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.
Size: 92KB Download file
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.
Size: 33KB Download file
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.
Size: 20KB Download file
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
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.
Size: 16KB Download file
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).