic factors • commonly called a wheal-and-flare reaction
and environmental triggers.
• caused by release of vasoactive mediators from mast
• Exposure to stress, variations in physical factors such as cells in the skin following contact with allergens such as
temperature, and contact with environmental pollutants pet dander or insect venom
such as cigarette smoke and diesel exhaust fumes can
• occur deeper in the dermal tissues → they are known as
intensify clinical manifestations of allergy in susceptible
angioedema
individuals.
b. Atopic eczema
• can take on a variety of forms, from erythematous, oozing
vesicles to thickened, scaly skin, depending on the stage
of activity and age of the individual

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• chronic, itchy skin rash that usually develops during • allergy immunotherapy (AIT)
infancy, persists during childhood, and is strongly o If environmental control measures and
associated with allergic rhinitis and asthma pharmacotherapy are not successful
o induce immune tolerance to a specific allergen by
administering gradually increasing doses of the
allergen over time
o administer allergens subcutaneously (i.e., under the
skin) over 3 to 5 years

F. Testing for Type I Hypersensitivity

In Vivo Skin Tests
• less expensive and more sensitive

Two types of skin tests

1. Percutaneous tests
• also known as prick or puncture tests
• can detect hypersensitivity to a wide variety of inhaled or
food allergens
• uses a needle or pricking device to introduce a small drop
of allergen extract into the upper layers of the individual’s
skin in the inner forearm or the back
• each applied to separate sites 2 to 2.5 cm apart.
• A negative control consisting of the diluent used for the
allergy extract and a positive control of histamine are also
included.
• After 15 to 20 minutes, the clinician examines the testing
spots and records the reaction.
• In a positive test, a wheal-and-flare reaction will appear at
the site where the allergen was applied
• Scoring of the reaction is based on the presence or
absence of erythema and the diameter of the wheal, with
a diameter larger than 3 to 4 mm correlating best with the
presence of allergy.

2. Intradermal tests
• use a greater amount of antigen and are more sensitive
than cutaneous tests
• usually performed only if prick tests are negative and
allergy is still suspected because they carry a larger risk
E. Treatment of Type I Hypersensitivity (0.05%) for anaphylactic reaction than prick tests (0.03%)
• First defense: avoidance of allergens • 1-mL tuberculin syringe is used to administer 0.01 to 0.05
• Localized allergic reactions, such as hay fever, hives, or mL of test solution between layers of the skin
rhinitis - antihistamines and decongestants • The test allergen is diluted 100 to 1,000 times more than
• Asthma - often treated with a combination of therapeutic the solution used for cutaneous testing.
reagents, including antihistamines and bronchodilators. • This test is performed on the inner forearm or upper arm
• Persistent asthma - leukotriene receptor antagonists and so that if a systemic reaction occurs, tourniquet can be
mast cell stabilizers applied to the arm to help stop the reaction.
o corticosteroids can be added to block recruitment of • After 15 to 20 minutes, the site is inspected for erythema
inflammatory cells and their ability to cause tissue and wheal formation, and the wheal diameter is measured
damage to determine a score.
• Systemic anaphylaxis - injection of epinephrine, a
powerful vasoconstrictor
• omalizumab
o anti-IgE monoclonal antibody
o recombinant humanized antibody that is composed
of human IgG framework genes recombined with
complementarity-determining region genes from
mouse anti-human IgE
o binds to the Cε3 domain of human IgE, which is the
site that IgE normally uses to bind to FcεRI
receptors.
o Blocking of this site prevents circulating IgE from
binding to mast cells and basophils and sensitizing
them

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Allergen-Specifific IgE Testing In Vitro Tests: Total IgE
• safer to perform than skin testing; are easier on some radioimmunosorbent test (RIST)
patients, especially children or apprehensive adults; and • first test developed for the measurement of total IgE
have excellent analytical sensitivity • used radiolabeled IgE to compete with patient IgE for
• useful in detecting allergies to a number of common binding sites on a solid phase coated with anti-IgE
trigger
noncompetitive solid-phase immunoassays
radioallergosorbent test (RAST) • anti-human IgE is bound to a solid phase such as
• original commercial testing method for determining cellulose, a paper disk, or a microtiter well.
specific IgE • Patient serum is added and allowed to react and then an
• patient serum was incubated with a paper disk to which enzyme-labeled anti-IgE is added to detect the bound
various allergens were covalently linked patient IgE.
• can be run with a single allergen or as a multiallergen • The second anti-IgE antibody recognizes a different
screen using a panel of allergens in a single run epitope than that recognized by the first antibody.
• commercial noncompetitive fluoroimmunoassay - • The resulting “sandwich” of solid-phase anti-IgE, serum
considered by most allergy specialists to be the method of IgE, and labeled anti-IgE is washed; a colorimetric,
choice fluorometric, or chemiluminescent substrate is then
• Allergen-specific IgE values added.
o reported in kilo international units (IU) of • The amount of reactivity detected is directly proportional
allergenspecific antibody per liter (kUa/L) to the IgE content of the serum.
o one unit is equal to 2.42 ng/mL of IgE.
o can detect IgE antibodies in the range of 0 to 100 NOTE
kU/L
o 0.35 kU/L - commonly used as the cut-off for a Total IgE values
positive test • reported in kilo international units (IU) per liter
• 1 IU = 2.4 ng of protein per milliliter
• typically lower than 1 kU/L in cord blood, and serum
IgE usually reaches adult levels at about 10 years of
age
• Cutoff value: 100 kU/L
• >100 kU/L - common in individuals with allergies

Total serum IgE

• ↑ helminth infections, and certain immunodeficiencies,
such as Wiskott-Aldrich syndrome, DiGeorge
syndrome, and hyper-IgE syndrome
• Endemic parasitic infections - > 1,000 kU/L
• hyper-IgE syndrome - extremely high IgE levels (2,000
50,000 kU/L)

Advanced biochemical and molecular techniques

• led to the development of a microarray format that allows TYPE II HYPERSENSITIVITY
for parallel detection of IgE antibodies to more than 100 A. Introduction
potential allergens using only 20 μL of patient serum • also known as antibody-mediated cytotoxic
• patient serum is incubated with a biochip containing hypersensitivity
miniature spots to which the purified allergenic • Mechanism: involves IgG and IgM antibodies directed
components have been applied. against antigens found on cell surfaces
• Chip • Binding of the antibody to a cell can have one of three
o contains a wide variety of antigens from foods, major
pollens, molds, fungi, latex, and insect venoms • effects, depending on the situation:
o scanned by a laser for fluorescence following (1) The cell can be destroyed;
addition of a fluorescent-labeled anti-IgE (2) the function of the cell can be inhibited; or
• highly sensitive and specific (3) the function of the cell can be increased above
normal
NOTE
Cell damage can occur by several different mechanisms,
Regardless of the format of the specific IgE test used, the some of which involve complement as well as antibodies:
results should always be interpreted in light of the (1) Activation of the classical pathway of complement can
patient’s medical history and clinical symptoms. This is lead to the formation of the membrane attack complex
because the presence of allergen-specific IgE antibody and cell lysis.
indicates sensitization to the allergen but not necessarily (2) Coating of the cell surface by antibodies can promote
the presence of a clinical allergy. opsonization and subsequent phagocytosis of the cells.
Opsonization can occur either through binding of IgG
antibody to Fc receptors on macrophages and

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 neutrophils or binding of cell surface C3b to complement • induce disseminated intravascular coagulation (DIC),
receptors on phagocytic cells. vascular collapse, and renal failure
(3) Cell damage can result from the mechanism of • Symptoms: fever, chills, nausea, lower back pain,
antibody-dependent cellular cytotoxicity (ADCC). ADCC tachycardia, shock, and hemoglobin in the urine
is mediated through binding of IgG antibody to its
corresponding antigen on the target cell and to Fc Delayed hemolytic reactions
receptors on macrophages or natural killer cells. This • occur within the first 2 weeks following a transfusion
binding stimulates the release of cytotoxic enzymes that • caused by an anamnestic response to the antigen to
destroy the cell. which the patient has previously been exposed
• IgG
• A second possible effect of type II hypersensitivity is that o type of antibody responsible
the cell surface antibody can inhibit the function of a cell. o initially present in such low titer that it was not
o This can occur when antibody blocks the binding detectable with an antibody screen
of a physiological ligand to its receptor, resulting in • Causes: Rh, Kell, Duffy, and Kidd blood groups
dysfunction of the cell.
o e.g. autoimmune disease myasthenia gravis, Hemolytic Disease of the Newborn (HDN)
which affects the neuromuscular junctions.
• appears in infants whose mothers have been exposed to
§ Patients with this disease produce
blood-group antigens on the baby’s cells that differ from
autoantibodies to receptors on muscle cells
their own
for the neurotransmitter acetylcholine
• mother makes IgG antibodies in response to these
(ACH).
antigens that cross the placenta to destroy the fetal RBCs
§ Normally, ACH is released from the nerve
endings and binds to its corresponding • erythroblastosis fetalis - severe HDN
receptors on muscle cells, stimulating • D antigen
contraction in the muscle fibers and muscle o a member of the Rh blood group
movement. o most common antigen involved in severe
§ However, in myasthenia gravis, attachment reactions
of the autoantibody to the ACH receptor • Exposure usually occurs during the birth process when
blocks the binding of ACH, leading to fetal cells leak into the mother’s circulation.
muscle weakness. • Typically, the first child is unaffected; however, the second
and later children have an increased risk of the disease
B. Clinical Examples of Type II Hypersensitivity because of an anamnestic response.
Transfusion Reactions • >20 mg/dL of bilirubin - associated with deposition in
• cell destruction that results from antibodies combining tissues such as the brain and result in a condition known
with heteroantigens as kernicterus
• Major groups involved in transfusion reactions: ABO, Rh, • Treatment: exchange transfusion to replace antibody-
Kell, Duffy, and Kidd systems. coated RBCs
• isohemagglutinins
o Anti-A and anti-B antibodies
o naturally occurring antibodies
o triggered by contact with identical antigenic
determinants on microorganisms
• Individuals do not make these antibodies to their own
RBCs.
• range from acute massive intravascular hemolysis to an
undetected decrease in RBC survival.
• The extent of the reaction depends on the following
factors:
o The temperature at which the antibody is most Hemolytic disease of the newborn (HDN) can develop in Rh positive
active babies who are born to Rh-negative mothers if no treatment is
o The plasma concentration of the antibody administered.
o The immunoglobulin class involved (A1) An Rh-negative mother may produce anti-Rh antibodies if an Rh-
positive baby’s RBCs enter her circulation late in gestation or during
o The extent of complement activation
the birth process. In this primary immune response, naïve B cells (NB)
o The density of the antigen on the RBC differentiate and the plasma cells secrete mainly anti-Rh IgM, which
o The number of RBCs transfused does not cross the placenta; therefore, a healthy baby is delivered.
o most important to detect antibodies that react at However,memory B cells (MB) are also produced and (A2) if the
37°C. mother becomes pregnant with another Rh positive baby, these
• If a reaction occurs only below 30°C, it can be memory cells produce a stronger secondary immune response and
disregarded because antigen–antibody complexes formed secrete mainly anti-Rh IgG, which crosses the placenta and destroys
the baby’s RBCs, resulting in HDN. (B) Alternatively, passive
at colder temperatures tend to dissociate at 37°C.
vaccination of the mother with an anti-Rh IgG called Rhogam during
pregnancy and upon delivery prevents the mother from mounting an
Intravascular hemolysis active immune response, allowing her future Rh-positive children to
• occurs because of complement activation be born normally.
• resulting in the release of hemoglobin and vasoactive and
procoagulant substances into the plasma

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Autoimmune Hemolytic Anemia Paroxysmal cold hemoglobinuria
• directed against self-antigens because individuals with • cause autoimmune hemolytic anemia
this disease form antibodies to their own RBCs • condition occurs most often after infection with certain
• Symptoms: malaise, lightheadedness, weakness, viral illnesses, including measles, mumps, chickenpox,
unexplained fever, pallor, and possibly mild jaundice and infectious mononucleosis
• Warm autoimmune hemolytic anemia • Patients produce a biphasic autoantibody that binds to the
o characterized by formation of IgG antibody RBCs at cold temperatures and activates complement at
o reacts most strongly at 37°C 37°C to produce an intermittent hemolysis.
• Idiopathic autoimmune hemolytic anemia
o underlying cause of antibody production is unknown Type II Reactions Involving Tissue Antigens
Goodpasture’s syndrome
Certain drugs • antibody produced during the course of this disease
• induce production of antibodies that can cause hemolytic reacts with basement membrane protein
anemia • Usually the glomeruli in the kidney and pulmonary
• capable of attaching to the RBCs directly or of forming alveolar membranes are affected.
immune complexes that attach to the RBCs • evenly bound linear deposition of IgG in the glomerular
• penicillins and cephalosporins basement membrane
o can act as haptens after binding to proteins on the o detected with fluorescent-labeled anti-IgG
RBC membrane o indicative of Goodpasture’s syndrome
o stimulate the production of anti-drug antibodies
that destroy the RBCs, primarily through Others
extravascular hemolysis • Hashimoto’s disease
§ cephalosporins - modify the RBC • myasthenia gravis
membrane by facilitating binding of • Insulin-dependent diabetes mellitus
immunoglobulins and complement
• quinidine and phenacetin C. Testing for Type II Hypersensitivity
o can stimulate the production of anti-drug Direct antiglobulin test (DAT)
antibodies that bind to the drug to form soluble • performed to detect transfusion reactions, HDN, and
immune complexes autoimmune hemolytic anemia
• methyldopa • Polyspecific anti-human globulin
o can induce hemolytic anemia by stimulating o a mixture of antibodies to IgG and complement
production of autoantibodies against the RBC components such as C3b and C3d
membrane o used for initial testing
o Positive: repeated using monospecific anti-IgG,
Cold agglutinins anti-C3b, and anti-C3d to determine which of these
• less frequent cause of immune hemolytic anemias is present
• autoantibodies that react with antigens on the RBC o If an autoimmune hemolytic anemia is caused by
membrane at cold temperatures IgM antibody, only the tests for complement
• reversible upon exposure to a warm temperature components would be positive.
• belong to the IgM class and most are specific for the Ii
blood groups on RBCs Indirect antiglobulin test (DAT)
• do not cause clinical symptoms unless the individual is • used in the crossmatching of blood to prevent a
exposed to the cold and usually have their maximal effect transfusion reaction
when the temperature in the peripheral circulation falls • can either determine the presence of a particular antibody
below 30°C in a patient or type patient RBCs for specific blood group
• areas of the body most affected are those having greatest antigens
exposure to the cold (fingers, toes, earlobes, and nose)
• Polyclonal cold agglutinins Process
o produced secondary to certain infections, most • a two-step process in which RBCs and antibody are
notably Mycoplasma pneumonia and infectious allowed to combine at 37°C and then the cells are
mononucleosis but also respiratory viruses and carefully washed to remove any unbound antibody.
HIV • Anti-human globulin is added to cause a visible reaction if
• Monoclonal cold agglutinins antibody has been specifically bound.
o associated with B- or plasma cell- • Any negative tests are confirmed by quality-control cells,
lymphoproliferative disorders including B-cell which are coated with antibody.
chronic lymphocytic leukemia (CLL), B-cell
lymphomas, Hodgkin disease, and To determine the titer of a cold agglutinin antibody
Waldenstrom’s macroglobulinemia as well as • Patient serum can be serially diluted and incubated
autoimmune diseases such as systemic lupus overnight at 4°C with a dilute suspension of washed
erythematosus (SLE) human type O RBCs.
• Chronic cold agglutinins • The tubes are then gently shaken and observed for
o produced as a primary characteristic of a disease agglutination.
entity of unknown origin, which is known as • The last tube with agglutination represents the titer.
chronic cold agglutinin disease (CCAD) or o Titers of 64 or higher are considered to be
chronic hemagglutinin disease (CHD) clinically significant.

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• The agglutination should disappear after warming the
tubes briefly in a 37°C water bath.
• Before testing, it is important to use prewarmed blood to
separate the serum or plasma from the patient’s RBCs.49
o Failure to do so can result in binding of the cold
agglutinins to the patient’s own RBCs, producing
false-negative results when the patient’s serum is
assayed for cold agglutinin reactivity against the C. Serum Sickness
reagent type O cells. • a generalized type III hypersensitivity reaction that results
from passive immunization of humans with animal serum
TYPE III HYPERSENSITIVITY o horse antiserums were used to treat infections
A. Introduction such as diphtheria, tetanus, and pneumonia
• similar to type II reactions in that IgG or IgM is involved o horse antivenoms - still used to treat people who
and destruction is complement-mediated have been bitten by poisonous snakes
• Difference: in the case of type III-associated diseases, the • also occur after treatment of patients with mouse
antigen is soluble monoclonal antibodies for diseases such as cancer or
• When soluble antigen combines with antibody, complexes autoimmune disorders
are formed that precipitate out of the serum. • Patients exposed to such animal sera can produce
• Complement binds to these complexes (glomerular antibodies against the foreign animal proteins.
basement membrane, vascular endothelium, joint linings, • These can combine with their corresponding antigen to
and pulmonary alveolar membranes in the tissues) form immune complexes that can deposit in the tissues
causing the release of mediators that increase and trigger the type III hypersensitivity response
vasodilation and vasopermeability, attract macrophages • Generalized symptoms of serum sickness appear 7 to 21
and neutrophils, and enhance binding of phagocytic cells days after injection of the animal serum
by C3b-mediated opsonization. o Include headache, fever, nausea, vomiting, joint
• If the target cells are large and cannot be engulfed for pain, rashes, and lymphadenopathy
phagocytosis to take place, granule and lysosome
contents are released by a process known as exocytosis. D. Autoimmune Diseases and Other Causes of Type III
Hypersensitivity
B. Arthus Reaction • SLE and rheumatoid arthritis (RA)
• classic example of a localized type III reaction o commonly produce antibodies against nuclear
• demonstrated by Maurice Arthus in 1903 constituents such as DNA and histones
• Using rabbits that had been immunized to produce an o may also produce an antibody against IgG called
abundance of circulating antibodies, Arthus showed that rheumatoid factor
when these rabbits were challenged with an intradermal o autoantibodies combine with their corresponding
injection of the antigen, a localized inflammatory reaction antigen to produce immune complexes that trigger
resulted. the type III hypersensitivity response
• erythema and edema • can also be caused by a number of other factors:
o reaction components of vaccines, bee stings, treatment with
o peaks within 3 to 8 hours and certain drugs (for example, penicillin and sulfonamides),
o followed by a hemorrhagic necrotic lesion that may and infections, such as viral hepatitis and Group A
ulcerate Streptococcus.
• The inflammatory response is caused by an antigen–
antibody combination and subsequent formation of E. Testing for Type III Hypersensitivity
immune complexes that deposit in small dermal blood • presence of antinuclear antibodies can be detected by a
vessels. variety of methods, including indirect
• can sometimes be seen in humans following booster immunofluorescence, enzyme-linked immunosorbent
injections with tetanus, diphtheria, or measles vaccines assay, and fluorescent microsphere multiplex
immunoassays
• Rheumatoid factor - can be detected by latex
agglutination, nephelometry, or other immunoassays

TYPE IV HYPERSENSITIVITY
A. Introduction
• first described in 1890 by Robert Koch
o He observed that individuals infected with
Mycobacterium tuberculosis (Mtb) developed a
localized inflammatory response after receiving
intradermal injections of a filtrate from the
organism.
• differs from the other three types of hypersensitivity in that
sensitized T cells, rather than antibodies, play the major
role in its manifestations
• Symptoms peak between 48 to 72 hours after exposure to
antigen
• also known as delayed hypersensitivity

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• classic type IV response involves the Th1 subclass of T o Cytokine production by the Th1 cells causes
helper (Th) cells macrophages to accumulate and release cytokines
and other substances that produce a local
Initial sensitization phase inflammatory response.
• 1 to 2 weeks • produces a skin eruption characterized by erythema,
• takes place after the first contact with antigen. swelling, and the formation of papules that appears from 6
• During this phase, Langerhans cells in the skin and hours to several days after the exposure
macrophages in the tissues capture antigen and migrate • can last for 3 to 4 weeks
to nearby lymph nodes, where they present the antigen to
naïve Th cells.
• The antigen-presenting cells (APCs) also release
cytokines that promote differentiation of the naïve T cells
into Th1 cells and other T-cell subsets and induce their
proliferation.
• The expanded Th1 cells then migrate to the site where the
antigen is located and the effector phase of the response
begins.
• At the site, the activated Th1 cells release cytokines.
o IL-3 and GM-CSF - induce hematopoiesis of cells
of the granulocyte-macrophage lineage and
chemokines such as monocyte chemotactic
protein (MCP-1/CCL2) recruit macrophages to
the site.
• In the tissues, the monocytes differentiate into
macrophages and are activated by IFN-γ and TNF-β
These activated macrophages release reactive oxygen
species, nitric oxide, and proinflammatory mediators that
recruit more macrophages to the site and stimulate them C. Hypersensitivity Pneumonitis
to become effective APCs, thus perpetuating the
• mediated predominantly by sensitized T lymphocytes that
response.
respond to inhaled allergens
• an allergic disease of the lung parenchyma characterized
Granulomas
by inflammation of the alveoli and interstitial spaces
• organized clusters of cells • caused by chronic inhalation of a wide variety of antigens
• caused by chronic persistence of antigen and is most often seen in individuals who are engaged in
• epithelioid-shaped and multinucleated fused work or hobbies involving exposure to the implicated
macrophages with an infiltrate of lymphocytes or other antigen
WBCs • other names: farmer’s lung, bird breeder’s lung disease,
• can release large amounts of lytic enzymes that can and humidifier or air conditioner lung disease
destroy surrounding tissue and promote fibrin deposition • most likely caused by microorganisms, especially
bacterial and fungal spores, which individuals are
Two types of antigens that can trigger the type IV exposed to from working with moldy hay, pigeon
hypersensitivity droppings, compost, moldy tobacco, infested flour, and
1. Intracellular pathogens moldy cheese
• can be bacteria, fungi, parasites, or viruses • Symptoms: dry cough, shortness of breath, fever, chills,
• Pathogens that commonly induce delayed hypersensitivity weight loss, and general malaise, which may begin 6 to 8
include Mycobacterium tuberculosis, Mycobacterium hours after exposure to a high dose of the offending
leprae, Pneumocystis carinii, Leishmania species,and antigen
herpes simplex virus. • Treatment: Systemic corticosteroid therapy

2. Contact antigens D. Skin Testing for Delayed Hypersensitivity

• come into direct contact with the skin • used clinically to detect delayed hypersensitivity
• e.g. plants such as poison ivy and poison oak, metals responses to a variety of antigens
such as nickel salts, and components of hair dyes and • based on a T-cell–mediated memory response
cosmetics • When antigen is injected intradermally or applied to the
surface of the skin, previously sensitized individuals
B. Contact Dermatitis develop a reaction at the application site.
• usually caused by low-molecular-weight compounds that • Reaction reaches a peak by 72 hours after exposure
touch the skin
o urushiol - a chemical released by poison ivy, poison Patch test
oak, and poison sumac • gold standard in testing for contact dermatitis
• Langerhans cells in the skin are thought to process the
• must be done when the patient is free of symptoms or at
hapten–protein complexes and migrate to the regional
least has a clear test site
lymph nodes where they present the antigen to Th1 cells.
• A nonabsorbent adhesive patch containing the suspected
o Sensitization of the Th cells takes several days;
contact allergen is applied on the patient’s back and the
however, once it occurs, its effects can last for
skin is checked for a reaction over the next 48 hours.
years because of immunologic memory.

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• Redness with papules or tiny blisters is considered a Cell lysosomal inflammati
positive test. function enzymes on or
• Final evaluation is conducted at 96 to 120 hours. inhibited that cause activate
byantibody tissue cytotoxic
binding damage. T cells to
Tuberculin skin test Cell cause
• uses an M tuberculosis antigen extract prepared from a function direct cell
purified filtrate of the organism’s cell wall, called a purified stimulated damage.
protein derivative (PPD) by
• routinely performed by the Mantoux method in which 0.1 antibody
binding
mL of 5 tuberculin units (TU) of PPD is injected
Clinical Anaphyla Transfusio Serum Contact
intradermally into the inner surface of the forearm using a Example xis, n sickness, dermatitis,
fine needle and syringe s allergic reactions, Arthus tuberculin
• examined between 48 and 72 hours for the presence of a rhinitis, autoimmu reaction, and
hardened, raised area called induration allergic ne lupus anergy
• Interpretation asthma, hemolytic erythemato skin tests,
o induration reaction of >15 mm - considered a food anemia, sus, hyper-
allergies, hemolytic rheumatoid sensitivity
positive test in individuals with no risk factors
urticaria disease arthritis, pneu-
o >10 mm or greater - considered positive in recent of the drug monitis
immigrants of high prevalence countries, newborn, reactions
intravenous drug users, employees of health-care drug
and other high-risk facilities, persons with certain reactions,
clinical conditions, and children younger than 5 myastheni
years of age a
o >5 mm - considered positive in persons who have gravis,
Goodpastu
HIV infection or other forms of
re’s
immunosuppression, features on a chest x-ray syndrome,
consistent with tuberculosis, or recent contact with Graves
tuberculosis patients disease
• Positive PPD test - indicates that the individual has
previously been exposed to M tuberculosis or a related Review Questions
organism, but it does not necessarily mean that he or she 1. Which of the following is a general characteristic of
has an active tuberculosis infection. hypersensitivity reactions?
• anergy a. The immune responsiveness is depressed.
o absence of positive reactions for all of the common b. Antibodies are involved in all reactions.
antigens used in the skin test c. An exaggerated immune response to an
o deficient cell-mediated immunity antigen occurs.
d. The antigen triggering the reaction is a harmful
Comparison of Hypersensitivity Reactions one.
Type I Type II Type III Type IV 2. Which of the following is associated with an increase in
Immune IgE IgG or IgM IgG or IgM T cells IgE production?
Mediator a. Transfusion reaction
s
b. Activation of Th2 cells
Synonym Anaphyla Antibody- Complex- Cell-
ctic mediated mediated mediated c. Reaction to poison ivy
cytotoxic or d. HDN
delayed 3. Which of the following would cause a positive DAT test?
type a. Presence of IgG on RBCs
Timing Immediat Immediate Immediate Delayed b. Presence of C3b or C3d on RBCs
e c. A transfusion reaction caused by preformed
Antigen Heterolog Cell Soluble: Autologou antibody
ous surface: autologous s or d. Any of the above
autologous or heterolog
4. All of the following are associated with type I
or heterologo ous
heterologo us hypersensitivity except
us a. release of preformed mediators from mast
Comple No Yes Yes No cells.
ment b. activation of complement.
Involvem c. cell-bound antibody bridged by antigen.
ent d. an inherited tendency to respond to allergens.
Immune Release Cell Antigen– Antigen- 5. Which of the following is associated with anaphylaxis?
Mechani of destruction antibody sensitized a. Buildup of IgE on mast cells
sm mediators caused by complexes Th1 cells
from antibody activate release
b. Activation of complement
IgE- and compleme cytokines c. Increase in cytotoxic T cells
sensitized compleme nt proteins. that d. Large amount of circulating IgG
mast cells nt, Neutrophils recruit 6. To determine if a patient is allergic to rye grass, the best
and opsonizati are macro- test to perform is the
basophils on, or recruited phages a. total IgE test.
ADCC and and b. skin prick test.
release induce

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 c. DAT. d. patient serum should be separated from whole
d. complement fixation. blood at 37°C and tested at 37°C.
7. Which condition would result in HDN?
a. Buildup of IgE on mother’s cells
b. Sensitization of cytotoxic T cells
c. Exposure to antigen found on both mother and
baby RBCs
d. Prior exposure to foreign RBC antigen
8. What is the immune mechanism involved in type III
hypersensitivity reactions?
a. Cellular antigens are involved.
b. Deposition of immune complexes occurs in
antibody excess.
c. Only heterologous antigens are involved.
d. Tissue damage results from exocytosis.
9. What is the immune phenomenon associated with the
Arthus reaction?
a. Tissue destruction by cytotoxic T cells
b. Removal of antibody-coated RBCs
c. Deposition of immune complexes in blood
vessels
d. Release of histamine from mast cells
10. Which of the following conclusions can be drawn about
a patient whose total IgE level was determined to be
150 IU/mL?
a. The patient definitely has allergic tendencies.
b. The patient may be subject to anaphylactic
shock.
c. Antigen-specific testing should be done.
d. The patient will never have an allergic reaction.
11. Which of the following explains the difference between
type II and type III hypersensitivity reactions?
a. Type II involves cellular antigens.
b. Type III involves IgE.
c. IgG is involved only in type III reactions.
d. Type II reactions involve no antibody.
12. Two days after administration of the PPD test, a female
health-care worker developed an area of redness and
induration 12 mm in size at the injection site. This result
means that she has
a. an active case of tuberculosis.
b. been exposed to M tuberculosis.
c. developed protective immunity against
tuberculosis.
d. a result in the normal range for her risk group.
13. A young woman developed red, itchy papules on her
wrist 2 days after wearing a new bracelet. This reaction
was caused by
a. IgE-sensitized mast cells in the skin.
b. antigen-antibody complexes in the skin.
c. damage to the skin cells by antibodies and
complement.
d. an inflammatory response induced by
cytokines released from Th1 cells.
14. Reactions to latex are caused by
a. type I hypersensitivity.
b. type IV hypersensitivity.
c. skin irritation.
d. all of the above.
15. To determine a cold agglutinin titer
a. patient serum should be separated from whole
blood at 4°C and tested at 4°C.
b. patient serum should be separated from whole
blood at 4°C and tested at 37°C.
c. patient serum should be separated from whole
blood at 37°C and tested at 4°C.

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Christine Joy R. Timbang | 0963-467-6983
IMMUNOSEROLOGY 14.

Autoimmunity
OUTLINE affinity for these self-antigens are deleted by
I. Introduction apoptosis, a physiological form of cell death
A. Definitions o Negative selection occurs with both the
II. Etiology of Autoimmune Disease immature, double-positive CD4+/CD8+ cells in
B. Self-Tolerance the cortex and with the more mature, single-
C. Genetics positive CD4+ or CD8+ cells in the medulla.
D. Other Endogenous and Environmental § During this process, some of the self-
Factors reactive CD4+ T cells are not deleted, but
III. Systemic Autoimmune Diseases instead differentiate into T regulatory
A. Introduction (Treg) cells that can specifically inhibit
B. Systemic Lupus Erythematosus (SLE) immune responses to self-antigens.
C. Antinuclear Antibodies (ANAs) o Similarly, as B cells mature in the bone marrow,
D. Antiphospholipid Antibodies those with receptors having a strong affinity for
E. Rheumatoid Arthritis (RA) self-antigens are eliminated by apoptosis. Some
F. Granulomatosis With Polyangiitis [GPA] self-reactive B cells are not deleted: rather, they
(Wegener’s Granulomatosis [WG]) are stimulated to rearrange their immunoglobulin
G. Antineutrophil Cytoplasmic Antibodies genes so that their B-cell receptors are no longer
(ANCAs) antigen specific.
IV. Organ-Specific Autoimmune Disease § This process is known as receptor
A. Autoimmune Thyroid Diseases (AITDs) editing. B cells that possess receptors
B. Type 1 Diabetes Mellitus (T1D) that only weakly recognize self-antigens
C. Celiac Disease are induced to downregulate the
D. Autoimmune Liver Diseases expression of their receptors and develop
E. Multiple Sclerosis (MS) a specific state of unresponsiveness to
F. Myasthenia Gravis (MG) the antigens known as anergy.
G. Goodpasture’s Syndrome
Peripheral tolerance
• lymphocytes that recognize self-antigens in the secondary
INTRODUCTION
lymphoid organs are rendered incapable of reacting with
A. Definitions those antigens
Horror autotoxicus
• can result from anergy caused by the absence of a
• Paul Ehrlich noted that the immune system could attack costimulatory signal from an antigen-presenting cell (APC)
the very host it was intended to protect, a phenomenon he or binding of inhibitory receptors such as CTLA-4 (a
referred to as “horror autotoxicus,” or “fear of self- molecule that prevents T-cell activation)
poisoning.” • can also result from inhibition by Tregs or death by
• Conditions in which this phenomenon occurred later apoptosis.
became known as autoimmune diseases. • Self-reactive B cells in the periphery can be deleted by
apoptosis, be rendered anergic after repeated stimulation
Autoimmune diseases with self-antigens, or receive inhibitory signals through
• disorders in which immune responses are targeted toward receptors such as CD22.
self-antigens and result in damage to organs and tissues
in the body
• can be caused by T-cell–mediated immune responses or
autoantibodies that are directed against host antigens

ETIOLOGY OF AUTOIMMUNE DISEASE

A. Self-Tolerance
• ability of the immune system to accept self-antigens and
not initiate a response against them
• type of immunologic tolerance, or a state of immune
unresponsiveness that is directed against a specific
antigen, in this case, a self-antigen

Central tolerance
• occurs in the central or primary lymphoid organs, the
thymus, and the bone marrow
• As T cells mature in the thymus, they encounter self-
antigens that are normally present on the surface of the
thymic epithelial cells.
o In a process called negative selection, T cells that
express T-cell receptors (TCRs) with a strong

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B. Genetics • Instead, the microorganism can induce a local
• Autoimmune diseases are often more prevalent among inflammatory response that recruits leukocytes and
family members than among unrelated individuals and are stimulates APCs to release cytokines that nonspecifically
more prevalent among monozygotic (genetically identical) activate T cells.
twins than dizygotic (non-identical) twins or siblings. • Some of the T cells that are activated may have specificity
• Examples for self-antigens.
o HLAB27 allele - development of ankylosing o This expansion of the immune response to
spondylitis (autoinflammatory disease that affects unrelated antigens has also been termed “epitope
the spine) spreading.”
o TPN22 gene,
o T- and B-cell - receptor signaling Superantigens
o IL2RA gene - T-cell activation and maintenance of • third way that microorganisms might induce autoimmunity
Tregs • proteins that are produced by various microbes that have
o CTLA4 gene - has an inhibitory effect on T-cell the ability to bind to both class II MHC molecules and
activation; BLK gene - involved in B-cell activation TCRs, regardless of their antigen specificity
and development • e.g. staphylococcal enterotoxins - cause food poisoning
o AIRE (autoimmune regulator) gene - promotes the and toxic shock syndrome
development of T-cell tolerance in the thymus
Epigenetics and Modification of Self-Antigens
C. Other Endogenous and Environmental Factors
Hormonal Influence • refers to modifications in gene expression that are not
• Women caused by changes in the original DNA sequence
o 2.7 times more likely to acquire an autoimmune • stable and can be inherited.
disease than men • triggered by exposure to environmental toxins, ingestion
o have higher absolute CD4+ T-cell counts and of harmful foods or drugs, or the aging process
higher levels of circulating antibodies than men • induce epigenetic changes by increasing or decreasing
• estrogens - tend to direct the immune system in favor of a methylation of cytosine bases, modifying histones, and
type 2 helper cell (Th2) response → more B-cell causing abnormal regulation by microRNAs
activation and antibody production
• androgens - favor a type 1 helper cell (Th1) response with post-translational modifications
activation of CD8+ T cells • exposure to environmental factors can lead to changes at
the protein level
Tissue Trauma and Release of Cryptic Antigens • may involve biochemical processes such as acetylation,
• When immunologic tolerance to self-antigens occurs lipidation, citrullination, and glycosylation
during the early development of lymphocytes in the o e.g. citrullination of collagen might play a role in
thymus and bone marrow, some self-antigens may be the pathogenesis of rheumatoid arthritis (RA) and
cryptic, or hidden within the tissues of the host. glycosylation of myelin may be involved in the
• Immunologic ignorance pathology of multiple sclerosis
o At a later time in life, inflammation or tissue trauma
could cause the cryptic antigens to be released and Interactions Between Factors
to suddenly be accessible to the uneducated
lymphocytes, triggering an immune response.
o Tissue damage could be caused by factors such as
infections, contact with environmental toxins, or
physical injury from exposure to ultraviolet (UV)
radiation.
o may be responsible for the production of
autoantibodies to the lens of the eye following an
ocular injury, autoantibodies to sperm after a
vasectomy, and autoantibodies to DNA following
damage to skin cells by overexposure to UV rays
from the sun
SYSTEMIC AUTOIMMUNE DISEASES
Microbial Infections A. Introduction
Molecular mimicry Systemic Autoimmune Diseases
• principal means by which microbes are thought to Disease Target Cells Associated
accomplish in developing of autoimmune disease And Tissues Autoantibodies
• refers to the fact that many bacterial or viral agents Systemic lupus Multiple cells and • Antibodies to
contain antigens that closely resemble the structure or erythematosus organs double-
amino acid sequence of self-antigens (SLE) throughout the stranded DNA
• e.g. Streptococcus pyogenes - rheumatic fever body, including and other
the skin, joints, nuclear
Bystander effect kidneys, brain, components,
• second way that microbes might trigger autoimmunity heart, lungs such as Sm
• microbial organism does not have to share structurally (ANAs)
similar antigens with the host.

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 • Phospholipid antibodies to the nuclear antigens SS-A/Ro and SS-
antibodies B/La
• Antibody to
RBCs Clinical Signs and Symptoms
• Antibody to • Nonspecific symptoms: fatigue, weight loss, malaise,
platelets fever, and anorexia
• Antibody to • Joint involvement - most frequently reported manifestation
lymphocytes o arthritis is symmetric and involves the small joints
• Antibody to of the hands, wrists, and knee
ribosomal • skin manifestations - next most common signs
components o erythematous rash
• Antibody to o may appear on any area of the body exposed to
endothelium UV light
• Rheumatoid o butterfly rash across the nose and cheeks
factor o lupus, derived from the Latin term meaning “wolf-
Rheumatoid Joints, bone; • Anti-CCP like”
arthritis (RA) other tissues in (cyclic • Systemic effects: cardiac involvement with pericarditis,
some cases citrullinated tachycardia, or ventricular enlargement
proteins) • Drug-induced lupus - most common drugs implicated are
• Rheumatoid procainamide, hydralazine, chlorpromazine, isoniazid,
factor quinidine, anticonvulsants such as methyldopa, and
• Antinuclear possibly oral contraceptives
antibodies
(ANAs)
Granulomatosis Upper • Antineutrophil
with polyangiitis respiratory cytoplasmic
(Wegener’s system, lungs, antibodies
granulomatosis) blood vessels (ANCA); c-
ANCA pattern
• Rheumatoid
factor
• ANAs

B. Systemic Lupus Erythematosus (SLE)

• a chronic systemic inflammatory disease that affects
between 40 and more than 200 persons per 100,000,
depending on the population
• Peak age of onset: 20 and 40 years

Etiology
• originate from complex interactions between
environmental factors, genetic susceptibility, and
abnormalities within the immune system
• Environmental factors: UV light, certain medications, and
possibly infectious agents
• People with certain HLA types (HLA-A1, B8, and DR3)
have an increased chance of developing lupus
11 Clinical Criteria and 6 Immunologic Criteria
Immunopathology Clinical criteria
1) acute cutaneous lupus
• B cells and the autoantibodies they produce 2) chronic cutaneous lupus
o play a central role in the pathogenic mechanisms 3) oral ulcers
o presence can precede the onset of disease by 9 to 4) non-scarring alopecia (thinning or fragility of the
10 years hair)
• Anti-dsDNA and complement proteins 5) synovitis
o deposited in organs such as the kidneys and skin 6) serositis
o thought to play a major role in the pathogenesis 7) renal involvement
o Accumulation of IgG to dsDNA - most pathogenic 8) neurological symptoms
because it forms complexes of an intermediate size 9) hemolytic anemia
that become deposited in the glomerular basement 10) leukopenia
membrane (GBM) 11) thrombocytopenia
• Phospholipid antibodies Immunologic criteria
o associated with increased miscarriage, stillbirth, 1) elevated antinuclear antibody titer
and preterm delivery in pregnant women with lupus 2) elevated anti-dsDNA titer
o Neonatal lupus - occurs in up to 8% of babies born 3) presence of antibody to the Sm nuclear antigen
to pregnant women with SLE, is associated with 4) presence of antiphospholipid antibody

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 5) low complement levels • Antibodies to dsDNA typically produce a peripheral or a
6) positive direct Coombs’ test in the absence of homogeneous staining pattern on indirect
hemolytic anemia immunofluorescence (IIF)

Treatment 3. Antihistone antibodies

• Mild: high dose of aspirin or other anti-inflammatory drug • Histones
may bring relief o nucleoproteins that are essential components of
• Skin manifestations: antimalarials such as chromatin.
hydroxychloroquine or chloroquine and topical steroids o Five major classes of histones: H1, H2A, H2B, H3,
• The key to successful treatment is to prevent organ and H4.
damage and achieve remission. § Antibodies to H2A and H2B - can be
detected in almost all patients with drug-
Laboratory Diagnosis of Systemic Lupus Erythematosus induced lupus.
• Complete blood count (CBC), platelet count and urinalysis • presence of antihistone antibody alone or combined with
o leukopenia and possible anemia and antibody to ssDNA supports the diagnosis of drug-induced
thrombocytopenia lupus
o ↑ erythrocyte sedimentation rate (ESR)
o ↓ or = C-reactive protein (CRP) level 4. Nucleosome antibodies
• stimulated by DNA-histone complexes, known as
• More specific: quantification of complement proteins and nucleosomes, or deoxyribonucleoprotein (DNP)
the detection of specific autoantibodies • directed only against the complexes and not against DNA
o C3 - most commonly measured complement or the individual histones
protein • found in about 85% of patients with SLE and their levels
• antinuclear antibodies (ANAs) correlate with disease severity
o first test typically done • typically produce a homogeneous pattern in the IIF assay
o screening test
o present in the majority of patients with the disease 5. Antibody to the Sm antigen
• specific for lupus because it is not found in other
C. Antinuclear Antibodies (ANAs) autoimmune diseases
• autoantibodies that are directed against antigens in the • produces a coarse speckled pattern of nuclear
nuclei of mammalian cells. fluorescence on IIF
• present in over 95% of patients with active lupus and are
used as a major marker for the disease 6. Anti-RNP antibody
• not specific for SLE • directed against RNP, which consists of several
• heterogeneous group of antibodies that have different nonhistone proteins complexed to a small nuclear RNA
antigen specificities called U1-nRNP (U for “uridine-rich”
• nuclear antigens they are directed against include double- • forms complexes with the Sm antigen in the nucleus, and
stranded (ds) and single-stranded (ss) DNA antisera to these antigens produce a pattern of partial
(deoxyribonucleic acid), histones, nucleosomes (DNA- identity when they are reacted in the Ouchterlony double
histone complexes), centromere proteins, and extractable immunodiffusion test
nuclear antigens (ENAs) • produces a coarse speckled pattern on IIF

Types of Antinuclear Antibodies 7. Anticentromere antibodies

1. Extractable nuclear antigens (ENAs) • bind to proteins in the middle region of a chromosome
• group of nuclear antigens that were so named because where the sister chromatids are joined
they were isolated in saline extracts of mammalian tissues • directed against three centromere antigens of molecular
• represent a family of small nuclear proteins that are weights 16kDa, 80kDa, and 120kDa
associated with uridine-rich RNA • found in 50% to 80% of patients with the CREST
• ribonucleoproteins (RNP), the Sm antigen, the SS-A/Ro syndrome, a subset of scleroderma named after its five
and SS-B/La antigens, Scl-70, Jo-1, and PM-1 major features: calcinosis, Raynaud’s phenomenon,
esophageal dysmotility, sclerodactyly, and telangiectasia
SS-A/Ro and SS-B/La • produce discrete speckled staining in the nuclei of the
• another family of ENAs cells
• consist of small, uridine-rich RNAs complexed to cellular
Methods of ANA Detection
proteins and were given the prefix of SS- because a large
percentage of patients with Sjögren’s syndrome (~70%) 1. Indirect Immunofluorescence (IIF)
possess antibodies to the antigens • Fluorescent antinuclear antibody (FANA) testing
o most widely used and accepted test because it is
2. Double-stranded DNA (dsDNA) antibodies highly sensitive, detects a wide range of antibodies,
• most specific for SLE because they are mainly seen in and is inexpensive and easy to perform
patients with lupus and their levels correlate with disease • IIF test
activity o gold standard for ANA testing
• presence of these antibodies is considered diagnostic for o uses a commercially prepared microscope slide
SLE onto which nucleated cells have been fixed
o especially when they are found in combination with o human epithelial cell line (HEp-2)
low levels of the complement component C3

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 o standard substrate for clinical laboratories
worldwide
o used because they have large nuclei with high
antigen expression, allowing for high sensitivity and
facilitating visualization of results
o A titer of ≥160 is generally considered to be
clinically significant

Patterns of Immunofluorescence
a. Homogeneous (diffuse)
• characterized by uniform staining of the entire nucleus in
interphase cells and of the condensed chromosomal
region in metaphase cells
• associated with antibodies to dsDNA (also known as
native or nDNA), histones, and deoxyribonuclear protein
(DNP). found in patients with SLE, drug-induced lupus,
and many other autoimmune diseases.

b. Peripheral (rim or outline Fluorescent ANA test principle.

• diffuse staining is seen throughout the nucleus, but there (A) Microscope slide coated with HEp-2 cells (the plasma and nuclear
is a greater staining intensity around the outer circle membranes have been permeabilized). (B) Patient serum (containing
surrounding the nucleus in interphase cells antinuclear antibodies) is applied. (C) After washing, fluorescent-
labeled anti-human Ig is added to detect bound autoantibodies. After
• Dividing cells show strong staining of the condensed
a final wash, the slide is read under a fluorescent microscope. Five
chromatin typical patterns in nondividing cells are shown on the right.
• primarily caused by antibodies to dsDNA and is highly
specific for SLE

c. Speckled
• characterized by discrete, fluorescent specks throughout
the nuclei of interphase cells
• Staining is absent in the nucleolus and in the chromatin
region in dividing cells
• can be fine or coarse, depending on the autoantibody
present
• associated with antibodies to ENAs and can be found in
patients with SLE, Sjögren’s syndrome, systemic
sclerosis, and other systemic autoimmune rheumatic
diseases

d. Nucleolar
• prominent staining of the nucleoli within the nuclei of
interphase cells
• size, shape, and number of the nucleoli per cell are
variable and staining can be smooth, clumpy, or speckled,
depending on the type of antibody present
• staining may or may not be present in the dividing cells
• caused by antibodies to RNA and RNP and is seen mainly
in patients with scleroderma, but can also be present in
patients with other connective tissue diseases

e. Centromere
• numerous discrete speckles are seen in the nuclei of
interphase cells and the chromatin of dividing cells
• most cells have 46 speckles, representing the number of
chromosomes
• caused by antibodies to proteins in the centromeres of the
chromosomes and is found mainly in patients with the
CREST syndrome

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2. ELISA and CLIA • If an outer well does not contain antibodies to any of the
• test for a broad range of antibodies if multiple nuclear ENAs, no precipitate is formed between that well and the
antigens are coated onto a single test well, or for specific center well.
ANAs if each well is coated with a single antigen • Positive: immunoprecipitation lines of serological identity
• large variation in the performance of tests produced by
different manufacturers, which is influenced by the antigen
preparation used

3. Microsphere Multiplex Immunoassay (MIA)

• amenable to automated, high throughput testing with
objective results
• Patient serum is incubated in a microtiter plate well
containing a suspension of polystyrene microspheres that
are coated with individual nuclear antigens or with a HEp-
2 extract. Beads containing specific antigens can be
differentiated by their unique shade of red created by a
specific combination of infrared and red fluorescent dyes.
• Antibodies in the patient serum will bind only to the beads
containing their specified antigens.
• Following a washing step to remove unbound antibodies,
a phycoerythrin-labeled anti-human IgG is added.
• The conjugate will bind only to the beads that have bound
patient antibodies and excess conjugate is removed by
washing.
• The bead suspension is analyzed for fluorescence by a
flow cytometer that has two lasers, one that identifies
each bead and another that detects the amount of
fluorescent conjugate attached.

4. Immunofluorescence Using Crithidia luciliae

• used to detect antibodies to dsDNA
• a purified antigen preparation that is free from single-
stranded DNA (ssDNA) must be used because antibodies
to ssDNA occur in many individuals with other
autoimmune or inflammatory diseases
• substrate: Crithidia luciliae
o hemoflagellate organism
o trypanosome has a circular organelle called a
kinetoplast that is composed mainly of dsDNA
• Patient serum is incubated on a microscope slide coated Common Antinuclear Antibodies
with C luciliae organisms and binding is detected with a Autoantib Characteristics of Immunoflu Disease
fluorescent-labeled anti-Ig conjugate. ody Antigen orescent Associati
• Washing of the slide is performed after each step to Pattern on
remove unbound antibody. Anti- dsDNA Peripheral SLE
• Positive: brightly stained kinetoplast dsDNA or
homogene
ous
Anti- Related to purines Not SLE,
ssDNA and pyrimidines detected many
on routine other
screen diseases

Antihiston Different classes Homogene Drug-

e of histones ous induced
SLE,
other
diseases
Anti-DNP DNA-histone Homogene SLE,
complex ous drug-
(nucleosomes) induced
SLE
Anti-Sm Extractable Coarse Diagnost
5. Ouchterlony test nuclear antigen speckled ic for
• solution containing ENA antigens is placed in a central (uridine-rich RNA SLE
well of an agarose plate and patient samples and controls component)
are placed in the surrounding wells

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 Anti-RNP Proteins Coarse SLE, Etiology
complexed with speckled mixed • RA and specific HLA-DRB1 alleles or PTPN22 gene
small connecti polymorphisms
nuclear RNA ve tissue o positive rheumatoid factor (RF) or antibodies to
diseases CCP
Anti–SS- Proteins Finely SLE, • cigarette smoking
A/Ro complexed to RNA speckled Sjögren’ o strongest environmental risk factor for RA
s o doubles the risk of developing the disease
syndrom
e, others Immunopathology
Anti–SS- Phosphoprotein Finely SLE, • caused by an inflammatory process that results in the
B/La complexed to speckled Sjögren’ destruction of bone and cartilage
RNA polymerase s • lesions in rheumatoid joints show an increase in cells
syndrom lining the synovial membrane and formation of a pannus
e, others o a sheet of inflammatory granulation tissue that
Antinucle RNA polymerase, Prominent SLE, grows into the joint space and invades the
olar fibrillarin, PM-1 staining of systemic cartilage
nucleoli sclerosis • Proinflammatory cytokines found in synovial fluid: IL-1, IL-
(can be 6, IL-17, and TNF-α
smooth, o TNF-α - conjunction with other cytokines and a
clumpy, or molecule called RANKL (receptor activator of
speckled) nuclear factor kappa-B ligand), induces the
Anti–Scl- DNA Atypical Systemi differentiation of osteoclasts and inhibits bone
70 topoisomerase I speckled c formation.
sclerosis • Local bone erosion
, o another feature that is characteristic of the
sclerode pathology in RA
rma o Multinucleated giant cells called osteoclasts are
Anti–Jo-1 Histidyl-tRNA Fine Polymyo central to the structural damage that is seen in the
synthetase cytoplasmi sitis bones.
c speckling o Osteoclasts absorb bone as part of the normal
bone remodeling process that occurs in the body
Autoantib Antigens Discrete CREST in response to growth and repair of damaged
ody– in the speckled syndrom bone.
Anti- chromosomecentr e • RF and anti-CCP
Centrome omeres o two key antibodies found in the disease
re § RF
• antibody that is most often of the IgM
D. Antiphospholipid Antibodies class and is directed against the FC
• heterogeneous group of antibodies that bind to portion of IgG
phospholipids alone or phospholipids complexed with • increasing macrophage activity and
protein enhancing antigen presentation to T
• can affect every organ in the body, but they are especially cells by APCs
associated with deep-vein and arterial thrombosis and § anticyclic citrullinated peptide antibody [anti-
with recurrent pregnancy loss CCP or ACPA]
• can be identified by their ability to cause false-positive • second major type of antibody
results in nontreponemal tests for syphilis, the lupus associated with RA
anticoagulant assay, and immunoassays for antibodies to • Citrullinated proteins
cardiolipin or other phospholipids o contain an atypical amino acid
called citrulline
Lupus anticoagulant o generated when the enzyme
• one of several types of antiphospholipid antibodies peptidyl arginine deiminase
• produces a prolonged activated partial thromboplastin (PAD) modifies the amino acid
time (APTT) and prothrombin time (PT) arginine by replacing an NH2
• have an increased risk of clotting and spontaneous group with a neutral oxygen
abortion
Clinical Signs and Symptoms
E. Rheumatoid Arthritis (RA) • Symptoms: joints, tendons, and bursae
• another example of a systemic autoimmune disorder • Extra-articular manifestations: formation of subcutaneous
• strikes individuals between the ages of 25 and 55 nodules, pericarditis, lymphadenopathy, splenomegaly,
• can be characterized as a chronic, symmetric, and erosive interstitial lung disease, or vasculitis.
arthritis of the peripheral joints that can also affect multiple • Nodules can also be found in the myocardium,
organs such as the heart and the lungs pericardium, heart valves, pleura, lungs, spleen, and
larynx.
• Felty’s syndrome - combination of chronic, nodular RA
coupled with neutropenia and splenomegaly

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• Cardiovascular disease - most common cause of death produces remission with resolution of the inflammatory
lesions in most patient.
Treatment • Anti-CD20 monoclonal antibody rituximab - effective
• primarily based on nonsteroidal anti-inflammatory drugs alternative to cyclophosphamide with reduced toxicity.
(NSAIDs) such as salicylates (aspirin) and ibuprofen
• Disease-modifying anti-rheumatic drugs (DMARDs) Clinical Criteria and Laboratory Diagnosis
o most notably methotrexate Criteria published by the ACR
o now prescribed at the time of diagnosis (1) nasal or oral inflammation with oral ulcers or purulent or
o inhibits adenosine metabolism and T-cell bloody nasal discharge;
activation (2) abnormal chest x-ray, showing presence of nodules,
• Agents that act against TNF-α are classified into two fixed infiltrates, or cavities;
categories: (3) urinary sediment with microhematuria or RBC casts; and
(1) monoclonal antibodies to TNF-α (e.g., infliximab, (4) granulomatous inflammation on biopsy
adalimumab, certolizumab, golimumab) (5) positive antineutrophil cytoplasmic antibody (ANCA)
(2) TNF-α receptors fused to an IgG molecule result
(etanercept)
• General laboratory findings: normochromic, normocytic
Laboratory Diagnosis of Rheumatoid Arthritis anemia; leukocytosis; eosinophilia; and an elevated ESR
• combination of clinical manifestations, radiographic • Urinalysis: microhematuria, proteinuria, and cellular casts
findings, and laboratory testing • Serological findings: elevated CRP, elevated
• Manual agglutination tests - using charcoal or latex immunoglobulin levels, positive ANCAs, and possibly
particles coated with IgG have been used for many years other autoantibodies, such as RF and ANAs
to detect RF.
• Laboratory testing for antibody to cyclic citrullinated G. Antineutrophil Cytoplasmic Antibodies (ANCAs)
peptides (anti-CCP) - has been added to the classification • autoantibodies that are produced against proteins that are
criteria to increase the specificity for RA; performed present in the neutrophil granules
mainly by ELISA • strongly associated with three syndromes involving
• ↑ CRP and ESR are elevated vascular inflammation: GPA or WG, microscopic
• ↑ or = levels of serum complement components because polyangiitis (MPA), and eosinophilic granulomatosis with
of increased acute-phase reactivity polyangiitis (EGPA; formerly known as Churg-Strauss
syndrome); these syndromes are collectively known as
F. Granulomatosis With Polyangiitis [GPA] (Wegener’s ANCA-associated vasculitides (AAV)
Granulomatosis [WG])
• rare autoimmune disease involving inflammation of the Ethanol-fixed leukocytes as the cellular substrate
small- to medium-sized blood vessels, or vasculitis • Ethanol treatment permeabilizes the granule membranes,
• usually begins with a localized inflammation of the upper allowing for migration of the contents.
and lower respiratory tract • In this method, patient serum is incubated with a
• Symptoms: fever, malaise, arthralgias, anorexia, and microscope slide containing ethanol-fixed leukocytes.
weight loss • Following incubation, the slide is washed to remove
• Symptoms of the upper airway: severe or persistent unbound serum and an anti-human IgG, FITC-labeled
rhinorrhea (“runny nose”), rhinitis, sinusitis, oral or nasal conjugate is added.
ulcers, and gingivitis • Following a second incubation and wash step, the slide is
• Chronic otitis media - cause perforation and scarring of viewed under a fluorescent microscope for staining of the
the eardrums, leading to hearing loss. neutrophils.
• Lymphocyte staining should not be present; if it is, it
Etiology should be minimal.
• unknown
Two patterns of fluorescence
• but multiple genes are thought to be involved
o HLA-DPB1*0401 allele - found to have a strong 1. Cytoplasmic
association with GPA in Caucasian patients • also known as c-ANCA
o HLA-DRB1*0901 and HLA-DRB*1501 alleles - • primarily caused by PR3-ANCA and appears as a diffuse,
associated with increased risk in Asian and African granular staining in the cytoplasm of the neutrophils
American populations • caused by antibodies against positively charged antigens
• S aureus - induce molecular mimicry because it contains such as MPO that migrate out of the granules after
peptides that bear similarity to the proteinase 3 (PR3) ethanol fixation and are attracted toward the negatively
autoantigen charged nucleus
• Antibody to the PR3 antigen
o play a role in the pathophysiology of the systemic 2. Perinuclear
vasculitis seen in GPA • p-ANCA
• true p-ANCA pattern is present, ANA testing using HEp-2
Treatment cells as the substrate in IIF should be negative
• Therapy for GPA is directed toward suppression of this • For more definitive differentiation, the test can be
inflammatory response. repeated using microscope slides with formalin-fixed
• Patients with severe forms of GPA are treated with a leukocytes.
combination of prednisone and cyclophosphamide, which

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• A positive ANCA test must therefore be combined with ing the antigens, Eosinophilic
clinical manifestations and histological findings of biopsy lobes of including granulomato
tissue to make an accurate diagnosis. the myeloperoxid sis with
neutrophil ase polyangiitis
nuclei, (MPO) (EGPA;
blending Churg-
them Strauss
together syndrome)

ORGAN-SPECIFIC AUTOIMMINE DISEASE

A. Autoimmune Thyroid Diseases (AITDs)
• Hashimoto’s thyroiditis and Graves disease
o distinctly different symptoms
o share some antibodies in common
o both interfere with thyroid function
• thyroglobulin (Tg)
o primary constituent of colloid
o a large iodinated glycoprotein from which the active
thyroid hormone triiodothyronine (T3) and its
precursor, thyroxine (T4), are synthesized
• thyroid peroxidase (TPO)
o plays an important role in the synthesis of these
hormones by oxidizing iodine ions, allowing for their
incorporation into the tyrosine residues of
thyroglobulin to produce the building blocks for the
hormones
• Thyrotropin-releasing hormone (TRH)
o secreted by the hypothalamus to initiate the
process that eventually causes release of
hormones from the thyroid
o acts on the pituitary gland to induce release of
thyroid stimulating hormone (TSH)
§ binds to receptors on the cells of the thyroid
gland, causing thyroglobulin to be broken
down into secretable T3 and T4
• If the levels of T3 and T4 become too high, they feed back
to the hypothalamus and pituitary to inhibit release of TRH
and TSH, resulting in decreased production of the thyroid
hormones.

Antineutrophil Cytoplasmic Antibodies (ANCAs)

Pattern on Appearance Autoantigens Associated
Indirect Diseases
Immunofluoresc
ence
With Ethanol-
Fixed
Leukocytes
c-ANCA Diffuse, PR3 antigen Granulomat
granular osis with
staining polyangiitis
in the (GPA;
cytoplasm Wegener’s
of the granulomato
neutrophil sis)
s, fading
toward the
outer
edges
of the
cells
p-ANCA Fluoresce Positively Microscopic
nce charged polyangiitis
surround- (MPA)

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Etiology Laboratory Diagnosis of Autoimmune Thyroid Diseases
Graves disease • measurement of circulating TSH levels
• HLA-DR3 - associated with Graves disease o TSH level is inversely related to the levels of T3 and
T4
Hashimoto’s thyroiditis o routinely measured with highly sensitive
• HLA antigens DR3, DR4, DR5, and DQ7 - association of chemiluminescent immunoassays that can detect
Hashimoto’s thyroiditis fewer than 0.1 mU/L
• high iodine intake - development of Hashimoto’s disease
Typical Laboratory Findings in
\ HLA-DR antigens are expressed on the surface of thyroid Autoimmune Thyroid Diseases
epithelial cells - unique feature of both Graves and Disease TSH Level Free T4 Autoantibodie
Hashimoto’s disease (FT4) s
Level
Clinical Signs and Immunopathology Hashimoto’ Normal or Decrease Antithyroglobuli
Graves disease s thyroiditis elevated d n
• characterized by hyperthyroidism (a state of excessive Antithyroid
thyroid function) peroxidase
• most common cause of hyperthyroidism (TPO)
• manifested as thyrotoxicosis (an excess of thyroid Graves Decreased Elevated Thyroid-
hormones, with a diffusely enlarged goiter that is firm disease or stimulating
• instead of rubbery) undetectabl hormone
• Symptoms: nervousness, insomnia, depression, weight e receptor
loss, heat intolerance, sweating, rapid heartbeat, antibodies
palpitations, breathlessness, fatigue, cardiac (TRAbs)*
dysrhythmias, and restlessness Antithyroglobuli
o exophthalmos - hypertrophy of the eye muscles n
and increased connective tissue in the orbit cause Antithyroid
the eyeball to bulge out so that the patient has a peroxidase
large-eyed staring expression (TPO
• thyroid-stimulating hormone receptor antibodies (TRAbs) - *diagnostic
major antibodies involved
o When TRAbs bind to the TSH receptor they mimic B. Type 1 Diabetes Mellitus (T1D)
the action of TSH, resulting in uncontrolled • characterized by
receptor stimulation with excessive release of o hyperglycemia (a high level of glucose in the blood)
thyroid hormones o complete or nearly complete deficiency in insulin
Hashimoto’s thyroiditis • previously known as juvenile onset diabetes
• aka chronic lymphocytic thyroiditis • usually develops in children or in young adults before the
• most common autoimmune disease age of 30
• patients develop an enlarged thyroid called a goiter, which • chronic autoimmune disease that involves selective
is irregular and rubbery. destruction of the beta cells of the pancreas
• Patients also produce thyroid-specific autoantibodies and • insulin
cytotoxic T cells o cells are located in clusters called the islets of
• hypothyroidism - immune destruction of the thyroid gland Langerhans and are responsible for the production
occurs, which results in a state of decreased thyroid and secretion of the hormone
function
• Symptoms: dry skin, decreased sweating, puffy face with Etiology
edematous eyelids, pallor with a yellow tinge, weight gain, • HLA-DR3 or DR4 gene
fatigue, and dry and brittle hair o ↑ risk when both of these genes are present
• Six forms of Hashimoto’s disease • HLA-DQ haplotypes
o Classic - thyroid shows hyperplasia with an • early exposure to cow’s milk
increased number of lymphocytes
Immunopathology
• Progressive inflammation of the islets of Langerhans in
the pancreas leads to fibrosis and destruction of most
beta cells.

Treatment
• Daily injectable insulin
• use of stem cells to produce islet cells in vitro and
methods to induce immunologic tolerance in recipients

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Laboratory Diagnosis of Type 1 Diabetes Mellitus selective IgA deficiency and will therefore test negative in
• A person is considered to have diabetes if he or she IgA-based assays
meets one of four criteria: o Resolution: IgG anti-tTG or for IgG antibodies to
(1) a fasting glucose greater than 126 mg/dL on more DGP
than one occasion (normal value is lower than 100 • Biopsy of the small intestine
mg/dL); o performed to confirm the diagnosis
(2) a random plasma glucose level of 200 mg/dL or o increase in the number of intraepithelial
more with classic symptoms of diabetes; lymphocytes, elongation of the intestinal crypts, and
(3) an oral glucose tolerance test of 200 mg/dL or more partial to total atrophy of the villi
in a
(4) 2-hour sample with a 75 g glucose load; or D. Autoimmune Liver Diseases
(5) a hemoglobin A1c value (HbA1c) greater than 6.5% Autoimmune Hepatitis (AIH)
• autoimmune process targets the hepatocytes
• Tests for antibodies to GAD and IA-2A - done to confirm • formerly known as chronic active hepatitis
the diagnosis • immune-mediated liver disease that can lead to end-stage
o If negative: liver failure if left untreated
§ ICA - children • HLA-DRB1 and HLA-DQB1 alleles - higher risk of
§ Insulin - antibodies in adults developing AIH
• Combined screening for IA-2A, ICA, and GAD antibodies -
most sensitivity and best positive predictive value for T1D Laboratory findings
in high-risk populations • ↑ liver enzymes, aspartate aminotransferase (AST) and
alanine aminotransferase (ALT)
C. Celiac Disease • less prominent increases in serum bilirubin and alkaline
• autoimmune disease affecting the small intestine and phosphatase
other organs • ↑ Serum immunoglobulin levels, particularly IgG
• unique in that it is associated with a known environmental • Autoantibodies present: ANAs, ANCAs, smooth muscle
trigger— dietary gluten antibodies (SMA), anti-liver kidney microsomal antibody
• Gluten (anti-LKM-1), anti-liver cytosol type 1 antibody (anti-LC-1),
o a protein complex found in wheat, barley, and rye and antimitochondrial antibodies (AMAs)
that is poorly digested by the upper • Liver biopsy
gastrointestinal system o necessary to confirm the diagnosis of AIH and to
o contains an alcohol-soluble component called assess the extent of liver damage
gliadin that is rich in the amino acids glutamine o detection of autoantibodies, elevated IgG, and
and proline exclusion of viral hepatitis
§ Gliadin - resistant to digestive enzymes in • interface hepatitis
the stomach, pancreas, and small o inflammation at the portal-parenchymal boundary
intestine and therefore remains intact in o typical of AIH
the lumen, or space within the intestines, o characterized by an infiltrate of lymphocytes,
after ingestion plasma cells, and histiocytes surrounding dying
• tissue transglutaminase (tTG) hepatocytes
o an intestinal enzyme that converts the glutamine
residues in gliadin to glutamic acid Two types of AIH
o enhances immunogenicity of gliadin 1. AIH-1
• HLA-DQ2 or HLA-DQ8 - presence of one of these two • accounts for two-thirds of all AIH cases and has a
HLA haplotypes is a necessary condition for developing female:male ratio of 4:1
celiac disease • positive for SMA or ANA
• Environmental factors: administration of gluten in the diet o SMAs - directed against actin and other
of an infant younger than 4 months in the absence of components of the cytoskeleton.
breastfeeding, rotavirus infection, and overgrowth of • They can be detected by IIF on rodent kidney, stomach,
pathogenic bacteria in the gut or liver sections, where they produce fluorescent staining
of the smooth muscle in the artery walls and other
Clinical Symptoms and Treatment components, such as the glomeruli and tubules of the
• Symptoms: diarrhea, abdominal distention, and failure to kidneys.
thrive, but may also experience vomiting, irritability, • Antibody titers:
anorexia, and constipation o >1:80 or higher - adults
• Treatment: Gluten-free diet o 1:20 - children

Laboratory Diagnosis of Celiac Disease 2. AIH-2

• based on clinical symptoms, serological findings, • has a female:male ratio of 10:1 and is seen mostly in
duodenal biopsy, and presence of the HLA-DQ2 or HLA- children
DQ8 haplotype • produce antibodies against LKM-1 or LC-1
• IgA antibodies to tTG - serological method of choice for • anti-LKM-1 - stains the cytoplasm of the hepatocytes and
initial testing the P3 portion of the kidney tubules
• Serum IgA levels - concurrently measured in these • Antibody titers:
individuals because a significant number also have o 1:40 - adults
o 1:10 - children

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• Antibodies to LC-1 - directed against a folate-metabolizing palsy, and numerous sensory abnormalities such as
enzyme in the liver and stain the cytoplasm of liver cells in tingling or “pins and needles” that run down the spine or
IIF. extremities, as well as flashes of light seen on eye
movement.
Treatment • Classified into four major subtypes based on the clinical
• Standard immunosuppressive treatment of prednisolone course
(+/azathioprine) to induce remission o relapsing-remitting MS
§ characterized by clearly defined episodes
Primary Biliary Cirrhosis (PBC) of neurological attacks with periods of full
• most common autoimmune liver disease or partial recovery in between
• HLA-DRB1, HLA-DQA1, HLA-DPB1, and HLA-DQB1
haplotypes Treatment for MS
• an autoimmune disease that involves progressive • Aimed at easing recovery from acute attacks and reducing
destruction of the intrahepatic bile ducts → cholestasis (a the risk of future relapses.
condition in which the flow of bile is slowed or blocked), • Acute exacerbations - corticosteroids to reduce
inflammation of the portal vein in the liver, and inflammation.
accumulation of scar tissue that can ultimately lead to • IFN-β1a and IFN-β1b - treat MS for the long term
cirrhosis and liver failure • Severe - reduced by therapy with natalizumab
• Symptoms: fatigue, pruritis (itchy skin), abdominal pain, o a humanized monoclonal antibody directed against
and dry eyes and mouth; in the later stages, symptoms an adhesion molecule of lymphocytes, preventing
include jaundice, ascites, and greasy stools them from binding to endothelial cells and crossing
the blood–brain barrier
Laboratory findings and diagnosis
• Three diagnostic criteria for the disease (two to diagnose) Laboratory Diagnosis of Multiple Sclerosis
(1) Anti-mitochondrial antibodies (AMAs) - found in the • based primarily on clinical symptoms, demonstration of
majority of patients with PBC disseminated lesions in the white matter of the brain and
(2) Serum alkaline phosphatase level - elevated at spinal cord by magnetic resonance imaging (MRI), and
least 1.5 times the upper limit of normal for 6 exclusion of other possible causes
months or more • Immunoglobulins are increased in the spinal fluid in 75%
(3) Liver biopsy showing nonsuppurative destructive to 90% of patients
cholangitis and interlobular bile duct injury o two or more distinct bands on protein
• detected by IIF with mitochondria-rich tissue substrates electrophoresis that are not seen in the serum
such as rodent liver, kidney, or stomach sections o referred to as oligoclonal and can be identified by
• These antibodies produce a bright, uniform granular isoelectric focusing with immunoblotting
cytoplasmic fluorescence in the distal renal tubules, • IgG index
gastric parietal cells, thyroid epithelial cells, and cardiac o ↑ calculated ratio of cerebral spinal fluid (CSF)
muscle IgG/albumin ÷ serum IgG/albumin
Treatment
• ursodeoxycholic acid (UDCA) F. Myasthenia Gravis (MG)
o a bile acid that helps move bile through the liver • an autoimmune disease that affects the neuromuscular
o helped to slow down disease progression and junction
increase patient survival • characterized by weakness and fatigability of skeletal
• Liver transplantation muscles
o only effective treatment for patients who have o Early-onset MG (EOMG)
reached end stage liver disease § occurs before the age of 40 and affects
predominantly females
Primary sclerosing cholangitis (PSC) § HLA haplotype, A1, B8, DR3
• affects the medium-sized, intra- and extrahepatic bile o Late-onset form of the disease (LOMG)
ducts § occurs after the age of 40 and is seen more
often in males
E. Multiple Sclerosis (MS) § HLA antigens B7 and DR2
• autoimmune disorder involving inflammation and • HLA-DR14-DQ5 may increase susceptibility to MuSK
destruction of the CNS antibody production in MG
• most closely associated with inheritance of a particular • antibody-mediated damage to the acetylcholine receptors
HLA molecule coding for the beta chain of the DR in skeletal muscle or to other proteins in the
subregion, namely DRB1*1501 neuromuscular junction leads to progressive muscle
• Environmental factors: reduced exposure to sunlight, weakness
vitamin D deficiency, and cigarette smoking o ptosis (drooping of the eyelids)
• characterized by the formation of lesions called plaques in o diplopia (double vision)
the white matter of the brain and spinal cord, resulting in o inability to retract the corners of the mouth, often
the progressive destruction of the myelin sheath of axons resulting in a snarling appearance

Clinical Symptoms and Treatment Immunopathogenesis

• Damage to the tissue of the CNS can cause visual • Have antibody to acetylcholine (ACH) receptors (ACHR) -
disturbances, weakness or diminished dexterity in one or contribute to the pathogenesis of the disease by three
more limbs, locomotor incoordination, dizziness, facial mechanisms

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 o ACH is released from nerve endings to generate Laboratory Diagnosis of Goodpasture’s Syndrome
an action potential that causes the muscle fiber to • Renal involvement: gross or microscopic hematuria,
contract. proteinuria, a decreased 24-hour creatinine clearance,
o When the antibody combines with the receptor and elevated blood urea and serum creatinine levels.
site, binding of ACH is thought to be blocked and Abnormally shaped RBCs and casts can be found in the
in the clustering of ACHRs on the muscle cell urine sediment
membrane, which enhances the transmission of • Pulmonary involvement: decreased total lung capacity
the signals from the nerve cells. and increased uptake of carbon monoxide
o Consequently, there is fragmentation of the • An iron deficiency anemia with decreased hemoglobin
postsynaptic ACHR clusters, resulting in muscle and hematocrit can develop if pulmonary hemorrhage is
weakness and atrophy. severe.
• Thymic hyperplasia - common in EOMG patients with • ↑ or = ESR and CRP level
ACHR antibodies • IIF assay
• Thymoma - for LOMG; a tumor of the thymus that may o uses frozen kidney sections that are incubated
contain autoreactive T cells with patient serum and then overlaid with a
fluorescein-labeled anti-IgG
Treatment • Western blot
• Anticholinesterase agents - prevent destruction of the o technique detects antibodies to the Goodpasture
neurotransmitter, acetylcholine, are used as the main basement membrane antigen as well as other
therapy human α chain proteins that have been separated
• Thymectomy - should be performed on patients who have by polyacrylamide gel electrophoresis, followed by
a thymoma transfer to nitrocellulose paper for immunoblotting
• Begins with high doses of corticosteroid drugs followed by o highly specific test that can be used for
other immunosuppressive drugs such as azathioprine or confirmation and minimization of false-positive
mycophenolate mofetil, to maintain the response.133 results
• Plasmapheresis or intravenous immunoglobulin - • Histological analysis
administered to patients in crisis. o confirmation of the diagnosis and for assessment
• Biological agents such as monoclonal antibodies or fusion of tissue damage
proteins - unresponsive to conventional therapies o indicated by formation of a smooth, linear,
ribbonlike pattern of fluorescence along the GBM
Laboratory Diagnosis of Myasthenia Gravis
• radioimmunoprecipitation (RIPA) assay Organ-Specific Autoimmune Diseases
o most commonly used procedure Disease Target Associated
o based on precipitation of the patient’s antibody Cells or Autoantibodies
with ACHRs isolated from human muscle Tissues
o complex is detected with a radio-labeled snake Addison’s disease Adrenal Antibody to adrenal
venom called α-bungarotoxin, which binds with glands cells
high affinity to a different site on the receptors Autoimmune Red blood Antibody to RBCs
• I-labeled human MuSK hemolytic cells
o used to detect antibody to MuSK anemia (RBCs)
• Immunofluorescence cell-based assays in which patient Autoimmune Liver AIH-1—smooth
serum is incubated with HEK293 cells expressing all four hepatitis (AIH) muscle antibodies;
ACHR subunits. ANAs
AIH-2—anti-liver
G. Goodpasture’s Syndrome kidney microsomal
• characterized by the presence of autoantibody to an antibody (anti-LKM-
antigen in the basement membranes in the glomeruli of 1); anti-liver cytosol
the kidneys and alveoli of the lungs type 1 antibody (anti-
• are disorder that is found mainly in Caucasians of LC-1)
European origin Autoimmune Platelets Antiplatelet antibody
• Treatment: administration of high dose corticosteroids to thrombocytopenic
stop inflammation, followed by immunosuppressive drugs purpura
such as cyclophosphamide to inhibit further production of Celiac disease Small Antitransglutaminase
autoantibodies intestine (tTG)
and other Antibodies to
Etiology and Immunopathology organs deamidated gliadin
• HLA-DRB1-15 antigen peptides (DGPs)
• Exposure to cigarette smoke and organic solvents has Endomysial
also been implicated in disease pathogenesis antibodies
• autoantibodies - produced in Goodpasture’s syndrome are Goodpasture’s Kidneys, Antibody to an
known to be specifically directed against the syndrome lungs antigen in the renal
noncollagenous domain of the alpha-3 chain of type IV and pulmonary
collagen basement
• reacts with collagen in the glomerular or alveolar membranes
basement membranes and causes damage by type II
hypersensitivity

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 Graves disease Thyroid Thyroid-stimulating 3. SLE can be distinguished from RA on the basis of which
gland hormone receptor of the following?
antibodies (TRAbs) a. Joint pain
Antithyroglobulin b. Presence of antinuclear antibodies
Antithyroid c. Immune complex formation with activation of
peroxidase (TPO) complement
Hashimoto’s Thyroid Antithyroglobulin d. Presence of anti-dsDNA antibodies
thyroiditis gland Antithyroid 3. Which of the following would support a diagnosis of
peroxidase (TPO) drug-induced lupus?
Multiple sclerosis Myelin Antibodies to myelin a. Antihistone antibodies
sheath of basic protein b. Antibodies to Smith antigen
nerves c. Presence of RF
Myasthenia gravis Nerve- Antibodies to d. Antibodies to SS-A and SS-B antigens
muscle acetylcholine 4. A peripheral pattern of staining of the nucleus on IIF is
synapses receptors (AChR) caused by which of the following antibodies?
Anti-muscle-specific a. Anti-Sm antibody
kinase (MuSK) b. Anti-SSA/Ro antibody
Antibody to the c. Centromere antibody
lipoprotein LRP4 d. Anti-dsDNA
Pernicious anemia Stomach Parietal cell 5. Which of the following would be considered a significant
antibody, intrinsic finding in Graves disease?
factor antibody a. Increased TSH levels
Poststreptococcal Kidneys Streptococcal b. Antibody to TSHR
Glomerulonephritis antibodies that c. Decreased T3 and T4
cross-react with d. Antithyroglobulin antibody
kidney tissue 6. Destruction of the myelin sheath of axons caused by the
presence of antibody is characteristic of which disease?
Primary biliary Intrahepatic Antimitochondrial
a. MS
cirrhosis bile ducts antibodies (AMA)
b. MG
c. Graves disease
Rheumatic fever Heart Streptococcal
d. Goodpasture’s syndrome
antibodies that
7. Blood was drawn from a 25-year-old woman with
cross-react with
suspected SLE. A FANA screen was performed and a
cardiac tissue
speckled pattern resulted. Which of the following actions
Scleroderma Connective Antinuclear
should be taken next?
tissue antibodies: anti-Scl-
a. Report out as diagnostic for SLE
70, anticentromere
b. Report out as drug-induced lupus
antibody
c. Perform an assay for specific ANAs
Sjögren’s Eyes, Antinuclear d. Repeat the test
syndrome mouth antibodies, 8. Which of the following is a mechanism used to achieve
rheumatoid factor, peripheral tolerance?
antisalivary duct a. Negative selection of autoreactive T cells in the
antibodies, thymus
antilacrimal gland b. Apoptosis of autoreactive B cells in the bone
antibodies marrow
Type 1 diabetes Pancreas Anti-insulin c. Editing of B-cell receptors that weakly
mellitus Islet cell antibodies recognize self-antigens in the bone marrow
Anti–IA-2 and anti– d. Lack of a costimulatory signal to autoreactive T
IA-2βA cells in the lymph nodes
Antibody to glutamic 9. Epitope spreading refers to
acid phosphatase a. post-translational modifications to self-
(GAD) antigens.
b. modifications in gene expression that are not
Review Questions caused by changes in DNA sequence.
1. All of the following may contribute to autoimmunity c. expansion of the immune response to
except unrelated antigens.
a. clonal deletion of self-reactive T cells. d. cross-reaction of the immune response to a
b. molecular mimicry. pathogen with a similar self-antigen.
c. increased expression of class II MHC antigens. 10. Anti-CCP (cyclic citrullinated proteins) is specifically
d. polyclonal activation of B cells. associated with which autoimmune disease?
2. Which of the following would be considered an organ- a. RA
specific autoimmune disease? b. MG
a. SLE c. Autoimmune hepatitis
b. RA d. Goodpasture’s syndrome
c. GPA 11. Which autoantibodies are strongly associated with
d. Hashimoto’s thyroiditis granulomatosis with polyangiitis (Wegener’s
granulomatosis)?

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 a. ANA
b. ANCA
c. AMA
d. SMA
12. A technologist performs an IIF test for ANCAs and
observes that there is an intense fluorescent staining of
the nuclear lobes of the neutrophils. How can this type
of staining be differentiated from a peripheral ANA
pattern?
a. Perform the test on formalin-fixed leukocytes
b. Perform IIF with HEp-2 cells
c. Perform an ELISA for ANCAs
d. All of the above
13. A 20-year-old woman made an appointment to see her
physician because she was experiencing intermittent
diarrhea. Laboratory testing revealed that she also had
an iron deficiency anemia. To determine if the patient
has celiac disease, her doctor should order which of the
following laboratory tests?
a. Anti-tTG
b. Antigliadin
c. Antigluten
d. All of the above
14. Antimitochondrial antibodies are strongly associated
with which disease?
a. Autoimmune hepatitis
b. Celiac disease
c. Primary biliary cirrhosis
d. Goodpasture’s syndrome

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Christine Joy R. Timbang | 0963-467-6983
IMMUNOSEROLOGY 15.

Transplantation Immunology
OUTLINE • HLA proteins
I. Introduction o encoded by a set of closely linked genes located
A. Transplantation on the short arm of chromosome 6 within the MHC
II. Histocompatibility Systems region
A. Major Histocompatibility Complex (MHC) o inherited as haplotypes from parental chromosome
Antigens o heterodimeric molecules consisting of two different
B. Minor Histocompatibility Antigens (mHAs) polypeptide chains chemically bound to each other
C. MHC Class I-Related Chain A (MICA) • Based on Mendelian inheritance
Antigens o 25% chance that any two siblings will inherit the
D. Killer Immunoglobulin-Like Receptors (KIRs) same two haplotypes (i.e., are HLA identical)
E. Self-Antigens o 50% chance of them being HLA haploidentical
III. Allorecognition (i.e., share one of two HLA haplotypes)
A. Classification by the genetic relatedness of o 25% chance of them being HLA nonidentical (i.e.,
the donor and the recipient share neither HLA haplotype)
B. Two distinct mechanism by which a
recipient’s immune system recognizes
foreign HLA proteins
IV. Transplant Rejection
C. Hyperacute Rejection
A. Acute Rejection (AR)
B. Chronic Rejection
V. Graft-Versus-Host Disease (GVHD)
VI. Immunosuppressive Agents
A. Introduction
B. Agents
VII. Clinical Histocompatibility Testing
A. HLA Typing
B. HLA Phenotyping
C. HLA Genotyping
D. HLA Antibody Screening, Identification, and
Crossmatching

INTRODUCTION
A. Transplantation
• potentially lifesaving treatment for end-stage organ failure,
cancers, autoimmune diseases, immune deficiencies, and
a variety of other diseases
• Significance
o graft-versus-host disease (GVHD)
o human leukocyte antigens (HLAs) and the
development of pharmacological agents that
promote graft survival by interfering with various
components of the immune system B. Minor Histocompatibility Antigens (mHAs)
• also observed that a “slower” rejection pace was mediated
HLA system by these transplantation antigens
• strongest immunologic barrier to successful allogeneic • non-HLA proteins that demonstrate variation in the amino
organ and HSC transplantation acid sequence between individuals
• consists of cell surface proteins that play a central role in • Transplanting one individual’s tissue or cells containing a
thymic education of T lymphocytes, initiation of adaptive polymorphic variant of one of these proteins into another
immune responses, and regulation of other immune individual possessing a different polymorphic variant can
system components induce a recipient’s immune response to the donor
variant. CD4 or CD8 T cells recognize the variant protein
HISTOCOMPATIBILITY SYSTEMS in an MHC-restricted fashion and mediate the immune
response.
A. Major Histocompatibility Complex (MHC) Antigens
o This response is analogous to the reaction to a
• major histocompatibility complex (MHC) antigens
microbial antigen.
• composed of the class I and class II proteins
• Several mHAs have been identified, including proteins
o Class I proteins - include HLA-A, HLA-B, and HLA-
encoded by the male Y chromosome, proteins for which
C
the recipient has a homozygous gene deletion, proteins
o Class II proteins - consist of HLA-DR, HLA-DQ,
that are autosomally encoded, and proteins that are
and HLA-DP
encoded by mitochondrial DNA.

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C. MHC Class I-Related Chain A (MICA) Antigens receptor,9 vimentin, K-alpha1 tubulin, collagen-v, and
• encodes a cell surface protein that is involved in gamma myosin.
or delta T-cell responses
• polymorphic, with over 50 allelic variants ALLORECOGNITION
• expressed on endothelial cells, keratinocytes, fibroblasts, A. Classification by the genetic relatedness of the
epithelial cells, dendritic cells, and monocytes, but they donor and the recipient
are not expressed on T or B lymphocytes • Autograft - transfer of tissue from one area of the body to
• serve as targets for allograft immune responses. another of the same individual.
• Antibodies to MICA antigens have been detected in as • Syngeneic graft (also known as an isograft) - transfer of
many as 11% of kidney-transplant patients and are cells or tissues between individuals of the same species
associated with rejection episodes and decreased graft who are genetically identical, for example, identical twins.
survival. • Allograft - transfer of cells or tissue between two
genetically disparate individuals of the same species.
D. ABO Blood Group Antigens • Xenograft - transfer of tissue between two individuals of
• only blood group system that affects clinical different species
transplantation
• incompatibility is a barrier to solid-organ transplantation
because these antibodies can bind to the corresponding • HLA disparity between donor and recipient that occurs
antigens that are expressed on the vascular endothelium with allografts and xenografts → vigorous cellular and
• Binding activates the complement cascade, which can humoral immune response to the foreign MHC antigens.
lead to very rapid rejection of the transplanted organ. o This response is the primary stimulus of graft
• hyperacute rejection rejection.
o occurs within minutes to hours after the vascular
supply to the transplanted organ is established B. Two distinct mechanism by which a recipient’s
• Transplantation approaches using plasma exchange and immune system recognizes foreign HLA proteins
intravenous immunoglobulin administration have allowed 1. Direct allorecognition
successful transplantation of kidneys from ABO- • recipient T cells bind and respond directly to foreign (allo)
incompatible donors. HLA proteins on graft cells
• The procedures reduce the ABO antibody to levels that • Although an individual T lymphocyte can recognize self-
significantly lower the risk of hyperacute rejection. HLA + peptide, foreign HLA proteins may mimic a self-
HLA + peptide complex because of similarities in the
E. Killer Immunoglobulin-Like Receptors (KIRs) structure of the allo-HLA protein itself or to structural
• affects allogeneic transplantation similarities of allo-HLA protein + peptide
• one of several types of cell surface molecules that
regulate the activity of NK lymphocytes mixed lymphocyte reaction (MLR)
• contain activating and inhibitory receptors that vary in • an in vitro correlate of direct allorecognition
number and type on any individual NK cell • In this assay, lymphocytes from an individual needing a
• Normally, an NK cell encounters self-class I MHC proteins transplant are incubated with lymphocytes from a potential
as it circulates in the body. donor that have been inactivated so they cannot
o This interaction between MHC protein and the proliferate.
inhibitory KIR maintains the NK cells in a • A disparity in the HLA-D antigens found on the two
quiescent state. If an NK cell encounters a cell populations of lymphocytes results in the proliferation of
with absent or decreased HLA class I expression, the recipient cells, which can be quantitated by
inhibitory receptors are not engaged and a loss of incorporation of radio-labeled (3H) thymidine into the DNA
negative regulatory activity occurs, resulting in NK of the proliferating cells
cell activation.
• The regulatory role of KIRs has been exploited in 2. Indirect allorecognition
haploidentical stem cell transplantation. • second pathway by which the immune system recognizes
• Stem cell donors have been selected for recipients who foreign HLA proteins.
lack a corresponding class I MHC protein for the donor’s • involves the uptake, processing, and presentation of
inhibitory KIR type. foreign HLA proteins by recipient APCs to recipient T cells
o This results in alloreactivity by NK cells that • analogous to the normal mechanism of recognition of
repopulate the recipient after transplant. foreign antigens
• These alloreactive NK cells have been shown to mediate • may play a predominant role in induction of alloantibody
a graft-versus-leukemia (GVL) reaction and prevent and chronic rejection
relapse after transplantation for certain types of
hematologic malignancies.
TRANSPLANT REJECTION
A. Hyperacute Rejection
F. Self-Antigens
• Humoral immune responses to self-antigens in transplant • occurs within minutes to hours after the vascular supply to
recipients have been associated with poor transplant the transplanted organ is established
outcomes, although a direct causal relationship has yet to • mediated by preformed antibody that reacts with donor
be firmly established. vascular endothelium
• Among the several proteins to which antibody has been • elicited by ABO, HLA, and certain endothelial antigens
described post-transplantation are angiotensin II type-1

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• Donor–recipient pairs are chosen to be ABO identical or • In mismatched allogeneic stem cell transplantation, the
compatible and patients awaiting transplantation are targets of GVHD are the mismatched HLA proteins,
screened for the presence of preformed HLA antibodies. • In matched stem cell transplantation mHAs are targeted.
• In addition, the absence of donor HLA-specific antibodies The infused T cells can mediate GVHD in several ways,
is confirmed before transplant by the performance of a including a massive release of cytokines because of
crossmatch test. large-scale activation of the donor cells by MHC-
mismatched proteins and by infiltration and destruction of
accelerated rejection tissue.
• Some individuals possess very low levels of donor-
specific antibody in the pretransplant period. IMMUNOSUPPRESSIVE AGENTS
• In these cases, antibody-mediated rejection may take A. Introduction
place over several days. • used in several ways, including induction and
maintenance of immune suppression and treatment of
B. Acute Rejection (AR) rejection.
• days to months after transplant • Combinations of different agents are frequently used to
• can be mediated by a cellular alloresponse (ACR) or by prevent graft rejection.
donor-specific antibody (also known as antibody-mediated
response; AMR) B. Agents
• characterized by parenchymal and vascular injury 1. Corticosteroid
• Antibody • potent anti-inflammatory and immunosuppressive agents
o also be involved in acute graft rejection by binding used for immunosuppression maintenance.
to vessel walls and activating complement • At higher doses, they are used to treat AR episodes.
o induces transmural necrosis and inflammation as Steroids act by blocking production and secretion of
opposed to the thrombosis typical of hyperacute cytokines, inflammatory mediators, chemoattractants, and
rejection adhesion molecules.
• Diagnostic criteria: characteristic histological findings, • These activities decrease macrophage function and alter
deposition of the complement protein C4d in the leukocyte-trafficking patterns.
peritubular capillaries, and detection of donor-specific • However, long-term use is associated with several
HLA antibody. complications, including hypertension and diabetes
mellitus.
C. Chronic Rejection
• results from a process of graft arteriosclerosis 2. Antimetabolites
characterized by progressive fibrosis and scarring with • interfere with the maturation of lymphocytes and kill
narrowing of the vessel lumen caused by proliferation of proliferating cells
smooth muscle cells • Azathioprine
• remains the most significant cause of graft loss after the o first such agent employed
first year post-transplant because it is not readily o has been replaced in large part by mycophenolate
amenable to treatment mofetil
• also thought to have an immunologic component, § more selective effect on lymphocytes
presumably a delayed-type hypersensitivity reaction to compared with azathioprine and thus fewer
foreign HLA proteins side effects.
• indicated in studies employing animal models of graft
arteriosclerosis in which mice lacking IFN gamma do not 3. Calcineurin inhibitors
develop graft arteriosclerosis
• Cyclosporine and FK-506 (tacrolimus)
• Cytokines and growth factor o compounds that block signal transduction in T
o secreted by endothelial cells, smooth muscle lymphocytes, resulting in impaired synthesis of
cells, and macrophages activated by IFN gamma cytokines such as IL-2, IL-3, IL-4, and interferon-
o stimulate smooth muscle cell accumulation in the gamma.
graft vasculature
• Inhibition of cytokine synthesis blocks the growth and
differentiation of T cells, impairing the antigraft response.
GRAFT-VERSUS-HOST DISEASE (GVHD) • Rapamycin (sirolimus)22
• unique allogeneic response o agent that inhibits T-cell proliferation by binding to
• lymphoid cells in the graft mount an immune response specific intracellular proteins, including mammalian
against the host’s histocompatibility antigens target of rapamycin (mTOR).
• Recipients of HSC transplants for hematologic § mTOR - intracellular molecule involved in
malignancies typically have depleted bone marrow before cellular functions such as proliferation
transplantation as a result of the chemotherapy used to and motility
treat the malignancy.
• The individual receives an infusion of donor bone marrow 4. Monoclonal antibodies
or, more commonly, peripheral blood stem cells. • some that bind to cell surface molecules on lymphocytes
• The infused products often contain some mature T cells. are used at the time of organ transplant and to treat
severe rejection episodes after transplantation.
Acute graft-versus-host disease (GVHD) • Two anti-CD25 monoclonal antibodies
• occurs during the first 100 days postinfusion and o available for use in transplant patients
manifests in the skin, gastrointestinal tract, and liver

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 o Basiliximab and daclizumab - both bind the CD25 • After incubation with the antisera, complement is added.
(IL-2 receptor) and thus interfere with IL-2– Binding of antibody occurs only if the lymphocytes
mediated T-cell activation. express the HLA antigen targeted by the antisera.
o may also deplete CD25-expressing cells • A complement reagent derived from rabbit serum is added
• alemtuzumab to each well. If the cells possess the HLA antigen defined
o an additional monoclonal antibody that targets the by the antibody in that well, complement is activated and
CD52 receptor found on T and B lymphocytes the cells are killed.
o may be used for induction therapy at the time of • A vital dye such as eosin red or trypan blue is added to
transplantation distinguish live cells from dead cells when they are viewed
• Disadvantage: Patients can develop anti-mouse antibody microscopically.
that may interfere with the effectiveness of these agents. • The dead cells, whose membranes have been made more
o Resolution: reduced with increased use of permeable, are able to take up the dye and appear
humanized and fully human monoclonal antibodies colored, whereas the live cells, whose membranes remain
intact, cannot take up the dye and remain colorless.
5. Polyclonal antibodies • The proportion of dead cells is estimated by microscopic
• Two polyclonal anti-T-cell antibody preparations have examination and scored according to the following scale,
been used as induction agents or to treat severe rejection. established by the American Society for Histocompatibility
o Thymoglobulin - antithymocyte antibody prepared and Genetics (ASHI):
in rabbits o 1 = 0% to 10% cell death; negative
o ATGAM - polyclonal antiserum prepared from the o 2 = 11% to 20% cell death; doubtful negative
immunization of horses. o 4 = 21% to 50% cell death; weak positive
§ Both are potent immunosuppressive o 6 = 51% to 80% cell death; positive
agents that deplete lymphocytes from the o 8 = 81% to 100% cell death; strong positive
circulation. o 0 = unreadable
• Disadvantage: Development of serum sickness because
of antibody responses to the foreign immunoglobulin

CLINICAL HISTOCOMPATIBILITY TESTING

A. HLA Typing
• phenotypic or genotypic identification of the HLA antigens
or genes in a transplant candidate or donor
• This information is used to find the most suitable donor–
recipient combination from an immunologic standpoint.
• It must be stressed that other factors must also be
considered when choosing a particular donor for any
given patient, be it a solid-organ or stem cell transplant.
o For example, ABO compatibility and infectious
disease status are important considerations in
donor selection.

B. HLA Phenotyping
• complement-dependent cytotoxicity (CDC) test
• classic procedure for determining the HLA phenotype

Procedure
• Panels of antisera or monoclonal antibodies that define
individual or groups of immunologically related HLA
antigens are incubated with lymphocytes from the person
to be HLA typed in separate wells of a microtiter plate.
• Each well of the plate contains a different antibody.
• It is important to note that multiple antisera are used for
HLA typing.
o This requirement is based on the presence of both
unique epitopes on HLA molecules (those that
define the phenotypic specificity of a specific HLA
antigen) and public epitopes (epitopes that are
present on more than one unique HLA protein).
• Because responses to public epitopes are common, many
sera must be used to define a pattern of reactivity that
correlates with a specific HLA antigen.
• T and B lymphocytes are used for HLA class I typing,
whereas purified B lymphocytes are used for HLA class II
typing because class II antigens are not found on most T
cells.

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 • amplified gene is then subjected to hybridization with a
panel of DNA probes, each specific for a unique HLA
allele or allele group.
• Only those probes that specifically hybridize to the
amplified DNA will be detected.
• The HLA genotype is determined by assessing which
probes hybridized.

C. HLA Genotyping
• use polymerase chain reaction (PCR)-based amplification
of HLA genes followed by analysis of the amplified DNA to SBT
identify the specific HLA allele or allele group
• third common method for HLA genotyping
• Three DNA-based HLA typing methods are in use:
o polymerase chain reaction with sequence-specific • which involves sequencing of PCR-amplified HLA genes
PCR (PCR-SSP) • typically carried out using Sanger dideoxy chain
o PCR-sequence-specific oligonucleotide probe terminator sequencing
hybridization (PCR-SSOP) • considered the gold standard and is able to detect new
o sequence-based typing (SBT) allelic variants because it interrogates all nucleotides in
the amplified target region
PCR amplification of HLA genes with panels of primer pairs
• most common approaches for analysis; each of which
amplifies specific alleles or related allele groups (PCR-
SSP)
• Only those primer pairs that bind perfectly to the target
gene result in detection of an amplification product.
• Amplification is detected by agarose gel electrophoresis.
• The HLA genotype is then identified by determining which
primers resulted in amplification.

PCR-SSOP
• second common approach for HLA genotyping
• involves a single PCR reaction that will amplify all HLA
gene variants at a specific locus (referred to as a generic
amplification)

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 nonidentical member other
member of the than an identical
same twin
species Grafts between
genetically
nonidentical
strains of mice
Xenograft Transfer of cells Transplant of a
or tissues to a pig valve into a
member of human heart
a different
species

Types of Graft Rejection

D. HLA Antibody Screening, Identification, and Type Timing (After Immunologic
Crossmatching Transplant) Mechanism
• Crossmatch test Hyperacute Minutes to Preformed antibodies to
o antibodies to HLA antigens can be detected in hours ABO, HLA, and certain
candidates and recipients of solid-organ endothelial antigens bind
transplants to donor vascular
o These antibodies can develop in response to endothelium, activating
multiple blood transfusions or to prior HLA- complement and clotting
mismatched transplants. factors. This leads to
o They can also be produced by women who have thrombus formation,
had multiple pregnancies in response to paternally ischemia, and necrosis
derived fetal antigens. of transplanted tissue.
o Because of the potential adverse impact HLA Accelerated Days Same as for hyperacute
antibodies can have on graft survival, patients rejection.
awaiting solid-organ transplantation are screened Acute Days to Cell-mediated response
periodically for their presence through an HLA months to foreign MHC-
antibody screen. expressing cells. CD4+ T
• percent panel reactive antibody (%PRA) - proportion of cells. Produce cytokines
lymphocytes in the panel that are killed by the patient’s and induce delayed type
serum hypersensitivity. CD8+ T
• Screening and identification of preformed HLA antibodies cells mediate cytotoxic
in transplant recipients can be performed by the CDC reactions. Antibodies
method through incubation of patient serum with panels of produced against HLA
lymphocytes with known HLA antigens, ELISA, traditional antigens bind to vessel
flow cytometry, or a flow cytometry-based multiplex bead walls, activate
array. complement, and induce
• Crossmatching is performed by incubation of recipient transmural necrosis and
serum with donor lymphocytes in a CDC assay to confirm inflammation.
the absence of donor-specific antibody. Chronic 1 year or more Delayed type
hypersensitivity
response, and possibly
Classification of Grafts
antibodies, to foreign
Type of Graft Definition Examples
HLA antigens on graft.
Autograft Transfer of tissue Skin graft from Graft arteriosclerosis and
within the same leg to face of a smooth muscle
individual burn patient proliferation occur,
Transfer of a resulting in fibrosis,
saphenous vein scarring, and narrowing
from the leg of a of vessel lumen.
cardiac
Graft- 100 days or T cells in HSC, lung, or
bypass patient to
versus-host more liver transplants react
his heart
disease against foreign HLA
Syngeneic (Iso) Transfer of cells Transplant (GVHD) proteins in the recipient’s
graft or tissues to a between identical cells, causing massive
genetically human twins cytokine release,
identical Grafts between inflammation, and tissue
individual genetically destruction in various
identical strains locations throughout the
of mice body.
Allograft Transfer of cells Human
or tissues to a transplant from a
genetically cadaver donor or
family

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 Review Questions 9. Phenotyping for HLA class II antigens requires B
1. Which of the following responses is the type of allograft lymphocytes because
rejection associated with vascular and parenchymal a. B lymphocytes express HLA class II antigens.
injury with lymphocyte infiltrates? b. B lymphocytes do not express HLA class I
a. Hyperacute rejection antigens.
b. Acute cellular rejection c. B lymphocytes are exquisitely sensitive to
c. Acute humoral rejection complement-mediated lysis.
d. Chronic rejection d. B lymphocytes represent the majority of
2. Antigen receptors on T lymphocytes bind HLA class II + lymphocytes in the peripheral blood.
peptide complexes with the help of which accessory 10. A renal transplant candidate was crossmatched with a
molecule? donor that was mismatched for only the HLA-B35
a. CD2 antigen. The candidate was known to have an antibody
b. CD3 specific for HLA-B35. Which of the following
c. CD4 combinations of T- and B-cell flow cytometric
d. CD8 crossmatch results would be expected?
3. Patients who have received the following types of grafts a. T cell negative, B cell negative
are at risk for graft-versus-host disease (GVHD) except b. T cell positive, B cell positive
for recipients of c. T cell negative, B cell positive
a. bone marrow transplants. d. T cell positive, B cell negative
b. lung transplants. 11. Which of the following HLA alleles differs from
c. liver transplants. A*02:01:02 by a synonymous nucleotide substitution?
d. irradiated leukocytes. a. A*01:01:01:01
4. Which of the following properties are not exhibited by b. A*02:01:03
HLA molecules? c. A*02:02
a. They belong to the immunoglobulin d. A*02:03:0
superfamily. 12. Which one of the following donors would be expected to
b. They are heterodimeric. elicit a positive mixed lymphocyte response in
c. They are integral cell membrane glycoproteins. lymphocytes from a patient who has the HLA-
d. They are monomorphic. DRB1*01:01, 01:03 alleles?
5. Kidney allograft loss from intravascular thrombosis a. DRB1*01:01, 01:03
without cellular infiltration 5 days post-transplant may b. DRB1*01:01, 01:01
indicate which primary rejection mechanism? c. DRB1*01:03, 01:03
a. Hyperacute rejection d. DRB1*01:01, 01:05
b. Accelerated humoral rejection 13. Which of the following donors would be the most
c. Acute humoral rejection appropriate, based on ABO compatibility, for a renal
d. Acute cellular rejection transplant candidate with the ABO type = O?
6. Which reagents would be used in a direct (forward) a. O
donor–recipient crossmatch test? b. A
a. Donor serum and recipient lymphocytes + c. B
rabbit serum complement d. AB
b. Recipient serum and donor lymphocytes + 14. Which of the following HLA antigens would be expected
rabbit serum complement to elicit an HLA antibody response in a kidney transplant
c. Donor stimulator cells + recipient responder recipient with the following HLA type: HLA-A*01,03;
cells + complete culture medium B*07,14; C*01,04N; DRB1*16,07?
d. Recipient stimulator cells + donor responder a. HLA-A*01
cells + complete culture medium b. HLA-B*14
7. The indirect allorecognition pathway involves which one c. HLA-C*04
of the following mechanisms? d. HLA-DRB1*16
a. Processed peptides from polymorphic donor 15. Suppose a 30-year-old man was found to be a suitable
proteins restricted by recipient HLA class II donor for a kidney transplant to his younger sister. This
molecules transplant would be an example of a(an)
b. Processed peptides from polymorphic recipient a. autograft.
proteins restricted by donor HLA class I b. allograft.
molecules c. isograft.
c. Intact polymorphic donor protein molecules d. xenograft.
recognized by recipient HLA class I molecules
d. Intact polymorphic donor protein molecules
recognized by recipient HLA class II molecules
8. Which immunosuppressive agent selectively inhibits IL-2
receptor-mediated activation of T cells and causes
clearance of activated T cells from the circulation?
a. Mycophenolate mofetil
b. Cyclosporine mofetil
c. Corticosteroids
d. Daclizumab

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Christine Joy R. Timbang | 0963-467-6983
IMMUNOSEROLOGY 17.

Tumor Immunology
OUTLINE C. Cancer
I. Introduction To Tumor Biology • named after the Latin word for “crab”
A. Tumor Immunology o property of invasiveness
B. Classifications of Tumor o resemble the legs of a crab when viewed in
C. Cancer microscopic tissue sections
II. Tumor Antigens • commonly exhibit metastasis
A. Tumor-Specific Antigens (TSAs) o ability of cells to break away from the original
B. Tumor-Associated Antigens (TAAs) tumor mass and spread through the blood to
III. Clinically Relevant Tumor Markers nearby or distant sites in the body
A. Tumor Markers • Classification of cancer
B. Clinical Uses of Tumor Markers: Benefits o carcinomas - skin or epithelial linings of internal
and Limitations organs or glands
C. Serum Tumor Markers o sarcomas.- bone or soft tissues such as fat,
IV. Laboratory Detection of Tumors muscles, tendons, cartilage, nerves, and blood
A. Tumor Morphology vessels
B. Immunohistochemistry • carcinogenesis - transformation of a cell into a malignant
C. Immunoassays for Circulating Tumor tumor; a multistep process involving a series of genetic
Markers mutations that cause the phenotype of a cell to be
D. Molecular Methods in Cancer Diagnosis changed over time
V. Interactions Between the Immune System And
Tumor Two major types of genes are involved in malignant
A. Introduction transformation
B. Immune Defenses Against Tumor Cells 1. proto-oncogenes
C. Innate Immune Responses • normal genes that have a positive influence on cell
D. Adaptive Immune Responses proliferation and development
VI. Immunoediting And Tumor Escape
A. Elimination 2. tumor suppressor genes
B. Equilibrium • inhibits cell division
C. Escape o gatekeeper genes - exert their effects by
VII. Immunotherapy controlling the entry of cells into the cell cycle and
A. Introduction preventing cells from completing the cell cycle if
B. Active Immunotherapy and Cancer they contain damaged DNA
Vaccines o caretaker genes - important in maintaining genetic
C. Passive Immunotherapy stability by recognizing and repairing damaged
D. Adoptive Immunotherapy DNA in a cell.

INTRODUCTION TO TUMOR BIOLOGY

A. Tumor Immunology Six Characteristics of a Cancerous Cell by Hanahan and
• study of the relationship between the immune system and Weinberg
cancer cells 1. Sustained signaling of proliferation
• Normally, cell growth and division are carefully regulated 2. Resistance to cell death
processes designed to rapidly produce new cells when 3. Ability to induce angiogenesis (development of new
necessary, inhibit cell division when enough cells are blood vessels to provide oxygen and nutrients to the
present, and limit cell life span through a normal tumor)
physiological process of cell death (apoptosis). 4. Immortality in terms of cell division
• Continue unchecked → excessive cell growth and division 5. Invasion and metastasis
→ abnormal cell mass called a tumor (from the Latin, 6. Ability to avoid suppressors of cell growth
meaning “to swell”) or neoplasm (from the Greek,
meaning “new growth”). Additional (2011)
7. Reprogramming of energy metabolism to support
B. Classifications of Tumor malignant proliferation
1. Benign 8. Ability to evade destruction by the immune system
9. Genomic instability and mutations
• composed of slowly growing cells that are well- 10. Inflammatory responses that promote tumor growth
differentiated and organized, similar to the normal tissue
from which they originated

2. Malignant
• cancer cells
• disorganized masses that are rarely encapsulated,
allowing them to invade nearby organs and destroy their
normal architecture

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 MUST KNOW Merkel cell polyomavirus Merkel cell carcinoma (a
type of skin cancer)
TNM system of the American Joint Committee on
Cancer (AJCC) TUMOR ANTIGENS
• most widely used staging scheme A. Tumor-Specific Antigens (TSAs)
• classified according to the • unique to the tumor of an individual patient or shared by a
o size and extent of the primary tumor (T0 to T4) limited number of patients with the same type of tumor
o degree of spread to adjacent lymph nodes (N0 • coded for by viral oncogenes or by host proto-oncogenes
to N3) or tumor suppressor genes that have undergone genetic
o presence or absence of distant metastases mutations
(M0 or M1) • fusion protein
§ e.g. T2N1M0 - tumor between 2 to 5 o produced in chronic myelogenous leukemia (CML)
cm in diameter that has spread to one
cells
to three regional lymph nodes but has
o result of a reciprocal chromosome translocation
not spread to distant sites
commonly known as the Philadelphia
chromosome, which involves the BCR (breakage
cluster region) on chromosome 9 and the c-ABL
gene on chromosome 22
§ C-ABL - cellular proto-oncogene that
codes for tyrosine
• kinase (a key enzyme in cell-
signaling pathways that promote
cell division)
§ During the translocation, the two
chromosomes break and exchange parts
so that the c-ABL gene is combined with
part of the BCR to produce a hybrid gene
that is constantly. expressed.
§ The BCR/ABL gene rearrangements
result in uncontrolled cell proliferation and
are found in the majority of CML patients.
• produced by mutations induced by carcinogenic
chemicals and radiation

Genetic mutations in the evolution of cancer.

As cells acquire an increasing number of mutations over time, they
develop more of the characteristics that allow them to evolve into
invasive cancer cells (numbers indicate mutations).
The BCR/ABL gene rearrangement characteristic of CML.
Human Viruses Associated With Cancer
Virus Cancer Associations B. Tumor-Associated Antigens (TAAs)
Epstein-Barr virus (EBV) Burkitt lymphoma • expressed in normal cells as well as in tumor cells
Hodgkin lymphoma
Leiomyosarcomas Categories of peptide TAAs that have been identified by the
Post-transplant Cancer Research Institute
lymphoproliferative disease 1. Shared TSAs
Nasopharyngeal carcinoma • aka cancer/testis antigens
Hepatitis B virus (HBV) Hepatocellular carcinoma • only normal cells in which they have been detected are
Hepatitis C virus (HCV) Hepatocellular carcinoma testicular germ cells
Human herpes virus 8 Kaposi sarcoma • become TAAs when the transformation process causes
(HHV-8) them to be expressed on tumors originating from other
Human papilloma virus Cervical cancer cell types
(HPV) Other genital and anal • identified on many tumors of epithelial or mesenchymal
cancers origin
Head and neck cancer o e.g. melanoma antigen gene (MAGE) proteins -
Human T-lymphotropic Adult T-cell leukemia or are expressed by melanoma tumors
virus I (HTLV-1) lymphoma

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2. Differentiation antigens • In order to improve the sensitivity and specificity of the
• expressed on immature cells of a particular lineage testing, multiple tumor markers could be examined or
o e.g. CD10 antigen (previously known as the testing for tumor markers could be combined with other
CALLA, or common acute lymphoblastic leukemia laboratory tests.
antigen)
• includes the oncofetal or embryonic antigens that are Prognosis Using Tumor Markers
normally expressed on developing cells of the fetus but • ↑ tumor marker at the time of diagnosis or increasing
not on cells in the adult levels of a tumor marker over time - indicate the presence
o e.g. carcinoembryonic protein (CEA), alpha- of an aggressive tumor that has metastasized and
fetoprotein (AFP), and prostate-specific antigen requires rigorous treatment
(PSA)
Monitoring Patient Response to Treatment Using Tumor
3. Overexpressed antigens Markers
• found in higher levels on malignant cells than on normal • level of a serum tumor marker = amount of tumor in the
cells patient
• levels up to 100 times greater than normal
o e.g. human epithelial growth factor receptor 2
(HER2) protein - a transmembrane receptor that
binds human epidermal growth factor

CLINICALLY RELEVANT TUMOR MARKERS

A. Tumor Markers
• biological substances that are found in increased amounts
in the blood, body fluids, or tissues of patients with a
specific type of cancer
• produced by the tumor itself or by the patient’s body in
response to the tumor or related benign conditions
• concentration of a tumor marker in the serum - depends
on the degree of tumor proliferation, the size of the tumor
mass, the proteolytic activities of the tumor, or release of
the marker from dying tumor cells
• can be proteins, carbohydrates, oncofetal antigens,
hormones, metabolites, receptors, or enzymes

Seven Characteristics of an Ideal Tumor Marker

Tumor marker analysis.
1. Be produced by the tumor itself or by the patient’s body A curve showing a sample scenario monitoring a cancer patient for
in response to the tumor tumor recurrence and for therapy efficacy using levels of a tumor-
2. Be secreted into a biological fluid, where it can be associated antigen.
inexpensively and easily quantified
3. Have a circulating half-life long enough to permit its C. Serum Tumor Markers
concentration to rise with increasing tumor load 1. Alpha-Fetoprotein (AFP)
4. Increase to clinically significant levels above the
• 70,000 MW glycoprotein
reference level while the disease is still treatable
• similar to albumin in its physical and chemical properties
5. Have a high sensitivity; in other words, it should easily
• classified as an oncofetal antigen because it is
detect the majority of individuals in the population who
synthesized by the fetal liver and yolk sac and is abundant
have a particular cancer
6. Have a high specificity; in other words, the marker • in fetal serum
should be absent from, or present at background levels • declines to low levels (10–20 ug/L) by 12 months of age
in all individuals without the malignant disease in • s marker to detect abnormalities in the fetus
question to minimize false-positive test results
Elevated AFP
B. Clinical Uses of Tumor Markers: Benefits and a. Primary hepatocellular carcinoma (HCC)
Limitations • most widely used tumor marker for HCC
Screening for Tumor Markers • screening for HCC with AFP is routinely performed in high
• can be detected by a simple blood test prevalence areas of the world such as China and
• effectiveness of a tumor marker in screening for cancer Southeast Asia
depends on the sensitivity and specificity of the marker, • isoform AFP-L3 - has a stronger correlation with HCC
as well as the cancer’s prevalence in the population • Other supporting tests: DCP (des-γ- carboxy-
• most effective when it is conducted in populations at a prothrombin), the liver enzyme ALT (alanine
high risk for developing the disease, such as certain aminotransferase), and platelet count
ethnic groups or those with a family history for a particular • ↑ levels: poor prognosis in patients with HCC
type of cancer
b. Nonseminomatous testicular cancer (NSGCT)
Diagnosis Using Tumor Markers • alongside with human chorionic gonadotropin and lactate
dehydrogenase (LDH)
• Follow-up staining of the biopsy for tumor markers could
help determine the neoplasm’s tissue origin

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c. Other types of cancers • first discovered in semen, where its function is to regulate
d. Nonmalignant conditions: pregnancy and hepatitis the viscosity of the seminal fluid to facilitate mobility of the
e. Open neural tube defects such as spina bifida - serum sperm cells.
or amniotic fluid of a pregnant woman • specific for prostate tissue, it is not specific for prostate
cancer
Decreased AFP • Levels
a. Good response to therapy o Normal total PSA value: 0 to 4.0 ng/mL
b. Down syndrome - serum or amniotic fluid of a pregnant o 4.0 ng/mL - prostate biopsy is recommended
woman • free PSA and the naturally occurring PSA-α-1-
antichymotrypsin complex, in addition to total serum PSA
2. Cancer Antigen 125 (CA 125) - increases the specificity of testing because the
• large, heavily glycosylated, mucinlike protein that is a proportion of free PSA is higher in benign conditions,
marker for ovarian cancer whereas the proportion of complexed PSA is greater in
• not unique to ovarian tumors because it is also found in prostate cancer
the normal ovary as well as other tissues, including the • Because free PSA quickly degrades at temperatures
endocervix, endometrium, fallopian tubes, pleura, above 4°C, it is important to perform testing within 3
pericardium, peritoneum, and epithelial tissues of the hours of sample collection or to store the sample at –70°C
colon, pancreas, lung, kidney, prostate, breast, stomach, if a longer time interval is required.
and gallbladder • PSA velocity (PSAV)
• best marker for ovarian cancer o rate of increase in PSA values over time
o PSA concentration divided by the number of years
Elevated CA125 spanning the interval between sequential tests
• >35 kU/L - above normal (reported as ng/mL/year).
• ↑ = recurrence of the disease o >0.75 ng/mL/year - prostate cancer
• >65 kU/L - poor prognosis • PSA density (PSAD)
o an increase in serum PSA is more likely to be
3. Carcinoembryonic Antigen (CEA) caused by the occurrence of cancer in a man with
• glycoprotein a small prostate gland versus a large prostate
• mw 180,000 to 200,000 gland
• was the first oncofetal antigen to be discovered o calculated as the ratio of total PSA to the prostate
gland volume
• most widely used marker for colorectal cancer
• Recommended: obtain a baseline CEA value from the
Elevated PSA
laboratory just before therapy, followed by CEA testing
• Prostate cancer
every 1 to 3 months during active treatment
• Benign prostatic hyperplasia (BPH) - enlargement of the
• not recommended for colon cancer screening because of
prostate gland that commonly occurs as men age, or
its low sensitivity and specificity in this situation
prostatitis, an inflammation of the gland occurring as a
result of infection or irritation
Elevated CEA
• If samples are collected shortly after ejaculation, digital
• Colorectal cancer
rectal examination (DRE), or prostate manipulation
• Cigarette smoking

4. Human Chorionic Gonadotropin (hCG) LABORATORY DETECTION OF TUMORS

A. Tumor Morphology
• pregnancy hormone
• Tumor marker antibodies, special stains, and nucleic acid
• rises during the first few weeks of gestation, when it can
probes - applied to the slides to enhance visible features.
be detected in the blood and urine of pregnant women
• choriocarcinoma - rare type of cancer that is caused by
B. Immunohistochemistry
malignant transformation of the trophoblast cells
• uses labeled antibodies to detect tumor antigens in
• 45,000 MW glycoprotein
formalin fixed or frozen tissue sections of tumor biopsy
• composed of an α subunit, which is shared by luteinizing material
hormone (LH), follicle-stimulating hormone (FSH), and
• formalin-fixed sections - must be treated with heat to
thyroid-stimulating hormone (TSH), and a β subunit that is
make the antigen epitopes accessible
unique to hCG
• The first step in immunohistochemistry is to broadly
classify the tumor into one of three major lineages:
Elevated hCG
o epithelial
• pregnancy
§ cytokeratins - intermediate filaments found
• malfunction of the testes in all types of epithelial; used as markers for
• gonadal suppression caused by chemotherapy tumors of epithelial lineage
o mesenchymal
5. Prostate-Specific Antigen (PSA) § vimentin - intermediate filament that is
• most widely used marker for prostate cancer found in most mesenchymal cells; used as
• 28,000 MW glycoprotein a marker to indicate the presence of a
• produced specifically by epithelial cells in the prostate sarcoma or melanoma, but its specificity is
gland low because it is expressed in some other
tumor types as well
o hematopoietic

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 § CD45 - WBC marker; used to identify • To confirm the presence of interfering antibodies, the
hematopoietic malignancies sample can be diluted and the linearity of the results can
be analyzed. Specimens with interfering antibodies tend
C. Immunoassays for Circulating Tumor Markers to exhibit nonlinear behavior.
Factors are that related to the use of antibodies as reagents
1. Antibody reagents from different manufacturers can vary D. Molecular Methods in Cancer Diagnosis
greatly in terms of what they detect, particularly if Genetic Biomarkers
monoclonal antibodies are used. • used for prospective and postdiagnostic evaluation of
2. Antibodies will cross-react with similar structures, which • Drugs target tumors with alterations in the
is particularly problematic when the cross-reacting o BRAF gene (metastatic melanoma)
substances are present in excessive amounts, as can o KRAS gene (colorectal cancer)
occur with cancer. o EGFR gene (non-small cell lung cancer; NSCLC)
3. Antigen excess. By virtue of their unchecked growth and o ALK gene (NSCLC)
aggressive metabolism, some neoplasms may produce o HER2 gene (breast cancer)
massive amounts of tumor marker molecules. The o ESR1 and PGR genes (breast cancer)
excess of tumor antigen as compared with reagent • Methods used to amplify the sequence that potentially
antibody can result in a postzone effect. When the contains the genetic mutation of interest, allowing tiny
measurements exceed the linear range of reportable changes in the sequence to be detected by the
results, this phenomenon is called the high-dose hook differences in fragment sizes that can be visualized by gel
effect because of the shape of the curve that depicts the electrophoresis.
relationship of the concentration of the analyte and the
intensity of the reaction signal. Cytogenetics
4. Interference can be caused by the presence of • studies play a large role in the diagnosis and
endogenous heterophile, anti-animal, or autoantibodies
management of cancer
in the patient sample.
• Karyotype analysis - used for many years to detect the
chromosomal abnormalities associated with many
cancers
• FISH - allowed chromosomal abnormalities to be
characterized more precisely at a molecular level;
interphase cells from the patient’s tumor are incubated
with fluorescent-labeled nucleic acid probes that are
complementary to the sequence of interest
• Each cell should have two red signals and two green
signals.
o If a translocation has occurred, a fusion probe
signal is generated, in which the red signal is
adjacent to the green signal, producing a yellow
color.

Microarrays
• test for panels of markers, rather than individual mutations
• single-stranded DNA or RNA from the tumor is tagged
with a fluorescent label and incubated with known nucleic
acid sequences that have been spotted onto different
areas of a membrane

Next Generation Sequencing (NGS)

• sequenced simultaneously in just a few hours to identify
genetic variations
• playing a major role in generating an enormous volume of
data for The Cancer Genome Atlas (TCGA)

Proteomics
• analysis of the entire protein complement of a cell
population
• done through the use of two-dimensional electrophoresis
coupled with tandem mass spectrometry (MS/MS),
surface-enhanced laser desorption/ionization mass
spectrometry (SELDI-TOF), or more recently, antibody
arrays
• Advantage: do not require fractionation or depletion of
high abundance proteins to detect proteins that are
present in lower concentrations

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 INTERACTIONS BETWEEN neoplasm and tumor growth progresses, even in the
THE IMMUNE SYSTEM AND TUMOR presence of anti-tumor immune responses.
A. Introduction
Theory of immunosurveillance
• states that the immune system continually patrols the
body for the presence of cancerous or precancerous cells
and eliminates them before they become clinically evident

B. Immune Defenses Against Tumor Cells

Immune defenses against tumor cells (MHC-R = MHC restricted

killing, MHC-unR =MHC unrestricted killing).

C. Innate Immune Responses

• NK cells and possibly macrophages - key cells involved in Relationship between the immune system and cancer:
innate immune responses to tumors Immunoediting and mechanisms of tumor escape.

D. Adaptive Immune Responses IMMUNOTHERAPY

• cytotoxic T lymphocytes (CTLs) - mediates the primary
A. Introduction
mechanism of adaptive immunity against tumors
• also known as biological therapy or biological response
modifier therapy, is to harness the ability of the immune
IMMUNOEDITING AND TUMOR ESCAPE system to destroy tumor cells
A. Elimination
• If the immunologic mechanisms involved in B. Active Immunotherapy and Cancer Vaccines
immunosurveillance are highly effective, they will likely • In 1891, the bone sarcoma surgeon, William Coley, began
result in complete elimination of the tumor. the first systematic study of immunotherapy.65 In his
• If the immune responses are not completely effective, review of the literature, he noted that cancer patients who
some of the tumor cells will remain in the body. developed an infection after surgery experienced tumor
• The immunoediting hypothesis suggests that these cells regression and had a better prognosis than patients who
will then enter the equilibrium phase. did not acquire an infection. Inspired by this knowledge,
he decided to inject one of his cancer patients with
B. Equilibrium Streptococcus pyogenes bacteria. To his amazement, the
• In this phase, tumor cells are thought to enter a state of patient’s tumor shrank and the patient became cancer
dynamic equilibrium with the immune system, which free.
keeps the altered cells under control so that they are not • Now, Coley’s toxins are illegal.
clinically evident. • The development of prophylactic cancer vaccines offers
• During this period, tumor cells may remain dormant or an effective approach to active immunotherapy. These
evolve slowly over time. vaccines have been generated for the purpose of
• Mutations can occur in the genetically unstable preventing virus-associated cancers.
transformed cells. Under selective pressure from • Unlike vaccines for infectious diseases, which are used to
immunologic forces of attack by cells in the tumor prevent infection, most cancer vaccines are
microenvironment, some of the tumor cells may develop immunotherapeutic, being administered after the disease
into genetic variants that are resistant to immune has occurred. They are frequently given to patients in the
defenses. advanced stages of disease when other treatment options
• These cells move past the equilibrium phase and enter have been exhausted.
the escape phase.
C. Passive Immunotherapy
C. Escape • involves the administration of soluble components of the
• During this phase, the balance between immunologic immune system to boost the immune response
control and tumor development is tipped in favor of the

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Cytokines gastroesophageal
• small proteins that play an important role in regulating tumors with
immune responses by serving as chemical messengers overexpressed
that affect the interactions between cells of the immune HER2
system Antigens Inhibit formation Bevacizumab, a
• Because chemotherapy drugs inhibit cell division, they involved in of blood vessels MAb directed
often adversely affect the development of hematopoietic angiogenesis necessary for against vascular
stem cells in the bone marrow, resulting in decreased delivery endothelial
production of WBCs, red blood cells (RBCs), and of oxygen and growth factor
platelets. Hematopoietic growth factors, also known as nutrients to the (VEGF); for
colony stimulating factors, can be administered to patients tumor treatment of
to help them recover from or prevent these toxicities. glioblastoma,
o IFN-α - most commonly used IFN in cancer colon, lung, and
therapy and has been approved by the FDA for the renal cancers
treatment of several types of cancer, including Molecules Enhance anti- Ipilimumab, a
malignant melanoma, hairy cell leukemia, chronic that block tumor-specific MAb directed
myeloid leukemia, and Kaposi’s sarcoma. T-cell activation T-cell responses against CTLA-4
o IL-2 - systemic administration of IL-2 as and proliferation by preventing T- (cytotoxic T-
immunotherapy was limited because of its short by binding to cell lymphocyte
half-life (fewer than 10 minutes) and serious molecules on inhibition antigen 4); for
adverse effects, including vascular leakage antigen- treatment of
syndrome, marked fluid retention, and shock presenting metastatic
cells melanoma
Monoclonal Antibodies Nivolumab and
• derived from a single clone of cells, providing for an Lambrolizumab,
abundant source of highly specific antibodies directed MAbs directed
toward one particular epitope of an antigen against PD-1
• cancer immunotherapy have been directed against seven (programmed
major categories of antigens: CD antigens, glycoproteins, death-1); used to
glycolipids, carbohydrates, vascular targets, stromal and treat melanoma,
extracellular antigens, and growth factors. colon cancer.
and other tumors
Approaches to Cancer Immunotherapy Using
Monoclonal Antibodies Antibody–drug Deliver potent Brentuximab
Target Mechanism of Examples conjugates toxic vedotin, an
of Therapy Action (immunotoxins) molecules immunotoxin
directed against directly to directed against
Surface Opsonization Rituximab, a
TSAs tumor cells the CD30
antigens Complement- MAb* directed
antigen; used to
on tumor mediated against the CD20
treat Hodgkin
cells cytotoxicity antigen on B
lymphoma and
ADCC cells; used
systemic
to treat B-cell
anaplastic
neoplasms
large cell
Alemtuzumab, a
lymphoma
MAb directed
Trastuzumab-
against mature
DM1, an
lymphocyte
immunotoxin
antigen,
directed against
CD52; used to
the HER2
treat chronic
antigen;
lymphocytic
for treatment of
leukemia and T-
HER2-positive
cell lymphomas
metastatic breast
Cell surface Block signaling Panitumumab, a
cancer
receptors pathways MAb directed
*MAb = monoclonal antibody
involved against epidermal
in cell growth factor
D. Adoptive Immunotherapy
proliferation receptor
and survival (EGFR), used to • Scientists have reasoned that because cell-mediated
treat colorectal immunity is so important in defense against tumors,
cancer transfer of cells of the immune system to cancer patients
Trastuzumab, a may effectively assist them in eliminating tumor cells.
MAb directed • These scientists isolated lymphocytes from surgically
against HER2, removed tumors of patients with metastatic melanoma
used to treat and grew them in the laboratory in the presence of IL-2.
breast and They found that these cells, referred to as tumor-

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 infiltrating lymphocytes (TILs), demonstrated potent c. can also be elevated in conditions other than the
cytolytic activity against autologous melanoma cells. cancer.
• Subsequent modifications of technique resulted in d. vary too much between patients belonging to
significantly improved patient outcomes. Instead of different ethnic populations.
administering the entire population of TILs, cells are 5. Both AFP and hCG exhibit serum elevations in
subcultured and individually tested for their reactivity to a. pregnancy.
the tumor. b. ovarian germ cell carcinoma.
c. nonseminomatous testicular cancer.
d. all of the above.
6. Suppose a patient with ovarian cancer had a serum CA
125 level of 50 kU/L at initial diagnosis. After her tumor
was surgically removed, her CA 125 level declined to 25
kU/L. She received chemotherapy drug #1; after 1 year,
her CA 125 level was 40 kU/L. She was then given
chemotherapy drug #2 and her CA 125 level rose to 60
kU/L. These results indicate that
a. surgery was effective in removing the patient’s
tumor.
b. chemotherapy drug #1 was more effective than
chemotherapy drug #2.
c. both chemotherapy drug #1 and chemotherapy
drug #2 were effective.
d. neither chemotherapy drug #1 nor
chemotherapy drug #2 were effective.
7. All of the following are recommended for cancer
screening in the groups indicated except
a. CA 125/women of reproductive age.
b. AFP/subjects at high risk for liver cancer.
c. PSA/men over 50 with at least 10 years of life
Adoptive immunotherapy with tumor-infiltrating lymphocytes (TILs).
The patient’s tumor is surgically removed and cut into fragments, expectancy.
which are cultured in vitro with IL-2. The cultures are screened for d. none of the above.
lymphocytes with potent anti-tumor activity. Positive cultures are 8. The best use of serum tumor markers is considered to
expanded further in the presence of IL-2 and are infused into the be in
cancer patient. Before infusion, the patient has been treated with high- a. screening for cancer.
dose chemotherapy or radiation to deplete immunosuppressive cells. b. initial diagnosis of cancer.
c. monitoring patients undergoing cancer
treatment.
Review Questions d. determining patient prognosis.
1. How can normal cells become malignant? 9. In order to use a tumor marker to monitor the course of
a. Overexpression of oncogenes
the disease, which of the following must be true?
b. Underexpression of tumor suppressor genes
a. The laboratory measures the marker with the
c. Viral infection
same method over the entire course of the
d. All of the above
patient’s treatment.
2. Which of the following best summarizes the concept of
b. The marker must be released from the tumor or,
tumor development via immunoediting?
because of the tumor, into a body fluid that can
a. Tumor cells produce cytokines that are toxic to T be obtained and tested.
cells. c. The marker’s half-life is such that the marker
b. Tumor cells that can escape the immune system
persists long enough to reflect tumor burden but
have a growth advantage over tumor cells that clears fast enough to identify successful
are destroyed during immunosurveillance. therapy.
c. T-cell activity causes an increase in MHC
d. All of the above.
expression on tumor cells that allows them to
10. Which of the following markers could be elevated in
escape the immune system. nonmalignant liver disease?
d. Secreted tumor-associated antigen saturates T- a. AFP
cell receptors and makes T cells incapable of
b. CEA
binding to tumor cells.
c. CA 15-3
3. Which of the following is an example of a tumor-specific d. All of the above
antigen?
11. Each of the following markers is correctly paired with a
a. BCR/ABL fusion protein
disease in which it can be used for patient monitoring
b. CEA except
c. CA 125 a. CEA/choriocarcinoma.
d. PSA b. CA 15-3/breast adenocarcinoma.
4. Most tumor markers are not used to screen the general
c. CA 125/ovarian adenocarcinoma.
population because they d. CA 19-9/pancreatic adenocarcinoma.
a. cannot be inexpensively quantified. 12. Which of the following is a marker used in
b. do not rise to high enough levels in the presence immunohistochemical staining to identify tumors of
of cancer.
epithelial origin?

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 a. Cytokeratins
b. Vimentin
c. CD45
d. CD10
13. Which of the following assays would you recommend to
test for chromosomal rearrangements such as the
BCR/ABL translocation seen in CML?
a. PCR
b. FISH
c. Microarray
d. Next generation sequencing
14. Innate immune responses thought to be involved in
defense against tumors include
a. NK cell-mediated apoptosis.
b. MHC I-restricted T-cell–mediated destruction.
c. ADCC.
d. all of the above.
15. A woman with breast cancer is treated with a
monoclonal antibody to HER2. This is an example of
a. a cancer vaccine.
b. an immunotoxin.
c. passive immunotherapy.
d. active immunotherapy.

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Christine Joy R. Timbang | 0963-467-6983
IMMUNOSEROLOGY 17.

Immunoproliferative Disease
OUTLINE § plasma cell leukemia - plasma cells may be
I. Lymphoid Malignancies found in the blood late in the course of
A. Definitions myeloma
II. Malignant Transformation of Hematologic Cells • Monoclonal gammopathy of
A. Cell Properties undetermined significance (MGUS) -
B. Genetic Changes premalignant condition that can
III. Classification of Hematologic Malignancies develop into multiple myeloma,
A. French-American-British (FAB) Cooperative Waldenström macroglobulinemia, or
Group other lymphoproliferative disorders
B. Revised European American Lymphoma over time.
(REAL)
IV. Leukemias MALIGNANT TRANSFORMATION OF
A. Introduction HEMATOLOGIC CELLS
B. Acute Lymphocytic Leukemia (ALL) A. Cell Properties
C. Chronic Lymphocytic Leukemia or • Hematologic malignancies are characterized by excessive
Lymphoma (CLL) accumulation of cells in the blood, bone marrow, or other
D. Hairy Cell Leukemia lymphoid organs.
V. Lymphomas • This accumulation may occur because of
A. Hodgkin Lymphoma (HL) o rapid proliferation of the cells (i.e., excess
B. Non-Hodgkin Lymphoma (NHL) production); or
VI. Plasma Cell Dyscrasias o failure of the cells to undergo apoptosis (a normal
A. Introduction physiological process of cell death).
B. Monoclonal Gammopathy of Undetermined
Significance (MGUS) B. Genetic Changes
C. Multiple Myeloma • Malignancies are generally multifactorial in origin.
D. Waldenström Macroglobulinemia
• Alterations in proto-oncogenes can convert them into
E. Heavy-Chain Diseases oncogenes, which are involved in malignant
VII. Role of the Laboratory In Evaluating
transformation.
Immunoproliferative Diseases
• The genetic alterations in malignant cells of hematopoietic
A. Introduction
origin include point mutations involving a change in a
B. Immunophenotyping by Flow Cytometry
single nucleotide base, duplications or deletions of
C. Evaluation of Immunoglobulins
specific genes, and chromosome translocations in which
D. Serum Protein Electrophoresis (SPE)
two different chromosomes break apart and exchange
E. Immunofixation Electrophoresis (IFE)
genetic material.
F. Serum Free Light Chain Analysis (sFLC)
G. Evaluation of Genetic and Chromosomal
Abnormalities CLASSIFICATION OF HEMATOLOGIC
MALIGNANCIES
LYMPHOID MALIGNANCIES A. French-American-British (FAB) Cooperative Group
A. Definitions • consensus criteria for leukemias and myelodysplastic
• Lymphoid malignancies - classified as leukemias, syndromes
lymphomas, and plasma cell dyscrasias
o Leukemias - malignant cells are primarily present in B. Revised European American Lymphoma (REAL)
the bone marrow and peripheral blood • classification for leukemias and lymphomas
o Lymphomas - malignant cells arise in lymphoid • widely accepted system is considered the “gold standard”
tissues, such as the lymph nodes, tonsils, or spleen in the classification of tumors for diagnosis and
§ There can be an overlap between the sites determination of appropriate therapy
affected by leukemias and lymphomas, • classifies hematologic malignancies into 12 major groups
especially when the malignancy is far and numerous subgroups.
advanced. However, it is generally most • These groupings are based on cell lineage; specific
useful to classify the malignancy according to cancers are further defined by their immunologic markers
the site where it first arose, rather than the and genetic features, as well as their morphological and
sites it can ultimately involve. cytochemical staining properties
o Plasma cell dyscrasias (disorders)
§ include multiple myeloma and Waldenström
macroglobulinemia
§ commonly involve the bone marrow, lymphoid
organs, and other nonlymphoid sites
§ considered biologically distinct and are not
classified as either leukemias or lymphomas

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 LEUKEMIAS o hyperdiploidy - malignant cells contain more than
A. Introduction 46 chromosomes; associated with a good
Two groups based on the cell type from which they prognosis
originated o hypodiploidy - poorer prognosis
1. Myelogenous leukemias 3. T-cell ALL
4. mature B-cell ALL - rare
• derived from the common myeloid precursor and
encompass the granulocytic, monocytic, megakaryocytic,
C. Chronic Lymphocytic Leukemia or Lymphoma (CLL)
and erythrocytic leukemias
• group of diseases almost exclusively of B-cell origin
2. Lymphocytic leukemias • include chronic lymphocytic leukemia (CLL) and small
• originate from mature lymphocytes or their precursors lymphocytic lymphoma (SLL)
o WHO considers CLL and SLL a single disease
Each of the two groups of leukemias can be further divided with different clinical presentations.
into acute or chronic types: o Both reveal the B-cell marker CD19 but weakly
express CD20.
1. Chronic leukemias
• common hematopoietic malignancy that involves the
• usually slowly progressive and compatible with extended
expansion of a clone of B cells that have the appearance
survival.
of small mature lymphocytes
• generally not curable with chemotherapy
• occurs in patients over 50 years of age with a two-to-one
male-to-female predominance
2. Acute leukemias
o most common leukemia in adults
• generally much more rapidly progressive but have a
• Laboratory findings:
higher response rate to therapy. o ↑ peripheral blood lymphocyte count
o Acute lymphoblastic leukemias - characterized by
o Anemia and thrombocytopenia - absent at the time
the presence of lymphoblasts in the peripheral
of diagnosis.
blood.
o ↑ malignant lymphocytes
o replacement of normal elements in the bone
marrow → anemia and thrombocytopenia
o Lymph node enlargement

D. Hairy Cell Leukemia

• rare, slowly progressive disease characterized by
infiltration of the bone marrow and spleen by leukemic
cells without the involvement of lymph nodes
• four-to-one male predominance and is seen in individuals
over 20 years of age
• clinical presentation can resemble several other B-cell
neoplasms including CLL, SLL, and splenic marginal zone
lymphoma
• malignant lymphocytes are round and often have irregular
“hairy” cytoplasmic projections from their surfaces
o not evident on bone marrow or spleen
preparations
o Nuclei: oval and occupy a large percentage of the
cell volume
• Markers
B. Acute Lymphocytic Leukemia (ALL) o CD19, CD20, and CD22 - malignant cells mature
• also known as acute lymphoblastic leukemia B-cell markers
• characterized by the presence of very poorly differentiated o CD103 - highly specific and sensitive for hairy cell
precursor cells (blast cells) in the bone marrow and leukemia
peripheral blood o CD123 - bright staining; cells characteristically
• infiltrate soft tissues → organ dysfunction contain acid phosphatase isoenzyme, which can
• usually seen in children between 2 and 5 years of age be detected histochemically and provides
o most common form of leukemia in this age group resistance to tartrate treatment
• treatable disease with a remission rate of 90% and a cure • Polymerase chain reaction (PCR) for the detection of
rate of 80% in children mutated gene BRAF-V600E - sensitive and specific test
• B-cell origin, t(12:21)(p13;q22) or TEL-AML-1 - associated for the diagnosis of hairy cell leukemia
with an excellent prognosis in children

Four types of ALL

1. CALLA (CD10)-expressing precursor B-cell ALL - most
common
2. pre–B-cell ALL without CALLA (CD10)
• second most common

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 LYMPHOMAS Hodgkin RS cells
A. Hodgkin Lymphoma (HL) • interact with numerous cells including CD4+ and CD8+ T
• one of the most common lymphomas cells, B cells, plasma cells, macrophages, and others
• often-curable disease occurs both in young adults and the • secrete cytokines and chemokines, some of which attract
elderly these cells to the tumor
• Peripheral lymph nodes are primarily involved, although • presence of this mixed population of cells is unique
numerous organs such as the liver, lung, and bone among lymphomas. The nontumor cells often account for
marrow can be affected 99% of the cells in the tumor
• HL often have elevated levels of antibody to Epstein-Barr • not usually found in the peripheral bloodstream
virus (EBV)
• infectious mononucleosis - associated with an increased B. Non-Hodgkin Lymphoma (NHL)
risk of H • Immunosuppression seems - greatest risk factor for NHL
• Other conditions associated with increased risk for NHL
Divided into: include certain autoimmune diseases, congenital
1. nodular lymphocytic-predominant HL (NLPHL) immunodeficiency disorders, organ transplantation, and
• characterized by large lymphocyte-predominant cells exposure to certain infectious agent

2. classic HL B-cell lymphomas

• includes nodular sclerosis, mixed cellularity, lymphocyte- • generally begin in the germinal centers of lymph nodes
rich, and lymphocyte-depleted HL
• characterized by the presence of Hodgkin and Reed- 1. Diffuse large B-cell lymphoma (DLBCL)
Sternberg (RS) cells in affected lymph nodes and • most common NHL
lymphoid organs • a heterogeneous group of diseases characterized by
o RS cells diffuse growth of large atypical cells without a hallmark
§ typically large with a bilobed nucleus and two pattern of surface markers
prominent nucleoli • derive from rapidly dividing cells
§ gives the cell an “owl’s-eyes” appearance.
§ Hodgkin cells resemble RS cells except that 2. Follicular lymphoma
the nuclei are not bilobed and have a single • originates in the follicles of the lymphoid organs and is
nucleolus. characterized by a much more aggressive course than
DLBCL
REAL/WHO classification • often disseminated at the time of diagnosis; the spleen,
1. classic HL liver, and bone marrow are frequently involved
• RS cells all are CD30+ and about 80% of the cases are
CD15+ Three broad groups for prognostic purposes
• CD20 - weak or absent
• Some cells also express T-cell markers 1. Low-risk first group: CLL, follicular lymphomas, and
mucosa-associated lymphoid tissue (MALT)
• nodular lymphocytic-predominant HL (NLPHL) lymphomas.
o RS cells 2. Intermediate-risk second group: DLBCL and Burkitt
§ referred to as lymphocytic and histiocytic lymphoma.
cells 3. High-risk third group: mantle cell lymphoma and
§ rarely express CD30 lymphoblastic lymphoma
§ express the B-cell antigens CD19 and
CD20 Three characteristics usually identify lymphomas as having a
B-cell origin:
2. Nodular sclerosis HL 1. surface immunoglobulin, which is found on no other cell
• most common subtype type;
• characterized by infiltration of a mixture of normal 2. other cell surface proteins such as CD19 and CD20 that
macrophages, lymphocytes, and granulocytes in affected are both sensitive and specific for B cells; and
tissues along with small numbers of RS cells 3. rearranged immunoglobulin genes.
• has marked fibrosis, dividing affected lymph nodes into
T-cell and NK-cell lymphomas
nodules
• morphologically not clearly malignant, no easy way exists
3. Mixed cellularity HL to assay their clonality
• a mixed infiltrate of normal cells but with less fibrosis and
greater numbers of RS cells Ann Arbor classification system
• Staging is based on the number of lymph nodes affected,
4. Lymphocyte-depleted HL their location, and if extranodal organs are involved
• diffuse fibrosis, few infiltrating normal cells, the greatest
number of RS cells
• worst prognosis compared with other HL subtypes

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 PLASMA CELL DYSCRASIAS Clinical Manifestations of Multiple Myeloma
A. Introduction 1. Skeletal
• characterized by the overproduction of a single • producing multiple lytic lesions, that often lead to bone
immunoglobulin component called a myeloma protein (M pain and pathological fractures
protein), or paraprotein, by a clone of identical plasma • Bone loss - caused by a complex interaction between the
cells. myeloma cells and normal cells of the bone.
• M protein - may also be rarely associated with other • The myeloma cells trigger
lymphoproliferative disorders, such as NHL or primary o ↑ osteoclast activity
amyloidosis. o ↓ osteoblast activity
• Hypercalcemia - very common because the myeloma
B. Monoclonal Gammopathy of Undetermined promotes bone reabsorption
Significance (MGUS)
• common premalignant condition 2. Hematologic
• People with MGUS produce a monoclonal • failure of the bone marrow to produce a normal number of
immunoglobulin but do not have symptoms of organ hematopoietic cells because myeloma cells progressively
damage or other laboratory findings that are associated replace them
with multiple myeloma or the other plasma cell dyscrasias • anemia, thrombocytopenia, and neutropenia
• usually diagnosed incidentally when patients with various
nonspecific symptoms have laboratory testing such as 3. Immunologic
serum protein electrophoresis (SPE) • When immunoglobulin levels in the blood are sufficiently
• MGUS patients who high, they may cause the formation of rouleaux
o produce an IgG or IgA monoclonal Ig - typically • Deficiency of normal antibody responses and a higher
progress to multiple myeloma • incidence of infectious diseases
o produce an IgM monoclonal Ig can develop • Hyperviscosity
Waldenström macroglobulinemia or other o can develop when the level of M protein in the
lymphoproliferative disorders plasma is high
o produce monoclonal light chains can develop light o sometimes seen with an IgG3-producing myeloma
chain multiple myeloma, amyloidosis, or light chain because IgG3 is the largest of the IgG subclasses
deposition disease

International Myeloma Working Group (IMWG) has identified

three criteria that define the presence of MGUS:
1. a serum monoclonal protein concentration of less than 3
g/dL;
2. a plasma cell count of lower than 10% of the total cells
in the bone marrow; and
3. the absence of signs or symptoms associated with
multiple myeloma, known as the CRAB features
(increased serum calcium, renal failure, anemia, lytic
bone lesions)

C. Multiple Myeloma
• aka plasma cell myeloma
• a malignancy of mature plasma cells
• most serious and common of the plasma cell dyscrasias
• usually diagnosed in persons between 40 and 70 years of
age with a peak age of 65 years
• thought to be preceded by MGUS or smoldering multiple
myeloma (SMM)
• have excess plasma cells in the bone marrow, a
monoclonal immunoglobulin component in the plasma or
urine, and lytic bone lesions
• plasma cells infiltrating the bone marrow may be
morphologically normal or may show atypical or bizarre
cytological features.
• Malignant plasma cells phenotypically express CD38,
CD56, and CD138

Bence Jones proteins

• monoclonal light chains can be found in the blood, but are
rapidly excreted in the urine
• directly toxic to tubular epithelial cells and can damage
the kidneys by precipitating in the tubules, causing
intrarenal obstruction.

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 • macroprotein or IgM paraprotein
o elevated serum monoclonal protein
o migrates in the gamma region during SPE
o not specific for Waldenström macroglobulinemia
o behave as cryoglobulins
§ Cryoglobulins
• precipitate at cold temperatures and
can occlude small vessels in the
extremities in cold weather
• detected when a blood or plasma
sample is refrigerated in the clinical
laboratory
• Serum IgM levels
o 5,000 mg/dL - asymptomatic
o 500 mg/dL - significant bone marrow infiltration and
pancytopenia
• ↑ Serum β2–macroglobulin levels

E. Heavy-Chain Diseases
• rare B-cell lymphomas that are characterized by the
production of a monoclonal immunoglobulin (Ig) heavy
chain
• Genetic mutations in the affected B cells result in the
production of abnormal heavy chains that have lost part of
their CH1 or variable domain so they are incapable of
binding to Ig light chains.
• These diseases are classified according to the type of
heavy chain produced, which can be alpha (α), gamma
(γ), or mu (μ).

1. Alpha heavy-chain disease

• most common of the three types
• associated with poor hygiene, poor nutrition, and chronic
bacterial and parasitic infections
• lymphoma that involves the MALT and can occur as one
of three forms: gastrointestinal, respiratory, or
lymphomatous
• Because of their abnormal structure and tendency to
Criteria for the diagnosis of multiple myeloma polymerize, they may not be evident by SPE, which can
appear normal or demonstrate a broad band that migrates
• Plasma cells comprising greater than 10% of bone
to the α-2 or β region
marrow cells
• Evidence of end-organ damage such as bone marrow 2. Gamma chain disease
lesions (detectable by radiographs)
• very rare disorder
• Hypercalcemia
• heterogeneous and can present in one of three forms:
• Renal insufficiency
o disseminated lymphoma with lymphadenopathy
• Anemia
and generalized symptoms such as fever and
• Serum M protein - >3 g/dl or more
weight loss;
• Urinary M protein - >200 mg/day o localized disease with lymphoma limited to the
bone marrow; or
D. Waldenström Macroglobulinemia o localized disease involving areas outside of the
• lymphoplasmacytic lymphoma lymph nodes, such as the skin.
• malignant proliferation of IgM-producing lymphocytes • abnormal gamma chains tend to migrate to the β region
• malignant cells: B cells or plasma cells on SPE, where they may be masked by other proteins
o B cells
§ produce the B-cell markers CD19, CD20, 3. Mu heavy-chain disease
CD22, and CD79a
• extremely rare disorder
• lymphoproliferative lymphoma involving the bone marrow
• majority of patients also have a lymphoid malignancy that
with IgM paraprotein at any concentration
resembles CLL or SLL
• Clinical signs and symptoms - caused by infiltration of the
• half of patients have a normal SPE pattern, but IFE
malignant cells into the bone marrow, spleen, and lymph
typically reveals μ polymers of various sizes that are not
nodes with the overproduction of monoclonal IgM
associated with κ or λ light chains
• Signs and symptoms: weakness, fatigue, anemia,
bleeding, and occasionally plasma hyperviscosity.

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 ROLE OF THE LABORATORY IN EVALUATING antigens that they characteristically express. This
IMMUNOPROLIFERATIVE DISEASES difference can occur in any of the following ways:
1. There may be a gain of antigens not usually expressed
A. Introduction
by the normal cell type or lineage.
• suggested by a patient’s medical history and clinical 2. There may be abnormally increased or decreased levels
symptoms and confirmed by laboratory testing
of the antigens expressed by the malignant cells, or in
• begins with performance of a CBC and differential and some cases a complete loss of normal antigens.
examination of the cell populations on a peripheral blood 3. The malignant cells may express antigens at
smear inappropriate times during the maturation process.
4. There may be a homogeneous expression of antigens
B. Immunophenotyping by Flow Cytometry that are typically heterogeneously expressed by the
• analysis of cell surface marker expression commonly normal counterpart.
used in the diagnosis and classification of leukemias and
lymphomas C. Evaluation of Immunoglobulins
• Flow cytometry - ideal for fluids (such as blood, in which • The initial tests used to screen for the presence of a
cells are naturally suspende; useful for lymphoid tissues, monoclonal gammopathy are serum immunoglobulin
from which single-cell suspensions can be easily made) levels and SPE.
• Quantitative measurement of immunoglobulin levels in the
Markers Commonly Detected by Flow Cytometry in serum is routinely performed by nephelometric methods,
the Analysis of Hematologic Malignancies* or in smaller laboratories, by radial immunodiffusion (RID).
Cell Type Associated Markers • Because each plasma cell produces only one type of
T cells CD1, CD2, CD3, CD4, CD5, CD7, immunoglobulin, the persistent presence of an elevated
CD8, TCR alpha-beta, TCR gamma- amount of a single immunoglobulin class suggests
delta malignancy.
B cells CD10, CD19, CD20, CD22, CD23, • In contrast, an increase in the amount of total
CD79a, CD103, kappa (surface and immunoglobulin, without an increase in any one specific
cytoplasmic), lambda (surface and class, is characteristic of nonmalignant conditions such as
cytoplasmic) infections or autoimmune diseases.
Myeloid cells CD11b, CD13, CD14, CD15, CD33,
and monocytes CD64, CD117, myeloperoxidase D. Serum Protein Electrophoresis (SPE)
Miscellaneous CD11c, CD16, CD25, CD26, CD30,
CD34, CD38, CD41, CD42b, CD45,
CD56, CD57, CD61, HLA-DR,
glycophorin, TdT, CD123, CD138,
CD200

Surface Markers Characteristic of Selected

Leukemias and Lymphomas
Hematopoietic Characteristic
Malignancy Surface Markers*
Classic Hodgkin lymphoma CD15+ (most), CD30+,
CD3 +/–, CD20 – or weak,
CD45–
Nodular lymphocytic CD19+, CD20+, CD45+,
predominant Hodgkin CD15–, CD30–
lymphoma (NLPHL)
B-cell acute lymphocytic CD10+, CD19+, CD22+,
leukemia (B-ALL) CD34+, TdT+
T-cell acute lymphocytic CD1a+, CD2+, CD5+,
leukemia (T-ALL) CD7+, TdT+
Chronic lymphocytic CD5+, CD19+, CD20
leukemia (CLL) (weak+), CD23+
Hairy cell leukemia CD19+, CD20+, CD22+,
CD25+, CD103+,
CD123+
Multiple myeloma CD38+, CD56+, CD138+,
~20% are CD20+
Waldenström CD19+, CD20+, CD22+,
macroglobulinemia CD79a+, CD3–, CD5–,
CD10–, CD103–
* CD = cluster of differentiation, + = positive, – = negative, TdT =
Terminal deoxynucleotidyl transferase.

• Laboratorians and clinicians must recognize that

malignant cells can differ from their normal counterparts
(e.g., B-cell ALL versus normal B cells) in terms of the

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E. Immunofixation Electrophoresis (IFE)
Serum Immunofixation Electrophoresis
• In IFE, serum samples are electrophoresed in six
separate lanes on an agarose gel and specific antisera
are applied directly to the lanes. The antisera used are
selected to detect the most common M proteins and are
directed against:
o Whole human serum (lane 1)
o Anti-γ (to detect IgG) (lane 2)
o Anti-α (to detect IgA) (lane 3)
o Anti-μ (to detect IgM) (lane 4)
o Anti-κ (to detect kappa light chains) (lane 5)
o Anti-λ (to detect lambda light chains) (lane 6)

Principle of immunofixation electrophoresis (IFE).

IFE involves two major steps. In step 1, proteins in the clinical sample
(serum, urine, or CSF) are applied to an agarose gel and separated
according to their surface charge under the influence of an externally
applied electrical field. At a pH of 8.6, the proteins acquire a negative
charge and move toward the positively charged anode. Five major
protein fractions result: γ globulins, β globulins, α1 globulins, α2
globulins, and albumin. Immunoglobulins are located primarily in the
γ globulin fraction, but can also migrate into the β globulin fraction. In
step 2, specific antisera are added to each one of the lanes and react
with their corresponding protein to produce a precipitin band. A
protein fixative is added to lane 1, which binds to all of the major
protein fractions. The precipitin bands can be visualized after staining
the gel with a protein stain and destaining with acetic acid to remove
background color.

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Immunosubtraction
• variation of immunofixation G. Evaluation of Genetic and Chromosomal
• a sensitive procedure that uses capillary electrophoresis Abnormalities
to identify monoclonal immunoglobulin components Cytogenetics Characteristic of
• In this technique, antibodies to each heavy or light chain Selected Leukemias and Lymphomas
isotype are added to separate capillary runs of the Hematopoietic Characteristic
patient’s specimen. Malignancy Cytogenetics*
Burkitt lymphoma Most commonly t(8;14)
[IgH/myc];
also t(2;8) and t(8;22)
Follicular lymphoma t(14;18) [IgH/Bcl2]
Mantle cell lymphoma t(11;14) [IgH/cyclin D1]
B-cell acute t(12;21) [TEL-AML-1]
lymphoblastic
leukemia (B-ALL)
Chronic myelogenous t(9;22) [bcr/abl]
leukemia (CML)
*t = translocation; IgH = immunoglobulin heavy-chain gene.

Immunotyping of a monoclonal IgG, kappa protein.

Panel A: Serum protein electrophoresis. The arrow points to the M-
spike in the gamma region. Panel B: Electrophoresis with anti-alpha
heavy chain. This pattern is overlaid with the original serum protein FISH (fluorescence in situ hybridization) demonstrating a
electrophoresis. The patterns are identical. Panel C: Electrophoresis chromosomal translocation.
with anti-kappa light chain. The M-spike has disappeared, indicating In this assay, interphase cells were hybridized with molecular probes
the peak contains a kappa light chain (see arrow). Panel D: complementary to specific regions on chromosomes 9 and 22. The
Electrophoresis with anti-gamma heavy chain. The M-spike has green signal represents the BCR (breakpoint cluster region) on
disappeared, indicating the peak contains IgG (see arrow). Panel E: chromosome 22 and the red signal represents the ABL1 proto-
Electrophoresis with anti-mu heavy chain. This pattern is overlaid with oncogene on chromosome 9. A yellow signal represents the
the original serum protein electrophoresis. The patterns are identical. BCR/ABL1 fusion caused by a 9;22 chromosome rearrangement. (A)
Panel F: Electrophoresis with anti-lambda light chain. This pattern is A normal test result, showing two green signals representing two
overlaid with the original serum protein electrophoresis. The patterns copies of chromosome 22 and two red signals representing two
are identical. (Courtesy of Dr. Thomas Alexander.) copies of chromosome 9. (B) Result from a patient with a translocation
between chromosomes 9 and 22 [t(9;22)(q34.1;q11.2)]. The yellow
signal indicates the fusion between the BCR and ABL1 loci on the
Urine Immunofixation Electrophoresis (UPE)
rearranged chromosome 22, which is also known as the Philadelphia
• Some patients with these disorders produce an excessive chromosome. The smaller red signal detects the residual ABL1
amount of free monoclonal Ig light chains (i.e., Bence sequences present on the rearranged chromosome 9. This
Jones proteins). translocation is found in patients with CML (chronic myelogenous
• Because these proteins are rapidly cleared from the leukemia) as well as some individuals with AML (acute myeloid
circulation, they may not be detectable on serum IFE. leukemia) and ALL (acute lymphoblastic leukemia). (Courtesy of the
Cytogenetics Laboratory, SUNY Upstate Medical University.)
• Urine samples for UPE and IFE are typically collected
over a 24-hour period.

F. Serum Free Light Chain Analysis (sFLC)

• latex-enhanced immunoassays that measure free kappa
and lambda light chains in the serum.
• The assays employ polyclonal antibody reagents that
recognize a diverse range of FLC epitopes that are
normally hidden when the light chains are bound to heavy
chains in intact immunoglobulins.
• This allows for quantitative measurement of free κ and
free λ chain concentrations, as well as calculation of a κ/λ
ratio (normally, 0.26–1.65).
• An abnormal κ/λ ratio outside of the reference range,
along with an increase of either κ and λ, is a sensitive
indicator for the presence of a malignant plasma cell
clone, which is characteristic of a monoclonal
gammopathy.

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 c. an incurable, rapidly progressive course.
d. the presence of Reed-Sternberg cells in
lymph nodes.
6. Chronic leukemias are characterized as
a. usually being of B-cell origin.
b. being curable with chemotherapy.
c. usually occurring in children.
d. following a rapidly progressive course.
7. Which of the following is characteristic of heavy-chain
diseases?
a. Usually of B-cell origin
b. Rare lymphomas
c. Production of abnormal Ig heavy chains
d. All of the above
8. Flow cytometry results on a patient reveal a decrease of
cells with CD2 and CD3. What does this indicate?
a. Lack of B cells
b. Lack of T cells
c. Lack of monocytes
d. Lack of natural killer cells
9. Which of the following is true of Waldenström
macroglobulinemia but not multiple myeloma?
a. Hyperviscosity syndrome is often present.
b. A single protein-producing clone is elevated.
c. The cancerous cell is a preplasma cell.
d. Bence Jones proteins are present in the urine.
10. The presence of anemia, bone pain, thrombocytopenia,
and lytic bone lesions is suggestive of
a. Hodgkin lymphoma.
b. hairy cell leukemia.
c. chronic lymphocytic leukemia.
d. multiple myeloma.
11. The presence of an M protein on immunofixation
electrophoresis (IFE) is indicated by
a. broad, diffuse banding.
b. a narrow, discrete band.
c. a few well-defined bands in the IgG lane.
d. a single band at the point of application in all
of the lanes.
12. Surface immunoglobulin on a leukemic cell indicates
Review Questions a(n)
1. Bence Jones proteins consist of a. B cell.
a. monoclonal IgG. b. T cell.
b. IgG–IgM complexes. c. macrophage.
c. free κ or λ light chains. d. autoimmune disease.
d. free μ heavy chains. 13. Which of the following is not a requirement for urine
2. Which of the following would be the best indicator of a testing by IFE?
malignant clone of cells? a. Collection of a 24-hour sample
a. Overall increase in antibody production b. Concentration of the sample
b. Increase in IgG and IgM only c. Dilution of the sample
c. Increase in antibody directed against a d. Removal of sediment
specific epitope 14. Multiple myeloma is characteristically preceded by
d. Decrease in overall antibody production a. chronic hypogammaglobulinemia.
3. All of the following are features of malignancy except b. Helicobacter pylori infection.
a. excess apoptosis. c. non-Hodgkin lymphoma.
b. rapid proliferation. d. monoclonal gammopathy of undetermined
c. clonal proliferation. significance.
d. chromosomal mutations. 15. Which serum free light chain (sFLC) assay result
4. All of the following features are commonly used to indicates presence of a malignant plasma cell clone?
classify lymphoid neoplasms except a. An abnormal κ:λ ratio
a. cell of origin. b. A decrease in κ and λ concentrations
b. presence of gene translocations. c. A decrease in IgG, IgA, and IgM
c. exposure of the patient to carcinogens. concentrations
d. morphology or cytology of the malignant cells. d. An increase in immunoglobulin concentrations
5. Hodgkin lymphoma is characterized by over a 24-hour period
a. proliferation of T cells.
b. excess immunoglobulin production.

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Christine Joy R. Timbang | 0963-467-6983
IMMUNOSEROLOGY 18.

Immunodeficiency Diseases
OUTLINE CLINICAL EFFECTS OF PRIMARY
I. Introduction IMMUNODEFICIENCIES
A. Definitions A. Introduction
II. Clinical Effects of Primary Immunodeficiencies
III. The Nine Categories of Primary Immunodeficiencies
A. Introduction
B. Category 3: Predominantly Antibody
Deficiencies
C. Category 1: Combined Immunodeficiencies
D. Category 2: Combined Immunodeficiencies
with Associated or Syndromic Features
E. Category 4: Diseases of Immune
Dysregulation
F. Category 5: Congenital Defects of
Phagocyte Number, Function, or Both
G. Category 6: Defects in Innate Immunity
H. Category 7: Autoinflammatory Disorders
I. Category 8: Complement Deficiencies
IV. Laboratory Evaluation of Immune Dysfunction
A. Screening Tests
B. Confirmatory Tests
C. Newborn Screening for
Immunodeficiencies
D. Evaluation of Immunoglobulins
E. Bone Marrow Biopsy
F. Family History

INTRODUCTION
A. Definitions
Immunodeficiencies • affect one or more parts of the immune system
• disorders in which a part of the body’s immune system is • The types of infection or symptoms displayed by a patient
missing or dysfunctional. can give important clues regarding the specific
• decreased ability to defend themselves against infectious immunodeficiency present
organisms and are more susceptible to developing certain • Defects in humoral immunity (antibody production)
types of cancer o result in pyogenic (i.e., pus-forming) bacterial
infections
Primary immunodeficiencies (PIDs) o upper and lower respiratory tract
o Recurrent sinusitis and otitis media
• inherited dysfunctions of the immune system
o clinical course of viral infections in patients with
predominantly antibody deficiencies is not
Secondary immunodeficiency
significantly different from that in normal hosts
• e.g. acquired immunodeficiency syndrome (AIDS) - o hepatitis B
caused by the human immunodeficiency virus (HIV) § have a fulminant course
§ agammaglobulinemias: conditions in which
antibody levels in the blood are significantly
decreased
• Defects in T-cell–mediated immunity
o result in recurrent infections with intracellular
pathogens such as viruses, fungi, and intracellular
bacteria
o congenital T-cell deficiencies
§ develop mucocutaneous candidiasis, a
yeast infection that involves the skin, nails,
and mucous membranes
§ prone to disseminated viral infections,
especially with latent viruses such as
herpes simplex, varicella zoster, and
cytomegalovirus
o more susceptible to developing certain types of
cancer

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• Defects in neutrophil function o Therefore, deficiencies of immunoglobulins have
o reflected in recurrent pyogenic bacterial infections been termed agammaglobulinemias.
or impaired wound healing • The mechanisms of the agammaglobulinemias include
• Abnormalities in macrophage function genetic defects in B-cell maturation or mutations leading
o affects both the innate and the adaptive defenses to defective interactions between B and T cells.
because macrophages are involved in the • A wide range of immunoglobulin deficiency states have
nonspecific phagocytosis of microorganisms been reported and involve virtually all combinations of
during inflammation as well as in the processing of immunoglobulins and all degrees of severity.
antigens and their presentation to T cells in • In some cases, only a single isotype of one
humoral and cell-mediated immune responses immunoglobulin class is deficient, whereas all of the other
• Reduction in the macrophage population by splenectomy isotypes are normal. Only the more common and well-
o associated with an increased risk of overwhelming characterized syndromes are described here.
bacterial infection accompanied by septicemia • In evaluating immunoglobulin deficiency states, it is
• Deficiencies of complement components important to remember that blood levels of
o result in recurrent bacterial infections and immunoglobulins change with age.
autoimmune-type manifestations o The blood level of IgG at birth is about the same
• Persistent infections continuously stimulate the available as adult level, reflecting transfer of maternal IgG
immune cells or because a compensatory mechanism has across the placenta.
been activated to correct for the deficient immune function o The IgG level declines over the first 6 months of
• Defect in Th2 cell function life as maternal antibody is catabolized.
o deficiency in CD40L (a molecule involved in o Levels of IgA and IgM are very low at birth.
binding to cell receptors during T-dependent § The concentrations of all
immune responses) immunoglobulins gradually rise when the
o removes or creates an imbalance in the regulation infant begins to produce antibodies at a
of those immune responses few months of age in response to
environmental stimuli.
THE NINE CATEGORIES OF PRIMARY § IgM reaches normal adult levels first,
around 1 year of age, followed by IgG at
IMMUNODEFICIENCIES
about 5 to 6 years of age.
A. Introduction § In some normal children, IgA levels do
1. Category 1: Combined Immunodeficiencies not reach normal adult values until
2. Category 2: Combined Immunodeficiencies With adolescence.
Associated or Syndromic Features § Therefore, it is important to compare a
3. Category 3: Predominantly Antibody Deficiencies child’s immunoglobulin levels to age-
4. Category 4: Diseases of Immune Dysregulation matched reference ranges.
5. Category 5: Congenital Defects of Phagocyte Number,
Function, or Both
Characteristics of Selected Predominantly Antibody
6. Category 6: Defects in Innate Immunity
Deficiencies (Category 3)
7. Category 7: Autoinflammatory Disorders Condition Deficiency Level of Presentation
8. Category 8: Complement Deficiencies Defect
9. Category 9: Phenocopies of Primary Transient All Slow 2–6
Immunodeficiencies Hypogamma- antibodies; developme months;
globulinemia especially nt of helper resolves by
• In the past, the immunodeficiencies have been broadly of infancy IgG function in 2 years
classified as defects in T cells, B cells, phagocytes, some
complement proteins, and other components of the innate patients
immune system. Selective IgA IgA; some IgA-B cell Often
• As scientific knowledge has been gained about the deficiency also with differentiati asymptoma
complexity of these disorders, experts have recognized reduced on tic
that such a broad classification is overly simplistic. IgG2
• In 2014, the International Union of Immunologic Societies Btk deficiency All Pre–B-cell Infancy
(IUIS) updated their classification of PIDs by grouping antibody differentiati
them into nine different categories based on their isotypes on
characteristic clinical features, immunologic defects, and reduced
genetic abnormalities.
• The IUIS has also published diagnostic flow charts to aid Common Reduced B cell; Usually
in classifying patients into a disease entity based on variable antibody; excess T 20–30
clinical symptoms and laboratory results immunodeficie many suppressio years
• Although the PID diseases are separated into these ncy different n of age
categories, some diseases are listed in more than one combinatio
category because they possess overlapping features. ns

B. Category 3: Predominantly Antibody Deficiencies Isolated IgG Reduced Defect of Variable

• This category encompasses conditions in which the main subclass IgG1, isotype with the
characteristic is low levels of serum immunoglobulins. deficiency IgG2, differentiati class
• Immunoglobulins migrate in the “gamma region” of the IgG3, or on
serum protein electrophoretic profile IgG4

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 and degree streptococci, meningococci, and
of Haemophilus influenzae
deficiency o Some patients also have a susceptibility to
CD154 Reduced B-cell Variable certain types of viral infections, including vaccine
deficiency IgG, IgA, switching associated poliomyelitis
IgE, with • X-linked hypogammaglobulinemia results from arrested
elevated differentiation at the pre–B-cell stage, leading to a
IgM complete absence of B cells and plasma cells
• The differentiation stops at T-Beta cell stage due to the
Transient Hypogammaglobulinemia of Infancy deficiency of Bruton’s tyrosine kinase
o This enzyme is responsible for the VH gene
• Late increase in the levels of Immunoglobulins
arrangement
o IgM and IgA are not affected
o IgG o Lack of the enzyme apparently causes a failure of
§ 2SD levels below normal immunoglobulin VH gene rearrangement
§ Lasts 9 to 15 months of age • Agammaglobulinemia: genetic effect in the B-cell
§ Pyogenic sinopulmonary and skin infection maturation or mutation leading to the defective interaction
• At birth, IgG is about the same as adult level cause of between B- and T-cell
maternal transfer but this decreases due to catabolism • The syndrome can be differentiated from transient
over 2-3 months hypogammaglobulinemia of infancy by the absence of
CD19+ B cells in the peripheral blood
• On the few months of age its own antibodies rises as a
response to environmental stimuli • Treatment: IM/IV Ig
• All infants experience low levels of immunoglobulins at o intramuscular or intravenous immunoglobulin
o vigorous antimicrobial treatment of infections
approximately 5 to 6 months of age; however, in some
babies the low levels persist for a longer time.
Selective IgA deficiency
• Because these children do not begin synthesizing
immunoglobulins promptly, they can experience severe • Most common congenital immunodeficiency
pyogenic sinopulmonary and skin infections as protective • Impaired differentiation of lymphocytes to become IgA-
maternal IgG is cleared. producing PCs
• Cell-mediated immunity is normal and there may be o 30-40% develop anti-IgA Abs
normal levels of IgA and IgM. o Most patients with a deficiency of IgA are
o IgG appears to be the most affected, dropping to asymptomatic
at least 2 standard deviations (SDs) below the o Those with symptoms usually have infections in
age-adjusted mean with or without a depression of respiratory and gastrointestinal tract and have
IgM and IgA. an increased tendency to develop autoimmune
• Immunoglobulin levels in infants with this condition usually diseases such as Systemic Lupus
normalize spontaneously, often by 9 to 15 months of age. Erythematosus, Rheumatoid Arthritis, and other
• The mechanism of this transient hypogammaglobulinemia immunodeficiency diseases
is not known. o Allergic disorders and malignancy are also more
common.
• These patients have normal numbers of circulating CD19+
B cells. • 20% of the IgA-deficient patients who develop infections
also have an IgG2 subclass deficiency
• This condition does not appear to be X-linked, although it
o If the serum IgA is lower than 5 mg/mL
is more common in males.
§ deficiency is considered severe
o The cause may be related to a delayed maturation
o If the IgA level is two SDs below the age-
of one or more components of the immune
adjusted mean but greater than 50 mg/dL
system, possibly Th cells
§ deficiency is partial
X-Linked Bruton’s Tyrosine Kinase (Btk) Deficiency • hypothesized that lack of IgA is caused by impaired
differentiation of lymphocytes to become IgA-producing
• first described in 1952 plasma cells
• X-linked • IgE antibodies specifically directed against IgA
o syndrome affects males almost exclusively o produced by 30% to 40% of patients with severe
• Deficiency /lack of all classes of immunoglobulins IgA deficiency
o Lack circulating mature CD19+ B cells o cause anaphylactic reactions when blood
§ Pre-B cells in bone marrow but no products containing IgA are transfused
peripheral cells in lymphoid tissues • IgA deficiency may not be detected until the patient
§ Because of the lack of B cells, the tonsils experiences a transfusion reaction
and adenoids are small or entirely absent o resulting in the production of anti-IgA antibodies
and lymph nodes lack normal germinal
• Most gamma globulin preparations contain significant
centers
amounts of IgA
• About half of the patients have a family history of the o However, replacement IgA therapy is not useful
syndrome. because the half-life of IgA is short
o develop recurrent bacterial infections beginning in § around 7 days
infancy as maternal antibody is cleared o intravenously or intramuscularly administered
§ patients most commonly develop IgA is not transported to its normal site of
sinopulmonary infections caused by secretion at mucosal surfaces
encapsulated organisms such as

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Common Variable Immunodeficiency (CVI) • IgG1, IgG3: vs protein Ags (eg., toxins)
• A heterogenous group of disorders • IgG2, IgG4: vs carbohydrate Ags
• Most common primary immune deficiency with a severe o (eg., S. pneumoniae, H. influenzae)
clinical syndrome characterized by • Recurrent infections: levels of the different subclasses
hypogammaglobulinemia that can lead to severe bacterial should be measured if the total IgG level is normal but the
infections particularly sinusitis and pneumonia clinical picture suggests immunoglobulin deficiency
o Patients usually begin to have symptoms in their • IgG against protein: IgG1 and IgG3
20s and 30s o Reduced response to toxins
o age at onset ranges from 7 to 71 years of age • IgG against carbohydrate: IgG2 and IgG4
• The disorder can be congenital or acquired, or familial or o Impaired response to polysaccharide antigens
sporadic, and it occurs with equal frequency in men and o Recurrent infections with S. pneumoniae and H.
women influenzae
• Deficiency in both IgA and IgG but selective IgG • Associated with heavy chain gene deletion and
deficiency may occur. transcriptional defects
o Recurrent bacterial infections (sinusitis,
pneumonia) Characteristics of Selected Predominantly
§ up to 20% of CVI patients develop Antibody Deficiencies (Category 3)
herpes zoster Condition Deficiency Level of Presentation
§ CVI is often associated with a spruelike Defect
syndrome characterized by Transient All Slow 2–6
malabsorption and diarrhea Hypogamma- antibodies; developme months;
§ CVI is also associated with an increased globulinemia especially nt of helper resolves by
risk of of infancy IgG function in 2 years
§ lymphoproliferative disorders, gastric some
carcinomas, and patients
§ autoimmune disorders Selective IgA IgA; some IgA-B cell Often
§ Most common autoimmune deficiency also with differentiati asymptoma
manifestations of CVI are immune reduced on tic
thrombocytopenia and autoimmune IgG2
hemolytic anemia. Btk deficiency All Pre–B-cell Infancy
• This is diagnosed by demonstrating a low serum IgG level antibody differentiati
in patients with recurrent bacterial infections isotypes on
o blood group isohemagglutinins, or the so-called reduced
naturally occurring antibodies, are typically
absent or low Common Reduced B cell; Usually
o most patients with CVI have normal numbers of variable antibody; excess T 20–30
mature B cells immunodeficie many suppressio years
• do not differentiate normally into ncy different n of age
immunoglobulinproducing combinatio
plasma cells ns
§ CVI is often a diagnosis of exclusion, Isolated IgG Reduced Defect of Variable
where an immunodeficiency is present subclass IgG1, isotype with the
with no specific genetic defect defined deficiency IgG2, differentiati class
o Can be treated with intramuscular or IgG3, or on and degree
intravenous immunoglobulin preparations. IgG4 of
§ However, because of their low levels of deficiency
secretory IgA, patients are still CD154 Reduced B-cell Variable
susceptible to respiratory and deficiency IgG, IgA, switching
gastrointestinal infections IgE, with
• clinician should be vigilant for elevated
these infections and treat them IgM
vigorously with antibiotics.
• Three cellular defects C. Category 1: Combined Immunodeficiencies
1) T cells or their products appear to suppress • Defects in both humoral and cell-mediated immunity
differentiation of B cells • Result from mutations that affect development or cause
2) T cells fail to help in B cell terminal defective interaction
differentiation • Severe defect of T cells will have effects on
3) Primary defect in B cell line immunoglobulin levels
• Treatment: IM/IV Ig • Combined deficiencies are referred to using a shorthand
notation of T+/–B+/–NK+/– with the + or – superscript
Isolated IgG Subclass Deficiency denoting whether or not each cell type is present in the
• 1 or more of the IgG subclasses is below mean age- deficiency
appropriate level (>2SD) • T cells: cell-mediated immunity
• IgG4: most commonly affected 1) DiGeorge anomaly
o Of least clinical significance 2) Purine Nucleoside Phosphorylase Deficiency
• IgG1: least common

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Severe Combined Immunodeficiency (SCID) • It produces a moderate to severe defect in cell-mediated
• Most serious of the congenital immune deficiencies immunity with normal or only mild impaired humoral
• Patients with SCID generally present early in infancy with immunity
infection by nearly any type of organism. • The number of T cells progressively decreases because
o Oral candidal yeast infections, pneumonia, and of the accumulation of deoxyguanosine
diarrhea are the most common manifestations • triphosphate, a toxic purine metabolite.
• Group of related diseases that all affect T and B cell • The levels of immunoglobulins are generally normal or
function increased.
o X-linked • About two thirds of PNP-deficient patients also have
o JAK3 deficiency neurological disorders, but no characteristic physical
o Adenosine deaminase (ADA) deficiency abnormalities have been described.
o Ommen’s syndrome • Because of the relatively selective defect in cell mediated
immunity, PNP deficiency can be confused with neonatal
X-linked SCID HIV infection.
• T- B+ NK+ / T-B+NK- cell • The two conditions can usually be distinguished by
o T cell, B cell, natural killer cell specific tests for HIV and by assays for PNP activity.
• Most common
• No Ab production or lymphocyte proliferation response Characteristics of Selected Combined
follows an Ag/ mitogen challenge Immunodeficiencies (Category 1)
• Abnormal Interleukin 2 Receptor Subunit Gamma (IL2RG) Condition Deficiency Level of Defect Presentation
o There are defect receptors that code for common CD40 T cells Defective 1–2 years
gamma chain, thus normal signal does not occur ligand with isotype of age
o Common to receptors for IL2, 4, 7, 9, 15, 21 deficiency effects on switching
§ There is no normal signalling that occurs antibody with increased
production or normal
JAK3 deficiency IgM but
• Autosomal Recessive decreased
• cell-B cell +NK cell- concentrations
• Deficiency in intracellular kinase JAK3 (required for of other
processing interleukin binding signal from cell membrane isotypes
to the nucleus) SCID Both T ADA, purine Infancy
o No signal transmission from IL2 and IL4 in and B metabolism;
lymphocytes cells RAG-1/RAG-2;
JAK3;
Adenosine Deaminase Deficiency common
• T cell-B cell +NK cell- gamma chain
• Impaired proliferation of T and B cells due to accumulation receptor; others
of toxic metabolites of purines PNP T cells; PNP, purine Infancy
o Decrease lymphocyte deficiency some metabolism
• Deficiency can be treated by maintaining high plasma secondary
levels of ADA effects
on
Ommen’s Syndrome antibody
• T-B-NK+Defect in RAG1/RAG2 (recombinase activating production
gene)
o Recombinase activating gene is for the DNA D. Category 2: Combined Immunodeficiencies With
rearrangement necessary for the production of Associated or Syndromic Features
functional T and B cell receptors • Diseases in this category are typically caused by defects
• Lymphocytopenia is due to failure of T and B cells to in cell-mediated immunity, whichindirectly lead to
produce functional TCR and Immunoglobulin problems with the other
• branches of the immune response.
Purine-Nucleoside Phosphorylase (PNP) Deficiency • Often, these diseases can result from abnormalities at
• For purine metabolism different stages of T-cell development.
• T cell count decrease due to accumulation of • Many different molecular defects can result in a similar
deoxyguanosine triphosphase which is a toxic purine clinical picture (as in SCID).
metabolite o This is because T cells provide helper functions
• this is an autosomal recessive trait present in infancy with: that are necessary for normal B-cell development
o recurrent or chronic pulmonary infections and differentiation.
o oral or cutaneous candidiasis • Some of the more common defects of cellular and
o diarrhea combined cellular and humoral immunity are summarized
o skin infections in Table 9.18.
o urinary tract infections • In general, defects in cellular immunity are more difficult to
o failure to thrive manage than defects in humoral immunity.
• Affects an enzyme involved in the metabolism of purines. • When immunoglobulin production is deficient,
replacement therapy is often very effective.

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• However, there is usually no soluble product that can be • The primary molecular defect in the syndrome appears to
administered to treat a deficiency of cell-mediated be an abnormality of the integral membrane protein CD43,
immunity. Transplantation of immunologically intact cells, which is involved in the regulation of protein glycosylation.
usually in the form of allogenic bone marrow, is often • WAS gene is located on the X chromosome, region p11.
required to reconstitute immune function. • Abnormalities cause defective actin polymerization and
• Patients with severe defects in cell-mediated immunity affect its signal transduction in lymphocytes and platelets.
may develop graft-versus-host disease • Platelets have a shortened half-life and T lymphocytes are
• Transfused lymphocytes are normally destroyed by the also affected, although B lymphocytes appear to function
recipient’s T-cell system. normally.
o However, a severe defect in the T-cell system • Splenectomy can be very valuable in controlling the
allows the donor lymphocytes to survive, thrombocytopenia.
proliferate, and attack the tissues of the recipient • Current treatment for this immunodeficiency is
as foreign. transplantation of bone marrow or cord blood stem cells
• GVHD can occur in any patient with a severe defect in from an HLA identical sibling.
cell-mediated immunity (e.g., in bone marrow transplant
recipients) and can be fatal. DiGeorge Anomaly
o Irradiation of cell-containing blood products • Abnormal development of 3rd and 4th pharyngeal
(platelet concentrates, packed RBCs, and whole pouches
blood) before transfusion destroys the ability of the o affects the thymus development in the embryo
donor lymphocytes to proliferate and prevents (all organs derived from these embryonic
development of GVHD in immunodeficient structures can be affected)
recipients.
• It should be noted that a defect in humoral immunity does Affects thymic development in the embryo
not predispose an individual to GVHD. ↓
• GVHD also occurs in patients who have received a bone Quantitative defect in Thymocytes
marrow transplant as therapy for a congenital most patients show a deletion in chromosome 22 region
immunodeficiency. q11:1:19
o The closer the match between the genetic ↓
constitution of the patient and the graft donor, the can lead to but not all Severe and persistent decrease in
less severe the GVHD is likely to be. mature T cells
• Thus, although bone marrow transplantation can
potentially cure the immune defect, it can also have
o severe, recurrent viral and fungal infections
serious, lifelong complications of its own.
• Associated abnormalities include mental retardation,
absence of ossification of the hyoid bone, cardiac
Wiskott-Aldrich Syndrome (WAS)
anomalies, abnormal facial development, and thymic
• A rare X-linked recessive syndrome that is defined by the hypoplasia.
triad of symptoms: • The severity and extent of the developmental defect can
o Immunodeficiency be quite variable.
§ Lymphocytes and platelet have abnormal • Many patients with a partial DiGeorge anomaly have only
CD43 (protein glycosylation) a minimal thymic defect and, thus, near normal immune
• Abnormal signal transduction and function.
defective actin polymerization • However, about 20% of children with a defect of the third
§ Absence of isohemagglutinins (e.g., IgM and fourth pharyngeal pouches have a severe and
Abs to ABO) persistent decrease in T-cell numbers.
o Eczema
• These children tend to have severe, recurrent viral and
o Thrombocytopenia (with ↓ size)
fungal infections.
• ↑ serum alpha-fetoprotein
• Severely affected children usually present in the neonatal
• Lethal in childhood because of infection, hemorrhage, or period with tetany (caused by hypocalcemia resulting from
malignancy. hypoparathyroidism) or manifestations of cardiac defects.
• Milder variants may be an X-linked form of • The possibility of immunodeficiency can be overlooked if
thrombocytopenia. the association between the presenting abnormality and a
• Lab features of WAS include a decrease in platelet possible thymic defect is not recognized.
number and size with prolonged bleeding time. • The immunodeficiency associated with the DiGeorge
• Bone marrow contains a normal or somewhat increased anomaly is a quantitative defect in thymocytes.
number of megakaryocytes.There are abnormalities in • Not enough mature T cells are made, but those that are
both the cellular and humoral branches of the immune present are functionally normal.
system related to a general defect in antigen processing.
• The q11 region of chromosome 22 deletion is also
• Patients display a severe deficiency of the naturally associated with velocardiofacial syndrome (VFS) and
occurring IgM antibodies to ABO blood group antigens other syndromes.
(isohemagglutinins).
• Treatment
• Absence of isohemagglutinins is the most consistent o Fetal thymus transplantation
laboratory finding in WAS and is often used diagnostically. o Bone Marrow transplantation
• Patients with WAS varied immunoglobulin levels, but they o Thymic hormone Transplantation
usually have low levels of IgM, normal levels of IgA and
IgG, and increased levels of IgE.

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Ataxia-Telangiectasia a, small
• Rare autosomal recessive syndrome platelets, and
• Blunt Ab response to Ags, especially polysaccharides eczema
o Low or absent IgG2, IgA, IgE AT Reduced DNA Infancy, with
• Decreased circulating T cells IgG2, IgA, instability involuntary
o Rearrangement of TCR and Ig genes does not IgE, and T muscle
occur lymphocyte movements and
o Death usually occurs in early adults from either s capillary swelling
pulmonary disease or malignancy
• Treatment: BM trans/ cord blood stem cells E. Category 4: Diseases of Immune Dysregulation
o Characterized by cerebellar ataxia (involuntary • many diseases with normal numbers of T or B cells but
muscle movements) and telangiectasias with reduced control over their functions
(capillary swelling resulting in red blotches on the
skin), especially on the earlobes and conjunctiva. Autoimmune lymphoproliferative syndrome (ALPS)
• Blood vessels in the sclera of the eyes may be dilated and • may involve mutations in genes coding for caspase
there may also be a reddish butterfly area on the face and enzymes involved in apoptosis.
ears. • Defective apoptosis in the thymus may lead to
o Ninety-five percent of patients exhibit increased autoreactive cells in the circulation.
levels of serum alpha-fetoprotein. • The CD25 deficiency is manifested by a lack of T
• The incidence of this disease is between 1:10,000 to regulatory (Treg) cells, which leads to lymphoproliferation
1:100,000, 1% is heterozygous for the gene. and autoimmunity.
• Abnormal genes produce a combined defect of both • Mutations in the FoxP3 gene, which is required for Treg
humoral and cellular immunity. differentiation, may show a similar clinical presentation.
• Antibody response to antigens, especially
polysaccharides, is blunted. Chediak-Higashi syndrome
• Patients with AT have a defect in a gene that is apparently • an immunodeficiency with hypopigmentation (loss of skin
essential to the recombination process for genes in the color) caused by a mutation in the LYST gene
immunoglobulin superfamily. • characterized by a reduced number of natural killer (NK)
o The AT gene is located on chromosome 11, cells and neutrophils, as well as an increased production
region q22. of inflammatory proteins.
• This abnormality results in a defective kinase involved in • Peripheral blood smears from patients with Chediak-
DNA repair and in cell cycle control. Higashi syndrome show granulocytic inclusions attributed
o Rearrangement of TCR and immunoglobulin to enlarged lysosomes
genes does not occur normally.
• Patients’ lymphocytes often exhibit chromosomal breaks F. Category 5: Congenital Defects of Phagocyte
and other abnormalities involving the TCR genes in T Number, Function, or Both
cells and immunoglobulin genes in B cells. Chronic Granulomatous Disease (CGD)
o These are sites of high levels of chromosomal
• a group of disorders involving inheritance of either an X-
recombination and errors that occur during gene
linked or autosomal recessive gene that affects neutrophil
rearrangements may not be repaired properly.
microbicidal function
• The syndrome is associated with an even greater risk of
• most common and best characterized of the neutrophil
lymphoid malignancy than other immunodeficiency
abnormalities
syndromes, presumably because the failure to properly
• the inability of the patient’s neutrophils to produce the
repair DNA damage leads to the accumulation of
reactive forms of oxygen necessary for normal bacterial
mutations.
killing
o The only effective therapy for AT is allogeneic
bone marrow transplantation. • symptoms of CGD include recurrent suppurative
infections, pneumonia, osteomyelitis, draining
adenopathy, liver abscesses, dermatitis, and
Characteristics of Combined Immunodeficiencies
hypergammaglobulinemia
With Associated or Syndromic Features (Category 2)
Condition Deficiency Level of Presentation
o catalase-positive organisms such as S aureus, B
Defect cepacia, and C violaceum are involved in addition
DiGeorg T cells; Embryologi Neonatal, with to fungi such as Aspergillus and Nocardia
e some c hypocalcemia • Infections usually begin before 1 year of age and the
anomaly secondary developme or cardiac syndrome is often fatal in childhood
effects nt defects if severe; • historically diagnosed by measuring the ability of a
on of the abnormal mental patient’s neutrophils to reduce the dye nitroblue
antibody thymus delay; tetrazolium (NBT)
production development; o NBT reduction is caused by the production of
incomplete forms hydrogen peroxide and other reactive forms of
may oxygen
present later with o reduction converts the nearly colorless NBT into
infection a blue precipitate that can be assessed visually
WAS Reduced CD43 Usually infancy; on a microscope slide
IgM and T- expression with • can also be diagnosed with flow cytometric assay
cell defect thrombocytopeni

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 o neutrophils are labeled with dihydrorhodamine o Early-onset IBD is caused by mutations in genes
(DHR) coding for IL-10 or its receptor
o DHR will fluoresce when it is reduced
o more objective and quantitative than the I. Category 8: Complement Deficiencies
traditional NBT technique • C1q, 2, 4: lupus-like syndrome
• C2: most common complement deficiencies
Leukocyte Adhesion Deficiency (LAD) • C3: lupus-like w/ recurrent encapsulated organism
• Abnormal adhesion, motility aggregation, chemotaxis, infections
endocytosis • C5-9: recurrent Neisserial infections
• Occurs when neutrophils fail to leave the vasculature to • C1 esterase inhibitor: hereditary angioedema
migrate to site of incipient infection
J. Category 9: Phenocopies of Primary
LAD I LAD II Immunodeficiencies
Deficiency in CD-18 - a Deficiency in CD15s or • new classification of PIDs
component of the adhesion sialyl Lewis X • Disorders in this category have an inherited genetic
receptor of neutrophils and component but also include an acquired component, such
monocytes (CD11b or as somatic mutations or autoantibody production
CD11c), and T cells • chronic mucocutaneous candidiasis
(CD11a) o induced by a genetic mutation in the AIRE gene,
Decreased CD11/CD18: Problem in neutrophils: but also involves an antibody to either (or both)
Leads to abnormal Recurrent infections, IL-17 and IL-22
adhesion, motility, Persistent leukocytosis, • Mutations in the nRAS or kRAS genes
chemotaxis , aggregation, and Severe mental and
and endocytosis growth retardation. LABORATORY EVALUATION OF IMMUNE
DYSFUNCTION
G. Category 6: Defects in Innate Immunity A. Screening Tests
• new part of the PID classification, mirroring the explosive • Evaluation starts with patient history, followed by a
increase in knowledge and understanding of the innate complete blood count (CBC) and white blood cell (WBC)
immune system differential
• Measurement of the levels of serum IgG, IgM, and IgA
Chronic mucocutaneous candidiasis
and levels of the subclasses of IgG for Ab production
• involving mutations in genes coding for IL-17 defect
Defects in TLR signaling pathways, such as IRAK4 • overall assessment of antibody-mediated immunity can be
deficiency, can also occur. made by measuring antibody responses to antigens to
• Both types of defects can lead to bacterial infections. which the population is exposed normally or following
vaccination
H. Category 7: Autoinflammatory Disorders • Delayed hypersensitivity-type skin reactions can be used
Inflammasome Conditions to screen for defects in cell-mediated immunity
• protein oligomer that contains caspase enzymes and
other proteins associated with apoptosis Tests
• located primarily in myeloid cells and may be activated by 1. Tuberculin Skin Test
various microbial substances. • Defects in cell-mediated (Tcell) immunity
• Once activated, the inflammasome stimulates the
production of the proinflammatory cytokines IL-1 and IL-
18.2 2. CH50 Assay
• Determination of level of functional complement
a. Hyper IgD syndrome
• aka periodic fever syndrome 3. Nitro-Blue Tetrazolium (NBT) Reduction Test
• caused by a deficiency of mevalonate kinase, an enzyme • Ability of neutrophils to reduce NBT (blue) to nearly
involved in a sterol synthesis pathway colorless through oxidative burst (production of oxygen
products)
b. Muckle-Wells syndrome • Chronic Granulomatous Disease (CGD) Patients → The
• caused by a mutation in the CIAS1 gene coding for NBT reaction will remain blue
cryopyrin, a component of the inflammasome.
• Patients may present with urticaria and amyloidosis. 4. Flow Cytometric Assay
• detects defects in neutrophil oxidative burst activity
Noninflammasome conditions
• Inclide tumor necrosis factor (TNF) receptor-associated B. Confirmatory Tests
periodic syndrome (TRAPS) and early-onset inflammatory • If the screening tests detect an abnormality or the clinical
bowel disease (IBD). suspicion is high, more specialized testing will probably be
o TRAPS - caused by a mutation in the TNFRSF1A necessary to precisely identify an immune abnormality.
gene, which codes for a TNF receptor and may • Some of the tests used for confirming an
result in recurrent fevers, as well as ocular and immunodeficiency state are listed in the table below.
joint inflammation.

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 Specialized Confirmatory Tests • However, in the actual clinical arena, secondary
for Immunodeficiencies immunodeficiencies are more common than primary
Suspected Specialized Tests immunodeficiencies (PIDs).
Disorder o For example, in the laboratory of the author, the
Humoral B-cell counts by flow cytometry most common cause of absent B cells is not Btk
immunity B-cell proliferation in vitro (e.g., deficiency, but patients treated with rituximab, a
mitogen assays) monoclonal anti-CD20 antibody.
Histology of lymphoid tissues § This antibody, used to treat leukemic,
Cell-mediated T-cell counts by flow cytometry (total transplant, and autoimmune patients,
immunity and T-cell subsets) destroys B cells, resulting in no detectable
T-cell function in vitro (e.g., mitogen CD19+ or CD20+ cells.
assays)
Quantiferon TB assay
Cylex ImmuKnow assay
Enzyme assays (ADA, PNP)
Phagocyte Leukocyte adhesion molecule
defects analysis
(CD11a, CD11b, CD11c, CD18)
Phagocytosis and bacterial killing
assays
Chemotaxis assay
Enzyme assays (myeloperoxidase,
glucose-6-phosphate
dehydrogenase,
components of NADPH oxidase)
Complement Specific component assays

Flow Cytometry
• Enumeration of classes of lymphocytes in the peripheral
blood is performed by flow cytometry.
• Even though types of lymphocytes cannot be
distinguished morphologically, they exhibit different
patterns of antigen or surface immunoglobulin expression
that correlate with functional characteristics.
• Before flow cytometric analysis, antibodies to antigens
specific for different types of lymphocytes are labeled with
• Most of the genes associated with PIDs have been
a fluorescent probe.
identified and localized. Thus, genetic testing is available
o These antigens are generally referred to by a
for many conditions, including:
cluster of differentiation (CD) number.
o the DiGeorge deletion
o The antibodies are allowed to react with the
o the Wiskott-Aldrich gene
patient’s peripheral blood mononuclear cells in a
o IL2RG mutations
direct immunofluorescence assay and RBCs in the
• Although genetic testing is useful to understand the
sample are lysed.
pathology of the disease, it is often not required for
• The flow cytometer is used to count the WBCs that are
making a diagnosis.
labeled with each fluorescent antibody.
• Genetic testing of family members of affected patients
may be helpful in determining who may be at risk of
Enumeration of classes and subclasses of lymphos through
developing the disease or passing it on to offspring.
CD markers:
• Lymphocytes can then be assigned to specific types
T-Cell Function
based on antigen expression:
o B cells (CD19) • T-cell function can be measured by assessing the ability
o T cells (CD3) of isolated T cells to proliferate in response to an antigenic
§ T helper (Th) cells (CD3/CD4) stimulus or to T-cell mitogens in culture, such as
§ cytotoxic T cells (CD3/CD8) phytohemagglutinin (PHA) or Concanavalin A (Con A).
o NK cells (CD16 or CD56) o A mitogen is a substance that stimulates mitosis in
• Flow cytometry is objective and quite reliable in detecting all T cells or all B cells, regardless of antigen
those defects that result in a decrease in one or more specificity.
types of lymphocytes. For example:
o DiGeorge syndrome T-Cell Function (Classical)
§ an absence or profound decrease in the • Classically, the T-cell response may be measured by
number of CD3 cells quantitating the uptake of radioactive thymidine, a
o Btk deficiency precursor of DNA.
§ an absence of CD19+ B cells o Increased thymidine uptake suggests cell division
§ ↓ CD19 → suggests Bruton’s and activation.
agammaglobulinemia or X-linked
agammaglobulinemia (XLA)

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 oThis assay requires experienced technologists, a • Whole exome sequencing
radioactive materials license, and laboratory- o Another technique not normally applied to
determined reference ranges. newborn screening
• Summary: o has been used to identify Ataxia-telangiectasia
o Thymidine uptake quantification (AT) in two infants and will likely see increased
o ↑ thymidine uptake: means cell division and use in the future
activation

T-Cell Function (Automated)

• More recently, antigen- or mitogen-stimulated T-cell
activation has been measured without the use of
radioactive materials.
• The FDA has cleared three assays for diagnostic use.
• (1) Quantiferon TB assay & (2) T-Spot assay
o measure an individual’s response to
Mycobacterium tuberculosis antigens
o Following overnight whole blood activation with
TB antigens, gamma interferon secreted by
activated Th1 cells, is quantitated by either an
enzyme-linked immunosorbent assay (ELISA) or
ELISPOT procedure.
o Either of these assays may be used as an in vitro
assessment of exposure to M. tuberculosis.
• (3) Cylex ImmuKnow assay
o measures total T-cell activity
o uses the mitogen phytohemagglutinin (PHA) to
activate T cells
§ PHA is also used as a positive control in
the Quantiferon and T-Spot procedures.
o Following incubation, ATP production is
measured by a fluorescent immunoassay
technique.
o This test is a general measurement of T-cell
function and is often used to monitor individuals
receiving immunosuppressive therapy.
o The assay may be used to determine overall T-
cell functional capabilities in an individual
suspected of a PID.
• Summary:
o QuantiFERON TB assay and T-spot
§ Measure response to MTB Antigen
o Cylex ImmuKnow
§ T cell response to phytohemagglutinin
(PHA)
• ATP production Newborn testing for T-cell receptor excision circles (TRECs) formed
by normal T-cell receptor gene rearrangement.
C. Newborn Screening for Immunodeficiencies
• Several states have begun to include primary D. Evaluation of Immunoglobulins
immunodeficiency (PID) testing as part of their newborn Serum protein electrophoresis
screening programs. • molecules are separated on the basis of their size and
• This testing is typically performed to look for the presence electrical charge
of TCR excision circles (TRECs). • Divided into five (5) regions
• TRECs o albumin
o identified by quantitative PCR o alpha1 globulins
o present in T cells that have undergone alpha-beta o alpha2 globulins
receptor gene rearrangements o beta globulins
o They are the genetic material that has been o gamma globulins
removed from the germline DNA during alpha VJ • Some laboratories now use six regions, dividing the beta
and beta VDJ recombination region into the beta1 (transferrin) and beta2 (complement
o Their absence indicates a lack of functional T component C3) regions
cells, allowing early identification of T-cell related • may be performed using capillary electrophoresis
defects leading to SCID. o eliminates the need for gels and the drying and
§ DiGeorge and other non-SCID staining processes
diseases, such as Omenn
syndrome, have also been
detected using this method.

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Immunofixation electrophoresis following findings are noted: prolonged bleeding time,
• Serum proteins are electrophoresed decreased platelet count, increased level of serum
• Specific Abs allow to bind on the separated proteins alpha-fetoprotein, and a deficiency of naturally occurring
• Interpret band formation isohemagglutinins. Based on these results, which is the
o Diffuse: polyclonal Immunoglobulin most likely diagnosis?
o Narrow, intense: monoclonal a. PNP deficiency
b. Selective IgA deficiency
E. Bone Marrow Biopsy c. SCID
• Monoclonal gammopathy d. Wiskott-Aldrich syndrome
8. Which of the following is (are) associated with ataxia-
• Immunodeficiency state
telangiectasia?
• analyzed microscopically and by flow cytometry
a. Inherited as an autosomal recessive
• Cytogenetic analysis may also be performed
b. Defect in both cellular and humoral immunity
o detect the specific genetic anomalies associated
c. Chromosomal breaks in lymphocytes
with the PID diseases
d. All of the above
9. A 4-year-old boy presents with recurrent wound and soft
F. Family History
tissue infections. Which of the following assays should
• Very important in PID diagnosis process be considered for diagnosing his presumed PID?
• disease may be X-linked, such as in the common gamma a. DHR reduction
chain mutation, WAS, or the CD154 deficiency; autosomal b. CD4 quantitation
dominant, such as in Hyper IgE or Muckle-Wells c. CD19 quantitation
syndrome; or autosomal recessive, such as in AT or LAD d. CD56 quantitation
• CGD may be either X-linked or autosomal recessive 10. A patient with a deficiency in complement component
C7 would likely present with
a. recurrent Staphylococcal infections.
Review Questions b. recurrent Neisserial infections.
1. Patients with which immunodeficiency syndrome should c. recurrent E coli infections.
receive irradiated blood products to protect against the d. recurrent Nocardia infections.
development of GVHD? 11. A FoxP3 gene mutation may lead to a deficiency of what
a. Bruton’s thymidine kinase (Btk) deficiency cell type?
b. Selective IgA deficiency a. T helper cells
c. SCID b. T cytotoxic cells
d. CGD c. B cells
2. T-cell subset enumeration by flow cytometry would be d. T regulatory cells
most useful in making the diagnosis of which disorder? 12. The Cylex ImmunoKnow assay is useful in determining
a. Bruton’s thymidine kinase (Btk) deficiency functional capabilities of which cell type?
b. Selective IgA deficiency a. T cells
c. SCID b. B cells
d. Multiple myeloma c. NK cells
3. What clinical manifestation would be seen in a patient d. Neutrophils
with myeloperoxidase deficiency? 13. Recurrent, periodic fevers may be associated with
a. Defective T-cell function increased production of which immunoglobulin?
b. Inability to produce IgG a. IgG
c. Defective NK cell function b. IgM
d. Defective neutrophil function c. IgD
4. Defects in which branch of the immune system are most d. IgE
commonly associated with severe illness after 14. Chronic mucocutaneous candidiasis, a PID that was
administration of live virus vaccines? previously thought to be a cell-mediated deficiency, is
a. Cell-mediated immunity now classified as which type of PID?
b. Humoral immunity a. Autoinflammatory disorder
c. Complement b. Complement deficiency
d. Phagocytic cells c. Predominantly antibody deficiency
5. Which of the following statements applies to Bruton’s d. Innate immunity deficiency
thymidine kinase (Btk) deficiency? 15. Prenatal screening for SCID involves detecting
a. It typically appears in females. a. Tregs.
b. There is a lack of circulating CD19+ B cells. b. TRECS.
c. T cells are abnormal. c. THELPS.
d. There is a lack of pre-B cells in the bone d. TCYTOS.
marrow.
6. DiGeorge anomaly may be characterized by all of the
following except
a. autosomal recessive inheritance.
b. cardiac abnormalities.
c. parathyroid hypoplasia.
d. decreased number of mature T cells.
7. A 3-year-old boy is hospitalized because of recurrent
bouts of pneumonia. Laboratory tests are run and the

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Christine Joy R. Timbang | 0963-467-6983
 SEROLOGICAL AND
MOLECULAR DIAGNOSIS
OF INFECTIOUS DISEASE

Christine Joy R. Timbang | 0963-467-6983

Christine Joy R. Timbang | 0963-467-6983
IMMUNOSEROLOGY 19.

Immunization and Vaccines

OUTLINE water emulsions. They are believed to enhance the
I. Summary immune response by promoting migration and antigen
uptake by APCs and inducing the release of
proinflammatory cytokines and chemokines.
SUMMARY • In the future, scientists will develop next generation
• Immunity is the condition of resistance to a disease. vaccines that will identify vaccine target antigens more
Immunization is the process by which immunity is efficiently by using genetic technologies. In developing
acquired. There are three types of immunization—active, these vaccines, researchers will attempt to induce broadly
passive, and adoptive. neutralizing antibodies and cell-mediated responses,
• Active immunization involves stimulation of an individual’s employ novel adjuvants, and investigate the use of
own immune system to mount an adaptive immune nonparenteral routes of delivery.
response to an antigen, as a result of either natural • The effectiveness of a vaccine is not only influenced by
infection or receipt of a vaccine. the nature of the vaccine, but also by the age and immune
• Passive immunization involves transfer of premade status of the host. To obtain an optimal immune response,
antibodies from an immune host to a nonimmune host. vaccines must be administered according to
This type of immunity can occur naturally, by transfer of recommended schedules.
maternal IgG antibodies through the placenta to the fetus, • Vaccines protect not only the individuals who have
or by transfer of IgA antibodies from a mother to her infant received the vaccines, but also their contacts, a
through breast milk. Passive immunity can also be phenomenon known as “community immunity” or “herd
transferred artificially through commercial antibody immunity.”
preparations such as human immune serum globulin. • Vaccines are considered to be one of the greatest
• Adoptive immunotherapy is achieved by transferring cells achievements of medicine. The benefits of vaccines
of the immune system to a nonimmune host. The cells greatly outweigh their risks. Rarely, vaccines can cause
can be derived either from a different (allogeneic) host or side effects such as hypersensitivity reactions. Live
they can be stimulated in vitro before re-introduction into vaccines should not be administered to patients with
the same individual (autologous). immunodeficiency disorders because they can replicate
• Active immunity takes time to develop, but provides long- uncontrollably and cause disseminated disease.
lasting protection to the host because of the production of • Passive immunization with preformed antibody
immunologic memory. Passive immunity provides preparations is administered in order to provide immediate
immediate protection, but is short lived because antibody humoral immunity to unimmunized persons.
titers decline at a rate that is determined by the biological • Patients with humoral immunodeficiencies are routinely
half-life of the immunoglobulins. Adoptive immunity treated with standard human immune serum globulin,
provides long-lasting protection if the cells become prepared from pooled serum of many donors, to provide
established in the host. protection against a variety of pathogens. Specific human
• A traditional vaccine is a microbially derived antigen immune serum globulins containing antibody against a
suspension that is administered to a healthy host in order particular pathogen can be used to treat nonimmune,
to stimulate an immune response that will prevent the host healthy individuals who have had contact with the
from developing an infectious disease. Thus, vaccines are pathogen to provide immediate protection.
used for immunoprophylaxis. • Antitoxins are antibodies directed against toxins from
• In 1796, Dr. Edward Jenner was the first to publicize the pathogenic bacteria. They can be isolated from the serum
procedure of vaccination by using material from cowpox of laboratory animals that have been injected with the
skin lesions to immunize against smallpox. In the 1800s, toxin and used to prevent diseases such as tetanus,
Louis Pasteur used the principle of attenuation, or diphtheria, and botulism.
weakened microorganisms, to produce vaccines against • Passive immunization of immunoglobulins can also
chicken cholera, anthrax, and rabies. beused as immunosuppressive therapy for certain
• In the 20th century, vaccine development grew autoimmune and chronic inflammatory disorders, as well
exponentially as a result of new methods to attenuate as to prevent hemolytic disease of the newborn.
microorganisms, production of toxoids, and technologies • Monoclonal antibodies have specificity for a particular
that allowed viruses to be grown in cell culture. In the antigenic epitope. They are being used as therapeutic
latter part of the 20th century, the first recombinant agents for a variety of cancers and autoimmune diseases.
vaccine, produced by genetic engineering, was These antibodies were originally 100% mouse derived
developed. because they were produced by hybridoma technology.
• Conventional vaccines may be composed of live, Advancement in genetic technologies has allowed
attenuated (weakened, nonpathogenic) microorganisms; scientists to synthesize hybrid antibodies that consist
inactivated (killed) microorganisms; or antigenic subunits partially of mouse protein and partially of human protein.
such as recombinant antigens or toxoids. Toxoids are These antibodies can be characterized as chimeric,
bacterial toxins that have been inactivated so that they are humanized, or fully human, depending on the amount of
no longer pathogenic, but are still immunogenic. the human component. Administration of these antibodies
• Adjuvants are substances that are often included in reduces the chance that the recipients will produce
vaccines in order to increase the immune response to human-anti-mouse antibodies (HAMA) and develop
thetarget antigens. Examples of adjuvants include alum hypersensitivity reactions.
(aluminum hydroxide and aluminum phosphate) and oil-in-

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• Adoptive immunotherapy involves the transfer of cells of Characteristics of Conventional Vaccines
the immune system to provide immunity. Examples Composi Example Advanta Limitations
include the treatment of melanoma patients with tion s ges
autologous, IL-2 activated tumor-infiltrating lymphocytes Attenuate Live BCG Induce Cannot be
d pathogen Typhoid both administered
(TIL) and the administration of hematopoietic stem cells to
s that fever humoral to
patients with hematologic malignancies who have been have Oral polio and cell- immunocom
treated with high doses of chemotherapy and irradiation. been (Sabin) mediated promised
weakene Measles immunity individuals
Major Features of Active Immunity, Passive Immunity, and d by (Rubeola) Effective Rare
Adoptive Immunity growth Mumps in potential to
Mechanis Examples Advanta Limitations under German inducing mutate
m ges modified measles immunity to a
Active Activation Natural Long- Delay in culture (Rubella) after a pathogenic
Immun of humoral infection term initiation of condition Chickenp single form
ity and with immunolo immune s ox dose Maternal
cell- pathogen gic response (Varicella antibodies
mediated Immunizati memory ) can
responses on with a to the Shingles interfere with
in an vaccine antigen is (Zoster) immune
individual’s generate Influenza response to
own d (nasal the vaccine
immune mist) in infants
system by Rotavirus Require
exposure to careful
an antigen handling
Passiv Transfer of Passage of Provides Immunity is and storage
e antibodies IgG through immediat temporary, Inactivate Killed Intramusc Can Stimulates
Immun from the e declining d microorg ular polio safely be humoral
ity immunized placenta, protectio with anisms (Salk) given immunity but
host(s) to from n to the the half-life Influenza to little or no
nonimmune pregnant recipient of the (intramus immunoc cell-
individuals woman to antibodies cular or ompro- mediated
her fetus Immunologi intraderm mised immunity
Passage of c memory is al) individual May require
IgA through not Hepatitis s two or
breast milk, generated A more
from Hypersensit booster
mother ivity can doses
to infant develop, to produce
Standard especially protective
human when sera immunity
immune of animal Subunit One or Toxoids Induces Requires two
serum origin are more Purified an or more
globulin used purified proteins immune booster
Specific compone Polysacc response doses to
human nts of a harides to the produce
immune pathogen Recombi pathogeni protective
serum nant c immunity
globulin antigens compone Requires an
Antitoxins nt(s) of adjuvant to
Rhogam a increase
Monoclonal microorg immunogeni
antibodies anism city
Safer Must be
Adopti Transfer of Adoptive Can Patient’s than multivalent if
ve cells of immunother transfer own adminis- a broad
Immun Adoptive apy with cell- immune tration of immune
ity immunother activated mediated cells must an intact response
apy with TILs to immunity be depleted organism is desired
activated cancer to increase a. Bacterial Diphtheri Requires two
TILs to patients chance of Toxoids toxins a or more
cancer Hematopoi successful that Tetanus booster
patients etic stem therapy have doses to
Hematopoi cell Allogeneic been produce
etic stem transplantat cells may chemicall protective
cell ion be rejected y immunity
transplantat inactivate Requires an
ion d so adjuvant to
the that they increase
immune are not immunogeni
system to pathogen city
nonimmune ic
individuals

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 b. Biochemi Pertussis 6. Which of the following describes the properties of a
Purified cally puri- (whoopin toxoid?
Compone fied g a. Both pathogenic and immunogenic
nts compone cough) b. Pathogenic but not immunogenic
nts of
a
c. Not pathogenic but immunogenic
microorg d. Neither pathogenic nor immunogenic
anism 7. Suppose a vaccine was available in two forms:
c. Biochemi Streptoco Requires attenuated and inactivated. What is an advantage of the
Polysacc cally puri- ccal conjugation attenuated form?
harides fied pneumoni to a a. It can be used in immunocompromised
polysacc a carrier patients.
haride Haemoph protein to b. It induces both humoral and cell-mediated
from ilus induce
immunity.
bacterial influenza IgG
capsule e type b production c. There is no interference of the immune
Neisserial and long- response in infants by maternal antibodies.
meningiti term d. It does not require special handling and
s immunity storage to maintain its effectiveness.
d. Protein Hepatitis Highly Cannot be 8. What factor(s) influence the effectiveness of a person’s
Recombi produced B purified used to immune response to a vaccine?
nant by Human protein produce a. Age of the recipient
Antigen geneticall papilloma that is antigens b. The individual’s immune status
y virus safer other
modified (cervical, than than proteins c. The nature of the vaccine
nonpatho anal, administr d. All of the above
genic genital ation 9. An oral vaccine may be advantageous over an
bacteria, cancers) of intact injectable vaccine for a pathogen because it
yeast, organism a. reduces the risk of transmitting bloodborne
or other pathogens in developing areas of the world.
cells b. avoids the pain associated with injections.
c. induces mucosal immunity.
d. all of the above.
Review Questions 10. When one individual becomes immunized by receiving a
1. Suppose an individual develops antibodies in response series of vaccine injections according to schedule, the
to a streptococcal pharyngitis infection. This is an resulting protection extends to that individual’s nearby.
example of contacts. This concept is known as
a. active immunity. a. immunologic memory.
b. passive immunity. b. neighborhood immunity.
c. adoptive immunity. c. herd immunity.
d. immunoprophylaxis. d. contagious immunity.
2. Which of the following illustrates passive immunity? 11. Which preparation would you recommend for treatment
a. Development of high antibody titers in a of a patient with an antibody deficiency?
healthy person after receipt of the hepatitis B a. Monoclonal antibody
vaccine b. Specific human immune serum globulin
b. Recovery of a patient from a hepatitis A c. Standard human immune serum globulin
infection d. Animal serum antitoxins
c. Passage of IgG antibodies through the 12. Immunoglobulins consisting of a mouse-derived variable
placenta of a pregnant woman to her fetus region combined with a human-derived constant region
d. Transfer of tumor-infiltrating lymphocytes to a are known as
cancer patient a. monoclonal antibodies.
3. Which of the following is not a characteristic of passive b. chimeric antibodies.
immunity? c. humanized antibodies.
a. Transfer of antibodies d. fully human antibodies.
b. Occurs naturally or as a result of therapy 13. HAMA are
c. Provision of immediate protection a. mouse-derived antibodies that have been used
d. Development of long-term memory for therapy.
4. What was one of the major contributions of Louis b. monoclonal antibodies with therapeutic
Pasteur to vaccine development? benefits.
a. Development of the smallpox vaccine c. human antibodies that are produced against
b. Use of attenuated microorganisms in vaccines mouse proteins.
c. Inactivation of bacterial toxins for vaccines d. antitoxins that can provide immediate
d. Discovery of recombinant vaccine antigens immunity.
5. The antigenic component of the hepatitis B vaccine 14. What is a major characteristic of adoptive
differs from those of many of the conventional vaccines immunotherapy?
in that it consists of a a. It involves the transfer of cells to deliver
a. live, attenuated virus. immunity.
b. inactivated virus. b. It involves the transfer of cytokines to deliver
c. cryptic antigen. immunity.
d. recombinant antigen.

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 c. It can only occur in the presence of autologous
cells.
d. Its purpose is to increase the humoral immune
response.
15. Infusion of TILs into a cancer patient is an example of
a. active immunity.
b. adoptive immunity.
c. passive immunity.
d. natural immunity

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