Lessons learned from inter-laboratory studies of carbon isotope analysis of honey

Forensic application of carbon isotope ratio measurements of honey and honey protein to investigate the degree of adulteration with high fructose corn syrup or other C₄ plant sugars is well established. These measurements must use methods that exhibit suitable performance criteria, particularly with regard to measurement uncertainty and traceability – low levels of adulteration can only be detected by methods that result in suitably small measurement uncertainties such that differences of 1‰ or less can be reliably detected. Inter-laboratory exercises are invaluable to assess the state-of-the art of measurement capabilities of laboratories necessary to achieve such performance criteria. National and designated metrology institutes from a number of countries recently participated in an inter-laboratory assessment (CCQM-K140) of stable carbon isotope ratio determination of bulk honey. The same sample material was distributed to a number of forensic isotope analysis laboratories that could not participate directly in the metrological comparison. The results from these studies have demonstrated that the majority of participants provided isotope delta values with acceptable performance metrics; that all participants ensured traceability of their results; and that where measurement uncertainties were reported; these were fit-for-purpose. A number of the forensic laboratories only reported precision rather than full estimates of measurement uncertainty and this was the major cause of the few instances of questionable performance metrics. Reporting of standard deviations in place of measurement uncertainties is common practice outside metrology institutes and the implications for interpretations of small differences in isotopic compositions are discussed. The results have also highlighted a number of considerations that are useful for organisers of similar inter-laboratory studies in the future.     

An initial evaluation of stable isotopic characterisation of post-blast plastic debris from improvised explosive devices

A number of two-way radios, similar to those which have been employed to initiate Improvised Explosive Devices (IEDs), were acquired from a commercial supplier and grouped into four pairs. Samples of plastic material were collected from five distinct regions of each radio and analysed by Infrared and Raman spectroscopy to identify the nature of the material. One radio of each pair was then subjected to detonation with a commercially available plastic explosive. The combination of radio and explosive was considered to be representative of the components of an IED. Following detonation, fragments were recovered and, where possible, identified as specific sampling points of the radio.A combination of δ²H and δ¹³C stable isotopic analysis of material from each of the five sampling points was found to provide a pattern which was characteristic of a given radio and provided a means to associate pairs of radios. When few fragments were recovered, no positive association could be made between the fragments and the paired, undamaged radio. This was attributed, in part, to manufacturing variation in the radios. However, when three or more post-blast fragments were recovered it was possible to associate these with the paired, undamaged radio with a high degree of certainty.     

Identification of a disinterred grave by molecular and stable isotope analysis

Confirmation of a potential disinterred grave was sought by GC and GC/MS analyses of lipid extracts of whole soils and white particulate matter. Fatty acid profiles and concentrations determined for three of the soils correlated with the deposition of a large amount of exogenous organic matter, most likely adipocere and/or decomposed body fat. Determination of C_16:0 and C_18:0 fatty acid δ¹³C values by GC/C/IRMS revealed the input to be isotopically distinct from common British domesticated animals, plotting closely to values determined for adipose fat obtained from of a murder victim. By considering the difference between δ¹³C values (Δ¹³C_18:0–16:0) a potential isotopic proxy for identifying the source of adipocere (human) and adipose tissue was proposed.     

FaSTR DNA: A new expert system for forensic DNA analysis

The automation of DNA profile analysis of reference and crime samples continues to gain pace driven in part by a realisation by the criminal justice system of the positive impact DNA technology can have in aiding in the solution of crime and the apprehension of suspects. Expert systems to automate the profile analysis component of the process are beginning to be developed. In this paper, we report the validation of a new expert system FaSTR DNA, an expert system suitable for the analysis of DNA profiles from single source reference samples and from crime samples. We compare the performance of FaSTR DNA with that of other equivalent systems, GeneMapper™ ID v3.2 (Applied Biosystems, Foster City, CA) and FSS-i³ v4 (The Forensic Science Service^® DNA expert System Suite FSS-i³, Forensic Science Service, Birmingham, UK) with GeneScan^® Analysis v3.7/Genotyper^® v3.7 software (Applied Biosystems, Foster City, CA, USA) with manual review. We have shown that FaSTR DNA provides an alternative solution to automating DNA profile analysis and is appropriate for implementation into forensic laboratories. The FaSTR DNA system was demonstrated to be comparable in performance to that of GeneMapper™ ID v3.2 and superior to that of FSS-i³ v4 for the analysis of DNA profiles from crime samples.     

Sex Estimation from Human Cranium: Forensic and Anthropological Interest of Maxillary Sinus Volumes

Sex estimation is a key objective of forensic science. We aimed to establish whether maxillary sinus volumes (MSV) could assist in estimating an individual's sex. One hundred and three CT scans were included. MSV were determined using three‐dimensional reconstructions. Two observers performed three‐dimensional MSV reconstructions using the same methods. Intra‐ and interobserver reproducibility were statistically compared using the intraclass correlation coefficient (ICC) (α = 5%). Both intra‐ and interobserver reproducibility were perfect regarding MSV; both ICCs were 100%. There were no significant differences between right and left MSV (p = 0.083). No correlation was found between age and MSV (p > 0.05). We demonstrated the existence of sexual dimorphism in MSV (p < 0.001) and showed that MSV measurements gave a 68% rate of correct allocations to sex group. MSV measurements could be useful to support sex estimation in forensic medicine.     

Forensic isoscapes based on intra-individual temporal variation of δ18O and 206Pb/207Pb in human teeth

Isotopic signatures used in the georeferencing of human remains are largely fixed by spatially distinct geologic and environmental processes. However, location-dependent temporal changes in these isotope ratios should also be considered when determining an individual’s provenance and/or trajectory. Distributions of the relevant isotopes can be impacted by predictable external factors such as climate change, delocalisation of food and water sources and changes in sources and uses of metals. Using Multi-Collector Inductively-Coupled Plasma Mass Spectrometer (MC-ICP-MS) analyses of ²⁰⁶Pb/²⁰⁷Pb in tooth enamel and dentin from a population of 21 ± 1-year-old individuals born circa 1984 and isotope ratio mass spectrometry (IRMS) of δ¹⁸O in their enamel, we examined the expected influence of some of these factors. The resulting adjustments to the geographic distribution of isotope ratios (isoscapes) found in tooth enamel and dentin may contain additional useful information for forensic identification, but the shifts in values can also impact the uncertainty and usefulness of identifications if they are not taken into account.

KEY POINTS

• Isoscapes of ²⁰⁶Pb/²⁰⁷Pb and δ¹⁸O used for geolocation are not static.
• Within a few years, the enamel and dentin of a person may exhibit measurable differences in ²⁰⁶Pb/²⁰⁷Pb even without changing locations.
• Changes in climatic patterns tied to rising temperatures are more significant than the direct effect of increasing temperature on δ¹⁸O fixed in tooth bioapatite.
• Third molar (M3) enamel mineralisation includes material incorporated from before formal amelogenesis takes place.

     

Perceptions of blind proficiency testing among latent print examiners

In recent years, scholars have levied multiple criticisms against traditional proficiency testing procedures in forensic laboratories. Consequently, on several occasions, authorities have formally recommended that laboratories implement blind proficiency testing procedures. Implementation has been slow, but laboratory management has increasingly expressed interest in initiating blind testing in at least some forensic disciplines, with some laboratories conducting blind testing in almost all disciplines. However, little is known about how a key population perceives blind proficiency testing, i.e., forensic examiners. We surveyed active latent print examiners (N = 338) to explore perceptions of blind proficiency testing and determine whether beliefs varied between examiners who work for laboratories with and without blind proficiency testing. Results suggest that examiners do not hold particularly strong beliefs about such procedures, but that examiners who work in laboratories with blind proficiency testing procedures view them significantly more positively than those who do not. Further, examiner responses provide insight into potential obstacles to continued implementation.     

Forensic applications of stable isotope analysis: Case studies of the origins of water in mislabeled beer and contaminated diesel fuel

This paper describes the use of oxygen (¹⁸O) isotope analysis of water contained in two different materials — beer and diesel fuel — involved in the resolution of two separate cases. In the first case study, it was possible to demonstrate that a sample of beer labelled as premium brand in fact belonged to a cheap brand. The second case related to the contamination of diesel fuel from a service station. The diesel fuel contained visible amounts of water, which caused vehicles that had been filled up with it to become defective. For insurance purposes, it was necessary to determine the source of water. The δ¹⁸O values for the water of nearly all samples of diesel was close to the δ¹⁸O of local tap water at the filling station.     

Analytical Characterization of Erythritol Tetranitrate,an Improvised Explosive

Erythritol tetranitrate (ETN), an ester of nitric acid and erythritol, is a solid crystalline explosive with high explosive performance. Although it has never been used in any industrial or military application, it has become one of the most prepared and misused improvise explosives. In this study, several analytical techniques were explored to facilitate analysis in forensic laboratories. FTIR and Raman spectrometry measurements expand existing data and bring more detailed assignment of bands through the parallel study of erythritol [¹⁵N₄] tetranitrate. In the case of powder diffraction, recently published data were verified, and ¹H, ¹³C, and ¹⁵N NMR spectra are discussed in detail. The technique of electrospray ionization tandem mass spectrometry was successfully used for the analysis of ETN. Described methods allow fast, versatile, and reliable detection or analysis of samples containing erythritol tetranitrate in forensic laboratories.     

Estimating Biological Characteristics With Virtual Laser Data

Laser scanning technology is increasingly being used in forensic anthropological research to obtain virtual data for archival purposes and post hoc measurement collection. This research compared the measurement accuracy of two laser scanners—the FARO Focus^3D 330X and the FARO Freestyle^3D—against traditionally obtained (i.e., by hand) control data (N = 454). Skeletal data were collected to address a novel question: the ability of laser scanning technology to produce measurements useful for biological characteristic estimation, such as sex and stature. Results indicate that both devices produced measurements very similar to control (c. 3‐mm average absolute error), but also illuminate a tendency to under‐measure. Despite these findings, the virtual data produced sex and stature estimates that varied little from control‐produced estimates, signifying the usefulness of virtual data for preliminary biological identification when the skeletal elements are no longer available for physical analysis.     

Post-injection hybridization of complementary DNA strands on capillary electrophoresis platforms: A novel solution for dsDNA artifacts

Several laboratories have reported the occurrence of a split or n − 1 peak at the vWA locus in PowerPlex^® 16 and PowerPlex^® ES amplification products separated on 4- and 16-capillary electrophoresis instruments. The root cause of this artifact is post-PCR reannealing of the unlabeled, unincorporated vWA primer to the 3′-end of the tetramethylrhodamine (TMR)-labeled strand of the vWA amplicon. This reannealing occurs in the capillary post-electrokinetic injection. The split peak is eliminated by incorporation into the loading cocktail of a sacrificial hybridization sequence (SHS) oligonucleotide that is complementary to the vWA primer. The SHS preferentially anneals to the primer instead of the TMR-labeled strand of the vWA amplicon. In addition, the n − 10/n − 18 artifact that may be seen at the vWA locus was determined to be due to double-stranded amplicon formed post-electrokinetic injection into the capillary. This was also eliminated by adding in two Complementary Oligo Targets (COT1 and COT2) in addition to the SHS oligonucleotide into the loading cocktail. These three oligonucleotides are complementary to the 33 bases at the 5′-end of the unlabeled vWA amplicon strand and the 60 bases at its 3′-end and therefore compete for hybridization to the TMR-labeled amplicon strand. Incorporation of these three oligonucleotides in the Internal Lane Standard 600 (ILS600) eliminate both the split peak and n − 10/n − 18 artifact in PowerPlex^® 16 and PowerPlex^® ES amplification products without affecting sizing of alleles at the vWA locus or any locus in the PowerPlex^® 16, PowerPlex^® Y, PowerPlex^® ES, AmpFlSTR^® Profiler Plus^® ID, AmpFlSTR^® Cofiler^®, and AmpFlSTR^® SGM Plus^® kits.     

Estimating Crime Laboratory Efficiency in the Testing of Sexual Assault Kits,

Over the past decade, the large numbers of untested sexual assault kits (SAKs) have been highlighted as a systematic problem that jeopardizes or delays justice for victims. Considering the benefits of testing SAKs, researchers have worked to shed light on why sexual assault evidence has not been effectively submitted to and processed by crime laboratories. Missing from this discourse has been an understanding of the types of practices or qualities that encourage efficiency in the testing of SAKs in crime laboratories. We analyzed results of a national survey administered to all publicly funded state and local crime laboratories (N = 132 respondents) to provide critical information about (i) the extent to which laboratories are testing all of the SAKs possible given the resources they have available; and (ii) the impact that staffing, equipment, policies, and other practices have on SAK testing efficiency. We find that the average laboratory tests only about 69% of the SAKs possible given the resources available to them. However, although technical inefficiencies explain a large proportion of the number of untested SAKs, the accumulation of untested SAKs must also be attributed to laboratories having insufficient resources (e.g., too few forensic analysts). Moreover, results from stochastic frontier models show that doubling the number of forensic analysts in the typical laboratory would allow them to expand their SAK testing capacity by nearly 50%. Implications of these findings are discussed as they relate to the prioritization of resources for crime laboratories, which often operate under strict budgetary realities.     

Developmental validation of the AmpF?STR SEfiler Plus™ PCR amplification kit: An improved multiplex with enhanced performance for inhibited samples

Since its introduction in 2002, the AmpF?STR^® SEfiler™ kit has provided a highly discriminating DNA profiling option to German forensic laboratories by combining the widely used SGM Plus^® Kit loci with the SE-33 locus required for the German DNA Database. Whilst proving successful on database samples, laboratories using the SEfiler™ kit have reported the need for chemistry better able to handle the ever-increasing number of casework samples.The new AmpF?STR^® SEfiler Plus™ kit contains the same loci and primer sequences as the SEfiler™ kit but uses improved synthesis and purification processes to minimize the presence of dye-labeled artifacts. Other improvements include modified PCR cycling conditions for enhanced sensitivity and a new buffer formulation that improves performance with inhibited samples when compared to the original SEfiler™ kit.Validation studies demonstrating the effectiveness of the multiplex are presented with emphasis on the models of inhibition and casework samples.     

Comparative analysis of 1-phenyl-2-propanone (P2P), an amphetamine-type stimulant precursor, using stable isotope ratio mass spectrometry: Presented in part as a poster at the 2nd meeting of the Joint European Stable Isotope User Meeting (JESIUM), Giens, France, September 2008

The isotope ratios of amphetamine type stimulants (ATS) depend as well on the precursor as the synthetic pathway. For clandestine production of amphetamine and methamphetamine, 1-phenyl-2-propanone (P2P, benzylmethylketone) is a commonly used precursor.Our aim was to determine the variation of the isotope ratios within precursor samples of one manufacturer and to compare seized samples of unknown sources to these values. δ¹³C_V-PDB, δ²H_V-SMOW and δ¹⁸O_V-SMOW isotope ratios were determined using elemental analysis (EA) and gas chromatography (GC) coupled to an isotope ratio mass spectrometer (IRMS). The comparison of all seized samples to the data of the samples of one manufacturer revealed considerable differences. The results show that IRMS provides a high potential in differentiating between precursors from different manufacturers for the clandestine production of ATS and identifying corresponding sources.     

Detection of counterfeit antiviral drug Heptodin™ and classification of counterfeits using isotope amount ratio measurements by multicollector inductively coupled plasma mass spectrometry (MC-ICPMS) and isotope ratio mass spectrometry (IRMS)

Isotope ratio mass spectrometry (IRMS) and multicollector inductively coupled plasma mass spectrometry (MC-ICP-MS) are highly important techniques that can provide forensic evidence that otherwise would not be available. MC-ICP-MS has proved to be a very powerful tool for measuring high precision and accuracy isotope amount ratios. In this work, the potential of combining isotope amount ratio measurements performed by MC-ICP-MS and IRMS for the detection of counterfeit pharmaceutical tablets has been investigated.An extensive study for the antiviral drug Heptodin™ has been performed for several isotopic ratios combining MC-ICP-MS and an elemental analyser EA-IRMS for stable isotope amount ratio measurements. The study has been carried out for 139 batches of the antiviral drug and analyses have been performed for C, S, N and Mg isotope ratios. Authenticity ranges have been obtained for each isotopic system and combined to generate a unique multi-isotopic pattern only present in the genuine tablets. Counterfeit tablets have then been identified as those tablets with an isotopic fingerprint outside the genuine isotopic range.The combination of those two techniques has therefore great potential for pharmaceutical counterfeit detection. A much greater power of discrimination is obtained when at least three isotopic systems are combined. The data from these studies could be presented as evidence in court and therefore methods need to be validated to support their credibility. It is also crucial to be able to produce uncertainty values associated to the isotope amount ratio measurements so that significant differences can be identified and the genuineness of a sample can be assessed.     

Developing STR databases on structured populations: The native South Siberian population versus the Russian population

Developing a forensic DNA database on a population that consists of local ethnic groups separated by physical and cultural barriers is questionable as it can be genetically subdivided. On the other side, small sizes of ethnic groups, especially in alpine regions where they are sub-structured further into small villages, prevent collecting a large sample from each ethnic group. For such situations, we suggest to obtain both a total population database on allele frequencies across ethnic groups and a list of θ-values between the groups and the total data. We have genotyped 558 individuals from the native population of South Siberia, consisting of nine ethnic groups, at 17 autosomal STR loci of the kit packages AmpFlSTR SGM Plus и AmpFlSTR Profiler Plus. The groups differentiate from each other with average θ-values of around 1.1%, and some reach up to three to four percent at certain loci. There exists between-village differentiation as well. Therefore, a database for the population of South Siberia is composed of data on allele frequencies in the pool of ethnic groups and data on θ-values that indicate variation in allele frequencies across the groups. Comparison to additional data on northeastern Asia (the Chukchi and Koryak) shows that differentiation in allele frequencies among small groups that are separated by large geographic distance can be even greater. In contrast, populations of Russians that live in large cities of the European part of Russia are homogeneous in allele frequencies, despite large geographic distance between them, and thus can be described by a database on allele frequencies alone, without any specific information on θ-values.     

The current status of forensic science laboratory accreditation in Europe

Forensic science is gaining some solid ground in the area of effective crime prevention, especially in the areas where more sophisticated use of available technology is prevalent. All it takes is high-level cooperation among nations that can help them deal with criminality that adopts a cross-border nature more and more. It is apparent that cooperation will not be enough on its own and this development will require a network of qualified forensic laboratories spread over Europe. It is argued in this paper that forensic science laboratories play an important role in the fight against crime. Another, complimentary argument is that forensic science laboratories need to be better involved in the fight against crime. For this to be achieved, a good level of cooperation should be established and maintained. It is also noted that harmonization is required for such cooperation and seeking accreditation according to an internationally acceptable standard, such as ISO/IEC 17025, will eventually bring harmonization as an end result. Because, ISO/IEC 17025 as an international standard, has been a tool that helps forensic science laboratories in the current trend towards accreditation that can be observed not only in Europe, but also in the rest of the world of forensic science. In the introduction part, ISO/IEC 17025 states that "the acceptance of testing and calibration results between countries should be facilitated if laboratories comply with this international standard and if they obtain accreditation from bodies which have entered into mutual recognition agreements with equivalent bodies in other countries using this international standard." Furthermore, it is emphasized that the use of this international standard will assist in the harmonization of standards and procedures. The background of forensic science cooperation in Europe will be explained by using an existing European forensic science network, i.e. ENFSI, in order to understand the current status of forensic science in Europe better. The Council of Europe and the European Union approaches to forensic science will also be discussed by looking at the legal instruments and documents published by these two European organizations. Data collected from 52 European forensic science laboratories will be examined and findings will be evaluated from a quality assurance and accreditation point of view. The need for harmonization and accreditation in forensic science will be emphasized. The steps that should be taken at the European level for increasing and strengthening the role of European forensic science laboratories in the fight against crime will be given as recommendations in the conclusion.     

Mutation rates at 14 STR loci in the population from Pernambuco, Northeast Brazil

Definition about mutation rates of short tandem repeats (STRs) loci used in forensic analysis are useful for the correct interpretation of resulting genetic profiles and the definition of criterions for exclusion in paternity testing. Germline mutation of 14 STR loci was studied for 54,105 parent–child allelic transfers from 2575 paternity testing cases carried out during 2000–2007 from the Pernambuco State, Northeast Brazil. The parenthood in each of these cases was highly validated (probability > 99.99%). We identified 43 mutations at 12 loci. Locus-specific mutation rate estimates varied between 2 × 10⁻⁴ and 2 × 10⁻³, and the overall mutation rate estimate was 8 × 10⁻⁴. Mutation events in the male germline were more frequent than in the female germline. The majority of the mutations could be explained by losses or gains of one repeat unit and there was no evidence for selection between insertion or deletion changes. Our data were compared with those of Portuguese and North-American populations for CSF1PO, D18S51, D21S11, D7S820, TH01, TPOX and demonstrated, despite the great difference in the size of the sample, that mutation rates of STR loci in a mixed population do not differ from that encountered in different populations.     

Lead isotope measurement of primer gunshot residues and likelihood ratio predictions for forensic cartridge discrimination and individualization in China

Gunshot residues (GSR), cartridge projectiles, and casings are frequently encountered evidence in gun-related forensic investigations. However, in circumstances where the investigation of striation marks is impossible, such as unrecovered or deformed projectiles and cartridge casings, GSR deposited on the hands or clothes of the shooter and victim-related items can provide information to establish a link between the suspect, the firearms used, and the victim. Since the formula of primers used by all cartridge manufacturers in China is identical, links based on the conventional morphological and compositional analysis of GSR are difficult to establish. However, the abundance of lead isotopes in primer components of lead styphnate varies significantly, and a fundamental understanding of these differences may facilitate the validation of primer (p)GSR evidence in forensic investigations. Here, 44 pGSR samples were characterized by Pb isotope ratios of ²⁰⁶Pb/²⁰⁴Pb, ²⁰⁷Pb/²⁰⁴Pb, and ²⁰⁸Pb/²⁰⁴Pb using laser ablation multicollector inductively coupled plasma mass spectrometry. There was no obvious mass fractionation of the lead isotope ratios of the primers from individual cartridges analyzed before and after the shooting process, thereby establishing a basis for the comparison of pGSR and unfired cartridges. Evaluation of the results using univariate likelihood ratio (LR) computations revealed low rates of misleading evidence (<0.53%) The results demonstrated that lead isotope ratio analysis of pGSR and LR predictions can provide a practicable method for forensic cartridge discrimination and individualization.