Electrospray and MALDI mass spectrometry in the identification of spermicides in criminal investigations.

Electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry have been used to examine evidence in a sexual assault investigation. Because condoms are being used increasingly by sexual assailants and some condom brands include the spermicide nonoxynol-9 (nonylphenoxy polyethoxyethanol) in the lubricant formulation, the recovery, and identification of nonoxynol-9 from evidence items may assist in proving corpus delicti. A method was developed for the recovery of nonoxynol-9 from internal vaginal swabs and for its identification by reverse phase liquid chromatography/electrospray ionization mass spectrometry (LC ESI-MS), nanoelectrospray ionization (nanoESI) mass spectrometry, and high resolution MALDI Fourier transform mass spectrometry (MALDI-FTMS). The method was tested on extracts from precoitus, immediate postcoitus, and four-hours postcoitus vaginal swabs provided by a volunteer whose partner does not normally use condoms, but for this trial used a condom having a water-soluble gel-type lubricant that includes 5% nonoxynol-9 in its formulation. Subsequently, LC ESI-MS was used to identify traces of nonoxynol-9 from the internal vaginal swab of a victim of a sexual assault.     

Information from penile swabs in sexual assault cases

A survey has been made to assess the evidential value of tests carried out on 660 casework penile swabs. Most were from suspects in sexual assaults and were examined to see if the donor had had recent anal, oral or vaginal intercourse. The swabs were tested for one or more of the following: blood, faeces, saliva, vaginal secretions, semen. Blood was seldom found, it was usually weak and insufficient for grouping. Faeces were only identified on a pair of swabs from a dead homosexual showing that proof of buggery by this means is rare. Amylase, suggestive of saliva and oral intercourse, was occasionally detected. Glycogen-rich epithelial cells were sometimes present indicating vaginal intercourse. Semen was frequently found but its presence may not result from a recent sexual act. An ABO group different from the donor was obtained from a fifth of the swabs typed. Grouping in other blood group systems was rarely attempted or successful. Penile swabs provided a means of detecting a victim's ABO blood group on a suspect when it would not have been possible to demonstrate the suspect's group on samples from the victim. They also had value in assaults involving more than one offender. The main limitation of penile swabs was the paucity of material on them and the sampling site affected the interpretation of the results.     

DYS STR analysis with epithelial cells in a rape case

We report here the application of Y-chromosomal DNA analysis in a rape case, which occurred in Stuttgart, Germany. Microscopic examination of the victim's vaginal swabs and her underwear showed no sperm cells. DNA was extracted from vaginal and epithelial cells and analysed with the autosomal systems SE33, THO1 (singleplex) and with the multiplex Profiler Plus (Applied Biosystems, Foster City, USA). The results of these autosomal STR analysis gave no hint at a mixed sample and failed to identify a male profile. DYS STR analysis with the systems DYS391, DYS392, DYS393, DYS19 and DYS389 I/II showed the same characteristic features as the suspect. We used this incomplete haplotype to search in the Y-STR Haplotype Reference Database via Internet. In a Caucasian population sample of 3589 minimal haplotypes we found 71 matches. The suspect confessed the crime and was finally condemned to 4 years imprisonment.     

DNA typing of forensic material with mixed genotypes using allele-specific enzymatic amplification (polymerase chain reaction).

Biological material in forensic casework frequently contains a mixture of genotypes, with a predominance of material from the victim and only trace amounts from the person committing the crime. Physical separation of the two genotypes or preferential lysis of different cell types may sometimes be possible. However, it is often difficult to achieve complete separation due to the lysis of cells or lack of material. We have developed an enzymatic amplification system for the HLA DQA1 locus, that will allow the presence of individual alleles in a sample with mixed genotypes to be determined, independent of their initial proportion in the sample. This system permits the identification of an allele representing less than 10(-4) of the background genotype. Use of polymerase chain reaction (PCR) with general primers allows only alleles representing more than about 1% to be detected, while the allele-specific amplification represents up to a 1000-fold increase in sensitivity. This method was applied to a rape case and a combined rape and murder case; in both cases the biological evidential materials contained a mixture of alleles from the victim and the rapist. Allele-specific PCR revealed the presence of alleles identical to those of the suspect using DNA from a vaginal swab taken after a rape incident, whereas by using general primers in the PCR only trace amounts of alleles other than those of the victim were found. Similarly, allele-specific amplification of DNA from vaginal swabs from the murder case revealed the presence of alleles identical to those of the suspect, while standard PCR only indicated the presence of genetic material from the victim.     

Combined Statistical Analyses of Forensic Evidence in Sexual Assault: A Case Report and Brief Review of the Literature

DNA analysis has been widely used in the forensic field in order to contribute to identifying the perpetrator of a crime. Forensic investigation in sexual assaults usually focuses on locating and identifying biological fluids, followed by DNA analysis. The identification of certain compounds present in condoms can be useful to reconstruct the occurred event, especially in cases of sexual assaults where the DNA analysis did not show the presence of a male profile and where RNA analysis did not show the presence of sperm markers. Herein we describe the case of a woman reporting to be victim of sexual assault, who was not able to provide accurate information concerning the dynamics of the event; she remembered only forced penile–vaginal penetration by a single perpetrator. We performed short tandem repeat (STR) analyses and mRNA typing for forensic genetics testing on vaginal and rectal swabs collected on the victim, and Fourier-transform infrared spectroscopy (FTIR) followed by chromatographic analyses for the detection of condom compounds on the same swabs. The STR analysis showed only the victim’s genetic profile, and RNA analysis showed only the presence of vaginal and skin markers. In this situation, the identification of condom compounds residues on vaginal swabs became important as it complemented other collected evidences allowing the Court to reconstruct the events. A proposal of likelihood ratio (LR) calculation for the assessment of the weight of evidence in this case is described.     

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).     

Identification and isolation of male cells using fluorescence in situ hybridisation and laser microdissection, for use in the investigation of sexual assault

In cases of sexual assault involving an azoospermic assailant, vaginal swabs taken from the victim may fail to provide an autosomal DNA profile with which to search a suspect database, as the signal from any male cells present would be masked by that from the overwhelming number of female cells collected on the swab. Here, we describe a method of visually identifying diploid male cells in such samples using fluorescence in situ hybridisation, and selectively harvesting them by means of laser microdissection. This combination of techniques was tested on 26 post-coital vaginal swabs taken at a range of times after intercourse; the collected cells were then subjected to a simple lysis procedure and DNA was amplified using the AmpFlSTR^® SGMPlus^® multiplex under low copy number conditions. Useful DNA profiles were generated from samples taken up to 24 h after intercourse.     

Is it possible to predict the origin of epithelial cells? – A comparison of secondary transfer of skin epithelial cells versus vaginal mucous membrane cells by direct contact

In alleged sexual assault and rape cases, the focus has often been to collect samples from the victim's body, for detection of body fluids or skin cells from the offender. But in many cases intimate body samples from the perpetrator(s) can also be informative. However, in cases where the female victim claims vaginal penetration, the defendant may display an alternative explanation to the DNA findings, i.e. that the victim’s skin cells has been secondarily transferred to his penis. We hypothesized that female DNA will be detected in a significantly greater amount on swabs from penis after intercourse than after secondary transfer by skin contact.Fourteen male-female couples were recruited to test the above hypothesis, by collecting penile swabs from 3 specified anatomical locations: Glans, shaft, and the coronal sulcus, after two different situations: Vaginal intercourse and secondary transfer of epithelial cells by skin contact. The results show that penile swabs following intercourse produce significantly higher DNA concentrations than after secondary transfer by skin contact. Our results, indicates which of the anatomical regions is best suited for sampling. The DNA profiling results show a preponderance of female profiles over male profiles following intercourse compared to secondary skin contact.Based on these data, it is possible to make a statistical model to distinguish between samples taken after intercourse and samples taken after secondary transfer by skin contact based on the amount of female DNA and mixture proportion (Mx) between female and male DNA in samples collected from penis swabs.     

Isolation and identification of female DNA on postcoital penile swabs

After sexual assault, cells originating from the assailant may be recovered from the victim. Through polymerase chain reaction (PCR)-based technology, positive scientific identification of the assailant may be made from these cells. Described is a prospective study describing a method for positively identifying cells from a female sex partner obtained from postcoital swabs of the penis of the male sex partner. Swabs were taken from the penis of a man at 1- to 24-hour intervals after coitus. DNA was isolated from each swab through standard organic extraction methods. The presence of female DNA was detected using the gender-specific amelogenin marker. Extracted DNA was amplified for eight different genetic loci using the Promega PowerPlex kit (Promega) and Amplitaq Gold (Perkin Elmer). Amplified samples were electrophoresed on precast sequencing gels (Hitachi) and were analyzed fluorescently using Hitachi's FMBIO 2 fluorescent scanner and software. Each sample obtained from a penile swab or condom was compared to male and female buccal controls. Female DNA was isolated from all postcoital penile swabs as determined by exclusive amplification of the X-chromosome specific 212 base pair amelogenin marker. In all cases, scientific identification of the female DNA from the swabs was determined by coamplification of eight STR loci (PowerPlex) and was compared to female and male control profiles. Cells shed from a female victim during sexual intercourse can be retrieved from the penis of a male offender after sexual intercourse during a 1- to 24-hour postcoital interval. DNA can be extracted from these cells and can be used to scientifically identify the female sexual participant through PCR-based technology. It is suggested that penile swabs be taken from alleged perpetrators of sexual assaults to associate them with a female victim.     

Prostaglandin E in vaginal smears--a possibility for sperm detection in azoospermia]

The identification of seminal traces is exceptionally difficult, if the semen of the assailant is azoospermic. The evident value of prostatic acid phosphatase (PAP) activity must be evaluated in such cases with caution. In a murder investigation of a 13 year old girl a positive PAP reaction was found in vaginal swabs and in her underpants. Spermatozoa could not be found. Using the gas-chromatographic method, described by Douse (1985) the presence of prostaglandin E could be demonstrated in the swabs as well as in the crotch of the underpants. The offender was found to be a man with azoospermia, who admitted intercourse but with the consent of the victim. The E prostaglandins are mainly synthesized in the vesiculae seminal and seen to be specific for semen. Swabs taken from mouth and rectum showed negative reactions for prostaglandins in this case. Prostaglandins could never be detected in vaginal swabs taken at least 7 days after intercourse. Conversely Douse could detect prostaglandins in swabs up to 58 hours after intercourse. Apparently the prostaglandin detection by Douse provides a suitable alternative besides to the quantitative and immunological PAP detection or the immunological detection of the protein p 30.     

The use of phosphate buffered saline for the recovery of cells and spermatozoa from swabs.

In the forensic science laboratory, the recovery of spermatozoa from vaginal swabs, or vaginal cells from penile swabs, can help determine if sexual intercourse may have taken place. There are several methods used to recover spermatozoa and cells from the swabs before visualisation on a microscope slide and most of these methods use water. Phosphate buffered saline (PBS) is a non-toxic solution used in many biological laboratories. Unlike water, PBS prevents cells rupturing or shrivelling up due to osmosis. This study demonstrates that PBS can be used for the extraction of spermatozoa and cells from swabs and that PBS does not affect subsequent DNA profiling.     

Efficiency of forensic mtDNA analysis. Case examples demonstrating the identification of traces

The paper presents results of forensic mitochondrial DNA analyses which were aimed at typing the traces caused by touching or abrasion of skin cells. Five cases of strangulation tool investigation are summarised. Two cases of homicide could be cleared up by identifying the mtDNA of both the victim and the suspect on cables which had obviously been used as strangulation tools. In eight of 10 cases, weapons could be reliably assigned to their users. The mtDNA of the users could be even detected on cartridges after firing. In one case, evidence of a suicide could be provided by means of mtDNA sequencing of the wiping traces on a suicide note.     

Examples for use of the STR systems in stain assessment with special reference to Y chromosome systems

The authors describe 3 cases where Y-chromosomal systems were used for typing the biological traces. In the first case, a murder, for the major amount of cell material found on a dish towel (and analysed two years after the crime) female persons were excluded for the system amelogenin and Y-chromosomal systems. A brother of the victim could not be excluded for autosomal STR-systems. Upon confrontation with the results of the DNA-analysis (among other things), this brother confessed the murder of his sister some days later. He was found guilty by the court. In the second case described, a rape of two girls, many traces were analysed parallely with Y-chromosomal and autosomal PCR-systems. The objects where male DNA matching the suspect were found (a paper tissue, a sweat shirt and the knickers of the girls), also showed small amounts of alleles matching with the suspect for autosomal systems, while the major part in these systems was from the girls. The suspect was sentenced to many years imprisonment. In the third case, a possible rape of a young woman, a stained microscope slide of a vaginal swab had to be examined. Microscopically a few sperm heads could be seen in a surplus of leucocytes. The male proportion could be analysed only in the Y-chromosomal systems, not in the autosomal ones. For the frequency calculation of the Y-chromosomal allele combination the haplotype data bank of the Institute for Legal Medicine of the Humboldt university in Berlin was indispensable.     

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.     

Experiences with single locus DNA probes in casework.

DNA profiling in this laboratory has been employed primarily in cases of sexual assault and the largest category of items examined has been internal vaginal swabs. 79% of these gave a profile which was different from that of the victim. Results have been obtained from swabs taken up to 70 h after intercourse. In cases where DNA results were obtained, one or more suspects were excluded in 29% of the cases.     

A rape case solved by mitochondrial DNA mixture analysis

In cases of stains that contain mixed DNA from different contributors, analyzing mitochondrial DNA (mtDNA) requires the use of cloning techniques. We developed an efficient cloning technique that was applied in a rape case. After a differential lysis-based DNA extraction from vaginal swabs, hypervariable region I and II (HVI, HVII) amplicons obtained from the male fraction were cloned. Although we mainly found the victim's haplotype, we were able to detect the suspect's haplotype in two clones for HVI and in one clone for HVII. As the midpiece of the flagellum, which contains mitochondria, can be lost during the differential lysis, we also investigated the female fraction by cloning to evaluate the proportion of victim/suspect mtDNA. Unfortunately, only clones presenting the victim's haplotype were found. This case highlights the need for an optimal differential lysis protocol to enrich the male fraction not only with nuclear but also mitochondrial DNA.     

ARREST FOR DOMESTIC AND OTHER ASSAULTS*

We use the National Crime Victimization Survey to examine whether a suspect's relationship to an assault victim affects whether the police make an arrest. The results indicate that in cases of minor assaults the police are less likely to make an arrest when the suspect is an intimate partner of the victim than when the suspect is an identifiable stranger. However, the police are not as lenient when the suspect is an intimate partner as they are when the suspect is someone else the victim knows. Intimate partner suspects avoid arrest in part because they are less likely to commit their crimes in front of witnesses. In addition, victims who know the suspect in any way are reluctant to sign complaints, and this reluctance inhibits arrest. Men are less likely than women to sign complaints, particularly when the suspect is a partner.     

DNA recovery from different evidences in 300 cases of sexual assault

Almost 60% of the DNA evidences analyzed in our laboratory correspond to sexual assault cases. With the aim to assess the efficiency of the DNA IQ System (Promega) in recovering the perpetrator DNA profile, the statistical analysis of results obtained in 300 casework was performed. In such cases, 850 evidence samples were processed. In 71% of the cases the perpetrator DNA profile was detected in at least one of the submitted casework samples, with a minimum of 13 STRs markers typed using the AmpFlSTR Identifiler PCR amplification kit (Applied Biosystem). When the suspect DNA profile was available, 67% matched with the evidence.With regard to the type of evidence, the best performance corresponded to panties, with more than 70% of success in recovering male profile, whereas the efficiency of vaginal swabs was almost 60%, with a higher incidence of victim/perpetrator mixed profiles.     

An unusual appearance of a common pollen type indicates the scene of the crime

Forensic palynology is a useful source of evidence in cases of violence committed in the open. A young girl was grabbed off the street, threatened and brutally raped. During the investigation the exact place of the rape became an issue. Growing around the scene identified by the victim were shrubs identified as Coprosma, a common New Zealand plant and one that produces abundant, easily wind-dispersed pollen. Abundant Coprosma pollen was found at the scene. The pollen were unusual in that the site was very damp, encouraging fungal growth, and fungal hyphae had penetrated the pores of many of the tricolporate pollen grains. Some grains had fungal spores inside. Coprosma pollen identical in preservational characteristics and morphology to those from the scene and containing fungal hyphae and spores were found in considerable numbers on the victim's clothes. This and rare Coprosma pollen grains and fungal remains recovered from vaginal swabs provided evidence that she had been at the scene where she claimed to have been raped. The diversity of pollen types recovered from the clothing in this case provides further evidence of the usefulness of clothing in picking up and retaining pollen from crime scenes and that obvious staining on clothes is not a pre-requisite for good pollen recovery. It also demonstrates the importance of collecting samples from different parts of the same garment in order to get a full picture of events since different parts of a garment can come into contact with different plants or different parts of the ground in a scuffle. It is also demonstrated that significant evidential material can be collected from the body, in this case from vaginal swabs from the victim. Forensic palynology should be considered in every case of violent assault, especially, but not exclusively, when having occurred in an open area subject to extensive pollen settlement.     

强奸致孕形成部分性葡萄胎的检验分析

目的探讨强奸致孕案葡萄胎组织的DNA检验和STR结果分析。方法用DNA Typer TM15试剂盒对犯罪嫌疑人、受害人血样和流产胚胎组织进行荧光复合STR基因座扩增检测。结果检测的一例流产胚胎组织DNA的STR峰谱表现出部分3条带,且3条带中有两个等位基因来源于犯罪嫌疑人,另外一个来源于受害人,推断为单卵双精子受精的部分性葡萄胎。结论应用STR分型技术可以推断葡萄胎的DNA来源和受精类型,为亲缘关系鉴定提供依据。