Identification of skeletal remains in Croatia and Bosnia and Herzegovina, including the homeland war - a 30-year review.

Over the past 30 years, forensic experts from Croatia and Bosnia and Herzegovina have embraced advanced technologies and innovations to enable great efficacy and proficiency in the identification of war victims. The wartime events in the countries of former Yugoslavia greatly influenced the application of the selected DNA analyses as routine tools for the identification of skeletal remains, especially those from mass graves. Initially, the work was challenging because of the magnitude of the events, technical aspects, and political aspects. Collaboration with reputable foreign forensic experts helped tremendously in the efforts to start applying DNA analysis routinely and with increasing success. In this article, we reviewed the most significant achievements related to the application of DNA analysis in identifying skeletal remains in situations where standard identification methods were insufficient.

Investigation of WWII/postwar mass burials in Croatia-The implementation of the Croatian model of searching for the imprisoned and missing persons.

The search for victims of World War Two (WWII) and the immediate aftermath period (postwar) in Croatia started together with the search for Homeland War victims in 1991. It continued through years, most often sporadically and in a non-homogenous way. It was just with the adoption of the Law on Research, Arrangement and Maintenance of Military Cemeteries, Cemeteries of Victims of WWII and Postwar Period in 2013 that the search became more structured and gained a formal governmental body responsible for the organization and supervision of the activities related to it. It was then that the well-established model of searching for Homeland War victims, based on many years of field work and research, started to be implemented in the search and analysis of WWII/postwar victims. The model represents a continuously growing and developing project which encompasses a wide variety of steps and procedures, from the investigation of alleged burial locations to the analysis and reburial of recovered mortal remains. From its implementation in 2016, it allowed the successful investigation of 1300 alleged burial locations, 484 field surveys, 42 exhumations and the recovery of remains of more than 1600 individuals. Besides, care for military cemeteries, marking of mass burial sites and arranging of international treaties are conducted in order to guarantee proper handling, relocation and repatriation of all those that perished during WWII and the postwar period.

A case of synthetic cannabinoid poisoning in Croatia.

The number of new psychoactive substances (NPS), synthetic cannabinoids (SCs) in particular, is growing constantly. Because of the insufficiently explored effects on consumer health, they have become a major problem in the emergency departments. They are difficult to identify, and there are no antidotes that could reverse their detrimental effects. We report a case of poisoning of a young man who used SCs. The patient was admitted to the emergency department of the Clinical Hospital Merkur, Zagreb (Croatia) after sniffing and smoking a herbal product bought on the street. He presented with severe cognitive difficulties and visible eye redness. Other symptoms included somnolence, disorientation, loss of coordination, unsteady gait, hyporeflexia, stiffness, cramps and cold limbs, blurred vision, teeth grinding, dry mouth, tinnitus, fear, suicidal thoughts, impaired focus, memory, and speech, sedation, fatigue, depression, thought blocking, and autistic behaviour. His skin was dry, and his mucosa dry and irritated. Herbal products "Rainbow Special" and "Luminated Aroma" used by the patient were qualitatively analysed with gas chromatography / mass spectrometry (GC/MS) after direct extraction with an organic solvent. Solid-phase extraction method was used to analyse serum and urine samples. Despite the negative findings of biological samples, mostly due to the limitations of GC/MS, the clinical picture infallibly pointed to the poisoning with SCs. This was confirmed by the findings of 5-fluoro AMB (methyl 2-(1-(5-fluoropentyl)-1H-indazole-3-carboxamido)-3-methylbutanoate) in the herbal products.

Methadone Maintenance Treatment: A 15-year Retrospective Study in Split-Dalmatia County, Croatia.

BACKGROUND: The aim of this retrospective study is to use existing epidemiological data on patients in methadone maintenance treatment in Split-Dalmatia County from 2001 to 2015 to evaluate the substitution treatment system and policy of opiate addicts treatment, the epidemiological trend, and risk of overdose mortality. In addition, we would like to highlight the problem of poor control of methadone therapy and thus the possibility of selling methadone. The result is a greater number of deaths caused by methadone in people who were not in methadone therapy. METHODS: The research included data collected from 3189 patients who had been in the drug abuse treatment program in Split-Dalmatia County during 2001-2015 and data gathered from autopsy examinations of patients in Split-Dalmatia County with emphasis on data for 186 overdosed patients during research period. RESULTS: The total number of patients in the methadone treatment program in Split-Dalmatia County during the research period, except in 2005 and 2006, has been stable, while the number of new opiate patients, being in first-time treatment, decreased by 62.5%. The number of addicts who were in a long-term maintenance program has increased by 198%, whereas the number of addicts who were in a short-term detoxification treatment has decreased by 96.4%. According to results obtained from performed autopsies, 186 death cases were determined as overdoses. Methadone was found in 56 of those cases and was declared as the cause of death in 39 cases (70%). Of the total number of autopsied patients with diagnosed methadone overdose, only 23 (59%) had been recorded to receive methadone therapy in Public Health Institute of Split-Dalmatia County database. CONCLUSIONS: The results of this study show the favorable epidemiological trend because of the decreasing number of new opiate patients in treatment. The retention of opiate patients in substitution therapy indicates the effectiveness of methadone maintenance programs. Our research did not determine any influence of methadone substitution therapy on an increasing risk of specific (overdose) mortality.

Anthropometric characteristics and traffic accident circumstances of patients with isolated whiplash injury in University Clinical Hospital Mostar.

Aim To investigate anthropometric characteristics and traffic accident circumstances of subjects with isolated whiplash injury. Methods This cross sectional study involved 75 subjects from traffic accidents with isolated whiplash injury classified by Quebec Task Force (QTF). Anthropometric data were collected as well as claims about circumstances of traffic accidents. Results Distribution of 1st (28; 37.3%), 2nd (25; 33.3%) and 3rd (22; 29.3%) grade of whiplash injury was almost equal. Females had smaller anthropometric measurements than males; neck circumference was the most significant difference between males and females in the context of whiplash injury. The most frequent collision mechanism was impact to front (26; 34.7%) or to rear end (26; 34.7%) of a small passenger's car. Assertions of participants were that their car damage was significant (37; 49.2%) or total (24; 32%). A total of 38 (50.7%) participants claimed that they were not wearing safety belt and 52 (69.3%) did not find themselves responsible for accident. Conclusion Driving habits of our participants facilitate incidence of whiplash injuries, especially in vulnerable groups such as women and elderly.

Blood alcohol stability in postmortem blood samples.

The effect of long-term storage on alcohol stability in postmortem blood samples was investigated. Seventy-nine blood samples were used in this study. For the first time, blood alcohol concentrations were measured within 1 to 4 days after being taken at the Laboratory of Forensic Toxicology. After storage (ranging from 191 to 468 days), each sample was reanalyzed for blood alcohol concentration, and results were compared with the original analysis. Results showed good agreement between the experimental measurements. About 90% of the results lie within 95% limits, and 10% were outside. Despite these positive correlations, the results of some blood samples showed a high variation in ethanol concentration. Deviation in alcohol concentration between replicates of up to 10% (observed in 39% samples) is not acceptable as precise forensic evidence.

Bone porosity and longevity in early medieval Southern Croatia.

Porosity of the skull and skeletal remains, especially of the orbital roof, are one of the most frequent pathological findings on ancient human skeletal remains. There are several presumed causes of this condition and anthropologists consider skull porosities as a marker of physical and nutritional stress. A total of 115 graves were discovered at the early-medieval graveyard near Zadar (Croatia) that contained 128 partially preserved skeletons. Average estimated age at death was 37.2 ± 12.6 years for men, 31.9 ± 13.9 for women, and 5.3 ± 3.6 years for subadults. Pathological bone porosity was analysed. Cribra orbitalia was observed on 21 skulls (28.7%), signs of temporal porosity were noticed on six skulls and signs of subperiosteal bleeding on three skulls. Nineteen skulls had bone porosities in other areas. There was a significant difference (p = 0.039) in achieved age of adults with and without cribra orbitalia as those with cribra orbitalia lived on average 8.1 years longer. The bone porosity was probably caused by malnutrition that might have had a beneficial effect on longevity of adults, similar to effects of restricted food intake on extending lifespan through epigenetic signatures influencing gene expression.

The role of alcohol in road traffic accidents with fatal outcome: 10-year period in Croatia Split-Dalmatia County.

OBJECTIVE: The aim of the study was to investigate the relationship between alcohol consumption and fatal road traffic accidents that took place in Split-Dalmatia County (Croatia) during a 10-year period (from August 2001 to August 2011). METHODS: A retrospective analysis of 474 autopsy reports was performed, with an emphasis on 337 toxicologal findings of blood alcohol concentration (BAC) and the mode of participation in traffic (driver, passenger, pedestrian, or cyclist). Relations between the BAC, the weekday on which death occurred, victim's age and gender, and differences according to 3 successive legislation periods within the observed time frame were analyzed. The BAC was measured by gas chromatography with headspace and flame ionization detection. RESULTS: Alcohol was regarded as a contributive death factor if BAC was positive; that is, higher than 0.5 g/kg. BAC was positive in 177 cases. Most often it was the car driver who had a positive BAC. Victims of traffic accidents were mostly male drivers, and those accidents were more often associated with alcohol consumption. Consumption of alcohol produced a significant increase in culpability. CONCLUSION: The results of our study show that alcohol remains one of the main contributing factors of traffic accidents in Split-Dalmatia County. Unfortunately, legislation changes regarding the allowed BAC did not cause any reduction in casualties among drivers driving under the influence of alcohol (DUIA). Chi-square test of the number of total driver victims and driver victims who were DUIA revealed no significant difference (P = .173).

Croatia is a safe tourist destination - study of foreign citizen mortality in Splitsko-dalmatinska and Primorsko-goranska County during the period 2001-2010.

AIM: To investigate the mortality rate of foreign citizens in Croatia. METHODS: Data were collected from the Departments for Forensic Pathology in Split and Rijeka, which are the autopsy centers of the counties with approximately 35% of total foreign visitors, as well as from the Croatian Central Bureau for Statistics for the period 2001-2010. The mortality rate (number of deaths of members of each nationality per 100000 entrances ratio) and standardized mortality ratio (ratio between the observed and expected number of deaths) were calculated, and χ(2) goodness of fit test was used for statistical analyses. RESULTS: There were 447 deaths (325 men, 72.7%) of foreign citizens (mortality rate of 0.0015%). A total of 207 deaths (46.3%) were by natural causes, more often among older people, and 240 deaths (53.7%) were injury deaths, more often among younger people, mostly by drowning or traffic-related (22.2% and 18.6% of all deaths, respectively). Most represented were citizens of German, Austrian, Czech, and Italian nationality, with 115 (25.7%), 59 (13.2%), 58 (13.0%), and 52 deaths (11.6%), respectively. Mortality rate by nationality showed no significant difference (P<0.05). The standardized mortality ratio was lowest in Hungarian and Czech citizens (0.17) and highest in US citizens (0.35). CONCLUSIONS: Croatia has low foreign citizens' mortality rate and could be considered a safe tourist destination.

Influence of war on quantitative and qualitative changes in drug-induced mortality in Split-Dalmatia County, Croatia.

AIM: To study drug-induced mortality and characteristics of overdose deaths in the war (1991-1995), pre-war (1986-1990), and post-war period (1996-2000) in Split-Dalmatia County. METHODS: We retrospectively searched through Databases of the Department of Forensic Medicine, University Hospital Split, the national register of death records, the archives of the Split-Dalmatia County Police, and the Register of Treated Drug Addicts of the Croatian National Institute of Public Health, covering the period from 1986 to 2000, according to drug poisoning codes IX and X of the International Classification of Diseases. The indicators were statistically analyzed. RESULTS: There were 146 registered drug-induced deaths, with 136 (93%) deceased being men. The median age of all cases was 27 years (interquartile range 8). Most of them were single (70.6%), unemployed (44.6%), and secondary school graduates (69.2%). In the war period, there were 4.8 times more deaths than in the pre-war period (P=0.014), and in the post-war period there were 5.2 times more deaths than in the pre-war period (P=0.008). The most common site of death was the deceased person's home. The toxicological analyses showed that 59 (61%) deaths were heroin related, alcohol use was found in 62 cases (42.5%), and multi-substance use was found in more than a half of the cases. In 133 (91.1%) cases, deaths were classified as unintentional, whereas 13 (8.9%) were classified as suicides. CONCLUSION; The war, along with other risk factors, contributed to unfavorable developments related to drug abuse in Split-Dalmatia County, including the increase in the drug-induced mortality rate.

Suicide and Emo youth subculture--a case analysis.

Depression and suicide present a serious health problem especially for teenagers as they are increasingly diagnosed with mood disorders of different severity, possibly leading to suicidal activity. Reported here is a misfortunate young girl who committed suicide by jumping from high altitude. She left a suicide note which, together with her behavior in the death-preceding period, pointed to her apparently belonging to an Emo subculture. Although few and scarce, most existing articles and reports on Emo subculture found that its members like to focus on negative things, dark premonitions and deprivation of enjoyment, like self harm and suicide but no scientific information are available about the characteristics, trends and possible suicidal tendencies of children and adolescents who belong to this subgroup. It is for the future researches to answer whether this type of behavior and music preference are causal factors for increased suicidal vulnerability, or personal characteristics and anamnesis, upbringing and mental health status are actual sources of the problem.

Y-chromosomal short tandem repeat haplotypes in southern Croatian male population defined by 17 loci.

AIM: To define the Y-chromosome genetic structure in a sample of men from southern Croatia. METHODS: Blood samples were collected from 166 unrelated healthy men from southern Croatia at the Department of Forensic Medicine and Biochemical Laboratory of University Hospital Split between 2004 and 2007. Genomic DNA was extracted using the standard procedures. Seventeen Y-chromosome short tandem repeat (Y-STR) polymorphic loci (DYS456, DYS389I, DYS390, DYS389II, DYS458, DYS19, DYS385, DYS393, DYS391, DYS439, DYS635, DYS392, GATAH4, DYS437, DYS438, and DYS448) were analyzed using AmpFlSTR Yfiler Polymerase Chain Reaction Amplification Kit. RESULTS: We observed 152 different haplotypes. Total haplotype diversity was 0.997289 and 141 haplotypes (84.49%) were unique. The most common haplotype was shared by only 4 men in the study sample. The locus diversity ranged between 0.21292 for DYS392 and 0.75546 for DYS439 locus. CONCLUSION: The Y-chromosome structure in men from southern Croatia is very diverse. Combination of Y chromosome 17 STR loci may be used as a powerful tool for individual identification and parentage analysis in the southern Croatian male population.

Interaction of humic acids with human DNA: proposed mechanisms and kinetics.

Human DNA quantification by quantitative real-time PCR (QRT-PCR) has gained great importance in forensic DNA and ancient DNA studies. However, in such samples, DNA quantification is impaired by the frequently present humic acid (HA). We have previously shown that the addition of synthetic HA inhibits QRT-PCR. In this study we investigated the possible mechanisms of HA interaction with human DNA, and kinetics of QRT-PCR inhibition. In QRT-PCR with pure human DNA and no HA added, VMAX was 40. With DNA sample containing 4 microg/mL of HA, VMAX was 30.30 while the addition of extra Taq polymerase to the same sample changed VMAX into 38.91, amplifying between 80 and 90% of input DNA. The KM/VMAX ratio in all the samples remained constant, indicating that the mechanism of HA inhibition of QRT-PCR is uncompetitive by nature. Moreover, HA shifts the human DNA melting temperature point (Tm) from 75 to 87 degrees C and inhibits DNase I-mediated DNA cleavage, most probably affecting the enzyme's activity.

Suicide by fentanyl.

Fentanyl is a potent, short-acting narcotic analgesic widely used as surgical anaesthetic. This article presents a case in which fentanyl was self-injected by a 41-year old nurse, an employee at the hospital emergency department, who was found dead at home. She had no known history of drug and alcohol abuse. Two syringes, one empty and one filled with a clear liquid, were found near the body, while a needle was stuck into her hand. Toxicological analysis showed fentanyl poisoning. Fentanyl overdose was declared the cause of death and the manner of death was classified as suicide. To our knowledge, death due to the intravenous injection of fentanyl has not previously been reported in Croatia.

Drug analysis in necrophagous flies and human tissues.

Necrophagous insects may provide useful information about the time, place and cause of death. In addition, they can serve as reliable alternative specimens for toxicological analysis in cases where human tissue and fluids, normally taken during autopsies, are not available, due to decomposition of the corpse. This paper reports the results of drug analysis of the larvae of two fly families, Calliphoridae and Sarcophagidae, collected from the body of a middle-aged man who had committed suicide approximately three weeks before his corpse was found. Multiple samples of decomposed human tissue, of the blowfly, and of the larval flesh were analysed using gas chromatography/mass spectrometry (GC/MS), and amphetamine was detected in all samples. While the screening results were beyond doubt, the quantitative analysis was less clear, and further research is needed in this area.

Fatal poisoning by alcohol and heroin.

Drug abuse with alcohol consumption have been on the rise in Split-Dalmatian County for a while now. This article reports two separate cases with three deaths due to fatal combinations of heroin and alcohol. The first case of poisoning is related to a young couple, a 30-year-old man and a 28-year-old woman, who were found dead in a car, surrounded by cans of a variety alcoholic drinks. Two needles were found beside the bodies as well. The victims were registered drug abusers who had been in withdrawal programs. The second case was a 29-year-old man who was found dead in a house. Three fresh injection marks were visible on his right arm, and two needles were near his body. He was not known as a drug addict, but he had tried to commit suicide recently. Carboxyhaemoglobin was found in blood samples of both victims from the first case. The concentration was 25% and that could contribute to their death. In both described cases blood alcohol concentration was higher then 1.60 g kg(-1). Toxicology tests were positive for heroin, meconin, acetaminophen, 6-acetylmorphine, codeine, noscapine and papaverine. Ethanol, being a respiratory depressant, combined with morphine drastically increases the risk of rapid death due to respiration failure.

Skeletal remains from World War II mass grave: from discovery to identification.

AIM: To present the process of identification of skeletal remains from a mass grave found on a Dalmatian mountain-range in 2005, which allegedly contained the remains of civilians from Herzegovina killed in the World War II, including a group of 8 Franciscan monks. METHODS: Excavation of a site in Dalmatian hinterland, near the village of Zagvozd, was accomplished according to archeological procedures. Anthropological analysis was performed to estimate sex, age at death, and height of the individuals, as well as pathological and traumatic changes of the bones. Due to the lack of ante-mortem data, DNA typing using Y-chromosome was performed. DNA was isolated from bones and teeth samples using standard phenol/chloroform/isoamyl alcohol extraction. Two Y-chromosome short tandem repeats (STR) systems were used for DNA quantification and amplification. Typing of polymerase chain reaction (PCR) products was performed on an ABI Prism 310 Genetic Analyzer. PCR typing results were matched with results from DNA analysis of samples collected from the relatives of supposed victims--blood samples from the living relatives and bone samples collected during further exhumation of died parents or relatives of the supposed victims. RESULTS: The remains contained 18 almost complete skeletons, with considerable post-mortal damage. All remains were men, mainly middle-aged, with gunshot wounds to the head. DNA analysis and cross-matching of the results with relatives' data resulted in three positive identifications using the Y-chromosomal short tandem repeat (Y-STR) systems. All of the positively identified remains belonged to the Franciscan friars allegedly killed in Herzegovina and buried at the analyzed site. CONCLUSION: Our analysis of remains from a mass grave from the World War II confirmed the value of patrilineal lineage based on Y-STRs, even when missing persons had left no offspring, as is the case with Franciscan monks. Although this report is primarily focused on the identification of remains from a mass grave, it also emphasizes the role of forensic approach in documenting human right violations.

Divers' deaths in Split-Dalmatian County, Croatia (cases study, 1994-2004).

The circumstances which lead to divers' death in Split-Dalmatian County in eleven-year period (1994-2004) were analyzed. The data were extracted from the files of autopsy reports of the Department of Forensic Medicine, Split University Hospital and School of Medicine, and the police reports of the Ministry of Internal Affairs, Split-Dalmatian County. A total of 31 cases were found during the study period. The number of diving deaths didn't vary significantly through the years 1994-1999 but it increased after year 2000, mostly as a result of domestic and foreign tourists who practiced diving during their summer vacation. The average age of victims was 37.65 years. Of the 31 cases, 13 (41.9%) were apnoea (skin) divers and rest of them (18; 58.1%) were scuba divers. The leading activity of the apnoea divers was underwater fishing, while the majority of scuba divers were in recreational diving. The major cause of death was drowning. The circumstances which had led to the death remained unknown in most cases, mainly due to inexistence of proper criminal investigation.

Juvenile perpetrators of homicides and attempted homicides--a case control study.

The aim of this study was to explore the influence of certain aspects of family dynamics, as well as some behavior and psychological development disorders on the occurrence of homicides and attempted homicides among juveniles in the Split Dalmatian County over a period of 10 years (1989-1998). A retrospective case-control study was performed to compare juvenile murderers and attempted murderers with minors who committed other offences, i.e. property crimes. The subjects were paired according to demographic and socio-economic background. The data obtained from County Court's files included social anamnesis, specialists' observation and psychiatric expertise results. There was a significant difference between the study group and their controls in the frequency of physical violence and parental rejection during childhood. Neuropsychological and neurophysiological abnormalities were more frequent in the experimental than in the control group. Stealing occurred more often in the control group. No significant differences were found for other studied risk factors. There is no unique type of juvenile murderer. A juvenile homicide is committed under the influence of various developmental characteristics, family milieu, and constitution combined with environmental factors and perpetrator's perception of the victim.

Twelve-year experience in identification of skeletal remains from mass graves.

AIM: To present twelve-year (1993-2005) experience in identification of human remains found in mass graves in Croatia and Bosnia and Herzegovina (BH), as well as remains that presumably belonged to Croatian citizens given by Serbia and Montenegro. The unique experience of identification of more than a thousand of skeletal samples is valuable for better organization of post-mortem identifications. METHODS: Standard forensic methods and methods based on DNA analysis were used for identification of human remains from mass graves. DNA was isolated using standard phenol/chloroform/isoamyl alcohol extraction. In some cases, decalcification and repurification were used prior to the extraction to overcome inhibition of amplification process. Different DNA systems were used for DNA quantitation and amplification (AluQuant, short tandem repeats (STR) commercial systems, Y chromosome STRs, and mitochondrial DNA [mtDNA]). Typing of PCR products was performed on AmpliType PM and AmpliType DQA1 DNA probe strips, ABI PRISM(R) 310 Genetic Analyzer and immobilized sequence-specific oligonucleotide (SSO) probes. RESULTS: Up-to-date analysis of 1,155 skeletal samples resulted in 703 positively identified bodies: 577 using standard forensic methods, 109 by DNA typing, and 17 by combination of these two methods. The majority of identifications from 1993 to 1999 was, as usual, achieved by standard forensic methods. Later on, these methods were not sufficient and DNA analysis was requested. It was performed in 42% of all cases in 12 years. The crucial step in DNA analysis is extraction of genomic DNA. Standard phenol/chloroform/isoamyl alcohol extraction, complemented with other methods and modifications, proved as the most successful method for this step. In certain cases, the quality and/or quantity of nDNA was not satisfying and the analysis of the mtDNA was performed. CONCLUSION: Our experience demonstrated that the advent of forensic DNA analysis methods greatly increased our ability to positively identify previously unknown skeletal remains by a comparative genetic analysis with presumptive relatives.