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1.  Risk-based classification of leukemia by cytogenetic and multiplex molecular methods: results from a multicenter validation study 
Blood Cancer Journal  2012;2(7):e78-.
Modern management of leukemia and selection of optimal treatment approaches entails the analysis of multiple recurrent cytogenetic abnormalities with independent diagnostic or prognostic value. We report the first multicenter validation of a multiplex molecular assay for 12 relevant fusion transcripts relative to cytogenetic methods. Performance was evaluated using a set of 280 adult and pediatric acute or chronic leukemias representative of the variety of presentations and pre-analytical parameters encountered in the clinical setting. The positive, negative and overall agreements were >98.5% with high concordance at each of the four sites. Positive detection of cases with low blast count or at relapse was consistent with a method sensitivity of 1%. There was 98.7% qualitative agreement with independent reference molecular tests. Apparent false negatives corresponded to rare alternative splicing isoforms not included in the panel. We further demonstrate that clinical sensitivity can be increased by adding those rare variants and other relevant transcripts or submicroscopic abnormalities. We conclude that multiplex RT-PCR followed by liquid bead array detection is a rapid and flexible method attuned to the clinical laboratory workflow, complementing standard cytogenetic methods and generating additional information valuable for the accurate diagnosis, prognosis and subsequent molecular monitoring of leukemia.
doi:10.1038/bcj.2012.24
PMCID: PMC3408638  PMID: 22852047
leukemia; diagnosis; prognosis; molecular classification; RT-PCR; multiplex
2.  Establishment of a standardized multiplex assay with the analytical performance required for quantitative measurement of BCR–ABL1 on the international reporting scale 
Blood Cancer Journal  2011;1(3):e13-.
Accurate and standardized methods for the quantitative measurement of BCR–ABL1 are a prerequisite for monitoring of treatment response in t(9;22)-positive leukemia. Here, we describe a novel multiplex assay system based on the proven TaqMan and Armored RNA technologies and optimized for sensitive detection of three BCR–ABL1 fusion transcripts and ABL1 in a single reaction. Analytical experiments confirmed the absence of significant competition between the simultaneous amplification reactions and established the sensitivity, linearity and precision of the assay. Comparative studies with 115 clinical specimens resulted in high qualitative and quantitative agreement with independent singleplex laboratory-developed tests routinely used in clinical testing. Direct comparison with a reference laboratory calibrated to the international scale (IS) demonstrated minimal analytical bias between methods and an overall accuracy and precision within the performance range required for quantitative measurement of BCR–ABL1 on the IS. We conclude that detection of e1a2, b2a2, b3a2 and ABL1 can be achieved in a multiplex assay format compatible with IS reporting. Further clinical validation of the assay could improve the operational efficiency of clinical laboratories, increase their adherence to current recommendations for b2a2/b3a2 reporting on the IS and provide for the first time an opportunity to standardize e1a2-monitoring results.
doi:10.1038/bcj.2011.10
PMCID: PMC3255280  PMID: 22829126
BCR–ABL1; monitoring; tyrosine kinase inhibitor; international scale; e1a2

Results 1-2 (2)