Studies on preliminary concentration methods for recovery of fetal nucleated red blood cells in maternal blood

Abstract

Non-invasive prenatal diagnostic methods posing no danger to the embryo have been desired for many years in the field of prenatal medicine. We are in the process of improving the lectin method that we developed for recovering fetus-derived nucleated red blood cells (NRBC) in the maternal peripheral blood. We previously used Ficoll density-gradient centrifugation for preliminary concentration in the lectin method. In the present study, we developed a molecular filter method, compared it with the Ficoll method, and tested its applicability to prenatal diagnosis. We tested the usefulness of a high molecular filter method for preliminary concentration. First, in a basic study, we prepared three kinds of non-woven cloth (NWC1-3) and a multi-porous filter (MP) to determine the optimal filter. Next, we compared the recovery rates of the Ficoll and filter methods as preliminary concentration methods in 34 normal pregnant women. Then, to examine whether the recovered NRBC were derived from the fetus, we attempted prenatal diagnosis by the fluorescence in situ hybridization (FISH) technique in 12 women pregnant with a male fetus (determined later by ultrasound) at between 8 and 16 weeks of gestation. Among the four devices used in the basic study of the high molecular filter method, NWC-2 had the best recovery rate. Therefore, we compared the numbers of NRBC recovered by the lectin method after preliminary concentration with NWC-2 or by Ficoll centrifugation, and found that the mean recovery rate of NWC-2 was 4.2 +/- 5.0 times as high as that of the Ficoll method, indicating that the NWC-2 filter method is superior as a preliminary concentration method. Next, FISH analysis of the 12 pregnant women with a male fetus for the Y chromosome showed that 19.5 +/- 12.8 NRBC were recovered, in 12.7 +/- 8.1 (63.6%) of which a Y signal was confirmed, suggesting the NWC-2 filter method can be applied to prenatal diagnosis. We consider the filter-lectin method to be a superior method for isolation and recovery of NRBC in the maternal blood which can be applied to prenatal diagnosis.

Basic investigation of the lectin method for separation and recovery of nucleated red blood cells in maternal blood, and a study into the frequency of nucleated red blood cells in fetomaternal disorders

“…In our studies (Kitagawa et al . 2002; Wachi & Kitagawa 2004; Wada & Kitagawa 2004), NRBCs were investigated by fluorescence in situ hybridization (FISH) using Y‐probe in women with male fetuses at 6–20 weeks of gestation, and about 60% of NRBCs were derived from fetuses in this period. This finding indicates that the number of NRBCs is useful for fetal diagnosis before 20 weeks of gestation, the important period for diagnosis.…”

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

“…Microfluidics is another promising method for collecting a large number of erythroblasts from maternal blood by exploiting the hydrodynamic size difference between nucleated and non‐nucleated cells, but there is a possibility of physical damage to erythroblasts by processing with a microchip designed to separate blood cells based on size and magnetic fields. In our previous study, we investigated physical isolation of erythroblasts by using a non‐woven filter or multi‐porous membranes . Although enrichment of erythroblasts was successful, some of the cells were resistant to FISH analysis.…”

Isolation and Enrichment of Circulating Fetal Cells for NIPD: An Overview

“…The fnRBCs are also less susceptible to ammonium chloride lysis than adult nRBCs because CA activity is five times lower and acetozolamide permeability approximately ten times greater. Several studies [ 70 , 71 ] have used soybean agglutinin (SBA), which recognizes N-acetylgalactosamine and galactose, demonstrating high specificity for erythroblasts and eliminating leukocytes from the sample.…”