Contemporary blood doping—Performance, mechanism, and detection

Blood doping is prohibited for athletes but has been a well‐described practice within endurance sports throughout the years. With improved direct and indirect detection methods, the practice has allegedly moved towards micro‐dosing, that is, reducing the blood doping regime amplitude. This narrative review evaluates whether blood doping, specifically recombinant human erythropoietin (rhEpo) treatment and blood transfusions are performance‐enhancing, the responsible mechanism as well as detection possibilities with a special emphasis on micro‐dosing. In general, studies evaluating micro‐doses of blood doping are limited. However, in randomized, double‐blinded, placebo‐controlled trials, three studies find that infusing as little as 130 ml red blood cells or injecting 9 IU × kg bw−1 rhEpo three times per week for 4 weeks improve endurance performance ~4%–6%. The responsible mechanism for a performance‐enhancing effect following rhEpo or blood transfusions appear to be increased O2‐carrying capacity, which is accompanied by an increased muscular O2 extraction and likely increased blood flow to the working muscles, enabling the ability to sustain a higher exercise intensity for a given period. Blood doping in micro‐doses challenges indirect detection by the Athlete Biological Passport, albeit it can identify ~20%–60% of the individuals depending on the sample timing. However, novel biomarkers are emerging, and some may provide additive value for detection of micro blood doping such as the immature reticulocytes or the iron regulatory hormones hepcidin and erythroferrone. Future studies should attempt to validate these biomarkers for implementation in real‐world anti‐doping efforts and continue the biomarker discovery.


| INTRODUCTION
For more than 5000 years, humans have used substances or methods with the purpose of enhancing exercise performance. 1However, in 1967, the International Olympic Committee introduced the first List of Prohibited Substances and implemented anti-doping testing at the Summer Olympic Games in Mexico in 1968. 1 Today, the World Anti-Doping Agency (WADA) is coordinating the anti-doping movement and developing anti-doping policies with the aim of providing equal opportunities, fair competition and protecting the health of athletes.One of the most well-known doping practices in endurance sports is blood doping, that is, the misuse of certain techniques and/or substances to increase one's red blood cell (RBC) mass, which enables the body to transport more O 2 to muscles and therefore increase performance.
Blood doping is primarily known for the use of blood transfusions, specifically autologous blood transfusion (ABT), that is, where athletes withdraw and later reinfuse their own blood, or recombinant human erythropoietin (rhEpo) injections.These methods have likely been "popular" in endurance sports as they represent relatively easy methods to improve aerobic exercise performance substantially.However, throughout the years, the available tests and their sensitivity for detection of blood doping has improved, which likely has prompted misusers of blood doping to change their behavior. 2Specifically, anecdotal evidence suggests that the blood doping regimes have shifted to utilize smaller amounts of blood doping, referred to as "micro-doses", 3 which in the present review is defined as reinfusing RBCs from less than 450 ml whole blood (i.e., one standard blood bag) or injecting 20 IU rhEpo × kg body weight (bw) −1 or less.Establishing whether micro-doses of blood doping are performance-enhancing and detectable is important for the development of future anti-doping strategies.If micro-dosing is performance-enhancing, it is important for anti-doping organizations to know whether existing detection methods are adequate or whether they should aim at improving these in the future.Accordingly, the aim of the present review is to examine the evidence to determine whether micro-doses of ABT or rhEpo injections are performance-enhancing and is detectable.In addition, the mechanisms responsible for a performanceenhancing effect following blood doping is discussed to improve the physiological understanding of how blood manipulation interacts with exercise capacity.

EFFECTS
In this section, we examine the evidence to determine whether ABT and rhEpo administration can be considered performance-enhancing.In addition, we consider the evidence of whether a micro-dose treatment regime is sufficient for improving endurance performance.

| Autologous blood transfusion
The effect of blood transfusions on the O 2 -carrying capacity of the blood and human exercise performance has been investigated for several decades.In the aftermath of World War II, Pace & colleagues infused 2 L of homologous whole blood, that is, blood from another compatible individual, which increased hematocrit by ~10 percentage points and reduced heart rate at a given submaximal workload when subjects were exposed to simulated altitude. 4n 1960, the first study using ABT reported an increased time-to-exhaustion exercise performance of ~4% after the reinfusion of ~600 ml whole blood. 5Since then, numerous studies have clearly demonstrated an acute, augmented, aerobic exercise capacity following ABT in volumes ranging from 450-3500 ml whole blood both in terms of increased peak oxygen uptake (VȮ 2 peak), time-toexhaustion as well as time trial performance in untrained to well-trained males during various aerobic dominated disciplines, [6][7][8][9][10]46 while a single study determined that reinfusing ~370 ml RBC does not improve repeated sprint ability. 11 or a thorough review on the performanceenhancing effects of ABT independent of dosage, please see elsewhere.12 However, as outlined previously, indications are that micro blood doping may be utilized.Accordingly, in the following, we evaluate the available evidence for the potential performance-enhancing effects after a micro-dose ABT.
Currently, only two studies have investigated the performance-enhancing effects of a micro-dose ABT. 11,13n 2018, the effect of reinfusing 135 ml RBCs was investigated on aerobic endurance performance in nine endurance trained males (mean VȮ 2 peak of ~60 ml × min −1 × kg −1 ) using a randomized, double-blinded, placebo-controlled crossover design. 11In a 650-kcal cycling time trial performed 3 days before and 2 h after the ABT, the mean power output and time to completion improved by 4.7% and 4.4%, respectively, when compared to placebo. 11Thus, the study indicates that even minor increases in RBC volume and O 2 carrying capacity can enhance endurance performance, but the results were debated due to a potential concern of incomplete recovery from the 900 ml phlebotomy performed 4 weeks prior to the reinfusion. 14,15evertheless, the initial findings were re-investigated in a subsequent study on 13 moderately trained individuals (mean VȮ 2 peak of ~58 ml × min −1 × kg −1 ) using a randomized, placebo-controlled double-blinded design. 13Endurance performance was assessed in a 650kcal cycling time trial before phlebotomy of 450 ml whole blood and 2-3 days before as well as 24 h and 6 days after reinfusion of 130 ml RBCs conducted 4 weeks after the phlebotomy.The study validated the initial findings, as the ABT increased mean power output both 24 h and 6 days after reinfusion by 6.4% and 5.6%, respectively, when compared to before reinfusion. 13Importantly, the latter study overcame the potential concern of an incomplete recovery between phlebotomy and reinfusion, as the mean power output following reinfusion also was improved when compared to before the phlebotomy.Collectively, the current evidence clearly shows that as little as 130 ml RBCs is sufficient to increase endurance performance.

| Recombinant human erythropoietin
Upon a successful implementation of rhEpo in Europe and USA in 1987-1989, rhEpo quickly proved useful for increasing the circulating amount of RBCs as well as the [Hb] and arterial oxygen content (CaO 2 ) in patients with end-stage renal anemia. 16However, the physiological effects quickly found resonance among athletes and their physicians, who attempted to glean the benefits of rhEpo as a performance-enhancing drug.This is best exemplified by the multiple Tour de France victor, Lance Armstrong, who in January 2013 admitted to rhEpo misuse among other performance-enhancing drugs throughout his career for which he received a life-long doping sentence. 17 clear physiological rationale for rhEpo administration to improve aerobic performance exists, and at least 20 studies involving >380 un-and well-trained individuals demonstrated improved VȮ 2 peak by 6%-10% after administrating 20-150 IU × kg bw −1 rhEpo for 2-12 weeks. 18vailable studies evaluating closed-end performance tests (e.g., time trial) using subcutaneous injections of 50-80 IU epoetin β × kg bw −1 for 4-8 weeks 19-21 demonstrated a 5%-6% improved 3000 m running performance 19,20 or unaltered cycling time trial performance. 21Additionally, one study found that 20-40 × kg bw −1 epoetin β injected subcutaneously for 7 weeks did not alter repeated sprint ability, which is in line with unaltered repeated sprint ability following increased carboxyhemoglobin levels. 22For a scientific debate on whether rhEpo can improve endurance performance, please see elsewhere. 18,23mportantly, whether micro-doses of rhEpo are performance-enhancing has only been investigated in one recent study with appropriate assessment of aerobic endurance performance.Using a counter-balanced, double-blinded, randomized, placebo-controlled study design in 48 participants (24 females with mean VȮ 2 peak of ~46 ml × min −1 × kg −1 , 24 males mean VȮ 2 peak of ~55 ml × min −1 × kg −1 ), 4 weeks with three weekly injections of 9 IU × kg bw −1 epoetin β administered intravenously in males and females, effectively increased total hemoglobin mass by ~7%, while improving 400kcal cycling time trial performance and VȮ 2 peak by ~4% independent of sex. 24Another study also evaluated the performance-enhancing effect following a microdose treatment of rhEpo following six injections of 9 IU × kg bw −1 epoetin α over 2 weeks, which allegedly increased VȮ 2 peak relative to body weight by ~20%. 25owever, VȮ 2 peak were estimated indirectly by changes in steady-state heart rate during a one-step cycling protocol performed at 100 watts for 6 min, and the results should therefore be interpreted with a large degree of caution indicated by the increase in VȮ 2 peak from 70 to 97 ml × min −1 × kg −1 in one participant, equivalent to one of the highest VȮ 2 peak ever recorded. 26n summary, the available evidence strongly indicates that rhEpo is a performance-enhancing drug with clear effects on specifically aerobic performance.Similarly, micro-doses of rhEpo can improve aerobic endurance performance, but the evidence is limited and should be validated.

| PHYSIOLOGICAL MECHANISMS
In this section, it is considered how rhEpo and ABT increases VȮ 2 peak.Next, it is considered whether rhEpo and ABT improves exercise performance by mechanisms other than increasing VȮ 2 peak.

| Peak oxygen uptake
VȮ 2 peak is defined by maximal cardiac output × maximal arterial-venous O 2 difference.To date, only one study has measured maximal cardiac output in healthy volunteers after subcutaneous epoetin β treatment (~60 IU × kg bw −1 ) over 13 weeks. 27Despite increased VȮ 2 peak, maximal cardiac output was unaffected.Likewise, reinfusion of stored RBCs does not alter maximal cardiac output. 8,9Unaltered maximal cardiac output after rhEpo is in line with the lack of blood volume expansion, despite the increase in red cell volume due to the concomitant reduction of plasma volume after rhEpo. 20,28Likewise, following ABT the total blood volume is largely unaffected. 29Thus, the established potential of blood volume expansion to increase cardiac output 30 appears irrelevant after rhEpo and ABT.However, an increase in blood pressure is evident after rhEpo 27 and possibly also-although less clear-ABT. 8,9An increased filling pressure would be expected to increase cardiac output.However, increased cardiac mechanical work because of increased blood viscosity 31 may explain why the increased filling pressure does not result in increased maximal cardiac output.Although maximal cardiac output is unaltered, it is possible that the distribution of blood flow to the contracting muscles can improve with increased CaO 2 due to a reduced blood flow requirement for meeting the O 2 demand in other tissues.However, the maximal leg blood flow was similar before and after subcutaneous epoetin β treatment (~60 IU × kg bw −1 ) over 13 weeks, but an absolute difference of ~1000 ml × min −1 indicates that minor undetected differences could exist. 27In summary, the rhEpo and ABT-induced increase of VȮ 2 peak cannot be related to a higher maximal cardiac output or an improved blood flow distribution.Therefore, it is also unlikely that smaller doses of rhEpo or ABT reinfusion than previously studied can increase VȮ 2 peak through these mechanisms.
Based on the above, increased maximal O 2 extraction explains how rhEpo and ABT increase VȮ 2 peak.Indeed, O 2 extraction during maximal exercise is increased after rhEpo 27 whereas the effect of ABT has not been directly investigated.However, ABT causes increased CaO 2 8 and unchanged 7,9 or even reduced 32 venous O 2 content and explains why ABT is likely to cause higher O 2 extraction after reinfusion.As such, the primary reason for an increased VȮ 2 peak after rhEpo and ABT is increased O 2 extraction due to elevated CaO 2. In this respect, it is important that even micro-doses of ABT (i.e., 135 ml RBCs 11,13 ) cause increased [Hb] and thereby CaO 2 .

| Exercise performance
Exercise performance is determined by numerous factors, including physiological variables setting the limit for O 2 delivery to contracting muscle groups. 33Some competitive events such as 2000 m rowing 34 and 3-5000 m running 35 are completed at intensities and durations (i.e., 1-6 min) expected to elicit VȮ 2 peak.In these disciplines, an increase of VȮ 2 peak caused by elevated CaO 2 and maximal O 2 extraction will translate directly into a performance gain.A simplified example can illustrate the impact; the world record set in 2020 in 5000 m outdoor track running is currently 12:35.3636 (mm:ss:ms) resulting in an average speed of ~23.8 km × h −1 .The O 2 uptake during running is 37 which for the world record pace corresponds to ~4.6 L × min −1 for a ~55 kg male runner closely resembling the runners VȮ 2 peak. 38With a microdose administration of rhEpo, VȮ 2 peak can be increased ~5%. 24According to the observed linear relationship between running velocity and O 2 uptake 38 micro-dose blood doping has the potential to improve the 5000 m world record by more than 30 s resulting in a new world record below 12 min (~11:57).Yet, many competitions, for example, football matches and road-race cycling, are completed at average intensities below that requiring VȮ 2 peak although maximal demanding periods occur. 39,40In these events, the maximal sustainable O 2 delivery and consumption will be less than VȮ 2 peak but will be one of several performance determining variables.The sustainable aerobic energy provision for any given exercise period is determined by a combination of the central cardiovascular capacity (i.e., maximal cardiac output) as well as peripheral components (e.g., muscle capillary density, mitochondrial volume) and hematological characteristics (e.g., [Hb], pH, O 2 -saturation) as reviewed elsewhere. 33As outlined above, it is well established that rhEpo and ABT can increase VȮ 2 peak.But an increased VȮ 2 peak does not improve the highest non-maximal sustainable O 2 uptake per se.So, what are the mechanism(s) by which ABT and rhEpo exert their performance-enhancing effects in events where VȮ 2 peak is not the sole limiting factor?O 2 delivery to contracting skeletal muscle is closely matched to demand. 41Since the O 2 demand at a given submaximal workload (exercise efficiency) is not improved by rhEpo 24,42 and ABT 13 the O 2 delivery may be expected to be constant after both manipulations.This may, however, not be the case.To illustrate this point, it is important to note that the increased O 2 delivery with increased exercise intensity 41 is not sufficient to counter a fatigue-inducing increase of anaerobic metabolism when intensities surpass ~60% of peak workload.The gradual increase of metabolic stress at even submaximal intensities is clearly indicated by gradual rise of muscular lactate release and augmented blood lactate concentrations even well below peak workload. 43An important aspect in matching O 2 delivery to demand is regulation of blood flow to the contracting muscle groups.When leg blood flow is measured directly during two-legged cycling exercise at increasing intensities, blood flow is often considered to increase linearly with intensity. 43,44However, above ~60% of peak workload the linearity does not continue during two-legged cycling.This phenomenon was described in detail by Mortensen et al in 2005, 44 where it was clear that a deflection from the linear increase in leg blood flow with increased cycling exercise intensity above ~75% of peak power was temporally associated with a similar cardiac output response.Despite the continued linear increase in heart rate with increased intensity, stroke volume did not increase above 80% of peak power and even exhibited a reduction at the highest intensities.This phenomenon was also apparent at constant load intense exercise lasting ~5-7 min where stroke volume became compromised in the final (i.e., last ~1 min) phase of exercise. 44An increased blood pressure as seen during submaximal exercise after rhEpo treatment 45 but not ABT 46 may alter such phenomenon, but that remains speculative.These observations demonstrate that any manipulation able to increase the O 2 delivery during intense exercise, despite the intensity being lower than corresponding to VȮ 2 peak, has a performance-enhancing potential because O 2 delivery is not adequate to cover the energy demand.In this respect, an increased CaO 2 will be able to support exercise at higher intensities in the face of inadequate blood flow if extraction is increased.Indeed, increased [Hb] and thereby CaO 2 after rhEpo 20,28 and ABT 8,11 is associated with increased O 2 extraction at high submaximal exercise intensities as demonstrated after rhEpo treatment. 42It remains unknown whether leg O 2 uptake is affected by ABT but the increase of CaO 2 and extraction 7,9,32 clearly indicates this to be the case.Additionally, when CaO 2 is increased by rhEpo and ABT the O 2 diffusion rate from capillaries to contracting muscles may increase. 47This could improve extraction and in susceptible subjects counter exercise induced arterial hypoxemia.
The direct effect of the ABT-induced augmentation of O 2 availability on muscle metabolism is further supported by altered plasma lactate response to exercise as we have previously reviewed. 12In brief, plasma lactate concentrations are reduced during submaximal exercise after larger ABTs 8 by up to 50% as observed during running at 70% of VȮ 2 peak. 10While this is the case after ABT, we surprisingly found increased plasma lactate levels after subcutaneous epoetin β treatment possibly secondary to the increased norepinephrine levels and reduced dilution space. 42However, a control group was not included, so it cannot be excluded that the findings were erroneous albeit it seems unlikely.Notably, following both a micro-dose of ABT (~130 ml) 13 and rhEpo (intravenous injections of 9 IU × kg bw −1 epoetin β) 24 no effects on blood metabolites or lactate concentrations have been found during submaximal endurance exercise.
Another unexpected finding was that leg blood flow was increased at an intensity ~80% of maximal power output after rhEpo. 42It is possible that leg blood flow can be elevated because the increase of CaO 2 allowed the O 2 demand of other tissues to be met by a lower perfusion and thereby allowing more blood to flow through the contracting muscle groups.In addition, rhEpo increase norepinephrine, 42 which causes general vasoconstriction and therefore may cause a higher fraction of cardiac output to be diverted to contracting tissues.Both effects would allow more O 2 to be supplied to the contracting tissue.
In summary, the increase of CaO 2 appears the most likely explanation for an increased ability to sustain a higher exercise intensity for a given period after rhEpo and ABT, as it enables a higher O 2 extraction in the working muscles and possibly a higher blood flow due to an improved distribution.

| Other mechanisms than increased [Hb]?
It must be acknowledged that other possibilities than an increased [Hb] and CaO 2 may explain the rhEpo and ABT induced performance gains.In 2011, Boning et al. provided an informative critical evaluation of [Hb]'s role in altering VȮ 2 peak after ABT. 31 In the present review, a critical perspective is provided for some of the arguments previously brought forward to question the exclusive role of increased [Hb] as the single physiological factor that can explain the performanceenhancing effect of ABT and rhEpo.One argument is that alterations with ABT and rhEpo does not resemble exercise training induced adaptations normally associated with improved performance.A key example is that endurance training causes a reduction in [Hb] 48 in contrast to the ABT and rhEpo mediated [Hb] increase.However, this does not prove that increased [Hb] is unlikely to improve performance but only demonstrates that not all adaptations to exercise training translate into performance gains.Regarding the apparent counterintuitive reduction of [Hb] with exercise training it is likely a result of increased plasma volume which compensates the training induced reduction in blood pressure. 49,50Thus, the mechanism is different from both ABT and rhEpo, which leaves blood pressure unaltered at rest. 11,13,24,51Another line of reasoning that appears to contradict [Hb] as an important factor for exercise performance is that no clear correlation exist between [Hb] and VȮ 2 peak whereas the correlation between VȮ 2 peak and total hemoglobin mass is strong. 49otably, a strong correlation exists between large-dose ABT induced changes in [Hb] and the simultaneous change in VȮ 2 peak, 6,8 while the relationship remains unknown for micro-doses.However, it is irrefutable that the mean [Hb] is increased whenever performance is increased by micro-doses of rhEpo or ABT, 11,13,24 indicating that a similar relationship exists when microdoses are applied.
With rhEpo and ABT, the absolute number of RBCs increase and thereby theoretically improves the capacity for extracellular buffering. 31However, the magnitude of changes with a micro-dosing protocol of rhEpo or ABT corresponds to an increase in red cell mass of ~6%. 11,13,24ndeed, improvement of extracellular buffer capacity has the potential to cause improved performance, but the required increase in extracellular buffer capacity is likely much higher than obtained with ABT or rhEpo as judged from oral intake of sodium bicarbonate where dosages of 0.3 g × kg −1 causes ~30% increases in venous levels of sodium bicarbonate. 52Accordingly, the buffer capacity is unaltered after prolonged subcutaneous injections of epoetin β. 27 Thus, augmented extracellular buffering does not appear important for the ABT and rhEpo induced performance gains and especially not so with micro-dosing protocols.Of possible importance is that non-lactate-coupled muscular H + efflux appeared to be increased after a relative high-dose rhEpo treatment but the impact on performance is unclear. 42The mechanism for improved H + efflux could be related to improved diffusion conditions due to increased [Hb] but this is speculative and remains unresolved.
For rhEpo treatment specifically, it needs to be considered that the Epo-receptor is expressed in several tissues.While expression of the Epo-receptor has been reported in endothelium and smooth muscle cells 53 it is unclear whether it is evident in skeletal muscle. 53,54hus, rhEpo treatment may have the potential to alter skeletal muscle phenotype.Indeed, rhEpo treatment activates skeletal muscle signal pathway AKT serine/threonine kinase-1 but does not stimulate protein synthesis more than exercise training alone, 55 which confirms that rhEpo treatment does not seem to affect muscle phenotype. 54Also, rhEpo has the potential to induce angiogenesis, 56 although contradicting evidence exists as epoetin β treatment for 14 weeks does not appear to alter skeletal muscle mean fiber area, capillaries per fiber or number of proliferating endothelial cells in humans. 53mportantly, most studies have not included a control group and therefore findings can also be ascribed to training induced changes.This is true for the observed increase in mitochondrial capacity. 57Thus, it appears unlikely that rhEpo causes performance-enhancement by affecting muscular phenotype or vascularization.Finally, rhEpo treatment may also have neurotrophic effects and affect hippocampal volume and mood disorders 58 but it appears unlikely that this is a part of the explanation for potential performance enhancement in healthy athletes.
In summary, the rhEpo and ABT induced increase of [Hb] appears as the most likely single explanation for the associated performance gains (Figure 1).

| DETECTION
In this section, we will discuss existing and future possibilities for detecting blood volume manipulation with an emphasis on micro-dosing.Anti-doping tests can be divided into direct and indirect testing.The aim of the direct tests is to identify the banned substance itself.In contrast, the principle of indirect detection is to measure selected biomarkers with high sensitivity to the treatment in question and use established cutoff values to identify when the treatment is performed.A major advantage of the indirect approach is that despite a continuous development of, for example, rhEpo analogs that may require different methods for direct detection, the physiological response can be assumed to be similar.The present review will focus on indirect testing methods.

| Athlete biological passport
0][61] In the early 2000s, more sophisticated ON-and OFF-models designed to be sensitive during and after rhEpo treatment were developed, [62][63][64] which was followed by a proposition to compare the athlete's hematological values to the athlete's own historical values causing a narrowing of individualized and dynamic cutoff values 65 hereby improving sensitivity. 66The research led to the implementation of the Athlete Biological Passport (ABP) in 2009 by WADA, which today is the only accredited indirect model for detection of blood doping.The ABP include a hematological module monitoring 12 hematological biomarkers, a calculated stimulation index "OFF-hr score" ([Hb] -60 × √reticulocyte percentage 62 ), and a multifactorial score denoted "Abnormal Blood Profile Score" (ABPS). 67The dynamic and individualized upper and lower thresholds are calculated by a Bayesian approach for the primary biomarkers [Hb] and OFF-hr score and the secondary biomarkers reticulocyte percentage and ABPS aiming at a specificity >99%.If an athlete surpasses a threshold or a sequence of samples deviates from the predicted ranges for a primary biomarker, an atypical passport finding (ATPF) is evident.
Blood doping with rhEpo or blood transfusions are detectable by the ABP, although available literature is limited.A treatment regime with 30-65 IU × kg bw −1 epoetin β induce an ATPF in up to 45% of the individuals, 68,69 and 2 weeks of 250 IU × kg bw −1 epoetin β cause an ATPF in 100% of the individuals, 70 albeit a purpose-built spreadsheet mimicking the official ABP software was applied in the latter.Similarly, blood transfusions of 450-1350 ml blood is detectable in 20% of the individuals by the ABP, 71 but using a blinded investigator throughout a cycling season improved detection to 80%. 71owever, applying micro-doses of blood doping is expected to reduce the hematological fluctuations, which may constitute a challenge for the passport to identify.In 2011, the ABP software was evaluated for the first time for micro-doses of rhEpo by administering 10-20 IU × kg bw −1 of epoetin β intravenously twice a week for 2-4 weeks, which was followed by 20 and 30-40 IU × kg bw −1 for 4 weeks each. 72Although the gradual increase in dose eventually exceeded micro-dosing levels, only 20% were identified with an ATPF for [Hb] or OFF-hr when blood sample results were evaluated every fourth week.A decade later, the ABP was evaluated using three weekly intravenous injections of 20 IU × kg bw −1 epoetin α for 3 weeks. 73Here, weekly blood sampling for up to 5 weeks after treatment resulted in an ATPF for [Hb] or OFF-hr in 56% of the individuals.Others have utilized 10-15 IU × kg bw −1 epoetin β injections three times per week for 3 weeks, but the injections were given after 2 weeks of injecting 250 IU × kg bw −1 and a purposebuilt spreadsheet rather than the official ABP software was applied. 70Although an ATPF was evident in 100% of the individuals 2 weeks into the micro-dose administration for [Hb] and OFF-hr, this was likely influenced by the prior boosting treatment of 250 IU × kg bw −1 as the control group only receiving the high doses were all identified with an ATPF 2 weeks later.
Studies evaluating the ABP for micro-doses of blood transfusion are similarly few.In fact, only a single study has evaluated the passport against ABT of less than one blood bag (i.e., RBCs from ~450 ml whole blood).While the donation of 450 ml blood caused an ATPF for 63% of the individuals, only 29% had an ATPF up to 6 days after reinfusion of ~130 ml RBCs when evaluating both primary and secondary ABP biomarkers. 13 few factors are important to consider when the strength of the ABP is interpreted.One is intelligent target testing, which implies that information from a suspicious blood profile, race calendar etc. is used to schedule doping controls at specific time points where the athlete is suspected to use a prohibited drug or method.This strategy led to 20 direct detections of rhEpo misuse within the first 2 years after the ABP was implemented.74 In addition, the ABP is not limited to the rate of ATPFs obtained with a given treatment.An ATPF is followed by a blood profile review by an expert within the field to determine whether the athlete is likely to have used doping or whether the analytical finding could be due to natural causes.75 Three experts must unanimously agree that the athlete is likely to have used doping, when assessing the ABP, the explanation provided by the athlete as well as a documentation package including information of the athlete, altitude exposure, competition schedule and more is presented to the experts.If so, a disciplinary case may be initiated.Therefore, it is complex to identify the true sensitivity of the ABP and evaluating the sensitivity of the ABP exclusively using the rate of ATPF may provide inaccurate results.In addition to detection, the ABP is also likely to have a deterrence effect on athletes and prevent doping use, likely driving the normalization of atypical blood values in professional cycling in the years after implementation.76 Other considerations include differences in study design, for instance timing and frequency of blood sampling and the administration regime, which complicate the comparison between studies.Likewise, iron stores must be considered as insufficient iron availability can diminish the response to rhEpo treatment.77 In summary, the ABP appears capable of identifying both micro-doses of rhEpo and blood reinfusions, but the rate of ATPFs at ~20%-60% and ~30%, respectively, for the primary biomarkers are highly dependent on the sample timing.In a future perspective, it is recommended that studies evaluating the biological passport apply a higher ecological valid approach by including one or more blinded experts to evaluate the established profiles or have blinded investigators requesting blood samples with the aim of providing a more accurate estimate of the true sensitivity and specificity.4.1.2| Reticulocytes and the Abnormal Blood Profile Score One option for improving the capability of the ABP to identify micro blood doping is to evaluate the potential of the two secondary biomarkers, reticulocyte percentage and ABPS, as primary biomarkers.
Reticulocyte percentage is known to be stable during storage and transportation within the ABP guidelines, 78 has no diurnal variation 79 and is not affected by acute exercise. 79In addition, a key advantage of the reticulocyte percentage is the independency of plasma volume fluctuations, which may occur often in athletes for instance due to exercise, altitude or heat exposure and consequently alter [Hb] and OFF-hr. 80,81The added value of including the reticulocyte percentage for detection of micro-doses of rhEpo has to our knowledge only been evaluated in a single study administering three weekly intravenous injections of 20 IU × kg bw −1 epoetin α for 3 weeks, which increased the rate of ATPFs from 56% for [Hb] and OFF-hr to 72%. 73For blood transfusions, including the reticulocyte percentage did not increase ABP sensitivity compared with [Hb] and OFF-hr alone for up to 6 days after reinfusing ~130 ml RBCs. 13In studies of larger doses, injections of 65 IU × kg bw −1 epoetin β every second day for two weeks 69 increased the ATPFs to 63% compared with 31% for [Hb] and OFF-hr at day 11 of the treatment.Furthermore, when injecting 390 IU × kg bw −1 epoetin β for three consecutive days, inclusion of the reticulocyte percentage increased the ATPFs to 67% compared with 33% at Day 11, corresponding to 8 days after the final injection. 69However, about 60% of the individuals were iron deficient, which may have influenced the fluctuations in [Hb] and OFF-hr score.
The ABPS was developed in 2006, 67 and is calculated by an algorithm based on two different classification techniques; a naive Bayesian classifier and a Support Vector Machine.Seven hematological parameters are included, and the outcome is an arbitrary score like the OFF-hr.In contrast to the reticulocyte percentage, ABPS is expected to be influenced by plasma volume fluctuations, but inclusion of seven variables in the calculation may improve the robustness.Only one study has to our knowledge assessed the additive value of ABPS as a primary biomarker following a moderate rhEpo dose.Specifically, when 30 IU × kg bw −1 epoetin β was injected subcutaneously three times per week for 3 weeks, adding the ABPS as a primary biomarker increased the ATPFs from 45% to 65% when evaluated 10 days later. 68Moreover, a case-report of doping with the HIF stabilizer FG-4592 indicates that the ABPS may be more sensitive than other ABP values. 82Following an ABT of ~130 ml, the inclusion of the ABPS increased sensitivity from 4% to 13% after reinfusion. 13 required trait of a primary biomarker is sufficient specificity, which in the current ABP is set to 99%.The reticulocyte percentage intraindividual variability is of concern as it exceeds that of [Hb] and OFF-hr, 83 and one study reported a specificity at ~91%.69 However, the decreased specificity may be explained by a potential carry-over effect and a low number (<40) of total samples collected.In recent years, studies collecting 100-350 samples have obtained a specificity above 99%.13,73 In addition, studies with ~250-350 samples obtained a specificity of 98.3%-100% 13 for ABPS, indicating that the specificity for reticulocyte percentage and ABPS is sufficient in large cohorts during sea-level exposure.
Collectively, compelling evidence suggests that inclusion of the reticulocyte percentage as a primary biomarker can improve the sensitivity of the ABP, even when microdoses are applied.The evidence for inclusion of ABPS as a primary biomarker is limited, but existing evidence also suggest an additive value.However, minor flaws in specificity could be of concern for ABPS, and an evaluation of reticulocyte percentage and ABPS as primary biomarkers should be continued.

| Immature reticulocytes
The immature reticulocyte is the earliest changing erythroid biomarker in the circulation following erythropoietic stress.It is often reported as the immature reticulocyte fraction (IRF), defined as the fraction of immature reticulocytes to the total number of reticulocytes.Immature reticulocytes are measured by the RNA content, which decrease with maturation, and are defined as the fraction of high and medium fluorescent cells in relation to all fluorescent cells.A previous point of critique for implementing IRF in detection of blood doping has been the imprecise measurement. 73,84However, the World Anti-Doping accredited laboratories recently changed from the Sysmex XE to the XN-series, which is expected to improve precision. 85Upon accelerated erythropoiesis, immature reticulocyte increases within 36 h 86 and appear to be released earlier from the bone marrow 87 and mature slower, 88 which are all properties indicating a sensitive biomarker for blood doping.Indeed, IRF is able to identify 88% of the participants administered intravenous injections of 20 IU × kg bw −1 epoetin α every second day for 3 weeks, 89 which appear superior to the 56% obtained by the ABP using [Hb] and OFF-hr as well as the 72% obtained by addition of the reticulocyte percentage. 73hile IRF is the fraction of the total reticulocyte count, a biomarker relating the immature reticulocyte count to the red blood cell count (IR/RBC) was recently proposed for detection of ABT and rhEpo administration using venous blood analysis 89 or the surrogate markers CD71/band3 in venous blood on dried blood spots 84,90 known to correlate with venous blood analysis. 91As stress erythropoiesis may increase RBC count by only ~10% and reticulocyte by >100%, 60 changes in IRF may be diminished by a simultaneous increase in reticulocyte percentage compared with IR/RBC.However, intravenous injections of 20 IU × kg bw −1 epoetin α every second day for 3 weeks provided a sensitivity of 92%, similar to the 88% obtained by IRF. 89Injecting a micro-dose of 900 IU epoetin α intravenously three times per week for 2 weeks also increased the IR/RBC surrogate marker CD71/band3 in dried blood spots by ~30%-40% and IRF by ~250% while the reticulocyte percentage was unaltered. 90However, the results should be interpreted carefully as the micro-doses were administered after a 3-week boosting phase of subcutaneous injections of 40 IU × kg bw −1 followed by a 10-day wash-out.
Whether IRF and IR/RBC are sensitive to micro-dose blood reinfusion remain unknown.When evaluated for up to 15 days after reinfusing RBCs from a larger dose of 500 ml blood, the IRF did not decrease although a statistical trend of numerical decreases was apparent. 92owever, reinfusing a similar blood volume provided detection possibilities in 7 out of 10 transfused participants based on experimentally defined criteria by the IR/RBC surrogate measure CD71/band3 ratio in dried blood spots, whereas the reticulocyte percentage detected 3 out of 10 participants. 84n summary, IRF and IR/RBC appear to be sensitive biomarkers of micro-dose rhEpo administration but have not been evaluated with a small volumes of blood reinfusion.However, evidence suggests that IR/RBC can be of value for detection of blood reinfusions.Despite the promising results, it must be noted that the ABP software does not calculate upper and lower thresholds for IRF or IR/RBC, and existing evidence are therefore based on threshold defined within each experimental study.The biological variability should therefore be explored to establish standardized thresholds across studies with the aim of developing data suitable for implementation in the ABP..1.4| Mean cell volume Another potential biomarker for blood doping is the erythrocyte mean cell volume (MCV), which increased with rhEpo administration (20-80 IU × kg bw −1 , one to three times per week for 3-8 weeks) or blood donation. 60,68,93he increased MCV has been attributed to the increased number of the large reticulocyte cells, rather than large RBCs.However, it was recently demonstrated that signaling of the erythropoietin receptor increase cell size in an erythropoietin dose-dependent manner during erythroid terminal differentiation, through a regulation of the number and speed of cell divisions as well as the duration of terminal differentiation, which in turn produces larger reticulocytes as well as larger RBCs in both mice and humans. 93Importantly, the reticulocyte number did not correlate with MCV and the increased MCV could not be accounted for by a skewed age distribution of circulating RBCs towards younger cells. 93Furthermore, the increased MCV was maintained beyond the period of the increased reticulocyte number, 93 indicating a potential for long-term detection of altered erythropoiesis.While the red cell size usually is reported as the mean cell volume of all RBCs, routine complete blood count analyses can perform single-cell analysis of more than 50,000 cells.A single-cell measurement of the red blood cell population allows a better understanding of the RBC population dynamics 94 and an investigation of the RBC size distribution.The size distribution could be speculated to be more sensitive to changes rather than the bulk mean volume, as a change within a fraction of the cell population is likely greater than the population mean.In addition, when larger RBCs are produced during rhEpo administration, the sub-population of rhEpo-induced large RBCs could in theory be identified for their entire lifetime of ~120 days, but that remains hypothetical.Accordingly, the MCV might prove valuable in the future as a biomarker for altered erythropoietic activity.

| Iron biomarkers
Iron is essential for de novo synthesis of RBCs and increased erythropoietic stress can increase the bone marrow iron consumption 10-fold and dietary iron absorption 20-fold. 77Thus, biomarkers of iron homeostasis have also been proposed for detection of blood doping.Indeed, soluble transferrin receptors increase and ferritin decrease with rhEpo treatment 61,63,64,95 or blood donation, although the latter is not always the case. 13In addition, transferrin saturation promptly increase ~150% while EDTA plasma iron increase 4-10 fold after reinfusing 280 ml RBCs, 96 yielding a 93% sensitivity at 100% specificity by a 45 μg/dl plasma iron threshold. 97The evidence of iron biomarkers' value for micro-dose blood doping is limited.Reinfusing ~130 ml RBCs, iron in lithium-heparin plasma provided a 17% sensitivity at a 99% specificity, while transferrin saturation was increased up to 6 h after reinfusion and ferritin was unaltered. 13In addition, 3 weeks with three weekly intravenous injections of 20 IU × kg bw −1 epoetin α decreased ferritin although no temporal differences were found and accentuated by concurrent altitude exposure at 2320 m. 51 Despite being affected by micro-dose manipulations, standard biomarkers of iron homeostasis are likely insufficient to appropriately strengthen the current panel of biomarkers alone.

| Erythroferrone and hepcidin
The coordination between erythropoietic activity and iron homeostasis is regulated by the two hormones hepcidin and erythroferrone (ERFE), which have been suggested as potential markers for blood doping.Hepcidin regulates body iron homeostasis by inhibiting iron from entering the circulation through the iron exporter ferroportin and is tightly regulated and proportional to body iron stores. 98Erythropoiesis and iron homeostasis is linked via ERFE, which is released from erythroblasts in response to erythropoietin and suppresses hepcidin. 98epcidin decreases and ERFE increases after donating ~450 ml blood, 13,96 and the reinfusion of ~280 ml RBCs augment plasma hepcidin levels 7-and 4-fold 12 and 24 h after reinfusion, respectively. 96Importantly, reinfusing a micro-dose of 130 ml RBCs increase hepcidin levels ~100% within 24 h, whereas a 40% numerical decrease in ERFE was insignificant. 13Using experimentally defined criteria, a combined sensitivity for blood donation and reinfusion of 30% and 61% for hepcidin and ERFE, respectively, was evident.Following only the reinfusion of blood, the corresponding sensitivities were 25% and 28%, respectively.
Hepcidin and ERFE are also sensitive to rhEpo injections.Hepcidin levels decrease rapidly ~25-90% up to 24 hours after a single injection with both subcutaneous and intravenous epoetin β (50-65 IU × kg bw −1 ), 99,100 while continuous subcutaneous injections with epoetin β (65 IU × kg bw −1 ) for 4 weeks promote a continuous decrease in urinary hepcidin during the first week but not after 4 weeks. 101Similarly, ERFE may increase ~300% with three injections of 5000 IU epoetin delta. 102Importantly, when six subcutaneous micro-doses of 20 IU × kg bw −1 epoetin α are injected, ERFE levels increase during treatment. 103In alignment, 11 intravenous injections of epoetin α (20 IU × kg bw −1 ) augmented ERFE levels by ~100% in 19 healthy men and women. 51 possible concern for hepcidin is that numerous factors normal for an elite athlete such as altitude 102 and iron supplementation 104 affect the systemic levels.In addition, the response for hepcidin may not be uniform, as a decrease following a blood donation can be transient and reverted within 6-24 h 96 or sustained for up to ~3 months in iron-replete donors. 105Thus, rigid standardization criteria upon blood sample collection at an anti-doping control is likely to be put in place if hepcidin is to be implemented as a biomarker for blood doping, whereas the impact similar confounding factors must be determined for ERFE.

| Omics
A strategy receiving an increasing interest in recent years for biomarker discovery is the use of "omics" analyses, referring to the analysis of changes within the genome, transcriptome, proteome, and metabolome.While changes within the genome are unlikely to occur with blood doping, the epigenetic of erythroblasts appear to differ from other cell types. 106Based on the methylation profile of erythroblasts, it was determined that about 30% of the circulating plasma DNA originates from erythroid cells.Importantly, the level of circulating DNA originating from erythroid cells is sensitive to alterations in the reticulocyte percentage, 106 and could therefore potentially serve as a biomarker of erythropoiesis.In contrast to the genome, the transcriptome appears affected by both rhEpo administration and ABT, and this information is thoroughly reviewed elsewhere. 107,108n altered gene expression has the potential to induce changes within the proteome as well.Indeed, alterations in the proteome of the RBCs is demonstrated during storage 109 and aging, 110 including proteins mainly located in the cytoskeleton such as spectrin β, band 4.2, anykrin-1, tropomodulin-1, band 4.9 and tropomyosin, but alterations are also present in transmembrane proteins including glycophorin C, aquaporin-1, and band 3. 109 Moreover, peroxiredoxin 2 was identified as a biomarker of ABT as a progressive migration from the cytosol to the RBC membrane was present after at least 14 days of storage at 4°C. 111 Peroxiredoxin 2 is therefore not detectable in the RBC membrane of freshly drawn blood but only in stored blood, even when diluted 1:10 with freshly drawn blood. 111Collectively, it appears evident that changes in the proteome of stored RBCs occur, but no study has investigated whether a change in the proteome is detectable in vivo following ABT.
in the proteome are expected to induce a shift within the metabolome, and the potential of metabolomics is highlighted by a shift in the metabolite profile during RBC storage in vitro. 112In addition, reinfusion of stored RBCs induces acute changes in vasoactive, pro-oxidative, proinflammatory, immune-modulatory, and plasticizer metabolites in human plasma. 113However, as urine is the most frequent matrix to collect in anti-doping, it would be highly valuable if metabolic changes were evident in urine as well.Using an untargeted metabolomics analysis, the urine metabolome was investigated following reinfusion of ~500 ml RBCs in a randomized double-blind placebo-controlled study. 114It was demonstrated that the strongest metabolites in human urine were plasticizers, which from previous investigations are known to be associated with ABT. 115,116Accordingly, it appears unlikely that other metabolites in urine are of interest for detection of ABT, although a small and uncontrolled study indicated that other urinary biomarkers could be of value. 117owever, metabolome changes within the plasma or RBC may prove valuable, especially as the total hemoglobin mass can remain elevated for more than 4 weeks following blood reinfusion, 118 indicating that the transfused cells are present for a long period of time.
Collectively, it appears that omics analyses are valuable for identification of biomarkers sensitive to blood doping.While transcriptomics has been investigated to some extent, the proteomics and metabolomics approach appear insufficiently investigated for their potential of biomarker discovery.Existing evidence is in its infancy and identified biomarkers should be further explored before they are included as a biomarker in real-life anti-doping sanctions.This includes determination of specificity, normal biological variance, confounding factors, and alignment of applied analytical methods.

| PERSPECTIVES
Micro-dosing constitutes a potential challenge for antidoping authorities, why continuous efforts to maintain the work for a fair and clean sport are needed.Although abductive reasoning and ethical considerations preclude the necessity of conducting studies for all potential performance-enhancing substances on the Prohibited List, 18 studies investigating the potential differences of blood doping between biological sexes are limited to one 13 and should be investigated further.Furthermore, efforts within emerging test methods such as the dried blood spots are of importance.Dried blood spots have the advantage of less strict storage and shipping conditions, the possibility for significantly increased collection-to-analysis time, as well as long-term stability a −20°C where protein abundance only decrease 7% after 10 years. 119In addition, the collection does not require a trained phlebotomist, as the sample is collected by a semi-automated device making the dried blood spot a time-efficient and athlete-friendly method.

F I G U R E 1
Overview of physiological variables and the impact on these when injecting recombinant human erythropoietin (rhEpo) or performing autologous blood transfusion (ABT).The green arrows indicate an increase, the yellow arrows indicate no changes, and the question mark indicates that it remains unresolved.The parenthesis indicates that the effect is uncertain.