2.2. ADA Assay

Pleural fluid ADA activity was evaluated using the Diazyme Assay (Diazyme Laboratories, San Diego (CA), United States). ADA irreversibly catalyzes the conversion of adenosine (or deoxyadenosine) to inosine (or deoxyinosine) and ammonia. The classical method of Giusti and Galanti is not easily automated because the ammonia is measured with Berthelot's reaction. Hopkinson et al. (1969) described an enzyme assay for ADA determination based on nucleoside phosphorylase (NP) and xanthine oxidase (XOD). The quantification of ADA was based on the measurement of uric acid after enzymatic reactions with NP, XOD, inosine, and other reagents. Briefly, the Diazyme ADA Assay uses a kinetic method. It is based on the enzymatic deamination of adenosine to inosine which is converted to hypoxanthine by NP. Hypoxanthine is then converted to uric acid and hydrogen peroxide by XO. One unit of total ADA was defined as the amount of the enzyme that generates one micromol of inosine per min at 37°C from the substrate adenosine [19]. P-ADA assay was performed independently of the proven diagnosis of the patient in a hospital with the same health professional and conducted according to the manufacturer's guidelines.