The ability to perform forensic DNA analysis in criminal investigations has become an important part of solving numerous types of crime. DNA testing has become especially important in criminal cases involving sexual assault and rape.

In these types of cases, spermatozoa collected from the evidence (e.g., underwear, clothing, bed linens, etc.) or from a post-coital collection device, such as a cotton-tipped applicator swab, can be used to help identify the perpetrator. Currently, one of the most common methods of cellular recovery from these types of samples involves soaking the evidence (or a portion thereof) in a neutral, isotonic solution, such as phosphate buffered saline (PBS) or citrate buffer, accompanied by periodic agitation, such as vortexing. Often times, in order to achieve optimal recovery, the samples require several hours or even an overnight incubation in the soaking solution. Internal findings and recently published studies have indicated that less than 10% of the spermatozoa present on a cotton swab are actually recovered using this type of procedure.¹ Due to this low recovery, it is possible to have sexual assault specimens incorrectly characterized as negative for sperm and therefore insufficient for DNA testing. Some of these samples could, in fact, contain an adequate number of sperm, and if properly screened, could yield a suitable DNA profile. Due to the importance of accurately assessing the presence of sperm, it is imperative that an improved method be developed that significantly improves the forensic scientist’s ability to recover more spermatozoa from the swab. Obviously, improving this recovery step would lead to a larger number of successful profiles generated, and thereforea larger number of forensic cases solved.

This article reviews the status of current methodologies used to elute spermatozoa from cotton swabs and to report a portion of the results obtained by our laboratory. A series of experiments designed to increase the efficiency of recovery of spermatozoa from post-coital sexual assault samples examined the following factors: 1) the type and pH of the buffer system, 2) incubation times and temperatures, 3) the use of various enzymes, and 4) a more stringent mechanical method of agitation to help remove sperm adhered to the swab.

BACKGROUND
Recent statistics show that in the United States, there are more than 225,000 victims of rape, attempted rape, or sexual assault each year.² DNA analysis performed in these types of cases can often lead to identification of the perpetrator, provided that sufficient material of male origin is collected from the victim or the crime scene. Often, the victim of a sexual assault/rape may undergo an examination by a health care professional, at which time a specimen may be collected using an approved collection device, typically a cotton-tipped applicator swab. When discussing the recovery of cellular material from post-coital sexual assault specimens, it is important to remind the reader that these types of specimens frequently consist of two cell populations; i.e., epithelial cells of victim origin, and spermatozoa of perpetrator origin. Any method that will ultimately be considered useful in the analysis of forensic case work should be one that enhances the ability of the forensic scientist to distinguish DNA profiles from these two distinct cell populations of unique origin.

Currently, there are two basic methods used by most forensic laboratories to elute spermatozoa or the DNA from spermatozoa from post-coital sexual assault swabs. The first method involves the soaking of the cotton swab or a portion of the swab in a pH-neutral buffered saline solution, such as phosphate-buffered saline or sodium citrate. Following a timed and temperature-controlled incubation with several steps of mechanical agitation by vortexing, the resulting mixture of cells that has been eluted from the cotton matrix is pelleted by centrifugation. At this point, a visual search for spermatozoa can be performed by removing a small aliquot of the cellular pellet and placing it on a microscope slide for staining. A differential stain, such as the Christmas tree stain³, can be used to easily distinguish epithelial cells from spermatozoa present in the preparation. The remainder of this pellet is then subjected to a process known as differential extraction (DE), whereby the two cell populations present in the mixture are differentially lysed and DNA profiles can be obtained from each lysed cell population. Initially, a mixture of lysis buffer containing Proteinase K (ProK) and an anionic detergent such as sodium dode-cylsulfate (SDS) are added to the cell mixture in appropriate concentrations and incubated long enough to specifically lyse the epithelial cells present while allowing the spermatozoa present to remain intact. Following a centrifugation step, the resultant supernatant can be used to generate a DNA profile that most often is specific for the victim of the sexual assault. The remaining cell pellet can then be used to generate DNA from the male contributor by the addition of dithiothreitol (DTT) and ProK at sufficient concentrations to effect lysis of the spermatozoa present. Ideally, the DNA fraction from the sperm pellet will yield a sufficient amount of DNA to produce a clean, male profile that can be used to compare to DNA profiles from known suspects involved in the sexual assault case or can be uploaded into a local, state, or national database for comparison to known convicted offenders.