Evaluation of flow cytometric enumeration of foetal erythrocytes in maternal blood

In pregnant women subject to abdominal trauma or other foetomaternal haemorrhage, foetal red blood cells containing haemoglobin-F (HbF) can be found in the circulation. Recently, a monoclonal antibody to HbF has become commercially available, enabling application of a flow cytometric immunofluorescence method for accurately determining the concentration of HbF+ red blood cells. We demonstrate that white blood cells are included in the cluster selected as red blood cells and that these white blood cells exhibit a level of autofluorescence that coincides with the fluorescence signal from HbF+ red blood cells. However, these white blood cells can be excluded from the analysis, thus preventing spuriously increased HbF+ red blood cell counts. We present the results of patient samples containing HbF+ red cells as illustrations of the technique and as a potential interference by HbF-containing cells of nonfoetal origin. Using samples spiked with cord blood, the method is exactly linear with a high coefficient of correlation (r=0.997). Furthermore, the assay has excellent precision (CV < 2.4%), a low limit of detection (0.12% HbF+ RBC), is independent of Rhesus D and can be completed within 1.5 h. This method is suitable for accurate determination of foetomaternal haemorrhage.