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Objectives: In patients presenting to the Emergency Department (ED) with potential acute myocardial infarction (AMI), elevated cardiac troponin (cTn) levels are indicative of myocardial necrosis. We assessed the accuracy of 'delta cTn' at 2h or 6h compared to the cTn concentration above the 99th percentile reference value for AMI in a prospective study of adult patients presenting to ED with symptoms suggestive of possible acute coronary syndrome., Methods: Blood was sampled for cardiac troponin I (cTnI) on presentation, and at 2h and 6h following presentation using a sensitive assay (Beckman AccuTnI). All clinical endpoints were adjudicated by a cardiologist who was blinded to the 2h cTn assay result., Results: Of the 874 patients, 70 (8%) were diagnosed with an AMI during their index presentation. The area under the ROC curve for diagnosing AMI at 2h was 0.89 [95%CI, 0.84-0.95] for absolute delta cTn versus 0.79 [95%CI 0.73-0.85] for the relative change. Specificity and PPV at 2h were optimized using a delta cTnI ≥ 0.03 μg/L (95.8% [95%CI 94.1-97.0] and 61.4% [95%CI 50.9-70.9] respectively). Sensitivity and NPV for AMI were optimized using the 99th percentile with the addition of a delta of<0.03 μg/L (97.1% [95%CI 90.2-99.2] and 99.7% [95%CI 99-99.9] respectively)., Conclusions: An algorithm incorporating cTnI concentration and delta cTn values with a sensitive troponin assay allows accurate diagnosis of AMI within 2h from presentation and earlier rule-out of AMI in the majority of patients., (Crown Copyright © 2013. Published by Elsevier Ireland Ltd. All rights reserved.)

Objectives: The study objective was to validate a new high-sensitivity troponin I (hs-TnI) assay in a clinical protocol for assessing patients who present to the emergency department with chest pain., Background: Protocols using sensitive troponin assays can accelerate the rule out of acute myocardial infarction in patients with low-risk (suspected) acute coronary syndrome (ACS)., Methods: This study evaluated 2 prospective cohorts of patients in the emergency department with ACS in an accelerated diagnostic pathway integrating 0- and 2-h hs-TnI results, Thrombolysis In Myocardial Infarction (TIMI) risk scores, and electrocardiography. Strategies to identify low-risk patients incorporated TIMI risk scores= 0 or ≤ 1. The primary endpoint was a major adverse cardiac event (MACE) within 30 days., Results: In the primary cohort, 1,635 patients were recruited and had 30-day follow-up. A total of 247 patients (15.1%) had a MACE. The finding of no ischemic electrocardiogram and hs-TnI ≤ 26.2 ng/l with the TIMI = 0 and TIMI ≤ 1 pathways, respectively, classified 19.6% (n = 320) and 41.5% (n = 678) of these patients as low risk; 0% (n = 0) and 0.8% (n = 2) had a MACE, respectively. In the secondary cohort, 909 patients were recruited. A total of 156 patients (17.2%) had a MACE. The TIMI = 0 and TIMI ≤ 1 pathways classified 25.3% (n = 230) and 38.6% (n = 351), respectively, of these patients as low risk; 0% (n = 0) and 0.8% (n = 1) had a MACE, respectively. Sensitivity, specificity, and negative predictive value for TIMI = 0 in the primary cohort were 100% (95% confidence interval [CI]: 98.5% to 100%), 23.1% (95% CI: 20.9% to 25.3%), and 100% (95% CI: 98.8% to 100%), respectively. Sensitivity, specificity, and negative predictive value for TIMI ≤ 1 in the primary cohort were 99.2 (95% CI: 97.1 to 99.8), 48.7 (95% CI: 46.1 to 51.3), and 99.7 (95% CI: 98.9 to 99.9), respectively. Sensitivity, specificity, and negative value for TIMI ≤ 1 in the secondary cohort were 99.4% (95% CI: 96.5 to 100), 46.5% (95% CI: 42.9 to 50.1), and 99.7% (95% CI: 98.4 to 100), respectively., Conclusions: An early-discharge strategy using an hs-TnI assay and TIMI score ≤ 1 had similar safety as previously reported, with the potential to decrease the observation periods and admissions for approximately 40% of patients with suspected ACS. (Advantageous Predictors of Acute Coronary Syndromes Evaluation [APACE] Study, NCT00470587; A 2 hr Accelerated Diagnostic Protocol to Assess patients with chest Pain symptoms using contemporary Troponins as the only biomarker [ADAPT]: a prospective observational validation study, ACTRN12611001069943)., (Copyright © 2013 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.)

Introduction: The uremic toxins indoxyl sulphate (IS) and p-cresyl sulphate (pCS) are absorbed bacterial metabolites of tryptophan and tyrosine respectively and may be predictive of clinical outcome. Long chromatography times, incomplete data on the reference ranges of the free and total fractions and the biological variation limit wider clinical application., Methods: An UPLC method with fluorescence detection was developed and reference ranges and biological variation were investigated in healthy volunteers., Results: Chromatography time was 3 min with excellent linearity, precision and low detection limits (IS of 0.02 μmol/L and pCS of 0.05 μmol/L). Both IS and pCS increased with a decrease in renal function and were moderately correlated with eGFR (R(2) 0.65 and 0.33 respectively). The serum reference ranges were (μmol/L): total IS of 0.7-6.3; free IS of 0.0-0.2; total pCS of 0.0-38.4; and free pCS of 0.1-2.4. The intra individual biological variation was estimated at 35.9% and 50.5% with a critical difference of 3.9 μmol/L (100%) and 20.7 μmol/L (141%) for total IS and pCS respectively., Conclusion: We describe a robust analytical method with a short chromatography time that quantifies both IS and pCS. The data on reference ranges and intra-individual biological variation need to be considered in clinical studies that investigate these uremic toxins., (Crown Copyright © 2012. Published by Elsevier B.V. All rights reserved.)

Objectives: To compare cardiac risk stratification using a 0 and 2 h point-of-care (POC) cardiac troponin (cTn), 0 and 2 h POC multi-biomarkers against the 0 and 6 h laboratory cTn reference standard., Methods: A prospective observational study of ED patients presenting with chest pain was performed. Patients were risk stratified and treated as per the Heart Foundation of Australia/Cardiac Society of Australia and New Zealand (HF-A/CS-ANZ) guidelines using the 0 and 6 h laboratory cTn (T6). Patients were further stratified using a 0 and 2 h POC cTn (T2) plus 0 and 2 h POC multi-biomarkers (cTn, creatine kinase-MB, myoglobin) (M2). The T6, T2 and M2 strategies were compared using the 30-day major adverse cardiac events as the primary outcome., Results: Seven hundred and four patients (median age 54 years, male 62.1%) were enrolled. Using the T6 reference standard, 2%, 61% and 37% were stratified as low, intermediate and high risk, respectively. The 30-day event rates were 0%, 3.5% and 28.6%, respectively. The T2 strategy stratified 1.5%, 57% and 41% as low, intermediate and high risk, respectively, with 30-day event rates of 0%, 4.2% and 24.8%, respectively. The M2 strategy resulted in significantly different risk distribution with 1%, 40% and 59% stratified as low, intermediate and high risk, respectively, with 30-day event rates of 0%, 3.9% and 18.8%, respectively., Conclusion: Using a 2 h POC cTn-only biomarker strategy with the HF-A/CS-ANZ guidelines accurately identified a population at intermediate risk of 30-day events in whom further objective testing might be accelerated, allowing subsequent early discharge of the majority of this cohort. Within 2 h of presentation a high risk population could be identified in whom early management, including admission, was required., (© 2012 The Authors. EMA © 2012 Australasian College for Emergency Medicine and Australasian Society for Emergency Medicine.)

Introduction: The diagnosis of myocardial infarction is in part predicated on a rise and/or fall in cardiac troponin (cTn). z-Values incorporate analytical and biological variation to standardize serial differences: z=Δ/√[2SD²(Analytical) + 2SD²(Biological)]. We investigated the theoretical distributions of actual differences (Δ), relative differences (%Δ) and z-values and compared the agreement in classification of differences measured on four contemporary platforms., Methods: cTn was measured in 575 sample pairs on the Abbott Architect cTnI, Beckman Coulter Access2 cTnI, Roche Cobas cTnT and Siemens ADVIA Centaur cTnI methods., Results: Good agreement was obtained amongst all methods with a universal z-value cut-off (κ>0.79) and method specific fixed Δ cut-offs (κ>0.82) while suboptimal agreement was obtained between cTnI and cTnT with fixed %Δ cut-offs (κ<0.50)., Conclusion: Fixed Δ cut-offs will perform well at low cTn concentrations while fixed %Δ cut-off values are predicted to perform poorly. z-Values are independent of the cTn concentration, present differences as a continuum of probability and a universal decision level can be used for all cTnI and cTnT methods., (Crown Copyright © 2012. Published by Elsevier B.V. All rights reserved.)

With the clinical imperative to further research in the area of optimising antibiotic dosing in the intensive care setting, a simple high performance liquid chromatography method was developed and validated for routinely determining the free (unbound) concentration of ten beta-lactam antibiotics in 200 μL of human plasma. Antibiotics determined include three cephalosporins (ceftriaxone, cephazolin and cephalotin); two carbapenems (meropenem and ertapenem); and five penicillins (ampicillin, piperacillin, benzylpenicillin, flucloxacillin and dicloxacillin). There was a single common sample preparation method involving ultracentrifugation and stabilisation. Chromatography was performed on a Waters XBridge C18 column with, depending on analytes, one of four acetonitrile-phosphate buffered mobile phases. Peaks of interest were detected via ultraviolet absorbance at 210, 260 and 304 nm. The method has been validated and used in a pathology laboratory for therapeutic drug monitoring in critically ill patients. The significant variability in the level of protein binding that is common with antibiotics traditionally considered to have high protein binding (e.g. ceftriaxone, cephazolin, ertapenem, flucloxacillin and dicloxacillin) suggests that this assay should be preferred for measuring the pharmacologically active concentration of beta-lactam antibiotics in a therapeutic drug monitoring programme., (Crown Copyright © 2012. Published by Elsevier B.V. All rights reserved.)

Background: Cardiac troponin (cTn) has become the standard biomarker for the diagnosis of acute coronary syndromes. False-positive cTnI results have previously been reported on the Beckman Coulter analysers, which were shown to be random, not reproducible and occurred more commonly than expected. Our investigation ensued after a patient sample with an inordinately elevated cTnI was analysed, followed by a series of false-positive results being reported. The implications of falsely elevated cTnI results on patient care could be considerable., Methods: Multiple experiments with patient sample pools with concentrations below the 99th percentile to extremely high (0.025, 15, 175 and 884 μg/L) were conducted in varying sequences of high and low samples on the Beckman Coulter Access2, UniCel DxI600 and UniCel DxI800 analysers., Results: Our results demonstrate a significant increase in cTnI concentrations in the negative pool after analysis of high pool samples in various sequences. This increase is sufficient to cause elevations above the 99th percentile cut-off and false-positive cTnI results. These findings were reproducible on all three analysers., Conclusions: Our study is highly suggestive of carryover and cTnI reagent pack contamination by the pipettors on the Access2, DxI600 and DxI800 analysers when patient samples with extremely high cTnI concentrations are analysed, leading to potential false-positive cTnI results on subsequent samples.

Published data on adrenocortical function in septic shock have enrolled patients at various stages of critical illness and predominantly used plasma total cortisol, with minimal information on serial changes. Moreover, plasma free cortisol and tissue corticosteroid activity may not be strongly associated; however, few published data exist. The aim of this prospective observational study was to investigate serial changes in plasma total and free cortisol and tissue cortisol activity in septic shock. Twenty-nine adult patients admitted with septic shock to a tertiary-level intensive care unit were enrolled. A low-dose corticotropin test was performed on day 1. Plasma total and free cortisol, cortisone, transcortin, and urinary free cortisol and cortisone were analyzed on days 1 to 5, 7, and 10. Urinary and plasma cortisol-cortisone ratios (F:E ratio) were calculated as indices of 11-β hydroxysteroid dehydrogenase 2 and global 11-β hydroxysteroid dehydrogenase activity, respectively. Baseline total and free plasma cortisol values from 10 healthy control subjects were obtained for comparative analysis. Baseline plasma total and free cortisol levels were significantly higher than controls (457.8 ± 193 vs. 252 ± 66 nmol/L, P = 0.0002; and 50.83 ± 43.19 vs. 6.4 ± 3.2, P < 0.0001, respectively). Plasma free cortisol rose proportionately higher than total cortisol (124% ± 217.3% vs. 40% ± 33.2%, P = 0.007) following corticotropin. Baseline plasma and urinary F:E ratios were elevated over the reference ranges (13.13 ± 1.5, 1.69 ± 2.8) and were not correlated with plasma free cortisol values (r = 0.2, 0.3 respectively). Over the study period, total cortisol levels and plasma F:E ratios remained elevated, whereas plasma free cortisol levels and urinary F:E ratio declined. At baseline, plasma free cortisol levels were higher in patients who subsequently survived (23.7 ± 10.5 vs. 57.9 ± 45.8 nmol/L, P = 0.04). In septic shock, there is a differential response of plasma total and free cortisol over time and in response to corticotropin. Changes in plasma and urinary F:E ratios suggest tissue modulation of 11-β hydroxysteroid dehydrogenase activity. Total plasma cortisol measurements may not reflect the global adrenal response in septic shock.

Background: Free cortisol (FC) can be calculated from measurements of total cortisol and binding proteins or measured after mechanical separation of unbound and bound fractions by equilibrium dialysis or ultrafiltration. FC can then be measured indirectly by 3H-cortisol dilution or directly by immunologic or tandem mass spectrometry assays., Methods: We compared FC measured with ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC MS/MS) with 3H-cortisol dilution in ultrafiltrates and dialysates and also with calculated FC (Coolens equation). An adult FC reference interval was established., Results: The UHPLC MS/MS and 3H-cortisol dilution methods were non-linearly related (Cusum linearity test p<0.001) but well correlated (R2=0.984). FC calculated with Coolens equation agreed with the UHPLC MS/MS method. Impurity of 3H-cortisol and non-specific adsorption were excluded as causes on non-linearity. Ultrafiltration was linearly related to equilibrium dialysis, simpler to perform and more repeatable. A gender non-specific FC reference interval of 2.1-19.1 nmol/L was established., Conclusions: In view of the non-linearity between measuring techniques and the variability of reported reference ranges, care should be exercised in adopting a reference range. The ultrafiltration UHPLC MS/MS method we described is robust and suitable for use in a routine laboratory., (Crown Copyright © 2011. Published by Elsevier B.V. All rights reserved.)

We describe an ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC MS/MS) method suitable for a routine laboratory to determine endogenous and exogenous glucocorticoids in plasma, plasma ultrafiltrate, urine and saliva in a single analytical run. After addition of a multi-analyte internal standard, a standardised sample preparation procedure with solid phase extraction followed, before injecting into a tandem mass spectrometer with positive mode electron spray ionisation and multiple reactant monitoring acquisition. The chromatography time was 3min. The limit of quantitation for cortisol and cortisone in plasma was 3.75nmol/L and linearity extended to 2000nmol/L. The limit of quantitation for cortisol in plasma ultrafiltrate and saliva was 0.6nmol/L. The limit of quantitation for 11-deoxycortisol and prednisolone was 5nmol/L and for dexamethasone 1nmol/L. The intra-assay CV was <5% and the inter-assay CV <10% for all analytes in all matrices. Comparison with an immuno-assay (IA) plasma cortisol method resulted in a regression equation of UHPLC=0.79×IA+31.12 with R(2)=0.960 (p<0.0001). Comparison with a high performance liquid chromatography (HPLC) cortisol method yielded a regression equation of UHPLC=1.06×HPLC+9.82, R(2)=0.992 (p<0.0001). The simultaneous measurement of endogenous and exogenous glucocorticoids contributed to patient care in cases with dexamethasone and metyrapone dynamic tests and unsuspected therapeutic glucocorticoid use., (Crown Copyright © 2010. Published by Elsevier B.V. All rights reserved.)

A simple and economical high performance liquid chromatography method was developed and validated for routine analysis of 12 Penicillin, Cephalosporin and Carbapenem antibiotics in 200 microL of human plasma. Antibiotics determined were Ceftazidime, Meropenem, Ceftriaxone, Ampicillin, Cefazolin, Ertapenem, Cephalothin, Benzylpenicillin, Flucloxacillin, Dicloxacillin, Piperacillin and Ticarcillin. There was a common sample preparation approach involving precipitation of proteins with acetonitrile and removal of lipid-soluble components by a chloroform wash. Separations were performed on a Waters X-bridge C18 column with, depending on analytes, one of three acetonitrile-phosphate buffer mobile phases. Detection was by UV at 210, 260 and 304 nm. Validation has demonstrated the method to be linear, accurate and precise. The method has been used in a pathology laboratory for therapeutic drug monitoring (TDM) of beta-lactams in critically ill patients., (Crown Copyright 2010. Published by Elsevier B.V. All rights reserved.)