How are laboratory tests performed to assess hormone levels?

How Laboratory Tests Are Performed to Assess Hormone Levels

Hormone testing requires blood samples collected at specific times under fasting conditions, with the choice of assay method and laboratory quality being critical for accurate results.

Sample Collection Fundamentals

Blood samples should be obtained in the morning (8-9 AM) under fasting conditions to account for circadian hormone variations 1. For women with regular menstrual cycles, samples must be collected during the first 7 days of the cycle (follicular phase) to ensure accurate interpretation 1, 2, 1. This timing is crucial because testosterone and other hormone concentrations vary significantly throughout the menstrual cycle.

Specific Collection Protocols

• Morning collection (8 AM): Essential for cortisol, ACTH, testosterone, and 17-hydroxyprogesterone 1, 3
• Fasting state: Required for most hormone panels to avoid interference from metabolic factors 1
• Multiple samples: Some protocols require three blood samples at 20-minute intervals, which are then pooled to provide a more representative baseline 1

Laboratory Assay Methods

Mass Spectrometry (Gold Standard)

Liquid chromatography-mass spectrometry (LC-MS/MS) is the reference method for measuring testosterone, DHEAS, and androstenedione 4, 5, 6. This technique separates and molecularly fragments hormones, eliminating interference from antibodies and providing superior specificity compared to immunoassays 4. LC-MS/MS is particularly critical for:

• Testosterone measurements in women (where levels are at the low end of detection)
• Distinguishing between cortisol and prednisolone
• Measuring multiple steroids simultaneously 4

Immunoassays

While widely available, most commercial immunoassays lack accuracy for low hormone concentrations typical in women and fail to detect mild androgen excess 1, 5, 7. The same testosterone value measured by different laboratories can be reported as normal to abnormally high depending on reference ranges 1.

Critical caveat: Many commercial laboratories provide inaccurate normal ranges for testosterone, with research publications reporting much lower normal-range levels than commercial labs 1.

Essential Hormone Panel Components

Initial Screening Tests

For suspected hormonal disorders, the following should be measured 1, 2, 3:

• Total and free testosterone
• DHEAS (dehydroepiandrosterone sulfate)
• LH and FSH (luteinizing and follicle-stimulating hormones)
• Prolactin
• TSH and free T4 (thyroid function)
• 17-hydroxyprogesterone (if congenital adrenal hyperplasia suspected)
• Estradiol (in women, as clinically indicated)
• Morning cortisol and ACTH

Free Testosterone Measurement

Free testosterone should be calculated rather than measured directly 5, 6. Direct immunoassays for free testosterone are notoriously inaccurate 5. The preferred approaches are:

1. Calculation from total testosterone and SHBG (sex hormone-binding globulin) - shows good concordance with reference methods 5
2. Equilibrium dialysis - reference method but cumbersome 5
3. Ammonium sulfate precipitation - determines bioavailable testosterone 5

Laboratory Quality Requirements

Selection Criteria

Choose laboratories with documented proficiency in hormone determinations 1. An acceptable clinical laboratory must provide:

• Precise results with intra-assay variability <20% 4
• Reliable reference ranges validated for the specific population
• Quality control ensuring stable values over time 1
• Preferably LC-MS/MS capability for steroid hormones 4, 6

Common Pitfalls to Avoid

Interassay variability is substantial for estradiol, testosterone, GH, free T4, and gonadotropins 4. Therefore:

• Use the same laboratory and assay for serial measurements 4
• Be aware that prednisolone cross-reacts in many cortisol assays 4
• Results inconsistent with clinical presentation should be rechecked using LC-MS/MS 6
• Biotin supplementation can interfere with multiple hormonal assays 8

Dynamic Testing Protocols

Dexamethasone Suppression Test

For determining the source of androgen excess 1:

Day 1: Obtain baseline pooled samples, then administer 0.5 mg dexamethasone at lunch, dinner, and bedtime

Day 2: Continue 0.5 mg dexamethasone four times daily

Day 3: Final dose at breakfast, then obtain post-suppression samples within 2 hours

Interpretation:

• Testosterone suppression >40% and DHEAS >60% = adrenal source
• Testosterone fails to suppress but DHEAS responds = ovarian source
• Testosterone suppression <40% = combined ovarian and adrenal contribution 1

ACTH Stimulation Test

For suspected 21-hydroxylase deficiency (indicated in Ashkenazi Jews, Hispanics, or with elevated screening 17-OHP) 1:

• Measure baseline morning cortisol and 17-OHP
• Administer 0.25 mg cosyntropin intravenously
• Repeat sampling at 60 minutes 1

Sample Handling Considerations

Sample integrity is critical 4. Clinicians must consider:

• Storage conditions (freeze-thaw cycles affect results)
• Time from collection to processing
• Interference from replacement hormonal therapies
• Cross-reactivity in steroid immunoassays 4

Monitoring and Follow-up

Repeat hormone evaluation at 1-2 years for pituitary lesions, or earlier if new symptoms develop 3. For patients on long-term therapy, serial measurements using the same assay are essential because long-term treatment for hormonal disorders is frequently necessary 1.