Andrea Verzeletti

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.

Population data for 12 Y-chromosome STRs in a sample from Brescia (northern Italy)

Twelve Y-chromosome STRs--DYS19, DYS389-I, DYS389-II, DYS390, DYS391, DYS392, DYS393, DYS385, DYS437, DYS438, DYS439--were typed in a population sample (n=104) of unrelated males from Brescia (northern Italy). A total of 91 haplotypes were identified by the 12 Y-STR loci. The haplotype diversity (98.68%), discrimination capacity (87.50%) and gene diversity were calculated.

Population data on 5 non-CODIS STR loci (D10S1248, D22S1045, D2S441, D1S1656, D12S391) in a population sample from Brescia county (Northern Italy)

Abstract Five autosomal STR loci (D10S1248, D22S1045, D2S441, D1S1656 and D12S391), unlinked to the core STR loci (non-CODIS loci), were typed in a population sample of 210 unrelated individuals from Brescia (Northern Italy). Allelic frequencies and statistical parameters of forensic interest were estimated. A comparison between our population data and others from Caucasian and Asian populations was performed.

Genetic variation at 5 new autosomal short tandem repeat markers (D10S1248, D22S1045, D2S441, D1S1656, D12S391) in a population-based sample from Maghreb region

Aim To investigate allele distribution and genetic parameters of a population-based sample from Maghreb region. Methods Allele frequencies for 5 new autosomal short tandem repeat (STR) markers (D10S1248, D22S1045, D2S441, D1S1656, and D12S391) and several forensic parameters were determined for 95 unrelated individuals. Results The combined power of discrimination and power of exclusion for the 5 loci were high (0.9999991 and 0.9954757, respectively). Allele frequencies were compared with previously published population data. Significant differences were found between Maghreb population and all other populations at the locus D2S441. Also, significant differences were found between the Maghreb and the African American population at the D22S1045, D1S1656, and D12S391 loci, between Maghreb and Caucasian population at the D1S1656 locus, and between Maghreb and Hispanic population at the D22S1045 locus. Conclusions Typing of the 5 new STR loci may provide a useful addition to the previously established sets of autosomal STRs.

Post-mortem injuries by a dog: a case report

Despite its unlikely occurrence, post-mortem animal depredation is not unknown to forensic pathologists. In the case at issue, the corpse of a dead woman presented extensive facial wounds, which were then traced back to the dog she owned. A small specimen of injured tissue was subjected to species diagnosis, and came back positive for human and canine antigens, which confirmed the presence of biological material of canine origin on the body. The less than usual post-mortal injury pattern described herein clearly highlights the possibility that animal depredation on a corpse may occur soon after death, and underscores the diagnostic potential posed by commonly available and low expensive testing methods such as serological species diagnosis.

Homicide–suicide in Brescia County (Northern Italy): A retrospective study from 1987 to 2012

From January 1987 to December 2012, 19 homicide-suicide events were registered at the Brescia Institute of Forensic Medicine (Northern Italy), leading to 39 deaths (20 homicide victims and 19 suicide victims). The homicide victims were females in the total of the cases (100%), while perpetrators were exclusively males (100%). Only one event involved foreigners as both victim and perpetrator, all the other cases regarded Italian people. The average age was 37.3 years for the homicide victims and 41.57 years for the offenders. Perpetrators usually used a firearm both for murder (65%) and suicide (84%). In 66% of the cases the homicide-suicide events occurred at home; homicide victims were strictly bound to their perpetrators (husband, boyfriend or ex boyfriend, father) in all the events.

Population data for 15 autosomal STRs loci and 12 Y chromosome STRs loci in a population sample from the Sardinia island (Italy).

One hundred twenty-five unrelated individuals (69 females and 56 males) from Sassari (Northern Sardinia) and Orgosolo (Central Sardinia) were typed for 15 STRs loci. The 56 males were typed for 12 Y chromosome STRs loci too. Frequency distribution is described.

An autopsy study of sudden cardiac death in persons aged 1-40 years in Brescia (Italy).

Introduction Sudden cardiac death (SCD), above all when occurring in young people, remains a major clinical problem. We have analysed the clinical and post mortem findings of patients who were evaluated for SCD. Methods and results We have analysed 54 cases of SCD which occurred in patients aged below 40 years during the period 1993–2012 and were studied at the Institute of Forensic Medicine of Brescia. The following variables were considered: sex, age, medical history, autopsy findings with special reference to macroscopic and histological evaluation of the heart and toxicological investigation. In all cases, we also performed the dissection of the cardiac conduction tissue with subsequent serial sampling and careful microscopic evaluation. Most SCD patients were men (76%), with a mean age of 27 years. The results of post mortem investigations have identified the following abnormalities: coronary artery disease (18.5%), arrhythmogenic right ventricular dysplasia (11.1%), hypertrophic obstructive cardiomyopathy (9.2%), severe valvular heart disease (7.4%) and myocarditis (7.4%). A case of persistence of the inter-atrial communication with cardiomegaly and right and left ventricular hypertrophy was also reported. Examination of the cardiac conduction tissue showed abnormalities in 12 cases (22.2%), in whom the heart was structurally normal at macroscopic examination. Despite all the investigations carried out, any pathogenic substrate that could have justified death was not found in 12 cases (22.2%). Conclusion Our study underlines the value of an accurate routine post mortem investigation that may show an otherwise unsuspected structural heart disease. The serial study of the conduction tissue may provide pathologic substrates that may be responsible for the arrhythmic cause of death. A meaningful percentage of cases (22%) had no evidence of any abnormality. Genetic testing can be indicated in these cases.

Sudden death from an asymptomatic phaeochromocytoma: A case report

A case of sudden death due to a phaeochromocytoma in an apparently healthy young man is presented. In cases of sudden death it is necessary to perform a complete and accurate autopsy also looking for alterations in other organs than the heart that could explain the death.

The molecular characterization of a depurinated trial DNA sample can be a model to understand the reliability of the results in forensic genetics

The role of DNA damage in PCR processivity/fidelity is a relevant topic in molecular investigation of aged/forensic samples. In order to reproduce one of the most common lesions occurring in postmortem tissues, a new protocol based on aqueous hydrolysis of the DNA was developed in vitro. Twenty‐five forensic laboratories were then provided with 3.0 μg of a trial sample (TS) exhibiting, in mean, the loss of 1 base of 20, and a molecular weight below 300 bp. Each participating laboratory could freely choose any combination of methods, leading to the quantification and to the definition of the STR profile of the TS, through the documentation of each step of the analytical approaches selected. The results of the TS quantification by qPCR showed significant differences in the amount of DNA recorded by the participating laboratories using different commercial kits. These data show that only DNA quantification “relative” to the used kit (probe) is possible, being the “absolute” amount of DNA inversely related to the length of the target region (r2 = 0.891). In addition, our results indicate that the absence of a shared stable and certified reference quantitative standard is also likely involved. STR profiling was carried out selecting five different commercial kits and amplifying the TS for a total number of 212 multiplex PCRs, thus representing an interesting overview of the different analytical protocols used by the participating laboratories. Nine laboratories decided to characterize the TS using a single kit, with a number of amplifications varying from 2 to 12, obtaining only partial STR profiles. Most of the participants determined partial or full profiles using a combination of two or more kits, and a number of amplifications varying from 2 to 27. The performance of each laboratory was described in terms of number of correctly characterized loci, dropped‐out markers, unreliable genotypes, and incorrect results. The incidence of unreliable and incorrect genotypes was found to be higher for participants carrying out a limited number of amplifications, insufficient to define the correct genotypes from damaged DNA samples such as the TS. Finally, from a dataset containing about 4500 amplicons, the frequency of PCR artifacts (allele dropout, allele drop‐in, and allelic imbalance) was calculated for each kit showing that the new chemistry of the kits is not able to overcome the concern of template‐related factors. The results of this collaborative exercise emphasize the advantages of using a standardized degraded DNA sample in the definition of which analytical parameters are critical for the outcome of the STR profiles.