[Correlations between pediatric emergency department and legal medicine in the field of child abuse: the usefulness of flow-charts]. / Correlazione tra il dipartimento d'urgenza pediatrico e la medicina legale in tema di abuso sui minori: utilità di linee-guida.

According to the World Health Organization, child abuse is one of the major and unrecognised problems affecting the well-being and impairing the harmonic development of children and adolescents. Official statistics, provided by Police and Courts are not useful for quantifying abuse because these numbers refer to cases actually brought to trial, a small part of the amount. In Europe and North America more effective measures and intervention programs were set up in order to get with a reliable description of this crucial social problem; for example, a "dedicated" team performing careful screenings on suspicious cases (Suspected Child Abuse and Neglect Teams, SCAN). A legal medical expert joins the team bringing his specific experience on lesions from the perspective of criminological interpretation, thereby giving a substantial contribution for an objective evaluation of available evidence. The multidisciplinary approach can give the greatest contribution especially in the initial steps of the evaluation of the child. Intervention protocols and guidelines are useful tools to achieve this goal, as shown by medical literature. We propose to introduce a flow-chart for the initial evaluation of cases, derived from that used in the Canadian Paediatric Emergency Departments. This is supposed to be experimentally used by health care workers in an Emergency Department setting and to be subsequently evaluated both as a diagnostic and a statistic instrument.

Full deflection profile calculation and Young's modulus optimisation for engineered high performance materials.

New engineered materials have critical applications in different fields in medicine, engineering and technology but their enhanced mechanical performances are significantly affected by the microstructural design and the sintering process used in their manufacture. This work introduces (i) a methodology for the calculation of the full deflection profile from video recordings of bending tests, (ii) an optimisation algorithm for the characterisation of Young's modulus, (iii) a quantification of the effects of optical distortions and (iv) a comparison with other standard tests. The results presented in this paper show the capabilities of this procedure to evaluate the Young's modulus of highly stiff materials with greater accuracy than previously possible with bending tests, by employing all the available information from the video recording of the tests. This methodology extends to this class of materials the possibility to evaluate both the elastic modulus and the tensile strength with a single mechanical test, without the need for other experimental tools.

Calcitonin serum levels in heroin addicts: effects of methadone and clonidine detoxication treatments.

Calcitonin (CT), a 32-aminoacid peptide, is secreted by the parafollicular 'C' cells of the thyroid which derive from the ultimobranchial body and, ultimately, from the neural crest. In man, the main role of this hormone is to protect the skeleton during periods of physiological stress, such as growth, pregnancy and lactation by reducing calcium loss [1]. Nevertheless, some recent data, such as the finding of immunoreactive CT-like material (iCT) in the central nervous system [2] and in the cerebrospinal fluid and the demonstration of its analgesic action in rabbits [4] and in man [5] indicate a relationship between CT and the neuroendocrine system. The purpose of the present study is to evaluate the levels of CT in a group of addicts to heroin both before and during detoxication treatment with methadone or a non-opioid drug like clonidine.

Hormone levels in methadone-treated drug addicts.

To test the effect of methadone on hormone release in man, plasma levels of luteinizing hormone (LH), follicle-stimulating hormone (FSH), 17-beta-estradiol (E2), estrone (E1), progesterone (P), testosterone (T), dehydrotestosterone (DHT), androstenedione (A), dehydroepiandrosterone sulphate (DS), and prolactin (PRL) have been evaluated in 25 male subjects in treatment wit 30 mg/day methadone per os for at least two months after a minimum of one year of heroin addiction. All subjects had a history of sexual difficulties. The levels of E1, E2, P and LH were always within normal values whereas FSH showed lower values than normal and A, DS and PRL noticeably increased in many subjects. Modest variations were noted for T and DHT. These data seem to indicate that methadone abuse affects the hypophysis, the testiculum and the adrenal cortex. More details will probably be obtained when the variations in hormone rates and opiate serum concentrations are checked at a later date.

Immune response to opiates: new findings in heroin addicts investigated by means of an original enzyme immunoassay and morphine determination in hair.

In the early 1970's, some papers appeared reporting an immune response to opiates in animals treated with morphine and in heroin addicts, but further researches failed to confirm these results in humans. The aim of the present work is investigating with a newly developed enzyme immunoassay the existence of specific antibodies to morphine in a group of opiate chronic users, controlled by means of the toxicological analysis of hair. Twenty five opiate addicts inpatients for detoxication treatments were investigated for the presence of morphine specific antibodies and for the morphine content in hair, as a marker of addiction to opiates. Antibodies to morphine were investigated using an original ELISA method using a morphine-human serum albumin conjugate immobilized into the wells of polystyrene microtiter plates. Morphine determinations in hair were accomplished by a radioimmunologic screening followed by HPLC confirmation of positive results. The group of opiate users, in which all the subjects resulted positive for morphine content in hair, showed in the ELISA test an average D OD% value significantly higher than the control group (p < 0.001); in particular, 16 out of 25 addicts could be classified positive for anti-morphine antibodies, which were identified as IgM. Inhibition studies demonstrated Ka's for morphine ranging from 10(4) to 10(10) M-1 and a high cross reactivity for codeine. The presence of circulating antibodies specific to morphine in chronic users of opiates is strongly supported by the present findings.

Reversed-phase high-performance liquid chromatographic determination of cocaine in plasma and human hair with direct fluorimetric detection.

A simple, but sensitive and specific, high-performance liquid chromatographic assay for cocaine with direct fluorimetric detection, particularly intended for the routine analysis of hair and blood samples, is described. Benzoylecgonine, eluting before cocaine in a completely resolved peak, is also detectable. Detection is based on the weak native fluorescence of cocaine and benzoylecgonine, depending on the benzene ring present in both molecules. Hair samples (20-200 mg) were incubated overnight in 2 ml of 0.25 M HCl at 45 degrees C and extracted with a commercial liquid-liquid method; the dried residue reconstituted with 500 microliters of 0.05 M NaH2 PO4 (PH 5.2) was injected. Blood plasma samples (200 microliters) were mixed with 150 microliters of 0.1 M Na2 HPO4 (pH 8.9) and extracted with 5 ml of chloroform-2-propanol (9:1); the organic phase was evaporated and the residue dissolved and injected as above. Isocratic reversed-phase liquid chromatography was carried out on a column (150 x 4.6 mm I.D.) packed with spherical 5-microns poly(styrene-divinylbenzene) particles; the mobile phase was 0.1 M potassium phosphate (pH 3)-methanol-tetrahydrofuran (70:25:5). The excitation and emission wavelengths were set at 230 and 315 nm, respectively. Under the described conditions, cocaine eluted in a symmetrical peak with a capacity factor of about 5. The limit of detection was about 1 ng/ml (0.2 ng injected), with a signal-to-noise ratio of 3.(ABSTRACT TRUNCATED AT 250 WORDS)

Complementary use of capillary zone electrophoresis and micellar electrokinetic capillary chromatography for mutual confirmation of results in forensic drug analysis.

The purpose of this work was to compare different CE separation modes namely capillary zone electrophoresis (CZE) and micellar electrokinetic capillary chromatography (MECC) for the analysis of drugs of forensic interest in order to assess the mutual degree of independence and consequently the possibility of complementary use for mutual confirmation of results. A panel of drugs including caffeine, morphine, barbital, pentobarbital, codeine, nalorphine, lidocaine, procaine, heroin, flunitrazepam, acetylcodeine, papaverine, amphetamine, narcotine, cocaine, diazepam, tetracaine, narceine, 6-monoacetylmorphine acetylcodeine and thebaine, were separated according to a MECC and two CZE methods. The MECC separation was carried out in a bare silica capillary (50 micron I.D.) with a buffer composed of 25 mM borate (pH 9.24)--20% methanol--100 mM sodium dodecyl sulphate; the applied voltage was 20 kV. The first CZE method (CZE1) was carried out in 50 mM phosphate buffer (pH 2.35) at 20 kV with a bare silica capillary (50 micron I.D.), and the second (CZE2) with 50 mM borate (pH 9.24) at 12 kV with the same capillary. The three methods were effective in the separation of the test drug mixture, but MECC was the only able to resolve all the components. Relative (to flunitrazepam), migration time RSDs ranged from 0.3 to 2.8% for the three methods were compared with Spearman's test and with principal component analysis, CZE1 and CZE2 were significantly and directly correlated (r = 0.749, p < 0.002), whereas MECC and CZE2 were also significantly, but inversely correlated (r = -0.865, p < 0.001). MECC and CZE1 (limitedly to the basic drugs) appeared non-correlated (r= -0.131, p = 0.630) and therefore the two techniques are suitable for combined use to increase the discriminatory power.

Potassium concentration differences in the vitreous humour from the two eyes revisited by microanalysis with capillary electrophoresis.

This paper presents a study of the variability of potassium concentrations in the vitreous humour of the two eyes of the same body at identical postmortem interval. The study was carried out by collecting microsample amounts (50 microl) of vitreous humour and by using an original method of capillary electrophoresis with indirect detection. The electrophoretic separations were carried out in a pH 4.5 running buffer composed of 5 mmol/l imidazole, 5 mmol/l 18-crown-6 ether and 6 mmol/l alpha-hydroxybutyric acid. Detection was by indirect UVabsorption at 214 nm. Vitreous humour samples were collected from 57 medico-legal autopsies or external examinations of cases of sudden natural or violent deaths. All samples prior to analysis were diluted 1:20 with a 40 microg/ml aqueous solution of barium, the used internal standard, and finally injected by nitrogen pressure. The mean concentrations of potassium measured in the two eyes of all the cases included in the present study ranged from 4.1 to 23.5 mmol/l with the postmortem interval values varying from 7 to 144 h. A highly significant (P<0.0001) linear correlation was found between these two parameters as described by the equation: y=0.1698x+2.3587, r=0.89. The intra-eye variability of potassium concentrations was low with an average RSD of 3.89% (+/- 1.83 SD) (48 eyes, five samples per eye). No statistically significant difference was found between the potassium concentrations in the two eyes of the same subject in a group of 24 cases, excepting a single case.

Hair analysis for abused drugs by capillary zone electrophoresis with field-amplified sample stacking.

The present paper describes the methodological optimisation and validation of a capillary zone electrophoresis method for the determination of morphine, cocaine and 3,4-methylenedioxymethamphetamine (MDMA) in hair, with injection based on field-amplified sample stacking. Diode array UV absorption detection was used to improve analytical selectivity and identification power. Analytical conditions: running buffer 100 mM potassium phosphate adjusted to pH 2.5 with phosphoric acid, applied potential 10 kV, temperature 20 degrees C, injection by electromigration at 10 kV for 10 s, detection by UV absorption at the fixed wavelength of 200 nm or by recording the full spectrum between 190 and 400 nm. Injection conditions: the dried hair extracts were reconstituted with a low-conductivity solvent (0.1 mM formic acid), the injection end of the capillary was dipped in water for 5 s without applying pressure (external rinse step), then a plug of 0.1 mM phosphoric acid was loaded by applying 0.5 psi for 10 s and, finally, the sample was injected electrokinetically at 10 kV for 10 s. Under the described conditions, the limit of detection was 2 ng/ml for MDMA, 8 ng/ml for cocaine and 6 ng/ml for morphine (with a signal-to-noise ratio of 5). The lowest concentration suitable for recording interpretable spectra was about 10-20-times the limit of detection of each analyte. The intraday and day-to-day reproducibility of migration times (n = 6), with internal standardisation, was characterised by R.S.D. values < or = 0.6%; peak area R.S.D.s were better than 10% in intraday and than 15% in day-to-day experiments. Analytical linearity was good with R2 better than 0.9990 for all the analytes.

Capillary electrophoresis: a new tool in forensic toxicology. Applications and prospects in hair analysis for illicit drugs.

Capillary electrophoresis, the modern approach to instrumental electrophoresis, is probably the most rapidly expanding analytical technique that has appeared in recent years. In the hands of forensic toxicologists, capillary electrophoresis (CE) represents a powerful new analytical tool, which has proved suitable for the investigation of illicit drugs in seized preparations and also in complex biological matrices, among which is hair. CE can be applied according to different separation mechanisms, and among those that are toxicologically relevant are capillary zone electrophoresis and micellar electrokinetic capillary chromatography, which display different selectivities. For the investigation of hair for drugs of abuse, capillary electrophoresis proved effective, providing simultaneous determinations of different drugs without derivatization, with acceptable sensitivity (typically better than 1 ng of drug per mg of hair). The possibility of carrying out determinations of the same analytes, based on different separation mechanisms (capillary zone electrophoresis and micellar electrokinetic chromatography) with the same instrumentation, simply changing the buffer composition, provides an interesting possibility of 'internal' confirmation of the results.

Integrated use of hair analysis to investigate the physical fitness to obtain the driving licence: a casework study.

According to the laws presently in force in Italy and the guidelines of the Driving Licence Enforcement Commission of Verona, applicants for the driving licence with a history of drug abuse undergo a medical examination, during which complete anamnestic and clinical data are recorded. On this occasion, a hair sample (50-200 mg) is collected and a urinalysis program is started consisting of EMIT controls for opiates, methadone, cocaine, barbiturates, amphetamines, cannabinoids, benzodiazepines and alcohol carried out on eight seriate samples, collected at random over about 40 days under direct supervision. The positive results from urine immunoassays are confirmed by standardized GC/MS methods. The hair samples are screened for morphine and cocaine, the most abused illicit substances in our region, using commercial RIAs adopting cut-off levels of 0.1 ng/mg. All positive samples and about 10% of negative are confirmed by HPLC. In case of confirmed positive results, the applicant is informed: if the subject denies use of opiates or cocaine in the recent months, he or she has the chance of submitting for analysis a new hair sample, which is analyzed in parallel with the hair remaining from the previous assay. In case of persisting denial, claiming analytical interferences by other drugs or endogenous substances, further confirmation of results can be carried out by CE and/or by qualitative MS/MS. In addition, hair sampling from multiple sites (scalp, axillary, pubic hair) with different susceptibility to contamination from the external sources can be carried out to rule out the possibility of passive contamination. At present, we investigate more than 700 subjects per year. The results of this integrated diagnostic strategy are presented and discussed.

Optimization of a simple method for the chiral separation of phenethylamines of forensic interest based on cyclodextrin complexation capillary electrophoresis and its preliminary application to the analysis of human urine and hair.

Because of the forensic importance of the chiral analysis of amphetamine and other phenethylamines for investigating their synthetic pathways and the metabolic patterns of these compounds, a capillary electrophoresis method has been developed based on the chiral selectivity of beta-cyclodextrin. The influence of different experimental conditions, such as cyclodextrin nature and concentration, voltage, temperature and buffer concentration and pH, on analytical performance has been studied. The optimized analytical conditions are: capillary: bare fused silica, 50 microns I.D., 40 cm effective length; buffer: 150 mM phosphate pH = 2.5, 15 mM beta-cyclodextrin; voltage: 10 kV; temperature: 17.5 degrees C; detection: UV absorption at 200 nm wavelength. Under these conditions, amphetamine, methamphetamine and ephedrine have been easily separated, with baseline resolution of the respective enantiomers. Sensitivity was better than 300 ng per ml. The average precision of migration times of the three analytes was good with RSD = 0.45% and 0.58% in intra-day and day-to-day tests, respectively. Reproducibility of peak heights was also good, with RSD = 2.51% and 3.14% in intra-day and day-to-day tests, respectively. The preliminary analysis of amphetamine in human urine and hair samples, subjected to a simple work-up procedure based on liquid-liquid extraction, showed clean blank electropherograms, excellent chiral resolution and sensitivity, suitable for the analysis of real samples from amphetamine users.

Italian population data for D1S1656, D3S1358, D8S1132, D10S2325, VWA, FES/FPS, and F13A01.

Allele frequencies for seven STRs loci were obtained from a sample of 215 unrelated healthy Italian individuals.

High-sensitivity low-cost methods for determination of cocaine in hair: high-performance liquid chromatography and capillary electrophoresis.

Free solution capillary electrophoresis and high performance liquid chromatography were applied to the analysis of cocaine in hair. Capillary electrophoresis conditions were as follows. Background electrolyte: 0.050 M borate, pH 9.2; capillary: bare silica, 40 cm long, 50 micrograms i.d.; potential: 15 kV; detection: UV at 238 nm wavelength. In addition, the separation was accomplished in a 50 cm x 75 microns capillary with an instrument with a photodiode array detector, recording on-line UV spectra of peaks from 200 to 400 nm. The isocratic high performance liquid chromatographic method with fluorimetric detection used 230 nm (ex.) and 315 nm (em.) wavelengths. Cocaine separation was carried out under conditions summarized as follows: column packed with spherical polystyrene-divinylbenzene 5 microns particles, mobile phase 0.1 M potassium phosphate (pH 3.0)/methanol/THF (70:25:5) at a flow rate of 0.5 ml/min and at room temperature. Liquid-liquid and solid-liquid sample preparation methods were used. Typically, levels of cocaine in hair as low as 0.15-0.30 ng/mg were detected by capillary electrophoresis, while HPLC allowed the determination of concentrations lower by one order of magnitude (0.015 ng/mg). Intra- and inter-assay precision data of the two techniques were comparable and characterized by relative standard deviations in the range from 3 to 7%. On the other hand, the sample throughput of capillary electrophoresis (7-10 injections/h) was higher than high performance liquid chromatography (2 injections/h). A good correlation of the results (r2 = 0.92) between the two techniques was observed in the assay of real cases.

Hair analysis by using radioimmunoassay, high-performance liquid chromatography and capillary electrophoresis to investigate chronic exposure to heroin, cocaine and/or ecstasy in applicants for driving licences.

The present paper describes an integrated diagnostic strategy to check the physical fitness of subjects, formerly users of illicit drugs, to obtain a driving license, after having quit their addiction. According to the Italian law, applicants for a driving license with a history of drug abuse must give evidence to have quit this behaviour and to show no risk of relapse in the future. To prove this, at our institute, they undergo medical examination, hair analysis and a urinalysis program on eight seriate samples, collected over about 40 days. About 700 subjects per year are investigated with this strategy. The hair samples are screened for opiates (morphine), cocaine and ecstasy, the most abused illicit substances in our region, by using commercial radioimmunoassays adopting cut-off levels of 0.1 ng/mg. All positive samples and about 10% of negatives are confirmed by high-performance liquid chromatography. Further confirmation of results can be carried out by capillary electrophoresis (and/or GC/MS or MS/MS). In 1998, the prevalence of positives for morphine, cocaine and ecstasy was 4.8, 11.3 and 2.6%, respectively. In this year, for the first time, the percentage of hair samples positive for cocaine was greater than that for opiates. The results of this integrated diagnostic strategy are presented and discussed, with particular emphasis on the comparison between hair analysis on a single sample and seriate urinalyses (on eight samples).

Determination of CDT, a marker of chronic alcohol abuse, for driving license issuing: immunoassay versus capillary electrophoresis.

The present work is aimed at a validation of the carbohydrate-deficient transferrin (CDT) determination in real cases by comparison between the a commercial immunometric method and a method based on capillary electrophoresis. Overall, 650 serum samples from subjects applying to re-obtain their driving license, previously withdrawn for "drunk driving", were investigated. A highly significant correlation (P < 0.001) was found between the results from immunoassay and capillary electrophoresis. However, particularly in the samples with CDT values around the cut-off or moderately elevated, a wide dispersion of the correlation data was found. This finding stresses the need to confirm by alternative techniques all the results from CDT immunoassays. For this purpose, capillary electrophoresis, because of its inherent characteristics of high selectivity, easy operation, high productivity and low operative costs looks well-suited for becoming the method of choice.

Suicide attempted by burning: a 10-year study of self-immolation deaths.

In a 10-year retrospective survey of 699 admissions to the Burns Centre of Verona, 31 patients (4.4 per cent) had self-inflicted burns. No sex difference was found, the mean age was 38 years, with 58 per cent of the patients in the 20-39-year age group. The mean burned surface area (BSA) was 41 per cent, and 12 patients (38.7 per cent) died: seven within the first 24 h. The other five patients died within 40 days (mean survival: 18 days). A large number of patients had a history of previous psychiatric treatment, six patients had previously attempted suicide. Four patients were drug addicts, three of whom were affected by AIDS and one had been HIV-positive for a long time. The patients had continuous psychiatric treatment during the long hospital stay. After discharge the patients were assigned to territorial staff for psychiatric treatment. Our experience shows that these patients have a higher mortality rate and they need constant psychiatric support, which can be useful in preventing further suicide attempts.

Determination of morphine in the hair of heroin addicts by high performance liquid chromatography with fluorimetric detection.

A procedure has been presented for the quantitative determination of morphine contained in the hair of heroin addicts, by means of heat-acid hydrolysis, pre-column dansyl derivatization, straight phase liquid chromatography, and fluorescence detection. External standardization was used. Intra-assay and day-to-day variation coefficients were 5.6 and 7.8%, respectively (n = 10), when hair containing 1 ng/mg of morphine was assayed. Hair samples of 22 heroin addicts showed positive results in the range 0.08 to 15.7 ng/mg. No false positive results were found in 20 control subjects. A close correlation was shown between high performance liquid chromatography and radioimmunoassay results (y = 0.97x + 0.26)(r = 0.997, n = 15). Morphine hair content results significantly correlated with the grade of heroin use roughly estimated by means of serial determinations of morphine in urines during the last months before hair sampling.

Northeast Italy population data using multiplex PCR (HUMCD4, HUMTH01, HUMTPOX, and HUMCSF1P0) loci.

Allele and genotype frequencies for four short tandem repeat (STR) loci (HUMCD4, HUMTH01, HUMTPOX, and HUMCSF1P0) were determined in 100 unrelated individuals from Veneto (Northeast Italy). After a multiplex polymerase chain reaction (PCR)-amplification, semi-automatic DNA profiling was performed using an A.L.F.express DNA Sequencer. Conditions were optimized for the PCR co-amplification of these four STR loci and the quadruplex PCR was performed on various forensic DNA samples such as whole blood, blood-stains, teeth, and saliva from Caucasians living in the Northeast Italy. The distribution of the genotype frequencies showed no significant deviation from Hardy-Weinberg expectations in the sampled population. The combined Power of Discrimination (PD) of the quadruplex was 0.9999.

Concentrations of phenobarbital, flurazepam, and flurazepam metabolites in autopsy cases.

In five cases of death resulting from acute intoxication with phenobarbital and flurazepam, the blood, urine, brain, lung, liver, and kidney levels of these drugs as well as the levels of N-1 hydroxyethyl, N-1 desalkyl, and N-1 desalkyl-3-hydroxy flurazepam metabolites were determined. Concentration of flurazepam and its metabolites was determined by using new gas chromatographic conditions employing a selective detector for nitrogen-containing substances and a column of 1% SP-1000. In addition, the EMIT technique was also employed on blood and urine samples and the results compared with GLC data.