What lab workup is recommended for a female patient presenting with hirsutism?

Laboratory Workup for Female Hirsutism

Measure total testosterone (TT) and free testosterone (FT) using liquid chromatography-tandem mass spectrometry (LC-MS/MS) as your first-line tests, drawn in the morning on cycle days 3-6, as these have the highest diagnostic accuracy with sensitivities of 74% and 89% respectively. 1, 2

First-Line Androgen Testing

• Total testosterone (TT) should be measured using LC-MS/MS methodology, which has a pooled sensitivity of 74% and specificity of 86% for detecting biochemical hyperandrogenism 1, 2

• Free testosterone (FT) should be assessed by equilibrium dialysis, ammonium sulfate precipitation, or calculated using the free androgen index (FAI = total testosterone/SHBG ratio) when direct measurement is unavailable, with FT showing superior sensitivity of 89% and specificity of 83% 1, 2

• Avoid direct immunoassay methods for FT due to poor accuracy at low serum concentrations 1

• Blood samples must be drawn in the morning (around 8 am) due to diurnal variation in testosterone levels 1, 2

• Timing should be on cycle days 3-6 for women with regular cycles 2

Second-Line Androgen Testing (If TT/FT Not Elevated)

• Androstenedione (A4) should be measured if TT or FT are not elevated, with sensitivity of 75% and specificity of 71% 1, 2

• DHEAS (dehydroepiandrosterone sulfate) should be measured to identify adrenal androgen production, with age-specific cutoffs: >3800 ng/mL for ages 20-29, >2700 ng/mL for ages 30-39 1, 2

• Very high DHEAS levels (>600 μg/dL) indicate an adrenal source and raise concern for adrenocortical carcinoma, warranting immediate imaging 1

• Note that A4 and DHEAS have poorer specificity than testosterone measurements 1

Essential Screening Tests to Rule Out Other Causes

• Thyroid-stimulating hormone (TSH) must be checked to exclude thyroid disease, which can present with similar symptoms 3, 1, 2

• Prolactin levels should be measured to exclude hyperprolactinemia (abnormal if >20 μg/L), as this can cause menstrual irregularity and hirsutism 3, 1, 2

• LH and FSH should be measured between cycle days 3-6, with an LH/FSH ratio >2 suggesting polycystic ovary syndrome (PCOS) 2

• Mid-luteal progesterone should be assessed to evaluate ovulation, with levels <6 nmol/L indicating anovulation 2

Metabolic Screening (Critical for PCOS Evaluation)

• Fasting glucose followed by 2-hour oral glucose tolerance test with a 75-gram glucose load should be performed to screen for type 2 diabetes and insulin resistance, as women with PCOS have demonstrated increased risk 3, 2

• Fasting lipid panel including total cholesterol, LDL cholesterol, HDL cholesterol, and triglyceride measurements should be obtained to assess cardiovascular risk 3, 2

• Calculate body mass index and waist-hip ratio as part of metabolic assessment 3

Red Flags Requiring Urgent Evaluation

• Very high testosterone levels (>2.5 nmol/L or >150 ng/dL) or rapidly developing symptoms over a few months should prompt immediate investigation for androgen-secreting tumors of the ovary or adrenal gland 2, 4

• Look for signs of virilization including clitoromegaly, deepening voice, male-pattern baldness, or increased muscle mass, which suggest tumor 1, 4

• Markedly elevated DHEAS warrants evaluation for adrenal pathology with imaging 1, 2

Additional Testing for Specific Clinical Scenarios

• 17-hydroxyprogesterone should be measured if non-classic congenital adrenal hyperplasia is suspected, particularly in certain ethnic populations where prevalence is 1-10% 5

• Pelvic ultrasound (transvaginal preferred) should be performed if hormonal tests suggest PCOS or to rule out ovarian pathology, looking for >10 peripheral cysts of 2-8 mm diameter with thickened ovarian stroma 2

• Consider 24-hour urinary free cortisol or overnight dexamethasone suppression test if clinical features suggest Cushing's syndrome (buffalo hump, moon facies, hypertension, abdominal striae, easy bruising) 3

Critical Technical Considerations

• LC-MS/MS methodology is strongly preferred over immunoassays for all androgen measurements due to superior specificity and accuracy, particularly at the low concentrations seen in women 1, 2

• SHBG fluctuations can affect TT and FAI results, influenced by age, weight, and medications like oral contraceptives 1

• The 2023 International PCOS Guidelines emphasize that LC-MS/MS has superior accuracy compared to direct immunoassays across all androgen measurements 2

Common Pitfalls to Avoid

• Do not rely on testosterone measurements alone if clinical suspicion is high—proceed to second-line testing with A4 and DHEAS 1, 2

• Do not use direct immunoassay methods for free testosterone, as they are inaccurate at low serum concentrations typical in women 1

• Do not forget to time blood draws appropriately—morning collection on cycle days 3-6 for androgens, mid-luteal phase for progesterone 2

• Do not overlook metabolic screening, as PCOS (the most common cause, accounting for 80-90% of hirsutism cases) carries significant cardiovascular and diabetes risk 3, 5