This document discusses various techniques used in blood banking and transfusion medicine, including: 1. Pretransfusion testing involves ABO/Rh typing, antibody screening, and crossmatching to select compatible blood and prevent hemolytic transfusion reactions. 2. Antibody identification uses a panel of red blood cells to identify the specific antibody in a patient's serum through various testing phases including immediate spin, LISS incubation, and antiglobulin. 3. Special techniques like elution, hemagglutination inhibition, and titration are used to further characterize antibodies or quantify their concentration.

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