Introduction to Antibody Investigating
The Science and the Art
or Otherwise Known as
Text Book vs. Real World Practise
Debra Lane, MD FRCPC
Medical Director, Canadian Blood Services Winnipeg
Associate Professor University of Manitoba
Initial Detection and Identification of
Alloantibodies
• Alloantibdies can be found in 0.3- 38% of the
population
• Allogenic antibodies react only with allogenic
cells
• Immunization can be from:
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Pregnancy
Transfusion
Transplantation
Injections with immunogenic material ( needle
sharing)
AB ID
• Rare cases no specific immunising even is
identified
• Antigens exposure may be:
– Environmental
– Bacterial
– Viral
Definition of a Clinically
Significant Antibody
• An antibody that shortens the lifespan of a
transfused red cell or has been associated
with Hemolytic Disease of the newborn.
• May be detected in serum or plasma
• Whenever possible, patients with clinically
significant alloantibodies should be
investigated, the antibody determined and
antigen negative red cells provided
Medical History
• Useful to know:
– Clinical diagnosis
– History of transfusion
– Pregnancy history
– In some cases the ethnic background
Screening
• Commercial group O cells are available for
screening
• Usually a two or three cells screen
• Up to the Lab Director to determine which
to use
• Reagent cells must contain:
– D,C,E,c,e,M,N,S,s,P1,Lea,Leb,K,k,,Fya,Fyb,Jka
,Jkb
Dosage
• Antigens in the Rh, MNSs, Kidd show
dosage
• Reagent cells should be refrigerated when
not in use
• Should not be used past their expiration
date
• Lot number must be documented
• If there is a reaction on the screen cells
then the next step is a panel test
Panels
• Most blood banks now use commercial
panels
• Some blood banks attached to donor
centers still prepare there own panels from
regular donors with known phenotypes
whose cells have been frozen
• Panels are usually set up to give the same
characteristic patterns for the most
common antibodies
Secondary Panels
• These panels have more cells, and are
much more expensive
• Used for exclusions and often have more
homozygous cells
• Panels come every 2 to 4 weeks
• Back of the panels often have the
extended phenotypes listed on the back
Antiglobulin Reagents
• Most antibody identifications include an
antiglobulin phase
• Either a polyspecific anti-human globulin
or a monospecific IgG anti-human globulin
is used
• IgG anti-human globulin is used to avid
detecting the in-vitro binding of
complement
Enhancement Media
• Many enhancement media have been
utilized and have included:
– Saline Albumin
– LISS( Low Ionic Strength Saline)
– PEG (Polyethylene Gycol)
Auto Control
• Cells that react with the enhancement
media alone are not the same as a
positive Direct Antiglobulin Test
• If the autocontrol is positive a DAT should
be done
• If the DAT is positive then further studies
such as an elution may need to be
performed
• Most places use the same technique or method for
antibody detection as identification and crossmatch
• We don’t because we implemented Galileo for our
screening [solid phase] and maintained the method our
tech’s were familiar with for manual testing [PEG]
• Some techs start with immediate centrifugation and
reading before adding enhancing media
• This is useful to detect M,N,P,I Lea, Leb
• We omit these steps since we want to avoid finding
antibodies that react at lower temperatures
• Some antibodies may even cause complete red cell
lyses such as anti-Lea, anti-Jkb
Special Notes
• Patients with known antibodies do not
require a full panel
• Some exclusion cells are done to confirm
the antibody
Interpretation
• Presence or absence of agglutination is
important
• Positive reactions include the phase and
the strength of the reaction
• Single alloantibodies tend to give clear
reaction patterns
• Negative reactions allow for exclusions of
antibodies to antigens expressed on the
nonreactive cells
Exclusions
• “crossout”- is the widely used first approach
where antibodies are excluded when there is no
reaction to a positive antigen cell
• If the antigen is present on the cell and the
serum or plasma did not react, that antigen is
excluded
• This usually leaves a group of antibodies that
have not been excluded
• Next the cells reactive for the presumed
antibody are evaluated and if the pattern
matches exactly, that is most likely the specificity
of the antibody in the serum
Exclusions
• Additional testing may be required to rule
out other specificities
• If it fits anti-E but K and S have not been
excluded, then one might test cells such
as:
• E neg, K pos, S neg
• E pos, K neg, S neg
• E neg, K neg, S pos
Probability
• Probability has been studied to ensure that the
reactivity is not due to chance
• Fischer’s exact method is to require three
antigen positive cells that react and three
antigen negative cells that d not react
• Harris and Hoschman have done calculations for
minimum requirements for a (p) value of 0.05
are met by having two positive and three
negative cells or one positive and seven
negative cells
• Sometimes identification is difficult
• We may resort to phenotyping the patient
if they have not been transfused and give
phenotypically matched blood
• Antibodies do not always react with all the
positive cells
– Technical error
– Weak antigens
– Weak antibody reactivity
Zygosity
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Reaction strength may vary due to dosage
React stronger with homozygous cells
Or double dose of the antigen
This is common in:
– Rh
– Duffy
– MNS
– Kidd
Antigens Present in Common
• Instead of excluding, one can look at what
the reacting cells have in common
cells reacting at room temp are P pos
• Except for one sometimes the panel will
indicate it is a P weak cell
Variable Reactivity
• May indicate HLA antibodies
• Anti-Bga vary between individuals
• Perhaps the manufacturer has incorrectly
labelled a reagent red cell
• May react with an antigen not routinely
listed-Yta
Multiple Antibodies
• Observed pattern does not fit a single
antibody
• Reactivity is present at different test
phases
• Unexpected reactions are obtained when
trying to confirm an antibody
• No pattern emerges
Other Steps for Antibody
Identification
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Exclusion method
Test serum against homozygous cells
Enhancement media
Phenotype the patient (if not transfused)
Methods to inactivate antigens
Adsorption elution
Sneaky Antibodies
• All cells reactive with a positive DAT may
not always be a warm auto
• Can be a transfusion reaction with an
antibody to a high frequency antigen
• Like Diego
• The race of the individual can be helpful at
this point
Antibodies to Ingredient in the
Preservative Solution
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Antibodies may be made to:
Chloramphenicol
Neomycin
Tetracycline
Hydrocortisone
EDTA
Sodium caprylate
These may agglutinate cells suspended in that
solution
• Can wash the reagent cells before testing
Antibodies to Enhancement
Media Ingredients
• Antibodies to LISS or albumin;
• Sodium caprylate, thimersol
Cold Autoantibodies
• Next step is usually to find antigen
negative units for the patient
Cold Autoantibodies
• Potent cold autoantibodies can create
problems clinically if they react at room
temperature
• May be benign or pathologic
• Thermal amplitude helps to determine if it
is significant
• The freshly collected serum must be kept
warm until the serum is separated
Determining the Specificities
• Requires ample to be warm until the
serum is removed
• Autologous red cells
• Reagents:
– Pool O I adult red cells
– Group O I cells
– Pts own washed autologous cells ( 37o saline)
– Red cells same group of the patient
– Saline or phosphate buffered saline
Method
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Prepare serial two fold dilutions in saline or PBS( 12 tubes)
Label 12 tubes with the dilution 2,4,8.. For each adult, I, autologous
Dispense two drops into each tube
Add I drop 3 to 5% red cells
Incubate RT for 30 to 60 min
Centrifuge for 15 to 20 sec 900 to 1000 g
Examine for agglutination
Grade and record
Transfer tubes to 4 C and incubate for 1 to 2 hours centrifuge
Place tubes in rack in ice water
Grade results
Interpretation
• This method helps to determine the specificity and the
titre
• Need to use separate pipette tips for dilutions
• Large volumes prepare better dilutions
• Potent examples do not show specificity until titration
studies are performed
• This can be used to determine the titre and sensitivity
• If readings are taken sequentially after each incubation
[37,30,4] the specificity, titre and thermal amplitude can
be determined
• From AABB technical manual
Cold Agglutinin Titres
• High titred cold agglutinins may be
pathologic
• May have overt hemolysis
• May be seen in B cell lymphomas
Cold Agglutinin Titres
• Specimen:
– serum separated at 37 from a sample that
clotted at 37
• or plasma from sample collected and
maintained at 37 with periodic inversion
• Reagents
– Pool of t2 examples washed group O I
– PBS ph 7.3
Cold Agglutinin Titres
• Prepare serial dilutions in PBS
• ½ to 1/4096
• Mix two drops dilution with 1 drop 3 to 5%red
cells
• Mix and incubate at 4c for 1 to 2 hours
• Centrifuge 15 to 20 sec at 900 to 1000g
• Place in ice water bath
• Examine for agglutination
• Start with tube at highest grade
Cold Agglutinin Titres
• Titre is the r reciprocal of the highest serum
dilution where agglutination occurs
• Titres above 64 are elevated
• Hemolytic anemia rarely occurs from colds
unless the titre is greater than 1000
• Titres may be lower with anti-I
• If the DAT is positive with complement only, and
the patient has symptoms of hemolytic anemia
• Specificity and thermal amplitude studies should
be performed.