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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.
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.
The second method that is routinely used in forensic laboratories to generate DNA profiles from sexual assault evidence is known as a direct extraction and is not strikingly different from the method described above. In this method, the spermatozoa and epithelial cells collected are not eluted from the cotton swab prior to performing the DE. The cotton swab is placed into the DE buffer and epithelial cells are lysed while at the same time, spermatozoa are being released from the cotton matrix. Centrifugation of the resultant lysate will yield epithelial cell DNA in the supernatant and the cell pellet will contain intact spermatozoa. Once again, the supernatant can be used to generate the DNA profile of the victim. As described above, the cell pellet can be treated with DTT and ProK to generate the DNA profile of the perpetrator.
Neither the sperm elution method nor the direct extraction method is without limitations. The sperm elution method in its current format is very inefficient, typically recovering less than 10% of the sperm present on the cotton swab. The direct extraction method is somewhat more efficient, resulting in recovery of between 30%– 40% of the sperm4(and data not shown). Both of these methods involve multiple handling steps and often may require extended incubation times for optimal recovery. Finally, obtaining a DNA profile from the sperm fraction using either method is critically dependent on the relative ratios of epithelial to sperm cells present in the sample. The higher the ratio of epithelial cells to spermatozoa, the more likely it will be that the male DNA profile generated from the sperm fraction will be obscured by the presence of “carry over” DNA from the victim or epithelial fraction. This problem is significant in samples that contain less than 1000 total sperm in the presence of hundreds of thousands or even millions of epithelial cells from the victim.
A number of forensic investigators have searched for alternative means of separating epithelial cells from spermatozoa that may co-exist in many sexual assault swabs. These methods include laser microdissection,5,6 separation based on physical characteristics such as size and density of the cells,7,8 and enzymatic and chemical release of cells from cotton swabs.1,4 While some of these methods have shown limited potential for success, a fast, reliable method for obtaining clean male DNA profiles from sexual assault swabs containing relatively few numbers of sperm is still very much needed. This present study will demonstrate the latest advances in this endeavor using a set of reagents and procedures designed at our laboratory to improve the recovery of spermatozoa from cottons swabs. This improved recovery, coupled with an added feature of a reduction in the number of epithelial cells present in the mixed cell pellet, allows for the production of clean male and victim profiles, even when the number of sperm cells is small in comparison to the number of epithelial cells present.
Based on the need to improve the elution of spermatozoa from cotton swabs in our own forensics laboratory, Orchid Cellmark’s research and development team investigated a number of physical and chemical approaches designed to enhance the recovery of sperm. The factors examined in our study were as follows: 1) the type and pH of the buffer system used, 2) incubation times and temperatures, 3) the use of various enzymes to aid in sperm removal and recovery, and 4) more stringent mechanical methods of agitation to help dislodge sperm adhered to the swab. Based on these studies and an extensive validation of the resulting procedure, the results from a reproducibility study shown in Table 1 demonstrate that the recovery of sperm from cotton applicator swabs can be increased from the traditional 5%-10% with the conventional sperm elution method to an average of >85% using the newly adopted procedure.
This new procedure makes use of a two-buffer system that has been specifically designed to differentially release the two cell types from the cotton swab in different phases. This process allows the specific release of epithelial cells during the first phase and primarily sperm during the second phase. As a result, the sperm phase contains only a limited number of epithelial cells. In addition to recovering more sperm cells using this new procedure, the number of epithelial cells present during the subsequent differential extraction of the sample has been significantly decreased. This reduction in epithelial cells present in the eluted sperm pellet leads to two very important benefits of the new method. First, the removal of excess epithelial cells results in the production of considerably cleaner sperm search slides compared to samples treated using the standard method. An example of this is demonstrated in Figure 1.
The image on the left in Figure 1 is a sperm preparation from a sexual assault swab subjected to the old elution method, while the image on the right is an identical swab subjected to the new elution method. The ability to visualize sperm on slides prepared using the new method has been significantly enhanced due to the absence of epithelial cells in the background. Removing the excess epithelial cells allows the forensic scientist to more quickly and accurately identify positive samples.
The second significant outcome of reducing the number of epithelial cells in the eluted sperm pellet is the reduction in carryover of female or victim DNA into the male profile. This feature is particularly important when dealing with samples that contain thousands of female epithelial cells and only a few hundred sperm. An example of this benefit is illustrated in the electropherograms shown in Figures 2 and 3.
The electropherograms shown in these two figures compare the results of profiling two sperm fractions following differential extraction of two sperm pellets obtained by using either the direct extraction method or the new sperm elution method. The swabs used for this comparison were duplicates and each contained approximately 400 total sperm. Figure 2 shows the profile obtained using the direct extraction method. The resulting profile is essentially of female (victim) origin with a few very minor peaks appearing where the male profile should be. The profile obtained using the new sperm elution method (Figure 3) is essentially of male (perpetrator) origin with a few minor peaks appearing where the female peaks should appear. The male profile produced in this particular experiment yielded above-threshold peaks (100 rfus) on 14 of 15 possible markers. Peak height ratios < 60% were observed at three loci, D2S1338, THO1, and D19S433, partially due to slight female carryover in a few shared alleles. In summary, had this new sperm elution method not been performed, the male DNA profile generated from this sample would not have been possible.
Current methods for eluting spermatozoa from sexual assault samples are lacking, particularly when the quantity of sperm present on the swab is far less than the number of epithelial cells present. Our laboratory has examined a number of physical and chemical parameters aimed at improving the recovery of sperm from cotton-tipped swabs used in the collection of forensic evidence following sexual assault/rape. Orchid Cell-mark has developed a proprietary sperm elution procedure that utilizes two buffer systems and increased physical agitation to remove sperm adhered to the cotton fibers of a swab. As a result, we have been able to demonstrate an improvement in recovery of sperm from less than 10% using current elution methods to greater than 85% with the new optimized method. Using this new method, the eluted sperm pellet contains many fewer epithelial cells and thereby greatly enhances the ability of the forensic scientist to perform microscopic sperm searches much more rapidly. As an added feature of having fewer epithelial cells in the sperm pellet, this work demonstrates that the differential extraction process can now produce much cleaner male profiles even when the total number of sperm available on the swab is less than 1,000. Producing cleaner cell populations with the new elution method has also led to studies aimed at reducing the incubation times required for differential extraction. These studies are currently underway and have produced very promising results.
Finally, it is feasible that this method could be integrated with existing automation tools to assist in reducing the already massive backlog of sexual assault cases that currently exists in the United States. Anyone interested in obtaining more information about this method should contact the author.
- Voorhees, JC, Ferrance, J, and Landers, J, “Enhanced elution of sperm from cotton swabs via enzymatic digestion for rape kit analysis.” J. Forensic Sci., pp. 574-579, May 2006.
- Rape, Abuse, & Incest National Network, 2006, RAINN, 28 Jan, 2008, [www.rainn.org/statistics/].
- Gaensslen, R, Mertens, J, Lee, H, Stolorow, M, “ Staining and extraction techniques,” Proc. of a Forensic Sci. Symposium on the Analysis of Sexual Assault Evidence, FBI Academy, 1983.
- Norris, JV, Manning, K, Linke, S, Ferrance, J, and Landers, J, “Expedited, chemically enhanced sperm cell recovery from cotton swabs for rape kit analysis.” J. Forensic Sci., pp. 800-805, July, 2007.
- Elliott, K, Hill, DS, Lambert, C, Burroughes, TR, and Gill, P, “Use of laser microdissection greatly improves the recovery of DNA from sperm on microscope slides.” Forensic Sci. Intl., pp. 28-36, 2003.
- Sanders, CT, Sanchez, N, Ballantyne, J, and Peterson, DA, “Laser microdissection separation of pure spermatozoa from epithelial cells for short tandem repeat analysis.” J. Forensic Sci., pp.748-757, July 2006.
- Horsman, KM, Barker, SLR, Ferrance, JP, Forrest, KA, Koen, KA, and Landers, JP, “Separation of sperm and epithelial cells on microfabricated devices: potential application to forensic analysis of sexual assault evidence.” Anal. Chem., pp. 742-749, 2005.
- Horsman, KM, Nilsson, M, Nilsson, J, Laurell, T, and Landers, JP, “Acoustic differential extraction: a novel alternative to conventional differential extraction.” Proceedings of the 58th Annual Meeting of the Amer. Acad. Of Forensic Sci. 2006, Seattle, WA.Colorado Springs, CO: Amer. Acad. Of Forensic Sci., 2006.
Robert C. Giles, Ph.D., is the Executive Director, Global Research and Development for Orchid Cellmark. Bob has more than 20 years of experience in DNA testing for genetic diseases, parentage, and forensics. Orchid Cellmark; 13988 Diplomat Drive, Farmers Branch, TX 75234; 214-271-8401; firstname.lastname@example.org.