Bernard W. Steele

Analytic bias of thyroid function tests: Analysis of a college of American pathologists fresh frozen serum pool by 3900 clinical laboratories

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.

Lack of specificity of cyclosporine immunoassays. Results of a College of American Pathologists Study.

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 Abbott TDx 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.

Cytofluorographic evidence that thymocyte dipeptidyl peptidase IV (CD26) activity is altered with stage of ontogeny and apoptotic status

CD26 is a multifunctional molecule implied to have a variety of roles in the immune response including its activity as a membrane exopeptidase (Dipeptidyl peptidase IV) which cleaves several protein molecules. In order to further define the expression and functional activity of CD26 in the developing thymus, we utilized a nondisruptive, cytofluorogenic assay which allowed simultaneous measurement of DPP IV activity with a fluorochrome-conjugated peptide substrate and surface staining of the T lymphocyte lineage antigens CD4 and CD8. Neonatal and adult murine thymi were examined using the three-color assay and significant differences in DPP IV activity were found among the thymocyte subsets defined by their CD4/CD8 phenotype. Single-positive cells bore higher activity than CD4-/CD8- cells and neonates had higher activity than adults. Thymocytes with characteristics consistent with apoptotic cells expressed higher DPP IV activity. Thus, DPP IV appears to be upregulated both as thymocytes mature and among thymocytes which are undergoing programmed cell death. These results suggest that CD26 is ontogenically controlled during T cell maturation and may play a role in thymic deletion of emerging clones.

Total long-term within-laboratory precision of cortisol, ferritin, thyroxine, free thyroxine, and thyroid-stimulating hormone assays based on a College of American Pathologists fresh frozen serum study: Do available methods meet medical needs for precision?

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.

A longitudinal replicate study of immunosuppressive drugs: A College of American Pathologists Study

OBJECTIVE To identify the sources of analytical variation for cyclosporine and tacrolimus in a 3-year longitudinal study. DESIGN Two pools of whole blood were spiked with cyclosporine and tacrolimus, respectively. One aliquot of cyclosporine and 2 of the tacrolimus pool were distributed in the first and last mailing for years 1999 to 2001. For both drugs, the total variance for each method was partitioned into within- and between-laboratory components. SETTING The A and C mailings of the 1999, 2000, and 2001 AACC/CAP [American Association for Clinical Chemistry/College of American Pathologists] Immunosuppressive Drugs (CS) Monitoring Survey. MAIN OUTCOME MEASURES For each drug, total variance was partitioned into specimen, mailing, year, and interlaboratory effects for each analytical method. PARTICIPANTS The 292 laboratories for cyclosporine and 177 laboratories for tacrolimus enrolled in the survey from 1999 to 2001. RESULTS For both cyclosporine and tacrolimus, the major source of imprecision came from within-laboratory factors, which accounted for nearly 85% (range, 77% to 90%) of the total variance. For cyclosporine, the major component of within-laboratory variance was between-mailing, within-year effect, whereas for tacrolimus it was the between-year, within-laboratory variation. CONCLUSION The major source of long-term survey imprecision for cyclosporine and tacrolimus is within-laboratory factors. The finding that 85% of the total variance was due to within-laboratory variation is similar to other therapeutic drugs.

Crossreactivity of bupropion metabolite with enzyme-linked immunosorbent assays designed to detect amphetamine in urine.

Background: Drug screening is rapid, inexpensive, and is often used in clinical, forensic, and workplace drug testing to gain informative results. This article seeks to determine if bupropion and/or its metabolites is resulting in false-positive amphetamine screening results in our case samples using commercially available enzyme-linked immunosorbent assay tests. Method: Fortified urine and forensic case samples were used to determine crossreactivity of bupropion and its main metabolite to four different amphetamine and methamphetamine enzyme-linked immunosorbent assay kits. Results: Two of the enzyme-linked immunosorbent assay kits used to screen for amphetamine may result in false-positive results if bupropion metabolites are present in concentrations greater than 500 ng/mL. Three case samples gave a positive screen results for amphetamine using Amphetamine ULTRA kits, yet no amphetamines were confirmed by gas chromatography-mass spectrometry and all samples were positive for bupropion and metabolites. Conclusions: Laboratory directors and clinicians should be aware of the characteristic of their chosen laboratory assay and should communicate this to physicians so that results can be interpreted accurately.

Rapid cocaine screening of urine in a newborn nursery

A rapid cocaine screening test, the Abuscreen OnTrak assay, was compared with the EMIT (enzyme-multiplied immunoassay technique) screening test to determine relative accuracy in 450 newborn infants sequentially tested for urinary cocaine during a 6-week period at a large urban hospital. The Abuscreen Ontrak screen had a sensitivity of 96% and a specificity of 100%.

Reversal of acute tacrolimus-induced renal vasoconstriction by theophylline in rats.

Objective To determine whether theophylline, a nonselective adenosine receptor antagonist and phosphodiesterase inhibitor, reverses the acute declines in renal blood flow and glomerular filtration rate induced by high-dose tacrolimus in rats. Design Prospective, randomized, placebo-controlled experimental study. Setting University-based basic science research laboratory. Subjects Adult male Sprague-Dawley rats. Interventions After mechanical ventilation and instrumentation under isoflurane and nitrous oxide anesthesia, animals received either tacrolimus 0.5 mg/kg intravenously or vehicle and 1 hr later either theophylline 4 mg/kg intravenously or vehicle. Measurements and Main Results By using radiolabeled microspheres, renal blood flow was measured in three groups: control (n = 5), tacrolimus plus vehicle (n = 6), and tacrolimus plus theophylline (n = 6) at four time points—baseline and 60, 75, and 90 mins after tacrolimus or vehicle (the latter two time points being 15 and 30 mins after theophylline or vehicle, respectively). Whole blood tacrolimus and serum theophylline concentrations were measured. In a separate group of animals, by using 51Cr-EDTA, glomerular filtration rate was measured in two groups: tacrolimus plus vehicle (n = 5) and tacrolimus plus theophylline (n = 5) at baseline and over two consecutive 20-min time periods beginning 61 mins posttacrolimus. Urine flow rate also was measured. Following tacrolimus, both renal blood flow and glomerular filtration rate declined in parallel by approximately 33% and 50% from baseline after 75 and 90 mins, respectively (p < .05 by two-way repeated-measures analysis of variance). Theophylline completely reversed these tacrolimus-induced decreases in renal blood flow and glomerular filtration rate. Urine flow rate also increased in response to theophylline. Conclusions Low-dose theophylline reverses tacrolimus-induced declines in renal blood flow and glomerular filtration rate observed in an acute model of tacrolimus toxicity. Theophylline’s effect in chronic toxicity remains to be determined.

Sources of variability : A College of American Pathologists Therapeutic Drug Monitoring Survey Study

○ 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.

A fluorescent immunoassay for theophylline: description and comparison to enzyme immunoassay, liquid chromatography and radioimmunoassay

A fluorescent immunoassay for theophylline is described and comparatively evaluated with radioimmunoassay, high-performance liquid chromatography, and enzyme immunoassay. Fifty sera were collected from 43 patients of a large acute-care medical facility, many of whom were suffering from other diseases in addition to bronchial asthma or apnea of the newborn, and were receiving other medication besides theophylline. Assays of theophylline in each serum sample were performed by each of the 4 procedures. The four methods showed comparable results, although each method had at least one unexplained outlier. Nevertheless, all methods seemed suitable for routine chemistry laboratory use. Three of the techniques have been available for several years, but unexplained erroneous levels are sometimes obtained for every procedure.