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Editorials |
Division of Endocrinology, Department of Medicine, Harbor-UCLA Medical Center and Los Angeles Biomedical Research Institute, Torrance CA
aAddress correspondence to this author at:, Chief, Division of Endocrinology, Harbor-UCLA Medical Center, Los Angeles Biomedical Research Institute, 1000 West Carson Street Box 446, Torrance, CA 90509-2910, Fax (310) 533-0627, E-mail swerdloff{at}labiomed.org
Fritz and coworkers (1) have provided data arguing against the validity of a popular analog-based direct assay for free testosterone (T). Their rigorous dissection of characteristics of the assay and use of carefully defined solutions demonstrates that the assay correlates generally with total T but does not measure dialyzable T; this observation is consistent with prior reservations about this class of assay by reported by the Endocrine Society (2)(3) and of other investigators (4)(5)(6).
why worry about free t?
Testosterone circulates in the blood of men and women in several forms. It is bound tightly to sex-hormone binding globulin (SHBG), loosely to albumin, and unbound to proteins (free) (7). In most, but not all, clinical conditions, a measurement of total T is adequate for the evaluation of a patient. It is widely believed that the SHBG-bound T is not readily available to most tissues, whereas albumin-bound and free T are bioavailable. Because SHBG concentrations can be influenced by many factors (e.g., decreased by obesity, testosterone treatment, and hyperandrogenic female conditions such as polycystic ovary syndrome and increased by aging, pregnancy, and estrogen therapy), there are clinical situations in which measured concentrations of total T may not reflect the bioavailable concentrations or the clinical status of the patient (3)(7)(8). In these circumstances a supplemental test assessing bioavailable or free T will be helpful in clinical decision-making.
how is free t or bioavailable t determined?
Free T has been estimated for some time by dialyzing serum across a semipermeable membrane under selected conditions. The T in the serum that crosses the membrane is believed to be unbound or free. Because free T accounts for only about 2%–3% of the total T in men and even less in women, direct measurement of T in the dialysate has been reported (9) but is technically difficult with most assays. To overcome this limitation of T assay sensitivity, most dialysis assays for free T use tracer amounts of radiolabeled T added to the serum, and the isotope is measured in the dialysate. The percentage of dialyzable tracer is determined and multiplied by the total T to calculate the free T (7). This method, although sensitive and reproducible, is cumbersome and not easily adaptable to automated methods. An alternative method is to estimate free T by measuring total T and SHBG and calculate a free T with an algorithm based on the law of mass action or by empirical equations (7)(10). This calculated free T gives excellent correlations with the free T measured with the dialysis method (5). A third approach is to measure bioavailable T as a free T surrogate by precipitating SHBG with ammonium sulfate and measuring the albumin-bound and free T in the supernatant (11). Although the latter approach is technically simple, care must be taken to assure the correct concentration of ammonium sulfate for complete precipitation of the SHBG-T complex. The fourth method is that studied by Fritz et al. (1). Although this method is used widely, the analog-based assay described in their report is an automated black-box package with a proprietary radioactive buffer provided by the manufacturer; this class of assay has been criticized as having poor accuracy, sensitivity, and between-assay comparability and being influenced by the dilution of serum (2)(3)(4)(5)(6). It is of note that the reference interval for free T by analog-based assay is much lower (concentrations about one-fifth as high) than that for the equilibrium dialysis assay (2). This calibration difference between assays is a major problem.
what did fritz and coworkers find in their studies?
These investigators dissected one analog-based free T assay by measuring total T and free T by analog methods both in the retentate and dialysate fractions of serum after equilibrium dialysis. Free T was also measured using the standard equilibrium assay method on the dialysate. The analog free T assay correlated with total T in the retentate but neither the total T assay nor analog free T assay measured any T in the dialysate, although the concentrations were in the reference range by equilibrium dialysis. Fritz et al. then used the same method on the sample while covarying concentrations of SHBG, protein-bound T, and total T through dilution of serum. The results showed that the analog-based assay tracked total T more closely than free T by the dialysis method. In the third experiment they diluted the serum while adding increased amounts of protein-free T to keep the total T in the serum constant. The results showed that the total T and free T by the analog method remained constant while the free T by the dialysis method increased as expected. Thus their studies showed that free T measurement with the analog-based free T assay has no advantage over total T measurements and does not measure unbound T. Fritz et al. conclude that the assay is not properly calibrated, lacks specificity, and does not measure free T.
what does the analog assay measure?
It is not entirely clear what constituent(s) are being measured, but free T measured by the analog-based assay correlates with total T. Fritz et al. (1) speculated that the assay nonspecificity may be due to protein-T complexes binding to the T antibody, leading to a 3-way competition between free testosterone, testosterone complexes, and testosterone conjugates (analogs) for binding to the same antibody. This explanation should be testable using liquid chromatography–tandem mass spectrometry (12).
do the findings of fritz and coworkers apply to other branded analog-based free t assays?
The authors are cautious, but given the information provided, other free T analog assays probably show similar characteristics of tracking total T and not free T; thus providing no advantage over total T measurements.
why are these analog-based free t assays so widely used?
The wide use of these assays is based in part on their ease of performance, automated nature, and lower cost than free T measurement by dialysis. The popularity of these assays may also be a result of lack of specific knowledge of physicians regarding the limitations of the assay methodology. Because the free T by analog method tracks total T, and because most men with total T concentrations that are very low do not require a free T to confirm the diagnosis of male hypogonadism, the results seem to fit the clinical impression. Because the experts in the field recommend free T measurements in equivocal cases of hypogonadism, the practicing physician often orders the correct test but the wrong assay.
should the analog-based free t assay be used in clinical practice?
Because the analog-based assay does not measure free T, it makes no sense to label it as such. Because it tracks total T but has not been shown to add to the values of total T results, we do not recommend its use.
are the current methods of total t assays adequate?
It is important to note that free T measured by equilibrium dialysis requires a sensitive, specific, precise, and accurate assay for total T. Recent studies have shown that the current methods of measurement of total T lacked the required sensitivity for samples with very low concentrations, as occur in severely testosterone deficient men, normal or T-deficient women, and children; this sensitivity limitation can be corrected by newer methods based on mass spectrometry (12)(13)(14). Furthermore, the free T analog–based assay may be only one of a number of widely used but poorly assessed and quality controlled hormone assays (2)(15). The CDC has provided quality control services for several analytes, including recent efforts to do so for total T by liquid chromatography–tandem mass spectrometry. It is time that physicians, investigators, clinical chemists, pathologists, and editors of medical journals insist on better surveillance of assays used in research and patient care.
References
The following articles in journals at HighWire Press have cited this article:
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  C. Wang, E. Nieschlag, R. Swerdloff, H. M. Behre, W. J. Hellstrom, L. J. Gooren, J. M. Kaufman, J.-J. Legros, B. Lunenfeld, A. Morales, et al. Investigation, Treatment, and Monitoring of Late-Onset Hypogonadism in Males: ISA, ISSAM, EAU, EAA, and ASA Recommendations J Androl, January 1, 2009; 30(1): 1 - 9. [Full Text] [PDF]
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 C Wang, E Nieschlag, R Swerdloff, H M Behre, W J Hellstrom, L J Gooren, J M Kaufman, J-J Legros, B Lunenfeld, A Morales, et al. Investigation, treatment and monitoring of late-onset hypogonadism in males: ISA, ISSAM, EAU, EAA and ASA recommendations Eur. J. Endocrinol., November 1, 2008; 159(5): 507 - 514. [Full Text] [PDF]
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