BACKGROUND: Fetomaternal haemorrhage (FMH) assessment by the Kleihauer-Betke test (KBT) is rapid but semi-quantitative and liable to false positive results. OBJECTIVES: To compare FMH estimated by KBT with flow cytometry (FC) quantitation for 37 patients with massive FMH, obstetric risk factors or technical problems. METHODS: 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. RESULTS: 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. CONCLUSION: 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.
BACKGROUND:Fetomaternal haemorrhage (FMH) assessment by the Kleihauer-Betke test (KBT) is rapid but semi-quantitative and liable to false positive results. OBJECTIVES: To compare FMH estimated by KBT with flow cytometry (FC) quantitation for 37 patients with massive FMH, obstetric risk factors or technical problems. METHODS: 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. RESULTS: 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. CONCLUSION: 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.