Subscribe to RSS
The Effects of a Single Freeze-Thaw Cycle on Concentrations of Nutritional, Noncommunicable Disease, and Inflammatory Biomarkers in Serum Samples
Background The stability of biological samples is vital for reliable measurements of biomarkers in large-scale survey settings, which may be affected by freeze-thaw procedures. We examined the effect of a single freeze-thaw cycle on 13 nutritional, noncommunicable diseases (NCD), and inflammatory bioanalytes in serum samples.
Method Blood samples were collected from 70 subjects centrifuged after 30 minutes and aliquoted immediately. After a baseline analysis of the analytes, the samples were stored at − 70°C for 1 month and reanalyzed for all the parameters. Mean percentage differences between baseline (fresh blood) and freeze-thaw concentrations were calculated using paired sample t-tests and evaluated according to total allowable error (TEa) limits (desirable bias).
Results Freeze-thaw concentrations differed significantly (p < 0.05) from baseline concentrations for soluble transferrin receptor (sTfR) (− 5.49%), vitamin D (− 12.51%), vitamin B12 (− 3.74%), plasma glucose (1.93%), C-reactive protein (CRP) (3.45%), high-density lipoprotein (HDL) (7.98%), and cholesterol (9.76%), but they were within respective TEa limits. Low-density lipoprotein (LDL) (− 0.67%), creatinine (0.94%), albumin (0.87%), total protein (1.00%), ferritin (− 0.58%), and triglycerides (TAG) (2.82%) concentrations remained stable following the freeze-thaw cycle. In conclusion, single freeze-thaw cycle of the biomarkers in serum/plasma samples after storage at − 70°C for 1 month had minimal effect on stability of the studied analytes, and the changes in concentration were within acceptable limit for all analytes.
Keywordsnutrition and noncommunicable diseases biomarkers - serum sample - bioanalytes - freeze-thaw - stability - analysis - storage
24 May 2021 (online)
© 2021. The Indian Association of Laboratory Physicians. This is an open access article published by Thieme under the terms of the Creative Commons Attribution-NonDerivative-NonCommercial-License, permitting copying and reproduction so long as the original work is given appropriate credit. Contents may not be used for commercial purposes, or adapted, remixed, transformed or built upon. (https://creativecommons.org/licenses/by-nc-nd/4.0/).
Thieme Medical and Scientific Publishers Pvt. Ltd.
A-12, 2nd Floor, Sector 2, Noida-201301 UP, India
- 1 Cuhadar S, Koseoglu M, Atay A, Dirican A. The effect of storage time and freeze-thaw cycles on the stability of serum samples. Biochem Med (Zagreb) 2013; 23 (01) 70-77
- 2 Tanner M, Kent N, Smith B, Fletcher S, Lewer M. Stability of common biochemical analytes in serum gel tubes subjected to various storage temperatures and times pre-centrifugation. Ann Clin Biochem 2008; 45 (Pt 4) 375-379
- 3 Flood A, Pfeiffer R, Mai V, Remaley A, Lanza E, Schatzkin A. # 103 The effects of freeze-thaw cycles on serum measurement of insulin and glucose in epidemiologic studies. Ann Epidemiol 2002; 12 (07) 528
- 4 Kale VP, Patel SG, Gunjal PS. et al. Effect of repeated freezing and thawing on 18 clinical chemistry analytes in rat serum. J Am Assoc Lab Anim Sci 2012; 51 (04) 475-478
- 5 Ministry of Health and Family Welfare (MoHFW), Government of India, UNICEF and Population Council. Comprehensive National Nutrition Survey (CNNS) National Report. New Delhi: MoHFW 2019
- 6 Ricos C, Alvarez V, Cava F, Garcia-Lario JV, Hernande A, Jimenez CV, Minchinela J, Perich C, Simon M. Current databases on biologic variation: pros, cons and progress.. Scand J Clin Lab Invest 1999; 59: 491-500 Updated 2014
- 7 Wielders JP, Wijnberg FA. Preanalytical stability of 25(OH)-vitamin D3 in human blood or serum at room temperature: solid as a rock. Clin Chem 2009; 55 (08) 1584-1585
- 8 Enko D, Kriegshäuser G, Stolba R, Worf E, Halwachs-Baumann G. Method evaluation study of a new generation of vitamin D assays. Biochem Med (Zagreb) 2015; 25 (02) 203-212
- 9 Riley D. Method Development and Validation of Vitamin D2 and Vitamin D3 Using Mass Spectrometry [dissertation]. University of Washington, Seattle, WA; 2016
- 10 Antoniucci DM, Black DM, Sellmeyer DE. Serum 25-hydroxyvitamin D is unaffected by multiple freeze-thaw cycles. Clin Chem 2005; 51 (01) 258-261
- 11 Lewis JG, Elder PA. Serum 25-OH vitamin D2 and D3 are stable under exaggerated conditions. Clin Chem 2008; 54 (11) 1931-1932
- 12 Clark ML, Humphreys SM, Frayn KN. Stability of plasma glucose during storage. Ann Clin Biochem 1990; 27 (Pt 4) 373-377
- 13 Zhang DJ, Elswick RK, Miller WG, Bailey JL. Effect of serum-clot contact time on clinical chemistry laboratory results. Clin Chem 1998; 44 (6 Pt 1) 1325-1333
- 14 Ugwuezumba PC, Nwankpa P, Emengaha FC, Ekweogu CN, Etteh CC, Chukwuemeka OG. Influence of Freeze-Thawing and Storage on Human Serum Lipid Analytes. Int J Curr Microbiol Appl Sci 2018; 7 (10) 3287-3294
- 15 Curb JD, Overturf ML, Harrist RB. Lability of HDL-cholesterol during serum storage. Atherosclerosis 1980; 37 (04) 641-645
- 16 Ignatius CM, Emeka EN, Ebele JI, Chinelo VM, Fidelis EE, Silas AU. The effect of sample storage on total cholesterol and Hdl-cholesterol assays. Current Research Journal of Biological Sciences 2009; 1 (02) 1-5
- 17 Doumatey AP, Zhou J, Adeyemo A, Rotimi C. High sensitivity C-reactive protein (Hs-CRP) remains highly stable in long-term archived human serum. Clin Biochem 2014; 47 (4-5) 315-318
- 18 Aziz N, Fahey JL, Detels R, Butch AW. Analytical performance of a highly sensitive C-reactive protein-based immunoassay and the effects of laboratory variables on levels of protein in blood. Clin Diagn Lab Immunol 2003; 10 (04) 652-657
- 19 Macy EM, Hayes TE, Tracy RP. Variability in the measurement of C-reactive protein in healthy subjects: implications for reference intervals and epidemiological applications. Clin Chem 1997; 43 (01) 52-58
- 20 Jansen EHJM, Beekhof PK. Stability of folate and vitamin B12in human serum after long-term storage: a follow-up after 13 years. J Nutr Metab 2018; 2018: 9834181
- 21 Jee P, Fernandez L, Perkins SL, Brooks SP. Effect of storage and repeated freeze/thaw on (S) vitamin B12. Clin Biochem 2014; 47 (18) 344
- 22 Gislefoss RE, Lauritzen M, Langseth H, Mørkrid L. Effect of multiple freeze-thaw cycles on selected biochemical serum components. Clin Chem Lab Med 2017; 55 (07) 967-973
- 23 Pfeiffer CM, Cook JD, Mei Z, Cogswell ME, Looker AC, Lacher DA. Evaluation of an automated soluble transferrin receptor (sTfR) assay on the Roche Hitachi analyzer and its comparison to two ELISA assays. Clin Chim Acta 2007; 382 (1-2) 112-116
- 24 Kachhawa K, Kachhawa P, Varma M, Behera R, Agrawal D, Kumar S. Study of the stability of various biochemical analytes in samples stored at different predefined storage conditions at an accredited laboratory of India. J Lab Physicians 2017; 9 (01) 11-15
- 25 Kronenberg F, Lobentanz EM, König P, Utermann G, Dieplinger H. Effect of sample storage on the measurement of lipoprotein[a], apolipoproteins B and A-IV, total and high density lipoprotein cholesterol and triglycerides. J Lipid Res 1994; 35 (07) 1318-1328