Optimizing Exosome Enrichment: A Comparative Study in Pediatric Acute Lymphoblastic Leukemia Patients

Objective(s): Pediatric acute lymphoblastic leukemia (pALL) is the most prevalent neoplasm in children. pALL diagnostic process is invasive, and children need to be anesthetized for bone marrow aspiration. Exosomes are nanoparticles that reflect the status of parental cells. Given the elevated levels of exosomes in the peripheral blood of pALL patients, they can be readily extracted from blood plasma. The aim of this study was to compare the reliability of two different techniques of exosome purification in order to select the best method for clinical use in pALL. Methods: Exosomes were isolated from plasma samples using two methods: ultracentrifugation and the ExoQuick exosome isolation (EQ) kit. The performances of these methods were compared based on the nanoparticle tracking analysis (NTA), field emission electron microscopy (FESEM), and immunoblotting assays. Results: NTA results showed that exosome fractions extracted by the EQ kit were more concentrated and homogeneous compared with the ultracentrifugation method. Electron microscopy depicted spherical morphology for the isolated exosomes in both methods; however, the appearance of exosomes enriched by the commercial kit was more intact. Following the immunoblotting assays investigating the exosomal biomarkers, densitometry analysis showed that the exosome populations related to the EQ kit had higher concentrations and extreme purity.  Conclusions: The data demonstrated that the commercial kit exhibited superior efficacy in isolating concentrated, intact, and pure exosomes from small quantities of patients’ plasma samples when compared to the standard ultracentrifugation method. According to those findings, the ExoQuick exosome segregation kit was preferred to be used in pALL diagnostic investigations.