DETECTION OF FETAL RED-CELLS IN FETOMATERNAL HEMORRHAGE USING A FETALHEMOGLOBIN MONOCLONAL-ANTIBODY BY FLOW-CYTOMETRY

BACKGROUND: The laboratory determination of the level of fetal cells i n maternal circulation remains an important support in the obstetrical management of women with suspected uterine trauma and in the proper d ose administration of anti-D for prevention of Rh hemolytic disease of the newborn. Limitations in the sensitivity and precision of the wide ly used manual Kleihauer-Betke test have prompted an increased utiliza tion of flow cytometric methods for fetal cell detection in maternal b lood samples. STUDY DESIGN AND METHODS: Murine monoclonal antibodies d irected against fetal hemoglobin (HbF) were developed, conjugated to f luorescein isothiocyanate, and used in a multiparametric flow cytometr ic assay developed for the quantitation of fetal red cells. A rapid in tracellular staining method using brief glutaraldehyde fixation and Tr iton X-100 permeabilization prior to mono-clonal antibody incubation w as developed, along with optimization of the flow cytometric analysis protocol for the analysis of 50,000 cells. The performance of tt-e ass ay was assessed for linearity and precision and correlated with the Kl eihauer-Betke acid elution method. RESULTS: The anti-HbF flow cytometr ic method showed good correlation with the Kleihauer-Betke method (r(2 ) = 0.86) and superior precision with a CV <15 percent for blood sampl es with >0.1 percent fetal cells. Analysis of 150 blood samples from n onpregnant adults, including individuals with elevated HbF due to hemo globinopathies and hereditary persistence of HbF, gave a mean value of 0.02 percent fetal cells, and all results were less than 0.1 percent. CONCLUSIONS: The anti-HbF flow cytometric method for detection of fet al cells offers a simple, reliable, and more precise alternative to th e Kleihauer-Betke manual technique for the assessment of fetomaternal hemorrhage. The method has additional potential applications for the s tudy of HbF levels or frequency of adult red cells with low levels of HbF (F cells) in individuals with hemoglobinopathies.