Effects of storage conditions on the stability of serum CD163, NGAL, HMGB1 and MIP2

• Published in: International journal of clinical and experimental pathology Vol. 8; no. 4; pp. 4099 - 4105
• Main Authors: Wang, Jing, Zhu, Hai Hong, Xue, Ji Hua, Wu, Shan Shan, Chen, Zhi
• Format: Journal Article
• Language: English
• Published: United States e-Century Publishing Corporation 01.01.2015
• Subjects: Acute-Phase Proteins; Adolescent; Adult; Aged; Antigens, CD - blood; Antigens, Differentiation, Myelomonocytic - blood; Biomarkers - blood; Chemokine CXCL2 - blood; Child; Child, Preschool; Cold Temperature; Enzyme-Linked Immunosorbent Assay; Female; Freezing; HMGB1 Protein - blood; Humans; Lipocalin-2; Lipocalins - blood; Male; Middle Aged; Original; Protein Denaturation; Protein Stability; Proto-Oncogene Proteins - blood; Receptors, Cell Surface - blood; Specimen Handling - methods; Time Factors; Young Adult; NGAL; CD163; repeated freezing-thawing; storage time; MIP-2; temperature; HMGB1

Several cytokines have been involved in the diagnosis and prognosis for the pathogenesis and severity of chronic hepatitis B (CHB) such as cluster of differentiation 163 (CD163), neutrophil gelatinase-associated lipocalin (NGAL), high-mobility group box 1 (HMGB1) and macrophage inflammatory protein-2 (MIP-2). Nevertheless, the stability and reliability of these cytokines can be greatly influenced by handling and storage processes. In this study, potential utility of serum samples of a CHB cohort was evaluated to investigate several processes that might impact cytokine profiles such as temperature, storage time and number of freeze-thaw cycles. Blood samples collected from 100 patients with CHB were separated immediately and divided into two groups. In one group, samples (n=50) stored at -80 °C were subject to 1-3 freeze-thaw cycles. In the other group, samples (n=50) were stored at 4 °C and 25 °C for 3 h, 9 h, 24 h, 48 h, 72 h, and 7 d time points, respectively. To assess the influence of different storage conditions on cytokines, the levels of CD163, NGAL, HMGB1 and MIP-2 were measured using enzyme-linked immunosorbent assays (ELISA) kits. No significant differences of these four cytokines after 1-3 repeated freeze-thaw cycles. Significant differences of NAGL levels were seen between 9 h and 7 d (P<0.05), and also in HMGB1 at 25 °C, while the other cytokines were relatively stable at the two storage temperatures over the various time points. This study indicated that these four cytokines remained stable within three freeze-thaw cycles and 7 d at 4 °C. No perceptible effects on CD163 and MIP-2 levels were presented under the storage condition of 7 d at room temperature, whereas the degradation of NGAL and HMGB1 were notable.