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* Clinicians often prefer bioavailable testosterone as it more closely resembles the total bioactive testosterone in circulation when compared to free testosterone. Validation to ensure clinical accuracy of our bioavailable testosterone method modification involved a comparison of the reference interval flags between this method and verified free and total testosterone flags.
* Recently, our bioavailable testosterone assay underwent a method modification to enhance calibrator storage and quality, which required subsequent validation to confirm adequate clinical performance. It has been shown that a strong correlation exists between bioavailable and free testosterone. As such clinical performance was validated by investigating the concordance between the reference interval (RI) flags (low, normal, or high) obtained using the modified BAT method and the RI flags from validated LC-MS/MS total and free testosterone assays.
MSACL 2025 Abstract
* Recently, our bioavailable testosterone assay underwent a method modification to enhance calibrator storage and quality, which required subsequent validation to confirm adequate clinical performance. It has been shown that a strong correlation exists between bioavailable and free testosterone. As such clinical performance was validated by investigating the concordance between the reference interval (RI) flags (low, normal, or high) obtained using the modified BAT method and the RI flags from validated LC-MS/MS total and free testosterone assays.
MSACL 2025 Abstract
To Free or Not to Free? An LC-MS/MS-Based Method Modification to Validate Clinical Performance of Bioavailable Testosterone Measurement Chelsea B. Swartchick, Jennifer V. Kemp, Stefan K. Grebe, Irina Bancos, and Anthony D. Maus Mayo Clinic, Rochester, MN Abstract INTRODUCTION Measurement of total testosterone is an invaluable method for assessing an individual’s androgen status. This assay is often performed in conjunction with free testosterone measurement, which should encompass approximately 1 to 2% of the total testosterone concentration. However, measurement of free testosterone is influenced by abnormalities in both the concentration and/or function of sex hormone binding globulin (SHBG), the primary transport and regulatory protein for testosterone, along with albumin, the most abundant plasma protein. Therefore, analysis of bioavailable testosterone, which accounts for both free and testosterone loosely bound to albumin, is a more reliable representation of an individual’s true testosterone status. Recently, our bioavailable testosterone assay underwent a method modification to enhance calibrator storage and quality, which required subsequent validation to confirm adequate clinical performance. It has been shown that a strong correlation exists between bioavailable and free testosterone. As such clinical performance was validated by investigating the concordance between the reference interval (RI) flags (low, normal, or high) obtained using the modified BAT method and the RI flags from validated LC-MS/MS total and free testosterone assays. METHODS Residual serum samples (n = 65) from patients with recent (< 2 weeks) total and free testosterone measurements were used, where 23 were from women and 42 were obtained from men. Reference interval flags were categorized as high, normal, or low as determined being above, within, or below established reference intervals, respectively. There were 14 specimens classified as high, 36 as normal, and 15 as low, by both their total and free testosterone measurements. Bioavailable testosterone sample preparation involved the differential precipitation of testosterone-bound SHBG with ammonium sulfate. All non-SHBG bound testosterone remained in the supernatant, which subsequently was spiked with internal standard, carbon-13 labeled testosterone. Liquid-liquid extraction was performed, and the resulting isolate was derivatized before being quantified by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). All 65 bioavailable testosterone results were then categorized as either high, normal, or low according to the RI dependent on the patient’s sex and age. Flags from the bioavailable testosterone assay and flags from the free/testosterone measurements were evaluated for agreement. Serum SHBG was quantified using a chemiluminescent sandwich immunoassay. Serum albumin concentration was determined using a colorimetric method using the absorbance at 570 nm. RESULTS 59 of the 65 (90.7%) samples were correctly categorized with a RI flag as either low, normal, or high by both the free/total testosterone assays and the modified bioavailable testosterone method. The 6 samples that had discrepant flags required further investigation in which serum SHBG and albumin concentrations were evaluated. Of the discordant samples, 4 had elevated free and total testosterone, but normal bioavailable testosterone. Follow-up of this group demonstrated high SHBG concentrations. Elevated SHBG would expectedly bind more testosterone and decrease the bioavailable testosterone concentration, which provides a rationale of the normal categorization in this population. In a similar manner, one sample had a slightly elevated free and total testosterone, but normal bioavailable testosterone. The SHBG concentration of this sample was nearing the upper limit of normal, which follows the same principle as above where the bioavailable testosterone would be flagged as normal. The remaining discrepant sample had normal free and total testosterone, but elevated bioavailable testosterone. This specimen had an SHBG at the lower end of normal and an albumin at the higher end of normal. As there is less SHBG to bind testosterone and more albumin, an elevated bioavailable testosterone is appropriate. CONCLUSION Clinicians often prefer bioavailable testosterone as it more closely resembles the total bioactive testosterone in circulation when compared to free testosterone. Validation to ensure clinical accuracy of our bioavailable testosterone method modification involved a comparison of the reference interval flags between this method and verified free and total testosterone flags. In theory, samples that flag normal by both free and total testosterone assays should flag similarly with the bioavailable assay. We observed concordance in 90.7% of our samples, while the remaining 9.3% specimens had abnormal SHBG and/or albumin levels that were reflected appropriately in bioavailable testosterone RI flagging. In summary, our method modification performs clinically and can reflect the true testosterone status within a patient. |
