Fluorescent Spot Screening Test for Galactosemia: Increased Sensitivity

Galactosemia, caused by an hereditary deficiency (1) of galactose-1-phosphate uridyltransferase (EC 2.7.7.12), can be treated easily and successfully if the condition is discovered in the newborn. Blood tests tailored to mass screening include microbiological (2, 3) and en2ymatic (4) assays of galactose as well as a simple spot test for the presence or absence of transferase (5). Principle of the method The latter test, originally described by BEUTLER & BALUDA (5) for liquid blood samples, has since been adapted to blood collected and dried on filter paper and is now widely used for large-scale screening. Individual tests are set up in the wells of hemagglu-tination trays. Filter paper discs of appropriate size containing dried blood are incubated with galactose-1-phosphate and uridine diphosphoglucose as the substrates, in the presence of EDTA, a hemolyzing agent and NADP, in buffer. When transferase is present, a sequence of secondary reactions is initiated. They are catalyzed by auxiliary enzymes also present in the blood spots: phosphoglucomutase (EC 2.7.5.1), aldose 1-epimerase (EC 5.1.3.3), glucose-6-phosphate dehydrogenase (EC 1.1.1.49), and phosphogluconate dehydrogenase (EC 1.1.1.44); In the last two steps, NADP is reduced to NADPH. When activated with long wave ultraviolet light (365 nm) the reduced nucleotide will emit blue fluorescence. In the test, it is detected by spotting a drop of the reaction mixture on filter paper which is air dried and then inspected under UV light. Thus, lack of fluorescence will indicate absence of transferase. The spot test In our screening laboratory, 2 paper discs measuring 3mm in diameter are incubated in 60 \ of the original reaction mixtur