Agglutination 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.

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

Other rare subgroups of A exist. These comprise less than 1% of the total pool of A genes. Of these rare types, A3 is the most common, but Ax, Aend, and Ael have also been identified. In subgroup A3, the red blood cells characteristically give a “mixed field” agglutination pattern when tested with anti-A and anti-A,B. Small clumps of agglutinated cells are present among large numbers of cells which absorb the antibody to their surface but are not agglutinated by it.

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

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.

Rouleaux formation correlates with an increased concentration of serum monoclonal proteins. Rouleaux may be seen as an artifact in the thicker portions of blood smears. The addition of a drop of saline to the blood smear will serve to disperse any artifactual rouleaux formation. The presence of rouleaux formation or RBC agglutination may result in a falsely decreased electronic red blood count and falsely increased MCV, as these clusters may be read as one cell.