SpermFACS: Validation of a Highly Sensitive Sperm Cell Sorting Method for Sexual Assault Casework Analysis

The identification and isolation of sperm cells from biological samples remain a pivotal component of forensic analysis in sexual offense investigations. Although differential lysis (DL) is the standard method for sperm cell separation, it is time-consuming and frequently inadequate for generating informative male DNA profiles, particularly when the epithelial-to-sperm cell ratio is unfavorable. These limitations have driven interest in alternative separation techniques, such as fluorescence-activated cell sorting (FACS), which enables the detection and isolation of individual cells from mixed populations based on phenotypic markers. This study therefore aimed to establish and systematically validate a FACS-based methodology, referred to as spermFACS, for the separation of sperm cells from vaginal swab samples for routine implementation, following ISO/IEC 17025 guidelines. Notably, spermFACS enabled the recovery of informative male autosomal DNA profiles from mixtures with sperm/epithelial cell ratios as low as 1:7500 and from postcoital samples collected up to 120 h after intercourse. Supported by the calculation of likelihood ratios, a superior separation efficiency and purity of the sperm fraction was thus demonstrated in a comparative analysis with DL. These findings underscore the utility of spermFACS in delayed-report sexual assault cases and highlight its potential in the deconvolution of complex mixtures, such as those encountered in multiple offender rapes. By overcoming key limitations of traditional methods, the integration of spermFACS into forensic workflows may enhance the ability to identify offenders and contribute meaningfully to the resolution of sexual assault cases.