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Natriuretic Peptides and Analytical Barriers.

Natriuretic peptides assays are essential to the evaluation and treatment of heart failure (HF) [3] (1). However, there is relatively poor harmonization of the various assays owing to the use of different antibodies and different analytical detectors (2). To understand the use of these assays, one needs a basic understanding of natriuretic peptide physiology and how this physiology interdigitates with the assays.

Basic Physiology

Atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) were described in the 1980s as circulating peptides released from the heart and involved in maintaining cardiorenal homeostasis by diuresis and natriuresis (3). ANP is produced predominantly in the atrium and is stored in intracellular granules where it is readily available for release. BNP is synthesized to a greater extent in the ventricular myocardium and is stored in only modest quantities. Thus, BNP requires more time to be synthesized and released and is a slower reacting peptide. Both peptides induce diuresis and natriuresis, and reduce vascular resistance and systemic blood pressure. C-type natriuretic peptide (CNP) is produced in the central nervous system and vascular endothelium, and acts as a paracrine regulator with little known implications for the cardiovascular system. Dendroaspis natriuretic peptide (DNP) is another member of the natriuretic peptide family and urodilantin is a renally secreted cleavage product of ANP (3).

All natriuretic peptides in humans contain a preserved ring structure of 17 amino acid residues and form an intramolecular disulfide bond, which presumably is responsible for receptor binding. Most BNP assays target this ring structure for 1 of their epitopes. The receptors, termed natriuretic peptide receptors (NPRs) are classified as types A and B. Binding of these guanylyl cyclase-coupled receptors leads to an increase in cGMP, which has downstream effects of diuresis and natriuresis, vasodilation, inhibition of the renin-angiotensin-aldosterone system, enhanced myocardial relaxation, inhibition of fibrosis and hypertrophy, promotion of cell survival, and inhibition of the inflammatory response. NPR type C lacks guanylyl cyclase activity and is thought to be a clearance receptor (3).

Conditions of the heart accompanied by volume and pressure overload, such as HF and myocardial infarction, lead to cardiac wall tension and stretch, volume overload, or ischemia and result in increased production and release of natriuretic peptides (4). The use of these biomarkers for diagnostic and prognostic purposes has been delineated (4) and endorsed by guideline authorities for heart care (5). Moreover, recombinant forms of human ANP (carperitide) and BNP (neseritide) were developed as therapeutic agents for HF (6, 7). However, clinicians have lost enthusiasm for these agents due to doubtful efficacy and safety issues (8).

BNP is more stable in vitro and has superior diagnostic performance to ANP, thus BNP and BNP-related peptides have established a role in clinical practice for the diagnosis of risk stratification and follow-up of patients with HF (1, 9).

Brief Relevant Molecular Biology of Natriuretic Peptides

ANP

ANP is synthesized as a 151-amino acid prepropeptide, preproANP, which is stored in atrial intracellular granules (10). PreproANP is secreted and cleaved to the mature peptide, ANP, in response to atrial stretch, angiotensin II, endothelin, or sympathetic-mediated stimulation. PreproANP is cleaved to proANP, which is processed by the convertase corin into 2 circulating peptides, ANP 1-28 and the N-terminal fragment, NT-proANP 1-98 (11). NT-proANP is subsequently cleaved into 3 fragments with biological importance. ProANP 1-30 is the long-acting natriuretic peptide, proANP 31-67 has vasodilation properties, and proANP 79-98 or kaliuretic peptide, is involved in potassium excretion. NT-proANP and its cleavage derivatives are all found in circulation. Another member of the ANP family, urodilantin can be isolated from human urine and increases diuresis (12).

BNP

BNP is synthesized as a 134-amino acid preproBNP precursor, which is cleaved to the 108-amino acid proBNP by removal of a 26-amino acid signal peptide (13). A peptide fragment of preproBNP containing amino acid residues 17-26 is present in normal individuals and patients with acute MI. It has been proposed as a biomarker of cardiac ischemia. Another peptide of the signal residue containing amino acid residues 16-25 may be a biomarker of ischemia (14).

BNP is encoded by an early response gene allowing transcription to reach maximal levels rapidly (15). Intracellular BNP storage is minimal and it is synthesized de novo when needed and released by ventricular cardiomyocytes.

The processing of proBNP forms an N-terminal proBNP (NT-proBNP) fragment with amino acid residues 1-76 and a C-terminal region active BNP with amino acid residues 77-108 (16). Because of proteolysis of proBNP, BNP and NT-proBNP are produced in a stoichiometric ratio of 1:1. NT-proBNP has no known biologic activity. Despite the 1:1 stoichiometric ratio, the molar plasma concentration of NT-proBNP is higher than the concentration of BNP likely because NT-proBNP has slower clearance from the circulation (17). ProBNP in nonprocessed form is found in healthy individuals and patients with HF (18). ProBNP is post-translationally modified in patients with HF by

0-glycosylation at several threonine and serine residues within the N-terminal region (amino acid residues

1-76), but not within the BNP-portion of proBNP (amino acid residues 77-108) (19). It appears that NT-proBNP is glycosylated in the central region (amino acid residues 28 -56), while the C-terminus region of the molecule (amino acid residues 61-76) is not post-translationally modified (19). ProBNP, however, is glycosylated both in the central region and in the region located close to the cleavage site, specifically at amino acid residues 63-76. This region can be blocked to site-specific antibodies because of steric impediment due to glycosylation (20). The degree of glycosylation of both proBNP and NT-proBNP is highly individual. It is also known to inhibit the activity of furin on the 76 -78 amino acid cleavage site (21).

The diversity of circulating proBNP-derived peptides is explained in part by proBNP processing, which occurs immediately before, or at the time of release. The processing is facilitated by prohormone convertases. Furin and corin are convertases currently thought to be proBNP-processing enzymes. The evidence is indirect from in vitro studies, which demonstrate the formation of BNP 1-32 (by furin) and BNP 4-32 (by corin). Since corin produces a short BNP form, BNP 4-32, this enzyme is unlikely to be the only one responsible for the processing of proBNP, suggesting that furin also plays a role (22).

Glycosylation residues in the region of the proBNP molecule close to the cleavage site inhibit the processing of proBNP. In in vitro studies, both furin- and corin-mediated processing of proBNP are suppressed by O-glycans located at Thr71. It appears that only proBNP molecules not glycosylated at Thr71 can be processed into BNP and NT-proBNP, probably due to access of enzyme to the unbound cleavage site. Inhibition of processing via glycosylation might be a pathologic phenomenon found in HF.

Degradation Fragments of BNP in Circulation

Although initially it was thought that there were only 2 circulating fragments of BNP, BNP 1-32 and NT-proBNP 1-76, contemporary data have challenged this concept recently (23). Mass spectrometry analyses showed only minute amounts of circulating BNP 1-32. BNP 1-32 is found in patients with advanced HF in only very low levels (14). In blood samples from HF patients multiple N- and C-truncated fragments of BNP are present such as BNP 1-32, BNP 3-32, BNP 4-32, BNP 5-32, BNP 5-31, BNP 1-26, and BNP 1-25 (24). The variety of such fragments is believed to be the result of proteolysis by dipeptidyl peptidase IV (DPP IV), which forms BNP 3-32 and neprilysin (NEP), which forms BNP 5-32. BNP can also be proteolyzed by insulin-degrading enzyme (IDE), but not by NEP and IDE implying that another enzyme is involved in cleavage (25). BNP 4-32 may be the result of corin activity (21). Another protease, peptidyl arginine aldehyde protease, can also degrade BNP, and meprin was shown to lyse BNP in animal models but not in humans (26). Due to BNP instability in EDTA plasma even at low temperatures, protease inhibitors in high concentrations should always be used (23).

ProBNP and Cross-Reactivity

Intact proBNP is found in healthy individuals and patients with HF. Immunoassays used in clinical practice to detect BNP show extensive cross-reactivity with proBNP. The extent of cross-reactivity is different for various assays and different forms of glycosylated or nonglycosylated proBNP. Notably, only BNP and not the precursor proBNP has been shown to have a natriuretic response in patients with HF (27). ProBNP is resistant to proteolysis and inactivation by human kidney membranes (28). The role of proBNP in healthy volunteers and patients with HF may be a physiologic or a pathologic process. ProBNP is a poor stimulator of guanylyl cyclase. There are differential cGMP activating properties of BNP forms and, notably, proBNP 1-108 and NT-proBNP 1-76 have reduced cGMP activity in vitro (29). Some speculate that unprocessed proBNP is a circulating "reserve" of BNP since in vivo cleavage of proBNP produces BNP. Glycosylation of Thr71 in proBNP has inhibitory effects since it prevents proteolysis by both furin and corin. Only proBNP that is not glycosylated at Thr71 can form active BNP1-32.

ProBNP Circulating Forms in Acute and Chronic HF

The highest percentage of glycosylated proBNP is present in patients with chronic HF compared to patients in the acute decompensated and nonacute decompensated groups (30). Another interesting finding is that furin activity but not its concentration is greater in the acute HF group than in the chronic HF group (30). Perhaps there is a differential mechanism of proBNP processing in disease progression in HF patients with increased BNP production in the acute decompensated group of patients with fluid overload. In the chronic HF group, proBNP degradation will

not occur since there is less acute fluid overload, implying that there might be regulatory mechanisms responsible for rapid increases in plasma BNP by degradation of the processing-susceptible proBNP. These findings demonstrate that there is a different natriuretic peptide spectrum in patients with different forms of HF. Thus, assays that detect glycosylated and nonglycosylated forms of proBNP might provide additional diagnostic information.

Immunoassays

There is a multitude of immunodetection platforms for natriuretic peptides that are used in clinical practice. All detect multiple forms and resulting interpretation is challenging. It is important to keep in mind both this fact and also the type of HF syndrome involved. Moreover, it appears that BNP and NT-proBNP have the same renal extraction fraction in humans, but there is marked extraction of NT-proBNP across the skeletal muscle (31). Additionally, there are handling differences associated with various assays. Analytical characteristics of currently available commercial assays for natriuretic peptides are presented in Table 1. The location of epitopes on BNP and NT-proBNP molecules is depicted in Fig. 1.

NT-proBNP ASSAYS

All NT-proBNP assays use the same antibodies and calibrators. These are distributed by Roche, thus there is an advantage of unifying these detection platforms. There are only minor differences between the commercial NT-proBNP assays with variation across methods that is <10%. Despite this standardization, however, assay harmonization remains incomplete (32). The International Collaborative of NT-proBNP Study involving 1256 patients proposed a cutoff value for NT-proBNP assays below or above 125 ng/L. A value >300 ng/L was optimal with the existing assays for the exclusion of acute HF (33, 34). This study included a blend of chronic and acute HF patients. Reference limits also vary by age and sex of patients analyzed (35).

While the first generation of NT-proBNP immunoassays uses polyclonal antibodies to amino acids 1-21 and 39-50, the second generation uses monoclonal antibodies (MAb) recognizing the central region of NT-proBNP: amino acids 22-28 and 42-46 (27-31). Many studies have demonstrated the negative effect of glycosylation for antibody recognition to the middle area of the NT-proBNP molecule. The NT-proBNP immunoassays currently available show important cross-reactivity with nonglycosylated proBNP and do not detect glycosylated NT-proBNP and proBNP peptides owing to the presence of O-glycans in the epitopes recognized by the antibodies in the nonglycosylated form. Clinicians should be aware that these detection platforms underestimate the concentration of circulating biomarker considerably. Some studies suggest that the underestimation might be up to 10-fold (19, 35). Some recommend that future NT-proBNP platforms use deglycosylation of blood samples before assay. Perhaps more robust deglycosylation explains why some patients such as those with renal failure have discordantly high values for NT-proBNP. If alternative assays for NT-proBNP are developed, they will need to assess carefully the extent to which the antibodies are influenced by glycosylation.

BNP ASSAYS

All BNP assays use different antibodies and materials for peptide detection; thus there is a paucity of standardization and reports show differences of up to 50% across various BNP detection platforms, even when the same antibodies are used on equipment (2).

The most frequent method used clinically is the "sandwich" assay that uses 2 antibodies specific for 2 distantly located epitopes of the BNP peptide. One of these antibodies is always specific for the intact cysteine ring, which is thought to be the active form, while the other antibody recognizes either the C-terminus of the peptide (Abbott AxSYM and Architect, Shionogi IRMA) or for the N-terminus (Alere Triage and Beckman Access). Assays using antibodies specific to the C-terminus will not detect BNP molecules that are degraded in this region, whereas assays using antibodies specific to the N-terminus of BNP molecule will not measure peptides processed in the N-terminus region. The "Single Epitope Sandwich" Immunoassay (SES-BNP[TM]) designed by HyTest and implemented by ET healthcare is different. The assay uses 1 MAb (24C5) specific to the relatively stable ring fragment of the BNP molecule (epitope 11- 17), which is located within the biologically active cysteine ring, and the second MAb (Ab-BNP2), which recognizes the immune complex of 24C5 with BNP only. There is no space between epitopes, and cleavage between the epitopes does not affect detection since only 1 epitope is needed. This approach stabilizes the immune complex increasing the affinity for its antigen and leads to a very highly sensitive assay, which recognizes both intact BNP and recombinant glycosylated and nonglycosylated forms of proBNP.

The cutoff value for BNP to exclude acute HF with high negative predictive value is 100 ng/L, with the caveat that each assay should, however, have its own cutoff value due to the diversity and lack of standardization of BNP immunoassays. There are systematic differences amongst various BNP assays which partly can be explained by the multitude of proBNP peptides. There is also a lack of reference materials for assay calibration. A common calibrator would improve the harmonization of these assays. Indeed, there is extensive variation depending upon the nature of the natriuretic peptide calibrators used and their glycosylation (Table 2). Cross-reactivity of BNP with proBNP was also demonstrated; this is particularly important in HF patients since proBNP is more prevalent in these patients (36). A summary of cross-reactivity of BNP and NT-proBNP assays currently in clinical use is presented in Fig. 2. Additionally, it was revealed that all BNP immunoassays share substantial cross-reactivity with proBNP, as proBNP shares the same structure (part of BNP) as BNP. Considering that proBNP is the major BNP-immunoreactive form in the circulation of HF patients, this cross-reactivity is clinically relevant. Moreover, some BNP assays will not measure proBNP when it is glycosylated due to spatial obstruction to antibody recognition. Thus, most of the BNP measured is either nonprocessed proBNP or degradation product of BNP.

The PARADIGM-HF trial [Prospective Comparison of ARNI (angiotensin receptor neprisylin inhibitor) with ACEI (angiotensin-converting enzyme inhibitor) to Determine Impact on Global Mortality and Morbidity in HF] showed better outcomes with the neprilysin inhibitor and angiotensin receptor blocker, LCZ696, compared to the angiotensin-converting enzyme inhibitor, enalapril alone in patients with HF with New York Heart Association functional class II (37).

Neprilysin is a ubiquitously expressed membrane-bound protease, found mostly in the kidney, which cleaves the amino bonds of hydrophobic residues. It is implicated in the cleavage of glucagon, enkephalins, substance P, neurotensin, oxytocin, bradykinin, and amyloid [beta]. Both human ANP and human CNP are substrates for neprilysin, while human BNP is not. Specific neprilysin inhibitors fail to block BNP degradation by human kidney membranes, suggesting that neprilysin does not regulate BNP. Thus, the effect of LCZ696 should be more prominent on ANP than BNP. Since proBNP is the major active form resulting from BNP synthesis, one should question whether proBNP rather than BNP is the substrate of LCZ696 activity; however, recent data suggest that proBNP is relatively resistant to neprilysin in vitro. of interest, in that study, glycosylation had no impact. In addition, assays specific to the amino terminus (amino acids 11-17) are less susceptible to BNP cleavage by neprilysin than epitopes specific for amino acids 14-21. Currently it is not clear how BNP and NT-proBNP concentrations are affected by treatment with LCZ696 in different conditions. The PARADIGM-HF trial showed that after treatment with valsartan-sacubitril BNP levels increased initially while NT-proBNP sustainably decreased. Why this occurred is unclear since human BNP appears not to interact with neprilysin. By 8 months, the increases in BNP had abated. It is claimed that because of this NT-proBNP should be used exclusively with valsartan-sacubitril (37). Such a suggestion is premature. We do not know how representative the changes in NT-proBNP with this novel agent are compared to other agents. In addition, it may be after additional studies that metrics about how to use BNP assays will be developed (38,39). Increases in circulating BNP might inhibit proBNP production, thus decreases in the NT-proBNP concentration might not be due to improvements in cardiac function. In addition, increases in BNP have been reported to reduce neprilysin activity acutely, which may provide a governor on its effects. Furthermore, measurement of BNP or NT-proBNP individually may not be sufficient for diagnostic and prognostic purposes with this new agent.

ProBNP IMMUNOASSAYS

HyTest designed an assay based on a capture MAb specific for the region 26-32 of the BNP peptide and a detection antibody specific for the fragment 13-20 of proBNP peptide (18). This highly sensitive immunoassay is not affected by the glycosylation of proBNP molecules, since the epitopes are not glycosylation sites.

A specific MAb recognizes the hinge region of the proBNP molecule 75-80. This antibody along with a polyclonal antibody recognizing the BNP part of the BNP peptide constitutes a sandwich immunoassay for the measurement of proBNP. This method was performed on the BioPlex 2200 Analyzer Multiplex System (Bio-Rad). However, O-glycan residues at Thr71 may impede the hinge-specific antibody interaction. Thus, measured proBNP may underestimate the amount of proBNP present. In acutely decompensated HF patients, proBNP assays are similar to NT-proBNP and BNP assays without incremental value.

Preanalytical Issues

Another reason why various assays may differ from one another is related to preanalytical issues. Proteolytic degradation of the BNP molecule appears immediately upon blood collection. The stability of the sample is method dependent with different stabilities of the epitopes targeted by the specific assay. BNP is unstable when collected in glass tubes because of activation of kallikreins and this degradation may be dependent on the specificities of antibodies used in the measurement method (40). It is recommended that BNP blood samples be collected in plastic tubes only. For BNP assays, EDTA plasma is the only recommended specimen. For the Elecsys NT-proBNP assay, serum is the recommended specimen. There are substantial differences between serum and plasma natriuretic peptide concentrations measured with various detection platforms. The type of anticoagulant used is also important. BNP may be stored at room temperature for 24 h or at 30 [degrees]C for 12 h. In EDTA, overall plasma BNP values are stable at 20 [degrees]C for 1 month but the forms may vary over time. The addition of the protease inhibitor aprotinin, increases the storage time for BNP samples. The NT-proBNP assay is relatively stable during storage in serum, heparinized plasma, or EDTA plasma when stored at room temperature or 4 [degrees]C for at least 72 h or for up to 1 year when stored at -80 [degrees]C. Additionally, for both BNP and NT-proBNP assays, validation for the effect of freeze-thaw cycles upon stability needs to be performed (40).

Conclusions

Natriuretic peptides are established biomarkers for the diagnosis and prognosis of patients with HF. Owing to a high diversity of natriuretic peptides circulating in patients with various forms of HF, along with a complex molecular biology and detection-related issues with the currently widely used clinical detection platforms, challenges arise when clinicians are confronted with result interpretation of these immunoassays. The introduction of novel therapeutic agents in HF which target natriuretic peptides requires a thorough understanding of issues related to biologic activity, detection platforms and pathophysiology of the HF spectrum. Moreover, there are various HF types associated with distinct and differential expression of specific forms of natriuretic peptides. There are large differences between assays that result from the lack of common reference material and antibody diversity. Given the lack of harmonization across assays, providers need to be cautious when interpreting results measured with different assays and ideally should use the same detection platform for meaningful follow-up and comparison. Furthermore, preanalytic issues should also be taken into account. Newer generation assays should try to improve these impediments.

Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 3 requirements: (a) significant contributions to the conception and design, acquisition Of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; and (c) final approval of the published article.

Authors' Disclosures or Potential Conflicts of Interest: Upon manuscript submission, all authors completed the author disclosure form. Disclosures and/or potential conflicts of interest:

Employment or Leadership: None declared.

Consultant or Advisory Role: A.S. Jaffe, Beckman-Coulter, Abbott, Alere, Roche, Diadexus, Siemens, Lpath, Dart Neurosciences, Neu roGenomeX, Inc., and theheart.org.

Stock Ownership: None declared.

Honoraria: None declared.

Research Funding: None declared.

Expert Testimony: None declared.

Patents: None declared.

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Vlad C. Vasile 1 and Allan S. Jaffe (1), (2) *

[1] Division of Cardiovascular Diseases, Department of Medicine, Rochester, MN; [2] Department of Laboratory Medicine and Pathology, Mayo Clinic College of Medicine, Rochester, MN.

* Address correspondence to this author at: Cardiovascular Division, Gonda Bldg. 5th Floor, Mayo Clinic, 200 First St. SW, Rochester, MN 55905. Fax 507-266-0228; e-mail jaffe.allan@mayo.edu.

Received June 8, 2016; accepted June 30, 2016.

Previously published online at DOI: 10.1373/clinchem.2016.254714

[3] Nonstandard abbreviations: HF, heart failure; ANP, Atrial natriuretic peptide; BNP, B-type natriuretic peptide; CNP, C-type natriuretic peptide; DNP, Dendroaspis natriuretic peptide; NPRs, natriuretic peptide receptors.

Caption: Fig. 1. Epitope locations on BNP and NT-proBNP utilized by commercially available assays.

Caption: Fig. 2. Cross-reactivity of NT-proBNP and BNP assays [data from Luckenbill et al. (36)]. M, Mitsubishi; S, Scios; P, Peptide Institute; H, HyTest; R, Roche.
Table 1. Analytical characteristics of commercially available
MR-proANP, BNP, and NT-proBNP assays per the manufacturer.

                          Capture antibody        Detection antibody
Assay BNP

Abbott Architect,      N[H.sub.2] terminus      COOH terminus, murine
AxSYM iSTAT            and part of the ring     MAb, aa 26-32
                       structure (Scios),
                       murine MAb, aa 5-13

Alere (b) Triage BNP   N[H.sub.2] terminus      BNP (Biosite), murine
                       and part of the ring     omniclonal AB, epitope
                       structure (Scios),       not characterized
                       murine MAb, aa 5-13

Beckman Coulter (b)    BNP (Biosite), murine    N[H.sub.2] terminus
Access, Access 2,      Omniclonal AB, epitope   and part of the ring
DxI                    not characterized        structure (Scios),
                                                murine MAb, aa 5-13

Siemens (Bayer) ACS    COOH terminus (BC-203)   Ring structure
180, Advia Centaur,    (Shionogi), murine       (KY-hBNPII)
Advia Centaur CP       MAb, aa 27-32            (Shionogi), murine MAb

Siemens (Dade          Ring structure           COOH terminus
Behring) Dimension     (KY-hBNPII) murine       (BC-203), murine MAb,
VISTA, Dimension ExL   MAb, aa 14-21            aa 27-32

Shionogi               COOH terminus            Ring structure
                       (BC-203), murine MAb,    (KY-hBNPII), murine
                       aa 27-32                 MAb

TosohSTAIA-PACK BNP    COOH terminus            Ring structure
                       (BC-203), murine MAb,    (KY-hBNPII), murine
                       aa 27-32                 MAb

Assay 1-76 NT-proBNP

Alere Triage           Murine MAb, aa 27-31     Sheep MAb, aa 42-46
NT-proBNP

bioMerieux             N[H.sub.2] terminus      Central molecule,
NT-proBNPI VIDAS       polyclonal sheep AB,     polyclonal sheep AB,
                       aa 1-21                  aa 39-50

NT-proBNP2 VIDAS       Murine MAb, aa 27-31     Sheep MAb, aa 42 -46

Mitsubishi Chemical    N[H.sub.2] terminus      Central molecule,
PATHFAST               polyclonal sheep AB,     polyclonal sheep AB,
                       aa 1-21                  aa 39-50

Nanogen LifeSign       Monoclonal (mouse) and   Polyclonal sheep AB
DXpress Reader         polyclonal (goat) Abs

Ortho Clinical         N[H.sub.2] terminus      Central molecule,
Diagnostics Vitros     polyclonal sheep AB,     polyclonal sheep AB,
ECi                    aa 1-21                  aa 39-50

Radiometer AQT90       N[H.sub.2] terminus      Central molecule,
FLEX NT-proBNP         polyclonal sheep AB,     polyclonal sheep AB,
                       aa 1-21                  aa 39-50

Response Biomedical    Murine MAb, aa 27-31     Central molecule,
RAMP                                            polyclonal sheep AB,
                                                aa 39-50

Roche NT-proBNP I      N[H.sub.2] terminus      Central molecule,
Elecsys, E170          polyclonal sheep AB,     polyclonal sheep AB,
                       aa 1-21                  aa 39-50

NT-proBNP II           MAb, aa 27-31            Sheep MAb, aa 42-46
Elecsys, E170

Siemens (Dade          N[H.sub.2] terminus      Central molecule,
Behring) Dimension     monoclonal sheep AB,     Sheep MAb, aa 42-46
RxL, Stratus CS,       aa 22-28
Dimension VISTA,
Dimension EXL with
LM

Siemens (DPC)          N[H.sub.2] terminus      Central molecule,
Immulite 1000,2000     polyclonal sheep AB,     polyclonal sheep AB,
2500                   aa 1-21                  aa 39-50

Assay MR-proANP

Thermo Fisher          Polyclonal sheep AB,     Monoclonal rat AB, aa
Scientific KRYPTOR     aa 50-72 of NT-proANP    73-90 of NT-proANP

                          Standard material       FDA (a) cleared-yes/
Assay BNP                                               no/claim

Abbott Architect,      Synthetic BNP 32          Assist in diagnosis of
AxSYM iSTAT                                      HF; assess severity of
                                                 disease

Alere (b) Triage BNP   Recombinant BNP           Aid in diagnosis and
                                                 severity assessment of
                                                 HF; risk
                                                 stratification of
                                                 patients with ACS and
                                                 HF; FDA cleared

Beckman Coulter (b)    Recombinant BNP           Diagnosis HF; assess
Access, Access 2,                                severity HF; risk ACS;
DxI                                              risk HF

Siemens (Bayer) ACS    Synthetic BNP             Aid in diagnosis and
180, Advia Centaur,                              assessment of severity
Advia Centaur CP                                 of HF; predict
                                                 survival and
                                                 likelihood of future
                                                 HF in ACS patients

Siemens (Dade          Synthetic BNP 32          Aid in diagnosis and
Behring) Dimension                               assessment of severity
VISTA, Dimension ExL                             of HF; predict
                                                 survival and
                                                 likelihood of future
                                                 HF in ACS patients;
                                                 pending FDA clearance

Shionogi               Synthetic BNP             Not FDA cleared

TosohSTAIA-PACK BNP    Synthetic BNP             Not FDA cleared

Assay 1-76 NT-proBNP

Alere Triage           Synthetic NTproBNP 1-76   Aid in diagnosis of
NT-proBNP                                        HF; risk
                                                 stratification of
                                                 patients with ACS and
                                                 HF; assessment of
                                                 increased risk of
                                                 cardiovascular events
                                                 and mortality in
                                                 patients at risk for
                                                 HF who have stable
                                                 CAD; not currently
                                                 available in the US

bioMerieux             Synthetic NTproBNP 1-76   Diagnosis HF
NT-proBNPI VIDAS

NT-proBNP2 VIDAS       Synthetic NTproBNP 1-76   Not FDA cleared

Mitsubishi Chemical    Synthetic NTproBNP 1-76   Aid diagnosis of CHF;
PATHFAST                                         assess severity CHF;
                                                 risk stratification in
                                                 ACS and stable CAD

Nanogen LifeSign       Synthetic 1-76 NTproBNP   Diagnosis HF
DXpress Reader

Ortho Clinical         Synthetic 1-76 NTproBNP   Aid diagnosis of CHF;
Diagnostics Vitros                               risk stratification of
ECi                                              ACS and CHF; risk
                                                 assessment of CV
                                                 events and mortality
                                                 in patients at risk
                                                 for HF with stable
                                                 CAD; assess severity
                                                 in HF

Radiometer AQT90       Synthetic 1-76 NTproBNP   Diagnosis HF; risk
FLEX NT-proBNP                                   stratification of
                                                 patients with ACS and
                                                 HF; not FDA cleared

Response Biomedical    Synthetic 1-76 NTproBNP   Diagnosis HF; assess
RAMP                                             severity HF

Roche NT-proBNP I      Synthetic 1-76 NTproBNP   Diagnosis HF; assess
Elecsys, E170                                    severity HF; risk ACS;
                                                 risk HF

NT-proBNP II           Synthetic 1-76 NTproBNP   Treatment monitoring
Elecsys, E170                                    in LVD

Siemens (Dade          Synthetic 1-76 NTproBNP   Aid in the diagnosis
Behring) Dimension                               of CHF and assessment
RxL, Stratus CS,                                 of severity; risk
Dimension VISTA,                                 stratification of
Dimension EXL with                               patients with ACS and
LM                                               HF

Siemens (DPC)          Synthetic 1-76 NTproBNP   Not FDA cleared
Immulite 1000,2000
2500

Assay MR-proANP

Thermo Fisher          Synthetic 50-90           Not FDA cleared
Scientific KRYPTOR

(a) FDA, US Food and Drug Administration; CHF, congestive heart
failure; ACS, acute coronary syndrome; CV, cardiovascular; CAD,
coronary artery disease; LVD, left ventricular dysfunction; aa, amino
acid; AB, antibody; MR-proANP, midregional pro-ANP.

(b) Both the Alere and Beckman systems use the same 2 antibodies but
due to their different assay formats, designation of the monoclonal
and omniclonal antibodies as capture and detection antibody is not
absolute.(Adapted with permission from IFCC.International Federation
of Clinical Chemistry and Laboratory Medicine, website http://www.
ifcc.org.)

Table 2. Percentage recoveries and cross-reactivities by BNP and
NT-proBNP assays for each peptide [data from Luckenbill
et al. (36)].

Assay              Sa BNP             P BNP        S proBNP

Architect     151 (142-160) (b)   98 (85-110)     38(37-38)
AxSYM         184(164-205)        124(117-130)    34 (28-39)
Centaur       194(189-199)        137(133-141)    17(17-18)
Access        199(192-205)        130(129-130)    24 (24-25)
Triage        135(115-156)        79(78-80)       19(18-20)
NT-proBNP
  Dimension   <1 (0-0)            <1 (0-0)        <1 (0-0)
  Vitros      <1 (0.6-0.8)        <1 (0.03-0.4)   2 (1.6-2.2)
  Elecsys     <1 (0.2-0.8)        <1 (0-0.04)     1 (0.7-2)

Assay            HproBNP       H NT-proBNP    R NT-proBNP

Architect     6(2-11)         <1 (0-0.8)      4 (4-5)
AxSYM         9(3-15)         <1 (0-0.5)      4 (3-5)
Centaur       14(10-17)       <1 (0-0.3)      7 (5-9)
Access        13(7-19)        <1 (0-0.4)      6 (5-7)
Triage        5(0-12)         <1 (0-0.2)      3 (2-4)
NT-proBNP
  Dimension   249 (230-267)   243 (235-251)   95 (91-99)
  Vitros      55 (52-59)      127(124-130)    71 (68-74)
  Elecsys     29 (28-30)      131 (126-137)   47 (45-49)

(a) S, Scios; P, Peptide Institute; H, HyTest; R, Roche.

(b) 95% CIs are in parentheses.
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Date:Jan 1, 2017
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