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Editorials |
1 Mayo Clinic, Rochester, MN
2 Hennepin County Medical Center, Minneapolis, MN
aAuthor for correspondence.
Kemperman et al. (1) have evaluated the relationship of B-type natriuretic peptide (BNP; measured by the Bayer assay) and N-terminal proBNP (NT-proBNP; measured by the Roche assay) in patients treated with a left ventricular assist device (LVAD). They report that the relationship of the results of the two assays changes after prolonged support on a LVAD. Before LVAD use, the ratio of NT-proBNP to BNP is higher than after improvements in hemodynamics with use of the LVAD. They suggest that the initial high ratio reflects impaired renal clearance of NT-proBNP because of abnormal hemodynamics and that the subsequent decrease in the ratio reflects improved renal clearance in response to LVAD support. This may be the case and is one reasonable interpretation of the data given that glomerular filtration was significantly, although modestly, reduced in most patients before use of the LVAD. This hypothesis is consistent with other data suggesting a relationship between BNP and NT-proBNP and renal function (2). However, other explanations are possible, relating to the characteristics of the assays themselves and to physiologic factors in this special group of patients. Many of the issues we will raise ideally should have been explored during the development of the natriuretic assays themselves or during their clinical validation. Thus, there is a need not only to reflect on the specific issues related to BNP and NT-proBNP in patients with impaired renal function but also to ask why, so often, the critical analytical and clinical data that would help answer questions like this are not available in the literature.
Initial analytical validation studies tend to be designed to allow an assay to be shown to be useful and/or to pass regulatory muster. There is then some attempt to define a normal reference interval and ultimately an attempt to find a clinical niche for the assay. When, on occasion, data are published from these studies, they often fail to answer many of the questions that need to be addressed regarding quality specifications of an assay. This is clearly the situation for the BNP and NT-proBNP assays, but not unique to them. Because of similar problems with cardiac troponin, the IFCC Committee for Standardization of Markers of Cardiac Damage (C-SMCD) defined in detail the analytical criteria for troponin assays to meet if they are to be suitable for clinical use (3). None of those criteria are unusual, but they are more than is currently mandated by regulatory agencies and far more than what is usually performed or reported in clinical studies using BNPs or cardiac troponins. The IFCC document also suggests, as recommended by the European Society of Cardiology/American College of Cardiology task force on the redefinition of acute myocardial infarction (4), that the characteristics of all assays for troponin be published in peer-reviewed journals. This needs to be the case for natriuretic peptide assays also. One can take the list from the quality-specification troponin report and define most of the relevant issues that one might want to know about preanalytical and analytical issues for BNP and NT-proBNP assays. They include but are not limited to the following:
Unfortunately, we lack published data in many of these areas for many of the BNP and NT-proBNP assays. It seems to us that if organizations developing guidelines, such as the National Academy of Clinical Biochemistry (NACB), IFCC, AACC, American College of Cardiology, American Heart Association, and European Society of Cardiology, insist on these criteria, these critical data would likely become more available, as has been the case with troponin assays. It would be ideal if regulatory agencies such as the US Food and Drug Administration also accepted these criteria for 510(K) and premarket approval clearance applications.
BNP and proBNP increase with volume overload, cardiac hypertrophy, and left ventricle dilation. Patients with renal failure often have these problems and thus should have some increases in BNP and NT-proBNP. Are the increases all physiologic, or are they related to the measurement of different BNP fragments with different BNP assays, all of which have potentially different clearances? In this regard, it would be helpful to know what the antibodies used really measure. Do the Bayer antibodies used in the study by Kemperman et al. (1) detect both BNP and its degradation products, as reported by Shimizu et al. (5)? Given the epitopes their antibodies recognize, might they even measure proBNP as well? And if that is the case, might they also measure the trimers and tetramers of proBNP as well as some NT-proBNP fragments? It is likely, although not reported, that the antibodies used in the Roche assay used to measure NT-proBNP may also detect many of these forms. Is the issue in renal failure that these other forms become more prevalent or that corin or other convertases act differently so that other forms are generated? If so, it could be one of these products rather than the putative leader sequence of NT-proBNP that accumulates and is detected only by the NT-proBNP assay but not the Bayer BNP assay. Adding still further complexity is the question of whether the antibodies used in the Biosite BNP assay, which detect different epitopes, demonstrate similar or different findings. The fact that there are good correlations reported overall between the Bayer and Biosite BNP assays does not answer the questions in this special subset of patients. Unfortunately, the assays are not sufficiently characterized for us to know this answer.
Another issue includes assay linearity at very high concentrations. The Roche proBNP assay has been known to increase markedly in response to severe heart failure, with values in the 20 000–30 000 ng/L range (6). That may be attributable in part to the physiology of heart failure or, as suggested by Kemperman et al. (1), by delayed renal clearance, but are the very high values that are measured analytically correct? Unfortunately, validations over the entire range of this assay are not available for scrutiny. In the one published report (7) that compared the Roche proBNP ELISA with a proBNP RIA, the relationship between the ELISA and the RIA was close except at very high values, where the ELISA values were much higher. This certainly could be attributable to the better precision of ELISAs compared with RIAs, but it also could be attributable to an analytical problem as yet unreported and undetected.
The issues above are only some of the difficulties that can occur when the data available from analytical assay validation studies are less than ideal. The field needs to codify what is needed in this important area. It seems to us that a wonderful opportunity to do exactly this for BNP and proBNP assays may have been missed initially at the recent NACB Beckman Conference. The group seemed to focus more on clinical criteria and seemed less directed in developing the depth needed for the validation of BNP and NT-proBNP assays. It may well be that the IFCC C-SMCD group, which took on this task for troponin assays in an extremely productive manner, will meet this challenge by both developing an independent report on the analytical criteria necessary for BNP and NT-proBNP assays and by collaborating with the NACB Biomarkers Committee. It may be that regulatory agencies will not accept the proposed criteria and/or will not insist on the level of rigor that will be advocated. However, until experts define such analytical criteria, the field will have difficulty with questions like the one posed by Kemperman et al. (1).
Finally, one needs to define both analytical and biological variability. At the recent NACB meeting, it was argued that because biological variability (8)(9) was so high, perhaps assay precision was not so essential for BNP and NT-proBNP assays. Because these problems are synergistic, it would be wise to do all we can to reduce overall variability. By any criteria, the changes in the values reported by Kemperman et al. (1) certainly exceed the criteria proposed by Wu et al. (8)(9). However, there needs to be continued emphasis on this critical area, lest clinicians decide that it is easy to interpret modest differences in serial values.
The issues posed above are only analytical ones. There is an equal need to develop criteria for clinical validation of assays. This starts with the determination of appropriate normal reference intervals. Often studies are done using convenient samples or samples collected by one study or another to validate age- and gender-specific ranges. Often ethnicity is ignored. Frequently, the individuals included are not ideally characterized and the extent to which they may be anemic, have underlying cardiovascular disease, ventricular hypertrophy, or even mild renal dysfunction may not be scrutinized. This is a critical issue for all cardiovascular biomarker assays. There is a need for all of us who are interested in this area, as well as governmental agencies, to develop a bank of samples taken from well-characterized healthy individuals with sufficient numbers of samples to allow for the validation of age-, gender-, and ethnicity-specific reference intervals. It would certainly help comparisons between assays if all assays used this approach.
If the reference interval issues are not well defined, then it is easy to see why there might be problems in defining appropriate clinical cutoff values in disease states or in screening asymptomatic populations. Are there real differences in the age and gender ranges for BNP and NT-proBNP (10)(11)(12)(13)? Does NT-proBNP increase less with age than does BNP, as suggested by some, or do those differences reflect the different characteristics of different convenience populations? Would it not be appropriate for the answer to be clear? This is a tremendous problem for BNP and NT-proBNP. What is the appropriate cutoff for the diagnosis of congestive heart failure? The cutoff values that have been advocated by studies would include large numbers of elderly females who may or may not have congestive heart failure. Indeed, one could argue that a possible reason that studies suggest that BNP and NT-proBNP values are more predictive than clinicians may be that clinicians have undervalued the potent effect of age. On the other hand, values may perhaps increase physiologically with age, or perhaps they reflect other concomitant comorbidities, such as changes in renal function. Nonetheless, this critical issue for clinical assay validation needs to be addressed. What should be done with patients with and without symptoms who have minor increases in natriuretic peptide concentrations? Is there any concentration at which the diagnosis of heart failure is secure? What cutoff values are appropriate for patients with concomitant relevant abnormalities such as renal dysfunction?
Defining of issues like these must be a shared responsibility among industry, clinical laboratorians, and clinicians. Tackling these issues is often not attractive to commercial interests; it is an area where we as clinicians and laboratorians must step up to the plate. Nowhere is this issue clearer than with the question of whether NT-proBNP concentrations are higher in patients with renal failure. Is the NT-proBNP assay more accurate in this group because of the known up-regulation of neutral endopeptidases (14), which increase the degradation of BNP in patients with renal dysfunction? Could there be degradation of parts of the BNP molecule in an atypical way that makes the epitopes for certain antibodies unavailable? In point of fact, it is very difficult to say that the observed increases in NT-proBNP are attributable to renal failure when no one, to the best of our knowledge, has reported NT-proBNP in the urine. These sorts of gaps in knowledge databases are critical to all as we move forward using these assays in clinical studies.
A commitment is needed on the part of the laboratory medicine community and those involved in the clinical use of these analytes (in this instance, the congestive heart failure and renal communities) to partner with industry to do these sorts of studies. Establishing criteria and pushing for standards would be helpful. We would recommend that the NACB Biomarkers Committee include and expand on such critical issues related to clinical assay validation in their recommendations. It is only then that we will be able to answer the question that the report by Kemperman et al. (1) tries to address. It is likely that many of the issues elucidated above will participate in that answer, perhaps including abnormal renal clearance of NT-proBNP. However, until we know the correct answer, we need to have open minds about the possibilities. In the interim, turning our attention to the more important generic issue of how to develop the appropriate paradigms and funding for accurate analytical and clinical validation studies is essential for all biomarkers if the field is to progress.
Note added in proof:
This editorial was prepared prior to the publication of the two articles listed below, which further support the importance of the questions raised by the editorial.
"Biochemistry of pro-B-type natriuretic peptide-derived peptides: the endocrine heart revisited." Goetze JP. Clin Chem 2004;50:1503-10. DOI: 10.1373/clinchem.2004.034272
"Molecular heterogeneity has a major impact on the measurement of circulating N-terminal fragments of A- and B-type natriuretic peptides." Ala-Kopsala M, et al. Clin Chem 2004;50:1576-88. DOI: 10.1373/clinchem.2004.032490
References
The following articles in journals at HighWire Press have cited this article:
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  B. Vaes, W. de Ruijter, J. Gussekloo, and J. Degryse The accuracy of plasma natriuretic peptide levels for diagnosis of cardiac dysfunction and chronic heart failure in community-dwelling elderly: a systematic review Age Ageing, November 1, 2009; 38(6): 655 - 662. [Abstract] [Full Text] [PDF]
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 R. R.J. van Kimmenade, J. L. Januzzi Jr, J. A. Bakker, A. J. Houben, R. Rennenberg, A. A. Kroon, H. J.G.M. Crijns, M. P. van Dieijen-Visser, P. W. de Leeuw, and Y. M. Pinto Renal clearance of B-type natriuretic peptide and amino terminal pro-B-type natriuretic peptide a mechanistic study in hypertensive subjects. J. Am. Coll. Cardiol., March 10, 2009; 53(10): 884 - 890. [Abstract] [Full Text] [PDF]
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 I. Giuliani, F. Rieunier, C. Larue, J.-F. Delagneau, C. Granier, B. Pau, M. Ferriere, M. Saussine, J.-P. Cristol, A.-M. Dupuy, et al. Assay for Measurement of Intact B-Type Natriuretic Peptide Prohormone in Blood Clin. Chem., June 1, 2006; 52(6): 1054 - 1061. [Abstract] [Full Text] [PDF]
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