The development and evaluation of a high-throughput automated liquid handling solution for the processing of samples for the thiopurine S-methyltransferase (TPMT) enzymatic assay

Abstract

Assessment of thiopurine S-methyltransferase (TPMT) status is advocated prior to commencing thiopurine treatment to reduce the potential for adverse drug reactions. Our laboratory provides a highly successful national TPMT phenotyping service, receiving over 36,000 specimens per year. The current manual enzyme assay is labour-intensive and so here we describe the development and validation of an automated method using the Biomek NXP and Biomek i5 liquid handling workstations. The performance of each step of the automated method was reviewed and optimised, including primary sample mixing and aliquoting, carry-over, cell lysis, mixing, incubation time and temperature. The automated method was validated to assess intra- and inter-assay precision, linearity and lower limit of quantitation. Furthermore, patient samples were analysed by both the manual and automated methods and the results were compared. Primary sample mixing and aliquoting were performed on the Biomek NXP. Of 1423 samples reviewed, 89% could be aliquoted directly from the primary tubes. On-board inversions (n = 10) could replace offline mixing and no carry-over was observed. Adequate cell lysis required a 45-minute incubation at minus 80 degrees Celsius. On the Biomek i5, optimum vortex mixing speed was 2,500 rpm. Intra-assay and inter-assay precision were excellent, with CVs of less than or equal to 2.3% and 7.4%, respectively for patient samples. The assay was linear up to 199 mIU/L (R2 = 0.992), with a lower limit of quantitation of 3.9 mIU/L. Excellent correlation was observed between the manual and automated methods (R2 = 0.955, n = 703), with the automated method having a mean bias of 0.6 mIU/L. The 37 degrees Celsius incubation time could be reduced from 60 to 10 minutes and produce comparable results, although interference was observed in samples were acetylsalicyclic acid, or metabolites, were present. The automated method was estimated to cost 7% less than the manual method, at 5.65 GBP and 6.10 GBP per sample, respectively. We have successfully developed and validated an automated method for the TPMT enzyme assay using the Biomek NXP and i5 liquid handling workstations. The TPMT results are interchangeable with those produced using the manual method. The two methods are essentially cost neutral, albeit with an inverse split between pay and non-pay. This work has demonstrated a novel use of equipment readily available to the laboratory community. There are other complex manual methods which may be candidates for this approach to automation in the future both within our own laboratory and elsewhere.

Date of Award	1 Aug 2021
Original language	English
Awarding Institution	The University of Manchester
Supervisor	William Newman (Supervisor)