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Changes in red blood cell volume, plasma volume, and total blood volume after autologous blood collections
... With respect to the effect of blood reinfusion on VO 2peak , it is important that total blood volume alterations appear to depend on the time of investigation after reinfusion. Total blood volume appears unaffected ≥ 24 h post-reinfusion of 800 to 2250 mL of refrigerated whole blood [22] as well as refrigerated [50] or freeze-preserved [31,32,50] RBCs reconstituted with physiological saline to a normal Hct. In contrast, 1 h after reinfusion of packed RBCs from 900 mL of whole blood, a 5% increase in total blood volume occurs together with increases in Hct (2%), [Hb] (4%), VO 2peak (7%), and cycling performance lasting 3-6 min (24%) [11]. ...
... With respect to the effect of blood reinfusion on VO 2peak , it is important that total blood volume alterations appear to depend on the time of investigation after reinfusion. Total blood volume appears unaffected ≥ 24 h post-reinfusion of 800 to 2250 mL of refrigerated whole blood [22] as well as refrigerated [50] or freeze-preserved [31,32,50] RBCs reconstituted with physiological saline to a normal Hct. In contrast, 1 h after reinfusion of packed RBCs from 900 mL of whole blood, a 5% increase in total blood volume occurs together with increases in Hct (2%), [Hb] (4%), VO 2peak (7%), and cycling performance lasting 3-6 min (24%) [11]. ...
This review critically evaluates the magnitude of performance enhancement that can be expected from various autologous blood transfusion (ABT) procedures and the underlying physiological mechanisms. The review is based on a systematic search, and it was reported that 4 of 28 studies can be considered of very high quality, i.e. placebo-controlled, double-blind crossover studies. However, both high-quality studies and other studies have generally reported performance-enhancing effects of ABT on exercise intensities ranging from ~70 to 100% of absolute peak oxygen uptake (VO2peak) with durations of 5–45 min, and the effect was also seen in well-trained athletes. A linear relationship exists between ABT volume and change in VO2peak. The likely correlation between ABT volume and endurance performance was not evident in the few available studies, but reinfusion of as little as 135 mL packed red blood cells has been shown to increase time trial performance. Red blood cell reinfusion increases endurance performance by elevating arterial oxygen content (CaO2). The increased CaO2 is accompanied by reduced lactate concentrations at submaximal intensities as well as increased VO2peak. Both effects improve endurance performance. Apparently, the magnitude of change in haemoglobin concentration ([Hb]) explains the increase in VO2peak associated with ABT because blood volume and maximal cardiac output have remained constant in the majority of ABT studies. Thus, the arterial-venous O2 difference during exercise must be increased after reinfusion, which is supported by experimental evidence. Additionally, it remains a possibility that ABT can enhance repeated sprint performance, but studies on this topic are lacking. The only available study did not reveal a performance-enhancing effect of reinfusion on 4 × 30 s sprinting. The reviewed studies are of importance for both the physiological understanding of how ABT interacts with exercise capacity and in relation to anti-doping efforts. From an anti-doping perspective, the literature review demonstrates the need for methods to detect even small ABT volumes.
... The possibility of performing the analysis of DEHP metabolites in urine opens the door not only to suspect allogeneic transfusion (verifiable by whole blood flow cytometry [10][11][12] ) but also the capability to suspect on any athletes being subjected to autologous transfusion (not easily detectable nowadays 19,20 ). The approach could be applicable to all urine samples submitted to doping control in any accredited laboratory being a useful tool to alert on any potential cheater in any antidoping program. ...
... [26] New parameters, such as the relationship of total RBC haemoglobin (RBCHb)/total reticulocytes haemoglobin (reticHb), have been proposed for greater sensitivity. [27] This relationship has the advantage that variations in plasma volume affect the numerator and denominator similarly and should cancel the effect on the scoring of the test. The re-infusion of blood originated supra-physiological concentrations of RBCHb and reduced RetHb, resulting in an increase in the proportion RBCHb/ reticHb. ...
The use of blood doping is forbidden by the World Anti‐Doping Agency. Several practices, such as blood transfusions are used to increase oxygen delivery to muscles and all of them are highly pursued. In this regard, the development of accurate methodologies for detecting these prohibited practices is one of the current aims of the anti‐doping control laboratories. Flow cytometry methods are able to detect allogeneic blood transfusions but there is no official methodology available to detect autologous blood transfusions.
This paper reviews protocols, including the Athlete Biological Passport, that use indirect markers to detect misuse of blood transfusions, especially autologous blood transfusions. The methods of total haemoglobin mass measurements and the detection of metabolites of blood bags plasticizers in urine are reviewed. The latter seems to be an important step forward because it is a fast screening method and it is based on urine, a fluid widely available for doping control. Other innovative approaches to blood transfusion detection are also mentioned. A combination of the reported methodologies and the implementation of the Athlete Biological Passport is becoming a promising approach. Copyright © 2012 John Wiley & Sons, Ltd.
... The mathematical basis for this is a larger ratio between the relative increase in [Hb] and Hbmass at lower dosages compared with at higher dosages of transfused blood, which relates to changes in plasma volume. Differences in the plasma volume response to autologous blood transfusions have previously been shown to relate to the amount of transfused blood (Morkeberg et al., 2008), and it can be speculated that the relatively low hemoconcentration after transfusion of large blood volumes could be a natural Table 2. Sensitivities and specificities during the whole measurement period for hemoglobin concentration ([Hb]), OFF-hr, hemoglobin mass (Hbmass), and Hbmr when assessed by the athletes passport (AP), third Generation (3G) and Absolutist approach after reinfusion of hemoglobin derived from 3 or 1 bag(s) of whole blood stored in a liquid (141C) Sensitivities and specificities for Hbmass and Hbmr were only assessed by the 3G and AP methods as no data or thresholds were presented on Hbmass by Sallet et al. (2008). ...
Blood passport has been suggested as an indirect tool to detect various kinds of blood manipulations. Autologous blood transfusions are currently undetectable, and the objective of this study was to examine the sensitivities of different blood markers and blood passport approaches in order to determine the best approach to detect autologous blood transfusions. Twenty-nine subjects were transfused with either one (n=8) or three (n=21) bags of autologous blood. Hemoglobin concentration ([Hb]), percentage of reticulocytes (%ret) and hemoglobin mass (Hbmass) were measured 1 day before reinfusion and six times after reinfusion. The sensitivity and specificity of a novel marker, Hbmr (based on Hbmass and %ret), was evaluated together with [Hb], Hbmass and OFF-hr by different passport methods. Our novel Hbmr marker showed superior sensitivity in detecting the highest dosage of transfused blood, with OFF-hr showing equal or superior sensitivities at lower dosages. Hbmr and OFF-hr showed superior but equal sensitivities from 1 to 4 weeks after transfusion compared with [Hb] and Hbmass, with Hbmass being the only tenable prospect to detect acute transfusions. Because autologous blood transfusions can be an acute practice with blood withdrawal and reinfusion within a few days, Hbmass seems to be the only option for revealing this practice.
Autologous blood transfusions (ABTs) has been used by athletes for approximately 4 decades to enhance their performance. Although the method was prohibited by the International Olympic Committee in the mid 1980s, no direct detection method has yet been developed and implemented by the World Anti-Doping Agency (WADA). Several indirect methods have been proposed with the majority relying on changes in erythropoiesis-sensitive blood markers. Compared with the first methods developed in 1987, the sensitivity of subsequent tests has not improved the detection of blood doping. Nevertheless, the use of sophisticated statistical algorithms has assured a higher level of specificity in subsequent detection models, which is a crucial aspect of antidoping testing particularly to avoid "false positives." Today, the testing markers with the best sensitivity/specificity ratio are the Hbmr model (an algorithm based on the total amount of circulating hemoglobin level [hemoglobin level mass] and percentage of reticulocytes, 4.51·ln(Hbmass)-√%ret) and the OFF-hr model (algorithm based on hemoglobin level concentration and percentage of reticulocytes, Hb(g/L)-60·√%ret). Only the OFF-hr model is currently approved by WADA. Recently, alternative indirect strategies for detecting blood doping have been proposed. One method is based upon a transfusion-induced immune-response resulting in specific changes in gene expression related to leukocytes such as T lymphocytes. Another method relies on detecting increased plasticizer metabolite levels in the urine caused by the leakage of plasticizers from the blood bags used during the blood storage. These methods need further development and validation across different types of transfusion regimes before they can be implemented. In addition, several research projects have been funded by WADA in recent years and are now under development including "Detection of Autologous Blood Transfusions Using Activated Red Blood Cells (the red blood cells eNOS system)" and "Detection of Autologous Blood Transfusion by Proteomic: Screening to find Unique Biomarkers, Detecting Blood Manipulation from Total Hemoglobin Mass using 15-nitric Oxide as a Tracer Gas, Storage Contamination as a Potential Diagnostic Test for Autologous Blood Transfusion and Test for Blood Transfusion (Autologous/Homologous) based on Changes of Erythrocyte Membrane Protome" (WADA, WADA Funded Research Projects. http://www.wada-ama.org/en/Science-Medicine/Research/Funded-Research-Projects/. 2010). Although strategies to detect autologous blood transfusion have improved, a highly sensitive test to detect small volumes of transfused autologous blood has not yet been implemented.
Aerobic sport performance may be strongly influenced by the number of red blood cells available for transport and delivery of oxygen from lungs to muscles. Often, athletes search for an acute increase in red blood cells by means of blood transfusions. This paper reviews the possibilities for detecting such prohibited practice. Flow cytometry methods are able to detect a double population of red blood cell membrane surface antigens, thus revealing an allogeneic transfusion. Other ingenious approaches for total hemoglobin mass measurements or to test for the metabolites of blood bag plasticizers in urine are new trends for facing the detection of autologous transfusions. Steady increase of red blood cell number may be obtained also by erythropoietic stimulant agents such as erythropoietin, analogs and mimetics. The challenge of detecting those substances has stimulated the development of indirect markers of altered erythropoiesis, leading to the consequent development of the hematological blood passport approach, which is gaining legal acceptance.
Subjects submitted to intravenous (IV) blood transfusions for medical reasons or blood doping to increase athletic performance are potentially exposed to the plasticizer di-(2-ethylhexyl)phthalate (DEHP) found in IV bags. Exposure to DEHP has been evaluated by measuring DEHP metabolites in selected groups of subjects.
Urinary DEHP metabolites, mono-(2-ethylhexyl)phthalate, mono-(2-ethyl-5-hydroxyhexyl)phthalate (MEHHP), and mono-(2-ethyl-5-oxohexyl)phthalate (MEOHP) were measured in a control group with no explicit known exposure to DEHP (n = 30), hospitalized patients receiving blood transfusions (n = 25), nontransfused hospitalized patients receiving other medical care involving plastic materials (n = 39), and athletes (n = 127). Patients were tested in the periods 0 to 24 and 24 to 48 hours after exposition.
Urinary concentrations of all three DEHP metabolites were significantly higher in patients receiving blood transfusion than in nontransfused patients and the control group, except for MEHHP and MEOHP in the period 24 to 48 hours. Samples from four athletes showed increased concentrations of DEHP metabolites comparable to urinary concentrations of patients receiving blood transfusion.
Elevated concentrations of urinary DEHP metabolites represent increased exposure to DEHP. High concentrations of DEHP metabolites present in urine collected from athletes may suggest illegal blood transfusion and can be used as a qualitative screening measure for blood doping.
In conscious mammals including humans, the neurohumoral and hemodynamic responses to progressive acute hypovolemia have two distinct phases. There is an initial arterial baroreceptor-mediated phase in which the fall in cardiac output is nearly matched by a sympathetically mediated increase in peripheral resistance so that arterial pressure is maintained near normal levels. In most species, adrenal catecholamines and vasopressin contribute little to this phase. Increased renin release appears to augment the sympathetically mediated vasoconstriction. When blood volume has fallen by a critical amount (approximately 30%), a second phase develops abruptly. This phase is characterized by withdrawal of sympathetic vasoconstrictor drive, relative or absolute bradycardia, an increase in release of adrenal catecholamines and vasopressin, and a profound fall in arterial pressure. In rabbits and rats the signal that initiates this phase appears to travel in cardiopulmonary afferents. In dogs and humans its origin is unknown. Central opioidergic and serotonergic mechanisms may be involved.
A routine method to determine total haemoglobin mass (tHb) in clinical practice and sports medicine is non-existent. Radioactive tracers or other dilution procedures like the common CO-rebreathing method (Proc(com)) are impractical, the latter in particular because of the relatively long time of respiration. According to the multicompartment model of Bruce and Bruce (J Appl Physiol 95:1235-1247, 2003) the respiration time can be considerably reduced by inhaling a CO-bolus instead of the commonly used gas mixture. The aim of this study was to evaluate this theoretical concept in practice. The kinetics of the HbCO formation were compared in arterialised blood sampled from an hyperaemic earlobe after inhaling a CO-bolus (Proc(new)) for 2 min and a CO-O(2) mixture (Proc(com)) for approximately 10 min. The reliability of Proc(new) was checked in three consecutive tests, and phlebotomy was used to determine the validity. VO(2max) was determined with and without previous application of Proc(new) and the half-time of HbCO was registered also in arterialised blood after resting quietly and after the VO(2max) test. Proc(new) yielded virtual identical tHb values compared to Proc(com) when HbCO determined 5 min after starting CO-rebreathing was used for calculation. The typical error of Proc(new) was 1.7%, corresponding to a limit of agreement (95%) of 3.3%. The loss of 95 g (19) haemoglobin was detected with an accuracy of 9 g (12). After application of Proc(new) VO(2max) was reduced by 3.0% (3.7) (P=0.022) and half-time was lowered from 132 min (77) to 89 min (23) after the VO(2max) test. Inhaling a CO-bolus markedly simplifies the CO-rebreathing method without reducing validity and reliability and can be used for routine determination of tHb for various indications.
An acute reduction of blood hemoglobin concentration ([Hb]), even when the circulating blood volume is maintained, results in lower (.)V(O(2)(max) and endurance performance, due to the reduction of the oxygen carrying capacity of blood. Conversely, an increase of [Hb] is associated with enhanced (.)V(O(2)(max) and endurance capacity, that is also proportional to the increase in the oxygen carrying capacity of blood. The effects on endurance capacity appear more pronounced and prolonged than on (.)V(O(2)(max). During submaximal exercise, there is a tight coupling between O(2) demand and O(2) delivery, such that if [Hb] is acutely decreased muscle blood flow is increased proportionally and vice versa. During maximal exercise with either a small or a large muscle mass, neither peak cardiac output nor peak leg blood flow are affected by reduced [Hb]. An acute increase of [Hb] has no effect on maximal exercise capacity or (.)V(O(2)(max) during exercise in acute hypoxia. Likewise, reducing [Hb] in altitude-acclimatized humans to pre-acclimatization values has no effect on (.)V(O(2)(max) during exercise in hypoxia.