Dissertations / Theses on the topic 'Machine perfusion'

Organ perfusion systems have successfully been applied to solid organ preservation and subsequent transplantation. However, their use in limb preservation for Vascularized Composite Tissue Allotransplantation (VCA) has yet to be thoroughly investigated. This thesis explores the potential for hypothermic machine perfusion in prolonging limb graft viability in a swine forelimb amputation model. The experiment was designed with the right and left forelimbs from the same pig randomly assigned to the treatment and control groups. Eighteen (18) limbs were procured from a local abattoir, vessels cannulated, and an initial flush of a modified phosphate buffered saline (PBS) solution was performed. Half of those limbs, assigned to the treatment group, were then preserved with continuous hypothermic machine perfusion for 12 hours. The perfusate was a PBS solution supplemented with 5% w/v dextrose. The remaining nine limbs, assigned to the control group, were placed into a plastic bag and kept at room temperature (ca. 20oC) for the entire duration of the experiment. Methylene blue was used to verify perfusion throughout limbs. Histopathological analysis revealed the presence of significantly greater deterioration of the perfused limbs compared to control. I concluded that PBS solution is not suitable for extended limb preservation. Inadequate perfusate volume and lack of solution replenishment resulted in metabolic waste build up, accelerating total organ damage. Continued research is needed in order to develop clinically relevant hypothermic machine perfusion devices capable of prolonged limb preservation.
Master of Science

The Banner University Medical Center's (BUMC) renal transplant program relies on the LifePort Kidney Transporter to optimize marginal kidney organs via hypothermic machine perfusion (HMP) prior to transplantation. Hemodynamic parameters produced by the device followed over the duration of support, combined with clinical experience, guide decisions in determining the acceptability of a donor kidney for implantation. Thus far, statistical evidence supporting ideal parameters remain undefined. The purpose of this study is to create a logistic model that will ascertain the post-implant sustainability of LifePort® supported kidneys and predict clinical outcomes. My hypothesis is that the statistical models constructed based on retrospective LifePort® parameters and clinical outcome data will successfully predict donor organ vascular health for transplantation and the optimal support duration. A successful model will contribute to increased efficiencies in the kidney transplant process as well as improved patient outcomes. An overview of the institution’s success was weighed using a survival analysis, with delayed graft function (DGF) as the endpoint. A logistic regression model and forecast model were built to predict the outcome for rejecting or accepting the organ for transplant, as well as to predict the hemodynamic parameters hours after the start of infusion. Results concluded a flow greater than 80 mL/min had a 90% probability of transplantation. The forecast model was capable of predicting flow for up to five hours. The calculated flow was in a 10 mL/min range of the actual flow, when up to one hour parameters were entered into the model. The study concluded practicality in the clinical setting, in kidney transplantation.

Introduction: The predominate issue in transplantation today is the inadequate supply of suitable organs for an increasing number of patients on the transplant waiting lists. In efforts to address this demand there has been an increasing use of expanded-criteria and donation-after-cardiac death donor kidneys and pancreases, however these organs are at higher risk for reperfusion injury. Advances in organ preservation thus need to focus on techniques to assess and optimise organ viability prior to transplantation. Methods: This research focuses on organ viability assessment during preservation using rapid sampling micro-dialysis (rsMD); preconditioning organs with novel endothelial localising anticoagulants to prevent micro-vascular thrombotic complications; development of both novel hypothermic machine perfusion (HMP) solutions to minimise reperfusion injury, and HMP strategies that offer superior preservation of organ integrity compared to static cold storage (SCS). Using HMP and normothermic-reperfusion models of porcine and human kidney and pancreatic grafts, this project has investigated strategies addressing these themes. Results: rsMD can successfully provide detailed real-time information on tissue and organ viability during both SCS and HMP. Pre-conditioning grafts with novel localising anticoagulant proteins has been successful in ameliorating disturbances in macro and micro-vascular perfusion and graft microcvascular thrombosis, processes which play key roles in reperfusion injury and organ dysfunction. Separately the application of a novel adenosine/lidocaine based preservation solution in renal HMP preservation can potentially ameliorate reperfusion injury seen using conventional solutions. Post-ischaemic HMP reconditioning has been investigated in the context of recovering organs with an extreme period of SCS which may be a potential option to expand donor organ pools. Finally models of pancreatic HMP have been successfully established, opening the potential for organ viability assessment and optimization. Conclusion: This research has been successful in its overall objective to develop novel translational strategies that have high potential for clinical implementation. In doing so the goal would be to enable an expansion of the pool of acceptable donor organs by improving the methods used to determine and optimise their viability, specifically more effective preservation techniques and by addressing specific post-operative complications through graft preconditioning.

Kidney Transplantation is the gold standard treatment for patients with end-stage renal failure. Most kidneys used for transplantation are from deceased donors and ensuring successful outcomes depends on many factors. One of these is organ storage. Hypothermic Machine Perfusion (HMP) of deceased donor organs has been shown to have several benefits. However, it has not been widely adopted and the underlying mechanism is poorly understood. The first section of this thesis examines the introduction of HMP into clinical practice. HMP outcomes were similar to those of standard storage techniques but with the additional benefit of increasing safe storage times. This was likely due to inherent benefits of the machine itself, improved recipient preparation and better peri-operative conditions. The second part of this study analysed HMP perfusate using metabolomics (Nuclear Magnetic Resonance) to identify potential predictors of graft outcome. Differences were identified in the metabolic profiles of perfusate from kidneys with immediate and delayed graft function. These may have a future role in viability assessment. Improved understanding of metabolism during storage may help target optimization strategies for deceased donor organs. The final part of this study describes the development of a porcine model of transplantation to test future hypotheses.

While machine perfusion of explanted kidneys is theoretically superior to standard cold storage, it may damage potential transplants unless machine-associated swelling is controlled. This thesis presents the effects of perfusate tonicity on renal swelling during hypothermic machine perfusion. Phosphate buffered solution (PBS) and PBS supplemented with 5% w/v mannitol were used as isotonic (289 mOsm/kg) and hypertonic (568 mOsm/kg) perfusates, respectively. Porcine kidney pairs were procured then flushed and machine perfused; the right and left kidneys were assigned opposite perfusates. An experimental methodology was developed to image porcine kidneys undergoing hypothermic machine perfusion (5 deg C) for 15 minutes followed by 120 minutes without perfusion to quantify surface displacement (renal swelling) with digital image correlation (DIC). Surface displacement and size (thickness) were compared between the right and left kidneys of each pair. In addition, discharged renal fluids (i.e., filtrate and venous outflow) and biopsies were collected. On average, kidneys perfused with the mannitol solution were smaller in size than the kidneys perfused with PBS (p < 0.05) at the start and end of each experiment; however, there was no significant difference between the renal sizes at the end of the 15 minute perfusion interval (p > 0.05). Thus, hypertonic and isotonic perfusates yielded different renal swelling outcomes (i.e., physical size and surface displacement), which suggests that perfusate tonicity influences renal swelling. These experiments are the first time ex vivo renal surface displacement measurements have been collected during machine perfusion.
Master of Science

The last 1-2 decades have seen remarkable advances in organ procurement and preservation practices, especially with renewed enthusiasm for machine perfusion (MP) technology. However, cold static storage (CS) remains the most popular world-wide approach for the preservation of organs such as the kidneys, liver, and pancreas, largely due to its simplicity. It is clear that CS techniques have limited potential for further improvement, and will likely be supplanted and/or supplemented with MP technologies over the coming years due to the reparative, resuscitative, and assessment capabilities afforded by MP. This is especially important as we increase our utilisation of marginal and/or donation after circulatory death (DCD) organs to meet the ever-increasing demand requirements for transplantation. This dissertation explores selected aspects of abdominal organ procurement and preservation as targets for improvement and/or modification with the aim to enhance recipient transplantation outcomes. The kidney is used as a model organ for the development and exploration of MP as a means to ameliorate transplant organ ischaemia-reperfusion injury (IRI), including through the targeted delivery of anti-IRI drugs. In contrast, the optimization of CS protocols, including identification of ideal perfusion fluids and in situ perfusion routes, forms the basis for liver and pancreas transplantation work in this thesis. Such investigations are necessary to promote uniformity of practice between centres, and allow appropriate comparisons between MP and CS. The kidney MP work was guided by a systematic review and meta-analysis comparing MP and CS in the clinical and pre-clinical setting. Although hypothermic MP (HMP) was shown to enhance short-term graft outcomes, results were equivocal with respect to graft survival, especially in the DCD setting. Preliminary evidence indicated the potential superiority of normothermic MP (NMP) above HMP or CS, which may be further enhanced by using NMP as a conduit for directed drug delivery to the kidney to ameliorate IRI. We therefore developed and optimized a local NMP set-up using a series of porcine kidneys, which was then utilized to deliver the anti-IRI agent CD47-blocking antibody (αCD47Ab) in a porcine DCD model. The significant potential of this agent was initially confirmed by testing in a murine model of severe warm IRI, including its comparative efficacy to two other promising IRI agents, soluble complement receptor 1 (sCR1), and recombinant thrombomodulin, and also sCR1 in combination with αCD47Ab. αCD47Ab was successfully delivered to porcine DCD kidneys using NMP, with subsequent downstream positive impacts upon renal perfusion, and some functional and IRI-related parameters. The clinical utilisation of renal NMP has so far been limited to the UK, and this modality has not been tested in human kidneys in Australasia. Furthermore, the mechanistic basis of brief renal NMP is not entirely clear. Therefore, and as a prelude to a phase I clinical trial, NMP was tested in discarded deceased donor human kidneys. Fifteen kidneys were obtained from 10 donors, and successfully underwent NMP. NMP was especially effective for assessing and improving DCD kidneys discarded for poor macroscopic perfusion at retrieval. Flow cytometry analyses showed evidence of a massive passenger leukocyte efflux during NMP. In paired kidney analyses, one hour of NMP was shown to be superior to CS alone after simulated transplantation using ex vivo whole allogeneic blood reperfusion, in terms of renal perfusion and functional parameters. Whole transcriptome RNA sequencing revealed NMP-mediated induction of protective stress and inflammatory-related pathways, in addition to a reduction in cell death pathways. Accordingly, immunofluorescence techniques confirmed a reduction in cell death and IRI in NMP kidneys compared to their CS counterparts. CS and procurement techniques formed the basis of liver and pancreas transplantation-related studies conducted for this thesis. Firstly, we showed that blood transfusion requirements can be significantly reduced in recipients if the pancreas is retrieved using ultrasonic shears (Harmonic Scalpel), implying a reduction in procedural risk and recipient sensitization. Two systematic reviews and meta-analyses were then conducted to ascertain optimal in situ perfusion/preservation fluids, and perfusion routes, during procurement of pancreatic and hepatic allografts. There was a lack of overwhelming evidence favouring any specific preservation fluid, although University of Wisconsin solution will likely remain the solution of choice, especially for the pancreas. Furthermore, in standard criteria donors, aortic-only perfusion was found to produce equivalent liver transplant outcomes in comparison to dual (aorto-portal perfusion). However, existing studies included small patient numbers and short periods of follow-up. We therefore compared aortic and dual perfusion during liver retrieval using the Australia and New Zealand Liver Transplant Registry, which provided a much larger patient cohort with prolonged follow-up. This study confirmed the equivalence of aortic-only and dual perfusion in standard criteria liver donors, however there was also evidence indicating the superiority of dual perfusion in a subset of suboptimal/higher risk donors. Overall, this thesis expounds upon the putative benefits of NMP in kidney transplantation, including by directed drug delivery targeting the IRI cascade, and also enhances our understanding of optimal perfusion routes and preservation fluids for the liver and pancreas. The ultimate aim is to facilitate expansion of the donor pool whilst simultaneously enhancing recipient transplantation outcomes through the evidence-based implementation of technologies and techniques in a unified and coordinated manner.

La pénurie d'organes a amené les équipes de transplantation à élargir l'acceptation des greffons à des organes provenant de donneurs marginaux.Le recours aux greffons marginaux impose de réduire les lésions induites durant l'IR, et doit conduire à une prise en charge optimisée de façon à limiter le risque de PNF et de RRF.C'est face à cette situation, que la question de la perfusion d'organe et de l'utilisation des machines de perfusion s'est posée et a justifié la réalisation de ce travail.Un modèle d'autotransplantation chez le porc a été choisi car il permet d'évaluer les effets de l'IR sur le rein.Les reins ont subi une ischémie contrôlée. La conservation des reins a été randomisée soit en incubation statique dans IGL-1 ou Belzer MPS soit sur machine de perfusion.Nous avons utilisé comme paramètre d'étude : la survie des animaux, différents dosages biologiques, une analyse histologique et une évaluation immunologique par RTqPCR des certains gènes impliqués dans le mécanisme lésionnel d'IR.Nos résultats montrent au total :-Que la machine de perfusion RM3 diminue le risque de RRF et PNF post greffe.-La supériorité de l'IGL-1 sur le Belzer MPS ; il existe un effet propre de la solution IGL-1 pour moduler les mécanismes inflammatoires et immunologiques liés aux lésions d'IR.-Que la reprise de fonction et la PNF dépendent du liquide utilisé et qu'il existe un effet d'addition entre l'IGL-1 et la machine de perfusion
Organ shortage has led transplant teams to re-evaluate their acceptance criteria and to increase their use of marginal donor organs (ECD and DDAC). For this reason, it is necessary to reduce the lesions due to IR. By optimizing organ conservation, the risk of PNF and RRF can be limited, and organ survival increased. The question of organ perfusion and the use of perfusion machines arose in this context, leading to the work we present here.We chose to evaluate the effects of IR on a porcine auto-graft model, that being the gold standard for the study of IR lesions.The kidneys were subject to hot ischemia for 60 minutes, and then to cold ischemia for 22 hours, during which they were randomly conserved either through static incubation in IGL-1 or Belzer MPS or on a perfusion machine.We studied the animal survival rate, various bioassays, histological analysis and immune reactions though RTqPCR of certain genes involved in IR lesion mechanisms.Our results show:-That the RM3 perfusion machine decreases the RRF and PNF post-graft risk and thus the perfusion machine conservation is better than static conservation.-IGL-1 superiority over Belzer MPS; IGL-1 solution independently modulates inflammatory and immunological mechanisms linked to IR lesions.-That function recovery and PNF depend on the liquid used where there is an additive effect between the use of IGL-1 and the use of a perfusion machine

Hypothermic machine perfusion (HMP) is well established in the practice of renal transplantation and is associated with improved short and long-term outcomes for selected organs. Despite the advantages of this therapy during the period prior to transplantation, the mechanisms by which these benefits occur are not entirely clear and are likely to include factors additional to improved flow dynamics. In the first part of this thesis, using both a local and national transplant dataset, I aim to establish the benefits (if any) and rationale for HMP relevant to current UK practice for both deceased and live donor kidneys. In the second part of this thesis the mechanisms by which HMP exert benefit are further interrogated, with a particular focus on metabolism. Nuclear magnetic resonance (NMR) spectroscopy is the principal modality through which this is investigated and in addition to established 1D 1H NMR protocols, glucose tracer studies using 2D 13C NMR are described. Whole organ ex vivo perfusion is studied in this work using human kidneys (both transplanted and non-transplanted) and porcine organs, with the porcine model validated for metabolic studies. In the final section, methods to modify metabolism during HMP are attempted, with the effects of supplemental oxygenation described.

Neurosurgery is a demanding medical discipline that requires a complex interplay of several neuroimaging techniques. This allows structural as well as functional information to be recovered and then visualized to the surgeon. In the case of tumor resections this approach allows more fine-grained differentiation of healthy and pathological tissue which positively influences the postoperative outcome as well as the patient's quality of life. In this work, we will discuss several approaches to establish thermal imaging as a novel neuroimaging technique to primarily visualize neural activity and perfusion state in case of ischaemic stroke. Both applications require novel methods for data-preprocessing, visualization, pattern recognition as well as regression analysis of intraoperative thermal imaging. Online multimodal integration of preoperative and intraoperative data is accomplished by a 2D-3D image registration and image fusion framework with an average accuracy of 2.46 mm. In navigated surgeries, the proposed framework generally provides all necessary tools to project intraoperative 2D imaging data onto preoperative 3D volumetric datasets like 3D MR or CT imaging. Additionally, a fast machine learning framework for the recognition of cortical NaCl rinsings will be discussed throughout this thesis. Hereby, the standardized quantification of tissue perfusion by means of an approximated heating model can be achieved. Classifying the parameters of these models yields a map of connected areas, for which we have shown that these areas correlate with the demarcation caused by an ischaemic stroke segmented in postoperative CT datasets. Finally, a semiparametric regression model has been developed for intraoperative neural activity monitoring of the somatosensory cortex by somatosensory evoked potentials. These results were correlated with neural activity of optical imaging. We found that thermal imaging yields comparable results, yet doesn't share the limitations of optical imaging. In this thesis we would like to emphasize that thermal imaging depicts a novel and valid tool for both intraoperative functional and structural neuroimaging.

Thermal imaging is a non-invasive and marker-free approach for intraoperative measurements of small temperature variations. In this work, we demonstrate the abilities of active dynamic thermal imaging for analysis of tissue perfusion state in case of cerebral ischemia. For this purpose, a NaCl irrigation is applied to the exposed cortex during hemicraniectomy. The cortical temperature changes are measured by a thermal imaging system and the thermal signal is recognized by a novel machine learning framework. Subsequent tissue heating is then approximated by a double exponential function to estimate tissue temperature decay constants. These constants allow us to characterize tissue with respect to its dynamic thermal properties. Using a Gaussian mixture model we show the correlation of these estimated parameters with infarct demarcations of post-operative CT. This novel scheme yields a standardized representation of cortical thermodynamic properties and might guide further research regarding specific intraoperative diagnostics.

Thermal imaging is a non-invasive and marker-free approach for intraoperative measurements of small temperature variations. In this work, we demonstrate the abilities of active dynamic thermal imaging for analysis of tissue perfusion state in case of cerebral ischemia. For this purpose, a NaCl irrigation is applied to the exposed cortex during hemicraniectomy. The cortical temperature changes are measured by a thermal imaging system and the thermal signal is recognized by a novel machine learning framework. Subsequent tissue heating is then approximated by a double exponential function to estimate tissue temperature decay constants. These constants allow us to characterize tissue with respect to its dynamic thermal properties. Using a Gaussian mixture model we show the correlation of these estimated parameters with infarct demarcations of post-operative CT. This novel scheme yields a standardized representation of cortical thermodynamic properties and might guide further research regarding specific intraoperative diagnostics.

Il est prouvé que la conservation des greffons rénaux marginaux en machine de perfusion (MP) est bénéfique. Cependant, cette méthode nécessite des améliorations afin de minimiser les lésions d’ischémie-reperfusion (I/R), par l’ajout d’oxygène et/ou d’un transporteur d’oxygène. Nous avons cherché à évaluer les effets de l’oxygénation et de l’ajout d’une hémoglobine de ver marin (HbAm, M101) durant la perfusion rénale hypothermique avant transplantation. Nos critères de jugement étaient basés sur la reprise de fonction du greffon et sur les lésions tardives de dysfonction rénale. Nous avons utilisé un modèle porcin : les reins ont été exposés à 1h d’ischémie chaude, puis perfusés dans une MP WAVES® pendant 23h à 4°C avant autotransplantation. Quatre groupes ont étudié : W (MP-21% O2), W-O2 (MP-100% O2), W-M101 (MP-21% O2 + 2g/L HbAm), W-O2+M101 (100% O2 + 2g/L HbAm), (n=6 per groupe). Les reins du groupe W-M101 ont montré un débit de perfusion plus élevé et une résistance rénale plus faible comparé aux autres groupes. Pendant la première semaine post-transplantation, les groupes W-O2 et W-M101 ont montré une créatininémie significativement plus faible et un meilleur taux de filtration glomérulaire (GFR). Les niveaux circulants de KIM-1 et IL-18 étaient plus faibles dans le groupe W-M101, tandis que les niveaux de NGAL et d’ASAT étaient plus faibles dans les groupes d’oxygénation active. Trois mois post-transplantation, la fraction excrétée de sodium et le ratio protéinurie/créatininurie étaient plus élevé dans le groupe W. La créatininémie était plus faible dans le groupe W-M101. La fibrose interstitielle a évalué à 3 mois post-transplantation étaient plus faible dans les groupes W-M101 et W-O2+M101. Nous avons révélé histologiquement que l’infiltration de mastocytes était significativement élevée dans le groupe W comparé aux autres groupes. Nous avons montré que la combinaison de 21% O2 + hémoglobine améliorent la reprise de fonction du greffon rénale
Introduction: It is proved that preservation of marginal kidney graft in machine perfusion (MP) is beneficial. However, this method requires improvement to minimize the ischemia-reperfusion injuries (IRI), as addition of oxygen and/or an oxygen carrier. We aimed to evaluate the effects of oxygenation (100% or 21%) and the addition of marine worm hemoglobin (HbAm, M101) during hypothermic renal perfusion before transplantation. Our endpoints were based on graft function recovery and late renal dysfunction. Method and materials: We use a porcine model where kidneys were submitted to 1h warm ischemia, followed by WAVES® MP preservation for 23h before auto-transplantation. Four groups were studied: W (MP-21% O2), W-O2 (MP-100% O2), W-M101 (MP-21% O2 + 2g/L HbAm), W-O2+M101 (100% O2 + 2g/L HbAm), (n=6 per group). Results: Kidneys preserved in W-M101 group showed a higher perfusion flow and lower renal resistance, compared to other groups. During the first week post-transplantation, W-O2 or W-M101 groups showed lower blood creatinine and better glomerular filtration rate. Blood levels of KIM-1 and IL-18 were lower in W-M101 group, while blood levels of AST and NGAL were lower in groups with 100% O2. Three months after transplantation, the fractional excretion of sodium and the proteinuria/ creatininuria ratio were higher in W group. Blood creatinine was lower in W-M101 group. Interstitial fibrosis evaluated at 3 months was lower in groups W-M101 and W-O2+M101. We showed that the combination 21% O2 + hemoglobin improves the kidney graft outcome.Conclusion: We showed that the combination of 21% O2 + hemoglobin improved the kidney graft outcome

Intra-dialytic haemodynamic instability is a common and disabling problem which may lead to morbidity and mortality though repeated organ ischaemia, but it has proven difficult to link any particular blood pressure threshold with hard patient outcomes. The relationship between blood pressure and downstream organ ischaemia during haemodialysis has not been well characterised. Previous attempts to predict and prevent intra-dialytic hypotension have had mixed results, partly due to patient and event heterogeneity. Using the brain as the indicator organ, we aimed to model the dynamic relationship between blood pressure, real-time symptoms, downstream organ ischaemia during haemodialysis, in order to identify the most physiologically grounded, prognostic definition of intra-dialytic decompensation. Following on from this, we aimed to predict the onset of intra-dialytic decompensation using personalised, probabilistic models of multivariate, continuous physiological data, ultimately working towards an early warning system for intra-dialytic adverse events. This was a prospective study of 60 prevalent haemodialysis patients who underwent extensive, continuous physiological monitoring of haemodynamic, cardiorespiratory, tissue oxygenation and dialysis machine parameters for 3-4 weeks. In addition, longitudinal cognitive function testing was performed at baseline and at 12 months. Despite their use in clinical practice, we found that blood pressure thresholds alone have a poor trade off between sensitivity and specificity for predicting downstream tissue ischaemia during haemodialysis. However, the performance of blood pressure thresholds could be improved by stratification for the presence or absence of cerebral autoregulation, and personalising thresholds according to the individual lower limit of autoregulation. For patients without autoregulation, the optimal blood pressure target was a mean arterial pressure (MAP) of 70mmHg. A key finding was that cumulative intra-dialytic exposure to cerebral ischaemia, but not to hypotension per se, corresponded to change in executive cognitive function over 12 months. Therefore we chose cerebral ischaemia as the definition of intra-dialytic decompensation for predictive modelling. We were able to demonstrate that the development of cerebral desaturation could be anticipated from earlier deviations of univariate physiological data from the expected trajectory for a given patient, but sensitivity was limited by the heterogeneity of events even within one individual. The most useful phys- iological data streams included peripheral saturation variance, cerebral saturation variance, heart rate and mean arterial pressure. Multivariate data fusion techniques using these variables created promising personalised models capable of giving an early warning of decompensation. Future work will involve the refinement and prospective testing of these models. In addition, we envisage a prospective study assessing the benefit of autoregulation-guided blood pressure targets on short term outcomes such as patient symptoms and wellbeing, as well as longer term outcomes such as cognitive function.

Background In liver transplantation one of the most important promblem which has not a solution yet is the large discrepancy between supply and demand for liver transplantation. To expand the pool of organs we should find the way to utilize marginal livers like the Non-heart-beating-donors ones and the fatty grafts. The use of these livers is associated with high incidence of post-operative primary dysfunction caused by the damage due to the Cold Storage preservation in the context of the process of ischaemia- reperfusion in extreme hypothermia (4° C). The utilization of new methods in the conservation of hepatic grafts like Machine Perfusion becomes necessary, especially for its ability to reduce the damage of ischaemia-reperfusion in hypothermia. The great potentiality of this Machine in sane and marginal organ preservation and the number of works which took place about it, stimulated us to the deepen this argument. Purpose We decided to create a experimental model of Machine Perfusion for pigs and to test it with different temperatures to obtain the best condition of perfusion and graft preservation. We based our study on the various works that can be find in literature and on the research developed by the Università di Pavia on rats. Methods We used in our experiments 15 Landrace pig of the weight of 22 kilos each. We divided them in three groups of study. Group C: after hepatectomy the pig liver was preserved in Cold Storage (4° C) for 8 hours. Group N: after hepatectomy the pig liver was perfused in Normothermic Machine Perfusion (37°C) for 8 hours. Group I: the pig liver was perfused in Moderate Hypothermic Machine (20°C). After the preservation, all the 15 livers were tested with a rewarming (37°C) of 2 hours. During the two hours of the experiment we performed samples of blood and biopsies and we also valuated the indocianine green clearance. Results The results obtained by the histology, the functional test and biochemical levels of AST, ALT and LDH, in the moderate hypothermic perfusion prove the superiority of this machine . Conclusion It is concluded after these experiments the great ability of 20°C Machine Perfusion in pig's liver transplantation. We think that this method of preservation will be able to improve the pool of donors using NHBD livers and fatty livers. In future our research will deepen the problematic connected with the preservation of marginal organs, in particular of steatotic livers.

La transplantation est la meilleure alternative en cas d'insuffisance rénale terminale. Face à la pénurie de greffons, les équipes de transplantation se sont tournées notamment vers les donneurs décédés par arrêt circulatoire (DDAC) non contrôlés. Ces greffons soumis à une période d'ischémie chaude sont plus fragiles. Des méthodes de reconditionnement chez le donneur par refroidissement in situ (RIS) et circulation régionale normothermique (CRN) se sont développées afin de réduire les lésions d'ischémie-reperfusion. Le choix de la méthode est laissé à l'appréciation de chaque équipe et il existe une grande hétérogénéité des pratiques. Après prélèvement, l'utilisation des machines de perfusion hypothermique (MPH) est généralement recommandée. L'optimisation de ces phases de reconditionnement chez le donneur et de conservation hypothermique apparait comme un enjeu majeur de santé publique. Concernant l'optimisation du mode de reconditionnement, la mise au point d'un modèle préclinique porcin parfaitement reproductible a permis de mettre en évidence une supériorité de la CRN sur le RIS. Une durée de CRN de 4 heures minimum sans dépasser 6 heures paraît optimale. Concernant la conservation hypothermique, les MPH permettent le maintien du niveau d'expression des gènes retrouvé en fin de CRN. L'ajout d'une oxygénation active en MPH ou de curcumine en solution statique améliore le devenir du greffon à court et long termes dans un modèle préclinique d'autogreffe. Ce travail pourrait s'étendre à l'étude d'autres organes, d'autres durées d'ischémie chaude et aux DDAC contrôlés afin d'élargir encore le nombre d'organes éligibles à la transplantation
Transplantation is the best alternative to end-stage renal disease. The shortage of grafts led the transplant teams to consider uncontrolled deceased donors after circulatory death (DCDs). These grafts suffered from a period of warm ischemia and are more vulnerable. Reconditioning methods in the donor by in situ cooling (ISC) and normothermic regional perfusion (NRP) have been developed to reduce the ischemia-reperfusion injuries. Each team has the choice as to the method and there are many different practices. After removal of kidneys, the use of hypothermic perfusion machines (HPM) is generally recommended. The optimization of reconditioning in the donor and hypothermic preservation appears as a major public health challenge. About optimization of the reconditioning method, the development of a high reproducible preclinical porcine model allowed to highlight the superiority of RNP over ISC. NRP duration of 4 hours minimum without exceeding 6 hours seems optimal. About hypothermic preservation, HPM allows to maintain the level of expression of the genes found at the end of RNP. The addition of active oxygenation to HPM or curcumin in static solution improves the graft outcomes in the short and long terms in a preclinical model of auto transplantation. This work could be extended to the study of other organs, other durations of warm ischemia and to controlled DCDs in order to further increase the number of transplantable grafts

A NEW LIVER AUTOTRANSPLANTATION TECHNIQUE USING SUBNORMOTHERMIC MACHINE PERFUSION FOR ORGAN PRESERVATION IN A PORCINE MODEL: CLINICAL INDICATION FOR THE TREATMENT OF UNRESECTABLE HEPATIC LESIONS WITH CONVENTIONAL SURGERY ! Abstract ! BACKGROUND: Hepatic resection is the gold standard of therapy for primary and secondary liver tumors, but few patients are eligible for this procedure because of the extent of their neoplasms. Improvements in surgical experience of liver transplantation (OLT), hepatic resection and preservation with sub-normothermic machine perfusion (MP) have prompted the development of a new model of large animal autotransplantation. This sperimental model allowed the clinical application of Ex situ ex vivo liver surgery to treat otherwise unresectable liver tumors. METHODS: Landrace pigs were used in this experiment. After intubation, hepatectomy was performed according to the classic technique. The intrahepatic caval vein was replaced with a homologous tract of porcine thoracic aorta. The liver was perfused with hypothermic Celsior solution followed by MP at 20 °C with oxygenated Krebs solution. An hepatectomy was performed during the period of preservation, which lasted 120 minutes, then the liver was reimplanted into the same animal in a 90° counterclockwise rotated position. The anastomoses were performed in the classic sequence. Samples of intravascular fluid, blood and liver biopsies were obtained at the end of the period of preservation in MP and again at 1 and 3 hours after liver reperfusion to evaluate graft function and microscopic damage. Then we report the clinical application of the model on 8 clinical cases described individually. After appropriate preoperative study and with the permission of the ethics committee, the patients underwent ex situ liver resection. RESULTS: All animals survived the procedure. The peak of aspartate aminotransferase was recorded 60 minutes after reperfusion and the peak of alanine aminotransferase and lactate dehydrogenase after 180 minutes. Histopathologic examination under the light microscope identified no necrosis or congestion. Intraoperative echo-color Doppler documented good patency of the anastomosis and normal venous drainage. Among the patients who underwent surgical treatment the overall mortality during the mean follow-up of 493 days , was 25 % ( 12.5 % for sepsis and 12.5 % for recurrence of disease ) . The 66.6 % of patients in life is alive and currently free of neoplastic disease , 33.3 % are alive with disease recurrence in chemotherapy and in good overall clinical condition . CONCLUSION: This system made it possible to perform hepatic resections and vascular reconstructions ex situ while preserving the organ with mechanical perfusion (ex vivo, ex situ surgery). Improving surgical techniques regarding autotransplantation and our understanding of ischemia-reperfusion damage may enable the development of interesting scenarios for aggressive surgical treatment (Ex situ, ex vivo liver surgery ) or radiochemotherapy options to treat primary and secondary liver tumors unsuitable for conventional in situ surgery.
NUOVA TECNICA DI AUTOTRAPIANTO DI FEGATO IN UN MODELLO SPERIMENTALE PORCINO CON PRESERVAZIONE DEL GRAFT IN MACHINE PERFUSION: APPLICAZIONE CLINICA PER NEOPLASIE EPATICHE NON RESECABILI TRAMITE CHIRURGIA CONVENZIONALE. !INTRODUZIONE : La resezione epatica rappresenta il gold standard per il trattamento dei tumori del fegato primitivi e secondari , tuttavia solo una minoranza di pazienti possono essere sottoposti a tale trattamento poichè spesso la diagnosi di neoplasia avviene in stadi di malattia neoplastica troppo avanzata. L’esperienza tecnica del nostro Centro legata alla chirurgia epatica resettiva e sostituitiva, associata ai nostri studi sul danno da ischemia-riperfusione e sull’allestimento di nuove metodiche di preservazione del graft tramite l’utilizzo della Machine Perfusion (MP), ci ha permesso di sviluppare un sistema di perfusione meccanica sub-normotermica per preservare il fegato, che consente di eseguire resezioni epatiche e complesse ricostruzioni vascolari ex-situ durante la perfusione meccanica (chirurgia ex-vivo, ex-situ). Questo modello sperimentale ha permesso l' applicazione clinica della chirurgia epatica ex vivo ex situ per il trattamento di tumori al fegato altrimenti non operabili . METODI : Per l’esecuzione dell’esperimento abbiamo utilizzato 5 maiali Landrace x Large White . Dopo l’ intubazione è stata eseguita l’ epatectomia secondo la tecnica classica . La vena cava intraepatica è stata sostituita con un omologo tratto di aorta toracica di suino. Il fegato è stato perfuso con soluzione di preservazione ipotermica (Celsior solution 60 mL/kg in aorta e 30 mL/kg in vena porta) a 4°C. Quindi il fegato veniva posto in un sistema di perfusione ossigenato e continuamente perfuso per 120 minuti con soluzione di Krebs (MP). Durante la preservazione in Machine Perfusion, veniva eseguita una epatectomia tramite dissezione a freddo con forbici. Dopo 120 minuti di preservazione in MP, il fegato veniva reimpiantato nello stesso animale (auto- trapianto) ruotato di 90° in senso antiorario. Le anastomosi sono state eseguite nella sequenza classica . Campioni sia di sangue che di liquido di preservazione venivano prelevati per determinare i livelli di AST, ALT, LDH e lattati dopo il termine del periodo di preservazione con MP e nuovamente a un ora e a tre ore dalla riperfusione epatica. Di seguito viene descritta l'applicazione clinica di questo modello sperimentale su 8 casi clinici descritti individualmente . Dopo adeguato studio preoperatorio e con il permesso del comitato etico , i pazienti sono stati sottoposti a resezione epatica ex situ. RISULTATI: Tutti gli animali sono sopravvissuti alla procedura . Il picco di aspartato aminotransferasi è stato registrato 60 minuti dopo la riperfusione e il picco di alanina aminotransferasi e lattico deidrogenasi dopo 180 minuti. L’indagine istopatologica alla microscopia ottica non ha documentato reperti significativi di necrosi e congestione all’analisi dei preparati esaminati. L’Eco color Doppler intraoperatorio ha documentato buona pervietà delle anastomosi e normale drenaggio venoso . Tra i pazienti sottoposti a trattamento chirurgico la mortalità globale su un follow-up medio di 493 giorni , è stata del 25 % ( 12,5 % per sepsi e 12,5% per recidiva di malattia ) . Il 66,6 % dei pazienti in vita è vivo e attualmente libero da malattia , il 33,3 % sono vivi con recidiva di malattia in trattamento chemioterapico. ! CONCLUSIONI : Questo modello sperimentale ha consentito di effettuare resezioni epatiche e ricostruzioni vascolari ex situ preservando l'organo con la perfusione meccanica ( chirurgia ex vivo-ex situ ) . Il miglioramento delle tecniche chirurgiche relative all’autotrapianto e la miglior comprensione del danno da ischemia-riperfusione potrebbe rendere possibile lo sviluppo di scenari interessanti per il trattamento di tumori epatici primitivi e secondari non suscettibili di trattamento chirurgico convenzionale.

Im Rahmen dieser Dissertation wurden anhand eines Ferkelmodells (8-15kg Schweine, 5 Gruppen: „nicht pulsatile HLM“: n=9, „Minozyklin+HLM“: n= 6, „pulsatile HLM“: n=7, „Minozyklin-Kontrolle: n=6, „Kontrolle“: n=8) während einer 120-minütigen extrakorporaler Zirkulation (EKZ) und einer darauffolgenden 90-minütigen Rekonvaleszenzzeit der physikalische Einflussfaktor des pulsatilen Flusses sowie der pharmakologische Effekt von Minozyklin auf die Niere jeweils unabhängig voneinander untersucht. In allen Gruppen wurden HE-Färbungen sowie immunhistochemische Färbungen (HIF-1-α, 3-Nitrotyrosin, PAR, AIF) durchgeführt um pathologische Veränderungen auf zellulärer Ebene zu detektieren. Zusätzlich wurden energiereiche Phosphate und ihre Abbauprodukte mittels High Pressure/Performance Liquid Chromatography (HPLC) bestimmt. Zur Beurteilung der klinischen Funktion der Niere wurden nierenspezifische Blutwerte (Serumkreatinin, Serumharnstoff) und Laktat im arteriellen Blut bestimmt. Mit der pulsatilen Perfusion konnte ein Abfall des O2-Partialdruckes nicht verhindert werden (HIF-1-α), allerdings konnte die ATP-Konzentration aufrecht erhalten werden. Dies spricht dafür, dass die pulsatile Perfusion im Gegensatz zu der nicht pulsatilen Perfusion keinen relevanten O2-Mangel verursachte. Auch die Ergebnisse der Nitrotyrosin-3-Auswertung zeigen, dass die Bildung von Peroxynitrit reduziert und somit der nitrosative Stress auf die Zellen begrenzt wurde. Die DNA wurde jedoch unabhängig vom gewählten Blutflussprofil geschädigt (PAR). Auch anhand der nierenspezifischen Blutparameter (Serumkreatinin, Serumharnstoff) ließ sich eine postoperative Beeinträchtigung der Nierenfunktion feststellen. Im Vergleich zu der nicht pulsatilen EKZ war hier jedoch eine geringfügige Verbesserung zu erkennen (Serumkreatinin). Zusammenfassend kann gesagt werden, dass durch die pulsatile EKZ der Grad der Ischämie beeinflusst werden konnte, allerdings waren insgesamt keine wesentlich positiven Auswirkungen auf zellulärer Ebene und auf die postoperative Nierenfunktion festzustellen. Der Einsatz des technisch anspruchsvollen pulsatilen Perfusionssystems scheint daher in Bezug auf die Niere in der routinemäßigen Herzchirurgie nicht unbedingt erforderlich zu sein. Durch die Gabe von Minozyklin wurde zwar der Grad der Ischämie (HIF-1-α, ATP) nicht beeinflusst, allerdings konnte Minozyklin durch seine antioxidativen bzw. antinitrosativen (3-Nitrotyrosin), PARP-1-hemmenden (PAR) sowie antiapoptotischen (AIF) Wirkmechanismen die Niere offenbar vor den Folgen einer Ischämie schützen. Anhand der nierenspezifischen Blutwerte (Serumkreatinin, Serumharnstoff) wurde erkenntlich, dass Minozyklin die Nierenfunktion positiv beeinflusst, was wiederum die histologischen Befunde bestätigt. Für die Humanmedizin ist somit der Einsatz von Minozyklin während der EKZ eine Möglichkeit die Auswirkungen des Ischämie/Reperfusionsschadens und deren klinische Folgen hinsichtlich der Niere zu begrenzen. Allerdings muss berücksichtigt werden, dass der einmalige Einsatz eines Antibiotikums auch negativen Einfluss auf den Körper ausübt (Resistenzentwicklung, Nebenwirkungen), sodass Minozyklin aufgrund der in dieser Versuchsreihe gezeigten positiven Eigenschaften, insbesondere die PARP-1-Inhibition, lediglich als Modellsubstanz für Weiterentwicklungen genutzt werden kann.

L'Accident Vasculaire Cérébral (AVC) - pathologie résultant d'une perturbation de l'apport sanguin dans le cerveau - est un problème de santé publique majeur, représentant la troisième cause de mortalité dans les pays industrialisés. Afin d'améliorer la prise en charge des patients atteints d'un AVC, il est important de posséder des méthodes efficaces pour l'identification des patients éligibles aux différentes thérapies et pour l'évaluation du rapport bénéfice/risque associé à ces thérapies. Dans ce contexte, l'Imagerie par Résonance Magnétique (IRM) dynamique de perfusion par contraste de susceptibilité, une modalité d'imagerie utile pour apprécier l'état de la perfusion cérébrale, peut aider à identifier les tissus à risque de s'infarcir. Cependant, l'intégralité de la chaîne de traitement, de l'acquisition à l'analyse et l'interprétation de l'IRM de perfusion demeure complexe et plusieurs limitations restent encore à surmonter. Durant ces travaux de thèse, nous contribuons à l'amélioration de la chaîne de traitement de l'IRM de perfusion, avec comme objectif final, l'obtention d'une meilleure prédiction de l'évolution de la lésion ischémique dans l'AVC. Dans une première partie, nous travaillons principalement sur l'étape de déconvolution des signaux temporels, une des étapes clefs à l'amélioration de l'IRM de perfusion. Cette étape consiste en la résolution d'un problème inverse mal-posé, et permet le calcul de paramètres hémodynamiques qui sont des biomarqueurs importants pour la classification de l'état final des tissus dans l'AVC. Afin de comparer de façon objective les performances des différents algorithmes de déconvolution existants et d'en valider des nouveaux, il est nécessaire d'avoir accès à une information sur la vérité terrain après déconvolution. Dans ce but, nous avons développé un simulateur numérique pour l'IRM de perfusion, avec une vérité terrain générée automatiquement. Ce simulateur est utilisé pour démontrer la faisabilité d'une automatisation du réglage des paramètres de régularisation, et établir la robustesse d'un algorithme de déconvolution avec régularisation spatio-temporelle d'introduction récente. Nous proposons également un nouvel algorithme de déconvolution globalement convergent. Enfin, la première partie de ces travaux se termine avec une discussion sur une autre étape de la chaîne de traitement en IRM de perfusion, à savoir, la normalisation des cartes de paramètres hémodynamiques extraites des images déconvoluées
Stroke – a neurological deficit resulting from blood supply perturbations in the brain – is a major public health issue, representing the third cause of death in industrialized countries. There is a need to improve the identification of patients eligible to the different therapies, as well as the evaluation of the benefit-risk ratio for the patients. In this context, perfusion Dynamic Susceptibility Contrast (DSC)-MRI, a prominent imaging modality for the assessment of cerebral perfusion, can help to identify the tissues at risk of infarction from the benign oligaemia. However, the entire pipeline from the acquisition to the analysis and interpretation of a DSC-MRI remains complex and some limitations are still to be overcome. During this PhD work, we contribute to improving the DSC-MRI processing pipeline with the ultimate objective of ameliorating the prediction of the ischemic lesion evolution in stroke. In a first part, we primarily work on the step of temporal signal deconvolution, one of the steps key to the improvement of DSC-MRI. This step consists in the resolution of an inverse ill-posed problem and allows the computation of hemodynamic parameters which are important biomarkers for tissue fate classification in stroke. In order to compare objectively the performances of existing deconvolution algorithms and to validate new ones, it is necessary to have access to information on the ground truth after deconvolution. To this end, we developed a numerical simulator of DSC MRI with automatically generated ground truth. This simulator is used to demonstrate the feasability of a full automation of regularization parameters tuning and to establish the robustness of a recent deconvolution algorithm with spatio-temporal regularization. We then propose a new globally convergent deconvolution algorithm. Then, this first part ends with a discussion on another processing step in the DSC-MRI pipeline, the normalisation of the hemodynamic parameters maps extracted from the deconvolved images. In a second part, we work on the prediction of the evolution of the tissue state from longitudinal MRI data. We first demonstrate the interest of modeling longitudinal MRI studies in stroke as a communication channel where information theory provides useful tools to identify the hemodynamic parameters maps carrying the highest predictive information, determine the spatial observation scales providing the optimal predictivity for tissue classification as well as estimate the impact of noise in prediction studies. We then demonstrate the interest of injecting shape descriptors of the ischemic lesion in acute stage in a linear regression model for the prediction of the final infarct volume. We finally propose a classifier of tissue fate based on local binary pattern for the encoding of the spatio-temporal evolution of the perfusion MRI signals

La technique de réalité augmentée basée sur la fluorescence permet de quantifier la dynamique d’un signal fluorescent et de superposer une cartographie de perfusion aux images laparoscopiques en temps réel. Un modèle d’ischémie colique a été choisi afin de différencier différents types d’ischémie et l’extension d’une zone ischémique dans les différentes couches de la paroi. L’évaluation de la dynamique de fluorescence assistée par logiciel et couplée à une approche d’apprentissage automatique a permis de faire la distinction entre une ischémie d’origine artérielle et d’origine veineuse avec un bon taux de prédiction. Dans la seconde étude colique, les cartographies de perfusion ont clairement mis en évidence que l’étendue d’ischémie était significativement plus large du côté muqueux et risquait d’être sous-estimée avec une analyse exclusive du côté séreux. Deux études ont démontré que la technique d’imagerie par fluorescence permet de guider le chirurgien en temps réel au cours d’une chirurgie mini-invasive des glandes surrénales et que l’analyse quantitative effectuée avec le logiciel facilite la distinction entre les segments vascularisés et ischémiques
The fluorescence-based enhanced reality approach is used to quantify fluorescent signal dynamics and superimpose the perfusion cartography onto laparoscopic images in real time. A colonic ischemia model was chosen to differentiate between different types of ischemia and determine the extension of an ischemic zone in the different layers of the colonic wall. The evaluation of fluorescence dynamics associated with a machine learning approach made it possible to distinguish between arterial and venous ischemia with a good prediction rate. In the second study, quantitative perfusion assessment showed that the extent of ischemia was significantly larger on the mucosal side, and may be underestimated with an exclusive analysis of the serosal side. Two further studies have revealed that fluorescence imaging can guide the surgeon in real time during minimally invasive adrenal surgery, and that quantitative software fluorescence analysis facilitates the distinction between vascularized and ischemic segments

There is a chronic lack of donor hearts to meet the need for heart transplant both in the US and worldwide. Further, the use of available hearts is limited by the short period between collection and implantation during which the heart can be safely preserved ex vivo. Using mid-thermic Langendorff machine perfusion, we have been able to preserve the metabolic function of a healthy heart for up to 8 hours, twice the limit for current static cold storage. We have also been able to preserve the metabolic function of a damaged DCD Heart collected 30 minutes after cardiac arrest for a period of 8 hours. We further investigated whether it was possible to improve the preservation of DCD heart using treatment with 10 μM Triiodothyronine to stimulate the tissue metabolism and we did find a reduction in damage markers in the treated DCD hearts as compared to the untreated group.

[ES] El futuro de la imagen médica está ligado a la inteligencia artificial. El análisis manual de imágenes médicas es hoy en día una tarea ardua, propensa a errores y a menudo inasequible para los humanos, que ha llamado la atención de la comunidad de Aprendizaje Automático (AA). La Imagen por Resonancia Magnética (IRM) nos proporciona una rica variedad de representaciones de la morfología y el comportamiento de lesiones inaccesibles sin una intervención invasiva arriesgada. Sin embargo, explotar la potente pero a menudo latente información contenida en la IRM es una tarea muy complicada, que requiere técnicas de análisis computacional inteligente. Los tumores del sistema nervioso central son una de las enfermedades más críticas estudiadas a través de IRM. Específicamente, el glioblastoma representa un gran desafío, ya que, hasta la fecha, continua siendo un cáncer letal que carece de una terapia satisfactoria. Del conjunto de características que hacen del glioblastoma un tumor tan agresivo, un aspecto particular que ha sido ampliamente estudiado es su heterogeneidad vascular. La fuerte proliferación vascular del glioblastoma, así como su robusta angiogénesis han sido consideradas responsables de la alta letalidad de esta neoplasia. Esta tesis se centra en la investigación y desarrollo del método Hemodynamic Tissue Signature (HTS): un método de AA no supervisado para describir la heterogeneidad vascular de los glioblastomas mediante el análisis de perfusión por IRM. El método HTS se basa en el concepto de hábitat, que se define como una subregión de la lesión con un perfil de IRM que describe un comportamiento fisiológico concreto. El método HTS delinea cuatro hábitats en el glioblastoma: el hábitat HAT, como la región más perfundida del tumor con captación de contraste; el hábitat LAT, como la región del tumor con un perfil angiogénico más bajo; el hábitat IPE, como la región adyacente al tumor con índices de perfusión elevados; y el hábitat VPE, como el edema restante de la lesión con el perfil de perfusión más bajo. La investigación y desarrollo de este método ha originado una serie de contribuciones enmarcadas en esta tesis. Primero, para verificar la fiabilidad de los métodos de AA no supervisados en la extracción de patrones de IRM, se realizó una comparativa para la tarea de segmentación de gliomas de grado alto. Segundo, se propuso un algoritmo de AA no supervisado dentro de la familia de los Spatially Varying Finite Mixture Models. El algoritmo propone una densidad a priori basada en un Markov Random Field combinado con la función probabilística Non-Local Means, para codificar la idea de que píxeles vecinos tienden a pertenecer al mismo objeto. Tercero, se presenta el método HTS para describir la heterogeneidad vascular del glioblastoma. El método se ha aplicado a casos reales en una cohorte local de un solo centro y en una cohorte internacional de más de 180 pacientes de 7 centros europeos. Se llevó a cabo una evaluación exhaustiva del método para medir el potencial pronóstico de los hábitats HTS. Finalmente, la tecnología desarrollada en la tesis se ha integrado en la plataforma online ONCOhabitats (https://www.oncohabitats.upv.es). La plataforma ofrece dos servicios: 1) segmentación de tejidos de glioblastoma, y 2) evaluación de la heterogeneidad vascular del tumor mediante el método HTS. Los resultados de esta tesis han sido publicados en diez contribuciones científicas, incluyendo revistas y conferencias de alto impacto en las áreas de Informática Médica, Estadística y Probabilidad, Radiología y Medicina Nuclear y Aprendizaje Automático. También se emitió una patente industrial registrada en España, Europa y EEUU. Finalmente, las ideas originales concebidas en esta tesis dieron lugar a la creación de ONCOANALYTICS CDX, una empresa enmarcada en el modelo de negocio de los companion diagnostics de compuestos farmacéuticos.
[EN] The future of medical imaging is linked to Artificial Intelligence (AI). The manual analysis of medical images is nowadays an arduous, error-prone and often unaffordable task for humans, which has caught the attention of the Machine Learning (ML) community. Magnetic Resonance Imaging (MRI) provides us with a wide variety of rich representations of the morphology and behavior of lesions completely inaccessible without a risky invasive intervention. Nevertheless, harnessing the powerful but often latent information contained in MRI acquisitions is a very complicated task, which requires computational intelligent analysis techniques. Central nervous system tumors are one of the most critical diseases studied through MRI. Specifically, glioblastoma represents a major challenge, as it remains a lethal cancer that, to date, lacks a satisfactory therapy. Of the entire set of characteristics that make glioblastoma so aggressive, a particular aspect that has been widely studied is its vascular heterogeneity. The strong vascular proliferation of glioblastomas, as well as their robust angiogenesis and extensive microvasculature heterogeneity have been claimed responsible for the high lethality of the neoplasm. This thesis focuses on the research and development of the Hemodynamic Tissue Signature (HTS) method: an unsupervised ML approach to describe the vascular heterogeneity of glioblastomas by means of perfusion MRI analysis. The HTS builds on the concept of habitats. A habitat is defined as a sub-region of the lesion with a particular MRI profile describing a specific physiological behavior. The HTS method delineates four habitats within the glioblastoma: the HAT habitat, as the most perfused region of the enhancing tumor; the LAT habitat, as the region of the enhancing tumor with a lower angiogenic profile; the potentially IPE habitat, as the non-enhancing region adjacent to the tumor with elevated perfusion indexes; and the VPE habitat, as the remaining edema of the lesion with the lowest perfusion profile. The research and development of the HTS method has generated a number of contributions to this thesis. First, in order to verify that unsupervised learning methods are reliable to extract MRI patterns to describe the heterogeneity of a lesion, a comparison among several unsupervised learning methods was conducted for the task of high grade glioma segmentation. Second, a Bayesian unsupervised learning algorithm from the family of Spatially Varying Finite Mixture Models is proposed. The algorithm integrates a Markov Random Field prior density weighted by the probabilistic Non-Local Means function, to codify the idea that neighboring pixels tend to belong to the same semantic object. Third, the HTS method to describe the vascular heterogeneity of glioblastomas is presented. The HTS method has been applied to real cases, both in a local single-center cohort of patients, and in an international retrospective cohort of more than 180 patients from 7 European centers. A comprehensive evaluation of the method was conducted to measure the prognostic potential of the HTS habitats. Finally, the technology developed in this thesis has been integrated into an online open-access platform for its academic use. The ONCOhabitats platform is hosted at https://www.oncohabitats.upv.es, and provides two main services: 1) glioblastoma tissue segmentation, and 2) vascular heterogeneity assessment of glioblastomas by means of the HTS method. The results of this thesis have been published in ten scientific contributions, including top-ranked journals and conferences in the areas of Medical Informatics, Statistics and Probability, Radiology & Nuclear Medicine and Machine Learning. An industrial patent registered in Spain, Europe and EEUU was also issued. Finally, the original ideas conceived in this thesis led to the foundation of ONCOANALYTICS CDX, a company framed into the business model of companion diagnostics for pharmaceutical compounds.
[CA] El futur de la imatge mèdica està lligat a la intel·ligència artificial. L'anàlisi manual d'imatges mèdiques és hui dia una tasca àrdua, propensa a errors i sovint inassequible per als humans, que ha cridat l'atenció de la comunitat d'Aprenentatge Automàtic (AA). La Imatge per Ressonància Magnètica (IRM) ens proporciona una àmplia varietat de representacions de la morfologia i el comportament de lesions inaccessibles sense una intervenció invasiva arriscada. Tanmateix, explotar la potent però sovint latent informació continguda a les adquisicions de IRM esdevé una tasca molt complicada, que requereix tècniques d'anàlisi computacional intel·ligent. Els tumors del sistema nerviós central són una de les malalties més crítiques estudiades a través de IRM. Específicament, el glioblastoma representa un gran repte, ja que, fins hui, continua siguent un càncer letal que manca d'una teràpia satisfactòria. Del conjunt de característiques que fan del glioblastoma un tumor tan agressiu, un aspecte particular que ha sigut àmpliament estudiat és la seua heterogeneïtat vascular. La forta proliferació vascular dels glioblastomes, així com la seua robusta angiogènesi han sigut considerades responsables de l'alta letalitat d'aquesta neoplàsia. Aquesta tesi es centra en la recerca i desenvolupament del mètode Hemodynamic Tissue Signature (HTS): un mètode d'AA no supervisat per descriure l'heterogeneïtat vascular dels glioblastomas mitjançant l'anàlisi de perfusió per IRM. El mètode HTS es basa en el concepte d'hàbitat, que es defineix com una subregió de la lesió amb un perfil particular d'IRM, que descriu un comportament fisiològic concret. El mètode HTS delinea quatre hàbitats dins del glioblastoma: l'hàbitat HAT, com la regió més perfosa del tumor amb captació de contrast; l'hàbitat LAT, com la regió del tumor amb un perfil angiogènic més baix; l'hàbitat IPE, com la regió adjacent al tumor amb índexs de perfusió elevats, i l'hàbitat VPE, com l'edema restant de la lesió amb el perfil de perfusió més baix. La recerca i desenvolupament del mètode HTS ha originat una sèrie de contribucions emmarcades a aquesta tesi. Primer, per verificar la fiabilitat dels mètodes d'AA no supervisats en l'extracció de patrons d'IRM, es va realitzar una comparativa en la tasca de segmentació de gliomes de grau alt. Segon, s'ha proposat un algorisme d'AA no supervisat dintre de la família dels Spatially Varying Finite Mixture Models. L'algorisme proposa un densitat a priori basada en un Markov Random Field combinat amb la funció probabilística Non-Local Means, per a codificar la idea que els píxels veïns tendeixen a pertànyer al mateix objecte semàntic. Tercer, es presenta el mètode HTS per descriure l'heterogeneïtat vascular dels glioblastomas. El mètode HTS s'ha aplicat a casos reals en una cohort local d'un sol centre i en una cohort internacional de més de 180 pacients de 7 centres europeus. Es va dur a terme una avaluació exhaustiva del mètode per mesurar el potencial pronòstic dels hàbitats HTS. Finalment, la tecnologia desenvolupada en aquesta tesi s'ha integrat en una plataforma online ONCOhabitats (https://www.oncohabitats.upv.es). La plataforma ofereix dos serveis: 1) segmentació dels teixits del glioblastoma, i 2) avaluació de l'heterogeneïtat vascular dels glioblastomes mitjançant el mètode HTS. Els resultats d'aquesta tesi han sigut publicats en deu contribucions científiques, incloent revistes i conferències de primer nivell a les àrees d'Informàtica Mèdica, Estadística i Probabilitat, Radiologia i Medicina Nuclear i Aprenentatge Automàtic. També es va emetre una patent industrial registrada a Espanya, Europa i els EEUU. Finalment, les idees originals concebudes en aquesta tesi van donar lloc a la creació d'ONCOANALYTICS CDX, una empresa emmarcada en el model de negoci dels companion diagnostics de compostos farmacèutics.
En este sentido quiero agradecer a las diferentes instituciones y estructuras de financiación de investigación que han contribuido al desarrollo de esta tesis. En especial quiero agradecer a la Universitat Politècnica de València, donde he desarrollado toda mi carrera acadèmica y científica, así como al Ministerio de Ciencia e Innovación, al Ministerio de Economía y Competitividad, a la Comisión Europea, al EIT Health Programme y a la fundación Caixa Impulse
Juan Albarracín, J. (2020). Unsupervised learning for vascular heterogeneity assessment of glioblastoma based on magnetic resonance imaging: The Hemodynamic Tissue Signature [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/149560
TESIS

A better understanding of tumour heterogeneity is central for accurate diagnosis, targeted therapy and personalised treatment of glioblastoma patients. This thesis aims to investigate whether pre-operative multi-parametric magnetic resonance imaging (MRI) can provide a useful tool for evaluating inter-tumoural and intra-tumoural heterogeneity of glioblastoma. For this purpose, we explored: 1) the utilities of habitat imaging in combining multi-parametric MRI for identifying invasive sub-regions (I & II); 2) the significance of integrating multi-parametric MRI, and extracting modality inter-dependence for patient stratification (III & IV); 3) the value of advanced physiological MRI and radiomics approach in predicting epigenetic phenotypes (V). The following observations were made: I. Using a joint histogram analysis method, habitats with different diffusivity patterns were identified. A non-enhancing sub-region with decreased isotropic diffusion and increased anisotropic diffusion was associated with progression-free survival (PFS, hazard ratio [HR] = 1.08, P < 0.001) and overall survival (OS, HR = 1.36, P < 0.001) in multivariate models. II. Using a thresholding method, two low perfusion compartments were identified, which displayed hypoxic and pro-inflammatory microenvironment. Higher lactate in the low perfusion compartment with restricted diffusion was associated with a worse survival (PFS: HR = 2.995, P = 0.047; OS: HR = 4.974, P = 0.005). III. Using an unsupervised multi-view feature selection and late integration method, two patient subgroups were identified, which demonstrated distinct OS (P = 0.007) and PFS (P < 0.001). Features selected by this approach showed significantly incremental prognostic value for 12-month OS (P = 0.049) and PFS (P = 0.022) than clinical factors. IV. Using a method of unsupervised clustering via copula transform and discrete feature extraction, three patient subgroups were identified. The subtype demonstrating high inter-dependency of diffusion and perfusion displayed higher lactate than the other two subtypes (P = 0.016 and P = 0.044, respectively). Both subtypes of low and high inter-dependency showed worse PFS compared to the intermediate subtype (P = 0.046 and P = 0.009, respectively). V. Using a radiomics approach, advanced physiological images showed better performance than structural images for predicting O6-methylguanine-DNA methyltransferase (MGMT) methylation status. For predicting 12-month PFS, the model of radiomic features and clinical factors outperformed the model of MGMT methylation and clinical factors (P = 0.010). In summary, pre-operative multi-parametric MRI shows potential for the non-invasive evaluation of glioblastoma heterogeneity, which could provide crucial information for patient care.

碩士
國立陽明大學
生物醫學影像暨放射科學系
102
Background: Brain perfusion parameters are altered by the presence of stenosis or occlusion of internal carotid artery. A lesion can’t be distinguished from normal tissue by using a single perfusion parameter perfectly because the parameter values varied with the components of different brain tissues. The distinct distributions of lesion and normal brain parenchyma in a scatter plot with two parameters can be separated by support vector machine (SVM) with an optimal dividing line. According to the algorithm, the mask of normal brain parenchyma can be identified. Material and method: We retrospectively analyzed 7 cases with severe unilateral internal carotid artery stenosis. Six combinations of perfusion parameters were choosing from all scatter plots for classification by SVM. Sifting the best combination for SVM to identify normal brain parenchymal mask, and then the mean values of perfusion parameters of the mask calibrated with standard values from literature. The scaling factors were applied to whole brain to calculate the absolute perfusion parameters. Two different approaches for identification of normal brain masks, the contralateral cerebral hemisphere and the median of time to peak (TTP), were compared with SVM to evaluate the accuracy of normal brain masks. Result: Tmax-TTP scatter plot provided the best result with classification by SVM. The percentage of difference between absolute values of perfusion parameters and standard values demonstrated the superiority of SVM. Conclusion: The accuracy of normal brain mask identified by SVM can improve the absolute values of brain perfusion parameters.

Neurosurgery is a demanding medical discipline that requires a complex interplay of several neuroimaging techniques. This allows structural as well as functional information to be recovered and then visualized to the surgeon. In the case of tumor resections this approach allows more fine-grained differentiation of healthy and pathological tissue which positively influences the postoperative outcome as well as the patient's quality of life. In this work, we will discuss several approaches to establish thermal imaging as a novel neuroimaging technique to primarily visualize neural activity and perfusion state in case of ischaemic stroke. Both applications require novel methods for data-preprocessing, visualization, pattern recognition as well as regression analysis of intraoperative thermal imaging. Online multimodal integration of preoperative and intraoperative data is accomplished by a 2D-3D image registration and image fusion framework with an average accuracy of 2.46 mm. In navigated surgeries, the proposed framework generally provides all necessary tools to project intraoperative 2D imaging data onto preoperative 3D volumetric datasets like 3D MR or CT imaging. Additionally, a fast machine learning framework for the recognition of cortical NaCl rinsings will be discussed throughout this thesis. Hereby, the standardized quantification of tissue perfusion by means of an approximated heating model can be achieved. Classifying the parameters of these models yields a map of connected areas, for which we have shown that these areas correlate with the demarcation caused by an ischaemic stroke segmented in postoperative CT datasets. Finally, a semiparametric regression model has been developed for intraoperative neural activity monitoring of the somatosensory cortex by somatosensory evoked potentials. These results were correlated with neural activity of optical imaging. We found that thermal imaging yields comparable results, yet doesn't share the limitations of optical imaging. In this thesis we would like to emphasize that thermal imaging depicts a novel and valid tool for both intraoperative functional and structural neuroimaging.

碩士
國立陽明大學
生物醫學影像暨放射科學系
104
Background and purpose: Computed tomography (CT) perfusion images are used for the diagnosis of cerebral stenosis or ischemic stroke. Hemodynamic parameter maps can help doctors to identify the regions of normal and abnormal perfusion. It is good for doctors to get reliable results automatically. This research used independent component analysis (ICA) and support vector machine (SVM) to determine the normal and abnormal brain regions from CT perfusion images. Material and methods： CT perfusion digital phantoms were generated from magnetic resonance images and CT perfusion images. Firstly, the simulated images were analyzed by using the ICA method. Secondly, hemodynamic parameters maps were analyzed by using the SVM method. The regions of normal or abnormal brain were calculated and the accuracy of two methods were compared using the tanimoto index. Finally, three clinical data were analyzed using the two methods. Results： The tanimoto index for ICA method is higher than all the results with SVM method. It demonstrates that ICA method is more accurate than the SVM method. ICA method is more sensitive in detecting abnormal areas. It is more stringent to find the abnormal area by SVM method. Conclusion： The result from ICA is more accurate than that of SVM for identifying normal and stenotic brain tissues on CT perfusion images.