Detection & Identification of Antibodies

Topic Content:
- Introduction
- Antibody Screen
1. Tube Method
2. Gel Method
3. Solid Phase Adherence Method
4. Interpretation
5. Limitations
- Antibody Identification
1. Patient History
2. Reagents
3. Exclusion
4. Evaluation of Panel Results
- Additional Techniques for Resolving Antibody Identification
1. Selected Cell Panels
2. Enzymes
3. Neutralization
4. Adsorption
- Direct AGT and Elution Techniques
1. Temperature-Dependent Methods
2. pH
3. Organic Solvents
- Antibody Titration
- Providing Compatible Blood Products
Introduction:
 The detection of antibodies directed against RBC antigen is critical in pretransfusion
compatibility testing
 Principal tool in investigating potential HTRs, IHA, and HDFN
 Antibody detection method focuses on detecting unexpected antibodies like:
o Immune alloantibodies – produced in response to RBC stimulation through
transfusion, transplantation, or pregnancy
o Natural occurring antibodies – form as exposure to foreign substances such as
pollen, fungus, or bacteria
o Passively acquired antibodies – antibodies produced in an individual then
transmitted to another (e.g.) occurs when a baby receives a mother's antibodies
through the placenta or breast milk. It can also occur when a person receives an
injection of antibodies to protect against the effects of a toxin such as snake
venom. Other example is “IVIG or Rhogam”.
 Clinically significant antibodies – IgG antibody reacts at 37˚C or AHG phase in IAT; cause
decreased survival due to RBC possesses target antigen
 Autoantibodies – directed against own self; may mask the presence of clinically
significant antibody (reacts all tested RBCs)
 Detection —> Antibody identification panel —> Transfusion consideration

Antibody Screen:
 AABB’s Standards for Blood Banks and Transfusion Services – to detect clinically
significant antibodies in both blood donor and recipient as part of pretransfusion
compatibility testing.
 Only a small percentage of population (0.2% and 2%) has detectable RBC antibodies
that’s why it requires careful and proper screening.
 Also evaluates the risk of
HDFN and to assess the
candidate for RHIG prophylaxis
 How to perform using Tube
Method?
o Traditional method for
detection is IAT
performed in test tube

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 o Degree of reactivity: 0 (no agglutination), w+ (agglutination barely visible to
naked eye), to 4+ (one solid agglutinate)
o All negative test will add Coombs’ control cells (check cells “Group O+ coated
with anti-D) to confirm negative result
o Used for sensitization: RBC reagents, Enhancement reagents, and AHG reagents
= formation of visible agglutinates
o RBC Reagents – group O cells are used so that anti-A and anti-B will not interfere
in the detection of antibodies to other BGS. Suspended in 2 and 5% preservative
diluent (maintains antigen’s integrity and prevents hemolysis)
 BGS with antibodies that exhibit dosage effects – Duffy, Rh (except D),
Lutheran, Kidd, and MNSs, (DR. Lu KiMNSs)
o Enhancement Reagents – “potentiators” increases sensitivity (reduces zeta
potential); add to cell/serum mixture before 37˚C incubation phase
 22% Albumin – reducing zeta potential and dispersing the charges = RBCs
contacts together and increases agglutination
 LISS – contains glycine in albumin sol. Increases uptake of antibody onto
RBC during sensitization phase = increases agglutination
 PEG – removes water in LISS sol = concentrating antibodies present and
increases RBC sensitization. Centrifugation after 37˚C incubation is not
performed due to nonspecific aggregation. Not use for elevated plasma
proteins (Multiple Myeloma).
o AHG Reagents – allows incomplete antibodies to agglutinate. Contains
antibodies to both IgG and complement components either C3 and C4 or C3b
and C3d (polyspecific). Any negative test after adding AHG reagent should be
controlled using Coomb’s control cells (check cells; O+ coated with anti-D) +
Reacts with anti-IgG = visible agglutination; proves that washing is adequate,
AHG reagents was added to tube, and AHG reagent is working properly. If fail to
agglutinate, perform antibody screen again (beginning). Disadvantage is
instability of reactions. Advantage is relative low cost.
 How to perform using Gel Method?
o A microtubule filled with a dextran acrylamide gel (controlled centrifugation red
cells)
o Suspended in LISS of 0.8%
o Patient’s serum or plasma specimen and screen cells are added to reaction
chamber that sits above the gel (up to 6 chamber contained in a plastic card;
about a size of credit card)
o Incubated at 37˚C for 15 mins – 1 hour and allow to sensitized then after,
centrifuged for 10 mins
o Gel contains IgG, if sensitization occurs it’ll react to antibody-coated RBCs

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 o If no agglutination occurs, RBCs will form a pellet at the bottom of the chamber
o 4+. Solid band of agglutinated red cells at the top of
the gel column. No red cell are visible in the bottom
of the microtube.
o 3+. Predominant agglutinated cells towards the top
of the gel column with a few agglutinates staggered
below the thicker band. Tophalf of the gel (majority
of agglutination are observed).
o 2+. Cells agglutinates dispersed throughout the gel
column with few agglutinates at the bottom.
Distributed through the upper and lower halves of
the gel.
o 1+. Cells agglutinate predominantly in the lower half
of the gel column with red cells also in the bottom.
o Mixed field. Layer of the cell agglutinates at the top of the gel accompanied by a
pellet of unagglutinated cells in the bottom.
o 0 / Neg. Cells forming a well-delineated pellet in the bottom of the microtube.
Gel above the red cell pellet is clear and free from agglutinates.
 How to perform Solid Phase Red Cell Adherence Method?
o Immucor’s Capture-R, RBC antigens coat microtiter wells rather than being
present on intact RBCs
o Patient’s serum or plasma is added to each well in the screen cell set along with
LISS
o Incubate at 37˚C
o Wells are washed to remove unbound antibodies
o Indicator red cell that have coated with anti-IgG are added
o Centrifuge for several minutes
o Forming a diffused pattern if sensitization occurs (indicator cells reacts to
antibodies bound to
antigen coated in
microtiter well)
o Indicator cells formed a
pellet in the bottom if
no sensitization
occurred

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 Summary of Gel and SPRCA:

 Interpretations
o In what phase did the reaction occur?
 IgM reacts best at room temperature or lower and capable of
agglutination at immediate spin phase

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  IgG reacts best at AHG phase
 Frequently IgM – anti-N, anti-I, and anti-P1
 Frequently IgG – directed against Rh, Kell, Kidd, Duffy and Ss antigens
 Mixture of IgG or IgM or both – Lewis and M antibodies
o Is the autologous control negative or positive?
 Autologous control is the patient’s RBCs tested against the patient’s
serum or plasma in the same manner as the antibody screen
 Detected alloantibody – pos antibody screen and neg autologous control
 Presence of autoantibody or antibody to medication and if patient has
been recently transfused (coats circulating donor RBCs) – pos autologous
control
o Did more than one screen cell sample react? If so, did they react at the same
strength and phase?
 More than one screen cell sample may be positive when:
1. Patient has multiple antibodies
2. Single antibody’s target antigen is found on more than one screen
cell
3. Patient’s serum contains autoantibodies
 Note: Single antibody specificity reacts at same phase and
strength while multiple antibodies reacts at different phases
or strengths. Autologous control positive = Autoantibody
o Is hemolysis or mixed-field agglutination present?
 Cause in vitro hemolysis – anti-Lea , anti-Leb , anti-PP1 PK , and anti-Vel
 Cause mixed-field agglutination – anti-Sda , and Lutheran antibodies
o Are the cells truly agglutinated, or is rouleaux present?
 Rouleaux – altered albumin-to-globulin ratios or having HMW plasma
expanders. Stacked coin appearance, observed in test contains patient’s
serum (autologous control and reverse ABO grouping), doesn’t interfere
with AHG phase (washed away serums), and dispersed by adding 1-3
drops of saline to the test tube.
 Limitations
o When using three-cell screen set, a negative result with all three cells gives the
technologist 95% confidence that there’s no clinically significant antibodies
o Screening reagents and methods are designed to detect clinically significant
antibodies
o Screen will not detect antibodies if:
 Antibody titer drops below the level of sensitivity

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  Directed against low-prevalence antigens that are not present on RBCs in
the screen cell set
 Antibodies showing dosage may not be detected if none of the screen
cells have homozygous expression of the target antigen
o Factors influence the sensitivity of the antibody screen:
 Cell-to-Serum-Ratio – prozone and postzone phenomenon (False neg). 2
drops serum and 1 drop RBC suspension gives balance for proper
sensitization and agglutination to occur
 Temperature and Phase of Reactivity – enhance reactivity by incubating
the screen at 18˚C or 4˚C

 Length of Incubation – saline environment incubates at 30mins to 1hr

while potentiators will shorten it to 10mins
 pH – reacts best at pH 6.8 and 7.2; anti-M enhanced reactivity at pH 6.5
Antibody Identification:
 Once antibody has been detected, additional testing is necessary to identify the
antibody and determine its clinical significance. Patient’s serum or plasma is tested
against additional RBCs possessing known antigens
 Patient History
o Age, sex, race, diagnosis, transfusion and pregnancy, medications, and
intravenous solutions
o Anti-U – African decent
 Reagents
o Antibody identification panel is a collection of 11 to 20 group O RBCs with
various antigen expression
 Exclusion
o When interpreting panel results, the first step is to exclude / rule-out antibodies
that could not be responsible for the reactivity seen

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 o Rule-out technique – homozygous expression of the antigen on the cell only
o Low-prevalence antigen that rarely expressed homozygously – K, Kpa , Jsa, Lua
o Sample Panel; Identify the possible antibody present:

SAMPLE EXERCISE:

ANSWER:

ANSWER:

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  Evaluation of Panel Results – after each negatively reacting cells have been evaluated,
the remaining antigens should be examined to see if the pattern of reactivity matches a
pattern of antigen-positive cells

Additional Techniques for Resolving Antibody Identification:

 Selected Cell Panels – the cells selected for testing should have minimal overlap in the
antigens they possess. Useful when patient has a known antibody and the technologist
is attempting to determine if additional antibodies are present.
 Enzymes – if multiple antibodies are present in sample, treating the panel cells with
enzymes will separate the specificities and allowing for identification
o Ficin is commonly used; Papain, Bromelin, or Trypsin may also be used
o Removes sialic acid residues and denaturing
glycoproteins
o Destroy certain antigen and enhances
expression of others
o Enzymes may be utilized in place of
enhancement media, such as LISS or PEG, in a
one-step enzyme test method. A second,
more sensitive method uses enzymes to treat
the panel RBCs first, and then the antibody
identification panel is performed using the treated cells.
 Neutralization - Other substances in the body and in nature have antigenic structures
similar to RBC antigens. These substances can be used to neutralize antibodies in serum,
allowing for separation of antibodies or confirmation that a particular antibody is
present.
o The patient’s serum is first incubated with the neutralizing substance, allowing
the soluble antigens in that substance to bind
with the antibody.
o An antibody identification panel is performed
using the treated serum.
o The neutralizing substance inhibits reactions
between the antibody and panel RBCs. Use of
a control (saline and serum) is necessary to
prove that the loss of reactivity is due to
neutralization and not to dilution of antibody
strength by the added substance.

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  Adsorption - antibodies may be removed from serum by adding the target antigen and
allowing the antibody to bind to the antigen, in a manner similar to the neutralization
technique.
o In the adsorption method, the antigen-antibody complex is composed of solid
precipitates and is removed from
the test system by centrifugation.
o The absorbed serum is tested
against an RBC panel for the
presence of unabsorbed
alloantibodies.
o The adsorbent is typically
composed of RBCs but may be
another antigen-bearing
substance.
 Commercial Reagents for Adsorption - Human platelet concentrate is
used to adsorb Bg-like anti- bodies from serum. The HLA antigens present
on platelets bind the HLA-related Bg antibodies, leaving other specificities
in the serum. Antibody identification can be performed on the adsorbed
serum.

 Autoadsorption - autoantibodies are commonly removed through

adsorption techniques. Perhaps the simplest method is adsorption using
the patient’s own RBCs. The autologous cells are first washed thoroughly
to remove unbound antibody. They may then be treated to remove any
autoantibody coating the RBCs.

 Homologous Adsorption - for homologous adsorption, the patient is

phenotyped, and then phenotypically matched RBCs are used for the
adsorption in place of autologous cells. (When a patient is so anemic that
there are not enough autologous RBCs available to perform an adequate
number of adsorptions or when a patient has been recently transfused
(donor RBCs in the specimen may adsorb alloantibodies), homologous or
differential adsorptions may be employed in place of autoadsorption).

 Differential Adsorption - When phenotyping the patient is difficult

because of a positive DAT or recent transfusion, differential absorption
may be performed. For this method, the patient’s serum sample is
divided into a minimum of three aliquots. Each aliquot is adsorbed using
a different cell.

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Direct Antiglobulin Test and Elution Techniques
 Detection of antibodies coating RBCs is valuable when investigating suspected hemolytic
transfusion reactions, HDFN, and autoimmune and drug-induced hemolytic anemias.
 The direct antiglobulin test (DAT) is used to detect in vivo sensitization of RBCs.
 In the tube method, the patient’s RBCs are washed thoroughly to remove any unbound
antibody, and then AHG reagent is added. If IgG antibodies or complement are coating
the RBCs, agglutination will be observed. If neither is present, no agglutination will be
observed. Coombs’ control cells are added to validate the negative test.
 The DAT may also be performed using solid phase adherence and gel methods.
 When IgG antibodies are detected, the next step is to dissociate the antibodies from the
RBC surface to allow for identification.
 Elution techniques are used to release, concentrate, and purify antibodies. The methods
used to remove the antibody change the thermodynamics of the environment, change
the attractive forces between antigen and antibody, or change the structure of the RBC
surface. The antibody is then freed into a solution known as an eluate. The eluate may
be tested against an RBC panel to identify the antibody. A total elution, in which
antibody is released and the RBC antigens are destroyed, is usually necessary when
performing antibody identification.
 Partial elution, in which antibody is removed but RBC antigens remain intact, is useful to
prepare RBCs for phenotyping and to use in autoadsorption procedures. Chloroquine
diphosphate, EGA, and ZZAP are examples of chemicals used for these purposes.

o Temperature-Dependent Methods - heat may be used to remove antibody. The

gentle heat method, performed at 45°C, allows for antibody removal while
leaving the RBC intact.32 Elution performed at 56°C is a total elution method,
allowing for antibody identification.33 The Lui freeze method34 also performs a
total elution. With this method, washed, coated RBCs suspended in saline or
albumin are frozen at –18°C or colder until solid. The mixture is then thawed
rapidly, causing the RBCs to burst, freeing the bound antibody. Temperature-
dependent elutions are best at detecting IgG antibodies directed against
antigens of the ABO system.
o pH - in this method, the washed antibody-coated cells are mixed with a glycine
acid solution at a pH of 3. The antigen-antibody bond is disrupted, and the
antibody is released into the acidic supernatant. Citric acid and digitonin acid are
also used.
o Organic Solvents - several organic solvents have been used in total elution
methods, including dichloromethane, xylene, and ether. The last wash should be
nonreactive, or the eluate results will be invalid.

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Antibody Titration:
 Performing an antibody titration can help determine antibody concentration levels.
Twofold serial dilutions of serum containing an antibody are prepared and tested
against a suspension of RBCs that possesses the target antigen. The titer level is the
reciprocal of the greatest dilution in which agglutination of 1+ or greater is observed.
 After determining the initial titer, the specimen should be frozen. When new specimens
are submitted for titer determinations, the initial titer specimen should be tested in
parallel to control variability among technologists’ technique (e.g., pipetting, grading
reactions) and the relative strength of the target antigen on the cells used in testing.
 A comparison of the current specimen’s results and the initial specimen’s current
results should be made. A fourfold or greater increase in titer (reactivity in two or more
additional tubes) or an increase in score of 10 or more is considered to be significant.
 Titer-level studies are useful in monitoring the obstetric patient who has an IgG
antibody that may cause HDFN. An increase in antibody titer level during pregnancy
suggests that the fetus is antigen-positive and therefore at risk of developing HDFN. An
increasing titer level may indicate the need for intrauterine exchange transfusion. An
antibody titer may also be used to help differentiate immune anti-D from passively
acquired anti-D due to RhIG administration.

Providing Compatible Blood Products:

 The relative difficulty in providing compatible blood products is determined by the
frequency of the antigen in the population and by the clinical significance of the
antibody.
 If the antibody does not cause decreased survival of antigen-positive RBCs, then use of
random blood products that are crossmatch- compatible is acceptable.
 When the patient sample contains clinically significant antibodies or the patient has a
history of clinically significant antibodies, units for transfusion must be antigen-negative.
 If the patient sample is plentiful, the technologist may choose to crossmatch units, then
antigen-type those that are crossmatch-compatible.
 If sample quantity is limited or if the antibody is no longer detectable in the serum, units
should be antigen-typed first, then crossmatched.

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