1. Grouping and compatibility issues and their resolution
3rd Basic Haematopathology Course
TMH
Mumbai

2. Discrepancy – A conflict or variation …between things that ought to be the same Collins dictionary
In the blood bank
Reverse typing discrepant with forward grouping
Patients phenotype inconsistent with antibody profile
Group/type inconsistent with previously assigned group/type
Crossmatch results discrepant with prior or subsequent compatibility testing

3. Classification of grouping discrepancies
Group I – Unexpected reactions in serum grouping due to weakly reacting or missing antibodies
Group II – Unexpected reaction in cell grouping due to weakly reacting or missing antigens
Group III – Unexpected reactions in cell and serum grouping caused by protein or plasma abnormalities
Group IV – Miscellaneous problems

4. General principles for resolution
Eliminate the chance of clerical error or misidentification by retesting where possible on a fresh sample.
Test with alternate platforms and reagents
Use washed cells and saline replacement/dilution to eliminate type III reactions
Use enhancement techniques to bring out weak or missing reactions
Review and analyse the patient’s history for a possible role

5. Lab tests that aid in resolution The tools to him that has the ability to handle them French proverb
Multiple platforms and reagents
Antibody screening and identification
Non-routine serological tests (enhancement techniques, secretor test, adsorption and elution, etc.)
Extended phenotyping
Genotyping

6. Case 1 - Where a discrepancy aided diagnosis!
A 55 year old lady slated to undergo an orthopaedic surgery elsewhere, was referred to our blood bank. Results of grouping done elsewhere were:-
Anti A
Anti B
Anti AB
A cells
B cells
O cells 4+ 2+
Forward group:AB
Reverse group:???

7. Microscopy: Rouleaux
Rouleaux – Stacking of erythrocytes in a coin like fashion (right) can be mistaken for agglutination (left)
Saline dilution/replacement will neutralise rouleaux formation

8. With saline dilution reverse group consistent with AB.
In view of the clinical history of fracture, patient’s clinician informed of need to exclude plasma cell dyscrasia.
On further investigation, patient diagnosed to have myeloma
The presence of a discrepancy may point to a significant clinical condition

9. Case 2 – Clinical history to the rescue!
Sample from a 1 year old child was sent for grouping
Grouping results
Anti A
Anti B
Anti AB
A cells
B cells
O cells
Neg
Forward group:O
Reverse group: AB?

10. Sample retested after incubation at 4 degrees Celsius showed similar results.
Clinical history enquired into:- history of recurrent infections; suspected case of severe combined immunodeficiency syndrome
Inference: Serum grouping negative because of hypogammaglobulinemia.
Clinical history can play an important role in resolving a discrepancy

11. Case 3 – A blood group makeover!
59 year old gentleman posted for surgery for carcinoma colon.
Previously documented A group
Repeat grouping:-
Anti A
Anti B
Anti AB
A cells
B cells
O cells 4+ 2+
Neg
Forward group: AB
Reverse group: A?

12. Patient’s serum tested against patient’s own cells gave a negative reaction
Acquired B suspected in view of the above, and clinical history
Confirmed by secretor test: Showed only anti A
Inference: A group with acquired B

13. Acquired B phenotype
Seen mostly in patients with gastrointestinal lesions, particularly carcinoma colon.
Always occur in group A patients, nearly always A1
Acquired B cells do not agglutinate with patient’s own anti B
Secretors do not secrete B substance
Reversible on treatment with acetic anhydride
Ph sensitive:- Anti B reagents with PH beyond the range of do not agglutinate acquired B cells

14. CH2OH
CH2OH OH O O OH
Bacterial deacetylating
enzymes
Bacterial deacetylating
enzymes OH O O O O o OH OH OH
NHCO-
NH2
A immunodminant sugar
(N- acetyl D galactosamine)
Anti B
Acquired B
(D galactosamine)
CH2OH OH O OH OH OH
B immunodominant sugar
(D galactose)

15. Case 4 – An argument in favour of different phases of compatibility testing!
A 19 year old youth posted for an elective surgery was grouped in our blood bank. The patient had received 16 uneventful transfusion prior to this
Grouping results
Anti A
Anti B
Anti AB
A cells
B cells
O cells 4+
Neg
Forward group:AB
Reverse group:AB
Antibody screen - Negative

16. Crossmatch results Phase of testing Donor 1 Group AB Donor 2 Group A
Group O
saline
Neg
37o C 3+
Coombs 4+

17. Patients cells tested with A1 lectin – Negative reaction signifying A2B group
Incompatible units tested with lectin and found to be positive signifying A1 phenotype.
Crossmatch performed with A2B unit was compatible
Inference: A2 with anti A1

18. Significance:- Anti A1 reactive at 37o C – clinically significant!!
Would have been missed if immediate spin crossmatch alone was done eg. type and screen/electronic crossmatch scenario

19. Case 5 – Dilemma after resolution!
55 year old lady slated for elective surgery referred from elsewhere. She had received 2 uneventful transfusions of O positive blood elsewhere one year previously.
Grouping performed elsewhere.
Anti A
Anti B
Anti AB
A cells
B cells
O cells
Neg 4+
Forward group: O
Reverse group: O

20. Case 5 – Dilemma after resolution!
55 year old lady slated for elective surgery referred from elsewhere. She had received 2 uneventful transfusions of O positive blood elsewhere one year previously.
Grouping performed elsewhere.
Anti A
Anti B
Anti AB
Anti H
A cells
B cells
O cells
Neg 4+

21. Our grouping Phase Anti A B AB H H lectin A cells B cells O
cells pooled
cells donor1
cells donor2
cells donor 3
IS/
LISS
Neg 4+
4O inc -
37O inc
Coombs 2+
Lewis typing: Le(a-b+) suggesting secretor positive status
Inference: Parabombay (Oh secretor)

22. Bombay categories Classification Gene Glycosyltransferase Antigens
Secretion
Antibodies
Bombay (Red cell H deficient, non secretor)
hh sese
A and/or B
None or A and/or B (depending on ABO genotype) in RBC or serum
None
Anti-A, anti-B, anti-H
Parabombay(Red cell H partially deficient, non secretor)
hh (weak variant)
sese
A and/or B (depending on genotype)
Weak A and/or B depending on genotype. Weak H in Oh.Residual H if A or B enzymatically removed in others
Anti H, anti A and/or anti B (depending on ABO genotype)
Parabombay(Red cell H deficient secretor) hh Se
A and/or B(depending on genotype), H in serum
Weak A and/or B depending on genotype and weak H
H, A and/or B
Weak IH, anti-A and or anti-B

23. Para-bombay (Oh secretor)
H deficient secretor
Little or no A,B and H antigens
RBCs may be agglutinated by strong anti-H.
Usually not agglutinated by anti-A and anti-B but may occasionally be agglutinated by potent antisera or anti-AB
Weak H like antibody (HI) reactive at low temperature almost always present in sera. This is not inhibited by secretor saliva and does not agglutinate cord cells.
Normal H levels in saliva
Ulex europaeus (common gorse)

24. Bombay blood group not available
Bombay blood group not available! Can I transfuse O group cells in Parabombay phenotype?
(In red cell H deficient secretors) ‘Anti-HI is unlikely to be active at 37oC. ABO-identical blood, compatible by ICT at 37oC, can be used for transfusion.’
(In H partially deficient non secretors) ‘little information exists on the clinical significance of anti H. Ideally Oh (Bombay) phenotype should be selected, but if not available red cells of the appropriate ABO group (A for Ah, B for Bh) compatible by ICT at 37oC, may be used.’
Source: The clinical significance of blood group alloantibodies and the supply of blood for transfusion. (NHSBT specification SPN214/1.1)

25. Case 6 – Transfusion and transplantation
39 year old male with Chronic Myeloid Leukaemia
Underwent bone marrow transplant using stem cells from HLA identical sibling
Blood group of patient – AB positive
Blood group of donor – O positive
Received 2 units of compatible O+ve red cells in CMC, 6 days and 3 days prior to transplant

26. 1 day after transplant blood request for 1 unit packed cells received.
Forward typing of patient sample – AB+ve
Backward typing of patient sample –AB+ve
Antibody screen – Negative
Issued 1 unit of O positive blood crossmatched and compatible till Coombs
1 day after transfusion haemoglobinuria reported.

27. DD of haemolysis following haematopoietic stem cell transplantation
Major ABO incompatibility between donor and recipient
Minor ABO incompatibility between donor and recipient
Major incompatibility for other blood group antigens
Minor incompatibility for other blood group antigens
Transfusion of erythrocytes incompatible with donor or patient
Other causes of immune haemolysis eg. auto immune haemolytic anaemia, drug induced
Non immune haemolysis - TTP

28. Major incompatibility between donor and recipient
Due to recipient-derived antierythrocyte antibodies
Haemolysis immediately after transplant
Should be suspected when DCT positive
Eluate must be tested for anti A and anti B to identify haemolysis due to ABO mismatch

29. Volume of erythrocytes in bone marrow HPC product can be equal to or greater than that of a unit of blood. Therefore, significant haemolysis is possible.
Erythrocytes can be removed from the donor's bone marrow by Hetastarch separation, mononuclear cell concentration by machine, or through density gradient separation.
During apheresis collection, haematocrit should be kept to less than 2%
Other methods of prophylaxis include plasma exchange using AB plasma or in vivo adsorption using donor group plasma blood group substances; carry risk of rebound and haemolytic transfusion reactions

30. Minor incompatibility
Usually delayed haemolysis
May be due to plasma or ‘passenger lymphocytes’
Plasma in a bone marrow product to be depleted depending on agglutinin tire.
Diagnosis - DCT positive with eluate showing anti A or anti B

31. Passenger lymphocyte syndrome
Donor derived lymphocytes in the HPC graft form blood group specific antibodies to patient erythrocytes
Usually ABO antibodies but can involve other blood group antibodies. (Kell, Kidd, Duffy reported)
Risk factors inclcude treatment with cyclosporine alone without an antproliferative agent like methotrexate, low intensity conditioning and ?PBSCT (theoretically, since it contains more B lymphocytes)
Usually begins 1-2 weeks after transplant, persists for 5-10 days, then subsides
Haemolysis can be severe
Can involve transfused compatible erythrocytes (bystander immune haemolysis)
Management - Transfusion of compatible erythrocytes at a pace that matches haemolysis
In massive haemolysis with risk of renal injury, exchange transfusion may be considered

32. Incompatibility due to other blood group antibodies
Supected when DCT is positive and antibody screen positive for donor or recipient
The eluate shows an irregular antibody

33. Investigations in our patient
DCT +ve (2+)
Post transfusion sample – ICT +ve; antibody screen positive (1+)
Antibody ID – anti Jkb (3+)
Transfused red cells – Jkb positive
Phenotype of stem cell donor- D+; C4+; E-; c4+; e4+; K-; Fya3+; Fyb -; Jka3+; Jkb3+
Phenotype of patient – D+; C4+; E-; c4+; e4+; K-; Fya3+mf; Fyb 3+mf; Jka3+mf; Jkb3+mf

34. Inference: Delayed haemolytic transfusion reaction due to anti-Jk(b) formed by patient reacting with transfused cells.
Subsequent transfusions with Jkb negative fully compatible units.
However, haemoglobinuria persisted until 54 days after transplant, when antibody screen turned negative.

35. Pure red cell aplasia Occurs in major mismatch
Recipient lymphocytes may remain and later produce antibodies to transplanted donor derived erythrocytes
Can occur early or late (>100 days)
Suppress erythropoiesis by destroying erythroid progenitors
Diagnosis – Based on reticulocytopenia persisting for more than 60 days with absence of erythroids in marrow
Delayed erythrocyte engraftment occurs in 20% of ABO mismatched transplants
?related to haemagglutinins

36. Transfusion support in mismatched transplants
Must be compatible with recipient and donor
Such compatible blood must be selected prior to transplantation (from the start of myeloablation)as red cells may persist for weeks

37. 3 phases of transplantation
RBC tx:DCT negative and recipient
isoagglutinins not detectable
Beginning of preparation
Phase 1
Phase 2
Phase 3
Transplant
Plasma and platelets tx: Recipient red cells not detectable
Recipient group
Compatible with
both donor and recipient
Donor group

38. Selection of blood products in phase 2
Recipient RBC group
Donor's RBC group
Category of ABO mismatch†
Erythrocyte transfusion
Platelet or plasma transfusion A O
Minor
A, AB B
B, AB AB
A, O
B, O
Major
Minor and major
None
AB, A, B, O
O, A, B, AB

39. Blood group chimerism
Forward or backward grouping discrepancies and mixed field agglutination
Intrinsic characteristic of mismatched transplantation eg. A group recipient transplanted with O group donor
Anti-A
Anti-B
Anti-AB
A cells
B cells
O cells
Inference MF
Neg 4+
Mixed field on front type may be due to transfused O cells/ conversion. Recipient red cells still seen. Reverse type is recipient group 2+
Front and reverse type donor group, indicating full donor engraftment

40. Case 7 – ABO mismatch in kidney transplant
A 21 year old gentleman of blood group O-positive, with end stage kidney disease
Only available related donor was mother whose blood group was A-positive.
HLA-crossmatch by Complement Dependent Cytotoxicity (CDC) was negative
Anti-A titre at presentation was 1:512.
Desensitization protocol involving Rituximab, plasmapheresis and Anti-thymocyte globulin initiated

41. Column agglutination test used for monitoring
Schedule of monitoring: Pre-pheresis, immediately post pheresis and 24 hours later to detect rebound
A group FFP used for replacement in order to quench anti A in recipient.
Rituximab interfered with HLA CDC crossmatch. HLA antibody monitoring supplemented with solid phase assays
Anti A titre reduced to 1:2 against which transplant performed successfully.
3 years post transplant - graft functional, patient well

43. A brief history of ABO incompatible kidney transplant (ABOi KT)
earliest efforts by Chung et al. 8/ 10 grafts failed within few days
1987 – Thielke et al transplanted A2 grafts into O donors. 12/20 retained long term function
1987- Alexandre et al used plasmapheresis in protocol and achieved 75% 1 year graft survival
From 1989 popularised in Japan (near absence of deceased donors and only 0.15% A2). 14 % of all transplants in Japan.
From 2000, gained acceptance in western countries
Today, outcome not different from other transplants*
*J. M. Gloor and M. D. Stegall, “ABO incompatible kidney transplantation,” Current Opinion in Nephrology and Hypertension, vol. 16, no. 6, pp. 529–534, 2007. 

44. Principles of ABOi KT
Removal of existing antibody – plasma exchange, double filtration plasmapheresis, immunoadsorption
Preventing recurrence of antibody – IVIg, immunosuppression, splenectomy/ Rituximab
Waiting until titres achieve safe levels for transplantation

45. Range of Plasmapheresis Treatments required Before and After ABOiKT based on initial antibody titre
Lipshutz GS et al. Arch Surg. 2011;146(4):

46. Monitoring of antibody titre
Baseline antibody levels (> 256) predict severity of antibody mediated graft injury and graft survival. Reports suggest this is no longer true in patients that received tacrolimus or mycophenolate mofetil for immunosuppression*
Titre cutoff for transplant 16– empirically chosen
Titre endpoint based on ?IgG or ?IgG and IgMB.
* H. Chung, J. Y. Lee, S. H. Kang et al., “Comparison of clinical outcome between high and low baseline anti-ABO antibody titers in ABO-incompatible kidney transplantation,” Renal Failure, vol. 33, no. 2, pp. 150–158, 2011.

47. Possible outcomes of ABOi KT
Rejection (2-5%)
Immunological tolerance – ABO antibodies against graft do not reaccumulate. ?an outcome of prolonged B cell/ T cell suppression
Accomodation – ABO antibodies recur, but the graft is not rejected. Due to ?change in quality of antibody eg. shift in IgG isotype ?change in allograft eg. change in balance of apoptotic proteins bcl-2 and bcl-xl, up regulation of complement inhibitory substance CD59

48. If we cannot end our differences, at least we can make the world safe for diversity John F. Kennedy
Acknowledgement : Dr. Mary Purna Chacko
Asst. Prof: Dept of Transfusion Medicine
So, to conclude, the HLA system play a major role in complications associated with transplantation and transfusion owing to its polymorphism and its crucial role in allo-recognition. Understanding the complex role of these molecules helps us to prevent and minimise these reactions.