28 Testing for Weak D
Principle
D antigen expression can vary on red cells .Weak D antigen refers to inheritance of the complete D antigen however less of it (quantity) so agglutination may be weak or not detected by direct antigen testing. Weak D expression is often detected by an indirect antiglobulin test .
Specimen
Red-top (clotted) or EDTA (pink/lavender anti-coagulated) blood sample.
Reagents
- Reagent anti-D: Suitable reagents must have an IgG component. (refer to the manufacturers’ instructions before performing testing).
- Antihuman globulin reagent, either polyspecific or anti-IgG.
- IgG-coated red cells (Coombs Control Cells- CC).
Procedure
- If the original, direct test with anti-D was performed by tube testing using anti-D (containing IgG anti-D), the same tube(s) may be used (continued) for weak D testing. If not, refer to the tube test for Rh typing of red cells to set up the testing. If only using “one” anti-D tube another drop of anti-D may be added to the original tube to insure the antibody was not omitted with the initial testing.
- Mix and incubate anti-D1 and anti-D2 tube(s) and the Rh control tube at 37ºC for 15-30 minutes (follow the manufacturers’ instructions).
- After incubation, wash the cells 4 times with saline.
- After the final wash, decant the saline completely, blot the rims of the tubes dry, and add 2 drops of anti-human globulin (AHG) reagent (follow the manufacturers’ instructions).
- Mix thoroughly but gently then centrifuge for 20 seconds.
- Gently suspend each cell button and examine macroscopically.
- Grade and record results immediately while holding the tubes in your hands.
- If the test result is negative, add 1 drop of known IgG-sensitized red cells (CC).
- Mix thoroughly, but gently and centrifuge for 20 seconds.
- Examine for agglutination and immediately record results while holding tube in hand.
Interpretation
- Agglutination in the anti-D tube(s) and none in the Rh control tube constitutes a positive test result and indicates the presence of the D antigen by the weak D test. When the appropriate Rh control test is negative, the red cells can be designated as D positive.
- Absence of agglutination in the tube with anti-D is a negative result, indicating that the cells do not express D and should be classified as D negative.
- If there is agglutination at any point in the control tube, no valid interpretation can be made of the weak D test.
Notes
- There must be agglutination in the last step with the IgG-sensitized red cells (CC) confirming the presence of active antiglobulin reagent in the test mixture. If there is no agglutination or the results are less than 2+ the negative testing must be repeated. Record results.
- It is permissible to use the DAT on the test cells as a control, but the Rh control tube is preferable as this ensures that all reagent components that might cause a false positive result are represented.
- Finding a weak D typing in a pregnant woman or one who recently gave birth may indicate a mixture of maternal Rh negative and fetal positive blood and may require further investigation.
- Red blood cells demonstrating a positive DAT cannot be accurately tested for weak D by this method (or any other testing requiring the use of antihuman globulin reagent).
- Some facilities may elect to do an additional reading after the 37ºC incubation and before completing the antiglobulin phase of testing.
- Weak D testing is done on all D negative first time blood donors and D negative babies of Rh negative mothers. In Nova Scotia there is a specific policy across the province for standardized testing of Rh negative mother’s, father’s and babies taking into consideration research on which mother’s are known to make immune anti-D.
- Weak D positive units of blood are labelled as Rh “positive”.