ORIGINAL ARTICLE Evaluation of the impact of density gradient centrifugation on fetal cell loss during enrichment from maternal peripheral blood Ahmed Emad and Régen Drouin* Division of Genetics, Department of Pediatrics, Faculty of Medicine and Health Sciences, Université de Sherbrooke, Sherbrooke, QC, Canada *Correspondence to: Régen Drouin. E-mail: Regen.Drouin@USherbrooke.ca ABSTRACT Objective Physical separation by density gradient centrifugation (DGC) is usually used as an initial step of multistep enrichment protocols for purification of fetal cells (FCs) from maternal blood. Many protocols were designed but no single approach was efficient enough to provide noninvasive prenatal diagnosis. Procedures and methods were difficult to compare because of the nonuniformity of protocols among different groups. Recovery of FCs is jeopardized by their loss during the process of enrichment. Any loss of FCs must be minimized because of the multiplicative effect of each step of the enrichment process. The main objective of this study was to evaluate FC loss caused by DGC. Methods Fetal cells were quantified in peripheral blood samples obtained from both euploid and aneuploid pregnancies before and after enrichment by buoyant DGC using Histopaque 1.119 g/mL. Results Density gradient centrifugation results in major loss of 60% to 80% of rare FCs, which may further complicate subsequent enrichment procedures. Eliminating aggressive manipulations can significantly minimize FC loss. Conclusion Data obtained raise questions about the appropriateness of the DGC step for the enrichment of rare FCs and argues for the use of the alternative nonaggressive version of the procedure presented here or prioritizing other methods of enrichments. © 2014 John Wiley & Sons, Ltd. Funding sources: This study was partly supported by an operating grant from the Canadian Institute for Health Research (CIHR) and MetaSystems through the University-Industry Program to R. D. and the Canada Research Chair Program to R. D. A. E. is a student scholar of the Public Health Ministry of Egypt. R. D. held a Canada Research Chair in Genetics, Mutagenesis, and Cancer. R. D. is member of the FRQS-funded Centre de Recherche Clinique Étienne-Le Bel. The Cell Imaging Facility of the Faculty of Medicine and Health Sciences, Université de Sherbrooke, was funded by grants from the Canadian Foundation for Innovation and from the Centre de Recherche Clinique Étienne-Le Bel. Conflicts of interest: None declared. INTRODUCTION A long sought goal of prenatal diagnosis has been the replacement of current invasive procedures of fetal sampling by noninvasive methods with virtually no risk to mother, fetus, and pregnancy. Data generated in different laboratories led to the conclusion that fetal materials (fetal cells and more recently, cell-free fetal DNA) are, undeniably, present in maternal blood. 1–5 Investigators envisaged that they could provide an alternative source of fetal genetic material for noninvasive prenatal diagnosis (NIPD). Although cell-free fetal DNA was successfully used with expensive technologies such as massive parallel and deep sequencing to diagnose fetal aneuploidies 6–9 and has largely eclipsed the use of purified fetal cells (FCs) in NIPD. FCs can potentially provide pure fetal genetic material, and their use can consequently be easier, affordable, and clinically acceptable approach for NIPD. Considering the extreme low frequency of FCs in maternal blood and relative abundance of contaminating maternal cells, 10–12 various purification and enrichment procedures have been employed. By far, the simplest was the buoyant density gradient centrifugation (DGC), which exploits the differing densities of nucleated FCs likely to be present and which separates and enriches a specific FC type from a heterogeneous cell population. 13 Because FCs, after DGC, were still dispersed between large amounts of maternal cells, further enrichment was required. Numerous methods of cell separation have been developed and various FC markers have been used in the isolation of FCs from maternal blood. Most published studies described the use of fluorescent-activated cell sorting, 14,15 magnetic-activated cell sorting, 16,17 selective maternal cell lyses, 18 charge flow separation, 17 and lectin base method. 19 Over the past two decades, investigators have devised and pursued different strategies that depend on combinations of two or more successive steps of enrichments to provide efficient isolation of FCs from maternal blood. Although a number of reports describe successful enrichment of FCs, a preferred protocol has not been established. 3,15,18,20,21 All of Prenatal Diagnosis 2014, 34, 878–885 © 2014 John Wiley & Sons, Ltd. DOI: 10.1002/pd.4387