The intra-individual variation of cardiac troponin I: the effects of sex, age, climatic season, and time between samples.

OBJECTIVES: Knowing the intra-individual variation (CVi), also termed within subject biological variation, of an analyte is essential to properly interpret apparent changes in concentration. While there have been many studies assessing the CVi of cardiac troponin (cTnI), they have been limited in looking at CVi in different settings, and there is no data available on whether CVi might change in different settings. METHODS: We used our large cTnI data bank to look at the CVi of cTnI in Emergency Department (ED) patients who had an acute myocardial infarction event excluded. We looked at the effects of gender, age, climatic season, and time between samples to assess whether CVi changed. To assess the effect of age, after exclusion, we collected two samples from each subject for each study which were used to calculate the CVi between those identified groups. There were 139 males and 98 females aged <65 years and 109 males and 98 females aged ≥65 years. For gender and season, there were 122 males and 94 females in the summer period and 126 males and 102 females in the winter period. To assess long term variation there were 195 males and 153 females who had further admissions after more than 12 months. RESULTS: For the four variables listed, there were no significant differences in within individual variation (CVi), but there was a significant difference in between individual variation (CVg) for men and women with regard to age. The Index of Individuality (II) was <0.20 for all conditions studied. We noted that >90% of subjects had an reference change value (RCV) <9 ng/L. CONCLUSIONS: Because troponin concentration in patients without an identified cardiac condition change so little, delta changes are potentially of great value in assessing patients in the ED. Significant delta changes in troponin can occur without the 99th percentile being exceeded.

Gamma-Glutamyl Transferase (GGT) Is the Leading External Quality Assurance Predictor of ISO15189 Compliance for Pathology Laboratories.

Pathology results are central to modern medical practice, informing diagnosis and patient management. To ensure high standards from pathology laboratories, regulators require compliance with international and local standards. In Australia, the monitoring and regulation of medical laboratories are achieved by conformance to ISO15189-National Pathology Accreditation Advisory Council standards, as assessed by the National Association of Testing Authorities (NATA), and an external quality assurance (EQA) assessment via the Royal College of Pathologists of Australasia Quality Assurance Program (RCPAQAP). While effective individually, integration of data collected by NATA and EQA testing promises advantages for the early detection of technical or management problems in the laboratory, and enhanced ongoing quality assessment. Random forest (RF) machine learning (ML) previously identified gamma-glutamyl transferase (GGT) as a leading predictor of NATA compliance condition reporting. In addition to further RF investigations, this study also deployed single decision trees and support vector machines (SVM) models that included creatinine, electrolytes and liver function test (LFT) EQA results. Across all analyses, GGT was consistently the top-ranked predictor variable, validating previous observations from Australian laboratories. SVM revealed broad patterns of predictive EQA marker interactions with NATA outcomes, and the distribution of GGT relative deviation suggested patterns by which to identify other strong EQA predictors of NATA outcomes. An integrated model of pathology quality assessment was successfully developed, via the prediction of NATA outcomes by EQA results. GGT consistently ranked as the best predictor variable, identified by combining recursive partitioning and SVM ML strategies.

Reference intervals for venous blood gas measurement in adults.

OBJECTIVES: Venous blood gas (VBG) analysis is becoming a popular alternative to arterial blood gas (ABG) analysis due to reduced risk of complications at phlebotomy and ease of draw. In lack of published data, this study aimed to establish reference intervals (RI) for correct interpretation of VBG results. METHODS: One hundred and 51 adult volunteers (101 females, 50 males, 18-70 years) were enrolled after completion of a health questionnaire. Venous blood was drawn into safePICO syringes and analysed on ABL827 blood gas analyser (Radiometer Pacific Pty. Ltd.). A non-parametric approach was used to directly establish the VBG RI which was compared to a calculated VBG RI based on a meta-analysis of differences between ABG and VBG. RESULTS: After exclusions, 134 results were used to derive VBG RI: pH 7.30-7.43, partial pressure of carbon dioxide (pCO2) 38-58 mmHg, partial pressure of oxygen (pO2) 19-65 mmHg, bicarbonate (HCO3-) 22-30 mmol/L, sodium 135-143 mmol/L, potassium 3.6-4.5 mmol/L, chloride 101-110 mmol/L, ionised calcium 1.14-1.29 mmol/L, lactate 0.4-2.2 mmol/L, base excess (BE) -1.9-4.5 mmol/L, saturated oxygen (sO2) 23-93%, carboxyhaemoglobin 0.4-1.4% and methaemoglobin 0.3-0.9%. The meta-analysis revealed differences between ABG and VBG for pH, HCO3-, pCO2 and pO2 of 0.032, -1.0 mmol/L, -4.2 and 39.9 mmHg, respectively. Using this data along with established ABG RI, calculated VBG RI of pH 7.32-7.42, HCO3- 23 - 27 mmol/L, pCO2 36-49 mmHg (female), pCO2 39-52 mmHg (male) and pO2 43-68 mmHg were formulated and compared to the VBG RI of this study. CONCLUSIONS: An adult reference interval has been established to assist interpretation of VBG results.

Choice of Statistical Tools for Outlier Removal Causes Substantial Changes in Analyte Reference Intervals in Healthy Populations.

BACKGROUND: Reference intervals are an important aid in medical practice as they provide clinicians a guide as to whether a patient is healthy or diseased.Outlier results in population studies are removed by any of a variety of statistical measures. We have compared several methods of outlier removal and applied them to a large body of analytes from a large population of healthy persons. METHODS: We used the outlier exclusion criteria of Reed-Dixon and Tukey and calculated reference intervals using nonparametric and Harrell-Davis statistical methods and applied them to a total of 36 different analytes. RESULTS: Nine of 36 analytes had a greater than 20% difference in the upper reference limit, and for some the difference was 100% or more. CONCLUSIONS: For some analytes, great importance is attached to the reference interval. We have shown that different statistical methods for outlier removal can cause large changes to reported reference intervals. So that population studies can be readily compared, common statistical methods should be used for outlier removal.

Age-related differences in hs-cTnI concentration in healthy adults.

BACKGROUND: Because the 99th percentile is of such importance in defining myocardial injury and myocardial infarction, it is important to know whether there are real age-related differences in troponin 99th percentiles. METHODS: We went to our database from the Canberra Heart Study where 1062 apparently healthy subjects were extensively screened for occult cardiac disease, and looking at persons aged <65 years and >65 years, for men and women separately, we compared a variety of cutpoints from the 99th percentile down to the 50th percentile. RESULTS: With our rigorous criteria for defining cardiac health, we excluded 67.2% of males aged >65 years and 53.8% of women aged 65 years and older. Even with these rigorous exclusions we found that at every cutpoint examined between the 99th percentile and the 50th percentile, persons aged <65 years had lower troponin I concentrations that persons aged 65 years and older. Similarly, at every cutpoint examined, women had lower troponin I concentrations than did men. For the 4 separate groups examined (men and women, age < 65 years and 65 years and older) after the exclusions of persons with subclinical cardiac disease, the distributions were not significantly different to a Gaussian distribution. CONCLUSIONS: With the rigorous exclusions of persons with subclinical cardiac disease, and the fact that our populations have a Gaussian distribution, our data suggests that age-related hs-cTnI concentrations are real. This has important implications particularly when assessing older persons in the Emergency Department.

The importance of low level QC for high sensitivity troponin assays.

BACKGROUND: With the advent of the new high-sensitivity troponin assays, it is becoming critical to measure troponin accurately to low concentrations. To ensure assay performance is acceptable, appropriate QC must be run. METHODS: In addition to the routine use of commercial QC material, we prepared pools of human QC material with low troponin concentrations close to the limit of quantitation, and ran these regularly on our laboratory analysers. RESULTS: Over 3 years we found no drift or shift in our hs-cTnI assay. We found that only the very low concentration human QC material gave warning of precision problems with the hs-cTnI assay. At the time of the documented poor assay precision, the higher concentration QC material indicated satisfactory performance. CONCLUSIONS: Choice of QC material with an appropriate concentration is important for any assay. For hs-cTn assays, it is of particular importance to use control material with a concentration near to the limit of quantitation.

An update report on the harmonization of adult reference intervals in Australasia.

The Australasian Association of Clinical Biochemists (AACB) has over the past 5 years been actively working to achieve harmonized reference intervals (RIs) for common clinical chemistry analytes using an evidence-based checklist approach where there is sound calibration and metrological traceability. It has now recommended harmonized RIs for 18 common clinical chemistry analytes which are performed in most routine laboratories and these have been endorsed by the Royal College of Pathologists of Australasia (RCPA). In 2017 another group of analytes including urea, albumin and arterial blood gas parameters were considered and suggested harmonized RIs proposed. This report provides an update of those harmonization efforts.

Missed detection of significant positive and negative shifts in gentamicin assay: implications for routine laboratory quality practices.

INTRODUCTION: A product recall was issued for the Roche/Hitachi Cobas Gentamicin II assays on 25th May 2016 in Australia, after a 15 - 20% positive analytical shift was discovered. Laboratories were advised to employ the Thermo Fisher Gentamicin assay as an alternative. Following the reintroduction of the revised assay on 12th September 2016, a second reagent recall was made on 20th March 2017 after the discovery of a 20% negative analytical shift due to erroneous instrument adjustment factor. MATERIALS AND METHODS: The practices of an index laboratory were examined to determine how the analytical shifts evaded detection by routine internal quality control (IQC) and external quality assurance (EQA) systems. The ability of the patient result-based approaches, including moving average (MovAvg) and moving sum of outliers (MovSO) approaches in detecting these shifts were examined. RESULTS: Internal quality control data of the index laboratory were acceptable prior to the product recall. The practice of adjusting IQC target following a change in assay method resulted in the missed negative shift when the revised Roche assay was reintroduced. While the EQA data of the Roche subgroup showed clear negative bias relative to other laboratory methods, the results were considered as possible 'matrix effect'. The MovAvg method detected the positive shift before the product recall. The MovSO did not detect the negative shift in the index laboratory but did so in another laboratory 5 days before the second product recall. CONCLUSIONS: There are gaps in current laboratory quality practices that leave room for analytical errors to evade detection.

The relationship of plasma creatinine (as eGFR) and high-sensitivity cardiac troponin and NT-proBNP concentrations in a hospital and community outpatient population.

OBJECTIVES: While persons with overt renal failure have a well-described rise in troponin and NT-proBNP, it is less well described what the relationship is between cardiac markers and persons with impaired renal function, not requiring dialysis. DESIGN & METHODS: We have collected ALL samples referred to our pathology practice over a 24h period and measured hs-cTnI, hs-cTnT, NT-proBNP, calculated the eGFR, and related our measurements to clinical outcomes. RESULTS: For both men and women, for all of hs-cTnI, hs-cTnT and NT-proBNP, there was a graded response, as renal function worsened, the concentration of the cardiac marker increased. CONCLUSIONS: There is a graded inverse relationship between eGFR and the concentrations of hs-cTnI, hs-cTnT and NT-proBNP. For women only there appeared to be an increase in mortality at lowest eGFR.

Statistical considerations for determining high-sensitivity cardiac troponin reference intervals.

The troponin 99th percentile is used as the laboratory decision point in the diagnosis of acute myocardial infarction. A recent publication has shown that the statistical treatment for outlier removal may dramatically change the calculated troponin 99th percentile. We have used our large database from the previously reported Canberra Heart Study to independently assess the effect of various methods for removing outliers on the calculated 99th percentile. We have performed the same exercise using the troponin 97.5th percentile as an exercise to assess how outlier removal may affect calculated upper reference intervals for any analyte which uses this boundary. For healthy males aged <75years the hs-cTnI troponin 99th percentile varied by a factor>3× depending upon the outlier removal method chosen and for the 97.5th percentile the variation was >50%. For women the variation in the hs-cTnI 99th percentile varied by a factor of nearly 2×. Qualitatively similar results were obtained forhs-cTnT. This is not simply a problem for troponin reference intervals. All analyte reference intervals have the potential to be significantly affected by the method chosen for outlier removal. To ensure that studies can be meaningfully compared, guidance on procedures for removing outliers needs to be standardized as a matter of urgency.

Using a thyroid disease-free population to define the reference interval for TSH and free T4 on the Abbott Architect analyser.

OBJECTIVE: Thyroid disease can be subtle in its presentation, and TSH reference intervals may be artefactually increased by including persons with subclinical thyroid disease. We have therefore used a thyroid disease-free population to determine TSH and fT4 reference intervals. DESIGN: Apparently healthy subjects were assessed by health questionnaire, drug history, clinical assessment and measurement of thyroid antibodies. PATIENTS: Healthy subjects in a community setting. MEASUREMENTS: TSH, free T4, antithyroglobulin and anti-TPO were measured on the Abbott Architect analyser. Subjects with clinical abnormalities, consumption of thyroid-active medications or with thyroid antibodies above the manufacturer-quoted reference intervals were excluded. TSH and fT4 data were log-transformed, and the central 95% was used to calculate reference intervals. We assessed whether these data were normally distributed. We compared samples spanning the reference intervals for both TSH and fT4 between different assays looking at biases. RESULTS: From a population of 1,606 subjects, 140 males (18%) and 284 females (34%) were excluded. The central population 95% for TSH was 0·43-3·28 mU/l and for fT4 10·8-16·8 pmol/l. There were no age- or sex-related differences. For both analytes, the distribution was not significantly different to a Gaussian distribution (P > 0·05). For 5 commonly used assays for TSH, the maximum difference in the upper limit of the TSH reference interval was 0·48 mU/l and for fT4 the maximum difference for the upper reference limit was 4·1 pmol/l. CONCLUSIONS: A substantial proportion of apparently healthy persons have subclinical thyroid disease. These subjects must be excluded for any thyroid hormone reference interval studies.

Cross-sectional study of high-sensitivity cardiac troponins T and I in a hospital and community outpatient setting.

OBJECTIVES: Cardiac troponins are specific for the heart, but not for the acute coronary syndrome. We wanted to assess how common elevated cardiac troponin concentrations were, in a population with significant non-cardiac disease. DESIGN & METHODS: We measured both hs-cTnT and hs-cTnI on all samples submitted to the laboratory during one 24h period, and assessed the magnitude of the cTn concentration with the location and severity of disease of the patient. RESULTS: Community patients and patients from the maternity ward had the lowest cTn concentrations with results above the 99th percentile being only 0-2% of the total. As expected, the highest proportion of results >99th percentile came from Coronary Care and Intensive Care. However, substantial numbers of persons on Medical and Surgical wards, without a primary diagnosis of cardiac disease, also had cTn >99th percentile. Particularly for cTnT, there was a highly significant odds ratio predicting mortality when results above and below the 99th percentile were compared. CONCLUSIONS: Significant illnesses apart from the acute coronary syndrome are important causes of a rise in cTn to above the 99th percentile, and appear to reflect the total body burden of disease. Even when the high hs-cTn concentration is not due to the acute coronary syndrome, there is a significant association with all-cause mortality.

Harmonising Reference Intervals for Three Calculated Parameters used in Clinical Chemistry.

For more than a decade there has been a global effort to harmonise all phases of the testing process, with particular emphasis on the most frequently utilised measurands. In addition, it is recognised that calculated parameters derived from these measurands should also be a target for harmonisation. Using data from the Aussie Normals study we report reference intervals for three calculated parameters: serum osmolality, serum anion gap and albumin-adjusted serum calcium. The Aussie Normals study was an a priori study that analysed samples from 1856 healthy volunteers. The nine analytes used for the calculations in this study were measured on Abbott Architect analysers. The data demonstrated normal (Gaussian) distributions for the albumin-adjusted serum calcium, the anion gap (using potassium in the calculation) and the calculated serum osmolality (using both the Bhagat et al. and Smithline and Gardner formulae). To assess the suitability of these reference intervals for use as harmonised reference intervals, we reviewed data from the Royal College of Pathologists of Australasia/Australasian Association of Clinical Biochemists (RCPA/AACB) bias survey. We conclude that the reference intervals for the calculated serum osmolality (using the Smithline and Gardner formulae) may be suitable for use as a common reference interval. Although a common reference interval for albumin-adjusted serum calcium may be possible, further investigations (including a greater range of albumin concentrations) are needed. This is due to the bias between the Bromocresol Green (BCG) and Bromocresol Purple (BCP) methods at lower serum albumin concentrations. Problems with the measurement of Total CO2 in the bias survey meant that we could not use the data for assessing the suitability of a common reference interval for the anion gap. Further study is required.

Harmonising Adult Reference Intervals in Australia and New Zealand - the Continuing Story.

Reference intervals (RIs) are used to help clinicians determine if a patient can be classified as being in a diseased or healthy state and there are often sound scientific and clinical reasons for differences in RIs. One of the current strategic priorities for the Australasian Association of Clinical Biochemists is to encourage and assist laboratories to achieve harmonisation of RIs for common clinical chemistry analytes where sound calibration and traceability are in place. This need is based on good laboratory practice, providing the clinician with results that allow appropriate and reliable clinical interpretation and progression further toward the national e-health framework and a single electronic health record. After reviewing and considering studies related to bias as well as both a priori and a posteriori RI studies nationally and internationally and the consideration of flagging rates and clinical relevance, an initial group of 12 harmonised RIs were endorsed by the Royal College of Pathologists of Australasia in 2014. In 2015, after further stakeholder consultation, a second group of six harmonised RIs for common chemistry analytes has been proposed for adults which includes ALT and AST where methods do not use pyridoxal-5'-phosphate as an activator and lipase excluding the Ortho Clinical Diagnostics and Siemens Dimension assays.

Opinion Paper: Deriving Harmonised Reference Intervals - Global Activities.

Harmonisation of reference intervals (RIs) refers to use of the same or common RI across different platforms and /or assays for a specified analyte. It occurs optimally for those analytes where there is sound calibration and traceability in place and evidence from a between-method comparison shows that bias would not prevent the use of a common RI. The selection of the RI will depend on various sources of information including local formal RI studies, published studies from the literature, laboratory surveys, manufacturer's product information, relevant guidelines, and mining of databases. Pre-analytical and partitioning issues, significant figures and flagging rates, are assessed for each analyte. Several countries and regions including the Nordic countries, United Kingdom, Japan, Turkey, and Australasia are using common RIs that have been determined either by direct studies or by a consensus process. In Canada, the Canadian Society of Clinical Chemists Taskforce is assessing the feasibility of establishing common reference values using the CALIPER (Canadian Laboratory Initiative on Pediatric Reference Intervals) and CHMS (The Canadian Health Measures Survey) databases as the basis. Development of platform-specific common reference values for each of the major analytical systems may be a more practical approach especially for the majority of analytes that are not standardised against a primary reference method and are not traceable to a primary or secondary reference material. We encourage laboratories to consider adopting reference intervals consistent with those used by other laboratories in your region where it is possible and appropriate for your local population. Local validation of the adopted reference interval is also recommended as per CLSI guidelines.

Influence of exercise intensity and duration on functional and biochemical perturbations in the human heart.

KEY POINTS: Strenuous endurance exercise induces transient functional and biochemical cardiac perturbations that persist for 24-48 h. The magnitude and time-course of exercise-induced reductions in ventricular function and increases in cardiac injury markers are influenced by the intensity and duration of exercise. In a human experimental model, exercise-induced reductions in ventricular strain and increases in cardiac troponin are greater, and persist for longer, when exercise is performed within the heavy- compared to moderate-intensity exercise domain, despite matching for total mechanical work. The results of the present study help us better understand the dose-response relationship between endurance exercise and acute cardiac stress/injury, a finding that has implications for the prescription of day-to-day endurance exercise regimes. ABSTRACT: Strenuous endurance exercise induces transient cardiac perturbations with ambiguous health outcomes. The present study investigated the magnitude and time-course of exercise-induced functional and biochemical cardiac perturbations by manipulating the exercise intensity-duration matrix. Echocardiograph-derived left (LV) and right (RV) ventricular global longitudinal strain (GLS), and serum high-sensitivity cardiac troponin (hs-cTnI) concentration, were examined in 10 males (age: 27 ± 4 years; V̇O2, peak : 4.0 ± 0.8 l min(-1) ) before, throughout (50%, 75% and 100%), and during recovery (1, 3, 6 and 24 h) from two exercise trials. The two exercise trials consisted of 90 and 120 min of heavy- and moderate-intensity cycling, respectively, with total mechanical work matched. LVGLS decreased (P < 0.01) during the 90 min trial only, with reductions peaking at 1 h post (pre: -19.9 ± 0.6%; 1 h post: -18.5 ± 0.7%) and persisting for >24 h into recovery. RVGLS decreased (P < 0.05) during both exercise trials with reductions in the 90 min trial peaking at 1 h post (pre: -27.5 ± 0.7%; 1 h post: -25.1 ± 0.8%) and persisting for >24 h into recovery. Serum hs-cTnI increased (P < 0.01) during both exercise trials, with concentrations peaking at 3 h post but only exceeding cardio-healthy reference limits (14 ng l(-1) ) in the 90 min trial (pre: 4.2 ± 2.4 ng l(-1) ; 3 h post: 25.1 ± 7.9 ng l(-1) ). Exercise-induced reductions in ventricular strain and increases in cardiac injury markers persist for 24 h following exercise that is typical of day-to-day endurance exercise training; however, the magnitude and time-course of this response can be altered by manipulating the intensity-duration matrix.

Multiple biomarkers including cardiac troponins T and I measured by high-sensitivity assays, as predictors of long-term mortality in patients with chronic renal failure who underwent dialysis.

There is a high cardiac mortality in patients on long-term renal dialysis. No studies have reported long-term outcomes relating to both high-sensitivity cardiac troponin T (hs-cTnT) and high-sensitivity cardiac troponin I (hs-cTnI) in these patients. Patients who underwent long-term dialysis at the Canberra Hospital had blood samples collected for both cardiac and other biomarkers. Samples were stored at -80°C until analysis. Mortality data were collected at 5 years, and univariate and multivariate analyses were performed to identify which biomarkers were predictive of mortality at 5 years. After multivariate analysis, albumin, C-reactive protein (CRP), and hs-cTnT remained independently predictive of all-cause mortality, with hs-cTnT having the highest hazard ratio. If hs-cTnT was excluded from the analysis, then hs-cTnI was independently predictive of mortality. For hs-cTnT, for both genders, the ninety-ninth percentile, derived from a population with subjects with subclinical disease excluded, served as an excellent partition between survivors and nonsurvivors. Receiver-operating characteristic curve analysis for hs-cTnT had area under the curve of 0.798 and for hs-cTnI of 0.774. Kaplan-Meier curves for the aggregation of albumin, CRP, and hs-cTnT showed a stronger predictive power with receiver-operating characteristic area under the curve of 0.805. The addition of echocardiographic data in an analysis of all patients who had an echocardiogram for clinical reasons (n = 105) did not alter the final observations in this subgroup. In conclusion, hs-cTnT retains a superior predictive power in a dialysis-dependent population for identifying those at risk for death and when aggregated with albumin and CRP also has substantial additive value for identifying mortality risk in a renal-dialysis population.

Altered ventricular mechanics after 60 min of high-intensity endurance exercise: insights from exercise speckle-tracking echocardiography.

Transient reductions in myocardial strain coupled with cardiac-specific biomarker release have been reported after prolonged exercise (>180 min). However, it is unknown if 1) shorter-duration exercise (60 min) can perturb cardiac function or 2) if exercise-induced reductions in strain are masked by hemodynamic changes that are associated with passive recovery from exercise. Left ventricular (LV) and right ventricular global longitudinal strain (GLS), LV torsion, and high-sensitivity cardiac troponin T were measured in 15 competitive cyclists (age: 28 ± 3 yr, peak O2 uptake: 4.8 ± 0.6 l/min) before and after a 60-min high-intensity cycling race intervention (CRIT60). At both time points (pre- and post-CRIT60), strain and torsion were assessed at rest and during a standardized low-intensity exercise challenge (power output: 96 ± 8 W) in a semirecumbent position using echocardiography. During rest, hemodynamic conditions were different from pre- to post-CRIT60 (mean arterial pressure: 96 ± 1 vs. 86 ± 2 mmHg, P < 0.001), and there were no changes in strain or torsion. In contrast, during the standardized low-intensity exercise challenge, hemodynamic conditions were unchanged from pre- to post-CRIT60 (mean arterial pressure: 98 ± 1 vs. 97 ± 1 mmHg, not significant), but strain decreased (left ventricular GLS: -20.3 ± 0.5% vs. -18.5 ± 0.4%, P < 0.01; right ventricular GLS: -26.4 ± 1.6% vs. -22.4 ± 1.5%, P < 0.05), whereas LV torsion remained unchanged. Serum high-sensitivity cardiac troponin T increased by 345% after the CRIT60 (6.0 ± 0.6 vs. 20.7 ± 6.9 ng/l, P < 0.05). This study demonstrates that exercise-induced functional and biochemical cardiac perturbations are not confined to ultraendurance sporting events and transpire during exercise that is typical of day-to-day training undertaken by endurance athletes. The clinical significance of cumulative exposure to endurance exercise warrants further study.