Carel J. Pretorius

Measurement of cortisol, cortisone, prednisolone, dexamethasone and 11-deoxycortisol with ultra high performance liquid chromatography–tandem mass spectrometry: Application for plasma, plasma ultrafiltrate, urine and saliva in a routine laboratory

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.

Neonatal complete generalized glucocorticoid resistance and growth hormone deficiency caused by a novel homozygous mutation in Helix 12 of the ligand binding domain of the glucocorticoid receptor gene (NR3C1).

CONTEXT Glucocorticoid resistance is a rare genetic condition characterized by reduced sensitivity to cortisol signaling and subsequent hyperactivation of the hypothalamic-pituitary-adrenal axis. OBJECTIVE The objective was to confirm the diagnosis of glucocorticoid resistance in the patient, to determine the degree of suppression of cortisol and ACTH levels in response to dexamethasone, and to determine the underlying genetic abnormality and functional consequences of the mutation. PATIENT AND METHODS The patient presented on the first day of life with profound hypoglycemia. Initial cortisol levels were appropriately elevated; however, the patient was found to have persistently elevated levels of both cortisol and ACTH. The baby developed a tanned appearance and severe hypertension and fatigued easily with feeding. Serial oral dexamethasone suppression tests were performed with doses escalating from 0.125 mg to 12 mg dexamethasone given at 2300 h. Sequencing of the glucocorticoid receptor gene was performed along with functional studies of the glucocorticoid receptor. GH secretion was assessed with an arginine glucagon stimulation test. RESULTS Cortisol and ACTH levels did not suppress with doses of up to 12 mg dexamethasone. A 2-bp deletion was found at amino acid position 773 of the glucocorticoid receptor ligand binding domain. A complete lack of dexamethasone binding and in vitro biological effect was demonstrated. GH stimulation testing was consistent with GH deficiency. CONCLUSION The homozygous mutation in the ligand-binding domain of the glucocorticoid receptor gene resulted in a functionally inactive glucocorticoid receptor and apparent complete glucocorticoid resistance with biochemical GH deficiency.

Concordance, Variance, and Outliers in 4 Contemporary Cardiac Troponin Assays: Implications for Harmonization

BACKGROUND Data to standardize and harmonize the differences between cardiac troponin assays are needed to support their universal status in diagnosis of myocardial infarction. We characterized the variation between methods, the comparability of the 99th-percentile cutoff thresholds, and the occurrence of outliers in 4 cardiac troponin assays. METHODS Cardiac troponin was measured in duplicate in 2358 patient samples on 4 platforms: Abbott Architect i2000SR, Beckman Coulter Access2, Roche Cobas e601, and Siemens ADVIA Centaur XP. RESULTS The observed total variances between the 3 cardiac troponin I (cTnI) methods and between the cTnI and cardiac troponin T (cTnT) methods were larger than expected from the analytical imprecision (3.0%-3.7%). The between-method variations of 26% between cTnI assays and 127% between cTnI and cTnT assays were the dominant contributors to total variances. The misclassification of results according to the 99th percentile was 3%-4% between cTnI assays and 15%-17% between cTnI and cTnT. The Roche cTnT assay identified 49% more samples as positive than the Abbott cTnI. Outliers between methods were detected in 1 patient (0.06%) with Abbott, 8 (0.45%) with Beckman Coulter, 10 (0.56%) with Roche, and 3 (0.17%) with Siemens. CONCLUSIONS The universal definition of myocardial infarction should not depend on the choice of analyte or analyzer, and the between- and within-method differences described here need to be considered in the application of cardiac troponin in this respect. The variation between methods that cannot be explained by analytical imprecision and the discordant classification of results according to the respective 99th percentiles should be addressed.

Evaluation of the N Latex FLC free light chain assay on the Siemens BN analyser: precision, agreement, linearity and variation between reagent lots.

Background The clinical utility of serum κ and λ free light chains (FLC) for the diagnosis and prognosis of plasma cell proliferative disorders is well established. We assessed the analytical performance of the N Latex FLC assays and compared it with the Freelite™ assays. Methods Analytical precision was assessed according to the CLSI EP5-A2 protocol. Method comparison was performed with 116 clinical samples and linearity was assessed on samples with monoclonal and polyclonal elevations of FLC. Analytical bias and variance was measured with two lots of N Latex FLC reagent. Results The N Latex FLC κ and λ assays had a total coefficient of variation of 5–7% across the analytical range. The slopes were 1.36 and 1.37 and the between-method variances were 40.0% and 45.4%, respectively, compared with the Freelite™. Good agreement in classification was observed for κ, λ and the κ/λ ratio (Cohens κ 0.84, 0.76 and 0.89). Statistical non-linearity occurred commonly, but clinically significant non-linearity was observed in only one instance. Between-reagent lot variation was 7.3% and 10% for κ and λ, respectively. Conclusions The N Latex FLC assay has good precision, did not exhibit gross antigen excess and can be used in clinical practice based on the analytical performance characteristics.

Outliers as a Cause of False Cardiac Troponin Results:Investigating the Robustness of 4 Contemporary Assays

BACKGROUND It is important that cardiac troponin be measured accurately with a robust method to limit false results with potentially adverse clinical outcomes. In this study, we characterized the robustness of 4 analytical platforms by measuring the outlier rate between duplicate results. METHODS We measured cardiac troponin concurrently in duplicate with 4 analyzers on 2391 samples. The outliers were detected from the difference between duplicate results and by calculating a z value: z = (result 1 - result 2) ÷ √(SD1(est)² + SD2(est)²), with z > 3.48 identifying outliers with a probability of 0.0005. RESULTS The outlier rates were as follows: Abbott Architect i2000SR STAT Troponin-I, 0.10% (0.01%-0.19%); Beckman Coulter Access2 Enhanced AccuTnI, 0.44% (0.25%-0.63%); Roche Cobas e601 TroponinT hs, 0.06% (0.00%-0.13%); and Siemens ADVIA Centaur XP TnI-Ultra, 0.10% (0.01%-0.19%). The occurrence of outliers was higher than statistically expected on all platforms except the Cobas e601 (χ² = 2.7; P = 0.10). A conservative approach with a constant 10% CV and z > 5.0 identified outliers with clear clinical impact and resulted in outlier rates of 0.11% (0.02%-0.20%) with the Architect i2000SR STAT Troponin-I, 0.36% (0.19%-0.53%) with the Access2 Enhanced AccuTnI, 0.02% (0.00%-0.06%) with the Cobas e601 TroponinT hs, and 0.06% (0.00%-0.13%) with the ADVIA Centaur XP TnI-Ultra. CONCLUSIONS Outliers occurred on all analytical platforms, at different rates. Clinicians should be made aware by their laboratory colleagues of the existence of outliers and the rate at which they occur.

Serial changes in plasma total cortisol, plasma free cortisol and tissue cortisol activity in patients with septic shock: An observational study

ABSTRACT 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-&bgr; hydroxysteroid dehydrogenase 2 and global 11-&bgr; 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-&bgr; hydroxysteroid dehydrogenase activity. Total plasma cortisol measurements may not reflect the global adrenal response in septic shock. ABBREVIATIONS 11&bgr;-HSD—11&bgr;-hydroxysteroid dehydrogenase APACHE—Acute Physiology and Chronic Health Evaluation CBG—corticosteroid-binding globulin FC—free plasma cortisol F:E ratio—cortisol-cortisone ratio SOFA—Sequential Organ Failure Assessment

Free cortisol method comparison: Ultrafiltation, equilibrium dialysis, tracer dilution, tandem mass spectrometry and calculated free cortisol

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.

Discordance with 3 Cardiac Troponin I and T Assays: Implications for the 99th Percentile Cutoff

BACKGROUND We compared the 99th percentile reference intervals with 3 modern cardiac troponin assays in a single cohort and tested the hypothesis that the same individuals will be identified as above the cutoff and that differences will be explained by analytical imprecision. METHODS Blood was collected from 2005 apparently healthy blood donors. Cardiac troponin was measured with Abbott Architect STAT high sensitive troponin I, Beckman Coulter Access AccuTnI+3, and Roche Elecsys troponin T highly sensitive assays. RESULTS The 99th percentile cutoff limits were as follows: Abbott cardiac troponin I (cTnI) 28.9 ng/L; Beckman Coulter cTnI 31.3 ng/L; and Roche cardiac troponin T (cTnT) 15.9 ng/L. Correlation among the assays was poor: Abbott cTnI vs Beckman Coulter cTnI, R(2) = 0.18; Abbott cTnI vs Roche cTnT, R(2) = 0.04; and Beckman Coulter cTnI vs Roche cTnT R(2) = 0.01. Of the results above the cutoff 50% to 70% were unique to individual assays, with only 4 out of 20 individuals above the cutoff for all 3 assays. The observed differences among assays were larger than predicted from analytical imprecision. CONCLUSIONS The 99th percentile cutoff values were in agreement with those reported elsewhere. The poor correlation and concordance amongst the assays were notable. The differences found could not be explained by analytical imprecision and indicate the presence of inaccuracy (bias) that is unique to sample and assay combinations. Based on these findings we recommend less emphasis on the cutoff value and greater emphasis on δ values in the diagnosis of myocardial infarction.

PTHrP-Mediated Hypercalcemia: Causes and Survival in 138 Patients

CONTEXT Hypercalcemia is a common complication of cancer with PTHrP an important mediator. Literature on the underlying causes of PTHrP-mediated hypercalcemia, in both malignant and benign conditions, is limited to small case series and case reports. OBJECTIVE The purpose of this study was to systematically identify a large series of cases of PTHrP-mediated hypercalcemia and to document differences in demographics and the clinical course between malignant and benign etiologies. DESIGN, SETTING, AND PATIENTS This was a hospital-based, retrospective case series that identified subjects from 1999 to 2010 from the public hospital system in Queensland, Australia. Included subjects were 18 years and older and had persistent hypercalcemia with simultaneously elevated PTHrP. RESULTS A total of 138 cases were identified. Solid organ malignancies made up 82.6% (n = 114) of cases, with squamous cell carcinoma (28.2% of total) and adenocarcinomas (27.5%) almost equally as common. Hematological malignancy and benign conditions made up 8.7% (n = 12) each. Squamous cell carcinoma of the lung was the single most commonly identified etiology (10.9%). Causes not previously identified included myxoid sarcoma, plasma cell leukemia, duodenal adenocarcinoma, metastatic Merkel cell carcinoma, and epithelioid hemangioendothelioma. Median survival was different among the groups (52 days [interquartile range, 21-132 days] for solid organ malignancy, 362 days [18-652 days] for hematological malignancy, and 906 days [16 days to undefined] for the apparently benign group; P < .0001). There were no differences in PTHrP among the groups. Although the mean corrected calcium level was lower in the benign group (3.03 mmol/L [2.80-3.29 mmol/L]) compared with that in the solid organ (3.11 mmol/L [2.89-3.46 mmol/L]) and hematological malignancy groups (3.60 mmol/L [3.01-3.79 mmol/L]) groups (P = .046), it was not a useful discriminator of etiology. CONCLUSION PTHrP-mediated hypercalcemia is most frequently caused by solid organ malignancy, and it portends a poor prognosis. Although the solid organ malignancy group had the shortest survival, the hematological malignancy and apparently benign causes groups still had relatively short overall survival.

ELF score ≥9.8 indicates advanced hepatic fibrosis and is influenced by age, steatosis and histological activity

There is increasing need to identify individuals with advanced liver fibrosis, who are at risk of complications such as hepatocellular carcinoma. The commercially available enhanced liver fibrosis (ELF) test provides a non‐invasive assessment of fibrosis severity. This study was designed to determine the diagnostic accuracy of the manufacturers cut‐off value (≥9.8) in identifying advanced fibrosis.