The measured mean decrease of Hb mass after donation was 89 ± 16 g in group I and 120 ± 14 g in group II. After the reinfusion, mean Hb mass increased significantly compared with after donation (group I: 70 ± 16 g; group II: 90 ± 9 g) but was slightly lower than at baseline (group I: −19 ± 17 g; group II: −30 ± 14 g). The TEM calculated from two consecutive measurements for each time point is displayed in Table 1 and ranged between 0.8% (6.4 g of Hb) and 3.1% (22.1 g of Hb) in group II for the after donation measurements. The individual differences between measured and calculated (expected) Hb mass changes are presented in Table 2. It was observed that Hb mass determination lead to under- as well as overestimation of the calculated (expected) Hb mass change. A Bland-Altman test did not reveal significant differences between measured and calculated Hb mass when comparing after donation versus baseline and after reinfusion versus after donation in both groups.
Changes in vascular compartments.
The changes of the vascular compartments are shown in Table 3. When summarized to the three time points, the greatest changes were observed in RCV, which was 13% lower after donation than at baseline in group I, and 16% lower in group II. RCV was lower after reinfusion compared with baseline in both groups (group I, −4%; group II, −6%), although the changes were only significant in group I. In contrast to RCV, PV was not significantly decreased after donation in neither of the groups. In group I, PV was significantly higher after reinfusion than after donation (+11%), and at baseline (+9%), no significant changes of PV were observed in group II. In summary, in both groups the alterations in BV mainly resulted from the changes in RCV.
Venous parameters and indirect detection models.
The results are summarized in Table 3. [Hb] and Hct decreased significantly in both groups after blood donation. The serum level of EPO had a significant mean 2.4-fold increase in group I and a 2.5-fold increase in group II. At the same time, reticulocytes did not change after donation in group I and increased in group II.
After reinfusion, [Hb] increased by 8.5% in group I and 9.6% in group II compared with after donation, and the indirect parameters almost returned to baseline levels in group I, whereas group II showed significantly lower levels of [Hb] and Hct after reinfusion compared with baseline. Additionally, the reticulocyte count was significantly higher after reinfusion than at baseline in group II. EPO serum levels did not reveal significant differences after reinfusion compared with baseline levels. The highest scores of the indirect models observed were those at baseline; for instance, OFF-hr at baseline was 100.2 in group I and 101.6 in group II. In summary, none of the scores exceeded the thresholds suggested for the indirect detection of blood manipulations.
The major finding of this study is that the withdrawal of whole blood and reinfusion of autologous PRC are detectable using the optimized CO-rebreathing method, because Hb mass changed significantly after these interventions.
Although detection methods exist for doping with rhEPO (16) and homologous blood transfusions (17), autologous blood transfusions remain virtually undetectable. The aforementioned practices target Hb mass as the key variable to improve performance. Measurement of this parameter, as performed in this study with the optimized CO-rebreathing method, would therefore allow monitoring of absolute changes triggered by doping techniques. In this context, two conditions have to be met. On the one hand, Hb mass has to be relatively stable under physiological circumstances so that conspicuous alterations can be ascribed to doping practices. On the other hand, the method for determination of Hb mass must be reliable, with a small error of measurements, and adequately reflect the absolute changes induced by blood manipulations.
Hb mass is altered by endurance training and physical performance, as can be concluded from the fact that endurance-trained subjects usually have an Hb mass of 12 g·kg−1 or above compared with untrained controls, who have approximately 8 g·kg−1 (14,21,23). In contrast, it has been shown that Hb mass is not subject to longitudinal variations in elite athletes who have a history of long-term training, especially those in a professional set-up. In this context, Gore and coworkers (11) have reported that Hb mass did not change after 12 wk of intense rowing training or 4 wk of heat training (32°C). Thus, it can be assumed that, if at all, changes in Hb mass develop over several months of specific training rather than weeks. Other influences such as altitude exposure could potentially lead to variations in Hb mass (2,5,28). Furthermore, it has been shown that athletes originating from altitude have a higher Hb mass than athletes from sea level (23), though we speculate that Hb mass individually is not subject to acute physiological changes in those athletes, analogous to sea level-born athletes.
To evaluate whether the optimized CO-rebreathing method adequately reflects the absolute changes induced by blood transfusion, quantitative assessment has to be performed for validation. Burge and Skinner (6) and Schmidt and Prommer (24) have validated their experiments comparing calculated and measured Hb mass loss induced by phlebotomy. Whereas Burge and Skinner state that measured Hb mass 1 wk after venesection was not different from the calculated estimate (although they do not provide absolute values of the difference of measured and calculated Hb mass), Schmidt and Prommer have calculated the amount of Hb loss according to (550 mL (donation volume) × [Hb]) and have described a mean difference between measured and calculated Hb values of +9 g. The present study has confirmed these results and also has used a reinfusion setting as an additional means of assessing the optimized CO method accuracy and precision when using capillary blood samples. Furthermore, a very thorough approach was chosen to quantify the amount of blood lost by donations, additional venous sampling and the PRC preparation process. In this context, the net and gross sums of Hb mass after donation and after reinfusion were determined. The measurements of Hb mass after donation showed the greatest agreement between calculated (expected) and measured values (see Table 2). From an individual perspective, underestimation as well as overestimation of "true" Hb mass changes by the optimized CO-rebreathing method were observed. This variation was found to be within the limits of the errors of measurement (see Tables 1 and 2). Interestingly, Hb mass was found to be significantly lower after reinfusion than at baseline in both groups. This shows that the optimized CO-rebreathing method has sufficient validity, allowing conclusive results with respect to the Hb mass additionally lost by venous sampling and PRC preparation. Another potential factor in this context might be that some of the Hb did not survive or was not viable after reinfusion.
The reliability expressed by the typical error (15) of 0.8-3.1% for each pair of measurements (capillary sampling, 95% CI) is in line with the previously published reliability measures of Schmidt and Prommer (24) of 1.9-2.0% (venous sampling, 95% CI) and the values described in the meta-analysis by Gore et al. (12) between 1.4 and 3.5% (CO rebreathing as described by Burge and Skinner (6), 90% CI).
The administered units of PRC contained, on average, either 59 g (one unit) or 108 g (two units) of Hb. On the basis of the TEM of 0.8-3.1% (12-43 g of Hb mass (95% CI)), the infusion of one unit of blood might be problematic to detect in an individual if only one baseline measurement is available. If the TEM is small (e.g., 0.8% (smallest TEM in our study)), the likelihood of reliable detection is high. On the other hand, the amount of Hb in two PRC distinctly exceeds the above mentioned range and therefore, in an antidoping setting, the infusion of two units would trigger changes in Hb mass that would allow identification by the current method. In this context, previous literature suggests that an increase in aerobic capacity (e.g., an improvement of V˙O2max by 3-5%) is only achieved if the equivalent of two units of blood are infused (see (10) for a concise review of the subject). Additionally, lay press articles and current informal opinion report that the amount of blood misused for doping practices is believed to be about 800 mL. Nevertheless, it seems feasible that abusers infuse only one PRC, which might also enhance physical performance with a smaller, but nevertheless profitable, effect that would be difficult, if not impossible, to assess using the optimized CO-rebreathing method.
A possible strategy to reveal autologous blood doping could be the long-term screening of Hb mass and calculations of the 95% confidence and 95% tolerance intervals, especially because a single baseline measurement might not be suited to reveal autologous transfusion with the next Hb mass determination. Suspicious alterations could be defined as values that exceed those intervals. In any case, Parisotto et al. (18) have shown that rhEPO administration significantly increased Hb mass by 6.9-12%, corresponding to a mean Hb mass of about 58-107 g. Thus, the same approach of long-term Hb mass screening may unmask other blood-doping procedures such as homologous transfusion and administration of rhEPO for which further detection methods exist (16,17). Because it is possible to maintain elevated [Hb] with microdose rhEPO administration after high-dose EPO injections, which reduces the window of urine detection to as little as 12-18 h (1), irregularities might be revealed by repeated Hb mass measurements.
It may seem to be a limitation that the time span between donation and reinfusion of 2 d does not reflect possible doping practice, where that range is most likely 4-6 wk. However, the aim of this study was to determine whether the absolute changes in Hb mass caused by blood donation and reinfusion might be identified using the optimized CO-rebreathing method regardless of the pre-reinfusion values.
As a final point, practical approaches to detect autologous transfusion by indirect indicators are discussed. First models were already published in 1987 by Berglund et al. (3), and further strategies were recently described by Damsgaard et al. (9). Berglund et al. (3) conclude that, after reinfusion of 1350 mL of blood 4 wk after withdrawal, a combination of measurements of Hb, bilirubin, iron, and erythropoietin in serum could detect 50% of the blood-doped athletes by a single test sample during the first week after reinfusion. In the study of Damsgaard et al. (9), withdrawal and reinfusion included a partly larger quantity of blood compared with that in our study, which triggered [Hb] changes above normal variation (26), suggesting autologous blood manipulations if variation in [Hb] exceeds 15% between samples obtained shortly before any major competition. The results of our study are in accordance with those investigations: [Hb] increased after reinfusion versus after donation by 8.5% in group I and 9.6% in group II compared with a 14% increase after reinfusion in Berglund et al.'s work. Although rapid alterations in EPO serum levels could be demonstrated after donation and after reinfusion (2.4- to 2.5-fold change) in our investigation, the study period was probably too short to induce changes in the reticulocyte count. Additionally, our results were in line with Damsgaard et al. in reference to the OFF-hr model by Gore et al. (13), because none of the subjects demonstrated positive OFF-hr scores. However, a critical appraisal of these presently used variables is necessary. The main weakness is the fact that they are measures based on concentrations and, thus, are highly affected by fluctuations in plasma volume, such as those induced by altitude, posture, hydration status, and exercise (20,22,25). For most erythropoietic manipulations, [Hb] and Hct return to baseline after the vascular compartments (plasma volume) have adapted to newly increased red cell volume. Therefore, this approach based on concentration related variables probably yields a high number of false-negative blood tests, and determination of RCV or Hb mass themselves would be the most sensitive parameters in this context.
In summary, this investigation supports the potential of Hb mass measurement as a screening tool for blood doping, with certain limitations. Changes in Hb mass induced by artificial blood loss and subsequent reinfusion can be detected using the optimized CO-rebreathing method. In this context, assessment of method precision and knowledge of the physiological variation are of utmost importance. Especially in longitudinal settings, Hb mass measurements could unveil suspicious constellations. Further studies are needed to assess the individual variability of Hb mass and the impact of other biological factors, such as age and training status, to improve the differentiation between artificially induced variation and physiological variability, as well as detection of supraphysiological elevation in Hb mass.
The authors are indebted to the subjects who participated in the study. The authors do not have a professional relationship with companies or manufacturers that may benefit from the results of this study.
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Keywords:©2007The American College of Sports Medicine
EXERCISE; HEMOGLOBIN MASS; CARBON MONOXIDE REBREATHING; BLOOD DOPING; ANTIDOPING