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Comparison of methodologies for the detection of BRAF mutations in bone marrow trephine specimens
  1. Beatrix Cardus1,
  2. Richard Colling2,3,
  3. Angela Hamblin4,5,
  4. Elizabeth Soilleux2,6
  1. 1 Medical School, University of Oxford, Oxford, UK
  2. 2 Nuffield Division of Clinical Laboratory Sciences, University of Oxford, Oxford, UK
  3. 3 Department of Cellular Pathology, Oxford University Hospitals NHS Foundation Trust, Oxford, UK
  4. 4 Department of Laboratory Haematology, Oxford University Hospitals NHS Foundation Trust, Oxford, UK
  5. 5 NIHR Oxford Biomedical Research Centre, Oxford, UK
  6. 6 Department of Pathology, University of Cambridge, Cambridge, UK
  1. Correspondence to Dr Richard Colling, University of Oxford, Oxford OX1 2JD, UK; rtcolling{at}


Aims BRAF V600E detection assists in the diagnosis of hairy cell leukaemia (HCL); however, testing practices vary. We evaluated the clinical utility of 5 BRAF mutation testing strategies for use on bone marrow trephines (BMT).

Methods 11 HCL, 5 HCL ‘mimic’, 2 treated HCL and 10 normal BMT specimens were tested for mutant BRAF, comparing Sanger sequencing, pyrosequencing, amplicon-based next generation sequencing (NGS), automated (Idylla) PCR and immunohistochemistry (IHC).

Results PCR and IHC were cheaper and identified V600E in 100 % of HCL cases. Pyrosequencing detected the mutation in 91%, NGS in 55% of cases and Sanger sequencing in 27%. All assays gave wild-type BRAF results in HCL mimics and normal BMT samples.

Conclusions PCR and IHC were most sensitive and cost-effective, but these have limited scope for multiplexing and are likely to be replaced by NGS gene panels or whole genome sequencing in the medium to long term.

  • Hairy cell leukaemia
  • bone marrow
  • BRAF
  • molecular pathology
  • immunohistochemistry

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  • BC and RC are joint first authors.

  • AH and ES are joint senior authors.

  • AH and ES contributed equally.

  • Handling editor Mary Frances McMullin.

  • Correction notice This article has been corrected since it was published Online First. Dr Elizabeth Soilleux's affiliations has been corrected.

  • Contributors All authors devised the project, carried out aspects of the practical work and contributed to the writing and editing of the manuscript.

  • Funding The research was supported by the National Institute for Health Research (NIHR) Oxford Biomedical Research Centre (BRC), by local departmental sources and by Oxford University. The Idylla system was kindly loaned by Biocartis. Biocartis contributed the BRAF test cartridges for free. BC was funded during part of the work by a student elective bursary from the Pathological Society of Great Britain and Ireland. This study was funded by Oxford University Hospitals NHS Trust, Oxford NIHR Biomedical Research Centre and Pathological Society of Great Britain and Ireland.

  • Disclaimer The views expressed are those of the author(s) and not necessarily those of the NHS, the NIHR or the Department of Health.

  • Competing interests None declared.

  • Patient consent Not required.

  • Provenance and peer review Not commissioned; externally peer reviewed.