Detection of group-specific antigens of the ABO system in fish blood

The subject of the case study was blood of 8 fish species. It was examined within the ABO system. 68 tests of stains of fish blood mixed with human blood were made. It was shown as possible to differentiate between human blood and fish blood in mixed stains.     

The detection of group-specific component from urine samples

The detection of group-specific component (Gc) from serum and bloodstains has been widely used in the forensic laboratory. A recent increase in substituted or adulterated urine samples in various drug screening programs has necessitated methods to determine the donor. This paper discusses the detection of Gc from urine samples. The samples were concentrated and applied to ultrathin polyacrylamide gel and focused for 150 min. This method separates the samples into the three common phenotypes found in all human populations. A nitrocellulose membrane blotting technique was used to detect the Gc bands. Serum and urine samples were collected from each individual and were typed for Gc. Urine samples tested after 6 months of storage (4 degrees C) were still readable. This method provides the forensic laboratory with an additional test from a body fluid which, until recently, provided little information.     

Analysis of hair after contamination with blood containing cocaine and blood containing benzoylecgonine

In post-mortem work, blood is a potential source of external contamination of hair. The present study was carried out to investigate the amount of drug absorbed into hair which has been contaminated with blood containing either cocaine or BE. Solutions were prepared containing 0.05, 0.1, 0.2, 0.5 and 3.0 μg/mL of either cocaine or BE in human blood. Samples of approximately 3.2 g of drug-free hair were contaminated by soaking in the blood solutions for 5 min. They were then removed and left at room temperature. Approximately 0.5 g of hair was collected from each of the blood soaked hair samples at 6 h, 1, 2, 4 and 7 days after contamination. As each hair sample was collected it was shampoo-washed to prevent further drug absorption. Hair samples were analysed in triplicate using a fully validated method described previously. EME and cocaethylene were also measured in order to find out if cocaine or BE was breaking down to these compounds. Both cocaine and BE were absorbed into hair in significant concentrations when the concentration in the blood was 0.5 μg/mL or greater; cocaine was more readily absorbed than BE. Cocaine broke down to EME (<LOQ) at 0.5 μg/mL and to EME (>LOQ) and BE (<LOQ) at 3.0 μg/mL. When the blood concentration of cocaine was 0.5 μg/mL or less, there was no evidence of it breaking down to form BE. From the samples soaked in blood containing BE, there was no evidence of the BE breaking down. The absorption of drug into hair did not increase as the contamination period increased from 6 h to 7 days.     

Analysis of hair after contamination with blood containing 6-acetylmorphine and blood containing morphine

The study was carried out to investigate external contamination of hair by blood in heroin-related post-mortem cases. Solutions were prepared containing 0.05, 0.1, 0.2, 0.5 and 3.0μg/mL of 6-monoacetylmorphine (6-AM) only or morphine only in human blood. Samples of approximately 3.2g of drug-free hair were contaminated by soaking in the blood solutions for 5min. They were then removed and left at room temperature. Approximately 0.5g of hair was collected from each of the blood soaked hair samples at 6h, 1, 2, 4 and 7 days after contamination. As each hair sample was collected it was shampoo-washed to prevent further drug absorption. Hair samples were analysed in triplicate using a fully validated method described previously. 6-AM broke down to morphine in all samples. In hair contaminated with blood containing 0.05, 0.1 and 0.2μg/mL 6-AM or morphine drug was either not detected or was detected below the limit of quantitation (0.2ng/mg hair) at all contamination times. In hair contaminated with blood spiked with 0.5μg/mL morphine, the concentration in hair ranged from 0.54 to 0.91ng/mg and in hair contaminated with blood spiked with 3.0μg/mL, from 3.25 to 5.77ng/mg. The concentrations of 6-AM ranged from 0.65 to 1.11ng/mg and morphine from 0.34 to 0.80ng/mg in hair contaminated with 0.5μg/mL 6-AM in blood. 6-AM ranged from 2.12 to 3.67ng/mg and morphine from 0.84 to 2.05ng/mg in hair contaminated with 3μg/mL 6-AM in blood. For 6-AM and morphine ANOVA statistical evaluation showed no significant difference among the concentrations over time.     

Micromethod for the detection of erythrocyte antigens in blood stains

A micromethod was developed to allow the analysis of blood stains of minor size by the absorption elution technique. The individual absorption, washing, and elution steps were carried out in Beckman tubes containing 5 microliter antiserum. The final agglutination reaction was read through the inverted microscope in microtest plates regularly used for HLA typing. For this final reaction, 2-4 microliter eluate was incubated with 2,000 red blood cells suspended in 1 microliter saline and supplement. For the purpose of standardization, the intensity of agglutination in the microtest plate had to be defined. In comparison to the standard method (tube test and centrifugation), the proposed method proved to be slightly more sensitive with regard to the Rhesus and slightly less sensitive with regard to the AB0 system. With the proposed method very small traces could be successfully analyzed. Thus, two cotton threads 1 mm in length were sufficient for testing antigens A and B, and two cotton threads 2.5 mm in length were enough to detect an Rh antigen.     

On the detection of minor Rhesus antigens in blood stains

A new method for the detection of minor C, Cw, c, E, e-antigens of the Rhesus system in blood stains has been developed based on the absorption-elution technique with the use of anti-C, anti-Cw, anti-c, anti-E, and anti-e sera and standard erythrocyte preparations preliminarily treated with highly active proteases (protease C or papain). This method makes it possible to determine complete Rhesus phenotype in blood stains and substantially extend the possibilities of their differentiation on material objects (evidence) for the purpose of forensic-biological examination.     

The typing of group-specific component (Gc protein) in human blood stains

It is known that the typing of group-specific component (Gc protein) in human blood stains is difficult since Gc protein of the extracts of blood stains migrates more anodally to the α₁-globulin region in agar-gel immunoelectrophoresis, while Gc protein in liquid blood normally migrates to the α₂-globulin region. We have reported that the Gc protein found in the α₁-region is the result of binding of actin to Gc protein (Shinomiya, K., Kimura, H., Yoshida, K., and Shinomiya, T., J. Biochem., 92 (1982) 1163–1171, which renders it difficult to determine the Gc-phenotypes in the blood stains. On the basis of the above findings, we developed the method of phenotyping the Gc protein of human blood stains by agar-gel immunoelectrophoresis. Since the binding activity of actin to Gc protein is lost after treatment with a high concentration of guanidine HCl, the extracts of blood stains were treated with 4 M guanidine HCl to dissociate Gc protein and actin and then dialyzed to remove guanidine HCl. By this method we are able to determine the phenotypes of Gc protein in blood stains. The method we have developed is a useful tool in the forensic laboratory.     

The detection of A and B antigens on human hair by the absorption-elution technique using LISS and papain-treated test cells

The absorption-elution technique with low ionic strength solution (LISS) and papain-treated test cells previously used for bloodstains was employed for the detection of ABO antigens on human hair. Antigen identification was always possible, with good intensity of agglutination, even in those cases where classic techniques had given false-negative results. It was possible to obtain positive results with fragments of human hair as small as 0.2 cm.     

毛发中海洛因及其代谢物的分析综述

对毛发中海洛因及其代谢物—6-单乙酰吗啡和吗啡的提取和检测分析进行了综述。其分析方法大致为6步:收集毛发,清洗毛发,分段,剪(磨)碎毛发,水解,提取净化,检测分析。     

The detection of sedatives in hair and nail samples using tandem LC-MS-MS

Drug screening methods were developed to detect alprazolam, clobazam, clonazepam, diazepam, midazolam, oxazepam, temazepam, triazolam, zopiclone, and selected metabolites in human hair and nail samples employing liquid-liquid extraction and tandem liquid chromatography-mass spectrometry (LC-MS-MS). Hair and nail samples were obtained from patients who had recently discontinued or were currently prescribed one or more of the targeted drugs. Prazepam was used as the internal standard for all compounds. Some components in the hair matrix gave the same transitions as some of the analytes but did not compromise the analyses because their retention times differed from those for the target compounds. The analytical run time was 8-10min. Results of the hair analysis of a DFSA victim are also presented.     

The combination of mixed agglutination and immunofluorescence reactions for the simultaneous detection of 2 ABO-system antigens in isolated cells]

A combination of two immunologic tests, mixed agglutination and immunofluorescence, is suggested, that permits the detection of two AB0 system antigens in the same cell within one process. Such mode better validates the conclusion on the group appurtenance of cells, for instance, in analyses of mixed stains originating from different subjects with different blood groups.     

The detection of cocaine in hair specimens using micro-plate enzyme immunoassay.

The analysis of hair for drugs of abuse is becoming increasingly popular and is under consideration by the Division of Health and Human Services as a possible alternative or adjunct to urinalysis in workplace programs. The detection of cocaine in human hair using a commercially available micro-plate enzyme immunoassay is described for the first time. Sample size and incubation time were the major variables in the optimization of the method. In order to validate the procedure, the method was applied to 105 consecutive hair samples routinely received into our laboratory. The samples were simultaneously analyzed by the Micro-Plate immunoassay (EIA), as well as our current fluorescence polarization immunoassay (FPIA) procedure and gas chromatography-mass spectrometry (GC/MS). The sensitivity of the EIA and FPIA assays were 75% and 67.8% respectively; specificity 97.4% and 80.5% respectively; and efficiency 91.4 and 77.1% respectively. The Micro-Plate EIA was shown to be a valid alternative to other immunoassay screening methods for the detection of cocaine in hair by demonstrating increased sensitivity, specificity and efficiency over our current technique.     

Evaluation of the One-Step ELISA kit for the detection of buprenorphine in urine, blood, and hair specimens

A solid-phase enzyme immunoassay involving microtiter plates was recently proposed by International Diagnostic Systems corporation (IDS) to screen for buprenorphine in human serum. The performance of the kit led us to investigate its applicability in other biological matrices such as urine or blood, and also hair specimens. Low concentrations of buprenorphine were detected with the ELISA test and confirmed by HPLC/MS (buprenorphine concentrations measured by HPLC/MS: 0.3 ng/mL in urine, 0.2 ng/mL in blood, and 40 pg/mg in hair). The intra-assay precision values were 8.7% at 1 ng/mL of urine (n = 8), 11.5% at 2 ng/mL in serum (n = 8), and 11.5% at 250 pg/mg of hair (n = 8), respectively. The immunoassay had no cross-reactivity with dihydrocodeine, ethylmorphine, 6-monoacetylmorphine, pholcodine, propoxyphene, dextromoramide, dextrometorphan at 1 and 10 mg/L, or codeine, morphine, methadone, and its metabolite EDDP. A 1% cross-reactivity was measured for a norbuprenorphine concentration of 50 ng/mL. Finally, the immunoassay was validated by comparing authentic specimens results with those of a validated HPLC/MS method. From the 136 urine samples tested, 93 were positive (68.4%) after the ELISA screening test (cutoff: 0.5 ng/mL) and confirmed by HPLC/MS (buprenorphine concentrations: 0.3-2036 ng/mL). From the 108 blood or serum samples screened, 27 were positive (25%) after the ELISA test with a cutoff value of 0.5 ng/mL (buprenorphine concentrations: 0.2-13.3 ng/mL). Eighteen hair specimens were positive (72%) after the screening (cutoff: 10 pg/mg) and confirmed by LC/MS (buprenorphine concentrations: 40-360 pg/mg). The ELISA method produced false positive results in less than 21% of the cases, but no false negative results were observed with the immunological test. Four potential adulterants (hypochloride 50 mL/L, sodium nitrite 50 g/L, liquid soap 50 mL/L, and sodium chloride 50 g/L) that were added to 10 positive urine specimens (buprenorphine concentrations in the range 5.3-15.6 ng/mL), did not cause a false negative response by the immunoassay.     

The detection of HIV antibodies in blood stains

Blood samples were collected on cotton wool and stored at +20 degrees C. These samples were tested in an enzyme linked immunosorbent assay and the immunoblotting test. HIV-antibodies could be detected in samples stored up to four month.