Unit 10 Identification of
Unexpected Antibodies
Terry Kotrla, MS, MT(ASCP)BB
Introduction
Unexpected alloantibodies are antibodies
other than anti-A or –B.
 Found in 0.3-2% of population.
 Immunization to RBC antigens may result
from:
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Pregnancy
Transfusion
Deliberate injection of immunogenic material
Event unknown
Introduction
Once antibody detected must determine
specificity and clinical significance.
 Clinically significant antibody will
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Shorten survival of antigen positive RBCs.
Cause HDFN if fetus inherits antigen.
Clinical significance varies
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RBC destruction may occur within minutes.
RBC survival may decrease by only a few days.
Use documented experience with other
examples of antibodies with same specificity.
Introduction
May be no data for some antibodies
 Consider that it is not clinically significant
if no reactivity at 37C or IAT
 Clinical significance important in donor
blood selection and prenatal testing.
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Must select antigen negative blood if clinically
significant.
During pregnancy may predict likelihood of
HDFN.
General Procedures
Need adequate quantity of serum/plasma
and RBCs to resolve.
 EDTA RBCs preferred for study of RBCs.
 Medical History CRUCIAL and include:
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Number of pregnancies
Transfusion history, especially if recent
Drug therapy
General Procedures
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Test serum/plasma at all phases of activity
initially detected.
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Additional antibodies may be detected at
different phases.
Reactivity may be increased by:
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Extending incubation time
Lowering temperature if cold antibody suspected
Increase serum:cell ratio
Use of enzyme treated cells.
General Procedures - Enzymes
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Enhance reactivity of Rh, P, I, Le and Jk
antibodies.
Denatures M, N, S and Fy antigens,
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if multiple antibodies present and one has MNS or Fy
specificity, aids in identification.
Reactivity of antibodies to M, N, S or Fy will go away
or weaken tremendously.
Allows underlying antibody activity to be detected.
General Procedures
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Serum/plasma tested against panel of 8 or more
Group O RBCs of KNOWN phenotype.
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Must be able to identify WITH CONFIDENCE most
frequently encountered, clinically significant
alloantibodies.
Distinct pattern should be apparent for single
alloantibodies.
Must be sufficient RBCs that lack and carry antigens
to which antibodies most frequently made- usually
three of each.
Autocontrol MUST be run
Considerations in Interpreting
Serological Results
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Alloantibodies of some blood group
specificities FREQUENTLY display
consistent serologic characteristics:
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Evaluate effects of temperature, suspending
medium or enzymes with sample.
Variation in strength of reactivity?
Hemolysis present?
Is autocontrol positive or negative?
Single Alloantibody
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Usually easy to recognize, clear cut pattern of
reactions.
Test phase suggestive of specificity BUT
exceptional samples will be encountered
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Varying strength of reactivity within 1 phase may
be due to:
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IS/RT think anti-P1, Le, M, N, I or Lua
37C or AHG think anti-Rh, Jk, K, Fy or Lub
Antibody showing dosage
Multiple antibodies present
ALWAYS look for additional antibodies
Special Considerations Rh Antibodies
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If anti-E identified look for anti-c
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Determine Rh phenotype of patient.
If patient is E and c negative (R1R1) transfuse
with R1R1 even if anti-c NOT detectable
Anti-c common cause of delayed HTR in
R1R1 patients with anti-E
 Reverse not a problem, almost all c
negative donors will be E negative.
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Phenotype Autologous Cells
After identification of alloantibody type
patient for corresponding antigen.
 If interpretation correct patient will be
NEGATIVE.
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Provides additional confirmation of specificity
If patient is positive indicates misinterpretation
of results.
CANNOT antigen type recently transfused
patient, use pretransfusion sample or
perform cell separation.
Probability of Identification
Three RBCs antigen positive that react
with antibody.
 Three RBCs antigen negative that DO
NOT react with antibody.
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Multiple Antibodies
May be very challenging.
 Clues
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Pattern of reactive and non-reactive RBCs do
not fit single antibody specificity.
Variable strength of reactions.
Different RBCs react at different test phases.
Antigen negative donor incompatible with
patient sample.
Antibody to High Frequency Antigen
Very difficult to work up.
 Most, if not all, RBCs on panel will be
antigen positive.
 Must find additional negative RBCs to rule
out “hidden” or “masked” antibodies.
 Selected Cell Panel
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There is only one e negative RBC on panel
Must find additional e negative to rule out
antibodies not ruled on with negative RBC.
Technique also used for multiple antibodies.
Antibodies to High Incidence Antigen
Suspected when ALL reagent RBCs are
positive BUT autocontrol is negative.
 Rule out multiple antibodies.
 Will most likely have to send to reference
laboratory which possess rare cells.
 Patient’s siblings most likely source of
antigen negative blood.
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Antibodies to Low Incidence Antigen
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Serum/plasma has negative screen but
ONE donor incompatible.
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Check for ABO incompatibility if reaction
at IS only.
Perform DAT on donor if reaction at AHG
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If positive, not the problem.
If negative most likely alloantibody.
Perform a panel, be sure to test cells
positive for low frequency antigens.
 Inappropriate to delay transfusion.
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Anomalous Serological Reactions
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Antibodies to drugs or additives may be
present.
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If drug induced autocontrol will be positive.
If additive suspected try different additive.
Some times nothing can be done to
increase strength of reactivity.
 Interpretation, “Unable to determine
antibody specificity”.
 Transfuse with serologically compatible
blood.
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Selected Serological Procedures
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When pattern fails to indicate specificity other
procedures may be performed:
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Enzyme Techniques
Temperature reduction
Increase serum to cell ratio
Increase incubation time
Decrease pH
Change enhancement media
Use of thiol reagents
Prewarmed technique
Inhibition tests
Titration
Adsorption
Elution
Enzyme Techniques
Treatment of RBCs with proteolytic
enzymes ENHANCES reactivity of Rh, P, I,
Lewis and complement binding
alloantibodies such as anti-Jka.
 Antigens of M, N, S and Fy are depressed
or destroyed, antibody CANNOT react.
 Enzyme techniques used whenever weakly
reactive antibody may be Rh antibody OR
when a patient has multiple antibodies
and one of them is a Fy.
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Temperature Reduction
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Alloantibodies (e.g., anti-M, -P1) that react better
at cold temperatures.
Specificity may become apparent at or below 22
C.
Auto-control especially important for tests at cold
temperatures, because many sera contain cold
reactive auto-antibodies.
Anti-I specificity confirmed by testing patient
serum with adult (I+) and cord (i+) cells at 4 C.
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Positives with adult cells and negative with cord cells
confirms anti-I specificity.
Increase Serum/Plasma to Cell Ratio
Increases amount of antibody in test
system which may increase the strength
of reactivity of antibodies present in low
concentrations.
 Increase to 4 or more drops.
 May have to add additional washes.
 NOTE: LISS requires EQUAL volume of
serum and RBCs
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Increase Incubation Time
Use 30-60 minutes to allow more time for
more antibody to attach to RBCs.
 NOTE: DO NOT exceed incubation time
recommended by enhancement media
manufacturer.
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Decrease pH
Decrease pH to 6.5 by addition of 0.2 N
HCl.
 Weak anti-M GREATLY enhanced.
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Change Enhancement Media
Some blood group antibodies react
preferentially in test systems utilizing Low
Ionic Strength Salt (LISS) solutions.
 A variety of LISS procedures have been
described.
 PEG
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Thiol Reagents
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Strong IgM class antibodies may have high
thermal amplitude and may mask other
antibodies.
Two reagents - dithiothreitol (DTT) and 2mercaptoethanol (2-ME)
Cleave inter-subunit disulfide bonds of IgM
molecules.
The IgG molecules relatively resistant to such
cleavage, treatment results in destruction of IgM
but leaves IgG intact able to react in-vitro.
Applications of DTT and 2-ME
Determining the immunoglobulin class of
an antibody, especially if the antibody has
potential to cause HFDN.
 Dissociating RBC agglutinates caused by
IgM antibodies (e.g., spontaneous
agglutination of RBCs caused by potent
cold reactive auto-antibodies).
 Identifying specificities in a mixture of IgM
and IgG antibodies, particularly when an
agglutinating IgM antibody masks the
presence of IgG antibodies.
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Prewarmed Technique
Cold auto-agglutinins may demonstrate
high thermal amplitude resulting in false
positive reactions at 37C and AHG.
 Can
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Confirm that only a cold auto-agglutinin is
present or
Detect the presence of clinically significant
underlying alloantibodies.
Prewarmed Technique
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Place RBCs to be tested in appropriate test tubes
and place in 37C heat block.
Prewarm tube of serum to 37C.
Add 3 to 4 drops of prewarmed serum to
prewarmed cells and incubate 1 hour.
Without removing tubes from heat block add
saline that has been prewarmed to 37C.
Immediately spin and wash 2 additional times
with the warm saline.
Perform the AHG procedure.
Inhibition Tests
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Some blood group antigens exist in soluble form
in such body fluids as saliva, urine or plasma.
These substances useful in antibody identification
studies, either to confirm antibody specificity by
inhibition or to neutralize antibodies that mask
the presence of concomitant non-neutralizable
antibodies.
The following soluble blood group substances can
be used in antibody identification tests.
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Lewis
P1
Sda
Lewis Inhibition Tests
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Lea and Leb substances present in saliva of
persons with appropriate Lewis phenotype
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Lewis substance can be prepared from saliva.
Most blood banks use commercially prepared
Lewis substance.
Lewis substance will neutralize Lewis
antibodies in a patient specimen, allowing
the detection of underlying, clinically
significant alloantibodies.
P1 Inhibition Tests
Soluble P1 substance is present in hydatid
cyst fluid as well as pigeon eggs.
 Anti-P1 may mask the presence of
underlying alloantibodies.
 Addition of P1 substance to the patient's
serum causes neutralization of the anti-P1,
allowing the detection of underlying
alloantibodies
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Sda Inhibition
Sda blood group substance is present in
soluble form in various body fluids, with
the most abundant source being urine.
 Urine from Sda positive individual added to
patient serum, causing neutralization of
the anti-Sda.
 Aids in the detection of underlying
alloantibodies.
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Titration
Titer of an antibody usually determined by
testing serial two-fold dilutions of the
serum against selected RBC samples.
 Results expressed as reciprocal of highest
serum dilution causing macroscopic
agglutination.
 Three applications:
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Prenatal Studies
Antibody identification
HTLA antibodies
Titration – Prenatal Studies
If antibody is of a specificity known to
cause HDFN OR when the clinical
significance of the antibody is unknown,
results of titration studies and outcome of
previous pregnancies are used to assess
the need for amniocentesis.
 Rising titers are indicative of active
immunization of the mother.
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Titration – Antibody Identification
Some antibodies cause agglutination of
virtually all reagent RBC samples, but
specificity is indicated by differences in the
strength of reactivity with each sample in
titration studies.
 Titration procedure is not used very
commonly for this purpose.
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Titration - HTLA
HTLA antibodies react very weakly in
undiluted state but, unlike most weakly
reactive antibodies (e.g., anti-D with a
titer of 4), react at a high dilutions (e.g., 1
in 2000).
 Such antibodies include anti-Ch, -Rg, -Cs,
-Yk, -Kna, -McCa and -JMH.
 When weak reactions are observed in the
IAT, titration studies may be used to
establish whether or not the reactions are
due to the presence of an HTLA antibody.
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Adsorption
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Antibody can be removed from a serum by
adsorption to RBCs carrying the
corresponding antigen.
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Incubate serum with antigen positive cell.
Antibody forms complex with RBC membranebound antigens.
When the serum and RBCs are separated, the
antibody remains attached to the RBCs.
Subsequent elution of the bound antibody can
often give additional useful information.
Use of Adsorption Techniques
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Removing auto-antibody activity to permit
detection of coexisting alloantibodies.
Removing unwanted antibody from serum that
contains an antibody suitable for reagent use.
Confirming the presence of antigens on RBCs
through their ability to remove a specific serum
antibody.
Confirming the specificity of an antibody by
showing that it can be absorbed only to RBCs of a
particular blood group phenotype.
Separating multiple antibodies present in a single
serum sample.
Elution
Elution techniques free antibody
molecules from sensitized RBCs so
the recovered antibody can be tested.
 A variety of methods are employed with
the primary objective being breaking the
bond between the antigen and the
antibody.
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Use of Elution Techniques
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Identification of an antibody coating a baby's
RBCs in the case of HDFN.
Identification of an antibody causing an acute or
delayed hemolytic transfusion reaction.
Investigation of a positive DAT.
Concentration and purification of antibodies, the
detection of weakly expressed antigens and the
identification of multiple antibodies.
Preparation of antibody-free intact RBCs for use
in phenotyping or autoabsorption.
Elution Technique – Technical Factors
Incorrect technique.
 Incomplete washing
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If cells are incompletely washed
contaminating serum antibody will cause false
positive reactions.
An aliquot of saline from the last wash is saved
and tested in parallel. Positive reactions with
the last wash invalidates the test.
Binding of proteins to glass surfaces.
 Dissociation of antibody before elution.
 Instability of eluates.
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Summary
If an unexpected antibody is detected in a
patient’s serum or plasma it must be
identified.
 Once identified the clinical significance
must be determined.
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Summary
If the antibody is clinically significant
antigen negative donors must be found
and crossmatched for the patient, a
Coomb’s crossmatch must be done.
 If the antibody is not clinically significant it
is not necessary to provide antigen
negative blood, but the donors must be
compatible by the Coomb’s crossmatch.
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References
 AABB
Technical Manual, 16th
edition, 2008
 Basic and Applied Concepts of
Immunohematology, 2nd edition,
2008