Overview of Flow Cytometry Blood Cell Identification
Flow cytometry is a method that helps determine the unique properties of individual cells. It does this by examining how these cells react to light. This method involves making cells flow in a stream and then analyzing them one by one. The process provides a lot of information about each cell, which makes it particularly useful for studying different types of cells from various sources within the body, like blood, bone marrow, other body fluids, or cells from a fresh biopsy.
This method is capable of studying a staggering 30,000 cells in just a second. Not only does it examine how cells scatter light, but it also measures one or more aspects of their fluorescence, which is a type of light cells naturally emit. This makes flow cytometry a fast, affordable, and widely accessible tool for medical use. It helps doctors and scientists understand cells better, supporting diverse medical applications.
Why do People Need Flow Cytometry Blood Cell Identification
Immunophenotyping, which is commonly done using a method called flow cytometry, is a testing process that operates by identifying markers on cells known as immunomarkers. This method can reveal many aspects about certain diseases. Let’s go through a few examples:
Detecting and Classifying Acute Leukemia:
Acute leukemia is a fast-growing type of cancer of the blood and bone marrow. Flow cytometry is used to identify abnormal cells, often called “blast cells”, by looking for specific markers on these cells. Based on a system devised by the World Health Organization (WHO), these cells can be categorised as being of B, T, or myeloid lineage. This is important because it helps doctors diagnose the type of leukemia a patient may have.
Acute promyelocytic leukemia, a subtype of acute leukemia, has its own unique pattern. In this scenario, doctors will typically look for characteristics such as negative expression of CD34 and HLA-DR, and positive expression of CD13, CD33, and CD117.
Diagnosing Chronic Leukemia:
Chronic leukemia is a slower growing type of blood and bone marrow cancer. Flow cytometry helps identifying unique characteristics on the abnormal cells predominantly found in chronic lymphocytic leukemia. These cells typically exhibit certain markers like CD5, CD19, CD20 and CD23, this can help doctors to diagnose the disease.
Plasma Cell Neoplasms:
In certain diseases, doctors might need to identify abnormal plasma cells in the bone marrow. Flow cytometry helps in this by identifying these cells based on certain markers they express.
Detecting Minimal Residual Disease:
Sometimes, even after treatment for leukemia, a few cancer cells, often less than 0.1% to 0.001% of all cells, may survive within the bone marrow. Flow cytometry can help identify these minimal residual disease cells.
Myelodysplastic Disorders:
This is a group of disorders caused by poorly formed blood cells in the bone marrow. Flow cytometry can help detect Myelodysplastic syndromes by identifying abnormal antigen expression patterns.
Monitoring AIDS:
Flow cytometry is used to count CD4 T-lymphocytes in the blood of patients with HIV. This count helps in monitoring disease progression and the response to antiretroviral therapy.
Atypical Cells in Body Fluids:
Flow cytometry can also be used to analyse atypical cells in certain body fluids to diagnose conditions.
HLA-B27 Assay:
Ankylosing spondylitis, a type of arthritis, can be diagnosed using flow cytometry to study the expression of HLA-B27 on T cells.
DNA Ploidy and S-Phase Fraction:
The measurement of DNA ploidy and S-phase fraction can act as prognostic markers in cases of various types of carcinomas such as breast and cervical carcinoma.
Diagnosing Paroxysmal Nocturnal Hemoglobinuria:
Paroxysmal nocturnal hemoglobinuria, a rare disorder that leads to the destruction of red blood cells, can be diagnosed using flow cytometry.
Detection of Fetal Hemoglobin:
For pregnant women with Rh-negative blood type, fetal red blood cells can be identified using flow cytometry. This is important to determine the appropriate dose of anti-D immunoglobulin treatment.
Transfusion and Stem Cell Transplant:
Flow cytometry can also be useful in transfusion medicine and in stem cell transplants by evaluating things like blood typing discrepancies and quality control of blood components.
Diagnosis of Common Variable Immunodeficiency Syndrome:
Common variable immunodeficiency is a disorder that impairs the immune system. Flow cytometry can aid in diagnosing this condition by analysing expression of certain markers on lymphocytes.
Equipment used for Flow Cytometry Blood Cell Identification
Flow cytometers come in multiple types, each with its own attributes and uses.
Traditional flow cytometers are a common type and use a fluid to direct the sample, allowing for precise analysis of cells or particles based on how light passes through them. They often assist in identifying various types of cells in suspension. Some modern machines can analyze up to 50 factors at the same time.
There’s a type of flow cytometer known as cell sorters. These machines separate and collect specific cells into separate tubes or slides based on unique cell characteristics. Cell sorters typically use electrostatic methods to split cells into droplets with an aim to isolate a single cell within a droplet. When a desired cell is detected, a charge is applied to the droplet. The charged droplet goes off course and lands in a collection tube, while unaffected droplets go into a waste container. There are other cell sorters that use different methods to isolate cells.
Imaging flow cytometers quickly analyze samples for shape and behavior, combining the principles of flow cytometry and fluorescence microscopy, a method that uses color dye and light to show the structures within cells. Cameras capture images of cells from different angles and put them together, allowing researchers to visualize where the fluorescent molecules are located within the cells.
Mass cytometers are another type. Cells are labeled with heavy metal ion-tagged antibodies and the cell population is analyzed using time-of-flight spectrometry, a method that measures the mass of molecules. This method doesn’t use fluorescence-based antibodies, instead, heavy metal ions are used to mitigate the overlapping which typically happens with fluorescing colors.
Spectral analyzers are designed to solve the problem of compensation and spectral overlap in traditional flow cytometers by capturing the full fluorescent emission spectrum for each color dye. Because of this, there’s more freedom in choosing and combining the color dyes.
Acoustic focusing cytometers use sound waves for improving the effectiveness of flow cytometry. Thanks to this technology, it can handle a wide variety of cell sizes, from small platelets to larger ones like heart cells.
Finally, there are cytometers for bead array analysis. Cytometric bead arrays can measure different factors at the same time. These use different bead groups, each with distinct sizes and color intensities.
Traditional flow cytometer is made up of three main parts: fluidics, optics, and electronics. The fluidics system uses a fluid to focus the cells in a stream, so they pass through one at a time, this is called hydrodynamic focusing.
A laser examines the stream of cells, which can scatter the light depending on the size and internal structures of the cells. In addition to scattering, fluorescence is used to differentiate cell types in flow cytometry. Fluorescing dye-labeled primary antibodies are applied to target specific cell proteins. These dyes are stimulated by the laser and emit light of a longer wavelength, which then passes through a series of lenses, mirrors, and filters that separate and aim the light at the right detectors. Captured light signals are then changed into electric pulses and then turned into digital data, which is analyzed using specialized software.
The software for flow cytometry data analysis typically uses charts to visualize the data. Cell groups of interest are found and separated by setting boundaries to single out cells with similar physical characteristics. The program then calculates statistics, such as frequency, within these boundaries.
In short, flow cytometry works by focusing a cell sample, allowing individual cells to pass through an opening. Each cell is then analyzed by a laser, and the resulting light scattering is processed by software for quantification and further analysis.
When it comes to clinical flow cytometry, having an effective quality management system is essential to ensure consistent test specifications, including precision, accuracy, sensitivity, specificity, reference ranges, and stability.
Preparing for Flow Cytometry Blood Cell Identification
Flow cytometry is a technique often used for testing the blood or bone marrow in the body. For the best results, doctors need a fresh sample of blood or bone marrow and should ideally test this within 18 hours after it has been collected. It’s also important that no more than 36 hours pass from the time of collection to the time of testing. It’s crucial that the sample is kept at room temperature; it should not be put in the fridge. Also, the doctors have to check the number of cells in the sample before they start the testing process.
To get the sample ready for testing, the doctors will follow a specific set of steps. First, they’ll expose the cells in the sample to special substances (fluorochrome-labeled primary antibodies), which will stick to the cells. Then, using a special liquid (lysis buffer), they’ll break open the red blood cells in the sample. Afterward, they’ll wash and spin the sample. This series of steps helps to color or stain the cells so that certain parts of the cells become visible. Those parts can then be recognized by a machine known as a flow cytometer.
In case the doctors need to examine some parts lying inside the cells, they have to perforate the cellular membrane to let the antibodies enter the cells and bind to the parts.
After preparing the sample, they will load it into the flow cytometer. This machine can classify and sort cells by detecting how light interacts with each stained cell. To ensure an optimal analysis, the flow cytometer is recommended to capture a specific range of cells at a certain rate. Proper checks are conducted before the analysis to ensure the experiment goes as planned.
How is Flow Cytometry Blood Cell Identification performed
Flow cytometry is a technology that helps identify different types of cells in the blood. This is achieved by examining various features of cells, like size and internal structure, and how they scatter light. Cells like lymphocytes, which are small, tend to scatter light less compared to larger cells such as granulocytes. A similar pattern can be noted with the granularity, or internal structure, where cells that possess a non-granular structure like lymphocytes display lower scattering compared to cells with granular content, such as granulocytes. Despite these observations, this method might not be completely accurate for classifying all types of cells.
Scientists have also figured out a way to increase the accuracy of cell identification through markers, which are substances used to paint or color cells for easy recognition. These markers attach themselves to specific types of cells. They’re often combined with substances that glow under certain types of light, allowing scientists to identify positive and negative expressions. The glow can vary from dim to bright or anywhere in between.
A commonly used marker named CD45, also known as a leukocyte common antigen, helps spot different types of white blood cells (leukocytes). Lymphocytes display a bright positive expression with low scattering, and they can be further identified as either T or B lymphocytes. T lymphocytes express a brighter CD45 marker compared to B lymphocytes. Monocytes, another type of leukocyte, also exhibit a bright positive expression for CD45 but with a higher scatter compared to lymphocytes. Neutrophils, a different type of white blood cell, display a moderate CD45 expression and high side scatter. Red blood cells and cell debris usually don’t express CD45 and scatter light minimally.
Depending on the examination objective, scientists also utilize other special markers to specifically identify each cell population. For instance, T lymphocytes can be identified by markers such as CD3, CD2, CD5, CD7, CD4, or CD8. B lymphocytes can be identified using markers like CD19, CD20, CD79a, and CD22. So on for other cell types. Therefore, even if the first method might not be completely accurate, using these additional markers can increase the precision of cell identification.
What Else Should I Know About Flow Cytometry Blood Cell Identification?
Immunology is the study of how the body defends its internal environment from disease-causing microorganisms. In this field, it’s often necessary to compare different types of cells. For instance, B cells (a type of white blood cell) may be useful in studying the presence of a certain protein called CD19. These B cells are said to have “positive expression” of CD19, essentially meaning they contain CD19 protein on their surface. Conversely, T cells (another type of white blood cell) do not contain CD19 on their surface, making them a “negative control” for studying this protein.
This process becomes particularly important when diagnosing blood cancers like acute lymphoblastic leukemia. When treating this disease, doctors need to be able to tell the difference between different types of cells found in bone marrow samples.
To do this, they often use a process called flow cytometry. This technique allows them to measure physical and chemical characteristics of cells. However, before using flow cytometry, it’s beneficial to visually examine the sample under a microscope to choose the most appropriate tests to run. This technique is particularly necessary when testing for diseases of the blood and lymphatic systems.
Nonetheless, it’s important to note that some cells may be lost during the preparation of the sample for testing. This can affect results and is especially problematic when diagnosing cancers like leukemia. So, in cases where test results are hard to interpret, additional examination of the cells under a microscope is recommended.
