Fluorescent flow cytometric assay: a new diagnostic tool for measuring beta-glucocerebrosidase activity in Gaucher disease

The purpose of this study was to determine glucocerebrosidase activity by a fluorescent flow cytometry method in patients and carriers of Gaucher disease, and in healthy controls, and correlate the results with the standard glucocerebrosidase assay in the same individuals. Biochemical diagnosis has heretofore been performed by measuring enzyme activity using a fluorimetric assay method with cell lysate. We present the results of a quantitative fluorescence-activated cell sorter (FACS) assay for diagnosis of Gaucher disease. Twenty-eight patients, 15 obligate carriers, and 21 healthy controls were tested by both the standard and FACS methods. Fluorescent products were obtained by intracellular hydrolysis of fluorescein di-beta-glucopyranoside and measured by fluorescent flow cytometer. Activity was then expressed as an index ratio of mean fluorescence of the patient sample relative to control. Specificity of the assay for lysosomal beta-glucocerebrosidase was demonstrated using conditurol-beta-epoxide (CBE), a specific irreversible inhibitor of beta-glucocerebrosidase. Both methods were performed on blood samples without knowledge of the genetic results. The mean +/- SD results using FACS were: controls 1.12 +/- 0.26, patients with Gaucher disease 0.21 +/- 0.09 and carriers 0.76 +/- 0.15. Using the standard method, mean enzyme activities were: controls 35.7 +/- 9.0 uU/mg protein, patients with Gaucher disease 8.4 +/- 4.5 uU/mg protein, and carriers 32.2 +/- 6.9 uU/mg protein. Thus, FACS is a reliable and easy to use method for determination of enzyme activity in Gaucher disease, with excellent correlation with the standard method. The FACS method allows single cell enzyme evaluation rather than global activity of cell lysate, and gives excellent separation between patients, carriers, and controls.