European Council of Legal Medicine (ECLM) on-site inspection forms for forensic pathology, anthropology, odontology, genetics, entomology and toxicology for forensic and medico-legal scene and corpse investigation: the Parma form.

Further to a previous publication by the European Council of Legal Medicine (ECLM) concerning on-site forensic and medico-legal scene and corpse investigation, this publication provides guidance for forensic medical specialists, pathologists and, where present, coroners' activity at a scene of death inspection and to harmonize the procedures for a correct search, detection, collection, sampling and storage of all elements which may be useful as evidence, and ensure documentation of all these steps. This ECLM's inspection form provides a checklist to be used on-site for the investigation of a corpse present at a crime or suspicious death scene. It permits the collection of all relevant data not only for the pathologist, but also for forensic anthropologists, odontologists, geneticists, entomologists and toxicologists, thus supporting a collaborative work approach. Detailed instructions for the completion of forms are provided.

Determining the time of death by morphological and immunohistochemical evaluation of collagen fibers in postmortem gingival tissues.

The estimation of the post mortem interval (PMI) is still one of the most challenging variables to determine and the different approaches currently used in its estimation generally yield to large post mortem windows. In the present study we combined morphological and immunohistochemical analysis in order to reach a more detailed knowledge on tissue organization and degradation after death. Ultrastructural cellular changes and the extracellular matrix of gingival tissues, collected at different post mortem intervals, were observed by a Transmission Electron Microscopy (TEM), in combination with the immunohistochemical detection of extracellular matrix proteins (i.e. collagen type I and collagen type III) as potential post mortem biochemical markers. The final goal was to find a correlation between morphological modifications, biomarkers expression and the time of death. Samples of gingival tissues obtained from 10 cadavers at different post mortem intervals (short post mortem interval, 1-3 days; mid post mortem interval, 4-6 days; long post mortem interval, 7-9 days) were processed for light microscopy and TEM and they were also immunostained with anti-collagen type I and type III antibodies. Results showed gradual degradation of extracellular matrix in the suboral connective tissue in relation to the different time of death. Moreover PMI was related to an increase of nuclear chromatin condensation and cytoplasmic vacuolization both in epithelial and connective tissues. In conclusion, in addition to traditional forensic approaches to estimate PMI, the combined analyses of cellular morphology, ultrastructure and immunohistochemical expression of collagen proteins allow to better infer the PMI.

In vitro gene and chromosome characterization of expanded bone marrow mesenchymal stem cells for musculo-skeletal applications.

OBJECTIVE: A number of studies have shown the role of expanded Bone Marrow-derived Mesenchymal Stem Cells in the repair and regeneration of musculo-skeletal tissues. The current European regulations define in vitro expanded cells for clinical purposes as substantially manipulated and include them in the class of Advanced-Therapy Medicinal Products to be manufactured in compliance with current Good Manufacturing Practice. Among the characteristics that such cells should display, genomic stability has recently become a major safety concern. The aim of this study is to perform a chromosomal and genetic characterization of Bone Marrow-derived Mesenchymal Stem Cells expanded in compliance with Good Manufacturing Practice for a potential clinical use in orthopaedics. MATERIALS AND METHODS: Mesenchymal Stem Cells, isolated from bone marrow, were expanded for six weeks in compliance with current Good Manufacturing Practice. DNA profiling analyses were applied to test cross-contamination absence. Genomic stability was evaluated by means of karyotyping, sequencing of TP53, p21/CDKN1A and MDM2 genes and the expression analysis of c-MYC and H-RAS oncogenes, p21/CDKN1A, TP53, p16/CDKN2A, RB1 and p27/CDKN1B tumor suppressor genes and hTERT gene. RESULTS: The DNA profiling analysis showed a unique genetic profile for each Mesenchymal Stem Cell culture, indicating the absence of cross-contamination. Karyotyping evidentiated some chromosomal abnormalities within the 10% limit set by the Cell Products Working Party review, except for one patient. In all cases, the molecular biology analyses did not revealed DNA point mutations, acquisition or changes in gene expression. hTERT levels were undetectable. CONCLUSIONS: Cultured Mesenchymal Stem Cells do not seem to be prone to malignant transformation. In fact, although some chromosomal aberrations were found, molecular biology analyses demonstrated that the expansion phase did not induce the acquisition of de novo genetic changes.

A novel DNA profiling application for the monitoring of cross-contamination in autologous chondrocyte implantation.

BACKGROUND: Autologous chondrocyte implantation (ACI) is a cell-based treatment that can be used to regenerate chondral defects. European legislation specifically classifies such produced chondrocytes as "medicinal for advanced cell therapy" that have to be manufactured in pharmaceutical factories according to specific rules, named Good Manufacturing Practices (GMPs). One main requirement of cell manipulation in advanced therapy is to prevent the risk of any contamination. AIM: The aim of this study was to verify if chondrocyte cultures suitable for ACI were free of cross-contamination by means of DNA profiling techniques. MATERIALS AND METHODS: Cell cultures were carried on in a Hospital Cell Factory in compliance with European current Good Manufacturing Practices. DNA profiling, by means of Short Tandem Repeats and miniShort Tandem Repeats analyses, was performed on expanded chondrocytes and their related control blood samples. Mitochondrial DNA was analysed to further confirm the results and to evaluate possible mutations occurred in the samples. RESULTS: Our findings demonstrated the absence of cross-contamination between chondrocyte cultures and, thus, their identity maintenance until the end of the manipulation. CONCLUSIONS: DNA profiling technique can be a suitable test for quality control not only for chondrocyte manipulation, but for cell therapy in general.

The 2011 GeFI collaborative exercise. Concordance study, proficiency testing and Italian population data on the new ENFSI/EDNAP loci D1S1656, D2S441, D10S1248, D12S391, D22S1045.

The 2011 collaborative exercise of the ISFG Italian Working Group GeFI was aimed at validating the five ENFSI/EDNAP miniSTR loci D1S1656, D2S441, D10S1248, D12S391 and D22S1045. The protocol required to type at least 50 multilocus profiles from locally resident individuals and two blind bloodstains in duplicate (i.e., using at least two different commercial kits), and to send the electropherograms to the Organizing Committee. Nineteen laboratories distributed across Italy participated, collecting a total of 960 samples. Full concordance was found for the five new miniSTRs as observed from the comparison of 13,150 alleles. The inspection of the electropherograms allowed the identification of a very limited number of mistypings in the miniSTR genotypes thus contributing to the establishment of an high quality Italian database of frequencies.

Multiplex mtDNA coding region SNP assays for molecular dissection of haplogroups U/K and J/T.

Mitochondrial DNA (mtDNA) U/K and J/T are sister haplogroups within the superhaplogroup R. They are both common in Europe, with a combined overall frequency similar to the one reported for H, the most common European haplogroup (40-50%). In this study, we selected 159 Italian subjects, already ascribed to U/K and J/T by RFLP typing, and assigned each mtDNA to specific clades/subclades by investigating at least one diagnostic coding region SNP. For each sister haplogroup, one multiplex PCR and one SNaPshot minisequencing reaction were set up targeting 16 U/K and 7 J/T coding region SNPs. Each mtDNA sample was clearly assigned to a specific subclade, which could be further subdivided into several minor sub-branches according to peculiar HVS I/II motifs. Such a molecular dissection of haplogroups U/K and J/T could be extremely useful to reduce the overall analysis time and labor intensive sequencing procedures in high volume forensic casework, for example when it is important to rapidly exclude samples in order to restrict the number of suspects.

Effects of HEMA on type I collagen protein in human gingival fibroblasts.

The cytotoxicity of dental composites has been attributed to the release of residual monomers from polymerized adhesive systems due to degradation processes or the incomplete polymerization of materials. 2-Hydroxyethyl methacrylate (HEMA) is one of the major components released from dental adhesives. Cytotoxic effects due to high concentrations of HEMA have already been investigated, but the influence of minor toxic concentrations on specific proteins such as type I collagen has not been studied in depth. The objective of this project was to study the effect of minor toxic concentrations of HEMA on human gingival fibroblasts (HGFs), investigating modification in cell morphology, cell viability, and the influence on type I collagen protein. Primary lines of human gingival fibroblasts were exposed to 3 mmol/L HEMA for different periods of time (24 h, 72 h, 96 h). The cell vitality was determined by MTT assay, and high-resolution scanning electron microscopy analysis was performed to evaluate differences in cell morphology before and after treatment. The presence and localization of type I collagen was determined by immunofluorescence in HGFs treated with HEMA for the same period of time. The vitality of the cells decreased after 72 h of exposure. The HGFs grown in monolayer and observed by field emission in-lens scanning electron microscopy demonstrated a preserved surface morphology after 24 h of treatment, while they showed an altered morphology after 96 h of treatment. Immunofluorescence demonstrated a reduction of type I collagen due to HEMA exposure after 96 h. From these results, we conclude that low concentrations of HEMA can significantly alter the morphology of human gingival fibroblasts and interfere with the presence of type I collagen protein.

Sex chromosome analysis in Turner Syndrome by a pentaplex PCR assay.

In this study, we describe a pentaplex PCR to determine the parental origin of the X chromosome and the presence of mosaicism, via amplification of four polymorphic markers located along the X chromosome (DXS10011, DXS6807, HUMARA, DXS101) and the X-Y amelogenin marker, in 41 families having a daughter with Turner Syndrome. Our results confirmed the cytogenetic findings and we found that the parental origin of the single X chromosome to be maternal in 84% of cases.

Different informativeness of the three hypervariable mitochondrial DNA regions in the population of Bologna (Italy).

Mitochondrial DNA (mtDNA) sequence variations at hypervariable regions HVI, HVII and HVIII were analysed in 100 unrelated Italians from Bologna. The values of the statistical parameters are in agreement with the range of European populations. We suggest that the less informative HVIII region may be useful to distinguish HVI-HVII identical sequences in forensic analysis especially when nuclear DNA cannot be investigated.

Automated fluorescence analysis of CAG repeats at the human androgen receptor gene (HUMARA): evaluation of polymorphism in an Italian sample and report of a new allele.

The HUMARA CAG repeats polymorphism was studied in an Italian population sample. Polymerase chain reaction amplification and automated fluorescent analysis were used. A total of 19 and 15 repeats was observed in female and male subjects, respectively, and one new allele was found. The authors conclude that this X-linked short tandem repeat, typed without ambiguity and with a heterozygosity of 0.902, is useful in parentage testing of female subjects.

The HUMFIBRA (FGA) polymorphism in an italian population and a world-wide frequency distribution analysis.

OBJECTIVE: The study investigated the highly polymorphic HumFGA short tandem repeat in a sample of 219 unrelated and native individuals from Bologna, and analysed a complete database of FGA allele frequency distributions in 57 world-wide populations collected from the literature. METHOD: The HumFGA polymorphism was screened by automated fluorescence analysis of PCR-amplified labelled sample fragments performed with an ABI PRISM 310 Genetic Analyser. Genetic distances (Dsw, delta mu2 and Fst) between populations were computed with the MSAT.2 program. Non-metric multidimensional scaling (nmMDS) and neighbour-joining trees (NJTs) were used to investigate patterns of population affinities. Correspondence analysis of the genetic relationships among populations was also performed. MAIN RESULTS AND CONCLUSIONS: The FGA microsatellite locus is a population marker with a high degree of polymorphism throughout the world. Fourteen HumFGA alleles, ranging in size from 18 to 29 repeats, were identified and sequenced in the Bologna population. The sample was in Hardy-Weinberg equilibrium and had a heterozygosity value of 0.86. Results obtained from the multivariate analyses were consistent in showing great similarity among Europeans. The few African populations investigated were characterized by an even higher level of polymorphism, probably related to the ancient peopling of that continent.

A method for sex assignment in mixed samples.

A method for male sex assignment in mixed samples by amplifying a specific amelogenin Y sequence is described. The specificity and the high sensitivity make it suitable for basic research, forensic evaluations and clinical applications.

Myotonic dystrophy CTG repeats in an Italian population sample: evaluation of the polymorphism for forensic applications.

The myotonic dystrophy (DM) CTG repeat polymorphism has been studied in an Italian population sample. Polymerase chain reaction (PCR) amplification, manual polyacrylamide gel electrophoresis (PAGE), and silver staining were employed. Alleles were typed by comparison with a sequenced allelic ladder. A total of 25 different alleles, spanning the range from 5 to 31 CTG triplets, was observed. The heterozygosity was 79%, and no significant deviation from Hardy-Weinberg equilibrium was found. Eighty-one meioses from parentage testing were also analyzed, and a Mendelian pattern of inheritance was observed in all cases. In addition, we could successfully type the DM locus in 20 laboratory-prepared bloodstains, with 1 ng of DNA allowing clear definition of alleles. We conclude that the CTG repeats at the DM locus may be useful for forensic applications.

The DRPLA CAG repeats in an Italian population sample: evaluation of the polymorphism for forensic applications.

The DRPLA CAG repeats polymorphism has been studied in an Italian population sample. PCR amplification, manual PAGE and silver staining were employed. A total of 16 different alleles, spanning the range from 5 to 21 CAG triplettes, was observed. The heterozygosity was 0.81 and no significant deviation from Hardy-Weinberg equilibrium was found 81 meioses from parentage testing were also analyzed and a Mendelian pattern of inheritance was observed in all cases. In addition, we could successfully type DRPLA locus in some forensic specimens, 1 ng of DNA allowing clear definition of alleles. The authors conclude that the DRPLA CAG repeats analysis may be useful for forensic applications.

Identification of anti-HIV-1 antibodies in bloodstains of various ages.

The persistence of anti-HIV-1 antibodies in bloodstains has been studied by ELISA and Western Blot (WB) analysis. The immunoblot technique was found to be specific and more sensitive and the antibodies could be detected in 0.7 mg of bloodstains for up to 6 months. The authors emphasize the importance of such an investigation on non-genetic markers in individual diagnosis for forensic purposes.

A common disinfectant used in condom processing inhibits endonuclease digestion of sperm DNA.

DNA recovered from a condom found at a crime scene appeared undigestible with restriction enzymes, preventing characterization by Southern blot and polymorphic probe hybridization. Several chemical substances used in the processing and treatment of condoms were tested for inhibitory action of restriction enzymes. In particular dibenzalkonium chloride appeared to promote enzyme inhibition at very low concentrations. The effectiveness of treatments to restore cleavage of sample DNA in the presence of such contaminants is discussed.

[Sex determination in hair roots without sheath: proposal of a method for preparation of specimens]. / La diagnosi di sesso sulle radici pilifere prive di guaina: proposta di un metodo per allestire i preparati.

Japanese authors have described a procedure for the sex determination of human hair roots without epithelial sheath, paraffin embedded and fluorescent stained for cortical cell nuclei Y or X sex chromatins. This method however is not sufficiently practical for forensic science purposes: it is difficult and not guaranteed to obtain suitable longitudinal hair sections with possible loss of evidence particularly serious when the questioned hair is only one. The authors, researching Y-bodies with Quinacrine, present an alternative technique of preparation of specimens without embedding which is simple and quick, reproducible and satisfying in results and that avoids loss of material.