Adult Sexual Assault Patients' Experience of the Physical Examination Component of the Medical Forensic Examination: A Scoping Review to Identify Gaps in the Research Literature.

We undertook a scoping review of published research literature that reported on adult sexual assault patients' experience of the physical examination component of the medical forensic examination (MFE). Eligible papers were those reporting data about the physical examination component of the MFE from the adult patient's perspective, published in the period January 2000 to March 2022 in peer reviewed journals and reports containing original research data published from a tertiary institution. Twelve papers were identified. The review identified a gap in the literature regarding the patient's experience of the physical examination component of the MFE. The existing literature is limited but suggests that some patients find the examination empowering and reassuring, restoring a sense of bodily control; however, for others it is an intrusive, violating experience that they endure. A more patient-centered sexual assault service appears to have a therapeutic value in itself, but more research is warranted as existing research is very limited and often from a proxy. In particular, more research on patients' self-reported experience of the MFE, including specific aspects of the examination and the experience of male and gender nonconforming victim-survivors, is needed. A better understanding, from the point of view of the patient, of the benefits of attending, may encourage victim-survivors to seek a health response and, possibly, report to police. It may also be time to assess the impacts of certain aspects of the forensic examination, rethink standard evidence collection processes, and consider enabling more acceptable options for victim-survivors including self-collection of intimate samples.

Differential Extraction with Purification via Organic/Microcon® and Promega DNA IQ™ Methods.

The differential extraction method allows for the separation of sperm cell DNA from non-sperm cell DNA by incorporating two separate lysis steps. This is crucial in forensic casework, as sexual assault samples frequently deal with a mixture of seminal fluid and other body fluids. After performing a differential lysis, DNA extraction can be completed through a variety of methods. In addition to the differential lysis, two methods will be described in this chapter for DNA purification: Organic (Phenol)/Microcon® purification and purification with the Promega DNA IQ™ System.

Improved resolution of mixed STR profiles using a fully automated differential cell lysis/DNA extraction method.

Sexual assault evidence often contains sperm cells, which are typically separated from nonsperm cells using manual differential lysis procedures. The goal of this study was to evaluate the automated QIAGEN QIAcube for this purpose and to compare it to manual QIAGEN and manual organic differential methods using DNA yields and STR profile data for assessment. DNA yields were determined by qPCR, followed by multiplex STR amplification, CE analysis, and mixture interpretation. The automated method was capable of effective cell separation, producing DNA yields sufficient for STR amplification. Further, sperm fraction human:male DNA ratios from the QIAcube samples were consistently closer to the desired 1:1 and STR profiles were less likely to result in mixtures, with 6-8× fewer female alleles detected (median 1.5 alleles). Ultimately, using the QIAcube for automated differential processing of semen-containing mixtures reduces the need for downstream mixture interpretation and improves STR profile quality with substantially less hands-on time.

Data driven optimization of sexual assault case processing.

In recent years, several forensic laboratories have noted an increase in the number of sexual assault cases submitted for testing, often leading to longer turnaround times. In that context, forensic laboratories may be interested in reviewing their procedures to increase productivity. Here, we present two different strategies that were put in place in our laboratory. First, we changed the way sexual assault evidence kits (SAEK) are processed by implementing an optimized workflow that prioritizes the internal samples (vaginal, anal, and oral). This new procedure allowed for a drastic decrease in turnaround time, while maintaining a similar investigative power. Secondly, we used data from casework to target cases and samples that were likely to yield biological material from the perpetrator, in an attempt to avoid dedicating time and effort to cases for which there is a very low probability of obtaining foreign DNA evidence. Among other things, we looked at the likelihood of obtaining DNA from the perpetrator when the complainant reported the use of a condom, has showered after the assault or when the complainant has no memory of the assault. Results show that those circumstances do not dramatically decrease the probability of finding DNA from the perpetrator.

Automation of the standard DNA differential extraction on the Hamilton AutoLys STAR system: A proof-of-concept study.

For several decades, a common approach for processing sexual assault evidence has been to use the "standard" differential extraction to separate the evidence into a non-sperm-cell fraction and a sperm-cell fraction for further analysis. In this standard approach (P. Gill et al., Nature 318 (1985) 577-579), an initial mild chemical lysis step preferentially digests the mainly epithelial cells, which allows for removing this lysate as a non-sperm-cell fraction. The undigested sperm cells in the remaining fraction may then be purified by a series of wash and centrifugation steps, after which more robust lysis conditions are used to digest this sperm-cell fraction. Although this standard approach has been generally effective, it has been difficult to fully automate, due to the variety of different types of manipulations required for sample processing (e.g., incubation, shaking, substrate separation by centrifugation, and multiple liquid transfers for sperm-pellet centrifugation and washing steps). We describe here a fully automated standard differential extraction procedure that uses the Hamilton AutoLys STAR liquid handling assay-ready workstation, which is configured with on-deck components for sample incubation, shaking and centrifugation steps, and works with unique AutoLys-A® sample tubes for front-end sample processing. In this proof-of-concept procedure, up to 24 samples may be processed, "hands-free," in a single automated workflow. The automated procedure was tested by performing differential extractions on mock sexual assault swabs. For comparison, manual differential extractions were performed on identically prepared swabs in a side-by-side manner. DNA quantification and STR typing results showed that similar levels of separation efficiency were achieved for the sperm-cell fractions using both automated and manual procedures, although the results suggest that somewhat higher male DNA yields may be achieved for samples with extremely low semen levels (<∼0.1 µL) using the manual processing procedure. In addition to these mock samples, automated differential extractions were also performed on a set of authentic post-coital swabs (24, 48, 72, and 96 hours, post-coitus); primarily male STR profiles for the sperm-cell fractions were obtained for each sample in this set.

Automation of the Differential Digestion Process of Sexual Assault Evidence.

Sexual assault evidence samples require the use of a specific process known as a differential digestion to separate sperm from nonsperm cells prior to DNA extraction. An automated differential digestion process was developed using a selective degradation technique, which uses DNase I to digest the remaining nonsperm DNA in the sperm fraction. The use of DNase on pristine samples, as well as aged and degraded samples, was assessed to ensure that the quantity and quality of the sperm DNA were not compromised or adversely affected. Samples processed using the selective degradation technique yielded comparable DNA yield and DNA typing data to the conventional differential digestion process. The automated process utilized 96-well plates for high throughput and incorporated microscope slide preparations for confirmation of sperm. It reduced processing time by about sixfold and was paramount in the elimination of the Oakland Police Department Criminalistics Laboratory's sexual assault kit backlog.

Improving the efficacy of the standard DNA differential extraction method for sexual assault evidence.

The efficacy of a DNA differential extraction procedure relies on reducing the amount of non-sperm female DNA carryover into the sperm fraction, while providing a sufficient recovery of male DNA from the sperm cell component. A standard approach to this extraction is to use a mild initial lysis step to digest the female (epithelial cell) component in the mixture, followed by a series of centrifugation and wash steps to further purify the resulting sperm-pellet fraction. This sperm fraction is then digested in the presence of a chemical reducing agent in preparation for DNA extraction. This method has been employed with relatively few changes since its introduction in the mid-1980s, despite numerous attempts to develop new or improved procedures. In this report, we demonstrate that it is possible to improve the efficacy of the standard differential extraction by applying simple modifications that can reduce the amount of female DNA carryover into the sperm fraction, with no adverse effects on the recovery of male DNA. In one modification, the addition of a second mild lysis step at the beginning of the differential extraction procedure improved the average male-to-female DNA ratio in the sperm fraction by 3- to 6-fold. In another modification, a "tube transfer" step was added to move the re-suspended sperm pellet to a new tube for the second mild lysis and subsequent wash steps. With this modification, the average male-to-female DNA ratio in the sperm fraction was improved by 4- to 90-fold, relative to results obtained for the non-modified differential extraction method. These modifications may be accomplished using tools and reagents that are already present in most forensic DNA laboratories, so that implementation should be relatively low-cost and practical.

Evaluating the efficacy of DNA differential extraction methods for sexual assault evidence.

Analysis of sexual assault evidence, often a mixture of spermatozoa and victim epithelial cells, represents a significant portion of a forensic DNA laboratory's case load. Successful genotyping of sperm DNA from these mixed cell samples, particularly with low amounts of sperm, depends on maximizing sperm DNA recovery and minimizing non-sperm DNA carryover. For evaluating the efficacy of the differential extraction, we present a method which uses a Separation Potential Ratio (SPRED) to consider both sperm DNA recovery and non-sperm DNA removal as variables for determining separation efficiency. In addition, we describe how the ratio of male-to-female DNA in the sperm fraction may be estimated by using the SPRED of the differential extraction method in conjunction with the estimated ratio of male-to-female DNA initially present on the mixed swab. This approach may be useful for evaluating or modifying differential extraction methods, as we demonstrate by comparing experimental results obtained from the traditional differential extraction and the Erase Sperm Isolation Kit (PTC©) procedures.

Enhanced DNA Profiling of the Semen Donor in Late Reported Sexual Assaults: Use of Y-Chromosome-Targeted Pre-amplification and Next Generation Y-STR Amplification Systems.

In some cases of sexual assault the victim may not report the assault for several days after the incident due to various factors. The ability to obtain an autosomal STR profile of the semen donor from a living victim rapidly diminishes as the post-coital interval is extended due to the presence of only a small amount of male DNA amidst an overwhelming amount of female DNA. Previously, we have utilized various technological tools to overcome the limitations of male DNA profiling in extended interval post-coital samples including the use of Y-chromosome STR profiling, cervical sample, and post-PCR purification permitting the recovery of Y-STR profiles of the male DNA from samples collected 5-6 days after intercourse. Despite this success, the reproductive biology literature reports the presence of spermatozoa in the human cervix up to 7-10 days post-coitus. Therefore, novel and improved methods for recovery of male profiles in extended interval post-coital samples were required. Here, we describe enhanced strategies, including Y-chromosome-targeted pre-amplification and next generation Y-STR amplification kits, that have resulted in the ability to obtain probative male profiles from samples collected 6-9 days after intercourse.

SPERM HY-LITER™ for the identification of spermatozoa from sexual assault evidence.

Accurate microscopic identification of human spermatozoa is important in sexual assault cases. We have compared the results of examinations with (1) a fluorescent microscopy method, SPERM HY-LITER™, and (2) Baecchi's method for identification of human spermatozoa. In 35 artificial, forensic type samples, spermatozoa were identified in 45.7% with SPERM HY-LITER™ in Copenhagen, in 54.3% in the laboratory of the manufacturer of SPERM HY-LITER™, and 40.0% of the samples with Baecchi's staining method. When differences occurred between the two methods, it was significantly more often that SPERM HY-LITER™ detected spermatozoa when Baecchi's method did not (ts=6.567, df=1, P=0.048). This trend was also seen in selected compromised or degraded samples and in selected adjudicative samples. The reactions with spermatozoa from dog, horse, pig and bull were negative with SPERM HY-LITER™, whereas Baecchi's method was non-selective. Data from forensic casework samples in Copenhagen from two years (2008 and 2009) are presented. The samples from 2008 were investigated using Baecchi's method, while those from 2009 were investigated using SPERM HY-LITER™. The frequencies of positive results were similar between the two methods for the two years (27.9% and 32.1% respectively). Analysis of acid phosphatase (ACP) activity for the positive results obtained for these two years does not support the use of a negative ACP result as a prescreen for microscopic analysis for spermatozoa.

A comparison of the effectiveness of swabbing and flossing as a means of recovering spermatozoa from the oral cavity.

This study examined whether flossing the teeth is a more effective collection method in recovering spermatozoa than conventional swabbing techniques. It was hypothesized that inclusion of flossing as a collection method would extend the recovery of spermatozoa to longer postcoital intervals (PCIs). Eighteen individuals provided 174 oral cavity samples. Successful recovery of spermatozoa was assessed with respect to the collection method and reported activity in the oral cavity during the PCI. Samples were subjected to a differential extraction procedure prior to microscopic evaluation of the extracted pellet. The results indicate that swabbing is more effective than flossing when the PCI falls within 1.5-12 h. However, spermatozoa were recovered from seven floss samples where the corresponding swabs gave negative results. When combining the results from the two collection methods, the percentage of subjects from whom spermatozoa are recovered increases for each PCI beyond the 0-h interval.

Hospital wet mount examination for the presence of sperm in sexual assault cases is of questionable value.

Many protocols for the examination of sexual assault victims include the preparation of vaginal wet mount slides to determine whether sperm are present and if so, whether the sperm are motile. We have reviewed findings in 501 case reports to compare the efficiency of sperm detection on wet mounts to subsequent crime laboratory results of sperm searches on vaginal swabs. Sperm were detected on wet mounts in only 41% of cases in which sperm were detected in the crime laboratory. Motile sperm were observed in only 12% of cases reporting a 0-9 h postcoital interval; in three cases, motile sperm were seen at 15 h and beyond, indicating that motile sperm are not reliable evidence of a short postcoital interval. These findings demonstrate that wet mount examinations are of little value in guiding subsequent analyses in the crime laboratory or in corroborating other investigative aspects of the case.

A Y-short tandem repeat specific DNA enhancement strategy to aid the analysis of late reported (≥ 6 days) sexual assault cases.

The ability to obtain an autosomal short tandem repeat (STR) profile of the semen donor from the reproductive tract of a living victim rapidly diminishes as the post-coital interval increases. This is of concern where victims of sexual assault provide vaginal samples several days after the incident. In order to overcome the technological impediments inherent in autosomal DNA typing with extended interval samples, we previously employed the use of Y chromosome STR profiling which, by targeting only male DNA, can eliminate masking of the male profile (by the victim's alleles) or critical polymerase chain reaction reagent titration (due to excessive female DNA). Thus employing Y-STR profiling and additional enhancement strategies, we reported the ability to recover Y-STR profiles from samples collected 5 to 6 days after intercourse. However, the reproductive biology literature indicates that spermatozoa are found in the human cervix up to 7 to 10 days post coitus. Thus, even with improved extraction and profiling techniques, we failed to routinely recover profiles from samples collected ≥ 6 days after intercourse. The aim of the present work was to develop additional strategies to permit the recovery of male donor DNA profiles from ≥ 6 post-coital samples. Using nested polymerase chain reaction and DNA concentration procedures that together maximize the recovery and targeting of male DNA, we demonstrate the ability to obtain semen donor Y-STR profiles in extended interval post-coital samples collected 6 to 9 days after intercourse. This approaches the recognized time limits of sperm residence in the cervico-vaginal canal as described in the clinical literature.