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Basics of Flow Cytometry. Prashant Tembhare. 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.

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Basics of flow cytometry

Basics of Flow Cytometry

Prashant Tembhare


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


Flow cytometry Chemical and Biological properties of individual cells (Cytometry) or particles flowing in a single stream (Flow) in a fluidic system.

  • 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


Principles of flow cytometry Chemical and Biological properties of individual cells (Cytometry) or particles flowing in a single stream (Flow) in a fluidic system.

1. Measurement of physical properties i.e. size and complexity (granularity).

Right Angle

Light Detector

Forward Light Detector

c

LASER BEAM


2 Chemical and Biological properties of individual cells (Cytometry) or particles flowing in a single stream (Flow) in a fluidic system.. 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


Instrument Components Chemical and Biological properties of individual cells (Cytometry) or particles flowing in a single stream (Flow) in a fluidic system.

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


Crosland-Taylor - Chemical and Biological properties of individual cells (Cytometry) or particles flowing in a single stream (Flow) in a fluidic system.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


Fluidics Chemical and Biological properties of individual cells (Cytometry) or particles flowing in a single stream (Flow) in a fluidic system.

↓D by 10-40 = ↑V by 100-1600 fold


HYDRODYNAMIC FOCUSING Chemical and Biological properties of individual cells (Cytometry) or particles flowing in a single stream (Flow) in a fluidic system.


Laser argon dichroic filters and mirrors b photodiode d pmt photo multiplier tubes

LASER (argon) Chemical and Biological properties of individual cells (Cytometry) or particles flowing in a single stream (Flow) in a fluidic system.

Dichroic Filters and Mirrors

(b) Photodiode

(d) PMT (photo multiplier tubes )

OPTICS


What is Fluorescence ? Chemical and Biological properties of individual cells (Cytometry) or particles flowing in a single stream (Flow) in a fluidic system.

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.


Mechanism of fluorochrome Chemical and Biological properties of individual cells (Cytometry) or particles flowing in a single stream (Flow) in a fluidic system.


Fluorescence Chemical and Biological properties of individual cells (Cytometry) or particles flowing in a single stream (Flow) in a fluidic system.

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


Emission Spectra Chemical and Biological properties of individual cells (Cytometry) or particles flowing in a single stream (Flow) in a fluidic system.

100%

FITC

APC

PE

PerCP

Normalized Intensity

0%

600

400

500

700

800

Wavelength (nm)


Emission Spectra Chemical and Biological properties of individual cells (Cytometry) or particles flowing in a single stream (Flow) in a fluidic system.

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)


Fluorescent Light absorption Chemical and Biological properties of individual cells (Cytometry) or particles flowing in a single stream (Flow) in a fluidic system.

Control

Absorption

No blue/green light

red filter


Dichroic Filters Chemical and Biological properties of individual cells (Cytometry) or particles flowing in a single stream (Flow) in a fluidic system.

Detector 1

Detector 2

Dichroic

Filter

  • 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.


Coulter optical system elite
Coulter optical system - Elite Chemical and Biological properties of individual cells (Cytometry) or particles flowing in a single stream (Flow) in a fluidic system.

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

The Elite optical system uses 5 side window PMTs and a number of filter slots into which any filter can be inserted


Optical Design Chemical and Biological properties of individual cells (Cytometry) or particles flowing in a single stream (Flow) in a fluidic system.

PMT 5

PMT 4

Sample

PMT 3

Dichroic

Filters

Flow cell

PMT 2

Scatter

PMT 1

Laser

Sensor

Bandpass

Filters


Electronics Chemical and Biological properties of individual cells (Cytometry) or particles flowing in a single stream (Flow) in a fluidic system.

  • 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: Chemical and Biological properties of individual cells (Cytometry) or particles flowing in a single stream (Flow) in a fluidic system.Triggering on a voltage pulse

Laser

Voltage

Time

Voltage

Laser

Time

Voltage

Laser

Time


Optical to Digital Chemical and Biological properties of individual cells (Cytometry) or particles flowing in a single stream (Flow) in a fluidic system.

PMT

Voltage

Signal

Out

Log amplification of signals

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


Data Analysis by Software Chemical and Biological properties of individual cells (Cytometry) or particles flowing in a single stream (Flow) in a fluidic system.

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


Sample processing Chemical and Biological properties of individual cells (Cytometry) or particles flowing in a single stream (Flow) in a fluidic system.

  • 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
    Clinical Applications of Flow Cytometry Chemical and Biological properties of individual cells (Cytometry) or particles flowing in a single stream (Flow) in a fluidic system.

    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 Chemical and Biological properties of individual cells (Cytometry) or particles flowing in a single stream (Flow) in a fluidic system.


    FCM in management of Acute Leukemia Chemical and Biological properties of individual cells (Cytometry) or particles flowing in a single stream (Flow) in a fluidic system.

    • Accurate diagnosis and classification

    • Knowledge of prognostic factors

    • Monitoring response

    • Diagnosis of early relapse at other sites like CNS


    ALL Chemical and Biological properties of individual cells (Cytometry) or particles flowing in a single stream (Flow) in a fluidic system.

    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


    FCM in diagnosis and classification Chemical and Biological properties of individual cells (Cytometry) or particles flowing in a single stream (Flow) in a fluidic system.

    • Identification of blasts

    • Enumeration of blasts

    • Assignment of blast lineage

      • Identification of abnormal blasts

      • Subclassification


    Identification of blasts
    Identification of blasts Chemical and Biological properties of individual cells (Cytometry) or particles flowing in a single stream (Flow) in a fluidic system.

    • 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 Chemical and Biological properties of individual cells (Cytometry) or particles flowing in a single stream (Flow) in a fluidic system.

    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


    Immunophenotypic markers
    Immunophenotypic markers Chemical and Biological properties of individual cells (Cytometry) or particles flowing in a single stream (Flow) in a fluidic system.

    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 Chemical and Biological properties of individual cells (Cytometry) or particles flowing in a single stream (Flow) in a fluidic system.

    (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 M0 Chemical and Biological properties of individual cells (Cytometry) or particles flowing in a single stream (Flow) in a fluidic system.


    Aml m2
    AML M2 Chemical and Biological properties of individual cells (Cytometry) or particles flowing in a single stream (Flow) in a fluidic system.

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


    AML M5a Chemical and Biological properties of individual cells (Cytometry) or particles flowing in a single stream (Flow) in a fluidic system.


    AML Monocytic differentiation (M5b) Chemical and Biological properties of individual cells (Cytometry) or particles flowing in a single stream (Flow) in a fluidic system.


    Aml m6
    AML M6 Chemical and Biological properties of individual cells (Cytometry) or particles flowing in a single stream (Flow) in a fluidic system.


    AML M7 Chemical and Biological properties of individual cells (Cytometry) or particles flowing in a single stream (Flow) in a fluidic system.


    B all
    B - ALL Chemical and Biological properties of individual cells (Cytometry) or particles flowing in a single stream (Flow) in a fluidic system.


    T all
    T - ALL Chemical and Biological properties of individual cells (Cytometry) or particles flowing in a single stream (Flow) in a fluidic system.


    Biphenotypic or mixed lineage leukemia
    Biphenotypic or mixed lineage leukemia Chemical and Biological properties of individual cells (Cytometry) or particles flowing in a single stream (Flow) in a fluidic system.

    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.


    Bi lineal leukemia
    Bi-lineal Leukemia Chemical and Biological properties of individual cells (Cytometry) or particles flowing in a single stream (Flow) in a fluidic system.

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


    Hematogones
    Hematogones Chemical and Biological properties of individual cells (Cytometry) or particles flowing in a single stream (Flow) in a fluidic system.


    All in various cluster patterns
    ALL in various cluster patterns Chemical and Biological properties of individual cells (Cytometry) or particles flowing in a single stream (Flow) in a fluidic system.


    Role of flow cytometry in clpd mm
    Role of flow cytometry in CLPD & MM Chemical and Biological properties of individual cells (Cytometry) or particles flowing in a single stream (Flow) in a fluidic system.

    • Diagnosis

    • Staging of lymphoma – Bone marrow involvement or body fluids

    • Prognostication eg Zap 70 in CLL

    • Minimal residual disease

    • Diagnosis of relapse


    Analysis approach
    Analysis Approach Chemical and Biological properties of individual cells (Cytometry) or particles flowing in a single stream (Flow) in a fluidic system.

    • 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
    Antibody panels- B CLPD Chemical and Biological properties of individual cells (Cytometry) or particles flowing in a single stream (Flow) in a fluidic system.

    • 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
    Disease oriented Chemical and Biological properties of individual cells (Cytometry) or particles flowing in a single stream (Flow) in a fluidic system.

    • 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


    Approach to immunophenotyping clpd
    Approach to immunophenotyping CLPD Chemical and Biological properties of individual cells (Cytometry) or particles flowing in a single stream (Flow) in a fluidic system.

    • 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 Chemical and Biological properties of individual cells (Cytometry) or particles flowing in a single stream (Flow) in a fluidic system.


    MANTLE CELL LYMPHOMA Chemical and Biological properties of individual cells (Cytometry) or particles flowing in a single stream (Flow) in a fluidic system.


    HAIRY CELL LEUKEMIA Chemical and Biological properties of individual cells (Cytometry) or particles flowing in a single stream (Flow) in a fluidic system.


    ATLL Chemical and Biological properties of individual cells (Cytometry) or particles flowing in a single stream (Flow) in a fluidic system.

    PERIPHERAL T CELL LYMPHOMA - NOS


    Immunophenotype of plasma cells
    Immunophenotype of plasma cells Chemical and Biological properties of individual cells (Cytometry) or particles flowing in a single stream (Flow) in a fluidic system.

    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 Chemical and Biological properties of individual cells (Cytometry) or particles flowing in a single stream (Flow) in a fluidic system.


    Immunophenotyping in myelodysplastic syndrome
    Immunophenotyping in Myelodysplastic Syndrome Chemical and Biological properties of individual cells (Cytometry) or particles flowing in a single stream (Flow) in a fluidic system.

    Normal Granulocytic Maturation

    Granulocytic dysplasia in MDS


    Immunophenotyping in myelodysplastic syndrome1
    Immunophenotyping in Myelodysplastic Syndrome Chemical and Biological properties of individual cells (Cytometry) or particles flowing in a single stream (Flow) in a fluidic system.

    Normal Monocytic Maturation

    Monocytic dysplasia in MDS


    Paraxysmal nocturnal hemoglobinuria pnh
    Paraxysmal Nocturnal Hemoglobinuria (PNH) Chemical and Biological properties of individual cells (Cytometry) or particles flowing in a single stream (Flow) in a fluidic system.


    Thank you
    THANK YOU! Chemical and Biological properties of individual cells (Cytometry) or particles flowing in a single stream (Flow) in a fluidic system.


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