Article

A model for harmonizing flow cytometry in clinical trials.

Institute for Immunity, Transplantation and Infection, Stanford University, Stanford, California, USA.
Nature Immunology (Impact Factor: 24.97). 11/2010; 11(11):975-8. DOI: 10.1038/ni1110-975
Source: PubMed

ABSTRACT Complexities in sample handling, instrument setup and data analysis are barriers to the effective use of flow cytometry to monitor immunological parameters in clinical trials. The novel use of a central laboratory may help mitigate these issues.

2 Bookmarks
 · 
212 Views
  • Cytometry Part B Clinical Cytometry 11/2014; 86(6). · 2.23 Impact Factor
  • Frontiers in Bioscience 01/2011; 16(1):3014. · 4.25 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Toxicity test of new chemicals belongs to the first steps in the drug screening, using different cultured cell lines. However, primary human cells represent the human organism better than cultured tumor derived cell lines. We developed a very gentle toxicity assay for isolation and incubation of human peripheral blood leukocytes (PBL) and tested it using different bioactive oligopeptides (OP). Effects of different PBL isolation methods (red blood cell lysis; Histopaque isolation among others), different incubation tubes (e.g. FACS tubes), anticoagulants and blood sources on PBL viability were tested using propidium iodide-exclusion as viability measure (incubation time: 60 min, 36°C) and flow cytometry. Toxicity concentration and time-depended effects (10-60 min, 36 °C, 0-100 μg /ml of OP) on human PBL were analyzed. Erythrocyte lysis by hypotonic shock (dH2O) was the fastest PBL isolation method with highest viability (>85%) compared to NH4Cl-Lysis (49%). Density gradient centrifugation led to neutrophil granulocyte cell loss. Heparin anticoagulation resulted in higher viability than EDTA. Conical 1.5 mL and 2 mL micro-reaction tubes (both polypropylene (PP)) had the highest viability (99% and 97%) compared to other tubes, i.e. three types of 5.0 mL round-bottom tubes PP (opaque-60%), PP (blue-62%), Polystyrene (PS-64%). Viability of PBL did not differ between venous and capillary blood. A gentle reproducible preparation and analytical toxicity-assay for human PBL was developed and evaluated. Using our assay toxicity, time-course, dose-dependence and aggregate formation by OP could be clearly differentiated and quantified. This novel assay enables for rapid and cost effective multiparametric toxicological screening and pharmacological testing on primary human PBL and can be adapted to high-throughput-screening.°z
    02/2014;

Full-text (2 Sources)

Download
70 Downloads
Available from
May 30, 2014