Transcript Serology Update - Transfusion Medicine

Serology Update
September 2007
Dr Debra Lane
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.