title: Immune cell subsets and their gene expression profiles from human PBMC isolated by Vacutainer Cell Preparation Tube (CPT™) and standard density gradient.
abstract: BACKGROUND
  High quality genetic material is an essential pre-requisite when analyzing gene expression using microarray technology. Peripheral blood mononuclear cells (PBMC) are frequently used for genomic analyses, but several factors can affect the integrity of nucleic acids prior to their extraction, including the methods of PBMC collection and isolation. Due to the lack of the relevant data published, we compared the Ficoll-Paque density gradient centrifugation and BD Vacutainer cell preparation tube (CPT) protocols to determine if either method offered a distinct advantage in preparation of PBMC-derived immune cell subsets for their use in gene expression analysis. We evaluated the yield and purity of immune cell subpopulations isolated from PBMC derived by both methods, the quantity and quality of extracted nucleic acids, and compared gene expression in PBMC and individual immune cell types from Ficoll and CPT isolation protocols using Affymetrix microarrays.
  
  
  RESULTS
  The mean yield and viability of fresh PBMC acquired by the CPT method (1.16 × 10(6) cells/ml, 93.3%) were compatible to those obtained with Ficoll (1.34 × 10(6) cells/ml, 97.2%). No differences in the mean purity, recovery, and viability of CD19+ (B cells), CD8+ (cytotoxic T cells), CD4+ (helper T cell) and CD14+ (monocytes) positively selected from CPT- or Ficoll-isolated PBMC were found. Similar quantities of high quality RNA and DNA were extracted from PBMC and immune cells obtained by both methods. Finally, the PBMC isolation methods tested did not impact subsequent recovery and purity of individual immune cell subsets and, importantly, their gene expression profiles.
  
  
  CONCLUSIONS
  Our findings demonstrate that the CPT and Ficoll PBMC isolation protocols do not differ in their ability to purify high quality immune cell subpopulations. Since there was no difference in the gene expression profiles between immune cells obtained by these two methods, the Ficoll isolation can be substituted by the CPT protocol without conceding phenotypic changes of immune cells and compromising the gene expression studies. Given that the CPT protocol is less elaborate, minimizes cells' handling and processing time, this method offers a significant operating advantage, especially in large-scale clinical studies aiming at dissecting gene expression in PBMC and PBMC-derived immune cell subpopulations.
authors: Molecular Virology and Hepatology Research Group, Division of BioMedical Sciences, Faculty of Medicine, Health Sciences Centre, Memorial University, St. John's, NL, A1B3V6, Canada. c.corkum@mun.ca.
authors: Christopher P
authors: CP
authors: Corkum
authors: Molecular Virology and Hepatology Research Group, Division of BioMedical Sciences, Faculty of Medicine, Health Sciences Centre, Memorial University, St. John's, NL, A1B3V6, Canada. dings@mun.ca.
authors: Danielle P
authors: DP
authors: Ings
authors: Novartis Oncology Companion Diagnostics, Cambridge, MA, 02139, USA. chris.burgess@novartis.com.
authors: Christopher
authors: C
authors: Burgess
authors: Novartis Oncology Companion Diagnostics, Cambridge, MA, 02139, USA. sylwia.karwowska@novartis.com.
authors: Sylwia
authors: S
authors: Karwowska
authors: Novartis Oncology Companion Diagnostics, Cambridge, MA, 02139, USA. werner.krol@quidel.com.|Present address: Quidel Corporation, San Diego, CA, 92130, USA. werner.krol@quidel.com.
authors: Werner
authors: W
authors: Kroll
authors: Molecular Virology and Hepatology Research Group, Division of BioMedical Sciences, Faculty of Medicine, Health Sciences Centre, Memorial University, St. John's, NL, A1B3V6, Canada. timich@mun.ca.
authors: Tomasz I
authors: TI
authors: Michalak
mesh_terms: D000328:Adult; D002469:Cell Separation / Q000295:instrumentation* / Q000379:methods*; D002470:Cell Survival; D002499:Centrifugation, Density Gradient; D015925:Cryopreservation; D004247:DNA / Q000302:isolation & purification; D005362:Ficoll; D020869:Gene Expression Profiling; D006801:Humans; D016131:Lymphocyte Subsets / Q000378:metabolism*; D020411:Oligonucleotide Array Sequence Analysis; D025341:Principal Component Analysis; D012313:RNA / Q000302:isolation & purification
chemical_list: D005362:Ficoll; D012313:RNA; D004247:DNA