F. De Ferrari

Application of alternative fixatives to formalin in diagnostic pathology.

Fixation is a critical step in the preparation of tissues for histopathology. The objective of this study was to investigate the effects of different fixatives vs formalin on proteins and DNA, and to evaluate alternative fixation for morphological diagnosis and nucleic acid preservation for molecular methods. Forty tissues were fixed for 24 h with six different fixatives: the gold standard fixative formalin, the historical fixatives Bouin and Hollande, and the alternative fixatives Greenfix, UPM and CyMol. Tissues were stained (Haematoxylin-Eosin, Periodic Acid Schiff, Trichromic, Alcian-blue, High Iron Diamine), and their antigenicity was determined by immunohistochemistry (performed with PAN-CK, CD31, Ki-67, S100, CD68, AML antibodies). DNA extraction, KRAS sequencing, FISH for CEP-17, and flow cytometry analysis of nuclear DNA content were applied. For cell morphology the alternative fixatives (Greenfix, UPM, CyMol) were equivalent to formalin. As expected, Hollande proved the best fixative for morphology. The morphology obtained with Bouin was comparable to that with formalin. Hollande was the best fixative for histochemistry. Bouin proved equivalent to formalin. The alternative fixatives were equivalent to formalin, although with greater variability in haematoxylin-eosin staining. It proved possible to obtain immunohistochemical staining largely equivalent to that following formalin-fixation with the following fixatives: Greenfix, Hollande, UPM and CyMol. The tissues fixed in Bouin did not provide results comparable to those obtained with formalin. The DNA extracted from samples fixed with alternative fixatives was found to be suitable for molecular analysis.

The labelling of gunpowder: An approach to improve gunshot residues determination

Abstract Modern physico-chemical methods allow reliable forensic analysis of gunshot residues (GSR) from the hands of suspected persons. Inorganic components of primer such as barium and antimony and such elements as lead and copper contained in the bullets can be accurately revealed by means of many analytical techniques. In order to improve the reliability of the method, we labelled some commercial cartridges with samarium oxide and tested, after shooting, its presence on the hands of the shooter. The results show that Sm has never been found on the hands of non-shooters, but it has been shown by neutron activation analysis to be on the hands of the person who fired the gun, therefore suggesting the use of samarium oxide to label gunpowder to be a reliable method in forensic research. The ballistic tests made using labelled and non-labelled cartridges, showed that the presence of samarium oxide does not change their properties.

Age determination by teeth examination: a comparison between different morphologic and quantitative analyses

Age determination by teeth examination is one of the main means of determining personal identification. Current studies have suggested different techniques for determining the age of a subject by means of the analysis of microscopic and macroscopic structural modifications of the tooth with ageing. The histological approach is useful among the various methodologies utilized for this purpose. It is still unclear as to what is the best technique, as almost all the authors suggest the use of the approach they themselves have tested. In the present study, age determination by means of microscopic techniques has been based on the quantitative analysis of three parameters, all well recognized in specialized literature: 1. dentinal tubules density/sclerosis 2. tooth translucency 3. analysis of the cementum thickness. After a description of the three methodologies (with automatic image processing of the dentinal sclerosis utilizing an appropriate computer program developed by the authors) the results obtained on cases using the three different approaches are presented, and the merits and failings of each technique are identified with the intention of identifying the one offering the least degree of error in age determination.

Typing of teeth with two different amplification systems

Abstract Two different automated procedures (capillary electrophoresis and infrared automated sequencer) for STR analysis on DNA obtained from highly degraded teeth were compared. Even with these two automatic protocols, negative or not much informative results were obtained. Our results confirm that in extreme conditions, it is necessary to perform more sensitive analysis (i.e. SNPs, mt-DNA) to obtain informative genetic profiles.

Italian population data on the loci MR, GYPA, HBGG, D7S8 and GC

Allele and genotype frequencies for the five loci LDLR, GYPA, HBGG, D7S8 and GC were determined for 374 unrelated Italians using a multiplex PCR-amplification and typing commercial kit. The distribution of the genotype frequencies showed no deviations from Hardy-Weinberg expectations. The combined power of discrimination and chance of exclusion for all five loci were 0.999 and 0.702, respectively. A test for homogeneity was performed and no significant differences were observed among the Caucasian population samples.

Genetic polymorphism of orosomucoid (ORM1 and ORM2) in Lombardy (Italy)

SummaryOrosomucoid subtypes have been analysed in 600 unrelated individuals from the Brescia Area (Lombardy-Italy) by the isoelectric focusing technique and immunoprinting. Five ORMI phenotypes were observed. The ORM2 pattern was found to be virtually monomorphic, but one variant with an additional single band was found. The estimated allele frequencies were: ORM1*F1 = 0.5992, ORM1*F2 = 0.0150, ORM1*S = 0.3858.ZusammenfassungBei 600 nicht verwandten Individuen aus Brescia (Italien) wurden die Orosomucoid-Subtypen mittels isoelektrischer Fokussierung und anschließender PrintImmunofixation bestimmt. Die beobachteten Allelefrequenzen waren: ORM1*F1 = 0.5992, ORM1*F2 = 0.0150, ORM1*S = 0.3858.

MtDNA: a successful identification case in burned remains

Abstract Some human carbonized remains were found in a wood near a lake. Investigations and a macroscopic analysis of the remains led suspicions to a killed and burned 20-year-old man. To identify with certitude the remains, nuclear DNA from the remains and from the alleged mother were analysed. Only at vWA locus an exclusion was found. Because of the delicacy of the case (a murder) and since only the alleged mothers DNA was available, a mt-DNA analysis was performed. It was found a good correlation in both HV1 and HV2 regions between the DNA from the remains and the mothers.

Allele frequency distribution of 13 STRs in an Italian and immigrant population sample

In the present study, we analysed allelic and genotypic distribution of 13 STR in the population of Brescia (North Italy) and in the immigrant population (North Africans and East Europeans) to create a database. DNA was extracted from 120 samples; STR amplification was carried out according to the AmpFiSTR Profiler Plus and SGM kit protocols. Amplified products were analysed using an ABI Prism 310 Genetic Analyzer. The distributions were also compared with those from other Italian populations.

De novo mutations at D3S1358, D8S1179 and D18S51 loci emerged during paternity testing: confirmation of biological paternal lineage by using a panel of Y-chromosome STRs

Abstract In three paternity tests performed in our laboratories with a battery of autosomal STRs, we discovered three separate incompatibilities for the loci D3S1358, D8S1179 and D18S51. Since the other employed markers confirmed the paternity of the alleged father, the observed incompatibility for these markers was probably due to mutation events. However, taking into account the mutation value for the markers, the paternity index (PI) and posterior probability ( W ) results were very low. In these cases, we normally analyse an extra panel of polymorphic markers to increase the PI value. However, in these three cases, since the disputed children were males and the Y-chromosome is transmitted exclusively to the male lineage, we studied a battery of 10 Y-STR in the children and in the alleged fathers. Evaluation of Y-STR haplotype frequencies was deduced from a European available database.

Allele Distribution of the Amplitype PM Coamplification System in a Population of Northern Italy

Forensic application of any genetic marker requires a study to carry out a data base of the relevant population for a correct use of the analysis results. According to this guideline the allele distribution of 5 different PCR polymorphisms (LDLR, GYPA, HBGG, D7S8 and GC) were investigated.