blood doping blood doping blood doping is a form of doping in which the number of red blood cells in the bloodstream is boosted in order to enhance athletic performance. because such blood cells carry oxygen from the lungs to the muscles, a higher concentration in the blood can improve an athlete's aerobic capacity (vo2 max) and endurance. blood doping can be achieved by making the body produce more red blood cells itself using drugs, giving blood transfusions either from another person or back to the same individual, or by using blood substitutes. part of a series ondoping in sport substances and types anabolic steroids blood doping gene doping stem cell doping mechanical doping technology doping cannabinoids diuretics painkillers sedatives stem cell doping stimulants beta2-adrenergic agonist clenbuterol ephedrine epo human growth hormone methylhexanamine sarms stanozolol tetrahydrogestrinone terminology abortion doping biological passport blood-spinning doping test 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anti-doping authority french anti-doping agency national anti-doping agency russian anti-doping agency uk anti-doping united states anti-doping agency many methods of blood doping are illegal, particularly in professional sports where it is considered to give an artificial advantage to the competitor. anti-doping agencies use tests to try to identify individuals who have been blood doping using a number of methods, typically by analyzing blood samples from the competitors. history fig. 1 achieving maximum aerobic capacity blood doping is defined as the use of illicit products (e.g. erythropoietin (epo), darbepoetin-alfa, hypoxia-inducible factor (hif) stabilizers) and methods (e.g. increase aerobic capacity by maximizing the uptake of o2) in order to enhance the o2 transport of the body to the muscles. the body undergoes aerobic respiration in order to provide sufficient delivery of o2 to the exercising skeletal muscles and the main determining factors are shown in figure 1. the rate of maximum o2 uptake (o2max) depends on cardiac output, o2 extraction and hemoglobin mass. the cardiac output of an athlete is difficult to manipulate during competitions and the distribution of cardiac output is at the maximum rate (i.e. 80%) during competitions. in addition, the o2 extraction is approximately 90% at maximal exercise. therefore, the only method to enhance the physical performance left is to increase the o2 content in the artery by enhancing the hemoglobin mass. in other words, hemoglobin concentration and blood volume contribute to hemoglobin mass. methods drug treatments many forms of blood doping stem from the misuse of pharmaceuticals. these drug treatments have been created for clinical use to increase the oxygen delivery when the human body is not able to do so naturally. erythropoietin erythropoietin (epo) is a glycoprotein hormone produced by the interstitial fibroblasts in the kidney that signal for erythropoiesis in bone marrow. the increased activity of a hemocytoblast (rbc stem cell) allows the blood to have a greater carrying capacity for oxygen. epo was first developed to counteract the effects of chemotherapy and radiation therapy for cancer patients. epo also stimulates increased wound healing. because of its physiological side effects, particularly increased hematocrit, epo has become a drug with abuse potential by professional and amateur cyclists. hypoxia-inducible factor (hif) stabilizer hypoxia-inducible factor stabilizer (hif stabilizer) is a pharmaceutical used to treat chronic kidney disease. like most transcription factors, the hif transcription factor is responsible for the expression of a protein. the hif stabilizer activates the activity of epo due to anemia induced hypoxia, metabolic stress, and vasculogenesis—the creation of new blood vessels. hif stabilizers as used by cyclists in combination with cobalt chloride/desferrioxamine stimulate and de-regulate the natural production of erythropoietin hormone. at physiologically low pao2 around 40 mmhg, epo is released from the kidneys to increase hemoglobin transportation. the combination of drugs consistently releases epo due to increased transcription at the cellular level. the effect wears off when the hif stabilizers, cobalt chloride/desferrioxamine is excreted and/or decayed by the body. myo-inositol trispyrophosphate (itpp) myo-inositol trispyrophosphate (itpp), also known as compound number oxy111a, is an allosteric effector of hemoglobin which causes a rightward shift in the oxygen–hemoglobin dissociation curve, increasing the amount of oxygen released from red blood cells into surrounding tissue during each passage through the cardiovascular system. itpp has been a subject of anti-doping research in both humans and racehorses. blood transfusion blood transfusions can be traditionally classified as autologous, where the blood donor and transfusion recipient are the same, or as allogeneic/homologous, where the blood is transfused into someone other than the donor. blood transfusion begins by the withdrawal of 1 to 4 units of blood (1 unit = 450 ml of blood) several weeks before competition. the blood is centrifuged, the plasma components are immediately reinfused, and the corpuscular elements, principally red blood cells (rbcs), are stored refrigerated at 4 °c or frozen at −80 °c. as blood stored by refrigeration displays a steady decline in the number of rbcs, a substantial percentage, up to 40%, of the stored rbcs may not be viable. the freezing process, conversely, limits the aging of the cells, allowing the storage of the blood for up to 10 years with a 10% to 15% loss of rbcs. stored rbcs are then reinfused, usually 1 to 7 days before a high-endurance event. as a significant amount of iron is removed by each autologous transfusion, an adequate time for recovery of not less than 3 days from the last donation, and appropriate iron supplements, are usually required for patients undergoing autologous donations. nearly 50% of autologous donations are not used by the donor and are discarded, as current standards do not allow transfusion of these units to another patient for safety reasons. blood substitutes biochemical and biotechnological development has allowed novel approaches to this issue, in the form of engineered o2 carriers, widely known as "blood substitutes". the blood substitutes currently available are chiefly polymerized haemoglobin solutions or haemoglobin-based oxygen carriers (hbocs) and perfluorocarbons (pfcs). hemoglobin-based oxygen carriers (hbocs) hemoglobin-based oxygen carriers are intra/ inter-molecularly engineered human or animal hemoglobins, only optimized for oxygen delivery and longer intravascular circulation. the presence of 2,3-diphosphoglycerate within erythrocytes maintains the normal affinity of hemoglobin for oxygen. hbocs do not contain erythrocytes and lose this interaction, thus, unmodified human hboc solutions have a very high oxygen affinity which compromises their function. chemical methods developed to overcome this problem have resulted in carriers that effectively release oxygen at the physiological po2 of peripheral tissues. a common feature of all hbocs is their resistance to dissociate when dissolved in media, which contrasts hemoglobin of natural dissociation under non-physiological conditions. hbocs may hypothetically supply greater benefits to athletes than those provided by the equivalent hemoglobin in traditional rbc infusion. recent developments have shown that hbocs are not only simple rbc substitutes, but highly effective o2 donors in terms of tissue oxygenation. additional effects include increases in blood serum iron, ferritin, and epo; up to 20% increased diffusion of oxygen and improved exercise capacity; increased co2 production; and lower lactic acid generation in anaerobic activity. hbocs have been shown in trials to be extremely dangerous in humans. because hbocs increase both the risk of death and risk of myocardial infarction clinical trials were ended. they are not commercially available in the us or europe and there is no approved use for them. perfluorocarbons (pfcs) pfcs, also known as fluorocarbons, are inert, water-insoluble, synthetic compounds, consisting primarily of carbon and fluorine atoms bonded together in strong c–f bonds. pfcs are substantially clear and colorless liquid emulsions that are heterogeneous in molecular weight, surface area, electronic charge, and viscosity; their high content of electron-dense fluorine atoms results in little intramolecular interaction and low surface tension, making such substances excellent solvents for gases, especially oxygen and carbon dioxide. some of these molecules can dissolve 100 times more oxygen than plasma. pfcs are naturally hydrophobic and need to be emulsified to be injected intravenously. since pfcs dissolve rather than bind oxygen, their capacity to serve as a blood substitute is determined principally by the po2 gradients in the lung and at the target tissue. therefore, their oxygen transport properties differ substantially from those of whole blood and, especially, from those of rbcs. at a conventional ambient po2 of 135 mmhg, the oxygen content of 900 ml/l perfluorocarbon is less than 50 ml/l, whereas an optimal oxygen content of 160 ml/l, which is still lower than that of whole blood in normal conditions, can be achieved only by a po2 greater than 500 mmhg. in practice, at a conventional alveolar po2 of 135 mmhg, pfcs will not be able to provide sufficient oxygenation to peripheral tissues. due to their small size, pfcs are able to permeate circulation where erythrocytes may not flow. in tiny capillaries, pfcs produce the greatest benefit, as they increase local oxygen delivery much more efficiently than would be expected from the increase in oxygen content in larger arteries. in addition, as gases are in the dissolved state within pfcs, it po2 promotes efficient oxygen delivery to peripheral tissues. since the mid-1980s, improvements in both oxygen capacity and emulsion properties of pfcs have led to the development of second-generation pfc-based oxygen carriers; two pfc products are currently being tested in phase iii clinical trials. cobalt chloride administration transition metal complexes are widely known to play important roles in erythropoiesis; as such, inorganic supplementation is proving to be an emerging technique in blood doping. particularly of note is the cobalt complex, cobalamin (vitamin b12) commonly used as a dietary supplement. cobalamin is an important complex used in the manufacture of red blood cells and thus was of interest for potential use in blood doping. experimental evidence, however, has shown that cobalamin has no effect on erythropoiesis in the absence of a red blood cell/oxygen deficiency. these results seem to confirm much of what is already known about the functioning of cobalamin. the signaling pathway that induces erythropoietin secretion and subsequently red blood cell manufacture using cobalamin is o2 dependent. erythropoietin is only secreted in the kidneys when there is an o2 deficiency, as such, rbc manufacture is independent of the amount of cobalamin administered when there is no o2 deficiency. accordingly, cobalamin is of little to no value in blood doping. more potent for use in blood doping is co2+ (administered as cobalt(ii) chloride, cocl2). cobalt chloride has been known to be useful in treating anemic patients. recent experimental evidence has proved the efficacy of cobalt chloride in blood doping. studies into the action of this species have shown that co2+ induces hypoxia like responses, the most relevant response being erythropoiesis. co2+ induces this response by binding to the n-terminus (loop helix loop domain) of the hypoxia inducing transcription factors hif-1α and hif-2α, and thus stabilizes these protein complexes. under normal o2 conditions, hifs are destabilized as proline and asparagine residues are hydroxylated by hif-α hydroxylases, these unstable hifs are subsequently degraded following a ubiquitin-proteosome pathway, as such, they cannot then bind and activate transcription of genes encoding erythropoietin (epo). with co2+ stabilization, degradation is prevented and genes encoding epo can then be activated. the mechanism for this co2+ n terminus stabilization is not yet fully understood. in addition to n-terminus binding, it has also been hypothesized that replacement of fe2+ by co2+ in the hydroxylase active site could be a contributing factor to the stabilizing action of co2+. it is understood however, is that co2+ binding permits ubiquitin binding but prevents proteosomal degradation. detection of blood doping detection for homologous blood doping in 2004, a test for detection allogeneic/homologous blood transfusion doping was implemented. flow cytometry is the method of choice. by examining markers on the