Assessment of immunoassay calibrators for T, E2, and P4 using UPLC-MS

[madman]

Manufacturer-supplied calibrators for sex steroid immunoassays often have inaccurate concentrations. Only a small percentage of testosterone, estradiol, and progesterone calibrators meet the 20% margin of stated label concentration. The assigned uncertainties of the calibrators don't encompass the discrepancies. Direct steroid immunoassays show method-specific bias compared to mass spectrometry-based methods. Standardization efforts have been made, but deviations persist. Direct steroid immunoassays have limitations in accuracy and validity. LC-MS-based steroid measurement is desired for better accuracy in clinical practice, but cost and expertise pose challenges.

 

[madman]

Table 1: Testosterone immunoassay calibrators.

 

[madman]

Figure 1: Bland-Altman deviance plot (left) and Passing-Bablok regression plot (right) for testosterone calibrators for five different commercial calibrators. The line of identity is shown as a dashed black line.

 

[madman]

Figure 2: Bland-Altman deviance plot (left) and Passing-Bablok regression plot (right) for estradiol calibrators for four different commercial calibrators. The line of identity is shown as a dashed black line.

 

[madman]

Table 2: Estradiol immunoassay calibrators.

 

[madman]

Figure 3: Bland-Altman deviance plot (left) and Passing-Bablok regression plot (right) for progesterone calibrators for four different commercial calibrators. The line of identity is shown as a dashed black line.

 

[madman]

Table 3: Progesterone immunoassay calibrator measurements.

 

[madman]

*Steroid measurements in clinical practice are primarily done using direct, non-extraction immunoassays on automated platforms

*The accuracy of these immunoassays is limited by cross-reactivity, matrix interference, and the accuracy of manufacturer-supplied calibrators

*The accuracy of calibrators for testosterone, estradiol, and progesterone immunoassays was investigated using liquid chromatography-mass spectrometry (LC-MS)

*A significant number of calibrators deviated from the label concentration, with some being outside the 20% margin

*The inaccurate calibration of manufacturer-supplied standards can contribute to the inaccuracy of commercial direct steroid immunoassays

 

[madman]

*Discrepancies persist in steroid measurements despite international standardization efforts

*The Joint Committee for Traceability in Laboratory Medicine (JCTLM) promotes traceability and provides reference materials, measurement procedures, and services for all three measurements

*Manufacturers provide information on reference methods and materials, with only one (Beckman-Coulter E2) explicitly stating it in their product information

*The CDC HoST program supports accuracy in steroid measurements, but currently, only Siemens Centaur testosterone is verified in the program

*Direct steroid immunoassays face difficulty meeting the accuracy-based standards of the CDC HOST program due to inherent method-specific bias

 

[madman]

Thread 'IMMUNOASSAY DISCREPANCIES IN MEASUREMENT OF T LEVELS: UNDERESTIMATION OF TRUE TESTOSTERONE MAY RESULT IN OVER-TREATMENT WITH TRT'

Mar 28, 2025

Introduction

AUA guidelines define testosterone deficiency as two levels < 300 ng/dL with signs/symptoms.Testosterone (T) measurement is performed using immunoassays (IA) or mass spectrometry (MS), with MS as the gold standard. However, limited data suggests there may be significant variation between assays, which may have implications for initiation and cessation of testosterone replacement therapy (TRT). The objective was to examine variation in testosterone assays and men at risk for unnecessary TRT treatment.


Methods

After IRB approval, we identified men...

• madman
• hypogonadism; t therapy; sexual health
• Replies: 2
• Forum: Testosterone and Men's Health Articles