Figure 2. RESULTS DATA ANALYSIS

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1 ANDROGEN PARAMETERS IN HIRSUTE AND NORMAL FEMALE PATIENTS: IS THERE A ROLE FOR THE FREE ANDROGEN INDEX (FAI)? Castracane VD 1, Childress E 1, Tawwater B 1, Vankrieken L 2, El Shami AS 2 ( 1 Department of Ob/Gyn, Texas Tech Health Sciences Center, Amarillo, TX; 2 Diagnostic Products Corporation, Los Angeles, CA) ABSTRACT INTRODUCTION METHODS SUBJECTS CONCLUSIONS A variety of androgen assays have been used as diagnostic tools to document hyperandrogenism in women. To date, however, no single test of this kind can discriminate hyperandrogenic patients from nonhirsute, normally cycling women with 1% accuracy, though some appear more effective than others. In the present study, we undertook to compare the value of key androgen measurements and derived parameters for making this discrimination. Subjects: The subjects consisted of 53 patients with moderate to severe hirsutism, and 55 normally cycling women serving as controls. Serum samples were collected in either the follicular or the luteal phase of the cycle: women in the periovulatory period were excluded, since there are reports indicating that androgen levels may be elevated during this interval. The parameters included total testosterone (TT) and sex hormone-binding globulin (SHBG), as well as the free androgen index (FAI), calculated as 1 times the molar ratio of TT to SHBG. Methods: Results were obtained using the IMMULITE Total Testosterone and IMMULITE SHBG assays (Diagnostic Products Corporation, Los Angeles, CA). These are solid-phase, chemiluminescent enzyme immunoassays designed for use on the IMMULITE automated analyzer. IMMULITE Total Testosterone is a competitive immunoassay that uses ligand-labeled testosterone and a polyclonal antibody specific for testosterone. IMMULITE SHBG is an immunometric assay that uses monoclonal and polyclonal antibodies for SHBG capture and detection, respectively. Results: Medians (and central 95% limits) calculated nonparametrically for the control group were as follows. TT:.35 ng/ml (.13.73), SHBG: 53 nmol/l ( ), FAI: 2.25% ( ). Using the estimated 97.5th centile as the cutoff, 19 (36%) and 24 (45%) of the 53 hirsute women were identified via the TT and FAI results, respectively (Figure 2). Correlation coefficients for FAI versus TT and SHBG were.615 and.648, respectively. Thus, in the present study, the calculated FAI based on IMMULITE results outperformed the measurement of TT alone in discriminating hirsute patients from normals, showing an improvement of about 25% in this task. A variety of androgen assays have been used to document hyperandrogenism in women. To date, however, no single test of this kind can discriminate hyperandrogenic patients from nonhirsute, normally cycling women with 1% accuracy, though some appear more effective than others. In the present study, we undertook to compare the value of two major approaches to making this discrimination, based on direct measurements and derived parameters available within the resources of an automated, random-access immunoassay system. Specifically, we compared total testosterone (TT) to the free androgen index (FAI). The latter is calculated as a simple ratio from the circulating levels of total testosterone and sex hormone-binding globulin (SHBG), the principal transport protein for this hormone. The FAI is often used as a proxy for free testosterone [Whe95], though its clinical utility may be somewhat context-dependent: thus, for example, it may be generally of less value in men than in women.[bra98] Indices of greater complexity for free or "bioavailable" testosterone have been proposed, but none has displaced the simple ratio of total testosterone to SHBG in clinical practice.[bli89, Whe95] Moreover, there exist manual assays for estimating the free testosterone concentration directly [ElS95, Ooi98], but not on the automated platform used for this study. Results were obtained using the IMMULITE Total Testosterone and IMMULITE SHBG solid-phase, chemiluminescent enzyme immunoassays, designed for use on the IMMULITE automated analyzer (Diagnostic Products Corporation, Los Angeles, CA; Figure 1. IMMULITE Total Testosterone is a competitive immunoassay based on ligand-labeled testosterone and a polyclonal antibody specific for testosterone. It has a calibration range of 2 1,6 ng/dl (.6 55 nmol/l) and a detection limit of 1 ng/dl (.3 nmol/l). IMMULITE SHBG is an immunometric assay utilizing monoclonal and polyclonal antibodies for SHBG capture and detection, respectively. It has a calibration range of up to 18 nmol/l, and a detection limit of.2 nmol/l. The free androgen index (FAI) was calculated as 1 times the molar ratio of total testosterone (TT) to sex hormone-binding globulin (SHBG). The study population consisted of 53 patients with moderate to severe hirsutism, and 55 normally cycling women serving as controls. Overall, the two groups were of comparable age. However, because the exact age in years was known for only some of the subjects, no attempt was made to take age into account as a covariate, even Figure 2. though normal levels of testosterone, free testosterone and other androgens (such as DHEA-SO 4 ) are to some extent age-related in women as well as in men.[kub86, Ooi98] Serum samples were collected in either the follicular or the luteal phase of the cycle: women in the periovulatory period were excluded, to avoid the possibility that androgen levels might be elevated during this interval. DATA ANALYSIS Discrete point cluster plots or "dotplots" [Sas96] are closely related to frequency plots and histograms, but provide a better sense of the granularity of the data, because each result is represented by a readily discernible point. As with histograms, there is an arbitrariness to the number, size and alignment of the "bins" involved, which can sometimes mislead. Nevertheless, dotplots provide a valuable, first-line method for surveying and comparing small-to-moderate sized data sets. For this presentation, dotplots were produced by coercing data into the form expected for a scatterplot and a stacked bar chart the latter serving to represent the upper limit of the central 95% range observed for the normals. Calculations were performed using simple spreadsheet templates; SigmaPlot v5.2 ( was used for the graphs. RESULTS Medians (and central 95% limits) calculated nonparametrically for the control group were as follows. Total testosterone: 35 ng/dl (13 73 ng/dl). SHBG: 53 nmol/l ( nmol/l). Free androgen index: 2.25% ( %). Using the estimated 97.5th centile for normals as the cutoff, 19 (36%) and 24 (45%) of the 53 hirsute women were identified via the total testosterone and free androgen index results, respectively. (Figures 1 and 2. The upper edge of the shading indicates the cutoff.) Correlation coefficients for free androgen index versus total testosterone and SHBG were.62 and.65, respectively; whereas total testosterone and SHBG were virtually uncorrelated: r =.14. (Figures 3 5. In these scatterplots, open circles represent normal controls, solid circles represent hirsute patients.) Figure 3. Figure 4. Figure 5. In the present study, the calculated free androgen index based on IMMULITE results outperformed the measurement of total testosterone alone in discri-minating hirsute patients from normal women, showing an improvement of about 25% in this task. This represents the added value of performing a second test an assay for SHBG in conjunction with the assay for total testosterone in this clinical setting. REFERENCES [Bra98] Brannian JD, Long P, Kreger DO. Is the free androgen index a useful clinical marker in male patients? So Dakota J Med 1998;51: [ElS85] El Shami AS, et al. First solidphase radioimmunoassay for free testosterone based on the analog method [abstract 4]. Clin Chem 1985;31:1. [Kub86] Kubasik NP, et al. Age and sex reference ranges for DHEA and DHEA-SO 4 [abstract 595]. Clin Chem 1986;32: [Ooi98] Ooi DS, et al. Establishing reference intervals for DPC s free testosterone radioimmunoassay. Clin Biochem 1998;31: [Sas96] Sasieni PD, Royston P. Dotplots. Appl Statist 1996;45: [Whe95] Wheeler MJ. The determination of bio-available testosterone. Ann Clin Biochem 1995;32:

2 Figure Normal Hirsute Total Testosterone, ng/dl

3 Figure Normal Hirsute Free Androgen Index

4 Figure 3 3 Free Androgen Index IMMULITE Total Testosterone, ng/dl

5 Figure 4 3 Free Androgen Index IMMULITE SHBG, nmol/l

6 Figure 5 16 IMMULITE SHBG, nmol/l IMMULITE Total Testosterone, ng/dl

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