To discriminate autologous blood doping procedures from normal conditions, we examined the hematological response to blood withdrawal and reinfusion. We found that biomarkers of erythropoiesis are primarily affected in the anemic period. Therefore, individual variations in [Hb] exceeding 15% between samples obtained shortly before any major competition would be indicative of autologous blood manipulation

Damsgaard, R

González-Alonso, J

Mortensen, SP

Munch, T

Mørkeberg, J

Haematologica

English

Brunel University Research Archive

Letters to the Editor| 1006 | haematologica/the hematology journal | 2005; 91(7)Blood DopingEffects of blood withdrawal and reinfusion onbiomarkers of erythropoiesis in humans:Implications for anti-doping strategiesTo discriminate autologous blood doping proce-dures from normal conditions, we examined thehematological response to blood withdrawal andreinfusion. We found that biomarkers of erythro-poiesis are primarily affected in the anemic period.Therefore, individual variations in [Hb] exceeding15% between samples obtained shortly before anymajor competition would be indicative of autolo-gous blood manipulation.Haematologica 2006; 91:1006-1008(http://www.haematologica.org/journal/2006/07/1006.html)Infusion of red blood cells in man to enhance oxygencarrying capacity of blood is a known practice since the1947.1 The release of recombinant human erythropoietin(rHu-EPO) most likely replaced blood transfusion prac-tices since rHu-EPO was found to be more efficient andeasy to manage by non-medical personnel. In year 2000,a urine test for rHu-EPO became available, which provid-ed a means for detecting its illegal used.2 Recently, ath-letes have been tested positive for allogeneic blood trans-fusion by means of flowcytometry3 indicating a return toformer blood transfusion practices. However, anunequivocal detection method revealing autologoustransfusion is still not available. Since accelerated or inhibited erythropoiesis leads tocharacteristic changes in peripheral blood parameters,irrespectively of the stimulating agent, an indirect detec-tion method of rHu-EPO use based on changes in certainhematological parameters4 has been implemented bysome international sport federations. Despite its use, dataon the effect of blood withdrawal and reinfusion in thesehematological parameters have never been reported infull. We hypothesize that autologous blood manipula-tions produce large variations in currently used biomark-ers of erythropoiesis making individual hematologicalprofiles an effective tool identifying this manipulation.We measured hemoglobin concentration [Hb], hemat-ocrit, reticulocytes, serum EPO and sTfR concentrationsin 10 healthy, male subjects at baseline and after thewithdrawal of 20±3% (1.3±0.2 L; mean±SEM) of the sub-jects’ blood volume on day 0, 1, 3, 7 14, 21 and 28 andafter reinfusion of 0.8±0.1 L of packed RBCs on day 0, 1,3, 7, 14 and 21. To maintain blood volume after bloodwithdrawal, 1.3 L of hydroxyl-ethyl-starch (Voluven®)was infused. All subjects were given daily oral iron sup-plementation (100 mg). [Hb], hematocrit, reticulocyteswas measured using an automatic hematology system(ADVIA 120, Bayer Diagnostics). Serum EPO and sTfRconcentrations were determined by ELISA (R&D systemsInc.). The indirect OFF-hr blood models for rHu-EPOdetection were applied.4 A one-way ANOVA for repeat-ed measurements was used to test for differencesbetween control values and samples taken during theanemic and polycytemic phase.We found that following blood withdrawal, [Hb]remained reduced on average by ~15% for 2 weeks(Table and Figure 1). Accordingly, s-EPO increased 4-foldwithin a day, declining exponentially thereafter.Reticulocyte count increased rapidly by 2.4-fold after 7days, remaining elevated for another 7 days whereassTfR increased by 60% by day 14 and remained elevateduntil 3 days after blood reinfusion. Following blood reinfusion, [Hb] increased acutely by8% returning to the initial baseline value after 2 days. s-EPO remained unchanged whereas reticulocyte countwas reduced by 25%-37% from day 7 to 21. sTfRdeclined progressively after reinfusion. The highest OFF-hr score was 126 at day 2 during the polycytemic periodcorresponding to a 1 in 1000 cut-off threshold. EvidenceTable 1. Hematological parameters after blood withdrawal and reinfusion.Control Blood withdrawal ReinfusionDays C 1 3 7 14 28 0 1 3 7 14 21Hb, g/dL 14.8±0.2 12.8±0.3* 12.4±0.2* 12.8±0.3* 13.2±0.2* 14.3±0.2 15.4±0.3*† 16.0±0.2*† 15.4±0.3*† 14.9±0.3 14.7±0.2 15.0±0.2Hct, % 42.9±0.6 36.3±0.9* 35.5±0.7* 36.9±0.7* 38.9±0.6* 41.9±0.5 45.4±0.8† 47.1±0.5*† 45.2±0.8† 44.0±0.5 43.4±0.5 43.6±0.6ret, % 1.1±0.1 1.6±0.1 1.9±0.1 3.0±0.2* 2.7±0.1* 1.3±0.1 1.3±0.1 1.1±0.1 1.1±0.1† 0.8±0.1*† 0.8±0.1*† 0.9±0.1†s-EPO, 7.9±0.9 33.3±3.6* 28.5±3.6* 20.7±1.9* 14.8±1.4 10.5±0.9 NA 5.4±1.0 NA 9.0±0.9 6.6±0.9 7.8±0.9mIU/mLsTfR, 17.5±1.3 17.2±1.7 19.0±2.5 23.9±3.1 28.3±3.3* 28.4±3.0* NA 33.3±3.7* NA 28.1±2.9* 25.9±2.6 20.9±2.0nmol/LOFF-hr 85±3 53±5 43±5 24±4 33±4 75±3 87±4 96±3 87±3 95±3 90±2 92±3Values are means±SEM for 10 male subjects. Hb, hemoglobin concentration; Hct, hematocrit; ret, reticulocytes; s-EPO, serum erythropoietin;sTfR, soluble transferrin receptors; NA, Not Available. *Significantly different from control (p<0.05). †Significantly different preinfusion value(p<0.05). Letters to the Editorhaematologica/the hematology journal | 2005; 91(7) | 1007 |indicates that the hematocrit within-subject biologicaland seasonal variation in man shows a maximal relativechange of 15% within a 95% confidence interval i.e. achange from 0.42-0.48.5 Data of a wider range of sportsalso show within subject estimates of variance in [Hb] of1.6 g/dL,6 which agree with a10% fluctuation found insoccer players.7 In the present study, however, all 10 sub-jects exceeded the normal variation, with [Hb] rangingfrom 19-39%. Even when the control [Hb] value wastaken as the mean [Hb] value, 9 of 10 subjects exceededtheir individual upper limit based on a 7.5% addition totheir control [Hb] value (range 3.0-11.5%). Importantly,alterations in other hematological biomarkers (s-EPO,reticulocytes and sTfR) were observed at all timesthrough this investigation, suggesting that determinationof these biomarkers could be used as supportive evidencefor erythropoietic manipulations with acute [Hb] increas-es of more than 7.5%.At present, only two blood tests are available. To pro-tect the health of the athlete, World Anti Doping Agencyhas implemented an upper limit of 17.0 g/dL, which – ifexceeded – elicits a no start sanction. However, only onesubject in this study showed [Hb] values higher than 17.0g/dL. When using the OFF-hr model of Gore et al. con-structed to convey the use of rHu-EPO4, we observedthat none of the subjects demonstrated positive OFF-scores, rendering this model ineffective detecting blooddoping.In conclusion, autologous blood procedures induce aclear pattern of accelerated erythropoiesis during the ane-mic period. Within the limitations of the study, it is sug-gested that variations in [Hb] exceeding 15% betweensamples obtained in top ranked endurance athletes dur-ing the anticipated anemic period and shortly before anymajor competition would be indicative of autologousblood manipulation.Rasmus Damsgaard,* Troels Munch,* Jacob Mørkeberg,*Stefan P. Mortensen,* José González-Alonso*°*The Copenhagen Muscle Research Centre, Rigshospitalet,University of Copenhagen, Denmark; °Centre for Sports Medicineand Human Performance, Brunel University, Uxbridge,Middlesex UB8 3PH, UKAcknowledgments: we give special thanks to the volunteer subjects.We also thank José Salado from the Rigshospitalet Blood Bank forhis outstanding help with the storage and handling of blood.This study was supported by the Danish Anti Doping Agency.JG-A. was supported by the Copenhagen Hospital System.Key words: blood doping, red blood cells, reticulocytes,erythropoietin, sTfR.Correspondence: Rasmus Damsgaard, Copenhagen MuscleResearch Centre, section 7652, Rigshospitalet, 9 Blegdamsvej,DK 2200 Copenhagen Ø, Denmark. Phone: international+45.35450441. Fax: international +45.35457634.E-mail: rdamsgaard@cmrc.dkFigure 1 (left).. Hemoglobin ([Hb]), serum erythropoietin, reticulo-cytes and serum transferrin receptors (sTfR) responses during the4 weeks after blood withdrawal of 20% of subjects’ blood volume(1.3±0.2 L) and during the 3 weeks after blood reinfusion of 0.8±2L of packed RBCs. Blood samples were collected before the inves-tigation (Control, C) and on day 0, 1, 3, 7 14, 21 and 28 after bloodwithdrawal and on day 0, 1, 3, 7, 14 and 21 after reinfusion.Horizontal lines illustrate control values. Data are means±SEM for10 subjects. * Significantly different from control (p<0.05). †Significantly different from preinfusion values (p<0.05).171615141312111093.53.02.52.01.51.00.50.0403530252015105040353025201510Hemoglobin(g/dL)Erythropoietin(mIU/mL)Retyculocytes(%)sTfR(nmol/L)Time (days)0 10 20 30 40 50Letters to the Editor| 1008 | haematologica/the hematology journal | 2005; 91(7)References1. Pace N, Lozner EL, Consolazio WV, Pitts GC, Pecora LJ. Theincrease in hypoxic tolerance of normal men accompanyingthe polycythaemia induced by transfusion of erythrocytes.Am J Physiol 1947;148:152-63.2. Lasne F, De Ceaurriz J. Recombinant erythropoietin in urine.Nature 2000; 405(6787):635.3. Nelson M, Popp H, Sharpe K, Ashenden M. Proof of homolo-gous blood transfusion through quantification of blood groupantigens. Haematologica 2003;88:1284-95.4. Gore CJ, Parisotto R, Ashenden MJ, Stray-Gundersen J, SharpeK, Hopkins W et al. Second-generation blood tests to detecterythropoietin abuse by athletes. Haematologica 2003;88:333-44.5. Thirup P. Haematocrit: within-subject and seasonal variation.Sports Med 2003;33:231-43.6. Sharpe K, Hopkins W, Emslie KR, Howe C, Trout GJ,Kazlauskas R, et al. Development of reference ranges in eliteathletes for markers of altered erythropoiesis. Haematologica2002;87:1248-57.7. Malcovati L, Pascutto C, Cazzola M. Hematologic passport forathletes competing in endurance sports: a feasibility study.Haematologica 2003;88:570-81.

Effects of blood withdrawal and reinfusion on biomarkers of erythropoiesis in humans: Implications for anti-doping strategies

http://bura.brunel.ac.uk/bitstream/2438/10439/1/Fulltext.pdf

Effects of blood withdrawal and reinfusion on biomarkers of erythropoiesis in humans: Implications for anti-doping strategies

Abstract

Similar works

Full text

Available Versions

Brunel University Research Archive