Cross transfusion and compatibility tests

2. Ahmad A. Al-Qudah
LM755 - ADVANCED DIAGNOSTIC IMMUNOHEMATOLOGY AND BLOOD BANKING
Cross transfusion and Compatibility

3. Cross transfusion and Compatibility
- Pretransfusion Testing .
- Pretransfusion testing elements :
- ABO & Rh
- Antibody Screening
- Cross Matching
- Antibody Identification .
- Special Techniques in Ab Identification :
- Elution
- Hemagglutination inhibition
- Titration

4. Pretransfusion Testing
History
- After Landsteiner described the ABO .
- Crossmatch :
- Major Crossmatch : Recipient Serum + Donor RBCs.
- Minor Crossmatch : Recipient RBCs + Donor Plasma (not required by AABB)
- RT ( IS Phase ) IgM , then Rh (IgG) and detection at various
Temperatures with or without enhancements .

5. Pretransfusion Testing
- To select blood components that will not cause harm to the
recipient and will have acceptable survival when transfused.
- ABO compatibility , Rh compatibility , clinically significant
Antibody Screening and Identification .

6. Indirect Antiglobulin Test Phases
- Immediate Spin ( IS ) Phase at Room Temperature .
Detection of ABO incompatibility and Cold Antibody (M,N, Lea+b,P1)
- 37° with Enhancement media (LISS)
Allows IgG and other complement to bind to RBCs .
- Anti Human Globulin Phase ( AHG )
Detection of IgG or complements binds to RBCs
- Check Cells : Approve the Results

7. ABO & Rh
- ABO & Rh typing for (recipient ) sample .
- ABO & Rh typing for Donor sample .

8. Antibody Screening
- Autoantibody : is an antibody manufactured by the immune
system that is directed against one or more of the individual's
own cells, Causes Many autoimmune disease.

9. Antibody Screening
- Alloantibody : is an immune response to foreign antigen,
Formed from previous transfusion or pregnancy.
- Antibody Screening test performed to detect auto-antibodies
/allo-antibodies using DAT (Direct Anti-human Globulin Test)

10. Antibody Screening
- Inclusion of auto control helps to rule out :
 Auto antibodies
 Allo antibodies
- Auto control :
Negative - alloantibody .
Positive – autoantibody .

11. Antibody Screening
- Antibody Screens use 2 or 3 Screening Cells to “detect” if
antibodies are present in the serum.
- If antibodies are detected, they must be identified .

12. Antibody Screening

13. Antibody Screening

14. Cross Matching
- Cross match is done to ensure there are no clinically significant
Abs in patient blood ( Serum ) against donor RBCs and so prevent
hemolytic reaction during or after transfusion .

15. Cross Matching
- Major cross-match test, consisting of mixing the patient’s
serum with donor RBCs.
- Minor cross-match test, consisting of mixing the donor’s
plasma with patient’s RBCs.
- Minor cross-match test has been completely eliminated in
most blood banks according to AABB SOPs since 1960.

16. Cross Matching
Prepare 5% suspension of donors RBCs

17. Cross Matching
Patient serum
2 drops
Donor RBC
1 drop, 5%
Immediate centrifuge
ABO incompatibility
22oC
• Detects only IgM antibody, reactive at 22oC.
• Clinically significant IgG antibody reactive at 37oC not detected

18. Cross Matching
Patient serum
2 drops
Incubation
37oC, 1 hr
Donor RBC
1 drop, 5%
3 washes
Centrifuge
2 drops AHG
Mix properly
No agglutination =
compatible
Detects clinically significant (IgG) antibody
Agglutination =
incompatible

19. Antibody Identification
- It is a set of commercially screening cells with different antigenic
expression corresponding to the most commonly encountered Abs.

20. Antibody Identification
- Screening Cells and Panel Cells are the same with minor differences:
- Screening cells
- Antibody detection
- Sets of 2 or 3 vials
- Panel cells
- Antibody identification
- At least 10 vials per set.
- Panel test is just an extended version of an antibody screen

21. Antibody Identification
- 11 panel cells of Group O , last one is auto control (Patient RBCs + Patient serum) .

22. Antibody Identification
- Each of the panel cells has been antigen typed .

23. Antibody Identification
- Immediate Spin Technique (IST)
Patient serum
2 drops
Panel Cell
1 drop
Immediate centrifuge
incompatibility
22oC

24. Antibody Identification
2+
0
0
2+
0
0
2+
0
0
2+
0
Record results

25. Antibody Identification
Incubation
37oC, 15 min
Centrifuge
2 drops LISS
Mix properly
Agglutination
No agglutination
Detects clinically significant (IgG) antibody
(LISS) 37°C Phase
Patient serum
2 drops
Panel Cell
1 drop

26. Antibody Identification

27. Antibody Identification
IAT Phase
Incubation 37oC Centrifuge
2 drops AHG
Mix properly
Agglutination
No agglutination
Patient serum
2 drops
Panel Cell
1 drop
Wash 3 times
saline

28. Antibody Identification
2+
0
0
2+
0
0
2+
0
0
2+
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0 0 0
- Record Results , Grade of Reaction .
- Add Check cells to any negative AHG .

29. Antibody Identification
- Read Results , Interpreting Antibody Panels

30. Antibody Identification
Phase-1: IS , Rx. at room temperature (24°C)
- Cold reactive antibodies (Ex. Anti-H, I, i, P)
Phase-2: LISS Rx at 37°C:
- Rh incompatibility
- wide thermal range IgM antibodies (Anti-Le, I, P)
Phase-3: AHG Rx. (at 37°C)
- will detect the vast majority of IgG antibodies

31. Antibody Identification
- Basic steps to follow when interpreting panels :
• “Ruling out” means crossing out antigens that did not react
• Circle the antigens that are not crossed out
• Look for a matching pattern
• Rule of Three .

32. Antibody Identification

33. Antibody Identification

34. Antibody Identification
- The rule of three must be met to confirm the presence of the
antibody.
- A p-value ≤ 0.05 must be observed.
- This gives a 95% confidence interval.
- Patient serum MUST be:
- Positive with 3 cells with the antigen
- Negative with 3 cells without the antigen

35. Antibody Identification
- Rule of three :

36. Antibody Identification
- P value = Num# of Positive RBCs! * Num# of Negative RBCs
Total Num# of RBCs used
P value = 3! * 4!
P value = (1*2*3) * (1*2*3*4)
(1*2*3*4*5*6*7)
P value = 2.85 , P value is ≤ 0.05
7

37. Special Techniques in Ab Identification
- Elution
Elution techniques “free” antibodies from the sensitized red
cells so that the antibodies can be identified and measured .
Y
Y
Y
Y
Sensitized
RBC
Positive DAT
Elution
Y
Frees antibody Antibody ID

38. Special Techniques in Ab Identification
- Elution
• Acid elution (glycine acid)
- Most common
- Lowers pH, causing antibody to dissociate
• Organic solvents (ether, chloroform)
- Dissolve bilipid layer of RBC
• Heat (conformational change)
• Freeze-Thaw (lyses cells)

39. Special Techniques in Ab Identification
- Haemagglutination inhibition:
based on the neutralization of certain antibodies with its
corresponding soluble substances (antigens).

40. Special Techniques in Ab Identification
- Haemagglutination inhibition :

41. Special Techniques in Ab Identification

42. Special Techniques in Ab Identification
Antibody titration
• semiquantitative determination of Ab’s concentrations
• serial 2-fold dilutions of tested serum are prepared
• serum testing against corresponding antigens
• TITER: is the reciprocal of the highest serum dilution that gives
macroscopic agglutination

43. Special Techniques in Ab Identification
Antibody titration