Nichols Institute Diagnostics Research Articles

Development and Application of a Novel Sensitive Immunometric Assay for Calcitonin in a Large Cohort of Patients with Medullary and Differentiated Thyroid Cancer, Thyroid Nodules, and Autoimmune Thyroid Diseases

Background: Serum calcitonin (sCT) is a useful biomarker for medullary thyroid cancer (MTC). Consensus has not been reached concerning sCT measurements in the evaluation of nodular thyroid disease (NTD). Objective and Methods: We developed a new immunofluorometric assay for sCT and have validated it in samples from 794 patients [203 with MTC, 205 with autoimmune thyroid disease (ATD), 248 with NTD, 80 with differentiated thyroid cancer (DTC) ‘free of disease', 58 with chronic renal failure (CRF)] and 178 normal individuals, including samples after pentagastrin tests and samples from the washout of 92 FNA procedures in patients with NTD or MTC. We also compared some samples from patients with low or high calcitonin levels using both this assay and the Nichols Institute Diagnostics (NID) assay. Results: The assay's analytical sensitivity was 1.0 pg/ml. Considering MTC patients prior to surgery, the cut-off values for the 95% reference range were 11.1 pg/ml for males and 5.5 pg/ml for females and employing the ROC curve were 18.4 pg/ml for males and 7.8 pg/ml for females. sCT in patients with MTC was strongly correlated with disease status. Patients with NTD and ATD did not present false-positive results. sCT measurements were significantly correlated with age (excluding MTC and CRF). The NID test had a strong correlation with our assay. A hook effect was observed only with concentrations >200,000 pg/ml. Conclusions: We developed a novel sCT assay and validated it in healthy subjects, as well as in a large cohort of patients with MTC, NTD, ATD, DTC, and CRF.

Response of different PTH assays to therapy with sevelamer or CaCO3 and active vitamin D sterols

Amino-terminally truncated parathyroid hormone (PTH) fragments are detected to differing degrees by first- and second-generation immunometric PTH assays (PTH-IMAs), and acute changes in serum calcium affect the proportion of these fragments in circulation. However, the effect of chronic calcium changes and different vitamin D doses on these PTH measurements remains to be defined. In this study, 60 pediatric dialysis patients, aged 13.9 ± 0.7 years, with secondary hyperparathyroidism were randomized to 8 months of therapy with oral vitamin D combined with either calcium carbonate (CaCO3) or sevelamer. Serum phosphorus levels did not differ between groups. Serum calcium levels rose from 9.3 ± 0.1 to 9.7 ± 0.1 mg/dl during CaCO3 therapy (p < 0.01 from baseline) but remained unchanged during sevelamer therapy. In the CaCO3 and sevelamer groups, baseline serum PTH levels (1st PTH-IMA; Nichols Institute Diagnostics, San Clemente, CA) were 964 ± 75 and 932 ± 89 pg/ml, and levels declined to 491 ± 55 and 543 ± 59 pg/ml, respectively (nonsignificant between groups). Patients treated with sevelamer received higher doses of vitamin D than those treated with CaCO3. The PTH values obtained by first- and second-generation PTH-IMAs correlated closely throughout therapy and the response of PTH was similar to both PTH-IMAs, despite differences in serum calcium levels.

Immulite vs Scantibodies IRMA Plasma ACTH Assay

The 2-site IRMA assay for adrenocorticotrophic hormone (ACTH),1 as produced by Nichols Institute Diagnostics, was considered the gold standard in the US (1). This assay is no longer available. It was recently demonstrated that a manual IRMA ACTH assay produced by Scantibodies yielded results equivalent to those obtained with the Nichols Institute Diagnostics assay (2). We have observed that the results from the College of American Pathologists (CAP) Proficiency Survey samples for ACTH using the Scantibodies IRMA yielded higher results than those from the Immulite platform (Siemens), which …

Influence of oral contraceptive use on growth hormone in vivo bioactivity following resistance exercise: Responses of molecular mass variants

The purpose was to examine effects of oral contraceptive (OC) use on plasma growth hormone (GH) responses to heavy resistance exercise. Sixty untrained women were placed into one of two groups: currently using OC (Ortho Tri-Cyclen®) (n=25; mean±SD: 24.5±4.2y, 160.4±7.1cm, 64.1±11.3kg) or not currently using OC (NOC) (n=35; 23.6±4.6y, 165.9±6.0cm, 65.7±10.3kg). Participants performed an acute heavy resistance exercise test (AHRET; six sets of 10 repetition squats; 2min rest between sets) during days 2–4 of the follicular phase (NOC group) or of inactive oral contraceptive intake (OC group). Plasma was obtained before and immediately after AHRET and subsequently fractionated based on apparent molecular weight (>60kD, 30–60kD, and <30kD). GH was determined in unfractionated plasma and each plasma fraction using 4 methods: (1) Nichols Institute Diagnostics immunoradiometric assay (Nichols), (2) National Institute of Diabetes and Digestive Kidney Diseases (NIDDK) competitive radioimmunoassay, (3) DSL immunofunctional enzyme-linked immunoabsorbent assay (IFA) and (4) rat tibial line bioassay. GH increased (P<0.05) in all fractions post-AHRET for the Nichols, NIDDK, and IFA. The OC group displayed higher resting GH for the NIDDK, and higher exercise-induced GH for the IFA, Nichols, and NIDDK in unfractionated plasma and >60kD subfraction compared to NOC group. No differences were observed for the tibial line bioassay. OC use augmented immunological GH response to AHRET in unfractionated plasma and >60kD molecular weight subfraction. However, OC use only increased biological activity of GH in one of two bioassays. These data demonstrated that GH concentrations at rest and following exercise are assay-dependent.

Measurement of calcitonin by immunoassay analyzers

Since Nichols Institute Diagnostics (NID) ended production of their automated calcitonin immunoassay, evaluation of an alternative calcitonin assay was necessary. Calcitonin measured by the NID procedure was compared with a test from Diagnostic Products Corporation (DPC). Calcitonin was also measured by assays from DiaSorin (DS) and Scantibodies (SC). In 187 samples, detection failed either by NID (39%) or DPC (96%) assay; in 35% of samples, the tests agreed for non-measurable calcitonin. The regression line DPC=0.78 x NID-0.3 (r=0.998) fitted results from 236 samples with detectable calcitonin. A linear relationship, albeit with scattering at low concentrations, was observed. Importantly, stimulation by pentagastrin above basal calcitonin concentrations yielded similar results with both assays. Comparisons (DPC=0.80 x DS-3.4 and DPC=0.81 x SC-1.1) confirmed an aberrant calibration of the DPC test. To overcome the method bias, we propose a multiplication factor of 0.8 to convert NID to DPC results. Apparently due to non-specific effects, the DS and SC assays produced calcitonin results in samples from completely thyroidectomized patients, while the DPC assay correctly failed to detect calcitonin. Thus, the DPC assay on an Immulite 2000 analyzer may be used as a more accurate substitute for NID if the different calibration is noted.

Parathyroid hormone, kidney disease, evidence and guidelines

Parathyroid hormone (PTH) is released from the parathyroid glands predominantly as an 84 amino-acid peptide (PTH (1--84)). Classical biological activity, mediated via interaction with the PTH receptor PTH1R, resides in the N-terminus of the molecule. C-terminal (CPTH) fragments are also directly secreted by the parathyroid gland. Following release, PTH (1--84) undergoes proteolytic degradation in Kupier cells in the liver: resulting CPTH, but not the N-terminal fragments, may re-enter the circulation. The variously derived PTH fragments and PTH (1--84) are then excreted renally. In the setting of chronic kidney disease (CKD), assessment of plasma PTH concentration is used as a surrogate measure of underlying renal bone disease. Measurement of PTH in the presence of renal impairment has always been problematic. In the 1970s, it became apparent that PTH radioimmunoassays crossreacted with CPTH fragments that accumulated in the presence of renal failure. The development of a two-site immunoassay byNichols Institute Diagnostics (Nichols Allegro immunoradiometric assay [IRMA]) in the 1980s, described as an ‘intact’ PTH assay, seemed to resolve this issue. This assay became the bench mark against which other assays were developed. Further, the somewhat limited evidence correlating renal bone disease and plasma PTH concentration, and upon which guidelines have been based, relates largely to experience with this prototype second-generation assay. Currently a range of manufacturers oier intact PTH assays on a variety of automated platforms. Notably, Nichols Institute Diagnostics can no longer be included in that list following the demise of the company in 2006. In the mid-1990s, a Canadian research group discovered that all was not as it might seem. In fact, intact PTH assays seemed to cross-react to a large degree with a CPTH fragment thought to be PTH (7--84) and other less prevalent fragments of PTH that accumulated in renal failure. It would appear that cross-reactivity with this fragment had been accounting all along for approximately 50% of the immunoreactive PTH we had been measuring in such patients, although the exact degree of cross-reactivity varies between methods. Putting aside the perhaps more trivial matter of whether you should continue to market your assay as an intact PTH assay when it is clear that it is not (many manufacturers still do so), the question of how well diierent PTH assays agree with each other and whether measuring PTH (7--84) has clinical relevance in kidney disease needs addressing. It is evident that second-generation PTH assays do not agree among kidney disease patients, either with each other or with the original Nichols intact PTH assay. Among dialysis patients, a recent study has shown that commonly used commercial assays may overor underestimate PTH by up to 50% compared with the Nichols Allegro IRMA. Such betweenmethod variability is also con¢rmed on a regular basis by external quality assessment data (Figure 1). This is due to a variety of factors including lack of common standardization, variation in recovery of PTH (1--84) (Figure 2) and diierences in the extent of crossreactivity with PTH (7--84). For example, data from a United Kingdom National External QualityAssessment Service (UK NEQAS) study in July 2004 estimated approximately 69% cross-reactivity with PTH (7--84) for the Diagnostic Products Corporation (DPC) method, 73% with the Roche ElecSys method, 102% with the Bayer Advia method and 95% with the former Nichols Advantage method. Indeed all 14 ‘intact PTH’assays tested appeared to cross-react with PTH (7--84). PTH results may be interpreted in the setting of a range of clinical recommendations by nephrologists. The UK Renal Association standard states: ‘PTH concentration should be less than four times the upper limit of normal of the assay used in patients beingmanaged for chronic renal failure or after transplantation and in patients who have been on haemodialysis or peritoneal dialysis for longer than three months’. Using a cut-oi based on the upper limit of normal requires that the upper limit of normal is accurate and appropriate for the assay used.There is evidence to suggest that this may not always be the case. A survey conducted by UK NEQAS in July 2005 indicated that the majority of participants have an upper limit of normal close to 65 ng/L, irrespective of the assay used (Figure 3). Is there evidence that variations in

AN ANALOGUE-BASED FREE TESTOSTERONE RADIOIMMUNOASSAY DETECTS AND TRACKS TOTAL TESTOSTERONE CONCENTRATIONS.

Introduction This free testosterone (FTe) assay is routinely applied to whole serum. In whole male serum 1.5 to 2.0% of total testosterone (TTe) is FTe. (The remainder is bound to Te binding serum proteins.) The FTe assay is based on a radiolabeled Te analogue (a Te conjugate) that competes with unlabeled Te for binding to a Te-specific antibody. Two questions were asked about the assay. First, how does the assay respond when only protein-bound Te and TTe are diluted while FTe is constant? Second, how does the assay respond when FTe replaces protein-bound Te while TTe remains constant? Analogue-based FTe estimates and TTe determinations were compared. Materials and Methods Equilibrium dialysis (Nichols Institute Diagnostics) was applied to normal adult male serum. The free Te assay was applied to dialysate. A total Te assay was applied to retentate. Te was added to an aliquot of dialysate until total Te matched retentate total Te. One aliquot of serum retentate was progressively diluted with serum dialysate (no Te added). Thus, protein-bound and total Te were decreased while free Te remained constant. Another aliquot of serum retentate was progressively diluted with the dialysate containing added Te. Thus, protein-bound Te decreased and FTe increased while TTe remained constant. The FTe RIA and the TTe RIA (both from Diagnostic Products Company, Los Angeles, CA) were applied to all Te solutions. Results Free Te was not detected in serum dialysate. When TTe and protein-bound Te were progressively reduced while free Te was constant, TTe determinations progressively declined 95%, from 581 to 29 ng/dL. The median was 167 ng/dL. The analogue-based FTe determinations also progressively declined ≈95% from 1.28 to r 2 .98). When protein-bound Te was progressively replaced with FTe while total Te was constant (648 ng/dL ± 14), free Te determinations were similarly constant (1.48 ng/dL ± 0.02). Conclusions These analogue-based free Te estimates were not traceable to serum free Te concentrations. Free Te determinations were traceable to total Te concentrations and total Te concentrations only. The forms of Te tracked and the concentrations of Te detected by this assay provide direct evidence of protein-bound Te cross-reactivity. No evidence was found to support the concept that this free Te RIA tracks or detects serum free Te.

Lack of Noggin Expression by Cancer Cells Is a Determinant of the Osteoblast Response in Bone Metastases

Prostate and mammary cancer bone metastases can be osteoblastic or osteolytic, but the mechanisms determining these features are unclear. Bone morphogenetic and Wnt proteins are osteoinductive molecules. Their activity is modulated by antagonists such as noggin and dickkopf-1. Differential expression analysis of bone morphogenetic and Wnt protein antagonists in human prostate and mammary cancer cell lines showed that osteolytic cell lines constitutively express in vitro noggin and dickkopf-1 and at least one of the osteolytic cytokines parathyroid hormone-related protein, colony-stimulating factor-1, and interleukin-8. In contrast, osteoinductive cell lines express neither noggin nor dickkopf-1 nor osteolytic cytokines in vitro. The noggin differential expression profile observed in vitro was confirmed in vivo in prostate cancer cell lines xenografted into bone and in clinical samples of bone metastasis. Forced noggin expression in an osteoinductive prostate cancer cell line abolished the osteoblast response induced in vivo by its intraosseous xenografts. Basal bone resorption and tumor growth kinetics were marginally affected. Lack of noggin and possibly dickkopf-1 expression by cancer cells may be a relevant mechanism contributing to the osteoblast response in bone metastases. Concomitant lack of osteolytic cytokines may be permissive of this effect. Noggin is a candidate drug for the adjuvant therapy of bone metastasis.

Automated Chemiluminescence-Immunoassay for Aldosterone during Dynamic Testing: Comparison to Radioimmunoassays with and without Extraction Steps

Measurements of aldosterone have become more common since the recognition that primary aldosteronism is a more frequent cause of hypertension than previously believed. Our aim was to compare concentrations reported by 4 assays for samples obtained after saline infusion during dynamic testing. We tested 104 participants (27 with primary aldosteronism, 30 with essential hypertension, and 47 healthy controls) with the intravenous saline infusion test (2.0 L isotonic saline over 4 h), with repetitive sampling. In all blood samples, aldosterone concentration was measured by an in-house RIA after extraction and chromatography, by 2 commercially available RIAs without extraction (Aldosterone Maia, Adaltis; Active Aldosterone, Diagnostics Systems Laboratories) and by an automated CLIA (Advantage, Nichols Institute Diagnostics). Correlation coefficients for results of pairs of assays ranged from 0.74 to 0.98. Agreement between commercial assays and in-house RIA was best at the low to intermediate concentrations after saline infusion. Mean (SD) Adaltis and DSL RIA results were 2- to 3-times higher [healthy participants: 78 (25) ng/L and 56 (18) ng/L, respectively] than those obtained by Nichols CLIA [17 (8) ng/L] and in-house RIA [23 (18) ng/L]. Aldosterone concentrations measured by the Nichols CLIA were below the limit of detection (limit of the blank) in 27 of 47 healthy participants. Aldosterone concentrations reported by the Adaltis and DSL nonextraction RIAs were consistently higher than those produced by the Nichols CLIA and the in-house RIA. The convenient Nichols CLIA showed better agreement with the in-house RIA, but the concentrations in healthy participants were frequently undetectable by this method. Uncritical application of cutoff values from the literature must be avoided.

Inter-method variability in PTH measurement: Implication for the care of CKD patients

The National Kidney Foundation/Kidney-Dialysis Outcome Quality Initiative guidelines recommend to maintain the serum intact parathyroid hormone (PTH) concentration between 150 and 300 ng/l in chronic kidney disease (CKD) stage 5 patients. As these limits were derived from studies that used the Allegro intact PTH assay, we aimed to evaluate whether they were applicable to other PTH assays. We compared the PTH concentrations measured with 15 commercial immunoassays in 47 serum pools from dialysis patients, using the Allegro intact PTH assay as the reference. We also evaluated the recovery of graded amounts of synthetic 1-84 and 7-84 PTH added separately to a serum pool. Although the assays were highly correlated, the concentrations differed from one assay to another. The median bias between the tested assays and the Allegro intact PTH assay ranged from -44.9 to 123.0%. When the PTH concentrations were 150 or 300 ng/l with the Allegro intact PTH assay, they ranged with other assays from 83 to 323 ng/l and from 160 to 638 ng/l, respectively. The tested assays recognized 7-84 PTH with various cross-reactivities, whereas a given amount of 1-84 PTH was recovered differently by these assays. We found important inter-method variability in PTH results owing to both antibody specificity and standardization reasons. The unacceptable consequence is that opposite therapeutic attitudes may be reached in a single patient depending on the PTH assay used. We propose to use assay-specific decision limits for CKD patients, or to apply a correcting factor to the PTH results obtained with a given assay.

Parathyroid Hormone Monitoring during Total Parathyroidectomy for Renal Hyperparathyroidism: Pilot Study of the Impact of Renal Function and Assay Specificity

Commonly used assays for intact parathyroid hormone (iPTH) detect not only the biologically active 84-amino acid hormone [PTH1-84], but cross-react with an N-terminal-truncated fragment. Because iPTH assays often fail to predict success of parathyroidectomy in patients with renal hyperparathyroidism (rHPT), we compared results of a 3rd-generation PTH1-84 assay (Bio-iPTH; Nichols Institute Diagnostics) with two 2nd-generation iPTH assays (from Nichols and Roche Diagnostics) by evaluating the PTH clearance kinetics during surgical treatment. 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. Twenty minutes after removal of the last gland, the PTH1-84 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 PTH1-84 was shorter than the half-lives obtained with the iPTH assays. 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 PTH1-84 gave more reliable results than the iPTH assays, which overestimated the concentration of PTH and hampered the intrasurgical diagnosis of resection sufficiency.

Changes in total parathyroid hormone (PTH), PTH-(1-84) and large C-PTH fragments in different stages of chronic kidney disease

Loss of renal function is accompanied by progressive increase in serum levels of intact parathyroid hormone (iPTH) in patients with end-stage renal disease (ESRD). There is a paucity of data regarding levels of PTH-(1-84) and its large carboxyl-terminal fragments (large C-PTH fragments) and progressive loss of kidney function in patients with chronic kidney disease (CKD). The current study was undertaken to describe the glomerular filtration rate (GFR)-dependent plasma concentrations of PTH-(1-84) and related large C-PTH fragments in adult patients with CKD by using different commercially available PTH assays. We studied 80 Caucasian patients with CKD stages 1-5 without renal replacement therapy. Creatinine clearance was calculated by the Modification of Diet in Renal Disease (MDRD) formula. Levels of iPTH were determined by second-generation assays (iPTH Elecsys system, Roche Diagnostics; DUO total iPTH, Scantibodies Laboratory, Inc.; iPTH, Nichols Institute Diagnostics). Third-generation assays were used to measure PTH-(1-84) (CAP (cyclase activating PTH), Scantibodies; Bio-Intact PTH, Nichols). Levels of large C-PTH fragments and ratios of PTH-(1-84)/large C-PTH fragments were calculated and statistical analyses performed. Levels of iPTH and PTH-(1-84) showed CKD stage-dependent increases. Variations among the assays increased with progressive loss of kidney function. The assay from Scantibodies showed a GFR-dependent decrease of the ratio 1-84 PTH / large C-PTH fragment that was not observed with the Nichols assay. Increasing variations among the assays with progression of CKD emphasize the fact that the interpretation of measurements must take into consideration the specific assay. We found evidence for a possible preferential increase of the level of large C-PTH fragments over 1-84 PTH in a CKD stage-dependent manner (Scantibodies). The clinical implications of this finding have to be further evaluated by bone biopsy studies.

Effects of Blood Collection Tubes, including Pediatric Devices, on 16 Common Immunoassays

The effects of different blood collection tubes on hormones and tumor markers, clinical chemistry, and therapeutic drug monitoring have been studied (1)(2)(3)(4), and only serum triiodothyronine (T3) was found to be significantly affected by the types of blood collection tubes(4). One of the interferents for immunoassays has been identified as an organosilicone surfactant(5). Daae et al.(6)(7) investigated the difference of hematology values in capillary and venous blood samples collected in Microtainers and Vacutainers, respectively. Clinical differences were observed for some analytes (6)(7). We compared the results obtained by 4 immunoassays for specimens from 20 adult volunteers collected in red-top Microtainers and Vacutainers during the same venous draw and observed clinically significant differences(8). The objective of present study was to investigate effects of various types of Vacutainer and Microtainer tubes on 16 widely used immunoassays performed on the Immulite 2000 (Diagnostic Product Corporation) or Advantage (Nichols Institute Diagnostics). Blood collection tubes were from BD Diagnostics. The Vacutainers were red-top (plain …

Comparison of 3 Third-Generation Assays for Bio-intact Parathyroid Hormone

Current second-generation parathyroid hormone (PTH) assays suffer from interference by circulating PTH fragments. Newer assays that measure bio-intact PTH, i.e., the biologically active whole PTH molecule (1–84 PTH), have overcome this problem (1)(2). Our study was designed to evaluate and compare 3 commercial third-generation assays based on a chemiluminescence immunoassay, ELISA, and IRMA for bio-intact PTH and to compare these with a second-generation PTH assay. EDTA-plasma samples were collected from 40 healthy blood donors (age range, 18–67 years; mean age, 39 years) and from 26 patients with predialysis chronic kidney disease (age range, 28–86 years; mean age, 65 years). Bio-intact PTH was measured by ( a ) a chemiluminescence immunoassay on the Nichols Advantage® platform (Nichols Institute Diagnostics) (2); ( b ) an IRMA using the Duo PTH reagents (Scantibodies Laboratory)(1); and ( c ) an ELISA using the Human …

Clinical Utility of an Automated Immunochemiluminometric Thyroglobulin Assay in Differentiated Thyroid Carcinoma

Thyroglobulin (Tg) measurements are important in the follow-up of patients with differentiated thyroid carcinoma (DTC). We evaluated the analytical and clinical performance of a new automated immunochemiluminometric assay for Tg (Tg-ICMA; Nichols Advantage Tg; Nichols Institute Diagnostics). We used the Tg-ICMA to measure Tg concentrations in serum samples from 110 Tg antibody-negative DTC patients undergoing thyroid-hormone suppression therapy. Disease state at the time of measurement was assessed on the basis of routine follow-up data. We compared the clinical performance of this assay with the routinely used IRMA (ELSA-hTG; CIS Bio International). The detection limit and functional sensitivity of the Tg-ICMA, based on direct calibration to CRM-457, were 0.05 and 0.6 microg/L, respectively. No Tg-IRMA-positive cases were missed by the Tg-ICMA. Tg was measurable by Tg-ICMA (0.6-8.6 microg/L) but undetectable by Tg-IRMA (<1.5 microg/L) in 12 patients (11%). Clinical data showed evidence of disease in 4 of 12 patients (33%). The Tg-ICMA is a sensitive and reproducible assay for identifying patients in follow-up for DTC with evidence of disease, but uncertainty remains with regard to interpreting findings of measurable serum Tg in patients with no evidence of disease. Follow-up data are required to determine the predictive value of these isolated Tg results. New concepts, i.e., serial Tg measurements and risk stratification of patients, need to be tested to confirm the applicability of this assay for clinical practice.

Novel Parathyroid Hormone (1-84) Assay as Basis for Parathyroid Hormone Monitoring in Renal Hyperparathyroidism

Cross-reactivity of parathyroid hormone (PTH) fragments with immunometric "intact" PTH assays limited the use of intraoperative PTH monitoring in renal hyperparathyroidism. A new assay generation measuring whole PTH (1-84) should be able to predict complete or incomplete resection of hyperfunctioning parathyroid tissue. Consecutive series for evaluation of intraoperative PTH monitoring using a second-generation assay. University hospital section of endocrine surgery. Twenty-two patients received hemodialysis; 9 patients showed good and 4 patients reduced graft function after kidney transplantation. Total parathyroidectomy, central neck dissection, bilateral thymectomy, and immediate autotransplantation was the standardized approach in 35 consecutive patients. Blood samples were drawn before incision and at 5-minute intervals after excision of the last gland. Stored samples were analyzed using a "second-generation" assay (Bio-Intact PTH [1-84]; Nichols Institute Diagnostics, San Clemente, Calif). Parathyroidectomy was classified as total, subtotal, or insufficient according to first-generation intact PTH values in the first postoperative week. Intraoperative ability to predict total, subtotal, or incomplete parathyroidectomy. Independent of renal function, Bio-Intact PTH dropped into the normal range in all patients with total and subtotal resections after a maximum of 20 minutes. It indicated insufficient parathyroidectomy in 4 (80%) of 5 patients. One failure was caused by devascularization of remaining parathyroid tissue. An intraoperative differentiation between total and subtotal resection was not possible. Intraoperative monitoring with quick, second-generation assays for PTH (1-84) seems to be a valuable new tool in surgery for renal hyperparathyroidism because a more accurate differentiation between sufficient and insufficient parathyroidectomy may be achieved. An intraoperative decision about the need for immediate or delayed autotransplantation seems impossible because a differentiation between total or subtotal parathyroidectomy cannot be made. Because of possible devascularization of parathyroid tissue, Bio-Intact PTH monitoring can only be interpreted in the context of the operative findings.

Comparative evaluation of a new immunoradiometric assay for corticotropin

We have characterized the performance of a commercial two-site immunoradiometric assay for manual in vitro diagnostic measurement of plasma corticotropin from Scantibodies Laboratory. We compared the results with those of a similar commonly used assay from Nichols Institute Diagnostics that has recently been withdrawn from production. The lower detection limit, range of the standard curve, cross-reactivity, and intra-assay and inter-assay imprecision of the two assays are very similar. Measurement of clinical samples and a series of samples from an experimental subject demonstrate high correlations between the two assays. These factors, together with recent clearance by the United States Food and Drug Administration for manual in vitro diagnostic measurement, make the Scantibodies corticotropin immunoradiometric assay an appropriate replacement for the Nichols assay.

Possible Neuroprotective Effect of Erythropoietin in Acute Inflammation Induced by Exhaustive Exercise.

Erythropoietin is a glycoprotein hormone produced mainly by interstitial fibroblasts in the kidneys of the adult and by hepatocytes of the foetus. Released into the circulation, erythropoietin makes its way to the bone marrow, where it regulates red cell production by preventing apoptosis of erythroid progenitor cells. Recently, erythropoietin has emerged as a multifunctional growth factor that plays a significant role in the central nervous system (CNS). Both erythropoietin and its receptor are expressed throughout the brain in glial cells, neurons and endothelial cells. Hypoxia and ischaemia have been recognised as important stimuli of erythropoietin expression in the brain. Erythropoietin has potent neuroprotective properties in vivo and in vitro and appears to act in a dual way by directly protecting neurons from ischaemic damage and by stimulating endothelial cells. S-100b, a glial-derived protein is a well-established biomarker for severity of neurological injury and prognosis for recovery. Cell-based and clinical studies have implicated S-100b protein in the initiation and maintenance of a pathological, glial-mediated pro-inflammatory state in the CNS. We studied the possible association between erythropoietin and S-100b in 18 athletes participated in the ultra-distance foot race of the 246Km “Sparthathlon”. This race consists of continuous, prolonged, brisk exercise. We have reported that IL-6, CRP SAA and free plasma DNA levels markedly increased (by 8000-, 152- 108- and 10-fold, respectively) over the baseline at the end of the race1. However, IL-6 levels returned to normal by 48h, while CRP, SAA and free plasma DNA remained elevated. Erythropoietin and S-100b protein levels were determined by means of chemiluminescence (Nichols Institute Diagnostics, CA, USA and Sangtec Medical AB, Bromma, Sweden respectively). The measurements were performed before (phase I), at the end (phase II) and 48h post-race (phase III). Erythropoietin levels at phase I (13.5±6.3 IU/L), were increased significantly at phase II (52.0±52.0 IU/L) and subsequently decreased at phase III (21.0±14.6 IU/L). At the same time period S-100b protein followed the same pattern (phase I: 0.13±0.01 mg/L, phase II: 0.29±0.01 and phase III: 0.13±0.01mg/L). A significantly positive correlation between erythropoietin and S-100b protein was found at phase II (r=0.521, p<0.03), while this correlation was absent in the other two phases (p>0.40 and p>0.08 respectively). Simultaneous measurements of haemoglobin levels showed only a significant drop of haemoglobin at phase III (143.4±12.4, 140.4±13.0 and 124.3±9.5 g/L respectively). Exhaustive exercise induces inflammation and CNS injury, promoting an erythropoietin anti-inflammatory response. We suggest that in this particular inflammatory process, erythropoietin secretion is associated with brain lesions, as they are expressed by the S-100b protein release in the circulation. This is strongly supported by the lack of any association between erythropoietin values and haemoglobin levels.

Third- or Second-Generation Parathyroid Hormone Assays: A Remaining Debate in the Diagnosis of Primary Hyperparathyroidism

Since the demonstration that the second-generation PTH assays, also called intact PTH assays, recognize a non-1-84 PTH fragment in addition to the intact 1-84 PTH, new PTH assays defined as third-generation assays have been commercialized. Two previous studies aimed at evaluating whether these third-generation PTH assays improved the diagnostic sensitivity for primary hyperparathyroidism (PHPT), but they yielded opposite results. In the present study we compared two second-generation PTH assays (the total intact PTH assay from Scantibodies Laboratory, Inc., and the intact PTH assay from Nichols Institute Diagnostics) with two third-generation assays (the cyclase-activating PTH assay also from Scantibodies Laboratory and the bio-intact PTH assay from Nichols Institute) in a series of 145 consecutive PHPT patients operated in our endocrine surgery department over a 10-month period. A group of 74 healthy subjects served as controls. The diagnostic sensitivities for PHPT of the total intact, the intact, the cyclase-activating, and the bio-intact assays were 93.8%, 97.3%, 84.2%, and 89.0%, respectively, with 95% confidence intervals in the control groups of 10-46, 11-60, 8.4-34, and 9-41 ng/liter, respectively. Our findings demonstrate that the diagnostic sensitivities of second- and third-generation PTH assays are similar. Third-generation PTH assays do not therefore improve the diagnosis of elevated serum PTH levels in PHPT, although there are numerical differences among the values.

Underestimation of Serum 25-Hydroxyvitamin D by the Nichols Advantage Assay in Patients Receiving Vitamin D Replacement Therapy

An author of one of the studies cited in the above letter and a representative of Nichols Institute Diagnostics respond: In their letter, Leventis et al. compare methods for 25-hydroxyvitamin D [25(OH)D] measurements. In light of recent reports, and given the complexities of accurately measuring the physiologically relevant species among metabolites of vitamin D, we consider that the title and the conclusions of their letter may be potentially misleading. One of the methods they studied is based on antibody recognition and isotopic detection …