Diagnosis of Gaucher disease by a flow cytometric assay

Abstract

Gaucher disease (GD) is an inherited autosomal recessive disorder, caused by a deficient lysosomal β-glucocerebrosidase (GC) resulting in the acumulation of the glycoslyceramide in the lysosomes of cells of the reticuloendothelial system. Measurement of GC activity during the emzyme replacement therapy and following allogeneic bone marrow transplantation or gene therapy would be useful. GC activity is commonly measured using radiolabeled glucocerebroside in cell lysates. This method of in vitro assay does not allow one to measure GC activity on a single cell basis. The fluorescence-activated cell sorter (FACS) has been used for the measurement of GC activity in single cells. To determine the presence of enzyme dificiency for the diagnosis of GD and monitor the efficacy of enzyme replacement therapy, we examed a flow cytometric assay for GC using 5'-pentafluorobenzoyllaminofluorescein-di-β-D-glucoside (PFBFDGlu) which has been reported to be a substrate for GC. The study group consisted of 11 normal individuals, 14 obligatory carriers (parents of patients) and 11 known Gaucher patients (8 of type 1, 1 of type 2, and 2 of type 3). The peripheral blood mononuclear cells obtained by ficoll density centrifugation were washed three times in RPMI 1640 containing 10 % FBS and immediately processed for substrate loading. To determine the optimal assay condition 5 × 105 cells were mixed with an equal volume of 1 mM PFBFDGlu (0.5 mM final) and incubated at 37°C for 0 to 60 min or cells were incubated for 30 min at 0.5, 1 and 2 mM PFBFDGlu. To terminate substrate loading, 0.5 ml of ice-cold RPMI 1640 containg 4 % FBS was added to the cell suspension. FACS analysis was done on a FACSTAR Plus. FACS analysis revealed that PFBFDGlu loaded normal or carrier cells show brighter median fluorescence (MF) levels than patients' cells at all incubation time points, the fluorescence distributions show minimal overlap with a 30-min incubation. With longer incubation times, however, fluorescence histograms of cells became increasingly broader, and the determination of MF was difficult. At our optimal assay condition (0.1 mM PFBFDGlu, 30 min incubation), the fact that fluorescence distributions of all the tested ceUs from patients (6.237±1.866, n=27) did not overlap with those from carriers (13.704 ± 4.232, n=27) or from normal individuals (11.857 ± 1.351 n=14) indicating this sample and fast flow cytometric GC assay will be useful for diagnosis of GD. Furthermore, in our study, some population of cells which had increased GC activity can be detectable in cells from Gaucher patient after treatment with Cerezyme® indicating this method can be useful for monitoring effective dosage and frequency of enzyme replacement therary from each patients, as well as the efficacy of gene therapy for Gaucher patient in future.