A fluorometric assay for detection of fluorescein in plasma

Background: Ingestion of ethylene glycol (EG) from antifreeze can lead to metabolic acidosis, renal injury and death. The yellow-green dye, fluorescein, is added to antifreeze to facilitate detection of cooling system leaks and help deter ingestion. Gas chromatography is the best method for quantitating serum EG concentrations but the method is difficult and few hospital laboratories perform this assay. A rapid and reliable diagnostic test to evaluate potential antifreeze poisoning victims is needed. Methods: A fluorometric assay for the detection of fluorescein in plasma was developed using a Hitachi F2000 fluorescence spectrophotometer. Blank human plasma from a date-expired banked single donor unit was spiked with fluorescein concentrations from 2.0-200ng/mL. The low concentration is over an order of magnitude less than calculated peak fluorescein concentration in a 70 kg adult with a 30 mL antifreeze ingestion. Results: A seven point calibration curve including a blank for these data was linear with [r.sup.2] = 0.999 using the average of triplicate determinations of each point. The limit of detection was determined to be 0.5 ng/mL with a limit of quantitation of 2.0 ng/mL. Previous reports examining fluorescence using a Wood's lamp suffered from light contamination as a result of the broad wavelength range of the light. We solved this problem by use of specific excitation (494nm) and emission (516nm) wavelengths. Analysis of blank samples demonstrated that typical plasma components did not interfere with the assay. Conclusions: A fluorometric assay can detect fluorescein in plasma as a surrogate marker for EG ingestion. The preliminary results obtained with this assay are promising and support further investigation of the use of this assay in actual antifreeze exposures.
Palmer RB (1,2), O'Campo Y (2), Dart RC (1). (1) Rocky Mountain Poison & Drug Center Denver Health, Denver, CO; (2) Dept. Chemistry & Biochemistry, University of Northern Colorado, Greeley, [...]