Reproducibility and efficiency of serum-derived exosome extraction methods

Objectives: Exosomes are emerging as a source of biomarkers with putative prognostic and diagnostic value. However, little is known about the efficiency, reproducibility and reliability of the protocols routinely used to quantify exosomes in the human serum. Design and methods: We used increasing amounts of the same serum sample to isolate exosomes using two different methods: ultracentrifugation onto a sucrose cushion and ExoQuick (TM). Quantitative analysis of serum-derived exosomes was performed by determining protein concentration (BCA assay) and the number of nanoparticles (Nanosight (TM) technology). Exosome quality was assessed by Coomassie staining and Western blotting for CD9, LAMP2 exosomal markers and a negative marker Grp94. Results: Correlation between serum volume and the number of isolated exosomes is significant for both methods when exosomes are quantified using protein concentration. However, when the number of nanoparticles is used to quantify exosomes, ExoQuick (TM) is the only reproducible and efficient method. CD9, LAMP2 and Grp94 exosomal markers are equivalently expressed in both methods. However, exosomes isolated using ultracentrifuge method are strongly contaminated with albumin and IgG. Conclusion: ExoQuick (TM) is an efficient and reproducible method to isolate exosomes for quantitative studies, whereas ultracentrifugation is not. Moreover, high albumin contamination of ultracentrifuged-derived exosomes impairs the use of protein concentration as a mean to quantify serum-derived exosomes. (C) 2014 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.