B L O O D D O NO OR RI GS IANNADL B AL RO TO ID C CLOEL L E C T I O N Time course for the recovery of physical performance, blood hemoglobin, and ferritin content after blood donation Andreas K. Ziegler,1 Johannes Grand,1 Ida Stangerup,1 Hans J. Nielsen,2,3 Flemming Dela,1 Karin Magnussen,4 and Jørn W. Helge1
BACKGROUND: It is widely accepted that blood donation negatively affects endurance performance, but data on physical recovery after a standard blood donation are scarce. This study aimed to elucidate the temporary impact of blood donation on endurance performance, measured as peak oxygen uptake (VO2peak) and time trial (TT) performance. STUDY DESIGN AND METHODS: VO2peak, TT performance, blood, iron, and anthropometric variables were determined before (baseline) and 3, 7, 14, and 28 days after blood donation in 19 healthy men. RESULTS: VO2peak was reduced by 6.5% from 49.7 ± 2 mL/kg/min at baseline to 46.3 ± 2 mL/kg/min on Day 3 (p < 0.001), and TT performance was reduced by 5.2% from 13:31 ± 00:42 to 14:13 ± 00:50 min:sec (p < 0.001). Both VO2peak and TT performance were back to baseline 14 days after blood donation. Blood hemoglobin (Hb) concentration declined 7.9% from 9.3 ± 0.11 mmol/L at baseline to 8.6 ± 0.1 mmol/L on Day 3 (p < 0.001) and was not different from baseline 28 days after blood donation. The hematocrit (Hct) was reduced from 43.8 ± 0.5% at baseline to 40.6 ± 0.6% on Day 3 (p < 0.001). On Day 28 Hct was 42.8 ± 0.5% and still reduced below baseline (p = 0.028). Ferritin concentration was reduced 46% from 113 ± 23 μg/L at baseline to a minimum of 61 ± 14 μg/L on Day 14 (p = 0.008). CONCLUSION: The individual recovery was variable, but physical performance was recovered 14 days after a standard blood donation, despite blood Hb concentration remaining lower than at baseline.
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n 1954, Balke and colleagues1 showed that maximal oxygen uptake was reduced 9% immediately after donation of 500 mL of blood and that full recovery of maximal oxygen uptake was seen already 3 days after blood donation. Subsequent studies have extended this finding and demonstrated a negative correlation between blood loss and endurance performance and a recovery of endurance performance that spans from 3 to 28 days after blood donation.1-5 In several studies a phlebotomy of 800 to 1200 mL of blood was used to study the influence of blood loss and only few studies have investigated the impact of a standard 450-mL blood donation on endurance performance.1,3,6,7 Furthermore, most of these studies did not thoroughly investigate the recovery of endurance performance and maximal oxygen uptake in the weeks after the blood donation. In addition, Brownlie and colleagues8 demonstrated that the iron status of an
ABBREVIATIONS: P-2,3-DPG = plasma 2,3-diphosphoglycerate; P-EPO = plasma erythropoietin; P-iron = plasma iron; S-SHBG = serum sex hormone–binding globulin; S-testosterone = serum testosterone; TT = time trial; VO2peak = peak oxygen uptake. From the 1Center of Healthy Aging, Department of Biomedical Sciences, and the 3Institute of Clinical Medicine, University of Copenhagen, Copenhagen, Denmark; the 2Department of Surgical Gastroenterology, Hvidovre Hospital, Hvidovre, Denmark; and the 4Department of Clinical Immunology/Blood Centre, Copenhagen University Hospital, Rigshospitalet/ Hvidovre Hospital, Copenhagen, Denmark. Address reprint requests to: Jørn Wulff Helge, Department of Biomedical Science, Faculty of Health Science, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark; e-mail:
[email protected]. The study was supported from AP Møller Foundation (Fonden til Lægevidenskabens fremme) and the Danish Agency for Culture (Sport Science). Received for publication August 21, 2014; revision received September 17, 2014, and accepted September 17, 2014. doi: 10.1111/trf.12926 © 2014 AABB TRANSFUSION **;**:**-**. 2015;55:898–905. Volume **, ** **
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individual is an important variable for endurance performance and recovery of red blood cell (RBC) mass after blood loss. Based on these data it is entirely possible that the wide range of recovery seen after blood donation is due to both differences in the amount of donated blood and an inadequate control of blood iron status before as well as during the recovery period. Therefore, the main aim of this study was to measure the temporary impact of blood donation on physical performance, measured as peak oxygen uptake (VO2peak) and time trial (TT) performance in physically active men. In addition a secondary aim was to assess the effect of an increased blood hemoglobin (Hb) concentration achieved through an autologous blood reinfusion on physical performance in active men.
MATERIALS AND METHODS Subjects
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Experimental protocol Before enrollment in the study participants were screened for pulmonary and cardiovascular abnormalities, and other conditions, that could complicate the physical test results. At the interview it was emphasized that the subjects during the experimental period should maintain their habitual lifestyle in regard to physical activity and nutrition, to maintain training status. Furthermore subjects were informed not to consume iron supplementation during the experimental period. Before the blood donation 20 subjects performed a bicycle VO2peak test and a 3-km treadmill TT on 2 separate days. After the bloodletting the same tests were repeated on Days 3, 7, 14, and 28 (Fig. 1). All blood donors were invited to donate blood after 90 days in the clinical setting under the normal blood donation schedule. In total 11 of the subjects accepted the invitation from the blood center and in these subjects’ blood data are available after 90 days. A reinfusion protocol, where an autologous reinfusion of 1 unit of RBCs was performed, was also implemented. Eight subjects also volunteered to participate in the reinfusion protocol. The blood donation for the autologous transfusion was done 117 to 205 days after the initial blood donation, as an additional part of the protocol (Fig. 1). Due to an unforeseen event (sickness, not related to the project) one subject was unable to participate in the reinfusion protocol and was replaced by a newly recruited subject (age, 31 years; height, 1.90 m; weight, 86.2 kg; VO2peak, 51.6 mL/kg/min; tissue fat, 22.1%; lean mass, 64.7 kg). In the reinfusion protocol the eight subjects were tested once before blood donation, 29 days after blood donation, and between 18 and 24 hours after reinfusion that was performed 30 days after blood donation (Fig. 1).
Twenty healthy men volunteered to participate in the blood donation study. All subjects were informed about the possible risks and discomfort involved before written consent to participate was obtained. The study was performed according to the Declaration of Helsinki and was approved by the Ethical Committee of Copenhagen Region (H4-2011-097). The participants were recruited through the blood donor register for the Capital Region Blood Center. Inclusion criteria were as follows: active blood donor, regular recreational running at least two times per week, no diagnosed cardiovascular disease, age 20 to 45 years, normal plasma ferritin status of more than 40 μg/L, body mass index from 18 to 30 kg/m, and no use of iron supplements or medications affecting physical performance. In addition a blood reinfusion protocol was performed for eight participants (follow-up), where the same inclusion criteria were used. Since this procedure is characterized as doping in sports the subjects abstained from participating in official competitions during the project and the Danish AntiDoping Agency was notified about the trial and the participating subjects. Before the study, the 3-km TT was tested and validated by eight physically active male subjects (height, 185 ± 2 cm; weight, 78.7 ± 3.3 kg; age, 25.9 ± 1.6 years; range, 22-36 years). Each subject was tested three times spaced at least 1 day apart. The test protocol was reliable and possible for the subjects and the validation yielded a coefficient of Fig. 1. Experimental design for the blood donation protocol (A) and blood reinfusion variance of 1.73%. protocol (B). DXA = dual-energy X-ray absorptiometry scanning. 2
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On the experimental days subjects came to the laboratory during the day (Fig. 1). After a 10-minute rest in upright, sitting position, a venous blood sample was obtained. After this a VO2peak test was performed on a bicycle ergometer (Ergo Metrics 800, Jaeger, Wurzburg, Germany) and pulmonary oxygen uptake, carbon dioxide excretion, and ventilation were measured using an online system (Jaeger Oxycon Pro, Viasys Healthcare, Hochberg, Germany). The VO2peak protocol consisted of a 6-minute 100-W warm-up period followed by incremental load addition of 35 W every minute until the subject was unable to sustain pedaling. Criteria for VO2peak were a leveling off and/or a respiratory exchange ratio of more than 1.20 indicating a substantial anaerobic contribution and with a marked hyperventilation. After the VO2peak test subjects rested for 30 minutes in the sitting position and had free access to water. After this, subjects completed a 5-minute warm-up on a treadmill (Wood Way XL pro WOODWAY GmbH, Weil am Rhein, Germany) at a self-selected pace. After this a self-paced 3-km treadmill TT was performed where subjects were asked to complete the distance as fast as possible.9,10 Before the TT subjects were advised how to perform an optimal TT. The subjects were verbally encouraged during the entire test and the rate of perceived exertion was measured using the Borg scale11 to secure a score of at least 17 before 11⁄2 km. The Borg scale is a measure of subjective exertion ranging from 6 (rest) to 20 (maximal exertion) and 17 is considered as very difficult work. During the TT subjects were blinded for time and speed, leaving heart rate and distance as the only visible variables during the test. Body composition was assessed at baseline and on Day 28 in the blood donation protocol and at baseline and on Day 29 in the reinfusion protocol to control for changes in body composition. This was done using dual-energy X-ray absorptiometry scanning (GE Lunar IDEXA, GE Healthcare, Buckinghamshire, UK). The dual-energy X-ray absorptiometry scanner was calibrated daily with a body composition phantom. Thirty-five days after blood donation 18 subjects performed a treadmill VO2max test. One subject was unable to perform this treadmill test. The protocol consisted of a 5-minute warm-up, followed by an incremental speed addition of 1 km/hr every minute for 3 minutes until reaching an individually determined speed calculated as 80% of the subject’s mean pace on his fastest 3 km TT. Hereafter, a 1% incline was added every minute until exhaustion.
Blood samples and analytical procedures The blood samples were obtained from an antecubital vein and were sampled anaerobically and distributed into tubes containing heparin, EDTA, trasylol-EDTA, or lithium 900 TRANSFUSION Volume 55, April 2015
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heparin. The heparinized samples were immediately analyzed for Hb, oxygen saturation, blood pH, CO2, O2 tension, lactate, and hematocrit (Hct; ABL 815 Flex, Radiometer, Copenhagen, Denmark). For hematologic profiling and ferritin the tubes were put on ice and immediately sent to the department of clinical biochemistry at Copenhagen University Hospital, Hvidovre, and analyzed on a hematology analyzer (Model XE5000, Sysmex Corp., Kobe, Japan). Tubes for plasma erythropoietin (P-EPO) and plasma 2,3diphosphoglycerate (P-2,3-DPG) were centrifuged at 2480 × g, plasma was immediately frozen, and tubes for serum testosterone (S-testosterone) were used to obtain serum that was also frozen at −80°C. In the reinfusion protocol 1 unit of RBCs (245 mL) was stored at the Capital Region Blood Center. The RBCs were produced by the top-and-bottom method, and SAGM was added.12 The quality control revealed that hemolysis after storage at the time of reinfusion was below 0.8%. Thirty days later the subjects were reinfused with the unit of RBCs. The blood reinfusion lasted 1 hour, and all subjects were observed in the hours after the procedure in accordance with Danish clinical guidelines for autologous blood transfusion.13 In the reinfusion protocol a blood sample was obtained before blood donation, 2 days before and 18 to 24 hours after reinfusion; blood sampling and experimental protocol were performed exactly as described above. P-EPO, 2,3-DPG, S-testosterone, and serum sex hormone–binding globulin (S-SHBG) were measured using high-sensitivity enzyme-linked immunosorbent assay (ELISA) kits. For EPO a Elisa kit was used (Quantikine IVD, R&D Systems, Minneapolis, MN). 2,3DPG was measured using a human 2,3-DPG ELISA kit (CUSABIO, Wuhan, China). A testosterone ELISA (IBL International GmbH, Hamburg, Germany) was used for S-testosterone measurements. Finally an SHBG ELISA kit (IBL International GmbH) was applied to determine S-SHBG. The analytical procedures followed the guidelines of the manufacturers.
Statistical analysis The statistics were calculated using computer software (SigmaPLOT, Version 11.0, Systat Software, Inc., San Jose, CA). To assess changes after blood donation a one-way repeated-measures analysis of variance using time as a factor was performed. Multiple comparisons versus baseline and Day 3 were made using the Holm-Sidak method. In the reinfusion study paired t tests were used when comparing Day 0 versus Day 29 as well as Day 29 versus Day 31. In all cases, p values of less than 0.05 was taken as the level of significance. Results are given as mean ± SEM if not otherwise stated. Volume **, ** **
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Oxygen uptake At the initiation of the experiment, subjects had a VO2peak of 49.7 ± 1.7 mL/kg/min (range, 35-63 mL/kg/min) and 3-km TT performance of 13:31 ± 00:42 min:sec. Three days after blood donation the VO2peak was reduced (p < 0.05) by 6.5% and all subjects remained below (p < 0.05) baseline on Day 7, and after 14 days VO2peak was similar to baseline values (Fig. 2A). In the reinfusion protocol VO2peak was measured on Day 29 after donation where VO2peak had returned to baseline (51.1 ± 3.0 mL/kg/min vs. 52.1 ± 3.2 mL/kg/min). After reinfusion of blood, VO2peak was increased (p < 0.05) by 4.8%, from 52.1 ± 3 to 54.6 ± 3 mL/kg/min. The treadmill-determined VO2peak (n = 18) was 56.0 ± 1.3 mL/kg/min and thus 12.7% higher (p < 0.05) than VO2peak determined during bicycle ergometer exercise (49.7 ± 1.7 mL/kg/min). In the determination of VO2peak, a leveling off was seen in 12 of 19 tests at baseline, 17 of 19 tests on Day 3, 18 of 19 tests on Day 7, 15 of 19 tests on Day 14, and 18 of 19 tests on Day 28. In all but two tests the respiratory exchange ratio exceeded 1.2 indicating a very good test with a marked anaerobic component and in all VO2peak tests a marked hyperventilation was achieved.
TT TT performance (n = 19) was reduced by 5.2% on Day 3 (Fig. 2B, p < 0.001). On Day 14 after blood donation TT performance was similar to baseline. During the TT all subjects reached a Borg score of 17 before 11⁄2 km in every test performed. In the reinfusion study TT performance on Day 29 was 12:42 ± 00:35 min:sec and not significantly different from baseline 12:50 ± 00:50 min:sec (p = 0.286). On Day 31, the TT performance 12:16 ± 00:43 min:sec was improved (p = 0.038) by 4.6% compared to Day 29. The average VO2peak (Fig. 3A) and the TT performance (Fig. 3B) at baseline and Day 3 in the blood donation study and on 4
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The 20 enrolled subjects were aged 33 ± 2 years (range, 24-45 years), height 182 ± 1 cm, and weight 82.1 ± 2.1 kg. Of the 20 participating subjects, one was excluded due to poor iron status (ferritin, 17 μg/L) and borderline anemia (8.3 mmol/L). The subjects were recreational joggers or participants in recreational sports and jogging and none were elite or competitive athletes. During the experimental period body weight (82.1 ± 2.1 and 81.8 ± 2.0 kg), body fat (21.5 ± 1.7 and 21.0 ± 1.7%), and lean body mass (62.2 ± 1.5 and 62.4 ± 1.5 kg) remained unchanged from baseline to Day 28, respectively.
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Day 29 and Day 31 in the reinfusion study show a positive and a negative correlation between blood Hb and VO2peak and TT performance, respectively.
Blood variables Hb concentration declined 7.9% from baseline to Day 3 (Fig. 2C, p < 0.001). On Day 14, Hb concentration remained 5.9% lower (p < 0.001) than baseline values. On Day 28 after blood donation, Hb concentration was borderline (p = 0.054) significantly reduced compared to Volume 55, April 2015 TRANSFUSION 901
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baseline. Hct was reduced on Day 3 compared to baseline (Table 1, p < 0.001). On Day 28 Hct remained 2.4% lower than values at baseline (Table 1, p = 0.028), but was significantly increased (p < 0.001) compared to the nadir value at Day 3. The ferritin concentration was declined by 46% to a nadir 14 days after blood donation (Table 1, p = 0.008). Twenty-eight days after the blood donation ferritin concentration was still 37% lower (p < 0.05) than Day 0. Compared to baseline ferritin concentration for the 11 subjects tested Day 90 remained significantly reduced (p = 0.025) from 89.8 ± 14.9 μg/L at baseline to 58.3 ± 9 μg/L 90 days after blood donation corresponding to a 35.1% reduction. Plasma iron (P-iron) concentration was reduced (p < 0.05) by 19% on Day 3 compared to baseline values. The P-iron concentration reached a minimum on Day 7, which was 33% below (p < 0.05) baseline (Table 1). The RBC count decreased by 7.6% from baseline to Day 3 (Table 1, p < 0.001). The RBC count remained below (p = 0.011) baseline values for the duration of the entire study. On Day 28, a significant increase (p < 0.001) versus Day 3 was observed. The reticulocyte count was not significantly increased from baseline to Day 3. On Day 7 the reticulocyte count was increased 44% compared to baseline (Table 1, p < 0.001). On Day 28 no significant difference from Day 3 was observed (p = 0.529). The white blood cell count (WBC) was not significantly decreased after blood donation (Table 1). Three days after blood donation P-EPO concentration had increased by 95.5% compared to baseline (Table 1, p < 0.001) and it remained elevated (p = 0.042) above baseline values until termination of the study on Day 28. The concentrations of testosterone, SHBG, and 2,3-DPG remained unchanged during the course of the study (Table 1). In the reinfusion protocol blood Hb concentration was increased by 6.4% after reinfusion (Table 2, p = 0.005). The RBC count was increased by 5.6% (Table 2, p = 0.009).
TABLE 1. Blood variables measured before and 3, 7, 14, and 28 days after blood donation in 19 healthy men* Variable Hct (%) Ferritin (μg/L) P-iron (μmol/L) P-EPO (mU/mL) P-2,3-DPG (μmol/mL) S-Testosterone (ng/mL) S-SHBG (nmol/L) Reticulocytes (×109/L) RBCs (×1012/L) WBCs (×109/L) MCHC (mmol/L) MCV (fL)
Day 0 (baseline) 43.7 ± 0.6 112.7 ± 22.6 22.7 ± 1.2 7.1 ± 0.6 1.8 ± 0.2 3.9 ± 0.3 20.6 ± 2.4 45.7 ± 3.1 4.9 ± 0.07 6.4 ± 0.3 21.3 ± 0.1 88.4 ± 0.8
Day 3 40.6 ± 0.6† 102.9 ± 21.2 18.4 ± 1.2† 13.9 ± 1.5† 1.9 ± 0.2 3.7 ± 0.3 24.2 ± 4.6 50.1 ± 3.2 4.6 ± 0.07† 6.2 ± 0.3 21.2 ± 0.1 88.6 ± 0.7
Day 7 40.8 ± 0.6† 72.2 ± 16.5†‡ 15.2 ± 1.1† 12.6 ± 1.1† 1.9 ± 0.2 3.8 ± 0.3 23.9 ± 3.2 65.8 ± 4.8†‡ 4.6 ± 0.08† 6.4 ± 0.4 21.2 ± 0.1 88.6 ± 0.7
Day 14 41.2 ± 0.4† 60.5 ± 14.5†‡ 17.6 ± 1.2† 12.0 ± 1.4†‡ 2.1 ± 0.3 3.7 ± 0.3 23.3 ± 5.2 62.1 ± 4.2†‡ 4.7 ± 0.07† 6.1 ± 0.3 21.3 ± 0.1 88.0 ± 0.9
Day 28 42.8 ± 0.6†‡ 82.2 ± 29.9† 19.8 ± 1.4† 8.8 ± 0.9†‡ 1.9 ± 0.2 3.7 ± 0.3 21.4 ± 3.4 48.0 ± 3.3 4.8 ± 0.07†‡ 5.9 ± 0.3 21.4 ± 0.2 88.2 ± 0.8
* Values are mean ± SEM. † p < 0.05 vs. Day 0 (baseline). ‡ p < 0.05 vs. Day 3. MCHC = mean corpuscular Hb concentration; MCV = mean corpuscular volume.
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TABLE 2. Blood variables measured before bloodletting, after 29 days before reinfusion, and 18 to 24 hours after an autologous transfusion of RBCs in eight healthy men* Variable Hb (mmol/L) Hct (%) Ferritin (μg/L) P-iron (μmol/L) P-EPO (mIU/mL) P-2,3-DPG(μmol/mL) S-Testosterone (ng/mL) S-SHBG (nmol/L) Reticulocytes (×109/L) RBCs (×1012/L) WBCs (×109/L) MCHC (mmol/L) MCV (fL)
Day 0 (baseline) 9.0 ± 0.2 43.3 ± 0.9 90.5 ± 9.6 34.2 ± 9.9 NA NA NA NA 45 ± 4.3 4.8 ± 0.09 5.6 ± 0.4 20.9 ± 0.1 89.5 ± 1.1
Day 29 (before reinfusion) 8.7 ± 0.1 41.2 ± 0.6‡ 48.8 ± 5.1 27.2 ± 4.3 7.6 ± 1.1 1.2 ± 0.2 3.7 ± 0.3 22.9 ± 3.7 41.4 ± 4.3 4.6 ± 0.09 6.1 ± 0.8 21.2 ± 0.1 88.8 ± 1.3
Day 31 (after reinfusion) 9.3 ± 0.1† 44.0 ± 0.7† 55.1 ± 6.2 24.1 ± 9.2 5.6 ± 0.7 1.3 ± 0.2 3.2 ± 0.4 20.4 ± 4.1 40.4 ± 4.2 4.9 ± 0.09† 6.0 ± 0.6 21.3 ± 0.1 88.9 ± 1.2
* Values are mean ± SEM. † p < 0.05, Day 31 vs. Day 29. ‡ p < 0.05, Day 29 vs. Day 0. MCHC = mean corpuscular Hb concentration; MCV = mean corpuscular volume; NA = not available.
The Hct was significantly lower (p = 0.046) when comparing baseline to Day 29 values before blood reinfusion. After reinfusion Hct was increased by 6.7% from Day 29 (Table 2, p = 0.005). Blood testosterone, SHBG, 2,3-DPG, or EPO was not significantly changed after blood reinfusion (Table 2).
DISCUSSION In this study we observed that physical performance was recovered 14 days after a standard blood donation despite the slightly lower blood Hb concentration. Furthermore, the observed recovery of physical performance after blood donation was paralleled by an EPO-mediated recovery of RBC mass and no measurable changes in 2,3-DPG. Finally we observed that ferritin concentration was 37% decreased after 28 days and remained 35% decreased compared to baseline after 90 days. The study results also showed that VO2peak was already returned to baseline after 14 days in active male subjects that before the study all had normal iron status as indicated by ferritin values of more than 40 μg/L. There was pronounced interindividual variation, and prior studies that extracted the same volume of blood, equivalent to that of a normal blood donation, have revealed a range of time for recovery of VO2peak between 3 and 21 days.1,6,7 However, in two of these former studies iron status was not controlled before inclusion, and this may have influenced the rate of recovery. In the present data set we observed a wide variation in the recovery of VO2peak and three of 19 subjects had not fully regained VO2peak after 4 weeks, thus implying that more than 4 weeks of recovery after blood donation is necessary if all individuals should be back to baseline values. 6
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The study included a validated 3-km TT performed on a treadmill to evaluate endurance performance and TT performance, again with pronounced interindividual variation, was recovered to baseline values after 14 days. Prior studies used the time to exhaustion during an incremental exercise protocol performed on cycle ergometer or a treadmill to evaluate the recovery of exercise performance after blood donation.1,6 However, this approach is somewhat problematic since the performance in an incremental exercise protocol in addition to being determined by endurance exercise capacity is also determined by the anaerobic capacity of the individual and the aerobic fitness and anaerobic capacity are not always directly associated. A further indication of the validity of the TT in this study is the increased performance observed after reinfusion, which suggest that the higher Hb content and thus higher oxygen-carrying capacity of the blood, at least in part had an impact on the outcome of the test. The improved TT performance and VO2peak after RBC reinfusion are consistent with the reported results in the literature.14-16 It is possible that the reinfusion effects are underestimated since storage of RBCs 30 days before reinfusion may have induced storage lesions in the RBCs that attenuate their functional properties when reinfused.17 It is interesting to note that with the application of a solid and validated test we observed a similar decrease in VO2peak and endurance exercise capacity, as estimated by the TT performance, where former studies found that the endurance exercise capacity was more attenuated than VO2peak (reviewed in Calbet et al.18). In this study the testing on Days 3, 7, and 14 could provide a potential high-intensity training stimulus that may exert a training effect, which would increase the speed of recovery and thus skew our results. However, the recovery of VO2peak was identical for the seven subjects that participated in Volume 55, April 2015 TRANSFUSION 903
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both protocols, thus indicating that the test after 3, 7, and 14 days did not exert a training effect influencing the recovery of VO2peak (data not shown). Interestingly we observed dissociation between physical performance and the recovery of blood Hb concentration that remained borderline significantly reduced on Day 28. The attenuated recovery of blood Hb after blood donation is well known. Already in 1952 Fowler and Barer19 found that recovery of blood Hb in 105 subjects spanned from 18 to 98 days with a mean of 49.6 days. A more recent study found that mean recovery time of total RBC mass after donation of 550 mL of whole blood averaged 36 days (range, 10-59 days),20 and therefore it is unlikely that total Hb mass was already recovered on Day 14 in this study. It thus appears that recovery of total RBC mass and thus a fully restored blood oxygen-carrying capacity is unlikely and therefore it cannot explain the observed dissociation between physical performance and blood Hb concentration. Interestingly the EPO concentration was significantly increased at all time points after the blood donation, and this resulted in a higher reticulocyte content both 7 and 14 days after donation. However, in this study this did not lead to a change in mean corporscular Hb concentration and/or mean corposcular volume, which could have mediated an improved oxygen-binding efficiency and/or deformation ability of the RBCs.21,22 In addition, plasma 2,3-DPG concentration was not significantly changed by the intervention in this study and therefore there is no indication of a higher extraction of O2 from arterial blood due to a rightward shift in the oxygen-binding curve. After blood donation, plasma expansion is composed by two phases, where the first phase is characterized by an acute hypovolemia mostly without major changes in Hct and the second phase is characterized by plasma expansion where the lost blood volume is replaced by water within 24 to 48 hours after blood donation.14,23 This later phase is hallmarked by beginning reticulocytosis, a reduction in both blood Hb and Hct7 as also observed in this study. In this study the Hct remained below baseline for the duration of the entire study, despite the increase in reticulocyte content on Days 7 and 14 and the recovery of blood Hb on Day 28. The blood viscosity was not measured in this study, but since there is a very strong linear correlation between Hct and blood viscosity,24 it is likely that blood viscosity was lower throughout this study. A lower blood viscosity is considered to result in better perfusion of the working muscle due to more effective flow in the microcirculation,25 although the exact mechanism remain unexplained. Overall we cannot directly pinpoint the explanation behind the observed dissociation between performance and blood Hb concentration recovery, but as indicated above the restored performance and VO2peak is most likely mediated by a contribution of the Hb concentration increasing toward baseline values, a lower 904 TRANSFUSION Volume 55, April 2015
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blood viscosity, and a possible blood volume expansion providing a better venous return and higher cardiac output. As expected, we observed a decrease (46%) in ferritin concentration and a decrease in serum iron 14 days after the blood donation, and interestingly ferritin concentration was found to remain 35% reduced 3 months after the blood donation (n = 11). In Denmark the blood donation interval is at least 90 days and the present finding suggests that blood donors without iron supplementation may potentially become iron-deficient by continuous donation. In the general population the prevalence of iron defieciency without anemia in US women of 18 to 44 years of age ranges from 11% to 13% and in male US population approximately 1%.26 This is a significant problem for the women who are active donors. There is evidence that the iron status is a variable that influences physical performance8 and it has been demonstrated that iron supplementation increased performance in 42 nonanemic but iron-depleted women compared to placebo.27 However, in this study all subjects had normal ferritin concentration at inclusion (one subject excluded due to poor iron status) and therefore iron status–related limitations in physical performance are not a likely explanation for the observed changes in TT performance and VO2peak observed in this study. In support of this no correlation between baseline iron status (ferritin) and rate of physical recovery was observed in this study. In conclusion, we demonstrate that average physical performance using a proper validated test is recovered 14 days after a standard blood donation despite the slightly lower blood Hb concentration. The observed positive effect of autologous blood reinfusion and thus higher Hb on endurance performance and VO2peak support the validity of our testing model. Training or physical activity can already be performed the day after blood donation albeit with an attenuated endurance capacity. Overall we recommend that in normal male recreational active blood donors at least 14 days are needed to allow full recovery after blood donation before participation in recreational sports competitions.
ACKNOWLEDGMENTS The skilled technical and logistical assistance of Thomas Nyegaard Beck, Lis Christensen, Lone Visby, Mogens Fenger, Eva Rømer, and Cathrine Hjorth Bachmann is acknowledged. The study was supported from AP Møller Foundation (Fonden til Lægevidenskabens fremme) and the Danish Agency for Culture (Sport Science).
CONFLICT OF INTEREST The authors have disclosed no conflicts of interest. Volume **, ** **
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