Method validation for preparing urine samples for downstream proteomic and metabolomic applications.
Author(s): Ammerlaan W, Trezzi JP, Mathay C, Hiller K, Betsou F
Publication: Biopreserv Biobank, 2014, Vol. 12, Page 351-7
PubMed ID: 25289566 PubMed Review Paper? No
Purpose of Paper
This paper sought to identify the optimal urine centrifugation protocol for determination of microparticle counts, measurement of creatinine and cystatin C, and metabolomic analysis by GC-MS.
Conclusion of Paper
Microparticle counts decreased significantly with increasing centrifugation and brake speed and when specimens were centrifuged once rather than twice, but were unaffected by centrifugation temperature. There was no effect of brake speed or centrifugation temperature on the measurement of cystatin C or creatinine and the majority of detected metabolites. The identified protocol (single centrifugation at 12,000 x g for 20 min at 4˚C with hard braking) resulted in a coefficient of variance of less than 20% for microparticle counts, creatinine, and cystatin C and only nonsignificant differences in 96.6% (399 of 413) of metabolites.
Studies
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Study Purpose
This study investigated the effects of centrifugation speed, temperature, duration, and braking on urine microparticle counts, measurement of creatinine and cystatin C, and metabolomic analysis by GC-MS. Mid-stream urine from two healthy volunteers collected into polypropylene collection cups was aliquoted into 50 mL conical tubes and centrifuged at 4˚C within 3 h, unless otherwise specified. Initially the effect of centrifugation speed was investigated by centrifuging specimens at room temperature at: 1) 1000 x g for 10 min with medium braking, 2) at 2000 x g for 10 min with medium braking or 3) at 10,000 x g for 20 min with hard braking. Then a single centrifugation at 12,000 x g at 4˚C for 20 min with medium braking was compared to double centrifugation at 2,000 x g at 4˚C for 10 min with medium braking followed by 12,000 x g at 4˚C for 10 min with hard braking. The effects of brake speed and temperature were then compared in specimens centrifuged at 12,000 x g for 20 min. Specimens were analyzed for microparticle counts using an automated cell counter, creatinine and cystatin C using ELISA, and metabolite profile using GC-MS.
Summary of Findings:
When centrifuged at room temperature, microparticle counts decreased significantly with increasing centrifugation speed (P<0.001). Single centrifugation (12,000 x g for 20 min at 4˚C) resulted in lower microparticle counts than double centrifugation (2000 x g then 12,000 x g, each for 10 min at 4˚C) for both specimens (P=0.003 and P=0.014). Microparticle counts declined with increasing brake speed (P=0.025), but there was no effect of brake speed on the measurement of cystatin C or creatinine and only 10 of 331 detected metabolites were affected by brake speed. Microparticle counts and the measurement of cystatin C, creatinine, and 308 of 310 metabolites were unaffected by centrifugation temperature. The identified protocol (single centrifugation at 12,000 x g for 20 min at 4˚C with hard braking) resulted in a coefficient of variance of less than 20% for microparticle counts, creatinine and cystatin C levels, and only nonsignificant differences in 96.6% (399 of 413) of metabolites.
Biospecimens
Preservative Types
- None (Fresh)
Diagnoses:
- Normal
Platform:
Analyte Technology Platform Cell count/volume Hematology/ auto analyzer Small molecule GC-MS Small molecule ELISA Protein ELISA Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Aliquots and Components Centrifugation Different number of centrifugation steps compared
Multiple brake speeds compared
Multiple durations compared
Multiple speeds compared
Multiple temperatures compared