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Basics of Flow Cytometry. Prashant Tembhare , MD Tata Memorial Center, Mumbai e mail:[email protected] What is Flow Cytometry?. Cyto = cells. Metry = Measurement. Flow = in a flow or a stream. Flow cytometry. Flow cytometer is an instrument that

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basics of flow cytometry

Basics of Flow Cytometry

Prashant Tembhare, MD

Tata Memorial Center, Mumbai

email:[email protected]

slide2

What is Flow Cytometry?

Cyto = cells

Metry = Measurement

Flow = in a flow or a stream

slide3

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

slide5

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

slide6

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

slide7

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

slide9

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

slide10

Fluidics

↓D by 10-40 = ↑V by 100-1600 fold

2 optics

2. OPTICS

LASER (argon)

Dichroic Filters and Mirrors

(b) Photodiode

(d) PMT (photo multiplier tubes )

slide13

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.
slide16

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

slide17

Emission Spectra

100%

FITC

APC

PE

PerCP

Normalized Intensity

0%

600

400

500

700

800

Wavelength (nm)

slide18

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)

slide19

Fluorescent Light Filteration

Control

Absorption

No blue/green light

red filter

slide21

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.
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

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

slide27

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
slide28

Electronics:Triggering on a voltage pulse

Laser

Voltage

Time

Voltage

Laser

Time

Voltage

Laser

Time

slide29

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

slide31

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

slide32

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
slide33

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

management of leukemia
Management of Leukemia
  • Accurate diagnosis and classification
  • Knowledge of prognostic factors
  • Monitoring response
  • Diagnosis of early relapse at other sites like CNS
slide36

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

slide37

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

slide39

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

slide41

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 m2
AML M2

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

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.

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
slide64

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
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