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Basics of Flow Cytometry

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  1. Basics of Flow Cytometry Prashant Tembhare, MD Tata Memorial Center, Mumbai email:docprt@gmail.com

  2. What is Flow Cytometry? Cyto = cells Metry = Measurement Flow = in a flow or a stream

  3. Flow cytometry • Flow cytometer is an instrument that - illuminates cells as they flow in front of a light source - detects and correlates the signals from illumination. • Unique Ability – rapid analysis of thousands of cells Analyze 500-5000 cells/second - simultaneous illustration of multiple antigens • Two major principles 1. Measurement of physical properties 2. Measurement of antigenic properties

  4. Principles of flow cytometry 1. Measurement of physical properties i.e. size and complexity (granularity). Right Angle Light Detector Forward Light Detector c LASER BEAM

  5. 2. Measurement of ANTIGENIC properties of cell surface and inside the cell with the help of antibodies labeled with different fluorochromes. Principles of flow cytometry c LASER BEAM

  6. Instrument Components Fluidics:Specimen, Sheath fluid, flow chamber. Optics: Light source(s), mirrors, filters, detectors, spectral separation Electronics:Controls pulse collection, pulse analysis, triggering, time delay, data display, gating, sort control, light and detector control Data Analysis: SOFTWARE- Data display & analysis, multivariate/simultaneous solutions, identification of sort populations, quantitation

  7. Crosland-Taylor - Hydrodynamic focussing= coaxial flow → a narrow stream of cells flowing in a core within a wider sheath stream Provides a highly controlled fluid stream. Provides exact location of a cell in three dimensions Maintains sample handling compartment (Flow Cell) Forced under pressure through a conical nozzle assembly geometrically designed to produce a laminar flow This fluid is SHEATH FLUID - Isotonic fluid Fluidics

  8. Fluidics ↓D by 10-40 = ↑V by 100-1600 fold

  9. HYDRODYNAMIC FOCUSING

  10. 2. OPTICS LASER (argon) Dichroic Filters and Mirrors (b) Photodiode (d) PMT (photo multiplier tubes )

  11. What is Fluorescence ? O HO  = 488 nm  = 520 nm C IncidentLight Energy CO2H Emitted Fluorescent Light Energy FluoresceinMolecule Antibody • The fluorochrome absorbs energy from the laser. • The fluorochrome releases the absorbed energy by: • vibration and heat dissipation. • emission of photons of a longer wavelength.

  12. Mechanism of fluorochrome

  13. Excitation & Emission

  14. Fluorescence Emitted fluorescence intensity is proportional to binding sites FITC FITC FITC FITC FITC FITC FITC FITC Number of Events FITC FITC 0 Log scale of Fluorescent Intensity

  15. Emission Spectra 100% FITC APC PE PerCP Normalized Intensity 0% 600 400 500 700 800 Wavelength (nm)

  16. Emission Spectra 100% Alexa 430 PI FITC APC PE-Cy7 PE PerCP PerCP-Cy5.5 Cascade Blue Normalized Intensity 0% 600 400 500 700 800 Wavelength (nm)

  17. Fluorescent Light Filteration Control Absorption No blue/green light red filter

  18. Filters

  19. Dichroic Filters Detector 1 Detector 2 Dichroic Filter • Can be a long pass or short pass filter • Filter is placed at a 45º angle to the incident light • Part of the light is reflected at 90º to the incident light, and part of the light is transmitted and continues on.

  20. PMT – Photomultiplier Tubes

  21. Coulter optical system - Elite PMT2 PMT1 PMT4 PMT3 555 - 595 575 BP 525 BP 488 BP PMT5 L L L D D D 632 BP 488 BK 675 BP 0 5 5 9 2 5 4 6 0 655 - 695 APC PMT6 TM PMT7 Purdue Cytometry Labs The Elite optical system uses 5 side window PMTs and a number of filter slots into which any filter can be inserted

  22. BD optical system – Canto-II

  23. Optical Design

  24. Electronics • Compute pulse height • Perform calculations for pulse area and pulse width • Calculate ratios • Convert analog signals to proportional digital signals • Interface with the computer for data transfer

  25. Electronics:Triggering on a voltage pulse Laser Voltage Time Voltage Laser Time Voltage Laser Time

  26. Optical to Digital PMT Log amplification of signals Voltage Signal Out Analog to Digital Converter 2 Options for SSC and fluorescence channels PhotonIn Linear amplification of signals Voltage In PMTPower Supply compensationcircuit Levels 0–1000V adjusted by slider control on computer Gain levels from 0–9.99 adjusted by slider control on computer Amplifier output voltage ranging between 10mV to 10V

  27. Optical to Digital

  28. Data Analysis by Software Display Plots Create Gates Display Statistics Analyze Statistics Plot Types:Gate Types:Statistics Types: Results: Histogram Polygon # of Events % positive for Dot Ellipse % of Gated particular markers: Contour Histogram % of Total -viable cells Density Quadrant -immunophenotype meanmean fluorescence intensity geometric mean DNA content standard deviation absolute counts

  29. Sample processing • Single cell suspension:all specimens with cells in suspension PB, BMA, CSF, PF, BAL Solid tissue • Fine needle aspirations • Tissue suspensions - slicing, mincing and teasing = Filtering • Sample stabilization:Anticoagulant - EDTA or Heparin • Enrichment of cells:For leucocytes - RBC Lysis - NH4CL or - Density gradient centrifugation – Ficoll medium • Antibody staining:Separate cells-wash-incubate with Ab-F in dark • Acquisition: Acquire the stained cells at earliest or Fixed and store in refrigerator • Data Analysis: VIMP – Needs experience and knowledge

  30. Common Clinical Applications Enumeration of lymphocyte subsets (CD4/CD8) Immunophenotyping of hematologic malignancies Minimal Residual Disease (MRD) Myelodysplatic Syndrome (MDS) HLA B27 typing PNH diagnosis (CD55-/CD59-) DNA/RNA analysis & Cell cycle studies Reticulocyte analysis Hemotopoietic stem cell (CD34+)analysis Platelet analysis Antigen quantitation e.g. CD20, CD22, CD33 etc Other uncommon Microbiology Determination of drug resistance to chemotherapy Cell Function analysis

  31. Analysis Approach

  32. Management of Leukemia • Accurate diagnosis and classification • Knowledge of prognostic factors • Monitoring response • Diagnosis of early relapse at other sites like CNS

  33. ALL naïve germinal center B-lymphocytes Plasma cells Lymphoid progenitor T-lymphocytes Neutrophils AML Myeloid progenitor Eosinophils Hematopoietic stem cell Basophils Monocytes Platelets Red cells AUL Mixed Lineage Leukemia

  34. FCM in diagnosis and classification • Identification of blasts • Enumeration of blasts • Assignment of blast lineage • Identification of abnormal blasts • Subclassification

  35. Identification of blasts • Low side light scatter • Weak CD45 expression • Markers of immaturity such as CD34 and TdT • Lack markers of maturation Myeloblasts - CD11b, CD15, CD16. B lymphoblasts – surface light chains kappa/lambda T lymphoblasts – Surface CD3

  36. Enumeration of Blasts Flow cytometric count lower than manual count • Dilution with peripheral blood • Some blasts lack expression of CD34 and CD117 • CD45 expression may very • Flow cytometric count higher than manual count • Loss of NRBCS during red cell lysis. • FicollHypaque separation • Blast identifications may be difficult due to poor • preservation or may be disrupted during smear • preparation

  37. Immunophenotypic markers Markers of Immaturity – TdT, CD34 Lineage Specific markers Myeloid - cMPO B cell - cCD22/cCD79a T cell - cCD3 Lineage Associated markers Myeloid - Common - CD13, CD33, CD117 - Other - CD11b, CD15 Monocytic - CD13, CD33, CD64, CD68, CD117, CD11b, CD14, CD4, cLysozyme Erythroid - CD36, CD71, CD105, CD235a (Glycophorin A), Hb Megakaryocytic - CD36, CD41, CD42, CD61 andCD62 B cell - CD19, CD22, CD20, cCD79a, CD10, cIgM, sIg T cell - Common - CD1a, CD2, CD5, CD7, CD10 - Other - CD4, CD8, CD3, NK cell - CD16, CD56, CD57, CD94, KIR PDC - CD123, CD4, CD56, CD68, CD33, CD43, BDCA, - Other on PB subset CD2, CD5, CD7

  38. Lineage Infidelity markers (Leukemia associated immunophenotype; LAIP) Lymphoid markers in AML - CD7, CD56, CD2, CD5 and CD19. Myeloid markers in ALL – CD13, CD33, CD117, CD15 Other Markers useful for MRD detection Associated with AML – CD38, CD45, CD68, HLADR Associated with ALL – CD9, CD24, CD25, CD52, CD58, CD81, CD123

  39. AML M0

  40. AML M2 t(8;21)(q22;q22) RUNX1-RUNX1T1

  41. AML M5a

  42. AML Monocytic differentiation (M5b)

  43. AML M6

  44. AML M7

  45. B - ALL

  46. T - ALL

  47. Biphenotypic or mixed lineage leukemia Borowitz M, Bene M, Harris N and Matutes E, (2008) Acute leukaemias of ambiguous lineage., World Health Organization Classification of Tumours IARC Press, Lyon, pp. 150–155.