title: Effects of storage conditions on the stability of serum CD163, NGAL, HMGB1 and MIP2.
abstract: BACKGROUND
  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.
  
  
  METHODS
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Disease, The First Affiliated Hospital, School of Medicine, Zhejiang University Hangzhou 310003, Zhejiang, China.
authors.forename: Jing
authors.initials: J
authors.lastname: Wang
authors.affiliation: State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Disease, The First Affiliated Hospital, School of Medicine, Zhejiang University Hangzhou 310003, Zhejiang, China.
authors.forename: Hai Hong
authors.initials: HH
authors.lastname: Zhu
authors.affiliation: State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Disease, The First Affiliated Hospital, School of Medicine, Zhejiang University Hangzhou 310003, Zhejiang, China.
authors.forename: Ji Hua
authors.initials: JH
authors.lastname: Xue
authors.affiliation: State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Disease, The First Affiliated Hospital, School of Medicine, Zhejiang University Hangzhou 310003, Zhejiang, China.
authors.forename: Shan Shan
authors.initials: SS
authors.lastname: Wu
authors.affiliation: State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Disease, The First Affiliated Hospital, School of Medicine, Zhejiang University Hangzhou 310003, Zhejiang, China.
authors.forename: Zhi
authors.initials: Z
authors.lastname: Chen
mesh_terms: D000209:Acute-Phase Proteins; D000293:Adolescent; D000328:Adult; D000368:Aged; D015703:Antigens, CD / Q000097:blood*; D015214:Antigens, Differentiation, Myelomonocytic / Q000097:blood*; D015415:Biomarkers / Q000097:blood; D054426:Chemokine CXCL2 / Q000097:blood*; D002648:Child; D002675:Child, Preschool; D003080:Cold Temperature; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D005615:Freezing; D024243:HMGB1 Protein / Q000097:blood*; D006801:Humans; D000071068:Lipocalin-2; D054834:Lipocalins / Q000097:blood*; D008297:Male; D008875:Middle Aged; D011489:Protein Denaturation; D055550:Protein Stability; D011518:Proto-Oncogene Proteins / Q000097:blood*; D011956:Receptors, Cell Surface / Q000097:blood*; D013048:Specimen Handling / Q000379:methods*; D013997:Time Factors; D055815:Young Adult; D000098902:CD163 Antigen
chemical_list: D000209:Acute-Phase Proteins; D015703:Antigens, CD; D015214:Antigens, Differentiation, Myelomonocytic; D015415:Biomarkers; D000098902:CD163 Antigen; C499706:CXCL2 protein, human; D054426:Chemokine CXCL2; D024243:HMGB1 Protein; C585491:HMGB1 protein, human; C489506:LCN2 protein, human; D000071068:Lipocalin-2; D054834:Lipocalins; D011518:Proto-Oncogene Proteins; D011956:Receptors, Cell Surface
keywords: CD163; HMGB1; MIP-2; NGAL; repeated freezing-thawing; storage time; temperature