DEVELOPMENT OF A PREPARATION AND STAINING METHOD FOR FETAL ERYTHROBLASTS IN MATERNAL BLOOD - SIMULTANEOUS IMMUNOCYTOCHEMICAL STAINING AND FISH ANALYSIS

In order to detect fetal nucleated red blood cells (NRBCs) in maternal blood, a protocol was developed which aimed at producing a reliable s taining method for combined immunocytochemical and FISH analysis. The technique had to be suitable for eventual automated screening of slide s. Chorionic villi washings, cord blood, and maternal blood samples we re used for this study. After a density gradient separation and centri fugal cytology, slides were stained either with 3,3-diaminobenzidin (D AB), a marker for heme, or with antibodies against the gamma-chain of fetal hemoglobin (HbF). FISH analysis for both X- and Y-chromosomes wa s performed on the same slides. Cytocentrifugation provided a controll ed cell density on the slides with good cell morphology, Both the DAB and HbF staining were suitable for manual screening of large numbers o f slides. The HbF staining, although supposed to be more specific for fetal NRBCs, appeared to be more sensitive to minor changes in prepara tion. We were eventually able to combine HbF staining with FISH analys is, and produced a detection efficiency of > 85% for both X- and Y-chr omosome signals. This preparation protocol simplifies the detection of NRBCs in maternal blood. Immunocytochemical staining and FISH analysi s can be performed on the same cell with good image contrast, thus fac ilitating both manual and automated image analysis. This will facilita te the use of this approach for prenatal diagnosis. (C) 1998 Wiley-Lis s, Inc.