Isolation and Detection of Extracellular Vesicles from Peripheral Blood of Chronic HCV Patients

Abstract

The chronic liver disease burden in Pakistan is due to high prevalence rate of Hepatitis C (HCV) infection . The reason of an increase in prevalence is due to unhygienic practices and unawareness amongst majority of the infected population. Most of these infections enter chronic phase and early detection is not possible leading to delayed treatment in end stages of liver disease. This highlights the importance of an effective line of treatment, which is based on reduction of mismanagement, misdiagnosis of disease stage and early diagnosis where possible. Great emphasis has to be laid on quality diagnostics and prognostic markers that are both very sensitive and specific to the disease condition as well as minimally invasive to non invasive and at an affordable cost. Currently used biomarkers in everyday clinical routine are not that sensitive and specific for liver disease staging and do not provide with the -omics background of pathologically diseased liver tissue. This makes tissue biopsy a gold standard for disease staging, genomic and proteomic profiling of the diseased liver tissue. However, the process is invasive, involving risk and cannot represent the heterogeneity of the tissue. Extracellular vesicles (EVs) can serve as a minimally invasive to a non-invasive approach to gather information regarding the diseased tissue and its microenvironment. These EVs may be: 1) secreted by the cells under normal or stimulation due to pathological condition in form of exosomes, 2) budded only under stimulation or stress from the cell membrane in form of microvesicles (also known as microparticles), 3) undergo programmed cell death to form apoptotic bodies. Currently, the gold standard for exosomes (EXO) and microvesicles (MV) isolation is sucrose density gradient or ultracentrifugation. These techniques are both technical and expensive. In this study, we have optimized simple and relatively inexpensive protocol for isolation of MV and EXO via differential centrifugation, ultrafiltration and isolation in low molecular weight 18 Peg phase from peripheral blood of chronic HCV patients. Blood samples of 40 chronic HCV patients with evaluated liver disease stage and 10 healthy controls were collected from Center of Liver Disease, Holy Family Hospital, Rawalpindi, Pakistan, while the rest of the study was performed at Atta-ur-Rehman School of Applied Biosciences, National University of Sciences and Technology, Islamabad, Pakistan. Ethical approval was obtained from both the institutions prior to commencement of the present study. We observed that by increasing the concentration of Peg, the total protein concentration in exosomal pellet increased (p=0.05) and that platelet poor plasma (PPP) was a better source of EVs as compared to platelet poor serum (PPS). The size of these isolated EVs were analyzed via laser particle size diffraction and scanning electron microscopy (SEM). The size of isolated in range of EXO, MV and apoptotic bodies from 87nm-100µm in diameter with the mode value of 183nm via laser particle size analysis. Whilst the size of EXO observed via SEM ranges from 30.00nm-150.00nm± 0.01 for EXOs and 150.00nm-400.00nm±0.01 for MVs. The morphology of EXOs was spherical, whereas MVs gave an irregular circular and cuboidal under SEM. The protein cargo of these vesicles was then resolved on 1D-SDS. The observation was similar to EVs isolated using gold standard isolation, thus validating our study. Better recovery of EVs was observed using 8% and 13% Peg, whereas better purity of isolated EVs was observed at 8% and 17 % Peg in final 1ml PPP. This makes 8% Peg to be the best concentration for isolation of EVs. The accuracy of currently used methods for liver disease staging have also been established via Area under the curve (AUC) of Receiver Operating Characteristic (ROC) curve analysis to differentiate between different stages. These can serve as a platform to compare when further studies are performed on EV for potential use in diagnostic and prognostic of HCV mediated liver disease. We selected ultrasound scoring and computed tomography (CT) scan as a 19 standard to compare with, for our further investigations. Imaging techniques gave us the highest AUC of ROC, 0.67 in fibrotic to 1.00 in end-stage liver disease, when compared with Alanine Aminotransferase, Alkaline Phosphatase and total bilirubin levels from sera. Further research on EV cargo will help in identifying potential biomarkers with high AUC of ROC, i.e. they are more sensitive and specific, when compared to current clinical practices in liver disease staging.