Analog Computer Research

Contains reports on two research projects

Analog Computer Research

Contains reports on three research projects

Drug Facilitated Sexual Assault: Detection and Stability of Benzodiazepines in Spiked Drinks Using Gas Chromatography-Mass Spectrometry

Benzodiazepines are detected in a significant number of drug facilitated sexual assaults (DFSA). Whilst blood and urine from the victim are routinely analysed, due to the delay in reporting DFSA cases and the short half lives of most of these drugs in blood and urine, drug detection in such samples is problematic. Consideration of the drinks involved and analysis for drugs may start to address this. Here we have reconstructed the ‘spiking’ of three benzodiazepines (diazepam, flunitrazepam and temazepam) into five drinks, an alcopop (flavoured alcoholic drink), a beer, a white wine, a spirit, and a fruit based non-alcoholic drink (J2O) chosen as representative of those drinks commonly used by women in 16–24 year old age group. Using a validated GC-MS method for the simultaneous detection of these drugs in the drinks we have studied the storage stability of the benzodiazepines under two different storage conditions, uncontrolled room temperature and refrigerator (4°C) over a 25 day period. All drugs could be detected in all beverages over this time period. Diazepam was found to be stable in all of the beverages, except the J2O, under both storage conditions. Flunitrazepam and temazepam were found not to be stable but were detectable (97% loss of temazepam and 39% loss of flunitrazepam from J2O). The recommendations from this study are that there should be a policy change and that drinks thought to be involved in DFSA cases should be collected and analysed wherever possible to support other evidence types

Expert Panel Review of alternative biological matrices for use as an evidential sample for drug driving

Review to recommend an alternative biological matrix that could be used as evidence for the new drug driving offence.Executive Summary 1 METHODOLOGY AND ANALYTICAL TECHNIQUES AVAILABLE FOR THE DETECTION OF DRUGS INCLUDED IN THE DRUG-DRIVING LEGISLATION It is standard practice in Forensic Science for the methodology employed for the screening test to be qualitatively different from the confirmation test. In the UK drug-driving context, a Point of Contact Test (POCT) for an oral fluid (OF) screening device is used as the initial test and whole blood for the confirmatory (evidential) test. The confirmatory test is carried out in the laboratory using liquid or gas chromatography and mass spectrometry (LC or GC-MS). It is a requirement of law that confirmatory (evidential) tests are undertaken. 1.1 SCREENING TESTS Immunoassays are the basis for initial screening tests (POCT) undertaken at the roadside or in the police station. Such testing offers advantages in simplicity and ease of performance but this may be offset by potential problems concerning the lack of a positive control. Those interpreting the test result to reach a conclusion with serious consequences such as driving under the influence of drugs need to be sure of the validity of the findings. Drug testing at the roadside can therefore only be carried out with equipment that has been type-approved by the Secretary of State after testing by the Home Office’s Centre for Applied Science and Technology (CAST). Although roadside screening to detect psychoactive drug use is carried out using an OF immunoassay, developments such as the portable Surface-Enhanced Raman Spectroscope (SERS) analyser may offer an alternative technique, particularly if a handheld version comes to market. In terms of roadside testing the Panel recommends that the Home Office could expand the list of type-approved screening tests to include, in addition to THC and cocaine (which also provides a route to the cocaine metabolite benzoylecgonine (BZE)), the amphetamine-type drugs (methamphetamine and MDMA) and ketamine to reflect the growing use of these compounds in driving populations. 1.2 CONFIRMATORY TESTS More sophisticated equipment is needed for the confirmatory test than the screening test. Sample preparation (the extraction of the drug from the biological matrix) is required in order to quantify the presence of a specific drug. The direct injection of neat biological sample into chromatographic systems is an emerging development and significantly speeds up the analytical process. The confirmatory test can only take place in an accredited Forensic Service Provider (FSP) laboratory. The Panel confirmed that the substances included in section 5A of the drug-driving legislation can be easily quantified in whole blood or OF in the laboratory using GC or LC-MS. 1.2.1 Relationship between whole blood, Oral Fluid (OF), urinary, and hair drug concentrations It is widely accepted that blood and, to a lesser degree, OF are likely to give the most accurate measurement of drugs currently active in the body. Urine provides a somewhat longer detection period (drug use over the last 2-3 days), but with less quantitative accuracy. Hair provides a substantially longer detection window but does not usually used to indicate recent drug use in relation to a road traffic incident. The Panel acknowledged that OF collected at the roadside for confirmatory purposes would be particularly advantageous in terms of reducing the time lag between the driving incident and evidential testing for illicit drugs such as THC, cocaine and heroin that are known to decline rapidly in blood after use. 1.2.2 The maturity of confirmatory analysis in blood and OF To date, OF tests have not been fully validated as an alternative tool to whole blood for ‘per se’ evidential testing but, has more potential when a Lowest-Limit-of-Quantification (LLOQ) approach is utilised. The scientific evidence suggests that commercial OF immunoassay POCT devices are not suitable for evidential testing (although some devices allow for the collection of OF for separate evidential testing at the laboratory). The Panel recognised maturity in the chromatographic methodology available for the analysis of drugs included in the section 5A offence and this was at the level of sophistication that enabled the measurement of low concentrations of drugs in various matrices including whole blood and OF. The Panel recommends that the validity of the evidential drug-test requires that the level of confidence in the analytical methodology be known. Uncertainty data following chromatographic analysis of different drugs in whole blood (summarised in Table 1), can be found in the literature and are presented in the full report. The Panel noted that for whole blood an analytical uncertainty based on 3 standard deviations (k = 3) was preferred by the Home Office and should be achievable for chromatographic assays (confirmatory tests) of the drugs included in the Section 5A offence as follows: Table 1 Uncertainty data following chromatographic analysis of different drugs in whole blood summarised from the scientific literature (section 2.7 full report) Drug % Uncertainty Drug % Uncertainty THC 16 – 30.0 Amphetamine 34 Cocaine 21 - 29.5 Morphine 33 – 45 BZE 17 - 30.5 Methadone 27-33 LSD 30 Diazepam 7-12 MDMA 24 Lorazepam <33 Methamphetamine <30 Oxazepam <30 6-MAM 42 Temazepam <30 Ketamine 35 Clonazepam <34 Flunitrazepam <36 2 IDENTIFICATION OF ALTERNATIVE BIOLOGICAL MATRICES FOR EVIDENTIAL TESTING OF DRIVERS APPREHENDED FOR DRUG DRIVING 2.1 BLOOD Whole blood is currently the matrix used for evidential testing in those suspected of committing a drug-driving offence in Great Britain. This is based on the sound principle that drug concentrations in blood provide an accurate picture of the amount of drug(s) present in the body at the time of sampling and presents the strongest scientific evidence in relation to impaired driving performance. It remains the gold standard in this regard. However, the Panel acknowledged that care needs to be taken when collecting blood to ensure that sample integrity is assured; appropriate use of a preservative and an anticoagulant is mandated. Standardisation of the sampling kit and blood collection tubes for evidential tests are warranted with attention given to temperature and light during storage, transportation, and the timeliness of sample collection in relation to the driving incident. The Panel recommends that where whole blood is used for evidential tests there should be a specification (minimum standard) for the sample collection kit and the blood collection tube that includes details of the amount of preservative and anticoagulant required. The Panel also recommends moving towards the use of a vacutainer blood sampling device for safer sampling and in keeping with current practice within the NHS. 2.2 URINE Urine reflects drug use over the previous few days or longer and in this sense is not helpful in the drug-driving context since a relationship between observed impairment or time when drug consumption last occurred cannot be determined. Despite the advantage of having a matrix that requires little laboratory preparation and that can be collected in large volumes, urinalysis presents difficulties as a confirmatory test for drug driving purposes when efforts are made to correlate drug concentration directly with impairment. Urine drug test results provide information regarding the manner in which a drug is eliminated from the body rather than an indication of drug activity in the body. Excretion patterns of drugs vary within and between individuals such that the setting of cut-off values with this in mind would be very difficult. Although the relationship between blood concentrations and urine concentrations have been researched over many years, the general consensus is that urine cannot be used to determine current pharmacological drug activity in the body. The other major limitation with urine testing is with regard to the inconvenience of urine sample collection and the potential lack of integrity. Unless voidance of urine is observed, the authenticity of the sample may be called into question since urine can easily be contaminated. The possibility of false negative results following adulteration of urine with chemicals or by dilution is well described. Special facilities must be provided to be able to observe the sample collection to avoid adulteration, which may be time consuming and impractical. Urine is only suitable for the confirmation that drug use has taken place at some point in the past. However, when a zero tolerance approach is used and a laboratory LLOQ analysis is employed as the cut-off, then urinalysis can be used to support an impairment test result, as is the case for section 4 legislation. 2.3 ORAL FLUID (OF) There are several potential advantages with OF not least that it is readily accessible, and requires no medical personnel for sampling. OF collected at the roadside for confirmatory purposes would remove the need for specialist personnel to attend the police station to carry out sampling and therefore reduce the delay between the driving incident and sample collection so that drug concentrations reflective of those at the time of the driving incident were more likely. A threshold-based approach where the intention is to detect the presence of a psychoactive substance using OF and relate it to a time when the driver had been apprehended deemed impaired and to be a high road safety risk would be problematic. For confirmatory testing purposes using a per se (threshold) approach, the usefulness of OF as a possible matrix would be dependent on consistent oral fluid-whole blood (OF: B) ratios. For OF concentrations to predict whole blood concentrations accurately, the OF: B ratio would need to be independent of drug concentration and consistent within and between individuals. However due to large individual variations, ratios have been difficult to agree and cannot be easily determined for most psychoactive drugs, although some correlation has been described. OF: blood ratios have been shown to vary from drug to drug, from person to person, and even intra-individually making efforts to relate OF drug concentration at an equivalent blood drug concentration very challenging. The Panel agreed that blood concentrations could not be correlated with those in OF so that the ‘risk threshold’ limits set in the Section 5A legislation for medicinal controlled drugs (UK Government, 2014) could not be translated into OF cut-offs. The Panel concluded that the wide range of the ratios recorded does not allow reliable estimation of blood drug concentrations from OF concentrations. OF could conceivably be utilised for evidential testing where a LLOQ approach was employed and in this regard OF testing would be much better suited to illicit substances than to medicinal controlled drugs. For evidential testing using OF, the Panel recommended the OF cut-off limits published in the ‘Guide to Type-Approval Procedures for Preliminary Drug Testing Devices Used for Transport Law Enforcement in Great Britain’ in 2012, could be used with illicit drugs (Table 2). These were based on the approach taken for whole blood, i.e. the lowest limit of quantification (LLOQ) that most laboratories would be able to detect, yet above the concentrations commonly associated with passive (accidental) exposure. Table 2 Lowest Limit of Quantification (Cut-offs) for illicit drugs detected (µg/L) in OF (above concentrations commonly associated with passive exposure). Drug Group Drug to be detected LLOQ Cut-off (µg/L) Cannabinoids Delta-9-THC 10 Cocaine Cocaine Benzoylecgonine 30 30 (as composite) Amphetamines Methamphetamine MDMA 25 25 Opiates (heroin) 6-MAM 10 Hallucinogens LSD Ketamine 1 20 This matrix has been successfully used for preliminary drug testing (screening) where immediate results are required and, to this end, a number of jurisdictions around the world have adopted OF as a roadside screening tool for the detection of illicit drugs and psychoactive medicines in those suspected of drug-driving offences. However, the scientific evidence suggests that commercial OF immunoassay POCT devices are not suitable for evidential testing (indeed they were not designed with this in mind). This should not defer efforts to explore this matrix using other methodology. Although many OF immunoassay POCT drug screening devices involve the collection of a small volume of OF, some have a facility to send part of the sample to the laboratory for evidential tests. Evidential tests using the OF Cozart® Rapiscan has been successfully used in the State of Victoria, Australia for the detection of illicit substances (MDMA, THC and methamphetamine) in apprehended drug drivers. There would be practical limitations to overcome in order to use POCT OF devices for evidential testing. Should such an approach be envisaged and given the variability in performance of the commercial OF POCT devices on the market, the Panel recommend that sensitivity, specificity and accuracy criteria should be specified for OF POCT device(s). Criteria used for evidential tests has been set at ≥95 % (accuracy), ≥90 % sensitivity and ≥90 % specificity in the State of Victoria, Australia. Minimum standards were also established by the European Integrated Project DRUID (Driving under the Influence of Drugs, Alcohol and Medicines) and was set at 80% for each parameter; the Dräger DrugTest (DDT) 5000® type-approved by the Home Office fulfilled the DRUID criteria for all drug classes included in the section 5A offence. Evidential testing to quantify up to 17 compounds would likely require significantly more OF than currently used in the POCT roadside screening test: estimates by FSPs suggest 2 4 mL OF would be required. The collection of OF would therefore need to involve an OF collection device. There are important differences between OF commercial collection devices currently on the market. Buffer solutions are varied and differing volumes of OF/buffer solutions are collected which require ‘correction’ before reporting quantitative results. If OF is to be collected using a commercial device for evidential purposes, then the Panel recommend that recovery of the analytes of interest and the overall reliability of the device would need to be specified for use at the roadside or in the police station. The collection of the OF needs to be used in conjunction with collection volume imprecision data (i.e. whether the OF collected was above, below or had achieved the minimum volume required), and uncertainty of measurement to provide the OF drug concentration in neat fluid to satisfy the criminal justice system. The specification (minimum standard) for the sample collection kit and the OF collection tube should include details of the amount of preservative, stabiliser and buffer required. Whilst full type-approval is unlikely to be necessary the equipment would need to be independently assessed so that it meets the above standards. This might be part of the FSP accreditation process (See Section 4 on page 11). Based on what is known, the Panel recommends that OF samples should be refrigerated (3 – 5 °C) as quickly as possible after collection and transported to the laboratory at a controlled temperature to avoid bacterial contamination and degradation of drugs. It is also recommended that OF samples should be stored in glass tubes, away from fluorescent light and direct sunlight. OF samples should be frozen (ideally at -20 oC), if not available for immediate analysis. OF as a possible matrix for confirmatory testing may have other limitations. Potential confounders include the effects of pH variation on the appearance of drugs in OF, the potential for buccal cavity contamination and dry mouth syndrome (hyposalivation). To conclude, there is a stronger argument for the use of OF as an evidential matrix when using laboratory based cut-offs (LLOQ) such as those suggested by the DRUID studies, as the concentration above which an offence would occur. This approach would be in line with a zero tolerance approach, rather than a road safety risk based approach. With regards to the drug cut-off levels in the section 5A regulations, OF limits could not be identified for the medicinal drugs where a risk-based approach underpins the cut-off concentrations in whole blood. 2.4 SWEAT The use of a sweat POCT screening device has been employed in Europe to test those apprehended at the roadside and thought to be under the influence of illicit drugs. This approach uses immunoassay devices also employed for OF collection. However, sweat testing per se has yet to be shown to be applicable for confirmatory drug-driving tests. Consideration would need to be given to the issue of external contamination and how this can be negated as part of the sample collection procedure. 2.5 HAIR It takes about 7 days for a drug to be incorporated into a hair follicle making any attempt to correlate hair drug concentration with driving behaviour extremely difficult. This factor and the inconvenience of sample collection and the requirement for enough hair sample to be collected to test for all 17 compounds in the schedule 5A legislation impact on the usefulness of hair as an evidential test matrix. However, hair testing has been used in many European countries to confirm abstinence from illicit drugs in persons whose driving licences have been suspended for drug-driving offences. The Panel recommends that hair testing is an appropriate matrix to use as an adjunct to medical assessment for re-licensing since hair testing provides a much longer window of detection than either blood, urine or OF and would enable the determination of a history of past exposures to illicit or medicinal controlled substances. 2.6 DRIED BLOOD SPOTS (DBS) AND SMALL LIQUID SAMPLES As yet the procedures and technology for either dried blood spot (DBS) sampling or small liquid samples has not progressed far enough to be used as an evidential test in an environment such as a police station or at the roadside. However, the development of commercial devices may be suitable in the future for use by law enforcement officers in those suspected of drug-driving offences. 2.7 LATENT FINGERPRINTS The analysis of drugs in latent fingerprints is an exciting new development that shows promise in a number of arenas that require flexible drug screening services. The Panel noted that quantitative analysis of drugs of interest is not currently well developed and therefore could not recommend the use of latent fingerprints as an alternative to blood for evidential testing. Consideration will need to be given to the issue of external contamination and how this can be negated as part of the sample collection procedure. 2.8 EXHALED BREATH CONDENSATE (EBC) Exhaled breath condensate (EBC) is a further innovation with regard to drug testing matrices and is based on the premise that therapeutic and illicit drugs are present as non-volatile components in human breath. In Sweden the EBC (SensAbues®) has been used as a screening tool to test those apprehended on suspicion of a drug driving offence but as yet this matrix cannot be collected in a manner that would make it suitable for evidential testing in the British criminal justice system. 3 INTERNATIONAL APPROACHES TO SETTING CONCENTRATION THRESHOLDS FOR DRUG DRIVING A brief review of international practice in terms of drug-driving has shown that countries take different approaches to roadside drug testing both from a legislative and an analytical point of view. The number of drugs targeted differs according to national prevalence, although OF is commonplace as a screening tool. The LLOQ and/or a zero tolerance (LOD) limit seems to be the consensus for illegal drugs, in some cases with the additional requirement of evidence of impairment. A more pragmatic approach is taken with medicinal controlled drugs. It is becoming increasingly well known that drivers who misuse psychoactive substances may take more than one psychoactive substance together at one time before driving. In many instances this includes the use of alcohol as highlighted in the Technical report ‘Driving under the influence of drugs’. The Panel recommends that some discussion is needed with regard to the approach taken when more than one substance is detected in the evidential sample and particularly, whether consideration should be given to substances with a known impairing effect that are present below the level currently set in legislation but may in combination with other psychoactive substances be a risk to driver safety. In some countries a limit has been set for a drug class (e.g., the amphetamines), such that an offence occurs if any combination of the different drugs within the class, when summed, exceed the cut-off. There is also growing awareness that drugs with similar pharmacological mechanisms of action to those included in the section 5A legislation, but which are not controlled other than through the provisions of the Psychoactive Substances Act 2016, pose similar impairing effects on driving performance. In addition, new evidence is emerging for some drugs controlled under the Misuse of Drugs Act (1971) and these have been included in drug driving legislation elsewhere. Gamma-hydroxybutyrate (GHB) with sedative and anaesthetic effects is a good example. The Panel recommends that the Department for Transport keeps a watchful brief on developments in other drug-driving communities as well as the scientific literature in order to make informed decisions about the addition of further drugs to the section 5A drug-driving legislation. 4 THE CAPABILITY OF UK FORENSIC LABORATORIES TO U

Damage to the salience network and interactions with the default mode network

Interactions between the Salience Network (SN) and the Default Mode Network (DMN) are thought to be important for cognitive control. However, evidence for a causal relationship between the networks is limited. Previously, we have reported that traumatic damage to white matter tracts within the SN predicts abnormal DMN function. Here we investigate the effect of this damage on network interactions that accompany changing motor control. We initially used fMRI of the Stop Signal Task to study response inhibition in humans. In healthy subjects, functional connectivity (FC) between the right anterior insula (rAI), a key node of the SN, and the DMN transiently increased during stopping. This change in FC was not seen in a group of traumatic brain injury (TBI) patients with impaired cognitive control. Furthermore, the amount of SN tract damage negatively correlated with FC between the networks. We confirmed these findings in a second group of TBI patients. Here, switching rather than inhibiting a motor response: (1) was accompanied by a similar increase in network FC in healthy controls; (2) was not seen in TBI patients; and (3) tract damage after TBI again correlated with FC breakdown. This shows that coupling between the rAI and DMN increases with cognitive control and that damage within the SN impairs this dynamic network interaction. This work provides compelling evidence for a model of cognitive control where the SN is involved in the attentional capture of salient external stimuli and signals the DMN to reduce its activity when attention is externally focused

Priority setting for new technologies in medicine: A transdisciplinary study

BACKGROUND: Decision makers in health care organizations struggle with how to set priorities for new technologies in medicine. Traditional approaches to priority setting for new technologies in medicine are insufficient and there is no widely accepted model that can guide decision makers. DISCUSSION: Daniels and Sabin have developed an ethically based account about how priority setting decisions should be made. We have developed an empirically based account of how priority setting decisions are made. In this paper, we integrate these two accounts into a transdisciplinary model of priority setting for new technologies in medicine that is both ethically and empirically based. SUMMARY: We have developed a transdisciplinary model of priority setting that provides guidance to decision makers that they can operationalize to help address priority setting problems in their institution

Hrk/DP5 contributes to the apoptosis of select neuronal populations but is dispensable for haematopoietic cell apoptosis

The pro-apoptotic BH3-only members of the Bcl2 family, crucial initiators of cell death, are activated by a diverse array of developmental cues or experimentally applied stress stimuli. We have investigated, through gene targeting in mice, the biological roles for the BH3-only family member HRK (also known as DP5) in apoptosis regulation. Hrk gene expression was found to be restricted to cells and tissues of the central and peripheral nervous systems. Sensory neurons from mice lacking Hrk were less sensitive to apoptosis induced by nerve growth factor (NGF) withdrawal, consistent with the induction of Hrk following NGF deprivation. By contrast, cerebellar granule neurons that upregulate Hrk upon transfer to low-K+ medium underwent apoptosis normally under these conditions in the absence of Hrk. Furthermore, loss of Hrk was not sufficient to rescue the neuronal degeneration in lurcher mutant mice. Despite previous reports, no evidence was found for Hrk expression or induction in growth-factor-dependent haematopoietic cell lines following withdrawal of their requisite cytokine, and haematopoietic progenitors lacking HRK died normally in res onse to cytokine deprivation. These results demo rate that HRK contributes to apoptosis signalling elic4itd by trophic factor withdrawal in certain neuronal populations but is dispensable for apoptosis of haematopoletic cells

Assessment of ABT-263 activity across a cancer cell line collection leads to a potent combination therapy for small-cell lung cancer

BH3 mimetics such as ABT-263 induce apoptosis in a subset of cancer models. However, these drugs have shown limited clinical efficacy as single agents in small-cell lung cancer (SCLC) and other solid tumor malignancies, and rational combination strategies remain underexplored. To develop a novel therapeutic approach, we examined the efficacy of ABT-263 across >500 cancer cell lines, including 311 for which we had matched expression data for select genes. We found that high expression of the proapoptotic gene Bcl2-interacting mediator of cell death (BIM) predicts sensitivity to ABT-263. In particular, SCLC cell lines possessed greater BIM transcript levels than most other solid tumors and are among the most sensitive to ABT-263. However, a subset of relatively resistant SCLC cell lines has concomitant high expression of the antiapoptotic myeloid cell leukemia 1 (MCL-1). Whereas ABT-263 released BIM from complexes with BCL-2 and BCL-XL, high expression of MCL-1 sequestered BIM released from BCL-2 and BCL-XL, thereby abrogating apoptosis. We found that SCLCs were sensitized to ABT-263 via TORC1/2 inhibition, which led to reduced MCL-1 protein levels, thereby facilitating BIM-mediated apoptosis. AZD8055 and ABT-263 together induced marked apoptosis in vitro, as well as tumor regressions in multiple SCLC xenograft models. In a Tp53; Rb1 deletion genetically engineered mouse model of SCLC, the combination of ABT-263 and AZD8055 significantly repressed tumor growth and induced tumor regressions compared with either drug alone. Furthermore, in a SCLC patient-derived xenograft model that was resistant to ABT-263 alone, the addition of AZD8055 induced potent tumor regression. Therefore, addition of a TORC1/2 inhibitor offers a therapeutic strategy to markedly improve ABT-263 activity in SCLC.United States. Dept. of Defense (Grant W81-XWH-13-1-0323)National Cancer Institute (U.S.) (Cancer Center Support Grant P30-CA14051