Isolation and genetic analysis of pure cells from forensic biological mixtures: The precision of a digital approach

Abstract

Latest genotyping technologies allow to achieve a reliable genetic profile for the offender identification even from extremely minute biological evidence. The ultimate challenge occurs when genetic profiles need to be retrieved from a mixture, which is composed of biological material from two or more individuals. In this case, DNA profiling will often result in a complex genetic profile, which is then subject matter for statistical analysis. In principle, when more individuals contribute to a mixture with different biological fluids, their single genetic profiles can be obtained by separating the distinct cell types (e.g. epithelial cells, blood cells, sperm), prior to genotyping. Different approaches have been investigated for this purpose, such as fluorescent-activated cell sorting (FACS) or laser capture microdissection (LCM), but currently none of these methods can guarantee the complete separation of different type of cells present in a mixture. In other fields of application, such as oncology, DEPArray™ technology, an image-based, microfluidic digital sorter, has been widely proven to enable the separation of pure cells, with single-cell precision. This study investigates the applicability of DEPArray™ technology to forensic samples analysis, focusing on the resolution of the forensic mixture problem. For the first time, we report here the development of an application-specific DEPArray™ workflow enabling the detection and recovery of pure homogeneous cell pools from simulated blood/saliva and semen/saliva mixtures, providing full genetic match with genetic profiles of corresponding donors. In addition, we assess the performance of standard forensic methods for DNA quantitation and genotyping on low-count, DEPArray™-isolated cells, showing that pure, almost complete profiles can be obtained from as few as ten haploid cells. Finally, we explore the applicability in real casework samples, demonstrating that the described approach provides complete separation of cells with outstanding precision. In all examined cases, DEPArray™ technology proves to be a groundbreaking technology for the resolution of forensic biological mixtures, through the precise isolation of pure cells for an incontrovertible attribution of the obtained genetic profiles.

DEPArray™ system: An automatic image‐based sorter for isolation of pure circulating tumor cells

“…In particular, whole genome amplification of single CTCs and WBCs was carried out to confirm the absence of WBC contamination by DNA finger printing . Besides, similar results have been published in other application domains, where direct DNA fingerprinting of individual and pools of cells collected by DEPArray™ from forensic evidence has shown achievement of 100% purity .…”

“…B). DEP cages are able to trap and move cells of different type and size ranging from small sperm cells to large epithelial cells . This electronic structure is integrated within an innovative microfluidic architecture that includes three micro‐chambers in fluidic connection (Fig .…”

Deconvolution of blood-blood mixtures using DEPArrayTM separated single cell STR profiling

“…For forensic applications, the DEPArray TM forensic sample prep kit (Menarini Silicon Biosystems), which enables the staining of epithelial cells, leucocytes and sperm cells, was specifically developed by Menarini Silicon Biosystems. The proof of principle was demonstrated by Fontana et al [8]. Moreover, Williamson et al used the DEPArray TM technology for the enhanced mixture deconvolution of sexual offence samples [21].…”

“…Therefore, special DNA extraction methods (e.g. single tube lysis and amplification) and modified amplification protocols (low template, LT-DNA protocols) were developed [8,13]. While improving the sensitivity through an enhancement of PCR, LT-DNA protocols often have an influence on the appearance of the profiles; for example, increased stutter peaks and imbalances of the two peaks of a heterozygote loci as well as increased drop-out rates were observed [20].…”

Most Common Medico-Legal Autopsy-Related Human and Nonhuman Biological Samples for DNA Analysis

“…Molecular autopsy methods such as lasercapture microdissection (LCM) [94][95][96][97], flow cytometry, fluorescence (FACS) or magnetic (MACS) activated cell sorting methodologies [98][99][100][101] or alternatives [102][103][104] are fields where ongoing research is ensuring the potential to enhance the generation of single source genetic profiles of desired target DNA from cellular mixtures obtained by the initial collection device. Recent advances may provide a successful method for the separation of cellular mixtures of small numbers of cells [105], but complete separation of different cells with same morphology and gender origin seems to be a challenge that is still to be adequately addressed [98,102].…”