The GC-MS detection and characterization of reticuline as a marker of opium use

Reticuline (a precursor of opium alkaloids) was detected and characterised as its trimethylsilyl ethers, acetyl esters and methyl ethers by GC-EIMS and GC-CIMS in opium and the urine of opium users after hydrolysis by acid or beta-glucuronidase as coextractive of morphine. Because this compound cannot be detected in heroin and poppy seeds, it is suggested as a differentiating marker between opium and heroin use, opium and poppy seeds use, or opium and "pharmaceutical" codeine use in cases when opiate use has been confirmed by detection of morphine and codeine in the urine. As well as being a constituent of opium, reticuline in the urine of opium users may also result from the metabolic demethylation of the three other benzyltetrahydroisoquinoline opium alkaloids: codamine, laudanosine and laudanine.     

Heart fatty acid binding protein as a marker for postmortem detection of early myocardial damage

Depletion of heart fatty acid binding protein (H-FABP) from cardiomyocytes with varying post-ischemia intervals was studied in acute myocardial infarction (AMI) model of rat, and 22 human autopsy cases were studied with streptavidin-peroxidase conjugated method (S-P). It was observed that as early as 15 min after ischemia, the depletion of H-FABP could be detected in model rats. With the ischemic time prolonged, the depletion of H-FABP was more and more evident. In all human cases with myocardial infarction, absent H-FABP staining could be found in infarcted area. And in some suspected early myocardial infarction cases, depletion of H-FABP staining could be demonstrated in areas that showed normal hematoxylin-eosin (HE) staining. The blood samples from model rats before ligation, at varying post-ischemia intervals and various postmortem time were measured for plasma concentration of H-FABP with enzyme-linked immuno-sorbent assay (ELISA) method. At 15 min after myocardial ischemia, the concentration of H-FABP was 4 times higher (546.0+/-85.3 microg/l) than that of the baseline level (103.7+/-94.1 microg/l). With the continuation of ischemic time, the concentration of H-FABP increased and peaked at 4 h (1953.5+/-405.3 microg/l), then decreased. The plasma concentration of H-FABP decreased slightly with postmortem time, but was still significant higher at any postmortem intervals than that of baseline level within 48 h after death. The results suggest that H-FABP staining can detect very early ischemic damages in human myocardium and the elevated plasma concentration of H-FABP in rat was an indicator of AMI, which was not affected by autolysis.     

The GC–MS detection and characterization of reticuline as a marker of opium use

Reticuline (a precursor of opium alkaloids) was detected and characterised as its trimethylsilyl ethers, acetyl esters and methyl ethers by GC–EIMS and GC–CIMS in opium and the urine of opium users after hydrolysis by acid or β-glucuronidase as coextractive of morphine. Because this compound cannot be detected in heroin and poppy seeds, it is suggested as a differentiating marker between opium and heroin use, opium and poppy seeds use, or opium and “pharmaceutical” codeine use in cases when opiate use has been confirmed by detection of morphine and codeine in the urine. As well as being a constituent of opium, reticuline in the urine of opium users may also result from the metabolic demethylation of the three other benzyltetrahydroisoquinoline opium alkaloids: codamine, laudanosine and laudanine.     

A study of ethyl glucuronide in post-mortem blood as a marker of ante-mortem ingestion of alcohol

The possibility of post-mortem production of ethanol makes correct interpretation of ethanol detection in forensic autopsy samples difficult. Even though the levels of ethanol formed post-mortem are generally low, this may be highly relevant in cases where intake of alcohol was forbidden, for instance for pilots, professional drivers and countries with low legal alcohol limits for driving. Different criteria are used to determine whether a finding of ethanol is of exogenous origin, but there is no marker for alcohol ingestion that has been studied in detail. In this study, we wanted to evaluate the sensitivity and specificity of ethyl glucuronide (EtG), a direct minor metabolite of ethanol, measured in blood, as a marker of ante-mortem alcohol ingestion. Forensic autopsy cases were divided into groups with and without ante-mortem alcohol ingestion, according to strict inclusion criteria. In 93 cases with information on ante-mortem alcohol ingestion, EtG was detected in blood in all cases, even when levels of ethanol were low. In another 53 cases where there were no indications of ante-mortem alcohol intake, EtG could not be detected in blood in a single case, also in 11 cases in which ethanol was detected and considered to be most probably formed post-mortem. In conclusion, blood EtG determination seems to be a reliable marker of ante-mortem ingestion of alcohol, and it could be considered in forensic autopsy cases when post-mortem formation of ethanol is questioned.     

直接扩增法在血痕、肋软骨和唾液检材中的应用

目的采用Identifiler Direct PCR试剂盒直接扩增法进行棉签擦拭血痕、肋软骨和烟蒂唾液斑DNA分型检验,并评价其应用价值。方法收集棉签擦拭血痕、烟蒂各20份,肋软骨10份,采用Identifiler Direct PCR试剂盒进行直接扩增及分型检验,以相同检材采用磁珠法/Chelex-100法提取模板DNA后扩增检验结果作为对照,对两组所得结果进行比较分析。结果棉签擦拭血痕和肋软骨一次检测完整分型率均为100%,分型结果与对照组一致;烟蒂上唾液斑有2份检材第一次未能完整分型,调整方法再次检验后获分型成功。结论实际检案中的棉签血痕、肋软骨和烟上唾液斑,采用直接扩增法检测,方法简单、快速、稳定、检材用量小,可在实际检案中选择使用。     

Morphine to codeine concentration ratio in blood and urine as a marker of illicit heroin use in forensic autopsy samples

A morphine to codeine ratio greater than unity (M/C>1) has been suggested as an indicator of heroin use in living individuals. The aim of this study was to examine the morphine to codeine ratio in a large population (N=2438) of forensically examined autopsy cases positive for 6-monoacetylmorphine (6-MAM) and/or morphine in blood and/or urine. Blood and urine concentrations of 6-MAM, morphine and codeine were examined using GC-MS and LC-MS/MS methods. In 6-MAM positive samples, the M/C ratio was greater than unity in 98% (N=917) of the blood samples and 96% (N=665) of the urine samples. Stratification of 6-MAM negative cases by M/C above or below unity revealed similarities in morphine and codeine concentrations in cases where M/C>1 and 6-MAM positive cases. Median blood and urine morphine concentrations were 8-10 times greater than codeine for both groups. Similarly to 6-MAM positive cases, 25-44 year-old men prevailed in the M/C>1 group. In comparison to cases where M/C ≤ 1, the M/C ratio was a hundred times higher in both 6-MAM positive and M/C>1 cases. The range of morphine concentration between the lowest and the highest quintile of codeine in M/C>1 cases was similar to that in 6-MAM positive cases. This range was much higher than for M/C ≤ 1 cases. Moreover, linear regression analyses, adjusted for age and gender, revealed a strong positive association between morphine and codeine in 6-MAM positive and M/C>1 cases. The M/C ratio appeared to be a good marker of heroin use in post-mortem cases. Both blood and urine M/C>1 can be used to separate heroin users from other cases positive for morphine and codeine.     

Vaginal fluid zinc concentration as a marker for intercourse

Zinc is present in high concentration in semen, but in low concentration in vaginal fluid. We evaluated vaginal zinc levels as a marker for intercourse by measuring precoital (greater than 11 h after intercourse) or postcoital (less than 5 h after intercourse) zinc and acid phosphatase levels in 26 specimens of vaginal fluid from 18 women. The approximate 95% reference range for zinc in precoital vaginal fluid was 1.2 to 15 micrograms/mL (mean 4.5), and in postcoital vaginal fluid 4.0 to 135 micrograms/mL (mean 24). There is an overlap between the precoital and postcoital reference ranges. Provided that the vaginal fluid zinc level is less than approximately 4.0 micrograms/mL or greater than approximately 15 micrograms/mL, vaginal fluid zinc concentration may be useful as an indicator of intercourse.     

A sandwich enzyme-linked immunosorbent assay for ABO blood typing of semen by using anti-p 84 monoclonal antibody as a marker of blood group substance in semen

A blood group substance (BGS), a protein with ABH antigenic activity, was isolated from human seminal plasma and designated as p 84 (Sato, 1995). We have developed a method for determining the ABO blood type of semen by performing a sandwich enzyme-linked immunosorbent assay (ELISA) in which p 84 is captured with an anti-p 84 monoclonal antibody, and evaluated the specificity and sensitivity of this method. Although BGS activity was detected in semen sensitively by this method, it was not detected in saliva, urine, breast milk, blood or vaginal secretions. Since the concentration of p 84 in semen was independent of the secretion status, the status can be determined as non-secretor when p 84 but not BGS activity was detected. To determine the stability of BGS activity on p 84, dried stains of semen on filter paper were kept at 4, 26, and 37 degrees C for 8 months, 2 years and 1 month, respectively, and their BGS activities were examined. After 8 months at 4 degrees C, over 60% of the original BGS activity was recovered from the stain. The activity could be detected even from a square as small as 0.25 by 0.25 cm. After 1 month at 37 degrees C and 2 years at 26 degrees C, 31 and 20% of the BGS activity, respectively, still remained. It could be detected from the pieces of 1.0 by 1.0 cm and 0.5 by 0.5 cm squares, kept for 1 month at 37 degrees C and 2 years at 26 degrees C, respectively. Finally, semen was mixed with saliva or blood at varying volumetric ratios and used for the sources of dried stains. The BGS activity of p 84 could be detected in the stains until the ratio between semen and saliva or blood reached 1:4. We conclude that this sandwich ELISA offers a more sensitive and specific method for determining the ABO blood type of semen samples obtained from sexual assault victims than existing methods, such as the conventional absorption-elution and classical hemagglutination-inhibition tests.     

PCR-based detection of HLA-DQ alpha and polymarker loci and their application in forensic science

Studies were conducted to evaluate the forensic applicability of multiplex amplification of HLA-DQ alpha and PM loci. These loci could be simultaneously typed by reverse dot blot approach where allele specific oligonucleotide probes were immobilized on a nylon membrane strip. Allele and genotype frequencies for HLA-DQ alpha and PM loci were determined in Chinese Han population. The frequency data can be used in individual identification and paternity test.     

Detection and use of salivary hemagglutinins for forensic blood grouping

A sensitive method for the detection of anti-A and anti-B hemagglutinins in fresh saliva has been developed. The method utilizes a bromelin treated erythrocyte suspension as indicator cells and includes a simple procedure to concentrate these hemagglutinins. Antiserum directed against immunoglobulin A enhances the hemagglutination assay. We find that these salivary hemagglutinins are present in over 90% of the population and that their titer remains stable over a period of two months. These hemagglutinins can be used to blood type the donor of a saliva sample and can be used in a confirmatory test that complements the commonly used absorption-inhibition test which is used to detect salivary blood group agglutinogens. In preliminary studies we have determined that hemagglutinins can be successfully isolated and analyzed from dried saliva stains.     

Psychiatric diagnosis as a risk marker for victimization in a national sample of children

Research examining childhood abuse has shown an association between victimization and psychiatric diagnoses (e.g., posttraumatic stress disorder, depression). Historically, psychiatric diagnoses have been emphasized as a consequence of victimization, with less research examining if it also functions as a risk factor for further victimization, perhaps making diagnoses a general victimization risk marker. In addition, much of this research has emphasized particular types of victimization such as childhood physical or sexual abuse. Researchers have given less attention to other forms of victimization (e.g., peer victimization, witnessed violence) or a diverse victimization history. Using the Juvenile Victimization Questionnaire (JVQ) we surveyed parents and children between the ages of 2 and 17 using a random digit dial (RDD) methodology. We examined the relationship between a number of different forms of victimization (termed poly-victimization ) in the preceding year and parent-reported lifetime psychiatric diagnosis. Results show that children with a psychiatric diagnosis have significantly higher rates of victimization than children without a psychiatric diagnosis. In addition, using logistic regression models, we find that psychiatric diagnosis was associated with increased risk for poly-victimization, conventional crime victimization, maltreatment, peer or sibling victimization, and witnessing violence, but not sexual abuse. The results highlight the need to consider psychiatric diagnoses as a risk marker for past and possible future victimization. In addition, the importance of obtaining a comprehensive and more diverse victimization history when working with children is highlighted.     

Frequencies of salivary genetic marker systems in the Japanese population and their application to forensic medicine

Seven salivary polymorphic systems were studied using whole and parotid saliva from random Japanese individuals. The gene frequencies obtained were: Pa⁺ = 0.212, Pb¹ = 1.000, Pb² = 0, Pr¹ = 0.763, Pr² = 0.237, Db⁺ = 0.051, Pm⁺ = 0.409, Ph⁺ = 0.026 and Amy₁^v = 0.013, respectively. Based on these gene frequencies, the chances for exclusion of falsely alleged fathers were calculated. The chance of exclusion on the basis of five salivary polymorphic systems was 0.305. The combined chance of exclusion utilizing only blood, serum and red-cell enzyme polymorphic systems among the Japanese population was 0.919; however, by applying salivary polymorphic systems to the calculation, the total exclusion rose to 0.944.     

Glycogenated squamous epithelial cells as a marker of foreign body penetration in sexual assault

Nonconsensual insertion of a foreign object into the vagina, anus, or mouth in some judicial jurisdictions is synonymous with rape, and elsewhere may constitute some degree of sexual assault or battery. Few techniques, however, are available to assist the criminalist in determining whether an object has been criminally inserted. Glycogenated epithelial cells have been used as a marker for vaginal epithelium, and as such, may indicate vaginal insertion if recovered from an object. This hypothesis was tested by studying orally and vaginally inserted objects from 42 volunteers and 20 rectally inserted objects recovered from cadavers. Glycogen positivity was assayed from smears of object swabbings stained with the periodic acid-Schiff (PAS) technique. More than 75 glycogen positive cells were recovered from 39 of 42 vaginally inserted objects. Glycogenated cells were recovered from 8 of 20 rectally inserted objects (5 with more than 100 positive cells). Of 42 orally inserted objects, 32 also contained glycogen positive cells, but none with more than 28 positive cells. No glycogen positive cells were seen in skin exposed but not inserted objects. Large numbers of glycogen cells were seen in dried saliva drops. Amylase activity was not seen on 5 of 20 orally inserted shields, and thus the possibility of noninsertional saliva contamination could not be ruled out with shields yielding only small numbers of positive cells. Recovery of large numbers of glycogenated cells from foreign objects is strongly suggestive of either vaginal or anal insertion assuming amylase negativity. Glycogen positive cells are not seen secondary to glabrous skin exposure.     

Gelatinase A in human dentin as a new biochemical marker for age estimation

Dentin proteins from 24-individual permanent molars from patients aged 15 to 73 years were sequentially extracted, with guanidinium chloride (G1-extract) and then with EDTA; after demineralization, the material was again extracted with guanidinium chloride (G2-extract). Extracts were analyzed by SDS-PAGE electrophoresis and the gels were processed for zymography to detect gelatinolytic activities. The patterns of gelatinase A distribution differed in the different dentin protein fractions, and the changes varied with age. Significant differences were detected in gelatinase A in G2-extracts between individual younger than 20 years old and the rest of the sample (chi2exp = 19.429; 1 d.f.; p < or = 0.001). The incidence of true and false positives and negatives, and sensitivity and specificity for the presence of gelatinase A in dentin extracts, were calculated. Determination of gelatinase A in human dentin may be a useful marker to estimate age, especially when other morphological methods are of limited usefulness.     

Cardiac blood pH as a possible indicator of postmortem interval

Postmortem changes in the pH of blood and selected tissues in rats were evaluated at intervals ranging from 2 min to 96 h. Cardiac blood pH was significantly and reproducibly decreased in all groups at all postmortem intervals, independent of the method of sacrifice used. A preliminary study using cardiac blood obtained at autopsy from a limited number (n = 11) of human subjects demonstrated a significant negative correlation (r = -0.908, P less than 0.01) between postmortem interval (range 2 to 20 h) and cardiac blood pH.     

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.