title: The effects of a freeze-thaw cycle and pre-analytical storage temperature on the stability of insulin-like growth factor-I and pro-collagen type III N-terminal propeptide concentrations: Implications for the detection of growth hormone misuse in athletes.
abstract: A method based on two serum biomarkers - insulin-like growth factor-I (IGF-I) and pro-collagen type III N-terminal propeptide (P-III-NP) - has been devised to detect growth hormone (GH) misuse. The aims of this study were to determine the stability of IGF-I and P-III-NP concentrations in serum stored at -20°C and to establish the effects of one freeze-thaw cycle. Blood was collected from 20 healthy volunteers. Serum aliquots were analyzed after storage for one day at 4°C and one day, one week, five weeks, and three months at -20°C. IGF-I and P-III-NP results were combined to calculate a GH-2000 discriminant function score for each volunteer. Inter-assay precision was determined by analysing one quality control sample at each time-point. A single freeze-thaw cycle, storage of serum at 4°C for one day and at -20°C for up to three months had no significant effect on IGF-I or P-III-NP concentration. Intra-sample variability for IGF-I was 6.8% (Immunotech assay) and 12.9% (DSL assay). Intra-sample variability for P-III-NP was 10.9% (Cisbio assay) and 13.7% (Orion assay). When IGF-I and P-III-NP results were combined, intra-sample variability of the GH-2000 score expressed as a standard deviation varied between 0.31 and 0.50 depending on the assay combination used. Variability in IGF-I and P--III-NP results of stored samples is largely determined by the characteristics of the assays. A single freeze-thaw cycle, storage of serum at 4°C for one day or at -20°C for up to 3 months does not result in a significant change in GH-2000 score.
authors.affiliation: Human Development and Health Academic Unit, Faculty of Medicine, University of Southampton, UK.
authors.forename: Nishan
authors.initials: N
authors.lastname: Guha
authors.forename: Ioulietta
authors.initials: I
authors.lastname: Erotokritou-Mulligan
authors.forename: Christiaan
authors.initials: C
authors.lastname: Bartlett
authors.forename: David A
authors.initials: DA
authors.lastname: Cowan
authors.forename: E Eryl
authors.initials: EE
authors.lastname: Bassett
authors.forename: Michael
authors.initials: M
authors.lastname: Stow
authors.forename: Peter H
authors.initials: PH
authors.lastname: Sönksen
authors.forename: Richard I G
authors.initials: RI
authors.lastname: Holt
mesh_terms: D000328:Adult; D056352:Athletes; D015415:Biomarkers / Q000097:blood / Q000737:chemistry; D001800:Blood Specimen Collection / Q000379:methods; D004300:Doping in Sports; D005260:Female; D005615:Freezing; D019382:Human Growth Hormone / Q000494:pharmacology*; D006801:Humans; D007334:Insulin-Like Growth Factor I / Q000032:analysis* / Q000737:chemistry; D008297:Male; D010446:Peptide Fragments / Q000097:blood* / Q000737:chemistry; D011347:Procollagen / Q000097:blood* / Q000737:chemistry; D055550:Protein Stability; D015813:Substance Abuse Detection / Q000379:methods*; D013696:Temperature; D013997:Time Factors; D055815:Young Adult
chemical_list: D015415:Biomarkers; D010446:Peptide Fragments; D011347:Procollagen; C041876:procollagen Type III-N-terminal peptide; D019382:Human Growth Hormone; D007334:Insulin-Like Growth Factor I