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High purity of tumour samples is a necessity for accurate genetic and …

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

Enrichment of tumour cells to a purity of more than 90% is highly desirable for accurate results in many applications, especially for RT-PCR and microarray based expression analysis [1,2]. In B-cell chronic lymphocytic leukaemia (CLL), such purities have usually been achieved by density gradient centrifugation (DGC) and subsequent fluorescent-activated cell sorting (FACS) or by magnetic cell sorting (MCS) for CD19 positive cells [1].

Studies focusing on expression analysis in CLL utilising microarrays report median purities of 88 and 90% of CD19 positive cells using DGC [3,4] though it is likely that selection occurred for samples with high purity. One study applying DGC and FACS of mononuclear cells reported purities of between 90 and 95% of CD5–CD19 co-expressing cells [5]. Three studies [6-8] reported purities higher than 97% of CD19 positive cells after DGC and MCS. Although high purity is achieved with FACS and MCS, both are time and cost intensive procedures which often are limited in terms of tumour cell yields and applicability, since they require expensive equipment and the processing time depends on the sample volume. Another potential disadvantage is that they are positive selection approaches which might alter gene expression through the activation of cell surface receptors [1].

Our study focused on adapting a negative selection method that could offer the required purity after the DGC step thereby markedly cutting down the time and cost of sample processing and reducing the risk of altering the gene expression pattern.

We used a bifunctional antibody cocktail for B-cell enrichment (RosetteSep™ (RS)) that binds erythrocytes (via glycophorin) on one side and white cell populations other than B-cells (via the CD2, CD3, CD16, CD36, CD56 and/or CD66b antigens) on the other side thus forming dense rosettes of erythrocytes surrounding the unwanted white blood cells when added to whole blood. The increased density of the rosetted cells results in their pelleting by subsequent DGC. This combination of RS incubation and subsequent density gradient centrifugation (RS+DGC) thus results in the depletion of undesired cells and leaves purified B-cells behind that can be harvested from the interface [9]. Here, we investigate whether RS+DGC can also effectively isolate CLL cells at high purity from peripheral blood (PB).

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