Presentation on theme: "Grouping and compatibility issues and their resolution"— Presentation transcript:
1 Grouping and compatibility issues and their resolution 3rd Basic Haematopathology CourseTMHMumbai
2 Discrepancy – A conflict or variation …between things that ought to be the same Collins dictionary In the blood bankReverse typing discrepant with forward groupingPatients phenotype inconsistent with antibody profileGroup/type inconsistent with previously assigned group/typeCrossmatch 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 antibodiesGroup II – Unexpected reaction in cell grouping due to weakly reacting or missing antigensGroup III – Unexpected reactions in cell and serum grouping caused by protein or plasma abnormalitiesGroup 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 reagentsUse washed cells and saline replacement/dilution to eliminate type III reactionsUse enhancement techniques to bring out weak or missing reactionsReview 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 proverbMultiple platforms and reagentsAntibody screening and identificationNon-routine serological tests (enhancement techniques, secretor test, adsorption and elution, etc.)Extended phenotypingGenotyping
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 AAnti BAnti ABA cellsB cellsO cells4+2+Forward group:ABReverse group:???
7 Microscopy: RouleauxRouleaux – 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 myelomaThe 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 groupingGrouping resultsAnti AAnti BAnti ABA cellsB cellsO cellsNegForward group:OReverse 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 syndromeInference: 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 groupRepeat grouping:-Anti AAnti BAnti ABA cellsB cellsO cells4+2+NegForward group: ABReverse 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 historyConfirmed by secretor test: Showed only anti AInference: A group with acquired B
13 Acquired B phenotypeSeen mostly in patients with gastrointestinal lesions, particularly carcinoma colon.Always occur in group A patients, nearly always A1Acquired B cells do not agglutinate with patient’s own anti BSecretors do not secrete B substanceReversible on treatment with acetic anhydridePh sensitive:- Anti B reagents with PH beyond the range of do not agglutinate acquired B cells
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 thisGrouping resultsAnti AAnti BAnti ABA cellsB cellsO cells4+NegForward group:ABReverse group:ABAntibody screen - Negative
16 Crossmatch results Phase of testing Donor 1 Group AB Donor 2 Group A Group OsalineNeg37o C3+Coombs4+
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 compatibleInference: 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 AAnti BAnti ABA cellsB cellsO cellsNeg4+Forward group: OReverse 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 AAnti BAnti ABAnti HA cellsB cellsO cellsNeg4+
21 Our grouping Phase Anti A B AB H H lectin A cells B cells O cells pooledcells donor1cells donor2cells donor 3IS/LISSNeg4+4O inc-37O incCoombs2+Lewis typing: Le(a-b+) suggesting secretor positive statusInference: Parabombay (Oh secretor)
22 Bombay categories Classification Gene Glycosyltransferase Antigens SecretionAntibodiesBombay (Red cell H deficient, non secretor)hh seseA and/or BNone or A and/or B (depending on ABO genotype) in RBC or serumNoneAnti-A, anti-B, anti-HParabombay(Red cell H partially deficient, non secretor)hh (weak variant)seseA 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 othersAnti H, anti A and/or anti B (depending on ABO genotype)Parabombay(Red cell H deficient secretor)hhSeA and/or B(depending on genotype), H in serumWeak A and/or B depending on genotype and weak HH, A and/or BWeak IH, anti-A and or anti-B
23 Para-bombay (Oh secretor) H deficient secretorLittle or no A,B and H antigensRBCs 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-ABWeak 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 salivaUlex 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 LeukaemiaUnderwent bone marrow transplant using stem cells from HLA identical siblingBlood group of patient – AB positiveBlood group of donor – O positiveReceived 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+veBackward typing of patient sample –AB+veAntibody screen – NegativeIssued 1 unit of O positive blood crossmatched and compatible till Coombs1 day after transfusion haemoglobinuria reported.
27 DD of haemolysis following haematopoietic stem cell transplantation Major ABO incompatibility between donor and recipientMinor ABO incompatibility between donor and recipientMajor incompatibility for other blood group antigensMinor incompatibility for other blood group antigensTransfusion of erythrocytes incompatible with donor or patientOther causes of immune haemolysis eg. auto immune haemolytic anaemia, drug inducedNon immune haemolysis - TTP
28 Major incompatibility between donor and recipient Due to recipient-derived antierythrocyte antibodiesHaemolysis immediately after transplantShould be suspected when DCT positiveEluate 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 haemolysisMay 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 erythrocytesUsually 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 subsidesHaemolysis can be severeCan involve transfused compatible erythrocytes (bystander immune haemolysis)Management - Transfusion of compatible erythrocytes at a pace that matches haemolysisIn 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 recipientThe 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 positivePhenotype 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 erythrocytesCan occur early or late (>100 days)Suppress erythropoiesis by destroying erythroid progenitorsDiagnosis – Based on reticulocytopenia persisting for more than 60 days with absence of erythroids in marrowDelayed erythrocyte engraftment occurs in 20% of ABO mismatched transplants?related to haemagglutinins
36 Transfusion support in mismatched transplants Must be compatible with recipient and donorSuch 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 recipientisoagglutinins not detectableBeginning of preparationPhase 1Phase 2Phase 3TransplantPlasma and platelets tx: Recipient red cells not detectableRecipient groupCompatible withboth donor and recipientDonor group
38 Selection of blood products in phase 2 Recipient RBC groupDonor's RBC groupCategory of ABO mismatch†Erythrocyte transfusionPlatelet or plasma transfusionAOMinorA, ABBB, ABABA, OB, OMajorMinor and majorNoneAB, A, B, OO, A, B, AB
39 Blood group chimerismForward or backward grouping discrepancies and mixed field agglutinationIntrinsic characteristic of mismatched transplantation eg. A group recipient transplanted with O group donorAnti-AAnti-BAnti-ABA cellsB cellsO cellsInferenceMFNeg4+Mixed field on front type may be due to transfused O cells/ conversion. Recipient red cells still seen. Reverse type is recipient group2+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 diseaseOnly available related donor was mother whose blood group was A-positive.HLA-crossmatch by Complement Dependent Cytotoxicity (CDC) was negativeAnti-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 reboundA 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 assaysAnti 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 days1987 – Thielke et al transplanted A2 grafts into O donors. 12/20 retained long term function1987- Alexandre et al used plasmapheresis in protocol and achieved 75% 1 year graft survivalFrom 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 countriesToday, 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 KTRemoval of existing antibody – plasma exchange, double filtration plasmapheresis, immunoadsorptionPreventing recurrence of antibody – IVIg, immunosuppression, splenectomy/ RituximabWaiting 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 chosenTitre 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 suppressionAccomodation – 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 ChackoAsst. Prof: Dept of Transfusion MedicineSo, 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.