DNA Identification of Sperm Cells Collected and Sorted by Flow Cytometry

Nunno, Nunzio Di; Melato, Mauro; Vimercati, Antonella; Nunno, Cosimo Di; Costantinides, Fulvia; Vecchiotti, Carla; Frezzini, Cristina; Cina, Steven; Vimercati, Francesco

doi:10.1097/01.paf.0000070224.58005.ac

Cited by 17 publications

(7 citation statements)

References 22 publications

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“…As they used traits specific to sperm in their FACS analysis, their protocol could not be applied to separate other cell types of forensic significance. Di Nunno et al [16] introduced a modified protocol for use of FACS in a case study, although no further case reports are presented in the literature. No studies have been published relating to mixed samples not containing sperm.…”

Section: Introductionmentioning

confidence: 97%

FACS separation of non-compromised forensically relevant biological mixtures

Verdon

Mitchell

Chen

et al. 2015

Forensic Science International: Genetics

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Section: Introductionmentioning

confidence: 97%

FACS separation of non-compromised forensically relevant biological mixtures

Verdon

Mitchell

Chen

et al. 2015

Forensic Science International: Genetics

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“…FACS is a method commonly used in biomedical research for cell sorting with fluorescent probes. In recent studies, flow cytometry has also been successfully applied to identify and separate sperm cells from vaginal epithelial cell mixtures in forensic science922. However, only scarce data has been published applying this approach to mixed samples from two or more donors with the same gender111223.…”

Section: Discussionmentioning

confidence: 99%

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

Xie

Chen

et al. 2016

Sci Rep

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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.

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“…This separation results in two distinct fractions, one containing the spermatozoa originating from the alleged offender (the sperm fraction), and the other, the non-spermatozoa biological material originating mainly from the victim (the non-sperm fraction). Several approaches, for the separation of spermatozoa from other cell types, have been developed over the years to process sexual assault samples (differential lysis [21][22][23][24], sieve-based filtration [25,26], laser microdissection [27][28][29][30][31][32], micro-fluidic devices [33,34], flow cytometry [35][36][37], acoustic trapping device [38], capillary zone electrophoresis [39], DEP-array sorting [40][41][42][43], antibody-based separation [44][45][46][47]). They can result in the autosomal STR profiles of the alleged offenders.…”

Section: Introductionmentioning

confidence: 99%

Comparison between MACSprep™ forensic sperm microbead kit and Erase Sperm Isolation kit for the enrichment of sperm fractions recovered from sexual assault samples

2022

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Sexual assault samples often contain mixtures of cells coming from at least two donors. Ideally, one would need to separate the cells into two cellular fractions: one consisting of the alleged aggressor’s spermatozoa (the sperm fraction) and the other containing the victim’s epithelial cells (the non-sperm fraction). This separation increases the probability of obtaining the alleged offender’s autosomal DNA profile. However, spermatozoa are often collected along with an excess of biological material originating from the victim, and with unfavorable male:female biological material ratios, the absence of separation could result in the PCR amplification of the victim’s DNA profile only. Several approaches are available to enrich/purify the spermatozoa present on sexual assault samples. In this paper, we compare a new method, the MACSprep™ Forensic Sperm MicroBead Kit (MACSprep, based on microbeads conjugated with antibodies bound to spermatozoa and their retention within a magnetic column) with the Erase Sperm Isolation Kit (Erase, a standard differential lysis separation procedure combined with a specific removal of free DNA) routinely used in our lab. The performance of both kits was tested using sets of vaginal and buccal swabs loaded with different dilutions of sperm, or azoospermic semen, representing a total of 120 independent samples. For the samples containing undiluted sperm, an average recovery of 58% was observed for the MACSprep’s sperm fractions and 43% for Erase’s. Significantly better recovery of azoospermic semen was observed in MACSprep’s non-sperm fractions (~ 85%) compared to Erase (~ 28%). Erase performed significantly better than MACSprep in terms of recovery for diluted sperm samples (1:10 to 1:800 sperm dilutions) in the presence of vaginal cells, while the purities of the achieved sperm fractions were in favor of MACSprep for the highest sperm dilutions tested. Similar trends were observed with buccal swabs loaded with 1:200 sperm dilutions. Increased sperm dilutions on vaginal swabs resulted in higher variability in the male material recovered, whatever the separation method used. Both methods were easy to perform and resulted in male DNA extracts ready to use in less than 2 h. Both kits showed their specificities in terms of recovery efficiency and purity of the sperm fractions. Ideally, additional experiments should be performed in different laboratories, using workflow and chemistries different than ours, to better define the peculiarities observed with MACSprep for high dilutions. Improving the recovery of MACSprep for diluted samples, in addition to its better purity observed in the experiments performed, could make it a method of choice for laboratory workflow, despite MACSprep’s current price per sample being about twice the price of Erase’s.

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DNA Identification of Sperm Cells Collected and Sorted by Flow Cytometry

Cited by 17 publications

References 22 publications

FACS separation of non-compromised forensically relevant biological mixtures

FACS separation of non-compromised forensically relevant biological mixtures

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

Comparison between MACSprep™ forensic sperm microbead kit and Erase Sperm Isolation kit for the enrichment of sperm fractions recovered from sexual assault samples

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