Thrombosis is the major global healthcare burden, accounting for high mortality and morbidity and severe economic impact. Addressing this issue requires concerted efforts in prevention, early detection, effective treatment, and management strategies to reduce the load of thrombotic diseases on individuals and society. As platelets are among the pivotal players in thrombosis, antiplatelet agents are being administered to an increasing percentage of population, in parallel to anticoagulant medications. A major problem in antiplatelet treatment is the high variability of patients responses to antiplatelet agents, with acetylsalicylic acid (i.e. aspirin) being a popular example. Platelet function testing (PFT) has been proposed as tool to help patients during their journey through the healthcare system and their daily life, with the goal of tailoring and managing the therapeutic treatments to prevent, or at least mitigate, adverse effects. To this day, a PFT assay able to successfully meet all the desired requirements is still missing: reliability, use of small blood volume, accessibility, cost-effectiveness, rapid outcomes, physiologically significant testing conditions. A number of PFT devices exist, but often do not agree on patients’ risk stratification between each other. In this PhD work, different PFT solutions have been explored, in search of an assay suitable to combine together as many requirements as possible. Electrical impedance aggregometry was elected as working principle to exploit, as it permits a label-free and real-time monitoring of biological phenomena. A first system, called MICELI aggregometer, was developed and validated. It proved to be sensitive to study the effect of different concentrations of several antiplatelet drugs, working on distinct activation pathways. In the effort of adding flow, a critical contributor to thrombus formation, to the assay, a meticulous study was performed to characterise assay-dependent variables in a basic flow-based platform. With fluorescence microscopy as readout, blood storage temperature, shear stress and collagen substrate concentration have been investigated with the aim of setting guidelines for flow-based protocols. Platelet reactivity after addition of two antiplatelet agents was successfully detected and optimised testing conditions were identified for each drug, depending on their mechanism of inhibition. Once established the benchmark, a prototype for an electrical impedance aggregometer integrating flow was designed and fabricated. Although the protocol had to be adjusted, preliminary testing showed promising results for patients’ thrombotic risk stratification. To account for the vascular microenvironment, a model encorporating all the key elements of haemostasis and thrombosis was developed. This model accounts not only for the blood components, but also for the presence of the vessel wall and its surroundings, and allows to apply shear rate as well as to co-culture endothelial cells with perivascular cells. The model was perfused with whole blood and used to mimic thrombosis in vitro, and is amenable to dissect the pro-thrombotic properties of patients’ own endothelial cells and blood in an autologous model, following the precision medicine approach. In this PhD thesis, technical solutions were employed to enhance the clinical relevance of in vitro thrombosis model. The devices presented enable the stratification of patients’ thrombotic risk and provide a platform to study platelet and endothelial dysfunctions in several diseases.
La trombosi rappresenta il principale onere sanitario globale, causando alta mortalità, morbidità e pesante impatto economico. Per affrontare questo problema è necessaria una azione concertata nella prevenzione, nella diagnosi precoce, nel trattamento efficace e nelle strategie di gestione per ridurre il carico delle malattie trombotiche sugli individui e sulla società. Poiché le piastrine sono tra i principali attori del fenomeno trombotico, farmaci antiaggreganti vengono somministrati a una percentuale sempre crescente della popolazione, parallelamente a farmaci anticoagulanti. Uno dei problemi principali legati al trattamento antiaggregante è l'alta variabilità delle risposte dei pazienti ai farmaci, con l'acido acetilsalicilico (ovvero aspirina) come esempio noto. I test della funzionalità piastrinica (PFT) sono stati proposti come strumento per aiutare i pazienti durante il loro percorso attraverso il sistema sanitario e nella vita quotidiana, con l'obiettivo di personalizzare e gestire i trattamenti terapeutici per prevenire, o almeno mitigare, effetti avversi. Ad oggi, un PFT in grado di soddisfare tutti i requisiti desiderati è ancora mancante: affidabilità, uso di minime quantità di sangue, accessibilità, costo-efficacia, risultati rapidi, condizioni di test fisiologicamente rilevanti. Esistono in commercio diversi dispositivi per PFT, ma spesso non concordano tra loro nella stratificazione del rischio dei pazienti. In questo lavoro di dottorato sono state esplorate diverse soluzioni di PFT, alla ricerca di un test adatto a combinare il maggior numero possibile di requisiti. L'aggregometria a impedenza elettrica è stata scelta come principio di funzionamento, poiché consente un monitoraggio in tempo reale e senza marcatori dei fenomeni biologici. Un primo dispositivo, chiamato MICELI, è stato sviluppato e validato. Si è dimostrato sensibile nello studio dell'effetto di diverse concentrazioni di vari farmaci antiaggreganti che agiscono su diversi percorsi di attivazione piastrinica. Con lo scopo di integrare nel modello il flusso, fattore critico per la formazione di trombi, è stato eseguito uno studio critico di caratterizzazione delle variabili saggio-dipendenti utilizzando una piattaforma basata sul flusso. Utilizzando la microscopia a fluorescenza come readout, sono state investigate la temperatura di conservazione del sangue, lo shear stress e la concentrazione del substrato di collagene con l'obiettivo di stabilire linee guida per i protocolli in vitro basati sul flusso. La reattività piastrinica dopo l'aggiunta di due agenti antiaggreganti è stata rilevata con successo e sono state identificate le condizioni di test ottimizzate per ciascun farmaco, a seconda del loro meccanismo di inibizione. Una volta stabilito il benchmark, è stato progettato e realizzato un prototipo per un aggregometro a impedenza elettrica integrato col flusso. Sebbene il protocollo dovesse essere riadattato, i test preliminari hanno mostrato risultati promettenti per la stratificazione del rischio trombotico dei pazienti. Per tenere conto del microambiente vascolare, è stato sviluppato un modello che incorpora tutti gli elementi chiave dell'emostasi e della trombosi. Questo modello tiene conto non solo dei componenti del sangue, ma anche della presenza della parete del vaso e dei suoi dintorni, e consente di applicare shear rate, nonché di co-coltivare cellule endoteliali con cellule perivascolari. Il modello è stato perfuso con sangue intero e utilizzato per simulare la trombosi in vitro, e risulta idoneo ad analizzare le proprietà pro-trombotiche delle cellule endoteliali e del sangue di pazienti in un modello autologo, seguendo l'approccio della medicina di precisione. In questa tesi di dottorato sono state impiegate soluzioni tecniche per migliorare la rilevanza clinica della modellazione di trombosi in vitro. I dispositivi presentati consentono la stratificazione del rischio trombotico dei pazienti e forniscono piattaforme per studiare le disfunzioni piastriniche ed endoteliali in diverse malattie.
Advanced microfluidic assays for thrombotic risk assessment: towards clinical translation
MENCARINI, TATIANA
2023/2024
Abstract
Thrombosis is the major global healthcare burden, accounting for high mortality and morbidity and severe economic impact. Addressing this issue requires concerted efforts in prevention, early detection, effective treatment, and management strategies to reduce the load of thrombotic diseases on individuals and society. As platelets are among the pivotal players in thrombosis, antiplatelet agents are being administered to an increasing percentage of population, in parallel to anticoagulant medications. A major problem in antiplatelet treatment is the high variability of patients responses to antiplatelet agents, with acetylsalicylic acid (i.e. aspirin) being a popular example. Platelet function testing (PFT) has been proposed as tool to help patients during their journey through the healthcare system and their daily life, with the goal of tailoring and managing the therapeutic treatments to prevent, or at least mitigate, adverse effects. To this day, a PFT assay able to successfully meet all the desired requirements is still missing: reliability, use of small blood volume, accessibility, cost-effectiveness, rapid outcomes, physiologically significant testing conditions. A number of PFT devices exist, but often do not agree on patients’ risk stratification between each other. In this PhD work, different PFT solutions have been explored, in search of an assay suitable to combine together as many requirements as possible. Electrical impedance aggregometry was elected as working principle to exploit, as it permits a label-free and real-time monitoring of biological phenomena. A first system, called MICELI aggregometer, was developed and validated. It proved to be sensitive to study the effect of different concentrations of several antiplatelet drugs, working on distinct activation pathways. In the effort of adding flow, a critical contributor to thrombus formation, to the assay, a meticulous study was performed to characterise assay-dependent variables in a basic flow-based platform. With fluorescence microscopy as readout, blood storage temperature, shear stress and collagen substrate concentration have been investigated with the aim of setting guidelines for flow-based protocols. Platelet reactivity after addition of two antiplatelet agents was successfully detected and optimised testing conditions were identified for each drug, depending on their mechanism of inhibition. Once established the benchmark, a prototype for an electrical impedance aggregometer integrating flow was designed and fabricated. Although the protocol had to be adjusted, preliminary testing showed promising results for patients’ thrombotic risk stratification. To account for the vascular microenvironment, a model encorporating all the key elements of haemostasis and thrombosis was developed. This model accounts not only for the blood components, but also for the presence of the vessel wall and its surroundings, and allows to apply shear rate as well as to co-culture endothelial cells with perivascular cells. The model was perfused with whole blood and used to mimic thrombosis in vitro, and is amenable to dissect the pro-thrombotic properties of patients’ own endothelial cells and blood in an autologous model, following the precision medicine approach. In this PhD thesis, technical solutions were employed to enhance the clinical relevance of in vitro thrombosis model. The devices presented enable the stratification of patients’ thrombotic risk and provide a platform to study platelet and endothelial dysfunctions in several diseases.| File | Dimensione | Formato | |
|---|---|---|---|
|
Tatiana_Mencarini_PhDthesis.pdf
solo utenti autorizzati a partire dal 19/09/2027
Descrizione: Tesi di dottorato
Dimensione
96.12 MB
Formato
Adobe PDF
|
96.12 MB | Adobe PDF | Visualizza/Apri |
I documenti in POLITesi sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
https://hdl.handle.net/10589/228272