NIH, National Cancer Institute, Division of Cancer Treatment and Diagnosis (DCTD) NIH - National Institutes of Health National Cancer Institute DCTD - Division of Cancer Treatment and Diagnosis

Pre-analytical handling conditions and protein marker recovery from urine extracellular vesicles for bladder cancer diagnosis.

Author(s): Lee J, Kim E, Park J, Choi S, Lee MS, Park J.

Publication: PLoS One, 2023, Vol. 18, Page e0291198

PubMed ID: 37676879 PubMed Review Paper? No

Purpose of Paper

 The purpose of this paper was to compare counts, morphology, and protein levels among urinary extracellular vesicles (uEV) that were isolated from urine stored at 20˚C and 4˚C for 0–6 days with and without centrifugation and filtration.

Conclusion of Paper

Significant effects of urine storage on protein concentrations; EV counts; and levels of CD63, CD9, A2M, and CLU were observed but statistical significance was dependent on the storage temperature and duration and not observed in all specimens. Only a change in protein concentration, which was reduced with storage of unprocessed urine at 20°C, was observed in all five specimens. Importantly, changes with storage were generally similar regardless of whether urine was preprocessed before storage.

Studies

  1. Study Purpose

    The purpose of this study was to compare counts, morphology and protein levels among uEVs that were isolated from urine stored at 20˚C and 4˚C for 0–6 days with and without centrifugation and filtration. First morning urine was collected from 5 men (53-78 years of age) with bladder cancer prior to transurethral resection of the tumor.  After collection, urine was divided. One aliquot (control) was centrifuged at 2000 g for 10 min, and filtered through a 0.45 µm filter before isolation of urine EVs using an Exodisc. Half of the remaining aliquots were immediately centrifuged at 2000 g for 10 min and filtered through a 0.45 µm filter before storage at 20˚C and 4˚C for 1, 2, 3, 4, 5, 6, and 7 days; the remaining aliquots were not centrifuged prior to storage. After storage, aliquots were frozen at -70°C.  Before EV isolation using the Exodisc protocol, aliquots were centrifuged at 2000 g for 10 min, and filtered through a 0.22 µm filter. uEVs size distribution was analyzed by nanoparticle tracking analysis and morphology was evaluated by transmission electron microscopy.  Proteins were quantified with a bicinchoninic acid (BCA) assay kit and levels of CD63, CD9, and IgG were quantified by western blot. Levels of A2M and CLU were quantified by ELISA.

    Summary of Findings:

    Protein concentration was unaffected by storage of centrifuged and filtered (preprocessed) urine at 4°C, but was significantly reduced in three of five specimens when preprocessed urine was stored at 20°C for 3 days. Storage of uncentrifuged urine at 4°C resulted in significantly higher protein concentration after ≥4 days in 2 of 5 specimens, but storage of uncentrifuged urine at 20°C resulted in a significant decline in 3 specimens after ≥2 days and in the other two specimens at 3 days.  CD63 and CD9 were stable when preprocessed and unprocessed urine were stored at 4°C, but were significantly reduced when urine was stored at 20°C. The number of uEVs was not consistently affected by storage of processed or unprocessed urine at 4°C (lower in matched processed and unprocessed urine from one patient on day 6 and lower in unprocessed urine from one patient on day 4 and 6).  When preprocessed urine was stored at 20°C, lower uEV counts were found in one specimen, but when unprocessed urine was stored at room temperature one specimen had higher uEV counts at three timepoints, one had lower uEV counts after ≥2 days of storage, and a third specimen had higher uEVs on day 3 of storage.  Despite the variability in EV counts, there was no effect of storage on the mean size, size distribution, or morphology of EVs.  A2M levels were not affected by storage of preprocessed urine at 4°C in three of the five specimens evaluated with significantly lower levels at one and three timepoints in the remaining two specimens, Similarly, there was no effect of storing unprocessed urine at 4°C for two of the five specimens evaluated, with significantly lower levels at one, two, and three timepoints in the remaining three specimens. Levels of A2M in two preprocessed specimens and one unprocessed specimen were unaffected by storage at 20°C, with significant reductions first observed after 2 days in two preprocessed and one unprocessed specimen, 3 days in one unprocessed specimen, 4 days in one preprocessed specimen, and 6 days in the remaining two unprocessed specimens.  CLU levels were unaffected by 4°C storage in four of the five preprocessed and unprocessed urine specimens evaluated, with significant reductions observed after ≥5 days in the remaining preprocessed and unprocessed specimen. CLU levels were unaffected by 20°C storage in two of the five preprocessed and one of the five unprocessed urine specimens evaluated, with significant reductions observed after ≥1 day in one unprocessed specimen, after  ≥2 days in two of the unprocessed and two of the preprocessed specimens, and after 4 days in the final preprocessed and unprocessed specimen.

    Biospecimens
    Preservative Types
    • Frozen
    Diagnoses:
    • Neoplastic - Carcinoma
    Platform:
    AnalyteTechnology Platform
    Morphology Electron microscopy
    Cell count/volume Light scattering
    Protein Colorimetric assay
    Protein ELISA
    Protein Western blot
    Cell count/volume Electron microscopy
    Pre-analytical Factors:
    ClassificationPre-analytical FactorValue(s)
    Biospecimen Aliquots and Components Centrifugation Centrifugation delays investigated
    Storage Storage duration 0-6 days
    Storage Storage temperature 20˚C
    4˚C

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