Medical Education - Research Publications

Permanent URI for this collection

http://hdl.handle.net/11343/190

Filters

2008 2
2
Reset filters

Settings
Statistics
Citations
Start date: 2000 × End date: 2009 × Author: Seymour, John × Author: Westerman, David ×

Search Results

Now showing 1 - 2 of 2
  • Item
    Thumbnail Image
    Negative selection of chronic lymphocytic leukaemia cells using a bifunctional rosette-based antibody cocktail
    Essakali, S ; Carney, D ; Westerman, D ; Gambell, P ; Seymour, JF ; Dobrovic, A (BMC, 2008-01-29)
    BACKGROUND: High purity of tumour samples is a necessity for accurate genetic and expression analysis and is usually achieved by positive selection in chronic lymphocytic leukaemia (CLL). RESULTS: We adapted a bifunctional rosette-based antibody cocktail for negative selection of B-cells for isolating CLL cells from peripheral blood (PB). PB samples from CLL patients were split into aliquots. One aliquot of each sample was enriched by density gradient centrifugation (DGC), while the other aliquot of each sample was incubated with an antibody cocktail for B-cell enrichment prior to DGC (RS+DGC). The purity of CLL cells after DGC averaged 74.1% (range: 15.9 - 97.4%). Using RS+DGC, the purity averaged 93.8% (range: 80.4 - 99.4%) with 23 of 29 (79%) samples showing CLL purities above 90%. RNA extracted from enriched CLL cells was of appropriately high quality for microarray analysis. CONCLUSION: This study confirms the use of a bifunctional rosette-based antibody cocktail as an effective method for the purification of CLL cells from peripheral blood.
  • Item
    Thumbnail Image
    Detection of NPM1 exon 12 mutations and FLT3-internal tandem duplications by high resolution melting analysis in normal karyotype acute myeloid leukemia
    Tan, AYC ; Westerman, DA ; Carney, DA ; Seymour, JF ; Juneja, S ; Dobrovic, A (BMC, 2008-07-29)
    BACKGROUND: Molecular characterisation of normal karyotype acute myeloid leukemia (NK-AML) allows prognostic stratification and potentially can alter treatment choices and pathways. Approximately 45-60% of patients with NK-AML carry NPM1 gene mutations and are associated with a favourable clinical outcome when FLT3-internal tandem duplications (ITD) are absent. High resolution melting (HRM) is a novel screening method that enables rapid identification of mutation positive DNA samples. RESULTS: We developed HRM assays to detect NPM1 mutations and FLT3-ITD and tested diagnostic samples from 44 NK-AML patients. Eight were NPM1 mutation positive only, 4 were both NPM1 mutation and FLT3-ITD positive and 4 were FLT3-ITD positive only. A novel point mutation Y572C (c.1715A>G) in exon 14 of FLT3 was also detected. In the group with de novo NK-AML, 40% (12/29) were NPM1 mutation positive whereas NPM1 mutations were observed in 20% (3/15) of secondary NK-AML cases. Sequencing was performed and demonstrated 100% concordance with the HRM results. CONCLUSION: HRM is a rapid and efficient method of screening NK-AML samples for both novel and known NPM1 and FLT3 mutations. NPM1 mutations can be observed in both primary and secondary NK-AML cases.