| 2ND SEM | PRELIM

IMH_100 3MT02
NATHANIEL VERON M. CASAMINA, RMT, ASCP, MSMT

LESSON #2: RELATIONSHIP BETWEEN IMMUNOLOGY AND IMMUNOHEMATOLOGY

IMMUNOGEN
OUTLINE ● Macromolecules capable of triggering an adaptive
immune system response by inducing the formation of
I. Blood Group Antigens antibodies or sensitized T cells in an immunocompetent
II. Immunogens Host
III. Immunogenicity ● Adaptive Immune Response requires: Prior exposure
IV. Antigen to that particular particle/molecule, the reaction of the
A. Epitope vs Paratope body is to produce an antibody or sensitized T cells in an
B. Linear Epitope vs Conformational immunocompetent Host.
Epitope ● Production of T-cells are from: Lymphocytes
V. Composition of Antigen ○ Two types of Lymphocytes:
A. Hapten ● T Cells - roaming around the circulation and
VI. Traits of an Immunogen when they see a foreign immunogen they will
VII. Antigen Frequency be agitated and be sensitized and proceed to B
VIII. Antibodies/Immunoglobulins cells.
A. Blood Group Immunoglobulin ● B cells - production of antibodies; it transforms
B. Naturally Occuring Antibodies to become plasma cells (appearance: cart
C. Immune Antibodies wheel appearance) and only the plasma cells
D. Immunoglobulin Classes can produce the antibodies.
E. Types of Antibodies Reaction Phases ● All immunogens are antigens, but not all antigens are
F. Reduction of Antibody immunogens.
1. Effect of the Enzyme Papain ○ Because there is a type of antigen that can’t induce
and Pepsin in a Monomer a production of antibodies while immunogen is
G. Factors that Influence capable of antibody production.
Antibody-Antigen Reaction
1. Antigen-Antibody Ratio IMMUNOGENICITY
2. Effect of Zonal REaction
3. Dosage effect Table No. 1 Immunogenicity
4. pH (6.5 - 7.5) BLOOD GROUP DEFINITION
5. Temperature (from most
immunogenic to
6. Immunoglobulin type
least immunogenic)
7. Centrifugation A, B ● Most immunogenic (upon exposure,
8. Zeta Potential your body will produce an antibody.
H. Cause of False-Positive Result (AHG) ● It has no O because there is no
I. Cause of False-Negative Result (AHG) antigen in Blood Group O.
IX. Positive Reactions in Blood Bank Rh D ● Rh+ : has D antigen in blood
○ A+ : Have A antigen and D
antigen
BLOOD GROUP ANTIGENS ● Rh- : has no D antigen in blood
○ A- : No D antigen
● Refers to the genetically ● D antigen is immunogenic
encoded antigen system on K (Kell Blood
the erythrocytes, leukocytes, Group)
thrombocytes, and plasma Fya (Duffy Blood
● Characteristics: Group)
○ Glycolipids Common Rh
■ ABO blood group antigens
○ Glycoproteins
■ Lewis Blood ANTIGEN
Group (Part of the ● Substance that reacts with antibody or sensitized T
Secretor status cells but may not be able to evoke an immune response
meaning it can't be in the first place.
seen in rbc, wbc, platelet and plasma and it ● All antigens can bind to Antibodies.
can be found in secretions such as saliva, ● Incomplete
urine, and other body fluids)
EPITOPE VS. PARATOPE (balikan)
Table No. 2 Epitope vs Paratope
EPITOPE PARATOPE
Seen in Antigen Seen in Antibody
Exact binding site of the Exact binding site of antigen to
antibody to your antigen your antibody

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LESSON #2: RELATIONSHIP BETWEEN IMMUNOLOGY AND IMMUNOHEMATOLOGY

Properties of Epitope: Antigen-Antibody Dependent on Dependent on the

● Linear Reactions linear structure of 6 3-dimensional
● Discontinuous amino acids structure
Availability for Become available Usually associated
antibody reaction upon denaturation with native proteins
of proteins

COMPOSITION OF IMMUNOGEN
● Complete (hapten + carrier)
○ Hapten comes before anemia
○ Hapten = incomplete, need a carrier
● Characteristics:
○ Ability to stimulate antibody production
○ Ability to react with its corresponding antibody

HAPTEN
LINEAR VS CONFORMATIONAL EPITOPE ● Incomplete/ partial antigen
● Low molecular weight substance, has the ability to react
Table No. 3 Linear vs Conformational Epitope with corresponding antibody but not able to stimulate
LINEAR EPITOPE CONFORMATIONAL EPITOPE antibody reaction
A stretch of continuous amino Key amino acid residues are ● It can be a protein; example is Albumin which is a major
acids are sufficient for binding. brought together by protein
carrier protein.
folding.
Consist of continuous Consist of residues that are
● It can only be complete if you found your carrier molecule
residues on a protein discontinuous in the protein that is why it is a macromolecule.
sequence. sequence yet come within close
proximity to form an antigenic
surface on the protein's
three-dimensional structure.
Preferred for applications in Preferred for applications
which the protein target is involving protein targets in their
wholly or partially denatured native state, such as therapeutic
during the sample preparation applications or flow cytometry.
prior to the immunoassay,
such as in Western blot (WB),
immunohistochemistry (IHC)
or immunofluorescence-based
confocal microscopy.

TRAITS OF AN IMMUNOGEN
● Macromolecular size
○ Because hapten + carrier = malaki na siya
● Chemical composition and molecular complexity
● Foreignness
○ For an immunogen to be able produce an antibody
response, dapat foreign siya. Kasi kung meron ka na
antigen na yun, your body will not produce an
antibody against that unless you have autoantibody
in your system.
● Ability to be processed and presented with MHC
a. Linear epitopes are short and continuous. After denaturation the molecules (antigen presentation)
linear epitopes may still be able to bind the antibody. ○ MHC (Major Histocompatibility Complex: T cells
and Dendritic cells): They are antigen presenting
b. Conformational epitopes are domains of proteins composed of cells, present the antigen/immunogen present in the
specific regions of protein chains. After denaturation the
body to the B cells so that B cells can memorize their
discontinuous epitope can no longer bind the antibody.
structure and will produce antibody against that
immunogen; capable of determining HLA (for organ
Table No. 4 Linear vs Conformational Epitope donation) .
PROPERTIES LINEAR EPITOPE CONFORMATIONAL ○ HLA (Human Leukocyte Antigen) Typing: Used in
EPITOPE organ donation to determine if the organ will not be
Location Most Most globular rejected by the recipient. If you have not done the
polysaccharides, proteins and native HLA typing, there is a high chance that the organ will
fibrillar proteins, nucleic acids be rejected. First done in organ transplantation in the
and patient is to undergo immunosuppression
single-stranded (pinapahina ang immune system ng recipient and
nucleic acids.
sinasaksakan ng steroids) and determine the HLA of
Composition Adjacent amino Amino acid residues
the recipient and the donor. Minsan kahit relative mo,
acid residues in the brought into proximity
covalent sequence to one another by there is an incompatible HLA, because antigens are
folding created unique and sometimes it can become your
immunogen if it has a carrier molecule.

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LESSON #2: RELATIONSHIP BETWEEN IMMUNOLOGY AND IMMUNOHEMATOLOGY

○ The MHC genes determine the human leukocyte

antigens (HLA) present on leukocytes and other
cells. HLA molecules have been known for many
years to be a major determinant of tissue graft
rejection.

ANTIGEN FREQUENCY
● Low Incidence: Less than 1% appearance of this type of
antigen in the population.
○ If you are exposed to low incidence antigen, medyo
mahirap magproduce ng antibodies because it is
very rare.
● High Incidences: 99% chance of appearing this antigen
in the population. Public antigens, almost all of the
populations are exposed.
○ SARS COV 2 (Before it is a low incidence that
caused high mortality rate but due to pandemic, it
became a high incidence) ● Gerald Edelman and Rodney Porter - described and
○ High Incidence Antigen = Herd Immunity discovered the antibody.

ANTIBODIES/IMMUNOGLOBULINS
● Products of antigenic stimulation and that demonstrate
specific antibody activity
● Humoral branch of immune response
○ Humoral response: Antibody
■ Innate Humoral: ABO antibody
■ Adaptive Humoral: Exposed to Immunogen
○ Cellular response: Phagocytes; counterpart of
humoral BLOOD GROUP IMMUNOGLOBULIN
● Graph Picture: Serum Electrophoresis
Table No. 6 Blood Group Antigen
○ Gamma region
AUTOANTIBODY ALLOANTIBODY
○ Immunoglobulins are sometimes called Gamma
Reacts with an antigen on Reacts with a foreign antigen not
Globulins because they are only seen in the Gamma the patient's own cells. present on the patient's own
Region erythrocytes.
● Structure of Antibodies: ● Example: Lupus is a ● Unexpected antibodies
○ Heavy Chains and Light Chains are connected by disease that occurs ● Common reason that will
disulfide bonds and have a paratope. when your body's produce alloantibody: Blood
○ Within the antibody, you have two regions: Fab immune system attacks Transfusion, Pregnancy
(antigen binding fragments) and Fc portion your own tissues and ● Example: A female that is RH -
(fragment crystallizable and its purpose is if the organs. will react to a male that is RH +
● Example of producing alloantibodies due to
antibody can’t remove the antigen, it will send
Autoantibody: the D antigen of the RH + which
chemotaxis to the cell mediated response which is Systemic lupus can result in Hemolytic Disease
the phagocytes so that the phagocytes will eliminate erythematosus (SLE) of the Fetus and
the foreign antigens) and Type 1 DM Newborn(HDFN).
● Example: May babae na RH -,
Table No. 5 Structure of an Antibody ung asawa ay RH+ nagboom
STRUCTURE OF AN CONFORMATIONAL EPITOPE boom pow sila, ung babae
ANTIBODY nagreact sa RH+. Ung RH+
Two Identical: The N-terminus of each heavy may D antigen kaya yung
● Heavy Chains chain forms an antigen-binding babae magpoproduce ng
● Light Chains domain with a light chain alloantibody kasi RH- siya.
Fab Region Antigen binding fragments Kaya un ang birth ng Hemolytic
disease of the fetus and
Fc Region The C-terminus of the heavy newborn(HDFN).
chains forms ‘fragment
crystallization’ (Fc) domain, which
helps in the interaction with the
NATURALLY OCCURING ANTIBODIES
effector cells. ● Antigen stimulus is unknown
Fragment crystallizable ● Example: ABO antibodies
All four polypeptide subunits are held together by disulfide and ● Non-RBC stimulated antibodies (Howard p. 105)
non-covalent bonds. ○ Individuals was exposed to biochemical structures
similar to A or B antigens present in the
The heavy chains of the antibodies contain a variable region and environment (Bacteria, pollen, plants)
three constant regions. Each antibody has two identical
antigen-binding sites and they differ in the antibodies IMMUNE ANTIBODIES
● Produced as the result of immunization to foreign
erythrocyte antigens by exposure through transfusion
of blood components or through pregnancy.

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LESSON #2: RELATIONSHIP BETWEEN IMMUNOLOGY AND IMMUNOHEMATOLOGY

IMMUNOGLOBULIN CLASSES
*See Table 7 at the last page.
● IgM
○ the best complement fixing antibody
because it is macromolecule and it is a
pentamer antibody (5 antigen binding site)
○ Primary immune response antibody
● IgG
○ Smallest antibody
○ Only capable of placental transfer
○ rH
○ Secondary immune response antibody
○ Monomer; single antibody
○ Requires antihuman globulin
● IgE
○ Anaphylactic Reaction and Allergy ● Detecting warm reacting antibodies - anti human globulin
phase
TYPES OF ANTIBODIES REACTION PHASES ● What type of antibody induced by ABO blood group? IgM
● Immediate Spin Phase (done in room temp)
○ IgM is a cold reacting antibody REDUCTION OF ANTIBODY
○ IgM antibodies usually react optimally at ambient ● Conversion of polymeric antibody into monomeric
temperatures or below 22°C at the immediate spin antibody
phase of testing ○ 2-mercaptoethanol (2-ME)
● Antiglobulin Testing (Warm Reacting) ○ dithiothreitol (DTT)
○ Detect IgG and incubated at 37 C ■ Reagents are used in order to know the cause
○ React optimally at the antihuman globulin (AHG) of antigen and antibody reaction because
phase of testing. IgG antibodies usually require 37°C sometimes antibodies in patient serum are
incubation because it only reacts at warm mixed.
temperature ● In BB, we first identify if the antibody is IgG or IgM. Once
we know the antibody class, we already know what blood
Table No. 8 Type of Antibodies Reaction Phase group it came from.
IMMEDIATE SPIN PHASE ANTIGLOBULIN PHASE ● Polymeric antibody: IgM
(37 C) ● Clinically significant antibody: IgG
Examples of IgM antibodies that IgG antibodies are those
have importance in blood directed against EFFECT OF THE ENZYMES PAPAIN AND PEPSIN IN A
banking include those against ● Ss
the: ● Kell (Kk, Jsa, Jsb,
MONOMER
● ABH Kpa, Kpb) PAPAIN
● Ii ● Rh (DCEce)
● MN ● Lutheran (Lub) ● Papain cleaves antibodies into two Fab fragments, which
● Lewis (Lea, Leb), ● Duffy (Fya, Fyb), recognize the antigen specifically with their variable
● Lutheran (Lua) ● Kidd (Jka, Jkb) region, and one Fc fragment.It cleaves above the hinge
● P blood group antigens region containing the disulfide bonds that join the heavy
antigens chains, but below the site of the disulfide bond between
Lewis Is the MaN for P1A Rich Daring Kidd can Kell the light chain and heavy chain.
(IgM/cold antibodies) Sir Lutheran B (IgG) This generates two separate monovalent (containing a
single antibody binding site) Fab fragments and an intact
Fc fragment. The fragments can be purified by gel
filtration, ion exchange, or affinity chromatography.
● Papain cleaves the heavy-chain CH2 domains above the
disulfide bonds that connect heavy chains and yields
three different fragments. The resultant pieces are
identical in structure and consist of constant and variable
regions of heavy (VH and CH1) and light (VL and CL)
chains linked by disulfide bonds. These two fragments,
each having a molecular weight of 60,000, are called Fab
(fragment antigen binding). Each Fab binds a single
antigen. The remaining fragment, which consists of CH2
and CH3 heavy-chain units, is easily crystallized and is
called Fc (fraction crystallized). Later studies showed that
the Fc portion of antibodies binds to receptors (FcR) on
immunocompetent cells.

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LESSON #2: RELATIONSHIP BETWEEN IMMUNOLOGY AND IMMUNOHEMATOLOGY

PEPSIN ● Another reason antigen amounts may be altered is due to

● Pepsin is one of the principal protein degrading or weak expression of antigen on RBCs (dosage effect).
proteolytic enzymes in the digestive system. During the Weak expression occurs as a result of the inheritance of
process of digestion, Pepsin acts on the complex dietary genotypes that give rise to heterozygous expression of
protein and breaks up into peptides and amino acids RBC antigens and resultant weaker phenotypes. For
which can be readily absorbed by the intestinal lining. example, M-positive RBCs from an individual having the
● Pepsin is a chain protein (monomer) composed of two genotype MM (homozygous) have more M antigen sites
similar folding domains separated by a deep cleft. The than M-positive cells from an individual having the MN
catalytic site of pepsin is formed at the junction of the (heterozygous) genotype.
domain, each domain contains two aspartic acid ○ In dosage effect: Homozygous expression = stronger
residues, Asp32 and Asp215. Under the catalysis of antibody-antigen reaction
pepsin, the water molecule helps the active carboxyl ○ In dosage effect: Heterozygous expression = weaker
group to bear positive and negative charges with aspartic antibody-antigen reaction
acid 215 and aspartic acid 32, respectively, which breaks
PH
the peptide bond in the protein.
● Pepsin cleaves IgG heavy chains at a point below the ● Ideal pH of a test system for antigen-antibody reactions
disulfide bond that links two heavy chains. The resulting ranges between 6.5 and 7.5.
fragment consists of two Fab linked together by ● Exceptions include some anti-M and some Pr(Sp1)
heavy-chain disulfide bonds. The large-molecular-weight group antibodies that show stronger reactivity below
fragments are called F(ab’)2. Each F(ab’)2 can bind two pH 6.5.
antigens. ○ Anti-M and anti-Pr(Sp1) reacts best at acidic pH.
● Acidification of the test serum may aid in distinguishing
FACTORS THAT INFLUENCE ANTIGEN-ANTIBODY anti-M and anti-Pr(Sp1) antibodies from other antibodies.
REACTION
● Antigen-Antibody Ratio TEMPERATURE
○ Effect of Zonal Reaction ● IgM antibodies usually react optimally at ambient
○ Dosage effect temperatures or below 22°C at the immediate spin
● pH (6.5 - 7.5) phase of testing.
● Temperature ● IgG antibodies usually require 37°C incubation and react
● Immunoglobulin type optimally at the antihuman globulin (AHG) phase of
● Centrifugation testing. Because clinically significant antibodies may be
in both temperature ranges, it is important to do testing
ANTIGEN-ANTIBODY RATIO with a range of temperatures.
● Antigen and antibody have optimal concentrations; in ● IgM antibodies are generally capable of agglutinating
ideal reactive conditions, an equivalent amount of antigen RBCs suspended in a 0.85% to 0.90% saline medium.
and antibody binds. Any deviation from this decreases The IgM antibody is 160 Å larger than an IgG molecule
the efficiency of the reaction and a loss of the zone of and approximately 750 times as efficient as IgG in
equivalence between antigen and antibody ratio, agglutination reactions. This allows it to easily bridge the
which is necessary for agglutination reactions to occur. distance between two RBCs.
An excess of unbound immunoglobulin leads to a ● Another factor that contributes to the difference in
prozone effect, and a surplus of antigen leads to a reactivity between the IgM and IgG molecules is the
postzone effect. number of antigen-combining sites on each type of
immunoglobulin.
○ IgM molecule has the potential to bind 10 separate
antigen sites, while an IgG molecule has only two
binding sites per molecule (bind two RBCs with only
one binding site on each cell.)
● Agglutination reactions involve more than one
● immunoglobulin molecule, and these conditions are
multiplied many times in order to represent the
agglutination reaction.

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LESSON #2: RELATIONSHIP BETWEEN IMMUNOLOGY AND IMMUNOHEMATOLOGY

Table No. 9 Type of Antibodies Reaction Phase

IMMEDIATE SPIN PHASE ANTIGLOBULIN PHASE
(37 C) TYPES OF ENHANCEMENT MEDIA/ POTENTIATOR
Examples of IgM antibodies that IgG antibodies are those
have importance in blood directed against 1. PROTEIN MEDIA
banking include those against ● Ss
● Increases the dielectric constant (a measure of electrical
the: ● Kell (Kk, Jsa,
conductivity), which then reduces the zeta potential of the
● ABH ● Jsb, Kpa, Kpb)
RBC
● Ii ● Rh (DCEce)
● Examples:
● MN ● Lutheran (Lub)
○ 22% or 30% bovine serum albumin (incubation: 15-60
● Lewis (Lea, Leb), ● Duffy (Fya, Fyb),
minutes)
● Lutheran (Lua) ● Kidd (Jka, Jkb)
○ Polyethylene Glycol (PEG)
● P blood group antigens
○ Polyvinylpyrrolidone (PVP)
antigens
○ Protamine
Lewis Is the MaN for P1A Rich Daring Kidd can KellS
(IgM/cold antibodies) Joke (IgG)
POLYETHYLENE GLYCOL
● Removes water molecule in the environment
● Hydrophilic polar heads of lipid molecules making up the
CENTRIFUGATION outer cell membrane bilayer attract water molecules. The
● Effective way to enhance agglutination reactions because water thus creates a surface tension that helps to keep the
it decreases reaction time by increasing the gravitational cells apart (Henry’s p.711)
forces on the reactants and bringing reactants closer ● Incubation: 10-30 minutes
together. ● More effective than albumin, LISS for detection of weak
antibodies
● High-speed centrifugation is one of the most efficient
methods used in blood banking. 2. LOW IONIC STRENGTH SOLUTION (LSS)
● Having RBCs in closer physical proximity allows for an ● Generally contain 0.2% sodium chloride
increase in antigen-antibody lattice formation, which ● Decreases the ionic strength of a reaction medium, which
results in enhanced agglutination. reduces the zeta potential and therefore allows antibodies to
react more efficiently with RBC membrane antigens
ZETA POTENTIAL ● Incubation time: 5 to 15 minutes
● Pulse repulsion between red cells due to their ● Disadvantage: prone to false positive result
difference in electrostatic surface of RBC; not normal
3. PROTEOLYTIC ENZYMES
because it will form agglutination
● Target the sialic component, protein molecule
● IgG cannot form agglutination (needs a potentiator, it
● Enhances: Rh, Kidd, P1, Lewis, and I antigens (Rich Kidd
will enhance the IgG para paglapitan ung dalawang red LIP)
cells ) while IgM can because of its size and ● Destroys: Fya, Fyb, M, N, S antigens (Duffy MeNS)
molecular weight (reason why it is a good ● Example:
complement fixation). ○ Papain (Papaya)
● Ionic Cloud: Responsible para hindi magdikit dikit ang red ○ Ficin (Fig plants)
cells. ○ Trypsin (Pig stomach)
○ Bromelain (Pineapple)

4. ANTIHUMAN GLOBULIN TEST

● 1945 Coombs, Mourant and Race
● Detects antibodies and complement
proteins that have attached to the RBC
● Involved the injection of human serum
into rabbits to produce anti human
serum

Schematic Diagram: Schematic representation of the ionic

cloud concept and its relevance to hemagglutination induced
by IgM and IgG antibodies. Compare the size of the IgG
antibody with that of the IgM molecule. The size of the IgG
molecule is not large enough to span the distance between two
adjacent RBC

ENHANCEMENT MEDIUM/POTENTIATOR
● Reagents that enhances the detection of IgG antibodies
by increasing their reactivity
● Reduces the zeta potential
● May enhance antibody uptake or promote direct
agglutination
● Only used when detecting IgG antibodies.
● Most common used potentiators in blood bank:
○ Polyethylene Glycol (PEG) Coomb’s Test
○ Polyvinylpyrrolidone (PVP)
○ Low Ionic Strength Solution

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LESSON #2: RELATIONSHIP BETWEEN IMMUNOLOGY AND IMMUNOHEMATOLOGY

● Patients RBC coated in vivo w/ IgG or complement ● Red Cell phenotyping

fractions + Anti-human globulin (AHG) = Agglutination ● Crossmatching
(Positive Reaction)
Sample: Patient Serum
● Patients RBC not w/ IgG or complement fractions +
Anti-human globulin (AHG) =No Agglutination (Negative Procedure of IAT: ● Add 2 drops of the patient's
Reaction) serum to the tube.
○ Anti-human globulin = Antibodies designed to react ● Label the tube with patients
name
to against IgG antibodies and complement proteins
● Add 1 drop of the reagent red
cell (KNOWN O SUSPENSION)
● DAT: in vivo sensitization : Patients Red Cells (EDTA to the tube
blood) ● Add the
○ Sensitized RBC + Wash 3x (to remove unbound potentiator/enhancement media
antibodies) + add AHG reagent = With ● Incubate at 37 C
Agglutination + add Check Cells (sensitized red ● Centrifuge the sample for 15
cells) = With Agglutination = +DAT seconds at 3400 rpm.
● Examine for agglutination or
○ Patients RBC not coated in vivo w/ IgG or
hemolysis using a good light
complement fractions + Wash 3x (to remove source.
unbound antibodies) + add AHG reagent = Without ● Wash the cells 3x using the
Agglutination + add Check Cells (sensitized red isotonic saline solution.
cells) = With Agglutination = -DAT (nagbind lang sa AHG REAGENTS
check cells )
Polyspecific AHG Contain antibody to human IgG and
to the C3d component of human
● IAT: in vitro sensitization: Patients Serum complement
○ Detect if the serum has antibdoies tht can sensitize
the RBCs. Monospecific AHG Contain only one antibody
specificity: either anti-IgG or
○ Patients serum + add Red Cell Reagent + add antibody to specific components of
Potentiator + Incubate anti-sera + Wash 3x + complement, such as C3b or C3d
AHG reagent = With Agglutination (to sensitized AHG PREPARATION
RBC) + add Check cells = With Agglutination =
Polyclonal ● Mixture of antibodies from
+iAT because the serum is has the antibodies that different plasma clones
are capable of sensitizing the red cells. ● Recognize different antigenic
○ Serum is incapable of sensitizing the RBC = -IAT determinant (epitopes), or the
because AHG bind to the check cells same portion of the antigen but
with different affinities
TYPES OF COOMB’S TEST ● Rabbits
Direct Antihuman Globulin Detects in vivo sensitization of RBC Monoclonal ● Derived from one clone plasma
(DAT) with IgG/ complement components. cells and recognize a single
epitope
Clinical Conditions: ● Diagnosis of HDFN
● Mice
● Investigation of HTR
● Diagnosis of AIHA
Polyclonal Antibody
Sample of Choice: EDTA Blood
Procedure of DAT ● Washed RBC + AHG reagent
○ Inadequate washing may
result in a false-negative
reaction because of
neutralization of the AHG
reagent by residual unbound
serum globulins.
○ 3 times washing so that it
removes the unbound
antibodies and AHG will react
to the coated antibodies.
● Does not require incubation
Monoclonal Antibody Reaction
What are Check Cells? ● The wash phase can be
(Both used in DAT and IAT) controlled using Check cells, or
group O cells sensitized with
IgG.
● Red cells with attached
antibodies.
● Sensitized red cells
● Also known as the “Coombs
Cell”
● Used if the AHG testing is
negative.
Indirect Antihuman Globulin Detects in vitro Sensitization of
RBC.
Used in the following: ● Antibody reaction
● Antibody Identification
● Antibody titration

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LESSON #2: RELATIONSHIP BETWEEN IMMUNOLOGY AND IMMUNOHEMATOLOGY

● Contamination of AHG by extraneous protein (i.e., glove,

MONO vs. POLY wrong dropper)
● High concentration of IgG paraproteins in test serum
● Early dissociation of bound IgG from RBCs due to
interruption in testing
● Early dissociation of bound IgG from RBCs due to
improper testing temperature (i.e., saline or AHG too cold
or hot)
● AHG reagent nonreactive because of deterioration or
neutralization (improper reagent storage)
● Excessive heat or repeated freezing and thawing of test
serum
● Polyclonal Antibody: React both to IgG and C3d, ● Serum non reactive because of deterioration of
● Monoclonal Antibody: Specific, kapag IgG ay IgG complement
lang, kapag C3d ay C3d lang. ● AHG reagent, test serum, or enhancement medium not
● Monoclonal antibodies are produced from one cell added
and all antibodies produced are the same whereas in ● Under centrifuged or over centrifuged
the Polyclonal antibodies, the same specificity but ● Cell suspension either too weak or too heavy
produced by different clones of cell antibodies and the ● Serum-to-cell ratios are not ideal.
good thing is it reacts with the same epitopes. ● Rare antibodies are present that are only detectable with
● Polyclonal Source: Rabbit polyspecific
● Monoclonal Source: Hybridomas from the mice, the ● AHG and when active complement is present.
fusion of spleen cells challenged with antigen + the ● Low pH of saline
myelomas cells will form the hybridomas and the ● Inadequate incubation conditions in the IAT
hybridomas will be cultured in a HAT medium and ● Poor reading technique
those who will survive will become monoclonal
antibodies. POSITIVE REACTION IN BLOOD BANK
● Agglutination
TAKE NOTE: ○ Antibody - mediated clumping of particles that
● DAT: Detect a level of 100 to 500 IgG molecules per express antigen on the surface
RBC and 400 to 1,100 molecules of C3d per RBC. ● Hemolysis
● IAT: There must be between 100 and 200 IgG or C3 ○ Rupture of red cells with release of intracellular
molecules on the cell to obtain a positive reaction. hemoglobin
○ By product of complement proteins, complement
FACTORS AFFECTING THE AHG TEST activation
● Cell: Serum = 1:40 or 1 gtt/2 gtt ● Precipitation
● Reaction medium ○ Formation of an insoluble complex when soluble
● Temperature antigen reacts with soluble antibody
● Incubation Time ○ End point of immunodiffusion and
● Washing immunoelectrophoresis
● Centrifugation (1000 RCF for 20 seconds) ● Specific hemagglutination is the single most important in
vitro immunologic reaction in blood banking because it is
CAUSE OF FALSE-POSITIVE RESULT IN AHG TESTING the endpoint of almost all test systems designed to detect
● Improper specimen (refrigerated, clotted) may cause in RBC antigens and antibodies (Henry’s p.711).
vitro complement attachment
● Over centrifugation and overreading Table No.10 Serologic Grading
● Centrifugation after the incubation phase when PEG or SEROLOGIC GRADING FOR HEM AGGLUTINATION
other positively charged polymers are used as an Grade Description Score
enhancement medium 4+ One solid agglutinate with clear background 10
● Bacterial contamination of cells or saline used in washing 3+ Several Medium to Large agglutinates with clear 8
● Dirty glassware background
● Presence of fibrin in the test tube may mimic 2+ Medium-sized agglutinates with clear 5
agglutination. background
1+ Small agglutinates with a turbid background 3
● Cells with a positive DAT will yield a positive IAT.
W+ Tiny agglutinates with turbid background 1
● Polyagglutinable cells
0 No agglutination of hemolysis 0
● Saline contaminated by heavy metals or colloidal silica
Mf Mixed Field na
● Using a serum sample for a DAT (use EDTA, ACD, or
H Hemolysis 10
CPD anticoagulated blood)
PH Partial Hemolysis
● Samples collected in gel separator tubes may have
unauthentic complement attachment.
● Complement attachment when specimens are collected
from infusion lines infusing dextrose solutions
● Preservative-dependent antibody directed against
reagents

CAUSE OF FALSE-NEGATIVE RESULT

● Inadequate or improper washing of cells
● Failure to wash additional times when increased serum
volumes are used

8 GONZALES, LIM, MANALO

LESSON #2: RELATIONSHIP BETWEEN IMMUNOLOGY AND IMMUNOHEMATOLOGY

POSITIVE RESULTS IN BLOOD BANK DIRECT TYPING/FORWARD TYPING

● Agglutination ● Antigen in red cells
● Kung saan nag react, yun ang antigen
○ Patients Red Cells + Anti Sera (Anti-A
agglutinates and Anti-B w/ no agglutinates) = A
antigen = Blood Type A

● Hemolysis

INDIRECT TYPING/REVERSE TYPING

● Antibody Patient Serum + Known A cells
(agglutinate) + Known B cells (no agglutinates) =
● Mixed-Field Blood Type A because of A antigen.
○ Small agglutinates within predominantly
unagglutinated red cells

DIRECT vs INDIRECT TYPING

● Prevent Rouleaux Formation: Add NSS (Normal

Saline Solution, to rule out rouleaux formation which
can be mistaken as agglutination and can cause
false-positive results.

REFERENCES

● Notes from the discussion by NATHANIEL VERON

M. CASAMINA, RMT, ASCP, MSMT
● Tankeshwar, A. (2021, June 1). Epitopes: Types,
Function, Epitope Spreading. Microbe Online.
https://microbeonline.com/epitope/

Agglutination Rouleaux formation (2nd and 3rd pic)

9 GONZALES, LIM, MANALO

LESSON #2: RELATIONSHIP BETWEEN IMMUNOLOGY AND IMMUNOHEMATOLOGY

Table No. 7 Immunoglobulin Classes

CHARACTERISTICS IgA IgD IgE IgG IgM
Heavy chain type Alpha Delta Epsilon Gamma Mu
Sedimentation coefficient (s) 7-15* 7 8 6.7 19
Molecular weight 160-500 180 196 150 900
Biologic half-life (d) 5.8 2.8 2.3 21 5.1
Carbohydrate content (%) 7.5-9 10-13 11-12 2.2-3.5 7-14
Placental transfer No No No Yes No
Complement Fixation (classical pathway) No No No +++
Agglutination in saline + 0 0 + ++++
Heavy chain allotypes Am None None Gm None
Proportion of total immunoglobulins (%) 13 1 0.002 80 6
● May occur in monomeric or polymeric structural forms
● d= Days; kD = kilodaltons; 0 = negative reactivity; + = weak reactivity; + = slight reactivity; +++ = strong reactivity,
++++= very strong reactivity

10 GONZALES, LIM, MANALO