Method Description

RNA is extracted from blood or bone marrow and reverse transcription is performed. Real time quantitative polymerase chain reaction (PCR) is performed from complementary DNA template using the LC480 instrument platform (Roche). This assay targets the most common recurrent NPM1 alterations in acute myeloid leukemia (A, B, and D insertion variants). The quantitative value of NPM1 messenger RNA copy number is determined relative to ABL1 as the reference transcript using the delta-delta CT method. The reproducible analytical sensitivity (limit of detection) of this part of the assay is approximately 0.01%.

DNA is extracted from blood or bone marrow, and a PCR assay is performed using primers that amplify a fragment of NPM1 DNA containing the region susceptible to insertion variant. One of the PCR primers contains a fluorescent label. The amplified fragments are size separated by capillary electrophoresis. Wild type NPM1 produces a fragment length of 187 base pairs (bp). PCR fragments containing an insertional variant are observed as larger fragments, most typically 191 bp, as the majority of alterations are 4 bp insertions. The analytical sensitivity (limit of detection) of this part of the assay is approximately 5%.(Unpublished Mayo method)