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Open Access Methodology article

Negative selection of chronic lymphocytic leukaemia cells using a bifunctional rosette-based antibody cocktail

Salim Essakali12, Dennis Carney13, David Westerman13, Peter Gambell13, John F Seymour13 and Alexander Dobrovic13*

Author Affiliations

1 Department of Pathology, Peter MacCallum Cancer Centre, St Andrews Place, Melbourne, Victoria 3002, Australia

2 Universitaetsklinik Duesseldorf, Molekulare Pathologie, Moorenstr. 5, 40215 Duesseldorf, Germany

3 Department of Pathology, University of Melbourne, Parkville, Victoria 3010, Australia

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BMC Biotechnology 2008, 8:6  doi:10.1186/1472-6750-8-6

Published: 29 January 2008

Abstract

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.