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 .
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 . 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 .
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 . Here, we investigate whether RS+DGC can also effectively isolate CLL cells at high purity from peripheral blood (PB).