Method of separation and concentration of fetal nucleated red blood cells in maternal blood and its application to fetal diagnosis.

Abstract

A fetal diagnostic method that is without risk to the embryo has been long awaited in the field of gene diagnosis. Establishment of non-invasive fetal diagnosis using maternal peripheral blood will greatly contribute to perinatal medical care. The lectin method that we have studied and developed selectively recovers nucleated red blood cells (NRBC) among fetal cells mixed in maternal peripheral blood. Maternal blood, 7 mL, was collected with ethylene diamine tetraacetic acid (EDTA) treatment in each week of gestation and subjected to preliminary concentration by density centrifugation (Histopaque [Histopaque-1077; Sigma Diagnostics, MO, USA], specific gravity: 1.095), and NRBC were separated and collected on slide glasses by the lectin method (soybean agglutinin [SBA]: 50 microg/mL). To investigate selective adhesion of the erythrocyte fraction, the SBA concentration was set to 50 microg/mL, and the cells were labeled with CD11a and CD33 (anti-white blood cell antibodies) and investigated by flowcytometry. Erythrocytes adhered at a high rate (87.0 +/- 9.7%) while the adhesion rates of granulocytes, monocytes, and lymphocytes were low, confirming the usefulness of the method for separation and recovery of the erythrocyte fraction. When recovery of NRBC was investigated using this method, a mean of 6.57 +/- 7.12 cells were recovered from 1 mL of maternal blood (May-Gluüwald-Giemsa stain). The number of recovered NRBC increased slowly with pregnancy, but differences were not significant. To confirm that recovered NRBC were derived from the fetus, NRBC were recovered by the lectin method in four patients suspected of 18 trisomy by echography and analyzed by fluorescence in situ hybridization. Three hybridization signals were detected in NRBC at a high frequency, showing that most cells were derived from the fetus and thus, fetal diagnosis may be possible. Since the procedure of the lectin method we have developed is simple, and high concentration efficiency can be obtained at a low cost, it may be clinically applicable.