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%.     

北京地区人群血清型α2HS频率调查与血痕中α2HS的检测

本文采用聚丙烯酰胺凝胶等电聚焦和免疫固定技术,调查北京地区随机人群的α2HS糖蛋白(α2HS)的频率分布。在185名无亲缘关系的健康人中,发现3种常见表型,即α2HS1-1型(99人)、2-1型(74人)、2-2型12人。未发现稀有型。基因频率为:α2HS~1=0.7351,α2HS~2=0.6490。室温中保存6个月的血痕,可检出其α2HS表型。     

Phenotyping of alpha-2-HS glycoprotein in bloodstains by isoelectric focusing and immunoblotting

A sensitive immunoblotting procedure has been applied to the detection of alpha-2-HS-glycoprotein (A2HS) phenotypes from control and casework bloodstains. A2HS phenotypes were separated by thin layer polyacrylamide gel isoelectric focusing (PAGIEF) in gels containing Pharmalyte pH 4.2-4.9. After transfer to nitrocellulose by a rapid capillary blot, the A2HS phenotypes were developed using a double antibody enzyme-immunoassay. The evaluation of A2HS phenotyping of casework material was undertaken in parallel with phosphoglucomutase (PGM) phenotyping by PAGIEF. A total of 598 water extracts from casework bloodstains have been tested. Positive results were obtained in 84% and 75% of samples for PGM and A2HS respectively. The A2HS gene frequencies A2HS*1 = 0.6420, A2HS*2 = 0.3530, and A2HS*3 = 0.0050 were determined from a survey of 1000 people in Brisbane.     

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.     

The phenotypic frequencies of group specific component and alpha-2-HS-glycoprotein in three ethnic groups. The use of these proteins as racial markers in forensic biology

The phenotypic frequencies of group-specific component (Gc) and alpha-2-HS-glycoprotein (A2HS) were determined in White European, Asian and Afro-Caribbean populations. Typical allele frequencies were observed for Gc, with Gc 1S being the major allele for the first two groups and Gc 1F being the major allele for Afro-Caribbeans. For all groups the dominant A2HS allele was A2HS 1, although Asians had a significantly higher proportion of this allele than the White Europeans. Gc and A2HS either singly or in combination with other blood grouping systems provide good discriminating potential. The A2HS 10 allele was detected with a very low frequency in the White European group (A2HS 10 = 0.0013) and was not detected in the Asian group, while the Afro-Caribbean group had a relatively high frequency of this allele (A2HS 10 = 0.0966). The different distribution of the Gc 1F and A2HS 10 alleles in White Europeans and Asians compared with Afro-Caribbeans, can be used to determine the likelihood of blood coming from an Afro-Caribbean.     

Routine phenotyping of native and desialyzed alpha 2HS-glycoprotein from blood stains

The application of a polyacrylamide gel isoelectric focusing (PAGIEF) and immunoblotting procedure for the identification of native alpha 2HS-glycoprotein (AHSG) in routine casework blood stains has produced reportable results on 57.2% of samples. This reporting rate is lower than that for group specific component (GC) (83.8%) and phosphoglucomutase (PGM 1) (72.8%) phenotyping of the same samples. Blood stain samples were desialyzed with 1 U/ml neuraminidase, overnight at room temperature prior to PAGIEF in gels containing pharmalyte pH 5-6 and 2.5 M urea. Simple AHSG patterns were developed by immunoblotting. This procedure was five times as sensitive as the native AHSG method and desialyzation was reproducible over a range of incubation times and neuraminidase concentrations. The application of the desialyzed AHSG analysis to routine casework samples has resulted in a significant increase in the number of reportable results (762 reported out of 1027 samples). This reporting rate (74.2%) compares favourably with that for GC (79.1%) and PGH 1 (69.6%) phenotyping of the same samples. The three AHSG alleles (AHSG*1, 2 and 3) are clearly resolved after sample desialyzation and separation in gels containing pharmalyte pH 5-6 and 2.5 M urea. The sensitivity of desialyzed AHSG phenotyping approaches that of GC and this technique is worthy of inclusion in blood stain screening protocols of forensic laboratories in regions where the population has a limited range of rare AHSG alleles.     

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.     

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 development of an enzyme-linked immunosorbent assay for 19-OH PG F1 alpha/F2 alpha

An enzyme-linked immunosorbent assay (ELISA) has been developed for the detection of semen-specific 19-OH prostaglandin F1 alpha/F2 alpha (PGF). The assay can reliably and reproducibly detect as little as 2 pg 19-OH PGF/100 microliters and allows for the rapid analysis of large numbers of swab and stain extracts for the presence of semen.     

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.     

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.     

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.     

PGM1 subtypes determined by agarose gel isoelectrofocusing

PGM1 subtypes were determined in red cell hemolysates by isoelectric focusing on agarose gel plates. By this modified procedure PGM1 subtypes may be readily classified. Nine of the 10 expected phenotypes were found in a sample of 470 unrelated individuals from Southern Germany. The frequencies for the four alleles were found to be: PGM1(1+) = 0.212, PGM1(1-) = 0.1224, PGM1(2+) = 0.2043, PGM1(2-) = 0.0521.     

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.     

Screening of amphetamine/methamphetamine and their derivatives in urine using FPIA and Triage 8 and the Scope and limits of a subsequent identification by means of the REMEDi HS system

This study describes screening and identifying amphetamines, methamphetamines, and their derivatives in urine using immunochemical (Triage, FPIA) and chromatographic techniques (REMEDi HS). Amphetamines, methamphetamines, MDMA (3,4-methylenedioxymethamphetamine), MDA (3,4-methylenedioxyamphetamine), MDE (3,4-methylenedioxyethyl-amphetainine), MBDB (N-methyl-1-(3,4-methylenedioxyphenyl)-2-butanamine), BDB (3,4-(methylenedioxyphenyl)-2-butanamine), PMA (4-methoxyamphetamine), DOM (2,5-dimethyloxy-4-methylamphetamine), DOB (4-bromo-2,5-dimethyloxyamphetamine), amphetaminil, pholedrine, fenfluramine, and amfepramone were subjected to a comparative study. For this, the substances were analyzed to determine their specific threshold concentration for a positive detection in the Triage test and their limit of detection and positive threshold concentration for the FPIA test and the results compared. Furthermore, the capabilities of a more detailed analysis with the REMEDi system were studied. This HPLC system was able to produce information on the single drugs and main metabolites found in the sample with the danger of false-positive or false-negative screening results greatly minimized.     

Report of the blind trial of the Cetus Amplitype HLA DQ alpha forensic deoxyribonucleic acid (DNA) amplification and typing kit

The AmpliType HLA DQ alpha forensic DNA amplification and typing kit is designed for the qualitative analysis of the human leukocyte antigen (HLA) DQ alpha alleles present in deoxyribonucleic acid (DNA) extracted from forensic samples. The AmpliType kit is the first forensic DNA typing product based on the GeneAmp polymerase chain reaction (PCR) process. The kit was evaluated by five forensic science laboratories (test sites) to assess their ability to perform DNA typing using PCR on sample types typically encountered by forensic laboratories. None of the DNA-containing samples was mistyped. Of the 180 DNA-containing samples analyzed, results were reported for 178 (98.9%). Of the 178 samples with results, all were correctly typed. Two sites did not report a result for one sample each. Four of the five laboratories experienced no significant levels of contamination in the DNA-containing samples. At the one site with the highest number of DNA-containing samples with contamination, the typing results were not compromised. This site was able to correct the contamination problem through simple procedural changes and stricter attention to sterile technique. Blank controls were important to monitor contamination. In conclusion, the trial demonstrated that forensic science laboratories are capable of setting up a PCR-based DNA typing laboratory and successfully using the AmpliType HLA DQ alpha forensic DNA amplification and typing kit to analyze forensic samples.     

HS/GC/ECD分析生物检材样品中的氰化物

目的 建立生物样品中氰化物的衍生化定性定量分析方法。方法 用氯胺T衍生化,HS/GC/ECD分析衍生物CICN。结果 在1ml血中,添加0.2μg氰化钾,回收率为84.6％,RSD为6.39％;在1g肝中添加0.5μg氰化钾,回收率为67.3％,RSD为5.05％;血中检出限为5ng/ml。结论 所建方法能定性定量分析生物样品中的氰化物。     

Gc subtypes determined by ultrathin-layer isoelectric focusing. Distribution in the Veneto population (Italy)

The distribution of Gc phenotypes in the population of Veneto was investigated by ultrathin-layer isoelectric focusing. In our sample (n = 732) the six common phenotypes, Gc 1S, 1F, 1S1F, 2, 2-1S, 2-1F and a further phenotype, GC 1S1C3, were observed and the following frequencies calculated: Gc 1S = 0.560792; GC 1F = 0.159153; Gc2 = 0.277323; Gc 1C3 = 0.002732. Our gene frequencies have been compared with those found in other populations.