Proper assaying of cortisol
There are few things I think we need to consider before proceeding in assaying of cortisol. First you must read and understand the principle, procedure of the test and don’t forget to wear the proper PPE (laboratory gown, gloves, mask , hair cap,& goggles), and after reading you may now prepare the materials needed in the method like the reagents, specimen and laboratory materials.
Make sure that all thing are set and properly labeled and organized to avoid confusion and inaccurate results. And most of all don’t forget the universal precaution “to treat all specimens as infectious and potentially hazardous that can transmit diseases”.
Proper assaying of cortisol requires preliminary extraction of free cortisol before measurement, because large number of cortisol metabolites in urine reacts with the cortisol antibody in the assay and since interfering substances are more soluble than cortisol in urine, the cortisol can be extracted with organic solvent.
Generally, the sample is mixed with organic solvent with the proper ratio (1:5), the layer allowed to separate and the aqueous layer is removed. An aliquot of the organic layer containing the free cortisol must be transferred to separate tube, evaporated to dryness, and redissolved in buffered solution that can be use as unknown sample in the assay for serum cortisol. Extraction efficiency can be monitored by adding known cortisol standard to an aliquot of the specimen and then extracting the spiked sample in parallel with an aliquot of specimen to which no standard has been added.
Extraction efficiency of cortisol can be calculated from the difference in measured cortisol between the two samples divided by the known amount of cortisol standard which is added to one of the samples.
The calculation of the amount of cortisol in an extracted sample must take into account the dilution with organic solvent and the extraction efficiency.
Reference:
http://www.flickr.com/photos/tym/1255655367/
Make sure that all thing are set and properly labeled and organized to avoid confusion and inaccurate results. And most of all don’t forget the universal precaution “to treat all specimens as infectious and potentially hazardous that can transmit diseases”.
Proper assaying of cortisol requires preliminary extraction of free cortisol before measurement, because large number of cortisol metabolites in urine reacts with the cortisol antibody in the assay and since interfering substances are more soluble than cortisol in urine, the cortisol can be extracted with organic solvent.
Generally, the sample is mixed with organic solvent with the proper ratio (1:5), the layer allowed to separate and the aqueous layer is removed. An aliquot of the organic layer containing the free cortisol must be transferred to separate tube, evaporated to dryness, and redissolved in buffered solution that can be use as unknown sample in the assay for serum cortisol. Extraction efficiency can be monitored by adding known cortisol standard to an aliquot of the specimen and then extracting the spiked sample in parallel with an aliquot of specimen to which no standard has been added.
Extraction efficiency of cortisol can be calculated from the difference in measured cortisol between the two samples divided by the known amount of cortisol standard which is added to one of the samples.
The calculation of the amount of cortisol in an extracted sample must take into account the dilution with organic solvent and the extraction efficiency.
Reference:
http://www.flickr.com/photos/tym/1255655367/
Tietz textbook of clinical chemistry 2nd edition edited by burtis & ashwood
