FAST AND COST-EFFECTIVE RNA EXTRACTION FOR BCR-ABL QUANTIFICATION IN ONCO-HEMATOLOGY LABORATORIES

Abstract

Background: Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm characterized by the presence of a BCR-ABL1 rearrangement; its detection and quantification by real-time quantitative PCR (RQ-PCR) plays a central role in CML diagnosis, therapy monitoring, and sequencing to identify resistance mutations in case of therapy failure. To evaluate the molecular response of CML, BCR-ABL1 quantification must be calculated on the international scale (IS%) for which RQ-PCR has the necessary sensitivity (up to 10−5). Currently, BCR-ABL1 quantifications are ubiquitous in onco-hematology laboratories (OHLs; ̃70% of tests performed in our OHL), whereby quality, cost, and turn-around-time are of critical importance. Today, the majority of OHLs in Brazil rely on laborious manual RNA-extraction methods (e.g., Trizol) that usually require 8 mL of peripheral blood (PB) to monitor CML. Here, we report a strategy that requires only 2 mL of PB in combination with the automated magnetic DNA/RNA-extraction kit Extracta-MPTA (Loccus do Brasil) for CML diagnosis and monitoring. Methods: BCR:ABL1 was quantified and its IS% determined using our developed kit, which was calibrated using ERM-AD263 and UK-NEQAS (external quality assessment). Total RNA was extracted from 40 PB samples, after erythrocyte lysis using two different methods: 1) Leukocytes from 8 mL of PB were extracted manually using Trizol; 2) leukocytes from 2 mL of PB were automatically extracted using Extracta-MPTA (Loccus do Brasil). BCR-ABL1 quantification, BCR-ABL1 IS%, and BCR quantification (internal control) were compared statistically (paired t-test). The reproducibility was evaluated by analyzing 20 replicates; the low limit of the leucocyte number to BCR:ABL1 quantification using Extracta-MPTA were also evaluated. Median and moving means of 135 unpaired samples were used to monitor BCR-ABL1 IS% between Trizol, Extracta-MPTA, while EQA from UK-NEQAS was also evaluated using both methods. In parallel, RNA was extracted by both methods from 20 samples for the diagnosis of acute lymphoblastic leukemia (ALL) (BCR-ABL p190 transcript) and acute promyelocytic leukemia (APL) (PML-RARA transcript). Results: A comparison of BRC-ABL1, BCR, and %IS in 40 paired samples showed that the results obtained from Trizol or Extracta-MPTA are comparable (paired t-test: p = 0,4222, p = 0,9263, and p = 0,071, respectively). The reproducibility was evaluated by BCR quantification (24%).. A comparison of the medians from 135 unpaired samples extracted using Trizol and Extracta-MPTA revealed that BCR-ABL1 IS% distribution among our patients is stable over time (p = 0,7704; Mann Whitney: r2 = 0,0012). EQA was extracted using both methods and the results were identical. A comparison between Trizol and EXTRACTA_MPTA for ALL and APL was 100% identical (Fischer; p = 1,000). Conclusions: Both RNA extraction methods are suitable for OHLs for PML-RARA and BCR-ABL (p190 and p210) detection and CML diagnosis/monitoring by BCR-ABL1 detection/quantification. By using Extracta-MPTA the amount of PB used to obtain RNA can be reduced by 75%, test results can be obtained within 4 h, and direct reagent costs are reduced by 25% with a minimum of hands-on time.