ABO Discrepacie-HA (1).pdf

ABO blood group discrepancy… From Modern blood banking… ABO Discrepancies:  ABO discrepancies occur when unexpected reactions are obtained in the forward and/or reverse grouping.  These can be due to problems with the:  patient’s serum (reverse grouping).  problems with the patient’s RBC...

ABO blood group discrepancy… From Modern blood banking… ABO Discrepancies:  ABO discrepancies occur when unexpected reactions are obtained in the forward and/or reverse grouping.  These can be due to problems with the:  patient’s serum (reverse grouping).  problems with the patient’s RBCs (forward grouping).  problems with both the serum and cells.  The unexpected reaction(s) may be due to an extra-positive reaction or a weak or missing reaction in the forward and reverse grouping.  All ABO discrepancies must be resolved prior to reporting a patient or donor ABO group  Forward grouping (front type):  is defined as using known sources of commercial antisera (anti-A, anti-B)to detect antigens on an individual’s RBCs.  Reverse grouping(back type):  is defined as detecting ABO antibodies in the patient’s serum by using known reagent RBCs, namely A1and B cells. Technical errors:  They can result in ABO discrepancies include: 1.Clerical error: Incorrect or inadequate identification of blood specimens, test tubes, or slides. 2. Technical errors: Sample: 1. A mix-up in samples. 2. Missed observation of hemolysis. Reagent: Expired or Contaminated reagents. Instrument: Uncalibrated centrifuge. Procedure: 1. Cell suspension either too heavy or too light. 2. Failure to add reagents. 3. Failure to add sample. 4. Failure to follow manufacturer’s instructions. 5. Warming during centrifugation. 6. Under or over centrifugation. Interpretation: incorrect recording of results (strong serum reaction may cause hemolysis).  Note:  serum and antiserum should always be added first,  followed by the patient or reagent RBCs to avoid both reagent contamination and potential omission of either patient sample or reagent.  Results must be recorded immediately after obtaining them to avoid transcription errors. Resolution of Technical Errors(4R):  Retest with saline and avoid technical error:  If initial testing was performed using RBCs suspended in serum or plasma, repeat testing of the same sample using a saline suspension of RBCs can usually resolve the ABO discrepancy.  Rule out clerical error.  Review patient history:  It is also essential to obtain adequate information regarding the patient’s age, diagnosis, transfusion history, medications, and history of pregnancy.  Request another sample:  If the discrepancy persists and appears to be due to an error in specimen collection or identification, a new sample must be drawn from the patient and all RBC and serum testing repeated. Do I have solve the discrepancy?  Yes, it should be resolved before labeling donated unit or issuing blood products.  If the blood is from a potential transfusion recipient, it may be necessary to administer group O, Rh-compatible RBCs and safest plasma (AB) before the discrepancy is resolved. How to suggest which is likely abnormal; cell(Ag) or serum (Abs)?  In general, when investigating ABO discrepancies, always remember that RBC and serum grouping reactions are very strong (3+ to 4+) and the weaker reactions typically represent the discrepancy.  Discrepancy of serum grouping is more common. Categories of ABO Discrepancies:  ABO discrepancies divided into four major categories:  Group I Discrepancies :  are associated with unexpected reactions in the reverse grouping due to weakly reacting or missing antibodies.  These discrepancies are more common than those in the other groups.  Reasons for the missing or weak isoagglutinins : 1.patient has depressed antibody production 2. patient cannot produce the ABO antibodies.  Note: normally, RBC and serum grouping reactions are very strong (4+),So when a reaction in the serum grouping is weak or missing ,a group 1 discrepancies. Populations with group1 discrepancies 1.Newborns (ABO antibody production is not detectable until 3 to 6 months of age). 2. Elderly patients (production of ABO antibodies is depressed). 3. Patients with a leukemia (e.g., CLL) or lymphoma (e.g.,malignant lymphoma) demonstrating Hypogammaglobulinemia. Patients using immunosuppressive drugs that yield hypogammaglobulinemia. 4. Patients with congenital or acquired agammaglobulinemia or immunodeficiency diseases. 6.Patients with bone marrow or hematopoietic progenitor stem cell (HPC) transplants (patients develop hypogammaglobulinemia from therapy & start producing a different RBC population from that of the transplanted bone marrow). 7.Patients whose existing ABO antibodies may have been diluted by plasma transfusion or exchange transfusion. 8.ABO subgroups (A or B) typed as O :characteristically show single Ab.  3.An auto control and O cell control must always be tested concurrently with the reverse typing when trying to solve the discrepancy since the lower temperature of testing will most likely enhance the reactivity of other commonly occurring cold agglutinins such as anti_I, that react with all adults RBCs.  Mixed-field agglutination: may look like small to large agglutinates with unagglutinated cells.  Mixed-field may also appear as a “halo” or “puff of smoke” of unagglutinated RBCs as the RBC button is dislodged from the test tube bottom. Common causes of mixed-field reactions include :  receiving non-ABO-type specific RBCs.  ABO subgroups (A3).  bone marrow or hematopoietic stem cell transplantation  A major ABO incompatibility occurs when the donor’s red RBCs are incompatible with the recipient’s plasma (e.g.,the donor is group B and the recipient is group O with naturally occurring anti-B).  Complications of major ABO incompatibility: 1.hemolysis of RBCs at time of infusion. 2.continued antibody production directed against erythroid progenitors and donor RBCs by the engrafted HPCs.  A minor ABO incompatibility occurs when the donor’s plasma is incompatible with the recipient’s RBCs (e.g., the donor is group O with naturally occurring anti-B and the recipient is group B).  Bidirectional ABO incompatibility :  occurs when both a major and minor incompatibility are present (e.g., the donor is group A and the recipient is group B).  Since ABO antigens are histo-blood group antigens present on many tissues, including the lung, pancreas, bowel, endothelium, heart, and kidney, recipients of ABO incompatible bone marrow or hematopoietic progenitor stem cell transplantation pose distinctive challenges to transfusion service staff when selecting blood components for transfusion.  Following an ABO-incompatible HPC transplant, the pre- transplant ABO type will remain in these tissues for the rest of the patient’s life.  Accordingly, the patient will never make antibody against the ABO type the body still sees as self (e.g., a group B recipient converted to group A will never make anti-B). Note:RBCs, platelets, and plasma products must be compatible with both the donor and recipient blood types.  Chimerism:  Is defined as the presence of two cell populations in a single individual.  It was discovered in twins born to a group O mother and group B father with a mixture of both B and O cells instead of the expected group of either B or O.  Detecting a separate cell population may be easy or difficult, depending on what percentage of cells of the minor population are present.  Reactions from chimerism are typically mixed-field. True chimerism:  occurs only in twins and is rarely found. The two cell populations will exist throughout the lives of the individuals.  In utero exchange of blood occurs because of vascular anastomosis. As a result, two cell populations emerge, both of which are recognized as self, and the individuals do not make anti-A or anti-B.  Expected isoagglutinins are not present in the reverse grouping, depending on the percentage of the population of RBCs that exist in each twin.  If the patient or donor has no history of a twin, then the chimera may be due to dispermy (two sperm fertilizing one egg) and indicates mosaicism. Artificial chimeras:  More commonly, which yield mixed cell populations as a result of the following: 1.Blood transfusions (e.g., group O cells given to an A or B patient). 2.Transplanted bone marrow or hematopoietic. progenitor stem cells (HPC) of a different ABO type. 3. Exchange transfusions. 4.Fetal-maternal bleeding. Group II Discrepancies: ► Associated with unexpected reactions in the forward grouping due to weakly reacting or missing antigens.It is least frequently encountered. The following are some of the causes of discrepancies in this group: 1.Subgroups of A or B may be present. 2.Leukemias may yield weakened A or B antigens , and Hodgkin’s disease has been reported in some cases to mimic the depression of antigens found in leukemia. 3.The “acquired B” phenomenon will show weak reactions with anti-B antisera and is most often associated with diseases of the digestive tract (e.g., cancer of the colon). Resolution of Group II Discrepancies:  The agglutination of weakly reactive antigens with the reagent antisera can be enhanced by :  1.incubating the test mixture at room temperature for up to 30 minutes to increase the association of the antibody with the RBC antigen  2.If the reaction is still negative, incubate the text mixture at 4°C for 15 to 30 minutes.  Include group O and autologous cells as controls.  3.RBCs can also be pretreated with enzymes and retested with reagent antisera. Acquired B phenomenon:  Arises when bacterial enzymes modify the immunodominant blood group A sugar (N-acetylD- galactosamine) into D-galactosamine, which is sufficiently similar to the group B sugar (D-galactose) to cross-react with anti-B antisera, this pseudo-B antigen is formed at the expense of the A1 antigen and disappears following the patient’s recovery.  The reaction of the appropriate antiserum with these acquired antigens demonstrates a weak reaction, often yielding a mixed-field appearance. Resolution of acquired B antigen by the followings:  1.Blood group reagents of a monoclonal anti-B clone (ES4)strongly agglutinate cells with the acquired B antigen.  The pH of reagents containing ES4 has been lowered, and consequently, only those cells with the strongest examples of acquired B antigen react with the antisera  2.Testing the patient’s serum or plasma against autologous RBCs gives a negative reaction, because the anti-B in the-serum does not agglutinate the patient’s RBCs with the acquired B antigen.  3.The acquired B antigen is also not agglutinated when reacted with anti-B that has a pH greater than 8.5 or less than 6.  4.Secretor studies can be performed when trying to characterize the acquired B phenomenon. If the patient is in fact a secretor, only the A substance is secreted in the acquired B phenomenon.  5.Treating RBCs with acetic anhydride re-acetylates the surface molecules, then markedly decreases the reactivity of the cells tested with anti-B.The reactivity of normal B cells is not affected by treatment with acetic anhydride.  Rare Group II Discrepancies  1. Excess amounts of blood group–specific soluble (BGSS) substances present in the plasma, which sometimes occurs with certain diseases, such as carcinoma of the stomach and pancreas.  Excess amounts of BGSS substances will neutralize the reagent anti-A or anti-B, leaving no unbound antibody to react with the patient cells and yields a false-negative or weak reaction in the forward grouping.  washing the patient cells free of the BGSS substances with saline should alleviate the problem, resulting in correlating forward and reverse groupings. 2.Antibodies to low-prevalence antigens in reagent anti- A or anti-B can also cause weakly reactive or missing reactions in RBC group.  It has been reported that this additional antibody in the reagent antisera has reacted with the corresponding low-prevalence antigen present on the patient’s RBCs.  This produces an unexpected reaction of the patient’s cells with anti-A or anti-B or both, mimicking the presence of a weak antigen. Resolution of discrepancy caused by low prevalence antibodies by:  Repeating the forward type, using antisera with a different lot number.  If the cause is a low-prevalence antibody in the reagent antisera reacting with a low-prevalence antigen on the patient’s cells, the antibody will most likely not be present in a different lot number of reagent.  This phenomenon is only seen when human source antiserum is used.  Note: most ABO reagents in use today are monoclonal antibodies and these reagents are free of contaminating antibodies to low-prevalence antigens. Group III Discrepancies:  These discrepancies between forward and reverse groupings are caused by protein or plasma abnormalities and result in rouleaux formation or pseudo agglutination, attributable to:  1.Elevated levels of globulin from certain disease states, such as: multiple myeloma, Waldenström’s macroglobulinemia, other plasma cell dyscrasia, and certain moderately advanced cases of Hodgkin’s lymphomas.  2.Elevated levels of fibrinogen.  3.Plasma expanders, such as dextran and polyvinylpyrrolidone.  4.Wharton’s jelly in cord blood samples.  5.Rouleuax formation. Resolution of Group III Discrepancies  Rouleaux: is a stacking of erythrocytes that adhere in a coin like fashion, giving the appearance of agglutination.  Resolution can be done by the followings: 1.Rouleaux formation can observed on microscopic examination. 2. saline replacement technique will free the cells in the case of rouleaux formation in the reverse type. Serum is removed and replaced by an equal volume of saline.  NOTE: true agglutination, RBC clumping will still remain after the addition of saline. NOTE:  The Cord blood samples can pose a problem in ABO testing, since cord cells may be contaminated with Wharton’s jelly(this substance is a viscous mucopolysaccharide material present on cord blood cells that may cause the red blood cells’ aggregation).  Washing cord cells six to eight times with saline should alleviate spontaneous rouleaux due to Wharton’s jelly and result in an accurate ABO grouping.  However, testing is usually not done on cord serum because the antibodies detected are usually of maternal origin, reverse grouping may still not correlate with the RBC forward grouping. Group IV Discrepancies:  These discrepancies between forward and reverse groupings (EXTRA REACTION) are due to miscellaneous problems that have th following causes: 1.Extra reaction with Positive AC: 1. Cold reactive autoantibodies in which RBCs are so heavily coated with antibody that they spontaneously agglutinate, independent of the specificity of the reagent antibody. 2. Rouleaux formation. 2. Extra reaction with Negative AC: 1.Unexpected ABO isoagglutinins.(ABO subgroups). 2.Unexpected non-ABO alloantibodies.(Cold alloabs). 3. Circulating RBCs of more than one ABO group due to RBC transfusion or marrow/stem cell transplant.(mixed field agglutination). Resolution of Group IV Discrepancies: 1. Extra positive reaction with positive AC: Potent cold autoantibodies can cause spontaneous agglutination of the patient’s cells.These cells often yield a positive direct Coombs’ or antiglobulin test.  If the antibody in the serum reacts with all adult cells, for example, anti-I, the reagent A1 and B cells used in the reverse grouping also agglutinate. To resolve this discrepancy by the following’s:  1. Worming at 37C: Patient’s RBCs could be incubated at 37°C for a short Period, then washed with saline at 37°C three times and retyped.  2. Enzyme treatment: if this is not successful in resolving the forward type, the patient’s RBCs can be treated with 0.01 M dithiothreitol (DTT) to disperse IgM-related agglutination.  As for the serum, the reagent RBCs and serum can be warmed to 37°C for 10 to 15 minutes, mixed, tested, and read at 37°C.  The test can be converted to the antihuman globulin phase if necessary.  Weakly reactive anti-A or anti-B may not react at 37° C, which is outside their optimum thermal range.  3. Cold auto absorption If the reverse typing is still negative (and a positive result was expected), a cold auto absorption (patient cells with patient serum) could be performed to remove the cold autoantibody from the serum. The absorbed serum can then be used to repeat the serum typing at room temperature. 2. Extra reaction with Negative AC: A-Unexpected ABO isoagglutinins(Abo subgroups):  In the patient’s serum react at room temperature with the corresponding antigen present on the reagent cell.  Examples discrepancy include A2 and A2B individuals, who can produce naturally occurring anti-A1, or A1 and A1B, individuals who may produce naturally occurring anti-H.  To resolve this discrepancy by the followings: 1. Serum grouping can be repeated using at least three examples of A1, A2, B cells; O cells 2. Anautologous control (patient’s serum mixed with patient’s RBCs). The specificity of the antibody can be determined by examining the pattern of reactivity (e.g., if the antibody agglutinates only A1 cells, it can most likely be identified as anti-A1). 3. The patient’s RBCs can be tested with Dolichos biflorus to confirm the presence of the ABO subgroup. Dolichos biflorus will agglutinate cells of the A1 but not the A2 phenotype. B. Unexpected alloantibodies in the patient’s serum other than ABO isoagglutinins (e.g., anti-M) may cause a discrepancy in the reverse grouping. Reverse grouping cells possess other antigens in addition to A1 and B, and it is possible that other unexpected antibodies present in the patient’s serum will react with these cells.  Resolved by: an antibody identification panel should be performed with the patient’s serum.  Once the unexpected alloantibodies are identified, reagent A1 and B cells negative for the corresponding antigen should be used in the reverse grouping. Rare Group IV Discrepancies:  Antibodies other than anti-A and anti-B may react to form antigen-antibody complexes that may then adsorb onto patient’s RBCs give extra positive reaction in forward grouping.  1. Individual with an antibody against acriflavine, the yellow dye used in some commercial anti-B reagents.  The acriflavine antiacriflavine complex attaches to the patient’s RBCs, causing agglutination in the forward type.  Resolving this discrepancy by washing the patient’s cells three times with saline and then retyping them. 2.Cis-AB:  refers to the inheritance of both AB genes from one parent carried on one chromosome and an O gene inherited from the other parent.This results in the offspring inheriting three ABO genes instead of two.  The designation cis-AB is used to distinguish this mode of inheritance from the more usual AB phenotype in which the alleles are located on different chromosomes.  The cis-AB phenotype was first discovered in 1964, when a Polish family was described in which the father was group O and the mother was group AB and gave birth to children who were all group AB.  It the fact that the A and B genes were inherited together and were both on the same, or cis, chromosome; thus the term cis-AB.  RBCs with the cis-AB phenotype (a rare occurrence) express a weakly reactive A antigen (analogous to A2 cells) and a weak B antigen.  Forward grouping: The B antigen usually yields a weaker reaction with the anti-B from random donors, with mixed-field agglutination typical of subgroup B3 reported in several cases.  Reverse grouping: Weak anti-B (present in the serum of most cis-AB individuals) leads to an ABO discrepancy in the reverse grouping.  The serum of most cis-AB individuals contains a weak anti-B, which reacts with all ordinary B RBCs, yet not with cis-AB RBCs. A and B transferase levels are lower than those found in ordinary group AB sera.  Many favor an unequal crossing over between the A and B gene with gene fusion and the formation of a new gene . B-specific sugars to the precursor molecule.  Note: Many families have been reported in other parts of the world, with a high incidence of cis-AB being found in Japan.  The banding pattern of the distal end of the long arm of chromosome 9 representing the ABO locus is normal.  There are other examples of cis-ABs.In which a point mutation at the ABO locus, and an enzyme was produced that was capable of transferring both A-specific and B-specific sugars to the precursor molecule.  Note: Many families have been reported in other parts of the world, with a high incidence of cis-AB being found in Japan. Acquired B phenomenon Extra positive reaction in reverse grouping with negative AC AB subgroup with anti A1 Extra positive reaction in reverse grouping with negative AC Cold Allo-antibodies Extra positive reaction in reverse grouping with positive AC Cold auto antibodies Or rouleaux 1.An example of a technical error that can result in an ABO discrepancy is: a. Acquired B phenomenon. b. Missing isoagglutinins. c. Cell suspension that is too heavy. d. Acriflavine antibodies. 2. An ABO type on a patient gives the following reactions: Patient cells With Patient Serum With Anti-A Ant-B A1 cells B cells O cells auocontrol 4+ Neg 2+ 4+ 2+ Neg These results are most likely due to: a. ABO alloantibody. b. Non-ABO alloantibody. c. Rouleaux. d. Cold autoantibody. 3.An ABO type on a patient gives the following reactions: Patient Cells With Patient Serum With Anti-A Anti-B Anti-A1 A1 cells B cells 4+ 4+ Neg 2+ Neg The reactions above may be seen in a patient who is: a. A1 with acquired B. b. A2B with anti-A1. c. AB with increased concentrations of protein in the serum. d. AB with an autoantibody Thank you