Not true. Lab Corp has more than six total and free testosterone assays. When my doctor writes a script for free testosterone, I always append the appropriate test number for the equilibrium ultrafiltration assay. Lab Corp also offers an equilibrium dialysis assay through an outside lab.
LabCorp Vs Quest free testosterone lab ranges: Why are they different?
- Thread starter GeorgieBoy
- Start date
As I said, the value for 'free T' you get is a test number specific for THAT test and does NOT give the absolute biological amount of free T. It is just some number from some scale that shows if you are normal or high/low with respect to the sample of people that was used to create the lab test.
It is completely logical and actually consistent that the top of the normal range for Quest, 15.5ng/dL should correspond to the top of normal range for Lab Corp 25 ng/dL if they used the same statistical definitions of 'normal' and had defining samples of similar means and standard deviations.
The problem arises if you actually take these numbers as the actual value of free testosterone. The number on a test only gives the relative position within that test scale. Online calculators for free T will give completely different values within their own scales.
It is completely logical and actually consistent that the top of the normal range for Quest, 15.5ng/dL should correspond to the top of normal range for Lab Corp 25 ng/dL if they used the same statistical definitions of 'normal' and had defining samples of similar means and standard deviations.
The problem arises if you actually take these numbers as the actual value of free testosterone. The number on a test only gives the relative position within that test scale. Online calculators for free T will give completely different values within their own scales.
T
tareload
Guest
You are truly confusing guys when you give these definitive statements that are in fact not true. No differentiation between RIA free T vs dialysis or ultrafiltration coupled with LCMS to measure free T? How do you think the calculated free T methods were created? Do you understand their basis?
If dialysis free T methods are not an assessment of actual free T (study the physics of the situation), then Total T by LCMS is just a proxy for Total T with your logic? Your position makes no sense in accepting Total T by LCMS as an “absolute” assay but casting dialysis in with RIA as a “proxy” measure for free T. The information you are sharing here is scientifically unsound and provides misinformation to guys who are trying to learn this stuff.
T
tareload
Guest
Really? No difference in reference ranges between RIA methods vs LCMS? Not true. Even differences between different labs for Total T by LCMS. Why? Measurement error.
Take a look at the two parity plots below. What variables is each plot showing? Notice anything about the slopes of these two parity plots? Is the top plot showing an absolute measure for Total T or a "proxy" for it?
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Measuring total testosterone is based on recognizing the testosterone molecule and there are relatively reliable lab methods for that, within 'calibration errors'. I never got one value in US an a completely different value in Europe.
Measuring free testosterone requires a method to physically separate the free testosterone from the bound testosterone and the dialysis method does that with a semi-permeable membrane. The concentration of free testosterone is vanishingly small and some part of it gets stuck in the dialysis membrane. So in all cases you are measuring some proxy of it, depending on the membrane used, lab/test conditions, methods used to determine amount free testosterone after dialysis etc. It is proxy first because the method of separation free from bound testosterone is not reliable and second because lab methods to measure free testosterone are unreliable at such small concentrations.
You can keep arguing semantics but the simple fact remains - total testosterone values have little variability between labs using the same method, while each free testosterone value is valid only in its own test scale because they are impossible to compare between different methods/labs - hence are proxy.
Measuring free testosterone requires a method to physically separate the free testosterone from the bound testosterone and the dialysis method does that with a semi-permeable membrane. The concentration of free testosterone is vanishingly small and some part of it gets stuck in the dialysis membrane. So in all cases you are measuring some proxy of it, depending on the membrane used, lab/test conditions, methods used to determine amount free testosterone after dialysis etc. It is proxy first because the method of separation free from bound testosterone is not reliable and second because lab methods to measure free testosterone are unreliable at such small concentrations.
You can keep arguing semantics but the simple fact remains - total testosterone values have little variability between labs using the same method, while each free testosterone value is valid only in its own test scale because they are impossible to compare between different methods/labs - hence are proxy.
Nelson Vergel
Founder, ExcelMale.com
Differences Between LabCorp and Quest Sensitive Estradiol and Testosterone Lab Test Ranges
The Labcorp results are from January and the Quest results are from this week. The ranges they use are different so I’m not sure how to compare them. Do these seem pretty much in line? Especially the “Free Test” difference. Quest is the one in color, FYI Also, how do you guys feel about the E2...
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What is a Normal Blood Level of Testosterone? What Do Guidelines Say?
Normal values may vary from lab to lab depending on what reference range they use. Depending on the country, ranges are in nanograms per deciliter (usually in the United States) or nanomoles per liter (Europe and other countries). The conversion factor is: Click here: What’s a “Normal”...
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T
tareload
Guest
I'll let the astute reader see if your comments (quoted above) are consistent with the data I've presented. Here I'll show the plot again below:
I asked you some simple questions about these two plots but you didn't answer:
Take a look at the two parity plots below.
What variables is each plot showing?
Notice anything about the slopes of these two parity plots?
Is the top plot showing an absolute measure for Total T or a "proxy" for it?
For the interested reader, try to answer these questions yourself and see if @sammmy 's comments follow from these data. Two labs measuring TT and free T on the same samples (TT by LCMS and fT by dialysis coupled with LCMS/MS).
And I'll let the 'astute reader' compare their total testosterone between different labs (success) and then try to compare their free testosterone between different labs or between a lab result and online calculators (total failure). Reality vs 'studies'.
If they all 'measure the same thing', labs for free testosterone and online calculators should have at least similar normal ranges. In reality they differ by FACTORS in the same units! So much for "not measuring a proxy of free testosterone".
If they all 'measure the same thing', labs for free testosterone and online calculators should have at least similar normal ranges. In reality they differ by FACTORS in the same units! So much for "not measuring a proxy of free testosterone".
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T
tareload
Guest
Already did it for you. Go to this thread (and the volume of information I presented there):
www.excelmale.com
FACTORs for dialysis/LCMS-MS? The lower ranges are extremely tight (and much lower than Tru-T calculator but very close to Vermeulen). High ranges have a factor of 2 difference but as we've discussed I think that may be difference between direct vs indirect dialysis method (I'd have to call each lab and confirm if they are using direct vs indirect). Besides the difference between Tru-T and lab ranges, where's the FACTORs?
Here I actually compared Vermeulen and Tru-T calculators with YOUR own results you reported....
www.excelmale.com
Vermeulen is within 20%. Tru-T way high. Where's the FACTORs difference between Vermeulen and your own dialysis free T result?
I posted a whole wall of these comparisons. What am I missing?
Calculate Free Testosterone with TruT by FPT
[0026] Fig. 7 depicts the thermodynamic Parameters associated with testosterone's binding to SHBG derived from the fit of binding isotherms and ITC data to new model. While this parameter set is not unique, together they consistently describe the binding isotherms, depletion curves and ITC data...
www.excelmale.com
FACTORs for dialysis/LCMS-MS? The lower ranges are extremely tight (and much lower than Tru-T calculator but very close to Vermeulen). High ranges have a factor of 2 difference but as we've discussed I think that may be difference between direct vs indirect dialysis method (I'd have to call each lab and confirm if they are using direct vs indirect). Besides the difference between Tru-T and lab ranges, where's the FACTORs?
Here I actually compared Vermeulen and Tru-T calculators with YOUR own results you reported....
Calculate Free Testosterone with TruT by FPT
[0026] Fig. 7 depicts the thermodynamic Parameters associated with testosterone's binding to SHBG derived from the fit of binding isotherms and ITC data to new model. While this parameter set is not unique, together they consistently describe the binding isotherms, depletion curves and ITC data...
www.excelmale.com
Vermeulen is within 20%. Tru-T way high. Where's the FACTORs difference between Vermeulen and your own dialysis free T result?
I posted a whole wall of these comparisons. What am I missing?
Last edited by a moderator:
Here is the ultimate reality check.
My recent Quest Diagnostics test results:
Test name: "Testosterone, Free (Dialysis), Total (MS) and Sex Hormone Binding Globulin"
Methodology: Chromatography/Mass Spectrometry • Equilibrium Dialysis • Calculation (CALC) • Immunoassay (IA)
Total Testosterone: 817 ng/dL
SHBG: 86 nmol/L
Albumin: 4.5 g/dL
Free Testosterone: 60.3 pg/mL = 6.03 ng/dL
Free Testosterone calculated by TruT = 21.93 ng/dL, which is about 3 times higher than the Quest Diagnostics value. That is the FACTOR difference that I am talking about.
I am not no TRT or any anabolic compounds.
My recent Quest Diagnostics test results:
Test name: "Testosterone, Free (Dialysis), Total (MS) and Sex Hormone Binding Globulin"
Methodology: Chromatography/Mass Spectrometry • Equilibrium Dialysis • Calculation (CALC) • Immunoassay (IA)
Total Testosterone: 817 ng/dL
SHBG: 86 nmol/L
Albumin: 4.5 g/dL
Free Testosterone: 60.3 pg/mL = 6.03 ng/dL
Free Testosterone calculated by TruT = 21.93 ng/dL, which is about 3 times higher than the Quest Diagnostics value. That is the FACTOR difference that I am talking about.
I am not no TRT or any anabolic compounds.
T
tareload
Guest
Yes, that's exactly my point about Tru-T and why I spent so much time daylighting the fact it's usually way high compared to Vermeulen. Here's your numbers with Vermeulen:
So about 50% high (typically 20-50%) with Vermeulen; point taken with Tru-T. This was the whole gist of my work over in the other post. As @Cataceous and I agree, using one of the dated empirical calculators or Vermeulen will typically get you much closer to your measured value. Great to find some common ground with you, thanks for the input.
www.excelmale.com
Sartorius (see link above) gives 9.07 ng/dL:
cFT (pMol/L) = 24.00314 * T / log10(SHBG) - 0.04599 * T * T
T and SHBG in nMol/L
T(ng/dL) = 28.8184 * T(nMol/L)
FT(ng/dL) = FT(pMol/L) / 1000 * 28.8184
Not all calculators are created equal when trying to fit actual data. Data wrong, Tru-T correct? That was my whole question. Tru-T has supposedly fit all the constitutive/thermo binding behavior but it's ability to fit dialysis free T data is poor.
See here and the walls of data:
Calculate free testosterone with TruT by FPT
So about 50% high (typically 20-50%) with Vermeulen; point taken with Tru-T. This was the whole gist of my work over in the other post. As @Cataceous and I agree, using one of the dated empirical calculators or Vermeulen will typically get you much closer to your measured value. Great to find some common ground with you, thanks for the input.
Calculate Free Testosterone with TruT by FPT
https://www.sciencedirect.com/science/article/pii/S0009912013000118 See method validation and method comparison sections: See panel E:
www.excelmale.com
Sartorius (see link above) gives 9.07 ng/dL:
cFT (pMol/L) = 24.00314 * T / log10(SHBG) - 0.04599 * T * T
T and SHBG in nMol/L
T(ng/dL) = 28.8184 * T(nMol/L)
FT(ng/dL) = FT(pMol/L) / 1000 * 28.8184
Not all calculators are created equal when trying to fit actual data. Data wrong, Tru-T correct? That was my whole question. Tru-T has supposedly fit all the constitutive/thermo binding behavior but it's ability to fit dialysis free T data is poor.
See here and the walls of data:
Calculate free testosterone with TruT by FPT
Last edited by a moderator:
bennettjc
Member
Thanks. Great info that Lab Corp sends their dialysis specimens to another lab! No wonder it takes ~ 7 days for the results to come back.
Nelson Vergel
Founder, ExcelMale.com
Hey @madman
I know I have seen a reference somewhere comparing Eq. Dialysis to Ultrafiltration for free testosterone measurement. Since you are our reference library, can you find it for me? Thanks!!
madman
Super Moderator
Direct measurement of serum-free testosterone by ultrafiltration followed by liquid chromatography-tandem mass spectrometry (2009)
Background: Currently there is no reliable method suitable for routine measurement of serum-free testosterone (FT).
Aim: To develop such a method involving liquid chromatography-tandem mass spectrometry (LC-IDMS/ MS) that directly detects and quantifies the FT present in serum.
Methods: Ultrafiltrate testosterone obtained from 0.5 mL of serum was partially purified by liquid/liquid extraction and quantified using an Agilent 1200 Series HPLC system coupled to an API 5000 mass spectrometer equipped with an atmospheric pressure chemical ionization ion source. Using split samples serum free testosterone was compared between direct ultrafiltration (UF) coupled LC-MS/MS, analog FT immunoassay, free testosterone calculated from mass action equations (cFT), and with equilibrium dialysis (ED) coupled LC-MS/MS.
Results: Total imprecision determined over twenty runs was b6% at 67 pmol/L and 158 pmol/L FT. The dynamic response was linear up to at least 2500 pmol/L while physical LLOQ (18 % CV) equaled 16 pmol/L. The UF method agreed poorly with analog immunoassay (correlation coefficient 0.667; bias −81%), somewhat better against cFT when total testosterone was determined by immunoassay (correlation coefficient 0.816, bias 21% ) and still better yet against cFT when total testosterone was determined by LCMS/MS (correlation coefficient 0.8996, bias 10%). The agreement was closest with the ED method (correlation coefficient 0.9779, bias 2.4%).
Conclusion: We present a relatively simple UF coupled LC-MS/MS definitive method that measures serum-free testosterone. The method is relatively fast, reliable, and is suitable for routine clinical laboratory practice.
Several approaches have been used to measure FT in circulation. The most reliable method physically separates the protein-bound from the free testosterone prior to quantifying the latter either through indirect measurement involving radioactively labeled tracer or direct measurement. The physical separation has traditionally been carried out by equilibrium dialysis (ED), a tedious technique for routine clinical practice [3]. Also problematic, tracer impurities can cause substantial errors when a radioactively labeled tracer is used to indirectly quantify the free fraction. A second approach has sought to calculate the free fraction from the amount of total testosterone, the binding capacity of SHBG and albumin, and the affinity constants of albumin and SHBG for testosterone. The calculated FT (cFT) usually correlates well with FT measured by the reference equilibrium dialysis method but is highly dependent on the accuracy of the total testosterone, SHBG, and albumin quantification [3,13,14]. The final approach, most widely used in clinical labs but fraught with inaccuracy, has utilized analog-based immunoassay to estimate the free fraction. Unfortunately, estimates by this approach reflect total testosterone levels more closely than they do the free fraction [6,15].
Recently, Van Uytfanghe et al. [16,17] reported a reference method for FT that separated the protein-bound and free fractions by ultrafiltration (UF) instead of by equilibrium dialysis. This was attractive insofar as ultrafiltration is inherently faster and less technically demanding than equilibrium dialysis. However, the solid phase purification and the GC-MS detection used by Van Uytfanghe et al. is cumbersome and time-consuming which makes it difficult for routine clinical testing. Previously we reported an LC-MS/MS procedure [4] for the measurement of serum total testosterone. Here we describe a new method using UF coupled with our testosterone LCMS/MS procedure for the measurement of FT offering further improvements in analytical sensitivity, convenience, and decreased sample requirement. A split sample comparison against analog immunoassay, cFT, and ED coupled LC-MS/MS is also presented.
Discussion
Reliable measurement of free steroid and thyroid hormones in the blood is inherently technically challenging and until recently has been difficult to achieve in routine clinical practice. However, the increased sensitivity and ease of use of LC-MS/MS technology has made this an attractive, if not superior, alternative to immunoassay, and when coupled to prior ultrafiltration (UF) or equilibrium dialysis (ED) to remove the protein-bound fraction, a powerful tool to measure free hormone and drug levels. Several methods using just this technology have recently been published for the measure of free thyroid hormone and unbound antiretroviral drugs [23-25]. Although both UF and ED are acceptable as reference procedures to separate protein-bound from circulating free ligands, UF is inherently better suited to the demands of the clinical lab because of its greater simplicity and speed, and accordingly, we chose to implement it in our proposed method. To the best of our knowledge, our proposed method is the first in the literature that directly measures FT by LC-MS/MS.
*The UF-LC-MS/MS method described here compares favorably to the UF coupled GC-MS reference method recently published by Van Uytfanghe et al. [16]. This method achieves equivalent LLOQ (16 pmol/L vs. 15–20 pmol/L) with less sample (0.5 mL vs. 1 mL serum), higher throughput (3.9 vs. 10.45 min LC elution), and no need for derivitization.
The Centrifree® ultrafiltration device is designed for the rapid separation of free ligands by the unhindered passage through the YM hydrophilic and nonabsorptive membrane (30 kDa cutoff) with a high degree of protein retention. This membrane was ideal in that it truly retained the protein present in the serum (less than 0.07 g/L, the limit of detection of our protein assay, in the ultrafiltrate) while not adsorbing FT (Table 1, Supplementary Data). Of note, the Centrifree® ultrafiltration device only worked when spun in a fixed angle rotor; swinging bucket rotors were unsuitable.
Our proposed method is attractive in that it agrees closely with ED and LC-MS/MS which must be considered the gold standard, but is much faster and easier to run, and better suited for the routine clinical laboratory, than the latter. The ultrafiltration procedure requires approximately 1 h to complete while equilibrium dialysis requires at least 16 h to complete the dialysis step. The dynamic range of the procedure described here easily accommodates FT levels expected in the adult male, and lipemia, icterus, and other steroids do not interfere, and imprecision is less than 6% at FT levels expected in the adult male. The limitation of the current LLOQ makes the method is not sufficient for all female samples at low levels. However, for female test application, it is the high end of the range most clinical interested, i.e. hyperandrogenic conditions such as polycystic ovary syndrome. The 16 pmol/L LLOQ needs to be reduced by at least half to make the method suitable to measure with the imprecision of less than 20% of the FT levels expected in many women. Reducing the functional sensitivity by 50% might be achieved by increasing the serum ultrafiltrate from 0.5 to 1 mL or by adopting atmospheric pressure photoionization, but these will require additional studies to substantiate. The overall fraction of total testosterone concentration measured by the present UF-LC/MS/MS method is 1.72%, which is very close to the reported before as 1.87% by UFGC/MS [17]. Further clinical studies are needed for the evaluation of clinical utilities of FT by this UF coupled LC-MS/MS method in subpopulations such as hypoandrogenic men and polycystic ovary syndrome women.
*In conclusion, we have developed a simple, rapid, highly selective, and sensitive method that accurately determines serum-free testosterone. The minimal sample preparation, reasonable throughput, and superior specificity and sensitivity may allow this method to serve both as a reference procedure and a routine method in the routine clinical laboratory practice.
Fig. 4. Method comparison of UF coupled LC-MS/MS with ED coupled LC-MS/MS (n= 26). The upper panel shows the correlation plot of UF vs. ED. The lower panel shows plots of the bias between free testosterone measurements by UF and ED as a percentage of ED values (Y-axis) against the ED measured FT concentration. UF, ultrafiltration coupled LC-MS/MS; ED, equilibrium dialysis coupled LC-MS/MS.
madman
Super Moderator
Free testosterone by LC/MSMS - Comparison of ultrafiltration and equilibrium dialysis (2010)
Introduction
Circulating testosterone (T) exists in the blood as free (FT) and protein-bound forms. Since FT is considered to be more reflective of the physiological actions of the hormone than total T, the measurement of FT is a better parameter to evaluate the androgen status and bioavailability.
Equilibrium dialysis (ED) is the gold standard method for the separation of FT. It is a time-dependent equilibrium process, that depends on various parameters, including temperature and pH. The objective of the project was to compare equilibrium dialysis and ultrafiltration as sample prep methods to separate FT and T. A practical LC/MSMS method needed to be validated, with an LLOQ of 1.0 pg/mL.
Conclusions
ED and UF methods were validated for the determination of FT in human plasma with acceptable accuracy and precision, with an LLOQ of 1 pg/mL. It was shown that FT can be reliably measured following filtration of buffered plasma, equilibrated at 37°C for 1hr, through passivated Amicon Ultra 4 (Millipore) filters at 37°C for 1hr. The UF process can be performed under controlled conditions, is simpler and faster than ED, and was, therefore, the method of choice for the bioanalysis of FT in a Phase I study of a new formulation of testosterone.
Introduction
Circulating testosterone (T) exists in the blood as free (FT) and protein-bound forms. Since FT is considered to be more reflective of the physiological actions of the hormone than total T, the measurement of FT is a better parameter to evaluate the androgen status and bioavailability.
Equilibrium dialysis (ED) is the gold standard method for the separation of FT. It is a time-dependent equilibrium process, that depends on various parameters, including temperature and pH. The objective of the project was to compare equilibrium dialysis and ultrafiltration as sample prep methods to separate FT and T. A practical LC/MSMS method needed to be validated, with an LLOQ of 1.0 pg/mL.
Conclusions
ED and UF methods were validated for the determination of FT in human plasma with acceptable accuracy and precision, with an LLOQ of 1 pg/mL. It was shown that FT can be reliably measured following filtration of buffered plasma, equilibrated at 37°C for 1hr, through passivated Amicon Ultra 4 (Millipore) filters at 37°C for 1hr. The UF process can be performed under controlled conditions, is simpler and faster than ED, and was, therefore, the method of choice for the bioanalysis of FT in a Phase I study of a new formulation of testosterone.
T
tareload
Guest
I know this isn't lost on you. Fig 3.
cfTV holding up very well against either UF-LC-MS/MS or ED-LC-MS/MS.
I haven't checked each data point but my observation is that cfTZ would be way higher than experimental data with LC-MS/MS.
Time will tell after harmonization work as you have stated.
T
tareload
Guest
Calculate Free Testosterone with TruT by FPT
[0026] Fig. 7 depicts the thermodynamic Parameters associated with testosterone's binding to SHBG derived from the fit of binding isotherms and ITC data to new model. While this parameter set is not unique, together they consistently describe the binding isotherms, depletion curves and ITC data...
www.excelmale.com
Required reading for everyone tonight. Homework, read the whole thread if you are interested. I'm proud to be almost as meticulous as @madman. I gotta find another hobby
.Scratch that, @madman is not human. Must be AI. Way to go **man**.
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Nelson Vergel
Founder, ExcelMale.com
Thanks, guys
T
tareload
Guest
If we are stating opinion, I have absolutely no issue estimating my free T using cfTV with TT (via RIA or better LC-MS/MS) and SHBG via RIA. cfTV may be flawed but based on the total data we have in our hands today, it appears less flawed than cfTZ (Tru-T). Of course that's because different clusters of literature data for ED-LC-MS/MS don't seem to agree well. Hence, some data agrees well with cfTV and other data agrees better with cfTZ.
Hence in head to head parity analysis, I'm still betting on cfTV vs cfTZ for superior predictive power. Come on harmonization data!
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