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Technical Briefs |
1
Institut für Klinische Chemie und Pathobiochemie, and
2
Medizinische Klinik I, Universitätsklinikum der RWTH Aachen, Pauwelsstrasse 30, 52074 Aachen, Germany
a author for correspondence: fax 49-241-8888512, e-mail yuriko.fischer{at}post.rwth-aachen.de
Congestive heart failure (CHF) is among the most frequently encountered cardiac diagnoses, with an estimated prevalence of 1% (1). Plasma concentrations of B-type natriuretic peptide (BNP) and its precursor, N-terminal pro-BNP (NT pro-BNP), are increased in patients with CHF and have been shown to accurately predict clinical severity and left ventricular ejection fraction (LVEF) as well as morbidity and mortality in those patients (2)(3). A major limitation in the routine determination of natriuretic peptides is the time-consuming nature of analytical techniques, e.g., extraction procedures, long incubation times, or radioactive labeling (4)(5)(6)(7)(8). A rapid bedside test for determination of BNP was introduced recently (Triage BNP; Biosite Diagnostics, San Diego, CA). We evaluated the assay and compared it, in samples from patients with suspected CHF, with other assay systems for determination of either BNP (Shionoria BNP; CIS Diagnostics) or NT pro-BNP (Roche Diagnostics, Tutzing, Germany).
The Triage BNP test is an immunofluorometric assay for quantitative determination of BNP in EDTA-anticoagulated whole blood or plasma (9)(10). A murine recombinant polyclonal antibody is bound to the fluorescent label, and a murine monoclonal antibody against the disulfide bond-mediated ring structure of BNP 32 is bound to the solid phase.
Briefly, after the addition of 250 µL of EDTA-anticoagulated whole
blood, the plasma is separated and allowed to react with fluorescent
antibody conjugates within a reaction chamber. After an incubation
period, complexes of the analyte and fluorescent antibody conjugates
are captured on a detection lane. The concentration of BNP in the
specimen is proportional to the fluorescence bound to the detection
lane, which is determined quantitatively by a handheld fluorescence
instrument (Triage Meter) as described in detail elsewhere
(11). The time from application of the sample until the
result is reported is 
15 min.
The Shionoria BNP test is a one-step immunoradiometric assay that uses
two different monoclonal antibodies that recognize the C-terminal
structure and the disulfide bond-mediated ring structure of BNP 32,
respectively. This test requires 20 h. NT pro-BNP(1–76) was
determined by a recently developed research enzyme immunoassay that
uses a polyclonal antibody specific for the N-terminal sequence (amino
acids 1–21) of NT pro-BNP as well as a polyclonal antibody specific
for the middle sequence (amino acids 30–38) of NT pro-BNP. The assay
time is 2 h, and no extraction is required. Both assays have been
described in detail elsewhere
(6)(12)(13)(14).
The within-run (n = 11) and between-day (n = 11 days) imprecision of the Triage BNP assay were determined at three different concentrations that were prepared by mixing patient samples with high and low BNP concentrations. Within-run and between-day studies used EDTA-anticoagulated whole blood and EDTA plasma, respectively, each with a single assay lot and unchanged calibration.
For determination of the detection limit, defined as the lowest concentration that could be differentiated from zero (mean + 3 SD), a zero calibrator was measured 20 times with four lots of reagents on 5 consecutive days.
Plasma samples from 50 apparently healthy individuals (30 males, 20 females; age range, 20–60 years) were measured, and the median and 97.5th percentile concentrations were calculated.
The study population consisted of 100 patients with underlying cardiac
disease and suspected CHF. Clinical severity was characterized
according to the functional classification system of the New York Heart
Association (NYHA) in stages I to IV (15). The LVEF was
evaluated with biplane transthoracic echocardiography by the modified
Simpson rule using second harmonic imaging (16). Five
patients were excluded because of insufficient sample material or
missing clinical data. A total of 95 patients remained in the study
group (Table 1
). All patients gave informed consent. The study was approved by
the local ethics committee.
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On the day of echocardiography, peripheral venous blood was collected into sampling tubes containing EDTA as the anticoagulant. Within 20 min after venipuncture, BNP concentrations were determined by the Triage system. The remaining sample material was centrifuged, and EDTA plasma was frozen at -80 °C until further determination.
Correlation of BNP Triage with Shionoria BNP and NT pro-BNP was
evaluated by the statistical procedure by Passing and Bablok
(17). For calculation of assay comparison, results of the 95
study patients and the 50 healthy subjects were included (n =
145). Results of ROC analysis are expressed as areas under the
individual ROC curves, including the 95% confidence
interval. The diagnostic sensitivity of each assay was
calculated as the number of test results with a BNP or NT pro-BNP value
equal to or higher than a specific cutoff value among all patients with
an LVEF 
50%, whereas diagnostic specificity was defined as the
number of test results below the cutoff value among all patients with
an LVEF >50%.
For imprecision, the within-run CVs of the Triage BNP assay were 9.4%, 13%, and 15% at 40, 450, and 800 ng/L, respectively. At the same mean concentrations, the between-day CVs were 11%, 13%, and 16%, respectively. The detection limit was 6 ng/L. The 97.5th percentile of the healthy subjects was 82 ng/L for the Triage BNP assay, 64 ng/L for the Shionoria BNP, and 57 pmol/L for the NT pro-BNP ELISA.
Comparison of results by regression analysis (Fig. 1
) revealed the following: Triage BNP = 1.579(Shionoria
BNP) - 2.947 (r = 0.963); Triage BNP =
1.198(NT pro-BNP) + 1.419 (r = 0.947); NT pro-BNP
= 1.309(Shionoria BNP) - 3.780 (r = 0.948). The
95% confidence intervals for slope and intercept were as follows:
Triage BNP vs Shionoria BNP, 1.490–1.659 and -4.909 to -1.884;
Triage BNP vs NT pro-BNP, 1.136–1.270 and -0.664 to 3.500; NT pro-BNP
vs Shionoria BNP, 1.251–1.388 and -6.493 to -1.004. Comparison of
BNP and NT pro-BNP concentrations with NYHA classes revealed only
moderate correlations for all assays:
rs = 0.689 (Triage BNP), 0.667
(Shionoria BNP) and 0.632 (NT pro-BNP); P <0.001; n =
95. Nevertheless, BNP and NT pro-BNP values showed a strong inverse
correlation with LVEF: rs = -0.816
(Triage BNP), -0.762 (Shionoria BNP), and -0.718 (NT pro-BNP);
P <0.001; n = 95.
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ROC analysis regarding impaired ventricular function, defined as LVEF
50%, revealed areas under the ROC curve (95% confidence interval)
of 0.91 (0.83–0.98) for Triage BNP, 0.88 (0.80–0.95) for Shionogi
BNP, and 0.86 (0.77–0.94) for NT pro-BNP, respectively. At a cutoff
value of 130 ng/L, the Triage assay had a sensitivity of 93%
and a specificity of 79% to detect patients with impaired LVEF.
Similar results were obtained for the other assays: for the Shionogi
BNP, at a cutoff of 100 ng/L, the sensitivity was 80% and the
specificity was 79%; for the NT pro-BNP assay, at a cutoff of 100
pmol/L, the sensitivity was 90% and the specificity was 66%.
The Triage BNP assay seems to be a sensitive screening method to decide which patient with suspected CHF warrants further investigation, particularly when assessment by echocardiography is not readily available. The major advantage of the Triage test system compared with the other investigated assays is its rapid and accurate measurement of BNP from whole blood with 24-h availability in a routine laboratory or at the point of care. Additional time-consuming preparation, centrifugation, extraction, and incubation steps can be omitted. Rapid measurement of either BNP or NT pro-BNP seems useful, especially in the triage of patients with suspected CHF presenting to the emergency room, where these results add important information for faster diagnostic evaluation (8)(18). It might also be useful in patients admitted with decompensated CHF as a previous study has shown that changes in BNP during treatment are a strong predictor for mortality and early readmission (19). Furthermore, the recently introduced BNP-guided therapy for chronic CHF will require faster determination of the analyte (20). Nevertheless, additional clinical trials need to be designed to clarify the detailed clinical benefit in patients diagnosis, management, and therapy as well as the cost-effectiveness of this marker (8).
Acknowledgments
We gratefully acknowledge VIVA Diagnostika Germany, Biosite Diagnostics Europe, and Roche Diagnostics Germany for providing reagents free of cost.
References
The following articles in journals at HighWire Press have cited this article:
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