Collection, storage, preservation, and normalization of human urinary exosomes for biomarker discovery.

Author(s): Zhou H, Yuen P S, Pisitkun T, Gonzales P A, Yasuda H, Dear J W, Gross P, Knepper M A, Star R A

Publication: Kidney Int, 2006, Vol. 69, Page 1471

PubMed ID: 16501490 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this experiment was to determine whether protease inhibitors are necessary for preservation of NKCC2 in urine.

   Summary of Findings:

   Specimens without protease inhibitors exhibited decreased or absent NKCC2 signal compared to those with protease inhibitors.

   Biospecimens

   • Bodily Fluid - Urine

   Preservative Types

   • None (Fresh)

   Diagnoses:

   • Normal

   Platform:

   Analyte	Technology Platform
   Protein	Western blot

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Analyte Extraction and Purification	Protease inhibitor	With protease inhibitors Without protease inhibitors

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2. Study Purpose

   The purpose of this study was to determine the influence of storage temperature and duration on urinary exosome proteins (NHE3, TSG101, ALIX, and AQP2), and to further determine whether 10 ml biospecimens provide sufficient starting material to detect urinary exosome proteins.

   Summary of Findings:

   Storage temperature robustly affected recovery of exosome proteins, 27% and 86% recovery at -20 and -80 degrees C for 1 week, respectively, when compared to storage of controls at 4 degrees C for 1 hour; however, vortexing for 90 seconds after thawing improved recovery significantly, 87-100%. Western blot analysis confirmed the results above, as the absence of NHE3, TSG101, and ALIX in urine stored at -20 degrees C was attenuated with extensive vortexing. Further, NHE3, TSG101, ALIX, and AQP2 were detectable in urine aliquots as small as 10 ml. Vortexing urine samples stored at -80 degrees for 7 months yielded exosome protein expression comparable to fresh samples, with the exception of ALIX, which displayed individual variability independent of storage duration.

   Biospecimens

   • Bodily Fluid - Urine

   Preservative Types

   • Frozen

   Diagnoses:

   • Normal

   Platform:

   Analyte	Technology Platform
   Protein	Western blot
   Protein	1D/2D gels

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Aliquots and Components	Aliquot size/volume	300 ml 10 ml
   Storage	Storage duration	1 week 7 months 1 h
   Storage	Storage temperature	4 degrees C -20 degrees C -80 degrees C

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3. Study Purpose

   The purpose of this study was to evaluate whether first morning urine was significantly different from second urine of the day in terms of the detectable level of exosomes.

   Summary of Findings:

   Total protein amounts were similar among first and second morning urine specimens. When normalized for urine flow rate or urine creatinine levels, the abundance of four exosome proteins (NHE3, TSG101, ALIX, and AQP2) were similar in first and second morning urines.

   Biospecimens

   • Bodily Fluid - Urine

   Preservative Types

   • None (Fresh)

   Diagnoses:

   • Normal

   Platform:

   Analyte	Technology Platform
   Protein	ELISA
   Protein	Western blot

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Aliquots and Components	Aliquot sequential collection	First urine of the day Second urine of the day

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