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Clinical Chemistry 48: 766-767, 2002;
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(Clinical Chemistry. 2002;48:766-767.)
© 2002 American Association for Clinical Chemistry, Inc.

Technical Briefs

Parathyroid Hormone Is More Stable in EDTA Plasma Than in Serum

Paul Glendenning1a, Leonie L. A. Laffer1, Hayley K. Weber1, Alexander A. Musk1 and Samuel D. Vasikaran1

1 Department of Core Clinical Pathology and Biochemistry, Royal Perth Hospital, Perth 6000, Western Australia

aauthor for correspondence: fax 618-9224-2491, email Paul.Glendenning{at}health.wa.gov.au

Measurements of circulating parathyroid hormone (PTH) are important in the evaluation of individuals with hyper- or hypocalcemia and for the investigation of PTH function in bone and mineral disorders (1)(2). Serum may be a good alternative to EDTA plasma in the assessment of PTH status and is an acceptable alternative to EDTA plasma in the reformulated IMMULITE 2000 intact PTH (iPTH) assay. However, reports on the stability of PTH in serum and EDTA plasma are conflicting (3)(4)(5).

Blood samples from 36 patients were collected in parallel into 10-mL tripotassium EDTA tubes, ensuring complete sample filling of the collection vessel (6), and into Becton Dickinson SST II gel separator tubes. PTH was assayed with the reformulated IMMULITE 2000 iPTH assay (beginning with lot L2KPP 106) within 3 h of collection and reassayed on both samples after storage for 3 days at room temperature.

The mean iPTH was 14.8 pmol/L (range, 0.9–27 pmol/L) for EDTA-plasma samples at baseline (target value). Serum iPTH values were 35% lower. After 3 days of storage at room temperature, the serum iPTH values were >60% lower than the baseline EDTA-plasma target values (Fig. 1A ), whereas the iPTH in EDTA was marginally higher (by 8%). Statistical analysis (repeated-measures ANOVA and Dunnett’s post hoc test) of all results indicated that serum values both at baseline and after 3 days were statistically lower than the target value (P <0.0001). In contrast, there was no significant difference between EDTA-plasma values at baseline and after 3 days at room temperature. The magnitude of difference between serum and EDTA-plasma iPTH was similar for high iPTH values and iPTH values within the reference interval (Fig. 1B ).



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  		Figure 1. Effect of sample type (EDTA or serum) and storage for 72 h at room temperature on iPTH values with the IMMULITE 2000 iPTH assay.

(A), Scatterplot of iPTH values at baseline and after 72 h of storage at room temperature. EDTA 0d and Serum 0d, baseline iPTH values in EDTA plasma and serum, respectively; EDTA 3d and Serum 3d, iPTH values in EDTA plasma and serum, respectively, after 72 h of storage at room temperature. Mean values are indicated by horizontal lines. Sample iPTH values in serum at baseline and after 72 h of storage at room temperature are significantly different from EDTA plasma at baseline (**, P <0.001; repeated-measures ANOVA and Dunnett’s post hoc test). (B), Bland–Altman analysis of data comparing EDTA-plasma and serum iPTH values at baseline with regression line and 95% confidence limits. Analysis of the proportional error noted between serum and EDTA plasma for iPTH indicated that the slope of the regression line was not significantly different from zero.

The Royal Australasian College of Pathologists Quality Assurance Program determines the allowable limits of performance for iPTH assays by two criteria: (a) <25% difference between the sample and the target value when iPTH is >10 pmol/L; and (b) 2.5 pmol/L absolute difference between the sample and target value when the target value is <10 pmol/L. When the Royal Australasian College of Pathologists Quality Assurance Program criteria were applied, 19 of 36 (52%) serum samples at baseline and 27 of 36 (75%) serum samples stored at room temperature for 3 days failed assurance criteria, in contrast to one EDTA sample stored at room temperature for 3 days.

We defined our reference interval for iPTH (0.8–8.0 pmol/L) according to the values in healthy, vitamin D- sufficient blood donors (7). When we applied this reference interval for iPTH as the diagnostic classification criterion, 6 of 36 (17%) serum samples at baseline and 13 of 26 (50%) serum samples stored at room temperature for 3 days were misclassified, in contrast to two EDTA samples stored at room temperature for 3 days.

Despite attempts to analyze serum samples expeditiously, serum values for iPTH were significantly lower than in EDTA-plasma samples. Thus, the IMMULITE 2000 iPTH assay does not give comparable results for serum and EDTA plasma. The further decline in iPTH values in serum at 3 days is consistent with the susceptibility of PTH to degrade in serum samples. As long as adequate sample volume during collection is ensured, EDTA samples are the most appropriate for iPTH measurement by the IMMULITE 2000 immunoassay (6). Use of serum samples for the measurement of iPTH by the IMMULITE 2000 iPTH assay will lead to high rates of diagnostic misclassification unless analysis is carried out promptly and the values are defined by a different reference interval.


Acknowledgments

We wish to acknowledge the help of Stephen Stewart, Dulcie King, and all of the staff in the central specimen reception area, Royal Perth Hospital, for collection of patient samples and their ongoing dedication to meticulous sample collection.


References

  1. Glendenning P, Gutteridge DH, Retallack RW, Stuckey BG, Kermode DG, Kent GN. High prevalence of normal total calcium and intact PTH in 60 patients with proven primary hyperparathyroidism: a challenge to current diagnostic criteria. Aust N Z J Med 1998;28:173-178.[ISI][Medline] [Order article via Infotrieve]
  2. Vasikaran SD, Sturdy G, Musk AA, Flicker L. Vitamin D insufficiency and hyperparathyroidism in Perth blood donors. Med J Aust 2000;172:406-407.[Medline] [Order article via Infotrieve]
  3. Omar H, Chamberlin A, Walker V, Wood PJ. Immulite 2000 parathyroid hormone assay: stability of parathyroid hormone in EDTA blood kept at room temperature for 48 h. Ann Clin Biochem 2001;38:561-563.[ISI][Medline] [Order article via Infotrieve]
  4. Walker KS, Seth J. Stability of parathyroid hormone in blood from renal patients on haemodialysis. Ann Clin Biochem 2000;37:800-801.
  5. Levin GE, Nisbet JA. Stability of parathyroid hormone-related protein and parathyroid hormone at room temperature. Ann Clin Biochem 1994;31:497-500.
  6. Glendenning P, Musk AA, Taranto M, Vasikaran SD. Preanalytical factors in the measurement of Intact parathyroid hormone using the DPC IMMULITE assay. Clin Chem 2002;48:566-567.[Free Full Text]
  7. Glendenning P, Vasikaran SD. Vitamin D status and redefining the serum PTH reference range in the elderly. J Clin Endocrinol Metab 2002;87:946-947.[Free Full Text]



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