Abstract
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.
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.
| Original language | English |
|---|---|
| Pages (from-to) | 354-360 |
| Number of pages | 6 |
| Journal | Annals of Clinical Biochemistry |
| DOIs | |
| Publication status | Published - 1 Sept 2006 |