Journal of Forensic and Legal Medicine 37 (2016) 50e54
Contents lists available at ScienceDirect
Journal of Forensic and Legal Medicine j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / j fl m
Original communication
Urine specimen collection following consensual intercourse e A forensic evidence collection method for Y-DNA and spermatozoa € a, *, Sari Tuomisto b, Mervi Seppa €la € b, Heini Huhtala c, Arja Ahola d, Minna Joki-Erkkila Pekka J. Karhunen e a
Departments of Gynecology and Obstetrics and Forensic Child Psychiatry, Tampere University Hospital, Finland Department of Forensic Medicine, University of Tampere, Finland School of Health Sciences, University of Tampere, Finland d Biomedical Laboratory Scientist, Bachelor of Health Care, School of Medicine, University of Tampere, Finland e Department of Forensic Medicine, School of Medicine, University of Tampere and Fimlab Laboratories, Tampere University Hospital Region, Finland b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 3 August 2015 Received in revised form 18 September 2015 Accepted 24 October 2015 Available online 2 November 2015
The purpose of the prospective research was to evaluate the benefit of urine specimen as a collection technique for biological forensic evidence in adult volunteers following consensual intercourse. For detecting Y-chromosomal material Buccal Swab Spin Protocol® was used in DNA extraction and purification and samples were analysed with Quantifiler Y Human Male DNA Quantification Kit®. The time frame for positive Y-DNA was evaluated. Immediate microscopy for detection of spermatozoa was performed. Y-DNA was detected in 173/205 (84.4%) urine samples. Of the 86 first post-coital void urine samples available, Y-DNA was detected in 83 (96.5%) specimens. Of the 119 urine samples from volunteers with post-coital activities Y-DNA was still measurable in 70 (58.8%) urine specimens. The male DNA amount was below 0.023 ng/ml in 28/153 (18.3%) urine samples. Of the 22 urine samples obtained after 24 post-coital hours, 9 (40.9%) were still Y-DNA positive. No associations were found between coital durance, coital frequency during the past two weeks prior to the study intercourse, post-coital activities, and the urine sample Y-DNA positivity. Of the 111 urine samples where the immediate microscopy was performed, in 66 (59.5%) samples spermatozoa were verified and one sample even contained motile spermatozoa. Microscopy detected 66 (67.3%) and failed to detect spermatozoa in 32 (32.7%) of Y-DNA positive samples. In addition to conventional invasive swab techniques, urine samples seem to be an effective biological trace collection method for Y-DNA and spermatozoa within 24 h following penile-vaginal penetration. Furthermore, it may be considered as a non-invasive collection method in suspected acute child sexual abuse cases to diminish time delay in forensic evidence collection and to improve patients' positive attitudes towards evidence collection. © 2015 Elsevier Ltd and Faculty of Forensic and Legal Medicine. All rights reserved.
Keywords: Consensual intercourse Sexual assault Urine samples Forensic evidence Y-DNA
1. Introduction In suspected sexual assaults biological trace evidence is most often collected by swabs from the anogenital area. The possibility of using urine for evidence collection has rarely been considered. The idea of urine sample collection for forensic purposes is based on human anatomy and physiology. In women, the urethral
* Corresponding author. P.O. Box 2000, 33521, Tampere, Finland. Tel.: þ358 500 5557760. €). E-mail address: minna.joki-erkkila@pshp.fi (M. Joki-Erkkila
orifice in the vulvar area is next to the hymenal opening of the vagina. During voiding, the vaginal outflow of secretions increases due to physiologic relaxation of the pelvic floor and due to the increase in abdominal pressure. The urine rinses the periuretral vulvar area and the vaginal orifice and it can be collected for further analysis. We found only one recent Australian study where the frequency of spermatozoa was analyzed from first void urine specimens.1 They detected spermatozoa in 35% of first void urine specimens collected following alleged penile-vaginal penetration.1 To our knowledge, there are no published studies analyzing the frequency
http://dx.doi.org/10.1016/j.jflm.2015.10.008 1752-928X/© 2015 Elsevier Ltd and Faculty of Forensic and Legal Medicine. All rights reserved.
€ et al. / Journal of Forensic and Legal Medicine 37 (2016) 50e54 M. Joki-Erkkila
of spermatozoa for a longer period than from the first void urine. Although DNA identification is used to obtain evidence in sexual assault, a Y-DNA analysis of urine has not been yet done for forensic purposes. The purpose of the study was to evaluate the detection rate of YDNA in post-coital urine samples and to evaluate the time frame for positive Y-DNA following consensual intercourse and the detection rate of spermatozoa in urine samples.
2. Material and methods 2.1. Participant characteristics Female volunteers, comprising medical or laboratory students, hospital personnel or acquaintances, were invited to participate in a research study collecting and analyzing urine samples following consensual vaginal intercourse from May 2008 to December 2009 by either a personal invitation or by collective invitation after a lecture. 90 volunteers took part in the study. Two volunteers were excluded because of misunderstandings in filling in the study information form. 88 volunteers were included in the study. Most of the study participants had participated also in other studies evaluating collection of forensic evidence.2,3 The women were instructed to record the time of their last intercourse before the studied urine collection, the urine sample collection time and which of the consecutive post-coital voidings (1st, 2nd etc.) was collected as a specimen. A medical history was given by the volunteer using a form. Contraception, time of previous intercourse before the intercourse for the sample collection, frequency of intercourse in the past two weeks prior to the sample collection, activities following the intercourse (wiping, washing, showering, going to the sauna, urinating, defecating) and duration of intercourse were recorded.
51
2.3. DNA extraction and quantification The amount of urine and the pH of the urine were measured. The entire volume in the urine samples were centrifuged for 10 min at 3000 rpm and the supernatant was decanted. After centrifugation the supernatant (urine) was poured out and the cellular deposit at the base of the centrifuge tube was collected for analysis. From 200 ml of cell pellet DNA was extracted using QIAamp DNA Mini Kit® (Qiagen, Germany). Centrifugation steps were carried out at room temperature according to the protocol. Quantifiler Y Human Male DNA Quantification Kit® (Applied Biosystems, USA) was used to quantify the total amount of amplifiable human male DNA. PCR reactions and analysis were done with AbiPrism® 7000 HT Sequence Detection System (AppliedBiosystems, USA) according to the instructions provided. The PCR of Quantifiler Y Human Male DNA Quantification Kit® method is standardized and we used recommended water as blank controls alongside the purification and amplification steps. A sample was considered positive if a measurable amount of DNA was detected. The cut-off limit for possible DNA identification of the male was considered to be 0.01 ng/ml and according to the recommended cutoff limit of 0.023 ng/ml for Quantifiler Y. 2.4. Microscopic analysis Immediate microscopy was performed in a subset of 111 (54.1%) urine samples from 47 volunteers in a forensic laboratory. The following parameters were examined: volume, motility and sperm density. The density was categorized to no sperm, a few sperm (1e10/slide), a moderate amount (10e50/slide), and many (>50/ slide). The examination was performed according to the WHO laboratory manual 1999.4 Each sample was suspended on a slide by mixing the sample with a small amount of a culture medium (5 ml). The covered preparation was examined under the microscope. All samples were examined by phase-contrast microscope (magnification of 10 20). 2.5. Contamination issues
2.2. Sample collection Following consensual intercourse, volunteers were advised to void into a collection bottle (volume 500 ml, Sarstedt prod. no 77.582, Germany) so that the rim of the collection bottle would lie tightly onto the perineum. The purpose was to collect the outflow of vaginal secretions as much as possible into the collection bottle when physiologically relaxing the pelvic floor muscles while urinating. The urine volume was collected as much as possible into the collection bottle. The volunteers were advised not to worry if the urine volume exceeded the volume of the collection bottle. The first post-coital voiding urine sample was instructed to be collected without washing or without any other possible post-coital activities. Post-coital activities were allowed for the following collected urine samples. A single-use collection bottle was used for each episode of voiding. The urine collection bottle(s) were used for preserving the urine in the refrigerator. The collection bottle was labelled by (1) the study number given to the volunteer earlier during invitation and the volunteer (2) numbered the post-coital voiding time and marked it on the side of the collection bottle. The collection bottle was returned to the examiner as soon as possible but not later than 5 days. Time of the studied intercourse, time of the collected urine sample, post-coital activities, and the numbering of post-coital voiding times were recorded on the study information form.
The possibility of contamination has to be considered. All the urine sample processing, extraction and amplification steps were performed by the same female experimenters to avoid secondary YDNA contamination. Disposable powder free gloves were used and changed frequently to minimize the contamination risk. 2.6. Ethical considerations The study protocol was approved by the Ethics Committee (#R08018) of Pirkanmaa Hospital District, Finland. Written consent was required for participation in the study, for forensic laboratory analysis and for publication purposes. No data from the patient's medical records were collected. Full anonymity was secured in storage and laboratory analysis. 2.7. Statistical considerations Results were summarised by descriptive statistics; medians with range or frequencies (n) and percentages (%). Differences between detection of Y-DNA and spermatozoa were analyzed using the McNemar test. The Chi-square test was used to compare our microscopy results on detected spermatozoa rate in first void urine to results from the study by Smith et al. (2014). SPSS 21 was used for data analysis (IBM Corp. Released 2012. IBM SPSS Statistics for Windows, Version 21.0. Armonk, NY: IBM Corp.).
€ et al. / Journal of Forensic and Legal Medicine 37 (2016) 50e54 M. Joki-Erkkila
52
3. Results
3.4. Immediate phase-contrast microscopy
3.1. Volunteer characteristics
Immediate phase-contrast microscopy was performed on a subset of 111 urine specimens from 47 volunteers. Immediate microscopy verified spermatozoa within 24 h in 66 (59.5%) samples; few in 29 (26.1%), moderate amount in 15 (13.5%) and many in 22 (19.8%) samples at 10 20 magnification. Spermatozoa was detected in 37/44 (84.1%) in first void specimens. A significantly higher rate of spermatozoa was detected when compared to a recent study of Smith et al. (2014) of first void urine specimen in alleged sexual assault cases, 84% vs 35% (p < 0.001).
Age range was 20e52 years (median age 26.5 years). Eightyeight female Caucasian volunteers were included in the study. All reported penile-vaginal penetration. Most of the volunteers participated in other clinical studies by the study group.2,3
3.2. Y-DNA detection in urine samples The study consists of 205 urine specimens. The median urine volume was 175 ml (range 2e550 ml, Q1 ¼ 90 ml, Q3 ¼ 290 ml). The mean urine pH was 6.3 (range 4.8e8.2). No association was found between urine volume, urine pH and measurements of male DNA. Y-DNA was measurable in 173/205 (84.4%) urine specimens, median 0.68 ng/ml (range 0.0009e121.8 ng/ml). Of the 86 first post-coital void urine specimens available, Y-DNA was detected in 83 (96.5%) specimens. Y-DNA was still measurable in 70/119 (58.8%) urine specimens of the volunteers with post-coital activities (Table 1). The median time from consensual intercourse to urine specimen collection was 5 h (range 0.08e96.33 h). The median time for measurable male DNA was 9.2 h (range 0.08e52.5 h). Most (89.3%) of the urine specimens were collected within 24 post-coital hours (Figs. 1 and 2). After 24 post-coital hours Y-DNA was measurable in 9/22 (40.9%) urine specimens. No associations were found between duration of intercourse, coital frequency during the past two weeks prior to the study intercourse, post-coital activities, and the Y-DNA specimen positivity.
3.5. Sensitivity and specificity immediate phase contrast microscopy compared to Y-DNA in urine specimens A significant (p < 0.001) association was found between positive Y-DNA and detection of sperm in immediate phase contrast microscopy. Microscopy detected 66 (67.3%) of positive Y-DNA samples and failed to detect spermatozoa in 32 (32.7%) of Y-DNA positive samples. Compared to Y-DNA, the sensitivity of phase contrast microscopy was 67.4% (95% CI; 57.1e76.5%), but specificity was 100% (95%CI; 82.2e100%). 4. Discussion To our knowledge, this is the first study evaluating the value of urine specimens following consensual intercourse as a clinical forensic collection method for identification of Y-DNA and detection of spermatozoa. 4.1. Detection of Y-DNA and spermatozoa
3.3. Cut-off limits for possibility of DNA-identification in urine samples The cut-off limit for possible DNA identification of the male was hypothetically considered to be 0.01 ng/ml. Y-DNA of 0.01 ng/ml was quantified in 153/205 (74.6%) urine specimens. The mean of 0.01 ng/ml Y-DNA quantity was 4.2 ng/ml (median 0.9 ng/ml; range 0.01e121.8 ng/ml) (Fig. 3). The hypothetical DNA identification was also estimated according to the recommended cut-off limit of 0.023 ng/ml for Quantifiler Y. The male DNA amount was below 0.023 ng/ml in 28/153 (18.3%) urine samples. After 24 post-coital hours Y-DNA remained measurable in 9/22 (40.9%; range 0.0013e6.6 ng/ml) urine specimen. And in five of these measurable specimens Y-DNA was quantified over 0.01 ng/ml. One of the urine specimens was positive even at 52.5 h containing 0.025 ng/ml of Y-DNA, making it still sufficient for possible DNA identification.
Table 1 Measurable male DNA in urine specimens collected following consensual intercourse according to the number of post-coital voiding. Y-DNA
Post-coital voiding samples First void
Positive Negative
Second void
Third void
Fourth void
Fifth void
Sixth to tenth void
n ¼ 86
n ¼ 43
n ¼ 27
n ¼ 11
n ¼ 10
n ¼ 28
n
%
n
%
n
%
n
%
n
%
n
%
83 3
96.5 3.5
31 12
72.1 27.9
15 12
55.6 44.4
8 3
72.7 27.3
8 2
80.0 20.0
8 20
28.6 71.4
The detection rate of Y-DNA in urine specimens was very high when collected within 18 post-coital hours. Overall, the time frame for positive Y-DNA detection was for over two post-coital days, whereas spermatozoa were detected up to 24 post-coital hours in collected urine specimens. Most of the urine specimens were collected within the 24 post-coital hours, but still in 40% of urine specimens Y-DNA was quantified after 24 h. Furthermore, the hypothetical possibility for male DNA identification was considered in 75% of urine specimens. Our results suggest that urine specimens for forensic purposes might be collected even up to three days in suspected sexual assaults. Our study detected a significantly higher rate of spermatozoa (84%) in first void urine specimen following consensual intercourse when compared to a recent reported study of alleged sexual assault cases where the detection rate of first void urine spermatozoa was 35%.1 The difference in the detection rate may at least partly be due to lower ejaculation rates in assault cases. The quantity of measurable male DNA (median 0.68 ng/ml) in the post-coital urine specimens seems to be higher when compared to vaginal swabs (median 0.06 ng/ml), cervical swabs (median 0.01 ng/ ml), and to cervical brush (median 0.02 ng/ml) samples analyzed by the same quantification method.2 The benefit of correctly collected urine specimens is that it collects a higher quantity of male DNA in the sample, and it is a non-invasive collection method for forensic purposes. Also it may be considered a good forensic evidence collection method in acute suspected child sexual abuse cases, to diminish the time delay, and to improve patients' positive attitudes towards evidence collection.
€ et al. / Journal of Forensic and Legal Medicine 37 (2016) 50e54 M. Joki-Erkkila
53
Fig. 1. Male DNA detected in urine samples according to the time from the consensual intercourse to urine specimen collection.
Fig. 2. Proportion of positive male DNA in urine samples according to the time from consensual intercourse to urine specimen collection.
4.2. Study limitations Confounding factors such as misunderstandings in filling in the study information form are possible. The collection bottle volume may have been too small for total volume collection which may have caused a bias in the urine volume. The volunteers were advised not to worry if the urine volume exceeded the volume of the collection bottle. We assumed that the volume of urine in sample collection is not as important as the collection outflow of vaginal secretions while urinating. The sample size at this point was rather small and there is a need for larger studies. The risk of contamination needs to be considered in this study. To diminish the risk of contamination, all the laboratory work was conducted by female experimenters. The quantified male DNA may also result from sources other than spermatozoa or semen such as male epithelial cells. A certain limitation of this study is the lack of using only single Y-DNA quantification PCR and not using Y-STR profiling. The detected male DNA of 0.0009 ng/ml was considered measurable because we assumed that Y-DNA cannot be detected from female
urine specimens otherwise than from a close contact with a male person, due to contamination, or due to rare XY-chromosomal syndrome in female. Detecting male DNA from female vaginal secretions most likely proofs a male contact. Detection of spermatozoa from vaginal secretions is a proof of sexual intercourse with a male person unless there has been an infertility treatment of intra uterine insemination performed shortly prior the specimen collection. We cannot rule out the presence of ‘non-sperm’ male DNA although no known vasectomy, azoo- or oligospermia were reported by the volunteers. We did not have the permission to evaluate medical records of the volunteers or their partners. Present study evaluated mainly the value of evidence collection technique of urine specimen for Y-DNA detection and not the laboratory quantitative PCR experiment techniques.5 Another limitation is the unsolved stability of DNA recovered from urine, due to differences in urine composition especially in long-term storage conditions, which is still under discussion. Unfortunately, the volunteers were not asked their opinion on possible ejaculation or known azoospermia, which may explain part of the negative Y-DNA results. We assumed voluntary penile vaginal penetration to include ejaculation.
54
€ et al. / Journal of Forensic and Legal Medicine 37 (2016) 50e54 M. Joki-Erkkila
Acknowledgements We thank the Finnish Crime Laboratory for their contribution to the study. Conflict of interest None declared. Funding This study was financially supported by the Competitive State Financing of the Expert Responsibility area of Tampere University Hospital (9S013) and Clinical research fund of Tampere University Hospital. Ethical approval The study protocoll was approved by the Ethics Committee (#R08018) of Pirkanmaa Hospital District, Finland. Fig. 3. Quantification of human male DNA in urine samples collected following consensual intercourse according to time and 0.01 ng/ml cut-off limit. One specimen (121.84 ng/ml, collected 50 min following intercourse) was omitted from the figure as an outlier.
5. Conclusions Along with conventional invasive swab techniques, urine specimen collection may be used as an additional biological trace collection method for forensic purposes. It could be considered as a non-invasive collection technique especially in acute suspected child sexual abuse cases to diminish time delay and to improve patients’ positive attitudes towards evidence collection. Higher amounts of male DNA detected in urine specimens would offer better chances for DNA identification when compared to conventional swabs.
References 1. Smith DA, Webb L, Fennel A, Nathan E, Bassindale C, Phillips M. Early evidence kits in sexual assault: an observational study of spermatozoa detection in urine and other forensic specimen. Forensic Sci Med Pathol 2014;10: 336e43. € M, Tuomisto S, Sepp€ 2. Joki-Erkkila anen M, Huhtala H, Ahola A, Rainio J, et al. Clinical forensic sample collection techniques following consensual intercourse in volunteers - cervical canal brush compared to conventional swabs. J Forensic Leg Med 2014;27:50e4. € M, Rainio J, Huhtala H, Salonen A, Karhunen PJ. Evaluation of ano3. Joki-Erkkila genital injuries using white and UV-light among adult volunteers following consensual sexual intercourse. Forensic Sci Int 2014;242:293e8. 4. WHO laboratory manual for the examination of human semen and sperm-cervical mucus interaction. 4th ed. Cambridge University Press and The World Health Organization; 1999. 5. Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M, et al. The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem 2009;55:611e22.
Contents lists available at ScienceDirect
Journal of Forensic and Legal Medicine j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / j fl m
Original communication
Urine specimen collection following consensual intercourse e A forensic evidence collection method for Y-DNA and spermatozoa € a, *, Sari Tuomisto b, Mervi Seppa €la € b, Heini Huhtala c, Arja Ahola d, Minna Joki-Erkkila Pekka J. Karhunen e a
Departments of Gynecology and Obstetrics and Forensic Child Psychiatry, Tampere University Hospital, Finland Department of Forensic Medicine, University of Tampere, Finland School of Health Sciences, University of Tampere, Finland d Biomedical Laboratory Scientist, Bachelor of Health Care, School of Medicine, University of Tampere, Finland e Department of Forensic Medicine, School of Medicine, University of Tampere and Fimlab Laboratories, Tampere University Hospital Region, Finland b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 3 August 2015 Received in revised form 18 September 2015 Accepted 24 October 2015 Available online 2 November 2015
The purpose of the prospective research was to evaluate the benefit of urine specimen as a collection technique for biological forensic evidence in adult volunteers following consensual intercourse. For detecting Y-chromosomal material Buccal Swab Spin Protocol® was used in DNA extraction and purification and samples were analysed with Quantifiler Y Human Male DNA Quantification Kit®. The time frame for positive Y-DNA was evaluated. Immediate microscopy for detection of spermatozoa was performed. Y-DNA was detected in 173/205 (84.4%) urine samples. Of the 86 first post-coital void urine samples available, Y-DNA was detected in 83 (96.5%) specimens. Of the 119 urine samples from volunteers with post-coital activities Y-DNA was still measurable in 70 (58.8%) urine specimens. The male DNA amount was below 0.023 ng/ml in 28/153 (18.3%) urine samples. Of the 22 urine samples obtained after 24 post-coital hours, 9 (40.9%) were still Y-DNA positive. No associations were found between coital durance, coital frequency during the past two weeks prior to the study intercourse, post-coital activities, and the urine sample Y-DNA positivity. Of the 111 urine samples where the immediate microscopy was performed, in 66 (59.5%) samples spermatozoa were verified and one sample even contained motile spermatozoa. Microscopy detected 66 (67.3%) and failed to detect spermatozoa in 32 (32.7%) of Y-DNA positive samples. In addition to conventional invasive swab techniques, urine samples seem to be an effective biological trace collection method for Y-DNA and spermatozoa within 24 h following penile-vaginal penetration. Furthermore, it may be considered as a non-invasive collection method in suspected acute child sexual abuse cases to diminish time delay in forensic evidence collection and to improve patients' positive attitudes towards evidence collection. © 2015 Elsevier Ltd and Faculty of Forensic and Legal Medicine. All rights reserved.
Keywords: Consensual intercourse Sexual assault Urine samples Forensic evidence Y-DNA
1. Introduction In suspected sexual assaults biological trace evidence is most often collected by swabs from the anogenital area. The possibility of using urine for evidence collection has rarely been considered. The idea of urine sample collection for forensic purposes is based on human anatomy and physiology. In women, the urethral
* Corresponding author. P.O. Box 2000, 33521, Tampere, Finland. Tel.: þ358 500 5557760. €). E-mail address: minna.joki-erkkila@pshp.fi (M. Joki-Erkkila
orifice in the vulvar area is next to the hymenal opening of the vagina. During voiding, the vaginal outflow of secretions increases due to physiologic relaxation of the pelvic floor and due to the increase in abdominal pressure. The urine rinses the periuretral vulvar area and the vaginal orifice and it can be collected for further analysis. We found only one recent Australian study where the frequency of spermatozoa was analyzed from first void urine specimens.1 They detected spermatozoa in 35% of first void urine specimens collected following alleged penile-vaginal penetration.1 To our knowledge, there are no published studies analyzing the frequency
http://dx.doi.org/10.1016/j.jflm.2015.10.008 1752-928X/© 2015 Elsevier Ltd and Faculty of Forensic and Legal Medicine. All rights reserved.
€ et al. / Journal of Forensic and Legal Medicine 37 (2016) 50e54 M. Joki-Erkkila
of spermatozoa for a longer period than from the first void urine. Although DNA identification is used to obtain evidence in sexual assault, a Y-DNA analysis of urine has not been yet done for forensic purposes. The purpose of the study was to evaluate the detection rate of YDNA in post-coital urine samples and to evaluate the time frame for positive Y-DNA following consensual intercourse and the detection rate of spermatozoa in urine samples.
2. Material and methods 2.1. Participant characteristics Female volunteers, comprising medical or laboratory students, hospital personnel or acquaintances, were invited to participate in a research study collecting and analyzing urine samples following consensual vaginal intercourse from May 2008 to December 2009 by either a personal invitation or by collective invitation after a lecture. 90 volunteers took part in the study. Two volunteers were excluded because of misunderstandings in filling in the study information form. 88 volunteers were included in the study. Most of the study participants had participated also in other studies evaluating collection of forensic evidence.2,3 The women were instructed to record the time of their last intercourse before the studied urine collection, the urine sample collection time and which of the consecutive post-coital voidings (1st, 2nd etc.) was collected as a specimen. A medical history was given by the volunteer using a form. Contraception, time of previous intercourse before the intercourse for the sample collection, frequency of intercourse in the past two weeks prior to the sample collection, activities following the intercourse (wiping, washing, showering, going to the sauna, urinating, defecating) and duration of intercourse were recorded.
51
2.3. DNA extraction and quantification The amount of urine and the pH of the urine were measured. The entire volume in the urine samples were centrifuged for 10 min at 3000 rpm and the supernatant was decanted. After centrifugation the supernatant (urine) was poured out and the cellular deposit at the base of the centrifuge tube was collected for analysis. From 200 ml of cell pellet DNA was extracted using QIAamp DNA Mini Kit® (Qiagen, Germany). Centrifugation steps were carried out at room temperature according to the protocol. Quantifiler Y Human Male DNA Quantification Kit® (Applied Biosystems, USA) was used to quantify the total amount of amplifiable human male DNA. PCR reactions and analysis were done with AbiPrism® 7000 HT Sequence Detection System (AppliedBiosystems, USA) according to the instructions provided. The PCR of Quantifiler Y Human Male DNA Quantification Kit® method is standardized and we used recommended water as blank controls alongside the purification and amplification steps. A sample was considered positive if a measurable amount of DNA was detected. The cut-off limit for possible DNA identification of the male was considered to be 0.01 ng/ml and according to the recommended cutoff limit of 0.023 ng/ml for Quantifiler Y. 2.4. Microscopic analysis Immediate microscopy was performed in a subset of 111 (54.1%) urine samples from 47 volunteers in a forensic laboratory. The following parameters were examined: volume, motility and sperm density. The density was categorized to no sperm, a few sperm (1e10/slide), a moderate amount (10e50/slide), and many (>50/ slide). The examination was performed according to the WHO laboratory manual 1999.4 Each sample was suspended on a slide by mixing the sample with a small amount of a culture medium (5 ml). The covered preparation was examined under the microscope. All samples were examined by phase-contrast microscope (magnification of 10 20). 2.5. Contamination issues
2.2. Sample collection Following consensual intercourse, volunteers were advised to void into a collection bottle (volume 500 ml, Sarstedt prod. no 77.582, Germany) so that the rim of the collection bottle would lie tightly onto the perineum. The purpose was to collect the outflow of vaginal secretions as much as possible into the collection bottle when physiologically relaxing the pelvic floor muscles while urinating. The urine volume was collected as much as possible into the collection bottle. The volunteers were advised not to worry if the urine volume exceeded the volume of the collection bottle. The first post-coital voiding urine sample was instructed to be collected without washing or without any other possible post-coital activities. Post-coital activities were allowed for the following collected urine samples. A single-use collection bottle was used for each episode of voiding. The urine collection bottle(s) were used for preserving the urine in the refrigerator. The collection bottle was labelled by (1) the study number given to the volunteer earlier during invitation and the volunteer (2) numbered the post-coital voiding time and marked it on the side of the collection bottle. The collection bottle was returned to the examiner as soon as possible but not later than 5 days. Time of the studied intercourse, time of the collected urine sample, post-coital activities, and the numbering of post-coital voiding times were recorded on the study information form.
The possibility of contamination has to be considered. All the urine sample processing, extraction and amplification steps were performed by the same female experimenters to avoid secondary YDNA contamination. Disposable powder free gloves were used and changed frequently to minimize the contamination risk. 2.6. Ethical considerations The study protocol was approved by the Ethics Committee (#R08018) of Pirkanmaa Hospital District, Finland. Written consent was required for participation in the study, for forensic laboratory analysis and for publication purposes. No data from the patient's medical records were collected. Full anonymity was secured in storage and laboratory analysis. 2.7. Statistical considerations Results were summarised by descriptive statistics; medians with range or frequencies (n) and percentages (%). Differences between detection of Y-DNA and spermatozoa were analyzed using the McNemar test. The Chi-square test was used to compare our microscopy results on detected spermatozoa rate in first void urine to results from the study by Smith et al. (2014). SPSS 21 was used for data analysis (IBM Corp. Released 2012. IBM SPSS Statistics for Windows, Version 21.0. Armonk, NY: IBM Corp.).
€ et al. / Journal of Forensic and Legal Medicine 37 (2016) 50e54 M. Joki-Erkkila
52
3. Results
3.4. Immediate phase-contrast microscopy
3.1. Volunteer characteristics
Immediate phase-contrast microscopy was performed on a subset of 111 urine specimens from 47 volunteers. Immediate microscopy verified spermatozoa within 24 h in 66 (59.5%) samples; few in 29 (26.1%), moderate amount in 15 (13.5%) and many in 22 (19.8%) samples at 10 20 magnification. Spermatozoa was detected in 37/44 (84.1%) in first void specimens. A significantly higher rate of spermatozoa was detected when compared to a recent study of Smith et al. (2014) of first void urine specimen in alleged sexual assault cases, 84% vs 35% (p < 0.001).
Age range was 20e52 years (median age 26.5 years). Eightyeight female Caucasian volunteers were included in the study. All reported penile-vaginal penetration. Most of the volunteers participated in other clinical studies by the study group.2,3
3.2. Y-DNA detection in urine samples The study consists of 205 urine specimens. The median urine volume was 175 ml (range 2e550 ml, Q1 ¼ 90 ml, Q3 ¼ 290 ml). The mean urine pH was 6.3 (range 4.8e8.2). No association was found between urine volume, urine pH and measurements of male DNA. Y-DNA was measurable in 173/205 (84.4%) urine specimens, median 0.68 ng/ml (range 0.0009e121.8 ng/ml). Of the 86 first post-coital void urine specimens available, Y-DNA was detected in 83 (96.5%) specimens. Y-DNA was still measurable in 70/119 (58.8%) urine specimens of the volunteers with post-coital activities (Table 1). The median time from consensual intercourse to urine specimen collection was 5 h (range 0.08e96.33 h). The median time for measurable male DNA was 9.2 h (range 0.08e52.5 h). Most (89.3%) of the urine specimens were collected within 24 post-coital hours (Figs. 1 and 2). After 24 post-coital hours Y-DNA was measurable in 9/22 (40.9%) urine specimens. No associations were found between duration of intercourse, coital frequency during the past two weeks prior to the study intercourse, post-coital activities, and the Y-DNA specimen positivity.
3.5. Sensitivity and specificity immediate phase contrast microscopy compared to Y-DNA in urine specimens A significant (p < 0.001) association was found between positive Y-DNA and detection of sperm in immediate phase contrast microscopy. Microscopy detected 66 (67.3%) of positive Y-DNA samples and failed to detect spermatozoa in 32 (32.7%) of Y-DNA positive samples. Compared to Y-DNA, the sensitivity of phase contrast microscopy was 67.4% (95% CI; 57.1e76.5%), but specificity was 100% (95%CI; 82.2e100%). 4. Discussion To our knowledge, this is the first study evaluating the value of urine specimens following consensual intercourse as a clinical forensic collection method for identification of Y-DNA and detection of spermatozoa. 4.1. Detection of Y-DNA and spermatozoa
3.3. Cut-off limits for possibility of DNA-identification in urine samples The cut-off limit for possible DNA identification of the male was hypothetically considered to be 0.01 ng/ml. Y-DNA of 0.01 ng/ml was quantified in 153/205 (74.6%) urine specimens. The mean of 0.01 ng/ml Y-DNA quantity was 4.2 ng/ml (median 0.9 ng/ml; range 0.01e121.8 ng/ml) (Fig. 3). The hypothetical DNA identification was also estimated according to the recommended cut-off limit of 0.023 ng/ml for Quantifiler Y. The male DNA amount was below 0.023 ng/ml in 28/153 (18.3%) urine samples. After 24 post-coital hours Y-DNA remained measurable in 9/22 (40.9%; range 0.0013e6.6 ng/ml) urine specimen. And in five of these measurable specimens Y-DNA was quantified over 0.01 ng/ml. One of the urine specimens was positive even at 52.5 h containing 0.025 ng/ml of Y-DNA, making it still sufficient for possible DNA identification.
Table 1 Measurable male DNA in urine specimens collected following consensual intercourse according to the number of post-coital voiding. Y-DNA
Post-coital voiding samples First void
Positive Negative
Second void
Third void
Fourth void
Fifth void
Sixth to tenth void
n ¼ 86
n ¼ 43
n ¼ 27
n ¼ 11
n ¼ 10
n ¼ 28
n
%
n
%
n
%
n
%
n
%
n
%
83 3
96.5 3.5
31 12
72.1 27.9
15 12
55.6 44.4
8 3
72.7 27.3
8 2
80.0 20.0
8 20
28.6 71.4
The detection rate of Y-DNA in urine specimens was very high when collected within 18 post-coital hours. Overall, the time frame for positive Y-DNA detection was for over two post-coital days, whereas spermatozoa were detected up to 24 post-coital hours in collected urine specimens. Most of the urine specimens were collected within the 24 post-coital hours, but still in 40% of urine specimens Y-DNA was quantified after 24 h. Furthermore, the hypothetical possibility for male DNA identification was considered in 75% of urine specimens. Our results suggest that urine specimens for forensic purposes might be collected even up to three days in suspected sexual assaults. Our study detected a significantly higher rate of spermatozoa (84%) in first void urine specimen following consensual intercourse when compared to a recent reported study of alleged sexual assault cases where the detection rate of first void urine spermatozoa was 35%.1 The difference in the detection rate may at least partly be due to lower ejaculation rates in assault cases. The quantity of measurable male DNA (median 0.68 ng/ml) in the post-coital urine specimens seems to be higher when compared to vaginal swabs (median 0.06 ng/ml), cervical swabs (median 0.01 ng/ ml), and to cervical brush (median 0.02 ng/ml) samples analyzed by the same quantification method.2 The benefit of correctly collected urine specimens is that it collects a higher quantity of male DNA in the sample, and it is a non-invasive collection method for forensic purposes. Also it may be considered a good forensic evidence collection method in acute suspected child sexual abuse cases, to diminish the time delay, and to improve patients' positive attitudes towards evidence collection.
€ et al. / Journal of Forensic and Legal Medicine 37 (2016) 50e54 M. Joki-Erkkila
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Fig. 1. Male DNA detected in urine samples according to the time from the consensual intercourse to urine specimen collection.
Fig. 2. Proportion of positive male DNA in urine samples according to the time from consensual intercourse to urine specimen collection.
4.2. Study limitations Confounding factors such as misunderstandings in filling in the study information form are possible. The collection bottle volume may have been too small for total volume collection which may have caused a bias in the urine volume. The volunteers were advised not to worry if the urine volume exceeded the volume of the collection bottle. We assumed that the volume of urine in sample collection is not as important as the collection outflow of vaginal secretions while urinating. The sample size at this point was rather small and there is a need for larger studies. The risk of contamination needs to be considered in this study. To diminish the risk of contamination, all the laboratory work was conducted by female experimenters. The quantified male DNA may also result from sources other than spermatozoa or semen such as male epithelial cells. A certain limitation of this study is the lack of using only single Y-DNA quantification PCR and not using Y-STR profiling. The detected male DNA of 0.0009 ng/ml was considered measurable because we assumed that Y-DNA cannot be detected from female
urine specimens otherwise than from a close contact with a male person, due to contamination, or due to rare XY-chromosomal syndrome in female. Detecting male DNA from female vaginal secretions most likely proofs a male contact. Detection of spermatozoa from vaginal secretions is a proof of sexual intercourse with a male person unless there has been an infertility treatment of intra uterine insemination performed shortly prior the specimen collection. We cannot rule out the presence of ‘non-sperm’ male DNA although no known vasectomy, azoo- or oligospermia were reported by the volunteers. We did not have the permission to evaluate medical records of the volunteers or their partners. Present study evaluated mainly the value of evidence collection technique of urine specimen for Y-DNA detection and not the laboratory quantitative PCR experiment techniques.5 Another limitation is the unsolved stability of DNA recovered from urine, due to differences in urine composition especially in long-term storage conditions, which is still under discussion. Unfortunately, the volunteers were not asked their opinion on possible ejaculation or known azoospermia, which may explain part of the negative Y-DNA results. We assumed voluntary penile vaginal penetration to include ejaculation.
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Acknowledgements We thank the Finnish Crime Laboratory for their contribution to the study. Conflict of interest None declared. Funding This study was financially supported by the Competitive State Financing of the Expert Responsibility area of Tampere University Hospital (9S013) and Clinical research fund of Tampere University Hospital. Ethical approval The study protocoll was approved by the Ethics Committee (#R08018) of Pirkanmaa Hospital District, Finland. Fig. 3. Quantification of human male DNA in urine samples collected following consensual intercourse according to time and 0.01 ng/ml cut-off limit. One specimen (121.84 ng/ml, collected 50 min following intercourse) was omitted from the figure as an outlier.
5. Conclusions Along with conventional invasive swab techniques, urine specimen collection may be used as an additional biological trace collection method for forensic purposes. It could be considered as a non-invasive collection technique especially in acute suspected child sexual abuse cases to diminish time delay and to improve patients’ positive attitudes towards evidence collection. Higher amounts of male DNA detected in urine specimens would offer better chances for DNA identification when compared to conventional swabs.
References 1. Smith DA, Webb L, Fennel A, Nathan E, Bassindale C, Phillips M. Early evidence kits in sexual assault: an observational study of spermatozoa detection in urine and other forensic specimen. Forensic Sci Med Pathol 2014;10: 336e43. € M, Tuomisto S, Sepp€ 2. Joki-Erkkila anen M, Huhtala H, Ahola A, Rainio J, et al. Clinical forensic sample collection techniques following consensual intercourse in volunteers - cervical canal brush compared to conventional swabs. J Forensic Leg Med 2014;27:50e4. € M, Rainio J, Huhtala H, Salonen A, Karhunen PJ. Evaluation of ano3. Joki-Erkkila genital injuries using white and UV-light among adult volunteers following consensual sexual intercourse. Forensic Sci Int 2014;242:293e8. 4. WHO laboratory manual for the examination of human semen and sperm-cervical mucus interaction. 4th ed. Cambridge University Press and The World Health Organization; 1999. 5. Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M, et al. The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem 2009;55:611e22.
