Amy K. Saenger

Multicenter analytical evaluation of a high-sensitivity troponin T assay.

BACKGROUND High-sensitivity cardiac troponin assays are being introduced clinically for earlier diagnosis of acute myocardial infarction (AMI). We evaluated the analytical performance of a high-sensitivity cardiac troponin T assay (hscTnT, Roche Diagnostics) in a multicenter, international trial. METHODS Three US and 5 European sites evaluated hscTnT on the Modular® Analytics E170, cobas® 6000, Elecsys 2010, and cobas® e 411. Precision, accuracy, reportable range, an inter-laboratory comparison trial, and the 99th percentile of a reference population were assessed. RESULTS Total imprecision (CVs) were 4.6-36.8% between 3.4 and 10.3 ng/L hscTnT. Assay linearity was up to 10,000 ng/L and the limit of blank and detection were 3 and 5 ng/L, respectively. The 99th percentile reference limit was 14.2 ng/L (n=533). No significant differences between specimen types, assay incubation time, or reagent lots existed. A substantial positive bias (76%) exists between the 4th generation and hscTnT assays at the low end of the measuring range (<50 ng/L). hscTnT serum pool concentrations were within 2SD limits of the mean of means in the comparison trial, indicating comparable results across multiple platforms and laboratories. CONCLUSION The Roche hscTnT assay conforms to guideline precision requirements and will likely identify additional patients with myocardial injury suspicious for AMI.

Diseased skeletal muscle: a noncardiac source of increased circulating concentrations of cardiac troponin T.

OBJECTIVES The purpose of this study was to determine whether there is immunoreactive cardiac troponin T (cTnT) expression in diseased skeletal muscle that might cause possible false-positive increases in cTnT. BACKGROUND Cardiac troponin (I or T) is the biomarker of choice for the diagnosis of cardiac injury. Recently, we were presented with a case that challenged the specificity of cTnT. METHODS Patients with myopathies seen in the Neuromuscular Clinic at the Mayo Clinic were screened for increases in cTnT. If present, an assay for cTnI was performed. If normal, skeletal biopsy tissue was obtained for Western blot analysis using the capture and detection antibodies from both the fourth-generation and high-sensitivity cTnT assays. Results were compared with findings in normal cardiac tissue. RESULTS Sixteen patients had increases in cTnT but not cTnI. All had a myopathy by clinical evaluation, clinical testing, and biopsy. Four residual biopsy samples were obtained. All 3 antibodies used in the cTnT (M11.7, M7) and high-sensitivity cTnT (5D8, M7) assays were immunoreactive with a 37- to 39-kDa protein in all 4 diseased skeletal muscle biopsy specimens and in cardiac tissue. A second immunoreactive isoform (34 to 36 kDa) was also found in 1 patient. None of the noncardiac control tissues expressed immunoreactive protein. CONCLUSIONS These results document that there are forms in diseased skeletal muscle that could cause increases in circulating levels of cTnT. These increases could reflect re-expressed isoforms. Clinicians need to be aware of the possibility that noncardiac increases in cTnT may occur and lead to a possible false-positive diagnosis of cardiac injury when skeletal muscle pathology is present.

Stroke biomarkers: progress and challenges for diagnosis, prognosis, differentiation, and treatment.

BACKGROUND Stroke is a devastating condition encompassing a wide range of pathophysiological entities that include thrombosis, hemorrhage, and embolism. Current diagnosis of stroke relies on physician clinical examination and is further supplemented with various neuroimaging techniques. A single set or multiple sets of blood biomarkers that could be used in an acute setting to diagnosis stroke, differentiate between stroke types, or even predict an initial/reoccurring stroke would be extremely valuable. CONTENT We discuss the current classification, diagnosis, and treatment of stroke, focusing on use of novel biomarkers (either solitary markers or multiple markers within a panel) that have been studied in a variety of clinical settings. SUMMARY The current diagnosis of stroke remains hampered and delayed due to lack of a suitable mechanism for rapid (ideally point-of-care), accurate, and analytically sensitive biomarker-based testing. There is a clear need for further development and translational research in this area. Potential biomarkers identified need to be transitioned quickly into clinical validation testing for further evaluation in an acute stroke setting; to do so would impact and improve patient outcomes and quality of life.

Biological and analytical variability of a novel high-sensitivity cardiac troponin T assay.

BACKGROUND High-sensitivity cardiac troponin assays will augment the frequency of increased results, making important the determination of reference change values to distinguish acute from chronic increases. We assessed short- and long-term biological variability of cardiac troponin T (cTnT) in healthy subjects with a novel high-sensitivity (hs) assay. METHODS We collected blood from 20 healthy volunteers at 5 time points for short-term study and biweekly at 4 times from the same volunteers for long-term study. We analyzed serum samples in duplicate with a hscTnT assay on the Roche Modular E170 and computed reference change values (RCVs) for analytical, intraindividual, interindividual, and total change values (CV(A), CV(I), CV(G), and CV(T), respectively) and the index of individuality (II). We calculated RCVs by using a log-normal approach, owing to the skewed results of the data. RESULTS Short- and long-term CV(A) values were 53.5% and 98%. CV(I) and CV(G) were 48.2% and 85.9%, respectively, for short-term studies and 94% and 94% for long-term studies. Mean delta values for the within-day study were 58% and -57.5%, and between-day mean delta values were 103.4% and -87%. Within- and between-day IIs were 0.8 and 0.14, respectively. CONCLUSIONS The biological variation demonstrated with the hscTnT assay is higher than prior data for cardiac troponin I. This may be attributed to differences in biology or assay imprecision at low concentrations. A short-term change (RCV log normal) of 85% and a long-term change of 315% is necessary to define a changing pattern.

Expedited PublicationDiseased Skeletal Muscle: A Noncardiac Source of Increased Circulating Concentrations of Cardiac Troponin T

OBJECTIVES The purpose of this study was to determine whether there is immunoreactive cardiac troponin T (cTnT) expression in diseased skeletal muscle that might cause possible false-positive increases in cTnT. BACKGROUND Cardiac troponin (I or T) is the biomarker of choice for the diagnosis of cardiac injury. Recently, we were presented with a case that challenged the specificity of cTnT. METHODS Patients with myopathies seen in the Neuromuscular Clinic at the Mayo Clinic were screened for increases in cTnT. If present, an assay for cTnI was performed. If normal, skeletal biopsy tissue was obtained for Western blot analysis using the capture and detection antibodies from both the fourth-generation and high-sensitivity cTnT assays. Results were compared with findings in normal cardiac tissue. RESULTS Sixteen patients had increases in cTnT but not cTnI. All had a myopathy by clinical evaluation, clinical testing, and biopsy. Four residual biopsy samples were obtained. All 3 antibodies used in the cTnT (M11.7, M7) and high-sensitivity cTnT (5D8, M7) assays were immunoreactive with a 37- to 39-kDa protein in all 4 diseased skeletal muscle biopsy specimens and in cardiac tissue. A second immunoreactive isoform (34 to 36 kDa) was also found in 1 patient. None of the noncardiac control tissues expressed immunoreactive protein. CONCLUSIONS These results document that there are forms in diseased skeletal muscle that could cause increases in circulating levels of cTnT. These increases could reflect re-expressed isoforms. Clinicians need to be aware of the possibility that noncardiac increases in cTnT may occur and lead to a possible false-positive diagnosis of cardiac injury when skeletal muscle pathology is present.

Relative Performance of the MDRD and CKD-EPI Equations for Estimating Glomerular Filtration Rate among Patients with Varied Clinical Presentations

BACKGROUND The Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equation was developed using both CKD and non-CKD patients to potentially replace the Modification of Diet in Renal Disease (MDRD) equation that was derived with only CKD patients. The objective of our study was to compare the accuracy of the MDRD and CKD-EPI equations for estimating GFR in a large group of patients having GFR measurements for diverse clinical indications. DESIGN, SETTING, PARTICIPANTS, AND MEASUREMENTS A cross-sectional study was conducted of patients who underwent renal function assessment for clinical purposes by simultaneous measurements of serum creatinine and estimation of GFR using the MDRD and CKD-EPI equations and renal clearance of iothalamate (n = 5238). RESULTS Bias compared with measured GFR (mGFR) varied for each equation depending on clinical presentation. The CKD-EPI equation demonstrated less bias than the MDRD equation in potential kidney donors (-8% versus -18%) and postnephrectomy donors (-7% versus -15%). However, the CKD-EPI equation was slightly more biased than the MDRD equation in native CKD patients (6% versus 3%), kidney recipients (8% versus 1%), and other organ recipients (9% versus 3%). Among potential kidney donors, the CKD-EPI equation had higher specificity than the MDRD equation for detecting an mGFR <60 ml/min per 1.73 m(2) (98% versus 94%) but lower sensitivity (50% versus 70%). CONCLUSIONS Clinical presentation influences the estimation of GFR from serum creatinine, and neither the CKD-EPI nor MDRD equation account for this. Use of the CKD-EPI equation misclassifies fewer low-risk patients as having reduced mGFR, although it is also less sensitive for detecting mGFR below specific threshold values used to define CKD stages.

Requiem for a Heavyweight The Demise of Creatine Kinase-MB

The development of rapid, automated, and accurate laboratory testing for creatine kinase MB (CK-MB) revolutionized the treatment of patients with acute cardiac events in the 1970s and 1980s.1 To clinicians, CK-MB values augmented a thorough history, physical, and ECG findings, and elevations rapidly became the gold standard for identifying cardiac injury.1 CK-MB allowed earlier diagnosis of acute myocardial infarction (AMI), and detection of reinfarction, and measurements could be used to provide a facile clinical estimate of infarct size. Elevations of CK-MB were never intended to be synonymous with myocardial infarction, only indicative of cardiac injury.1 However, because of the relative insensitivity of measurements, increased concentrations occurred predominantly with larger insults such those associated with acute ischemic heart disease. For that reason, AMI was rarely diagnosed, assuming appropriate timing of the samples, in the absence of a CK-MB elevation.2,3 CK-MB assays initially relied on the measurement of enzyme activity, but over time, improved accuracy and ease of use were established by the use of mass assays. Mass assays allowed earlier detection of abnormal values and improved both clinical sensitivity and specificity. However, mass assays unmasked an increased frequency of CK-MB elevations due primarily to skeletal muscle injury because of their increased sensitivity.4–6 Clinical use of the percent relative index was then initiated. This approach improved the specificity of elevations for cardiac muscle injury but was insensitive when concurrent cardiac injury and skeletal muscle injury were present because elevations from skeletal muscle often are of a large magnitude.7–10 A large number of analytical confounds such as macrokinases and interfering substances also were substantial problems with these assays.10,11 Attempts to standardize assays12 have been partially successful, but differences still exist between manufacturers and even between the same testing antibodies used on different analytical platforms (ie, …

High sensitivity cardiac troponin T in patients with immunoglobulin light chain amyloidosis

Objectives To define whether the high sensitivity cardiac troponin T (hs-cTnT) assay in patients with immunoglobulin light chain amyloidosis (AL) improves risk prediction. Background Cardiac involvement is the major cause of death in patients with AL amyloidosis. Risk stratification is facilitated by cardiac biomarkers such as cardiac troponin T (cTnT) and N-terminal pro B-type natriuretic peptide (NT-proBNP). Methods Stored serum from patients with newly diagnosed AL was used to measure hs-cTnT, cTnT, and NT-proBNP. Survival modelling was performed. Results The direct numeric result from hs-cTnT measurement cannot merely be substituted for a cTnT measurement in the Mayo AL staging system. The performance of the receiver operator curve derived an hs-cTnT cut-point of 54 ng/L which improves on the value of 35 ng/L validated with the prior iteration of the assay. An alternate staging option using hs-cTnT alone—using the two thresholds 14 ng/L and 54 ng/L—performs as well as either the original Mayo AL staging system or other systems incorporating hs-cTnT. On multivariate analysis, an hs-cTnT alone staging system was independent of period of diagnosis, type of therapy, and NT-proBNP value, the last of which dropped out of the model. Alternate models were explored, but none performed better than the original system or the new hs-cTnT system. Thus, hs-cTnT can be used alone for the staging of disease prognosis. Conclusions A survival model based on hs-cTnT improves the prognostic staging of patients with AL amyloidosis, relegating NT-proBNP to a measure of cardiac response.

Biologic Variation of a Novel Cardiac Troponin I Assay

To the Editor: Cardiac troponin is the marker of choice for evaluating myocardial injury (1). High-sensitivity assays improve analytical detection limits, thereby allowing concentrations to be measured in the majority of healthy individuals. This capability allows an assessment of biologic variation (BV)1 to determine what constitutes a clinically important change in the cardiac troponin concentration, a critical metric for identifying acute events. Such an event is often a myocardial infarction, but any acute cardiac injury can cause increasing and/or decreasing values (1). Accordingly, we evaluated BV for a recently developed high-sensitivity cardiac troponin I (hs-cTnI) assay (2) from Beckman Coulter. We performed this study with the same cohort used to define BV for the high-sensitivity cardiac troponin T (hs-cTnT) assay (3) and according to a protocol approved by our institutional review board. For assessment of short-term BV, we collected blood into serum separator tubes (Becton Dickinson) at 0, 1, 2, 3, and 4 h, centrifuged the tubes, and stored the serum samples immediately at −70 °C. …

Catecholamine Interference in Enzymatic Creatinine Assays

BACKGROUND Enzymatic creatinine assays are routinely used in clinical laboratories to provide more accurate estimated glomerular filtration rates and to avoid a perceived lack of analytical specificity associated with picrate (Jaffe) methods. Negative interferences with the enzymatic creatinine assay, which we noted in several patients on dopamine or dobutamine, prompted our further investigation into interference of catecholamines with enzymatic methods. METHODS Spiked solutions of dopamine, dobutamine, epinephrine, and norepinephrine were added to pooled sera at catecholamine concentrations consistent with clinically relevant dosing. Creatinine was measured enzymatically on the Roche P-Modular, Ortho Clinical Diagnostics Vitros 350, and Abbott i-STAT. Jaffe methods were performed on the Roche P-Modular and Siemens Dimension RxL. In 10 patients receiving dopamine and/or dobutamine via a venous or arterial line we evaluated and compared the extent of in vivo creatinine interference in paired serum samples obtained by venipuncture and from indwelling catheters. RESULTS All catecholamines caused significant negative interference with the Roche enzymatic creatinine assay, most pronounced for dopamine and dobutamine. The Vitros enzymatic assay demonstrated slight negative interferences, and i-STAT enzymatic and Jaffe methods were unaffected by the presence of catecholamines. Significant (P < 0.001) differences in creatinine concentrations by Roche enzymatic vs Jaffe methods were observed in venipuncture specimens compared with arterial or venous catheter specimens, suggesting dopamine and dobutamine reversibly adhere to the catheter lumen. CONCLUSIONS Negative interferences were pronounced for Roche enzymatic results in blood samples obtained from indwelling catheters, a phenomenon not observed in peripheral draws. Physicians and laboratorians should be alert to the possibility of a falsely low creatinine result and reevaluate questionable samples using a method unaffected by catecholamines.