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Saliva, an alternative to blood-based testing

For many years, saliva has been used as a biological fluid for the detection of different human biomarkers, such as electrolytes, hormones, drugs and antibodies.

There are clear advantages for choosing saliva over blood collection. Saliva sampling is:

  • Non-invasive
  • Painless and very convenient, especially for patients
  • Collecting saliva samples is possible any time – day or night
  • A better alternative when blood collection is difficult or inadvisable (for children and the elderly)

How do fractions differ in blood and saliva?


Figure 1: Steroids in blood and saliva.


Steroids in blood are either conjugated or unconjugated. The conjugated forms – where steroids have, sulfates or glucuronides attached– are excreted by the kidneys. The unconjugated forms of the steroids are mostly bound to various binding proteins, such as CBG (corticosteroid-binding globulin) or SHBG (sex hormone-binding globulin). This bound fraction represents the vast majority of unconjugated steroids – 95-99 % of the total hormone fraction – in plasma or serum.

The bound fraction is biologically inactive; it is sometimes described as a biological inactive reservoir of the steroid. Only a small fraction, about 1-5 % of steroids in serum, is free and as such biologically active. Any measurement of steroid hormones in serum or plasma will therefore mainly reflect the inactive hormones.

However, steroid hormone assessment in saliva allows a specific determination of the biologically active or ‘free’ fraction of the target hormone. Only this free fraction can pass through the space between the acinar cells of the salivary gland membrane. This means that the free fraction of steroids in saliva correlates to the fraction in free blood, and therefore represents the biological active fraction.

Who is using saliva samples so far?

  • Traditional medical care labs
  • Academic labs
  • Complementary medicine

There is ever-increasing demand for health measurement and monitoring. With the ability to evaluate a pooled saliva sample from multiple collections, it is possible to get a reliable assessment of hormone concentrations, even if distinct diurnal fluctuations occur.

See the stories of real labs working with Saliva diagnostics

How do you measure steroids in saliva?

Assays for salivary steroid determination have to be highly sensitive, as the concentrations of biologically active free fractions are significantly lower than the total analyte concentrations in serum. Immunoassays provide these characteristics in order to measure the low concentrations of hormones to be measured in saliva.

Which products does Tecan offer?

Tecan offers an extensive range of immunoassays for measurement of saliva samples, including steroid assays for cortisol, progesterone, testosterone, estradiol, estriol, estrone, DHEA, DHEA-S, 17-OH-progesterone but also further parameters like Melatonin and alpha Amylase.

Tecan provides you with highly sensitive luminescence immunoassays and ELISAs, which have been specially developed and validated for saliva.

Reasons to choose Tecan assays

We are careful to develop, manufacture and market products with:

  • Very high analytical sensitivity, with limits of quantification from 2.1-50 pg/ml (e.g. estradiol = 2.1 pg/ml))
  • Luminescence and ELISA formats
  • Low saliva sample volumes of 20-100 μl
  • No extraction step necessary
  • Kit reagents are ready to use (except wash buffer)
  • 2 controls per kit – normal and elevated
  • Easy test procedure
  • Bulk packages of 10x 96 tests available


Our goals are to provide:

  • A fast, reliable and professional service
  • Capable scientific and technical support
  • Worldwide assistance through a tightly-knit international distribution network

What do you need to know about saliva sampling?

1. The importance of the saliva flow rate

The flow rate of saliva may influence the concentration of salivary hormones. This is the case if the hormone has a high molecular weight, or if the molecule is polar or ionic, making it difficult to accurately determine hormone levels. On the other hand, the concentration of small and non-polar molecules has not been shown to be dependent on the flow rate. This is a very important aspect in the utility of salivary testing. A classic example is the measurement of DHEA and DHEA-S in saliva. DHEA is a non-polar and small molecule, therefore the free fraction will pass from the vascular system into the salivary system. The salivary DHEA concentration specifically reflects the free fraction found in blood and, consequently, the hormone activity. DHEA-S is highly polar, due to its ionic composition, and cannot pass through the membrane passively. As a result, most of the DHEA-S present in saliva originates either from wounds in the mouth or possible active transporters discussed in reasearch, which would skew the results.

In blood, DHEA-S and DHEA are loosely bound to albumin, while in saliva, only the free active hormone is present. The concentration of DHEA in saliva is around 1:30 of that in serum, while the concentration of DHEA-S in saliva is even lower (1:500 to 1:1000), and is dependent on the saliva flow rate.

2. Sampling device

The preanalytical phase and choice of sampling device is crucial to obtaining reliable results, and should not interfere with the salivary analyte under investigation. Unlike serum or plasma, saliva contains very low analyte levels, which makes interactions between the surface of the sampling device and the analyte much more likely. Due to these low concentrations, any retention by the material should be avoided.

Any saliva sampling device needs to be validated by measuring the recovery of the analyte in saliva. Tecan provides an ultrapure polypropylene sampling device – the SaliCap – to minimize surface interactions and keep differences to a minimum (e.g. <5 %), even for progesterone. If alternative devices are used for saliva sample collection and transport, the individual laboratory will need to validate its chosen collection device with regard to analyte absorption rates.

3. Influence of blood contamination

It is crucial to avoid any blood contamination, as the total steroid hormone concentrations in blood can be up to 100 times higher than in saliva. Every patient should be aware that they must discard any sample that shows even a slight tint of red. If blood contamination occurred, the sampling device should be rinsed twice with tap water, followed by a 10 minute wait before sampling again.

Saliva sample collection for hormone testing is as easy as one-two-three

This vivid two-minute video will walk you through the simple steps and rules to follow and show you the best products used in labs for accurate and reliable results from saliva testing.

Dos and don’ts of saliva testing

  • Choice of collection device
    Tecan’s ultra-pure polypropylene SaliCap ensures very good recovery of all the steroids in saliva, as well as melatonin and alpha-amylase.
  • Timing
    A clear sampling strategy will lead to reproducible results. The concentration of many analytes in saliva is highly dynamic e.g. cortisol
  • Food and beverage consumption
    It is recommended that individuals fast for a minimum of 30 minutes before the collection of saliva samples. It is best to rinse the mouth with water before saliva sampling, especially after eating.
  • Saliva collection
    In order to ensure convenient and consistent collection of samples, the passive drool method is recommended for collection. Simply allow saliva to flow to the base of the mouth before drooling into the tube.
  • Volumes for analysis
    The more analytes you plan to measure from one sample, the larger the volume that needs to be collected. Volumes of around 20-100 µl, depending on the analyte, are required for testing.
  • Blood contamination
    Samples with the slightest red tint should not be used, as the total steroid concentration in blood can be up to 100 times higher than in saliva.
  • Measurement using analytical assays
    Make sure a sufficiently sensitive analytical assay is used. Tecan saliva assays are highly sensitive, and have been evaluated for this application. This is essential for detecting the very low concentration of hormones in saliva. Sensitivity, specificity, reproducibility, reference values and usefulness of the testing method for the intended medical purpose should all be checked.
  • Pretreatment of saliva
    It is recommended that saliva samples are subjected to a freeze-thaw cycle and centrifuged, to break down mucins and provide better measurement precision.
  • Drugs and medication
    Some drugs or medications might influence hormone concentrations and, therefore, testing results. This should be kept in mind when evaluating results.

Fields of saliva application

Saliva testing is already an integral part in some fields, such as endocrinology, psychology and stress research. The advantages of saliva as a matrix are well established. This includes generating precise daily profiles by collecting many samples in a short time span.

What is happening in the body under stress?


Figure 2: Stress and the Hypothalamus pituitary gland.


Adrenal stress is a general term for a great number of symptoms that indicate how the body is coping with stress.

Stress is any kind of physical, mental or emotional pressure. The body reacts by starting to produce ACTH (adrenocorticotropic hormone) in the pituitary gland, which then activates the production of other stress markers, such as cortisol or DHEA in the adrenal glands. Their role is to manage the flow of stress-related hormones and regulate various bodily functions, including sleep and mood, and fat and protein metabolism. The higher the continual level of adrenal stress, the lower the hormone concentration.

Cortisol helps to evaluate the stress response and generally has a catabolic effect, whereas DHEA represents the counterpart as an anti-stress hormone, and determines stress adaptation. Adrenal imbalance may be a factor in many related clinical conditions, including fibromyalgia, hypothyroidism, chronic fatigue syndrome and depression. The adrenal stress index includes the determination of levels of both hormones – cortisol and DHEA – which may reveal adrenal dysfunction.

How do you measure stress?

Psychological research is related to the science of behavior and mental processes, and includes areas such as biological, cognitive and social psychology. In many cases, the patient’s psychobiological status is established by means of validated self-assessment questionnaires. However, it is also highly important to know and understand the patient’s biological status. Many of the hormones in the body maintain a roughly 24-hour rhythm. The hypothalamus-pituitary-adrenal (HPA) axis is one of the most important circadian rhythms in the body, with a great influence on health, vitality and the immune system.

Abnormal hormone rhythms have been implicated in many diseases, and more research is devoted to understanding them and finding appropriate medications. The levels of cortisol, DHEA, testosterone and alpha-amylase exhibit relationships with chronic stress, post-traumatic stress disorder, behavior, cognitive function and health. Leading research psychologists are therefore very interested in studying, measuring and understanding the levels of these hormones in relation to circadian patterns.


Figure 3: Cortisol responses to moderate psychosocial stress.


Many studies test individuals’ endocrinological response to various kinds of stress, and cortisol concentrations are found to be higher when the individual is subjected to a stressful situation. The Trier Social Stress Test (TSST) is a standardized test that is widely applied as a method of choice in research, to differentiate the varying nature of individual reactions to stress.

It is worth mentioning that other factors – for example, gender and the menstrual cycle – can influence the stress response and cortisol. The application of drugs, such as oral contraceptives, also seem to diminish the cortisol response to stress.

What happens when there is a hormone imbalance?

Hormones are chemical messengers in very low concentrations, and affect organs and tissues. A proper hormone balance is key to good health, and the measurement of the hormone levels is an indicator of patient health. Any excess or deficiency in hormone concentrations might lead to serious medical conditions, and should be treated.

The free, biologically active proportion of hormones can be detected in saliva, and it is recommended that the levels of individual hormone are measured to enable optimal treatment, including hormone replacement therapy. Most often, these treatments involve administering a drug to artificially restore hormone balance.

Which hormones dictate your sleep?

Occupational diseases – such as sleep disorders in shift workers –can lead to psychological and physical stress. In this type of disorder, it is helpful to evaluate individual circadian rhythms, combining measurements of melatonin and cortisol concentrations, which are produced according to circadian rhythms with contrary patterns.


Figure 4: Melatonin and cortisol diurnal pattern.


Melatonin is the key biomolecule for promoting nighttime sleep. The melatonin levels increases soon after the onset of darkness, peak in the middle of the night between 2 and 4 a.m. and gradually fall during the second half of the night. The melatonin levels during the day are about 10 times lower. Cortisol levels, on the other hand, are highly dynamic during the day, and typically peak in the early morning. However, the timing of this cortisol peak depends on the individual’s waking time, and not on actual time or daylight. Both cortisol and melatonin are regarded as stress markers. Recent studies have shown that melatonin can influence cortisol levels. These results are useful in the testing of sleep disorders.

What happens when cortisol is too high?

Cushing’s syndrome is a result of excessive cortisol secretion, and is characterized by the failure of cortisol release to reach the circadian nadir late at night. This endocrine pathology is difficult to diagnose, because the observed phenotype can be due to a multitude of reasons. The endocrine system consists of several glands – in various locations within the body – that secrete hormones directly into the blood. Many hormones have multiple different functions and modes of action; one hormone may have several effects on a number of different target organs, and one target organ can be affected by more than one hormone. Many endocrine disorders also involve more than one hormone, making them complex to understand. Patients often, therefore, suffer from obesity, hypertension, depression, alcoholism and others.


Figure 5: Diagnosis of Cushing’s syndrome.
Source: Raff H, Pituitary 2012; 15:64-70


Late-night salivary cortisol measurement is a well-accepted and recommended as a test for Cushing’s syndrome. This method shows high specificity and sensitivity, and can be easily handled using ELISAs or luminescence assays. Tecan’s Cortisol Salivary Luminescence Assay only requires 20 μl of saliva, making it easily practicable, even for collecting samples from children or the elderly.

Why measure testosterone?

The measurement of free testosterone is an ideal testing tool for androgen levels in both men and women, but accurate measurement of testosterone remains a challenge, as determination in serum by conventional immunoassays is inaccurate. Directly measuring testosterone in saliva is a better alternative to blood measurement, as only the free, biologically active fraction is measured.

The Tecan Testosterone Saliva Luminescence Immunoassay and Testosterone Saliva ELISA show high analytical and functional sensitivity, and allow quantification of very low concentrations in saliva. Studies show that free salivary testosterone levels in morning samples correlate well with calculated free testosterone in blood, both in healthy men and in patients with androgen deficiency. Our assays are well suited to differentiating between healthy adults and individuals with androgen dysfunctions e.g. hypogonadism or hypergonadism.

Our quality assessment scheme (QAS) – an extra service for you

Tecan’s QAS was established in 2005 as a standard for steroids in saliva, as there was none previously available. The QAS is conducted twice a year, with each round of sample distribution three samples are included and send out to the customers. The multiparameter samples contain the following in various concentrations:

  • Cortisol
  • Testosterone
  • Estradiol
  • Progesterone
  • DHEA

Each participant can send their results to Tecan, either directly by email or via a form available on our homepage. Tecan analyzes the results, and provides each participant with a QAS results sheet that is sent back to them by email. Confidentiality is assured throughout the process by the use of an ID number known only to Tecan and the individual participant.

For further details, or to participate, click here.

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