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

L Ford, V Graham, J Berg

Research output: Contribution to JournalArticlepeer-review



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.


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).


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%.


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.
Original languageEnglish
Pages (from-to)354-360
Number of pages6
JournalAnnals of Clinical Biochemistry
Publication statusPublished - 1 Sept 2006


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