The comparison of enrichment effects of different screening schemes for fetal nucleated red blood cells from maternal blood by magnetic-activated cell sorting

Abstract:

Objective To isolate fetal nucleated red blood cells（FNRBC）from maternal umbilical cord blood through density gradient centrifugation（DGC）and magnetic-activated cell sorting（MACS） by four different screening schemes，and to evaluate the screening effect of different methods.Methods Ten samples of umbilical cord blood（12 mL/case）were collected. Each sample was divided into 4 groups：Group 1：DGC； Group 2：DGC+MACS negative screening（CD45^-）；Group 3：DGC+MACS positive screening（CD71⁺）；Group 4：DGC+MACS negative/positive screening（CD45^-/CD71⁺）. After screening，the number of cells was counted，and the t-test was carried out to evaluate the enrichment efficiency of each method. K-B staining was used to identify FNRBC count，and then the number and the proportion of FNRBC were calculated.Results ①Compared with the other three groups，DGC method had the largest total cell number（0.95×10⁷/mL），the largest FNRBC number（39.87/mL） and the lowest proportion of FNRBC（0.004‰），with statistically significant differences（P=0.000）；②DGC+MACS negative / positive screening（CD45^-/CD71⁺） method got the least total cells number（0.81×10⁶/mL）and the least FNRBC number（7.47/mL），with statistically significant differences（P=0.000）；③In the DGC+MACS single antibody screening method，the total number of cells（1.99×10⁶ /mL）obtained by DGC+MACS positive screening（CD71⁺） was lower than that by DGC+MACS negative screening（CD45^-）（2.56×10⁶/mL）. The difference was statistically significant（P=0.000）. However，there was no significant difference in the number of FNRBC between the two methods（P=0.910）；④There was no significant difference in the proportion of FNRBC between DGC+MACS negative screening（CD45^-），DGC+MACS positive screening（CD71⁺） and DGC+MACS negative/positive screening（CD45^-/CD71⁺）（P=0.030，0.050，0.210）.Conclusion The number of FNRBC obtained by DGC is the largest. DGC+MACS negative/positive screening（CD45^-/CD71⁺）has the strongest ability to remove mixed cells. The effect of enriching FNRBC by DGC+MACS positive screening（CD71⁺） is the best without reducing the number of target cells.