Flow Cytometry is a technology used to measure and analyze the physical characteristics of single particles (cells) flowing in a fluid through a beam of light. The multiple characteristics measured include the particle size, the internal complexity of granularity, and relative fluorescence intensity. An optical-to-electronic coupling system is used to determine and record the characteristics of the single-particle and how it scatters in laser light as well as how it emits fluorescence (BD Biosciences).
The principle of flow cytometry
The basic principle of flow cytometry is the passage of cells in single-cell suspension in front of a laser beam to be detected, counted and sorted. (Indian Institute of Technology, IIT)
How does a flow cytometry work?
Through cytometry, researchers are able to get specific information on an individual cell such as cell count, cell size, or cell cycle in a single-cell suspension. The volume of cells is measured in a rapidly flowing fluid stream passing in front of a viewing vent.
The instruments have detectable fluorescent parameters that are measured simultaneously on the same cell. Flow cytometry offers the statistical power to the cell biologist due to its speed and ability to analyze a cell rapidly.
The instrument has three major components that include fluids, optics, and electronics. The fluidic part transports the sample from the sample tube to the flow cell where it is sorted or transported to waste. The optical part consists of sources of excitation light, the lenses, and filters that are used to collect and move the light around and generate photocurrent.
In addition, the electronics control the flow cytometer by digitalizing the photocurrent from the detection system and processing it to be saved for future analysis.
During the experiment, the cells are fluorescently labelled in a single-cell suspension and the sample placed on a flow cytometer. Once the sample is taken up in the instrument, the cells are surrounded by physiological buffers known as the sheath fluid.
Where the sample is organized into a single stream of cells as they flow through the cytometer for subsequent analysis. At the interrogation point (the point where the cells interact with the laser light) the cell is illuminated by the laser beam.
The light strikes the internal physical structures of the cell and excites all the fluorophores of the cell and in return produce a fluorescence emission. Then the light is collected by the detection system and processed through the electronic system of the cytometer.
Cells that pass through the interrogation point are not needed and are therefore carried by the fluidic system to the waste container. However, a sorter passes a cell to the collection tube where it is used for subsequent analysis (ThermoFisherScientific)
Clinical Applications of flow cytometry
Flow Cytometry is used in medical diagnosis and in scientific research particularly in cell biology, pathology, and immunology. Examples of applications include
Hematological malignancies diagnosis
Flow cytometry is used in the immunophenotypic analysis, and in the classification of acute leukemia’s, chronic lymph proliferative disease and malignancies including disease management. Flow cytometry assists in monitoring the progress of patients after chemotherapy or transplantation. In addition, flow cytometry assist in detecting the minimal residual disease (MRD).
Application in the detection of minimum residual disease (MRD)
MRD is the persistent malignant cells in the bone marrow of patients suffering from hematological malignancies at levels that are below the limit of detection by conventional morphological assessment.
The residual malignant cells are responsible for disease relapse in most patients. Flow cytometry predicts the outcome in a simple and rapid way and provides quantitative data in most leukemia cases.
Antibodies in the flow cytometric panels define unique immunophenotypic signs of leukemia cells and distinguish leukemic blasts from their normal counterparts.
Application in the measurement of the efficacy of cancer chemotherapy
The process of selecting the most optimal chemotherapeutic agent is a challenge faced by the majority of oncologists. Flow cytometry is among the multi-therapeutic trials that are being carried out to determine chemotherapeutic efficacy and individual variability of tumor characteristics.
Application in multiple drug resistance
Multiple drug resistance is the major cause of failure in chemotherapeutic agents. Flow cytometry has been used to determine the expression of cell surface and intracellular markers of multidrug resistance as well as the intracellular accumulation and efflux of chemotherapeutic drugs. To identify whether the multidrug resistance is intrinsic or acquired is a potential factor with major clinical value in planning chemotherapy and developing drug efflux blockers.
Detection of the sensitivity of tumor cells to radiation
Flow cytometry is used to detect a tumor cell`s sensitivity to radiation before therapy to measure the viability of the cell. The process is important when designing protocols for drug treatment to improve both accuracy and reliability.
Application in Ligand binding affinity
Flow cytometry is being used prior to treatment with monoclonal antibodies to confirm whether the specific antigens are expressed by the aberrant cells. It is used during and after treatment to verify antibodies binding and to monitor the efficacy of tumor eradication.
Flow cytometry offers the most reliable and precise option for accurate quantitation of anti-D immunoglobulin in the fetal-maternal hemorrhage.
Lymphocyte subset enumeration in AIDS patients
Flow cytometry determines the absolute count of CD4+, CD3+, and CD8+ T lymphocytes. Basically, Human immunodeficiency virus (HIV) infection is characterized by depletion of CD4Tcells, expansion of CD8 T cells, and chronic activation of the immune system leading to immune dysfunction. Therefore, over the years, flow cytometry has proven to be significant in the diagnosis and prognostication of HIV infections in addition to managing people receiving antiviral therapy.
Identification of stem cell
Stem cells are responsible for tissue regeneration and engineering. CD34 cell subsets contain sustainable multilineage engraftment. The flow cytometry system provides a simultaneous enumeration of viable dual-positive CD34+/CD45+ in CD34+ absolute counts and in total viable leukocyte count (Choudhury, Mohania, Dinand, & Taneja, 2013).
Flow Cytometry has been used to provide a well-established method to identify cells in solution. It is most commonly used for the evaluating peripheral blood, bone marrow, and other body fluids. Hence, flow cytometry is a critical component in evaluation of major immunodeficiency disorders thus an important method for the characterization of the immune function.
Biosciences. The USA. (2002, December). Introduction to flow cytometry: A learning guide. Retrieved from https://www.bu.edu/flow-cytometry
Choudhury, S., Mohania, D., Dinand, V., & Taneja, V. (2013, August). Application of flow cytometry in healthcare: An update. Retrieved from https://www.researchgate.com
Indian Institute of Technology. (n.d.). Principles of flow cytometry. Retrieved from https://www.iitk.ac.in
ThermoFisher Scientific. (n.d.). How a flow cytometer works. Retrieved from https://www.thermofisher.com