8 Color, 10-parameter flow cytometry to elucidate complex leukocyte heterogeneity

Abstract

We developed the chemistry, instrumentation, and software technologies needed to measure, simultaneously and independently, eight different fluorescent molecules on individual cells. Conjugation of these fluorochromes to monoclonal antibodies is straightforward; all immunofluorescence staining is accomplished with direct stains only. We built a hybrid flow cytometer with eight fluorescence detectors and two light scatter channels, with excitation provided by three spatially separated laser beams emitting at 407 nm, 488 nm, and 595 nm. The fluorescence compensation required to make the data orthogonal is of sufficient complexity that it cannot be performed manually; thus, we use software to compensate the data post hoc, based on data collected from singly stained compensation control samples. In this report, we evaluate the 8 color staining technology. Of the seven fluorochromes other than fluorescein, six have a useful brightness at least as great as fluorescein. Three of the fluorochromes (phycoerythrin, allophycocyanin, and the Cy5 resonance energy tandem of phycoerythrin) are considerably brighter than fluorescein and are useful for detecting antigens expressed at low levels. Finally, we show the power and utility of the 8 color, 10-parameter technology using staining experiments on both human and murine immune systems.

Sensitivity measurement and compensation in spectral imaging

“…Poor compensation can compromise the spatial integrity of the data and degrade sensitivity, thereby reducing the efficacy of these measurements. Like compensation in conventional flow cytometry, spectral compensation of multispectral imagery requires the application of a crosstalk compensation matrix (9). Unlike conventional flow cytometry, the compensation process in a multispectral imaging system involves the added dimension of space, so the compensation matrix is applied on a pixel-by-pixel basis.…”

Discriminant function analysis as decision support system for the diagnosis of acute leukemia with a minimal four color screening panel and multiparameter flow cytometry immunophenotyping

“…1,2 Recent developments in flow cytometry technologies including fluidics, lasers, optics, analog and digital electronics, computers, software, fluorochromes and antibodies have facilitated the comprehensive use of multiparametric flow cytometry immunophenotyping, allowing for an objective analysis of high numbers of cells on a single cell level in a relatively short period of time. 3,4 With the use of three-and four-color immunophenotyping, simultaneous measurement of five or six different parameters is now routinely applied for the diagnosis of AL in most clinical laboratories. 5,6 The complexity of five-and sixparameter analyses and the interpretation of the data rely to a great extent on standardization and validation of the instrument, the reagents and the procedure.…”

Nine‐color flow cytometry for accurate measurement of T cell subsets and cytokine responses. Part I: Panel design by an empiric approach

“…Using appropriate controls, software compensation algorithms can correct spillover problems post acquisition. However, due to the increased number of spectral overlaps in polychromatic reagent combinations, even properly compensated data can exhibit unwanted spreading into other measurement channels, complicating data analysis and interpretation (3,4). For example, dimly expressed markers, such as cytokines, are difficult to measure in channels where spreading in properly compensated data increases the background in the cytokine measurement channel.…”

“…Counting errors are influenced by signal intensity and the degree of spillover, and are minimized by using bright dyes with as little spectral overlap as possible. While relative dye brightness (4) and spectral overlaps can be predicted (5), these factors are also influenced by the density of antigen expression. Thus, the counting errors generated by particular antibody and fluorochrome combinations may promote a high degree of spreading into spillover channels, limiting the usefulness of certain reagent combinations.…”