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ABO Discrepancies & other problems

Reneé Wilkins , PhD, MLS(ASCP) CM CLS 325/435 School of Health Related Professions University of Mississippi Medical Center. ABO Discrepancies & other problems. It is important to recognize discrepant results and how to (basically) resolve them

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ABO Discrepancies & other problems

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  1. ReneéWilkins, PhD, MLS(ASCP)CM CLS 325/435 School of Health Related Professions University of Mississippi Medical Center ABO Discrepancies & other problems

  2. It is important to recognize discrepant results and how to (basically) resolve them Remember, the ABO system is the most important blood group system in relation to transfusions Misinterpreting ABO discrepancies could be life threatening to patients Importance

  3. A discrepancy occurs when the red cell testing does NOT match the serum testing results In other words, the forward does NOT match the reverse Discrepancies

  4. Reaction strengths could be weaker than expected Some reactions may be missing in the reverse or forward typings Extra reactions may occur Why?

  5. Identify the problem • Most of the time, the problem is technical • Mislabeled tube • Failure to add reagent • Either repeat test on same sample, request a new sample, or wash cells • Other times, there is a real discrepancy due to problems with the patient’s red cells or serum What do you do?

  6. If a real discrepancy is encountered, the results must be recorded However, the interpretation is delayed until the discrepancy is RESOLVED Discrepancy ?

  7. Errors

  8. Clerical errors • Mislabeled tubes • Patient misidentification • Inaccurate interpretations recorded • Transcription error • Computer entry error • Reagent or equipment problems • Using expired reagents • Using an uncalibrated centrifuge • Contaminated or hemolyzed reagents • Incorrect storage temperatures • Procedural errors • Reagents not added • Manufacturer’s directions not followed • RBC suspensions incorrect concentration • Cell buttons not resuspended before grading agglutination Technical Errors

  9. Serum that does not clot may be due to: • Low platelet counts • Anticoagulant therapy (Heparin, Aspirin, etc) • Factor deficiencies • Serum that does not clot completely before testing is prone to developing fibrin clots that may mimic agglutination • Thrombin can be added to serum to activate clot formation Clotting deficiencies

  10. Sample contamination • Microbial growth in tube • Reagent contamination • Bacterial growth causes cloudy or discolored appearance…do not use if you see this! • Reagents contaminated with other reagents (don’t touch side of tube when dispensing) • Saline should be changed regularly Contaminated samples or reagents

  11. Routine maintenance should be performed on a regular basis (daily, weekly, etc) • Keep instruments like centrifuges, thermometers, and timers calibrated • Uncalibrated serofuges can cause false results Equipment problems

  12. Detected in serum after centrifugation (red) • Important if not documented • Can result from: • Complement binding • Anti-A, anti-B, anti-H, and anti-Lea • Bacterial contamination Hemolysis Red supernatant

  13. ABO discrepancies

  14. Problems with RBCs • Weak-reacting/Missing antigens • Extra antigens • Mixed field reactions • Problems with SERUM • Weak-reacting/Missing antibodies • Extra antibodies ABO Discrepancies

  15. Grouping Forward Reverse Missing/Weak Extra Mixed Field Missing/Weak Extra A/B Subgroup Acquired B O Transfusion Young Elderly Immunocompromised Cold Autoantibody Disease (cancer) B(A) Phenotype Bone Marrow Transplant Cold Alloantibody Rouleaux Rouleaux Anti-A1 May cause all + reactions

  16. Forward Grouping Problems

  17. Affect the forward grouping results • Missing or weak antigens • Extra antigens • Mixed field reactions Red Cell Problems

  18. ABO Subgroups Disease (leukemia, Hodgkin’s disease) Forward Grouping:Missing or Weak antigens Group O Group A • Since the forward and reverse don’t match, there must be a discrepancy (in this case, a missing antigen in the forward grouping)

  19. Subgroups of A account for a small portion of the A population (B subgroups rarer) These subgroups have less antigen sites on the surface of the red blood cell As a result, they show weakened (or missing) reactions when tested with commercial antisera Resolution: test with Anti-A1, Anti-H, and anti-A,B for A subgroups Subgroups of A (or B)

  20. Acquired B B(A) phenotype Rouleaux Wharton’s Jelly Forward Grouping:Extra Antigens EXAMPLE

  21. Acquired B Phenotype • Limited mainly to Group A1 individuals with: • Lower GI tract disease • Cancer of colon/rectum • Intestinal obstruction • Gram negative septicemia (i.e. E. coli)

  22. Bacteria (E. coli) have a deacetylating enzyme that effects the A sugar…. Acquired B Acquired B Phenotype Group A individual N-acetyl galactosamine Galactosamine now resembles D-galactose (found in Group B) Bacterial enzyme removes acetyl group

  23. Check patient diagnosis: Infection? • Some manufacturers produce anti-B reagent that does not react with acquired B • Test patients serum with their own RBCs • The patients own anti-B will not react with the acquired B antigen on their red cell (autologous testing) Resolving Acquired B

  24. Similar to acquired B Patient is Group B with an apparent extra A antigen The B gene transfers small amounts of the A sugar to the H antigen Sometimes certain anti-A reagents will detect these trace amount of A antigen Resolution: test with another anti-A reagent from another manufacturer B(A) phenotype

  25. Polyagglutination – agglutination of RBCs with human antisera no matter what blood type • Due to bacterial infections • Expression of hidden T antigens react with antisera • Rouleaux – extra serum proteins • Wharton’s Jelly – gelatinous substance derived from connective tissue that is found in cord blood and may cause false agglutination (Remember: only forward typing is performed on cord blood) • Wash red cells or request new sample from heel, etc Other reasons for “extra” antigens

  26. Results from two different cell populations Agglutinates are seen with a background of unagglutinated cells All groups transfused with Group O cells Bone marrow/stem cell recipients A3 phenotype (sometimes B3) Forward Grouping:Mixed Field Agglutination

  27. Mixed Field Agglutination (Post transfusion) ~ (ABO Testing) Can be seen in A, B and AB individuals who have received O units. The antisera reacts with the patient’s RBCs, but not with the transfused O cells. ~ (Antibody screen) Can also be seen post transfusion if a person makes an antibody to antigen on donor cells; antibody agglutinates with donor cell, but not their on cells.

  28. Reverse Grouping Problems

  29. Affect the reverse grouping results • Missing or weak antibodies • Extra antibodies Reverse Grouping

  30. Newborns • Do not form antibodies until later • Elderly • Weakened antibody activity • Hypogammaglobulinemia • Little or no antibody production (i.e. immunocompromised) • Often shows NO agglutination on reverse groupings Reverse Grouping:Missing or Weak antibodies

  31. Determine patients age, diagnosis Incubate serum testing for 15 minutes (RT) to enhance antibody reactions If negative, place serum testing at 4°C for 5 minutes with autologous control (Autocontrol, AC) This is called a “mini-cold” panel and should enhance the reactivity of the antibodies Resolving Weak or Missing antibodies

  32. Cold antibodies (allo- or auto-) • Cold antibodies may include anti-I, H, M, N, P, Lewis • Rouleaux • Anti-A1 in an A2 or A2B individual Reverse Grouping:Extra Antibodies

  33. Sometimes a patient will develop cold-reacting allo- or auto-antibodies that appear as “extra” antibodies on reverse typing • Alloantibodies are made against foreign red cells • Autoantibodies are made against ones own red cells. Cold reacting antibodies cause agglutination with red cells at room temperature and below. The autocontrol will be positive. • Resolution: warming tube to 37° and washing red cells can disperse agglutination; breaking the IgM bonds with 2-ME will also disperse cells Cold antibodies

  34. Can cause both extra antigens and extra antibodies • “stack of coins” appearance • May falsely appear as agglutination due to the increase of serum proteins (globulins) • Stronger at IS and weak reaction at 37°C and no agglutination at AHG phase • Associated with: • Multiple meloma • Waldenstrom’s macroglobulinemia (WM) • Hydroxyethyl starch (HES), dextran, etc Rouleaux

  35. Remove proteins! • If the forward grouping is affected, wash cells to remove protein and repeat test • If the reverse grouping is affected, perform saline replacement technique (more common) • Cells (reagent) and serum (patient) centrifuged to allow antigen and antibody to react (if present) • Serum is removed and replaced by an equal volume of saline (saline disperses cells)* • Tube is mixed, centrifuged, and reexamined for agglutination (macro and micro) Resolving Rouleaux

  36. Sometimes A2 (or A2B) individuals will develop an anti-A1 antibody A2 (or A2B) individuals have less antigen sites than A1 individuals The antibody is a naturally occurring IgM Reacts with A1 Cells, but not A2 Cells Anti-A1 + A1 cells AGGLUTINATION Anti-A1 from patient + A2 cells NO AGGLUTINATION

  37. 2 steps: Typing patient RBCs with Anti-A1 lectin Repeat reverse grouping with A2 Cells instead of A1 Cells Both results should yield NO agglutination Resolving anti-A1 discrepancy

  38. The Bombay phenotype (extremely RARE) results when hh is inherited These individuals do not have any antigens and naturally produce, anti-A, anti-B, anti-A,B, and anti-H Basically, NO forward reaction and POSITIVE reverse Resolution: test with anti-H lectin (Bombay’s don’t have H and will not react) Others…

  39. Finding the problem… • Forward type tests for the antigen (red cell) • Reverse type tests for the antibody (serum) • Identify what the patient types as in both Forward & Reverse Groupings • Is there a weaker than usual reaction? • Is it a missing, weak, or extra reaction??

  40. Get the patient’s history: • age • Recent transplant • Recent transfusion • Patient medications • The list goes on…. Resolving ABO Discrepancies

  41. Let’s practice !

  42. Example 1 Problem: Causes: Resolution:

  43. Example 2 Problem: Causes: Resolution:

  44. Example 3 Problem: Causes: Resolution:

  45. Example 4 Problem: Causes: Resolution:

  46. Example 4 • Probably a subgroup of A (Ax) • if the result was negative (0), adsorption or elution studies with anti-A could be performed (these will help determine what A antigens)

  47. Example 5 Problem: Causes: Resolution:

  48. Example 6 Problem: Causes: Resolution:

  49. Example 7 Problem: Causes: Resolution:

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