title: hGH isoform differential immunoassays applied to blood samples from athletes: decision limits for anti-doping testing.
abstract: OBJECTIVE
  To detect hGH doping in sport, the World Anti-Doping Agency (WADA)-accredited laboratories use the ratio of the concentrations of recombinant hGH ('rec') versus other 'natural' pituitary-derived isoforms of hGH ('pit'), measured with two different kits developed specifically to detect the administration of exogenous hGH. The current joint compliance decision limits (DLs) for ratios derived from these kits, designed so that they would both be exceeded in fewer than 1 in 10,000 samples from non-doping athletes, are based on data accrued in anti-doping labs up to March 2010, and later confirmed with data up to February-March 2011. In April 2013, WADA asked the authors to analyze the now much larger set of ratios collected in routine hGH testing of athletes, and to document in the peer-reviewed literature a statistical procedure for establishing DLs, so that it be re-applied as more data become available.
  
  
  METHODS
  We examined the variation in the rec/pit ratios obtained for 21,943 screened blood (serum) samples submitted to the WADA accredited laboratories over the period 2009-2013. To fit the relevant sex- and kit-specific centiles of the logs of the ratios, we classified 'rec/pit' ratios based on low 'rec' and 'pit' values as 'negative' and fitted statistical distributions to the remaining log-ratios. The flexible data-driven quantile regression approach allowed us to deal with the fact that the location, scale and shape of the distribution of the modeled 'rec/pit' ratios varied with the concentrations of the 'rec' and 'pit' values. The between-kit correlation of the ratios was included in the fitting of the DLs, and bootstrap samples were used to quantify the estimation error in these limits. We examined the performance of these limits by applying them to the data obtained from investigator-initiated hGH administration studies, and in athletes in a simulated cycling stage race.
  
  
  RESULTS
  The mean and spread of the distribution of the modeled log-ratios depended in different ways on the magnitude of the rec and pit concentrations. Ultimately, however, the estimated limits were almost invariant to the concentrations, and similar to those obtained by fitting simpler (marginal) log-normal and Box-Cox transformed distributions. The estimated limits were similar to the (currently-used) limits fitted to the smaller datasets analyzed previously. In investigator-initiated instances, the limits distinguished recent use of rec-hGH from non-use.
  
  
  CONCLUSIONS
  The distributions of the rec/pit ratios varied as a function of the rec and pit concentrations, but the patterns in their medians and spreads largely canceled each other. Thus, ultimately, the kit- and sex-specific ratio DL obtained from the simpler model was very close to the 'curve of DLs' obtained from the more complex one. Both were close to previously established limits.
authors: Department of Epidemiology, Biostatistics and Occupational Health, McGill University, Montreal, Canada; Department of Mathematics and Statistics, McGill University, Montreal, Canada. Electronic address: james.hanley@mcgill.ca.
authors: James A
authors: JA
authors: Hanley
authors: Department of Epidemiology, Biostatistics and Occupational Health, McGill University, Montreal, Canada.
authors: Olli
authors: O
authors: Saarela
authors: Department of Mathematics and Statistics, McGill University, Montreal, Canada.
authors: David A
authors: DA
authors: Stephens
authors: Paris Descartes University, MAP5, UMR CNRS 8145, Paris, France; Endocrine Gynaecology Unit, Hôpital Cochin, Paris, France.
authors: Jean-Christophe
authors: JC
authors: Thalabard
mesh_terms: D056352:Athletes / Q000706:statistics & numerical data; D001774:Blood Chemical Analysis / Q000379:methods* / Q000592:standards / Q000706:statistics & numerical data; D003657:Decision Making; D004300:Doping in Sports / Q000706:statistics & numerical data; D005006:Ethnicity / Q000706:statistics & numerical data; D005260:Female; D019382:Human Growth Hormone / Q000032:analysis / Q000097:blood*; D006801:Humans; D007118:Immunoassay / Q000379:methods; D057230:Limit of Detection; D008297:Male; D020033:Protein Isoforms / Q000097:blood; D011933:Reagent Kits, Diagnostic / Q000592:standards; D012016:Reference Values; D012680:Sensitivity and Specificity; D015813:Substance Abuse Detection / Q000592:standards* / Q000706:statistics & numerical data*
chemical_list: D020033:Protein Isoforms; D011933:Reagent Kits, Diagnostic; D019382:Human Growth Hormone
keywords: Decision limits; Doping; Human Growth Hormone; Isoforms; Quantile; Regression