Flow cytometry was first developed in 1965 by Mack Fulwyler. It is generally used for the determination of leukemia and also for the research purposes. It is a semi – quantitative strategy that permits laser-based innovation to analyze the frequency and furthermore to check, sort and profile cells stained in the specific fluorochrome conjugated antibodies. Particles pass through a laser light beam, one by one in a single file, which is identified by the indicator and afterward the data is exchanged to the PC. (Lakschevitz, 2016).
PRINCIPLE OF FLOW CYTOMETRY
It uses the principle of laminar flow sheath, where by red blood cells suspension is introduced into a faster-moving stream of fluid, resulting in the formation of sheath. Which surrounds and aligns the cells and it applies hydrodynamic focusing on the cells, allowing then to pass one by one in a single file. As the cells flow through the tube, it passes through a laser bean light which is a central spot of intense illumination. Lasers opposes optical and electrical signals which reflects the biological properties of the cells such as cell estimate, volume, inward qualities. (Woo, Baumann, & Arguello, 2014)
Laser gives extraordinary light to the stream cytometry, which goes through a few focal points. The light hits the cells prompting the interruption of the laser light, which makes light diffusing in forward and side direction. Forward scattered light is specifically relative to the extent of blood cells. Side scattered light is corresponding to the inner structures of the cells, for example, the morphology, N/C proportion, granularity. (Woo, Baumann, & Arguello, 2014)
Light scattered hits unique dichroic mirrors known as beam splitters which allows certain wavelength to pass through while reflecting others. Each dichroic reflect has a relating bandpass channel exceptional to a foreordained wavelength which limits wavelength cover. The light force is then estimated by the PMT. The data is gathered and handled with the guide of computer produced algorithms, grouping the data into spot plots and histograms. These illustrations reflect particular properties of the cells in light of inward cell structures, size, and fluorescence or other extraordinary color. One would then be able to center around particular locales of the histogram and limit information introduction to only those parameters of enthusiasm. For instance, lymphocytes, granulocytes, and monocytes can be effortlessly isolated in light of size and interior cell structures. Dead cells and cellular debris are electronically excluded. (Woo, Baumann, & Arguello, 2014)
Today, numerous fluorochromes are accessible that emits at various wavelength, demonstrates few fluorochromes with their excitation and emanating wavelengths. This permits user to create panels. Multi-color flow cytometry is the present best in class indicative apparatus in flow cytometry and offers altogether more data identified with subsets inside a secluded populace. From a solitary tube containing various antibodies, each named with an alternate fluorochrome, more information is gotten, notwithstanding sparing specialized and systematic time, diminishing example necessities, and diminishing reagent utilize. (Jain et al., 2018)
CLASSIFICATION OF LEUKEMIA
Flow cytometry analysis is requested when there is presence of immature blast cell, atypical lymphocytes in peripheral blood, bone marrow or enlarged lymph nodes with presence of cytopenia. Hence, flow cytometry diagnosis must be parallel with morphologic evaluations for the lymphoma and leukemia.
• Hematopoietic stem cells -CD34, CD43, CD17 (c-kit), Sca1, CD133, CD45
• B cells – CD19, CD20, CD22, CD24 (early B cell), CD27 (memory B cell), CD40, CD57 (activated B cell), CD79, CD79a, CD45
• T cells – CD2 (early T cell), CD3, CD4 (Th1), CD5, CD7, CD8 (cytotoxic T cell), CD25 (activated T cell), CD38 (activated T cell), CD73, CD101, IL-7Ra, CTLA4, CD45
• NK cells – CD16, CD56. (Ahuja et al., 2018)
• Granulocytes – CD13, CD14, CD15, CD33, CD64, CD10, CD114, CD182, MPO, CD45.
• Monocytes – CD13, CD14, CD33, CD64, CD68, CD69, CD163, CD45
• Megakaryocytes/platelets – CD31 (platelet), CD36 (platelet), CD41, CD61, CD71, CD45
• Erythrocytes – CD36, CD47, CD71, CD235, glycophorin A
• Myeloblasts – CD13, CD33, CD117, CD38, HLA-DR, CD34, CD45
• Promyelocytes – D13, CD15, CD33, CD117, MPO, CD45
• Leukemia, lymphoma, myeloma phenotyping -Leukocyte surface antigens, TdT, MPO, Kappa/lambda light chains
• Paroxysmal nocturnal hemoglobinuria – CD55, CD59.
• Pro-B ALL: lymphoblasts are CD19, CD34, CD22, CD79a positive and CD10 negative
• Pre-B ALL: lymphoblasts are CD22, CD34, CD19(Falay et al., 2018)
• Analyses at higher speed
• Large number of cells can be measured
• Can be done in small population
• Quantification of flurescence intensities
• Sorts predefined cells populations
• It is portable. (Illoh, 2004)
• It is less sensitive because of the similarities between the normal cells and malignant cells
• Limited standardization
• Requires experienced worker
• QC is not run
• Limited in diagnosis of Acute lymphocytic lymphoma. (Illoh, 2004)