Bone and Tooth: Substrates for determining Drowning: A new diagnostic procedure in forensic medicine Practice?

INTRODUCTION: Diatom tests are rarely used during autopsy to confirm drowning as the cause of death (COD) because of limitations of the current literature involving these techniques. Instead, experts rely on physical examination by the pathologist. Due to interpretive concerns regarding Diatom tests, they are often insufficient in establishing a diagnosis, but offer the potential to be an extremely useful diagnostic tool with further understanding. The aim of study is to optimize "Diatom Tests" for use in forensic medicine in Bosnia and Herzegovina. METHODS: A randomized prospective experimental study was conducted, using albino Wistar rat models (Rattus norvegicus), at the Veterinary Facility, University of Sarajevo. Thirty-two adult albino rats, were used and distributed into groups as follows: Group A (6 deceased rats with COD other than drowning, but due to mechanical asphyxia, which were then submerged for 1 h after death); Group B (6 deceased rats with COD other than drowning, but due to mechanical asphyxia, which were then submerged for 72 h after death); Group C (6 rats that were immediately autopsied after drowning, with COD determined as drowning); Group D (6 rats that underwent a 48-hour postmortem period after drowning); Group E (COD: drowning, post-mortem 72hrs after death, remained submerged in water until PM). Live algological material was collected for the research of the systematics of algae from the Bosna river, Sarajevo, and transported to the University of Sarajevo (Department of Biology, Faculty of Science). Periphyllon, epiphyllon and epipelon were used to collect phytobenthos. The material was fixed with 4% formalin solution. Laboratory processing of diatoms was performed using the methods described by Hustedt (16). In the process of obtaining pure diatom valves, part of the material is digested with potassium permanganate (KMnO4), sulfuric acid (H2SO4), and oxalic acid (C2H2O4). In the next step, the cleaned diatom valves were mounted in Canadian balsam. A light microscope under 1000x magnification (Best Scope 2020) was used to evaluate and analyze the species. The identification of diatoms was performed using the reference of Cantonati et al (17). The nomenclature of diatom species was performed according to Guiry & Guiry's worldwide electronic internet database. RESULTS: No diatoms were found in Groups A and B. However, Navicula sp. and Sellaphora sp. cf., were discovered during bone analysis of Group C where rats were immediately autopsied after drowning. Hantzschia amphioxus taxon was present in Group D, which underwent a 48-hour postmortem period after drowning and before samples were taken. In Groups C and D, where drowning was the COD, Diatoma vulgaris i Pinnularia major, Achnanthidium minutissimum i Melosira varians were present in the tooth samples. CONCLUSION: Optimization of the "Diatom Test" method could potentially lead to its future use as a routine method within experimental settings. This experimental study is a starting point that guides forenscic medicine pracitioners towards the optimization of tests and sampling in cases of unexplained etiology, where preserved soft tissue structures is not available. In these cases, teeth and bones serve as accessible materials for diagnosing COD, alongside standardized nonspecific findings in the absence of organs for micro- and macroanalysis.

Sex estimation based on foramen magnum: a three-dimensional geometric morphometrics approach / Estimación del sexo basado en el foramen magno: un enfoque de morfometría geométrica tridimensional

SUMMARY: The foramen magnum is an important topographic opening which connects cranial cavity and spinal canal. The analysis of the bone material established that there are differences in the shape of the foramen magnum between individuals. The aim of this study was to determine sex based on shape and size of foramen magnum using geometric morphometrics method. A study was performed on three-dimensional models (3D models) of 214 human skulls of known sex and known age (141 male skulls and 73 female skulls). The skulls are located at the museum of Medical Faculty, University of Sarajevo. Skulls belong to Bosnian population from the mid-twentieth century. All examined skulls were scanned with a laser scanner to obtain their 3D models. On 3D models of the examined skulls, four landmarks were marked on foramen magnum. Analysis of sex determination was performed using the MorphoJ program. Results of this study showed that there are sex differences in the shape and size of the foramen magnum. Sex determination based on the shape and size of the foramen magnum was showed 65.25 % accuracy for male and 63.01 % accuracy for female using geometric morphometrics method. Examination of the effect of size of foramen magnum on sexual dimorphism of shape of foramen magnum showed a statistically significant effect. Sex determination based just on the shape of foramen magnum using geometric morphometrics method was possible with 62.41 % accuracy for male and 58.90 % accuracy for female on examined sample. Sex differences on shape and size of foramen magnum were found using geometric morphometrics method on three-dimensional models of the examined skulls. The percentage of accuracy was higher for male based on the shape and size of the foramen magnum than for female.

El foramen magno es una importante abertura topográfica que conecta la cavidad craneal y el canal espinal. El análisis del material óseo estableció que existen diferencias en la forma del foramen magno entre individuos. El objetivo de este estudio fue determinar el sexo en función de la forma y el tamaño del foramen magno utilizando morfometría geométrica. El estudio se realizó en modelos tridimensionales (modelos 3D) de 214 cráneos humanos de sexo y edad conocidos (141 cráneos masculinos y 73 cráneos femeninos). Los cráneos se encuentran en el museo de la Facultad de Medicina de la Universidad de Sarajevo. Los cráneos pertenecen a población bosnia de mediados del siglo XX. Todos los cráneos examinados fueron escaneados con un escáner láser para obtener sus modelos 3D. En los modelos 3D de los cráneos examinados, se marcaron cuatro puntos de referencia en el foramen magno. El análisis de determinación de sexo se realizó utilizando el programa MorphoJ. Los resultados de este estudio mostraron que existen diferencias de sexo en la forma y el tamaño del foramen magno. La determinación del sexo basada en la forma y el tamaño del foramen magno mostró una precisión del 65,25 % para los hombres y del 63,01 % para las mujeres utilizando morfometría geométrica. El examen del efecto del tamaño del foramen magno sobre el dimorfismo sexual de la forma del foramen magno mostró un efecto estadísticamente significativo. La determinación del sexo basada solo en la forma del foramen magno utilizando morfometría geométrica fue posible con una precisión del 62,41 % para los hombres y del 58,90 % para las mujeres en la muestra examinada. Se encontraron diferencias de sexo en la forma y el tamaño del foramen magno utilizando morfometría geométrica en modelos tridimensionales de los cráneos examinados. El porcentaje de precisión fue mayor para los hombres en función de la forma y el tamaño del foramen magno que para las mujeres.

Challenges associated with investigating a mass grave at the Koricani cliffs in central Bosnia.

More than 200 men were killed at the Koricani Cliffs on Mount Vlasic in central Bosnia during the Bosnian war. The location of this mass grave remained unknown for a long time following the war, until 2008, when the Missing Persons Institute discovered a site containing the remains of approximately 60 individuals. Later, in September 2017, a new mass grave was identified at this location that had not been robbed and skeletal remains remained close to the location where the victims had died. This grave was also unique, by definition, as it was a primary inhumation site, but with a high degree of commingling and disarticulation, typical of secondary inhumation locations. The exhumation team found the first remains in this grave approximately one and a half meters beneath the rocks, while the extent of the commingling resulted in necessary modifications to standardized exhumation protocols. The search and recovery process primarily focused on skulls, groups of bones that remained in clothing, and any bones that were still connected to each other. In total, 86 skulls, 137 groups of bones that had at least 2 bones connected, and a couple of hundred small bones that could not be appointed to individuals, were retrieved. The material was taken to the Sejkovaca Identification Centre where the team took over 1,300 DNA samples for analysis and are now awaiting the results.

Histological observations on adipocere in human remains buried for 21 years at the Tomasica grave-site in Bosnia and Herzegovina.

The Tomasica grave-site near Prijedor in the north of Bosnia is reported to be the largest primary mass grave discovered thus far relating to the 1992-95 war. A total of 275 complete bodies and 125 body parts were exhumed from it in 2013. Post mortem examinations of the victims showed that nearly all had died from gunshot injuries but an additional striking feature was the degree of preservation of many of the bodies, even 21 years on, with skin, soft tissues and internal organs still present in abundance and gross structures clearly identifiable. Histology was performed on 68 samples of soft tissue from a total 13 bodies, on both skin and internal organs, and the degree of preservation was assessed in terms of the ability to recognize microscopic structure. Further comparison was made with samples taken a month or so later (56 tissue samples from 9 bodies, all but one different from the first group), after the bodies had been covered in salt as a means of general preservation. Generally, at a microscopic level, skin and subcutaneous tissues were better preserved than internal organs, while tissues sampled at the time of autopsy were better preserved than those sampled weeks later.

Secular trends in body height in Balkan populations from 1945 to 1995.

The aim of this study was to look for any secular trend in the stature of Balkan populations from the time of World War II (1939-1945) to the Balkans War (1991-1995). The research was based on the examination of exhumed skeletons of 202 men killed in World War II in the area of the Republic of Slovenia, and 243 men killed in the Bosnian War in Bosnia and Herzegovina. The length measurements of the right and left humerus, femur, tibia and fibula were taken. Since the results revealed no significant differences and the left-sided bones were more complete and recurrent in the sample, the bones of the left side were used in the analysis. Since the increase in height depends mostly on the increase in length of the long bones, with an average absolute change of about 0.28 cm for humerus, 0.55 cm for femur, 0.49 cm for tibia and 0.20 cm for fibula per decade in our case, these results suggest a significant increase of the height of the Balkans population. The difference of the sum of the average femur and tibia length for the study period was 4.13 cm. Recalculated average length increase of the sum length of femur and tibia per decade was 0.88 cm for the left side. Our study revealed that there was a trend towards increased long bone lengths, at least in the male population analyzed.

Cluster analyses of association of weather, daily factors and emergent medical conditions.

The goal of this study was to evaluate associations between the meteorological conditions and the number of emergency cases for five distinctive causes of dispatch groups reported to SOS dispatch centre in Uppsala, Sweden. Center's responsibility include alerting to 17 ambulances in whole Uppsala County, area of 8,209 km2 with around 320,000 inhabitants representing the target patient group. Source of the medical data for this study is the database of dispatch data for the year of 2009, while the metrological data have been provided from Uppsala University Department of Earth Sciences yearly weather report. Medical and meteorological data were summoned into the unified data space where each point represents a day with its weather parameters and dispatch cause group cardinality. DBSCAN data mining algorithm was implemented to five distinctive groups of dispatch causes after the data spaces have gone through the variance adjustment and the principal component analyses. As the result, several point clusters were discovered in each of the examined data spaces indicating the distinctive conditions regarding the weather and daily cardinality of the dispatch cause, as well as the associations between these two. Most interesting finding is that specific type of winter weather formed a cluster only around the days with the high count of breathing difficulties, while one of the summer weather clusters made similar association with the days with low number of cases. Findings were confirmed by confidence level estimation based on signal to noise ratio for the observed data points.

Morphological characteristics of pubic symphysis for age estimation of exhumed persons.

Suchey-Brooks method based on morphological features of pubic symphysis is a common method of age evaluation of human skeletal remains. The aim of the study was the classification of the examined samples into a particular phase according to Suchey-Brooks method, comparing the estimated age according to Suchey-Brooks with living age of the identified persons. The study was conducted on 120 pairs of pubic bones symphysis of male who disappeared from the area of north-west Bosnia during previous war, who were exhumed and the process of their identification was previously finished. The youngest person was 19 years old and the oldest 86. The study was retrospective, made by comparing the morphological characteristics of pubic symphysis pairs of the examined sample with Suchey-Brooks standards. The statistical analysis of data obtained by the survey showed that the smallest deviations from the average were in phase II by Suchey-Brooks, while the maximum deviation was in phase IV. The standard deviation generally increases from phase to phase, which is to be expected. The span of age obtained for Bosnian population is smaller at all stages in relation to Suchey Brooks-standards, as well as standard deviation, except in phases IV and VI. There was a significant difference noticed in phase V in the estimation of age between the original method and Bosnian population. In general, Suchey-Brooks method leads to underestimation of age, but the underestimation or overestimation in relation to the actual age is possible at each stage, which indicates the individual biological variation.

Aging quantification on alveolar bone loss.

Objectives were to measure labial alveolar resorption using Lamendin's method; to correlate the measured values of resorption with age at death; to determine whether age influences alveolar resorption, and if so, to quantify this impact. The study was performed during the 1992-1995 period in identified war casualties in Bosnia and Herzegovina. Data on the date, month and year of birth, and on the month and year of death were known in all cases. Measurements were carried out in 845 anterior monoradicular maxillary and mandibular teeth from male bodies (n=198) aged 23-69, divided into five age groups of 23-29, 30-39, 40-49, 50-59 and 60-69 years. Teeth with macroscopic pathologic lesions were excluded from analysis. Lamendin's method was used to measure the alveolar bone level on the labial aspect of the extracted teeth. Results are presented in tables. Regression analysis was used to determine the alveolar resorption increase with age. Results are also presented by the factor of alveolar resorption, where the youngest age group was divided by older age groups. In the total sample of 845 teeth, alveolar bone level of up to 3.49 mm was recorded in 740 (87.76%), of 3.50-5.99 mm in 99 (11.79%), and of > 6.00 mm in 6 (0.71%) teeth. In anterior monoradicular teeth, labial alveolar resorption increased with age and showed a regular age dependent pattern toward older age groups in mandibular but not in maxillary teeth. A > 6-mm pocket was very rarely recorded. Study results contribute to clinical practice, demonstrating that periodontology is a preventive discipline in dental medicine.

Diverse stature estimation formulae applied to a Bosnian population.

Since 1996, the Trotter formulae, developed on American Whites, have been used almost without exception to determine the stature of the population in Bosnia and Herzegovina. In 2002, Ross and Konigsberg presented new formulae for stature estimation for Balkans, using bones from unidentified Bosnian and Croatian males, victims of the recent war. The same year, 2002, in his master thesis, Sarajlic also presented formulae for stature estimation of Bosnian population. The research was undertaken on male cadavers. The cadaver length was measured directly and the length of the long bones was obtained from radiographs. However, none of these formulae were tested on the bones from exhumed persons. This study compares all three methods on a large independent sample in order to determine which formulae provide the most accurate stature estimation for the male population in Bosnia and Herzegovina. The sample for this research consists of long bones (humerus, femur, tibia and fibula) and was obtained from 369 exhumed and identified Bosnian males. Bones from the left side of the body were tested. Not all of them were present in each case: 211 left humeri, 270 left femura, 273 left tibiae and 175 left fibulae. Data about height was recollected by family members. Apart of whole sample, all three methods were applied separately on individuals taller then 180 cm. Of all three tested methods formulae proposed by Sarajlic et al. folowed by Ross and Konigsberg formulae, produced significantly better results in stature estimation of tall persons.

Two-criteria dental aging method applied to a Bosnian population: comparison of formulae for each tooth group versus one formula for all teeth.

The purpose of this study was to obtain age estimation formulae using the length of periodontosis, transparency of the root and root height in each tooth group for the current male population in Bosnia and Herzegovina and to compare these formulae with Lamendin's and Prince's formulae. The research was undertaken on 847 single rooted teeth from 200 identified deceased persons, individuals who died between the ages of 23 and 85 years. Periodontosis, transparency and root height were measured according to Lamendin's procedure. All teeth were grouped in two ways: according to the tooth groups and to the age groups. The highest coefficients of correlation are obtained for maxillary canines (R = 0,731) and mandibular canines (R = 0,706) and the maxillary lateral incisors showed the lowest mean error (ME = 6,63 years). In age groups, the lowest mean error was obtained in the 40-49 years age group (ME = 5,15 years). Equations developed in this study give statistically significantly better age estimations in comparison to the original Lamendin and Prince formulae for the whole sample as well as for each tooth group, except for mandibular central incisors. Age estimation with models developed for each tooth group (except for mandibular lateral incisors) was statistically significantly better than models based on the whole sample.

Stature estimation for Bosnian male population.

Since 1996, the Trotter and Gleser formulae to determine the stature of recovered missing persons in Bosnia and Herzegovina have been used. The purpose of this study is to develop appropriate stature estimation formulae from the length of the femur, tibia and fibula for use in the Bosnia and Herzegovina to help in identifications of the victims. Research was undertaken on 50 male cadavers, of individuals who died between the ages of 23 to 54 years. The cadaver length was measured and the length of the long bones was obtained from X-ray photographs. The length of the cadavers of the individuals who died after age of 45 years was corrected according to Giles' table. This study established that using Trotter and Gleser's formulae underestimate stature of tall people in the current population of Bosnia and Herzegovina. Smallest standard error of estimate is observed in the formula that uses the sum of the length of femur and fibula. There are no statistically significant differences between the length of the bones from the left and right sides of the body. Therefore, formulae developed from the average length of bone pairs are recommended for use.

[Age estimation based on sternal rib ends changes in Bosnian male population]. / Procjena starosti bosanske populacije muskog spola na osnovu faznih promjena steranlih krajeva rebara.

One of the methods used for age estimation of exhumed remains of the missing persons from the recent war in Bosnia and Herzegovina is Iscan et al. rib phase analysis method, developed o n American population. Several researches have tested this method on other populations and proposed modifications for age estimation in a certain population. The purpose of this study is to test the applicability of the mentioned method for age assessment on Bosnian male population. The sample consistS of ribs from 410 identified deceased persons, from the recent war in Bosnia and Herzegovina. Whenever possible, the 4th rib has been used. In other cases 3rd, 5th or 6th rib has been used. Adjusted age ranges for each phase were developed specifically for the Bosnian male population. Up to the phase 7 Bosnians reach and finish each phase a little later t han Americans. However phases 7 and 8 were achieved andended earlier in Bosnian population.