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Collection, Processing, and Storage Consideration for Urinary Biomarker Research

Published: October 21st, 2021



1Department of Pediatrics, University of Pittsburgh

This protocol aims to provide considerations for urine sample collection, processing, and storage for urine tract infection biomarker studies.

There are several urinary proteins that show promise as novel markers of urinary tract infections. The identification of a novel biomarker that has greater predictive accuracy compared to current diagnostic methods has the potential to greatly improve the ability to manage patients with urinary tract infections. However, sample collection, processing, and storage can all potentially impact the results of biomarker research. Understanding the effects of each of these stages on biomarker studies is necessary to inform future, high-quality research in this area, as well as to critically review other studies in this area. Here, the study reviews the literature regarding the effects of each stage of urine sample processing and reports the effects of various conditions on urinary proteins. The protocol will focus on collection techniques, time and temperature of storage, processing techniques, use of reagents, and long-term freezing on biomarker stability. It will focus on proteins but will briefly discuss other materials that may be utilized in biomarker research. In doing so, this protocol will provide a guide to future researchers to assist in the design of urinary biomarkers studies.

Urinary tract infections (UTI) are one of the most common bacterial infections in both children and adults1. While the diagnosis of UTI in some populations can be uncomplicated, it can be more complex in others, such as those with neuropathic bladders2. The ability to accurately diagnose UTIs will help improve antibiotic stewardship efforts by decreasing the use of unnecessary antibiotics and potentially aid in the earlier diagnosis of UTI, thus decreasing the risk of morbidity. Given the prevalence of UTIs, there is significant interest in improving the management of this common infection.


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This protocol follows the guidelines of the institution's human research ethics committee. Ensure that the approval is obtained from the institutional review board (IRB) prior to the collection and utilization of biological specimens for research.

1. Collection

  1. Obtain urine sample in a sterile specimen cup. Decide the type of urine sample, as well as specific inclusion and exclusion criteria, based on the specific study design. For UTI studies, use either the clean-catch method.......

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Centrifugation had a small impact on NGAL levels. Centrifuged samples stored at -80 °C had lower levels of NGAL than non-centrifuged samples (2.17 ng/mL ± 0.32 ng/mL, 2.77 ng/mL ± 0.21 ng/mL). Freeze cycles also had an impact on NGAL levels after the third freeze-thaw cycle. (Figure 2). Of the conditions studied (centrifugation, freeze-thaw cycles, and storage temperature), storage temperature had the greatest impact on NGAL levels. Storage at -80 °C immediately after sam.......

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The importance of producing consistent and reproducible results is not limited to the success of individual studies but will also enable a better comparison of results within the literature9. Variation between studies in key procedural steps can introduce irreversible bias that may affect biomarker signals and their interpretation, which may be responsible for discrepancies among several studies10. This demonstrates the need to establish a more standardized approach to proc.......

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No external funding was obtained for this work. Institutional funds were used to obtain the data in this work.


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Name Company Catalog Number Comments
Boric acid Sigma-Aldrich B6768 To be considered for samples that cannot be rapidly processed and frozen
Freezer boxes Fisher Scientific 03-395-464
Microcentrifuge tubes Thomas scientific 1149X93
NGAL ELISA Kit R&D Systems DLCN20 Used to create representative results
Pipette and tips Dependent on pipette size and volume of fluid.
Sodium azide Sigma-Aldrich S2002 To be considered for samples that cannot be rapidly processed and frozen
Urine collection cups Thermo Scientific 3122B03ORG Sterile cups not required unless needed for other studies

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