To isolate fetal cells from maternal blood, we developed a new method based on galactose-bearing conjugation. Nucleated red blood cells (NRBCs), which highly express galactose on their surface, were selectively attached to a substrate coated with a galactose-containing polymer via soybean agglutinin (SBA), a galactose-specific lectin. Cord blood samples were used to evaluate enrichment efficacy of NRBCs by this method. Blood samples were obtained from 131 pregnant women between 6 and 27 gestational weeks. After preliminary condensation of fetal cells by Ficoll gradient centrifugation, NRBCs were enriched using galactose-positive selection by adjusting SBA concentration. We isolated one to several hundred NRBCs (mean+/-SD, 7.8+/-8.5) in 2.3 ml of peripheral blood samples from 96% of pregnant women. The isolated NRBCs were analyzed by a Y-chromosome FISH probe in eight cases carrying male fetuses. Y-signals were detected in all eight cases and more than half of the NRBCs were off fetal origin. The study demonstrates that our new method using galactose-specific lectin provides effective enrichment of fetal NRBCs allowing non-invasive prenatal diagnosis.
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We previously reported the separation and recovery of nucleated red blood cells (NRBCs) in maternal blood using the lectin method. In the present study, we verified the lectin method and investigated the appearance of NRBCs during pregnancy. For the concentration of lectin soy bean agglutinin, 7 mL of maternal peripheral blood was collected from 20 subjects, and the relative fluorescence intensity was measured using flowcytometry; 50 mg/mL, used in previous studies, was the optimal concentration. The number of cells recovered at each step of the lectin method was also investigated by FACS using fluorescence-labeled CD11a and CD33, and the results showed the usefulness of the method. Next, 7 mL of maternal peripheral blood was collected from 292 women with a normal single pregnancy (389 specimens), and NRBCs were separated and recovered using the lectin method. NRBCs slightly increased over the course of pregnancy (y = 4.29x + 5.03, r2 = 0.11). When blood was collected multiple times in the same subjects, NRBCs increased in 63 of 77 subjects (83.1%, percent change: 2.4 +/- 19.0). No NRBCs were recovered in 17 subjects (4.7%). Regarding the relationship between fetomaternal disorders and the frequency of NRBCs, 89.4 +/- 92.6 cells appeared per 10 mL of maternal blood in the normal group, but NRBCs increased in patients with 18 trisomy, placenta previa, pre-eclampsia, intrauterine fetal death, and 21 trisomy. NRBC examination may play an assisting role not only in fetal diagnosis but also in fetomaternal diagnosis.
Fetal nucleated cells in maternal peripheral blood are a non-invasive source of fetal DNA for prenatal genetic diagnosis. However, the number of fetal cells present in maternal peripheral blood is very small. Therefore, fetal cell enrichment is generally considered necessary to allow detection and subsequent genetic analysis of the rare fetal cells. In the study presented here, we performed fetal cell separation from maternal blood using galactose-specific lectin to concentrate fetal nucleated red blood cells (FNRBCs), and attempted paternal diagnosis using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP). Fetal cell separation was performed using galactose-specific lectin on a PV-MeA coated slide. Twenty cells consisting of an NRBC and its surrounding 19 maternal cells were collected using laser microdissection for stable DNA amplification. DNA analysis was performed using three sequence tagged site markers (D13S270, D17S5, and D18S474) by PCR-SSCP. All seven cases were informative because they showed heterozygosity at least one locus in D13S270, D17S5, or D18S474, and paternal-specific bands were detected in all cases. These results suggest that our proposed lectin-laser-micromanipulation-PCR-SSCP method may contribute to the development of prenatal diagnosis.
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