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.     

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.     

Improved detection of semen by use of direct acid phosphatase testing

Acid phosphatase (AP) reagent (Fast Black) is used as a presumptive test for the presence of seminal fluid on exhibits submitted in allegations of sexual assault. Research was carried out to determine whether the direct application of AP reagent to exhibits is a viable alternative to the traditional indirect (blot) testing method used routinely in the laboratory. The relative sensitivity of the indirect and direct testing methods was investigated as was the effect of AP reagent on histological staining of spermatozoa, the incidence of false positives from vaginal material and saliva, and the effect of AP reagent on subsequent DNA testing. Also included are the results of specificity studies from validations of the direct AP testing method. The results of this research show that, provided the incidence of false positives is borne in mind, direct AP testing can be especially useful when screening exhibits which are difficult to indirectly (blot) AP test or when it is problematic to relocate an AP positive stain. Direct application of AP reagent can also be beneficial for locating dilute semen stains. Three case examples are given which illustrate the use of direct AP testing in laboratory casework.     

Developmental validation of RSID™-Semen: a lateral flow immunochromatographic strip test for the forensic detection of human semen

Abstract: Tests for the identification of semen commonly involve the microscopic visualization of spermatozoa or assays for the presence of seminal markers such as acid phosphatase (AP) or prostate‐specific antigen (PSA). Here, we describe the rapid stain identification kit for the identification of semen (RSID?‐Semen), a lateral flow immunochromatographic strip test that uses two antihuman semenogelin monoclonal antibodies to detect the presence of semenogelin. The RSID?‐Semen strip is specific for human semen, detecting <2.5 nL of semen, and does not cross‐react with other human or nonhuman tissues tested. RSID?‐Semen is more sensitive with certain forensic evidence samples containing mixtures of vaginal secretions and semen than either of the commercially available PSA‐based forensic semen detection tests or tests that measure AP activity that were tested in parallel. The RSID?‐Semen kit also allows sampling a fraction of a questioned stain while retaining the majority of the sample for further processing through short tandem repeat analysis.     

人类岩藻糖基转移酶5特异性分布及其在精细胞的表达与定位

目的研究人类岩藻糖基转移酶5(fucosyltransferase 5,FUT5)的特异性分布及在精细胞的表达与定位。方法收集健康志愿者的精液(分离精细胞并提取精细胞膜蛋白)、阴道拭子、唾液及静脉血,应用免疫印迹方法检测FUT5在人类精细胞膜、精浆、阴道液、唾液及血清中的表达量,采用免疫荧光技术检测FUT5在精细胞中的表达与定位。结果免疫印迹方法结果显示FUT5在精细胞膜及血清中有较高表达,但在精浆、阴道液及唾液中未被检测到。免疫荧光实验结果显示FUT5主要存在于精细胞头部。表明人类精细胞膜存在一定表达量的特异性FUT5,可采用抗原-抗体反应分离精液和阴道液混合斑中的精细胞。结论人类FUT5表现出分布特异性,可应用到法医学性犯罪案件中混合斑的鉴定。     

用ELISA检测精液及精斑中精子抗原的研究

本实验应用单克隆抗人精子抗体和酶标记羊抗人精子抗体,采用ELISA方法确定精子抗原成份的存在。对10份新鲜精液,15份精斑进行了验测,其结果阳性率为100%。新鲜精液(精子数约10,000万个/ml)稀释100万倍,精斑浸出液稀释50万倍,均可出现阳性。对唾液斑、尿斑、乳汁斑、阴道斑、汗斑及输精管结扎的精液均为阴性。实验结果表明,本法检验精子抗原具有灵敏度高,特异性好的优点。     

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.     

A sandwich enzyme-linked immunosorbent assay for human seminal γ-glutamyl transpeptidase

A sandwich enzyme-linked immunosorbent assay (ELISA) was developed for detecting human seminal γ-glutamyl transpeptidase (γ-GTP) using a combination of anti-seminal γ-GTP monoclonal antibodies. These monoclonal antibodies did not react with human ovary or uterus in immunohistochemical study. Optimal assay condition, resulting in a sensitive assay with a low background, is presented. The detection limit of this assay was estimated to be 1 ng/ml of seminal γ-GTP corresponding to about 100 000 times dilution of seminal sample. This ELISA was specific for seminal γ-GTP, without cross-reactivity to renal or hepatic gg-GTP, normal blood serum, non-coital vaginal fluid or saliva. The recovery of seminal γ-GTP added to various biological fluids were also examined.     

混合斑中精斑ABO血型的检验

<正> 1988年,壹岐裕志等报告了用吸附抗α_2-SGP 血清的硝化纤维素膜(NCF)检验混合斑中的精斑 ABO 血型,但耗时。本文作者通过对此方法的改进,采用常彩琴等研制的抗人精特异蛋白血清(anti-human seminalpeculiar protein,ASPP),采用蛋清粘片热解离法检验混合斑中精斑 ABO 血型,耗时短,效果好。现介绍如下。     

A multi-enzyme electrophoretic system for the identification of seminal fluid from postmortem specimens

The screening of autopsy specimens, vaginal, buccal, and rectal swabs, for the presence of seminal fluid in rape homicide investigations utilizing classical techniques can lead to erroneous results. In the absence of spermatozoa, techniques are needed which can help to identify seminal fluid. This report illustrates the use of a multi-enzyme electrophoretic approach identifying seminal acid phosphatase (SAP) and lactic acid dehydrogenase (LDH-X) as an initial screening procedure. Subsequent analyses for the presence of acid and alkaline phosphatase (semiquantitative) yield information which can help identify false-positive SAP's. Additionally, salivary amylase can be tentatively identified using the same multi-enzyme procedure which informs the investigator of possible salivary contamination of the sample and possible erroneous PGM results. Statistics utilizing the multi-enzyme approach in case work are also presented.     

Evaluation of three rapid detection methods for the forensic identification of seminal fluid in rape cases

We sought to discover whether spermatozoa concentration and the delay between ejaculation and test influence the results of seminal fluid fast detection tests. Two hundred and twenty-seven anonymous samples divided into four groups (normospermia, oligospermia, azoospermia, and controls) after a semen analysis were subjected to three fast detection semen tests: Diff-Quick fast coloration, Phosphatesmo Km Paper for acid phosphatases (AP) detection, and PSA-Check 1 for prostate specific antigen (PSA) detection. The study was performed at three time points (0, 48, and 72 h). Unlike cytology, results obtained with AP and PSA were not influenced by spermatozoa concentration. PSA detection results remained constant up to 72 h and were more reliable after 48 h than those obtained by AP detection.     

自制与商业化抗人精液蛋白P30金标试剂条的法医学应用比较

目的　检测比较自制和商业销售的抗人精液蛋白P30 金标试剂条 ,探讨它们法医学应用的可靠性。方法　以人精液和人前列腺特异蛋白检测效价及灵敏度 ,以人阴道分泌物、人血清、人唾液和人初乳检测特异性 ,以保存不同年限的陈旧人精斑和斑痕类检材测定检验实际检材的能力。结果　自制抗人精液蛋白P30 金标试剂条效价及灵敏度与商业销售的试剂条中较好的基本相当 ,特异性则相当或好于商业试剂条 ,对非特异性测定均为阴性 ,而商业化试剂条则多有非特异性反应。结论　使用商业金标试剂条 ,要确定其是否合乎法医学检验标准与要求。     

精浆r—谷氨酰转移酶多态性研究

本次实验采用聚丙烯酰胺阶段梯度凝胶电泳方法,对武汉地区135名无关男性精液γ—GT进行了遗传多态性调查,根据γ—GT的电泳差异,分为1型、2型、2—1型三种常见表现型,1型为靠近正极的单一带,2型为靠近负极的单一带,2—1型为1型带和2型带组成的杂合双带。表现型频率分别为0.156,0.356,0.488,基因频率为γ—GT~1=0.400,γ—GT~2=0.600(P>0.5)。室温下保存8周的精斑,也能检测出较清晰的谱带。本次实验还探讨了单纯阴道分泌物及精液、阴道分泌物混合斑进行γ—GT分型的可能性。     

PCR-RFLP法检验混合斑中精斑ABO血型及基因型

运用PCR-RFLP检验方法,对21份精斑样品、5份混合斑检材进行了检验,结果表明该方法能够很好地检测混合斑中精斑的ABO血型及基因型,有效地解决了非分泌型精斑的ABO血型及基因型,拓宽了混合斑中精斑的检验范围.     

Advanced statistical analysis of Raman spectroscopic data for the identification of body fluid traces: Semen and blood mixtures

Conventional confirmatory biochemical tests used in the forensic analysis of body fluid traces found at a crime scene are destructive and not universal. Recently, we reported on the application of near-infrared (NIR) Raman microspectroscopy for non-destructive confirmatory identification of pure blood, saliva, semen, vaginal fluid and sweat. Here we expand the method to include dry mixtures of semen and blood. A classification algorithm was developed for differentiating pure body fluids and their mixtures. The classification methodology is based on an effective combination of Support Vector Machine (SVM) regression (data selection) and SVM Discriminant Analysis of preprocessed experimental Raman spectra collected using an automatic mapping of the sample. This extensive cross-validation of the obtained results demonstrated that the detection limit of the minor contributor is as low as a few percent. The developed methodology can be further expanded to any binary mixture of complex solutions, including but not limited to mixtures of other body fluids.     

The fallacy of the two-minute acid phosphatase cut off

Research was carried out to determine whether the likelihood of obtaining a positive Acid Phosphatase (AP) test result is affected by the make and type of paper used. Also, we aimed to investigate the frequency of AP positive reactions occurring after 2min using a series of known semen dilutions and to determine whether spermatozoa transfer onto the paper during the act of AP screening. In this research, most brands of paper tested were able to detect a 1 in 40 semen dilution within 2min. Leaving AP test papers for longer will allow the detection of greater dilutions of semen and as the amount of ejaculation is not reliably known in most casework situations and levels of AP activity can vary in different men, this will increase the seminal detection rate in sexual offence allegations.     

Vaginal fluid zinc concentration as a marker for intercourse

Zinc is present in high concentration in semen, but in low concentration in vaginal fluid. We evaluated vaginal zinc levels as a marker for intercourse by measuring precoital (greater than 11 h after intercourse) or postcoital (less than 5 h after intercourse) zinc and acid phosphatase levels in 26 specimens of vaginal fluid from 18 women. The approximate 95% reference range for zinc in precoital vaginal fluid was 1.2 to 15 micrograms/mL (mean 4.5), and in postcoital vaginal fluid 4.0 to 135 micrograms/mL (mean 24). There is an overlap between the precoital and postcoital reference ranges. Provided that the vaginal fluid zinc level is less than approximately 4.0 micrograms/mL or greater than approximately 15 micrograms/mL, vaginal fluid zinc concentration may be useful as an indicator of intercourse.     

A case study on forensic polymer analysis by DIOS-MS: the suspect who gave us the SLIP

New technology was used to identify traces of a commercial barrier/spermicide in evidence from a case of a man accused of rape of a minor. Examination of vaginal swabs performed by another laboratory had been negative for seminal fluid or other sources of DNA from the suspect and we were asked to examine the remaining swabs for any traces that might have originated from the commercial product. Encare consists of vaginal inserts having a suppository-like shape. They contain the spermicide, nonoxynol-9, in a matrix consisting of approximately two parts polyethylene glycol (PEG) 1000 to one part PEG 1450, plus minor inorganic components added to produce foaming. Portions of the cotton from vaginal swabs from the victim and penile swabs from the suspect were extracted with methanol and subsequently examined by desorption ionization on silicon time-of-flight mass spectrometry (DIOS TOF MS). Low levels of PEG in the same mass range as Encare were found on two separate vaginal swabs from the victim and one penile swab from the suspect. Subsequent to these findings, the suspect (through his attorneys) provided us with a sample of SLIP Plus, a commercial sexual lubricant that also contains nonoxynol-9. Traces of PEG in the same mass range as Encare were found in this sample, while no PEG was found in a sealed sample of SLIP Plus provided by the manufacturer. At trial the suspect's attorneys stipulated that their client had added some Encare to the SLIP Plus sample he had provided.     

Optimisation of choline testing using Florence Iodine reagent,including comparative sensitivity and specificity with PSA and AP tests

The detection of semen in forensic science is essential in cases of sexual assault but can be problematic in the absence of spermatozoa. Choline is known to occur in high concentrations in seminal fluid and the Florence Iodine test for its detection has been used in forensic science for many years, however very little is documented regarding its sensitivity and specificity in forensic casework. This paper describes the optimisation of the choline Florence Iodine test (FI) and investigates the sensitivity and specificity of the test against different body fluids, food and drink substances, cleaning products and laboratory chemicals. Comparative testing against Acid Phosphatase (AP) and Prostate Specific Antigen (PSA Seratec®) tests is described and shows that the FI test has greater specificity than the PSA test which cross reacts with a number of body fluids.