ChronoBiologyLETTER Spring 2012 ▎ 11th Edition nothing, or they may contain a variable Diagnos-Techs™ amount of unknown substances to Obtaining Adequate Clinical & Research Laboratory be analyzed. The Diagnos-Techs™ Saliva for Testing Quarterly Newsletter laboratory assessment is then compared HIGHLIGHTS IN THIS EDITION to the known test sample contents by John J. White, MD, CM the external testing service. Ronda Nelson, PhD 2 Saliva Hormone Testing One such area of proficiency testing Adequate saliva for testing 5 The Therapy Corner: is our GP-2 assay, the O&P (ova and (to fill testing vials to required Clostridium difficile Protocol parasites) assay performed on the stool levels) may be difficult to sample submitted as part of our GI obtain in some circumstances. 6 “Saliva Testing” In Wikipedia Health panel. In this assay, samples Some patients have difficulty from two stool samples submitted producing saliva; other patients have 7 Resources for You -Insurance Billing are examined three times under the difficulty collecting saliva into the vials. -Technical Services microscope by a parasitologist. Any A few suggestions may be of help in -Shipping & Contact Info organisms or parasite eggs discovered either case. are then listed on the report. For patients who have problems Diagnos-Techs™ We are accustomed to receiving salivating, saliva production may be 100% scores on these proficiency facilitated by chewing a piece of dental Laboratory Excels in tests from the external testing source. grade paraffin wax (obtainable from Proficiency Testing On recent testing, however, our O&P a pharmacist); sniffing pickles, a cut examinations of the test sample proved lemon, vinegar, or any tart substance; Ning Cigielska, MS even superior. Our parasitologist or preparing a favorite savory dish Michael Everitt, PhD recognized not only the test organisms, (remember to collect saliva prior to Like most well but several additional parasites present enjoying!). run laboratories, in the test specimen – which had not For cortisol testing (ASI/TAP), a cotton Diagnos-Techs™ been noted by the testing facility. When roll placed under the tongue is used. participates called to the facility’s attention, the To ensure adequate saturation of the in periodic presence of these other parasites was roll, do not remove roll and place back proficiency recognized and acknowledged. into the vial until the mouth has refilled testing. This MORAL: A highly trained, with its usual amount of saliva. process involves analysis of test knowledgeable pair of examining eyes In other instances, such as advanced samples provided by an external is essential to an accurate O&P. age, mental or physical incompetence, source. These samples may contain or disinclination (an unruly or reluctant child), it may be difficult to Diagnos-Techs™ collect saliva into the vial. In such circumstances, use of a sterile syringe is going Paperless! (3cc is the ideal size) to siphon saliva from the cheek pouches of In an effort to be more mindful of the environment, we will now the patient can help. The saliva then be distributing an electronic version of our quarterly newsletter, ChronoBiology Letter. If you do not have an email address listed on is transferred to the proper vial. An your account, please call Client Services at 1.800.878.3787 to add unused (preferably sterile) syringe is your email address. necessary to ensure an uncontaminated sample. Such syringes generally can be If you prefer to receive a printed copy of our ChronoBiology Letter, obtained from a cooperative pharmacy. please email us at [email protected]. ChronoBiology LETTER constant interchange between “bound” and “free” hormones in the blood, Saliva Hormone Testing a dynamic that calls into question A number of new developments in saliva testing have occurred over the whether any measured serum “free” past several years, coupled with serious reappraisals of serum hormone values actually are meaningful. testing. In view of these, and requests from our clients, an updated These shortcomings have been review of saliva testing is presented in this issue of Chronobiology. recognized by The Endocrine Society for testosterone, the hormone with John J. White, MD, CM the lowest “free” fraction (<3%). In 2008, The Endocrine Society issued In the past, clinical laboratory analysis the benefits and availability of salivary a position statement stating that “the of circulating body fluids has been testing; the majority of laboratories manner in which most [serum] assays done using serum, which necessitates have made significant investments in for TT [total testosterone] and FT [free a venipuncture. Since the latter part of blood analysis systems and equipment; testosterone] are currently performed is 3 the 20th century, saliva has been used there is an innate reluctance to waiver decidedly unsatisfactory.” Recognizing increasingly for this purpose. Recently, from serum testing, which has been that “important discrepancies and a consortium of three research groups the basis for most research and clinical inconsistencies in measurements are categorized 1,116 proteins secreted in decision making; and, until recently, widespread,” in 2010 The Endocrine saliva and stated that “[such] studies there has been little criticism of Society paired with the Center for can be used to translate blood-based existing serum testing. Disease Control (CDC), other clinical societies, and commercial laboratories clinical laboratory tests into a format Hormone testing has been carried that utilizes saliva.”1 Saliva has been to endorse “accuracy-based testing out traditionally by assaying levels of testosterone and calibration of all categorized as a “valuable biofluid… in serum. This type of testing is with the potential to extract more data methods traceable to a single high-level beleaguered by nature’s mechanism reference material.”4 These methods than is possible currently with other of transport of hormones in the diagnostic methods.”2 A non-inclusive are being developed by the CDC using blood: over 90% of the hormones in mass spectrometry. This process will list of current saliva testing is presented blood are bound to proteins (binding in Table 1. measure not free testosterone, but total globulins) and are inactive. The small (free and protein bound) testosterone. Background percentage of hormones remaining At the time of this writing, only a few are considered “free,” or available to laboratories appear to have been so Laboratory testing for hormones using traverse the capillary membranes into certified; those in the United States saliva was verified in the 1980’s and the tissues and have their effect on use expensive mass spectroscopy. 1990’s, and saliva testing is becoming target cells. Only the free form of a In light of this overall problem, the increasingly more utilized clinically, hormone is biologically active. There editors of the Journal of Clinical particularly for endocrinologic and are major problems inherent in serum Endocrinology and Metabolism have immunologic investigations. The slow hormone testing: there is no assurance raised the question whether “all of pace of its acceptance appears to have that the “free” hormone measured our other routinely employed [serum] been the result of a variety of factors: actually reaches the tissue fluid to hormonal assays provide truly accurate most clinicians are unfamiliar with affect its target cells; and there exists a and precise measurements.”5 Faced Some Clinical Conditions Detected Through Saliva Testing with major imperfections in and (List not comprehensive) doubts about serum hormone testing, clinicians are seeking other forms of • Adrenal conditions • Metabolic disturbances (e.g. Addison’s/Cushing’s, adrenal (e.g. insulin resistance, diabetes) hormone testing that don’t have protein fatigue) • Neoplasms (e.g. benign, metastatic) binding and capillary transfer problems. • Altered female hormone states • Infectious conditions A widespread and accurate form of (e.g. PCOS, menopause, hormonal (e.g. HIV, hepatitis, Helicobacter pylori) hormone testing, one that is not subject alterations, infertility) • Allergic states (e.g. asthma, mold, food) to these limitations, utilizes saliva. • Altered male hormone states (e.g. hypogonadism, hyperestrogenism) • Drugs (e.g. marijuana, cocaine, alcohol) Table 1: Some clinical conditions detected through salivary testing. 2 © 2012 Diagnos-Techs, Inc. All rights reserved. Spring 2012 ▎ 11th Edition A L S E N T R R E D S S Laboratory Improvement Act) and A Saliva Testing Adrenal Cortisol COLA (major laboratory accreditation I N L Salivary testing is eminently suitable D E Testing E X P A N organization) approved. The salivary for hormone testing because it does One principal application of test panel offered by Diagnos-Techs™ not entail the problems inherent to chronobiology is circadian cortisol truly constitutes a chronobiologic serum testing: it measures hormone output assessment. Assessment of investigation. It includes four cortisol levels in the tissue fluid, where there the adrenal gland’s diurnal/circadian evaluations throughout the day to are no binding proteins. The 2007 rhythm using multiple cortisol samplings detect possible stressful disturbances edition of Greenspan’s textbook, Basic throughout the day can only be in the circadian cortisol rhythm. and Clinical Endocrinology, states that accomplished, with absolute accuracy, Chronobiologic salivary cortisol “salivary cortisol reflects ‘free’ cortisol, via non-stressful saliva testing.