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Technical Briefs |
1 Department of Pathology and Laboratory Medicine, University of Texas-Houston Medical School, 6431 Fannin, MSB 2.292, Houston, TX 77030
2 Memorial-Hermann Laboratory Services, Houston, TX 77030
aauthor for correspondence: fax 713-500-0730, e-mail Amitava.Dasgupta{at}uth.tmc.edu
Atrial natriuretic peptides, B-type natriuretic peptide (BNP), and C-type peptides play important roles in the cardiovascular system (1)(2)(3). ProBNP is a 108-amino acid peptide that is secreted by the ventricle and is cleaved to physiologically active BNP (amino acids 77–108) and N-terminal fragment NT-proBNP (amino acids 1–76) (3). NT-proBNP is a useful marker for heart failure, including congestive heart failure (4)(5)(6). Recently, Roche Diagnostics marketed an automated NT-proBNP assay on the Elecsys analyzer. The assay uses polyclonal antibodies against NT-proBNP. The concentration of NT-proBNP in healthy individuals depends on both age and gender, with females and elderly patients having higher concentrations. The clinical sensitivity and specificity of NT-proBNP at a cutoff of 125 ng/L for male patients younger than 75 years are 81.6–91.7% (sensitivity) and 86.7–95.7% (specificity), depending on age. The corresponding sensitivity and specificity for female patients are 86.7–94.3% (sensitivity) and 57.8–84.9% (specificity), respectively. The negative predictive value of this test is almost 100% at a clinical threshold of 125 ng/L, according to the package insert (NT-proBNP; Roche Diagnostics).
A common practice in clinical laboratories is to draw blood in serum-separator tubes because of the advantage of the barrier gel, which facilitates rapid separation of serum from cellular constituents of blood and prevents hemolysis during prolonged storage. However, measured concentrations of certain drugs, such as phenytoin, phenobarbital, carbamazepine, quinidine, and lidocaine, can be reduced if specimens are stored in Becton Dickinson SSTTM tubes (7). Bush et al.(8) reported reductions in estradiol concentrations when specimens were stored in SST tubes. The purpose of the current study was to determine the stability of NT-proBNP in specimens stored in SST tubes.
We designed our first experiment with healthy volunteers because the expected NT-proBNP concentrations should be within the reference interval. Blood specimens were simultaneously collected in two plain red-top tubes and in two SST tubes (to have duplicate results). Both plain red-top tubes and SST tubes were purchased from Becton Dickinson. Samples were centrifuged immediately after clotting, and NT-proBNP concentrations were measured on an Elecsys 2010 analyzer. The initial determinations were performed within 1 h of blood collection (0–1 h). Specimens were then stored at room temperature, and NT-proBNP concentrations were again determined at 2 and 5 h. Specimens were then stored refrigerated at 4 °C, and NT-proBNP concentrations were again determined at 24 and 48 h to evaluate long-term stability.
In another experiment, blood was collected in SST tubes from patients with suspected cardiac problems. These specimens are routinely submitted to our laboratory for measuring NT-proBNP concentrations. An aliquot of specimen was also stored in a plain glass tube to serve as the control. The concentrations of NT-proBNP in both tubes were measured within 1 h of blood collection. Specimens were then stored at room temperature for 5 h, followed by storage up to 48 h at 4 °C to measure NT-proBNP concentrations at 24 and 48 h (duplicate measurement of each specimen).
We validated the performance of the NT-proBNP assay by evaluating the precision, linearity, and detection limit. The within- and between-run imprecisions (as the CV) for the low control were 1.3% [mean (SD), 279 (3.6) ng/L; n = 10] and 3.0% [278 (8.3) ng/L; n = 16], respectively. The total imprecision (CV; obtained by combining raw data from within- and between-run precision studies) was 2.4% [mean (SD), 278 (6.6) ng/L; n = 26]. The within- and between-run CVs for the high control were 1.4% [6607 (95.8) ng/L; n = 10] and 3.4% [6414 (218.6) ng/L; n = 16], respectively. The total imprecision (CV) for the high control was 3.1% [6489 (202.9) ng/L; n = 26]. The assay is linear up to 35 µg/L. The linearity of the assay was verified with use of the NT-proBNP CalCheck supplied by Roche Diagnostics.
The detection limit was established by analyzing the lowest CalCheck specimen 20 times in a single batch. This CalCheck specimen is free of NT-proBNP, according to the package insert. The observed mean (SD) was 4.3 (4.8) ng/L (range, 0–11.4 ng/L). The detection limit (mean + 2 SD) was 13.9 ng/L. In this calculation, a value <5 ng/L was considered as "none detected" or a zero value. The manufacturer defined the detection limit as 5 ng/L, which is lower than our observation. The manufacturer also defined functional sensitivity of the assay (lowest concentration with a CV <20%) as 50 ng/L. However, in our experience, NT-proBNP values <45 ng/L are not reliable because when Diluent Universal (sample diluent for all Elecsys assay; Roche Diagnostics) was analyzed 10 times in a single batch with this assay, we observed a range of readings between 33 and 44 ng/L [mean (SD), 37 (4.1) ng/L; mean + 2 SD, 45.2 ng/L]. The within-run CV was 11%. In contrast, we observed excellent precision for the NT-proBNP assay in a specimen with a low NT-proBNP concentration of 60 ng/L (total CV, 4.9%; n = 10). Precision was further improved in another specimen with a NT-proBNP concentration of 100 ng/L (total CV, 2.1%; n = 10).
We observed no adsorption of NT-proBNP by the gel of the SST tubes in the 10 volunteers we studied (NT-proBNP range, 64–302 ng/L; five males and five females). The NT-proBNP values obtained in the specimens collected in the red-top tubes did not differ from those for the specimens collected in SST tubes at 0–1, 2, 5, 24, or 48 h. For example, the mean (SD) NT-proBNP concentration in the first volunteer was 64 (1.1) ng/L in specimens collected in the plain red-top tubes. The corresponding NT-proBNP value in the specimens collected in SST tubes was 61 (3.0) ng/L. After 48 h, the mean (SD) NT-proBNP concentration in the specimens collected in plain red-top tubes was 63 (1.0) ng/L, and the NT-proBNP concentration in the specimens collected in SST tubes was 64 (1.2) ng/L. These values are not significantly different by the two-tailed Student t-test at 95% confidence. A comparison of NT-proBNP concentrations in specimens collected in plain red-top tubes and in SST tubes from two volunteers with NT-proBNP concentrations of 125 ng/L (decision point) and 302 ng/L is shown in Fig. 1
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We also observed no adsorption of NT-proBNP by the gel in the 10 patients we studied. We studied nine patients with high NT-proBNP concentrations (range, 1424–12 851 ng/L) and in one patient near the cutoff value of 125 ng/L. In all cases, we observed no statistically significant difference in NT-proBNP values within 1 h of blood collection and after 48 h of storage of the specimens (both plain red-top tubes and SST tubes). For example, in one patient, the mean (SD) original NT-proBNP concentration of 5820 (47) ng/L was not statistically different from the mean (SD) NT-proBNP value of 5908 (100) ng/L observed after 48 h in a specimen stored in a SST tube. Another patient with an original mean (SD) NT-proBNP concentration of 120 (3.5) ng/L had a mean (SD) NT-proBNP value of 121 (4.2) ng/L after storage of specimen in a SST tube for 48 h at 4 °C. However, one limitation of this study is that blood was collected in SST tubes and parallel specimens were not collected in plain glass tubes in these patients.
Our previous studies on the stability of therapeutic drugs in specimens stored in SST tubes demonstrated that reductions in the values were more significant at low drug concentrations. Because adsorption of an analyte by the gel is time dependent, any adsorption of NT-proBNP by the gel should be reflected in significant decreases in NT-proBNP concentrations as a function of storage time. The adsorption of therapeutic drugs by the gel is time dependent, and a significant decrease in drug concentration can be observed within 2–4 h of storage (7)(9). We observed no statistically significant decrease in NT-proBNP concentrations over a 48 h period in specimens collected in SST tubes.
In conclusion, we believe that SST tubes manufactured by Becton Dickinson can be used for blood collection for routine analysis of NT-proBNP in both fresh and stored samples.
Acknowledgments
We thank Roche Diagnostics (Indianapolis, IN) for providing the NT-proBNP assays.
References
The following articles in journals at HighWire Press have cited this article:
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  D.-e. van der Merwe, R. Henley, G. Lane, R. Field, M. Frenneaux, F. Dunstan, and I. McDowell Effect of Different Sample Types and Stability after Blood Collection of N-Terminal Pro-B-Type Natriuretic Peptide as Measured with Roche Elecsys System Clin. Chem., April 1, 2004; 50(4): 779 - 780. [Full Text] [PDF]
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) and SST tubes (
) from volunteers 4 and 10 and stored over 48 h.