The Origin of CD45-CD71+ Cells Enriched by MACS Technology
Journal Title: Biomedical Journal of Scientific & Technical Research (BJSTR) - Year 2018, Vol 8, Issue 3
Abstract
Fetal nucleated cells within maternal blood represent a potential source of fetal genes obtainable by venipuncture. In our previous study, we have enriched CD45-CD71+ cells from maternal blood. To confirm the potential usefulness of our method, we analyzed the origin of the enriched CD45-CD71+ cells using a real-time polymerase chain reaction (PCR) system. The SRY (sex-determining region Y) gene was detected only in cells extracted from the blood of the pregnant woman with the male fetus. The relative dose of the SRY gene to the TFRC (transferrin receptor) gene was 0.001, indicating that 99.9% of the separated cells were from the mother and only 0.1% were of fetal origin. Our method using MACS technology was insufficient to analyze the fetal genome, methylome, or transcriptome, as some additional enrichment is needed, such as single cell technology.Fetal nucleated cells within maternal blood represent a potential source of fetal genes obtainable by venipuncture. An analysis of nuclear red blood cells (NRBCs) in pregnant woman is believed to be useful for understanding prenatal fetal status. However, the extreme scarcity of fetal cells puts strong demands on sensitivity and specificity. We have effectively used a simple MACS technology without density gradient centrifugation or lysis of RBCs [1].Briefly, in our previous study, we analyzed two patients. Patient 1 was an 18-year-old woman bearing a single male fetus. Patient 2 was a 42-year-old woman bearing a single female fetus. These two blood samples (2 mL each) containing anticoagulant were stored at room temperature and analyzed within 12 hours after sampling. In patient 1, CD45-CD71+ cells were enriched from 0.3% in the analyzed cells to 93% in the enriched cells (310-fold). The yield of CD45-CD71+ cells was 2.5 x 106 cells from 2 mL of maternal blood (Figure 1). In patient 2, CD45-CD71+ cells were also enriched from 0.3% to 93%. The yield was 3.7 x 106 cells from 2 mL of maternal blood. To confirm the potential usefulness of our method, we analyzed the origin of the enriched CD45-CD71+ cells using a real-time polymerase chain reaction (PCR) system.a) Genomic DNA was extracted from frozen enriched CD45- CD71+ cells using the DNeasy® Blood & Tissue Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. The extracted DNA (20 ng) was used in the following reaction. b) We used three pairs of primers for the real-time PCR: TFRC-F; 5'-ACGGAATATGAAGATCTCAGCAAGG-3'; TFRC-R; 5'-GCGCAGATCACTAACGAGATGGA-3'; HPRT1-F; 5'-GTTGGCTGAAATAGTTGAACAGCTT-3'
Authors and Affiliations
Masato Kantake
Keywords
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- EP ID EP591033
- DOI 10.26717/BJSTR.2018.08.001648
- Views 151
- Downloads 0
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