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Test Code TESTF Testosterone, Total, Bioavailable, and Free, Serum

Reporting Name

Testosterone, Total, Bio, Free, S

Useful For

Second- or third-order test for evaluating testosterone status (eg, when abnormalities of sex hormone-binding globulin are present)

Performing Laboratory

Mayo Clinic Laboratories in Rochester

Specimen Type

Serum Red

Advisory Information

The preferred test for diagnosis of mild abnormalities of testosterone homeostasis, particularly if abnormalities in sex hormone-binding globulin (SHBG) function or levels are present, is TTBS / Testosterone, Total and Bioavailable, Serum.

Necessary Information

Patient's age and sex are required.

Specimen Required

Container/Tube: Red top (serum gel/SST are not acceptable)

Specimen Volume: 3.5 mL

Specimen Minimum Volume

2 mL

Specimen Stability Information

Specimen Type Temperature Time Special Container
Serum Red Refrigerated (preferred) 14 days
  Frozen  60 days

Reference Values



0-5 months: 75-400 ng/dL

6 months-9 years: <7-20 ng/dL

10-11 years: <7-130 ng/dL

12-13 years: <7-800 ng/dL

14 years: <7-1,200 ng/dL

15-16 years: 100-1,200 ng/dL

17-18 years: 300-1,200 ng/dL

≥19 years: 240-950 ng/Dl

Tanner Stages*

I (prepubertal): <7-20

II: 8-66

III: 26-800

IV: 85-1,200

V (young adult): 300-950



0-5 months: 20-80 ng/dL

6 months-9 years: <7-20 ng/dL

10-11 years: <7-44 ng/dL

12-16 years: <7-75 ng/dL

17-18 years: 20-75 ng/dL

≥19 years: 8-60 ng/dL

Tanner Stages*

I (prepubertal): <7-20

II: <7-47

III: 17-75

IV: 20-75

V (young adult): 12-60


*Puberty onset (transition from Tanner stage I to Tanner stage II) occurs for boys at a median age of 11.5 (±2) years and for girls at a median age of 10.5 (±2) years. There is evidence that it may occur up to 1 year earlier in obese girls and in African American girls. For boys, there is no definite proven relationship between puberty onset and body weight or ethnic origin. Progression through Tanner stages is variable. Tanner stage V (young adult) should be reached by age 18.



Males (adult):

20-<25 years: 5.25-20.7 ng/dL

25-<30 years: 5.05-19.8 ng/dL

30-<35 years: 4.85-19.0 ng/dL

35-<40 years: 4.65-18.1 ng/dL

40-<45 years: 4.46-17.1 ng/dL

45-<50 years: 4.26-16.4 ng/dL

50-<55 years: 4.06-15.6 ng/dL

55-<60 years: 3.87-14.7 ng/dL

60-<65 years: 3.67-13.9 ng/dL

65-<70 years: 3.47-13.0 ng/dL

70-<75 years: 3.28-12.2 ng/dL

75-<80 years: 3.08-11.3 ng/dL

80-<85 years: 2.88-10.5 ng/dL

85-<90 years: 2.69-9.61 ng/dL

90-<95 years: 2.49-8.76 ng/dL

95-100+ years: 2.29-7.91 ng/dL


Males (children):

<1 year: Term infants


1-15 days: 0.20-3.10 ng/dL*

16 days-1 year: Values decrease gradually from newborn (0.20-3.10 ng/dL) to prepubertal levels

*Citation: J Clin Endocrinol Metab 1973;36(6):1132-1142


1-8 years: <0.04-0.11 ng/dL

9 years: <0.04-0.45 ng/dL

10 years: <0.04-1.26 ng/dL

11 years: <0.04-5.52 ng/dL

12 years: <0.04-9.28 ng/dL

13 years: <0.04-12.6 ng/dL

14 years: 0.48-15.3 ng/dL

15 years: 1.62-17.7 ng/dL

16 years: 2.93-19.5 ng/dL

17 years: 4.28-20.9 ng/dL

18 years: 5.40-21.8 ng/dL

19 years: 5.36-21.2 ng/dL


Females (adult):

20-<25 years: 0.06-1.08 ng/dL

25-<30 years: 0.06-1.06 ng/dL

30-<35 years: 0.06-1.03 ng/dL

35-<40 years: 0.06-1.00 ng/dL

40-<45 years: 0.06-0.98 ng/dL

45-<50 years: 0.06-0.95 ng/dL

50-<55 years: 0.06-0.92 ng/dL

55-<60 years: 0.06-0.90 ng/dL

60-<65 years: 0.06-0.87 ng/dL

65-<70 years: 0.06-0.84 ng/dL

70-<75 years: 0.06-0.82 ng/dL

75-<80 years: 0.06-0.79 ng/dL

80-<85 years: 0.06-0.76 ng/dL

85-<90 years: 0.06-0.73 ng/dL

90-<95 years: 0.06-0.71 ng/dL

95-100+ years: 0.06-0.68 ng/dL


Females (children):

<1 year: Term infants


1-15 days: 0.06-0.25 ng/dL*

16 days-1 year: Values decrease gradually from newborn (0.06-0.25 ng/dL) to prepubertal levels

*Citation: J Clin Endocrinol Metab 1973;36(6):1132-1142


1-4 years: <0.04 ng/dL

5 years: <0.04-0.07 ng/dL

6 years: <0.04-0.14 ng/dL

7 years: <0.04-0.23 ng/dL

8 years: <0.04-0.34 ng/dL

9 years: <0.04-0.46 ng/dL

10 years: <0.04-0.59 ng/dL

11 years: <0.04-0.72 ng/dL

12 years: <0.04-0.84 ng/dL

13 years: <0.04-0.96 ng/dL

14 years: <0.04-1.06 ng/dL

15-18 years: <0.04-1.09 ng/dL

19 years: 0.06-1.08 ng/dL




≤19 years: not established

20-29 years: 83-257 ng/dL

30-39 years: 72-235 ng/dL

40-49 years: 61-213 ng/dL

50-59 years: 50-190 ng/dL

60-69 years: 40-168 ng/dL

≥70 years: not established


Females (non-oophorectomized)

≤19 years: not established

20-50 years (on oral estrogen): 0.8-4.0 ng/dL

20-50 years (not on oral estrogen): 0.8-10 ng/dL

>50 Years: not established

Day(s) and Time(s) Performed

Monday through Saturday; 1 p.m.

Test Classification

This test was developed and its performance characteristics determined by Mayo Clinic in a manner consistent with CLIA requirements. This test has not been cleared or approved by the U.S. Food and Drug Administration.

CPT Code Information




LOINC Code Information

Test ID Test Order Name Order LOINC Value
TTFB Testosterone, Total, Bio, Free, S 58716-2


Result ID Test Result Name Result LOINC Value
82978 Testosterone, Bioavailable, S 2990-0
3631 Testosterone, Free, S 2991-8
8533 Testosterone, Total, S 2986-8

Clinical Reference

1. Manni A, Pardridge WM, Cefalu W, et al: Bioavailability of albumin-bound testosterone. J Clin Endocrinol Metab. 1985;61:705

2. New MI, Josso N: Disorders of gonadal differentiation and congenital adrenal hyperplasia. Endocrinol Metab Clin North Am. 1988;17:339-366

3. Dumesic DA: Hyperandrogenic anovulation: a new view of polycystic ovary syndrome. Postgrad Obstet Gynecol. 1995 June;15(13)

4. Morley JE, Perry HM 3rd: Androgen deficiency in aging men: role of testosterone replacement therapy. J Lab Clin Med. 2000;135:370-378

5. Goldman AL, Bhasin S, Wu FCW, et al: A reappraisal of testosterone's binding in circulation: physiological and clinical implications. Endocr Rev. 2017;38(4):302-324. doi: 10.1210/er.2017-00025

Method Description

Total Testosterone:

Deuterated stable isotope (d3-testosterone) is added to a 0.2-mL serum sample as an internal standard. Protein is precipitated from the mixture by the addition of acetonitrile. The testosterone and internal standard are extracted from the resulting supernatant by an online extraction utilizing high-throughput liquid chromatography (HTLC). This is followed by conventional liquid chromatography and analysis on a tandem mass spectrometer equipped with a heated nebulizer ion source.(Wang C, Catlin DH, Demers LM, et al: Measurement of total testosterone in adult men: comparison of current laboratory methods versus liquid chromatography-tandem mass spectrometry. J Clin Endocrinol Metab. 2004;89:534-543; Taieb J, Mathian B, Millot F, et al: Testosterone measured by 10 immunoassays and by isotope-dilution gas chromatography-mass spectrometry in sera from 116 men, women, and children. Clin Chem. 2003;49:1381-1395)


Free Testosterone:

This method is based on equilibrium dialysis in which free-labeled testosterone is allowed to pass through a semipermeable membrane, whereas testosterone bound to the sex hormone-binding globulin (SHBG) remains inside the dialysis tubing. After dialysis, radioactivity is measured both inside and outside the tubing; the free testosterone results are expressed as a percentage of total testosterone. The resulting percentage is multiplied by the total testosterone concentration obtained by liquid chromatography-tandem mass spectrometry (LC-MS/MS), and an absolute free testosterone value is obtained.(Vermeulen A, Stoica T, Verdonck L: The apparent free testosterone concentration, an index of androgenicity. J Clin Endocrinol Metab. 1971;33:759-767; Bammann BL, Coulam C, Jiang NS: Total and free testosterone during pregnancy. Am J Obstet Gynecol. 1980;137:293-298)


Bioavailable Testosterone:

The method is based on the differential precipitation of SHBG by ammonium sulfate following equilibration of the serum specimen and tracer amounts of tritium-labeled testosterone. The results are expressed as the percent of testosterone free or albumin bound (not precipitated with SHBG) compared to an albumin standard. The product of this percentage and the total testosterone measurement is the total bioavailable testosterone.(Wheeler MJ: The determination of bioavailable testosterone. Ann Clin Biochem. 1995;32:345-357)

Analytic Time

3 days

Reject Due To

Gross hemolysis Reject
Gross lipemia Reject
Gross icterus Reject

Method Name

FRTST: Equilibrium Dialysis

TTST: Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS)

BATS: Differential Precipitation

Profile Information

Test ID Reporting Name Available Separately Always Performed
TTST Testosterone, Total, S Yes Yes
FRTST Testosterone, Free, S No Yes
BATS Testosterone, Bioavailable, S No Yes

Secondary ID