Abo Information and Courses from MediaLab, Inc.

In 1900, a German scientist, Karl Landsteiner, discovered that blood groups differ from one individual to another. He took blood samples from five associates and himself, allowed them to clot, and then separated the serum from the cells. Landsteiner found that when he mixed the serum and red cells from different individuals, some samples clumped and some didn’t. Our present day classification of the ABO system is based on Landsteiner’s realization that agglutination occurred because of highly reactive antigens present on the red blood cell which corresponded to antibodies present in the serum. Landsteiner isolated and named the red cell antigens “A” and “B” and the corresponding antibodies “Anti-A” and “Anti-B.” If the red cells contained neither antigen, he called these cells “O”, representing zero antigens present. The fourth type of red cells, “AB”, was discovered in 1902 by Von Decastello and Sturli, associates of Landsteiner. “AB” cells contained both A and B antigens on their surface.

Antigen on Red Cells Antibodies in Serum ABO Blood Group A Anti-B A B Anti-A B Neither A nor B Anti-A, Anti-B, Anti-A,B O A and B Neither Anti-A nor Anti-B AB

It has been demonstrated that antibodies occur predictably in the sera of all normal adults in association with the ABO antigens. Demonstration of these antibodies is therefore necessary for definitive classification of an individual’s ABO cell type. The individual’s serum is therefore tested against reagent red cells containing known antigens. Patient ABO Blood Group Patient Serum Tested with Known Reagent Cells A Cells B Cells A 0 4+ B 4+ 0 O 4+ 4+ AB 0 0 + = agglutination (graded 1+ to 4+)0 = no agglutination or hemolysis

It is also important to note that in addition to red cells, ABO antigenic determinants (epitopes) are found in many tissues, body fluids, and other cells including endothelial cells and platelets. Because ABO antigens are so widely expressed, ABO antigens are also a major consideration in solid organ and bone marrow transplants.

Specific sugars, attached to the red cell membrane in unvarying linkage conformations, determine ABO antigenic activity. Galactose resides at the end of this specific sugar chain. This configuration constitutes the ABO antigen precursor substance.

The ABO locus is on chromosome number 9. There are three major allelic genes and numerous rare genes. The three principle genes are A, B, and O. The A gene determines the product N-acetylgalactosaminyltranferase activity. The B gene determines galactosyltransferase activity. The O gene does not produce a functional enzyme. The enzyme products of the A and/or B genes act on H substance to convert it to A and/or B antigens. Not all H substance is converted; thus, all cells normally contain some H substance along with the A and/or B antigens. If both the A and B genes are present, some H antigen sites are converted to A antigen and other H antigen sites are converted to B antigen. (A single antigen site does not have both A and B antigens.) The O gene is an amorph and doesn’t act on H substance, therefore group O cells contain only H substance. See the diagram on the next page.

The mating of an A individual with another A individual can produce AA, AO, or OO offspring, depending on the genotype of the parents. This is illustrated by the Punnett squares on the next page. You can determine all possible offspring from ABO mating using these straightforward genetic principals.   

In most other blood group systems, antibody may be formed after an individual has been immunized by an antigen that is missing from his or her red cells; perhaps as the result of pregnancy or transfusion. In the ABO system, when the antigen is missing from the cells, the corresponding antibody will predictably be found in the serum and must be found before determining the ABO type. There are few exceptions to this rule and any exception must be explained before the true ABO blood type can be determined.

A person exposed to a specific immunizing event may produce “immune” ABO antibodies of the same specificity as the “naturally” occurring antibody, but with different biological behavior. Such immunizing events include pregnancy with an ABO incompatible fetus or transfusion of ABO incompatible red cells. After immunization, the subject’s antibody may increase in titer and/or avidity, develop powerful hemolyzing properties, or become more active at 37ºC.

A subgroup antigens are inherited, as are other ABO antigens with A1 being dominant over A2. A phenotypically A1 individual may be genotypically A1O, A1A1, or A1A2. A phenotypically A2 individual may be genotypically A2A3. These alleles are passed to offspring in the same manner as other ABO antigens. Weak variant forms of the B antigen (B3, Bx, and Bel) exist but are so rare that they do not warrant discussion here.

Antibodies of the ABO system cause agglutination of saline-suspended red cells at 4°C to 20°C. Heating to 37° weakens the reaction. “Naturally” occurring ABO antibodies may not be strong enough to agglutinate cells without centrifugation. Thus, testing serum for the presence of anti-A or anti-B has classically been performed using the tube system in which serum and cells added to a test tube are centrifuged and then evaluated for agglutination. A slide test has also been performed for forward reactions. Although tube tests are still in wide use, newer systems utilizing other technology such as gel agglutination are becoming more prevalent. The image on this page illustrates agglutination reactions observed with the tube system, from 4+ in the topmost image, to 0 in the lowest image. ABO reactions should be strong. Weak or missing reactions occur, but must be "resolved" before blood products can be released.4+ agglutination: Red blood cell button is a solid agglutinate; clear background.3+ agglutination: Red blood cell button breaks into several large agglutinates; clear background.2+ agglutination: Red blood cell button breaks into many medium-sized agglutinates; clear background; no free red blood cells.1+ agglutination: Red blood cell button breaks into many small clumps barely visible macroscopically; background is turbid; many free red blood cells.Negative: No agglutinated red blood cells present; red cells are observed flowing off the red blood cell button during the process of grading.Other reaction which may occur are the mixed-field reaction, in which mixtures of agglutinated and unagglutinated red blood are present; and hemolysis, in which red cells are hemolyzed by the antibody. Both of these patterns are considered positive reactions.

Patient Red Cells Tested With Known Antisera ABO Antigens Present on Red Cell Anti-A Anti-B Anti-A,B 4+ 0 4+ A 0 4+ 4+ B 0 0 0 Neither A nor B 4+ 4+ 4+ A and B + = agglutination (graded 1+ to 4+) 0 = no agglutination or hemolysis

Patient Serum Tested With Known Reagent Red Cells Antibodies Present in Serum A1 Cells B Cells 0 4+ Anti-B 4+ 0 Anti-A 4+ 4+ Anti-A and Anti-B 0 0 No ABO antibodies present + = agglutination (graded 1+ to 4+) 0 = no agglutination or hemolysis

An increasing number of transfusion services are using automated blood banking systems. These systems may employ either solid phase or gel techniques. Use of automation may increase productivity, reduce costs, and, by decreasing the number of manual steps in the testing process, potentially reduce errors.

The composite image shown on the right illustrates the ABO typing reactions that were obtained for a patient. This particular case illustrates an ABO discrepancy. An ABO discrepancy occurs when the results of forward and reverse typing do not match. The reactions shown are described below in descending order:Patient red cells with reagent anti-A: negative reaction.Patient red cells with reagent anti-B: 4+ agglutination.Patient red cells with reagent anti-D: 4+ agglutination.Patient serum with reagent A1 red cells: negative reaction.Patient serum with reagent B red cells: negative reaction.This patient forward types as a group B, but reverse types as a group AB. (A group B patient should have anti-A. This patient demonstrates neither anti-A nor anti-B, similar to an AB patient). Further workup is necessary to determine the ABO type since the forward and back typing do not match. In this case, incubation at 40 C demonstrated the presence of weakened anti-A. The patient was therefore typed as group B. This case is an example of an ABO discrepancy which was due to a "missing" anti-A antibody. This could be due to old age, severe illness or immunosuppression. Although evaluation of ABO discrepancies is beyond the scope of this course, it is important to note that all ABO discrepancies must be resolved before blood products can be released for transfusion.This patient is Rh (D) positive, as evidenced by the strong agglutination of his cells with reagent anti-D antibody.