Serum Testosterone Assays—Accuracy Matters

[Megatron28]

All of us rely so heavily on blood work as a tool to validate "how we feel." I came across this study regarding the accuracy of Testosterone Blood Tests and thought I would share it. There is a lot to take in, but here are a few points I found interesting. I know it was published in 2004, but based on anecdotal information it seems like the issues identified here may still exist to some degree.

Serum Testosterone Assays—Accuracy Matters. JCEM Volume 89 Issue 2 - February 1, 2004

It has been disturbing to clinicians and researchers alike that the lower limit of normal in men for some of these assays has fluctuated and drifted down to as low as 132 ng/dl (4.6 nmol/liter) (3). How can this be? It appears that a major contributor to this variation and decline has been a lack of attention to validation of accuracy for many of these assays. Accuracy is a measure of the closeness of agreement between values measured in an assay to a “gold standard” or accepted method of measurement. For total T assays, the most appropriate gold standard for comparison of assay measurements of samples is steroid-free serum that is spiked with a range of gravimetrically determined amounts of T, or an independent method such as liquid or gas chromatography with mass spectrometry that has been validated in this way. At present, approval of T immunoassays by regulatory agencies is based on demonstrating that results obtained from them are comparable to previously approved assays that may or may not have been validated using gravimetrically determined gold standards.

careful examination of their results also seems to reveal relatively consistent underestimation of total T levels by the automated immunoassays compared with LC-MSMS in samples falling within the mild to moderate hypogonadal range (100–300 ng/dl). Potentially, this could result in problems in distinguishing eugonadal from mildly hypogonadal males, and it reinforces the need to establish normal reference ranges for adult males in each individual laboratory. In addition to the accuracy of T immunoassays, another factor that likely contributes to the extreme variability in the normal range for T is the lack of attention to differences in the adequacy and standardization of populations used to establish reference ranges, both during assay validation and during implementation of the assays in individual laboratories.

An important finding of the paper by Wang et al. (2) is that the currently widely used immunoassays tested were not sufficient to measure total T concentrations accurately in females and prepubertal males.

For younger, otherwise healthy men with classical manifestations of androgen deficiency, very low total T concentrations are usually adequate to confirm the diagnosis of hypogonadism. Because total T assays measure both free and protein-bound T, total T levels may be influenced by alterations in SHBG concentrations. For example, total T levels are decreased in conditions associated with reduced SHBG levels (e.g. moderate obesity, hypothyroidism, androgen, glucocorticoid or progestin use, nephrotic syndrome) and increased in situations associated with elevated SHBG levels (e.g. aging, hyperthyroidism, androgen deficiency, estrogen or anticonvulsant use, hepatic cirrhosis) (9). If clinical conditions associated with alterations in SHBG levels are suspected, measurements of free or bioavailable T should be used to assess gonadal status.

In men, calculated free T levels (derived from total T, SHBG, and albumin measurements or assuming a constant albumin concentration) were found to be nearly identical with values measured by equilibrium dialysis.

it is unclear how many T measurements are needed to confidently confirm the diagnosis of hypogonadism. Approximately 30–35% of men who were classified as hypogonadal on the basis of a single low total T level were found subsequently to have average T levels over 24 h within the normal adult male range (15). Other studies using frequent blood sampling demonstrated that 15% of young normal men had total T levels below the normal range within a 24-h period (16). The intrasubject variation in T levels is particularly problematic in older men who exhibit T levels that fluctuate between the lower part of the normal range and slightly below normal. Because the diagnosis of hypogonadism usually implies a need for and commitment to long-term T treatment, experts in the field recommend that at least two low T values be obtained to confirm the diagnosis of hypogonadism.

Few would dispute that for T or other hormone assays, accuracy matters. In the last 30 yr, there have been remarkable improvements in the sensitivity, specificity, efficiency, rapidity, and cost of T assays. What is needed now is refocusing of attention to more rigorous validation and standardization of the accuracy and normal reference ranges for these assays to alleviate the confusion that has arisen in the clinical and research community as a result of the variability and discrepancies in T assays.