The application of a simple RIA technique for the detection of 19-OH F1 alpha/F2 alpha prostaglandin, a specific semen marker, in semen contaminated vaginal swabs: time since intercourse studies

The specificity of the 19-OH F1 alpha/F2 alpha prostaglandin antisera for the detection of semen in seminal/vaginal mixtures, has been evaluated. Using a parallel curve test we found that the antibody showed a high specificity for these seminal prostaglandins in seminal/vaginal mixtures at concentrations of between 2 pg and 40 pg/100 microliter. The precision of the assay has been improved by the use of a donkey-anti-rabbit ferritin-bound second antibody. The application of this detection system makes it possible to complete an assay within 4.5 h. A survey of 50 semen-free vaginal swabs obtained from 3 donors, taken throughout the menstrual cycle, showed no trace of these prostaglandins. They were also not detected in the vaginal secretions of two further donors who were undergoing medication. Using only 10-microliter aliquots of a seminal/vaginal swab extract, prepared in 500 microliter, it was possible to detect semen up to approx. 80 h after one sexual act.     

Prostaglandin E in vaginal smears--a possibility for sperm detection in azoospermia]

The identification of seminal traces is exceptionally difficult, if the semen of the assailant is azoospermic. The evident value of prostatic acid phosphatase (PAP) activity must be evaluated in such cases with caution. In a murder investigation of a 13 year old girl a positive PAP reaction was found in vaginal swabs and in her underpants. Spermatozoa could not be found. Using the gas-chromatographic method, described by Douse (1985) the presence of prostaglandin E could be demonstrated in the swabs as well as in the crotch of the underpants. The offender was found to be a man with azoospermia, who admitted intercourse but with the consent of the victim. The E prostaglandins are mainly synthesized in the vesiculae seminal and seen to be specific for semen. Swabs taken from mouth and rectum showed negative reactions for prostaglandins in this case. Prostaglandins could never be detected in vaginal swabs taken at least 7 days after intercourse. Conversely Douse could detect prostaglandins in swabs up to 58 hours after intercourse. Apparently the prostaglandin detection by Douse provides a suitable alternative besides to the quantitative and immunological PAP detection or the immunological detection of the protein p 30.     

The development of an enzyme-linked immunosorbent assay for 19-OH PG F1 alpha/F2 alpha

An enzyme-linked immunosorbent assay (ELISA) has been developed for the detection of semen-specific 19-OH prostaglandin F1 alpha/F2 alpha (PGF). The assay can reliably and reproducibly detect as little as 2 pg 19-OH PGF/100 microliters and allows for the rapid analysis of large numbers of swab and stain extracts for the presence of semen.     

Radioimmunoassay detection limits for 19-OH F1 alpha/F2 alpha prostaglandin in normal, infertile and vasectomized semen stains. Analysis of saliva, sweat and urine for possible non-specific or matrix effects

The sensitivity of a simple radioimmunoassay (RIA) for the detection of 19-OH prostaglandin F1 alpha/F2 alpha (PG F) has been evaluated on a number of semen samples from vasectomized, infertile and fertile donors. The specificity of the test has been examined by testing saliva, sweat and urine from a number of male and female donors. The assay technique could readily detect the PG in semen stains prepared from 0.2 microliter of semen from normal, infertile and vasectomized donors. The detection limit of the assay system, based on the observed displacement, was calculated to be approximately 0.05 microliter semen. The assay could be conducted over a pH range of 7.5-10.5 even after the PG has been heated to 100 degrees C. The 19-OH series of PG were absent from sweat, saliva and female urine using the normal assay protocol; volumes in excess of 100 microliters of some urines particularly from women in labour and those with acute urinary tract infection showed some displacement. Low levels of PG were detected in 50% of the male urine analysed. However, urine samples from men who had engaged in recent sexual activity contained relatively high concentrations of PG which could be readily detected in 10 microliters of urine. These results emphasise the potential of these compounds as specific and sensitive markers for the presence of human semen.     

The evaluation of an ELISA for semen-specific 19-OH prostaglandin F1 alpha/F2 alpha using ex-casework swabs and stains

The application of an enzyme-linked immunosorbent assay (ELISA) for 19-OH Prostaglandin F1 alpha/F2 alpha (19-OH PG F) to casework analysis of seminal contamination of swabs and stains is reported. The results are compared to those where the identification of semen was based on the presence of acid phosphatase and spermatozoa. Five hundred and one samples were analysed and there was good agreement between the results of ELISA and conventional methods. The determination of 19-OH PG F confirmed both the presence of semen where spermatozoa were absent and indicated semen was present when acid phosphatase and spermatozoa were both absent. The results indicate that 19-OH PG F represents a useful marker for the casework identification of semen and is particularly valuable where spermatozoa are absent.     

A survey of the concentration of 19-OH F1 alpha/F2 alpha prostaglandins in the semen of fertile, infertile and vasectomized men and their stability in both liquid semen and semen stains

The levels of 19-hydroxy-prostaglandins F1 alpha/F2 alpha (PG F) in the semen of 19 vasectomized, 44 infertile and 8 fertile men were determined using a simple RIA technique. The mean concentrations observed in this survey were 45 micrograms/ml, 49.5 micrograms/ml and 59 micrograms/ml, respectively. No significant difference was recorded between the vasectomized and infertile groups; there were too few fertile samples available to undertake a meaningful statistical comparison. No reduction was observed in the levels of this PG in a liquid semen sample retained at room temperature over a 4 week period in the presence of a bacteriostat (sodium azide). However, a 30% reduction in the levels of 19-OH PG F occurred over the same time period when aliquots of the same semen sample were retained at either room temperature or at 4 degrees C without azide. Finally, no reduction was observed in the concentration of 19-OH PG F in a series of 10-microliters semen stains stored over a period of 6 weeks at room temperature.     

Changes in the amount of prostaglandin F2 alpha in incision wounds of the skin with different time intervals after injury

A radioimmunological method is presented for the determination of the quantity of prostaglandin F2 alpha in cut wounds on the skin of guinea pigs. Rapidly increasing quantities of prostaglandin F2 alpha can be found in skin cuts, and the level reaches 71 ng/g within the 1st hour after the injury. In the postmortem period, the quantities calculated in the cuts while the animals were still alive gradually decreased and reached a value of 17 ng/g in the 6th h after death. In postmortem cuts, inflicted in the 8th h after death, the prostaglandin was 14-18 ng/g. In later postmortem cuts the quantity was about 9-10 ng/g. Establishing the dynamics of the quantitative changes permits investigation of the prostaglandin to be used to certify whether the victim was alive or not, as well as when the skin damage was inflicted.     

An enzyme immunoassay for prostate-specific p30 antigen detection in the postcoital vaginal tract

A sandwich enzyme immunoassay test utilising a mouse monoclonal antibody has been adapted for the sensitive detection of the p30 prostatic antigen in semen and in postcoital vaginal swabs. In liquid semen, p30 was still detectable at a 1/1,000,000 dilution, and it could still be detected on vaginal swabs 24 h postcoitus.     

The relevance of false positive acid phosphatase reactions indicative of the presence of seminal fluid from oral and vaginal samples

The Body Fluid Forum of the Association of Forensic Science Providers recognised concerns raised by forensic practitioners regarding the lack of data to inform on the incidence of significant AP (Acid Phosphatase) reactions from vaginal and oral samples, i.e. those which might be misinterpreted as indicating the presence of semen. This is particularly relevant in the light of appeal court rulings regarding the need for data to support evaluations. This paper presents collaborative data on the nature and incidence of AP reactions from vaginal and oral swabs provided by donors. The results demonstrate that caution is required with mid to strong purple AP reactions from direct testing of oral swabs and with mid purple reactions from vaginal swabs. The use of a Bayesian approach to assist with the provision of opinions regarding the presence of seminal fluid is highlighted.     

制备抗人精液特异蛋白P30血清的研究

作者以精浆特异蛋白P30为抗原免疫新西兰白兔、豚鼠和鸡三种实验动物,制备了抗P30血清。用双向琼脂扩散法检测兔和豚鼠的抗 P30血清,其特异性和敏感性均达到目前国外同类产品的水平。抗 P30血清与阴道分泌物,血清、唾液、尿液、初乳以及羊精液、鸡精液均不出现交叉反应。用抗 P30血清检测混合的人精浆,其抗原效价为1:160;P30含量可测到12.5ug/ml。在三种动物的抗血清中,豚鼠抗 P30血清的抗体效价最高。以不同浓度的 P30测豚鼠、兔和鸡抗 P30血清抗体效价豚鼠平均滴度可达52.50,兔次之,鸡的抗 P30血清最差。经作者制备的抗 P30血清可用来确证精液。     

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.     

Studies on glyoxalase 1 isoenzymes in semen stains: polymorphism in Himachal (India) population

One hundred and ten pairs of blood and semen samples and their stains were studied to type glyoxalase 1 (GLO 1) isoenzymes using agarose-starch medium. A good agreement was observed between the phenotypes expressed in blood and semen samples of the same donor. No GLO 1 activity however could be demonstrated in the vaginal swabs tested. The gene frequencies of GLO 1 polymorphs in Himachal population has been worked out and their stability studies carried out at -12 degrees C and at room temperature.     

Evaluation of prostate-specific antigen (PSA) membrane test assays for the forensic identification of seminal fluid.

Prostate specific antigen (PSA, also known as p30), a glycoprotein produced by the prostatic gland and secreted into seminal plasma, is a marker used for demonstrating the presence of seminal fluid. Methods for the detection of PSA include Ouchterlony double diffusion, crossover electrophoresis, rocket immuno-electrophoresis, radial immunodiffusion, and ELISA. The extremely sensitive ELISA technique can detect PSA in concentrations as low as approximately 4 ng/mL. However, all these techniques are cumbersome and time consuming to perform in forensic laboratories, especially when only a few samples per week are processed. Various membrane tests are currently used in clinical settings to screen a patient's serum for the presence of PSA at levels greater than 4 ng/mL. In this study we evaluated three immunochromatographic PSA membrane tests by analyzing semen stains stored at room temperature for up to 30 years, post-coital vaginal swabs taken at different time after intercourse, semen-free vaginal swabs, and various female and male body fluids, including urine. The data demonstrate that PSA membrane test assays offer the same sensitivity as ELISA-based tests and provide a rapid approach for the forensic identification of seminal fluid. Furthermore, when the supernatant from a DNA extraction is used for the assay, there is essentially no DNA consumption for determining the presence of PSA in a forensic sample.     

PCR－RFLP技术鉴定ABO基因型的法医学应用研究

目的建立PCR－RFLP、非变性PAG胶垂直电泳和银染技术进行ABO基因分型的方法体系,并对200名广东汉族人群ABO基因型频率进行了调查。方法用Chelex－100和酚、氯仿抽提法处理样本,PCR扩增后用非变性聚丙烯酰胺凝胶垂直电泳和银染技术检测分型。结果ABO位点特异性扩增片段长度为175bp～210bp,6种基因型频率分布为0.0250～0.4300,杂合度H值为0.5162,个体识别力DP值为0.7111。结论该方法可成功运用于血液、血痕、精斑、毛发、骨组织和混合斑等检材的个体识别及亲权鉴定的检验。     

Validation of the use of a commercially available kit for the identification of prostate specific antigen (PSA) in semen stains

PSA is currently being used to detect and monitor quantitatively the development of prostate cancer by serum levels of PSA and has also been found to be present in high concentrations in semen. Elegantly simple, sensitive, and reproducible methods have been developed for analysis of the presence of PSA, including the Tandem-E PSA Immunoenzymetric Assay. The most common procedures for the forensic identification of semen have focused on the microscopic detection of sperm, acid phosphatase activity, and immunoelectrophoretic methods for the detection of PSA. Although these methods have been used for many years, there are problems associated with each method. The Tandem-E PSA Immunoenzymetric Assay detected PSA in 100% of the forensic casework fabric samples, 80% of the forensic casework vaginal swabs and 100% of the vasectomized individuals tested. The cut-off value was determined to be 1.77 ng/mL. These results indicate that this method can be used to identify the presence of semen in forensically significant specimens.     

A survey of the concentration of 19-OH F1α/F2α prostaglandins in the semen of fertile, infertile and vasectomized men and their stability in both liquid semen and semen stains

The levels of 19-hydroxy-prostaglandins F₁α/F₂α (PG F) in the semen of 19 vasectomized, 44 infertile and 8 fertile men were determined using a simple RIA technique. The mean concentrations observed in this survey were 45 μg/ml, 49.5 μg/ml and 59 μg/ml, respectively. No significant difference was recorded between the vasectomized and infertile groups; there were too few fertile samples available to undertake a meaningful statistical comparison. No reduction was observed in the levels of this PG in a liquid semen sample retained at room temperature over a 4 week period in the presence of a bacteriostat (sodium azide). However, a 30% reduction in the levels of 19-OH PG F occurred over the same time period when aliquots of the same semen sample were retained at either room temperature or at 4°C without azide. Finally, no reduction was observed in the concentration of 19-OH PG F in a series of 10-μl semen stains stored over a period of 6 weeks at room temperature.     

Use of a Y chromosome probe as an aid in the forensic proof of sexual assault

Currently, the most common procedures for the forensic identification of semen that may be present due to a sexual assault include the microscopic identification of spermatozoa, acid phosphatase activity, or the detection of PSA. However, not all cases of sexual assault result in the deposit of semen. Fluorescent In Situ Hybridization (FISH) has been found to be a very sensitive and specific method for detection of the Y chromosome from male cells. This study was undertaken to demonstrate the presence of epithelial cells of male origin in the postcoital vaginal tract using a commercially available probe. Results identified Y chromosome in intact epithelial cells on postcoital Days 1 through 4, and on Day 7. Additionally, Y chromosome positive epithelial cells were identified in vaginal swabs obtained following intercourse with no ejaculation. The method developed in this study demonstrates that FISH is a sensitive method for the identification of the presence of male epithelial cells in the postcoital vagina.     

An evaluation of the polymerase chain reaction method for forensic applications

This paper describes the use of polymerase chain reaction (PCR) for amplifying small amounts of DNA obtained from samples of interest to the forensic scientist. A region of the HLA DQalpha (DQa) locus was amplified in DNA prepared from the following: hair roots, liquid blood, blood-stains, semen and vaginal swabs (semen free and semen contaminated). A population study was conducted using DNA from 78 unrelated individuals. The observed distribution of HLA DQa alleles varied from that reported for an American population but obeyed the Hardy-Weinberg equilibrium. Interpretation problems associated with the PCR technique are discussed.     

Effects of presumptive test reagents on the ability to obtain restriction fragment length polymorphism (RFLP) patterns from human blood and semen stains.

Some of the commonly used presumptive test reagents for identification of blood and semen could potentially affect the recovery of intact high-molecular-weight deoxyribonucleic acid (DNA) from evidentiary samples. Thus, the capability of performing restriction fragment length polymorphism (RFLP) analysis on evidentiary samples could be compromised. In order to investigate the potential effects of presumptive test reagents on the DNA present in these samples, bloodstains on cotton and glass were exposed directly to luminol, benzidine, phenolphthalein, o-tolidine, and leucomalachite green, while semen stains and vaginal swabs containing semen were exposed directly to bromochloroindolyl phosphate (BCIP) and sodium thymolphthalein monophosphate (STMP) reagents. The yield gels for DNA quality and quantity and RFLP results indicated that bloodstains exposed to luminol, benzidine dissolved in ethanol, and phenolphthalein, as well as semen stains and vaginal swabs exposed to BCIP and STMP yield RFLP patterns consistent with that of the uncontaminated control. Except for the phenolphthalein treatment, the quantity of extractable, high-molecular-weight DNA obtained was comparable with that of untreated stains. Therefore, evidentiary material purposely or inadvertently contaminated with these reagents can be successfully typed. However, stains exposed to benzidine dissolved in glacial acetic acid, leucomalachite green, and o-tolidine failed to yield high-molecular-weight DNA or to produce any RFLP patterns.