Laboratory Testing: Flow Cytometry’s Working Principle and Components

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Cell biology methods like flow cytometry use laser beams for cell counting, sorting, and profiling involving a fluid mixture of various components. A flow cytometry instrument is used to direct a laser light on particles suspended in a liquid stream in a single file to measure their interaction through fluorescence intensity and light scatter. Today, flow cytometry has emerged as a powerful tool for researchers for its ability to analyze thousands of particles’ chemical and physical traits rapidly. Due to this, experts rely on this quantitative method for the analysis and purification of the suspended cells. To increase the functionality of this system, they have also started using the fluorescence-activated cell sorting (FACS) technique, which enables them to sort and gather data from a biological sample according to diverse parameters.

Users determine how to sort cells, and the flow cytometry machines impose electrical charges on each cell to sort them in different vessels as they exit the flow chamber. Scientific fields, including clinical and research, greatly rely on this technology to efficiently separate different cell groups from the heterogeneous mixture faster. Hence, it makes sense to learn more about this system and the equipment in detail.

  • Flow Cytometer Overview

As mentioned, flow cytometry measures cells’ physical and chemical makeup when they pass in a single file through a laser beam. The cells distribute laser light in diverse directions from side to forward, allowing users to examine their intrinsic properties, including cytoplasmic complexity and size. For example, a flow cytometry instrument can efficiently examine human blood cells like granulocytes, monocytes, and lymphocytes as they deflect laser beams uniquely. The application of this technology is growing with time in basic research and clinical environments, encompassing stem cells, virology, microbiology, systems biology, cell biology, oncology, neuroscience, immunology, hematology, transplantation, drug discovery, and neuroscience. These machines are also used for clinical trials and patient monitoring. Some clinics use the method to distinguish recipient cells from donor cells before transplant. It allows them to control risks associated with transplantation.

  • Components of A Flow Cytometer

A flow cytometry instrument comprises three core parts: electronics, fluidics, and optics. Fluidics contain a flow cell where sample fluid is introduced. The flow cell uses carrier fluid to move and arrange particles or cells in a single file so that they smoothly navigate a slender path and reach the light beam. Due to this precise process, analyzing every cell one by one becomes possible. As for the optics system, you will find light detectors, light sources, and filters here. An optics system can be described as a laser line that creates a single light wavelength at a chosen frequency. Each particle must face at least one light beam at this stage. Interestingly, one can find various wavelengths of lasers, including far-red to ultraviolet. Their power levels can also be determined by their photon output over time.

When a laser beam is directed, compatible fluorescent molecules bound to antibodies become excited, leading to light emission at a specific wavelength. Likewise, the electronics component detects and converts emitted light into electrical signals before processing and analyzing them through hardware and software systems.

  • Steps Involved in Flow Cytometry Protocol or Workflow

Flow cytometry testing requires a sophisticated approach as it deals with different types of blood or cancer cell detection and characterization. A researcher performs various crucial steps when using this diagnostic tool. It can start with sample collection and preparation, whether tissue, body fluid, bone marrow, blood, or tissue cell is used. One has to maintain sample quality and integrity for accurate analysis. The testing can be smooth if you use the right flow cytometry machine. Many companies offer this instrument. However, only reputable brands can be trusted. Their solutions come equipped with advanced laser technology. When lasers interact with particles, they help determine cell characteristics or fluorescent markers. At the same time, their machines can offer reliable data generated through the interactions between laser beams and cells.

Fortunately, evolved technologies also enable researchers to determine various light-emitting markers of a cell, leading to a better understanding of cell characteristics. Having this data is essential for the experts as it gives them insight into the effects of those cells on health and illness. However, data needs to be correctly analyzed to avoid anomalies or errors. That’s why popular life sciences companies offer special software solutions for data interpretation. You can customize your analysis based on the research purpose, such as cancer cell detection, specific cell counting, immune function evaluation, etc. Ensuring quality control at every step is also critical to obtaining accurate results. If there is an abnormality, you can conduct more tests to corroborate findings and decide further actions related to research and clinics.

  • Flow Cytometry Standardization and Quality Control

In the context of quality control, a flow cytometry lab must meet some requirements to ensure the result’s accuracy. It begins with recording steps that help reproduce the results. So, researchers must follow this step right from the sample preparation stage. Another critical consideration includes checking the flow cytometry instrument’s settings and performance. One must also ensure they are well-maintained, especially those used in clinical labs. Stringent steps should be taken with reagent QC during flow cytometric assessment to reduce the risk of false positives. What’s more? During specimen testing, adequate measures must be taken to avoid cell visibility and count issues.

  • Flow Cytometry Applications

The advancement in this cell biology method has led to its application in various fields, such as immunology, pathology, virology, and molecular biology, as mentioned earlier. Experts use this tool in clinical labs to detect malignant bodily fluids like leukemia. Cytometers are also used to sort and separate select cells in distinct tubes. With the help of fluorescent markers, the method makes it easy to study DNA content. Replicated cells can be analyzed through fluorescent dye during the four cell cycles. The instrument can also examine cell behavior to track its stages, including necrosis, apoptosis, and death.

The benefits of using flow cytometry tools in research and clinical settings are manifold. However, technicians need proper training to achieve the best results.