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Parathyroid hormone measurement in CKD

2009; Elsevier BV; Volume: 77; Issue: 2 Linguagem: Inglês

10.1038/ki.2009.374

1523-1755

Jean-Claude P. Souberbielle, Hubert Roth, Denis Fouque,

Magnesium in Health and Disease

The Kidney Disease Outcomes Quality Initiative (K/DOQI) guidelines recommend that serum parathyroid hormone (PTH) concentration of patients with chronic kidney disease (CKD) should be measured regularly and maintained within target ranges that are defined according to the stage of CKD (e.g., 150–300 pg/ml in patients with CKD stage 5). The quality of the PTH assay is of paramount importance, as it contributes to the therapeutic decision. Indeed, when the PTH concentration is above these target values, drugs that decrease PTH secretion, such as active vitamin D compounds or calcimimetic agents, may be given and the doses are then adapted according to the evolution of the PTH concentration. By contrast, if the PTH concentration is below the target range, any treatment that may decrease PTH secretion is stopped to avoid adynamic bone disease and associated extra-skeletal calcifications. The aim of this article is to discuss the main features and pitfalls related to PTH measurement in the setting of CKD. The Kidney Disease Outcomes Quality Initiative (K/DOQI) guidelines recommend that serum parathyroid hormone (PTH) concentration of patients with chronic kidney disease (CKD) should be measured regularly and maintained within target ranges that are defined according to the stage of CKD (e.g., 150–300 pg/ml in patients with CKD stage 5). The quality of the PTH assay is of paramount importance, as it contributes to the therapeutic decision. Indeed, when the PTH concentration is above these target values, drugs that decrease PTH secretion, such as active vitamin D compounds or calcimimetic agents, may be given and the doses are then adapted according to the evolution of the PTH concentration. By contrast, if the PTH concentration is below the target range, any treatment that may decrease PTH secretion is stopped to avoid adynamic bone disease and associated extra-skeletal calcifications. The aim of this article is to discuss the main features and pitfalls related to PTH measurement in the setting of CKD. Parathyroid hormone (PTH) is a single-chain 84-amino-acid peptide hormone encoded by a gene on the short arm of chromosome 11 and produced by the parathyroid glands in response to a decrease in the extracellular concentration of ionized calcium (Ca++). Its half-life is very short (2–4 min) and its main role is to increase serum Ca++, which is achieved by stimulating the release of calcium from bone and its renal re-absorption in the distal tubule. In the renal proximal tubule, PTH also stimulates the synthesis of calcitriol which in turn increases intestinal absorption of calcium and exerts an endocrine feed-back on the secretion of PTH at the parathyroid level. PTH also decreases the renal re-absorption of phosphate in the proximal tubule, thereby decreasing serum phosphate. Furthermore, PTH stimulates bone formation, and this property is now used in clinical practice for treatment of osteoporosis. It has been demonstrated that the very first N-terminal amino acids of the PTH molecule are indispensable for this interaction. Besides full-length 1–84 PTH, various PTH fragments are present in blood, whose exact composition and possible function are not yet fully elucidated. Apart from the well-known bone and renal effects, PTH also acts in a number of other cells, such as the cardiomyocyte, adipocyte, pancreas beta cell, among others. The dysfunction of these cells or tissues observed in response to an increase in serum PTH has led to the concept of PTH being a uremic toxin.1.Fadda G.Z. Thanakitcharu P. Smogorzewski M. et al.Parathyroid hormone raises cytosolic calcium in pancreatic islets: study on mechanisms.Kidney Int. 1993; 43: 554-560Abstract Full Text PDF PubMed Scopus (33) Google Scholar, 2.Ni Z. Smogorzewski M. Massry S.G. Effects of parathyroid hormone on cytosolic calcium of rat adipocytes.Endocrinology. 1994; 135: 1837-1844Crossref PubMed Scopus (45) Google Scholar, 3.Tanaka H. Smogorzewski M. Koss M. et al.Pathways involved in PTH-induced rise in cytosolic Ca2+ concentration of rat renal proximal tubule.Am J Physiol. 1995; 268: F330-F337PubMed Google Scholar The Kidney Disease Outcomes Quality Initiative (K/DOQI) for bone metabolism and disease in chronic kidney disease (CKD) guidelines4.National Kidney Foundation K/DOQI clinical practice guidelines for bone metabolism and disease in chronic kidney disease.Am J Kidney Dis. 2003; 42: S1-201Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar recommend that serum PTH concentration of patients with CKD should be measured regularly and maintained within target ranges that are defined according to the stage of CKD (i.e., 150–300 pg/ml in patients with CKD stage 5). The quality of the PTH assay is of paramount importance as it contributes to the therapeutic decision. Indeed, when the PTH concentration is above these target values, drugs that decrease PTH secretion such as active vitamin D compounds or calcimimetic agents may be given and the doses are then adapted according to the evolution of the PTH concentration. By contrast, if the PTH concentration is below the target range, any treatment that may decrease PTH secretion is stopped to avoid adynamic bone disease and associated extra-skeletal calcifications. The aim of this study is to discuss the main features and pitfalls related to PTH measurement in the setting of CKD. First-generation PTH assays were radioimmunoassays5.Berson S.A. Yalow R.S. Immunochemical heterogeneity of parathyroid hormone in plasma.J Clin Endocrinol Metab. 1968; 28: 1037-1047Crossref PubMed Scopus (265) Google Scholar using polyclonal antibodies directed mainly toward synthetic C-terminal (such as 53–84 PTH) or mid-region (such as 44-68 PTH) PTH fragments. In addition to PTH 1–84, these assays were known to measure fragments, which are mainly produced in the liver by the catabolism in the Kupffer cells, are eliminated by the kidney, have a longer half-life than 1–84 PTH, and accumulate in CKD patients.6.D'Amour P. Brossard J.H. Carboxyl-terminal parathyroid hormone fragments: role in parathyroid hormone physiopathology.Curr Opin Nephrol Hypertens. 2005; 14: 330-336Crossref PubMed Scopus (35) Google Scholar The consequence was that, in CKD patients PTH concentrations measured with these first-generation assays were always greatly increased. Furthermore, these assays had poor analytical sensitivity in the low concentrations, rendering discrimination between low and normal levels difficult. For these reasons, the first-generation PTH assays are currently considered obsolete for the clinical practice. During the mid 1980's, the first second-generation PTH assay, the Allegro intact PTH assay, became available.7.Nussbaum S.R. Zahradnik R.J. Lavigne J.R. et al.Highly sensitive two-site immunoradiometric assay of parathyrin, and its clinical utility in evaluating patients with hypercalcemia.Clin Chem. 1987; 33: 1364-1367PubMed Google Scholar This immunoradiometric assay used two different antibodies. The capture antibody coated to a plastic bead was directed toward the 39–84 portion of the PTH molecule, whereas the 125-I-labeled antibody recognized mainly the 15–20 portion of the PTH molecule.8.D'Amour P. Brossard J.H. Rakel A. et al.Evidence that the amino-terminal composition of non-(1–84) parathyroid hormone fragments starts before position 19.Clin Chem. 2005; 51: 169-176Crossref PubMed Scopus (46) Google Scholar This assay was, thus, unable to measure the C-terminal or mid-fragments (such as 53–84 or 44–68), which were measured with the first-generation assays. During the following years, several similar assays, either immunoradiometric assay or 'non-radioactive' immunometric assays, became available,9.Endres D.B. Villanueva R. Sharp Jr, C.F. et al.Immunochemiluminometric and immunoradiometric determinations of intact and total immunoreactive parathyrin: performance in the differential diagnosis of hypercalcemia and hypoparathyroidism.Clin Chem. 1991; 37: 162-168PubMed Google Scholar, 10.Ratcliffe W.A. Heath D.A. Ryan M. et al.Performance and diagnostic application of a two-site immunoradiometric assay for parathyrin in serum.Clin Chem. 1989; 35: 1957-1961PubMed Google Scholar, 11.Blind E. Schmidt-Gayk H. Scharla S. et al.Two-site assay of intact parathyroid hormone in the investigation of primary hyperparathyroidism and other disorders of calcium metabolism compared with a midregion assay.J Clin Endocrinol Metab. 1988; 67: 353-360Crossref PubMed Scopus (102) Google Scholar some of them using fully automated immunoanalyzers allowing better analytical performances and shorter turnaround time.12.Michelangeli V.P. Heyma P. Colman P.G. et al.Evaluation of a new, rapid and automated immunochemiluminometric assay for the measurement of serum intact parathyroid hormone.Ann Clin Biochem. 1997; 34: 97-103Crossref PubMed Scopus (34) Google Scholar,13.Hermsen D. Franzson L. Hoffmann J.P. et al.Multicenter evaluation of a new immunoassay for intact PTH measurement on the Elecsys System 2010 and 1010.Clin Lab. 2002; 48: 131-141PubMed Google Scholar Some of these assays use an anti-N-terminal antibody directed, like in the Allegro assay, toward the proximal 15–20 portion of the hormone, whereas others, like the Elecsys/Modular intact PTH assay, recognize a more distal epitope in the 26–32 portion.8.D'Amour P. Brossard J.H. Rakel A. et al.Evidence that the amino-terminal composition of non-(1–84) parathyroid hormone fragments starts before position 19.Clin Chem. 2005; 51: 169-176Crossref PubMed Scopus (46) Google Scholar These second-generation assays were globally called 'intact' PTH assays as they were thought to measure only the full-length 1–84 PTH. Although producing far more clinically satisfying data than first-generation assays (i.e., very low to very high values instead of always high, and significant correlation with bone biopsy parameters in CKD patients; clearly low values in 'non-parathyroid hypercalcemia or hypoparathyroidism), they were rapidly shown to present some limitations. In particular, several reports suggested that they overestimated the degree of secondary hyperparathyroidism in CKD patients,14.Quarles L.D. Lobaugh B. Murphy G. Intact parathyroid hormone overestimates the presence and severity of parathyroid-mediated osseous abnormalities in uremia.J Clin Endocrinol Metab. 1992; 75: 145-150Crossref PubMed Scopus (232) Google Scholar,15.Wang M. Hercz G. Sherrard D.J. et al.Relationship between intact 1–84 parathyroid hormone and bone histomorphometric parameters in dialysis patients without aluminum toxicity.Am J Kidney Dis. 1995; 26: 836-844Abstract Full Text PDF PubMed Scopus (203) Google Scholar leading to an increased effort to suppress PTH with its consequent concern for the development of adynamic bone disease and erroneous referrals for parathyroidectomy. Indeed, it was not understood why a hemodialysis patient with histological features of low-turnover bone disease may have an 'intact' PTH concentration as high as 400–500 pg/ml. One possible explanation came from the demonstration that several 'intact' PTH assays recognized with various cross-reactivities (from approximately 50 to 100%) a PTH molecule, different from 1–84 PTH, which co-eluted in high-performance liquid chromatography with a synthetic 7–84 PTH fragment.16.Lepage R. Roy L. Brossard J.H. et al.A non-(1–84) circulating parathyroid hormone (PTH) fragment interferes significantly with intact PTH commercial assay measurements in uremic samples.Clin Chem. 1998; 44: 805-809Crossref PubMed Scopus (305) Google Scholar In 1999, the first third-generation PTH assay was developed by Scantibodies Laboratories.17.John M.R. Goodman W.G. Gao P. et al.A novel immunoradiometric assay detects full-length human PTH but not amino-terminally truncated fragments: implications for PTH measurements in renal failure.J Clin Endocrinol Metab. 1999; 84: 4287-4290Crossref PubMed Google Scholar This immunoradiometric assay, called Whole PTH assay, uses an anti C-terminal antibody similar to that of the 'intact' PTH assays, but an anti N-terminal antibody directed against the very first amino-acids (1–4), and, thus, does not measure the 7–84 PTH.18.Gao P. Scheibel S. D'Amour P. et al.Development of a novel immunoradiometric assay exclusively for biologically active whole parathyroid hormone 1–84: implications for improvement of accurate assessment of parathyroid function.J Bone Miner Res. 2001; 16: 605-614Crossref PubMed Scopus (312) Google Scholar Although highly correlated to the Allegro 'intact' assay, it was shown to produce lower serum concentrations (approximately 50%) but similar values in solutions of synthetic full-length 1–84 PTH.18.Gao P. Scheibel S. D'Amour P. et al.Development of a novel immunoradiometric assay exclusively for biologically active whole parathyroid hormone 1–84: implications for improvement of accurate assessment of parathyroid function.J Bone Miner Res. 2001; 16: 605-614Crossref PubMed Scopus (312) Google Scholar It was, thus, believed that when serum PTH is measured with these two assays, the difference between the two measured values corresponds to the concentration of 7–84 PTH. Assuming this, it was shown that the percentage of 7–84 PTH increases when the glomerular filtration rate decreases,19.Brossard J.H. Lepage R. Cardinal H. et al.Influence of glomerular filtration rate on non-(1–84) parathyroid hormone (PTH) detected by intact PTH assays.Clin Chem. 2000; 46: 697-703PubMed Google Scholar and is variable from one patient to another.20.Salomon R. Charbit M. Gagnadoux M.F. et al.High serum levels of a non-(1–84) parathyroid hormone (PTH) fragment in pediatric haemodialysis patients.Pediatr Nephrol (Berlin, Germany). 2001; 16: 1011-1014Crossref PubMed Scopus (20) Google Scholar Since then, several studies have demonstrated that, in animal or cellular models, 7–84 PTH exerts effects that are opposite to those of 1–84 PTH (decrease in serum calcium and urine phosphate, inhibition of bone resorption), and is produced by the parathyroid glands in response to an increase in serum calcium levels.21.Slatopolsky E. Finch J. Clay P. et al.A novel mechanism for skeletal resistance in uremia.Kidney Int. 2000; 58: 753-761Abstract Full Text Full Text PDF PubMed Scopus (330) Google Scholar,22.Divieti P. John M.R. Juppner H. et al.Human PTH-(7–84) inhibits bone resorption in vitro via actions independent of the type 1 PTH/PTHrP receptor.Endocrinology. 2002; 143: 171-176Crossref PubMed Scopus (160) Google Scholar There is now convincing evidence that these inhibitory effects of the 7–84 PTH fragment are mediated through a receptor different of the PTH/PTHrP receptor (PTHR1) as reviewed in23.Murray T.M. Rao L.G. Divieti P. et al.Parathyroid hormone secretion and action: evidence for discrete receptors for the carboxyl-terminal region and related biological actions of carboxyl-terminal ligands.Endocr Rev. 2005; 26: 78-113Crossref PubMed Scopus (214) Google Scholar and through a desensitization of PTHR1 in some cells.24.Friedman P.A. PTH revisited.Kidney Int Suppl. 2004; : S13-S19Abstract Full Text Full Text PDF PubMed Google Scholar An important question is whether the third-generation PTH assays improve the detection of altered bone turnover compared with the second-generation assays. To answer this question, bone biopsy studies are necessary. To date, four studies have addressed this question for dialysis patients with bone biopsies. In the first one,25.Monier-Faugere M.C. Geng Z. Mawad H. et al.Improved assessment of bone turnover by the PTH-(1–84)/large C-PTH fragments ratio in ESRD patients.Kidney Int. 2001; 60: 1460-1468Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar it was found that the ratio 1–84 PTH/7–84 PTH (obtained by measuring PTH with two different assays, a second- and a third-generation assay) discriminated between high- and low-turnover bone disease significantly better than PTH measured by either a second- or third generation assay alone. However, the three other studies26.Coen G. Bonucci E. Ballanti P. et al.PTH 1–84 and PTH '7–84' in the noninvasive diagnosis of renal bone disease.Am J Kidney Dis. 2002; 40: 348-354Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, 27.Salusky I.B. Goodman W.G. Kuizon B.D. et al.Similar predictive value of bone turnover using first- and second-generation immunometric PTH assays in pediatric patients treated with peritoneal dialysis.Kidney Int. 2003; 63: 1801-1808Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 28.Lehmann G. Stein G. Huller M. et al.Specific measurement of PTH (1–84) in various forms of renal osteodystrophy (ROD) as assessed by bone histomorphometry.Kidney Int. 2005; 68: 1206-1214Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar did not find any improvement in the diagnosis of bone turnover anomalies with either this ratio or the third-generation PTH alone compared with a second-generation assay. As treatment with active vitamin D may greatly modify the relationship between histomorphometric indices of bone turnover and PTH levels, it must be stressed that the patients studied in these four studies differed in terms of past or actual vitamin D therapy, and that this may represent a possible explanation for the discrepancies between these studies. Another aspect, which should be controlled in such studies, is the ethnic origin of the enrolled patients. Indeed, it has been shown that second-generation PTH levels correlated with bone turnover indexes in Caucasian but not African-American dialysis patients.29.Sawaya B.P. Butros R. Naqvi S. et al.Differences in bone turnover and intact PTH levels between African American and Caucasian patients with end-stage renal disease.Kidney Int. 2003; 64: 737-742Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar Furthermore, in CKD African-American patients, the PTH levels are usually found to be higher than those in Caucasians despite near-similar levels of bone alkaline phosphatise, suggesting that optimal bone turnover may be associated with different levels of PTH in these two ethnic groups. Nevertheless, even if the group of experts who published the K/DOQI guidelines acknowledged the potential advantage of the third-generation PTH assays, they recommended measuring PTH in CKD patients by means of a second-generation assay until more definitive bone biopsy studies have been published.4.National Kidney Foundation K/DOQI clinical practice guidelines for bone metabolism and disease in chronic kidney disease.Am J Kidney Dis. 2003; 42: S1-201Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar,30.Martin K.J. Olgaard K. Coburn J.W. et al.Diagnosis, assessment, and treatment of bone turnover abnormalities in renal osteodystrophy.Am J Kidney Dis. 2004; 43: 558-565Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar However, besides the evaluation of altered bone turnover, other outcomes may be considered when comparing the relative merits of both generations of PTH assays. In a recent publication31.Melamed M.L. Eustace J.A. Plantinga L.C. et al.Third-generation parathyroid hormone assays and all-cause mortality in incident dialysis patients: the CHOICE study.Nephrol Dial Transplant. 2008; 23: 1650-1658Crossref PubMed Scopus (50) Google Scholar for example, a third-generation PTH assay (but not a second-generation assay, or the 1–84 PTH/7–84 PTH ratio) was predictive of all-cause mortality in a cohort of incident dialysis patients. We, thus, think, like the experts of the K/DOQI group, that further research on the third-generation assays is mandatory, but that there is currently no need to ask the laboratory to switch from a second-generation to a third-generation PTH assay. Finally, it should also be mentioned that a new PTH species called amino-PTH (N-PTH), measured with the Whole PTH assay (third-generation assay) and the Elecsys assay (second-generation, which uses an anti 26–32 N-terminal antibody), but not with an 'intact' PTH assay, which used an anti 15–20 N-terminal antibody, has been described recently, a finding, which adds more complexity to this already highly complicated topic.32.D'Amour P. Brossard J.H. Rousseau L. et al.Amino-terminal form of parathyroid hormone (PTH) with immunologic similarities to hPTH(1–84) is overproduced in primary and secondary hyperparathyroidism.Clin Chem. 2003; 49: 2037-2044Crossref PubMed Scopus (88) Google Scholar Even if its exact structure is still unknown, N-PTH should, thus, contain the 1–4 amino-acids, but should be different from the 1–84 PTH in the 15–20 portion of the molecule. Whereas the amount of N-PTH is approximately one-tenth of that of 1–84 PTH in normal subjects,32.D'Amour P. Brossard J.H. Rousseau L. et al.Amino-terminal form of parathyroid hormone (PTH) with immunologic similarities to hPTH(1–84) is overproduced in primary and secondary hyperparathyroidism.Clin Chem. 2003; 49: 2037-2044Crossref PubMed Scopus (88) Google Scholar it has been shown to be excessively produced in rare patients with either a parathyroid carcinoma33.Rubin M.R. Silverberg S.J. D'Amour P. et al.An N-terminal molecular form of parathyroid hormone (PTH) distinct from hPTH(1 84) is overproduced in parathyroid carcinoma.Clin Chem. 2007; 53: 1470-1476Crossref PubMed Scopus (59) Google Scholar,34.Caron P. Maiza J.C. Renaud C. et al.High third generation/second generation PTH ratio in a patient with parathyroid carcinoma: clinical utility of third generation/second generation PTH ratio in patients with primary hyperparathyroidism.Clin Endocrinol (Oxf). 2009; 70: 533-538Crossref PubMed Scopus (31) Google Scholar or a severe primary hyperparathyroidism.35.Rakel A. Brossard J.H. Patenaude J.V. et al.Overproduction of an amino-terminal form of PTH distinct from human PTH(1–84) in a case of severe primary hyperparathyroidism: influence of medical treatment and surgery.Clin Endocrinol (Oxf). 2005; 62: 721-727Crossref PubMed Scopus (41) Google Scholar In these patients, the PTH concentration measured with a third-generation assay was higher than when measured with a second-generation assay with a proximal (15–20) epitope. This atypical profile became normal (second-generation higher than third-generation PTH) after parathyroidectomy in patients with either primary hyperparathyroidism35.Rakel A. Brossard J.H. Patenaude J.V. et al.Overproduction of an amino-terminal form of PTH distinct from human PTH(1–84) in a case of severe primary hyperparathyroidism: influence of medical treatment and surgery.Clin Endocrinol (Oxf). 2005; 62: 721-727Crossref PubMed Scopus (41) Google Scholar or parathyroid cancer,34.Caron P. Maiza J.C. Renaud C. et al.High third generation/second generation PTH ratio in a patient with parathyroid carcinoma: clinical utility of third generation/second generation PTH ratio in patients with primary hyperparathyroidism.Clin Endocrinol (Oxf). 2009; 70: 533-538Crossref PubMed Scopus (31) Google Scholar suggesting that it was produced by the abnormal glands. Table 1 and Figure 1 summarize what the different PTH immunoassays measure. This table also highlights the need for a revision of the nomenclature of the different PTH molecules and PTH assays. Indeed, when one understands that the 'intact' assays do not measure only the intact PTH molecule, whereas BioIntact assay measures the intact molecule and another molecule whose biological activity is unknown, one can imagine the confusion for the nonspecialist.Table 1The main circulating fragments of PTH, and whether they are measured (Yes) or not (No) by the various PTH assay generationsFirst-generation assaysSecond-generation assaysThird-generation assaysMost common identificationsC-PTH assays, Mid-PTH assays'Intact' PTH assaysWhole PTH assay, Ca-PTH assay, BioIntact PTH assayMethodologyCompetition (mostly RIA)Immunometry ('sandwich' assays)Immunometry ('sandwich' assays)1–84 PTHYesYesYes7–84 PTHYesYes (with various cross-reactivity)NoC-terminal fragmentsYesNoNo'Amino' PTHYesDepends on the epitope of the anti-N-terminal Ab: No if the epitope is proximal (13–24) and Yes if the epitope is distal (26–32)YesAb, antibody; Ca-PTH, calcium-PTH; PTH, parathyroid hormone; RIA, radioimmunoassay. Open table in a new tab Ab, antibody; Ca-PTH, calcium-PTH; PTH, parathyroid hormone; RIA, radioimmunoassay. The second- and third-generation assays are overall of good analytical quality with within-run and inter-day coefficients of variation typically in the range of 1 to 10%, automated assays giving generally moderately lower coefficients of variation than manual assays. It is important that the chosen assay presents a detection limit that is sufficiently low to avoid overlap with low-normal values, say at least below 3–5 pg/ml. From the pre-analytical point of view, serum PTH measured either with the second-36.Joly D. Drueke T.B. Alberti C. et al.Variation in serum and plasma PTH levels in second-generation assays in hemodialysis patients: a cross-sectional study.Am J Kidney Dis. 2008; 51: 987-995Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar or third-generation assays18.Gao P. Scheibel S. D'Amour P. et al.Development of a novel immunoradiometric assay exclusively for biologically active whole parathyroid hormone 1–84: implications for improvement of accurate assessment of parathyroid function.J Bone Miner Res. 2001; 16: 605-614Crossref PubMed Scopus (312) Google Scholar have been shown to be stable when serum is left standing at room temperature for up to 6 h before being frozen. For longer delay, PTH seems slightly more stable in ethylenediaminetetraacetic acid (EDTA) plasma or in serum kept at +4 °C.37.Parent X. Alenabi F. Brignon P. et al.[Delayed measurement of PTH in patients with CKD: storage of the primary tube in the dialysis unit, which temperature? Which kind of tube?].Nephrol Ther. 2009; 5: 34-40Crossref PubMed Scopus (13) Google Scholar However, if EDTA sampling tubes are used instead of dry tubes, it should be kept in mind that EDTA PTH values may be higher than serum values by 10–30% depending on the assay used.36.Joly D. Drueke T.B. Alberti C. et al.Variation in serum and plasma PTH levels in second-generation assays in hemodialysis patients: a cross-sectional study.Am J Kidney Dis. 2008; 51: 987-995Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 37.Parent X. Alenabi F. Brignon P. et al.[Delayed measurement of PTH in patients with CKD: storage of the primary tube in the dialysis unit, which temperature? Which kind of tube?].Nephrol Ther. 2009; 5: 34-40Crossref PubMed Scopus (13) Google Scholar, 38.Omar H. Chamberlin A. Walker V. et al.Immulite 2000 parathyroid hormone assay: stability of parathyroid hormone in EDTA blood kept at room temperature for 48 h.Ann Clin Biochem. 2001; 38: 561-563Crossref PubMed Scopus (28) Google Scholar, 39.Holmes D.T. Levin A. Forer B. et al.Preanalytical influences on DPC IMMULITE 2000 intact PTH assays of plasma and serum from dialysis patients.Clin Chem. 2005; 51: 915-917Crossref PubMed Scopus (36) Google Scholar There is no clear explanation for this phenomenon, but the most probable one is a calcium dependency of the epitope recognized by some antibodies. It is, thus, important to verify that the reference values have been established with the correct samples. Furthermore, compared with EDTA plasma, serum samples have the advantage to allow calcium measurement and to be valid even in poorly filled tubes (see Table 2 for the relative advantages of serum or EDTA samples). Finally, PTH concentration is not affected by four18.Gao P. Scheibel S. D'Amour P. et al.Development of a novel immunoradiometric assay exclusively for biologically active whole parathyroid hormone 1–84: implications for improvement of accurate assessment of parathyroid function.J Bone Miner Res. 2001; 16: 605-614Crossref PubMed Scopus (312) Google Scholar,39.Holmes D.T. Levin A. Forer B. et al.Preanalytical influences on DPC IMMULITE 2000 intact PTH assays of plasma and serum from dialysis patients.Clin Chem. 2005; 51: 915-917Crossref PubMed Scopus (36) Google Scholar or even six40.Inaba M. Nakatsuka K. Imanishi Y. et al.Technical and clinical characterization of the Bio-PTH (1–84) immunochemiluminometric assay and comparison with a second-generation assay for parathyroid hormone.Clin Chem. 2004; 50: 385-390Crossref PubMed Scopus (63) Google Scholar freeze–thaw cycles.Table 2Summary of the advantages/disadvantages of serum and EDTA plasma as the sample of choice for the measurement of PTH in CKD patientsSerumEDTAStability of PTH during 4 h at RTGoodGoodStability of PTH during 18 h at RTConcentration does not change or decreases by up to 20% (usually less)Concentration does not change or increases by up to 12% (usually less)Stability of PTH during 24 h at 4 °CGoodGoodPossibility to measure calcium in the same sampleYesNoNecessity to fill the tube sufficiently (>50%)NoYesNecessity to delay centrifugation to allow blood to clotYesNoAb, antibody; Ca-PTH, calcium-PTH; CKD, chronic kidney disease; EDTA, ethylenediaminetetraacetic acid; PTH, parathyroid hormone; RIA, radioimmunoassay; RT, room temperature. Open table in a new tab Ab, antibody; Ca-PTH, calcium-PTH; CKD, chronic kidney disease; EDTA, ethylenediaminetetraacetic acid; PTH, parathyroid hormone; RIA, radioimmunoassay; RT, room temperature. A constant finding when comparing different immunoassays is that, although the values obtained from different assays are generally highly correlated, absolute concentration may greatly differ. As suggested above, this may be due to differences in cross-reactivity with 7–84 PTH. However, PTH assays also suffer a lack of standardization as evidenced by D'Amour et al.8.D'Amour P. Brossard J.H. Rakel A. et al.Evidence that the amino-t