madman
Super Moderator
Reassessing Free-Testosterone Calculation by Liquid Chromatography-Tandem Mass Spectrometry Direct Equilibrium Dialysis (2018)
Assessment of FT might improve precision in the diagnosis of hyperandrogenism in women and hypogonadism in men, in particular when total T levels are borderline and in situations known to alter SHBG levels (9, 10). Equilibrium dialysis (ED)– or ultrafiltration (UF)-based methods, considered the reference for determination of FT, were, in the past, mostly indirect, with the addition of a labeled T tracer, determination of the percentage of free labeled T and calculation of FT from the percentage of free labeled T and total T. ED and UF methods are technically challenging and have potential pitfalls; reliable implementation is labor-intensive and poorly suited for high throughput (4, 8).
Therefore, there has been widespread use of easier-to-implement surrogate estimates of FT. The direct analog tracer-based immunoassays do not reliably reflect true FT and should not be used (9). Most frequently used in clinical and research settings are calculated estimates of serum FT levels based on serum total T and SHBG levels with or without the actual serum albumin concentrations (11). The simple FT index of total T over SHBG is less reliable and now generally abandoned in favor of alternative calculations (9, 12). Equations derived from the general law of mass action, with association constants for T binding to SHBG and albumin values derived from in vitro experiments (8, 13, 14), are frequently used in clinical practice. The version proposed by Vermeulen et al. (8) has been the most widely applied. Equations empirically developed by regression modeling on large sets of values for FT as determined with a reference method have also been used (11, 15). Although calculations seem to have performed well in many studies, acknowledging that they all have inherent limitations, there is ongoing controversy about the accuracy of calculated estimates of FT. It has been suggested that equations derived from the law of mass action are based on an inaccurate model of T binding to SHBG and/or that the chosen set of binding constants is not appropriate (4, 16). In this context, the alternative dynamic allosteric model of T binding to SHBG recently put forward (4, 17) may have far-reaching implications. If confirmed, this new model not only invalidates most of the methods commonly used for calculating FT levels but also suggests that the percentage of FT data obtained by most established methods including ED- or UF-based assays is incorrect.
All this translates into uncertainty as to how to best calculate FT and its true measured value. In the current study, we used a state-of-the-art direct ED method to reassess FT in sets of representative serum samples. This method takes advantage of the ability of a highly sensitive and accurate measurement of T by liquid chromatography-tandem mass spectrometry (LC-MS/MS) to reliably measure the low FT concentration directly in the dialysate after ED. This more straightforward method avoids potential sources of inaccuracy in indirect ED, such as those resulting from tracer impurities or from measures to limit their impact (e.g., sample dilution). We then used the measured FT results to re-evaluate some characteristics of two more established and more recently proposed calculations for the estimation of FT.
Calculated estimations of FT
FT level was calculated from total T, SHBG, and albumin serum levels according to the three following methods: (1) an equation based on the law of mass action as published by Vermeulen et al. (8) (cFT-V); (2) two empirically derived formulae (for men, the formula for T > 5 nM was used; for women, the formula <5 nM was used) as published by Ly and Handelsman (15) (cFT-L); and (3) according to a calculation based on a multistep, dynamic, allosteric model of testosterone binding to SHBG as published by Zakharov et al. (17) (cFT-Z). The values for cFT-Z from the EMAS samples (calculated with original T and SHBG from EMAS) were provided by Dr. R. Jasuja, Boston, MA, to the EMAS investigators. We had no direct access to the algorithm for cFT-Z; therefore, we were unable to present any data on cFT-Z for the samples from SIBLOS and from women.
Discussion
In this study, we reassessed FT with a state-of-the-art direct ED assay. The findings support and further validate the basic tenets of FT in men and women as previously established with traditional indirect ED and UF methodologies. They further highlight important differences in commonly used or recently proposed algorithms for deriving calculated FT values from serum total T and SHBG (and albumin) levels compared with FT measurement by direct ED.
Calculated estimates of FT
Calculated FT estimates are critically dependent on the reliability and calibration of the T and SHBG assays (9, 34, 35). Furthermore, they are based on the assumption of a normal steady-state protein-binding characteristic for T, which is not the case in every individual. Any equation will incorrectly estimate FT in situations such as the presence of large concentrations of competing steroids, large deviations from physiological protein concentrations, or rare genetic variants of SHBG affecting T binding affinity (3, 8). Besides these occasional problems, more systematic differences between FT estimates depending on the used equation and as compared with measured FT have been reported (11, 12, 28).
Our results show that cFT-V is strongly correlated to FT measured by direct ED but systematically overestimates FT by 20% to 30%. This confirms our prior findings (22, 23) and those of others (28). The relation between cFT-V and measured FT is linear and independent of serum T, SHBG, and albumin levels. This is a strength of the cFT-V approach for clinical use because the assessment of FT is most relevant in patients with high or low SHBG levels. This also indicates that the equation derived from the mass-action law predicts the binding behavior of T to serum proteins quite well. The systematic positive bias observed for cFT-V has to be taken into account when comparing FT levels across different methods. This bias likely reflects that the in vitro determined association constants used in the equation are imperfect approximations of the actual in vivo association constants for binding of T to SHBG and albumin. FT measured with our prior in house indirect ED, which involves a correction for serum dilution effect with the use of the same basic equation and set of association constants as in cFT-V (1, 8), shows a similar positive bias compared with FT measured by direct ED (data not shown). This explains our prior findings of correlation without a bias between cFT-V and FT by indirect ED (8, 35).
Our results for cFT-L show that median cFT-L approximates closely median FT by direct ED in men and women, as calculated with the equations intended for high and low T levels, respectively (16). However, the agreement between cFT-L and measured FT was found to be strongly dependent on SHBG and T levels. Thus, cFT-L performs differently depending on serum T and SHBG levels and increasingly underestimates FT at low SHBG or low T levels. This may limit the accuracy of cFT-L in hypogonadal men with low T levels and in obese men or women with PCOS with low SHBG levels.
The cFT-Z values reported here have been supplied by the authors who reported data for cFT-Z in the EMAS cohort in the publication describing their multistep, dynamic, allosteric model to calculate FT (17). We requested access to the cFT-Z algorithm from the research group that developed this allosteric model algorithm. However, at the time of completion of this work, we had not been able to gain direct access to the algorithm. Therefore, it was not possible to make comparisons with cFT-Z for all three cohorts. This is a limitation of the current study that is beyond our control. We felt it important to evaluate cFT-Z in the current study because the results obtained by the authors according to their allosteric model to replicate the dimeric binding of T to SHBG differed substantially from the model based on the law of mass action (4, 17). Using the allosteric model, they reported higher FT% in men of 3% to 5% and that cFT-V substantially underestimated FT compared with their findings for FT by dialysis (17). Our results for the EMAS samples, indeed, do reproduce their finding that cFT-Z values are markedly higher than cFT-V values. Similarly, cFT-Z values are much higher compared with cFT-L. However, in contrast to their findings, our results also show that cFT-Z is markedly higher (about double) compared with FT measured by direct ED. Moreover, the accuracy of cFT-Z as reflected in the ratio of cFT-Z over measured FT was strongly dependent on serum SHBG levels and, to a lesser degree, on T and albumin levels. At present, it is unclear what underlies the apparent discrepancy between the results reported by Zakharov et al. (17) and the findings in the current study performed on the same set of samples. A factor involved may be differences in ED methods between laboratories giving discrepant measured FT results. The descriptive nature of this study does not allow us to address possible merits or demerits of basic assumptions on which the allosteric model is based.
In summary, for none of the three evaluated equations does calculated FT perfectly match FT measured with a state-of-the-art direct ED assay. However, there are distinct differences in how the respective equations behave. cFT-Z appears far off target relative to the results of direct ED in this study as well as compared with a substantial body of published data obtained with a variety of ED- or UF-based methods. Although cFT-L performs well in the midrange levels of serum T and SHBG, the dependence of its accuracy on T and SHBG levels has clinical implications (e.g., underestimation by cFT-L of FT at low SHBG concentrations could impair the ability to detect hyperandrogenism in PCOS or lead to overdiagnosis of hypogonadism in obese men). Although a systematic positive bias affects the external comparability of cFT-V with other methods, the consistency of its performance compared with directly measured FT, independent of serum T and SHBG levels, ensures strong internal validity. This is an important asset for clinical applications of FT assessments in patients with widely different T and SHBG levels. The cFT-V equation admittedly is a simplified representation of the binding of T to its binding proteins. Moreover, there is room for refinement of the association constants implemented in the equation. Nevertheless, contrary to what has been suggested (4, 17, 28, 36), our results do confirm that cFT-V is based on a valid model of T binding to SHBG.
In conclusion, calculated estimates of FT have inherent limitations with distinct and clinically important differences in the performance of different algorithms. Of the three methods, we evaluated in this study, cFT-V, albeit systematically overestimating FT, most robustly approximated directly measured FT in samples representative of a broad range of T and SHBG levels. There is a need for collaborative efforts to further validate and harmonize methods to measure and calculate FT levels.
Assessment of free testosterone concentration (2019)
ABSTRACT
Testosterone (T) is strongly bound to sex hormone-binding globulin and measurement of free T may be more appropriate than measuring total serum T, according to the free hormone theory. This view remains controversial and it has its detractors who claim that little extra benefit is gained than simply measuring total T, but it is endorsed by recent clinical practice guidelines for investigation of androgen disorders in both men and women. Free T measurement is very challenging. The gold standard equilibrium dialysis methods are too complex for use in routine clinical laboratories, assays are not harmonized and consequently, there are no common reference intervals to aid result interpretation. The algorithms derived for calculating free T are inaccurate because they were founded on faulty models of testosterone binding to SHBG, however, they can still give clinically useful results. To negate the effects of differences in binding protein constants, some equations for free T have been derived from an accurate measurement of testosterone in large population studies, however, a criticism is that the equations may not hold true in different patient populations. The free androgen index is not recommended for use in men because of inaccuracy at extremes of SHBG concentration, and in women, it can also give inaccurate results when SHBG concentrations are low. If the free hormone hypothesis is to be believed, then calculated free testosterone may offer the best way forward but better equations are needed to improve accuracy and these should be derived from detailed knowledge of testosterone binding to SHBG. There is still much work to be done to improve the harmonization of T and SHBG assays between laboratories because these can have a profound effect on the equations used to calculate free testosterone.
If the free hormone hypothesis is to be believed, then SHBG-bound T is not biologically active, and therefore some estimate of the fraction not bound to SHBG may be a more suitable marker. Methods of determining free T include measurement of the non-protein bound T testosterone concentration by a variety of different assays. The older, direct analog RIA methods have been discredited and are no longer recommended for use [6–8].
3. Equilibrium dialysis
T Equilibrium dialysis and ultrafiltration methods have been considered the reference methods for quantification of free T. Historically, dialysis- and ultrafiltration-based methods have mostly been indirect, using a labeled T tracer to determine the percentage free labeled T. Dialysis and ultrafiltration methods are analytically difficult with numerous technical issues and as such, they are not suitable for use in routine clinical laboratories [12,14].
The classical way to determine the quantity of true free testosterone was to adopt a three-stage approach. Unbound and bound T in undiluted serum were separated by equilibrium dialysis. T in the dialysate was extracted and then column chromatography used to separate T from similar steroids which may cross-react in the sensitive radioimmunoassay used for T quantification. The radioimmunoassay detection limit was said to be acceptable and the detection limit of the overall method was found to be 6 pmol/L [15], although this is open to doubt. Whilst it is true that detection limits can be applied appropriately to the various LC-MS/MS or immunoassay assays used for detecting T the same cannot be said for the overall measurement of free T because there are no free T reference standards or QCs available.
This method has been updated in recent years with the introduction of LC-MS/MS. Serum-free T can now be determined by the direct dialysis of undiluted serum followed by LC-MS/MS assay of free T in the dialysate. Equilibrium dialysis for 24 h at 37 °C with protein-free buffer was performed using relatively large volumes of serum, 500 μL, or 1000 μL of serum for male and female samples respectively [16]. Free T concentrations are lower in females compared to males, but the percentages of free T are similar, with a range of 0.9–2.9% in men and 0.4–2.8% in women. As expected, free T in both women and men is positively associated with total T, and there is a strong negative association between percentage free T and serum SHBG. Observed ranges for free T measured by a state-of-the-art LC-MS/MS-based direct dialysis method are in full agreement with earlier findings obtained using the older indirect equilibrium dialysis [12,17–19] and ultra-filtration methods [20,21].
Equilibrium dialysis’ is often named as the ‘gold standard’ method for the determination of free T but there are many technical difficulties with the method and it is therefore not surprising that there should be poor inter-laboratory agreement [22,23]. The poor agreement is caused by methodological differences in temperature, pH, or equilibrium shifts between free and bound T caused by sample dilution effects [12,24,25]. Indirect measurement of bioavailable T using a radioactive tracer [23], or using an LC-MS/MS method which has not been standardized against the gold standard can also increase total error. It is not difficult to see why these many technical challenges render equilibrium dialysis unsuitable for use in routine clinical laboratories, but as with many other areas of laboratory medicine, there is still a need to standardize methods between reference laboratories. It has been suggested that this should probably be achieved using direct equilibrium dialysis (and/or ultrafiltration) methods [16], using high-quality highly sensitive fully validated mass spectrometry-based assays for T [26]. It is also important that standardized reference ranges for free testosterone are used for the diagnosis of androgen disorders in women and men.
The latest endocrine society guidelines recommend that T should be measured using a CDC-certified assay or an assay which has been verified by an external quality control program using accuracy based target values [27]. If this approach is taken and assays have been calibrated to a reference measurement procedure, then harmonized male population reference ranges for testosterone can be used [28]. Although how this can be truly applied to immunoassays that demonstrate method-specific bias remains to be seen. And crucially to aid result interpretation there are no harmonized reference intervals based on large population studies currently available for free T.
4. Calculated free T
As the equilibrium dialysis reference method is not practical for use in the routine clinical laboratory, several equations have been advocated to estimate free T. Most commonly applied are equilibrium binding equations derived from the law of mass action using estimates of the association constants for binding of T to SHBG and albumin respectively [12,29]. Equations have also been developed empirically, modeled on large sets of free T concentrations, and free of assumptions about theoretical binding equilibria [30,31]. The weakness of these methods, apart from reliance on the accuracy of T and SHBG measurements, is that best-fit parameters in the test population may not be the same as in the patient population.
The calculation proposed by Vermeulen et al has been the most widely used but the main criticism of all these equations is that the model of binding of T to SHBG may not be accurate, and in addition, the set of binding constants used may not be appropriate [14,32]. SHBG is a homodimeric glycoprotein with a molecular weight of approximately 90 kDa [33] and the distribution of testosterone bound to SHBG is different in males and females. When Estradiol is present, approximately 20% of SHBG binding sites are occupied by testosterone [34]. It was previously thought that the two binding sites on the SHBG molecule are equivalent, but using modern biophysical techniques it is now known that the binding sites are not equivalent, and they each bind SHBG with a different affinity [22]. As a result of this, alternative models of binding of T to SHBG have been advocated [14,22].
Calculated FT (cFT) estimates are based on the assumption that there is normal steady-state protein binding for T and the equations are dependent on the reliability and accuracy of both the T and SHBG assays [6,35,36]. All of the equations will estimate free T incorrectly when the protein concentrations differ widely from physiological values, if there are large concentrations of competing steroids, or if the SHBG binding affinity is affected by a rare genetic variant [12,37]. Depending on the equation used, systematic differences between free T estimates have been reported compared with measured free T [18,21,30]. Discrepancies between free T measured using equilibrium dialysis and cFT are most likely caused by erroneous modeling of testosterone binding to SHBG.
Recent work shows that cFT-Vermeulen is strongly correlated to free T measured by the reference method (direct equilibrium dialysis), but free T is overestimated by 20–30%, thus agreeing with previous work [21,38,39]. However, the relationship between cFT-Vermeulen and measured free T was found to be linear and independent of serum T, albumin, and SHBG concentrations. The lack of reliance on SHBG in the Vermeulen equation was thought to be the strength of the cFT-Vermeulen since the assessment of free T is especially important in patients at the extremes of the SHBG concentration range. The bias is probably due to imperfect estimations of the association constants for the binding of T to SHBG and albumin, as discussed above, and this would need to be allowed for when comparing different methods for cFT.
The cFT-Ly equation shows good performance in the mid-range of serum T and SHBG, but its accuracy depends on T and SHBG levels and this has clinical consequences: e.g. the underestimation of free T by the cFT-Ly equation at low SHBG concentrations could potentially misdiagnose hyperandrogenism in women with PCOS or over-diagnosis hypogonadism in obese men. CFT-Zakharov showed discrepant results relative to direct equilibrium dialysis and also compared to other published data obtained with equilibrium dialysis or ultra-filtration based methods. It should be noted that the Zakharov equation is patented and not available for independent scrutiny. The many different versions of formulas and methods for calculating and measuring free T have been shown to cause problems with the poor inter-laboratory agreement due to the use of different methods with different reference intervals [40,41].
A recent study in our laboratory (Adaway J unpublished data) has shown that commonly used assays can give different results for SHBG, even when traceable to the same international standard. Anonymized surplus male and female serum samples were analyzed on four different immunoassay platforms (Abbott Architect, Roche, Beckman, and Siemens). The results were used to calculate free testosterone using the Vermeulen equation, with a constant testosterone concentration of 10 nmol/L for males and 1.5 nmol/L for females, keeping the albumin concentration fixed at 40 g/L. The Abbott Architect and Siemens Advia Centaur assays were both traceable to the WHO 2nd international standard 08/266 but there was a mean difference of 4.6 nmol/L between the results. The Roche E170 and Beckman Access were both traceable to the WHO 1st International Standard 95/560 but there was a mean difference of 3.4 nmol/L between their results. In contrast, although the Beckman Access and Siemens Advia Centaur assays are traceable to different international standards, the mean difference between their results was only 1.59 nmol/L. Although these differences may seem small, the widespread use of universal reference ranges for cFT means that the SHBG assay used can cause the difference between male patients receiving or being denied testosterone replacement, or results supporting or being inconsistent with the diagnosis of PCOS in a female patient. The difference between the Abbott Architect and the Roche E170 results were the greatest, with the Roche E170 results being a mean 9.449 nmol/L higher than the Abbott Architect results. Moving between analyzers, for instance, if a patient was being followed up in Primary and Secondary Care and the care providers use different laboratories, or if a laboratory changes immunoassay platform could cause diagnostic confusion if the same reference ranges for cFT were used. The differences in SHBG assay results are consistent with those found on the UKNEQAS scheme (Rachel Marrington-personal communication).
7. Conclusion
The estimation of free T in both men and women has always been based on the central dogma of the free hormone hypothesis. As discussed in this review, the hard evidence for this hypothesis is scant and the two most recent and largest studies in men provide contradictory evidence. Nevertheless, the Endocrine Society still recommends the measurement of free T for the investigation of hypogonadism in men although this view is not supported by the Australian Endocrine Society. The use of free T in women is also recommended by the Endocrine society and it is still widely measured in clinical laboratories to support clinical practice and research into hyperandrogenism, although the evidence for this is also weak. The measurement of free testosterone is fraught with difficulty. Equilibrium dialysis methods are too complex for routine clinical use; they lack harmonization and consequently common reference intervals. Equations for calculating FT are inaccurate because they were founded on faulty models of T binding to SHBG. Calculated FT methods offer are simple and inexpensive and may offer the best way forward, but more accurate equations are needed and these must be based on more detailed knowledge of the complicated binding testosterone to SHBG. There is also still much work to be done to improve the harmonization of T and SHBG assays between laboratories because these can have a profound effect on the cFT equations. This calls for closer co-operation between regulatory bodies, EQA schemes, and laboratories. Necessary requirements to improve the measurement of free T include the availability of commutable human serum based reference materials, internal standards for the calibration of assays, and standardization of sample preparation and chromatography techniques. With the relatively weak evidence available to support the measurement of free T and the difficulty of its measurement, there still remains a question mark as to the usefulness of this test or indeed if an accurate measurement of T using highly specific LC-MS/MS methods may not be more useful.
-------------------------------------------------------------------------------------------------------------------------------
A critical point made here regarding the TruTTM algorithm.
*In this context, the alternative dynamic allosteric model of T binding to SHBG recently put forward (4, 17) may have far-reaching implications. If confirmed, this new model not only invalidates most of the methods commonly used for calculating FT levels but also suggests that the percentage of FT data obtained by most established methods including ED- or UF-based assays is incorrect.
*Any equation will incorrectly estimate FT in situations such as the presence of large concentrations of competing steroids, large deviations from physiological protein concentrations, or rare genetic variants of SHBG affecting T binding affinity (3, 8). Besides these occasional problems, more systematic differences between FT estimates depending on the used equation and as compared with measured FT have been reported (11, 12, 28).
*At present, it is unclear what underlies the apparent discrepancy between the results reported by Zakharov et al. (17) and the findings in the current study performed on the same set of samples. A factor involved may be differences in ED methods between laboratories giving discrepant measured FT results. The descriptive nature of this study does not allow us to address possible merits or demerits of basic assumptions on which the allosteric model is based.
*Although a systematic positive bias affects the external comparability of cFT-V with other methods, the consistency of its performance compared with directly measured FT, independent of serum T and SHBG levels, ensures strong internal validity. This is an important asset for clinical applications of FT assessments in patients with widely different T and SHBG levels. The cFT-V equation admittedly is a simplified representation of the binding of T to its binding proteins. Moreover, there is room for refinement of the association constants implemented in the equation. Nevertheless, contrary to what has been suggested (4, 17, 28, 36), our results do confirm that cFT-V is based on a valid model of T binding to SHBG.
*As the equilibrium dialysis reference method is not practical for use in the routine clinical laboratory, several equations have been advocated to estimate free T. Most commonly applied are equilibrium binding equations derived from the law of mass action using estimates of the association constants for binding of T to SHBG and albumin respectively [12,29]. Equations have also been developed empirically, modeled on large sets of free T concentrations, and free of assumptions about theoretical binding equilibria [30,31]. The weakness of these methods, apart from reliance on the accuracy of T and SHBG measurements, is that best-fit parameters in the test population may not be the same as in the patient population.
*Discrepancies between free T measured using equilibrium dialysis and cFT are most likely caused by erroneous modeling of testosterone binding to SHBG.
*Recent work shows that cFT-Vermeulen is strongly correlated to free T measured by the reference method (direct equilibrium dialysis), but free T is overestimated by 20–30%, thus agreeing with previous work [21,38,39]. However, the relationship between cFT-Vermeulen and measured free T was found to be linear and independent of serum T, albumin, and SHBG concentrations. The lack of reliance on SHBG in the Vermeulen equation was thought to be the strength of the cFT-Vermeulen since the assessment of free T is especially important in patients at the extremes of the SHBG concentration range. The bias is probably due to imperfect estimations of the association constants for the binding of T to SHBG and albumin, as discussed above, and this would need to be allowed for when comparing different methods for cFT.
-------------------------------------------------------------------------------------------------------------------------------
Unfortunately, the Zakharov equation is patented as it is going to be commercialized (TruTTM).
The Zakharov equation is patented and not available for independent scrutiny.
The TruTTM algorithm is still in phase II.
From a previous thread where I stated:
Need to look further..... thinking this will be the set reference range for the TruT calculated free testosterone method: "Based on the new data on the distribution of free testosterone levels in healthy men the target range of free testosterone has been determined to be 164 to 314 pg/ml (mean+/−1SD)"
Which would convert to 16-31 ng/dl
From what I understand once Phase II is completed it is just a matter of time before....."commercialization of the TruTTM algorithm using a HIPAA compliant infrastructure for its clinical adoption"
Phase II: Research and Commercialization of TruT Algorithm for Free Testosterone
ABSTRACT
- the measurement of testosterone(T) levels is central to the diagnosis of androgen disorders, such as hypogonadism in men and polycystic ovary syndrome (PCOS) in women
- circulating t is bound with high affinity to sex hormone-binding globulin (SHBG) and with substantially lower affinity to albumin; only the free fraction is biologically active
- conditions that affect SHBG concentrations, such as aging and obesity, alter total T but not free T concentrations; in these conditions, the determination of free t is necessary to obtain an accurate assessment of androgen status
- tracer analog method, the most widely used method for free T, has been shown to be inaccurate
- equilibrium dialysis method is, technically difficult to implement and standardize, and is not available in most hospital laboratories, leading the Endocrine Society's Expert Panel to conclude that?? the calculation of free testosterone is the most useful estimate of free testosterone in plasma??
- therefore, there is an unmet need for algorithms that provide accurate estimates of free T that match those derived from equilibrium dialysis
- we have designed a novel and accurate TruTTM algorithm for the determination of free T, based on the characterization of testosterones' binding to SHBG using modern biophysical techniques
- we have discovered that testosterone's binding to SHBG is a dynamic multistep process that includes allosteric interaction between the two binding sites on an SHBG dimer
- our computational frame-work incorporates the correct binding parameters derived experimentally in these studies, the non-linear dynamics in T: SHBG association, and allostery
- in phase I studies, we demonstrated that the TruTTM algorithm provides accurate free T values that match those obtained using the equilibrium dialysis in healthy and hypogonadal men
- we have also shown that the binding parameters that have formed the basis of previous equations (e.g., Vermeulen) are incorrect and that free T values derived using these equations deviate substantially from free T measured by equilibrium dialysis
- the phase I studies have led to the adoption of the TruTTM algorithm at several institutions
- the phase II program will continue the development of the TruTTM algorithm by validating it in common conditions characterized by altered SHBG concentration, such as obesity and aging (AIM 1), in healthy women across the menstrual cycle, and in women with PCOS (AIM 2)
- we will generate population-based reference ranges for free T (AIM 3)
- phase II also includes plans for commercialization of the TruTTM algorithm using a HIPAA compliant infrastructure for its clinical adoption
-the phase II program will provide validation of the TruTTM algorithm in the two most common clinical indications for free T measurement? men suspected of hypogonadism and altered SHBG levels, and women with hyperandrogenic disorders
- it will also enable the development of a HIPAA compliant platform that can be embedded into the electronic medical record for wider clinical adoption and for improving clinical care
Project Start 2014-09-15
Project End 2019-05-31
Phase II: Research and Commercialization of TruT Algorithm for Free Testosterone
-------------------------------------------------------------------------------------------------------------------------------
How the CDC Clinical Standardization Programs Are Improving Hormone Tests
Congress Funds AACC-Led Harmonization Initiative - AACC.org
The $1.3 trillion omnibus spending bill passed by Congress in March will fund efforts to harmonize laboratory tests, the culmination of more than a decade of AACC advocacy efforts. AACC worked closely with Rep. Kevin Yoder (R-Kan.) and other lawmakers, as well as a partnership of 17 clinical associations. The bill provides $2 million to the Centers for Disease Control and Prevention (CDC). The agency plans to use the new funding for materials and monitoring that will enable the harmonization of tests for free testosterone, thyroid-stimulating hormone, and estrogen.
Key point being:
* The agency plans to use the new funding for materials and monitoring that will enable harmonization of tests for free testosterone
The commercialization of the TruT platform is going to be a huge part of this.
You remember this paper:
Harmonized Reference Ranges for Circulating Testosterone Levels in Men of Four Cohort Studies in the United States and Europe
Thomas G. Travison, Hubert W. Vesper, Eric Orwoll, Frederick Wu, Jean-Marc Kaufman, Ying Wang Bruno Lapauw, Tom Fiers, Alvin M. Matsumoto, and Shalender Bhasin
Notice who played a big part in this? (S.B.)
Acknowledgments
This work was supported primarily by National Institutes of Health Grant 1RO1AG31206 to S.B. Additional support was provided by the Endocrine Society and Boston Claude D. Pepper Older Americans Independence Center Grant 5P30AG031679 from the National Institute on Aging. The Framingham Heart Study was supported by National Heart, Lung, and Blood Institute Framingham Heart Study Contract N01-HC-25195. The European Male Aging Study (EMAS) was supported by the Commission of the European Communities Fifth Framework Programme “Quality of Life and Management of Living Resources” Grant QLK6-CT-2001-00258. The Osteoporotic Fractures in Men Study (MrOS) was supported by the National Institutes of Health. The following institutes provided support: National Institute on Aging, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Center for Advancing Translational Sciences, and National Institutes of Health Roadmap for Medical Research under Grants U01 AG027810, U01 AG042124, U01 AG042139, U01 AG042140, U01 AG042143, U01 AG042145, U01 AG042168, U01 AR066160, and UL1 TR000128. The Belgian Sibling Study of Osteoporosis was supported by a grant from the Fund for Scientific Research–Flanders (FWO–Vlaanderen Grant G.0662.08) and by a grant from the Hercules Foundation, Flanders.
Disclaimers: The findings and conclusions in this manuscript are those of the authors and do not necessarily represent the official views or positions of the Centers for Disease Control and Prevention/Agency for Toxic Substances and Disease Registry. All individuals listed as authors agreed to be co-authors.
Disclosure Summary: S.B. has received research grant support from AbbVie Pharmaceuticals, Transition Therapeutics, Takeda Pharmaceuticals, and Eli Lilly for investigator-initiated research unrelated to this study. S.B. has served as a consultant to AbbVie, Regeneron, Novartis, and Eli Lilly. S.B. has a financial interest in Function Promoting Therapies, a company aiming to develop innovative solutions that enhance precision and accuracy in clinical decision making and facilitate personalized therapeutic choices in reproductive health. S.B.’s interests were reviewed and are managed by Brigham and Women’s Hospital and Partners HealthCare in accordance with their conflict of interest policies. A.M.M. has received research grant support from AbbVie and GlaxoSmithKline and has served as a consultant to AbbVie, Endo, Lilly, and Lipocine. F.W. has received research grant support from Besins Healthcare and Eli Lilly and has served as a consultant to Besins Healthcare and Repro Therapeutics. Other authors have nothing to disclose.
Let me be very clear here when I tell you that the man behind the efforts that are underway to standardize the procedures for free testosterone measurement and to generate harmonized reference ranges is the same man who has big plans for the commercialization of the TruTTM algorithm using a HIPAA compliant infrastructure for its clinical adoption.
Notice who the inventors are behind TruTTM.
Function Promoting Therapies
Function Promoting Therapies, LLC, and Tesvgen, the commercialization arm of FPT, LLC, was established in 2012. The company’s goals are to provide advanced technologies that improve accuracy in the diagnosis and rational management of testosterone replacement therapy for male reproductive disorders. FPT strives to innovate at a high level, maintain our access to academic research and pre-eminence in this content area, and to optimally balance research and commercial success. We are based in the suburbs of Boston and within reach of some of the most renowned endocrine research and clinical centers in the world. Our research partners are spread throughout the USA and we have research collaborations with leading institutions in Europe. These networks provide us the opportunity to share and validate our findings and enable us to leverage from a global palette of resources.
About Function Promoting Therapies
Function Promoting Therapies, LLC, and Tesvgen, the commercialization arm of FPT, LLC, was established in 2012. The company’s goals are to provide:
We are based in the heart of Boston within reach of some of the most renowned endocrine research and clinical centers in the world. Our research partners are spread throughout the USA and we have research ties with leading institutions in Europe. These networks provide us the opportunity to share and validate our findings and enable us to leverage from a global palette of resources.
Our access to a network of academic experts at leading academic institutions also means that we are in tune with the upcoming changes in treatment patterns or recommendations of professional societies.
Partners and Collaborations
We continue to work with collaborators in academia and the wider industry for exciting new applications and further development of TruT™. Some of our existing partnerships include:
Johns Hopkins University ‐ Diagnosis and management of hypogonadism in HIV and co-infections.
The Mayo Clinic ‐ Personalized algorithm and therapy development.
Karolinska Institute ‐ Dynamics of free testosterone levels after surgical interventions in men and women.
regionh.dk - Examining population reference ranges of free testosterone for Danish cohorts.
UCLA School of Nursing ‐ Examining the dynamic role of multiple hormones in altering transport and bioavailability.
Boston IVF ‐ Developing a novel platform for rational treatment and management of in-vitro fertilization interventions.
Myosyntax ‐ Dynamics of testosterone bioavailability during caloric restriction.
Investors
Tesvgen is funded through the seed investment round in Function Promoting Therapies LLC. FPT successfully closed the seed round in 2016 and is currently in discussions with strategic partners for further commercialization of innovations.
Assessment of FT might improve precision in the diagnosis of hyperandrogenism in women and hypogonadism in men, in particular when total T levels are borderline and in situations known to alter SHBG levels (9, 10). Equilibrium dialysis (ED)– or ultrafiltration (UF)-based methods, considered the reference for determination of FT, were, in the past, mostly indirect, with the addition of a labeled T tracer, determination of the percentage of free labeled T and calculation of FT from the percentage of free labeled T and total T. ED and UF methods are technically challenging and have potential pitfalls; reliable implementation is labor-intensive and poorly suited for high throughput (4, 8).
Therefore, there has been widespread use of easier-to-implement surrogate estimates of FT. The direct analog tracer-based immunoassays do not reliably reflect true FT and should not be used (9). Most frequently used in clinical and research settings are calculated estimates of serum FT levels based on serum total T and SHBG levels with or without the actual serum albumin concentrations (11). The simple FT index of total T over SHBG is less reliable and now generally abandoned in favor of alternative calculations (9, 12). Equations derived from the general law of mass action, with association constants for T binding to SHBG and albumin values derived from in vitro experiments (8, 13, 14), are frequently used in clinical practice. The version proposed by Vermeulen et al. (8) has been the most widely applied. Equations empirically developed by regression modeling on large sets of values for FT as determined with a reference method have also been used (11, 15). Although calculations seem to have performed well in many studies, acknowledging that they all have inherent limitations, there is ongoing controversy about the accuracy of calculated estimates of FT. It has been suggested that equations derived from the law of mass action are based on an inaccurate model of T binding to SHBG and/or that the chosen set of binding constants is not appropriate (4, 16). In this context, the alternative dynamic allosteric model of T binding to SHBG recently put forward (4, 17) may have far-reaching implications. If confirmed, this new model not only invalidates most of the methods commonly used for calculating FT levels but also suggests that the percentage of FT data obtained by most established methods including ED- or UF-based assays is incorrect.
All this translates into uncertainty as to how to best calculate FT and its true measured value. In the current study, we used a state-of-the-art direct ED method to reassess FT in sets of representative serum samples. This method takes advantage of the ability of a highly sensitive and accurate measurement of T by liquid chromatography-tandem mass spectrometry (LC-MS/MS) to reliably measure the low FT concentration directly in the dialysate after ED. This more straightforward method avoids potential sources of inaccuracy in indirect ED, such as those resulting from tracer impurities or from measures to limit their impact (e.g., sample dilution). We then used the measured FT results to re-evaluate some characteristics of two more established and more recently proposed calculations for the estimation of FT.
Calculated estimations of FT
FT level was calculated from total T, SHBG, and albumin serum levels according to the three following methods: (1) an equation based on the law of mass action as published by Vermeulen et al. (8) (cFT-V); (2) two empirically derived formulae (for men, the formula for T > 5 nM was used; for women, the formula <5 nM was used) as published by Ly and Handelsman (15) (cFT-L); and (3) according to a calculation based on a multistep, dynamic, allosteric model of testosterone binding to SHBG as published by Zakharov et al. (17) (cFT-Z). The values for cFT-Z from the EMAS samples (calculated with original T and SHBG from EMAS) were provided by Dr. R. Jasuja, Boston, MA, to the EMAS investigators. We had no direct access to the algorithm for cFT-Z; therefore, we were unable to present any data on cFT-Z for the samples from SIBLOS and from women.
Discussion
In this study, we reassessed FT with a state-of-the-art direct ED assay. The findings support and further validate the basic tenets of FT in men and women as previously established with traditional indirect ED and UF methodologies. They further highlight important differences in commonly used or recently proposed algorithms for deriving calculated FT values from serum total T and SHBG (and albumin) levels compared with FT measurement by direct ED.
Calculated estimates of FT
Calculated FT estimates are critically dependent on the reliability and calibration of the T and SHBG assays (9, 34, 35). Furthermore, they are based on the assumption of a normal steady-state protein-binding characteristic for T, which is not the case in every individual. Any equation will incorrectly estimate FT in situations such as the presence of large concentrations of competing steroids, large deviations from physiological protein concentrations, or rare genetic variants of SHBG affecting T binding affinity (3, 8). Besides these occasional problems, more systematic differences between FT estimates depending on the used equation and as compared with measured FT have been reported (11, 12, 28).
Our results show that cFT-V is strongly correlated to FT measured by direct ED but systematically overestimates FT by 20% to 30%. This confirms our prior findings (22, 23) and those of others (28). The relation between cFT-V and measured FT is linear and independent of serum T, SHBG, and albumin levels. This is a strength of the cFT-V approach for clinical use because the assessment of FT is most relevant in patients with high or low SHBG levels. This also indicates that the equation derived from the mass-action law predicts the binding behavior of T to serum proteins quite well. The systematic positive bias observed for cFT-V has to be taken into account when comparing FT levels across different methods. This bias likely reflects that the in vitro determined association constants used in the equation are imperfect approximations of the actual in vivo association constants for binding of T to SHBG and albumin. FT measured with our prior in house indirect ED, which involves a correction for serum dilution effect with the use of the same basic equation and set of association constants as in cFT-V (1, 8), shows a similar positive bias compared with FT measured by direct ED (data not shown). This explains our prior findings of correlation without a bias between cFT-V and FT by indirect ED (8, 35).
Our results for cFT-L show that median cFT-L approximates closely median FT by direct ED in men and women, as calculated with the equations intended for high and low T levels, respectively (16). However, the agreement between cFT-L and measured FT was found to be strongly dependent on SHBG and T levels. Thus, cFT-L performs differently depending on serum T and SHBG levels and increasingly underestimates FT at low SHBG or low T levels. This may limit the accuracy of cFT-L in hypogonadal men with low T levels and in obese men or women with PCOS with low SHBG levels.
The cFT-Z values reported here have been supplied by the authors who reported data for cFT-Z in the EMAS cohort in the publication describing their multistep, dynamic, allosteric model to calculate FT (17). We requested access to the cFT-Z algorithm from the research group that developed this allosteric model algorithm. However, at the time of completion of this work, we had not been able to gain direct access to the algorithm. Therefore, it was not possible to make comparisons with cFT-Z for all three cohorts. This is a limitation of the current study that is beyond our control. We felt it important to evaluate cFT-Z in the current study because the results obtained by the authors according to their allosteric model to replicate the dimeric binding of T to SHBG differed substantially from the model based on the law of mass action (4, 17). Using the allosteric model, they reported higher FT% in men of 3% to 5% and that cFT-V substantially underestimated FT compared with their findings for FT by dialysis (17). Our results for the EMAS samples, indeed, do reproduce their finding that cFT-Z values are markedly higher than cFT-V values. Similarly, cFT-Z values are much higher compared with cFT-L. However, in contrast to their findings, our results also show that cFT-Z is markedly higher (about double) compared with FT measured by direct ED. Moreover, the accuracy of cFT-Z as reflected in the ratio of cFT-Z over measured FT was strongly dependent on serum SHBG levels and, to a lesser degree, on T and albumin levels. At present, it is unclear what underlies the apparent discrepancy between the results reported by Zakharov et al. (17) and the findings in the current study performed on the same set of samples. A factor involved may be differences in ED methods between laboratories giving discrepant measured FT results. The descriptive nature of this study does not allow us to address possible merits or demerits of basic assumptions on which the allosteric model is based.
In summary, for none of the three evaluated equations does calculated FT perfectly match FT measured with a state-of-the-art direct ED assay. However, there are distinct differences in how the respective equations behave. cFT-Z appears far off target relative to the results of direct ED in this study as well as compared with a substantial body of published data obtained with a variety of ED- or UF-based methods. Although cFT-L performs well in the midrange levels of serum T and SHBG, the dependence of its accuracy on T and SHBG levels has clinical implications (e.g., underestimation by cFT-L of FT at low SHBG concentrations could impair the ability to detect hyperandrogenism in PCOS or lead to overdiagnosis of hypogonadism in obese men). Although a systematic positive bias affects the external comparability of cFT-V with other methods, the consistency of its performance compared with directly measured FT, independent of serum T and SHBG levels, ensures strong internal validity. This is an important asset for clinical applications of FT assessments in patients with widely different T and SHBG levels. The cFT-V equation admittedly is a simplified representation of the binding of T to its binding proteins. Moreover, there is room for refinement of the association constants implemented in the equation. Nevertheless, contrary to what has been suggested (4, 17, 28, 36), our results do confirm that cFT-V is based on a valid model of T binding to SHBG.
In conclusion, calculated estimates of FT have inherent limitations with distinct and clinically important differences in the performance of different algorithms. Of the three methods, we evaluated in this study, cFT-V, albeit systematically overestimating FT, most robustly approximated directly measured FT in samples representative of a broad range of T and SHBG levels. There is a need for collaborative efforts to further validate and harmonize methods to measure and calculate FT levels.
Assessment of free testosterone concentration (2019)
ABSTRACT
Testosterone (T) is strongly bound to sex hormone-binding globulin and measurement of free T may be more appropriate than measuring total serum T, according to the free hormone theory. This view remains controversial and it has its detractors who claim that little extra benefit is gained than simply measuring total T, but it is endorsed by recent clinical practice guidelines for investigation of androgen disorders in both men and women. Free T measurement is very challenging. The gold standard equilibrium dialysis methods are too complex for use in routine clinical laboratories, assays are not harmonized and consequently, there are no common reference intervals to aid result interpretation. The algorithms derived for calculating free T are inaccurate because they were founded on faulty models of testosterone binding to SHBG, however, they can still give clinically useful results. To negate the effects of differences in binding protein constants, some equations for free T have been derived from an accurate measurement of testosterone in large population studies, however, a criticism is that the equations may not hold true in different patient populations. The free androgen index is not recommended for use in men because of inaccuracy at extremes of SHBG concentration, and in women, it can also give inaccurate results when SHBG concentrations are low. If the free hormone hypothesis is to be believed, then calculated free testosterone may offer the best way forward but better equations are needed to improve accuracy and these should be derived from detailed knowledge of testosterone binding to SHBG. There is still much work to be done to improve the harmonization of T and SHBG assays between laboratories because these can have a profound effect on the equations used to calculate free testosterone.
If the free hormone hypothesis is to be believed, then SHBG-bound T is not biologically active, and therefore some estimate of the fraction not bound to SHBG may be a more suitable marker. Methods of determining free T include measurement of the non-protein bound T testosterone concentration by a variety of different assays. The older, direct analog RIA methods have been discredited and are no longer recommended for use [6–8].
3. Equilibrium dialysis
T Equilibrium dialysis and ultrafiltration methods have been considered the reference methods for quantification of free T. Historically, dialysis- and ultrafiltration-based methods have mostly been indirect, using a labeled T tracer to determine the percentage free labeled T. Dialysis and ultrafiltration methods are analytically difficult with numerous technical issues and as such, they are not suitable for use in routine clinical laboratories [12,14].
The classical way to determine the quantity of true free testosterone was to adopt a three-stage approach. Unbound and bound T in undiluted serum were separated by equilibrium dialysis. T in the dialysate was extracted and then column chromatography used to separate T from similar steroids which may cross-react in the sensitive radioimmunoassay used for T quantification. The radioimmunoassay detection limit was said to be acceptable and the detection limit of the overall method was found to be 6 pmol/L [15], although this is open to doubt. Whilst it is true that detection limits can be applied appropriately to the various LC-MS/MS or immunoassay assays used for detecting T the same cannot be said for the overall measurement of free T because there are no free T reference standards or QCs available.
This method has been updated in recent years with the introduction of LC-MS/MS. Serum-free T can now be determined by the direct dialysis of undiluted serum followed by LC-MS/MS assay of free T in the dialysate. Equilibrium dialysis for 24 h at 37 °C with protein-free buffer was performed using relatively large volumes of serum, 500 μL, or 1000 μL of serum for male and female samples respectively [16]. Free T concentrations are lower in females compared to males, but the percentages of free T are similar, with a range of 0.9–2.9% in men and 0.4–2.8% in women. As expected, free T in both women and men is positively associated with total T, and there is a strong negative association between percentage free T and serum SHBG. Observed ranges for free T measured by a state-of-the-art LC-MS/MS-based direct dialysis method are in full agreement with earlier findings obtained using the older indirect equilibrium dialysis [12,17–19] and ultra-filtration methods [20,21].
Equilibrium dialysis’ is often named as the ‘gold standard’ method for the determination of free T but there are many technical difficulties with the method and it is therefore not surprising that there should be poor inter-laboratory agreement [22,23]. The poor agreement is caused by methodological differences in temperature, pH, or equilibrium shifts between free and bound T caused by sample dilution effects [12,24,25]. Indirect measurement of bioavailable T using a radioactive tracer [23], or using an LC-MS/MS method which has not been standardized against the gold standard can also increase total error. It is not difficult to see why these many technical challenges render equilibrium dialysis unsuitable for use in routine clinical laboratories, but as with many other areas of laboratory medicine, there is still a need to standardize methods between reference laboratories. It has been suggested that this should probably be achieved using direct equilibrium dialysis (and/or ultrafiltration) methods [16], using high-quality highly sensitive fully validated mass spectrometry-based assays for T [26]. It is also important that standardized reference ranges for free testosterone are used for the diagnosis of androgen disorders in women and men.
The latest endocrine society guidelines recommend that T should be measured using a CDC-certified assay or an assay which has been verified by an external quality control program using accuracy based target values [27]. If this approach is taken and assays have been calibrated to a reference measurement procedure, then harmonized male population reference ranges for testosterone can be used [28]. Although how this can be truly applied to immunoassays that demonstrate method-specific bias remains to be seen. And crucially to aid result interpretation there are no harmonized reference intervals based on large population studies currently available for free T.
4. Calculated free T
As the equilibrium dialysis reference method is not practical for use in the routine clinical laboratory, several equations have been advocated to estimate free T. Most commonly applied are equilibrium binding equations derived from the law of mass action using estimates of the association constants for binding of T to SHBG and albumin respectively [12,29]. Equations have also been developed empirically, modeled on large sets of free T concentrations, and free of assumptions about theoretical binding equilibria [30,31]. The weakness of these methods, apart from reliance on the accuracy of T and SHBG measurements, is that best-fit parameters in the test population may not be the same as in the patient population.
The calculation proposed by Vermeulen et al has been the most widely used but the main criticism of all these equations is that the model of binding of T to SHBG may not be accurate, and in addition, the set of binding constants used may not be appropriate [14,32]. SHBG is a homodimeric glycoprotein with a molecular weight of approximately 90 kDa [33] and the distribution of testosterone bound to SHBG is different in males and females. When Estradiol is present, approximately 20% of SHBG binding sites are occupied by testosterone [34]. It was previously thought that the two binding sites on the SHBG molecule are equivalent, but using modern biophysical techniques it is now known that the binding sites are not equivalent, and they each bind SHBG with a different affinity [22]. As a result of this, alternative models of binding of T to SHBG have been advocated [14,22].
Calculated FT (cFT) estimates are based on the assumption that there is normal steady-state protein binding for T and the equations are dependent on the reliability and accuracy of both the T and SHBG assays [6,35,36]. All of the equations will estimate free T incorrectly when the protein concentrations differ widely from physiological values, if there are large concentrations of competing steroids, or if the SHBG binding affinity is affected by a rare genetic variant [12,37]. Depending on the equation used, systematic differences between free T estimates have been reported compared with measured free T [18,21,30]. Discrepancies between free T measured using equilibrium dialysis and cFT are most likely caused by erroneous modeling of testosterone binding to SHBG.
Recent work shows that cFT-Vermeulen is strongly correlated to free T measured by the reference method (direct equilibrium dialysis), but free T is overestimated by 20–30%, thus agreeing with previous work [21,38,39]. However, the relationship between cFT-Vermeulen and measured free T was found to be linear and independent of serum T, albumin, and SHBG concentrations. The lack of reliance on SHBG in the Vermeulen equation was thought to be the strength of the cFT-Vermeulen since the assessment of free T is especially important in patients at the extremes of the SHBG concentration range. The bias is probably due to imperfect estimations of the association constants for the binding of T to SHBG and albumin, as discussed above, and this would need to be allowed for when comparing different methods for cFT.
The cFT-Ly equation shows good performance in the mid-range of serum T and SHBG, but its accuracy depends on T and SHBG levels and this has clinical consequences: e.g. the underestimation of free T by the cFT-Ly equation at low SHBG concentrations could potentially misdiagnose hyperandrogenism in women with PCOS or over-diagnosis hypogonadism in obese men. CFT-Zakharov showed discrepant results relative to direct equilibrium dialysis and also compared to other published data obtained with equilibrium dialysis or ultra-filtration based methods. It should be noted that the Zakharov equation is patented and not available for independent scrutiny. The many different versions of formulas and methods for calculating and measuring free T have been shown to cause problems with the poor inter-laboratory agreement due to the use of different methods with different reference intervals [40,41].
A recent study in our laboratory (Adaway J unpublished data) has shown that commonly used assays can give different results for SHBG, even when traceable to the same international standard. Anonymized surplus male and female serum samples were analyzed on four different immunoassay platforms (Abbott Architect, Roche, Beckman, and Siemens). The results were used to calculate free testosterone using the Vermeulen equation, with a constant testosterone concentration of 10 nmol/L for males and 1.5 nmol/L for females, keeping the albumin concentration fixed at 40 g/L. The Abbott Architect and Siemens Advia Centaur assays were both traceable to the WHO 2nd international standard 08/266 but there was a mean difference of 4.6 nmol/L between the results. The Roche E170 and Beckman Access were both traceable to the WHO 1st International Standard 95/560 but there was a mean difference of 3.4 nmol/L between their results. In contrast, although the Beckman Access and Siemens Advia Centaur assays are traceable to different international standards, the mean difference between their results was only 1.59 nmol/L. Although these differences may seem small, the widespread use of universal reference ranges for cFT means that the SHBG assay used can cause the difference between male patients receiving or being denied testosterone replacement, or results supporting or being inconsistent with the diagnosis of PCOS in a female patient. The difference between the Abbott Architect and the Roche E170 results were the greatest, with the Roche E170 results being a mean 9.449 nmol/L higher than the Abbott Architect results. Moving between analyzers, for instance, if a patient was being followed up in Primary and Secondary Care and the care providers use different laboratories, or if a laboratory changes immunoassay platform could cause diagnostic confusion if the same reference ranges for cFT were used. The differences in SHBG assay results are consistent with those found on the UKNEQAS scheme (Rachel Marrington-personal communication).
7. Conclusion
The estimation of free T in both men and women has always been based on the central dogma of the free hormone hypothesis. As discussed in this review, the hard evidence for this hypothesis is scant and the two most recent and largest studies in men provide contradictory evidence. Nevertheless, the Endocrine Society still recommends the measurement of free T for the investigation of hypogonadism in men although this view is not supported by the Australian Endocrine Society. The use of free T in women is also recommended by the Endocrine society and it is still widely measured in clinical laboratories to support clinical practice and research into hyperandrogenism, although the evidence for this is also weak. The measurement of free testosterone is fraught with difficulty. Equilibrium dialysis methods are too complex for routine clinical use; they lack harmonization and consequently common reference intervals. Equations for calculating FT are inaccurate because they were founded on faulty models of T binding to SHBG. Calculated FT methods offer are simple and inexpensive and may offer the best way forward, but more accurate equations are needed and these must be based on more detailed knowledge of the complicated binding testosterone to SHBG. There is also still much work to be done to improve the harmonization of T and SHBG assays between laboratories because these can have a profound effect on the cFT equations. This calls for closer co-operation between regulatory bodies, EQA schemes, and laboratories. Necessary requirements to improve the measurement of free T include the availability of commutable human serum based reference materials, internal standards for the calibration of assays, and standardization of sample preparation and chromatography techniques. With the relatively weak evidence available to support the measurement of free T and the difficulty of its measurement, there still remains a question mark as to the usefulness of this test or indeed if an accurate measurement of T using highly specific LC-MS/MS methods may not be more useful.
-------------------------------------------------------------------------------------------------------------------------------
A critical point made here regarding the TruTTM algorithm.
*In this context, the alternative dynamic allosteric model of T binding to SHBG recently put forward (4, 17) may have far-reaching implications. If confirmed, this new model not only invalidates most of the methods commonly used for calculating FT levels but also suggests that the percentage of FT data obtained by most established methods including ED- or UF-based assays is incorrect.
*Any equation will incorrectly estimate FT in situations such as the presence of large concentrations of competing steroids, large deviations from physiological protein concentrations, or rare genetic variants of SHBG affecting T binding affinity (3, 8). Besides these occasional problems, more systematic differences between FT estimates depending on the used equation and as compared with measured FT have been reported (11, 12, 28).
*At present, it is unclear what underlies the apparent discrepancy between the results reported by Zakharov et al. (17) and the findings in the current study performed on the same set of samples. A factor involved may be differences in ED methods between laboratories giving discrepant measured FT results. The descriptive nature of this study does not allow us to address possible merits or demerits of basic assumptions on which the allosteric model is based.
*Although a systematic positive bias affects the external comparability of cFT-V with other methods, the consistency of its performance compared with directly measured FT, independent of serum T and SHBG levels, ensures strong internal validity. This is an important asset for clinical applications of FT assessments in patients with widely different T and SHBG levels. The cFT-V equation admittedly is a simplified representation of the binding of T to its binding proteins. Moreover, there is room for refinement of the association constants implemented in the equation. Nevertheless, contrary to what has been suggested (4, 17, 28, 36), our results do confirm that cFT-V is based on a valid model of T binding to SHBG.
*As the equilibrium dialysis reference method is not practical for use in the routine clinical laboratory, several equations have been advocated to estimate free T. Most commonly applied are equilibrium binding equations derived from the law of mass action using estimates of the association constants for binding of T to SHBG and albumin respectively [12,29]. Equations have also been developed empirically, modeled on large sets of free T concentrations, and free of assumptions about theoretical binding equilibria [30,31]. The weakness of these methods, apart from reliance on the accuracy of T and SHBG measurements, is that best-fit parameters in the test population may not be the same as in the patient population.
*Discrepancies between free T measured using equilibrium dialysis and cFT are most likely caused by erroneous modeling of testosterone binding to SHBG.
*Recent work shows that cFT-Vermeulen is strongly correlated to free T measured by the reference method (direct equilibrium dialysis), but free T is overestimated by 20–30%, thus agreeing with previous work [21,38,39]. However, the relationship between cFT-Vermeulen and measured free T was found to be linear and independent of serum T, albumin, and SHBG concentrations. The lack of reliance on SHBG in the Vermeulen equation was thought to be the strength of the cFT-Vermeulen since the assessment of free T is especially important in patients at the extremes of the SHBG concentration range. The bias is probably due to imperfect estimations of the association constants for the binding of T to SHBG and albumin, as discussed above, and this would need to be allowed for when comparing different methods for cFT.
-------------------------------------------------------------------------------------------------------------------------------
Unfortunately, the Zakharov equation is patented as it is going to be commercialized (TruTTM).
The Zakharov equation is patented and not available for independent scrutiny.
The TruTTM algorithm is still in phase II.
From a previous thread where I stated:
Need to look further..... thinking this will be the set reference range for the TruT calculated free testosterone method: "Based on the new data on the distribution of free testosterone levels in healthy men the target range of free testosterone has been determined to be 164 to 314 pg/ml (mean+/−1SD)"
Which would convert to 16-31 ng/dl
From what I understand once Phase II is completed it is just a matter of time before....."commercialization of the TruTTM algorithm using a HIPAA compliant infrastructure for its clinical adoption"
Phase II: Research and Commercialization of TruT Algorithm for Free Testosterone
ABSTRACT
- the measurement of testosterone(T) levels is central to the diagnosis of androgen disorders, such as hypogonadism in men and polycystic ovary syndrome (PCOS) in women
- circulating t is bound with high affinity to sex hormone-binding globulin (SHBG) and with substantially lower affinity to albumin; only the free fraction is biologically active
- conditions that affect SHBG concentrations, such as aging and obesity, alter total T but not free T concentrations; in these conditions, the determination of free t is necessary to obtain an accurate assessment of androgen status
- tracer analog method, the most widely used method for free T, has been shown to be inaccurate
- equilibrium dialysis method is, technically difficult to implement and standardize, and is not available in most hospital laboratories, leading the Endocrine Society's Expert Panel to conclude that?? the calculation of free testosterone is the most useful estimate of free testosterone in plasma??
- therefore, there is an unmet need for algorithms that provide accurate estimates of free T that match those derived from equilibrium dialysis
- we have designed a novel and accurate TruTTM algorithm for the determination of free T, based on the characterization of testosterones' binding to SHBG using modern biophysical techniques
- we have discovered that testosterone's binding to SHBG is a dynamic multistep process that includes allosteric interaction between the two binding sites on an SHBG dimer
- our computational frame-work incorporates the correct binding parameters derived experimentally in these studies, the non-linear dynamics in T: SHBG association, and allostery
- in phase I studies, we demonstrated that the TruTTM algorithm provides accurate free T values that match those obtained using the equilibrium dialysis in healthy and hypogonadal men
- we have also shown that the binding parameters that have formed the basis of previous equations (e.g., Vermeulen) are incorrect and that free T values derived using these equations deviate substantially from free T measured by equilibrium dialysis
- the phase I studies have led to the adoption of the TruTTM algorithm at several institutions
- the phase II program will continue the development of the TruTTM algorithm by validating it in common conditions characterized by altered SHBG concentration, such as obesity and aging (AIM 1), in healthy women across the menstrual cycle, and in women with PCOS (AIM 2)
- we will generate population-based reference ranges for free T (AIM 3)
- phase II also includes plans for commercialization of the TruTTM algorithm using a HIPAA compliant infrastructure for its clinical adoption
-the phase II program will provide validation of the TruTTM algorithm in the two most common clinical indications for free T measurement? men suspected of hypogonadism and altered SHBG levels, and women with hyperandrogenic disorders
- it will also enable the development of a HIPAA compliant platform that can be embedded into the electronic medical record for wider clinical adoption and for improving clinical care
Project Start 2014-09-15
Project End 2019-05-31
Phase II: Research and Commercialization of TruT Algorithm for Free Testosterone
-------------------------------------------------------------------------------------------------------------------------------
How the CDC Clinical Standardization Programs Are Improving Hormone Tests
Congress Funds AACC-Led Harmonization Initiative - AACC.org
The $1.3 trillion omnibus spending bill passed by Congress in March will fund efforts to harmonize laboratory tests, the culmination of more than a decade of AACC advocacy efforts. AACC worked closely with Rep. Kevin Yoder (R-Kan.) and other lawmakers, as well as a partnership of 17 clinical associations. The bill provides $2 million to the Centers for Disease Control and Prevention (CDC). The agency plans to use the new funding for materials and monitoring that will enable the harmonization of tests for free testosterone, thyroid-stimulating hormone, and estrogen.
Key point being:
* The agency plans to use the new funding for materials and monitoring that will enable harmonization of tests for free testosterone
The commercialization of the TruT platform is going to be a huge part of this.
You remember this paper:
Harmonized Reference Ranges for Circulating Testosterone Levels in Men of Four Cohort Studies in the United States and Europe
Thomas G. Travison, Hubert W. Vesper, Eric Orwoll, Frederick Wu, Jean-Marc Kaufman, Ying Wang Bruno Lapauw, Tom Fiers, Alvin M. Matsumoto, and Shalender Bhasin
Notice who played a big part in this? (S.B.)
Acknowledgments
This work was supported primarily by National Institutes of Health Grant 1RO1AG31206 to S.B. Additional support was provided by the Endocrine Society and Boston Claude D. Pepper Older Americans Independence Center Grant 5P30AG031679 from the National Institute on Aging. The Framingham Heart Study was supported by National Heart, Lung, and Blood Institute Framingham Heart Study Contract N01-HC-25195. The European Male Aging Study (EMAS) was supported by the Commission of the European Communities Fifth Framework Programme “Quality of Life and Management of Living Resources” Grant QLK6-CT-2001-00258. The Osteoporotic Fractures in Men Study (MrOS) was supported by the National Institutes of Health. The following institutes provided support: National Institute on Aging, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Center for Advancing Translational Sciences, and National Institutes of Health Roadmap for Medical Research under Grants U01 AG027810, U01 AG042124, U01 AG042139, U01 AG042140, U01 AG042143, U01 AG042145, U01 AG042168, U01 AR066160, and UL1 TR000128. The Belgian Sibling Study of Osteoporosis was supported by a grant from the Fund for Scientific Research–Flanders (FWO–Vlaanderen Grant G.0662.08) and by a grant from the Hercules Foundation, Flanders.
Disclaimers: The findings and conclusions in this manuscript are those of the authors and do not necessarily represent the official views or positions of the Centers for Disease Control and Prevention/Agency for Toxic Substances and Disease Registry. All individuals listed as authors agreed to be co-authors.
Disclosure Summary: S.B. has received research grant support from AbbVie Pharmaceuticals, Transition Therapeutics, Takeda Pharmaceuticals, and Eli Lilly for investigator-initiated research unrelated to this study. S.B. has served as a consultant to AbbVie, Regeneron, Novartis, and Eli Lilly. S.B. has a financial interest in Function Promoting Therapies, a company aiming to develop innovative solutions that enhance precision and accuracy in clinical decision making and facilitate personalized therapeutic choices in reproductive health. S.B.’s interests were reviewed and are managed by Brigham and Women’s Hospital and Partners HealthCare in accordance with their conflict of interest policies. A.M.M. has received research grant support from AbbVie and GlaxoSmithKline and has served as a consultant to AbbVie, Endo, Lilly, and Lipocine. F.W. has received research grant support from Besins Healthcare and Eli Lilly and has served as a consultant to Besins Healthcare and Repro Therapeutics. Other authors have nothing to disclose.
Let me be very clear here when I tell you that the man behind the efforts that are underway to standardize the procedures for free testosterone measurement and to generate harmonized reference ranges is the same man who has big plans for the commercialization of the TruTTM algorithm using a HIPAA compliant infrastructure for its clinical adoption.
Notice who the inventors are behind TruTTM.
Function Promoting Therapies
Function Promoting Therapies, LLC, and Tesvgen, the commercialization arm of FPT, LLC, was established in 2012. The company’s goals are to provide advanced technologies that improve accuracy in the diagnosis and rational management of testosterone replacement therapy for male reproductive disorders. FPT strives to innovate at a high level, maintain our access to academic research and pre-eminence in this content area, and to optimally balance research and commercial success. We are based in the suburbs of Boston and within reach of some of the most renowned endocrine research and clinical centers in the world. Our research partners are spread throughout the USA and we have research collaborations with leading institutions in Europe. These networks provide us the opportunity to share and validate our findings and enable us to leverage from a global palette of resources.
About Function Promoting Therapies
Function Promoting Therapies, LLC, and Tesvgen, the commercialization arm of FPT, LLC, was established in 2012. The company’s goals are to provide:
- Advanced diagnostic algorithms that improve accuracy in the diagnosis of male reproductive disorders
- Enabling tools to enhance compliance and improve treatment outcomes for male reproductive disorders
- Advanced tools for dosimetry to optimize testosterone therapy
We are based in the heart of Boston within reach of some of the most renowned endocrine research and clinical centers in the world. Our research partners are spread throughout the USA and we have research ties with leading institutions in Europe. These networks provide us the opportunity to share and validate our findings and enable us to leverage from a global palette of resources.
Our access to a network of academic experts at leading academic institutions also means that we are in tune with the upcoming changes in treatment patterns or recommendations of professional societies.
Partners and Collaborations
We continue to work with collaborators in academia and the wider industry for exciting new applications and further development of TruT™. Some of our existing partnerships include:
Johns Hopkins University ‐ Diagnosis and management of hypogonadism in HIV and co-infections.
The Mayo Clinic ‐ Personalized algorithm and therapy development.
Karolinska Institute ‐ Dynamics of free testosterone levels after surgical interventions in men and women.
regionh.dk - Examining population reference ranges of free testosterone for Danish cohorts.
UCLA School of Nursing ‐ Examining the dynamic role of multiple hormones in altering transport and bioavailability.
Boston IVF ‐ Developing a novel platform for rational treatment and management of in-vitro fertilization interventions.
Myosyntax ‐ Dynamics of testosterone bioavailability during caloric restriction.
Investors
Tesvgen is funded through the seed investment round in Function Promoting Therapies LLC. FPT successfully closed the seed round in 2016 and is currently in discussions with strategic partners for further commercialization of innovations.
