Fig 1.
Time lines showing blood donation and re-infusion, blood sampling and VO2max testing from Part A of the Study and B of the study. In total, 19 men and ten women have participated, and 430 blood samples were taken (257 in Part A of the Study and 173 in Part B of the Study). D; Donation of one unit (450 mL) blood (for women in Part B of the Study at Week 3 only). R; Re-infusion of RBC. T; Testing of physical performance. ▲Clean samples from the Transfusion group; ■ Clean samples from Control group; ●Samples after RBC re-infusion.
Table 1.
Hematological variables analyzed in blood samples.
Table 2.
Subject characteristics (Part A of the Study).
Table 3.
Subject characteristics (Part B of the Study).
Fig 2.
Change in performance and VO2max (mL·min-1) with blood donation and RBC re-infusion in Part A of the Study.
Data is presented as percent change from baseline, with a line connecting means. The graph shows the effects of blood donation and RBC re-infusion on repeated testing for time to exhaustion and VO2max in the Transfusion group, and changes over time with repeated testing only in the Control group. For effects of blood donation baseline was set to Week 0, and changes in data on VO2max and time to exhaustion calculated to 48 h and 16 weeks after donation. For effects of RBC re-infusion baseline was set to Week 16. MANOVA demonstrated a difference over time between groups, and differences between groups at each time point (oneway ANOVA, p<0.1) are indicated by brackets. Changes at week seven is not shown, and results from tests weeks 19–21 collated. Control as dashed line (-—-), Transfusion as solid line (—). Difference between groups over time investigated by repeated measures MANOVA was significant (F = 12, p-value by Greenhouse-Geisser ε = 0.03), and one-way ANOVA at each time-point subsequently calculated.
Fig 3.
Individual blood [Hb] (g·L-1). Results from analysis of 161 blood samples from transfused men (N = 11, Nr 1–17) in Panel A, and 93 samples from control men in Panel B (N = 7, Nr 9–19). All samples were taken in parallel during 22 weeks (Fig 1). Transfused subjects donated two units (900 mL) blood and were re-infused with the washed, cryopreserved RBC fraction at week 17 (Fig 1). Control subjects were sampled and tested at the same time intervals, except no sample was taken at one hour before and after the RBC reinfusion. For reference, a dashed black line at [Hb] ± 15% from the first sample (noted as Clean in both groups) is suggested to be the individual upper and lower limits for indication of autologous blood doping [41]. Upper limit for [Hb] set at 170 g·L-1 for men by some sport federations such as FIS, is indicated by dash-dot purple lines.
Fig 4.
Individual blood reticulocyte count per RBC (%). Results from analysis of 161 blood samples from transfused men (N = 11, Nr 1–17) in Panel A, and 93 samples from control men in Panel B (N = 7, Nr 9–19). All samples were taken in parallel during 22 weeks (Fig 1). Transfused subjects donated two units (900 mL) blood and were re-infused with the washed, cryopreserved RBC fraction at week 17 (Fig 1). Control subjects were sampled and tested at the same time intervals, except one hour before and after the RBC reinfusion. For reference, a dashed black line at 0.5% and 2% is suggested to be the individual upper and lower limits for indication of autologous blood doping [60].
Fig 5.
Individual blood OFF-hr score. Results from analysis of 161 blood samples from transfused men (N = 11, Nr 1–17) in Panel A, and in Panel B 93 samples from control men (N = 7, Nr 9–19). All samples were taken in parallel during 22 weeks (Fig 1). Transfused subjects donated two units (900 mL) blood and were re-infused with the washed, cryo-preserved RBC fraction at week 17 (Fig 1). Control subjects were sampled and tested at the same time intervals, except one hour before and after the RBC reinfusion. For reference, a dashed black line at OFF-hr = 129 (OFF-hr = ) is one suggested limits for indication of blood doping with EPO [47] but used by others for investigation of blood manipulation in general.
Fig 6.
Individual blood [Hb] (g·L-1) from ten men and nine women during a ten week period before and after autologous blood transfusion. One (women) and two (men) units (450 mL each) whole blood was withdrawn, with re-infusion of the washed, cryo-preserved RBC fraction five weeks later (Fig 1). A total of 190 samples are included from women (N = 80) and Men (N = 110). Over time, samples are group into Clean (N = 57), Donation (N = 19) and Doped (N = 114. Upper limit for [Hb] set by some some sport federations, such as FIS, for women at 160 g·L-1 and for men a 170 g·L-1 is indicated by dash-dot purple lines. ▲(green) Clean (triplicate samples), ■ (blue) After blood donation, ● (red) After RBC re-infusion.
Fig 7.
Part A of the Study and Part B of the Study. PCA overview comparing scores scatter plot of Control, Clean, Donated and Transfused and subjects. Observations close to each other are more similar than observations distant from each other. Each marker represents the sum of all hematological variables. Panel A is data from 323 blood samples taken from the Control and Transfusion groups, over a period of 21 week (Fig 1). A PCA analysis including all data from nine hematological variables (Table 5) with N = 323 blood samples and X = 2272 data points (individual hematological data) indicated a separation between Control and Transfusion groups (R2X = 0.56; Q2 = 0.38; significant by R1). Panel B is data from 210 blood samples taken over 10 weeks (Fig 1) where Clean samples are taken before blood donation (green squares), Post donation samples are indicated by blue squares and Doped samples by red circles. R 2 = 0.60, and Q 2 = 0.31. Clean samples from the Control group (▲); Transfusion group before donation (■), Transfusion group after donation (■), Transfusion group after RBC re-infusion (●). Ellips indicates 95% CI.
Fig 8.
Part A of the Study and B. OPLS X/Y overview plot of the effect of blood donation and RBC re-infusion over time. The OPLS X/Y overview plot displays the individual cumulative Regression (R2) and Prediction (Q2) for every time point of blood sampling in Part A of the Study and B. Part A of the Study: R2X = 0.60; Q2 = 0.04. Part B of the Study: R2X = 0.49; Q2 = -0.01. Neither part (A and B) of the study reached a significant separation of Clean and Doped subjects based on hematological variables. D; Blood donation, R; RBC re-infusion.
Fig 9.
An OPLS-DA analysis at 48 h after blood donation. A) Scores Scatter plot showing separation of Control and Transfusion groups 48 h after blood donation. This plot shows how the modeled observations in X space are situated with respect to each other. Observations close to each other are more similar than observations distant from each other. Regression (R 2) = 0.76, and prediction by cross-validation (Q 2) = 0.59. B) VIP rank for included variables. C) Misclassification table showing one incorrectly predicted Transfused (Subject H, circled in red) to the Control group (p < 0.001, Fisher’s exact probability test).
Fig 10.
An OPLS-DA analysis at 48 h after blood re-infusion. A) Scores Scatter plot showing separation of Control and Transfusion groups 48 h after RBC re-infusion. Observations close to each other are more similar than observations distant from each other. Regression (R 2) = 0.40, and prediction by cross-validation (Q 2) = 0.01. B) VIP rank for included variables. C) Misclassification table showing one incorrectly predicted Transfused (Subject T) to the Control group, and one Control (Subjects L), predicted as Transfused (both circled in red; p = 0.004, Fisher’s exact probability test).
Table 4.
Physical performance and maximal aerobic power as an effect of blood transfusion (Part A of the Study).
Table 5.
Hematological variables.
Table 6.
Hematological variables.
Table 7.
Misclassification table.
Table 8.
Misclassification table.