23 Tube Tests for Rh Typing of Red Cells

Principle

Rh Testing determines the presence or absence of D antigen. Patient red cells are tested for D antigen using commercial Anti-D reagent(s). Agglutination indicates D is present (Rh positive) lack of agglutination indicates its’ absence (Rh negative).

Specimen

Clotted( red-top) or  EDTA (pink/lavender )anti-coagulated blood samples.

Reagents

1. Two types of reagent anti-D: Suitable reagents include low protein monoclonal reagents (these may be a blend of IgG and IgM) and polyclonal high-protein reagents (refer to instructions of manufacturer before performing testing).
2. The manufacturer’s instructions will indicate if an Rh control is required (i.e Rh control or 6% albumin etc).

Procedure

1. Label tubes with patient initials, MRN and reagent (antisera) identification.
2. Place 1 drop of anti-D (D1 and D2) in the appropriate clean, labeled test tubes (D1 and D2).
3. Place 1 drop of the appropriate control reagent (if required) in a clean, labeled test tube.
4. Add 1 drop of the 2-5% cell suspension of patient red cells to each tube.
5. Mix the contents of the tubes thoroughly but gently.
6. Centrifuge tubes for 20 seconds.
7. Gently suspend the cell button and examine macroscopically.
8. Grade and record results immediately while holding tubes in your hand.
9. Confirm that both anti-D results are the same and both are similar in reaction strength.

Interpretation

1. Agglutination in the anti-D tubes, combined with a smooth suspension in the control tube, indicates that the test red cells are D positive and have the D antigen.
2. A smooth suspension of red cells in the anti-D tubes, as well as the control tube is a negative result. Further testing for weak D may be required.
3. If the control tube has agglutination the results for the anti-D are invalid and can’t be reported without further testing. Methods for removal of IgG antibody from the red cells may be required.

Notes

1. Most commercially prepared anti-sera provide a 2+ or greater agglutination with D positive cells. Additional testing may be necessary on results that are less than 2+.
2. A negative tube test with anti-A and/or anti-B serves as a valid control when a low protein anti-D reagent has been used.