The PGM1 polymorphism as revealed by ultrathin-layer isoelectric focusing in the population of Padua

The occurrence of PGM₁ phenotypes in 589 samples from the population of Padua was investigated by ultrathin-layer isoelectric focusing. All ten phenotypes were observed. Frequencies of the PGM₁ alleles (1+ = 0.6180; 1? = 0.1163; 2+ = 0.2122; 2? = 0.0535) have been compared to those found in other populations.     

Phosphoglucomutase types in blood and hair roots taken from post-transfusion subjects

Pre- and post-transfusion blood samples were collected from 22 subjects together with the corresponding plucked hair samples taken 2 days and 2 weeks after the transfusion. The phosphoglucomutase1 (PGM1) subphenotypes of blood and hair were determined by isoelectric focusing and the phenotypes confirmed by gel electrophoresis. Many of the post-transfusion blood samples showed an alteration in the PGM1 bands when compared with the pre-transfusion samples. However, the PGM1 types determined from the hair samples were identical to the corresponding pre-transfusion samples in all cases.     

Possibilities of DNA sex determination in hair roots

Species- and sex-determination on hair roots were simultaneously performed using extracted DNA and a human Y chromosomespecific probe. (pH Y 2.1) After Southern hybridization, Hae III-digested DNA fragments were detected by non-radioactive digoxigenin detection system. DNA was extracted from one to five freshly plucked hair roots. The specific 2.12 kb fragment was successfully detected in male DNA samples from a single hair root. A positive identification of female DNA was more difficult. The hair root DNA was revealed to be stable at room temperature for at least 2 weeks (examination time) and produced the same specific band pattern as the DNA of fresh hair roots. In the blind tests with DNA samples from randomly plucked one to four hair roots, the rate of successful sex-determination was 95.8% on male samples (23 out of 24 samples) and 25% on female samples (4 out of 16 samples).     

Detection of meperidine and its metabolites in the hair of meperidine addicts

The presence of meperidine and its metabolites in the hair of meperidine addicts was investigated using GC–MS (EI, PCI). Meperidine and its three metabolites – normeperidine, N-methoxy meperidine and acetyl normeperidine, were found in hair samples from addicted subjects. Methods for the simultaneous determination of meperidine and its metabolites by GC–MS-SIM were also established for human hair samples. After the addition of d₄-meperidine as an internal standard, hair samples weighing 5 mg were incubated in 0.1 M HCl at 45°C overnight, and the resulting digests were extracted with ether. The recoveries were greater than 80%, with coefficients of variation (CVs) between 4.48 and 8.31%. The calibration curves for meperidine and normeperidine in hair were linear over a concentration range of 1 to 500 ng per mg of hair, with correlation coefficients of r=0.9990 and r=0.9992, respectively. Values less than 0.25 ng/mg of hair were cut off. Hair samples obtained from 60 drug addicts were analyzed using this method, and the content of meperidine and normeperidine was determined to be 103±130 and 117±143 ng/mg, respectively. Sectional analysis revealed that meperidine was present and stable in hair for at least 20 months, but normeperidine content at the level of the hair root was higher compared to the tip of the hair shaft. The results also revealed that there was a correlation between the subject’s drug abuse history and the distribution of drug along the hair shaft, and between the doses of meperidine and drug content presented in hair.     

Temperature‐dependent Development of Parasarcophaga similis (Meade 1876) and its Significance in Estimating Postmortem Interval

Flesh flies are commonly found insects on decaying corpses that appears slightly later than blowflies, and their development patterns are significant indicators for minimum postmortem interval (PMI_min) estimation. In this study, the flesh fly Parasarcophaga similis (Meade 1876) was reared at nine constant temperatures ranging from 15°C to 35°C to examine indicators for estimating their age. We generated three development models, including isomorphen diagram, isomegalen diagram, and thermal summation model. Larval body length at different rearing temperatures was fit into an L = a + bT + cT² + dT³ equation with which the relationship between the larval body length (L) and the time after larviposition (T) was confirmed. The pupal stage was categorized into 13 substages according to intrapuparial morphological changes, and a detailed table was generated of the pupal developmental stages at five rearing temperatures, 15°C, 20°C, 25°C, 30°C, and 35°C. This study provides fundamental data in supporting P. similis as an indicator for PMI_min estimation.     

Polymorphism of EsD by isoelectric focusing: phenotyping in human tissues, dental pulps, hair roots, and semen

The polymorphism of EsD was investigated in tissues of various human organs, dental pulps, hair roots, and seminal stains by isoelectric focusing. The method yielded an excellent resolution of the isoenzyme components. The time limits of determination were: in organ tissues 3 weeks, in dental pulps 1 week, and in hair roots several days. The 7-1 type was less stable than the common types. Phenotyping was possible from fresh semen samples, but was unsuccessful from dried seminal stains after storage. The results show that the EsD typing by isoelectric focusing is of practical use for medicolegal individualization of organs, teeth, and hairs.     

The Use of Forensic Tests to Distinguish Blowfly Artifacts from Human Blood,Semen, and Saliva

This study investigated whether routinely used forensic tests can distinguish 3‐day‐old or 2‐week‐old fly artifacts, produced after feeding on human blood, semen, or saliva, from the biological fluid. Hemastix^®, Hemident^?, and Hemascein^? were unable to distinguish blood from artifacts. Hemastix^® returned false positives from negative controls. ABAcard^® Hematrace^® and Hexagon OBTI could distinguish blood from 3‐day‐old artifacts, but not 2‐week‐old artifacts. Phadebas^® and SALIgAE^® were unable to distinguish saliva from artifacts. RSID^?‐Saliva was able to distinguish saliva from 3‐day‐old artifacts, but not 2‐week‐old artifacts. Semen tests Seminal Acid Phosphatase, RSID^?‐Semen, and ABAcard^® p30 were all able to distinguish semen from 3‐day‐old artifacts, but not 2‐week‐old artifacts. The tests investigated cannot be relied upon to distinguish artifacts from biological fluids. However, if an artifact is identified by its morphology, a positive result may indicate which biological fluid the fly consumed, and this knowledge may prove useful for investigators searching for DNA.     

PGD phenotyping in bloodstains, organ tissues, dental pulps and hair roots by isoelectric focusing

Polymorphism of PGD was investigated in bloodstains, organ tissues, dental pulps, hair roots and semen by isoelectric focusing. This technique provided much higher resolution of PGD isoenzymes than starch gel electrophoresis. Phenotyping was possible from bloodstains for 5 weeks, from organ tissues (except pancreas) for 1-3 weeks, from dental pulps for 2 weeks and from hair roots for 2 weeks when they were stored at room temperature. The method is simple, rapid, reliable and therefore useful in medicolegal individualization of bloodstains, organ tissues, teeth and hairs.     

DIA3 phenotyping in human tissues, dental pulps, and hair roots by isoelectric focusing

The polymorphism of DIA3 was investigated in tissues of various human organs, dental pulps, and hair roots by isoelectric focusing. DIA3 types were demonstrated from tissues of brain, prostate, testis, ovary, and uterus, but not from tissues of spleen, pancreas, heart, liver, muscle, lung, skin, and kidney. Determination was possible from dental pulps stored at room temperature for up to 2 weeks and from fresh hair roots. The results show that the DIA3 typing by isoelectric focusing is useful for medicolegal individualization of brain, reproductive organs, teeth, and hairs.     

Stability of Morphine,Codeine, and 6‐Acetylmorphine in Blood at Different Sampling and Storage Conditions

The stability of drugs in biological specimens is a major concern during the evaluation of the toxicological results. The stability of morphine, codeine, and 6‐acetyl‐morphine in blood was studied after different sampling conditions: (i) in glass, polypropylene or polystyrene tubes, (ii) with addition of dipotassium ethylene diamine tetraacetic acid (K₂EDTA) or sodium oxalate (Na₂C₂O₄), and (iii) with or without the addition of sodium fluoride (NaF). Spiked blood samples were stored at two different temperatures (4 and ?20^°C), analyzed after different storage times and after three freeze–thaw cycles. Opiate concentrations were decreased in all conditions, but the most unstable was 6‐acetyl‐morphine. The addition of NaF as preservative improved the stability of opiates at all conditions studied, whereas the type of anticoagulant did not affect the stability of opiates. It was concluded that blood samples should be stored at ?20^°C in glass tubes containing oxalate and NaF for maximum stability.     

Disappearance of cocaine from human hair after abstinence

In this work the study of the disappearance of cocaine in hair is reported. The subject of the study is a woman who stopped the consumption of cocaine after a period of drug abuse of over 1 year. Hair samples were collected over a period of 10 months. During this time the absence of cocaine intake was monitored by the toxicological analysis of urine, performed every 2 days. After decontamination with methanol, the hair sample, cut in two segments (0-1.5 and 1.5-3 cm from the hair root) was added with cocaine-D(3) (internal standard), hydrolyzed and extracted with chloroform/isopropanol (9:1). The extract was evaporated to dryness, reconstituted in 25 microl of ethyl acetate and analyzed by GC-MS in SIM mode. The obtained results show that the incorporation of cocaine in hair decreased during the first 3 months after the last consumption and after this period of time no cocaine was found in the hair sections closest to the root.     

Phenobarbital in hair and drug monitoring

Phenobarbital analysis was performed in vertex hair of patients by gas chromatography mass spectrometry (GC/MS). After washing with dichloromethane, about 250 mg were ground to dust in a ball mill. A 50-mg sample was stirred mechanically for 10 min with 3 ml of NH₄Cl/HCl buffer (pH 2.0) containing phenobarbital D₅. A solid phase extraction was performed (extrelut Merck) and elution was achieved with chloroform/isopropanol/n-heptane (50:17:33; v/v). A full scan (40–240 uma) acquisition was realized by GC/MS with an ion trap (ITD 700 Finnigan) using a DB5-MS chromatographic column. Quantification was achieved by integrating dominants ions (phenobarbital, 204; phenobarbital D₅, 209). Compared to serum, hair concentrates phenobarbital during anti-epileptic therapy (average value 36.4 ng/mg, n = 40 vs. 18.7 mg/l, n = 23). A group correlation exists between phenobarbital in hair and phenobarbital in serum, and between phenobarbital in hair and clinic observation in some typical cases. Phenobarbital in hair yields good information over a long period, especially when blood collection has not been made, when clinical disorders are observed on long-term therapeutic observance.     

The polymorphism of transferrin by ultrathin-layer isoelectric focusing. Tf phenotypes and TfC subtypes in the population of Padua

The distribution of Tf phenotypes in the population of Padua was investigated by ultrathin-layer isoelectric focusing. In our sample (n = 618) nine phenotypes, Tf C₁, C₂, C₃, C_3?1, C_2?1, C_3?2, C₁B, C₂B and C₁D, were observed and the following frequencies calculated: TfC₁ = 0.77837; TfC₂ = 0.1804; TfC₃ = 0.03641; TfB = 0.0040; TfD = 0.0008. These gene frequencies have been compared to those found in other populations. Analysis of 101 mother-child pairs was in agreement with an autosomal codominant mode of inheritance.     

Effect of Temperature on the Visualization by Digital Color Mapping of Latent Fingerprint Deposits on Metal

Visualization of fingerprint deposits by digital color mapping of light reflected from the surface of heated brass, copper, aluminum, and tin has been investigated using Adobe^® Photoshop^®. Metals were heated to a range of temperatures (T) between 50°C and 500°C in 50°C intervals with enhancement being optimal when the metals are heated to 250°C, 350°C, 50°C, and 300°C, respectively, and the hue values adjusted to 247°, 245°, 5°, and 34°, respectively. Fingerprint visualization after color mapping was not degraded by subsequent washing of the metals and color mapping did not compromise the visibility of the fingerprint for all values of T. The optimum value of T for fingerprint visibility is significantly dependent of the standard reduction potential of the metal with Kendall's Tau (τ) = 0.953 (p < 0.001). For brass, this correlation is obtained when considering the standard reduction potential of zinc rather than copper.     

Simultaneous analysis of six amphetamines and analogues in hair, blood and urine by LC-ESI-MS/MS: Application to the determination of MDMA after low Ecstasy intake

A rapid and sensitive method using LC-MS/MS triple stage quadrupole for the determination of traces of amphetamine (AP), methamphetamine (MA), 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxymethamphetamine (MDMA, “ecstasy”), 3,4-methylenedioxyethamphetamine (MDEA), and N-methyl-1-(3,4-methylenedioxyphenyl)-2-butanamine (MBDB) in hair, blood and urine has been developed and validated. Chromatography was carried out on an Uptisphere ODB C₁₈ 5 μm, 2.1 mm × 150 mm column (Interchim, France) with a gradient of acetonitrile and formate 2 mM pH 3.0 buffer. Urine and blood were extracted with Toxitube A^® (Varian, France). Segmented scalp hair was treated by incubation 15 min at 80 °C in NaOH 1 M before liquid–liquid extraction with hexane/ethyl acetate (2/1, v/v). The limits of quantification (LOQ) in blood and urine were at 0.1 ng/mL for all analytes. In hair, LOQ was <5 pg/mg for MA, MDMA, MDEA and MBDB, at 14.7 pg/mg for AP and 15.7 pg/mg for MDA. Calibration curves were linear in the range 0.1–50 ng/mL in blood and urine; in the range 5–500 pg/mg for MA, MDMA, MDEA and MBDB, and 20–500 pg/mg for AP and MDA. Inter-day precisions were <13% for all analytes in all matrices. Accuracy was <20% in blood and urine at 1 and 50 ng/mL and <10% in hair at 20 and 250 pg/mg. This method was applied to the determination of MDMA in a forensic case of single administration of ecstasy to a 16-year-old female without her knowledge during a party. She suffered from hyperactivity, sweating and agitation. A first sample of urine was collected a few hours after (T + 12 h) and tested positive to amphetamines by immunoassay by a clinical laboratory. Blood and urine were sampled for forensic purposes at day 8 (D + 8) and scalp hair at day 60 (D + 60). No MDMA was detected in blood, but urine and hair were tested positive, respectively at 0.42 ng/mL and at 22 pg/mg in hair only in the segment corresponding to the period of the offence, while no MDA was detectable. This method allows the detection of MDMA up to 8 days in urine after single intake.     

Simultaneous quantification of opiates, cocaine and cannabinoids in hair

The present paper describes a sensitive method developed in our laboratory for the simultaneous analysis of opiates (morphine, codeine and monoacetylmorphine), cocainics (cocaine and benzoylecgonine) and cannabinoids (Δ⁹-tetrahydrocannabinol and 11-nor-Δ⁹-tetrahydrocannabinol-9-carboxylic acid) in hair samples. After decontaminating the sample with dichloromethane, two consecutive hydrolyses were performed in order to achieve the best conditions for extracting the three kinds of drugs from the protein matrix. First the opiate and cocainic compounds were extracted by means of a soft acidic hydrolysis with 0.1 N HCl at 50 °C overnight and organic solvent extraction at pH 9.2. The cannabinoids need a stronger basic hydrolysis with 11.8 N KOH for 10 min at laboratory temperature. After adding maleic acid, the cannabinoids were extracted with an organic solvent. The derivatization was carried out with heptafluorobutyric anhydride and hexafluoropropanol. Calibration curves were linear between 0.5–100 ng/mg of hair. Recovery and reproducibility were assured. The quantification limits ranged between 0.04–0.26 ng/mg of hair. Seventy hair samples from known drug abusers were cut into 1-cm segments and analyzed by this method. The ranges of measured concentrations (ng/mg) were 0.31–89 for cocaine, 0.1–5.76 for benzoylecgonine, 0.34–45.79 for morphine, 0.45–39.59 for codeine, 0.09–48.18 for monoacetylmorphine, 0.06–7.63 for THC and 0.06–3.87 for THC---COOH. The results of sectional analyses agreed with the self reported drug histories. The usefulness of this method is in assessing earlier drug consumption, and also at the same time obtaining a chronological profile of the consumption of these three types of drugs.     

Forensic determination of hair deposition time in crime scenes using electron paramagnetic resonance

Several types of biological samples, including hair strands, are found at crime scenes. Apart from the identification of the value and the contributor of the probative evidence, it is important to prove that the time of shedding of hair belonging to a suspect or victim matches the crime window. To this end, to estimate the ex vivo aging of hair, we evaluated time‐dependent changes in melanin‐derived free radicals in blond, brown, and black hairs by using electron paramagnetic resonance spectroscopy (EPR). Hair strands aged under controlled conditions (humidity 40%, temperature 20–22°C, indirect light, with 12/12 hour of light/darkness cycles) showed a time‐dependent decay of melanin‐derived radicals. The half‐life of eumelanin‐derived radicals in hair under our experimental settings was estimated at 22 ± 2 days whereas that of pheomelanin was about 2 days suggesting better stabilization of unpaired electrons by eumelanin. Taken together, this study provides a reference for future forensic studies on determination of degradation of shed hair in a crime scene by following eumelanin radicals by utilizing the non‐invasive, non‐destructive, and highly specific EPR technique.     

The Stability of Prostate-Specific Antigen in Semen Under Various Temperatures

Prostate-specific antigen (PSA) is most commonly used for identifying semen, especially in the absence of sperm. However, PSA concentration varies according to storage temperature and duration, and little is known about its stability in semen. This study was therefore aimed to determine the stability under five different temperatures: −80, −20, 4, 25, and 37°C; and nine different durations: 1, 2, 3, 5, 7, 14, 30, 90, and 180 days. All samples were stored at −80°C after being secreted from the volunteers' body until analyzed. Results showed that the PSA concentration declined significantly over time under all temperatures studied except −80°C. At −20 and 4°C, PSA was still detectable on Day 180 with 50% and 70% decrease from its original concentration, respectively. At 25 and 37°C, PSA was detected up to Day 7 and 3, respectively. This information might assist forensic scientists understand more about PSA nature and integrate it into their works.     

Esterase D phenotyping of bloodstains and hair roots by low voltage isoelectric focusing

A new isoelectric focusing method is described for phenotyping of esterase D in blood stains and hair roots. It permitted easy and rapid discrimination of six phenotypes determined by ESD*1, ESD*2 and ESD*7. Experiments showed it to be practicable in forensic stain work. In addition, this technique was also usable in phenotyping of ESD 5.     

Detection and Identification of Psilocybe cubensis DNA Using a Real‐Time Polymerase Chain Reaction High Resolution Melt Assay,

Psilocybe cubensis, or “magic mushroom,” is the most common species of fungus with psychedelic characteristics. Two primer sets were designed to target Psilocybe DNA using web‐based software and NBCI gene sequences. DNA was extracted from eighteen samples, including twelve mushroom species, using the Qiagen DNeasy^® Plant Mini Kit. The DNA was amplified by the polymerase chain reaction (PCR) using the primers and a master mix containing either a SYBR^® Green I, Radiant? Green, or LCGreen Plus^® intercalating dye; amplicon size was determined using agarose gel electrophoresis. The PCR assays were tested for amplifiability, specificity, reproducibility, robustness, sensitivity, and multiplexing with primers that target marijuana. The observed high resolution melt (HRM) temperatures for primer sets 1 and 7 were 78.85 ± 0.31°C and 73.22 ± 0.61°C, respectively, using SYBR^® Green I dye and 81.67 ± 0.06°C and 76.04 ± 0.11°C, respectively, using Radiant? Green dye.