Identification and quantification of fetal red blood cells in maternal blood by a dual‐color flow cytometric method: evaluation of the Fetal Cell Count kit

Abstract

As an alternative to the cumbersome Kleihauer-Betke test (KBT), flow cytometry represents a powerful method for the identification and quantification of fetal red blood cells (RBCs) in maternal circulation.The aim of this study was to evaluate the Fetal Cell Count kit (IQ Products), an innovative flow cytometric method, based on the combination of antibodies directed, respectively, against fetal hemoglobin (HbF) and carbonic anhydrase (CA), a marker expressed after birth, to discriminate fetal RBCs from adult F cells containing HbF. The investigation was performed by two French laboratories that compared the data obtained by flow cytometry and KBT in 455 pregnant or just-delivered women as well as in 124 artificial mixtures containing from 0.01 to 5.00 percent cord cells.The FL1/FL2 histogram allowed distinction between fetal RBCs (HbF+, CA-), F cells (HbF+, CA+), and adult RBCs (HbF-, CA+). The limits of detection and quantification were determined at 0.03 and 0.10 percent or 0.02 and 0.05 percent when analyzing 100,000 or 200,000 events, respectively. Linearity was demonstrated between 0.01 and 5.00 percent fetal cells in the mixtures (r = 0.95, p < 0.01). A good correlation between fluorescence-activated cell sorting (FACS) and KBT results was obtained with artificial mixtures (r = 0.94, p < 0.01). From the 405 Kleihauer-negative samples, none were identified as positive by FACS. Among the 50 Kleihauer-positive samples, 6 were shown not to contain fetal cells but F cells by FACS.With this new dual-color flow cytometric method, accurate evaluation of fetomaternal hemorrhage was achieved even in the face of HbF of maternal origin.