Y-STR DNA amplification as biological evidence in sexually assaulted female victims with no cytological detection of spermatozoa

Identification of spermatozoa is the biological evidence most often sought in specimens from rape victims. Absence of spermatozoa usually terminates biological investigations, and the victim's testimony can be contested. We assessed the utility and reliability of PCR amplification using Y-chromosomal STR polymorphisms in specimens from female victims of sexual assault with negative cytology.One hundred and four swabs without spermatozoa detected by cytology were collected from 79 alleged sexually assaulted female victims and amplification of Y-STR and of amelogenin was performed.Overall, Y-chromosome was detected and evidenced sexual penetration in 28.8% of swabs. In the population of victims examined more than 48 h after the sexual assault, Y-STR were still evidenced in 30% of the cases. These results show that swabs should be taken from victims for Y-chromosome DNA typing even after long delays between sexual assault and medical examination.     

Persistence of spermatozoa and prostatic acid phosphatase in specimens from deceased individuals during varied postmortem intervals

The survival of spermatozoa and the persistence of prostatic acid phosphatase has been an area of interest for investigators of sexual assault. However, not much documentation exists concerning the examination of a deceased individual with regard to the postmortem interval and presence of such evidence. The authors reviewed cases referred to the medical examiner's office during a 10-year period. During this time, 199 cases were both autopsied and examined for sexual assault. In particular, these examinations included procurement of swabs for Papanicolaou staining of smears and for quantitation of prostatic acid phosphatase. Most of the victims were female, although a few were male. In the majority of cases, the swabs for smears and prostatic acid phosphatase were taken from oral, vaginal, and anorectal areas in females and oral and anorectal areas in males. The smears all were stained with the routine Papanicolaou stain, and intact spermatozoa and spermatozoan heads were sought. The prostatic acid phosphatase was analyzed by the microparticle enzyme immunoassay method and reported as ng/ml. A level of greater than 100 ng/ml was considered positive. The cases were analyzed with respect to postmortem interval; presence or absence of intact spermatozoa or spermatozoan heads; presence of an elevated prostatic acid phosphatase; body location of the specimen; the time of year; location of the victim; and physical injury (anogenital) of sexual assault. The authors hope that by examining the laboratory evidence of sexual assault, a correlation can be drawn between the presence or absence of such evidence and the aforementioned variables.     

Biological and DNA evidence in 1000 sexual assault cases

Using a Filemaker-based database (DNA Pro-FILES, Synchrone Infosystème Inc.), we have conducted a large-scale study on 1000 sexual assault (SA) cases where a standardized kit was submitted to our laboratory alone or with other types of exhibits. We looked at the likelihood of obtaining good quality DNA evidence, allegedly from the assailant, according to a number of parameters.The overall proportion of SA cases with DNA evidence is nearly 50%. A little more than 30% of SA kits provided DNA evidence while for 16% of cases DNA evidence could be obtained only from other exhibits.The likelihood of obtaining DNA evidence is approximately 50% in teenager and adult SA cases, but much lower for children 10 years old or younger (15%). In children cases, profiles were found mostly on clothing or skin swabs.The likelihood of obtaining DNA evidence from vaginal swabs remains good for up to 3 days after the assault (from 35% on the first day to 23% on the third day). A DNA profile was obtained from approximately 22% of anal/rectal swabs and 41% of skin swabs taken less than 1 day after the assault. Less than 10% of oral washes provided DNA evidence, all having been collected within 24 h of the assault.We found that in bodily samples, a negative result for acid phosphate (AP) is a poor predictor of the likelihood of obtaining good quality DNA evidence. Approximately 15% of vaginal swabs and 8% of anal swabs negative for AP nevertheless provided good quality DNA evidence.     

Evaluation of usefulness of further Y-STR analysis in sexual assault cases on PSA positive samples resulting in female autosomal STR profiling

Samples from male to female sexual assault cases that are positive for the presumptive prostate specific antigen (PSA) test often do not result in a male autosomal STR profile. Due to highly unequal proportions of female and male DNA in typical sexual assault samples, routine autosomal STR analysis often fails to detect the DNA of the assailant, even after differential extraction of the samples. Previous studies have already shown the value of Y-STR analysis in such cases [1]. In Belgium, forensic DNA laboratories are only allowed to perform Y-STR profiling on sexual assault samples by a specific requisition, after routine autosomal STR analysis has been performed. However, a request for additional Y-STR analysis is rather exceptional.In this study, we evaluated the usefulness of further Y-STR analysis. For 100 PSA positive rinses and swabs from male to female sexual assault cases resulting in female autosomal STR profiling, 7% resulted in a full or partial Y-STR profile useful for comparison, using the 23-loci Y-STR PowerPlex Y23 System (Promega). The success rate raised to 12.5% with a higher DNA input in the PCR mix. In conclusion, these results support the usefulness of performing Y-STRs analysis on the sperm DNA extracts to identify the alleged assailant in sexual assault cases.     

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.     

Background levels of body fluids and DNA on the shaft of the penis and associated underpants in the absence of sexual activity

This study examines the background of blood, saliva, semen and autosomal DNA on penile swabs and underpants from males in the absence of recent sexual activity. Based on the data collected by the AFSP Body Fluid Forum, the results of this study show that; there is a very low expectation of detecting blood on penile swabs and male underpants; a low expectation of detecting saliva on penile swabs and male underpants; and spermatozoa would be expected in less than a quarter of penile swabs and three quarters of male underpants. As none of the samples had detectable levels of DNA which were suitable for meaningful comparison that did not match the donor or their partner, the expectation of detecting a DNA profile from the cellular background on penile swabs or underpants from a male who has not been involved in recent sexual intercourse is very low. The results of this study are extremely informative when evaluating the significance of blood, saliva, semen and DNA detected on the penile swabs and underpants of males in cases of alleged sexual assault.     

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.     

The Persistence of Sperm and the Development of Time Since Intercourse (TSI) Guidelines in Sexual Assault Cases at Forensic Science Ireland,Dublin, Ireland

The persistence of sperm using confirmatory microscopic analysis, the persistence of sperm with tails, time since intercourse (TSI) analysis, and results from the acid phosphatase (AP) reaction from approximately 5581 swabs taken from circa 1450 sexual assault cases are presented. The observed proportions of sperm in the vagina and anus declines significantly after 48 h TSI, and sperm on oral swabs were observed up to 15 h TSI. The AP reaction as a predictor of sperm on intimate swabs is questioned. All AP reaction times gave a low true positive rate; 23% of sperm‐positive swabs gave a negative AP reaction time. We show the AP reaction is an unsafe and an unreliable predictor of sperm on intimate swabs. We propose that TSI not AP informs precase assessment and the evaluative approach for sexual assault cases. To help inform an evaluative approach, TSI guidelines are presented.     

An evaluation of gamma-glutamyl transpeptidase (GGT) and p30 determinations for the identification of semen on postcoital vaginal swabs

The following study entails the investigation of gamma-glutamyl transpeptidase (GGT) and p30 for the identification of seminal stains in sexual assault cases. A commercial kit was used to test for GGT activity, while p30 was demonstrated with a crossed electrophoresis technique. Specificity, sensitivity, and stability of both markers were studied. Postcoital swabs from lab staff were tested for GGT and p30. In addition, 144 postcoital swabs from case material were tested for p30, spermatozoa, and acid phosphatase. Results show p30 to be a useful semen marker particularly in cases of azoospermia. However, GGT was found to be unsuitable for forensic science casework.     

How many DNA analyses are performed on adult sexual assault victims in Milan (Italy): A ten-year review

The aim of this study is to evaluate the actual use of serological/DNA analyses in the investigations carried out on adult sexual violence victims in Italy during the years 2006–2015.The victims were assisted in the largest Italian rape center, in Milan (Soccorso Violenza Sessuale e Domestica – SVSeD - Service for Sexual and Domestic Violence).The total number of sexual violence victims examined during the years 2006–2015 (adults and minors) was 3521, in 1697 of cases, biological evidence had been collected, while the number of adult victims (>18 y.o.) examined was 2300, in 1211 of cases biological evidence had been collected.Biological evidence was collected from the victims’ bodies using two swabs in five anogenital areas (labia maiora, labia minora, perineum, perianal and anal/rectum regions) and two swabs in all other skin areas suggested by the victims as areas of possible contact (double swab technique). Clothes were also collected on a case by case basis for the search of biological stains. Despite the proper collection, handling and chain of custody for all the swabs/items collected, serological/DNA analyses were requested in 86 cases out of 1211 only (710%). This percentage dropped to 190% when considering adolescent victims (13–19 y.o.).The reason why Italian Magistrates make little use of the powerful tool of DNA analyses in sexual assault cases, still remains unclear. Legal and procedural aspects are therefore also discussed.     

Sperm elution: An improved two phase recovery method for sexual assault samples

This report describes the validation of a two phase cell recovery technique for the elution of two common cell types, epithelia and spermatozoa, from frequently examined items submitted as part of sexual assault casework. Furthermore, separation of cell types prior to microscopic examination of cell pellets improves the scientist's confidence in observing and scoring spermatozoa that may be present. During the validation, Orchid Cellmark's Sperm Elution© method consistently recovered a greater number of spermatozoa from simulated sexual assault items and swabs taken following consensual sexual intercourse compared to a water extraction technique. On average the Sperm Elution method recovered over twice the number of spermatozoa compared to the water method. The ability to separate the cell types present allows a rapid microscope slide search for spermatozoa and faster DNA extraction protocol in comparison to Cellmark's previous preferential 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.     

Diagnostic Value of PSA and AP Tests for the Detection of Spermatozoa in Postmortem Swabs from the Genital and Anal Region in Males

The aim of this study was to clarify whether positive results for prostate‐specific antigen (PSA) and acid phosphatase (AP) occur in postmortem swabs from the genito‐anal region in males (n = 80; 4 regions) and females (n = 20; 3 regions) and to calculate the positive predictive value (PPV) concerning the presence of spermatozoa. In male subjects, the highest incidence of positive test results was found in urethral swabs (PSA 76%, AP 71%) and the lowest frequencies appeared in perianal and rectal swabs (15–20%). Microscopic evaluation for spermatozoa was positive between 39% in urethral swabs and 1% in rectal swabs. PPV regarding positive identification of spermatozoa was 33.3% for PSA and 31.5% for AP. The combination of both tests yielded a PPV of 38.2%. In female cases, no spermatozoa were identified, and one case was PSA‐ and AP‐positive in perianal swabs. Our findings indicate that PSA and AP tests are of limited value for the postmortem detection of spermatozoa in male subjects.     

Cold Case Homicides: DNA Testing of Retained Autopsy Sexual Assault Smears

Archival medical examiner specimens may contain perpetrator DNA evidence useful in unsolved (“cold case”) homicides. The Office of the Chief Medical Examiner (OCME) histology slide archives were searched for sexual assault smears for all 376 female homicides from 1990 to 1999. Of these, the OCME had sexual assault smears on 84 of which 13 slides had sperm. Of these 13, six were still unsolved. DNA profiles were obtained on all six (5 from smears and one from swabs). Combined DNA Index System ( submission resulted in two matches (“hits”) for new suspects. In addition, three suspects were eliminated in two cases. Our review of archival sexual assault smears resulted in DNA profiles that were able to assist in the investigation of four cold case homicide investigations. It may be worthwhile for medical examiner offices to search their archival histology slides for sexual assault smears on previously unsolved cases particularly those prior to the mid‐1990s when DNA testing was less widely available.     

Analysis of non-suspect samples lacking visually identifiable sperm using a Y-STR 10-plex

Y-STRs are valuable in the investigation of sexual assaults in which autosomal STR genotype interpretation is challenging. To detect male DNA from compromised sexual assault evidence, 45 non-suspect samples were differentially extracted and analyzed with 10 Y-STRs. These samples were positive for the presence of human seminal fluid, but were negative for spermatozoa by microscopic examination. Y-STR data were obtained in approximately 86.2% of the epithelial or sperm fractions. On samples yielding incomplete profiles, results were obtained on an average of 5 loci per sample. The inability to obtain results may be due to insufficient amplifiable male DNA, PCR inhibition, or unfounded accusations of sexual assault. This study indicates that it is possible to obtain a male STR profile even in the absence of visually identifiable spermatozoa. Furthermore, Y-STR loci should become components of CODIS if they are to be used in solving non-suspect sexual assaults.     

Frequency of azoospermia

The frequency of azoospermia was found to be 1.9% in seminal stains on swabs examined in sexual assault cases.     

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.     

Epidemiological characteristics of male sexual assault in a criminological database

Sexual assault among males, compared with females, is understudied, and may also be significantly underreported. Past studies have relied primarily on population-based survey data to estimate the prevalence of sexual assault and associated health outcomes. However, survey-based studies rely primarily on self-reports of victimization and may not accurately estimate the true prevalence of male sexual assault victimization. In order to obtain a detailed assessment of sexual assault among males, criminological databases like the National Incident Based Reporting System (NIBRS) may provide an important and unique source of information. The objective of the current study was to use data from the 2001-2005 NIBRS to construct an epidemiological profile of sexual assault among males. Our results suggest that the incidence of sexual assault was higher among young males (less than 19 years of age), with approximately 90% of all cases being reported among members of this age group. Among males of all ages, forcible fondling and sodomy were the most prevalent forms of sexual assault. Results from additional analyses include age- and race-specific rates of male sexual assault, the prevalence and severity of injury, and time trends detailing incidence by time of the day and location of the incident. Our analyses show that sexual assault is experienced by males of all age groups. However, the rate of sexual assault is higher among younger males. Despite some limitations, results from this study suggest that NIBRS data may provide a important complement to survey data for understanding breadth and consequences of male sexual assault.