Thiopurine methyltransferase enzyme activity determination: Measurement of TPMT activity
Direct health care costs, including the cost of the TPMT assay, scheduled laboratory investigations and nonscheduled diagnostic investigations were recorded. The TPMT assay was valued at $50.00, and the local regional laboratory and provincial billing guidelines were used to determine the value of laboratory and diagnostic investigations (Table 2). Telephone calls to nurses and visits to a physician because of side effects or consequences of AZA treatment were also recorded and valued according to local provincial billing guidelines (Table 2).
Heparinized blood samples were centrifuged within 2 h of collection, and the plasma was removed. The erythrocytes were then washed with saline and resuspended in 2 mL of saline to determine the hematocrit. The cells were then lysed with ice-cold water, cellular debris was removed, and the supernatant was assayed for TPMT activity using the method previously described by Weinshilboum et al. Briefly, the erythrocyte lysate was treated with Chelex-100 (BioRad, USA) to remove magnesium before the assay. An aliquot was then added to a reaction mixture containing 6-MP, S-adenosyl-L-[14C]methionine, dithiotre-itol and allopurinol in a phosphate buffer (pH 7.5), and the resulting mixture was incubated for 1 h at 37°C. The reaction was then stopped by the addition of a borate buffer; the resulting 6-MMP was extracted into isoamyl alcohol and quantitated through scintillation counting of the 14C buy-asthma-inhalers-online.com.
TABLE 2 Direct costs for laboratory and diagnostic investigations
|Thiopurine methyltransferase assay*||50.00|
|Complete blood count*||15.05|
|Hepatitis A, B and C serology*||15.00|
|Phone call with nurse*||22.50|
|Family practitioner visit*||28.97|
*Values represent direct cost based on Alberta Provincial Laboratory fee codes; fValue represents direct (salary plus benefit) cost based on 30 min time interval; *Values represent direct reimbursement based on Alberta Medical Association fee codes