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.     

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.     

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.     

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.     

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.     

Background Levels of Salivary-α-amylase Plus Foreign DNA in Cases of Oral Intercourse: a Female Perspective

Saliva plus DNA from a suspect is commonly encountered in sexual assault cases on bodily swabs. However, without background knowledge, the weight of this evidence is unknown. It may indicate the presence of saliva resulting from cunnilingus, or it may represent indirect transfer. In this study, females who refrained from cunnilingus donated 43 items of underwear and 19 vaginal swabs. The samples were subjected to Phadebas^®, RSID^™-Saliva and mRNA profiling and were subsequently DNA-profiled to determine the prevalence of background saliva in the female population. The results report that 15.8% of females who refrained from cunnilingus were positive for saliva and a further 10.5% also had DNA from unknown source(s). These findings of the rate of indirect transfer were evaluated with the Bayesian approach, and it was found that the evidence of saliva plus a high foreign DNA source adds moderately strong support to the allegation of cunnilingus.     

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.     

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.     

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.     

The relevance of false positive acid phosphatase reactions indicative of the presence of seminal fluid from oral and vaginal samples

The Body Fluid Forum of the Association of Forensic Science Providers recognised concerns raised by forensic practitioners regarding the lack of data to inform on the incidence of significant AP (Acid Phosphatase) reactions from vaginal and oral samples, i.e. those which might be misinterpreted as indicating the presence of semen. This is particularly relevant in the light of appeal court rulings regarding the need for data to support evaluations. This paper presents collaborative data on the nature and incidence of AP reactions from vaginal and oral swabs provided by donors. The results demonstrate that caution is required with mid to strong purple AP reactions from direct testing of oral swabs and with mid purple reactions from vaginal swabs. The use of a Bayesian approach to assist with the provision of opinions regarding the presence of seminal fluid is highlighted.     

Results of forensic medicine victim/perpetrator studies following sex offenses

39.1% of all clinical examinations performed at the institute for legal medicine in Hannover during a period of 9 years were carried out after sexual assault (229 out of 585 cases between 1979-1987). In 74.4% of all women extragenital injuries and in 26.2% of the cases genital lesions could be observed. Vaginal swabs showed sperm at a rate of 44%; spermatozoa could be observed at a maximum delay between assault and examination of 36 hours. Three male victims of sexual assaults and 37 male defendants were examined. On male victim presented superficial lesions of the anus. In 27 penis swabs there could be found spermatozoa (4x), vaginal epithelia (2x) and red blood cells (1x; after intercourse during menstruation).     

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.     

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.     

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.     

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.     

A case study on forensic polymer analysis by DIOS-MS: the suspect who gave us the SLIP

New technology was used to identify traces of a commercial barrier/spermicide in evidence from a case of a man accused of rape of a minor. Examination of vaginal swabs performed by another laboratory had been negative for seminal fluid or other sources of DNA from the suspect and we were asked to examine the remaining swabs for any traces that might have originated from the commercial product. Encare consists of vaginal inserts having a suppository-like shape. They contain the spermicide, nonoxynol-9, in a matrix consisting of approximately two parts polyethylene glycol (PEG) 1000 to one part PEG 1450, plus minor inorganic components added to produce foaming. Portions of the cotton from vaginal swabs from the victim and penile swabs from the suspect were extracted with methanol and subsequently examined by desorption ionization on silicon time-of-flight mass spectrometry (DIOS TOF MS). Low levels of PEG in the same mass range as Encare were found on two separate vaginal swabs from the victim and one penile swab from the suspect. Subsequent to these findings, the suspect (through his attorneys) provided us with a sample of SLIP Plus, a commercial sexual lubricant that also contains nonoxynol-9. Traces of PEG in the same mass range as Encare were found in this sample, while no PEG was found in a sealed sample of SLIP Plus provided by the manufacturer. At trial the suspect's attorneys stipulated that their client had added some Encare to the SLIP Plus sample he had provided.     

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.     

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.     

Improved detection of semen by use of direct acid phosphatase testing

Acid phosphatase (AP) reagent (Fast Black) is used as a presumptive test for the presence of seminal fluid on exhibits submitted in allegations of sexual assault. Research was carried out to determine whether the direct application of AP reagent to exhibits is a viable alternative to the traditional indirect (blot) testing method used routinely in the laboratory. The relative sensitivity of the indirect and direct testing methods was investigated as was the effect of AP reagent on histological staining of spermatozoa, the incidence of false positives from vaginal material and saliva, and the effect of AP reagent on subsequent DNA testing. Also included are the results of specificity studies from validations of the direct AP testing method. The results of this research show that, provided the incidence of false positives is borne in mind, direct AP testing can be especially useful when screening exhibits which are difficult to indirectly (blot) AP test or when it is problematic to relocate an AP positive stain. Direct application of AP reagent can also be beneficial for locating dilute semen stains. Three case examples are given which illustrate the use of direct AP testing in laboratory casework.     

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.