# Analysis of Antibody Hybridization and Autofluorescence in Touch Samples by Flow Cytometry: Implications for Front End Separation of Trace Mixture Evidence

## TL;DR

This study explores how touch samples can be analyzed using flow cytometry to separate cells from different contributors in biological mixtures.

## Contribution

The study introduces optical and biochemical features for differentiating cell populations in touch mixtures using flow cytometry.

## Key findings

- HLA surface antigens on touch-transferred cells are largely unreactive, limiting their use for differentiation.
- Red autofluorescence profiles show donor-specific variation, suggesting potential for contributor separation.
- FACS sorting based on autofluorescence successfully separated DNA profiles from a two-person touch mixture.

## Abstract

The goal of this study was to survey optical and biochemical variation in cell populations deposited onto a surface through touch or contact and identify specific features that may be used to differentially label and then sort cell populations from separate contributors in a trace biological mixture. Cell characterizations initially focused on two different protein systems, Human Leukocyte Antigen (HLA) complex and cytokeratin (CK) filaments. Hybridization experiments using pan and allele-specific HLA antibody probes showed that surface antigens on cells transferred from the palmar surface of volunteers are largely unreactive, suggesting that they cannot be used to differentiate cell populations in a touch mixture. Touch samples were also hybridized with the pan-CK probe AE1, which targets CK proteins 10, 14, 15, 16 and 19. Fluorescence levels of AE1 hybridized cells were observed to vary across donors, although these differences were not consistent across all sampling days. We then investigated variations in red autofluorescence profiles (650-670nm) as a potential signature for distinguishing contributor cell populations. Although distinct differences in red autofluorescence profiles were observed ‐‐ with one donor consistently exhibiting higher levels of fluorescence than others ‐‐ some variation was also observed in touch samples collected from the same individual on different days. While this suggests that contributor touch samples cannot be defined by a discrete level of autofluorescence, this attribute may still be a useful means of isolating contributors to some touch mixtures. To test whether these observed optical differences could potentially be used as the basis for a cell separation workflow, a controlled two person touch mixture was separated into two fractions via Fluorescence Activated Cell Sorting (FACS) using gating criteria based on intensity of 650-670nm emissions, and then subjected to DNA analysis. STR typing of the sorted fractions provided partial profiles that were consistent with separation of individual contributors from the mixture.