THE INNATE AND ADAPTIVE IMMUNITY IMMUNOLOGY

SHERWIN ALVARO, RMT MARCH 07, 2022 AND SEROLOGY

TABLE OF CONTENTS TABLE OF CONTENTS

Significance Breakthroughs 1-4

Immunity 5

Adaptive Immunity 5

2 Types of Adaptive Immunity 5

Convetional Vaccines 6

Types of Vaccines 6

Types of CoVid19 Vaccines 7

2 Arms of Adaptive Immunity 8

Thinking Out of the Box 8

Natural Immunity 9 Legends: PPT, AUDIO, BOOK, ONLINE SOURCES

Significant breakthroughs
Name of Scientist/s or Year Contribution/ Discovery
Reported on Chinese practice of Variolation
- Inhalation of powdered small pox scabs.
Voltaire (1773)
- Variolation (inoculation)- method of scratching the skin and
applying the pulverized powder from a small pox scab.
Small pox vaccinations using cow pox virus.
- English country doctor
- Relationship between exposure to cowpox and immunity to small
pox.
! Edward Jenner (1798) - Deliberately inject a material from cow pox lesion and expose them
to small pox.
- Cross Immunity- exposure to one agent (cow pox) produced
protection against another agent (small pox)

! Albert Calmette and Developed the first successful vaccine against Tuberculosis
Camille Guerin (1921) Bacillus Calmette–Guérin (BCG) vaccine

Ernst Haeckel (1862) Phagocytosis

1881-1881: live, attenuated chicken cholera (Pasteurella multocida) and
anthrax vaccines (by accident)
! Louis Pasteur (Attenuation-weakening)
(Father of immunology) 1885: live attenuated vaccine for rabies
3 types of Vaccine
Cellular theory of immunity through phagocytosis (cells eat cells)
- Foreign objects were introduced to a starfish larva. It became
! Elie Metchnikoff (1883-1905) surrounded by motile cells that attempted to destroy these invaders.
- Noble Prize champion of Cellular Immunity.
Emil Von Behring, Humoral theory of immunity
Kitasato (1890) -Champions of humoral immunity

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 THE INNATE AND ADAPTIVE IMMUNITY IMMUNOLOGY
SHERWIN ALVARO, RMT MARCH 07, 2022 AND SEROLOGY

Demonstration of cutaneous hypersensitivity granuloma (specific reaction

! Robert Koch (1891) to Tuberculosis)
- Discovered vaccine for TB.
Gruber and Disvover agglutination reactions
Durham (1896) Agglutination- combination of antibody and a particulate antigen.
Devised an agglutination reaction for the diagnosis of typhoid fever
Ferdinand Widal (1896) -Widal Test- Uses H (Flagellar) and O (sOmatic) antigen.

Paul Ehrlich (1900) Antibody formation theory

Karl Landsteiner (1900) Discovery of the ABO Blood Group
P. Portier and Immediate hypersensitivity reaction
Charles Richet (1902) (Anaphylaxis- most severe form of allergy)
Discovered opsonins and described its relation to phagocytosis
- Awarded with Noble Prize
! Almoth Wright, - Opsonins- substances that coats the microbes to make them more
Stephen Douglas, prone to phagocytosis.
Joseph Denys (1903) - Opsonization- the recognized antigen are coated by an antibody
and/or complement to trigger the phagocytosis.
Describe the relationship of immunity and hypersensitivity.
Von Pirquet and -basis of mantoux test.
Schick (1906) - Mantoux Test- Screening test for TB.
Complement mediated Cytolysis
Pfeiffer, -Cytolysis-destruction of cells.
Buchner (1894-1895) - Complement- principal soluble mediator of inflammation.
Received Nobel Prize for his
pioneering works on
! Jules Bordet (1920) complement.
- Discovered the end product of complement activation which is Mac.

! Jhonas Salk, Development of polio vaccine

Albert Sabin
Worked out the genetics of the murine major histocompatibility complex
(MHC) and generated the congenic strains needed for its biologic
George Snell (1903) analysis.
-MHC- set of genes found in chromosome 6. They express the production
of Human Leukocyte antigen (HLA) which are found in all nucleated cells.

Developed the hemolytic plaque assay and several important

Niel Jerne (1911)
immunological theories including an early version of clonal selection

Peter Medawar( 1915-1987) Made studies on acquired immunologic tolerance.

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 THE INNATE AND ADAPTIVE IMMUNITY IMMUNOLOGY
SHERWIN ALVARO, RMT MARCH 07, 2022 AND SEROLOGY

Jean Dausset (1906) An early pioneer in the study of MHC or HLA.

Discovered immune response genes and collaborated in the first

Baruj Benacerraf (1920)
demonstration of MHC restriction

Worked out the polypeptide structure of the antibody molecule

Rodney Porter (1920-1985) (Y-shaped antibody), laying out the groundwork of its analysis by
protein sequencing

Developed RIA of peptide hormones

! Rosalyn Yalow (1921) -RIA- used for the diagnosis of diabetes.
- Noble Prize awardee
Described the action of lysozymes
Alexander Flemming (1922) -Discovered Penicillin

James Gowan (1924) Discovered that adaptive immunity is mediated by lymphocytes

! Cesar Milstein (1927) and Developed the hybridoma technique of monoclonal antibody formation.
Georges Kohler (1946) -Noble Prize Awardees

Made crucial discoveries about the structure of Ig, including the first
Gerald Edelman (1929)
complete sequence of antibody molecule.

Developed the quantitative precipitin assay.

Michael Heidelberger (1888-1991)
In 1930-1935, collaborated with Kendall in conducting quantitative
precipitin assay studies on antigen-antibody reactions (agglutination)

! Max Theiler (late 1930) Developed a vaccine against yellow fever

Discovered antibody diversity and T cell receptors diversity

- Discovered the somatic recombination of immunological receptors
Susumu Tonegawa (1939) genes that underlies the generation of diversity in human and murine
antibodies and T Cell receptors.

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 THE INNATE AND ADAPTIVE IMMUNITY IMMUNOLOGY
SHERWIN ALVARO, RMT MARCH 07, 2022 AND SEROLOGY

discovered interferons
- Interferons-Substances with antiviral proteins. They are serum
molecules which concentrations increase rapidly at the onset of
inflammatory disease.
- Mechanism of action of interferon: They block the translation of
viral proteins.
Types of interferons
Alic Isaacs and - Alpha Interferons (Type 1 interferon)- they are produced by
Jean Lindemann (1957) leukocytes type 1. They stimulate the production of natural Killer
Cells. NK cells are responsible in fighting for leukemia, non-
Hodgkin’s lymphoma and Kaposi sarcoma.
- Beta Interferons- produced by fibroblasts. They fight against
multiple sclerosis
- Gamma interferons (Type 2 interferon)- Produced by T cytotoxic
cells. Enhances the function of Natural Killer Cells to fight against
chronic granulomatous disease.

Isolated a retrovirus from a non-immune deficient homosexual man with

Luc Montaigner (1980) lymphadenopathy and called the virus lymphadenopathy associated
virus (LAV).

Robert Gallo (1980) Renamed the retrovirus as Human Immunodeficiency Virus (HIV)

Mosmann (1986) Th1 versus Th2 model of T helper function

E. Dounal Thomas and

Introduced the concepts of Transplantation
Joseph Murray (1991)
Identification of Toll-Like Receptors (TLRS)
1996-1998
- TLRS- involved in direct phagocytosis
Work on the mechanism of cellular immune response mediated by T-cells
towards virally infected cells.
- Noble Prize Awardee
- Principle of Double Recognition on how T lymphocytes responds
against the infected and cancer cells
! Peter Doherty and - B lymphocytes- responsible for humoral immunity. They are the
Rolf Zinkernagel (1996) major producers of antibodies.
- T Lymphocytes are responsible for cellular immunity.
- Cellular Immunity- there must be a presentation of antigen
presenting cells and an an association with MHC Class 1 or Class 2
Molecules.

Discovery of FOX3p, the gene

2001 directing regulatory -T-cell
development

discovery of the HPV (Human papilloma virus) vaccine

! Ian Frazer (2005) - Associated with warts.

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 THE INNATE AND ADAPTIVE IMMUNITY IMMUNOLOGY
SHERWIN ALVARO, RMT MARCH 07, 2022 AND SEROLOGY

IMMUNITY
! Complex reaction involving many different cells,
molecules and genes aimed essentially in ! The capacity to recognize materials as foreign to
maintaining the genetic integrity of an individual, itself and to neutralize, eliminate or metabolize
protecting it from invasion of substances that can them with or without injury to its own tissues.
bear the imprint of a foreign genetic code. ! It is part of a complex system of defense reactions
! The body’s ability to resist foreign organisms and of the body which can be classified by as either
toxins (poisons) that damage tissues & organs . innate immunity or acquired immunity.
Adaptive Immunity
TYPES OF IMMUNITY
2 TYPES OF ADAPTIVE IMMUNITY
NATURAL/ INNATE/ NON-SPECIFIC/ ACQUIRED/ ADAPTIVE/
NON-ADAPTIVE IMMUNITY SPECIFIC IMMUNITY ACTIVE PASSIVE
it is the product of the it is an immunity in which
Ability of an individual to resist
individual's own immune antibodies produce
infections by means of normally
system in response to elsewhere, which are
present body functions.
foreign antigen or given to the individual.
Nonspecific host responses provide
foreign substances
an effective barrier that prevents
microorganism from penetrating A reaction resulting Natural Active Natural Passive
from the invasion of a is the immunity that refers to the antibodies
and inhibits or destroys the invader
foreign substance. comes from infections transferred from the
if it gains access to your tissues The opposite of
and even your organs. encountered in life mother to the fetus
natural immunity. across the placenta and
Furthermore, it not only destroys Responsible for the
but also eliminates or neutralizes also to the newborn first
3rd line of defence few months of life
any toxic substances produced by
acquired from colostrum
that infectious agent. (breast milk)
Responsible for 1st and 2nd line of Artificial Active Artificial Passive
defence is stimulated by initial is the introduction of
HALL MARK FEAUTURES exposure to specific antibodies that are
foreign macro molecules formed by an animal or
LYMPHOCYTES through the use of a human to an individual
Reinforcement vaccines to artificially to prevent or treat an
Mechanisms involved are non- Inducibility establish a state of infection
specific Specificity immunity
Mechanisms that pre-exist the Diversity
invasion of foreign agents. Memory - it has the
Components are pre-formed. ability to recall
They are non-adaptive, has a previous exposure
standardized magnitude of with the same antigen
response. or micro organism
Lacks immunologic memory Specialization
Self-Limitation
Discrimination

Both of these systems are essential to maintain good health. They operate in concert and are dependent upon one
another for maximum effectiveness.

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 THE INNATE AND ADAPTIVE IMMUNITY IMMUNOLOGY
SHERWIN ALVARO, RMT MARCH 07, 2022 AND SEROLOGY

Conventional Vaccines
Vaccine is an antigen suspension derived from a pathogen that would be routinely administered to healthy
individuals to stimulate an immune response to an infectious disease; thus, vaccination is a type of immuno
prophylaxis or disease prevention through immunization.
TYPES OF VACCINES
Attenuated- being weakened. Not applicable to immunocompromised. Virus is injected, alive but weakened or
lessened.
Inactivated- produced by killing the organism but they are capable. Typhoid fever vaccine.
Toxoid- no longer toxin but are immunogenic and can be used ass Vaccine. Diphtheria Toxoid Vaccine.
Purified components - There is a biochemically purified component of the micro organism that is similar to toxoids
okay, such as a vaccine for pertussis or whooping cough.
Polysaccharide type of vaccine, in which they are also biochemically purified polysaccharide from a bacterial
capsule, specifically from the bacterial capsule, and one example of this type of conventional vaccine is the vaccine
for streptococcal pneumonia, and so on.
Recombinant antigen - this one contains a protein produced by genetically modified nonpathogenic bacteria, yeast,
or sets okay.
There are different ways to inoculate the vaccine, it can be through nonparenteral routes such as oral (are likely to
stimulate mucosal immune mucosal immunity, as well as humoral antibody production and cell mediated responses.),
intranasal, aerosol, transcutaneous, intradermal or rectal.
Vaccination and vaccines has a significant effect in transfusion medicine.

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 THE INNATE AND ADAPTIVE IMMUNITY IMMUNOLOGY
SHERWIN ALVARO, RMT MARCH 07, 2022 AND SEROLOGY

TYPES OF COVID-19 VACCINES

Pfizer Oxford Sinovac Gamaleya Bharat
Janssen MOderna Novavax
BioNTech AstraZeneca CoronaVac Sputnik V BioTech
Viral Vector Viral Vector
Technology Inactivated Viral Vector Inactivated Protein
mRNA (non- (non- mRNA
Platform Virus (non-replicating) Virus Subunit
replicating) replicating)
Philippines
January 14, January 28, February 22, March 19, April 19, May 5,
FDA EUA April 19, 2021 -
2021 2021 2021 2021 2021 2021
Approval [A]
2 doses, 21 2 doses, 2 doses, 28 2 doses, 3 2 doses, 28 2 doses, 2 doses,
Dose and days apart 4-12 weeks days apart weeks apart 1 dose [A] days apart 28 days 21 days
Frequency [A] apart [A] [A] [A] [A] apart [B] apart [C]
-25 to -15
-18C and C
Stroage -80C to 2 to 8C (3
2 to 8 C [A] 2 to 8 C [A] below (frozen 2 to 8C [A] 2-8C 2-8 C [H]
Requirements -60C months [A]
solution) [A] (30days
[E]
94.1%
70.4% 66.1-66.9%
91.6% agansint
against against
against symptomat Awaiting
Vaccine symptomatic 65-91% confirmed80.6%
95% symptomatic ic official
Efficacy COVID-19 (based on moderate to
against PCR- COVID-19
against COVID-19 [B] Phase III
Based on [A][B[ Brazil, severe/critical
confirmed
symptomatic [B] Interim
Phase III 100% Indonesia, COVID -19
synptomatic
COVID-19 100% against 100% JOurnal
Clinical Trial against and Turkey COVID-19
[A][B] moderate or against Publicatio
(CT) severe Trials) [A] 77-85% against [E]
severe cases severe n
COVID-19 severe
[B] COVID-19
[B] COVID-19 [J] [B]
! Local
lymphadeno ! Pain on
injection site,
pathy at the ! Pain/
hyperthermia,
injection site erythema
swelling [B]
! Allergic / swelling
reaction that ! Headache, on
asthenia,
may be injection
! Short – muscle/joint
caused by ! Injection site ! Headache site,
term, mild- ! Injection site pain ,
any pain, redness, ! Fatigue axiallry Awaiting
Common to- pain and malaise, sore
component swelling ! Fever lymphade official
Adverse Events moderate tenderness throat,
of thhe ! Tiredness, ! Body ache nopathy Phase III
Reported pain at the ! Fatigue, diarrhea,
vaccine headache, ! Abdominal [B] Interim
Observed in injection site headache, rhinorrhea,
(hives, muscle pain, pain ! Fever, Journal
Phase III CT ! Fatingue, feverishness, loss of
allergic chills, fever, ! Nausea headache Publication
headache myalgia [B] apetite, pain
rashes and nausea [G] ! Vomiting [E] , fatigue,
[B] in the
purpura, myalgia,
oropharynx,
anaphylactic arthralgia
nasal
shock) , nausea,
congestion,
! Convulsion vomiting,
colds,
(with or chills [B]
sneezing,
without
cough [B]
fever) [I]

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 THE INNATE AND ADAPTIVE IMMUNITY IMMUNOLOGY
SHERWIN ALVARO, RMT MARCH 07, 2022 AND SEROLOGY

Thinking out of the box 2 ARMS OF ADAPTIVE IMMUNITY

Can we donate blood? If we got COVID? Can CELL MEDIATED (CELLULAR)

HUMORAL (HI)
(CMI)
we donate blood if we just got if we just got vaccine a
few days ago, three days ago, etc.?: It depends on the Mechanism - Antibody
Cell mediated
technology platform of the vaccines response to mediated
transfusion medicine. Cell type - B-
T-lymphocytes
lymphocytes
Can we administer vaccine to previously
infected individual (ex. CoVID-19 infection): Yes. Direct cell-to-cell contact
(Research for more info). (Antigen Presenting Cells
Mode of Action -
[APC] and Major
Antibodies in biologic
Is it safe to administer two/or more different fluid
Histocompatibility complex
vaccines?: it depends on the interaction of the two [MHC]) or soluble products
vaccines, the individuals health status who will receive secreted by cells
the vaccine and other factors should be considered. Defense against viral and
Function - Primary
fungal infections, intracellular
Are the number of booster shots will be defense against
organisms, tumor antigens,
administered equally to all the individuals who bacterial infections
and graft rejection, parasites
received vaccination?: It depends on the individuals
progress of immunity, reaction and other factors. Nature of infecting
antigen - Circulating Intracellular organism
(Research for more info). Extracellular Antigen
Is it necessary to take the booster shot?: Yes, Chronic, granulomatous
because it adds additional protection against infection. Type of infection -
infection, neoplasm, fungal,
Acute pyogenic infection
During a pandemic, vaccination is priority to achieve parasitic diseases
herd immunity. Contact sensitivity, DTH,
Are the dosage of vaccines administered to Variants - Ab-mediated Allograft rejection, GVH
hypersensitivities response, elimination of
children similar to adult vaccine dosage? Yes. The only Autoimmunity tumors, formation of chronic
difference for the children and adult vaccine are the granulomas
following; type of vaccine and what is reaction of the
immune system to the vaccine. *Take note of the action

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 THE INNATE AND ADAPTIVE IMMUNITY IMMUNOLOGY
SHERWIN ALVARO, RMT MARCH 07, 2022 AND SEROLOGY

NATURAL IMMUNITY

(a) Intact Skin (healthy skin, no portal of entry) Sweat (Lactic acid=pH 5.6)
(b) Mucous membranes of the respiratory and GI tract
A. Structural/
(c) Ciliated Epithelium
Physical Barriers
(d) Lacrimal apparatus (Tears=Lysozyme vs. Gram positive bacteria)
(e) Sebaceous glands (sebum and fatty acids)
EXTERNAL DEFENSE (a) Peristaltic movement of intestine BOTULIN (Clostridium botulinum) canned
MECHANISMS good poisoning
B. Mechanical
Prevents most of the Barriers (b) Shedding of cells
infectious agents to (c) Coughing and sneezing
(d) Flushing action of urine
enter the body
(a) Acid pH
" Normal flora (Lactobacillus acidophilus)
C. Chemical " Competitive exclusion
Barriers " Pathogen and opportunistic
(b) Lysozyme
(c) Lactoferrin
(a) Body temperature
A. Physiologic
(b) Oxygen tension
Factors
(c) Hormonal balance
B. Basic (a) Spermin
polypeptides (b) Defensin
Types:
(a) Alpha IFN
C. Interferons
(b) Beta IFN
(c) Gamma IFN
D. Complement -
! Most commonly requested acute phase
protein in the lab. To determine if there
is inflammation.
! Primitive antibody against C
polysaccharide of Pneumococcus (S.
pneumoniae)
" 0.5 MG/DL
" up to 1000X
(a) CRP " Males: up to 1.5 mg/L
" Females: up to 2.5 mg/L
! CLINICAL USES:
" Marker of acute inflammation
" monitor antibiotic/chemo-
therapy

# CRP Agglutination Test

E. Acute phase # hsCRP (Quantitative)
proteins/Reactants
(b) Serum Amyloid A
(c) Mannose Binding Protein/
Binds with mannose
Lectin

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 IMMUNOLOGY
NATURAL IMMUNITY AND SEROLOGY
SHERWIN ALVARO, RMT MARCH 15, 2022

TABLE OF CONTENTS C. Interferons

External Defense Mechanisms 1 Group of molecules that limit the spread the viral
Internal Defense Mechanisms 1 infections by blocking the translation of viral proteins
Phagocytosis 2
Pathways of killing pathogens by 3 Types:
Phagocytes c. Alpha IFN
Inflammation 4 d. Beta IFN
e. Gamma IFN
NATURAL IMMUNITY D. Complement
Principle soluble mediator of inflammatory response
Components:
E. Acute phase proteins/Reactants
1. External Defense Mechanisms
A. CRP (C reactive protein)
A. Structural/Physical Barriers
a. Intact Skin -Most commonly requested acute phase protein in the
b. Mucous membranes of the respiratory and GI lab. To determine if there is inflammation.
tract -Primitive antibody against C polysaccharide of
c. Ciliated Epithelium Pneumococcus (S. pneumoniae)
d. Lacrimal apparatus - 0.5 MG/DL
e. Sweat, sebaceous glands - up to 1000X
-Males: up to 1.5 mg/L
B. Mechanical Barriers -Females: up to 2.5 mg/L
a. Peristaltic movement of intestine BOTULIN
(Clostridium botulinum) Canned-good CLINICAL USES:
poisoning -Marker of acute inflammation
b. Shedding of cells
-monitor antibiotic/chemo-therapy
c. Coughing and sneezing
d. Flushing action of urine
 CRP Agglutination Test (Qualitative)
- Positive/negative results
C. Chemical Barriers - No specific value of CRP
a. Acid pH  hsCRP (Quantitative)
 Normal flora (Lactobacillus ) - gives the exact number of the increased
CRP on the sebum due to inflammation
 Competitive exclusion
- early predictor of myocardial infarction
 Pathogen and opportunistic and cerebral vascular accidents
b. Lysozyme
c. Lactoferrin (found in Human Milk) b. Serum Amyloid A
- remove cholesterol
c. Mannose Binding Protein/ Lectin
2. Internal Defense Mechanisms
 Binds with mannose
d. Alpha-1-antitrypsin
A. Physiologic Factors
a. Body temperature (36.5-37.5°C)  General inhibitor of protease
b. Oxygen tension  Neutralizes elastase (digestive enzymes found
c. Hormonal balance in the granules of neutrophils; degrades
elastin and collagen)
B. Basic polypeptides e. Haptoglobin
a. Spermin (pH dependent; found in semen) - binds hemoglobin released during intravascular
b. Defensin (Human neutrophil) hemolysis
f. Fibrinogen
- primary and most abundant coagulation factor

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g. Ceruloplasmin F. Macrophages
- binds and transports copper or ferroxidase - larger version of monocytes on tissues. Major
(converts ferrous to ferric) phagocyte of the body, active against
h. Alpha-1-acid glycoprotein intravascular organisms
i. Endogenous pyrogens: IL-1 - granules contain no peroxidase at all compared
with monocytes.
F. Cellular Defense Mechanisms - Monocyte-macrophage system functions in
A. Neutrophils microbial killing, tumoricidal activity, killing of
- 50-70% of circulating WBCs intracellular parasites, phagocytosis, secretion of
Primary granules cell mediators and antigen presentation (APC)
-contain myeloperoxidase, elastase, proteinase
3, lysozyme, cathepsin G, defensins Macrophages are named based on their location:
Lungs = Alveolar macrophage
Secondary granules Liver = Kupffer macrophage
-contain collagenase, lysozyme, lactoferrin, Brain = Microglia cell
plasminogen activators, ALP, NADPH Connective tissues = Histiocytes
Bones = Osteoclast
Tertiary granules Kidney= Mesangial cells
-contain gelatinase and plasminogen activator
-Capable of the process of diapedesis G. Dendritic cells
- Major antigen-presenting cell
B. Eosinophils - function is to phagocytosed antigen and
- 1-3% of circulating WBCs present it to T-helper cells
- increases in allergic reactions in parasitic - most potent phagocytic cell in the tissues.
diseases
- Primary granules Dendritic cells are named based on their
-contain ACP, arylsulfatase location:
-Secondary granules Skin and mucous membrane = Langerhans cells
-contain Major Basic Protein, eosinophil Vital organs = Interstitial cells
cationic protein, eosinophil peroxidase, Lymphoid organs = Interdigitating cells
eosinophil- derived neurotoxin,
phospholipase, histaminase, aminopeptidase PHAGOCYTOSIS
and ribonuclease.
- engulfment of cells and particulate matter by leukocyte,
C. Basophils macrophage
- Less than 1% of circulating WBCs and other cells.
- bluish-purple granules contain histamine,
eosinophil chemotactic factor of anaphylaxis, and Steps:
heparin.
- involved in hypersensitivity reactions 1. Physical contact between the WBC and the foreign
particle
D. Mast cells
-Granules contain ACP, ALP, and protease 2. Formation of phagosome

E. Monocytes 3. Fusion with lysosome

-largest WBC,
-constitutes 4-10% of circulating WBCs 4. Digestion and release of debris to the outside
- granules contain peroxidase, ACP, arylsulfatase
- other type of granule contains B-glucuronidase,
lysozyme and lipase
-ground glass and kidney-bean shaped
appearance

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Creation of oxygen radicals in the phagocytic cell. The

hexose monophosphate (HMP) shunt reduces NADP to
NADPH. NADPH reduces oxygen to superoxide (2O2-)
when the NADPH oxidase complex (NOC) is assembled in
the membrane of the phagolysosome. Superoxide
dismutase (SOD) catalyzes the conversion of superoxide to
hydrogen peroxide (H2O2). Myeloperoxidase (MPO)
catalyzes formation of hypochlorite (OCl_), a very powerful
oxidizing agent. Hydroxyl radicals (•OH), which are also
powerful oxidizing agents, may also be formed if iron ions
are present

A. Adherence: physical contact between the 2. Oxygen Independent

phagocytic cell and the microorganism occurs,
aided by opsonin. - Production of nitric oxide from oxidation of L-
B. Engulfment: outflowing of cytoplasm to surround arginine by NO synthase which is produced by
the microorganism. IFN-gamma activated cells.
C. Formation of phagosome: microorganism is - It uses activated phagocytic cells which also
completely surrounded by a part of the cell synthesizes lysozymes in various hydrolytic
membrane. enzymes like elastase whose degradative
D. Formation of the phagolysosome: contents of the enzymes that do not require oxygen.
lysosome are emptied into this membrane-bound
space.
E. Digestion of the microorganism by hydrolytic TYPES OF PHAGOCYTOSIS
enzymes. INDERECT DIRECT
F. Excretion: contents of phagolysosome are Via opsonin receptors that Via Pattern Recognition
expelled to the outside by exocytosis. recognize opsonins such as Receptors that recognize
IgG, CRP, and C3b bound lipid and carbohydrate
PATHWAYS OF KILLING PATHOGENS BY to microorganisms. sequences on
PHAGOCYTES microorganisms.

G. Toll-like receptors (TLRs)

1. Oxygen Dependent - is a protein originally discovered in the fruit fly
Drosophila; where it plays an important role in
o Respiratory Burst- occurs when the antifungal immunity in the adult fly. Very similar
cytoplasmic pseudopods enclosed the molecules were found on human leukocytes and
particle within a vacuole. some other cell types.
- The highest concentration of these TLRs occurs on
monocytes, macrophages, and neutrophils.
- There are 11 slightly different TLRs in humans. Each
of these receptors recognizes a different microbial
product.
- For example, PRRs = pathogen-associated
molecular patterns (PAMPs)

RECEPTOR SUBSTANCE TARGET

RECOGNIZED MICROORGANISM
TLR1 Lipoprotein Mycobacteria
TLR2 Teichoic acid, Gram-positive
Peptidoglycans bacteria
TLR4 Lipopolysaccharide Gram-negative
bacteria
TLR5 Flagellin Mycobacteria

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Cardinal Signs:
INFLAMMATION
1. Rubor - redness
- the overall reaction of the body to injury or invasion by 2. Calor – presence of heat
an infectious agent. 3. Tumor - swelling
4. Dolor - pain
TYPES OF INFLAMMATION 5. Functio Laesa – loss of function
1. Acute inflammation (mediated by
neutrophils)
2. Chronic inflammation (mediated by REFERENCES:
monocytes or macrophages that leads to
permanent tissue damage)
 Turgeon, Mary Louise. (2014). Immunology
Major Events: &serology in laboratory medicine, (5th ed.).
Missouri :Elsevier.
1. Tissue damage cause release of vasoactive and  Male, David. (2013). Immunology, (8th ed.).
chemotactic factors that trigger a local increase in blood St.Louis, Missouri : Elsevier.
flow and capillary permeability.  Stevens, Christine Dorresteyn. (2010).
Clinicalimmunology & serology : a laboratory
2. Permeable capillaries allow the influx of fluids and perspective,3rd ed. Philadelphia ; F.A. Davis
cells.

3. Phagocytes migrate to the site of the inflammation.

“For I know my plans for you, declares by the Lord, plans to
prosper you and plans not to harm you, gives you future and
4. Phagocytes and anti-bacterial exudates destroy
pathogen. hope.”
- Isaiah 29:11

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 IMMUNOLOGY
THE IMMUNE SYSTEM AND SEROLOGY
SHERWIN V. ALVARO, RMT , MPH March 15, 2022

TABLE OF CONTENTS
IMMUNE SYSTEM 1
ACQUIRED IMMUNITY 1
THE LYMPHOID SYSTEM 1
NATURAL KILLER CELLS 5
MECHANISMS OF NK CELL CYTOTOXICITY 5
LABORATORY IDENTIFICATION OF 5
LYMPHOCYTES

Immune System
- our body's major safeguard against infection,
illness, and disease. 2 MAJOR STAGES OF LYMPHOCYTE
- This system is a vast network of cells, tissues and DEVELOPMENT
organs that coordinate as our body defenses 1. Antigen Independent Stage of Lymphopoiesis
against any threats to our health. -takes place on primary lymphoid organs
2. Antigen Dependent Stage of Lymphopoiesis
• Lymphatic system -takes place on secondary lymphoid organs
• Computerized war machine
• “Defense department of the body” THE LYMPHOID SYSTEM

CELLS OF THE IMMUNE SYSTEM 1. Primary/Central Lymphoid Organs

1. Myeloid Lineage • developmental sites; maturation site of
2. Lymphoid Lineage lymphocytes

Major Functions of the Immune System: a. Bone Marrow

1. Block harmful agents ✓ main source of hematopoietic stem cells
2. Seek-out invasive pathogens ✓ Maturation site for B cells
3. Isolate and neutralize activity of that antigen ✓ Center for antigen-independent lymphopoiesis.

ACQUIRED IMMUNITY b. Thymus

✓ Maturation site for T cells
• type of resistance that is characterized by ✓ small, flat bi-lobed organ found in thorax or
specificity for each individual pathogen chest cavity right below the thyroid gland and
• the ability to remember a prior exposure overlying the heart.
(MEMORY) which results in an increased response • An endocrine gland - produces hormone=Thymosin
upon repeated exposure. • Cortex- where thymocytes can be found (85% of
population of T cells)
LYMPHOCYTES • Medulla - where mature T cells can be found.
• the key cell involved in adaptive immunity. • Thymic stromal cells - include epithelial cells,
macrophages and dendritic cells
• Represents approximately 20% of the circulating
WBCs.
• Has a large rounded nucleus which contains a
dense nuclear chromatin

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SHERWIN V. ALVARO, RMT , MPH March 15, 2022

SPLENECTOMY
- removal of spleen
- lead to increased platelet count
- prone to infection with encapsulated bacteria

Because the spleen is removed, 1/3 of the produced

platelets will go to the blood vessel, added to the
circulating platelets.

b. White pulp - contains lymphoid tissue that is

arranged around arterioles as PALS
(Periarteriolar Lymphoid Sheath)

o T cells - found near the central arteriole.

o Naïve B cells/ Virgin B-
cells/Unstimulated/Inactivated B-cells - are found
on primary follicles
o Activated B cells (Plasma Cell And Memory Cell)
2. Secondary/Peripheral Lymphoid Organs
- are found on Secondary Follicles
• activation sites for lymphocytes
(germinal centers)
• What is the cause of the activation of the o Marginal zone contain macrophages
lymphocyte? Antigen
A. Encapsulated Organs
1. Spleen - largest secondary lymphoid organ that
SITES OF ENTRY OF LYMPHOCYTE/ANTIGEN INTO
is found on the ULQ of the abdomen just below
SPLEEN:
the diaphragm.
a. Specialized Capillaries on marginal sinus
b. Via trabecular artery

2. Lymph Nodes
• junctional filter of the lymphoid system.
• Sizes may range from 1mm to 25 mm.
• Cortex - B-cell area; also contains
primary follicles which small amount of T
cell and follicular dendritic cells and
secondary follicles that contains germinal
center.
• Paracortex - T-cell area
• Medulla- contains differentiated cells
and APCs
Main Types of Splenic Tissue:
o Axillary Lymph Nodes
A. Red Pulp - involved in culling process, pitting
o Inguinal Lymph Nodes
AND platelet sequestration
o Cervical Lymph Nodes
- Culling- process of removing old or synesent RBC
- Pitting- procees of removing abnormal inclusions
of RBC

• 1/3 PRODUCED PLATELETS - stored in spleen

• 2/3 CIRCULATING PLATELETS (Blood vessel)

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 IMMUNOLOGY
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SHERWIN V. ALVARO, RMT , MPH March 15, 2022

o CD MARKERS FOR T-CELL: CD2, CD3, CD4, CD8

o CD MARKERS FOR B-CELLS; CD19, CD21, CD23
o CD MARKERS FOR NK (NATURAL KILLER) CELLS:
CD16, CD56, CD94

Lymphocyte traffic/recirculation

• movement of lymphocytes from blood to

lymphoid organs and back to the blood.
How will T and B cells find its way to your lymph nodes?
Through the post-capillary traffic endothelial venule or
High endothelial venules (HEV)
HEV has addressins and other adhesion molecules which
includes the CD34 and CD102 marker STAGES OF B-CELL DIFFERENTIATION
T and B cells in HEV have homing receptors which is the L- 1. Pro-B Cells
selectin
- markers include CD19, CD45R, TdT, RAG-1and RAG-2
Site of Entry and Exit of Lymphocytes in the lymph enzymes
node:

• Afferent Arteriole
• Tdt - terminal deoxyribonucleotide transferase
• Efferent arteriole
• RAG - recombinase activating gene
B. Non-encapsulated organs
• Mucosal Associated Lymphoid Tissue
2. Pre-B cells
- small masses of lymphoid tissue found in intestinal,
genitourinary tract and respiratory tract. - contains pre-B cell receptor that is made up of 2 mu
heavy chains and a surrogate light chain whose function is
BALT (BRONCHUS ASSOCIATED LYMPHOID TISSUE) to transmit signals to prevent rearrangement of any other
heavy chain genes.
- includes the tonsils, adenoids
B cells are major producers of antibodies.
GALT (GUT ASSOCIATED LYMPHOID TISSUE)
ANTIBODY
- Peyer’s patches, appendix
o made of monomer as basic unit
CALT (CUTANEOUS ASSOCIATED LYMPHOID TISSUE)
MONOMER
- includes intraepidermal lymphocytes
o y-shaped molecule
CD – CLUSTER OF DIFFERENTIATION o made of 2 heavy chains and 2 light chains
-membrane proteins that serve as surface marker for HEAVY CHAINS
certain blood cells
o GAMMA, ALPHA, MU, DELTA, EPSILON

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 IMMUNOLOGY
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SHERWIN V. ALVARO, RMT , MPH March 15, 2022

LIGHT CHAIN marker: you have dengue in the past, and still
experiencing dengue in the present)
o KAPPA, LAMBDA
• marker of lifelong immunity
Mnemonic:
Ig(M)= Maaga 🌞 – early, or present/ right
3. Immature B-cells
now
- recognized by the appearance of complete IgM Ig(G)= Gabi 🌚– late, or past
molecules on their surface. • CD 19- present in all population of B cells.
- Specificity of the surface immunoglobulin to be
synthesized can already be predicted or noted. DENGUE IgM: +
DENGUE IgG: +
- Surface markers that can be seen include receptors for • convalescent stage/recovery phase of
complement components such as C3d, CD21. infection
- B-cells capable of producing antibody to self-antigens
are deleted in the marrow through APOPTOSIS

4. Mature B cells
- exhibit IgD and more IgM on their surface as well as
MHC Class II products

5. Activated B cells
- exhibit CD25 on surface. CD25 in turn acts as receptor
for IL-2
- CD25 + IL-2 --------→ proliferation of activated
lymphocytes

6. Plasma Cells
-large spherical, ellipsoidal cells that contain abundant
cytoplasmic immunoglobulins and little to no surface
immunoglobulins.
- also called short-lived cells

7. Memory B Cells STAGES OF T CELL DIFFERENTIATION

- progeny of antigen stimulated B-cells that are
capable of responding to antigen with increased 1. Pro-thymocyte
speed and intensity.
- possess CD44 and TdT
• -SURFACE IgG
• -long-lived cells 2. Double Negative Thymocyte

IgM - marker of acute/present/active infection -lack CD4 and CD8 antigens.

(currently experiencing dengue) -possess CD2, CD5, CD7, CD45R
IgG - marker of chronic/past infection (has
past exposure/infection to dengue; Chronic 3. Double Positive Thymocyte

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 IMMUNOLOGY
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SHERWIN V. ALVARO, RMT , MPH March 15, 2022

- express both CD4 and CD8 antigens on their surface as - NKG2D - binds to MICA and MICB proteins on
well as CD3-αβ (TCR) diseased or cancerous cells

- would undergo Positive and Negative Selection - If an inhibitory signal is not produced, NK cells will
release PERFORINS AND GRANZYMES
4. Mature T cells
- represents those population of thymocytes that had
survived positive and negative selection. 2. Antibody Mediated Cell Cytotoxicity

- express only 1 of either CD4 or CD8 on their surface. - Through binding of IgG-coated cell with CD 16

5. Activated T cells
- express receptors for IL-2 and produce cytokines. LABORATORY IDENTIFICATION OF LYMPHOCYTES

• Cytokines functions include assisting B cells in the

commencement of antibody production, killing of 1. Density Gradient Centrifugation with Ficoll-Hypaque
tumors and target cells, rejection of grafts, 2. Flourescence Microscopy
stimulation of hematopoiesis in the bone marrow
and initiation of delayed hypersensitivity allergic a. Direct Immunofluorescence - use
reactions. monoclonal antibodies with a fluorescent
tag (e.g. fluorescein and phytoeryhtrin
6. T memory cells [490 nm]; rhodamine [545 nm].
b. Indirect immunofluorescence - uses
unlabeled antibody that first combines
NATURAL KILLER CELLS with the antigen by itself and a second
antibody that is complexed with a dye.
- Larger than T and B cells; contains a kidney shaped 3. Cell Flow Cytometry/Fluorescence Activated Cell
nucleus with condensed chromatin and prominent nucleoli. Sorter
- Has high cytoplasmic:nuclear ratio - an automated system for identifying cells based on the
scattering of light as cells flow in single file through a
- Cytoplasm has many azurophilic granules.
laser beam. Fluorescent antibodies are used to screen of
- Constitutes 5-15% of the circulating lymphoid pool; subpopulation of T and B cells.
found mainly in spleen and blood.
Components:
- No specific surface markers
a. Sample delivery system
- Possess CD16, CD56, CD 94 b. A laser for cell illumination
c. Photodetectors for signal detection
- Lack CD3, 4 and 8 d. Computer based management system
- Play a complementary role to CD8+ T cells 4. Rosetting
- Become LAK cells in response to IL-2 5. ELISA
COMPARISON OF T AND B CELLS
MECHANISMS OF NK CELL CYTOTOXICITY T cells B cells
Developed in the thymus Developed in the bone
marrow
1. It is brought about by the balance between activating
and inhibitory signals that enables NK cells to distinguish Found in blood (60-70% Found in the bone marrow,
healthy cells from infected or cancerous cells. of circulating spleen and lymph nodes
lymphocytes), thoracic duct
- Killer cell inhibitory receptors (KIRs) fluids, lymph nodes

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 IMMUNOLOGY
THE IMMUNE SYSTEM AND SEROLOGY
SHERWIN V. ALVARO, RMT , MPH March 15, 2022

Identified by rosette Identified by surface

formation with sheep RBC immunoglobulins

End product of activation Antibody

are cytokines

Antigens include CD2, 3, CD19, 20, 21, 40 and

4, and 8 MHC Class II

Located in the Located in the cortical

paracortical region of region of lymph nodes
lymph nodes

REFERENCES:

• Turgeon, Mary Louise. (2014). Immunology &

serology in laboratory medicine, (5th ed.). Missouri :
Elsevier.
• Male, David. (2013). Immunology, (8th ed.). St. Louis,
Missouri : Elsevier.
• Stevens, Christine Dorresteyn. (2010). Clinical
immunology & serology : a laboratory perspective,
3rd ed. Philadelphia ; F.A. Davis

“Commit to the LORD whatever you do, and he will

establish your plans.”
- Proverbs 16:3

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 ANTIGEN, ADJUVANTS, AND MHC IMMUNOLOGY
SHERWIN ALVARO, RMT MARCH 22, 2022 AND SEROLOGY

TABLE OF CONTENTS 4. Health status of the host

Immunocompetent /Immunosuppressed
Antigens 1
5. Genetics
2 Main Parts of Antigen 2 ◇ Involves the espond to a particular immunogen
Types of Antigens 3 ◇ MHC/ Major Histocompatility Complex-has a
clinical significance to the receptors generated
Adjuvants 4 by T and B cells (part of immune system,
another factors influencing immune response)
Major Histocompatibility Complex 4
Characteristics of an Immunogen:
Nature of HLA Antigens 4
ability of an immunogen to stimulate a host response
Structure Of MHC Class I and II Molecules 4 1. Foreignness
Antigen Presentation 4 Ability of the immunogen to stimulate a host response
kapag mas foreign sya
MHC Restriction or MHC Restricted Recognition 5
2. High Molecular Weight
HLA Disease Association 6
• >10,000 Daltons
Histocompatibility Testing 7
-The greater the molecular weight the better
-Should be macromolecular
ANTIGENS -immunogenic
a substance that reacts with antibody or sensitized T cells but • < 10,000 Daltons- non-immunogenic
may or not may provoke an immune response.
3. Molecular/Chemical Complexity
It may or may not cause antibody formation/ may or may not
provoke an immune response More complex structure-more immunogenic
IMMUNOGEN • Proteins –more
immunogenic complex
as an antigenon structure, more
a macromolecule capable of eliciting the formation of • Carbohydrates
immunoglobulins or sensitized T cells in an immunocompetent • Glycoproteins
host. • Glycolipids
• Lipids
◇ Refers to a substance that reacts with an antibody or a • Nucleic Acids –poor immunogens
sensitized T cells that MAY evoke immune response
4. Digestability
◇ Ideal term for foreign substances in capable of eliciting
formation of immunoglobulins ◇ Ability of the antigens to be processed ans to be
presented with your MHC molecules
◇ Causes antibody formation (ibig sabihin may immune
response) 5. Availability and Accessibility of Reactive Sites
◇ All immunogens are antigens but not all antigens are 2 Main Parts Of Antigen
immunogens.
Factors Influencing the Immune Response:
A. Antigenic Determinant/Epitope
1. Age
molecular shape or configurations that are recognized by
◇ Babies and Elderlies/Geriatrics-lower immune antibody or T cells. May be either linear or conformational,
response or may have repeating units or different specificities.
◇ 4-6yrs. Fully developed antibodies, immune
system, immunity. Protein epitopes-most likely to produce an immune response
2. Dose recognized by B cells.
◇ Small amount of immunogen/ foreign substances Kung san kumakapit yung B cells or antibodies for an immune
-1st and 2nd line of defense (Innate/ Natural response
Immunity)
◇ Larger dose of immunogen -3rd line of defense
(Adaptive immunity)
3. Route of Inoculation
a. Intravenous –through veins
b. Intramuscular -muscles
c. Intradermal –through the skin
Subcutaneous-beneath the skin
Parenteral/ oral

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 ANTIGEN, ADJUVANTS, AND MHC IMMUNOLOGY
SHERWIN ALVARO, RMT MARCH 22, 2022 AND SEROLOGY

• HAPTEN drug protein conjugate that can result in a life

threatening allergic response
Ex: Penicillin
1917 (The Specificity of Serological Reactions)
- publication of one of the well-known book which study
how Hapten work
- by Karl Landsteiner
HAPTEN

Types Of Epitope:
1. Linear Epitope
◇ Also called sequential
◇ Amino acids follow one another on a single chain
◇ There is only one single chain
Illustration refers to how hapten works.
2. Conformational epitope 1st Illustration- Reacts with an antibody but with no complexes
to the carrier
Result from the folding of one chain or multiple chains
bringing certain amino acids from different segments If that hapten is complexed to a carrier at multiples sites there
of a linear sequence/s into close proximity with each would be an agglutination/immune complex that will take
other so they can be recognized together place.
✓ B-cells: Surface antibody on B cells may react B. Carrier
with both linear and conformational epitopes
present on the surface of an immunogen. responsible to give the antigen its required size
✓ T-cells: T-cell epitopes are linear pinaglalagyan ng antigenic determinants
yung malaking oval in the picture
Antigenic Determinant/Epitope
• VALENCE
• HAPTEN - number of combining sites.
- non-immunogenic materials that , when combined with a
carrier, create new antigenic determinants.
TYPES OF ANTIGENS
- Some substances are too small (< 10,000 Daltons), to
be recognized by themselves but if they are complex to 1. Autoantigens/SELF-ANTIGEN
larger molecules they are then able to stimulate an
immune response - antigens that belongs to the host.
- New antigenic determinants-dito didikit yung mga - Abonormal in cases of rheumatoid arthritis, systemic
antibodies glucose erythematosus/ SLE, Type 1 DM, multiple
sclerosis (also called autoimmine diseases)
- they only become immunogenic when they bind with 2. Alloantigens/ISOANTIGEN
particles
- derived from the body of other individuals of the same
Examples: species.
(1) poison ivy –RHUS RADICANS- contain chemical - Capable of eliciting an immune response
substances CATECHOLS which are haptens
- Important to consider in tissue transplantation and
when they are bind on the skin they become blood transfusions
immunogenic that will lead to a condition called
CONTACT DERMATITIS
(2) drug related –coupling with normal proteins in the
body to provoke an immune response with a certain

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 ANTIGEN, ADJUVANTS, AND MHC IMMUNOLOGY
SHERWIN ALVARO, RMT MARCH 22, 2022 AND SEROLOGY

3. Heteroantigens ADJUVANTS
- derived from other species. (animals, plants,
microorganisms) - a substance administered with an immunogen that
- individuals exposed to these antigens that he/she lacks increases immune response.
will have an immune response - Acts by producing a local inflammatory response that
4. Heterophile antigens attracts a large number of immune system cells to the
injection site (intramuscular)
- those that exist in unrelated plants or animals but
which are either identical or closely related in structure Examples:
so that that antibody to one will crossreact with • aluminum salts
antigen of the other.
Ex. Polysaccharide type XIV of pneumococcus reacting –only approved for clinical use in US
with anti-A antisera. Used to complex to immunogen to increase its size and
OTHER CATEGORIES OF ANTIGENS prevents rapid scape from tissues
• complete Freund’s adjuvant (not FDA approve, produces
granulomas or large areas of scar tissues)
Thymus dependent Thymus Independent Composition:
antigen antigen o mineral oil
o emulsifier
Capable of mounting Can mount immune o killed Mycobacteria or Bordetella (0.5 g/ml)
immune response response without the help
provided there's from T-helper cells Ways of Enhancing the immune response by adjuvants:
assistance from T cells
• It prolongs the existence of immunogen in the area
Capable of stimulating Capable of stimulating B
B cells cells • It increases the effective size (high molecular weight)
of an immunogen
Capable of inducing Not capable of inducing • It increases the number of macrophages involved in
production of memory proliferation of memory antigen processing. (stimulate phagocytic cells)
cells cells • Stimulate T-cells enhances CMI
Can induce B cell to Only IgM can be • Stimulate B-cells enhances humoral immunity
form different produced
immunoglobulin classes MAJOR HISTOCOMPATIBILITY COMPLEX
Examples: Examples:
viral hemagglutinin,
• Large multi-gene locus consisting of several thousand
kilo base pair of DNA on a single chromosome.
diphtheria toxin, PPD Type 1:
(Purified Protein - bacterial • MHC complex forms a group of closely linked genes
polysaccharide, that controls not only the exchange of tissues (tissue
Derivative) compatibility) but also the myriads cellular interaction
Brucella abortus of immune cells, the production of certain serum
- HAVE INHERENT proteins and the production of some cytokines and
MITOGENIC enzymes.
CAPACITY
Type 2:
• In humans , it is referred to as Human Leukocyte
Antigen (HLA) complex located at (short arm,
- pneumococcal autosomal dominant gene, inherited in both parents)
polysaccharide, chromosome # 6
salmonella
polymerized
• This is found in all nucleated cells in the body
flagellin.hapten • Has a vital role in humoral and cellular immunity
conjugated ficoll
(polysucrose), dextran
• P arm-short arm; q arm-long arm
- HAVE LINEARY Jean Dausset-MHC
ARRANGED
MONOTONOUSLY
REPEATING EPITOPES

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 ANTIGEN, ADJUVANTS, AND MHC IMMUNOLOGY
SHERWIN ALVARO, RMT MARCH 22, 2022 AND SEROLOGY

NATURE OF HLA ANTIGENS: STRUCTURE OF MHC CLASS I and II MOLECULES

1. Glycoprotein component of cell membrane MHC CLASS II

2. Present in all nucleated cells • Much more restricted than MHC CLASS I
3. Products of the genes of MHC
• Inheritance of HLA genes CLASS I CLASS II
o controlled by a MHC located in the short
arm of chromosome # 6 1. Genetic loci HLA-A, B, and C HLA SB (DP), DC
• Major regions in the MHC: D, B, C and A A-580 diff. (DQ), DR
alleles
ORGANIZATION BASED ON B-921 diff. alleles
THEIR: BIOCHEMICAL TRAITS UNDER THEIR
CONTROL: C-312 diff.
COMPOSITION alleles
1. Class I 2. Chain Structure α chain + β2 α chain + β chain
- A, B, C, E, F, G, H, X microglobulin
transplantation antigens, (C#15)
Composed of serologically detected
HLA-A,B,C membrane antigens, cellular 3. Cell distribution all nucleated cells APC (antigen
target antigens for cell presenting cells )
Non-classical: mediated lympholysis (macrophage,
HLA-E,F,G dendritic cells,
Langerhan cells, T
*coded at 3 different loci / and B cells,
locations endothelial,
epithelial and
2. Class II stromal cells)
- DP, DQ, DR, DM, DN , DO • I Region leukocyte
antigen 4. Presents antigen to T-cytotoxic cells T-helper cells
*situated at the D region • T and B cell interaction (CD8+) (CD4+)
• Immune response
Major Class II molecules: • Mixed Leukocyte 5. Source of Proteins made in Endocytosed
DP, DQ, DR, Reaction peptide the cytosol plasma membrane
• Graft versus Host fragments and extracellular
DR: expressed at the highest reaction proteins
level • Tumor virus
½ of all class II molecules in the susceptibility 6. Polymorphic α1 and α2 α1 and β1
particular cell • Peptide transport domains
• Generation of peptides
Non-classical: from cytosolic proteins
HLA-DM,DN & DO
ANTIGEN PRESENTATION
3. Class III
- complement components (C4a,
C4b, C2), cytochrome p450, MAIN ROLE OF CLASS I AND CLASS I MOLECULES
21-hydroxylases and TNF - Is to bind peptides within cells and transport them to the
(tumor necrosis factor) plasma membrane where T cells can recognize them in a
phenomenon called ANTIGEN PRESENTATION.
*located in between MHC Class • CLASS I molecules
• serum protein molecule
I and MHC Class II regions • complement levels - Mainly present peptides that have been synthesized
• Cytokines and enzymes within the cell to CD8+/T-cytotoxic cell
ONE FUCTION: To produce
complimentary proteins (C2, C4, • CLASS II molecules
& factor B); part of compliment
system) - Mainly bind exogenous proteins and present it to CD4+/
T-helper cell
Antigen recognition ▪ Class I process those infectious agents that attacks
from inside
at each loci there is a possibility
of presence of multiple alleles ▪ Class II process those infectious agents that attack
from the outside
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 ANTIGEN, ADJUVANTS, AND MHC IMMUNOLOGY
SHERWIN ALVARO, RMT MARCH 22, 2022 AND SEROLOGY

Probability that any individual will have the same MHC 4. Forensic Medicine and Anthropology
molecules is yes but very low.
5. Basic research in Immunology
An individual inherits 2 copies of chromosome 6 and thus
there is a possibility of 2 different alleles for each gene 6. Studies of Racial Ancestry and Migration
unless that person is homozygous.
Testing for MHC genes
MHC molecules-co dominant, always expressed in phenotypic
level MHC Class I and Class II can induce a response that
leads to GRAFT REJECTION
ROLE OF Class I and Class II MOLECULES
HLA-Human Leukocyte Antigens
Both are synthesized in Rough ER because of the presence of
ribosome. -gene complex encoding the MHC proteins in humans
-surface proteins responsible in regulation of the
CLASS I HLA CLASS II HLA immune system in humans
Effective for endogenous Effective for exogenous HLA DISEASE ASSOCIATION
antigens antigens
• Tumors • bacteria
• Viruses
• Parasites HLA- B27 • Ankylosing
Spondylitis
Presented in CD8+ T Presented in CD4+ T • Reiter’s Syndrome
cytotoxic cells helper cells • Acute anterior uveitis
Proteasomes/ Invariant chain • Reactive arthritis
proteasome • maintain the (Yersinia,
• Cylindrical class 2: not yet Salmonella,
contain enzymes invariant chain Campylobacter)
that process peptide • Psoriatic arthritis
antigen (central) - B28
• Psoriatic arthritis
Transporting peptides (peripheral) – B38
• TAPI • Juvenile Rheumatoid
• TAP2 arthritis
HLA- B47 Congenital Adrenal
Hyperplasia

MHC Restriction or MHC Restricted Recognition

HLA- B5 Behcet’s Disease
HLA-CW6 Psoriasis Vulgaris
- the process whereby the MHC control interactions
between cells. It involves the recognition of foreign HLA- DR3 Type 1 DM
antigen in association with Class I or Class II molecules.
Reactions considered as MHC Restriction: HLA- DR4 Rheumatoid Arthritis
1. Antigen Presentation HLA-DR5 • Chronic Lymphatic
2. T-cell & B-cell cooperation Leukemia
3. Cytotoxic T-cell interaction with target cell
• Gold-induced
Nephropathy
Application of HLA Typing/Matching: • Kaposi’s Sarcoma
• Juvenile Rheumatoid
1. Organ Transplantation arthritis
Pauciarticular
Low immune response
2. Paternity Testing
3. For Diagnostic and Genetic counselling

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SHERWIN ALVARO, RMT MARCH 22, 2022 AND SEROLOGY

HLA DISEASE ASSOCIATION Disease HLA Relative

Antigen Risk
Disease HLA Relative
Antigen Risk Endocrine Subacute B35
(con't) thyroiditis 13.7
(deQuervain)
Rheumatic Rheumatoid DR4 3.8
arthritis
Hashimoto's DR5 3.2
Sjogren's syndrome DR3 5.7 thyroiditis
(SLE)
Congenital Bw47 15.4
Systemic Lupus DR2/ 2.6 adrenal
Erythematosus DR3 hyperplasia
Neurologic Myasthenia B8 3.3
Gastro- Gluten sensitive DR3 11.6 gravis (without
Intestinal Enteropathy thymoma)
Chronic Active DR3 6.8 Multiple sclerosis DR2 2.7
Hepatitis
Ulcerative colitis B5 3.8 Bipolar affective B16 2.3
disorder
Hematologic Idiopathic A3 6.7 Narcolepsy DR2 130.0
Hemochromatosis B14 25.7
A3, B14 90.0 Schizophrenia A28 2.3

Pernicious Anemia DR5 5.4 Disease HLA Relative

Antigen Risk

Disease HLA Relative Renal Idiopathic DR3 5.7

Antigen Risk membranous
glomerulonephritis
Skin D e r m a t i t i s DR3 17.3
Goodpasture's DR2 15.9
herpetiformis syndrome
Psoriasis 813, 817, 7.5 M i n i m a l c h a n g e DR7 4.2
vulgaris Bw57, nephrotic syndrome
Cw6
IgA nephropathy DR4 3.1
Behcet's disease B5 3.8
Multiple sclerosis DRZ 2.7
Endocrine Insulin DR4 DR3 3.6
dependent DR2 4.8 Polycystic kidney B5 2.6
diabetes 0.2 disease)
mellitus
Infectious Tuberculoid leprosy B8 6.8
Grave's disease B8 2.5 (Indians
DR3 3.7
Paralytic polic B16 4.3
Addison's Dw3 10.5
disease Low vs high reponse Cw3 12.7
to vaccinia virus

LEC MERCADO A., MINA M.J., PADRE A.C. 6

 ANTIGEN, ADJUVANTS, AND MHC IMMUNOLOGY
SHERWIN ALVARO, RMT MARCH 22, 2022 AND SEROLOGY

Methods for Detecting HLA Antigens:

Histocompatibility Testing

A. Tissue Typing
1. Serological approach
• Lymphocyte Microtoxicity Method ϒ
• For determination of Class I antigen
2. Cellular approach
• Mixed Lymphocyte Reaction
• For determinatiosn of Class II antigen
3. Molecular approach
• PCR (polymerase chain reaction) and RFLP
(Restriction fragment length polymorphism)

LEC MERCADO A., MINA M.J., PADRE A.C. 7

 IMMUNOLOGY
CYTOKINES AND SEROLOGY
SHERWIN V. ALVARO, RMT , MPH March 29, 2022

TABLE OF CONTENTS
CYTOKINES 1
FEATURES OF CYTOKINES 1
DIFFERENT TYPES/FAMILIES CYTOKINES 2
COLONY STIMULATING FACTOR (CSF) 2
IL-1 2
TUMOR NECROSIS FACTOR 2
IL-6 2
CHEMOKINES 2
TGF-B (transforming growth factor beta) 3
IFN-α and IFN-β 3
TH1 CYTOKINES 3
IFN- ɣ 3
IFN- ɣ Production 3
IL-2 3 • cytokines have these different characteristics, so
IL-4 3 cytokines can induce a stimulus wherein cytokines will
IL-10 3
act only on cells bearing specific receptors
ERYTHROPOIETIN AND COLONY 3
STIMULATING FACTORS • the expression of cytokines enters receptors or highly
INTERLEUKINS (IL) 5 regulated for example interleukin 2, cytokines can
CHEMOKINES 6 induce stimulus which is one of its features, just like
INTERFERONS 6 interleukin 2 receptor.
CYTOTOXIC / IMMUNOMODULATORY/ 6
GROWTH FACTORS (TNF FAMILY)

CYTOKINES

• are small soluble proteins that regulate the immune

system, orchestrating both innate immunity and the
adaptive response to infection.
- Secreted, low molecular weight proteins that
regulate the nature intensity and duration of the
immune response by exerting a variety of effects
on lymphocytes and on other cells. So, both innate
and adoptive immunity is the effect of these
cytokines.
• are induced in response to specific stimuli—such as
bacterial lipopolysaccharides, flagellin, or other
bacterial products— through the ligation of cell
adhesion molecules or through the recognition of
foreign antigens by host lymphocytes. • cytokine is that it can act in an autocrine, it can act
Originally, , cytokines are called as lymphocytes with the same cell.
because they were initially taught to be produced • it can also act in a paracrine action meaning it can
only by lymphocyte. And then they are also called act in a close proximity, it can also act in an
monocytes because they were secreted by endocrine manner or in a long distance
monocytes and macrophages and then afterwards • close proximity meaning that it affects a target cell
they were called as interleukin and then became in a close proximity while if it’s in an endocrine
cytokines action it means that cytokines will also exert
systemic activities.
• occasionally cytokines will also exert systemic
activities.

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 IMMUNOLOGY
CYTOKINES AND SEROLOGY
SHERWIN V. ALVARO, RMT , MPH March 29, 2022

FEATURES OF CYTOKINES
5. Cascade effect.
1. Cytokines are pleiotropic.
• nature of cytokines activity relates to the
widespread distribution of cytokine receptors;
one cytokine can have different effects on the
different cells.
• activated T helper cell with this interleukin 4
can have different effects on different cells

2. Cytokines can be redundant.

• Cytokines here can stimulate the
production of other cytokines
• it can act in networks to stimulate the
release of other cytokines and not only
that but in this characteristic of
cytokine, it can act as a growth factor
for hematopoietic cells meaning it
modulates the number and composition
• Different cytokines can often have very similar of cells.
effects
• example your interleukin-2, interleukin -4, and
DIFFERENT TYPES/FAMILIES CYTOKINES
interleukin-5 different cytokines but they the same
effect, which is the proliferation of B cell.
• TNF
3. Cytokines can synergize with each other. • IFN
• Chemokines
• TGF

INTERLUKINS
• Synergize – there is that cooperative effect of • These are class of glycoproteins produced by
multiple cytokines and it is different from leukocytes for regulating immune response
redundancy • These interleukins can induce stimulus
• Ex. Interleukin-4 its target is B cells for the
4. Cytokines can antagonize each other. promotion of class switch or it enhances the
immunoglobulin 1 and IgE production.
• there is an inhibition of one cytokine effects by
another cytokine
• Just like interleukin-4 wherein it blocks class switch
to IgE induced by IL-4. But having this interferon
gamma that inhibits the effect of interleukin-4.

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 IMMUNOLOGY
CYTOKINES AND SEROLOGY
SHERWIN V. ALVARO, RMT , MPH March 29, 2022

CHEMOKINES CYTOTOXIC/ IMMUNOMODULATORY/

• derived from their ability to induced directed GROWTH FACTORS (TNF FAMILY)
chemotaxis in near life responsive cells. - Substances capable of stimulating from the
CHEMOKIN SOURCE TARGET PRINCIPAL name itself, stimulating cellular growth,
ES EFFECT proliferation killing and cellular differentiation.
IL-8 (NAP-1) Monocytes T- CELLS CHEMOTACTI
Macrophag NEUTROPHI C TNF/ SOURCE PRINCIPAL
TARGET
es LS TGF EFFECT
Keratinocyt
TUMOR Th1 Macrophag -local
es
Fibroblasts NECROSIS Some Th2 e inflammatio
MCP-1 Monocytes Memory T Chemotactic FACTOR NK Cells n
MCAF( Macrophag Cells ALPHA Macrophag -activates
es Monocytes e induces no
Keratinocyt production
es TUMOR Th1 T cells -Inhibits B
Fibroblasts NECROSIS Some of B cells cells
MIP 1 Alpha Macrophag T-Cells Chemoattracta FACTOR CTL Macrophag - enhances
e Monocytes nt BETA e T cell Killing
Eosinophil
(Lymphotoxi Neutrophils -Activates
MIP-1 Beta Monocytes CD 8 T Cells Chemoattacta
Macrophag
n) neutrophils
nt
es -Activates
Neutrophil induces no
Endothelium reaction
RANTES T-Cells CD 4 T cells Chemoattracta Transformin T Cells B Cells -Inhibits B
Platelets Monocytes nt g Growth Macrophag NK Cells cells, T cells
Memory Factor Beta e Macrophag and NK
Cells Monocyte e Cells
Neutrophils activity
INTERFERONS -Activates
- they are the family of glycoproteins that are neutrophils
classically defined by their ability to protect -promotes
cells against viral infections. matrix
formation
INTERFERO SOURCE TARGET PRINCIPA
(wound
NS L EFFECT healing)
IFN ALpha T CELLS MACROPHA Anti-viral angioenesis
B CELLS GE Increased
MACROPHAG MHC
ES expressio COLONY STIMULATING FACTOR (CSF)
LEUKOCYTES n - Not cerebrospinal fluid
IFN BETA Fibroblast MACROPHA Anti-viral
Increased
One of the families of cytokines and their
GE
MHC production is boosted by endotoxin antigens and
expressio horbol esters.
n • synthesized by bone marrow stromal cells,
IFN LAMBDA T cells Macrophage Cell endothelial cells, fibroblast, macrophages, &
NK Cells Activation
lymphocytes
Increased
MHC • growth glycoproteins controlling the proliferation
expressio and differentiation of committed hematopoietic
n cells.
Granulocyte-Macrophage CSF (GM-CSF)
Granulocyte CSF (G-CSF)
Macrophage CSF (M-CSF)
IL-3
IL-7

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 IMMUNOLOGY
CYTOKINES AND SEROLOGY
SHERWIN V. ALVARO, RMT , MPH March 29, 2022

IL-6
CYTOKINES INVOLVED IN IMMUNE RESPONSE - Expressed by a variety and normal and
- responsible for many of the physical symptoms transformed cells including D cells, b cells,
attributed inflammation such as fever, swelling, monocytes, and macrophages as well as fibroblast,
pain and even cellular infiltrates into damage hepatocytes, keratinocytes, astrocytes, vascular
tissues. endothelial cells and various tumor cells.
Ex. Inflammatory agents (attributed to • IL-6 is a single protein produced by both lymphoid
inflammation) -the main function of your innate and nonlymphoid cell types.
immune response is to recruit a vector cell to the • It is part of the cytokine cascade released in
area and one is the function cytokines in terms of response to lipopolysaccharide and plays an
being an inflammatory agent important role in acute phase reactions and the
- Cytokines is involved in triggering this response adaptive immune response.
which includes IL-1, tumor necrosis factor, alpha, IL • PLEOMORPHIC ACTIVITIES OF IL-6
6, chemokines, transforming growth factor beta - Inflammation
and Interferons alpha and beta.
- Acute Phase Reactions - immediately
increase if there is an inflammation or
IL-1
• This helps to regulate the physiological response infection.
to IL-1 and turn off the response when no longer - Immunoglobulins synthesis
needed. - Activation states of B cells and T-cells
• Mediator of the innate immune response - Proliferation and differentiation of B cells into
plasma cells
• Types of IL-1:
- IL-1α
CHEMOKINES
- IL-1β
• Classified into 4 families based on the position of
- IL-1RA (IL-Receptor antagonist)
the N thermal cysteine residue
• also produced by monocytes and macrophages.
• Chemokines are a family of cytokines that enhance
motility and promote migration of many types of
TUMOR NECROSIS FACTOR
white blood cells toward the source of the
chemokine (chemotaxis).
• Principal mediator of the acute inflammatory
- alpha, or CXC, chemokine: contains a single
response to gram-negative bacteria and other
amino acid between the first and second
infectious microbes.
cysteines.
• 1st isolated from tumor cells. - Beta or CC, chemokines: has adjacent cysteine
• Named because they induced the lysis in this cells residues.
• Stimulates gene transcription or induces apoptosis - C chemokines: lacks one of the cysteines.
- CX3C: has three amino acids between the
• TNF-α cysteines
- exists in both membrane-bound and
soluble forms and causes vasodilation and TGF-B (transforming growth factor beta)
increased vasopermeability
• TGF- β was originally characterized as a factor
that induced growth arrest in tumor cells.

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 IMMUNOLOGY
CYTOKINES AND SEROLOGY
SHERWIN V. ALVARO, RMT , MPH March 29, 2022

IFN- ɣ Production
IFN-α and IFN-β - 2 stepS
- They literally interfere with viral replication • (1) ligation of the T-cell receptor (TCR) by MHC-
process in uninfected cell. peptide antigen presentation IFN-ɣ Production can
- They block viral proteins that’s why they are called be stimulated in mature T-helper 1 cell
interferons. • (2) cytokine stimulation by IL-12 and IL-18- help
each other in order for them to stimulate the
• also used in Immunoregulation interferon gamma production.
• Type 1 IFN or non-immune IFN • IL-12 and IL-18 act synergistically to stimulate IFN-
- IFN-α ɣ production
- IFN-β
produced primarily during the initial innate response to IL-2
viral infection
• Also secreted by Th1 cells.
CYTOKINES IN THE ADAPTIVE IMMUNE RESPONSE • IL-2 is also known as the T-cell growth factor.
- In adaptive immune response it is mainly secreted • It drives the growth and differentiation of both T
by B cells especially your B helper cells and it and B cells and induces lytic activity in NK cells
affects your T and B cell function.

IL-4
TH1 CYTOKINES belong to the TH2 cytokines (IL-4, IL-5 and IL10)
the dendritic cells in damaged tissues produce the - Triggers activation, proliferation and
interleukin 12 response to certain stimuli such as differentiation of B-cells- increase the expression
your mycobacteria, intercellular bacteria, and of MHC class 2 on resting B cell.
even viruses. • Responsible in allergic reactions, parasitic
- Produced by : infections and autoimmune disease-t also induces T
• Macrophages and B cell proliferation
- B cells has multiple effects on both T cells and
natural killer cell IL-10
• IL-12 binds to its receptor on naïve T cells and
causes the expression of a new set of genes, • Inhibits production of pro-inflammatory cytokines
including those that determine maturation into the by mononuclear phagocytes.
Th1 lineage • Inhibits the accessory functions of mononuclear
phagocytes for T-cell activation.
IFN- ɣ
Stimulates antigen presentation by class 2 MHC molecules ERYTHROPOIETIN AND COLONY STIMULATING
• Produced mainly by Th1 cells FACTORS
• Genes involved in regulation and activation of
CD4+ Th1 cells, CD8+ cytotoxic lymphocytes, NK • IL-3
cells, bactericidal activities, IL-12R and IL18R are o EPO: Erythropoietin
all regulated by IFN-ɣ o TPO: Thrombopoietin
o G-CSF: Granulocyte-colony stimulating
factor
o M-CSF: Megakaryocyte-colony stimulating
factor
o GM-CSF: Granulocyte-macrophage-
colony stimulating factor
WBC Maturation under the influence of the Colony
Stimulating factor

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 IMMUNOLOGY
CYTOKINES AND SEROLOGY
SHERWIN V. ALVARO, RMT , MPH March 29, 2022

LEC LIM, J.M., MARCAIDA, V.P., PROGRESO, L.C.

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 IMMUNOLOGY
CYTOKINES AND SEROLOGY
SHERWIN V. ALVARO, RMT , MPH March 29, 2022

INTERLEUKINS (IL)

INTERLEUKIN SOURCE TARGET PRINCIPAL EFFECTS

IL-1α Lymphocytes, Macrophages, - lymphocyte co-stimulation

Macrophage, Fibroblast Lymphocytes, - phagocyte activation
Endothelium ↑ endothelial adhesion molecules

IL-1β Epithelial cells, Astrocytes Others - induces sleep or fever

↑ Prostaglandin synthesis
1L-2 T-cells T-cells, B-cells, NK- - T-cell & NK-cell growth & activation
cells - co-stimulates B-cell differentiation
IL-3 T-cells, Thymic Epithelium Stem cells - multi-lineage Haematopoietic factor

IL-4 Th2 cells, Bone Marrow, B-cells - co-stimulates B-cell differentiation

Stroma - stimulates MHC class II expression on B-cell &
Macrophages
- promotes class switch
- enhances IgG 1 & IgE production
IL-5 Th2 cells B-cells, Eosinophils - enhances eosinophil differentiation
- enhances IgA production by stimulated B-cells
- stimulates In Vitro Ab responses
IL-6 Th2 cells, Macrophages, T-cells, B-cells, - lymphocyte growth
Endothelium Hepatocytes - B-cell differentiation
- Acute-phase protein synthesis
IL-7 Bone Marrow, Stroma Pre-T cells, Pre-B-cells - cell activation

IL-8 Monocytes, Fibroblast, T-cells, Monocytes, - stimulates chemotaxis of target cells

Endothelium Neutrophils - stimulates granulocytes activity
IL-9 CD4 T-cells T-cells, Mast cells - cell division
- promotes development
IL-10 Th2 cells Th1 cells - inhibits cytokine synthesis

IL-11 Bone Marrow, Stroma Plasma cells - cell division & proliferation
Stem cells - stimulates maturation of hematopoietic cells
IL-12 B-cells Macrophages T-helper cells - Th1 cell development & activation
NK cells - induces IFN-y production from target cells
- augments cytotioxic activity of NK cells
IL-13 Th2 cells B-cells - division & proliferation
Macrophages - blocks inflammatory monokine production
IL-14 T-cells B-cells - cell proliferation
↓ Ig synthesis
IL-15 Monocytes T-cells, B-cells - cell division: shares IL-2 bioactivity

IL-16 CD8* T-cells CD4* T-cells Chemotactic

IL-17 T-cells Many cells Proinflammatory

LEC LIM, J.M., MARCAIDA, V.P., PROGRESO, L.C.

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 IMMUNOLOGY
CYTOKINES AND SEROLOGY
SHERWIN V. ALVARO, RMT , MPH March 29, 2022

CHEMOKINES

CHEMOKINES SOURCE TARGET PRINCIPAL EFFECTS

IL-8 (NAP-1) Monocytes, T-cells Chemotactic
Macrophages, Neutrophils
Keratinocytes,
Fibroblasts
MCP-1 (MCAF) Monocytes, Memory T-cells, Monocytes Chemotactic
Macrophages,
Keratinocytes,
Fibroblasts
MIP-1α Macrophages T-cells, Monocytes, Eosinophil Chemoattractant
MIP-1β Monocytes, CD 8* T-cells Chemoattractant
Macrophages,
Neutrophil,
Endothelium
RANTES T-cell, Platelets CD 4* T-cells, Monocytes, Chemoattractant
Memory cells

INTERFERONS

INTERFERONS SOURCE TARGET PRINCIPAL EFFECTS

IFN-α T-cells, B-cells, Macrophage - anti-viral
Macrophages, ↑MHC expression
Leukocytes
IFN-β Fibroblast Macrophage - anti-viral
↑MHC expression
IFN-γ T-cells, NK- Macrophage - cell activation
cells ↑MHC expression

CYTOTOXIC / IMMUNOMODULATORY/
GROWTH FACTORS (TNF FAMILY)

TNF/TGF SOURCE TARGET PRINCIPAL EFFECTS

Tumor Necrosis Th1, some Th2, Macrophage - local inflammation
Factor-α NK-cells, - activates induce NO production
Macrophage
Tumor Necrosis Th1, some CTL T-cells, B-cells, Macrophage, - inhibits B-cell
Factor -β Neutrophils - enhances T-cell killing
(Lymphotoxin) - activates neutrophil
-activates induces NO production
Transforming T-cells, B-cells, NK-cells, - inhibits T-cell, B-cell, & NK-cell activity
growth factor-β Macrophage, Macrophage, Monocyte -activates Neutrophil
Monocyte -promotes matrix formation (wound healing)
- angiogenesis

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 IMMUNOLOGY
ANTIBODY AND SEROLOGY
SHERWIN V. ALVARO, RMT , MPH March 15, 2022

TABLE OF CONTENTS B. According to temperature at which they react:

ANTIBODIES 1 1. Cold antibodies
CLASSIFICATION OF ANTIBODIES 1 - 4 degrees C to RT (24-27 degrees C)
THE STRUCTURE OF IMMUNOGLOBULIN 2 - ABO antibodies
ANTIBODIES 2
IMMUNOGLOBULIN VARIABILITY 3 2. Warm antibodies
CHARACTERISTICS OF THE DIFFERENT 3 - Best reacts @ 37 degrees C
TYPES OF IMMUNOGLOBULINS - Rh antibodies
THEORIES OF ANTIBODY PRODUCTION 4
C. According to occurrence:
1. Natural antibodies
ANTIBODIES - antibodies that are produced even without
previous antigenic stimulation
- ABO antibodies
 glycoprotein substances synthesized by plasma
cells in response to antigenic stimulation. Composed 2. Immune/Acquired antibodies
of: - antibodies that can only be produced
o 2-14% carbohydrate given that there is exposure with
o 82-96% proteins corresponding antigen.
 Immunoglobulins are considered to be the humoral Needs the presence of an antigen for it to
branch of the immune response. be produced
Structurally, all antibodies are immunoglobulins or - Rh antibodies
gamma globulins Can produce anti D- provided tha there is
Functionally, not all immunoglobulins are an exposure, either from transfusion or
antibodies, organ transplantation and pregnancy
 PROPERTIES OF AN ANTIBODY: + Presence of an antigen on the
1. Protein in nature Red cells
2. With high molecular weight - Absence/ does not have of
antigen on the red cell
3. Present in serum/plasma, saliva, semen, CSF
and other body fluids.

CLASSIFICATION OF ANTIBODIES: D. According to the species which produce them:

 IgG, IgA, IgM, IgD, IgE 1. Isoantibodies/Alloantibodies
- antibodies produced in response to
A. According to its sedimentation constant/ antigens of other individual of the same
coefficient: species.
Unit used to express the rate of sedimentation of 2. Heterophile antibodies
an immunoglobulin when subjected to ultra- - antibodies produced in response to
centrifugation antigens coming from other species.

Immunoglobulin Sedimentation Molecular Weight E. According to its reaction with an antigen

Coefficient(in (in daltons) 1. Agglutinins
Svedverg units) - involved during agglutination reactions
IgG 7s 150,000 (antigen involved is cellular or particulate)
Serum IgA 7s 160,000 2. Precipitins
Secretory IgA 9s; 11s; 13s 170,000 - involved in precipitation reactions (antigen
involved is soluble antigen)
IgM 19s 900,000
3. Agglutinoids
IgD 7s 180,000
- agglutinins that are modified by HEAT
IgE 8s 190,000
4. Hemagglutinins
- antibodies that can clump RBCs
5. Lysins
- antibodies that can cause lysis
LEC 4 TRANSCRIBERS: MARCAIDA, VP., LIM, J., PROGRESO,LC
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 IMMUNOLOGY
ANTIBODY AND SEROLOGY
SHERWIN V. ALVARO, RMT , MPH March 15, 2022

6. Opsonins  Tetrapeptide made up of:

7. Neutralizing antibodies 1. Heavy chain - with five principal antigenic
- antibodies that neutralize certain reactions types with their corresponding Ig. (Gamma,
8. Allergic antibodies alpha, mu, delta, epsilon)
- involved during hypersensitivity reactions 2. Light chain- has two antigenically defined
9. Antitoxins types: kappa and lambda
- neutralize harmful effects of toxins o Hinge region
10. Complement fixing antibodies - flexible part of the antibody located in the
- can activate the complement system. heavy chains.
- It is more exposed to enzymes and
F. According to their in-vitro behavior: chemicals thus papain acts here to produce
Fab and Fc fragments.
1. Complete 2. Incomplete - The light chains are each linked to one-half
antibody (IgM) antibody (IgG) of a heavy chain by disulfide bonds at the
Synonyms Bivalent; Saline Univalent; proximal end.
acting blocking; co- o Proline
agglutinating; - responsible for the flexibility of the hinge.
conglutinating
o Disulfide bonds
Response to Thermolabile Thermostable
temperature - are chemical bonds essential for the
Ability to cross Cannot cross the Can cross the normal 3-dimensional structure of Ig.
placenta placenta (too big; placenta
high molecular 2 types of disulfide bonds:
weight) a. Interchain
Occurrence Early in Late in - connects light chains and heavy
immunization immunization chains
Reaction Saline acting Albumin acting b. Intrachain
- connects domains of the heavy
THE STRUCTURE OF IMMUNOGLOBULIN chain
 Monomer - connects domains of the light chain
- Basic structural unit of an antibody  DOMAINS
- Y shape - are globular regions on polypeptide chain
- Epitope of antigen to the antibody stabilized by intrachain disulfide bonds.
a. Domains on the heavy chain - VH, CH1,
CH2, CH3, (CH4-- IgE)
b. Domains in the light chain - VL, CL
VL + CL bind with antigen
CH1 binds with C4b
CH2 binds with C1q (if IgG)
CH3 binds with C1q (if IgM)
Binding site for T and B cells,
platelets, mast cells, monocytes,
macrophage
CH2 + CH3 binds with NK cell, placental
syncitiotrophoblast and neutrophils

ANTIBODIES

 Functions of antibodies:
1. Binding with antigen
2. opsonization
3. complement fixation and activation

LEC 4 TRANSCRIBERS: MARCAIDA, VP., LIM, J., PROGRESO,LC

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 IMMUNOLOGY
ANTIBODY AND SEROLOGY
SHERWIN V. ALVARO, RMT , MPH March 15, 2022

 Regions on polypeptide chain

1. Variable region IMMUNOGLOBULIN VARIABILITY
a. Amino acid sequence subject to change
b. Amino terminals (NH2) 1. Isotypic Variation (isotypes)
c. Concerned with binding to antigen - refers to the different heavy and light chain classes
2. Constant 2. Allotypic variation (allotypes)
a. Amino acid sequence is fixed and - refers to the genetic variation within a species
unchanging involving different alleles at a given locus
b. carboxyl terminal (COOH) (subclasses within a class of Ig.)
c. concerned with binding to host tissue 3. Idiotypic variation (idiotypes)
 Polymer - refers to the diversity at the binding site and in
- Ig composed of more than a single basic particular relates to the hypervariable segments of
monomeric unit. the antibody combining site (paratope).
 J chain
- polypeptide chain which normally holds
polymeric Ig. IMMUNOGLOBULIN VARIABILITY
1. Isotypic Variation (isotypes)
 Secretory component - refers to the different heavy and light chain classes
- a substance attached to polymeric Ig
found on secretions. 2. Allotypic variation (allotypes)
 Protect secretory IgA from acid digestion - refers to the genetic variation within a species involving
in the stomach.
different alleles at a given locus (subclasses within
 Facilitated the transport of secretory IgA
a class of Ig.)
across mucosal surfaces
3. Idiotypic variation (idiotypes)
 Fragments of Ig:
- 2 Fab (antigen binding fragment) - refers to the diversity at the binding site and in
- 1 Fc (crystallizable fragment) particular relates to the hypervariable segments of
the antibody combining site (paratope).
 ENZYME DIGESTION
1. Cleavage with papain enzyme
CHARACTERISTICS OF THE DIFFERENT
- cleaves before the hinge
TYPES OF IMMUNOGLOBULINS:
- lead to 2 Fab + 1 Fc
2. Cleavage with pepsin enzyme 1. IgG - major antibody in SECONDARY/ANAMNESTIC
- cleaves after the hinge IMMUNE RESPONSE
- lead to 1 F(ab)2 + 1 Fc’ a) Predominant Ig among humans comprising 75-80%
3. Reduction using of the total Ig pool.
mercaptoethylamine/mercaptoethanolamine b) Has 4 major sublasses: IgG1, IgG2, IgG3, IgG4
- disrupts disulfide bonds c) Equally distributed in the different fluid
- leads to presence of 2 HC + 2 LC compartments with detectable amounts in CSF and
urine
 GENETICS OF IG: d) Readily diffusible
- Genes coding for the synthesis of heavy chains are e) IgG antibody response appears later than IgM in
located in C#14 primary response but they form the major antibody
- Genes coding for the synthesis of kappa light of the secondary immune response.
chains are located in C#2 f) Maternal IgG is actively and selectively
- Genes coding for the synthesis of lambda light transferred across the placenta to the fetus and
chains are located in C#22 imparts passive protection to the newborn for 6-9
1. V gene variable months
2. D gene diversity g) Functions of IgG:
3. J gene joining  Provides immunity for the newborn
4. C gene constant

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ANTIBODY AND SEROLOGY
SHERWIN V. ALVARO, RMT , MPH March 15, 2022

 Complement fixation IgG3, IgG1, IgG2 IgG4 pathway of complement system and only in the
(cannot activate the complement via classical presence of lysozyme
pathway)
 Opsonization
 Neutralization of toxins and viruses
 Participation in agglutination and precipitation
reactions
2. IgM
a) The largest of the immunoglobulin molecule it has
high molecular weight containing 900 000 daltons,
accounting for 5-10% of the total immunoglobulin
pool.
b) Star-shaped in the free state; crab-like in antigen- 4. IgD marker for mature B-cells
antibody reaction. a) Heat labile immunoglobulin, accounts for less than
c) The earliest antibody to appear in the primary 1% of the total serum Ig but is known to be present
immune response but it does not persist for long. in large quantities on the membrane of many
d) Maternal IgM does not cross the placenta for it is circulating immunocompetent B lymphocytes.
too big. b) Detectable by highly sensitive assay requiring
e) IgM detection in newborn is a useful indicator of radio-labelled antisera
intrauterine infection c) Precise biological action is not known but it may
f) A powerful agglutinator of a particulate antigen play a role in antigen-triggered lymphocyte
g) Functions of IgM: differentiation.
 Complement fixation 5. IgE
 Agglutination a) Heat labile immunoglobulin. Least abundant Ig in
 Opsonization the serum accounting for only 0.004% to the total
 Neutralization of toxins serum Ig.
 Surface receptor for antigens (on B-cells)* b) Synthesized locally by plasma cell present in the
mucous membrane of the Gi and respiratory tracts.
c) It is unable to fix the complement via the classical
pathway.
d) It is homocytotropic due to its affinity for cells of
the host species, particularly for tissue mast cells
and blood basophils.
e) Because of its ability to attach to the human skin, it
is associated with immediate hyper sensitivity
3. IgA reactions but also, apparently, with immunity to
a) Represent 15-20% of human serum Ig pool. certain helminthic parasites.
b) Found in serum in small amounts but predominant in f) Also known as reaginic antibody/ nuisance
sero-mucous secretions of the respiratory tract, antibody
genito-urinary tract and GI tracts. It is also found in
tears, sweat, saliva, colostrum and breastmilk. THEORIES OF ANTIBODY PRODUCTION
c) Forms of IgA
1. Ehrlich’s Side Chain Theory
 Serum IgA (IgA1)
 Certain cells have had specific surface receptors
- can agglutinate motile infectious agents thus
for antigen that were present before contact with
promoting their phagocytosis but they cannot
antigen occurred. Once antigen was introduced, it
activate the complement system
would select the cell with proper receptors,
 Secretory IgA (IgA2)
combination would take place and then receptors
- a polymeric form stabilized a short
will break off and enter the circulation as antibody
polypeptide chain. It is known as the “antiseptic
molecules. New receptors will be formed in place
paint” of mucous membranes. It can activate
of those broken off and this process could be
the bacteriolytic activity through the alternate
repeated.

LEC 4 TRANSCRIBERS: MARCAIDA, VP., LIM, J., PROGRESO,LC

4
 IMMUNOLOGY
ANTIBODY AND SEROLOGY
SHERWIN V. ALVARO, RMT , MPH March 15, 2022

2. The Template Theory/Instructive Theory

o Felix Haurowitz
 Antibody –producing cells are capable of
synthesizing a generalized type of antibody, and
when contact with an antigen occurs, the antigen
serves as a mold or template and alters protein
synthesis so that antibody with a specific fit is
made. The “molded” antibody then enters the
circulation, while the antigen remains behind to
direct further synthesis
3. Selective Theory
o Replication----Transcription -----Translation
 Assumes that antibodies are synthesized in a
manner similar to that of other proteins. Instructions
for their synthesis are provided by genetic
elements in the nucleus of the cell rather from the
antigen
4. Clonal Selection Theory
o Jerne and MacFarlane Burnet
 Individual lymphocytes are genetically pre-
programmed to produce one type of
immunoglobulin, and that specific antigen finds or
selects those particular cells capable of responding
to it, causing these to proliferate. Repeated contact
with the antigen would continually increase a
lymphocyte pool

REFERENCES:

 Turgeon, Mary Louise. (2014). Immunology & serology

in laboratory medicine, (5th ed.). Missouri : Elsevier.
 Male, David. (2013). Immunology, (8th ed.). St. Louis,
Missouri : Elsevier.
 Stevens, Christine Dorresteyn. (2010). Clinical
immunology & serology : a laboratory perspective,
3rd ed. Philadelphia ; F.A. Davis

“So do not fear, for I am with you; do not be dismayed, for I

am your God. I will strengthen you and help you; I will uphold
you with my righteous right hand.F
- Isaiah 41: 10

LEC 4 TRANSCRIBERS: MARCAIDA, VP., LIM, J., PROGRESO,LC

5
 COMPLEMENTS IMMUNOLOGY
SHERWIN ALVARO, RMT APRIL 19, 2022 AND SEROLOGY

TABLE OF CONTENTS “complemented or augmented” antibodies in the killing of

General Information 1
bacteria.
Consists of serum and cell surface proteins involved in defense
General properties 1 against pathogens and tissue damage mediated by
antibodies.
Effects of complement activation 2
The Complement system is the major effector of cellular and
Routes or Pathways of complement activation 2 humoral branch of immune system.
Acute phase reactants 2 Plays major role in both innate and adaptive immunity.
Complement system represents a group of about 30 proteins
Proteins of the complement system 2 which augment or complement the immune response.
Vital functions 3 Most of these proteins are found in serum or on cell surfaces.
Classical pathway 3 Synthesized in liver as inactive precursors and are activated
by proteolysis during their interaction in a sequential manner.
Stages of Complement Activation 4 Also produced by blood monocytes, tissue macrophages and
Alternative pathway 4
epithelial cells of he gastrointestinal and genitourinary tract.

Alternate pathway activation 5

General properties
Mannose-binding lectin pathway 5 • Present in serum of all animals but its concentration is
maximum in serum of guinea pig.
Regulation of the complement pathway 6
• Complement of one species are able to react with
Comparison of Classical and alternative pathway 6 antibodies of other species but not to the same extent.
Main source of complement 6 • C- proteins (Complement proteins) constitute about 5% of
normal serum protein .
In-vitro destruction of complement 7 • Are glycoproteins.
Diagnostic evaluation of complement 7 • Are synthesized rapidly in inflammatory responses – hence
are called acute phase proteins.
Deficiencies of complement component 7
• Heat labile and lost activity at 560 C for 30 mins and
Receptors on cell membranes for complement components 8 inactivated. Immunoglobulins are not inactivated at this
temperature.
• Binds with Fc potion of immunoglobulins.
General Information
Complement System • Over 30 serum and cell surface proteins.
- Army of billions/ trillions of tiny bombs which work • Complement components
together in a complex and elegant chance to stop the • Components are designated by numbers (E.g. C1 –
intruders in the body. C9) or letters (E.g. Factor D).
- Enhances or complements the immunity (Adaptive and • C in complements are not “complement”. They are
Innate). called “Component”. Example is C1- Complement
Complement Pathway Component 1.
It is a complex series of more than30 soluble and cell-bound • in serum inactive, activated sequentially as a
proteins that interact in a very specific way to enhance host cascade)
defense mechanisms against foreign cells. • Complement receptors (cell surface, recognize activated
- Group of non-immunoglobulin circulating in blood in components)
biologically inactive form through a zymogen. When • Regulatory proteins of complement (both in serum and cell
active is converted to serine proteases. surface, inhibit activated components) system
It is a complex series of more than30 soluble and cell-bound • Complement proteins: are proenzymes - activation by
proteins that interact in a very specific way to enhance host cleavage. Example: C4
defense mechanisms against foreign cells. • C4a : a = smaller fragment; Diffusion
Heat-labile substance. (Breaks down easily through heat) • C4b : b= larger fragment; remains bound to microbe
• Exception: C2:
Research on complement began in the 1890s, when Jules • C2a = large fragment
Bordet at the Institut Pasteur in Paris showed that sheep
antiserum to the bacterium Vibrio cholerae caused lysis of the • C2b = small fragment
bacteria and that heating the antiserum destroyed its
bacteriolytic activity. He named those substances as Alexins.
Jules bordet was awarded in 1919
Paul Ehrlich (1980’s) coined the term complement.
It is named “complement system” because it was first
identified as a heat-labile component of serum that
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 COMPLEMENTS IMMUNOLOGY
SHERWIN ALVARO, RMT APRIL 19, 2022 AND SEROLOGY

Effects of complement activation Acute phase reactants

Complement activation promotes: Protein Response Normal Increase Function
• Activation of immune system Time Concentration
C-Reactive 6-10 0.5 1000x Opsonization
• Opsonization (C3b, C4b, C5b) and; protein
• Opzonization (recognized antigen coated by an Serum amyloid A 24 3.0 1000x Removal of
antibody that complement to trigger phagocytosis.)
cholesterol
• Opsonin- coats the microbes to make them more Alpha1- 24 200-400 2-5X Protease inhibitor
prone to phagocytosis antirypsin
• Eventually, after the activation of complement will result to Fibrinogen 24 110-400 2-5x Clot formation
LYSIS of foreign cells and immune complexes
Haptoglobin 24 40-200 2-10x Binds hemoglobin
Chronic activation: Ceruloplasmin 48-72 20-40 2x Binds copper and
• Leads to inflammation and tissue damage oxidezes iron
Routes or Pathways of Complement Activation: Complement C3 48-72 60-140 2x Opsonization, lysis
results in antigen antibody reaction/ formation of the immune Manose-binding ? 0.15-1.0 ? Complement
complex protein activation
1. Classical pathway - C reactive protein together with Serum Amyloid A is
- Is antibody dependent pathway and triggered by very useful to determine if there are inflammation but
formation of soluble antigen-antibody complex or by are non-specific. Produced primarily by the
binding of the antibody to the antigen present on the target hepatocytes within 12-24 hours in response to an
cell surface. increase in certain intracellular signaling
polypeptides- cytokines.
- major mechanism of adaptive humoral activity
- Most complement proteins are synthesized in the liver
- discovered by Pillemer and colleagues. They except C1 component
discovered the antibody dependent pathway and
antibody independent pathway in 1950s and Proteins of the complement system
displays a major role in natural defense mechanism
which involves the proteins triggered by the antigen- SERUM MOLECULAR CONCENTRATI FUNCTION
antibody combination PROTEIN WEIGHT (KD) ON (µG/ML)
2. Alternative pathway
Classical Pathway
- Is antibody independent pathway stimulated by antigen
directly C1q 410 150 Binds to Fc region of
e.g. Bacterial cell surface components. IgM and IgG

- effector of innate immunity C1r 85 50 Activates C1s

- stabilize by C3 convertase of alternative pathway C1s 85 50 Cleaves C4 and C2
- It is called the Properdin Pathway because the protein
Properdin is thought to initiate this pathway. C4 205 300–600 Part of C3 convertase
(C4b)
3. Lectin Pathway
- Also antibody independent but resembles classical pathway. C2 102 25 Binds to C4b—forms
C3 convertase
- results to Mannose Binding Lectin
- effector of innate immunity C3 190 1,200 Key intermediate in
all pathways. present
- activated when there is the mannose sugar on the cell in all pathways.
membrane.
- Major constitute is mannose/mannan binding lectin C5 190 80 Initiates membrane
(MBL) attack complex

C6 110 45 Binds to C5b in MAC

C7 100 90 Binds to C5bC6 in

MAC

C8 150 55 Binds c5b67. Starts

pore formation on
membrane and c9
polymerization

C9 70 60 Polymerizes C5b678
to cause cell lysis

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 COMPLEMENTS IMMUNOLOGY
SHERWIN ALVARO, RMT APRIL 19, 2022 AND SEROLOGY

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 0.0002–10 Binds to mannose

MASP-1 93 1.5–12 Unknown

MASP-2 76 Unknown Cleaves C4 and C2 ————————————————————————

- C1 is a trimolecular complex: C1q. C1r and C1s.
VITAL FUNCTIONS - C1 is an aggregate of 18 polypeptides that binds with
the Fc potion of IgM and IgG
1. Host defense mechanism
- C1 is multivalent and it can crosslink several
• a. Opsonization immunoglobulin molecules.
• b. Chemotaxis and leukocyte activation - The inactive form is Zymogen and is activated to serine
• c. Lysis of bacterial and mammalian cells proteases.
- There is antigen-antibody complex that activates the C1
• d. Stimulation of inflammatory response to form the Protease which is the C1s
2. Interface between innate and adaptive immunity - C1s cleaves (breakdown) to form active component
• - augmentation of antibody response (protease)
• - enhancement of immunologic memory - C1s cleaves the C2 and C4 to form C4b2a/ C4bC2a
complex. (C3 convertase)
3. Disposal of wastes - C4b2a complex will cleave C3 to form C3a and C3b
• a. Clearance or removal of immune (Antigen-antibody) - C4b2a complex and C3b will combine to produce a new
complexes from tissues enzyme which is the C4b2a3b complex (C5 convertase)
• Spleen- organ involved in removing immune complexes - C4b2a3b complex (C5 convertase) will cleave to form
C5a and C5b.
C-activation: alteration of C proteins such that they - Cb5 will combine to C6 and C7 to form a complex
interact with the next component (C5b67) that interacts with C8 and C9
C-fixation: utilization of C by Ag-Ab complexes - C5b67 will interact with C8 and C9 to form C5b6789
which is the membrane attack complex which causes the
C-inactivation: denaturation (usually by heat) of an lysis of Cell (Cytolysis)
early C-component resulting in loss of hemolytic - “b” fragments always bind because “b” fragments
continue in the mean pathways whereas the “a”
activity fragment split off and has other activities.
Convertase/esterase: altered C-protein which acts - The exemption of C2a which binds to produce the C3
convertase while the C2b fixes the complement.
as a proteolytic enzyme for another C-component
- “a” fragments are smaller and “b” fragments are
CLASSICAL PATHWAY/CASCADE larger (except for C2a and C2b)
- part of acquired immunity - C4a, C3a and C5a- mediators of inflammation
- Antibody dependent (anaphylatoxin).
- Not all immunoglobulins can activate this pathway. - Opsonin- C3b
- immunoglobulins IgG and IgM are able to activate - Chemotaxin- C5a
this pathway - C5a-not only an anaphylatoxin but also a chemotaxin
- Most efficient immunoglobulin G: IgG3, IgG1, IgG2 (stimulate the movement of white blood cells)
- IgG 4 cannot activate the complement system. - C9 polymerizes to cause the lysis of the target cell.
- Main trigger is the interaction of antigen and
antibody

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 COMPLEMENTS IMMUNOLOGY
SHERWIN ALVARO, RMT APRIL 19, 2022 AND SEROLOGY

STAGES OF COMPLEMENT ACTIVATION ————————————————————————

C4b2a - This complex is formed when activated C1s cleave
the C4 and C2 complement component during the first step
in activation
- The C2 Gene is closely associated with the gene for
Factor B (Alternative Pathway on Chromosome 6 in the
Major Histocompatibility Complex (MHC) Class II)
- Half-life is estimated to be between 15 secs to 3 minutes
(not very stable)
- most significant step in the complement activation.
- If binding does occur, C3 is cleaved into two parts, C3a
and C3b
Formation of the MAC.
C3b act as an opsonin.
Creates a new enzyme known as C5 convertase. Written as
“C4b2a3b” in classical pathway
1. Initiation or Recognition MEMBRANE ATTACK COMPLEX
Involves C1q, C1r and C1s. - C5 convertase will cleave the C5 into C5a and C5b at
2. Amplification or Enzymatic Activation another site on the cell membrane
- C5b is extremely labile. It is rapidly inactivated when no
Once the C1 complement component are already binding of C6 occurs.
fixed, the nect components to be activated are C4, C6 binds to C5b to form C5b6 (unstable)
C2 and C3- Activation unit.
C7 binds to C5b6 to form C5b67
3. Formation of MAC (Membrane Attack Complex)
C8 binds to C5b67 to form C5b678
Leads to cell destruction/lysis C7 and C8 interacts with the cell membrane to form a small
Involves C5 through the C9 components hole in the membrane
RECOGNITION UNIT C8 is inserted into the lipid bilayer of the cell membrane
Pore formation - formation of the hole which then the lysis of
The first complement to bind is C1. the cell starts
Three subunits Contents of the cell leaks (potassium, amino acids, proteins,
ribonucleotides)
(1) C1q
Pore formed are small (able to lyse the RBC but not the
(2) C1r nucleated cells)
(3) C1s C9 binds to C5b678 = complete lysis of the cell
C1r and C1s subunits- generate enzyme activity to begin the ALTERNATIVE/ALTERNATE/BYPASS/ PRIMITIVE/
cascade PROPERDIN
Triggering substances for the alternative pathway include:
C1q - is composed of six strands that form six globular heads ✓ Bacteria
with a collagen like tail portion.
✓ Fungi
- C1q “recognizes” the fragment crystallizable (FC) region of ✓ viruses
two adjacent antibody molecules
✓ tumor cell lines
- -Binds to the Fc portion of the antibody.
✓some parasites (Trypanosomes)
Activation unit - No C1, C2 and C4 in this pathway
C4 - Is the second most abundant complement protein - Main trigger is bacterial endotoxin, a
- Cleaves by C1 to form C4a and C4b lipopolysaccharide compound found in the outer
membrane of the gram negative bacteria
C4b must bind to a protein of a carbohydrate within a few - Other triggering factors: yeast cell wall, cobra venom
seconds or it will react to a water molecule to form the factor.
inactive form.
- C4b must be activated to form C3 convertase Complement components:
———————————————————————— - The normal serum proteins are termed as Factors
- Symbolized by letters essential in the initiation of the
C2 - Next component to be activated step by step process
- When combined with C4b in the presence of magnesium ions - Example: First step: Spontaneous hydrolysis of factor C3
C2 is cleaved by C1s to formC2a and C2b which is present in the blood plasma.
- C2a binds to C4b to form C3 convertase
- Written as “C4b2a” in classical pathway
- The C2 Gene is closely associated with the gene for
Factor B (Alternative Pathway on Chromosome 6 in the
Major Histocompatibility Complex (MHC) Class II)

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 COMPLEMENTS IMMUNOLOGY
SHERWIN ALVARO, RMT APRIL 19, 2022 AND SEROLOGY

1. Soluble C3- major factor in the complement system.

Present in classical and alternative pathway
2. Factor B
- analogous to C2
- bind to C3 to form C3 convertase
- Binds to activated C3 (C3b)
3. Factor D
- believed to be similar to C1s
- cleaves the factor B that are bounded to C3b
- it cleaves the factor B to form Bb and Ba.
- Bb will remain and will attach to C3b forming
C3bBb (C3 Convertase)
4. Properdin
- a gamma globulin which when complexed with C3b
stabilizes the alternate pathway C3 convertase
Alternate pathway activation

Components of the alternative pathway

Active
Native component Function(s)
component(s)
Mediates inflammation;
C3a anaphylatoxin
C3 Binds cell surfaces for
- Antibody independent C3b opsonization and activation
- Immune complex is not needed for it to be triggered. of alternate pathway
- There are many unrelated cell substances (bacterial Binds membrane bound C3b.
lipopolysaccharides, fungal cell wall, fungi, viral B Cleaved by Factor D
envelopes, viruses that can initiate the process by binding
C3 and Factor B.
Ba Unknown
- This complex is cleaved by Factor B (a protease) to Factor b
produce C3bBb (C3 Convertase) with the help of Factor Cleaved form stabilized by P
D. Bb produces C3 convertase.
- C3 convertase generate more C3b Attaches to C3b
- Factor Ba causes a takeover. The spontaneous activation Cleaves Factor B when bound
of C3 Factor D D to C3b
- The alternative pathways are more important; the first Binds and stabilizes
time we are infected by a microorganism. Properdin P
- C3bBb will cleave additional C3 to form C3a and C3b. membrane bound C3bBb.
- Some C3b will attach to cellular surfaces and will act as MANNOSE - BINDING LECTIN (MBL)
a binding site for more Factor B.
- Properdin- stabilizes the C3 convertase which increases
its half-life from 90 seconds to several minutes.
- C3bBb will combine with C3b to form C3bBb3b (C5
convertase)
- C3bBb will cleave to form C5a and C5b.
- Same with Classical Pathway until the formation of
C5b6789 (MAC).
- At first, they know that Properdin triggers this pathway
but later found out that it only stabilizes the C3
convertase.

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 COMPLEMENTS IMMUNOLOGY
SHERWIN ALVARO, RMT APRIL 19, 2022 AND SEROLOGY

- MBL binds to the surface of the microbes bearing a Decay accelerating factor
mannan.
• Also known as CD55
- Mannan- polymer of the sugar mannose
- Mannose- building block (Singular) • Cleaves C3b or C4b
- Binding causes the activation of MASP (MBL Associated Comparison of Classical and alternative pathway
Serine Proteases). Classical Alternative
- MASP-2 cleaves o C2 and C4 and activates the Classical
Pathway. Immunologic activators: Aggregated IgA and in some
- The purpose of MBL Pathway is to activate the classical IgM, IgG3, IgG1, IgG2 bund instances IgG4 and IgE
pathway.
- This process bypasses the antibody requiring step and so to antigen = immune complex
is protective early in infection before the antibody is Non-Immunologic Activators: Bacteria and plant
formed. Apoptotic cells, polysaccharide, LPS,
- No antibody requirement. staphylococcal protein A, CRP zymosan, inulin, cobra vemon
- Mannose is just bind with a microbe with Mannan and the bound t ligand, certain factor, viruses and tumor cells,
pathway is activated already
viruses and gram-negative some parasites like
————————————————————————
bacteria, DNA trypanosomes, shistosomes
- Called as Collecting family of molecules where there is
the structurally similar to C1q in its function as an Prossess a recognition unit Bo recognition unit
opsonin. (C1q)
- Interaction of MBL with a carbohydrate on the surface of Requires presence of Ca and Lack dependence on Ca and
polysaccharide of microbes that leads to the formation
of enzymatic complex that binds and activates the C4 requires C1, C2 and C4 for activation immidiately starts
and C2. its activation with C3 (bypass pathway)
- Provides an additional link between the innate and the
acquired immune response because it involves non- MAIN SOURCES of COMPLEMENT
specific recognition of carbohydrates that are common
constituents of microbial cell wall that are distinct from PROTEINS: hepatocytes, intestinal and urogenital
human cell surfaces.
epithelial cells, blood monocytes and macrophages.
MBL is considered an acute phase protein.
- Produced in the liver and is normally present in serum Homeostatic maintenance of complement activation is
but increases during an initial inflammatory response. mediated by regulatory proteins.
- Mannose- carbohydrate Examples:
- Lectins- proteins that binds to carbohydrates. 1. On plasma (Fluid based inhibitors): anaphylatoxin inhibitor,
This pathway provides an additional link between the C1 inhibitor, Factors H and I, C4 binding protein, S protein
innate and acquired immune response. and S 40-40.
(1) MASP-1
(2) MASP-2 2. On cells (Cell Bound regulators): C3b/C4b receptor (CR1),
(3) MASP-3 Decay accelerating factor (CD55), membrane co-factor
MASP (MBL Associated Serine Protease) protein and CD 59 (MIRL- Membrane Inhibitor of Reactive
REGULATION OF THE CLASSICAL AND LECTIN PATHWAYS Lysis)
C1 inhibitor (C1INH) - Inactivates C1 and MASP-2;
disassociates C1s and C1r from C1q Serum Molecular Concentration
Function
- C1q will remain bound to the antibody but all enzymatic protein weight (mg/mL)
C1 105 240 Dissociates C1r and C1s
activity seizes.
inhibitor from C1q
a glycoprotein with a molecular weight of 105,000 that (C1-INH)
inhibits activation at the first stages of both the classical and Factor I 88 35 Cleaves C3b and C4b
lectin pathways
C3 convertase Inhibited by four main regulators Factor H 150 300-450 Cofactor with I to
C4b-binding protein (C4BP) inactivate C3b; prevents
• Works with factor 1 binding of B to C3b
C4-binding 520 250 Acts as cofactor with I to
• They inactivate C3b and C4b protein inactivate C4b
Complement receptor 1 (CR1) (C4BP)
• Also known as CD35 S protein 84 500 Prevents attachment of the
• Works also with factor 1 (vitronectin C5b67 complex to cell
membranes
• Cleaves C3b or C4b
Membrane Cofactor protein
• Most efficient cofactor for factor I
• Cleaves C3b or C4b
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 COMPLEMENTS IMMUNOLOGY
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In vitro destruction of complement: • Sheep RBC coated with Hemolysin

External Source Of Complement: GUINEA PIG SERUM
(complement is mostly present in Guinea pig serum)
COMPLEMENT FIXATION TEST

Deficiencies of complement components

Deficient component Associated disease
C1 (q, r, or s) Lupuslike syndrome; recurrent infections
C2 Lupuslike syndrome; recurrent
infections; atherosclerosis
C3 Severe recurrent infections;
glomerulonephritis
C4 Lupuslike syndrome
C5-C8 Neisseria infections
C9 No known disease association
C1-INH Hereditary angioedema
DAF Paroxysmal nocturnal hemoglobinuria
MIRL Paroxysmal nocturnal hemoglobinuria
Factor H or Factor I Recurrent pyogenic infections
MBL Pneumococcal diseases, sepsis,
Neisseria infections
Properdin Neisseria infections
Patient serum and antigen are incubated, and then a MASP-2 Pneumococcal diseases
measured amount of complement is added. After a C1-INH = C1 inhibitor; DAF = decay-accelerating factor;
second incubation, sheep red blood cells, which are MASP-2 = mannose-associated serine protease; MBL =
coated with antibody, are added to the reaction tube. If mannose-binding lectin; MIRL = membrane inhibitor of
patient antibody to the specific antigen is present, no reactive lysis.
hemolysis occurs. Lack of patient antibody allows the
complement to combine with coated sheep red blood
cells and lysis occurs.
1. Addition of chelating agents (EDTA)
2. HEAT Inactivation of serum: Heat serum at 56 degrees
Celsius for 30 minutes
3. Treatment with Zymosan
Diagnostic evaluation of Complement:
A. Serum/Plasma assay (AH50 Assay)
o C3 and C4 by Nephelometry
B. C1 binding assay (CH50 Assay)
o measures the binding of immune complexes containing
IgG1, IgG2 or IgG3 and/or IgM to C1q.
o It can be useful as a prognostic tool at diagnosis and during
remission of acute myelogenous leukemia.
example of secondary immunologic/serologic test
Sample Used: Inactivated Serum with destroyed complement
proteins
SYSTEMS:
1. TEST SYSTEM
• antigen and antibody
2. INDICATOR SYSTEM

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 COMPLEMENTS IMMUNOLOGY
SHERWIN ALVARO, RMT APRIL 19, 2022 AND SEROLOGY

Receptors on Cell Membranes for Complement Components

RECEPTOR LIGAND CELL TYPE FUNCTION
CR1 (CD35) C3b, iC3b, C4b RBCs, neutrophils, monocytes, Cofactor for Factor I; mediates transport
macrophages, eosinophils, B and T cells, of immune complexes
Follicular dendritic cells
CR2 (CD21) C3dg, C3d, iC3b B cells, follicular dendritic cells, epithelial B-cell co-receptor for antigen with CD19
cells
CR3 (CD11b/CD18) iC3b, C3d, C3b Monocytes, macrophages, neutrophils, Adhesion and increased activity of
NK cells phagocytic
cells
CR4 (CD11c/CD18) iC3b, C3b Monocytes, macrophages, neutrophils, Adhesion and increased activity of
NK cells, activated T and B cells, phagocytic cells
dendritic cells
DAF (CD55) C3b, C4b RBCs, neutrophils, platelets, monocytes, Dissociates C2b or Bb
endothelial cells, fibroblasts, T cells, from binding sites, thus preventing
B cells, epithelial cells formation of C3 convertase
MIRL (CD59) C8 RBCs, neutrophils, platelets, monocytes, Prevents insertion of C9 into cell
endothelial cells, epithelial cells membrane
MCP (CD46) C3b, C4b Neutrophils, monocytes, macrophages, Cofactor for Factor I cleavage of C3b
platelets, T cells, B cells, endothelial cells and C4b

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 IMMUNOLOGY
IMMUNODULATION AND SEROLOGY
SHERWIN V. ALVARO, RMT , MPH May 10, 2022

TABLE OF CONTENTS
IMMUNOMODULATION 1 IMMUNOSUPRESSION
IMMUNOENHANCEMENT 1 TYPES OF IMMUNOSUPPRESSION:
IMMUNOSUPRESSION 1 1. SPECIFIC
PHAGOCYTIC CELL DEFICIENCIES 1 • Refers to Immune Tolerance
B LYMPHOCYTES DEFICIENCIES 2 TOLERANCE
T LYMPHOCYTE DEFICIENCIES 3 - inability of body to mount immune response to a
COMBINATION OF T AND B LYMPHOCYTES 3
DEFICIENCIES substance that is potentially immunogenic.
2. NON-SPECIFIC
-The body’s immune system is normally occurring 2.1. ARTIFICIALLY INDUCED
protective mechanism that helps the body defend 2.1.A. PHYSICAL MEANS
itself against potentially harmful agents. o Irradiation
-However, sometimes the immune system perceives o Thoracic Duct Drainage
normally harmless substances such as allergens or o Thymectomy
person’s own body tissues or organs as harmful o Splenectomy
invaders. o Bursectomy
- They try to eliminate those harmless substances.
- Inappropriate activation of the immune response 2.1.B. CHEMICAL MEANS
is a major factor in allergic and autoimmune o Corticosteroids
disorders. o Alkylating Agents
o Anti-metabolites
o Antibiotics
2.1.C. BIOLOGICAL MEANS
IMMUNOMODULATION o Anti-lymphocyte antibodies
o Anti-thymocyte antibodies
Modifying the immune response o Antigen desensitization
• Positive Immunomodulation: 2.2. NATURALLY INDUCED
IMMUNOENHANCEMENT/ Examples are different IMMUNODEFICIENCY
IMMUNOPOTENTIATION DISORDERS.
• Negative Immunomodulation: Deficiencies of the immune system include:
IMMUNOSUPPRESSION • PHAGOCYTIC CELL DEFICIENCIES
IMMUNOENHANCEMENT • B LYMPHOCYTES DEFICIENCIES
• Accomplished through adjuvants. • T LYMPHOCYTE DEFICIENCIES
ADJUVANTS - substance added to antigens before • COMBINATION OF T AND B
administration. LYMPHOCYTES DEFICIENCIES

• Purpose of adjuvants: to prolong exposure of PHAGOCYTIC CELL DEFICIENCIES

antigen to immune system • Result in a decreased ability to phagocytize and kill
bacteria.
COMPONENTS OF ADJUVANTS a. Chronic granulomatous disease (CGD)
1. Muramyl dipeptide is a genetic disease characterized by ineffective
2. Cell wall constituents of microorganisms (M. killing of catalase positive bacteria by neutrophils?
tuberculosis, B. pertussis, Brucella sp.) Cause:
3. Other components: mineral oil, lanolin, alum, CGD is caused by a defect in cytochrome b
detergent, acrylic particles, polynucleotides oxidase, which results in decreased hydrogen
peroxide production.
EXAMPLES OF ADJUVANTS
1. Aluminum hydroxide Diagnosis:
2. Complete Freund’s adjuvant (made up of killed M. The nitro blue tetrazolium (NBT) reductase
tuberculosis, mineral oil, lanolin) test is used to detect impaired neutrophil
3. Incomplete Freund’s adjuvant

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SHERWIN V. ALVARO, RMT , MPH May 10, 2022

phagocytosis. The neutrophils of CGD patients e. Specific granule deficiency

fail to reduce the NBT dye.
• is inherited as an autosomal recessive trait.
Symptoms: • Neutrophils fail to develop specific granules
Patients with CGD suffer from recurrent during myelopoiesis, and as a result, patients
infections caused by catalase-positive bacteria who have this disorder experience severe
and yeast and fungi. recurrent bacterial infections.
f. Chédiak-Higashi syndrome
Treatment:
includes the use of GSM-CSF or G-CSF and • is an inherited disorder that is characterized by
IFN-y. the abnormal fusion of primary granules in
neutrophils.
b. MPO deficiency • Patients who have Chédiak- Higashi syndrome
• is inherited as an autosomal recessive trait and have recurrent bacterial infections and are
is one of the most common inherited disorders. also characterized by albinism and extreme
• The MPO in the primary granules of neutrophils photosensitivity.
is decreased or absent, and although • During phagocytosis, the granulation is
phagocytosis takes place normally, bacterial impaired or no MPO is released into the
killing is inefficient. phagosome.
• Fungal killing is more seriously impaired than
bacterial killing. Healthy patient with MPO g. Lazy leukocyte syndromes include:
deficiency do not have an increase frequency 1. Job syndrome, also known as
of infection as well as diabetic patient who hyperimmunoglobulin E, is characterized by
have this disorder may have candida species poor chemotaxis and recurrent skin infections and
infection. abscesses.
c. Glucose-6-phospate dehydrogenase (G6PD) 2. Tuftsin deficiency. Tuftsin is a chemotaxin that also
deficiency improve phagocyte motility, engulfment and
oxidative metabolism. Affected persons
• is another inherited disorder in which the experience recurrent bacterial infections.
aerobic system of neutrophils is impaired.
• This deficiency results in a substantial decrease 3. Actin dysfunction. A deficiency of the cytoskeletal
in the amount of hydrogen peroxide protein actin can result in decreased bacterial
produced during phagocytosis, and thus motility and chemotaxis. Patients experience
decreased bacterial killing efficiency. recurrent bacterial infections.
• Patients with G6PD deficiency experience B LYMPHOCYTES DEFICIENCIES
recurrent bacterial infections. • may be inherited or acquired and account for
d. CR3 (iC3b receptor) deficiency more than half of all immunodeficiencies.
• Affectedness varies widely, depending on the
• is a rare, autosomal recessive trait class of immunoglobulin that is deficient.
characterized by a decrease or absence of • A deficiency of a minor immunoglobulin such as
specific complement component receptors on IgE causes little in any increase and the incidence
neutrophils, monocytes and lymphocytes. of the bacterial infections.
• Responsible for adherence related function. • However, 75%- 80% of the total
• Abnormalities result in defective margination immunoglobulins IgG, at individual deficient in
and diapedesis of neutrophils, impaired IgG would be significantly affected.
chemotaxis, and ineffective phagocytosis.
• T lymphocytes adhere to target cells • 1. Bruton’s agammaglobulinemia
• Chemically there is an increase frequency of • is a sex-linked disorder that primarily affects
bacterial infection, a decrease inflammatory men. Recognized in early in life where antibodies
response as well as neutrophilia fail to develop.

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SHERWIN V. ALVARO, RMT , MPH May 10, 2022

• B cells may be found in the bone marrow but they COMBINATION OF T AND B LYMPHOCYTES
do no mature. Few mature B cells are found in DEFICIENCIES
the peripheral blood wherein • are the most serious of the immunodeficiencies,
• Gamma globulin levels are markedly because both cell-mediated and humoral immune
decreased. responses are affected.
• This disorder may be treated with gamma 1. Bare-lymphocyte syndromes
globulin preparations. • Are characterized by defects in Class I MHC
• due to deficiency in Bruton’s thymidine kinase antigen expression,
• Class II MHC antigen expression, or a combination
2. Common variable hypogammaglobulinemia of both.
• is an acquired disorder in which one or two
immunoglobulin classes are deficient 2. Severe combined immunodeficiency
• Total immunoglobulin levels are normally because • disease may be inherited as autosomal recessive or
increase in one immunoglobulin is often X-linked traits
compensated by an increase in the production of • All are characterized by ______ decrease
another. stoppers of both A and B lymphocyte.
•
• Selective IgA deficiency is one of the most 3. ACQUIRED IMMUNODEFICIENCY SYNDROME (AIDS)
common of these deficiencies. • is caused by the human immunodeficiency virus 1
• Typically, only those patients whose disease (HIV-1) or the human immunodeficiency virus 2
includes IgG deficiency suffer from increased (HIV-2). Contracted by from an infected person to
bacterial infections. the whole mark of these syndrome is that it
weakens the body immune system wherein it
3. Neonatal hypogammaglobulinemia invades the Helper T cells or the CD4 cells making
• is cause by the normal immaturity of the neonate’s the infected person vulnerable to various
immune system. It corrects itself between ages of 6 opportunistic life threatening infection and as well
to 12 months as well as infants immune system. as cancers. Due to progressive failure of immune
system.
T LYMPHOCYTE DEFICIENCIES • So, AIDS predisposes our body to other
• without an accompanying loss of B-cell function are opportunistic infection.
rare, composing only 7% of all immunodeficiencies. • The CD4-positive T lymphocytes are the primary
This orders or immunodeficiency’s may be target cells.
acquired or inherited. • Approximately 5% of B-lymphocytes are also
1. DiGeorge syndrome infected.
• results when the thymus gland develops abnormally
during embryogenesis. REFERENCES:
• T-lymphocytes are usually decreased, but may be i. Turgeon, Mary Louise. (2014). Immunology &
normal. Serology in laboratory medicine, (5th ed.). Missouri :
• Most patients have high CD4-CD8 ratio. Antibody Elsevier.
responses may be normal cell mediated immune ii. Male, David. (2013). Immunology, (8th ed.). St. Louis,
responses are impared. Missouri : Elsevier.
iii. Stevens, Christine Dorresteyn. (2010). Clinical
2. Nezelof syndrome immunology & serology : a laboratory perspective, 3rd
ed. Philadelphia ; F.A. Davis
• is an autosomal recessive disorder.
• Patients are especially susceptible to viral and
fungal infections, which can be fatal in these
patients. “Be still and know that I am with you.”
- Psalm 46:10

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SHERWIN ALVARO, RMT MAY 24, 2022 AND SEROLOGY

TABLE OF CONTENTS
release of pre-formed vasoactive mediators such as
Hypersensitivity 1 histamine, ECF-A (eosinophil chemotactic factor of
anaphylaxis), neutrophil chemotactic factor and
Major Sensitivity Reactions 1 tryptase as well as newly synthesized mediators
Type I Hypersensitiivity 1
such as prostaglandins and leukotrienes.
-also called Anaphylactic hypersensitivity
Type II Hypersensitiivity 2 -Immediate (antibody mediated)
-Reacts with antigen to release
Type III Hypersensitiivity 2
-mediated by antibody -Immunoglobulin E
Type IV Hypersensitiivity 3 -involves secondary exposure to an offending antigen that
bonds to your mast cell fix immunoglobulin E
Hypersensitivity Comparison 3 -It is attached to your cell bound antibody and it reacts with
Diagnosis of Hypersensitivity Reactions 3 antigen to release physiologically active substances
-Key reactant: Immunoglobulin E
Summary 4 -Allergens or atopic antigens- antigens that trigger
Legends: PPT, AUDIO, BOOK, ONLINE SOURCES
formation of Immunoglobulin E
*In Immediate hypersensitivity- patients produce large
amount of IgE in response to a small concentration of
HYPERSENSITIVITY antigen
• heightened state of immune responsiveness. *Eosinophils- commonly present during allergic and parasitic
• It is an exaggerated response to an innocuous reactions
antigen that results in gross tissue changes that are Short lag time-early phase of allergic reactions that
deleterious to the host. appears within minutes upon exposure to antigen (Signs and
-end goal of immune system is to protect from potential symptoms would appear within minutes after exposure but
antigens foreign substances some may begin after hours (rare))
-it is reaction that ends up causing damage to tissues *Pinaka-late is within hours
-STEPS
1. Sensitize before exposure of antigen IMMUNOLOGIC ACTIVITY
2. Symptoms and signs of hypersensitivity • MAST CELLS
-OA/exaggerated response of immune response to attack o Are cellular receptors for IgE, which attaches to
harmless antigens their outer surface
-There are antigens that are harmless, some are regarded o Basophils- approx. 1%, contain histamine
as antigens granules, high affinity receptors for IgE just as in
-MOST of them are harmful, but SOME are harmless the mast cells
-Effect: Tissue damage, disease and death o IgE-number of receptors has been found to
-exaggerated or uncontrolled response to an antigen that increase indicating possible mechanism of
can produce inflammation, cell destruction, or tissue injury regulation during an allergic reactions
-Normal but exaggerated immune response
CLASSIFICATION • IMMEDIATE HYPERSENSITIVITY
• IMMEDIATE-antibody mediated hypersensitivity, much o Basis of acute allergic reactions caused by
dangerous molecules released by mast cells when an
• DELAYED –cell-mediated hypersensitivity allergen interacts with membrane-bound IgE.
MAJOR HYPERSENSITIVITY REACTIONS o Major mediators of allergies of asthma
PGH GEL& RRA COOMBS-british immunologist, 1. Histamine
device a classification system for such reactions on 2. Leukotriens
4 different categories 3. C4
*PGH GeL-Philip George Houthem Gell 4. IL 4
• Type I Hypersensitivity 5. IL 3
• Type II Hypersensitivity • ANAPHYLACTIC REACTION
• Type III Hypersensitivity o Is the clinical response to immunoglobulin
• Type IV Hypersensitivity formation and fixation between a specific
Type I Hypersensitivity antigen and a tissue-fixing antibody.
• It is an immediate hypersensitivity, sudden allergic o most dangerous form of type I hypersensitivity
responses mediated by antibodies, primarily IgE. o other term: anaphylaxis
• The antigen cross-links two adjacent IgE molecules,
leading to degranulation of the mast cells, with
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SHERWIN ALVARO, RMT MAY 24, 2022 AND SEROLOGY

• ANAPHYLACTOID REACTION • A 1mL tuberculin syringe is used to administer 0.01

o Anaphylaxis-like to 0.05mL of test solution between layers of the
o anaphylaxis like, clinically similar to anaphylaxis skin.
which are not mediated by antigen-antibody • The test allergen is diluted 100 to 1000 times more
reaction, than the solution used for cutaneous testing.
o there are these offending substances that act • After 15 to 20 minutes, the site is inspected for
directly at mast cells causing release of erythema and wheal formation.
mediators on tissues (Anaphylatoxins of • A wheal 3mm greater than the negative control is
complement cascade) considered a positive test.
• ATOPIC REACTION • -usually performed only if cutaneous test or prick
o Exaggerated response characterized by test are negative but still suspect an allergic
production of allergen specific IgE antibodies reaction to patient
o inherited tendency to respond to naturally IN VITRO:
occurring inhaled and ingested allergens with Radioimmunosorbent test (RIST)
continued production of IgE • measures total IgE
o Allergy –any altered reaction to external Competitive RIST
substances • Uses radiolabeled IgE to compete with patient IgE
EXAMPLE for binding sites on a solid phase coated with anti-
Bagoong- in your second exposure, signs and symptoms IgE.
appear, the body recognize bagoong as antigen Radioallergosorbent assay (RAST)
knowing that it is harmless. • measures antigen specific IgE.
There is uncontrolled response because once or • Principle: Fluorescent label
twice exposure only.
*Signs and Symptoms- erythema, wheal and flare, increase Type II Hypersensitivity
mucus production, increase vascular permeability, increase Reactions that produce cell damage which is mediated by
acid production in stomach. complement-fixing antibodies directed against cell surface
Etiologic agents: antigens.
1. Venom from bees, wasps, hornets. • Transfusion reactions (incompatibility, HDN)
2. Food allergies • Autoimmune hemolytic anemia
3. Drugs • Drug-induced (purpura)
4. Latex •Antibodies involve: IgM and IgG
5. Other allergens •they are triggered by antigens found in cell surfaces
Clinical states: •antibodies can induce complement dependent lysis or
1. Hay fever engage macrophages
2. Asthma •____________ antibodies “cellular surfaces and promote
3. Eczema phagocytosis by both opsonization and activation of
4. Anaphylactic shock complement cascade”
5. Helminthic infection

TEST FOR IMMEDIATE HYPERSENSITIVITY Type III Hypersensitivity

In Vitro • Also called immune complex media ted
–Indirect skin testing, least expensive and most specific, hypersensitivity.
measures total and specific immunoglobulin E • They are due to the deposition of Ag-Ab complexes
In Vivo in tissues and blood vessels.
-Direct skin testing • These complexes can destroy the surrounding tissue
Cutaneous testing directly or indirectly by attracting neutrophils to the
• in cutaneous testing, or a prick test, a small drop of site of complex deposition that release hydrolytic
material is injected into the skin at a single point. enzymes, causing local damage.
• After 15 minutes, the spot is examined, and the • similar to Type II reactions in that it involves IgM
reaction is recorded. and destruction is complement mediated
• A positive reaction is formation of a wheal that is • difference from TYPE II: ANTIGEN is SOLUBLE
3mm greater in diameter than the negative control. • When soluble antigens combines with an antibody,
Intradermal testing there are complexes
• use a greater amount of antigen and are more
sensitive than the negative control.

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• Complexes are cleared by phagocytic cells, but if HYPERSENSITIVITY COMPARISON

immune system is overwhelmed this complexes HUMORAL MEDIATED CELL-MEDIATED
deposit in your tissues.
• Deposition of Antigen-antibody complexes in tissues HYPERSENSITIVITY HYPERSENSITIVITY
REACTIONS REACTIONS
due to soluble antigens
Arthus reaction Includes Types I, II, II I and Include type IV reactions
• a necrotic dermal reaction considered to be a local V reactions
immune complex deposition phenomenon.
• Demonstrated by Maurice Arthus in 1903 Imm une reactions are Reactions are delayed in
Serum sickness observed minutes after time
• it results from passive immunization with animal serum antigen exposure
usually horse or bovine serum, used to treat such
infections as diphtheria, tetanus and gangrene. Inflammatory reactions are Reactions are
• generalized symptoms appear 7 to 21 days after characterized by more characterized by
injection of animal serum ( fever, headache, nausea, fluid and erythema significant cell infiltratioin
etc.) (Wheal and Flare reaction) with
resultant induration
Type IV Hypersensitivity
• It is a hypersensitivity reaction mediated by sensitized T DIAGNOSIS OF HYPERSENSITIVITY REACTIONS
cells releasing lymphokines, attracting macrophage to - TYPE 1: in vivo skin tests
the site and activating them.
• Once the macrophage arrives, they begin to cause • RIST (Radioimmunosorbent Test)
tissue damage that may develop into a chronic - measures total IgE
granulomatous reaction if antigen persists. • RAST (Radioallergosorbent Test)
• It is also called delayed type hypersensitivity - measures allergen specific IgE
-appear weeks and months (more than 24hrs) for signs and 1. TYPE 2
symptoms to appear • DAT (Direct Antiglobulin Test)
-sensitized cells-are usually sub population of TH1 cells that • INDIRECT ANTIGLOBULIN TEST –first incubate
plays a major role in this manifestation 3. TYPE 3
-Initial Sensitization Phase-1 and 2 Weeks, takes place • immunoassays and certain agglutination reactions
after first contact of antigen 4. TYPE 4
-T cytotoxic cells • Tuberculin Test/Mantoux test
Types of Cell-Mediated or Type IV Hypersensitivity
reactions:
a. Delayed type hypersensitivity
• effector cells are Tdth cells that recruit other cells such
as macrophages.
b. Cell-mediated Cytotoxic reactions
• effector cells are T cytotoxic cells.

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 HYPERSENSITIVITY IMMUNOLOGY
SHERWIN ALVARO, RMT MAY 24, 2022 AND SEROLOGY

SUMMARY
I- Anaphylactic • Allergic rhinitis, urticarial, hives • Plant pollen, house dust mites, foods IgE, Mast Cells
or Immediate • Bronchial Asthma (e.g. Fish, eggs, chocolate
• Drugs or Insect Bites • Fungal or mold spores, animal
• Tropical Eosinophilla danders, animal hairs
• Loffler’s Syndrom • Penicililin, antiseptics, anesthetics, bee
sting wasp
• Parasites (Wuchereira bancrofti)
• Chitin on Larva of Ascaris
lumbricoides
II- Cytotoxic • Hemolytic Transfusion Reactions (HTR) • Antigens on RBC IgG, IgM,
• Hemolytic Disease of the Newborn • Antigens on RBC Complement (IgA)
• Acquired Immune Hemolytic Anemia • Penicillin, quinidine coating RBC
• Idiopathic Thrombocypenic Purpura • Ags on surface of platelets
• Good Pasteur Syndrome • Ags on basement membrane of the
glomeruli of the Kidney
III- Immune • SLE (Systemic Lupus Erythematosus) • Nuclear Material IgG, IgM, IgA
Complex • RA (Rheumatoid Arthritis) • IgG Complement
• Arthus- like Reaction • Aspergillus fumigatus
• Serum Sickness • Horse Serum
IV- Delayed or • PTB • M. tuberculosis Lymphocytes
Tuberculin • Leprosy • M. leprae
• Contact dermatitis • Dyes, metals, cathecol, rubber
V- Antireceptor • Grave’s Disease • TSH Receptor acinar cells IgG but
or Stimulatory • Myasthenia gravis • acetylcholine Complement Fixing
VI- • Gram negative endotoxic Shock • LPS- Bacteria
Miscellaneous • PNH (Paroxysmal nocturnal • Defective RBC Membrane
hemoglobinuria)

“Be still and know that I am with you.”

- Psalm 46:10

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 IMMUNOLOGY
TRANSPLATATION AND SEROLOGY
SHERWIN V. ALVARO, RMT , MPH May 31, 2022

TABLE OF CONTENTS RA 7170

HISTORY 1
HISTOCOMPATIBILITY ANTIGENS 1 - Organ Donation Act of 1991
RA 7170 1 - January 7, 1992, approved by 8th congress
HLA APPLICATIONS 1 - An act authorizing the legacy or donation of
DIVERSITY OF TRANSPLANTATION (HLA) 1 all or part of a human body after death for
ANTIGENS specified purposes.
TYPES OF TRANSPLANTATION 2 - Transplantation is defined as the transfer of
TYPES OF GRAFT 2 one cell, tissue or organ from one site to
TRANSPLANTATION 2 another within an individual or between other
ORGANS/BODY PARTS THAT CAN BE 3 species whether genetically identical or not.
TRANSPLANTED
- The transferred organ or tissue is called
GRAFT ACCEPTANCE 3
transplant organ or graft.
GRAFT REJECTION 3
- The individual from whom the organ came from
STAGES OF GRAFT REJECTION 3
is called the donor and the individual to whom
TYPES OF GRAFT REJECTION 4
GRAFT VERSUS HOST DISEASE 4 the organ is applied or given is called the
TESTS ON DONOR TESTING & TISSUE TYPING 5 recipient/ acceptor.
TRANSPLANT REJECTION 5 - After transplantation if the graft survived
TRANSPLANTATION inside the recipient graft is said to be accepted
-transfer of cells, tissues, or even organ - If the graft does not survive it is rejected/ white
graft.
ORGANIZATION OF HUMAN BODY
- CELLS HLA APPLICATIONS
- TISSUES
- ORGANS • It is obligatory to select HLA- identical donors for
- ORGAN SYSTEM bone marrow transplantation to reduce frequency
- ORGANISM of graft- versus host disease.
• -that’s why part of the serological tests before
HISTORY transplantation is HLA typing/ matching

• DR. JOSEPH MURRAY • HLA-matched platelets are useful for patients who
- Performed the first organ transplantation, are refractory to treatment with random donor
using a kidney from an identical twin, in 1954 platelets.
at Peter Bent Brigham Hospital in Boston • Paternity Testing
- Won a noble prize in 1990 in medicine • Forensic Medicine
- The recipient survived for 9 years • Anthropology
• Basic research in immunology
HISTOCOMPATIBILITY ANTIGENS
• Diagnostic and genetic counselling
• The Major Histocompatibility Complex (MHC)
- Is a cluster of genes found on the short arm of Diversity Of Transplantation (HLA) Antigens:
chromosome 6 at band 21 (6p21)
- These genes code for the proteins that have a
• 6 different types of Class I HLA molecules
role in immune recognition (HLA)
expressed/cell
- HLA → the molecular basis for T-cell
• 12 different types of Class II HLA molecules
discrimination of self from non-self expressed/cell
- Transplanted tissue may trigger a destructive
• Huge number of Class I and Class II alleles in
mechanism (rejection), if the patient’s cells
humans *
recognize the MHC protein products on the
- * there are 13,680 Class I HLA and 5091 Class
surface of the transplanted tissue as foreign, or
II HLA alleles
if immunocompetent cells transplanted on the
donor tissue target the foreign cells of the • TOTAL number of HLA Class I and Class II alleles =
recipient for elimination. 18,771 across the human population

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SHERWIN V. ALVARO, RMT , MPH May 31, 2022

TYPES OF TRANSPLANTATION: • XENOGRAFT/ HETEROGRAFT

- Transfer of tissue between two individuals
1. ORTHOTOPIC of different species (ex: pig heart valve to
- transfer of tissue/organ from the site of the a human heart)
donor to the site of the recipient Considered a successful operation
2. HETEROTOPIC in immunology research, because
- transfer of tissue/organ from the site of the this was the first xenograft
donor to different site of the recipient transplantation that happened to a
3. HOMEOSTATIC senile person.
- no tissue growth after transplantation
4. HOMOVITAL
- there is tissue growth after transplantation

TYPES OF GRAFT:

• AUTOGRAFT
- Transfer of tissue from one area of the body
to another of the same individual TRANSPLANTATION
- It can be the skin, the most common, aside from
that, is a tissue transferred to the nose.

• SYNGRAFT/ ISOGRAFT
- Tissue transferred between genetically
identical individuals.
- or within breed mouse population, because it is
the most common study population specially in
experimental research is mouse or rat
population. Graft between identical twins

T cell is responsible for transplantation, especially in organ

rejection and acceptance during transplantation.

• ALLOGRAFT/ HOMOGRAFT In here we have the recipient APC and the donor APC and
- Transfer of cells or tissues between two that circle on the other side is the Recipient CD4 T cell which
individuals of the same species is linked via foreign MHC derived peptide for the indirect
- Grafted donor tissue or organ contains allorecognition. While for direct allorecognition we have
antigens not present in recipient here a peptide, they are almost similar in terms of antigen
- done in genetically non identical. Most presentation, however they are different in terms of direct,
common human transplantation wherein this is from the donor itself while for indirect is for
recipient.

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SHERWIN V. ALVARO, RMT , MPH May 31, 2022

ORGANS/BODY PARTS that can be TRANSPLANTED - both Delayed-type Hypersensitivity & Cell-
mediated Cytotoxicity reactions have been
• Blood vessels implicated
• Liver - Necrosis occurs
• Cornea - No vascurization happened, in means graft
rejection
• Heart
• Kidneys
• Bone marrow or stem cells
• Bone
• Lungs
• Middle ear
• Pancreas
• Skin
In the Philippines the DOH lists the human transplantable
organs that includes:
Kidneys- most common transplanted organ
Liver
Heart
Lungs
Skins
Pancreas
Eye tissue (Sclera, cornea and et.)
Bones
Blood vessels
Stages of Graft Rejection:
1. SENSITIZATION STAGE
GRAFT ACCEPTANCE
- when vascularization & healing lead to a repaired - Where CD-4 & CD-8 T-cells recognize Alloantigens
site in about 2 weeks. expressed on cells of the foreign graft &
- connection of blood vessels proliferate in response
- The host T-helper cell becomes activated when it
interacts with an Antigen presenting cell
- Depending upon the tissue, different population of
cells within a graft may function as Antigen
presenting cells. Migration of passenger
Leukocytes from a donor graft to regional Lymph
nodes of the recipient results in the activation of T-
helper cells in response to different Class-II MHC
antigens expressed by the passenger Leukocytes
- These activated T-helper cells then induce
generation Cytotoxic-T cells of which mediate graft
rejection

GRAFT REJECTION
- caused by cell-mediated immune response
to Alloantigens expressed on cells of the
graft

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SHERWIN V. ALVARO, RMT , MPH May 31, 2022

2. EFFECTOR STAGE • ETIOLOGY:

- most are Cell-mediated reactions involving - Immunocompetent T lymphocytes are
Delayed-type Hypersensitivity & CTL-mediated transfused from a donor to an immunodeficient
cytotoxicity- less common mechanisms are or immunosuppressed recipient, the transfused
Antibody-plus-Complement lysis & destruction by or grafted lymphocytes recognized that the
Antibody-dependent cell-mediated cytotoxicity antigens of the host are foreign and react
(ADCC) immunologically against them.
- The whole mark of graft rejection involving cell • 2 Types:
mediated reactions is the influx of t cells and o ACUTE GVHD
macrophages into the graft. ➢ Occurs during the first 100 days post
- Instead na magkaroon ng vascularization, infusion and targets the skin, GIT, and liver
nagkakaroon ng influx of macrophages of t cells
and antigen presenting cells making the graft to be o CHRONIC GVHD
rejected. ➢ Beyond 100 days post-transplant;
resembles autoimmune disease, with
fibrosis affecting the skin, eyes, mouth, and
other mucosal surfaces
• Signs and Symptoms:

- Post-transfusion symptoms begin within 3

to 30 days after transfusion
- Ulcerative skin and mouth lesions,
diarrhea, and liver destruction
- Jaundice, fever, anemia, weight loss, skin
rash, splenomegaly
Types of Graft Rejection o ACUTE GVHD
1. FIRST-SET REJECTION - Erythematous maculopapular skin rash →
- first time a graft is encountered & rejected diarrhea, often with abdominal pain, liver
- the immune system attacks & ultimately disease; fever, granulocytopenia, and
destroys the “non-self- tissue” which occurs bacteremia
10-14 days after transplantation
- pag 1st time mag receive ng organ sa o CHRONIC GVHD
isang tao at nireject ng katawan nito ang - Resembles collagen vascular disease; skin
organ, ito and 1st set rejection changes: erythema and cutaneous ulcers
→ liver dysfunction; susceptible to
2. SECOND-SET REJECTION bacterial infections
- the second time the same set of “non-self- • Immunologic Manifestation:
tissue” is encountered
- which is usually rejected within 6 days ✓ Laboratory evidence of immunosuppression or
- kapag may nag donate ulit ng organ sa immunodeficiency
taon iyon at nireject ulit ito ng katawan ✓ Evidence of inflammation
niya, ito ang tinatawag na 2nd set ✓ Complication of anemia and liver disease
rejection.
✓ Presence of opportunistic pathogens
GRAFT VERSUS HOST DISEASE
• Prevention:
• Graft-versus-host Disease (GVHD)
- Can be unintentional consequence of blood ✓ Lymphocyte reduction- leuko reduced- most
transfusion or transplantation in severely in common given to those with GVHD
severely immunocompromised or
✓ Irradiation - irradiated RBC
immunosuppressed patients
✓ Immunosuppression

LEC TRANSCRIBERS LIM, J.M., MARCAIDA V.P., PROGRESO, L.C.

4
 IMMUNOLOGY
TRANSPLATATION AND SEROLOGY
SHERWIN V. ALVARO, RMT , MPH May 31, 2022

✓ Most immunogenic organ: Cells of Langerhans,

skins, kidneys, bone marrows, bone, middle ear
✓ Least immunogenic organ: cornea

Tests on Donor Testing & Tissue Typing:

1. ABO/Rh Typing
2. CMV, EBV, HPV-B19 (Cytomegalovirus, Epstein
Barr Virus, Human Papilloma Virus-B19)
3. Anti-HIV 1 & 2, HTLV-I & II
4. HBsAg, HBcAg, HCV
5. RPR, FTA (Rapid Plasma Reagin, Fluorescent
Treponemal Absorption test)
6. HLA matching

REFERENCES:

• Turgeon, Mary Louise. (2014). Immunology & serology

in laboratory medicine, (5th ed.). Missouri : Elsevier.
• Male, David. (2013). Immunology, (8th ed.). St. Louis,
Missouri : Elsevier.
• Stevens, Christine Dorresteyn. (2010). Clinical
immunology & serology : a laboratory perspective,
3rd ed. Philadelphia ; F.A. Davis

“Be still and know that I am with you.”

- Psalm 46:10
Transplant Rejection
Type Onset
Mechanism & Vessel Histology Type of Hypersensitivity
Hyperacute Immediate
Preformed antibodies directed against the donor
1st 24 hours Humoral
tissue. Caused by accidental ABO blood type
after incompatibility which is very rare. Presents while still in II
transplantation surgery with thrombosis and occlusion of graft vessels
Acute
Weeks to T-Cell mediated immune response directed against the
“ Host vs. Graft”

months foreign MHC. Inflammation and leukocyte infiltration IV

of graft vessels results. Most common type.
Chronic Months to T-Cell mediated process resulting from the foreign MHC
years "looking like" a self MHC carrying an antigen. Results III & IV
in intimal thickening and fibrosis of graft vessels as
well as graft atrophy
Graft vs. Host Varies Donor T-Cells in the graft proliferate and attack the
recipient's tissue. Most commonly seen in bone marrow IV
transplants. Presents with diarrhea, rash and
jaundice.
Accelerated First 5
hours
* The most important thing to remember is the timeframe for the onset of symptoms as you can often determine what type
of rejection it is based on time alone

LEC TRANSCRIBERS LIM, J.M., MARCAIDA V.P., PROGRESO, L.C.

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 AUTOIMMUNITY IMMUNOLOGY
SHERWIN ALVARO, RMT JUNE 07, 2022 AND SEROLOGY

TABLE OF CONTENTS
❑ Polyclonal B-cell activation - some gram negative
AUTOIMMUNITY 1
bacteria and several viruses like CMV and EBV can
trigger the auto-reactive.
CLINICAL TYPES OF AUTOIMMUNITY 1

RHEUMATOID ARTHRITIS
CLINICAL TYPES OF AUTOIMMUNITY:
1
1. ORGAN SPECIFIC AUTOIMMUNITY
SYSTEMIC LUPUS ERYTHEMATOSUS 2 • Lesions from damaged tissue and autoantibodies
are directed towards a single target organ or
COMPARISON OF ORGAN SPECIFIC AND SYSTEMIC AUTOIMMUNITY 4
cell inside the human body.
GENERAL SIGNS OF AUTOIMMUNE DISEASES THAT MAY HAVE DIAGNOSTIC VALUE 4 AUTOIMMUNE DISEASES ANTIGENS/TARGET
OTHER FACTS ABOUT AUTOIMMUNE DISORDERS 4 ORGANS OR CELLS
POSITIVE PATTERS OBSERVED IN FLUORESCENT MICROSCOPY 4 Microsomal proteins of adrenal
Addison’s disease cells
INTERPRETATION OF IMMUNOFLUORESCENT PATTERS 4
Acute disseminated
Basic protein of myelin
IMMUNOPROLIFERATIVE DISEASES 5 encephalomyelitis

Legends: PPT, AUDIO, BOOK, ONLINE SOURCES Thyroglobulin, Microsomal

Hashimoto’s thyroiditis
antigen

thyroid peroxidase Islet cells of

AUTOIMMUNITY Type 1 DM pancreas
• Antinuclear antibodies (ANA) - autoantibodies
• conditions in which damage to organs and or tissues
results from the presence of autoantibody or Type IV collagen of basement
Goodpasteur’s syndrome
membrane
autoreactive T cells.
• T-cells can't recognize its own antibody and antigen Grave’s disease TSH receptors
resulting to abnormal response of the immune system.
ETIOLOGY Myasthenia gravis Acetylcjoline receptors
• defect in mechanisms underlying self-recognition Autoimmune chronic active
Smooth muscles (ASMA)
POSSIBLE MEHCANISMS: hepatitis
❑ Sequestered antigens - antigens that do not
Gastric parietal cell antigens,
usually circulate in the blood which leads to failure Pernicious anemia
intrinsic factor
of immune tolerance.
❑ Foreign antigens - may trigger or cross react with Sjogren’s syndrome
Salivary gland nucleolar
self-antigens by the virtue of molecular mimicry. antigens
Molecular mimicry - refers to the similarity between an Primary Biliary Cirrhosis Mitochondria
infectious agent and a self antigen that causes antibody
formation in response to the infectious agent to cross-react Autoimmune Myocarditis Striated cardiac muscle
with self. Example: Pemphigus vulgaris Epidermal antigens
Polio virus (VP2) - it has the resembled self-antigen same
as acetyl choline receptors. Bullous Pemphigoid Skin basement region antigens
Micelles Virus (P3) - it has the resembled self-antigen
Autoimmune rheumatic fever Heart and joint tissue antigen
same as myelin basic proteins
Papilloma Virus (E2) - it has the resembled self-antigen Glomerular basement
Autoimmune glomerulonephritis
same as insulin receptors. membrane
❑ Altered antigens - antigens become denatured or
mutated because of physical, chemical, and 2. SYSTEMIC OR NON-ORGAN SPECIFIC
biologic changes. • Lesions from damaged tissue and autoantibodies
❑ Mutation of immunocompetent cells - cells may are not confined to any organ but is present in
become responsive to self-antigens the system of a human body. Example:
❑ Dysfunction of T cells - specifically T-regulatory
cells or T-regulatory cells are responsible for auto A. RHEUMATOID ARTHRITIS (RA)
reactive T-cells and B-cells suppression and - Chronic inflammatory joint disease with systemic
regulation. involvement. RA can cause joint damage and joint pain
which is seen throughout your body. Damage usually affects
both sides of the body.
LEC MERCADO A., MINA M.J., PADRE A.C. 1
 AUTOIMMUNITY IMMUNOLOGY
SHERWIN ALVARO, RMT JUNE 07, 2022 AND SEROLOGY

Its hallmark feature is the presence of rheumatoid factors The Stages of Rheumatoid Arthritis
(RF). RF is an anti-IgG Ig that is produced by B-cells and STAGE 1 The body mistakenly attacks its own joint tissue.
plasma cells in the synovial membrane STAGE 2 The body makes the antibodies and the joints start
Anti-CCP (cyclic citrullinated peptides) - the most specific swelling up.
antibody for RA. STAGE 3 The joints start becoming bent and deformed, the
HLA DR-1 and HLA DR-4 -has disease association with RA fingers become crooked. These misshapen joints can press on
the nerves and can cause nerve pain as well.
CLINICAL SIGNS OF RHEUMATOID ARTHRITIS STAGE 4 If not treated, the disease will progress to the last
• Morning stiffness around the joints lasting for at least 1 stage, in which there's no joint remaining at all and the joint
hour is essentially fused.
• Swelling of the soft tissue around 3 or more joints
• Swelling of the proximal interphalangeal, LABORATORY DIAGNOSIS OF RHEUMATOID ARTHRITIS:
metacarpophalangeal, or wrist joints AGGLUTINATION TEST TO DETECT RF
• Symmetric arthritis • Two types:
• Subcutaneous nodules ❑ Sheep cell agglutination (Rose-Waaler Test) - Use
• Positive test for rheumatoid factor of sheep red blood cells coated with IgG
• Radiographic evidence of erosion in the joints of the ❑ Latex test (Latex Fixation Test/ Singer & Plotz
hands, wrists, or both Test) - has greater sensitivity. Has passive or indirect
• Swan neck deformity agglutination principle. Titter grading:
• Pannus (in the joints)- is the organized mass of cells made ! 80 or greater - positive reaction.
up of lympocytes, macrophages and fibroblasts. ! 20-40 - weakly positive reaction
! 20 - negative reaction
*According to the American College of Rheumatology, at
least 4 of these must be present for 6 weeks or more for the • Positive result is not specific for RA, because RF can be
diagnosis to be made found in other diseases such as syphilis, SLE, chronic active
hepatitis, tuberculosis, leprosy, IM, malaria, and Sjorgren’s
syndrome
• Negative result does not rule out RA, because it only
detect IgM isotype that is present in 75% of the
patients
Anti-CCP
• more reliable indicator of RA than the RF test

TREATMENT
• Anti-inflammatory drugs
• Disease-modifying anti-rheumatic drugs (DMARDS)
• Corticosteroids

B. SYSTEMIC LUPUS ERYTHEMATOSUS (SLE)

• Chronic, inflammatory multi-organ disorder that
predominantly affects young women of
childbearing age.
• HLA DR-2 and HLA DR-3
• has anti-nuclear antibodies (ANA) - that group of
antibodies reacting with the components of the
nucleus.
• Anti-Sm and anti-dsDNA - most specific
antibodies for SLE
Lupus is a disease that occurs when your body's immune
system attacks your own healthy tissues and organs
(autoimmune disease). Inflammation caused by lupus can
affect many different body systems — including your joints,
skin, kidneys, blood cells, brain, heart and lungs.

LEC MERCADO A., MINA M.J., PADRE A.C. 2

 AUTOIMMUNITY IMMUNOLOGY
SHERWIN ALVARO, RMT JUNE 07, 2022 AND SEROLOGY

There are 4 kinds of Lupus and the most common is the ❑ Fatigue, weight loss, malaise, fever, and anorexia - this
Systemic Lupus Erythematosus non-specific symptoms are the first to appear in SLE
❑ Joint involvement (small joints of the hands, wrists, and
1. Systemic Lupus Erythematosus (SLE) - most common knees)
2. Cutaneous Lupus (CLE)- limited to the skin ❑ Erythematous rash - Maculopapular rash appear when
3. Drug-induced Lupus (DIL) - lupus like disease exposed to UV light. Responsible for its name lupus,
4. Neonatal Lupus - are condition that affects fetus if the from latin meaning "wolf-like"
mother has the disease. ❑ Renal failure – most frequent cause of death in patients
with SLE - 1/2 - 2/3 of patients exhibits this symptom.
Major cause of lupus and other autoimmune disease is The most frequent cause of death of SLE patients.
unknown but genetics plays a major role in autoimmune • Diffuse proliferative glomerulonephritis (DPGN) – most
disorders in short it is hereditary. Experts also suggest that dangerous, there is cellular proliferation in at least 50%
autoimmune disorders are caused by some certain hormones of the glomeruli
and other environmental triggers that can induce the • Deposition of immune complexes in the subendothelial
symptoms of lupus or contribute to its severity. tissue
• Thickening of the basement membrane
Lupus Erythematosus (LE) phenomenon ❑ Cardiac involvement with pericarditis, tachycardia,
• It was observed that when peripheral blood from a ventricular enlargement
patient is incubated at 37 degrees Celsius for ❑ Pleuritis with chest pain
30-60 minutes, the lymphocytes swell and extrude ❑ Neuropsychiatric manifestations such as seizures, mild
their nuclear material. The nuclear materials are cognitive dysfunction, psychoses, or depression
opsonized by anti-dsDNA Ab and complement, and ❑ Anemia, leukopenia, thrombocytopenia, lymphopenia
phagocytized by neutrophils (LE cell).
• LE cell (Lupus Erythematosus cell), also known as
Hargraves cell, is a neutrophil or macrophage that
has phagocytized (engulfed) the denatured nuclear
material of another cell.

9 years after the discovery of the LE cells the first anti-

dsDNA Ab was identified (1957)

Anti-dsDNA antibodies can be isolated from lupus kidneys in

both humans and mice.
Elevated anti-double-stranded (ds) DNA antibody titers are
diagnostic and prognostic markers of systemic lupus
erythematosus (SLE), and their presence is well documented
to correlate with lupus nephritis.

CLINICAL TYPES OF SLE

Most common signs and symptoms of SLE
Fatigue
Pain or swelling in the joints CLINICAL DIAGNOSIS of SLE
Swelling of hands, feet or around the eyes ❑ Malar rash
Head-aches ❑ Discoid rash
low fevers ❑ Photosensitivity
Photosensitivity (sunlight or florescent light) ❑ Oral ulcers
Chest pain when breathing ❑ Arthritis
Hair loss ❑ Serositis
Mouth or Nose ulcers ❑ Renal disorders
Feeling numb in cold and stress ❑ Neurological disorders
Skin rash - ex. Butterfly rash ❑ Hematologic disorders
Raynaud's Phenomenon - fingers turning white and/or blue ❑ Immunologic disorders
in the cold ❑ Presence of antinuclear antibodies
The last two is the most unique for SLE
SLE shares similar signs and symptoms with Arthritis and DM

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 AUTOIMMUNITY IMMUNOLOGY
SHERWIN ALVARO, RMT JUNE 07, 2022 AND SEROLOGY

4 out of 11 specific criteria must be present for clinical POSITIVE PATTERNS OBSERVED UNDER FLUORESCENCE
diagnosis of SLE and 1 immunologic findings in the PBS MICROSCOPE:
(presence of LE cell). IMMUNOFLUORESCENT ANTIBODY
MLS role is to detect the presence of anti-nuclear antibodies AUTOIMMUNE DISEASE
PATTERN INVOLVED
COMPARISON OF ORGAN SPECIFIC AND SYSTEMIC
AUTOIMMUNITY Rheumatoid arthritis
Homogenous
Chronic active hepatitis
Fluorescence
ORGAN-SPECIFIC SYSTEMIC Anti-nucleoprotein Sjogren’s syndrome
(staining of the
Drug-induced SLE
Antibodies and lesions are Antibodies and lesions are not entire nucleus)
Myasthenia gravis
directed towards a single target confines to any organ
organ Anti-DNA/native
Membranous/
DNA (double
Clinical and serological overlap Overlap of SLE, RA, and other Shaggy/Peripheral
stranded) Anti- SLE
(e.g. thyroid, stomach, adrenal connective tissue disorders Staining (staining
nucleoprotein
glands, kidney) around the nucleus)
(soluble)
Antigens are only available to Antigens are accessible at higher Speckled
lymphoid system in low concentration fluorescence Non-DNA nuclear
concentration SLE
(numerous minute constituents/anti-
Scleroderma
fluorescence ENA (extractable
Antigens evoke organ-specific No antibodies produced inanimals Mixed connective tissue
(numerous minute nuclear antigen)
antibodies in normal animals with comparable stimulation disease
fluorescent points (a) Anti-Sm
with complete Freund’s adjuvant Sjogren’s syndrome
throughout the (b) Anti-RNP
nucleus)
Familial tendency to develop Familial tendency to develop
organ-specific autoimmunity connective tissue disease Nucleolar
fluorescence only SLE
Lymphoid invasion, parenchymal Questionable abnormalities in Ig Anti-RNA
(fluorescence on the Scleroderma
destruction by questionable cell- synthesis in relatives
nucleolus only)
mediated hypersensitivity or
antibodies Lesions caused by deposition of CREST syndrome (a
antigen-antibody (immune variant of SLE)
complexes) Fine Speckled Calcinosis cutis
Anti-centromere
pattern of Raynaud's phenomenon,
Tendency to develop cancer in Tendency to develop antibody
fluorescence Esophageal dysmotility
organ lymphoreticular neoplasia
Sclerodactyly
GENERAL SIGNS OF AUTOIMMUNE DISEASES THAT MAY Telangiectasia
HAVE DIAGNOSTIC VALUE:
1. Elevated serum gamma globulin INTERPRETATION of IMMUNOFLOURECENT PATTERNS
2. Presence of diverse antibodies HOMOGENOUS (solid, diffuse)
3. Depressed levels of serum complement • Whole nucleus fluoresces evenly gold
4. Immune complexes in serum • Detects antibodies to nDNA, dsDNA, ssDNA, DNP, or
5. Depressed levels of T suppressor cells histones
6. Lesions detected on biopsy resulting from deposition • High titers are suggestive of SLE
of immune complexes • Low titers are found in SLE, RA, Sjogren’s syndrome,
OTHER FACTS ABOUT AUTOIMMUNE DISEASES: MCTD
1. Patient may have one autoantibody and in fact, may
suffer from multiple autoimmune diseases.
2. Although SLE is associated primarily with anti-nuclear
antibodies and rheumatoid arthritis primarily with RF,
both types of antibodies may be found in both
diseases.
3. Autoantibodies are not unique to autoimmune diseases
4. Autoimmune diseases are also grouped into two:
(a) Humoral or antibody-mediated
(b) Cell-mediated or T-cell mediated

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 AUTOIMMUNITY IMMUNOLOGY
SHERWIN ALVARO, RMT JUNE 07, 2022 AND SEROLOGY

PERIPHERAL (ring, membranous, shaggy, thready) ANTI-CENTROMERE ANTIBODY (ACA)

• Sharp, ring-gold fluorescence of outer edge on • Discrete and speckled pattern
nucleus with a gradually darkening inner border • Antibodies to centromeric chromatin of metaphase
blending with a dark nuclear center and interphase cells
• Antibodies to nDNA, dsDNA, DNP • Highly selective of CREST variant of progressive
• Seen in active stage of SLE systemic sclerosis
• Infrequently found in SLE and other MCTD

SPECKLED (mottled)
• Numerous round speckles of green-gold nucleoli of DIAGNOSIS of AUTOIMMUNE DISEASES:
various sizes against a dark background; “pepper 1. ELISA
dots” 2. Indirect Immunofluorescence (IIF) - (FANA)
• Antibodies to ENA (anti-Sm and anti-RNP) 3. RIA
• Anti-RNP is indicative of other rheumatic diseases 4. WESTERN BLOT/ Immunoblotting - gold standard
for ANA
5. Immunodiffusion, Binding Assays

IMMUNOPROLIFERATIVE DISEASES
1. LEUKEMIAS
• malignant cells are present in the bone marrow and
peripheral blood.

a. Acute Lymphocy c Leukemia

NUCLEOLAR • characterized by the presence of very poorly

• Multiple, round, smooth, green gold fluorescing differentiated blast cells in the bone marrow and
nucleoli of various sizes the peripheral blood.
• Antibodies to Nrna • FAB classification includes L1, L2 and L3
• Present in 50% patients with scleroderma, Sjogren’s
syndrome and SLE b. Chronic Lymphoid Leukemic Disorders
• Also observed in illnesses manifesting Raynaud’s
phenomenon ❑ Chronic Lymphocytic Leukemia/Lymphoma (CLL)
• a common hematopoietic malignancy that
involves the expansion of a clone of B cells
that have the appearance of small mature
lymphocytes
❑ Prolymphocytic Leukemia
• a variant of CLL that is composed of larger
cells with round to oval nuclei and coarser
chromatin pattern.
❑ Hairy Cell Leukemia
• a r a r e, s l ow l y p ro g r e s s i ve d i s e a s e
characterized by the infiltration of the bone
marrow and spleen by leukemic cells, without
involvement of lymph nodes.

LEC MERCADO A., MINA M.J., PADRE A.C. 5

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 AUTOIMMUNITY IMMUNOLOGY
SHERWIN ALVARO, RMT JUNE 07, 2022 AND SEROLOGY

2. LYMPHOMAS
• malignant cells arise in lymphoid tissues such as
lymph nodes, tonsils or spleen

(a) Hodgkin’s lymphoma

• characterized by the presence of
• Reed-Sternberg cells (a large cell with bilobe
nucleus and 2 prominent nucleoli that resembles
owls eye) in affected lymph nodes and
lymphoid organs. - presence of owl eye
appearance cell.
(b) Non-Hodgkin’s lymphoma (NHL or Lymphoma
[only])
• anti-It - specific for individuals with this antigen
• associated with blood group I

3. PLASMA CELL DYSCRASIAS

• malignancies that involved the bone marrow,
lymphoid organs and non-lymphoid sites that are
not classified as either leukemias or lymphomas

(a) Multiple myeloma/Kahler’s disease

• a malignancy of plasma cells. - most common
and most serious
• Patients excrete Bence Jones proteins (light
chains) in urine. - rarely found in urine and; an
abnormal buildup of tissues and proteins in the
urine.
• Bence Jones protein is a monoclonal globulin
protein or immunoglobulin light chain found in
the urine, with a molecular weight of 22–24
kDa. Detection of Bence Jones protein may be
suggestive of m ultiple myeloma or
Waldenström's macroglobulinemia.
(b) Waldentrom’s macroglobulinemia/
cryoglobulinemia
• is a malignant proliferation of IgM producing
lymphocytes

REFERENCES:
i. Turgeon, Mary Louise. (2014). Immunology &
serology in laboratory medicine, (5th ed.).
Missouri :Elsevier.
ii. Male, David. (2013). Immunology, (8th ed.). St.
Louis, Missouri : Elsevier. “Many are the plans in a man’s heart, but it is the
iii. Stevens, Christine Dorresteyn. (2010). Clinical Lord’s purpose that prevails.”
i m m u n o l o gy & s e ro l o gy : a l a b o ra t o r y
perspective,3rd ed. Philadelphia ; F.A. Davis - Proverbs 19:21

LEC MERCADO A., MINA M.J., PADRE A.C. 6

 LABELED IMMUNOASSAYS IMMUNOLOGY
SHERWIN ALVARO, RMT JUNE 22, 2022 AND SEROLOGY

LABELED IMMUNOASSAYS • This provides a simple way to separate bound and free
• designed for antigens and antibodies that may be small in size reactants.
or present in very low concentrations. In most assays, once the reaction between Ag-Ab has taken place,
The presence of such antigens or antibodies are determined there must be a partitioning step or separation between reacted
indirectly by using Labeled reactant to detect whether or not from unreacted analytes.
specific binding has taken place. Antigen or antibody is attached by physical absorption. When
• The substance to be measured is known as the analyte. specific binding takes place, complexes attach to the solid phase.
Examples: Bacterial antigens, hormones, drugs, tumor markers and ○ The bound and unbound fractions are usually separated by
others. physical means, including decanting, centrifugation, or
filtration
Characteristics on Immunoassay This method is followed by a washing step to remove any
There are current techniques that include the use of fluorescent, remaining unbound analyte. Incomplete washing leads to removal
radioactive, chemiluminescence and enzyme assay. of incomplete analyte and will result to inaccurate immunoassay.

COMPETITIVE IMMUNOASSAY DETECTION OF THE LABEL

all the reactants are mixed together simultaneously, and labeled
antigen competes with unlabeled patient antigen for a limited ● RADIOIMMUNOASSAYS
number of antibody-binding sites. ○ counting radioactivity
• The amount of bound label is inversely proportional to the ● For other labels such as enzymes, fluorescence, or
concentration of the labeled antigen. chemiluminescence
• The more label detected, the less there is a patient antigen. ○ a change in absorbance in a substrate is measured by
spectrophotometry.
NONCOMPETITIVE IMMUNOASSAY
• An antibody first passively absorbed to a solid phase. QUALITY CONTROL
• Unknown patient antigen is then allowed to react with and be
captured by the antibody. ● To run a blank tube, usually phosphate-buffered saline, with
• After washing, to remove the unbound antigen, a second every test.
antibody with a label is added to the reaction. • A negative control and a high and a low positive control should
• The amount of label measured is directly proportional to the be run in addition. Which serves as a check on the quality of
amount of patient antigen the reagent to make sure that the label is readily detectable
In both immunoassay, the label should not alter the reaction and under current testing conditions.
must remain stable for the shelf life of the reagent. ● All controls and the patient sample are usually run in duplicate.
If any control is are out of range, test values should not
ANTIBODIES be_______(audio was unclear)

It Is essential for the antibody used to have a high affinity for the RADIOIMMUNOASSAY
antigen. Competitive Binding Assays
● AFFINITY is the strength of the primary interaction between a RADIOIMMUNOASSAY (RIA)
single antibody-combining site and an antigenic determinant or • first type of immunoassay developed
epitope. The higher the affinity of antibody for antigen, the • pioneered by Yalow and Berson in the late 1950s
larger the amount of antigen bound to antibody and the more • It was used to determine the level of insulin–anti-insulin
accurately specific binding can be measured. complexes in diabetic patients.
Radioactive elements have nuclei that decays spontaneously
STANDARDS OR CALIBRATORS emitting matter and energy.
• radioactive substance is used as a label
● Standards, also known as calibrators, are unlabeled analytes ○ 125I: most popular; half-life of 60 days It was originally
that are made up in known concentrations of the substance to be based on the principle of competitive binding
measured. ○ 131I
● They are used to establish a relationship between the labeled
analyte measured and any unlabeled analyte that might be ○ tritiated hydrogen, or 3H
• was originally based on the principle of competitive binding.
present in patient specimens
• the analyte being detected competes with a radiolabeled
Deferring amounts of standards are added to an antibody-
analyte for a limited number of binding sites on a high-affinity
antigen mixture
antibody. The concentration of the radioactive analyte in in
SEPARATION METHODS excess. All the binding sites on an antibody will be occupied. If
the patient antigen is present, some of the binding sites will be
filled with unlabeled analytes thus decreasing the amount of
SOLID-PHASE
bound radioactive level.
• most commonly used in immunoassays
• polystyrene test tubes, microtiter plates, glass or polystyrene
beads, magnetic beads, and cellulose membranes

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 LABELED IMMUNOASSAYS IMMUNOLOGY
SHERWIN ALVARO, RMT JUNE 22, 2022 AND SEROLOGY

ENZYMES
• are naturally occurring molecules that catalyze certain
biochemical reactions. They react with suitable substrates to
produce breakdown products that may be chromogenic,
fluorogenic, or luminescence.
• As labels for immunoassay, they are cheap and readily
available, have a long shelf life, are easily adapted to
automation, and cause changes that can be measured using
inexpensive equipment Sensitivity can be achieved without
disposal problems or the health hazard of radiation
• Assays based on the use of enzymes can be found in such
diverse settings as clinical laboratories, doctors’ offices, and at-
home testing. It can also be used qualitatively to identify the
presence of antigen and antibody or quantitatively to
determine the actual concentration of an analyte in an unknown
specimen.
• Enzymes used as labels in colorimetric reactions:
• Horseradish peroxidase
• Glucose-6-phosphate dehydrogenase
• Alkaline phosphatase
• β-D-galactosidase
Horseradish peroxidase and Alkaline phospatase- has the highest
turnover/highest conversion of substrate rates. It also has the
highest sensitivity. Most often used in EIA.

Heterogeneous enzyme immunoassays

● require a step to physically separate free from bound analyte.
In the Diagram, the competitive radioimmunoassay The RIA has a
solid phase antibody. Ther Labeled antigen has radioactive
COMPETITIVE EIA
substances and when the solid phase antibody is combined with
• The first enzyme immunoassays (EIAs) were competitive assays
labeled antibody, if patient antigen is not present or very little,
based on the principles of RIA.
there will be a high radioactivity.
• Enzyme-labeled antigen competes with unlabeled patient
If there are more unlabeled antigen or patient antigen, there will
antigen for a limited number of binding sites on antibody
be more competitors for the labeled antigen with radioactivity.
molecules that are attached to a solid phase.
They will compete with the solid phase antibody making it to have
• This method is typically used for measuring small antigens that
less radioactivity.
are relatively pure, such as insulin and estrogen.
• Enzyme activity is inversely proportional to the concentration of
● Advantages of Radioimmunoassay
the test substance. The more patient antigen is bound, the less
• Examples of substances that are measured by RIA include
enzyme labeled antigen is attached.
thyroid-stimulating hormone and total serum IgE.
• RIA is an extremely sensitive and precise technique for
determining trace amounts of analytes that are small in size. NONCOMPETITIVE EIA
• are often referred to as indirect enzyme-linked
● Disadvantages of Radioimmunoassay
immunosorbent assays (ELISA) because the enzyme labeled
• The health hazard involved in working with radioactive reagent does not participate in the initial Ag-ab binding.
substances. • This type of assay is one of the most frequently used
• more difficult and expensive to maintain a license and to immunoassays in the clinical laboratory due to its sensitivity,
comply with federal regulations. specificity, simplicity, and low cost.
• disposal problems, short shelf life, and the need for expensive • solid-phase supports: microtiter plates, nitrocellulose membranes,
equipment and magnetic latex beads.
• This type of assay has been used to measure antibody
Enzyme Immunoassay production to infectious agents that are difficult to isolate in the
or Enzyme as a label, Have largely replaced RIA because of laboratory and has been used for autoantibody testing.
their comparable sensitivity and availability of automated • This technique remains the preferred screening method for
instrumentation that allows for processing of a large number of detecting antibody to HIV, hepatitis A, and hepatitis C. It is also
sample in less time. used to identify Epstein-barr specific antibody produced in
infectious mononucleosis.

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 LABELED IMMUNOASSAYS IMMUNOLOGY
SHERWIN ALVARO, RMT JUNE 22, 2022 AND SEROLOGY

PROCESS OF NON COMPETITIVE EIA

• When the antigen is bound to the solid phase (microtiter plates,
nitrocellulose membranes, and magnetic latex beads) patient
serum with the unknown antibody is added, given time to react.
• After the wash step, an enzyme labeled anti globulin is added.
The second antibody reacts with patient antibody that is bound
with the solid phase.
• If no patient antibody is bound to the solid phase, the second
labeled antibody will not be bound. • Sandwich assays are also subject for hook effect which
• After a second wash step (to remove any unbound antiglobulin), is an unexpected fall in the amount of the measured
the enzyme substrate is added and the amount of enzyme label analyte when an extremely high concentration is
detected is directly proportional to the amount of the antibody present. This typically occurs in excess antigen where the
in the specimen. majority of binding sites field and the remainder of the
patient antigen has no place to bind.
Color development is directly proportional to the amount of • If this condition is suspected, serum dilution must be
antibody present. made and retested.

Homogeneous immunoassays
• Antigen-antibody system in which no separation step is
necessary
• No washing steps are necessary
• It is less sensitive than the Heterogenous assay but they are
rapid, simple to perform and adapt easily to automation
• Based on the principle change in the enzyme activity as specific
antigen antibody combination ________
• Reagent antigen is labeled with an enzyme tag and when an
antibody bind to specific determinant sites on the antigen, the
active site on the enzyme is BLOCKED resulting in a
measureable loss of activity
• There is a free analyte or antigen that competes with the
enzyme labelled analyte for a limited number of antibody
binding site
• This is a competitive assay
• Enzyme activity is directly proportional to the concentration of
the patients antigen or hapten present in the test solution
• Physical separation of bound or free analyte is thus not
necessary. (No washing steps necessary )
CAPTURE ASSAYS • It is used for the determination of low-molecular-weight analytes
• If antibody is bound to the solid phase such as hormones, therapeutic drugs, and drugs of abuse in both
• sandwich immunoassays aka capture assays. serum and urine.
• Enzymatic activity is directly proportional to the amount of • Enzyme multiplied immunoassay technique (EMIT)
antigen in the test sample • frequently used enzymes are malate dehydrogenase and
• Antigens captured in these assays must have multiple epitopes. glucose-6-phosphate dehydrogenase
Excess antibody attached to a solid phase is allowed to
combine with a test sample to capture any antigen present. The sensitivity of homogenous assay is determined by the
After incubation period, enzyme labeled antibody is added the following:
second antibody recognizes a different epitope than the solid • Detectability of enzymatic activity
phase antibody and complete the sandwich. • Change in that activity when antibody binds to antigen
• Capture assays are best suited to antigens that have multiple • Strength of the antibody!s binding
determinants (epitopes), such as antibodies, polypeptide • Susceptibility of the assay to interference from endogenous
hormones, proteins, tumor markers, and microorganisms, enzyme activity, cross-reacting antigens, or enzyme inhibitors
especially viruses.
• Rotavirus in stool and respiratory syncytial virus in respiratory Advantages
tract secretions are two examples of capture assays • Achieved a sensitivity similar to that of RIA without creating a
• major use of capture assays is in the measurement of health hazard or causing disposal problems.
immunoglobulins. Especially those in certain classes. For instance: • No need for expensive instrumentation.
• the presence of IgM can be specifically determined thus • Reagents are inexpensive and have a long shelf life.
indicating an acute infection. • They are simple and require no separation step.
• Measurement of IgE including allergen specific IgE which
appears in minute quantity in serum

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 LABELED IMMUNOASSAYS IMMUNOLOGY
SHERWIN ALVARO, RMT JUNE 22, 2022 AND SEROLOGY

Disadvantages FLUORESCENT IMMUNOASSAY

• Some specimens may contain natural inhibitors. FLUORESCENT IMMUNOASSAY
• the size of the enzyme label may be a limiting factor in the
design of some assays. • Albert Coons demonstrated that antibodies could be labeled
• Nonspecific protein binding with molecules that fluoresce.
• Fluorophores or Fluorochromes
Rapid Immunoassays • Fluorescent compounds
• They can absorb energy from an incident light source
and convert that energy into light of a longer
wavelength and lower energy as the excited electrons
return to the ground state.
• Typically organic molecules with a ring structure
• Each has a characteristic has optimum absorption range
• Time interval between absorption of energy and
emission of fluorescence is very short and can be
measure in nanoseconds.
• fluorescein (absorbed maximally in 490-495 nanometers, emits
green color at 517-520 nm, high intensity, good photo stability,
high quantum yield) and rhodamine (tetra methyl rhodamine,
absorb light at 550 nm, emits red light at 580-585nm)
MEMBRANE-BASED CASSETTE ASSAYS
• because their Absorbance and emission patterns differ, Both can
• Are relatively new type of enzyme immunoassays
be used together
• They are rapid, easy to perform, and give reproducible results
• immunofluorescent assay (IFA) (other name FI)
• Designed primarily for point-of-care or home testing
• Fluoresent tags or labels were first used for histochemical
• Many of these have been modified for in case sensitivity and
localization of antigen in tissues.
can be made semi quantitative for the use of clinical laboratory
• Is used for rapid identification of microorganisms in cell culture
• designed as single-use, disposable assays in a plastic cartridge
or infected tissue, tumor specific antigens on neoplastic tissue
• The membrane is usually nitrocellulose, which is able to easily
and transplantation antigen and CD antigen on t and B cells
immobilize proteins and nucleic acids.
with the use of cell flow cytometry.
• The rapid flow through the membrane and its large surface
• The appearance of a specific antigen is determined by the
area enhance the speed and sensitivity of ELISA reactions
appearance of localized color against a dark background
• In here, either antigen or antibody can be coupled to the
membrane. The reaction is read by looking for the presence of
colored reaction product and some test device require the Direct Immunofluorescent Assays
separate addition of patient sample, wash reagent, labelled • Fluorescent staining can be categorized as Direct or indirect
antigen/ antibody, and the substrate. depending whether the original antibody has a fluorescent tag
is attached to it.
IMMUNOCHROMATOGRAPHY • antibody that is conjugated with a fluorescent tag is added
(combines all previously mentioned steps into one) directly to unknown antigen that is fixed to a microscope slide.
The analyte is applied at one end of the strip and migrates • After incubation and a washed tip, the slide is read using a
toward the distal end, where there is an absorbent pad to Fluorescence microscope
maintain a constant capillary flow rate. • Antigens are visualize as bright apple green or orange yellow
(labelling and detection zones are set between The two ends and objects against dark background
as the sample is loaded, it reconstitutes the labeled antigen/ • best suited to antigen detection in tissue or body fluids, while
antibody and the two form a complex that migrates towards the indirect assays can be used for both antigen and antibody
detection zone. An antigen/antibody immobilize in the detection identification
zone captures the immune complex and forms a colored line for a • Legionella pneumophila, Pneumocystis carinii, Chlamydia
positive test (may be in a form of plus sign). Excess labelled trachomatis, and respiratory syncytial virus (RSV)
immunoreactant migrates to the absorbent pad. Test results are
often qualitative rather than quantitative.)
This type of test device has been used to identify microorganisms
such as Streptococcus pyogenes and Streptococcus agalactiae and
has been used to test for pregnancy, for troponin in a heart
attack, and for hepatitis B surface antigen
A colored line indicates a positive test.

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 LABELED IMMUNOASSAYS IMMUNOLOGY
SHERWIN ALVARO, RMT JUNE 22, 2022 AND SEROLOGY

Indirect Immunofluorescent Assays Fluorescence polarization immunoassay (FPIA)

• involve two steps, the first of which is incubation of patient

serum with a known antigen attached to a solid phase.
• Then the slide is washed and then an anti-human immunoglobulin
containing a fluorescent tag is added and this combines with
first antibody to form a sandwich which localizes the
fluorescence
• In this manner one antibody conjugate can be used of many
different types of reaction eliminating the need for numerous
______labelled reagent antibodies
• Indirect assays result in increased staining, because multiple the
molecules can bind to each primary molecule, thus making this a
more sensitive technique.
• useful in antibody identification and have been used to detect
treponema, antinuclear, chlamydial, and toxoplasma antibodies,
as well as antibodies to such viruses such as herpes simplex,
Epstein- Barr, and cytomegalovirus
• In fluorescent immunoassay,In general face the issue of
subjectivity in reading the slides. Only experienced clinical
laboratorians are responsible in reporting slide results.

• The degree of polarized light reflects the amount of labelled

analyte that is bound
• based on the change in polarization of fluorescent light emitted
from a labeled molecule when it is bound by antibody.
• Incident light directed at the specimen is polarized with a lens or
prism so the waves are aligned in one plane.
• the degree of fluorescence polarization is inversely proportional
to concentration of the analyte.
Other Fluorescent Immunoassays • used mainly to determine concentrations of therapeutic drugs
There are Quantitative fluorescent immunoassay that are and hormones.
classified as heterogenous and homogenous assays. The degree of polarized light reflects the amount of labelled
Solid-phase heterogeneous fluorescent assays analyte that is bound
• developed for the identification of antibodies to nuclear Advantages
antigen, toxoplasma antigen, rubella virus, and numerous other • high sensitivity and versatility
virus antigens. • The methodology is fairly simple, and there is no need to deal
• used to detect such important biological compounds as cortisol, with and dispose of hazardous substances.
progesterone, and serum thyroxine (T4).
Disadvantages
Homogeneous FIA • separation of the signal on the label from autofluorescence
• requires no separation procedure, so it is rapid and simple to produced by different organic substances normally present in
perform. serum.
• As binding of the patient antigens increases, the binding of the • nonspecific binding to substances in serum can cause quenching
fluorescent analyte decreases. There is more fluorescent or diminishing of the signal and change the amount of
observed. It has a direct relationship between the two. fluorescence generated.
• There is a direct relationship between the amount of
fluorescence measured and the amount of antigen in the patient
• Fluorescence polarization has been developed to
sample. overcome some of these problems
• It des however expensive dedicated instruments which
may limit its use in smaller laboratories

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 LABELED IMMUNOASSAYS IMMUNOLOGY
SHERWIN ALVARO, RMT JUNE 22, 2022 AND SEROLOGY

CHEMILUMINESCENT IMMUNOASSAYS REFERENCES:

i. Turgeon, Mary Louise. (2014). Immunology & serology in
Chemiluminescence laboratory medicine, (5th ed.). Missouri : Elsevier.
• Uses chemiluminescence ii. Male, David. (2013). Immunology, (8th ed.). St. Louis, Missouri :
• It is the production of light energy by certain compounds when Elsevier.
they are oxidized iii. Stevens, Christine Dorresteyn. (2010). Clinical immunology &
• It is the emission of light caused by a chemical reaction, serology : a laboratory perspective, 3rd ed. Philadelphia ; F.A.
typically an oxidation reaction, producing an excited molecule Davis
that decays back to its original ground state.
• most common substances used are luminol, acridinium esters,
ruthenium derivatives, and nitrophenyl oxalates. "Many are the plans in a man!s heart, but it is the Lord!s purpose
• When these substances are oxidized, typically using hydrogen that prevails.”
peroxide and an enzyme for a catalyst. Intermediates are - Proverbs 19:21
produced that are of a higher energy states.
• These intermediates spontaneously turn to their original state
giving off energy in the form of light
• Light emissions ranges from a rapid flash of light to a more
continuous flow lasting for how many hours
• This type of labelling can be used for heterogenous and
homogenous assays because labels can be attached to an
antigen or antibody
• in heterogenous assays, competitive and sandwich formats are
the ones most often used
• smaller analytes such as therapeutic drugs and steroid hormones
are measured using competitive assays
• sandwich formats are used in larger analytes such as proteins
hormones

Advantages
• excellent sensitivity
• the reagents are stable and relatively non toxic
• quite inexpensive to perform (little reagent used)
• high speed of detection also means a faster turnaround
time

Disadvantages
• false results may be obtained if there is lack of precision
in injection of the hydrogen peroxide or if some
biological materials such as urine or plasma cause
quenching of the light emission.

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TUMOR IMMUNITY AND SEROLOGY
SHERWIN V. ALVARO, RMT , MPH June 14, 2022

TABLE OF CONTENTS RELATED TERMINOLOGIES

TUMOR IMMUNOLOGY 1 Normal cell growth and division
RELATED TERMINOLOGIES 1 - Regulated processes designed to rapidly
METASTASIS 2 produced new cells, when necessary, inhibit cell
LIVER FUNCTION ALTERATIONS DURING 2 division when sufficient cells are present, and
DISEASE limit cell lifespan.
CLASSIFICATION OF TUMORS BY THE TNM 3 Cancer
SYSTEM - Uncontrolled growth of cells that often forms a
CANCER PROGRESSION 3
solid mass or tumor (neoplasm) and spreads to
CANCER STAGING 3
CARCINOGENS 4
other areas of the body.
CLASSIFICATION OF CANCER 4 Protooncogenes
TUMOR ANTIGENS 4 - Regulatory genes that promote cell division,
TSTAs 4 and can cause inhibited cell division if their
TATAs 5 expression is altered or if they are mutated
TUMOR ASSOCIATED ANTIGENS 5 into oncogenes.
IDEAL TUMOR MARKER 5 o Oncogenes = cancer genes that are
CRITERIA FOR DIAGNOSTIC MARKER 5 often found in viral genomes
USES 5 - Cellular counterpart of oncogene
CLASSIFICATION 5
TYPES OF TUMOR MARKERS 5 Mutations or malfunctions in tumor suppressor genes
APPLICATION OF TUMOR MARKER 6 - remove growth inhibitory signals can cause
DETECTION tumors.
LABORATORY CONSIDERATIONS FOR 6
TUMOR MARKER MEASUREMENT Tumors
FREQUENTLY ORDERED TUMOR MARKERS 7 - Composed of cells that possess many of the
SUMMARY OF TUMOR MARKERS 8 attributes of the normal cells from which they
TUMOR MARKERS 9
arose but have accelerated or dysregulated
Tumor- abnormal lump; abnormal growth of cells growth. 
- When cells in the tumor are normal, it is called Angiogenesis
benign - Development of new blood vessels to supply
- When normal cells overgrew, they produce a oxygen and nutrients to cells
lump. Apoptosis
- After months/years, it will become malignant. - Programmed cell death
- Our immune system cannot control the growth Neoplasm
of tumor. - Uncontrolled tissue growth “Gr. For new
- When cells are abnormal, grow uncontrollably formation”
and divides continuously, they are called Oncogene
cancerous cells (malignant). - Encodes a protein that, when mutated,
promotes uncontrolled cell growth
TUMOR IMMUNOLOGY Tumor suppressor gene
- Study of the antigens associated with tumors, - Encodes a protein involved in protecting cells
the immune response to tumors, the tumor’s from unregulated growth
effect on the host’s immune status, and the use Metastasis
of the immune system to help eradicate the - Malignant cells travel through the body,
tumor. causing new foci of malignancy until body
- Best understood with a background on the function is so disrupted that death occurs.
differences of normal and abnormal cells. o Malignant cells are metabolically more
(Histopathology/histology) active to support their growth and
expressed differently on the different
level of gene products as compared to
the normal cells.
- Spreading of the tumors or cancerous cells

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SHERWIN V. ALVARO, RMT , MPH June 14, 2022

Induction phase
- Cells are exposed to a variety of
environmental insults
o Includes:
▪ Chemical carcinogens
▪ Oncogenic viruses
▪ Radiation (Ionizing and
Ultraviolet lights)
- Start of the formation of tumors
Tumorigenesis
- formation of tumors

METASTASIS

During the induction phase:

DYSPLASIA
- Abnormal growth that is not yet considered.
NEOPLASIA
- Consistent with a tumor
- from the neoplasm; wherein it is synonymous
with a tumor
In situ phase
- Neoplastic cells have formed but are confined
to the tissue of origin.

Invasion Phase
- If these cells are malignant, the cancer
- Invasive tumors cells; enters the blood vessels proceeds to this phase and then disseminate
allowing metastasis to occur throughout the body via blood and lymphatics.
- The progeny of the cells that undergoes
transformation are monoclonal origin.
LIVER FUNCTION ALTERATIONS DURING DISEASE - As rapid uncontrolled proliferation continues,
mistakes can occur in DNA replication, causing
TUMORS cellular phenotypic and genotypic
- Can be classified as primary or metastatic heterogeneity to develop.
- Can be classified as benign or malignant - treatment is far more effective the earlier the
- Similarity to normal tissue is one of the malignancy is detected.
reasons why the immune system does not - Mortality rate of cancer worldwide: 12.7
automatically eradicate all the tumor million
- As there are fewer cells to be detected, they
A. Benign are more closely resembled with the normal
- if a tumor does not invade the surrounding tissue cells that they are dysplastic rather than
and normal body function is largely reserved neoplastic
- Hepatocellular adenoma and hemangioma Well –differentiated tumors
B. Malignant - More similar to normal tissue
- Hepatocellular carcinoma (HCC) aka
hepatocarcinoma or hepatoma and bile duct
carcinoma, hepatoblastoma Poorly differentiated tumors
- tumors can surround and can invade the - Are more aggressive and lead to poorer
surrounding tissue; wherein not only the liver is patients’ prognosis.
affected, but also the bile duct will be affected.
- It will greatly disrupt the normal body functions

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SHERWIN V. ALVARO, RMT , MPH June 14, 2022

- In pathology, tumors are more similar to fetal NX Regional lymph nodes cannot be assessed
or embryonic tissue; these are classified as N0 No regional lymph nodes metastasis
poorly differentiated or anaplastic N1 Metastasis in 1-3 regional lymph nodes
N2 Metastasis in 4 or more regional lymph
CLASSIFICATION OF TUMORS BY THE TNM SYSTEM: nodes
Classification of Tumors by the TNM System: M- DISTANT METASTASIS
T–the size of the primary tumor - There is the metastasis or the extent of
N–the involvement of adjacent lymph nodes tumors spreading from one tissue to
M –the detection of metastasis another
MX Distant metastasis cannot be assessed
M0 No distant metastasis
CLASSIFICATION OF TUMORS BY THE TNM M1 Distant metastasis
SYSTEM:
CANCER PROGRESSION

CANCER STAGING

TNM CLINICAL CLASSIFICATION

INTERNATIONAL UNION AGAINST CANCER
SYSTEM
T - Primary Tumor
- tumor size and involvement
- also refers to the invasion of the nearby tissue
TX Primary tumor cannot be assessed
T0 No evidence of primary tumor
Tis Carcinoma in situ intraepithelial or
invasion of lamina popria
T1 Tumor invades submucosa
T2 Tumor invades muscularis popria
T3 Tumor invades through submucularis
popria into subsero or into non Cancer remains as one of the leading causes of death
pritonealized periodic or perirectal globally with an estimated of 12.7 million cases
tissues around the world, affecting both sexes equally. This
T4 Tumor directly invades other organs or number is expected to raise to 21 million up until 2030.
structures and/ or perforates visceral
peritoneum
N- REGIONAL LYMOH NODES
- Regional lymph node involvement

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SHERWIN V. ALVARO, RMT , MPH June 14, 2022

Chronic leukemia: much less aggressive,

develops slowly. Will affect mature cells (e.g.
CARCINOGENS CLL, CML )

Radiation: TUMOR ANTIGENS

- Ultraviolet light, sunshine; X-rays, radioactive TSTAs

elements induce DNA damage and - Tumor Specific Transplantation Antigens
chromosome breaks. TATAs
- X-rays (digital imaging) are only allowed -Tumor Associated Transplantation Antigens
twice (2x) a year.

Chemical:

- smoke and tar, countless chemicals that

damage DNA (mutagens).

Oncogenic viruses:
- insert DNA or cDNA copies of viral oncogenes
into the genome of host target cells.

Hereditary: TSTAs
- certain oncogenes are inheritable.
- These do not occur on normal cells in the body
- Unique to tumor cells
- Novel proteins created my mutation presented
on class I MHC
- Can either be chemically/physically induced or
virally induced tumor antigens

Chemically/Physically Induced:
- Specific Immunologic Response that can
protect against later challenge by live cells of
the same line but not other tumor-line cells.
- Methylcholanthrene (Chemical)/ UV light
(Physical)
CLASSIFICATION OF CANCER

- Carcinomas – epithelial origin; involves skin,

mucous tissues, epithelial cells, endodermal
and ectodermal tissue
- Sarcomas – cancer of the connective tissues or
mesodermal connective tissue
- Lymphomas – b and t cells, Hodgkin’s and
Burkitt’s lymphoma; wherein there is presence
of solid tumors; came from hematopoietic cells
- Leukemia – disseminated tumor that maybe
lymphoid, myeloid, chronic or acute;
hematopoietic in origin
Acute leukemia: appears suddenly; progress
rapidly; arise in less mature cells (e.g. ALL,
AML)

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SHERWIN V. ALVARO, RMT , MPH June 14, 2022

 - Screening, diagnosis, histopathologic

 evaluation, staging, monitoring, localization,
Virally Induced: and immunotherapy of various malignancies.
- Express tumor antigens shared by all tumors
induced by the same virus IDEAL TUMOR MARKER
- Burkitt’s Lymphoma
*Epstein Barr - Must be produced by tumor cells.
- HPV - Should not be present in normal or healthy or
benign condition.
- Must be detectable in body fluids. (Blood,
urine, semen etc.)
- It could be used for screening the presence of
cancer in asymptomatic patients.

CRITERIA FOR DIAGNOSTIC MARKER

- Highly specific
- Highly sensitive
- Able to differentiate between neoplastic and
non-neoplastic diseases.
- Increase in level should precede neoplastic
process.
o The cancer staging is directly
proportional to the level of tumor
marker in the body fluid
TATAs
- NOT unique to tumor cells USES

So where’s the problem? Diagnosis/ Case finding

- Fetal/adult presence - Staging/ Prognosis
- Concentration of Growth Factors and Growth - Detecting reoccurrence
Factor Receptors - Monitoring treatment
Oncofetal Tumor Antigens (AFP & CEA)
- Normally appear in fetus before CLASSIFICATION
immunocompetence Tumor markers are classified into various groups:
- Later recognized as non-self - Enzymes
Oncogene Proteins - Hormones
- Proteins
- Human Melanomas - Oncofetal antigen
 - Genes
Virally induced tumors have the same antigens for - Carbohydrate antigen
each tumor caused by that virus. - Blood antigen
- HPV - Receptors
- TUMOR ASSOCIATED ANTIGENS
TYPES OF TUMOR MARKERS
- Antigens present in the tumor tissue in higher
amounts than in normal tissue. -Enzyme tumor markers
- Often the products of mutated genes and - Serum protein tumor markers
viruses, but they can also arise from aberrant - Endocrine tumor markers
expression of normal genes. - Carbohydrate and Cancer Antigen tumor
markers
- Receptor tumor markers

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TUMOR IMMUNITY AND SEROLOGY
SHERWIN V. ALVARO, RMT , MPH June 14, 2022

and that phenomenon is called the antigen excess or

APPLICATIONS OF TUMOR MARKER hook effect
DETECTION Hook effect
Ideal tumor marker should be: - Refers to the shape of the concentration-signal curve
- Specific when the reagents are saturated with excess antigen.
- Absent in healthy individuals
- Readily detectable in body fluids
It should be direct proportional with the cancer staging
and cancer progression
* Clinically, tumor markers are used in combination
with clinical signs, symptoms, and histology to facilitate
clinical decision-making.

SCREENING AND SUSCEPTIBILITY TESTING

* No tumor marker identified to date can be used to
effectively screen asymptomatic populations (except
PSA). Prostate-Specific Antigen

APPLICATION OF TUMOR MARKERS DETECTION Take Note: there is excess antigen in hook effect and
if clinical suspicion is high for an elevated tumor
PROGNOSIS marker, it can be identified by laboratory with the
- Tumor marker concentration generally dilution and repeat testing. There is excess antigen,
increases with tumor progression, reaching you will just dilute your sample and then you will
their highest levels when tumor metastasize. repeat the testing.
- High concentrations might indicate the
presence of malignancy and possible You have the equivalence zone; you have the equal
metastasis, which is associated with POOR number of the antigen and antibody is present.
PROGNOSIS. Therefore, we use 2-4% red cell suspension so that
there would be false negative and false positive
MONITORING EFFECTIVENESS OF THERAPY AND results in blood bank laboratory as well as in
DISEASE RECURRENCE immuno-serology laboratory. To prevent the excess
- In patients with elevated tumor markers at antigen or excess antibody.
diagnosis, effective therapy results in dramatic
decrease or disappearance of the tumor 2. HIGH-PERFORMANCE LIQUID
marker. CHROMATOGRAPHY
- If the initial treatment is effective, the - Used to detect small molecules, such as endocrine
reappearance of circulating tumor markers metabolites
can then be used as highly sensitive marker for 3. IMMUNOHISTOCHEMISTRY AND
recurrence. IMMUNOFLUORESCENCE
4. ENZYME ASSAYS
LABORATORY CONSIDERATIONS FOR TUMOR
MARKER MEASUREMENT
1. IMMUNOASSAYS
Most commonly used method to measure tumor
markers
Linearity
- The linear range is the span of analyte concentrations
over which a linear relationship exists between the
analyte and signal.
- Excessively high linearity tumor marker
concentrations can result in falsely low measurement

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TUMOR IMMUNITY AND SEROLOGY
SHERWIN V. ALVARO, RMT , MPH June 14, 2022

FREQUENTLY ORDERED TUMOR MARKERS

– most common tumor
1. α-Fetoprotein
- This test is very pricey. (3k-10k for each tumor
marker)
- Belongs to special chemistry section
- For each tumor marker 15ml of serum is need

- Often elevated in cases of hepatocellular

carcinoma (HCC) and germ cell tumors
- Major use is for classification and monitoring therapy
for testicular CA.
- Abundant serum protein normally produced or
sensitized by the fecal liver; re-expressed in certain
types of tumors
- Carcinoembryonic protein
- Also useful for tumor staging

STAGE AFP INCREASE

I 10-20%
II 50-80%
III 90-100%

2. CANCER ANTIGEN 125

- First defined by murine monoclonal antibody raised
against a serous ovarian CA
- Useful in detecting ovarian tumors
- Should not be used to screen for ovarian CA among
asymptomatic individuals.

- CA 125 is elevated in high percentage of ovarian

tumors and it is recommended as an annual test for
women to the history of ovarian cancer

3. CARCINOEMBRYONIC ANTIGEN (CEA)

- Example of an oncofetal antigen
- Marker for colorectal CA and is frequently elevated
in lung, breast, and gastrointestinal tumors

4. HUMAN CHORIONIC GONADOTROPIN (HCG)

- Dimeric hormone normally secreted by trophoblasts
to promote implantation of the blastocyst and the
placenta to maintain corpus luteum through the first
trimester of pregnancy.

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TUMOR IMMUNITY AND SEROLOGY
SHERWIN V. ALVARO, RMT , MPH June 14, 2022

- Elevated in trophoblastic tumors, mainly REFERENCES:

choriocarcinoma and germ cell tumors of the ovary
and testis. 
- also used in pregnancy test, therefore, when results 
are positive (+), it doesn’t automatically mean you - i. Turgeon, Mary Louise. (2014). Immunology &
are pregnant. It may indicate the presence of a serology in laboratory medicine, (5th ed.).
tumor Missouri : Elsevier.
- Testicular Cancer = positive (+) from men - ii. Male, David. (2013). Immunology, (8th ed.).
St. Louis, Missouri : Elsevier.
5. PROSTATE-SPECIFIC ANTIGEN - iii. Stevens, Christine Dorresteyn. (2010).
- Produced only by the epithelial cells of the acini Clinical immunology & serology : a laboratory
and ducts of the prostatic ducts of the prostate perspective, 3rd ed. Philadelphia ; F.A. Davis
- iv. Bishop, Michael L (2013). “Clinical
- A serine protease of the kallikrein gene family Chemistry: Principles, Techniques, and
Correlations”. Philadelphia, USA.
- Mostly, patients who test for PSA are priests. - v. McPherson, Richard A et. al (2011). “Henry’s
Clinical Diagnosis and Management by
SUMMARY OF TUMOR MARKERS Laboratory Methods”. Philadelphia, USA.
- vi. Rodriguez, Maria Teresa (2014). “Clinical
TUMOR ASSOCIATED CANCER Chemistry Review Handbook for Medical
MARKERS Technologists”. Sampaloc, Manila.
AFP Hepatic & testicular Cancer - vii. Llanera, Frederick (2007). “Lecture Notes
Placental ALP Lung CA on Clinical Chemistry 2 (2007)” . College of
Amylase Pancreatic CA Pharmacy, Department of Medical
BRCA-1 Breast and ovarian CA Technology. University of Santo Tomas,
CA-125 Ovarian CA Sampaloc, Manila.
CA 15-3 Breast Cancer
CA 19-9 Gastric, Pancreatic & Colorectal
CA
CA- 50 Gastric & Pancreatic CA
CA 27-29 Breast CA “Many are the plans in a man’s heart, but it is the
Calcitonin Medullary Thyroid Lord’s purpose that prevails.”
Cathepsin-D Breast CA -Proverbs 19:21
CEA Breast, Lung, Colorectal &
Stomach CA
CK-1 Small cell lung CA, Prostate CA
Estrogen Breast CA
Receptor (ER)
GGT Hepatoma
HER- 2/ neu Breast CA (efficiency of
transtuzumab or Herceptin
therapy)
Nuclear Urinary Bladder CA
Matrix
Protein
(MNP)

LEC TRANSCRIBERS LIM, J.M., MARCAIDA V.P., PROGRESO, L.C.

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 IMMUNOLOGY
TUMOR IMMUNITY AND SEROLOGY
SHERWIN V. ALVARO, RMT , MPH June 14, 2022

***
TUMOR MARKERS
TUMOR MARKER TUMOR TYPE METHOD SPECIMEN CLINICAL UTILITY
ENZYME TUMOR MARKERS
Prostate- Specific Prostate Cancer IA Serum Prostate CA screening, therapy
Antigen monitoring and recurrence
Lactate Hematologic EA Serum Prognostic indicator, elevated
dehydrogenase malignancies nonspecifically in numerous
cancers
Alkaline Metastatic EA Serum Determination of liver and
Phosphatase Carcinoma of bone, bone involvement, nonspecific
hepatocellular elevation in many bone-
carcinoma, related and liver cancers
osteosarcoma,
lymphoma,
leukemia
Neuron- specific Neuroendocrine RIA, IHC Serum Prognostic indicator and
enolase tumors monitoring disease progression
for neuroendocrine tumors
SERUM PROTEIN TUMOR MARKERS
Serum M Protein Plasma cell SPE/IFE Serum Diagnosis, therapeutic
dyscraiasis monitoring of plasma cell
malignancies
Serum-Free light Plasma Cell IA Serum Diagnostic therapeutic
chains dyscrasias monitoring of plasma cell
malignancies
Beta 2 Hematologic IA Serum Prognostic Marker for
Microglobulin Malignancies lymphoproliferative disorders
ENDOCRINE TUMOR MARKERS
ACTH Pituitary adenoma, IA Serum Diagnosis of ectopic ACTH
(Adrenocorticotropic ectopic ACTH- producing tumor
Hormone) producing tumor
ADH Posterior pituitary IA Serum Diagnosis of SIADH
(Antidiuretic tumor
Hormone)

C Peptide Insulin secreting ELISA, IA Serum Diagnosis of Insulinoma

tumors
Calcitonin MTC and IA Serum Screening, response to
Neuroendrocrine therapy, and monitoring
tumors recurrence of MTC
Chromogranin A Pheochromacytoma, ELISA, RIA Serum Aid in diagnosis of carcinoid
neuroblastoma, tumors, pheochromacytomas
carcinoid tumors, and neuroblasts
small cell lung
cancers
Cortisol Adrenal Tumors IA Serum or Diagnoses of Cushing’s
Urine syndrome, adrenal adenoma
Gastrin Neuroendocrine IA Serum Zollinger-Ellison syndrome;
Tumor gastrinoma

LEC TRANSCRIBERS LIM, J.M., MARCAIDA V.P., PROGRESO, L.C.

9
 IMMUNOLOGY
TUMOR IMMUNITY AND SEROLOGY
SHERWIN V. ALVARO, RMT , MPH June 14, 2022

GH Pituitary adenoma, IA Serum Diagnosis and post monitoring

ectopic GH- of acromegaly
secreting Tumors
HVA Neuroblastoma, HPLC 24-H urine Diagnosis of neuroblastoma
pheochromacytoma,
paraganglioma
5-HIAA Carcinoid Tumors HPLC 24-H urine Diagnosis of Carcinoid Tumors
Metanephrines Pheochromocytoma, HPLC 24-H urine Screening and Diagnosis of of
(Fractionated) paraganglioma, or Plasma pheochromocytoma
neuroblastoma
PTH Parathyroid IA Serum Diagnosis and post surgical
adenoma monitoring of parathyroid
adenoma
PRL Pituitary adenoma IA Serum Diagnosis and post surgical
monitoring of prolactinoma
VMA Pheochromocytoma, HPLC 24-H Urine Diagnosis of neuroblastoma
paraganglioma,
neuroblastoma
USE OF SERUM ALPHA FETOPROTEIN AND HCG FOR TESTICULAR CANCER CLASSIFICATION
GERM CELL AFP HCG
TUMOR (Alpha
Fetoprotein)
Non seminomatous Yolk sac Tumor Increased no
tumors (endodermal sinus
tumor)
Choriocarcinoma No Increased
Embryonal Increased +/-
Carcinoma
Teratoma No No
Seminoma Not +/-
elevated
pure tumors
CARBOHYDRATE AND CANCER ANTIGEN TUMOR MARKERS
TUMOR MARKER TUMOR TYPE METHOD SPECIMEN CLINICAL UTILITY
CA 19-9 Gastrointestinal Immunoassay Serum Monitoring pancreatic cancer
(Cancer Antigen) cancer and
Adrenocarcinoma
CA 15-3 Metastatic breast Immunoassay Serum Response to therapy and
cancer detecting recurrence
CA 27-29 Metastatic breast Immunoassay Serum Response to therapy and
carcinoma detecting recurrence
CA 125 Ovarian cancer Immunoassay Serum Monitoring therapy
RECEPTOR TUMOR MARKERS
Estrogen receptor Breast Cancer IHC Biopsy Hormonal therapy indicator
Progesterone Breast Cancer IHC Biopsy Hormonal therapy indicator
receptor
Her-2/neu Breast, Ovarian ICH, FISH, Biopsy Prognostic and hormonal
gastrointestinal ELISA therapy
tumors

LEC TRANSCRIBERS LIM, J.M., MARCAIDA V.P., PROGRESO, L.C.

10
 IMMUNOLOGY
TUMOR IMMUNITY AND SEROLOGY
SHERWIN V. ALVARO, RMT , MPH June 14, 2022

Epidermal growth Head, neck, IHC Biopsy Prognostic indicator

factor receptor ovarian, cervical
cancers

Mnemonic:
CA 15-3 – yung # 3 ay gagawing letter B (Breast)
CA 125 – ilan yun letter sa OVARY (5)
CA 19 – 9 –yung 9 is letter G (gastrointestinal) – adenocarcinoma (Baliktad na letter a)

LEC TRANSCRIBERS LIM, J.M., MARCAIDA V.P., PROGRESO, L.C.

11
 BASIC IMMUNOLOGY PROCEDURES IMMUNOLOGY
SHERWIN ALVARO, RMT JUNE 22, 2022 AND SEROLOGY

LAB MERCADO A., MINA M.J., PADRE A.C. 1

 BASIC IMMUNOLOGY PROCEDURES IMMUNOLOGY
SHERWIN ALVARO, RMT JUNE 22, 2022 AND SEROLOGY

LAB MERCADO A., MINA M.J., PADRE A.C. 2

 BASIC IMMUNOLOGY PROCEDURES IMMUNOLOGY
SHERWIN ALVARO, RMT JUNE 22, 2022 AND SEROLOGY

LAB MERCADO A., MINA M.J., PADRE A.C. 3

 BASIC IMMUNOLOGY PROCEDURES IMMUNOLOGY
SHERWIN ALVARO, RMT JUNE 22, 2022 AND SEROLOGY

TECHNIQUE APPLICATION SENSITIVITY COMMENT

CAUSE CORRECTION

LAB MERCADO A., MINA M.J., PADRE A.C. 4