Evidence that "vaginal peptidase" is a bacterial gene product

A peptidase has been described in vaginal samples, termed "vaginal peptidase." This enzyme has been proposed as a tissue specific marker for vaginal debris. We have explored the presence of this enzyme in vaginal swabs from alleged sexual assault victims and volunteer donors as well as bacterial cultures. These studies reveal that "vaginal peptidase" is composed of a family of peptidase isozymes that originate from several bacterial species. The characterization of "vaginal peptidase" as a tissue specific marker for vaginal debris is premature.     

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.     

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.     

A demonstration of spermatozoa on vaginal swabs after complete destruction of the vaginal cell deposits

The proteolytic enzyme, proteinase K, has been found to destroy all vaginal cells though it does not have the same effect on spermatozoa. In cases of sexual offenses, in which a swab has been used to wipe out the vagina, the female cells and their nuclei on that swab may also contain the heads of spermatozoa. After as short a time as 30 min of proteinase K treatment, the spermatozoa that had separated from the enzymatically destroyed vaginal cells were recovered. This proteinase destruction furnishes some spermatozoa with deformed heads and a somewhat greater number of isolated tails though a sufficient number of spermatozoan heads still remain for a reliable diagnosis. For detection of spermatozoa from a vaginal swab after proteinase K pretreatment, the heads of the spermatozoa are distinctly stained by Oppitz's method. Further, on prior treatment with proteinase K, the ABO blood grouping of the spermatozoa could also be determined on the vaginal swab by using the absorption-elution technique. The resistance of the spermatozoa to proteinase K is the basis for this method.     

Alternative direct-to-amplification cell lysis techniques for forensically relevant non-sperm cells

While efforts have been made to reduce the pervasive backlog of sexual assault evidence collection kits, the actual laboratory process remains very time-consuming due to the requirement of a differential lysis step before DNA purification, as well as intricate mixture analysis towards the end of the DNA workflow. Recently, an alternative, direct-to-amplification sperm lysis method (using 1 M NaOH) was identified. However, a direct cell lysis method for non-sperm cells has not been identified yet. Thus, the primary objective of this work was to find an alternative method that is quick, inexpensive, and does not require multiple purification steps for the lysis of non-sperm cells in sexual assault samples. In this study, vaginal swab samples were lysed with the control method, prepGEM™, as well as six alternative reagents: alkaline buffer with 25–200 mM NaOH, high-salt stain extraction buffer, modified radioimmunoprecipitation assay (RIPA) buffer, mammalian protein extraction reagent (M-PER™), digitonin buffer, and urea/thiourea buffer. Quantification using Quantifiler® Trio of vaginal and semen lysates revealed that the alkaline (25 mM NaOH) and M-PER™ methods were efficient for the lysis of vaginal epithelial cells without substantial sperm cell lysis. Following quantification, analysis of STR profiles from vaginal lysates revealed that the M-PER™ method showed promising results across all metrics examined, including the percentage of detected STR alleles, mean peak heights, peak height ratio, and interlocus balance. Thus, this method was recommended as an alternative to the traditional differential lysis method for non-sperm cells given its ability to produce amplification-ready lysates without any DNA purification step.     

ASTM standards for fire debris analysis: a review

The American Society for Testing and Materials (ASTM) recently updated its standards E 1387 and E 1618 for the analysis of fire debris. The changes in the classification of ignitable liquids are presented in this review. Furthermore, a new standard on extraction of fire debris with solid phase microextraction (SPME) was released. Advantages and drawbacks of this technique are presented and discussed. Also, the standard on cleanup by acid stripping has not been reapproved.Fire debris analysts that use the standards should be aware of these changes.     

Information from penile swabs in sexual assault cases

A survey has been made to assess the evidential value of tests carried out on 660 casework penile swabs. Most were from suspects in sexual assaults and were examined to see if the donor had had recent anal, oral or vaginal intercourse. The swabs were tested for one or more of the following: blood, faeces, saliva, vaginal secretions, semen. Blood was seldom found, it was usually weak and insufficient for grouping. Faeces were only identified on a pair of swabs from a dead homosexual showing that proof of buggery by this means is rare. Amylase, suggestive of saliva and oral intercourse, was occasionally detected. Glycogen-rich epithelial cells were sometimes present indicating vaginal intercourse. Semen was frequently found but its presence may not result from a recent sexual act. An ABO group different from the donor was obtained from a fifth of the swabs typed. Grouping in other blood group systems was rarely attempted or successful. Penile swabs provided a means of detecting a victim's ABO blood group on a suspect when it would not have been possible to demonstrate the suspect's group on samples from the victim. They also had value in assaults involving more than one offender. The main limitation of penile swabs was the paucity of material on them and the sampling site affected the interpretation of the results.     

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.     

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。     

Comparison of enzyme assay and radioimmunoassay for the measurement of human acid phosphatase in cases of sexual assault

We investigated whether a radioimmunoassay for prostatic acid phosphatase might be used as a more specific test for the identification of semen in samples from cases of sexual assault than the measurement of total acid phosphatase enzyme activity. The results of the measurement of acid phosphatase by enzyme assay in semen and vaginal swab extracts were compared with the results of the radioimmunoassay. It was found that the radioimmunoassay is a sensitive and more specific method than the enzymic determination of acid phosphatase. Incidentally we have found that a low concentration of an immunological cross reacting acid phosphatase is present in semen free vaginal swab extracts.     

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.     

Saliva identification in forensic samples by automated microextraction and intact mass analysis of statherin

The enzyme α-amylase has long been a commonly targeted protein in serological tests for saliva. While being especially abundant in saliva, α-amylase is detectable in vaginal secretions, sweat, fecal matter, breast milk and other matrices. As a result, assays for α-amylase only provide a presumptive indication of saliva. The availability of mass spectrometry-based tools for the detection of less abundant, but more specific, protein targets (e.g., human statherin) has enabled the development of high confidence assays for human saliva. Sample throughput, however, has traditionally been low due to multi-step workflows for protein extraction, quantitation, enzymatic digestion, solid phase cleanup, and nano-/capillary-based chromatography. Here, we present two novel “direct” single-stage extraction strategies for sample preparation. These feature immunoaffinity purification and reversed-phase solid-phase microextraction in conjunction with intact mass analysis of human statherin for saliva identification. Mass analysis was performed on the Thermo Scientific Q-Exactive™ Orbitrap mass spectrometer with a 10-min analytical run time. Data analysis was performed using Byos^® from Protein Metrics. Two sample sets were analyzed with a population of 20 individuals to evaluate detection reliability. A series of casework-type samples were then assayed to evaluate performance in an authentic forensic context. Statherin was confidently identified in 92% and 71% of samples extracted using the immunoaffinity purification and solid phase microextraction approaches, respectively. Overall, immunoaffinity purification outperformed the solid phase microextraction, especially with complex mixtures. In toto, robotic extraction and intact mass spectrometry enable the reliable identification of trace human saliva in a variety of sample types.     

Cytochemical detection of ABH antigens in human body fluids

The use of the peroxidase-anti-peroxidase (PAP) technique has been described previously for the detection of cellular antigens and in particular ABO antigens from tissue samples (Pedal and Hülle 1984; Pedal and Baedeker 1985; Pedal et al. 1985). In this survey, the PAP method has been employed to study the detection of ABO antigens in cells from body fluids of particular interest to forensic science, namely buccal cells and vaginal cells. Also tested, but in a limited number, were mixtures of body fluids and semen samples. No false reactions were obtained from buccal cells, all samples corresponding to the ABO blood type of the donor. Preliminary results from vaginal cells, vaginal/buccal cell mixtures, and semen were encouraging but must be treated with caution due to the limited number tested. Vaginal smears contaminated with semen showed varying degrees of nonspecificity.     

Evaluation of a Headspace Solid‐Phase Microextraction Method for the Analysis of Ignitable Liquids in Fire Debris

The chemical analysis of fire debris represents a crucial part in fire investigations to determine the cause of a fire. A headspace solid‐phase microextraction (HS‐SPME) procedure for the detection of ignitable liquids in fire debris using a fiber coated with a mixture of three different sorbent materials (Divinylbenzene/Carboxen/Polydimethylsiloxane, DVB/CAR/PDMS) is described. Gasoline and diesel fuel were spiked upon a preburnt matrix (wood charcoal), extracted and concentrated with HS‐SPME and then analyzed with gas chromatography/mass spectrometry (GC/MS). The experimental conditions—extraction temperature, incubation and exposure time—were optimized. To assess the applicability of the method, fire debris samples were prepared in the smoke density chamber (SDC) and a controlled‐atmosphere cone calorimeter. The developed methods were successfully applied to burnt particleboard and carpet samples. The results demonstrate that the procedure that has been developed here is suitable for detecting these ignitable liquids in highly burnt debris.     

Comparison of gasolines using gas chromatography-mass spectrometry and target ion response

Gas chromatography-mass spectrometry was used to compare gasoline samples obtained from different sources based on the difference in amounts of certain components found in the headspace of gasoline using target response data. Many suspected arson cases involve comparing an ignitable liquid extracted from fire debris to a liquid found in a suspect's possession to determine if they could have had a common source. Various component ratios are proposed for determining if an unevaporated gasoline sample could have originated from the same source as an evaporated gasoline extracted from fire debris. Fifty and 75% evaporated gasoline samples were both found to contain similar ratios of certain components when compared with its unevaporated source gasoline. The results of the comparisons in this study demonstrate that for cases involving gasoline that has been evaporated up to 50% and extracted from pine, it is possible to eliminate comparison samples as originating from the same source. The results of the 75% comparisons suggest it may be possible to apply the same type of comparison to cases involving 75% evaporated gasoline.     

酶消化联合强酸消化法的硅藻检验CSCD

目的采用酶消化联合强酸消化法对溺死尸体的器官进行硅藻检验,并评价其应用价值。方法收集40例本地区确证为溺死的尸体,每例提取肺、肝、肾组织及现场水样,分别采用强酸消化法、酶消化联合强酸消化法、酶消化法对组织样本进行硅藻检验,从消化时间、消化能力、硅藻检出率等方面进行比较分析。结果在消化时间、消化能力方面,酶消化联合强酸消化法明显优于酶消化法;在硅藻检出率方面,酶消化联合强酸消化法明显优于强酸消化法。结论酶消化联合强酸消化法结合了强酸消化法和酶消化法的优点,并且操作安全,环境污染小,具有一定的推广和实践价值。     

用等电聚焦酶免疫分析法检测人类精斑GC表现型

本文用IEF结合使用过氧化物酶标记第二抗体的酶免疫分析法检测了17名键康成年男性的精液(斑)和唾液斑及10名健康成年女性的阴道分泌液中的GC表现型。结果发现17份人类精斑均可测出三种GC蛋白普通表现型。在10份阴道液中测出一份样本的GC表现型,3份样本有不甚清楚的GC带,不能定型,其他样本均无GC带。17份唾液斑未测出GC。本法的灵敏度(0.675ng)比文献报道的用过氧化物酶标记第二抗体的酶免疫分析(5.6ng)高。     

Identification of saliva stains by determination of the specific activity of amylase.

The specific activity (enzyme activity/protein concentration) of amylase was determined for the identification of saliva stains. The specific activity of amylase in saliva stains rapidly decreased during the first hour but, from 1 to 28 days, this decrease was much less when the stains were kept at room temperature. Stains of various human biological materials, breast milk, nasal secretion, meconium and vaginal secretion showed comparatively high amylase activity, but the saliva stains could be differentiated by their high specific activity of amylase, over 2 I.U./mg. When saliva stains were contaminated with blood or vaginal secretions at various ratios, the specific activity of amylase decreased with increase in the ratio of contaminant, especially when the contaminant was blood. However, the specific activity of amylase was still higher than 2 I.U./mg even after one fifth volume of blood was added or after five volumes of the extract of the stains of vaginal secretions were added.     

Optimization of solid-phase microextraction (SPME) for the recovery of explosives from aqueous and post-explosion debris followed by gas and liquid chromatographic analysis

Solid-phase microextraction (SPME) has been evaluated for the recovery of explosives residues from aqueous samples and real post-explosion solid debris samples and optimized using gas chromatography with an electron capture detector (GC-ECD) and high-performance liquid chromatography with ultraviolet detection (HPLC-UV). A modified SPME/HPLC interface utilizing dual six-port valves allowed for independent optimization of SPME desorption and injection variables that provided improved chromatographic resolution and sensitivity. A unique combination of cyano and octadecyl columns resulted in the complete separation of the 14 explosives in EPA method 8330 mixture using HPLC with good quantitative results. At the optimum SPME conditions, the limits of detection (LOD) were found to be of 5 ng/mL to 16 ng/mL of explosives in water and 10 microg/kg to 40 microg/kg of explosives from soil. The technique has been successfully applied to the analysis of real post-explosion debris and can be adapted for use in the field utilizing portable chromatographic instruments.     

Vaginal "fisting" as a cause of death

We describe the death of a young girl that resulted from the insertion of a clenched hand and forearm into her vagina during heterosexual activity. (The male homosexual practice of rectal fist insertion has been described previously.) We believe this death to be the first reported case of a "fisting" death due to vaginal fist insertion during heterosexual activity. This death is reported to alert forensic pathologists, medicolegal death investigators, and coroners aware of the role of aberrant sexual activity and its potential to cause death.