CDC Hormone Standardization Program (CDC HoSt) Certified Free Testosterone Procedures is coming!
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madman
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THE IMPORTANCE OF ACCURATE HORMONE TESTS
https://www.hormoneassays.org/ Watch our video to learn about the importance of accurate hormone testing. Tests to measure the level of a specific hormone in the body are the third most common diagnostic in medicine today yet some current hormone tests are not sufficiently accurate or...
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madman
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*Measuring FT is technically challenging and shows high variability. The CDC clinical standardization program is developing a high throughput method using the gold-standard equilibrium dialysis (ED) procedure with isotope dilution ultra-high-performance liquid chromatography-tandem mass spectrometry (ID-UHPLC-MS/MS).
post #21
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Centers for Disease Control and Prevention, Atlanta, GA, USA.
Abstract
Free testosterone (FT) has been used as a biomarker in clinical patient care and public health research to assess and manage patients with androgenic abnormalities. The latest Endocrine Society clinical practice guideline for testosterone therapy in men with hypogonadism recommends measuring FT for those with borderline and low total testosterone concentrations, or those who have conditions that change SHBG concentrations, such as some metabolic or hormonal diseases, certain medication use, or SHBG genetic polymorphisms. Measuring FT is technically challenging and shows high variability. The CDC clinical standardization program is developing a high throughput method using the gold-standard equilibrium dialysis (ED) procedure with isotope dilution ultra-high-performance liquid chromatography-tandem mass spectrometry (ID-UHPLC-MS/MS).
A serum sample was dialyzed against a protein-free HEPES buffer (pH 7.4) at 37 °C until equilibrium. After isolating endogenous FT from protein-bound testosterone by ED, isotope-labeled internal standard (13C3-testosterone) was added to the dialysate for quantification. Certified pure primary reference material (National Measurement Institute M914) was used to prepare calibrators, enabling traceable quantitation and ensuring measurement trueness. FT was further isolated from the dialysate matrix using supported liquid extraction and a chromatographic separation from interfering compounds and quantitation by tandem MS. The dialysis step requires maintaining the endogenous free hormone equilibrium so that results in dialysate reflect FT concentrations in the blood without influence from dilution, temperature, or pH. The dialyzer system has a 1:1 sample-to-buffer volume and has been used in reference measurement procedures for free hormone measurements, serving as the standard for method performance comparison. Four commercially available devices designed for high throughput in multiple well-plate formats, requiring respective sample-to-buffer ratios, were evaluated for their recovery, speed, ease of automation by a liquid handling system, repeatability, and robustness. Preliminary results showed that a device with 1:1 sample-to-buffer volume had the most comparable results to those obtained from the standard dialyzer, with the mean bias less than 15%. The device with the highest sample-to-buffer ratio showed bias as high as 50%. These data suggest that controlling the sample-to-buffer ratio is a critical step in the ED FT method.
post #21
CDC STANDARDIZED TOTAL T and ESTRADIOL ASSAYS and soon to be FREE TESTOSTERONE!
How the CDC Clinical Standardization Programs Are Improving Hormone Tests - AACC.org WHAT PROGRESS HAS CDC MADE WITH STANDARDIZING TOTAL TESTOSTERONE AND ESTRADIOL TESTS? Since HoSt began in 2010, CDC has had more than 350 participants in 15 countries. Participants have shown measurable...
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Measurement of Free Testosterone in Serum Using Equilibrium Dialysis Coupled With ID-UHPLC-MS/MS: Comparison Between Equilibrium Devices (2021)
Hui Zhou, Ph.D., Ashley Ribera, BS, Amonae Dabbs-Brown, BS, Uliana Danilenko, Ph.D., Hubert W. Vesper, Ph.D.Centers for Disease Control and Prevention, Atlanta, GA, USA.
Abstract
Free testosterone (FT) has been used as a biomarker in clinical patient care and public health research to assess and manage patients with androgenic abnormalities. The latest Endocrine Society clinical practice guideline for testosterone therapy in men with hypogonadism recommends measuring FT for those with borderline and low total testosterone concentrations, or those who have conditions that change SHBG concentrations, such as some metabolic or hormonal diseases, certain medication use, or SHBG genetic polymorphisms. Measuring FT is technically challenging and shows high variability. The CDC clinical standardization program is developing a high throughput method using the gold-standard equilibrium dialysis (ED) procedure with isotope dilution ultra-high-performance liquid chromatography-tandem mass spectrometry (ID-UHPLC-MS/MS).
A serum sample was dialyzed against a protein-free HEPES buffer (pH 7.4) at 37 °C until equilibrium. After isolating endogenous FT from protein-bound testosterone by ED, isotope-labeled internal standard (13C3-testosterone) was added to the dialysate for quantification. Certified pure primary reference material (National Measurement Institute M914) was used to prepare calibrators, enabling traceable quantitation and ensuring measurement trueness. FT was further isolated from the dialysate matrix using supported liquid extraction and a chromatographic separation from interfering compounds and quantitation by tandem MS. The dialysis step requires maintaining the endogenous free hormone equilibrium so that results in dialysate reflect FT concentrations in the blood without influence from dilution, temperature, or pH. The dialyzer system has a 1:1 sample-to-buffer volume and has been used in reference measurement procedures for free hormone measurements, serving as the standard for method performance comparison. Four commercially available devices designed for high throughput in multiple well-plate formats, requiring respective sample-to-buffer ratios, were evaluated for their recovery, speed, ease of automation by a liquid handling system, repeatability, and robustness. Preliminary results showed that a device with 1:1 sample-to-buffer volume had the most comparable results to those obtained from the standard dialyzer, with the mean bias less than 15%. The device with the highest sample-to-buffer ratio showed bias as high as 50%. These data suggest that controlling the sample-to-buffer ratio is a critical step in the ED FT method.
madman
Super Moderator
When Binding Proteins Interfere With Immunoassays
https://www.aacc.org/cln/articles/2022/june/when-binding-proteins-interfere-with-immunoassays WHY ARE ABNORMAL BINDING PROTEIN CONCENTRATIONS AN ISSUE FOR IMMUNOASSAYS? Immunoassay detection relies on noncovalent binding interactions between the receptor and the target analyte. These...
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madman
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What's Coming? Accurate measurement of total and free testosterone levels for the diagnosis of androgen disorders
Accurate measurement of total and free testosterone levels for the diagnosis of androgen disorders (7/2022) Ezgi Caliskan Guzelce, MD, Clinical/ Research Fellow, Francesca Galbiati, MD, Fellow, Anna L. Goldman, MD, Assistant Professor of Medicine, Arijeet K. Gattu, MD, Fellow, Shehzad Basaria...
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madman
Super Moderator
Measurement of FT in Serum using ED Coupled with ID-UHPLC-MS/MS: in Comparison to cFTV
Reproductive Endocrinology ODP419 Measurement of Free Testosterone in Serum using Equilibrium Dialysis Coupled with ID-UHPLC-MS/ MS: in Comparison to Calculated FT Hui Zhou, Ph.D., Ashley Ribera, BS, Uliana Danilenko, Ph.D., Otoe Sugahara, BS, and Hubert Vesper, PhD Assessment of free...
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madman
Super Moderator
Comparability of Free Testosterone Measurements- Results of the Interlaboratory Study
https://academic.oup.com/clinchem/article/70/Supplement_1/hvae106.074/7760719 Assessment of free testosterone (FT) has been recommended as a biomarker for diagnosis and management of hypogonadism and female hyperandrogenism. Reliable laboratory measurements are essential for correctly...
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