Confounders of haemoglobin mass in athletes: implications for anti-doping

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Doctor of Philosophy of the University of Canberra Clare E. Humberstone BSc.

1 Confounders of haemoglobin mass in athletes: implications for anti-doping A thesis submitted in the fulfilment of the requirements for the degree of Doctor of Philosophy of the University of Canberra Clare E. Humberstone BSc. (Hons) Sports Science and Physiology (Ind), University of Leeds, 2006 May 2012

2 Abstract The Athlete Biological Passport (ABP) is used for the indirect detection of blood doping in athletes. The calculations within the ABP (Adaptive model) monitor longitudinal changes in characteristics of an athlete s blood, specifically haemoglobin concentration ([Hb]) and percent reticulocytes (%Ret), and flag unusual variations that may be characteristic of doping. The inclusion of total haemoglobin mass (Hb mass ) as a marker in the ABP may improve the Passport s sensitivity. However, concerns have been expressed about the suitability of Hb mass measurements for this purpose. This thesis investigated two potential barriers to including Hb mass in the ABP: the lack of a quality control system for Hb mass measurement and the potential confusion between longitudinal Hb mass profiles of doped and non-doped athletes. The use of custom-made quality control solutions eliminated the majority of betweenlaboratory differences in Hb mass measures. Analytical error associated with making successive Hb mass measurements in three different laboratories was reduced from 2.4% to 1.7% when the quality controls were used. These findings demonstrated that using quality control solutions would ensure that Hb mass results from different laboratories were equivalent if Hb mass was included as a marker in the ABP. The effects of various confounders on Hb mass in non-doped athletes were quantified. These confounders were investigated specifically for their potential to increase the biological variability of Hb mass. For detection of blood doping, these confounders represent a worstcase-scenario for the variability of Hb mass in non-doped athletes. Ultra-endurance triathlon racing (+3.2%), Classical altitude training (+3.8%) and Live High:Train Low altitude training (two estimates, +4.0% and +4.3%) each caused substantial mean increases in Hb mass. Conversely, reduced training (-2.3%) and surgery (-2.7%) in injured / ill athletes caused ii

3 substantial mean decreases in Hb mass. Acute Intermittent Hypoxic Exposure did not substantially affect Hb mass (-0.3%). The effects of microdoses of recombinant human erythropoietin (rhuepo) on Hb mass were also examined to represent a worst-case-scenario for the detection of doped athletes using Hb mass in the ABP. Over a 12 week period, rhuepo microdosing caused a mean increase of Hb mass of 11.0%, with individual responses ranging from increases of 1.4% to 19.2%. Finally, an investigation was carried out to determine which of six different Hb mass Adaptive models might be suitable for inclusion in the ABP. The sensitivities and specificities of these models were compared in a sample of 159 non-doped and 18 doped athletes. In models that used Hb mass as a single marker, the sensitivity and specificity of the model was heavily influenced by the estimate of Hb mass biological within-subject (BioWS) variance included in the calculations. These models were each named after the first author of a publication in which the BioWS variance of Hb mass in athletes was estimated. Due to their low specificities in non-doped athletes, neither of two Hb m (Prommer) models would be suitable for inclusion in the ABP. In contrast, based on specificities close to 100%, any of the Hb m (Pottgiesser), Hb m (Morkeberg) or Hb m (Eastwood) models may be suitable for inclusion, although each model only offered ~20% sensitivity to rhuepo doping. The novel ON hm+ret model, which combined Hb mass and %Ret into a single marker, would not be useful in the ABP due to its low specificity. Overall, the inclusion of Hb mass may improve the sensitivity of the ABP, particularly to microdose rhuepo doping. The sensitivities of the Hb m (Pottgiesser), Hb m (Morkeberg) and Hb m (Eastwood) models should be examined in a larger sample of doped athletes. Unfortunately, these models may be susceptible to recording false-positive results in some extreme cases of Hb mass perturbation in ill or injured athletes. iii

4 Acknowledgements I initially had real doubts about whether a PhD was the right path for me, but with encouragement from the lovely Dan and our friend, Jo Vaile, I embarked on a journey that has enriched me with a level of skill, knowledge and a confidence that I never anticipated. It has been an experience that I will look back on with great fondness and I have many people to thank for getting me to this point. To the lovely Dan. Thank you for being the super boyfriend and now the super husband. Your support for me is unwavering and I couldn t have achieved all that I have over the past few years without you. Everything is more fun when you re there. I love you. Thank you xxx To Mum, Dad, Catherine and Mel. We have a wonderful family and the support you have all given me my whole life has got me to this point and beyond. Thank you for all your love, generosity, and teaching, and for having faith in me to make the right decisions. I m sad that we now live so far apart, but the distance is only physical and I think we are all closer than ever. To my good friend Jo Vaile. I feel so privileged to be your buddy. The encouragement, support, walks, enforced runs, and fun you contribute to my life are all equally important and without you I wouldn t have had the confidence to embark on the PhD in the first place. Thanks! To my fantastic supervisors Prof. Chris Gore, Dr. Judith Anson and Dr. Philo Saunders. The guidance you have given me has been invaluable and I m constantly amazed by the time you v

5 dedicate to helping me. The opportunity to work with you has been a real highlight of the PhD experience and the skills I have learned from you will benefit me for a long time to come. The advice of my Hb mass PhD predecessors, Dr. Laura Garvican, Dr. Eileen Robertson and Dr. Annette Eastwood, has been invaluable and I want to thank you all for giving up your time for me to ask questions, compare notes and help with testing throughout my PhD. To Dr Ken Sharpe, your willingness to advise me, help me investigate the data, and generally explain mathematical concepts to me was invaluable; thank you. To Dr. Mike Ashenden, thank you for being available to discuss my ideas and offer critical bits of advice. To Dr Pierre-Edouard Sottas, thank you for granting me access to the Athlete Biological Passport software and for offering your guidance and advice about its use. To all the athletes who volunteered to take part in my studies, I can t thank you enough. You all participated with enthusiasm and great humour which made the hours of sitting at the OSM3 analysing blood worthwhile. To everyone who helped me with the testing, thank you so much. I couldn t have done it without you: Nicola Bullock, Darrell Bonetti, Laura Garvican, Nicole Prommer, Melissa Arkinstall, Kiara Johnson, Jesse Featonby, Graeme Albon, Wei Chung, Lizzie Wraith, Peter Fowler, Annette Eastwood, Jo Vaile and Dean Higham. To Maz, thanks for all your work behind the scenes, for the hugs and encouragement and for all times you said, You know how I like to tell you.... Finally, the attitude towards excellence demonstrated by all my colleagues at the Australian Institute of Sport is infectious and AIS Physiology will hold a cherished place in my heart for a long time to come. vi

6 Declaration In this thesis I detail the findings from research carried out between January 2009 and May The research studies described in Chapters 3, 4, 5, 6, 7 and 8 were carried out in collaboration with my co-authors, the names of whom are listed at the start of each chapter. For each of these studies I took a lead role in the experimental design, subject recruitment, data collection and analysis, and I wrote the manuscripts. Note, I got married in August 2011 and subsequently I changed my name from Clare E. Gough to Clare E. Humberstone. Consequently, I used my maiden name for the submission of publications from this thesis, whilst for this dissertation I have used my married name. vii

7 Publications and Presentations relevant to this thesis Peer-reviewed journal articles Gough C.E., Saunders P.U., Fowlie J., Savage B., Pyne D.B., Anson J.M., Wachsmuth N., Prommer N. and Gore C.J. Influence of altitude training modality on performance and total haemoglobin mass in elite swimmers. European Journal of Applied Physiology [Epub ahead of print]. DOI: /s Gough C.E., Sharpe K., Ashenden M.J., Anson J.M., Saunders P.U., Garvican L.A., Bonetti D.L., Gore C.J. and Prommer N. Quality control technique to reduce the variability of longitudinal measurement of hemoglobin mass. Scandinavian Journal of Medicine and Science in Sports (6): e DOI: /j x. Gough C.E., Eastwood A., Saunders P.U., Anson J.M. and Gore C.J. Spurious Hb mass increases following exercise. International Journal of Sports Medicine (In press). Gough C.E., Sharpe K., Garvican L.A., Anson J.M., Saunders P.U. and Gore C.J. The effects of injury and illness on haemoglobin mass. (Submitted to International Journal of Sports Medicine in March 2012; In Review) Gough C.E., Saunders P.U., Bonetti D.L., Stephens S., Bullock N., Anson J.M. and Gore C.J. Comparison of Acute Intermittent Hypoxic Exposure and Live High:Train Low altitude. (Submitted to International Journal of Sports Physiology and Performance in January 2012; In Review) viii

8 Ashenden M., Gough C.E., Garnham A., Gore C.J. and Sharpe K. Current markers of the Athlete Blood Passport do not flag microdose EPO doping. European Journal of Applied Physiology (9): Peer reviewed conference proceedings Gough C.E., Bullock, N., Bonetti D.L., Saunders P.U., Anson J., Stephens S. and Gore, C.J. Acute Intermittent Hypoxic exposure is inferior to live high/train low altitude for haemoglobin adaptation. Proceedings of the 15 th Annual Congress of the European College of Sport Science. 2010, p 59. Gough C.E., Eastwood A., Saunders P.U., Anson J. and Gore C.J. Ultra-endurance triathlon racing spuriously increases haemoglobin mass. Proceedings of the 15 th Annual Congress of the European College of Sport Science. 2010, p 364. Conference presentations Gough C.E., Eastwood A., Saunders P.U., Anson J. and Gore C.J. Ultra-endurance triathlon racing spuriously increases haemoglobin mass (Oral presentation). Congress of the European College of Sports Science, Turkey, 2010 Gough C.E., Bullock N., Bonetti D.L., Saunders P.U., Anson J., Stephens S. and Gore C.J. Acute Intermittent Hypoxic exposure is inferior to live high/train low altitude for haemoglobin adaptation (Poster presentation). Congress of the European College of Sports Science, Turkey, 2010 ix

9 Table of Contents ABSTRACT... II CERTIFICATE OF AUTHORSHIP OF THESIS... IV ACKNOWLEDGEMENTS... V DECLARATION... VII PUBLICATIONS AND PRESENTATIONS RELEVANT TO THIS THESIS... VIII PEER-REVIEWED JOURNAL ARTICLES... VIII PEER REVIEWED CONFERENCE PROCEEDINGS... IX CONFERENCE PRESENTATIONS... IX LIST OF TABLES... XVI LIST OF FIGURES... XIX LIST OF ABBREVIATIONS... XXI CHAPTER INTRODUCTION... 1 AIMS... 3 CHAPTER LITERATURE REVIEW... 6 INTRODUCTION... 6 INDIRECT DETECTION OF BLOOD DOPING AND THE ATHLETE BIOLOGICAL PASSPORT PRACTICALITIES OF INCLUDING HB MASS IN THE ABP Time efficiency Quality control between laboratories SOURCES OF HB MASS VARIATION IN NON-DOPED ATHLETES Analytical variability Normal BioWS variance in athletes Additional BioWS variance: Acute effect of exercise Additional BioWS variance: Injury and illness Additional BioWS variance: Training and detraining Additional BioWS variance: Hypoxia SOURCES OF HB MASS VARIATION IN DOPED ATHLETES Blood transfusion x

10 rhuepo doping ABP MODELS INCORPORATING HB MASS Models using Hb mass as a single marker Models combining Hb mass with %Ret SUMMARY CHAPTER QUALITY CONTROL TECHNIQUE TO REDUCE VARIABILITY OF LONGITUDINAL MEASUREMENT OF HAEMOGLOBIN MASS ABSTRACT INTRODUCTION MATERIALS AND METHODS Overview CO re-breathing procedures Quality Control measurements Analysis RESULTS DISCUSSION Limitations Perspectives ACKNOWLEDGEMENTS CHAPTER SPURIOUS HB MASS INCREASES FOLLOWING EXERCISE ABSTRACT INTRODUCTION METHODS CO re-breathing Serum haptoglobin concentration Urine haemoglobin concentration Data analysis RESULTS DISCUSSION CONCLUSION xi

11 ACKNOWLEDGEMENTS CHAPTER THE EFFECTS OF INJURY AND ILLNESS ON HAEMOGLOBIN MASS ABSTRACT INTRODUCTION METHODS Subjects Procedures STATISTICS Normal within-subject variation Additional biological variation RESULTS Normal within-subject variation Additional biological variation DISCUSSION Normal within-subject variation Additional biological variation: Reduced training Additional biological variation: Surgery, body mass, iron supplementation and altitude Application to anti-doping CONCLUSION ACKNOWLEDGEMENTS CHAPTER INFLUENCE OF ALTITUDE TRAINING MODALITY ON PERFORMANCE AND TOTAL HAEMOGLOBIN MASS IN ELITE SWIMMERS ABSTRACT INTRODUCTION METHODS Parallel groups trial Swimming performance Blood testing Training load xii

12 Season-long comparison Statistical analysis RESULTS Race performance Hb mass and haematology Training DISCUSSION Comparison of Classic and LHTL Time course of performance changes Season-long comparison Limitations CONCLUSIONS ACKNOWLEDGEMENTS CHAPTER COMPARISON OF ACUTE INTERMITTENT HYPOXIC EXPOSURE AND LIVE HIGH:TRAIN LOW ALTITUDE ABSTRACT INTRODUCTION METHODS Study design Hypoxic exposure Blood parameters Incremental treadmill test Participation variations Statistical analysis RESULTS Blood parameters Running parameters DISCUSSION Limitations CONCLUSIONS ACKNOWLEDGEMENTS xiii

13 CHAPTER DOES THE INCLUSION OF HAEMOGLOBIN MASS IN THE ATHLETE BIOLOGICAL PASSPORT IMPROVE DETECTION OF MICRODOSE RHUEPO DOPING? ABSTRACT INTRODUCTION METHODS Participants Study design Haemoglobin mass estimation Venous blood analysis CALCULATIONS AND STATISTICS Haemoglobin mass models RESULTS DISCUSSION Limitations CONCLUSION ACKNOWLEDGEMENTS CHAPTER SPECIFICITY OF HAEMOGLOBIN MASS IN THE ATHLETE BIOLOGICAL PASSPORT INTRODUCTION METHODS Study design Sources of the data Adaptive models Sensitivity and specificity calculations RESULTS DISCUSSION Specificity Sensitivity BioWS variance Test frequency Combination of Hb mass and %Ret xiv

14 Limitations CONCLUSION ACKNOWLEDGEMENTS SUMMARY PRACTICAL RECOMMENDATIONS FUTURE DIRECTIONS REFERENCES APPENDIX ESTIMATES OF BIOWS VARIANCE INCLUDED IN FIVE ADAPTIVE MODELS BASED ON HB MASS 202 STUDY 1: PROMMER ET AL. (2008) STUDY 2: POTTGIESSER ET AL. (2012) STUDY 3: MORKEBERG ET AL. (2011) STUDY 4: EASTWOOD ET AL. (2011B) Male Female xv

15 List of Tables CHAPTER 5 Table 5.1 Table 5.2 Table 5.3 The number of athletes and observations included in the analysis of the effects of reduced training, surgery, altitude and iron supplementation on total haemoglobin mass (Hb mass )...78 Estimates of normal within-subject standard deviation of Hb mass (SD within ) and analytical standard deviation (SD analytical ) in male and female athletes, with 95% confidence intervals, after the influences of reduced training, changes in body mass, surgery, altitude exposure and iron supplementation were removed...82 Effects on Hb mass of reduced training, surgery, body mass, altitude and iron supplementation, with 95% confidence intervals Table 5.4 Effects on Hb mass of categorised decreases in current cardiovascular training intensity (ccvi), with 95% confidence intervals CHAPTER 6 Table 6.1 Subject characteristics...96 Table 6.2 Percent changes in swimming performance from pre-training to 1, 7, 14 and 28 days after 3 weeks of Classic altitude, LHTL altitude or sea-level (Race Control) training Table 6.3 Percent difference in the change in swimming performance from pre-training to 1 and 7 days after Classic altitude or LHTL altitude compared to Race Control xvi

16 CHAPTER 7 Table 7.1 Table 7.2 Physical characteristics of participants at baseline Percent change in blood and running parameters after either Live High:Train Low (LHTL) altitude training, Intermittent Hypoxic Exposure (AcIHE), or Placebo Table 7.3 Percent difference in the changes in blood and running parameters after either Live High:Train Low altitude training (LHTL) or Intermittent Hypoxic Exposure (AcIHE) compared with Placebo CHAPTER 8 Table 8.1 Number of athletes flagged by Hb m (Prommer - small), Hb m (Prommer - large), Hb m (Pottgiesser), Hb m (Morkeberg), Hb m (Eastwood) and ON hm+ret models during 12 weeks of a recombinant human erythropoietin (rhuepo) or Placebo microdosing regimen CHAPTER 9 Table 9.1 Table 9.2 Description of studies included in the analyses of six Adaptive models that include Hb mass The sensitivities and specificities of six Adaptive models at the 99% and 99.9% levels, incorporating separate analyses for the inclusion of all test results and for the inclusion of only results separated by at least 21 days xvii

17 List of Figures CHAPTER 2 Figure 2.1 Methods to increase oxygen delivery to the tissues by direct action on haemoglobin...7 Figure 2.2 Figure 2.3 Screen shot from the ABP software of a male subject engaged in autologous blood doping...14 Percent change in Hb mass after 5, 11 and 13 weeks of rhuepo...33 CHAPTER 3 Figure 3.1 %HbCO of High and Low quality controls measured on 16 occasions over a 22-day period using a separate OSM3 analyser in each of laboratories A, B and C...51 Figure 3.2 The difference between high and low quality controls (Δ%HbCO) measured on 16 occasions over a 22-day period using a separate OSM3 analyser in each of laboratories A, B and C CHAPTER 4 Figure 4.1 Scatter plot showing individual values for percent change in (a) Hb mass and (b) [Hap] for Racers and Controls...66 CHAPTER 6 Figure 6.1 Figure 6.2 Schematic timeline of study design...97 Percent changes in total haemoglobin mass (Hb mass ) from pretraining to 1 and 14 days after 3 weeks of Classic altitude or xix

18 LHTL altitude Figure 6.3 Training volume, intensity and load during three weeks (Alt 1, Alt 2, Alt 3) of Classic and LHTL training CHAPTER 7 Figure 7.1 Outline of the study design illustrating the sequence of carbon monoxide (CO) re-breathing tests, incremental treadmill tests and venepuncture blood sampling CHAPTER 8 Figure 8.1 Percent changes in haemoglobin mass (Hb mass ) and percent reticulocytes (%Ret) over 12 weeks of a microdose recombinant human erythropoietin (rhuepo) or Placebo regimen Figure 8.2 Exemplar graphs of two athletes, rhuepo subject #2 and Placebo subject #5, illustrating the differences between two models, Hb m (Prommer - small) and Hb m (Eastwood), in the expected ranges and the incidence of flagged results CHAPTER 9 Figure 9.1 An exemplar graph depicting the Hb mass results of one athlete from the injury/illness study along with 99% and 99.9% limits from the Hb m (Pottgiesser) model xx

19 List of Abbreviations ABP AcIHE BioWS variance Classic CI CL CO CV ccvh ccvi ctth CO 2 EPO ESAs [Ferr] FINA FIS [Hap] Hb Athlete biological passport Acute intermittent hypoxic exposure Biological within-subject variance Classical altitude training Confidence interval Confidence limits Carbon monoxide Coefficient of variation Current cardiovascular training hours Current cardiovascular training intensity Current total training hours Carbon dioxide Erythropoietin Erythropoiesis-stimulating agents Serum ferritin concentration Federation Internationale de Natation Federation Internationale de Ski Serum haptoglobin concentration Haemoglobin xxi

20 [Hb] Hb mass Hct Haemoglobin concentration Haemoglobin mass Haematocrit HIF-1 Hypoxia inducible factor 1 HR IPS [Lac] LHTL ln Mb O 2 %Ret rhuepo RPE SD SpO 2 [STfR] SWC TE TRIMP TTE Heart rate International point score Blood lactate concentration Live High:Train Low altitude training Natural-log transfromed Myoglobin Oxygen Percent reticulocytes Recombinant human erythropoietin Rating of perceived exertion Standard deviation Peripheral oxygen saturation Serum soluble transferrin receptor concentration Smallest worthwhile change Typical error Training impulse Time to exhaustion xxii

21 U23 UCI VO 2 VO 2peak Under-23 Union Cycliste Internationale Volume of oxygen consumed Peak oxygen consumption vvo 2peak WADA Velocity at VO 2peak World Anti-Doping Agency xxii

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