A dot-blot-immunoassay for semen identification using a polyclonal antibody against semenogelin, a powerful seminal marker

Among various seminal plasma proteins, semenogelin (Sg), produced in the seminal vesicle, has been considered a candidate for demonstrating the presence of semen. Sg consists of two proteins, one 52 kDa (Sg-I) in size, and the other a mixture of 71 and 76 kDa proteins (Sg-II). Recombinant Sg-I and Sg-II proteins were obtained using a baculovirus system and then injected into a rabbit to produce the respective antibodies [Characterization of recombinant precursor proteins of the human seminal plasma sperm motility inhibitor synthesized in insect cells, Int. J. Mol. Med. 2 (1998) 693]. When liquefied seminal plasma was immunoblotted with the anti-Sg-I and Sg-II antibodies, the anti-Sg-II antibody identified a wider range of the polypeptides originating from Sg than did the anti-Sg-I antibody. A dot-blot-immunoassay using anti-Sg-II antibody revealed a clear immunoreactive spot even when the semen was diluted 6400-fold. However, this assay showed that the Sg antigen was undetectable in saliva, urine, vaginal secretions, sweat, nasal secretions and serum. To determine the stability of Sg antigenic activity, filter paper with dried semen stains were kept at 37, 4 and 22 degrees C for 1, 6 and 18 months, respectively, and the Sg antigenic activity was examined. The activity was detectable in an area not less than 0.5 cm x 0.5 cm under all of the above environmental conditions during each period. Finally, semen was mixed with saliva or blood at various volumetric ratios, and used as a source of dried stains. The Sg antigenic activity was detectable in the stains until the ratio of semen to saliva or blood reached 1:8. These results suggest that Sg may be useful as a marker for semen identification.     

Isotachophoretic analysis of bloodstains: differentiation of human, menstrual, bovine, and ovine bloods

Isotachophoresis, a technique to separate components by constant current electrophoresis, was used to differentiate between bloodstains of male, female, menstrual, bovine, and ovine bloods on cotton cloth and filter paper. Bloodstain analysis by isotachophoresis of stains from male and female subjects showed identical cationic patterns, but gave different profiles in the anionic system. Plasma had one extra peak in the anionic system when compared to the profile of serum. This extra peak is due to the presence of fibrinogen in plasma. Some hemoglobin peaks overlapped with serum protein peaks, but these could be identified by comparisons at lower concentrations. Menstrual blood had a much different pattern than normal human blood as was expected since many more compounds are found in menstrual blood than in normally circulating blood. Human, bovine, and ovine bloodstains showed different profiles both in the cationic and anionic systems. These results indicate that isotachophoresis can be used for the rapid and simple analysis of bloodstains to differentiate reliably human male, female, and menstrual blood and also to distinguish human bloodstains from those of cattle or sheep.     

The analysis of biological samples from crime scene for a future human DNA profile confrontation. Effects of presumptive test reagents on the ability to obtain STR profiles for human identification

Because of the increase of evidence of blood stains, that have been washed or cleaned in an attempt to mask the analysis of DNA profiles, there is also an increase in the use of presumptive tests on samples sent to laboratories. Some of the presumptive tests, used to identify blood and semen stains, could potentially affect the recovery of high molecular weight DNA from the samples, or extinguish them, especially those already present in small quantities. After the presumptive tests, often these samples are discarded. This study aimed to examine the possibility of obtaining a DNA profile from samples submitted for presumptive testing and cleaned with bleaches with and without chlorine. Two different protocols were conducted: (a) A unique sample of human blood in natura (5 μL), already typed through the DNA techniques with the genetic profile previously known (control), was distributed onto cotton fabrics and dried at room temperature. Four samples of fabric were macerated in saline solution and Coombs serum and then stored for three months (room temperature and freezer −20 °C). (b) Another sample of human blood, type A, in natura, already typed through the techniques of DNA (control) was used. Aliquots of 200 μL were distributed in: cotton, denim and synthetic fabric. The samples were dried at room temperature for 24 h. The blood stains in those fabrics (cotton, denim and synthetic) were then divided into three groups: unwashed, cleaned with chlorine bleach and cleaned with chlorine bleach and soap powder. The samples were again dried at room temperature for 24 h, before the use of luminol. The DNA were extracted with Chelex 100 and amplified with the Identifiler Kit (Applied Biosystems). The blood stains exposed to saline and Coombs serum had DNA profiles consistent with untreated samples (controls). This result shows that the experts should keep and store the samples treated with saline and Coombs serum for future DNA confrontation when necessary. Also discussed in this paper the pattern of blood stains after washing with bleaching solutions, as well as the quantity of DNA obtained from these samples.     

Detection of human proteins in buried blood using ELISA and monoclonal antibodies: towards the reliable species indentification of blood stains on buried material.

The survival of human proteins in blood stains on fragments of cloth buried in exposed soil was examined in a 15-month investigation carried out from September 1990 to December 1991. During this period there was a wide variety of weather conditions. Samples were exhumed at 4-weekly intervals for 16 weeks and finally at 65 weeks; extracts of the stains were tested for albumin and IgG using a highly specific and sensitive enzyme-linked immunosorbent assay (ELISA) performed with monoclonal antibodies. Human albumin survived well throughout the 15 months of study, but IgG could be detected only in the 4- and 8-week samples. The reactions for IgG were weaker than those for albumin, although the method's sensitivity (10 ng) was the same for each protein. Appropriate buried and non-buried control experiments were carried out using cloth, either unstained or stained with human blood or animal sera; there was no cross-reactivity between human and the other species investigated and soil did not affect the assay; under laboratory conditions, IgG and albumin survived equally well. The system's versatility was illustrated by using monoclonal anti-bovine-albumin to detect specific albumin in the extracts of buried cloth which has been stained with bovine serum. It was concluded that ELISA performed with monoclonal antibodies could be of great value in identifying blood stains for forensic purposes.(ABSTRACT TRUNCATED AT 250 WORDS)     

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。     

The application of visible wavelength reflectance hyperspectral imaging for the detection and identification of blood stains

Current methods of detection and identification of blood stains rely largely on visual examination followed by presumptive tests such as Kastle–Meyer, Leuco-malachite green or luminol. Although these tests are useful, they can produce false positives and can also have a negative impact on subsequent DNA tests. A novel application of visible wavelength reflectance hyperspectral imaging has been used for the detection and positive identification of blood stains in a non contact and non destructive manner on a range of coloured substrates. The identification of blood staining was based on the unique visible absorption spectrum of haemoglobin between 400 and 500 nm. Images illustrating successful discrimination of blood stains from nine red substances are included. It has also been possible to distinguish between blood and approximately 40 other reddish stains. The technique was also successfully used to detect latent blood stains deposited on white filter paper at dilutions of up to 1 in 512 folds and on red tissue at dilutions of up to 1 in 32 folds. Finally, in a blind trial, the method successfully detected and identified a total of 9 blood stains on a red T-shirt.     

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。     

Specimen preparation for ABO determination of various kinds of blood stains

Trying to optimize the preparation of blood stains, we found methanol fixation not to produce very good results for the determination of ABO blood group antigens. It is advantageous to transfer blood stains before testing to cotton cloth. This transfer is also of practical use if blood stains are to be saved on a smooth surface for lateral determination. We testet on 35 different carrier materials, on which blood stains in casework often were found, whether blood grouping gave better results on either the original material or after transfer. Results are shown on a table. The test revealed, that solubility of the stain in aqua dest is a good sign for a successful transfer. Blood stains on pine-wood soil, soil and loam were not suited for ABO grouping.     

Identification of fetal hemoglobin in blood stains by high performance liquid chromatography

A new method for the identification of fetal hemoglobin (Hb F) in blood stains by reverse-phase high performance liquid chromatography is described. Differentiation between fetal and adult blood stains is based on the existence of gamma-chain peaks which are characteristics of Hb F. Very few gamma chains appeared on chromatograms of all the adult blood stains examined. The level of Hb F could be determined by measuring the total of chromatogram gamma-globin chain areas, and expressing it as a percentage of total Hb. Levels in six cord blood stains on filter paper ranged from 81.1% to 91.3% and remained constant for at least 12 weeks. This method is of great value for its simplicity, sensitivity and speed, and most importantly for its reliability in the field of forensic medicine.     

The influence of fabric surface characteristics on satellite bloodstain morphology

Bloodstains on fabrics such as clothing, soft furnishings or carpets are often encountered in casework. These stains often have a distinctive morphology that includes satellite stains, thought to be a highly sensitive feature that is a function of surface roughness. This study presents the findings of experimental studies conducted with proxy blood on two fabrics, similar in labeled composition, to assess the influence of fabric type on satellite stain generation. The morphology of proxy blood stains on the two fabric types were found to be statistically distinguishable from one another, with the volume of satellite stains generated being dependent upon the surface roughness of the fabric. These findings provide an initial step that illustrates the viability of providing an empirical evidence base for the interpretation of satellite stains in forensic blood pattern analysis (BPA).     

抗人精浆特异蛋白血清的制备与纯化

本文报导检验混合斑中精斑ABO 血型所用抗人精浆特异蛋白(anti-human seminal peculiarprotein,ASPP)血清的制备及用溴化氢活化琼脂糖4B 为载体的亲和层析纯化抗血清方法,并指出纯化时的最佳条件。该血清可准确地分离混合斑中精斑的ABO 血型。     

Identification and age estimation of blood stains on colored backgrounds by near infrared spectroscopy

Non-destructive identification and subsequent age estimation of blood stains are significant steps in forensic casework. The latter can provide important information on the temporal aspects of a crime. As previously shown, visible spectroscopy of blood stains on white backgrounds can successfully be used for their identification and age estimation. The use of this technique however, is hampered by dark backgrounds. In the present study the feasibility to use near infrared (NIR) spectroscopy was evaluated for blood stain identification and age estimation on dark backgrounds. Using NIR reflectance spectroscopy, blood stains were distinguished from other substances with 100% sensitivity and 100% specificity. In addition, Partial Least Squares Regression analysis was applied to estimate the age of blood stains on colored backgrounds. The age of blood stains up to 1 month old was estimated successfully with a root mean squared error of prediction of 8.9%. These findings are an important step toward the practical implementation of blood stain identification and age estimation in forensic casework, where a large variety of backgrounds can be encountered.     

Human genotyposcopy: the identification of the species, sex and individual by DNA genetic imprints in a case connected with a murder attempt]

Blood stains on a knife were identified by DNA genotyposcopy. The statistical validation method has confirmed that the blood stains on material evidence belonged to the victim, the probability of random coincidence being less than 10(-11). The efficacy of using hypervariable locus-specific DNA probes and the possibility of detecting DNA impressions in blood stains stored for more than 3 months have been demonstrated.     

Determination of phenotypes of haptoglobin and GLM(1) factor of human GM system in stains with admixtures of blood of some fish strains

It was shown that the phenotypes of haptoglobin (Hg) can not be detected in stains of dried blood of salmon, roach and bream. The results of experimental research of blood of the above fish species are described according to the Cm system. It was proven as possible to identify the human blood in stains with admixtures of blood of the above fish by using the Hp and Cm systems.     

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.     

用国产两性电解质进行血痕的种属鉴定

本文应用国产两性电解质等电点聚焦法(PAGIEF)对1个月的40种动物血痕浸出液及经PCMB处理后的血痕浸出液的谱型进行了研究。根据谱型特征,成人除与人胎儿及猴难以鉴别外,与其它动物均可鉴别。动物间除鸟纲,鱼纲内有五组动物组内难以鉴别外,其它均能鉴别。经PCMB处理后,成人与人胎儿的谱型仍无差别,与其它动物的鉴别则更容易。结果表明,国产两性电解质用于等电聚焦进行血痕的种属鉴别是完全可能的。     

Sensitivity of various study technics in blood stains

Quadratic pieces of fleece measuring 16 mm2 were soaked with 10 different blood-samples in the dilution steps of 1:1, 1:10, 1:100, 1:1000, respectively, and were tested in blood group typing and identification tests of forensic serology. The above spezified dilutions correspond with 5 microliters, 0.5 microliter, 0.05 microliter and 0.005 microliter of blood, respectively. The detection limit of the microspectrometric test for blood was the dilution 1:10, of the porphyrine test a dilution above 1:100, whereas the preliminary test for blood (peroxidase) succeeded always up to a dilution of 1:1000 and the species determination by the radial immunodiffusion test in agar gels succeeded in most cases op to a dilution of 1:1000. The detection limit of the anti-human globulin inhibition test was between the dilution steps 1:10 and 1:100 when non-titrated and undiluted anti-human globulin serum was used. Gc- and ABO-grouping were possible up to a dilution of 1:100 and were thus the most sensitive grouping systems. Phenotyping of the enzyme-systems and the Gm/Km-system usually required stains with considerably higher blood concentrations i.e. stains of undiluted blood.     

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 Lea substance in saliva stains by enzyme-linked immunosorbent assay (ELISA) using anti-gum arabic serum

It is known that rabbit anti-gum arabic (GA) serum has cross-reactivity with Lea antigen, and that, by using this cross-reactive anti-Lea antibody, the presence of Lea antigen in red blood cells and saliva can be demonstrated with accuracy. We have devised a rapid and highly sensitive method for detecting Lea substance in human saliva by the enzyme-linked immunosorbent assay (ELISA) method using an anti-Lea antibody isolated from anti-GA serum by affinity chromatography on Synsorb Lea. The ELISA plate, coated with the specific anti-Lea antibody, adsorbed the Lea substance in saliva which was subsequently identified by adding enzyme labeled anti-Lea IgG in that order. The method could detect the Lea substance in Le(a+) saliva stains as small as 0.1 by 0.1 cm in size that had been stored at room temperature for three weeks and in Le(a+) saliva stains 0.7 by 0.7 cm in size that had been stored for ten years. This method seems to be useful for quantitative analyses of the Lea substance in various body fluids.     

Determination of MN blood group from blood stains by electrophoresis and immunoblotting

The determination of blood groups from blood stains is extremely important in medicolegal practice, but there is the possibility of an error in the determination of MN phenotypes by the absorption-elution test. We investigated a new method applying electrophoresis and immunoblotting. As a consequence of various experiments, the most appropriate pretreatment of blood stains was as follows. Blood stains were immersed in physiological saline for 0.5 to 1 h and centrifuged. The supernatant was discarded. The sediment was dissolved in sample buffer (TRIS-buffered physiological saline containing 2% sodium dodecyl sulfate) and followed by thermodegradation. It was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). After transfer to a nitrocellulose membrane by Western blotting, MN phenotypes could be determined accurately from blood stains by an enzyme immunoassay (EIA) using commercially available polyclonal anti-M and anti-N sera. For blood stains more than 1 month old it was not easy to determine the MN phenotypes.