Rh Grouping. Practical Aspects of Rh Grouping. Rh grouping in routine use for donors and patients involves testing for Rh (D) antigen only However tests for other important Rh antigens e.g. C,c,E and e may be done for Rh genotyping.
These antibodies are highly specific, react equally well at 20°C as well as 37°C and are reliable for slide and rapid test tube technique.
IgM anti-D monoclonal reagent cannot be used for Du testing by indirect antiglobulin test (IAT) while IgM + IgG monoclonal reagent and blend of IgMmonolconal and IgG polyclonal can be used for Du testing.
In most of the blood transfusion laboratories, Rh (D) grouping is performed along with the ABO grouping and same techniques as used for ABO grouping may also be employed for Rh typing
a. Saline Agglutination test for Rh (13) TypingProcedure1. Prepare 2-5% washed red cell suspension of test sample. 2. Place 1 drop of anti-D in cleaned tube labelled D.3. Place 1 drop of 22% bovine albumin/control reagent in another tube labelled C. 4. Add 1 drop of 2-5% test cell suspension to each tube. 5. Mix well and centrifuge at 1000 rpm for 1 minute (in case of using IgG anti-D, incubate at 37°C for 10mm. and centrifuge (spin tube method) or incubate at 37°C for 60 minutes (sedimentation method). 6. Resuspend the cell button and look for agglutination. All negative results must be confirmed under microscope
Principle Albumin increases the dielectric constant of the medium and thus reduces the zeta potential. Due to this effect, the electrical repulsion between the red blood cells is less and the cells agglutinate. Mostly 22% bovine albumin is used, as higher concentrations can cause rouleaux formation.
In haemolytic disease of the newborn, the baby’s red cells may be coated with immunoglobulin and a saline reactive Rh antiserum is usually necessary for testing. When the cells are heavily coated with antibody, no free antigenic sites remain for reaction, resulting in a negative test. This is suspected when the infant’s cells show a positive direct antiglobulin test (DAT) and a negative test with anti-D reagent. In such instances it is recommended that the antibody should be eluted by gentle elution (heating at 45°C for 30 minutes) to expose the antigenic sites before testing.