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

Abstract

Summary. 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 R 1 r or R 2 r phenotype, with antigen site numbers per cell (SPC) of between 9900 and 16 000. 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-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 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.

The role of RhD agglutination for the detection of weak D red cells by anti-D flow cytometry

“…Lloyd‐Evans et al. (1999) found that weak D red cells with <1000 RhD sites per red cell were not detectable using quanti‐D (BTSN10 clone) flow cytometry.…”

“…However, the minimum volume of weak D red cells required for an immune response is not known. Lloyd‐Evans et al. (1999) suggested that the routine administration in the UK of a 500 IU dose of prophylactic anti‐D postpartum was probably sufficient to prevent anti‐D immunization when the FMH is much greater than 4 ml of weak D fetal red cells.…”

“…, 2003). However, it has been reported that this technique will not detect weak D red cells with fewer than 1000 RhD sites per red cell (Lloyd‐Evans et al. , 1999).…”

Immunocytometric quantitation of foeto-maternal haemorrhage with the Abbott Cell-Dyn CD4000 haematology analyser

“…, 1996; Kumpel, 2001). This antibody also discriminates D‐variant RBC when they express more than 1000 D‐sites per red cell (Lloyd‐Evans et al. , 1999).…”

Flow Cytofluorometric Analysis in Red Blood Cell Immunology

“…3) [86]. A further disadvantage of the directly labeled anti-D method is that it may not be able to distinguish weak D samples from D− cells [48,87]; this is not true for the indirect anti-D method [69]. In 1998, Davis et al [88] described an FC method using FITC anti-HbF for FMH quantitation.…”