Experimental forensic studies of the preservation of pollen in vehicle fires

The implications of the recent recommendations of the Law Commission regarding the use of admissibility tests have the potential to be far reaching for forensic disciplines that rely on the expertise of highly qualified expert witnesses. These disciplines will need a concomitant body of peer-reviewed experiments that provides a basis for the interpretations of such evidence presented in court. This paper therefore, presents such results from two experiments which were undertaken to address specific issues that were raised in cases presented in the British courtroom. These studies demonstrate that there is a variability in the persistence of Lily, Daffodil and Tulip pollen when exposed to high temperatures between 0.5 min and 1440 min (24 h). It was possible to identify all three pollen types after 30 min of exposure to 400 °C, and after shorter time frames the threshold for successful identification was 700 °C after 0.5 min for all pollen types tested and 500 °C for Daffodil and Lily after 5 min of heat exposure. Over longer time periods (18 h (1080 min)) the different pollen types were found to persist in a viable form for identification at 300 °C (Lily), 200 °C (Daffodil) and 50 °C (Tulip). These findings, albeit from a small sample of pollen types, provide empirical contextual information that would contribute to such evidence having sufficient scientific weight to meet admissibility criteria and be viable evidence for a court. These studies demonstrate the value in seeking pollen evidence from even such extreme crime scenes as encountered in vehicular fires.     

Post-cremation taphonomy and artifact preservation

Contemporary commercial cremation is a reductive taphonomic process that represents one of the most extreme examples of postmortem human alteration of bone. The thorough reduction and fragmentation of cremated human remains often leaves little biological evidence of diagnostic value. Instead, non-osseous artifacts often provide the best evidence of the origin of the cremated remains, the identity of the decedent, and commingling of the remains of more than one individual. Once human remains have been cremated they are most commonly placed into a processor and reduced into small fragments and fine ash suitable for inurnment or scattering. The type of processor determines the size and utility of the particulates and artifacts available for analysis. The newest type of processors have changed the manner and degree of postmortem bone modification and altered the preservation of diagnostic bone fragments and cremation artifacts. This paper addresses the impact of the newest cremation procedures on forensic analysis of cremated remains.     

Detection of acute fentanyl exposure in fresh and decomposed skeletal tissues part II: The effect of dose–death interval

The effects of dose–death interval on the detection of acute fentanyl exposure in fresh and decomposed skeletal tissues (marrow and bone), by automated enzyme-linked immunosorbent assay (ELISA) are described. Rats (n = 14) were administered fentanyl acutely at a dose of 0 (n = 2) or 60 μg/kg (n = 12) by intraperitoneal injection, and euthanized within 20, 45, 135, or 225 min. Femora and tibiae were extracted from the fresh corpses and marrow was isolated from the femoral and tibial medullary cavities. The remains were then allowed to decompose outdoors to the point of complete skeletonization, and vertebrae, pelvi and miscellaneous (humeri and scapulae) were recovered for analysis. In all cases, bones were cleaned in alkaline solution and then ground into a fine powder. Marrow was homogenized in alkaline solution. Fentanyl was extracted from ground bone by methanolic extraction. Extracts were adjusted to pH 6 and analyzed by ELISA. Perimortem heart blood was also collected and diluted in phosphate buffer prior to screening by ELISA. The effect of tissue type on ELISA response was examined through determination of binary classification test sensitivity and the relative decrease in absorbance (%DA, drug-positive tissues vs. drug-free controls) in each tissue type. Overall, the %DA varied significantly between extracts from different skeletal tissues at a given dose–death interval, according to the general order of marrow > decomposed bone > fresh bone. Binary classification test sensitivity values for fentanyl in marrow, fresh epiphyseal (femoral and tibial) bone, fresh diaphyseal (femoral and tibial) bone, decomposed vertebrae, decomposed pelvic bone, and decomposed miscellaneous bone were 67–100%, 0–33%, 0–33%, 0–67%, 0–67% and 0–33%, respectively, over all dose–death intervals. Although group mean %DA values showed a strong negative correlation with dose–death interval in marrow, fresh epiphyseal bone, decomposed vertebrae, pelvic and miscellaneous bone (r = ?0.989, ?0.930, ?0.955, ?0.903, and ?0.974, respectively), the high variability in both fresh and decomposed bone precluded differentiation of the dose–death intervals based on %DA value alone. Overall, the results suggested that the type of skeletal tissue sampled may not be as important as the amount of residual marrow remaining in skeletonized remains.     

The neuroendocrine effects of the TASER X26®: A brief report

IntroductionLaw enforcement officers use conducted electrical weapons (CEW) such as the TASER X26^® to control violently resistive subjects. There are no studies in the medical literature examining the effects of these weapons on the human stress response. This is the first study to compare the human stress response to conducted electrical weapons, oleoresin capsicum (O.C.), a cold-water tank immersion, and a defensive tactics drill.MethodsSubjects were randomized to one of the four interventions studied. Subjects received either a 5-s exposure from the TASER X26 CEW with the probes fired into the back from 7 ft, a 5-s spray of O.C., a skin and mucous membrane irritant, to the eyes, a 45-s exposure of the hand and forearm in a 0 °C cold water tank, or a 1-min defensive tactics drill.ResultsAlpha-amylase had the greatest increase from baseline at 10–15 min with the defensive tactics drill. Cortisol had the greatest increase at 15–20 min with O.C. Cortisol remained most elevated at 40–60 min in the defensive tactics drill group.ConclusionsOur preliminary data suggests that physical exertion during custodial arrest may be most activating of the human stress response, particularly the sympathetic–adrenal–medulla axis. This may suggest that techniques to limit the duration of this exertion may be the safest means to apprehend subjects, particularly those at high-risk for in-custody death. Conducted electrical weapons were not more activating of the human stress response than other uses of force.     

Duplex-specific nuclease (DSN): A method for the rehabilitation of low-copy number DNA profiles

A continual challenge in the field of forensic DNA analysis is the amplification and interpretation of degraded and low-copy number (LCN) DNA obtained from amounts of limited biological evidence. It has been well established that DNA profiles obtained from the amplification of low quality, degraded, and/or LCN DNA samples are often of limited value due to the frequent occurrence of preferential amplification during polymerase chain reaction (PCR). The by-products of preferential PCR amplification are often observed as inter- and intra-locus peak imbalance, allelic dropout, and/or locus dropout. These are all artifacts that are identified during the interpretation phase of analysis rather than by improving the quality of the DNA present. While it is theoretically possible to obtain a complete DNA profile from a single cell, in reality, profiles obtained from suboptimal amounts of DNA are difficult to interpret and frequently inconsistent when replicated. Inspired by advances in next-generation sequencing techniques, we propose a methodology for simultaneously normalizing the abundance of PCR products across all short tandem repeat (STR) loci using the DNA exonuclease, duplex-specific nuclease (DSN). DSN is an enzyme isolated from the hepatopancreas of Red King (Kamchatka) crab that possesses a strong affinity for digesting double stranded DNA (dsDNA) and has limited activity toward single stranded DNA (ssDNA). Degraded DNA known to display peak imbalance and allele dropout was amplified using AmpFlSTR^® Identifiler^® Plus for 28 cycles. Following amplification, samples were denatured at 99.9 °C for 5 min and incubated with one unit of DSN at 62 °C in a 28 μl volume for 1 min. Nuclease activity was terminated through the addition of equal volume of 10 mM EDTA and 95 °C incubation for 2 min. Following DSN treatment, 21 of 30 alleles within the known profile exhibited some improvement in peak height balance. The findings obtained support the potential use of DSN treatment as a method for normalizing STR profiles and improving the quality of data from degraded and low quantity DNA samples.     

Rapid GC–MS confirmation of amphetamines in urine by extractive acylation

Amphetamine and related derivatives are widely abused central- and psychostimulants. Detection of certain derivatives, such as methcathinone, by commonly available immunoassay screening techniques is insufficient. Multi-analyte confirmations for amphetamine type stimulants are therefore required, but traditional gas chromatography–mass spectrometry methods necessitate lengthy analytical procedures with prolonged sample turn-around times. A validated rapid GC–MS assay for urinary confirmation of amphetamine, methamphetamine, methcathinone, ephedrine, norephedrine, methylenedioxyamphetamine, methylenedioxymethamphetamine, methylenedioxyethylamphetamine and N-methyl-1-(3,4 methylenedioxyphenyl)-2-butanamine is reported. The method entailed in situ derivatization of urine specimens by extractive acylation with pentafluoropropionic anhydride, followed by rapid chromatography on a microbore capillary column. Analytes were separated in less than 3 min and quantified simultaneously by selected-ion monitoring using stable isotope substituted internal standards. The total instrument cycle-time was 6 min per sample. The limits of detection were between 1.5 ng/mL and 6.25 ng/mL for the various analytes. Intermediate precision and accuracy were in the range of 6.3–13.8% and 90.5–107.3% for the respective analytes at the lower limit of quantitation, and between 5.8–12.6% and 95.4–103.1% for the high control. Long-term storage of methcathinone positive specimens at ?20 °C proved insufficient stability of this analyte. The proposed assay is precise and accurate for confirmation of amphetamine and derivatives in urine. The complementary approach of extractive-derivatization and fast GC–MS analysis is especially applicable in routine clinical settings where reduced sample turn-around times are required. Further investigation of cathinone as a possible metabolite of methcathinone is warranted, based on results from analyzed authentic urine samples.     

Determination of amphetamine-type stimulants,ketamine and metabolites in fingernails by gas chromatography–mass spectrometry

A gas chromatography–mass spectrometry (GC–MS) method was developed and validated for the simultaneous qualification and quantification of methamphetamine (MA), amphetamine (AP), 3,4-methylenedioxy-N-methylamphetamine (MDMA), 3,4-methylenedioxy-N-amphetamine (MDA), ketamine (KET) and norketamine (NKT) in fingernails. Fingernail samples (20 mg) were washed with distilled water and methanol, digested with 1.0 M sodium hydroxide at 95 °C for 30 min, and then extracted with ethyl acetate. Extract solutions were evaporated to dryness, derivatized using heptafluorobutyric anhydride (HFBA) at 60 °C for 30 min, and analyzed by GC–MS. The linear ranges were 0.1–20.0 ng/mg for AP, MDMA and NKT, 0.2–20.0 ng/mg for MA and MDA, and 0.4–20.0 ng/mg for KET, with the coefficients of determination (r² ≥ 0.9989). The intra- and inter-day precisions were within 7.1% and 10.6%, respectively. The intra- and inter-day accuracies were ?10.9% to 0.8% and ?4.3% to 4.5%, respectively. The limits of detections (LODs) and the limits of quantifications (LOQs) for each analyte were lower than 0.094 ng/mg and 0.314 ng/mg, respectively. The recoveries were in the range of 72.3–94.9%. The average fingernail growth rates of two subjects for three years and six subjects for two months were 3.12 mm/month and 3.16 mm/month, respectively. The method proved to be suitable also for the simultaneous detection and quantification of MA, MDMA, KET and their metabolites in fingernails.     

A Case of Contested Cremains Analyzed Through Metric and Chemical Comparison

Since the 1980s, cremation has become the fastest growing area of the U.S. funeral industry. At the same time, the number of litigations against funeral homes and cremation facilities has increased. Forensic anthropologists are often asked to determine whether the contents of an urn are actually cremated bone, and to address questions regarding the identity of the remains. This study uses both metric and chemical analyses for resolving a case of contested cremains. A cremains weight of 2021.8 g was predicted based on the decedent's reported stature and weight. However, the urn contents weighed 4173.5 g. The urn contents also contained material inconsistent with cremains (e.g., moist sediment, stones, ferrous metal). Analysis using XRD and SEM demonstrated that the urn contained thermally altered bone as well as inorganic material consistent with glass fiber cement. Although forensically challenging, cremains cases such as this one can be resolved using a multidisciplinary approach.     

Freezing skeletal muscle tissue does not affect its decomposition in soil: Evidence from temporal changes in tissue mass,microbial activity and soil chemistry based on excised samples

The study of decaying organisms and death assemblages is referred to as forensic taphonomy, or more simply the study of graves. This field is dominated by the fields of entomology, anthropology and archaeology. Forensic taphonomy also includes the study of the ecology and chemistry of the burial environment. Studies in forensic taphonomy often require the use of analogues for human cadavers or their component parts. These might include animal cadavers or skeletal muscle tissue. However, sufficient supplies of cadavers or analogues may require periodic freezing of test material prior to experimental inhumation in the soil. This study was carried out to ascertain the effect of freezing on skeletal muscle tissue prior to inhumation and decomposition in a soil environment under controlled laboratory conditions. Changes in soil chemistry were also measured. In order to test the impact of freezing, skeletal muscle tissue (Sus scrofa) was frozen (?20 °C) or refrigerated (4 °C). Portions of skeletal muscle tissue (～1.5 g) were interred in microcosms (72 mm diameter × 120 mm height) containing sieved (2 mm) soil (sand) adjusted to 50% water holding capacity. The experiment had three treatments: control with no skeletal muscle tissue, microcosms containing frozen skeletal muscle tissue and those containing refrigerated tissue. The microcosms were destructively harvested at sequential periods of 2, 4, 6, 8, 12, 16, 23, 30 and 37 days after interment of skeletal muscle tissue. These harvests were replicated 6 times for each treatment. Microbial activity (carbon dioxide respiration) was monitored throughout the experiment. At harvest the skeletal muscle tissue was removed and the detritosphere soil was sampled for chemical analysis. Freezing was found to have no significant impact on decomposition or soil chemistry compared to unfrozen samples in the current study using skeletal muscle tissue. However, the interment of skeletal muscle tissue had a significant impact on the microbial activity (carbon dioxide respiration) and chemistry of the surrounding soil including: pH, electroconductivity, ammonium, nitrate, phosphate and potassium. This is the first laboratory controlled study to measure changes in inorganic chemistry in soil associated with the decomposition of skeletal muscle tissue in combination with microbial activity.     

Recovery of human DNA profiles from poached deer remains part 2: Improved recovery protocol without the need for LCN analysis

Although poaching is a common wildlife crime, the high and prohibitive cost of specialised animal testing means that many cases are left un-investigated. We previously described a novel approach to wildlife crime investigation that looked at the identification of human DNA on poached animal remains (Tobe, Govan and Welch, 2011). Human DNA was successfully isolated and amplified from simulated poaching incidents, however a low template protocol was required which made this method unsuitable for use in many laboratories. We now report on an optimised recovery and amplification protocol which removes the need for low template analysis.Samples from 10 deer (40 samples total — one from each leg) analysed in the original study were re-analysed in the current study with an additional 11 deer samples. Four samples analysed using Chelex did not show any results and a new method was devised whereby the available DNA was concentrated. By combining the DNA extracts from all tapings of the same deer remains followed by concentration, the recovered quantity of human DNA was found to be 29.5 pg ± 43.2 pg, 31 × greater than the previous study. The use of the Investigator Decaplex SE (QIAGEN) STR kit provided better results in the form of more complete profiles than did the AmpF?STR® SGM Plus® kit at 30 cycles (Applied Biosystems). Re-analysis of the samples from the initial study using the new, optimised protocol resulted in an average increase of 18% of recovered alleles. Over 17 samples, 71% of the samples analysed using the optimised protocol showed sufficient amplification for comparison to a reference profile and gave match probabilities ranging from 7.7690 × 10^? 05 to 2.2706 × 10^? 14.The removal of low template analysis means this optimised method provides evidence of high probative value and is suitable for immediate use in forensic laboratories. All methods and techniques used are standard and are compatible with current SOPs. As no high cost non-human DNA analysis is required the overall process is no more expensive than the investigation of other volume crime samples. The technique is suitable for immediate use in poaching incidents.     

Forensic oral imaging quality of hand-held dental X-ray devices: Comparison of two image receptors and two devices

Recently, different portable hand-held and battery-powered dental X-ray units have become available. Especially for forensic odontological purposes, they offer diverse advantages such as for use in disaster areas and crime-scene locations as also in autopsy rooms and mortuaries. For any application, the most important feature of these hand-held devices is the delivered image quality. The aim of this study is to evaluate the radiographic image quality acquired by two portable X-ray devices in combination with two types of image receptors and to compare the findings with the image quality of a standard intra-oral X-ray device.Eleven samples consisting of eight teeth, two dry skeletal specimens and one formalin-fixed mandible part were mounted on blocks for standardised (re)positioning. Radiological images were acquired with two hand-held (AnyRay^® 60 kVp, 0.02–4.00 mAs and NOMAD^® 60 kVp, 0.023–2.277 mAs) and one wall-mounted (MinRay^® 60/70 kVp 0.14–22.4 mAs) X-ray device combined with two image receptor systems (VistaScan^® phosphor storage plate (PSP) and SIGMA^® M CMOS Active Pixel technology sensor). The effect of X-ray source-to-object distance (SOD) was checked at 20 cm in conjunction with object to image receptor distances (OIDs) of 0.8 and 2.5 cm. For each parameter setup, the exposure times were run from low till high. An expert consent statement was achieved by agreement of four expert observers selecting the optimal images based on a developed four point quality rating system. Next, a selection of the images was assembled in a set of 198 observation screens and scored by seven observers. The observation screens were designed to compare observer scores, relations between devices, receptors and OIDs and images obtained from the different devices at equal exposure levels (mAs). All results were statistically analysed.Radiological image quality was significantly higher for phosphor plate compared with the CMOS digital receptor system (p < 0.0001). Furthermore, a significantly superior image quality was obtained for OID = 0.8 than for OID = 2.5 (p = 0.039). A significant difference in image quality between the three devices was also established (p = 0.02). The present study demonstrated the feasibility of portable X-ray systems for forensic odontological applications based on rendering optimal image quality, provided an in vitro guideline of optimal parameter settings and offered a radiological image database usable in further research.     

X‐ray Scattering Evaluation of Ultrastructural Changes in Human Dental Tissues with Thermal Treatment

Micro‐ and ultrastructural analysis of burned skeletal remains is crucial for obtaining a reliable estimation of cremation temperature. Earlier studies mainly focused on heat‐induced changes in bone tissue, while this study extends this research to human dental tissues using a novel quantitative analytical approach. Twelve tooth sections were burned at 400–900°C (30‐min exposure, increments of 100°C). Subsequent combined small‐ and wide‐angle X‐ray scattering (SAXS/WAXS) experiments were performed at the Diamond Light Source synchrotron facility, where 28 scattering patterns were collected within each tooth section. In comparison with the control sample, an increase in mean crystal thickness was found in burned dentine (2.8‐fold) and enamel (1.4‐fold), however at a smaller rate than reported earlier for bone tissue (5–10.7‐fold). The results provide a structural reference for traditional X‐ray scattering methods and emphasize the need to investigate bone and dental tissues separately to obtain a reliable estimation of cremation temperature.     

Pressing need for national governmental recognition of forensic anthropology in South Africa as illustrated in a medico-legal case

Forensic anthropology in South Africa is well developed in the higher education sector, with advanced training and research programmes. Despite this and decades of academic involvement in casework, forensic anthropology still lacks a defined framework and mandate at a governmental level. Therefore, the involvement of forensic anthropologists’ expertise varies markedly between cases, provinces, and among various stakeholders within the country, to the detriment of dispensation of social and criminal justice. The lack of clearly defined guidelines for the rendering of the service was exemplified and demonstrated through a recent forensic case. Here, contextual information was absent, and the remains posed a challenge to analyse, ostensibly due to missing information. Numerous questions were raised during the analysis of the remains, and broader concerns about the investigative involvement of a forensic anthropologist within South African casework were brought to the fore. Through the analysis of this case, we describe the deductive processes that led to the formation of an opinion that the skeletal linear defects were the result of taphonomic changes. In addition, we highlight how these efforts where constrained and each step in the process unnecessarily hindered. Finally, we demonstrate the capacity and willingness of forensic anthropology practitioners to be involved, and how, without governmental support, it is a great potential lamentably untapped.     

An investigation into the effects of decomposition on the post-mortem location of trans-dermal artefacts

A search area of a crime scene to recover trans-dermal artefacts (here, earrings) was conducted using 12 pig carcasses, of various sizes, with pierced ears; 6 were buried and 6 deposited on the surface in a wooded area. After 28 months, the remains were excavated and recovered, and the final location of the earrings recorded. The furthest recorded earring from its associated surface remains was 119 cm. In the buried remains, on three occasions earrings were found located 6 cm below the recorded base of the grave. Formal recommendations for the search area of a crime scene have been established as 120 cm radius from the originally located remains, whether surface deposition or burial, and confirmation of excavation below the assumed floor of the grave in burials is essential, at least to 10 cm.     

Transfer of fibres on the hands of living subjects and their persistence during hand washing

Textile fibres were transferred to the hands of ten living subjects and their persistence was determined after hand washing. Average number of fibres transferred was 300 ± 133 (female 288 ± 92, male 311 ± 163) per 100 cm² hand area in the 100 experiments. However the number of fibres transferred was not gender dependent but individual dependent. The hand texture of subjects was compared with the number of fibres transferred but the relationship was not observed. The number of fibres transferred varied significantly for the 10 repeated experiments performed under the same conditions for the same subject.The subjects were then asked to wash their hands with water. One test group washed their hands with standing water, and the other with running tap water. Afterwards, the number of fibres remaining on the test subjects' hands were investigated. Migration of the fibres on the surface of the observed hands did occur but total loss of transferred fibre after hand washing did not occur. The average number of fibres remaining per 100 cm² hand area was 14 ± 10 (range = 3–72) for hand washing with standing water, and 10 ± 12 (range = 0–79) for washing with running tap water. The results of this study show the possibility of finding fibres on the hands of a person involved in a criminal case even after hand washing before fibre collection.     

Changes in illicit cocaine hydrochloride processing identified and revealed through multivariate analysis of cocaine signature data

For nearly 30 years, the methods utilized in illicit cocaine hydrochloride production have remained relatively consistent. Cocaine hydrochloride is typically produced one kilogram at a time. As a result, each individual kilogram is unique and distinct from other kilograms in any particular seizure based on the total alkaloid profile, occluded solvent profile, and isotopic signature. Additionally, multi-kilogram cocaine seizures are often comprised of cocaine from several different coca growing regions. There has been a documented shift in this type of processing based on the recent analysis of a large cocaine seizure in the Eastern Pacific. Signature analyses of samples from 21 kg randomly selected from a 517 kg seizure were virtually identical. Triplicate analyses of each sample via gas chromatography with flame ionization detection, static headspace gas chromatography mass spectrometry, and isotope ratio mass spectrometry were completed. An initial outlier evaluation of the data and an in-depth univariate analysis indicated there was no statistically significant difference among the 21 samples at the 95% confidence interval. Principal components analysis did reveal consistent minor deviations between the samples and known authentic data from the Nariño coca growing region of Colombia. These deviations were only observed on the latter principal components and could be explained by differences in solvent selection during cocaine hydrochloride processing. Chemical analyses in addition to a thorough statistical evaluation suggest a shift in the traditional small-batch method of cocaine processing to a multi-kilogram, high throughput approach.     

Validity of Demirjian and Willems methods for dental age estimation for Malaysian children aged 5–15 years old

BackgroundOne of the most commonly used method for dental age assessment is the method reported by Demirjian and coworkers in 1973. It was later modified by Willems and coworkers whereby they &ldquo;performed a weighted ANOVA&rdquo; in order to adapt the scoring system.AimTo evaluate the applicability of Demirjian and Willems methods for dental age estimation for Malaysian children and to correlate the accuracy of the findings with the chronology of tooth development of premolars and second molars.Materials and methodsA total of 991 dental panoramic radiographs of 5–15-year-old Malaysian children were included in the study. The mean Demirjian and Willems estimated ages were compared to the mean chronological age.ResultsThe mean chronological age of the sample was 10.1 ± 2.8 and 9.9 ± 3.0 years for males and females respectively. Using the Demirjian method, the mean estimated dental age was 10.8 ± 2.9 years for males and 10.5 ± 2.9 years for females. For Willems method, the mean estimated age was 10.3 ± 2.8 years males and 10.0 ± 3.0 years respectively.ConclusionsWillems method was more applicable for estimating dental age for Malaysian children. Overestimation in Demirjian method could be due to advanced development of second bicuspids and molars.     

The age estimation of blood stains up to 30 days old using visible wavelength hyperspectral image analysis and linear discriminant analysis

A novel application of visible wavelength hyperspectral image analysis has been applied to determine the age of blood stains up to 30 days old. Reflectance spectra from selected locations within the hyperspectral image, obtained from a portable instrument, were subjected to spectral pre-processing. This was followed by the application of a linear discriminant classification model, making estimations possible with an average error of ± 0.27 days for the first 7 days and an overall average error of ± 1.17 days up to 30 days. This is also the first reported study of the determination of the age of fresh blood stains (less than one day old) with an error of ± 0.09 h. The studies have been made under controlled conditions and represent, at this stage, proof of concept results but also are the most accurate age estimation results for measurements between 0 and 30 days reported to date. The results are consistent with well-established kinetic processes suggesting that the pre-processing stages described are revealing spectroscopic changes which are reliably following the time dependent oxidation of HbO₂. The potential for parameterisation of environmental factors to make the method generally applicable at crime scenes is discussed, along with the developments required to further improve classification and to make the instrument genuinely portable.