A New Approach to the Investigation of Sexual Offenses—Cytoskeleton Analysis Reveals the Origin of Cells Found on Forensic Swabs*

Abstract: There are forensic inquiries in which an identification of epithelial cell types would provide important probative evidence. In cancer diagnosis, this information is yielded by histological examination of cytokeratin (Ck). Therefore, we tested 19 antibodies against different Cks (Ck1, Ck2e, Ck4, Ck5‐6, Ck7, Ck8, Ck9, CK10, Ck13, Ck14, Ck15, Ck16, Ck17, Ck18, Ck19, Ck20, Ck903, PanCkAE1_3, and CAM5‐2) on histological sections of epidermis, buccal mucosa, vaginal mucosa, penis, urogenital tract, and rectum and could identify two antigens unique to buccal‐cell and vaginal‐cell (Ck4) and skin epithelial‐cell (Ck10) cytokeratin. Subsequently, we developed an immunocytological technique for distinguishing swabbed skin and mucosal cells up to at least 1 year after sampling. By the detection of the Ck4 and Ck10 mRNAs in biopsy and laser capture microdissection collected samples via quantitative real‐time polymerase chain reaction, we were able to confirm our immunological findings. Hence, this study offers techniques to discriminate between skin and mucosal cells (buccal and vaginal) in forensic casework.     

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.     

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.     

MMP-11多克隆抗体的制备及其法医学意义

目的制备兔抗人基质金属蛋白酶-11(MMP-11)多克隆抗体,建立用MMP-11抗体检测月经血的可行方法,探讨其法医学意义。方法将用基因工程制备的人MMP-11融合蛋白免疫新西兰白兔,饱和硫酸铵法进行抗体纯化。运用蛋白印迹法检测月经血痕、外周血痕、阴道液斑、精液斑、唾液斑和尿液斑,盲测验证该方法的可靠性。结果高效价的兔抗人MMP-11多克隆抗体检测月经血MMP-11蛋白的阳性率为90.48%(93/105),而外周血痕、精液斑、唾液斑、尿液斑和阴道液斑均未检出MMP-11。结论用自制的抗MMP-11多克隆抗体所建立的蛋白印迹法检测月经血中的MMP-11特异性好,灵敏有效,可用于月经血及外周血的鉴别。     

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.     

Evaluation of the DNA stability of forensic markers used in betel-quid chewers' oral swab samples and oral cancerous specimens: implications for forensic application

Chewed betel-quid (BQ) residues are often considered vital biological evidence at crime scenes, since the human DNA extracted from the residues is actually from buccal epithelial cells and can be associated with suspects. BQ-chewing is also a risk factor for oral diseases and/or cancers. Archived medical oral-specimens can be used to identify specific individuals under adverse conditions, although STR markers are known to be unstable in various tumor tissues. This study evaluates the DNA stability of forensic marker systems in BQ-chewers' oral epithelial cells, and in archived clinical specimens of oral cancer patients. The genotypes of oral and paired peripheral blood samples in 200 subjects were compared, using the commercialized typing systems of HLA-DQA1, PM (including LDLR, GYPA, HBGG, D7S8, and GC loci), and AmpFlSTR markers (including 9 STR loci and the Amelogenin gene). The 100 healthy BQ-chewers had consistent oral swab and paired blood sample genotypes analyzed withboth DQA1/PM and STR marker systems. In the 100 oral cancer patients, one discordant result at D7S8 was found in the 600DQA1/PM-marker loci, and 25 allelic alterations with expansion or contraction were detected in the 900 STR loci. The findings herein suggest that when cancerous specimens were tested, the HLA-DQA1/PM system with point polymorphism appears more reliable than the STR system with length polymorphism. Our results also indicate that healthy BQ-chewers' oral cotton swabs containing buccal epithelial cells are useful for forensic purposes using the HLA-DQA1, PM, and STR marker systems.     

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).     

Potential Forensic Application of Receptor for Advanced Glycation End Products (RAGE) as a Novel Biomarker for Estimating Postmortem Interval

Studies which estimate the postmortem interval (PMI) based on diatoms or bacterial communities have been well‐conducted. However, predicting the PMI based on microorganisms has limited accuracy. The objective of this study was to discover specific biomarkers for estimating the PMI for drowning. The in vivo experimental model consisted of rats classified into nine groups (seven drowned groups from Day 1 to Day 7 and two control groups). Their lung tissues were analyzed for the receptor for advanced glycation end products (RAGE) mRNA expression determined by qRT‐PCR. RAGE protein levels were determined by Western blotting and immunohistochemical (IHC) staining. RAGE mRNA and protein levels gradually degraded over time. Immunostaining analysis revealed that RAGE in the Day 1 sample was significantly higher than that in the Day 6 or Day 7 samples. These results indicate that RAGE in drowned lungs could be a potential biomarker for the determination of the PMI after drowning.     

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.     

Evaluation of Tamm‐Horsfall Protein and Uroplakin III for Forensic Identification of Urine

Abstract: In this study, Tamm‐Horsfall protein (THP), a major component of urinary protein, and uroplakin III (UPIII), a transmembrane protein widely regarded as a urothelium‐specific marker, were evaluated for forensic identification of urine by ELISA and/or immunohistochemistry. THP was detected in urine, but not in plasma, saliva, semen, vaginal fluid, or sweat by the simple ELISA method developed in this study. In addition, most aged urine stains showed positive results. The urine specificity of THP was confirmed by gene expression analysis. Therefore, as reported previously, ELISA detection of THP can be used as a presumptive test for urine identification. UPIII was specific for immunohistochemical staining of cells in centrifuged precipitate of urine. However, ELISA and RT‐PCR for UPIII were not specific for urine. UPIII may be applicable for forensic urine identification by immunohistochemistry.     

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.     

A validation study of mRNA markers for skin cell identification

The objectives of this study were to develop and optimize a multiplex of three skin specific gene markers; loricrin (LOR), corneodesmosin (CDSN) and keratin 9 (KRT9) and 1 house-keeping marker, β-actin (ACTB) using an endpoint PCR assay to analyze expression data from a range of relevant samples. Marker specificity and suitability were evaluated for their inclusion in future forensic casework. The presence of the three skin mRNA markers was successfully confirmed from swabs of human skin obtained from 20 individuals at each of 6 different body sites (forehead, neck, arm, palm, leg and sole). Significant variation was observed in the relative expression of the three genes across the body sites, with some individuals consistently failing to express one or more of the targets. Inter-individual variation was also evident. Accordingly, these markers must be used with caution in the identification of skin in forensic samples.     

The modified method of two-step differential extraction of sperm and vaginal epithelial cell DNA from vaginal fluid mixed with semen

This investigation was undertaken as an efficient method for isolating sperm DNA from a mixed fluid sample which contains vaginal epithelial cells in a greater amount. The modified method of the two-step differential extraction procedure was found to be suitable for separating sperm DNA and vaginal epithelial cell DNA from the mixed stains. As the first step of digestion, vaginal epithelial cells in the mixed stains were lysed with Proteinase K and SDS, and sperm heads remaining in the lysed solution were collected by centrifugation. As the second step digestion, the sperm heads were lysed with the buffer containing Proteinase K, SDS and DTT as reducing agent. DNA fractions extracted from the two lysed solutions were enriched, one with sperm DNA and the other with vaginal epithelial cell DNA. MCT118(D1S80), ApoB VNTR and HLADQα types of sperm DNA were detected and were confirmed by matching with corresponding male blood DNA. In the case of vaginal secretion mixed with semen of two males, the mixture of MCT118 types of the two males was detected in sperm DNA fraction.     

Developmental validation of RSID™-Semen: a lateral flow immunochromatographic strip test for the forensic detection of human semen

Abstract: Tests for the identification of semen commonly involve the microscopic visualization of spermatozoa or assays for the presence of seminal markers such as acid phosphatase (AP) or prostate‐specific antigen (PSA). Here, we describe the rapid stain identification kit for the identification of semen (RSID?‐Semen), a lateral flow immunochromatographic strip test that uses two antihuman semenogelin monoclonal antibodies to detect the presence of semenogelin. The RSID?‐Semen strip is specific for human semen, detecting <2.5 nL of semen, and does not cross‐react with other human or nonhuman tissues tested. RSID?‐Semen is more sensitive with certain forensic evidence samples containing mixtures of vaginal secretions and semen than either of the commercially available PSA‐based forensic semen detection tests or tests that measure AP activity that were tested in parallel. The RSID?‐Semen kit also allows sampling a fraction of a questioned stain while retaining the majority of the sample for further processing through short tandem repeat analysis.     

Developmental validation of the SPERM HY-LITER™ kit for the identification of human spermatozoa in forensic samples

Abstract: With sexual assault evidence, the visualization of spermatozoa confirms that ejaculation has occurred. However, microscopic examination of spermatozoa is a laborious process and can sometimes result in sperm cells being overlooked. Here, we present the developmental validation of the SPERM HY‐LITER? kit, which contains a human sperm–specific mouse monoclonal antibody coupled to a fluorescent Alexa 488 dye. The kit was tested using samples of human semen, saliva, blood, and urine, various animal semen extracts, sexual lubricants, and a commercially available spermicidal film. Postcoital vaginal swabs, degraded semen samples, and samples prepared with sample fixation techniques that deviated from the kit‐provided protocol were also tested. In each case, the SPERM HY‐LITER? kit was demonstrated to bind only to human sperm cell heads. Limitations to this fluorescent staining procedure include nonspecific staining and increased background fluorescence with extreme heat fixation in some samples.     

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.