ABO discrepancies 24 July 2019 Dr. Rujirek Chaiwongsa Division of Transfusion Sciences Department of Medical Technology Faculty of Associated Medical Sciences Chiang Mai University 1
Landsteiner’s rule 1868-1943 “Normal, healthy individuals possess ABO Abs to ABO Ags 2 absent from their RBCs”
Importance - Accurate donor and patient ABO blood groups are fundamental to transfusion safety because of presence of ABO Abs in individual with no exposure to human RBCs -Transfusion of ABO-incompatible blood to patient may result in intravascular hemolysis and other serious consequence of acute hemolytic transfusion reaction (HTR) -Misinterpreting ABO discrepancies could be life threatening to patients 3
ABO discrepancies Are recognized when the reactions obtained in the forward type do not match the reactions obtained in the reverse type. ABO Typing Results Cell grouping Serum grouping Anti-A Anti-B A1 Cells B Cells 4+ 1+ Neg 4+ Group AB Group A 4
ABO discrepancies •Reaction strengths could be weaker than expected •Some reactions may be missing in the reverse or forward typing •Extra reactions may occur Sample errors Technical errors -Patient cause -Treatment cause ØIdentification or documentation errors ØReagent or equipment errors ØStandard operating procedure errors
Guidelines for investigating ABO technical errors Identification or documentation errors - Correct sample identification on all tubes - Results are properly recorded - Interpretation are accurate and properly recorded Interpretation Identification http://link.springer.com/chapter/10.1007/978-1-4613-2835-3_21 6
Reagent or equipment errors - Daily quality control on ABO typing reagents in satisfactory - Inspect reagent for contamination and hemolysis - Centrifugation time and calibration are confirmed 7
Standard operating procedure errors - Procedure follows manufacturer’s directions - Correct reagents were used and added to testing - RBC suspensions are at correct concentration -Cell buttons are completely suspended before grading reaction Performance http://link.springer.com/chapter/10.1007/978-1-4613-2835-3_21 8
If transfusion is necessary before resolving the ABO discrepancy the recipient should receive Group O Group AB Plasma Red blood cell 9
ABO discrepancies RBC testing Serum testing Extra Ags Missing/Weak Ag Mixed Field Extra Abs Missing/Weak Ab Acquired B phenotype A/B Subgroup O Transfusion A subgroup Newborn with Anti-A1 Elderly B(A) or A(B) Phenotype Pathogenic Bone Marrow/ Pathology etiology Stem Cell Cold etiology Transplants Alloantibodies Immunosuppressive Polyagglutination A3 or B3 Cold therapy phenotype Autoantibodies Rouleaux Rouleaux May cause false positive reaction Hematopoietic progenitor cell transplants 10
Resolution of ABO discrepancy • The problem is related with RBC testing or serum testing? 11 • Patient’s history? - Diagnosis? - Been transfused? - Age? • What is an additional test? • Interpretation
Cell Grouping Problem 12
Case 1 ABO Typing Results Cell grouping Serum grouping Anti-A Anti-B A1 Cells B Cells 4+ 1+ Neg 4+ Group AB Group A Extra antigen 13
Evaluation of ABO testing results ABO Typing Results Cell grouping Serum grouping Anti-A Anti-B A1 Cells B Cells 4+ 1+ Neg 4+ 1. The agglutination of the patient’s red cells with anti-A is strong (4+). 2. The agglutination of the patient’s red cells with anti-B is weaker (1+) than usually expected (3+ to 4+). 3. The results of serum testing reactions are typical of a group A individual. Acquired B phenotype 14
Acquired B phenotype nUsually group A individual, with disease of … nLower gastrointestinal tract nCancers of colon and rectum nIntestinal obstruction nGram negative septicemia (i.e. E.coli) https://www.fairview.org/HealthLibrary/Article/82149 Most common mechanism: 15 associated with a bacterial deacetylating enzyme
https://labmedicineblog.files.wordpress.com/2014/09/acqb.png 16
Resolution of ABO discrepancies (Acquired B phenotype) 1. Determine patient’s diagnosis and transfusion history 2. Test patient’s serum against autologous RBCs: Neg 3. Test RBCs with additional monoclonal anti-B reagents from other manufacturers that are documented not to react with acquired B Ag or source of human polyclonal anti-B 17
Case 2 ABO Typing Results Cell grouping Serum grouping Anti-A Anti-B A1 Cells B Cells 1+ 4+ 4+ Neg Group AB Group B Extra antigen 18
Evaluation of ABO testing results ABO Typing Results Cell grouping Serum grouping Anti-A Anti-B A1 Cells B Cells 1+ 4+ 4+ Neg 1. The agglutination of the patient’s red cells with anti-A is weak (1+). 2. The agglutination of the patient’s red cells with anti-B is strong (4+). 3. The results of serum testing are typical of a group B individual. 19
ABO Typing Results B(A) phenotypes Cell grouping Serum grouping Anti-A Anti-B A1 Cells B Cells 1+ 4+ 4+ Neg nD-galactosyl transferase transfers trace amount of N-acetylgalactosamine to H antigens ABO Typing Results A(B) phenotypes Cell grouping Serum grouping n N-acetylgalactosaminyl transferase transfers trace amount of Anti-A Anti-B A1 Cells B Cells D-galactose to H antigens 4+ 1+ neg 4+ 20
B(A) and A(B) phenotypes Result of increased sensitivity of potent monoclonal reagents for ABO phenotyping Detect trace amount of either A or B Ag that are nonspecifically transferred by glycosyltransferase enzymes 21
Resolution of ABO discrepancies (B(A) phenotype) 1. Determine patient’s diagnosis and transfusion history 2. Test RBCs with additional monoclonal anti-A reagents from other manufacturers or source of human polyclonal anti-A 22
Other potential explanations for extra antigens • Polyagglutination •Agglutination of RBCs by most human sera •Exposure of hidden T antigen on RBC membrane because of a bacterial infection •Non specific aggregation of serum suspended RBCs •Wharton’s jelly in cord blood •Abnormal concentration of serum proteins (Rouleaux) 23
Case 3 ABO Typing Results Cell grouping Serum grouping Anti-A Anti-B A1 Cells B Cells Neg Neg Neg 3+ Group O Group A Missing or weakly expressed antigens Subgroup A? 24
Resolution of ABO discrepancies (Subgroup A) 1. Determine patient’s diagnosis and transfusion history 2. Repeat RBC typing with extended incubation times and include human polyclonal anti-A,B or monoclonal blend anti-A,B 25
Anti-A: Neg Additional testing results Anti-A,B Reaction with Anti-A,B Interpretation 1+ Ax (Agglutination with anti-A,B only; anti-A1 almost always present in serum) neg Ael (Adsorbs and elutes anti-A; anti-A1 usually present in serum) 26
Reaction with Anti-A antibody Y YY Y Y YY Y YY Elution Expect to be EYlYuYaYtYeY group A Y Y Prove with A cells Y Y Agglutination if it is A blood group 27 It’s named Ael blood group
Case 4 ABO Typing Results Cell grouping Serum grouping Anti-A Anti-B A1 Cells B Cells Neg 2+mf* 3+ Neg Group B Group B Mixed field reactions Group B transfused with group O RBCs? 28
Resolution of ABO discrepancies (Group B transfused with group O RBC) 1. Determine patient’s diagnosis and recent transfusion history 2. Determine if patient is recently bone marrow or stem cell transplanted 3. Investigate pretransfusion ABO phenotype history, if possible 29
Mixed field reactions •Agglutination pattern where a population of the RBCs shows agglutinates with mass of unagglutinated RBCs http://www.traqprogram.ca/images/micro-mfa.jpg https://www.researchgate.net/figure/6240211_fig1_Monoclonal-gel-card-demonstrating-the- mixed-field-agglutination-arrows-presents-on-the 30
Unexpected or extra reactions in the cell grouping 1. Perform patient history check 2. Repeat the ABO grouping using -transfused with non-group specific 3-5% washed cell suspension RBC -received an ABO mismatched stem cell or BM transplant If the problem is resolved If the problem is not resolved -Perform a final clerical check -Suspect a cold agglutinin and -Interpret the ABO grouping perform a cold agglutinin investigation 31
Incubate the washed Enzyme patient cells with anti- treated cell A and anti-B for 15 minutes at 18-25O C Adsorption and elution To enhance the detection of weakly expressed antigens: 32
Serum Grouping Problem 33
Case 5 ABO Typing Results Cell grouping Serum grouping Anti-A Anti-B A1 Cells B Cells 4+ Neg 2+ 4+ Group A Group O Additional antibodies in serum testing 34 Subgroup A2 with anti-A1?
Resolution of ABO discrepancies (Subgroup A2 with anti-A1) 1. Determine patient’s diagnosis and recent transfusion history 2. Test patient’s RBCs with anti-A1 lectin (Dolichos biflorus) to ascertain if subgroup of A is present Neg Subgroup of A: suspect anti-A1 35 http://medplants.blogspot.com/2012/05/vigna- unguiculata-dolichos-biflorus.html
3. Test patient’s serum with 3 examples of A1 and A2 cells to confirm presence of anti-A1 Additional testing results Patient serum testing with A1 Cells A1 Cells A1 Cells A2 Cells A2 Cells A2 Cells 2+ 2+ 2+ Neg Neg Neg Conclusion: 36 Agglutination is observed with A1 red cells providing for anti-A1 Anti-A1 may be present in 1-8% of subgroup A2
Case 6 ABO Typing Results Cell grouping Serum grouping Anti-A Anti-B A1 Cells B Cells 4+ 4+ Neg 1+ Group AB Group A Additional antibodies in serum testing Cold alloantibody or cold autoantibody? 37
Patients may possess antibodies to other blood group system RBC antigens in addition to those of ABO system Cold alloantibodies Cold Autoantibodies Anti-P1 Anti-M Anti-N Anti-Lea Anti-Leb Anti-I Anti-IH 38
Resolution of ABO discrepancies (Cold alloantibody or cold autoantibody) 1. Determine patient’s diagnosis and recent transfusion history 2. Test patient’s RBCs with screening cells and autocontrol at RT Interpretation of testing results Sc cells Ac cells Conclusion Patient serum Pos Neg Cold alloantibody Patient serum Pos Pos Cold autoantibody 39
3. If alloantibody is detected, Ab identification can be performed 4. If autoantibody is detected, special techniques to identify Ab (a minicold panel) and remove Ab reactivity (prewarming techniques) can be used 40
Prewarm technique To prevent reactions of cold Abs binding at RT and activating complement • Incubate patient’s whole blood at 37°C for 10 min • Wash patient’s RBCs 3 times with prewarmed saline Cell grouping Serum Autoadsorption Serum grouping 41
Case 7 ABO Typing Results Cell grouping Serum grouping Anti-A Anti-B A1 Cells B Cells 4+ 4+ 2+ 2+ Group AB Group O Additional antibodies in serum testing? Rouleaux? 42
“Rouleaux formation” - Abnormal serum protein e.g. liver disease, multiple myeloma, Waldenstrom’s macroglobulinemia - Present of high-molecular-weight dextran or other plasma expander - Wharton jelly (gelatinous tissue contaminant in cord blood samples) http://www.medical-labs.net/autoagglutination-vs-rouleaux-formation-2605/ 43 RBCs appear as stacked coins
Resolution of ABO discrepancies (Rouleaux) 1. Determine patient’s diagnosis and recent transfusion history 2. Wash RBC suspension and repeat phenotyping 3. Perform saline replacement technique to help distinguish true agglutination from rouleaux 44
Saline replacement technique Incubate test serum and RBCs Centrifuge for 1 min and remove serum with a pipette Replace test serum with an equal volume of saline Mix Centrifuge for 15 sec and resuspend cell button gently No agglutination Agglutination Rouleaux True agglutination 45
Case 8 ABO Typing Results Cell grouping Serum grouping Anti-A Anti-B A1 Cells B Cells Neg Neg Neg Neg Group O Group AB Missing or weak antibodies in serum testing Newborn, eldery, lower immunoglobulin level? 46
Resolution of ABO discrepancies (Missing or weak antibodies in serum testing) 1. Determine patient’s diagnosis, age and immunoglobulin levels if available • Newborn or elderly • Lower immunoglobulin level • Hypogamma globulinemia • Agamma globulinemia • Immunosuppressive therapy • Serum protein electrophoresis may be useful 47
Serum protein electrophoresis albumin α1 α2 β g Normal pattern Hypogammaglobulinemia http://pro2services.com/lectures/Winter/Proteins/protein.htm 48
2. Incubate serum testing for 15 min at RT and centrifuge and examine for agglutination Neg Place serum testing at 4°C for 5 min with Ac control Additional testing results 4°C A1 Cells B Cells Ac Cells Conclusion Patient serum Pos Pos Neg Group O Patient serum Pos Pos Pos Cold autoantibody 49
Unexpected or extra reactions in the serum grouping Repeat the reverse grouping using saline replacement method Extra reaction Extra reaction are not are eliminated eliminated Multiple myeloma, Discrepancy caused by an Anti- macroglobulinemia, infusion A1 and/or a cold agglutinin of dextran Perform Anti-A1 lectin Record results testing neg Pos A2 or A2B with Cold agglutinin anti-A1 50
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