Body Fluids Commonly Used for Testing Steroid Hormones Blood Serum (venipuncture) Plasma (venipuncture) Capillary Blood (finger/heel stick) Urine Saliva
Limitations of Hormone Testing in Different Body Fluids Hormones Tested Saliva Blood Urine Estradiol Yes Yes Yes Progesterone Yes Yes Yes Testosterone Yes Yes Yes DHEA(S) Yes Yes Yes Cortisol Yes Yes Yes FSH No Yes Yes LH No Yes Yes ft3 No Yes Yes ft4 No Yes No TSH No Yes No IGF1 No Yes No Insulin No Yes No Hs-CRP No Yes No Vitamin D3 No Yes No PSA No Yes No
A Guide to Steroid Hormone Testing in Different Body Fluids Following Different Routes of Hormone Administration Type of Body Fluid None Endogenous Steroids Oral Steroids Topical Steroids Vaginal Steroids Troche Steroids Pellet/IM Steroids Serum Yes Yes (1) No (2) No (2) Yes Yes Saliva Yes Yes (3) Yes (3) Yes No (4) Yes Urine Yes Yes (1) No (2) No (5) No (2) Yes (1) DBS Yes Yes Yes (6) Yes Yes Yes 1) Overestimation: Metabolites interfer with immunoassays 2) Underestimation: Hormone levels not reflective of tissue uptake 3) Overestimation: Requires range adjustment 4) Overestimation: Direct contamination of oral mucosa/saliva 5) Overestimation: Direct contamination of urine 6) Overestimation: IF fingertips contaminated with topical hormones
HORMONE TESTING IN VENIPUNCTURE SERUM OR PLASMA
Serum/Plasma (Advantages) Wide range of hormone tests available Automated FDA-approved methodology Covered by most insurance carriers Familiarity with levels and ranges
Serum/Plasma (Disadvantages-1) Invasive-sharp needle in arm Stressful-inconvenient to patient-requires driving to blood draw station Phlebotomist required Processing of specimen-centrifugation Shipment-Biohazard labeling and requires cold packs
Serum/Plasma (Disadvantages-2) Normal ranges (+/- 2 SD) are too wide-more geared for disease than failing health Difficult to measure multiple times during day due to collection logistics Usually measures TOTAL and not BIOAVAILABLE fraction of hormone in bloodstream Not valid method for measuring hormones delivered topically -gross underestimation of hormone distribution to tissues
Serum Progesterone Following Oral Progesterone Delivery Problem with high level of progesterone metabolites that are bioinert, but cross-react with antibodies used in immunoassays
Orally administered progesterone results in non-active progesterone metabolites that are detected by immunoassays (false-high caused by cross-reactivity), but not by Mass Spectrometry. These metabolites/conjugates are too large to pass through the salivary glands and enter saliva; therefore, saliva a good matrix to measure the free/unconjugated fraction of progesterone following oral delivery. LC-MS Immunoassay
HORMONE TESTING IN URINE
Urine (Advantages) Non-invasive Simple Collection Procedure Measures total daily output of steroids Measures steroid metabolites More likely covered by insurance carriers
Urine (Disadvantages) More cumbersome collection procedure-all urine over 24 hours Mostly measures total excreted hormone metabolites-not bioavailable hormones Hormone testing restricted mostly to steroid, hormones (exceptions LH, FSH) Levels may vary depending on degree of hydration (level/l urine or g creatinine)
Urine (Disadvantages) Problem with 24 hr urine collections: It is well known that collecting 24-hour urine samples other than in hospital, though it seems easy, is in effect rather difficult. The individual creatinine excretion figures at the start and end of the investigation led to the conclusion that unfortunately not all subjects were as accurate as possible in urine collection. Sangster B, et.al. Fd Chem Toxic. 21, 409-419, 1983.
Urinary Hormone Levels Throughout the Menstrual Cycle Reproductive State ng/ mg Cr PgM/E1M PgM/E1M ratio Mid-Reproductive 15,000/50 300 Peri-Menopausal 5000/100 50
Urine Progesterone Following Topical Progesterone Serum results from Carey British J. Ob & Gyn 2000 & O Leary Clin. End. 2000 E.P. data from Leonetti F&S 2003
HORMONE TESTING IN SALIVA
Davenport HW.The Digestive Tract, Year Book Medical Publishers, Chicago, 1971. S S S S
Saliva (Advantages-1) Simple Stress-free Noninvasive (no needles) More convenient for health care provider and patient
Saliva (Advantages-2) Optimized for collection any time of day/month, any place No special processing (eg, centrifugation, icepacks) prior to shipment Hormones stable in saliva for prolonged period of time Convenient shipment by regular US mail
Saliva Disadvantages Restricted to STEROID hormones-no thyroid or peptide (eg. FSH) hormones Technically more challenging: need 10-20x sensitivity-more problematic for hormones at very low concentration (i.e. estrogens) Interfering substances-food, beverages Sublingual use of hormones leads to spurious high test results (direct contamination of the oral mucosa) Problematic for those with poor saliva production
Salivary Sex Hormones (E2, Pg, T) = 1-3% Serum Hormones e.g. Estradiol: Reported 1.5-2% Average Premeno Serum = 100 pg/ml Average Premeno Saliva = 2.0 pg/ml @ 2% Actual Serum E2 Range: 43-180 pg/ml Calculated Saliva Range @ 2%: 0.9-3.6 pg/ml Actual Saliva Range: 1.3-3.3 pg/ml
Assay Challenge: Relative Lowest Expected Concentrations of Steroids in Saliva 1000 Relative Concentrations of Saliva Steroids 1000-2000 pg/ml 800 Minimum Concentration (pg/ml) 600 400 200 0 <0.5-1.5 pg/ml Estradiol Testosterone Progesterone Cortisol DHEA-S
This poses a technical challenge for testing hormones present at very low levels-e.g. estradiol in postmenopausal women and men
UNEXTRACTED SALIVA Cortisol DHEAS Progesterone Testosterone Estradiol MATRIX = BACKGROUND
EXTRACTED SALIVA Cortisol DHEAS Progesterone Testosterone Estradiol MATRIX = BACKGROUND Extraction Advantages Decrease Matrix Effects (lower background) Increase concentration = Increase sensitivity
RELIABILITY BASED ON ACCURACY AND PRECISION Control Specimens Help Determine The Accuracy And Precision Of A Given Method Inter-laboratory Proficiency Studies Help Determine Consistency Among Labs Running Similar Tests
Testing for low levels of salivary testosterone in women and men also problematic without extraction-matrix effects may cause false-high levels
Testosterone in Saliva Correlates with Free Serum T (Saliva Extracted) Cutoff for Symptom-Based Hypogonadism = 55 pg/ml Salivary T Arregger et. Al. Clin. Endoc. 2007
Testosterone in Saliva Does Not Correlate with Free Serum T ( Saliva Not Extracted) Flyckt RL Menopause 16(4) 680-690, 2009
Clinical Utility of Salivary Hormone Testing Do salivary hormone levels show expected relationships with: Menstrual Cycle (Estradiol and Progesterone) Age: (e.g. Premenopausal vs Postmenopausal) Diurnal Patterns-High Morning/Low Night Symptoms of Hormone Imbalance-Deficiency and Excess Hormone Supplementation
Menstrual Cycle Variation
Comparing Estradiol Immunoassays 4 Wong (Extracted RIA) 20 Gandia (Extracted RIA) 3 15 2 10 1 5 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 15 IBL (Direct LIA) 15 Chatterton (Direct RIA) 10 10 5 5 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26
Monthly Cycle Serum Estradiol Serum Estradiol (pg/ml) 120 100 80 60 40 20 0 0 5 10 15 20 25 30 Day of Cycle 7 Salivary Estradiol Salivary Estradiol (pg/ml) 6 5 4 3 2 1 0 0 5 10 15 20 25 30 Day of Cycle
Age Variation
Estradiol levels relatively stable during premenopause, fluctuate erratically at peri-menopause, and decline gradually to lower steady state at postmenopause N = 18,663 NO HORMONES EXPECTED RANGE TOPICAL PROGESTERONE
Androgen (DHEAS) levels decline progressively and steadily with age (N = 19, 270) N = 18,663 DHEAS NO HORMONES CORTISOL TOPICAL PROGESTERONE ZRT DATABASE 6-04
Median Salivary Pg (pg/ml) 60 50 40 30 20 Progesterone in Women n = 48,169 Premenopausal Irregular Cycles Postmenopausal 10 0 0 10 20 30 40 50 60 70 Age
Symptom Association with Salivary Hormone Levels
Salivary Estradiol & Hot Flashes 1.6 Estradiol vs. Hot Flash Severity n = 39,570 1.5 Average Hot Flash Severity (0-3) 1.4 1.3 1.2 1.1 1 0.9 0.8 OPTIMAL REFERENCE RANGE 0.7 0.6 0 1 2 3 4 5 6 7 8 9 Salivary Estradiol (pg/ml)
Dose Response to Exogenous Hormone Therapy
Salivary Estradiol: Monitoring Supplementation 5 Oral Estradiol vs. Saliva E2 Results n = 3,023 Saliva Estradiol Results (pg/ml) 4 3 2 1 Premenopausal Range 0 0 0.2 0.4 0.6 0.8 1 Daily Dosage of E2 (mg)
Blood Spot and Plasma Levels of Estradiol, Progesterone, LH, and FSH Over a Normal Menstrual Cycle Validation of Blood Spot Sampling for Gonadotropins and Ovarian Hormone Levels in Reproductive Age Women. Fertility and Sterility, November 2007 A. Edelman, R. Stouffer, D. Zava, J. Jensen
18 16 Progesterone Blood Spot/Serum Correlation Serum Progesterone (ng/ml) 14 12 10 8 6 4 y = 1.05x - 0.6 R 2 = 0.99 2 0 0 2 4 6 8 10 12 14 16 18 Blood Spot Progesterone (ng/ml)
Blood Spot vs. Serum Correlations Blood Spot 7000 6000 5000 4000 3000 2000 1000 0 Testosterone R 2 = 0.99 0 1000 2000 3000 4000 5000 6000 7000 8000 Serum Blood Spot(U/L) 80 70 60 50 40 30 20 10 0 R 2 = 0.98 LH 0 10 20 30 40 50 Serum(U/L) FSH Blood Spot(U/L) 250 200 150 100 50 R 2 = 0.97 SHBG 0 0 50 100 150 200 Serum(U/L)
Why Blood Spot and Not Saliva? Able to test peptide hormones (e.g. TSH, insulin) not present in saliva and urine Most accurate test for measuring steroid hormone levels in individuals supplementing with a sublingual or troche steroid hormone-gross overestimation with saliva testing due to local saturation of oral mucosa Allows for measurement of bioavailable fraction of steroid in blood when combined with measurement of blood binding protein (e.g. ABI = T/SHBG)
Capillary Dried Blood Spot-Advantages-1 Convenient-Simple collection procedure More latitude in collection timing than serum-i.e. night time sampling for cortisol & insulin Dried serum analytes very stable for at least a month at ambient temperature-shipping simplified-allows for international shipment without need for Biohazard Label
Capillary Dried Blood Spot-Advantages-2 Wider range of analytes can be tested-similar to serum/plasma Results & ranges equivalent to serum/plasma for endogenously produced hormones More accurately reflects tissue uptake of exogenously used hormones than serum/plasma (gross underestimation)