flowcytometry-basic principle of flowcyto

flowcytometry-basic principle of flowcyto

1. Flow Cytometry - Definition, Principle, Parts, Types, Uses Dr. Ajit Kumar Singh MD ( Lab medicine )PGT Department of laboratory medicine Chittaranjan national cancer institute Kolkata - 7000160 Moderator Dr. Subhajit Hajra DM Specialist Grade-II Department of laboratory medicine Chittaranjan national cancer institute Kolkata - 7000160

2. Contents Introduction Basic Principles of Flow Cytometry Working of Flow Cytometer Applications of Flow Cytometry Types of Flow Cytometry Limitations

3. 1. Introduction Quantitative single cell analysis First described by Wallace Coulter in the 1950s Flow cytometer was developed in the 1970’s Flow cytometer count, examine and sort cells based on their optical properties (Scattering and fluorescence). The present flow cytometers can analyze up to 26 parameters

4. 1.1 Components of flow cytometer : Lasers Dichroic mirrors Filters Detectors

5. 1.2 Definition Flow cytometry laser-based technology Detection and measurement of physical and chemical characteristics of cells or particles Provides a rapid analysis of multiple characteristics (both qualitative and quantitative) of the cells The properties that can be measured Particle’s size Granularity or internal complexity and Fluorescence intensity.

6. 2. Basic Principle of Flow Cytometry Single cell or particle suspension Aspirated into a channel surrounded by a narrow fluid system They pass one at a time through a focused laser beam Light is either scattered or absorbed when it strikes a cell

7. Light scattering is dependent on Internal structure of the cell Size and shape. Fluorochrome-labelled antibodies are attached to surface or internal cell structures These antibodies absorb and emit light of specific wave length Basic Principle of Flow Cytometry

8. Light and/or fluorescence scatter signals are detected by a series of photodiodes/PMT and amplified. Optical filters are essential to block unwanted light and permit light of the desired wavelength to reach the photodetector. Fluorescein isothiocyanate (FITS) 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 (>1,000/sec). Basic Principle of Flow Cytometry

9. Basic Principle of Flow Cytometry

10. 3. 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 lasers 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.

11. 3.1 Fluidics System Flow cytometers use the principle of hydrodynamic focusing The fluidics system consist Central channel Outer sheath

12. 3.2 Optics Light scattering Forward Scatter Sides scatter Fluorescence emission

13. The FSC intensity roughly equates to the particle’s size Dead cells have lower FSC and higher SSC than living cells. Side scatter is based on the granularity or internal complexity What is FSC and SSC ?

14. Fluorescence measurements Quantitative Qualitative data Fluorescent dye is conjugated to a monoclonal antibody Lineage identification The stating pattern combined with FSC and SSC data Helps to identify cell type and quantity What is fluorescence ?

15. Optical detectors Scattered and emitted light from cells are converted to electrical pulses by optical detectors Detectors Silicon photodiodes: Detects FSC Photomultiplier tubes (PMTs): Detect SSC and fluorescence

16. Filters Signals are routed to their detectors via a system of filters and dichroic mirrors

17. Types of filters: There are three major type of filters: Longpass Filter: Equal to or greater than the spectral band of the filter Shortpass Filter: Equal to or shorter than the spectral band of the filter Bandpass Filter: Within a specific range of wavelengths LP 500 SP 500 Longpass 480 500 520 Shortpass 480 500 520 Bandpass 480 520 460 500 540 BP 500

18. 3.3 Electronics (Signal Processing) Scattered and emitted light data is converted to electrical pulses by optical detectors. Flow cytometry data is commonly represented as histograms or dot plots

19. Histogram: A histogram quantifies the intensity of a single parameter Dot plot: A dot plot is a two parameter representation Histograms vs Dot Plots

20. Other types of plots Density plots Continuous and smoothed version of a histogram. Uses Kernel Density Estimation (KDE) to create a smooth curve Represents the density of data points across a continuous range Useful for visualizing the distribution of data Contour plots Visualizes a 3D surface by displaying in a 2D format Contour lines represent areas with the same value of the response variable(Z). Useful for understanding relationships between two independent variables (X and Y)

21. Density plot vs Contour plot

22. Gating Gating – To interrogate cells of interest while eliminating results from unwanted cells and debris.

23. SSC vs FSC density plot

24. CD45 vs SSC Gating

25. 4.Applications of Flow Cytometry

26. 4.1 Cell Sorting (FCAS) Separation of sub-population of cells of interest from a heterogeneous population New drop Positive Charge Negative Charge No Charge

27. 4.2 Cell Cycle Study The duplication of the DNA occurs during the S-phase of cell cycle. There are two differnet methods to measure the DNA content: The cells have to be stained with a fluorescent dye that binds DNA in a stoichiometric manner. Incorporation of thymidine analog bromodeoxyuridine ( BrdU ) during new DNA synthesis.

28. 4.3 Clinical Applications of Flow Cytometry Diagnosis of Hematologic Malignancies Detection of Minimal Residual Disease Lymphocyte Subset Enumeration (HIV,PID) Efficacy of Cancer Chemotherapy Reticulocyte Enumeration Cell Function Analysis Application in Organ Transplantation DNA degradation (apoptosis) Cytoplasmic Ca++ Gene expression

29. 5.Types of Flow Cytometry There are different types of flow cytometers based on the purpose and precision of the process: Traditional flow cytometers Imaging flow cytometer Cell sorters Acoustic Focusing Cytometers Spectral Flow Cytometers

30. 5.1 Traditional cytometers The traditional cytometers are the common cytometer using sheath fluid for focusing the sample stream. The most common lasers used in traditional flow cytometers are 488 nm (blue), 405nm (violet), 532nm (green), 552nm (green), 561 nm (green-yellow), 640 nm (red) and 355 nm (ultraviolet).

31. 5.2 Imaging flow cytometer Imaging cytometers are traditional cytometers combined with fluorescence microscopy. Imaging cytometer allows for rapid analysis of a sample for morphology and multi-parameter fluorescence at both a single cell and population level.

32. 5.3 Cell sorters Cell sorters are a category of traditional flow cytometers which allows the user to collect samples after processing. The cells that are positive for the desired parameter can be separated from those that are negative for the parameters.

33. 5.4 Acoustic Focusing Cytometers In these cytometers, ultrasonic waves are used to focus the cells for analysis. This prevents sample clogging and also allows higher sample inputs.

34. 5.5 Spectral Flow Cytometers Spectral flow cytometry differs from conventional flow cytometry as it uses prisms to capture all the emitted light from laser excited fluorophores across a set of detectors Array of channels, rather than detecting emitted photons that are collected into individual detectors.

35. 6.Limitations This process doesn’t provide information on the intracellular location or distribution of proteins. Over time, debris is aggregated, which might result in false results. The pre-treatment associated with sample preparation and staining is a time-consuming process. Flow cytometry is an expensive process that requires highly qualified technicians.

36. 7. Summary Flow cytometry integrates electronics, fluidics, computer, optics, software, and laser technologies in a single platform. Provides a rapid analysis of multiple characteristics (both qualitative and quantitative) of the cells. Clinical Applications of Flow Cytometry are diverse.

37. Thank You !!!

38. The following features of the apoptotic cascade can be observed using flow cytometry: Altered phospholipid composition in the plasma membrane Activation of caspases Chromation condensation DNA fragmentation Expression of proteins involved in apoptosis Changes in mitochondrial membrane potential Decrease cytosolic pH Altered membrane permeability

39. Detection of apoptotic cells based on changes in forward scattering During apoptosis there is an initial increase in SSC (probably due to the chromatin condensation) with a reduction in FSC (due to cell shrinkage). Drawback: In many cases, the forward light scattering histograms of apoptotic and live cells overlap and make it difficult to discriminate apoptotic cells based solely on this parameter.

40. Detection of apoptotic cells based on Annexin V binding During early apoptosis cell lose symmetry, phosphatidylserine on the outer leaflet of the plasma membrane. Annexin V is a calcium-dependent phospholipid-binding protein that binds preferentially to negatively-charged phosphatidylserine.

41. The assay involves incubating cells briefly in a solution containing fluorochrome conjugated Annexin V (FITC – Annexin V) in a buffer that facilitates its binding. Apoptotic cells can be detected easily by flow cytometry on the basis of fluorescence due to increased binding of FITC-conjugated Annexin V. Drawback: Unfortunately, it is not specific only for apoptosis because whenever cell membrane integrity is disrupted (even non-ionic detergents), cells may stain with Annexin V.

42. Detection of apoptotic cells based on PI binding The intact plasma membrane of live cells have a tendency to exclude cationic dyes such as propidium iodide (PI) and 7-amino-actinomycin D (7-AMD). PI is a good staining method to distinguish apoptotic, necrotic and normal live cells. Apoptotic cells show an uptake of PI that is much lower than that of necrotic cells. It is therefore possible to distinguish live (PI-negative), apoptotic (PI-dim) and necrotic (PI-bright).

43. Thus, the combined use of cationic dyes (e.g. PI) with annexin V allows the discrimination between: Live cells = Annexin V negative/PI negative Early apoptotic cells = Annexin V positive /PI negative Late apoptotic = Annexin V positive / PI positive Necrotic cells = Annexin V negative/ PI positive

44. Detection of apoptotic cells based on DNA Fragmentation The late stages of apoptosis are characterized by changes in nuclear morphology, including DNA fragmentation, chromatin condensation, degradation of nuclear envelope, nuclear blebbing and DNA strand breaks. Cells undergoing apoptosis display an increase in nuclear chromatin condensation. As the chromatin condenses, cell-permeable nucleic acid stains become hyperfluorescent , thus enabling the identification of apoptotic cells.

45. Assessment of mitochondrial membrane potential and caspases level Assessment of mitochondrial membrane potential and caspases level within the cell through flow cytometry is also used to analyse apoptotic cells. Cells undergoing apoptosis often lose the electric potential that normally exists across the inner mitochondrial membrane. A distinctive feature of early stages of apoptosis is the activation of caspases enzymes. These enzymes can be labelled with fluorophore which can easily be detected by flow cytometry.

46. Fluorescent dye that binds DNA in a stoichiometric manner The cells are treated with a fluorescent dye that stains DNA quantitatively. The fluorescence intensity of the stained cells at certain wavelengths will therefore correlate with the amount of DNA they contain. Dyes have different binding mechanism: Intercalative binding dyes – Propidium iodide A.T rich regions binding dyes – DAPI (4,6- Diamidion-2-phenylindole) G.C rich regions binding dyes – Chromomycin A 3

47. Incorporation of thymidine analog bromodeoxyuridine ( BrdU ) during new DNA synthesis An accurate method for detection of cell cycle progression also uses the incorporation of thymidine analog bromodeoxyuridine ( BrdU ) during new DNA synthesis. The incorporated BrdU is then stained with specific fluorescently labelled anti- BrdU antibodies, and the levels of cell-associated BrdU measured using flow cytometry. By this method, the number of cells that are proliferating rapidly & the duration of S-phase can be calculated.

48. References Biotech, M. (2018). Flow cytometry instrumentation – an overview. Current Protocols in Cytometry, e52. DOI: 10.1002/cpcy.52 McKinnon K. M. (2018). Flow Cytometry: An Overview. Current protocols in immunology, 120, 5.1.1–5.1.11. https://doi.org/10.1002/cpim.40 Dean, P.N. and Hoffman, R.A. (2007), Overview of Flow Cytometry Instrumentation. Current Protocols in Cytometry, 39: 1.1.1-1.1.8. DOI:1002/0471142956.cy0101s39 https://enquirebio.com/flow-cytometry

49. Flow Cytometry

50. Applications/Uses Flow Cytometry is used in several fields including molecular biology, pathology, immunology, virology, plant biology, and marine biology. It is used in clinical labs for the detection of malignancy in bodily fluids like leukemia. Cytometers like cell sorters can be used to separate the cells of interest in separate collection tubes physically. It can be used for the detection of the content of DNA by using fluorescent markers. Flow cytometers allow the analysis of replication cells by using fluorescent dye for four different stages of the cell cycle. Acoustic flow cytometers are used in the study of multi-drug resistant bacteria in the blood and other samples. The different stages of cell death, apoptosis, and necrosis can be detected by flow cytometers based on the differences in the morphological and biochemical changes.

51. Flow Cytometry Principle The basic principle of flow cytometry is based on the measurement of light scattered by particles, and the fluorescence observed when these particles are passed in a stream through a laser beam.