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Flow cytofluorometric analysis of enzyme reactions based on quenching of fluorescence by the final reaction product: detection of glucose-6- phosphate dehydrogenase deficiency in human erythrocytes

CJ Van Noorden, F Dolbeare and J Aten

Laboratory of Cell Biology and Histology, University of Amsterdam, The Netherlands.

We developed a method for accurate cytofluorometric analysis of the final reaction product of enzyme reactions in individual cells. Glucose- 6-phosphate dehydrogenase (G6PD) activity in human erythrocytes was demonstrated cytochemically, and the amount of final reaction product (formazan) per cell was detected indirectly by quenching of autofluorescence generated by glutaraldehyde fixation. Formazan quenches fluorescence in a dose-dependent manner. The method has been used for detection of G6PD deficiency. Heterozygous and homo(hemi)zygous deficiency could easily be established, even in cases of extreme "Lyonization" where microscopic inspection failed to discriminate between either normal individuals and heterozygously deficient patients or heterozygously and homozygously deficient patients. The principle of quenching of fluorescence by final reaction products of enzymes can be applied to flow cytofluorometric analysis of enzyme activity in individual cells in general.

Volume 37, Issue 9, pp. 1313-1318, 09/01/1989
Copyright © 1989 by The Histochemical Society


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