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Letters to the Editor |
Departments of1 Medical Biochemistry and Immunology,2 Cardiology, and3 Medical Statistics, University Hospital of Wales and University of Wales College of Medicine, Cardiff CF14 4XW, United Kingdom
aAuthor for correspondence. Fax 44-2920-74-8383; e-mail DaElene.VanDerMerwe{at}CardiffandVale.wales.nhs.uk.
To the Editor:
N-Terminal pro-B-type natriuretic peptide (NT-proBNP; 76-amino acid peptide) is cosecreted with BNP and can be measured by automated immunoassay (Elecsys 2010; Roche UK). This is a fully automated electrochemiluminescence "sandwich" immunoassay based on capture (biotinylated) and detection (ruthenium) polyclonal antibodies against NT-proBNP amino acids 1–21 and 39–50 (1), respectively. Reports on in vitro stability are conflicting (2)(3)(4). We assessed the effect of different sample types on measurement of NT-proBNP and evaluated the effect of delayed separation of serum from cells on stability. We compared plasma in lithium heparinate (14 IU/mL of blood; Greiner Labortechnik; cat no. 9045-22-1) and serum in serum separator (SST) tubes from patients attending a heart failure clinic. For sample type comparison, blood from 60 patients was collected into SST and lithium heparin tubes. Blood was also collected in plain glass and SST tubes (n = 10) to investigate whether there was significant adsorption of NT-proBNP by the gel separator (plastic tubes; Greiner Labortechnik Bio-One GmbH).
To assess stability, blood from 12 individuals was collected in four SST tubes. The first tube was centrifuged, aliquoted, and stored at -20 °C for baseline measurement. The remaining serum was left on the gel at 22 °C; aliquots were taken and stored at -20 °C after 24, 48, and 72 h. This simulates the situation in the laboratory after the sample has been received. The second, third, and fourth samples were centrifuged after 24, 48, and 72 h at 22 °C, aliquoted, and frozen until assay. Samples from the same patient were thawed and analyzed within the same run. The between-day CV were 8.4% (34 ng/L), 3.3% (421 ng/L), and 3.6% (15 938 ng/L; n = 20) for pooled serum and 2.7% (216 ng/L) and 1.5% (4087 ng/L; n = 20) for the Elecsys PreciControl proBNP calibrator. All data followed a gaussian distribution after log transformation. NT-proBNP was 4% lower in lithium heparin plasma than in serum by the paired t-test (P <0.001; n = 60), with a regression equation of: Log hep = 1.006 log SST - 0.085 (r >0.99). The 95% confidence interval (CI) for the slope was 0.99–1.01, and for the intercept was -0.13 to -0.03 ng/L (r >0.99). A Bland–Altman plot is shown in Fig. 1
to aid interpretation. We found no significant difference between serum from plain glass and SST tubes (P = 0.15; n = 10). NT-proBNP was stable in serum left on top of the SST gel at room temperature for 72 h with no significant mean decrease from baseline at 24 h (0.2%; 95% CI, -1.8 to 1.5%), 48 h (0.8%; -2.6 to 0.9%), or 72 h (1.2%; 95% CI, -3.0 to 0.6%; n = 12). For serum that was left unseparated in the SST tube up to 72 h before separation, NT-proBNP decreased by a mean of 4.4% (-6.2 to -2.5%) at 24 h, 6.9% (-9.3 to -4.6%) at 48 h, and 9.6% (-12.2 to -7.1%) at 72 h (n = 12; all P <0.001 relative to baseline). Dasgupta et al. (5), found that NT-proBNP is stable for 48 h in SST tubes (Becton Dickinson) after centrifugation. We have demonstrated stability up to 72 h. However, we observed a decrease when blood was left unseparated for 24 h or longer. The manufacturers and Sokoll et al. (6) state that there is no difference in results obtained with serum and heparin plasma, whereas we have demonstrated a difference of 4%. Sokoll et al. (6) used lithium and ammonium heparin compared with the lithium heparinate tubes used here. We conclude that NT-proBNP can be precisely measured and that it is stable if serum has been separated from cells within 24 h. There is a small but statistically significant difference in results between heparin plasma and serum. This is unlikely to be of clinical importance because of the large intraindividual biological variation of NT-proBNP (33.3%) (7).
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Acknowledgments
We thank Roche Diagnostics (Lewes, East Sussex, UK) for reagents and quality-control material.
References
The following articles in journals at HighWire Press have cited this article:
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  B Cauliez, J Guignery, S Marinier, I Mariau, and A Lavoinne Two-year stability of NT-proBNP in frozen samples using the Roche Elecsys system Ann Clin Biochem, May 1, 2008; 45(3): 318 - 319. [Abstract] [Full Text] [PDF]
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 F. H. Rutten, M.-J. M. Cramer, N. P.A. Zuithoff, J.-W. J. Lammers, W. Verweij, D. E. Grobbee, and A. W. Hoes Comparison of B-type natriuretic peptide assays for identifying heart failure in stable elderly patients with a clinical diagnosis of chronic obstructive pulmonary disease Eur J Heart Fail, June 1, 2007; 9(6-7): 651 - 659. [Abstract] [Full Text] [PDF]
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 F. S. Apple, M. Panteghini, J. Ravkilde, J. Mair, A. H.B. Wu, J. Tate, F. Pagani, R. H. Christenson, A. S. Jaffe, and on Behalf of the Committee on Standardization of M Quality Specifications for B-Type Natriuretic Peptide Assays Clin. Chem., March 1, 2005; 51(3): 486 - 493. [Abstract] [Full Text] [PDF]
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