Practical Blood Bank

1. ABO Discrepancies Practical Blood Bank

2. ABO Discrepancy When the results of the forward grouping (patient cells) do not match to the results of the reverse grouping (patient serum) or abnormal reactivity is present (i.e. Mixed Field) then we called this ABO discrepancy. The Discrepancy will be noticed by: Strength of reaction Weak or missing. Additional reactions Abnormal reactions

3. HINT ABO forward and reverse reactions are typically very strong: 3+ to 4+. Weaker reactions should immediately send up red flags indicating that something is wrong. Since production of ABO antigens is genetically controlled they are less vulnerable to problems than does the production of ABO antibodies. Therefore we see more problems in which grouping: Forward or Reverse?

5. Forward Grouping Problems

6. Missing or Weak antigens Subgroups of A and B. Solution: test with Anti-A1, Anti-H, and anti-A,B for A subgroups

7. Extra Antigens Acquired B B(A) phenotype Rouleaux Polyagglutination Wharton’s Jelly

8. Solutions: Acquired B 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) B(A) phenotype Test with another anti-A reagent from another manufacturer Polyagglutination, Rouleaux, Wharton’s Jelly Wash red cells or request new sample from heel, etc

9. Can be seen in A, B and AB individuals who have received O units. Can also be seen post transfusion if a person makes an antibody to antigen on donor cells. Mixed Field Agglutination (Post transfusion)

10. Reverse Grouping Problems

11. Unexpectedly Weakened Antibodies Immunodeficient due to therapy or disease Immunosuppressive drugs Certain leukemia’s (CLL) or lymphoma’s (malignant lymphomas) have hypogammaglobulinemia (Little or no antibody production) Age related Very young: <6 months of age (Newborns) Very old: >65 years of age (Weakened Abs Activity) Dilutional Effect Plasma Exchange, Transfusion, etc. dilutes out patient antibodies Hypogammaglobulinemia: Often shows NO agglutination on reverse groupings Hypogammaglobulinemia: Often shows NO agglutination on reverse groupings

12. Resolving Weak or Missing antibodies 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 (a.k.a. Autocontrol, AC) This is called a “mini-cold” panel and should enhance the reactivity of the antibodies

13. Extra Antibodies Cold antibodies (allo- or auto-) Cold antibodies may include anti-I, H, M, N, P, Lewis 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 Rouleaux Stronger at IS and weak reaction at 37° C and no agglutination at AHG phase Solutions Anti-A1 in an A2 or A2B individual Rouleax : Multiple meloma, Waldenstrom’s macroglobulinemia (WM), Hydroxyethyl starch (HES), dextran, etc Rouleax : Multiple meloma, Waldenstrom’s macroglobulinemia (WM), Hydroxyethyl starch (HES), dextran, etc

14. Resolving Rouleaux 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)

15. Anti-A1 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

16. Resolving anti-A1 discrepancy 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

17. Others… 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)

18. Popular LAB CAUSES Of ABO Discrepancies Poorly labeled specimen OR test tubes Patient RBC suspension too heavy or light Wrong specimen put in Patient’s labeled test tubes Oh? Is hemolysis really a Pos. Rx’n? Wrong results recorded on Pt. Form Didn’t follow manufacturer’s instructions Poor centrifugation: over or under!

19. Popular LAB CAUSES Of ABO Discrepancies Didn’t add: Patient Serum Reagents Correct Reagent Reaction Reading: Shaking tubes while looking elsewhere Shaking tubes too hard Shaking tubes too gently or not completely re- suspending cell button

20. ABO Discrepancy When an ABO Discrepancy is encountered: Results must be recorded, but interpretation of the ABO group must be delayed until the discrepancy is resolved…by you! Begin follow up by getting an accurate patient history – age, medications, diagnosis, etc. Repeat testing to rule out tech errors such as mislabeling, adding reagents, wrong patient sample, etc.

21. Resolving ABO Discrepancies Repeat testing on the same sample… Repeat testing using saline suspended and/or washed patient red blood cell’s. Saline Replacement. From the beginning: re-label tubes, re-drop patient and reagent drops, etc. Many labs make the patients red blood cell suspension with the patient’s serum/plasma. If the patient has increased plasma proteins it can cause non-specific red cell aggregation.

22. Weak or missing reactions? Mislabeled or contaminated specimen: Incubate test system at room temperature for 15-30 minutes! Get patient history. Redraw Patient!! ALL of the above: any labeling error may account for the problem and needs to be redrawn. Drawn above an IV?

23. Resolving ABO Discrepancies Call the floor!!! Get patient history. Recent transplant: two cell populations Recent transfusion: two cell populations and/or dilutional effect Patient medication etc., etc., etc.

24. Test patient cells with anti-A1 (Dolichos biflorus), anti-A,B or anti-H (Ulex europaeus) Test patient serum with A1 or A2 cells For suspected subgroups of A Ditto!

25. Review Antibody Screening tests Allo antibody or cold reactive allo or auto Ab Incubate tests and controls for 10-30 minutes room temperature Can react with reagent A1 and B cells Should strengthen weakened ABO antibody reactivity! WHY?

29. Let’s practice !

30. EXAMPLES of ABO Discrepancies and Possible Resolution

31. Example 1 Problem: Reverse grouping, weakened patient antibody Causes: Age related or weakened immune system Resolution: Incubate at Room Temperature 15-30 minutes and respin. Check patient history.Problem: Reverse grouping, weakened patient antibody Causes: Age related or weakened immune system Resolution: Incubate at Room Temperature 15-30 minutes and respin. Check patient history.

32. Example 2 Problem: 1+ reaction with anti-B. Appears to have additional antigens. Causes: Acquired B antigen Resolution: Patient history – bowel obstruction, carcinoma of colon/rectum. (E. coli)Problem: 1+ reaction with anti-B. Appears to have additional antigens. Causes: Acquired B antigen Resolution: Patient history – bowel obstruction, carcinoma of colon/rectum. (E. coli)

33. Example 3 Problem: Weak forward with anti-A and 1+ reaction with A1 cells Causes: 1) Subgroup of A (A2 with anti-A1) 2) unexpected cold reacting antibody to antigen on reagent A1 cells Resolution: 1) test patient cells with anti-A1 lectin and with patient serum test with A2 cells 2) an unexpected cold antibody would be detected in the antibody screenProblem: Weak forward with anti-A and 1+ reaction with A1 cells Causes: 1) Subgroup of A (A2 with anti-A1) 2) unexpected cold reacting antibody to antigen on reagent A1 cells Resolution: 1) test patient cells with anti-A1 lectin and with patient serum test with A2 cells 2) an unexpected cold antibody would be detected in the antibody screen

34. Example 4 Problem: missing antigen in forward grouping. Patient appears as group A in reverse grouping Causes: A subgroup Resolution: extend incubation time because this may enhance the reaction. Test with a polyclonal or monoclonal blend of anti-A,B (may contain subgroup antigens)…..Problem: missing antigen in forward grouping. Patient appears as group A in reverse grouping Causes: A subgroup Resolution: extend incubation time because this may enhance the reaction. Test with a polyclonal or monoclonal blend of anti-A,B (may contain subgroup antigens)…..

35. Example 4

36. Example 5 Problem: strength of anti-B is weaker than expected; reverse indicates a group B individual Causes: Group B individual transfused with group O cells Resolution: recent transfusion? Bone marrow/stem cell transplant? Find what ABO type the patient was prior to transfusionProblem: strength of anti-B is weaker than expected; reverse indicates a group B individual Causes: Group B individual transfused with group O cells Resolution: recent transfusion? Bone marrow/stem cell transplant? Find what ABO type the patient was prior to transfusion

37. Example 6 Problem: Forward shows AB individual, Reverse shows weaker “extra” reaction with B cells (looks like a group A) Causes: Possible cold allo- or autoantibody (patient may have an antibody to another blood group system; A1 and B cells may have the antigens to these antibodies) (allo: P, M, N, Lewis) (auto: I or IH) Resolution: screen for antibodies using Screening Cells and an autocontrol (we’ll talk later about Ab screens)Problem: Forward shows AB individual, Reverse shows weaker “extra” reaction with B cells (looks like a group A) Causes: Possible cold allo- or autoantibody (patient may have an antibody to another blood group system; A1 and B cells may have the antigens to these antibodies) (allo: P, M, N, Lewis) (auto: I or IH) Resolution: screen for antibodies using Screening Cells and an autocontrol (we’ll talk later about Ab screens)

38. Example 7 Problem: Reverse grouping, missing patient antibody (probably group O with no antibodies) Causes: Age related or weakened immune system Resolution: Incubate at Room Temperature 15-30 minutes and respin. Check patient history.Problem: Reverse grouping, missing patient antibody (probably group O with no antibodies) Causes: Age related or weakened immune system Resolution: Incubate at Room Temperature 15-30 minutes and respin. Check patient history.