Basic science for the practicing physician: flow cytometry and cell sorting

INTRODUCTION Flow cytometry is a technique used to evaluate multiple parameters of individual cells (or other particles) by measuring the photons they scatter or emit as they stream individually through a light source. Fluorescence-activated cell sorting (FACS) refers to the same technology with the additional use of 1 or more measured parameters to physically separate subsets of cells. In conjunction with fluorochrome-labeled monoclonal antibodies (mAbs) to specific cell targets, one can routinely measure 6 to 8 targets simultaneously using a modern flow cytometer; research laboratories may measure more than a dozen. The fundamental strength of the technology lies in its ability to capture many parameters per cell, combined with its high throughput. This distinguishes it from “bulk” analysis methods, which measure the sum of the responses of all subsets in a given population, such as the amount of interleukin 4 (IL-4) secreted from whole peripheral blood mononuclear cells (PBMCs), and instead allows for subset analyses of mixed populations, such as the percentage of IL-4–secreting cells in the memory CD4 T-cell population present in whole PBMCs. Flow cytometry, especially as it has evolved together with mAb technology and the chemistry of fluorescent molecules (fluorochromes), has fundamentally transformed our understanding of immunology in the past 2 decades. There are many excellent references for in-depth review.1 This review focuses primarily on research applications of flow cytometry used in the investigation and evaluation of allergic disease in humans and on background technical information useful for the critical evaluation of flow cytometry data as it is typically presented in the literature. We also make reference to applications of flow cytometry that are currently available for the clinical evaluation of allergy (Table 1). BACKGROUND There are several basic components of any flow cytometer. Initially, a suspension of cells is injected into surrounding nonturbulent (laminar flow) fluid through a flow cell that focuses the cells into a single file stream, where they pass through 1 or more light sources. The light sources are typically lasers, allowing them to be distinguished from emitted light by wavelength and direction. Light that is scattered by diffraction (forward scatter) or by reflection/refraction (side or orthogonal scatter) or that is emitted (eg, after excitation of a fluorochrome) is then collected using a series of detectors, usually photomultiplier tubes (PMTs), that amplify and convert the information to a digital signal. This signal is then processed and analyzed using a computer in real time or is saved for later analysis. Any particle that triggers a PMT signal is referred to as an event, a jargon term that underscores the need to apply critical analysis to the data to determine which events are truly caused by the cells of interest. Forward scatter (principally diffracted light), aptly named as it is measured nearly in line with the light source ( 5°), is an approximate measure of cell size, whereas side scatter increases with granularity, membrane roughness, and other features of cell “complexity.” Lymphocytes, therefore, occupy the low forward and side scatter portion of a bivariate plot of data from PBMCs, which is often referred to as the lymphocyte gate. For most flow cytometers, the side-scattered light is separated by wavelength using a series of bandpass filters so that the PMT distal to each filter will detect light emitted primarily from a single fluorochrome. In this way, particular events can be identified as members of a particular population by using multiple characteristics, including the fluorescence signals that are expected based on which fluorochromes were labeled to which mAbs. Because the emission spectra of commonly used fluorochromes overlap significantly, some signal detected by a specific PMT will be the result of “spillover.” This spillover of light signal into other channels must be compensated for mathematically (Fig 1). For experiments with multiple fluorescence parameters, this is best done by using a series of single-color compensation controls, which can then be used to determine the exact amount of spillover of each fluorochrome being used for the given instrument, fluorochromes, and PMT settings. Compensation will correct the central Affiliations: * Division of Allergy and Immunology, Department of Pediatrics, Jaffe Food Allergy Institute, Mount Sinai School of Medicine, New York, New York; † Immunology Institute, Mount Sinai School of Medicine, New York, New York. Disclosures: Authors have nothing to disclose. Funding Sources: This study was supported by the National Institute of Allergy and Infectious Diseases, the Pediatric National Institutes of Health Loan Repayment Program, and the Food Allergy Initiative (Dr Shreffler). Received for publication February 20, 2008; Received in revised form April 16, 2008; Accepted for publication April 30, 2008.