Biological research and medical diagnostics increasingly rely on methods that analyze individual cells to understand their characteristics and functions. This helps identify different cell types within complex biological samples. By examining specific markers on cell surfaces or within cells, scientists and clinicians gain insights into cellular health, disease states, and treatment responses. This detailed analysis helps pinpoint changes not visible when looking at cells in bulk.
Understanding CD45
CD45 is a protein found on the surface of most white blood cells, also known as leukocytes. It belongs to a family of protein tyrosine phosphatases involved in regulating cell growth, differentiation, and other cellular processes. CD45 plays a role in cell signaling, particularly in the activation of lymphocytes like T and B cells. It works by removing phosphate groups from specific proteins, influencing how immune cells respond to signals.
CD45 expression levels serve as a marker for identifying and distinguishing various immune cell populations. Different isoforms of CD45 exist, found on different types or stages of immune cells. For instance, naive T cells express CD45RA, while memory T cells express CD45RO. This variation helps categorize immune cells.
How Flow Cytometry Works
Flow cytometry analyzes individual cells or particles suspended in a fluid as they pass through a laser beam. The instrument measures multiple physical and chemical characteristics of each cell simultaneously. As cells flow in a single file, they encounter one or more laser light sources.
When a cell intercepts the laser, it scatters light in different directions, and any fluorescent markers on or within the cell emit light. Two main types of light scatter are measured: forward scatter (FSC) and side scatter (SSC). FSC provides information about cell size, while SSC indicates internal complexity or granularity. Fluorescence detectors also capture light emitted by stained markers, allowing identification of specific molecules on or within cells.
CD45 and Flow Cytometry in Practice
CD45 and flow cytometry combine to precisely identify and quantify immune cell populations. The process begins by preparing a single-cell suspension from a biological sample, such as blood or bone marrow. Cells are then stained with fluorescently labeled antibodies that bind to the CD45 protein on the cell surface. Different fluorescent tags can also identify other cell-specific markers simultaneously.
Once labeled, cells enter the flow cytometer, passing one by one through a laser beam. The instrument measures light scatter (FSC and SSC) and CD45-associated fluorescence intensity for each cell. Lymphocytes, monocytes, and granulocytes exhibit distinct levels of CD45 expression and light scatter. Lymphocytes show high CD45 expression and low side scatter, while granulocytes have high side scatter and lower CD45 expression. Monocytes fall in between, with intermediate CD45 expression and side scatter. Plotting CD45 fluorescence against side scatter allows researchers and clinicians to create distinct “gates” on a scatter plot, isolating and quantifying these immune cell types, and distinguishing them from debris or abnormal cells.
Why CD45 Flow Cytometry Matters
CD45 flow cytometry has broad applications in clinical diagnostics and biological research, providing insights into immune system health and disease. In diagnosing and monitoring conditions like leukemia and lymphoma, this technique identifies abnormal cell populations. For example, leukemic blast cells show lower or different CD45 expression patterns compared to normal white blood cells, aiding in distinguishing acute myeloid leukemia (AML) from acute lymphoblastic leukemia (ALL) and assessing disease progression. Precisely identifying these aberrant cells, even in small numbers, aids diagnosis and tracking treatment response.
Beyond cancer, CD45 flow cytometry is used for immune monitoring. This includes tracking changes in specific lymphocyte subsets, such as T cells and B cells, in individuals with autoimmune diseases like multiple sclerosis or lupus, or in transplant patients to monitor graft-versus-host disease. The technique provides detailed information on cell numbers, differentiation states, and activation patterns, helping clinicians evaluate immunotherapy effectiveness. This detail supports personalized treatment strategies and improved patient outcomes.