Quantitative Beutler test for newborn mass screening of galactosemia usinga fluorometric microplate reader

Background: The Beutler enzyme spot test is an effective assay for newborn mass screening of galactosemia, but it is qualitative and relies on visual interpretation. We describe a quantitative, instrumental modification of th e assay. Methods: We modified the macroscopic visual Beutler enzyme spot test by add ing extraction of blood components from filter paper, deproteinization with acetone-methanol, and quantification and recording by a fluorescent microp late reader and personal computer. All handling was performed in microplate s. The measurement time was 90 min. Results: Fluorescence intensity (FI) of healthy controls correlated with he matocrit and gal actose-1-phosphate uridyltransferase (GALT) activity. Pati ents with GALT deficiency were distinguished clearly from healthy subjects and heterozygous carriers by Fl. FI decreased to 75% of the initial activit y after storage at 25 degrees C for 3 days and to 40% after storage at 37 d egrees C for 7 days. Screening of 46 742 newborns yielded I false-positive result (in a heterozygous carrier), I patient with glucose-6-phosphate dehy drogenase deficiency, and no apparent false negatives as judged by concurre nt measurements of galactose and galactose-l-phosphate. Conclusions: The quantitative Beutler test can provide precise GALT activit y in newborn mass screening, and can take into consideration the influence of high temperature and humidity, duration between sampling and testing, an d anemia. This method is clinically useful, simple, automated, and highly r eliable for newborn mass screening of galactosemia. (C) 2000 American Assoc iation for Clinical Chemistry.