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Testosterone Replacement, Low T, HCG, & Beyond
Blood Test Discussion
Calculate Free Testosterone with TruT by FPT
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<blockquote data-quote="tareload" data-source="post: 189343"><p>Here we go:</p><p></p><p></p><p><strong>Zakharov (2015)</strong></p><p>[URL unfurl="true"]https://www.sciencedirect.com/science/article/abs/pii/S030372071400272X?via%3Dihub[/URL]</p><p></p><p></p><p>[ATTACH=full]11198[/ATTACH][ATTACH=full]11200[/ATTACH]</p><p></p><p></p><p>Frozen samples never thawed:</p><p>[ATTACH=full]11201[/ATTACH]</p><p></p><p>[ATTACH=full]11196[/ATTACH]</p><p></p><p><strong>Fiers 2018:</strong></p><p>[URL unfurl="true"]https://academic.oup.com/jcem/article/103/6/2167/4956600[/URL]</p><p></p><p><span style="font-size: 18px"><strong>Assay procedures</strong></span></p><p></p><p>Assay of serum SHBG levels was performed on an E170 Modular immunoassay analyzer and of albumin was performed on the Cobas 8000 (Roche Diagnostics; Indianapolis, IN). Assay of total T in serum and direct measurement of FT in dialysate after ED against serum as described later in Methods, was done by LC-MS/MS on an AB Sciex 5500 triple-quadrupole mass spectrometer (Toronto, ON, Canada), as previously described (<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">22</a>). This method was validated for T against an isotope dilution mass spectrometry candidate reference method (<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">23</a>) using a common serum panel, and it also has recently been compared with a reference method at the Centers for Disease Control and Prevention (Atlanta, GA) (<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">24</a>).</p><p></p><p>Serum FT level was determined by a direct dialysis method (<em>i.e.</em>, ED of undiluted serum against buffer with direct LC-MS/MS assay of FT in the dialysate). ED was performed using Fast Micro-Equilibrium dialyzer cartridges and regenerated cellulose 25 kDa membranes (Harvard Apparatus, Holliston, MA). Serum (men, 500 μL; women, 1000 μL) was dialyzed at 37°C for 24 hours at pH 7.28 using protein-free buffer prepared according to Yue <em>et al.</em> (<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">25</a>).</p><p></p><p>For assay of total T, a liquid–liquid extraction was performed on 100 µL serum samples. Interassay coefficient of variation for T is 6.5% at 3 ng/dL (104 pmol/L; n = 30) with a limit of quantitation of 1 ng/dL (35 pmol/L). For measurement of FT by direct ED, interassay coefficient of variability was 13.5% at 0.18 ng/dL (6.2 pmol/L) with a limit of quantitation of 0.07 ng/dL (2.4 pmol/L). Experiments on various buffer and serum volumes, nonspecific binding, and mass spectrometry ion suppression all yielded excellent target recoveries within 5% tolerance. Details are provided in a validation summary in the <a href="https://oup.silverchair-cdn.com/oup/backfile/Content_public/Journal/jcem/103/6/10.1210_jc.2017-02360/1/jc.2017-02360.sd1.docx?Expires=1605834098&Signature=CVJ3MQD2heIBDMu9IvFoAaYl3XSxMfX93gsIsJAkKncya4KKhI1l6wu4pA6Z-cJdE8wDiyCIfxE42xmYKsviwTAbVEC9I8SWbknfx1gf1N68HVqR18J~SkbBdQPrGxeTxXZXFlgKxQNzF3INuyDVzQl-UsoyOjBEivwCp4VE-ewfjKgRfeN2NpD33D0Azk8PaPLZqPSoSpq20xLfUt8-MNbBApu-oNxpSJ4k1hOJ46RqDdG3CYakRL-4JlzlTwVoHBN69v34L~LF1uW6SSM5vYn5~otX1eyTYsFtha3UyPYWmxg1hQSaZ9uH7-p6q5IrvZ-WzL-WIX1bNET-SDqozw__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA" target="_blank">Supplemental Validation Summary</a>.</p><p></p><p></p><p>Check out the Validation summary:</p><p>[URL unfurl="true"]https://oup.silverchair-cdn.com/oup/backfile/Content_public/Journal/jcem/103/6/10.1210_jc.2017-02360/1/jc.2017-02360.sd1.docx?Expires=1605834098&Signature=CVJ3MQD2heIBDMu9IvFoAaYl3XSxMfX93gsIsJAkKncya4KKhI1l6wu4pA6Z-cJdE8wDiyCIfxE42xmYKsviwTAbVEC9I8SWbknfx1gf1N68HVqR18J~SkbBdQPrGxeTxXZXFlgKxQNzF3INuyDVzQl-UsoyOjBEivwCp4VE-ewfjKgRfeN2NpD33D0Azk8PaPLZqPSoSpq20xLfUt8-MNbBApu-oNxpSJ4k1hOJ46RqDdG3CYakRL-4JlzlTwVoHBN69v34L~LF1uW6SSM5vYn5~otX1eyTYsFtha3UyPYWmxg1hQSaZ9uH7-p6q5IrvZ-WzL-WIX1bNET-SDqozw__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA[/URL]</p><p></p><p></p><p></p><p></p><p></p><p></p><p>[ATTACH=full]11197[/ATTACH]</p><p></p><p></p><p>Now superimpose the % free T data from these two papers and plot vs SHBG or TT. What am I missing? Who didn't properly validate their free T ED assay? If you sent a sample and ran it on these two machines you would get drastically different free T by "gold standard" ED method.</p></blockquote><p></p>
[QUOTE="tareload, post: 189343"] Here we go: [B]Zakharov (2015)[/B] [URL unfurl="true"]https://www.sciencedirect.com/science/article/abs/pii/S030372071400272X?via%3Dihub[/URL] [ATTACH type="full" alt="1603342841770.png"]11198[/ATTACH][ATTACH type="full" alt="1603342900697.png"]11200[/ATTACH] Frozen samples never thawed: [ATTACH type="full" alt="1603343700489.png"]11201[/ATTACH] [ATTACH type="full" width="585px" alt="1603342700496.png"]11196[/ATTACH] [B]Fiers 2018:[/B] [URL unfurl="true"]https://academic.oup.com/jcem/article/103/6/2167/4956600[/URL] [SIZE=18px][B]Assay procedures[/B][/SIZE] Assay of serum SHBG levels was performed on an E170 Modular immunoassay analyzer and of albumin was performed on the Cobas 8000 (Roche Diagnostics; Indianapolis, IN). Assay of total T in serum and direct measurement of FT in dialysate after ED against serum as described later in Methods, was done by LC-MS/MS on an AB Sciex 5500 triple-quadrupole mass spectrometer (Toronto, ON, Canada), as previously described ([URL='https://www.excelmale.com/forum/javascript%3A;']22[/URL]). This method was validated for T against an isotope dilution mass spectrometry candidate reference method ([URL='https://www.excelmale.com/forum/javascript%3A;']23[/URL]) using a common serum panel, and it also has recently been compared with a reference method at the Centers for Disease Control and Prevention (Atlanta, GA) ([URL='https://www.excelmale.com/forum/javascript%3A;']24[/URL]). Serum FT level was determined by a direct dialysis method ([I]i.e.[/I], ED of undiluted serum against buffer with direct LC-MS/MS assay of FT in the dialysate). ED was performed using Fast Micro-Equilibrium dialyzer cartridges and regenerated cellulose 25 kDa membranes (Harvard Apparatus, Holliston, MA). Serum (men, 500 μL; women, 1000 μL) was dialyzed at 37°C for 24 hours at pH 7.28 using protein-free buffer prepared according to Yue [I]et al.[/I] ([URL='https://www.excelmale.com/forum/javascript%3A;']25[/URL]). For assay of total T, a liquid–liquid extraction was performed on 100 µL serum samples. Interassay coefficient of variation for T is 6.5% at 3 ng/dL (104 pmol/L; n = 30) with a limit of quantitation of 1 ng/dL (35 pmol/L). For measurement of FT by direct ED, interassay coefficient of variability was 13.5% at 0.18 ng/dL (6.2 pmol/L) with a limit of quantitation of 0.07 ng/dL (2.4 pmol/L). Experiments on various buffer and serum volumes, nonspecific binding, and mass spectrometry ion suppression all yielded excellent target recoveries within 5% tolerance. Details are provided in a validation summary in the [URL='https://oup.silverchair-cdn.com/oup/backfile/Content_public/Journal/jcem/103/6/10.1210_jc.2017-02360/1/jc.2017-02360.sd1.docx?Expires=1605834098&Signature=CVJ3MQD2heIBDMu9IvFoAaYl3XSxMfX93gsIsJAkKncya4KKhI1l6wu4pA6Z-cJdE8wDiyCIfxE42xmYKsviwTAbVEC9I8SWbknfx1gf1N68HVqR18J~SkbBdQPrGxeTxXZXFlgKxQNzF3INuyDVzQl-UsoyOjBEivwCp4VE-ewfjKgRfeN2NpD33D0Azk8PaPLZqPSoSpq20xLfUt8-MNbBApu-oNxpSJ4k1hOJ46RqDdG3CYakRL-4JlzlTwVoHBN69v34L~LF1uW6SSM5vYn5~otX1eyTYsFtha3UyPYWmxg1hQSaZ9uH7-p6q5IrvZ-WzL-WIX1bNET-SDqozw__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA']Supplemental Validation Summary[/URL]. Check out the Validation summary: [URL unfurl="true"]https://oup.silverchair-cdn.com/oup/backfile/Content_public/Journal/jcem/103/6/10.1210_jc.2017-02360/1/jc.2017-02360.sd1.docx?Expires=1605834098&Signature=CVJ3MQD2heIBDMu9IvFoAaYl3XSxMfX93gsIsJAkKncya4KKhI1l6wu4pA6Z-cJdE8wDiyCIfxE42xmYKsviwTAbVEC9I8SWbknfx1gf1N68HVqR18J~SkbBdQPrGxeTxXZXFlgKxQNzF3INuyDVzQl-UsoyOjBEivwCp4VE-ewfjKgRfeN2NpD33D0Azk8PaPLZqPSoSpq20xLfUt8-MNbBApu-oNxpSJ4k1hOJ46RqDdG3CYakRL-4JlzlTwVoHBN69v34L~LF1uW6SSM5vYn5~otX1eyTYsFtha3UyPYWmxg1hQSaZ9uH7-p6q5IrvZ-WzL-WIX1bNET-SDqozw__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA[/URL] [ATTACH type="full" alt="1603342765919.png"]11197[/ATTACH] Now superimpose the % free T data from these two papers and plot vs SHBG or TT. What am I missing? Who didn't properly validate their free T ED assay? If you sent a sample and ran it on these two machines you would get drastically different free T by "gold standard" ED method. [/QUOTE]
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