FLOW CYTOMETRY: PRINCIPLES AND APPLICATIONS. By: Douaa Moh. Sayed

FLOW CYTOMETRY:

PRINCIPLES AND

APPLICATIONS

By:

Definition

• Flow cytometry is a technique

for

counting

,

examining

, and

sorting

microscopic

particles

suspended in a stream of fluid.

• It

allows

simultaneous

multiparametric analysis

of the

physical

and/or

chemical

characteristics of

single cells

flowing through an

optical

and/or

BASIC PRINCIPLES

• Flow cytometry involves the

analysis of the

fluorescence

and

light scatter

properties of

single

particles (e.g. cells,

nuclei, chromosomes) during

their passage within a narrow,

precisely

defined

liquid

Flow Cytometry - Principles

Target:

Evaluation of single cell

Solution:

• When conditions are right, sample

fluid flows in a central core that

does not mix with sheath fluid.

Lasers

• Produce a single wavelength of high

intensity

• Nearly all instruments fitted with an

argon laser Gives blue light at

488 nm.

• Second or third laser may be fitted:

- He-Ne- red light at 633 nm

- He-Cd- UV at 325 nm

Light Scatter

Laser

Forward angle light scatter:

in a narrow angle from the

direction of the laser beam (FALS or

FS).

Light Scatter

Laser

Right angle light

scatter

at right angles

to the laser beam

Light Scatter

• FS tends to be more sensitive to the

size and surface properties

can be used to distinguish live

from dead cells.

• SS tends to be more sensitive to

inclusions within cells

can be used to distinguish

granulated cells from non-granulated

Instrument Parameters

Y

Laser

Fluorochromes

Dye Excitation Emission Molecular

Weight FITC 488 nm 520 nm 389 Da PE 488 nm 578 nm 240 000 Da ECD 488 nm 613 nm 250 000 Da PC5 488 nm 668 nm 105 000 da PerCP 488 nm 688 nm 35 000 Da APC 613 nm 665 nm 105 000 Da

Common Fluorochromes

for 488nm Excitation

FITC = Fluorescein Isothiocyanate PE = Phycoerythrin (RD1)

ECD = Energy Coupled Dye PI = Propidium Iodide

PC5 = Phycoerythrin Cyanin 5 (PC5)

<390 λ 400-450 λ 450-500 500-570 λ 570-590 λ 590-620 λ 620-750 λ >750 λ

ultra- violet blue green yellow orange red

infra-violet red

PE FL FITC FL

488nm Sct

Laminar Fluidic Sheath

Core Sheath

Outer Sheath

• Each cell generates a quanta of fluorescence

PE FL FITC FL 488nm Sct Confocal Lens Dichroic Lenses Photomultiplier Tubes (PMT’s) Discriminating Filters Forward Light Scattering Detector

Multiparametric Analysis

Y

• Simultaneous

Detection of up to

20 Parameters

– Cell size

– Cell structure

– Dyes

– Time (follow enzyme

reactions)

Flow Cytometry - Principles



Light source: argon laser

488



Fluidics



Detectors/Computer

system for analysis and

storage of digitized data.



A typical

flow

cytometer

consists of:

Amplification

• The electrical pulses originating from light

detected by the PMTs are then processed

by a series of linear and log amplifiers.

• Logarithmic amplification is most often

used to measure fluorescence in cells. This

type of amplification expands the scale for

weak signals and compresses the scale for

“

strong” or specific fluorescence signals.

Data display

• Univariate histogram

•Dot plot

Data display

Surface Plot

Tomogram

The Tomogram may be:

• rotated

• viewed in color

precedence

Light Scattering

,

2 Parameter Histogram

Forward Light Scatter (FLS) 90 degree Light Scatter Bigger More Granular Live Cells Bigger Cells Dead Cells Apoptotic Cells X Axis Y Axis

1 Parameter Histogram 1 2 3 4 6 7 150 160 170 .. 190 Channel Number Positive Negative Brighter Dimmer Count 1 4 6

Fluorescence picked up from the FITC PMT

Signal Analysis

• Statistics

• % Negative & % Positive Cells

• Antigen density ~ Mean

Fluorescence Intensity ~ Channel

number

2 Parameter Histogram FITC FL PE FL Negative Population Single Positive FITC Population Single Positive PI Population Double Positive Population

Compensation

–

Spectral overlap of emission

Compensation

Simple rule for Adjustment of Color Compensation

Fluorescence 1 F lu o rescen ce 2 B1 B2 B3 B4

• Internal Checks

– Internal Consistency

• X % CD3+ _{cells if all tubes +/- 3 %}

– Lymphocytes sums (Purity?)

• % CD3+ _{cells + % CD19}+ _{cells + % CD16}+_/CD56+ _cells

• n lymphocyte gate = 100 % (+/- 5%)

• % CD3+_/CD4+ _{cells + % CD3}+_/CD8+ _{cells = % CD3}+ _cells

Gating

• Set a region on a histogram or

cytogram

• If cell in region then show another

property cell selection

•Dual Parameter

Correlation:

Gating-Option

gated

Analysis of Cell

Sub-populations

Flow Cytometry Data Smaller Region, Live cells mostly Larger Region includes all cells

Cell sorting

• Flow cytometry can be used to select

and purify a specific subset of cells

within a population

• cell sorting based on physical,

biochemical and antigenic traits.

Measurable parameters

• volume and morphological complexity of cells • cell pigments such as chlorophyll or

phycoerythrin

• DNA (cell cycle analysis, cell kinetics, proliferation etc.)

• RNA

• chromosome analysis and sorting (library construction, chromosome paint)

• protein expression and localization

• transgenic products in vivo, particularly the Green fluorescent protein or related

fluorescent proteins

• cell surface antigens (Cluster of differentiation (CD) markers)

• intracellular antigens (various cytokines, secondary mediators etc.)

Measurable parameters

• enzymatic activity

• pH, intracellular ionized calcium, magnesium, membrane potential

• membrane fluidity

• apoptosis (quantification, measurement of DNA degradation, mitochondrial membrane potential, permeability changes, caspase activity)

• cell viability

• monitoring electropermeabilization of cells • oxidative burst

• characterising multidrug resistance (MDR) in cancer cells

• glutathione

• various combinations (DNA/surface antigens etc.)

Flow Cytometry Applications

• molecular biology, The specific antibodies bind to antigens on the target cells and help to give

information on specific characteristics of the cells • pathology,

• immunology,

• plant biology and

• marine biology: the auto-fluorescent properties of photosynthetic plankton can be exploited by flow cytometry in order to characterise abundance and community structure.

• protein engineering: flow cytometry is used in

conjunction with yeast display and bacterial display to identify cell surface-displayed protein variants with • desired properties.

Clinical applications in flow cytometry

•

and lymphoma

• DNA & cell cycle analysis • Minimal residual disease • PNH-diagnostics

• RNA content (reticulocytes) • Immune-deficiencies

Clinical applications in flow cytometry

Platelet Function Analysis:

•Platelet associated immunoglobulins

•platelet-associated IgG quantitation for the diagnosis of immune thrombocytopenias

•platelet cross-matching in transfusion

•reticulated platelet assay to detect “stress” platelets

•fibrinogen receptor occupancy studies for monitoring the clinical efficacy of platelet-directed anticoagulation in thrombosis

•detection of activated platelet surface markers, cytoplasmic calcium ion measurements, and

platelet microparticles for the assessment of hypercoagulable states.

Clinical applications in flow cytometry

Measurement of the Efficacy of Cancer Chemotherapy

•Multi-Drug-Resistance

•assays of proliferative survival using bromodeoxyuridine (BrdU) incorporation •Ligand, antigen, or molecule-targeted biological therapy utilizing monoclonal antibodies

Clinical applications in flow cytometry

Cell Function Analysis

every event that occurs during the process of lymphocyte activation can be measured by flow cytometry [tyrosine phosphorylation

(multiplex bead technology), calcium flux, oxidative metabolism, neoantigen expression (CD11b/CD18 and CD154), and cellular

proliferation)

Applications in Transfusion Medicine

Fetal red cells in maternal blood Quality control of blood products

Clinical applications in flow cytometry

Organ Transplantation and Hematopoietic Cell Therapy

•HLA-typing and cross- matching,

•enumeration of CD34+ hematopoietic stem cells

•pre-transplantation determinations of the efficacy of ex vivo T-cell graft depletion, •post-transplantation evaluation of immune recovery, graft rejection, graft-versus host disease, and the graft-versus-leukemia effect.

Clinical applications in flow cytometry

Applications in Microbiology

•detection of bacteria, fungi, parasites and viruses

•quantitative procedures to assess antimicrobial susceptibility and drug cytotoxicity

sperm sorting in IVF Apoptosis

PI incorporation TdT Assay

Annexin V APO2.7

Advantages

• high speed analysis (>100.000 s

)

• Measures single cells

• Measures large number of cells

• simultaneous analysis of multiple

parameters (up to 20)

• Identifies small subpopulations

• quantification of fluorescence

intensities

• sorting of predefined cell populations (up

to 70.000 s

Disadvantages

• very expensive and sophisticated

instruments

• Need single particle

• Tissue architecture is lost

• Little information about intra-cellular

distributions

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