Multi-Site Validation of a Newly FDA-Cleared Automated Methotrexate Immunoassay for Therapeutic Drug Monitoring

Methotrexate (MTX), a synthetic antimetabolite and folic acid antagonist, has become a cornerstone in the management of malignant diseases. However, MTX toxicity can be severe and potentially life-threatening, especially with high-dose regimens used in oncology. Given its narrow therapeutic index, precise monitoring of MTX levels is critical to optimize efficacy while minimizing toxicity.

We validated the performance of the Roche ONLINE therapeutic drug monitoring (TDM) Methotrexate Assay on Cobas c502 and Cobas pro c503 platforms across 2 clinical sites, focusing on key analytical parameters such as linearity, accuracy, precision, dilution, carryover, and method comparison.

The assay was linear across the analytical measurement interval (AMI) of 0.04-1.2 µmol/L. No significant carryover was observed (i.e., <5% bias). Automatic and manual dilutions demonstrated <10% bias, and the coefficients of variation (CVs) for precision and percentage bias for accuracy were <10%. A method comparison using 40 patient samples showed a strong correlation between the Roche and ARK MTX assays, with a Deming regression slope of 0.9634, an intercept of -0.0095, and Pearson correlation coefficient (r) = 0.9935, with a mean percentage difference (%Bias) of -12. Comparison with LC-MS/MS demonstrated high agreement for both assays. The ARK assay yielded a slope of 0.8873, an intercept of 0.0145, r = 0.9978, and %Bias of -6, while the Roche assay yielded a slope of 0.8541, an intercept of 0.0047, r = 0.9919, and %Bias of -18.

The Roche ONLINE TDM MTX assay is a robust and clinically adequate method for methotrexate quantification and therapeutic drug monitoring.