Document Title: Separation of Sperm and Epithelial Cells in a Microfluidic Device: An Automated Method for High Efficiency, High Purity Separations

The proven utility of forensic DNA evidence has increased the demand for DNA analysis services. Although conventional DNA analysis techniques are effective, they are time-consuming and laborious, which contributes to the overall cost and to an overwhelming backlog of forensic casework samples with possible biological evidence. Research efforts have focused on the development of more rapid and efficient analytical methods using microdevices to reduce the time and cost of forensic analysis as well as the magnitude of the existing casework backlog. Successful microfluidic methods for separating sperm and epithelial cells have previously been demonstrated by our laboratory.1, 2 These methods circumvent a time-consuming step in DNA analysis of sexual assault evidence, the conventional differential extraction procedure. The presented research will focus on a separation technique utilizing acoustic trapping (Acoustic Differential Extraction; ADE), which was developed for the isolation of male and female fractions from sexual assault evidence. The acoustic trapping microdevice consists of piezoceramic microtransducers imbedded into a printed circuit board layer, which serves as the bottom of a microchannel, and a glass fluidic layer containing the microchannel structure. Upon application of an alternating voltage to the microtransducers, a standing ultrasonic acoustic wave is formed within the microchannel, generating pressure minima in the center. Particles and cells, in a sizeand density-dependent manner, are drawn to these minima by acoustic forces generated from the standing wave; the magnitude and direction of the forces are dictated by the physical characteristics of the specific particles or cells. The ADE system has been optimized to immobilize sperm cells, while free DNA (primarily from epithelial cell lysate) will not be trapped. A biological sample containing a mixture of sperm and epithelial cell lysate is infused through the microfluidic channels and transported over the transducers. Upon activation of the ultrasound, sperm cells are retained in the acoustic trap, while free DNA is directed towards the female DNA outlet. Laminar flow valving is used to redirect the flow towards the male outlet, and the ultrasound is terminated, resulting in the release of sperm cells from the acoustic trap and subsequent movement of cells towards the male outlet. The male and female products from the ADE system were collected from their respective outlets and analyzed off-chip; DNA purification, amplification, and separation were performed using conventional laboratory methods. The results show that highly purified male and female fractions can be obtained within the ADE microdevice. This document is a research report submitted to the U.S. Department of Justice. This report has not been published by the Department. Opinions or points of view expressed are those of the author(s) and do not necessarily reflect the official position or policies of the U.S. Department of Justice. Landers Final Report USDJ Award Number 2006-DN-BX-K021 Pg -3Executive Summary Genetic analysis of mixed profile DNA samples obtained from vaginal swabs is a well-established technique in the investigation of sexual assault and rape cases.3-5 To obtain independent short tandem repeat (STR) profiles of both the victim and the perpetrator, it is necessary to separate the male and female components of the recovered genetic material. The current separation protocol, differential extraction (DE) is a wellestablished technique; however, it is one of the most time-consuming sample preparation steps of sexual assault evidence analysis, contributing to a substantial forensic casework sample backlog.6 In addition, conventional DE requires extensive sample handling, and often results in inefficient separation of female DNA from the male fraction.7, 8 Research efforts have focused on the development of more rapid and efficient analytical methods to reduce the time and cost of forensic analysis. Microfluidic technology provides the opportunity to automate forensic DNA sample processing, and all forensic genetic analysis steps have been demonstrated on microdevices.9-11 Techniques performed on microchips are advantageous because they can be integrated with other analytical steps on a single microfluidic device.12 The laborious, time-consuming centrifugation and wash steps of conventional DE are not easily amenable to a microdevice; therefore, the development of a microfluidic device for the separation of recovered biological material from rape case evidence is of interest. Trapping based on acoustic forces offers a simple, efficient way of retaining particles in a microfluidic system. This report focuses on the development and characterization of an acoustic differential extraction (ADE) microfluidic device. ADE This document is a research report submitted to the U.S. Department of Justice. This report has not been published by the Department. Opinions or points of view expressed are those of the author(s) and do not necessarily reflect the official position or policies of the U.S. Department of Justice. Landers Final Report USDJ Award Number 2006-DN-BX-K021 Pg -4relies on acoustic trapping of sperm cells in the presence of epithelial cell lysate (which is unretained). Laminar flow valving (LFV) was utilized to direct the male and female fractions to separate outlets. Preliminary results showed suboptimal purity of the male fraction after ADE separations.13 Improvements in fluidic control and trapping on the ADE device enabled efficient separation of mixed biological samples, resulting in independent male and female DNA profiles. The ADE microdevice consists of piezoceramic microtransducers imbedded into a printed circuit board layer, which serves as the bottom of a microchannel, and a glass fluidic layer containing the microchannel structure. The depth of the microfluidic channels was determined based on the number of desired pressure nodes (trapping sites) in the channel upon activation of the transducer. The fluidic layer conducting the cell suspension was enclosed by an air-backed miniature transducer. Regardless of the number of desired pressure nodes, the thickness of the reflector layer above the channel corresponded to an odd number of quarter wavelengths [(2n + 1)λ/4], as calculated by the speed of sound in borosilicate glass. ADE was performed in a sealed microdevice. A waveform generator was used to actuate the transducers with a sinusoidal signal at the specified frequency and amplitude. Samples and buffer were infused into the chip using pressure-driven flow from syringe pumps. Polytetrafluoroethylene (PTFE) tubing was utilized to connect the microdevice to the buffer and sample syringes. Outlets were comprised of capillaries [250 μm ID, bare or dynamically-coated with poly-N-hydroxyethylacrylamide (PHEA)] or 0.3 mm ID PTFE tubing. Samples consisted of prepurified human genomic DNA, semen diluted in This document is a research report submitted to the U.S. Department of Justice. This report has not been published by the Department. Opinions or points of view expressed are those of the author(s) and do not necessarily reflect the official position or policies of the U.S. Department of Justice. Landers Final Report USDJ Award Number 2006-DN-BX-K021 Pg -5phosphate buffered saline (PBS) solution, epithelial cell lysate, or a combination of sperm and epithelial cell lysate to simulate a sexual assault sample. PBS or conventional DE buffer was used for hydrodynamic focusing and laminar flow valving. During the sample infusion, the ultrasound was activated at a specific frequency and voltage using the waveform generator. Hydrodynamic focusing was used to direct the infused sample towards the center of the transducer to ensure a high trapping efficiency, while sample was infused through the sample inlet. Cells were trapped above the transducer in the pressure nodes of the standing wave, while the unretained material (female lysate) was directed towards the female outlet using laminar flow valving (LFV). During the wash step, the sample infusion was terminated; the ultrasound remained activated, allowing the sperm cells to be levitated in the acoustic trap, while the focusing buffer perfused the trapped cells and removed any residual lysate from the microchannel and outlet tubing. Upon completion of the washing, LFV was used to direct flow towards the male outlet, and the ultrasound was deactivated, resulting in a release of the sperm cells and subsequent collection in the male outlet. Microcentrifuge tubes were attached to each outlet for sample collection. DNA extraction of the ADE fractions was completed using commercially-available purification kit. Real time quantitative PCR (qPCR) was utilized to determine the quantity of human genomic (hg)DNA in the ADE fractions. To assess DNA purity, short tandem repeat (STR) profiles were generated using conventional PCR amplification and separation techniques. STR profiles were obtained from the male sperm cell and female buccal epithelial cell donors, and compared with those obtained This document is a research report submitted to the U.S. Department of Justice. This report has not been published by the Department. Opinions or points of view expressed are those of the author(s) and do not necessarily reflect the official position or policies of the U.S. Department of Justice. Landers Final Report USDJ Award Number 2006-DN-BX-K021 Pg -6from ADE separations in order to determine purity. Purity was calculated based on the peak area ratios of the male and female contributors for all seven STR loci. ADE was initially demonstrated using a mock sexual assault sample consisting of 10 μm polystyrene beads to simulate sperm cells and Evans Blue Dye to simulate female epithelial cell lysate. Upon infusion of the mixed sample, the ultrasound was activated, and the polystyrene particles were trapped in the acoustic standing wave over the transducer; the unretained dye was directed to the female outlet using LFV. After sample infusion, sample flow was terminated, and the channels were washed with buffer to remove any inadvertently-trapped Evans Blue Dye from the trapped particles, as well as residual dye from the microchannel. LFV was then implemented such that the direction of flow was switched to the male outlet. The ultrasound was then deactivated to give a controlled release of the microbeads towards the male outlet channel. It was originally concluded that LFV could be used to effectively separate sperm and epithelial cell lysate on the ADE microdevice; however, closer inspection of the still photomicrographs revealed a potential female DNA contamination source. During the ADE wash step, as sample flow was terminated, the infusion of focusing buffer unintentionally “pulled” sample (including residual blue dye) from the sample inlet. This “pull” of sample continued during the fluidic control and release steps, and blue dye was directed towards the male outlet, indicating a potential cause of female DNA contamination in the male ADE fractions. The suspected source of female DNA contamination in male ADE fractions was verified by initial attempts at separating sperm cells from epithelial cell lysate. ADE of This document is a research report submitted to the U.S. Department of Justice. This report has not been published by the Department. Opinions or points of view expressed are those of the author(s) and do not necessarily reflect the official position or policies of the U.S. Department of Justice. Landers Final Report USDJ Award Number 2006-DN-BX-K021 Pg -7mock sexual assault samples was performed as previously described; samples collected from the female outlet during sperm cell trapping and from the male outlet during the release were subsequently analyzed off-chip using commercially-available purification and STR amplification kits. The data obtained from these experiments consistently resulted in poor separation efficiencies. Comparison of the profiles from the sperm cell donor with the male ADE fraction indicated recovery of sperm cell DNA; however, the presence of additional alleles consistent with the epithelial cell donor signified poor purity (55% or less) of the male fraction. The data showed that additional efforts towards improving fluidic control were necessary to eliminate female lysate contamination of the male ADE separation product, and to demonstrate better purity levels. To prevent unwanted sample infusion during the wash and release steps, the experimental conditions were modified so that the sample was withdrawn from the device by the sample syringe (at a lower flow rate relative to that of the focusing buffer). Different colored dyes were used in the initial characterization of the modified sample withdrawal method. Sample was infused through sample inlet, while hydrodynamic focusing was applied. The sample and focusing buffer were directed towards the female outlet using LFV. During the wash step, withdrawal from the sample inlet (1 μL/m) effectively stopped the sample from entering the center channel, and the focusing buffer effectively removed residual sample from the remainder of the channel, including the female outlet. LFV valving was implemented to simulate fluidic control prior to the release step of ADE. The results show that LFV was not adversely affected by sample withdrawal, as no sample was observed entering the male outlet. The reliability of the This document is a research report submitted to the U.S. Department of Justice. This report has not been published by the Department. Opinions or points of view expressed are those of the author(s) and do not necessarily reflect the official position or policies of the U.S. Department of Justice. Landers Final Report USDJ Award Number 2006-DN-BX-K021 Pg -8modified fluidic control method was further assessed using prepurified DNA and subsequent quantitation of the collected fractions. A sample consisting of prepurified hgDNA used to simulate female epithelial cell lysate was infused through the sample inlet and hydrodynamically focused; the ultrasound was not initiated. LFV was used to direct the flow of the DNA towards the female outlet. After sample infusion, the sample flow was switched to withdraw from the sample inlet (1 μL/m), and the channels were washed with focusing buffer while maintaining flow towards the female outlet. Once the wash step was completed, the outlet flow was switched, such that the net flow was directed towards the male outlet. After each step the eluted fractions were collected from each outlet, and total genomic DNA of each sample was measured using a qPCR assay. The results show that fractions collected from the female outlet after sample infusion contained the majority of the recovered DNA; DNA was not detected in fractions collected from the female outlet, and more importantly, the male outlet, after the release step. The results verify that the use of sample withdrawal during the wash and release steps eliminates a source of contamination, as it ensures that sample (containing epithelial cell lysate) would not be “pulled” into the device and towards the male outlet during the release of the trapped material (sperm cells). Similar studies using pre-purified hgDNA were performed to optimize the sample withdrawal flow rate (while incorporating hydrodynamic focusing); it was determined that 0.5 μL/m sample withdrawal was more suitable for future experiments, as it provided consistent recoveries of hgDNA in the fractions collected from the female outlet after sample infusion. This document is a research report submitted to the U.S. Department of Justice. This report has not been published by the Department. Opinions or points of view expressed are those of the author(s) and do not necessarily reflect the official position or policies of the U.S. Department of Justice. Landers Final Report USDJ Award Number 2006-DN-BX-K021 Pg -9To ensure that the ADE microdevice was suitable for the separation of mock sexual assault samples, acoustic trapping conditions of sperm and epithelial cell lysate were separately assessed. Lysate trapped in the acoustic standing wave is a potential source of contamination, as it would be released upon termination of the ultrasound and directed towards the male outlet; therefore, it was important to establish that epithelial cell lysate (in the absence of sperm cells) was not trapped. Female epithelial cell lysate was infused through the sample inlet and hydrodynamically focused; trapping was initiated, and LFV was used to direct the flow of the DNA towards the female outlet. The wash step was performed as previously described; once completed, the outlet flow was switched such that the net flow was directed towards the male outlet, and the acoustic trapping was terminated. After each step, the eluted fractions were recovered from each outlet and purified off-chip using a commercially-available extraction kit. Total genomic DNA was measured using a qPCR assay. The results show that fractions collected from the female outlet after sample infusion contained the most DNA, and therefore, the most lysate; DNA was not detected in fractions collected from the female outlet, and more importantly, the male outlet, after the release step, confirming that epithelial cell lysate was not trapped in the absence of sperm cells, (as predicted by acoustic force theory). In addition, the data correlate with the results obtained using pre-purified hgDNA, and further verify the effectiveness of incorporating sample withdrawal to eliminate sample bleed towards the male outlet. The number of sperm cells captured and released from the acoustic trap, and consequently, the amount of DNA recovered in the male fraction after ADE, is critical to This document is a research report submitted to the U.S. Department of Justice. This report has not been published by the Department. Opinions or points of view expressed are those of the author(s) and do not necessarily reflect the official position or policies of the U.S. Department of Justice. Landers Final Report USDJ Award Number 2006-DN-BX-K021 Pg -10obtaining a male STR profile. Studies were therefore performed to assess sperm cell trapping and release using the ADE device, without interference from epithelial cell lysate. Preliminary results were obtained with acoustic trapping utilizing a single node fluidic layer; to increase capacity of the trap, a triple node fluidic layer was implemented for the remainder of the experiments. Samples consisting of semen (6, 12, or 24 μL) added to PBS were infused through the sample inlet and hydrodynamically-focused; acoustic trapping, wash and release steps were performed as previously described. The concentrations of DNA recovered from four consecutive runs for each prepared sample was determined via off-chip conventional purification and qPCR. Samples prepared with 6 μL of neat semen provided insufficient concentrations to obtain full STR profiles. For samples prepared with 12 μ L of semen, an increase in DNA concentration was observed over the course of the four trials; samples prepared with 24 μ L of semen provided inconsistent recoveries of DNA. It was concluded that the inconsistent recoveries of DNA obtained from samples prepared with higher volumes of semen were a result of poor release of larger clusters of cells from the outlet tubing. Similar studies were performed with devices containing capillary outlets. Samples consisted of neat semen (6, 12, 24, or 48 μ L) added to PBS; acoustic trapping, wash, and release steps were performed as previously described. The data further support the theory that more acoustically-trapped cells resulted in larger clusters of cells that were not released cleanly from the outlet tubing. In addition, lower numbers of trapped cells, coupled with off-chip conventional purification and amplification techniques, did not provide sufficient DNA for full STR profiles. Sample preparation and trapping conditions for the remainder of This document is a research report submitted to the U.S. Department of Justice. This report has not been published by the Department. Opinions or points of view expressed are those of the author(s) and do not necessarily reflect the official position or policies of the U.S. Department of Justice. Landers Final Report USDJ Award Number 2006-DN-BX-K021 Pg -11the reported studies were adjusted such that approximately 2000-2400 cells were acoustically trapped and released towards the male outlet. Using the current microchip design, it may be necessary to estimate and measure the sperm content of the sample prior to acoustic separation. In order to improve the release of biological material from outlets, focusing and LFV buffers were changed from PBS to a conventional DE buffer. Acoustic trapping of sperm cells added to conventional DE buffer was performed. Samples were infused through the sample inlet and hydrodynamically-focused; acoustic trapping, wash, and release steps were performed as previously described. Samples were recovered from the male outlet (250 μm ID capillary) and purified off-chip using a commercially-available extraction kit; conventional amplification and separation of STR loci were performed. The results show that samples prepared in DE buffer consistently provided full STR profiles, indicating that the use of DE buffer was more effective than PBS for the removal of cells from the outlet tubing; therefore, LFV and focusing buffers were changed to conventional DE buffer for all future studies. Once run-to-run reproducibility of acoustic trapping was established, ADE separations of mock sexual assault samples were performed. Sperm cells were added to female epithelial cell lysate; acoustic trapping, wash, and release steps were performed as previously described. The results indicate that the release of biological material was problematic for samples prepared in epithelial cell lysate, as STR profiles were not obtained. In an effort to further facilitate removal of biological material from the outlets, the capillaries were dynamically-coated with 0.1% (w/v) PHEA, chosen due to its This document is a research report submitted to the U.S. Department of Justice. This report has not been published by the Department. Opinions or points of view expressed are those of the author(s) and do not necessarily reflect the official position or policies of the U.S. Department of Justice. Landers Final Report USDJ Award Number 2006-DN-BX-K021 Pg -12ability to reduce protein interactions with silica surfaces. Samples were prepared in epithelial cell lysate as previously described. STR profiles of the mixed biological sample prior to ADE, the sperm cell donor, and the female epithelial cell donor were obtained using conventional methods; comparison of the original sample profile with those of the male and female donors indicated that the mixed biological sample was 40 ± 3.9% male. ADE was performed as previously described. Samples were recovered from the male outlet and purified off-chip using a commercially-available extraction kit; conventional amplification and separation of STR loci were performed. Comparison of the resultant profile with those of the male and female donors indicate that the sample is 79 ± 16% male, with seven of the core CODIS STR loci detected. The results show that ADE, performed on a device with coated capillary outlets, provided over 50% enhancement of male purity. However, due to issues with recovery of the separated biological material from the outlets; two out of six of the subsequent trials did not result in STR profiles. As samples prepared in epithelial lysate did not consistently provide STR profiles, the effects of varying the operating frequency of the transducer on the trapping pattern of particles were investigated. A suspension of particles was infused through the sample inlet with hydrodynamic focusing. Trapping was initiated at 11.6 MHz (the same operating frequency used for previous mock separation studies) and observed using light microscopy; the results show that particles were trapped in a large cluster near the center of the channel, suspended above the transducer. Using the same particle suspension, transducer, and fluidic layer, the frequency was increased to 12.1 MHz. The change in This document is a research report submitted to the U.S. Department of Justice. This report has not been published by the Department. Opinions or points of view expressed are those of the author(s) and do not necessarily reflect the official position or policies of the U.S. Department of Justice. Landers Final Report USDJ Award Number 2006-DN-BX-K021 Pg -13trapping frequency resulted in a change in the particle distribution; smaller clusters of particles that were spread out over a larger area of the transducer were observed. It was hypothesized that the change in particle distribution would allow for more effective removal of inadvertently-trapped female lysate from the trapped cells, and that the smaller clusters of cells trapped using the higher frequency would be less likely to clog the outlet tubing upon release compared to a single, larger cluster of cells obtained with the lower frequency. To test this, mock sexual assault samples were prepared in epithelial cell lysate. The STR profile of the mixed biological sample prior to ADE was obtained using conventional methods; the sample was determined to be 26 ± 2.0% male. ADE was performed as previously described. Samples were recovered from the male and female outlets (0.3 mm ID PTFE tubing) and purified off-chip using a commercially-available extraction kit; conventional amplification and separation of STR loci were performed. Comparison of the STR profile obtained from the male fraction with those of the male and female donors indicate that the sample is 92 ± 7.9% male, a nearly four-fold enhancement in male purity in comparison with the original sample. All seven core STR loci, devoid of alleles contributed from the female, were detected, allowing for interpretation of the male profile from the original mixture. Comparison of the STR profile obtained from the female fraction with those of the male and female donors indicates that the sample is 5.7 ± 4.7% male. The results suggest that the particle distribution in the acoustic trap may play an important role in the effective separation of sperm and female epithelial cell lysate using the ADE device. This document is a research report submitted to the U.S. Department of Justice. This report has not been published by the Department. Opinions or points of view expressed are those of the author(s) and do not necessarily reflect the official position or policies of the U.S. Department of Justice. Landers Final Report USDJ Award Number 2006-DN-BX-K021 Pg -14The higher operating frequency and its resultant trapping pattern were applied to ADE separations of a mock sexual assault samples containing lower levels of male DNA. Samples were prepared by addition of sperm cells to epithelial cell lysate generated from a larger number of epithelial cells than used in the previous study. The STR profile of the mixed biological sample prior to ADE was obtained using conventional methods; through comparison of this profile with those of the male and female donors, the sample was determined to be 13 ± 5.9% male. ADE was performed as previously described. Samples were recovered from the male and female outlets, and purified off-chip using a commercially-available extraction kit; conventional amplification and separation of STR loci were performed. Comparison of an STR profile obtained from the male fraction the profile with those of the male and female donors indicate that the sample is 73 ± 17% male, with all seven core STR loci detected – a nearly sevenfold enhancement of male purity compared to the original sample. The STR profile obtained from the female fraction with those of the male and female donors indicates that the sample is 3.1 ± 3.9% male – effectively a pure female profile without presence of male alleles. The enrichment of male DNA in the sample (as compared to the original mixture), combined with the STR profile obtained from the female donor, allows for interpretation of the male profile from the original mixture. However, the presence of female alleles in the profile obtained from the male outlet indicates a source of female DNA contamination. Since fluidic control was proven to be reliable and reproducible, these results are most likely due to excess female lysate inadvertently trapped within the sperm cell clusters. Further studies may require optimization of the wash step to effectively This document is a research report submitted to the U.S. Department of Justice. This report has not been published by the Department. Opinions or points of view expressed are those of the author(s) and do not necessarily reflect the official position or policies of the U.S. Department of Justice. Landers Final Report USDJ Award Number 2006-DN-BX-K021 Pg -15remove female DNA and prevent it from contaminating the post-ADE male sample. It is important to note that, while samples collected from the male outlet following subsequent ADE separations of the same sample provided male purities that did not differ considerably from the ones shown; samples collected from the female outlet did not consistently provide STR profiles, indicating that consistent release of female lysate from outlets is an issue. The results show that highly purified male and female fractions can be obtained with improvements to the ADE technique. It is reasonable to expect that this technique can be integrated with on-chip downstream sample processing steps. To implement ADE into forensic laboratories, further optimization and engineering improvements to the system are required. Developments are currently underway to produce disposable ADE devices; these devices will potentially consist of a sealed channel structure and an external transducer, as opposed to the current system where the transducer is in direct contact with the sample, and will reduce the possibility of sample contamination – a necessity for forensic laboratories. In addition, to obtain more consistent recoveries of DNA after ADE separations, different outlets will be designed and tested. The time savings associated with a fully-integrated analysis system incorporating ADE provides the potential to significantly alter the means by which sexual assault evidence is processed in crime laboratories. In parallel with the ADE studies, effort was exerted on pre-acoustic trapping processes. Effective utilization of the ADE method or other alternative DE methods relies on efficient elution of sperm cells from the vaginal swab, and the comprehensive This document is a research report submitted to the U.S. Department of Justice. This report has not been published by the Department. Opinions or points of view expressed are those of the author(s) and do not necessarily reflect the official position or policies of the U.S. Department of Justice. Landers Final Report USDJ Award Number 2006-DN-BX-K021 Pg -16lysis of epithelial cells, all in a timely manner. Regardless of the method utilized for the separation of female epithelial DNA and male sperm cells, the overall effectiveness of the procedure is also dependent on the efficiency with which material can be eluted and recovered from a cotton swab this is paramount for obtaining an STR profile from a low copy number sample. We have devised a number of improvements over the standard Gill buffer approach used in DE, including the use of enzymaticand chemical-enhancements to improve the yield of sperm cells removed from the swab. While most of the DE protocols currently in use in crime labs have an associated sperm cell yield of ~40% using Gill buffer and Proteinase K, we have shown that this can be improved substantially 1a. A two-step method for cell elution and recovery from mock sexual assault samples was developed by our laboratory. The first step involves detergent-mediated elution of intact sperm and epithelial cells from a cotton swab matrix; several detergent types were assessed, and detergent concentrations were investigated for swabs that were dried and stored for various periods of time. The second step involves preferential lysis of epithelial cells that were recovered using the detergent-mediated cell elution method. The effects of detergent and enzyme concentration on cell lysis were evaluated for mock casework samples prepared with various numbers of sperm cells. The results show that the two-step cell elution/preferential lysis method provided nearly twofold improvement in sperm cell recoveries compared to a conventional DE buffer, while providing comprehensive epithelial cell lysis, in a total incubation time of one hour – up to an order of magnitude decrease in incubation time than traditionally performed in some forensic 1a Voorhees, et al., 2006. J. Foren. Sci. 51(3):574-79; Voorhees, et al., J. Foren. Sci. 52(4):800-5; This document is a research report submitted to the U.S. Department of Justice. This report has not been published by the Department. Opinions or points of view expressed are those of the author(s) and do not necessarily reflect the official position or policies of the U.S. Department of Justice. Landers Final Report USDJ Award Number 2006-DN-BX-K021 Pg -17laboratories. The improvement in sperm cell recoveries using the SDS elution buffer is ideal for increasing the efficiency of obtaining accurate DNA profiles from samples containing low numbers of sperm cells, while comprehensive epithelial cell lysis is ideal for use in conjunction with the microfluidic ADE platform. This document is a research report submitted to the U.S. Department of Justice. This report has not been published by the Department. Opinions or points of view expressed are those of the author(s) and do not necessarily reflect the official position or policies of the U.S. Department of Justice. Landers Final Report USDJ Award Number 2006-DN-BX-K021 Pg -18Detailed Technical Report