Flow cytometry is a method of sorting and measuring types of cells by fluorescent labeling of markers on the surface of the cells. Flow cytometric analysis is most often clinically used to help determine the type of leukemia or lymphoma a patient has and to assess the prognosis.
Description
The physician will select a sample based on the type of cancer the patient is thought to have. In the case of lymphoma, the sample may be collected by fine needle aspiration biopsy, then the tissue sample will be separated into single cells. Analysis of leukemia will require a patient to give a blood sample.
The sample will be mixed with a variety of different antibodies that can interact with antigens on te surface of the cells. Different types of cells have characteristic markers on their cell surfaces, so a particular cell type can be identified by the antibodies that bind to it. The antibodies are labelled so that they will give off fluorescent light (glow) as they pass through the laser beam in the cytometer.
The cytometer also measures the size of the cell and some information about the interior of the cell. The physician uses this information to determine the specific type of leukemia such as myelogenous or lymphocyctic which in turn, helps to determine the type of treatment that wll be best suited to the patient.
Cytometer
A standardized glass slide or small glass chamber of known volume, used in counting and measuring cells, especially blood cells.
Preparation
Flow cytometry is usually performed on blood, body fluids, or bone marrow. If bone marrow aspiration is necessary or a biopsy is required from a solid tumor, the patient should be appropriately prepared for these procedures. However, the flow cytometry itself does not require any additional preparation on the part of the patient.
How Does A Flow Cytometer Work
Flow chamber - cells flow through the flow chamber one at a time very quickly. about 500 cells per second.
Laser - a small laser beam of very bright light hits the cells as they pass through the flow chamber. The way the light reflects off each cell gives information about the cell's physical characteristics.
Light detector - the light detector processes the light signals and sends the information to the computer. Forward scatter tells the size of the cell (FSC). Side scatter (SSC) tells is the cell contains granules. Each type of cell in the immune system has a unique combination of forward and side scatter measurements allowing count the number of each type of cell.
Color detectors - as the cells pass through the laser, the fluorochromes attached the cells absorb light and then emit a specific color of light depending on the type of fluorochrome. The fluorochromes on the cells act like a cod. In this case there are two types of fluorescent markers: yellow and green. Any one cell can have one, both or none of the markers on its surface. The color detectors collect the different colors of light emitted by the fluorochromes, The fluorochrome data signal is also sent to the computer.
Computer - the data from the light detector and the color detectors is sent to a computer and plotted on a graph called a histogram. The x-axis is the amount of green fluorescence. The more green fluorescence a cell emits, the farther to the right the cell data will appear on the histogram. The y-axis is the amount of yellow fluorescence a cell emits, The cell data will appear closer to the top on the histogram.
Normal Results
Normal result will indicate that there is no increase in the number of any particular type of immune cell. The pathologist will see several different types of cells, but no one type will be present in increased numbers.
Abnormal Results
An unusually large number of one particular cell type. the types of marker present on the cell will give further information about the type of leukemia or lymphoma and may indicate the patient's prognosis. For example, leukemic cells that have markers that are normally found on less mature cell types may suggest a poorer prognosis, and therefore more aggressive therapy may be recommended.
Analysis
Forward scattered light (FSC) is proportional to cell surface area or size.
Side scattered light (SSC) is proportional to cell granularity or internal complexity.
