Solving the two-decades-old murder case through joint application of ZooMS and ancient DNA approaches.

Bones are one of the most common biological types of evidence in forensic cases. Discriminating human bones from irrelevant species is important for the identification of victims; however, the highly degraded bones could be undiagnostic morphologically and difficult to analyze with standard DNA profiling approaches. The same challenge also exists in archaeological studies. Here, we present an initial study of an analytical strategy that involves zooarchaeology by mass spectrometry (ZooMS) and ancient DNA methods. Through the combined strategy, we managed to identify the only biological evidence of a two-decades-old murder case - a small piece of human bone out of 19 bone fragments - and confirmed the kinship between the victim and the putative parents through joint application of next-generation sequencing (NGS) and Sanger sequencing methods. ZooMS effectively screened out the target human bone while ancient DNA methods improve the DNA yields. The combined strategy in this case outperforms the standard DNA profiling approach with shorter time, less cost, as well as higher reliability for the genetic identification results. HIGHLIGHTS: • The first application of zooarchaeology by mass spectrometry technique in the forensic case for screening out human bones from bone fragment mixtures. • Application of ancient DNA technique to recover the highly degraded DNA sequence from the challenging sample that failed standard DNA profiling approaches. • A fast, sensitive, and low-cost strategy that combines the strengths of protein analysis and DNA analysis for kinship identification in forensic research.

Whose blood is it? Application of DEPArray™ technology for the identification of individual/s who contributed blood to a mixed stain.

The interpretation and statistical evaluation of mixed DNA profiles often presents a particular challenge in forensic DNA investigations. Only in specific combinations can single cellular components of a mixture be assigned to one contributor. In this study, the DEPArray™ technology, which enables image-assisted immunofluorescent-sorting of rare single cells using dielectrophoretic (DEP) forces, was applied together with different preliminary tests to identify the individual/s who contributed blood to a given mixture. The technique was successfully applied in two routine casework samples. In order to ascertain how old a stain can be and still be processed successfully, white blood cells from two 10- and one 27-year-old stains were investigated. Depending on the stain's age, the associated DNA degradation level and the number of target cells successfully isolated, the final profile reflects a compromise between the gain of information due to isolation of pure cells of a specific cell type from a single contributor and the loss of discriminatory power due to incomplete profiles caused by DNA degradation.

Envisioning Justice: The Complex Journey of Cold Case Homicide Survivors.

While police make an arrest in the majority of homicide cases occurring annually in the United States, a portion remain unsolved and are eventually classified as "cold cases." Family members of the victims are not only left grieving the loss of their loved ones, but also plagued by the knowledge that the murderer has yet to be officially identified or held accountable. How do these family members-cold case homicide survivors-navigate their open-ended journey through grief? Using a social constructivist approach, we analyze in-depth interviews with 24 cold case homicide survivors to describe the unique dimensions of their experience, including how their hopes are tied to understandings of achieving justice for their loved ones. Three themes emerge from their narratives: a certainty that the killers will be identified; a demand for the harshest punishment possible; and an underlying anxiety about what the identification of the offender will ultimately mean for them. We consider the implications of survivors' expectations for the future, especially for their relationships with the police, other family members, and the criminal justice process in general.

The Cause of Death of a Child in the 18th Century Solved by Bone Microbiome Typing Using Laser Microdissection and Next Generation Sequencing.

The history of medicine abounds in cases of mysterious deaths, especially by infectious diseases, which were probably unresolved because of the lack of knowledge and of appropriate technology. The aim of this study was to exploit contemporary technologies to try to identify the cause of death of a young boy who died from a putative "infection" at the end of the 18th century, and for whom an extraordinarily well-preserved minute bone fragment was available. After confirming the nature of the sample, we used laser microdissection to select the most "informative" area to be examined. Tissue genotyping indicated male gender, thereby confirming the notary's report. 16S ribosomal RNA sequencing showed that Proteobacteria and Actinobacteria were more abundant than Firmicutes and Bacteroidetes, and that Pseudomonas was the most abundant bacterial genus in the Pseudomonadaceae family. These data suggest that the patient most likely died from Pseudomonas osteomyelitis. This case is an example of how new technological approaches, like laser microdissection and next-generation sequencing, can resolve ancient cases of uncertain etiopathology. Lastly, medical samples may contain a wealth of information that may not be accessible until more sophisticated technology becomes available. Therefore, one may envisage the possibility of systematically storing medical samples for evaluation by future generations.

Ground penetrating radar and electrical resistivity tomography surveys with a subsequent intrusive investigation in search for the missing Beaumont children in Adelaide, South Australia.

Jane, Arnna, and Grant Beaumont went missing from Glenelg Beach in Adelaide, South Australia on 26 January (Australia Day) 1966. Despite multiple land and sea searches over nearly 60 years, the children have not been found. New credible eyewitness testimony led to a site of interest at the now disused New Castalloy factory in North Plympton, Adelaide. This site has a complex stratigraphy of anthropogenic fill, which made ground penetrating radar (GPR) investigations unpromising. Electrical resistivity tomography (ERT), while not commonly used in a forensic capacity, provided an alternative approach that allowed suitable depth penetration to resolve a feature of interest, which was subsequently excavated by the South Australia Police. This feature did contain organic, and animal remains but, sadly, not the grave of Jane, Arnna, and Grant Beaumont. However, this investigation highlights the potential to use ERT in a forensic capacity, as well as the limitations of using geophysical techniques for covert burial detection.

A controlled method for the identification of forensic traces from clandestine grave fill.

Deceased human remains are often buried as a forensic countermeasure or method of disposal by homicide perpetrators. Owing to this, the excavation of clandestine grave sites is a task that forensic crime scene teams may only encounter a few times a year. Not all crime scene units have specialised teams for this task, and even those that do, may not have specific protocols for the optimal recovery of forensic traces retained within grave fill as procedures such as sieving require optimisation for the specific soil conditions of the jurisdiction. This study aimed to define the optimal sieving conditions for a sandy environment when searching for minute traces of paint, glass, hair and fibres. Furthermore, this study justifies the practice of retaining grave fill and examining it under controlled laboratory conditions, rather than in-situ adjacent to the grave site. The results demonstrate that using sieve mesh sizes as fine as 0.1 mm can recover up to 82% of the deposited traces and almost all paint, hair and glass traces. The processing of grave fill in the laboratory lead to increased yield of forensic evidence, which on a case-basis may warrant the increased time needed. These findings merit consideration for clandestine grave crime scenes where evidence is scarce or the case is likely to become cold.

Joining forces: Combining police and external expertise for cold case reviews.

Cold case reviews within police and law enforcement agencies are challenging, not the least owing to the amount of time required to carefully review documentation, forensic exhibit holdings and various other casefile information. Most federal and state agencies are time poor, meaning there are very few dedicated cold case teams fortunate enough to have an abundance of police and expert staff resources. Universities and education organisations, however, have large troves of various expertise, alongside expansive human resources, by way of their academic and student body. In certain circumstances, the academic expertise and course offerings of a university may be well suited to assisting law enforcement in reviewing cold cases. There is growing desire for university courses to generate job ready graduates. In the field of law enforcement and policing this is difficult, as safety and the security of sensitive material and evidence is paramount. Educators strive to create workplace simulations, and with the correct mix of academic expertise, course offerings and industry linkages, the emerging opportunity for real cold case collaboration is possible. One such example is the Cold Case Review @ Murdoch (CCR) initiative. Since 2020, CCR has worked with the Lower Saxony Police Academy in Germany to develop the novel International Cold Case Analysis Project (ICCAP), now incorporating over 25 member institutions, to assist in solving real cases from both Niedersachsen (Lower Saxony) and federal police jurisdictions. One case, known as "The North Sea Man" has shown great success and demonstrates the power of joining forces between law enforcement and external agencies to help advance cold cases.

An unsolved case in a culturally diverse community in Italy.

The number of foreign citizens resident in Italy is approximately 5 million, making them the 8.7% of the resident population of the country. However, over the past 20 years, foreign citizens accounted for 24% of the total number of defendants involved in homicides, especially associated with robbery, disputes and assaults, generally connected to prostitution. Investigations of cases in the context of culturally diverse or minority communities can be particularly complex. This is due to the education, the cultural background and the cohesiveness of the community, alongside a negative view of law enforcement. These factors determine a relatively low resolution rate of these violent crimes in these communities. The investigation of the suspicious death of an Indian citizen living in Italy is presented in this paper. The body was discovered in a cattle shed, and despite the initial assumption of an accidental death, the cranial lesions were not consistent with the view that the death was caused by a kick by a cow. The results of a multidisciplinary analysis, including imaging, pathology, toxicology and veterinary forensics, were directed towards a homicide, with the manner of death ultimately filed as undetermined. During the course of the investigation, the collaboration between law enforcement and the community was thwart with difficulty, and as a consequence this case is currently unsolved. In order for law enforcement to continue to play a constructive and supportive role, it is imperative that every country invest in civil education of local population and of any ethnic groups, minorities and culturally diverse people.

Operational psychology and cold case investigations in New Zealand.

Purpose: The purpose of this paper is to examine the application of psychology to the investigation of cold cases. The paper reviews the development of the discipline of operational psychology and reviews the role of the Behavioural Science Unit in assisting with cold case investigations within New Zealand. Design/methodology/approach: The paper integrates theory, academic principles, and practical investigative experience. Findings: As a field, operational psychology has a wide application to cold case investigations. The main areas where expertise can be provided, includes, indirect assessment and personality profiles, offender profiling, crime analysis, victimology and equivocal death analysis, and in the interviewing and engagement of offenders. Operational psychology advice should be based upon sound reasoning, evidenced based conclusion, and within the bounds of practitioner competence. Psychologists should seek to educate investigators on probabilities, likelihoods and error rates, and endeavour to indicate the strength of conclusions and statements provided within a report. Originality/value: The review aims to provide the necessary and relevant impetus for integrating operational, or forensic psychology expertise into cold case investigations. The application of psychological science to police investigations has been subject to numerous academic commentaries, however, there is a dearth of involvement from practitioners practicing in the field.

Finding the missing and unknown: Novel educational approaches to warming up cold cases.

In recent years, students in police academies and higher education institutions around the world have worked together to analyse cold cases including long-term missing persons cases in collaboration with investigators and prosecutors. In 2020, three European organisations, the Police Expert Network on Missing Persons (PEN-MP), AMBER Alert Europe and Locate International, succeeded in connecting these educational organisations enabling them to work collectively on cases and conduct cold case analyses (CCA) across international borders. The International Cold Case Analysis Project (ICCAP) learning objectives were to 1) collect the necessary information about the victim, 2) reconstruct the crime, and 3) investigate trace control. In a learning objective-based evaluation using Computer-Assisted Web Interviewing, 76 participating students from the German and International ICCAP teams were asked to complete a pre- and post-review questionnaire to self-assess their personal competence development. Participants reported significant increases in competence in all evaluated areas, thus demonstrating that authentic and relevant collaborations can enrich the learning environment, promote the use of professional skills, and provide significant knowledge exchange opportunities between academia and industry. Drawing on case studies of cold case missing persons' investigations and unidentified found remains, this article shares how university academics, students and community volunteers can work together nationally and internationally to find out what has happened to missing people and how we can more effectively identify the previously unidentified. In so doing, we share the expertise required to progress these cold cases and provide recommendations to support other institutions and organisations in adopting this innovative approach.

Identification of a decedent in a 103-year-old homicide case using forensic anthropology and genetic genealogy.

Anthropologists are often the custodians of long-term unidentified human remains though their positions as curators of university or museum skeletal collections. Various factors decrease the solvability of these legacy cases including the passage of time, the loss of provenience for specific cases, and lack of documentation or case records. While anthropologists can contribute important information toward identification, it is often necessary to explore novel and cross-disciplinary strategies to resolve difficult cold cases. In long cold cases, the postmortem interval, in particular, may be difficult to estimate leading to further challenges in achieving identification. Modern advances in radiocarbon bomb pulse dating, isotope analysis, and actualistic studies have contributed to positive identification of unidentified human remains in some legacy cases, but may not be available to all forensic practitioners and law enforcement from resource-poor agencies. Pooling resources, as well as collaborating with professionals outside of forensic anthropology, is a useful strategy to pursue when anthropological methods are exhausted.The case study presented here demonstrates a collaborative approach between forensic anthropologists, forensic genetic genealogists, and law enforcement in a century-old homicide. The dismembered and mummified parts of a male body were recovered in a remote cave in 1979 and again in 1991. Despite forensic anthropologists creating and updating the biological profile over the decades from recovery to present, no identification was made until the application of forensic genetic genealogy (FGG) to the case in 2019. New interpretations of bone microstructure and trauma analysis are presented for the case, alongside the historical documentation and "proof of life" evidence used by the genealogy team. A review of the FGG methods underscores the challenges in this case (e.g. significant endogamy, multiple aliases used by the victim) and the steps taken toward resolution. Ultimately, a combined anthropology and genealogy approach resulted in a confirmed identity for a man who was murdered in 1916.Key pointsForensic scientists should leverage a collaborative, interdisciplinary approach toward human identification.When combined with forensic anthropology methods, forensic genetic genealogy is a valuable tool linking biological and cultural-historical aspects of identity.Forensic anthropologists should review challenging cases in their labs as new methods are introduced and new resources become available.

Can a reference 'match' an evidence profile if these have no loci in common?

Cold case reinvestigations are a common occurrence. Occasionally some of the original work was conducted up to 30 years ago using profiling systems of the early 1990s, which targeted HLA-DQA1, ApoB, D1S80 and D17S5. When contemporary work is carried out, if a suspect is identified they will be profiled in contemporary profiling kits such as GlobalFiler. It would be common to then also attempt to profile the evidence profiles in the same contemporary profiling kit. Imagine a scenario where two evidence samples, E1 and E2, had previously produced single-source profiles, but only E2 had any DNA extract left to re-profile with GlobalFiler. At the old loci E1 matched E2, and at the new loci E2 matched the suspect reference. Of interest to the investigation was whether anything could be said about the suspect being a donor of DNA to E1 even though the reference of the suspect and the profile from E1 had no loci in common, by using the information from the profile of E2. This paper explores that possibility.

Exploring Cold Case Homicide Survivors' Perceptions of and Relationships With Law Enforcement.

Given the nature of unsolved homicide, a complicated dynamic exists between survivors of cold case homicides and law enforcement personnel. The current study utilizes interviews with 24 survivors to explore the perceptions associated with this relationship. Employing a grounded theory framework, we find several themes depicted within these narratives that guide survivors' overall perceptions of their relationship with law enforcement, including investigative and communication efforts. Results indicate that the survivors universally perceive original detectives were incompetent or ineffective in their original investigation, thus leading to the unsolved status of their case. Over time, secondary and current investigators were also perceived as unable to properly investigate, due to challenges such as insufficient evidence or simply not conducting a comprehensive examination of the case. Regardless of this negative view of police activity on the case, relationships were defined by the level and nature of communication survivors had with their detective. Families who felt listened to, included, and/or valued reported a positive relationship with police that allowed them to trust their case was in good hands. Participants who perceived communication to be secretive, misleading, or completely absent noted strained relationships and further disappointment. Survivors may adopt roles, such as investigator, spokesperson, or advocate, to help compensate for their perception that the case is not properly cared for by police. With regard to grief progress and health of cold case homicide survivors, it is possible treatment by law enforcement outweighs the significant of case resolution years after the homicide. Recommendations for more productive survivor/police interactions are included.

Enhancing the evidential value of textile fibres Part 2: Application of a database-driven fibre comparison strategy to a cold-case investigation.

This paper describes the involvement of our laboratory in a Western Australian 'cold-case' investigation that spanned 24 years. The investigation was widely considered to be the largest in Australian history. During the investigative phase, our laboratory was tasked with the collection of trace evidence of all types in connection with a sexual assault and two homicides that were suspected to be related. Textile fibres represented the vast majority of trace evidence recovered. A much greater quantity of fibres (>10,800) was collected than would be typical for a routine case, as fibres of any colour or type were potentially of investigative value. The investigation was unprecedented in its scale, and presented numerous challenges in terms of evidence recovery, analysis, interpretation, reporting, and provision of testimony. A textile fibre microspectrophotometric (MSP) database was developed specifically for the interpretation of data in connection with the investigation. The database currently contains over 25,000 normalised and first derivative spectra of casework, validation and reference textile fibres. A fibre comparison strategy was devised, involving the identification of preliminary fibre groups on the basis of corresponding/similar MSP spectra, and verification of these groups via brightfield and fluorescence comparison microscopy. A potential link to an automotive source was identified for one of the homicide victims during the investigative phase. After identification of a suspect, a total of 98 fibres recovered from victims and from a seized motor vehicle were found to correspond in properties to six different fibre types from known textile sources in connection with the suspect. A highly publicised criminal trial was held, and textile fibre evidence provided a major contribution to the trial findings, in which the accused was found guilty of two homicides.

Methods for ensuring the highest DNA concentration and yield in future and retrospective trace DNA extracts.

In forensic laboratories, increased extraction efficiency of trace evidence is paramount because analytical success is intrinsically dependent on the quantity of DNA recovered. Moreover, highly concentrated nucleic acids are vital for effective downstream analysis and high quality results. This study investigated the efficiency of extraction with the Qiagen® QIAamp® DNA Investigator kit, and explored improvements to the methodology that would maximise the recovery of low concentration forensic samples. Controlled amounts of starting cellular material were used to mimic trace (or low level) DNA deposits prior to DNA extraction with the Investigator kit. Addition of the provided carrier RNA along with conducting two successive elutions of 50 µL improved the net recovery of DNA to 95%. Concentration with centrifugal filters post-extraction were able to concentrate DNA but a large net loss was observed. For the concentration of historic, retrospectively extracted DNA, centrifugal methods are able to concentrate DNA extracts previously too dilute for analysis. These concentrated volumes, however are small, allowing for minimal downstream analysis attempts before the sample is exhausted.

Technical note: Next generation identification - A powerful tool in cold case investigations.

Ever changing technology is allowing law enforcement/crime laboratories the ability to use new advances to solve old cases. Exonerations through advances in DNA testing have become increasingly common. However, other forensic disciplines are also utilizing new technologies, which are providing investigative leads and case closures. One advancement is the Federal Bureau of Investigation's Next Generation Identification (NGI) biometric system, which has enhanced the field of print identification. The Oklahoma State Bureau of Investigation (OSBI) Latent Evidence Unit is utilizing NGI to assist in solving "Cold Cases." While the OSBI cold case project has been successful, reviewing old evidence and case files, while keeping up with current casework, can be challenging. However, the recognition and application of such powerful technology is warranted, as demonstrated by the success that has been achieved.

The CSI effect on cold case investigations.

Much has been made about the potential impact of fictional crime scene shows in the media on jury verdicts. However, there has been little discussion about how those programs, and the myriad of other true crime shows and documentaries may otherwise influence the actual investigative process. The intent of this paper is to discuss ways that such shows may impact the investigative process. The focus will be on the investigation of old, unsolved cases, commonly referred to as cold cases, because of the increasing focus on forensics in all aspects of the investigation and the number of television shows that feature such cases. This paper is not intended to be an academic study of the issue, as it relies much on anecdotal evidence and the experience of the author and others. The purpose is to hopefully generate further discussion and research with the goal of improving the investigative process.

The Search for "Fred": An Unusual Vertical Burial Case.

Police witness intelligence stated a murdered adult male "Fred" had been vertically buried in wooded hilly terrain 30 years ago in the Midlands, U.K. Conventional search methods were unsuccessful; therefore, the police requested a geophysical investigation to be undertaken to determine whether "Fred" could be detected. A multiphased geophysical approach was conducted, using bulk ground conductivity and metal detectors, then follow-up magnetics and ground penetrating radar (GPR) survey profiles on electromagnetic (EM) anomalous areas. A tight grid pattern was used to account for the reduced target size. Relatively high-resolution EM and GPR techniques were determined optimal for this terrain and sandy soil. Geophysical anomalies were identified and the most promising intrusively investigated, and this was found to be a large boulder and tree roots. Study implications suggest careful multiphase geophysical surveys are best practice and give confidence in cold case searches. This study yielded a no-body result, effectively saving police time and costs from further investigations.

The role of the gunshot residue expert in case review - A case study.

This paper details the review of the gunshot residue (GSR) evidence relating to a 2002 murder involving the discharge of a pistol where the Prosecution used the GSR findings together with voice identification evidence and obtained a conviction in the case of R v Dwaine George. Mr George was subsequently represented by Cardiff University Law School's Innocence Project who made representations to the Criminal Cases Review Commission (CCRC) based on issues relating to the GSR evidence. The CCRC commissioned their own expert review and based on these findings referred the conviction to the Court of Appeal stating that in their opinion the weight of the GSR evidence was not appropriately conveyed to the jury at the original Trial and there should have been a warning relating to the limited significance that could be attached to the evidence. The Court of Appeal concluded that had the jury been aware of the full extent of the potential alternative sources of GSR in the case and the limited number of particles found on a jacket related to Mr George then this would have affected the decision of the Trial jury. The appeal was allowed and the convictions were quashed.

Cold Case Homicides: DNA Testing of Retained Autopsy Sexual Assault Smears.

Archival medical examiner specimens may contain perpetrator DNA evidence useful in unsolved ("cold case") homicides. The Office of the Chief Medical Examiner (OCME) histology slide archives were searched for sexual assault smears for all 376 female homicides from 1990 to 1999. Of these, the OCME had sexual assault smears on 84 of which 13 slides had sperm. Of these 13, six were still unsolved. DNA profiles were obtained on all six (5 from smears and one from swabs). Combined DNA Index System ( submission resulted in two matches ("hits") for new suspects. In addition, three suspects were eliminated in two cases. Our review of archival sexual assault smears resulted in DNA profiles that were able to assist in the investigation of four cold case homicide investigations. It may be worthwhile for medical examiner offices to search their archival histology slides for sexual assault smears on previously unsolved cases particularly those prior to the mid-1990s when DNA testing was less widely available.