Profiling of signal transduction in human memory T cells

Résumé pour un large public: La vaccination a eu un impact énorme sur la santé mondiale. Mais, quel est le principe d'un vaccin? Il est basé sur la 'mémoire immunologique', qui est une particularité exclusive des systèmes immunitaires des organismes évolués. Suite à une infection par un pathogène, des cellules spécialisées de notre système immunitaire (les lymphocytes) le reconnaissent et initient une réaction immunitaire qui a pour but son élimination. Pendant cette réaction se développent aussi des cellules, appelées cellules lymphocytaires mémoire, qui persistent pour longue durée et qui ont la capacité de stimuler une réaction immunitaire très efficace immédiatement après une seconde exposition à ce même pathogène. Ce sont ces cellules mémoires (lymphocytes B et T) qui sont à la base de la 'mémoire immunologique' et qui sont stimulées lors de la vaccination. Chez l'homme, deux populations distinctes des lymphocytes T mémoires ont été identifiées: les cellules centrales (CM) et effectrices (EM) mémoires. Ces populations sont fonctionnellement hétérogènes et exercent des rôles distincts et essentiels dans l'immunité protectrice. Typiquement, les cellules effectrices mémoires sont capables de tuer immédiatement le pathogène tandis que les cellules centrales mémoires sont responsables d'initier une réponse immunitaire complète. Pourtant, les mécanismes biochimiques qui contrôlent les fonctions de ces cellules ont été jusqu'à présent peu étudiés à cause de la faible fréquence de ces cellules et de la quantité limitée de tissus humains disponibles pour les analyses. La compréhension de ces mécanismes est cruciale pour la réalisation de vaccins efficaces et pour le développement de nouveaux médicaments capables de moduler la réponse immunitaire lymphocytaire. Dans cette thèse, nous avons d'abord développé et amélioré une technologie appelée 'protéine array en phase inverse' qui possède un niveau de sensibilité beaucoup plus élevé par rapport aux technologies classiquement utilisées dans l'étude des protéines. Grâce à cette technique, nous avons pu comparer la composition protéique du système de transmission des signaux d'activation des cellules CM et EM humaines. L'analyse de 8 à 13 sujets sains a montré que ces populations des cellules mémoires possèdent un système de signalisation protéique différent. En effet, les cellules EM possèdent, par rapport aux cellules CM, des niveaux réduits d'une protéine régulatrice (appelée c-Cbl) que nous avons démontré comme étant responsable des fonctions spécifiques de ces cellules. En effet, en augmentant artificiellement l'expression de cette protéine régulatrice dans les cellules EM jusqu'au niveau de celui des cellules CM, nous avons induit dans les cellules EM des capacités fonctionnelles caractéristiques des cellules CM. En conclusion, notre étude a identifié, pour la première fois chez l'homme, un mécanisme biochimique qui contrôle les fonctions des populations des cellules mémoires. Résumé en Français: Les cellules mémoires persistent inertes dans l'organisme et produisent des réactions immunitaires rapides et robustes contre les pathogènes précédemment rencontrés. Deux populations distinctes des cellules mémoires ont été identifiées chez l'homme: les cellules centrales (CM) et effectrices (EM) mémoires. Ces populations sont fonctionnellement hétérogènes et exercent des rôles distincts et critiques dans l'immunité protectrice. Les mécanismes biochimiques qui contrôlent leurs fonctions ont été jusqu'à présent peu étudiés, bien que leur compréhension soit cruciale pour le développement des vaccins et des nouveaux traitements/médicaments. Les limites majeures à ces études sont la faible fréquence de ces populations et la quantité limitée de tissus humains disponibles. Dans cette thèse nous avons d'abord développé et amélioré la technologie de 'protéine array en phase inverse' afin d'analyser les molécules de signalisation des cellules mémoires CD4 et CD8 humaines isolées ex vivo. L'excellente sensibilité, la reproductibilité et la linéarité de la détection, ont permis de quantifier des variations d'expression protéiques supérieures à 20% dans un lysat équivalent à 20 cellules. Ensuite, grâce à l'analyse de 8 à 13 sujets sains, nous avons prouvé que les cellules mémoires CD8 ont une composition homogène de leur système de signalisation tandis que les cellules CD4 EM expriment significativement de plus grandes quantités de SLP-76 et des niveaux réduits de c-Cbl, Syk, Fyn et LAT par rapport aux cellules CM. En outre, l'expression réduite du régulateur négatif c-Cbl est corrélée avec l'expression des SLP-76, PI3K et LAT uniquement dans les cellules EM. L'évaluation des propriétés fonctionnelles des cellules mémoires a permis de démontrer que l'expression réduite du c-Cbl dans les cellules EM est associé à une diminution de leur seuil d'activation. En effet, grâce a la technique de transduction cytosolique, nous avons augmenté la quantité de c-Cbl des cellules EM à un niveau comparable à celui des cellules CM et constaté une réduction de la capacité des cellules EM à proliférer et sécréter des cytokines. Ce mécanisme de régulation dépend principalement de l'activité d'ubiquitine ligase de c-Cbl comme démontré par l'impact réduit du mutant enzymatiquement déficient de c-Cbl sur les fonctions de cellules EM. En conclusion, cette thèse identifie c-Cbl comme un régulateur critique des réponses fonctionnelles des populations de cellules T mémoires et fournit, pour la première fois chez l'homme, un mécanisme contrôlant l'hétérogénéité fonctionnelle des ces cellules. De plus, elle valide l'utilisation combinée des 'RPP arrays' et de la transduction cytosolique comme outil puissant d'analyse quantitative et fonctionnel des protéines de signalisation. Summary : Memory cells persist in a quiescent state in the body and mediate rapid and vigorous immune responses toward pathogens previously encountered. Two subsets of memory cells, namely central (CM) and effector (EM) memory cells, have been identified in humans. These subsets display high functional heterogeneity and assert critical and distinct roles in the control of protective immunity. The biochemical mechanisms controlling their functional properties remain so far poorly investigated, although their clarification is crucial for design of effective T-cell vaccine and drug development. Major limitations to these studies lie in the low frequency of memory T cell subsets and the limited amount of human specimen available. In this thesis we first implemented the innovative reverse phase protein array approach to profile 15 signalling components in human CD8 and CD4 memory T cells isolated ex vivo. The high degree of sensitivity, reproducibility and linearity achieved, allowed an excellent quantification of variations in protein expression higher than 20% in as few as 20-cell equivalent per spot. Based on the analysis of 8 to 13 healthy subjects, we showed that CD8 memory cells have a homogeneous composition of their signaling machinery while CD4 EM cells express statistically significant increased amounts of SLP-76 and reduced levels of c- Cbl, Syk, Fyn and LAT as compared to CM cells. Moreover, in EM but not CM cells, reduced expression of negative regulator c-Cbl correlated with the expression of SLP-76, PI3K and LAT. Subsequently, we demonstrated that the higher functional properties and the lower functional threshold of EM cells is associated with reduced expression of c-Cbl. Indeed, by increasing c-Cbl content of EM cells to the same level of CM cells using cytosolic transduction, we impaired their proliferation and cytokine production. This regulatory mechanism was primarily dependent on c-Cbl E3 ubiquitin ligase activity as evidenced by the weaker impact of enzymatically deficient c-Cbl C381A mutant on EM cell functions. Together, these results identify c-Cbl as a critical regulator of the functional responses of memory T cell subsets and provides, for the first time in humans, a mechanism controlling the functional heterogeneity of memory CD4 cells. Moreover it validates the combined use of RPP arrays and cytosolic transduction approaches as a powerful tool to quantitatively analyze signalling proteins and functionally assess their roles.

Cryo-electron microscopy of vitreous sections of native biological cells and tissues : methodology and applications

Résumé L'eau est souvent considérée comme une substance ordinaire puisque elle est très commune dans la nature. En fait elle est la plus remarquable de toutes les substances. Sans l'eau la vie sur la terre n'existerait pas. L'eau représente le composant majeur de la cellule vivante, formant typiquement 70 à 95% de la masse cellulaire et elle fournit un environnement à d'innombrables organismes puisque elle couvre 75% de la surface de terre. L'eau est une molécule simple faite de deux atomes d'hydrogène et un atome d'oxygène. Sa petite taille semble en contradiction avec la subtilité de ses propriétés physiques et chimiques. Parmi celles-là, le fait que, au point triple, l'eau liquide est plus dense que la glace est particulièrement remarquable. Malgré son importance particulière dans les sciences de la vie, l'eau est systématiquement éliminée des spécimens biologiques examinés par la microscopie électronique. La raison en est que le haut vide du microscope électronique exige que le spécimen biologique soit solide. Pendant 50 ans la science de la microscopie électronique a adressé ce problème résultant en ce moment en des nombreuses techniques de préparation dont l'usage est courrant. Typiquement ces techniques consistent à fixer l'échantillon (chimiquement ou par congélation), remplacer son contenu d'eau par un plastique doux qui est transformé à un bloc rigide par polymérisation. Le bloc du spécimen est coupé en sections minces (d’environ 50 nm) avec un ultramicrotome à température ambiante. En général, ces techniques introduisent plusieurs artefacts, principalement dû à l'enlèvement d'eau. Afin d'éviter ces artefacts, le spécimen peut être congelé, coupé et observé à basse température. Cependant, l'eau liquide cristallise lors de la congélation, résultant en une importante détérioration. Idéalement, l'eau liquide est solidifiée dans un état vitreux. La vitrification consiste à refroidir l'eau si rapidement que les cristaux de glace n'ont pas de temps de se former. Une percée a eu lieu quand la vitrification d'eau pure a été découverte expérimentalement. Cette découverte a ouvert la voie à la cryo-microscopie des suspensions biologiques en film mince vitrifié. Nous avons travaillé pour étendre la technique aux spécimens épais. Pour ce faire les échantillons biologiques doivent être vitrifiés, cryo-coupées en sections vitreuse et observées dans une cryo-microscope électronique. Cette technique, appelée la cryo- microscopie électronique des sections vitrifiées (CEMOVIS), est maintenant considérée comme étant la meilleure façon de conserver l'ultrastructure de tissus et cellules biologiques dans un état très proche de l'état natif. Récemment, cette technique est devenue une méthode pratique fournissant des résultats excellents. Elle a cependant, des limitations importantes, la plus importante d'entre elles est certainement dû aux artefacts de la coupe. Ces artefacts sont la conséquence de la nature du matériel vitreux et le fait que les sections vitreuses ne peuvent pas flotter sur un liquide comme c'est le cas pour les sections en plastique coupées à température ambiante. Le but de ce travail a été d'améliorer notre compréhension du processus de la coupe et des artefacts de la coupe. Nous avons ainsi trouvé des conditions optimales pour minimiser ou empêcher ces artefacts. Un modèle amélioré du processus de coupe et une redéfinitions des artefacts de coupe sont proposés. Les résultats obtenus sous ces conditions sont présentés et comparés aux résultats obtenus avec les méthodes conventionnelles. Abstract Water is often considered to be an ordinary substance since it is transparent, odourless, tasteless and it is very common in nature. As a matter of fact it can be argued that it is the most remarkable of all substances. Without water life on Earth would not exist. Water is the major component of cells, typically forming 70 to 95% of cellular mass and it provides an environment for innumerable organisms to live in, since it covers 75% of Earth surface. Water is a simple molecule made of two hydrogen atoms and one oxygen atom, H2O. The small size of the molecule stands in contrast with its unique physical and chemical properties. Among those the fact that, at the triple point, liquid water is denser than ice is especially remarkable. Despite its special importance in life science, water is systematically removed from biological specimens investigated by electron microscopy. This is because the high vacuum of the electron microscope requires that the biological specimen is observed in dry conditions. For 50 years the science of electron microscopy has addressed this problem resulting in numerous preparation techniques, presently in routine use. Typically these techniques consist in fixing the sample (chemically or by freezing), replacing its water by plastic which is transformed into rigid block by polymerisation. The block is then cut into thin sections (c. 50 nm) with an ultra-microtome at room temperature. Usually, these techniques introduce several artefacts, most of them due to water removal. In order to avoid these artefacts, the specimen can be frozen, cut and observed at low temperature. However, liquid water crystallizes into ice upon freezing, thus causing severe damage. Ideally, liquid water is solidified into a vitreous state. Vitrification consists in solidifying water so rapidly that ice crystals have no time to form. A breakthrough took place when vitrification of pure water was discovered. Since this discovery, the thin film vitrification method is used with success for the observation of biological suspensions of. small particles. Our work was to extend the method to bulk biological samples that have to be vitrified, cryosectioned into vitreous sections and observed in cryo-electron microscope. This technique is called cryo-electron microscopy of vitreous sections (CEMOVIS). It is now believed to be the best way to preserve the ultrastructure of biological tissues and cells very close to the native state for electron microscopic observation. Since recently, CEMOVIS has become a practical method achieving excellent results. It has, however, some sever limitations, the most important of them certainly being due to cutting artefacts. They are the consequence of the nature of vitreous material and the fact that vitreous sections cannot be floated on a liquid as is the case for plastic sections cut at room temperature. The aim of the present work has been to improve our understanding of the cutting process and of cutting artefacts, thus finding optimal conditions to minimise or prevent these artefacts. An improved model of the cutting process and redefinitions of cutting artefacts are proposed. Results obtained with CEMOVIS under these conditions are presented and compared with results obtained with conventional methods.

First systematic chemical profiling of cocaine police seizures in Finland in the framework of an intelligence-led approach

For the first time in Finland, the chemical profiling of cocaine specimens was performed at the National Bureau of Investigation (NBI). The main goals were to determine the chemical composition of cocaine specimens sold in the Finnish market and to study the distribution networks of cocaine in order to provide intelligence related to its trafficking. An analytical methodology enabling through one single GC-MS injection the determination of the added cutting agents (adulterants and diluents), the cocaine purity and the chemical profile (based on the major and minor alkaloids) for each specimen was thus implemented and validated. The methodology was found to be efficient for the discrimination between specimens coming from the same source and specimens coming from different sources. The results highlighted the practical utility of the chemical profiling, especially for supporting the investigation through operational intelligence and improving the knowledge related to the cocaine trafficking through strategic intelligence.

Postmortem diagnosis of diabetes mellitus and its complications.

Diabetes mellitus has become a major cause of death worldwide and diabetic ketoacidosis is the most common cause of death in children and adolescents with type 1 diabetes. Acute complications of diabetes mellitus as causes of death may be difficult to diagnose due to missing characteristic macroscopic and microscopic findings. Biochemical analyses, including vitreous glucose, blood (or alternative specimen) beta-hydroxybutyrate, and blood glycated hemoglobin determination, may complement postmortem investigations and provide useful information for determining the cause of death even in corpses with advanced decompositional changes. In this article, we performed a review of the literature pertaining to the diagnostic performance of classical and novel biochemical parameters that may be used in the forensic casework to identify disorders in glucose metabolism. We also present a review focusing on the usefulness of traditional and alternative specimens that can be sampled and subsequently analyzed to diagnose acute complications of diabetes mellitus as causes of death.

Use of the dried blood spot sampling process coupled with fast gas chromatography and negative-ion chemical ionization tandem mass spectrometry: application to fluoxetine, norfluoxetine, reboxetine, and paroxetine analysis.

The objective of this work was to combine the advantages of the dried blood spot (DBS) sampling process with the highly sensitive and selective negative-ion chemical ionization tandem mass spectrometry (NICI-MS-MS) to analyze for recent antidepressants including fluoxetine, norfluoxetine, reboxetine, and paroxetine from micro whole blood samples (i.e., 10 microL). Before analysis, DBS samples were punched out, and antidepressants were simultaneously extracted and derivatized in a single step by use of pentafluoropropionic acid anhydride and 0.02% triethylamine in butyl chloride for 30 min at 60 degrees C under ultrasonication. Derivatives were then separated on a gas chromatograph coupled with a triple-quadrupole mass spectrometer operating in negative selected reaction monitoring mode for a total run time of 5 min. To establish the validity of the method, trueness, precision, and selectivity were determined on the basis of the guidelines of the "Société Française des Sciences et des Techniques Pharmaceutiques" (SFSTP). The assay was found to be linear in the concentration ranges 1 to 500 ng mL(-1) for fluoxetine and norfluoxetine and 20 to 500 ng mL(-1) for reboxetine and paroxetine. Despite the small sampling volume, the limit of detection was estimated at 20 pg mL(-1) for all the analytes. The stability of DBS was also evaluated at -20 degrees C, 4 degrees C, 25 degrees C, and 40 degrees C for up to 30 days. Furthermore, the method was successfully applied to a pharmacokinetic investigation performed on a healthy volunteer after oral administration of a single 40-mg dose of fluoxetine. Thus, this validated DBS method combines an extractive-derivative single step with a fast and sensitive GC-NICI-MS-MS technique. Using microliter blood samples, this procedure offers a patient-friendly tool in many biomedical fields such as checking treatment adherence, therapeutic drug monitoring, toxicological analyses, or pharmacokinetic studies.

Review of quantitative phase-digital holographic microscopy: promising novel imaging technique to resolve neuronal network activity and identify cellular biomarkers of psychiatric disorders.

Quantitative phase microscopy (QPM) has recently emerged as a new powerful quantitative imaging technique well suited to noninvasively explore a transparent specimen with a nanometric axial sensitivity. In this review, we expose the recent developments of quantitative phase-digital holographic microscopy (QP-DHM). Quantitative phase-digital holographic microscopy (QP-DHM) represents an important and efficient quantitative phase method to explore cell structure and dynamics. In a second part, the most relevant QPM applications in the field of cell biology are summarized. A particular emphasis is placed on the original biological information, which can be derived from the quantitative phase signal. In a third part, recent applications obtained, with QP-DHM in the field of cellular neuroscience, namely the possibility to optically resolve neuronal network activity and spine dynamics, are presented. Furthermore, potential applications of QPM related to psychiatry through the identification of new and original cell biomarkers that, when combined with a range of other biomarkers, could significantly contribute to the determination of high risk developmental trajectories for psychiatric disorders, are discussed.

Comparison between Radiographic (2-dimensional and 3-dimensional) and Histologic Findings of Periapical Lesions Treated with Apical Surgery.

INTRODUCTION: The aim of this study was to evaluate the concordance of 2- and 3-dimensional radiography and histopathology in the diagnosis of periapical lesions. METHODS: Patients were consecutively enrolled in this study provided that preoperative periapical radiography (PR) and cone-beam computed tomographic imaging of the tooth to be treated with apical surgery were performed. The periapical lesional tissue was histologically analyzed by 2 blinded examiners. The final histologic diagnosis was compared with the radiographic assessments of 4 blinded observers. The initial study material included 62 teeth in the same number of patients. RESULTS: Four lesions had to be excluded during processing, resulting in a final number of 58 evaluated cases (31 women and 27 men, mean age = 55 years). The final histologic diagnosis of the periapical lesions included 55 granulomas (94.8%) and 3 cysts (5.2%). Histologic analysis of the tissue samples from the apical lesions exhibited an almost perfect agreement between the 2 experienced investigators with an overall agreement of 94.83% (kappa = 0.8011). Radiographic assessment overestimated cysts by 28.4% (cone-beam computed tomographic imaging) and 20.7% (periapical radiography), respectively. Comparing the correlation of the radiographic diagnosis of 4 observers with the final histologic diagnosis, 2-dimensional (kappa = 0.104) and 3-dimensional imaging (kappa = 0.111) provided only minimum agreement. CONCLUSIONS: To establish a final diagnosis of an apical radiolucency, the tissue specimen should be evaluated histologically and specified as a granuloma (with/without epithelium) or a cyst. Analysis of 2-dimensional and 3-dimensional radiographic images alike results only in a tentative diagnosis that should be confirmed with biopsy.

Mini invasive skeletal muscle biopsy technique with a tri-axial end cut needle.

OBJECTIVE: Skeletal Muscle Biopsy is a minor surgical procedure for the diagnosis of different neuromuscular pathological conditions and has recently gained popularity also in the research field of age-related muscular modifications and sarcopenia. Few studies focused on the application of mini-invasive muscular biopsy in both normal and pathological conditions. The aim of our study was to describe a mini invasive ultrasound-guided skeletal muscular biopsy technique in complete spinal cord injured (SCI) patients and healthy controls with a tri-axial end-cut needle. PATIENTS AND METHODS: Skeletal muscle biopsies were collected from 6 chronic SCI patients and 3 healthy controls vastus lateralis muscle with a tri-axial end cut needle (Biopince© - Angiotech). Muscle samples were stained for ATPase to determine fibers composition, moreover, gene expression of cyclooxygenase-1 (COX-1) and prostaglandin E2 receptor has been analyzed by Real Time RT-PCR. RESULTS: All the procedures were perfomed easily without failures and complications. Control tissue was macroscopically thicker than SCI one. Control specimen displayed an equal distribution of type I and type II fibers, while SCI sample displayed a prevalence of type II fibers SCI specimen displayed a significant reduction in COX-1 gene expression. This mini-invasive approach was easy, accurate and with low complication rate in performing skeletal muscle biopsy in both SCI patients and controls. CONCLUSIONS: This technique could be useful in conditions in which the overall quantity of specimen required is small like for molecular biology analysis. For histological diagnostic purposes and/or conditions in which the original tissue is already pathologically modified, this technique should be integrated with more invasive techniques.

Comparison of Inoculation with the InoqulA and WASP Automated Systems with Manual Inoculation.

The quality of sample inoculation is critical for achieving an optimal yield of discrete colonies in both monomicrobial and polymicrobial samples to perform identification and antibiotic susceptibility testing. Consequently, we compared the performance between the InoqulA (BD Kiestra), the WASP (Copan), and manual inoculation methods. Defined mono- and polymicrobial samples of 4 bacterial species and cloudy urine specimens were inoculated on chromogenic agar by the InoqulA, the WASP, and manual methods. Images taken with ImagA (BD Kiestra) were analyzed with the VisionLab version 3.43 image analysis software to assess the quality of growth and to prevent subjective interpretation of the data. A 3- to 10-fold higher yield of discrete colonies was observed following automated inoculation with both the InoqulA and WASP systems than that with manual inoculation. The difference in performance between automated and manual inoculation was mainly observed at concentrations of >10(6) bacteria/ml. Inoculation with the InoqulA system allowed us to obtain significantly more discrete colonies than the WASP system at concentrations of >10(7) bacteria/ml. However, the level of difference observed was bacterial species dependent. Discrete colonies of bacteria present in 100- to 1,000-fold lower concentrations than the most concentrated populations in defined polymicrobial samples were not reproducibly recovered, even with the automated systems. The analysis of cloudy urine specimens showed that InoqulA inoculation provided a statistically significantly higher number of discrete colonies than that with WASP and manual inoculation. Consequently, the automated InoqulA inoculation greatly decreased the requirement for bacterial subculture and thus resulted in a significant reduction in the time to results, laboratory workload, and laboratory costs.

Non-small cell lung cancer: the prevalence of oncogenic driver mutations in Switzerland

Background. Predictive molecular marker analyses are standard of care in order to select non-small cell lung cancer (NSCLC) patients for targeted therapies. The aim of this study was to determine the prevalence of targetable oncogenic driver mutations including EGFR, KRAS, BRAF, HER2, ALK and ROS1 in Switzerland. Methods. Eight Swiss pathology institutions provided retrospective and anonymized data on their predictive molecular marker results performed on NSCLC from January 2012 to December 2014. Clinico-pathological data were recorded including age, gender, histological NSCLC-subtype and specimen type (biopsy, conventional cytology and cell block, respectively) used for molecular analyses. The prevalence of oncogenic mutations were calculated and compared between the centres. Results. A total of 4187 NSCLC were included into the study. The median age was 67 years and 55% were male patients. The tumor specimens for molecular analysis were mostly derived from biopsies (69%), 26% were from conventional cytology specimens and only in 5% from cell blocks. The most prevalent gene mutation was KRAS with 30.6% (range: 27.3-33.9%), followed by EGFR, BRAF and HER2 mutations in 12.2% (range: 10.2-13.1%), 3.9% (range: 2.5-5.6%) and 1.1% (range: 0.9-4.0%), respectively, without significant differences between the eight centers. Concomitant EGFR and KRAS mutations were detected in only 3/2027 NSCLC. In contrast the prevalence of ALK (mean 6.5%, range: 2.8-11.7%) and ROS1 (mean 2.4%, range: 1.5-6.2%) rearrangements varied significantly between centers. Conclusions. The Prevalence of EGFR, KRAS, BRAF and HER2 mutations are well in line with data from other West European populations. Concomitant EGFR, KRAS, BRAF or HER2 mutations are exceptional. ALK FISH results vary significantly between the eight centres. Concomitant ALK FISH positive results in NSCLC harbouring other oncogenic driver mutation have only been observed in two smaller centres, highlighting the difficulty in ALK-FISH interpretation.

Study of criteria influencing the success rate of DNA swabs in operational conditions: a contribution to an evidence-based approach to crime scene investigation and triage

DNA is nowadays swabbed routinely to investigate serious and volume crimes, but research remains scarce when it comes to determining the criteria that may impact the success rate of DNA swabs taken on different surfaces and situations. To investigate these criteria in fully operational conditions, DNA analysis results of 4772 swabs taken by the forensic unit of a police department in Western Switzerland over a 2.5-year period (2012-2014) in volume crime cases were considered. A representative and random sample of 1236 swab analyses was extensively examined and codified, describing several criteria such as whether the swabbing was performed at the scene or in the lab, the zone of the scene where it was performed, the kind of object or surface that was swabbed, whether the target specimen was a touch surface or a biological fluid, and whether the swab targeted a single surface or combined different surfaces. The impact of each criterion and of their combination was assessed in regard to the success rate of DNA analysis, measured through the quality of the resulting profile, and whether the profile resulted in a hit in the national database or not. Results show that some situations - such as swabs taken on door and window handles for instance - have a higher success rate than average swabs. Conversely, other situations lead to a marked decrease in the success rate, which should discourage further analyses of such swabs. Results also confirm that targeting a DNA swab on a single surface is preferable to swabbing different surfaces with the intent to aggregate cells deposited by the offender. Such results assist in predicting the chance that the analysis of a swab taken in a given situation will lead to a positive result. The study could therefore inform an evidence-based approach to decision-making at the crime scene (what to swab or not) and at the triage step (what to analyse or not), contributing thus to save resource and increase the efficiency of forensic science efforts.

Accuracy of cognitive MRI-targeted biopsy in hitting prostate cancer-positive regions of interest.

PURPOSE: Prostate cancer (PCa) diagnosis relies on clinical suspicion leading to systematic transrectal ultrasound-guided biopsy (TRUSGB). Multiparametric magnetic resonance imaging (mpMRI) allows for targeted biopsy of suspicious areas of the prostate instead of random 12-core biopsy. This method has been shown to be more accurate in detecting significant PCa. However, the precise spatial accuracy of cognitive targeting is unknown. METHODS: Consecutive patients undergoing mpMRI-targeted TRUSGB with cognitive registration (MRTB-COG) followed by robot-assisted radical prostatectomy were included in the present analysis. The regions of interest (ROIs) involved by the index lesion reported on mpMRI were subsequently targeted by two experienced urologists using the cognitive approach. The 27 ROIs were used as spatial reference. Mapping on radical prostatectomy specimen was used as reference to determine true-positive mpMRI findings. Per core correlation analysis was performed. RESULTS: Forty patients were included. Overall, 40 index lesions involving 137 ROIs (mean ROIs per index lesion 3.43) were identified on MRI. After correlating these findings with final pathology, 117 ROIs (85 %) were considered as true-positive lesions. A total of 102 biopsy cores directed toward such true-positive ROIs were available for final analysis. Cognitive targeted biopsy hit the target in 82 % of the cases (84/102). The only identified risk factor for missing the target was an anterior situated ROI (p = 0.01). CONCLUSION: In experienced hands, cognitive MRTB-COG allows for an accuracy of 82 % in hitting the correct target, given that it is a true-positive lesion. Anterior tumors are less likely to be successfully targeted.

Poorly differentiated clusters (PDC) in colorectal cancer: what is and ought to be known.

BACKGROUND: The counting of poorly differentiated clusters of 5 or more cancer cells lacking a gland-like structure in a tumor mass has recently been identified among the histological features predictive of poor prognosis in colorectal cancer. MAIN BODY: Poorly differentiated clusters can easily be recognized in the histological sections of colorectal cancer routinely stained with haematoxylin and eosin. Despite some limitations related to specimen fragmentation, counting can also be assessed in endoscopic biopsies. Based on the number of poorly differentiated clusters that appear under a microscopic field of a ×20 objective lens (i.e., a microscopic field with a major axis of 1 mm), colorectal cancer can be graded into malignancies as follows: tumors with <5 clusters as grade 1, tumors with 5 to 9 clusters as grade 2, and tumors with ≥10 clusters as grade 3. High poorly differentiated cluster counts are significantly associated with peri-neural and lympho-vascular invasion, the presence of nodal metastases or micrometastases, as well as shorter overall and progression free survival to colorectal cancer. CONCLUSION: The morphological aspects and clinical relevance of poorly differentiated clusters counting in colorectal cancer are discussed in this review.

Robotic-Assisted Surgery Improves the Quality of Total Mesorectal Excision for Rectal Cancer Compared to Laparoscopy: Results of a Case-Controlled Analysis.

BACKGROUND: The use of a robotic surgical system is claimed to allow precise traction and counter-traction, especially in a narrow pelvis. Whether this translates to improvement of the quality of the resected specimen is not yet clear. The aim of the study was to compare the quality of the TME and the short-term oncological outcome between robotic and laparoscopic rectal cancer resections. METHODS: 20 consecutive robotic TME performed in a single institution for rectal cancer (Rob group) were matched 1:2 to 40 laparoscopic resections (Lap group) for gender, body mass index (BMI), and distance from anal verge on rigid proctoscopy. The quality of TME was assessed by 2 blinded and independent pathologists and reported according to international standardized guidelines. RESULTS: Both samples were well matched for gender, BMI (median 25.9 vs. 24.2 kg/m(2), p = 0.24), and level of the tumor (4.1 vs. 4.8 cm, p = 0.20). The quality of the TME was better in the Robotic group (complete TME: 95 vs. 55 %; p = 0.0003, nearly complete TME 5 vs. 37 %; p = 0.04, incomplete TME 0 vs. 8 %, p = 0.09). A trend for lower positive circumferential margin was observed in the Robotic group (10 vs. 25 %, p = 0.1). CONCLUSIONS: These results suggest that robotic-assisted surgery improves the quality of TME for rectal cancer. Whether this translates to better oncological outcome needs to be further investigated.

Chemical profiling: a tool to decipher the structure and organisation of illicit drugs markets: an 8-year study in Western Switzerland

Illicit drug analyses usually focus on the identification and quantitation of questioned material to support the judicial process. In parallel, more and more laboratories develop physical and chemical profiling methods in a forensic intelligence perspective. The analysis of large databases resulting from this approach enables not only to draw tactical and operational intelligence, but may also contribute to the strategic overview of drugs markets. In Western Switzerland, the chemical analysis of illicit drug seizures is centralised in a laboratory hosted by the University of Lausanne. For over 8 years, this laboratory has analysed 5875 cocaine and 2728 heroin specimens, coming from respectively 1138 and 614 seizures operated by police and border guards or customs. Chemical (major and minor alkaloids, purity, cutting agents, chemical class), physical (packaging and appearance) as well as circumstantial (criminal case number, mass of drug seized, date and place of seizure) information are collated in a dedicated database for each specimen. The study capitalises on this extended database and defines several indicators to characterise the structure of drugs markets, to follow-up on their evolution and to compare cocaine and heroin markets. Relational, spatial, temporal and quantitative analyses of data reveal the emergence and importance of distribution networks. They enable to evaluate the cross-jurisdictional character of drug trafficking and the observation time of drug batches, as well as the quantity of drugs entering the market every year. Results highlight the stable nature of drugs markets over the years despite the very dynamic flows of distribution and consumption. This research work illustrates how the systematic analysis of forensic data may elicit knowledge on criminal activities at a strategic level. In combination with information from other sources, such knowledge can help to devise intelligence-based preventive and repressive measures and to discuss the impact of countermeasures.