DOI 10.1515/cclm-2013-0767      Clin Chem Lab Med 2014; 52(6): 911–918

Kevin M. Goode*, Rachel Nicholls, Pierpaolo Pellicori, Andrew L. Clark and John G.F. Cleland

The in vitro stability of novel cardiovascular and sepsis biomarkers at ambient temperature Abstract Background: The in vitro stability of a biomarker can determine whether it should be used in clinical practice where long delays between sampling and assay are common. We measured the in vitro stability of five novel biomarkers that are being evaluated for their diagnostic and/or prognostic utility in patients with heart failure: mid-regional pro-atrial natriuretic peptide (MR-proANP), mid-regional pro-adreno-medullin (MR-proADM), C-terminal pro-endothelin-1 (CT-proET-1), C-terminal pro-arginine vasopressin (copeptin) and ultrasensitive procalcitonin (PCT). Methods: Peripheral venous blood samples were obtained from 19 patients with chronic heart failure into four EDTA tubes. The first tubes were centrifuged immediately at 4°C with the other tubes stored at 20°C for 4, 24 or 72 hours (h) before centrifuging. Supernatant plasma was frozen and stored at −80°C until assay. The levels of analyte in samples processed with and without delay were compared using correlation analysis, paired t-tests and Bland-Altman plots. Results: Copeptin and PCT were stable up to 72 h at 20°C in whole blood and MR-proANP and MR-proADM up to 24  h. However, CT-proET-1 showed some signs of degradation after only 4 h with 94% of analyte recovered after 24 h, dropping to 80% after 72 h. Conclusions: MR-proANP, MR-proADM, copeptin and PCT are stable biomarkers and therefore suitable for introduction into routine clinical practice in a primary or secondary care setting where delays in sample preparation and assay are likely. Ideally, samples for measurement of CTproET-1 should be centrifuged soon after venepuncture but the analyte is stable enough for most routine clinical purposes. Keywords: C-terminal pro-arginine vasopressin; C-terminal pro-endothelin-1; in vitro stability; mid-regional pro-adreno-medullin; mid-regional pro-atrial natriuretic peptide; ultrasensitive procalcitonin. *Corresponding author: Dr. Kevin M. Goode, Faculty of Health and Social Care, University of Hull, Room 101, Aire Building, Cottingham Road, Hull, HU6 7RX, UK, E-mail: [email protected]

Rachel Nicholls, Pierpaolo Pellicori, Andrew L. Clark and John G.F. Cleland: Hull York Medical School, Department of Cardiology, University of Hull, Castle Hill Hospital, Hull, UK

Introduction The role of B-type natriuretic peptide (BNP) and aminoterminal pro B-type natriuretic peptide (NT-proBNP) in the diagnosis and prognosis of heart failure is now well established, appearing in the UK, European and American guidelines on the management of heart failure [1–3]. The widespread adoption of NT-proBNP has been helped, in part, by its apparent stability in vitro in whole blood at room temperature [4–6], although the evidence is patchy. Despite this, it is now routinely used in settings (such as primary care) where the needle-to-assay times can be protracted due to transport delays and the lack of availability of non-emergency laboratories at weekends. BNP is considered less stable, although the actual stability observed depends on the form of BNP, the matrix and the type of sample tube used, and can vary between 6 and 72 h when stored as whole blood [7–13]. This either excludes its use in primary care or requires point-of-care assays, with their attendant advantages and disadvantages. Equally, knowing that samples will not degrade in transport at room temperature increases the opportunities for research where blood samples may be sent as whole blood from different institutions to a central core laboratory for preparation and analysis. With advances in modern medicine and assay research, there is an increasing array of biomarkers related to heart failure and its associated pathophysiology that might improve diagnosis, risk stratification or help in targeting specific treatments [14, 15]. Understanding the stability of emerging biomarkers is important to ensure they are not used in settings where sample degradation may make affect the accuracy of assay results. We evaluated the in vitro stability of five biomarkers that may be useful for the evaluation of patients with suspected acute or chronic heart failure (Table 1): midregional pro-atrial natriuretic peptide (MR-proANP),

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912      Goode et al.: Stability of novel biomarkers Table 1 Role of biomarkers. Biomarker



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Mid-regional pro-atrial natriuretic peptide (MR-proANP)



Atrial natriuretic   peptide

Involved in natriuresis, diuresis, vasodilation and cardiovascular homeostasis. Elevated levels indicate cardiovascular stress.

Mid-regional pro-adrenomedullin (MR-proADM)



Adrenomedullin  

A vasodilator responsible for volume regulation and electrolyte homeostasis. Elevated levels can indicate endothelial dysfunction and/or sepsis.

C-terminal pro-endothelin-1 (CT-proET-1)



Endothelin-1

A pulmonary and systemic vasoconstrictor.



Pathophysiological role

C-terminal pro-arginine vasopressin  (CT-proAVP or copeptin)

Vasopressin (anti-  diuretic hormone)

Increase in response to pain, opiates, cardiac stress and dehydration. May be inappropriately elevated in patients with hyponatraemia

Procalcitonin (PCT)

Calcitonin

Produced in response to endotoxin or mediators released in response to bacterial infections. Marker of the severity of infection. Also increased in patients with HF and probably related to pneumonia.





mid-regional pro-adreno-medullin (MR-proADM), C-terminal pro-endothelin-1 (CT-proET-1), C-terminal pro-arginine vasopressin (CT-proAVP or copeptin) and ultrasensitive procalcitonin (PCT).

Materials and methods Study population Blood samples for the study were obtained from 19 patients attending a specialist heart failure outpatient clinic (Kingston-upon-Hull, UK) with known or suspected heart failure. All participants provided written informed consent for their data to be used and the study was carried out in accordance with the Helsinki Declaration II and the European Standards for Good Clinical Practice. Ethical approval was granted by the Hull and East Yorkshire Local Research Ethics Committee. All patients had a physical examination and electro- and echo-cardiograms.

Collection and processing of samples Patients, non-fasting, were asked to sit in a relaxed position in an air-conditioned room for 15  min prior to venepuncture. Peripheral venous blood was collected into four polypropylene tubes containing ethylene-diamine-tetra-acetic acid (EDTA). The first tube was centrifuged immediately after collection (i.e., 0 h) at 1666 g for 15 min at 4°C. The plasma was removed and stored immediately in a cryotube at −80°C for subsequent batch analysis. The second, third and fourth tubes were each left to stand at room temperature for 4, 24 and 72 h, respectively, before centrifuging. The plasma was then removed and stored in a cryotube at −80°C in the same way as the first tube. Room temperature was 20°C. Samples were stored at −80°C until shipped on dry-ice with temperature logging to Thermo Scientific Biomarkers, Hennigsdorf, Germany for assay. Samples were kept at −80°C for no more than 2 months. Samples were allowed to thaw at room temperature immediately prior to assay.

Assay The plasma concentrations of MR-proANP, CT-proET-1, MR-proADM, copeptin and PCT were measured using an automated sandwich immunoassays (KRYPTOR®; Thermo Scientific Biomarkers, Hennigsdorf, Germany) based on time-resolved amplified cryptate emissions (TRACE) technology. All samples were measured in duplicate to ensure a coefficient of variation of   1.5  nmol/L in only two patients, and these out­ liers could have affected the analysis. Excluding these measurements did not alter the results significantly (bias ± limits of agreement at 4 h  

The in vitro stability of novel cardiovascular and sepsis biomarkers at ambient temperature.

The in vitro stability of a biomarker can determine whether it should be used in clinical practice where long delays between sampling and assay are co...
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