ABO Discrepancies & other problems Prepared By Ahmad Shihada Silmi Msc, FIBMS Medical Technology Dept Islamic University of Gaza

Importance It is important for students to recognize discrepant results and how to (basically) resolve them Remember, the ABO system is the most important blood group system in relation to transfusions Misinterpreting ABO discrepancies could be life threatening to patients

Discrepancies A discrepancy occurs when the red cell testing does NOT match the serum testing results In other words, the forward does NOT match the reverse

Why? Reaction strengths could be weaker than expected Some reactions may be missing in the reverse or forward typing Extra reactions may occur

Patient Anti-A Anti-B A1 Cells B Cells 1 4+ 1+ 2 3 4 3+

What do you do? Identify the problem Most of the time, the problem is technical Mislabeled tube Failure to add reagent Either repeat test on same sample, request a new sample, or wash cells Other times, there is a real discrepancy due to problems with the patient’s red cells or serum

Discrepancy ? If a real discrepancy is encountered, the results must be recorded However, the interpretation is delayed until the discrepancy is RESOLVED

Errors

Technical Errors Clerical errors Reagent or equipment problems Mislabeled tubes Patient misidentification Inaccurate interpretations recorded Transcription error Computer entry error Reagent or equipment problems Using expired reagents Using an uncalibrated centrifuge Contaminated or hemolyzed reagents Incorrect storage temperatures Procedural errors Reagents not added Manufacturer’s directions not followed RBC suspensions incorrect concentration Cell buttons not resuspended before grading agglutination Clerical errors can result in fatalities. Please develop a set order when performing tests. This will eliminate mix-ups with tubes and prevent serious errors in reporting results.

Clotting deficiencies Serum that does not clot may be due to: Low platelet counts Anticoagulant therapy (Heparin, Aspirin, etc) Factor deficiencies Serum that does not clot completely before testing is prone to developing fibrin clots that may mimic agglutination Thrombin can be added to serum to activate clot formation OR, tubes containing EDTA can be used Protamine sulfate can also be added to neutralize heparin in the serum.

Contaminated samples or reagents Sample contamination Microbial growth in tube Reagent contamination Bacterial growth causes cloudy or discolored appearance…do not use if you see this! Reagents contaminated with other reagents (don’t touch side of tube when dispensing) Saline should be changed regularly

Equipment problems Routine maintenance should be performed on a regular basis (daily, weekly, etc) Keep instruments like centrifuges, thermometers, and timers calibrated Uncalibrated serofuges can cause false results

Hemolysis Detected in serum after centrifugation (red) Important if not documented Can result from: Complement binding Anti-A, anti-B, anti-H, and anti-Lea Bacterial contamination Red supernatant

ABO discrepancies

ABO Discrepancies Problems with RBCs Problems with serum Weak-reacting/Missing antigens Extra antigens Mixed field reactions Problems with serum Weak-reacting/Missing antibodies Extra antibodies

May cause all + reactions

Forward Grouping Problems

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

Forward Grouping: Missing or Weak antigens ABO Subgroups Disease (leukemia, Hodgkin’s disease) Anti-A Anti-B A1 Cells B Cells 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) 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-A1, Anti-H, and anti-A,B for A subgroups

Forward Grouping: Extra Antigens Acquired B B(A) phenotype Rouleaux Polyagglutination Wharton’s Jelly Anti-A Anti-B A1 Cells B Cells 4+ 1+ EXAMPLE

Acquired B Phenomenon Cause: there are two causes of acquired B phenomenon: In vivo, patients with bacterial infections and often cancer of the colon or rectum may develop a false B-like antigen. The mechanism: The bacterial produce a deacetylase (enzyme) which chemically alters the terminal sugar of A antigens (N-acetyl-D-galactosamine) into D-galactosamine. Because the terminal sugar of the B antigen is galactose, anti-B antisera will cross react with the B-like D-galactosamine antigen. Because of this, in vivo, only group A people can develop an acquired B-like antigen. The condition is transient and disappears when the infection is cured.

Acquired B Bacteria (E. coli) have a deacetylating enzyme that effects the A sugar…. Acquired B Phenotype Group A individual Galactosamine results from the deacetylating reaction, resembling D-galactose (found in Group B individuals). This sugar cross-reacts with the reagent anti-B, giving a weak reaction (but still technically it is “extra”). Patients should receive Group A units. Acquired B usually goes away when the condition resolves. N-acetyl galactosamine Galactosamine now resembles D-galactose (found in Group B) Bacterial enzyme removes acetyl group

Another mechanism In vitro, blood specimens can get an acquired B-like antigen if they are bacterially contaminated. This is because the membranes of some bacteria (e.g., E. coli and P. vulgaris ) have determinants which are chemically similar to the B antigen. In this case, anti-B antisera is actually reacting with the bacterial antigens which have attached to the red cells. In vitro, both group O and group A cells can acquire the B-like antigen. Note: most examples of acquired B phenomenon detected in the blood bank happen in vivo to group A people only.

ES4 Anti-B antisera The use of monoclonal ABO typing antisera (specifically an anti-B clone designated "ES4") initially caused an increase in acquired B phenomenon. because The ES4 monoclonals can detect even a small number of galactosamine molecules on red cells. However, the reactions are particularly sensitive to pH and can be reduced (not eliminated totally) if the pH is lowered, something that the manufacturers have done.

Typical reaction pattern The reactions with anti-B are weaker than expected (e.g., 1+ or 2+). The patient's autocontrol is negative even though anti-B is present (patient is group A). The patient's own anti-B will not recognize and agglutinate the B-like antigen, but everyone else's anti-B (including the typing sera) will.

Resolution of acquired B Check the past records in case the patient is a known group A. Check the diagnosis for bacterial infection (with or without Cancer of the colon or rectum). Test the red cells with anti-B reagent acidified to pH 6.0 If using human polyclonal reagents, redo the ABO group using monoclonal anti-A and anti-B typing sera, which may resolve the problem. If using monoclonal reagents, redo the ABO group using human polyclonal anti-A and anti-B typing sera. Do autologous control it should give negative result. Try secretor status studies (usually not necessary). If the patient is group A and a secretor, he will secrete A and H antigens only.

Implication in blood transfusion Group A people (especially children with a small blood volume) who have acquired B phenomenon should receive group A washed red cells (or group O washed red cells). The red cells should be washed to remove all traces of donor anti-B which can react with the patient's B-like antigens.

Acquired B Phenotype Limited mainly to Group A1 individuals with: Lower GI tract disease Cancer of colon/rectum Intestinal obstruction Gram negative septicemia (i.e. E. coli)

Resolving 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 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 Polyagglutination – agglutination of RBCs with human antisera no matter what blood type 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) Wash red cells or request new sample from heel, etc

Forward Grouping: Mixed Field Agglutination Results from two different cell populations Agglutinates are seen with a background of unagglutinated cells All groups transfused with Group O cells Bone marrow/stem cell recipients A3 phenotype Anti-A Anti-B A1 Cells B Cells 2+ 4+

Mixed Field Agglutination

Reverse Grouping Problems

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

Reverse Grouping: Missing or Weak antibodies Newborns Do not form antibodies until later Elderly Weakened antibody activity Hypogammaglobulinemia Little or no antibody production (i.e. immunocompromised) Often shows NO agglutination on reverse groupings

Missing or Weak antibodies Example Anti-A Anti-B A1 cells B cells Tentative group #1 +4 - A #2 B or AB #3 O #1: Patient is a newborn: Anti-A and anti-B are not present at birth and develop about 3-6 months of age. (Usually the reverse group is not done when grouping newborns.) #2: Patient is very elderly: Anti-A and anti-B levels decrease in old age because levels of immunoglobulins decrease. Because the levels may only be decreased and not totally missing, further investigation can be done. (Note: It would be unusual for an elderly person to totally lack ABO antibodies in the absence of an immune disorder.) #3: Patient has a- or hypogammaglobulinemia: Anti-A and anti-B will be weak or missing in patients with a gammaglobulinemia or hypogammaglobulinemia.

Resolution Check the age of the patient Repeat the ABO group at 4°C [anti-A and anti-B react best at 4°C]. QC required: because all persons have a harmless auto-anti-I reactive at 4°C, include an autocontrol. (Auto-anti-I may agglutinate the A1 cells, the B cells, and the patient's own cells at 4°C.) Check the diagnosis. If undiagnosed, have gammaglobulin levels tested

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

Reverse Grouping: Extra Antibodies Cold antibodies (allo- or auto-) Cold antibodies may include anti-I, H, M, N, P, Lewis Rouleaux Anti-A1 in an A2 or A2B individual

Cold antibodies Sometimes a patient will develop cold-reacting allo- or auto-antibodies that appear as “extra” antibodies on reverse typing 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. Resolution: warming tube to 37° and washing red cells can disperse agglutination; breaking the IgM bonds.

Rouleaux 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: Multiple meloma Waldenstrom’s macroglobulinemia (WM) Hydroxyethyl starch (HES), dextran, etc Agglutination at AHG should not occur because cells have been washed three times

Resolving Rouleaux 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) 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.

Extra Antibodies Example Anti-A Anti-B A1 cells B cells Tentative group #1 +4 - +1 A #2 +2 AB #3 B Anti-A1 in A2 or A2B people: examples #1 and #2 illustrate the presence of anti-A1. The autocontrol (not shown) would be negative. Irregular IgM Alloantibodies: All three examples could represent the presence of irregular IgM alloantibodies such as anti-M, -N, -Lea, -Leb, or -P1. The A1 cells (or B cells) may be agglutinating because they are positive for the corresponding antigen. The autocontrol (not shown) would be negative.

Example Anti-A Anti-B A1 cells B cells Tentative group #1 +4 - +1 A #2 +2 AB #3 B Rouleaux: providing both cells in the reverse grouping show agglutination (examples #1 and #3), the discrepancy could be due to Rouleaux. The autocontrol (not shown) would be positive. Causes: Rouleaux is a type of false agglutination caused by an increase in serum globulins. This can occur in diseases such as multiple myeloma or macroglobulinemia or can be caused by infusion of macromolecular substances such as dextran or polyvinyl pyrollidone (PVP), which are used as blood volume expanders. Autoanti-I: Many people have a harmless autoanti-I that is IgM and reacts best at 4°C. The harmless autoanti-I of most people will not react above 10°-15°C, but some people have an autoanti-I that can react at RT and cause unexpected agglutination in both cells of the reverse serum group (examples #1 and #3).

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 + A1 cells AGGLUTINATION Anti-A1 from patient + A2 cells NO AGGLUTINATION

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 Anti-A Anti-B A1 Cells B Cells 4+ 2+

Resolution of discrepancies caused by anti-A1 First step: We must determine if the person is group A1 or group A2. (If group A1, the discrepancy with the A1 cells is NOT due to anti-A1). To do this, we antigen type the person's red cells with the anti-A1 lectin which is Dolichos biflorus . If the red cells agglutinate, the person is group A1. If the red cells do not agglutinate, the person is not group A1, and probably is group A2 assuming the red cells reacted strongly (3+ or 4+ with anti-A).

Resolution of discrepancies caused by anti-A1 Second step: If the person appears to be group A2, we must prove that the extra antibody is anti-A1, and not some other IgM irregular antibody. To do this we test the person's serum against a panel of 3 A1 cells and 3 A2 cells. If anti-A1 is present, only the A1 cells should agglutinate.

Resolution of discrepancies caused by anti-A1 NOTE: 3 A1 and 3 A2 cells are required in order to ensure that the antibody reacting is anti-A1 and not some other antibody. With 3 cells of each group we can achieve a statistical probability of 95% that the right antibody has been identified. For example, if only one A1 cell and one A2 cell were tested, by chance, another antibody like anti-M or anti-P1 could react with the A1 cells (if they were M+ or P1+), but not the A2 cells (if they were M- or P1-).

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)

Finding the problem… Forward type tests for the antigen (red cell) Reverse type tests for the antibody (serum) Identify what the patient types as in both Forward & Reverse Groupings Is there a weaker than usual reaction? Is it a missing, weak, or extra reaction??

Resolving ABO Discrepancies Get the patient’s history: age Recent transplant Recent transfusion Patient medications The list goes on….

Let’s practice !

Example 1 Anti-A Anti-B A1 Cells B Cells 3+ 1+ Problem: Causes: 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: Causes: Resolution:

Example 2 Anti-A Anti-B A1 Cells B Cells 3+ 1+ 4+ Problem: Causes: 4+ 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: Causes: Resolution:

Example 3 Anti-A Anti-B A1 Cells B Cells 2+ 0+ 1+ 4+ Problem: Causes: 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 screen Problem: Causes: Resolution:

Example 4 Anti-A Anti-B A1 Cells B Cells 3+ Problem: Causes: 3+ 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: Causes: Resolution:

Example 4 Anti-A,B Patient RBC 1+ Probably a subgroup of A (Ax) if the result was negative (0), adsorption or elution studies with anti-A could be performed (these will help determine what A antigens)

Example 5 Anti-A Anti-B A1 Cells B Cells 2+mf 3+ Problem: Causes: 2+mf 3+ 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 transfusion Problem: Causes: Resolution:

Example 6 Anti-A Anti-B A1 Cells B Cells 4+ 1+ Problem: Causes: 1+ 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 Problem: Causes: Resolution:

Example 7 Anti-A Anti-B A1 Cells B Cells Problem: Causes: Resolution: 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: Causes: Resolution:

Screening Cells (I and II) Example 8 Screening Cells (I and II) Autocontrol (AC) Conclusion Patient Serum 1 Pos Neg Cold alloantibody Patient Serum 2 Cold autoantibody if alloantibody – antibody ID techniques if autoantibody – special procedures (minicold panel, prewarming techniques

References Rudmann, S. V. (2005). Textbook of Blood Banking and Transfusion Medicine (2nd Ed.). Philadelphia, PA: Elsevier Saunders. Blaney, K. D. and Howard, P. R. (2000). Basic & Applied Concepts of Immunohematology. St. Louis, MO: Mosby, Inc.