How Does Flow Cytometry Work?

Overview

Flow cytometry is an advanced rapid technology that provides a multi-parametric analysis of cells present in a solution. It uses lasers as a source of light to produce scattered and fluorescent light signals. These light signals are read by photomultiplier tubes and photodiodes.

The light signals are converted into electrical signals, which can be easily analysed by a computer. Flow cytometry helps to analyse and purify the cell population depending on their fluorescence and light scattering characteristics.

The usage of reagents depends on the type of cells used. Some of the used reagents include fluorescently conjugated antibodies, viability dyes, DNA binding dyes, ion indicators and florescent expression proteins.

Flow cytometry is an effective and powerful tool used in the field of molecular biology, immunology, virology, cancer biology and in infectious disease monitoring. The usage of flow cytometry in the field of immunology and cell biology has provided minute details that will help the research to move forward.

How does flow cytometry work?

The major advantage of flow cytometry is it allows for multiple characterisations in a mixed population of cells. For example, a mixture of blood, blood marrow, lymph nodes, spleen, solid tumour and mucosal tissues can disassociate the solid tumour cells and small cells at the same time. Additionally, it is a multi-parametric tool.

Traditional flow cytometry will have three systems—fluidics, optics and electronics. The fluidics system will contain a buffer solution which is usually saline. The saline buffer solution will be enforced to pressure in such a way as to focus and deliver the sample to the laser intercept, where the sample will be analysed.

The optical system will have lasers which will be excited when a sample hits in. The optic system also includes photomultiplier tubes (PMTs) and photodiodes. These photomultipliers and laser lights will produce fluorescent and visible light depending on the filter used.

The electronic system will help to convert digital signals into a standard format that can be read by a computer.

Some of the multiple parametric analysis that can be done by flow cytometry are listed below.

• Cell granularity
• Cell size
• DNA gene expression
• Total DNA
• Intracellular proteins
• Transient signal and
• Surface receptors.

These parameters can be performed simultaneously at the same time.

A solution of cells will be passed through the narrow, rapidly flowing stream of liquid. The flow will be arranged in such a way that there is a separation of cells depending on their diameter.

A vibrating mechanism is involved, which forces the cells to break into individual droplets. The system will be adjusted in such a way that there is less probability of more than one cell in a droplet.

Before the cell stream can break into droplets, the cells will be passed through a fluorescence measuring station. This will measure the cells that have an interest in fluorescent characteristics.

A ring that is electrically charged is placed at the point where the cell stream breaks into droplets. The charged ring will capture the cells that have an opposite charge from the stream of cells as it breaks into droplets.

In advanced flow cytometry systems, the charge will be directly applied to the cell stream. When the cell stream breaks, the droplet that has the same charge will be retained, whereas the opposite charge will pass through.

Types of flow cytometry cell

Flow cytometry is used in clinical research across various fields. It provides multiple results at the same time. Some of the common uses are listed below.

Cancer cells

Flow cytometry analysis can determine the presence of cancer cells and the growth of cancer cells if the cells are doubling aggressively or subtly.

Sperm Cells

Sperm cell count can be done with semen collection from the male patient. It analyses the sperm health of males.

Immune Cells

Immune cells can be analysed with flow cytometry. The immune competence of the cell will be analysed. The ability of the lymphocytes to produce antibodies is called immune competence.

The lymphocytes will be isolated from the cell population, and the immune competence will be analysed with the help of flow cytometry.

Uses of flow cytometry

Flow cytometry is used in many research fields as it can sort and analyse multi parameters. Some of the uses are discussed below.

Immune function with HIV infection

The flow cytometric analysis is used to detect the immune functions of patients affected with HIV (human immunodeficiency virus). The flow cytometry that is used in clinical grade can analyse at least four samples at a time with multi-parametric analysis.

The mononuclear cells will be separated from the blood obtained from the HIV patients.

The HIV antigen in the blood sample will be detected indirectly with an immunofluorescence method.

After the immunofluorescence method, the collected blood will be subjected to certain laboratory methods, and then it will be analysed with the flow cytometry method. The flow cytometry will spot HIV antibodies.

The result determined by flow cytometry is a rapid, sensitive and quantitative assay to determine the antigens of HIV-seropositive patients. These results can indicate whether the disease is progressing or in the initial stages of infection.

Bone marrow and anaemia

The flow cytometry can count the immature red blood cells, also called as recticulocytes, that are present in the bone marrow.

When the ratio of immature bone marrow cells are detected in the bone marrow, the cause of anaemia can be determined.

Flow cytometry can also analyse the bone marrow cells if a person wants to do a bone marrow transplant. The technique can also be used to check the health after a bone marrow transplant or after chemotherapy.

This analysis can be done with a sample, and multiple sample collection will not be required.

Organ transplants

For organ transplantation, certain test will be required. Flow cytometry can help with detection methods.

Histocompatibility or HLA testing is the preferred test to check for compatibility of the donated organ. This type of test can be utilised whether it is from the donator or to the recipient. The blood sample will be collected and analysed with flow cytometry.

Sperm Count

The sperm count can be analysed with the help of flow cytometry. The semen sample collected from the patient will be analysed for the total count of sperm cells in the semen.

The sperm count is usually done to test for infertility check up or after a vasectomy to check for the fertility rate.

Blood related cancers

Flow cytometry is used to diagnose any health illness related to blood like leukemia or lymphoma. The flow cytometry diagnostic method can effectively diagnose blood cancer and any defect in the red blood cells.

The sample collection can be different. A sample of bone marrow, blood sample or tissue collection will be required when flow cytometry diagnosis is used.

Blood clotting disorders

Blood clotting disorders can be diagnosed with flow cytometry. It can assess whether the platelets or the blood cells are performing their activities properly or if there is any lag in the body functions.

However, the flow cytometry diagnosis will depend on the patient’s health history and the doctor’s requirement to diagnose a particular disease.

Immunophenotyping

Immunophenotyping is a technique that is used to count specific antibodies with the help of fluorescent compounds to detect the expression of proteins within a cell population.

Immunophenotyping measures the CD4 count in a cell population with the help of flow cytometry. Flow cytometry uses scattered light signals and chooses the fluorescent compounds present in the cell population.

Flow cytometry has the ability to determine the presence or absence of fluorescent compounds and reports the expression of desired proteins.

Before Flow Cytometry Test

Before the flow cytometric analysis, your doctor will give certain instructions. If there is no specific advice from your doctor, then no preparation will be required.

For example, if the tissue or bone sample is to be collected. You can visit the diagnostic centre, and the sample will be collected by your healthcare provider.

After Flow Cytometry Test

After the analysis of flow cytometry, the findings will be discussed with your doctor. If further treatment is needed, it will be provided in accordance with the diagnostic findings.

When to see a doctor?

After collecting your test report, visit your doctor. The doctor will explain the test results and findings.

Conclusion

Flow cytometric analysis is an advanced technique to determine the presence of any faulty red blood cells. It can also effectively diagnose cancer cells.

The analysis method also tells about the progression of a disease and how severe the disease is.

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