Protocol for a laboratory-developed chromogenic anti-Xa assay for rivaroxaban monitoring

Rivaroxaban monitoring is essential in specific clinical scenarios, such as renal impairment or emergency surgery. While LC-MS/MS is the gold standard, its high-cost limits accessibility. This study describes the development and validation of a cost-effective, laboratory-developed chromogenic anti-Xa assay suitable for automated platforms. The assay utilizes the competitive inhibition of Factor Xa, where residual enzyme activity is measured via the cleavage of a specific chromogenic substrate, with absorbance being inversely proportional to the rivaroxaban concentration. Dual-Range Quantification: Validated for both routine therapeutic levels (30-500 ng/mL) and an extended range (500-1500 ng/mL) via automated dilution. Kinetic Optimization: Employs a specific 180-second incubation and a stabilized measurement window (20-80 s) to maximize linearity. Reference Comparability: Demonstrates high correlation with LC-MS/MS (r = 0.997) and meets CLSI precision requirements.