A comparison of high resolution melting, allele-specific priming and Sanger sequencing for the detection of BRAFV600E mutation in hairy cell leukaemia from different haematological specimens.

The BRAFV600E mutation is a highly sensitive and specific marker for the diagnosis of hairy cell leukaemia (HCL) and a potential therapeutic target. We assessed the performance of high resolution melting (HRM), allele-specific priming (ASP) and Sanger sequencing (SS) for BRAFV600E detection in 17 unenriched samples from 15 HCL patients: blood (n = 7), marrow aspirate (n = 3), ethylenediaminetetraacetic acid (EDTA)-decalcified trephine biopsy (n = 2), formic acid (FA)-decalcified trephine biopsy (n = 5). Our results showed that for blood and marrow aspirate samples, both HRM and ASP had a very high analytical sensitivity (1%) in clinical specimens and excellent diagnostic sensitivity (100%) and specificity (100%) in analysable samples. Sanger sequencing had a lower analytical sensitivity (4%), resulting in false-negative analysis in samples with a low tumour cell percentage. High resolution melting was technically the simplest and had the shortest turn-around time (2 hours). Analysis of decalcified trephine biopsies was more difficult because of suboptimal DNA preservation. Although Sanger sequencing was least demanding on sample DNA quality for a successful analysis, none of the three techniques showed satisfactory diagnostic performance on trephine biopsies. Therefore, careful selection of a suitable sample type and testing platform is important to optimise the detection of this important mutation in HCL.