1. September 2007 Dr Debra Lane
Serology Update
September 2007
Dr Debra Lane

2. Initial detection and Identification of Alloantibodies
Alloantibdies can be found in % of the population
Allogenic antibodies react only with allogenic cells
Immunization can be from:
Pregnancy
Transfusion
Transplantation
Injections with immunogenic material ( needle sharing)

3. AB ID Rare cases no specific immunising even is identified
Antigens exposure may be:
Environmental
Bacterial
Viral

4. 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

5. Medical History Useful to know: Clinical diagnosis
History of transfusion
Pregnancy history
In some cases the ethnic background

6. 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

9. 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

11. 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

13. 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

15. 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

16. Enhancement Media
Many enhancement media have been utilized and have included:
Saline Albumin
LISS( Low Ionic Strength Saline)
PEG (Polyethylene Gycol)

17. 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

18. 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

19. Special notes
Patients with known antibodies do not require a full panel
Some exclusion cells are done to confirm the antibody

20. 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

21. 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

22. 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

23. 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

25. 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

26. Zygosity 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

27. 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

28. 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

29. 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

30. Other steps for Antibody Identification
Exclusion method
Test serum against homozygous cells
Enhancement media
Phenotype the patient (if not transfused)
Methods to inactivate antigens
Adsorption elution

31. 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

32. Antibodies to Ingredient in the Preservative solution
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

33. Antibodies to enhancement media Ingredients
Antibodies to LISS or albumin;
Sodium caprylate, thimersol

34. Cold Autoantibodies

35. Next step is usually to find antigen negative units for the patient

38. 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

39. 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

40. Method 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

41. 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

42. Cold Agglutinin Titres
High titred cold agglutinins may be pathologic
May have overt hemolysis
May be seen in B cell lymphomas

43. 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

44. 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

45. 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.