The ABC's of Pediatric Laboratory Medicine- T is for Testosterone

Shannon Haymond, PhD, DABCC
Director, Clinical Chemistry and Mass Spectrometry, Ann & Robert H. Lurie Children’s Hospital of Chicago

Testosterone is the primary androgenic hormone, which is synthesized from cholesterol via the steroid pathway. The most prominent production of testosterone is by the Leydig cells in the testis. However, other sources of testosterone synthesis include the thecal cells of the ovaries, placenta, adrenal cortex (zona reticularis), adipose tissue, brain, muscle and skin. This may occur via de novo synthesis or by peripheral conversion of precursors. The quantities produced by these alternate sources are significantly smaller than that of the testis. In males, the regulation of testosterone production occurs via the hypothalamus-pituitary-testicular axis. To increase testosterone, gonadotropin-releasing hormone (GnRH) from the hypothalamus exerts effect on the pituitary to secrete lutenizing hormone (LH) and follicle stimulating hormone, which act in the testis to increase both the number of Leydig cells and the synthesis of testosterone in the Leydig cells. Rising concentrations of testosterone then provide negative feedback to the hypothalamus and pituitary to inhibit further synthesis.

Testosterone circulates in blood as free (unbound, <5%), tightly bound (~45%) to sex hormone binding globulin (SHBG) and loosely bound (~55%) to albumin and corticosteroid binding globulin. The free and loosely-bound forms are considered the ‘bioavailable’ fractions, as the SHBG-bound form is not available to target tissues. Free testosterone enters cells via passive diffusion and either binds to androgen receptors in the nucleus to exert its effects or may also be converted into a different hormone. Testosterone can be a precursor for a weaker androgen (androstenedione), a hormone with different activities (estradiol) or for a more potent hormone having similar activities (5α-dihydrotestosterone (DHT)).

The nuclear testosterone-receptor complex alters gene expression and related protein transcription to produce androgenic and anabolic effects in cells. It is through this mechanism that testosterone regulates the growth and development of male reproductive organs, as well as the development and establishment of secondary ‘male’ sex characteristics during puberty. The anabolic effects include increased muscle mass and strength and maintenance of bone mass, density and strength. The effects and concentrations of testosterone vary according to age and gender to regulate necessary androgenic and anabolic functions from gestation through adulthood. This fact creates challenges in the measurement of testosterone, as it requires assays that precisely and accurately detect a wide range of concentrations (e.g., <5 to >500 ng/dL). Lowest concentrations of testosterone are found in pre-pubertal children and women. Hypogonadal men and those with ‘castrate’ levels of testosterone will also have concentrations in this problematic range (e.g., <50 ng/dl). Testosterone is an important diagnostic tool in these populations but most commercially available, direct immunoassays (IAs) are not reliable in these cases.

Table 1 lists examples of clinical scenarios involving measurement of testosterone.

Table 1.

Clinical Scenarios Involving Testosterone Measurement




Puberty disorders
Monitor response to therapy
Testosterone replacement
Anti-androgen or GnRH-analogs




Irregular or no menses
Ambiguous genitalia
Polycystic ovary syndrome (PCOS)
Non-classical congenital adrenal hyperplasia (NCCAH)
Idiopathic hirsuitism
Androgen-secreting tumor
Monitor response to therapy
Testosterone replacement
Anti-androgen or GnRH-analogs

There are a variety of methods used to measure testosterone and they have been in existence for decades. The most common are either IA- or mass spectrometry (MS)-based. The earliest of these methods utilized a radioimmunoassay (RIA) platform and incorporated labor-intensive sample pre-treatment. The sample preparation steps served to release the hormones from binding proteins and to remove potential interferents from the sample matrix and chromatography steps further enhanced the selectivity. Due to the up-front processing, these assays are commonly referred to as ‘indirect’. As the need for higher-throughput, reduced complexity and the availability of non-RIA platforms increased, automated IAs gained popularity. These so-called ‘direct’ IA methods eliminated the sample purification steps and relied on improved performance of reagents and antibodies to directly measure testosterone in patient serum. They were validated for use in primarily normal adult male samples and met performance specification, as such. Reports emerged demonstrating the severe limitations of direct IAs for accurate and precise measurement of testosterone in children, women and hypogonadal men (1-3). Additionally it was becoming clear that there was a lack of standardization across IA platforms. However, even decades later, presumably due to the availability, cost and turn-around-time of these methods, they are still used, despite limitations leading to misidentification and misclassification of diseases in these select populations. Alternative, mass spectrometry-based methods are increasingly available in clinical labs and although these methods have improved sensitivity and specificity over most direct IAs, they lack standardization.

Mass spectrometry-based methods for testosterone measurement have been in use since the 1960s. These early methods incorporated sample preparation steps similar to the indirect RIAs. They also included derivitization with separation by gas chromatography (GC) and detection by MS, based on an isotope-labeled internal standard. Therefore, these methods were highly sensitive and specific but due to the complexity, low throughput and high sample volume requirements they were better suited as reference or comparator methods than for routine use in clinical care. The advancement of liquid chromatography (LC) tandem mass spectrometry (MS/MS) methods for clinical use enabled testosterone measurements to become widely available in reference/specialty labs and also in many hospital labs. LC-MS/MS methods, due to their improved sensitivity and specificity over IAs were favored and deemed suitable for measurements in situations with low (e.g., <50 pg/mL) concentrations. However, due to differences in sample preparation, calibration and other methodological differences in these methods that are developed and validated by individual labs, there was variability reported between LC-MS/MS methods.(4)

The mounting evidence and concern about IA performance at low testosterone concentrations with the high degree of variability noted across all methods led to a workshop in 2010, focused on the need for standardization of testosterone methods. This included perspectives from researchers, clinical laboratorians, clinicians, professional and governmental organizations and industry. The Endocrine Society issued a consensus document outlining the limitations with testosterone assays and recommending actions for improvement. (5) The recommendations included the need for technical improvement and standardization of testosterone assays. The CDC developed an initiative for ensuring testosterone results are traceable to a single source and would, thus, be comparable across all methods and performing labs and over time.(6) The program consists of 3 steps: developing a reference system, calibrating individual assays, and verifying end-user test performance. The CDC’s candidate reference method was published in 2013 and has been used to assign values to single-donor sera. These commutable samples are intended for use by assay developers and manufacturers as calibrators and/or trueness controls. (7) Labs and manufacturers participating in the Hormone Standardization (HoSt) Program perform a calibration/calibration verification with the CDC method and samples and their performance is monitored on a quarterly basis. Laboratories with a mean bias ± 6.4% from 4 consecutive challenges are considered sufficiently accurate and standardized to CDC. To date, there are 9 methods certified as standardized to CDC (8 LC-MS/MS and 1 IA). (8) End-user performance is verified by CDC’s collaborations with proficiency testing manufacturers to provide accuracy-based materials. The reference values for these materials are assigned using the CDC’s reference method. The efforts to standardize testosterone are beginning to show positive impact as the inter-laboratory variability for MS-based methods decreased (mean absolute bias decreased by ~50%) in a recent comparison of measurements in 2007 and 2011.(9) The need for standardization in clinical measurements is clear and the progress made to date with the testosterone program is promising, however, smaller, lower volume clinical labs performing testosterone measurement by LC-MS/MS face challenges in participating in the HoSt program.

Measurement of testosterone is an important diagnostic tool in the evaluation of androgen inadequacy and excess. Most direct IA methods do not meet accuracy and precision requirements for use in women, children and hypogonadal men, due to the extremely low circulating testosterone concentrations in these populations. It is recommended that testosterone measurements in these populations be performed using a validated LC-MS/MS method. Although generally more sensitive and specific than direct immunoassays, LC-MS/MS methods still lack standardization. A standardization initiative to address the measurement variability for testosterone was proposed and is underway with recent reports of success among its participants. It is important to note that the limitations described herein about testosterone measurement (e.g., lack of standardization, commercially available direct IAs with inadequate accuracy and precision at low end concentrations) are also true for estradiol. Mounting evidence indicates that most commercially available direct IAs are not suitable for measurement of estradiol at concentrations typically found in children, post-menopausal women and those on aromatase inhibitors.(10) Standardization efforts, parallel to those for testosterone, are in progress for estradiol measurement, as the available methods also show high variability.


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  6. CDC. CDC Hormone Standardization Project. Standardization of serum total testosterone measurements. http://www.cdc.gov/labstandards/pdf/hs/HoSt_Protocol.pdf (Accessed April 2014).
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  8. CDC. CDC Hormone Standardization Project website: http://www.cdc.gov/labstandards/hs.html (Accessed April 2014)
  9. Challenges and improvements in testosterone and estradiol testing. Vesper HW, Botelho JC, Wang Y. Asian Journal of Andrology (2014) 16, 178–184.
  10.  Rosner W, Hankinson SE, Sluss PM, Vesper HW, Wierman ME. Challenges to the measurement of estradiol: an endocrine society position statement. J Clin Endocrinol Metab 2013; 98: 1376–87
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