EDITOR DR. YAKUP GUL EE

INNOVATIVE APPROAGH

iyi

BZT TURAN PUBLISHING HOUSE

INNOVATIVE APPROACHES IN FORENSIC SCIENCE: SCIENTIFIC METHODS, TECHNOLOGY, AND EVIDENCE MANAGEMENT

EDITOR: _ DR YAKOR GUEEKGCI

Dr. Yakup GULEKGI, an expert in crime scene investigation, fingerprint development laboratory, and underwater crime scene investigation, has worked as a specialist within the Turkish National Police, particularly within the Criminal Department, for many years. He has also served as an expert witness innumerous high-profile cases, including the "Kasik¢i" murder, and continues to hold a faculty position at Kitahya Health Sciences University in the Department of Forensic Sciences. Between 2009 and 2020, he served within the Istanbul Police Department, where he gained extensive experience in fingerprint development methods, bloodstain pattern analysis, and firearm trajectory reconstruction. Furthermore, he served as an instructor in specialized training programs during his tenure.

He completed his undergraduate studies in 2007 at an education faculty and received specialized training in crime scene investigation and fingerprint research at the Police Academy in 2012. He completed his master's degree in 2012 and his doctorate in 2017 at Istanbul University's Institute of Forensic Sciences. His master's thesis focused on "Evaluation of Fingerprint Evidence Obtained from Underwater Crime Scene Investigations through Modeling," while his doctoral dissertation centered on "Examination of Fingerprints and Biological Evidence on Homemade Fire Extinguishers Used in Explosion and Molotov Cocktail Throwing Incidents at Crime Scenes."

He has published 6 articles in international peer-reviewed journals, presented 32 papers at international scientific conferences, served as editor for 3 books, contributed chapters to 6 books, and published 10 articles in national peer-reviewed journals. Additionally, he has served as a reviewer for two journals and has been a member of the scientific and organizing committees for one symposium and three national and international congresses.

Dr. GULEKG! endeavors to transform forensic sciences into practical tools for everyday use and to develop fingerprint enhancement methods for crime scenes. He has initiated a patent study in this regard.

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INNOVATIVE APPROACHES IN FORENSIC SCIENCE: SCIENTIFIC METHODS, TECHNOLOGY, AND EVIDENCE MANAGEMENT

Yakup GULEKGI

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BZT TURAN PUBLISHINGHOUSE Certificate Number: 202401 Delaware, United States

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INNOVATIVE APPROACHES IN FORENSIC SCIENCE: SCIENTIFIC METHODS, TECHNOLOGY, AND EVIDENCE MANAGEMENT

YAKUP GULEKGI

Language: English

Publication Date: December 2024

Cover designed by Kagan SARGI

Print and digital versions typeset by BZT TURAN Media Co. Ltd.

E-ISBN:978-9952-8541-4-5

DOI: https://doi.org/10.30546/19023.978-9952-8541-9-0.2024.211.

OPEN ACCESS Suggested Citiation:

GUlekci, Y. (2024). INNOVATIVE APPROACHES IN FORENSIC SCIENCE: SCIENTIFIC METHODS, TECHNOLOGY, AND EVIDENCE MANAGEMENT. BZT TURAN PUBLISHING HOUSE.

DOI: https://doi.org/10.30546/19023.978-9952-8541-9-0.2024.211.

re)

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Editor

Dr. Yakup GULEKGCI, an expert in crime scene investigation, fingerprint development laboratory, and underwater crime scene investigation, has worked as a specialist within the Turkish National Police, particularly within the Criminal Department, for many years. He has also served as an expert witness in numerous high-profile cases, including the "Kasikci" murder, and continues to hold a faculty position at Kiitahya Health Sciences University in the Department of Forensic Sciences. Between 2009 and 2020, he served within the Istanbul Police Department, where he gained extensive experience in fingerprint development methods, bloodstain pattern analysis, and firearm trajectory reconstruction. Furthermore, he served as an instructor in specialized training

programs during his tenure.

He completed his undergraduate studies in 2007 at an education faculty and received specialized training in crime scene investigation and fingerprint research at the Police Academy in 2012. He completed his master's degree in 2012 and his doctorate in 2017 at Istanbul University's Institute of Forensic Sciences. His master's thesis focused on "Evaluation of Fingerprint Evidence Obtained from Underwater Crime Scene Investigations through Modeling," while his doctoral dissertation centered on "Examination of Fingerprints and Biological Evidence on Homemade Fire Extinguishers Used in Explosion and Molotov Cocktail

Throwing Incidents at Crime Scenes."

He has published 6 articles in international peer-reviewed journals, presented 32

papers at international scientific conferences, served as editor for 3 books, contributed chapters to 6 books, and published 10 articles in national peer- reviewed journals. Additionally, he has served as a reviewer for two journals and has been a member of the scientific and organizing committees for one symposium and three national and _ international congresses.

Dr. GULEKGI endeavors to transform forensic sciences into practical tools for everyday use and to develop fingerprint enhancement methods for crime scenes. He

has initiated a patent study in this

re

BZT TURAN

PUBLISHING HOUSE

Il

IV

Editor

Uzun yillar olay yeri inceleme, parmak izi gelistirme laboratuvari ve sualti olay yeri inceleme _ alanlarinda Emniyet Genel Mudurliigii / Polis Kriminal Daire Baskaligi bunyesinde uzman olarak calisan, basta “kasikei” cinayeti olmak Uzere pek cok dikkat ceken Onemli olaylarda_bilirkisilik yapan ve Kitahya Saglik Bilimleri Universitesi, Adli Bilimler bdliimiinde ogretim Uyeligine devam eden Yakup GULEKGI, 2009-2020 yillari arasinda Istanbul Emniyet Midiirliigi'nde gérev yapt. Istanbul Olay Yeri Inceleme ekiplerinde ve Istanbul Adli Polis Laboratuvarinda alistigi sire icinde parmak izi gelistirme yontemleri, kan lekesi model analizi ve atisin yeniden yapilandirilmas konularinda genis tecrubeye sahip oldu) ve uzmanlik

egitimlerinde egitmen olarak gérev yapti.

Lisans egitimini 2007 yilinda egitim fakiiltesinde, kriminal uzmanlik egitimlerini (olay yeri inceleme ve parmak_izi arastirmalart vb.) 2012 yilinda_ polis akademisinde tamamladi. Yuksek lisansin! 2012 yilinda, doktorani ise 2017 yilinda Istanbul Universitesi Adli _Bilimler Enstitust'nde tamamladi. Yuksek lisans tezi "Sualtt Olgu Calismasindan Elde Edilen Parmak izi Delillerinin Modelleme Ile Degerlendirilmesi", doktora tezi ise "Olay

Yerinde Elde Edilen Patlama ve Molotof

Kokteyli Atma Olayinda Kullanilan El Yapimi Yangin S6ndiiriictiler Uzerindeki Parmak Izlerinin ve

Biyolojik Bulgularin

Incelenmesi" konularinda yapti.

Uluslararas! hakemli dergilerde yayimlanan 6 adet makalesi vardir. Uluslararasi bilimsel toplantilarda sunulan ve bildiri kitaplarinda basilan 32 adet bildirisi vardir. 3 (Ug) adet kitap editérliigii, 6 (alti) adet kitap bélumut vardir. Ulusal hakemli §dergilerde yayimlanan 10 adet makalesi vardir. Iki adet dergide hakemlik yapmistir. Bir adet sempozyum, 3 adet ulusal ve uluslararasi Kongrenin bilim ve dizenleme kurullarinda

goren yapmistir.

Gtlekci, calismalarimla adli bilimleri gunlik

hayatta, adli olaylarin cdzUminde kullanilabilecek araclara ddntstirmeye calismaktadir. Ayrica; olay yerlerinde kullanilabilecek parmak izi_ iyilestirme yontemleri gelistirmeye alisiyor. Bu

konuda bir patent calismasi mevcuttur.

rey)

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PUBLISHING HOUSE

Preface

Forensic science is an interdisciplinary field that plays a crucial role in the pursuit of truth, a fundamental requirement of justice. Rapid advancements in science and technology have introduced significant innovations in methods and techniques for solving crimes. The effective application of these innovations in forensic processes requires the continuous updating of educational and research _ practices. | Consequently, adopting a multidisciplinary approach in forensic science while embracing innovative methods instead of relying solely on established knowledge is essential for introducing novel solutions to the evidence collection and analysis

process.

Evidence Dynamics seeks to transcend the traditional understanding of forensic science by addressing the criminal phenomenon holistically, with a focus on innovative methods for evidence research. Every stage of the process from the meticulous collection and preservation of crime scene findings to their analysis through the latest technologies plays a critical role in

ensuring scientific accuracy. This book

thoroughly examines how new techniques and analytical methods in criminal investigations contribute to solving crimes and explores which innovative approaches can enhance the

reliability of evidence.

The book does not limit itself to classical methods in evidence management but also emphasizes recent innovations, such as advanced analytical techniques and the application of artificial intelligence in forensic science. These methods not only accelerate the evidence evaluation process but also stand out as powerful tools that help ensure the proper administration of

justice.

Designed primarily for students of forensic science, judges, prosecutors, and experts working in the criminal divisions of law enforcement agencies, this work provides a clear, accessible, and _ practical guide to evidence management. By translating theoretical knowledge into practical application, it offers a roadmap for how innovative approaches can be _ integrated into criminal investigations. The use of visual aids further supports the simplifying

explanations, complex

VI

concepts and illustrating how evidence

can be scientifically analyzed.

In conclusion, Evidence Dynamics introduces an innovative perspective on evidence research in crime resolution, enabling forensic science to be applied more effectively and efficiently. We

hope this book will contribute both to

academic research and the practical application of forensic science, fostering the adoption and dissemination of new

methodologies.

We trust that this work will serve as an inspiring resource for all researchers and professionals seeking to advance

their expertise in forensic science...

DR. YAKUP GULEKGI

Ons6z

Adli _Bilimler, temel gereksinimlerinden biri olan gercegin katki

saglayan, hizla gelisen bir disiplinler

hukukun en

ortaya_ cikarilmasina _ bilimsel aras! alandir. Bilim ve teknolojideki hizli ilerlemeler, suclarin gc6zUmune yonelik yontem ve tekniklerde 6nemli yenilikleri beraberinde getirirken, bu yeniliklerin etkili bir

egitim ve

adli sureclerde Sekilde

uygulanmasl, arastirma sureclerinin de surekli gUncellenmesini gerekli kilmaktadir. Bu nedenle, adi bilimler alaninda multidisipliner bir yaklasim_ gelistirmek, sadece gecmis bilgilerle yetinmek yerine inovatif yontemleri benimseyerek delillendirme surecine yenilikgi cozumler sunmay!

kacinilmaz kilmaktadir.

Delillendirme Dinamikleri kitabi, klasik adli bilim anlayisinin 6tesine gecerek, suc olgusunu tum yonleriyle ele almay! inovatif

ve delil arastirmalarinda

yontemleri merkeze koymay!

amaclamaktadir. Olay yerinden elde edilen bulgularin titizlikle toplanmasi ve korunmasindan baslayarak, bu bulgularin en glincel teknolojilerle analiz edilmesine kadar gecen surecin her dogrulugun

asamasl, bilimsel

saglanmasi adina bilywk 6nem tasir. Bu

kitap, Ozellikle sug arastirmalarinda kullanilan yeni tekniklerin ve analitik yontemlerin sugun cozUmtne nasil katki sagladigint detayli bir bicimde ele almakta ve delillerin giivenilirligini artirmak icin

hangi yenilikci

yaklasimlarin kullanilabilecegini

tartismaktadir.

Kitap, delil yonetimi sUrecinde sadece klasik yontemlere degil, ayn! zamanda son yillarda gelisen analiz teknikleri ve yapay zekanin ileri duzeyde kullanimi gibi yenilikci yOntemlere de genis yer ayirmaktadir. Bu yontemler, delillendirme sUrecini hizlandirmanin yan sira, adaletin dogru sekilde tecelli etmesine katki saglayan glicli araclar

olarak 6n plana cikmaktadir.

Ozellikle adli

Ogrencilere, hakim ve savcilara, kolluk

bilimler egitimi alan

kuvvetlerinin kriminal birimlerinde calisan uzmanlara yonelik hazirlanan bu eser, delillendirme streclerine dair acik, anlasilir ve uygulamali bir rehber olma nitelidi

tasimaktadir. Teorik _ bilgileri

pratige donisttirerek, SUG arastirmalarinda yenilikci yaklasimlarin nasil uygulanacagina dair yol gésterici bir kaynak sunmay! amaclamaktadir. desteklenen

Gorsellerle anlatim,

okuyuculara karmasik kavramlari sade

VI

VUl

bir dille aciklamakta ve delillerin bilimsel temellerle nasil analiz edilecegini

gostermektedir.

Sonus olarak, Delillendirme Dinamikleri, suclarin cozumunde delil arastirmalarina yenilikci bir perspektif kazandirarak, adli bilimlerin daha verimli ve etkili bir sekilde uygulanmasina olanak saglamaktadir. Bu kitabin, hem akademik calismalara hem de_ adi bilimlerin uygulama alanlarina katkida bulunarak, yeni yontemlerin benimsenmesine ve _ yayginlasmasina

isik tutmasi en buyiik dilegimizdir.

Adli bilimler alaninda ilerleme kaydetmek isteyen tum arastirmaci ve uzmanlar icin ilham verici bir kaynak

olmasi temennisiyle...

CONTENTS

CNA UGE A ictipnecosti eae ieco eee aoe 1 Identification of New Psychoactive Substances (NPSs) in Biological Materials:

Evidence in Forensic Cases

DUYGU YESIM OVAT, MELIKE AYDOGDU, SERAP ANNETTE AKGUR

©) 17: ] 0] <1? ee nn ner ee nO Pn OE nO eT OE eT ee 33 Skeletal Anatomy Analysis and Evidence Collection at the Crime Scene: Forensic

Anthropology and Artificial Intelligence Applications MERT OCAK , CUMALI CATAK

INA ccs saccade cca cactercisteiceecnceans abcdnelocteens io tatecncteaaecdetevenceroat 65 Simultaneous Analysis Of Organic And Inorganic Gunshot Residue

Harun SENER, Hatice SOYTURK

CON resort teeta es: 84 The Significance of Postmortem Imaging In The Evidence Analysis CEREN AKAGUNDUZ AYRANCIOGLU

© 1 F-| 0] <1 een Ene Pe OPER noe re eC ee 118 Novel Techniques Used In Identifications Through Dental Structures: The Role Of

Forensic Odontology In Crime Scene

MEHMET ALI KILICARSLAN

IX

CRI G sosriscsisdscoca secs tase sicsnescustessnsesocssieestaseviuyerianeiesmdaeiaedanaiedeneniaks 151 Evaluating Strategies For Detecting Drugs In Impregnated Materials Current And

Future Trends FATMA CAVUS YONAR, YAKUP GULEKCI

Chapter 7 Pe meee cree rr ccc cccccccccc cece rs ee er eeee esse sees eres ress sess sees eserseeesseeereseerseeeeseseseseesses 17 4

Transnational Strategies: Evidential Approaches to NPS Detection in Prisons ASENA AVCI AKCA

IDENTIFICATION OF NEW PSYCHOACTIVE SUBSTANCES (NPSs) IN BIOLOGICAL MATERIALS: EVIDENCE IN FORENSIC CASES

Duygu Yesim OVAT, Melike AYDOGDU, Serap Annette AKGUR

Chapter 1 Identification of New Psychoactive Substances (NPSs) in Biological Materials: Evidence in

Forensic Cases

DUYGU YESIM OVAT!, MELIKE AYDOGDU2, SERAP ANNETTE AKGUR?

Overview

The emergence of a vast amount of “New Psychoactive Substances (NPSs)” represents a considerable risk to public health and = safety. Due to their abundance, structure and composition, NPSs pose significant challenges to substance analysis researchers and forensic toxicologists. They also pose many challenges to global drug policy. NPSs have been described as a “growing worldwide epidemic” (Shafi et al., 2020a). NPSs, commonly so-called designer or synthetic drugs, are

commercially known as “legal highs”,

' PhD, Ege University, Institute on Drug Abuse, Toxicology and Pharmaceutical Science, Department of Addiction Toxicology, Tiirkiye, duygu.yesim.ovat @ege.edu.tr, https://orcid.org/0000-0003-1310-0118

> PhD, Ege University, Institute on Drug Abuse, Toxicology and Pharmaceutical Science, Department of Addiction Toxicology, Tiirkiye,

“research compounds”, “herbal highs”, “path salts”, “party pills”, or “internet drugs” (Peacock et al., 2019; Zuba, 2014). The regulatory bodies for NPSs include “the United Nations Office on Drugs and Crime (UNODC), the European Union Drugs Agency (EUDA), the National Institute on Drug Abuse (NIDA), and the World Health Organization (WHO)”. These global organizations aim to intervene at the regulatory and policy-making levels to control or completely ban a particular substance (Al-Imam & AbdulMajeed, 2017). However, agencies working on this topic have different definitions of NPSs. The most comprehensive term, NPS, is defined by the UNODC as “a new narcotic or psychotropic drug, in pure form or in preparation, that is not controlled by the United Nations drug conventions, but which may pose a public health threat comparable to that posed by substances listed in these conventions” (UNODC, 2021). NPSs may be analogues of already regulated

drugs or pharmaceutical products, or

melike.aydogdu @ege.edu.tr, https://orcid.org/0000- 0002-6324-0234

3 Prof. Dr., MD., Ege University, Institute on Drug Abuse, Toxicology and Pharmaceutical Science, Department of Addiction Toxicology, Tiirkiye, serap.akgur @ege.edu.tr, https://orcid.org/0000- 0001-9638-23 11

they tend to be “new” chemicals designed to mimic the effects of, particularly their psychoactive effects, licensed medicines and/or other restricted controlled substances (Shafi

et al., 2020a).

These compounds can achieve similar effects as conventional drugs or drugs that have already been synthesized and are being used in new ways. While some NPSs_ were first synthesized several decades ago, the term “new” refers to recently becoming commercially available chemicals. It is a phrase used to represent a group, although it does not always refer to new

inventions (Hasan & Sarker, 2023).

They are synthesized by clandestine chemists in illegal laboratories to evade international and national controls, mimicking the pharmacological effects of restricted substances and altering their chemical structure to enhance the desired effect. The laboratories are mostly located in Far Eastern countries. Organized crime groups are constantly shifting toward these substances to establish black markets because of the decrease in traditional drug use by NPSs. Due to the deficiencies in the definition of NPSs, the chemical

components used to produce such

compounds require very small changes to make them completely legal (Hagan & Smith, 2017). With these minor modifications, an unlimited variety of substances can be designed, produced through quick and easy synthesis steps and distributed to end users. On the other hand, scientific studies are being conducted to prove that the substances seized within this rapidly changing, infinite variety of substances have evidentiary value. This section of the book aims to provide information about NPSs, their mechanisms, and_ their detectability in biological materials. It also aims to present current studies that present ways to use data on these

substances as forensic evidence. Historical Perspective

Today, the types of NPSs used or seized vary according to countries’ substance use trends, and these substances are classified according to their different physicochemical or pharmacological characteristics. UNODC divided NPSs into subgroups as_ “aminoindanes, benzodiazepines, fentanyl analogues, lysergamides, nitazenes, other substances, phencyclidine-type substances, phenethylamines, phenidates, phenmetrazines,piperazines plant-based

substances, synthetic

cannabinoids, synthetic cathinones and tryptamine” (United Nations Office on Crime., 2024). The

substances classified according to their

Drugs and

effects are as _ follows; synthetic stimulants,cannabinoids, hallucinogens, depressants, opioids (Shafi et al., 2020). Synthesis of

non-controlled analogs of popular drugs

benzodiazepines, and

is not new. The first morphine analogs were prepared in the 1920s. In the 1980s-1990s, many phenethylamines and tryptamines were sythesied and introduced to the market, expanding the field of so-called “designer drugs” (Zuba, 2014). Nitazenes were initially studied by researchers nearly 60 years ago as an alternative to morphine but were never marketed due to the potential for overdose. Nitazenes have been linked to several overdose deaths

worldwide.

Mephedrone was first synthesized in China in 1929 but did not become widely available until 2003. The use of mephedrone has also increased rapidly due to its affordable price. In 2008 mephedrone was connected to several fatalities. The increase in mephedrone use meant that it had gained a new foothold in the NPS market. By 2010,

mephedrone had become illegal (Hagan & Smith, 2017).

Fentanyl was developed in the 1960s as a highly’ potent intravenous anesthetic that is about 50 to 100 times more effective than morphine. Illicitly produced fentanyl and fentanyl analogs have been implicated in overdose deaths over the last few decades. In a 1979 study in California, fentanyl was reported to be the cause of death in two intravenous heroin users. Drug residues were present in the deaths, and recent injection sites were found on the bodies; however, interestingly, toxicology results were negative. Fifteen deaths were recorded in 1980 following these cases, and similarly, toxicology analyses showed _ no evidence of drug use. Because of these suspicious deaths, law enforcement officers seized the drugs and determined that the

contained no known drugs. They

substance

identified it as a-methyl fentanyl, a powerful narcotic that has not

undergone scientific evaluation. Between 1979 and 1988, 112 deaths related to fentanyl and/or 10 different fentanyl analogs were reported across various states in the United States. In

1988, a chemist in Pennsylvania was

reported to have manufactured and distributed 3-methyl fentanyl, which caused numerous deaths soon after it entered the illicit drug market (Pearson et al., 2015).

The main active compound in the production of synthetic cannabinoids is THC. The first “classic cannabinoid” analog synthesized in Israel in 1988 was “HU-210”, and this was followed by the production of cannabinoid families chemically unlike to THC have been produced, including both non-classic and aminoalkylindole. further categorized by JWH compounds (Hagan

& Smith, 2017).

Aminoalkylindoles —are

In Cambodia, sassafras oil from tree roots is used to convert methylenedioxymethamphetamine (also known as_ ecstasy) into its precursor chemical. In 2008, tons of sassafras oil were seized and destroyed in Cambodia and Australia. Because of this destruction, piperazines, which mimic the effects of ecstasy, have been synthesized and produced and sold illegally. These novel compounds became common among users because they were affordable, available, had high purity, and were egal (Hagan & Smith, 2017). Abuse was first recorded

in the US and Scandinavia in the end of the 1990s, but abuse has been documented in several other countries since 2000 (Elliott, 2011).

Early Warning System

Several countries and regions have embraced different strategies to monitor NPSs through research and case reports. In 1997, the EUDA established the Early Warning System (EWS) to determine the global prevalence, elimination, market stability and market continuity of NPSs, detect trends and identify new and emerging threats. With this system, threats can be identified, monitored, detected early and intervened in a timely response. It also provides important evidence to develop new policies and interventions to address existing threats. UNODC established the Early Warning Advisory (EWA) in 2013 to increase international collaboration on the detection and reporting of NPSs, not only in Europe but also globally. The scope of work has been expanded to include identifying threats to public health and safety, determining the rate of emergence of substances, demonstrating diversity and heterogeneity in terms of substance types, and identifying problems in

specific regions.

In its 2024 EWA report, it published the latest information on NPSs and an analysis of more than 2800 cases submitted by toxicology laboratories in 2023. According to this report, 1245 individual NPSs were reported to the UNODC by 142 countries and territories worldwide. In recent years, the number of emerging substances has decreased significantly; 44 NPSs were reported in 2022, and 31 NPSs were reported in 2023 for the first time (United Nations Office on Drugs and Crime (UNODC) Research and Trend Analysis Branch, 2019).

Legal and Criminal Aspects

NPS definitions vary between countries and regions, depending not only on pharmacological/structural classifications but also on social and cultural perspectives. Today, the main purpose of changing the structures is to bypass the existing anti-drug laws that emerged in most countries by ratifying

the two conventions on narcotic drugs

(1961) and psychotropic substances (1971). According to the conventions, a certain number of compounds is controlled in the annexes. The banning of the substance detected in the preparations was similar to that of a “cat and mouse game”. New derivatives appear on the market when a substance is prohibited (Zuba, 2014).

However, under the umbrella of UN agreements, many NPSs are under global surveillance. As of December 2021, UNODC EWS reported that the majority of NPS were stimulants, followed by synthetic cannabinoids and drugs with hallucinogen receptor agonists. There has also been a recent significant increase in synthetic opioids. The rapidly altering NPS landscape has led to growing concerns about the social, mental and physical health hazards associated with the uncertainty and ambiguity of their metabolic, toxic and chemical profiles (Awuchi et al., 2023).

Figure 1. Timeline of the European Early Warning System in Europe (EU Early Warning System, 2023)

Trends in NPSs

Before 2008, the NPS market was characterized by some NPSs and a few groups of consumers, mostly from high- income countries. Accordingly, the

amount, quantity, type and accessibility

of NPSs have increased dramatically

worldwide.

The globalization of the pharmaceutical manufacturing industries and new technologies, such as the use of the Internet, have fueled this growth market. This has enabled the industrial- scale production, supply of NPSs and the materials and supplies used to

produce, package and distribute the precursors required for production. The spread of NPSs is endemic for many different reasons, including the availability, ease of production and diversity of the chemicals used in the production phase. Today, laboratories to produce illicit substances located in the Far East are known to supply the world with vary quantities of the final products and basic chemicals required for the synthesis of NPSs. A survey by UNODC reported that these substances are trafficked by airways or by mail. Usually, when they reach European or American countries, they are repacked and redistributed by dealers via online or offline sources. The accessibility of these goods once they reach their ultimate destination is very extensive, but they basically fall into three groups: Internet, high street retailers and non-retail sellers. The Internet has become a key platform for NPS sales. The appearance and expansion of this community are becoming a concern for control agencies. High-street retailers are shops specializing in drug paraphernalia. Non-retailers are categorized as “street vendors” who

source their products online or from

high street retailers (Hagan & Smith, 2017).

Another problem with the production and marketting of NPS is the general instability of the products. In one study, analysis of 32 separate commercial plant materials sent to a laboratory found extreme variability in compounds, with concentrations of the common synthetic cannabinoid JWH- 018 ranging from 0.8% to >30%. Any dosage _ instructions can lead to inconsistent and unpredictable health outcomes. In addition to dosage uncertainty,

although many NPS

packages contain ingredient _ lists consistent with the ingredients in the products, few products have been found to contain illegal substances that are not listed on the label. This poses a great risk to users who may unknowingly consume illegal products (Hagan & Smith, 2017). In a study conducted in Turkiye in 2021, 500 urine samples that were negative for enzymatic immunoassay were selected and studied via chromatographic analysis. Of the 500 urine samples analyzed, 108 (21.6%) were reported to be positive for 20 different synthetic cannabinoids and their metabolites

(Atasoy et al., 2021). Organizations

working in this field report that NPSs are present worldwide. Although numerous NPSs have been reported to date, they can vary in their nature and pharmacological effects (Tettey et al., 2018).

In general, there is little known about the potential health effects and social threats of NPSs, which poses a challenge _ for Published reports on NPSs indicate that

users often present to hospital with

preventive action.

severe intoxication due to the composition and_ purity/impurity of NPSs. NPSs have a range of adverse effects, ranging from seizures to agitation, aggression, acute psychosis, and potential addiction. However, data on the toxicity of many NPS are not available and information on the long- term side effects and risks of these drugs is limited. (UNODC, 2024).

NPSs Prevalence

Communities use traditional methods to determine NPS use, utilizing surveys and extrapolating from reported data. These data suggest that 3% or less of the adult population reported using substances in the past year. These survey-based estimates are generally

greater than estimates derived from

adult populations, with some countries reporting (almost one in ten) youth having used substances recently. Some countries report a higher prevalence of NPS use compared to more traditional drugs (excluding cannabis) (United Nations Office on Drugs and Crime, 2023).

The Pharmacokinetics, Metabolism and Biological Materials for NPSs

Knowledge of the metabolism of NPSs is important for toxicological _ risk assessment for medical purposes and for developing toxicological analyses for forensic purposes. However, information on their metabolism is lacking to identify potential metabolic targets for analysis (Gampfer et al.,

2024; Shafi et al., 2020a).

The testing of NPSs in clinical and forensic contexts can be a complicated task, as testing for such compounds in individuals presenting for drug testing is often not routinely performed, and the reliability and accuracy of existing test kits vary considerably in identifying these many novel compounds (Grafinger et al., 2020; Shafi et al., 2020a). To explain the criminal case,

the analysis and detection of NPSs in

10

various biological materials is a complex and evolving field. This complexity arises from the rapid emergence of NPSs, which often mimic the effects of traditional controlled substances. The analysis and detection of NPSs_ in biological samples as forensic evidence involve sophisticated methodologies aimed at identifying and quantifying these substances, which are increasingly prevalent in the illicit drug market (Ameline et al., 2024; Richter et

al., 2017; J. Tettey & Crean, 2015).

The analysis of NPSs has some significant limitations, primarily due to their various chemical structure, rapid emergence and the insufficiencies of

current testing methodologies. The

following sections summarize the importance/relevance of pharmacokinetics, metabolism and

biological materials for the detection of

NPSs in forensic science.

Pharmacokinetics and Metabolism

Pharmacokinetics and metabolism play a crucial role in forensic toxicology. Pharmacokinetics is the study of the ‘absorption, distribution, metabolism, and excretion (ADME) processes of a

drug. Understanding how drugs are

metabolized in the body can provide important information in a variety of legal investigations, including evaluating the case in antemortem or postmortem forensic cases, determining death,

impairment in criminal cases, and

the cause of assessing

determining drug abuse.

ADME is an important part of the study of the post-administration process of a drug molecule. This is a complex procedure comprising transporters and metabolic

various enzymes with

physiological outcomes on pharmacological and __ toxicological effects. With this process drugs are different

metabolites. The goal is to convert

structurally modified to

drugs into metabolites that more easily excreted from the organism. The cytochrome P450s (CYPs) isozymes are primarily responsible for phase I metabolic reactions, which involve the introduction of functional groups to drugs, making them more polar and water-soluble. This process is essential for the subsequent elimination of these substances from the body. The metabolism of drugs typically involves both phase I (e.g., oxidation, reduction) and phase II (e.g., conjugation)

reactions. Enzymes such as CYPs play a

crucial role in phase I metabolism, while phase II reactions often involve uridine diphosphate-glucuronosyltransferase

(UGT) which

metabolites to enhance their solubility

enzymes, conjugate and facilitate excretion. Conjugation may also occur through acetylation or sulfoconjugation (Benoit et al., 2008; Drummer, 2008; Shafi et al., 2020b).

The liver is the main site of drug metabolism, and the first-pass effect can significantly reduce the bioavailability of orally administered drugs. This can impact the efficacy of medications and contribute to addiction by altering the levels of active compounds in the body. Excretion is the final step in drug elimination and can occur in different ways: The kidneys has an important role’ in excretion, especially of water-soluble drugs. Various diseases and conditions can affect kidney function. Impaired kidney function can lead to drug accumulation and toxicity. Some drugs are excreted in bile, as a parent drug or its metabolites. The bile then enters the gastrointestinal system where the drugs may be excreted in the feces or reabsorbed into the bloodstream. Small amounts of medicines can also be

eliminated via saliva, sweat, breast milk

and exhaled air (Negrusz & Jickells, 2008).

Genetic factors significantly influence drug metabolism. Variations in genes, such as those coding for cytochrome P450 enzymes, can lead to differences in how individuals metabolize drugs. affect the

interpretation of toxicological results,

This variability can especially in autopsy cases where determining the metabolic state of a deceased individual is critical (Zhou & 2022),

metabolites of the parent drug, can

Lauschke, Analysis of have significant legal consequences. For instance, it can help establish whether a person was under the effect of a drug at the time of an incident, which can influence charges in criminal cases. In addition, understanding the metabolism of drugs can be helpful in cases of drug- facilitated crime, where the presence of certain drugs in a convict, offender or victim system can be crucial for

investigation.

Drug metabolism is essential in forensic settings as it aids in identifying the parent drugs and metabolites, understanding individual differences in drug effects, toxicity, and persistence of drugs in the body conducting forensic

toxicological analyses, and influencing

11

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legal implications. The integration of advanced techniques like metabolomics into forensic toxicology continues to enhance the accuracy and reliability of these assessments. Metabolites, which are the products of drug metabolism, can serve as biomarkers for drug intoxication or abuse. Forensic toxicologists analyze these metabolites to confirm the presence of specific drugs in biological samples, such as blood or urine. This is particularly important in cases involving new psychoactive substances, where traditional testing methods may not be

effective. Biological Materials

Forensic toxicology utilizes biological materials for analysis to investigate drug abuse or _ toxic substance exposure. Various biological materials are used in analytical methods for the sensitive and specific identification of NPSs, whose diversity, number and usage are increasing. The biological

samples used in this field include:

Blood: Blood is the most frequently used sample for forensic toxicological analysis, particularly in postmortem investigations. It is essential for

determining the presence and

concentration of drugs and poisons in the body. In addition, a blood sample allows the assessment of whether a person is under the influence of a

substance in antemortem cases.

Urine: Urine is another standard sample, often collected for drug screening due to its ease of collection

and ability to detect recent drug use.

Hair: Hair analysis is valuable for assessing long-term exposure to drugs. It allows for the detection of substances over extended periods, making it useful in cases where historical drug use is

relevant.

Nails: Similar to hair, nails can provide a record of drug exposure over time and are increasingly used in toxicological

assessments.

Bile and Gastric Contents: These samples can be analyzed to determine the presence of substances ingested death,

understanding the

shortly before aiding in circumstances

surrounding a death.

Liver and Brain Tissue: These tissues are often analyzed in postmortem examinations to ensure insights into the

metabolic effects of drugs.

Alternative Matrices:

e Recent advancements have led to the use of alternative biological

matrices such as:

e Oral Fluid: Useful for detecting recent drug use (Busardo et al., 2024; Marchei et al., 2024).

e Sweat: Can provide information on continuous drug exposure (Busardo et al., 2024; Gomes et al., 2024).

e Meconium: Analyzing this can

indicate prenatal drug exposure

(L6pez-Rabufial et al., 2019).

e Breast Milk: Important for assessing drug transfer to infants (Baker et al., 2023).

e Vitreous Humor: Its unique

properties make it _ particularly suitable for detecting substances after death, is less susceptible to postmortem changes compared to blood and urine (Andrade et al.,

2016; Campos et al., 2022).

Data are already existing on detection in alternative matrices for more conventional drugs (morphine, cocaine, etc.). For newly emerging drugs such as NPSs, alternative biological

limited (Campos et al., 2022).

data on detection NPSs_ in

matrices are

In forensic cases involving drug abuse, both blood and urine are the most commonly used biological materials for drug detection, but they have different advantages and disadvantages. The detection window for drugs in blood is generally shorter compared to urine. This implies that blood is the better material for detecting recent drug use, typically within a few hours to a few days after ingestion. Blood tests are more invasive and expensive compared to urine tests, which can limit their use in some settings and provide a direct measure of drug concentration in the body. It allows for precise analysis and identifying of drugs and metabolites. Urine accumulates drug metabolites over time, allowing for the detection of past drug use. Urine tests have a longer detection window compared to blood. They can detect drug metabolites for several days to weeks after use, making them suitable for drug testing. Urine is non-invasive and easier to collect compared to blood and it is more susceptible to tampering and adulteration, which can lead to false- negative or false-positive results. However, urine is commonly used for routine drug screening and monitoring

of substance use.

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In summary, blood provides more accurate information about recent drug exposure and intoxication, while urine is better for detecting past drug use, offers a longer detection window and is more practical for routine screening with more convenient collection. Forensic drug testing often utilizes both blood and urine samples to obtain a more complete picture of an individual § drug use history and current state. As a result, it is valuable to identify NPSs together with their metabolites in human biological materials with reliable and clinically validated tests. Different techniques based on_ colorimetric, immunoassay or chromatographic are used in NPSs detection. It is reported that very few NPSs can be detected today, and this situation has vital importance in clinical and especially

forensic processes.

Analytical Strategies

The transformation of drugs_ into metabolites with various chemical and physical properties as a consequence of metabolism in the body and the identification of these metabolites is very important not only for the

pharmaceutical and medical field but

also for providing evidence in forensic toxicology. In the last years, it is known that forensic toxicology has suffered an influx of NPSs (Drug Enforcement Administration & Control Division, 2020; European Monitoring Center for Drugs and Drug Addiction (EMCDDA), 2022; 2021).

traditional substances, NPSs are more

Luki¢é et al., Apart from commonly used for (i) to produce equivalent psychoactive responses by targeting similar systems in the body, (ii) to avoid detection by using a substance that is known and likely to be detected in drug tests, and/or (iii) because of analytical limitations in identification (Soussan et al., 2018). At the same time, clandestine laboratories steadily create NPSs to circumvent legal efforts,

toxicological analyses challenging and

classification making complex. This poses new _ analytical challenges in the detection of these chemically variable NPSs, in terms of the identification and classification of the substance in seized material. The metabolic fate of NPSs is not entirely predictable, however, since metabolic studies have been conducted extensively, 'NPSs profiling provides a significant amount of hard evidence,

not just a prediction.

Emerging NPSs present a significant challenge for drug testing laboratories, as new substances cannot be identified methods.

by current _—_ analytical

Quantitative and robust liquid chromatography-high-resolution mass (LC-HRMS) analysts to go further beyond targeted

analysis (Karabulut & Ertas, 2021). New

spectrometers allow

high resolution mass spectrometers are capable of detecting large numbers of molecules (from 100 to 1000) at low levels in the same analysis. These new detectors mainly consist of HR-MS, Time-Of-Flight-MS.

Especially, HR-MS allows discrimination

Orbitrap and

among highly similar mass spectrometers such as, CisHi403N or CisHicOS can be easily distinguished by their m/z values: 257.10464 and 257.09946 Da (delta = 5 mDa), respectively. HR-MS analysis showed ‘with = full scan

acquisition, while 1000s of ions were

excellent selectivity

recorded in the LC-MS analysis “(Rochat et al., 2014).

In general, in laboratories using HR-MS, both global and non-targeted data are simply obtained, but the data are often processed in a targeted way to identify the compounds that are expected,

however, when considered non-

targeted, these big data can possibly

reveal unexpected compounds of interest in specific samples (Rochat et al., 2014; Wu & Colby, 2016). Therefore, laboratories are challenged to improve and validate outdated methods to identify NPSs. Currently, non-targeted screening methods are often used for the identification of NPSs, based on LC-HRMS and retrospective searches of previously obtained data for the identification of NPSs (Di Trana et al., 2021; Stork et al., 2020). An untargeted screening method is beneficial for the identification of NPSs either known or unknown at the point of testing. To target the ever-changing class of substances to be identified, there is a_ significant endeavor to develop HR-MS screening methods that are fast with all non-targeted ion detection and can be updated more rapidly with new drugs (Diao & Huestis, 2017a; Kronstrand et al., 2014). High- resolution MSs efficiently first identify and then validate substance-specific fragment ion formulas by providing accurate mass measurement, and also achieve

enable laboratories to

metabolite definition with improved efficiency and quality (Marchei et al.,

2018; Stork et al., 2020). Virtually the

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only restriction of targeted screening methods is the fast-changing NPS family.

The analysis of NPSs_in_ biological samples is compelling, especially NPSs that are rapidly metabolized at low active doses. Therefore, it is essential to biomarkers _ of

investigate specific

substance use. In _ these studies, metabolite profiling studies of NPS are usually performed first. Then, human and/or animal hepatocyte incubations are performed and, _ if possible, completed with the analysis of real human samples (Di Trana et al., 2021; Diao & Huestis, 2017b). Urine, a very important material in forensic toxicology, is the most widely used matrix in NPS profiling due to its sample volume, reflecting high drug concentrations and offering a longer drug detection window, versus to blood (Scheidweiler et al., 2015). NPSs can

also be detected in blood and oral fluids

especially and oral fluid

if the laboratory has knowledge of the molecular structure and molecular weight of the substance of interest or uses a

non-targeted _ analytical

approach. However, the detection

windows for these potent substances

are short and therefore it is critical to

target marker metabolites in urine.

The elucidation of the pharmacological and pharmacokinetic processes and the discovery of metabolism products provide important evidence. The fate of the substances, delivered directly to our bodies is mainly managed by the three phases of drug metabolism: “phase I, addition of a reactive group (by means such as_ oxidation, reduction, or hydrolysis); phase II, conjugation with diverse bonds; and _ phase _ III, elimination of drugs and metabolites intestinal cells 2017). The

metabolism of substances is commonly

from liver = and

(Mackenzie et al.,

predicted based on their interactions (CYP450)

enzymes and their metabolic endpoints

with cytochrome P450

(Tan et al., 2017). In cases, urine samples are usually hydrolyzed under appropriate conditions using the beta- glucuronidase enzyme. As a result of this process, characteristic phase I metabolites are selected as biomarker metabolites in the urine to confirm substance use. Most psychoactive substances/drugs are excreted by phase II metabolism, as in NPSs, with the formation of glucuronide or sulfate

metabolites. However, the

is that the

glucuronides and sulfates formed in

disadvantage of this

phase II are not as stable as phase I metabolites (Scheidweiler et al., 2015; Wohlfarth et al., 2013). Therefore, strategies for the detection of possible metabolites, especially after phase I reactions, resulting from the use of a new generation substance such as NPSs, which have not been previously identified or whose newly identified metabolites are unknown, are a necessity. Multiple approaches have been used to estimate urine marker

metabolites including;

-in vitro incubation in human liver

microsome (HLM),

-in_ vitro. incubation in human

hepatocytes,

-in vivo application of controlled drugs studies on rats or mice and also

rodents, zebrafish larvae, -in silico prediction.

Alternative approaches recommend the most abundant and typical metabolites as “marker metabolites”, which can then be validated in real urine samples from suspected NPS cases. In vitro modeling for substance metabolism has to exactly mimic in vivo biotransformation. Over the last few

decades various in vitro models of the human liver have been developed in the pharmaceutical industry, the most commonly utilized and well-established in vitro incubation methods are liver microsomes and hepatocytes, which are also approved by relevant officials such as “the US Food and_ Drug Administration” (Brandon et al., 2003). The use of human __ hepatocyte incubation and HR-MS to elucidate NPS metabolites is the most favored coupled approach. Hepatocytes, isolated living cells, are a physiological system that enables to simulate human metabolism with the presence of extensive phase I and phase IT enzymes and drug-binding proteins (Costa et al., 2014). HLM is the most widespread /n vitro model due to its lower expense and ease of handling. Albeit, hepatocytes present a metabolic environment more similar to liver physiology than HLM, with some

drawbacks. The main benefit of hepatocytes versus HLM is that robust liver cells generate either phase I and phase II metabolites and inappropriate abundance; however, the abundance of HLM metabolites may not reflect the prevalence in real liver cells (Brandon et al., 2003; Diao & Huestis, 2017c). For

that reason, metabolites can be used as

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a reference for verifying metabolites

found in urine.

Early estimation of metabolic pathway is crucial to pre-clinical, clinical and decision-making. For drug discovery, it is essential to have trustworthy data on exactly how a compound reacts in the presence of metabolizing enzymes. This demands considerable experimentation effort. As a consequence, the ability to estimate possible sites of metabolism in any synthesized or virtual compound will be highly useful and time-efficient (Afzelius et al., 2007; Diao & Huestis, 2017c). In silico-based applications are progressively being adopted to estimate the metabolic transformation of drugs and are therefore recognized as the best approach to predict the metabolic transformation of drugs, allowing for lower costs, timesaving and hence reduced rates of late drug discovery (Kazmi et al., 2019). To date, forensic toxicology studies investigate the metabolic pathway of substances using in silico predictions, human hepatocyte incubations and LC-HRMS/MS detection in an original workflow to identify relevant consumption markers. Jn vitro incubations with human hepatocytes have been shown to be appropriate for

the estimation of NPS pathways in

former research and LC-HRMS/MS has evolved into the gold standard method for exhaustive scanning of sophisticated materials. In addition, novel data mining based on dual- targeted/untargeted analysis is often used, allowing for fast and semi- automated characterization of NPS "NPSs

‘mzCloud and

HighResNPS ~ The general workflow of

metabolites via spectrum

libraries” such as freely available mass spectrum databases is well suited for rapid implementation for NPS metabolite identification researches, given the market dynamics and underground 2018).

Furthermore, ‘metabolite samples from

production (Carlier et al.,

human hepatocytes” can be used as reference for verifying metabolites identified in urine, but their precise structure (i.e. the position of hydroxyl groups) may not be obtained by MS alone, for precise structural elucidation further techniques such as ‘huclear magnetic resonance spectroscopy “may be required (Gandhi et al., 2013).

In vitro and In _ silico Practices to Provide

Evidence

In this process, although the flow chart may vary according to the infrastructure of the laboratories, the purpose of the analysis and the target analyte, the workflow chart is generally carried out

in the following order. I. JInvitroPractices

The workflow for metabolism research of NPSs is generally recommended as follows: (i) incubate NPSs in human hepatocytes; (ii) define the most abundant and typical metabolites in hepatocyte samples by HR-MS; and (iii) acquire true positive urine samples and

validate hepatocyte metabolite markers. II. Jn-silicoPractices

"In silico prediction also aids metabolite definition without any reference standard and proposes metabolic sites and potential metabolites” Jn silico software tools are widely applied for the estimation of NPSs metabolites. These software programs essentially aim at CYP450-mediated _biotransformations, specifically considering phase I and substance

phase II metabolizing

enzymes. These are metabolites that may form through processes such as hydroxylation, acetylation, demethylation, dealkylation, and glucuronidation. Jn silico estimation is an important evidential tool for metabolite elucidation, although there are sometimes discrepancies between

urinary metabolite profiles. > Data Mining

Databases and_ related software prepared for /n silico studies enable the prediction and ranking of metabolites via the integration of 'machine learning- based” reaction estimation fields to determine reaction norms (Solimini et al., 2018). The list of metabolites is also created by related custom software. Usually, a match score is determined and metabolites higher than this score are selected. The data is re-processed to mimic the ‘second-generation

metabolism reaction; the second-

generation metabolite score _ is

multiplied by the _ first-generation metabolite score, and scores higher than the current score “are considered. The specific workflow commonly used and developed for data mining is given

in Figure 2.

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Input raw data files

Samples and controls (neg. and

pos. ion mode)

ae Spectra prosessing ee

Expected Compound Identification

Comparison and control with

the expected metabolite list

Mass | Mass monitoring

Unexpected Compound Identification

Elemental composition

prediction

Library research ——a . Librar. | Library research research

| Mergethedata | the data

Final specialist evaluation

Figure 2. Raw data mining workflow using LC-HRMS/MS

Pre-processing: Following analysis of the “raw data of samples and controls, the retention times of peaks in the mass spectra are evaluated with a maximum shift of 0.1 min and a mass tolerance of 5 ppm”. In full-scan HRMS, data are processed in the same way in both positive and negative ion selection

modes.

Untargeted data mining: It is usually evaluated based on peaks with an intensity above 10°, a signal-to-noise ratio above “3 and a 30% intensity tolerance for isotopes” (these values may vary according to the analyte) (Di Trana et al., 2021; Kazmi et al., 2019;

Taoussi et al., 2024). Mass spectra and

molecular formulas are then checked

with chosen libraries.

Targeted data mining. For targeted data mining, a theoretical metabolite list is produced by combining probabilities. Usually, “peaks with intensity above 5x10?, signal-to-noise ratio above 3 and intensity tolerance of 30% for isotopes, observed peaks with a mass tolerance of 5 ppm” are compared to the compound list. Compounds are finally sorted between the data files based on a retention time tolerance of 0.1 min and compared to the libraries

(Gergov, 2004; Taoussi et al., 2024).

Findings from non-targeted and targeted data mining applications are combined and compounds identified in

controls and with equal or greater

abundances than those detected in incubations are screened out. Outcomes are ultimately evaluated by the expert for final definition and

structure clarification.

Literature review on utilize in vivo, in vitro and in silico

approaches for NPS profiling

In this section, a brief literature review was conducted on studies that utilize in vivo, in vitro and in silico approaches for NPS profiling to elucidate biomarkers that may indicate the use of known or unknown target substances of interest in forensic toxicology. In this research, studies conducted in the last decade aimed at the analysis of substances from

the NPS class were discussed.

Methods: The research design was prepared in accordance with the PRISMA guidelines. The findings of the systematic review are provided in the flow diagram of the literature review (Fig. 3). After the

exclusion criteria were determined by

inclusion and

the researchers, the data obtained were evaluated separately by the authors and then cross-checked. The research was conducted using the keywords “in vivo”,

“in vitro”, “in silico”, and which were

matched separately with the terms

“New Psychoactive Substance” on PubMed, Wos and Scopus databases in the last decade. The search by keywords was aimed at preventing possible data loss by covering all sections, such as title, keywords, and with the “title,

keywords” search option in databases.

abstract, abstract, The data obtained was stored in .x/s extension on the computers of the researchers and the data obtained from the three databases were combined. and DOI

numbers of the studies included in the

Considering the names

study, duplicate publications were

manually removed by removing duplicates in the Excel file. Following the removal of duplicate publications, the researchers evaluated the remaining publications by reviewing the abstracts in terms of content appropriateness. If the title and abstract were assessed as the study

publications.

suitable, included these

Inclusion Criteria: The study included research articles and case reports published in English between January 2014 and September 2024, which met the search criteria based on keywords. Research articles, reviews, books, book chapters, Letters to the editor, and

Viewpoints were included.

21

oe

Exclusion Criteria: Studies not written in english and Erratums were technically excluded. Studies filtered after technical screening according to the title. Studies that did not serve the purpose of the review; (i) study titles that addressed the receptor activity, behavioral effects and addiction formation of NPSs in the field of clinical toxicology and (ii) studies that did not mention the use of in vivo, in vitro or in silico methods were

eliminated.

Results: A total of n=1247 studies were filtered with the keywords in three separate databases. One of these was excluded due to being an Erratum, and n=755 were eliminated from the study as duplicates. The remaining studies were excluded due to incompatibility with the designed research topic specified in items (i) and (ii) of the exclusion criteria. Studies with titles incompatible with the purpose (n=179) were eliminated. The abstracts of the remaining studies were read by two researchers. According to the abstracts

of the studies that were incompatible

with the objective, n=113 studies were excluded. After eliminating n=15 studies without full text access from the

remaining studies, 173 were included.

Challenges in monitoring NPS use can be overcome with different analysis techniques to learn more about the prevalence and expansion of NPS use. In the literature review, 173 out of 1237 studies used /n vivo, in vitro and/or in silico methods to detect NPSs, identify possible metabolites indicating their use and elucidate their metabolic pathways. With these alternative methods, it will be able to figure out further the pathways by which the NPS metabolism can be~ estimated with higher confidence. Not only will this contribute to improving better methods to manage NPS intoxication, but it will also be useful in assisting in compiling forensic and medico-legal reports for the

jurisdiction.

Records identified from:

PubMed, Web of Science and Scopus Databases (n =1237)

Records screened (n = 480)

Reports screened for content (n =301)

Reports sought for retrieval (n =188)

Studies included the review (n =173)

Figure 3. Flow diagram of the literature search (Prisma flow diagram)

Conclusion

Future Perspectives

Novel Psychoactive Substances consist of a broad variety of synthetic substances specially designed to produce psychoactive effects. They are typified by a structural multiplicity

produced by altering the molecular

Records removed before screening: Duplicate records (n=755) and erratum (n=1) removed

Records excluded (n =179) Unrelated content screened on title

Reports excluded (n =113) Unrelated content screened on abstract

Reports not retrieved (n=15)

structure of available substances or

conventional ones. However, such structural diversity poses difficulties for regulatory authorities and makes it harder to keep a close monitor on the proliferation and abuse of these substances. The problem is by the

challenges of analyzing NPSs. With the

compounded analytical

current information provided, new

analytical methods other than traditional ones are presented to obtain

forensic evidence to detect NPSs. Thus,

23

24

NPSs, which are increasingly being used in an uncontrolled manner and non-

detection of NPSs creates forensic

problems, can be prevented from posing a risk to human health and public Safety.

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SKELETAL ANATOMY ANALYSIS AND EVIDENCE COLLECTION AT THE CRIME SCENE: FORENSIC ANTHROPOLOGY AND ARTIFICIAL INTELLIGENCE APPLICATIONS

Mert OCAK , Cumali Catak

34

Chapter 2

Skeletal Anatomy Analysis and Evidence Collection at the Crime Scene: Forensic Anthropology and Artificial Intelligence

Applications

MERT OCAK?! , CUMALI CATAK2

Introduction

Integrating artificial intelligence (AI) models and machine learning into forensic

processes represents an

effective approach to victim identification, particularly by utilizing advanced biomedical imaging techniques alongside anthropological and anatomical measurements. The most common imaging methods in forensic identification are X-rays and computed tomography, both of which provide critical information about Skeletal remains and _ facilitate the extraction of anatomical features necessary for age estimation and sex These

determination. imaging

techniques are increasingly recognized 1 Asst. Prof. Dr., Ankara University, Tiirkiye,

mert.ocak @ankara.edu.tr, https://orcid.org/0000-0001-6832- 6208

for their effectiveness in forensic

investigations, enabling detailed analysis of skeletal materials, especially in scenarios such as mass disasters where traditional identification methods

may be inadequate (Vaswani, 2023).

The application of AI, especially through deep learning methodologies such as Convolutional Neural Networks (CNN), has revolutionized imaging data processing. CNN are adept’ at performing feature extraction and classification tasks necessary for

automatically analyzing anatomical measurements from processed images 2023).

imaging data, including standardization

(Nasien, Preprocessing of of dimensions, noise reduction, and contrast enhancement, improves the learning capacity § and _ prediction accuracy of the model. Furthermore, the application of data augmentation techniques and transfer learning has proven to be effective in improving model generalization, thereby increasing the robustness of age- and sex-related predictions (Shorten et al.,

2021).

2 PhD(c), Ankara University, Tiirkiye,ccatak@ankara.edu.tr , https://orcid.org/0000-0003- 1721-5182

In forensic anthropological studies, a systematic review of existing studies on structures such as the mandible, femur, and pelvis provides a rich dataset for training machine learning models (Woon & Stringer, 2012). Researchers continue to work on new models and develop methods to obtain more accurate and reliable prediction and identification results by analyzing anthropological measurements and demographic

information.

The implications of using AI and machine learning in forensic sciences go beyond identification, covering the ethical and practical dimensions of Skeletal

evidence collection and

analysis. AI tools provide a more scientific basis for forensic evidence by minimizing human error and subjectivity (Vaswani, 2023). Moreover, the fast processing capabilities of AI Significantly speed up_ identification processes in emergencies, thus improving the effectiveness of forensic investigations (Thurzo et al., 2021). The target audience of this research is professionals and academics in the fields of forensic sciences, forensic anthropology, medicine, and AI. The

research aims to increase the interest of

experts and relevant stakeholders in forensic science methods integrated with evolving technology, encouraging them to contribute to the field by critically evaluating studies conducted on the subject (Baryah et. al., 2019).

The Role of Forensic Anthropology

in Crime Scene Investigations

Forensic anthropology plays a crucial role in crime scene _ investigations, identification, and analysis of human remains. This speciality combines the principles of anthropology and forensic science to assist law enforcement in solving crimes involving unidentified bodies or skeletal remains. When soft tissues are distorted and decomposition has taken place, the skeletal structure becomes compromised and the remains fragmented. Forensic anthropologists have the expertise to extract crucial information from the evidence to identity and circumstances of death. (Sharma et al., 2011; Devraj et al., 2022).

determine the

They create a biological profile by studying bones to determine sex, estimate age, and analyze trauma for unidentified individuals. This profile is

then compared with antemortem

a5

36

information and DNA analysis to aid in

identification. Working alongside

forensic pathologists and law enforcement, forensic anthropologists provide vital details about the deceased, such as age, sex and height, which are crucial for the identification process. (Kahana & Hiss, 2009; Boer et

al., 2018).

Collecting evidence at crime scenes is a meticulous process that requires a thorough understanding of both anthropological techniques and forensic protocols. Forensic anthropologists specialize in recognizing and recovering skeletal ~—_ remains, ensuring that the context of evidence is preserved. Lack of sufficient expertise in this area can lead to problems in cases such as fires, and the integrity of evidence can be compromised. In short, the anthropological approach to crime scene investigation emphasizes the importance of a multidisciplinary team, including forensic archaeologists, to ensure that all potential evidence is documented and recovered systematically (Porta et al., 2013;

Schultz & Dupras, 2008).

In complex cases such as natural

disasters, the stress experienced by

crime scene investigation teams can affect their performance and the quality of evidence’ collected. In such situations, it is important to increase methods integrated with technological innovations that will reduce stress for crime scene investigation teams and improve the accuracy of their work (Adderley et al., 2012; Eeden et al.,

2018).

Forensic anthropologists also have a major role in broader societal events, such as human rights violations along with major disasters. Forensic anthropologists use their expertise to identify victims in these cases and take their place to assist in the subsequent legal processes. Challenges such as the need for rapid response’ and management of large-volume remains to emphasize the critical importance of forensic anthropology in contemporary forensic sciences (Boer et al., 2018; Ferllini, 2017).

Biological Profiling

The biological profile typically includes estimates of sex, age at death, ancestry, and stature. Each of these components plays a_ vital role in narrowing down the identity of skeletal

remains. For example, SEX

determination is often prioritized due to its significant impact on the accuracy of subsequent biological assessments (Winburn & _ Algee-Hewitt, 2021; Belcher et al., 2021). While ancestry estimation is challenging, it is equally important as it can provide critical individual's

context about an

background and_ potential familial connections (Passalacqua et al., 2023;

Boyd & Boyd, 2011).

Sex determination is one of the

most critical stages of biological profiling. Traditional methods rely on morphological features of the pelvis and skull, which are known to exhibit sexual dimorphism. The pelvis is particularly useful due to its reproductive function, making it the most sexually dimorphic

skeletal element (Francisco et al., 2017;

Barbieri et al., 2018). Recent advancements have introduced quantitative methods, such as

geometric morphometrics and three-

dimensional imaging, which have

increased the accuracy of sex estimation (Soriano et al., 2022; Goldstein et al., 2022). Additionally, the reliability of sex determinations has been further enhanced by _ using

machine learning algorithms to analyze

skeletal features (Gorka & Mazur, 2021; Keyes, 2023).

Age estimation in _ forensic anthropology has become an important field, especially with studies on skeletal remains of individuals in childhood and adolescence. In this context, "non- adult" remains to cover individuals from birth to 18 years of age, and age estimation of these remains is based on factors such as tooth development, bone fusion (ossification), and skeletal maturity. In adult remains, skeletal maturity is among the most commonly used indicators. This involves assessing skeletal features such as epiphyseal fusion in long bones and the ossification of the iliac crest, clavicle, and sacrum to determine whether the individual has reached at least 18 or 20 years of age. The pubic symphysis is considered one of the most reliable methods for age estimation, especially for individuals under 40. In-situ photography of the pelvis at the crime scene, along with careful packaging, is recommended to ensure the integrity of the study. Another frequently used method is age estimation from the sternal end of the ribs developed by Iscan et al. (Iscan et al., 1984; Iscan et al., 1985; Iscan et al., 1987).

Imaging methods

of

38

(conventional radiography, computed tomography (CT)) and _ histological analyses have recently played an important role in age estimations. Researchers have achieved successful results through the histological analysis of long bones. Molecular and chemical methods, particularly techniques like aspartic acid racemization, also show promising potential in age estimation (Crowder & Stout, 2011).

Estimating the age at death from

skeletal remains involves various

methodologies, including dental

analysis, epiphyseal fusion, and examination of bone

Each method has

histological microstructure. strengths and limitations, and often, a multifaceted approach is necessary to obtain accurate results (Chatterjee et al., 2020; Nasien, 2023). For example, while tooth wear patterns can provide insight into an individual's age, the fusion of skeletal elements can indicate developmental stages (Rizos, 2023; Baryah et al., 2019). Recent studies have also explored the use of advanced imaging techniques, such as dual- energy X-ray absorptiometry, to assess age-related changes in bone density (Zhang, 2024; Bethard, 2016).

Stature estimation is another critical component of the biological profile, usually derived from long bone measurements. Various regression formulas based on population-specific data have been developed to estimate height from skeletal remains (Diac et al., 2021; Yang et al., 2020). However, the accuracy of these estimates can be affected by factors such as population diversity and environmental conditions. Recent advances in imaging technology and statistical modeling have improved the precision of height estimates, allowing for more reliable applications in forensic cases (Pilloud et al., 2022;

Robles et al., 2023).

Biomedical Imaging Methods Used

in Forensic Anthropology

Forensic anthropologists also use

biomedical imaging techniques to analyze skeletal remains and fragments recovered from crime scenes. These techniques include X-ray and CT scans to visualize internal structures without damaging evidence, in addition to the application of classical osteometric methods (Porta et al., 2013). While these techniques help identify remains in investigations, they can also provide

valuable information about the cause of

death through analysis of trauma on bones (Eeden et al., 2018).

Forensic anthropology is

increasingly integrating advanced biomedical imaging methods to improve the accuracy and_ reliability of investigations involving human remains. The application of imaging techniques such as CT, magnetic resonance imaging (MRI), and three- dimensional (3D) surface

revolutionized the field by offering non-

scanning has invasive alternatives to traditional These

facilitate the

autopsy methods. imaging methods not only examination of skeletal remains but also allow for assessing soft tissue injuries and other pathological conditions that may not be visible through traditional

methods.

CT and MRI have emerged as essential tools in forensic imaging, providing detailed information about complex body structures and allowing for the

decomposed or contaminated remains.

examination of highly Chen highlighted the advantages of post-mortem typically traditional

imaging in examining

areas inaccessible during autopsies, such as_ the

thoracic cavity and brain. These

imaging techniques can also be applied to cases involving infectious diseases or toxic substances where _ traditional methods may pose health risks to forensic personnel (Chen, 2017; Singh 2022). Additionally, the

integration of CT angiography allows for

et al.,

the visualization of vascular structures, which can be crucial in cases of trauma

or homicide.

The application of imaging techniques extends beyond examining skeletal remains; they also play a Significant role in assessing soft tissue injuries. Zhang emphasized the utility of imaging in sex estimation by analyzing cranial and pelvic structures, which can be critical in forensic investigations where biological sex is a determining factor in identification. The ability to visualize and analyze these structures non-invasively increases the accuracy of forensic assessments and reduces the

potential for damaging remains.

Imaging methods contribute to diagnostic capabilities and the legal and forensic

ethical dimensions of

anthropology. The non-destructive nature of these techniques aligns with the increased emphasis on respecting

the dignity of the deceased and the

39

40

wishes of their families, particularly in cultures where traditional autopsy methods may conflict with religious beliefs (Ahuja & Ansari, 2022). The use of imaging promotes a more respectful approach to forensic investigations, allowing forensic experts to gather necessary information while minimizing physical intervention with the remains. Integrating AI and machine learning into forensic imaging is a promising new area of research for enhancing the capabilities of forensic anthropologists. AI algorithms can analyze imaging data to identify patterns and abnormalities that may not be easily visible to human observers, thus potentially increasing the accuracy of forensic assessments (Yang et al., 2020; Camacho &Wang, 2021). For example, deep learning techniques have been used to detect and classify various image manipulations, which can be particularly useful in

cases_ involving digital

evidence. This intersection of technology and forensic science is likely to continue evolving, offering new tools and methodologies for forensic anthropologists. Challenges in interpreting imaging data should also be acknowledged. The complexity of

human anatomy and the variability in

individual cases can make the analysis of imaging findings difficult. Therefore, forensic anthropologists must be cautious in their interpretations, considering the broader context of the case and the limitations of the imaging techniques used (Chen, 2017; Bolliger & Thali, 2015). Continuous education and training in the latest imaging technologies and methodologies are essential for forensic experts to maintain their expertise and make

accurate assessments.

The Application of Machine Learning and Deep Learning Algorithms in Forensic

Anthropology

1, Machine Learning Applications

Machine learning is a component of AI that can acquire knowledge from data and utilize this knowledge to forecast outcomes. It is categorized into supervised, unsupervised, and semi- supervised learning, each with distinct data structures and (Ongsulee, 2017).

applications

1,1. Supervised learning

In supervised learning, the model learns from labeled data to create a link between input data and their corresponding outputs. Through this training process, the model acquires the capability to predict outcomes for new data based on its learning from the provided data. This type of learning is commonly employed for solving classification and regression problems. While classification allows data to be divided

categories, regression aims to predict a

into specific

continuous value. This type of learning is widely used in many fields, such as health, finance, and marketing (Hlad et al., 2021).

Machine learning algorithms have become increasingly common in forensic anthropology, especially in analysing skeletal remains. One of the main applications of machine learning in this field

biological sex from skeletal features.

is the determination of

Classification techniques in machine learning have significantly enhanced ability to accurately determine biological profiles,

forensic anthropologists’

particularly in estimating sex and age

from skeletal remains. Supervised

machine learning classification methods applied in forensic anthropology have

yielded the following results:

Binary and Multinomial Logistic Regression (BLR and MLR)

Logistic regression is a_ statistical

technique used to examine the relationship between a binary outcome variable and one or more independent variables. In forensic anthropology, binary logistic regression is mainly used to estimate sex, where the outcome variable is binary (male or female), while multiple logistic regression is used for estimating age when considering multiple age categories. The flexibility of logistic regression allows researchers to include various Skeletal measurements as predictors, increasing the accuracy of their estimates. Malatong et al., in their study using deep learning techniques, employed a morphometric approach to analyze lumbar vertebrae and demonstrated the effectiveness of logistic regression in determining sex from skeletal remains (Table 1.) (Malatong et al., 2022). The study emphasized the importance of using precise measurements and statistical modeling to improve the

accuracy of sex estimations.

4]

42

Table 1. Sex determination accuracies obtained in the study (Comparison of the

measurement made while preparing the datasets and the prediction made by the

deep learning model) (Malatong et al., 2022).

Endplate Type Sex Accuracy (%) Superior endplate | Female 92.5

Superior endplate | Male 92.5

Inferior endplate | Female 88.5

Inferior endplate | Male 88.5

Superior and Female 91.0

inferior endplate

Superior and Male 91.0

inferior endplate

Kurniawan's research offers a comprehensive analysis of artificial intelligence applications in dental age estimation, focusing on_ integrating machine learning models with logistic to enhance

regression techniques

prediction accuracy (Kurniawan, 2024).

Despite the progress made in sex and age estimation using logistic regression, some challenges persist. One significant issue is the variability in skeletal morphology across different which affects _‘the

generalizability of models developed

populations,

from specific datasets. Researchers are increasingly focusing on developing population-specific models that account for morphological differences.

Additionally, advanced imaging

techniques such as _ cone-beam computed tomography (CBCT) and 3D modeling are expected to improve the precision of measurements used _ in logistic regression analyses (Dédouit et

al., 2014). Linear Discriminant Analysis (LDA)

LDA is a statistical technique used to determine the most effective linear combination of features that enhances the distinction between several object categories. In forensic anthropology, Linear Discriminant Analysis (LDA) is especially advantageous for sex estimation when the dependent variable is categorical (male or female), and the independent variables are continuous Skeletal

measures obtained from

characteristics. The method

assumes that predictors are normally distributed and that classes share a common covariance matrix, making it suitable for analyzing anthropometric data (Liebenberg et al., 2015; Bertsatos & Athanasopoulou, 2019).

Bidmos et al. conducted a study on sex estimation using 100 patellae in South Africa. They identified significant differences between male and female measurements using six different measurement parameters and applied discriminant

stepwise and _ direct

function analyses to model these differences. The study also employed classical machine learning techniques and feature ranking methods to

determine the optimal feature combinations. As a result, the stacking machine learning technique achieved a 90.8% accuracy rate in sex estimation. These findings are consistent with similar studies conducted in other countries, demonstrating the achievement of high accuracy rates

(Table 2.) (Bidmos et al., 2023).

Table 2. Comparing cross-validated performance of various Classifiers and stacked models

(Bidmos et al., 2023). Classification | Method Accuracy (%) Age NB 76 Classification Sex NB 77.5 Classification

Naive Bayes Classification (NB)

NB is a classification method that uses Bayes' theorem and assumes that the presence of a specific feature in a class is not affected by the presence of any other feature. This "naive" assumption simplifies the calculation of

probabilities, making NB particularly

efficient for classification tasks. In forensic anthropology, NB is used to classify skeletal remains into categories such as male or female (for sex estimation) and various age groups (for age estimation) based on_ skeletal measurements and morphological

features. In their study, Hemalatha et

43

44

al. used NB to estimate sex and age from dental radiographs, achieving an

accuracy rate of 77.5% for sex

determination estimation (7ab/e 3.) (Hemalatha et al., 2023).

and 76% for age

Table 3. Performance comparison (Hemalatha et al., 2023).

Classifier Mean Standard Deviation

LDA 83.85 + 4.47

RF 89.23 3.77

LR 83.85 4.47

K neighbors 83.08 4.56

classifier

The variability in skeletal morphology across populations can negatively affect the generalizability of this method, as with other classification methods. As emphasized in the study by Winburn & Algee-Hewitt, it is important to develop population-specific models that account for morphological

differences to increase _ prediction accuracy (Winburn & Algee-Hewitt,

2021). Decision Trees (DT)

DT belong to the category of supervised learning algorithms that create decision models based on data attributes. Their operational mechanism involves iteratively dividing the dataset into

smaller subsets according to the values

of input attributes. This process results in a_ tree-structured model where

internal nodes represent attribute- based decisions, branches signify the outcomes of these decisions, and leaf nodes denote class labels. For instance, in the context of forensic anthropology, these labels could be 'male' or 'female' for sex estimation or specific age ranges for age estimation tasks. The simplicity and visual structure of DT make them particularly attractive for forensic applications where interpretability is

crucial (Brown et al., 2018).

Traditional methods often rely on morphological assessments that can be subjective and variable in accuracy. However, the application of machine

learning algorithms, especially DT,

shows promise in improving the reliability of these predictions. DT allows for clear visualization of decision- making processes by classifying data according to feature values, particularly useful in forensic cases where transparency is crucial (Mohammad et al., 2022; Austin & King, 2016). In this context, machine learning algorithms like DT offer an effective method for classifying and predicting data. Particularly in forensic anthropological applications like sex determination,

using such algorithms increases the

N=284.834 [0.982]

N=4.088 [0.016]

accuracy and reliability of the obtained data (Nasien, 2023; Oner et al., 2021).

The study by Oner et al. aimed to analyze the accuracy of sex determination using the DT method based on patellar morphometry. With the measurements made, the prediction rate was calculated to be 98.2% for male individuals and 98.4% for female individuals. As a result of the study, they achieved sex determination with high accuracy in the patellar DT analysis

(Figure 1.) (Oner et al., 2021).

N=5.332 [0.018]

Figure 1. Confusion Matrix for the test set (Oner et al., 2021).

Random Forest (RF)

During its training phase, RF employs a collective learning technique to produce trees. For

multiple decision

classification tasks, it produces an

output based on the most frequent class

prediction among individual trees. In regression scenarios, it yields the average of predictions from all trees in the ensemble. This technique is particularly advantageous in processing high-dimensional data and is robust

against overfitting, making it suitable

45

46

for complex datasets often encountered in forensic anthropology. The RF algorithm works by creating numerous DTs from random subsets of the training data, thus increasing the accuracy and generalizability of the

model (Schonlau & Zou, 2020).

Balan et al. evaluated panoramic radiographs using deep learning techniques and compared classification methods. The success percentages of the classification methods used for age estimation in their study are shown in

Table 4, (Balan et al., 2022).

Table 4. Comparison of classification methods used in age and sex estimation (Balan

et al., 2022).

Method Age Accuracy Sex Accuracy (%) (%)

RF 91.0 90.0

NB 76.0 775

CNN Tis 78.0

SVM 83.0 82.5

Deep CNN 91.0 91.7

SNCNN 99.6 93.8

In the study conducted by Farhadian et al., data obtained from

mastoid process images acquired

through CBCT were evaluated to assess differences between sexes. As a result of the evaluation, RF achieved the highest performance percentage at 97% (Table 5.) (Farhadian et al., 2020).

Table 5. Evaluation of performance accuracy among various Classification models employed

in sex determination (Farhadian et al., 2020).

Prediction model Test Accuracy SVM 0.927 ANN 0.841 RF 0.969 K-NN 0.909 LR 0.917 NB 0.925 LDA 0.919

Despite the advantages of using RF for sex and age estimation, various challenges persist. One significant issue is the potential for overfitting, where the model becomes too complex and captures noise in the data rather than the underlying pattern. This limitation affects the accuracy of predictions, especially when dealing with small datasets or datasets with high variability (Liebenberg et al., 2024).

Artificial Neural Networks (ANN)

ANN are models inspired by the neural networks found in the human brain. ANN are made up of linked neurons that work together to recognize patterns and anticipate results based on input data. They excel at representing intricate, non-linear connections between input

factors and results, which makes them

well-suited for tasks like classification

and regression.

In forensic anthropology, ANN are used to classify skeletal remains into sex categories and age groups based on various morphological features. In the study by Anic-Milosevic et al., an ANN model was developed using orthodontic measurements of teeth, demonstrating the effectiveness of ANN in sex determination. The study achieved over 80% success rate (Anic-Milosevic et al., 2023).

In another study, Bewes et al. developed a deep-learning ANN model with images obtained from CT scans. The model showed a 95% accuracy rate in sex determination (Figure 2.) (Bewes et al., 2019).

47

48

Males

Figure 2. Prediction accuracy percentages of the developed model by sex (Bewes et al., 2019)

Patil et al. examined machine and ANN

panoramic radiographs in their study.

learning created with

The Deep Learning Model created with

ANN achieved an 87.2% success rate

(Table 6.) (Patil et al., 2023).

Table 6. Classification Performance Analysis (Patil et al., 2023).

Class Model Accuracy Division 2-Class Classification | 87.2

using Deep

Learning

While artificial neural networks (ANN) offer benefits for

estimating sex and age, there are still

several

several challenges to address. One major obstacle is the requirement for extensive, top-notch datasets to efficiently train the models. The of ANN is

effectiveness greatly

influenced by the quality and quantity of training data, and inadequate data can result in overfitting or underfitting. (Sevim et al., 2016).

Support Vector Machines (SVM)

SVM are used in supervised learning for

classifying and analyzing data for

regression. These models work by finding the best hyperplane in a multi- dimensional space to separate different classes of data points. The primary goal is to maximize the margin between classes, and this is calculated based on support vectors, which are the data points closest to the hyperplane. Due to its ability to utilize kernel functions for data transformation into higher

dimensions, SVM is especially efficient

for classifying data that is not linearly separable (Shan et al., 2022).

Darmawan et al. compared

different classification methods to determine sex from finger bone lengths by dividing individuals under 19 years old into age groups. The results of the study are shown in 7ab/e 7. (Darmawan

et. al., 2015).

Table 7. Highest accuracy percentage (%) obtained from three classification

techniques for each age group (Darmawan et al., 2015).

Group of age SVM SVM SVM ANN ANN ANN Male Female Average Male Female Average

Newborn—19 63.86 72.46 68.17 69.28 63.47 66.37 16-19 96.67 90.0 93.33 96.67 96.67 96.67 13-15 81.08 68.42 74.67 63.25 69.46 66.37 10-12 70.45 65.85 68.24 65.91 68.29 67.06 7-9 61.11 86.96 75.61 83.33 78.26 80.49 4-6 70.0 21.05 46.15 50.0 52.63 51.28 Newborn-3 76.47 18.75 48.48 64.71 68.75 66.67

Toneva et al. used SVM and ANN to create classification models for sexse prediction based on skull measurements. The results of the

evaluation of the measurements

obtained from the skull with CT are shown in 7ab/e 8. The accuracy results for all three methods were over 95%, and the SVM had the highest accuracy at 96.1%. (Toneva et al., 2021).

49

50

Table 8. Classification accuracies obtained by SVM, ANN and LR (Toneva et al., 2021).

Algorithm Selection Accuracy Accuracy Males Females SVM Full 95.6 + 1.0 96.5 + 0.6 BestFirst 93.7 + 1.0 96.0 + 0.7 GeneticSearch 92.2 + 1.0 95.8 + 0.4 ANN Full 94.2 41.2 95.9 + 0.7 BestFirst 91.7413 95.4 + 0.7 GeneticSearch 91.9 + 1.2 94.5 + 1.2 LR Full 93.5 + 0.7 93.5 + 0.7 BestFirst 94.7 + 0.7 95.7 + 0.6 GeneticSearch 92.2 + 1.2 95.7 + 0.5 1,2, Semi-Supervised develop a classifier that can predict

Learning

Semi-Supervised Learning is a type of learning where both labelled and unlabelled data are used together. In this type, the model is trained with a limited number of labelled data while improving the learning process by using more unlabelled data. Semi-supervised learning is generally preferred when labelled data is difficult or costly to obtain. This type of learning stands out as an effective method to improve the overall performance of the model, especially in areas with large data sets 2022).

approaches are either transductive

(Mousavi, Semi-supervised methods, which aim to assign labels to a collection of unlabelled images, or

inductive methods, which attempt to

labels for any image in the input space (Jesper et al., 2020). Semi-supervised includes _semi-

learning mainly

supervised classification, semi- supervised clustering, semi-supervised regression, and semi-supervised

dimensionality reduction. 1,3. Unsupervised Learning

Unsupervised Learning is a

method in machine learning that discovers hidden structures, patterns, or groupings in data by operating on unlabeled data. Unsupervised learning techniques can be listed as K-Means Clustering, Hierarchical DBSCAN, Fuzzy C-Means Clustering, Principal Component Analysis (PCA), t-

Distributed

Clustering,

Stochastic § Neighbour

Embedding (t-SNE) (Naeem et. al., 2023).

K-Means clustering divides the data points into K number of predetermined clusters and determines the centre of each cluster. In forensic anthropology, this method can be used to analyze the morphometric properties of bones. For example, morphometric data of bones such as the femur and pelvis can be grouped with the K-Means algorithm, and sex estimation studies can be- performed. Hierarchical Clustering creates a dendrogram, a tree-like structure, by combining or dividing the data step by step. This structure visually presents the relationships between the data. In forensic anthropology, Hierarchical Clustering can be useful in comparing of different individuals. DBSCAN groups data points density and

distinguishes low-density regions as

the bone © structures

according to their

noise. In forensic anthropology, this method can be used to make age estimates by analyzing the density distributions of bones. Fuzzy C-Means Clustering allows each data point to belong to more than one cluster. This flexibility can be useful in forensic better

anthropology to manage

uncertainties in the age and sex

estimates of individuals. Principal Component Analysis (PCA) extracts the of high

dimensional data by reducing its size. In

most important features forensic anthropology, PCA can help to obtain more meaningful results by reducing the size of the data set when analyzing the morphometric characteristics of bones. For example, age and sex estimates can be made by analyzing the size and_ shape characteristics of bones with PCA. Autoencoders compress the input data through a series of hidden layers and then use this compressed representation to produce outputs as similar as possible to the original data. This method can be used in forensic anthropology to minimize data loss when analyzing the morphometric t-SNE

technique that helps visualize complex

properties of bones. is a data in a _ lower-dimensional space, ensuring that similar data points are clustered together and dissimilar points are spread apart. This method can be useful for visual analyses in forensic anthropology, making the morphometric properties of bones more

understandable, and for age and sex

sal

52

estimation (de la paz-Marin et al., 2015).

1.4, Transfer Learning

Transfer Learning is a technique that enables a model to use the knowledge learnt in one task in another task. For example, features learnt by a model in one dataset can be applied in a similar dataset. This is particularly useful when working with limited data sets. Transfer learning is often used in deep learning and enables pre-trained

models to be reused for new tasks. This

method allows the model to learn quickly and effectively and use the previously learnt information in the new task.

Atas conducted a study comparing deep transfer learning architectures and concluded that the DenseNeti21 model, using deep transfer learning and a fully automatic technique, could be employed to analyze panoramic dental X-ray images, especially for sex estimation research in forensic sciences (Table 9.) (Atas, 2022).

Table 9. Deep Transfer Learning Models (Atas, 2022).

Model

Accuracy

VGG16

0.8220

ResNet50

0.9260

EfficientNetB6

0.9400

DenseNet121

0.9725

2. Deep Learning Applications

Deep learning, a branch of machine learning, has become a revolutionary technology that employs multi-layer neural networks to understand intricate data representations. This method enables the model to automatically extract features from raw data, allowing it to identify complex patterns and

relationships that traditional machine

learning techniques may not be able to detect. Deep learning models typically comprise multiple layers, with each layer progressively transforming the input data into higher-level abstractions. The development of network architectures in artificial intelligence has significantly impacted various fields, including forensic anthropology, especially in the study of

age and sex estimation. Starting with

classical architectures such as LeNet-5, AlexNet and VGG16,

evolved with more advanced models

studies have

such as Inception, ResNet, ResNeXt and DenseNet. Each of these architectures

has unique features that can be used to

analyze skeletal remains, which is very important in forensic anthropology (Mohamed et al., 2023). The taxonomic distribution of DCNN architectures is as

shown in Figure 3. (Khan et al., 2020).

Figure 3. Developmental distribution tree of CNN architectures (Khan et al., 2020).

Deep learning relies on the

concept of deriving representations from data without requiring manual feature engineering. In image processing, for instance, the _ initial layers of a CNN can recognize simple features like edges and textures, while subsequent layers can combine these

features to identify more _ intricate

structures such as shapes and objects (Najafabadi et al., 2015). In a study conducted by creating CNN models, Cao et al. performed sex determination from images of coxae bones obtained by CT (Table 10.) (Cao et al., 2022).

53

54

Table 10. Performance of CNN models in independent sex prediction

Pelvic region

Accuracy

Ventral pubis

0.979 7 1.000

Dorsal pubis

0.959 - 1.000

notch

Greater sciatic | 0.881 -

0.963

Pelvic inlet

0.926 . 0.987

Ischium

0.800 - 0.909

Acetabulum

0.666 ; 0.802

The automatic evaluation of radiographs constitutes a_ practical application for bone age determination and an ideal target for deep learning. Age estimation based on comparing one or several radiographic images with a

reference standard has been used for

several decades. Senel et al. evaluated X-ray films of wrist bones with different deep-learning architecture methods and compared the performance of classifiers (Table 11.) (Senel et al., 2021).

Table 11. Performance comparisons of classifiers (Sene/ et al., 2021).

Classifier Training-Testing Metrics Mean Data Splitting GoogleNET 70%-30% MSE 0.0072 MAE 0.0107 Acc 0.9137 80%-20% MSE 0.0046 MAE 0.0077 Acc 0.9406 AlexNET 70%-30% MSE 0.0148 MAE 0.0204

Acc 0.8199

80%-20% MSE 0.0090

MAE 0.0128

Acc 0.8942

Vgg19 70%-30% MSE 0.0235 MAE 0.0300

Acc 0.7263

80%-20% MSE 0.0125

MAE 0.0163

Acc 0.9281

Conclusion

The role of AI in forensic anthropology is increasingly recognized in the context of Disaster Victim Identification. Forensic anthropologists play a crucial role in mass disasters where rapid identification of victims is required. The integration of AI _ technologies automates the comparison of skeletal remains with antemortem records, accelerating the identification process and making the resolution of time- intensive cases more practical (Piraianu, 2023). This capability is especially important in scenarios such as natural disasters or terrorist attacks, as timely identification allows forensic processes to be concluded both quickly and accurately, ensuring justice and preventing potential victimization of the

victim's relatives.

Despite the promising developments in AI and machine learning applications in forensic anthropology, challenges remain. Since the effectiveness of machine learning algorithms is highly dependent on the quality and quantity of data used for training, the demand for high-quality training datasets increases as multiple fields of study continue to diversify. In addition, ethical considerations surrounding the use of AI in forensic cases are discussed, particularly in the context of bias issues and _ the implications of automated decision- making on the legal landscape (Ahmed, 2023). The potential for algorithmic bias raises concerns about the fairness and reliability of Al-assisted forensic analyses. Continuous research and development are required to ensure these technologies are used responsibly

and ethically.

55

56

Furthermore, integrating AI into forensic anthropology raises questions about the role of human expertise in the While AI can

improve the efficiency and accuracy of

analytical process. forensic investigations, it is important to remember that human bias and experience remain critical components of forensic analysis. Collaboration between forensic anthropologists and

AI technologies can enable algorithms

to capture contexts and structures that human experts cannot fully capture with classical methods, leading to more robust and reliable results (Mladenovic 2023). This

approach will help reduce the risks

et al., collaborative

associated with’ over-reliance on

automated systems, performing

forensic analyses with the highest

scientific rigour Figure 4.

Figure 4, Machine learning training material for the mandibular identification model,

which will enable sex determination and stone prediction through the collaboration of

machine learning and panoramic radiography in our ongoing work.

Human-AI collaborations in forensic identification studies, conducted after events such aS wars and mass murders—regardless of the passage of time—in both murder cases and mass disasters, will provide significant benefits to humanity and society as

research advances and developments

progress. Such collaborations will help prevent law and human rights violations under all circumstances and enable law enforcement officers, crime scene investigators, forensic scientists, and lawyers involved in the judicial process

to work in much healthier conditions.

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Vaswani, V., Caenazzo, L. & Congram, D. (2024). Corpse identification in mass disasters and other violence: The ethical challenges of a humanitarian approach. Forensic Sciences Research, 9(1), owad048.

Winburn, A. P. & Algee-Hewitt, B. (2021). Evaluating population affinity estimates in forensic anthropology: Insights from the forensic anthropology database for assessing methods accuracy (FADAMA). Journal of Forensic Sciences, 66(4), 1210-1219.

Woon, J. T., & Stringer, M. D. (2012). Clinical anatomy of the coccyx: A systematic review. Clinical anatomy, 22), 158-167.

Yang, W., Zhou, M., Zhang, P., Geng, G., Liu, X. & Zhang, H. (2020). Skull sex estimation

based on wavelet transform and Fourier transform. BioMed Research International, 2020, 8608209.

Zhang, M. (2024). The application of forensic imaging to sex estimation: Focus on skull and

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SIMULTANEOUS ANALYSIS OF ORGANIC AND INORGANIC GUNSHOT RESIDUE

Harun SENER, Hatice SOYTURK

66

Chapter 3

Simultaneous Analysis Of Organic

And Inorganic Gunshot Residue

HARUN SENER}, HATICE SOYTURK2

Introduction

Gunshot (GSR)

constitutes a significant type of trace

Residue

evidence essential for investigating firearm-related offenses. GSR consists of a mixture of particles originating from both burned and unburned primer, propellant gases, bullet components, and material expelled from the firearm itself during the discharge (Basu, 1982; Carneiro et al., 2023; Wolten, n.d.; Lucas et al., 2019). These particles, due to their minuscule size, can transfer to the shooter's hands, clothing, face, and hair. They can also deposit on surrounding surfaces, including those of victims and the environment near the discharge site (Lucas et al., 2019). Factors that affect the quantity of gunshot residue (GSR) and its dispersal

range encompass the type of firearm,

1 Assist. Prof. Dr., Kiitahya Health Sciences University, Tiirkiye, harun.sener @ksbu.edu.tr, https://orcid.org/0000-0003-352 1-0684

the movement of the weapon, the type of ammunition utilized, the number of shots discharged, and the existing environmental conditions

2020).

(Sener,

In forensic investigations, detecting GSR on individuals or objects can aid in linking residues to a specific firearm incident (Rosengarten et al., 2021).

between a

Establishing a_ relationship

suspect and_ actions connected to a shooting event is crucial in cases involving firearms. GSR collected from suspects, victims, or the crime scene provides valuable insights. However, because samples are often collected at varying times post-incident, forensic scientists must consider factors such as GSR transfer and persistence during evidence Additionally,

formation of GSR and its presence in the

interpretation. understanding the ambient environment is _ essential.

Nonetheless, significant challenges remain in identifying and interpreting GSR, presenting ongoing difficulties for the forensic science community (Sener,

2020).

2 MSC Student, Ankara University, Tiirkiye, haticesoyturk1999 @ gmail.com, https://orcid.org/0009-0002- 6796-0172

GSR refers to inorganic gunshot residues (IGSR) and organic gunshot residues (OGSR). The current analytical method for detecting IGSR relies on identifying particles of specific size and morphology that contain various combinations of elements, including barium, antimony, lead, gadolinium, titanium etc. (Dalby et al., 2010; Saverio Romolo & Margot, 2001). The residues originate from the primary source, and the analysis is performed using scanning electron microscopy (SEM) with X-ray spectroscopy. In recent years, OGSR analyses have been conducted alongside heavy metal

analyses to evaluate ammunition designed to be safe for both the shooter and the environment, as it is devoid of heavy metals. International standards have not yet validated a standardized analysis method for OGSR analyses

(Sener, 2021).

Forensic scientists must have a solid understanding of GSR_ traces, including their transfer, persistence, and prevalence, to develop reliable methodologies for detecting, collecting, analyzing, and_ interpreting these residues in forensic investigations. This chapter seeks to summarize the current

knowledge regarding GSR, focusing on

residues transferred to individuals or objects not directly involved with the shooter, such as_ bystanders’ or surrounding surfaces. Additionally, it outlines the existing methods for GSR detection and analysis, evaluates the of the

mentioned approaches. Also, it provides

strengths and _ limitations

recommendations for future research to

advance forensic GSR studies.

1. GSR Production and

Composition

Gunshot

released from firearms in the form of

residues (GSR) are

inorganic particles and organic vapors 2018; Vander Pyl,

Feeney, et al., 2023). The two main

(Blakey et al.,

types of GSR are distinguished for methodological purposes, as different methods are typically utilized for their detection and analysis. IGSR originates mainly from the firearm's capsule and several metallic elements, including the barrel, bullet, and cartridge case, while OGSR is a product of the incomplete combustion of the propellant. After a shooting incident, projectile residue accumulates on surfaces near the firearm,

discharged including the

shooter's hands and face, as well as on

67

68

the intended targets (Vander Pyl, Feeney, et al., 2023).

1.1. Inorganic GSR

In forensic science, Inorganic Gunshot Residue (IGSR) consists mainly of metallic particles derived from the primer, which is a mixture of fuel, oxidizer, and explosive that reacts during firearm discharge. The particles may originate from the bullet core, cartridge casing, and the mechanical wear of the _ firearm's (Szakas & Gundlach-

Graham, 2024). The primer's explosive

moving

components

reaction comprises three essential components: lead styphnate (CeHN3OsPb) as the primary explosive, barium nitrate (Ba(NO3)z2) functioning as an oxidizer, and antimony trisulfide (Sb2S3) as a fuel (Feeney et al., 2020; Vander Pyl, Feeney, et al., 2023). Forensic laboratories commonly employ Scanning Electron Microscopy with Energy-Dispersive X-ray Spectroscopy (SEM/EDX) to analyze IGSR. This technique demonstrates high sensitivity and specificity, enabling forensic scientists to identify inorganic particles within a sample and ascertain their

elemental composition and morphology

with remarkable precision (White, 1987; Redouté Minziére et al., 2023).

While conventional sinoxid-type primers, which release toxic elements like lead, barium, and antimony, remain in widespread use, there is a growing shift toward heavy metal-free primers (Figure 1).

friendly primers incorporate elements

These environmentally

like sulfur (S), aluminum (Al), potassium (K), titanium (Ti), gadolinium (Gd), gallium (Ga), silicon (Si), and zinc (Zn) 2000; Gunaratnam & Himberg, 1994; Romano et al., 2020). This transition reflects

increased awareness of environmental

(Charpentier & Desrochers,

and health concerns related to releasing toxic substances from __ traditional

ammunition. 1.2. Organic GSR

Organic gunshot residue (OGSR) mostly originate from the smokeless powder and primer contained in the cartridge casing. Smokeless powders consist of intricate combinations of various

explosive materials and

additives, including stabilizers, plasticizers, flash inhibitors, coolants, deterrents, surface lubricants, dyes, enhance

and other agents that

performance (Vander Pyl, Feeney, et

al., 2023). Upon the discharge of a

firearm, incomplete combustion,

evaporation, and condensation processes result in the presence of smokeless

unburned powder

constituents and their degradation

products in gunshot residue (Minziére et

Gunpowder-Smokless powder

Nitrocellulose (NC) Nitroglycerine (NG) Nitroguanidine (NGU) Diphenylamine (DPA) 2-nitrodiphenylamine (2-nDPA) 4-nitrodiphenylamine (4-nDPA) N-Nitrosodiphenylamine(N-nDPA) Dibutylphthalate (DBP) Dimethylephthalate (DMP) Diethylphthalate (DEP) Akardite I-II-III

Dinitrotoluene

al., 2023). The unburned particles

possess significant forensic value, offering insights into the type of ammunition utilized and the circumstances surrounding the discharge.

Primer (Sinoxid)

Lead styphnate Barium nitrate Lead peroxide Lead dioxide Antimony sulfate Calcium silicate Tetrazene

Primer (Sintox/Heavy Metal Free)

Diazole

Tetrazene

Zinc peroxide Titanium chloride Gadolinium (III) oxide Gallium-copper-tin Samarium oxide Titanium oxide

Figure 1. Composition of firearm ammunition

The principal propellants in firearms are smokeless powders, categorized into three types according to their

chemical composition:

e Single-base powders: Consisting exclusively of nitrocellulose (NC) asthe

singular explosive

element.

e Double-base powders comprise both nitrocellulose (NC) and nitroglycerin (NG).

e Triple-base powders comprise nitrocellulose (NC), nitroglycerin (NG), and nitroguanidine (NQ) (Taudte et al., 2015).

Notable volatile organic compounds

used as additives in smokeless powders

69

70

include stabilizers such as diphenylamine (DPA) and its isomers; (2-nDPA), 4-

nitrodiphenylamine (4-nDPA), and N-

2-nitrodiphenylamine

nitrosodiphenylamine (N-nDPA) along with methyl centralite (MC) and ethyl centralite (EC) (Figure 1) (Taudte et al., 2015). These compounds are critical for extending the shelf life of smokeless powder and minimizing the risk of accidental ignition, thereby enhancing

the stability and safety of ammunition.

Forensic scientists concentrate on elucidating the composition of organic residues to enhance methodologies for detecting and residue (GSR). gunshot

analyzing gunshot Examining organic residue enables forensic specialists to identify the type of ammunition employed, assess the firing distance, and _ possibly connect a suspect to a shooting incident. This analytical methodology is essential in criminal investigations pertaining to

firearms.

2. The Evidence of Gunshot Residue

2.1- Transfer

Gunshot residue (GSR) can be transferred through various mechanisms. Primary transfer occurs

directly after a firearm is discharged,

leading to the deposition of GSR on the shooter's hands, which can then spread to other body parts or clothing. Secondary transfer happens when a clean surface comes into contact with a surface already contaminated with GSR, such as handling a fired weapon, shaking hands with a shooter, or touching other contaminated surfaces. involves _ further

Tertiary transfer

contamination, where a surface affected by secondary transfer contacts another, continuing the spread of GSR. The behavior of GSR _ particles is influenced by their physicochemical properties, including how they interact with surfaces like human skin (Blakey et al., 2018; Vander Pyl, Dalzell, et al.,

2023).

The factors influencing GSR transfer encompass the elapsed time since the shooting, the specific firearm and ammunition utilized, the distance to the target, and subsequent activities following the shooting. Research demonstrates that organic gunshot (OGSR) is

concentrated on the

residue frequently hands and forearms owing to its volatile and lipophilic properties, facilitating absorption into the the skin epidermal

layer. As a result, OGSR_ exhibits

reduced vulnerability to secondary transfer (Demircioglu et al., 2024; Feeney et al., 2020, 2022; Hofstetter et al., 2017; Moran, 2013). In contrast, (IGSR)

particles, which adhere to the external

inorganic gunshot residue surfaces of skin and clothing, are more susceptible to secondary and tertiary transfers when exposed to physical disturbances (Blakey et al., 2018; Feeney et al., 2020, 2022; French et al., 2014). If the shooter is not identified and samples are not collected promptly after the discharge, the increased possibility of secondary transfer and the depletion of GSR particles poses a significant challenge for the

investigation (Demircioglu et al., 2024).

Research shows that OGSR is not

easily transferred through casual contact but can be lost during activities that involve prolonged or intense contact, such as rubbing hands or washing with soap (Vander Pyl, Dalzell, 2023). Furthermore, the

hand, which _ typically

accumulates more GSR, is also more

et al.,

dominant

vulnerable to transfer than the non- dominant hand (Maitre, Horder, et al., 2018; Maitre, Kirkbride, et al., 2018).

An important consideration in GSR analysis is the memory effect—the potential impact of the firearm’s shooting history on collected samples. The type of ammunition last fired, whether it produces OGSR or IGSR, can influence the results, particularly in the initial shots after changing ammunition types, though this effect gradually diminishes over time (Donghi et al., 2024; 2001).

Interpreting GSR analyses requires

MacCrehan et al.,

caution because the exact type of ammunition used is unknown, and prior ammunition can significantly influence

the findings. 2.2. Persistence (t > 0)

Gunshot residue (GSR) does not persist indefinitely on the shooter's hands or other surfaces, gradually diminishing over time. Criminals may take deliberate actions such as washing hands, changing clothes, showering, or wearing gloves to remove or prevent GSR _ contamination. Therefore, it is imperative to obtain samples from suspects promptly following a shooting and to implement necessary measures to prevent the destruction of potential evidence (Arndt et al., 2012; Maitre, Horder, et al., 2018).

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72

Comprehending the persistence of GSR is essential for enhancing the detection and analysis of OGSR. This knowledge aids various stakeholders engaged in forensic investigations. Forensic investigators and laboratories offer critical insights into the probability of obtaining positive gunshot residue (GSR) results, particularly when there is a temporal gap between the shooting incident and sample acquisition. It assists forensic specialists in analyzing OGSR outcomes in situations requiring differentiation between individuals who discharged a firearm and_ those uninvolved, as well as in evaluating the participation of individuals in a firearm- related event (Maitre, Horder, et al.,

2018).

Most research indicates a rapid decline in GSR levels within the first few hours following a shooting (Gassner & Weyermann, 2016; Maitre, Horder, et al., 2018; Minziére et al., 2022). In 1975, it was shown that neutron activation analysis (NAA) could detect barium (Ba) and antimony (Sb) residues on shooters' hands for up to six hours post-discharge (JW, 1995; Romano et al., 2020). Another study by Chavez Reyes et al. observed a decrease in GSR

levels within four hours of the shooting

using nasal stubs as a collection method (Chavez Reyes et al., 2018). Maitre, Horder, and colleagues, as well as Gassner & Weyermann, reported a significant decrease in OGSR levels within the first-hour post-discharge, though they were still detectable up to four hours later through _ liquid chromatography-mass spectrometry (LC-MS/MS) analyses. They also noted that handwashing could lead to partial or complete removal of OGSR, with the lipophilic nature of certain OGSR compounds playing a key role in their persistence on the skin (Bonnar, 2023; Gassner & Weyermann, 2016; Maitre, Horder, et al., 2018). Additionally, Rosengarten et al. found that GSR could be detected on towels, even if suspects attempted to wash off residues by

showering (Rosengarten et al., 2021).

The findings suggest that the behavior of OGSR depends heavily on the physicochemical properties of the compounds, while the behavior of (IGSR) is more influenced by physical properties. IGSR

inorganic GSR

particles tend to persist unless mechanically disturbed, whereas OGSR is more easily diminished through daily

activities and can be more readily

transferred (Vander Pyl, Dalzell, et al., 2023).

2.3. Prevalence

Prevalence refers to the frequency with which gunshot residue (GSR) is detected in a_ specific population or environment, including cases where GSR is found on individuals not directly involved in firearm-related incidents. This can occur due to secondary contamination, such as through contact with police officers or other individuals who may have handled firearms or GSR-contaminated surfaces during custody’ or _ investigation procedures (Minziere et al., 2022). Understanding the levels of GSR prevalence among law enforcement personnel is crucial. It enables forensic experts and police departments to adjust their operational procedures to find ways to minimize the risk of contamination during arrests (Cook, 2016; Stamouli et al., 2021). Studies analyzing IGSR among _ civilian populations in various countries have generally detected a low number of characteristic particles (Minziére et al.,

2022).

3. GSR Collection and Analysis 3.1. Collection

Selecting the most suitable method for collecting gunshot residue (GSR) from various surfaces is essential to maximize the efficiency of collection and ensure reliable forensic analysis (Minziére et al., 2023; Shrivastava et al., 2021). Common tools for collecting GSR include alcohol wipes, adhesive tapes, stubs, and cotton swabs. The optimal method for collecting gunshot residue (GSR) depends on both the type of surface being sampled and the analytical technique that will be employed for residue detection. A well- chosen collection method should be simple to use, quick, and accurate, while also being portable and adaptable

for crime scene use (Sener, 2021).

When gathering gunshot residue evidence, it's vital to follow specific protocols to maintain the integrity of the

samples:

« Personal Hygiene and Glove Use: The forensic specialist should thoroughly wash. their hands before starting the

collection process. It's crucial to

change gloves between handling

74

each of the suspect's hands to prevent cross-contamination.

« Preventing Evidence Loss: Ensure the suspect does not wash their hands or touch other surfaces. Such actions can result in the loss or contamination of GSR evidence.

« Documenting Dominant

Hand and Sample Collection:

Record whether the suspect is

right-handed or left-handed in

the official

swabbing from the palm of the

report. Begin

dominant hand, as it's more

likely to contain significant residue.

« Preserving Protective Bags: If the suspect's hands were secured in protective bags to prevent contamination, do not discard these bags. Place them in separate, labeled containers for

further examination.

3.2. Analysis

Gunshot residue analysis can help us link the suspect to the incident, show the origin of the incident (homicide, suicide, etc.), determine the distance and direction of fire, and

confirm the veracity of a statement

(Hofstetter et al., 2017).

analysis methods for GSR mainly focus

Existing

on the analysis of inorganic GSR consisting of metallic particles from primers,

bullets, cartridges, and

weapons. 3.2.1. IGSR Analysis

In the past, dermal nitrite testing, paraffin testing, and Harrisson and Gilroy Method Neutron Activation Analysis (NAA) were used. Today, Inductively Coupled Plasma-Mass (ICP-MS),

Scanning Energy Dispersive X-ray

Spectrometry Electron Spectrometry (SEM-EDX), and atomic absorption spectrometry (AAS) are used (Meng H.H., 1997; Krishnan, Gillespie, 1971).

Today, Inorganic § Gunshot Residue (IGSR) is analyzed using Scanning Electron Microscopy with Energy Dispersive X-ray Spectrometry (SEM/EDX) following the ASTM E1588- 20 standard guidelines. This method involves evaluating GSR and GSR-like particles from environmental sources based on their morphology and elemental composition. The chemical composition is categorized into GSR characteristic particles, which include

lead (Pb), barium (Ba), and antimony

(Sb); particles consistent with GSR, such as PbBa or BaSb; particles commonly associated with GSR, such as Ba, Pb, or Sb; and combinations like GdTiZn or GaCuSn found in heavy metal free ammunitions. This highly sensitive and specific technique allows for the detection of inorganic particles within a sample, providing detailed information regarding their elemental composition and morphology (R.S White, 1987;

Redouté Minziére et al., 2023).

The SEM-EDX method has two

advantages:

1- It allows the shot residue to

retain its particle structure

and allows’ the particle morphology to be photographed.

2- All elements emit x-ray

radiation at characteristic wavelengths when excited by incident electrons. This makes it possible to record the chemical composition of

each particle (Bonnar, 2023).

However, the time required for the characterization of a single sample via SEM-EDX,

increased use of heavy metal-free

combined with the

ammunition that contains elements

commonly found in the environment, Additionally, the

and persistence of IGSR

poses challenges. transfer particles complicate the interpretation of results. To address these limitations and improve the efficiency and reliability of GSR evidence, research on OGSR analyses is expanding. OGSR analyses aim to overcome the complexities associated with IGSR by focusing on organic compounds, potentially providing more robust and

specific forensic insights. 3.2.2. OGSR Analysis

Raman Spectrometry, Fourier

Transform Infrared (FTIR), Ion (IMS), Gas

Spectrometry

Spectrometry Mobility Spectroscopy Chromatography-Mass (GC-MS), Chromatography-Mass

Liquid Spectrometry (LC-MS), Infrared Spectroscopy (IR), and Capillary Electrophoresis (CE) are commonly used to identify organic gunshot residues (OGSR). However, an internationally recognized standard method for OGSR analysis has not yet been established. There are, however, standard practice recommendations for the analysis of OGSR using LC-MS or

GC-MS (OSAC, 2022a, 2022b).

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76

Recent studies show that OGSR

offers supplementary insights in

examining shooting incidents, particularly with ammunition lacking heavy metals (Feeney et al., 2020; Minziére et al., 2022; Redouté Minziére et al., 2023). OGSR has also been studied to contribute to estimating the distance or time of a shot, but there is no standardized method, such as the sodium-rhodizonate test, based on the lead presence (LOpez-Lopez & Garcia- Ruiz, 2014; Redouté Minziére et al.,

2023; Zeichner, 2003).

Compounds like methyl centralite (MC) and ethyl centralite (EC) are primarily used in the production of smokeless gunpowder, making them some of the most _ distinctive constituents of firearm shot residues. (DPA) has

industrial applications; however, it is

Diphenylamine various considered a_ defining feature of gunshot residue (GSR) when detected alongside its nitrated derivatives like N- (N-nDPA), 2- nitrodiphenylamine (2-nDPA), or 4-

nitrosodiphenylamine

nitrodiphenylamine (4-nDPA) (Maitre, Horder, et al., 2018).

Conclusion

Gunshot residue (GSR) analysis provides crucial information that can link a suspect to a crime, confirm whether a weapon was fired, identify bullet entry and exit holes, and help determine the cause of death, whether it be homicide, self-defense, or suicide (Krishna & Ahuja, 2024; Lopez-Ldopez et al., 2013). However, interpreting GSR results requires careful consideration to avoid wrongful convictions. The primary aim of GSR detection and analysis is to determine whether the detected traces are indeed _ firearm-related = shot residues. If confirmed as shot residue, the level of activity, specifically the transfer level, must be established. It is critical to distinguish between primary transfer (direct firearm discharge) and secondary or higher-level transfers (French et al., 2014; Hofstetter et al., 2017; Minziére et al., 2022; Werner et al., 2020). With the advent of heavy metal-free ammunition, current standard procedures are insufficient for evaluating the transfer and persistence mechanisms of organic gunshot residue (OGSR), particularly when determining firing distance. To address these challenges, research into OGSR analysis

has increased, focusing on conducting

simultaneous or sequential analyses of inorganic GSR (IGSR) and OGSR. Data on transfer, persistence, and prevalence are essential for developing interpretation models and incorporating OGSR analysis into routine forensic laboratory practices (Redouté Minziére 2023). It is

et al., strongly

recommended that IGSR and OGSR analyses be performed together to ensure more comprehensive forensic results (Taudte et al., 2014).

7a

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OSAC, 2022-S-0002. (2022a). OSAC 2022-S-0002 Standard Practice for the Identification of Compounds Related to Organic Gunshot Residue (OGSR) by Gas Chromatography-Mass Spectrometry (GC-MS). OSAC 2022-S-0007, 11.

OSAC, 2022-S-0003. (2022b). Standard Practice for the Identification of Compounds Related to Organic Gunshot Residue (OGSR) by Liquid Spectrometry (LC-MS) OSAC. OSAC 2022-S-0003, 1-12.

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Redouté Minziére, V., Robyr, O., & Weyermann, C. (2023). Should inorganic or organic gunshot residues be analysed first? Forensic Science International, 348, 111600. https://doi.org/10.1016/j.forsciint.2023.111600

Romano, S., De-Giorgio, F., D’Onofrio, C., Gravina, L., Abate, S., & Romolo, F. S. (2020). Characterisation of gunshot residues from non-toxic ammunition and their persistence on the shooter’s hands. International Journal of Legal Medicine, 1343), 1083-1094. https://doi.org/10.1007/s00414-020-02261-9

Rosengarten, H., Israelsohn, O., Sirota, N., & Mero, O. (2021). Finding GSR evidence

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THE SIGNIFICANCE OF POSTMORTEM IMAGING IN THE EVIDENCE ANALYSIS

Ceren AKAGUNDUZ AYRANCIOGLU

84

Chapter 4

The Significance of Postmortem Imaging in the Evidence Analysis

CEREN AKAGUNDUZ AYRANCIOGLU!

1. Introduction

Postmortem imaging has

become a crucial tool in forensic medicine, particularly in the field of Although

conventional autopsies have long been

forensic pathology. the standard for death investigations, postmortem imaging adds a

noninvasive, detailed layer of information that can both complement and, in substitute

some Cases,

traditional methods. Postmortem imaging with technologies such as digital radiography (DR), computed (CT), and

resonance imaging (MRI) offers a visual

tomography magnetic

roadmap of _ internal anatomical structures and preserves the integrity of

the body while providing critical

| Ph. D. Candidate, izmir Tinaztepe Univesity, Turkey, 0000-0002-0124-6534

ceren.ayrancioglu@tinaztepe.edu.tr,

insights. In modern forensic science, the accuracy and precision of evidence analysis are paramount. Postmortem imaging has emerged as a transformative tool in this arena, reshaping how forensic pathologists and medical examiners deaths. The ability to capture detailed,

three-dimensional, and reconstructed

investigate

images of a body using advanced imaging technologies has gathering and Additionally,

imaging supports legal

revolutionized the analysis of evidence. postmortem proceedings by _ providing visual evidence and documentation, making it an essential tool in modern forensic pathology. Furthermore, the digital nature of the images allows for

repeated review, consultation with experts, and preservation of evidence for future reference, ensuring that no overlooked

critical details are

(Clemente, La Mattera, Guglielmi, 2017; Decker et al., 2019; Elifritz, Nolte, Hattch, Adolphi, Gerranrd, 2014; O’Donnell, Woodford,

2008)

Tegola,

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This chapter aims to provide essential information on postmortem imaging techniques and overview the existing literature on medical imaging from a forensic viewpoint. This chapter is going to assess the definition, purpose, and importance of postmortem imaging, its applications, and techniques and compare various imaging methods that enhance forensic

investigations in detail.

2. Definition, Purpose, and Importance of Postmortem

Imaging

Postmortem imaging refers to the use of radiological techniques, such as ultrasonography (US), DR, CT and MRI, to examine a body after death. These technologies allow for detailed tissue analysis without the need for dissection (Clemente et al, 2017; Decker et al., 2019, Ellifritz et al., 2014; ODonnell, Woodford, 2008).

The primary purpose_ of postmortem imaging is to provide a noninvasive, accurate, and_ rapid assessment of the internal structures of the body to assist in determining the cause, origin, and mechanism of death. Unlike traditional autopsies, which incisions and

require physical

manipulation of the body, postmortem imaging allows medical examiners to view internal structures without intervention. Moreover, it can reveal evidence that

critical might be

overlooked during conventional autopsy. Beyond forensic applications, postmortem imaging serves as a valuable tool in medical research, offering insights into disease progression, congenital abnormalities, and undiagnosed or underlying medical conditions that may have contributed to the individual’s death (Clemente et al, 2017; Decker et al., 2019, Ellfritz et al.,

2014; ODonnell, Woodford, 2008).

The importance of postmortem

imaging lies in its ability to revolutionize

the way forensic and_ medical professionals approach death investigations and pathology.

Postmortem imaging preserves the integrity of the body, making it particularly valuable in situations where the nature of a case limits the use of standard dissection, such as disintegrated corpses like decomposed or burned bodies. Most postmortem

imaging technologies help improve

diagnostic accuracy by providing precise, high-resolution, three- dimensional, cross-sectional, and

reconstructed body images, offering detailed death

investigation without the need for

insight into the

invasive procedures. In addition to

forensic applications, | postmortem imaging plays an essential role in medical research and education. This enables the examination of disease progression in ways that may not have been detectable during life. It will contribute to the advancement of medical knowledge and the development of new diagnostic and treatment strategies. Furthermore, postmortem imaging enhances disaster victim identification efforts by enabling rapid, noninvasive assessment of bodies during mass casualty events, where rapid and efficient identification is paramount. Its ability to store and archive digital data for future analysis also ensures that cases can be reviewed or reanalyzed long after the _ initial examination between antemortem and postmortem data. Considering that a healthy autopsy can only be performed once after death, postmortem imaging is crucial for the re-evaluation of cases. Overall, is a

postmortem imaging

crucial tool that bridges forensic science, clinical practice, and medical

education, offering greater accuracy

and efficiency (Clemente et al., 2017; Decker et al., 2019, Elifritz et al., 2014; O‘Donnell, Woodford, 2008).

3. Applications of

Postmortem Imaging

Postmortem imaging is

particularly useful in various cases where traditional autopsy methods are limited, impractical, or in need of enhancement. Applications of

postmortem imaging in some _ key

scenarios are especially useful, such as

the detection of foreign bodies, determination of age, victim identification in mass _ disasters,

examination of disintegrated corpses bodies), deaths,

(decomposed or burnt

pediatric and neonatal asphyxiation cases, violent deaths,

homicides and abuse, accidental deaths, clinical research, and missed

diagnoses.

Detection of Foreign Bodies: Postmortem imaging plays a crucial role in detecting foreign bodies within a deceased individual. It offers a noninvasive method to identify the anatomic location, shape, and number of objects that may have contributed to or resulted in the death. Technologies

that, in particular, allow X-ray imaging,

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such as DR and CT, are particularly

effective for visualizing foreign materials or medical devices that may

be embedded in the body.

In forensic cases involving gunshot wounds, postmortem imaging can clearly identify the trajectory of a bullet, its exact location, and any internal damage caused along its path, providing critical evidence without the need for an autopsy. Similarly, in cases explosions,

involving stabbings or

postmortem imaging can _ reveal fragments of knives or debris, allowing forensic experts to reconstruct the event and analyze the impact of these objects on the body. This is particularly important when dealing with fragmented foreign bodies that may be dispersed throughout various tissues or organs (Magnin,Grabberr, Michaud., 2020; Wozniak, Moskata, Rzepecka-

Wozniak., 2015).

Postmortem imaging is also beneficial for identifying medical devices like pacemakers, implants, and surgical instruments left inside the body, helping to differentiate accidental causes from intentional harm (De

Angelis et al., 2020).

In the

smuggling cases, particularly when

investigation _ of

identifying the presence of _ illicit materials or contraband hidden in a body. Postmortem imaging technologies are

deceased individual’s highly effective in detecting foreign objects that may have been ingested, inserted, or surgically implanted during smuggling operations. These objects range from drug packets, capsules, and balloons to high-value items like precious stones, currency, and electronics. Postmortem imaging can identify the exact

condition (ruptured or not), and

number, _ size,

location of packages inside the gastrointestinal tract, body cavities, or Surgically created (Abedzadeh, Iqbal, Bastaki, Pierre- Jerome, 2019; Hamid, Rashid, Saini,

2012).

compartments

In cases of choking, a blockage in the air passages, often due to food or foreign material, leads to asphyxiation mechanisms _ like

or fatal events

asphyxia or stimulation of the autonomic nerve plexus. Advanced postmortem imaging techniques, such as CT and MRI are commonly used to detect obstructions in the trachea,

bronchi, and upper

airways. These modalities can visualize the size, location, and type of the obstructing material, offering valuable insight into the case and helping to detect secondary signs of asphyxia, such as pulmonary edema and aspiration, which can further support the diagnosis. These imaging findings complement traditional autopsy techniques, aiding in confirming airway obstruction as the primary cause of death and information for legal and medical ODonnell, 2010; Oesterhelweg, Bolliger, Thali, Ross,

2009).

providing —_ essential

inquiries (Zino,

Another significant benefit of postmortem imaging in cases of foreign bodies is its ability to preserve digital records for further examination and consultation with experts in forensic medicine, law enforcement, and customs. These images can be used as evidence in court, providing a clear, objective visualization of the items and

their impact on the deceased.

Determination of Age: Medical imaging is a powerful tool in the determination of age, both antemortem and postmortem, and it provides

forensic experts with noninvasive

methods to estimate age based on skeletal and dental analysis. Using X- ray imaging techniques, such as DR and CT, the development and degeneration of bones, dentition, closure of sutures, ossification center development, epiphyseal plate maturation, and the condition of articular surfaces can be assessed to estimate an individual’s age

(Barszcz, Wozniak, 2024)

The degree of bone growth and tooth development are key indicators of age. For example, the presence or absence of certain’ primary § and permanent teeth, the stage of the tooth

wear, and the apposition of secondary

eruption, root development, dentin can help estimate the age from dental radiographs (Panchbhai, 2011; Limdiwala, Shah, 2013; Chulamanee, Panyarak, 2023)

Additionally, epiphyseal plate maturation in long bones is a reliable marker. As the body matures, these plates fuse, and the timing of this fusion can be tracked using imaging to

estimate age. (Khatam-Lashgari,

Harying, Villa, Lynnerup, Larsen, 2024;

Lopatin, Barszcz, Wozniak, 2023)

In adults, age-related

degenerative changes in_ bones,

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particularly in the spine and joints, and the condition of articular surfaces can help forensic experts estimate age (Adams, Butler, Fuehr, Olivares-Pérez, Tamayo et al., 20274)

Changes in the symphysis pubis and sternal rib ends are also commonly used indicators of age. The symphysis pubis undergoes predictable morphological changes throughout life, which can be visualized through imaging and correlate with established age ranges. Similarly, the ribs exhibit progressive ossification, and_ their degree of calcification and changes in shape over time can serve as additional markers for age determination (Chiba et al., 2014; Sodhi et al., 2016; Monum,

2019)

Medical images also allow for the preservation of digital records, facilitating further consultation or comparison with pre-existing medical images. The noninvasive nature of medical imaging also respects the integrity of the body while providing detailed

necessary for forensic age estimation,

anatomical information

making it an essential tool in modern

forensic practice.

Victim Identification in Mass Disasters: Postmortem imaging is an indispensable tool in mass disaster scenarios, where rapid, efficient, and accurate identification of victims is essential for humanitarian and legal purposes. In the aftermath of large- scale disasters such as earthquakes, floodings, tsunamis, plane crashes, terrorist attacks, or industrial accidents, the condition of victims’ bodies may be severely compromised due to trauma, burns, or decomposition, making traditional visual identification methods unreliable or impossible. Advanced imaging techniques, like CT and MRI, allow forensic teams to examine bodies, even under extreme conditions, non- invasively. In mass __ disasters, postmortem imaging offers the ability to scan multiple bodies quickly, preserving crucial anatomical details such as dental records, bone structure, and the presence of medical devices like pacemakers, prosthetics, and implants (De Angelis et al. 2020; Argo et al.,

2020; Jackowski, Thali, 2009).

Dental images like DR, CT and cone beam computed tomography (CBCT) play a critical role in disaster victim identification due to tooth

durability. Detailed images of the teeth,

jaws, and palatal rugae reveal the individual dental work, such as fillings, crowns, or root canals, and provide a reliable match with antemortem dental records,

making dental images a

cornerstone of postmortem identification. (De Angelis et. al. 2020; Jackowski, Thali, 2009; Viner, Robson;

2017; Forrest, 2019)

Scans of sinus _ cavities, particularly DR and CT, are frequently used in forensic identification because their shape, dimensions, and patterns vary from person to person, allowing for comparison with pre-existing medical records to confirm identity (De Angelis et al., 2020; Ruder, 2012;

Deloire, 2019).

In patients who have undergone orthopedic surgeries or medical implants, postmortem imaging can reveal the presence of medical devices implanted in the body, such as pacemakers, stents, joint replacements, plates, screws, pins, or other prosthetic devices. These medical devices often have serial numbers or other identifying features that can be traced back to medical records,

providing another method for

identifying individuals, especially in

cases in which decomposition has progressed to the point where other

identification methods are less

effective. (De Angelis e. al., 2020;

Jackowski, Thali, 2009; Sidler, Jackowski, Dirnhofer, Vock, Thali., 2007; Numata, Makinae, Yoshida,

Daimon, Murakami., 2017)

Beyond identification, assists in injury forensic

postmortem imaging trauma and

which

documenting patterns, helps reconstruct the of death. This is

particularly important when the cause

pathologists

circumstances

of a disaster is under investigation, such as airplane crashes, terrorist attacks, or industrial accidents (Kahana, Ravioli, Urroz, Hiss., 1997).

The ability to quickly process and examine multiple bodies using imaging reduces the need for immediate invasive autopsies, which can be time-consuming and stressful in mass casualty situations. Furthermore, digital data captured through imaging can be stored and reviewed later, which is particularly valuable in large-scale investigations that may take months or years to complete. The digital nature of

postmortem imaging also enables

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forensic experts to store and share findings with other professionals for second opinions or further analysis, thereby enhancing the overall efficiency of the identification process. Overall, postmortem imaging accelerates victim identification in mass disaster situations and preserves critical forensic evidence, ensuring the investigation is thorough

and accurate for legal and humanitarian

purposes. Examination of Disintegrated Corpses

(Decomposed or Burnt Bodies): Traditional autopsies can be challenging when’ bodies are in advanced stages of decomposition or have been subjected to fire. In such cases, x-ray imaging techniques such as DR and CT can reveal bone fractures, dental conditions, medical devices, or the presence of foreign objects that aid

in identification and evidence collection.

In cases of advanced decomposition, in which the external

tissues have deteriorated to the point of

obscuring anatomical landmarks, postmortem imaging can _ reveal distinguishing anatomical characteristics, such as _ healed

fractures, surgical implants, and dental

condition, all of which can be used to match the deceased to antemortem records. Imaging allows forensic experts to visualize bones, remaining soft tissues, and internal organs, which may offer clues to the cause of death or help identify the

instance, CT scans can clearly show

individual. For skeletal injuries, fractures, or the presence of foreign objects, even when the body is extensively decomposed. Imaging can also visualize gas accumulation and other postmortem changes in tissues and organs, which is common during decomposition, and identify patterns of fluid distribution or blood pooling. This is also crucial for determining the postmortem interval and enables experts to gather critical evidence despite the severe state of decomposition (Cartocci et al., 2019; Levy, Harcke, Mallak, 2010; Hussein et al., 2022; Wagensveld et al., 2017; klein et al., 2015, Shen et al., 2024; De- Giorgio et al., 2021; Wang, Zheng, Zhang, Ni, Zhang, 2017)

In the case of burnt bodies,

postmortem imaging is_ invaluable because of its ability to penetrate charred and damaged tissues, which are often fragile and difficult to handle

during traditional autopsy. Burn injuries

typically destroy the soft tissues and outer layers of the body; however, postmortem imaging can still capture detailed images of bones, remaining internal structures, and any foreign materials. Postmortem imaging is essential to exclude the cause of death in burned corpses, especially when foul play is suspected. In severely burned corpses, determining the cause of death can be particularly challenging because of the extensive damage inflicted on both the external and internal structures of the body. CT scans are particularly effective in such cases, as they can detect and differentiate between thermal and traumatic fractures, assess the extent of damage to the skeletal system, and assess the state of internal organs and other critical structures that might still be preserved despite the burns. Imaging can help detect signs of pre- existing medical conditions, such as heart disease or stroke, that may have death

Additionally, it can reveal other key

caused before the fire. indicators, such as the presence of fire products in the airways, which helps determine whether the individual was alive when the fire started or if the

individual was already deceased

(Aydogdau et al., 2021; Coty et al, 2018; De Bakker, Roelandt, Soerdjbalie-Maikoe, Van Rijn, De Bakker, 2019).

In cases of dismemberment, images of dismembered limbs and organs enable forensic experts to analyze the methods and tools used for dismemberment. Imaging can reveal the characteristics of the cuts or breaks in bones, such as whether they were made with a saw, knife, or another sharp object, which can _ help investigators link the crime to a specific weapon or perpetrator. Postmortem imaging is particularly useful when multiple body parts are discovered at different times or locations because it enables forensic teams to compare anatomical structures and confirm that the parts belong to the same individual (Matzen, Ondruschka, Fitzek, Plischel,

Well, 2022; Maiese et al., 2020)

Overall, in cases involving

decomposed or _ burned _ bodies, postmortem imaging is a critical tool that provides comprehensive insights into the cause of death, aids in identification and preserves valuable forensic evidence that might otherwise

be lost. Imaging also preserves digital

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records of body parts for future reference, ensuring a comprehensive

forensic investigation.

Examinations of Pediatric and Neonatal Deaths: Postmortem imaging is particularly beneficial in cases involving pediatric or neonatal deaths, where the size and fragility of the body make traditional autopsy challenging. Imaging technologies such as DR, CT, MRI, ultrasonography (US), and even mammography due to its magnification feature, allow forensic specialists to examine the body in detail without the need for dissection.

Postmortem imaging is highly effective for detecting congenital and developmental anomalies or undiagnosed medical conditions that may have contributed to death in neonates and young _ children. Postmortem DR provides information about the

regarding skeletal dysplasia diagnosis.

Skeleton, _ particularly Postmortem MRI is especially useful for assessing soft tissues, providing a clearer view of potential causes, such as malformations, congenital anomalies, hemorrhages, and infections (Ashby et al., 2022; Arthurs, Van Rijn, Taylor, Sebire., 2015; Gould

et al, 2019; Thayyil, Robertson, Sebire, Taylor, 2010)

In cases of sudden infant death syndrome (SIDS), postmortem imaging can exclude the cause of death and provide critical insights into potential explanations, for instance, subtle abnormalities in the airways, lungs, or heart that might indicate suffocation, infection, a congenital issue, or ruling out traumatic injuries or signs of abuse

(Van Goethem et al., 2024).

In suspected cases of child abuse and shaken baby syndrome (SBS), both postmortem CT and MRI are particularly

antemortem and

useful for detecting internal injuries like organ damage, hemorrhages and other traumatic injuries (McGraw, Pless, Pennington, White, 2022; Cartocci et al., 2021)

Postmortem imaging also allows for repeated analysis and the opportunity for pediatry, radiology, and pathology specialists to collaborate on a case, providing a _ multidisciplinary approach to understanding the cause of death. Overall, postmortem imaging in pediatrics and neonates provides enhanced information to clarify the

cause of death, making it an essential

tool in modern pediatric forensic

investigations.

Examinations of Asphyxiation Cases: Postmortem imaging plays a crucial role in the forensic investigation of asphyxiation cases, such as_ strangulation and drowning, by providing noninvasive insights into the internal and external injuries associated with these types of

death.

In cases of strangulation, CT and MRI

examining the

scans are valuable for throat

anatomy. CT and MRI scans can

neck and

provide detailed images of internal hemorages in the neck muscles, arteries, and veins, which are often key indicators of manual or ligature strangulation. CT scans, in particular, can provide bone and cartilage damage in the neck and throat, allowing forensic experts to assess damage to the larynx, trachea, and hyoid bone, which may fractured Additionally, 3D

reconstruction of CT images can help

have been during

strangulation.

detect external injuries, such as ligature marks around the neck. By identifying these _ internal

signs of trauma,

postmortem imaging can corroborate

evidence of strangulation, even in cases in which external bruising or marks are minimal (Gascho, Heimer, Tappero, Schaerli., 2019; Deininger-Czermak, Heimer, Tappero, Thali, Gascho, 2020; Nagai et al., 2024; Yen et al., 2005)

In cases of drowning, CT scans

reveal key physiological changes associated with water inhalation. CT imaging of the lungs can reveal fluid in trachea, bronchi, pleural effusion, pulmonary emphysema, frothy fluid or debris in the lungs, as well as gastric and duodenal dilatation, fluid in the sinuses and _ pericardial effusion— findings that are characteristic of drowning. It can also help distinguish drowning from other causes of death by analyzing fluid accumulation in the sinuses, airway obstructions, and water in the stomach due to drowning (Van Hoyweghen, Jacobs, Op de Beeck, Parizel, 2015; Ogawara, Usui, Homma, Funayama, 2022; Wang et al., 2020,

Plaetsen et al., 2015)

By providing a_ noninvasive, detailed examination of the body's internal structures, postmortem imaging enhances the ability of forensic experts to detect subtle signs of

asphyxiation, ultimately contributing to

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more accurate cause-of-death determinations in cases of strangulation

and drowning.

Examinations of Violent Deaths, Homicides, and Abuse: In cases of gunshot, stab, or blunt force trauma, imaging can identify internal

injuries without the need for dissection.

In firearm and gunshot injuries, postmortem imaging offers a detailed, noninvasive method to examine and document the number and_ exact anatomic location of the bullets, bullet fragments, or shrapnel. Imaging is especially useful for determining the trajectory of bullets and the extent of tissue damage (Van Kan, Haest, Lahaye, Hofman, 2017; Usui et al., 2016)

CT scans are particularly valuable in firearm and gunshot injuries due to their ability to produce high- resolution, high-contrast, three- dimensional, cross-sectional images, allowing forensic experts to trace the trajectory of bullets, bullet fragments, or shrapnel and assess the injuries caused along their trajectory. CT imaging can also detect and localize small metallic fragments, which are

often dispersed within the body after a

gunshot, providing crucial evidence in of the shooting, determining the angle of the

reconstructing the events

missile trajectory, and even information

about shooting distance and determining the caliber of the bullet based on the size and shape of fragments left in the body (Oehmichen et al., 2003; Junno, Kotiaho, Oura, 2022; Alves et al., 2020, Wozniak,

Moskata, Rzepecka-Wozniak, 2015).

In puncturing, penetrating, and

perforating injuries, postmortem imaging provides information about the depth, trajectory, and impact of these internal

injuries on organs and

structures. For perforating injuries where the object fully penetrates the body, postmortem imaging provides critical insights into both entry and exit wounds and internal damage caused along the trajectory (A/, Mourtzinos, 2022; Schnider et al., 2009; WoZniak et

al, 2015).

In the investigation of abuse cases, DR, CT, and MRI can reveal different aspects of injuries. For example, DR and CT scans can clearly show fractures, especially those in delicate areas, such as the skull, ribs,

and long bones, which are often

associated with physical abuse. Additionally, these imaging modalities can identify patterns of bone healing, such as multiple fractures occurring at different stages of healing, which are indicative of repetitive trauma and a key marker of chronic abuse (Wozniak et al., 2015; Van Wijk, Vester, Arthurs, Van Rijn, 2017). MRI is particularly useful for detecting soft tissue injuries, including internal hemorrhage and organ damage, which may result from shaking, blunt force trauma, or other forms of abuse (Hart, Dudley, Zumwalt,

1996).

The digital

postmortem imaging allows for detailed

nature of

documentation and preservation of evidence, repeated examinations, and

consultations with forensic experts,

ensuring thorough and_ accurate analysis of evidence. Overall, postmortem imaging is crucial for

understanding the full extent of violent

deaths, homicides, and abuse, enhancing forensic investigations by providing precise, detailed, and noninvasive visualizations of internal

damage.

Examination of Accidental Deaths: Motor vehicle accidents, falls traumatic

from height, and _ other

incidents often result in complex

injuries. Postmortem imaging can capture the extent and nature of trauma, fractures, dislocations, internal hemorrhage, and organ damage, enabling investigators to reconstruct the sequence of events leading to fatal injuries (Obertova et al, 2019; Wijetunga et al., 2020; Jalalzadeh et

al., 2015).

Postmortem imaging may provide information for understanding the biomechanics of injury. For instance, analyzing the forces exerted on the body during a crash or fall can help forensic experts determine factors like the direction and intensity of the impact (Fukuda et al., 2024). Another example is the distribution and severity of injuries, which can _ provide information to determine whether the victim was a driver, passenger, or pedestrian and whether the injuries were consistent with the vehicle's speed, point of impact, or position of the body at the time of the crash (Breen, Naess, Gaarder, Stray- Pedersen, 2021; Moskata, WoZniak,

kluza, Romaszko, Lopatin, 2017).

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Additionally, injuries like whiplash or dashboard injuries help forensic experts reconstruct the accident and provide insights into how the collision occurred and the likelihood of survival based on injury patterns (Johansson, 2006; Van Goethem, Biltjes, Van Den Hauwe, Parizel, De Schepper, 1996; Aiker et al., 1975).

In legal contexts, postmortem imaging is especially useful when there are conflicting eyewitness accounts or when a detailed biomechanical analysis is required to understand the accident’s cause. Furthermore, postmortem imaging helps identify additional factors that might have contributed to the accident, such as pre-existing medical conditions, which can be detected through scans and linked to sudden incapacitation, such as heart attacks or Additionally, imaging enhances the accuracy and depth of

providing clear, comprehensive insights

strokes. postmortem

forensic investigations,

that help reconstruct the accident, identify

support the legal processes.

contributing factors, and

Examination of Clinical Research and Missed Diagnoses:

Postmortem imaging is a noninvasive

method for _ investigating and understanding medical conditions that may have been overlooked’ or misinterpreted in cases in which missed diagnoses are suspected or in which further clinical research is essential. In cases in which the patient’s symptoms were not fully understood or properly diagnosed, postmortem imaging allows for retrospective investigation and provides crucial insights into conditions that may have gone undetected during clinical care (Sonnemans, Kubat, Prokop, klein, 2018; Inai et al., 2016; Roberts et al., 2012).

These imaging studies can help identify the missed condition and its better

understanding of disease progression

evolution, providing a and patient outcomes (Ko/lasinski et al., 2012).

Postmortem imaging is

essential for improving diagnostic accuracy for future patients because researchers and clinicians can learn from these postmortem findings to refine clinical practices and enhance early detection strategies. Postmortem imaging is crucial for quality assurance and medical education because it allows healthcare institutions to investigate

potential diagnostic errors. It also

enables clinicians and pathologists to review complex cases in which the cause of death is unclear, thereby facilitating a more detailed analysis of medical

treatment efficacy and

decision-making. Additionally, digital records produced by postmortem imaging can be stored and shared for further

research or educational

purposes, thereby contributing to spreading knowledge to improve patient care and _ prevent similar diagnostic errors in the future. Overall, postmortem imaging plays a pivotal role in clinical research by helping uncover missed diagnoses, enhancing medical knowledge, and driving advancements

in diagnostic and treatment strategies.

3. Postmortem Imaging

Modalities Postmortem imaging encompasses various __ radiological

modalities, such as ultrasonography,

digital radiography, fluoroscopy, angiography, computed tomography, and magnetic resonance imaging, that provide different types of information Each

modality has strengths and applications

about the body in question.

in forensic analysis, which are briefly

described below.

Postmortem Ultrasonography (PM-US):

Postmortem ultrasonography is a modality that uses sound waves to examine the internal structures of the body after death.

supplementary diagnostic technique to

It serves aS a

the traditional autopsy, allowing for the visualization of organs, tissues, and any potential pathological changes. This method is particularly useful in forensic investigations to determine the cause of death, identify trauma or hemorrhage, and detect signs of disease. It also has applications in pediatric and perinatal cases (Shelmerdine, Sebire, Arthurs, 2071; Shelmerdine, Sebire, Arthurs, 2019 (a); Shelmerdine, Sebire, Arthurs, 2019 (b)). PM-US can be considered a high-risk postmortem procedures, particularly in

safer alternative to

case of infectious diseases, where minimizing exposure risks is Shrestha, Krishan, 2021). PM-US can detect

pathological

paramount. (Kanchan,

findings like cardiac

hypertrophy, pericardial tamponade,

aneurysm of the abdominal aorta, pleural effusions, subphrenic abscess, ascites or intra-abdominal bleeding, liver metastasis and cirrhosis, fatty

change of liver, parenchyma, bile

99

100

stones, renal cysts, diverticulum of the urinary bladder, prostate hyperplasia, myoma uteri, intracranial hemorrhage in infants, bone fractures, and implants in breasts (Uchigasaki, 2006).

Postmortem Digital Radiography (PM-DR): PM-DR is one of the x-ray imaging modalities in postmortem imaging and is particularly valuable for visualizing bones, dental structures, foreign objects like bullets, shrapnel, smuggling packages, and

medical devices.

PM-DR

CaSes

is widely used in

forensic involving trauma, dismemberment, skeletal fractures, or joint dislocations (Hughes-Roberts,

Arthurs, Moss, Set, 2012)

PM-DR is also beneficial in perinatal cases, as it aids in estimating gestational age and intrauterine growth by detecting ossification centers and measuring the length of long bone shafts (Fuente, Dornseiffen, Noort, Laurini, 1988; Shelmerdine, Arthurs, 2023; Shelmerdine, Sebire, Arthurs, 2021)

DR is especially useful for identifying victims by crosschecking antemortem and postmortem dental

records (Viner, Robson, 2017; Heinrich,

Guittler, Schenkl, Wagner, Teichgraber, 2020).

Although PM-DR lacks detailed soft-tissue visualization and superposition problems due to its 2D nature, it remains an essential tool for fast and efficient skeletal analysis and detection of foreign objects in forensic

investigations.

Postmortem Fluoroscopy (PM-F): PM-F is a dynamic imaging technique that provides real-time x-ray images. PM-F is useful for whole-body scanning to evaluate skeletal structures, foreign bodies, and medical devices like in PM-DR. Although PM-F has limited soft tissue resolution, its ability to provide continuous dynamic

imaging makes it a unique and

important technique for certain postmortem examinations. For example, real-time visualization

capability makes fluoroscopy a valuable tool for guiding procedures, such as locating foreign objects like bullets, projectiles, or shrapnel, in the body and assisting in the retrieval of these items

without extensive dissection. Postmortem Angiography (PM-A); PM-A is a

fluoroscopy or computed tomography

specialized

technique in which contrast agents are injected into the vascular system to visualize blood vessels and provide detailed

system in deceased individuals. This

images of the circulatory

technique is particularly useful in cases of suspected vascular abnormalities or trauma, such as aneurysms, vascular blockages, hemorrhage, varices, and traumatic injuries. Additionally, it is critical for detecting small ruptures or microbleedings that may not be visible with conventional autopsy methods. By providing a clear visualization of the vascular system, PM-A helps forensic experts understand the cause and mechanism of death in cases in which vascular trauma or disease play a significant role (Grabherr, Djonov, Yen, Thali, Dirnhofer, 2007; Grabherr et al., 2018, Ross et al., 2014; Franckenberg, Flach, Gascho, Thali, Ross, 2015).

Postmortem Tomography (PM-CT): PM-CT is one

of the most effective X-ray imaging

Computed

techniques, offering high-resolution,

high-contrast, three-dimensional, cross-sectional, and reconstructed

images of the body.

PM-CT scans are _ particularly

valuable for viewing _ fractures,

hemorrhages, internal damage, and the presence and trajectory of foreign objects (Adelman et al., 2018; Willaume et al, 2018; Rutty, 2020; Burton, kitsanta, 2020). Additionally, PM-CT is essential in mass disaster situations where multiple bodies need to be quickly scanned for victim identification (Brough, Morgan, Rutty, 2015; Sidler, Jackowski, Vock, Thali, 2007). PM-CT scan is also crucial in

pediatric and neonatal cases because it

Dirnhofer,

can detect subtle injuries or congenital abnormalities (Gould et al, 2019; Thayyil, Robertson, Sebire, Taylor, 2010). PM-CT can be used as a replacement to invasive autopsy practice in the investigation of high-risk autopsies, such as in cases of infectious diseases (Roberts, Traill, 2021; De- Giorgio et al., 2021; Filograna et al.,

2022),

The digital nature of CT images allows the post-processing, reconstruction, and measurement of different parameters on the target image, such as distance, area, volume, and density. It also allows for easy and long-term storage, enabling ongoing analysis and collaboration with other forensic experts, making it one of the reliable

most comprehensive and

101

102

imaging techniques for postmortem

investigations.

Postmortem Magnetic Resonance Imaging (PM-MRI): MRI is a non-ionizing imaging modality, unlike techniques that use X-rays for

imaging.

MRI is excellent for detecting subtle tissue changes, such as brain hemorrhage, edema, infarcts, tumors or neurodegenerative conditions that may have contributed to death even in putrefied bodies Gerlach, Scheurer, Lenz, 2022; Saito et al., 2024; Ringger, Schwendener, Klaus, Jackowski, Zech, 2023) In

pediatric and neonatal deaths, MRI is

(Bauer, Berger,

invaluable for identifying congenital anomalies, brain malformations, and undetected infections (Arthurs et al, 2015; Pérez-Serrano et al., 2021; Addison, Arthurs, Thayyil, 2014; Thayyil et al, 2013) In addition, it plays a critical role in forensic investigations involving strangulation, suffocation, and other asphyxiation deaths by visualizing soft tissue injuries in neck and airway structures (Gascho, Heimer, Tappero, Schaerli, 2019; Deininger- Czermak, Heimer, Tappero, Thali,

Gascho 2020; Yen et al., 2005)

MRI provides exceptional soft- tissue contrast, which is particularly useful for examining internal organs. Although MRI is less efficient than CT for skeletal analysis or cases involving foreign objects, it remains an essential tool for investigating deaths where soft

tissue pathology is the primary concern.

Conclusion Postmortem imaging _ has become a_ critical adjunct to

conventional autopsy due to its ability to offer detailed, noninvasive insights into the deceased’s internal anatomy that complement the findings of While

conventional autopsy remains the gold

traditional dissection. standard for determining the cause of death, it can be limited in certain scenarios, such as when disintegrated, decomposed, or burned bodies are involved or pediatric or neonatal deaths occur, where the size and fragility of the body challenging.

make traditional autopsy

Postmortem CT or MRI provide three-dimensional and cross-sectional images of the body, allowing for an in- depth

dislocations,

examination of _ fractures,

tissue damage, organ

abnormalities, or the presence of

foreign objects. This makes postmortem imaging an essential tool in forensic and medical investigations, where data accuracy and precision are paramount. Additionally, postmortem imaging can detect certain subtle pathologies that may be missed or misinterpreted during __ traditional autopsy. For example, microfractures, gas embolisms, or small brain lesions may not be easily visible during manual dissection but can be effectively captured and

analyzed = through

imaging.

Postmortem imaging also allows for the repeated review of the same evidence without further invasive procedures, which is _ especially beneficial in complex cases that require multidisciplinary consultation. Furthermore, when time is critical, such as during mass casualty’ events, imaging is a faster alternative that can quickly assess multiple bodies. Another key benefit of postmortem imaging is

the ability to store digital images for

future reference, which makes it possible to perform retrospective crosschecking. Additionally,

postmortem imaging allows forensic

experts to examine missed diagnoses retrospectively and contributes to a deeper understanding of medical conditions, improving forensic and

clinical outcomes.

Combining postmortem imaging with conventional autopsy not only ensures a more comprehensive investigation but also provides a higher level of accuracy, helping to rule out or confirm certain diagnoses or causes of death that might otherwise remain ambiguous. This integration is particularly crucial in modern forensic practice and precise medical Thus,

imaging is a must-process alongside

examinations. postmortem

conventional autopsy to enhance diagnostic accuracy and to provide a complete understanding of the circumstances surrounding death. As technology continues to advance, the role of postmortem imaging in death investigation and research will only become more significant, providing an essential complement to traditional autopsy methods and advancing the field of

pathology.

forensic medicine and

103

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