Microfluidic isolation of leukocytes from whole blood for phenotype and gene expression analysis.
Technologies that enable the isolation of cell subtypes from small samples of complex populations will greatly facilitate the implementation of proteomics and genomics to human diseases. Transcriptome analysis of blood requires the depletion of contaminating erythrocytes. We report an automated microfluidic device to rapidly deplete erythrocytes from whole blood via deionized water lysis and to collect enriched leukocytes for phenotype and genomic analyses. Starting with blood from healthy subjects, we demonstrate the utility of this microfluidic cassette and lysis protocol to prepare unstimulated leukocytes, and leukocytes stimulated ex vivo with Staphylococcal enterotoxin B, which mimics some of the cellular effects seen in patients with severe bacterial infections. Microarrays are used to assess the global gene expression response to enterotoxin B. The results demonstrate that this system can isolate unactivated leukocytes from small blood samples without any significant loss, which permits more information to be obtained from subsequent analysis, and will be readily applicable to clinical settings.
Sethu
Palaniappan
P
Surgical Services and Center for Engineering in Medicine, Massachusetts General Hospital, Harvard Medical School and Shriners Hospital for Children, Boston, Massachusetts 02114, USA.
Moldawer
Lyle L
LL
Mindrinos
Michael N
MN
Scumpia
Philip O
PO
Tannahill
Cynthia L
CL
Wilhelmy
Julie
J
Efron
Philip A
PA
Brownstein
Bernard H
BH
Tompkins
Ronald G
RG
Toner
Mehmet
M
D002469:Cell Separation / Q000295:instrumentation*; D004768:Enterotoxins / Q000494:pharmacology; D015870:Gene Expression / Q000187:drug effects; D020869:Gene Expression Profiling / Q000379:methods; D006801:Humans; D007962:Leukocytes / Q000737:chemistry* / Q000166:cytology* / Q000187:drug effects; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation* / Q000379:methods; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D010641:Phenotype; D012680:Sensitivity and Specificity
D004768:Enterotoxins; C031818:enterotoxin B, staphylococcal