What are the guidelines for evaluating total and free testosterone levels in patients?

Evaluating Total and Free Testosterone Levels

Diagnose testosterone deficiency by obtaining two separate morning (8-10 AM) total testosterone measurements showing levels <300 ng/dL on both occasions, combined with clinical symptoms of hypogonadism. 1, 2, 3

Diagnostic Algorithm for Total Testosterone

Initial Testing Requirements

• Measure total testosterone between 8-10 AM on two separate mornings using the same laboratory and methodology to confirm diagnosis 1, 2
• Both measurements must be <300 ng/dL to meet diagnostic criteria 1, 2, 3
• Fasting samples are preferred for accurate measurement 4, 5
• The morning timing is critical due to diurnal variation—never measure testosterone at random times of day, as afternoon or evening measurements will be physiologically lower and lead to false-positive diagnoses 1

Laboratory Methodology Standards

• Use liquid chromatography-tandem mass spectrometry (LC-MS/MS) in a laboratory certified by the CDC's Hormone Standardization (HoST) Program for the most accurate total testosterone measurement 4
• This method provides superior accuracy compared to immunoassays, particularly at lower testosterone concentrations 4, 6
• Harmonized reference ranges can only be reliably applied to laboratories certified by the HoST Program 4

When to Measure Free Testosterone

Specific Clinical Scenarios Requiring Free Testosterone

• Measure free testosterone by equilibrium dialysis in patients with obesity, as obesity-related low total testosterone may be due solely to low sex hormone-binding globulin (SHBG) with normal free testosterone 1, 2
• Measure free testosterone in older patients where SHBG abnormalities commonly occur 2
• Measure free testosterone when total testosterone levels are borderline (slightly above or slightly below 300 ng/dL) 5
• Measure free testosterone when alterations in binding protein concentrations are suspected 5

Free Testosterone Measurement Standards

• Equilibrium dialysis is the gold standard method for measuring free testosterone 1, 4, 7
• If free testosterone is also low on at least 2 separate assessments, consider testosterone replacement after completing the hypogonadism workup 1
• Calculated free testosterone using algorithms is an alternative when equilibrium dialysis is unavailable, though less accurate 7

Essential Adjunctive Laboratory Testing

Determining Etiology

• Measure serum LH and FSH in all patients with confirmed low testosterone to distinguish primary (testicular) from secondary (hypothalamic-pituitary) hypogonadism 1, 2
• Measure serum prolactin if testosterone is low AND LH is low or low-normal to screen for hyperprolactinemia 1, 2
• Measure SHBG when free testosterone is being evaluated, especially in obese or older patients 1, 2

Additional Workup for Secondary Hypogonadism

• Consider iron saturation, comprehensive pituitary function testing, and MRI of the sella turcica if secondary hypogonadism is confirmed 1

Clinical Symptoms That Must Be Present

Required Symptom Assessment

• Diagnosis requires BOTH low testosterone levels AND clinical symptoms—never diagnose based solely on laboratory values 1, 3
• Assess for decreased libido and erectile dysfunction (key symptoms) 1, 3
• Evaluate reduced energy, endurance, physical performance, fatigue, and diminished work performance 1, 3
• Screen for depression, reduced motivation, poor concentration, and impaired memory 1
• Examine for loss of muscle mass, body hair changes, hot flashes, gynecomastia, or infertility 1, 3

Physical Examination Components

• Evaluate body habitus, BMI, and waist circumference 1, 3
• Assess virilization status (body hair patterns in androgen-dependent areas) 1, 3
• Examine testicular size and consistency 3

High-Risk Populations Requiring Screening

Test Even Without Typical Symptoms

• Measure testosterone in all patients with unexplained anemia, even without symptoms 1, 3
• Screen patients with bone density loss or osteoporosis 1, 3
• Test patients with diabetes mellitus 1, 3
• Evaluate patients with HIV/AIDS 1

Critical Pitfalls to Avoid

Common Diagnostic Errors

• Using a single testosterone measurement instead of two separate measurements leads to inaccurate diagnosis due to natural fluctuations in testosterone levels 1, 3
• Measuring testosterone at non-standard times (not 8-10 AM) results in false-positive diagnoses 1
• Diagnosing testosterone deficiency based solely on laboratory values without considering clinical symptoms leads to overdiagnosis or underdiagnosis 3
• Using immunoassays in non-certified laboratories produces variable and unreliable results—reference ranges vary significantly among laboratories (lower limits ranging from 160-300 ng/dL) 8
• Failing to measure free testosterone in obese patients may miss cases where total testosterone is falsely low due to low SHBG but free testosterone is normal 1