A Silicon-based Coral-like Nanostructured Microfluidics to Isolate Rare Cells in Human Circulation: Validation by SK-BR-3 Cancer Cell Line and Its Utility in Circulating Fetal Nucleated Red Blood Cells.

Gwo-Chin Ma,Ting-Yu Chang,Chung-Er Huang,Ming Chen,Wen-Hsiang Lin,Yun-Shiang Chang,Wan-Ju Wu,Mei-Hui Lee,Ya-Jun Yang,Jia-Yun Liu

doi:10.3390/mi10020132

Abstract

Circulating fetal cells (CFCs) in maternal blood are rare but have a strong potential to be the target for noninvasive prenatal diagnosis (NIPD). “Cell RevealTM system” is a silicon-based microfluidic platform capable to capture rare cell populations in human circulation. The platform is recently optimized to enhance the capture efficiency and system automation. In this study, spiking tests of SK-BR-3 breast cancer cells were used for the evaluation of capture efficiency. Then, peripheral bloods from 14 pregnant women whose fetuses have evidenced non-maternal genomic markers (e.g., de novo pathogenic copy number changes) were tested for the capture of circulating fetal nucleated red blood cells (fnRBCs). Captured cells were subjected to fluorescent in situ hybridization (FISH) on chip or recovered by an automated cell picker for molecular genetic analyses. The capture rate for the spiking tests is estimated as 88.1%. For the prenatal study, 2–71 fnRBCs were successfully captured from 2 mL of maternal blood in all pregnant women. The captured fnRBCs were verified to be from fetal origin. Our results demonstrated that the Cell RevealTM system has a high capture efficiency and can be used for fnRBC capture that is feasible for the genetic diagnosis of fetuses without invasive procedures.

Highlights

Since the first report of circulating fetal cells (CFCs) in maternal blood in 1959 [1], Circulating fetal cells (CFCs) have been expected as the potential target of noninvasive prenatal diagnosis (NIPD)
There are two directions to solve this hurdle: one is to explore more fetal specific antigens to undoubtedly identify fetal nucleated red blood cells (fnRBCs) [25,26,27] and the other is to optimize the efficiency of the cell capture platform used. We adopted the latter strategy to overcome this difficulty by demonstrating that at least a significant proportion of the captured nucleated red blood cells (nRBCs) are fetal origin, in contrast to most previous reports that showed a rarity of fnRBCs by their capturing methodologies [3,28,29]
Our results demonstrated that by capturing fnRBCs and using the subsequent well-established comprehensive genomic approaches, a true NIPD with resolutions similar to the invasive sampling is closer to reality

Summary

Introduction

Since the first report of circulating fetal cells (CFCs) in maternal blood in 1959 [1], CFCs have been expected as the potential target of noninvasive prenatal diagnosis (NIPD). One major criticism of the previous studies is that very few fetal specific antigens are available since nucleated red blood cells (nRBCs) in maternal circulation can be of both maternal and fetal origin [19,20]. We have verified our captured circulating nRBCs were of fetal origin using whole genome amplification (WGA) followed by subsequent short tandem repeat (STR) analyses, with a limited sample size (n = 5) [19]. We adopted the latter strategy to overcome this difficulty by demonstrating that at least a significant proportion of the captured nRBCs are fetal origin, in contrast to most previous reports that showed a rarity of fnRBCs (one in 30 mL maternal blood) by their capturing methodologies [3,28,29]. Our results demonstrated that by capturing fnRBCs and using the subsequent well-established comprehensive genomic approaches, a true NIPD with resolutions similar to the invasive sampling is closer to reality

Materials

Coral Chip Manufacture

Capture Efficiency Estimated by Cell Spiking Test

Circulating fnRBC Captured by Coral Chip

STR Analysis

Conclusions