title: Gene expression profiling of immunomagnetically separated cells directly from stabilized whole blood for multicenter clinical trials.
abstract: BACKGROUND
  Clinically useful biomarkers for patient stratification and monitoring of disease progression and drug response are in big demand in drug development and for addressing potential safety concerns. Many diseases influence the frequency and phenotype of cells found in the peripheral blood and the transcriptome of blood cells. Changes in cell type composition influence whole blood gene expression analysis results and thus the discovery of true transcript level changes remains a challenge. We propose a robust and reproducible procedure, which includes whole transcriptome gene expression profiling of major subsets of immune cell cells directly sorted from whole blood.
  
  
  METHODS
  Target cells were enriched using magnetic microbeads and an autoMACS® Pro Separator (Miltenyi Biotec). Flow cytometric analysis for purity was performed before and after magnetic cell sorting. Total RNA was hybridized on HGU133 Plus 2.0 expression microarrays (Affymetrix, USA). CEL files signal intensity values were condensed using RMA and a custom CDF file (EntrezGene-based).
  
  
  RESULTS
  Positive selection by use of MACS® Technology coupled to transcriptomics was assessed for eight different peripheral blood cell types, CD14+ monocytes, CD3+, CD4+, or CD8+ T cells, CD15+ granulocytes, CD19+ B cells, CD56+ NK cells, and CD45+ pan leukocytes. RNA quality from enriched cells was above a RIN of eight. GeneChip analysis confirmed cell type specific transcriptome profiles. Storing whole blood collected in an EDTA Vacutainer® tube at 4°C followed by MACS does not activate sorted cells. Gene expression analysis supports cell enrichment measurements by MACS.
  
  
  CONCLUSIONS
  The proposed workflow generates reproducible cell-type specific transcriptome data which can be translated to clinical settings and used to identify clinically relevant gene expression biomarkers from whole blood samples. This procedure enables the integration of transcriptomics of relevant immune cell subsets sorted directly from whole blood in clinical trial protocols.
authors.affiliation: Biomarker Development, Novartis Institutes for BioMedical Research (NIBR), Basel, Switzerland.
authors.forename: Martin
authors.initials: M
authors.lastname: Letzkus
authors.affiliation: Biomarker Development, Novartis Institutes for BioMedical Research (NIBR), Basel, Switzerland.
authors.forename: Evert
authors.initials: E
authors.lastname: Luesink
authors.affiliation: Biomarker Development, Novartis Institutes for BioMedical Research (NIBR), Basel, Switzerland.
authors.forename: Sandrine
authors.initials: S
authors.lastname: Starck-Schwertz
authors.affiliation: Biomarker Development, Novartis Institutes for BioMedical Research (NIBR), Basel, Switzerland.
authors.forename: Marc
authors.initials: M
authors.lastname: Bigaud
authors.affiliation: Scientific Capability Development, Pharma-Development, Novartis Pharma AG, Basel, Switzerland.
authors.forename: Fareed
authors.initials: F
authors.lastname: Mirza
authors.affiliation: Biomarker Development, Novartis Institutes for BioMedical Research (NIBR), Basel, Switzerland.
authors.forename: Nicole
authors.initials: N
authors.lastname: Hartmann
authors.affiliation: Miltenyi Biotec GmbH, Bergisch Gladbach, Germany.
authors.forename: Bernhard
authors.initials: B
authors.lastname: Gerstmayer
authors.affiliation: Miltenyi Biotec GmbH, Bergisch Gladbach, Germany.
authors.forename: Uwe
authors.initials: U
authors.lastname: Janssen
authors.affiliation: Spheromics, Kontiolahti, Finland.
authors.forename: Andreas
authors.initials: A
authors.lastname: Scherer
authors.affiliation: Biomarker Development, Novartis Institutes for BioMedical Research (NIBR), Basel, Switzerland.
authors.forename: Martin M
authors.initials: MM
authors.lastname: Schumacher
authors.affiliation: Biomarker Development, Novartis Institutes for BioMedical Research (NIBR), Basel, Switzerland.
authors.forename: Aurelie
authors.initials: A
authors.lastname: Verles
authors.affiliation: Biomarker Development, Novartis Institutes for BioMedical Research (NIBR), Basel, Switzerland.
authors.forename: Alessandra
authors.initials: A
authors.lastname: Vitaliti
authors.affiliation: Biomarker Development, Novartis Institutes for BioMedical Research (NIBR), Cambridge, MA, USA.
authors.forename: Nanguneri
authors.initials: N
authors.lastname: Nirmala
authors.affiliation: Biomarker Development, Novartis Institutes for BioMedical Research (NIBR), Cambridge, MA, USA.
authors.forename: Keith J
authors.initials: KJ
authors.lastname: Johnson
authors.affiliation: Biomarker Development, Novartis Institutes for BioMedical Research (NIBR), Basel, Switzerland.
authors.forename: Frank
authors.initials: F
authors.lastname: Staedtler
keywords: Cell sorting; Clinical; Transcriptomics