Context.-It is important that the total long-term precision of laboratory methods meet the medical needs of the patients being served.Objectives.-To determine the long-term within- and between-laboratory variation of cortisol, ferritin, thyroxine, free thyroxine, and thyroid-stimulating hormone measurements using commonly available methods and to determine if these variations are within accepted medical needs.Design.-Two vials of pooled frozen serum were mailed 6 months apart to laboratories participating in 2 separate College of American Pathologists surveys. The data from those laboratories that analyzed an analyte in both surveys were used to determine for each method the total variance and the within- and between-laboratory components. Setting.-The study included the A mailing of the 2003 College of American Pathologists Ligand Survey and the C mailing of the Chemistry Survey.Main Outcome Measures.-For each analyte, total variance was partitioned into within- and between-laboratory components for each analytic method. The within-laboratory variations were then compared with imprecision criteria based on biological variation.Participants.-The laboratories that reported results on the same analyte using the same method in both surveys.Results.-For each analyte, the median of the long-term within-laboratory variances of each peer group was 78% to 95% of its total-survey variance, and the median long-term within-laboratory coefficients of variation varied from 5.1% to 7.6%. The number of methods that met within-laboratory imprecision goals based on biological criteria were 5 of 5 for cortisol; 5 of 7 for ferritin; 0 of 7 for thyroxine and free thyroxine; and 8 of 8 for thyroid-stimulating hormone.Conclusions.-For all analytes tested, the total within-laboratory component of variance was the major source of variability in this study. In addition, there are several methods, especially for thyroxine and free thyroxine, that may not meet analytic goals in terms of their imprecision.

Context.-In proficiency testing surveys, there are differences in the values reported by users of various analytic methods. Two contributors to this variation are calibrator bias and matrix effects of proficiency testing materials.Objectives.-(1) To quantify the biases of the analytic methods used to measure thyroid-stimulating hormone, thyroxine, triiodothyronine, free thyroxine, and free triiodothyronine levels; (2) to determine if these biases are within allowable limits; and (3) to ascertain if proficiency testing materials correctly identify these biases.Design.-A fresh frozen serum specimen was mailed as part of the 2003 College of American Pathologists Ligand and Chemistry surveys. The means and SDs for each analytic method were determined for this sample as well as for a proficiency testing sample from both surveys. In the fresh frozen serum sample, target values for thyroxine and triiodothyronine were determined by isotope dilution/liquid chromatography/tandem mass spectrometry. All other target values in the study were the median of the means obtained for the various analytic methods.Main Outcome Measures.-Calibration biases were calculated by comparing the mean of each analytic method with the appropriate target values. These biases were evaluated against limits based on intra- and interindividual biological variation. Matrix effects of proficiency testing materials were assessed by comparing the rank of highest to lowest analytic method means (Spearman rank test) for each analyte.Participants.-Approximately 3900 clinical laboratories were enrolled in the College of American Pathologists Chemistry and Ligand surveys.Results.-The number of methods in the Ligand Survey that failed to meet the goals for bias was 7 of 17 for thyroid-stimulating hormone and 11 of 13 for free thyroxine. The failure rates were 12 of 16 methods for thyroxine, 8 of 11 for triiodothyronine, and 9 of 11 for free triiodothyronine. The means of the analytic method for the proficiency testing material correlated significantly (P < .05) only with the fresh frozen serum means for thyroxine and thyroid-stimulating hormone in the Chemistry Survey and free triiodothyronine in the Ligand Survey.Conclusions.-A majority of the methods used in thyroid function testing have biases that limit their clinical utility. Traditional proficiency testing materials do not adequately reflect these biases.

CONTEXT:The College of American Pathologists (CAP) provides proficiency testing (PT) surveys to laboratories around the world.OBJECTIVES:To compare diagnostic assay methods for serum/plasma cortisol and immunoglobulin (Ig) E in terms of their bias and precision, to determine how well CAP PT specimens simulate human serum, and to reassess proficiency test grading criteria in light of these findings.DESIGN:A participant-blinded, prospective trial. One vial of pooled fresh frozen serum (FFS) and 4 different admixtures of PT material (PTM) were sent to laboratories participating in PT surveys.PARTICIPANTS:Laboratories providing cortisol (>1000) or IgE (>230) results among the subscribers to the CAP surveys, Ligand (General) 2003, set K/KN-A and Chemistry 2003, set C-C.MAIN OUTCOME MEASURES:The main outcome measures were (1) bias among laboratories using the same method (peer groups), defined relative to the median of method means (MedMM); (2) imprecision as measured by the SD and coefficient of variation (CV) about each method mean; and (3) total error across laboratories for the FFS cortisol results, defined as |Bias Relative to Reference Method| + 2 SD.RESULTS:Cortisol method biases, relative to MedMM, ranged from -22% to 9% for the FFS challenge and from -24% to 36% for comparable PTM challenges. The method biases, relative to the reference method, ranged from -3% to 19% for the FFS challenge. The cortisol method CVs ranged from 4.2% to 13.6% for the FFS challenge and from 4.7% to 12.7% for comparable PTM challenges. Total error across laboratories ranged from 1.4 to 6.9 microg/dL (39 to 190 nmol/L) for the FFS challenge. Immunoglobulin E method biases, relative to MedMM, ranged from -8% to 9% for the FFS challenge and from -7% to 5% for comparable PTM challenges. The IgE method CVs ranged from 3.6% to 6.7% for the FFS challenge and from 3.4% to 9.8% for comparable PTM challenges.CONCLUSIONS:The bias for cortisol results was less with FFS than with PTM, but imprecision was comparable. The FFS MedMM was 8.5% higher than the reference value. Fresh frozen serum and PTM bias and imprecision for IgE methods were each less than 10%. Because some of the methods demonstrated greater bias when analyzing PTM than FFS, peer group grading of both these analytes is appropriate.

Objective.-To evaluate the cross-reactivity of the 6 most abundant cyclosporine A (CsA) metabolites in commonly used assays for CsA. Design.-Whole blood samples containing either only 62 ng/mL CsA (A) or 62 ng/mL CsA and between 49 and 86 ng/ mL of 1 of the 6 most abundant CsA metabolites (B) were lyophilized. One sample of A and 1 of B were mailed to each of the laboratories participating in the College of American Pathologists Proficiency Testing Program quarterly during a 3-year period (1999-2001). Method means and coefficients of variation were calculated for each mailing. Results.-The study showed significant cross-reactivity of metabolites in all the immunoassay systems studied. Overall degree of interference decreased from AbbottTDx polyclonal > Abbott TDx monoclonal > DiaSorin > Syva EMIT. High-performance liquid chromatography methods gave results close to those found using mass spectrometric techniques. Conclusions.-Significant metabolite interference was found to occur with the immunoassay systems studied.

The biggest landmark in the treatment of epilepsy was the discovery of phenytoin. Phenytoin was introduced in 1938 and is still the most widely used anticonvulsant drug, probably because of its nonsedative properties. It is commonly used to treat general motor (tonic-clonic, grand mal) and focal seizures and is less effective in the treatment of complex partial seizures.The mechanism of action of phenytoin is not clear. However, phenytoin stabilizes membranes in the brain (and thereby suppresses seizures) and in the heart (and hence suppresses arrhythmias). It has been suggested that phenytoin suppresses seizures by blocking posttetanic potentiation by influencing synaptic transmission. The mechanisms postulated for this effect include alteration of (a) ion fluxes associated with depolarization, (b) repolarization, (c) membrane stability, (d) calcium uptake in presynaptic terminals, and (e) sodium/potassium adenosine triphosphate–dependent ionic membrane pump.12The usual dose of phenytoin prescribed to adults is 4 to 6 mg/kg/d. Prepubertal children require somewhat higher doses (5–10 mg/kg/d) to achieve the same steady-state concentrations, owing to the greater activity of the hepatic microsomal enzyme system in this age range. Phenytoin undergoes saturation kinetics; that is, each individual has a threshold serum/plasma concentration beyond which the enzymes involved in its metabolism become “saturated.” Any further slight increase in dose can cause the serum concentration to increase out of proportion to the dose administered. When saturation occurs, the metabolism of phenytoin changes from a first-order (drug-concentration–dependent) process to a zero-order (drug-concentration–independent) process.12Phenytoin protects against seizures at serum/plasma concentrations of 10 to 20 μg/mL (40–79 μmol/L), although higher levels may be needed in some cases.2 It can precipitate seizures at concentrations greater than 40 μg/mL (>158 μmol/L).1 Because the relationship between serum/plasma concentration and clinical efficacy and toxicity is good, therapeutic drug monitoring and optimizing the dosage regimen to achieve a therapeutic level and avert toxicity is an important adjunct to therapy. The pharmacokinetics of phenytoin are shown in Table 1.Toxic side effects of phenytoin include nystagmus, dysarthria, diplopia, ataxia, and exacerbation of seizures. Furthermore, chronic use can lead to hirsutism and gum hypertrophy. Taking phenytoin during pregnancy is contraindicated and can lead to the fetal hydantoin syndrome. Folate and vitamin D deficiency may necessitate vitamin supplementations and, if uncorrected, leads to megaloblastic anemia. High concentrations lead to drowsiness, confusion, and coma.12Absorption rate and bioavailability depend on the formulation used. Approximately 90% of the oral dose is absorbed. Peak concentrations are achieved in 2 to 8 hours. For treatment of status epilepticus, phenytoin is usually administered via the intravenous route as fosphenytoin.3The major biotransformation pathway consists of metabolism to arene oxide via the cytochrome oxidase system enzyme arene oxidase. Arene oxide is spontaneously converted to 5-p-hydroxyphenyl-5-phenylhydantoin (HPPH). This pathway accounts for 60% to 80% of phenytoin elimination. Saturation of this enzyme occurs at low concentrations of phenytoin, a substrate whose Michaelis-Menten constant is low, resulting in saturation of the system usually within the therapeutic range of phenytoin. Once saturation has occurred, any further slight increase in dose results in a disproportionate increase in serum/plasma concentration with concomitant toxic side effects. The activity of this hepatic microsomal system is very age dependent, being low at 0 to 3 months of age, approximately double that of the adult from 6 months to puberty, and declining to adult values after puberty. It follows that patients approaching puberty need to be observed closely with frequent monitoring of serum/plasma concentrations and appropriate downward adjustment of dose when required. Less than 5% of the dose is excreted unchanged in the urine, with 60% to 70% being excreted as HPPH conjugated with glucuronic acid. HPPH is converted by the enzyme epoxide hydrolase to the dihydrodiol. The diol accounts for 7% to 11% of phenytoin metabolites recovered from urine.12Numerous drugs (valproic acid, salicylates, thiazides, and endogenous compounds in patients with renal failure) displace phenytoin from its binding site on albumin. Phenytoin is usually 90% protein bound. However, in renal failure, by-products and phenytoin metabolites build up in the blood and displace phenytoin from its protein-binding sites. In these cases, it is necessary to make dosage adjustments to bring the free phenytoin concentration into the therapeutic range (1–2 mg/L). In the presence of these compounds, the free fraction of phenytoin increases, placing the patient at increased risk for toxicity, and symptoms of toxicity become apparent. Free phenytoin concentrations can be measured by using either equilibrium dialysis or, more commonly, devices with molecular cutoff filters, such as the Amicon Centrifree micropartition system (Millipore Corporation, Bedford, Mass) or the Worthington Diagnostics “ultrafree” system (Worthington Diagnostics, Jacksonville, Fla). Temperature affects the degree of binding of phenytoin to albumin, with drug binding decreasing as temperature increases. Control of temperature is therefore important. A 10-degree drop in temperature (37°C–27°C) can result in an approximately 25% decrease in the free fraction.4 Because the patient's temperature is usually 37°C, it is recommended to use this temperature when assessing free phenytoin concentrations.The 3 commonly used methods of analysis for phenytoin include immunoassays, high-performance liquid chromatography, and gas-liquid chromatography. High-performance liquid chromatography and gas-liquid chromatography methods have the advantage of separating phenytoin from its major metabolite, HPPH. Some of the immunoassays cross-react with the metabolite. Nevertheless, in surveys such as the College of American Pathologists' (CAP) proficiency testing programs, by far the most laboratories are using immunoassays to monitor phenytoin concentrations in patients.5Patients with renal failure and on dialysis often receive phenytoin to treat their seizures. It would be expected that the concentration of HPPH, which is excreted renally, would be significantly elevated in these individuals. For this reason, we sought to ascertain whether the major phenytoin metabolite interfered with current phenytoin immunoassays.To evaluate whether the phenytoin metabolite HPPH cross-reacts in current immunoassay systems, Z-01 and Z-02 specimens of the CAP 2003 Z-A TDM Survey had identical concentrations of phenytoin (target 5 μg/mL [20 μmol/L]). However, Z-01 contained an additional 5 μg/mL HPPH.Table 2 shows the survey results obtained on Z-01 and Z-02 for phenytoin. The concentration of phenytoin metabolite chosen relative to the parent drug accurately reflects the expected concentration in patients with renal failure (unable to secrete the metabolite), because 60% to 80% of phenytoin is metabolized to HPPH. As can be seen from the table, both the total and free phenytoin concentrations were significantly affected by the presence of metabolite, an interference of 7% and 16%, respectively. When the interference was broken down by method, the Abbott AxSYM and Abbott TDx/TDxFLX (Abbott Diagnostics, Abbott Park, Ill) are the most significantly affected methods. The 2 methods alone account for approximately 1225 laboratories in North America or 30% of laboratories currently enrolled in the CAP proficiency testing program for phenytoin. The interference in these 2 methods is 17% to 19% for total phenytoin and 19% to 37% for free phenytoin. Errors of this magnitude could easily result in inappropriate dosage adjustments and errors in patient management. DPC Immulite (6.25%) (Diagnostic Products Corporation, Flanders, NJ) and Roche Cobas Integra (3.48% total, 4.07% free) (Roche Diagnostic Systems, Branchburg, NJ) showed a smaller degree of cross-reactivity with the metabolite, while several methods (Bayer Immuno-1, Bayer Corporation, Tarrytown, NY; Beckman Synchron RGT, Beckman Coulter Inc, Fullerton, Calif; Dade Dimension, Dade Behring, Newark, Del; and Vitros, Ortho Clinical Diagnostics, Raritan, NJ) showed minimal (<2%) interference at the concentration tested.

OBJECTIVE:To determine if the levels of imprecision of the commonly used analytic methods for drug measurements are suitable for long-term therapeutic drug monitoring.DESIGN:In 1996, 4 identical lyophilized samples (2 in the first mailing and 2 in the second mailing 4 months later) were sent to laboratories participating in a nationwide proficiency testing program. Similarly, in 1999, replicates from a liquid pool of spiked sera were mailed 3 times, 4 months apart, to participating laboratories. For each of 11 drugs regulated under the Clinical Laboratory Improvement Amendments of 1988 and 1 metabolite, the total variance for each method was partitioned into within- and between-laboratory components. The total within-laboratory and the total survey coefficients of variation (CVs) for each method were then compared with the "acceptable" precision criteria of Glick, Burnett, and Fraser for each drug.SETTING:The first 2 mailings of the College of American Pathologists Therapeutic Drug Monitoring surveys for 1996, sets Z and ZM, and the 3 mailings of 1999, sets ZM, Z, and Z2.MAIN OUTCOME MEASURES:For each drug studied, the CV of each method was compared with the various imprecision criteria, and if greater than any of the criteria, the method was then tabulated as not meeting that specific criterion.Participants.-The approximately 5000 participants of the survey.RESULTS:The number of methods deemed as not having acceptable total long-term within-laboratory precision by the various criteria ranged from 35% to 88% in 1996 and from 22% to 77% in 1999.CONCLUSION:The number of failures possibly indicates that many of the reagent assays being utilized are not precise enough for long-term therapeutic drug monitoring of chronically administered drugs or that the published criteria used to evaluate the data in this study are too stringent.

Objective.-To determine the magnitudes and sources of analytic variation in testing for therapeutic drugs. Specifically, among laboratories using the same analytic method, to compare the within-laboratory variation (including both short- and long-term variation) with the between-laboratory variation.Design.-Four identical challenges were prepared from a lyophilized pool of spiked sera and were sent in pairs 4 months apart to laboratories participating in a nationwide proficiency-testing program. For each of 25 drugs, the variability in reported results from laboratories using the same method was investigated using nested analysis of variance.Setting.-The first 2 mailings of the College of American Pathologists Therapeutic Drug Monitoring Survey, 1996, sets Z and ZM.Main Outcome Measures.-For each drug, total variance was partitioned into within- and between-laboratory components for common methods. The within-laboratory component was further partitioned into short- and long-term components.Participants.-The approximately 5000 laboratories enrolled in the survey.Results.-For the 25 drugs, the average percentages of the total variance due to short-term, within-laboratory variance; long-term, within-laboratory variance; between-laboratory variance; and total laboratory variance were 25.0% (range, 8.8-50.6%), 57.8% (35.3-73.7%), 17.3% (5.0-35.4%), and 82.7% (64.6-95.0%), respectively.Conclusion.-For all drugs tested, the within-laboratory component of variance was greater than the between-laboratory component of variance. Within laboratories, the magnitude of the long-term component was generally greater than the magnitude of the short-term component. This information will be helpful in determining the clinical utility of various drug assays and in evaluating the appropriateness of regulations involving therapeutic drug testing.