Testosterone, Free, Direct

Additional Information:

UFHPL Epic order code: LAB5318

The concentration of free testosterone is very low, typically less than two percent of the total testosterone concentration. In most men and women, more than 50 percent of total circulating testosterone is bound to sex hormone-binding globulin, SHBG, and most of the rest is bound to albumin.1 Routinely available assay methods used to measure total testosterone are not sensitive enough to accurately quantitate the free testosterone fraction directly. Free testosterone is estimated in this test by a direct, analogue radioimmunoassay method. This assay uses a labeled testosterone analogue that has a low binding affinity for both SHBG and albumin but is bound by antitestosterone antibody used in the assay. Since the analogue is unbound in the plasma, it competes with free testosterone for binding sites on an antitestosterone antibody that is immobilized on the surface of the polypropylene tube.2

Several authors have found that the analogue method has good correlation with equilibrium dialysis,1 – 4 but have found that the analogue results were only about one-fourth as high. Another group found that the analogue method produced results directly comparable to equilibrium dialysis without multiplication by a factor.5 More recently, Winters and coworkers have found the analogue method to correlates better with total testosterone levels than with bioavailable testosterone determined by the ammonium sulfate precipitation method.6 They suggested that the analogue-free testosterone results might be misleading in men with low SHBG concentration.6 Ooi suggested that the problems observed by Winters6 might, in large part, be resolved by simply using a more appropriate population-based reference interval.7 Vermeulin and coworkers found that the analogue-free testosterone method correlated well with free testosterone by equilibrium dialysis, but did not agree well with a free testosterone calculated from total testosterone and SHBG.3


  1. Wheeler MJ. The determination of bio-available testosterone. Ann Clin Biochem. 1995 Jul; 32(Pt 4):345-357 (review). PubMed 7486793
  2. Cheng RW, Reed MJ, James VH. Plasma free testosterone: Equilibrium dialysis vs direct radioimmunoassay. Clin Chem. 1986 Jul; 32(7):1411. PubMed 3719956
  3. Vermeulen A, Verdonck L, Kaufman JM. A critical evaluation of simple methods for the estimation of free testosterone in serum. J Clin Endocrinol Metab. 1999 Oct; 84:3666-3672. PubMed 10523012
  4. Wilke TJ, Utley DJ. Total testosterone, free-androgen index, calculated free testosterone, and free testosterone by analog RIA compared in hirsute women and in otherwise-normal women with altered binding of sex-hormone-binding globulin. Clin Chem. 1987 Aug; 33(8):1372-1375. PubMed 3608155
  5. Déchaud H, Lejeune H, Garoscio-Cholet M, Mallein R, Pugeat M. Radioimmunoassay of testosterone not bound to sex-steroid-binding protein in plasma. Clin Chem. 1989 Aug; 35(8):1609-1614. PubMed 2758628
  6. Winters SJ, Kelley DE, Goodpaster B. The analog free testosterone assay: Are the results in men clinically useful? Clin Chem. 1998 Oct; 44:2178-2182. Erratum: 1999 Mar; 45(3):444. PubMed 9761253
  7. Ooi DS, Donnelly JG. More on the analog free-testosterone assay. Clin Chem. 1999 May; 45(5):714-716. PubMed 10223785

CPT Code(s):


Specimen Requirements:

Important: Indicate the patient’s age and gender when ordering this test.

Type: Serum

Container/Tube: Red-top tube or gel-barrier tube

  • If a red-top tube is used, transfer the separated serum to a plastic transport tube.

Sample Volume: 0.5 mL

Minimum Volume: 0.3 mL (Repeat testing is not possible with this specimen volume.)

Storage: Refrigerate specimens after collection.

Stability (collection to time of analysis/testing):

  • Ambient: 14 days
  • Refrigerated: 14 days
  • Frozen: 14 days
  • Freeze/Thaw cycles: Stable (x3)
Rejection Criteria:

  • Gross lipemia (may yield erroneously high free testosterone results)
  • Plasma specimen


This test is used to evaluate hirsutism and masculinization in women, as well as testicular function in clinical states where the testosterone binding proteins may be altered (obesity, cirrhosis, thyroid disorders).


A pediatric reference interval has not been established for this analyte. Gross lipemia may yield erroneously high free testosterone results.


Direct analog enzyme immunoassay (EIA)

Reference Values:


Range (pg/mL)


0 − 19 years old

Not established

20 − 29 years old

9.3 − 26.5

30 − 39 years old

8.7 − 25.1

40 − 49 years old

6.8 − 21.5

50 − 59 years old

7.2 − 24.0

> 59 years old

6.6 − 18.1


0 − 19 years old

Not established

> 19 years old

0.0 − 4.2