Automation of the Whole-Blood Thiopurine -Methyltransferase (TPMT) Phenotyping Assay Using the Biomek NX and Biomek i5 Liquid-Handling Workstations.

Abstract

Assessment of thiopurine S-methyltransferase (TPMT) status is required before commencing thiopurine treatment to reduce the potential for adverse drug reactions. Our laboratory has provided a national TPMT phenotyping service since 2003. Our assay uses 6-thioguanine as substrate and detection of 6-methylthioguanine via high-performance liquid chromatography (HPLC) fluorescence. Here, we report the automation of this complex, labor-intensive, manual assay using the Biomek NXP and Biomek i5 liquid-handling workstations. We optimized assay performance and validated for precision, linearity, and lower limit of quantitation. We also compared results from the manual and automated methods. Primary sample mixing and aliquoting were performed on the Biomek NXP. On-board inversions (n = 10) replaced offline mixing. No carryover was observed. Eleven percent of primary sample tubes were incompatible with the Biomek NXP, and these were assayed manually. The Biomek i5 was used to automate the enzyme assay. Optimum vortex mixing was achieved at 2500 rpm for 60 s, and the temperature was set to 37.0 °C for the 60 min enzyme incubation. Intra- and inter-assay precision were excellent, with coefficients of variation (CVs) of ≤2.3% and ≤7.4%, respectively, for patient samples. Linearity was demonstrated up to 199 mIU/L (R2 = 0.992), with a lower limit of quantitation of 3.9 mIU/L. Correlation between the manual and automated methods was good (R2 = 0.979, n = 405), with results being interchangeable. We have successfully developed and validated a novel automated method for the TPMT phenotyping enzyme assay. The two methods are cost-neutral. Automation of other complex enzyme assays may be possible using this approach.