title: High-throughput flow cytometry: validation in microvolume bioassays.
abstract: BACKGROUND
  We recently reported an automated sample handling system, designated HyperCyt, by which samples are aspirated from microplate wells and delivered to the flow cytometer for analysis at rates approaching 100 samples per minute. In this approach, an autosampler and peristaltic pump introduce samples into a tubing line that directly connects to the flow cytometer. Air bubbles are inserted between samples to prevent sample dispersion. In the present work, we compare results of HyperCyt with those of conventional manual flow cytometric analysis in representative flow cytometric bioassays and describe a cell suspension method in which HyperCyt exploits the use of microvolume wells.
  
  
  METHODS
  Human eosinophils and neutrophils were treated with trypsin to generate a wide (>25-fold) range of membrane P-selectin glycoprotein ligand-1 (PSGL-1) expression and then stained with fluorescent anti-PSGL-1 antibodies. Human peripheral blood mononuclear cells were stained with fluorescein isothiocyanate- and phycoerythrin-conjugated monoclonal antibodies for multiparameter immunophenotype analysis. U937 cells labeled with PKH62GL were used to assess cell settling in microplate wells.
  
  
  RESULTS
  Differences in PSGL-1 expression levels were detected by HyperCyt autosampling of leukocytes from 96-well plates at an analysis rate of approximately 1.5 s/well. HyperCyt measurements linearly correlated with parallel manual measurements (r(2) = 0.98). Lymphocyte subpopulations were accurately distinguished and reproducibly quantified in multiparameter immunophenotyping assays performed over a range of HyperCyt analysis rates (1.4-5.5 s/sample). When assay volumes were reduced to 10 microl/well in 60-well Terasaki plates, cells could be maintained in uniform suspension for up to 30 min by periodically inverting plates on a rotating carousel before HyperCyt analysis. HyperCyt analysis of five fluorescence-level Cyto-Plex beads sampled from Terasaki plate microwells at 2.5 s/well produced highly reproducible results over a wide range of input bead concentrations (from 7 x 10(5) to 20 x 10(6) beads/ml) that linearly correlated with manual analysis results.
  
  
  CONCLUSIONS
  The HyperCyt autosampling system enabled a 10-fold or greater increase in sample throughput compared with conventional manual flow cytometric sample analysis, with comparable analysis results. Assays were performed efficiently in 10-microl volumes to enable significant reagent cost savings, use of quantity-limited reagents at otherwise prohibitive concentrations, and maintenance of uniform suspensions of cells for prolonged periods.
authors.affiliation: Cancer Research and Treatment Center, Department of Pathology, University of New Mexico Health Sciences Center, 2325 Camino de Salud, Albuquerque, NM 87131, USA.
authors.forename: Sergio
authors.initials: S
authors.lastname: Ramirez
authors.forename: Charity T
authors.initials: CT
authors.lastname: Aiken
authors.forename: Brett
authors.initials: B
authors.lastname: Andrzejewski
authors.forename: Larry A
authors.initials: LA
authors.lastname: Sklar
authors.forename: Bruce S
authors.initials: BS
authors.lastname: Edwards
mesh_terms: D001681:Biological Assay / Q000379:methods*; D004305:Dose-Response Relationship, Drug; D004804:Eosinophils / Q000166:cytology*; D005434:Flow Cytometry / Q000379:methods*; D016650:Fluorescein-5-isothiocyanate / Q000494:pharmacology; D005456:Fluorescent Dyes / Q000494:pharmacology; D006801:Humans; D016130:Immunophenotyping; D020014:K562 Cells; D007963:Leukocytes, Mononuclear / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000378:metabolism; D009504:Neutrophils / Q000166:cytology*; D010799:Phycoerythrin / Q000494:pharmacology; D013997:Time Factors; D014357:Trypsin / Q000494:pharmacology; D020298:U937 Cells
chemical_list: D005456:Fluorescent Dyes; D008562:Membrane Glycoproteins; C084882:P-selectin ligand protein; D010799:Phycoerythrin; D014357:Trypsin; D016650:Fluorescein-5-isothiocyanate