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
- 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).
- The manufacturer’s instructions will indicate if an Rh control is required (i.e Rh control or 6% albumin etc).
Procedure
- Label tubes with patient initials, MRN and reagent (antisera) identification.
- Place 1 drop of anti-D (D1 and D2) in the appropriate clean, labeled test tubes (D1 and D2).
- Place 1 drop of the appropriate control reagent (if required) in a clean, labeled test tube.
- Add 1 drop of the 2-5% cell suspension of patient red cells to each tube.
- Mix the contents of the tubes thoroughly but gently.
- Centrifuge tubes for 20 seconds.
- Gently suspend the cell button and examine macroscopically.
- Grade and record results immediately while holding tubes in your hand.
- Confirm that both anti-D results are the same and both are similar in reaction strength.
Interpretation
- 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.
- 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.
- 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
- 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+.
- 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.