Implementation of plate reader-based indooxine and Nessler protocols for monitoring L-asparaginase serum activity in childhood acute lymphoblastic leukaemia.

Abstract

Monitoring the blood serum activity of L-asparaginase in children with acute lymphoblastic leukaemia (ALL) has been highly recommended to detect enzyme inactivation that can cause relapse and to avoid unwanted toxicity. Nevertheless, perhaps at least partially due to the lack of clinically approved commercially available kits or standardized and independently reproduced and validated in-house protocols, laboratory assay-based determination of the optimal doses of L-asparaginase is not carried out routinely. In this study, we adapted previously published protocols for two plate reader-based colorimetric methods, indooxine and Nessler, to measure asparaginase activity. Mock samples with dilutions of the enzyme for initial optimization steps, and patient samples were used as a proof of principle and to compare the two protocols. For the first time the indooxine and the Nessler methods are adapted for a plate reader and L-asparaginase serum activity levels are compared by both protocols. Passing-Bablok and Bland-Altman's statistical analyses found very little difference, strong correlation (r = 0.852), and bias of only 6% between the data from the two methods when used for fresh patient samples. Furthermore, we demonstrate that the Nessler method could also be applied for frozen sera as the results, compared to fresh samples, showed little difference, strong correlation (r = 0.817), and small bias (9%). We successfully adapted and validated two methods for measuring L-asparaginase activity in cALL and provided the most detailed description to date on how to reproduce and implement them in other clinical laboratories.