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Letters to the Editor |
Departments of1 Clinical Biochemistry and2 Renal Medicine East Kent Hospitals NHS Trust Kent, UK
aAddress correspondence to this author at: Department of Clinical Biochemistry, Kent and Canterbury Hospital, Canterbury, Kent CT1 3NG, UK. E-mail edmund.lamb{at}ekht.nhs.uk.
To the Editor:
Measurement of circulating parathyroid hormone (PTH) is used in the differential diagnosis of disorders of calcium metabolism. Many samples come from patients with end-stage renal disease (ESRD), in whom PTH is used as an indicator of bone turnover (1). The instability of PTH in serum or plasma ex vivo has long been a concern. Use of EDTA-containing tubes for sample collection (2)(3)(4) reportedly improves stability, presumably as a result of protease inhibition (5). Several manufacturers recommend EDTA plasma for PTH measurement, but no information is available on the stability of PTH in ESRD samples measured with newer assays.
The new full-length (also called "whole molecule" or "Bio-Intact") PTH assays appear to recognize only forms containing the N-terminal serine. They are thought not to cross-react with truncated C-terminal fragments of PTH, in particular PTH(7–84), that increase in ESRD and cross-react in older assays. Because of their improved specificity, these assays will likely replace older assays.
We collected blood from 17 patients (12 males and 5 females; median age, 61 years; range, 37–86 years), immediately before hemodialysis, into VacutainerTM Tubes (Becton Dickinson Ltd.) that contained either no additives (plain) or potassium EDTA. Tubes were filled with the recommended volume of blood. Aliquots of serum and plasma were frozen rapidly after centrifugation (zero time). The remaining serum/plasma was removed from the cells and left at room temperature, with further aliquots being frozen (–20 °C) at 2, 4, 8, 24, and 48 h.
Samples were analyzed with an automated two-site immunochemiluminescent assay (Bio-Intact PTH; Nichols Institute Diagnostics; manufacturer’s reference interval, 3–51 ng/L) on a Nichols AdvantageTM Specialty System analyzer. One of the antibodies used in the assay is directed toward the six N-terminal amino acids; therefore, the assay does not cross-react with the PTH(7–84) fragment (6). Within-batch imprecision (CV; n = 7) was 5.1% in pooled uremic serum (mean, 166 ng/L) and 4.1% in pooled uremic plasma (mean, 180 ng/L). All of the samples from each individual patient were analyzed in a single batch to eliminate any effects of between-batch imprecision.
In 11 patients, plasma PTH was also measured with a second-generation "intact" PTH assay supplied by the same manufacturer, which uses antibodies raised against the N-terminal (amino acids 1–34) and C-terminal (amino acids 39–84) portions of the molecule (7). All patients gave informed consent, and the study had the approval of the Local Research Ethics Committee. Data were analyzed with the Friedman one-way ANOVA and the Wilcoxon matched-pairs signed-ranks test.
At zero time, PTH concentrations in plain serum did not differ (P = 0.431) from those in EDTA plasma. Time delay before freezing had a significant effect on stability in plain serum (P = 0.0106), but not in EDTA plasma (P = 0.642). The PTH concentration decreased significantly after 24 h in plain serum (Table 1
). As would be expected, plasma PTH concentrations measured with the second-generation intact PTH assay (median, 193 ng/L; range, 10–709 ng/L) were significantly (P = 0.0098) higher than those measured with the third-generation assay (median, 97 ng/L; range, 34–397 ng/L).
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Our data are broadly in keeping with our earlier results obtained with the second-generation assay (4), although intact PTH decreased significantly in plain serum after only 2 h in that study. Perhaps the Bio-Intact PTH measured by the third-generation assay is more stable than the fragments detected by the second-generation assay. Alternatively, it could reflect increased stability of a recently proposed phosphorylated N-terminal fragment (8) that may be detected by third-generation, but not second-generation, immunoassays and is thought to be present at appreciable concentrations in uremic plasma. However, our study was not designed to specifically test this aspect, and the relatively small number of valid plain serum samples we had available at the 4- and 8-h time points limit the strength of this conclusion.
Bio-Intact PTH measured by the Nichols assay demonstrates excellent stability in blood collected into tubes containing EDTA. This stability may be particularly useful for dialysis patients and whenever samples are collected at inconvenient times (e.g., twilight dialysis clinics) or in settings remote from the main laboratory (e.g., satellite dialysis units).
Acknowledgments
We are grateful to Nichols Institute Diagnostics Ltd. for the provision of reagents used in this study and to T. Anslow from the Department of Clinical Biochemistry for technical assistance.
References
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