Saliva Diagnostics

 

Introduction

For many years saliva has been used as a biological fluid for the detection of different biomarkers such as electrolytes, hormones, drugs and antibodies in human and veterinary medicine. Sample collection is non-invasive, painless and very convenient especially for the patients. Collecting saliva samples is possible any time, day or night because of the overall convenience of collection. Saliva collection can be accomplished under circumstances where blood collection is difficult or inadvisable.

Saliva is the specimen of choice in a variety of the subsets of traditional medical care as well as emerging areas of 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 concentration even if distinct diurnal fluctuations occur.

Steroid hormone assessment from saliva also allows the specific determination of biologically active or “free” fraction of target hormone.

Assays for salivary steroid determination must be extremely sensitive as the concentrations of biologically active free fractions are significantly lower than the concentrations of total analyte in serum.

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

Advantages of working with IBL International

We are careful to develop, manufacture and market products with

  • very high analytical sensitivity
  • luminescence and ELISA format
  • low sample volumes (20-100 μl saliva)
  • no sample pretreatment (no extraction)
  • kit reagents that are ready to use
  • 2 kit controls (normal and elevated)
  • easy test procedure
  • bulk packages available (10 x 96 tests)

Our goals are to provide:

  • fast, reliable and professional service
  • capable scientific and technical support
  • worldwide assistance though a tightly-knit international distribution network
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Free, bound and conjugated steroids

 
free steroid
 

Steroids are found in blood in conjugated and in unconjugated form. The conjugated form is excreted by the kidneys, and the steroids may be linked with sulfates, glucuronides etc. The unconjugated form of the steroids is mostly bound to various binding proteins like CBG, SHBG etc. This bound fraction represents the vast majority of the unconjugated steroid fraction (between 95% and 99% of the total hormone) present in plasma or serum.

The bound fraction is biologically inactive; it is sometimes described as a biological inactive reservoir of the steroid hormone. The only biologically active hormone is the small fraction of free steroid and represents between 1% and 5% of the total steroid concentration in serum. Any measurement of steroid hormones in serum or plasma will therefore mainly reflect the inactive hormone: only a small (and indeterminate) percentage of the resulting measurement will be the biologically active hormone.

Steroid hormone assessment from saliva allows specific determination of the biologically active or “free” fraction of the target hormone. Only the free fraction passes through the space between the acinar cells of the salivary glands membrane. The salivary steroid concentration specifically reflects the free fraction in the blood, and consequently the hormone activity.

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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 even ionic. Under these circumstances the concentration of the hormone in saliva will be influenced by the flow rate. On the other hand, the concentration of small and non-polar molecules in saliva has not been shown to be dependent on the flow rate. This is a very important aspect in a discussion of the utility of salivary diagnostics. If the concentration of the target analyte is dependent on the flow rate it is difficult to accurately determine hormone levels. A classical 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 to the salivary system. The salivary DHEA concentration reflects specifically the free fraction found in blood and consequently the hormone activity. DHEA-S is highly polar due to the ionic composition and thus cannot pass the membrane passively. As a result, most of the DHEA-S present in saliva originates from micro bleeding.

In blood, DHEA-S and DHEA are loosely bounded to albumin. In saliva, only the free active hormone is found. While the concentration of DHEA in saliva is around 1/30 of those in sera, the concentration of DHEA-S in saliva is even lower (1/500 to 1/1000) and is dependent on the saliva flow rate.

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Preanalytics

1. Sampling device

The preanalytic is of utmost importance for obtaining reliable results. It is important to use a sampling device which does not interfere with the salivary analyte under investigation. Unlike serum or plasma, saliva contains very low analyte levels, so interactions between the surface of the sampling device and the analyte are much more likely.

Any saliva sampling device needs to be validated by measuring the recovery of the analyte in saliva. IBL International has accordingly introduced a special ultra-pure polypropylene sampling device, the SaliCap, featuring absorption of less than 5% even for low concentrations of Progesterone in saliva. If IBL International’s SaliCap is not used for saliva sample collection and transport, then the individual laboratory will need to validate its own collection device in some other way, paying critical attention to analyte absorption rates.

2. Influence of blood contamination

It is of utmost importance to avoid any significant blood contamination, as the total steroid hormone concentration in blood can be up to 100 times higher than in saliva. Blood contamination is saliva is a critical issue. Every patient should be aware that they must discard any sample which shows even a slightly red color. In this case the patient should rinse the sampling device 2 times with tap water, wait for another 10 minutes and sample again.

3. Hints for saliva testing

Tips for a successful collection and analysis of saliva samples:

  • Use the right collection device according to the analyte of interest IBL International offers an ultra-pure polypropylene collection device named Salicap, which ensure a very good recovery of all the steroids in saliva but also of Melatonin and alpha-Amylase a.o.
  • Think about a meaningful timing of the sampling procedure The concentration of many analytes in saliva is highly dynamic. A real sampling strategy will result in reproducible results.
  • Collect Saliva fasting or earliest 30 minutes after eating or drinking Rinsing of the oral cavity with water is recommended prior saliva collection (especially after eating).
  • Ensure a convenient and consistent collection of samples We recommend using the passive drool method for collection. Just allow the saliva to flow to the base of the mouth before drooling into the tube.
  • Volume of saliva needed to complete all of analyses The more analytes you plan to measure from one sample, the larger collection volume you will need for your laboratory work
  • Do not use even the slightest red saliva samples The total steroid hormone concentration in blood can be up to 100 times higher than in saliva.
  • Send the samples to the laboratory in the right way Saliva samples can be easily dispatched per mail to the laboratory without cooling elements when steroids should be measured.
  • Make sure about the high quality of the analytical assay

The IBL International assays are high sensitive and have been very well evaluated for saliva. This is of utmost importance for detecting the very low concentration of hormones in saliva. Sensitivity, specificity, reproducibility, reference values and usefulness of diagnostic method for the intended medical purpose should be checked

  • Pretreatment of saliva We recommend freezing, thawing and centrifuging saliva samples before measurement, in order to break the mucins and reach better precision.
  • Medication of the patient Some medicine might have an influence on the hormone concentration and as such on the testing results.
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Products

IBL International Products

IBL International offers an extensive range of immunoassays for measurement of saliva samples. Assays for salivary hormone determination must be extremely sensitive as the concentrations of biologically active free fractions are significantly lower than the concentrations of total analytes in serum. We provide you with highly sensitive luminescence immunoassays and ELISAs which have been specially developed and validated for saliva.

Luminescence Immunoassays vs. ELISA

Chemiluminescence assays were considered to be approximately ten times more sensitive than enzyme immunoassays for some years. For this reason IBL International developed first saliva assays using glow luminescence in microtiter format. Chemiluminescence is a specific term describing light emission as a release of energy created during chemical reaction. This technology allows exact measurements below the picogram range. Instrumentation for measuring glow luminescence is relatively inexpensive (compared to flash luminescence) as no injectors are needed. There are many manufacturers of luminometers on the market. Please contact us if you should need any recommendation. As an example, the multimode reader Infinite PRO200 from Tecan® is perfectly appropriated for the reading of our Luminescence assays.

The ELISA technique remains one of the diagnostic methods of choice for the majority of laboratories. Although the luminescence method offers high sensitivity, few laboratories own a luminometer.

In order to offer our customers reliable salivary ELISA kits, IBL International developed assays by use of an enzymatic signal amplification system. This new technology make possible the achievement a very good sensitivity also with an ELISA test. The composition and performance of the ELISA and luminescence immunoassays are very similar. The main difference is the substrate reaction which is responsible for the colorimetric or luminescence signal generation.

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Other products

Of course not only steroids can be measured in saliva. IBL International also offers assays for determining the concentration of melatonin, alpha amylase activity, IgA and other parameters such as cytokines and also neopterin. Please contact us with any particular requirements: we may be developing the very product you are looking for!

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Applications for salivary diagnostics

Saliva is the specimen of choice in a variety of traditional medical care situations as well as in the emerging areas of 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 concentration even in cases of marked diurnal fluctuation.

Advantages of working with saliva samples

  • Non-invasive
  • Painless
  • Available at any time, day or night, because of the overall convenience of collection
  • Multiple specimens possible
  • Saliva can be collected in circumstances where blood collection is difficult or inadvisable.
  • Saliva can be collected anywhere, without medical assistance
  • Saliva appears to be the right option for diagnostic in your routine or research applications in many different fields.
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Adrenal Stress

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

 
Stress assessment Cortisol DHEA
 

Stress is any kind of physical, mental or emotional pressure. It begins by producing ACTH (adrenocorticotropic hormone) in the pituitary gland, and activates the production of other stress markers such as cortisol or DHEA in the adrenal glands located just above the kidneys. Their role is to manage the flow of stress-related hormones and regulate various of body functions such as control of the immune system, sleep and mood levels, fat and protein metabolism etc. 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 the counterpart DHEA is an anti-stress hormone and determines the stress adaptation. Adrenal imbalance may be a factor in many related clinical conditions, including fibromyalgia, hypothyroidism, chronic fatigue syndrome and depression, for example. The adrenal stress index includes the determination of levels of both hormones (cortisol and DHEA) that may reveal adrenal dysfunction.

Relationship to immune status

Adrenal function is also closely coupled with immune system control. Acute or chronic stress can lead to inflammatory disorders. For this reason there is currently much research aimed at understanding the relation between the HPA axis and the immune system. IBL International offers enzyme immunoassays for the measurement of IgA and cytokines such as IL-1, IL-6, TNF-α, TGF-ß, MMP9, … in various biological samples. Please contact us for any details you may require and we shall be happy to give you the support you look for in your research applications.

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Hormone Balance

Hormones are chemical messengers in a very low concentration which affect organs and tissues. A proper hormone balance is the key to good health: any excess or deficiency in hormone concentration should be treated, or serious medical conditions may appear.
The measurement of saliva offers many advantages. Only the free, biologically active proportion of the hormone is detected, which makes the reading highly significant and strongly representative of the patient’s health.
Determining optimal hormone levels for each individual patient may lead to an indication of hormone replacement therapy. Most often estrogen, progesterone and testosterone are involved in such treatments, in which a set of drugs is administered to artificially boost hormone levels.
Hormone replacement treatment may readily be adjusted by measuring salivary hormones.
All IBL International assays comprise a broad and physiologically relevant measuring range.

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Occupational medicine

Occupational medicine is the branch of clinical medicine most actively involved in occupational health. Occupational health aims at the promotion and maintenance of the greatest physical, mental and social well-being of workers in all occupations.

Burnout

Recent stress research indicates that more and more managers are concerned about workrelated stress; this makes occupational stress as important for companies as workplace accidents. The World Health Organization (WHO) has described stress as “one of the major threats of the 21st century”. For this reason more and more institutes and physicians are involved in identifying and measuring biological parameters which can reveal individuals’ psychobiological and physiological status.

Cortisol is a well-known marker of hypothalamuspituitary-adrenal (HPA) axis activity. This steroid can easily be measured in saliva, enabling patients to collect samples in comfort at any time of day, even in stressful situations.
Alpha amylase is a sympathetic system marker and can also be used in research projects. IBL International also offer assays for measuring catecholamines (adrenaline, noradrenaline, dopamine, etc.) in plasma and urine samples. Please visit our homepage www.ibl-international.com for more details of assays of other biological fluids besides saliva, or contact us directly for more information.

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Sleep Disorders

Occupational diseases include sleep disorders, occurring mostly in shift workers and leading to psychological and physical stress. In this kind of disorder it is helpful to evaluate individual circadian rhythms, combining measurements of melatonin and cortisol concentration: both hormones are produced according to a circadian rhythm, with contrary circadian patterns.

 
sleep disorders
 

Melatonin is the key substance promoting night-time sleep; it is extremely low during the day and rises rapidly around 11-12 p.m. (up to ten or twenty times daytime levels). Normal cortisol concentration in human saliva during the day is highly dynamic. Cortisol typically peaks in the early morning: the timing of this cortisol peak does not depend on clock time nor is it influenced by daylight: it is set by the individual’s time of waking. Both hormones are regarded as stress markers. Recent studies have shown that melatonin can influence cortisol levels. These results are useful in the diagnosis of sleep disorders.

Inflammation

The study of inflammation markers is also becoming more and more important nowadays, because the immune system is also strongly related to the HPA axis and has an impact on the health balance. Combining measurements of cytokines such as IL-6, Il-1a, IL-8, TNF-α provides additional help in diagnoses of health status.
IBL International offers cytokine measurement assays which are validated for serum, plasma and other biological fluids. Measurement of salivary samples is also possible and may be highly useful in enhancing your patient records.

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Psychological research

Psychological research is related to the science of behavior and mental processes; it includes areas such as biological psychology, cognitive psychology and social psychology. In many cases the patient’s psychobiological status is established by means of validated selfassessment questionnaires, but 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 and more research is devoted to understanding them and finding medication. Parameters such as levels of cortisol, DHEA, testosterone, alpha amylase exhibit relationships with chronic stress, posttraumatic stress disorder, behavior, cognitive function, and health. For this reason leading research psychologists are very interested in studying, measuring and understanding of these substances’ concentrations and circadian patterns. Cortisol concentrations are found to be higher when the individual is subject to a stress situation.

Stress research

 
Stress research cortisol amylase
 

Many studies test individuals’ endocrinological response to various kinds of stress. To differentiate the varying nature of individual reactions to stress a standardized test is needed which can be applied to all participants. The Trier Social Stress Test (TSST) is generally accepted as such a method.
There are suggestions that other factors can influence the level of cortisol in response to stress in humans: gender and the menstrual cycle seem to have an impact on this hormone profile, and the application of drugs such as oral contraceptives seems to diminish the cortisol response to stress.

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Endocrinology

Endocrinology is related to the biology and medicine of the endocrine system, its diseases and specific secretion products, known as “hormones”. It studies the biosynthesis, storage and physiological function of hormones and their pathology, along with the cells of the endocrine glands and tissues that secrete them.
The endocrine system consists of several glands, in various locations within the body, that secrete hormones directly into the blood rather than into a duct system. Hormones have many different functions and modes of action; one hormone may have several effects on a number of different target organs, and conversely one target organ may be affected by more than one hormone. There are many endocrine disorders which are quite often complex due to hyposecretion or hypersecretion of one or more particular hormones.

Cushing´s Syndrome

Cushing´s Syndrome, for example, results from excessive secretion of one or all of the adrenocortical hormones, mainly cortisol. This disease is characterized by failure to reach the circadian nadir in cortisol secretion late at night. This is quite a difficult endocrine pathology to diagnose because of the very common phenotype: obesity, hypertension, depression, alcoholism, etc. Furthermore it is relatively unusual, though growing increasingly common.

 

 

Late-night salivary cortisol is now a well-accepted and recommended screening test for Cushing’s syndrome. This method shows high specificity and sensitivity, and ELISA or luminescence assays are very easy to handle. Our Cortisol salivary luminescence assay only requires 20μL of saliva, making it easily practicable even with difficult patients such as children or the elderly.

Androgen Status

Free Testosterone is an ideal diagnostic tool for androgen levels in both men and women, but accurate measurement of testosterone remains a challenge. The determination of the blood testosterone concentrations in serum by conventional immunoassays is inaccurate in men, and even more so in women and children. Directly measuring testosterone in saliva is a better alternative to blood measurement: only the free, biologically active fraction is represented in saliva.
The IBL International luminescence enzyme immunoassay shows high analytical and functional sensitivity, and allows quantification of the very low concentration in saliva, as well as in serum, after 1/40 dilution.
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 assay is particularly well suited to differentiating between healthy men and patients with androgen status disorders.

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Sports Medicine

Saliva sampling is very easy, non-invasive and no medical assistance is required. Samples can be collected under circumstances where blood collection is difficult or inadvisable. For these reasons saliva measurement is the right choice for evaluating physical conditions during exercise. Salivary cortisol, DHEA and testosterone determinations are useful in sports medicine research. The use of these biological markers is based on the assumption that these salivary steroids may reasonably be taken to reflect the hypothalamic pituitary- adrenal (HPA) axis function. Indeed, in the diagnostic setting, salivary cortisol levels parallel those in plasma following ACTH and CRH stimulation, and following exercise-induced stress. During physical exercise, free salivary cortisol, DHEA and testosterone concentrations increase with the intensity of the exercise. It is particularly important in sports medicine to note that hormone levels and exercise-induced hormone changes vary among subjects with different types of training.

Physical activity on a bicycle ergometer

 
physical activity test non-trained
 

 
physical activity test well trained
 

Whereas endurance-trained subjects display a less pronounced hormone response to exercise, there are some differences between endurance and resistance sessions:

  • Resistance-trained subjects demonstrate a more pronounced hormone response to exercise
  • Increased cortisol may indicate an intensity- rather than a mode-dependent response
  • Monitoring hormone levels during sport sessions is found to be a useful tool for optimizing athletes’ training status and identifying any excessive exercise resulting in overtraining syndrome.
  • Measurement of IgA in saliva samples will also provide information on the athlete’s immune status.

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Support

Training

We are proud to provide you with high quality products for your specific purposes, and with the professional support you expect. Would you like to start a saliva laboratory or implement saliva measurement in your laboratory? Our technicians can help you to do this, either in your own lab or by offering you training in our facilities!

During this training you will learn the basics for establishing a reliable and trustworthy saliva analytics.
It is essential to appreciate the importance of the preanalytic aspects:

  • Proper collection of saliva samples
  • The dynamic nature of steroid secretion
  • Effects of the blood contamination
  • Volume of saliva needed to complete all of analyses
  • Pretreatment of saliva (freezing, thawing, centrifugation)
  • Medication of the patient
  • Validation and evaluation of the analytical method (sensitivity, specificity, reproducibility, reference values, usefulness of diagnostic method for the intended medical purpose)

Furthermore you will be able to run an assay on your own, and we can help if you have any queries or comments. Of course we will also give you the necessary background for the analyte(s) of interest, providing you with state of the art information as required for setting up your laboratory activities.

Do you need to refresh your skills or those of your laboratory staff? Please contact us, and we will happily take time to give you the support you expect in an efficient way!

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Equipment support

We also can provide you with laboratory equipment needed for ELISA and/or Luminescence IA. Please just contact us for tips and hints on pipettes, orbital shakers, washers or microplate readers.
We offer a broad range of luminescence IA for steroid testing. Assays for salivary steroid determination must be extremely sensitive, as concentrations of biologically active free fractions are significantly lower than the concentrations of total analyte in serum.
Luminescence technology offers many advantages:

  • High sensitivity
  • Accurate detection results
  • Stable signal
  • Broad linear range
  • Low background noise
  • Well established
  • Non isotopic

For the measurement of Luminescence you will need e.g. a microplate luminometer. The infinite PRO200 from Tecan® is a very fast, reliable and highly sensitive device which we recommend and can supply. Please do not hesitate to contact us directly for further details: we shall be happy to help.

Quality Assessment Scheme

The IBL International quality assessment scheme was established in 2005 since there was no standard available for steroids in saliva. The quality assessment scheme is conducted twice a year. Each distribution provides three samples. The multiparameter samples contain:

  • Cortisol
  • Testosterone
  • Estradiol
  • Progesterone and
  • DHEA

in various concentrations.

Each participant sends back the results to IBL International, either by fax using the results form available on our homepage or by email. IBL International statistically analyses the results and provides the participant with a QAS result sheet. Confidentiality is assured by the use of an ID number known only to the participant and to IBL International. The results are sent back per Email.

For further details or to take part, click here

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