Lecture 6 - ABO Discrepancies PDF

SCIE 2040 – Transfusion Medicine 1 Textbook Chapter 4 Lecture 6 - ABO Discrepancies 4.12 ABO Grouping In adults, ABO testing consists of two components: testing for antigen(s) on the person’s red blood cells and antibody(ies) in the person’s plasma. Antig...

SCIE 2040 – Transfusion Medicine 1 Textbook Chapter 4 Lecture 6 - ABO Discrepancies 4.12 ABO Grouping In adults, ABO testing consists of two components: testing for antigen(s) on the person’s red blood cells and antibody(ies) in the person’s plasma. Antigen is identified by combining the unknown (patient or donor) red blood cells with known antibody (anti-A or anti-B). The results of this testing is called a forward grouping. The presence of antibody is identified by combining the unknown (patient or donor) plasma with known ABO red blood cells (A1 or B). This results in a reverse grouping. A positive test, seen as agglutination, indicates the presence of the corresponding antigen or antibody. A negative test or no agglutination indicates the absence of the corresponding antigen or antibody. Although the actual blood type assigned to a patient technically relates to the antigens present on patient red blood cells, the results of the forward and reverse ABO grouping should always correlate to confirm an accurate ABO is reported. Forward Group Unknown Reverse Group Unknown Interpretation cells tested with plasma tested with commercial sera commercial cells Anti-A Anti-B A1 cells B cells + 0 0 + A 0 + + 0 B 0 0 + + O + + 0 0 AB Anti-A,B reagent is also frequently used in testing, and will agglutinate A, B or AB red cells. This serves as a check on the forward reaction and can help identify some ABO subgroups. 4.13 ABO Discrepancies ABO discrepancies typically involve the forward and reverse reactions not agreeing. Detecting mixed field agglutination or obtaining different results with current testing as compared to historical testing are also considered discrepancies. When identifying discrepancies, weaker reactions are more likely to be aberrant than stronger reactions. 4.13.1 Missing or weak reactions in the Forward Group Missing or weak reactions in the forward group may indicate: The patient/donor is a subgroup of A or, less frequently, B. The very rare cis- AB genotype (where both the A and B gene are found on the same 1 SCIE 2040 – Transfusion Medicine 1 Textbook Chapter 4 chromosome with no ABO gene on the other) may also result in weak expression of ABO antigens. Leukemia and other malignancies may result in alteration of the A or B antigen on the red blood cells. The altered antigens do not react normally with routine antisera, resulting in a missing or weak reaction. Some people may have excessive amounts on soluble A and/or B antigen in their plasma. If plasma suspended cells are used in the testing, the soluble antigens can neutralize the reagent antiserum, resulting in a false-negative reaction. Using washed cells will avoid this. There are several methods that can be used to enhance the detection of a weak ABO antigen, including: Modifying the testing procedure. This may include extended room temperature or 4°C incubation, or enzyme treating the patient’s cells (as ABO antigens are enhanced by enzymes). Must be done in accordance with the directions of the reagent manufacturer. Saliva inhibition test may be used instead (if the patient is a secretor) Genetic analysis can be performed Mixed field agglutination is seen when a sample has two different cell populations. This is most often seen caused by transfusion with non-ABO identical cells. Most commonly, a nongroup O patient has received an emergency transfusion of group O cells. To avoid the chance of intravascular hemolysis, only group O red blood cells are transfused in emergency situations when testing on a current sample is not complete. Other causes of mixed-field agglutination include: ABO nonidentical bone marrow or hematopoietic progenitor cell transplantation can also result in two cell populations. This is usually transient but persists in a subgroup of patients. Weak ABO subgroups, such as A3. Chimerism - fraternal twins exchange blood-forming cells in utero or when two different populations of cells (donor and recipient) reside together in the bone marrow after transplantation. Mosaicism occurs when two sperm fertilize one egg (dispermy) resulting in two distinct cell populations. Mixed-field agglutination due to chimerism or mosaicism exists throughout the patient/donor’s life. In addition, antigens on both cell populations are considered self by the person’s immune system and therefore no corresponding ABO antibodies are made. 2 SCIE 2040 – Transfusion Medicine 1 Textbook Chapter 4 Example #1) Possible causes: A3, Chimera, Blood transfusion Anti-A Anti-B Anti-A,B A1 cells B cells 2+MF 0 2+MF 0 4+ 4.13.2 Missing or Weak Reaction in the Reverse Group Discrepant results in the reverse group are more common than in the forward group. The usual first step when a missing reaction in the reverse group is suspected is to check the patient’s age. If the patient is younger than 4 months of age, the reverse group should not be tested and the ABO group should be interpreted based on the forward group alone. Elderly patients may also exhibit decreased antibody concentration. Transfusion and transplantation histories, as well as other medical conditions, are also important in resolving ABO discrepancies: ABO antibodies may be diluted to a nondetectable level in a patient who has recently received therapeutic plasma exchange therapy. Patients who are on aggressive immunosuppressant therapy can have decreased expression of their ABO antibodies. Patients who have had a nonidentical ABO bone marrow transplant will exhibit various ABO discrepancies depending on their stage of engraftment. Certain diseases such as agammaglobulinemia, hypogammaglobulinemia, and some leukemias and lymphomas may decrease ABO antibody production. However, in adult patients the ABO blood group should not be based solely on the forward grouping. All attempts should be made to enhance ABO antibodies to a detectable level. If the antibodies cannot be detected even with enhancement techniques, then the ABO group remains discrepant. Some resolution techniques include: Serum/Cell Ratio: In tube testing, adding 1-2 additional drops of patient/donor plasma can sometimes enhance a weak reaction. Temperature: ABO antibodies are IgM and that IgM antibodies are generally cold-reactive. The reverse group can be incubated for 15–30 minutes at room temperature. If the room temperature incubation does not resolve the discrepancy, the testing temperature can be lowered to 4° C, where group O cells and an autocontrol should also be tested to control for the presence of other cold antibodies. Enzymes: Proteolytic enzyme treatment of the reverse grouping cells can sometimes help enhance a weakly reactive ABO antibody. Enzyme treatment may be done using ficin (figs), papain (papaya), bromelin (pineapple) or trypsin (hog’s stomach). To ensure that any reactivity observed is due to an ABO 3 SCIE 2040 – Transfusion Medicine 1 Textbook Chapter 4 antibody, controls of group O and autologous cells should be enzyme-treated and tested at the same time. Example #2) Possible causes: Newborn, Elderly, Agammaglobulinemia or hypogammaglobulinemia Anti-A Anti-B Anti-A,B A1 cells B cells 4+ 0 4+ 0 0 4.13.3 Unexpected Positive Reactions in the Forward Group: Unexpected positive reactions in the forward group can be due to substances in the plasma, especially if the testing is done using unwashed cells: Wharton’s jelly is a gelatinous, intercellular substance in umbilical cords, rich in hyaluronic acid. Samples obtained by stripping the cord are often contaminated with Wharton’s jelly, causing nonspecific agglutination in ABO tests. Abnormally elevated globulin plasma protein can cause rouleaux (clumping of red blood cells resembling a “stack of coins”) that may be interpreted as agglutination. Cold autoagglutinins (cold-reacting antibodies), Ph-dependent autoantibodies, antibodies to a constituent of the antiserum (preservatives or dye), or the presence of infused high-molecular weight substances All of these discrepancies are usually resolved by repeating the tests using washed, saline-suspended cells. In the case of Wharton’s jelly, cells may need to be washed multiple times in warm saline. Differences in the red blood cell membrane from the norm may also cause unexpected reactions in the forward group. Some patient/donors may have acquired or inherited changes to the red blood cell membrane that causes their cells to agglutinate with all plasma. This is called polyagglutination, and is the result of exposure of an antigen on the red blood cell membrane that is usually hidden. These antigens are called cryptantigens. Most normal adult plasma contains naturally occurring antibodies against various cryptantigens. It should be suspected when a patient experiences a hemolytic transfusion reaction with no explanation (no antibodies are found in the patient’s plasma directed against antigens on the donor cells). Patients with polyagglutinating cells generally do not have the corresponding antibody in their plasma. However, the normal human plasma in the transfused unit will contain antibody against the patient’s exposed cryptantigen and can cause extravascular or intravascular hemolysis. 4 SCIE 2040 – Transfusion Medicine 1 Textbook Chapter 4 Polyagglutinating cells can be classified using a panel of lectins with various cryptantigen specificity. These classifications include: Cause Type(s) of Polyagglutination Bacterially acquired T, Th, Tk, Tx, acquired B, possible VA (transient) Inherited (permanent) Cad, hemoglobin M-Hyde Park, HEMPAS, NOR Somatic mutation (persistent) Tn Most examples of polyagglutination are acquired due to bacterial infections. Acquired B antigen, for example, has been identified in patients with gastrointestinal disease where enzymes from enteric bacteria modify some A antigens to antigens that are similar enough to the B antigen to react with some clones of Anti-B sera. Today, polyagglutination is rarely discovered on routine ABO testing because clonal rather than human source antisera are used. However, this has resulted in the detection of “new” phenotypes. An example of this is the B(A) phenotype, in which some group B patient/donor RBCs react weakly with monoclonal anti-A made from certain clones. Most B(A) people have a minor mutation which causes their B enzyme to be slightly more similar to the A enzyme. This can result in a small amount of A-like antigens being made by the B-specific enzyme. Example #3) Possible causes: Polyagglutination (acquired B), patient antibody to dye/preservative in anti-B reagent Anti-A Anti-B Anti-A,B A1 cells B cells 4+ 1+ 4+ 0 4+ Example #4) Possible cause: Warton’s Jelly Anti-A Anti-B Anti-A,B A1 cells B cells 4+ 1+ 4+ 0 0 4.13.4 Unexpected Positive Reactions in the Reverse Group ABO antibodies passively acquired by transfusion of intravenous immune globulin (IVIG) or, less frequently, by ABO nonidentical plasma or platelet transfusions may cause unexpected reverse reactions. However, unexpected positive reactions in the reverse group may be due to the presence of non-ABO antibodies in the patient/donor plasma that are cross-reacting in the ABO test. These antibodies 5 SCIE 2040 – Transfusion Medicine 1 Textbook Chapter 4 could be cold autoagglutinins, alloantibodies that reacts at room temperature to an antigen present on the reverse cell(s), or, rarely, an antibody to one of the constituents of the reverse cells’ suspension medium. A positive reaction with A1 cells in a suspected group A or AB person could indicate that the person is a subgroup of A2 with an anti-A1. Much rarer weak subgroups of A or B or who are cis-AB (a condition where the A and B gene are on the same chromosome) may produce unexpected ABO antibodies. Additionally, the expression of their A and/or B antigens may be weakened. A suspected anti-A1 should be distinguished from cold antibodies against other antigen systems: A1 Positive O Interpretation Cells Cells + 0 Likely Anti-A1. Confirm by phenotyping the patient with Dolichious biflorus. + + Antibody other than anti-A1 alone or in combination with anti-A1. Confirm/rule-out presence/absence of anti-A1 by phenotyping the patient with Dolichious biflorus and testing patient plasma against multiple different A1 cells. 0 + Antibody other than anti-A1. Anti-P1 and anti-M are two relatively common other cold alloantibodies that often result in discrepancy. Cold autoantibodies that can interfere in ABO testing include anti-I, anti-H and anti-IH. Cold-reactive antibodies can sometimes be neutralized in the ABO testing by prewarming the plasma and cells before combining them. Rather than centrifuging the tests, allow the cells to settle to the bottom of the tube by incubating at 37° C. Strong ABO antibodies should still react, but interfering reactions are inhibited. Additionally, cold-reactive antibodies can be identified by testing panels of cells with known phenotypes using the patient/donor’s plasma. Then, reverse grouping A1 and B cells that phenotype as negative for the offending antigen can be used to resolve the discrepancy. Adsorbtions and elutions are additional techniques that may be used to resolve discrepancies and will be discussed in the next course. These are especially useful when plasma agglutinates all cells it is tested against (panagglutination) so the antibody cannot easily be identified. 6 SCIE 2040 – Transfusion Medicine 1 Textbook Chapter 4 Weak positive could also be due to substances in the patient/donor plasma that are causing nonspecific agglutination of the red blood cells. Rouleaux can be caused by diseases that greatly increase serum protein concentrations, such as multiple myeloma and Waldenström’s macroglobulinemia, or by the transfusion of high- molecular-weight substances, such as dextran. Suspected rouleaux can be investigated with the saline replacement technique, where cells are reacted with the patient’s plasma but then the plasma is carefully removed and replaced with an equal amount of saline before reading. True agglutination will persist but rouleaux will be dispersed. Example #5) Probable causes listed under each reaction. Anti-A Anti-B Anti-A,B A1 cells B cells O cells Auto 4+ 0 4+ 1+ 4+ 1+ 0 Cold alloantibody (such as anti-M or anti-P1). Anti-A1 may or may not also be present Anti-A Anti-B Anti-A,B A1 cells B cells O cells Auto 4+ 0 4+ 0 4+ 1+ 0 Cold alloantibody (such as anti-M or anti-P1) Anti-A Anti-B Anti-A,B A1 cells B cells O cells Auto 4+ 0 4+ 1+ 4+ 0 0 A2 patient with an anti-A1 Anti-A Anti-B Anti-A,B A1 cells B cells O cells Auto 4+ 0 4+ 1+ 4+ 2+ 1+ Cold autoantibody (such as anti-I, anti-H or anti-IH) causing panagglutination, or rouleaux misinterpreted as agglutination 4.13.5 Technical Errors No matter which type of discrepancy is discovered, a good first step in the resolution is to repeat the testing in case a technical error was made in the procedure. Always consult with your laboratory’s procedure, as it may vary from lab to lab. Also observe the manufacturer’s instructions for reagents in use. 7 SCIE 2040 – Transfusion Medicine 1 Textbook Chapter 4 It is also good laboratory practice to check the reagent control results when a discrepancy occurs. Decreasing strength of reaction over time could indicate a contaminated or expired reagent. Also check the expiration date and appearance of reagents. Signs of contamination can include cloudiness, changes in color, or hemolysis. Similar ABO discrepancies on sequential samples may indicate an equipment problem, for example, centrifuge overdue for calibration. An ABO discrepancy, especially between current and historical results, can also be a warning that a specimen has been mislabeled. Drawing and testing a new sample can reveal the problem. This may require a larger investigation to determine the scope and impact of the clerical error, which may extend outside the blood bank. 8