hrvatski

Usporedba osjetljivosti ugnježđenog PCR i kvantitativnog PCR u određivanju Bcr/Abl p210 transkripta kronične mijeloične leukemije

INTRODUCTION: For minimal residual disease (MRD) detection in chronic myelogenous leukemia (CML) patients who achieved complete clinical remission and complete cytogenetic response, nested PCR and quantitative real-time PCR (Q-PCR) can be used. Achieving of molecular remission is the goal of therapy, so it is of critical importance for clinical utility to obtain high sensitivity of molecular testing. AIM: Compare the level of sensitivity of nested PCR and Q-PCR in detection of Bcr/Abl p210 transcripts in Bcr/Abl-positive cell dilution model. MATERIALS AND METHODS: For determination of sensitivity level serial dilutions of K562 cell line (Bcr/Abl-positive) in NB4 cell line (Bcr/Abl-negative) have been made (range of dilution of positive cells: 10-3 – 10-7). Isolated RNA samples were transcribed into cDNA and tested for p210 transcript by nested PCR (Biomed1, Leukemia 1999:13) and Q-PCR-Taqman (EAC, Leukemia 2003:17). Bone marrow and peripheral blood samples of two CML patients on Imatinib mesylate therapy, were also tested by both methods. RESULTS: In cells dilution test nested PCR showed sensitivity for detecting Bcr/Abl positive cell of 10-4, and Q-PCR showed sensitivity of 10-6. Both methods detected p210 transcripts in bone marrow samples of CML patients. However, Q-PCR detected also transcripts in peripheral blood samples, with significantly lower level of transcription in comparison to bone marrow samples. CONCLUSION: Although frequently quoted as nested-PCR being aprox. 1 log more sensitive than Q-PCR, in our marker-positive cell line K562 dilution experiment we documented higher sensitivity for standardized Europe Against Cancer Program Q-PCR method.

Bcr/Abl p210; ugnježđeni PCR; kvantitativni PCR; osjetljivost

nije evidentirano

engleski

Comparison of sensitivity of nested PCR and quantitative PCR in Bcr/Abl p210 transcript deztection in chronic myelogenous leukemia

INTRODUCTION: For minimal residual disease (MRD) detection in chronic myelogenous leukemia (CML) patients who achieved complete clinical remission and complete cytogenetic response, nested PCR and quantitative real-time PCR (Q-PCR) can be used. Achieving of molecular remission is the goal of therapy, so it is of critical importance for clinical utility to obtain high sensitivity of molecular testing. AIM: Compare the level of sensitivity of nested PCR and Q-PCR in detection of Bcr/Abl p210 transcripts in Bcr/Abl-positive cell dilution model. MATERIALS AND METHODS: For determination of sensitivity level serial dilutions of K562 cell line (Bcr/Abl-positive) in NB4 cell line (Bcr/Abl-negative) have been made (range of dilution of positive cells: 10-3 – 10-7). Isolated RNA samples were transcribed into cDNA and tested for p210 transcript by nested PCR (Biomed1, Leukemia 1999:13) and Q-PCR-Taqman (EAC, Leukemia 2003:17). Bone marrow and peripheral blood samples of two CML patients on Imatinib mesylate therapy, were also tested by both methods. RESULTS: In cells dilution test nested PCR showed sensitivity for detecting Bcr/Abl positive cell of 10-4, and Q-PCR showed sensitivity of 10-6. Both methods detected p210 transcripts in bone marrow samples of CML patients. However, Q-PCR detected also transcripts in peripheral blood samples, with significantly lower level of transcription in comparison to bone marrow samples. CONCLUSION: Although frequently quoted as nested-PCR being aprox. 1 log more sensitive than Q-PCR, in our marker-positive cell line K562 dilution experiment we documented higher sensitivity for standardized Europe Against Cancer Program Q-PCR method.

Bcr/Abl p210; nested PCR; quantitative PCR; sensitivity

nije evidentirano