Artifacts in Plasma Volume Changes due to AHA-Derived HCT
Purpose: This investigation quantified the effect of changes in plasma osmolality on the measurement of hematocrit (Hct) and the implications for the subsequent use of these data to calculate changes in plasma volume and application to the World Anti-Doping Agency Athlete Biological Passport.
Methods: Two groups of eight male volunteers visited the laboratory after an overnight fast. In study 1, a 20-mL blood sample was collected and aliquoted into collection tubes containing saline of varying concentrations to alter the sample osmolality. In study 2, plasma osmolality was manipulated in vivo through prolonged exercise. Samples were analyzed for hemoglobin concentration and Hct using manual methods and using an automated hematology analyzer (AHA).
Results: Changes in blood, plasma, and red cell volumes were calculated. Although AHA Hct values did not change (P = 0.652), spun packed cell volume fell progressively as the osmolality of the sample increased (P < 0.001, study 1). Consequently, there was a significant increase in apparent plasma volume as osmolality increased (P < 0.001): regression analysis revealed that a 10 mOsm·kg change in plasma osmolality produced a difference of 0.8 Hct units and a 1.6% change in plasma volume. In study 2, exercise produced a 12 ± 3 mOsm·kg increase in plasma osmolality. No difference in Hct was apparent at rest (P = 0.659), but spun packed cell volume was 1.0 ± 0.9 Hct units lower during exercise compared with AHA data (P < 0.001). There was a difference in the degree of plasma volume change calculated, with a reduction of 8.7% ± 3.4% and 11.3% ± 3.5% reported with the manual and AHA methods, respectively (P = 0.002).
Conclusions: Conditions or interventions that result in a marked change in plasma osmolality produce a discrepancy in Hct measured using an AHA, consequently introducing errors into any calculation of changes in plasma volume using these data. These findings may also have implications for the measurement of Hct by World Anti-Doping Agency-accredited laboratories.
Direct measurement of blood and plasma volumes can be made using a variety of dilution techniques, typically requiring the serial measurement of a tracer substance after allowing sufficient time for uniform distribution throughout the compartment. Such techniques often require access to labeling facilities and are not amenable to repeated measurements over a relatively short time frame. For these reasons, it has become common to measure changes in hemoglobin (Hb) concentration as an index of blood volume changes. Hb is present within the erythrocyte and does not enter or leave the circulation in appreciable quantities over the time scale of most human physiology experiments, so a change in Hb concentration can be used to calculate changes in blood volume. Dye dilution methods for plasma volume measurement are also limited because of the impossibility of making repeated measurements on a short time scale. Changes in plasma volume can be calculated, however, if the Hb concentration and hematocrit (Hct) are known. The Hct reflects the volume of red blood cells as a fraction of the total volume of a sample, usually expressed as a percentage. The possibility of using these measures has long been recognized, but Dill and Costill published an improved method for the calculation of changes in blood, red cell, and plasma volumes from serial measurements of Hb concentration and Hct. Because a wide variety of exercise, environmental, nutritional, and pharmacological interventions produces marked changes in blood and plasma volumes, this method remains in common use today.
The introduction of automated hematology analyzers (AHA) has allowed the rapid analysis of both Hb and Hct, along with several other hematological measures of clinical interest. These instruments typically require only a small volume of blood and offer results within a short time frame, making them attractive in a variety of clinical and research settings. Typically, AHA uses a spectrophotometric technique to determine Hb concentration similar to that used in manual procedures (i.e., cyanmethemoglobin assay), but the analytical method used by these instruments to determine Hct differs markedly from that used in conventional laboratory settings. Before the introduction of AHA, Hct was determined by centrifugation of anticoagulated blood in a capillary tube, with the height of the column of erythrocytes compared with the total height of the column of blood in the tube. This is called the Hct or spun packed cell volume (PCV), with an appropriate correction typically applied to account for plasma trapped between the red cells. Most AHA determines Hct by measuring the electrical impedance of a sample of cells suspended in a conductive reagent. Because red blood cells have a lipid membrane, they account for the majority of the electrical resistivity of the whole blood. The impedance variation generated by the passage of cells through a small aperture is used to determine the number and the size of the red blood cells. A value for the Hct is calculated from these two measures.
The volume of each individual red blood cell is highly responsive to changes in extracellular osmolality. In individuals who are acutely hyperosmotic, red cell volume will rapidly and reversibly decrease because of free water movement out of the cells to balance intracellular and extracellular osmotic force. Stott et al. previously demonstrated this expected inverse relationship between the osmolality of a sample and the spun PCV, but this response was not apparent in Hct values determined by an AHA method. These differences are explained by the sample handling procedure used by most commercially available AHA (e.g., COULTER® AC·T™ 5diff (Beckman Coulter, High Wycombe, UK) or Sysmex XT-2000i (Sysmex Corp., Kobe, Japan)); samples of whole blood are subjected to a series of dilution steps in relatively large volumes of "isotonic" reagent, which effectively normalizes the sample osmolality and results in the measurement of an "isotonic Hct", which may be very different from the in vivo osmolality. These Hct measurements will not reflect the true circulating red cell volume present in vivo if the plasma osmolality is markedly different from that of the "isotonic" diluent. Calculated changes in the plasma volume made using values derived from AHA will therefore also contain an unknown degree of error if plasma osmolality has changed during an experiment.
The Athlete Biological Passport (ABP) was introduced by the World Anti-Doping Agency (WADA) in 2009. The fundamental principle of the ABP is based on the monitoring of selected biological parameters over time that will indirectly reveal the effects of doping rather than detect a particular doping substance or method. Unexplained or abnormal changes in passport data can be used to identify and target athletes for specific analytical testing, as well as can be used directly to pursue antidoping rule violations. The hematological module of the ABP, which is used to detect the use of prohibited methods to enhance oxygen transfer, relies on the measurement of the following parameters: Hct, Hb, red blood cells count, percentage of reticulocytes, reticulocyte count, mean corpuscular volume, mean corpuscular Hb, and the mean corpuscular Hb concentration. Hct is used along with these other measures to determine an "Abnormal Blood Profile Score". In an effort to standardize analytical results, WADA-accredited laboratories are required to use analyzers with comparable technical characteristics, and these instruments are subject to regular internal and external quality assessment tests (WADA Technical Document TD2010BAR 2011). Antidoping laboratories use a commer cially available AHA (typically, Sysmex XT-2000i) to produce these data, and consequently, ABP samples are subject to the analytical artifacts described previously. Because presample conditions are not standardized in doping control, distinct variation in sample osmolality is likely, and this may limit the reliability of the ABP to detect the use of prohibited methods to enhance oxygen transfer.
Many laboratories routinely measure Hb and Hct using either traditional assay methods (Hb by cyanmethemoglobin method and Hct by spun PCV) or an AHA. The aim of the present study, therefore, was to determine the influence of changes in plasma osmolality on the measurement of Hb and Hct using these different analytical approaches. Given the importance of Hct to the equations described by Dill and Costill, any discrepancy between these measures is likely to introduce error into the calculation of changes in red cell and plasma volumes. In addition, Hct is monitored by antidoping authorities using an AHA-based approach as part of the ABP. Because many exercise, nutritional, and pharmacological interventions result in marked changes in plasma osmolality, this will likely invalidate the use of AHA data to determine changes in plasma volume and may have significant implications for reliability of the ABP. If Hct values obtained from hematology analyzers are influenced by osmolality in a predictable manner, then it should be possible to apply a correction factor to these data to account for the use of an "isotonic Hct" produced by AHA devices. These objectives were addressed through the completion of two separate, but interrelated, studies. First, the effect of changes in sample osmolality on measured Hb and Hct was directly determined through the addition of known concentrations of saline to blood samples drawn at rest (study 1). To illustrate this response in vivo, a separate study examined Hb and Hct measured during prolonged exercise in the heat, an intervention where a significant change in sample osmolality was expected (study 2).
Abstract and Introduction
Abstract
Purpose: This investigation quantified the effect of changes in plasma osmolality on the measurement of hematocrit (Hct) and the implications for the subsequent use of these data to calculate changes in plasma volume and application to the World Anti-Doping Agency Athlete Biological Passport.
Methods: Two groups of eight male volunteers visited the laboratory after an overnight fast. In study 1, a 20-mL blood sample was collected and aliquoted into collection tubes containing saline of varying concentrations to alter the sample osmolality. In study 2, plasma osmolality was manipulated in vivo through prolonged exercise. Samples were analyzed for hemoglobin concentration and Hct using manual methods and using an automated hematology analyzer (AHA).
Results: Changes in blood, plasma, and red cell volumes were calculated. Although AHA Hct values did not change (P = 0.652), spun packed cell volume fell progressively as the osmolality of the sample increased (P < 0.001, study 1). Consequently, there was a significant increase in apparent plasma volume as osmolality increased (P < 0.001): regression analysis revealed that a 10 mOsm·kg change in plasma osmolality produced a difference of 0.8 Hct units and a 1.6% change in plasma volume. In study 2, exercise produced a 12 ± 3 mOsm·kg increase in plasma osmolality. No difference in Hct was apparent at rest (P = 0.659), but spun packed cell volume was 1.0 ± 0.9 Hct units lower during exercise compared with AHA data (P < 0.001). There was a difference in the degree of plasma volume change calculated, with a reduction of 8.7% ± 3.4% and 11.3% ± 3.5% reported with the manual and AHA methods, respectively (P = 0.002).
Conclusions: Conditions or interventions that result in a marked change in plasma osmolality produce a discrepancy in Hct measured using an AHA, consequently introducing errors into any calculation of changes in plasma volume using these data. These findings may also have implications for the measurement of Hct by World Anti-Doping Agency-accredited laboratories.
Introduction
Direct measurement of blood and plasma volumes can be made using a variety of dilution techniques, typically requiring the serial measurement of a tracer substance after allowing sufficient time for uniform distribution throughout the compartment. Such techniques often require access to labeling facilities and are not amenable to repeated measurements over a relatively short time frame. For these reasons, it has become common to measure changes in hemoglobin (Hb) concentration as an index of blood volume changes. Hb is present within the erythrocyte and does not enter or leave the circulation in appreciable quantities over the time scale of most human physiology experiments, so a change in Hb concentration can be used to calculate changes in blood volume. Dye dilution methods for plasma volume measurement are also limited because of the impossibility of making repeated measurements on a short time scale. Changes in plasma volume can be calculated, however, if the Hb concentration and hematocrit (Hct) are known. The Hct reflects the volume of red blood cells as a fraction of the total volume of a sample, usually expressed as a percentage. The possibility of using these measures has long been recognized, but Dill and Costill published an improved method for the calculation of changes in blood, red cell, and plasma volumes from serial measurements of Hb concentration and Hct. Because a wide variety of exercise, environmental, nutritional, and pharmacological interventions produces marked changes in blood and plasma volumes, this method remains in common use today.
The introduction of automated hematology analyzers (AHA) has allowed the rapid analysis of both Hb and Hct, along with several other hematological measures of clinical interest. These instruments typically require only a small volume of blood and offer results within a short time frame, making them attractive in a variety of clinical and research settings. Typically, AHA uses a spectrophotometric technique to determine Hb concentration similar to that used in manual procedures (i.e., cyanmethemoglobin assay), but the analytical method used by these instruments to determine Hct differs markedly from that used in conventional laboratory settings. Before the introduction of AHA, Hct was determined by centrifugation of anticoagulated blood in a capillary tube, with the height of the column of erythrocytes compared with the total height of the column of blood in the tube. This is called the Hct or spun packed cell volume (PCV), with an appropriate correction typically applied to account for plasma trapped between the red cells. Most AHA determines Hct by measuring the electrical impedance of a sample of cells suspended in a conductive reagent. Because red blood cells have a lipid membrane, they account for the majority of the electrical resistivity of the whole blood. The impedance variation generated by the passage of cells through a small aperture is used to determine the number and the size of the red blood cells. A value for the Hct is calculated from these two measures.
The volume of each individual red blood cell is highly responsive to changes in extracellular osmolality. In individuals who are acutely hyperosmotic, red cell volume will rapidly and reversibly decrease because of free water movement out of the cells to balance intracellular and extracellular osmotic force. Stott et al. previously demonstrated this expected inverse relationship between the osmolality of a sample and the spun PCV, but this response was not apparent in Hct values determined by an AHA method. These differences are explained by the sample handling procedure used by most commercially available AHA (e.g., COULTER® AC·T™ 5diff (Beckman Coulter, High Wycombe, UK) or Sysmex XT-2000i (Sysmex Corp., Kobe, Japan)); samples of whole blood are subjected to a series of dilution steps in relatively large volumes of "isotonic" reagent, which effectively normalizes the sample osmolality and results in the measurement of an "isotonic Hct", which may be very different from the in vivo osmolality. These Hct measurements will not reflect the true circulating red cell volume present in vivo if the plasma osmolality is markedly different from that of the "isotonic" diluent. Calculated changes in the plasma volume made using values derived from AHA will therefore also contain an unknown degree of error if plasma osmolality has changed during an experiment.
The Athlete Biological Passport (ABP) was introduced by the World Anti-Doping Agency (WADA) in 2009. The fundamental principle of the ABP is based on the monitoring of selected biological parameters over time that will indirectly reveal the effects of doping rather than detect a particular doping substance or method. Unexplained or abnormal changes in passport data can be used to identify and target athletes for specific analytical testing, as well as can be used directly to pursue antidoping rule violations. The hematological module of the ABP, which is used to detect the use of prohibited methods to enhance oxygen transfer, relies on the measurement of the following parameters: Hct, Hb, red blood cells count, percentage of reticulocytes, reticulocyte count, mean corpuscular volume, mean corpuscular Hb, and the mean corpuscular Hb concentration. Hct is used along with these other measures to determine an "Abnormal Blood Profile Score". In an effort to standardize analytical results, WADA-accredited laboratories are required to use analyzers with comparable technical characteristics, and these instruments are subject to regular internal and external quality assessment tests (WADA Technical Document TD2010BAR 2011). Antidoping laboratories use a commer cially available AHA (typically, Sysmex XT-2000i) to produce these data, and consequently, ABP samples are subject to the analytical artifacts described previously. Because presample conditions are not standardized in doping control, distinct variation in sample osmolality is likely, and this may limit the reliability of the ABP to detect the use of prohibited methods to enhance oxygen transfer.
Many laboratories routinely measure Hb and Hct using either traditional assay methods (Hb by cyanmethemoglobin method and Hct by spun PCV) or an AHA. The aim of the present study, therefore, was to determine the influence of changes in plasma osmolality on the measurement of Hb and Hct using these different analytical approaches. Given the importance of Hct to the equations described by Dill and Costill, any discrepancy between these measures is likely to introduce error into the calculation of changes in red cell and plasma volumes. In addition, Hct is monitored by antidoping authorities using an AHA-based approach as part of the ABP. Because many exercise, nutritional, and pharmacological interventions result in marked changes in plasma osmolality, this will likely invalidate the use of AHA data to determine changes in plasma volume and may have significant implications for reliability of the ABP. If Hct values obtained from hematology analyzers are influenced by osmolality in a predictable manner, then it should be possible to apply a correction factor to these data to account for the use of an "isotonic Hct" produced by AHA devices. These objectives were addressed through the completion of two separate, but interrelated, studies. First, the effect of changes in sample osmolality on measured Hb and Hct was directly determined through the addition of known concentrations of saline to blood samples drawn at rest (study 1). To illustrate this response in vivo, a separate study examined Hb and Hct measured during prolonged exercise in the heat, an intervention where a significant change in sample osmolality was expected (study 2).
SHARE
