The evaluation of an ELISA for semen-specific 19-OH prostaglandin F1 alpha/F2 alpha using ex-casework swabs and stains

The application of an enzyme-linked immunosorbent assay (ELISA) for 19-OH Prostaglandin F1 alpha/F2 alpha (19-OH PG F) to casework analysis of seminal contamination of swabs and stains is reported. The results are compared to those where the identification of semen was based on the presence of acid phosphatase and spermatozoa. Five hundred and one samples were analysed and there was good agreement between the results of ELISA and conventional methods. The determination of 19-OH PG F confirmed both the presence of semen where spermatozoa were absent and indicated semen was present when acid phosphatase and spermatozoa were both absent. The results indicate that 19-OH PG F represents a useful marker for the casework identification of semen and is particularly valuable where spermatozoa are absent.     

Radioimmunoassay detection limits for 19-OH F1 alpha/F2 alpha prostaglandin in normal, infertile and vasectomized semen stains. Analysis of saliva, sweat and urine for possible non-specific or matrix effects

The sensitivity of a simple radioimmunoassay (RIA) for the detection of 19-OH prostaglandin F1 alpha/F2 alpha (PG F) has been evaluated on a number of semen samples from vasectomized, infertile and fertile donors. The specificity of the test has been examined by testing saliva, sweat and urine from a number of male and female donors. The assay technique could readily detect the PG in semen stains prepared from 0.2 microliter of semen from normal, infertile and vasectomized donors. The detection limit of the assay system, based on the observed displacement, was calculated to be approximately 0.05 microliter semen. The assay could be conducted over a pH range of 7.5-10.5 even after the PG has been heated to 100 degrees C. The 19-OH series of PG were absent from sweat, saliva and female urine using the normal assay protocol; volumes in excess of 100 microliters of some urines particularly from women in labour and those with acute urinary tract infection showed some displacement. Low levels of PG were detected in 50% of the male urine analysed. However, urine samples from men who had engaged in recent sexual activity contained relatively high concentrations of PG which could be readily detected in 10 microliters of urine. These results emphasise the potential of these compounds as specific and sensitive markers for the presence of human semen.     

A survey of the concentration of 19-OH F1 alpha/F2 alpha prostaglandins in the semen of fertile, infertile and vasectomized men and their stability in both liquid semen and semen stains

The levels of 19-hydroxy-prostaglandins F1 alpha/F2 alpha (PG F) in the semen of 19 vasectomized, 44 infertile and 8 fertile men were determined using a simple RIA technique. The mean concentrations observed in this survey were 45 micrograms/ml, 49.5 micrograms/ml and 59 micrograms/ml, respectively. No significant difference was recorded between the vasectomized and infertile groups; there were too few fertile samples available to undertake a meaningful statistical comparison. No reduction was observed in the levels of this PG in a liquid semen sample retained at room temperature over a 4 week period in the presence of a bacteriostat (sodium azide). However, a 30% reduction in the levels of 19-OH PG F occurred over the same time period when aliquots of the same semen sample were retained at either room temperature or at 4 degrees C without azide. Finally, no reduction was observed in the concentration of 19-OH PG F in a series of 10-microliters semen stains stored over a period of 6 weeks at room temperature.     

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.     

Evaluation of the 190H analogues of prostaglandins E1, E2, F1 alpha and F2 alpha as specific markers for the identification of human semen in body fluid mixtures

The specificity of antisera raised against each of the prostaglandin series 190H E1/E2 and 190H F1 alpha/F2 alpha, produced in males, was evaluated by radioimmunoassay. Further, the ability of these antisera to detect semen specific prostaglandins in mixtures of body fluids was examined. Antisera directed against the 190H E1/E2 series cross-reacted with prostaglandin E1 and marginally with E2. Antisera raised to the 190H F1 alpha/F2 alpha series were, however, highly specific to the semen specific prostaglandins 190H F1 alpha/F2 alpha and 190H E1/E2. It was possible to detect picogramme quantities of contaminating 190H F1 alpha/F2 alpha on vaginal swabs taken up to 72 h after intercourse and on vaginal swabs stored at room temperature for up to 2 years. These prostaglandins were not detected on semen free vaginal swabs, in faecal material, saliva, urine or in a sample of human milk (stain). A limited study of casework material is also described. Detection of the 190H F series, as a group, has considerable potential in the identification of human semen at picogramme levels, eliminating the need for alternative chemical tests and extensive microscopic examination.     

Changes in the amount of prostaglandin F2 alpha in incision wounds of the skin with different time intervals after injury

A radioimmunological method is presented for the determination of the quantity of prostaglandin F2 alpha in cut wounds on the skin of guinea pigs. Rapidly increasing quantities of prostaglandin F2 alpha can be found in skin cuts, and the level reaches 71 ng/g within the 1st hour after the injury. In the postmortem period, the quantities calculated in the cuts while the animals were still alive gradually decreased and reached a value of 17 ng/g in the 6th h after death. In postmortem cuts, inflicted in the 8th h after death, the prostaglandin was 14-18 ng/g. In later postmortem cuts the quantity was about 9-10 ng/g. Establishing the dynamics of the quantitative changes permits investigation of the prostaglandin to be used to certify whether the victim was alive or not, as well as when the skin damage was inflicted.     

A survey of the concentration of 19-OH F1α/F2α prostaglandins in the semen of fertile, infertile and vasectomized men and their stability in both liquid semen and semen stains

The levels of 19-hydroxy-prostaglandins F₁α/F₂α (PG F) in the semen of 19 vasectomized, 44 infertile and 8 fertile men were determined using a simple RIA technique. The mean concentrations observed in this survey were 45 μg/ml, 49.5 μg/ml and 59 μg/ml, respectively. No significant difference was recorded between the vasectomized and infertile groups; there were too few fertile samples available to undertake a meaningful statistical comparison. No reduction was observed in the levels of this PG in a liquid semen sample retained at room temperature over a 4 week period in the presence of a bacteriostat (sodium azide). However, a 30% reduction in the levels of 19-OH PG F occurred over the same time period when aliquots of the same semen sample were retained at either room temperature or at 4°C without azide. Finally, no reduction was observed in the concentration of 19-OH PG F in a series of 10-μl semen stains stored over a period of 6 weeks at room temperature.     

Classification of alpha 2-HS-glycoprotein (alpha 2HS) types by isoelectric focusing

A sample of 300 sera from unrelated individuals from Northern Japan was examined by isoelectric focusing on polyacrylamide gels. Three common types, alpha 2HS 1-1, 2-1, and 2-2 were differentiated. The frequencies of the alpha 2HS alleles in our sample were found to be: alpha 2HS1 = 0.7250 and alpha 2HS2 = 0.2750. Analysis of 16 parents with 21 children did not show deviations from the expected mode of inheritance.     

Comparability of the MMPI-2 F(p) and F scales and the SIRS in clinical use with suspected malingerers

Relationships among Structured Interview of Reported Symptoms (SIRS) scores and Minnesota Multiphasic Personality Inventory--2 (MMPI-2) F(p) and F scores were examined for 63 suspected malingerers evaluated at either of two psychiatric facilities. Despite differences between facilities in terms of seriousness of subjects' offenses, mean scores on the malingering tests were similar. Cutting scores for F(p) and F resulting in substantial correspondence between these scales and the SIRS were derived. Use of the cut score for F(p) proposed by Arbisi and Ben-Porath (1995) resulted in less agreement with the SIRS than did a lower cut score. No substantial difference between F(p) and F in each scale's overall agreement with the SIRS was observed. A principal components analysis of the SIRS primary scales produced two factors, interpreted as Overreporting of Symptoms and Implausible Symptoms. F(p) was observed to correlate significantly with Implausible Symptoms but not with Overreporting of Symptoms; F was significantly correlated with both factors.     

Genetic polymorphism of desialyzed alpha 2 HS-glycoprotein by ultrathin isoelectric focusing

The genetically determined polymorphism of alpha 2 HS-glycoprotein was analyzed by immunoblotting ultrathin-layer polyacrylamide gel isoelectric focusing in the pH range 4-6.5 and neuraminidase pretreated sera. In a Libyan population sample from Tripoli (n = 110) three common phenotypes, alpha 2 HSG 1-1, 2-1, and 2-2, were observed. The allele frequencies were alpha 2 HSG1 = 0.8364 and alpha 2 HSG2 = 0.1636. The theoretical exclusion rate in cases of disputed paternity is 11.8%.     

Validated method for the simultaneous determination of Delta9-THC and Delta9-THC-COOH in oral fluid, urine and whole blood using solid-phase extraction and liquid chromatography-mass spectrometry with electrospray ionization

A fully validated, sensitive and specific method for the extraction and quantification of Delta(9)-tetrahydrocannabinol (THC) and 11-nor-9-carboxy-Delta(9)-THC (THC-COOH) and for the detection of 11-hydroxy-Delta(9)-THC (11-OH THC) in oral fluid, urine and whole blood is presented. Solid-phase extraction and liquid chromatography-mass spectrometry (LC-MS) technique were used, with electrospray ionization. Three ions were monitored for THC and THC-COOH and two for 11-OH THC. The compounds were quantified by selected ion recording of m/z 315.31, 329.18 and 343.16 for THC, 11-OH THC and THC-COOH, respectively, and m/z 318.27 and 346.26 for the deuterated internal standards, THC-d(3) and THC-COOH-d(3), respectively. The method proved to be precise for THC and THC-COOH both in terms of intra-day and inter-day analysis, with intra-day coefficients of variation (CV) less than 6.3, 6.6 and 6.5% for THC in saliva, urine and blood, respectively, and 6.8 and 7.7% for THC-COOH in urine and blood, respectively. Day-to-day CVs were less than 3.5, 4.9 and 11.3% for THC in saliva, urine and blood, respectively, and 6.2 and 6.4% for THC-COOH in urine and blood, respectively. Limits of detection (LOD) were 2 ng/mL for THC in oral fluid and 0.5 ng/mL for THC and THC-COOH and 20 ng/mL for 11-OH THC, in urine and blood. Calibration curves showed a linear relationship for THC and THC-COOH in all samples (r(2)>0.999) within the range investigated. The procedure presented here has high specificity, selectivity and sensitivity. It can be regarded as an alternative method to GC-MS for the confirmation of positive immunoassay test results, and can be used as a suitable analytical tool for the quantification of THC and THC-COOH in oral fluid, urine and/or blood samples.     

Identification of fetal blood stains by radioimmunoassay of alpha 1-fetoprotein.

Radioimmunoassay of alpha 1-fetoprotein(AFP) for medico-legal identification of fetal blood stains using a commercial kit is described. The AFP content in fetal blood stains on filter paper ranged from 21--320 ng/9 mm2. The protein was detected in stains of adult blood and retroplacental blood in only negligible amounts. Aging of the blood stains did not influence the values up to 1 month. The method is simple and sensitive enough for application to medico-legal-practice.     

STR-typer 10G/F试剂在单亲鉴定中的应用

目的观察和分析STR-typer 10G/F试剂盒在单亲鉴定中的作用。方法随机抽样湖北地区汉族个体206人静脉血样,Chelex-100法提取基因组DNA。采用STR-typer 10G/F试剂盒进行复合扩增,分型并计数基因型并计算基因频率、基因座杂合度(H)、标准三联体非父排除率(PE3)和二联体非父排除率(PE2)。结果 9个STR基因座等位基因数9～14个,基因型分布观察值与Hardy-Weinberg平衡理论值的差异均无显著性(P>0.05),杂合度在0.75以上,平均非父排除率0.57～0.75;STR-typer 10G/F试剂盒累积非父排除率(PE3)0.999 973,合并使用Identifiler Plus系统,累积非父排除率可达0.999 999 967 6。结论 STR-typer 10G/F系统多态性程度基本接近Identifiler Plus系统,在单亲鉴定时,可作为Identifiler试剂盒的有效补充。     

Study of the skeleton of Admiral F. F. Ushakov

Reports the results of examination of the skeleton of Admiral F. F. Ushakov, carried out in connection with canonization in the Russian Orthodox Church. Heretofore unknown data on the somatic characteristics of F. F. Ushakov and his diseases are presented. Special attention is paid to uneven age involution of the skeleton and the hypoergic aging velocity. A detailed morphological similarity between the skulls of F. F. Ushakov and monk Feodor (Admiral's uncle, I. I. Ushakov) evidenced common features inherited through the masculine line. Modern computer technologies showed that the appearance of F. F. Ushakov, determined by the skull and postcranial skeleton, agree with his appearance on the life-time portrait, in spite of Professor M. M. Gerasimov's opinion (1949). In this connection, the possibility of repeated reconstruction of Admiral Ushakov's appearance is proven.     

Application of immunoblotting to serum protein phenotyping with reference to alpha 2HS-glycoprotein (AHS) typing of bloodstains

Sera and bloodstain extracts were subjected to isoelectric focusing in polyacrylamide gels. The focused proteins were transferred to nitrocellulose membranes by diffusion or electrophoretically, then allowed to react with specific antiserum and, after washing, with peroxidase-labeled anti-rabbit IgG. The immune complexes formed on the membranes were detected with 4-chloro-1-naphthol and hydrogen peroxide. Serum group-specific component, alpha 2HS-glycoprotein, the sixth and the seventh component of complement, factor 13B, and plasminogen could be phenotyped with high sensitivity. Bloodstains as old as 6 months could be correctly typed for alpha 2HS-glycoprotein by the blotting technique.     

Validated method for the simultaneous determination of Δ-THC and Δ-THC-COOH in oral fluid, urine and whole blood using solid-phase extraction and liquid chromatography–mass spectrometry with electrospray ionization

A fully validated, sensitive and specific method for the extraction and quantification of Δ⁹-tetrahydrocannabinol (THC) and 11-nor-9-carboxy-Δ⁹-THC (THC-COOH) and for the detection of 11-hydroxy-Δ⁹-THC (11-OH THC) in oral fluid, urine and whole blood is presented. Solid-phase extraction and liquid chromatography–mass spectrometry (LC–MS) technique were used, with electrospray ionization. Three ions were monitored for THC and THC-COOH and two for 11-OH THC. The compounds were quantified by selected ion recording of m/z 315.31, 329.18 and 343.16 for THC, 11-OH THC and THC-COOH, respectively, and m/z 318.27 and 346.26 for the deuterated internal standards, THC-d₃ and THC-COOH-d₃, respectively. The method proved to be precise for THC and THC-COOH both in terms of intra-day and inter-day analysis, with intra-day coefficients of variation (CV) less than 6.3, 6.6 and 6.5% for THC in saliva, urine and blood, respectively, and 6.8 and 7.7% for THC-COOH in urine and blood, respectively. Day-to-day CVs were less than 3.5, 4.9 and 11.3% for THC in saliva, urine and blood, respectively, and 6.2 and 6.4% for THC-COOH in urine and blood, respectively. Limits of detection (LOD) were 2 ng/mL for THC in oral fluid and 0.5 ng/mL for THC and THC-COOH and 20 ng/mL for 11-OH THC, in urine and blood. Calibration curves showed a linear relationship for THC and THC-COOH in all samples (r² > 0.999) within the range investigated.The procedure presented here has high specificity, selectivity and sensitivity. It can be regarded as an alternative method to GC–MS for the confirmation of positive immunoassay test results, and can be used as a suitable analytical tool for the quantification of THC and THC-COOH in oral fluid, urine and/or blood samples.     

Validation of a high performance liquid chromatographic method for alpha amanitin determination in urine

The objective of the present study was to develop and validate a liquid chromatographic method with electrochemical detection to measure alpha amanitin concentrations in urine after sample pretreatment with double mechanism (reversed phase/cation exchange) solid-phase extraction cartridges. The urine samples (10 ml) were purified and concentrated to 1 ml with elimination of matrix contaminants. The extracts were then separated by isocratic reversed-phase chromatography using a C18 column (4.6 mm×25 cm) with a mobile phase composed of 0.005 M phosphate buffer (pH 7.2) and acetonitrile (90:10). Coulometric detection was performed by applying an oxidation potential of +500 mV to a porous graphite electrode in a low-volume analytical cell. The limit of quantitation was 10 ng/ml with a signal-to-noise ratio=25. The linearity studied on spiked urine was satisfactory (r=0.9966) from 10 ng/ml to 200 ng/ml. The average extraction recovery of alpha amanitin was 78%, determined using spiked urine samples ranging from 10–300 ng/ml. The intra-assay precision was checked at 10, 50 and 100 ng/ml levels (n=10) in spiked urine samples, with resulting coefficients of variation of 3.6%, 2% and 1.5%, respectively.     

Use of polymerase chain reaction of the HLA-DQ alpha system in forensic trace analysis]

The polymerase chain reaction (PCR) can be used for genetic typing of minute amounts of biological stains. This is achieved by in vitro amplification of a well-defined and genetically polymorphic human genomic DNA sequence. Using the HLA-DQ alpha system, a population study was carried out among 212 unrelated individuals of German origin. The usefulness of this system is discussed by presenting examples of its application in forensic casework, i.e. the analysis of mixed (male/female) body fluids as well as segregation studies on embryonic and paraffin-embedded tissue samples.     

The BF F subtypes are detectable in the Ba fragment of factor B

The unanimous recognition of the two subtypes FA and FB of the BF*F allele has repeatedly been challenged. In the present investigation we are reporting about the unequivocal and simple detection of the subtypes on the Ba fragment of factor B by immunofixation isoelectric focusing after conversion with inulin. The common BF phenotypes F, S, and FS could be diagnosed in addition to the subtypes of BF*F which were observed in two regions acidic of the F major band. By comparison of standard phenotypes the subtypes in the Ba fragment corresponded to those of native factor B. All BF bands could be attributed to the Ba fragment by developing Western Blots with monoclonal antibodies directed against Ba. The distribution of the major BF phenotypes and alleles and the BF F subtypes in a population sample of 527 unrelated individuals from F.R.G. was in Hardy-Weinberg equilibrium. The allele frequency was determined to be 0.0731 for BF*FA, and 0.1053 for BF*FB. The advantages of determining the subtypes on the Ba fragment are: broadening of the FA/FB corridor, a more reliable diagnosis of phenotypes, improved distinction between homozygous FA and heterozygous FAFB types, and recognition of common BF phenotypes as well as subtypes in aged sera. It is suggested that the problem in the designation of BF F subtypes by different groups should be resolved by an international reference typing.