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<blockquote data-quote="madman" data-source="post: 196777" data-attributes="member: 13851"><p>Forget the lab.....it's the assay!</p><p></p><p></p><p><strong>Methodology for measuring testosterone, DHT, and SHBG in a clinical setting (2004)</strong></p><p><em>M.Simoni</em></p><p></p><p></p><p><strong>21.4.4 Assessment of free testosterone</strong></p><p></p><p>The direct measurement of free testosterone in serum is based on the same principles governing the assay of free thyroid hormones and has been extensively considered and reviewed by R. Ekins in the past (Ekins 1990). As indicated above, <strong><em><u>serum testosterone exists in an equilibrium between free and protein-bound fractions, an equilibrium which is invariably disturbed by all methods of free hormone measurement, a factor that should be kept in mind when choosing a method and analyzing the data</u>.</em></strong> <strong><em><u>The methods of reference for free hormone analysis are equilibrium dialysis and ultrafiltration, which should be used for research purposes and to validate other systems</u>.</em></strong></p><p><strong></strong></p><p><strong></strong></p><p><strong>21.4.4.3 Direct free testosterone RIA</strong></p><p><strong></strong></p><p><strong><em><u>These methods are based on the concept that if an antibody is added to a serum sample, only free hormone will bind to it and the antibody occupancy will depend on the free hormone concentration</u>.<u>As a result, however, the protein-bound hormone will dissociate and a new equilibrium will be established</u>. Therefore, the antibody concentration should be kept “small” enough to minimize the depletion of the protein-bound pool, i.e. not more than 1% of total hormone should be displaced from the binding proteins to the antibody.</em></strong> Quantification of the antibody-bound hormone, i.e. the free hormone concentration, can be achieved indirectly by knowing the total hormone concentration, adding labeled hormone, and measuring the fraction of it which is taken up by the antibody (“labeled hormone antibody uptake”). Alternatively, a two-step approach involves adding the sample to a solid phase antibody, washing off the unbound serum components, and adding labeled hormone which will be bound by the residual, unoccupied antibody binding sites. Since the amount of antibody is limited, the higher the free hormone concentration, the lower the number of unoccupied antibody sites at the end of the first incubation, the lower the antibody occupancy by labeled hormone at the end of the second incubation. In order for this method to work, a two-step approach, with the removal of serum after the first reaction of the antiserum with the free hormone, is necessary because if the labeled hormone interacts with the serum binding proteins, this would impair the estimation of antibody occupancy by the tracer. <em><strong><u>The two-step approach, however, is not necessary if one uses a labeled-compound that is totally non-reactive with serum proteins but can be recognized by the solid phase antibody present in a limited amount and which competes for binding with the free hormone in the sample</u>.</strong></em> <strong><em><u>This is the principle of the free “analog” testosterone assay on which some popular commercially available kits are based</u>. </em></strong></p><p><strong><em></em></strong></p><p><strong><em><u>The direct measurement of free testosterone in serum based on the labeled hormone “analog” is valid, provided that the analog does not interact with the serum proteins, a condition which is currently not met by commercial kits</u>. </em></strong>In fact, neither</p><p>the identity of the analog tracer, nor the validation of the kit (showing the absence of interactions with the serum protein) is usually disclosed by the manufacturer<em><strong>.</strong></em><strong><em> <u>On the contrary, the “analog” principle is often not even mentioned or is misrepresented in the instruction accompanying the kits,which are often validated only against other kits and not against dialysis or ultrafiltration</u>. <u>It should be kept in mind that, in practice, finding a hormone analog totally unreactive with serum proteins is very difficult and several studies have shown that such an interaction indeed occurs, resulting in inaccurate measurements of free testosterone</u>. <u>In this respect it is interesting that serum-free testosterone measured by the analog method accounts for 0.5–0.65% of total testosterone, while equilibrium dialysis and ultrafiltration give values of 1.5–4%, revealing inconsistencies between the different approaches</u> (Rosner 1997; Winterset al. 1998).</em></strong><em><strong> <u>In a direct comparison, free testosterone values measured by a bestseller “analog” kit were only 20–30% of those measured by equilibrium dialysis</u> (Vermeulen et al. 1999).</strong></em>For these reasons it is recommended that if an “analog” method is to be considered for routine free testosterone determination, in-house validation of the kit should involve comparison with dialysis or ultrafiltration and estimation of the binding of the analog tracer to endogenous proteins e.g. by adding exogenous SHBG (e.g. serum from pregnant women) and by estimating tracer binding to concanavalin A-bound SHBG after chromatography of the serum samples (Winterset al. 1998). <strong><em>Unfortunately, the kits for direct free testosterone measurement presently available do not measure what they claim to do and give inaccurate results (Rosner 2001). Their use should be discouraged.</em></strong></p><p>--------------------------------------------------------------------------------------------------</p><p></p><p></p><p><strong>Methodology for measuring testosterone, dihydrotestosterone and sex hormone-binding globulin in a clinical setting (2008)</strong></p><p><em>Manuela Simoni, Flaminia Fanelli, Laura Roli, and Uberto Pagotto</em></p><p></p><p></p><p><strong>4.3.4.3 Direct free testosterone assays </strong></p><p></p><p>The principles of the direct free testosterone methods have been described in detail in the previous edition of this book (Simoni 2004). <strong><em>In practice such assays are based on a labeled compound to be added to the sample, the “analog,” which is totally non-reactive with serum proteins, but can be recognized by the solid-phase antibody present in a limited amount and which competes for binding with the free hormone in the sample.</em></strong></p><p><strong><em></em></strong></p><p><strong><em>Several commercially available kits are based on this principle and are widely used to assess free testosterone in clinical samples. <u>Unfortunately, there is evidence that these methods are totally inaccurate</u> (Fritz et al. 2008; Chen et al. 2010). </em></strong>In fact, the direct measurement of free testosterone in serum based on the labeled hormone “analog” is valid only if the analog does not interact with the serum proteins: a condition which is currently not met by commercial kits. In fact, neither the identity of the analog tracer, nor the validation of the kit (showing the absence of interactions with the serum protein) is usually disclosed by the manufacturer. On the contrary, the “analog” principle is often not even mentioned or is misrepresented in the instructions accompanying the kits, which are often validated only against other kits and not against dialysis or ultrafiltration. It should be kept in mind that, in practice, finding a hormone analog totally unreactive with serum proteins is very difficult, and several studies have shown that such an interaction indeed occurs, resulting in inaccurate measurements of free testosterone. In this respect it is interesting that serum-free testosterone measured by an “analog” method accounts for 0.5–0.65% of total testosterone, while equilibrium dialysis and ultrafiltration give values of 1.5–4%, revealing inconsistencies between the different approaches (Rosner 1997; Winters et al. 1998). In a direct comparison, free testosterone values measured by a bestseller “analog” kit was only 20–30% of those measured by equilibrium dialysis (Vermeulen et al. 1999).</p><p></p><p><strong><em><u>For these reasons the kits for direct free testosterone measurement presently available do not measure what they claim and should not be used</u> (Rosner 2001; Swerdloff and Wang 2008). <u>If LC-MS/MS coupled to ultrafiltration becomes technically affordable, this approach is likely to become the method of choice for assessment of free testosterone in clinical serum samples</u>.</em></strong></p></blockquote><p></p>
[QUOTE="madman, post: 196777, member: 13851"] Forget the lab.....it's the assay! [B]Methodology for measuring testosterone, DHT, and SHBG in a clinical setting (2004)[/B] [I]M.Simoni[/I] [B]21.4.4 Assessment of free testosterone[/B] The direct measurement of free testosterone in serum is based on the same principles governing the assay of free thyroid hormones and has been extensively considered and reviewed by R. Ekins in the past (Ekins 1990). As indicated above, [B][I][U]serum testosterone exists in an equilibrium between free and protein-bound fractions, an equilibrium which is invariably disturbed by all methods of free hormone measurement, a factor that should be kept in mind when choosing a method and analyzing the data[/U].[/I][/B] [B][I][U]The methods of reference for free hormone analysis are equilibrium dialysis and ultrafiltration, which should be used for research purposes and to validate other systems[/U].[/I] 21.4.4.3 Direct free testosterone RIA [I][U]These methods are based on the concept that if an antibody is added to a serum sample, only free hormone will bind to it and the antibody occupancy will depend on the free hormone concentration[/U].[U]As a result, however, the protein-bound hormone will dissociate and a new equilibrium will be established[/U]. Therefore, the antibody concentration should be kept “small” enough to minimize the depletion of the protein-bound pool, i.e. not more than 1% of total hormone should be displaced from the binding proteins to the antibody.[/I][/B] Quantification of the antibody-bound hormone, i.e. the free hormone concentration, can be achieved indirectly by knowing the total hormone concentration, adding labeled hormone, and measuring the fraction of it which is taken up by the antibody (“labeled hormone antibody uptake”). Alternatively, a two-step approach involves adding the sample to a solid phase antibody, washing off the unbound serum components, and adding labeled hormone which will be bound by the residual, unoccupied antibody binding sites. Since the amount of antibody is limited, the higher the free hormone concentration, the lower the number of unoccupied antibody sites at the end of the first incubation, the lower the antibody occupancy by labeled hormone at the end of the second incubation. In order for this method to work, a two-step approach, with the removal of serum after the first reaction of the antiserum with the free hormone, is necessary because if the labeled hormone interacts with the serum binding proteins, this would impair the estimation of antibody occupancy by the tracer. [I][B][U]The two-step approach, however, is not necessary if one uses a labeled-compound that is totally non-reactive with serum proteins but can be recognized by the solid phase antibody present in a limited amount and which competes for binding with the free hormone in the sample[/U].[/B][/I] [B][I][U]This is the principle of the free “analog” testosterone assay on which some popular commercially available kits are based[/U]. [U]The direct measurement of free testosterone in serum based on the labeled hormone “analog” is valid, provided that the analog does not interact with the serum proteins, a condition which is currently not met by commercial kits[/U]. [/I][/B]In fact, neither the identity of the analog tracer, nor the validation of the kit (showing the absence of interactions with the serum protein) is usually disclosed by the manufacturer[I][B].[/B][/I][B][I] [U]On the contrary, the “analog” principle is often not even mentioned or is misrepresented in the instruction accompanying the kits,which are often validated only against other kits and not against dialysis or ultrafiltration[/U]. [U]It should be kept in mind that, in practice, finding a hormone analog totally unreactive with serum proteins is very difficult and several studies have shown that such an interaction indeed occurs, resulting in inaccurate measurements of free testosterone[/U]. [U]In this respect it is interesting that serum-free testosterone measured by the analog method accounts for 0.5–0.65% of total testosterone, while equilibrium dialysis and ultrafiltration give values of 1.5–4%, revealing inconsistencies between the different approaches[/U] (Rosner 1997; Winterset al. 1998).[/I][/B][I][B] [U]In a direct comparison, free testosterone values measured by a bestseller “analog” kit were only 20–30% of those measured by equilibrium dialysis[/U] (Vermeulen et al. 1999).[/B][/I]For these reasons it is recommended that if an “analog” method is to be considered for routine free testosterone determination, in-house validation of the kit should involve comparison with dialysis or ultrafiltration and estimation of the binding of the analog tracer to endogenous proteins e.g. by adding exogenous SHBG (e.g. serum from pregnant women) and by estimating tracer binding to concanavalin A-bound SHBG after chromatography of the serum samples (Winterset al. 1998). [B][I]Unfortunately, the kits for direct free testosterone measurement presently available do not measure what they claim to do and give inaccurate results (Rosner 2001). Their use should be discouraged.[/I][/B] -------------------------------------------------------------------------------------------------- [B]Methodology for measuring testosterone, dihydrotestosterone and sex hormone-binding globulin in a clinical setting (2008)[/B] [I]Manuela Simoni, Flaminia Fanelli, Laura Roli, and Uberto Pagotto[/I] [B]4.3.4.3 Direct free testosterone assays [/B] The principles of the direct free testosterone methods have been described in detail in the previous edition of this book (Simoni 2004). [B][I]In practice such assays are based on a labeled compound to be added to the sample, the “analog,” which is totally non-reactive with serum proteins, but can be recognized by the solid-phase antibody present in a limited amount and which competes for binding with the free hormone in the sample. Several commercially available kits are based on this principle and are widely used to assess free testosterone in clinical samples. [U]Unfortunately, there is evidence that these methods are totally inaccurate[/U] (Fritz et al. 2008; Chen et al. 2010). [/I][/B]In fact, the direct measurement of free testosterone in serum based on the labeled hormone “analog” is valid only if the analog does not interact with the serum proteins: a condition which is currently not met by commercial kits. In fact, neither the identity of the analog tracer, nor the validation of the kit (showing the absence of interactions with the serum protein) is usually disclosed by the manufacturer. On the contrary, the “analog” principle is often not even mentioned or is misrepresented in the instructions accompanying the kits, which are often validated only against other kits and not against dialysis or ultrafiltration. It should be kept in mind that, in practice, finding a hormone analog totally unreactive with serum proteins is very difficult, and several studies have shown that such an interaction indeed occurs, resulting in inaccurate measurements of free testosterone. In this respect it is interesting that serum-free testosterone measured by an “analog” method accounts for 0.5–0.65% of total testosterone, while equilibrium dialysis and ultrafiltration give values of 1.5–4%, revealing inconsistencies between the different approaches (Rosner 1997; Winters et al. 1998). In a direct comparison, free testosterone values measured by a bestseller “analog” kit was only 20–30% of those measured by equilibrium dialysis (Vermeulen et al. 1999). [B][I][U]For these reasons the kits for direct free testosterone measurement presently available do not measure what they claim and should not be used[/U] (Rosner 2001; Swerdloff and Wang 2008). [U]If LC-MS/MS coupled to ultrafiltration becomes technically affordable, this approach is likely to become the method of choice for assessment of free testosterone in clinical serum samples[/U].[/I][/B] [/QUOTE]
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