Elemental Analysis of Variably Contaminated Cremains Using X‐ray Fluorescence Spectrometry

Analyzing and identifying skeletal remains becomes increasingly difficult when remains have been cremated, especially in cases where the cremated material may have been intentionally contaminated with nonskeletal material. This study examined the potential of X‐ray fluorescence spectrometry (XRF) to detect the presence of nonskeletal contaminants in samples of cremains. Eleven samples of cremains were variably combined with concrete mix and analyzed using XRF. Photon counts of elements in each sample were analyzed, and the coefficient of determination (R²) using unweighted linear regression as a function of percent cremains was calculated. Results showed that with changes in the proportion of skeletal material and contaminant, there were significant (R² > 0.90) changes in detected levels of phosphorus, potassium, zinc, aluminum, and sulfur. The use of XRF is concluded to be a valid approach in the identification of the presence of nonskeletal material in potentially contaminated cremains.     

Validation of X-ray fluorescence spectrometry for determining osseous or dental origin of unknown material

Forensic anthropological examinations typically involve the analysis of human skeletal remains, but in cases where samples are very small and/or physically compromised, it may first be necessary to determine whether the material is even osseous or dental in origin. X-ray fluorescence spectrometry (XRF) is a technique that reveals the elemental composition of materials and is hypothesized to have utility in such cases. XRF analysis was conducted on a variety of tissues and materials in unaltered and altered (damaged) states. With few exceptions, osseous and dental tissues in unaltered and altered conditions contained characteristic levels of calcium and phosphorus, while other materials did not. Materials could be accurately identified as osseous or dental in origin based on the calcium and phosphorus levels identified by XRF, and we therefore conclude that XRF analysis is a valid and effective means of determining osseous or dental origin of unknown material.     

Practical Considerations in Trace Element Analysis of Bone by Portable X‐ray Fluorescence

Forensic anthropologists are more often turning to nondestructive methods to assist with skeletal analyses, specifically for trace elemental analyses. Portable XRF (pXRF) instruments are versatile and are able to be used in diverse settings or for specimens of a shape and size that cannot be accommodated by laboratory‐based instruments. Use of XRF requires knowledge of analysis parameters such as X‐ray penetration and exit depth. Analysis depth was determined by examining pure elements through known thicknesses of equine bone slices. Correlation between the element's X‐ray emission energy and the depth of reading was observed. Bone surfaces from a small unknown historic cemetery were analyzed before and after sanding of the periosteal surface to observe possible changes in XRF readings based on potential diagenesis. Results validate the pXRF device as a powerful and convenient instrument for nondestructive analysis, while highlighting limitations and considerations for the analysis of osseous materials.     

Limitations of ZAF correction factors in the determination of calcium/phosphorus ratios: important forensic science considerations relevant to the analysis of bone fragments using scanning electron microscopy and energy-dispersive x-ray microanalysis

A series of calcium phosphate standards having calcium/phosphorus (Ca/P) molar ratios of 0.50, 1.00, 1.50, and 1.67, respectively, was prepared for bulk specimen analysis using scanning electron microscopy (SEM) and energy-dispersive X-ray microanalysis (EDXA). The standards were mounted on carbon planchettes as either pure crystals or crystals embedded in epoxy resin. Ten different samples of each embedded and non-embedded standard were analyzed in a JEOL 100 CX electron microscope interfaced with a Kevex 8000 EDXA system using a lithium-drifted silicon detector and a multichannel analyzer. The Ca/P ratios were determined by calculating both net peak intensities without matrix corrections and atomic kappa-ratios using the MAGIC V computer program with ZAF correction factors for quantitative analysis. There was such extensive absorption of phosphorus X-rays in standards embedded in an epoxy matrix that the observed Ca/P ratios were statistically compatible with four different standards ranging in theoretical Ca/P ratios from 1.0 to 1.67. Although the non-embedded crystals showed a greater separation in the Ca/P ratios, both methods of preparation produced serious flaws in analysis. Direct application of the discovery of this caveat to the identification of suspected bone fragments for forensic science purposes is discussed.     

Lead Theft—A Study of the “Uniqueness” of Lead from Church Roofs

In the United Kingdom, theft of lead is common, particularly from churches and other public buildings with lead roofs. To assess the potential to distinguish lead from different sources, 41 samples of lead from 24 church roofs in Northamptonshire, U.K, have been analyzed for relative abundance of trace elements and isotopes of lead using X‐ray fluorescence (XRF) and inductively coupled plasma mass spectrometry, respectively. XRF revealed the overall presence of 12 trace elements with the four most abundant, calcium, phosphorus, silicon, and sulfur, showing a large weight percentage standard error of the mean of all samples suggesting variation in the weight percentage of these elements between different church roofs. Multiple samples from the same roofs, but different lead sheets, showed much lower weight percentage standard errors of the mean suggesting similar trace element concentrations. Lead isotope ratios were similar for all samples. Factors likely to affect the occurrence of these trace elements are discussed.     

Analytical survey of restorative resins by SEM/EDS and XRF: databases for forensic purposes

Frequently in forensic cases, unknown substances must be identified. Automated databases can ease the burden of comparison as materials may be compared against many known standards in a relatively short period of time. It has been shown that dental resins can be named according to brand or brand group even in conditions as harsh as cremation. Databases are already in use for many materials, but no such database exists for dental resins. Thus, two databases were generated. One utilized a laboratory-based method, scanning electron microscopy with energy dispersive X-ray spectroscopy (SEM/EDS), in conjunction with the Spectral Library Identification and Classification Explorer (SLICE) software. The other was based on portable X-ray fluorescence (XRF). The ability to perform database comparison with portable instrumentation can thus be brought directly to the field. Both the SLICE and XRF databases were evaluated by testing unknown resins. EDS is a well-established technique and the SLICE program was demonstrated to be a good tool for unknown resin identification. Portable XRF is a relatively new instrument in this regard and its databases have been constructed mostly for metal alloy comparison and environmental soil testing. However, by creation of a custom spectral library, it was possible to distinguish resin brand and bone and tooth from other substances.     

Identification through X-ray fluorescence analysis of dental restorative resin materials: a comprehensive study of noncremated, cremated, and processed-cremated individuals

Tooth-colored restorative materials are increasingly being placed in the practice of modern dentistry, replacing traditional materials such as amalgam. Many restorative resins have distinct elemental compositions that allow identification of brand. Not only are resins classifiable by elemental content, but they also survive extreme conditions such as cremation. This is of significance to the forensic odontologist because resin uniqueness adds another level of certainty in victim identification, especially when traditional means are exhausted. In this three-part study, unique combinations of resins were placed in six human cadavers (total 70 restorations). Simulated ante-mortem dental records were created. In a blind experiment, a portable X-ray fluorescence (XRF) unit was used to locate and identify the resin brands placed in the dentition. The technique was successful in location and brand identification of 53 of the restorations, which was sufficient to enable positive victim identification among the study group. This part of the experiment demonstrated the utility of portable XRF in detection and analysis of restorative materials for victim identification in field or morgue settings. Identification of individuals after cremation is a more difficult task, as the dentition is altered by shrinkage and fragmentation, and may not be comparable with a dental chart. Identification of processed cremains is a much greater challenge, as comminution obliterates all structural relationships. Under both circumstances, it is the nonbiological artifacts that aid in identification. Restorative resin fillings can survive these conditions, and can still be named by brand utilizing elemental analysis. In a continuation of the study, the cadavers were cremated in a cremation retort under standard mortuary conditions. XRF was again used to analyze retrieved resins and to identify the individuals based on restorative materials known to exist from dental records. The cremains were then processed and the analysis was repeated to determine whether restorative resins could be found under this extreme condition. Under both circumstances, sufficient surviving resin material was found to distinguish positively each individual in the study group. This study showed the utility of XRF as an analytical tool for forensic odontology and also the significance of the role of restorative resins in victim identification, even after cremation.     

微束X射线荧光光谱法检测电流损伤皮肤金属化

目的建立微束X射线荧光光谱（μ-probe X-ray fluorescence,μ-XRF）检测电流损伤皮肤金属化的方法。方法新西兰大白兔32只,随机分为黄铜电击组、紫铜电击组、铁电击组、铝合金电击组,每组8只。电极一极固定于左后腿中部,另一极固定于左前腿,建立电击模型。提取左后腿触电部位皮肤,以及对侧右后腿相应部位皮肤作为对照,应用μ-XRF光谱仪对电流损伤皮肤内金属元素进行测定。结果正常对照组皮肤中检测出磷、氯、钾、钙元素成分;在电击组皮肤中,除正常皮肤检出的元素外,黄铜电击组检测出铜、锌元素,紫铜电击组检出铜元素,铁电击组检出铁元素,铝合金电击组检出铝元素。渗透到电流损伤皮肤内的金属元素呈不均匀分布。结论μ-XRF光谱法检测皮肤金属化可作为诊断电流损伤的特征性指标,并可为触电材料的推断提供依据。     

The Use of an Alternate Light Source for Detecting Bones Underwater

When searching underwater crime scenes or disaster scenes for fragmentary human remains, it may be advantageous for forensic divers to be able to detect the presence of bones and teeth among other marine materials (such as shells and rocks). In terrestrial environments, this can typically be accomplished by visual and instrumental methods, but underwater conditions make it difficult to employ detection and sorting techniques in these environments. This study investigates fluorescence of bones and teeth and other marine materials using a submersible alternate light source (ALS) and concludes that an ALS can be a useful tool for detecting bones and teeth in underwater searches as well in terrestrial searches and laboratory environments. The results could impact the methods and equipment used by forensic divers and forensic anthropologists when searching for skeletal remains, potentially increasing the quantity and efficiency of forensic evidence recovered.     

SEM/EDS法和XRF法在电工胶带检验中的应用

目的探索电工胶带带基的元素检验方法。方法利用扫描电镜/能谱法（SEM/EDS）对电工胶带带基中的主体元素进行定量分析,通过SPSS软件对SEM/EDS定量分析法检测出的主体元素（Cl和Ca）相对含量进行统计分析;利用X射线荧光光谱法（XRF）对电工胶带带基中的微量元素进行定性分析。结果34个品牌红色电工胶带组成的561组样品对通过SEM/EDS定量分析法可以区分539组,区分率达到96.1%;根据XRF法定性分析测出的Mg、Al、Si、S、Ti、Sb、Ba、Pb微量元素的不同组合可将34种电工胶带分成11组,两种方法结合可使不同品牌红色电工胶带的区分率达到98.4%。结论SEM/EDS法和XRF法结合可以实现同种颜色、不同品牌电工胶带的高效区分。     

Use of 2D:4D Digit Ratios to Determine Sex

Second to fourth digit ratios (2D:4D) are sexually dimorphic in human hands and established by the 13th gestational week. Application of 2D:4D for determining sex in living individuals by Kanchan et al. (Forensic Sci Int, 181, 2008, 53.e1) produced classification rates of 80% for males and 74–78% for females. Few studies have explored the use of 2D:4D for sexing skeletal remains. We test estimated finger lengths, phalanx lengths, and 2D:4D derived from hand bones for determining sex. Maximum phalanx length was collected using a mini‐osteometric board from 451 individuals of known age, sex, and ancestry in four skeletal collections. Logistic regression of 2nd and 4th digit finger and phalanx lengths produced classification rates greater than 80%. Digit ratios, however, failed to reach classification rates greater than 59%. Our results support those of Voracek (Forensic Sci Int, 185, 2009, e29) and suggest that 2D:4D may be population‐specific and thus inappropriate for universal application as a means of determining sex.     

Evaluation of postmortem serum calcium and magnesium levels in relation to the causes of death in forensic autopsy

There appears to be very poor investigation of postmortem serum calcium (Ca) and magnesium (Mg) for diagnostic evidence to determine the cause of death. The aim of the present study was a comprehensive analysis of the serum levels in relation to the causes of death in routine casework. Autopsy cases (total, n=360; 5-48 h postmortem), including blunt injury (n=76), sharp injury (n=29), asphyxiation (n=42), drownings (n=28: freshwater, n=11; saltwater, n=17), fire fatalities (n=79), methamphetamine (MA) poisoning (n=8), delayed death from traumas (n=37), and acute myocardial infarction/ischemia (AMI, n=61), were examined. In total cases, there was no significant postmortem time-dependent rise in serum Ca and Mg. Both Ca and Mg levels in the heart and peripheral blood were significantly higher in saltwater drowning compared with those of the other groups. In addition, a significant elevation in the Ca level was observed in freshwater drowning and fire fatalities, and in the Mg level in fatal MA intoxication and asphyxiation. Topographic analyses suggested a rise in serum Ca and Mg due to aspirated saltwater in drowning, that in serum Ca in freshwater drowning and fire fatalities of peripheral skeletal muscle origin and that in serum Mg in MA fatality and asphyxiation of myocardial and/or peripheral origin. These markers may be useful especially for diagnosis and differentiation of salt- and freshwater drownings and may be also helpful to determine the causes of death involving skeletal muscle damage, including burns and MA intoxication.     

中介素（IMD）对大鼠心肌细胞缺氧-复氧损伤的作用

目的探讨中介素（IMD）对大鼠体外培养心肌细胞缺氧-复氧损伤的作用。方法用大鼠H9c2心肌细胞株制作实验模型,样本分为对照组、缺氧-复氧组（缺氧1h、复氧30min）、IMD组（缺氧-复氧前30min加入10-7mol/L IMD）。采用MTT比色法检测心肌细胞活力;透射电镜观察细胞超微结构;激光共聚焦显微镜观察并测定细胞内钙离子浓度;流式细胞术测定细胞凋亡率。结果与对照组比较,缺氧-复氧组和IMD组细胞存活率显著降低,而IMD处理组明显升高细胞存活率（P〈0.01）;在形态学上,IMD预处理可明显减轻缺氧-复氧对大鼠心肌细胞的损伤;缺氧-复氧组细胞[Ca2＋]i荧光强度和细胞凋亡率比对照组显著升高,IMD预处理可明显降低上述升高的比率（P〈0.01）。结论 IMD对大鼠心肌细胞缺氧-复氧损伤有一定保护作用,提高心肌细胞存活率、减轻心肌细胞钙超载和抑制凋亡是其作用途径。     

Indoor Wet Screening of Exhumed Skeletal Remains: A Suggested Procedure for the Preparation of Fragile Evidence for Anthropological Analysis

Abstract: The 2007 exhumation of three children’s graves, located in rural upstate New York and dating to 1979 and 1980, was warranted as their mother had come under suspicion for the death of a child she had been babysitting in late 2006. The local March weather conditions had been wet, and heavy rains fell during the 2‐day process of casket removal. The extremely wet soil and the poor preservation of two wooden caskets increased the likelihood of damage to evidence. After remains’ transport to the forensic center, an indoor wet‐screening station was established so that skeletal elements could be (i) separated from soil matrix and (ii) preserved carefully for analysis. Not only were the remains relatively small and fragile in comparison with those of an adult, but two of the three remains were known to be fire damaged, thus the use of special laboratory preparation techniques was crucial.     

DNA Typing for the Identification of Old Skeletal Remains from Korean War Victims*

Abstract: The identification of missing casualties of the Korean War (1950–1953) has been performed using mitochondrial DNA (mtDNA) profiles, but recent advances in DNA extraction techniques and approaches using smaller amplicons have significantly increased the possibility of obtaining DNA profiles from highly degraded skeletal remains. Therefore, 21 skeletal remains of Korean War victims and 24 samples from biological relatives of the supposed victims were selected based on circumstantial evidence and/or mtDNA‐matching results and were analyzed to confirm the alleged relationship. Cumulative likelihood ratios were obtained from autosomal short tandem repeat, Y‐chromosomal STR, and mtDNA‐genotyping results, and mainly confirmed the alleged relationship with values over 10⁵. The present analysis emphasizes the value of mini‐ and Y‐STR systems as well as an efficient DNA extraction method in DNA testing for the identification of old skeletal remains.     

Elemental analysis of glass by laser ablation inductively coupled plasma optical emission spectrometry (LA-ICP-OES)

The elemental analysis of glass evidence has been established as a powerful discrimination tool for forensic analysts. Laser ablation inductively coupled plasma optical emission spectrometry (LA-ICP-OES) has been compared to laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) and energy dispersive micro X-ray fluorescence spectroscopy (μXRF/EDS) as competing instrumentation for the elemental analysis of glass. The development of a method for the forensic analysis of glass coupling laser ablation to ICP-OES is presented for the first time. LA-ICP-OES has demonstrated comparable analytical performance to LA-ICP-MS based on the use of the element menu, Al (Al I 396.15 nm), Ba (Ba II 455.40 nm), Ca (Ca II 315.88 nm), Fe (Fe II 238.20 nm), Li (Li I 670.78 nm), Mg (Mg I 285.21 nm), Sr (Sr II 407.77 nm), Ti (Ti II 368.51 nm), and Zr (Zr II 343.82 nm). The relevant figures of merit, such as precision, accuracy and sensitivity, are presented and compared to LA-ICP-MS. A set of 41 glass samples was used to assess the discrimination power of the LA-ICP-OES method in comparison to other elemental analysis techniques. This sample set consisted of several vehicle glass samples that originated from the same source (inside and outside windshield panes) and several glass samples that originated from different vehicles. Different match criteria were used and compared to determine the potential for Type I and Type II errors. It was determined that broader match criteria is more applicable to the forensic comparison of glass analysis because it can reduce the affect that micro-heterogeneity inherent in the glass fragments and a less than ideal sampling strategy can have on the interpretation of the results. Based on the test set reported here, a plus or minus four standard deviation (± 4s) match criterion yielded the lowest possibility of Type I and Type II errors. The developed LA-ICP-OES method has been shown to perform similarly to LA-ICP-MS in the discrimination among different sources of glass while offering the advantages of a lower cost of acquisition and operation of analytical instrumentation making ICP-OES a possible alternative elemental analysis method for the forensic laboratory.     

Assessing the Utility of Soil DNA Extraction Kits for Increasing DNA Yields and Eliminating PCR Inhibitors from Buried Skeletal Remains

DNA identification of human remains is often necessary when decedents are skeletonized; however, poor DNA recovery and polymerase chain reaction (PCR) inhibition are frequently encountered, a situation exacerbated by burial. In this research, the utility of integrating soil DNA isolation kits into buried skeletal DNA analysis was evaluated and compared to a standard human DNA extraction kit and organic extraction. The soil kits successfully extracted skeletal DNA at quantities similar to standard methods, although the two kits tested, which differ mechanistically, were not equivalent. Further, the PCR inhibitors calcium and humic acid were effectively removed using the soil kits, whereas collagen was less so. Finally, concordant control region sequences were obtained from human skeletal remains using all four methods. Based on these comparisons, soil DNA isolation kits, which quickened the extraction process, proved to be a viable extraction technique for skeletal remains that resulted in positive identification of a decedent.     

Review of analytical methods for screening and quantification of fentanyl analogs and novel synthetic opioids in biological specimens

Fentanyl, fentanyl analogs, and other novel synthetic opioids (NSO), including nitazene analogs, prevail in forensic toxicology casework. Analytical methods for identifying these drugs in biological specimens need to be robust, sensitive, and specific. Isomers, new analogs, and slight differences in structural modifications necessitate the use of high-resolution mass spectrometry (HRMS), especially as a non-targeted screening method designed to detect newly emerging drugs. Traditional forensic toxicology workflows, such as immunoassay and gas chromatography mass spectrometry (GC–MS), are generally not sensitive enough for detection of NSOs due to observed low (sub-μg/L) concentrations. For this review, the authors tabulated, reviewed, and summarized analytical methods from 2010–2022 for screening and quantification of fentanyl analogs and other NSOs in biological specimens using a variety of different instruments and sample preparation approaches. Limits of detection or quantification for 105 methods were included and compared to published standards and guidelines for suggested scope and sensitivity in forensic toxicology casework. Methods were summarized by instrument for screening and quantitative methods for fentanyl analogs and for nitazenes and other NSO. Toxicological testing for fentanyl analogs and NSOs is increasingly and most commonly being conducted using a variety of liquid chromatography mass spectrometry (LC–MS)-based techniques. Most of the recent analytical methods reviewed exhibited limits of detection well below 1 μg/L to detect low concentrations of increasingly potent drugs. In addition, it was observed that most newly developed methods are now using smaller sample volumes which is achievable due to the sensitivity increase gained by new technology and new instrumentation.     

The Utility of Skeletal and Surgical Features for the Personal Identification Process: A Pilot Study

This pilot study provides a conceptual framework for the application of the anthropological analysis of skeletal features and surgical interventions for the purpose of identification in cases of unknown deceased individuals with unavailable fingerprint, genetic or odontological antemortem data. The study sample includes 276 individuals with known demographic and clinical information from the Italian CAL Milano Cemetery Skeletal Collection. In the sample, 124 (45%) individuals showed one or more skeletal features that may be potentially individualizing. Of these, 79% showed two and more features, which occurred in a multitude of different combinations. Skeletal findings may provide useful postmortem information that can be compared with antemortem witness statements and clinical imaging. However, more research into the utility of dry bone findings and the availability of comparative material, including imaging, and epidemiological data needs to be undertaken before skeletal features can be implemented into identification protocols and databases.     

Classification of black powder toners on the basis of integrated analytical information provided by Fourier transform infrared spectrometry and X-ray fluorescence spectrometry

Differentiating between black powder toners used in laser printers and copiers can be challenging for forensic examiners. One hundred and sixty-two samples from 82 different types of cartridges produced by 21 manufacturers that are currently available on the Polish market were studied using Fourier transform infrared spectrometry (FT-IR) and energy-dispersive X-ray fluorescence spectrometry (XRF). The spectra obtained by these two methods were classified into 14 groups and 28 groups using FT-IR and XRF, respectively. These classifications served as a basis for the development of an analytical scheme for differentiating black powder toners. This scheme can be used with any two methods that supply different information about an examined sample. The research will show that if two samples are similar in polymer composition (e.g., FT-IR spectra), additional quantitative elemental composition from XRF analyses may provide more discrimination. It was possible to differentiate 82.5% pairs of examined samples using only FT-IR, 90.8% pairs of examined samples using only XRF, and 95.8% pairs of examined samples using these two methods. Obtained spectra and all the available information could be used to create/build a database. The results obtained confirm the multiplicity and compatibility of toners. Additionally, it was stated that not all the samples were original (OEM).