Detection of weak D and D(VI) 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 16000. If the fetus is of weak D or partial D(VI) 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-ELSA 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 D(VI) were also analysed. None of these samples could be distinguished: SPC were all <1000. Although one of the reagents used reacts with D(VI) cells, quantitation of a D(VI) 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.