Testing for the D zygosity with three different methods revealed altered Rhesus boxes and a new weak D type.

Abstract

The discrimination of D+/D+ from D+/D- partners of D- mothers with anti-D is important to estimate the risk for HDN. This may be achieved if the presence or absence of the hybrid Rhesus box in the father can be demonstrated. A new PCR-SSP method specific for the hybrid Rhesus box comprising an internal amplification control was compared with two published PCR-based methods (PCR-SSP and PCR-RFLP) in 83 D+, 13 D-, and 37 weak D samples. The deletion of RHD was detectable in all D- and weak D samples. By all three methods, concordant results were obtained in 82 of 83 D+ samples, with one sample showing discrepant results. The control band in the PCR-RFLP method, specific for the downstream Rhesus box, was missing in two weak D samples, namely a weak D type 4.0 and a novel weak D type dubbed weak D type 29. Further investigations revealed an altered downstream Rhesus box in the weak D type 29 sample. In the weak D type 4.0 sample, no amplicon was achieved with any primer specific for the upstream and downstream Rhesus box. A PCR-SSP method with internal control was established for the detection of the hybrid Rhesus box. Polymorphisms in the downstream Rhesus box may interfere with the detection of RHD.