The use of vitreous humour as a potential source of DNA for postmortem identification in forensic science.

Vitreous humour (VH) is routinely collected for toxicological analyses. However, only a few studies have examined its possible use in genotyping. Because of its isolation from the rest of the body, later onset of postmortem changes, and availability, VH could be a potential source of DNA for postmortem identification and other molecular analyses in forensic genetics. During forensic autopsies, samples of different VH volumes (5 ml, 4 ml, 3 ml, 2 ml, 1 ml, and 0.5 ml) were taken from 66 cadavers 24-48 h after death. DNA was also isolated from the samples immediately after collection and after a specific time of storage (1, 2, and 6 months). DNA was isolated using phenol‒chloroform‒isoamyl alcohol (PCI), and the yield and purity of the obtained DNA were determined spectrophotometrically using a FastGene NanoView Photometer. The integrity of the isolated DNA molecule was determined by PCR amplification of the hTERT (113 bp) gene. The results showed that VH could be a reliable source of genetic material for forensic analysis, and the method used for DNA extraction was effective. The yield of the isolated DNA ranged from 6.20 to 609.5 ng/µl, and the purity of the samples was 1.24-2.34. The isolated DNA concentration and integrity depend on the sample volume, but the DNA purity does not. It is also shown that DNA can be extracted from VH samples that have been stored for up to 6 months at - 20 °C. Therefore, using VH can be a valuable material for DNA identification.

The quality of DNA isolated from autopsy formalin-fixed and formalin-fixed paraffin-embedded tissues: study of 1662 samples.

BACKGROUND: There are enormous formalin-fixed paraffin-embedded tissue archives and a constantly growing number of methods for molecular analyses but, the isolation of DNA from this tissue is still challenging due to the damaging effect of formalin on DNA. To determine the extent to which DNA purity, yield and integrity depend on the process of fixation in formalin, and to what extent on the process of tissue paraffin embedding, we compared the quality of DNA isolated from fixed tissues and DNA isolated from tissues embedded in paraffin blocks after fixation. METHODS AND RESULTS: Heart, liver and brain tissues obtained from healthy people who suddenly died a violent death were fixed in 10% buffered formalin as well as in 4% unbuffered formalin for 6 h, 1-7 days (every 24 h), 10, 14, 28 days and 2 months. Additionally, the same tissues were fixed in 4% unbuffered formalin embedded in a paraffin block and stored from a few months to 30 years. The yield and purity of the DNA samples isolated from these tissues were measured using spectrophotometry. PCR amplification of the hTERT gene was performed to evaluate the degree of DNA fragmentation. Although the purity of the DNA isolated from almost all tissue samples was satisfactory, the DNA yields changed significantly. There was a decrease in successful PCR amplification of the hTERT gene in DNA samples isolated from tissue fixed in buffered and unbuffered formalin for up to 2 months from 100% to 8.3%. Archiving the tissue in paraffin blocks for up to 30 years also impacts the integrity of DNA, so there was a decrease in PCR amplification of the hTERT gene from 91% success to 3%. CONCLUSION: The largest decrease in DNA yield was observed after tissue formalin fixation after 14 days of fixation in buffered and unbuffered formalin. DNA integrity depends on the time of tissue formalin fixation, especially after 6 days for tissue fixed in unbuffered formalin, while for tissue fixed in buffered formalin the time is prolonged up to 28 days. The age of paraffin blocks also impacted DNA integrity, after 1 year and 16 years of archiving the paraffin blocks of tissues, there was a decrease in the success of PCR amplification.

DNA isolated from formalin-fixed paraffin-embedded healthy tissue after 30 years of storage can be used for forensic studies.

Tissue formalin fixation and paraffin embedding (FFPE) is a standard method for long-term preservation and morphological and molecular analysis. The aim of this study was to analyze the effect of storage time on the integrity of DNA isolated from three different healthy FFPE tissues. DNA was isolated from FFPE heart, liver and brain tissues obtained from autopsy and archived from 1988 to 2017 using two different methods of DNA isolation: phenol-chloroform-isoamyl alcohol (PCI) and PureLink Genomic DNA Kit. The quantification and purity of DNA was measured spectrophotometrically at 260 nm and 280 nm. The quality of isolated DNA was evaluated by PCR amplification of GPD1 (150 bp), ACTB (262 bp) and RPL4 (407 bp) genes. The histomorphological characteristics of FFPE tissues were not significantly changed during 30 years of storage. Higher yield (272.9 ± 10.3 µg) and purity (A260/280 = 2.05) of DNA was obtained using the PCI method for DNA isolation from FFPE liver tissue. The PCI extraction method showed reproducible and consistent results in PCR amplification of all of three examined genes. The GPD1 gene can be amplified up to 30 years, the ACTB gene in the same samples up to 26 years and the RPL4 gene up to 6 years of storage in FFPE blocks. Although the best yield and purity of isolated DNA (using both isolation methods) was obtained from FFPE liver tissue, the DNA with the most preserved integrity was obtained from brain tissue archived up to 30 years. This is the first report using long-term archived healthy FFPE tissues (up to 30 years) that shows that the DNA isolated from these tissues is of preserved integrity and can be used in molecular autopsy.

How Vulnerable Are the Elderly in Road Traffic-Autopsy Study?

Background: The aim of this study was to analyze characteristic and pattern of road traffic injuries in this special aging group, as well to examine the relevance of certain injuries or risk factors to outliving the injuries. Methods: This epidemiological, analytical, retrospective, autopsy study investigated the road traffic deaths for period between 2001 and 2022. The subjects were divided into two age groups, elderly (65 years and older) and middle aged (16-64 years old). Results: We included 213 (32%) elderly participants out of all 665 RTA subjects who died from sustained injuries or complications of the injuries. The majority of elderly subjects (61%) were pedestrians, and elderly participants in RTA were more likely to survive the injuries. The most common injuries among the elderly in all group of participants were chest injuries 81% (and among them, rib fractures were present in 76%). Cranio-cerebral injury is the most common cause of death in RTA subjects, singular (45%) or in combination with other causes. In the group of elderly RTA subjects complication of injury is represented in 36 subjects (17%), while as cause of death is represented in 22 subjects (10%). Elderly are less likely to have positive BAC, and even in cases when BAC was increased, the levels were on the lower scale. Conclusion: More effort should be made to enhance road safety for elderly pedestrians, especially considering the expected rapid growth in the elderly population.

Laboratory Puzzle of Oxidative Stress, Parameters of Hemostasis and Inflammation in Hospitalized Patients with COVID-19.

Bearing in mind that coronavirus disease (COVID-19) is associated with a wide range of laboratory abnormalities, the aim of this study was to examine the importance of determining the parameters of oxidative stress and antioxidant protection as well as markers of inflammation and hemostasis in hospitalized patients with COVID-19. The study population included 105 patients with severe COVID-19 and 65 healthy control subjects. The parameters of oxidative stress and the activity of enzymes of the antioxidant system were determined from the obtained samples using spectrophotometric methods. Standard laboratory methods were performed for the determination of the biochemical and hematological parameters. Patients with COVID-19 showed a significantly higher level of pro-oxidative parameters (hydrogen peroxide (H2O2) and the index of lipid peroxidation in the form of thiobarbituric acid-reactive substances (TBARSs)) and a significantly lower activity of the antioxidant system (catalase (CAT)). Patients with COVID-19 had significantly higher values of inflammation parameters (C-reactive protein (CRP), procalcitonin (PCT), ratio of the number of neutrophils to lymphocytes (NLR), and ratio of the number of platelets to lymphocytes (PLR)) and parameters of hemostasis (activated partial thromboplastin time (aPTT), prothrombin time (PT), D-dimer, fibrinogen) than the control healthy subjects. In addition, changes in hemostatic parameters correlated positively with inflammatory markers in the group of patients with COVID-19. The early determination of hemostasis parameters and the parameters of inflammation can help in the prediction of poor prognosis in COVID-19 patients.

Beyond the virus: Examining the impact of COVID-19 on violent fatalities.

INTRODUCTION: The COVID-19 pandemic has had a significant impact on various aspects of society, including crime rates. In Serbia, it is important to examine the changes in violent deaths before and during the pandemic to inform public health and safety policies. MATERIALS AND METHODS: We conducted a retrospective, epidemiological, cross-sectional analytical study of medico-legal autopsies in the Department of Forensic Medicine and Toxicology at the University Clinical Center of Kragujevac. Our study sample comprises all forensic autopsy cases examined from January 2017 to December 2019 (151 cases), labeled as "Before," and from January 2020 to December 2022 (192 cases), labeled as "During" the pandemic period. Natural deaths, skeletal remains, and undetermined cases were excluded from our sample. RESULTS: The data show an increase in the total number of incidents reported from 152 in the "Before" period to 191 in the "During" period. The proportion of incidents involving males remained relatively stable at around 70%, while the proportion of incidents involving females increased. There was no statistically significant change in the proportion of incidents classified as accidental, while the proportion of incidents classified as homicide and suicide increased. The results show a statistically significant association between gender and incident type for both the "Before" and "During" periods. Deaths due to domestic violence have increased by 22.2% during the pandemic, which is cause for concern. In terms of demographic characteristics, males and younger individuals were more likely to be victims of violent deaths both before and during the pandemic. CONCLUSIONS: The COVID-19 pandemic had a significant impact on violent deaths in the Sumadija region (Central Serbia), with an overall increase in the number of violent deaths and a major impact on deaths due to domestic violence. Policies to address domestic violence should be prioritized during the pandemic and beyond, and strategies should be developed to mitigate the effects of future pandemics or lockdowns.

From suicide to accident - Case report - The significance of the medico-legal autopsies.

INTRODUCTION: Suicide is the intentional and voluntary act of destroying one's own life, while an accident is an unintended event that involuntarily causes injury to one's health or destruction of life. CASE PRESENTATION: We report a case of a 54-year-old male who was found in a forest in late November, approximately 500 m away from his car. He was positioned bent over a trunk of a tree with his head beneath the rest of his body. His pants were down to his knees, and there were soiled blades of grass and leaves on his body. Investigation of the case circumstances revealed that he had attempted suicide by carbon monoxide poisoning, using a hose connected to the exhaust pipe and running it through the window into the cabin. Window on the driver's side was broken with glass particles on the driver's seat. Wrappers from "Rivotril" tablets, a generic benzodiazepine, were also found in the car. Autopsy revealed the following: postmortem hypostasis was of a cherry red color and well pronounced on the upper part of the front of the body and face. Numerous bruises, contusions, and erosions were present all over the body. Frostbites were especially pronounced in the knees and elbows area. The synovial membranes were partially bloodstained and reddish in color. Opening the stomach revealed erosions of the gastric mucosa (Wischnewsky sign). Chemical toxicological analysis detected presence of benzodiazepines and carboxyhemoglobin (25%). CONCLUSION: Based on the autopsy findings, chemical toxicological analyses, and investigation of the case circumstances, it has been concluded that the death occurred due to the combined effects of hypothermia, postural asphyxia and carboxyhemoglobin and benzodiazepine intoxication. The manner of death in this case is a combination of accidental and suicidal, as the victim attempted suicide but ultimately died due to exposure to low external temperature.

The changes in tissue histomorphology and quality of DNA in healthy human autopsied tissues stored at -20 °C and -150 °C.

The aim of this study was to compare the changes in tissue histomorphology and DNA quality in six different healthy tissues (brain, heart, lung, liver, spleen and kidney) exempted during autopsy of healthy individuals and storage at -20 °C and -150 °C three month. Tissue samples were obtained, divided by tissue and temperature group, and for each sample, tissue histomorphology and DNA (isolated from all tissues in duplicated - 72 samples of DNA) quality were analysed. Morphology of tissue samples was studied using H&E staining. DNA was isolated using the phenol-chloroform-isoamyl alcohol method. To assess the concentration and purity of the DNA samples, we used a spectrophotometer to measure absorbance at wavelengths of 280 nm and 260 nm. The fragments of human telomerase reverse transcriptase (hTERT) gene were amplified from the DNA using PCR reaction and then visualised using the 2 % agarose gel. Samples stored at -150 °C sustained the highest degree of histomorphological damage, while samples stored at -20 °C were less degraded, compared to control. The liver samples stored at -20 °C had a mean DNA concentration (1030.4 ± 51.5 ng/µl) higher than the samples of liver tissue stored at -150 °C (497.4 ± 167.1 ng/µl) (p < 0.001). Other tissues did not have statistically significantly different DNA concentration at both temperatures. Liver samples at -20 °C had degraded DNA, showed as the absence of hTERT gene in most of samples. Other tissue samples in both temperature groups had unfragmented DNA. Storing tissue samples at -20 °C is not inferior in terms of DNA yield and integrity, and possibly superior for tissue histomorphology, comparing with samples stored at -150°C.

Socioeconomic inequalities in the use of dental health care among the adult population in Serbia.

Objectives: The aim of this paper is to assess the association of demografic and socioeconomic determinants with utilization of dental services among Serbian adults. Materials and methods: The study is a part of the population health research of Serbia, conducted in the period from October to December 2019 by the Institute of Statistics of the Republic of Serbia in cooperation with the Institute of Public Health of Serbia "Dr. Milan JovanovicBatut" and the Ministry of Health of the Republic of Serbia. The research was conducted as a descriptive, cross-sectional analytical study on a representative sample of the population of Serbia. For the purposes of this study, data on the adult population aged 20 years and older were used. Results: Men were approximately 1.8 times more likely than women to not utilize dental healthcare services (OR = 1.81). The likelihood of not utilizing dental healthcare protection rises with increasing age, reaching its peak within the 65-74 age range (OR = 0.441), after which it declines. Individuals who have experienced marital dissolution due to divorce or the death of a spouse exhibit a higher probability of not utilizing health protection (OR = 1.868). As the level of education and wealth diminishes, the probability of abstaining from health protection increases by 5.8 times among respondents with an elementary school education (OR = 5.852) and 1.7 times among the most economically disadvantaged respondents (OR = 1.745). Regarding inactivity, respondents who are not employed have a 2.6-fold higher likelihood of not utilizing oral health care compared to employed respondents (OR = 2.610). Conclusion: The results suggest that individual sociodemographic factors influence utilization of dental services by Serbian adults and confirmed the existence of socioeconomic disparities.

Mental health status and coping strategies during COVID-19 pandemic among university students in Central Serbia.

Background: The objective of this research was to evaluate the occurrence of depression, anxiety, and stress, as well as the level of life contentment and coping mechanisms employed by college students amidst the COVID-19 pandemic. Methods: A total of 588 students of the Faculty of Mechanical Engineering and the Higher School of Medicine, Kraljevo, University of Kragujevac, Central Serbia, participated in an online cross-sectional survey in the period September-October 2022. Depression Anxiety Stress Scales (DASS-21) was used to measure the levels of depression, anxiety, and stress. The Coping Questionnaire-a shorter version (Brief Cope Inventories) assesses the coping strategies a person uses in stressful situations, and the 5-item Life Satisfaction Scale is used to examine the level of life satisfaction. Results: The symptoms of depression were reported by 34.9%, of anxiety by 47.1% and of stress by 44.2%. The type of faculty (p = 0.001), and place of residence (p = 0.036) correlated with depression, gender with anxiety (p = 0.001) and stress (p = 0.015). In terms of coping mechanisms, the most frequently mentioned strategies, based on average scores, were acceptance (5.76), positive interpretation (5.55), humor (5.46). Conclusion: The adverse impact of COVID-19 was a risk factor for depression, anxiety and stress symptoms. A negative correlation exists between the levels of depression, anxiety, and stress, and the overall life satisfaction of university students. Offering the necessary assistance through psychological interventions and effective coping techniques is crucial in ensuring the optimal mental health of university students.

Clinical - pathological significance of leptin receptor (LEPR) expression in squamous cell carcinoma of the skin.

Adipokine leptin functions through its transmembrane receptors (LEPR). In many malignant tumors it stimulates the growth, migration and invasion of malignant cells. The aim of our work is to examine the effect of LEPR expression on the clinical-morphological properties of squamous cell carcinoma of the skin (cSCC). The biopsy material obtained by excision of squamous cell skin cancer was used. The test group consisted of excision biopsies of squamous cell carcinoma of the skin (n = 62), and the control group (n = 62) consisted of excision biopsies of non-tumor tissue of the skin (from the tumor environment) from an operative preparation delivered to the Pathohistology Department. After routine processing and paraffin molding, histochemical Hematoxylin-Eosin and immunohistochemical ABC method with anti LEPR and Ki67 antibodies were applied at 4 µm sections. The statistical software package SPSS for Windows (26.0) was used to analyze obtained results. Intracytoplasmic and intramembranous LEPR expression was found in 100 % of examined cSCCs. LEPR expression was statistically significantly associated with proliferation index and histologic grade of tumors. Pronounced LEPR expression was associated with a high proliferation index in 66.7 % of cases and with poorly differentiated cSCC in 94.4 %. Multivariate regression analysis showed that cSCCs with pronounced LEPR expression were seven times more often poorly differentiated than tumors with moderate or LEPR expression in trace. Our results indicate that LEPR expression is a predictor of the malignant potential of cSCC, so that based on LEPR expression, it is possible to identify an aggressive cSCC phenotype, which provides the possibility of individualizing anti-tumor treatment using LEPR antagonists.

Effect of formalin fixation on pcr amplification of DNA isolated from healthy autopsy tissues.

The aim of this study is to investigate the effects of formalin fixation on the degradation of DNA molecules in five different healthy tissues exempted during the autopsy, as well as the selection of the method that is most suitable for the DNA isolation. Heart muscle, liver, brain, lung and kidney tissue obtained from the healthy people who suddenly died from a violent death were used. The parts of tissue were fixed in 10% phosphate-buffered formalin as well as in 4% unbuffered formalin at room temperature. Morphology of tissue was studied using H&E staining. The DNA was isolated 6 h, 1-7 days (every 24 h), 10, 14, 28 days and 2 months after fixation using two different methods: extraction with phenol-chloroform-isoamyl alcohol as well as with PureLink Genomic DNA Kit. Yield and purity of the DNA samples were measured spectrophotometrically at 260 nm and 280 nm. The PCR amplifications of the glycerol-3-phosphate dehydrogenase 1 (GPD1, 150 bp), ß actin (ACTB, 262 bp) and ribosomal protein L4 (RPL4, 407 bp) genes were performed to evaluate the degree of DNA fragmentation. The RPL4 gene was amplified up to 72 h, ACTB gene up to 14 days and GPD1 gene up to 28 days from tissue fixed in phosphate-buffered formalin using phenol-chloroform-isoamylalcohol protocol for DNA isolation. Liver and kidney gave better results of PCR amplification, but statistical significance between tissues was not found. Preserving period, fixative and DNA extracting method are important factors for successful PCR amplification. The healthy tissue, fixed in phosphate-formalin up to 28 days, can be useful source in molecular studies. Tissues fixed in unbuffered formalin are suitable for molecular analysis up to 7 days.