Possibility of the species identification using blood stains located on the material evidences and bone fragments with the method of solid phase enzyme immunoassay with "IgG general-EIA-BEST" kit and human immunoglobulin G

The authors give the comparative analysis of Russian and foreign forensic medical methods of species character identification of the blood from the stains on the material evidences and bone fragments. It is shown that for this purpose it is feasible to apply human immunoglobulin G (IgG) and solid phase enzyme immunoassay (EIA) with the kit "IgG general-EIA-BEST". In comparison with the methods used in Russia this method is more sensitive, convenient for objective registration and computer processing. The results of experiments shown that it is possible to use the kit "IgG general-EIA-BEST" in forensic medicine for the species character identification of the blood from the stains on the material evidences and bone fragments.     

Detection of blood in stains on material evidence by fluorescent hemotest and spectrofluorometry

The efficiency of detection of blood in stains on material evidence by spectrofluorometry and fluorescent hemotest developed at Bureau for Forensic Medical Expert Evaluations of the Leningrad region and by traditional methods used in Russia and abroad is compared. The proposed methods are 1000 times more effective than the methods routinely used in Russia; moreover, they allow objective computer recording and processing. These methods are 50-70 times cheaper than the methods used in foreign countries and are virtually as sensitive. Fluorescent hemotest persuasively proves the presence of blood in stains on material evidence under laboratory conditions, at the site of accident, and even under field conditions (express analysis).     

Detection of semen and blood stains using polilight as a light source.

Photoluminescence spectra of dry untreated semen have been measured and a suggested method for rapid detection of untreated semen stains is derived from these measurements. The method is presented in the form of a flow chart to cover most crime scene situations. The absorption spectrum of dry untreated blood has also been measured and a suggested method for enhancement and photography of blood stains is derived from this measurement. The method is presented in the form of a flow chart. Both methods are based on the use of a high intensity light source such as the Polilight.     

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.     

The use of Polilight in the detection of seminal fluid, saliva, and bloodstains and comparison with conventional chemical-based screening tests

Biological stains can be difficult to detect at crime scenes or on items recovered from crime scenes. The use of a versatile light source may assist in their detection. The ability of Polilight to locate potential semen, saliva, and blood stains on a range of substrates and at different dilutions was tested. We also tested the use of Polilight in comparison with conventional chemical-based presumptive screening tests such as acid phosphatase (AP), Phadebas, and luminol, often used in casework for detecting potential semen, saliva, and blood stains, respectively. The Polilight was able to locate stains that were not apparent to the naked eye. The color of the material on which a stain is deposited can have an effect on the detectibility of the stain. The Polilight was found to be comparable with the AP and Phadebas tests in terms of its sensitivity. In a comparative study between the AP test and Polilight on 40 casework exhibits, one false-negative result was observed when using the Polilight. On a series of mock casework exhibits it was determined that the Polilight can be used successfully to locate saliva stains for DNA analysis. The sensitivity of luminol for detecting potential bloodstains was greater than that of Polilight; however the Polilight has particular application in instances where a bloodstain may have been concealed with paint. Overall, the Polilight is a relatively safe, simple, noninvasive, and nondestructive technique suitable for use in forensic casework.     

Status and prospects of developing the Forensic Medical Service in the Russian Federation

Forensic medical service is constantly updated, which is confirmed by introduction of new modern methods, such as detection of blood or semen with test strips, new modifications of methods for handling bone material, differentiation of blood of the same group within ABO system, identification of blood groups in mixed stains of blood of humans and some animals, use of gel plates, etc. Methodological and information letters have been issued and field seminars are regularly carried for upgrading expert biologists. The overwhelming majority of expert biologists have received specialized education and topical upgrading.     

Development of a Real‐Time PCR‐Based Method for Analyzing Semen‐Specific Unmethylated DNA Regions and Methylation Status in Aged Body Fluid Stains

The detection of semen in forensic investigation is considered important evidence of sexual assault. In this study, we report the development of a real‐time polymerase chain reaction‐based method for identifying semen that can simply and rapidly analyze the semen‐specific unmethylated region of the DACT1 gene. Using two fluorescent probes designed for the methylated or unmethylated status, this method could perform quantitative analysis of the methylation status in this region. Furthermore, this method was used to analyze various body fluid samples, including 29‐year‐old semen and blood stains. The results showed that this method can detect almost exclusively semen or nonsemen signals even in highly decomposed samples, while a few semen or nonsemen samples showed slight signals of the other fluorescence probe. Although there is still a need for further analysis such as setting thresholds to analyze unknown samples, this method could be a useful supplementary tool for identifying semen, especially in old stains such as those in cold‐case investigations.     

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.     

Improving the effectiveness of fluorescence for the detection of semen stains on fabrics

The paper describes the use of the Polilight, a light source based on a xenon arc lamp, to exploit the fluorescence properties of semen as an aid to searching fabrics for stains in sexual assault cases. The broad excitation spectrum of semen allows the fluorescence to be generated at a range of wavelengths. This permits the excitation and emission conditions to be selected that minimize interference from background fluorescence of the fabric and thereby optimizes the contrast between the fabric and the stain. A common method for the observation of fluorescence is the use of colored plastic goggles or filters. The paper shows that the detection of fluorescence from semen stains is significantly enhanced using appropriate interference filters.     

New possibilities of detecting blood and seminal fluid in traces on material evidence using Fluorat-02-3M analyzer

Fluorat-02-3M liquid analyzer essentially extends the range of hemoglobin measurements, increasing its concentration in the studied extract from 0.1 to 100 micrograms in a 100 microliters sample (the lower threshold concentration remains unchanged: 0.005 in a 100-microliters sample) in detection of the blood in stains on material evidence pieces by fluorescent hemotest. This allows detection of blood in both washed and clearly seen blood stains on pieces of material evidence. Examples of using this method for measuring mass concentrations of zinc in water extracts on a Fluorat-02-3M analyzer are presented. The analyzer was developed at the Bureau of Forensic Medical Expert Evaluations of the Leningrad region for tentative detection of seminal fluid in stains on material evidence pieces. The results of this method are objectively recorded.     

Transferrin (TF) typing from semen stains using isoelectric focusing and immunoblotting: correlation of TF types among blood, semen, urine, and vaginal secretion.

We describe a method for obtaining nondistorted and reproducible transferrin (TF) typing from liquid semen and semen stains. Isoelectric focusing of TF isoproteins on polyacrylamide gel (IEF-PAGE, pH 4 to 6.5) was accomplished using a 0.5 mm thick gel. The separated isoproteins were then visualized by immunoblotting with TF-specific antibody. Pretreatment of semen samples with neuraminidase enhanced the TF band resolution. The method was reliable, sensitive and simple, with a high resolution. When maintained at room temperature, laboratory-prepared semen stains were TF-typable for up to at least 50 weeks. The TF types in semen stains were correlated with the types found in the corresponding blood and urine samples. TF typing could therefore provide an additional discriminant characteristic in the forensic examination of semen stains. An evaluation of TF typing by IEF-PAGE and immunoblotting was also performed on casework samples submitted to our laboratory.     

Acid phosphatase screening — Wetting test paper or wetting fabric and test paper?

Detecting and locating semen stains is addressed by identifying the associated acid phosphatase activity of semen. The recovery of semen stains is critical as it can, via DNA testing, address the possible source(s) of the semen and may aid in the interpretation of a case. The purpose of these experiments, carried out on behalf of the Body Fluids Forum, was to consider whether wetting the test paper alone or wetting the semen stained fabric and the test paper affected the detection and location of the semen stains on various fabric types, or the subsequent recovery of spermatozoa from these fabrics. It became evident that the preferred approach varied depending on the fabric type being tested but that more often than not, wetting both the fabric and the test paper had a detrimental effect on the recovery of spermatozoa.     

Comparison of laser and ultraviolet techniques used in the detection of body secretions

Evaluation of the detection capabilities of both laser and ultraviolet light sources was performed. The Spectra-Physics Model 171-19 argon ion laser was used in a comparison with the hand held Mineralight multiband ultraviolet lamp, Model UVSL-58 and the Fotodyne Foto UV 410, Model 3-4100. Both techniques were evaluated as to their detection limits for various biological stains. A serial dilution was made from semen, saliva, and sweat samples and their corresponding stains were examined under laser and ultraviolet light sources. The techniques were also evaluated as to possible interferences which may arise based on the type of fabric the stains were made on. The advantages and disadvantages of each technique in relationship to their initial costs are discussed.     

Application of an immunological method to detect trace amounts of dried human semen

An immunological assay based on a monoclonal antibody was used for identification of trace amounts of dried human semen in forensic science evidence. The monoclonal antibody (Mab 4E6) produced recognizes a human sperm-coating antigen which is specific to human seminal plasma. This antigen seems to be a protein secreted by the epithelial cells of the ejaculatory duct, which is stable indefinitely at room temperature. Mab 4E6 reacts positively with semen samples from individuals independently to their ABO group or secretory status, but does not react with semen from bull, ram, boar, horse, rabbit and dog. In the assay system developed, Mab 4E6 can detect human seminal plasma at concentrations of 0.5 micrograms/ml total protein. A similar sensitivity is found when human semen stains are eluted from forensic science samples and tested by the same assay. This method shows a good correlation with the microscopic methods routinely used. The method described is very sensitive and reproducible, it is time saving and special laboratory equipment is not needed.     

An evaluation of an enzymatic choline determination for the identification of semen in casework samples

This study compares the detection of choline in seminal stains by both an enzymatic method and by the standard Florence crystal test. The tests were conducted on 293 actual casework samples. In those samples identified as containing semen, choline was detected twice as often by the enzymatic method compared to the Florence method (84.6 versus 40.3%). The choline results were correlated with spermatozoa and acid phosphatase tests. The enzymatic detection of choline in seminal stains was found to be a fast, easy, sensitive, and reliable test.     

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.     

Comparison of laser and high-intensity quartz arc tubes in the detection of body secretions

The detection capabilities of both laser and high-intensity quartz arc tubes were evaluated. The Spectra-Physics Model 171-19, 18-W argon ion laser and Laser Sonics Model CS-2, 200-mW air-cooled argon ion laser were compared with Payton Scientific's Luma-Print, high-intensity quartz arc tube. The light sources were evaluated as to their detection limits for various biological stains. The stains that were evaluated had been made during prior research. These stains had been stored at room temperature for approximately two years. The stains were a serial dilution made from semen, saliva, and sweat specimens and were examined using both laser light sources and the high-intensity quartz arc tube. The advantages and disadvantages of each light source in relationship to its initial costs and potential use in forensic serology 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.     

Gm/Km determination in sperm traces

In the forensic laboratories of the Federal Republic of Germany and West-Berlin 23 different semen stains and in our own laboratory 20 semen stains were typed in the gm/km-system doing 125 and 61 (own) test respectively. Examination was carried out by means of the haemagglutination method, which has been used successfully in typing bloodstains. Our critical assessment based on earlier experiences with semen stains was now confirmed and statistically evaluated: typing was successful in about 35-50% of the tests, but besides false-negative results, there was also a considerable percentage (4-10%) of false-positive ones. Therefore for the present it seems best to exclude the gm/km-typing of secretion stains from forensic investigations in order to avoid false incriminations or exonerations of suspects.     

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.