Do you happen to have an albumin measurement to go with these?
It's interesting that with default albumin both Tru-T and Vermeulen are suggesting above-range free T.
@madman: Because the calculated methods use albumin, total testosterone and SHBG, when these parameters are the same then computed free testosterone is the same each time. Are there any reasons why this should not be true with equilibrium dialysis tests, setting aside measurement errors and the small dependence on other hormones? That is, if a guy has a free T measurement at some time, then if he repeats the measurement at some later date and happens to have the same SHBG, TT and albumin, then is it expected that the free T measurement will be the same as the earlier measurement?
Seems off as FT level should be higher when looking at where his TT/SHBG sit.
Unfortunately could be a lab error and it would be interesting to see numerous results using the same lab/assay.
Herein lies the problem although Equilibrium Dialysis is considered the gold standard for measuring FT there will always be a chance of lab error due to the numerous issues listed below.
Until we have standardization let alone a harmonized reference range for FT there will always be issues even when using what would be considered the gold standard reference methods.
As we know this s**t show should be coming to an end soon enough.
*The Centers for Disease Control and Prevention’s (CDC’s) hormone standardization program is invested in improving clinical assays and minimizing factors that affect measurement variability
Until such happens there will always be a chance of inconsistent results using various labs/assays (even accurate) when testing FT.
Equilibrium dialysis and its various embodiments
Equilibrium dialysis is widely considered the reference method against which other methods are compared.
It is technically demanding, and its performance is affected by assay conditions, which can result in high assay variability (192). Typically, the equilibrium dialysis procedure involves the dialysis of serum or plasma samples across a semipermeable cellulose membrane with a low-molecular-weight cutoff; protein-bound testosterone is retained, whereas free testosterone equilibrates across the dialysis membrane and can be measured in the dialysate either directly using a liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay or immunoassay or indirectly using a tracer. Indirect methods require adding a trace amount of radioactively labeled testosterone to the sample, and after equilibrium has been achieved, the proportion of tracer in the dialysate provides a measure of the percentage of free testosterone. Because free testosterone concentration can then be calculated by multiplying the percentage of the free fraction with the total testosterone concentration obtained from the same sample in a separate assay, accurate determination of total testosterone levels is necessary for accurate determination of free testosterone levels by this method.
Although a diligently conducted equilibrium dialysis assay accurately measures free testosterone level, the method is fraught with operator-dependent errors. The protocol itself is labor-intensive, requiring repeated purification of the radioactive tracer, and is not readily amenable to high throughput. Even some large commercial diagnostic laboratories have stopped offering this assay.
Although equilibrium dialysis is widely considered to be the gold standard for measuring free testosterone, this method is subject to various sources of error that may contribute to inaccuracy and imprecision. For instance, the dilution of serum or plasma may disturb the equilibrium between SHBG and its ligands. (
193).
Results may also be altered when solutes become attached to the dialysis apparatus or membrane or when there is an unequal distribution of free ligands between the two compartments as a result of (1) inadequate time to reach equilibrium; (2) release of materials from the plate or membrane that interferes with the determination of concentration; and (3) the Donnan effect at low ionic strengths, which alters the distribution of charged particles near a semipermeable membrane so that they may not distribute evenly across the two sides of the membrane (194, 195). The ionic strength and pH of the dialysis buffer and the temperature at which dialysis is performed affect the equilibrium and the estimates of binding parameters. The batch-to-batch variability in adsorption characteristics of dialysis plates from different manufacturers may be an additional source of interassay variation.
*The Centers for Disease Control and Prevention’s (CDC’s) hormone standardization program is invested in improving clinical assays and minimizing factors that affect measurement variability (196).
Lack of Standardization of Free Testosterone Measurement Methods and Unavailability of Harmonized Reference Ranges for Free Testosterone
Le et al
.(
222) surveyed 120 academic and community laboratories in the United States to characterize the distribution of assays and the associated reference values for free testosterone.
In all, 84% of the surveyed laboratories sent their samples for free testosterone measurement to larger centralized reference laboratories (222). These large commercial laboratories offered a variety of methods, including ultracentrifugation, radioimmunoassay, and calculation-based algorithms, as well as equilibrium dialysis (222). Many clinical laboratories used calculated free testosterone based on published linear equations (3). The laboratories reported wide variations in the reference ranges. Only 30 of the laboratories surveyed would confirm that validation studies had been performed, and the authors advised that reference ranges provided by manufacturers and laboratories should be interpreted with caution.
In a survey of 12 academic laboratories, 12 community medical laboratories, and one national laboratory, Lazarou et al. (223) found 17 and 13 different sets of reference values for total and free testosterone, respectively, which were established largely without clinical considerations. Recently, Bhasin
et al. (
224) reported reference ranges for calculated free testosterone concentrations in a large, rigorously collected sample of community-dwelling men. In healthy young men of the Framingham Heart Study who were 19 to 40 years of age, the lower limit of the normal range, defined as the 2.5th percentile of calculated free testosterone, was 70 pg/mL (242.7 pmol/L) (
198).
Clinical Implications and Recommendations
Male hypogonadism is a clinical condition characterized by the presence of typical signs and symptoms in the setting of consistently low serum testosterone concentrations. The Endocrine Society guidelines currently suggest measuring free testosterone levels in men in whom total testosterone concentrations are near the lower limit of the normal range and in men with conditions that affect SHBG concentrations and render total testosterone a less reliable index of gonadal function (
206).
If the free hormone hypothesis is correct, free testosterone should serve as the benchmark for biochemical confirmation of hypogonadism. Accurate determination of free testosterone values is therefore central to an accurate diagnosis of hypogonadism.
The direct analog assays for free testosterone determination are inaccurate and should not be used. However, a confluence of factors related to the regulatory process, economic considerations, and difficulties in performing equilibrium dialysis methods in many hospital laboratories has led to their surprising endurance despite their known inaccuracy.
Historically, laboratory-certifying bodies, such as the Clinical Laboratory Improvement Amendments, have certified laboratories and assays mostly on the basis of process measures; unlike the CDC and its Hormone Assay Standardization program for testosterone, these bodies have generally not required accuracy-based benchmarks. Similarly, the requirement in the assay approval process for the demonstration of comparability to a previously approved assay enables new tracer analog assays to be approved because they can demonstrate comparability to previously approved analog methods.
Equilibrium dialysis is the reference method for free testosterone determination, but this assay is not always available to clinicians in all hospital laboratories; in addition, there are substantial interlaboratory variations because of the lack of standardization of assay conditions, making it difficult for practicing endocrinologists to interpret free testosterone levels. Mechanisms to harmonize the equilibrium dialysis procedure across laboratories are needed. Until equilibrium dialysis methods can be standardized across laboratories, a computational framework that accurately captures the dynamics of testosterone to SHBG and HSA interactions in calculating free testosterone values is an unmet need for precise clinical diagnosis. The EAM appears to be an accurate and testable model for calculating free testosterone levels, but this model needs further validation in large populations.
