Free Testosterone Tests

DJ87

Member
Hey Guys,

I received two different sets of free testosterone labs and am trying to make sense of them. The first was LabCorp's Equilibrium FT test and the second was their Direct FT test. What is the difference and which labs would be more accurate in this case? For reference my SHBG is 20 and my protocol is .25 MWF of 200mg cyp with 200IUs of HCG at the time of injection with no AI. Thanks for any input!

Equilibrium:

TT - 914 ng/dL (264 - 916)
FT - 44.97 ng/dL (5 - 21)
E2 - 29.5 pg/mL (8 - 35)
Free Test Percent - 4.92 (1.50 - 4.2)

Direct:

TT - 777 ng/dL (264 - 916)
FT - 24.6 ng/dL (8.7 - 25.1)
E2 - 29.5 pg/mL (8 - 35)
 

ERO

Member
Not to be purposely obtuse by answering a question with a question, but how do you feel? I ask because if you feel good on your current protocol then it doesn't really matter.

Of course if you are trying to get dialed in then, yes, it would be best to understand the differences in these tests before adjusting anything.
 

madman

Member
Hey Guys,

I received two different sets of free testosterone labs and am trying to make sense of them. The first was LabCorp's Equilibrium FT test and the second was their Direct FT test. What is the difference and which labs would be more accurate in this case? For reference my SHBG is 20 and my protocol is .25 MWF of 200mg cyp with 200IUs of HCG at the time of injection with no AI. Thanks for any input!

Equilibrium:

TT - 914 ng/dL (264 - 916)
FT - 44.97 ng/dL (5 - 21)

E2 - 29.5 pg/mL (8 - 35)
Free Test Percent - 4.92 (1.50 - 4.2)

Direct:

TT - 777 ng/dL (264 - 916)
FT - 24.6 ng/dL (8.7 - 25.1)
E2 - 29.5 pg/mL (8 - 35)

Your first set of labs that tested FT using an accurate assay (Equilibrium Ultrafiltration) is the one you want to rely on.

The only way to know where your FT level truly sits is to have it tested using an accurate assay such as the gold standard Equilibrium Dialysis or Ultrafiltration (next best).

Forget using/relying on the piss poor direct immunoassay.

Even if you would have only tested TT and SHBG I would have guessed that with a TT 900s and lowish SHBG 20 nmol/L that your FT level would have been high.

As you can clearly see with a TT 914ng/dL and SHBG 20 nmol/L that your FT is very high roughly 45 ng/dL and if these are your trough levels then keep in mind that levels 8-12 hrs post-injection will be higher.

Regardless as ERO stated if you feel well overall and blood markers are healthy then I would not change a thing.

If you are struggling on such protocol let alone dealing with any sides whether cosmetic/blood markers then you easily have room to lower your dose slightly and bring down your FT.

Next time save your $$$ and forget using piss-poor assays when testing hormones!
 
Last edited:

madman

Member
Hey Guys,

I received two different sets of free testosterone labs and am trying to make sense of them. The first was LabCorp's Equilibrium FT test and the second was their Direct FT test. What is the difference and which labs would be more accurate in this case? For reference my SHBG is 20 and my protocol is .25 MWF of 200mg cyp with 200IUs of HCG at the time of injection with no AI. Thanks for any input!

Equilibrium:

TT - 914 ng/dL (264 - 916)
FT - 44.97 ng/dL (5 - 21)
E2 - 29.5 pg/mL (8 - 35)
Free Test Percent - 4.92 (1.50 - 4.2)

Direct:

TT - 777 ng/dL (264 - 916)
FT - 24.6 ng/dL (8.7 - 25.1)
E2 - 29.5 pg/mL (8 - 35)

Post your full labs!

Where does your RBCs/hemoglobin/hematocrit sit?
 

DJ87

Member
Not to be purposely obtuse by answering a question with a question, but how do you feel? I ask because if you feel good on your current protocol then it doesn't really matter.

Of course if you are trying to get dialed in then, yes, it would be best to understand the differences in these tests before adjusting anything.
Thanks for the response ERO, I feel good on this protocol and have been on it for the past year and half without changes. My FT(direct) and TT dropped from around 29(FT direct) and 1000(TT) recently and was curious what another assay would pull.
 

DJ87

Member
Post your full labs!

Where does your RBCs/hemoglobin/hematocrit sit?
Thanks Madman, appreciate the insight as always! Here are some of the relevant labs below.

RBC - 5.91 x10E6/uL (4.14 - 5.80)
Hemoglobin - 16.2 g/dL (13.0 - 17.7)
Hematocrit - 49.7 % (37.5 - 51)
Albumin - 4.7 g/dL (4.0 - 5.0)
Globulin - 2.2 g/dL (1.5 - 4.5)
AST - 22 IU/L (0 -40)
ALT - 23 IU/L (0 - 44)
Cholesterol, Total - 129 mg/dL (100 - 199)
Triglycerides - 113 mg/dL (0 - 149)
HDL - 41 mg/dL (>39)
LDL - 21 mg/dL (0 - 99)
DHEA - 154 ug/dL (102.6 - 416.3)
Prostate Ag - .4 ng/mL (0 - 4.0)
SHBG - 21 nmol/L (16.5 - 55.9)
 

bennettjc

Member
Hey Guys,

I received two different sets of free testosterone labs and am trying to make sense of them. The first was LabCorp's Equilibrium FT test and the second was their Direct FT test. What is the difference and which labs would be more accurate in this case? For reference my SHBG is 20 and my protocol is .25 MWF of 200mg cyp with 200IUs of HCG at the time of injection with no AI. Thanks for any input!

Equilibrium:

TT - 914 ng/dL (264 - 916)
FT - 44.97 ng/dL (5 - 21)
E2 - 29.5 pg/mL (8 - 35)
Free Test Percent - 4.92 (1.50 - 4.2)

Direct:

TT - 777 ng/dL (264 - 916)
FT - 24.6 ng/dL (8.7 - 25.1)
E2 - 29.5 pg/mL (8 - 35)
I recently got double Free Testosterone tested as well. LabCorp

MS/Dialysis
TT - 1175 ng/dl ( 264-916)
FT - 200 pg/ml (52-280)

Direct:
FT - 33.4 pg/ml (7.2 - 24)

Also:
SHBG - 37.9 nmol/L (19.3 - 76.4)
E2 Sensitive - 52.6 pg/ml (8-35)
Hematocrit - 47.5 (37.5-51)
Hemoglobin - 16 (13-17.7)
Red Blood Count - 5.19 (4.1-5.8)

My regimen is 60mg IM of Testosterone Enanthate twice a week. HCH 250iu sq three times a week.

I feel pretty good. More energy and libido since starting TRT.

Observations:
-The contrasting result between the "Direct" and the more accurate dialysis (the "gold standard" as per madman above) methods is striking. No more "Direct" Free T levels for me. Waste of money. For what it's worth, this is the most popular Free T test providers order.

- I've had a great deal of communication with someone at LabCorp about the confusing units on their Direct Free Testosterone test. I was met with arrogance and poor
consumer relations.
 
Last edited:

bennettjc

Member
Your first set of labs that tested FT using an accurate assay (Equilibrium Ultrafiltration) is the one you want to rely on.

The only way to know where your FT level truly sits is to have it tested using an accurate assay such as the gold standard Equilibrium Dialysis or Ultrafiltration (next best).

Forget using/relying on the piss poor direct immunoassay.

Even if you would have only tested TT and SHBG I would have guessed that with a TT 900s and lowish SHBG 20 nmol/L that your FT level would have been high.

As you can clearly see with a TT 914ng/dL and SHBG 20 nmol/L that your FT is very high roughly 45 ng/dL and if these are your trough levels then keep in mind that levels 8-12 hrs post-injection will be higher.

Regardless as ERO stated if you feel well overall and blood markers are healthy then I would not change a thing.

If you are struggling on such protocol let alone dealing with any sides whether cosmetic/blood markers then you easily have room to lower your dose slightly and bring down your FT.

Next time save your $$$ and forget using piss-poor assays when testing hormones!
Madman - Could you share a bit of knowledge, on a technical level, why LabCorp's Direct Free Testosterone test is so lousy. What leads to it's inaccuracy? Does its mismeasurement tend to overstate the Free Testosterone or vice-versa.
Thanks!
 

madman

Member
Madman - Could you share a bit of knowledge, on a technical level, why LabCorp's Direct Free Testosterone test is so lousy. What leads to it's inaccuracy? Does its mismeasurement tend to overstate the Free Testosterone or vice-versa.
Thanks!

Forget the lab.....it's the assay!


Methodology for measuring testosterone, DHT, and SHBG in a clinical setting (2004)
M.Simoni


21.4.4 Assessment of free testosterone

The direct measurement of free testosterone in serum is based on the same principles governing the assay of free thyroid hormones and has been extensively considered and reviewed by R. Ekins in the past (Ekins 1990). As indicated above, serum testosterone exists in an equilibrium between free and protein-bound fractions, an equilibrium which is invariably disturbed by all methods of free hormone measurement, a factor that should be kept in mind when choosing a method and analyzing the data. The methods of reference for free hormone analysis are equilibrium dialysis and ultrafiltration, which should be used for research purposes and to validate other systems.


21.4.4.3 Direct free testosterone RIA

These methods are based on the concept that if an antibody is added to a serum sample, only free hormone will bind to it and the antibody occupancy will depend on the free hormone concentration.As a result, however, the protein-bound hormone will dissociate and a new equilibrium will be established. Therefore, the antibody concentration should be kept “small” enough to minimize the depletion of the protein-bound pool, i.e. not more than 1% of total hormone should be displaced from the binding proteins to the antibody.
Quantification of the antibody-bound hormone, i.e. the free hormone concentration, can be achieved indirectly by knowing the total hormone concentration, adding labeled hormone, and measuring the fraction of it which is taken up by the antibody (“labeled hormone antibody uptake”). Alternatively, a two-step approach involves adding the sample to a solid phase antibody, washing off the unbound serum components, and adding labeled hormone which will be bound by the residual, unoccupied antibody binding sites. Since the amount of antibody is limited, the higher the free hormone concentration, the lower the number of unoccupied antibody sites at the end of the first incubation, the lower the antibody occupancy by labeled hormone at the end of the second incubation. In order for this method to work, a two-step approach, with the removal of serum after the first reaction of the antiserum with the free hormone, is necessary because if the labeled hormone interacts with the serum binding proteins, this would impair the estimation of antibody occupancy by the tracer. The two-step approach, however, is not necessary if one uses a labeled-compound that is totally non-reactive with serum proteins but can be recognized by the solid phase antibody present in a limited amount and which competes for binding with the free hormone in the sample. This is the principle of the free “analog” testosterone assay on which some popular commercially available kits are based.

The direct measurement of free testosterone in serum based on the labeled hormone “analog” is valid, provided that the analog does not interact with the serum proteins, a condition which is currently not met by commercial kits.
In fact, neither
the identity of the analog tracer, nor the validation of the kit (showing the absence of interactions with the serum protein) is usually disclosed by the manufacturer. On the contrary, the “analog” principle is often not even mentioned or is misrepresented in the instruction accompanying the kits,which are often validated only against other kits and not against dialysis or ultrafiltration. It should be kept in mind that, in practice, finding a hormone analog totally unreactive with serum proteins is very difficult and several studies have shown that such an interaction indeed occurs, resulting in inaccurate measurements of free testosterone. In this respect it is interesting that serum-free testosterone measured by the analog method accounts for 0.5–0.65% of total testosterone, while equilibrium dialysis and ultrafiltration give values of 1.5–4%, revealing inconsistencies between the different approaches (Rosner 1997; Winterset al. 1998). In a direct comparison, free testosterone values measured by a bestseller “analog” kit were only 20–30% of those measured by equilibrium dialysis (Vermeulen et al. 1999).For these reasons it is recommended that if an “analog” method is to be considered for routine free testosterone determination, in-house validation of the kit should involve comparison with dialysis or ultrafiltration and estimation of the binding of the analog tracer to endogenous proteins e.g. by adding exogenous SHBG (e.g. serum from pregnant women) and by estimating tracer binding to concanavalin A-bound SHBG after chromatography of the serum samples (Winterset al. 1998). Unfortunately, the kits for direct free testosterone measurement presently available do not measure what they claim to do and give inaccurate results (Rosner 2001). Their use should be discouraged.
--------------------------------------------------------------------------------------------------


Methodology for measuring testosterone, dihydrotestosterone and sex hormone-binding globulin in a clinical setting (2008)
Manuela Simoni, Flaminia Fanelli, Laura Roli, and Uberto Pagotto


4.3.4.3 Direct free testosterone assays

The principles of the direct free testosterone methods have been described in detail in the previous edition of this book (Simoni 2004). In practice such assays are based on a labeled compound to be added to the sample, the “analog,” which is totally non-reactive with serum proteins, but can be recognized by the solid-phase antibody present in a limited amount and which competes for binding with the free hormone in the sample.

Several commercially available kits are based on this principle and are widely used to assess free testosterone in clinical samples. Unfortunately, there is evidence that these methods are totally inaccurate (Fritz et al. 2008; Chen et al. 2010).
In fact, the direct measurement of free testosterone in serum based on the labeled hormone “analog” is valid only if the analog does not interact with the serum proteins: a condition which is currently not met by commercial kits. In fact, neither the identity of the analog tracer, nor the validation of the kit (showing the absence of interactions with the serum protein) is usually disclosed by the manufacturer. On the contrary, the “analog” principle is often not even mentioned or is misrepresented in the instructions accompanying the kits, which are often validated only against other kits and not against dialysis or ultrafiltration. It should be kept in mind that, in practice, finding a hormone analog totally unreactive with serum proteins is very difficult, and several studies have shown that such an interaction indeed occurs, resulting in inaccurate measurements of free testosterone. In this respect it is interesting that serum-free testosterone measured by an “analog” method accounts for 0.5–0.65% of total testosterone, while equilibrium dialysis and ultrafiltration give values of 1.5–4%, revealing inconsistencies between the different approaches (Rosner 1997; Winters et al. 1998). In a direct comparison, free testosterone values measured by a bestseller “analog” kit was only 20–30% of those measured by equilibrium dialysis (Vermeulen et al. 1999).

For these reasons the kits for direct free testosterone measurement presently available do not measure what they claim and should not be used (Rosner 2001; Swerdloff and Wang 2008). If LC-MS/MS coupled to ultrafiltration becomes technically affordable, this approach is likely to become the method of choice for assessment of free testosterone in clinical serum samples.
 

Cataceous

Well-Known Member
I recently got double Free Testosterone tested as well. LabCorp

MS/Dialysis
TT - 1175 ng/dl ( 264-916)
FT - 200 pg/ml (52-280)

Direct:
FT - 33.4 pg/ml (7.2 - 24)

Also:
SHBG - 37.9 nmol/L (19.3 - 76.4)
E2 Sensitive - 52.6 pg/ml (8-35)
Hematocrit - 47.5 (37.5-51)
Hemoglobin - 16 (13-17.7)
Red Blood Count - 5.19 (4.1-5.8)
...
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?
 

madman

Member
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.
 

madman

Member
This sums it up nicely!



Total testosterone, which can be measured with high accuracy using LC-MS/MS assays in CDC-certified laboratories, and free testosterone are highly correlated, and it is only in individuals with altered binding-protein concentrations that the associations begin to diverge. For the time being, we, therefore, suggest continuing to follow the Endocrine Society’s guidelines to measure total testosterone level and, in circumstances of suspected alterations in SHBG and albumin concentrations and/or binding, checking free testosterone level by equilibrium dialysis. Efforts are underway to standardize the procedures for free testosterone measurement and to generate harmonized reference ranges. Until that time, clinicians should be aware that inaccuracies in free testosterone measurements and calculations and poorly defined reference ranges can increase the risk of misclassification in the diagnosis of androgen disorders.
 

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