Basics of Flow Cytometry Prashant Tembhare. Page 2 Flow Cytometry is the automated measurement of...

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 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

Flow cytometry

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

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

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

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

Fluidics

↓D by 10-40 = ↑V by 100-1600 fold

HYDRODYNAMIC FOCUSING

(a) LASER (argon)

(b) Dichroic Filters and Mirrors

(b) Photodiode

(d) PMT (photo multiplier tubes )

OPTICS

· 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 EnergyAntibody

IncidentLight Energy Fluorescein

Molecule

= 520 nmHO

CO2H

O

C

What is Fluorescence ?

Mechanism of fluorochrome

FITC FITC

FITC

FITC

FITC

FITC

FITC

FITC

FITC

Emitted fluorescence intensity is proportional to binding sites

FITC

Log scale of Fluorescent Intensity

Num

ber

of E

vent

s

Fluorescence

0

Emission Spectra

APC PerCP

Wavelength (nm)400 500 600 700

100%

0%

Nor

mal

ized

Int

ensi

ty

FITC PE

800

Emission Spectra

APC PerCPPI

Wavelength (nm)400 500 600 700

100%

0%

Cascade Blue

Nor

mal

ized

Int

ensi

ty

FITC PEAlexa 430 PerCP-Cy5.5

800

PE-Cy7

Absorption

Control

No blue/green light red filter

Fluorescent Light absorption

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

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

555 - 595

PMT4

APC 655 - 695

PMT6

PMT7

49

0

DL

488

BK

05

5

DL

62

5

DL

675

BP

488 BP525 BP575 BP

632

BP

TM

PMT3 PMT2 PMT1

PMT5

Optical Design

PMT 1

PMT 2

PMT 5

PMT 4

DichroicFilters

BandpassFilters

Laser

Flow cell

PMT 3

Scatter

Sensor

Sample

·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

Electronics:Triggering on a voltage pulse

Laser

Laser

Laser

Time

Volt

age

Time

Volt

age

Time

Volt

age

Voltage In

PMTPower Supply

Levels 0–1000Vadjusted by slider control on computer

PhotonIn

VoltageSignal Out Analog to

Digital Converter

Log amplification of signalsPMT

Linear amplification of signals

Gain levels from 0–9.99adjusted by slider control on computer

2 Options for SSC and fluorescence channels

Amplifier output voltage ranging between 10mV to 10V

compensationcircuit

Optical to Digital

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

Display Plots

Create Gates

Display Statistics

Analyze Statistics

Data Analysis by Software

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

Sample processing

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

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

Hematopoieticstem cell

Neutrophils

Eosinophils

Basophils

Monocytes

Platelets

Red cells

Myeloidprogenitor

Lymphoidprogenitor

B-lymphocytes

T-lymphocytes

Plasmacells

naïve

ALL

AML

AUL

Mixed Lineage Leukemia

Identification of blasts

Enumeration of blasts

Assignment of blast lineage

Identification of abnormal blasts

Subclassification

FCM in diagnosis and classification

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

CD45 PerCP

SS

C-A

102

103

104

105

0

65536

131072

196608

262144

CD45-ECD

cyT

dT

-FIT

C

100

101

102

103

10410

0

101

102

103

104

0.00% 84.98%

0.00% 15.02%

CD45 FITC

CD

34

Pe

rCP

100

101

102

103

10410

0

101

102

103

104

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

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.

Bi-lineal Leukemia

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

Hematogones

Antigens Early(St-1)

Intermediate(st 2 & 3)

MatureB cells

TdT + - -

CD34 + - -

CD10 bright dim -

CD19 dim intermediate bright

CD22 dim dim intermediate

CD20 - (-/+) weak intermediate

CD38 bright bright variable

CD45 dim intermediate bright

CD58 dim dim dim

CD81 bright bright intermediate

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

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

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 – 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

MANTLE CELL LYMPHOMA

HAIRY CELL LEUKEMIA

PERIPHERAL T CELL LYMPHOMA - NOS

ATLL

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

CD19 APC

CD

56 P

C7

102 103 104 105

102

103

104

105 10.83%1.20%

78.34%

9.63%

CD19 PerCP Cy55

CD

81 F

ITC

102 103 104 105

102

103

104

105Br CD81+

Dim CD81+

8c icKappa PE

CD

19 A

PC

102 103 104 105

102

103

104

105 12.73% 20.32%

3.05% 63.90%

CD45 V500

CD

19 A

PC

102 103 104 105

102

103

104

105

32.66%

0.15%

67.02%

0.18%

8c icPC 38v450+ 19+45+

CD19 neg PCs

8c icLambda FITC

CD

19 A

PC

102 103 104 105

102

103

104

105

3.76%63.35%

12.39%20.49%

Multiple Myeloma

Immunophenotyping in Myelodysplastic Syndrome

Normal Granulocytic Maturation

Granulocytic dysplasia in MDS

Normal Monocytic Maturation

Monocytic dysplasia in MDS

Immunophenotyping in Myelodysplastic Syndrome

Paraxysmal Nocturnal Hemoglobinuria (PNH)

THANK YOU!

Basics of Flow Cytometry Prashant Tembhare. Page 2 Flow Cytometry is the automated measurement of...

Documents

Transcript of Basics of Flow Cytometry Prashant Tembhare. Page 2 Flow Cytometry is the automated measurement of...