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™ 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 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, 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 , 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 , 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 . 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. , ) hormone testing that don’t have protein fatigue) • (e.g. benign, metastatic) binding and capillary transfer problems. • Altered female hormone states • Infectious conditions A widespread and accurate form of (e.g. PCOS, , hormonal (e.g. HIV, hepatitis, ) 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. , hyperestrogenism) • Drugs (e.g. marijuana, , alcohol) Table 1: Some clinical conditions detected through salivary testing.

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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. testing has been used clinically most and saliva testing directly measures prominently by the United States active (emphasis added) steroid In 2008, The Endocrine Society government. Panels that measure hormone (‘free’ hormone).”6 published guidelines for the diagnosis cortisol and other hormones have of Cushing’s Syndrome.20 One of the Salivary hormone testing was fully been relied upon clinically to assess four recommended initial screening verified in the 1980’s, at which time objectively the circadian adaptation tests is late night salivary cortisol 21 any problems with laboratory methods, of aircrew to transmeridian flight; measurement (on two consecutive specimen collection, and specimen to create physiologic profiles of days). Ample references are provided transport were settled.7,8,9,10,11 In 1994, soldiers experiencing military survival to show that “free cortisol in the 22 it was reported that more than 2500 training; and to evaluate circadian blood is in equilibrium with cortisol clinical and research articles dealing shifts in astronauts before flight in the saliva”; that “the concentration (including the proposed MARS M AL with the accuracy and application FE E of salivary cortisol does not appear 23,24 of salivary diagnostic tests had been project astronauts).

to be affected by a change in the H 12 L published. These papers emphasized O E R N rate of salivary production”; and M A that close correlation exists between Cycling Woman’s O N E P that “an increase in blood cortisol is salivary testing (“free” fraction) and reflected by a change in the salivary Hormone Assessment the “free” fraction of the serum. Since cortisol concentration within a Another prime example of chronobi- then, several review articles have few minutes.” The accuracy of a ologic evaluation is a cycling female further verified and vindicated salivary single salivary cortisol assay has hormone evaluation. Because of the hormone analysis.13,14,15,16,17,18,19 been attested to and validated. It wide variations of the two principal Chronobiologic logically follows that cortisol samples cycling hormones, and collected at other times of the day , throughout the follicular Testing must be equally valid, using proper and luteal phases of a woman’s Not only is saliva testing more accurate technology. Such technology can be monthly cycle, no single day’s inasmuch as it selectively measures by either (costly) mass spectrometry or hormonal sample can in any the active “free” hormone, but it also (economical) ELISA techniques; both way be representative of the cycle. allows chronobiologic testing, which is “yield a 92%-100% sensitivity and a The Diagnos-Techs™ Cycling Female not feasible with serum testing with its 93%-100% specificity.”20 Homone Panel (FHP™) entails a single saliva collection every 2-4 attendant . Championed Diagnos-Techs™ salivary hormone days depending on the length of the by Franz Halberg of the University of testing laboratory was the first woman’s cycle. A representative graph Minnesota, chronobiology is defined in the United States to provide, illustrating the vagaries of a woman’s as the field of science that examines commercially, a temporal adrenal periodic (cyclic) phenomena in living cortisol profile for clinical use hormones throughout her cycle is organisms. That is, not just a single (offered since 1987). Hormone depicted in Figure 1. Ordering the measurement, but serial measurements assays are carried out using the ELISA expanded version of the FHP™ adds 7 over time of dynamically varying technology, and results and standards measurements of pituitary hormones vital hormones can be obtained. It are checked regularly using mass (follicular stimulating hormone is the difference between looking at spectrometry. Diagnos-Techs™ has [FSH] and [LH]) a snapshot of a changing scene and earned the Joint Commission’s Gold which are measured around the watching an entire movie. Seal of Approval and is CLIA (Clinical Continued on page 4

www.diagnostechs.com  1.800.878.3787 3 ChronoBiology LETTER

• The accuracy of saliva testing was established during the 1980’s, and since then has been verified continually by, literally, thousands of scientific papers; • The most accurate laboratory methodologies for saliva analysis are ELISA techniques and mass spectrometry; • Salivary hormone testing for Figure 1: Female Hormone Panel (FHP™) result for a cycling 32-year-old woman. clinical use has been available The pre-ovulatory estradiol surge is seen, followed by elevated progesterone commercially since 1987 (from during the of the cycle. Diagnos-Techs™). It is not merely a research tool. It has been used time of . These FSH and LH A representative and thorough by the United States government, assessments are valuable particularly investigation that can provide a sound and it is recommended by The for women with fertility problems, basis for therapy recommendations is Endocrine Society for the diagnosis pre-menopausal symptoms, and the Expanded Male Hormone Panel™ of Cushing’s Syndrome. There menopausal distress. (eMHP™) from Diagnos-Techs™. has been a steady increase in A L M E saliva testing laboratories and Andropause Inasmuch as andropause saliva hormone testing since the H symptomatology can result not only L O E technique was introduced over two R N M Assessment P A from testosterone deficiency, but O N E decades ago by Diagnos-Techs™; It is becoming more evident that from vascular, psychologic, and • Salivary testing is an accurate and investigation of hypogonadism in men, neurological problems as well, a accessible method for sometimes termed “andropause,” broad and thorough investigation assessment which is an important cannot be limited to a single serum should include an adrenal stress element in the investigation of food testosterone assessment. Such a assessment (ASI™). and intestinal parasitic measurement is unreliable and fraught diseases; with error as previously noted. As well, Summary overall problems with deficiencies • Serum hormone testing, for years • Salivary hormone testing is of predecessor hormone supply and the staple and standard for hormone non-invasive, economic, private, reduced activity of the underlying testing, has been called into serious accurate, and non-stressful. It is the testosterone pathway co-reactions, question by endocrine experts due only feasible method to carryout the amount present of the potent to uncertainty in distinguishing chronobiologic testing, which is male hormone dihydrotestosterone, protein-bound from “free” forms of very valuable in assessing stress and the dynamics of the controlling a hormone, and uncertainty in how and the vagaries in hormone pituitary hormones (FSH and LH), much “free” or available hormone cycling. Such testing is nearly should be taken into account prior actually crosses the capillary barrier impossible in clinical practice using to any possible recommendations for into the tissue fluid; serum testing and its requisite serial venipuncture; testosterone therapy. Aromatization • Saliva hormone testing has of testosterone to estradiol and attracted considerable interest • In view of the demonstrable conversion of the androstenedione because it measures hormones in benefits of saliva hormone testing, pathway to estriol and estradiol the tissue fluid itself (i.e., the “free” and the doubts regarding the “Gold (common in older men) are not form of the hormone), thereby Standard” serum testing, saliva accounted for without measuring providing more accurate results hormone testing deserves to be these other hormones. All these values than serum testing; considered the “Platinum Standard” should be known and addressed prior for hormone testing. to pronouncing the testes unresponsive and prescribing testosterone therapy. For references please see back page.

4 © 2012 Diagnos-Techs, Inc. All rights reserved. Spring 2012 ▎ 11th Edition

substantive therapy with high quality The Therapy Corner probiotics. Overall, probiotic therapy In an effort to further broaden the perspective and service provided by is the mainstay in CDI except in those Diagnos-Techs™, we are adding a new section to the Chronobiology Newsletter cases involving immunocompromised or critically ill patients (administration called The Therapy Corner. In this section, we will provide background material, in these cases may result in interpretive guidelines, and therapeutic strategies for a variety of conditions, with of the bloodstream).4 Benefit has been particular focus on conditions identified by our tests. In this issue, we discuss demonstrated with supplementation Clostridium difficile infection. of Saccharomyces boulardii, bifidobacteria, and Lactobacillus persistence rates for C. difficile are species in cases of C. difficile.4,5 One Clostridium difficile estimated to be 20-30%).2 key to successful probiotic therapy January 2012 Recent evidence underscores is proper dosing; aim for 100 billion the importance of screening for CFUs per day for at least four weeks to John J. White, MD, CM Clostridium difficile infection (CDI) in achieve adequate colonization in the Brandy Webb, ND non-hospitalized patients. Community- GI tract (lower doses of probiotics may In our Gastrointestinal Health Panel acquired CDI cases comprise up to be prescribed for several additional (regular or expanded), Diagnos-Techs™ 41% of total CDI cases, and patients weeks). Some forms of fiber, especially provides analysis of submitted are more likely to be younger, female, fructooligosaccharides (FOS) and desiccated stool specimens for the and have no recent hospitalization or inulin, frequently are included in Clostridium difficile antigenic toxins (A exposure to antibiotics.3 Moreover, probiotic formulas to act as a food and B). This assay has a sensitivity of they are more likely to experience source for beneficial gut flora and 93.1% and a specificity of 99.2%.1 It is milder symptoms and thus go promote their proliferation; in this an accurate and valuable test when C. undiagnosed.3 context, these forms of fiber are often 5 difficile proliferation is present. The most current definition of termed “prebiotics”. Clostridium difficile is a spore-forming Clostridium difficile infection is Antimicrobial herbs are another bacterium with broad distribution. the presence of symptoms (usually mainstay of comprehensive It comprises a usually manageable diarrhea) and a positive stool test Clostridium difficile treatment portion of the normal bowel flora. for C. difficile toxins.4 The protocols, since they can combat Occasionally, an increase in C. immunoassay stool test for C. infection without further predisposing difficile population may occur, difficile toxins A and B offered by to C. difficile. Antimicrobial herbs usually accompanied by diarrhea. Diagnos-Techs™ (lab code “GP3Cd”) possess antiseptic activity, but their An estimated three million cases of is a convenient option for C. difficile primary action is to augment the C. difficile infection occur in the U.S. testing, and it is available as part of the body’s own defense mechanisms to each year, with rising mortality due to more comprehensive GI panels offered assist in fighting the infection. Helpful increasingly virulent strains and rising by Diagnos-Techs™ (“GI-1” and herbs include echinacea, calendula, host vulnerability.2 “GI-2”) for cases where screening for myrrh, garlic, artemisia, berberis, and hydrastis; these can be taken as Clostridium difficile infection has other infectious causes is prudent. tinctures or encapsulated preparations. four clinical forms: 1) short term Upon detection of Clostridium Many of these herbs have the added over-colonization (frequently occurs difficile toxins, treatment must be benefit of anti-inflammatory or after a stay in a healthcare facility selected appropriate to the severity tonifying (strengthening) effects. Any or following an intense antibiotic of presentation. In all cases, any drugs and herbs designed to halt course); 2) acute diarrhea; 3) fulminant offending antimicrobial agent should peristalsis (e.g., some antidiarrheals) diarrhea (may be associated with be discontinued as soon as possible should be avoided, because these pseudo-membranous colitis and (unless very strongly indicated can mask symptoms and possibly leukocytosis, and may lead to death); otherwise). In the mildest form (clinical result in toxic megacolon.4 In view of and 4) recurrent or persistent infection signs and symptoms are few and mild), the 20-30% incidence of persistent (defined as C. difficile infection non-pharmacologic therapies designed or recurrent infection, routine occurring within sixty days of initial to rebalance the intestinal flora usually clinical and laboratory follow-up is diagnosis and treatment; recurrence/ are sufficient. The first step is to initiate Continued on page 6

www.diagnostechs.com  1.800.878.3787 5 ChronoBiology LETTER

Therapy Corner Continued (typically dosed at 500-1000mg PO recommended two weeks following BID for 10-14 days).9,10 Recent studies “Saliva Testing” completion of probiotic and have shown that proton pump inhibitor In Wikipedia antimicrobial therapy. (PPI) use is a significant risk factor for For more severe cases of Clostridium recurrence of C. difficile-associated John J. White, MD, CM and 11 difficile infection—those that present diarrhea. Discontinuing PPI use in Brandy Webb, ND favor of alternative treatments with acute diarrhea and the distinct Until recently, Wikipedia, the online (dietary and lifestyle modification, possibility of fulminant infection and encyclopedia, contained little death—pharmacologic therapy must herbs, etc.) should be advised in about saliva testing, and that little be considered. The current initial recalcitrant patients. was erroneous and incomplete. It was an evident lapse that needed to be recommendation is the use of: References addressed, updated, and corrected. Metronidazole (Flagyl) 250mg PO QID 1. Diagnos-Techs ™, Inc. Validation We are very pleased to report that a (or 500mg PO TID) for 10-14 days4,6 Summary. Clostridium Difficile Toxin A&B. April 2011. new and more complete overview of saliva testing has been up and running OR 2. DuPont HL. The Search For since October 2011 on Wikipedia. Vancomycin (Vancocin) 125mg PO Effective Treatment of Clostridium difficile .New Eng J Med QID for 10-14 days4,6 It reviews saliva testing from many points (2011)364:473-475. of view. It highlights some of the more Metronidazole typically is used 3. Khanna S, Pardi DS, Aronson SL, et than 1200 different substances in saliva for mild-to-moderate CDI, while al. The Epidemiology of Community- for which testing has been done, and it vancomycin generally is reserved for Acquired Clostridium difficile Infection: discusses the various conditions that can A Population-Based Study. Am J 4 severe CDI. A newer drug, fidaxomicin Gastroenterol. 2012 Jan;107(1):150. be evaluated using saliva . These include metabolic disturbances, (Dificid), dosed at 200mg PO BID for 4. Cohen S, Gerding D, Johnson S, et 10 days, can be prescribed in severe al. Infection Control and Hospital neoplasms, infectious conditions, drugs and allergic states, as well as aberrations cases as an alternative to metronidazole Epidemiology. Clinical Practice Guidelines for Clostridium difficile in male and female hormones, stress 6,7 or vancomycin. Fidaxomicin was Infection in Adults: 2010 Update by the hormones, and familiar approved by the FDA in May 2011 Society for Healthcare Epidemiology to our clients at Diagnos-Techs™. and has been demonstrated to be of America (SHEA) and the Infectious Several links to other appropriate topics Diseases Society of America (IDSA) two- to eight-fold more potent than have been added, and there are over 5. Nadeau DA. Intestinal Warfare: The Role 8 sixty peer-reviewed, scientific articles metronidazole and vancomycin. of Short-Chain Fructooligosaccharides in Administration of antibiotics should Health and Disease. Nutrition in Clinical referenced within the article. be accompanied or followed by Care. 9 Oct 2008. We recommend this addition to four weeks of potent probiotics as 6. Aberra F, Katz J, et al. Medscape Wikipedia to you as a source of described previously. Again, clinical Reference. Clostridium Difficile Colitis additional knowledge about saliva Medication. Sept 2011. and laboratory follow-up should be testing, and as a resource for you and 7. Louie TJ, Miller MA, Mullane KM, et your patients. It contains a wealth of obtained about two weeks following al. Fidaxomicin Versus Vancomycin For well-referenced information to help completion of therapy. Clostridium difficile Infection.New Eng J you with your practices. Med (2011)364:422-431. As mentioned, Clostridium difficile 8. Blondeau JM. Expert Rev Clin Pharmacol. L & R E S C A E A has a significant rate of persistence or I R N 5(1), 9-11 (2012). Macrocyclic antibiotics: I C L H C recurrence; moreover, patients who a novel class of drug for the treatment of TM have one recurrence are far more Clostridium difficile infection. DiagnosTechs L A Y THE LEADING LAB IN SALIVA TESTING SINCE 1989 B R likely to have additional recurrent 9. Musher DM, Logan N, Hamill RJ, et O R A T O 4 al. Nitazoxanide for the treatment of Issue #11 Chronobiology Letter is published episodes. Recommended treatment Clostridium difficile colitis.Clin Infect Dis quarterly by Diagnos-Techs™ Laboratory, Inc. for the first recurrence of CDI is 2006;43:421-427. in Kent, WA, USA as an educational resource usually the same regimen used for for our health care clients. The content in this 10. Stuppy WD. Nitazoxanide and Treatment newsletter is for informational purposes only the initial episode, but it may need of Gastrointestinal Infections/Parasitosis. and is not to be construed as medical advice. Am J Gastroenterology (2006)101:382. to be modified according to the Diagnos-Techs™, Inc. severity of the clinical presentation.4,7,9 11. Kim JW, Lee KL, Jeong JB, et al. 19110 66th Avenue S. Proton pump inhibitors as a risk Building G Nitazoxanide (Alinia) has been shown factor for recurrence of Clostridium- Kent, WA 98032 USA to have activity against C. difficile difficile-associated diarrhea.World J Printed on recycled paper. and may be considered in some cases Gastroenterol. 2010 Jul 28;16(28):3573-7. 

6 © 2012 Diagnos-Techs, Inc. All rights reserved. Spring 2012 ▎ 11th Edition DIAGNOS-TECHS ™ RESOURCES FOR YOU ▼

Billing Client Services Contact Us Courtesy Service Tips for Success How to Reach Us FROM OUR TEAM OF n Have your account number or Business Hours INSURANCE SPECIALISTS accession number ready before 7:30am–5:00pm Our dedicated team of Insurance you call. Pacific Standard Time (PST) Specialists is trained to assist you n Sign up to access results online. Monday through Friday, with insurance-related questions. Call Client Services for password, except major holidays We avoid payment processing 1-800-878-3787. Corporate Address challenges by helping you and n We must have at least two forms 19110 66th Avenue S., Building G your patients navigate the maze of of written patient ID: name Kent, WA 98032 USA test codes and fees. and date of birth on all vials and Customer Service As a courtesy, we will submit order forms to process samples. p 800-878-3787 f 425-656-2871 claims to most insurance n Patients should not contact the p 425-251-0596 companies at our clients’ and lab with test related questions. Accounting patients’ requests. In addition, Please advise patients to consult f 425-264-0612 we are able to bill insurance with you if they have any questions email [email protected] carriers for all referring doctors, pertaining to collection. Medical Shipping nurse practitioners and registered Support is available for provider nurses. We are able to advise you questions about results or p 800-878-3787 f 253-398-2449 on which services are billable. treatment suggestions at Technical Services Although we are a non-contracted 1-800-878-3787. p 800-878-3787 f 425-251-0637 provider with all insurance n Review patient medication list. Lab Address companies (with the exception of This will help your patients to Medicare), most insurance carriers Sample Processing understand what to avoid before 6620 S. 192nd Place, Building J-106 offer coverage on our services taking our tests. We do not discuss Kent, WA 98032 USA and are billable. Depending on a medications directly with patients. patient’s benefit plan, insurance companies usually cover our tests n Tips for saliva production. Please visit us online at at the maximum allowable rates so In order to produce enough www.diagnostechs.com there are no out-of-pocket expenses. saliva, suggest to your patients to www.facebook.com/diagnostechs Patient Insurance Disclaimer chew dental grade paraffin wax. Diagnos-Techs™, Inc. is a non-contracted provider with all insurance companies. Please verify your out-of-network benefits (including out-of-network deductibles and co-insurances) by contacting your insurance carrier. Diagnos-Techs™, Inc. will Shipping bill your insurance at the retail price per line item. If deductibles and/or co-insurances are applied, Diagnos-Techs™, Inc. is obligated by law in the Free UPS Return Shipping State of Washington to collect. on returned DOMESTIC test kits STORAGE & MAILING INSTRUCTIONS FOR ALL SPECIMENS HIPAA Compliance n INTERNATIONAL Ship samples on the same day as last sample collection (preferred). COURIER SHIPPING n If not possible, refrigerate samples and ship within 3 days. The Health Insurance Portability and Diagnos-Techs™, Inc. Accountability Act of 1996 (HIPAA) requires No ice bags are required during shipping. Sample Processing n us to protect and maintain the privacy of our Write the patient’s name and address on the outside of the box. 6620 S. 192nd Pl., #J-106 patients’ identifiable health information. The n Include all samples, test form and, if applicable, a check or a copy of the Kent, WA 98032 standards are meant to improve the efficiency front and back of insurance card together inside the supplied box. Please p 425-251-0596 and effectiveness of the nation's health care be sure to seal the box with clear tape OR the UPS shipping label (US only). system. We are committed to implementing • US Domestic: Deliver completed test kit box to any UPS location. www.UPS.com/dropoff appropriate administrative, technical and Return shipping to Diagnos-Techs™ is PRE-PAID. Kits will arrive within three business days of shipment. physical safeguards to protect the privacy of Protected Health Information. For more •  International: Delivery charges apply. Visit our website for access to discounted return information, go to www.diagnostechs.com shipping via UPS. Deliveries can also be made Monday through Friday via a private and click About Us/Notice of HIPAA courier of your choice. International deliveries should be addressed to the physical Privacy Practices. address only, as noted above and to the right. Do not address to the PO Box.

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DiagnosTechsTM L A Y THE LEADING LAB IN SALIVA TESTING SINCE 1989 B R O R A T O 19110 66th Avenue South, Building G Kent, Washington 98032 USA www.diagnostechs.com

Saliva Hormone Testing Continued 9. Vining RF, McGinley RA, Symons RG. 18. Gozansky WS, Lynn JS, Laudenslager ML, et “Hormones in Saliva: Mode of Entry and al. “Salivary Cortisol Determined By Enzyme Consequent Implications for Clinical Interpre- Immunoassay is Preferable to Serum Total References tation.” Clin Chem (1983) 29:1752-1756. Cortisol For the assessment of Dynamic 1. Denny P, Hagen FK, Hardt M, et al. 10. Read GF, Walker RF, Wilson DW, et al. Hypothalamic—Pituitary—Adrenal Axis The Proteomes of Human Parotid and “Steroid Analysis in Saliva for the Assessment Activity.” Clin Endocrinol (Oxf). (2005) Submandibular/Sublingual Gland Saliva of Endocrine Function.” Ann N Y Acad Sci 63:336-341. Collected as the Ductal Secretions. J Proteome (1990) 595:260-274. 19. Gavrilova N, Lindau ST. “Salivary Sex Res. (2008)10:1012. 11. Malamud D. “Saliva As A Diagnostic Fluid.” Hormone Measurement in a National, 2. Wong DT. “Salivary Diagnostics Powered Brit Med J (1992) 305:207-208. Population-Based Study of Older Adults.” by Nanotechnologies, Proteomics, J Gerontol B Psychol Sci Soc Sci (2009) 64 12. Ellison PT. “Salivary Steroids and Natural Suppl 1:i94-105. and Genomics” J Am Dent Assoc Variation in Human Ovarian Function.” Ann (2006)137:313-321. N Y Acad Sci (1994) 709:287-298. 20. Nieman LK, Biller BM, Findling JW, et al. “The Diagnosis of Cushing’s Syndrome: An 3. Rosner W, Auchas RJ, Azziz R. POSITION 13. Collins JJ. “Salivary Hormone Testing: Science, STATEMENT: “Utility, Limitations, and Pitfalls Endocrine Society Clinical Practice.” J Clin Benefits, Limitations & Clinical Applications.” Endocrinol Metab (2008)93:1526-1540. in Measuring Testosterone: An Endocrine Anti-Aging Medical News (Winter 2000). Society Position Statement.” J Clin Endocrinol 21. French J, Bisson RU, Neville KJ, et al. 14. Lewis JG. “Steroid Analysis in Saliva: An Metab (2008) 92:405-413. “Circadian Adaptation of Aircrew to Overview.” Clin Biochem Rev (2006) Transmeridian Flight.” Aviat Space Environ 4. Rosner W, Vesper H. “Toward Excellence in 27:139-146. Testosterone Testing: A Consensus Statement.” Med (1994)65(5Suppl):A1-6. 15. Gröschl M. “Current Status of Salivary J Clin Endocrinol Metab (2010) 95:4542-4548. 22. Morgan CA 3rd, Wang S, Mason J, et al. Hormone Analysis.” Clin Chem (2008) 54:11 “Hormone Profiles in Humans Experiencing 5. Wartofsky L, Handelsman DJ. “Standard- 1759-1769. ization of Hormonal Assays for the 21st Military Survival Training.” Bio Psychiatry 16. Raff H. “Utility of Salivary Cortisol Century.” J Clin Endocrinol Metab (2010) (2000)47:891-901. Measurements in Cushing’s Syndrome and 95:5141-5143. 23. Whitson PA, Putcha L, Chen YM, et al. Adrenal Insufficiency.”J Clin Endocrinol “ and Cortisol Assessment of 6. Greenspan Basic and Clinical Endocrinology Metab (2009) 94:3647-3655. (2007) Eighth Edition. Circadian Shifts in Astronauts Before Flight.” 17. Arafah BM, Nishiyama FJ, Tlaygeh H, J Pineal Research (1995)18:141-147. 7. Peters JR, Walker RF, Riad-Fahmy D, et et al. “Measurement of Salivary Cortisol al. “Salivary Cortisol Assays for Assessing 23. Assessing Group Dynamics in A Mars Concentration in the Assessment of Adrenal Simulation (2006) Project Site: The NASA Pituitary-Adrenal Reserve.” Clinical Function in Critically Ill Subjects: A Surrogate Endocrinology (1982) 17:583-592. Haughton-Mars Project (HMP) on Devon Marker of the Circulating Free Cortisol.” J Island, Canada. 8. Vining RF, et al. “Salivary Cortisol: A Better Clin Endocrinol Metab (2007) 92:2965-2971. Measure of Adrenal Cortical Function Than Serum Cortisol.” Ann Clin Biochem (1983) (Pt6):329-335.

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