Autofluorescence (AF) is defined as the fluorescence emission observed when certain cell molecules are excited by UV or visible light of suitable wavelenghts. When a biologic molecule is illuminated at an excitation wavelength within the absorption spectrum of that molecule, it will absorb this energy and be activated from its ground state to an excited state. The molecule (fluorophore) can then relax back from the excited to the ground state by generating energy in the form of fluorescence, at emission wavelengths, which are longer than that of the excitation wavelength. The most important endogenous fluorophores are molecules widely distributed in cells and tissues, like proteins containing aromatic aminoacids, flavins and lipopigments. The main fluorophores of healthy skin are located in the epithelium (eg. keratin, nicotinamide adenine dinucleotide or NADH and flavin adenine dinucleotide or FAD) and the submucosa (e.g. collagen and elastin). These molecules when irradiated between the wavelengths from 375 and 440 nm, show fluorescence in the green spectral range. Nonmelanoma skin cancer (NMSC) is the most common malignancy worldwide. The developement of NMSC is accompanied by histopathological changes in epidermis such as loss of cellular maturation, alteration in keratin production, overall thickening of the epithelial layer and biochemical alterations (NADH decrease). NMSC is also accompanied by histopathological changes in the underlying stroma and submucosa, including neovascularization and destruction of the collagen cross-link by proteases. These alterations lead to a general decrease in AF due the alteration in distribution of the fluorochromes and in particular to NADH and collagen. In the last two decades, studies concerning cell and tissue AF has had a dramatic increase. AF studies have been performed both in vitro and in vivo, for the study of normal tissue and for the discrimination between normal tissues and neoplastic lesions of oral mucosa, skin, esophagus, colon, lung, bronchi, brain and bladder. The methods used are both direct visual fluorescence examination (DVFE) and spectrophotometry. In particular, DVFE has been widely used for clinical studies on oral mucosa. Regarding AF of the skin, this has been studied more frequently by using spectrophotometry. The principle is scanning and analyzing reflected light from the skin after exposure to an activating light source. AF spectroscopy is a very sensitive technique for quantitative measurements of tissue constituents. However, to date no methods have emerged that can be translated into clinical practice. The primary objective of this study is to investigate the correlation between spectral mesurement of cutaneous AF and the histopathological characteristics of malignant and pre-malignant skin in NMSC. Following surgical removal of the cancer, an ex vivo evaluation of the AF will be performed. The specimen will be irradiated with a probe that emits a light in the blue spectrum (wavelength 400-440 nm) and the fluorescence emitted by the tissue will be measured using a spectrophotometer in a standardized spot modality. Any changes detected will be reported on the surgical specimen with the application of a surgical mark. Histopathological examination of the lesion will be performed and any changes in the fluorescence pattern will be correlated with possible alterations in the histopathological pattern, referring to surgical marks. Alterations in AF spectral measurement correlate with histopathological alterations in NMSC. the spectral measurement can be a new support for the early diagnosis of NMSCs, a guide for the targeted incisional biopsies, a tool for the definition of the intraoperative surgical margins, and for the follow-up of treated patients.

L'autofluorescenza (AF) è definita come l'emissione di fluorescenza osservata quando determinate molecole sono eccitate da raggi UV o luce visibile di lunghezze d'onda adeguate. Quando una molecola viene illuminata ad una lunghezza d'onda di eccitazione, assorbirà questa energia e verrà attivata dal suo stato fondamentale a quello eccitato. La molecola (fluoroforo) può quindi rilassarsi dallo stato eccitato a fondamentale generando energia sotto forma di fluorescenza, a lunghezze d'onda di emissione più lunghe di quella di eccitazione. I fluorofori endogeni sono molecole ampiamente distribuite in cellule e tessuti, come proteine ​​contenenti aminoacidi aromatici, flavine e lipopigmenti. I principali fluorofori della cute sana si trovano nell'epitelio (ad es. cheratina, nicotinamide adenine dinucleotide o NADH e flavin adenine dinucleotide o FAD) e nella sottomucosa (ad es. collagene ed elastina). Queste molecole, quando irradiate tra le lunghezze d'onda da 375 a 440 nm, mostrano fluorescenza nell'intervallo spettrale del verde. Il non-melanoma skin cancer (NMSC) è il tumore maligno più comune al mondo. Lo sviluppo dei NMSC è accompagnato da cambiamenti istopatologici nell'epidermide come perdita di maturazione cellulare, alterazione della produzione di cheratina, ispessimento generale dello strato epiteliale e alterazioni biochimiche (riduzione del NADH). I NMSC sono anche accompagnati da cambiamenti istopatologici nello stroma e nella sottomucosa sottostanti, tra cui la neovascolarizzazione e la distruzione del legame crociato di collagene da parte delle proteasi. Queste alterazioni portano ad una generale riduzione dell’AF dovuta all'alterazione della distribuzione dei fluorocromi e in particolare al NADH e al collagene. Negli ultimi due decenni, gli studi riguardanti ll’AF cellulare e tissutale hanno avuto un notevole aumento. Sono stati condotti studi sull’AF sia in vitro che in vivo, per lo studio dei tessuti normali e per la discriminazione tra tessuti normali e lesioni neoplastiche di mucosa orale, cute, esofago, colon, polmone, bronchi, cervello e vescica. I metodi utilizzati sono sia il direct visual fluorescence examination (DVFE) sia lo spettrofotometria. In particolare, il DVFE è stato ampiamente utilizzato per studi clinici sulla mucosa orale. Per quanto riguarda l’AF della cute, questa è stata studiata più frequentemente usando lo spettrofotometria. Il principio è la scansione e l'analisi della luce emessa dalla cute dopo l'esposizione a una fonte di luce attivante. Tuttavia, ad oggi non sono emersi metodi che fossero traducibili nella pratica clinica. L'obiettivo principale di questo studio è quello di analizzare la correlazione tra la misurazione spettrale dell’AF cutanea e le caratteristiche istopatologiche della cute maligna e pre-maligna nel campo dei NMSC. Dopo la rimozione chirurgica, verrà eseguita una valutazione ex vivo dell’AF. Il campione verrà irradiato con una sonda che emette una luce nello spettro blu (lunghezza d'onda 400-440 nm) e la fluorescenza emessa dal tessuto verrà misurata mediante uno spettrofotometro in modalità spot standardizzata. Eventuali modifiche rilevate verranno riportate sul campione chirurgico con l'applicazione di un repere. Verrà eseguito un esame istopatologico della lesione e eventuali cambiamenti nel pattern di fluorescenza saranno correlati con possibili alterazioni del pattern istopatologico, facendo riferimento ai reperi chirurgici. Le alterazioni delle misure spettrali sono correlate alle alterazioni istopatologiche dei NMSC. La misurazione spettrale può essere un nuovo supporto per la diagnosi precoce dei NMSC, una guida per le biopsie incisionali mirate, uno strumento per la definizione dei margini chirurgici intraoperatori e per il follow-up dei pazienti trattati.

Quantificazione spettrofotometrica dell'autofluorescenza come potenziale strumento diagnostico per lesioni maligne della cute e della mucosa orale / Ilaria Giovannacci , 2020 Sep 28. 32. ciclo, Anno Accademico 2018/2019.

Quantificazione spettrofotometrica dell'autofluorescenza come potenziale strumento diagnostico per lesioni maligne della cute e della mucosa orale

GIOVANNACCI, ILARIA
2020

Abstract

Autofluorescence (AF) is defined as the fluorescence emission observed when certain cell molecules are excited by UV or visible light of suitable wavelenghts. When a biologic molecule is illuminated at an excitation wavelength within the absorption spectrum of that molecule, it will absorb this energy and be activated from its ground state to an excited state. The molecule (fluorophore) can then relax back from the excited to the ground state by generating energy in the form of fluorescence, at emission wavelengths, which are longer than that of the excitation wavelength. The most important endogenous fluorophores are molecules widely distributed in cells and tissues, like proteins containing aromatic aminoacids, flavins and lipopigments. The main fluorophores of healthy skin are located in the epithelium (eg. keratin, nicotinamide adenine dinucleotide or NADH and flavin adenine dinucleotide or FAD) and the submucosa (e.g. collagen and elastin). These molecules when irradiated between the wavelengths from 375 and 440 nm, show fluorescence in the green spectral range. Nonmelanoma skin cancer (NMSC) is the most common malignancy worldwide. The developement of NMSC is accompanied by histopathological changes in epidermis such as loss of cellular maturation, alteration in keratin production, overall thickening of the epithelial layer and biochemical alterations (NADH decrease). NMSC is also accompanied by histopathological changes in the underlying stroma and submucosa, including neovascularization and destruction of the collagen cross-link by proteases. These alterations lead to a general decrease in AF due the alteration in distribution of the fluorochromes and in particular to NADH and collagen. In the last two decades, studies concerning cell and tissue AF has had a dramatic increase. AF studies have been performed both in vitro and in vivo, for the study of normal tissue and for the discrimination between normal tissues and neoplastic lesions of oral mucosa, skin, esophagus, colon, lung, bronchi, brain and bladder. The methods used are both direct visual fluorescence examination (DVFE) and spectrophotometry. In particular, DVFE has been widely used for clinical studies on oral mucosa. Regarding AF of the skin, this has been studied more frequently by using spectrophotometry. The principle is scanning and analyzing reflected light from the skin after exposure to an activating light source. AF spectroscopy is a very sensitive technique for quantitative measurements of tissue constituents. However, to date no methods have emerged that can be translated into clinical practice. The primary objective of this study is to investigate the correlation between spectral mesurement of cutaneous AF and the histopathological characteristics of malignant and pre-malignant skin in NMSC. Following surgical removal of the cancer, an ex vivo evaluation of the AF will be performed. The specimen will be irradiated with a probe that emits a light in the blue spectrum (wavelength 400-440 nm) and the fluorescence emitted by the tissue will be measured using a spectrophotometer in a standardized spot modality. Any changes detected will be reported on the surgical specimen with the application of a surgical mark. Histopathological examination of the lesion will be performed and any changes in the fluorescence pattern will be correlated with possible alterations in the histopathological pattern, referring to surgical marks. Alterations in AF spectral measurement correlate with histopathological alterations in NMSC. the spectral measurement can be a new support for the early diagnosis of NMSCs, a guide for the targeted incisional biopsies, a tool for the definition of the intraoperative surgical margins, and for the follow-up of treated patients.
Autofluorescence spectrophotometric quantification as potential diagnostic tool for malignant lesions of skin and oral mucosa
28-set-2020
MAGNONI, Cristina
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11380/1211519
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