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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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)
BZT TURAN
PUBLISHING HOUSE
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)
BZT TURAN
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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20
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,
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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.
White, R.S. A. D. O. (1987). Automation of gunshot residue detection and analysis by scanning electron microscopy/energy dispersive X-ray analysis (SEM/EDX). J Forensic Sci., 32, 1595-1603.
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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Sener, H., Anilanmert, B., Mavis, M. E., Gursu, G. G., & Cengiz, S. (2021). LC-MS/MS monitoring for explosives residues and OGSR with diverse ionization temperatures
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Stamouli, A., Niewdhner, L., Larsson, M., Colson, B., Uhlig, S., Fojtasek, L., Machado, F., & Gunaratnam, L. (2021). Survey of gunshot residue prevalence on the hands of individuals from various population groups in and outside Europe. Forensic Chemistry, 2XOctober 2020), 1-8. https://doi.org/10.1016/j.forc.2021.100308
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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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