How reliable are salivary hormone assays compared with serum measurements for evaluating endocrine function?

Reliability of Saliva Tests for Hormones

Saliva testing is highly reliable for measuring unconjugated steroid hormones (cortisol, testosterone, estradiol, progesterone, DHEA) because these hormones enter saliva by passive diffusion through salivary gland cells, making their concentration independent of saliva flow rate and directly reflective of the biologically active (free) hormone fraction in blood. 1, 2, 3

When Saliva Testing is Reliable and Recommended

Unconjugated Steroid Hormones

• Cortisol: Salivary cortisol is the gold standard for detecting Cushing's syndrome, with late-night salivary cortisol showing 92-100% sensitivity and 93-100% specificity 4
• The Endocrine Society recommends collecting at least 2-3 late-night salivary cortisol samples on different nights to account for cyclic hypercortisolism and variability 4
• Salivary cortisol reflects the free (bioactive) fraction in plasma because unconjugated steroids diffuse through salivary gland cells independent of saliva flow rate 2, 3

Other Unconjugated Steroids

• Testosterone, estradiol, progesterone, and DHEA measurements in saliva accurately reflect free hormone levels in blood 1, 5
• These hormones are useful for monitoring hormone replacement therapy, assessing climacteric changes in perimenopausal women, evaluating androgen deficiency, and tracking age-related hormone decline 1, 5

Practical Advantages Over Serum

• Collection is noninvasive, stress-free, painless, and can be performed at home without medical staff 1, 6, 5
• Hormones remain stable in saliva for three weeks at room temperature, allowing inexpensive postal transport 1
• Multiple daily samples can be easily collected to establish diurnal rhythms and perform dynamic testing (e.g., dexamethasone suppression test) 5
• Saliva collection timing is more controllable than blood draws, critical for hormones with circadian variation 1

When Saliva Testing is NOT Reliable

Conjugated Steroids

• Conjugated steroids (e.g., estrogen sulfate, testosterone glucuronide) enter saliva via ultrafiltration through tight junctions between acinar cells, making their concentration highly dependent on saliva flow rate 2, 3
• Salivary concentrations of conjugated steroids do not reliably reflect plasma levels and lack clinical utility 2, 3

Protein Hormones and Thyroid Hormones

• Protein hormones (e.g., human chorionic gonadotropin) and thyroid hormones (thyroxine) enter saliva via ultrafiltration or contamination from plasma/gingival fluid 2
• Their salivary concentrations do not reflect plasma levels in any clinically useful way 2, 3
• The molecular weight, polarity, and protein-binding characteristics of these hormones make them unsuitable for salivary measurement 3

Critical Care Settings

• The Society of Critical Care Medicine and European Society of Intensive Care Medicine specifically recommend AGAINST using salivary cortisol instead of serum cortisol for diagnosing critical illness-related corticosteroid insufficiency (CIRCI) 4

Critical Factors Affecting Reliability

Pre-analytical Variables

• Current salivary biomarker studies often fail to control for confounding pre-analytical variables during sampling, leading to inaccurate readings and conflicting findings 7
• Factors affecting results include gender, age, timing and site of sampling, saliva volume, and flow rate 4
• Low-affinity plastic collection tubes (high-grade polypropylene) are essential to prevent hormone binding to tube walls 7

Collection Standardization

• Assisted collection by trained staff ensures adherence to pre-collection instructions, controls timing and duration, measures flow rates, and detects gross contamination (blood, food, lipstick) 7
• Patients should refrain from eating, drinking, smoking, and oral hygiene for at least 30 minutes to 2 hours before collection 7
• Samples should be placed on ice immediately and centrifuged before storage at -80°C 7

Analytical Methods

• Liquid chromatography-tandem mass spectrometry (LC-MS/MS) provides superior sensitivity and specificity compared to immunoassays 4
• Enzyme immunoassays are commonly used but may not be routinely available in most centers 4
• Lack of standardized analytical tools, defined reference intervals, and round-robin trials remains a major limitation 6

Common Pitfalls to Avoid

• Never use a single salivary cortisol sample for Cushing's syndrome diagnosis: Multiple samples are required due to variability and cyclic hypercortisolism 4
• Never use salivary hormone testing in critically ill patients: Serum cortisol remains the standard for evaluating adrenal insufficiency in this context 4
• Never interpret results without considering collection conditions: Strict adherence to pre-collection instructions is essential to avoid false results 4
• Never assume saliva testing works for all hormones: Only unconjugated steroids and certain amines (melatonin) provide clinically useful data 2, 6, 3
• Avoid contamination: Visual inspection should exclude samples with visible blood contamination 7

Current Limitations

• Insufficient saliva volume is common in many patients, particularly older adults and those with dry mouth 7
• Patient compliance with home collection protocols can be problematic without strict standardization 6
• Many insurance plans cover salivary hormone testing, but availability varies 1
• The field lacks widespread clinical acceptance, requiring continued development of standardized protocols and validation studies 6