Short Tandem Repeat (STR) analysis has become 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. This report describes the use of laser microdissection (LMD) for the separation of pure populations of spermatozoa from two-donor cell mixtures. In this study, cell separation was demonstrated by microscopic identification of histologically stained spermatozoa and female buccal cell mixtures, and STR analysis of DNA obtained from the separated sperm cells. Clear profiles of the male donor were obtained with the absence of any additional alleles from the female donor. Five histological stains were evaluated for use with LMD and DNA analysis: hematoxylin/eosin, nuclear fast red/picroindigocarmine, methyl green, Wright's stain, and acridine orange. Hematoxylin/eosin out-performed all other stains however nuclear fast red/picroindigocarmine could be used satisfactorily with STR analysis. In addition, three DNA isolation methods were evaluated for LMD collected cells: QIAamp (Qiagen), microLYSIS (Microzone Ltd.) and Lyse-N-Go (Pierce Chemical Co.). MicroLYSIS performed poorly, yielding low levels of PCR product. Lyse-N-Go extraction was effective for the recovery of DNA from LMD collected sperm cells while QIAamp isolation performed best for the recovery of DNA from LMD collected epithelial cells. LMD is shown to be an effective, low-manipulation separation method that enables the recovery of sperm while excluding epithelial cell DNA.

Over time, criminals have become more aware of the different types of trace evidence that are capable of being identified by forensic analysis. As a result, the frequency of actions taken to prevent the transmission of evidence, specifically biological fluids and subsequent DNA evidence, with the usage of condoms in the commission of sexual assaults, has increased. With the increased use of condoms, comes the increased awareness and probative nature of forensic analysis of the potentially unique chemical profiles residues may leave behind. This includes the identification of lubricant type and of any additives that may be present, such as spermicides, flavoring or topical anesthetics. The two predominate condom lubricants are polydimethylsiloxane (PDMS) and polyethylene glycol (PEG): PEG, a water-soluble lubricant, is soluble in polar solvents, such as methanol, while PDMS, a silicone-based lubricant, is reported to be soluble in non-polar solvents, such as hexane. A total of thirty condoms representing eight brands, each of a different type, were evaluated by Attenuated Total Reflectance - Fourier Transform Infrared Spectroscopy (ATR-FTIR). It has been reported that PDMS is the more prevalent condom lubricant compared to PEG; this trend was reflected in this small subset of products. Of the thirty condom lubricants analyzed, twenty-five were PDMS (approximately 83%), six PEG (approximately 7%), six glycerol (approximately 7%) and one “other” (approximately 3%). A direct extraction method was developed to isolate the lubricant from the condoms. Following the direct extraction of the condom lubricants from ten condoms of different brands and types containing PDMS, the extraction capabilities of three solvents: hexane, methanol and methylene chloride, in the presence of blood and saliva separately, were evaluated. Two different biological fluid/lubricant sample types were created: liquid suspensions of lubricant, biological fluid and solvent; and contrived casework samples consisting of a mixture of lubricant and biological fluid dried onto a cotton swab. Hexane was capable of isolating only the PDMS lubricant in the presence of biological fluids. In an effort to extract and identify the water-soluble lubricant PEG and any additional additives, two Trojan condoms marketed as containing the spermicide nonoxynol 9 (N9) and one Durex condom marketed as containing the topical anesthetic benzocaine were obtained. Methanol was used as the direct extraction solvent as much of the published literature has determined that additives, such as spermicides and topical anesthetics, are often found in combination with PEG, which must be extracted in a polar solvent. Although capable of extracting the lubricant PEG, PDMS, and the N9 from the condoms directly, the presence of biological fluids prevented the successful isolation of any condom lubricants or additives with the use of methanol. This extraction study established the solubility of PDMS in both methanol and hexane as well as the limited solubility of PEG in methanol. To identify a solvent capable of extracting both lubricant types as well as the spermicide additive N9 in the presence of biological fluids, the extraction capabilities of methylene chloride were assessed. In the literature, methylene chloride is often used to eliminate a two-step, or two-solvent, extraction for condom lubricants. The isolation of PDMS and N9 had mixed results when using methylene chloride as a solvent. PDMS and N9 were successfully isolated and identified in one of the Trojan brand spermicidal condoms, but not the other, most likely due to a difference in concentration of N9 in the two condoms. Only PEG could be isolated in the Durex condom marketed as containing benzocaine using methylene chloride. In the blood and saliva/lubricant contrived casework samples extracted with methylene chloride, the PDMS and PEG in the respective condoms were isolated but N9 was not. An evaluation of solvent extraction efficiency was made by comparing the ability of each solvent to isolate condom lubricant and additives in the presence of biological fluids. Methylene chloride was found to be the most effective solvent when compared to hexane and methanol for this purpose.

In the field of forensic biology, the term “time since intercourse (TSI)” is used to describe the approximate time elapsed between an alleged sexual assault and the collection of a Sexual Assault Evidence Collection Kit (SAECK) from a victim. The estimation of TSI, or Post Coital Interval (PCI), can be crucial information for particular cases in which the time between offense and the collection of a SAECK is in question. Oftentimes, forensic scientists must evaluate the significance of biological test results from evidence in SAECKs, but the variability in current literature complicates interpreting these results. Developing a reliable framework to estimate TSI based on a more extensive review of forensic casework would provide investigators with a fundamental tool for estimating a general timeline in which the offense occurred. This information may play an important role in supporting or refuting a narrative, or weighing the significance of the evidence at hand. This study aims to develop a dependable framework for estimating TSI in living victims based on casework received by Boston Police Department (BPD) Crime Laboratory, Boston, MA. Additionally, this study seeks to determine if any significance exists between the victim’s reported post coital activities and the collection of evidence, including the presence of intact sperm cells. The need to expand research on estimating TSI for sexual assault victims using actual forensic casework is crucial to provide a more reliable method for TSI estimation, compared to previous studies, which have generally been based on fertility studies. Between the years of 2009 and 2017, over 1,800 reported SAECKs were submitted to the Boston Police Department for evidence processing. More than 500 of these kits met the qualifications for this study, including: a living victim, smear slides prepared by a medical professional, and the identification of sperm cells during original kit processing. In order to estimate TSI, the smear slides from these cases were microscopically examined for the presence of intact sperm cells with the aid of Kernechtrot Picroindigocarmine (KPIC) stain. Based on casework received by the BPD, the maximum TSI reported for observing intact spermatozoa on vaginal smear slides was 105 hours, with an average collection time of 15 hours. The maximum TSI in which intact spermatozoa were observed on anorectal smear slides was 17.75 hours, with an average collection time of 7.9 hours. The average collection time in which intact spermatozoa on oral smear slides were observed was 6.9 hours, with a maximum reported TSI of 13.5 hours. Moreover, data from this study indicates a positive relationship between the total number of post coital activities completed before kit collection and the passage of time. Overall, this study provides reliable evidence based on actual casework samples for more accurately estimating the timeframe in which sperm evidence can be recovered after intercourse in living victims of sexual assault crimes.

The typical differential extraction procedure utilized by the forensic science community to extract male deoxyribonucleic acid (DNA) from the sperm cells of the perpetrator separately from female DNA from the epithelial cells of the victim is both time-consuming and labor-intensive. This has contributed greatly to the backlog of unanalyzed sexual assault evidence collection kits (SAECK) seen in many laboratories today and has encouraged research in new methods that are more efficient and more effective in achieving better sperm DNA recovery. The Cotton Lab has developed a Temperature-Controlled Differential Extraction (TCDE) procedure geared towards attaining better sperm recovery and better distribution of male DNA in the sperm fraction (SF) to generate a single source or distinguishable male profile. The TCDE protocol is a direct-lysis procedure that utilizes highly temperature-controlled enzymes, or enzymes that are active at or near their optimal temperatures. This procedure has been previously shown to decrease extraction time significantly and to extract samples that are suitable for downstream analysis. This research specifically attempted to modify the TCDE procedure in the hopes of obtaining higher sperm DNA recovery and eliminating previous concerns of too much sperm being retained by the cotton swab material. It also compared a slightly modified TCDE procedure where the material fraction (MF) and SF are kept as separate fractions (the Separate Method) and a method that results in a recombined MF and SF (Recombined Method) to see if there was a greater distribution of the total male DNA eluted into the SF. Preliminary experimentation with swabs prepared with semen was performed to help make effective modifications. Then, vaginal swabs from eight different female donors were prepared with semen to mimic forensic casework samples and extracted using the Separate and Recombined Methods for comparison of the two extraction methods. Despite unusual epithelial cell lysis results for some samples, the quantitation of the fractions by quantitative polymerase chain reaction (qPCR) showed that for approximately half of the samples extracted using the Separate Method, a majority of total male DNA was eluted into the SF. For these samples, a single source or distinguishable male profile can be generated. However, it was also demonstrated that even with good separation, a very small proportion of the female DNA in the SF still overwhelms the male DNA that is present in much smaller amounts, particularly for the Recombined Method where there are only two fractions. Though further experimentation is necessary, these modifications proved effective in achieving high sperm recovery in the SF and generating a distinguishable male profile when extracting samples using the Separate Method. This research has confirmed that the TCDE procedure can be faster and less labor intensive while still producing clean DNA profiles in downstream analysis, and thus has the potential to be implemented in forensic laboratories after some of the concerns are addressed.