Practical Blood Bank Lab 2 ABO Discrepancies ABO

Practical Blood Bank Lab 2 ABO Discrepancies

ABO Discrepancy n 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): 1. Strength of reaction n Weak or missing 2. Additional reactions 3. Abnormal reactions

HINT n 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. n 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 Anti-A Anti-B A 1 -Cells B-Cells A 4+ 1+ 0 4+ B 0 4+ 1+ 0 C 4+ 4+ 1+ 0 D 0 3+ 0 0 Patient A: Additional reaction with anti-B and patients cells. Patient B: Weak reaction with patients serum and A 1 -cells. Patient C: Additional reaction with patients serum and A 1 -cells. Patient D: Missing reactions with patients serum A 1 -cells

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Red Cell Problems n Affect the forward grouping results q q q Missing or weak antigens Extra antigens Mixed field reactions

Mixed Field reactions n n n Chimera: Two cell populations 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 1. Increased serum proteins: Multiple Myeloma patient 2. Contamination in cord blood sample: Wharton’s jelly 3. Infusion of macromolecular solutions: Dextran, etc.

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.

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

Missing or Weak antigens Anti-A Anti-B A 1 Cells B Cells 0 0 0 4+ 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)

Subgroups of A (or B) n n 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-A 1, Anti-H, and anti-A, B for A subgroups

Extra ABO antigens 1. Acquired ‘B’ Antigen a) Microbial deacetylating enzymes such as E. coli cleave off the N-Acetyl of the Group A Nacetyl-D-galactosamine immunodominant sugar. The remaining D-galactosamine becomes similar enough to the Group B Dgalactose immunodominant sugar that it does react with reagent anti-B. 2. Secondary to bowel obstruction or carcinoma of the bowel

3. Polyagglutinable state q q 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.

Forward Grouping: Extra Antigens n n n Acquired B B(A) phenotype Rouleaux Polyagglutination Wharton’s Jelly Anti-A Anti-B 4+ 1+ A 1 Cells 0 EXAMPLE B Cells 4+

Acquired B Phenotype n Limited mainly to Group A 1 individuals with: q q Lower GI tract disease Cancer of colon/rectum Intestinal obstruction Gram negative septicemia (i. e. E. coli)

Acquired B n Bacteria (E. coli) have a deacetylating enzyme that effects the A sugar….

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

B(A) phenotype n n n 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

Other reasons for “extra” antigens n Polyagglutination – agglutination of RBCs with human antisera no matter what blood type q q n n 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) q Wash red cells or request new sample from heel, etc

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Reverse Grouping n Affect the reverse grouping results q q Missing or weak antibodies Extra antibodies

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

Resolving Weak or Missing antibodies n Determine patients age, diagnosis n n n 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

Reverse Grouping: Extra Antibodies n Cold antibodies (allo- or auto-) q n n Cold antibodies may include anti-I, H, M, N, P, Lewis Rouleaux Anti-A 1 in an A 2 or A 2 B individual

Extra Antibodies : Cold antibodies Sometimes a patient will develop cold-reacting allo- or auto-antibodies that appear as “extra” antibodies on reverse typing n q q n n 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. q Resolution: warming tube to 37° and washing red cells can disperse agglutination; breaking the Ig. M bonds with 2 -ME will also disperse cells

Rouleaux n n n 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: q q q Multiple meloma Waldenstrom’s macroglobulinemia (WM) Hydroxyethyl starch (HES), dextran, etc

Resolving Rouleaux n n n 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) q q q 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) *some procedures suggest only 2 drops of saline (UMMC)

Anti-A 1 n n Sometimes A 2 (or A 2 B) individuals will develop an anti-A 1 antibody A 2 (or A 2 B) individuals have less antigen sites than A 1 individuals The antibody is a naturally occurring Ig. M Reacts with A 1 Cells, but not A 2 Cells

Resolving anti-A 1 discrepancy n 2 steps: q q q Typing patient RBCs with Anti-A 1 lectin Repeat reverse grouping with A 2 Cells instead of A 1 Cells Both results should yield NO agglutination Anti-A Anti-B 4+ 0 A 1 Cells 2+ B Cells 4+

Others… n n 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)

Popular LAB CAUSES Of ABO Discrepancies 1. 2. 3. 4. 5. 6. 7. 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!

Popular LAB CAUSES Of ABO Discrepancies Didn’t add: 1. 2. 3. Patient Serum Reagents Correct Reagent Reaction Reading: 1. 2. 3. Shaking tubes while looking elsewhere Shaking tubes too hard Shaking tubes too gently or not completely resuspending cell button

ABO Discrepancy When an ABO Discrepancy is encountered: 1. 2. 3. 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 1. Repeat testing on the sample… 2. Repeat testing using saline suspended and/or washed patient red blood cell’s. Saline Replacement. 1. From the beginning: relabel tubes, re-drop patient and reagent drops, etc. 2. 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.

3. 4. Weak or missing reactions? Mislabeled or contaminated specimen: 3. 4. Incubate test system at room temperature for 1530 minutes! Get patient history. Redraw Patient!! a) ALL of the above: any labeling error may account for the problem and needs to be redrawn. b) Drawn above an IV?

Resolving ABO Discrepancies Call the floor!!! 1. Get patient history. a) b) c) d) Recent transplant: two cell populations dilutional effect Patient medication etc.

5. Test patient cells with anti-A 1 (Dolichos biflorus), anti-A, B or anti-H (Ulex europaeus) 6. Test patient serum with A 1 or A 2 cells 5. For suspected subgroups of A 6. Ditto!

7. Review Antibody Screening tests 1. 8. 7. Can react with reagent A 1 and B cells 8. Should strengthen weakened ABO antibody reactivity! WHY? Allo antibody or cold reactive allo or auto Ab Incubate tests and controls for 10 -30 minutes room temperature

Anti-A 3+ Anti-B 0 A 1 -Cells 0 B-Cells 1+ Problem: Reverse grouping - weakened patient antibody Causes: Age related (>65, infant), immunosuppressed or immunocompromised, Resolution: Incubate Room Temperature 1530 minutes and respin. Check Patient history.

Anti-A 3+ Anti-B 1+ A 1 -Cells 0 B-Cells 4+ 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. )

Anti-A 2+ Anti-B 0 A 1 -Cells 1+ B-Cells 4+ Problem: Weak forward anti-A and 1+ reaction with A 1 Cells. Causes: 1. Subgroup of A – A 2 with anti-A 1. 2. Unexpected cold reacting antibody to antigen on reagent A 1 cells. Resolution: 1. Test patient cells with anti-A 1 lectin and with patient serum test A 2 cells 2. Antibody screen should demonstrate unexpected cold reacting antibody.

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EXAMPLES of ABO Discrepancies and Possible Resolution Forward: Anti-A 1 2 3 4 5 6 0 4+ 4+ 3+ 0 4+ Anti-B 0 4+ 0 2+ Reverse: A 1 Cells 0 2+ 1+ 1+ 4+ 0 B Cells 0 2+ 4+ 0 4+ 4+ Screening Cells 0 2+ 0 0 4+ 0 Autocontrol: Possible Causes Possible Resolutions 0 Group O newborn; elderly patient; low immunoglobulin levels Incubate tests at 4°C, check age of patient 2+ 0 0 Rouleaux; cold autoantibody Probable A 2 subgroup with anti-A 1 Wash RBCs and repeat testing; test for cold antibodies Test with anti-A 1 and anti. H lectins and A 2 cells Probable A 2 B subgroup with anti-A 1 Test with anti-A 1 and anti. H lectins and A 2 cells Probable Oh (Bombay) Test with anti-H lectin; may sent to reference lab for confirmation Investigate patient history; test with anti-B lectin if available Perform antibody identification (antibody panel) Test for cold antibodies and identify if appropriate 0 0 Probable acquired B phenotype Probable alloantibody 7 8 4+ 0 4+ 4+ 2+ 4+ 0 1+ 2+ 1+ 0 1+ Probable group B with cold autoantibody Adapted from Table 3 -11: Flynn, J. C. (1998). Essentials of Immunohematology. Philadelphia: W. B. Saunders Company.

Example 1 Anti-A Anti-B A 1 Cells B Cells 3+ 0 0 1+ Problem: Causes: Resolution:

Example 2 Anti-A Anti-B A 1 Cells B Cells 3+ 1+ 0 4+ Problem: Causes: Resolution:

Example 3 Anti-A Anti-B A 1 Cells B Cells 2+ 0+ 1+ 4+ Problem: Causes: Resolution:

Example 4 Anti-A Anti-B A 1 Cells B Cells 0 0 0 3+ Problem: Causes: Resolution:

Example 4 Anti-A, B Patient RBC Problem: Causes: Resolution: 1+

Example 5 Anti-A Anti-B A 1 Cells B Cells 0 2+mf 3+ 0 Problem: Causes: Resolution:

Example 6 Anti-A Anti-B A 1 Cells B Cells 4+ 4+ 0 1+ Problem: Causes: Resolution:

Example 7 Anti-A Anti-B A 1 Cells B Cells 0 0 Problem: Causes: Resolution: