Disease mechanisms of RA are not yet fully understood, especially when considering subclinical phases. US-guided joint biopsies are the least invasive method to collect synovial tissue, which can be used for histological and single-cell high-dimensional analysis. In this study, we aimed to better assess the feasibility and the performance of a protocol designed for single-cell separation of cryopreserved US-guided synovial biopsy. The possibility of obtaining a cellular yield even from tissues with low inflammatory grade was also tested. The secondary exploratory objective was the analysis of predictors of total cell number output. The protocol produced highly viable single cells. From knee-derived biopsies, 4 tissue fragments were sufficient to obtain >100.000 cells, even when tissues with a low inflammatory grade were processed. Preliminary results indicated that the same amount of cells were sufficient to run a flow cytometry panel. The parallel analysis of clinical, histological and ultrasound parameters revealed the potential applicability of histopathological analysis as a predictor of total cell separation output (LAI). Further analysis on a larger population of patients are required to optimize the performance of the current protocol on wrist biopsies as well as to improve our understanding of predictors of total cell number output.
I meccanismi di malattia nell’artrite reumatoide sono ancora largamente incompresi, specialmente se si considera fasi di malattia subclinica. Le biopsie eco-guidate sono il metodo meno invasivo per ottenere tessuto sinoviale, il quale può essere utilizzato per le analisi istologiche e per più avanzate analisi a livello delle singole cellule. In questo studio, abbiamo valutato la fattibilità e performance di un protocollo ideato per la separazione di tessuto sinoviale criopreservato, ottenuto mediante biopsie eco-guidate, in singole cellule a partire. Abbiamo testato la possibilità di isolare le cellule anche da tessuto poco infiammato. L’obiettivo secondario è stato di indagare sui predittori dell’output cellulare. Il protocollo permette di isolare cellule vitali. Da biopsie di ginocchio, 4 frammenti di tessuto sono stati sufficienti per ottenere >100.000 cellule, anche a partire dalla processazione di tessuti a basso grado infiammatorio. Risultati preliminari indicano che la stessa quantità di cellule è stata sufficiente per svolgere un pannello di citofluorimetria. Le analisi parallele dei parametri clinici, istologici e ultrasonograci hanno rivelato il possibile utilizzo dell’analisi istopatologica (LAI) per predire l’output della separazione cellulare. Ulteriori analisi su una popolazione più ampia sono necessarie per ottimizzare la performance dell’attuale protocollo sulle biopsie di polso e per migliorare la comprensione dei predittori del output cellulare.
CELL SEPARATION OF CRYOPRESERVED ULTRASOUND-GUIDED SYNOVIAL BIOPSIES: A PILOT ANALYSIS OF THE OUTPUT AND ITS PREDICTORS FOR RESEARCH APPLICATION IN SUB-CLINICAL PHASES OF CHRONIC ARTHRTIDES
GIURCO, MARIANNA
2023/2024
Abstract
Disease mechanisms of RA are not yet fully understood, especially when considering subclinical phases. US-guided joint biopsies are the least invasive method to collect synovial tissue, which can be used for histological and single-cell high-dimensional analysis. In this study, we aimed to better assess the feasibility and the performance of a protocol designed for single-cell separation of cryopreserved US-guided synovial biopsy. The possibility of obtaining a cellular yield even from tissues with low inflammatory grade was also tested. The secondary exploratory objective was the analysis of predictors of total cell number output. The protocol produced highly viable single cells. From knee-derived biopsies, 4 tissue fragments were sufficient to obtain >100.000 cells, even when tissues with a low inflammatory grade were processed. Preliminary results indicated that the same amount of cells were sufficient to run a flow cytometry panel. The parallel analysis of clinical, histological and ultrasound parameters revealed the potential applicability of histopathological analysis as a predictor of total cell separation output (LAI). Further analysis on a larger population of patients are required to optimize the performance of the current protocol on wrist biopsies as well as to improve our understanding of predictors of total cell number output.È consentito all'utente scaricare e condividere i documenti disponibili a testo pieno in UNITESI UNIPV nel rispetto della licenza Creative Commons del tipo CC BY NC ND.
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https://hdl.handle.net/20.500.14239/17542