Human Blood Exosomes: Isolation and Characterization Methods, Variability, and the Need for Standardized Protocols—A Review

<b>Background/Objectives</b>: As bioactive extracellular vesicles, exosomes participate in cellular communication and disease mechanisms, yet their structural complexity continues to challenge standard analytical methodologies. This review summarizes published studies reporting exosome concentrations in human plasma, serum, and platelet-rich plasma from healthy individuals and highlights methodological differences. <b>Methods</b>: A comprehensive PubMed search (1986-31 August 2025) was performed using terms related to exosomes and their quantification, excluding cancer- and disease-related studies. Eligible articles reported exosome concentrations in plasma, serum, or platelet-rich plasma using particle-counting techniques such as nanoparticle tracking analysis, flow cytometry, or tunable resistive pulse sensing. <b>Results</b>: Twenty-two articles, including 167 healthy donors, met the inclusion criteria. The following mean concentration ranges were reported: plasma (n = 18), ranged from 4.50 × 10⁸ to 6.70 × 10¹¹ particles/mL with differences by quantification method; serum (n = 10), from 5.30 × 10⁸ to 2.13 × 10¹¹ particles/mL; non-activated platelet-rich plasma (n = 1), 7.52 × 10⁹ particles/mL; activated platelet-rich plasma (n = 3), 4.87 × 10¹⁰ to 7.16 × 10¹⁰ particles/mL; and preconditioned platelet-rich plasma with photothermal biomodulation (n = 2), 2.53 × 10¹¹ to 2.99 × 10¹¹ particles/mL. <b>Conclusions</b>: Isolation and quantification methods exhibit high variability, which strongly influences the overall quantity and quality of the exosomes obtained. Characteristics, including cargo composition, purity, and exosome integrity, must be considered when developing validated methods. Furthermore, emerging evidence suggests that PTBM preconditioning can increase exosome release from cells. In summary, rigorous standardization of protocols is essential to advance the scientific understanding and the clinical potential of exosome-based therapies.