RESUMEN
Individuals with acute promyelocytic leukemia (APL) usually express 1 of 3 primary hybrid transcripts associated with a t(15;17). The 3 fusion transcripts are the result of heterogeneous breakpoint cluster regions (bcr) within the promyelocytic leukemia (PML) gene and are denoted bcr1 (long), bcr2 (variant), and bcr3 (short) forms. Many researchers have shown that real-time quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) of the involved transcript is a valuable tool for monitoring APL and its treatment. In addition, some research suggests that identification of a specific breakpoint region may be used to predict an individual's likelihood of relapse and possibly their response to all-trans retinoic acid treatment. We describe the first reported 1-step multiplex RT-PCR assay capable of t(15;17) fusion transcript real-time relative quantitation and simultaneous transcript form identification in 2 reactions. This assay uses a novel dual-probe technique to achieve what has required a laborious procedure of 2 or more reactions followed by postamplification analysis. We found a correlation of 100% in detection and breakpoint determination of the long, short, and variant forms with a breakpoint 5' to nucleotide 1709 compared with results from traditional methods.
Asunto(s)
Rotura Cromosómica/genética , Cromosomas Humanos Par 15 , Cromosomas Humanos Par 17 , Proteínas de Fusión bcr-abl/genética , Leucemia Promielocítica Aguda/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Translocación Genética/genética , Cartilla de ADN/química , Sondas de ADN/química , ADN de Neoplasias/análisis , Humanos , Leucemia Promielocítica Aguda/patología , ARN Neoplásico/análisisRESUMEN
Clinical diagnostic assays, may be classified as quantitative, quasi-quantitative or qualitative. The assay's description should state what the assay needs to accomplish (intended use or purpose) and what it is not intended to achieve. The type(s) of samples (whole blood, peripheral blood mononuclear cells (PBMC), bone marrow, bone marrow mononuclear cells (BMMC), tissue, fine needle aspirate, fluid, etc.), instrument platform for use and anticoagulant restrictions should be fully validated for stability requirements and specified. When applicable, assay sensitivity and specificity should be fully validated and reported; these performance criteria will dictate the number and complexity of specimen samples required for validation. Assay processing and staining conditions (lyse/wash/fix/perm, stain pre or post, time and temperature, sample stability, etc.) should be described in detail and fully validated.