Immuno hematology Practical -2- Rh Grouping Dr: Dalia Kamal Eldien

1. Immuno hematology Practical -2- Rh Grouping Dr: Dalia KamalEldien

2. Rh (D) Grouping 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

3. Slide Technique • This technique may be used in emergency Rh (D) typing if a centrifuge is not available. • The slide test is not recommended for routine test as it may not pick up weak reactions, thus giving negative results.

5. Tube Technique a. Saline Agglutination test for Rh TypingProcedure1. Prepare 2-5% washed red cell suspension of test sample. 2. Place 1 drop of anti-D in cleaned tube labeled D as test tube.3. Place 1 drop of 22% bovine albumin/control reagent in another tube labeled C as control tube.

6. 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

7. Tube Technique • InterpreationPositive test : Agglutination in anti-D and smooth suspension in control tube.Negative test : Smooth suspension in all the tubes (test and control) • Test is considerable invalid if both test and control tubes show a positive reaction.In such case, the test should be repeated using saline IgM anti-D. • For all microscopically negative reactions in donor grouping, Du testing should be performed.

9. Du method Some red cells possess the D antigen but it is expressed so weakly that the cells are not agglutinated directly by anti-D sera. An indirect antiglobulin test is necessary to identify patients with the Weak D (formerly known as Du )phenotype. Weak D testing is done on Rh negative donors to ensure they are truly D negative.

10. SAMPLE Any sample satisfactory for ABO and Rh testing is acceptable. REAGENTS, EQUIPMENT, AND SUPPLIES - 37oC incubator -Wash bottle with physiologic saline -Coombs serum - either polyspecific or monospecific

11. PROCEDURE -Prepare a washed 3% suspension of patient cells, and set up the test and DC (Rh Control) tubes, if not already done -Record the test and DC immediate spin results. If the Rh test is negative, continue the method.   -Incubate both tubes at 37oC for 15 to 30 minutes. -Centrifuge and read for agglutination as usual. If the Rh test is negative. -Wash both tubes 3-4 times with saline. -Immediately after the last wash, add one drop Coombs serum to each tube and centrifuge. -Immediately resuspend gently and examine for agglutination. -Confirm all negative results by adding one drop Coombs control cells to all tubes showing no agglutination and centrifuge 15-30 seconds at high speed. -Gently resuspend and examine for agglutination. Agglutination should be present in this step or the test is invalid.

12. INTERPRETATION - A negative result in the immediate spin phase but agglutination in the D tube following incubation indicates a positive test for weak D. - Lack of agglutination is a negative test and the patient is considered truly D negative. - Agglutination in the DC tube invalidates the test.

13. Thank you