Dealing with ABO
By Ruth E. Ross, MHS, MT(ASCP)SBB
Accurate Blood Typing of Donors and Recipients
at Heart of Transfusion Medicine
In 1900 Karl Landsteiner discovered that by mixing blood samples from himself and others he was able to obtain agglutination, and from these observations he categorized them into three blood groups–A, B and O.1 Thus, the first blood group system was developed.
Today, almost 100 years later, the ABO blood group system remains the most important one for transfusion purposes.
Many advances have been made throughout the century and as a result we have different methods available for determining ABO types of blood donors and recipients. These vary from slide testing to tube testing and microplate testing.
Despite all of the modern technologies and reagents available to blood bankers today, ABO discrepancies still occur. An ABO typing discrepancy means that the interpretations of the red cell (forward) testing do not agree with the serum (back) testing. Since the ABO blood group system is the only blood group where we find non-immune antibodies to the antigens lacking in an individual, the transfusion of ABO incompatible blood can result in antibody-mediated hemolysis, which can lead to death.
Discrepancy Factors to Consider
On routine testing, ABO blood grouping appears as indicated in Table 1. When a discrepancy occurs, there are important factors to consider–the patient’s age, diagnosis, transfusion history, the specimen used for testing and technique. Repeat testing must be performed first to ascertain that proper technique was used. Then, if the results are still the same, the sample should be examined. If the serum or plasma used contains fibrin clots, it could mask as a false positive reaction. If the sample is hemolyzed, interpretation cannot be made because it is difficult to see reactions if they are weak or if a hemolyzing antibody is present. Using a fresh, unhemolyzed specimen in this situation is best, so that proper interpretation can be carried out.
One should not forget that there are hemolyzing, anti-A and anti-B antibodies and that hemolysis is a positive reaction. The patient’s age is an important factor to consider. In the newborn, back typing will not result in any reactions, as antibodies are not fully developed yet and the antibodies present have been passively acquired from the mother. Routinely, serum from newborns is not tested. In the elderly, sometimes antibodies weaken with age, and this may result in a discrepancy. Incubating for a longer period of time–30 minutes at room temperature or even at 4 degrees Celsius–may resolve the problem, but if tested at a low temperature an autocontrol and screening cells also need to be added because there could be cold antibodies present.
The patient’s diagnosis is very important in resolving discrepancies as patients with malignancies are often treated with chemotherapy that causes im-munosuppression, giving antibodies a weak appearance. Patients who are diagnosed with multiple myeloma will usually have large amounts of proteins present, and these will cause rouleaux formation or abnormal aggregation of the red cells, which can mask true agglutination. To correct this problem, cell suspensions must be carefully washed, and the serum sample should be diluted with saline to the point where rouleaux is no longer present.2
A problem that seems to be appearing more and more in our blood banks today involves patients who have received a hematopoietic stem cell transplant of an ABO group other than their own. This results in a transplantation chimera, meaning that there are two distinct populations of red cells present in the same sample. The mixed field agglutination present when testing usually disappears when the donor cells have taken over and the host is no longer producing red cells of his own type.3 Transfusion needs of hematopoietic stem cell transplant patients are normally taken care of before the transplant. The type of red blood cells and plasma the patient is to receive and both the donor and the recipient’s blood types are known by the blood bank where the patient receives the transplant. However, if the patient has been transferred from another hospital, it is essential to obtain his/her history to resolve typing discrepancies and avoid giving the wrong type of red blood cells or plasma products.
Mixed field agglutination also can be seen in patients who have been recently transfused. This can occur in the emergency room or in the operating room where massive transfusion with group O red cells can take place due to unforeseen circumstances. This type of mixed field usually will persist only for the life of the transfused red cells.
Cold autoantibodies can be present with a wide thermal amplitude, causing problems at room temperature testing and interfering with ABO typing. In this situation, pre-warming of all reagents and blood samples used will resolve the problem. There are other antibodies that react at room temperature, which may bind with the cells used for back typing if these cells contain the corresponding antigen. These include antibodies to the M and P1. Once all other factors have been ruled out it may be necessary to resolve the discrepancy by testing with various lot numbers of back typing cells to see if different reactions are obtained, and testing screening cells or even a panel of cells at room temperature to arrive at an antibody specificity.
There are a few other, less common, problems that may be encountered. For example, a very high concentration of A or B blood group substance in the serum can neutralize the reagents used and cause false negative reactions. Also, patients can occasionally present with antibodies to the dyes used for the forward typing reagents, which may cause false positive reactions.4
Another serological problem occasionally encountered involves the acquired B and acquired A antigens. The acquired B problem is encountered when bacterial enzymes deacetylate the A1 antigen, causing it to react weakly when tested with anti-B antisera. Usually associated with patients diagnosed with carcinoma, this problem is not commonly seen as most blood banks use monoclonal reagents, and acquired B may or may not react with a monoclonal anti-B.
Acquired A occurs when Tn activated cells are encountered because of a somatic mutation which results in some hematopoietic cells incapable of synthesizing certain oligosaccharide side chains entering the circulation in the Tn polyagglutinable state. The Tn and A cells have the end sugar N-acetyl-galactosamine and will be agglutinated by the anti-A antisera. The use of proteolytic enzymes in this situation would be beneficial as this would degrade the acquired A antigen.5
Within the “A” blood group are several subgroups which may at times react with the A1 used in back typing, resulting in a discrepancy. Most of the time it will be an A2 or an A2B individual. Typing the cells with anti-A1 lectin (Dolichos biflorus) will help resolve this problem.
A phenotype that is rarely encountered is the Bombay or Oh phenotype. The cells of a Bombay individual will forward and back type as a group O, because they lack the antigens A, B and H , and have anti-A, anti-B and anti-H antibodies. While a discrepancy does not appear at first, the problem surfaces when the patient’s serum is tested against O cells. Since anti-H is present they will agglutinate these cells.
This is a very rare blood type to encounter; mainly found in India, it is the result of consanguineous marriage (both partners descending from the same ancestor). For transfusion purposes, it is recommended that these individuals have their own blood frozen, or that another Bombay individual be found. This can be done by contacting the rare donor registry of the American Red Cross.6
Standards to Be Followed
The last issue to be discussed is that of transfusion practice. As recommended by the American Association of Blood Banks (AABB), when a discrepancy is encountered during ABO typing, the following must be done:
* If a donor unit is being tested, this unit must not be released for use until the ABO discrepancy has been resolved or, if the problem is not resolved, the unit must be destroyed (AABB Std E1.000).
* If a potential recipient is being tested and a discrepancy is encountered, group O red blood cells of the appropriate Rh type should be given if blood is needed before the discrepancy is resolved (AABB Std 14.100).
* When giving blood products such as fresh frozen plasma, cryoprecipitate or platelets, where there may be a large volume of plasma present, it is advisable to use ABO type specific.7
In summary, ABO typing discrepancies can appear when we least expect them, and we need to be aware of the many factors that influence the outcome of ABO typing. This can be useful when out-of-the-ordinary elements are encountered. Table 2 summarizes all aspects reviewed.
Last but not least, we must never let our guard down, but always be well informed and prepared so that we can be assertive when approaching these circumstances head on without hesitation. *
1. Landsteiner, K. Blood group antigens and antibodies as applied to the ABO and Rh sytem. Ortho Dianostics, 1969, p. 3.
2. Issit, P.D. Applied blood group serology. Montgomery Scientific Publications, 3rd ed., 1985, pp. 604-5.
3. Ross, R.E. Immunohematology No I-2(1005) Tech sample. American Society of Clinical Pathology, 1995.
4. American Association of Blood Banks, Technical Manual, Bethesda, MD, 12th ed., 1996, p. 239.
5. Issit, P.D. Applied blood group serology. Montgomery Scientific Publications, 3rd ed., 1985, pp. 153-55.
6. Issit, P.D. Applied blood group serology. Montgomery Scientific Publications, 3rd ed., 1985, pp. 144-5.
7. Standards for Blood Banks and Transfusion Services. American Association of Blood Banks, 19th ed., Bethesda, MD, 1999.
Ruth Ross is adjunct professor at Old Dominion University, Norfolk, VA.
Resolving ABO blood typing discrepancies–Related Websites
ABO Blood Group System
ABO Blood Types and Parentage
Blood Type and Rh Information
Human Genetic Diversity