Peripheral blood samples collected for measuring asparaginase activity were maintained at room temperature and processed within 24 hours.
The resulting plasma aliquots were stored immediately at −80°C and tested in batches. Asparaginase activity in banked plasma samples was analysed using a modified indooxine assay.14 (link) Replicates of plasma aliquots were incubated with excess L-aspartic acid ß-hydroxamate (AHA; Sigma) at 37°C in 96-well microtitre plates. Hydroxylamine generated from ASNase hydrolysis of AHA was condensed with 8-hydroxyquinoline (Sigma) and the resulting indooxine quantified spectrophotometrically at 710 nm (Spectramax, Molecular Devices). A trough asparaginase activity ≥100 IU/L was considered adequate.7 (link) The dynamic linear range of the assay was 100–1000 IU/L. For ASNase activity levels below the dynamic range, plasma samples were retested using a modification of the assay with a dynamic range of 5–100 IU/L. The assay standard curve was developed using serial dilutions of the reference asparaginase in pooled human plasma. Assay performance was monitored by including plasma samples with known asparaginase activity (low, mid and high activity) as calibrators.
The resulting plasma aliquots were stored immediately at −80°C and tested in batches. Asparaginase activity in banked plasma samples was analysed using a modified indooxine assay.14 (link) Replicates of plasma aliquots were incubated with excess L-aspartic acid ß-hydroxamate (AHA; Sigma) at 37°C in 96-well microtitre plates. Hydroxylamine generated from ASNase hydrolysis of AHA was condensed with 8-hydroxyquinoline (Sigma) and the resulting indooxine quantified spectrophotometrically at 710 nm (Spectramax, Molecular Devices). A trough asparaginase activity ≥100 IU/L was considered adequate.7 (link) The dynamic linear range of the assay was 100–1000 IU/L. For ASNase activity levels below the dynamic range, plasma samples were retested using a modification of the assay with a dynamic range of 5–100 IU/L. The assay standard curve was developed using serial dilutions of the reference asparaginase in pooled human plasma. Assay performance was monitored by including plasma samples with known asparaginase activity (low, mid and high activity) as calibrators.