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.

Practicability of prenatal testing using lectin‐based enrichment of fetal erythroblasts

“…Blood was subjected to density gradient centrifugation (1.095 g/mL) to deplete maternal erythrocytes . Then CD45 panning using dishes coated with an immobilized anti‐CD45 antibody (Fujikura Kasei) was conducted at 18°C for 30 min to remove contaminating leukocytes.…”

“…In our previous research, we performed lectin‐based erythroblast isolation (LBEI) with May–Grünwald–Giemsa (MGG) staining to identify fetal erythroblasts . We used soybean agglutinin (SBA) and galactose‐bound vinyl polymer to enrich erythroblasts.…”

Validation of automatic scanning of microscope slides in recovering rare cellular events: application for detection of fetal cells in maternal blood

“…Although there is general agreement about the presence of FCs, their routine use is not yet feasible. Most researchers agree that the presence of FCs in maternal blood is a rare event with only one FC per 10 5 –10 9 of maternal cells . This makes their isolation difficult but not impossible.…”

“…The presence of fetal cells (FCs) in maternal blood was first described more than a century ago and has since been confirmed by many investigators. [6][7][8][9][10] The use of these cells as an alternative source of fetal genetic material can provide a non-invasive, risk-free opportunity for detection of fetal chromosomal abnormalities. Although there is general agreement about the presence of FCs, their routine use is not yet feasible.…”

“…Most researchers agree that the presence of FCs in maternal blood is a rare event with only one FC per 10 5 -10 9 of maternal cells. 8,10,12 This makes their isolation difficult but not impossible. Manual screening was used for the retrieval of FCs from maternal blood after targeting a specific genetic marker exclusive to the FCs.…”