FLOW CYTOMETRYIn 1965 Mack Fulwyler first developed the flow cytometry.
It is widely used for the diagnosis of leukemia and also for research purposes. It is a semi – quantitative method that allows laser-based technology to analyze the frequency and also to count, 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 detected by the detector and then the information is transferred to the computer. (Lakschevitz, 2016).PRINCIPLE OF FLOW CYTOMETRYIt gives a quick and synchronous investigation of numerous qualities of single cells. Measures the cell size, cytoplasmic complexity, nucleic acid content and various intracellular/membrane proteins.
It uses the principle of laminar flow sheath. When the red blood cells suspension is injected into a faster-moving stream of fluid, a sheath is form. Which surrounds and aligns the cells and it applies hydrodynamic focusing on the cells, allowing then to pass one by one. As the cells flow in the orifice, it passes through a laser bean which is a focal spot of intense illumination. Lasers opposes optical and electrical signals which reflects the biological properties of the cells such as cell size, volume, internal characteristics. (Woo, Baumann, & Arguello, 2014)Laser provides intense light for the flow cytometry, which passes through several lenses. The light hits the cells leading to the disruption of the laser light, which creates light scattering in forward and side direction. Forward scattered light is directly proportional to the size of the blood cells.
Side scattered light is proportional to the internal structures of the cells such as the morphology, N/C ratio, 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.CD Markers • 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)ADVANTAGE • 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)DISADVANTAGE • It is less sensitive because of the similarities between the normal cells and malignant cells• Limited standardization• Time-consuming• Expensive• Requires experienced worker • QC is not run• Limited in diagnosis of Acute lymphocytic lymphoma.