The Proportion of Fetal Nucleated Red Blood Cells in Maternal Blood: Stimation by FACS Analysis

Sohda, Satoshi; Arinami, Tadao; Hamada, Hiromi; Nakauchi, Hiromitsu; Hamaguchi, Hideo; Kubo, Takeshi

doi:10.1002/(sici)1097-0223(199708)17:8<743::aid-pd144>3.3.co;2-v

Cited by 25 publications

(29 citation statements)

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“…Although various recovery methods focusing on the physical properties and surface markers of cells such as Ficoll-Hypaque density gradient centrifugation (Takabayashi et al . 1995), methods using a cell sorter (Zheng 1997;Sohda et al . 1997), and methods using magnetic beads (Ganshirt et al 1998) have been investigated as the separation/concentration of fetal NRBC in maternal blood, these methods were not practical, and the development of a single method with high recovery and reproducibility has been awaited.…”

Section: Introductionmentioning

confidence: 99%

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

Wada

Kitagawa

2004

Congenital Anomalies

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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.

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Section: Introductionmentioning

confidence: 99%

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

Wada

Kitagawa

2004

Congenital Anomalies

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“…Fetal erythroblasts are rare in the peripheral blood from pregnant women. Their number has been estimated at 8.1 10 5 to 1.6 10 5 (Sohda et al, 1997) or 1 fetal to 10 5 maternal cells in normal primigravidae (Hamada et al, 1993).…”

Section: Introductionmentioning

confidence: 99%

A comparison of different density gradients and antibodies for enrichment of fetal erythroblasts by MACS

1999

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The enrichment of fetal cells, in particular fetal erythroblasts from the blood of pregnant women offers a promising non‐invasive alternative for prenatal diagnosis. The purpose of this study was to compare the retrieval of erythroblasts by different density gradients and different antibodies against erythroid surface antigens, in both a model test system and in blood samples of pregnant women. We enriched erythroblasts from artificial mixtures of cord and adult blood (1:50) and from 16 ml of peripheral blood from pregnant women at a mean gestational age of 14+2 weeks. The yield of erythroblasts was calculated and compared using Wilcoxon's matched‐pairs signed‐rank test. In the artificial mixture most erythroblasts were retrieved using the heaviest density gradient (specific density of 1119) followed by antibody‐labelled magnetic cell sorting (MACS). With anti‐GPA the yield of erythroblasts from the artificial mixture was highest (9362.5 erythroblasts) compared with anti‐CD36 (5164.3), anti‐i (3455.2), anti‐CD71 (3055.8) and HAE9 (2364.2). The difference between anti‐GPA and anti‐CD71, HAE9 and anti‐i was significant (p=0.0277). The enrichment of erythroblasts from peripheral blood of pregnant women showed similar results. The yield of erythroblasts using anti‐GPA was the highest. These results enable us to simplify our enrichment protocols to a single density gradient of 1119 specific density followed by MACS, with anti‐GPA. Copyright © 1999 John Wiley & Sons, Ltd.

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“…Culturing peripheral blood lymphocytes from 30 pregnant women with a male fetus, Walknowska and Conte reported in 1969 the detection of a 46,XY karyotype in 21 women (Walknowska & Conte 1969). Many researchers followed suit (Herzenberg 1979;Iverson & Bianchi 1981;Sohda et al . 1997), and in 1990, Lo et al .…”

Section: Introductionmentioning

confidence: 99%

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

Wachi

Kitagawa

2004

Congenital Anomalies

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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.

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The Proportion of Fetal Nucleated Red Blood Cells in Maternal Blood: Stimation by FACS Analysis

Cited by 25 publications

References 0 publications

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

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

A comparison of different density gradients and antibodies for enrichment of fetal erythroblasts by MACS

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

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