Flow cytometry is a laboratory method used to analyze the characteristics of individual cells or particles as they pass in a fluid stream through a beam of light. It measures features like cell size, internal complexity, and the presence of specific markers. By rapidly assessing these properties, flow cytometry provides detailed information about cell populations within a sample, allowing for both counting and sorting of cells.
Why Flow Cytometry is Performed
Flow cytometry serves various purposes across medical and research fields. In clinical settings, it is frequently used to diagnose and monitor conditions affecting the immune system and blood, such as identifying specific types of leukemia and lymphoma by characterizing abnormal cell populations. It also helps monitor immune system function, like tracking HIV progression by measuring specific immune cell counts. Researchers use this method to study cell proliferation, cell death, and protein expression, providing insights into biological processes and disease mechanisms.
Getting the Sample Ready
Before analysis, samples undergo several preparation steps to ensure accurate results. Flow cytometry can analyze various biological samples, including whole blood, bone marrow, cerebrospinal fluid, and disaggregated solid tissues. For solid tissues, cells must first be separated into a single-cell suspension, often involving mechanical methods or enzymatic digestion.
Once suspended, cells are labeled with fluorescent dyes or antibodies. These labels bind specifically to cell components, such as proteins on the cell surface or inside the cell. For example, fluorescently tagged antibodies attach to unique protein markers, allowing the flow cytometer to identify and quantify specific cell types. This labeling ensures the machine can distinguish different cell populations based on emitted fluorescent signals.
How the Flow Cytometer Works
A flow cytometer operates through three main systems: fluidics, optics, and electronics. The fluidics system precisely delivers the prepared sample to the laser detection point. Cells are suspended in a fluid and focused into a single-file stream by a sheath fluid, ensuring individual cells pass one by one through the laser beam, preventing multiple cells from being analyzed simultaneously.
The optics system uses lasers to illuminate cells as they flow. When a cell intercepts the laser, it scatters light in different directions and, if labeled, emits fluorescent light. Detectors capture this scattered light, providing information on cell size (forward scatter) and internal complexity (side scatter). Separate detectors measure emitted fluorescence, with specific filters directing different wavelengths to sensors.
The electronics system converts these optical signals into digital data for computer processing. As each cell passes, light signals transform into electronic pulses, with intensity correlating to signal strength. This allows the flow cytometer to analyze thousands of cells per second, generating a comprehensive dataset for each cell based on its light scattering and fluorescent properties.
Understanding the Results
After data collection, flow cytometry results are visualized using graphical representations like scatter plots and histograms. Scatter plots, often two-dimensional, display cells as individual dots based on two measured parameters, such as size versus granularity or fluorescent marker intensity. This allows for identifying and differentiating various cell populations within a complex sample.
Histograms display a single parameter, showing signal intensity (like fluorescence) on one axis and the number of cells exhibiting that intensity on the other. These plots help assess specific marker expression levels. Trained professionals interpret this visual data, often using “gating” to isolate and analyze specific cell subsets, providing quantitative information about cell types and their proportions.