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7. 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.
The method of Rh grouping mainly depends on the type of reagents available, for which the manufacturers’ instructions have to be strictly followed.
Reagents for Rh (D) Grouping
I Polyclonal human anti-D serum (IgG)
Potentiating or enhancing substances such as albumin, enzymes and AHG reagent are used to bring about agglutination with human IgG anti-D.
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.
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.
a. Saline Agglutination test for Rh (13) Typing
1. Prepare 2-5% washed red cell suspensionof test sample.
2. Place 1 drop of anti-D (Dl) in cleaned tube labelled Dl and place 1 drop .of anti-D (D2) from a different manufacturer in a clean tube labelled D2.
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.
Positive test : Agglutination in anti-D (both tubes) 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.
- conflicting results in tubes Dl and D2.
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, hereas some workers suggest that if the two anti D reagents used are potent and specific, it is not necessary to perform Du testing.
b. Albumin technique for Rh (D) typing
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.
Albumin can be used in
= Albumin addition technique additive to serum & cell mixture.
- Albumin layering technique layered on cell button
Albumin addition technique Procedure 1. Add 1 drop of anti-D to a labelled tube. 2. Add 1 drop of 2-5% test red cell suspension. 3 Add 1 drop of 22% bovine albumin. 4. Mix and incubate at 37°C for 15-20 minutes. 5. Centrifuge at 1000rpm for 1 minute. 6. Gently resuspend the cell button and observe for agglutination. 7. Confirm all negative reactions under microscope. Albumin layering technique
1. Place 1 drop of anti-D in a labelled tube.
2. Add 1 drop of 2-5% test red cell saline suspension.
3. Incubate at 37°C for 45-60 minutes.
4. Allow 1 drop of 22% albumin to run down the inside wall of the tube. Albumin will form a layer on top of the red cells. Do not mix.
5. Incubate further at 37°C for 15-20 minutes.
6. Examine for agglutination after gentle shaking and confirm all negative results under microscope.
c. Enzyme agglutination technique for Rh (D) typing
Proteolytic enzymes such as papain, trypsin, bromelain and ficin digest the cell membrane partially and expose Rh antigens to react with IgG antibodies. When the membrane is partially removed, it brings about a loss of negative electric charge on the red cell which is responsible for keeping the cells a set distance apart, hence small IgG molecules are able to span the gap between cells and bring about agglutination. (for further details see Subsection 3.6 : Specialized serological techniques.)
One stage enzyme technique (papain-cystein)
1. To 1 drop of anti-D (IgG), add 1 drop 1% papain-cystein enzyme.
2. Add 1 drop of 2-5% saline suspension of test red cell (put appropriate controls).
3. Mix and incubate at 37°C for 45 minutes.
4. Centrifuge at 1000 rpm for 1 minute and record the results.
5. Agglutination of test cells indicates positivity while no agglutination shows a negative test (Read macroscopically only).
Two-stage enzyme technique (Pre-treatment of test red cells with exzyme)
1. Prepare a 1% papain solution and dilute it to 1:3 using normal saline.
2. To .1 drop of washed packed test cells, add 2 drop of diluted papain solution.
3. Incubate at 37°C for 15 minutes.
4. Wash the red cells 3-4 times with normal saline and prepare a 2-5% cell suspension.
5. To 1 drop of anti-D (IgG), add I drop of enzyme-treated red cell suspension.
6. Mix & incubate at 37°C for 45 minutes.
7. Centrifuge at 1000 rpm for 1 minute and record the result.
8. Agglutination of cells indicates a positive reaction and no agglutination indicates a negative reaction (Ready only macroscopically)
d. Indirect antiglobulin test (Du Testing)
If using IgG anti-D in routine saline agglutination Rh (D) grouping proceed from step 6 in all negative reactions otherwise perform the following procedure :
1. Take 1 drop of anti-D (IgG) in a cleaned labelled test tube (T).
2. Take 1 drop of appropriate diluent control in another tube (C).
3. Add 2-5% washed test red cell suspension to both the tubes.
4. Mix and incubate at 37°C for 45-60 minutes.
5. Centrifuge at 1000 rpm for 1 minute.
6. Gently suspend the cell button and look for agglutination, if positive test, no need to proceed as the sample is Rh (D) positive.
7. If negative, wash the cells 3-4 times with saline and decant the last washing.
8. Add 1-2 drop of anti-human globulin reagent (AI-IG-Coombs’ reagent). Mix gently and centrifuge at 1000 rpm for 1 minute.
9. Resuspended the cell button gently, examine for agglutination and record the results.
10. All negative reactions should be confirmed by adding known IgG sensitized cells, re-centrifuge and re-examine for agglutination. The presence of agglutination confirms the test result. (For details, refer to Subsection 3.3 Antiglobulin test)
Agglutination in test sample and negative reaction in control sample shows a positive test and the sample is labelled Rh (d) positive.
Microplate technique for Rh (D) grouping
As described in ABO grouping
Rh(D) Grouping In Haemolytlc Disease of the Newborn
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.
Erroneous Results in Rh Grouping
Inaccurate or incorrect results in Rh grouping may occur due to technical errors. These can result from defects in equipment, reagents, specimen and techniques or through wrong interpretation.
Resolving Rh Problems
1. Perform clerical checks for validity of labels and requisition forms.
2. Obtain a fresh blood sample of patient.
3. Check patient rçcords for history, diagnosis, pregnancy, medication and previous transfusion.
4. Check equipment and reagents for proper quality control.
5. Check the antisera and controls.
6. Perform alternative procedures such as washing of red cells with warm saline, enzyme treatment of red cells, absorption / elution studies.
7. Carry out family studies.
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