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

Essakali S, Carney D, Westerman D, Gambell P, Seymour JF, Dobrovic A - BMC Biotechnol. (2008)

Bottom Line: 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pathology, Peter MacCallum Cancer Centre, St Andrews Place, Melbourne, Victoria 3002, Australia. salim.essakali@qiagen.com

ABSTRACT

Background: 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).

Results: 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.

Conclusion: 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.

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Related in: MedlinePlus

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.
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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.

Mentions: 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).


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

Essakali S, Carney D, Westerman D, Gambell P, Seymour JF, Dobrovic A - BMC Biotechnol. (2008)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2254389&req=5

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.
Mentions: 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).

Bottom Line: 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pathology, Peter MacCallum Cancer Centre, St Andrews Place, Melbourne, Victoria 3002, Australia. salim.essakali@qiagen.com

ABSTRACT

Background: 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).

Results: 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.

Conclusion: 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.

Show MeSH
Related in: MedlinePlus