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Comparison of intact parathyroid hormone (iPTH) assays in the diagnostic laboratory: Roche Modular E170 and Advia Centaur.

Introduction

Parathyroid hormone (PTH) is synthesized by four parathyroid glands which are located close to or on the posterior surface of the thyroid gland. However, additional parathyroid glands may be located elsewhere such as in the neck or within the thymus in the superior mediastinum (1). PTH is a single-chain polypeptide of 84 amino acids with molecular weight of 9500 Daltons. Intact PTH (iPTH) is the form stored in the glands and the principal form secreted into the blood stream. The plasma half-life (t1/2) of iPTH is less than 10 minutes on which iPTH is cleaved in the region of amino acids 33-37 and elsewhere in peripheral tissues, principally the liver and kidney, to an amino acid (N-terminal) fragment of at least 34 amino acids, carboxy-terminal (C-terminal), and midpeptide fragments (1,2). The biological activity of iPTH resides in the first 34 amino acids of the N-terminal, therefore, both iPTH and N-terminal fragments posses full biological activity.

Together with vitamin D and calcitonin, iPTH binds to type 1 PTH receptors on target tissues and regulates the ionized or free calcium (Cai) (1). The level of iPTH also affects the concentration of plasma ionized phosphate (Pi), even though the plasma Pi concentration does not control iPTH secretion directly. Elevated plasma iPTH which causes hypercalcaemia is found in primary hyperparathyroidism and secondary hyperparathyroidism. On the other hand, low plasma iPTH, which causes hypocalcaemia, is found in primary hypoparathyroidism and secondary hypoparathyroidism.

The plasma concentration of iPTH can be measured by noncompetitive immunoassay on either the Roche Modular E170 or Advia Centaur analyzers. On the Roche Modular E170 analyzer, electrochemiluminescent technology is used to measure iPTH level, on which a biotinylated monoclonal antibody and a monoclonal antibody labelled with ruthenium are applied to bind the N-terminal fragment (1-37) and C-terminal fragment (38-84) respectively. After the addition of streptavidin-coated microparticles, the complex becomes bound to the solid phase via interaction of biotin and streptavidin. The reaction mixture is aspirated into the measuring cell where the microparticles are magnetically captured onto the surface of the electrode. Unbound substances are then washed off, followed by the application of a voltage to the electrode to induce chemiluminescent emission which is measured by a photomultiplier.

The Advia Centaur analyzer uses a similar principle as the Roche Modular E170 analyzer. However, instead of using electrochemiluminescent detection technology, the Advia Centaur analyzer uses chemiluminometric detection technology on which dispensing of both acid reagent and base reagent induces the chemiluminescent emission. The Advia Centaur analyzer uses a polyclonal goat anti-human PTH antibody labelled with an acridium ester and a biotinylated polyclonal goat anti-human PTH antibody to bind the N-terminal (1-34) and the C-terminal (39-84) respectively.

The objective of this study was to compare the reliability of iPTH assays between the Roche Modular E170 and the Advia Centaur analyzers. and to determine whether both analysers can be used interchangeably.

Methods

K3-EDTA plasma samples were collected over the period of four weeks from 24 different patients reported by Canterbury Health Laboratory (CHL) Christchurch to have iPTH levels between 1.7 and 135.0 pmol/L. The whole blood samples were centrifuged at 3700 rpm for seven minutes, plasma separated, and stored at -20[degrees]C in plastic Kahn tubes. On the day of analysis, the samples were thawed and mixed using a vortex mixer then centifuged 3700 rpm for seven minutes prior to analysis.

Calibrations and quality controls were analyzed for both analysers according to the manufacturers' instructions. The Roche Modular E170 was calibrated using Elecsys PTH CalSet (0.005 pmol/L and 477 pmol/L), while the Advia Centaur was calibrated using intact PTH (iPTH) calibrator (2.68 pmol/L and 88.30 pmol/L). The quality control materials used were PreciControl Bone 1, 2, and 3 for the Roche Modular E170, and Ligand plus 1, 2, and 3 for the Advia Centaur. The patients K3-EDTA plasma samples were assayed in duplicate on both analysers. The coefficient of variance (CV) was calculated from the paired duplicate data. The correlation coefficient and regression analysis was used to determine the association of the results measured by two different analysers. The difference or bias between two analysers was evaluated by plotting the Bland and Altman difference graph and by performing 95% confidence interval (p<0.05) paired t-test using n-1 degree of freedom.

Results

The paired duplicate data from the Roche Modular E170 and the Advia Centaur analyzers gave the internal reliability (CV) of 7% and 3% respectively. The correlation between the two analysers is shown in Figure 1; a significant difference was demonstrated between the two analyzers (t=3.77, p<0.05).

[FIGURE 1 OMITTED]

From the slope value of 1.6047, it can be concluded that the Advia Centaur gave higher results compared to the Roche Modular with the average difference between two means of 12.32 pmol/L. This difference increases as the iPTH concentration increases (Figure 2).

[FIGURE 2 OMITTED]

Discussion

Even though the correlation analysis (r2 = 0.995) showed that the results from both analysers were associated, it is clear from the regression equation in Figure 1 that iPTH assays had higher values on the Advia Centaur analyzer when compared with the Roche Modular E170 analyzer As correlation analysis measures association rather than agreement (3), this demonstrates that both analysers are designed to measure the same substance but not necessarily be in numerical agreement. Therefore, paired t-test was undertaken to show the agreement between two data sets. The paired t-test (t = 3.77, p<0.05)) indicated that the average difference between two methods was significant. Therefore, both analysers should not be used interchangeably. As expected, the paired t-test between iPTH levels obtained by Roche Modular E170 and CHL Christchurch (who use this method) showed no significant difference between two results (t=1.89, p<0.05). Furthermore, the paired t-test between iPTH levels obtained by Advia Centaur and CHL Christchurch showed that there was a significant difference between two results (t=3.64, p<0.05) confirming the results in this study.

The significant difference in results obtained using both analysers may be explained by the fact that both analysers use different calibrator materials thereby producing differences in the expected values of iPTH. This is reflected in the reference ranges of the two analyzers where the Roche Modular E170 analyzer reference range was 1.6-6.9 pmol/L while on the Advia Centaur, the reference range was 1.18-8.43 pmol/L.

The difference in these results between both analysers may be caused by two different features found in both assays. First, unlike the Roche Modular E170 that uses electrochemiluminescent technology, the Advia Centaur uses direct chemiluminometric technology and second, the Roche Modular E170 uses monoclonal antibodies while the Advia Centaur uses polyclonal antibodies. A previous study on iPTH assays by Santini et al (2004) indicated that the use of more standardized method of calibration and antibodies that recognize only the biologically active PTH molecule may decrease the wide gap between results obtained from a range of different analysers (4).

This study was limited by a small number of samples as well as low sample volume as the the Advia Centaur required a 200[micro]L sample volume while the Roche Modular E170 required only 50 [micro]L of sample. This limited the opportunity to undertake further evaluation such as a sensitivity analysis and a wiser range of iPTH values.

In conclusion, iPTH assays on either analyzer took approximately 18 minutes per sample and assay costs were comparable. However, despite a good correlation, the average difference of iPTH assay between the Roche Modular E170 and the Advia Centaur is significantly different from zero. Therefore, they should not be used interchangeably. Comparison of the two analysers showed that the Advia Centaur had better reliability. Despite a higher plasma volume requirement, the Advia Centaur can be the analyser of choice for iPTH assay in the clinical biochemistry laboratory.

Acknowledgments

This research project was undertaken as a requirement for the Bachelor of Medical Laboratory Science degree at the University of Otago, Dunedin, New Zealand. I would like to thank Trevor Rollinson who supervised this project and all the staff in Biochemistry department at Southern Community Laboratories (SCL) Dunedin for their assistance and advice in this research project.

References

(1.) Fraser D, Jones G, Kooh SW, Radde IC. Calcium and phosphate metabolism. In: Tietz NW. Fundamentals of Clinical Chemistry. 3rd Ed. WB Saunders Company, Philadelphia, USA, 1987; pp 705-28.

(2.) Calbreath DF, Ciulla A. Clinical Chemistry; a Fundamental Textbook. WB Saunders Company, Philadelphia, USA, 1992; pp 386-94.

(3.) Bland JM, Altman DG. Statistical methods for assessing agreement between two methods of clinical measurement. Lancet 1986; 1 (8476): 307-10.

(4.) Santini SA, et al. Assessment of parathyroid function in clinical practice: which parathyroid hormone assay is better? Clin Chem 2004; 50: 1247-50.

Address for correspondence: Christian Christian, Automation Department, SCL-Otago Southland, Southland Hospital, PO Box 828, Kew Road, Invercargill. Email: Christian.Christian@sdhb.govt.nz

Christian Christian, BMLSc; Medical Laboratory Scientist Biochemistry Department, Southern Community Laboratories, Dunedin
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Author:Christian, Christian
Publication:New Zealand Journal of Medical Laboratory Science
Article Type:Report
Geographic Code:8NEWZ
Date:Aug 1, 2008
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