Should we depend on reference intervals from manufacturer package inserts? Comparing TSH and FT4 reference intervals from four manufacturers with results from modern indirect methods and the direct method.

OBJECTIVES: Correct interpretation of thyroid function tests relies on correct reference intervals (RIs) for thyroid-stimulating hormone (TSH) and free thyroxine (FT4). ISO15189 mandates periodic verification of RIs, but laboratories struggle with cost-effective approaches. We investigated whether indirect methods (utilizing historical laboratory data) could replace the direct approach (utilizing healthy reference individuals) and compared results with manufacturer-provided RIs for TSH and FT4. METHODS: We collected historical data (2008-2022) from 13 Dutch laboratories to re-establish RIs by employing indirect methods, TMC (for TSH) and refineR (for FT4). Laboratories used common automated platforms (Roche, Abbott, Beckman or Siemens). Indirect RIs (IRIs) were determined per laboratory per year and clustered per manufacturer (>1.000.000 data points per manufacturer). Direct RIs (DRIs) were established in 125 healthy individuals per platform. RESULTS: TSH IRIs remained robust over the years for all manufacturers. FT4 IRIs proved robust for three manufacturers (Roche, Beckman and Siemens), but the IRI upper reference limit (URL) of Abbott showed a decrease of 2 pmol/L from 2015. Comparison of the IRIs and DRIs for TSH and FT4 showed close agreement using adequate age-stratification. Manufacturer-provided RIs, notably Abbott, Roche and Beckman exhibited inappropriate URLs (overall difference of 0.5-1.0 µIU/mL) for TSH. For FT4, the URLs provided by Roche, Abbott and Siemens were overestimated by 1.5-3.5 pmol/L. CONCLUSIONS: These results underscore the importance of RI verification as manufacturer-provided RIs are often incorrect and RIs may not be robust. Indirect methods offer cost-effective alternatives for laboratory-specific or platform-specific verification of RIs.

Analytical performance of a single epitope B-type natriuretic peptide sandwich immunoassay on the Minicare platform for point-of-care diagnostics.

Point-of-care B-type natriuretic peptide (BNP) testing with adequate analytical performance has the potential to improve patient flow and provide primary care givers with easy-to-use advanced diagnostic tools in the management of heart failure. We present the analytical evaluation of the Minicare BNP immunoassay under development on the Minicare I-20 platform for point-of-care testing. Analytical performance was evaluated using EDTA venous whole blood, EDTA plasma and capillary whole blood. Method comparison with a lab-testing system was performed using samples from 187 patients. Normal values were determined based on 160 healthy adults, aging from 19 to 70 years. Limit of blank (LoB), limit of detection (LoD) were determined to be 3.3 ng/L, 5.8 ng/L. Limit of quantitation (LoQ) in whole blood at 20% and 10% coefficient of variation (CV) was found < 9 ng/L and <30 ng/L respectively without significant differences between EDTA whole blood and EDTA plasma. Total CV was found to be from 6.7% to 9.7% for BNP concentrations between 92.6 and 3984 ng/L. The sample type comparison study demonstrated correlation coefficients between 0.97 and 0.99 with slopes between 1.03 and 1.09 between the different samples. Method comparison between Minicare BNP and Siemens ADVIA Centaur BNP demonstrated a correlation coefficient of 0.92 with a slope of 1.06. The 97.5% URL of a healthy population was calculated to be 72.6 ng/L. The Minicare BNP assay is a robust, easy-to-use and sensitive test for rapid determination of BNP concentrations that can be used in a near-patient setting.

False positive troponin result caused by a true macrotroponin.

BACKGROUND: A 35-year-old female presented with complaints of tiredness, shortness of breath and recent onset of chest pain. The cardiac troponin I (cTnI) concentration was elevated at 6.4 µg/L (Abbott Architect reference value: <0.03 µg/L) with a normal ECG. Physical examination, radiological imaging and routine laboratory investigations did not provide an explanation for the elevated cTnI concentration. METHODS: Use of different troponin assays, dilutions with assay diluent, addition of mouse serum, polyethylene glycol (PEG) precipitation and gel filtration chromatography (GFC) were used to investigate possible interference. RESULTS: Troponin concentrations were below the level of detection when measured using all other troponin immunoassays. Serial dilutions of sample and addition of mouse serum did not alter the results. However, PEG precipitation and GFC showed the presence of a high molecular weight immunoreactive protein. Using GFC and protein-A IgG precipitation, the interference could be identified as a macrocomplex containing IgG and (fragments of) cTnI. CONCLUSIONS: We report a false positive cTnI result caused by a true macrotroponin, containing IgG and (fragments of) cTnI. This macrotroponin was only immunoreactive in the Abbott Architect cTnI immunoassay. Clinicians should be aware of analytical interference when troponin results are constantly elevated in the absence of (non)coronary causes of an increased troponin.

Reference population and marathon runner sera assessed by highly sensitive cardiac troponin T and commercial cardiac troponin T and I assays.

BACKGROUND: Endurance exercise can increase cardiac troponin (cTn) concentrations as high as those seen in cases of minor myocardial infarction. The inability of most cTn assays to reliably quantify cTn at very low concentrations complicates a thorough data analysis, and the clinical implications of such increases remain unclear. The application of recently developed highly sensitive cTn immunoassays may help resolve these problems. METHODS: We evaluated the precommercial highly sensitive cardiac troponin T (hs-cTnT) assay from Roche Diagnostics and the Architect cardiac troponin I (cTnI-Architect) assay from Abbott Diagnostics by testing samples from a reference population of 546 individuals and a cohort of 85 marathon runners. We also measured the samples with the current commercial cTnT assay for comparison. RESULTS: Although the hs-cTnT and cTnI-Architect assays were capable of measuring cTn concentrations at low concentrations (<0.01 microg/L), only the hs-cTnT assay demonstrated a CV of <10% at the 99th percentile of the reference population and a near-gaussian distribution of the measurements. After a marathon, 86% of the runners had cTnT concentrations greater than the 99th percentile with the hs-cTnT assay, whereas only 45% of the runners showed increased concentrations with the current cTnT assay. cTn concentrations remained significantly increased the day after the marathon. A multiple regression analysis demonstrated marathon experience and age to be significant predictors of postmarathon cTn concentrations (P < 0.05). CONCLUSIONS: The hs-cTnT assay was the only assay tested with a performance capability sufficient to detect cTn concentrations in healthy individuals. The number of runners with increased cTn concentrations after a marathon depends highly on an assay's limit of detection (LOD). The assay with the lowest LOD, the hs-cTnT assay, showed that almost all runners had increased cTn concentrations. The clinical implications of these findings require further investigation.

The diagnostic value of serum and urinary NT-proBNP for heart failure.

BACKGROUND: Serum B-type natriuretic peptide (BNP) and the amino-terminal cleavage product of the prohormone (NT-proBNP) have been shown to be valuable parameters for the diagnosis of heart failure (HF) in the general population. Urinary BNP and NT-proBNP have also been suggested for diagnosis of HF. The present study investigated the diagnostic value of both serum and urinary NT-proBNP in selected groups of controls and patients diagnosed with HF. METHODS: Creatinine clearance and serum and urinary NT-proBNP were measured in 76 controls and in 47 patients diagnosed with HF (NYHA III and IV). Echocardiography was used to exclude cardiac dysfunction in the control population by the combined normality of left ventricular ejection fraction (LVEF), E/A ratio (echocardiographic early [E] and late, or atrial [A] phases of ventricular filling), deceleration time and LV mass index. All patients diagnosed with HF had LVEF <40%. RESULTS: NT-proBNP measurements in urine samples are subject to high variability. Receiver-operating characteristic area under the curve (AUC) for serum, urinary NT-proBNP and their products were 0.94, 0.72 and 0.93, respectively. Correction of urinary NT-proBNP for urinary creatinine content improved the AUC from 0.72 to 0.80. Negative predictive values for ruling out HF were 0.94, 0.67 and 0.89, respectively. Linear regression analysis revealed that creatinine clearance was more important in determining serum NT-proBNP concentrations than age. CONCLUSIONS: Serum NT-proBNP is the best parameter to rule out HF. The product of the serum and urinary concentrations has equal value. Urinary NT-proBNP alone performs rather poorly. Renal function influences NT-proBNP concentrations more than age in this selected population.

Cardiac troponin T release after prolonged strenuous exercise.

Over the past 2 decades, there has been a large interest in cardiac troponin T (cTnT) elevations, which are often seen following endurance sport events. There have been many reports on this topic, although sometimes with different approaches. We reviewed the available literature on cTnT elevations after prolonged strenuous exercise and discovered profound differences in the percentage of subjects reported to have elevated cTnT concentrations. This could partly be attributed to differences in immunoassay characteristics, such as cross-reactivity with skeletal troponin T, and the use of different cut-off values used in the different studies. The elevations were transient, with levels decreasing to pre-event concentrations within 24-48 hours. This might be explained by the relatively short half-life of cTnT, or water imbalance during and after the event. The release mechanism of cTnT, as well as the long-term positive or negative effects, remains unclear. Future research should therefore be aimed at clarifying the release mechanism of cTnT. Furthermore, the benefits and the possible long-term negative aspects of prolonged exercise should be evaluated.

A specific immunoprecipitation method for isolating isoforms of insulin-like growth factor binding protein-3 from serum.

BACKGROUND: This report describes an in-house developed immunoprecipitation method to isolate insulin-like growth factor binding protein-3 (IGFBP-3) and its isoforms from serum. The method was compared to other existing immunoprecipitation methods. The study of IGFBP-3 isoforms is relevant for further studies on congenital defects in glycosylation (CDG), galactosemia, and alcoholic liver cirrhosis. METHODS: Monoclonal and/or polyclonal anti-human IGFBP-3 antibodies were covalently immobilised on protein-A Sepharose beads using dimethyl pimelimidate as cross-linker. By incubation with these immobilised antibodies, intact IGFBP-3 and fragments of IGFBP-3 were isolated from serum. Enzyme-linked immunosorbent assay (ELISA) and one-dimensional gel electrophoresis (1-DE) experiments were performed to define the optimal immunoprecipitation method. Isolated proteins were separated by 1-DE and two-dimensional gel electrophoresis (2-DE) and visualised by Western blotting. RESULTS: ELISA and 1-DE results illustrated that an optimal isolation was performed using PBS for the incubation with serum. Laemmli sample buffer, containing 2-amino-2-(hydroxymethyl)-1,3-propanediol hydrochloride and sodium dodecyl sulfate, or urea/CHAPS was optimal for the elution. Clinical validation was performed using CDG-Ia serum samples. The 2-DE experiments showed characteristic isoform patterns for CDG-Ia. CONCLUSIONS: The optimized in-house developed immunoprecipitation method resulted in specific detection of IGFBP-3 isoforms and is suitable for further studies on glycosylation defects.

Investigation of release and degradation of cardiac troponin T in patients with acute myocardial infarction.

OBJECTIVES: Cardiac troponin T (cTnT) degradation after tissue release is still under debate. Because degradation of cTnT might have consequences on clearance of the molecule from the circulation, but also on the assay performance, the aim of this study was to investigate cTnT release and degradation in serum of AMI patients. DESIGN AND METHODS: Serum samples were collected from 20 patients with AMI diagnosis undergoing rapid revascularization. Intact cTnT and fragments were detected using a combination of immunoprecipitation, SDS-PAGE and Western blotting. RESULTS: The intact cTnT protein was detected only during the first 12 h after the cTnT concentration started to increase above the AMI cut-off value of 0.03 microg/L. Thereafter only fragments with molecular weights ranging from 10 to 30 kDa were detected, with two fragments being most prominent (15 and 25 kDa). CONCLUSIONS: Intact cTnT rapidly disappears from the circulation during the early hours after AMI, but immunoreactive fragments remain present longer. The current cTnT immunoassay detects both intact cTnT and fragments.

Interpretation of cardiac troponin T behaviour in size-exclusion chromatography.

BACKGROUND: Knowledge about the presence of intact cardiac troponin T (cTnT) and/or its immunoreactive fragments is of great value for the interpretation of cTnT clearance from the circulation. Until now there has been a lot of controversy about cTnT fragmentation. To provide an answer to this controversy, we investigated fragmentation of cTnT with size-exclusion chromatography (SEC), and confirmed our data using mass spectrometry. METHODS: A highly purified human cTnT standard, characterised using mass spectrometry as a single peak of 34,377 Da and using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) as a single immunoreactive band (37 kDa), was incubated in serum for 0, 24 and 48 h at 37 degrees C and analysed using SEC. A troponin TIC complex standard, used in an earlier study, was also investigated. RESULTS: We demonstrated that, because of its rod-like shape, the molecular weight of cTnT cannot be estimated from SEC using the molecular weight of globular proteins as a reference. The Stokes radius of intact cTnT was calculated to be 33.7 A. Incubation of both cardiac troponin standards in troponin-free serum resulted in a time-dependent decrease in intact cTnT and a simultaneous increase in smaller immunoreactive fragments (13.4 and 22.4 A). CONCLUSIONS: cTnT has a Stokes radius of 33.7 A. Compared with globular calibrator proteins, intact cTnT elutes earlier than expected based solely on its molecular weight. For non-globular or uncharacterised proteins, Stokes radii should be used for correct interpretation of SEC data. By doing so, we were able to clearly demonstrate cTnT fragments.

Superior T-cell suppression by rapamycin and FK506 over rapamycin and cyclosporine A because of abrogated cytotoxic T-lymphocyte induction, impaired memory responses, and persistent apoptosis.

Immunosuppressive therapy is best achieved with a combination of agents targeting multiple activation steps of T cells. In transplantation, cyclosporine A (CsA) or tacrolimus (FK506) are successfully combined with rapamycin (Rap). Rap and CsA were first considered for combination therapy because FK506 and Rap target the same intracellular protein and thus may act in an antagonistic way. However, in clinical studies, FK506+Rap proved to be effective. To date, there is no in vitro data supporting these in vivo findings, and it is unclear whether the observed effects are T-cell mediated. In a human polyclonal allogeneic in vitro model, we found that although combined drug treatment markedly reduced expansion of naive T cells, T-cell activation occurred irrespective of the drug combination used. The induction of cytotoxic effector T cells was reduced by CsA+Rap but completely abolished by FK506+Rap. Importantly, combined immunosuppression allowed generation of memory CD4+ and CD8+ T cells and hence did not result in T-cell anergy. However, FK506+Rap treatment resulted in a reduced number of allospecific memory T cells showing a decreased cell-cycle turnover and cytokine producing capacity. In contrast, CsA+Rap treatment led to increased memory T-cell numbers responding with elevated kinetics. The ability of Rap to promote apoptosis, which contributes to T-cell suppression, remained unaffected upon combination with FK506 or CsA. These data support the combined use of FK506+Rap over CsA+Rap for immunosuppressive therapy.