Laboratory and Clinical Aspects of B-Type Natriuretic Peptides

B-type natriuretic peptide (BNP) is a cardiac neurohormone that is primarily synthesized in ventricular myocardium in proportion to the end diastolic pressure and ventricular volume. The measurement of the BNP concentration in plasma was introduced to the clinical laboratory and approved by the Food and Drug Administration (FDA) approximately 3 years ago as a test to identify cardiac failure (ie, congestive heart failure) among other causes of dyspnea. During the past 3 years there have been more than 500 publications about BNP listed in Index Medicus that have established it as a key laboratory test for the diagnosis of congestive heart failure. We will answer some of the important questions about the laboratory and clinical aspects of BNP.It has been more than 23 years since de Bold1 reported that intravenous administration of atrial extracts into rats resulted in a marked increase in sodium excretion. This sentinel observation initiated intense research into the endocrine role of the heart that led eventually to the characterization of a family of structurally similar but genetically distinct natriuretic peptides. Of interest, BNP was originally named brain-type natriuretic peptide, since it was initially discovered in the brain. To date, the following 4 peptides have been identified in the family: atrial natriuretic peptide (ANP), C-type natriuretic peptide (CNP), urodilatin, and BNP. Structurally, the natriuretic peptides include a 17–amino acid disulfide-bonded ring with N-terminal and C-terminal arms of various lengths and sequences. In the ring, 11 amino acids are conserved among the 4 natriuretic peptides. BNP is composed of 32 amino acids and has a molecular weight of 3472 d. ANP is produced primarily by the atria of the heart under similar conditions of BNP production, but elevations in ANP do not correlate as well with congestive heart failure as BNP elevations. CNP is released from the endothelium in response to increases in shear stress to act locally as a vasodilator. CNP has no systemic natriuretic or diuretic effects. Urodilatin is released from the renal, distal, convoluted tubule cells into the tubular lumen and does not circulate in blood.The synthesis of BNP occurs through a preprohormone. The BNP gene encodes preproBNP, a 134–amino acid residue precursor in which the bioactive 32–amino acid sequence is at the C-terminus.2 Once produced in myocardial cells from preproBNP, proBNP (108 amino acids) is cleaved into a 76–amino acid N-terminal fragment (NT-proBNP, amino acids 1 to 76) and the 32–amino acid BNP fragment (amino acids 77 to 108). NT-proBNP is not biologically active. BNP and NT-proBNP are secreted in a 1:1 ratio. However, their molar ratio in plasma is not 1:1, because BNP has a half-life of approximately 20 minutes, whereas NT-proBNP has a half-life of approximately 90 minutes. The difference is probably related to size, since NT-proBNP has molecular weight more than 2.5 times BNP. The shorter half-life of BNP allows more rapid monitoring of the patient's status with therapy. However, there are currently no definitive data that show that the monitoring of BNP or NT-proBNP for the treatment of acute congestive heart failure alters clinical outcome.Currently, there are 3 analytical systems available to measure BNP or NT-proBNP. A point-of-care assay for BNP was initially introduced by Biosite (San Diego, Calif) in 2000 using the Triage meter with either EDTA plasma or whole blood. In 2002, Roche Diagnostics Corporation (Indianapolis, Ind) introduced an assay for NT-proBNP with either serum or heparinized plasma using the Elecsys 1010/2010 and the Modular Analytics E170 immunoassay analyzers. With EDTA plasma, the results for NT-proBNP were approximately 10% lower than serum results. In 2003, Bayer Healthcare (Tarrytown, NY) introduced a BNP assay using EDTA plasma on their ADVIA Centaur system. At least 2 other instrument manufacturers have indicated plans to offer one of these assays in the near future. There appears to be a good correlation between BNP and NT-proBNP. BNP measured with the Shionogi immunoradiometric assay compared with NT-proBNP as measured with the Roche electrochemiluminescent method showed a correlation coefficient of 0.97.3 The following equation related the concentration of the 2 analytes: NT-proBNP = 3.48pM × BNP − 19pM. A recent multicenter study compared the Roche NT-proBNP assay with the Biosite triage BNP assay and reported “reasonable correlation” but with a substantially higher slope bias for the NT-proBNP assay.4 The correlation coefficients ranged from 0.54 to 0.62 and were statistically significant.The College of American Pathologists (CAP) began offering proficiency testing for BNP and NT-proBNP in 2003. For the first challenge, there were 975 Biosite Triage users and 52 submissions from Roche users. The coefficients of variation for the 2 challenges were 17.8% with a mean of 99.2 pg/mL and 15.0% with a mean of 586.8 pg/mL for the Biosite Triage system, and 7.2% with a mean of 106.3 pg/mL and 4.8% with a mean of 644.7 pg/mL for the Roche systems. In the second challenge, 83 laboratories reported results with the Bayer ADVIA Centaur for BNP with a coefficient of variation of 5.1% with a mean of 693.0 pg/mL and 5.6% with a mean of 2,831.8 pg/mL. Even though the Centaur was calibrated by Bayer to match the Biosite Triage results, the results for the Centaur were approximately double that of the Triage. It is highly likely that this is due to a matrix effect of the proficiency testing material. Further, a preliminary study performed by the CAP showed that the BNP material loses approximately 35% of its immunoreactivity after 4 hours at 4°C, whereas the NT-proBNP material showed no decline in immunoreactivity.Stability, medication, and renal function are important preanalytical factors. NT-proBNP is more stable in the sample than BNP. Biosite recommends testing for BNP immediately or within 4 hours of sample collection. If the testing cannot be completed within 4 hours, the sample should be stored at −20°C until it can be tested. Bayer recommends testing within 24 hours with storage of the sample at 2°C to 8°C. The average percentage of BNP recovery in EDTA plasma after 24-hour storage at 2°C to 8°C was 91% per the Bayer package insert. Plastic blood collection tubes are recommended for BNP sample collection by Biosite. However, a recent study found no statistical difference between results obtained for NT-proBNP or BNP when EDTA anticoagulated blood was aliquoted and stored at room temperature or 4°C in glass or plastic tubes for up to 48 hours.4 Roche recommends testing for NT-proBNP within 2 hours of blood collection because of possible evaporation effects but indicates NT-proBNP is stable for 72 hours at 20°C to 25°C. BNP is available as the prescription drug Natrecor (nesiritide, recombinant DNA-produced BNP) for the treatment of heart failure. Unless 4 or 5 half-lives of the drug have been allowed to pass, Natrecor will be detected by the BNP assay but not the NT-proBNP assay. Lastly, preliminary studies have shown a graded increase in both of these analytes with decreasing glomerular filtration rate.56 Further studies are needed to define the importance of renal insufficiency on the interpretation of results for these analytes.Age, sex, and body mass index influence the reference interval for these 2 analytes. Biosite defines the reference interval for BNP as less than 100 pg/mL. The Bayer BNP assay was harmonized to match the Biosite decision threshold. Roche Diagnostics defines their reference interval for NT-proBNP as less than 125 pg/mL for people younger than 75 years and less than 450 pg/mL for individuals 75 years and older. The plasma concentrations of BNP and NT-proBNP are slightly higher in women than men and rise progressively with age.7 The study by Redfield et al7 proposes reference intervals by sex and decade beginning at the age of 45 years. Further, a recent study has documented that obesity (ie, increased body mass index) significantly lowers the BNP concentration.8 Thus, age, sex, and body mass index should be taken into consideration for the appropriate interpretation of a BNP or NT-proBNP result by the physician, but currently the effect of these factors on the reference interval requires further study and quantification.Originally, the FDA approved the use of BNP or NT-proBNP for the evaluation of dyspnea to assist in differentiating a cardiac cause for dyspnea (eg, congestive heart failure) from a noncardiac origin for dyspnea (eg, chronic obstructive pulmonary disease). A recent, prospective, randomized, controlled study of 452 patients who presented to the emergency department with acute dyspnea showed that the measurement of BNP improved the evaluation and treatment of patients with a reduced time to discharge and total cost of treatment.9 Recently, NT-proBNP was approved by the FDA for use in assessing the prognosis of patients with congestive heart failure and acute coronary syndromes, whereas the Biosite BNP assay is also approved for risk stratification in acute coronary syndromes. There are extensive studies in the medical literature that document the excellent ability of both BNP and NT-proBNP to diagnose heart failure and indicate the severity of heart failure. In a recent study, BNP, NT-proBNP and the cardiac ejection fraction were measured in 150 patients hospitalized for cardiac disease.10 The data showed sensitivities of 94% and 100% for both BNP and NT-proBNP for patients with an ejection fraction of less than 60% and less than 40%, respectively. Further, the negative predictive values for both tests were 96% and 100% with an ejection fraction of less than 60% and less than 40%, respectively. There is a direct relationship between the severity of heart failure as assessed by the New York Heart Association Classification System and the concentration of BNP or NT-proBNP.11 Thus, BNP and NT-proBNP seem to be equally effective in diagnosing heart failure and estimating the severity of the heart failure. However, when and how frequently to order these tests for diagnosis and management of heart failure require additional study.12Further, a recent review of the literature by Clerico and Endin13 concluded that “several aspects of cardiac natriuretic peptide assays are still to be elucidated, and further work is needed to carefully assess their diagnostic accuracy and prognostic value in cardiac disease.” This is still a relatively new test that needs additional study of the pattern of results in the disease process plus the several factors that influence the reference interval. This is best illustrated by a recent study that identified 40 patients of 717 with no history of congestive heart failure with a BNP concentration of more than 500 pg/mL who were confirmed by 2 cardiologists to not have congestive heart failure.14 The final diagnoses for these patients were the following: asthma or chronic obstructive pulmonary disease with right ventricular strain, pneumonia, pulmonary embolism, anemia, cardiac ischemia, and sepsis. Thus, there are diseases other than congestive heart failure that may effect a significant elevation of BNP.Several exciting uses for these tests have been identified in the past few years. These tests have been used effectively to assess prognosis after myocardial infarction. In a recent study, BNP, NT-proBNP, and the left ventricular ejection fraction (LVEF) were measured 1 to 4 days after myocardial infarction in 666 patients with a mean 3-year follow-up.15 The authors evaluated NT-proBNP, BNP, and LVEF as predictors of death or heart failure. The results showed BNP or NT-proBNP were comparable and independent predictors of major adverse events. Furthermore, both markers were better predictors of adverse events than LVEF. However, combining BNP or NT-proBNP with LVEF provided risk stratification substantially better than that provided by either test alone. BNP showed a sensitivity of 71%, a specificity of 76%, and an area under the curve (AUC) of 0.81 at an optimum cutoff of 30pM (104 ng/L), and NT-proBNP showed a sensitivity of 80%, a specificity of 72%, and an AUC of 0.81 at an optimum cutoff of 162pM (146 ng/L). The authors concluded that BNP and NT-proBNP levels measured within a few days of acute MI independently predict death and heart failure in the presence or absence of preserved ejection fraction and are related to the risk of new ischemic events.In acute coronary syndromes, BNP was found to provide unique prognostic information when compared with troponin I and C-reactive protein.16 BNP has been used as a marker of risk for thromboembolism in patients with nonvalvular atrial fibrillation.17 This study documented a significant increase in BNP in those patients with a history and/or echocardiographic evidence of a thrombus. Gerber et al18 evaluated 74 patients with isolated aortic stenosis by echocardiography and measurement of ANP, BNP, and NT-proBNP. The results were similar among the 3 analytes and showed a significant elevation in symptomatic patients when compared with asymptomatic patients with aortic stenosis. The authors concluded that these tests may complement clinical and echocardiographic evaluation of this patient population. In a recent study, the prognostic significance of BNP and NT-proatrial natriuretic peptide were evaluated in a prospective study of 3346 persons without heart failure.19 The study showed that plasma natriuretic peptide levels predict the risk of death and cardiovascular events after adjustment for traditional risk factors. In fact, excess risk was apparent at natriuretic peptide concentrations well below the current thresholds used to diagnose heart failure. Further, an increase in BNP has been found to predict an adverse outcome in patients with pulmonary embolism.20 Thus, the determination of BNP or NT-proBNP seems to have diagnostic and predictive value for several cardiac diseases and pulmonary embolism.The determination of BNP or NT-proBNP is one of the modern success stories in laboratory medicine. Many studies have documented the unquestionable clinical value of these tests for the diagnosis and assessment of congestive heart failure, in addition to their application to several other cardiovascular disease entities. Future observations will help clarify the analytical and diagnostic differences between BNP and NT-proBNP and the application of these tests to other diseases. Laboratories not currently offering BNP or NT-proBNP measurements should consider adding them to their clinical armamentarium.We thank Bonnie Deppert for her clerical help with the preparation of the manuscript.