An appraisal of the practice of duplicate testing for the detection of irregular analytical errors.

OBJECTIVES: Our study aimed to determine the usefulness of duplicate testing in identifying irregular analytical errors and subsequent prevention of patient mismanagement. METHODS: In our laboratory, all requests for Na+, Ca2+, alkaline phosphatase (ALP), and high-sensitivity cardiac-troponin-I (hs-cTnI) are run in duplicate. Data from four separate weeks for Na+ (n=21,649), Ca2+ (n=14,803) and ALP (n=19,698); and a full year for hs-cTnI (n=17,036) were gathered. For each test, pre-defined limits for differences between duplicates were used to identify erroneous results (Fliers). We further characterised a subset of such fliers as "critical errors", where duplicates fell on opposing sides of a reference/decision making threshold. The costs/benefits of running these tests in duplicate were then considered in light of increased number of tests analysed by this approach. RESULTS: For Na+, 0.03 % of duplicates met our flier defining criteria, and 0.01 % of specimens were considered critical errors. For Ca2+ requests, 4.58 % of results met our flier defining criteria and 0.84 % were critical errors. For ALP, 0.22 % of results were fliers, and 0.01 % were critical errors. For hs-cTnI, 1.58 % of results were classified as fliers, whilst 0.14 % were classified as a critical error. Depending on the test in question, running all analyses in duplicate increased annual costs by as little as €1,100 (for sodium), and as much as €48,000 (for hs-cTnI). CONCLUSIONS: Duplicate testing is effective at identifying and mitigating irregular laboratory errors, and is best suited for assays predisposed to such error, where costs are minimal, and clinical significance of an incorrect result can justify the practice.

In control? IQC consensus and statutory regulation.

Purpose - Internal quality control (IQC) represents an essential risk management tool within the total testing pathway (TTP) that contributes to the overall objective of assuring the quality of results produced in medical laboratories. Controlling analytical phase quality alone requires significant expertise and input by scientifically trained staff. This effort has escalated exponentially following the publication of the International Organisation for Standardisation (ISO)15189:2012 requirements for quality and competence in medical laboratories. The reported inconsistency and diversity to IQC approaches in diagnostic laboratories is definitive evidence that international guidance in IQC programme design and implementation is long overdue. The paper aims to discuss these issues. Design/methodology/approach - Herein, the authors define, describe and critically examine the essential elements four stages of an IQC programme and suggest a template to inform both design and ease of implementation. For practical application, the authors have stratified the proposed methodology into four stages: staff education and training; IQC material; IQC targets; and IQC procedure, and provide recommendations that meet ISO15189:2012 requirements. Findings - These recommendations are informed by the published literature together with the collective experience working in clinical biochemistry and diagnostic endocrinology laboratories. The authors note that the laboratory staff's effort on IQC is a continuous process, driven by changes within each IQC stage, in response to risk analysis, maximising economic value or through professional leadership and central to IQC programme implementation and delivery. Practical implications - The authors offer a template that laboratories can use to inform the design and implementation of their IQC programme. Originality/value - The proposed IQC programme is user friendly, flexible and pragmatic with the potential to harmonise practice. The authors have provided a template to potentially harmonise IQC practice nationally. Given the central and critical role that IQC practice plays in ensuring the quality of patient results' importance, the authors contend that the time has come for international consensus and statutory regulation regarding the minimally acceptable criteria for its implementation, monitoring and review.

Laboratory services: regaining and maintaining control.

Purpose - After implementing an internal quality control (IQC) programme, the purpose of this paper is to maintain the requisite analytical performance for clinical laboratory staff, thereby safeguarding patient test results for their intended medical purpose. Design/methodology/approach - The authors address how quality can be maintained and if lost, how it can be regained. The methodology is based on the experience working in clinical laboratory diagnostics and is in accord with both international accreditation requirements and laboratory best practice guidelines. Findings - Monitoring test performance usually involves both prospective and retrospective IQC data analysis. The authors present a number of different approaches together with software tools currently available and emerging, that permit performance monitoring at the level of the individual analyser, across analysers and laboratories (networks). The authors make recommendations on the appropriate response to IQC rule warnings, failures and metrics that indicate analytical control loss, that either precludes further analysis, or signifies deteriorating performance and eventual unsuitability. The authors provide guidance on systematic troubleshooting, to identify undesirable performance and consider risk assessment preventive measures and continuous quality improvement initiatives; e.g., material acceptance procedures, as tools to help regain and maintain analytical control and minimise potential for patient harm. Practical implications - The authors provide a template for use by laboratory scientific personnel that ensures the optimal monitoring of analytical test performance and response when it changes undesirably. Originality/value - The proposed template has been designed to meet the International Organisation for Standardisation for medical laboratories ISO15189:2012 requirements and therefore includes the use of External Quality Assessment and patient results data, as an adjunct to IQC data.

Total plasma homocysteine measurement: Evaluation of the Abbott immunoassay, comparison with the JEOL ion exchange chromatography and investigation of its clinical utility.

Objectives: Homocysteine is an intermediary amino acid formed in methionine metabolism, with elevated total homocysteine (tHCY) being a biomarker of cardiovascular and cerebrovascular diseases. We evaluated the Abbott ARCHITECT tHCY immunoassay, compared it with the current established JEOL ion exchange chromatography (IEC) method and evaluated its clinical utility. Design and methods: Following immunoassay method verification, plasma samples of 91 patients were analysed for tHCY using immunoassay and IEC. Results: For the Abbott immunoassay, accuracy was assessed, with UK NEQAS EQA specimens, by the correlation of our Abbott immunoassay measurements to the Abbott ARCHITECT immunoassay mean (bias = 1.6%), and to the overall immunoassay mean (bias = 2.0%). The total imprecision was 2.7% (11.00 µmol/L), 2.4% (16.80 µmol/L) and 2.8% (24.30 µmol/L) respectively. Bias in linearity assessment was 0.12%-2.58%. The inter-method correlation was strong in Passing-Bablok regression: immunoassay = IEC x0.857 + 2.445 (95% CI: slope = [0.742,0.947], intercept = [1.340,3.582]), with Spearman correlation = 0.803 (p < 0.001). The Bland-Altman plot showed an average difference of -0.284 µmol/L (95% CI: [-1.043,0.474]) with limits of agreement (mean ± 1.96SD) from -7.425 µmol/L to 6.857 µmol/L.No significant difference in tHCY was found using both methods in patients with cerebrovascular diseases and cardiovascular diseases. Most tHCY measurements were within the reference ranges of both methods. All homocystinuria patients had tHCY values above the reference ranges of both methods. Conclusions: The immunoassay demonstrated robust performance in its verification and showed good comparability with the IEC but with some biases so caution is needed if both are used interchangeably. The immunoassay offers an automated alternative to IEC in the assessment of hyperhomocysteinaemia.

Effects of an endothelin receptor antagonist on a model of hypertensive retinopathy.

Hypertensive retinopathy manifests itself as progressive retinal microvascular pathology in response to aberrant blood flow. The current study sought to evaluate whether dysfunction of the vasoactive endothelin-1 (ET-1) system is involved in the pathogenesis of hypertension-induced retinopathy in an animal model of systemic hypertension. The endothelin receptor antagonist, bosentan, was administered to spontaneously hypertensive rats (SHRs) and comparisons were made with untreated SHRs and normotensive Wistar Kyoto (WKY) rats. The retinal mRNA expression of ET-1, ET-converting enzyme-1, ET(A) and ET(B) receptors and the basement membrane proteins, laminin beta1, collagen IV and fibronectin was quantified using real-time RT-PCR. In addition, retinal arteriole and/or capillary bed damage was assessed by qualitative and quantitative microscopy. mRNA for the ET(A) receptor was increased in SHRs, when compared to WKY control animals (p < 0.001). Treatment with bosentan in SHRs significantly reduced the expression of ET-1 (p < 0.05), and both the ET(A) (p < 0.0001) and ET(B) (p < 0.05) receptor subtypes. The laminin beta1, collagen IV and fibronectin mRNA expression was significantly higher in SHRs when compared to WKY control animals (p < 0.001). Treatment with bosentan abolished these responses and also the appearance of various microvascular lesions. ET-mediated vasoregulation abnormalities in the retinal microvasculature could play an associative role in lesion formation during hypertensive retinopathy.

Generating method-specific Reference Ranges - A harmonious outcome?

OBJECTIVES: When laboratory Reference Ranges (RR) do not reflect analytical methodology, result interpretation can cause misclassification of patients and inappropriate management. This can be mitigated by determining and implementing method-specific RRs, which was the main objective of this study. DESIGN AND METHODS: Serum was obtained from healthy volunteers (Male + Female, n > 120) attending hospital health-check sessions during June and July 2011. Pseudo-anonymised aliquots were stored (at - 70 °C) prior t° analysis on Abbott ARCHITECT c16000 chemistry and i2000SR immunoassay analysers. Data were stratified by gender where appropriate. Outliers were excluded statistically (Tukey method) to generate non-parametric RRs (2.5th + 97.5th percentiles). RRs were compared to those quoted by Abbott and UK Pathology Harmony (PH) where possible. For 7 selected tests, RRs were verified using a data mining approach. RESULTS: For chemistry tests (n = 23), Upper or Lower Reference Limits (LRL or URL) were > 20% different from Abbott ranges in 25% of tests (11% from PH ranges) but in 38% for immunoassay tests (n = 13). RRs (mmol/L) for sodium (138-144), potassium (3.8-4.9) and chloride (102-110) were considerably narrower than PH ranges (133-146, 3.5-5.0 and 95-108, respectively). The gender difference for ferritin (M: 29-441, F: 8-193 ng/mL) was more pronounced than reported by Abbott (M: 22-275, F: 5-204 ng/mL). Verification studies showed good agreement for chemistry tests (mean [SD] difference = 0.4% [1.2%]) but less so for immunoassay tests (27% [29%]), particularly for TSH (LRL). CONCLUSION: Where resource permits, we advocate using method-specific RRs in preference to other sources, particularly where method bias and lack of standardisation limits RR transferability and harmonisation.

Transitioning high sensitivity cardiac troponin I (hs-cTnI) into routine diagnostic use: More than just a sensitivity issue.

OBJECTIVES: High sensitivity cardiac troponin T and I (hs-cTnT and hs-cTnI) assays show analytical, diagnostic and prognostic improvement over contemporary sensitive cTn assays. However, given the importance of troponin in the diagnosis of myocardial infarction, implementing this test requires rigorous analytical and clinical verification across the total testing pathway. This was the aim of this study. DESIGN AND METHODS: Analytical verification included assessment of critical outlier frequency, for hs-cTnI and cTnI assays. Concordance for paired cTnI and hs-cTnI measurements (n=1096) was verified using 99th percentiles for both genders (cTnI: 30 ng/L, hs-cTnI: 25 ng/L) and for men and women separately (hs-cTnI: M: 34;F: 16 ng/L). Discordant data was correlated with clinical and laboratory information. Diagnosis of Acute Coronary Syndrome (ACS) or Non-ACS was adjudicated by two cardiologists independently. RESULTS: The hs-cTnI assay showed a lower (10-fold) critical outlier rate (0.091%) and more detectable results above the limit of detection (LOD) (23.4%) and 99th percentile (2.4%), compared to cTnI. Analytical concordance between the two assays was high (94.5%) but decreased (91.7%) when gender-specific hs-cTnI cut-offs were used. The hs-cTnI assay gave fewer false negatives (up to 1.0%) but disproportionately more false positives (up to 6.7%) overall, which improved (3.9%) for serial measurements. CONCLUSIONS: Laboratories should analytically and clinically verify hs-cTn assays before use, with attention to performance and the clinical and diagnostic algorithms that support appropriate testing and result interpretation. Work in the pre- and post-analytical phases is necessary to augment the analytical improvement in the new era of troponin testing.

Temporal characteristics of cardiomyocyte hypertrophy in the spontaneously hypertensive rat.

BACKGROUND: The spontaneously hypertensive rat (SHR) is frequently used as model of cardiovascular disease, with considerable disparity in reported parameters of hypertrophy. The aim of this study was to assess the temporal changes occurring during the development and progression of cardiomyocyte hypertrophy in SHR, subsequent to pressure overload, compared to changes associated with normal aging using the normotensive Wistar-Kyoto (WKY) rat. METHODS: Ventricular cardiomyocytes were isolated from rats at 8, 12, 16, 20 and 24 weeks, and parameters of hypertrophy (cell dimensions, protein mass, de novo protein synthesis, and gene expression) and function (contraction and hypertrophic responsiveness in vitro) were assessed. RESULTS: Hypertension was evident at > or =7 weeks in SHRs. Heart:body mass ratio, cardiomyocyte protein mass and width were elevated (P<.05) in SHRs at 16-20 weeks compared to WKYs. In SHRs compared to WKYs at 16 weeks, there was a transient increase (P<.05) in protein synthesis, enhanced hypertrophic responsiveness to phorbol-12-myristate-13-acetate, and induced hypertrophic responsiveness to isoprenaline. Skeletal-alpha-actin mRNA was detected in SHR but not WKY cells at all ages. ANP mRNA was lower in SHR than in WKY cells at 8-20, but progressively increased (P<.05) from 12 to 24 weeks within SHRs. Contractile function increased (P<.05) at 20 weeks in SHR compared to WKY rats. CONCLUSION: Structural and functional changes occurring at the cellular level in the myocardium of SHR follow a distinct pattern, such that pressure overload was initially accompanied by expressional changes (8-12 weeks), followed by active hypertrophic growth and enhanced function (16-20 weeks), which subsequently decelerated as stable compensation was attained.

Peri-operative troponin monitoring using a prototype high-sensitivity cardiac troponin I (hs-cTnI) assay: comparisons with hs-cTnT and contemporary cTnI assays.

BACKGROUND: Non-cardiac surgery is associated with major vascular complications and higher incidences of elevated plasma troponin (cTn) concentration. Goal-directed therapy (GDT) is a stroke volume (SV)-guided approach to intravenous (IV) fluid therapy that improves tissue perfusion, oxygenation and reduces post-operative complications. In patients undergoing major gastro-intestinal surgery, we compared high sensitive and contemporary troponin assays and correlated results with patient outcome. METHODS: Patients (n = 135) were randomized to receive IV fluid, guided by either the central venous pressure (CVP group, n = 45) or SV (± dopexamine inotrope, n = 45 per group). Serum was obtained pre- and post-operatively (0, 8 and 24 h) for troponin analysis by a prototype hs-cTnI assay (Abbott Laboratories), hs-cTnT (Roche Diagnostics) and contemporary cTnI (Beckman Coulter) assays. RESULTS: All troponin measurements were increased (P ≤ 0.05) post-operatively but there was no difference (P > 0.05) amongst treatments. Post-operative increases were reported more frequently (P ≤ 0.05) and earlier with hs-cTnI. Temporal increases (P ≤ 0.05) were reported in patients with and without complications for hs-cTnI/T assays but only in the complications group for cTnI measurements. Elevations ≥99th centile occurred most often (P ≤ 0.05) for hs-cTnT measurements but with similar frequency for both outcome groups (all assays). Only the hs-cTnI assay showed an increased relative risk of mortality (P ≤ 0.05) for elevations ≥99th centile CONCLUSIONS: Our study may suggest a possible preference for the hs-cTnI assay in the peri-operative setting; however, our findings should be verified for larger cohort studies where emerging reference range data is incorporated for improving risk prediction with hs-cTn assays.

A comparison of plasma-free metanephrines with plasma catecholamines in the investigation of suspected pheochromocytoma.

OBJECTIVE: To compare the diagnostic performance of plasma metanephrines by ELISA and plasma catecholamine measurements by HPLC in patients selected for clonidine suppression testing. METHODS: Plasma catecholamines adrenaline (ADR) and noradrenaline (NOR) were measured by HPLC and metanephrine with normetanephrine (NMN) by ELISA (n = 67). The diagnostic performance of metanephrines was determined by receiver operating characteristic (ROC) curve analysis. RESULTS: Phaeochromocytoma was confirmed by histological analysis in 14 patients and excluded in 53 patients by a negative clonidine suppression test (CST), abdominal computerized tomography scan and clinical follow-up (median 2.5 years). A sensitivity and specificity of 100 and 96%, respectively, was obtained by using our current CST diagnostic criteria for ADR and NOR values. ROC curve analysis revealed optimum sensitivity and specificity for plasma-free metanephrines using a threshold of 784 pmol/l at baseline and 663 pmol/l at 180 min. Baseline measurements of metanephrine with NMN showed 100% sensitivity and 98% specificity, as assessed by ROC curve analysis-derived criteria or when evaluated against published decision thresholds. A sensitivity and specificity of 100% was obtained for the combined measurements of metanephrine with NMN at 180 min. CONCLUSION: Plasma metanephrines (metanephrine with NMN) were equally effective as plasma catecholamines during CST. This study supports the use of measuring plasma metanephrines by ELISA as a less labour-intensive and equally effective biochemical test for phaeochromocytoma in patients with a high clinical suspicion. There was still overlap between groups with and without phaeochromocytoma at baseline under controlled conditions and clinically some patients still need to undergo clonidine suppression testing.

Evidence for altered ETB receptor characteristics during development and progression of ventricular cardiomyocyte hypertrophy.

The hypothesis that endothelin (ET) receptor mechanisms are altered during development and progression of left ventricular hypertrophy (LVH) in vivo was tested using spontaneously hypertensive rats (SHRs). Ventricular cardiomyocytes were isolated from SHRs before onset (8 and 12 wk) and during progression (16, 20, and 24 wk) of LVH and compared with age-matched normotensive Wistar-Kyoto (WKY) rats. PreproET-1 mRNA expression was elevated in SHR (P < 0.05) relative to WKY cardiomyocytes at 20-24 wk. ET binding-site density was twofold greater in SHR than WKY cells at 12 wk (P < 0.05) but normalized at 20 wk. ET(B) receptors were detected on SHR cardiomyocytes as early as 8 wk and their affinity increased progressively with age (P < 0.05), whereas ET(B) receptors were not detected on WKY cells until 20 wk. ET-1 stimulated protein synthesis with similar maximum responses between strains (21-30%), in contrast with sarafotoxin 6c, which stimulated protein synthesis in SHR (13-20%) but not WKY cells at 12-20 wk. In SHR but not WKY cells, the ET(B) receptor-selective ligand A-192621 increased protein synthesis progressively with the development of LVH (15% maximum effect). In conclusion, the presence of ET(B) receptors (8-12 wk) coupled with functional responsiveness of SHR cells but not WKY cells to sarafotoxin 6c at 12 wk supports the involvement of ET(B) receptors before the onset of cardiomyocyte hypertrophy, whereas altered ET(B) receptor characteristics during active hypertrophy (16-24 wk) indicate that ET(B) receptor mechanisms may also contribute to disease progression.