ABO Typing Discrepancies

Function

Specific antigens on the surface of RBCs and the corresponding antibodies in serum determine the 4 main blood types. These blood groups are inherited based on the ABO gene (OMIM 616093), which encodes enzymes that modify carbohydrate molecules on RBCs. The backbone carbohydrate, the H antigen, encoded by FUT1 (OMIM 211100), is a fucosyltransferase to which the sugars N-acetylgalactosamine for the A antigen and d-galactose for the B antigen are added.[5] More specifically, the A antigen occurs on RBCs when the enzyme α-1,3-N-acetyl-D-galactosyltransferase adds N-acetylgalactosamine to the H antigen. The B allele of ABO encodes for the glycosyltransferase enzyme that adds d-galactose to the H antigen, creating the B antigen on the RBC surface. The O allele encodes for a nonfunctional enzyme, leaving the H antigen unmodified.

Individuals with type A blood have A antigens on the RBC surface and anti-B antibodies, whereas those with type B blood have B antigens and anti-A antibodies. Individuals with type AB blood possess both A and B antigens without anti-A or anti-B antibodies, making them universal plasma donors and recipients of RBCs. Individuals with type O blood lack A and B antigens but have anti-A and anti-B antibodies, making them universal RBC donors but restricted recipients. 

An additional component of blood type is the Rh blood group system. The Rh antigens, located on proteins in the RBC membrane and encoded by RHD (OMIM 111690) and RHCE (OMIM 617970 ), further refine blood compatibility. The most common Rh antigens include D, C, c, E, and e. Individuals whose RBCs lack the RhD antigen are classified as Rh-negative

Unlike most antibodies, anti-A and anti-B antibodies develop naturally without prior exposure to blood products. As the gut becomes colonized in early infancy, infants between 4 and 6 months develop antibodies against bacterial antigens that closely resemble A and B antigens through a process known as molecular mimicry. In contrast, antibodies to Rh antigens typically develop only after exposure to foreign RBCs through transfusion or pregnancy.

Clinicians perform blood group typing or ABO typing with both forward and reverse typing, whereas Rh testing only involves forward-type testing. The forward grouping reaction, which detects the antigen on the surface of the patient's RBCs, should match the results of the reverse grouping reaction, which detects the antibodies in the patient's serum. Naturally occurring anti-A and anti-B antibodies should be present in individuals lacking the corresponding RBC antigens. Clinicians expect the forward and reverse grouping reactions to be strong (level 3+ or 4+) (See Table 1. Expected ABO Typing Results). In cases of ABO discrepancies, weak reactions (level 1+ or 2+) occur.[6]

Table 1. Expected ABO Typing Results

If discrepancies arise between the forward and reverse blood type results, clinicians must determine the reason before determining the blood type. Until it is resolved, the patient can receive O-negative blood. Clinicians must not ignore ABO discrepancies.[1][7] With the automatization and standardization of testing procedures, the incidence of ABO discrepancies has reduced considerably due to decreased technical errors. Excluding technical errors should be the first step when faced with discordant results. Clinicians should verify accurate sample labeling, adherence to proper testing techniques, absence of contamination, and validity of reagents. Routine training of laboratory staff and continuous monitoring of quality standards help mitigate these errors.[7] 

Classification of ABO Discrepancies

Experts classify ABO discrepancies into 4 categories.[8][9]

Group I

Group I discrepancies occur when decreased antibody levels in the patient's serum lead to weak or absent reactions in reverse grouping.

The following scenarios may lead to this type of result: 

• ABO typing in newborns
• Occasionally, in older patients
• Patients on immunosuppressant medications leading to decreased lymphocyte function and antibody production
• Congenital immunodeficiency
• Hypogammaglobulinemia
• Children in a sterile environment and those on artificial sterile nutrition cause an underdeveloped gut microbiome
• Natural chimerism [10][11][12][13]

Chimerism is a rare phenomenon in which an individual has two sets of RBCs with different blood groups circulating in the body. Testing recognizes both red cell antigens as self-antigens; therefore, no antibodies against them exist in the serum. This scenario gives rise to mixed field reactions in forward grouping reactions and absent reactions in reverse grouping resulting in an ABO discrepancy, often occurring in fraternal twins. More rarely, chimerism occurs in individuals who are not fraternal twins due to mosaicism.[13][14]

The patient's history determines the underlying cause of most group I reactions. To confirm this discrepancy, clinicians must augment the missing or weak antibody by incubating the serum with reagent RBCs at room temperature for 15 to 30 minutes, after which, in most cases, an acceptable reaction is evident. Some instances require centrifuging the sample to bring the antibodies and cells closer. Clinicians then incubate the sample, and an appropriate reaction should follow. If a reaction is still not evident, clinicians incubate a mixture of the patient's cells and the reagent cells at 4 °C for 30 minutes.

Group II

Patients with group II discrepancies experience an unexpected reaction in the forward grouping. These discrepancies occur in patients with weakly reacting antigens due to a low antigen burden on the RBC surface, the presence of another antigen similar to AB antigens on the RBC surface reacting with the antibody, or partial deletion of antigens on the surface of cells. These antigens yield a weaker-than-expected reaction in the forward typing, which remains unmatched in the reverse typing reaction.

Clinical scenarios that may lead to Group II discrepancies include:

• A or B subgroups
• B(A) phenotype causing a weak expression of A on B RBCs
• Acquired B phenomenon—a temporary condition caused by bacterial enzymes that modify the A antigen on the cell surface, making them temporarily look like B antigens
• Hematological malignancies such as acute and chronic leukemias, myelodysplasia, non-Hodgkin's lymphoma, multiple myeloma, and myeloproliferative disease can alter the antigens present on the surface of cells
• Pregnancy
• Post-transfusion of a different blood group
• Post-allogenic bone marrow transplant with an ABO-incompatible donor 
• Fetomaternal hemorrhage 
• Tn polyagglutination syndrome [3][9][15][16][17][18][19][20]

Clinicians can enhance the weak reaction in forward typing by extending the incubation time to up to 30 minutes at room temperature. If enhancement does not occur with more prolonged incubation, then the mixture should be incubated at 4 °C for up to 30 minutes. Reviewing a patient's clinical history can provide important insights into the cause of the discrepancy.

Subgroups: Blood group A subgroups are among the most common causes of ABO discrepancies. Group B subgroups are very rare. The most common subgroup within group A is A1, followed by A2, which accounts for more than 99% of the A blood group. Other A subgroups, such as A3 and Ax, are very rare. Clinicians can resolve discrepancies caused by the A subgroups using Dolichos bilflorus plant seed extract lectin, which agglutinates A1 but not A2 cells. Detection of A1 antibodies in the patient's serum using A1 RBC reagent cells, detection of secretion A, B, and H substances in the patient's saliva, and molecular genotyping studies can help resolve ABO subgroups.[21][22] Weaker variants may require additional testing, such as subgroup A quantification or molecular analysis.[4]

Acquired B antigen: Acquired B antigen is a rare phenomenon in which certain gram-negative bacterial infections produce enzymes that alter the A antigen on RBCs in an individual with group A blood. These enzymes convert N-acetyl-D-galactosamine into D-galactosamine, which closely resembles D-galactose, the B antigen, and can cross-react with anti-B reagents.[23] This phenomenon is temporary and resolves once the infection clears. This effect may also occur with colorectal carcinomas, intestinal obstruction, and gastrointestinal infections.

To confirm the acquired B phenomenon, clinicians incubate the cells with the monoclonal anti-B clone, yielding a strong reaction. In contrast, the regular B antiserum can yield a weak mixed-field reaction only. In addition, mixing the patient's sera, which contains anti-B antibodies, with the patient's RBCs expressing acquired B does not yield any reaction. Acetic anhydride can also be helpful in these cases as it re-acetylates the modified antigen and decreases its reaction to anti-B antibodies. The patient's original blood group remains A, and their serum contains anti-B antibodies. If transfusion is required, they should receive group A-compatible blood products.[24][25]

Tn polyagglutination syndrome: Tn polyagglutination syndrome is a rare, acquired, permanent disorder involving a mutation in C1GALT1C1 (OMIM 300611) located on chromosome xq24.[26][27] Anti-Tn antibodies are naturally occurring in the serum of most adult humans. This disorder affects pluripotent stem cells, causing all cell lineages to express Tn-antigen on their surfaces. As a result, patients may develop mild hemolytic anemia, thrombocytopenia, and leukopenia, which typically do not require treatment. Tn polyagglutination syndrome is associated with some blood disorders, such as leukemia, myelodysplasia, and myeloproliferative disorders.[20]

B(A) phenotype: The B(A) phenotype is a rare autosomal dominant condition in which the patient's RBCs predominantly express B antigen, but occasional A antigens are also expressed on the surface.[28] These individuals have anti-A antibodies in the serum. The A antigens on the red cells are very few, so no significant hemolysis occurs, and the affected individuals are asymptomatic. However, this variation can cause an ABO discrepancy with an unexpected reaction in forward grouping during ABO typing.[9]

The resolution of the remaining discrepancies depends on the underlying cause; for example, if the discrepancy is due to a recent blood transfusion, then it is reasonable to repeat ABO typing at a later date and use a Kleihauer-Betke test when fetomaternal hemorrhage is suspected. This test measures the amount of fetal hemoglobin in the maternal blood.

Group III

Patients with group III discrepancies exhibit an unmatched reaction between the forward and reverse grouping due to protein or plasma abnormalities. These abnormalities result in rouleaux formation or pseudo-agglutination, leading to false or uninterpretable results.

Conditions that cause rouleaux formation include:

• Hypergammaglobulinemia
• Hyperfibrinogenaemia
• Plasma expanders such as dextran or hydroxyethyl starch
• Wharton jelly contamination of cord blood [8][29][30][31]

ABO typing identifies antigens and antibodies through agglutination. Rouleaux formation closely resembles the agglutination of RBCs and can lead to false-positive results. When rouleaux formation is suspected, technicians should wash the sample with normal saline to remove excess antibodies and proteins responsible for unwanted RBC stacking. Repeating the test with washed cells typically resolves the discrepancy.

In cases of reverse typing discrepancies, clinicians may utilize a saline replacement technique. In this method, after centrifuging the patient's blood, the plasma is removed and replaced with an equal volume of saline, followed by repeating the test. This process removes excess protein in the patient's serum that is responsible for rouleaux formation. The test can then be conducted without protein interference, yielding an interpretable result.

Group IV

Group IV discrepancies are unmatched reactions between the forward and reverse grouping due to miscellaneous causes. An agglutination reaction occurs independent of a specific reagent antibody. Potential causes of a group IV discrepancy are:

• Cold-reactive autoantibodies
• ABO isoagglutinin
• Non-ABO alloantibodies
• Recent intravenous immunoglobulin infusion
• Reaction to rare antigens or antibodies in the reagent used [32][33]

When unexplained positive results occur in both forward and reverse grouping during ABO typing, clinicians should consider cold autoantibodies in the patient's serum. A direct Coombs test may be positive. Clinicians should incubate the patient's RBCs at 37 °C, which typically inactivates immunoglobulin M (IgM) antibodies. The sample should then be washed 3 times in saline at 37 °C before repeating the test. This process helps eliminate the effects of the antibodies on the results. In rare cases, if the previous steps are unsuccessful, 0.01 M dithiothreitol can be added to the patient's RBCs, preventing agglutination due to IgM antibodies.[34]

If the discrepancy arises in the reverse grouping test, the testing should be repeated at 37 °C as this typically stops the interference of the autoantibodies. If an unexpected result persists, a cold autoabsorption test should be performed. The autoabsorption process involves incubating the patient's RBCs with their serum,  leading to a reaction of autoantibodies with the patient's RBCs and reducing their concentration in the serum. Before testing, the sample is centrifuged to remove autoantibody-coated RBCs, and the remaining serum is used for reverse blood grouping.

A review of the patient's clinical condition and medication history can help identify the cause of the discrepancy in this group. If the reagent is the suspected cause of the reaction, clinicians should repeat the test using a different lot number of the reagent.

H-Substance: The H-antigen is the foundational compound from which the ABO system originates.[29] A deficiency of this antigen is most commonly found in South Asia and is called the Bombay phenotype.[35] Patients with the Bombay phenotype lack fucosyltransferase activity.[3] Because the H-antigen is necessary for the formation of A and B antigens, patients with the Bombay phenotype lack A, B, and H antigens regardless of their ABO genotype. In addition, they form antibodies to A, B, and H, placing them at high risk of severe hemolytic transfusion reactions when transfused in anything other than Bombay blood. During ABO typing, patients with Bombay blood essentially appear like type O blood due to the absence of A, B, and H antigens on their RBCs. Confirmation of the Bombay blood type requires evaluation for anti-H antibodies, checking the secretor status, such as saliva analysis, and molecular genotyping of the ABO gene, where exons 6 and 7 account for a significant portion of the glycosyltransferase activity.[36] If a patient with the Bombay blood type needs emergency blood products, they must receive Bombay donor or artificial blood unless autologous blood was donated before a surgical procedure.                                                   

Issues of Concern

The primary issues of concern regarding ABO discrepancies are acute hemolytic transfusion reactions, hemolytic disease of the fetus or newborn, or rejection of tissue or organ transplants. Transfusing blood incompatible with the recipient's ABO blood group may cause hemolysis, shock, kidney failure, and death. In addition to major ABO and Rh incompatibility, clinicians must be vigilant for potential errors in typing due to technical factors, the presence of ABO subgroups that can lead to weaker reactions during testing, and the possibility of ABO incompatibility in pregnancy, mainly when the mother is type O and the baby is type A or B. ABO incompatibility can cause hemolytic disease of the fetus or newborn though this tends to be less severe than Rh incompatibility. 

To mitigate these concerns, all healthcare team members must collaborate to ensure proper verification of the patient's identity when collecting the patient's blood and before administering any blood products. Team members must adhere to standard protocols for collecting, transporting, and processing blood products. In addition, cross-matching must occur to ensure the blood is compatible with the recipient's blood, and a thorough investigation of ABO typing discrepancies is necessary before proceeding with a transfusion. 

Transfusion and pregnancy are the most common causes of alloimmunization in Rh-negative individuals, resulting in the formation of antibodies against Rh antigens. These antibodies can potentially cause hemolytic transfusion reactions and place Rh-positive infants born to Rh-negative mothers at high risk of hemolytic disease of the fetus or newborn. Healthcare professionals can mitigate this risk by ensuring that Rh-negative individuals are not inadvertently exposed to Rh-positive blood or platelets. If this occurs, the patient can receive prophylactic anti-D immune globulin. Similarly, pregnant patients also receive prophylactic anti-D immune globulin. Please see StatPearls' companion, "Rh Blood Group System," for more information.

Incompatible ABO antigens on epithelial and endothelial cells of transplanted organs raise the risk of transplant rejection. Ideally, transplanted organs have identical ABO antigens. However, in some cases, clinicians may elect to transplant incompatible organs using desensitization and immunosuppression. Generally, this occurs with a living kidney donor or in the case of fulminant hepatic failure. Because cornea, bone, and tendon do not contain an appreciable quantity of RBCs, ABO compatibility is not required.[37][38]  

Additional clinically significant blood group systems such as the Duffy, Kell, Kidd, Lewis, and MNS can also play a role in hemolytic transfusion reactions and hemolytic disease of the fetus or newborn. Please see StatPearls' companions, "Hemolytic Disease of the Fetus and Newborn," " Kell Blood Group System," and "Duffy Blood Group System," for more information.                                                      

Clinical Significance

Although some ABO discrepancies can be quickly resolved by reviewing the patient's history, others may be more challenging and require extensive evaluation. Further evaluation and resolution of some discrepancies may be time-consuming, risking a significant delay in care in critical and unstable patients with an immediate need for blood products. To avoid compromised patient care, clinicians should administer universal donor products such as O-negative blood while working to resolve the discrepancy (see Table 2. Summary of ABO Types).

Table 2. Summary of ABO Types

Lab personnel should not report a blood type until testing documents the patient's results without any discrepancy.[1]

Enhancing Healthcare Team Outcomes

ABO discrepancies occur when the results of forward and reverse blood typing do not match, leading to uncertainty in determining a patient's actual blood group. These discrepancies can arise due to technical errors, weak antigen expression, unexpected antibodies, or recent transfusions. If not correctly identified and resolved, ABO discrepancies can result in incompatible blood transfusions, leading to severe hemolytic reactions and death. To prevent these risks, laboratory professionals should carefully review test procedures, verify sample integrity, use additional serological testing if necessary, and collaborate with clinicians to assess the patient's medical history. Prompt recognition and resolution of ABO discrepancies are critical to ensuring safe transfusion practices and patient care.

The effective management of ABO discrepancies requires a collaborative approach involving clinicians, advanced practitioners, pharmacists, and laboratory professionals. Clinicians and advanced practitioners are critical in reviewing patient history, identifying potential causes of discrepancies, and making informed clinical decisions. Nurses contribute by accurately collecting and labeling samples, monitoring patients for adverse reactions, and communicating findings with the healthcare team. Pharmacists assist by evaluating medication interactions that may influence serological results and ensuring safe transfusion practices. Laboratory professionals are responsible for conducting precise blood typing, identifying discrepancies, and coordinating confirmatory testing. Clear interprofessional communication and structured care coordination are essential to resolving ABO discrepancies efficiently. Implementing standardized protocols, using electronic health records for documentation, and engaging in regular team training can enhance patient-centered care, improve outcomes, and strengthen team performance. By fostering a culture of interprofessional collaboration and communication, healthcare teams can mitigate risks associated with ABO discrepancies and promote patient safety.