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  • Title: Improved sensitivity for methotrexate analysis using enzyme multiplied immunoassay technique on the Siemens Viva-E instrument.
    Author: Borgman MP, Hiemer MF, Molinelli AR, Ritchie JC, Jortani SA.
    Journal: Ther Drug Monit; 2012 Apr; 34(2):193-7. PubMed ID: 22377747.
    Abstract:
    BACKGROUND: The available assay kit for methotrexate (MTX) using the Syva enzyme multiplied immunoassay technique (EMIT) reagents on the Siemens Viva-E instrument allows for the detection of MTX in serum or plasma to concentrations as low as 0.3 μmole/L. Current clinical decision points for MTX therapeutic drug monitoring and leucorvorin rescue exist at concentrations below that limit. OBJECTIVE: The goal of this study was to lower the limit of MTX quantitation to 0.05 μmole/L using the EMIT assay technology. METHODS: EMIT MTX assay parameters were modified on the Viva-E instrument to increase the sample volume, alter the calibration method, and employ an alternate calibrator set created to achieve lower detection. Intraassay and interassay precision was assessed for MTX controls. RESULTS: We observed a CV of 9.4% for intraassay precision with a bias of <0.01% and a CV of 15.7% for interassay precision with a bias of 22.5% for the 0.05 μmole/L control. Precision data for all other controls were <4%. The modified EMIT MTX assay and the unmodified approved assay were compared with a high sensitivity fluorescence polarization immunoassay method. Linear regression of correlation data revealed that both the modified and the commercial EMIT assays produced positive bias compared with the high sensitivity fluorescence polarization immunoassay method (y-int = 0.03 and 0.08, respectively). However, the modified EMIT assay had the best correlation in the low range (0.03-2 μmole/L). Additionally, endogenous and chemical interference testing demonstrated that the modified assay was not affected to a clinically significant extent. CONCLUSIONS: The described modifications have enhanced the sensitivity of the Syva EMIT assay for MTX measurements down to 0.05 μmole/L with acceptable precision that can be used in clinical practice for monitoring MTX therapy.
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