Touch or trace evidence consists of epidermal cells deposited by contact with items such as handled objects, touched surfaces, or worn clothes. This type of evidence has surpassed most other sample types submitted to forensic labs and typically consists of low quantities of DNA and multiple contributors. In this study epithelial skin cells, i.e., “touch/trace evidence,” were used as they are estimated to constitute approximately half of the casework evidence items submitted for DNA analysis. For the optimization of antibody staining, male and female skin epithelial samples from donors were incubated and hybridized with antibodies of various concentrations of Alexa 488-conjugated anti-testosterone antibody (7.00E-4 µg/µL), FITC-conjugated anti-DHT antibody (4.10E-4 µg/µL) Alexa 647-conjugated anti-estradiol antibody (2.00E-4 µg/µL), and Alexa 647-conjugated anti-testosterone (5.00E-4 µg/µL) separately at varying volumes (1.25, 2.5, 5, and 10 µL). They were also hybridized with combined Alexa 488-conjugated anti-testosterone and FITC-conjugated anti-DHT antibody (1.11E-3 µg/µL) at varying volumes (2.5 and 5 µL). Antibody binding efficiency was assessed by analyzing stained single-source male, female, and control epithelial skin cells through flow cytometry to determine if the staining to the specific target was significant when compared to the unstained control. The objective was to maximize differential binding between contributors, and to increase fluorescent signal versus noise for antibody binding. Once a staining condition was established, male and female samples were collected from different individuals and stained to determine if the staining conditions were consistent with different individuals. It was determined that not all individuals could be differentiated after staining. However, if an improved signal was observed as demonstrated by an increased median fluorescence and separation between male and female samples visualized by overlaying the histograms, then the testing moved forward to FACS analysis. The results from this study demonstrate that certain contributor cell populations derived from the epidermis may be differentiated by targeting testosterone, dihydrotestosterone, and estradiol sex hormones within cell populations as demonstrated by flow cytometry. This study resulted in a protocol for differentially labeling contributors with anti-steroid antibodies when compared to the unstained controls. This study has potential application for casework samples to simplify complex trace mixtures prior to DNA profiling
