Quantitation of fetomaternal haemorrhage and F cells in unusual maternal blood samples by flow cytometry using anti‐D and anti‐HbF

Abstract

Fetomaternal haemorrhage (FMH) assessment by the Kleihauer-Betke test (KBT) is rapid but semi-quantitative and liable to false positive results. To compare FMH estimated by KBT with flow cytometry (FC) quantitation for 37 patients with massive FMH, obstetric risk factors or technical problems. Maternal blood was sent for analysis by FC after KBT. A variety of reagents including anti-haemoglobin F (HbF), anti-D and combined anti-HbF/anti-carbonic anhydrase (CA) were used. Eight cases of massive FMH (>100 mL fetal cells) causing fetal death or severe neonatal anaemia in late gestation were confirmed by FC. Anti-HbF FC identified maternal F cells and fetal cells. In some cases these red cell populations merged but they could be differentiated by anti-CA, labelling F cells only. Using KBT, false positive FMH results were obtained for 12 patients, who had strongly stained cells that were then shown by FC to be maternal F cells. All these patients had increased F cells (>5% of total red cells) whereas only 16% of patients with FMH and 22% of donors had elevated F cells. In contrast, anti-D FC was simple and rapid, quantitating D-positive FMH in all 15 D-negative patients except one with massive FMH of weak D fetal cells. Leucocytes in four samples bound anti-D, variably, giving erroneously high FMH, but they could be eliminated from FC analysis using combined anti-D/anti-CD45. FMH quantitation using anti-D by FC is suitable for the majority of maternal samples and could enable accurate targeted dosing of anti-D prophylaxis.