Comparison between paramagnetic and CD71 magnetic activated cell sorting of fetal nucleated red blood cells from the maternal blood

Abstract

Background Fetal nucleated red blood cells (NRBC) from maternal circulation are rare events but can be enriched and used to evaluate the genetics of the fetus. We compared two simplified selection methods of the fetal cells from the maternal blood. Methods We isolated fetal cells from maternal blood through double‐density gradient centrifugation followed either by magnetic cell selection, based on the paramagnetic proprieties of the NRBC hemoglobin, converted to methemoglobin, or by a positive magnetic‐activated cell sorting (MACS) enrichment, using anti‐CD71 monoclonal antibodies. Finally, the cells were identified through fluorescence in situ hybridization (FISH) with specific chromosome X and Y probes. Results We processed 10 mL of peripheral blood samples from 27 pregnant women with singleton normal male fetuses. Hemoglobin‐based enrichment isolated significantly more NRBCs: 29.7 × 104 cells than anti‐CD71 MACS: 10.1 × 104 cells (P < .001). The FISH analysis found at least one XY cell in 81.5% and 61.5% of cases, respectively, for paramagnetic and anti‐CD71 selection. Also, the average number of XY cells identified through paramagnetic selection was 5.09 ± 2.5, significantly higher than those observed through CD71 sorting: 3.38 ± 1.7 cells (average ± SE) (P = .03). Conclusion The combination of density gradient centrifugation with paramagnetic selection has the advantage of simplicity and achieves a minimal manipulation and treatment of cells. It yields an increased number of NRBCs and FISH confirmed fetal cells, compared to the anti‐CD71 sorting.

Isolation and characterization of fetal nucleated red blood cells from maternal blood as a target for single cell sequencing‐based non‐invasive genetic testing

“…The frequencies of those cells per milliliter of blood ranged from 0 to 1.7 (median 0.3). This median value was consistent with the average number of male (XY) NRBC cells in maternal blood isolated by CD71 antibody selection and identified by FISH (3.38 +/− 1.7 cells from 10 ml of blood), recently reported by Nemescu et al 16 …”

Fetal Nucleated Red Blood Cells Preferable Than Cell-Free Fetal DNA for Early Determination of Gender Among Invasive and Non-Invasive Source Using Novel Four Genes Multiplex PCR

“… 25 , 26 Both studies reported full accuracy in detected Y chromosome in the enrichment fNRBC as like the present study using double enrichment process. 17 …”

A Computer-Aided Diagnosis System of Fetal Nucleated Red Blood Cell With Convolutional Neural Network

“…As a simple, fast, and economical bench-top technique with the capability to process large cell numbers, MACS is usually selected as a further enrichment method 46 . Transferrin receptor (CD71) is a common positive marker 47,48 , which was expressed on the entire erythroid lineage, activated lymphocytes, monocytes, trophoblasts, any cell incorporating iron and definitive fNRBCs in maternal and cord blood. In our study, DGC and MACS (anti-CD71) method were used to separate f{Choolani, 2003 #119}{Ganshirt, 1998 #69}NRBCs from maternal cord blood.…”