The Phenylalanine Microwell Enzyme Assay (PHE-MW EA) is designed for the quantitative determination of phenylalanine in neonatal blood spots. It is for use as an aid in screening newborns for elevated phenylalanine levels.The PHE-MW EA is an enzyme assay which follows an acid extraction of the phenylalanine contained in a 3/16” (5 mm) blood spot. In this assay, a single blood spot is extracted in a special semi-porous membrane bottom plate. Following extraction, all 96 extracts are simultaneously transferred to a conventional microwell plate, by means of a vacuum manifold. A neutralizing buffer is added to each acid extract, followed by addition of a combination enzyme-substrate reagent. This rapidly oxidizes the phenylalanine to phenylpyruvate, reducing NAD to NADH+ in the process. The NADH+ reduces a colorless dye to form a colored end-product with an absorbance maximum at 570 nm. The absorbance read is directly proportional to the amount of phenylalanine in the sample.