An unusual appearance of a common pollen type indicates the scene of the crime

Forensic palynology is a useful source of evidence in cases of violence committed in the open. A young girl was grabbed off the street, threatened and brutally raped. During the investigation the exact place of the rape became an issue. Growing around the scene identified by the victim were shrubs identified as Coprosma, a common New Zealand plant and one that produces abundant, easily wind-dispersed pollen. Abundant Coprosma pollen was found at the scene. The pollen were unusual in that the site was very damp, encouraging fungal growth, and fungal hyphae had penetrated the pores of many of the tricolporate pollen grains. Some grains had fungal spores inside. Coprosma pollen identical in preservational characteristics and morphology to those from the scene and containing fungal hyphae and spores were found in considerable numbers on the victim's clothes. This and rare Coprosma pollen grains and fungal remains recovered from vaginal swabs provided evidence that she had been at the scene where she claimed to have been raped. The diversity of pollen types recovered from the clothing in this case provides further evidence of the usefulness of clothing in picking up and retaining pollen from crime scenes and that obvious staining on clothes is not a pre-requisite for good pollen recovery. It also demonstrates the importance of collecting samples from different parts of the same garment in order to get a full picture of events since different parts of a garment can come into contact with different plants or different parts of the ground in a scuffle. It is also demonstrated that significant evidential material can be collected from the body, in this case from vaginal swabs from the victim. Forensic palynology should be considered in every case of violent assault, especially, but not exclusively, when having occurred in an open area subject to extensive pollen settlement.     

Limitations and recommendations for successful DNA extraction from forensic soil samples: A review

Soil is commonly used in forensic casework to provide discriminatory power to link a suspect to a crime scene. Standard analyses examine the intrinsic properties of soils, including mineralogy, geophysics, texture and colour; however, soils can also support a vast amount of organisms, which can be examined using DNA fingerprinting techniques. Many previous genetic analyses have relied on patterns of fragment length variation produced by amplification of unidentified taxa in the soil extract. In contrast, the development of advanced DNA sequencing technologies now provides the ability to generate a detailed picture of soil microbial communities and the taxa present, allowing for improved discrimination between samples. However, DNA must be efficiently extracted from the complex soil matrix to achieve accurate and reproducible DNA sequencing results, and extraction efficacy is highly dependent on the soil type and method used. As a result, a consideration of soil properties is important when estimating the likelihood of successful DNA extraction. This would include a basic understanding of soil components, their interactions with DNA molecules and the factors that affect such interactions. This review highlights some important considerations required prior to DNA extraction and discusses the use of common chemical reagents in soil DNA extraction protocols to achieve maximum efficacy. Together, the information presented here is designed to facilitate informed decisions about the most appropriate sampling and extraction methodology, relevant both to the soil type and the details of a specific forensic case, to ensure sufficient DNA yield and enable successful analysis.     

Bacterial Profiling of Soil For Forensic Investigations: Consideration of Ex Situ Changes in Questioned and Known Soil Samples

Soil, being diverse and ubiquitous, can potentially link a suspect or victim to a crime scene. Recently scientists have examined the microbial makeup of soil for determining its origin, and differentiating soil samples is well-established. However, when soil is transferred to evidence its microbial makeup may change over time, leading to false exclusions. In this research, “known” soils from diverse habitats were stored under controlled conditions, while evidence soils were aged on mock evidence. Limited quantities of soil were also assayed. Bacterial profiles were produced using next-generation sequencing of the 16S rRNA gene. Overall, known soils stored open at room temperature were more similar to evidence soils over time than were known soils stored bagged and/or frozen. Evidence soils, even as little as 1 mg, associated with the correct habitat 99% of the time, accentuating the importance of considering ex situ microbial changes in soil for its successful use as forensic evidence.     

泥土中植物花粉的提取与检验

目的建立泥土中植物花粉的提取和检验方法。方法用去二甲苯、18%盐酸溶液处理提取泥土中的花粉 ,然后用扫描电镜进行检验。结果从不同的泥土中检出不同花粉。结论本方法能有效分离提取泥土中花粉。对其它混合物中花粉的提取有参考价值。本文介绍一种泥土中花粉提取的简捷方法 ,以及利用扫描电镜 /能谱仪对提取的植物花粉进行检验的方法。     

The use of crossover immunoelectrophoresis to detect human blood protein in soil from an ambush scene in Kosovo

This study examines the survivability of human blood proteins in soils from a year and a half old ambush scene in Kosovo. A total of 72 soil samples were collected, a number of which were directly associated with bone fragments or bullet projectiles. The samples were examined using crossover immunoelectrophoresis (CIEP) to determine the presence of blood protein and species affiliation. Human blood proteins were identified in 44 of the 72 samples (61%) with the majority of the positive observations (29 of 44) found 0.0-4.5 cm below ground surface (65%). Chi-squared and two-sample difference of proportions tests confirmed significant differences between samples with and without associated physical evidence and the presence and depth of human blood proteins. While DNA has largely replaced immunological analysis in forensic analyses, our results suggest that in particular situations, CIEP may still be a valuable tool in criminology.     

Forensic palynology: current status of a rarely used technique in the United States of America

The United States of America would seem to be an excellent location for using pollen data in forensic applications. The vegetation within the region is highly diverse ranging from areas of Arctic tundra to some of the most inhospitable deserts anywhere in the Western Hemisphere. The highly varied ecology, great plant diversity, thousands of vegetational microhabitats, and extensive published pollen records for the region provide an ideal setting for these types of analyses. This diversity, often characterized in most locations by unique combinations of pollen types, makes the use of forensic pollen a reliable technique that can often be used to associate individuals with a unique crime scene or geographical region. Nevertheless, forensic pollen studies in the United States of America are currently one of the most highly under utilized techniques available to assist in solving criminal and civil cases. During the past century there has been a very limited attempt to use pollen evidence in either criminal or civil cases, for a variety of reasons, including a lack of available information about the technique, a very limited number of specialists trained to do forensic pollen work, and an almost total absence of academic centers able to train needed specialists or forensic facilities able, or willing, to fund research in this area. Hopefully, this paucity of use will change if certain steps are taken to encourage the routine collection and use of pollen evidence in both criminal and civil cases.     

Sub-sampling and preparing forensic samples for pollen analysis

The main forensic application of palynology is in providing associative evidence, assisting to prove or disprove a link between people and objects with places or with other people. Although identification and interpretation of pollen is a specialist job, sub-sampling and preparing pollen samples for analysis may be carried out by non-specialists. As few forensic laboratories have residing palynologists, laboratories may wish to reduce the cost of analysis or risk of contamination by doing their own sub-sampling and preparation. Presented is a practical guide for sub-sampling and preparing forensic samples for pollen analysis, providing a complete standard procedure for both the palynologist and non-specialist. Procedures for sub-sampling include a wide variety of materials commonly collected for forensic analysis (soil, clothing and other fabrics, footwear, twine and rope, firearms, granulated materials, plant and animal material, and illicit drugs), many of which palynologists will not be familiar with. Procedures for preparation of samples (pollen concentration) are presented as a detailed, step-by-step method. Minimizing the risks of laboratory and cross-sample contamination during sub-sampling and preparation is emphasized.     

Pollen on grass clippings: putting the suspect at the scene of the crime.

In a case of alleged sexual assault, the pollen content of samples of grass clippings and soil from the suspect's clothing and shoes was compared to that of a sample of grass clippings from the alleged crime scene (a grassy area) to determine whether or not the suspect had been at the scene. The clothing and shoe samples showed a very strong correlation with each other and with the sample from the alleged crime scene in the combination of the different types of pollen present, very strongly supporting the contention that the suspect had been at the scene.     

Time Radically Alters Ex Situ Evidentiary Soil 16S Bacterial Profiles Produced Via Next‐Generation Sequencing,,

Previous research has revealed the potential of soil bacterial profiling for forensic purposes; however, investigators have not thoroughly examined fluctuations in microbial profiles from soil aged on evidence. In this research, soils collected from multiple habitats were placed on evidence items and sampled over time, and then bacterial profiles were generated via next‐generation sequencing of the 16S rRNA locus. Bacterial abundance charts and nonmetric multidimensional scaling plots provided visual representation of bacterial profiles temporally, while supervised classification was used to statistically associate evidence to a source. The ex situ evidence soils displayed specific, consistent taxonomic changes as they aged, resulting in their drift in multidimensional space, but never toward a different habitat. Ninety‐five percent of the 364 evidentiary profiles statistically classified to the correct habitat, with misclassification generally stemming from evidence type and increased age. Ultimately, understanding bacterial changes that occur temporally in ex situ soils should enhance their use in forensic investigations.     

Hair as a source of forensic evidence in murder investigations

Obtaining palynological and other botanical evidence from murder victims is becoming part of routine mortuary protocol in the United Kingdom. Forensic pathologists are often keen to cooperate in the collection of classes of material that have, in the past, been considered to be of little importance in criminal investigation. Work over the last eight years has demonstrated the great value in scrutinising cadavers for the presence of plant material and/or soil stains. Macroscopic plant remains and palynomorphs (pollen, spores and other microscopic entities) retrieved from skin and hair have allowed the differentiation of murder scenes from places of eventual deposition. Furthermore, although the opportunity has not yet presented itself, obtaining palynological evidence from the hair of suspects is feasible. During an offence, the offender might have had physical contact with foliage or the ground. Pollen and spore assemblages picked up by hair during that activity might provide forensic evidence for contact. Brief details of some aspects of case histories are presented to demonstrate the value of sampling cadavers. One case has been through the courts while the other is ongoing and, therefore, cannot be identified.     

Particle size analysis of soils under simulated scene of crime conditions: the interest of multivariate analyses

Two simulated scenes of crime have been studied. Soil traces adhering to boots, sport shoes and tissues have been compared with control samples using particle size analysis. Comparisons of percentage of particles per class interval and multivariate analyses were used to determine how the size distribution of each suspect sample varied compared to the original distribution. A loss of coarse fractions characterized most of the suspect samples even though they were derived from soils having different distributional patterns. However, the differences between control and suspect samples were found to be more important for soils dominated by extreme size classes than for soils rich in medium size particles. The differences among soil samples from the same source were found to be restricted to the extreme size classes. The finding of significant differences within size classes ranging from 1 to 0.063 mm should be then considered as a dissimilarity sign between trace sample and hypothetic control soil.     

Macroscopic assessment of environmental trace evidence dynamics in forensic settings

Environmental trace evidence offers useful circumstantial intelligence to link persons and scenes of forensic interest. An increasing empirical research base is dedicated towards understanding the transfer and persistence dynamics of environmental indicators including pollen, soils, and diatoms, within a diverse range of experimental frameworks. This paper presents two discrete studies exploring transfer and persistence of soils and sediments on footwear and diatomaceous earth adhered to clothing in forensically pertinent scenarios. Variables including sediment type, foot position, clothing type, and body positioning were also explored throughout. Both experiments incorporated a field-based methodology during the sampling effort. Photographs were collected of an initial transfer sample and of a retained assemblage following hours, days, and up to one-week of wear, facilitating macroscopic assessment of trace evidence dynamics. All images were processed using accessible, open-source software before spatial analysis of evidence distribution within and temporal assessment (% retention) upon each evidential surface. The results highlighted consistent loss of transferred sediment from footwear with significantly greater retention of loamy clay soil than dune sand which was absent beyond 24 h of wear. Loss was not influenced by wearer gait but was more rapid from those areas of the shoe sole in direct contact with the ground. Diatomaceous earth was retrieved from all three clothing types tested after one week – significant losses of material occurred before 48 h with a consistent assemblage identified beyond this. Denim was significantly more effective than acrylic and fleece for diatomaceous earth retention and significantly more material was lost from clothing worn on the lower body. These findings highlight the value of using visual environmental markers and a macroscopic analytical approach during the investigation of environmental trace dynamics. The methodology offers a novel, non-destructive assessment of soil and diatom transfer and persistence, complementing more extensive laboratory-based examinations to ensure the development of a well-rounded research base within the forensic sciences.     

Particle size analysis of soils under simulated scene of crime conditions: the interest of multivariate analyses

Two simulated scenes of crime have been studied. Soil traces adhering to boots, sport shoes and tissues have been compared with control samples using particle size analysis. Comparisons of percentage of particles per class interval and multivariate analyses were used to determine how the size distribution of each suspect sample varied compared to the original distribution. A loss of coarse fractions characterized most of the suspect samples even though they were derived from soils having different distributional patterns. However, the differences between control and suspect samples were found to be more important for soils dominated by extreme size classes than for soils rich in medium size particles. The differences among soil samples from the same source were found to be restricted to the extreme size classes. The finding of significant differences within size classes ranging from 1 to 0.063 mm should be then considered as a dissimilarity sign between trace sample and hypothetic control soil.     

Some factors affecting soil sieve analysis in forensic science. 1. Dry sieving

The results reported in this study indicate that careful control of the applied torque settings and instrumental modification can play an important part in the determination of particle size distributions of soils using the dry sieving technique. Determinations were highly repeatable and increased the sensitivity of the method for comparative soil analyses used to test for similarity. Experimental results also suggested that equal weights of soils should be compared in the determinations of both control and questioned samples.     

The recovery of pollen evidence from documents and its forensic implications

Three experiments were undertaken to establish the potential for forensic palynological analysis in cases of suspected document fraud. The first study tested 6 different types of paper and 9 different types of ink (n = 54) and it was established that the best retainer of particulates (in this case a proxy was used in the form of UV powder) was medium biro ink and Wove and Connoisseur paper. It was found that for the different paper types 42–52% of the particulates collected were found in the ink and thus both the paper and the ink are potentially valuable sources of trace evidence in a forensic investigation. The second study sought to address the differences in the spatial distribution of particulates on documents when writing took place before or after the paper was treated with UV particulates. Ninety-six observations were made for each piece of paper tested and it was found that when the writing took place after the particulates were applied to the paper; more particulates were retained on the paper in contrast to when the writing took place before the particulate treatment. The spatial distribution of particulates was also affected, with particulates being retained in the folds of the paper when the writing took place before particulate treatment in contrast to a more erratic pattern that emerged due to the pressure of the hand of the writer when the writing took place after the particulate treatment. The third study utilised lily (Lilium) pollen grains and the findings broadly concurred with the second study. The main difference identified was when the writing took place before the particulates were applied; when UV powder was used the particulates were retained in the folds of the paper whereas this pattern was not seen to the same degree when pollen grains were used due to their ‘stickier’ nature. Envelopes and the pen nibs were also found to be rich sources of pollen grains after the experiments were undertaken.These studies have implications for the application of forensic palynology in cases of suspected document fraud. Pollen grains may well be present, and their analysis has the potential to reveal not only the timing of the generation of the document, but the spatial trends revealed indicate that it may well be possible to establish the sequence of significant events for forensic reconstruction. As such forensic palynology is demonstrated to have great potential in aiding forensic investigations, and is as yet an under-utilised form of trace evidence.     

The role of forensic geoscience in wildlife crime detection

The increase in both automation and precision in the analysis of geological materials has had significant impact upon forensic investigations in the last 10 years. There is however, a fundamental philosophical difference between forensic and geological enquiry. This paper presents the results of forensic geoscientific investigations of three cases of wildlife crime. Two cases involve the analysis of soils recovered after incidents of illegal badger baiting in the United Kingdom. The third case involves the illegal importation of Eleonora's Falcon (Falco eleonorae) into the United Kingdom from the Mediterranean. All three cases utilise the analysis of soils by a variety of physical, chemical and biological techniques. These involve mineral and grain size analyses, cation and anion compositions, pH, organic content and pollen analysis. The independent analysis undertaken by specialists in each of these three main fields conclude firstly, that there is a significant similarity between sediments taken at the crime site at both badger setts and with sediments recovered from various spades, shovels and clothing belonging to suspects and secondly, that the soils analysed associated with the removal of the falcon eggs in the Mediterranean contained characteristics similar in many respects to the soils of the breeding areas of F. eleonorae on the cliffs of Mallorca. The use of these independent techniques in wildlife crime detection has great potential given the ubiquitous nature of soils and sediments found in association with wildlife sites.     

Understanding Variations of Soil Mapping Units and Associated Data for Forensic Science

Soil samples have potential to be useful in forensic investigations, but their utility may be limited due to the inherent variability of soil properties, the wide array of analytical methods, and complexity of data analysis. This study examined the differentiation of similar soils based on both gross (texture, color, mineralogy) and explicit soil properties (elemental composition, cation exchange, Fe‐oxyhydroxides). Soils were collected from Fallbrook and adjacent map units from Riverside and San Diego Counties in California. Samples were characterized using multiple techniques, including chemical extracts, X‐ray diffraction (XRD), and Fourier transform infrared spectroscopy. Results were analyzed using multiple analytical approaches to compare counties and land uses. Some analyses (XRD, extractions) were better at distinguishing among samples than others (color, texture). Ratios of rare earth elements were particularly useful for distinguishing samples between counties. This potential to “fingerprint” soils illustrates the usefulness of a comprehensive soil database for criminal investigators.     

High‐throughput Sequencing of Trace Quantities of Soil Provides Reproducible and Discriminative Fungal DNA Profiles

High‐throughput sequencing (HTS) offers improved resolution between forensic soil samples by characterizing individual taxa present; however, the heterogeneous distribution of taxa in soils, and limited quantity of material available, may hinder the reliability of HTS in casework. Using HTS of the internal transcribed spacer, we examined the effect of soil mass (50, 150, and 250 mg) on fungal DNA profiles, focusing on reproducibility and discriminatory power between close proximity soils, and samples with similar textural classification. The results show that reduced soil mass had no significant effect on sample differentiation and that 150 mg soil provides the most reproducible DNA profiles across different soil types. In addition, Ascomycota was identified as a robust fungal target for forensic intelligence as this phylum was detected consistently across all samples regardless of sample quantity. Overall, this study highlights the value of trace quantities of soil for use in forensic casework.