Rh(D) typing and weak D testing

Principle

• Patient red cells are tested with monoclonal anti-D. Agglutination shows the D antigen is present.
• Weak D (reduced D antigen quantity) may not react at immediate spin; an indirect antiglobulin test detects IgG anti-D bound to the cells.

Specimen

• EDTA or clotted blood; 2–5% red cell suspension.

Procedure

• Add 1 drop anti-D to a labelled tube and 1 drop cell suspension; centrifuge and read macroscopically.
• Use a control: the reagent manufacturer's control, or the negative anti-A/anti-B results for low-protein monoclonal reagents. Group AB D-positive samples need a separate control.
• Weak D test (if immediate spin is negative and required): incubate the same tube at 37 °C (commonly 15–30 min), wash 3–4 times with saline, add anti-human globulin, centrifuge and read.
• Add IgG-sensitised check cells to negative AHG tubes; they must agglutinate.

Results

• Positive at immediate spin: D positive.
• Negative at immediate spin but positive by IAT with a negative control: weak D.
• Negative at all phases: D negative.

Quality control and pitfalls

• A positive DAT makes the weak D test invalid (false positive); check the DAT or the AHG control.
• Mixed field: after transfusion of D-negative cells, fetomaternal haemorrhage or stem cell transplant.
• Partial D (e.g. DVI) individuals lack parts of the D antigen and can form anti-D. Patient-typing monoclonal reagents are often chosen not to detect DVI so these patients are treated as D negative.
• Donor units must be tested for weak D (AABB); a weak D donor unit is labelled D positive.
• Recipient weak D testing is often not required; such patients may receive D-negative blood. RHD genotyping is recommended for serological weak D in women of childbearing age (weak D types 1, 2 and 3 can be managed as D positive).

Clinical use

• Transfusion compatibility, antenatal testing and decisions about RhIG prophylaxis.