- Clinical Relevance and Reliability
- Diurnal Rhythm and Cortisol Awakening Response (CAR)
- Additional Advantages of Saliva Testing Dim Light Melatonin Onset (DLMO)
- Female Hormones and Hormone Replacement Therapy
Hormone measurements are used for a variety of clinical interpretations including the screening for Cushing’s disease, capturing dysfunction of the hypothalamic-pituitary-adrenal (HPA) axis, and mapping male and female sex hormones. Hormones can be tested using saliva, blood (serum), and urine (with fingernail and hair analysis coming on strong for cortisol). A patient’s health and well-being depends on the accuracy and reliability of the laboratory data and health professionals need to be wary of using misleading testing methods. The following information explains some of the key differences between the use of saliva and serum specimens for the laboratory assessment of hormones.
Clinical Relevance and Reliability
A number of significant advantages that saliva assessment has over serum have resulted in tremendous growth in medical research using saliva as well as salivary assays. Saliva has the primary advantage of ease of collection. Scheduled saliva collections are simple, non-invasive, result in increased compliance especially when multiple collections are required in a day. When looking to test the primary stress hormone, cortisol, saliva collection provides improved accuracy because it causes much less stress and anxiety during collection as compared to a venipuncture. Because venipuncture collection can significantly increase the production of cortisol, collection of blood is not representative of a normal day’s stress load and is clinically irrelevant. Conversely, cortisol concentrations in saliva reflect real-life physiological conditions and responses.
Furthermore, around 80-90% of circulating cortisol is bound to cortisol binding globulin and 10% is bound to albumin (1). The remaining fraction is unbound, free hormone and is considered to be biologically active and available for tissue uptake (2). This low molecular weight, free hormone is the fraction that is present in saliva. Saliva testing detects unbound (free) cortisol and provides more clinically useful information on how much hormone is available to enter the tissues (3).
For blood hormone tests, the blood sample contains total hormone which includes transport proteins and the non-biologically active, bound form of the hormone in addition to free hormone. Mathematical algorithms and filtering techniques exist to derive serum free hormone fractions but the reliability is questionable (4). Increasingly sensitive and specific methods of salivary hormone analysis combined with precise timing of saliva collection will contribute to the accuracy and relevance of the patient’s hormone data.
Diurnal Rhythm and Cortisol Awakening Response (CAR)
Numerous hormones exhibit marked circadian variations; therefore timing of specimen collection greatly affects the results. The time-specific capture of cortisol concentrations circulating in the body is essential to the valid interpretation of HPA axis reactivity. Although diurnal hormone pattern measurements are possible with blood, having a venipuncture performed by a health professional for multiple collections, especially bedtime samples or waking samples, is impractical. Saliva collection is easily completed at specific time points and is the ideal medium for evaluating the distinct circadian pattern of cortisol secretion.
This cortisol diurnal pattern is comprised of two separate phases. First, the Cortisol Awakening Response (CAR) is the rise in cortisol levels observed at 30 minutes post-awakening followed by an expected decline one hour after awakening. The behavior of cortisol is an important physiological response to anticipation of the day ahead. Saliva provides the ability to measure the waking, 30 minutes post-waking, and 60 minute post-waking cortisol results and capture this dynamic rise and fall (5). Measuring the CAR response is not feasible with blood because this specific phenomenon is largely influenced by environmental factors and timing of collection. Clinically relevant results are only derived from a saliva collection because it can be performed at home during a normal daily awakening routine which best reflects the patient’s regular physiological state (6).
The second phase of cortisol diurnal pattern is the steady decrease in cortisol production throughout the day. While blood can be collected during various time points throughout the day, this would be extremely inconvenient and further increase cortisol when combined with the already stressful venipuncture collection. As discussed, the results should reflect the overall clinical picture and mapping time-specific values is necessary for visualizing cortisol’s unique pattern and identifying disease states and conditions listed in the figure below (7).
|Axis Dysfunction Increased activity of the HPA axis||Decreased activity of the HPA axis|
Additional Advantages of Saliva Testing Dim Light Melatonin Onset (DLMO)
Dim Light Melatonin Onset (DLMO) is a key technique for the diagnosis and treatment of circadian rhythm sleep disorders. For this test, patients collect samples in 30 minute intervals before bedtime while in an environment with dimmed light (to minimize suppression of melatonin production). The accuracy of this test depends on the environment and timing of collections. Saliva collection is a simplified and proven method for measurement of melatonin for DLMO patterns (8,9). Serum collections often don’t provide controlled surroundings and the standard bedtime routine of the patient should be followed to produce valid results. The testing of melatonin for this circadian rhythm assessment is a specific clinical application that is best suited for use of a saliva collection.
Female Hormones and Hormone Replacement Therapy
Moreover, measurement of female hormones to check for imbalances and replacement hormone therapy is a well established facet of saliva testing. For measuring estrogens and progesterone in women, saliva testing is an invaluable tool for measuring the dynamics of hormone patterns throughout the menstrual cycle. Though possible with serum, logistics involved with collecting multiple samples makes saliva a better collection method. Additionally, when following up on topical hormone regimens saliva has several distinct benefits to serum.
Serum and urine assays often significantly underestimate the effect of topical hormones such as skin creams and patches. This can result in harmful effects to the patient if overdosing is the result of misinterpretation of the laboratory data (10). A paper by Stanczyk et al. describes how progesterone transdermal creams are absorbed through the skin into the blood stream and the hormones bind to red blood cells due to their afinity to lipids (fat) over aqueous (water) interaction. Prior to analysis, blood cells (and hormones) are spun out of the serum sample and removed prior to testing. As a result, the serum results report little to no increase in hormone levels after many weeks of application (11). There is a serious lack of scientific documentation for long term serum assessment of topical hormone use as it relates to tissue specific levels. Saliva hormones more accurately reflect tissue uptake and response to replacement hormones delivered through these topical methods (10,12). The response in saliva is significant and prompt which indicates that the monitoring of topical or transdermal hormone replacement therapy is most affectively done with salivary assays.
Summary Comparison of Common Sample Type
|Criteria||Saliva||Wet Urine||Blood||Dried Urine|
|Peer reviewed studies to support testing of hormones||Yes||Yes||Yes||No|
|Assay validation criteria met||Yes||Yes||Yes||Maybe*|
|Clinical applications supported by research||Yes||Yes||Yes||No|
|Stability of analytes demonstrated||Yes||Yes||Yes||No|
|Ability to test free, bioactive hormones||Yes||Yes||Yes||Yes|
|Measures total hormones load over 24 h||No||Yes||No||No|
|Measures real-time hormones levels||Yes||No||Yes||No|
*While the other sample types are well-established globaly with common validations, dried urine’s validations remain unavailable given their LTD status.
**Urinating on paper may be convenient, however the quality of the paper and risk of contamination while the sample is drying are of concern, as explained in the text
Peer reviewed literature to support hormone testing in different sample types
|Criteria||Saliva||Wet Urine||Blood||Dried Urine|
|Free diurnal rhythm of Cortisol||> 1000 citations||2 citations by same group||Not feasible||0|
|Measurement of Cortisol Awakening Response||> 450 citations||Not feasible||Not feasible||0|
|Melatonin for DLMO patterns||> 75 citations||Not feasible||~ 39 citations||0|
|Relevant clinical applications of free cortisol||> 900 citations||> 800 citations||> 5600 citations||0|
|Relevant clinical applications of hormone metabolites||Not feasible||80 citations||> 100 citations||0|
Health providers must make careful decisions when selecting the most clinically applicable testing method. Interpretation of laboratory data must be put in context of the overall clinical picture to ensure the highest quality of care for patients. It is important to look at the advantages and disadvantages of each technique and specimen type.
While serum testing has specific applications in clinical diagnostics and medical research; several significant limitations should be acknowledged. Complications with venipuncture affecting true baseline hormone levels, ability to detect the active (free) hormone, limitations on collection timing, and potentially harmful overdosing that can result from the inability of serum to capture certain hormone replacement treatments are a few key concerns. Salivary hormone testing avoids many of these obstacles and is an instrumental tool in the assessment of numerous conditions and diseases.
1 Galbois A, et al. Assessment of adrenal function in cirrhotic patients: salivary cortisol should be preferred. J Hepatol. 2010; 52: 839-45.
2 Gozansky WS, Lynn JS, Laudenslager ML, Kohrt, WM. Salivary cortisol determined by enzyme immunoassay is preferable to serum total cortisol. Clin Endocrinol. 2005; 63(3):336-41.
3 Kaufman E, Lamster I. The Diagnostic Applications of Saliva – A Review. Crit Rev Oral Biol Med. 2002; 13(2):197-212.
4. Ly LP, Handelsman DJ, Empirical estimation of free testosterone from testosterone and sex hormone-binding globulin immunoassays. Eur J Endocrinol. 2005; 152:471-478.
5 Clow A, et al., The cortisol awakening response: More than a measure of HPA axis function. Neurosci Biobehav. Rev. 2010.
6 Gozansky WS, et al. Salivary cortisol determined by enzyme immunoassay is preferable to serum total cortisol for assessment of dynamic hypothalamic-pituitary-adrenal axis activity. Clin Endocrinol (Oxf). 2005; 63: 336-41.
7 Chrousos GP. Stress and disorders of the stress system. Nat Rev Endocrinol. 2009; 5:374-381.
8 Burgess HJ, et al. Home dim light melatonin onsets with measures of compliance in delayed sleep phase disorder. J Sleep Res. 2016; 25(3): 314-317.
9 Burgess HJ, et al. Home Circadian Phase Assessments with Measures of Compliance Yield Accurate Dim Light Melatonin Onsets. J Sleep Res. 2015; 38(6): 889-897.
10 Du JY, et al. Percutaneous progesterone delivery via cream or gel application in postmenopausal women: a randomized cross-over study of progesterone levels in serum, whole blood, saliva, and capillary blood. Menopause. 2013; 20(11):1169-75.
11 Stanczyk FZ, Paulson RJ, Roy S. Percutaneous administration of progesterone: blood levels and endometrial protection. Menopause. 2005; 12(2):232-7.
12 O’Leary P, Feddema P, Chan K, Taranto M, Smith M, Evans S. Salivary, but not serum or urinary levels of progesterone are elevated after topical application of progesterone cream to pre and postmenopausal women. Clin Endocrinol. 2000; 53:615-620.