Negative selection of chronic lymphocytic leukaemia cells using a bifunctional rosette-based antibody cocktail
© Essakali et al; licensee BioMed Central Ltd. 2008
Received: 17 October 2007
Accepted: 29 January 2008
Published: 29 January 2008
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).
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.
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.
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).
Results and Discussion
A preliminary experiment was used to assess the optimal RS concentration that resulted in the best purity. Aliquots of three CLL samples were treated with 50, 60, 70 and 80 μl RS/ml PB to monitor the effect on the resulting purity. The experiments indicated that a concentration of 70 μl RS/ml PB resulted in the best purity (see Additional File 1). This was the concentration used to subsequently enrich all CLL samples.
Comparison of the discussed four different enrichment approaches
ca. 70% a
ca. 93% a
> 98% d
90 – 95% f
> 93% e
86% (± 14%) b
90% (± 7%) c
Time of procedure
< 60 mins.
< 90 mins.
> 120 mins (increasing with sample volume)
> 120 mins (increasing with sample volume)
highly depending on WBC count
dependent on WBC count when < 20 × 106 cells/ml PB
long process time and expensive
long process time and very expensive
centrifuge + MCS Sorter
centrifuge + FACS sorter
Not surprisingly, the cell yield also depended on the WBC count (see Figure 1B and Additional File 4). The number of cells harvested can be regulated by increasing (or decreasing) the volume of blood to be processed and by adjusting the volume of added RS antibody cocktail accordingly without any effect on the processing time of 90 minutes. This is another potential advantage over FACS and MCS where increased cell numbers require increased processing time.
The RNA extracted from enriched cells using RS+DGC displayed an average RIN of 8.9 (ranging from 7.7 to 9.5), indicating high-quality RNA (see Additional File 3) that subsequently gave excellent results on microarray analysis (data not shown).
This study shows that negative selection using a bifunctional rosette-based antibody cocktail is an effective method to isolate CLL cells of high purity, especially in samples with a WBC count above 20 × 106 cells/ml. The short purification time, the independence from expensive and time consuming procedures, such as FACS and MCS (see Table 1), and the flexible adjustment of cell yields makes RS+DGC an attractive purification method for a wide spectrum of downstream applications, particularly expression analysis utilising microarrays, in which a CLL purity of >90% is desirable.
Peripheral blood (PB) samples of CLL patients were obtained as part of a study approved by the Peter MacCallum Ethics of Human Research Committee. The white blood cell (WBC) count ranged from 7.81 to 437.08 × 106/ml and averaged 76.1 × 106/ml. The diagnosis of CLL was based on earlier examination of the patients' blood film and immunophenotyping for CD3, CD4, CD5, CD8, CD10, CD16, CD19, CD20, CD22, CD23, CD38, CD45, CD56, FMC7 and surface immunoglobulin light chain expression.
Blood from CLL patients was incubated with RosetteSep™ (StemCell Technologies Inc., Vancouver, British Columbia, Canada) (RS) at a concentration of 70 μl/ml PB in the dark at room temperature for 20 minutes with gentle manual swirling every 5 minutes. As a control, an aliquot of the same sample was processed the same way except without addition of RS. After incubation, both aliquots of blood were diluted with 4 volumes (rather than 2 volumes as recommended by the manufacturer) of Dulbecco's phosphate buffered saline (PBS) containing 2% foetal bovine serum as otherwise we found that blood samples with high WBC counts were insufficiently diluted for efficient separation. The samples were then underlaid with 3 ml Lymphocyte Separation Medium (MP Biomedicals, Aurora, OH) and centrifuged for 20 minutes at 1,200 g. The enriched cells were subsequently harvested from the interface and washed once in 2 volumes Dulbecco's PBS with 2% foetal bovine serum by centrifuging for 10 minutes at 200 g.
The purity of the enriched cell population was analysed by staining with a panel of fluorescently labelled antibodies (BD Biosciences, San Jose, CA) in three different tubes. (Tube 1: CD5-FITC, CD10-PE, CD19-PerCP and CD45-APC. Tube 2: FMC7-FITC, CD23-PE, CD19-PerCP and CD45-APC. Tube 3: CD22-FITC, CD38-PE, CD20-PerCP and CD45-APC). Data acquisition was carried out using a FACScalibur-cytometer (BD Biosciences) and Cell Quest software (BD Biosciences). Ungated data analysis was conducted using Cytomics RXP software (Beckman Coulter, Fullerton, CA).
The diagnosis of CLL was reconfirmed by assessing positivity for CD5, CD19, CD23 and CD45, weak positivity for CD20; weak positivity or negativity for CD22 and FMC7 and negativity for CD10. After confirming the CLL diagnosis, the CLL purity was assessed by the co-expression of CD5 and CD19.
RNA was extracted from RS+DGC enriched CLL cells by applying a combination of the Trizol-protocol (Invitrogen, Carlsbad, CA, USA) and the RNeasy Micro Kit (Qiagen, Hilden, Germany). The RNA Integrity Number (RIN) of the extracted RNA was determined using the 2100 Bioanalyzer (Agilent, Santa Clara, CA) according to the manufacturer's protocol.
Chronic Lymphocytic Leukaemia
density gradient centrifugation
fluorescent-activated cell sorting
magnetic cell sorting
Dulbecco's phosphate buffered saline
polymerase chain reaction
RNA Integrity Number
addition of RosetteSep B-cell enrichment cocktail prior to density gradient centrifugation
reverse transcription polymerase chain reaction
white blood cell
We thank Nancy Messino for assisting with the immunophenotyping and Sophia Halley, Ida Candiloro, Angela Tan, Lasse Kristensen, Michael Krypuy and Chelsee Hewitt for critical reading of the manuscript. Funding for this study was from grants from the CLL Global Research Foundation to AD and JFS and Reece Australia to DW and AD. SE also acknowledges his academic supervisors at the Heinrich Heine University, Prof. Dr. Heinz Mehlhorn and Prof. Dr. Christopher Poremba.
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