Fluorescence- and magnetic-activated cell sorting strategies to separate spermatozoa involving plural contributors from biological mixtures for human identification.

Yan Xu,Dan Wu,Lihua Gu,Huaigu Zhou,Yuan Ping,Wei Hu,Jianhui Xie,Jiang Zhong,Rui Li,Ronghua Chen,Xin Chen,Yu Cao,Ziqin Zhao,Qingwen Xu

doi:10.1038/srep36515

Abstract

No effective method has been developed to distinguish sperm cells originating from different men in multi-suspect sexual assault cases. Here we combined MACS and FACS to isolate single donor sperm cells from forensic mixture samples including female vaginal epithelial cells and sperm cells from multiple contributors. Sperms from vaginal swab were isolated by MACS using FITC-conjugated A kinase anchor protein 3 (AKAP3) antibody; target individual sperm cells involving two or three donors were separated by FACS using FITC-labeled blood group A/B antigen antibody. This procedure was further tested in two mock multi-suspect sexual assault samples and one practical casework sample. Our results showed that complete single donor STR profiles could be successfully obtained from sperm/epithelial cell and sperm mixtures from two contributors. For unbalanced sperm/epithelial cells and sperm cells mixtures, sensitivity results revealed that target cells could be detected at as low as 1:32 and 1:8 mixed ratios, respectively. Although highly relies on cell number and blood types or secretor status of the individuals, this procedure would still be useful tools for forensic DNA analysis of multi-suspect sexual assault cases by the combined use of FACS and MACS based on sperm-specific AKAP3 antigen and human blood type antigen.

Highlights

No effective method has been developed to distinguish sperm cells originating from different men in multi-suspect sexual assault cases
In order to solve the problem, the present study describes two novel cell-sorting strategies for forensic analysis of male sperm DNA from mixture samples, eg
To isolate male sperm cells from mixtures involving female epithelial cells, previous studies by Anslinger et al.[5] and Xue-Bo Li et al.[4] showed that magnetic beads coupled to an antibody against testicular angiotensin-converting enzyme or motile sperm domain-containing protein 3 (MOSPD3), can successfully separate sperm cells from cell mixtures

Summary

Introduction

No effective method has been developed to distinguish sperm cells originating from different men in multi-suspect sexual assault cases. We combined MACS and FACS to isolate single donor sperm cells from forensic mixture samples including female vaginal epithelial cells and sperm cells from multiple contributors. Sperms from vaginal swab were isolated by MACS using FITC-conjugated A kinase anchor protein 3 (AKAP3) antibody; target individual sperm cells involving two or three donors were separated by FACS using FITC-labeled blood group A/B antigen antibody. This procedure was further tested in two mock multi-suspect sexual assault samples and one practical casework sample. Previous data showed AKAP3 located primarily in the sperm head and flagella, which may function as a regulator of both motility- and head-associated functions activities such as capacitation and the acrosome reaction[8]

Methods

Results

Conclusion