Microfluidic leukocyte isolation for gene expression analysis in critically ill hospitalized patients.
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.
Russom
Aman
A
Surgical Services and Center for Engineering in Medicine, Massachusetts General Hospital, Harvard Medical School and Shriners Hospital for Children, Boston, MA, USA.
Sethu
Palaniappan
P
Irimia
Daniel
D
Mindrinos
Michael N
MN
Calvano
Steve E
SE
Garcia
Iris
I
Finnerty
Celeste
C
Tannahill
Cynthia
C
Abouhamze
Amer
A
Wilhelmy
Julie
J
López
M Cecilia
MC
Baker
Henry V
HV
Herndon
David N
DN
Lowry
Stephen F
SF
Maier
Ronald V
RV
Davis
Ronald W
RW
Moldawer
Lyle L
LL
Tompkins
Ronald G
RG
Toner
Mehmet
M
D002056:Burns / Q000473:pathology; D002469:Cell Separation; D016638:Critical Illness; D020869:Gene Expression Profiling; D006760:Hospitalization; D006801:Humans; D007962:Leukocytes / Q000737:chemistry / Q000166:cytology*; D046210:Microfluidic Analytical Techniques; D020411:Oligonucleotide Array Sequence Analysis; D012313:RNA / Q000302:isolation & purification; D014949:Wounds, Nonpenetrating / Q000473:pathology
D012313:RNA