Approximately 70 percent of extremely low birth weight infants survive to hospital discharge; of these about 30 percent develop chronic lung disease requiring supplemental oxygen to breathe. The lungs of these premature infants are easily injured by mechanical ventilators, oxygen, and infection. OBJECTIVE: To validate the Luminex technology as a method of measuring inflammatory cytokines in biological samples. METHOD: Discarded tracheal aspirates (TA) were collected from children on mechanical ventilation with full informed parental consent. The samples were obtained from the normal saline wash used to clear the tracheal lines. The cell debris in the TA was removed by centrifugation and the superanant frozen at - 80C. The samples were thawed once for protein determination, aliquoted, and then frozen. Each aliquot was thawed only once for use. IL-1B, IL-6, IL-8, and IL-10 were measured using ELISA kits from Pierce Endogen (Rockford, IL). TNF alpha was measured using a duo kit from R & D Systems (Minneapolis, MN). For the Luminex assays, all the cytokines were measured using the LincoPlex kit from Linco Research (St. Charles, MO). RESULTS: IL-1B, IL-6, IL-8, IL-10 , and TNF alpha in TA were measured in duplicate using both commercial ELISA and Luminex kits. The TA from the children in this pilot study showed elevations of cytokines typical to any inflammatory response: elevated IL-1B, IL-6, IL-8, TNF alpha, and low IL-10. In addition, Luminex assay showed that IL-2, IL-4, IL-5, IL-12, GM-CSF, and TNF-gamma were not elevated in any of the pilot samples. The five commercial ELISA kits cost US $ 865 more than the single LincoPlex kit, measured only five cytokines compared to eleven in the standard LincoPlex, and required five times the volume to measure less than half as many analytes. The elevations observed with the ELISA kits were also observed when processed by the Luminex. Additionally, five ELISA kits required two full days of sample processing to complete the pipetting, plate washing, etc, associated with handling five ELISA kits, while the Luminex kit required only five hours to complete the processing. CONCLUSIONS: Multiplexed Luminex-based fluorescent assays allow the rapid determination of eleven human cytokines in as little as 25 ul of aqueous human samples and compares favorable with ELISA in terms of cost, sensitivity, and processing speed (Thanks to Dr. Carl Bose, of UNC Chapel Hill, for providing the TA).
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