Detection and Identification of Antibodies

ANTI-HUMAN GLOBULIN TEST

ANTI-HUMAN GLOBULIN TEST
- Used to detect RBCs sensitized with IgG
- Only IgM was detected by direct alloantibodies, IgG autoantibodies, and
agglutination prior to AHG test complement components.
● Coombs, Mourant, Race
- Used antiglobulin test for detection of ● Indirect antiglobulin test (IAT)
week and non-agglutinating Rh - Use of AHG to detect in vitro
antibodies in serum sensitization of RBCs is a two-stage
technique
● Moreschi
- Used rabbit anti-goat serum to ● Direct antiglobulin test (DAT)
agglutinate rabbit RBCs that were - In vivo sensitization is detected
sensitized with low nonagglutinating
doses of goat anti-rabbit RBC serum.
ANTI-HUMAN GLOBULIN TEST REAGENTS
● Dacie 1. Polyspecific AHG
- First evidence of another antibody - Contain antibody to human lgG and to
activity present that influenced final the C3d component of human
reaction of reagents complement.
- Different reaction patterns when dilutions - Facilitate agglutination when RBCs have
of AHG were used to test cells sensitized been sensitized with IgG or C3d or both
with warm (IgG, 37°C) as compared with - Little activity against IgA and IgM heavy
cold antibodies (IgM <22°C). chains.
- May contain antibody activity to
ANTI-HUMAN GLOBULIN TEST kappa and lambda light
- Also known as Coomb's Test - Reacts with some IgA or IgM
- Principle: Anti-human globulins obtained molecules.
from immunized nonhuman species bind
to human globulins such as IgG or 2. Monospecific AHG
complement, either in free serum or - Contain only one antibody specificity:
attached to antigens on RBCs either anti-IgG or antibody to specific
- Essential testing for transfusion medicine components of complement, such as
C3b or C3d
Two major blood group antibodies:
- IgM and IgG - Anti-IgG
- Specific for the Fc fragment of
● IgM the gamma heavy chain of the
- Large pentameric structure IgG molecule
- Direct agglutination in saline
- Anticomplement
● IgG - Reactive against only the
- Single monomer structure designated complement
- Too small to directly agglutinate components
sensitized - No activity against human
- + AHG reagent causes hemagglutination immunoglobulins

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PREPARATION OF AHG REAGENT
Use of Polyspecific Versus Monospecific
AHG in the IAT
- Most clinically significant antibodies
detected during antibody screening are
IgG

- Polyspecific AHG - Unwanted positive

reactions not caused by clinically
Antibodies Required in AHG significant antibodies.
1. ANTI-IGG - False positive - 93% is caused
- Must contain antibody activity to by C3 on the cells
nonagglutinating blood group antibodies - Repeat using prewarmed
- mixture of IgG1 and IgG3 technique - false positive
- Only anti-IgG activity is required becomes negative

- Anti-light chain activity allows detection Principles of the Antiglobulin Test

of all immunoglobulin classes.
- Antibody molecules and complement
2. Monospecific IgG AHG (Gamma-clone components are globulins.
anti-IgG) - Injecting an animal with human globulin
- Specific stimulates the animal to produce
- Detects IgG1, IgG2, and IgG3 antibody to the foreign protein
- Fails to react to IgG4
- IgG4 antibodies - uncommon, Direct Antiglobulin Test
not associated with acute
● Detects in vivo sensitization of RBCs
hemolytic reactions, high-titer,
with IgG or complement components.
low-avidity-type, clinically
- Hemolvtic disease of the fetus and
insignificant
newborn (HDFN)
- Hemolvtic transfusion reaction (HTR)
3. Anticomplement
- Autoimmune and drug-induced
- "fix" complement components to the
hemolytic anemia (AIHA)
RBC membrane after complexing of the
antibody with its corresponding antigen.
● Initial DATs - 1 drop of 3% to 5%
- Anticomplement activity enhances the
suspension of washed RBCs with
reactions of clinically significant
polyspecific reagent
antibodies

● Positive results - DAT panel using

monospecific anti-IgG and anti-C3d
- determine specific type of protein
sensitizing the cell

● Polyspecific and monospecific reagents

may be run together with saline control

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● Investigation of HDFN
- protein sensitizing the newborn RBCs is
presumed to be maternal IgG
- If the DAT is positive due to IgG and the
immediate spin for D typing is negative, a
test for weak D cannot be performed.
- The antibody must be removed
from the RBCs for accurate
phenotyping.

Evaluation of a Positive DAT

● Positive DAT may occur without clinical
manifestations of immune mediated
hemolysis
- DAT alone is not diagnostic of hemolytic
anemia

● Review patient history:

- Recent drug use, pregnancy, transfusion
history, hematopoietic transplant therapy,
acquired or unexplained hemolytic
anemia

Indirect Antiglobulin Test

● Determine in vitro sensitization of RBs
and is used in the following situations:

- Detection of incomplete
(nonagglutinating) antibodies to potential
donor RBCs (compatibility testing) or to
screening cells (antibody screen) in
serum

- Determination of BC phenotype using

known antisera (e.g., weak D, any other
antigen testing that requires IAT)

- Titration of incomplete antibodies

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 - PEG - Remove water molecules
surrounding the RBC

● Temperature
- Optimum temperature for complement
activation and IgG reaction

● Incubation Time
● Washing of RBCs
- Removes free unbound serum globulins.
- Saline
- pH of 7.2 to 7.4

● Addition of AHG
- Minimize the chance of antibody eluting
from the cell and subsequently
neutralizing the AHG reagent
-
● Centrifugation for Reading
- Use of higher RCFs yields more
sensitive results inadequate
resuspension - false positive
- Too vigorous - false negative

Factors Affecting the Antiglobulin Test

Modified and Automated Antiglobulin Test
● Ratio of Serum to Cells Techniques
- Increasing the ratio of serum to cells
increases the sensitivity of the test ● Solid-Phase Technology
system. - Occurs in small microwells, where
antigens are bound to the bottom of the
● Reaction Medium well and the patient's plasma is
- Albumin - Allow antibody-coated cells to incubated in the well.
come into closer contact with each other
so that agglutination occurs. ● Gel Test
- LISS - Enhance antibody uptake and - Detects BC antigen-antibody reactions
allow incubation times to be decreased by means of a chamber filled with
polyacrylamide gel.

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 Antibody Testing
Why do we need to detect and identify
antibodies? - Tube Method
- Investigating potential hemolytic - Gel Technology
transfusion reactions and - Solid-Phase Technology
immunehemolytic anemias.
Tube Method
- Detecting and monitoring patients who
are at risk of delivering infants with - Traditional method
hemolytic disease of the fetus and
newborn (HDFN) ● Utilizes:
- RBC reagents (2-5% cell suspension)
- Enhancement reagent
Terms - AHG reagent
Unexpected antibodies - Any non-ABO - Sensitizes reagent RBCs with
antibody present in a recipient or donor. the patient's antibodies

Immune alloantibodies - antibody formed in ● Advantages:

response to pregnancy, transfusion, or - flexibility, availability of required testing
transplantation reagents, low cost

Naturally occurring alloantibodies - form as a ● Disadvantage:

result of exposure to environmental sources (ex: - Instability of reactions
pollen, fungus, bacteria) - Subjective
- Time consuming
Passively acquired antibodies - produced in - Failure of washing phase
one individual and then transmitted to another
individual via plasma-containing blood
components (ex: Ivlg)

Autoantibodies - directed against antigens on

one's own RBCs

Clinically significant alloantibodies - causes

decreased survival of RBCs processing that
target, antigen

RBC Reagents
Limitations
● Type O
- Will not detect antibodies when antibody ● Screen cells
titer dropped below level of sensitivity for ● R1R1, R2RZ, rr
screening method ● Pooled screening reagent
- For donor screening only
- Antibodies formed in response to - RBCs from two different
transfusion may no longer be detectable individuals
- Observe for mixed-field
- Cannot detect antibodies directed agglutination
against low-prevalence antigens not
present on RBCs in the screen

- Antibodies with dosage effect may not be

detected if screen cells are not
homozygous

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 - Are the cells truly agglutinated, or is
rouleaux present?

In what phase(s) did the reaction(s) occur?

● IgM: Anti-N, anti-I, anti-P1
● IgG: Rh, Kell, Kidd, Duffy, Ss
● Both: Lewis and M antibodies

Is the autologous control negative or

Enhancement Reagents positive?
● Rule out presence of an autoantibody
● Increases sensitivity
● History needs to be evaluated
● Allows for shortened incubation time
● 22% albumin
Did more than one screen cell sample react?
● LISS
If so, did they react at the same strength and
● PEG
phase(s)?
● Presence or absence of multiple
AHG Reagents antibodies
- Agglutination of incomplete antibodies
Is hemolysis or mixed-field agglutination
● Monospecific - either anti-IgG or present?
antibodies to complement components ● Hemolysis: anti-Lea, anti-Leb,
● Polyspecific (Coomb's reagent) - both anti-PP1Pk, and anti-Vel
anti-IgG and antibodies to complement ● Mixed-field: anti-Sda and Lutheran
components antibodies
- May lead to detection of clinically
insignificant antibodies reacting Are the cells truly agglutinated or is rouleaux
to complement present?

● Any tube test negative after AHG - Antibody identification

controlled by adding Coomb's control
● Review history
cells
- Sex, age, race, diagnosis, transfusion hx,
- Rh-positive RBCs coated by
OB hx, medications, IV solutions
anti-D
- Exposure to non-self RBC via transfusion
- Negative: antibody coated cells
or pregnancy - more likely to produce
will react with anti-IgG in AHG
immune antibodies
reagent - visible agglutination
- Proves adequate washing = no
● Test antibody identification panel with
unbound antibodies
auto cont or DAT

Interpretation of Results
Antibody identification panel
- In what phase(s) did the reaction(s)
● 11 to 20 group O reagent RBCs with
occur?
various antigen expression
- Is the autologous control negative or
● A profile sheet specifying antigens on
positive?
each cell provides a place to record
- Did more than one screen cell sample
reactions
react? If so, did they react at the same
● Indicates presence of rare cells
strength and phase(s)?
● Lot specific - do not interchange
- Is hemolysis or mixed-field agglutination
present?

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 Exclusion
● First step: exclude antibodies that could
not be responsible for the reactivity seen.
● "Rule-outs"
- If results all give a negative reaction,
they are not the antibody's target
- Only if there is homozygous expression
on the antigen of the cell
- Exception: low-prevalence antigens (K,
Kpa, Jsa, and Lua)

Evaluation
Do all positive cells react to the same
degree?
● Strength does not indicate significance
● Influenced by dosage effect
● Presence of more than one antibody

Do all of the positive cells react at the same

phase, or do any react at different or multiple
phases?
● May indicate multiple antibodies

Does the serum reactivity match any of th

cource or orreläining specificities?

Are all commonly encountered BC antibodies

excluded?

Is the autologous control (last row in panel

antigen profile) positive or negative?
● Rule out autoantibodies

Is there sufficient evidence to prove the

suspected antibody?
● Test patient's serum with at least three
antigen-positive and three
antigen-negative cells (3 and 3 rule)

Does the patient lack the antigen

corresponding to the antibody?

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 2.

EXERCISES
1.

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3.
Phenotyping
● Antigen typing the patient red cells to
confirm an antibody
● Only performed if patient has not been
recently transfused
● Remove coated antibody prior to testing
○ Glycine EDTA
- Kell antigens are denatured in
this method
- 2 minutes incubation at room
temp
○ Chloroquine disphosphate
- 30 minutes - 2 hours incubation
at room temp
- 5-30 minutes at 37C

● If treated cells: Negative DAT = may be

phenotyped

Selected Cell Panels

● Additional cells from different panel or
panels
● Useful when a patient has a known
antibody and the technologist is
attempting to determine if additional
antibodies are present.

Enzymes
● Use of proteolytic enzymes (ficin, papain,
bromelin, and trypsin)
● Modify RBC surface
- Destruction of certain antigens
- Enhances expression of others
● Useful in identification when multiple
antibodies are present
● May be utilized instead of enhancement
media
○ One stage: Untreated panel BC,
enzyme, and patient serum are
added to test tube prior to
incubation
○ Two stage: enzyme treatment of
panel RBCs prior to addition of
patient serum to test tube
● Compare pretreated and post-treated
results

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 ● When autoantibodies are present, BC
Neutralization
adsorptions can be performed using
● Some antibodies may be neutralized as allogeneic or autologous RBCs
a way of confirmation ● Only if patient has not been transfused
● Useful when multiple antibodies are within the last 3 months
suspected and one of the suspected ● Only if volume of RBC is adequate
antibodies appears to be an antibody ● Autoadsorption
that can be neutralized. ○ Simplest method
○ Uses patient's own RBCs
○ Requires 3-6 aliquotes of
autologous RBCs.

● Incubation of patient serum with ● Allogenic Adsorption - if autologous

neutralizing substance RBC is insufficient or if with recent
● Perform antibody identification panel transfusion
using the treated serum ● Phenotypically matched Adsorption -
● Control - proves that loss of reactivity is if patient's phenotype cannot be
due to neutralization and not dilution determined
● Differential Adsorption
Adsorption ○ If patient's phenotype is
unknown and performing
● Antibodies are removed or adsorbed
antigen-typing is not an option.
from serum by incubating the specimen
○ Uses group O cells with the
with the corresponding antigen.
following phenotype: R1R1,
R2R2, and ir
● Antigen-antibody complex is separated
from adsorbed serum by centrifugation.
Temperature-Dependent
● Adsorbed serum tested against BC panel
● Simple method
cells for presence of unadsorbed
● Changing the temperature of
alloantibodies.
antigen-antibody environment
● 56C (heat method)
● Commercial Reagents for Adsorption
● -18C or colder (Lui freeze-thaw)
○ Human platelet concentrate -
● RBCs are destroved and cannot be used
Bg-like antibodies
for further testing

○ RESt - I, H, and IH-like

structures pH method
- Incubate at 4C to ● Detection of non-ABO lqG antibodies
remove insignificant ● Acid elution
antibodies ● Washed antibody-coated cells are mixed
with a glycine acid solution at pH 3
RBC Adsorption ● Disruption of antigen-antibody bond,
releasing antibody into acidic
supernatant

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 Clinical application
Organic Solvent Method
● If antibody present does not caused
● Dichloromethane, Xylene, Ether decreased survival of antigen-positive
● Act on lipids in RBC membrane to RBCs
reduce surface tension ○ Crossmatch-compatible is
○ Reversal of Van der Waals acceptable
forces ○ Anti-M, anti-N, anti-P1, anti-Lea,
● Time consuming and anti-Leb
● Solvents can be health and safety
hazards ● If clinically significant
○ Units for transfusion MUST BE
Antibody Titration ANTIGEN NEGATIVE
○ Must also demonstrate
● Quantifies amount or concentration of compatibility at AHG phase
antibody identified
● Flow cytometry, radioimmunoassay, or
ELISA 2 fold serial dilutions of serum ● Sickle cell and beta-thalassemia patients
tested against reagent RBCs with target ○ May receive units that are
antigen phenotypically matched even if
● Titer level is reciprocal of the greatest they have not formed
dilution (1 + or greater is observed) alloantibodies
○ Score may also be assigned ○ More likelv to make
based on strength of reactivity. alloantibodies due to repeated
transfusion
○ Transfuse units that are
phenotypically matched for Rh
(C,c,E,and e), and Kell antigens

● After initial titer - specimen frozen

● Initial specimen tested in parallel to
control variability
● Comparison of current and initial
specimen's result must be made
● 4-fold or greater increase in titer or
increase in score of 10 or more
○ Clinically significant

● Useful in monitoring Obstetric patients

with IgG antibody causing HDFN
● Increase in antibody titer during
pregnancy
○ Fetus is antigen-positive
○ At risk for HDFN

● Differentiates immune anti-D from

passively acquired anti-D
○ Titer level of Rhlg administration
is rarely above 4.

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