A novel histological technique for distinguishing between epithelial cells in forensic casework

There are a number of forensic cases in which the identification of the epithelial cell type from which DNA originated would provide important probative evidence. This study aimed to develop a technique using histological staining of fixed cells to distinguish between skin, buccal and vaginal epithelium. First, 11 different stains were screened on formalin-fixed, wax-embedded cells from five women. Samples were analysed qualitatively by examining staining patterns (colour) and morphology (absence or presence of nuclei). Three of the staining methods – Dane's, Csaba's and Ayoub-Shklar – were successful in distinguishing skin epithelial cells from buccal and vaginal. Second, cells were smeared directly onto slides, fixed with one of five fixatives and stained with one of the three stains mentioned above. Methanol fixation, coupled with the Dane's staining method, specific to keratin, was the only technique that distinguished between all three cell types. Skin cells stained magenta, red and orange and lacked nuclei; buccal cells stained predominantly orange–pink with red nuclei; while vaginal cells stained bright orange with orange nuclei and a blue extracellular hue. This staining pattern in vaginal cells was consistent in samples collected from 50 women aged between 18 and 67. Identification of cell type from unlabelled micrographs by 10 trained observers showed a mean success rate of 95%. The results of this study demonstrate that histological staining may provide forensic scientists with a technique for distinguishing between skin, buccal and vaginal epithelial cells and thus would enable more conclusive analyses when investigating sexual assault cases.     

正常月经周期中阴道液蛋白SDS-PAGGE谱型及特异性

采用 SDS-PAGGE,研究了由21个个体提供的共25份(其中4人提供两个周期)月经周期的阴道液蛋白电泳谱型。结果发现阴道液电泳谱型变化与月经周期无关。通过与其它体液对比发现,阴道液电泳具有特异蛋白带,有器官特异性。     

Differentiation of vaginal cells from epidermal cells using morphological and autofluorescence properties: Implications for sexual assault casework involving digital penetration

This work explores morphological and autofluorescence differences between vaginal and epidermal cells detectable through Imaging Flow Cytometry (IFC), a non-destructive, high-throughput technique. These differences were used to build a predictive framework for classifying unknown cells as originating from vaginal or epidermal tissue, which was tested on hand swabbings with and without digital penetration. Many more cells possessing a vaginal signature (median posterior probability ≥0.90) were detected in digital penetration samples than control hand swabbings. Minimum interpretation thresholds were developed to minimize/eliminate false positives; these thresholds were also effective when screening licked hands, indicating the potential utility of this method for a variety of biological mixture types and depositional events relevant to forensic casework.     

Comparison of p30 and acid phosphatase levels in post-coital vaginal swabs from donor and casework studies

The application of p30 detection to casework analysis of seminal traces on vaginal swabs is reported and compared with the levels of acid phosphatase determined.A simple crossed-over immunoelectrophoresis system was used for batch identification of swab extracts using a commercially obtained anti-p30 serum.Positive p30 results were obtained in less than 20% of the casework swab extracts, but they provide conclusive proof of the presence of semen which is a substantial advantage over the quantitative determination of acid phosphatase.     

The use of bacteria for the identification of vaginal secretions

We have used the 16S–23S rRNA intergenic spacer region for identifying vaginal specific bacteria. Lactobacillus crispatus and Lactobacillus gasseri were detected in vaginal secretions but not in semen, blood or saliva. Our data indicated that both L. crispatus and L. gasseri were detected in vaginal secretions from women with different levels of expression of hormonal genes including pregnant, pre- and post-menopausal women, and a woman who has had a hysterectomy. Therefore, we have demonstrated that these Lactobacilli are promising new markers for the forensic identification of vaginal secretions. We have incorporated the Lactobacilli markers into a mRNA multiplex system to produce an 11-plex assay that can identify circulatory blood, menstrual blood, saliva, semen (in the presence and absence of spermatozoa) and vaginal secretions.     

Lactate dehydrogenase isozymes in vaginal swab extracts: A problem for the identification of menstrual blood

Extracts of vaginal swabs free of both blood and semen, collected from three donors throughout several menstrual cycles, have been examined by cellulose acetate membrane electrophoresis for the presence of lactate dehydrogenase (LDH) isozymes. LDH activity, predominantly in the form of LDH-5 was detected in some extracts. Mixtures of these particular extracts with peripheral blood gave LDH patterns similar to those observed for menstrual blood. The results indicate that LDH isozyme patterns cannot be used to differentiate between stains of menstrual blood and bloodstains that have become mixed or contaminated with vaginal secretions.     

Use of RGB values in the Periodic Acid-Schiff color test to determine the presence of vaginal fluid

This study demonstrates how RGB color values from microscopic smears stained with the Periodic Acid-Schiff reagent under standardized microscopy conditions can be used to indicate the presence of vaginal secretions. Based on data obtained in the study, a numeric threshold determined from the sum of separate values for red, blue and green was determined to differentiate vaginal-based samples with other body fluids. Using this threshold, 55 of 57 vaginal-based samples tested positive for the presence of vaginal secretion. Conversely, 27 of 29 smears prepared from other body fluids yielded negative results. However, when graphing RGB sum values against a calculated RGB integer no overlap in data was obtained between all vaginal-based samples and other body fluid samples, clearly differentiating them. One-way ANOVA testing with a 95% confidence interval indicated that vaginal samples from different age groups showed no difference in RGB sum values. Similarly, the location that vaginal swabs were collected (from the outside of a condom or a vaginal swab) also showed no statistical difference using one-way ANOVA at 95% confidence. Furthermore, refrigerated test swabs aged up to 15 months showed no demonstrable differences. Pair-wise t-testing using RGB sum values, however, did show significant differences between vaginal samples and all other body fluids tested. Finally, the method successfully differentiated between pre-and post-coital penile swabs and finger swabs taken before and after digital vaginal penetration in anecdotal comparisons using the method.     

Application of fragment analysis based on methylation status mobility difference to identify vaginal secretions

Identifying vaginal secretions attaching or adhering to a suspect’s belongings would be beneficial for reconstructing the events that have taken place during a sexual assault. The present study describes a novel approach to identify vaginal secretions by fragment analysis using capillary electrophoresis, based on the mobility differences of PCR amplicons from bisulfite-treated DNA depending on methylation status. We targeted three genome regions including each of three vaginal secretion-specific methylated CpG sites reported previously: cg25416153, cg09765089, and cg14991487. In all three genome regions, the amplicon peaks for methylated genomic DNA (gDNA) sequences were only detected in vaginal samples, whereas samples of other body fluids (blood, saliva, semen, and deposit on skin surface) only showed amplicon peaks for unmethylated gDNA sequences. In vaginal secretions, the methylation ratio of each of the three targeted regions between samples was variable, while the ratios at the three regions in each sample were similar. Furthermore, commercial vaginal epithelial cells were completely methylated at the three regions. Therefore, vaginal secretion-specific methylation may derive from vaginal epithelial cells present in the sample.In forensic cases with a limited amount of DNA, the reproducibility of a detected peak using the present method is not high due to degradation of DNA by bisulfite treatment and subsequent stochastic PCR bias. However, it was possible to detect peaks from methylated DNA sequences by performing PCR and capillary electrophoresis in triplicate after bisulfite treatment, even when bisulfite treatment was performed using 0.5 ng of gDNA from vaginal secretions. In addition, the level of methylation at each targeted region was found to be stable in vaginal secretions stored for 1 year at room temperature. Therefore, we conclude that detection of the visual peak from vaginal secretion-specific methylated DNA sequence is useful to prove the presence of vaginal secretions. This approach has the potential to analyze multiple marker regions simultaneously, and may provide a new multiplex assay to identify various body fluids.     

Evaluation of vaginal mRNA markers in women from different age groups: A GeFI collaborative study

Forensic mRNA profiling assays normally include a set of vaginal-specific markers. Although it is known that vagina undergoes characteristic age-related morphological and physiological changes over a lifetime, few studies have evaluated the efficacy of proposed forensic vaginal mRNA markers in women from different age groups.In this collaborative study involving ten GeFI (Italian working group of ISFG) laboratories, a 19-plex mRNA profiling assay including three vaginal-specific markers (CYP2B7P1, MUC4, MYOZ1) was tested in a collection of vaginal swabs obtained from female volunteer donors in their reproductive years (n = 84) and postmenopausa (n = 55).Differential expression of vaginal markers in the two age categories was assessed by means of: a) overall success rate of mRNA profiling (vaginal mucosa “observed” in the tested sample according to scoring protocol) b) average peak height ratios between vaginal-specific markers within mRNA profiling replicates.Other factors potentially influencing mRNA profiling outcomes, like time interval between vaginal swab collection and analysis, concurrence of menstrual cycle and recent sexual activity at the time of sampling were also investigated.