Viability Methods Of Assessing Leukocyte

Francis J Hornicek and George I Malinin, Department of Orthopaedics and Rehabilitation (R-12), University of Miami School of Medicine, Miami, USA

The advent of cell culture methodology in the early twentieth century imposed a requirement for rapid and reliable methods to assess the viability of cells maintained in vitro. In 1914, by establishing a positive correlation between the capacity of intact cells to exclude exogenous dyes and their viability, Evans and Schuleman laid the foundation of all viability assays based on the dye exclusion principle. Soon thereafter Pappenheimer determined the viability of isolated thymocytes by microscopic enumeration of trypan blue stained and unstained cells. Although many dyes have been used for viability assays, the anionic dye trypan blue (961 Da) is the one most frequently employed.

The viability of leukocytes as assessed by trypan blue exclusion is usually determined by suspending the cells either in a balanced salt solution or in phos-phate-buffered saline. After diluting the cell suspen sion with the corresponding salt solution, it is mixed with 0.4% w/v freshly filtered trypan blue dissolved in an identical salt solution. Thereafter, leukocytes are counted on a hemacytometer, and the percentage viability is calculated by dividing the number of viable (unstained) cells by the number of all counted cells (stained and unstained). Until the middle of the twentieth century, microscopic assessment of dye exclusion was the sole practical method of determining viability. Rapid strides in particle counting, computing and ancillary technologies made in the 1960s spawned a number of commercially produced, versatile flow cytometric systems. In contrast to microscopic viability assays, numerically large cell samples can be analyzed by flow cytometry within minutes. In this case the fundamental principle of viability assessment, i.e. viability implies plasma membrane integrity, remains inviolate. In flow cytometric procedures, fluorochromes such as fluorescein diacetate, 4'-6-diamidino-2-phenylindole, acridine orange and propidium iodide are commonly used instead of non-fluorescent dyes employed in microscopic assays.

All cell populations assayed for viability invariably contain dead and dying cells. Regardless of whether necrosis or apoptosis is the underlying cause of cell death, dead and dying cells undergo progressive degradation of their macromolecular components, such as RNA, and consequently lose their affinity for supravital dyes. For this reason any leukocyte population to be assayed for viability must be regarded from the onset as consisting of viable dye excluding cells, morphologically intact nonviable stained cells, and dead chromophobic cells. The failure to include all three categories of cells in viability determinations may yield a significantly higher viability index than its true value.

The identification of nonviable chromophobic cells is practically unattainable with a hemacytometer. They can, however, be identified and counted in fixed leukocyte smears after staining at acidic pH with a cationic dye such as toluidine blue. Dead chromophobic cells in these smears are recognized by the absence of cytoplasmic and/or nuclear basophilia.

Analogous determinations can be performed more expeditiously by flow cytometric methods. In such cases, unfixed cells stained with acridine orange and ethidium bromide are quantified by single-laser multiparametric flow cytometry. Moreover, cells with damaged plasma membranes (nonviable)

undergoing progressive structural degradation (apoptosis) have a lower refractive index than viable cells. As a consequence, optical properties and especially forward light scatter of such cells differ from their viable counterparts. These differences may also be used for flow cytometric assessment of cell death by forward and right-angle light scatter.

Viability assays performed with a hemacytometer and by flow cytometry usually yield higher viability values with the latter method. This quantification disparity is generally attributed to the higher sensitivity of flow cytometric systems as compared with visual observation. A real possibility that laser illumination of cells stained with photosensitizers may inflict lethal photosensitization of analyzed cells is essentially ignored in reporting results. For all practical purposes there is no ideal method of cell viability determination. Therefore, a choice of a specific assay procedure is frequently a matter of personal preference and the accessibility of requisite instrumentation and facilities.

See also: Apoptosis; Cell separation techniques; Cytotoxicity, assays for; Flow cytometry; Leukocyte culture.

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