DETECTION AND IDENTIFICATION OF
Passively acquired antibodies:
ANTIBODIES (ANTIBODY SCREENING)
LEARNING OUTCOMES:
1. Differentiate between the following antibodies: expected
and unexpected, immune, naturally occurring, passive,
autoantibody, alloantibody, warm and cold.
2. Explain what factors make an antibody clinically significant.
3. Describe which patient populations require an antibody
screen.
4. Select appropriate cells to include in a screen cell set.
5. Interpret the result of an antibody screen.
6. List the limitations of the antibody screen.
7. Interpret the results of an antibody identification panel.
8. Summarize the exclusion and inclusion methods.
9. Correlate knowledge of the serologic characteristics of
commonly encountered antibodies with antibody
identification panel findings.
10. Identify the situations in which additional panel cells should
be tested and select appropriate cells.
11. Describe antigen-typing techniques.
12. Calculate the number of red blood cell (RBC) units that must
be antigen-tested to fulfill a provider’s request for
crossmatching.
13. Explain the principles behind enzyme and neutralization
techniques.
14. Given a patient’s history and initial results, choose the
correct method for performing adsorption.
15. Describe elution methods and give an example of when
each would be used.
16. Outline the procedure for determining the antibody titer
level, including reporting of results.
17. Given a patient scenario, identify additional steps that could
be employed to resolve a complex antibody identification
problem.
INTRODUCTION:
Applications of Antibody Screening:
▪ The detection of antibodies directed against red blood cell
antigens is critical in pretransfusion compatibility testing.
▪ It is one of the principal tools for investigating potential
hemolytic transfusion reactions and immune hemolytic
anemias.
▪ In addition, it aids in detecting and monitoring patients who
are at risk of delivering infants with hemolytic disease of the
fetus and newborn (HDFN).
The Scope of Antibody Screening/Detection Methods:
▪ The focus of antibody detection methods is on “irregular”
or “unexpected” antibodies.
What are the Different Categories of Unexpected Antibodies
Commonly Encountered in Blood Banking/Transfusion
Medicine?
1. Immune alloantibodies:
- Unexpected antibodies of primary importance in blood
banking or transfusion medicine.
- These are antibodies formed in response to pregnancy,
transfusion, or transplantation, targeted against a
blood group antigen that is not present on the person’s
red blood cells.
- The process of forming an alloantibody is called
alloimmunization.
2. Naturally occurring alloantibodies:
- Form as a result of exposure to environmental sources,
such as pollen, fungus, and bacteria.
- Some of these exogenous or environmental antigens
have similar structures to some RBC antigens.
3.
- Antibodies that are produced in one individual and then
transmitted to another individual via plasma-containing
blood components or derivatives such as intravenous
immunoglobulin (IVIG).
4. Autoantibodies:
- Autoantibodies are antibodies directed against antigens
expressed on one’s own RBCs and generally react with
all RBCs tested.
What are Clinically Significant Antibodies?
▪ Making an alloantibody is important, but only certain
antibodies can cause clinical harm to a patient. We call such
antibodies “clinically significant” because they may either
lead to destruction (i.e., hemolysis) of transfused red blood
cells or because they may harm a fetus or newborn when
a mother carries an alloantibody against an antigen on the
baby’s red cells.
▪ These antibodies are typically IgG antibodies that react best
at 37°C and the antihuman globulin (AHG) phase of the
indirect antiglobulin test (IAT).
Blood Group Antibodies:
▪ They can be categorized as follows:
o Group I: Clinically significant antibodies
▪ ABO (A, B, A,B)
▪ Rh (D, C, c, E, e)
▪ Kell (K, k, Jsa, Jsb, Kpa, Kpb)
▪ Duffy (Fya, Fyb)
▪ Kidd (Jka, Jkb)
▪ MNS (S, s)
o Group II: Benign antibodies
▪ Xg (Xga)
▪ HTLA (high-titer, low-avidity)
• Chido/Rodgers (Cha/Rga)
• Knops (Csa, Kna, McCa, Yka)
▪ HLA (Bga, Bgb, and Bgc)
▪ John Milton Hagen (JMH)
o Group III: Clinically insignificant if not reactive at 37°C;
possibly significant when reacting at 37°C
▪ Lewis (Lea–rarely significant, Leb–insignificant)
▪ P (P1–insignificant)
▪ MNS (M–rarely significant, N–insignificant)
▪ Lutheran (Lua, Lub)–rarely significant
▪ ABO (A1)
o Group IV: Antibodies that are sometimes clinically
significant
▪ Cartwright (Yta)
▪ Vel
▪ Gerbich
▪ Dombrock (Gya, Hy)
▪ Sid (Sda)
What is an Antibody Identification Panel?
▪ Antibodies to blood group antigens must be detected and
their clinical significance assessed. Clinically significant
antibodies have been associated with red blood cell
destruction, hemolytic transfusion reactions, and hemolytic
disease of the fetus and newborn (HDFN).
▪ When an unexpected antibody is detected in the antibody
screen, an antibody identification panel is performed to
determine the specificity of the antibody (or antibodies)
present.
▪ Once identified, the antibody’s clinical significance can be
ascertained and the appropriate transfusion considerations
put into place.
▪ The FDA mandates that red blood cells used in antibody
detection tests must express the following antigens: D, C,
E, c, e, K, k, Fya, Fyb, Jka, Jkb, Lea, Leb, P1, M, N, S, and s.
ANTIBODY DETECTION METHODS:
Identification of RBC antigens and antibodies has been the basis
of pretransfusion compatibility testing and the safe transfusion
practices used today and also can provide insights into
understanding the etiology of hemolytic disease of the fetus and
the newborn.
Indirect Antihuman Globulin Test (IAT, Tube Method):
▪ Indirect Coombs’ test (tube method) is the traditional
testing method to detect clinically significant IgG antibodies.
▪ Principle:
The patient’s serum (containing the unexpected antibody or
antibodies) is mixed with reagent red blood cells that have
known antigen content. The test mixture is then incubated
at 37°C. During the incubation phase, IgG antibodies will
bind to (sensitize) any reagent antibodies that possess the
target antigen if present in the patient's serum. An
enhancement reagent may be added before incubation to
increase the degree of sensitization. After adequate
incubation, the tubes are centrifuged and observed for
agglutination and/or hemolysis (which are considered
positive results for IAT).
▪ Steps or Phases of IAT/Indirect Coombs’ Test:
1. Immediate Spin Phase:
- Inclusion of this phase in the IAT is optional.
- It is used to detect antibodies that optimally react
at room temperature or colder (i.e., clinically
insignificant cold-reacting antibodies).
- An enhancement reagent (e.g., LISS, PEG, or BSA)
may be added at this phase before proceeding to
the incubation phase.
2. 37°C Incubation Phase:
- It is used to detect warm-reacting clinically
significant IgG antibodies.
- During this phase, specific warm-reacting IgG
antibodies bind to its corresponding antigen found
on the reagent RBC.
3. AHG Phase:
- It involves the addition of the Coombs’ reagent.
- If the RBCs are coated with IgG antibodies, the
anti-IgG antibody in the AHG reagent will create a
bridge between sensitized reagent RBCs, resulting
in observable agglutination.
- If there are no antibodies directed against any of
the antigens present on the reagent RBCs, the
RBCs will not be sensitized, and there will be no
agglutination.
▪ Coombs’ Test Quality Control:
- All negative IAT and DAT tests must have Coombs’
control cells (IgG-coated group O, Rh-positive RBCs;
also known as check cells) added to confirm the
validity of the negative result.
- When an IAT or DAT is negative, the anti-human
globulin (AHG) reagent is free in the tube (it did find
any coated RBCs with which to bind).
- Coombs control cells (check cells) are added. The free
AHG should attach to the IgG-coated check cells and
give a visible agglutination after centrifugation.
- This required step ensures that the AHG reagent was
added and is working properly.
▪ Disadvantages of the Conventional IAT (Tube Method):
- Instability of the reactions;
- Subjective nature of grading by the medical
technologist;
- Amount of hands-on time for the technologist; and
- Problems related to the failure of the washing phase to
remove all unbound antibodies.
Column Agglutination Technology:
▪ Red blood cells and the patient’s serum are allowed to
interact in a chamber that has an air bubble barrier to keep
unbound plasma from neutralizing the antiglobulin reagent
that is located in the lower gel column.
▪ Because unbound plasma and antiglobulin reagent are
prevented from mixing, antiglobulin control cells are not
needed to confirm the presence of antiglobulin reagent in
negative tests.
▪ IAT Column Agglutination / Gel Test Principle:
The gel test, which is performed in a specially designed
microtube, is a hemagglutination test based on controlled
centrifugation of RBCs through a dextran-acrylamide gel
that contains pre-dispensed reagents. Each microtube is
composed of an upper reaction chamber that is wider than
the tube itself and a long, narrow portion referred to as the
column. In the gel test, a plastic card with microtubes is
used instead of test tubes. A gel card consists of six or eight
columns. Each microtube contains pre-dispensed gel,
diluent, and reagents, if applicable. Measured volumes of
RBCs and serum or plasma are dispensed into the reaction
chamber of the microtube. If appropriate, the card is
incubated and then centrifuged.
The reaction chamber is where RBCs may be sensitized
(antigen-antibody binding) during incubation. The column
of each microtube contains dextran-acrylamide gel particles
suspended in a diluent with reagents added, if applicable.
The shape and length of the column provide a large surface
area for prolonged contact of the RBCs with the gel particles
during centrifugation. The gel particles are porous, and they
serve as a reaction medium and filter, sieving the RBC
agglutinates according to size during centrifugation. Large
agglutinates are trapped at the top of the gel and are not
allowed to travel through the gel during the centrifugation
of the card. Agglutinated RBCs remain trapped in the gel,
while non-agglutinated RBCs travel unimpeded through the
length of the microtube, forming a pellet at the bottom
following centrifugation.
▪ Interpretation of Result:
Agglutination reactions in the gel test are graded similar to
the reactions in the test tube hemagglutination technique.
Illustration Grading Description
A negative reaction is characterized
by non-agglutinated RBCs forming
0 a well-delineated pellet at the
bottom of the gel column.
Characterized by red cell
agglutinates that are predominantly
1+ observed in the lower half of the
gel column.
Characterized by red cell
agglutinates that are dispersed
throughout the entire length of the
2+ gel column. Few agglutinates may
be observed at the bottom of the
gel column.

Characterized by red cell
agglutinates that are predominantly
3+ observed in the upper half of the
gel column.
Characterized by a solid band of
4+ red cell agglutinates on the top
portion of the gel column.
A mixed-field (mf) reaction is
characterized by a band of red cell
agglutinates on the top of the gel
mf column, accompanied by a pellet of
non-agglutinated red cells at the
bottom of the gel column.
▪ Before interpreting a reaction as “mixed-field,” the clinical
history of the patient should be considered. For example,
recently transfused patients or bone marrow transplant
recipients are expected to have mixed populations of RBCs,
and their RBCs commonly produce mixed-field reactions.
▪ Hemolysis is seen as a clear red liquid in the chamber.
▪ Applications of the Column Agglutination / Gel Test:
- ABO forward and reverse grouping
- Rh typing
- Direct Coombs’ Test/DAT
- Indirect Coombs’ Test/IAT/Antibody screen
- Antibody identification
- Antibody titration
- Antigen typing
- Compatibility testing
▪ Advantages of the Column Agglutination / Gel Test:
- Simple, standardized procedure; no washing steps,
and no need for antiglobulin control cells.
- There is no need to shake or resuspend the RBC
pellet/button following centrifugation.
- Provides well-delineated endpoints of the agglutination
reaction that can be observed or reviewed for up to
three days.
- More objective, consistent, and reproducible
interpretation of the test results.
- Offers improved productivity when compared to
traditional tube testing, especially when combined with
automation.
▪ Disadvantages of the Column Agglutination / Gel Test:
- Sample restrictions and the need for special
equipment.
- Hemolyzed or grossly icteric blood samples should not
be used because the results may be difficult to
interpret visually.
- Grossly lipemic samples containing particulates may
clog the column, as indicated by diffuse blotches of red
blood cells. Such samples must be clarified by
centrifugation or filtration and retested.
- Rouleaux formation caused by serum or plasma with
abnormally high concentrations of protein (such as in
patients with multiple myeloma or Waldenström’s
macroglobulinemia or from patients who have received
plasma expanders of high molecular weight) may
produce hazy reactions or false-positive result.
- Centrifuging serum that is not completely clotted may
cause the formation of fibrin in the serum, which may
render the sample unusable for testing and produce a
false-positive result.
Solid-Phase Red Cell Adherence (SPRCA) Technology:
▪ In SPRCA, one of the test reactants (either antigen or
antibody) is bound to a solid support (usually a microplate
well that is made of polystyrene plastic) before the test is
started.
▪ Principle:
The patient’s serum or plasma and low-ionic-strength saline
(LISS) are added to microwells that are pre-coated with the
target antigen (RBC stroma). The microplates are incubated
at 37°C, allowing time for possible antibodies to attach to
the antigens in the well. After incubation, the wells are
washed with pH-buffered isotonic saline to remove
unbound serum proteins. Next, anti-IgG–coated indicator
RBCs are added, and the microwells are centrifuged.
Centrifugation forces the indicator RBCs into close contact
with IgG antibodies (from the test serum/plasma) that are
bound to the immobilized target antigen, that is, the
sandwich technique. If the antibody is attached to the
antigen during the incubation phase, the indicator cells form
a monolayer of RBCs. If no antibody is present, nothing is
attached to the antigen, and the indicator cells form a clearly
delineated button at the center of the microplate well during
centrifugation.
▪ Advantages of the SPRCA Technology:
- SPRCA provides stable, well-defined endpoints, which
makes cross-training of technologists with rotating
schedules easy.
- No predilution of reagents is required, and it is possible
to test hemolyzed, lipemic, or icteric samples.
- LISS in the test process provides enhanced sensitivity
for detecting weak antibodies.
- Because the reagent red blood cells in SPRCA are dried
and precoated onto the surface of the wells, the test
plates have a long shelf life.
▪ Disadvantages of the SPRCA Technology:
- The need for specialized equipment: a microplate
centrifuge, a 37°C incubator for microplates, and a light
source for reading the final results.
- The user’s technique affects the outcome of the
testing, especially the manual handling of the test
strips.
- The increased sensitivity may also be a disadvantage
inasmuch as SPRCA may detect weak autoantibodies
that other systems miss.
Positive reaction Negative reaction

RED BLOOD CELL (RBC) REAGENTS:
Overview:
▪ Blood group antigens are structures on the outer surface of
human red blood cells (RBCs) that can be recognized by the
immune system of individuals who lack that particular
structure.
▪ RBC reagents used in antibody screen testing come from
group-O individuals who have been typed for the most
common and the most significant blood group antigens:
o Rh (D, C, c, E, e)
o Kell (K, k, Jsa, Jsb, Kpa, Kpb)
o Duffy (Fya, Fyb)
o Kidd (Jka, Jkb)
o Lewis (Lea, Leb)
o P (P1)
o MNS (M, N, S, s)
▪ The US-FDA mandates that red blood cells used in antibody
detection tests must express the following antigens: D, C,
E, c, e, K, k, Fya, Fyb, Jka, Jkb, Lea, Leb, P1, M, N, S, and s.
▪ Group O cells are used so that the anti-A and anti-B
isoagglutinins will not interfere in the detection of
antibodies to other blood group systems.
Variation in Expression of RBC Blood Group Antigens:
▪ RBCs from individuals who are homozygous for an allele
typically have a greater number of antigen sites than do
RBCs from individuals who are heterozygous.
Consequently, their RBCs can react more strongly with
antibodies. This difference in expression and antigen-
antibody reactivity because of zygosity is known as dosage.
▪ Common blood group antigens that show dosage:
o Rh (except D antigen) o MNSs
o Kidd o Lutheran
o Duffy
▪ Antibodies showing dosage may not be detected if none of
the screen cells have a homozygous expression of the
target antigen.
▪ Antithetical antigens commonly show a dosage effect.
o Antithetical is a term used to describe a pair (and
occasionally more than a pair) of antigens that are
coded by different alleles of a single gene. The
products of allelic genes, therefore, are referred to as
antithetical antigens.
o Examples:
o Rh: C and c; E and e
o MNSs: M and N; S and s
o Kidd: Jka and Jkb
▪ Dosage is less obvious with the following antigens:
o Rh: D
o Kell: K and k
o Lutheran: Lua and Lub
▪ Dosage within the Duffy system also may not be
serologically obvious because Fy(a+b–) or Fy(a–b+)
phenotypes are seen in either homozygous (FyaFya or
FybFyb) or hemizygous (FyaFy or FybFy) individuals.
▪ Haplotype pairings and gene interaction (either cis or trans)
also can affect phenotypic expression. For example:
o In the Rh system:
- The pairing of RHCE*C in trans position to RHD
can result in a weak expression of D antigen.
- RHCE*E in cis position with RHD is associated with
a strong expression of D antigen.
- Among the common Rh phenotypes, R2R2 RBCs
carry the strongest expression of D antigen.
o In the Kell system:
- Kpa is associated with weakened expression of in
cis k and Jsb antigens.
The expression of blood group antigens can be homozygous or
heterozygous.
▪ Homozygous Expression:
o A cell with homozygous antigen expression is from an
individual who inherited only one allele at a given
genetic locus.
o Their red cell surface has a “double dose” of that
antigen. Thus, they typically have a greater number of
antigen sites than do RBCs from individuals who are
heterozygous.
o Allows for the detection of antibodies to the blood
group antigens that may show dosage.
o For example, RBCs from a homozygous MM individual
carry a double dose of M antigen and react more
strongly with anti-M than do RBCs from an MN
heterozygous individual carrying only a single dose of
M antigen.
o Certain antibodies, such as those of the Kidd system,
may only be detected when tested against RBCs
expressing one allele (homozygous).
o The following are examples of phenotypes with a
homozygous expression:
✓ Kidd:
o Jk(a+b–) → homozygous for Jka only
o Jk(a–b+) → homozygous for Jkb only
✓ Duffy:
o Fy(a+b–) → homozygous for Fya only
o Fy(a–b+) → homozygous for Fyb only
✓ MNSs:
o M+ N– → homozygous for M only
o M– N+ → homozygous for N only
▪ Heterozygous Expression:
o A cell with heterozygous antigen expression is from an
individual who inherited two different alleles at a given
genetic locus (i.e., a pair of chromosomes possess
different alleles).
o When this occurs, the alleles “share” the available
antigen sites on the cell surface. Thus, their RBCs react
weakly with antibodies.
o The following are examples of phenotypes with a
heterozygous expression:
✓ Kidd: Jk(a+b+) → expression of both Jka and Jkb
✓ Duffy: Fy(a+b+) → expression of both Fya and Fyb
✓ MNSs: M+ N+ → expression of both M and N
Reagent Screen Cells:
▪ Reagent Screen Cells for PATIENT Antibody Screening:
o The screen cells used for patient antibody screening
are manufactured as a set and are comprised of two
(R1R1 and R2R2) or three (R1R1, R2R2, rr) cells, each
having a unique combination of antigens.
o A pooled (mixed or combined) RBC screening reagent
cannot be used for patient antibody screening.
- Since pooled reagent RBCs contain more than
one RBC population, reactions should be
carefully observed for mixed-field
agglutination, as only one RBC in the pool may
express the target antigen.
▪ Reagent Screen Cells for DONOR Antibody Screening:
o For blood donor antibody screening, it is acceptable to
use a pooled screening reagent that contains RBCs
from at least two different individuals.
INTERPRETATION OF RESULTS:
▪ A positive test result (i.e., agglutination or hemolysis) at any
phase of testing (immediate spin, 37°C, or AHG) indicates
the need for antibody identification studies.
▪ Evaluation of antibody screen results, including the
autologous control if tested at this time, and patient history
(i.e., age, sex, race, diagnosis, pregnancy and transfusion
history, current medications, and intravenous solutions)
can provide clues and give direction for identification and
resolution of the antibody or antibodies present.
▪ The medical technologist should consider the following
conditions in complex cases:
A. Optimum reaction temperature or IAT phase(s) of the
antibody (or antibodies) involved
o Antibodies of the IgM class react best at room
temperature or colder and are capable of causing
agglutination of saline-suspended RBCs
(immediate spin reaction).
o Antibodies of the IgG class react best at the 37°C
and AHG phases.
o Refer to the table on the next page (Serologic
Activity of Some Common Blood Group
Antibodies).
B. The reaction of the autologous control (auto-control)
o The autologous control is the patient’s own RBCs
tested against the patient’s serum in the same
manner as the antibody screen.
o A positive antibody screen and a negative
autologous control indicate that an alloantibody
has been detected.
o If the autologous control is positive, transfusion
history needs to be evaluated.
o If the patient has not been transfused within the
last 3 months, a positive autologous control may
indicate the presence of autoantibodies or
antibodies to medications.
o If the patient has been recently transfused (i.e.,
within the previous 3 months), the positive
autologous control, which may exhibit a mixed-
field appearance, may be caused by alloantibodies
coating the circulating donor RBCs.
o Evaluation of samples with a positive autologous
control or positive direct antiglobulin test (DAT)
result is often complex and may require a great
deal of time and experience on the part of the
investigator.
o Autocontrol should be included when performing
antibody identification studies.
C. Positive reaction with one or more screen cells
o More than one screen cell may be positive when
the patient has multiple antibodies, when a single
antibody’s target antigen is found on more than
one screen cell, or when the patient’s serum
contains an autoantibody.
o A single antibody specificity should be suspected
when all screen cells yielding a positive reaction
react at the same phase(s) and strength. Multiple
antibodies are most likely present when screen
cells react at different phases or strengths.
o Autoantibodies should be suspected when the
autologous control or DAT is positive and all
screen cells tested yield a positive reaction.
D. Presence of hemolysis or mixed-field agglutination
o Certain antibodies, such as anti-Lea, anti-Leb, anti-
PP1Pk, and anti-Vel, are known to cause in vitro
hemolysis in the presence of complement.
o Mixed-field agglutination is associated with anti-
Sda and Lutheran antibodies.
E. True agglutination versus pseudo-agglutination caused
by rouleaux formation
o Serum from patients with altered albumin-to-
globulin ratios (e.g., patients with multiple
myeloma, Waldenström’s macroglobulinemia) or
those who have received high molecular weight
plasma expanders (e.g., dextran) may cause
nonspecific aggregation of RBCs, known as
rouleaux.
o Rouleaux is not a significant finding in antibody
screening tests; however, it is easily confused with
antibody-mediated agglutination.
o Knowing the following characteristics of rouleaux
helps in differentiating between rouleaux and
agglutination:
✓ Cells have a “stacked coin” appearance when
viewed microscopically.
✓ Rouleaux is observed in all tests containing
the patient’s serum, including the autologous
control and the reverse ABO grouping.
✓ Rouleaux does not interfere with the AHG
phase of testing in the tube method or solid-
phase technology because the patient’s
serum is washed away before adding the AHG
reagent.
✓ Unlike agglutination, rouleaux disperses by
adding one to three drops of normal saline
solution (NSS) to the test tube when
performing tube testing.
 SEROLOGIC ACTIVITY OF SOME COMMON CLINICALLY SIGNIFICANT (AND INSIGNIFICANT) BLOOD GROUP ANTIBODIES

Antibody Reactivity Papain/ DTT Associated with

Antibody
Class 4°C 22°C 37°C AHG Ficin (200 mM) HDFN HTR
Anti-M IgG > IgM Most Most Rare Sensitive Resistant Rare Rare
Anti-N IgM > IgG Most Most Rare Sensitive Resistant No Rare
Anti-S IgG > IgM Most Most Variable Resistant Yes Yes
Anti-s IgG > IgM Most Variable Resistant Yes Yes
Anti-U IgG Most Resistant Resistant Yes Yes
IgM
Anti-P1 Most Most Most Resistant Resistant No Rare
(IgG rare)
IgG > IgM
Anti-D Some Some Most Resistant Resistant Yes Yes
(IgA rare)
Anti-C IgG > IgM Some Some Most Resistant Resistant Yes Yes
Anti-E IgG > IgM Some Some Most Resistant Resistant Yes Yes
Anti-c IgG > IgM Some Some Most Resistant Resistant Yes Yes
Anti-e IgG > IgM Some Some Most Resistant Resistant Yes Yes
Resistant or
Anti-Lua IgM > IgG Most Most Variable No Mild
weakened
Resistant or
Anti-Lub IgG > IgM Some Most Variable No Mild
weakened
Anti-K IgG > IgM Some Most Resistant Sensitive Yes Yes
Anti-k IgG > IgM Most Resistant Sensitive Yes Yes
Anti-Kpa IgG Most Resistant Sensitive Yes Yes
Anti-Kpb IgG > IgM Most Resistant Sensitive Yes Yes
Anti-Jsa IgG > IgM Most Resistant Sensitive Yes Yes
Anti-Jsb IgG Most Resistant Sensitive Yes Yes
Anti-Lea IgM > IgG Most Most Most Most Resistant Resistant No Rare
Anti-Leb IgM > IgG Most Most Most Most Resistant Resistant No No
Anti-Fya IgG > IgM Most Sensitive Resistant Yes Yes
Anti-Fyb IgG > IgM Most Sensitive Resistant Yes Yes
Anti-Jka IgG > IgM Most Resistant Resistant Rare Yes
Anti-Jkb IgG > IgM Most Resistant Resistant Rare Yes

HDFN: hemolytic disease of the fetus and newborn; HTR: hemolytic transfusion reaction; AHG: antihuman globulin; DTT: Dithiothreitol

ANTIBODY IDENTIFICATION
The following information concerning the patient’s history may
Overview of Pretransfusion Compatibility Testing: provide valuable clues in antibody identification studies,
▪ Several universal procedures in immunohematology apply particularly in complex cases:
these principles in the testing of patient samples before ▪ Age and sex
transfusion with blood products containing red cells or in the ▪ Race
testing of donor samples: ▪ Clinical diagnosis (e.g., infections, autoimmune diseases)
1. ABO and Rh typing for the detection of the A, B, and D ▪ Blood transfusion and pregnancy history
antigens. ▪ Medications and intravenous solutions administered
2. ABO serum/plasma testing for the detection of ABO
antibodies, anti-A, and anti-B. Examples of Antibody Screen Results with Possible Causes
3. Determination of the presence or absence of red cell
antigens from other blood group systems in both patient Screen Cell IS 37°C AHG
and donor samples (e.g., testing a donor unit for the C, SC I 0 0 0
E, c, or e antigens of the Rh blood group system). SC II 0 0 2+
4. Antibody screen (antibody detection) for the detection of Autocontrol 0 0 0
preformed antibodies to red cell antigens as a result of
previous exposure to red cells through transfusion and Possible interpretation:
pregnancy. • Single alloantibody
5. Antibody identification for determination of the red cell • Two alloantibodies, antigens only present on the SC II
antibody specificity after detection with the antibody • Probably an IgG alloantibody
screen.
6. Crossmatch for the serologic check of the donor unit and
patient compatibility before a transfusion. Screen Cell IS 37°C AHG
SC I 0 1+ 3+
▪ Once an antibody has been detected in the antibody screen, SC II 0 0 1+
additional testing/review is necessary to identify the antibody Autocontrol 0 0 0
and determine its clinical significance.
▪ Clinically significant antibodies are warm (IgG) antibodies Possible interpretation:
associated with hemolytic transfusion reactions (HTR) and • Multiple alloantibodies
hemolytic disease of the fetus and newborn (HDFN). • Single alloantibody that demonstrates dosage
• Probably an IgG alloantibody
 Screen Cell IS 37°C AHG STEPS IN ANTIBODY IDENTIFICATION:
SC I 1+ 0 0
SC II 2+ 0 0 1. Review the patient’s medical history (e.g., history of
Autocontrol 0 0 0 previous blood transfusions, pregnancy, therapeutic drugs,
or medications taken).
Possible interpretation:
• Single or multiple alloantibodies
2. Interpret the ABO (forward and reverse typing) and Rh
• Probably an IgM alloantibody
typing results.

3. Review the antibody screening (IAT) result.

Screen Cell IS 37°C AHG
SC I 2+ 0 1+ 4. If the antibody screening result is positive, then proceed to
SC II 3+ 1+ 2+ the next step, which is antibody identification.
Autocontrol 0 0 0
5. Interpret or review the antibody panel results for positive
Possible interpretation: and negative results:
• Multiple alloantibodies, warm (IgG) and cold (IgM) 5.1. See whether there are reactions at the immediate spin
• Potent cold (IgM) alloantibody binding to complement in phase – a positive reaction is most likely caused by an
polyspecific AHG reagent (Anti-IgG/C3b/C3d) IgM antibody, which optimally reacts at room
temperature or colder (4°C).
✓ Anti-Lea and Anti-Leb
Screen Cell IS 37°C AHG ✓ Anti-M, Anti-N
SC I 0 0 1+ ✓ Anti-Lua
SC II 0 0 1+ ✓ Anti-P1
Autocontrol 0 0 0 5.2. See whether there are reactions at the thermal or
incubation phase – a positive reaction is most likely
Possible interpretation: caused by an IgG antibody, which optimally reacts at
• Single warm (IgG) alloantibody, an antigen is present on warmer temperatures (37°C).
both screening cells ✓ Potent cold (IgM) antibodies (especially those
• Antibody to a high-prevalence antigen may be present causing hemolysis)
o High-prevalence antigens are those that are present in ✓ Some warm (IgG) antibodies, if high in titer
almost all individuals. (e.g., Anti-D, Anti-E, Anti- K)
o Suspect an antibody to a high-prevalence antigen when 5.3. See whether there are reactions at the antihuman
all screen and panel cells are positive, with reactions in globulin (AHG) phase – a positive reaction is most likely
the same phase and at the same strength, along with caused by the presence of unbound antibodies in the
a negative autocontrol. patient’s serum.
• Complement binding by a cold alloantibody not detected at ✓ Anti-Rh (D, C, E, c, e)
immediate spin (IS) phase ✓ Anti-S and Anti-s
✓ Anti-Lub
✓ Anti-Kell (K, k, Jsa, Jsb)
✓ Anti-Duffy (Fya and Fyb)
Screen Cell IS 37°C AHG
✓ Anti-Kidd (Jka and Jkb)
SC I 0 0 3+
6. Exclusion technique: Rule out or cross out the antibodies
SC II 0 0 3+
that could NOT be responsible for the reactivity seen.
Autocontrol 0 0 3+ 6.1. To perform exclusions or “rule-outs,” the RBCs that
gave a NEGATIVE reaction in all phases of testing are
Possible interpretation: examined; the antigens on these negatively reacting
• Warm alloantibodies cells are probably not the antibody’s target.
• Transfusion reaction 6.2. Perform this rule-out technique only if there is a
• Probably a warm (IgG) alloantibody homozygous expression of the antigen on the cell. This
• A warm (IgG) autoantibody may be present → 3+ reaction avoids excluding a weak antibody that is showing
with the autologous control dosage.

Common blood group antigens that show dosage:

THE ANTIBODY IDENTIFICATION PANEL
▪ An antibody identification panel is a collection of 11 to 20
group O reagent RBCs, with various antigen expressions of
known specificity or antigenicity.
▪ This is used during an Antibody Screening Test.
o It detects alloantibodies or autoantibodies to non-ABO
red blood cell antigens (e.g., Rh, MNSs, Kell, Duffy,
Kidd, Lewis, Lutheran, etc.) in the recipient's serum or
plasma.
▪ When an antibody screen is POSITIVE: further identification
of the specificity of any antibody detected (using panels of
red cells of known antigenicity) makes it possible to test
donor blood for the absence of the corresponding antigen.
▪ Primary response to first antigen exposure requires 20 to
120 days; antibody is largely IgM (small quantity of IgG).
▪ Secondary response requires 1 to 14 days.
✓ Rh (only C, E, c, e – except D and Cw antigens)
✓ Kidd (Jka, Jkb)
✓ Duffy (Fya, Fyb)
✓ MNSs
✓ Lutheran (Lua, Lub)
6.3. Exceptions are made for low-prevalence antigens that
are rarely expressed homozygously, such as K, Kpa,
Jsa, and Lua.
7. Inclusion technique: After each negatively reacting cell has
been evaluated, the remaining antigens that were not
excluded should be examined to see if the pattern of
reactivity matches a pattern of antigen-positive cells.
 Reagent RBC Antigenic Profile / Phenotype (provided by the manufacturer; LOT-specific)

Reagent Panel RBCs

Test Phases (IS, 37°C, AHG) and Coombs’ Control

Patient’s RBC Antigenic Profile / Phenotype

ADDITIONAL TECHNIQUES FOR RESOLVING ANTIBODY
IDENTIFICATION
There may be times when the initial antibody identification panel
does not reveal a clear-cut specificity. When multiple
specificities remain following the exclusion and inclusion
process, additional testing is necessary.
1. Selected Cell Panels
▪ The simplest step to take is to test additional cells from
a different panel or panels.
▪ The cells selected for testing should have minimal
overlap in the antigens they possess.
▪ Selected cell panels are also useful when a patient has
a known antibody and the technologist is attempting to
determine if additional antibodies are present.
2. The Use of Proteolytic Enzymes
▪ Proteolytic enzymes (ficin, papain, bromelin, and
trypsin) modify the RBC surface by removing sialic acid
residues and by denaturing or removing glycoproteins.
▪ This modification results in the destruction of certain
antigens and the enhanced expression of others.
▪ The use of enzyme-treated panel cells may aid in the
identification process when multiple antibodies appear
to be present or help narrow down possible
specificities when a high-prevalence antibody appears
to be present.
▪ Effect of proteolytic enzymes (at 37°C) on select
antigen-antibody reactions:
Markedly Enhanced:
✓ ABO system: Anti-A, Anti-B, Anti-A,B, and Anti-A1
✓ H system: Anti-H
✓ P system: Anti-P, Anti-P1, and Anti-PP1Pk
✓ Rh system: Anti-D, Anti-C, Anti-E, Anti-c, Anti-e,
and Anti-Cw
✓ Lewis: Anti-Lea and Anti-Leb
✓ I system: Anti-I and Anti-i
✓ Vel system: Anti-Vel
Enhanced:
✓ Kidd system: Anti-Jka and Anti-Jkb
✓ Dombrock system: Anti-Doa and Anti-Dob
Unaffected to Enhanced:
✓ Kell system: Anti-Jsa and Anti-Jsb
Unaffected:
✓ MNS system: Anti-U
✓ Kell system: Anti-K, Anti-k, Anti-Kpa, and Anti-Kpb
✓ Diego system: Anti-Dia, Anti-Dib, Anti-Wra
✓ Colton system: Anti-Coa and Anti-Cob
✓ Cost system: Anti-Csa and Anti-Csb
Unaffected to Weakened:
✓ Lutheran system: Anti-Lua and Anti-Lub
Weakened:
✓ Chido/Rodgers system: Anti-Ch1 and Anti-Rg1
✓ Knops system: Anti-Kna, Anti-McCa, and Anti-Yka
Denatured:
✓ MNS system: Anti-M and Anti-N
✓ Duffy system: Anti-Fya and Anti-Fyb
✓ Xg system: Anti-Xga
Variable Reaction:
✓ MNS system: Anti-S and Anti-s
✓ Cartwright system: Anti-Yta and Ytb
3. Neutralization Test
▪ Substances in the body and in nature have antigenic
structures similar to certain RBC antigens.
▪ Consequently, these substances can be used to
neutralize the corresponding RBC antibodies in serum,
allowing for the exclusion of the remaining antibodies
or confirmation that a particular antibody is present.
▪ This technique is especially helpful when multiple
antibodies are suspected and one of the suspected
antibodies appears to be an antibody that can be
neutralized.
▪ To perform neutralization, the patient’s serum is first
incubated with the neutralizing substance, allowing the
soluble antigens in that substance to bind with the
corresponding antibody, if present.
Sources of Neutralizing Substances:
✓ Anti-P1: hydatid cyst fluid, pigeon droppings, or
turtledoves’ egg white
✓ Anti-Lea/Anti-Leb: plasma, serum, or saliva
✓ Anti-Ch1/Anti-Rg1: serum (has complement)
✓ Anti-Sda: urine
✓ Anti-I: human breast milk
▪ An antibody identification panel is then performed
using the treated serum.
▪ Use of a control (saline and serum) is also required to
prove that loss of reactivity is due to neutralization and
not to dilution of the antibody by the added substance.
4. Adsorption Tests
▪ Adsorption is used by medical technologists in the
blood bank in order to bind autoantibodies to red blood
cells in order to remove them from the plasma and
better analyze the antibodies that might remain behind.
▪ Antibodies may be removed or adsorbed from serum
by incubating the specimen with the corresponding
antigen, thus allowing the antibody to bind to that
antigen.
▪ The adsorbent used in an adsorption procedure is
typically RBCs but may be another antigen-bearing
substance. In the adsorption method, the antigen-
antibody complex is composed of solid precipitates
and is separated from the adsorbed serum by
centrifugation.
▪ Once the desired antibody has been completely
adsorbed from the specimen, the adsorbed serum can
be tested against RBC panel cells for the presence of
non-adsorbed alloantibodies.
▪ Autoantibodies are commonly removed using RBC
adsorption techniques so that any underlying, clinically
significant antibodies can be detected. When
autoantibodies are present,
▪ RBC adsorptions can be performed using allogeneic or
autologous RBCs; autologous RBCs can only be used
when the patient has not been transfused within the
last 3 months (donor cells may adsorb alloantibodies)
and the volume of RBCs is adequate.
▪ RBCs used to perform autoantibody adsorption can be
enzyme-treated prior to adsorption to enhance
adsorption of the autoantibody.
▪ Human platelet concentrate is used to adsorb Bg-like
antibodies from the serum.
5. Elution Methods
▪ In general, to remove or extract one material from
another. In the blood bank practice, the term refers to
removing (or “dissociating”) an antibody that is
attached to the surface of a red blood cell.
i. Altering the reaction temperature (warm/cold Ab)
ii. Altering the pH (i.e., glycine acid solution, pH 3)