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

1. Reagent anti-D: Suitable reagents must have an IgG component. (refer to the manufacturers’ instructions before performing testing).
2. Antihuman globulin reagent, either polyspecific or anti-IgG.
3. IgG-coated red cells (Coombs Control Cells- CC).

Procedure

1. 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.
2. 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).
3. After incubation, wash the cells 4 times with saline.
4. 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).
5. Mix thoroughly but gently then centrifuge for 20 seconds.
6. Gently suspend each cell button and examine macroscopically.
7. Grade and record results immediately while holding the tubes in your hands.
8. If the test result is negative, add 1 drop of known IgG-sensitized red cells (CC).
9. Mix thoroughly, but gently and centrifuge for 20 seconds.
10. Examine for agglutination and immediately record results while holding tube in hand.

Interpretation

1. 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.
2. 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.
3. If there is agglutination at any point in the control tube, no valid interpretation can be made of the weak D test.

Notes

1. 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.
2. 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.
3. 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.
4. 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).
5. Some facilities may elect to do an additional reading after the 37ºC incubation and before completing the antiglobulin phase of testing.
6. 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.
7. Weak D positive units of blood are labelled as Rh “positive”.