Impact of RNA Stabilization on Minimal Residual Disease Assessment in Chronic Myeloid Leukemia.

Abstract

Background and objective: Accurate quantification of BCR-ABL mRNA is of critical importance for managing chronic myeloid leukemia (CML) patients receiving Imatinib therapy. RNA degradation thus constitutes a potential problem for laboratories quantifying minimal residual disease (MRD). Patient samples with long transport times between the hospital and 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. Design and methods: The aim of the study was to investigate the performance of the PAXgene Blood RNA Kit in CML follow-up peripheral blood samples and compared the results to unstabilized parallel Trizol extracted samples. The different sample processing methods were evaluated by real-time PCR. Results: RNA isolated with the PAXgene system gave a superior yield per ml blood than with the routine Trizol extraction method. However, although of comparable quality, the RNA did not PCR-amplify as efficiently compared to equal amounts of RNA from routinely processed samples. Therefore, RNA processed with the PAXgene system showed to decreased sensitivity for MRD detection, resulting in false negatives. The sensitivity was comparable to 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.

Molecular monitoring and mutation analysis of patients with advanced phase CML and Ph+ ALL receiving dasatinib

“…Peripheral blood or bone marrow mononuclear cells were used for RNA isolation. One microgram of total RNA was reverse transcribed into cDNA as described in detail elsewhere (7, 8), using pd(N)6 random hexamers (GE Healthcare, Little Chalfont, UK) and Moloney Murine Leukemia Virus‐Reverse Transcriptase enzyme (Invitrogen, Carlsbad, CA, USA). GUS was used as control gene to correct for variations in RNA quality and quantity (7).…”

Best Practices in Chronic Myeloid Leukemia Monitoring and Management

“…Because progressive degradation of RNA starting soon after blood collection is an inevitable physiological phenomenon [56], samples should be delivered to the laboratory and processed within 24 hours, which mandates avoiding sampling on Fridays and holiday eves unless agreed on with the laboratory. Whole blood can be shipped at room temperature or refrigerated, but it must never be frozen unless specific vials containing RNA stabilizing solutions [57][58][59]…”

Early landmark analysis of imatinib treatment in CML chronic phase: Less than 10% BCR‐ABL by FISH at 3 months associated with improved long‐term clinical outcome

“…From October 2008 and onwards, total blood leukocytes were used for RNA isolation. RNA (1 μg) was reverse‐transcribed into cDNA as described elsewhere using pd(N)6 random hexamer (GE Healthcare, UK) and M‐MLV enzyme (Invitrogen, Carlsbad) [17]. ABL and GUS were used as control genes to correct for variations in RNA quality and quantity [18].…”