Image Cytometry Measures Mononuclear Cell Concentration

The viability and concentration of isolated peripheral blood mononuclear cells (PBMCs) are traditionally measured by manual counting with trypan blue (TB) using a hemacytometer, but red blood cell (RBC) contamination can be an issue.

Scientists at the Nexcelom Bioscience Laboratories, (Lawrence, MA, USA) compared their Cellometer Vision instrument with both manual counting and automatic method for accurately measuring the concentration of PBMCs in prepared blood samples. Fifteen freshly isolated samples were stained with acridine orange and propidium iodide (AO/PI) to identify RBC contamination. The five different methods were manual counting of TB-stained PBMCs in hemacytometer; manual counting of PBMCs in brightfield images; manual counting of acetic acid lysing of RBCs with TB-stained PBMCs; automated counting of acetic acid lysing of RBCs with PI-stained PBMCs; and AO/PI dual staining method.

Each of the 15 samples measured was categorized into low, medium, or high RBC contamination. Five samples showed less than 10% of RBC contamination, six samples showed 10% to 40% of RBC contamination, and four samples showed RBC contamination greater than 40%. The total particles counted in brightfield increased due to the addition of RBCs, while AO/PI staining showed consistent measurement of PBMCs, which again demonstrated the robustness of the method despite RBC contamination. Although inherent RBC contaminants may have existed in the sample, the purpose was to observe the increasing difference between fluorescently stained nucleated cells and total brightfield cell count including RBCs.

The authors concluded that fluorescence-based image cytometry can be utilized to eliminate the RBC-induced error in patient samples, which can improve accuracy and efficiency of PBMC measurement. Cellometer image cytometry has also demonstrated fluorescence-based cell population analysis such as apoptosis detection, cell cycle, as well as surface marker labeling. The system can be used to perform immunophenotyping of collected PBMCs, and can quickly characterize incoming patient samples, further simplifying PBMC characterization protocol. The study was published in the February 2013 issue of the Journal of Immunological Methods.

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