Is it possible to predict the origin of epithelial cells? – A comparison of secondary transfer of skin epithelial cells versus vaginal mucous membrane cells by direct contact

In alleged sexual assault and rape cases, the focus has often been to collect samples from the victim's body, for detection of body fluids or skin cells from the offender. But in many cases intimate body samples from the perpetrator(s) can also be informative. However, in cases where the female victim claims vaginal penetration, the defendant may display an alternative explanation to the DNA findings, i.e. that the victim’s skin cells has been secondarily transferred to his penis. We hypothesized that female DNA will be detected in a significantly greater amount on swabs from penis after intercourse than after secondary transfer by skin contact.Fourteen male-female couples were recruited to test the above hypothesis, by collecting penile swabs from 3 specified anatomical locations: Glans, shaft, and the coronal sulcus, after two different situations: Vaginal intercourse and secondary transfer of epithelial cells by skin contact. The results show that penile swabs following intercourse produce significantly higher DNA concentrations than after secondary transfer by skin contact. Our results, indicates which of the anatomical regions is best suited for sampling. The DNA profiling results show a preponderance of female profiles over male profiles following intercourse compared to secondary skin contact.Based on these data, it is possible to make a statistical model to distinguish between samples taken after intercourse and samples taken after secondary transfer by skin contact based on the amount of female DNA and mixture proportion (Mx) between female and male DNA in samples collected from penis swabs.     

Persisting fetal microchimerism does not interfere with forensic Y-chromosome typing

Forensic Y-chromosome typing applies Y-chromosomal polymorphisms to the analysis of male/female mixed stains such as vaginal swabs in rape cases. The sensitivity of this approach exceeds that of cytological techniques combined with autosomal DNA typing. Y-chromosome typing is based on the assumption that Y-chromosomal DNA found in tissue or secretions of women must originate from a male individual, usually the perpetrator. Nevertheless, it was shown recently that fetal cells can migrate into the female body during pregnancy and can persist for decades ("persisting fetal microchimerism"). The body of a woman after a pregnancy with a male embryo can thus display a small fraction of fetal cells with Y-chromosomes. Using high sensitivity PCR protocols (reamplification with nested primers and up to 60 PCR cycles) fetal cells were previously identified in a number of maternal tissues including skin, blood, muscle and solid organs. It is, however, not clear at present, whether these cells can occur in vaginal secretions, and whether they are capable of producing false positive results in forensic Y-chromosome typing. To evaluate these questions, 66 blood samples of women with at least one son and nine vaginal swabs of women without sexual intercourse in the last 2 weeks were amplified for a stretch of the SRY gene. Eight thyroid gland tissues with already established male fetal microchimerism were used as positive control samples. Blood samples of 10 young girls without history of pregnancy were used as negative controls. Using a PCR with 10 ng of extracted DNA and 30 PCR cycles ("routine sensitivity assay") none of the samples yielded positive results. However, in a PCR with 200 ng of extracted DNA and 45 PCR cycles ("high sensibility assay"), 14% of the blood samples of mothers and 33% of the vaginal swabs amplified for SRY. Our results thus show that increasing the sensitivity of the PCR method and the amount of template DNA produce positive results while protocols used for routine Y-chromosomal typing with small amounts of DNA (approximately 10 ng of DNA) and with a limited number of PCR cycles (approximately 30) can clearly eliminate this peril.     

The use of phosphate buffered saline for the recovery of cells and spermatozoa from swabs.

In the forensic science laboratory, the recovery of spermatozoa from vaginal swabs, or vaginal cells from penile swabs, can help determine if sexual intercourse may have taken place. There are several methods used to recover spermatozoa and cells from the swabs before visualisation on a microscope slide and most of these methods use water. Phosphate buffered saline (PBS) is a non-toxic solution used in many biological laboratories. Unlike water, PBS prevents cells rupturing or shrivelling up due to osmosis. This study demonstrates that PBS can be used for the extraction of spermatozoa and cells from swabs and that PBS does not affect subsequent DNA profiling.     

Isolation and identification of female DNA on postcoital penile swabs

After sexual assault, cells originating from the assailant may be recovered from the victim. Through polymerase chain reaction (PCR)-based technology, positive scientific identification of the assailant may be made from these cells. Described is a prospective study describing a method for positively identifying cells from a female sex partner obtained from postcoital swabs of the penis of the male sex partner. Swabs were taken from the penis of a man at 1- to 24-hour intervals after coitus. DNA was isolated from each swab through standard organic extraction methods. The presence of female DNA was detected using the gender-specific amelogenin marker. Extracted DNA was amplified for eight different genetic loci using the Promega PowerPlex kit (Promega) and Amplitaq Gold (Perkin Elmer). Amplified samples were electrophoresed on precast sequencing gels (Hitachi) and were analyzed fluorescently using Hitachi's FMBIO 2 fluorescent scanner and software. Each sample obtained from a penile swab or condom was compared to male and female buccal controls. Female DNA was isolated from all postcoital penile swabs as determined by exclusive amplification of the X-chromosome specific 212 base pair amelogenin marker. In all cases, scientific identification of the female DNA from the swabs was determined by coamplification of eight STR loci (PowerPlex) and was compared to female and male control profiles. Cells shed from a female victim during sexual intercourse can be retrieved from the penis of a male offender after sexual intercourse during a 1- to 24-hour postcoital interval. DNA can be extracted from these cells and can be used to scientifically identify the female sexual participant through PCR-based technology. It is suggested that penile swabs be taken from alleged perpetrators of sexual assaults to associate them with a female victim.     

The evidential value of peptidase A as a semen typing system

Human erythrocyte peptidase A (Pep A) displays a genetic polymorphism in blacks. Its occurrence in human semen was examined for its possible use as a semen typing system. Studies by starch gel electrophoresis, in which the Pep A was located by an improved method, were carried out on semen, semen stains, and vaginal swabs taken at known times after intercourse. In addition, a large number of vaginal swabs, negative for semen, were taken from females throughout their menstrual cycles and examined for Pep A activity. The results indicated that Pep A typing could be carried out on semen and semen stains. However, it was possible to determine the Pep A type on vaginal swabs only when they had been taken within about 3 h after intercourse.     

Evaluation of Skin Surface as an Alternative Source of Reference DNA Samples: A Pilot Study

An acceptable area for collecting DNA reference sample is a part of the forensic DNA analysis development. The aim of this study was to evaluate skin surface cells (SSC) as an alternate source of reference DNA sample. From each volunteer (n = 10), six samples from skin surface areas (forearm and fingertips) and two traditional samples (blood and buccal cells) were collected. Genomic DNA was extracted and quantified then genotyped using standard techniques. The highest DNA concentration of SSC samples was collected using the tape/forearm method of collection (2.1 ng/μL). Cotton swabs moistened with ethanol yielded higher quantities of DNA than swabs moistened with salicylic acid, and it gave the highest percentage of full STR profiles (97%). This study supports the use of SSC as a noninvasive sampling technique and as a extremely useful source of DNA reference samples among certain cultures where the use of buccal swabs can be considered socially unacceptable.     

Experiences with single locus DNA probes in casework.

DNA profiling in this laboratory has been employed primarily in cases of sexual assault and the largest category of items examined has been internal vaginal swabs. 79% of these gave a profile which was different from that of the victim. Results have been obtained from swabs taken up to 70 h after intercourse. In cases where DNA results were obtained, one or more suspects were excluded in 29% of the cases.     

Background levels of body fluids and DNA on the shaft of the penis and associated underpants in the absence of sexual activity

This study examines the background of blood, saliva, semen and autosomal DNA on penile swabs and underpants from males in the absence of recent sexual activity. Based on the data collected by the AFSP Body Fluid Forum, the results of this study show that; there is a very low expectation of detecting blood on penile swabs and male underpants; a low expectation of detecting saliva on penile swabs and male underpants; and spermatozoa would be expected in less than a quarter of penile swabs and three quarters of male underpants. As none of the samples had detectable levels of DNA which were suitable for meaningful comparison that did not match the donor or their partner, the expectation of detecting a DNA profile from the cellular background on penile swabs or underpants from a male who has not been involved in recent sexual intercourse is very low. The results of this study are extremely informative when evaluating the significance of blood, saliva, semen and DNA detected on the penile swabs and underpants of males in cases of alleged sexual assault.     

Biological and DNA evidence in 1000 sexual assault cases

Using a Filemaker-based database (DNA Pro-FILES, Synchrone Infosystème Inc.), we have conducted a large-scale study on 1000 sexual assault (SA) cases where a standardized kit was submitted to our laboratory alone or with other types of exhibits. We looked at the likelihood of obtaining good quality DNA evidence, allegedly from the assailant, according to a number of parameters.The overall proportion of SA cases with DNA evidence is nearly 50%. A little more than 30% of SA kits provided DNA evidence while for 16% of cases DNA evidence could be obtained only from other exhibits.The likelihood of obtaining DNA evidence is approximately 50% in teenager and adult SA cases, but much lower for children 10 years old or younger (15%). In children cases, profiles were found mostly on clothing or skin swabs.The likelihood of obtaining DNA evidence from vaginal swabs remains good for up to 3 days after the assault (from 35% on the first day to 23% on the third day). A DNA profile was obtained from approximately 22% of anal/rectal swabs and 41% of skin swabs taken less than 1 day after the assault. Less than 10% of oral washes provided DNA evidence, all having been collected within 24 h of the assault.We found that in bodily samples, a negative result for acid phosphate (AP) is a poor predictor of the likelihood of obtaining good quality DNA evidence. Approximately 15% of vaginal swabs and 8% of anal swabs negative for AP nevertheless provided good quality DNA evidence.     

Use of RGB values in the Periodic Acid-Schiff color test to determine the presence of vaginal fluid

This study demonstrates how RGB color values from microscopic smears stained with the Periodic Acid-Schiff reagent under standardized microscopy conditions can be used to indicate the presence of vaginal secretions. Based on data obtained in the study, a numeric threshold determined from the sum of separate values for red, blue and green was determined to differentiate vaginal-based samples with other body fluids. Using this threshold, 55 of 57 vaginal-based samples tested positive for the presence of vaginal secretion. Conversely, 27 of 29 smears prepared from other body fluids yielded negative results. However, when graphing RGB sum values against a calculated RGB integer no overlap in data was obtained between all vaginal-based samples and other body fluid samples, clearly differentiating them. One-way ANOVA testing with a 95% confidence interval indicated that vaginal samples from different age groups showed no difference in RGB sum values. Similarly, the location that vaginal swabs were collected (from the outside of a condom or a vaginal swab) also showed no statistical difference using one-way ANOVA at 95% confidence. Furthermore, refrigerated test swabs aged up to 15 months showed no demonstrable differences. Pair-wise t-testing using RGB sum values, however, did show significant differences between vaginal samples and all other body fluids tested. Finally, the method successfully differentiated between pre-and post-coital penile swabs and finger swabs taken before and after digital vaginal penetration in anecdotal comparisons using the method.     

Development of STR profiles from firearms and fired cartridge cases

Fired cartridge cases are a common type of evidence found at crime scenes. However, due to the high chamber temperatures and touch nature of this evidence, DNA testing is not commonly sought because it is believed DNA is only present in low levels, whether it is due to initial low levels of DNA and/or DNA degradation from the heat or inhibition of the PCR reaction. Moreover, very few laboratories report STR typing success with fired cases. This study focused on obtaining STR profiles from fired cartridge cases using the AmpFℓSTR^® MiniFiler™ kit, which is designed to amplify DNA from low level, inhibited, and degraded samples. Comparisons to other STR amplification kits were also conducted. In attempt to simulate casework, random individuals loaded cartridges into a firearm. DNA was recovered from the fired cartridge cases using the double swab technique and extracted using an automated large volume DNA IQ™ method. Initially, testing focused on known shedders handling cartridges for 30 s prior to firing. A significantly greater number of alleles was obtained following amplification with the MiniFiler™ kit versus the PowerPlex^® 16 BIO kit. No alleles were observed using the Identifiler^® kit. In an attempt to better simulate casework, a random selection of laboratory personnel handled shotshells for as long as needed to load and fire the weapon. In this mock sample study, the MiniFiler™ kit successfully amplified an average of 22% of expected alleles from DNA recovered from shotshell cases versus the PowerPlex^® 16 BIO kit where an average of 7% of alleles were observed. However, the total number of alleles obtained from the two kits was not significantly different. The quality of the DNA obtained from fired cases was studied with evidence of inhibition in at least 11% of shotshell case samples. After swabbing the head and the hull of three shotshell cases separately, a significantly greater number of alleles was obtained from the hull as opposed to the head of the fired shotshell case. In addition, after firing, various internal firearm surfaces were swabbed, including the chamber of barrel, ejection port, and breechface, in an attempt to obtain amplifiable DNA. DNA was obtained from the chamber of the barrel and was amplifiable using the MiniFiler™ kit, although mixtures were obtained with extensive drop-in and drop-out making this analysis unlikely to aid an investigation.     

The NucleoSpin DNA Clean-up XS kit for the concentration and purification of genomic DNA extracts: An alternative to microdialysis filtration

Traditionally, DNA extracts from biological evidence items have been concentrated and rinsed using microdialysis filtration units, including the Centricon^® and Microcon^® centrifugal filter devices. As an alternative to microdialysis filtration, we present an optimized method for using NucleoSpin^® XS silica columns to concentrate and clean-up aqueous extracts from the organic extraction of DNA from biological samples. The method can be used with standard organic extraction and dithiothreitol (DTT)-based differential extraction methods with no modifications to these methods prior to the concentration and clean-up step. Extracts from laboratory-prepared bloodstains, saliva and semen stains have been successfully amplified with both qPCR and STR assays. Finally, the total time to process a set of samples with the NucleoSpin^® XS column is approximately 30 min vs. approximately 1.5 h with the Centricon^® YM-100 filter device.     

Y-STR profiling in extended interval (> or = 3 days) postcoital cervicovaginal samples

Depending upon specific situations, some victims of sexual assault provide vaginal samples more than 36-48 h after the incident. We have tested the ability of commercial and in-house Y-STR systems to provide DNA profiles from extended interval (> or =3 days) postcoital samples. The commercial Y-STR systems tested included the AmpFlSTR Yfiler (Applied Biosystems), PowerPlex Y (Promega) and Y-PLEX 12 (Reliagene) products whereas the in-house systems comprised Multiplex I (MPI) and Multiplex B (MPB). Three donor couples were recruited for the study. Postcoital cervicovaginal swabs (x2) were recovered by each of the three females at specified intervals after sexual intercourse (3-7 days). Each time point sample was collected after a separate act of sexual intercourse and was preceded by a 7-day abstention period. As a negative control, a precoital swab was also recovered prior to coitus for each sampling and only data from postcoital samples that demonstrated a lack of male DNA in the associated precoital sample was used. A number of DNA profile enhancement strategies were employed including sampling by cervical brushing, nondifferential DNA extraction methodology, and post-PCR purification. Full Y-STR profiles from cervicovaginal samples recovered 3-4 days after intercourse were routinely obtained. Profiles were also obtainable 5-6 days postcoitus although by this stage partial profiles rather than full profiles were a more likely outcome. The DNA profiles from the sperm fraction of a differential lysis were superior to that obtained when a nondifferential method was employed in that the allelic signal intensities were generally higher and more balanced and exhibited less baseline noise. The incorporation of a simple post-PCR purification process significantly increased the ability to obtain Y-STR profiles, particularly from 5- to 6-day postcoital samples. Remarkably an 8 locus Y-STR profile was obtained from a 7-day postcoital sample, which is approaching the reported time limit for sperm detection in the cervix.     

Generating DNA profiles from immunochromatographic cards using LCN methodology

The aim of this research was to obtain DNA profiles from immunochromatographic test devices which have already yielded positive results with body fluids obtained from fourteen volunteers. Three different immunochromatographic cards for the identification of human blood and one for the identification of human saliva were used for this research. Each body fluid was detected using the appropriate immunochromatographic card. The used cards were kept at room temperature for various lengths of time. The membranes were removed at the end of the designated times and the entire strip was extracted using low copy number (LCN) extraction procedure. The extracted DNA was amplified using reduced amplification volume and higher PCR cycle numbers. Autosomal STR profiles were detected using AmpFℓSTR^® Identifiler™ PCR Amplification Kit from Applied Biosystems (AB). Additionally, DNA extracted from the male volunteers was amplified using the AB AmpFℓSTR^® Yfiler™ PCR Amplification Kit. Analysis of the amplified products was carried out by capillary electrophoresis injection on the AB 3130xl Genetic Analyzer. The generated DNA data was analyzed using the SoftGenetics GeneMarker^® HID Version 1.7 software.Autosomal and Y-STR DNA profiles were obtained from most of the cards which were stored at room temperature for up to three months. DNA profile was obtained from all four types of the immunochromatographic cards used in this study. These profiles were concordant with the profiles obtained from the donors’ reference samples.     

Differentiation of epithelial cell types by cell diameter

Genital swabs play an important role in cases of alleged sexual assault. The aim of our study was to see if epithelial cells from the vagina, glans penis, or mouth could be distinguished on the basis of size. Vaginal swabs were taken from 12 women in different phases of their menstrual cycles; penile swabs were taken from 5 men, and mouth swabs were taken from 6 men and 6 women. For each swab, a sample was smeared across a microscope slide and allowed to dry. The dried epithelial samples were then viewed without any further processing with a "SteReoLumar.V12" stereo microscope. The microscope slide surfaces were divided into grids and all single epithelial cells whose contours could be clearly distinguished were photographed. The maximum diameter for each photographed cell was digitally determined using the Axiovision software. In total, 995 vaginal epithelial cells, 211 penile epithelial cells, 329 male oral epithelial cells, and 525 female oral epithelial cells were measured. Menstrual cycle phase did not affect vaginal epithelial cell diameter. The mean vaginal epithelial cell diameter was 63.95 microm (min. = 28.08 microm, max. = 108.06 microm, s = 11.50 microm). The mean penile epithelial cell diameter was 39.24 microm (min. = 28.38 microm, max. = 51.02 microm, s = 4.84 microm). The diameter of oral epithelial cells hardly differed for both sexes, although the female cells were, on the whole, slightly larger. On the basis of these results, it is not possible to conclude that epithelial cells of less than a certain diameter found in the assessment of a vaginal swab must be of penile origin. It is also not possible to usefully distinguish vaginal epithelial cells from male or female oral epithelial cells on the basis of the diameter. However, finding epithelial cells with a diameter distinctly greater than 50 microm in a penile swab sample suggests the presence of vaginal or oral epithelial cells. Epithelial cells examined with the presented method can be used without restrictions for further examinations, such as single-cell DNA analysis after single-cell picking with the micromanipulator developed by Aura Optik (Jena).     

The use of DNA stabilizing solution to enable room temperature storage and transportation of buccal and trace sample swabs

It is proposed that a DNA stabilizing solution (DNA Genotek Inc.) designed to preserve DNA in saliva samples at room temperature can be extrapolated to the storage of swab heads. The aim of this study was to evaluate the effectiveness of the solution for the preservation of reference swabs (buccal) and trace samples (facial swabs). To this end, the solution was used during a twin-site DNA transfer project assessing background levels of carer DNA present in children. Tubes containing 400 μl of solution were used to store and transport swab heads. At the laboratory, samples were extracted using the QIAamp DNA Mini Kit (Qiagen), quantified using the Quantifiler Duo Kit and profiled using the AmpF?STR^® SGM Plus^® PCR Amplification Kit (both Applied Biosystems). Twenty-eight PCR cycles were applied to all samples. Thirty-four cycles or a longer electrophoresis injection time was applied to trace samples where necessary. All Reference swabs produced high quantities of DNA and full DNA profiles after 28 cycles. Profile morphology indicated good quality DNA with no degradation. Of the trace samples, sufficient profiles were achieved to study the transfer of carer DNA making the solution fit for continued use in this project. DNA stabilizing solution enables the storage and transportation of swabs without freezing. This is convenient, reduces transportation costs and enables instant analysis of samples upon arrival at the laboratory. This is a useful alternative for a multi-site research project as well as a reliable storage tool for use in remote areas.     

FaSTR DNA: A new expert system for forensic DNA analysis

The automation of DNA profile analysis of reference and crime samples continues to gain pace driven in part by a realisation by the criminal justice system of the positive impact DNA technology can have in aiding in the solution of crime and the apprehension of suspects. Expert systems to automate the profile analysis component of the process are beginning to be developed. In this paper, we report the validation of a new expert system FaSTR DNA, an expert system suitable for the analysis of DNA profiles from single source reference samples and from crime samples. We compare the performance of FaSTR DNA with that of other equivalent systems, GeneMapper™ ID v3.2 (Applied Biosystems, Foster City, CA) and FSS-i³ v4 (The Forensic Science Service^® DNA expert System Suite FSS-i³, Forensic Science Service, Birmingham, UK) with GeneScan^® Analysis v3.7/Genotyper^® v3.7 software (Applied Biosystems, Foster City, CA, USA) with manual review. We have shown that FaSTR DNA provides an alternative solution to automating DNA profile analysis and is appropriate for implementation into forensic laboratories. The FaSTR DNA system was demonstrated to be comparable in performance to that of GeneMapper™ ID v3.2 and superior to that of FSS-i³ v4 for the analysis of DNA profiles from crime samples.     

Combined DNA Typing and Protein Identification from Unfired Brass Cartridges,,,

Biological evidence analysis from contact traces is adversely affected by low quantity and quality of DNA. Proteins in these samples contain potentially individualizing information and may be particularly important for difficult surfaces such as brass, where DNA may yield incomplete profiles. In this study, touched unfired brass cartridges were sampled using dry tape or wet swabs and analyzed by separating DNA and protein from the same collected material, thus producing both genomic and proteomic information. DNA recovery was similar for both collection methods, with tape yielding an average of 1.36 ± 1.87 ng and swabs, 1.34 ± 3.04 ng. Analysis by mass spectrometry identified 95 proteins, with the two collection methods showing no significant difference (p = 0.76) in the average number of collected proteins: 44.5 ± 10.9, (tape) versus 47.9 ± 20.4 (swabs). Proteins can be collected from fingerprints at levels necessary to provide identifying information, thus expanding information obtained from challenging evidence.     

Analysis of Δ-tetrahydrocannabinol in oral fluid samples using solid-phase extraction and high-performance liquid chromatography–electrospray ionization mass spectrometry

An analytical method using solid-phase extraction (SPE) and high-performance liquid chromatography–mass spectrometry (LC–MS) has been developed and validated for the confirmation of Δ⁹-tetrahydrocannabinol (THC) in oral fluid samples. Oral fluid was extracted using Bond Elut LRC-Certify solid-phase extraction columns (10 cm³, 300 mg) and elution performed with n-hexane/ethyl acetate. Quantitation made use of the selected ion-recording mode (SIR) using the most abundant characteristic ion [THC + H⁺], m/z 315.31 and the fragment ion, m/z 193.13 for confirmation, and m/z 318.00 for the protonated internal standard, [d₃-THC + H⁺]. The method proved to be precise for THC, in terms of both intra-day and inter-day analyses, with coefficients of variation less than 10%, and the calculated extraction efficiencies for THC ranged from 76 to 83%. Calibration standards spiked with THC between 2 and 100 ng/mL showed a linear relationship (r² = 0.999). The method presented was applied to the oral fluid samples taken from the volunteers during the largest music event in Portugal, named Rock in Rio-Lisboa. Oral fluid was collected from 40 persons by expectoration and with Salivette^®. In 55% of the samples obtained by expectorating, THC was detected with concentration ranges from 1033 to 6552 ng/mL and in 45% of cases THC was detected at concentrations between 51 and 937 ng/mL. However, using Salivette^® collection, 26 of the 40 cases had an undetectable THC.     

Novel Y-STR typing strategies reveal the genetic profile of the semen donor in extended interval post-coital cervicovaginal samples

For a variety of reasons, some victims of sexual assault provide vaginal samples more than 24-36 h after the incident. In these cases, the ability to obtain an autosomal STR profile of the semen donor from the living victim diminishes rapidly as the post-coital interval is extended. We have used a number of carefully selected Y-STR loci in a variety of multiplex or monoplex formats to extend the post-coital interval from which a genetic profile of the semen donor can be obtained. The proposed Y-STR typing strategies enable the routine detection of the male donor Y-STR haplotype in cervicovaginal samples recovered up to 4 days post-coitus. We attribute our success to a number of factors that significantly improve the sensitivity and specificity of the analysis. Firstly, we utilize a subset of Y-STR loci that have been carefully selected for their superior performance under stressed conditions in both multiplex and monoplex formats. Specifically these loci function with low copy number templates in the presence of a vast excess of potentially confounding female DNA. Secondly, sperm and non-sperm DNA is co-extracted without a differential extraction process to prevent the unnecessary loss of the small number of structurally fragile sperm remaining in the cervicovaginal tract several days after intercourse. Thirdly, low copy number detection is facilitated by increasing the cycle number to 34-35 cycles and by the ability to input up to 450 ng of co-extracted sperm/non-sperm DNA into the PCR reaction without the appearance of confounding female artifacts. Lastly, the proper collection of post-coital cervicovaginal samples, instead of the lower or mid-vaginal tract samples often taken, is required for optimal recovery of sperm for analysis. In this report we demonstrate that our previously described 19 Y-STR loci systems (MPI and MPII) permit a reliable high resolution haplotype determination of the semen donor in cervicovaginal samples taken up to 48 h after intercourse. However, as the post-coital interval is extended further, dramatic loss of signal is observed and haplotype determination of the male donor is no longer possible with MPI and MPII. Nonetheless, subsets of these 19 loci (MPA and MPB) have been developed specifically to detect the male haplotype in samples recovered 4 days after intercourse. Thus, it is possible to derive an 11-19 locus Y-STR profile of the semen donor in cervicovaginal samples recovered 2-4 days after intercourse.