CCA-13944; No of Pages 4 Clinica Chimica Acta xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Clinica Chimica Acta journal homepage: www.elsevier.com/locate/clinchim
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Janka Franeková a,d, Martin Bláha b, Jiří Bělohoubek c, Markéta Kotrbatá a, Peter Sečník Jr. a, Zdeněk Kubíček a, Jiří Kettner b, Antonín Jabor a,d,⁎
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Article history: Received 29 January 2015 Received in revised form 23 February 2015 Accepted 2 March 2015 Available online xxxx
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Keywords: Troponin Heterophile antibodies Immunoassay interference
Department of Laboratory Methods, Institute for Clinical and Experimental Medicine, Vídeňská 1958/9, 140 21 Prague 4, Czech Republic Department of Cardiology, Institute for Clinical and Experimental Medicine, Vídeňská 1958/9, 140 21 Prague 4, Czech Republic Department of Preventive Cardiology, Institute for Clinical and Experimental Medicine, Vídeňská 1958/9, 140 21 Prague 4, Czech Republic d Charles University, 3rd Faculty of Medicine, Ruská 87, 100 00 Prague 10, Czech Republic b
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A clinical and laboratory approach used to elucidate discordant results of high-sensitivity troponin T and troponin I
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Case report
Background: Careful interpretation of discordant results in high-sensitivity troponin measurements is necessary in cases of suspect immunoassay interferences. We describe several procedures taken in a case of a polymorbid patient with chest pain, without clear evidence of myocardial necrosis and with increased high-sensitivity cardiac troponin T (hs-cTnT). We checked the Vafaie's algorithm for the evaluation of suspect interference in troponin measurements. Methods: We conducted a case report analysis, additional measurements, a dilution test and pretreatment of plasma with blocking agents. Results: Concentration of hs-cTnT (99th percentile of “healthy” population 14 ng/L) increased from 120.1 ng/L to 280.4 ng/L during an 8-month period and decreased to 216.3 ng/L during the following month with repeatedly negative troponin I (TnI), hs-cTnI, myoglobin and creatine kinase MB (CK-MB). Suspected false positivity of hs-cTnT was further confirmed by treatment of plasma with an antiheterophile blocking agent (hs-cTnT before treatment 280.4 ng/L, after 16.53/16.23 ng/L). This outcome was further confirmed by the manufacturer's experiments. Conclusions: The false-positive results of hs-cTnT were caused by the presence of extremely rare high molecular weight protein, presumably IgM, most likely HAMA (human anti-mouse antibody). Only the pre-treatment of plasma with a blocking agent provided a reliable indication of the interference. Cooperation among clinicians, laboratory personnel and the manufacturer is essential. © 2015 Published by Elsevier B.V.
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1. Introduction
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The introduction of high-sensitivity cardiac troponin (hs-cTn) measurements tightens requirements on the interpretation of borderline increases of troponin concentrations as well as of unambiguously increased but stable concentrations due to different influences. The high quality of hs-cTn measurement does not exclude rare situations of falsely increased (or decreased) concentrations of troponin; e.g., as a result of analytical interference. Here, we describe a rare case of discordant levels of hs-cTn measured by two routine systems in which the interference was found in only
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⁎ Corresponding author at: Department of Laboratory Methods, Institute for Clinical and Experimental Medicine, Vídeňská 1958/9, 140 21 Prague 4, Czech Republic. Tel.: +420 236 055 236. E-mail addresses: [email protected] (J. Franeková), [email protected] (M. Bláha), [email protected] (J. Bělohoubek), [email protected] (M. Kotrbatá), [email protected] (P. Sečník), [email protected] (Z. Kubíček), [email protected] (J. Kettner), [email protected] (A. Jabor).
one immunoassay, and we characterize a series of steps taken to 52 clarify the reason for this discrepancy. We also tested the effective- 53 ness of Vafaie's recommendation for these situations [1]. 54 2. Materials and methods
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2.1. Patients
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The study was approved by IKEM Ethic Committee. The patient gave written informed consent approving all examinations and procedures (IKEM F-39). All additional examinations including testing at the manufacturer's facilities were blinded.
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2.2. Materials and methods
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All measurements were performed in an accredited clinical 62 laboratory (ISO 15189:2007). Hs-cTnT was measured with the 63 Elecsys Troponin T hs STAT kit on Cobas 6000 (both Roche Diagnostic 64
http://dx.doi.org/10.1016/j.cca.2015.03.046 0009-8981/© 2015 Published by Elsevier B.V.
Please cite this article as: Franeková J, et al, A clinical and laboratory approach used to elucidate discordant results of high-sensitivity troponin T and troponin I, Clin Chim Acta (2015), http://dx.doi.org/10.1016/j.cca.2015.03.046
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J. Franeková et al. / Clinica Chimica Acta xxx (2015) xxx–xxx
Table 2 Other laboratory examinations and procedures with the potential to show falsely increased troponins.
t2:3 t2:4 Q2
Possible causes of false-positive troponin results [1,2; modified]
t2:5 t2:6
Heterophile antibodies (HAAA, human anti-animal antibodies; Concentrations of other analytes with the same type of potential interference were measured (Cobas 6000): HAMA, human anti-mouse antibodies) PSA: under limit of detection CEA: 0.6 μg/L (reference range b3.8 μg/L) CA 19-9: 11.9 kU/L (b27 kU/L) CA 72-4: 1.2 kU/L (b6.9 kU/L) CA 125: 3.6 kU/L (b35 kU/L) Autoantibodies ANCA negative, anti-myocardium antibodies negative Rheumatoid factor Negative (20.1 kIU/L, reference range up to 30 kIU/L) Fibrin clots, microparticles in specimen Excluded, careful separation of blood cells, measured both in serum and lithium-heparin plasma Interference caused by endogenous components in blood Bilirubin total: 9.1–11.6 μmol/L (reference range 3.4–20 μmol/L) (bilirubin, hemoglobin, lipemia) Lipemia: excluded, lipemic index measured on every sample, lipid profile: total cholesterol 3.7 mmol/L, HDL cholesterol 1.31 mmol/L, LDL cholesterol 1.8 mmol/L, triglycerides 1.31 mmol/L Hemoglobin: excluded, hemolysis index measured in every sample High concentration of alkaline phosphatase 0.91–0.97 μkat/L (reference range 0.58–1.75 μkat/L) Immunocomplex formation Circulating immunocomplexes: 0.284 (index of positivity, values b0.9 are negative), C1q: negative Analyzer malfunction Excluded, results repeatedly measured, results confirmed in another laboratory (hs-cTnT) Use of another analytical system Mitsubishi Pathfast: cTnI 2 ng/L (cut-off 2 ng/L; system uses 2 monoclonal mouse antibodies)
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Our results
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ANCA = anti-neutrophil cytoplasmic antibodies; CA = cancer antigen; CEA = carcinoembryonic antigen; HAAA = human anti-animal antibodies; HAMA = human anti-mouse antibodies; LDL cholesterol = low-density lipoprotein cholesterol; PSA = prostate specific antigen.
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Ltd., Rotkreuz, Switzerland). Hs-cTnI and cTnI were measured on an Abbott Architect i2000SR system with ARCHITECT hs Troponin I and ARCHITECT STAT Troponin-I kits, respectively (all designed by Abbott Diagnostics, Lake Forest, Illinois, US). Skybio HBT-50 Heterophilic Blocking Tubes (Skybio Ltd., Bedfordshire, UK) were used to block the expected heterophile antibodies according to the manufacturer's instructions.
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The patient was polymorbid with diabetes mellitus type 2, treated arterial hypertension, treated dyslipidemia, vertebrogenic algic syndrome (VAS), excised breast fibroadenoma, osteoporosis, chronic left bundle branch block, gastroesophageal reflux disease (GERD) and a small diaphragm hiatal hernia. In 2007, a skin melanoma was diagnosed and excised and an oncological follow-up was performed with no signs of dissemination. There was no evidence of myocardial infarction, ischemic/hemorrhagic stroke or severe infectious diseases. The patient does not smoke or drink alcohol and has no allergies.
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3.2. Recent patient history and laboratory measurements
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Heidelberg group to elucidate an explanation for these discordant 82 results [1]. 83
t1:1 t1:2 t1:3 Q1
Table 1 Time course of troponin and BNP concentrations. TnI was below 0.03 μg/L throughout the examination period. Both BP and heart rate were within reference ranges during the entire follow-up.
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Laboratory data and clinical presentation are shown in Table 1.
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A 72-year-old female (BMI: 30.1 kg/m ) was repeatedly examined in the emergency department due to chest pain. Increased concentrations of high-sensitivity troponin T (hs-cTnT, Roche) were measured without objective evidence of acute myocardial injury. During follow-up, concentrations of hs-cTnT continuously increased. In contrast, other cardiac biomarkers, high-sensitivity troponin I (hs-cTnI, Abbott, cut-off 26.2 ng/L), troponin I (“conventional,” Abbott, cut-off 0.03 μg/L), CKMB mass and myoglobin, were normal or below the detectable cut-off. Due to this discrepancy, we followed the recommendation of the
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Date
hs-cTnT (ng/L)
Clinical presentation
BNP (ng/L)
hs-cTnI (ng/L)
t1:5
January, 2013
120.1/118.0/115.5
18.6
14
t1:6
April–June, 2013
143.0/135.0
t1:7
August 4–6, 2013
272.0/280.4/259.5a
t1:8
August 30, 2013
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September 3, 2013
Repeatedly atypical chest pain. Electrocardiogram (ECG) and echocardiogram (ECHO): negative. Computed tomography (CT): 50% stenosis on the ramus interventricularis anterior. Coronarography: insignificant 30–40% stenosis of the proximal ramus interventricularis anterior. Musculoskeletal etiology (VAS, on-going physiotherapy) was noted as the most likely cause. Repeatedly examined due to occasional chest pain. Transthoracic ECHO and ECG: negative. Hs-cTnT elevated without a rise/fall pattern. Symptoms were consistent with an earlier diagnosis of VAS and were most likely of musculoskeletal origin. Presented in Emergency with sharp, burning chest pain. ECG: intermittent sinoatrial block. Hs-cTnT elevated without a rise/fall pattern. ECHO: normal myocardial structure and function. The symptoms were most likely non-cardiac due to GERD. A follow-up ECG: elevation of estimated arterial pulmonalis systolic pressure. Scintigraphic ventilation/perfusion lung scan: subclinical acute bilateral segmental pulmonary embolism of unknown etiology. Neither clinical nor laboratory signs of pulmonary embolism were expressed: stable New York Heart Association (NYHA) Functional Classification I class, negative D-dimers, normal values (measured several times) of B-type natriuretic peptide (BNP) and TnI. ECHO: small tricuspidal and mitral regurgitation, ventricles not dilated, left ventricular ejection fraction (LVEF) 50–55%, side-wall hypokinesis. Overall satisfactory heart function.
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BNP = B-type natriuretic peptide; hs-cTnT = high-sensitivity cardiac troponin T; hs-cTnI = high-sensitivity cardiac troponin I; TnI = troponin I. a This sample was measured in another laboratory on the same day (The General University Hospital in Prague) with a result of 255 ng/L.
Please cite this article as: Franeková J, et al, A clinical and laboratory approach used to elucidate discordant results of high-sensitivity troponin T and troponin I, Clin Chim Acta (2015), http://dx.doi.org/10.1016/j.cca.2015.03.046
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Table 3 Possible actions to exclude or confirm false positive troponin results.
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Recommended action [1,2; modified]
Result
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Repeat measurement to avoid WBIT-type error, check type of tube used
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Use most sensitive and specific diagnostic procedures (echocardiography, if inconclusive, perform contrast-enhanced high resolution MRI) Endomyocardial biopsy Serial blood sampling ECG evidence of ischemia Clinical evidence
Results were repeatedly confirmed by careful selection of test tubes, both lithium-heparin plasma and serum were tested and yielded similar results Results of echocardiography and MRI were without any conclusive findings
t3:11
Other laboratory signs of ischemia
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Renal failure
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Structural heart disease Re-expression of cTnT in the diseased skeletal muscle or cross reactivity with skeletal muscle
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Not performed, no clinical indication Performed, there was an increase of hs-cTnT over the 8 months Negative Except for pulmonary embolism at the end of follow-up, there were no clear clinical explanations for significantly elevated concentrations of troponins Myoglobin 31 μg/L (reference range for women 40–106 μg/L), CK-MB mass 1.92 μg/L (reference range for women up to 3.4 μg/L) Serum creatinine (enzymatic method) 79.2–92.4 μmol/L (reference range for women 49–90 μmol/L) MRI revealed a thin stripe of fibrosis (scar) in the septal region Not likely due to normal total CK 0.89 μkat/L (reference range for women 0.48–2.80 μkat/L)
CK-MB = creatine kinase MB; cTnT = cardiac troponin T; ECG = electrocardiogram; MRI = magnetic resonance imaging; WBIT = wrong blood in tube.
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An attempt to explain the causes of discordant results was made according to Vafaie and Lum [1,2]. Description is given in Table 2 and Table 3.
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3.4. Further experiments to exclude interference
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We performed a dilution test to exclude falsely positive results of hs-cTnT. We also mixed serum with increased concentration of hscTnI with serum of our patient to exclude falsely negative results of hs-cTnI. The results were inconclusive.
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3.5. Treatment of the sample with blocking agents
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We treated the plasma sample of the patient with blocking agents directed against heterophile antibodies (Skybio blocking tubes). Results are given in Table 4. For comparison, we used a sample from a patient with acute myocardial infarction (male, final diagnosis of NSTEMI) and concentration of hs-cTnI of 1462.9 ng/L. To ensure that all heterophilic antibodies were blocked, we treated the sample twofold (after the first blocking treatment, the sample was transferred to the second blocking tube and the second treatment was performed).
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3.6. Report from the manufacturer
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Two serum samples and one plasma sample from the patient were sent to the Roche Diagnostics GmbH Case Resolution Unit (CRU). The manufacturer reproduced both our high hs-cTnT and low cTnI results with CRU retention kits. Therefore, the manufacturer forwarded our samples to Roche Research & Development for further investigation
t4:1 t4:2
Table 4 Concentrations of troponin and other analytes before and after treatment of the sample in Skybio tubes containing blocking agent. Some measurements conducted in duplicate.
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and reported: “Sample was fractionated by size exclusion chromatography. TnT could be measured with TnT hs kit in those fractions with molecular weight where IgM is expected. Interference caused by an IgM molecule can be confirmed.” For this chromatography method the high molecular weight compounds elute faster than those with lower molecular weight. All fractions were measured with hs-cTnT and marker substances in parallel and the fraction showing reactivity in hs-cTnT was of a similar molecular weight to IgM.
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Concentration after 1st treatment in ng/L (% of original value)
Concentration after 2nd treatment in ng/L (% of original value)
t4:4
Patient with suspect interference
hs-cTnT
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hs-cTnI
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hs-cTnT
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hs-cTnI
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16.53/16.23 (5.8%) 3.8 (90.5%) 263.9/265.1 (97.6%) 1474.5 (100.8%)
15.66/15.58 (5.6%) 2.9 (69.0%) 253.0/255.2 (93.8%) 1416.7 (96.8%)
t4:8
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Baseline concentration in ng/L
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We report discordant results of hs-cTnT and hs-cTnI levels in a polymorbid patient with repeated episodes of chest pain with repeatedly increased concentrations of hs-cTnT. Clinicians accepted the possibility of increased concentrations of hs-cTnT, but there were some clues for taking an interference into account (chest pain with increased hs-cTnT, but without a rise/fall pattern, cTnI and hs-cTnI levels close to the detection limit). Therefore we aimed to elucidate the potential cause of this discrepancy. Common reasons for falsely increased troponin results include increased concentrations of rheumatoid factor, heterophile antibodies, human anti-mouse antibody (HAMA) and autoantibodies [3–5]. Excellent reviews on this topic have been recently published by Lippi and Bolstad [6,7], other papers describe the role of heterophile antibodies as well [7–11]. Analytical characteristics of troponin assays have been recently summarized by Apple and Collinson [12]. The Elecsys Troponin T hs STAT assay employs two monoclonal antibodies specifically directed against human cardiac troponin T. The antibodies recognize two epitopes (amino acid position 125–131 and 136–147) located in the central part of the cardiac troponin T protein, which consists of 288 amino acids. The influence of heterophile
Measured analyte
Control patient (male with NSTEMI)
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Patient
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hs-cTnI = high-sensitivity cardiac troponin I; hs-cTnT = high-sensitivity cardiac troponin T; NSTEMI = non-ST elevation myocardial infarction.
Please cite this article as: Franeková J, et al, A clinical and laboratory approach used to elucidate discordant results of high-sensitivity troponin T and troponin I, Clin Chim Acta (2015), http://dx.doi.org/10.1016/j.cca.2015.03.046
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Acknowledgments
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We express our thanks to Roche representatives in the Czech Republic, namely Dr. Tatiana Godarska and Mr. Igor Klimicek for their valuable co-operation and support. We also appreciate the excellent co-operation with our Penzberg colleagues in Roche Company, with special thanks to Dr. Karl Hallermayer.
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References
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[1] Vafaie M, Biener M, Mueller M, et al. Analytically false or true positive elevations of high sensitivity cardiac troponin: a systematic approach. Heart 2014;100:508–14. [2] Lum G, Solarz DE, Farney L. False positive cardiac troponin results in patients without acute myocardial infarction. Lab Med 2006;37:546–50. [3] Dupont J, Fromonot J, Franceschi F, et al. A case of false positive troponin elevation: role of the biological laboratory. Int J Cardiol 2013;162:e66–7. [4] Ntelios D, Mpei E, Gousi E, Potolidis E, Fanourgiakis P, Mandros C. Are troponin assays occasionally deceiving us? Am J Emerg Med 2013;31(997):1–2. [5] Pernet P, Bénéteau-Burnat B, Hermand C, Vaubourdolle M. Point-of-care testing: false elevation of cardiac troponin I assayed in the emergency department. Am J Emerg Med 2008;26(969):e1–2. [6] Lippi G, Aloe R, Meschi T, Borghi L, Cervellin G. Interference from heterophilic antibodies in troponin testing. Case report and systematic review of the literature. Clin Chim Acta 2013;426:79–84. [7] Bolstad N, Warren DJ, Nustad K. Heterophilic antibody interference in immunometric assays. Best Pract Res Clin Endocrinol Metab 2013;27:647–61. [8] Emerson JF, Lai KKY. Endogenous antibody interferences in immunoassays. Lab Med 2013;44:69–73. [9] Sturgeon CM, Viljoen A. Analytical error and interference in immunoassay: minimizing risk. Ann Clin Biochem 2011;48:418–32. [10] Emerson JF, Ngo G, Emerson SS. Screening for interference in immunoassays. Clin Chem 2003;49:1163–9. [11] Tate J, Ward G. Interferences in immunoassay. Clin Biochem Rev 2004;25:105–20. [12] Apple FS, Collinson PO. Analytical characteristics of high-sensitivity cardiac troponin assays. Clin Chem 2012;58:54–61. [13] Ghali S, Lewis K, Kazan V, et al. Fluctuation of spuriously elevated troponin I: a case report. Case Rep Crit Care 2012;2012:585879. http://dx.doi.org/10.1155/2012/ 585879. [14] Ungerer JP, Marquart L, O'Rourke PK, Wilgen U, Pretorius CJ. Concordance, variance, and outliers in 4 contemporary cardiac troponin assays: implications for harmonization. Clin Chem 2012;58:274–83.
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The following limitations should be considered regarding our research. Due to the very low concentration of hs-cTnI, it is difficult to interpret some of results due to concentrations close to the limit of detection. We used a dilution factor of 8 in the dilution test, while a dilution factor of up to 21 was recommended [9]; therefore, interference elimination could appear with higher dilution factors.
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In sum, we repeatedly measured and noted increased concentrations of hs-cTnT with low concentrations of other markers of cardiac injury (cTnI, hs-cTnI, CK-MB mass and myoglobin) without clear clinical evidence of myocardial necrosis. Extremely rare positive analytical interference of high molecular weight protein was demonstrated, most likely caused by IgM human anti-mouse antibody. To our knowledge this has only been the second reported case since 2009, proving the efficacy of chimeric antibodies at minimizing the probability of interference. In such cases of discordant findings, we suggest following the recommendation of Vafaie with a special emphasis on the use of tubes with blocking agents in cases of suspect heterophile/
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HAMA interference [1]. Co-operation between the laboratory and 196 clinicians is essential, and communication with the manufacturer is 197 advisable. 198
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antibodies is minimized in this diagnostic kit (the mouse-human chimeric detection antibody is used to decrease the potential influence of heterophile antibodies); however, as the manufacturer claims in the product instructions, this interference cannot be completely excluded. On the other hand, the use of chimeric antibodies is the reason for the very low level of interference with the Elecsys Troponin T hs STAT assay. It was rather unclear whether the discordant results in hs-cTn measurements in our patient were caused by positive interference in hs-cTnT or negative influence on hs-cTnI. However, the clinical course and additional measurement in the follow-up showed evidence of positive interference in hs-cTnT measurement. However, dilution test to exclude falsely positive results of hs-cTnT was inconclusive and following experiment to exclude falsely negative interference of hscTnI was not very informative. Also several recommended steps taken to show falsely increased troponins [1,2] were inconclusive (Tables 2 and 3). The only indicative results were those obtained from the sample treated with blocking agents (Table 4). The blocking was sufficient after the first treatment, and the second treatment was not necessary. Nevertheless, discrepant results occasionally occur and screening for heterophile antibodies is doubtful [10]. Interfering substances may react differently in different systems and with different analytes [11, 13,14]. It is, therefore, advisable to try several procedures for interference testing in clinical practice in co-operation with the manufacturer. Thanks to manufacturer co-operation, we received a final manufacturer's statement: “The observed TnT value is most likely false positive. An interference caused by a high molecular weight protein (presumably IgM) can be assumed, most likely HAMA. This type of interference is highly specific and extremely seldom for the Elecsys Troponin T hs assay as this assay contains the most up-to-date measures for interference elimination (e.g., mouse-human chimeric detection antibody). This type of interference is the second reported since launch of the assay in 2009. However, rare interferences by extremely high titers of HAMA cannot fully be excluded. This complaint is covered by our corresponding disclaimer in the method sheet.”
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Please cite this article as: Franeková J, et al, A clinical and laboratory approach used to elucidate discordant results of high-sensitivity troponin T and troponin I, Clin Chim Acta (2015), http://dx.doi.org/10.1016/j.cca.2015.03.046
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Contents lists available at ScienceDirect
Clinica Chimica Acta journal homepage: www.elsevier.com/locate/clinchim
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Janka Franeková a,d, Martin Bláha b, Jiří Bělohoubek c, Markéta Kotrbatá a, Peter Sečník Jr. a, Zdeněk Kubíček a, Jiří Kettner b, Antonín Jabor a,d,⁎
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Article history: Received 29 January 2015 Received in revised form 23 February 2015 Accepted 2 March 2015 Available online xxxx
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Keywords: Troponin Heterophile antibodies Immunoassay interference
Department of Laboratory Methods, Institute for Clinical and Experimental Medicine, Vídeňská 1958/9, 140 21 Prague 4, Czech Republic Department of Cardiology, Institute for Clinical and Experimental Medicine, Vídeňská 1958/9, 140 21 Prague 4, Czech Republic Department of Preventive Cardiology, Institute for Clinical and Experimental Medicine, Vídeňská 1958/9, 140 21 Prague 4, Czech Republic d Charles University, 3rd Faculty of Medicine, Ruská 87, 100 00 Prague 10, Czech Republic b
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A clinical and laboratory approach used to elucidate discordant results of high-sensitivity troponin T and troponin I
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i n f o
a b s t r a c t
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Case report
Background: Careful interpretation of discordant results in high-sensitivity troponin measurements is necessary in cases of suspect immunoassay interferences. We describe several procedures taken in a case of a polymorbid patient with chest pain, without clear evidence of myocardial necrosis and with increased high-sensitivity cardiac troponin T (hs-cTnT). We checked the Vafaie's algorithm for the evaluation of suspect interference in troponin measurements. Methods: We conducted a case report analysis, additional measurements, a dilution test and pretreatment of plasma with blocking agents. Results: Concentration of hs-cTnT (99th percentile of “healthy” population 14 ng/L) increased from 120.1 ng/L to 280.4 ng/L during an 8-month period and decreased to 216.3 ng/L during the following month with repeatedly negative troponin I (TnI), hs-cTnI, myoglobin and creatine kinase MB (CK-MB). Suspected false positivity of hs-cTnT was further confirmed by treatment of plasma with an antiheterophile blocking agent (hs-cTnT before treatment 280.4 ng/L, after 16.53/16.23 ng/L). This outcome was further confirmed by the manufacturer's experiments. Conclusions: The false-positive results of hs-cTnT were caused by the presence of extremely rare high molecular weight protein, presumably IgM, most likely HAMA (human anti-mouse antibody). Only the pre-treatment of plasma with a blocking agent provided a reliable indication of the interference. Cooperation among clinicians, laboratory personnel and the manufacturer is essential. © 2015 Published by Elsevier B.V.
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1. Introduction
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The introduction of high-sensitivity cardiac troponin (hs-cTn) measurements tightens requirements on the interpretation of borderline increases of troponin concentrations as well as of unambiguously increased but stable concentrations due to different influences. The high quality of hs-cTn measurement does not exclude rare situations of falsely increased (or decreased) concentrations of troponin; e.g., as a result of analytical interference. Here, we describe a rare case of discordant levels of hs-cTn measured by two routine systems in which the interference was found in only
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20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36
⁎ Corresponding author at: Department of Laboratory Methods, Institute for Clinical and Experimental Medicine, Vídeňská 1958/9, 140 21 Prague 4, Czech Republic. Tel.: +420 236 055 236. E-mail addresses: [email protected] (J. Franeková), [email protected] (M. Bláha), [email protected] (J. Bělohoubek), [email protected] (M. Kotrbatá), [email protected] (P. Sečník), [email protected] (Z. Kubíček), [email protected] (J. Kettner), [email protected] (A. Jabor).
one immunoassay, and we characterize a series of steps taken to 52 clarify the reason for this discrepancy. We also tested the effective- 53 ness of Vafaie's recommendation for these situations [1]. 54 2. Materials and methods
55
2.1. Patients
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The study was approved by IKEM Ethic Committee. The patient gave written informed consent approving all examinations and procedures (IKEM F-39). All additional examinations including testing at the manufacturer's facilities were blinded.
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2.2. Materials and methods
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All measurements were performed in an accredited clinical 62 laboratory (ISO 15189:2007). Hs-cTnT was measured with the 63 Elecsys Troponin T hs STAT kit on Cobas 6000 (both Roche Diagnostic 64
http://dx.doi.org/10.1016/j.cca.2015.03.046 0009-8981/© 2015 Published by Elsevier B.V.
Please cite this article as: Franeková J, et al, A clinical and laboratory approach used to elucidate discordant results of high-sensitivity troponin T and troponin I, Clin Chim Acta (2015), http://dx.doi.org/10.1016/j.cca.2015.03.046
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J. Franeková et al. / Clinica Chimica Acta xxx (2015) xxx–xxx
Table 2 Other laboratory examinations and procedures with the potential to show falsely increased troponins.
t2:3 t2:4 Q2
Possible causes of false-positive troponin results [1,2; modified]
t2:5 t2:6
Heterophile antibodies (HAAA, human anti-animal antibodies; Concentrations of other analytes with the same type of potential interference were measured (Cobas 6000): HAMA, human anti-mouse antibodies) PSA: under limit of detection CEA: 0.6 μg/L (reference range b3.8 μg/L) CA 19-9: 11.9 kU/L (b27 kU/L) CA 72-4: 1.2 kU/L (b6.9 kU/L) CA 125: 3.6 kU/L (b35 kU/L) Autoantibodies ANCA negative, anti-myocardium antibodies negative Rheumatoid factor Negative (20.1 kIU/L, reference range up to 30 kIU/L) Fibrin clots, microparticles in specimen Excluded, careful separation of blood cells, measured both in serum and lithium-heparin plasma Interference caused by endogenous components in blood Bilirubin total: 9.1–11.6 μmol/L (reference range 3.4–20 μmol/L) (bilirubin, hemoglobin, lipemia) Lipemia: excluded, lipemic index measured on every sample, lipid profile: total cholesterol 3.7 mmol/L, HDL cholesterol 1.31 mmol/L, LDL cholesterol 1.8 mmol/L, triglycerides 1.31 mmol/L Hemoglobin: excluded, hemolysis index measured in every sample High concentration of alkaline phosphatase 0.91–0.97 μkat/L (reference range 0.58–1.75 μkat/L) Immunocomplex formation Circulating immunocomplexes: 0.284 (index of positivity, values b0.9 are negative), C1q: negative Analyzer malfunction Excluded, results repeatedly measured, results confirmed in another laboratory (hs-cTnT) Use of another analytical system Mitsubishi Pathfast: cTnI 2 ng/L (cut-off 2 ng/L; system uses 2 monoclonal mouse antibodies)
F
O
t2:12 t2:13 t2:14 t2:15
R O
t2:7 t2:8 t2:9 t2:10 t2:11
Our results
t2:16 t2:17
ANCA = anti-neutrophil cytoplasmic antibodies; CA = cancer antigen; CEA = carcinoembryonic antigen; HAAA = human anti-animal antibodies; HAMA = human anti-mouse antibodies; LDL cholesterol = low-density lipoprotein cholesterol; PSA = prostate specific antigen.
65 66
Ltd., Rotkreuz, Switzerland). Hs-cTnI and cTnI were measured on an Abbott Architect i2000SR system with ARCHITECT hs Troponin I and ARCHITECT STAT Troponin-I kits, respectively (all designed by Abbott Diagnostics, Lake Forest, Illinois, US). Skybio HBT-50 Heterophilic Blocking Tubes (Skybio Ltd., Bedfordshire, UK) were used to block the expected heterophile antibodies according to the manufacturer's instructions.
72 73
P
The patient was polymorbid with diabetes mellitus type 2, treated arterial hypertension, treated dyslipidemia, vertebrogenic algic syndrome (VAS), excised breast fibroadenoma, osteoporosis, chronic left bundle branch block, gastroesophageal reflux disease (GERD) and a small diaphragm hiatal hernia. In 2007, a skin melanoma was diagnosed and excised and an oncological follow-up was performed with no signs of dissemination. There was no evidence of myocardial infarction, ischemic/hemorrhagic stroke or severe infectious diseases. The patient does not smoke or drink alcohol and has no allergies.
85 86
3.2. Recent patient history and laboratory measurements
95
D
84
E
71
3.1. Patient history
T
69 70
3. Results 2
C
67 68
Heidelberg group to elucidate an explanation for these discordant 82 results [1]. 83
t1:1 t1:2 t1:3 Q1
Table 1 Time course of troponin and BNP concentrations. TnI was below 0.03 μg/L throughout the examination period. Both BP and heart rate were within reference ranges during the entire follow-up.
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Laboratory data and clinical presentation are shown in Table 1.
O
78 79
C
76 77
E
80 81
A 72-year-old female (BMI: 30.1 kg/m ) was repeatedly examined in the emergency department due to chest pain. Increased concentrations of high-sensitivity troponin T (hs-cTnT, Roche) were measured without objective evidence of acute myocardial injury. During follow-up, concentrations of hs-cTnT continuously increased. In contrast, other cardiac biomarkers, high-sensitivity troponin I (hs-cTnI, Abbott, cut-off 26.2 ng/L), troponin I (“conventional,” Abbott, cut-off 0.03 μg/L), CKMB mass and myoglobin, were normal or below the detectable cut-off. Due to this discrepancy, we followed the recommendation of the
74 75
Date
hs-cTnT (ng/L)
Clinical presentation
BNP (ng/L)
hs-cTnI (ng/L)
t1:5
January, 2013
120.1/118.0/115.5
18.6
14
t1:6
April–June, 2013
143.0/135.0
t1:7
August 4–6, 2013
272.0/280.4/259.5a
t1:8
August 30, 2013
t1:9 t1:10
September 3, 2013
Repeatedly atypical chest pain. Electrocardiogram (ECG) and echocardiogram (ECHO): negative. Computed tomography (CT): 50% stenosis on the ramus interventricularis anterior. Coronarography: insignificant 30–40% stenosis of the proximal ramus interventricularis anterior. Musculoskeletal etiology (VAS, on-going physiotherapy) was noted as the most likely cause. Repeatedly examined due to occasional chest pain. Transthoracic ECHO and ECG: negative. Hs-cTnT elevated without a rise/fall pattern. Symptoms were consistent with an earlier diagnosis of VAS and were most likely of musculoskeletal origin. Presented in Emergency with sharp, burning chest pain. ECG: intermittent sinoatrial block. Hs-cTnT elevated without a rise/fall pattern. ECHO: normal myocardial structure and function. The symptoms were most likely non-cardiac due to GERD. A follow-up ECG: elevation of estimated arterial pulmonalis systolic pressure. Scintigraphic ventilation/perfusion lung scan: subclinical acute bilateral segmental pulmonary embolism of unknown etiology. Neither clinical nor laboratory signs of pulmonary embolism were expressed: stable New York Heart Association (NYHA) Functional Classification I class, negative D-dimers, normal values (measured several times) of B-type natriuretic peptide (BNP) and TnI. ECHO: small tricuspidal and mitral regurgitation, ventricles not dilated, left ventricular ejection fraction (LVEF) 50–55%, side-wall hypokinesis. Overall satisfactory heart function.
t1:11 t1:12
U
N
t1:4
216.3
19.6–63.9 2.5
–
2.3–2.6
–
–
–
–
BNP = B-type natriuretic peptide; hs-cTnT = high-sensitivity cardiac troponin T; hs-cTnI = high-sensitivity cardiac troponin I; TnI = troponin I. a This sample was measured in another laboratory on the same day (The General University Hospital in Prague) with a result of 255 ng/L.
Please cite this article as: Franeková J, et al, A clinical and laboratory approach used to elucidate discordant results of high-sensitivity troponin T and troponin I, Clin Chim Acta (2015), http://dx.doi.org/10.1016/j.cca.2015.03.046
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3
Table 3 Possible actions to exclude or confirm false positive troponin results.
t3:3 Q3
Recommended action [1,2; modified]
Result
t3:4
Repeat measurement to avoid WBIT-type error, check type of tube used
t3:5 t3:6 t3:7 t3:8 t3:9 t3:10
Use most sensitive and specific diagnostic procedures (echocardiography, if inconclusive, perform contrast-enhanced high resolution MRI) Endomyocardial biopsy Serial blood sampling ECG evidence of ischemia Clinical evidence
Results were repeatedly confirmed by careful selection of test tubes, both lithium-heparin plasma and serum were tested and yielded similar results Results of echocardiography and MRI were without any conclusive findings
t3:11
Other laboratory signs of ischemia
t3:12
Renal failure
t3:13 t3:14 t3:15
Structural heart disease Re-expression of cTnT in the diseased skeletal muscle or cross reactivity with skeletal muscle
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Not performed, no clinical indication Performed, there was an increase of hs-cTnT over the 8 months Negative Except for pulmonary embolism at the end of follow-up, there were no clear clinical explanations for significantly elevated concentrations of troponins Myoglobin 31 μg/L (reference range for women 40–106 μg/L), CK-MB mass 1.92 μg/L (reference range for women up to 3.4 μg/L) Serum creatinine (enzymatic method) 79.2–92.4 μmol/L (reference range for women 49–90 μmol/L) MRI revealed a thin stripe of fibrosis (scar) in the septal region Not likely due to normal total CK 0.89 μkat/L (reference range for women 0.48–2.80 μkat/L)
CK-MB = creatine kinase MB; cTnT = cardiac troponin T; ECG = electrocardiogram; MRI = magnetic resonance imaging; WBIT = wrong blood in tube.
97
3.3. Other laboratory data and extra-laboratory procedures
98 99 100
An attempt to explain the causes of discordant results was made according to Vafaie and Lum [1,2]. Description is given in Table 2 and Table 3.
101
3.4. Further experiments to exclude interference
102
105
We performed a dilution test to exclude falsely positive results of hs-cTnT. We also mixed serum with increased concentration of hscTnI with serum of our patient to exclude falsely negative results of hs-cTnI. The results were inconclusive.
106
3.5. Treatment of the sample with blocking agents
107 108
113 114
We treated the plasma sample of the patient with blocking agents directed against heterophile antibodies (Skybio blocking tubes). Results are given in Table 4. For comparison, we used a sample from a patient with acute myocardial infarction (male, final diagnosis of NSTEMI) and concentration of hs-cTnI of 1462.9 ng/L. To ensure that all heterophilic antibodies were blocked, we treated the sample twofold (after the first blocking treatment, the sample was transferred to the second blocking tube and the second treatment was performed).
115
3.6. Report from the manufacturer
116
119 120
Two serum samples and one plasma sample from the patient were sent to the Roche Diagnostics GmbH Case Resolution Unit (CRU). The manufacturer reproduced both our high hs-cTnT and low cTnI results with CRU retention kits. Therefore, the manufacturer forwarded our samples to Roche Research & Development for further investigation
t4:1 t4:2
Table 4 Concentrations of troponin and other analytes before and after treatment of the sample in Skybio tubes containing blocking agent. Some measurements conducted in duplicate.
117 118
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E
T
C
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R
111 112
N C O
109 110
and reported: “Sample was fractionated by size exclusion chromatography. TnT could be measured with TnT hs kit in those fractions with molecular weight where IgM is expected. Interference caused by an IgM molecule can be confirmed.” For this chromatography method the high molecular weight compounds elute faster than those with lower molecular weight. All fractions were measured with hs-cTnT and marker substances in parallel and the fraction showing reactivity in hs-cTnT was of a similar molecular weight to IgM.
U
103 104
R O
t3:16
Concentration after 1st treatment in ng/L (% of original value)
Concentration after 2nd treatment in ng/L (% of original value)
t4:4
Patient with suspect interference
hs-cTnT
280.4
hs-cTnI
4.2
hs-cTnT
272.4/269.4
hs-cTnI
1462.9
16.53/16.23 (5.8%) 3.8 (90.5%) 263.9/265.1 (97.6%) 1474.5 (100.8%)
15.66/15.58 (5.6%) 2.9 (69.0%) 253.0/255.2 (93.8%) 1416.7 (96.8%)
t4:8
128
130 131
Baseline concentration in ng/L
t4:7
126 127
We report discordant results of hs-cTnT and hs-cTnI levels in a polymorbid patient with repeated episodes of chest pain with repeatedly increased concentrations of hs-cTnT. Clinicians accepted the possibility of increased concentrations of hs-cTnT, but there were some clues for taking an interference into account (chest pain with increased hs-cTnT, but without a rise/fall pattern, cTnI and hs-cTnI levels close to the detection limit). Therefore we aimed to elucidate the potential cause of this discrepancy. Common reasons for falsely increased troponin results include increased concentrations of rheumatoid factor, heterophile antibodies, human anti-mouse antibody (HAMA) and autoantibodies [3–5]. Excellent reviews on this topic have been recently published by Lippi and Bolstad [6,7], other papers describe the role of heterophile antibodies as well [7–11]. Analytical characteristics of troponin assays have been recently summarized by Apple and Collinson [12]. The Elecsys Troponin T hs STAT assay employs two monoclonal antibodies specifically directed against human cardiac troponin T. The antibodies recognize two epitopes (amino acid position 125–131 and 136–147) located in the central part of the cardiac troponin T protein, which consists of 288 amino acids. The influence of heterophile
Measured analyte
Control patient (male with NSTEMI)
125
129
Patient
t4:6
123 124
4. Discussion
t4:3
t4:5
121 122
hs-cTnI = high-sensitivity cardiac troponin I; hs-cTnT = high-sensitivity cardiac troponin T; NSTEMI = non-ST elevation myocardial infarction.
Please cite this article as: Franeková J, et al, A clinical and laboratory approach used to elucidate discordant results of high-sensitivity troponin T and troponin I, Clin Chim Acta (2015), http://dx.doi.org/10.1016/j.cca.2015.03.046
132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149
175 176 177 178 179 180 181 182
187 188 189 190 191 192 193 194 195 244
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173 174
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171 172
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169 170
R
167 168
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165 166
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Acknowledgments
202 203 204 205 206
We express our thanks to Roche representatives in the Czech Republic, namely Dr. Tatiana Godarska and Mr. Igor Klimicek for their valuable co-operation and support. We also appreciate the excellent co-operation with our Penzberg colleagues in Roche Company, with special thanks to Dr. Karl Hallermayer.
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References
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[1] Vafaie M, Biener M, Mueller M, et al. Analytically false or true positive elevations of high sensitivity cardiac troponin: a systematic approach. Heart 2014;100:508–14. [2] Lum G, Solarz DE, Farney L. False positive cardiac troponin results in patients without acute myocardial infarction. Lab Med 2006;37:546–50. [3] Dupont J, Fromonot J, Franceschi F, et al. A case of false positive troponin elevation: role of the biological laboratory. Int J Cardiol 2013;162:e66–7. [4] Ntelios D, Mpei E, Gousi E, Potolidis E, Fanourgiakis P, Mandros C. Are troponin assays occasionally deceiving us? Am J Emerg Med 2013;31(997):1–2. [5] Pernet P, Bénéteau-Burnat B, Hermand C, Vaubourdolle M. Point-of-care testing: false elevation of cardiac troponin I assayed in the emergency department. Am J Emerg Med 2008;26(969):e1–2. [6] Lippi G, Aloe R, Meschi T, Borghi L, Cervellin G. Interference from heterophilic antibodies in troponin testing. Case report and systematic review of the literature. Clin Chim Acta 2013;426:79–84. [7] Bolstad N, Warren DJ, Nustad K. Heterophilic antibody interference in immunometric assays. Best Pract Res Clin Endocrinol Metab 2013;27:647–61. [8] Emerson JF, Lai KKY. Endogenous antibody interferences in immunoassays. Lab Med 2013;44:69–73. [9] Sturgeon CM, Viljoen A. Analytical error and interference in immunoassay: minimizing risk. Ann Clin Biochem 2011;48:418–32. [10] Emerson JF, Ngo G, Emerson SS. Screening for interference in immunoassays. Clin Chem 2003;49:1163–9. [11] Tate J, Ward G. Interferences in immunoassay. Clin Biochem Rev 2004;25:105–20. [12] Apple FS, Collinson PO. Analytical characteristics of high-sensitivity cardiac troponin assays. Clin Chem 2012;58:54–61. [13] Ghali S, Lewis K, Kazan V, et al. Fluctuation of spuriously elevated troponin I: a case report. Case Rep Crit Care 2012;2012:585879. http://dx.doi.org/10.1155/2012/ 585879. [14] Ungerer JP, Marquart L, O'Rourke PK, Wilgen U, Pretorius CJ. Concordance, variance, and outliers in 4 contemporary cardiac troponin assays: implications for harmonization. Clin Chem 2012;58:274–83.
N
161 162
200 201
U
159 160
The following limitations should be considered regarding our research. Due to the very low concentration of hs-cTnI, it is difficult to interpret some of results due to concentrations close to the limit of detection. We used a dilution factor of 8 in the dilution test, while a dilution factor of up to 21 was recommended [9]; therefore, interference elimination could appear with higher dilution factors.
F
In sum, we repeatedly measured and noted increased concentrations of hs-cTnT with low concentrations of other markers of cardiac injury (cTnI, hs-cTnI, CK-MB mass and myoglobin) without clear clinical evidence of myocardial necrosis. Extremely rare positive analytical interference of high molecular weight protein was demonstrated, most likely caused by IgM human anti-mouse antibody. To our knowledge this has only been the second reported case since 2009, proving the efficacy of chimeric antibodies at minimizing the probability of interference. In such cases of discordant findings, we suggest following the recommendation of Vafaie with a special emphasis on the use of tubes with blocking agents in cases of suspect heterophile/
157 158
199
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185 186
156
5.1. Limitations
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5. Conclusions
154 155
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184
152 153
HAMA interference [1]. Co-operation between the laboratory and 196 clinicians is essential, and communication with the manufacturer is 197 advisable. 198
T
183
antibodies is minimized in this diagnostic kit (the mouse-human chimeric detection antibody is used to decrease the potential influence of heterophile antibodies); however, as the manufacturer claims in the product instructions, this interference cannot be completely excluded. On the other hand, the use of chimeric antibodies is the reason for the very low level of interference with the Elecsys Troponin T hs STAT assay. It was rather unclear whether the discordant results in hs-cTn measurements in our patient were caused by positive interference in hs-cTnT or negative influence on hs-cTnI. However, the clinical course and additional measurement in the follow-up showed evidence of positive interference in hs-cTnT measurement. However, dilution test to exclude falsely positive results of hs-cTnT was inconclusive and following experiment to exclude falsely negative interference of hscTnI was not very informative. Also several recommended steps taken to show falsely increased troponins [1,2] were inconclusive (Tables 2 and 3). The only indicative results were those obtained from the sample treated with blocking agents (Table 4). The blocking was sufficient after the first treatment, and the second treatment was not necessary. Nevertheless, discrepant results occasionally occur and screening for heterophile antibodies is doubtful [10]. Interfering substances may react differently in different systems and with different analytes [11, 13,14]. It is, therefore, advisable to try several procedures for interference testing in clinical practice in co-operation with the manufacturer. Thanks to manufacturer co-operation, we received a final manufacturer's statement: “The observed TnT value is most likely false positive. An interference caused by a high molecular weight protein (presumably IgM) can be assumed, most likely HAMA. This type of interference is highly specific and extremely seldom for the Elecsys Troponin T hs assay as this assay contains the most up-to-date measures for interference elimination (e.g., mouse-human chimeric detection antibody). This type of interference is the second reported since launch of the assay in 2009. However, rare interferences by extremely high titers of HAMA cannot fully be excluded. This complaint is covered by our corresponding disclaimer in the method sheet.”
D
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4
Please cite this article as: Franeková J, et al, A clinical and laboratory approach used to elucidate discordant results of high-sensitivity troponin T and troponin I, Clin Chim Acta (2015), http://dx.doi.org/10.1016/j.cca.2015.03.046
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