Obtain total testosterone and SHBG to calculate free testosterone where (a) a symptomatic Service member's total testosterone falls within the reference range, or (b) an SHBG-altering condition is present. Record the method used. The reference interval is 66 to 309 pg/mL across adult ages.[4]
REFERENCES (DHA testosterone guideline 2026)
4. Jasuja R, Pencina KM, Spencer DJ, et al. Reference intervals for free testosterone in adult men measured using a standardized equilibrium dialysis procedure. Andrology. 2023. [Verify against primary text — see drafting note.]
I think there is a methodological problem in the DHA testosterone guideline regarding the most critical blood marker free testosterone.
They need to fix this.
DHA says obtain TT and SHBG to calculate FT and then gives 66–309 pg/mL (6.6-30.9 ng/dL) as the FT reference interval.
Although they state record the testing method used that 66–309 pg/mL (6.6-30.9 ng/dL) range comes from Jasuja et al. (2023) but Jasuja did not establish that range for cFT.
Jasuja measured FT using standardized equilibrium dialysis followed by CDC HoST-certified LC-MS/MS.
The Jasuja paper specifically states that these reference ranges should not be applied to other assays/laboratories without appropriate cross-calibration.
Look over the limitations in the paper that I posted back in 2022 before it was. released.
I have pointed this out numerous times on the forum
As I stated in that thread the reference ranges given in that paper only apply to that specific assay (ED device/procedure).
The reference ranges in that paper are not set in stone!
As of now there is no standardized ED procedure/assay or harmonized reference range for free testosterone!
Also need to understand that when it comes to the confusion of the currently used/relied upon ED methods between laboratories for testing free testosterone everyone needs to keep in mind that not all ED devices/procedures are the same.
This is key!
* Limitations of using free testosterone by equilibrium dialysis and calculated free testosterone concentrations in practice are the lack of assay standardization, an accuracy-based quality control program, and a harmonized reference range. Until these limitations are addressed, free testosterone by equilibrium dialysis and calculated free testosterone should use reference ranges established by individual laboratories or their specific assay method
* The lack of standardization of the equilibrium dialysis method among laboratories has been a barrier to the generation of a harmonized reference range for free testosterone levels; until such rigorously-derived harmonized reference ranges become available, the clinicians currently must rely on reference ranges provided by a laboratory or those published from the analyses of large epidemiologic studies
Take home point!
* Assays that are standardized are designed to provide accurate results, traceable to “true” value-assigned certified reference materials and gold-standard reference methods. Results obtained using standardized methods can be compared across assays, institutions, populations, and past and future test results, thereby improving diagnosis, treatment, and outcomes of patients.
The paper!
Again you heard it here first only at Nelson's
ExcelMale!
@Nelson Vergel
Was going to drop this bomb a few days ago but you let down the curtains early LOL!
Reference Intervals for Free Testosterone in Adult Men Measured Using a Standardized Equilibrium Dialysis Procedure (2022)
Ravi Jasuja, Ph.D.; Karol M. Pencina, Ph.D.; Daniel J. Spencer, BS; Liming Peng, MS; Fabiola Privat, BS; Waljit Dhillo; MD, Ph.D.; Channa Jayasena, MD, Ph.D.; Frances Hayes, MD; Bu B. Yeap, MB, BS, Ph.D.; Alvin M. Matsumoto...
An expert panel of the Endocrine Society9 reviewed the various methods for determining FT4, 5, 9, 21-32 and concluded that each method has some inherent limitations but that the equilibrium dialysis method is the reference standard against, which all other methods should be compared.33 However, substantial heterogeneity in the procedures used by various laboratories for performing the equilibrium dialysis assay has contributed to variability in the reported FT values. For instance, the equilibrium dialysis can be performed by adding 3H-labeled testosterone to serum sample and FT derived from the total testosterone concentration and the dialyzed fraction of 3H-testosterone; the procedures that use 3H-testosterone are susceptible to error due to tracer impurities and the potential for tritium exchange with water.9 Furthermore, the determination of FT concentration by equilibrium dialysis is affected greatly by the assay conditions, including the buffer composition, the incubation time, and the temperature. Historically, some laboratories have included a carrier protein such as gelatin or bovine serum albumin in the dialysis buffer, which affects the partitioning of testosterone among binding proteins and the measured FT concentration. The equilibrium dialysis procedure can also be performed by direct measurement of testosterone on both sides of the dialysis chamber.24, 25 The accuracy and precision of the total testosterone assay also affects the precision and accuracy of the measured FT concentration. Most commercial laboratories do not report the buffer composition and other dialysis conditions, which renders it difficult to evaluate their methods; the procedures for equilibrium dialysis have varied even in published reports from academic research laboratories.2, 24, 32-37 Because of the wide variation in the equilibrium dialysis procedures, the reference ranges are not generalizable across laboratories.
In an effort to generate reference intervals for FT concentrations in men that can be used across laboratories, we report here a detailed description of a nonproprietary, standardized equilibrium dialysis procedure that can be replicated in any qualified laboratory using commercially available reagents. Instead of using a tritium-labeled tracer, we used liquid chromatography tandem mass spectrometry (LC-MS/MS) to directly measure testosterone concentrations on both sides of the dialysis chamber. We employed standardized dialysis conditions at 37°C for a duration of time shown to be sufficient for reaching equilibrium. Using this validated equilibrium dialysis method, we evaluated the distribution of FT concentrations in a prospectively collected sample of community-living healthy nonobese men, 19 years or older. The blood samples were collected in the morning before 10 a.m. after an overnight fast. Total testosterone levels in the serum as well in the dialysate were measured using a validated LC-MS/MS assay that is certified by the Hormone Standardization Program for Testosterone (HoST) of the US Centers for Disease Control and Prevention.42 We report the distribution of FT concentrations in healthy young men, 19–39 years, as well by age groups, generated using this standardized equilibrium dialysis procedure.
This study also has some limitations. These reference ranges were derived from single morning samples, which discount the pulsatile and diurnal secretory rhythms. Previous analyses show that early morning testosterone levels, obtained in a manner similar to that used by physicians in practice, are associated cross-sectionally with symptoms and clinical outcomes.38-40, 48 We report reference ranges in men <40 years of age but also provide the distribution of FT levels by age groups. This approach of generating the reference range in healthy young men is analogous to the use of T-scores for bone mineral density. Although the sample included men of various races and ethnicities, the number of nonwhites was not large enough to offer sufficient power to detect meaningful differences in FT levels among racial or ethnic subgroups. The data on geographic and racial differences in total testosterone levels are inconsistent,50, 51 and no study has examined racial and ethnic differences in FT levels using equilibrium dialysis in different geographic regions of the world. Additional investigations of multiethnic cohorts to evaluate the generalizability of the proposed reference limits to men of other races and ethnicities in different regions of the world are important. Although the sample size was within the IFCC guidelines for analytes with normal distribution, there were relatively small numbers of men within each decade of age, and a larger sample size would provide more robust estimates of the reference ranges by age decades.
Our normative ranges are similar to those reported by pioneers of this field using the legacy method,2 but the percent FT in our reference sample differs from that reported by another laboratory 10, 52; this difference in percent FT from that reported by another research laboratory (<2.8%) could be due to differences in buffer composition or in other assay conditions that are not apparent in the published methods.10, 52 The normative ranges of most commercial laboratories have changed substantially in recent years suggesting changes in their procedures over time53, 54; because procedures used by the commercial laboratories and the details of how reference ranges were derived are not published, an evaluation of these procedures was not feasible.
These reference ranges, generated in a reference sample of healthy men, should not be applied to other assays in other laboratories without appropriate cross-calibration of assays. Differences in study populations, time of sample collection, and testosterone assays can contribute to the differences in reference ranges. The adoption of a standardized procedure for measuring FT and cross-calibration of the testosterone assays against an accuracy-based benchmark such as the CDC's HoST program will facilitate the application of these reference ranges across laboratories.
The data here define reference intervals from a population of healthy nonobese men using a standardized equilibrium dialysis procedure coupled to a HoST-certified LC-MS/MS assay and represent an important first step. Further studies are needed to determine how well these reference limits can be applied to the diagnosis of androgen deficiency in clinical populations and in people of different races and ethnicities in different geographic regions. The association of low FT defined using these criteria with incident outcomes in epidemiologic studies should be studied. Importantly, randomized trials are needed to determine whether testosterone therapy improves outcomes in men, who have FT below these reference limits.
Dr. Shalender Bhasin
* Equilibrium Dialysis is the reference method which I recommend highly, the problem is that the Equilibrium Dialysis method has not been harmonized across laboratories and the reference ranges vary across laboratories so there's a great need for standardizing the procedure and generating population-based harmonized reference ranges and we have done this and published a standardized procedure for Equilibrium Dialysis and published the reference ranges in this paper that you can look up
Nelson's house u heard it here first!
Dr. Shalender Bhasin!
* over the past 4 decades he has become a global leader in the biology of androgens, sarcopenia and aging related functional decline, his research has shaped clinical guidelines, its informed public health policy particularly around testosterone therapy, anabolic interventions and then the development of function promoting therapies
Pay attention to 25:40-34:36 of the presentation where he explains the importance of measuring free testosterone using a standardized Equilibrium Dialysis method!
Explains the...
www.dha.mil
