Errors and Discrepancies in ABO Typing · 9 頁
[[p.1]] In the ABO system of blood groups, if an antibody is present in the serum, the reciprocal antigen will be absent from the red cell membrane and vice versa is true. When the cell grouping does not correlate with the serum grouping, there is said to be a discrepancy in ABO grouping. Mistyping either a donor or recipient can lead to transfusion with ABO-incompatible blood. ABO antibodies can activate complement and result in vivo haemolysis. The clinical conse- quences of this in vivo destruction can be significant and may even result in death of the recipient. Most errors being technical errors and clerical errors, other ABO discrepancies can be classified into 4 categories: Category I: Weakly reacting or missing antigens Category II: Weakly reacting or missing antibodies Category III: Unexpected antigen reactions Category IV: Unexpected antibody reactions Technical errors and clerical errors 1. Incorrect labeling 2. Mix-up of samples 3. Poor quality/expired reagents/red cells 4. Contaminated reagents 5. Inappropriate storage temperature 6. Failure to add reagents 7. Failure to follow manufacturer’s instructions 8. Missed observation by untrained staff 18 Errors and Discrepancies in ABO Typing 162 [[p.2]] Errors and Discrepancies in ABO Typing 163 9. Equipment variables 10. Uncalibrated instruments 11. Contaminated glassware 12. Wrong entry of results 13. Transcription error 14. Computer entry error Resolution: Re-check the label. Ask for new sample. Check for red cell suspension or freshly prepare the red cell suspension. Check QC of reagents. Check equipment. Verify the patient data for previous blood group report and history of transfusion. Category I: Weakly Reacting or Missing Antigens ABO subgroups (subgroups of A or B): Rare subgroups of a like A2, Ax, Am, Ae, etc. have weak antigens. Sometimes associated with presence of Anti-A1 antibodies. In forward grouping appears as O, but reverse grouping appears as A or B. A example is given below. Antibodies Cells Anti-A Anti-B Anti-AB A1 A2 B O Auto 1 min spin 0 0 0 0 0 4+ 0 0 RT 15 min 0 0 1+ 0 0 4+ 0 0 4°C 30 min 1+ 0 2+ 1+ 0 4+ 0 0 Note: Weak subgroup of A (A2), reaction can be enhanced by incubation at room temperature and at 4°C. Anti-A1 can be demonstrated in serum at 4°C. Antibody screen for O cells and auto control should be performed. Resolution: Incubation at room temperature and 4°C will enhance the reactivity of antibodies. ABO antibodies at cold temperature of 4°C incubation can demonstrate weak reacting antigen. ABO subgroups can be classified based on strength of agglutination with ABO antisera. [[p.3]] 164 Essentials of Blood Banking and Transfusion Medicine Pathologic causes: In acute leukemia there is weakened expression of A and B antigens. Carcinoma of stomach and pancreas: There can be excess of soluble blood group substances in the plasma which may neutralize the anti-A and anti-B antibodies causing a false negative reaction. Resolution: Washing removes blood specific substances. Only red cells will remain with washing which gives correct grouping. Antibodies Cells Anti-A Anti-B Anti-AB A1 B O Auto Unwashed RBCs 0 0 0 4+ 0 0 0 Washed RBCs 0 4+ 4+ Note: With washed RBCs, it is B blood group. Category II: Weakly Reacting or Missing Antibodies Antibodies Cells Anti-A Anti-B Anti-AB A1 cells B cells O cells Auto 0 0 0 0 0 0 0 Note: In the above example, with cell grouping it is O blood group, but reverse grouping no antibodies are detected with A1, B and O cells. Possible cause is—infant, elderly or patho- logical cause such as hypogammaglobulinemia. In such case check the age and diagnosis of the case. Weakly reacting or missing antibodies are found in the following conditions. • Newborns: Missing antibodies in reverse grouping are found in neonatal specimens as infants do not demonstrate anti-A and anti-B antibodies (IgM) at birth. They can have passively acquired maternal antibodies and these are usually IgG antibodies. Titers of anti-A and anti-B increase by 6 months of age. Resolution: Reverse grouping is not done in neonates. [[p.4]] Errors and Discrepancies in ABO Typing 165 • Elderly: Elderly patients demonstrate decreased reactivity in reverse grouping due to decrease in antibody production. • Pathologic causes: Congenital agammaglobulinemia, hypogammaglobulinemia and X-linked Wiskott-Aldrich syndrome have low concentration of anti-A and anti-B antibodies. CLL, multiple myeloma, Waldenstrom’s macroglobulinemia patients who are on immunosuppressive therapy or plasma exchange. Serum protein electrophoresis and immunoglobulin levels should be done to confirm the possibility of the diseases. • Bone marrow transplant: Immunosuppression produces temporary hypogammaglobulinemia. These cases have reduced titers of ABO antibodies. • Prozoning: 1. Prozone phenomenon is associated with antibody excess. High antibody titers may not cause the agglutination of A or B cells, as all antigen sites are bound and visual agglutination is not produced. 2. High titres of anti-A or anti-B are not detected due to steric hindrance by complement C1. Resolution: Repeat with a1:10 dilution of the serum with saline or 5 minutes incubation at room temperature. Category III: Unexpected Antigen Reactions • When strong serum group reactions are contradicted by weak antigen reactions of the same specificity, unexpected antigen reaction should be suspected. • When an individual appears to be group AB, careful evalua- tion with auto controls, bovine albumin, etc. should be used for validation the result. Causes of unexpected antigen reactions include: • Polyagglutination • Antibody coated red cells • Acquired B antigens • Rouleaux formation • Multiple myeloma [[p.5]] 166 Essentials of Blood Banking and Transfusion Medicine • Waldenstrom’s macroglobulinemia • Plasma cell dyscrasiasis • Wharton’s jelly and serum separator tubes containing gels • Chimerism Washing the cells, free of serum allows a valid cell typing. Antibodies Cells Anti-A Anti-B Anti-AB A1 B O Auto Unwashed RBCs 4+ 2+ 4+ 2+ 4+ 2+ 2+ Washed RBCs 4+ 0 4+ Saline replacement 0 4+ 0 0 Note: In the above example, there is discrepancy with forward and reverse grouping and autocontol has reaction. With washed RBCs it is A blood group. Saline replacement disperses the rouleaux formation. Antibody coated red cells can be found in following situations: • Cold autoantibody • Warm autoantibody • Haemolytic transfusion reaction and HDN Resolution: Washing red cells or warming tube to 37°C will disperse the agglutination. Polyagglutination Bacterial infection particularly due to Clostridium species exposes the hidden T antigen. This is due to the action of bacterial neuraminidase which removes sialic acid from the red cell surface. These Tn activated cells are agglutinated by adult human anti-sera as they have antibody to this antigen, giving false positive results. • Cells are agglutinated by ABO compatible adult human sera but not by cord sera. • Due to IgM polyagglutinins present in normal adult sera. • They react with abnormal membrane structure exposed by microbial enzymes (T, Tk, Th, VA) or a somatic mutation. [[p.6]] Errors and Discrepancies in ABO Typing 167 • Using monoclonal antisera can be used to obtain valid results. • These antibodies can be classified using lectins. Antibodies Cells Anti-A Anti-B Anti-AB A1 B O Auto Polyclonal antisera 1+ 1+ 1+ 4+ 4+ 0 0 Monoclonal antisera 0 0 0 Note: Above example shows T-activation which fails to react with monoclonal sera. Resolution: 1. Treat the cells with proteolytic enzymes before testing which can destroy Tn exposed RBCs. 2. Use monoclonal anti-sera to obtain valid results. Acquired B Antigen Patient with bacterial infections, often cancer of colon or rectum may have false B like antigen. Due to action of microbial enzymes causing deacetylation of A antigen or adsorption of bacterial polysaccharide. Removal of acetyl group, the N-acetyl galactosamine, resulting in galactosamine which crossreacts with anti-B giving the cells to appear as B cells (Fig. 18.1). By altering the pH (lowering) of the anti-B reagent to the antigen–antibody reaction can be avoided. Diagram to show acquired B antigen Fig. 18.1: Formation of acquired B [[p.7]] 168 Essentials of Blood Banking and Transfusion Medicine Resolution: 1. Check the patient’s diagnosis. 2. Test the patient’s serum with his or her own red cells. No agglutination is seen if acquired B determinant is present, i.e. anti-B in the serum of acquired B person does not aggluti- nate autologous red cells. 3. Test the RBCs with monoclonal anti-B anti-sera. The agglutination is fragile and easily dispersed • In vitro immune complex attachment – Formed by dyes, drugs and reagents preservatives which get adsorbed onto the red cells. These are removed by washing the cells. • Contaminating antibody in the sera – Anti-Yt, anti-Co antibodies to low incidence antigens – Repeat testing with a different lot of reagents gives valid results. Agents causing non-specific agglutination • Serum separator tubes containing gels may cause non- specific aggregation of red cells. • Wharton’s jelly in cord blood: Wharton’s jelly in cord blood can trap the RBCs appearing like agglutination. Resolution: Wash cells minimum two times before testing. Problems are less, if the blood sample is taken with needle and syringe. Using autologous controls, 6–8% albumin and washing the red cells with warm saline may yield a valid typing. Chimerism • This is temporary or genetic condition with simultaneous presence of two or more populations of red cells. • Seen with – Intrauterine transfusion – Exchange transfusion – Allogenic bone marrow transplants – Feto-maternal haemorrhage – Dispermy – Mosiacism [[p.8]] Errors and Discrepancies in ABO Typing 169 Genetic chimerism: Exchange of hematopoietic tissue between fraternal twins. Mosiacism: Occurs with dispermy when one ovum is fertilized by two sperms. Two populations of cells are recognised as self. Category IV: Unexpected Antibody Reactions • This is the most common cause of ABO discrepancies. • Autologous controls and serum grouping with O cells are needed to detect the discrepancy. • Causes include: – Rouleaux and pseudoagglutination – A subgroups with anti-A1 antibodies – Cold autoantibodies – Cold alloantibodies – Passively acquired anti-A or anti-B • Rouleaux and pseudoagglutination: Elevated levels of globins from multiple myeloma, Waldenstrom’s macro- globulinemia, elevated level of fibrinogen, high molecular weight expanders, etc. Resolution: True agglutination is stable in saline, whereas rouleaux and pseudoagglutination will disperse with saline • A subgroup with anti-A1 – Anti-A1 is found in 1–8% of A2 group individuals, 22–35% of A2B group individuals. – Anti-A1 antibodies are usually not reactive at 37°C, how- ever, if reactive at 37°C, a compatible blood which is A1 negative should be issued. Antibodies Cells Anti-A Anti-B Anti-AB Anti-A1 A1 A2 B O Auto 4+ 0 4+ 4+ 0 0 Additional 4+ 0 4+ 0 2+ 0 4+ 0 0 test Note: In this example, it is blood group A2 with anti-A1 antibodies, cold antibodies. • Autoanti-I and anti-IH cause strong agglutination of all adult cells at room temperature. [[p.9]] 170 Essentials of Blood Banking and Transfusion Medicine • Warm saline washing of red cells gives valid results • Cold alloantibodies – React with A1, B and O cells – An immediate spin reaction with group O screening cells is indicative of anti-M, anti-N, anti-P1 – Autologous controls are negative – Repeat serum grouping with known M negative A1 and B cells. Antibodies Cells Anti-A Anti-B Anti-AB A1 B O Auto Cord O Unwashed RBCs 3+ 4+ 4+ 4+ 3+ 3+ 2+ 1+ Washed RBCs 2+ 4+ 4+ DTT 0+ 4+ 4+ Auto-adsorbed 4+ 0 0 0 0 serum Note: For IgM cold antibody (anti-I, H, M, N, P and Lewis), washing the cells with warm saline or dithiothreitol (DTT) or 2-mercaptoethenol treatment yields immunoglobulin free cells. In the above example, it is B blood group with autoanti-I in serum. For IgG antibodies, using low protein reagents allow valid grouping. They can also be removed by 45°C elution or treat- ment with chloroquine diphosphate. Antibodies Cells Anti-A Anti-B Anti-AB Anti-H A1 cells B cells O cells O cells Auto (cord) 0 0 0 0 4+ 4+ 4+ 4+ 0 Note: No agglutination with anti-A, and anti-B antisera. It looks apparently as O blood group in forward blood grouping. In reverse grouping not only with A1, B cells, but also with O cells and O cord cells, agglutination is seen. There is no auto- agglutination. Anti-H lectin resolves the problem and this blood is of Bombay Blood group. If anti-H is added to any of the normal O, A, B or AB cells, they will agglutinate. Only Bombay blood group red cells will not show any agglutination.