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ABO Blood Grouping - Methods and Procedures

BY: Shikha Sharma | Category: Others | Submitted: 2010-11-12 07:26:08

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ABO blood grouping system is the most widely known and medically important blood type because of its importance in transfusion medicine. Its discovery goes back to 1900, when Karl Landsteiner was trying to solve the mystery that some blood transfusions are life-threatening while others are life-saving. In 1930, he was crowned with Nobel Prize for his excellent discovery.
The four basic ABO phenotypes are A, B, AB and O. Apart from man, this system is also found in apes, gorillas and chimpanzee.

Importance in Forensic Biology

In forensic science, blood typing is one of the preliminary tests, which is done to exclude some suspected sources of bloodstains. Although this test doesn't clearly indicate the suspect involved because many people share the same blood type.

Methods of grouping
In fluid blood samples, the presence or absence of an antigen is ascertained by whether or not the red cells are agglutinated. In dried bloodstains, the cells have ruptured and therefore, direct agglutination tests are no longer feasible. However, the antigens are not immediately denatured upon drying. Indeed, in the ABO system, they survive for many years and retain the capability of combining with specific antibodies. The formation of these antigen-antibody complexes is the basis of all methods employed in the detection of red cell antigens in dried bloodstains. Three methods, which are routinely used for ABO blood grouping, are described below:

1) Mixed agglutination
A high proportion of bloodstains, which are grouped occur on textile fabrics and the corpuscular debris containing the antigenic material adheres tenaciously to the fibres. By this method, finely teased bloodstained fibres are submerged in antiserum and, after a period of absorption, the unbound antibody is washed away leaving behind only antibody, which has combined specifically with the antigens. The fibres then suspended in indicator cell suspension of appropriate group and rotated. During this period, agglutination occurs between the absorbed antibody and the indicator cells so that, in the presence of a specific antigen, the fibres when viewed with a microscope are seen to be coated with a layer of red cells of the same specificity as the stain.
This method has proved very satisfactory for the ABO system and can be used on extremely small stains, but, for maximum sensitivity, it is necessary to use specially selected high titre immune anti-A, anti-B and the group O indicator cells, must be pretreated with enzyme papain in order to detect H antigen. The method has not been found suitable for Rh and MN typing.

2) Absorption-inhibition
It is the classical method of detection of antigens in dried bloodstains. Aliquots of antisera are added to portions of stains or extracts thereof and, after a suitable period of absorption, the residual sera are tested with red cell suspensions of appropriate groups (e.g., group A cells are added to anti-A serum) and the degree of agglutination is compared with that of controls tested in parallel from the unstained substrate. A substantial reduction in titre of serum added to stained material compared with that of serum in the control indicates the presence of an antigen. For this test, the antisera are usually diluted, but nevertheless, it is necessary for a very high proportion of the antibody content to be absorbed by the red cell antigens in order to obtain a significant reduction in titre. This method is relatively insensitive when compared with more recent techniques and, in practice, has proved satisfactory only for grouping in the ABO system.

3) Absorption-Elution method
Another means of direct testing for absorbed antibody is that of absorption-elution. After absorption and subsequent washing away of unbound serum, the combined antibody is eluted, usually by raising the temperature to about 56ÂșC. The elute is then tested with indicator cells of the appropriate group; agglutination indicates the presence of an antigen of the same specificity as that of the indicator cells.
This method has proved highly satisfactory for ABO grouping where it is possible to group stains of 1mm diameter. It is also in routine use for Rh and MN Typing in dried bloodstains. This method is both more sensitive and more versatile than mixed agglutination. One possible reason for the greater sensitivity is that in mixed agglutination, reactions are only obtained from the antigenic sites exposed on the surface whereas in absorption-elution, the submerged sites are also included. It is also likely that red cells agglutinate more readily with each other than with debris adhering to fibres.

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