1. An unpleasant visitor? The challenge of having a red cell antibody visit the lab
Dr. Aseem Kumar Tiwari 1 1

2. Talk - Organization Expected Antibodies and ABO blood group system
Other blood group systems and unexpected antibodies
Detection of unexpected antibodies
Antibody identification and Interpretation
Rule of Three and Phenotyping
Magnitude of thse problem
Antibody Screen Negative – Change in algorithm
Antibody Screen Positive – Approach to Patient 2

3. Expected Antibody

4. Laboratory Work-up – ABO system
Expected Antibody (Regular Antibody/Typical Antibody)
Forward(cell) Reverse(serum) Interpretation
-A -B AC BC A B AB O
Slide(1900)/Tube(1960)/Micro-titer plate(1980)/Solid phase adhesion/Column Agglutination Technique

5. Other Blood Group Systems
Other blood group typing is not done routinely – ‘immunogenicity’ is considerably less than ABO and Rh

6. Unexpected Antibody – Unpleasant Visitor

7. Other Blood Group Systems and Unexpected Antibody
Unexpected (Irregular Antibody/Atypical)
Irregular Antibody – Develops either because of Transfusion/Pregnancy

8. Unexpected Antibody – Detection

9. Antibody Detection/Screening - Screening Cells
Each vial (donor) has been phenotyped for each antigen
18 antigens are required on at least one of the vials: D, C, E, c, e, M, N, S, s, P1, Lea, Leb, K, k, Jka, Jkb, Fya, Fyb
Single Vial (Reagent Pooled ‘O” cells)
Two Cells (Selectogen)
Three Cells (Surgiscreen)
Protocol
Reagent Pooled ‘O” cells for donors
Three Cells (Surgiscreen) for patients

10. Antigram- An example
An antigram (2 or 3 cells) will list the antigens present in each vial
A reaction to one or more cells indicates the presence of an unexpected antibody
AHG 37 IS P1 N M s S
Leb
Lea
Jkb
Jka
Fyb
Fya k K e c E C D
Antigens 2+ - + I II
Principle of testing ‘Unknown’ with ‘Known’

11. Antigram

12. If If an antibody is detected, it must be identified!

13. Antibody Panel - Identification
An antibody panel usually includes at least 10 panel cells:

14. Antibody Panel - Identification

15. Antibody Panel - Identification

16. Antibody Panel - Identification
Group O red blood cells

17. Antibody Panel - Identification
Each of the panel cells has been antigen typed (shown on antigram)
+ refers to the presence of the antigen
0 refers to the absence of the antigen
Example: Panel Cell #1 has 14 antigens present: C, c, e, f, M, N, S, s, P1, Lea, k, Fya,, Fyb and Jka

18. Antibody Panel - Identification
The same phase (s) used in an antibody screen is used in a panel
AHG

19. AHG Phase - Identification3 3 3

20. Interpreting Antibody Panels
Few 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
Consider antibody’s usual reactivity
Look for a matching pattern

21. 1. Ruling Out 2+    2+    2+   2+    
Cross out antigens that show NO REACTION in any phase; do NOT cross out heterozygous antigens that show dosage.

22. 2. Circle antigens not crossed out2+    2+    2+   2+    

23. 3. Consider antibody’s usual reactivity2+    2+    2+   2+    
Lea is normally a Cold-Reacting antibody (IgM), so it makes sense that we see the reaction in the IS phase of testing; The E antigen will usually react at warmer temperatures

24. 4. Look for a matching pattern
E doesn’t match and it’s a warmer rx Ab 2+    2+    2+   2+    
…Yes, there is a matching pattern!

25. Interpretation
anti-Lea

26. Rule of three! Patient serum MUST be:
Rule of three must be met to confirm the presence of the antibody
Patient serum MUST be:
Positive with 3 cells with the antigen
Negative with 3 cells without the antigen

27. Same example fulfills the “rule of three”2+  
3 Positive cells  2+    2+  
3 Negative cells 2+    
Panel Cells 1, 4, and 7 are positive for the antigen and gave a reaction at immediate spin
Panel Cells 8, 10, and 11 are negative for the antigen and did not give a reaction at immediate spin

28. Screening and Identification only in AHG Phase (Customization !)

29. Antibody Screen Expected Antibody with 3 cell panel are
(c, k, kpb, Jsb, Leb, N and s)

30. Antibody Identification
In 11 cell panel identified anti-”c” in the patient.
Confirmed patient red blood cell with rare antisera ”c”

31. Antibody Panels - Interpretation
Few 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

32. Another Example

33. Another Example

34. Phenotyping
Antigen typing the patient red cells can also confirm an antibody
If reagent antisera (of the suspected antibody) is added to the patient RBCs, a negative reaction should result…Why?
Individuals DO NOT make allo-antibodies against antigens they have
Patient has NOT been recently transfused (donor cells could react)

35. Magnitude of Problem

36. Scale of Problem 40/32560 (0.12%) or approximately 1 in 1000
15 out of 2026 (0.75%)
18/531 (3.4%) or approximately 34 in 1000
(multi-transfused patients)
Rajendra Chaudhary, Nitin Agarwal.Safety of type and screen method compared to conventional antiglobulin crossmatch procedures for compatibility testing in Indian setting. 2011:5 (2):157-15
Thakral B, Saluja K, Sharma RR, Marwaha N. Red cell alloimmunization in a transfused patient population: a study from a tertiary care hospital in north India. Hematology Oct;13(5):313-8.

37. Rh System 14 anti-D (35%) anti-D (5.6%), 6 anti-E (15%) anti-E (22.2%)
3 anti-c (7.5%)
anti-c (38.8%)
2 anti-e (5%)
1 anti-C (2.5%)
3 anti-D with anti-C (7.5%)
Kidd System
1 anti-Jk(b) (2.5%)
anti-Jk(a) (5.6%)
Lewis System
1 anti-Le(a) (2.5%)
anti-Le(a) (11.1%)
1 anti-Le(b) (2.5%)
anti-Le(b) (5.6%)
MNS System
5 anti-M (12.5%)
anti-M (11.1%)
1 anti-s (2.5%)
INCONCLUSIVE
2 (5%)
Total
40/32560
18/531
Thakral B, Saluja K, Sharma RR, Marwaha N. Red cell alloimmunization in a transfused patient population: a study from a tertiary care hospital in north India. Hematology Oct;13(5):313-8.

38. Antibody Screen Antibody Screen Screened Negative (999/1000)
Screened Positive (1/1000)

39. Antibody Screen Negative
999/1000

40. Type & Screen Vs Cross-match
45373 patients for whom a total of units of packed red blood cells (PRBC) were cross-matched in the AHG phase using DiaMed; ID cards. An antibody screen was carried out in all the patients using the DiaMed; ID-DiaCell I+II+III. The AHG cross-match was also carried out for all recipients, irrespective of the result of the antibody screen. The results were compared to see if there were any cases where the antibody screening was negative but the AHG cross-match showed incompatibility. Not a single case was found where the antibody screen was negative and AHG cross-match showed incompatibility. Type and screen policy can be safe, efficient, cost-effective, and beneficial to the transfusion service in India.
Pathak S, Chandrashekhar M, Wankhede GR. Type and screen policy in the blood bank: Is AHG cross-match still required? A study at a multispecialty corporate hospital in India. Asian J Transfus Sci 2011;5:153-6

41. Type & Screen Vs Cross-match
11000 patients were cross-matched in the AHG phase Simultaneous Type and Screen. Not a single case was found where the antibody screen was negative and AHG cross-match showed incompatibility.
Dr. Dolly Daniel – Personal Communication

42. Not as yet!
Evaluated safety of T & S procedure for PTT in comparison with conventional test tube cross match.
Study did not show the T and S to reach the expected safety level of 99%, it has achieved a reasonable level of safety (91.6%). Only one sample gave a negative antibody screen while conventional crossmatch was incompatible. This may be due to a rare antibody in the patient sera against which the corresponding antigen was not present on the screening cells but present on the donor red cell. A transfusion reaction would have occurred in this case since it was reacting at 37΀C in AHG. Since, the screening panel used in the present study was not from Indian subcontinent, therefore, it would be advisable to prepare screening cell panels that include RBC antigens which are prevalent within the local/regional population e.g, In, Mi a etc.
Rajendra Chaudhary, Nitin Agarwal. Safety of type and screen method compared to conventional antiglobulin crossmatch procedures for compatibility testing in Indian setting. 2011:5 (2):157-15

43. Can Cross-match be Omitted?
Over the 2-year interval of the study 9128 patients were entered. There were 8936 patients (97.9%) with a negative antibody screen were transfused a total of 10,899 red cell concentrates. Antiglobulin crossmatch performed after the transfusion indicated that 168 red cell concentrates (1.5%) would have been incompatible if the antiglobulin crossmatch had been performed pretransfusion. 79.2% (103/130) were false positive laboratory results. There were 27 transfusion episodes where the antiglobulin crossmatch on blood transfused was positive due to an IgG antibody. Even though these transfused red cell concentrates were designated incompatible by the antiglobulin crossmatch, none of the patients receiving this blood had clinical or serological evidence of haemolysis. We concluded that the antiglobulin phase of the crossmatch can be omitted from pretransfusion testing without putting patients at risk.
Heddle NM, O'Hoski P, Singer J, McBride JA, Ali MA, Kelton JG. A prospective study to determine the safety of omitting the antiglobulin crossmatch from pretransfusion testing. Br J Haematol Aug;81(4):579-84

44. Antibody Screen Positive
1/1000

45. Antigen negative compatible blood
Coomb’s Cross-match

46. Antigen Negative Blood?
Random Cross-match
Type the stocked blood units
Rare Donor Registry

47. Case study Case 1 Patient Name: Master S , (5 yr/M)
Hospitsl- RML hospital, New Delhi
Problem: Grouping discrepancy
Blood group: forward- “A”
Reverse- “O”
Plan of Action
Repeat grouping
Screening with 3 cell panel
Identification with 11 cell panel
Finding ‘antigen negative blood
using rare antisera, if red cell blood transfusion needed

48. Provisional diagnosis

49. Findings… Result of repeat blood grouping =Forward- “A”, Reverse- “O”
Result of antibody detection(screening)
=Probability of Anti-M and Anti- Fyb
Result of antibody identification
= Anti- “M”
Confirmation of absence of M antigen on patients red cells
= No agglutination with Antisera-”M”

51. Conclusion:
Anti- “M” antibody present in the patient. Out of 21 unit got only one “M” negative unit, preserved for Master S.

52. Comparative study of frequency of Ag
Antigen
Pos. donor (%)
Neg. donor (%)
PGI Chandigarh
(Pos %)
(Neg %) D 85
93.4 6
6.59
93.39
6.61 C 77
84.60 14
15.38
84.76
15.24 c 57
62.63 34
37.36
52.82
47.18 E 23
25.27 68
74.79
17.9
82.1 e 88
96.79 3
3.29
98.3
1.7 K 4
4.39 87
95.6
5.56
94.44 k 91
100
Fya 79
86.81 12
13.18
86.75
13.25
Fyb 62
68.13 29
31.86
56.15
43.85
Jka 72
79.12 19
20.87
82.65
17.35
JKb 53
58.24 38
41.75
66.56
33.44
Lea 13
14.28 78
85.71
20.82
79.18
Leb 54
59.34 37
40.65
60.57
43.53 S 59
64.83 32
35.16
56.47 s
87.38
12.62 M 81
89.01 10
10.98
75.39
24.61 N
61.51
38.49 P1
71.92
28.08

53. Acknowledgements
Renee Wilkins, PhD, MLS(ASCP)cm CLS 325/435; School of Health Related Professions; University of Mississippi Medical Center

54. Thank You

55. Antibody screen Crossmatch Cause Resolution Pos Neg
Antibody directed against antigen on screening cell
ID antibody, select antigen negative blood
Antibody directed against antigen on donor cell which may not be on screening cell OR donor unit may have IgG previously attached
ID antibody, select antigen negative blood OR perform DAT on donor unit
Antibodies directed against both screening and donor cells
Antibody ID, select antigen negative blood