Know Your Samples – How Resilient are your Biomarkers?

Posted by Debra A. Barnes, Ph.D. on May 6, 2014 9:30:00 AM

biobanking samples

For all types of biomarker research, whether academic studies or clinical trials, the target analytes within your samples are the most important elements to safeguard. No matter if it’s as simple as the pH of a serum sample or as specific as the quaternary structure of a protein complex, any biomarker can be affected and potentially compromised by a number of external factors, most easily temperature.

The optimal storage temperature of your samples varies depending on both the type of biospecimen and the length of storage, but that is a topic we have already discussed in the past. However, I have seen many researchers investigate on their own impetus the stability of their biomarkers in various conditions, and therefore determine the best temperature at a far more specific level. Below are a few examples, all stability studies testing for biomarkers in human serum:

  • Jansen et al (2012) investigated the stability of two vitamins (folate/B9 and B12), at three temperatures (-20⁰C, -70⁰C, and -196⁰C) for one year. While one vitamin is stable at all temperatures, the other is unstable at -20⁰C, leading to a concrete decision of the optimal storage temperature for serum samples containing both.
  • Another study by Jansen et al (2013) tested stability of several iron-related biomarkers from human serum and plasma at the same temperatures (-20⁰C, -70⁰C, and -196⁰C) for one year. In this investigation, all of the biomarkers, including multiple proteins, were shown to be stable at all temperatures tested.
  • Baleriola et al (2011) compared the deterioration of viral nucleic acid visibility over time, covering HCV, HBV, and HIV at both -20⁰C and -70⁰C. While some concentrations differed significantly before and after frozen storage, the researchers concluded that the differences could be attributed to intra-assay variation as well as storage temperature.
  • Kisand et al (2011) investigated serum concentrations of certain new tissue biomarkers, comparing not only the effects of storage at -20⁰C vs. -75⁰C, but also their resilience to the stress induced by undergoing freeze/thaw cycles. Among the four biomarkers investigated, there was substantial variability in stability; one was estimated to remain stable for centuries at -75⁰C while two others demonstrated instability at both tested temperatures. Interestingly, one of the "non-temperature-stable" biomarkers proved to be highly resilient to freeze/thaw stress, while the other was substantially compromised by a single freeze-thaw cycle. Two of the biomarkers withstood a single freeze-thaw cycle but showed deterioration after subsequent cycles.

These four studies illustrate the range of the possible results of stability testing at different temperatures. The paper by Jansen et al (2012) led quite clearly and concretely to a specific decision on the ideal mutual storage temperature, even though one of the vitamins demonstrated high stability at warmer temperatures. The second and third examples both concluded that either of the temperatures would be suitable, and also demonstrated admirable scientific rigor. The final study demonstrated the potential difficulty of working with multiple target biomarkers with very different stability profiles, and the potential effect on the results of various downstream assays.

laboratory processing biomarker analysis
Even these studies, however, do not necessarily delve as deeply into the topic of optimal storage temperature as they could have. If you are interested to learn more about optimal storage temperature, click here to view our Biobank Storage Temperatures InfoPoster (shown below). An example is for "cryogenic" temperatures. The term can describe a whole range of temperatures, generally between -135⁰C to -196⁰C: what exact temperature within that range is best for your product or samples? Any of the “typical” storage temperatures are just points on a band, with “ambient” describing 15⁰C to 30⁰C, and ultra-low temperatures include -70⁰C to -86⁰C. How thoroughly have you tested the stability of your target biomarkers within the upper and lower limits of the potential range?

For all that can be said regarding this subject, it is always paramount to quantify to the extent possible the characteristics of a target analyte, and to investigate the optimal conditions for maintaining sample integrity before proceeding with further biomarker research and risking spurious results.

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1) Jansen, E.H. et. al.  (2012) ‘Long-term (in)stability of folate and vitamin B12 in human serum’, Clinical Chemistry and Laboratory Medicine, 50 (10), pp. 1761-1763.

2) Jansen, E.H. Beekhof, P.K. & Schenk, E. (2013) ‘Long-term stability of biomarkers of the iron status in human serum and plasma’, Biomarkers, 18 (4), pp. 365-368.

3) Baleriola, C. et. al. (2011) ‘Stability of hepatitis C virus, HIV, and heptatis B virus nucleic acids in plasma samples after long-term storage at -20⁰C and -70⁰C’, Journal of Clinical Microbiology, 49 (9), pp. 3163-3167.

4) Kisand, K. et. al., (2011) ‘Impact of cryopreservation on serum concentration of matrix metalloproteinases (MMP)-7, TIP-1, vascular growth factors (VEGF) and VEGF-R2 in biobank samples’, Clinical Chemistry and Laboratory Medicine, 49 (2), pp. 229-235.