Anti-a Information and Courses from MediaLab, Inc.

Landsteiner, knowing that none of his subjects had been immunized, realized that “natural” antibodies must develop which are directed against antigens not present on the red cells. Individuals with “A” antigens on their red cells had sera containing “Anti-B” antibody. Individuals with “B” antigens had sera containing “Anti-A.” “AB” individuals had sera with no ABO antibodies present and “O” individuals’ sera contained “Anti-A” and “Anti-B.” Sera from group O individuals may contain a separate antibody, “Anti-A,B.” Anti-A,B possesses serologic activity not found in mixtures of Anti-A and Anti-B. Anti-A,B sera will agglutinate A, B, and AB cells. It is particularly useful in detecting weak A and B antigens. See the table on the next page.

In routine practice, specially prepared blood grouping sera containing anti-A, anti-B, (and optionally anti-A,B) are used to identify the four types of red cells. These sera will agglutinate cells with the corresponding antigen. This is called forward typing. ABO Blood Group Patient Red Cells Tested with Known Antisera Anti-A Anti-B Anti-A,B A 4+ 0 4+ B 0 4+ 4+ O 0 0 0 AB 4+ 4+ 4+ + = agglutination (graded 1+ to 4+)0 = no agglutination

ABO antibodies are not usually produced by an infant until 3 to 6 months of age. Antibodies found in the sera of newborns are almost always IgG, passively acquired from the mother. Thus, serum testing of newborns is not performed. Anti-A and anti-B titers are highest at ages 5-10 years and then they gradually decrease. Thus, in elderly patients, ABO antibodies may be difficult to detect. In patients with hypogammaglobulinemia, some leukemias, lymphomas or patients who are taking immunosuppressive drugs, the expected antibodies may be weak or even absent, reflecting the low levels of gamma globulin in the patient’s serum. As previously mentioned, these and other ABO typing discrepancies must be resolved before true ABO type can be determined.

The most common classifications are A1 and A2. These account for over 99% of group A bloods. Of this 99%, A1 compromises approximately 80%. Commercial anti-A typing serum does not differentiate between A1 and A2 cells. A1 cells contain “A” antigen and “A1” antigen. A2 is not really a unique antigen. It is thought to be simply “A” antigen with no “A1” antigen. Several preparations are available that will react with A1 cells, but not other subgroups of A. An extract of the seeds of the plant, Dolichos biflorus has specific anti-A1 activity. “Absorbed anti-A” serum can also be prepared. To do this, the anti-A from group B people is absorbed with A2 cells. Anti-A is removed and a second antibody that reacts only with A1 cells remains. Anti-A1 can also be found as a separate antibody in the sera of A2 and A2B individuals.

For the most part, subgroups are merely of academic interest, but occasionally they present clinical problems. The antigen may be so weak that it is not detected and the red cells are mistyped as group O. This is especially dangerous if the cells are those of a donor. Problems may arise because the serum of an A2 or A2B, A3 or Ax individual might contain anti-A1. This antibody may be detected in serum typing and cause confusion. You would not expect to find a person with A antigen on his red cells and anti-A in his serum. Anti-A1 is produced by about 1-2% of group A2 persons and about 25% of group A2B persons. Subgroups may be determined by reactions with antisera as seen in the table on the next page.

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 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

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.