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Lessons Learned/Challenges Corticotropin Releasing Hormone (CRH) Why Measure CRH? • 41 amino acid neuropeptide, plays a major role in the physiological response to stress • The principal hypothalamic regulator of the pituitary-adrenal axis • Higher levels of CRH associated with adverse pregnancy outcomes What Medium and Collection Method to Use? • Medium – Plasma • Collection Method – Collection tube • EDTA • Protease inhibitor – Time • Timing of sample collection – no circadian rhythm • Effects on plasma samples-process within 12 hrs • Timing during pregnancy – Temperature • Continuously chilled at 4°C Considerations when Processing • Sample Processing – Refrigerated Centrifugation • 3,000-4,500 rpm for 15 minutes • Aliquots siphoned and analyzed or stored – Storage • -80°C for < 2 years – Plasma extraction • Removes CRH from plasma • Methanol vs C18 Sep-Column • Immediately prior to assay Factors in Selection of Assay • Factors to consider – Cost – Ease – Reliability/sensitivity of tests • ELISA vs RIA – Elisa (easier, rapid turnover time) – RIA (well documented in literature, greater sensitivity) Lessons Learned Challenges • Designing research study – Timing of collection in relation to EGA – Stratifying for ethnicity/race • Identify published protocol, consult with author/experts • Consider role of lab manager • Evaluate each plate result with consultantassess standardized curve • Which method of assay? References • Glynn, L.M., Dunekl Schetter, C., Chicz-DeMet, A., Hobel, C.J., & Sandman, C.A. (2006). Ethnic differences in adrenocorticotroic hormone, cortisol and corticotropinreleasing hormone during pregnancy. Peptides, 28, 11551161. • Latendress, G. & Ruiz, J. (2008). Bioassay research methodology: measuring CRH in pregnancy. Biological Research for Nursing, 10, 54-62. • Strong, E.F., Kleinman, K.P., Gillman, M.W. et al. (2006). Measuring corticotropin-releasting hormone in pregnant women-comparing blood collection protocols for epidemiological studies. Paediatric and Perinatal Epidemiology, 20, 67-71.
