The effect of storage temperature fluctuations on the stability of biochemical analytes in blood serum

Helmuth Haslacher 1 , Thomas Szekeres 1 , Marlene Gerner 1 , Elisabeth Ponweiser 1 , Manuela Repl 1 , Oswald F. Wagner 1  and Thomas Perkmann 2
  • 1 Department of Laboratory Medicine, Vienna, Austria
  • 2 Department of Laboratory Medicine, Waehringer Guertel 18-20, 1090 Vienna, Austria
Helmuth HaslacherORCID iD:, Thomas Szekeres, Marlene Gerner, Elisabeth Ponweiser, Manuela Repl, Oswald F. Wagner and Thomas Perkmann
  • Corresponding author
  • Department of Laboratory Medicine, Medical University of Vienna, Waehringer Guertel 18-20, 1090 Vienna, Austria
  • Email
  • Search for other articles:
  • degruyter.comGoogle Scholar



Irreproducibility of scientific results constitutes an undesirably onerous economic burden and is in many cases caused by low-quality materials. Therefore, researchers are increasingly devoting their attention to the bioresources they use. In turn, those bioresources are required to validate their preanalytical processes in order to ensure best possible quality. The present study thus aimed to evaluate the impact of repeated temperature fluctuations, as they occur in most research biobanks due to repetitive opening and closing of freezer doors, on the stability of 26 biochemical analytes.


Serum of 43 individuals was randomly assigned to a fluctuation (n=21) and a control group (n=22). Serum of the fluctuation group underwent controlled temperature fluctuations (30 fluctuations <−75°C – <−65°C – <−75°C under real-life freezer conditions within 21 days). Control sera were stored at constant conditions. After 10, 20, and 30 fluctuations, results derived from the fluctuation group were compared to baseline and to the control group by means of general linear models.


Sixteen biomarkers showed statistically significant changes over time, whereas only seven of those presented with diagnostically/clinically relevant changes at certain time points (aspartate aminotransferase, amylase, calcium, uric acid, creatinine, inorganic phosphate and total protein). However, there was no difference between the fluctuation and the control group.


Some serum analytes are influenced by storage, even at temperatures as low as <−70°C. In contrast, we found no evidence that complex temperature fluctuations produced by storage of and access to biospecimens in biobank freezers generate any additional variability.

  • 1.

    Freedman LP, Cockburn IM, Simcoe TS. The Economics of Reproducibility in Preclinical Research. PLoS Biol 2015;13:e1002165.

  • 2.

    Ellervik C, Vaught J. Preanalytical variables affecting the integrity of human biospecimens in biobanking. Clin Chem 2015;61:914–34.

  • 3.

    Lippi G, Guidi GC, Mattiuzzi C, Plebani M. Preanalytical variability: the dark side of the moon in laboratory testing. Clin Chem Lab Med 2006;44:358–65.

  • 4.

    Craig WY, Ledue TB. The effects of long term storage on serum Lp(a) levels. Atherosclerosis 1992;93:261.

  • 5.

    Wigmore JG. Blood ethanol concentrations are less stable than serum or plasma upon storage because of oxyhemoglobin-mediated oxidation of ethanol to acetaldehyde. J Anal Toxicol 2009;33:182–3.

  • 6.

    Gaye A, Peakman T, Tobin MD, Burton PR. Understanding the impact of pre-analytic variation in haematological and clinical chemistry analytes on the power of association studies. Int J Epidemiol 2014;43:1633–44.

  • 7.

    Ikeda K, Ichihara K, Hashiguchi T, Hidaka Y, Kang D, Maekawa M, et al. Evaluation of the short-term stability of specimens for clinical laboratory testing. Biopreserv Biobank 2015;13:135–43.

  • 8.

    Ollier W, Sprosen T, Peakman T. UK Biobank: from concept to reality. Pharmacogenomics 2005;6:639–46.

  • 9.

    Zander J, Bruegel M, Kleinhempel A, Becker S, Petros S, Kortz L, et al. Effect of biobanking conditions on short-term stability of biomarkers in human serum and plasma. Clin Chem Lab Med 2014;52:629–39.

  • 10.

    Polestar Cooling Ltd. UF 756 Ultra Cold Freezer [updated 2014-04-29]. Available from:

  • 11.

    Benjamini Y, Hochberg Y. Controlling the false discovery rate: a practical and powerful approach to multiple testing. J Royal Stat Soc Series B (Methodological) 1995;57:289–300.

  • 12.

    Zander J, Bruegel M, Kleinhempel A, Becker S, Petros S, Kortz L, et al. Effect of biobanking conditions on short-term stability of biomarkers in human serum and plasma. Clin Chem Lab Med 2014;52:629–39.

  • 13.

    Fraser CG. Reference change values. Clin Chem Lab Med 2012;50:807–12.

  • 14.

    Huang GT. An integrated, module-based biomarker discovery framework. Doctoral dissertation, University of Pittsburgh, 2014.

  • 15.

    Taylor EC, Sethi B. Stability of 27 biochemistry analytes in storage at a range of temperatures after centrifugation. Br J Biomed Sci 2011;68:147–57.

  • 16.

    Wilson SS, Guillan RA, Hocker EV. Studies of the stability of 18 chemical constituents of human serum. Clin Chem 1972;18:1498–503.

  • 17.

    Cuhadar S, Koseoglu M, Atay A, Dirican A. The effect of storage time and freeze-thaw cycles on the stability of serum samples. Biochemia Medica 2013;23:70–7.

  • 18.

    Alberghina D, Casella S, Giannetto C, Marafioti S, Piccione G. Effect of storage time and temperature on the total protein concentration and electrophoretic fractions in equine serum. Can J Vet Res 2013;77:293–6.

  • 19.

    Jansen EH, Beekhof PK, Cremers JW, Viezeliene D, Muzakova V, Skalicky J. Long-term stability of parameters of antioxidant status in human serum. Free Radic Res 2013;47:535–40.

  • 20.

    Askenazi DJ, Moore JF, Fineberg N, Koralkar R, Clevenger S, Sharer JD. Comparison of methods, storage conditions, and time to analysis of serum and urine creatinine measured from microsamples by liquid chromatography mass spectrometery (lc/ms) vs. Jaffe. J Clin Lab Anal 2014;28:405–8.

  • 21.

    Comstock GW, Burke AE, Norkus EP, Gordon GB, Hoffman SC, Helzlsouer KJ. Effects of repeated freeze-thaw cycles on concentrations of cholesterol, micronutrients, and hormones in human plasma and serum. Clin Chem 2001;47:139–42.

Purchase article
Get instant unlimited access to the article.
Log in
Already have access? Please log in.

Journal + Issues

Clinical Chemistry and Laboratory Medicine ( CCLM) publishes articles on novel teaching and training methods applicable to laboratory medicine. CCLM welcomes contributions on the progress in fundamental and applied research and cutting-edge clinical laboratory medicine. It is one of the leading journals in the field, with an impact factor of over three. CCLM is the official journal of nine national clinical societies and associated with EFLM.