BLOOD

Reproductive Ecology Lab Seminar: BIOA 455 Spring Quarter 2008 BLOOD, URINE, SALIVA and FECES BLOOD Venous Blood Draw—this is the **Gold Standard** Any blood collection has complicated ethical issues Advantages      Disadvantages Can detect pulsatile patterns Can detect circadian patterns Can assess immediate responses to drugs/challenges Very broad range of hormones can be assessed, in bioactive forms. Collection is fast Can’t collect daily or more frequent samples (difficulties with prospective research) Hard to get at average daily levels? Highly invasive Biohazard risk to researchers Health risk to subjects (especially in non-clinical and remote settings) Self collection is difficult; low compliance Long term storage significantly affects sample integrity, precluding additional research on samples (varies by biomarker) Essentially unavailable from wild animals unless very invasive methods (darting, capture) are used. Ethical Issues with Blood Many interfering substances in blood for assay purposes (e.g. binding proteins)           Dried Blood Spots Advantages        Disadvantages Can assess immediate responses to drugs/challenges Collection is fast Blood spots on filter paper can be stored at room temp up to 2 weeks or more, depending upon biomarker. Blood spots on filter paper do not use leaky, bulky, breakable vials; easy to transport Blood spots can be transported at room temp; venous blood requires optimal storage conditions Less invasive than blood draw; doesn’t require centrifuging, separation or immediate freezing Can get many biomarkers not available in urine (e.g. CRP) or saliva (e.g. proteins) Can’t collect daily or more frequent samples (difficulties with prospective research) Self collection is difficult; low compliance. Hard to obtain an adequate DBS sample; need evenly dispersed blood spot and enough but not too much volume. Long term storage significantly affects sample integrity, precluding additional research on samples (varies by biomarker) Assay availability is limited; need documentation with respect to gold standard for clinical, comparative and performance reasons. Essentially unavailable from wild animals unless very invasive methods (darting, capture) are used. Ethical Issues with Blood Many interfering substances in blood for assay purposes (e.g. binding proteins)         1 Reproductive Ecology Lab Seminar: BIOA 455 Spring Quarter 2008 SALIVA Collected using passive drool, or stimulated salivation (with gum or sugary drink)—the latter are not recommended for testosterone). Can use salivettes (cotton rolls), but these need centrifuging to get saliva out, and may interfere with hormone recovery (e.g. testosterone). Advantages          Can detect circadian patterns Can detect pulsatility Can measure acute responses as well as baseline levels Non-invasive Can collect multiple samples across short (minutes, hours) and long (days, months) periods of time (prospective research easily done) Storage at room temp is possible for long periods of time (up to 6 mos) for most steroid hormones (with preservative) BUT NOT TESTOSTERONE Self-collection is easy Samples can be transported at room temp. BUT NOT TESTOSTERONE Stability, sample collection and treatment may vary across hormones in saliva—for example, salivary cortisol seems pretty robust and salivary testosterone is pretty wimpy. The verdict is still out for salivary progesterone and estrogen (we will see what the grad student presentations have to say about this!). Can’t assess any protein hormones High risk of confounded samples due to blood/food contamination of saliva (a problem for testosterone, but apparently not a big problem for cortisol. Progesterone?) Need highly sensitive assays due to low amounts of steroids in saliva hard or impossible to use for individuals with very low levels of hormones (children, menopausal women) (a problem for testosterone?) Can only detect unbound hormones (a very underappreciated fact; see next comment) Have to assess or control for variation in carrier protein levels, which influence unbound levels. Even one freeze/thaw can have deleterious effects (depending on hormone) Time required for sample collection on the part of subjects is inconvenient, unless salivette is used. Long term storage not ideal? Essentially unavailable from wild animals unless invasive methods used; been used with captive animals. Sex differences in association of serum/saliva correlation for testosterone—assay limits of detection? Should we be concerned about other biomarkers? Disadvantages            URINE Can be collected in a vial, via a whiz-pop, or on filter paper. The latter two require validation for each hormone assayed. Advantages         Disadvantages Self collection is easy and fast; high compliance Non-invasive Integrated hormone values; minimal or no effects from confounding pulses Can detect circadian patterns Can collect multiple samples across long periods of time (prospective research easily done) Long term storage (frozen) has little effect on sample integrity (varies with biomarker) Broad range of hormones can be assessed Relatively easily available in some contexts from wild animals Can’t easily assess acute responses Correction for hydration status and time since last urination is required (using specific gravity or creatinine) Storage requires refrigeration or freezing within 1-2 days after collection Mulitple freeze/thaws can affect levels of some hormones Can’t detect pulses easily Metabolic effects confounding measurement of urinary hormone metabolites (unknown) Transport requires cold storage if transport last longer than 1-2 days. Hard to avoid contamination and identify appropriate subject when collected for wild animals. 2         Reproductive Ecology Lab Seminar: BIOA 455 Spring Quarter 2008 FECES Advantages • Can be collected relatively easily from wild animals • noninvasive Disadvantages • Requires special preservation and extraction methods • Long lag time between hormone secretion and excretion of hormone in feces • Hard to avoid contamination and identify appropriate subject when collected for wild animals • Requires correction factors PROBLEMS COMMON TO ALL COLLECTION METHODS           ethical issues cost participant and researcher burden assay sensitivity assay performance presentation and interpretation of results subject selection and bias specimen volume storage conditions non-comparability across studies and across fluid types o o o assay differences in protocol or performance normal range of error clinical cut offs 3