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Biology Articles » Biotechnology » Red Biotechnology » Negative selection of chronic lymphocytic leukaemia cells using a bifunctional rosette-based antibody cocktail » Results and Discussion

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

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.

Additional file 1. CD5/CD19 immunophenotyping of sample CLL22 (1), CLL35 (2) and CLL36 (3) after RS+DGC enrichment using either 50 μl (A), 60 μl (B), 70 μl (C) or 80 μl (D) RosetteSep/ml whole blood. Data indicates that a concentration of 70 μl RosetteSep/ml whole blood gives the highest purities of CD5+ CD19+ cells.

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Enrichment with RS+DGC was performed in less than 90 minutes and showed higher purities of CD5/CD19 co-expressing cells for every sample compared to the enrichment with DGC alone. The analysed PB samples of CLL patients showed an average CLL cell purity of 74.1% (ranging from 15.9 to 97.4%) after DGC (see Figure 1A and Additional Files 2 and 3). After RS+DGC enrichment, the same samples exhibited an average CLL cell purity of 93.8% (ranging from 80.4 to 99.4%). The average purity of CD5/CD19 co-expressing cells was raised from 74.1% after DGC to 93.8% after RS+DGC. The average percentage of CD5- CD19+ (normal B-cells), CD5- CD19- (natural killer cells and monocytes) and CD5+ CD19- cells (T-cells) was reduced from 1.4, 10.1 and 14.4 to 1.0, 3.5 and 1.6% respectively after RS+DGC (see Additional File 3).

Additional file 2. CD5/CD19 immunophenotyping of sample CLL2 after DGC (left) and after RS+DGC enrichment (right). Figure shows the CD5/CD19 cell surface expression of one sample (CLL2) post DGC, and post RS+DGC enrichment determined by immunophenotyping. The RS+DGC enrichment lead to a significant increase in the proportion of the CD5+ CD19+ cells.

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Additional file 3. White Blood Cell (WBC) counts of fresh CLL peripheral blood samples and their examination for CLL purity after density gradient centrifugation DGC and RosetteSep incubation prior to DGC (RS+DGC) enrichment. The data is sorted by ascending WBC count. RNA Integrity Numbers (RIN) for RNA extracted from CLL cells after RS+DGC enrichment are shown. The figure shows the WBC counts of all CLL peripheral blood samples and the respective purity of the CD5- CD19+, CD5+ CD19+, CD5- CD19- and CD5+ CD19- fractions after DGC and after RS+DGC. This table also displays RNA Integrity Numbers for RNA extracted from purified CLL cells using RS+DGC.

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Figure 1. purity of CD5+ CD19+ cells in the same sample after DGC or RS+DGC enrichment (A) and absolute yield of WBCs after RS+DGC enrichment (B) plotted against the respective white blood cell count.

The purity of the enriched CLL cells (based on CD5/CD19 co-expression) increased with the WBC count of the samples (see Figure 1A). RS+DGC enrichment resulted in a CLL purity of greater than 90% for all 23 of the 29 samples that showed a WBC count higher than 20 × 106cells/ml PB, while the 6 samples with a WBC count between 7 and 20 × 106cells/ml PB showed CLL purity between 80 and 90% after RS+DGC enrichment. The consistently higher purities achieved with RS+DGC in all 29 samples compared to DGC alone show the efficacy of the rosette based enrichment method, and is comparable with purities achieved by MCS and FACS and superior in terms of time and cost (see Table 1).

Table 1. Comparison of the discussed four different enrichment approaches

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.

Additional file 4. White blood cell (WBC) counts of fresh CLL peripheral blood (PB) samples and yields and purities after RosetteSep incubation prior to density gradient centrifugation (RS+DGC) enrichment. The data is sorted by ascending WBC count. The figure shows the WBC counts of all CLL peripheral blood samples and the respective cell yield and purity of the CD5+ CD19+ fractions after DGC and after RS+DGC.

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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).


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