Special modifications of the fluorescent screening method for glucose-6-phosphate dehydrogenase deficiency.

for glucose-6-phosphate dehydrogenase (G-6-PD) deficiency.1 This method depends upon the fluorescence of reduced triphosphopyridine nucleotide (TPNH ) as an indicator of G-6-PD activity. Ten j.d. of whole blood are added to 100 jil. of a screening solution containing glucose-6-phosphate, TPN, buffer, and saponin. A spot is made on ordinary filter paper after 5 minutes incubation at room temperature. The spot is permitted to dry, and is then examined for fluorescence under long-wave UV light. We now introduce modifications of the procedure which may enhance its usefulness under certain circumstances. It has been found that when the test reagent originally described is used the fluorescence on filter paper fades relatively rapidly. It is difficult to differentiate normal from enzyme-deficient samples after more than 24 hours. While this does not pose any problem for the ordinary clinical laboratory, it may limit the usefulness of the technic for field surveys. In such studies it may be desirable to examine the results of tests under ultraviolet light in a distant central laboratory, possibly after several days’ delay. It has been found that the stability, and indeed the initial intensity, of fluorescence can be greatly enhanced by substituting an appropriate trishydrochloride buffer for phosphate buffer. A second modification makes it possible to eliminate almost entirely the slight fluorescence which sometimes appears when blood from individuals with the mildest forms of glucose-6-phosphate dehydrogenase deficiency are examined, especially using a reaction mixture buffered with tris. This fluorescence can be eliminated by adding oxidized glutathione (GSSG) to the reaction mixture. When GSSG is present, TPNH is oxidized through the action of glutathione reductase (GSSG-R). But the activity of glutathione reductase is normally only approximately one-quarter of that of the activity of the glucose-6-phosphate dehydrogenase-phosphogluconic dehydrogenase sys-