Flow Cytometry - An Overview

Introduction

Flow cytometry is a quick and effective technique that studies individual cells in a liquid by exposing them to lasers. By measuring light scatter, it is possible to assess many aspects of cells, such as internal complexity and size. This approach advances scientific knowledge of cancer, infectious illnesses, and the immune system. Sorting cells for more research is another use for it.

Flow cytometry equipment has advanced, incorporating specialized instruments and several lasers. With developments in proteins and fluorescent dyes, the number of parameters (characteristics) evaluated has increased, enabling tests with more than 30 parameters.

Data analysis after the experiment is essential. Newer algorithms such as PCA, SPADE, and tSNE assist in extracting useful information from the complicated data provided by flow cytometry, even though traditional methods are still employed.

What Are the Uses of Flow Cytometry?

Flow cytometry is a research tool employed for various purposes:

• Cell Counting: Counting cells quickly is helpful.

• Sorting Cells: It divides several cell types according to their properties.

• Determining Cell Function: It can determine the functions and mechanisms of cells.

• Determining Cell Characteristics: This method pinpoints particular features of cells.

• Identifying Microbes: It may locate microscopic organisms like fungi or bacteria.

• Finding Biomarkers: It finds characteristics that indicate typical cell function.

• Identifying and Managing Malignancies of the Blood and Bone Marrow: It identifies and treats certain bone marrow and blood tumors.

What Are the Different Types of Cytometers in the Field of Flow Cytometry?

• Traditional Flow Cytometer: The three primary components of a traditional flow cytometer are electronics, optics, and fluidics. The fluidics system concentrates and delivers cells for analysis using a solution. The optical system produces signals for analysis using detectors and lasers. Filters direct and choose particular light wavelengths for every fluorescent signal. The electronic system transforms These impulses into digital data for a computer.

• Conventional Flow Cytometers: Conventional flow cytometers often have multiple lasers, which enable the analysis of up to 20 parameters. Even more lasers and parameters are possible with newer equipment. Different types of lasers emit certain wavelengths of light, including blue, violet, green, green-yellow, red, and ultraviolet.

Other cytometer types consist of:

• Acoustic Cytometers With Focus: For improved analysis, employ ultrasonic waves to focus cells, enabling greater sample entry and less clogging.

• Cells Sorter: A particular flow cytometer used to gather and purify samples for additional research. Using charges and oscillating liquid streams, it guides cells according to their properties.

• Cytometer Imaging: Fluorescence microscopy and conventional flow cytometry can be combined to study fluorescence and cell morphology quickly at the single-cell and population levels.

• Mass Cytometers: Use heavy metal-labeled antibodies to combine mass spectrometry and flow cytometry. Their lack of light detection and sample destruction during analysis restricts the acquisition rate.

• Bead Array Analysis Cytometers: For large-scale analyte detection in limited sample volumes, analyze multiplex bead arrays.

• Spectral Analyzers: Measure the full fluorescence emission spectrum for every fluorochrome to overcome the problem of spectral overlap in multi-parameter flow cytometry.

• New Technologies for Detectors: Avalanche photodiodes (APDs) and silicon photodiodes (SiPDs) are two new technologies that are emerging in addition to the classic photomultiplier tubes (PMTs).

These cytometers have a wide range of uses, from straightforward cell analysis to specialized methods for particular types of study.

What Are the Key Features of Various Dyes Used in Flow Cytometry?

• Small Organic Molecules: Small compounds that bind to antibodies include fluorescein, Texas Red, Alexa Fluor 488, Alexa Fluor 647, Pacific Blue, and Cy5. They constantly release light, although there is a slight variation between the light that excites them and the light they release. They attach to antibodies with ease and stability. Certain more recent dyes, such as Alexa Fluor, are better for imaging and have a higher fading resistance.

• Phycobiliproteins: These are big proteins that come from algae and bacteria. Because of their size, they provide a 1:1 ratio with fluorochromes during conjugation, making them ideal for flow cytometry use. They are steady because significant disparities exist between the excitation and emission of light. They are not recommended for prolonged exposure to light since they can fade.

• Quantum Dots: Tiny crystals of semiconductors with well-defined emission spectra. They work best with UV (ultraviolet) or violet lasers. However, polymer dyes have taken their place because of problems in multi-parameter research and difficulties attaching them to antibodies.

• Polymer Dye: Dyes with steady polymer chains and the ability to emit and absorb certain wavelengths of light. They have superior photostability than phycobiliproteins and are very stable. They circumvent the problems that quantum dots have with multi-laser excitation.

• Tandem Dyes: These increase the number of fluorochromes that can be ignited with a single laser by combining tiny organic fluorochromes with either phycobiliproteins or polymer dyes. They can vary in efficiency between batches and are bright, but they are less stable, which makes correction more difficult.

• Conjugates of Metal for Mass Cytometry: Antibodies are connected to heavy metal ions for mass cytometry, enabling several isotopes. They are exclusively utilized in mass cytometry and are not fluorescent.

• Fluorescent Proteins: As gene expression reporters, proteins like GFP, CFP, YFP, and more recent variations are employed. They are widely used in flow cytometry and come in various colors.

• Nucleic Acid Dyes: Colors that bind to DNA or RNA and are employed in various applications, such as stem cell sorting and cell cycle studies.

• Proliferation Dyes: Cell divisions are tracked for long-term proliferation investigations using dyes such as CFSE, which do not impact cell growth.

• Viability Dyes: Dyes that either bind to or exclude cells, indicating the integrity of the membrane, are used to test the viability of cells. Some are fixable with additional examination.

• Calcium Indicator Dyes: Color-changing dyes that bind to calcium and change color to show signaling and cell activation. The information is presented as a ratio of the two wavelengths corresponding to the dye and bound and unbound calcium.

When Is It Required to Do a Flow Cytometry Test?

When medical professionals want to learn more about the cells in the human body, they need to perform a flow cytometry test. It is useful for determining the quantity and characteristics of various white blood cell types, particularly for assessing immunodeficiencies, autoimmune illnesses, and infectious diseases. Additionally, this test is used to identify and categorize specific malignancies, such as lymphoma or leukemia.

Usually performed as a follow-up to other tests, such as a white blood cell scan (WBC) or complete blood count (CBC), flow cytometry is performed when the results of the original tests indicate aberrant cell counts, elevated lymphocyte counts, or the presence of immature blood cells. The outcomes of flow cytometry can reveal crucial details on the aggressiveness of cancer and assist in predicting how the disease will react to particular therapies. It is also helpful in determining whether a condition has returned following treatment.

Flow cytometry is used by medical professionals when they require more precise information on the cells in the human body. Testing of this kind can determine the quantity of immune cells in the human body, examine the cell cycle, spot cancer cells, and even examine genetic material. Researchers also use flow cytometry to better understand the intricacies of illnesses and medical situations.

What Are the Applications of Flow Cytometry?

Flow cytometry is a potent method for learning more about cells in various fields. Here are a few important uses, presented more easily:

• Immunology:

  • Immunophenotyping: Flow cytometry is most frequently used for immunophenotyping. Using fluorescent antibodies that adhere to particular cell surface indicators (such as CD numbers) aids in the identification and counting of various immune cell types.

  • Lineage Markers: Different cell types, including T cells, B cells, monocytes, and NK (natural killer) cells, can be identified by specific markers.

  • Additional Markers: Activation markers, memory markers, tissue homing markers, and chemokine receptor markers are some additional markers that aid in the further characterization of cells.

• Antigen-Specific Reactions: Assessing the immune system's reaction to a particular antigen. T cells are bound by MHC multimers containing the antigen, indicating the response degree.

• Intracellular Cytokine Analysis: This examines how cytokines are generated within cells. Intracellular cytokines are detected by staining cells after they have been treated with inhibitors.

• Analysis of Proliferation: Cell growth and division, or proliferation, is quantified using flow cytometry. Various techniques, such as dyes or antibodies, monitor cell division.

• Apoptosis Analysis: Various techniques, including Annexin V staining, the TUNEL assay, and the detection of caspase activity, apoptosis, or programmed cell death, are investigated.

• Molecular Biology:

  • Fluorescent Protein Analysis: GFP and other fluorescent proteins are utilized as indicators of gene expression. This helps monitor cells and research relationships.

  • Cell Cycle Analysis: By staining DNA (deoxyribonucleic acid), flow cytometry facilitates the study of the many cell cycle phases.

• Transduction of Signals Flow Cytometry: This technique uses antibodies against resting and phosphorylated signaling molecules to study cell signaling pathways.

• RNA Flow Cytometry: This method detects protein and RNA (ribonucleic acid) expression by combining fluorescent in situ hybridization and flow cytometry.

• Cell Sorting: Based on their fluorescence, flow cytometers equipped with sorting features are used to separate and filter cells in preparation for additional examination.

• Other Uses:

  • Absolute Cell Counting: Counting the total number of cells in a sample is known as absolute cell counting.

  • Quantitative Flow Cytometry: Beads are used in quantitative flow cytometry to measure the fluorescence of cells.

  • Multiplexed Bead Array Assays: Several analytes can be detected simultaneously using multiplexed array assays using beads coated with antibodies.

  • Assays for Phagocytosis: Identify particles that devour cells.

  • Analysis and Sorting of Small Particles: Identifies and arranges exosomes and viruses among other small particles.

How Are the Findings of Flow Cytometry Interpreted?

Pathologists are medical specialists who interpret results from flow cytometry. The pathologist examines a report containing details from the flow cytometry test, medical history, symptoms, and a recent physical examination. The primary focus is on cell markers or antigens. Antigen patterns in healthy cells are according to their kind and maturity. An aberrant cell will exhibit distinct patterns that could point to diseases like lymphoma or leukemia. Diseases such as multiple myeloma (cancer affecting bone marrow's plasma cells), non-Hodgkin lymphomas (lymphatic system cancer), acute myeloid leukemia (rapid bone marrow cancer, excessive immature cell production, and a decrease in healthy blood cells), acute lymphoblastic leukemia (rapidly spreading bone marrow malignancy that affects immature lymphocytes and causes an excessive overproduction of cells), and chronic lymphocytic leukemia (mature lymphocytes are impacted by slow bone marrow cancer, which causes abnormal cells to build up in lymph nodes and blood) are frequently linked to abnormal results.

What to Expect While Undergoing a Flow Cytometry Test?

• Before the Test:

  • Different preparations are required depending on the test's goal and the medical history.

  • Sample collection can occur immediately or necessitate additional preparations, such as quitting certain drugs or activities. Let the physician know if one is expecting any allergies to materials or medications and how the prescriptions might affect the test.

  • Semen samples take longer, and bone marrow samples could require anesthesia; blood samples are comparable to standard draws.

  • Depending on the type of sample, a surgical center or the provider's office may be the best place to collect it.

• During the Test:

  • Depending on the required sample, multiple healthcare practitioners may be engaged.

  • Simple blood draws and more complex bone marrow aspirations are among the procedures performed.

  • Following the sample, a bandage is put on, pressure is used, and the patient might need to lie motionless for a bit.

  • Pain management may be suggested if necessary.

• After the Test:

  • Routine adjustments are typically not necessary following a blood draw.

  • One might need to restrict their activities or skip bathing for 24 hours after a bone marrow biopsy.

  • Pay attention to the healthcare provider's specific instructions.

  • Contact the healthcare professional if one has a fever, continuous bleeding, increasing pain, edema, or redness at the biopsy site.

  • Over-the-counter drugs can treat mild to moderate pain, but always listen to the doctor's advice, especially if there are bleeding problems.

Conclusion

Flow cytometry is a potent tool in biology and medicine that allows for the simultaneous examination of several properties of a single cell or particle. Cell sorting, counting, and profiling are common uses, and they offer important insights into the make-up, functionality, and disease states of cells. By utilizing the principles of light scattering and fluorescence, researchers and doctors may accurately and efficiently evaluate enormous cell populations. Flow cytometry is an essential technique in the modern life sciences, with various applications from fundamental research to clinical diagnosis.