Is “Controlling Pre-analytical Variability” an Elusive Target in Biobanking Urine Samples?

Posted by Abdul Ally on Oct 22, 2015 11:00:00 AM

Urine_SamplesThe wealth of information in urine samples makes this material critical for research, and for biobanking. The value of this humble waste product is evident from the fact that it is the third most-tested sample in diagnostic laboratories, after serum/plasma profiles and complete blood cell (CBC) counts. However, researchers collecting urine samples for storage and downstream analysis face the same issues as diagnostic laboratories—determining how reliable a test result can be, given the difficulty in controlling  pre-analytical variability.

Urine samples are in a unique category of biospecimens from a number of perspectives, beginning with collection.  Unlike blood and other samples, urine is typically collected by the patient, and the testing lab assumes that the verbal instructions provided to the patient are adequate and that the collection was performed properly. Proper collection most often means mid-stream, first morning collection into a sterile container. However, second morning samples differ significantly and may be more suitable, depending on the study.  

Most information given to donors is in regard to the practical elements of urine collection. Possible confounding elements such as dietary supplements, residue from bath soap and similar products, and even exercise are generally not provided. 

The ability of the donor to clearly follow writer instructions is probably the most significant issue: collection of a urine sample for diagnostic purposes often involves a person who is above age 65 and suffering from a chronic condition. Depending on the study, participants who are donating a sample for freezing and repository storage may be in better health. However, there is ultimately no way to determine if the sample was collected properly.

Another way in which urine differs from other specimens is that we currently do not have a preservative that will stabilize the sample for all downstream analyses. Means exist to prevent bacterial growth and preserve specific analytes, but refrigeration of a sample prior to performing a diagnostic test, and freezing of the sample for long-term storage is the only means we have of preserving its integrity.

When contamination is a critical issue, a vacuum system that allows aspiration of a sample directly into a secondary container can be used. However, this technology has limitations, as the resulting sample is useful only for chemical analyses. Particulate and cellular matter can be disrupted by the aspiration process.

Flow cytometry for routine urinalysis dramatically increases the reliability of diagnostic tests, and metabolomics is giving new value to urine samples that are less affected by pre-analytical variables. However, for reliable results, the samples must be processed correctly. For instance, urine should be first centrifuged to remove particulates, but  excessive centrifugal speed can rupture cells, releasing cellular components which alter the nuclear magnetic resonance (NMR) profile of the sample being analyzed.

The bottom line: If you are relying on urine samples for current or downstream research, you must have standard operating procedures (SOPs) for collection, processing, and storage that will ensure their suitability for use. If you are only getting started, Delanghe and Speeckaert(1) published a review of the literature summarizing confounding factors, including a list of guidelines for managing pre-analytical variables. This review focused on the issues facing diagnostic laboratories, but provides information from a biobanking perspective.  For instance, if you know your target analyte, the SOP for handling urine should include an appropriate time limit from collection through processing. The time span between collection and processing should be documented for each sample, and the sample discarded if the limit is exceeded.

Bernini et al. (2) proposed SOPs for handling urine (and blood) to be stored for downstream metabolic studies.  Likewise, Yuille (3) et al. listed the basics of biobanking of urine samples: 

  • No additives, unless for a specific downstream analysis, and the additive should be documented
  • Samples should be collected mid-stream
  • Centrifuge to remove particulates and cells
  • Aliquot to minimize freeze-thaw cycles
  • Store at -80°C or below, preferably in liquid nitrogen

Without SOPs, training in these SOPs, and uniform collection supplies, you will know very little about the quality of the sample, as well as the reliability of your assay results.

Biospecimen-based research depends on controlling pre-analytical variability, maintaining sample integrity, and on annotating the biosamples with the appropriate data. Collection kits not only assist in controlling variability, but can also serve as an ideal starting point for data collection. To learn more, download your free eBook Standardizing Biosample Management: Why Use Collection Kits?

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  1. Delanghe, J. &  Speeckaert. M. (2014)  Preanalytical requirements of urinalysis. Biochemia Medica, 24, 89–104.
  2. Bernini, P.; Bertini, I.; Luchinat, C.; Nincheri, P. & Staderini S, et al. (2011). Standard operating procedures for pre-analytical handling of blood and urine for metabolomic studies and biobanks. J Biomol NMR, 49, 331-43.
  3. Yuille, M.; Illig, T.; Hveem,K.;  Schmitz, G. & Hansen, J. et al. (2010).   Laboratory management of samples in biobanks: European Consensus Expert Group Report. Biopreservation and Biobanking, 8, 65-69.