32 Resolving ABO Anomalies

The results of ABO grouping should follow Landsteiner’s Rule; the forward grouping is confirmed by/ or matches the reverse grouping. Unexpected findings are known as anomalies or discrepancies. Anomalies may occur in either reverse or forward grouping.

• We never “assume “ the patient’s ABO
• The discrepancy must be resolved
• If blood is needed before the discrepancy is resolved, Group O red cells and group AB plasma must be transfused.

There are four major types of anomalies:

1. An expected antibody may be weak or missing
2. An unexpected antibody may be present
3. An expected antigen may be weak or missing
4. An unexpected antigen may be present

There may be “multiple” explanations for an anomaly. The technologist must perform history checks and follow- up testing to determine the cause. Every effort to determine the actual ABO group of the patient, in keeping with organizational policy and procedure, should be performed.

Obtain and review the following:

1. Sample identity, i.e., verify the plasma and cell suspension are from the same person.
2. Repeat the tests to rule out technical error.
3. Obtain a patient history record. This should include details about previous transfusion, pregnancy, previous ABO grouping results, diagnosis, drug therapy, etc.

Reverse Grouping Anomalies

Anomalies Involving A Weak or Missing Antibody

Hypogammaglobulinemia or agammaglobulinemia

Problem

Expected anti-A and/or anti-B are missing or weak.

Explanation

Some individuals fail to make gamma globulins, or produce then in such small quantities that they are below the levels detectable in routine tests. The disorder is called hypogammaglobulinemia if the amount of gamma globulin formed is reduced, or agammaglobulinemia if it is not detectable.

Resolution

1. Check the patient’s history (diagnosis; presence of recurring infections; regular injections of immune serum globulin or IVIG).
2. This condition can be caused by leukemia, immunosuppressive drugs, bone marrow or stem cell transplants, dilution of ABO antibodies by plasma transfusion, ABO subgroups
3. Serum protein levels or immunoelectrophoresis can show decreased or absent gamma globulins
4. Repeat reverse grouping using 4-6 drops of serum; incubate initially for 15-30 minutes at room temperature. If there is still no agglutination after centrifugation then incubate the tubes (including group O and auto cells in parallel to detect cold/auto antibodies) at 4°C, centrifuge and read. The group O and auto control should be negative.

Neonatal or Geriatric Samples

Problem

Expected anti-A and/or anti-B are missing or weak.

Explanation

Infants do not make their own ABO antibodies until three to six months of age and any antibody present would be the result of placental transfer of maternal IgG antibody. ABO reverse grouping is not reliable and not performed on infants for this reason.

Elderly patients may show weakened expression of the expected antibody, because of poor nutrition or because of a chronic disease state. The expected antibody may, in some cases, be totally absent.

Resolution

1. check patient’s history; confirm age
2. for elderly patients repeat reverse grouping using 4-6 drops of serum; incubate initially for 15-30 minutes at room temperature. If there is still no agglutination after centrifugation then incubate the tubes (including group O and auto cells in parallel to detect cold/auto antibodies) at 4°C, centrifuge and read. The group O and auto control should be negative.
3. for infants, verify identity of sample
4. rule out hypogammaglobulinemia or agammaglobulinemia

Chimera (missing antibody or additional antigen)

Problem

Expected anti-A and/or anti-B are missing in the reverse grouping or mixed field agglutination is present in the forward grouping.

Explanation

Chimeras have two populations of red cells in their circulation. In a natural chimera, the individual would have exchanged hematopoietic tissue with a fraternal twin while still in utero. For some reason, the circulations of the twins were  joined for a period of time, and each bears a majority of his/her own hematopoietic cells and a minority of his/her twin’s. The minor population of cells is recognized as self, and so no antibody develops even though the antigen is not present in “full” strength. This same type of reaction can be seen in Dispermy where fertilization of the ovum occurs with two genetically different sperm.

A transient chimera occurs whenever there is a massive transfusion of group O blood to a group A, B or AB individual, or when bone marrow or peripheral stem cells of a different ABO type are transplanted in a person whose marrow is inactive due to disease or irradiation.

Resolution

1. Obtain the patient’s history; look for twinning or recent transfusion or transplantation. Most chimeras are of the artificial transfusion type.
2. Try to separate the suspected cell populations by differential agglutination using anti-M or anti-N, if you suspect a natural chimera. No further investigation would be required if the patient’s history showed a massive transfusion of group compatible red cells.

Anomalies Involving an Extra Antibody

Anti-A1 in A2 or A2B Individuals

Problem

Unexpected reaction with A1 cells in reverse grouping.

Explanation

Some individuals who are group A2 or A2 B develop anti-A1.

Resolution

1. Test patient’s red cells with anti-A1; expected result is negative.
2. Test patient’s serum with at least 3 examples of A1, A2, B cells; O cells and an auto control; expected results with A2 cells are negative.

Anti-H in Bombay Persons

Problem

Unexpected reactions with reverse grouping cells or group O cells in antibody screening test.

Explanation

Individuals with the Bombay phenotype lack H antigen and develop a potent anti-H which will react “H” antigen positive red cells.

Resolution

1. Test patient’s cells with Ulex europaeus lectin (anti-H);expected result negative
2. It may be necessary to run a differential “cold” panel to isolate what type of cold antibody this is (Adult O, Adult A1, Adult A2, Cord O and Cord A cells).

Unexpected cold allo-antibody

Problem

Unexpected reactions with some or all of the cells in reverse grouping.

Explanation

A small percentage of patients have antibodies directed against red cell antigens other than A or B e.g. anti-M, -N, -P1, -Lea, -Leb. These antibodies will react with red cells that have the corresponding antigen. They may react with the A1 and B cells if the cells  have the corresponding antigen.

Resolutions

1. Identify the cold allo-antibody using a panel of group O reagent red cells at 4^oC.
2. Test the patient’s cells with the appropriate antiserum to confirm the absence of the antigen corresponding to the antibody identified.

Unexpected cold auto-antibody

Problem

Unexpected reactions with some or all of the cells in reverse grouping. Sometimes the patient’s cells are heavily coated with the auto-antibody and may spontaneously agglutinate regardless of the reagent antibody so there might be interference in the forward grouping as well.

Explanation

Cold reactive auto-antibodies are often detected in reverse grouping but may interfere in the forward grouping if the cells are already coated with antibody (+DAT). These antibodies are not clinically significant.

Resolution

1. If the discrepancy is interfering with the reverse grouping (using the patient’s serum): identify auto-antibody using a “cold panel” (Adult O, Adult A1, Adult A2, Cord O and Cord A cells) or use antibody screen/ID with panel.
2. If the discrepancy is interfering with the forward grouping (using patient’s cells): incubate the patient’s red cells at 37°C for a short period then wash them with warm saline 3 times before re-typing (pre-warm). If this does not work the patient’s rbc’s can be treated with DTT to disperse the IgM agglutination.

Rouleaux

Problem

All cells in the reverse grouping show agglutination. This might also show up in the forward grouping when using “unwashed” cell suspensions.

Explanation

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 polyvinylpyrollidone (PVP), which are used as blood volume expanders.

Rouleaux will be present wherever the patient’s serum is present, e.g., in the reverse serum group, with unwashed cell suspensions and all phases of the antibody screen and auto control except the antiglobulin phase where the serum is washed away. Macroscopically, rouleaux usually presents as weak agglutination. Microscopically, rouleaux can appear as coin stacks.

Resolution

1. Check all serum tests are “positive” (2+ or less) microscopically for “coin stacks”.
2. Check the diagnosis for diseases such as multiple myeloma.
3. Check for infusions of substances such as dextran or PVP.
4. Try to disperse the rouleaux using the following saline replacement method: centrifuge the test tube, remove the serum and replace it with an equivalent amount of saline. Re-read the test. Rouleaux should disperse to give a negative result. (in some cases saline can be added to the slide while observing microscopically and the rouleaux can be seen dispersing). True agglutination remains positive even after saline replacement is performed.

Extra antibody (weak anti-B) with weak B antigen or “CIS AB”.

Problem

RBC’s with CIS “AB phenotype” express a weakly reactive A antigen and weak B antigen.  The person may make a “weak” anti-B so will react weakly with the group B cells.

Explanation

(NOTE: This is an extremely rare condition)

Serum of most CIS-AB people contains a weak anti-B, which reacts with all ordinary B red cells but NOT with CIS-AB red cells. It has been suggested the B antigen may represent only a piece of the normal B antigen. There is probably unequal crossing-over between the two chromosomes and A antigen and B antigen end up on one chromosome.

Resolution

Since this is such a rare condition a resolution would have to be decided upon by the policy and procedures in the institution at that time.

Forward Grouping Anomalies

 Anomalies Involving a Missing/Weak Antigen

Subgroups of A or B (excluding A1 and A2) or Leukemia

Problem

Presence of anti-B in reverse group indicates red cells should have A antigen; reaction with anti-A and/or anti-A,B in forward grouping is/are much weaker than expected.

Explanation

There are several classes of A subgroups weaker than A1 and A2. Subgroups of B exist, but these are rarely encountered. Cells from adults suffering from certain kinds of leukemia may sometimes show a similar weakened expression of the A or B antigen.

Resolutions

1. Initially, repeat the test and incubate at room temperature for up to 30 minutes to allow sufficient time for the antibody and antigen to connect. If still negative/weak, incubate the tubes at 4oC for 15 to 30 minutes with group O and auto tubes as controls. The group O and auto control should be negative.
2. It may be necessary to incubate well-washed patient’s cells with anti-A, and prepare an eluate from these cells. Cells belonging to a subgroup of A will adsorb and elute anti-A, although they may not show agglutination in standard tube tests.
3. Test the patient’s saliva for the group presence of A, B and H blood group substances (can only be used if they are a secretor).
4. Test the patient’s serum for the presence of anti-A1 using A1 and A2 cells.

Weak Expression of Antigens

Problem

The reactions of the patient’s cells in forward grouping are weaker than expected.

Explanation

The A and B antigens in infants up to two years of age are not fully developed, and a weaker reaction is therefore seen when these cells are tested with anti-A and anti-B.

Resolution

Check the patient’s diagnosis and confirm the patient’s age.

Presence of Excessive Amounts of Blood Group Substances in Plasma

Problem

The expected antigen is not detected.

Explanation

The presence of large amounts of A (or B) blood group substance in serum is characteristic of some tumors. The tumor secretes a mucopolysaccharide similar to blood group substances which inhibits the reactivity of the antibodies in the antisera. These substances would not interfere if the tests were performed with well washed red cells. (many labs use suspensions of unwashed red cells as a time saving measure).

Resolution

1. Confirm patient’s clinical history.
2. Repeat tests using suspension of well washed red cells.

Anomalies Involving an Extra Antigen

Acquired B Antigen

Problem

Unexpected reaction with anti-B.

Explanation

Patients with cancer of the colon or severe bowel infections may acquire a “B-like” antigen on their red cells. This happens when the N-acetyl group on the GalNAc immundominant sugar of group A1 cells is digested by acetylase enzymes secreted by enteric bacteria. The remaining Gal portion is very similar to the B antigen. This “B-like” antigen will react with some but not all high titer anti-B sera. This condition will disappear when the patient recovers (or the tumor is removed).

Resolution

1. Check the patient’s clinical history and previous grouping results.
2. Often, monoclonal anti-B reagents will strongly agglutinate with this acquired B antigen. Testing the patient’s cells and serum will yield a negative result as the anti-B in their serum does not agglutinate with the acquired B antigen. Secretor studies may also be helpful. Also a different lot/clone number of anti-B may be used.

T or Tn activation (Polyagglutination)

Problem

Unexpected reactions with anti-A, anti-B and anti-A,B sera.

Explanation

The patient’s cells are polyagglutinable. Latent antigens (hidden below the cell membrane) are exposed when the membrane is digested by enzymes produced by bacteria or viruses causing an infection. The latent antigens are called T or may also be Tn. Anti-T and anti-Tn are present in the serum of all normal adults. The condition of T activation is transient. The patient’s cells are agglutinated by the anti-T or anti-Tn in the grouping sera IF the anti-sera being used is of “human” origin and not monoclonal. Because forward grouping reagents are often monoclonal now, this condition is rarely identified.

T_n activation is rarer than T activation; the same reactions are seen. Tn activation seems to be permanently acquired.

Resolution

1. Check patient’s clinical history and transfusion records (to confirm ABO group).
2. Test patient’s cell with the following lectins:
   1. Arachis hypogea (unroasted peanuts): anti-T
   2. Salvia sclarea: anti-T_n
   3. Salvia horminum: anti-T_n
   4. Dolichos biflorus: anti-T_n

To obtain a reliable ABO group:

• use monoclonal reagents (do not contain anti-T or anti-T_n)

Wharton’s Jelly

Problem

Wharton’s jelly will cause unexpected false agglutination in the forward grouping if the red cells are not well washed. Reactions may vary from 1+ to 4+ depending on the size of the jelly clumps that have red cells attached to them.

Explanation

Wharton’s jelly is the jelly-like substance present in the umbilical cord. If cord blood samples are improperly drawn, i.e., cut and “milked”, wharton’s jelly will be present. Although not really an extra antigen, wharton’s jelly can mimic extra antigens.

Resolution

If cord samples are used for ABO grouping, the red cells for the forward grouping must be well washed with warm saline, at least 3 times, to remove the wharton’s jelly that may be present. The forward grouping alone will determine the ABO group. Alternatively, a fresh blood sample (e.g., a heel prick) may be drawn for ABO grouping.

Antibodies to Dyes

Problem

Unexpected reaction with forward grouping anti-B.

Explanation

Some patients have antibodies (anti-acriflavine) directed to the coloring dyes used in the anti-B (acriflavine-used sometimes as yellow dye in anti-B). This causes false positives with the anti-B when unwashed cells are used.

Resolution

Wash patient’s red blood cells three times with saline to get rid of anti-acriflavine.