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1 Hennepin County Medical Center, University of Minnesota School of Medicine, Minneapolis, MN
2 University of Maryland School of Medicine, Baltimore, MD
3 Mayo Clinic and Mayo College of Medicine, Rochester, MN
4 Department of Internal Medicine, Clinical Division of Cardiology, Innsbruck Medical University, Austria
5 Department of Biochemistry, Hospital de la Santa Creu i Sant Pau and Universitat Autonoma, Barcelona, Spain
6 Laboratorio Analisi Chimico Cliniche, Dipartimento di Medicina di Laboratorio, Azienda Ospedaliera ‘Spedali Civili’, Brescia, Italy
7 Chemical Pathology Department Pathology, Queensland, Royal Brisbane and Women’s Hospital, Brisbane, Australia, 8
University of California at San Francisco, San Francisco General Hospital, San Francisco, CA
aAddress correspondence to this author at: Hennepin County Medical Center, Clinical Laboratories P4, 701 Park Ave, Minneapolis, MN 55415, Fax 612-904-4229, e-mail apple004{at}umn.edu
To the Editor:
B-type natriuretic peptide (BNP) is a 32 amino acid cardiac-synthesized hormone that reduces blood pressure and increases sodium excretion (1). Following proteolytic cleavage of proBNP, a 108-amino acid precursor, an N-terminal fragment (NT-proBNP) and BNP are released (2). Increased concentrations of BNP and NT-proBNP can be used clinically to monitor heart failure, but a lack of alignment between commercial BNP and NT-proBNP assays (3) can lead to confusion when clinicians or laboratorians compare results measured for the same analyte on different instruments. Some of this confusion arises from variable assay specificity regarding what peptides are being measured. We studied whether (a) BNP assays demonstrated cross-reactivity with NT-proBNP or proBNP, and (b) whether NT-proBNP assays demonstrated cross-reactivity with BNP or proBNP, by using 5 commercial BNP and 3 commercial NT-proBNP assays with 2 BNP, 2 NT-proBNP, and 2 proBNP materials.
The NPs studied were: Peptide Institute synthetic BNP (aa 77–108), Scios human recombinant BNP (aa 77–108), HyTest human recombinant NT-proBNP (aa 1–76), Roche modified (amidated for stabilization) synthetic NT-proBNP, HyTest human recombinant proBNP (aa 1–108), and Scios glycosylated human recombinant proBNP. BNP assays evaluated were Abbott Architect, Abbott AxSYM, Bayer Centaur, Biosite Triage, and Beckman Access (Biosite assay packaged for use by Beckman). NT-proBNP assays (all based on Roche reagents) were Dade-Behring Dimension, Ortho-Clinical Diagnostics Vitros, and Roche Elecsys 2010. All assays, for which epitopes of the antibodies used have been previous described (3), were run according to the manufacturers’ guidelines. BNP, NT-proBNP, and proBNP materials were diluted with normal (low NP concentration) EDTA-plasma pools and lithium-heparin plasma (Dade assay only) pools, collected from healthy donors after institutional review board approval was obtained, to achieve target concentrations of 250, 500, and 1000 ng/L. Baseline BNP and NT-proBNP were quantified first in the pools and then after the pools were spiked with NP peptides. All measurements were performed in duplicate. Baseline BNP or NT-proBNP concentrations were subtracted from each spiked pool measurement. Percent cross-reactivity was calculated by dividing the measured concentration for the spiked pool into the expected peptide concentration, multiplying by 100, and then averaging across all 3 expected concentrations.
Recoveries and cross-reactivity percentages between peptides and BNP and NT-proBNP assays are displayed in Table 1
. The BNP assays were more specific for the BNP peptides, with recovery ranging from 79% to 199%, compared to 5% to 38% cross-reactivity to the proBNP peptides and <1% to 7% cross-reactivity to the NT-proBNP peptides. Similarly, the NT-proBNP assays were more specific for the NT-proBNP peptides, showing 47% to 243% recovery, with substantial cross-reactivity to proBNP peptides (<1% to 249%), and no cross-reactivity to the BNP peptides (<1% across all assays).
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This study demonstrates that the BNP peptides used do not substantially cross-react with NT-proBNP assays, and that the NT-proBNP peptides do not substantially cross-react with BNP assays. We confirm that there is substantial cross-reactivity between proBNP peptides and commercially available BNP and NT-proBNP assays. Variations depended on the different sources and types of peptide tested in each assay. We observed minimal cross-reactivity with the glycosylated Scios proBNP peptide, compared with substantial cross-reactivity to the nonglycosylated HyTest proBNP peptide with the NT-proBNP assays. Glycosylation likely interfered with peptide antibody binding. The mechanisms responsible for different reactivities between the HyTest and Roche NT-proBNP peptides using different NT-proBNP assays, which use the same antibodies but different assay architectures, cannot be explained presently. The modest differences in reactivities for the recombinant (Scios) and synthetic (Peptide Institute) BNP materials using different BNP assays also requires additional study; with different assay architectures for the same reagents (Biosite, Beckman) showing diverse recovery.
Little is known about which NP forms are circulating physiologically. The clinical significance of measured cross-reactivities will also depend on the relative quantities of different molecular forms found in individual patient blood. Mass spectrometry has shown that mature BNP1–32 is absent in severe heart failure patients, but a commercial assay still detected the presence of BNP1–32 (4). Glycosylated forms of proBNP in heart failure patients have also been found, along with uncleaved proBNP (1)(5). Expression of the multiple forms that circulate during acute and chronic heart failure must be investigated in future studies to ensure that commercial assays are measuring the appropriate diagnostic biomarkers for heart failure. Our preliminary observations challenge the analytical field to better characterize what immunoassays measure and challenge the clinical field to better understand what molecules are measured and how to best interpret both BNP and NT-proBNP findings in regard to patient care.
Acknowledgments
Grant /funding Support: Support for this project was from the IFCC Committee for Standardization of Markers of Cardiac Damage through generous donations by the in vitro diagnostic manufacturers of BNP and NT-proBNP assays.
Financial Disclosures: FSA has consulted for Abbott and Ortho Clinical Diagnostics; served on advisory boards for Ortho, Biosite, and Beckman Coulter; and received research grant funding from Abbott, Beckman, bioMeriuex, Biosite, Dade-Behring, MKI, Ortho Clinical Diagnostics, Response Biomedical, Siemens, and Roche. ASJ has received research support and consultation from Dade-Behring, Beckman Coulter, and Ortho Clinical Diagnostics and consulting from Critical Diagnostics, Singulex, Intermune, and Celladon. JO-L has received research funding from Roche. He has received honoraria for speaking from Roche and bioMerieux. RHC has received research funding from Biosite, Dade Behring, Response Biomedical, and Roche; received honoraria from Biosite, Inverness Medical Solutions, and Response Biomedical; and served on advisory boards for Biosite, Dade Behring, Inverness Innovations, and Response Biomedical. JM has received research funding and honoraria from Roche, Abbott, and Siemens. AHBW, KNL, FP, and JT have no declarations.
Acknowledgments: We thank the Peptide Institute, Scios, HyTest, and Roche for their generous donation of peptides.
References
The following articles in journals at HighWire Press have cited this article:
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  J. E. Rame, S. W. Tam, D. McNamara, M. Worcel, M. L. Sabolinski, A. H. Wu, and D. L. Dries Dysfunctional Corin I555(P568) Allele Is Associated With Impaired Brain Natriuretic Peptide Processing and Adverse Outcomes in Blacks With Systolic Heart Failure: Results From the Genetic Risk Assessment in Heart Failure Substudy Circ Heart Fail, November 1, 2009; 2(6): 541 - 548. [Abstract] [Full Text] [PDF]
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 J. Mair Clinical Significance of Pro-B-Type Natriuretic Peptide Glycosylation and Processing Clin. Chem., March 1, 2009; 55(3): 394 - 397. [Full Text] [PDF]
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J. P. Goetze, U. Dahlstrom, J. F. Rehfeld, and U. Alehagen Impact of Epitope Specificity and Precursor Maturation in Pro-B-Type Natriuretic Peptide Measurement Clin. Chem., November 1, 2008; 54(11): 1780 - 1787. [Abstract] [Full Text] [PDF]
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