Practical Blood Bank

Practical Blood Bank ABO Discrepancies Lab 2

ABO Discrepancy • When the results of the forward grouping (patient cells) is not matching 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

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?

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

Forward Grouping Problems

Missing or Weak antigens • Subgroups of A and B. • Solution: test with Anti-A1, Anti-H, and anti-A,B for A subgroups • Since the forward and reverse don’t match, there must be a discrepancy (in this case, a missing antigen in the forward grouping)

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

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

Mixed Field agglutination Mixed Field Agglutination (Post transfusion) 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.

Reverse Grouping Problems

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

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

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

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

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

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)

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.

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.

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?

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!

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?

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: • Subgroup of A – A2 with anti-A1. • Unexpected cold reacting antibody to antigen on reagent A1 cells. • Resolution: • Test patient cells with anti-A1 lectin and with patient serum test A2 cells • Antibody screen should demonstrate unexpected cold reacting antibody.

Let’s practice !

EXAMPLES of ABO Discrepancies and Possible Resolution Adapted from Table 3-11: Flynn, J. C. (1998). Essentials of Immunohematology. Philadelphia: W.B. Saunders Company.

Example 1 Problem: Causes: Resolution:

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