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Update on parathyroid hormone: New tests and new challenges for external quality assessment
Clinical Biochemistry 40 (2007) 585 – 590
Review
Update on parathyroid hormone: New tests and new challenges for external quality assessment David E.C. Cole a,b,c,d , Sharon Webb e , Pak-Cheung Chan a,b,⁎ a
e
Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada b Department of Clinical Pathology, Sunnybrook Health Sciences Center, Toronto, ON, Canada c Department of Medicine, Ontario Medical Association, Toronto, ON, Canada d Department of Pediatrics, University of Toronto, Ontario Medical Association, Toronto, ON, Canada Quality Management Program–Laboratory Services, Ontario Medical Association, Toronto, ON, Canada Received 3 November 2006; received in revised form 16 March 2007; accepted 18 March 2007 Available online 6 April 2007
Abstract It is now 43 years since Berson and Yalow published the first radio-immunoassay (RIA) for parathyroid hormone (PTH) [S.A. Berson, R.S. Yalow, G.D. Aurbach, J.T. Potts, Immunoassay of bovine and human parathyroid hormone. Proc Natl Acad Sci U S A 49 (1963) 613–617] [1]. Since then, there have been marked advances in our understanding of this peptide hormone, its mechanism of action and biological regulation [J.T. Potts, Parathyroid hormone: past and present. J. Endocrinol. 187 (2005) 311–325] [2]. PTH has become a routine assay in tertiary care hospitals and is an essential element in the management of chronic kidney disease, parathyroid disorders and the investigation of abnormalities in calcium homeostasis. Despite continuing technological advances in PTH measurement, analyte heterogeneity remains a problem, while improved turnaround time and better precision are constantly escalating clinical demands. This mini-review begins with a brief update of current knowledge on PTH, followed by a summary of a recent Ontario-wide External Quality Assurance (EQA) survey, and concludes with comments on utilization trends, current and future. © 2007 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved. Keywords: Parathyroid hormone (PTH); Calcium homeostasis; Utilization
Contents Introduction . . . . . . . . . . . . . . . . Biology of PTH. . . . . . . . . . . . . . Measurements of PTH . . . . . . . . . . A province-wide survey of PTH assays . Utilization—an area for future continuing Conclusions . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . .
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Introduction Parathyroid hormone (PTH) is a polypeptide hormone that plays a central role in the maintenance of calcium and phosphate ⁎ Corresponding author. Rm B204, Sunnybrook Health Sciences Center, 2075 Bayview Avenue, Toronto ON, Canada M4N 3M5. Fax: +1 416 4806120. E-mail address: [email protected] (P.-C. Chan).
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homeostasis and bone health. It acts to maintain bone and mineral homeostasis largely through four mechanisms: (i) elevation of blood calcium by increasing osteoclastic bone resorption, (ii) enhancement of renal calcium reabsorption, (iii) stimulation of renal 1,25-dihydroxyvitamin D synthesis, leading to increased intestinal calcium absorption, and (iv) promotion of phosphaturia via inhibition of renal tubular transepithelial phosphate reabsorption [3]. These biologic responses are down-
0009-9120/$ - see front matter © 2007 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved. doi:10.1016/j.clinbiochem.2007.03.019
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stream actions following the binding of the NH2-terminal portion of the mature PTH molecule or fragments to a common PTH/PTH related protein (PTHrP) receptor, PTH1R, on the plasma membranes of target tissue cells [3–5]. Signals of PTH1R activation following ligand binding may be transduced via either the classic pathway involving G-protein (Gαq/11), adenylyl cyclase and phospholipase C or the non-classic pathway involving G-protein (GαS), Erk, phospholipase A2 and phospholipase D under the influence of a Na+/H+ exchange regulatory factor (NHERF1) in a cell specific manner [34]. While the first two amino acid residues of PTH are required for PTH1R signaling through adenylyl cyclase, evidence now exists for distinct carboxyl- or C-terminal PTH receptors, which confer antagonistic or hypocalcemic effects that oppose some of the actions of intact PTH or NH2-terminal fragments, with potential clinical and pharmacological implications [3,6]. While other types of N-terminal PTH receptors (non-PTH1R) and nonclassical actions of PTH have been described, they are beyond the scope of this mini-review and will not be discussed here. Interested readers may refer to other recent publications [3,36]. Biology of PTH Hypocalcemia stimulates PTH secretion into the circulation through intracellular signals from the calcium-sensing receptor embedded in the plasma membrane of the parathyroid glandular cells. Even small fluctuations in extracellular ionized calcium can generate intracellular signals via the G-protein complex that inhibit PTH release when blood calcium rises [7]. PTH is synthesized by the chief cells of the parathyroid gland as a pre-pro-peptide of 115 amino acids, but the signal fragment (25 amino acids) is cleaved as the nascent peptide enters the lumen of the endoplasmic reticulum. The resultant pro-peptide is subjected to further proteolysis (loss of another 6 amino acids) as it passes through the endoplasmic reticulum and matures in the secretory vesicles. There are generally two forms of secretory vesicles in parathyroid cells: one contains the active, full-length 84-amino acid PTH or PTH 1–84, while the other type contains both PTH 1–84 and proteases cathepsins B and H. Cathepsin B cleaves PTH 1–84 to PTH 37–84 and a mixture of NH2-truncated fragments that are released under the control of calcium-sensing receptor in response to elevated blood calcium [8,37]. This occurs in conjunction with the inhibition of the secretion of PTH 1–84 from the other type of secretory vesicle, resulting in a reduction of circulatory PTH 1–84 and a concomitant increase in C-terminal fragment production. PTH 1–84 has a very short half-life in the circulation (t1/2 ∼3 to 8 min) [9]. It is mostly degraded by the liver, generating a mixture of N-terminal, mid-molecule and C-terminal fragments. Unlike C-terminal fragments, Kupffer cell-generated N-terminal fragments are degraded rapidly. Since N-terminal PTH fragments are not produced and accumulated in any appreciable amount from parathyroid tissues, most (up to 88%) plasma PTH bioactivity resides with intact PTH 1–84 [10,37]. Renal clearance is also important in PTH metabolism, and relatively small reductions in renal function may be accompanied by significant increases in both the intact hormone and its fragments. Al-
though it was once thought that PTH 1–84 was the only biologically active peptide secreted by the parathyroid glands, it is now apparent that some long C-terminal fragments e.g., PTH 7– 84 have potentially significant inhibitory properties [11,12], presumably through specific C-terminal PTH receptor actions mentioned above. Nevertheless, removal of one or more amino acids from the amino-terminus of PTH 1–84 renders the hormone less active, and large NH2-terminal truncated fragments (e.g., PTHs 7–84, 19–34, 37–84) are largely inactive as PTH1R agonists. Measurements of PTH Initially, diagnostic accuracy of PTH immunoassays was limited by the cross-detection of inactive C-terminal fragments. So-called ‘mid-molecule assays’ (first-generation) were supplemented by more expensive NH2-terminal assays that tended to yield more accurate estimates of biologically active PTH, especially in patients with renal failure. Thus, two-site assays (second generation) were a significant advance and remain the methods most commonly used today [13]. Although it was initially thought that the detection antibody in the two-site assays targeted the key NH2-terminal amino acids (PTH 1–5), it became clear that the PTH fragments containing amino acids between 7 and 84 were also immunoreactive and the epitopes have been localized to the stretch of residues between amino acid 7 and 34 [14]. Subsequently, assays that target the whole PTH 1–84 peptide (‘bio-intact PTH’, see Fig. 1) have been developed and made available commercially [15]. However, under physiological conditions and in chronic renal disease, the ratio of PTH 7–84 fragment and whole PTH 1–84 is roughly constant [16], although whole PTH 1–84 has been argued to provide better sensitivity for detecting primary hyperparathyroidism [17]. DOQI Guidelines from the National Kidney Foundation in the United States indicate that the advantages of these third-generation assays are insufficient to warrant wholesale replacement of the second-generation ones for the purpose of monitoring chronic kidney disease [18]. Although the results from second- and third-generation PTH assays generally correlate well and their normal reference ranges are reasonably comparable, the overall differences do not allow these results to be fully transferable numerically [19]. In Ontario, this problem is compounded by the use of different units of measurement. There are obvious reasons for adoption of molar units whenever possible, but this ideal is difficult or impossible to implement when there is microheterogeneity of the PTH molecular species. Currently, it is considered acceptable to use the calculated MW for PTH 1–84 in second-generation assays, so that the conversion of mass units in nanograms per liter (ng/L), or picograms per milliliter (pg/ mL), to molar units (pmol/L) is a single factor of about 0.105. As yet, there is no standardization sufficiently reliable to harmonize results among methods. By way of example, a recent comparison of 15 different assays (13 second- and third-generation assays) found that the median bias ranged from − 44% to +123%, using the Nichols Allegro® intact-PTH assay as the reference [20]. Thus, clinical laboratories are dependent on
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Fig. 1. Two-site (2nd and 3rd generation) PTH assays. The numbers refer to the amino acid residues of PTH 1–84.
manufacturers' stated reference intervals unless they are willing to undertake the laborious task of setting their own [21]. But then, analytically comparable second-generation assays such as the Beckman Access® and Roche Elecsys® (slope = 0.984, intercept = − 1.25, N = 50, r = 0.996, unpublished personal observation) do not necessarily report comparable reference intervals: 1.6–6.9 (Elecsys) versus 1.6–9.3 pmol/L (Access). Perhaps it should not be surprising that clinicians question the consistency of national guidelines that rely on method-dependent serum PTH cut-offs for clinical decisions [22]. There are other issues to bear in mind when considering reference intervals for second- and third-generation assays. Reference intervals offered by manufacturers are usually based on young and middle-aged adults, but upper and lower PTH limits differ for certain patient groups. In the elderly, there is an agedependent rise in PTH concentration that correlates roughly with the physiological decline in renal function. In pregnancy, there is a physiological decline of maternal serum PTH in the first trimester followed by a rise toward normal adult values at term. In the fetus, the parathyroid gland is suppressed. As a result, there is a postnatal delay in glandular responsiveness that can manifest as short-lived and mild physiological hypocalcemia in full-term newborns, but a more prolonged hypocalcemic phase in premature infants [23]. An equally important issue with reference intervals for PTH is that of interpretation. While it is safe to say that serum PTH concentrations outside the quoted reference intervals require some sort of follow-up, it is not true that a PTH result within the reference interval rules out a parathyroid gland abnormality [24]. In a patient with symptomatic hypocalcemia, a so-called “normal” PTH value could suggest a lack of parathyroid gland reserve and might be an early indicator of functional hypoparathyroidism. Similarly, serum PTH may be within the reference intervals in some cases of primary hyperparathyroidism, perhaps because of various inhibitory fragments being released by the abnormal gland(s). Consequently, it is the failure of suppression of PTH (to below normal) in the face of hypocalcemia that should alert the clinician to the diagnosis [25]. In summary, PTH results should ideally be interpreted in conjunction with matching values for serum calcium, phosphate and, if indicated, urinary calcium excretion. A province-wide survey of PTH assays In April 2006, the Endocrinology EQA Survey distributed by Quality Management Program-Laboratory Services (QMP-LS),
Ontario Medical Association (http://www.qmpls.org/) included parathyroid hormone. This was the second time that laboratory performance for this analyte was surveyed in Ontario. The survey material consisted of three vials of lyophilized serum purchased from a commercial supplier. There were no target values assigned to the vials. Of the twenty-one laboratories that participated, the most common assays used were those marketed by Roche™ for the Elecsys® and Modular® instruments (Table 1). The Immulite® and Immulite 2000® platforms from Diagnostic Products Corporation (DPC) were represented by multiple users while methods based on the Bayer Advia Centaur® system and the DiaSorin N-tact® immunoradiometric assay were each represented by one laboratory. For all three vials, there was significant variability among assays (Fig. 2). Most evident and statistically significant was the upward bias of the DPC-based assays relative to the Roche systems. Using non-parametric comparisons (Mann–Whitney test), the difference was statistically significant (p < 0.001) for each of the three vials. With only single laboratories using Bayer and DiaSorin methods, further analysis was not possible. In Vial A, the all methods' median (dotted line) was 12.7 pmol/ L, but the median for the Roche assay was 11% below and the DPC assay 42% above this level. The lowest reported value (9.6 pmol/L) was close to the upper reference limit, while the highest value (22.9 pmol/L) was 2.4-fold higher. However, all laboratories would have identified the sample as falling above the manufacturers' upper reference limit. For vial B, the all methods' median (3.90 pmol/L) was clearly in the middle of the reference intervals, and all laboratories reported a value that fell within their own manufacturer's interval. However, interpretation of the lowest value (2.2 pmol/ Table 1 PTH assays assessed in the ENDO-0604 QMP-LS Survey
1. 2. 3. 4. 5. 6.
Platform (manufacturer)
Method
Reference interval (pmol/L)
No. of laboratories
BMC Elecsys (Roche) Modular (Roche) Immulite 2000 (DPC) Immulite (DPC) Advia Centaur (Bayer) N-tact PTH (DiaSorin)
Electrochemiluminescence
1.6–6.9
7
Electrochemiluminescence Chemiluminescence
1.4–7.6 1.0–7.0
5 6
Chemiluminescence Immunochemiluminometry
1.1–7.3 1.2–8.5
1 1
Immunoradiometry
1.2–4.9
1
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to those reported from the QMP-LS or the UK NEQAS EQA scheme. Analytically, it would be of considerable interest to have EQA survey samples characterized for their respective proportion of PTH 1–84 and other fragments. The UK NEQAS has reported significant method biases (> 50%) between highest and lowest reporting methods based on the recovery of added synthetic human PTH 1–84 [26], indicating a pressing need for assay standardization even in terms of PTH 1–84 alone. The presence of variable amounts of different PTH fragments and their largely undefined effects (cross-reactivity) on different methods will continue to be the sources of discordance among PTH assays. Utilization—an area for future continuing quality initiative (CQI)
Fig. 2. Results of a recent province-wide survey of EQA samples, assayed by different methods and platforms. Solid lines represent method-group medians while dotted lines represent all-method medians.
L) with a calcium concentration at or slightly above the upper reference limit (a common occurrence) might be quite different from the interpretation of the highest value (6.2 pmol/L). For Vial C, the all methods' median (10.7 pmol/L) was above the upper reference limits stated by the manufacturers (see Table 1), and all laboratories reported values that would be interpreted as elevated. The between-laboratory imprecision was about the same as for vial A, suggesting a relatively smaller variation among different assays at or near normal PTH concentrations than at the extremes. At a future date, it would be interesting to assess assay performance with a sample from a stage 5 (end-stage) chronic kidney disease patient, where the “expected PTH” is 16.5– 33 pmol/L (150–300 ng/L). In this group of patients, management of progressive secondary hyperparathyroidism and renal osteodystrophy can be difficult, and any improvement in assay precision or accuracy will have a material effect on patient well-being. By way of comparison, the College of American Pathologists (CAP) EQA Program typically surveys with samples in the 20+ and 40+ pmol/L (200+ and 400+ ng/L) range and routinely reports a ∼ 1.8-fold difference between the highest and the lowest reporting method-groups. The imprecision (CV) within each method-group varies from 5 to 9% and is quite comparable
For many outpatient laboratories and small hospital laboratory facilities, serum PTH is used primarily as a diagnostic test to determine the cause of abnormal serum calcium values or a disorder of skeletal architecture or metabolism. With the steady growth of effective treatments for primary osteoporosis, exclusion of bone disease due to primary hyperparathyroidism and monitoring of secondary hyperparathyroidism in vitamin D insufficiency will be increasingly important. As the population ages, this will be a source of increasing serum PTH testing. In a small number of patients who are surgically hypoparathyroid or already diagnosed with a parathyroid gland abnormality, serum PTH will be a test used on a regular basis to monitor patient health. In such cases, the clinician will be tempted to compare results over time, but rarely is the laboratory called upon to suggest what the least significant difference in consecutive test results may be. As with other tests which are monitored over time (e.g. PSA), it is essential that the laboratories inform clinicians when a change in assay performance occurs. This is another area for future study and improvement. In tertiary care hospitals, it is the management of renal diseases that will remain the greatest source of demand for routine PTH testing. Although management of renal osteodystrophy continues to improve, and current guidelines are revised and refined in the coming years, there is no obvious alternative to PTH screening in patients with progressive renal failure, and the number of such patients will increase, in part because of the aging demographic and the accelerating epidemic of diabetes with its nephropathic complications. Of similar concern to laboratories is the increasing demand for PTH assay results to follow patients through surgical procedures aimed at removing the thyroid or parathyroid glands. There are essentially three types of assays: (i) intraoperative PTH to localize specific parathyroid glands; (ii) intraoperative PTH to ensure removal of all parathyroid glands; and (iii) post-operative PTH to assess risk of symptomatic hypocalcemia. With continued improvements in neck ultrasonography and radionuclide scans (Sestamibi), monitoring by intraoperative PTH has become less crucial, if not redundant [27,38]. Nevertheless, there are some surgeons who consider that the report of a decreased peripheral PTH is an important consideration in their decision wheteher or not to stop the search
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for parathyroid glands or tumors and helps ensure operative success [28]. Intraoperative PTH testing dates back to 1994 and has become relatively widely available in the United States and well established in a few Canadian centers. Several manufacturers provide an automated “rapid” assay that relies on a kinetic endpoint, but most of these procedures still require at least 15 min from sample insertion to result reporting. A survey conducted under the auspices of the College of American Pathologists found that nearly threequarters of all responding laboratories opt to transfer the sample to the central laboratory rather than offering point-ofcare testing [29]. High cost and relatively poor analytical performance (typical imprecision or CV of ∼ 14% according to CAP Surveys) are main reasons. While enthusiasm about intraoperative PTH testing remains [35,39], unbiased prospective studies showing improved outcomes are hard to come by, and derived clinical benefits, if any, are marginal at best. In a series of 35 consecutive renal failure patients requiring total parathyroidectomy, Kaczirek et al. [30] regarded the frequency of false negatives in the serum PTH measured 15 min after removal of the last gland as unacceptable. Third-generation assays may prove to be better for this type of program because of shorter half-life of whole PTH 1–84 [9], but the current standard of laboratory service should probably include a serum PTH available the morning after surgery [31]. There are other reasons to measure PTH in the immediate post-operative period. The most common metabolic complication of a total thyroidectomy is symptomatic hypocalcemia due to transient suppression (rarely permanent loss) of parathyroid gland function. In a prospective series of 40 total or complete thyroidectomies, Lam and Kerr [32] found that a serum PTH of less than 0.7 pmol/L (8 ng/L) measured 1 h post-operatively was a strong predictor for clinically significant hypocalcemia occurring 18 to 42 h later. Similarly, a rapid post-operative PTH result less than 1.1 pmol/L (12 ng/L) has been shown to be both more sensitive and specific in predicting symptomatic hypocalcemia following thyroidectomy than an equivalent intraoperative PTH value [33]. With ever-increasing pressure to reduce post-operative length-of-stay, the availability of a predictor for rapid asymptomatic recovery should be attractive to hospital administrators and would offset the extra effort required to offer a “STAT” service — that is, a serum PTH result with same-day turnaround time. Conclusions Despite continuing changes in methodology and the persistence of sizable method-to-method variations, Ontario laboratory performance for serum PTH appears to be acceptable for primary diagnosis of calcium and parathyroid disorders. Increasing use of specific serum PTH values for therapeutic intervention in progressive renal failure remains a challenge however, and laboratory performance of PTH for such indications remains untested. Although intraoperative PTH assays are unlikely to be widely used in Ontario, there will be increasing
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requests for within-day turnaround of PTH measurements after parathyroidectomy to assess the success of surgery, and after thyroidectomy, to assess the recovery of parathyroid function. References [1] Berson SA, Yalow RS, Aurbach GD, Potts JT. Immunoassay of bovine and human parathyroid hormone. Proc Natl Acad Sci U S A 1963;49:613–7. [2] Potts JT. Parathyroid hormone: past and present. J Endocrinol 2005;187: 311–25. [3] Murray TM, Rao LG, Divieti P, Bringhurst FR. Parathyroid hormone secretion and action: evidence for discrete receptors for the carboxyterminal region and related biological actions of carboxyl-terminal ligands. Endocr Rev 2005;26:78–113. [4] Rosenblatt M, Segre GV, Tregear GW, Shepard GL, Tyler GA, Potts Jr JT. Human parathyroid hormone: synthesis and chemical, biological and immunological evaluation of the carboxyl-terminal region. Endocrinology 1978;103:978–84. [5] Abou-Samra AB, Juppner H, Force T, et al. Expression cloning of a common receptor for parathyroid hormone and parathyroid hormonerelated peptide from rat osteoblast-like cells: a single receptor stimulates intracellular accumulation of both cAMP and inositol triphosphates and increases intracellular free calcium. Proc Natl Acad Sci U S A 1992;89: 2732–6. [6] Slatopolsky E, Finch J, Clay P, et al. A novel mechanism for skeletal resistance in uremia. Kidney Int 2000;58:753–61. [7] Thompson MD, Burnham WM, Cole DEC. The G-protein coupled receptors: pharmacogenetics and disease. Crit Rev Clin Lab Sci 2005;42: 311–89. [8] Hashizume Y, Waguri S, Watanabe T, Kominami E, Uchiyama Y. Cysteine proteinases in rat parathyroid cells with special reference to their correlation with parathyroid (PTH) in storage granules. J Histochem Cytochem 1993;41:273–82. [9] Bieglmayer C, Kaczirek K, Prager G, Niederle B. Parathyroid hormone monitoring during total parathyroidectomy for renal hyperparathyroidism: pilot study of the impact of renal function and assay specificity. Clin Chem 2006;52:1112–9. [10] MacGregor RR, Jilka RL, Hamilton JW. Formation and secretion of fragments of parathyroid hormone. Identification of cleavage sites. J Biol Chem 1986;261:1929–34. [11] D'Amour P, Brossard JH. Carboxyl-terminal parathyroid hormone fragments: role in parathyroid hormone physiopathology. Curr Opin Nephrol Hypertens 2005;14:330–6. [12] Friedman PA, Goodman WG. PTH(1–84)/PTH(7–84): a balance of power. Am J Physiol: Renal Physiol 2006;290:F975–84. [13] Juppner H, Potts Jr JT. Immunoassays for the detection of parathyroid hormone. J Bone Miner Res 2002;17(Suppl 2):N81–6. [14] Lepage R, Roy L, Brossard JH, 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: 805–9. [15] Goodman WG. The evolution of assays for parathyroid hormone. Semin Dial 2005;18:296–301. [16] Chang JM, Lin SP, Kuo HT, et al. 7–84 Parathyroid hormone fragments are proportionally increased with the severity of uremic hyperparathyroidism. Clin Nephrol 2005;63:351–5. [17] Gao P, Scheibel S, Amour D'P, John MR, Rao SD, Schmidt-Gayk H, Cantor TL. 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(4):605–14. [18] National Kidney Foundation. K/DOQI clinical practice guidelines for bone metabolism and disease in chronic kidney disease. Am J Kidney Dis 2003;42(4 Suppl 3):S1–201. [19] Ljungdahl N, Haarhaus M, Linder C, Magnusson P. Comparison of 3 thirdgeneration assays for bio-intact parathyroid hormone. Clin Chem 2006; 52:903–4.
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Review
Update on parathyroid hormone: New tests and new challenges for external quality assessment David E.C. Cole a,b,c,d , Sharon Webb e , Pak-Cheung Chan a,b,⁎ a
e
Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada b Department of Clinical Pathology, Sunnybrook Health Sciences Center, Toronto, ON, Canada c Department of Medicine, Ontario Medical Association, Toronto, ON, Canada d Department of Pediatrics, University of Toronto, Ontario Medical Association, Toronto, ON, Canada Quality Management Program–Laboratory Services, Ontario Medical Association, Toronto, ON, Canada Received 3 November 2006; received in revised form 16 March 2007; accepted 18 March 2007 Available online 6 April 2007
Abstract It is now 43 years since Berson and Yalow published the first radio-immunoassay (RIA) for parathyroid hormone (PTH) [S.A. Berson, R.S. Yalow, G.D. Aurbach, J.T. Potts, Immunoassay of bovine and human parathyroid hormone. Proc Natl Acad Sci U S A 49 (1963) 613–617] [1]. Since then, there have been marked advances in our understanding of this peptide hormone, its mechanism of action and biological regulation [J.T. Potts, Parathyroid hormone: past and present. J. Endocrinol. 187 (2005) 311–325] [2]. PTH has become a routine assay in tertiary care hospitals and is an essential element in the management of chronic kidney disease, parathyroid disorders and the investigation of abnormalities in calcium homeostasis. Despite continuing technological advances in PTH measurement, analyte heterogeneity remains a problem, while improved turnaround time and better precision are constantly escalating clinical demands. This mini-review begins with a brief update of current knowledge on PTH, followed by a summary of a recent Ontario-wide External Quality Assurance (EQA) survey, and concludes with comments on utilization trends, current and future. © 2007 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved. Keywords: Parathyroid hormone (PTH); Calcium homeostasis; Utilization
Contents Introduction . . . . . . . . . . . . . . . . Biology of PTH. . . . . . . . . . . . . . Measurements of PTH . . . . . . . . . . A province-wide survey of PTH assays . Utilization—an area for future continuing Conclusions . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . .
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Introduction Parathyroid hormone (PTH) is a polypeptide hormone that plays a central role in the maintenance of calcium and phosphate ⁎ Corresponding author. Rm B204, Sunnybrook Health Sciences Center, 2075 Bayview Avenue, Toronto ON, Canada M4N 3M5. Fax: +1 416 4806120. E-mail address: [email protected] (P.-C. Chan).
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homeostasis and bone health. It acts to maintain bone and mineral homeostasis largely through four mechanisms: (i) elevation of blood calcium by increasing osteoclastic bone resorption, (ii) enhancement of renal calcium reabsorption, (iii) stimulation of renal 1,25-dihydroxyvitamin D synthesis, leading to increased intestinal calcium absorption, and (iv) promotion of phosphaturia via inhibition of renal tubular transepithelial phosphate reabsorption [3]. These biologic responses are down-
0009-9120/$ - see front matter © 2007 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved. doi:10.1016/j.clinbiochem.2007.03.019
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stream actions following the binding of the NH2-terminal portion of the mature PTH molecule or fragments to a common PTH/PTH related protein (PTHrP) receptor, PTH1R, on the plasma membranes of target tissue cells [3–5]. Signals of PTH1R activation following ligand binding may be transduced via either the classic pathway involving G-protein (Gαq/11), adenylyl cyclase and phospholipase C or the non-classic pathway involving G-protein (GαS), Erk, phospholipase A2 and phospholipase D under the influence of a Na+/H+ exchange regulatory factor (NHERF1) in a cell specific manner [34]. While the first two amino acid residues of PTH are required for PTH1R signaling through adenylyl cyclase, evidence now exists for distinct carboxyl- or C-terminal PTH receptors, which confer antagonistic or hypocalcemic effects that oppose some of the actions of intact PTH or NH2-terminal fragments, with potential clinical and pharmacological implications [3,6]. While other types of N-terminal PTH receptors (non-PTH1R) and nonclassical actions of PTH have been described, they are beyond the scope of this mini-review and will not be discussed here. Interested readers may refer to other recent publications [3,36]. Biology of PTH Hypocalcemia stimulates PTH secretion into the circulation through intracellular signals from the calcium-sensing receptor embedded in the plasma membrane of the parathyroid glandular cells. Even small fluctuations in extracellular ionized calcium can generate intracellular signals via the G-protein complex that inhibit PTH release when blood calcium rises [7]. PTH is synthesized by the chief cells of the parathyroid gland as a pre-pro-peptide of 115 amino acids, but the signal fragment (25 amino acids) is cleaved as the nascent peptide enters the lumen of the endoplasmic reticulum. The resultant pro-peptide is subjected to further proteolysis (loss of another 6 amino acids) as it passes through the endoplasmic reticulum and matures in the secretory vesicles. There are generally two forms of secretory vesicles in parathyroid cells: one contains the active, full-length 84-amino acid PTH or PTH 1–84, while the other type contains both PTH 1–84 and proteases cathepsins B and H. Cathepsin B cleaves PTH 1–84 to PTH 37–84 and a mixture of NH2-truncated fragments that are released under the control of calcium-sensing receptor in response to elevated blood calcium [8,37]. This occurs in conjunction with the inhibition of the secretion of PTH 1–84 from the other type of secretory vesicle, resulting in a reduction of circulatory PTH 1–84 and a concomitant increase in C-terminal fragment production. PTH 1–84 has a very short half-life in the circulation (t1/2 ∼3 to 8 min) [9]. It is mostly degraded by the liver, generating a mixture of N-terminal, mid-molecule and C-terminal fragments. Unlike C-terminal fragments, Kupffer cell-generated N-terminal fragments are degraded rapidly. Since N-terminal PTH fragments are not produced and accumulated in any appreciable amount from parathyroid tissues, most (up to 88%) plasma PTH bioactivity resides with intact PTH 1–84 [10,37]. Renal clearance is also important in PTH metabolism, and relatively small reductions in renal function may be accompanied by significant increases in both the intact hormone and its fragments. Al-
though it was once thought that PTH 1–84 was the only biologically active peptide secreted by the parathyroid glands, it is now apparent that some long C-terminal fragments e.g., PTH 7– 84 have potentially significant inhibitory properties [11,12], presumably through specific C-terminal PTH receptor actions mentioned above. Nevertheless, removal of one or more amino acids from the amino-terminus of PTH 1–84 renders the hormone less active, and large NH2-terminal truncated fragments (e.g., PTHs 7–84, 19–34, 37–84) are largely inactive as PTH1R agonists. Measurements of PTH Initially, diagnostic accuracy of PTH immunoassays was limited by the cross-detection of inactive C-terminal fragments. So-called ‘mid-molecule assays’ (first-generation) were supplemented by more expensive NH2-terminal assays that tended to yield more accurate estimates of biologically active PTH, especially in patients with renal failure. Thus, two-site assays (second generation) were a significant advance and remain the methods most commonly used today [13]. Although it was initially thought that the detection antibody in the two-site assays targeted the key NH2-terminal amino acids (PTH 1–5), it became clear that the PTH fragments containing amino acids between 7 and 84 were also immunoreactive and the epitopes have been localized to the stretch of residues between amino acid 7 and 34 [14]. Subsequently, assays that target the whole PTH 1–84 peptide (‘bio-intact PTH’, see Fig. 1) have been developed and made available commercially [15]. However, under physiological conditions and in chronic renal disease, the ratio of PTH 7–84 fragment and whole PTH 1–84 is roughly constant [16], although whole PTH 1–84 has been argued to provide better sensitivity for detecting primary hyperparathyroidism [17]. DOQI Guidelines from the National Kidney Foundation in the United States indicate that the advantages of these third-generation assays are insufficient to warrant wholesale replacement of the second-generation ones for the purpose of monitoring chronic kidney disease [18]. Although the results from second- and third-generation PTH assays generally correlate well and their normal reference ranges are reasonably comparable, the overall differences do not allow these results to be fully transferable numerically [19]. In Ontario, this problem is compounded by the use of different units of measurement. There are obvious reasons for adoption of molar units whenever possible, but this ideal is difficult or impossible to implement when there is microheterogeneity of the PTH molecular species. Currently, it is considered acceptable to use the calculated MW for PTH 1–84 in second-generation assays, so that the conversion of mass units in nanograms per liter (ng/L), or picograms per milliliter (pg/ mL), to molar units (pmol/L) is a single factor of about 0.105. As yet, there is no standardization sufficiently reliable to harmonize results among methods. By way of example, a recent comparison of 15 different assays (13 second- and third-generation assays) found that the median bias ranged from − 44% to +123%, using the Nichols Allegro® intact-PTH assay as the reference [20]. Thus, clinical laboratories are dependent on
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Fig. 1. Two-site (2nd and 3rd generation) PTH assays. The numbers refer to the amino acid residues of PTH 1–84.
manufacturers' stated reference intervals unless they are willing to undertake the laborious task of setting their own [21]. But then, analytically comparable second-generation assays such as the Beckman Access® and Roche Elecsys® (slope = 0.984, intercept = − 1.25, N = 50, r = 0.996, unpublished personal observation) do not necessarily report comparable reference intervals: 1.6–6.9 (Elecsys) versus 1.6–9.3 pmol/L (Access). Perhaps it should not be surprising that clinicians question the consistency of national guidelines that rely on method-dependent serum PTH cut-offs for clinical decisions [22]. There are other issues to bear in mind when considering reference intervals for second- and third-generation assays. Reference intervals offered by manufacturers are usually based on young and middle-aged adults, but upper and lower PTH limits differ for certain patient groups. In the elderly, there is an agedependent rise in PTH concentration that correlates roughly with the physiological decline in renal function. In pregnancy, there is a physiological decline of maternal serum PTH in the first trimester followed by a rise toward normal adult values at term. In the fetus, the parathyroid gland is suppressed. As a result, there is a postnatal delay in glandular responsiveness that can manifest as short-lived and mild physiological hypocalcemia in full-term newborns, but a more prolonged hypocalcemic phase in premature infants [23]. An equally important issue with reference intervals for PTH is that of interpretation. While it is safe to say that serum PTH concentrations outside the quoted reference intervals require some sort of follow-up, it is not true that a PTH result within the reference interval rules out a parathyroid gland abnormality [24]. In a patient with symptomatic hypocalcemia, a so-called “normal” PTH value could suggest a lack of parathyroid gland reserve and might be an early indicator of functional hypoparathyroidism. Similarly, serum PTH may be within the reference intervals in some cases of primary hyperparathyroidism, perhaps because of various inhibitory fragments being released by the abnormal gland(s). Consequently, it is the failure of suppression of PTH (to below normal) in the face of hypocalcemia that should alert the clinician to the diagnosis [25]. In summary, PTH results should ideally be interpreted in conjunction with matching values for serum calcium, phosphate and, if indicated, urinary calcium excretion. A province-wide survey of PTH assays In April 2006, the Endocrinology EQA Survey distributed by Quality Management Program-Laboratory Services (QMP-LS),
Ontario Medical Association (http://www.qmpls.org/) included parathyroid hormone. This was the second time that laboratory performance for this analyte was surveyed in Ontario. The survey material consisted of three vials of lyophilized serum purchased from a commercial supplier. There were no target values assigned to the vials. Of the twenty-one laboratories that participated, the most common assays used were those marketed by Roche™ for the Elecsys® and Modular® instruments (Table 1). The Immulite® and Immulite 2000® platforms from Diagnostic Products Corporation (DPC) were represented by multiple users while methods based on the Bayer Advia Centaur® system and the DiaSorin N-tact® immunoradiometric assay were each represented by one laboratory. For all three vials, there was significant variability among assays (Fig. 2). Most evident and statistically significant was the upward bias of the DPC-based assays relative to the Roche systems. Using non-parametric comparisons (Mann–Whitney test), the difference was statistically significant (p < 0.001) for each of the three vials. With only single laboratories using Bayer and DiaSorin methods, further analysis was not possible. In Vial A, the all methods' median (dotted line) was 12.7 pmol/ L, but the median for the Roche assay was 11% below and the DPC assay 42% above this level. The lowest reported value (9.6 pmol/L) was close to the upper reference limit, while the highest value (22.9 pmol/L) was 2.4-fold higher. However, all laboratories would have identified the sample as falling above the manufacturers' upper reference limit. For vial B, the all methods' median (3.90 pmol/L) was clearly in the middle of the reference intervals, and all laboratories reported a value that fell within their own manufacturer's interval. However, interpretation of the lowest value (2.2 pmol/ Table 1 PTH assays assessed in the ENDO-0604 QMP-LS Survey
1. 2. 3. 4. 5. 6.
Platform (manufacturer)
Method
Reference interval (pmol/L)
No. of laboratories
BMC Elecsys (Roche) Modular (Roche) Immulite 2000 (DPC) Immulite (DPC) Advia Centaur (Bayer) N-tact PTH (DiaSorin)
Electrochemiluminescence
1.6–6.9
7
Electrochemiluminescence Chemiluminescence
1.4–7.6 1.0–7.0
5 6
Chemiluminescence Immunochemiluminometry
1.1–7.3 1.2–8.5
1 1
Immunoradiometry
1.2–4.9
1
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to those reported from the QMP-LS or the UK NEQAS EQA scheme. Analytically, it would be of considerable interest to have EQA survey samples characterized for their respective proportion of PTH 1–84 and other fragments. The UK NEQAS has reported significant method biases (> 50%) between highest and lowest reporting methods based on the recovery of added synthetic human PTH 1–84 [26], indicating a pressing need for assay standardization even in terms of PTH 1–84 alone. The presence of variable amounts of different PTH fragments and their largely undefined effects (cross-reactivity) on different methods will continue to be the sources of discordance among PTH assays. Utilization—an area for future continuing quality initiative (CQI)
Fig. 2. Results of a recent province-wide survey of EQA samples, assayed by different methods and platforms. Solid lines represent method-group medians while dotted lines represent all-method medians.
L) with a calcium concentration at or slightly above the upper reference limit (a common occurrence) might be quite different from the interpretation of the highest value (6.2 pmol/L). For Vial C, the all methods' median (10.7 pmol/L) was above the upper reference limits stated by the manufacturers (see Table 1), and all laboratories reported values that would be interpreted as elevated. The between-laboratory imprecision was about the same as for vial A, suggesting a relatively smaller variation among different assays at or near normal PTH concentrations than at the extremes. At a future date, it would be interesting to assess assay performance with a sample from a stage 5 (end-stage) chronic kidney disease patient, where the “expected PTH” is 16.5– 33 pmol/L (150–300 ng/L). In this group of patients, management of progressive secondary hyperparathyroidism and renal osteodystrophy can be difficult, and any improvement in assay precision or accuracy will have a material effect on patient well-being. By way of comparison, the College of American Pathologists (CAP) EQA Program typically surveys with samples in the 20+ and 40+ pmol/L (200+ and 400+ ng/L) range and routinely reports a ∼ 1.8-fold difference between the highest and the lowest reporting method-groups. The imprecision (CV) within each method-group varies from 5 to 9% and is quite comparable
For many outpatient laboratories and small hospital laboratory facilities, serum PTH is used primarily as a diagnostic test to determine the cause of abnormal serum calcium values or a disorder of skeletal architecture or metabolism. With the steady growth of effective treatments for primary osteoporosis, exclusion of bone disease due to primary hyperparathyroidism and monitoring of secondary hyperparathyroidism in vitamin D insufficiency will be increasingly important. As the population ages, this will be a source of increasing serum PTH testing. In a small number of patients who are surgically hypoparathyroid or already diagnosed with a parathyroid gland abnormality, serum PTH will be a test used on a regular basis to monitor patient health. In such cases, the clinician will be tempted to compare results over time, but rarely is the laboratory called upon to suggest what the least significant difference in consecutive test results may be. As with other tests which are monitored over time (e.g. PSA), it is essential that the laboratories inform clinicians when a change in assay performance occurs. This is another area for future study and improvement. In tertiary care hospitals, it is the management of renal diseases that will remain the greatest source of demand for routine PTH testing. Although management of renal osteodystrophy continues to improve, and current guidelines are revised and refined in the coming years, there is no obvious alternative to PTH screening in patients with progressive renal failure, and the number of such patients will increase, in part because of the aging demographic and the accelerating epidemic of diabetes with its nephropathic complications. Of similar concern to laboratories is the increasing demand for PTH assay results to follow patients through surgical procedures aimed at removing the thyroid or parathyroid glands. There are essentially three types of assays: (i) intraoperative PTH to localize specific parathyroid glands; (ii) intraoperative PTH to ensure removal of all parathyroid glands; and (iii) post-operative PTH to assess risk of symptomatic hypocalcemia. With continued improvements in neck ultrasonography and radionuclide scans (Sestamibi), monitoring by intraoperative PTH has become less crucial, if not redundant [27,38]. Nevertheless, there are some surgeons who consider that the report of a decreased peripheral PTH is an important consideration in their decision wheteher or not to stop the search
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for parathyroid glands or tumors and helps ensure operative success [28]. Intraoperative PTH testing dates back to 1994 and has become relatively widely available in the United States and well established in a few Canadian centers. Several manufacturers provide an automated “rapid” assay that relies on a kinetic endpoint, but most of these procedures still require at least 15 min from sample insertion to result reporting. A survey conducted under the auspices of the College of American Pathologists found that nearly threequarters of all responding laboratories opt to transfer the sample to the central laboratory rather than offering point-ofcare testing [29]. High cost and relatively poor analytical performance (typical imprecision or CV of ∼ 14% according to CAP Surveys) are main reasons. While enthusiasm about intraoperative PTH testing remains [35,39], unbiased prospective studies showing improved outcomes are hard to come by, and derived clinical benefits, if any, are marginal at best. In a series of 35 consecutive renal failure patients requiring total parathyroidectomy, Kaczirek et al. [30] regarded the frequency of false negatives in the serum PTH measured 15 min after removal of the last gland as unacceptable. Third-generation assays may prove to be better for this type of program because of shorter half-life of whole PTH 1–84 [9], but the current standard of laboratory service should probably include a serum PTH available the morning after surgery [31]. There are other reasons to measure PTH in the immediate post-operative period. The most common metabolic complication of a total thyroidectomy is symptomatic hypocalcemia due to transient suppression (rarely permanent loss) of parathyroid gland function. In a prospective series of 40 total or complete thyroidectomies, Lam and Kerr [32] found that a serum PTH of less than 0.7 pmol/L (8 ng/L) measured 1 h post-operatively was a strong predictor for clinically significant hypocalcemia occurring 18 to 42 h later. Similarly, a rapid post-operative PTH result less than 1.1 pmol/L (12 ng/L) has been shown to be both more sensitive and specific in predicting symptomatic hypocalcemia following thyroidectomy than an equivalent intraoperative PTH value [33]. With ever-increasing pressure to reduce post-operative length-of-stay, the availability of a predictor for rapid asymptomatic recovery should be attractive to hospital administrators and would offset the extra effort required to offer a “STAT” service — that is, a serum PTH result with same-day turnaround time. Conclusions Despite continuing changes in methodology and the persistence of sizable method-to-method variations, Ontario laboratory performance for serum PTH appears to be acceptable for primary diagnosis of calcium and parathyroid disorders. Increasing use of specific serum PTH values for therapeutic intervention in progressive renal failure remains a challenge however, and laboratory performance of PTH for such indications remains untested. Although intraoperative PTH assays are unlikely to be widely used in Ontario, there will be increasing
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requests for within-day turnaround of PTH measurements after parathyroidectomy to assess the success of surgery, and after thyroidectomy, to assess the recovery of parathyroid function. References [1] Berson SA, Yalow RS, Aurbach GD, Potts JT. Immunoassay of bovine and human parathyroid hormone. Proc Natl Acad Sci U S A 1963;49:613–7. [2] Potts JT. Parathyroid hormone: past and present. J Endocrinol 2005;187: 311–25. [3] Murray TM, Rao LG, Divieti P, Bringhurst FR. Parathyroid hormone secretion and action: evidence for discrete receptors for the carboxyterminal region and related biological actions of carboxyl-terminal ligands. Endocr Rev 2005;26:78–113. [4] Rosenblatt M, Segre GV, Tregear GW, Shepard GL, Tyler GA, Potts Jr JT. Human parathyroid hormone: synthesis and chemical, biological and immunological evaluation of the carboxyl-terminal region. Endocrinology 1978;103:978–84. [5] Abou-Samra AB, Juppner H, Force T, et al. Expression cloning of a common receptor for parathyroid hormone and parathyroid hormonerelated peptide from rat osteoblast-like cells: a single receptor stimulates intracellular accumulation of both cAMP and inositol triphosphates and increases intracellular free calcium. Proc Natl Acad Sci U S A 1992;89: 2732–6. [6] Slatopolsky E, Finch J, Clay P, et al. A novel mechanism for skeletal resistance in uremia. Kidney Int 2000;58:753–61. [7] Thompson MD, Burnham WM, Cole DEC. The G-protein coupled receptors: pharmacogenetics and disease. Crit Rev Clin Lab Sci 2005;42: 311–89. [8] Hashizume Y, Waguri S, Watanabe T, Kominami E, Uchiyama Y. Cysteine proteinases in rat parathyroid cells with special reference to their correlation with parathyroid (PTH) in storage granules. J Histochem Cytochem 1993;41:273–82. [9] Bieglmayer C, Kaczirek K, Prager G, Niederle B. Parathyroid hormone monitoring during total parathyroidectomy for renal hyperparathyroidism: pilot study of the impact of renal function and assay specificity. Clin Chem 2006;52:1112–9. [10] MacGregor RR, Jilka RL, Hamilton JW. Formation and secretion of fragments of parathyroid hormone. Identification of cleavage sites. J Biol Chem 1986;261:1929–34. [11] D'Amour P, Brossard JH. Carboxyl-terminal parathyroid hormone fragments: role in parathyroid hormone physiopathology. Curr Opin Nephrol Hypertens 2005;14:330–6. [12] Friedman PA, Goodman WG. PTH(1–84)/PTH(7–84): a balance of power. Am J Physiol: Renal Physiol 2006;290:F975–84. [13] Juppner H, Potts Jr JT. Immunoassays for the detection of parathyroid hormone. J Bone Miner Res 2002;17(Suppl 2):N81–6. [14] Lepage R, Roy L, Brossard JH, 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: 805–9. [15] Goodman WG. The evolution of assays for parathyroid hormone. Semin Dial 2005;18:296–301. [16] Chang JM, Lin SP, Kuo HT, et al. 7–84 Parathyroid hormone fragments are proportionally increased with the severity of uremic hyperparathyroidism. Clin Nephrol 2005;63:351–5. [17] Gao P, Scheibel S, Amour D'P, John MR, Rao SD, Schmidt-Gayk H, Cantor TL. 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(4):605–14. [18] National Kidney Foundation. K/DOQI clinical practice guidelines for bone metabolism and disease in chronic kidney disease. Am J Kidney Dis 2003;42(4 Suppl 3):S1–201. [19] Ljungdahl N, Haarhaus M, Linder C, Magnusson P. Comparison of 3 thirdgeneration assays for bio-intact parathyroid hormone. Clin Chem 2006; 52:903–4.
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