madman
Super Moderator
ABSTRACT
Accurate measurement of testosterone is important for the diagnosis of gonadal disorders in men, women, and children. Testosterone measurement has limited accuracy at low concentrations by most commercially available immunoassays. We aimed to develop an LC-MS/MS assay to address the inaccuracy of the in-house immunoassay observed over the past decade and to replace it with the new assay. Testosterone in serum/plasma was extracted with commercial-supported liquid extraction plates. Method validation was performed following the CLSI C62-A guideline. A total of 126 samples were used for method comparison between the Beckman UniCel DxI immunoassay and LC-MS/MS. Results by immunoassay were 20% lower compared with LC-MS/MS and had minimal correlation (R2 = 0.403) with LC-MS/MS below 100 ng/dL. When comparing specimens from the AccuracyBased Survey from the College of American Pathologists, the newly developed assay agreed well with the CDC reference measurement procedure. In summary, immunoassay measurement of testosterone can be significantly inaccurate, especially at low concentrations. The newly developed LC-MS/MS assay provides accurate results across the entire measurable range.
1. Introduction
Testosterone is a cholesterol-derived androgen (sex hormone) with a molecular mass of 288.4 Da. Testosterone is primarily produced by the Leydig cells in the testes and by the theca cells in the ovaries, with small quantities produced by the adrenal glands in both sexes. Testosterone is responsible for primary sexual development and regulation of secondary male characteristics. In addition, it also plays a systemic role in the maintenance of bone density, muscle mass, and erythropoiesis [1]. Measurement of testosterone is central in the workup of hypogonadism in men, hyperandrogenism in women (e.g., idiopathic hirsutism, congenital adrenal hyperplasia, polycystic ovarian syndrome, and androgen-secreting ovarian or adrenal tumors), and delayed or precocious puberty in children [2–4]. It is also a useful biomarker to monitor testosterone supplementation in patients with hypogonadism and transgender men, as well as to monitor testosterone suppression in specific patients with prostate cancer [2,5].
In circulation, testosterone binds to sex hormone-binding globulin (SHBG) with high affinity and binds to albumin with low affinity. Only a small fraction of testosterone is present as free testosterone. Because the concentration of total testosterone can be affected by the abundance of SHBG and albumin, the concentration of free or bioavailable testosterone (the sum of free testosterone and that loosely bound to albumin) may be more informative in cases where SHBG or albumin are abnormal [2].
Immunoassay measurements of total testosterone can be significantly inaccurate, especially at low concentrations (i.e., <100 ng/dL) [6,7]. This is partially due to the technical challenges around making the signal of low concentrations of analyte statistically different from noise. In addition, different testosterone immunoassays use different approaches to calibration, which contributes to a lack of concordance of results between platforms and laboratories.
*The CDC formally harmonized the reference range for total testosterone in men in 2017 [8]. However, the application of this reference range to the diagnosis of hypogonadism in adult males across all laboratories requires that testosterone results be comparable to those generated by the CDC reference measurement procedure
Our laboratory has been using the testosterone immunoassay from Beckman Coulter (UniCel DxI). Clinical demand from our endocrinologists prompted us to set up a better in-house assay for the accurate measurement of testosterone. Given the cost of purchasing another immunoassay platform, we decided to develop a testosterone assay on our existing mass spectrometry platform to replace the immunoassay. The assay described in this manuscript only measures testosterone; however, it has the capacity for a multi-plex panel covering other clinically relevant steroid hormones in the future. Our method takes advantage of supported liquid extraction (SLE) to simplify sample preparation, which can be easily automated. We confirmed the inaccuracy of the Beckman UniCel DxI immunochemiluminescent total testosterone assay observed over the past decade.
4. Discussion
As with immunoassays in general, testosterone immunoassays are prone to interferences such as anti-reagent antibodies, structurally similar compounds, and alkaline phosphatases [12–16]. In addition to these sample-specific interferences that could cause spurious results on certain immunoassay platforms, testosterone immunoassays, in general, have limited accuracy, especially at low concentrations (e.g., <100 ng/ dL). The results from a large survey involving 142 certified clinical laboratories using 16 immunoassays showed that the bias was high as 73.1% compared to the reference method [6]
*In summary, this newly developed LC-MS/MS testosterone assay has improved accuracy and precision and agrees well with the reference method. We believe that this assay will allow clinicians to follow the diagnostic and treatment guideline with cutoffs established based on LCMS/MS-based assays. It will also benefit different patient populations including males, females, and pediatrics.
Accurate measurement of testosterone is important for the diagnosis of gonadal disorders in men, women, and children. Testosterone measurement has limited accuracy at low concentrations by most commercially available immunoassays. We aimed to develop an LC-MS/MS assay to address the inaccuracy of the in-house immunoassay observed over the past decade and to replace it with the new assay. Testosterone in serum/plasma was extracted with commercial-supported liquid extraction plates. Method validation was performed following the CLSI C62-A guideline. A total of 126 samples were used for method comparison between the Beckman UniCel DxI immunoassay and LC-MS/MS. Results by immunoassay were 20% lower compared with LC-MS/MS and had minimal correlation (R2 = 0.403) with LC-MS/MS below 100 ng/dL. When comparing specimens from the AccuracyBased Survey from the College of American Pathologists, the newly developed assay agreed well with the CDC reference measurement procedure. In summary, immunoassay measurement of testosterone can be significantly inaccurate, especially at low concentrations. The newly developed LC-MS/MS assay provides accurate results across the entire measurable range.
1. Introduction
Testosterone is a cholesterol-derived androgen (sex hormone) with a molecular mass of 288.4 Da. Testosterone is primarily produced by the Leydig cells in the testes and by the theca cells in the ovaries, with small quantities produced by the adrenal glands in both sexes. Testosterone is responsible for primary sexual development and regulation of secondary male characteristics. In addition, it also plays a systemic role in the maintenance of bone density, muscle mass, and erythropoiesis [1]. Measurement of testosterone is central in the workup of hypogonadism in men, hyperandrogenism in women (e.g., idiopathic hirsutism, congenital adrenal hyperplasia, polycystic ovarian syndrome, and androgen-secreting ovarian or adrenal tumors), and delayed or precocious puberty in children [2–4]. It is also a useful biomarker to monitor testosterone supplementation in patients with hypogonadism and transgender men, as well as to monitor testosterone suppression in specific patients with prostate cancer [2,5].
In circulation, testosterone binds to sex hormone-binding globulin (SHBG) with high affinity and binds to albumin with low affinity. Only a small fraction of testosterone is present as free testosterone. Because the concentration of total testosterone can be affected by the abundance of SHBG and albumin, the concentration of free or bioavailable testosterone (the sum of free testosterone and that loosely bound to albumin) may be more informative in cases where SHBG or albumin are abnormal [2].
Immunoassay measurements of total testosterone can be significantly inaccurate, especially at low concentrations (i.e., <100 ng/dL) [6,7]. This is partially due to the technical challenges around making the signal of low concentrations of analyte statistically different from noise. In addition, different testosterone immunoassays use different approaches to calibration, which contributes to a lack of concordance of results between platforms and laboratories.
*The CDC formally harmonized the reference range for total testosterone in men in 2017 [8]. However, the application of this reference range to the diagnosis of hypogonadism in adult males across all laboratories requires that testosterone results be comparable to those generated by the CDC reference measurement procedure
Our laboratory has been using the testosterone immunoassay from Beckman Coulter (UniCel DxI). Clinical demand from our endocrinologists prompted us to set up a better in-house assay for the accurate measurement of testosterone. Given the cost of purchasing another immunoassay platform, we decided to develop a testosterone assay on our existing mass spectrometry platform to replace the immunoassay. The assay described in this manuscript only measures testosterone; however, it has the capacity for a multi-plex panel covering other clinically relevant steroid hormones in the future. Our method takes advantage of supported liquid extraction (SLE) to simplify sample preparation, which can be easily automated. We confirmed the inaccuracy of the Beckman UniCel DxI immunochemiluminescent total testosterone assay observed over the past decade.
4. Discussion
As with immunoassays in general, testosterone immunoassays are prone to interferences such as anti-reagent antibodies, structurally similar compounds, and alkaline phosphatases [12–16]. In addition to these sample-specific interferences that could cause spurious results on certain immunoassay platforms, testosterone immunoassays, in general, have limited accuracy, especially at low concentrations (e.g., <100 ng/ dL). The results from a large survey involving 142 certified clinical laboratories using 16 immunoassays showed that the bias was high as 73.1% compared to the reference method [6]
*In summary, this newly developed LC-MS/MS testosterone assay has improved accuracy and precision and agrees well with the reference method. We believe that this assay will allow clinicians to follow the diagnostic and treatment guideline with cutoffs established based on LCMS/MS-based assays. It will also benefit different patient populations including males, females, and pediatrics.
