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 LearnedChallenges

• 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 consultant-

assess 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 corticotropin-releasing hormone during pregnancy. Peptides, 28, 1155-1161.

• 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.