Immunohistochemical staining as a potential method for the identification of vaginal epithelial cells in forensic casework

There is currently no accurate method to identify vaginal epithelial cells uniquely. This study aimed to use a cell extraction procedure compatible with routine forensic sampling methods, and to investigate the expression of cytokeratin (CK), estrogen receptor-alpha (ERalpha), and phosphodiesterase 5 (PDE5) in order to distinguish between skin, buccal, vaginal, and external penile epithelial cells. Seminal fluid samples were also examined. Epithelial cell samples were fixed in formalin, embedded in agarose, and processed using histological methods. Antigen-antibody reactions were detected using the DAKO Envision+ detection system. CK was present in all cells from all five sources confirming the origin of cells as epithelial. Both ERalpha and PDE5 positively labeled vaginal, buccal, and skin epithelial cells. Although an antigen unique to vaginal epithelial cells was not identified, we have described a cell extraction procedure for use in the immunohistochemical detection of a wide range of antigens, an approach compatible with forensic diagnostics.     

Efficacy of Several Candidate Protein Biomarkers in the Differentiation of Vaginal from Buccal Epithelial Cells*

Abstract: Currently, there is no accurate method to differentiate vaginal epithelial cells from buccal epithelial cells in biological samples typically encountered in forensic casework. This study tested the expression of a selection of candidate proteins in buccal and vaginal epithelial cells. We investigated six candidate biomarkers, such as loricrin, vimentin, stratifin, cytokeratin 4, cytokeratin 13, small proline‐rich protein 2, and involucrin, using Western blot analysis on whole protein extracts and immunohistochemistry (IHC) on intact cells in an attempt to identify cell‐specific markers that would differentiate these cells by microscopy. Involucrin, loricrin, and stratifin showed differential expression during Western blot analysis and were carried through to IHC. Although proteins unique to vaginal epithelial cells and buccal epithelial cells were not identified from among the proteins tested, the increased expression levels of two proteins, loricrin and stratifin in vaginal cells, when compared to buccal cells, do provide encouraging results in the search for epithelial cell‐specific markers.     

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.     

Cytochemical detection of ABH antigens in human body fluids

The use of the peroxidase-anti-peroxidase (PAP) technique has been described previously for the detection of cellular antigens and in particular ABO antigens from tissue samples (Pedal and Hülle 1984; Pedal and Baedeker 1985; Pedal et al. 1985). In this survey, the PAP method has been employed to study the detection of ABO antigens in cells from body fluids of particular interest to forensic science, namely buccal cells and vaginal cells. Also tested, but in a limited number, were mixtures of body fluids and semen samples. No false reactions were obtained from buccal cells, all samples corresponding to the ABO blood type of the donor. Preliminary results from vaginal cells, vaginal/buccal cell mixtures, and semen were encouraging but must be treated with caution due to the limited number tested. Vaginal smears contaminated with semen showed varying degrees of nonspecificity.     

Laser microdissection separation of pure spermatozoa from epithelial cells for short tandem repeat analysis

Short tandem repeat (STR) analysis is a valuable tool in identifying the source of biological stains, particularly from the investigation of sexual assault crimes. Difficulties in analysis arise primarily in the interpretation of mixed genotypes when cell separation of the sexual assailant's sperm from the victim's cells is incomplete. The forensic community continues to seek improvements in cell separation methods from mixtures for DNA typing. The feasibility of applying laser microdissection (LMD) technology to precisely separate sexual assault cell mixtures by visual inspection coupled with laser dissection was assessed through three experiments. First, various histological stains were evaluated for use with LMD and DNA analysis. Second, different DNA isolation methods were evaluated on LMD-collected cells. Finally, STR analysis was performed on LMD-separated sperm cells from mixtures of semen and female buccal epithelial cells. The results indicated (a) hematoxylin/eosin staining performed best in its ability to differentiate sperm and epithelial cells while exhibiting the least negative effect on further downstream analysis; (b) both QIAamp and Lyse-N-Go methods were useful for recovery of DNA from LMD-collected sperm cells; and (c) LMD separation provided clear STR profiles of the male donor with the absence of any additional alleles from the female donor. This report describes an efficient, low-manipulation LMD method for the efficient separation of spermatozoa from two-donor sperm/epithelial cell mixtures.     

Swollen Lips After a Night of Partying—An Allergic Reaction to Ecstasy?

Ecstasy (MDMA) is a mood‐lifting drug with numerous somatic side effects, for example, dehydration or continuous chewing and biting. We describe the case of a young woman who underwent a forensic medical examination for suspected sexual assault. She claimed to have suffered from a memory lapse, and she had a painful swelling of her lips with a plaque‐like coating on her lips and buccal mucosa. The attending physician suspected that these findings might have been caused by strong sucking pressure on her lips within the context of a sexual assault. A toxicological examination of a blood specimen verified that she had been under the influence of an extremely high dose of ecstasy (1.456 mg/L MDMA and 0.0213 mg/L MDA). Pursuant to the forensic medical assessment, the described findings on her lips, and buccal mucosa were interpreted as an allergic and mechanical reaction (through continuous chewing and biting) to ecstasy.     

Use of a Y chromosome probe as an aid in the forensic proof of sexual assault

Currently, the most common procedures for the forensic identification of semen that may be present due to a sexual assault include the microscopic identification of spermatozoa, acid phosphatase activity, or the detection of PSA. However, not all cases of sexual assault result in the deposit of semen. Fluorescent In Situ Hybridization (FISH) has been found to be a very sensitive and specific method for detection of the Y chromosome from male cells. This study was undertaken to demonstrate the presence of epithelial cells of male origin in the postcoital vaginal tract using a commercially available probe. Results identified Y chromosome in intact epithelial cells on postcoital Days 1 through 4, and on Day 7. Additionally, Y chromosome positive epithelial cells were identified in vaginal swabs obtained following intercourse with no ejaculation. The method developed in this study demonstrates that FISH is a sensitive method for the identification of the presence of male epithelial cells in the postcoital vagina.     

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.     

单细胞检验技术用于混合上皮细胞的分离和检验

目的建立单细胞显微捕获联合低体积扩增技术,用于混合上皮细胞检材分离检验。方法取5名男性口腔上皮细胞拭子浸泡液30μL,分别滴加到5份含同一女性皮肤表皮细胞拭子上,制成5份混合上皮细胞样本为实验组,同时制备同样的5份样本为对照组。实验组样本采用显微捕获单个口腔上皮细胞,并使用低体积扩增技术进行扩增;对照组用M48纯化试剂盒提取DNA,Identifiler试剂盒复合扩增,扩增体系为10μL。所有产物均用ABI 3130遗传分析仪进行STR分型。结果 5份实验组样本均得到男性STR分型结果,5份对照组样本则均仅得到混合分型结果。将该方法应用于1例强奸杀人案例检验,取得了满意效果。结论单细胞显微捕获联合低体积扩增技术可用于混合上皮细胞样本的分离检验。     

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.     

The determination of buccal epithelial cells in forensic medical cytological research]

The morphology of isolated cells of multi-layer squamous non-horny epithelium (buccal, vaginal, urethral, rectal), stained with 0.05% nigrosine solution, was under study, and the sizes of these cells and their nuclei were measured. Specific markers were detected in the buccal epitheliocytes, that permit their reliable differentiation from other epitheliocytes studied by the size of the cells and nuclei, by a characteristic structure of the cytoplasm, etc. The findings may be used for the diagnosis of the origin of isolated cells of the buccal mucosa.     

Mitochondrial DNA sequence alterations observed between blood and buccal cells within the same individuals having betel quid (BQ)-chewing habit

There are hundreds of millions of betel quid (BQ) lovers widely spreading around the world. Compositions in BQ may generate reactive oxygen species, which would induce DNA damage. However, oral epithelial cells as well as blood have often been used as reference samples in comparison with the mitochondrial DNA (mtDNA) sequence of hairs. The main purpose of this study was to investigate the extent of mtDNA sequence variation in regular BQ-chewers' oral epithelial cells, and thus to evaluate the forensic availability of the buccal cells from BQ-chewers using the mtDNA markers. The hypervariable segments I and II in the D-loop control region of mtDNA between paired samples of blood and buccal scrape cells from 75 non-BQ-chewers (to be a control group), 60 BQ-chewers, and 67 oral cancerous patients were DNA sequenced and compared. Among the three groups, the alteration rates of 1.3% (1 out of 75), 10% (6 out of 60), and 61% (41 out of 67) were identified from the control, BQ-chewers, and the cancerous group, respectively. In the cancerous group, as expected, high rate of DNA alteration between blood and buccal samples was found. In the BQ-chewers, one and five individuals had the length and point alterations, respectively. Interestingly, most of point alteration sites, e.g., mtDNA positions 153, 16189, 16093 identified from BQ-chewers, were also observed in previous literatures. As for the control subjects, one case with point alteration, and none with length alteration, was identified. For all the three groups, not only the oral cells but also the normal blood samples exhibited high frequency (>55%) of length heteroplasmy at poly-(C)n track. Statistical analyses revealed that significance was observed between the severity of mtDNA alteration in BQ-chewers' oral epithelial cells and the history of BQ-chewing (p = 0.02), with a tendency of positive association. Based on the guidelines by Carracedo et al., we suggest that the interpretation of mtDNA variations between criminal evidences and the oral epithelial cells (as a reference or known sample) from BQ-chewers should be performed with particular caution using the PCR-based mtDNA sequencing. Our findings would be valuable in mtDNA analysis of hair evidence, especially for those countries where the habit of BQ-chewing is popular.     

Evaluation of Skin Surface as an Alternative Source of Reference DNA Samples: A Pilot Study

An acceptable area for collecting DNA reference sample is a part of the forensic DNA analysis development. The aim of this study was to evaluate skin surface cells (SSC) as an alternate source of reference DNA sample. From each volunteer (n = 10), six samples from skin surface areas (forearm and fingertips) and two traditional samples (blood and buccal cells) were collected. Genomic DNA was extracted and quantified then genotyped using standard techniques. The highest DNA concentration of SSC samples was collected using the tape/forearm method of collection (2.1 ng/μL). Cotton swabs moistened with ethanol yielded higher quantities of DNA than swabs moistened with salicylic acid, and it gave the highest percentage of full STR profiles (97%). This study supports the use of SSC as a noninvasive sampling technique and as a extremely useful source of DNA reference samples among certain cultures where the use of buccal swabs can be considered socially unacceptable.     

Separation of sperm and epithelial cells based on fluorescence-activated cell sorting

The detection and separation of spermatozoa is crucial in the forensic investigation of alleged sexual assault cases. Differential lysis and microscopy-based techniques are conventional methods for the isolation of spermatozoa, though are time-consuming and frequently fail with samples containing an unfavourable sperm/epithelial cell ratio. Successful separation by means of fluorescence-activated cell sorting (FACS) has previously been reported, yet little efforts have been dedicated towards the further improvement or routine implementation of this technique. With this ongoing research, a methodology is being developed to sort sperm from epithelial cells by combining the Sperm Hy-Liter™ staining kit and FACS. Sorted sperm cells are then subjected to a direct lysis and low volume PCR (LV-PCR) protocol. Preliminary results demonstrate the successful separation of both cell types at a sperm/epithelial cell ratio of up to 1:500. The direct lysis and LV-PCR protocol allows to produce full haploid profiles from single sperm cells and mostly full diploid profiles from 10 spermatozoa. These data suggest that the proposed methods are potentially viable for forensic casework, yet additional testing is required for validation purposes.     

Perineal‐Vaginal Injuries in Children: Accident or Abuse?

Abstract: A large number of conditions have been mistaken for abuse. Differentiating accidental injuries from inflicted injuries is important in the management of injured children. In this work, the authors describe two cases of accidental perineal‐vaginal injury in children. In case 1, a 4‐year‐old girl suffered a vaginal tear caused by violent stretching during play; in case 2, a 3‐year‐old girl had minor lacerations of labia minora and majora. The intervention of a multi‐specialist team including a forensic pathologist and forensic psychiatrist was the key factor in being able to exclude abuse by third parties in the described cases.. This leads to the appropriate recommendations to be adopted in the forensic medicine setting.     

3种DNA提取法在污染严重混合斑分型中的应用比较

目的比较Chelex-100法、酚/氯仿法和二氧化硅膜法3种DNA提取法在污染严重混合斑分型中的应用效果。方法从日常案例中收集污染严重的混合斑25份,差异消化法分离精子后同时用Chelex-100法、酚/氯仿法和二氧化硅膜技术3种方法提取DNA,采用PCR-STR技术对D19S253、FGA和CSF1PO 3个基因座进行分型,Gel-Pro软件处理电泳图谱,SPSS软件分析比较不同方法之间的差异。结果采用Chelex-100法提取DNA,25份检材分型结果均未成功;采用酚/氯仿法,25份检材中10份分型成功,3份检材FGA和CSF1PO基因座可分型,4份检材CSF1PO基因座可分型;采二氧化硅膜纯化法,25份检材均成功分型;酚/氯仿法和二氧化硅膜法两种方法比较,结果存在显著性差异(P<0.05)。结论二氧化硅膜纯化技术可以有效去除PCR抑制物,提取的DNA扩增效果明显优于Chelex-100法和酚/氯仿法,具有较高的应用价值。     

Differentiation of epithelial cell types by cell diameter

Genital swabs play an important role in cases of alleged sexual assault. The aim of our study was to see if epithelial cells from the vagina, glans penis, or mouth could be distinguished on the basis of size. Vaginal swabs were taken from 12 women in different phases of their menstrual cycles; penile swabs were taken from 5 men, and mouth swabs were taken from 6 men and 6 women. For each swab, a sample was smeared across a microscope slide and allowed to dry. The dried epithelial samples were then viewed without any further processing with a "SteReoLumar.V12" stereo microscope. The microscope slide surfaces were divided into grids and all single epithelial cells whose contours could be clearly distinguished were photographed. The maximum diameter for each photographed cell was digitally determined using the Axiovision software. In total, 995 vaginal epithelial cells, 211 penile epithelial cells, 329 male oral epithelial cells, and 525 female oral epithelial cells were measured. Menstrual cycle phase did not affect vaginal epithelial cell diameter. The mean vaginal epithelial cell diameter was 63.95 microm (min. = 28.08 microm, max. = 108.06 microm, s = 11.50 microm). The mean penile epithelial cell diameter was 39.24 microm (min. = 28.38 microm, max. = 51.02 microm, s = 4.84 microm). The diameter of oral epithelial cells hardly differed for both sexes, although the female cells were, on the whole, slightly larger. On the basis of these results, it is not possible to conclude that epithelial cells of less than a certain diameter found in the assessment of a vaginal swab must be of penile origin. It is also not possible to usefully distinguish vaginal epithelial cells from male or female oral epithelial cells on the basis of the diameter. However, finding epithelial cells with a diameter distinctly greater than 50 microm in a penile swab sample suggests the presence of vaginal or oral epithelial cells. Epithelial cells examined with the presented method can be used without restrictions for further examinations, such as single-cell DNA analysis after single-cell picking with the micromanipulator developed by Aura Optik (Jena).