Function
The basic principle of ABO typing is that the forward grouping reaction, which detects the antigen present on the surface of the RBCs of the patient, should match the results of the reverse grouping reaction, which detects the antibodies present in the patient’s serum. The Anti-A and Anti-B, universally occurring antibodies are expected to be detected in the serum of all persons who lack those antigens on the surface of red blood cells. Both the forward and reverse grouping reactions are expected to be strong (3+ or 4+) reactions.
If there are any unmatched results between the two grouping reactions, ABO discrepancies arise. It is essential for patient safety that no ABO discrepancies are ignored. All discrepancies should be investigated and explained before determining the patient’s blood group and issuing group-compatible blood products.[3][1]
Expected normal ABO typing results appear in the following table.[4]
|
Blood Group
|
Forward reaction (patient’s RBCs with reagent antibodies)
|
Reserve reaction (Patient’s serum with reagent RBCs )
|
|
|
Anti A
|
Anti B
|
A group RBCs
|
B group RBC
|
|
A
|
3+ or 4+
|
-
|
-
|
3+ or 4+
|
|
B
|
-
|
3+ or 4+
|
3+ or 4+
|
-
|
|
O
|
-
|
-
|
3+ or 4+
|
3+ or 4+
|
|
AB
|
3+ or 4+
|
3+ or 4+
|
-
|
-
|
Issues of Concern
With the automatization and standardization of testing procedures, the incidence of ABO discrepancies has reduced considerably due to decreased technical errors. However, when these discrepancies occur, they need careful work up for resolution and explanation. When faced with these discrepancies, it is best to rule out technical errors first, which include:
- Incorrect labeling of samples,
- A wrong technique of the procedure,
- Reagent contamination, or expiry.
These can be avoided by regular training of lab staff and quality checks of the testing. Any discrepant test result must be repeated with the same or fresh sample if required, and further workup should be done if the discrepancy persists.[3]
For ease of understanding and work up, ABO discrepancies have been divided into four headings; group I, group II, group III, and group IV.[5][6]
Group I: Weak or absent reaction in the reverse grouping
These discrepancies occur when there are decreased antibodies in the patient's serum leading to weak or absent reaction in the reverse grouping.
The situations which fall in this group include:
- ABO typing in newborns[7]
- Occasionally encountered in elderly patients
- Patients on immunosuppressant medications which lead to decreased lymphocyte function and antibody production
- Congenital immunodeficiency[8]
- Hypogammaglobulinemia
- The absence of anti-B in serum can also be encountered in children in a sterile environment and those on artificial sterile nutrition as they remain unexposed to bacteria.[9]
- Natural chimerism[10]
Chimerism is a rare phenomenon when an individual has two sets of red cells with different blood groups circulating in the body. Both types of red cell antigens are recognized as self-antigens; therefore, no antibodies against them exist in the serum. This gives rise to mixed field reactions in forward grouping reactions and absent reactions in reverse grouping resulting in an ABO discrepancy. This usually occurs in fraternal twins. Most rarely, chimerism can be found in individuals without fraternal twins due to mosaicism.[10][11]
Workup and resolution: Most causes of these discrepancies will be obvious from the patient's history. To confirm this discrepancy, the missing or weak antibody has to be augmented by incubating the serum with reagent RBCs at room temperature for up to 15 to 30 minutes, after which, in most cases, an acceptable reaction will be recorded. In some cases, if there is no reaction, the sample can be centrifuged to bring the antibodies and cells closer and should be incubated for some time for a reaction.
If, despite these steps, a reaction is still not obvious, then the mixture of the patient's serum and reagent cells should be incubated at 4 degrees C for up to 30 minutes. Once an expected reaction is recorded, the discrepancy is resolved, and the patient's blood group can be reported with certainty.
Group II: Unexpected reaction in the forward grouping
These discrepancies occur in patients with weakly reacting antigens (due to low antigen burden on the RBC surface or 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 will yield a weaker than expected reaction in the forward typing, which will be unmatched in the reverse typing reaction.
The situations which can fall into this group include:
- A or B subgroups (among the most common causes of discrepancies)[12]
- B(A) phenotype[6]
- Acquired B phenomenon
- Hematological malignancies can alter the antigens present on the surface of cells (like leukemia and multiple myeloma).[13][14]
- Pregnancy is also reported to cause weakened ABO expression.
- Post-transfusion of a different blood group of red cells
- Post allogenic bone marrow transplant with an ABO incompatible donor
- Fetomaternal hemorrhage[15]
- Tn syndrome[16]
Workup and resolution: the weak reaction in forward typing can be augmented by incubating the mixture for a longer time, up to 30 minutes, at room temperature. If the reaction is not enhanced with longer incubation, then the mixture should be incubated at 4 degrees Celsius for up to 30 minutes. Reviewing a patient's clinical can provide important explanations of the cause of the discrepancy.
Blood group A subgroups are among the most common causes of ABO discrepancies. Group B subgroups are very rare. In group A, the most common subgroup is A1, followed by A2, which accounts for more than 99% of the A blood group. Other A subgroups like A3 and Ax are very rare. The resolution of discrepancies caused by the A subgroups can be achieved by using Dolichos bilflorus plant seed extract lectin, which agglutinates A1, but not A2, cells. Detection of the presence of A1 antibody in the patient's serum using A1 RBC reagent cells, Detection of secretion A, B, and H substances in patient's saliva, and molecular genotyping studies can also be of value in resolving ABO subgroups.[17][18]
The acquired B antigen is a special situation that occurs when a group A patient during an episode of infection when certain gram-negative bacteria secrete enzymes that can modify the A antigen on the RBC surface, which is N-acetyl-D-galactosamine to D-galactosamine that resembles D-galactose (the B antigen) and will cross-react with anti-B reagents. This phenomenon will resolve once the patient recovers.
To prove the acquired B phenomenon, the cells should be incubated with the monoclonal anti-B clone, yielding a strong reaction, while the regular B antiserum will yield a weak mix-field reaction only. Also, mixing the patient's sera which contains anti-B antibodies, with the patient's RBCs, with acquired B will 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. The patient's original blood group will be A, and their serum will contain anti-B antibodies. They should receive group A-compatible blood products when needed.[19][20]
Tn syndrome is a rare, acquired, permanent disorder of unknown origin. Anti Tn antibody is a naturally occurring antibody in the serum of most adult humans. In this disorder, the pluripotent stem cells are affected, which causes all cell lineage cells to express Tn-antigen on their surfaces. This leads to mild hemolytic anemia, thrombocytopenia, and leukopenia which usually do not require treatment. This disorder is associated with some blood disorders like leukemia and myelodysplastic disorders, including myeloproliferative neoplasms.[16]
The B(A) phenotype is a rare but autosomal dominant condition in which the patient's red cells predominantly express B antigen, but occasional A antigens are also expressed on the surface. 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. But this leads to ABO discrepancy with an unexpected reaction in forwarding grouping during ABO typing.[6]
The resolution of the rest of the discrepancies will depend on the underlying cause; for example, if the discrepancy is due to a recent blood transfusion, then it is reasonable to repeat the ABO typing at a later date, employ a Kleihauer-Betke (K-B) test when fetomaternal hemorrhage is suspected, etc.
Group III: Unmatched reaction between the forward and reverse grouping due to plasma abnormalities
These discrepancies occur in patients with proteins or plasma abnormalities resulting in rouleaux formation or pseudo agglutination, which will cause false and uninterpretable results.
Conditions that cause rouleaux formation include the following:
- Hypergammaglobulinemia (as in cases of multiple myeloma)[5]
- Hyperfibrinogenaemia
- Certain plasma expanders like dextran or hydroxyethyl starch[21][22]
- Contamination with Wharton jelly in cord blood
Work Up and Resolution
The ABO typing detects antigens and antibodies based on agglutination. Rouleaux formation closely resembles the agglutination of red cells and can lead to false positive results. When all reactions result positive in forward and reverse typing, it is important to perform a microscopic examination of the blood sample to document rouleaux. If that is confirmed, washing the sample with normal saline will eliminate other antibodies and other proteins, causing unwanted stacking of red blood cells. It will lead to the resolution of the discrepancy on repeated testing of washed cells.
In reverse typing discrepancies, it is worthwhile to perform a saline replacement technique, in which, after centrifugation of the patient's blood, the plasma is removed and replaced with saline of equal volume, and the test is performed again. This will remove the excess protein in the patient's serum that is causing rouleaux formation. The test can then be performed without the interference of those proteins and will provide an interpretable result.
Group IV: Unmatched reaction between the forward and reverse grouping due to miscellaneous causes
Discrepancies due to miscellaneous causes that do not fall into the above groups will fall in group IV. These will be seen in cases when there is an agglutination reaction independent of a specific reagent antibody.
Conditions that cause this include the following:
- Cold reactive autoantibodies
- ABO isoagglutinin
- Non-ABO alloantibodies
- Recent IVIG infusion[23]
- Reaction to rare antigens or antibodies in the reagent used[24]
Workup and Resolution
When unexplained results are encountered in ABO typing that are mainly positive in both forward and reverse grouping reactions, the presence of cold autoantibodies in the patient's serum should be considered. A direct Coomb's test could be positive. In these cases, the patient's RBCs should be incubated at 37 degrees C. In most cases, this will lead to the IgM antibodies becoming inactive. The sample should then be washed three times in saline at 37 degrees C, and the test should then be repeated. This will rid the effect of these antibodies on the test. Rarely, if the previous steps are unsuccessful, then 0.01 M dithiothreitol (DTT) can be added to the patient's RBCs, preventing agglutination due to IgM antibodies.[25]
If the discrepancy is in the reverse grouping test, then the testing should be repeated at 37 degrees C as this will usually stop the interference of the autoantibodies. If an unexpected result is still encountered, a cold autoabsorption test should be performed. The process of autoabsorption involves incubating the patient's RBCs with the patient's serum which will lead to a reaction of autoantibodies with the patient's RBCs and reduce their concentration in the serum. This sample should then be centrifuged to remove the autoantibody-coated RBCs, and the serum can be used for reverse blood grouping.
A review of the patient's clinical condition and history of medications administered can provide a clue as to the cause of the discrepancy in this group as well. If the reaction is suspected due to the reagent, then testing should be repeated with a different numbered lot of the reagent.
ABO Typing Discrepancies