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.     

A medicolegal study on enzymic fluorometry of choline in human semen

Studies have been conducted on an enzymic fluorometric method based on an initial rate of reaction for the determination of choline. The reaction system consists of choline oxidase coupled to peroxidase and homovanillic acid. Concentrations of choline as low as 0.1 nmol could be detected by this procedure. The concentration of free choline in normal semen was 18.7 to 29.5 mumol/mL. Free choline in other body fluids was negligible. The choline concentrations in seminal stains maintained at room temperature were not changed during a 30-day period. Those concentrations in seminal fluids kept at room temperature were detected until at least the fifth day.     

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。     

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。     

Immunologic characterization of human seminal leucine aminopeptidase (LAP) and its medicolegal use

A new method for identification of seminal stains is described, based on the immunologic demonstration of leucine aminopeptidase (LAP), which is extremely abundant in human semen and specific for the prostate as well as semen. An antiserum against human seminal plasma was obtained by repeated immunization of rabbits with seminal plasma and Freund's adjuvant. Ouchterlony's double immunodiffusion test and Culliford's precipitin electrophoresis were performed to demonstrate specific proteins of seminal plasma. LAP activity was visualized with L-leucyl-beta-naphthylamide as substrate and with Fast Garnet GBC as coupler. The immunologic analysis of LAP produced two precipitin lines with enzyme activity. One was observed in kidney, jejunum, pancreas, prostate, as well as in semen, and was completely absorbed with kidney homogenates. The other was found only in semen and the prostate and was not absorbed with kidney homogenates. When the anti-seminal plasma serum absorbed with the kidney was used, the semen-specific LAP could be demonstrated by precipitin electrophoresis only in seminal stains stored for up to 2 months, whereas it was not demonstrated in stains from other human body fluids. By means of precipitin electrophoresis the detection of the semen-specific LAP was possible at semen dilutions of up to 1:32. The method described here greatly enhances the value of semen identification and is quite recommendable for the examination of stains in medico-legal practice.     

Detection of human seminal γ-glutamyl transpeptidase in stains using sandwich ELISA

A sensitive and specific sandwich ELISA for human seminal γ-glutamyl transpeptidase (γ-GTP) was developed using a combination of monoclonal antibodies, SG1 and SG3, which we produced. For semen identification in forensic samples, we modified the assay so as to be more sensitive and to establish efficient extracting conditions. After testing the extracting abilities of several detergents, CHAPS and deoxy-BIGCHAP were chosen as the solubilizer. Polystyrene beads coated with SG1 were incubated with samples extracted by the detergents, and further with biotinylated SG3, followed by peroxidase-labeled streptavidin. γ-GTP was detected only in seminal samples. The sensitivity of this assay was 0.01 ng/ml of seminal γ-GTP equivalent to 10⁷ times diluted semen, which was ten times as compared with the previous plate assay. No significant seminal γ-GTP was detected in other biological stains such as blood, saliva and vaginal smear. The extract of a 500 fold diluted seminal stain, 8 months old, showed the detection limit. Seminal γ-GTP was detectable even in 14-year-old stains.     

Identification of human semenogelin in membrane strip test as an alternative method for the detection of semen

Semenogelin (Sg), a protein originating in the seminal vesicles and a substrate for prostate specific antigen (PSA or p30), is a useful marker for the identification of semen. And detection of Sg has been available commercially in a membrane test recently. PSA is commonly used to detect semen in forensic significant samples taken from sexual assault cases. The strip PSA test has been available commercially from various manufacturers for many years. In this study, we evaluated two immunochromatographic membrane tests, one for Sg and the other for PSA by analyzing human semen, other human bodily fluids/materials including urine, blood, saliva, sweat, breast milk, vaginal secretion and fecal materials, semen from various animals and forensic casework samples. The data demonstrate that both Sg and PSA strip tests provide rapid and sensitive method for identification of seminal plasma. These results show that the immunochromatographic method for Sg detection is useful for the identification of seminal plasma in forensic samples, an alternative to the method for PSA detection.     

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.     

Comparison of Catalytic and Immunological Amylase Tests for Identifying of Saliva from Degraded Samples

The stability of salivary α‐amylase is a critical factor in both catalytic and immunological method‐based forensic saliva identification. This study aimed to assess the sensitivity of catalytic and immunological tests on degraded saliva samples. Degraded saliva stains were prepared by microbial decomposition using humid soil. Salivary α‐amylase activity was catalytically detected both qualitatively and quantitatively using the Phadebas^® amylase test. As immunological methods, we conducted qualitative and quantitative tests using the RSID?‐saliva test and ELISA, respectively. Salivary α‐amylase activity of degraded samples (incubated at 37°C for 12 h) was significantly lower than that of controls in the quantitative tests. All the degraded samples obtained by the humid soil produced negative results in the Phadebas^® tests, but showed positive results in the RSID?‐saliva test and ELISA. These results suggest that immunological tests are effective for testing degraded saliva samples that have lost their enzymatic activity.     

Comparative study of the sensitivity and specificity of the zinc and acid phosphatase spot tests for the detection of seminal stains

The sensitivity and specificity of a zinc spot test for the detection of semen were compared with those of an acid phosphatase detection method. As screening techniques both tests were found to be very sensitive, but the zinc test was more specific and was more reliable in older and especially in deteriorated specimens. It is concluded that the zinc spot test deserves at least the same place as the acid phosphatase test in the primary investigation of suspected semen stains and might well be the test of choice in older and poorly preserved stains.     

The detection of protein p30 in seminal stains by means of thin-layer immunoassay

The detection of p30 by means of an indirect thin-layer immunoassay (TIA) is described. Extracts from 20 samples can be analyzed in approximately 2 h with a detection limit of approximately 50 ng. The p30 protein was detected in seminal stains which had been stored at room temperature for six months and at 130 degrees C for 4 h. Blood, saliva, urine, perspiration, and tears did not interfere with the method. The reliability of the method was demonstrated in a blind study.     

Practical value of LDH-X determination for the detection of sperm traces

The examination of 38 stains collected in sexual offences provided spermatozoa-positive, LDH-X-negative results in 4 samples and spermatozoa-negative, LDH-X-positive results in 6 samples. The results suggest that besides the microscopic detection of spermatozoa the demonstration of LDH-X should be performed in medicolegal identification of seminal stains.     

The LAP test. A new method for identification of seminal stains by a qualitative color reaction of leucine aminopeptidase.

A new simple method for identification of seminal stains is described. It employs a qualitative color reaction based on histochemical technique for demonstration of leucine aminopeptidase (LAP), which is extremely abundant in human semen. The method herein reported (the LAP test) is quite suitable for medicolegal examination of seminal stains as a preliminary test.     

Amylase levels in semen and saliva stains

Amylase levels were determined for 148 semen samples and 20 saliva samples as well as for their corresponding stains. The effect of aging on the detectability of amylase activity in these stains was also investigated. The Phadebas amylase test was used for the quantitative assay of amylase. High levels of amylase in fluid saliva resulted in high levels being detected in saliva stains. Lower levels present in most seminal fluids produce little or no detectable amounts of amylase in stains. Interpretations are made as to the possible sources of amylase activity found in stains from laboratory casework based on both the amylase concentration and the elapsed time between collection and analysis. The evidential value of the presence or absence of amylase activity in casework stains is also discussed.     

The establishment of the presence of sperm in stains by an agar gel electrophoretic method]

Comparative investigation of seminal stains and other secretions from human body and blood as well as objects of plant origin by gel electrophoresis using alkaline values of pH buffer solutions and subsequent enzymography for acid phosphatase made it possible to develop optimal conditions for detection of seminal presence in stains. Good preservation of seminal acid phosphatase in stains during several years is shown.     

gamma-Glutamyltransferase test as a new tool for identification of seminal stains

A simple qualitative method for identification of seminal stains based on a high activity of gamma-glutamyltransferase (gamma-GTP) in human semen is described. It employs the release of alpha-naphthylamine from N-gamma-glutamyl-alpha-naphthylamide by the gamma-GTP action: alpha-naphthylamine couples with Fast Garnet GBC salt to produce a strong brownish-red color. The data on its simplicity, specificity, and stability show that the present method is suitable for medicolegal examination of seminal stains as a preliminary test.     

型特异性沉淀素血清检验人唾液斑精斑ABO血型的研究与应用

本文介绍了利用型特异性沉淀素血清环状沉淀法检验人唾液斑、精液斑ABO血型的方法与实验结果,并与中和试验及解离试验进行了比较。实验结果表明,本法操作简便,对多种干扰条件下的唾液斑、精斑均具有高度的型特异性,并能从分泌液与血液的混合斑中准确地鉴别出分泌液的血型。本法仅需0.4cm的分泌斑纱线即可进行血型鉴定,其灵敏度高于中和试验而略低于热解离试验,并能有效地检出陈旧分泌液斑中的型物质,因此适于在实际检案中应用。     

Polymorphism of EsD by isoelectric focusing: phenotyping in human tissues, dental pulps, hair roots, and semen

The polymorphism of EsD was investigated in tissues of various human organs, dental pulps, hair roots, and seminal stains by isoelectric focusing. The method yielded an excellent resolution of the isoenzyme components. The time limits of determination were: in organ tissues 3 weeks, in dental pulps 1 week, and in hair roots several days. The 7-1 type was less stable than the common types. Phenotyping was possible from fresh semen samples, but was unsuccessful from dried seminal stains after storage. The results show that the EsD typing by isoelectric focusing is of practical use for medicolegal individualization of organs, teeth, and hairs.     

用斑点ELISA方法检测p30确证人类精斑

作者建立了检测精浆特异性抗原p30的斑点ELISA试验方法,确证人类精斑。p30最小检出量为0.4ng。用该法盲测了室温存放1年的生物性斑痕180例,无一例假阳性和假阴性,证明用此法确证人类精斑的敏感性和特异性均优于传统的方法,具有操作简便、快速,检材用量小,结果准确等优点。