Paramètres spéciaux

Erythrocytes Thiopurine Methyltransferase Activity Immunoassay

Venous blood in EDTA tube

Whole blood must be stored at 2-8 °C and must be processed within 3 days. Reconstituted RBCs have to be processed within 8 hours or may be stored at -70 °C for up to 14 days. RBC lysate is stable at 2-8 °C for 8 hours. Activated RBC lysate is stable for 1 hour at room temperature.

TPMT activity definition: One unit of TPMT is defined as a formation
of 1 nmol of 6-MMP per ml of packed red blood cells per 60 min incubation at 37 °C.

Human

Thiopurine Methyltransferase (TPMT; EC 2.1.1.1.67) is a cytoplasmic enzyme that catalyzes the Smethylation of aromatic and heterocyclic sulfhydryl compounds. In the human population, TMPMT enzyme activity varies considerably from one individual to another; and this variation is both genetically determined and affected by some other conditions that have not been well characterized. About 85 % of people have relatively high TPMT levels. The remaining ~11 % have partial or complete TPMT deficiency. TPMT deficient individuals cannot tolerate thiopurine drugs such as 6-mercaptopurine or azathioprine, resulting in haemopoietic toxicity or sometimes death. At the same time, the majority of the patient population may not receive enough dosage of medicine and suffer from much lower therapeutic efficacy. These thiopurines are commonly used in the treatment of leukemia, inflammatory bowel disease, myasthenia gravis, autoimmune hepatitis, a variety of dermatological disorders, or as an immunosuppressant in heart/kidney transplant patients. Therefore, it is important for a clinician to measure TPMT activity in patients before these drugs are administered.

Method Description:
The TPMT Activity assay is a competitive enzyme immunoassay for the semi quantitative determination of TPMT activity in erythrocytes prepared from lysed red blood cells. TPMT catalyzes the S-methylation of 6-mercaptopurine (6-MP) with the presence of S-adenosylmethionine (SAM), the methyl donor, yielding 6-methylmercaptopurine (6-MMP). 6-MMP in the sample and 6-MMP conjugated with enzyme compete for binding with its specific antibody, which is coated on the surface of the well. The absorbance produced is inversely proportional to the amount of 6-MMP in the sample, calibrator or control. Using calibrators of known TPMT activities plotted against their response, the TPMT activity in the unknown samples can be quantified.

Phenotyping-genotyping:
A knowledge of almost all occurring mutations in a defined population is necessary when genotyping is considered to be the method of choice to predict the correct patient's phenotype. To date, molecular diagnostic methods (PCR) for the three most common alleles TPMT*2, *3A and *3C are able to identify 80-95 % of heterozygous and homozygous mutant patients. The lack of a higher concordance rate between TPMT genotype and phenotype may be caused by the existence of further rare so far unknown mutations at the human TPMT locus. For reason to define all patients with Low Moderate and High TPMT levels, phenotype is the method of choice. Phenotype testing is able to quantify the biological enzyme activity and is hence thought to be a more reliable reflection of in vivo events.

TPMT, ELISA, Thiopurine Methyl Transferase Activity, Erythrocytes TPMT, Azathioprine intolerance, Thiopurine drugs, TPMT genotype, TPMT phenotype