A microfluidics approach for the isolation of nucleated red blood cells (NRBCs) from the peripheral blood of pregnant women - PubMed (original) (raw)

A microfluidics approach for the isolation of nucleated red blood cells (NRBCs) from the peripheral blood of pregnant women

R Huang et al. Prenat Diagn. 2008 Oct.

Abstract

Objective: Nucleated red blood cells (NRBCs) have been identified in maternal circulation and potentially provide a resource for the monitoring and diagnosis of maternal, fetal, and neonatal health and disease. Past strategies used to isolate and enrich for NRBCs are limited to complex approaches that result in low recovery and less than optimal cell purity. Here we report the development of a high-throughput and highly efficient microfluidic device for isolating rare NRBCs from maternal blood.

Material and methods: NRBCs were isolated from the peripheral blood of 58 pregnant women using a microfluidic process that consists of a microfluidic chip for size-based cell separation and a magnetic device for hemoglobin-based cell isolation.

Results: The microfluidic-magnetic combination removes nontarget red blood cells and white blood cells at a very high efficiency (approximately 99.99%). The device successfully identified NRBCs from the peripheral blood of 58/58 pre-termination samples with a mean of 37.44 NRBC/mL (range 0.37-274.36 NRBC/mL). These results were compared with those from previous studies.

Conclusion: The microfluidic device results in an approximate 10- to 20-fold enrichment of NRBCs over methods described previously. The reliability of isolation and the purity of the NRBC product have the potential to enable the subsequent application of molecular diagnostic assays.

Copyright (c) 2008 John Wiley & Sons, Ltd.

PubMed Disclaimer

Figures

Figure 1

Figure 1

Isolation of NRBCs from whole blood using a microfluidic device. (a) Schematic of flow logic of the microfluidic system. (b) The CSM (left) separates cells based on size through the microfluidic chip into nucleus+ and nucleus− fractions. The blood sample is continually mixed on a rocker, and pumped through the chip using a pneumatic-pressure regulated pump. The HE module (right) collects hemoglobin-containing cells (NRBCs) on a magnetic column that are eluted off the column when the run is complete. (c) (1) A photograph of the microfluidic device etched in silicon, (2) an example of whole blood running through the device, and (3) a schematic of one of 24 sets of buffer inlets and paired array sets on the microfluidic device showing the flow stream of NRBCs and WBCs, deflected by microposts (circles), into the buffer stream. RBCs are small and run into the waste outlets at the bottom of each chip. Colored lines with arrows represent the flow direction of RBCs (red lines), NRBCs (blue lines), and WBCs (green lines)

Figure 2

Figure 2

Engineering optimization of system performance. (a) Cell separation performance of the microfluidic chip shows WBC and RBC retention for three devices of differing gap size. (b) Flow rate dependence of RBC retention during magnetic hemoglobin separation. (c) Retention of WBCs in the NRBC product after 1, 2, and 3 cycles of magnetic hemoglobin separation

Figure 3

Figure 3. Cell types identified after microfluidic cell separation using Wright/Giemsa stain

Figure 4

Figure 4

Confirmation of NRBCs by differential staining with Wright/Giemsa followed by benzidine stain. All cells labeled ‘1’ are Wright/Giemsa, and photos labeled ‘2’ are Wright/Giemsa/Benzidine. Cells with a golden brown cytoplasm are hemoglobin-containing cells (NRBC and RBC), and nonhemoglobin-containing cells (WBC and platelets) have a blue cytoplasm. Cells ‘A’ and ‘B’ are representative NRBCs, cell ‘C’ is a polymorphonuclear neutrophil, and cell ‘D’ is an apoptotic WBC

Similar articles

Cited by

References

    1. Babochkina T, Mergenthaler S, Lapaire O, et al. Evaluation of a soybean lectin-based method for the enrichment of erythroblasts. J Histochem Cytochem. 2005;53:329–330. - PubMed
    1. Bianchi DW, Flint AF, Pizzimenti MF, Knoll JH, Latt SA. Isolation of fetal DNA from nucleated erythrocytes in maternal blood. Proc Natl Acad Sci U S A. 1990;87:3279–3283. - PMC - PubMed
    1. Bianchi DW, Simpson JL, Jackson LG, et al. Fetal gender and aneuploidy detection using fetal cells in maternal blood: analysis of NIFTY I data. Prenat Diagn. 2002;22:609–615. - PubMed
    1. Bianchi DW, Williams JM, Sullivan LM, Hanson FW, Klinger KW, Shuber AP. PCR quantitation of fetal cells in maternal blood in normal and aneuploid pregnancies. Am J Hum Genet. 1997;61:822–829. - PMC - PubMed
    1. Busch J, Huber P, Pfluger E, Miltenyi S, Holtz J, Radbruch A. Enrichment of fetal cells from maternal blood by high gradient magnetic sorting (double MACS) for PCR-based genetic analysis. Prenat Diagn. 1994;14:1129–1140. - PubMed

MeSH terms

LinkOut - more resources