Investigative Techniques in Blood Banking

1. Investigative Techniquesin Blood Banking Deborah BaudlerMS, MT(ASCP) SBB Assistant Professor University of Illinois-Springfield Patchwork Conference April 15, 2014

2. University of Illinois-Springfield

3. UIS CLS Students

4. Objectives • Identify common problems that occur in day to day blood banking • Discuss various techniques for problem-solving • Apply new knowledge to case studies for resolution

5. Sherlock Holmes “The science of deduction and analysis is one which can only be acquired by long and patient study...”

6. Common Problems That Can Occur • ABO Discrepancies • Weak Positive Antibody Screen……no antibody identified • Miscellaneous Reactivity showing up on the antibody panel • Incompatible Crossmatch when antibody screen is negative

7. Common Solutions Available • Cry a little • Start over, hoping the problem will just go away • Shake the tubes harder • Pretend the weak reactions don’t exist • Call your blood bank supervisor at 2 am to see if she/he is reading a good book • Leave it for the next shift to resolve!

8. ABO Discrepancy • A discrepancy occurs when the red cell testing does NOT match the serum test results • In other words, the forwardtype does NOT match the reverse. • What is the discrepancy here?

9. What we do know…. • Recall: the production of ABO antigens is controlled by the genes we inherit • ABO forward and reverse reactions are typically very strong: 3+ to 4+. • Where do we start?

10. Discuss the Possibilities • Most of the time, the problem is technical • Failure to add patient plasma • Reversed the A1 and B cells in the rack • Reagent contamination • Incubation time too short • Clot in specimen • Interpretations not accurately recorded

11. Grouping Forward Reverse Missing/Weak Extra Mixed Field Missing/Weak Extra A/B Subgroup Acquired B O Transfusion Cold Autoantibody Young Elderly Immunocompromised Disease (cancer) B(A) Phenotype Bone Marrow Transplant Cold Alloantibody Rouleaux Rouleaux Anti-A1 Forward vs Reverse Courtesy of School of Health Related Professions University of Mississippi Medical Center

12. Reasons for Red Cell Discrepancies • If you have extra reactivity: • Recent Bone Marrow/ Stem Cell Transplant: check medical history • Excess protein coating red cells or Rouleaux: Wash red cells and retest • Strong cold agglutinin coating cells: Treat cells with 0.01 M DTT • Antibody coated red cells causing autoagglutination: can be seen in HDFN. Perform DAT and Incubate cells and wash several times with 37°C saline

13. Reasons for Red Cell Discrepancies Acetyl group D-Galactose Enzyme cleaves off acetyl group • Acquired B antigen: occurs in Group A individuals with gram neg sepsis. True group A cells will not agglutinate with patient’s own Anti-B in plasma

14. Reasons for Red Cell Discrepancies • If you have missing or weak reactivity: • Subgroup of A: test cells with A1Lectin, Anti-A,B and Anti-H • Massive red cell transfusion: check transfusion history • Cancer or Chemotherapy: require longer incubation period

15. Reasons for Plasma Discrepancies • If you have extra reactivity: • Rouleaux: Check for “stack of coins” and perform Saline Replacement • Cold or RT alloantbody: Antibody ID and repeat reverse cells with antigen negative cells • Cold or RT autoantbody: Antibody ID and pre-warm plasma and reverse cells in separate tubes, combine and read

16. Reasons for Plasma Discrepancies • If you have extra reactivity: • Issoagglutinins: Passive ABO antibodies: check recent transfusion history • Subgroup of A: A1Lectin and antibody ID with A1 cells

17. Reasons for Plasma Discrepancies • If you have missing or weak reactivity: • Check age of patient: • Newborn: no antibody production until 4 mos • Elderly: extend incubation or increase serum/cell ratio • Hypogammaglobulinemia: extend incubation or increase serum/cell ratio • Chemotherapy or recent Bone Marrow Transplant: check medical history

18. Back to Our Case We have extra reactivity on the plasma side Most frequent cause for ABO discrepancy

19. In This Case • Patient has never been transfused and is not pregnant • Patient is here for elective surgery • Antibody Screen is negative, Auto control = 0 • Checked under the scope, no Rouleaux • What’s left to do?

20. Solution A1 cells Agglutination A2 cells No Agglutination 1. Recall: 20% of group AB individuals are actually A2B. 25% of A2B will make an alloantibody called Anti-A1 2. Test patient’s red cells with A1Lectin. A2B will not agglutinate with A1Lectin 3. Test patient’s plasma with several lots of A1 cells to confirm that the antibody is Anti-A1

21. In This Case Patient is an A2B Pos with Anti-A1 Solution: Use A2 reverse cells to eliminate extra reactivity and resolve discrepancy

22. Weak Antibody Activity • 2. Weak Positive Screen: Negative Antibody ID • Get the Patient’s Medical History • Possible Solutions: • Repeat antibody screen and ID by a second method • Check expiration dates of reagents • Increase serum/cell ratio • Increase incubation time • Contact the manufacturer • How should this be reported?

23. Miscellaneous Antibody Activity • 3. Positive Screen: No specific antibody identified • All alloantibodies have been ruled out!

24. What Should You Do?

25. Miscellaneous Antibody Activity • 3. Positive Screen: No specific antibody identified • Possible Solutions • Check lot number of antigrams! • Check expiration dates of reagents • Repeat antibody screen and ID by a second method • Increase serum/cell ratio • Increase incubation time

26. Miscellaneous Antibody Activity Highlight positive reactions Check for Dosage

27. Miscellaneous Antibody Activity • Additional Suggestions: • Get the Patient’s Medical History • Enzyme panel • Check Direct Coombs • Perform an Eluate

28. Benefits of an Eluate • For a DAT to become positive: > 200 molecules of IgG on red cell • Purpose of an eluate: • Removes an antibody that’s coating the red cell • Concentrates antibody • Allows identification of newly forming or weak antibodies • Can be positive even when DAT is negative

29. Miscellaneous Antibody Activity • What antibody is detected?

30. Incompatible Crossmatch When Antibody Screen is Negative • Possibilities: • Perform clerical check on specimen • Check agglutination under scope if <2+ • Specimen at room temp or out of refrigerator • Age of specimen: protein precipitation • If Reactivity is 3-4+ • Repeat patient’s blood type • Strong Cold Agglutinin

31. Incompatible Crossmatch When Antibody Screen is Negative • Other Possibilities: • Patient has an antibody to a Low Incidence antigen on unit • Unit has positive DAT • Most likely • Return unit to blood center • Solution: Try another unit

32. Let’s do some Investigating

33. Case 1 71 yr. old woman comes through the ER on a Friday night with a 6 g Hb. While her antibody screen is incubating, you get the following blood type:

34. Thoughts? ABO discrepancy present Most probably blood type? What should we do next?

35. Oh No! Next thing you know, her antibody screen comes up positive, YIKES!

36. Let’s Do the Cross-out Technique Which Antibodies Could Possibly be Present?

37. Antibody ID Results Which Antibody is Present?

38. Are We Done? • Antibody identified as Anti-M • Anti-M can possess both IgM and IgG components • Phenotype patient for M if not recently transfused • Test B Neg, M Neg cells with patient plasma

39. Case 2 62 yr. old man comes through the ER on a Saturday night with abdominal pain. He is rushed to surgery for a possible bowel obstruction While his antibody screen is incubating, you get the following blood type:

40. The Antibody Screen Results Patient’s medical history indicates he had cardiac by-pass surgery 4 weeks ago and received 3 units of prbcs Perform cross-out technique Which antibodies can not be ruled out?

41. The Antibody Screen Results Anti-C, e, Fya, Jka, N and Anti-S are not ruled out

42. Antibody ID Results Conclusion? What is the next step?

43. Antibody ID Results Repeated panel with PeG. Who is the culprit?

44. Case 3 27 yr. old man is medevac to your facility from a small community hospital. The patient has been in a motor vehicle accident and is bleeding internally. He is being prepped for the OR.

45. Case 3 So you perform a STAT Type and Screen While the antibody screen is incubating, you record the following results for the blood type: Any problems?

46. The Antibody Screen Results The patient’s antibody screen results are Negative! Now what?

47. Check Medical History Patient received: 10 units Group O Negative rbcs 4 Group O Single Donor Platelets

48. Resolution Patient’s ABO discrepancy was due to massive transfusion of out of group blood products. Important Clue: Mixed-field agglutination Information from transferring hospital confirmed that patient was AB Positive What blood type of rbcs should be transfused?

49. You did it!