Imprecision Profile Research Articles

Processing Validation Metrics of Syva Enzyme-Multiplied Immunoassay Technique (EMIT) Methotrexate Assay for Beckman Coulter System.

High-dose methotrexate (HDMTX), defined as a dose greater than 500 mg/m2, is used to treat a variety of cancers; and though safe, it can cause major toxicity. Syva enzyme-multiplied immunoassay technique (EMIT) methotrexate (MTX)assay(Gurgaon, India: Siemens Healthcare Diagnostics Ltd.) uses a homogeneous enzyme immunoassay method. Low-end precision performances are very important for laboratory methods, especially when their results have clinical significance at these levels. A total of 25 replicates (five replicates per run, for five runs) were analyzed for profiling. Precision, accuracy, linearity, limit of blank, limit of detection, and limit of quantification were determined using existing guidelines. Imprecision profile and limit of quantitation (LoQ) at 10% were determined by fitting data with hyperbolic regression. The coefficient of variation percentage (CV%) for low, mid, and high-level internal quality control (IQC) was 1.25%, 3.45%,and 1.55%, respectively. Similarly, estimated bias was -4.58%, -3.54%, -7.21% for each level. The assay linearity was maintained from a range of 0.041-1.993 mmol/L with an R2 of 0.959. The limit of detection was estimated to be 0.07 mmol/L. Syva EMIT MTX assay can be precisely and accurately used to measure low levels of serum methotrexate at levels lower than claimed by the manufacturer, aiding in the monitoring of toxicity in patients.

Automation on an Open-Access Platform of Alzheimer's Disease Biomarker Immunoassays.

The lack of (inter-)laboratory standardization has hampered the application of universal cutoff values for Alzheimer's disease (AD) cerebrospinal fluid (CSF) biomarkers and their transfer to general clinical practice. The automation of the AD biomarker immunoassays is suggested to generate more robust results than using manual testing. Open-access platforms will facilitate the integration of automation for novel biomarkers, allowing the introduction of the protein profiling concept. A feasibility study was performed on an automated open-access platform of the commercial immunoassays for the 42-amino-acid isoform of amyloid-β (Aβ1-42), Aβ1-40, and total tau in CSF. Automated Aβ1-42, Aβ1-40, and tau immunoassays were performed within predefined acceptance criteria for bias and imprecision. Similar accuracy was obtained for ready-to-use calibrators as for reconstituted lyophilized kit calibrators. When compared with the addition of a standard curve in each test run, the use of a master calibrator curve, determined before and applied to each batch analysis as the standard curve, yielded an acceptable overall bias of -2.6% and -0.9% for Aβ1-42 and Aβ1-40, respectively, with an imprecision profile of 6.2% and 8.4%, respectively. Our findings show that transfer of commercial manual immunoassays to fully automated open-access platforms is feasible, as it performs according to universal acceptance criteria.

The 99th percentile and imprecision of point-of-care cardiac troponin I in comparison to central laboratory tests in a large reference population

ObjectivesDetermination of cardiac troponin (cTn) is central in the emergency department (ED) for the diagnosis of myocardial infarction. In view of adverse effects of long waiting time on patient outcome, implementation of point-of-care-testing (POCT) is suggested if the turn-around-time is longer than 60min. The present study aimed to determine the 99th percentile and imprecision of two POCT in a healthy population measuring cTnI and cTnT and compare these analytical characteristics against three central laboratory test (CLT) for cTnI. Design & MethodsCTnI and cTnT were determined in parallel by means of the AQT90 FLEX analyzer in about 2250 plasma samples from individuals with known health status. Results were compared to previously determined performance data of three CLT. ResultsThe 99th percentile of cTnI in the POCT was determined at 19ng/L, the lowest concentration with an imprecision of 10% was reached at 22ng/L while an imprecision of 20% was reached at 13ng/L. Age, sex, or physical activity did not affect the 99th percentile of cTnI. Compared to CLT the AQT90 cTnI POCT the analytical performance was equivalent. The cTnT POCT could not be assessed due a considerable number of high values and an inadequate imprecision profile. ConclusionWhile the cTnI POCT showed analytical performance comparable to CLT, the results of the cTnT assay on the same device did not suffice to determine a reliable 99th percentile. The present evaluation supports the usage of the cTnI POCT, but application of the cTnT POCT needs further evaluation.

The clinical and diagnostic performance characteristics of the high sensitivity Abbott cardiac troponin I assay

ObjectivesThe aim of this study is to determine the imprecision profile, 99th percentile and diagnostic efficiency of a new high sensitivity cardiac troponin I (cTnI) assay. MethodsTotal imprecision was assessed by following CLSI protocol EP15-A.14. Serum pools prepared from sera of known high cardiac troponin concentrations were adjusted by dilution with serum considered to be troponin free. Determination of the 99th-percentile reference value examined a fully characterized population that had undergone non-invasive cardiac imaging. Diagnostic accuracy utilised samples from the point of care arm of the RATPAC trial (Randomised Assessment of Treatment using Panel Assay of Cardiac markers), set in the emergency departments of six hospitals. Blood samples were taken on admission and 90min from admission. Diagnosis was based on the universal definition of myocardial infarction utilising laboratory measurements of cardiac troponin performed at the participating sites together with measurements performed in a core laboratory and compared by construction of receiver operator characteristic curves. ResultsTotal imprecision was 4%–12.1% with 10% CV of 7ng/L. cTnI was measureable in 99.5% of the samples. Troponin values were influenced by gender but not by age. The 99th percentile was 14.8ng/L (18.1 males, 8.6 females). Progressive filtering of the population reduced the 99th percentile. For the diagnosis of MI on admission the area under the curve was 0.92, statistically indistinguishable from four other assays studied (0.90–0.94). ConclusionThe analytical performance of the new assay meets the criteria for a high sensitivity troponin assay.

Comparison of ordinary linear regression, orthogonal regression, standardized principal component analysis, Deming and Passing-Bablok approach for method validation in laboratory medicine

Abstract A well-accepted tool for method validation is a method comparison study. Results are usually assessed on a scatter plot of which the fitting line is calculated by several approaches, for example, ordinary (vertical) linear regression (OLR), orthogonal regression (OR), Deming regression (DR), Passing-Bablok method (PBR) or standardized principal component regression (SPCR). DR was applied in its general form (gDR), requiring information of the imprecision of at least two different quantities and as simple DR (sDR) with imprecision information of only one quantity. The equation of the regression line calculated by these concepts varies depending on range of measurement, analytical variation and on imprecision ratio (s AY /s AX ). There is still a global debate about which statistical concept is the most adequate for validating purposes. Various paired random samples with a size of 100 were simulated in 5000 replicates and evaluated with different regression models. The behavior of the slope and intercept of the regression lines were compared under various conditions. Two extreme ranges of measurement and several variance ratios in the absence and presence of bias were studied. The results clearly demonstrated that DR is the only model which can be applied without any precautions under conditions which usually occur in method comparison studies, and therefore should be preferred in laboratory medicine. Other models require restrictions with regard to range of measurement and/or imprecision profile. Differences of the concentrations at different positions of the measurement interval calculated with regression coefficients of both DRs did not deviate more than the permissible bias. Therefore, the advantage of using gDR does not justify its greater disadvantages in comparison with sDR.

Analytical characteristics of the Roche highly sensitive troponin T assay and its application to a cardio-healthy population

It is desirable that current assays for cardiac troponin (cTn) are able to meet the recommended criterion that the diagnosis and risk assessment of patients present with symptoms of myocardial infarction requires a rise and fall in cTn with at least one point above the 99th percentile of a reference population. We have evaluated the analytical characteristics of the new highly sensitive troponin T (hs-TnT) assay to see if it meets this criterion and applied it to a carefully defined, cardio-healthy Australian reference population. An imprecision profile was determined for the Roche hs-TnT assay (Roche Diagnostics, Sydney, Australia) using multiple samples analysed on nine separate occasions. The distribution of troponin T was determined using 104 samples from a cardio-healthy population. The new hs-TnT assay meets the specifications of a coefficient of variation of 10% at the 99th percentile of our cardio-healthy reference population. Of the 104 samples analysed 44 showed troponin T concentrations above the manufacturer's quoted limit of detection. Age and gender differences in the median and 99th percentile troponin T concentration were observed. The new hs-TnT assay shows improved precision and sensitivity at very low troponin concentration. We have shown that a significant number of individuals in this cardio-healthy population had detectable circulating troponin concentration. With many apparently healthy people having detectable troponin, clinical judgement will become more important in interpreting troponin results.

The determination of the 99th centile level for troponin assays in an Australian reference population

Current guidelines for the diagnosis and risk assessment of patients presenting with myocardial infarction recommend a single decision cut-off point for cardiac troponin (cTn) based on the 99th centile of a reference population. The 99th centile level for eight troponin assays was determined in an apparently cardio-healthy Australian reference population. Nine laboratories measured troponin in serum and plasma collected from 111 reference individuals. An imprecision profile was determined using up to 10 serum samples analysed on 10 separate days. Method comparison using 100 routinely tested plasma samples was performed to estimate method concordance. Generally 99th centile values determined in this study were lower than, or the same as manufacturers' levels, except for cTnI by Architect (0.020 vs. 0.012 microg/L), and imprecision at the 99th centile was 20% coefficient of variation (CV) or higher. Troponin concentrations at 10% CV were greater than those quoted in the manufacturer's package insert except by AxSYM, 0.06 vs. 0.16 microg/L cTnI, and by E-170, 0.02 vs. 0.03 microg/L cTnT. In the method comparison 74, 70, 65, 75, 58, 66, 58 and 77 samples measured by Access, Architect, AxSYM, Centaur, Dimension RxL, E-170, i-STAT and Vitros ECi assays, respectively, had troponin concentrations above the study 99th centile. Depending on the selected reference population for troponin, there is likely to be variability in the 99th centile as shown in this study. Some differences in sample concordance at the 99th centile cut-off were observed between cTn methods and may result in different clinical classification.

Performance of the Advia Centaur second-generation troponin assay TnI-Ultra compared with the first-generation cTnI assay

A cardiac troponin concentration above the 99th percentile limit of a reference population is a sensitive marker of myocardial necrosis. Current guidelines require troponin assays to have a total imprecision of < or =10% at the 99th percentile limit. In this study, the Advia Centaur second-generation TnI-Ultra assay was validated and compared with its predecessor the cardiac troponin I (cTnI) assay, with a focus on the current guidelines for diagnosis of acute myocardial damage. An imprecision profile of the TnI-Ultra assay was evaluated by analysing different pools over 20 days. The imprecision of the cTnI assay was used as comparison. The reference range was established based on TnI-Ultra analysis in 221 individuals. The cTnI concentration that could be determined with a total imprecision of 10% was 0.05 microg/L for the TnI-Ultra assay and 0.3 microg/L for the cTnI assay. The 99th percentile limit in the distribution of a reference population was 0.06 microg/L as determined with the TnI-Ultra assay. The TnI-Ultra assay provides significantly improved sensitivity when compared with the cTnI assay and a total imprecision of < or =10% is obtained at the 99th percentile limit of value distribution of a reference population. Using the TnI-Ultra assay, slightly increased cTnI concentration can be detected reliably following the current guidelines.

Evaluation of analytical performance of Advia ® TnI ultra immunoassay and comparison with Access ® AccuTnI™ method

We evaluated and compared the analytical and clinical performance of the TnI-Ultra immunoassay for cardiac Troponin I (cTnI) measurement, carried out on the fully automated ADVIA Centaur and ADVIA Centaur CP® platforms (Siemens Medical Solutions Diagnostics SrL), and that of Access AccuTnI™, carried out on the UniCell® DxI 800 platform (Beckman Coulter, Inc., Fullerton). We assayed 645 plasma Li-heparin samples of healthy subjects and 230 of patients with cardiovascular diseases (70 with a cTnI value higher than the decision level for acute myocardial infarction) by TnI-Ultra and Access AccuTnI™ immunoassays. The total imprecision (CV) profile of TnI-Ultra method was determined by repeatedly measuring 19 plasma pools, with different concentrations of TnI, in 20 different runs using two reagent lots; the CV ranged from 25% (at 0.02 μg/L) to less than 5% (for levels greater than 0.4 μg/L). On average, functional sensitivity (i.e. concentration measured with an imprecision of 10% CV) of TnI-Ultra method was 0.057 μg/L. The 99th percentile of normal distribution, calculated by enrolling a control group of 645 healthy subjects (278 males and 367 females; mean age of 47.4 ± 16.2 years, range 15–89 years), was 0.072 μg/L. Therefore, the ratio between functional sensitivity and 99th percentile values was less than 1 (i.e., 0.057/0.072 = 0.79), while for Access AccuTnI™ resulted 0.07/0.06 = 1.16. A close linear regression was found between the cTnI values obtained with the two methods only when the samples of patients with cardiac disease were taken into account (TnI-Ultra = −0.192 + 1.434 AccuTnIT; R = 0.976; n = 230). A significant bias was found between the two methods by means of Bland-Altman plot (mean difference 38.9%), but when 368 healthy subjects were considered, no significant linear regression was found between these two methods (R = 0.002, p = 0.9624). Our results indicate that the TnI-Ultra immunoassay carried out on the fully automated ADVIA Centaur platforms shows analytical characteristics well in agreement with the quality specifications recommended for cardiac troponin assay. Nous avons évalué et comparé les prestations analytiques et cliniques du dosage immunologique TnI-Ultra pour la quantification de la troponine I cardiaque effectué sur les plateformes complètement automatiques ADVIA Centaur et ADVIA Centaur CP (Siemens Medical Solutions Diagnostics SrL) ainsi que la performance d’Access AccuTnI™, conduite sur la plateforme de UniCell® DxI 800 (Beckman Coulter, Inc., Fullerton). Nous avons analysé 645 échantillons de plasma Li-héparine de sujets sains et 230 patients atteints de maladies cardiovasculaires (70 avec une valeur de cTnI plus élevée que la valeur-seuil pour l’infarctus du myocarde) à l’aide des dosages TnI-Ultra et Access AccuTnI™. Le profil d’imprécision de la méthode TnI-Ultra a été déterminé en mesurant plusieurs fois 19 pools de plasma à différentes concentrations de TnI, dans 20 différentes courses utilisant deux lots de réactifs ; le CV s’étendait entre 25 % (à 0,02 μg/L) et inférieur à 5 % (pour les valeurs supérieur à 0,4 μg/L). En moyenne, la sensibilité fonctionnelle (c’est-à-dire la concentration mesurée avec une imprécision de 10 % CV) de la méthode TnI-Ultra était 0,057 μg/L. Le 99e percentile de la distribution normale, calculée par recrutement d’un groupe témoin des 645 sujets sains (278 hommes et 367 femmes ; moyenne d’âge 47,4 ± 16,2 ans, compris entre 15–89 ans), était 0,072 μg/L. Donc, le rapport entre la sensibilité fonctionnelle et les 99es valeurs percentiles était moins que 1 (0,057/0,072 = 0,79), pendant que pour Access AccuTnI™ il était 0,07/0,06 = 1,16. Une forte régression linéaire était détectée entre les valeurs cTnI obtenues avec les deux méthodes seulement quand les échantillons de patients atteints de maladie cardiaque étaient pris en considération (TnI-Ultra = −0,192 + 1,434 AccuTnIT ; R = 0,976 ; n = 230). Un biais significatif était détecté entre les deux méthodes par le plot de Bland-Altman (différence moyenne 38,9 %), mais quand les 368 sujets sains étaient considérés, on ne trouvait aucune régression linéaire significative entre ces deux méthodes (R = 0,002, p = 0,9624). Nos résultats indiquent que le dosage immunologique TnI-Ultra effectué sur les plateformes complètement automatiques ADVIA Centaur démontre des caractéristiques analytiques très en accord avec les spécifications de qualité recommandés pour le dosage de la troponine cardiaque.

Redefining functional sensitivity of thyroglobulin assay on Immulite platform: implications in thyroid cancer management

Circulating thyroglobulin (Tg) measurement after thyrotropin stimulation is a pivotal tool in the management of patients affected by differentiated thyroid carcinoma. The Tg assay on Immulite platform has a declared functional sensitivity of 0.9 ng/mL and it is widely used in clinical practice and research on thyroid carcinoma. Recently, thyroglobulin measured during thyroxine treatment was found to be adequate for thyroid carcinoma follow-up if functional sensitivity at 0.2-0.3 ng/mL was reached by assay methods. Thus, the present study was then undertaken to evaluate the imprecision of Immulite Tg assay at very low concentrations. The detection limit was calculated on zero calibrator and Tg-free pooled sera replicates and was 0.14 and 0.16 ng/mL, respectively. Different serum pools with Tg concentrations ranging from 0.16 to 2.50 ng/mL were assayed according to the National Academy of Clinical Biochemistry guidelines to estimate functional sensitivity. By interpolating the imprecision profile with a coefficient of variation of 20%, the functional sensitivity was 0.36 ng/mL. The Immulite-Tg assay was directly compared with a high-sensitive immunoradiometric Tg assay in sera from 93 patients with thyroid carcinomas. No significant differences in sensitivity and specificity were observed by using the newly defined functional sensitivity. In conclusion, the Immulite Tg assay showed a lower functional sensitivity than expected and performed comparably to high-sensitive immunoradiometric assay in patients with thyroid carcinoma.

Validation of the 99th Percentile Cutoff Independent of Assay Imprecision (CV) for Cardiac Troponin Monitoring for Ruling Out Myocardial Infarction

By definition, the probability that a single measured cardiac troponin result exceeds the estimated 99th percentile is equal to 0.01 (1%) for a person randomly selected from the general (reference) population. This is irrespective of the imprecision profile of the analytical method as well as the lack of standardization of cardiac troponin assays (1)(2)(3)(4)(5)(6)(7). However, the probability that a measured result exceeds the 99th percentile limit for a specific person from the reference population will vary depending on that person’s true concentration and on the imprecision profile of the specific analytical method. Furthermore, the overall probability that at least one of multiple measured values (the second or third measured cardiac troponin concentration in a timed series of cardiac troponin orders) exceeds the 99th percentile also will vary depending on the imprecision of the analytical method. To investigate the influence of assay imprecision on the likelihood of misclassifying healthy individuals or patients without myocardial injury, we simulated the distribution of cardiac troponin I (cTnI) results in a general population and added random analytical error reflecting different assay imprecision profiles. One imprecision profile assumes a CV of 37.5% at a cTnI of 0.05 μg/L, decreasing to a CV of 25% at a cTnI of 0.07 μg/L, …

Usefulness of high‐sensitivity IL‐6 measurement for clinical characterization of patients with coronary artery disease

Interleukin 6 (IL-6) may represent an early marker of inflammatory activation and may be useful to ameliorate risk stratification in patients with ischemic heart disease. The aim of this study was to verify the performance characteristics of an ultrasensitive immunoassay (Biosource International, Camarillo, CA) for high-sensitivity (hs)-IL-6 measurement in comparison with hs-R&D Systems (Abingdon, United Kingdom) and Immulite System (Diagnostic Products Corporation [DPC], Los Angeles, CA) methods in patients with ischemic heart disease. In addition, hs-C-reactive protein (hs-CRP) concentrations were measured, to evaluate the correlation with hs-IL-6 levels. We measured IL-6 and CRP serum levels in 39 patients with ischemic heart disease and in 12 controls. Out of the 39 patients studied, 13 were affected by unstable angina, 13 by post-acute myocardial infarction (AMI) unstable angina, and 13 by stable angina. The imprecision profile and functional sensitivity were performed measuring 9 different serum pools in 10 runs. The Biosource method had the best performance characteristics as compared to the others. Mean IL-6 level was higher in patients with unstable and post-AMI unstable angina with respect to controls. CRP levels were elevated in patients with post-AMI. In the whole population a high significant linear regression was observed between Biosource hs-IL-6 and hs-CRP serum levels. The Biosource method for IL-6 measurement is characterized by a high functional sensitivity that allows a better stratification of patients with ischemic heart disease.

A method to estimate the uncertainty of measurements in a conglomerate of instruments/laboratories

A comparison of the performance, at two concentrations, of the measurement procedures for 23 common components is presented. For each component the within‐, pure between‐ and total variations have been calculated and an imprecision profile indicated. Thus a pragmatic, readily and generally applicable method is advised to describe the performance of laboratories under repeatability and reproducibility conditions. The calculated total variation is compared with the results estimated from the biological variation of the properties. In most cases the within‐laboratory variation is better than that given by the reference database, whereas in about half of the procedures the total variation exceeds that of the reference database. The outcome illustrates the transferability problems that multicentre studies and the diagnosis of patients face by measuring samples using a conglomerate of instruments/laboratories that are not aligned. The calculation procedures are detailed in an appendix and the calculation and presentation of the results are made with a custom‐made worksheet program.

Quality specifications for biochemical markers of myocardial injury

Background: The current approach to the diagnosis and monitoring of myocardial damage, recognizes to biochemical markers, and in particular to troponins, a key role being well demonstrated that all elevated values were associated with a worsened prognosis. In 2001, the IFCC Committee on Standardization of Markers of Cardiac Damage published guidelines addressing the quality specifications for troponin assays in order to guarantee an analytical performance satisfying medical requirements and to standardize the quality of commercial methods. We describe how the application of quality specifications may be useful in daily practice, in order to provide advice to clinicians in the investigations of complex clinical cases of patients suffering from myocardial damage. Materials and methods: The samples from three patients (cases 1–3) admitted to the hospital with symptoms suggestive of cardiac disease, showing high troponin I (cTnI) values not correlated with clinical condition, were investigated in order to verify the accuracy of the laboratory data. The standard of quality specifications related to assay specificity, imprecision and interferences were evaluated using different platforms for cTnI assays, carrying out imprecision profile and specific studies on more common interferents in immunoassays. Results: The obtained results allow us to demonstrate two cases of false-positive cTnI values attributable to a macrocomplex between a modified “in vivo” cTnI and immunoglobulin G (case 1) and to a presence of heterophilic antibodies affecting the RxL Dimension procedure (case 3). Instead, the accuracy of data obtained in case 2 was evidenced by the imprecision profile obtained in our laboratory and by the comparison of results between different laboratories using same platform. Conclusions: The lack of standardization as well as the wide differences in the development of each assay give rise to major concerns regarding cTnI determinations. The laboratory must therefore check the compliance between the analytical characteristics of the method utilised against recommended quality specifications for a reliable understanding of the frequency of false-positive results as well as other serious analytical errors.

Imprecision of cerebrospinal fluid net bilirubin absorbance.

Analysis of cerebrospinal fluid (CSF) has an important role to play in the diagnosis of subarachnoid hemorrhage (SAH) (1). Computed tomography, the first line of investigation for suspected SAH, shows a decrease in diagnostic accuracy as the time interval between the suspected hemorrhage and the time of investigation increases. A computed tomography scan would be positive in 98% of patients presenting within 12 h after an event, but positivity decreases to ∼50% in patients presenting after 1 week, 30% after 2 weeks, and 0% after 3 weeks (2). An increase in bilirubin in the CSF is the key finding supporting the occurrence of SAH (3). Guidelines for the analysis of CSF for bilirubin in suspected SAH have recently been published in the United Kingdom (3). The presence of bilirubin is assessed by calculating the net bilirubin absorbance (NBA) according to Chalmers’ modification (4) to the original method of Chalmers and Kiley (5). A single NBA cut point of 0.007 absorbance units (AU) is recommended in the decision tree for interpretation and reporting of results (3). The interpretative comment “No evidence to support SAH” is advised if the NBA value is ≤0.007 AU and no oxyhemoglobin is detected. In contrast, if the corrected NBA is >0.007 AU and no oxyhemoglobin is detected, the interpretative comment “Consistent with SAH” is recommended (3). Laboratories and clinicians need to define and be aware of the quality of the tests they provide when these tests are the basis for clinical decisions. To the best of our knowledge, the analytical imprecision profile for NBA has not been published to date. Our laboratory has performed 92 analyses of CSF for suspected SAH in the last year. Of …

Estimating imprecision profiles in biochemical analysis

We describe a computer program IMPROFIL which determines an imprecision profile of an analytical method from replicated measurements of samples. It calculates the variance function, the coefficient of variation, the power of definition, the critical limit, the limit of detection and the lower limit of quantification. The primary property, the variance function, is determined by two alternative methods: the conventional maximum approximate conditional likelihood method and the newly developed weighted absolute deviation method. For all quantities, confidence intervals are obtained using the bootstrap procedure. The program combines the use of robust numerical techniques, user-friendliness and integration into a spreadsheet program for data pre- and post-processing. The algorithms used are described in detail. Tests with synthetic data sets are used to validate the method and to establish its powers and limitations. Finally, its application to a practical analytical task (tumor marker CA 15-3 in human sera) is reported. For the method to yield a reliable estimate of the variance function and the derived properties, certain minimum requirements on the raw data must be met: They have to be spread throughout the concentration range of interest, there should not be less than three replicates per specimen, and there must be at least of the order of 25 (better at 50) specimens.

The imprecision profile in immunoassay: A study using a resampling technique

A resampling ('bootstrap') technique was applied to assess the reliability of the calculated imprecision profile (IP), as obtained from the dose/response curve and the response/error relationship (RER) using the cumulative data relative to two assays, i.e. a T4 radioimmunoassay (RIA) and a TSH immunofluorometric assay (IFMA), both run in duplicate. Mean values and the related uncertainty of the estimated dose errors were compared for different RER fitting conditions and different sizes of the duplicate response sets. The following observations were made: (a) compared to the maximum-likelihood procedure, the least-square fit proved to be unsuitable for estimating the parameters in the general RER equation variance(R) = aRb (where R indicates the response), (b) the simplifying assumption of a within-method constancy of the exponent in the RER equation, while acceptable for the T4 RIA, did not hold in the case of the TSH IFMA implying a much wider response range, (c) for both assays, response sets of ca. 100 duplicates were apparently compatible with an acceptable definition of the IP (+/- 10 to +/- 20% uncertainty).

Radioimmunoassay of epidermal growth factor in human saliva and gastric juice

A sensitive radioimmunoassay was developed for human epidermal growth factor (hEGF) in saliva and gastric juice. This method was sufficiently sensitive for an accurate measurement of hEGF in these biological fluids. The minimal detectable concentration of EGF was 30 ng/L. The imprecision profile of EGF standard curve had a CV less than 10% in the range of 0.1-3.0 micrograms/L. Serial dilution curves of saliva and gastric juice paralleled that of standard EGF. The antibody to hEGF showed no cross-reactivity with a large excess of growth factors, such as human transforming growth factor alpha, human insulin-like growth factor I, and platelet-derived growth factor (c-sis). No detectable cross-reactivity was observed with some biological gut peptides: somatostatin, gastrin, secretin or pancreatic polypeptide. The intra-assay CV for saliva and gastric juice was less than 10%, and the recoveries were 93.9 +/- 8.7% and 93.7 +/- 11.3%, respectively for saliva and gastric juice. Gel exclusion chromatography revealed hEGF-like substances, heterogeneous in size in saliva and gastric juice, the origins and physiological functions of which are unknown.

Use and abuse of imprecision profiles: some pitfalls illustrated by computing and plotting confidence intervals

Published estimates of the imprecision characteristics of immunoassays are often based on quantities of data that seem to be inadequate. The increasing use of imprecision profiles has not necessarily improved the situation. We describe and illustrate a method of computing 95% confidence intervals for profiles estimated directly from replicated assay results. The data used were chosen to mimic the sort of data that are typically available in practice, either within laboratories or from external quality-assessment programs. Use of confidence intervals is an effective pictorial way of incorporating information about the quantity and the distribution of the data used for estimation. This is in keeping with an important property of imprecision profile plots: potentially complex information is summarized in a readily comprehensible way. A computer program is available for estimating and plotting profiles and their 95% confidence intervals.

Estimation of imprecision in immunoassay quality assessment programmes.

A computer was used to produce and store a large body of simulated thyroxine and thyrotropin radioimmunoassay results over wide concentration ranges. Sets of 24 results, designed to represent results returned to an external quality assessment survey, were randomly drawn from the stored results in order to compare limited data survey estimates of imprecision with the 'true' values obtained from the population data. Results show that estimation of an imprecision profile has advantages over the usual index of imprecision, but that neither form of estimate is particularly reliable when the available data is limited. Results suggest that survey organisers should place considerably less emphasis on results from any one survey and greater emphasis on analysing cumulative data and method comparison data.