Identification and quantification of the alveolar compartment by confocal laser endomicroscopy in patients with interstitial lung diseases

Tese de mestrado integrado, Engenharia Biomédica e Biofísica (Biofísica Médica e Fisiologia de Sistemas), Universidade de Lisboa, Faculdade de Ciências, 2018Doenças Intersticiais Pulmonares (DIP) é um termo que inclui mais de 200 doenças que afectam o parênquima pulmonar, partilhando manifestações clínicas, radiográficas e patológicas semelhantes. Este conjunto de doenças é bastante heterogéneo, apresentando cada tipo de DIP em diferente grau os elementos de inflamação e fibrose: enquanto a inflamação é reflectida pelo aumento de células inflamatórias e presença de nódulos ou edema, a fibrose reflecte-se pelas fibras adicionais de colagénio e elastina. Identificar o tipo de DIP de um doente é um processo difícil, sendo a Discussão Multidisciplinar o actual método de diagnóstico "gold standard": vários médicos especialistas compõem uma equipa multidisciplinar que vai ter em conta os dados clínicos, radiológicos e patológicos disponíveis para chegar a uma conclusão. Estes dados incluem imagens de tomografia computorizada de alta resolução (TCAR), a descrição da lavagem broncoalveolar e, quando possível, dados de biópsias. Apesar do esforço e competência da equipa multidisciplinar, 10% dos pacientes são categorizados como inclassificáveis devido a dados inadequados ou discrepância entre os dados existentes. A maior causa para DIP inclassificáveis é a ausência de dados histopatológicos associada aos riscos das biópsias cirúrgicas. É muito importante determinar a DIP específica de um doente, dadas as suas implicações no tratamento e gestão do mesmo. É particularmente crítica a distinção entre doentes com Fibrose Pulmonar Idiopática (FPI) e doentes sem FPI, dado que há terapias anti-fibróticas – como o Pirfenidone – indicadas para FPI que são extremamente dispendiosas, exigindo certeza no diagnóstico antes de serem prescritas. Além disso, o tratamento com agentes imunossupressores pode funcionar com o grupo dos não-FPI mas aumenta a morte e hospitalizações nos doentes com FPI. A discussão multidisciplinar pode beneficiar da informação adicional oferecida pelo Confocal Laser Endomicroscopy (CLE), uma técnica de imagiologia que torna possível visualizar os alvéolos pulmonares com resolução microscópica de forma minimamente invasiva, através de uma broncoscopia. O laser do CLE tem um comprimento de onda de 488 nm que permite observar a autofluorescência das fibras de elastina. Há evidências de que a quantidade de fibras de elastina é aumentada e a arquitectura destas fibras é alterada na presença de fibrose pulmonar, a qual está associada a algumas doenças intersticiais pulmonares incluindo a fibrose pulmonar idiopática. Até à data, os vídeos de Confocal Laser Endomicroscopy são, na maioria dos casos, analisados apenas visualmente, e pouca informação objectiva e consistente foi conseguida destes vídeos em doentes de DIP. No entanto, é possível obter informação mais relevante dos mesmos, convertendo-os em frames, pré-processando as imagens e extraindo atributos numéricos. Neste projecto, foram obtidas imagens dos alvéolos pulmonares de doentes de DIP através de CLE. O principal objectivo do projecto é melhorar a técnica de CLE e aumentar a sua usabilidade para que no futuro possa contribuir para facilitar a estratificação de doentes com DIP e eventualmente reduzir o número de biópsias pulmonares nestes doentes. Como mencionado, o instrumento de Confocal Laser Endomicroscopy emite uma luz laser azul de 488nm, a qual é reflectida no tecido e reorientada para o sistema de detecção pela mesma lente, passando por um pequeno orifício (pinhole). Isto permite que a luz focada seja recolhida e que feixes provenientes de planos fora de foco sejam excluídos, originando uma resolução microscópica que permite imagens ao nível celular. Quando o CLE é aplicado a imagem pulmonar, é possível observar as paredes alveolares pela autofluorescência natural presente nas fibras de elastina. No estudo clínico subjacente a este estudo, o protocolo de CLE foi aplicado a 20 pacientes, embora 8 tenham sido posteriormente excluídos da análise. Os vídeos de CLE obtidos sofreram duas selecções: uma com base na região onde uma biópsia (usada como referência) foi tirada e outra com base na qualidade técnica das imagens. Depois, os dados foram pré-processados: geraram-se imagens mosaico com um campo de visão alargado e, paralelamente converteram-se as sequências de vídeo em frames. A qualidade da imagem foi melhorada, filtrando o ruído electrónico para que posteriormente pudesse ser aplicada a análise de imagem. Esta análise extraiu valores numéricos que reflectem o estado do espaço alveolar, nomeadamente, variáveis de textura e medições relacionadas com as fibras de elastina. As imagens de CLE obtidas mostraram-se muito interessantes. A resolução é superior à tomografia computorizada de alta resolução e a tridimensionalidade acrescenta informação às biópsias. O facto de permitir feedback em tempo real e observar ao vivo os movimentos naturais da respiração contribui para a análise do estado do doente. A análise de textura feita às imagens serviu-se de um algoritmo de extracção de variáveis de Haralick a partir de uma Gray-Level Co-occurence Matrix (GLCM). Foram extraídas as variáveis de textura Momento Angular Secundário (Energia), Entropia, Momento de Diferença Inversa, Contraste, Variação e Correlação. O algoritmo de Ridge Detection (detecção de linhas) identificou a maior parte das fibras de elastina detectáveis por um observador humano e mediu o Número de Fibras, o seu Comprimento e Largura e o Número de Junções entre fibras, permitindo também calcular a Soma dos Comprimentos de todas as fibras. Estes algoritmos devolveram valores consistentes num processo mais eficiente comparado com um observador humano, conseguindo avaliar em poucos segundos múltiplas variáveis para todo o conjunto de dados. As medições relacionadas com as fibras de elastina pretendiam ajudar a identificar os doentes fibróticos. Era esperado que as fibras dos doentes fibróticos fossem mais largas, mas isso não se observou. Também se previa que este grupo de doentes apresentasse maior número de fibras e junções, mas não houve uma diferença significativa entre grupos. No entanto, quando o grupo fibrótico foi segregado, o número de fibras e junções parece separar a fibrose moderada da fibrose severa. Este resultado é interessante na medida em que sugere que a monitorização do número de fibras/junções com CLE pode potencialmente ser usado como medida de eficácia de medicação anti-fibrótica. Em relação às variáveis de textura, esperava-se que os doentes fibróticos apresentassem valores mais elevados de Entropia, Contraste e Variância e valores inferiores de Momento de Diferença Inversa, dado que o seu tecido pulmonar deveria corresponder a imagens mais complexas e heterogéneas com mais arestas presentes. No entanto, ainda não foi possível estabelecer diferenças significativas entre grupos. Apesar dos resultados com o conjunto de dados usado não ter demonstrado correlações fortes entre as conclusões do CLE e da TCAR/histopatologia, os valores das variáveis em si já contribuem para o estudo das DIP, nomeadamente da sua fisiologia. De facto, a amostra de doentes deste estudo era reduzida, mas com uma amostra maior, espera-se que algumas das varáveis se correlacionem com outras técnicas usadas no diagnóstico e permitam segregar os pacientes em grupos e eventualmente aplicar classificação de dados. Neste momento, é possível especular que algumas variáveis seriam melhores candidatas para um classificador, nomeadamente os Números de Fibras e Junções, a Soma dos Comprimentos das fibras e as variáveis de Haralick Entropia e Energia. O projecto apresentado nesta dissertação foi desenvolvido através de um estágio de 6 meses no departamento de Pneumologia no Academic Medical Center em Amsterdão, Países Baixos. No Academic Medical Center (AMC), fui acompanhada pelos estudantes de doutoramento Lizzy Wijmans - médica - e Paul Brinkman - engenheiro biomédico - e supervisionada pelo Dr. Jouke Annema, MD, PhD, Professor de endoscopia pulmonar. Este grupo de investigação do AMC está focado em técnicas inovadoras de imagiologia do sistema pulmonar e teve a oportunidade de reunir com a empresa MKT –que produz a tecnologia de Confocal Laser Endomicroscopy –, o que enriqueceu a discussão aqui apresentada. Do Departamento de Física da Faculdade de Ciências da Universidade de Lisboa, fui orientada pelo Prof. Nuno Matela.Interstitial Lung Diseases (ILD) is a heterogeneous group of more than 200 diseases which affect the lung parenchyma. To identify the type of ILD a patient suffers from is a difficult process, and 10% of the patients are categorized as unclassifiable, mostly due to the absence of histopathological data associated with the risks of lung biopsies. The patient specific diagnosis is important because of its implications to the patient treatment and management, being particularly relevant to identify lung fibrosis. The Confocal Laser Endomicroscopy (CLE) can add information to this process. CLE allows to image the lung tissue with a micrometer resolution in a minimally invasive way, through a bronchoscopy. The elastin fibers from the lung alveoli are visible with this technique due to their autofluorescence. Since there is evidence that the amount of elastin fibers increases, and their architecture is altered in lung fibrosis, CLE should be used to extract values reflecting this condition. Thus, the main goal of this project was to improve the CLE technique and increase its usability, by extracting numerical values from the images which would reflect the state of the alveolar space, particularly the elastin fibers. The ILD patients recruited for the study had their lung alveoli imaged with CLE. The CLE movies were selected, pre-processed – were converted into frames, had their image quality enhanced and some mosaics were obtained – and then analyzed. The ridge detection algorithm detected most fibers recognized by a human observer. It allowed the measurement of the Number of Detected Fibers, their Length and Width, the Number of Junctions between fibers and to calculate the Sum from all Fibers’ Lengths. The Gray-Level Co-occurrence Matrix allowed the extraction of the Haralick texture features: Angular Second Moment (Energy), Entropy, Inverse Difference Moment, Contrast, Variance and Correlation. These algorithms produced consistent and unbiased numerical features, in an efficient process which can analyze the entire data set in a few seconds. Regarding the fiber related measurements, it was expected for the fibrotic patients to have wider fibers and a higher number of fibers and junctions. In terms of texture variables, it was expected from the fibrotic patients to present higher values of Entropy, Contrast and Variance, and lower values of Inverse Difference Moment, given their lung tissue should correspond to more complex and heterogeneous images with more ridges present. Due to the small sample size, it was still not possible to stratify patients with this data set. Nevertheless, the measurements presented here already contribute to the study of ILD, helping to understand the disease physiology. It is hoped that in the future, these measurements will aid the diagnosis process specially in those cases when patients cannot undergo a surgical biopsy. Additionally, CLE could potentially be used as an anti-fibrotic medication efficiency measurement tool

Transbronchial cryobiopsy and Neutrophil Lymphocyte Ratio - new precision medicine tools and markers in Interstitial Lung Disease

The interstitial lung diseases (ILDs) are a group of over 200 disease that may lead to progressive fibrosis and respiratory failure. ILDs are heterogenous, with varying amounts of inflammation and fibrosis, and differ in response to therapy and outcome. The most severe fibrotic (f) ILD, idiopathic pulmonary fibrosis (IPF), has a median survival of just three years. Progressive fILD may respond to antifibrotic treatments which slow down, but do not reverse, fibrosis albeit often with significant side effects. Better treatments or delivery of treatments are needed. Diagnosis of ILD relies on clinical history, imaging and, in some cases lung biopsy, with associated risks. Better diagnostic and prognostic biomarkers in ILD are urgently needed. This thesis examines the approach to diagnosis, prognostication, and treatment in fILDs, and, in particular IPF. It begins with the finding that Neutrophil Lymphocyte Ratio (NLR), derived from a simple, widely available blood test, is a prognostic biomarker in IPF. The role of lung biopsy in the diagnostic pathway is considered and the use of a relatively new minimally invasive technique of transbronchial cryo lung biopsy (TBCB) as an alternative to surgical lung biopsy (SLB) is described. The value of TBCB to obtain lung tissue for research is demonstrated with evaluation of the distribution of inhaled ipratropium in fILD. Using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) on samples of lung taken using TBCB, it was demonstrated that inhaled medication was able to reach the fibrotic lung, presenting a new approach to drug delivery in fILD. Further discussion focusses on the current role of SLB in the diagnostic pathway in ILD, the presentation of a systematic literature review, and a discussion of future trials to assess the potential benefits of a wider use of TBCB

Prototypes for Content-Based Image Retrieval in Clinical Practice

Content-based image retrieval (CBIR) has been proposed as key technology for computer-aided diagnostics (CAD). This paper reviews the state of the art and future challenges in CBIR for CAD applied to clinical practice

Biomedical Image Processing and Classification

Biomedical image processing is an interdisciplinary field involving a variety of disciplines, e.g., electronics, computer science, physics, mathematics, physiology, and medicine. Several imaging techniques have been developed, providing many approaches to the study of the human body. Biomedical image processing is finding an increasing number of important applications in, for example, the study of the internal structure or function of an organ and the diagnosis or treatment of a disease. If associated with classification methods, it can support the development of computer-aided diagnosis (CAD) systems, which could help medical doctors in refining their clinical picture

Development and Application of Gadolinium Free Cardiac Magnetic Resonance Fibrosis Imaging for Multiscale Study of Heart Failure in Patients with End Stage Renal Disease

Cardiac magnetic resonance (CMR) is a powerful tool to noninvasively image ventricular fibrosis. Late gadolinium enhancement (LGE) CMR identifies focal and, with T1 mapping, diffuse fibrosis. Despite prevalent cardiac fibrosis and heart failure, patients with end stage renal disease (ESRD) are excluded from LGE. Absence of a suitable diagnostic has limited the understanding of heart failure and obstructed development of therapies in the setting of ESRD. A quantitative, gadolinium free fibrosis detection method could overcome this critical barrier, propelling the advancement of diagnostic, monitoring, and therapy options. This project describes the development of a gadolinium free CMR technique and application for cardiac fibrosis measurement in patients with ESRD. Magnetization transfer (MT) occurs during standard cine balanced steady state free precession (bSSFP) CMR, where extracellular matrix protons exchange magnetization with water molecules. Extracellular water volume expansion, concomitant with fibrosis, reduces MT and subtly elevates signal intensity. Our technique, 2-pt bSSFP, extracts endogenous contrast sensitive to tissue fibrosis by obtaining pairs of high and low MT-weighted images and calculating normalized signal differences, denoted by ΔS/So. We tested 2-pt bSSFP in patients referred for CMR and found excellent agreement spatially with LGE and quantitatively with extracellular volume fraction. Diagnostic and clinical application of 2-pt bSSFP was comparable to LGE. We applied 2-pt bSSFP to patients with ESRD for multiscale comparison with correlates of fibrosis ranging from blood biomarkers to whole organ function. Patients with ESRD displayed hypertrophy with reduced contraction, but elevated ΔS/So and fibrosis. Some biomarkers correlated with both hypertrophy and fibrosis, highlighting the need to distinguish between hypertrophic and fibrotic remodeling. We monitored fibrosis over 1 year using 2-pt bSSFP in a cohort of patients with ESRD. ΔS/So and fibrotic burden increased substantially, despite minor changes in structure and function. Collectively these studies validate and apply 2-pt bSSFP for gadolinium free fibrosis CMR in patients with ESRD. While ventricular structure and function are commensurate with progression toward heart failure, it is now possible to specifically describe global and focal patterns of cardiac fibrosis in ESRD, along with comparisons to blood biomarkers which may lead to improved diagnostics and molecular treatment targets

Diseases of the Chest, Breast, Heart and Vessels 2019-2022

This open access book focuses on diagnostic and interventional imaging of the chest, breast, heart, and vessels. It consists of a remarkable collection of contributions authored by internationally respected experts, featuring the most recent diagnostic developments and technological advances with a highly didactical approach. The chapters are disease-oriented and cover all the relevant imaging modalities, including standard radiography, CT, nuclear medicine with PET, ultrasound and magnetic resonance imaging, as well as imaging-guided interventions. As such, it presents a comprehensive review of current knowledge on imaging of the heart and chest, as well as thoracic interventions and a selection of "hot topics". The book is intended for radiologists, however, it is also of interest to clinicians in oncology, cardiology, and pulmonology

Quantification of chronic fibrosing interstitial pneumonia on computed tomography

The chronic fibrosing interstitial pneumonias (CFIPs) are diseases which cause progressive and often fatal progressive scarring of the lungs. The recent discovery of the first effective pharmacological therapies for this condition have increased interest in the monitoring of this disease. Due to the complex appearance of the CFIPs on computed tomography, visual quantification of disease severity and extent is limited. The purpose of this thesis was to develop and test a computer algorithm for the automated quantification of pulmonary fibrosis on CT using textural measures known as Minkowski functionals. A computer algorithm was successfully developed and this thesis presents initial results of testing the algorithm on a series of normal scans and on 24 prospectively recruited patients who also underwent a series of other tests including pulmonary function tests and a patient reported symptom questionnaire. The computer output was also compared with the visual assessment of two radiologists. Significant correlations were found between computer calculated lung volume and total lung capacity as measured on pulmonary function tests. We also found a significant correlation between computer calculated fibrosis volume and both gas transfer and forced vital capacity. The radiologists’ visual assessment of fibrosis and the computer estimated fibrosis volume were highly correlated. The novel computer algorithm represents a promising method for quantifying pulmonary fibrosis on CT with potential roles in monitoring disease progression and effects of therapeutic interventions

Diseases of the Chest, Breast, Heart and Vessels 2019-2022

This open access book focuses on diagnostic and interventional imaging of the chest, breast, heart, and vessels. It consists of a remarkable collection of contributions authored by internationally respected experts, featuring the most recent diagnostic developments and technological advances with a highly didactical approach. The chapters are disease-oriented and cover all the relevant imaging modalities, including standard radiography, CT, nuclear medicine with PET, ultrasound and magnetic resonance imaging, as well as imaging-guided interventions. As such, it presents a comprehensive review of current knowledge on imaging of the heart and chest, as well as thoracic interventions and a selection of "hot topics". The book is intended for radiologists, however, it is also of interest to clinicians in oncology, cardiology, and pulmonology