Basics of flow cytometry
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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


Basics of flow cytometry

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


Basics of flow cytometry

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


Basics of flow cytometry

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


Basics of flow cytometry

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


Basics of flow cytometry

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


Basics of flow cytometry

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


Basics of flow cytometry

Fluidics

↓D by 10-40 = ↑V by 100-1600 fold


Basics of flow cytometry

HYDRODYNAMIC FOCUSING


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

LASER (argon)

Dichroic Filters and Mirrors

(b) Photodiode

(d) PMT (photo multiplier tubes )

OPTICS


Basics of flow cytometry

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.


Basics of flow cytometry

Mechanism of fluorochrome


Basics of flow cytometry

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


Basics of flow cytometry

Emission Spectra

100%

FITC

APC

PE

PerCP

Normalized Intensity

0%

600

400

500

700

800

Wavelength (nm)


Basics of flow cytometry

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)


Basics of flow cytometry

Fluorescent Light absorption

Control

Absorption

No blue/green light

red filter


Basics of flow cytometry

Dichroic Filters

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

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


Basics of flow cytometry

Optical Design

PMT 5

PMT 4

Sample

PMT 3

Dichroic

Filters

Flow cell

PMT 2

Scatter

PMT 1

Laser

Sensor

Bandpass

Filters


Basics of flow cytometry

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


Basics of flow cytometry

Electronics:Triggering on a voltage pulse

Laser

Voltage

Time

Voltage

Laser

Time

Voltage

Laser

Time


Basics of flow cytometry

Optical to Digital

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


Basics of flow cytometry

Data Analysis by Software

Display Plots

Create Gates

Display Statistics

Analyze Statistics

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


Basics of flow cytometry

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

    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


    Basics of flow cytometry

    Analysis Approach


    Basics of flow cytometry

    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


    Basics of flow cytometry

    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


    Basics of flow cytometry

    FCM in diagnosis and classification

    • Identification of blasts

    • Enumeration of blasts

    • Assignment of blast lineage

      • Identification of abnormal blasts

      • Subclassification


    Identification of blasts

    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


    Basics of flow cytometry

    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


    Immunophenotypic markers

    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


    Basics of flow cytometry

    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 M0


    Aml m2

    AML M2

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


    Basics of flow cytometry

    AML M5a


    Basics of flow cytometry

    AML Monocytic differentiation (M5b)


    Aml m6

    AML M6


    Basics of flow cytometry

    AML M7


    B all

    B - ALL


    T all

    T - ALL


    Biphenotypic or mixed lineage leukemia

    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.


    Bi lineal leukemia

    Bi-lineal Leukemia

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


    Hematogones

    Hematogones


    All in various cluster patterns

    ALL in various cluster patterns


    Role of flow cytometry in clpd mm

    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


    Analysis approach

    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

    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

    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


    Approach to immunophenotyping clpd

    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


    Basics of flow cytometry

    CLL


    Basics of flow cytometry

    MANTLE CELL LYMPHOMA


    Basics of flow cytometry

    HAIRY CELL LEUKEMIA


    Basics of flow cytometry

    ATLL

    PERIPHERAL T CELL LYMPHOMA - NOS


    Immunophenotype of plasma cells

    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


    Basics of flow cytometry

    Multiple Myeloma


    Immunophenotyping in myelodysplastic syndrome

    Immunophenotyping in Myelodysplastic Syndrome

    Normal Granulocytic Maturation

    Granulocytic dysplasia in MDS


    Immunophenotyping in myelodysplastic syndrome1

    Immunophenotyping in Myelodysplastic Syndrome

    Normal Monocytic Maturation

    Monocytic dysplasia in MDS


    Paraxysmal nocturnal hemoglobinuria pnh

    Paraxysmal Nocturnal Hemoglobinuria (PNH)


    Thank you

    THANK YOU!


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