Institute for Quality in Laboratory Medicine Series - Controversies in Laboratory Medicine: Insights Into B-type Natriuretic Peptide and N-terminal pro-B-type Natriuretic Peptide Measurements

Natriuretic Peptide Testing – A Laboratorian's Perspective Robert H. Christenson, PhD

The Institute for Quality in Laboratory Medicine (IQLM) is a new organization formed to engage the healthcare community in improving the use of laboratory tests and services. This series, brought to you by the IQLM, is intended to highlight issues and topics within the field of laboratory diagnostics that may have 2 or more points of view.

The Institute is unique. It is the only organization bringing together clinicians, laboratory professionals, patients, manufacturers, payers, government, and accrediting bodies to work on advancing substantial improvements in quality, effectiveness, safety, and appropriateness for the broad universe of laboratory testing. We hope that you find this series, published in Medscape General Medicine, equally unique and forthcoming in its multiview perspective on laboratory issues that affect us all.

The second articles in the series – “Controversies in Laboratory Medicine: Insights into B-type Natriuretic Peptide and N-terminal pro-B-type Natriuretic Peptide Measurements” – were written by W.H. Wilson Tang, MD, Assistant Professor in Medicine, Cleveland Clinic Lerner College of Medicine of Case Western Reserve University and Staff, Section of Heart Failure and Cardiac Transplantation Medicine at the Cleveland Clinic Foundation in Cleveland Ohio, and by Robert H. Christenson, PhD, Professor of Pathology and Professor of Medical and Research Technology and Director of the Rapid Response Laboratories at the University of Maryland School of Medicine in Baltimore, Maryland.

Introduction

Human cardiocytes manufacture a family of structurally related peptide hormones that include atrial natriuretic peptide (ANP), brain (or B-type) natriuretic peptide (BNP), and their metabolically associated peptides. Release of the natriuretic peptides is stimulated by hemodynamic stress, such as wall stretch, ventricular dilation, and/or increased pressures resulting from fluid overload. The natriuretic peptides have powerful natriuretic, diuretic, antimitotic, and vascular smooth muscle relaxing actions. Also, the natriuretic peptides are antagonists for the sympathetic nervous system and the renin-angiotensin-aldosterone axis.[1–3]

BNP has emerged as a superior biomarker to ANP for clinical applications involving heart failure and left-ventricular dysfunction. In a study by Cowie and colleagues,[4] BNP showed a greater predictive power as an indicator of heart failure when compared with either ANP or its metabolites. Contributing are BNP's longer half-life, activation at the gene level and greater quantity in left ventricular tissue.[5–7] Also, BNP has a 2- to 3-fold more powerful natriuretic and blood pressure lowering effect compared to ANP.[8] Thus in vitro diagnostics for BNP and associated metabolites have been the focus for clinical applications.

Biosynthesis and Secretion of BNP and NT-proBNP

Human BNP is derived from 134 amino-acid precursor termed pre-proBNP_1-134. Ventricular stretch or other hemodynamic stress activates cleavage of a 26-aa signal peptide sequence from the N-terminus of pre-proBNP_1-134. As indicated in Figure 1, the remaining proBNP_1-108 prohormone is further cleaved by corin, a membrane-bound serine protease; the N-terminal pro-BNP_1-76 (NT-proBNP) fragment and active 32-peptide, C-terminal BNP_77-108 (BNP) hormone are then released into circulation. BNP and the metabolically inert NT-proBNP are released in a 1:1 molar ratio.

Figure 1.

Mode for release of BNP77-108 and NT-proBNP1-76 into circulation after cleavage of proBNP1-108 by corin.

Although Figure 1 is useful for conceptualizing the model release of BNP and NT-proBNP, the actual metabolism is far more complicated. The intact prohormone proBNP_1-108 may be released into circulation and may indeed cross-react with immunoassays for BNP and NT-proBNP. Also, N-terminal proBNP_1-76 in blood may be comprised of numerous fragments and not a single 77 amino-acid molecular entity. In addition, BNP appears be undergo metabolism following release into circulation. Truncation of the 32-amino acid peptide at either the C-terminal or N-terminal arms to an inactive form of BNP has been reported.[9] Furthermore, the presence and extent of any structural changes may be disease-specific, occurring in some conditions but not in others.[9] Circulating forms of BNP and NT-proBNP are potentially of substantial importance from the laboratory medicine perspective, particularly because all current assays are based on immunological reagents that recognize specific amino acid sequences. Achieving consistent agreement or “harmony” among the BNP and NT-proBNP results is of paramount importance for interpretation of these results in patient care.

There is compelling evidence using nonimmunoassay technologies[9] that immunoassays intended for detection of the active BNP hormone may, in fact, be measuring inactive metabolic forms in patients with severe (NYHA class IV) heart failure. Knowledge in this area is evolving, but clearly such analytical “misinformation” could disrupt the thinking of clinicians caring for these very sick individuals.

In addition, collection of the appropriate specimen type is also a critical variable for appropriate BNP or NT-proBNP measurement. Methods for BNP use blood specimens collected in phlebotomy tubes containing EDTA as an anticoagulant. The preferred specimen for NT-proBNP measurements by methods currently available is lithium-heparinized plasma. Although serum and EDTA plasma may be also be used for measurement with some NT-proBNP assays, lower values may be observed.

In vitro stability of BNP and NT-proBNP after patient collection is also an important consideration, particularly when samples are referred to an outside laboratory for testing. Reports indicate that BNP is stable in either EDTA whole blood or plasma for up to 24 hours if the specimen is refrigerated. Frozen plasma specimens for BNP measurement are reportedly stable for approximately 3 months. NT-proBNP is stable for up to 3 days when plasma or serum is stored refrigerated; NT-proBNP appears to be stable for years when frozen at −70° C. Overall, available data indicate that in vitro measurements of NT-proBNP are more stable compared to those of BNP.

Should we Measure BNP or NT-proBNP? A Laboratory Medicine Viewpoint

Figure 2 displays assays for BNP and NT-proBNP that are either available or in development. From a practical viewpoint, an institution's selection for measurement of BNP or NT-proBNP and which assay to implement will be driven, in part, by the analytical equipment used or approved by the laboratory. Note in Figure 2 that assays from a number of manufacturers use the same immunological reagents.

Figure 2.

Assays that are available and under development for measurement of BNP and NT-proBNP. Dade Behring, Dade Behring, Inc; OCD, Ortho Clinical Diagnostics; DPC, Diagnostics Products Corporation, Inc.; Scios, Scios, Inc.; Bayer, Bayer Diagnostics, Inc.; Abbott, Abbott Diagnostics, Inc.; Biosite, Biosite, Inc.; Beckman Coulter, Beckman Coulter, Inc.

Currently available and in-development NT-proBNP assays use the same immunologic reagents licensed through Roche Diagnostics, Inc. as indicated in Figure 2. This situation is conducive to good agreement among these assays; however, it is well known that use of the same reagents is not the only factor necessary for achieving harmonization of measurements. Therefore, demonstrating that results from each new NT-proBNP method are congruent with data from the Roche Diagnostics technology throughout the analytical and clinical measurement ranges is necessary. Because access to all NT-proBNP immunologic reagents is exclusively through Roche, acceptable analytical and clinical agreement is a condition of licensing before marketing and sale is allowed. Thus after validation and appropriate monitoring to assure stability of measurements, the laboratory and clinical communities can be reasonably assured that NT-proBNP measurements will be harmony.

As illustrated in Figure 2, available BNP assays use immunologic reagents from several different sources. This situation raises potential for methodological differences that may be clinically important. A recent report in dialysis patients demonstrated that the Bayer assay yielded proportionately lower values compared to the Abbott assay by a factor of 0.53, 95% confidence interval, 0.50–0.56.[10] A separate study showed that compared with the Biosite Triage assay, measurements with the Beckman assay differed by a factor of 0.96, the Bayer assay by a factor of 0.77, and the Abbott BNP assay by a factor of 1.13.[11]

Consider the example illustrated in Table 1 and Figure 3 regarding the potential clinical impact of methodological differences in BNP.

Table 1.

Potential Effect of BNP Methodologic Differences for a Patient Having a Biosite Result of 500 pg/mL

Method(Slope)*	Value From Alternate Method	Estimated Posttest Probability
Biosite(1.00)	500 pg/mL	85%
Beckman(0.96)	480 pg/mL	82%
Bayer(0.77)	385 pg/mL	79%
Abbott(1.13)	565 pg/mL	88%

Pretest probability (prevalence) was 50% for the ED population; the posttest probability is estimated by connecting a line from the pretest probability (50%) through the BNP value in, pg/mL and estimating the probability from the right-most line.

^*Linear regression slopes from references.

Figure 3.

Nomogram for estimating posttest probability of decompensated heart failure in an emergency department population from pretest probability (prevalence) and Biosite BNP result. (Adapted from McCullough et al. Circulation 2002;106;416-422 with permission.)

Case: A 67-year-old woman presents to the emergency department with dyspnea and other symptoms and signs suggestive of decompensated heart failure (DHF). The attending physician orders a BNP test knowing that the prevalence of DHF in similar patients is 50%. The Biosite BNP result is 500 pg/mL and the physician uses the nomogram in Figure 3 to estimate the posttest probability (see Table 1). Knowing that there are differences between BNP methods, the physician questions what impact a different BNP method would have on his clinical impression.

Estimation of the pretest probability for an individual patient rests with the clinician. However given the pretest probability, the laboratory can assist the clinician in determining an approximate posttest probability using displays like Figure 3.

This is done by connecting a line from the clinician-provided pretest probability (or prevalence) of 50% through the BNP value of 500 pg/mL and then reading the posttest probability (85%) from the rightmost line. Table 1 illustrates that the posttest probability can range between 79% and 88% using method relationships for other BNP assays[11] and repeating the estimation process. Now consider what would happen if Figure 3 had been generated using Abbott data, and then the Bayer method had been used to measure BNP for the patient. If the true Abbott value was 500 pg/mL, then the Bayer value would have been 265 pg/mL and the estimated posttest probability with the Bayer test would have been 72%, rather than 85% with the Abbott test. This may have had an effect on the attending physician's clinical impression and management.

Proportional differences between methods that are reported in the literature may not fully reflect the degree of harmony between BNP assays. However, the purpose of this case was to provide insight into the possible clinical implications of BNP methodological differences. Because NT-proBNP methods are in harmony, no effect should result from using different methods.

Point-of-care (POC) testing for any analyte is driven by a “need for speed” to improve important patient, economic, or other outcomes. There are some data regarding the turnaround time needed for BNP or NT-proBNP measurement; however, no formal recommendations are yet published by professional organizations. One development that may broaden access and availability of BNP measurements is the Clinical Laboratories Improvement Act (CLIA)-waived status for the Biosite Triage BNP method. CLIA-waived status does not exempt performance of manufacturer-recommended quality control and documentation activities; however, a healthcare venue holding a CLIA license can have BNP measurements performed by individuals with any educational and experience background provided they are adequately trained. An important caveat is that local regulatory and accreditation requirements for testing vary widely, and the more stringent requirements “trump” CLIA-waived status. For this reason, managers and directors contemplating POC testing for BNP must educate themselves on all applicable local requirements.

Summary and Conclusion

The blood concentrations of BNP and NT-proBNP, which are heart-selective molecules, reflect hemodynamic stress. Their metabolism is complicated and not fully elucidated; interpretation of values may show important differences depending on disease status. The quality of results for clinical care depends on laboratory medicine considerations such as specimen type, in vitro stability, and the potential for cross-reactivity with proBNP. NT-proBNP values from different methods will be in harmony, whereas BNP values may show methodological differences due to use of different immunologic reagents. Performance requirements for most BNP and NT-proBNP methods fall in the moderately complex CLIA category, but 1 BNP method has CLIA-waived status. The quality of BNP and NT-proBNP interpretation for patient care is enhanced by availability of well-informed laboratory medicine staff.

Readers are encouraged to respond to George Lundberg, MD, Editor of MedGenMed, for the editor's eye only or for possible publication via email: glundberg@medscape.net

Natriuretic Peptide Testing – A Clinician's Perspective W.H. Wilson Tang, MD

Natriuretic peptide testing has been likened to “a novel white count for heart failure.[12]” Animal studies have demonstrated that circulating natriuretic peptides are expressed in response to myocyte stretch, which is caused by hemodynamic derangements in the heart failure syndrome.[13] Two main types of assays are clinically available, the active moiety BNP and its amino-terminal counterpart, NT-proBNP. Although they are produced in a 1:1 fashion from their propeptide, proBNP, they differ significantly in their plasma levels. In particular, BNP is metabolized by neutral endopeptidases and cleared by C- or “clearance” receptors, where NT-proBNP is primarily excreted by the kidneys. Over the past 5 years we have witnessed a wide range of studies that illustrate the robust diagnostic and prognostic value of testing for both BNP and NT-proBNP even beyond the diagnosis of heart failure.[14] For example, long-term sequential plasma BNP testing predicts clinical outcomes in patients with acute coronary syndromes[15] and in chronic heart failure.[16] Elevated levels of plasma BNP also confer high event rates in patients without underlying cardiac dysfunction.[17]

Nevertheless, we have also learned over the years that elevated plasma levels of natriuretic peptide may not be specific for heart failure and cardiac dysfunction. Several cardiac and noncardiac confounders have become apparent in the literature (such as renal dysfunction and obesity). Natriuretic peptide expression can also be variable, especially in the nonacute setting,[18] and individual and interassay variability makes a single-point determination difficult to interpret. Responses to treatment may only be loosely associated with changes in blood volume,[19] intracardiac filling pressures,[20] or changes in health status.[21] Therefore, the debate of the true clinical value of natriuretic peptide testing continues, despite numerous publications.

What do clinicians really want from a biomarker? First and foremost, we want biomarkers that can define a disease (not necessarily a disease process), and potentially reflect on the underlying etiology that can be targeted for therapy. Determining the presence and extent of the heart failure syndrome is hardly a binary decision, and finding “cut-off” values for these purposes may be self-limiting because the gold standard is difficult to define. Because high plasma levels of natriuretic peptides certainly provide a unique signal of myocardial distress, the question then becomes: should plasma BNP testing become the gold standard for “myocardial stress” rather than a biomarker for heart failure? That being said, at this stage of our understanding, even the strongest advocate for BNP testing will agree that an elevated plasma BNP level without any change in signs and symptoms would only prompt a more in-depth evaluation rather than a reflexive increase in diuretic therapy. One must therefore understand that plasma BNP testing may be useful in certain instances where ambiguity is present or decision-making is promptly needed. Patients presenting with acute dyspnea are a prime example, which explains why studies in this setting have yielded the most consistent results.[22,23] In particular, an intermediate pretest probability will yield a higher posttest probability if the plasma level of BNP or NT-proBNP is high. To date, there has only been 1 clinical trial that has a prospective randomized design, which found that using plasma BNP in this setting in the emergency department provided cost savings in heart failure hospitalizations by reducing length of stay.[24]

If plasma BNP has outperformed all other clinical and laboratory variables for predicting mortality and parallel measures of disease burden related to dysfunctional myocardium, it would be a useful and objective gauge in an otherwise very subjective disease. Making quantitative definitions are more likely to provide an “industry standard' for communications regarding the presence and disease severity of the heart failure syndrome. However, it is as important to point out that information regarding the use of biomarkers for risk stratification is rarely relayed directly to the patient, and will seldom change clinical practice unless specific drug therapy is being indicated based on the information. The optimal timing of repeating measures is also unclear, but daily measurements may not be necessary.[25]

As important as making an accurate diagnosis, clinicians also want biomarkers that can justify a medical intervention, and even better when changes in biomarker levels resulting from the intervention reflect the corresponding benefits. Although it may be tempting for healthcare providers to use plasma natriuretic peptide testing in a similar manner to other common endocrine biomarkers such as glycosylated hemoglobin or thyroid-stimulating hormone, this strategy may not always be clinically appropriate. A few examples may illustrate this important point. A patient who comes into the emergency department with signs and symptoms of fluid retention that are highly suspicious of clinical heart failure will not likely benefit from a routine confirmatory BNP test, and an ambiguously low plasma BNP level may even impede the appropriate treatment. In contrast, a patient with no clinical evidence of heart failure on diuretic therapy with rising plasma BNP level beyond the expected variations may be asked to increase his or her diuretic dose. However, increasing plasma levels may also be caused by intravascular volume depletion and azotemia.

Clinicians are often not aware of the diverse variation of plasma BNP levels obtained from different commercially available assays, and some may not even appreciate the differences between BNP and NT-proBNP (which may have 5- to 10-fold differences in plasma levels). Clinical differences between the assays have been the focus of marketing strategies by different diagnostic companies, but the true clinical impact of assay differences is virtually unknown. What is unavoidable is that this variation allows a potential point of confusion – for example, when a patient is being evaluated in an outside emergency department with a BNP" of 250 pg/mL that prompts admission for decompensated heart failure, when actually it was plasma NT-proBNP that was reported. Therefore, harmonization of these reported values will be of vital importance in guiding the interpretation of BNP and NT-proBNP results.

A special mention of plasma BNP and NT-proBNP assessment in the setting of acute coronary syndrome is worthwhile. Due to the availability of many clinical trials of different strategies and therapeutics in this area, we have had a wealth of data that validates the robust diagnostic and prognostic role of BNP[26] and NT-proBNP,[27] both by itself as well as in combination with other biomarkers (such as high-sensitivity C-reactive protein and cardiac troponin).[28] There have also been data suggesting the association between plasma BNP or NT-proBNP levels in degrees of severity of ischemia[29] as well as atherosclerotic burden and clinical outcomes in stable coronary artery disease.[30,31] However, future challenge to support broad adoption in this setting will rely on demonstrating that treatment indicated by elevated plasma BNP or NT-proBNP levels may directly lead to improved clinical outcomes.

From a clinician's point of view, BNP and NT-proBNP measurements have changed the way clinicians identify and manage acute and chronic heart failure. Plasma BNP and NT-proBNP may identify a patient population that is at heightened cardiovascular risk for further evaluation and intervention. However, we still have to recognize that there are a number of clinical limitations: (1) high plasma BNP or NT-proBNP level can only be suggestive of clinical decompensation or heightened severity of heart failure, but clinical correlation remains vital to accurate diagnosis and treatment decisions; (2) there are different assay characteristics and individual patient variability that may need to be factored into the interpretation of a plasma BNP or NT-proBNP level; and (3) at this time, although BNP or NT-proBNP levels may influence how we treat a patient, there is still no single cutoff or level that may tell us when to start or stop a drug, or whether or not to perform a procedure. Future studies in these areas will hopefully refine our understanding and enhance their clinical utility, allowing quality data to support broad clinical uses.

Key Quality Issues: Achieving Confluence With Clinical and Laboratory Perspectives?

1. Do disease states and comorbidities such as renal insufficiency make a difference in use of BNP or NT-proBNP values? Should an institution perform BNP measurements, NT-proBNP measurements, or both?

Laboratorian (Christenson): Any disease state that causes hemodynamic stress will increase circulating BNP and NT-proBNP values. Renal insufficiency will affect both BNP and NT-proBNP values so that higher cutoffs must be used. However, the diagnostic ability for decompensated heart failure is the same for both tests. The bottom line is that differences between BNP and NT-proBNP are dwarfed by the similarities.

Clinician (Tang): Clinically, we see very similar effects on both BNP and NT-proBNP by the same confounders (age, gender, obesity, renal insufficiency). For the current indication of BNP or NT-proBNP testing, any of the commercial assays will be adequate.

2. Who should make the decision as to which biomarker, BNP or NT-proBNP, is measured, and where the test is performed?

Laboratorian (Christenson): Provided that the appropriate cutoff points and education on age and gender are available to clinicians with results, based on current knowledge, there is no material difference in quality between BNP and NT-proBNP for clinical applications. Therefore availability measurement systems, cost of reagents, and local needs for availability of BNP or NT-proBNP data are primary considerations.

Clinician (Tang): Clinicians as “consumers” usually take whatever assay is available at their local laboratory or hospital, and usually do not have a choice regarding the assay offered to them. While there is a theoretical advantage over immediate results reporting at the POC, the need for phlebotomy even with the POC assay limits “real-time” testing at the bedside or in the office.

3. How sophisticated are laboratories in the area of natriuretic peptides? Should the laboratory (a clinical laboratorian) be available for informed consultation regarding differences in clinical application of differing assays?

Laboratorian (Christenson): Fairly sophisticated, but biased information from manufacturers has clouded the picture. Laboratory medicine involves more than in-laboratory quality monitoring. Quality data reporting implies that information is available about the test; however, covering all scenarios in a single message of reasonable length is impossible. Also, expecting clinicians to remain current on laboratory-related issues is unrealistic. Thus in dealing with interpretation of data, knowledgeable laboratory input can help maximize information provided by BNP and NT-proBNP measurements.

Clinician (Tang): Perhaps one of the most overlooked issues in natriuretic peptide testing is the fact that no matter how much data support the diagnostic and prognostic value of BNP and NT-proBNP testing, congestive heart failure remains a clinical diagnosis. No single laboratory test to date can define the presence or absence of the condition. At this time, there is only 1 clinical application that is approved by the US Food and Drug Administration – as an aid to the diagnosis of acute heart failure. Like checking a total leukocyte count in the setting of a fever, clinicians should aware that the data may help to confirm a diagnosis or to describe the risk profile that may affect the treatment regimen. Knowledgeable laboratory input is always helpful particularly where there are peculiar results, but it may not be necessary (and likely impractical) for every case.

4. Are clinicians aware of the differing laboratory assays for BNP and NT-proBNP?

Laboratorian (Christenson): With the exception of thought leaders in various clinical specialties, there is little experience and knowledge among physicians practicing among different hospitals. This is why laboratory medicine is important.

Clinician (Tang): The short answer is no. When a clinician orders a test, he or she usually is not aware of what particular assay is used for the measurement. A clinician has trust in the laboratory that the result reflects a reliable and reproducible measurement, and if interassay discrepancies exist, these should be minimal and perhaps inconsequential to the interpretation. Harmonization of different assay results is therefore of paramount importance, particularly when it comes to the role of BNP- or NT-proBNP-guided management. If there is a specific “cut-off” and 1 assay gives a much higher level than the other, then how does a clinician respond to such heterogeneity? The development of the International Normalization Ratio is a very good example of how harmonization can be achieved for vastly different results from different assays the purpose of unifying test results for effective clinical application.

5. What are the clinical limitations of BNP and NT-proBNP? What are the predictive values of the biomarkers for different clinical conditions and which applications are most cost-effective?

Laboratorian (Christenson): There is a huge difference between a biomarker being elevated in association with a disease condition and utilization of that biomarker clinically. Evidence-based laboratory medicine mandates the use of the best data available to generate unbiased informed decisions for help guide diagnosis, treatment, and care of patients. The limitation of BNP and NT-proBNP is that enough good evidence is simply not available for many potentially important clinical applications. The use of BNP or NT-proBNP for diagnosis and risk stratification of patients with decompensated heart failure is cost effective.

Clinician (Tang): The major clinical limitation is that BNP or NT-proBNP does not define a disease process. BNP and NT-proBNP perform very well under statistical testing, and give very nice P values and good receiver operator characteristic curves. However, when used in an individual basis, there are continuing challenges particularly with regard to the lack of specificity with respect to the degree of disease severity or treatment responses. For example, if a patient has a BNP level of 500 pg/mL, one can only tell that the patient may have relatively advanced or more decompensated heart failure, but cannot possibly tell what the functional class is, what degree of severity the underlying systolic or diastolic dysfunction is, how responsive to medications the patient is, or whether the patient would need a defibrillator. If the same patient has a subsequent BNP level of 200 pg/mL, I may consider the “risk profile” to be improved, but still cannot answer any of the previously mentioned questions. The observational nature of all the 4000+ papers in the literature will not advance our understanding unless we start to test by changing our clinical practices based on specific plasma BNP or NT-proBNP levels – these will hopefully be the results of some upcoming trials.