CLS 3311 Advanced Clinical Immunohematology

CLS 3311Advanced Clinical Immunohematology ABO Discrepancy’s

ABO Discrepancy • Definition: When the results of the forward grouping (patient cells) do not correspond to the results of the reverse grouping (patient serum) or abnormal reactivity is present (i.e. Mixed Field): • Strength of reaction • Weak or missing • Additional reactions • Abnormal reactions

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. HINT • 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?

Patient A: Additional reaction with anti-B and patients cells. Patient B: Weak reaction with patients serum and A1-cells. Patient C: Additional reaction with patients serum and A1-cells. Patient D: Missing reactions with patients serum A1-cells

Problems with The Forward Grouping Chimera: Two cell populations • Mixed Field reactions observed • Causes: Recent transfusion (O cells to an A patient), Bone marrow transplant Testing using Serum or plasma suspended Patient RBCs • Can cause non specific aggregation of rbc’s • Increased serum proteins: Multiple Myeloma patient • Contamination in cord blood sample: Wharton’s jelly • Infusion of macromolecular solutions: Dextran, etc.

Problems with The Forward Grouping Potent cold reacting autoagglutinins • Antibody coats patient RBC and agglutinate spontaneously in the diluent Weakened Antigen Expression • Subgroups of A or B • Some leukemia’s, Hodgkin's disease • Excess soluble A and B blood group substances • Carcinoma of the stomach and/or carcinoma of the pancreas

Extra ABO antigens Acquired ‘B’ Antigen Microbial deacetylating enzymes such as E. coli cleave off the N-Acetyl of the Group A N-acetyl-D-galactosamine immunodominant sugar. The remaining D-galactosamine becomes similar enough to the Group B D-galactose immunodominant sugar that it DOES react with reagent anti-B. Secondary to bowel obstruction or carcinoma of the bowel Problems with The Forward Grouping:

Problems with The Forward Grouping:Extra ABO antigens Polyagglutinable state • Exposure of ‘crypt’ or buried antigens (T, Tk, etc.) by inheritance or bacterial enzymes – RBC’s agglutinate with most sera. • Exposure of T, Tn and Tk (etc.) antigens. Antibodies to these antigens are present in virtually all human antisera. If using human source anti-A and anti-B these cells will agglutinate.

Plasma or incompletely clotted serum Non specific red cell aggregation Still has fibrinogen and can cause fibrin formation (rbc’s can be caught up and appears as clumping) Increased serum proteins (Multiple Myeloma, etc.), infusion of high molecular weight plasma expanders Problems with TheReverse Grouping

Unexpected antibodies to reagent RBCs Antibodies to constituents of the reagent RBC’s diluent Cold reacting antibodies such as anti-M, -N or -I Antibody reacts with diluent and non-specifically involves red blood cells in reaction causing clumping. Problems with TheReverse Grouping

Problems with The Reverse Grouping:Unexpectedly Weakened Antibodies Immunodeficient due to therapy or disease • Immunosuppressive drugs • Certain leukemia’s (CLL) or lymphoma’s (malignant lymphomas) have hypogammaglobulinemia Age related • Very young: <6 months of age • Very old: >65 years of age Dilutional Effect • Plasma Exchange, Transfusion, etc. dilutes out patient antibodies

PopularLAB CAUSESOf 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!

PopularLAB CAUSESOf 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

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.

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. ResolvingABO Discrepancies

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? ResolvingABO 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. ResolvingABO Discrepancies

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! ResolvingABO Discrepancies

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? ResolvingABO Discrepancies

Problem: Reverse grouping - weakened patient antibody Causes: Age related (>65, infant), immunosuppressed or immunocompromised, Resolution: Incubate Room Temperature 15-30 minutes and respin.Check Patient history.

Problem: 1+ Reaction with Anti-B. Appears to have additional antigens. Causes: Acquired ‘B’ antigen. Resolution: Patient history – bowel obstruction, carcinoma of the bowel. (E. coli deacetylation of the Group A antigen.)

Problem: Weak forward 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 A2 cells2) Antibody screen should demonstrate unexpected cold reacting antibody.

ABO Discrepancy Cases • The following slides are patient reactions including the ABO forward and reverse grouping using monoclonal reagent anti-A and anti-B and reagent red blood cells. The Indirect Antiglobulin Test (IAT) is included. • You need to email me your results PRIOR to posting them on the discussion board. All Students must post their cases on the same day to insure integrity of work. I will email the deadline to each student. Following student posting I will post my results. We can then discuss any questions you may have.

Case #1 MF stands for mixed field reaction.

Case #1 Write up. • List the abnormal results. • How does the Forward grouping correlate with the IAT? • What is the suspected ABO Group of the patient? Support you answer. • What action would you take to resolve this case?

Case #2

Case #3

Case #3 Write Up • List the abnormal results. • How does the Forward grouping correlate with the IAT? • What is the suspected ABO Group of the patient? Support you answer. • What action would you take to resolve this case? Explain your choice of tests.

Case #4

Case #5

CLS 3311 Advanced Clinical Immunohematology