title: Whole-blood thiopurine S-methyltransferase activity with genotype concordance: a new, simplified phenotyping assay.
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%.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Clinical Biochemistry Department, City Hospital, Birmingham, UK. Loretta.ford@swbh.nhs.uk
authors.forename: Loretta
authors.initials: L
authors.lastname: Ford
authors.forename: Valerie
authors.initials: V
authors.lastname: Graham
authors.forename: Jonathan
authors.initials: J
authors.lastname: Berg
mesh_terms: D001741:Black or African American / Q000235:genetics; D000483:Alleles; D044466:Asian People / Q000235:genetics; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D004912:Erythrocytes / Q000201:enzymology / Q000378:metabolism; D005787:Gene Frequency; D005838:Genotype; D006801:Humans; D007527:Isoenzymes / Q000097:blood / Q000235:genetics / Q000378:metabolism; D008780:Methyltransferases / Q000097:blood* / Q000235:genetics* / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D010641:Phenotype; D015203:Reproducibility of Results; D013866:Thioguanine / Q000097:blood / Q000378:metabolism; D044465:White People / Q000235:genetics
chemical_list: D007527:Isoenzymes; D008780:Methyltransferases; C022745:thiopurine methyltransferase; D013866:Thioguanine