Flow cytometry is a technology that provides rapid, multi-faceted analysis of individual cells suspended in a liquid medium. The technique is capable of assessing multiple physical and chemical characteristics simultaneously, processing up to 10,000 cells in less than one minute. This speed and precision make it a useful tool for characterizing complex cell populations, particularly the various immune cells found circulating in the peripheral blood. The system illuminates these cells with laser light, measuring how the light is scattered and how fluorescent molecules attached to the cells emit light. This process collects data on cell size, internal complexity, and the presence of specific surface proteins to identify and quantify different cell types.
How the Flow Cytometer Operates
The flow cytometer functions through the synchronized action of three integrated systems: fluidics, optics, and electronics. The fluidics system focuses the cell sample into a narrow, single-file stream using a surrounding buffer liquid known as sheath fluid. This hydrodynamic focusing ensures that each cell passes individually through the instrument’s detection point.
When the cells reach the interrogation point, the optics system uses focused laser beams to illuminate each cell. The interaction between the laser light and the cell generates two types of signals: light scatter and fluorescence. Light scatter provides information about the cell’s physical properties, while fluorescence reveals molecular characteristics.
Light scatter is measured in two distinct ways: forward scatter (FSC) and side scatter (SSC). The FSC detector measures light scattered along the laser’s path, correlating with the cell’s relative size. Conversely, the SSC detector collects light scattered at a 90-degree angle, providing data on the cell’s internal complexity, or granularity.
The electronics system converts the optical signals into digital data that a computer can process. Specialized detectors capture the scattered and fluorescent light, generating electrical currents proportional to the light intensity. Software then analyzes this data to create scatter plots and histograms. This allows researchers to visualize and selectively analyze specific cell populations based on their unique size, complexity, and fluorescence profiles.
Preparing Peripheral Blood for Analysis
Peripheral blood analysis requires preparatory steps to isolate the desired cells and tag them for detection by the cytometer. Samples are collected into anticoagulant tubes, such as those containing EDTA or heparin, to prevent clotting. Red blood cells must be removed before analysis because they are numerous and would obscure the white blood cells.
The removal process is called lysis, which involves adding a specialized buffer. This buffer selectively breaks open the red blood cell membranes while leaving the white blood cells largely intact. After lysis, the sample is centrifuged to pellet the white blood cells, and the supernatant containing the debris is discarded.
The remaining leukocyte pellet is then stained with fluorescent dyes, or fluorochromes, typically attached to antibodies. These labeled antibodies bind specifically to target molecules on or inside the white blood cells. Following staining, a washing step removes any unbound fluorescent antibodies.
Identifying Cells Through Immunophenotyping
Immunophenotyping analyzes the prepared blood sample using the specific binding of fluorescent antibodies to identify cell types. This technique relies on immune cells expressing unique combinations of surface or intracellular proteins, known as Cluster of Differentiation (CD) markers. By using a panel of antibodies, each labeled with a different color, researchers can create a unique fluorescent signature for every cell type present.
For example, all T lymphocytes express the CD3 marker, while B lymphocytes are identified by markers like CD19 and CD20. T cells are further subdivided into helper T cells (CD4 positive) and cytotoxic T cells (CD8 positive). The flow cytometer simultaneously measures the distinct light scatter profile and the multiple fluorescence signals for each passing cell.
Combining the physical data from light scatter with the molecular data from fluorochromes allows for highly specific identification of cell subsets. This multi-parameter analysis generates a comprehensive cellular map of the peripheral blood. This enables precise enumeration and characterization of even rare cell populations.
Diagnosing and Monitoring Diseases
Flow cytometry is an established diagnostic method in clinical hematology and immunology due to its ability to rapidly and precisely identify cell populations. Its primary application is the diagnosis and classification of blood cancers, such as leukemia and lymphoma, where it is used to detect abnormal cells. Cancer cells often display aberrant combinations of CD markers not found on healthy cells, such as the co-expression of markers associated with different stages of maturity. Identifying these abnormal immunophenotypes is crucial for subclassifying the cancer, which directly influences treatment decisions and prognosis.
The technology is also used to detect minimal residual disease (MRD), identifying very small numbers of remaining cancer cells after therapy. This helps monitor treatment effectiveness and predict the risk of relapse. For example, in acute myeloid leukemia, the blast cell population is often identified by the expression of immaturity markers like CD34.
Flow cytometry is also widely used to monitor the immune status of patients with acquired immunodeficiency syndrome (AIDS) caused by HIV. The HIV virus preferentially targets CD4-positive T lymphocytes, and the flow cytometer accurately counts the number of these cells in the peripheral blood. A CD4 count below a specific threshold, typically 200 cells per microliter, is used as a diagnostic criterion for AIDS. Regular monitoring helps physicians track disease progression and the efficacy of antiretroviral therapy.
The technique is also employed to evaluate primary immunodeficiency disorders by quantifying the various lymphocyte subsets, such as T cells, B cells, and natural killer (NK) cells. Abnormal numbers or ratios of these cell types can point toward underlying genetic or acquired immune defects. Other applications include evaluating immune status after organ transplantation to detect early signs of rejection and confirming diagnoses of rare conditions like paroxysmal nocturnal hemoglobinuria (PNH).