CVs Range Research Articles

Evaluating the Performance of Methods Used to Estimate Growth Parameters from Subsampled Age Data

AbstractDue to time and resource limitations, the growth of many fish species is evaluated with age and length data in a length‐stratified subsample of a sample from a population. Some fisheries professionals draw inferences about growth using age and length data from only those fish that were actually aged (aged‐only method). Alternatively, growth may be evaluated by employing a method of incorporating the length‐stratified aged subsample with information from the entire sample of fish collected, such as the weighted‐mean and assigned‐age methods, which are commonly used in inland fisheries management. More recently, two prominent alternatives to those methods have emerged: the reweighting and empirical proportion methods. In this study, we used observed data from three freshwater fish species with different life history traits (short‐ to long‐lived) to inform a simulation model comparing estimated von Bertalanffy growth model parameters and predicted mean lengths at age calculated using the five aforementioned methods across a range of sample sizes per length‐group and a range of CVs. As expected, our results demonstrated that the aged‐only method is unreliable and biased when estimating von Bertalanffy parameters and mean lengths at age. The weighted‐mean method performed better than the aged‐only method but was not among the top‐performing methods. The assigned‐age, reweighting, and empirical proportion methods all performed well and produced similar estimates, although the empirical proportion method generally resulted in slightly more precise and less biased estimates. We recommend that fisheries professionals discontinue their use of the aged‐only and weighted‐mean methods and instead use the assigned‐age, reweighting, or empirical proportion method.

Field susceptibility of almond cultivars to the four most common aerial fungal diseases in southern Spain

From 2004 to 2012, the incidence and severity of the four most common almond aerial diseases were evaluated in ten experimental fields in the main almond-growing areas of Andalusia, southern Spain. The studied experimental fields were representative of the different geographic locations, agronomic characteristics and farming systems in Andalusia. A total of 14 early-flowering and 26 late-flowering cultivars (cvs.) currently grown in the new almond plantings of Andalusia and the Mediterranean basin were evaluated. The incidences of red leaf blotch (RLB), shot hole (SH), blossom blight (BB), and leaf curl (LC), which were caused by Polystigma amygdalinum, Thyrostroma carpophilum, Monilia laxa and Taphrina deformans, respectively, were monitored in each field and year. RLB was the most prevalent disease, although its severity varied markedly among fields and years. BB caused the higher outbreaks, but this was only the case when rainfall coincided with flowering, and fungicide treatments were not applied. The early-flowering cvs. were more affected than the late-flowering cvs. by BB, although most of the early-flowering cvs. were less susceptible than the reference cv. Marcona to BB. However, ‘Marcona’ was less susceptible than most of the early-flowering cvs. to RLB. Among the late-flowering cvs., the reference cv. Guara was susceptible to the four monitored diseases. The responses of the cvs. to the diseases varied markedly among fields and years, with significant differences among cvs. for all diseases. This work represents the first study to evaluate a broad range of almond cvs. infected with the main almond foliar diseases in natural field conditions, making an important contribution to the understanding of varietal susceptibility of almonds to four significant foliar diseases. Moreover, this study highlights the need to consider cultivar resistance in the development and selection of plant material for new almond plantings and in integrated pest management.

Validation of the Intrinsic Hepcidin IDxTM Test, an LDT for Clinical Hepcidin Assessment

▪Introduction: Hepcidin is the principal regulator of iron absorption and iron recycling, and is required to provide iron for erythropoiesis. Hepcidin is a peptide hormone that binds to the sole iron channel ferroportin (Fpn), and blocks iron efflux by degrading Fpn, trapping iron stored in the cell. Dysregulation of hepcidin by genetic mutations affecting hepcidin expression in hepatocytes is responsible for hereditary hemochromatosis where too little hepcidin leads to iron-overload disease, and iron refractory iron deficiency anemia (IRIDA) where hepcidin expression is inappropriately high leading to anemia. Hepcidin itself is regulated by iron and the inflammatory cytokine IL-6. Thus, hepcidin is dysregulated in most inflammatory diseases and chronic infections. Interest in developing diagnostic applications for hepcidin in iron disorders is high.Objective: We developed a high-affinity monoclonal antibody (mAb) to hepcidin and used it to produce a competitive ELISA (C-ELISA) similar to our first RUO test (Ganz et al. 2008). We determined the performance characteristics using CLIA analytical method validation guidelines. The Intrinsic Hepcidin IDx Test is used for serum and Li-Heparin plasma. We determined the reference range in adult first time blood donors (n=292) and studied an iron deficient (ID) sub-set of the donors.Methods: We developed mouse anti-human hepcidin monoclonal antibodies and coated, blocked, and dried the plates in a temperature and humidity controlled incubator. We used highly purified synthetic hepcidin as reference standard and a biotinylated hepcidin analog as the competitive tracer for the C-ELISA (Peptides International, Louisville, KY). The Intrinsic Hepcidin IDx Test is fully automated on the Biomek FX platform. Serum from first time blood donors (n=292) was obtained, and ferritin, transferrin, plasma iron, CRP, and hepcidin were measured. The reference range for hepcidin was determined and the normal iron status range determined by eliminating iron deficient (ID) donors or those shown to be iron overloaded. Receiver operator characteristic curves (ROC) were plotted to determine the utility of hepcidin and to identify optimal cutoffs of hepcidin as a test of iron deficiency.Results: The mAb used in the Intrinsic Hepcidin IDx Test is specific for human hepcidin. The analytical measurement range (AMR) is 4 - 200 ng/ml. The intra-assay precision CVs range between 7% (at 12 ng/ml) and 4% (at 99 ng/ml). The between-day precision CVs range between 7% (12 ng/ml) and 9% (100 ng/ml). The mean spike recovery across the AMR is 3%. Hepcidin was stable at 4oC for up to 7 days and if specimens are stored at -20oC, for at least 6 months. Hepcidin concentrations determined in blood donors were highly correlated with those measured using our polyclonal RUO C-ELISA (r = 0.95, p<0.001). The reference range is 4.4-54.1 ng/ml for women and 6.1-91.2 ng/ml for men. Clear gender differences were observed: hepcidin was higher in men (median 28.5 ng/ml, n = 143) than in women (median 13.1 ng/ml, n = 149). In addition, the ID group had lower hepcidin (median 6.7 ng/ml) than the normal iron status group (median 21.4 ng/ml, p<0.0001). The area under the ROC curve for hepcidin compared with various ferritin concentrations was greater than 0.90. An optimal hepcidin cutoff less than 10 ng/ml was determined to predict ID with a correct classification rate of greater than 86%.Conclusion: We developed and validated a sensitive, accurate and reproducible C-ELISA, the Intrinsic Hepcidin IDx test, using CLIA validation guidelines. We determined the hepcidin reference ranges by analyzing first-time blood donor serum. We found a significant difference in median hepcidin values between genders. Also, our data show that hepcidin was lower in donors with ID compared to iron replete, healthy donors. DisclosuresGutschow:Intrinsic LifeSciences: Employment, Equity Ownership. Westerman:Intrinsic LifeSciences: Employment. Han:Intrinsic Lifesciences.: Employment, Equity Ownership. Ostland:Intrinsic LifeScienc s: Employment, Equity Ownership. Olbina:Intrinsic LifeSciences: Employment, Equity Ownership. Westerman:Intrinsic LifeSciences: Employment, Equity Ownership.

Inflation of Type I Error in the Evaluation of Scaled Average Bioequivalence, and a Method for its Control.

To verify previously reported findings for the European Medicines Agency's method for Average Bioequivalence with Expanding Limits (ABEL) for assessing highly variable drugs and to extend the assessment for other replicate designs in a wide range of sample sizes and CVs. To explore the properties of a new modified method which maintains the consumer risk ≤0.05 in all cases. Monte-Carlo simulations of three different replicate designs covering a wide range of sample sizes and intra-subject variabilities were performed. At the switching variability of CV wR 30% the consumer risk is substantially inflated to up to 9.2%, which translates into a relative increase of up to 84%. The critical region of inflated type I errors ranges approximately from CV wR 25 up to 45%. The proposed method of iteratively adjusting α maintains the consumer risk at the desired level of ≤5% independent from design, variability, and sample size. Applying the European Medicines Agency's ABEL method at the nominal level of 0.05 inflates the type I error to an unacceptable degree, especially close to a CV wR of 30%. To control the type I error nominal levels ≤0.05 should be employed. Iteratively adjusting α is suggested to find optimal levels of the test.

Measurement of serum aldosterone in picomolar level by LC-MS/MS using charge-tagged technique.

Aldosterone is a mineralocorticoid steroid hormone, the measurement of which in the clinical laboratory is principally performed for the investigation of primary hyperaldosteronism. Primary hyperaldosteronism is a specifically treatable and potentially curable form of hypertension, which typically presents as drug-resistant hypertension and, in up to 37% of cases, hypokalemia. Accurate measurement of aldosterone concentration is essential for correct diagnosis. The serum concentrations of aldosterone are in the picomolar range and therefore sensitive aldosterone assays are required. With the advancement in instrumentation of LC-MS/MS, the picomolar range of aldosterone can be easily measured by the newer models, but for those with a less sensitive instrument, special technique for sample preparation to enhance assay sensitivity is required. This work described the use of charge-tagging for the picomolar measurement of serum aldosterone in a less sensitive LC-MS/MS instrument. The assay was linear up to 3000 pmol/L with lower limit of quantitation at 80 pmol/L. The mean relative recovery was 96.5% with a range of 89.3-101.6% for aqueous calibrators and the mean relative recovery was 94.8% with a range of 87.5-101.4% for serum calibrators. Intra-assay CVs range from 8.2% to 11.3%, and inter-assay CVs ranged from 8.5% to 13.5% at concentration range from 229 to 1720 pmol/L. The LC-MS/MS method compared well (y = 1.04x + 8.97) with the in-use radioimmunoassay method. There was no significant difference found (p = 0.7135) between results determined by LC-MS/MS and radioimmunoassay method.

A practical approach for the validation and clinical implementation of a high-sensitivity cardiac troponin I assay across a North American city

ObjectivesDespite several publications on the analytical performance of high-sensitivity cardiac troponin (hs-cTn) assays, there has been little information on how laboratories should validate and implement these assays into clinical service. Our study provides a practical approach for the validation and implementation of a hs-cTn assay across a large North American City. Design and methodsValidation for the Abbott ARCHITECT hs-cTnI assay (across 5 analyzers) consisted of verification of limit of blank (LoB), precision (i.e., coefficient of variation; CV) testing at the reported limit of detection (LoD) and within and outside the 99th percentile, linearity testing, cTnI versus hs-cTnI patient comparison within and between analyzers (Passing and Bablok and non-parametric analyses). Education, clinical communications, and memorandums were issued in advance to inform all staff across the city as well as a selected reminder the day before live-date to important users. All hospitals switched to the hs-cTnI assay concurrently (the contemporary cTnI assay removed) with laboratory staff instructed to repeat samples previously measured with the contemporary cTnI assay with the hs-cTnI assay only by physician request. ResultsAcross the 5 analyzers and 6 reagent packs the overall LoB was 0.6ng/L (n=60) with a CV of 33% at an overall mean of 1.2ng/L (n=60; reported LoD=1.0ng/L), with linearity demonstrated from 45,005ng/L to 1.1ng/L. Precision testing with a normal patient-pool QC material (mean range across 5 analyzers was 3.9–4.4ng/L) yielded a range of CVs from 7% to 10% (within-run) and CVs from 7% to 18% (between-run) with the high patient-pool QC material (mean range across 5 analyzers was 29.6–36.3ng/L) yielding a range of CVs from 2% to 5% (within-run) and CVs from 4% to 8% (between-run). There was agreement between hs-cTnI versus cTnI with the patient samples (slope ranges: 0.89–1.03; intercept ranges: 1.9–3.8ng/L), however, the median CV on patient samples <100ng/L across the analyzers was 5.6% for hs-cTnI versus 18.7% for the contemporary assay (p<0.001). Following the switch to hs-cTnI testing, no requests for repeat measurements were received. ConclusionsValidation and implementation of hs-cTnI testing across multiple sites requires collaboration within the laboratories and between hospital laboratories and clinical staff.

The Need for Standardization of Tacrolimus Assays

Owing to the lack of an internationally recognized tacrolimus reference material and reference method, current LC-MS and immunoassay test methods used to monitor tacrolimus concentrations in whole blood are not standardized. The aim of this study was to assess the need for tacrolimus assay standardization. We sent a blinded 40-member whole-blood tacrolimus proficiency panel (0-30 μg/L) to 22 clinical laboratories in 14 countries to be tested by the following assays: Abbott ARCHITECT (n = 17), LC-MS (n = 9), and Siemens Dade Dimension (n = 5). Selected LC-MS laboratories (n = 4) also received a common calibrator set. We compared test results to a validated LC-MS method. Four samples from the proficiency panel were assigned reference values by using exact-matching isotope-dilution mass spectrometr at LGC. The range of CVs observed with the tacrolimus proficiency panel was as follows: LC-MS 11.4%-18.7%, ARCHITECT 3.9%-9.5%, and Siemens Dade 5.0%-48.1%. The range of historical within-site QC CVs obtained with the use of 3 control concentrations were as follows: LC-MS low 3.8%-10.7%, medium 2.0%-9.3%, high 2.3%-9.0%; ARCHITECT low 2.5%-9.5%, medium 2.5%-8.6%, high 2.9%-18.6%; and Siemens/Dade Dimension low 8.7%-23.0%, medium 7.6%-13.2%, high 4.4%-10.4%. Assay bias observed between the 4 LC-MS sites was not corrected by implementation of a common calibrator set. Tacrolimus assay standardization will be necessary to compare patient results between clinical laboratories. Improved assay accuracy is required to provide optimized drug dosing and consistent care across transplant centers globally.

Interunit and Intraunit Reliability of the RT3 Triaxial Accelerometer

Interest in the quantification of physical activity is on the rise. Triaxial accelerometry has frequently been used; however, research on the reliability of these devices is limited. We examine the interunit and intraunit reliability of 22 RT3 triaxial accelerometers using a performance-documented laboratory agitator. The RT3 units were tested while moving in 2 directions (antero-posterior, medio-lateral) and speeds (150 and 275 RPM) on a shaker with simultaneous documented performance output for three 24-hour periods. Minimal shaker variance was recorded for all trials (coefficients of variation [CVs] < 0.52%). Our data demonstrate good reliability within RT3s (CVs < 1.81%) but poor reliability among the 22 units (CVs range = 9.5% to 34.7%). In longitudinal studies, each subject should use the same RT3 unit at each assessment. The use of multiple RT3 units in cross-sectional studies is not recommended because data interpretation would be compromised by the high between-unit variability.

A one-step enzyme immunoassay for total plasminogen activator inhibitor-1 antigen in human plasma

A one-step enzyme immunoassay (EIA) has been developed for plasminogen activator inhibitor-1 (PAI-1) antigen. The assay is based on polyclonal antibodies, which were found to be slightly more reactive with tissue-type plasminogen activator (t-PA)/PAI-1 complexes and latent PAI-1 than with active PAI-1. To correct this, active PAI-1 is converted to t-PA/PAI-1 complexes. Latent PAI-1 and tPA/PAI-1 complexes were equally reactive. The EIA is specific (PAI-2 and PAI-3 are not detected), precise (CVs range from 1.8% to 11.1%, depending on the PAI-1 concentration), fast (assay time less than 3 h), and easy to perform. It is compatible with the use of Stabilyte plasma. The assay is calibrated against the putative international standard of the National Institute of Biological Standards and Control (NIBSC; lot 87/512). The normal ranges found with this EIA were 13.2-88 ng/ml; one apparently normal donor had a value of 100 ng/ml.

Comparison of serum copper determination by calorimetric and atomic absorption spectrometric methods in seven different laboratories

An interlaboratory collaborative trial was conducted on the determination of serum copper using two different methods, based on colorimetry (test combination Copper, Boehringer Mannheim, Mannheim, Germany) and flame atomic absorption spectrometry (FAAS). The general performance of the colorimetric method was below that of FAAS, except for sensitivity and linear range, as assessed by detection limit (0.44 versus 1.32 mumol/L) and upper limit of linearity (150 versus 50 mumol/L). The range of the between-run CVs and the recovery of standard additions were, respectively, 2.3-11.9% and 92-127% for the colorimetric method and 1.1-6.0% and 93-101% for the FAAS method. Interferences were minimal with both methods. The two techniques correlated satisfactorily (the correlation coefficients ranged from 0.945-0.970 among laboratories) but the colorimetric assay exhibited slightly higher results than the FAAS method. Each method was transferable among laboratories.

Direct Enzyme Immunoassay of Estradiol in Serum of Women Enrolled in an in Vitro Fertilization and Embryo Transfer Program

We present a fast and simple direct enzyme immunoassay for the sexual steroid hormone estradiol-17 beta in serum of women enrolled in an in vitro fertilization and embryo transfer program. We added 25 microL of standards or samples and 125 microL of a mixture of monoclonal antibody and displacing agents to the wells of a microtiter plate previously coated with a second antibody. After 30 min 50 microL estradiol-horseradish peroxidase conjugate is added. Total incubation time is 75 min. The immunoassay is stopped by washing the microtiter plate. The amount of bound conjugate is then measured at 450 nm with hydrogen peroxide as a substrate and 3,3',5,5'-tetramethylbenzidine as chromogen. The detection limit is 0.24 nmol/L. The assay is thus limited in its application. Analytical recovery is 90.8% (range 80.4-102.9%); within- and between-assay CVs range between 1.3% and 15.4%. Total assay time, including preparation of reagents and data reduction, is only 2.5 h. Results of this enzyme immunoassay compare well with those obtained with four different commercial radioimmunoassays (r = 0.88-0.93).

Rapid Microdetermination of Amiodarone and its Metabolite N. Desethylamiodarone in Plasma by High-Performance Liquid Chromatography

Abstract A rapid microdetermination of amiodarone (A) and its metabolite, N. Desethylamiodarone (DA) in plasma by HPLC is described. 100μL of plasma are deproteinized by 200μL of acetonitrile containing triflupromazine as an internal standard. After centrifugation, 20pL of supernatant aliquot are injected into a Nucleosil octyl analytical column and eluted with a mixture of acetonitrile, 0.01 M sodium acetate pH 3.3 and triethylamine (84 : 16 : 0.1 v/v) at a flow rate of 2.0 mumin. Detection limit is 50 ng/mL for both compounds. Between run CVs range from 2.5 to 4.2%. No interference is observed either with endogenous substances or with a variety of drugs frequently used in cardiovascular therapy. This simple procedure is suitable for routine determination of plasma levels of amiodarone and desethylamiodarone in therapeutic monitoring or emergency toxicology.

Micro Method for Liquid Chromatographic Determination of Cyclosporin A in Whole Blood with Use of a Rapid Extraction Procedure

In this precise, reversed-phase liquid chromatographic procedure, Cyclosporin A (Cy A) is extracted from 300 microL of whole blood with cyclosporin D as internal standard by liquid-liquid extraction. A 20-microL aliquot of the extract, injected onto a Nucleosil octyl analytical column heated at 72 degrees C, is eluted with a mixture of acetonitrile and 0.01 M phosphate buffer, pH 5.5 at a flow rate of 1 mL/min. Detection is set at 210 nm. The chromatography is complete within 10 min. The detection limit is 25 micrograms/L. Between-run CVs range from 2.3 to 6.2% and recovery is 92.1 +/- 6.3%. The major advantages of our extraction procedure are the rapid clean-up method and the small volume required. This procedure is especially suitable for cyclosporine determination in pediatric transplantations.

Direct chemiluminescence immunoassay of estradiol in saliva

A sensitive and simple direct solid-phase chemiluminescence immunoassay is described for estradiol in saliva. In this assay, a second antibody is bound to the wells of microtiter plates. Either buffer with standards or saliva (100 microL) is incubated in these wells with monoclonal anti-estradiol antibody and with estradiol-isoluminol conjugate. Incubation time is 2 h. Chemiluminescence of the bound fraction is measured in a manually operated luminometer (Biocounter). The assay has a detection limit of 3.8 pmol/L; analytical recovery of added estradiol is 96.8% (SD 7.0%); within- and between-assay CVs range between 2.5% and 12.7%. Forty unknown saliva samples can be assayed and results calculated within 4.5 h. Results of a slightly modified procedure-with black microtiter plates and a prototype of an automated plate reader (Luminoskan)--compare well with those of the described method (r = 0.97). Because steroid-binding globulins have been found in saliva, the effect of displacing agents on the results of the direct chemiluminescence assay is described.

Determination of Chlorinated Hydrocarbon Pesticides by Solid-Phase Extraction and Capillary GC with Electron Capture Detection*

Pesticides and their metabolites are extracted and concentrated from serum using C18 solid-phase extraction cartridges. The internal standard aldrin is added to 4 mL of serum or plasma, treated with 2 mL of methanol, and the resultant supernatant applied to the C18 cartridge. After several washes, pesticides are eluted from the column with isoctane and the eluate quantified with capillary gas chromatography using electron capture detection. Extraction efficiency is from 70 to 75% and the method detection limit ranges from 0.1-0.7 ng/mL depending on the analyte. Precision studies demonstrate that CVs range from 3.5 to 25.2%. Standard curves are linear to at least 7 ng/mL for lindane and chlordane isomers, heptachlor, heptachlor epoxide, oxychlordane, trans-nonachlor, dieldrin, dichlorodiphenyldichloroethylene (p,p'-DDE), dichlorodiphenyldichloroethane (p,p'-DDD), and dichlorodiphenyltrichloroethylene (p,p'-DDT).

Analyst and Observer Variability in Density Estimates from Spot Mapping

We studied variation in density estimates from spot mapping that was attributable to analysts and observers, using expert birders with little or no prior experience with spot mapping. Three observers independently spot mapped one 42-ha plot (Markwood) in mixed-conifer forest, and four others independently spot mapped another (Teakettle). All observers analyzed all maps. Consistency among analysts and observers in estimating the numbers of territories of breeding species on each plot was generally poor. Across all combinations of analysts and maps, 71% of all ANOVAs had significant analyst and/or observer effects. Observer effects were generally greater than analyst effects. When observers analyzed their own species maps, CVs of individual species ranged from 0% to 173% (mean 41%) at Markwood and from 0% to 188% (mean 60%) at Teakettle. As in similar studies, mean CVs from pooled totals of all species were less than those from individual species and were within the range of variation found by other researchers. Based on the range of CVs observed among species in this study, the number of sample plots needed to detect a statistical difference in density of a given species between samples is probably prohibitive for most studies. Instead, practitioners need to design studies to control observer and analyst variability.

Multilayer fluorescent immunoassay technique.

We describe a new multilayer immunoassay element for the determination of haptens in undiluted serum and plasma. Polysaccharide layers are coated onto a plastic base. The signal layer contains an immobilized antibody and a fluorescent-labeled hapten. A second layer, containing a pigment, acts as an optical screen. Sample spreading is achieved by a molded grid in contact with the upper layer of the immunoassay element. After sample is added to the element, endogenous analyte competes with the labeled hapten for the binding sites of the immobilized antibody; equilibrium is reached in 4-12 min. Because of the relative liquid-holding capacities of the layers and the grid, only a small amount of the free components remains in the signal layer. The signal is measured by front-surface fluorimetry. This technology has been applied to theophylline and thyroxin assays. Within- and between-run CVs range from 3% to 6%. Comparisons with fluorescent polarization immunoassays (Abbott TDx) showed excellent correlation (theophylline: r = 0.98, slope = 1.07, intercept = 0.3; thyroxin: r = 0.97, slope = 0.91, intercept = 0.8). The new method requires only one pipetting step (sample delivery) and is potentially applicable to a wide range of analytes.

Turbidimetric latex immunoassay of placental lactogen on microtiter plates.

In this latex immunoassay for human placental lactogen, microtiter plates are used as the reaction vessel and the absorbance at 405 nm is measured to quantify the reactions. This 30-min assay necessitates only one serum dilution and two pipetting steps. The calibration curve extends from 0.5 to 15 mg/L. CVs range from 4.2% to 7.0% for within-run determination and from 7.0% to 11.2% for between-run determinations. A correlation coefficient of 0.949 was obtained for 84 sera when the method was compared with a commercial radioimmunoassay.

Nonisotopic "sandwich" immunoassay of thyroglobulin in serum by the biotin-streptavidin technique: evaluation and comparison with an immunoradiometric assay.

In this sequential assay, the thyroglobulin in serum binds to polystyrene beads coated with two mouse monoclonal antibodies. These beads then react with a rabbit polyclonal antibody, biotinylated sheep anti-rabbit IgG, streptavidin-horseradish peroxidase, and a peroxidase substrate to yield a colored product that is measured spectrophotometrically at 492 nm. The range of the standard curve is 1.6 to 100 micrograms/L. The detection limit of the assay is 1.2 micrograms/L. The interassay coefficient of variation is 14.0% at 6.2 micrograms/L, 5.7% at 32.7 micrograms/L; the intra-assay CVs range from 7.8% to 14.8%. The reference intervals are 2.7 to 42.1 micrograms/L for euthyroid persons and less than or equal to 5 micrograms/L for athyreotic patients not on thyroxin replacement therapy. Some autoantibodies to thyroglobulin cause thyroglobulin values to be falsely low. The concentration of autoantibodies is not correlated with the analytical recovery of human thyroglobulin. The coefficient of correlation between the measurement of thyroglobulin by this assay and by an immunoradiometric assay was 0.969 for 186 autoantibody-negative samples, 0.963 for 37 autoantibody-positive samples.

New one-step enzyme immunoassay for free thyroxin.

This enzyme immunoassay for free thyroxin (FT4) involves simultaneous incubation of sample and thyroxin conjugated to horseradish peroxidase (EC 1.11.1.7) in antibody-coated tubes at room temperature. The test can be performed in 90 min. It is not influenced by variations in concentrations of thyroxin-binding globulin (TBG), thyroxin-binding prealbumin, or albumin. With increased concentrations of non-esterified fatty acids, the measured FT4 equivalent concentrations increase immediately, indicating displacement of endogenous thyroxin from its binding sites. FT4 values obtained with this assay for 110 samples, including sera with low albumin and high TBG as well as sera of patients treated with heparin, agreed well with those measured by a two-step radioimmunoassay and with the T4/TBG ratio. The measurable range of FT4 with our assay is 2.5 to 65 ng/L. Intra-assay CVs range from 3.4% to 2.2%, interassay CVs from 6.6% to 2.6% at concentrations of 9 to 45 ng of FT4 equivalent per liter.