TY - JOUR
T1 - A miniature cytometry platform for capture and characterization of T-lymphocytes from human blood
AU - Zhu, He
AU - Macal, Monica
AU - Jones, Caroline N.
AU - George, Michael D.
AU - Dandekar, Satya
AU - Revzin, Alexander
N1 - Funding Information:
We thank Prof. Louie's lab in the Department of Biomedical Engineering at UC Davis for providing assistance with confocal microscopy. Technical advice provided Dr. David Verhoeven is greatly appreciated. In addition, we thank Dr. Kazuhiko Sekine, Department of Emergency & Critical Care Medicine, Saiseikai Yokohamashi Tobu Hospital, Yokohama, Japan for the technical advice concerning leukocyte panning experiments. This publication was supported in part by the California Research Center for the Biology of HIV in Minorities, California HIV/AIDS Research Program #CH05-D-606. HZ was supported through an NIH fellowship grant (EB003827).
PY - 2008/2/11
Y1 - 2008/2/11
N2 - Given the clinical and diagnostic importance of blood analysis, there is considerable interest in developing novel miniature devices for rapid characterization of blood constituents. The present paper describes development of a miniature cytometry platform aimed at analysis of T-lymphocytes from peripheral human blood. Microarrays of T-cell-specific antibodies (Abs), including anti-CD3, -CD4, -CD8 and mouse IgG (negative control) were robotically printed onto glass slides coated with a non-fouling poly(ethylene glycol) (PEG) hydrogel. The glass substrates containing Ab arrays were incubated with 100 μL of red blood cell (RBC)-depleted whole human blood for 15 min and then exposed to a controlled shear of ∼2 dyn cm-2 for additional 10 min. This process led to the removal of non-specific leukocytes and "development" of patterns of T-cells captured on the Ab spots. The immunofluorescent staining of the surface-bound cells revealed the presence of purified CD4+ and CD8+ T-cells (purity >94%) on their respective Ab spots. Importantly, the proportions of CD4+ and CD8+ T-cells captured on the Ab spots correlated closely (R2 - 0.9) with flow cytometry analysis of T-cell subsets in blood. Overall, this cytometry platform allowed to rapidly (under 30 min) capture pure T-cell subsets from minimally processed human blood. Significantly, our device provided quantitative information about subset abundance solely based on the location of cells within the microarray. This cytometry platform is envisioned as a miniature immunology tool for determination of T-cell phenotype and will have immediate applications in HIV diagnostics and research.
AB - Given the clinical and diagnostic importance of blood analysis, there is considerable interest in developing novel miniature devices for rapid characterization of blood constituents. The present paper describes development of a miniature cytometry platform aimed at analysis of T-lymphocytes from peripheral human blood. Microarrays of T-cell-specific antibodies (Abs), including anti-CD3, -CD4, -CD8 and mouse IgG (negative control) were robotically printed onto glass slides coated with a non-fouling poly(ethylene glycol) (PEG) hydrogel. The glass substrates containing Ab arrays were incubated with 100 μL of red blood cell (RBC)-depleted whole human blood for 15 min and then exposed to a controlled shear of ∼2 dyn cm-2 for additional 10 min. This process led to the removal of non-specific leukocytes and "development" of patterns of T-cells captured on the Ab spots. The immunofluorescent staining of the surface-bound cells revealed the presence of purified CD4+ and CD8+ T-cells (purity >94%) on their respective Ab spots. Importantly, the proportions of CD4+ and CD8+ T-cells captured on the Ab spots correlated closely (R2 - 0.9) with flow cytometry analysis of T-cell subsets in blood. Overall, this cytometry platform allowed to rapidly (under 30 min) capture pure T-cell subsets from minimally processed human blood. Significantly, our device provided quantitative information about subset abundance solely based on the location of cells within the microarray. This cytometry platform is envisioned as a miniature immunology tool for determination of T-cell phenotype and will have immediate applications in HIV diagnostics and research.
KW - Antibody microarrays
KW - Blood-based diagnostics
KW - Cell microarrays
KW - T-cell capture
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U2 - 10.1016/j.aca.2007.12.021
DO - 10.1016/j.aca.2007.12.021
M3 - Article
C2 - 18215650
AN - SCOPUS:38349060533
SN - 0003-2670
VL - 608
SP - 186
EP - 196
JO - Analytica Chimica Acta
JF - Analytica Chimica Acta
IS - 2
ER -