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Basics of Flow Cytometry Prashant Tembhare. Page  2 Flow Cytometry is the automated measurement of Physical, Chemical and Biological properties of individual.

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Presentation on theme: "Basics of Flow Cytometry Prashant Tembhare. Page  2 Flow Cytometry is the automated measurement of Physical, Chemical and Biological properties of individual."— Presentation transcript:

1 Basics of Flow Cytometry Prashant Tembhare

2 Page  2 Flow Cytometry is the automated measurement of Physical, Chemical and Biological properties of individual cells (Cytometry) or particles flowing in a single stream (Flow) in a fluidic system. What is Flow Cytometry? Cyto = cells Metry = measurement Flow = in a flow or a stream

3 Page  3 Flow cytometer is an instrument that - illuminates cells as they flow in front of a light source & - detects and correlates the signals from the illumination. Unique Ability – rapid analysis of thousands of cells cells flow at a velocity of 5–50 m/s Analyze cells/second - simultaneous illustration of multiple antigens Two major principles 1. Measurement of physical properties 2. Measurement of antigenic properties Flow cytometry

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5 Page  5 Principles of flow cytometry Right Angle Light Detector Forward Light Detector LASER BEAM 1. Measurement of physical properties i.e. size and complexity (granularity). c

6 Page  6 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 LASER BEAM c

7 Page  7 1.Fluidics: Specimen, Sheath fluid, flow chamber. 2.Optics: Light source(s), mirrors, filters, detectors, spectral separation 3.Electronics: Controls pulse collection, pulse analysis, triggering, time delay, data display, gating, sort control, light and detector control 4.Data Analysis: SOFTWARE - Data display & analysis, multivariate/simultaneous solutions, identification of sort populations, quantitation Instrument Components

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9 Page  9  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

10 Page  10 Fluidics ↓D by = ↑V by fold


12 Page  12 (a)LASER (argon) (b)Dichroic Filters and Mirrors (b) Photodiode (d) PMT (photo multiplier tubes ) OPTICS

13 Page  13  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. = 488 nm Emitted Fluorescent Light Energy Antibody Incident Light Energy Fluorescein Molecule = 520 nm HO CO 2 H O C What is Fluorescence ?

14 Page  14 Mechanism of fluorochrome

15 Page  15 FITC Emitted fluorescence intensity is proportional to binding sites FITC Log scale of Fluorescent Intensity Number of Events Fluorescence 0

16 Page  16 Emission Spectra APC PerCP Wavelength (nm) % 0% Normalized Intensity FITC PE 800

17 Page  17 Emission Spectra APC PerCP PI Wavelength (nm) % 0% Cascade Blue Normalized Intensity FITC PE Alexa 430 PerCP-Cy PE-Cy7

18 Page  18 Absorption Control No blue/green light red filter Fluorescent Light absorption

19 Page  19  Can be a long pass or short pass filter or band pass  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. DichroicFilter Detector 1 Detector 2 Dichroic Filters

20 Page  20 Coulter optical system - Elite  The Elite optical system uses 5 side window PMTs and a number of filter slots into which any filter can be inserted PMT4 APC PMT6 PMT D L 488 BK D L D L 675 BP 488 BP525 BP575 BP 632 BP TM PMT3 PMT2 PMT1 PMT5

21 Page  21

22 Page  22 Optical Design PMT 1 PMT 2 PMT 5 PMT 4 Dichroic Filters Bandpass Filters Laser Flow cell PMT 3 Scatter Sensor Sample

23 Page  23  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 Electronics

24 Page  24 Electronics: Triggering on a voltage pulse Laser Laser Laser Time Voltage Time Voltage Time Voltage

25 Page  25 Voltage In PMT Power Supply Levels 0–1000V adjusted by slider control on computer Photon In Voltage Signal Out Analog to Digital Converter Log amplification of signals PMT Linear amplification of signals Gain levels from 0–9.99 adjusted by slider control on computer 2 Options for SSC and fluorescence channels Amplifier output voltage ranging between 10mV to 10V compensation circuit Optical to Digital

26 Page  26 Plot Types:Gate Types:Statistics Types: Results: HistogramPolygon# of Events% positive for DotEllipse% of Gatedparticular markers: ContourHistogram % of Total -viable cells DensityQuadrant -immunophenotype meanmean fluorescence intensity geometric meanDNA content standard deviationabsolute counts Display Plots Create Gates Display Statistics Analyze Statistics Data Analysis by Software

27 Page  27  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 – Transport at RT  Enrichment of cells: For leucocytes - RBC Lysis - NH 4 CL or - D ensity 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 Sample processing

28 Page  28  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

29 Page  29 Analysis Approach

30 Page  30  Accurate diagnosis and classification  Knowledge of prognostic factors  Monitoring response  Diagnosis of early relapse at other sites like CNS FCM in management of Acute Leukemia

31 Page  31 Hematopoietic stem cell Neutrophils Eosinophils Basophils Monocytes Platelets Red cells Myeloid progenitor Lymphoid progenitor B-lymphocytes T-lymphocytes Plasma cells naïve ALL AML AUL Mixed Lineage Leukemia

32 Page  32  Identification of blasts  Enumeration of blasts  Assignment of blast lineage  Identification of abnormal blasts  Subclassification FCM in diagnosis and classification

33 Page  33 Identification of blasts LLow side light scatter WWeak CD45 expression MMarkers of immaturity such as CD34 and TdT LLack markers of maturation Myeloblasts - CD11b, CD15, CD16. B lymphoblasts – surface light chains kappa/lambda T lymphoblasts – Surface CD3

34 Page  34 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.  Ficoll Hypaque separation  Blast identifications may be difficult due to poor preservation or may be disrupted during smear preparation Enumeration of Blasts

35 Page  35 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

36 Page  36 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

37 Page  37 AML M0

38 Page  38 AML M2 t(8;21)(q22;q22) RUNX1-RUNX1T1

39 Page  39 AML M5a

40 Page  40 AML Monocytic differentiation (M5b)

41 Page  41 AML M6

42 Page  42 AML M7

43 Page  43 B - ALL

44 Page  44 T - ALL

45 Page  45 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.

46 Page  46

47 Page  47 Bi-lineal Leukemia EG Weir and MJ Borowitz. Leukemia (2007) 21, 2264–2270.

48 Page  48 Hematogones AntigensEarly (St-1) Intermediate (st 2 & 3) Mature B cells TdT+-- CD34+-- CD10brightdim- CD19dimintermediatebright CD22dim intermediate CD20-(-/+) weakintermediate CD38bright variable CD45dimintermediatebright CD58dim CD81bright intermediate Cyt IgM-++ K/L--/++

49 Page  49 ALL in various cluster patterns

50 Page  50 Role of flow cytometry in CLPD & MM  Diagnosis  Staging of lymphoma – Bone marrow involvement or body fluids  Prognostication eg Zap 70 in CLL  Minimal residual disease  Diagnosis of relapse

51 Page  51 Analysis Approach I.Isolation of cells using lineage specific markers like CD19 for B cells and CD3 for T cells II.detection of abnormal immunophenotype III.Clonality evaluation eg kappa or lambda IV.Note size of cells – FSC

52 Page  52 Antibody panels- B CLPD  Mature B cells  CD19, CD20, CD22, cyto79a, CD79b  Mature T cells  CD2, CD3, CD4, CD5, CD7, CD8, TCR αβ/γδ  NK cells  CD2, cytoCD3, CD7, vCD8, CD16, CD56, vCD57, CD94, CD158 (KIRs)  Plasma cells  CD138, bCD38, CD19, cyto79a, cyto-Kappa, cyto-Lambda  Clonality markers –B cells - sKappa, sLambda, –PCs - cyto-Kappa, cyto-Lambda –T cells – TCR V beta repertoire  Other important Markers  CD45, CD38, HLADR, Granzyme, Perforin, TIA

53 Page  53 Disease oriented  B CLPD –CLL – CD19,CD5, CD23, d-n CD20, d-n CD22, d-n FMC7, CD43, CD81, CD200 –HCL – CD11c, CD25, CD103, CD123 –FCL/DLBCL – CD10 –MCL – CD5 & CCD  MM – CD19, CD20, CD27, CD45, CD56, CD81, CD117  T CLPD –ATLL/CTCL – CD25, CD26, CD27 –AILT – CD10 –ALCL – CD30 –EATCL – CD103

54 Page  54 Approach to immunophenotyping CLPD  Identification of lineage: expression of lineage specific markers.  B cell lineage- CD 19 or CD20 (CD20 may be lost after treatment with rituximab).  Immunoglobulin Light chain restriction  T cell lineage- CD7, CD3, CD2, CD5 (many markers may be lost in null cell phenotype)  TCR V beta repertoire restricted usage  NK cell – CD7, cytoCD3, CD2, CD16, CD56, CD57

55 Page  55 CLL


57 Page  57



60 Page  60 Immunophenotype of plasma cells Normal plasma cells  Specific markers- CD138, CD38 (strong)  B cell lineage – weak CD19, strong CD27  Moderate expression of CD45 Neoplastic plasma cells  Aberrant expression- CD20, bCD56, CD28, CD117, CD200  Loss of CD19, CD27, CD45, CD81  Surface/Cytoplasmic light chain restriction

61 Page  61 Multiple Myeloma

62 Page  62

63 Page  63 Immunophenotyping in Myelodysplastic Syndrome Normal Granulocytic Maturation Granulocytic dysplasia in MDS

64 Page  64 Normal Monocytic Maturation Monocytic dysplasia in MDS Immunophenotyping in Myelodysplastic Syndrome

65 Page  65 Paraxysmal Nocturnal Hemoglobinuria (PNH)

66 Page  66 THANK YOU!

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