Flow cytometry

flowcytometry

1. Chandigarh college of Pharmacy Assignment of FLOW CYTOMETRY Submitted to : Miss. Simerjeet kaur chahal Submitted by : Vishali Roll No. CCP212111

2. FLOW CYTOMETRY

3. INTRODUCTION Flow : the motion characteristics of fluids. Cyto : combining form of cells or cells. Metry : measurement / count. Hence , measurement of physical and /or chemical characteristics of cells,or in general,biological articles is known as CYTOMETRY. When such measurements are performed while the cells or the biological material pass in a fluid stream across an illuminated light path,the process is known as FLOW CYTOMETRY.

4. FLOW CYTOMETRY This method allows the quantitative and qualitative analysis of several properties of cell populations from virtually any type of fresh unfixed tissue or body fluid. The properties measured include a particles relative size , relative granularity or internal complexity , and relative fluorescence intensity. Most commonly analyzed materials are : Blood Bone marrow aspirate and Lymph node suspensions

5. PRINCIPLE The basic principle of flow cytometry is the passage of cells in single file in front of a laser so they can be detected, counted and sorted. Cell components are fluorescently labelled and then excited by the laser to emit light at varying wavelengths.The fluorescence can then be measured to determine the amount and type of cells present in a sample.

6. Prepare single cell or particle suspension is necessary for flow cytometric analysis. The suspension of cells or particles is aspirated into a channel surrounded by a narrow fluid system. They pass one at a time through a Focused laser beam. The light is either scattered or absorbed When it strikes a cell. Light scattering is dependent on the Internal structure of the cell and its s ize and shape.

7. Absorbed light of the appropriate wavelength may be re-emitted as fluorescence. Light and fluorescence scatter signals are detected by a series of photodiodes and amplified. Optical filters are essential to block the unwanted light and permit light of the desired wavelength to reach the photodetector. Fluorescein isothiocynate (FITS),Texas red and phycoerythrin (PE) are the most common fluorescent dyes used in the biomedical sciences. Large number of cells are analysed in a short period of time (>1000/sec).

8. Flow chamber Light source scaterring/absorption Light detectors Sample loading Computer

9. WORKING OF FLOW CYTOMETER A flow cytometer is composed of three main systems : FLUIDICS : Transport cells in a stream to the laser beam for interrogation. OPTICS : Consist of laser to illuminate the cells in the sample stream and optical filters to direct the resulting light signals to the appropriate detectors. ELECTRONICS : Converts the detected light signals into electrical signals that can be processed by computer.

10. FLUIDICS SYSTEM When a sample is injected into a flow cytometer,it is ordered into a stream of of single particles. The fluidic system consist of a FLOW CELL Central core : through which the sample is Injected. Outer sheath : contains faster flowing fluid k/a sheath fluid (0.9% saline /PBS), Enclosing the central core.

11. Hydrodynamic focusing Once the sample is injected into a stream of sheath fluid within the flow chamber , they are forced into the center of the stream forming a single file by the PRINCIPLE OF HYDRODYNAMIC FOCUSING. ‘Only one cell or particle can pass through the laser beam at a given moment’. The sample pressure is always higher than the sheath fluid pressure, ensuring a high flow rate allowing more cells to enter the stream at a given moment. High flow rate -Immunophenotyping analysis of cells. Low flow rate - DNA analysis.

12. OPTICS After the cell delivery system, the need is to excite the cells using a light source. The light source used in a flow cytometer : Laser (more commonly) Arc lamp Why Lasers are more common ? They are highly coherent and uniform. They can be easily focused on a very small area (like a sample stream). They are monochromatic ,emitting single wavelengths of light.

13. ARGON Lasers -488nm wavelength (blue to blue green). When a light intersects a laser beam at the so called ‘interogation point’ two events occur : Light scattering Emission of light (fluorescence). Fluorescence is light emitted during decay of excited electron to its basal state.

14. LIGHT SCATTER When light from a laser interrogates a cell, that cell scatters light in all directions. The scattered light can travel from the interrogation point down a path to a detector.

15. OPTICS - FORWARD SCATTER (FSC) Light that is scattered in the forward direction (along the same axis the laser is traveling)is detected in the forward scatter channel. The intensity of this signal has been attributed to Cell size , refractive index .

16. OPTICS - SIDE SCATTER (SSC) Laser light that is scattered at 90 degrees to the axis of the laser path is detected in the side scatter channel. The Intensity of this signal is proportional to the amount of cytosolic structure in the cell (eg:granules,cell inclusions etc.)

17. Why FSC and SSC? Study of FSC and SSC allow us to know the differentiation of different type of cells.

18. Commonly used Fluorochromes FLUOROCHROMES EMISSION MAXIMUM Fluorescein Isothiocynate (FITC) 530nm Phycoerythrin (PE) 576nm Peridin-chlorophyll alpha complex (PerCP) 680nm Allophycocyanin (APC) 660nm Texas red 620nm ECD (PE - Texas Red Tandem) 615nm PC5 (PE-cyanin 5 dye Tandem) 667nm

19. b) Emission of light (fluorescence) As the fluorescent molecule present in or on the particle is interrogated by the laser light , it will absorb energy from the laser light and release the absorbed energy at longer wavelengths. Emitted photons pass through the collection lens and are split and steered down specific channels with the use of filters.

20. Optics : Filters Different wavelengths of light scattered simultaneously from a cell. Need to split the light into its specific wavelengths in order to measure and quantify them independently.This is done with filters. The system of filters ensures that each photodetector receives light bands of various wavelengths. Optical filters are designed such that absorb or reflect some wavelengths of light,while transmitting others. Types of filters : Long pass Band pass Short pass Dichroic

21. Long pass filters Transmit all wavelengths greater than specified wavelengths. Example :500LP will transmit all wavelengths greater than 500nm.

22. Short pass filters Transmits all wavelengths less than specified wavelengths. Example :600SP will transmit all wavelengths less than 600nm.

23. Band pass filters Transmits a specific band of wavelengths. Example : 500/20BP filters will transmit wavelengths of light between 540nm and 560nm .

24. Dichroic filters Long pass or short pass filters. Placed at a 45degree angle of incidence. Part of light is reflected at 90degree , and part of the light is transmitted and continues.

25. OPTICS :DETECTORS The photodetectors convert the photons to electrical impulses. Two common types of detectors used in flow cytometry: Photodiodes:used for strong signals,when saturation is potential problem(eg:forward scatter detector). Photomultiplier tube (PMT) : more sensitive than photodiode but can be destroyed by exposure to too much light. Used for side scatter and fluorescent signals.

26. ELECTRONICS The electronic subsystem converts photons to photoelectrons. Measure amplitude,area and width of photoelectron pulse. It amplifies pulse either linearly or logoarithmically and then digitalizing the amplified pulse.

27. Data Analysis -PLOT TYPES There are several plot choices : Single color Histogram : Fluorescence intensity (FI) versus the number of cells counted. Two color dot plot : FI of parameter 1 versus FI of parameter 2. Two color contour plot : concentric rings form around populations. The more dense the population , the closer the rings are to each other. Two color density plot : Areas of higher density will have a different color than other areas.

28. PLOT TYPES

29. APPLICATIONS Analysis Immunophenotyping Dyes that bind to nucleic acid (DNA,RNA) Functional assays Cell counting Cell sorting

30. HIV / AIDS Absolute CD4 counts Absolute joint pain HLA B27 Assay Hematological malignancies Diagnosis and classification Detection of MRD Bleeding disorders Platelet receptor assay(platelet count Fetal Hb detection Platelet receptor as Feto materno hemorrhage Treatment response in sickle cell anaemia

31. THANK YOU