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Microfluidic Leukocyte Isolation for Gene Expression Analysis in Critically Ill Hospitalized Patients

¹ Surgical Services and Center for Engineering in Medicine, Massachusetts General Hospital, Harvard Medical School and Shriners Hospital for Children, Boston, MA; ² Department of Surgery, University of Florida College of Medicine, Gainesville, FL; ³ Stanford Genome Technology Center, Palo Alto, CA; ⁴ Department of Surgery, UMDNJ-Robert Wood Johnson Medical School, New Brunswick, NJ; ⁵ Department of Surgery, University of Washington, Harborview Medical Center, Seattle, WA; ⁶ Department of Surgery, University of Texas Medical Branch, Shriners Burns Hospital, Galveston, TX; ⁷ Department of Molecular Genetics and Microbiology, University of Florida College of Medicine, Gainesville, FL.

^aAddress correspondence to this author at: 114 16th St., Charlestown, MA 02129. Fax: (617) 523-1684; e-mail: mtoner{at}hms.harvard.edu.

Background: Microarray technology is becoming a powerful tool for diagnostic, therapeutic, and prognostic applications. There is at present no consensus regarding the optimal technique to isolate nucleic acids from blood leukocyte populations for subsequent expression analyses. Current collection and processing techniques pose significant challenges in the clinical setting. Here, we report the clinical validation of a novel microfluidic leukocyte nucleic acid isolation technique for gene expression analysis from critically ill, hospitalized patients that can be readily used on small volumes of blood.

Methods: We processed whole blood from hospitalized patients after burn injury and severe blunt trauma according to the microfluidic and standard macroscale leukocyte isolation protocol. Side-by-side comparison of RNA quantity, quality, and genome-wide expression patterns was used to clinically validate the microfluidic technique.

Results: When the microfluidic protocol was used for processing, sufficient amounts of total RNA were obtained for genome-wide expression analysis from 0.5 mL whole blood. We found that the leukocyte expression patterns from samples processed using the 2 protocols were concordant, and there was less variability introduced as a result of harvesting method than there existed between individuals.

Conclusions: The novel microfluidic approach achieves leukocyte isolation in <25 min, and the quality of nucleic acids and genome expression analysis is equivalent to or surpasses that obtained from macroscale approaches. Microfluidics can significantly improve the isolation of blood leukocytes for genomic analyses in the clinical setting.

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