Detection of weak D and DVI red cells in D‐negative mixtures by flow cytometry: implications for feto‐maternal haemorrhage quantification and D typing policies for newborns

Quantitation of feto-maternal haemorrhage (FMH) by flow cytometry (FC) has been shown to be more accurate than the Kleihauer-Bekte test. Fetal cells will be predominately of R1r or R2r phenotype, with antigen site numbers per cell (SPC) of between 9900 and 16 000. If the fetus is of weak D or partial DVI phenotype, fewer SPC will be present. Red cells from 20 adult weak D samples were mixed with rr red cells to give 1% mixes. Mixtures were stained and analysed by FC, using two different monoclonal reagents. The SPC of each sample was measured using SOL-ELISA with Scatchard plot analysis. 18 samples could not be distinguished and had <1000 SPC. Two samples that could be distinguished had 1350 and 3000 SPC. Red cells from seven samples of DVI were also analysed. None of these samples could be distinguished; SPC were all <1000. Although one of the reagents used reacts with DVI cells, quantitation of a DVI FMH would not be possible due to low SPC. The ability of fetal red cells with low Rh D SPC to cause immunization is questionable; failure to measure FMH in these cases is unlikely to cause clinical problems, as long as suitably sensitive serological reagents and techniques are used to type all weak D and D variant babies as Rh D positive, and thus ensure that the mother is given the appropriate dose of anti-D.