The use of growth hormone (GH)-dependent markers in the detection of GH abuse in sport: Physiological intra-individual variation of IGF-I, type 3 pro-collagen (P-III-P) and the GH-2000 detection score.

BACKGROUND: Growth Hormone is abused by athletes for its lipolytic and anabolic properties. Its use is prohibited by the World Anti-Doping Agency. The GH-2000 project developed a methodology to detect its abuse using the concentrations of two GH-dependent biomarkers, IGF-I and type 3 procollagen (P-III-P). The sensitivity of this method may be improved by considering intra-individual variability. AIM: The aim of this study was to examine the intra-individual variability of IGF-I, P-III-P and the GH-2000 score. SUBJECTS AND METHODS: IGF-I, P-III-P and GH-2000 score were evaluated in four longitudinal studies involving 303 elite and 78 amateur athletes. Samples were collected over a period of up to 12 months from a total of 238 men and 143 women aged between 17 and 53 years (mean 24.2). RESULTS: The four studies showed good agreement with no apparent difference in within-individual variation between amateur and elite athletes. The intra-individual variability for IGF-I ranged between 14-16% while the variability for P-III-P was 7-18%. No athlete tested positive for growth hormone during any of the studies. The overall mean intra-individual variability of the GH-2000 score was less than 0.6 units in all studies. CONCLUSIONS: The high stability of marker levels suggests that concentrations are largely genetically determined. Adopting a test based on the concept of an athlete's 'passport' or 'profiling' would take advantage of this and most likely increase the sensitivity of the test. These data also provide strong evidence that a positive test result for GH abuse would not occur as a result of chance variability.

Influence of ethnicity on IGF-I and procollagen III peptide (P-III-P) in elite athletes and its effect on the ability to detect GH abuse.

CONTEXT: A method based on the two GH dependent markers, IGF-I and procollagen III peptide (P-III-P) has been proposed to detect exogenously administered GH. As previous studies involved predominantly white European elite athletes, it is necessary to validate the method in other ethnic groups. OBJECTIVE: To examine serum IGF-I and P-III-P in elite athletes of different ethnicities within 2 h of competing at national or international events. DESIGN: Cross-sectional observational study. SETTING: National and International sporting events. SUBJECTS: 1085 elite athletes of different ethnicities. INTERVENTION: Serum IGF-I and P-III-P were measured and GH-2000 discriminant function score was calculated. Effect of ethnicity was assessed. RESULTS: In men, IGF-I was 21.7 +/- 2.6% lower in Afro-Caribbeans than white Europeans (P < 0.0001) but there were no differences between other ethnic groups. In women, IGF-I was 14.2 +/- 5.1% lower in Afro-Caribbeans (P = 0.005) and 15.6 +/- 7.0% higher in Orientals (P = 0.02) compared with white Europeans. P-III-P was 15.2 +/- 3.5%, 26.6 +/- 6.6% and 19.3 +/- 5.8% lower in Afro-Caribbean (P < 0.0001), Indo-Asian (P < 0.0001) and Oriental men (P = 0.001), respectively, compared with white European men. In women, P-III-P was 15.7 +/- 4.7% lower in Afro-Caribbeans compared to white Europeans (P =0.0009) but there were no differences between other ethnicities. Despite these differences, most observations were below the upper 99% prediction limits derived from white European athletes. All GH-2000 scores lay below the cut-off limit proposed for doping. CONCLUSIONS: The GH-2000 detection method based on IGF-I and P-III-P would be valid in all ethnic groups.

The effect of sports injury on insulin-like growth factor-I and type 3 procollagen: implications for detection of growth hormone abuse in athletes.

CONTEXT: A method to detect exogenously administered growth hormone (GH) based on the measurement of two GH-dependent markers, IGF-I and type 3 procollagen (P-III-P) has been proposed. Skeletal or soft tissue injury may alter these markers. Elevations in either of these proteins after injury might lead to a false accusation of doping with GH. OBJECTIVE: The objective of the study was to assess the effect of musculoskeletal or soft tissue injury on IGF-I and P-III-P concentrations in amateur and elite athletes and assess the effect of injury on the proposed GH detection method. DESIGN: This was a longitudinal observational study after sporting injury. SETTING: The study was conducted at Southampton General Hospital and British Olympic Medical Centre. SUBJECTS: Subjects included elite and amateur athletes after an injury. INTERVENTION: Interventions included measurement of IGF-I and P-III-P and application of the GH-2000 discriminant function score up to 84 d after an injury as well as classification of injury by type and severity. OUTCOME MEASURES: IGF-I and P-III-P concentration and ability to detect GH abuse in athletes without the risk of false accusation because of an injury were measured. RESULTS: There was no change in IGF-I concentration after an injury. By contrast, P-III-P concentrations rose by 41.1 +/- 16.6%, reaching a peak around 14 d after an injury. The rise in P-III-P varied according to injury type and severity. This rise had a trivial effect on the GH-2000 discriminant function score, and no subject reached the threshold needed for a doping offense. CONCLUSIONS: Although there was a rise in P-III-P after injury, this was insufficient to invalidate the GH-2000 detection method based on IGF-I and P-III-P concentrations.

A correction to the age-adjustment of the GH-2000 score used in the detection of growth hormone misuse.

OBJECTIVE: The GH-2000 biomarker test has been introduced by the World Anti-Doping Agency as a method of detecting growth hormone misuse in professional sport. The test involves the measurement insulin-like growth factor-I and the amino-terminal pro-peptide of type III collagen (P-III-NP) which increase in a dose-dependent manner in response to GH. These measurements are combined in sex specific formulae that include an age adjustment. The original age adjustment overcorrects the effect of age in male athletes and could potentially place older men at a disadvantage. The purpose of this note is to investigate the performance of a previously suggested correction term in two new and larger data sets. RESULTS: The GH-2000 score was calculated for 7307 samples obtained from 15 accredited WADA laboratories in 2017 and 3916 samples measured at Drug Control Centre, King's College London, UK between 2013 and 2017. The GH-2000 scores were investigated for positive age effects using standard regression modelling. As previously, all analyses confirmed a positive age effect. Applying the earlier suggested correction term of 0.032 × age showed a significant over-correction leading to a negative association of the GH-2000 score with age. We now suggest a smaller age correction of 0.020 × age, which corresponds to the smallest effect found in the earlier studies.

Why do endocrine profiles in elite athletes differ between sports?

BACKGROUND: Endocrine profiles have been measured on blood samples obtained immediately post-competition from 693 elite athletes from 15 Olympic Sports competing at National or International level; four were subsequently excluded leaving 689 for the current analysis. METHODS: Body composition was measured by bioimpedance in a sub-set of 234 (146 men and 88 women) and from these data a regression model was constructed that enabled 'estimated' lean body mass and fat mass to be calculated on all athletes. One way ANOVA was used to assess the differences in body composition and endocrine profiles between the sports and binary logistical regression to ascertain the characteristic of a given sport compared to the others. RESULTS: The results confirmed many suppositions such as basketball players being tall, weightlifters short and cross-country skiers light. The hormone profiles were more surprising with remarkably low testosterone and free T3 (tri-iodothyronine) in male powerlifters and high oestradiol, SHBG (sex hormone binding globulin) and prolactin in male track and field athletes. Low testosterone concentrations were seen 25.4% of male elite competitors in 12 of the 15 sports and high testosterone concentrations in 4.8% of female elite athletes in 3 of the 8 sports tested. Interpretation of the results is more difficult; some of the differences between sports are at least partially due to differences in age of the athletes but the apparent differences between sports remain significant after adjusting for age. The prevalence of 'hyperandrogenism' (as defined by the IAAF (International Association of Athletics Federations) and IOC (International Olympic Committee)) amongst this cohort of 231 elite female athletes was the highest so far recorded and the very high prevalence of 'hypoandrogenism' in elite male athletes a new finding. CONCLUSIONS: It is unclear whether the differences in hormone profiles between sports is a reason why they become elite athletes in that sport or is a consequence of the arduous processes involved. For components of body composition we know that most have a major genetic component and this may well be true for endocrine profiles.

Increases in Serum Growth Hormone Concentrations Associated with GHB Administration.

The administration of gamma-hydroxybutyrate (GHB) has been reported to augment the increase in growth hormone (GH) secretion associated with the onset of sleep. The ability of GHB to stimulate GH production in the absence of sleep in both male and female volunteers was investigated as part of a GHB administration study. Twelve healthy volunteers (six men and six women) were given a small oral dose (25 mg/kg) of GHB (as Xyrem®) at 10:00 h. Basal blood samples (as serum) were taken 10 min prior to GHB administration, with additional samples taken at 10, 15, 20, 25, 30, 45, 60, 90, 120, 150, 180, 240, 360 and 480 min post-administration. The serum concentrations of GHB were measured by GC-MS and GH by immunometric assay. Following GHB administration, volunteers exhibited effects consistent with mild sedation, i.e., relaxed with normal responses to verbal stimuli. Despite none being asleep, an increase in serum GH concentration occurred in 11 out of the 12 volunteers (5 women and 6 men). In these volunteers, peak GH concentrations occurred 45-60 min post-administration compared with a mean serum tmax for GHB of 23 min (SD = 5.4 min). The absolute increase in GH was similar for men and women, averaging 3.4 and 3.7 ng/mL, respectively. The mean intra-individual increase in GH was much greater in males (29 times) compared with females (2 times), as males had (as expected) smaller basal GH concentrations (mean = 0.26 ng/mL) compared with females (mean = 5.4 ng/mL). After maximizing, the GH concentration decreased rapidly (in agreement with GHB concentrations), returning to basal concentrations at ~90-120 min post-administration. GHB administration at a small therapeutic dose results in increases in serum GH concentrations in healthy male and female volunteers in the absence of sleep onset.

Novel markers to detect recombinant human insulin-like growth factor-I (rhIGF-I)/rhIGF binding protein-3 (rhIGFBP-3) misuse in athletes.

Insulin-like growth factor-I (IGF-I) is abused by elite athletes for its metabolic and anabolic effects. We have previously shown that it is possible to detect IGF-I misuse by measuring serum IGF-I and procollagen type III amino-terminal propeptide (P-III-NP) but a pilot study suggested measuring IGF-II, IGF binding protein-2 (IGFBP-2) and acid-labile subunit (ALS) may improve the detection of IGF-I administration. The aim of the study was to assess this in a randomized controlled trial. Twenty-six female and 30 male recreational athletes were randomized to 28 days' treatment with placebo or recombinant human (rh)IGF-I/rhIGF binding protein-3 (IGFBP-3) complex (30 mg/day or 60 mg/day), followed by 56 days' washout. IGF-II, IGFBP-2 and ALS (women only) were measured using commercial immunoassays. IGFBP-2 increased and IGF-II decreased in response to both low and high dose rhIGF-I/rhIGFBP-3 in both women and men while ALS decreased in women in response to high dose rhIGF-I/rhIGFBP-3. Two days after discontinuing treatment, significant differences remained between the three treatment groups in IGFBP-2 and IGF-II, but not ALS. Thereafter there were no significant differences between the three treatment groups in any of the markers. Combining IGF-I with IGF-II and/or IGFBP-2 improved the performance of the test to detect rhIGF-I/rhIGFBP-3 administration in both women and men. Copyright © 2016 John Wiley & Sons, Ltd.

Detection of the administration of human erythropoietin (HuEPO) to canines.

Recombinant human erythropoietin (rHuEPO) doping is prohibited in animal (canine and equine) sport. The effectiveness of a range of immunoassay screening methods for the detection of rHuEPO in canine urine was evaluated. The excretion profiles following rHuEPO administration to dogs were investigated. The presence of rHuEPO in postadministration samples was confirmed using the World Anti-Doping Agency (WADA)-approved isoelectric focusing immunoblotting confirmatory technique. Following the administration study, a screening program involving approximately 6000 greyhound sport (mostly racing) samples was undertaken for rHuEPO. This resulted in the detection of the first rHuEPO positives in the world of canine or equine sport. In an additional case, endogenous HuEPO was detected in a sample submitted as greyhound urine. It was determined that this arose from the submission to control stewards, as greyhound urine, of a substance that was, in fact, human urine. This was a particularly welcome development as definitive confirmatory evidence of such sample switching can be difficult to obtain in the case of greyhounds.

The development of decision limits for the GH-2000 detection methodology using additional insulin-like growth factor-I and amino-terminal pro-peptide of type III collagen assays.

The GH-2000 and GH-2004 projects have developed a method for detecting GH misuse based on measuring insulin-like growth factor-I (IGF-I) and the amino-terminal pro-peptide of type III collagen (P-III-NP). The objectives were to analyze more samples from elite athletes to improve the reliability of the decision limit estimates, to evaluate whether the existing decision limits needed revision, and to validate further non-radioisotopic assays for these markers. The study included 998 male and 931 female elite athletes. Blood samples were collected according to World Anti-Doping Agency (WADA) guidelines at various sporting events including the 2011 International Association of Athletics Federations (IAAF) World Athletics Championships in Daegu, South Korea. IGF-I was measured by the Immunotech A15729 IGF-I IRMA, the Immunodiagnostic Systems iSYS IGF-I assay and a recently developed mass spectrometry (LC-MS/MS) method. P-III-NP was measured by the Cisbio RIA-gnost P-III-P, Orion UniQ™ PIIINP RIA and Siemens ADVIA Centaur P-III-NP assays. The GH-2000 score decision limits were developed using existing statistical techniques. Decision limits were determined using a specificity of 99.99% and an allowance for uncertainty because of the finite sample size. The revised Immunotech IGF-I - Orion P-III-NP assay combination decision limit did not change significantly following the addition of the new samples. The new decision limits are applied to currently available non-radioisotopic assays to measure IGF-I and P-III-NP in elite athletes, which should allow wider flexibility to implement the GH-2000 marker test for GH misuse while providing some resilience against manufacturer withdrawal or change of assays.

Biochemical markers of insulin-like growth factor-I misuse in athletes: the response of serum IGF-I, procollagen type III amino-terminal propeptide, and the GH-2000 score to the administration of rhIGF-I/rhIGF binding protein-3 complex.

CONTEXT: The GH-2000 and GH-2004 research groups developed a method for detecting GH misuse in athletes based on the measurement of serum IGF-I and procollagen type III amino-terminal propeptide (P-III-NP). There are reports that IGF-I is also misused by athletes, but currently there is no internationally recognized test designed to detect recombinant human IGF-I misuse. OBJECTIVE: The objective of the study was to examine the response of serum IGF-I, P-III-NP, and the GH-2000 score to recombinant human (rh) IGF-I/rhIGF binding protein-3 (IGFBP-3) administration in recreational athletes. DESIGN AND SETTING: This was a randomized, double-blind, placebo-controlled rhIGF-I/rhIGFBP-3 administration study at Southampton General Hospital (Southampton, United Kingdom). PARTICIPANTS: Fifty-six recreational athletes (26 women, 30 men) participated in the study. INTERVENTION: Participants were randomized to treatment with low-dose (30 mg/d) or high-dose (60 mg/d) rhIGF-I/rhIGFBP-3 complex or placebo for 28 days. Blood was collected throughout the drug administration and washout periods. Serum IGF-I and P-III-NP were measured using commercial immunoassays and GH-2000 scores were calculated. RESULTS: IGF-I, P-III-NP, and the GH-2000 score rose in response to both low- and high-dose rhIGF-I/rhIGFBP-3 administration. The relative maximum response of IGF-I (approximately 4-fold increase in women and men) was greater than that of P-III-NP (40%-50% increase in women, 35%-50% increase in men). The GH-2000 formulae, which incorporate IGF-I and P-III-NP results, detected up to 61% of women and 80% of men in the rhIGF-I/rhIGFBP-3 groups but, using IGF-I concentrations alone, the sensitivity increased to 94% in both women and men during the administration period. CONCLUSIONS: The rise in P-III-NP after rhIGF-I/rhIGFBP-3 administration is small compared with that after rhGH administration. Although rhIGF-I/rhIGFBP-3 administration can be detected using the GH-2000 score method, a test based on serum IGF-I alone provides better sensitivity.

Biochemical markers of recombinant human insulin-like growth factor-I (rhIGF-I)/rhIGF binding protein-3 (rhIGFBP-3) misuse in athletes.

Insulin-like growth factor-I (IGF-I) is reportedly misused by elite athletes, either alone or with growth hormone (GH). The GH-2000 and GH-2004 research groups previously developed a method for detecting GH misuse based on the GH-sensitive markers IGF-I and procollagen type III amino-terminal propeptide (P-III-NP). Both markers increase in response to rhIGF-I/rhIGF binding protein-3 (rhIGFBP-3) administration in recreational athletes. The aim of this pilot study was to assess the effect of rhIGF-I/rhIGFBP-3 administration on other serum markers of the GH-IGF axis and on other bone and collagen markers. Twenty-six female and 30 male recreational athletes were randomized to 28 days' treatment with placebo or rhIGF-I/rhIGFBP-3 complex, followed by 56 days' washout. GH-IGF axis markers (IGFBP-2, IGFBP-3, acid-labile subunit (ALS) and IGF-II) and bone and collagen markers (procollagen type I carboxy-terminal propeptide (PICP), type I collagen cross-linked carboxy-terminal telopeptide (ICTP) and osteocalcin) were measured using commercial immunoassays. In women in the high dose treatment group, mean IGF-II decreased by 53% (P=0.0028) on Day 21. Mean IGFBP-2 increased by 119% (P=0.0039) and mean ALS decreased by 40% (P=0.0022) on Day 21. There were no significant changes in IGFBP-3, osteocalcin, ICTP or PICP. In men in the high dose group, mean IGF-II decreased by 51% on Day 21 (P<0.0001). Mean IGFBP-2 increased by 125% on Day 21 (P=0.0003). There were no significant changes in IGFBP-3, ALS, osteocalcin, ICTP or PICP. Serum IGFBP-2 and IGF-II may be useful markers of rhIGF-I/rhIGFBP-3 administration in both women and men while ALS may also be a useful marker in women; these markers are now undergoing further evaluation.

The development of decision limits for the implementation of the GH-2000 detection methodology using current commercial insulin-like growth factor-I and amino-terminal pro-peptide of type III collagen assays.

BACKGROUND: The GH-2000 project developed a method for detecting GH misuse based on the measurement of insulin-like growth factor-I (IGF-I) and the amino-terminal pro-peptide of type III collagen (P-III-NP). The objective of this study was to develop decision limits for the GH-2000 score to detect GH misuse in elite athletes using two currently available commercial assays for each analyte. SUBJECTS: 404 male (mean age 23.9 yrs, range 12-37 yrs) and 94 female elite athletes (mean age 24.5 yrs, range 18-34 yrs) participated. Blood samples were collected according to World Anti-Doping Agency (WADA) guidelines at various sporting events including 238 samples collected as part of the UK Anti-Doping Testing Programme. Laboratory analysis: IGF-I was measured by Siemens Immulite IGF-I assay and Immunotech A15729 IGF-I IRMA. P-III-NP was measured by RIA-gnost P-III-P and the UniQ™ PIIINP RIA. STATISTICAL ANALYSIS: The GH-2000 score decision limits were developed through the analysis of the elite athlete samples. RESULTS: For males and females separately, the distributions of GH-2000 scores were consistent with Normal distributions. Using a specificity of 99.99% new decision limits were determined which included an allowance for uncertainty associated with calculations based on a finite sample size. One outlier was identified with results incompatible with normal physiology and tested positive with the current isoform GH test. CONCLUSIONS: We have developed decision limits using currently available commercial assays to measure IGF-I and P-III-NP in elite athletes. This should allow the introduction of a test for GH misuse based on the measurement of these GH sensitive biomarkers.

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.

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.

The GH-2004 project: the response of IGF1 and type III pro-collagen to the administration of exogenous GH in non-Caucasian amateur athletes.

CONTEXT: The GH-2000 team proposed a method based on IGF1 and type III pro-collagen (P-III-P) to detect exogenously administered GH. As previous studies involved predominantly white European athletes, it is important to assess whether the response of these markers to recombinant human GH (rhGH) differs with ethnicity. OBJECTIVE: To examine the response of serum IGF1 and P-III-P and GH-2000 score to rhGH in non-Caucasian amateur athletes. DESIGN: Double-blind placebo-controlled rhGH administration study. SETTING: Wellcome Trust Clinical Research Facility, Southampton General Hospital. SUBJECTS: The study included 31 male and 14 female amateur athletes of different ethnicities. Intervention The subjects were assigned to treatment with placebo or 0.1 IU/kg per day (low dose) or 0.2 IU/kg per day (high dose) rhGH for 28 days. Blood was collected weekly during treatment and on days 35, 42 and 84 during the washout period. Serum IGF1 and P-III-P were measured, and GH-2000 score was calculated. RESULTS: IGF1, P-III-P and GH-2000 score rose in response to both low- and high-dose GH in both men and women. When compared with the Caucasian volunteers of the previous GH-2000 study, mean baseline and placebo-treated P-III-P and GH-2000 score were lower in GH-2004 men and women. Post-GH, however, peak IGF1 or P-III-P did not differ between studies but the peak GH-2000 score was lower in GH-2004 men. There was no difference between studies in the maximal change in IGF1, P-III-P and GH-2000 score in response to GH in either gender. CONCLUSIONS: These data do not support a significant ethnic effect on the peak or maximal response to rhGH.

Serum insulin-like growth factor-I and pro-collagen type III N-terminal peptide in adolescent elite athletes: implications for the detection of growth hormone abuse in sport.

CONTEXT: A method based on two GH-dependent markers, IGF-I and pro-collagen type III N-terminal peptide (P-III-P), has been devised to detect exogenously administered GH. Because previous studies on the detection of GH abuse involved predominantly adult athletes, the method must be validated in adolescent athletes. OBJECTIVE: The aim of the study was to examine serum IGF-I and P-III-P concentrations in elite adolescent athletes and to determine whether the method developed in adults is appropriate to detect GH abuse in this population. DESIGN AND SETTING: We conducted a cross-sectional observational study at national sporting organization training events. SUBJECTS: A total of 157 (85 males, 72 females) elite athletes between 12 and 20 yr of age participated in the study. INTERVENTION: Serum IGF-I and P-III-P were each measured by two commercially available immunoassays. GH-2000 discriminant function scores were calculated. RESULTS: Both IGF-I and P-III-P rose to a peak during adolescence, which was earlier in girls than in boys. All GH-2000 scores lay below the proposed cutoff limit of 3.7 (although some scores were close to this value), indicating that none of these athletes would be accused of GH doping if the GH-2000 discriminant formulae were used. The results between the two immunoassays for IGF-I and P-III-P were closely aligned. CONCLUSIONS: The GH-2000 score rises in early adolescence, reaches a peak in athletes aged 13-16 yr, and then falls. We have found no evidence that the proposed GH-2000 score developed in adults would lead to an unacceptable rate of false-positive results in adolescent athletes, but caution may be required around the time of peak growth velocity.

Laboratory issues in the implementation of the marker method.

Two important biomarkers for the identification of growth hormone or IGF-I administration are IGF-I and P-III-P. These substances are determined in plasma or preferably in serum. There are a number of assays on the market for IGF-I but only two for P-III-P. The principles behind these assays and the choice of assays for doping control purposes are discussed. The future possibility of quantification by mass spectrometry is also briefly discussed.

Moving one step closer to catching the GH cheats: The GH-2004 experience.

Growth hormone is abused by athletes for its anabolic and lipolytic properties. The detection of GH abuse is challenging because it is an endogenous hormone whose concentration varies widely in any one day. The GH-2000 project proposed a test based on the measurement of IGF-I and type III pro-collagen (P-III-P). When the results of the GH-2000 project were presented to an expert workshop, the method was supported but it was felt that several issues needed to be resolved before the method could be adopted. The first was a potential effect of ethnicity as most subjects in the GH-2000 were white Europeans and the second was a possible effect of injury as P-III-P is a marker of soft tissue turnover. The GH-2004 project was conceived to address these concerns. The GH-2004 project has shown that while there are minor differences in IGF-I and P-III-P between ethnicities, these are small and do not affect the performance of the test. Injury leads to a small rise in P-III-P but again this is not of sufficient magnitude to affect the performance of the test. The GH-2004 project has provided further support for the marker approach as a means of detecting GH abuse in athletes. As WADA have not developed their own immunoassays, however, further work is needed to validate newer commercial assays measuring IGF-I and P-III-P to establish reliable conversion factors to the original GH-2000 units to allow the published formulae to be used.

A determination of the pre-analytical storage conditions for insulin like growth factor-I and type III procollagen peptide.

OBJECTIVE: IGF-I and type III procollagen (P-III-P) have been proposed as markers to detect GH abuse. This study aims to determine whether the pre-analytical storage temperature or delayed centrifugation affect the measured IGF-I and P-III-P concentrations. DESIGN: Observational study. SETTING: Wellcome Trust Clinical Research Facility, Southampton. SUBJECTS: Nineteen healthy volunteers. INTERVENTION: Blood was collected into bottles containing a clotting agent, lithium heparin or EDTA. One sample from each group was centrifuged and stored at -80 degrees C (control sample). The remaining samples from each group were stored as either serum or whole blood at 4 degrees C or room temperature for up to five days prior to storage at -80 degrees C. OUTCOME MEASURES: IGF-I and P-III-P. RESULTS: The storage temperature or timing of centrifugation did not appear to affect IGF-I concentration. In contrast, the measured P-III-P concentration rose by 6.5-7% per day in clotted and lithium heparin samples when stored as whole blood (p<0.006) or serum (6.2-6.5% per day) at room temperature (p<0.001). P-III-P did not change when the samples were stored at 4 degrees C. Although collection into EDTA inhibited the rise in P-III-P, the baseline measured values were significantly higher than in other media and spiking experiments demonstrated that EDTA exerted a significant matrix effect on the assay. CONCLUSION: While the optimum collection method is immediate centrifugation and storage at -80 degrees C, it would seem acceptable to store serum or clotted blood samples at 4 degrees C, but not ambient temperature, for up to five days. It is incumbent on the anti-doping authorities to provide facilities to allow this.