ABSTRACT
OBJECTIVE: We wanted to obtain statistically relevant data about the efficiency of our method for the isolation of fetal nucleated red blood cells (NRBCs) from the maternal circulation. METHODS: More than 600 samples were investigated using a triple density gradient followed by magnetic separation of anti-CD71-labeled cells, and yields and purities of recovered NRBCs were determined. RESULTS: The enrichment effectivity as well as the morphological condition of cells was reproducibly good, if blood samples were enriched within 48 h after sampling. The efficacy was independent of various methodological parameters and our technique was superior to other magnetic cell-sorting techniques. Mean yields and purities of NRBCs increased with increasing gestational age, ranging from 100 to 1,000 cells per 40-ml blood sample and from 0.1 to 1%, respectively, from the 6th week of gestation to term. In pregnancies with preeclampsia NRBCs were increased by a factor of 10. CONCLUSION: Our enrichment technique proved to be optimized with respect to various methodological parameters, which were compared in the present study, and it is efficient and reproducible for the enrichment of NRBCs from the maternal circulation in all three gestational trimesters.
Subject(s)
Cell Separation/methods , Centrifugation, Density Gradient , Erythrocytes , Fetal Blood/cytology , Magnetics , Prenatal Diagnosis , Antigens, CD/analysis , Antigens, Differentiation, B-Lymphocyte/analysis , Cell Nucleus , Embryonic and Fetal Development , Erythrocytes/ultrastructure , Female , Flow Cytometry , Gestational Age , Humans , Leukocyte Common Antigens/analysis , Pregnancy , Receptors, TransferrinSubject(s)
Erythroid Precursor Cells/cytology , Immunomagnetic Separation/methods , Pregnancy/blood , Prenatal Diagnosis/methods , Age Factors , Cell Nucleus/ultrastructure , Female , Fetal Blood/cytology , Gestational Age , Humans , Maternal-Fetal Exchange , Parity , Pre-Eclampsia/blood , Trisomy/diagnosisABSTRACT
PROBLEM: The need for an inexpensive and reproducible technique for noninvasive prenatal diagnosis by fetal cell isolation from maternal blood. METHOD: For enrichment of fetal cells we used a combination of triple density gradient and magnetic sorting (MACS) of (anti-CD71) transferrin receptor antibody labeled cells followed by fluorescence in situ hybridization (FISH) with chromosome-specific DNA probes for detection of fetal aneuploidies. We identified 15 cases of fetal trisomy (five cases with a trisomy 18 and ten cases with a trisomy 21) with subsequent chromosome-specific FISH. RESULTS: We found in all of the aneuploid pregnancies that the percentage of cells with three hybridization signals did not overlap with those of normal controls independent from gestational ages and previous invasive procedures. CONCLUSIONS: Our new approach for noninvasive prenatal diagnosis has proven to be reliable in this first series.
