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Testosterone Replacement, Low T, HCG, & Beyond
Blood Test Discussion
Direct Measurements of Free Hormones Using Mass Spectrometry-Based Methods
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<blockquote data-quote="madman" data-source="post: 260322" data-attributes="member: 13851"><p><strong><em>*Because FHs are present in biological samples in trace concentrations, <u>highly sensitive and specific methods are required for accurate measurement of FH concentrations</u>.</em></strong></p><p><strong><em></em></strong></p><p><strong><em>*A limitation of calculation-based methods is in the <u>assumption of uniform hormone affinity to binding proteins among individuals</u>. <u>However, binding protein variants, posttranslational modifications, and other factors may alter binding affinity, causing erroneous results and biases</u> (10). Direct immunoassay methods show discrepancy among methods of different manufacturers and <u>often perform poorly when binding protein concentrations are very elevated or decreased</u>, with some methods suffering from biotin interference (9).</em></strong></p><p><strong><em></em></strong></p><p><strong><em>*To standardize testing and to harmonize among-laboratory quantitative agreement, it is important to use <u>high-quality reference materials for preparation of the calibration standards used in methods for measurement of FHs</u>.</em></strong></p><p><strong><em></em></strong></p><p><strong><em>*When a binding protein abnormality is suspected (15). <u>Equilibrium dialysis (ED) followed by LC–MS/MS is considered the gold standard methodology</u>, with a number of LC–MS/ MS methods developed and introduced in routine patient testing (12, 16–22).</em></strong></p><p><strong><em></em></strong></p><p><strong><em>*<u>The observed concentration of FH after ED depends on</u> (a) volume of the sample, (b) volume of the dialysis buffer, (c) the buffer composition, (d) size of the molecules being separated, material and MWC of the membrane, (e) incubation temperature, (f) agitation of the device during the dialysis process (e.g., rotation), (g) surface area of the membrane, and (h) adsorption of the FH of interest by the material of the membrane and the housing.</em></strong></p><p><strong><em></em></strong></p><p><strong><em>*<u>Potential pitfalls of ED are that</u> (a) a membrane or seal may break during the pipetting, resulting in a contamination of the dialysate with a protein-bound analyte; (b) as FH is distributed between the 2 chambers, the equilibrium between protein-bound and FH is continuously reestablished (in the chamber containing biological samples) with new fractions of FH released from the carrier proteins to compensate for the FH migrated into the dialysis buffer; this may result in overestimation of the FH concentration; and (c) some hormones are stable in biological samples while bound to the carrier proteins and may get adsorbed by the surfaces when present in solution in a free form (in the dialysis buffer), resulting in underestimation of the FH concentration.</em></strong></p><p><strong><em></em></strong></p><p><strong><em>*<u>A number of potential pitfalls could affect performance of UF-based devices and the observed concentrations of FH</u>: (a) the full surface of the membrane is accessible for filtration only during the initial stages of the UF process; as filtration continues, the membrane gets clogged with high MW proteins significantly reducing the filtration rate and resulting in only a fraction of FH being filtered from the initial sample; (b) because every patient sample is unique, clogging may occur at different times during the UF process, resulting in variability in the volume of the filtrate and the resultant fraction of FH that is filtered; and (c) the centrifugation process generates heat, which shifts equilibrium between the free- and the protein-bound hormone present in the processed samples. </em></strong></p><p></p><p><em><strong>*<u>FH concentrations measured by UF-based methods often do not agree with ED-based methods because of UF conditions</u> (temperature, time, centrifugation speed), <u>the type of the UF device</u> (MWC membrane material, material of the of the housing, seal around the membrane, etc.), and <u>inconsistencies in the filtration rate</u>. <u>Therefore, reference intervals are typically not interchangeable across methods for measurement of the same FH</u>.</strong></em></p></blockquote><p></p>
[QUOTE="madman, post: 260322, member: 13851"] [B][I]*Because FHs are present in biological samples in trace concentrations, [U]highly sensitive and specific methods are required for accurate measurement of FH concentrations[/U]. *A limitation of calculation-based methods is in the [U]assumption of uniform hormone affinity to binding proteins among individuals[/U]. [U]However, binding protein variants, posttranslational modifications, and other factors may alter binding affinity, causing erroneous results and biases[/U] (10). Direct immunoassay methods show discrepancy among methods of different manufacturers and [U]often perform poorly when binding protein concentrations are very elevated or decreased[/U], with some methods suffering from biotin interference (9). *To standardize testing and to harmonize among-laboratory quantitative agreement, it is important to use [U]high-quality reference materials for preparation of the calibration standards used in methods for measurement of FHs[/U]. *When a binding protein abnormality is suspected (15). [U]Equilibrium dialysis (ED) followed by LC–MS/MS is considered the gold standard methodology[/U], with a number of LC–MS/ MS methods developed and introduced in routine patient testing (12, 16–22). *[U]The observed concentration of FH after ED depends on[/U] (a) volume of the sample, (b) volume of the dialysis buffer, (c) the buffer composition, (d) size of the molecules being separated, material and MWC of the membrane, (e) incubation temperature, (f) agitation of the device during the dialysis process (e.g., rotation), (g) surface area of the membrane, and (h) adsorption of the FH of interest by the material of the membrane and the housing. *[U]Potential pitfalls of ED are that[/U] (a) a membrane or seal may break during the pipetting, resulting in a contamination of the dialysate with a protein-bound analyte; (b) as FH is distributed between the 2 chambers, the equilibrium between protein-bound and FH is continuously reestablished (in the chamber containing biological samples) with new fractions of FH released from the carrier proteins to compensate for the FH migrated into the dialysis buffer; this may result in overestimation of the FH concentration; and (c) some hormones are stable in biological samples while bound to the carrier proteins and may get adsorbed by the surfaces when present in solution in a free form (in the dialysis buffer), resulting in underestimation of the FH concentration. *[U]A number of potential pitfalls could affect performance of UF-based devices and the observed concentrations of FH[/U]: (a) the full surface of the membrane is accessible for filtration only during the initial stages of the UF process; as filtration continues, the membrane gets clogged with high MW proteins significantly reducing the filtration rate and resulting in only a fraction of FH being filtered from the initial sample; (b) because every patient sample is unique, clogging may occur at different times during the UF process, resulting in variability in the volume of the filtrate and the resultant fraction of FH that is filtered; and (c) the centrifugation process generates heat, which shifts equilibrium between the free- and the protein-bound hormone present in the processed samples. [/I][/B] [I][B]*[U]FH concentrations measured by UF-based methods often do not agree with ED-based methods because of UF conditions[/U] (temperature, time, centrifugation speed), [U]the type of the UF device[/U] (MWC membrane material, material of the of the housing, seal around the membrane, etc.), and [U]inconsistencies in the filtration rate[/U]. [U]Therefore, reference intervals are typically not interchangeable across methods for measurement of the same FH[/U].[/B][/I] [/QUOTE]
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Testosterone Replacement, Low T, HCG, & Beyond
Blood Test Discussion
Direct Measurements of Free Hormones Using Mass Spectrometry-Based Methods
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