BACKGROUND AND OBJECTIVES: Accurate quantification of BCR-ABL mRNA is of critical importance for managing patients with chronic myeloid leukemia (CML) who are receiving imatinib therapy. RNA degradation thus constitutes a potential problem for laboratories quantifying minimal residual disease (MRD). Patients' samples that take a long time to be transported from the hospital to the analyzing laboratory may be subject to RNA degradation with a corresponding loss in sensitivity and possible generation of false negative results. Recently, RNA preservation systems have been developed in order to improve RNA stability. The aim of the present study was to investigate such a system. DESIGN AND METHODS: We evaluated the performance of the PAXgene Blood RNA Kit in follow-up CML peripheral blood samples and compared the results to those from unstabilized parallel Trizol extracted samples. The different sample processing methods were evaluated by real-time polymerase chain reaction (PCR) analysis. RESULTS: RNA isolated with the PAXgene system gave a superior yield per milliliter of blood than did the routine Trizol extraction method. However, although of comparable quality, the RNA did not PCR-amplify as efficiently as equal amounts of RNA from routinely processed samples. Therefore, RNA processed with the PAXgene system showed decreased sensitivity for MRD detection, resulting in false negative results. The sensitivity was comparable to that of samples processed routinely 20-30 hours after phlebotomy. INTERPRETATION AND CONCLUSIONS: We conclude that routinely processed, i.e. unstabilized, peripheral blood that reaches the laboratory and is processed within 30 hours is preferable for MRD detection. Optimal results were achieved with fresh samples processed within 5 hours with the Trizol method. However, RNA stabilization may be useful if sample transit is expected to exceed 30 hours.
BACKGROUND AND OBJECTIVES: Accurate quantification of BCR-ABL mRNA is of critical importance for managing patients with chronic myeloid leukemia (CML) who are receiving imatinib therapy. RNA degradation thus constitutes a potential problem for laboratories quantifying minimal residual disease (MRD). Patients' samples that take a long time to be transported from the hospital to the analyzing laboratory may be subject to RNA degradation with a corresponding loss in sensitivity and possible generation of false negative results. Recently, RNA preservation systems have been developed in order to improve RNA stability. The aim of the present study was to investigate such a system. DESIGN AND METHODS: We evaluated the performance of the PAXgene Blood RNA Kit in follow-up CML peripheral blood samples and compared the results to those from unstabilized parallel Trizol extracted samples. The different sample processing methods were evaluated by real-time polymerase chain reaction (PCR) analysis. RESULTS: RNA isolated with the PAXgene system gave a superior yield per milliliter of blood than did the routine Trizol extraction method. However, although of comparable quality, the RNA did not PCR-amplify as efficiently as equal amounts of RNA from routinely processed samples. Therefore, RNA processed with the PAXgene system showed decreased sensitivity for MRD detection, resulting in false negative results. The sensitivity was comparable to that of samples processed routinely 20-30 hours after phlebotomy. INTERPRETATION AND CONCLUSIONS: We conclude that routinely processed, i.e. unstabilized, peripheral blood that reaches the laboratory and is processed within 30 hours is preferable for MRD detection. Optimal results were achieved with fresh samples processed within 5 hours with the Trizol method. However, RNA stabilization may be useful if sample transit is expected to exceed 30 hours.
Authors: Laura Kennedy; J Keith Vass; D Ross Haggart; Steve Moore; Michael E Burczynski; Dan Crowther; Gino Miele Journal: Biomark Insights Date: 2008-08-25