Basics of Flow Cytometry Prashant Tembhare 1

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

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 the illumination. Unique Ability – rapid analysis of thousands of cells cells flow at a velocity of 5–50 m/s Analyze 500-5000 cells/second - simultaneous illustration of multiple antigens Two major principles 1. Measurement of physical properties 2. Measurement of antigenic properties

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Principles of flow cytometry Forward Light Detector 1. Measurement of physical properties i.e. size and complexity (granularity). Right Angle Light Detector Forward Light Detector c Forward Scatter (FSC): Diffracted light FSC is related to cell surface area. FSC is detected along axis of incident light in the forward direction. Side Scatter (SSC): Reflected and Refracted light SSC is related to cell granularity and complexity. SSC is detected at 90° to the laser beam. Factors that affect light scatter: Shape, surface, size, granularity, internal complexity, refractive index. Notice that the forward scatter signal is the light that is diffracted (bends around the cell) and the side scatter is the light that is refracted at greater angles, typically off internal components of the cell. LASER BEAM 5

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

Fluidics 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 9

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

HYDRODYNAMIC FOCUSING

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

Emitted Fluorescent Light Energy What is Fluorescence ? HO O  = 488 nm  = 520 nm C Incident Light Energy CO2H Emitted Fluorescent Light Energy Fluorescein Molecule Antibody A fluorochrome absorbs laser light (energy) and gets excited. The excited fluorochrome releases that energy in three ways, vibration, heat dissipation, and photon emission. These photons are of a longer wavelength (different color) than the incident laser light. The photon emission is referred to as fluorescence. 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. 13

Mechanism of fluorochrome

Fluorescence Emitted fluorescence intensity is proportional to binding sites FITC FITC FITC FITC FITC FITC FITC FITC Number of Events Fluorescence intensity emitted from a cell which binds fluorochrome-labeled antibody is proportional to the quantity of binding sites. FITC FITC Log scale of Fluorescent Intensity 15

Emission Spectra 100% Normalized Intensity 0% 400 500 600 700 800 FITC PE APC PerCP Normalized Intensity Fluoroscence Chart of Fluorochromes used in immunofluorescence measurements on the FACSCalibur. This graphic shows the emission spectra of fluorochromes for which the FACSCalibur has been optimized to detect. With the use of optical filters, the majority of the signal from a fluorochrome can be routed to its respective detector. But notice how each fluorochrome emits a spectrum of wavelengths that overlap each other. Because of this overlap we must electronically compensate the detected signals. 0% 400 500 600 700 800 Wavelength (nm) 16

Emission Spectra 100% Normalized Intensity 0% 400 500 600 700 800 Cascade Blue FITC Alexa 430 PE PI APC PerCP PerCP-Cy5.5 PE-Cy7 Normalized Intensity Fluoroscence Chart of Fluorochromes used in immunofluorescence measurements on the BD LSR II. 0% 400 500 600 700 800 Wavelength (nm) 17

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

Dichroic Filters 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. Dichroic Filter Detector 1 Detector 2

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

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

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 FACSCalibur Electronics. We’ll discuss how the optical signal becomes an electronic signal, how the electronic pulse is evaluated, and how the pulse values are converted to digital values so that the computer can work with the data. 23

Electronics: Triggering on a voltage pulse Laser Voltage Time Laser Voltage The creation of a pulse applies to all channels, scatter or fluorescence. When the cell first encounters the laser beam it starts to scatter light or in the case of fluorescence, starts to fluoresce. When the cell is in the center of the beam, the maximum amount of scatter or fluorescence, is achieved. As the cell leaves the laser the signal drops back off. Time Voltage Laser Time 24

Optical to Digital PMT Voltage Signal Out Log amplification of signals Analog to Digital Converter 2 Options for SSC and fluorescence channels Photon In Linear amplification of signals Voltage In Photons strike PMT (Photo Multiplier Tube). PMT output current is converted to a voltage pulse by a preamplifier (not shown). The resultant voltage pulse is proportional to the number of photons impinging upon the detector. PMT gain can be adjusted from 0 volts to 1000 volts. The operator of a flow cytometer adjusts PMT voltage until the population is clearly visible on the display. Before the signals are passed to the ADC, they are routed through compenstion circuits, log or linear amplifiers (operator determined), Q: If you increased the FL2 PMT voltage, in which direction would you expect the FL2 population to move, right or left? A: The population would move to the right. Once the voltage pulse is generated, it is digitized and sent to memory. PMT Power Supply compensation circuit 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 25

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 mean mean fluorescence intensity geometric mean DNA content standard deviation absolute counts Data analysis consists of displaying data from a list-mode file in a plot, and then measuring the distribution of the events within the plots. Gates and statistics can be used to obtain information about subset populations. Plot Types: Gate Types: Statistics Types: Results: Histogram Polygon # of Events % positive for Dot Ellipse % of Gated particular markers Contour Histogram % of Parent -viable cells Density Quadrant % of Total -immunotphenotye mean mean fluorescence intensity geometric mean DNA content standard deviation absolute counts 26

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 – Transport at RT 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

Clinical Applications of Flow Cytometry 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

Analysis Approach 29

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

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

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

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

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. Ficoll Hypaque separation Blast identifications may be difficult due to poor preservation or may be disrupted during smear preparation

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

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

AML M0

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

AML M5a

AML Monocytic differentiation (M5b)

AML M6

AML M7

B - ALL

T - ALL

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.

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

Hematogones Antigens Early (St-1) Intermediate (st 2 & 3) Mature B cells TdT + - CD34 CD10 bright dim CD19 intermediate CD22 CD20 (-/+) weak CD38 variable CD45 CD58 CD81 Cyt IgM K/L -/+

ALL in various cluster patterns

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

detection of abnormal immunophenotype Analysis Approach Isolation of cells using lineage specific markers like CD19 for B cells and CD3 for T cells detection of abnormal immunophenotype Clonality evaluation eg kappa or lambda Note size of cells – FSC

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

Disease oriented B CLPD MM – CD19, CD20, CD27, CD45, CD56, CD81, CD117 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

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

CLL

MANTLE CELL LYMPHOMA

HAIRY CELL LEUKEMIA

ATLL PERIPHERAL T CELL LYMPHOMA - NOS

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

Multiple Myeloma

Immunophenotyping in Myelodysplastic Syndrome Normal Granulocytic Maturation Granulocytic dysplasia in MDS

Immunophenotyping in Myelodysplastic Syndrome Normal Monocytic Maturation Monocytic dysplasia in MDS

Paraxysmal Nocturnal Hemoglobinuria (PNH)

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