Flow cytometers (FC or FCM) is versatile technology, which use for quantification of analyses properties of single cells stained with specific fluorochrome-conjugated antibodies. Flow cytometry have the ability to analyses many properties of cell in very little time. They can measure cell size, cell granularity, the amounts of cell components such as total DNA, newly synthesized DNA, gene expression as the amount messenger RNA for a particular gene, amounts of specific surface receptors, amounts of intracellular proteins, or transient signalling events in living cells.
Moldavan in 1934, reported the first application in the automatic counting of cells during flow, by forcing a suspension of red blood cells (RBCs) through a capillary tube at a microscope stage and each passing cell was counted by a photoelectric device attached to the ocular lens.
In 1949, Wallace Coulter (patent issued in 1953) in “Means for Counting Particles Suspended in a Fluid ” which leading to “Coulter Counter”. The Particles (RBCs) in suspension were accurately counted using information generated when a cell passes a restricted path (opening). The presence or absence of a particle led to a detectable change in the electrical characteristics of the path and the machine allowed particle or cell size to take place. The ‘Coulter Principle’ is still used in many haematological machines today.
The first fluorescent device was ICP11, developed by Wolfgang Göhde of the University of Münster in 1968. It was commercially available in 1968 and 1969 when it was produced by the German developer Partec in Göttingen via Phywe AG. Traditional absorption methods were often preferred to fluorescence techniques at the time.
In 1965, Mack Fulwyler developed the flow cytometer. He was in particular responsible for the development of an electronic cell volume separator. This instrument was able to separate cells based on electronic cell volume and also used electrostatic deflection to separate and sort. He was in particular responsible for the development of an electronic cell volume separator. It was called the Los Alamos Flow Microfluorometer.
In 1969, Phywe AG of Gottingen produced the first commercial flow cytometer built around a Zeiss fluorescent microscope.
In 1970, the Cytograph commercial cytometer used the He-Ne laser system for scattering at 633 nm and became the first commercial instrument to incorporate a laser. This instrument could segregate and sort live and dead cells depending on the uptake of Trypan blue. This was followed by a fluorescence version called the Cytofluorograph, which was further developed into a device with an air-cooled argon laser at 488 nm.
The cytometer based on fluorescence was previously referred to in German as pulse cytophotometry or impulszytophotometry. In 1976, eight years after of the first fluorescence-based flow cytometer, at the American Engineering Foundation Conference in Pensacola, Florida, it was agreed that the term “flow cytometry” could be used and it quickly became popular.
Flow cytometry component include, fluidics, optics, laser light, electronics and fluorochrome-conjugater monoclonal antibodies. The ?uidics system includes a flow cell, where the sample fluid is injected. Flow cell requires sheath fluid to transports particles in a stream to the laser beam for interrogation. Laser light is the most common because of the properties of intensity, stability and monochromatism. The optics system consists of lasers to illuminate the particles in the sample stream and optical ?lters to direct the resulting light signals to the appropriate detectors. The electronics system converts the detected light signals into electronic signals that can be processed by the computer. Also, the electronics system is also capable of initiating sorting decisions to charge and de?ect particles.
Principle of fluorescence is the emitted light, heat or chemical agents by an atom or molecule that has absorbed light or electromagnetic radiation from another source. In absorption, high energy excites the electron or molecule, in which case the electrons to transition from the ground state, to an excited state. The electrons in higher energy state (excited state) is unstable because of the collision and they will quickly return to the lowest available energy state. Once this state is reached and after a lifetime of fluorescence, the electrons relax on the ground and release their stored energy as a photon. The emitted light usually has less energy than the absorbed radiation. Devices called fluorometers are used to measure fluorescence.
Data analysis are plotted on a histogram. Group of similar cell form a cluster of scatter when the forward and side scatters are plotted against each other. Most hematological sample contain multiple cell population. Before analysis it is necessary to identify the population. Whole blood is use to assay platelet and RBCs. Flow cytometry can displayed the collecting data as 1-parameter histogram or 2-parameter plots. A 1-parameter histogram is described as either the percentage of cell in a set of markers or the mean intensity of the fluorescent population. 2-paeameter is typically divided into four quadrant, every one containing of the total population and this is use to distinguish between fluorescent and nonfluorescent cells. Also, it defines of a cell molecule marked by fluorescent antibody or other fluorochrome. It is usually necessary to analyze one population within many population and debris. Gating is a software use to restrict analysis to a particular population. A gate is made by drawing a graphic boundary around a population of cell. Quadrant markers is divide two parameter plot into four section know as quadrants. To distinguish a negative, single-positive and double population they use the quadrants.
The flow cytometry protocols provide detailed procedures for the treatment and staining of cells prior to using a flow cytometer, is available for:
Direct staining of cells applicable where the fluorophore is directly linked to the primary antibody, Indirect staining of cells applicable when using unconjugated or biotin-conjugated monoclonal and polyclonal antibodies, Intracellular staining methods for intracellular antigens and cytokines, DNA staining for cell cycle analysis and Cell preparation. There are many protocols such as:
1-Direct Staining Flow Cytometry Protocols is one of the simplest and most common staining methods, where live or fixed cells are incubated with directly labeled antibodies against cell surface antigens.
2- Indirect Staining Flow Cytometry Protocols is use when there is not a directly labeled antibody available, or they wish to amplify a signal that can do indirect staining. This is where the stain cell with a primary antibody against the antigen of interest and visualize using a labeled secondary antibody which recognizes the primary.
3- Intracellular Staining Flow Cytometry Protocols use in order to detect antigen not present on the cell surface, cells have to be fixed and permeabilized to disrupt the cell membrane and allow entry of the antibody. Antigens can be then directly or indirectly labeled. Various methods are optimal depending on the antigen and antibody used.
4- Cell Cycle Staining Flow Cytometry Protocols use to measuring DNA content for cell cycle analysis requires fixation and permeabilization of the nuclear membrane. The protocols for staining using DNA binding dyes with and without antibody staining and a protocol for BrdU staining.
5- Cell Preparation Flow Cytometry Protocols use for harvesting cells from various sources to obtain healthy cells, essential for optimal staining and analysis.
6- Product Specific Flow Cytometry Protocols is specific protocols to use with our antibodies.
7- Cell Activation Protocols is general activation protocols using pharmacological reagents and antibodies. Ideal to determine immune competence, marker upregulation, cytokine release and proliferation by flow cytometry.
Clinical application of flow cytometry is counting of reticulocyte and platelets.
Counting of reticulocyte by flow cytometry is accurate and precise. Flow cytometry provide additional reticulocyte parameters of IRE or reticulocyte maturity index (RMI), and measurement of reticulocyte maturity. Different stain is use by different manufacturer for reticulocytes count for residual RNA in RBC such as; new methylene blue, thiazole orange and oxazine 750. Also, they use fluorescent dye CD4K530 and coulter system use neomethylene blue and sulfuric acid as reagent. Automated flow cytometry specifically designed for reticulocyte count by optical light scatter.
Platelets is measurement by counting the platelets that stain with RNA day and labeled with PE-conjugated CD41 antibody to distinguish them from other blood cell. Other platelets assays, platelets associated immunoglobulin G for diagnosis immune thrombocytopenia and platelet surface receptor for diagnosis of platelet function.
Other cellular application include:
Most flow cytometry use Immunophenotyping by using monoclonal antibodies identified by a cluster designation (CD or marker). Markers can be grouped into several categories. Such as cells that are specific for particular lineages(CD4+lymphocytes) or maturation pathway(CD34+ progenitor stem cells)and other varies according to the state of activation or differentiation of same cell.
Flow cytometry may be used to analyses cellular proliferation by determining the stages of the cell cycle within a population of cells. This can be carried out on fixed or live cells using different fluorescent DNA binding dyes in conjunction with monoclonal antibodies to analyse antigen expression or with FP expressing cells.
Cell functioning analysis is use flow cytometry for different clinical significant measurement such as tyrosine phosphorylation, calcium flux and cellular proliferation. Also, flow cytometry can use in measured event that occur during the lymphocyte activiation. Moreover, they can used in karyotype analysis.
Some flow cytometry have additional hardware that allows them to act as cell sortes. After measurement, the computer can sort or isolate a single cell by applying a charge to that cell as it leaves the flow cell. They can be also sterilely sorted and recovered any cell based on any combination of light scatter and fluorescence measurement. Addition, they can separate any type of cell from a complex mixture.
Hematological malignancy can be diagnosis and classification by flow cytometry with immu-neophnotyping. Antibodies and their various combination used in flow cytometry for rapid and reliable identification of neoplasma. Intracellular staining is use to diagnosis of acute leukemia and lymphoma. Also, it help in detect surface antigen. In addition, multiple cell surface and intercellular antigen may be studies in the same time.
Apoptosis measurement by using flow cytometric methods they can be stained with Annexin V or 7ADD to detect apoptosis.
Flow cytometry is a powerful tool that use in clinical laboratory for a variety of analyses. It can be used in a significant number of cell analysis applications ranging from phenotyping to cell health, viability and apoptosis. The two greatest advantages of flow cytometry are its ability to measure a large number of parameters on the same sample and its ability to collect information from millions of cells in a matter of seconds.