Comparison of haemoglobin F detection by the acid elution test, flow cytometry and high‐performance liquid chromatography in maternal blood samples analysed for fetomaternal haemorrhage

Abstract

All pregnant women undergoing blood grouping at Southmead Hospital are offered haemoglobinopathy screening by high-performance liquid chromatography (HPLC). RhD-negative women who deliver RhD-positive infants are tested for fetomaternal haemorrhage (FMH) by acid elution (AE). The effectiveness of these two assays for quantitation of FMH was compared with flow cytometry (FC). The relationship between expression of haemoglobin F (HbF) in individual cells by AE and FC and quantitation of HbF in haemolysates by HPLC was investigated, using maternal samples with unusually high levels of HbF-positive maternal cells (F cells) or with large FMH (fetal cells). Standard anti-D FC was performed to quantitate fetal D-positive cells in D-negative women and compared with FMH estimated by AE and HbF FC. AE overestimated FMH when maternal F cells were increased. HbF FC distinguished F cells from fetal cells. Values of HbF determined by HPLC were less than the level of 5% used for investigation of raised fetal haemoglobin, even in the maternal samples with elevated F cells or massive FMH. To quantitate FMH, measurement of HbF using FC was more sensitive and accurate than AE or HPLC. HbF FC is the method of choice when results from routine investigation using AE or standard anti-D FC are discrepant or when there is maternal and fetal RhD compatibility.