1. ¹Section of Experimental Haematology, Division of Cancer Sciences & Molecular Pathology, University of Glasgow, Glasgow, UK
2. ²Department of Haematology, Imperial College, Hammersmith Hospital, London, UK

Correspondence: Professor T Holyoake, Section of Experimental Haematology, Division of Cancer Sciences & Molecular Pathology, University of Glasgow, Level 3 Queen Elizabeth Building, Royal Infirmary, 10 Alexandra Parade, Glasgow G31 2ER, UK. E-mail: tlh1g@clinmed.gla.ac.uk

Received 3 November 2005; Revised 10 February 2006; Accepted 16 February 2006; Published online 30 March 2006.

Abstract

In chronic myeloid leukaemia, CD34⁺ stem/progenitor cells appear resistant to imatinib mesylate (IM) in vitro and in vivo. To investigate the underlying mechanism(s) of IM resistance, it is essential to quantify Bcr-Abl kinase status at the stem cell level. We developed a flow cytometry method to measure CrkL phosphorylation (P-CrkL) in samples with <10⁴ cells. The method was first validated in wild-type (K562) and mutant (BAF3) BCR-ABL⁺ as well as BCR-ABL^- (HL60) cell lines. In response to increasing IM concentration, there was a linear reduction in P-CrkL, which was Bcr-Abl specific and correlated with known resistance. The results were comparable to those from Western blotting. The method also proved to be reproducible with small samples of normal and Ph⁺ CD34⁺ cells and was able to discriminate between Ph^-, sensitive and resistant Ph⁺ cells. This assay should now enable investigators to unravel the mechanism(s) of IM resistance in stem cells.