Short Communication Reproducibility Studies and Interlaboratory Concordance for Androgen Assays of Male Plasma Hormone Levels

To help us identify appropriate techniques and laboratories for measuring hormones, we studied the variability and reproducibility of assay measurements of androstanediol glucuronide, androstenedione, dehydroepiandrosterone (DHEA), DHEA sulfate, dihydrotestosterone, testosterone, androstanediol, androsterone glucuronide, and androsterone sulfate for five men. Four sets of two aliquots from each sample were sent to participating laboratories, and one set was used for analyses monthly for four consecutive months. For each assay, estimates of components of variance were then used to estimate the coefficient of variation, the intraclass correlation between measurements on different days from a given individual, and the minimum detectable relative difference for a standard design. These data indicate that for at least one of the laboratories a single sample with two laboratory replicates per sample of androstanediol glucuronide, androstenedrone, DHEA, DHEA sulfate, and dihydrotestosterone yields an intraclass correlation coefficient exceeding 0.80 and can be used to discriminate reliably among men. The results for testosterone, androstanediol, androsterone, glucuronide, and androsterone sulfate do not meet this test. These data do not allow us to estimate the component of variation that corresponds to repeated blood samples taken over time from the same man. This reliability study design is, however, entirely appropriate for the typical case-control study which utilizes only one sample per subject. Introduction The NCI obtained plasma samples as part of several field studies to evaluate hormone levels and the risk of cancer. We have studied the reproducibility of hormone assays used by laboratories with the capability of performing large numbers of tests. Gail et al. (1) estimated the variability and reproducibility of assay measurements of estrone, estradiol, estrone sulfate, and progesterone using plasma from women. Fears et al. (2) reported results for nine androgens obtained using plasma samples from women: ADIOL G; ADION; DHEA; DHEA S; DHT; TESTO; ADIOL; ANDRO G; and ANDRO S. In this final report of the series, we present results for the androgen assays using plasma samples from men. Materials and Methods Each of the four participating laboratories was asked to use their standard assay procedures and to perform only those assays with which they had experience. Laboratory 1. For ADIOL G, unconjugated steroids were removed by organic extraction, followed by incubation with -glucuronidase, enzyme hydrolysis, and celite chromatography and, finally, measurement of ADIOL by RIA. ADION, DHEA, TESTO, and DHT were measured by extracting plasma with ethyl acetate (20%) in hexane, celite column chromatography, and RIA. DHEA S was measured by RIA. Laboratory 2. For ADIOL G, plasma was extracted with a polar solvent that was then subjected to complete enzymatic hydrolysis, followed by extraction with hexane:ethyl acetate, purification by high performance liquid chromatography, and measurement by RIA. ADION, DHEA, and TESTO were measured after extraction with hexane:ethyl acetate, followed by RIA. For DHEA S, the sulfate was removed by overnight hydrolysis with sulfatase, after which the procedures for measuring DHEA were followed. For DHT, extraction with hexane: ethyl acetate was followed by treatment with a strong oxidizer to destroy unsaturated steroids and purification on alumina columns. Laboratory 3. DHEA S was quanitified by direct RIA after a 1000-fold dilution of the plasma sample with assay buffer. DHEA, ADION, TESTO, DHT, and ADIOL were measured by RIA after extraction of plasma with diethyl ether and purification by celite column chromatography. ADIOL G was quantified directly in plasma using a commercial kit. ANDRO S and ANDRO G were measured after unconjugated steroids were removed by extraction with diethyl ether. The conjugated steroids were hydrolyzed utilizing hydrochloric acid or glucuronidase followed by extraction with ethyl acetate and purification by celite column chromatography. Received 2/6/01; revised 4/22/02; accepted 4/25/02. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 To whom requests for reprints should be addressed at, Biostatistics Branch, National Cancer Institute, 6120 Executive Boulevard, EPS/8040, Bethesda, MD 20892. 2 The abbreviations used are: NCI, National Cancer Institute; ADIOL G, androstanediol glucuronide; ADION, androstenedione; DHEA, dehydroepiandrosterone; DHEA S, DHEA sulfate; DHT, dihydrotestosterone; TESTO, testosterone; ADIOL, androstanediol; ANDRO G, androsterone glucuronide; ANDRO S, androsterone sulfate; CV, coefficient of variation; ICC, intraclass correlation coefficient; MDRD, minimum detectable relative difference; CI, confidence interval; SD, standard deviation. 785 Vol. 11, 785–789, August 2002 Cancer Epidemiology, Biomarkers & Prevention on April 30, 2017. © 2002 American Association for Cancer Research. cebp.aacrjournals.org Downloaded from Laboratory 4. ADION was measured by carbon tetrachloride extraction of plasma followed by a RIA kit. DHEA was measured by dichloromethane extraction and a RIA. DHEA S and TESTO were assayed directly using RIA. Further details of the assay procedures are provided in Fears et al. (2). A blood sample was obtained from each of five male volunteers from NCI, ages 31, 45, 47, 50, and 67 years. Each volunteer was in good health, with no known hormonal abnormality. Within 24 h of draw, the plasma was separated, aliquoted, and stored at 70°C (see Ref. 1). Each participating laboratory received four batches of samples, with one batch to be assayed on each of four consecutive months. Each batch contained two aliquots from each of the five subjects. Each aliquot was assayed in duplicate. The order of the 10 aliquots within each batch was randomly assigned. A nested components of variance analysis was performed using logarithmically transformed measurements to stabilize variances (1). Components were estimated for subjects, days, aliquots, and replicates. We used three measures of reproducibility derived from these components. The CV, which is the SD divided by the mean, is the usual measure of reproducibility. The sum of the components associated with day, aliquot, and replicate is a good estimate of the square of the CV. The ICC is the correlation between measurements on different days from a given sample. It is the ratio of the component associated with subjects to the sum of all components. The MDRD is the minimum difference that is reliably detected with a given number of cases and controls. The CV is of primary interest to the laboratory for quality control, whereas the ICC and MDRD are more important to the epidemiologist in determining the feasibility of an epidemiological study. As in the earlier studies, for each hormone and laboratory, we examined graphs of grand means, daily means and aliquot means (available on request). The Friedman rank order statistic was used to compare subject means across laboratories, and Spearman rank correlations were used to measure concordance of the participants’ grand means across laboratories. The estimated components of variance are given in the “Appendix.” The components of variance (Table A1) were used to obtain estimates of the CVs, ICCs, and MDRDs that were compared among the laboratories. To calculate ICCs and CVs, we assumed that the measurement used was the mean of the two logarithmic-transformed replicates. To calculate MDRDs, we assumed n1 300 cases and n2 600 controls, as in our earlier reports. Further details of these statistical methods are presented in Fears et al. (2). The estimates and their 95% CIs are given in Table 1.