Annals of Clinical Biochemistry > Volume 43, Number 5 > Pp. 354-360

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Bayer Award

Whole-blood thiopurine S-methyltransferase activity with genotype concordance: a new, simplified phenotyping assay

Loretta Ford, Valerie Graham and Jonathan Berg

Clinical Biochemistry Department, City Hospital, Dudley Road, Birmingham B18 7QH, UK

Background: We have developed a new thiopurine S-methyltransferase (TPMT) phenotyping method that measures TPMT activity in whole blood. To evaluate this assay, we compared it with conventional TPMT phenotyping, which uses a red blood cell (RBC) lysate and genotyping for analysis of common TPMT mutations.

Methods: Whole-blood and RBC lysates were prepared from 402 patients' samples received for routine analysis. The TPMT activity of lysates was determined using 6-thioguanine as substrate with high-performance liquid chromatographic (HPLC) analysis and fluorimetric detection. DNA was extracted from buffy coats using phenol-chloroform extraction. A multiplex amplification refractory mutation system (ARMS) strategy was used to screen for the common TPMT mutations TPMT*2 and TPMT*3 (TPMT*3A, TPMT*3C and TPMT*3D).

Results: TPMT activities were higher in the whole-blood (mean TPMT activity 51 nmol 6-MTG/gHb/h) compared with the RBC lysate (37 nmol 6-MTG/gHb/h). Overall, concordance with TPMT genotypic analysis was 97% for both the new whole-blood and standard RBC lysate methods. Between low TPMT activity and heterozygotes, both phenotypic methods gave a concordance of 79%.

Conclusion: Using multiplex ARMS testing for TPMT*2 and 3*C mutations to define the cut-off between low and normal TPMT activity, we have demonstrated that the new whole-blood TPMT phenotyping method performs as well as the conventional RBC lysate assay. This new method overcomes the need to prepare a RBC lysate, a process which is time consuming and increases analytical variation. The resulting assay is better suited to a regional or national TPMT phenotyping service.

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				L. Ford, P. Kampanis, and J. Berg Thiopurine S-methyltransferase genotype-phenotype concordance: used as a quality assurance tool to help control the phenotype assay Ann Clin Biochem, March 1, 2009; 46(2): 152 - 154. [Abstract] [Full Text] [PDF]