flow cytometry english - PTE

2015.03.4.

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UNIVERSITY OF PÉCSMEDICAL SCHOOL

www.medschool.pte.hu

FLOW CYTOMETRY AND CELL SEPARATION

BIOPHYSICS 2.2015 4th MarchDr. Beáta BugyiDepartment of Biophysics


Flow cytometry and cellseparation� FLOWFLOWFLOWFLOW = STREAM OF FLUID

in a flowing heterogeneous particle suspension

� CYTOCYTOCYTOCYTO = CELL

separate particles (e.g. cells)

� METRYMETRYMETRYMETRY = MEASUREMENT

and their individual characteristics can be measured based on optical (light) signals (number, size, complexity, presence and amount of special markers)

� SEPARATION SEPARATION SEPARATION SEPARATION = SORTINGon the basis of the measured parameters subsets of interesting particles can be separated from each other for further purposes(morphological, biochemical analysis, cell culturing)


Workflow

� investigated object: suspension of particles in size of 0.2 – 150 µm (biological particles: cells, microorganisms, blood-, bone-marrowsample…)

� instrument: flow cytometer

� measured quantities: optical signal: light scattering, fluorescence emission

� information/result: physical, chemical, biological features (size, complexity, presence of special markers)


Flow cytometer

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Flow cytometer

1. FLUIDICSflow and hydrodynamic focusing

of particles

LIGHT SOURCE

lasers

2. OPTICSlight source, filters, mirrors, detectors

FORWARD SCATTER

(FSC)

3. ELECTRONICS, DATA ANALYSISconversion of light signal to digital

data, analysis

4. SORTINGseparation of cells

SIDE SCATTER (SSC)

FLUORESCENCE

EMISSION (FL)


Flow cytometer

� 1. Flow Flow Flow Flow systemsystemsystemsystem, , , , fluidicsfluidicsfluidicsfluidics: flow and

focusing of particles

� 2. OpticsOpticsOpticsOptics: light source, optical elements

(filters, mirrors), detectors

� 3. Electronics and Electronics and Electronics and Electronics and datadatadatadata analysisanalysisanalysisanalysis:

conversion of light to digital signal

(numbers), analysis

� 4. Cell Cell Cell Cell sortingsortingsortingsorting: : : : separation of cells


Flow systemhydrodynamic focusing

sample fluid(particle suspension,

105 -106 cell/ml, v=

10 m/s)

FLOW HEAD

LAMINAR FLOW!(the two fluids cannot

mix)

particles are separated and aligned in a

single layer of fluid

particles are passed through the light

beam one at a time (104-105 particle/s)

interrogation

point

nozzle aperture d = 50-200 µm

sheath fluid

(surrounds the sample fluid to protect cells

from damage, distilled water, isotonic solution)


Hydrodynamic focusing

� Both the size and the geometry of the

flow head (sample-, sheath fluid, nozzle aperture), and

the characteristics of the flow (pressure) are

set to establish a stable laminar flow (the two

fluid do not mix), which alignes the particles

and they leave the flow head one after

another.

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Flow head


Optics: measurable quantitiesligh scattering, fluorescence emission

FORWARD SCATTERFSC: forward angle light

scatter

smaller angles

� size

� index of refraction

SIDE SCATTERSSC: side light scatter

larger angles

� intracellular

structural complexity

� granularity

FLUORESCENCE

EMISSIONFL1, FL2, …

� special parameter

� fluorophore

specifically bound to

cells, cellular

components

LIGHT SCATTERING


Optics: detectionforward angle-, side light scatter

90o

FORWARD ANGLE SCATTERdetector parallel to the light source

SIDE SCATTERdetector perpendicular to the light

source

LIGHT SOURCE

(laser)

FSC

SSC

DETECTORphotodiode, photoelectron multiplyer


Optics: detectionforward angle light scatter

plate

obscuration bar

detectorcell

laser

forward scatter

Animáció: http://media.invitrogen.com.edgesuite.net/tutorials/4Intro_Flow/player.html

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Optics: detectionfluorescence emission

FLUORESCENCE EMISSIONdetector perpendicular to the light

source

FL1

FL2

FL3

FL4DICHROIC MIRRORS: wavelength

dependent absorption and

reflection

OPTICAL FILTERS: wavelenght

dependent absorption

Separation of different fluorescence

emission: particular wavelenght

ranges are deliverd to the

appropriate detector.

LIGHT SOURCE

(laser)

90o


Data processinglist mode

� LIST MODE: presentation of all the measured datacorresponding to every single particle (raw data stored in

the computer)

cell

↓

…

measured parameter→…


Data processingsingle parametric representation – histogram

� HISTOGRAM: plot of the number of

particles characterized by a given parameter

number of

particles

measured parameter (FSC, SSC, FL)

more

less


Data processingsingle parametric representation – histogram

növekvő méret

FSC SSC FLUORESCENCE

size intracellular

complexity

fluorescent

amount of fluorofor

nonfluorescent

smaller

larger

less complex

more complex

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Data processingmultiparametric representation – dot plot

� DOT PLOT:

correlation method

corresponding values of two measured

parameters (x, y axis) are displayed as a dot

(each dot corresponds to a single particle).



size

intracellular

complexity

number of cells

nu

mb

er

of

cell

s

forward scatter: size

sid

esc

att

er:

co

mp

lexi

ty



FL1 +

FL2 +

FL1 -

FL2 -

number of cells

flu

ore

sce

nce

FL1

fluorescence FL2

nu

mb

er

of

cell

s

+/++/-

-/+-/-



GRANULOCYTEneutrofil (50-70 %), eosinophil (2-5

%), basophil (< 1%)

~ 75 %

size~ 10-15 µµµµm

high complexity

LYMPHOCYTE~ 20-40 %

size~ 7-8/12-15 µµµµm

low complexity

MONOCYTE~ 3-6 %

size~ 12-15 µµµµm

medium complexity

Example:

dot plot of white blood cell

sample(sample preparation: Sedimentation,

electrophoresis)


sid

esc

att

er:

co

mp

lexi

ty

LEUKOCYTE

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Data processingothers

DENSITY PLOT

color ~ number of

cells

CONTOUR PLOT

dots with the

same values are

bound with lines

3D PLOT

z axis ~ number

of cells


Gating

� GATING: definition of subpopulation of

cells/particles in one-, or multiparametric

representation for detailed

investigation/analysis.


Gating

Example: ratio of helper/cytotoxic T lymphocytes

B cell: CD19+

T cell: CD3+

CD3+/CD4+ helper

CD3+/CD8+ cytotoxic

natural killer cell: CD3-/CD16+


sid

esc

att

er:

co

mp

lexi

ty


Cell separation

� CELL SEPARATION: separation of cell

populations characterized by a given

parameter (FSC, SSC, FL) for further

investigation (morphological, biochemical analysis, cell

culturing).

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++

Cell sortingPrinciples

1. Data collection

2. Decision: based on gating, the cell

population to be sorted is defined.

3. Drop formation: the flow head is shaken

by a piezoelectric vibrator (30-40 kHz) to

decompose the fluid that leaves the head

into droplets (1 droplet = 1 cell).

4. Electrostatic separation: the droplet to

be separated is charged at the place of

droplet formation and deflected by an

electric field.

5. Separation: 3000 – 5000 cell/s

+ - --

flow head

1

delay time2

3

4

5


Applications

� BroadBroadBroadBroad fieldsfieldsfieldsfields of of of of applicationapplicationapplicationapplication inininin

medicalmedicalmedicalmedical biologybiologybiologybiology (basic and applied research)

clinicalclinicalclinicalclinical practicepracticepracticepractice (diagnostic, therapeutic monitoring,prognosticapproaches)

� quantitative, qualitative analysis of cell

population

� calculation of absolute cell number and

cell ratio

� identification and quantification of

markers

� functional description of cells:

cell signalling

cell-cell interactions

morphological studies

DNA damage and repair

cell cycle

autophagy

cell death

stem cell differentiaion

parasitology

microbiology

oncology

hematology

clinical immunology (targeted

immunotherapy, immune deficiency,

autoimmune diseases, infections)


ImmunophenotypingLymphocyte associated diseases

Analysis of a blood sample from an HIV infected patientT-helper lymphocytes are targetted by the virus, which results in a decline in the number of T-helper cell

Markers:

CD4+ helper T cell

CD8+ cytotoxic T cell



� abnormal FSC-SSC dot plot

� decrease in T lymphocyte (CD3+)

� Increase in B lymphocyte (CD20+)

� increase in human leukocyte antigen-DR+ cells

Analysis of a blood sample from a leukemia patent suffering from a B cell

lineage leukemia or lymphomaB lymphocytes grow out of control and accumulate in the bone marrow and blood, where they crowd out

healthy blood cells

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Immunophenotyping

� CDCDCDCD ((((clusterclusterclustercluster ofofofof differentiationdifferentiationdifferentiationdifferentiation)))) protokolprotokolprotokolprotokol

� using antigens specifically recognising cell surface marker

molecules (CD) different cell populations can be identified

� CD molecules can have diverse role (receptor, ligand,

signalling pathways, antigen recognition, growth factor, cell

adhesion)

� + / - symbol: marker is expressed/not expressed

� human cells > 360 CD marker


Immunophenotyping

Example: lymphoid cell differentiation CD markers

lymphocytes (T cells, B cells), NK cells and dendritic cells


Immunophenotyping

Example: myeloid cell differentiation CD cell markers

monocytes, granulocytes, platelets, erythrocytes, macrophages, dendritic cells



markers:

T cells CD3+

CD3+/CD8+ cytotoxic T cells

CD3+/CD4+ helper T cells

Selection of lymphocytes from leukocytes

FSC/SSC dot plot, gating

gatinggatinggatinggating

FSC

SS

C

LYMPHOCYTES

leukocyte

lymphocyte

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fluorescent

T cells

nonfluorescent

B & NK cells

Selection of CD3+ T-lymphocytes from lymphocytes

CD3 fluorescence emission histogram, gating

CD3 fluorescence emsission

lymphocyte

T-lymphocyte



Selection of CD4+ helper T-lymphocytes from T-lymphocytes

CD4-CD8 fluorescence dot plot



Analysis of a blood sample from an HIV infected patient


Flow cytometry and cell sortin -keywords� HydrodynamicHydrodynamicHydrodynamicHydrodynamic focusingfocusingfocusingfocusing

� OriginOriginOriginOrigin and and and and importanceimportanceimportanceimportance of of of of ppticalppticalppticalpptical signalssignalssignalssignals ((((lightlightlightlight scatteringscatteringscatteringscattering, , , , fluorescencefluorescencefluorescencefluorescence emissionemissionemissionemission))))

� Data Data Data Data processingprocessingprocessingprocessing: : : : listlistlistlist modemodemodemode, , , , histogramhistogramhistogramhistogram, , , , correlationcorrelationcorrelationcorrelation methodsmethodsmethodsmethods: : : : dotdotdotdotplotplotplotplot

� GatingGatingGatingGating

� Cell Cell Cell Cell sortingsortingsortingsorting

Keywords from previous lectures:

� laminar flow, photomultiplier, laser, light scattering, fluorescence

Keywords to coming lectures:

� Fluorescence microscopy: optical filters, dichroic mirrors

� Ultrasound: piezoelectric effect

� Sedimentation: preparation of blood samples

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Suggested web resources

� https://www.youtube.com/watch?v=2P7YsJ0Zkio

� http://media.invitrogen.com.edgesuite.net/tutorials/4Intro_Flow/play

er.html

� http://media.invitrogen.com.edgesuite.net/tutorials/5Data_Analysis/

player.html


Supplemental material


Optical filters, dichroic mirrors


Photelectron multiplyer

photon reaching the photocathode�photoelectron (photoelectric

effect)�amplification by a dynode chain�anode�electric current

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Conversion of optical signal toelectronic signal

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flow cytometry english - PTE