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Endocrinology and Metabolism |
1 Clinical Institute for Medical and Chemical Laboratory Diagnostics, and2 Section of Surgical Endocrinology, Division of General Surgery, General Hospital of the Medical University and City of Vienna, Vienna, Austria.
aAddress correspondence to this author at: Clinical Institute for Medical and Chemical Laboratory Diagnostics, EB05, General Hospital of the Medical University and City of Vienna, Waeringer Guertel 18-20, A 1090 Vienna, Austria. Fax 43-1-40400-6752; e-mail christian.bieglmayer{at}meduniwien.ac.at.
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Abstract |
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Methods: We collected blood samples in short time intervals from 35 consecutive surgical patients with rHPT. Three patients had to be excluded from further calculations; the remainder were grouped according to kidney function and postoperative outcome. All samples were analyzed with the 3 automated PTH assays, which have different specificities.
Results: Twenty minutes after removal of the last gland, the PTH(1–84) values decreased to within the reference intervals in all patients with total and subtotal resection; however, iPTH concentrations normalized in only one half of these patients. In patients with poor renal function, the half-life of PTH(1–84) was shorter than the half-lives obtained with the iPTH assays.
Conclusions: The accuracy of PTH monitoring during surgery for rHPT depends on renal function and assay specificity. All assays tested showed similar effectiveness in detecting missed glands, but the assay for PTH(1–84) gave more reliable results than the iPTH assays, which overestimated the concentration of PTH and hampered the intrasurgical diagnosis of resection sufficiency.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The management of patients with renal failure requires accurate and specific measurements of the bioactive PTH(1–84). Competitive (1st-generation) assays for N-terminal, C-terminal, and midregion fragments were limited by cross-reactivity, which was eliminated in 2nd-generation 2-site immunometric assays, which measured only intact PTH (iPTH) by use of 2 antibodies directed against the N- and C-terminal regions of PTH(1–84) (6).
In renal failure, iPTH is a predictive marker of different forms of renal osteodystrophy. Because of its short half-life, measurements of iPTH have become a standard tool for intraoperative analysis in minimally invasive surgery for primary hyperparathyroidism (HPT). Whereas primary HPT involves at least 1 adenomatoid gland, secondary (renal) hyperparathyroidism (rHPT) involves stimulation of all parathyroid tissues. To avoid adynamic bone disease, sufficient reduction in the amount of hyperfunctioning parathyroid tissue without causing a hypoparathyroid state can be achieved by subtotal (3
–) parathyroidectomy or by total parathyroidectomy (PTX) with immediate or delayed autotransplantation of fresh or cryopreserved parathyroid tissue.
The slower decrease in iPTH concentrations during the surgical treatment of rHPT (7)(8) may be partly explained by a lack of iPTH assay specificity (7). Common immunometric assays might overestimate the real PTH(1–84) values because of cross-reactivity with PTH fragments containing amino acids 7–84 [PTH(7–84)], which accumulate in renal failure and behave similar to synthetic PTH(7-84) (9)(10)(11). The questionable accuracy of iPTH measurements forced the development of 3rd-generation assays. These assays do not detect PTH(7–84) or other peptides lacking one or several of the N-terminal amino acid residues of PTH(1–84), but instead use antibodies directed to an epitope consisting of the first 4 to 6 amino acids of the N-terminal portion of the molecule (3)(12)(13)(14).
Intraoperative monitoring of PTH may be helpful to confirm total PTX for treatment of rHPT, but iPTH assays often fail to predict total PTX in patients with rHPT (7)(15). We compared two 2nd-generation iPTH assays with a 3rd-generation PTH(1–84) assay, evaluated the influence of renal function on kinetic data of PTH clearance, and calculated the sensitivity and specificity of the assays, the 3rd-generation assay in particular, in predicting surgical success from intraoperative data.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To assess postoperative outcomes, we measured iPTH concentrations and other biochemical indices. Because autotransplanted parathyroid glands do not secrete PTH during the first postoperative week, only these early PTH concentrations can be used to judge surgical success, i.e., total/subtotal/insufficient removal of parathyroid tissue. After the first postoperative week, the onset of PTH production by the autotransplanted glands will interfere. Total PTX was characterized by iPTH concentrations <15 ng/L during the first postoperative week, and subtotal PTX was assumed if iPTH was between 15 and 65 ng/L, the reference interval of the assay. Undetected ectopic glands left in situ caused insufficient PTX, defined as a measured iPTH concentration >65 ng/L. Cure of patients was assessed by a 6-month follow-up.
method of surgery
A standard bilateral neck exploration with macroscopic identification of all parathyroid glands was performed in all patients independent of renal function. Subsequently, the parathyroid glands were removed (PTX) in 
10-min intervals. Central neck dissection, bilateral transcervical thymectomy, and immediate autotransplantation into the brachioradialis muscle of the forearm were performed according to routine procedures (7). In 30 of 32 patients, 4 parathyroid glands were removed; 1 patient presented with 5 glands, and 1 patient had only 3 glands.
blood sampling
Approximately 18 blood samples per patient were drawn from a peripheral artery and collected in EDTA-containing tubes. The first blood sample was drawn immediately after induction of anesthesia but before any kind of neck manipulation, and the PTH concentration of this sample was defined as the baseline value. The next samples were drawn at the same times as the stepwise removal of parathyroid glands and between excisions, in 5-min intervals. After removal of the last gland, additional samples were collected every 5 min over a mean period of 50 min. In a few patients, blood sampling intervals were irregular, but the exact time points were documented. Plasma iPTH was measured intraoperatively (see below), and sample aliquots were stored at –80 °C.
pth immunoassays and other laboratory methods
We measured iPTH with immunoassays from Roche Diagnostics on the Elecsys® 1010 (iPTH-R) and from Nichols Institute Diagnostics (NID) on the Nichols Advantage® system (iPTH-N), respectively.
We routinely use the iPTH-R assay for intraoperative monitoring on fresh plasma samples in primary HPT (16), as well as in the postsurgical follow-up. To monitor the success of the surgery, blood samples collected by the surgical team were assayed in a room adjacent to the operation theater equipped with a mobile laboratory, which we also used during surgeries for rHPT. In minimally invasive surgery for primary HPT, the criterion of a decrease in iPTH-R to <50% of baseline within 10 min after resection is helpful in differentiating between single- and multiple-gland disease (17). In contrast, in rHPT, the iPTH-R values cannot be used to guide the exploration (15). The intraoperative laboratory data and plasma sample aliquots were collected only for retrospective analyses in rHPT.
The iPTH-R test has a turnaround time of 9 min and uses 2 monoclonal antibodies that bind to epitopes composed of amino acids 26–32 and 55–64 of PTH. The analytical sensitivity (detection limit, defined by a concentration 2 SD above the mean of 21 within-run measurements of the zero calibrator) was 1.2 ng/L, and the functional sensitivity (lowest concentration with a between-run CV <20% measured on at least 10 different days) was 6 ng/L. The reference interval was 15–65 ng/L.
The iPTH-N is based on affinity-purified polyclonal antisera specific for amino acid sequence 39–84 and the N-terminal portion of PTH (residues 12–34). The manufacturer reported an analytical sensitivity (detection limit) of 1 ng/L; intra- and interassay imprecision <0.7% and <10%, respectively; and a reference interval of 10–65 ng/L.
A previous study showed 68% cross-reactivity toward the PTH(7–84) fragments for the iPTH-R on the Elecsys and 100% cross-reactivity for iPTH-N on the NID Advantage (18).
The third automated PTH test was the NID Bio-Intact PTH(1–84) assay (Bio-iPTH), which according to the manufacturer detects the biologically active PTH(1–84) molecule without cross-reactivity to PTH fragments. Assay specificity is provided by the use of a polyclonal antibody toward the first 6 amino acids of the N-terminal region instead of the less-specific antibody against the far N-terminal epitope of the iPTH-N assay. The Bio-iPTH had an analytical sensitivity (detection limit) of 1.5 ng/L; a functional sensitivity of 4 ng/L; intra- and interassay CVs <4% and <10%, respectively; and a reference interval of 8–50 ng/L. The Bio-iPTH assay uses a 40-min protocol, but a "quick" modification with a 9-min turnaround time suitable for intraoperative monitoring is also available. In this pilot study, however, we used the 40-min protocol on samples that had been stored deep frozen. Measurements of fresh and thawed samples as well as with the regular and quick test variants of the Bio-iPTH yielded comparable results (quick = 1.00 x regular – 1.1 ng/L; r = 0.99; n = 130), but the quick Bio-iPTH assay was costlier. Concentrations of the large, N-truncated PTH(7–84) fragments had to be calculated by subtracting Bio-iPTH results from iPTH-N results. Both the iPTH-N and the regular Bio-iPTH tests took 40 min. Thus, it was beneficial to run both assays simultaneously later with thawed samples.
Serum creatinine and total calcium concentrations were measured with routine methods on a Modular System automated analyzer (Roche Diagnostics).
kinetic calculations and statistical analysis
We used nonlinear regression analyses to model the decay kinetics of PTH concentrations measured with the different assays and performed calculations with GraphPad Prism (Ver. 3.03) software.
For evaluation of kinetic data, we used a 1-phase exponential decay plus plateau model with the equation ct = c0e–kt + cP, where ct and c0 are the concentrations at a variable time t and at time zero (start of the exponential decay), respectively; k is the decay constant; and cP is an additional residual plateau concentration (16). c0 + cP was the starting concentration of the exponential decay, just at the last PTX. The half-life was calculated by ln(2)/k.
The significance of differences (P <0.05, two-tailed t-test) was evaluated by Student t-test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). The mean PTH concentrations are shown as percentages from baseline PTH (set as 100%), which was measured at the induction of anesthesia. At excision of the first and most enlarged parathyroid gland, PTH had already decreased to 70% of baseline. At the time of excision of the last (4th) gland, PTH values were still nearly one half of baseline PTH values. Toward the end of intraoperative surveillance, PTH had decreased to a few percentages of baseline concentrations. Throughout the surgery, the Bio-iPTH values were lower than the iPTH values (P <0.05, two-tailed t-test). For a method comparison of the 3 assays using 556 study samples, iPTH assay results (ranges, 9.5–2320 ng/L for the iPTH-R and 6.4–2588 ng/L for iPTH-N; regression results, iPTH-N = 1.08 x iPTH-R – 19 ng/L; r = 0.99) and Bio-iPTH concentrations (range, 1.3–1578 ng/L) were highly correlated but differed in slope and intercept as calculated by Deming regression analyses: iPTH-R = 1.75 x Bio-iPTH + 57 ng/L (r = 0.98) and iPTH-N = 1.86 x Bio-iPTH + 43 ng/L (r = 0.99).
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Results for patient groups established to assess the impact of renal function on PTH concentrations and clearance rate are shown in Table 1
. Preoperative iPTH-R concentrations were lower (P <0.0004, two-tailed t-test) in patients with good renal function (group B) than in patients on hemodialysis and/or with impaired renal function (groups A and C). Postoperative outcomes relative to iPTH-R concentrations obtained within the first postoperative week (Table 1
) showed that persistent HPT (PTH concentrations >3-fold higher than the upper limit of the reference interval plus hypercalcemia within the first 6 months after surgery) occurred in all 5 of 32 patients classified as "insufficient PTX" based on increased iPTH-R result. In all cases, persistent HPT was caused by parathyroid tissue left in the neck (15), whereas the 6-month follow-up revealed cure in 27 of 32 patients. iPTH-R concentrations below or within the reference interval several days after surgery suggested that in all cured patients, sufficient PTX had been achieved by either total or subtotal resections.
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PTH concentrations at resection of the last gland (L-PTX) and concentrations obtained 20 min thereafter were normalized to baseline PTH (Table 2
). At L-PTX, the mean PTH concentration had decreased to approximately one half of the baseline value in group A, to one third of baseline in group B, and to at least one fifth of baseline in group C. Approximately one half of group A patients and all of the group B and C patients presented with PTH <50% of baseline concentration at L-PTX. A decrease in PTH(1–84) concentrations to <50% of baseline within 20 min after L-PTX has been reported to be predictive of cure in rHPT (8). Twenty minutes after L-PTX, all study patients attained this criterion according to results obtained with all 3 assays (Table 2
). The decrease in PTH concentration after L-PTX contrasted with the postoperative outcome of the 5 patients with insufficient PTX (Table 1
).
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We used the disappearance of PTH after L-PTX and PTH clearance to within reference values during surgery for outcome prediction. We normalized concentrations to the values at excision of the last gland (L-PTX; t = 0) and graphically analyzed PTH clearance (Fig. 2
). The decrease in iPTH concentrations was similar with both the Roche and NID assays. In all groups, clearance rates of iPTH, Bio-iPTH, and PTH(7–84) differed from one another (P = 0.04 to <<0.001, two-tailed t-test). PTH as measured by all assays decreased to plateau-like concentrations 30 min after L-PTX. In all groups, the percentage of PTH(7–84) remained higher than the percentage of Bio-iPTH (P <<0.001, two-tailed t-test), whereas the percentage of iPTH was in between (P <0.05, two-tailed t-test). Bio-iPTH showed the most rapid elimination rate, particularly compared with the slow decrease in the concentration of the PTH(7–84) fragments (Fig. 2
). By fitting the measured concentrations to the exponential model, we calculated the apparent half-life of PTH clearance. Comparisons between the raw data and fitted results revealed excellent conformity (slopes of regression lines were 0.99–1.00, y-intercepts were 0.5–2 ng/L, and correlation coefficients were 0.990–0.998). In the 3 kidney-function groups, we observed differences in both preoperative iPTH concentrations (Table 1
) and PTH half-lives (Table 3
). Group B patients had the lowest iPTH values and a similar PTH half-life with all 3 PTH assays (3.8–4.4 min; difference not significant). Group C presented with high iPTH concentrations and prolonged half-lives vs the other groups (P <0.05, two-tailed t-test). In group A, the half-lives differed (P <0.05, two-tailed t-test) between iPTH, the PTH(7–84) fragments, and Bio-iPTH; Bio-iPTH showed a short and the PTH(7–84) fragments a prolonged half-life. Half-lives did not differ among subgroups of total, subtotal, and insufficient resections (not shown).
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To measure the contributions of Bio-iPTH and the PTH(7–84) fragments to iPTH during surgery, we normalized the respective concentrations to iPTH-N at baseline, L-PTX, and plateau (Fig. 3
). All groups showed a similar trend: PTH(7–84) fragment fractions accumulated significantly, whereas Bio-iPTH decreased. On average, the Bio-iPTH fraction contributed a mean (SD) of 55 (9)% to iPTH at baseline and 24 (10)% at the end of surgery.
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In 12 patients, the surgical goal of total PTX was not achieved (Table 1
), and none of the assays provided useful distinctions between total and subtotal resections (not shown). Sufficient PTX (total plus subtotal resections) and insufficient PTX corresponded to the long-term outcome and were used to compare data from groups A, B, and C (Table 4
). Residual plateau concentrations, calculated by the kinetic model, resembled an endpoint of intraoperative PTH monitoring (see Fig. 2
). Plateaus were 20% lower than concentrations 20 min after L-PTX.
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The corresponding results of both iPTH assays did not differ within groups, but the Bio-iPTH results were significantly lower in each group. In cured patients, the relevant PTH concentrations were matched in patients with impaired kidney function [sufficient PTX (groups A and C), P >0.05, two-tailed t-test]. The PTH concentrations of group B patients were significantly lower than in groups A and C and did not overlap with PTH in patients with insufficient PTX (Table 4
). Intraoperative PTH concentrations decreased significantly from baseline to L-PTX and so forth. Exceptions were observed with the Bio-iPTH results for groups B and C and with all assays for the subgroup of patients with insufficient PTX only at the end of surgery, when concentrations 20 min after L-PTX matched plateau concentrations.
Insufficient PTX leading to persistent rHPT was observed only in 5 patients of group A, who had 2- to 3-fold higher PTH plateau concentrations than group A patients who had sufficient PTX (P = 0.024–0.00002, two-tailed t-test). Even 20 min after L-PTX, Bio-iPTH results did not overlap between sufficient and insufficient resections and correctly reached reference values in all 15 cured patients. Bio-iPTH concentrations for 5 patients in group B reached reference values at L-PTX, but only 2 also had iPTH concentrations within reference values. In group C, similar to group A, iPTH concentrations remained increased despite sufficient PTX and did not differ from those in patients with insufficient PTX after surgery. Nevertheless, Bio-iPTH values had decreased to within the reference interval and were significantly lower compared with values for patients with insufficient PTX (P = 0.023, two-tailed t-test). At the end of surgery, Bio-iPTH concentrations were <50 ng/L in 28 of 32 patients. Twenty minutes after L-PTX, Bio-iPTH values for all of these patients already were below the upper reference limit, but in group A, only 3 patients had sufficient PTX on the basis of values within the reference intervals for both the iPTH-R and iPTH-N assays (Table 4
). Concentrations of iPTH were increased in the other 17 patients in group A, regardless of whether they had sufficient or insufficient PTX. Bio-iPTH reached the assay-specific reference interval in all 15 patients with total and subtotal PTX, but still remained increased (>50 ng/L) in 4 of 5 patients with insufficient resection. In group B, all patients had PTH concentrations below the upper reference limits of the respective assays. In group C, iPTH concentrations did not decrease to within the reference intervals for any of the 4 patients, but they all had Bio-iPTH results within the reference interval (Table 4
).
Bio-iPTH failed to predict patient outcome in only 1 of 32 patients. In this patient, iPTH values remained increased (plateaus at 99 and 79 ng/L for the iPTH-R and iPTH-N assays, respectively), and the iPTH-R value increased to 170 ng/L within the first postoperative week, confirming insufficient removal of all parathyroid tissue (Fig. 4
). Apparently, in this patient the decrease in PTH was disturbed by surgical manipulations near the undetected ectopic gland. Results for all assays indicated atypical increases in PTH toward the end of surgery in this patient, but the intraoperative iPTH-R results were not used to guide the operation.
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When we used the criterion of PTH decreasing to below the upper reference limit at least 20 min after L-PTX, differentiation between sufficient and insufficient resections was more accurate with the Bio-iPTH assay than with the iPTH assays in groups A and C. The positive predictive values of intraoperative PTH monitoring to forecast persistence of rHPT because of insufficient PTX in these 24 patients reached 24% (5 of 21) with the iPTH assays and 100% (4 of 4) with the Bio-iPTH assay. The negative predictive values were 100% (3 of 3) with the iPTH assays and 95% (19 of 20) with the Bio-iPTH assay, predicting cure by sufficient PTX (total and subtotal resections).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our pilot study demonstrated that the inadequate decrease and insufficient normalization of PTH values after total and subtotal PTX occurred with iPTH assays but not with the Bio-iPTH assay, independent of the initial PTH concentration. Compared with Bio-iPTH, the iPTH-N and iPTH-R assays had low predictive values for insufficient PTX and failed to predict cure intraoperatively in most patients with impaired renal function. The Bio-iPTH assay, which is available in a quick variant suitable for intraoperative monitoring, can measure a PTH decrease to within reference intervals in an adequate timeframe of 20–40 min after L-PTX. The criterion of PTH normalization together with specific measurements of PTH(1–84) seem to be highly predictive.
In conclusion, accurate PTH measurement depends on both renal function and assay specificity. In patients with renal failure, reduced clearance of the cross-reacting PTH(7–84) fragments leads to overestimation of the PTH concentration with the 2nd-generation iPTH assays. Although intraoperative monitoring in rHPT with the 2nd- and 3rd-generation PTH assays identified failed surgeries with similar effectiveness, the quick 3rd-generation PTH assay seemed to provide superior data for guiding surgery. In surgically treated rHPT patients with falsely increased iPTH concentrations attributable to impaired renal clearance, PTH(1–84) monitoring will avoid the need for unnecessarily prolonged surgical explorations.
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Acknowledgments |
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Footnotes |
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References |
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The following articles in journals at HighWire Press have cited this article:
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  S.-K. Meyer, M. Zorn, K. Frank-Raue, M. W Buchler, P. Nawroth, and T. Weber Clinical impact of two different intraoperative parathyroid hormone assays in primary and renal hyperparathyroidism Eur. J. Endocrinol., February 1, 2009; 160(2): 275 - 281. [Abstract] [Full Text] [PDF]
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
), iPTH-N (
), and Bio-iPTH (•) values normalized to the respective baseline concentrations of 29 patients during surgery on rHPT.
, PTH(7–84). Group A, hemodialysis patients; Group B, kidney transplantation with normal renal function (creatinine concentration within reference values); Group C, kidney transplantation with impaired renal function (increased creatinine).
) and PTH(7–84) fragment (
) concentrations relative to the concentrations of iPTH-N at baseline, L-PTX, and plateau in the 3 different kidney-function groups.