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Life is a journey and your words have

been a guiding light throughout.

FLOW
CYTOMETRY
NATASHA

SAWHNEY

Supervised byDR. VARSHA A.SINGH

CYTO - CELLS

METRY MEASUREMENT

FLOW CYTOMETRY
MEASUREMENT OF CELLS IN
FLOW

Flow cytometry is a laser


based, biophysical technology
employed in cell counting,
sorting, biomarker
detection and protein
engineering, by suspending
cells in a stream of fluid and
passing them by an electronic
detection apparatus.

GUCKER IN 1947
Developed a flow cytometer for detection
of bacteria in aerosols
By using sheath of filtered air flowing
through a dark-field flow illuminated
chamber.
L. Kamentsky, and M. Fulwyler
experimented with fluidic switching and
electrostatic cell sorters respectively. Both
described cell sorters in 1965.(size
measurements only)
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WOLFGANG
GOHDE.19
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The first fluorescence-based


pulse cytometry device was
developed in 1968 by
Wolfgang Gohde from the
University of Munster
first commercialized in
1968/69 by German
developer and manufacturer
Partec
1978Conference of the
American Engineering
Foundation in Pensacola,
Florida FLOW
CYTOMETER

FLUIDIC SYSTEM..
Which presents sample to the
interrogation point

THE ILLUMINATION SYSTEM


Which are the light source for scatter
and fluorescenceLASER LIGHT/
ARC LIGHT
THE OPTICS..
Which gather and direct the light
THE DETECTORS..
Which receive the light

THE ELECTRONICS & COMPUTER


SYSTEM..
Which converts the signals from detectors
into digital data.

1. ONLY SAMPLE IS USED


Flow Cell

HYDRO DYNAMIC
FOCUSSING
Fluid dynamics.
Lamella in the
middle of the
channel takes
smaller
crossection

Image from SonyInsider

FORWAR
D
The
amount of
SCATTER

light that is
scattered in the
forward
direction.

Magnitude of
FS is
proportional to
the SIZE of the
cell.

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HISTOGRAM OF FORWARD
SCATTER

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SIDE
The amount of
SCATTER
light that is
scattered
usually at 90
degrees..

Magnitude of
SS is
proportional to
the granularity
and inner
complexity of
the cell.

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HISTOGRAM OF BLOOD

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FSC v. SSC
Since FSC ~ size and SSC ~ internal
structure, a correlated measurement
between them can allow for differentiation of
cell types in a heterogenous cell population

Granulocytes
SSC

Lymphocytes

Monocytes

RBCs, Debris,
Dead Cells
FSC

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2. FLUOROCHROME LABELLED
SAMPLE
FLUOROPHORE

Added to cell

Laser light strikes the


fluorophore

Fluorescent signal
emitted

Detected by detectors

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FLUOROCHROMES
1. Whose fluorescence increases with

binding to

PROTEINS
FITC
RED
TEXAS RED

NUCLEIC ACIDS

LIPIDS

ETHIDIUM BROMIDE

NILE

YOYO 1
HOECHST 3325
CHROMOMYCIN A3
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2. Whose fluorescence depend


on physiological parameters

pH
membrane integrity
SNARF 1
bromide

Ethidium

propidium iodide
sytox

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FLOW CYTOMETRY
&
MICROBIOLOGY

A LONG TIME TOGETHER

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DETECTION OF MICROBES
IDENTIFICATION OF SPECIFIC
MICRO ORGANISMS
GRAM VARIABILITY
CELL VIABILITY
ANTIMICROBIAL AGENTS

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DETECTION OF MICROBES
By combining nucleic acid dyes with light
scatter measurements
YOYO 1 ..nonfluorescent unless bound
to nucleic cids
Samples with dust, pollen, fungal spores,
and unknown bacteria were tested
Fluorescence producing. BACTERIA
Pollens, molds, fungal spores.large size
Dust particles.dont have nucleic acid
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1st study..MANSOUR J.D et al


1985
The first study detecting of microbes
in blood by using FCM was done with
ethidium bromide as the detecting
fluorochrome
Blood cells were lysed, and the
remaining bacteria were stained with
ethidium bromide
as few as 10 E. coli cells/ml were
detected
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IDENTIFICATION OF
MICRO ORGANISMS
LIGHT SCATTER MEASUREMENTS
DNA CONTENT
IMMUNOFLUORESCENCE
APPROACH

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Light scattering
measurements
Cell size, shape, inner complexity & RI.
To study morphological features like
rods
Cocci
Spirillas
Spirochaetes
Vibrios
Spores give forward scatter signal out
of proportion to size .high RI.
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DNA content.
Used comb. of DNA specific fluorochromes to
analyse diff species of organisms.
HOECHST 3325
DNA rich in AT base
pair
CHROMOMYCIN A3
rich in GC base
pair
Resolved ind. Sp. Of staph.aureus, E. coli &
Ps.aerug
Each formed a distinct cluster within the
histogram.
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IMMUNOFLUORESCEN
CE APPROACH
FLUOROPHORE labelled antibody

Added to cell

Laser light strikes the


fluorophore

Fluorescent signal
emitted

Detected by
detectors
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Immunofluorescence approach.
Seo et al.1998.
Target E. coli O157:H7
cells.identified
By using FITC conjugated rabbit anti
E. coli O157:H7 polyclonal antibodies
Within few minutes of obtaining the
sample

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27

28

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GRAM
VARIABILITY
By using fluorescently labelled
lectin mol.
Bind specifically to N- ACETYL
GLUCOSAMINE in outer
peptidoglycan layer of gram
positive bacterias.
Gram ve bacteria have an outer
membrane covering yhe
peptidoglycan layer.prevents
lectin binding.
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CELL VIABILITY
MEMBRANE INTEGRITY Ethidium bromide
Propidium iodide
Sytox green
Passively enter stressed, injured, or dead
cells& intercalate into DNA & RNA.
FLUORESCENCE indicates loss of
membrane integrity & hence the viability.
No. of fluorescent cells counted I/ no of
viable cells
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ANTIMICROBIAL AGENTS
By using membrane potential
senstive dye & membrane integrity
dyes.
Membrane integrity & potential of
antibiotic treated dyes will collapse
However, untreated cells will
maintain their integrity.

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Ethidium
bromide

E .Coli
Ps. aeruginosa
S .Aureus
Str. pyogenes

amikacin

Cohen and
sahar et
al.1989

Ethidium
bromide &
mithramycin

e. coli

Ceftazidine
Ciprofloxacin
gentamicin

Walburg et al
1997

Acridine
orange

e. coli

Gentamicin

Mason & lloyd


1997

Propidium
iodide

e. coli

Gentamicin
Cefotaxime
Ampicillin
ciprofloxacin

Gant et al 1993

Sytox green

e. Coli
s. Aureus
b. cereus

Ampicilin
Amoxicillin
Penicillin
vancomycin

Roth et al 1997

Rhodamine
123

e. coli

vancomycin

Perter et al
1995

FITC

e. Coli
s. aureus

Ceftazidime
vancomycin

Suller & lloyd


1999

Di BAC 4

e. coli

Azithromycin

Jepras et al

33

PARASITOLOGY
NAEGLERIA FOWLERI &
ACANTHAMOEBA

FLORES et al.1999

GIARDIA LAMBLIA

DIXON et al..1997

MALARIA

JOUIN H et al.1995

CRYPTOSPORIDIUM PARVUM

LUIS M et al.1991

TOXOPLASMA GONDII

GREGOIRE et al.1993

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MYCOLOGY
CANDIDA ALBICANS

GROSHEN et al.1983
CHAFFIN et al.1998
HAN et al..1997

PNEUMOCYSTIS CARINII

LIBERTIN et al.1984

CRYPTOCOCCUS
NEOFORMANS

BAUTERS TG et al.2003

DERMATOPHYTES
(ONYCHOMYCOSIS)

GERALD E. PIERARD.1993

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VIROLOGY
HIV

FARMER et al..1954

VZV

SNOECK R.D et al.1992

INFLUENZA

LYDY S.L et al.1993

PARAINFLUENZA

LYDY S.L et al.1993

RABIES

WHITT M.A et al.1991

SV 40

HORAN M et al.1975

HCMV

ELMENDORF et al.1988

EBV

MILLER C.L et al.1993


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DETECTION OF MICROBES
Light scatter
measurements
+
Nucleic acid dyes

GRAM STAINING

ID. OF MICROORGANISMS
Light scatter
measurements
+
Dna specific dye
+
Immunofluorescencre
approach

CELL VIABILITY
Membrane
integrity specific
dyes

ANTIMICROBIAL
SUSCEPTIBILITY
Membrane
integrity
Specific dyes
37

DNA content analysis


By using fluorescent dyesDNA
aneuploidy
Rhabdomyosarcoma
Neuroblastoma
Multiple myeloma
Acute lymphoblastic leukemia
Myelodysplastic syndromes
38

Platelet analysis

Identification of inherited disorders


Monitoring of anti-platelet therapy
Monitoring clinical course of disease
Monitoring platelet production in
thrombocytopenia
Identification of patients at risk of
thrombosis
Diagnosis of heparin induced
thrombocytopenia
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Solid organ transplantation.


T cell cross match
Post operative monitoring
Diagnosing immunodficiencies
Diagnosing PNH
Reticulocyte analysis
Sperm sorting for sex preselection.

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Automated classification &


identification techniques
ARTIFICIAL NEURAL NETWORKS.
Are computing technologies that can be used to
discriminate between different cell types based
on flow cytometry data
A computer is TAUGHT how to recognize data
pattern & to analyse cell populations.
Developed for
Chromosomal classification
Leukemia subsets
Food analysis
Microbiology samples
Biological warfare agents id.
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ADVANTAGES
Sample heterogeneity can be quantified.
It is a rapid technique.
Can make physiological measurements like
DNA content of a particle
Thousands of cells can be measured in a
realistic time scale.
Measures single cells
Measures large number of cells
simultaneous analysis of multiple parameters
Identifies small subpopulations
42

DISADVANTAGES
EXPENSIVE
EXPERTISE ASSISTANCE AND
TECHNICAL SUPPORT REQUIRED
INSTRUMENT MAINTENENCE

43

44

Analyser a

new milestone
in flow

cytometry

Miltenyi Biotec Ltd,


Germany
Is a completely new 7
colour flow cytometer.

Powerful: Multisample
& multiparameter cell
analysis
Capable: Fast and
sensitive rare cell
analysis
Accurate: Volumetric
absolute cell counting
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46

47

FLOW CYTOMETRY
&
HEMATOLOGY
48

New microbial species analysed by flow


cytometry
Prokaryotes
Bacteria
Gram-negative bacteria
Azotobacter vinelandii
Aeromonas sahnonicida
A zospirillum brasilense
Bacteroides fragilis
Brueella abortus
HaemophUus influenzae
Nitrosomonas
Proteus vulgaris
Pseudomonas cepacia
Pseudomonas fluorescens
Sahnonella paratyphi
Sahnonella typhi
Shigella flexneri
Spirillum lipoferun
Gram-positive bacteria
Leuconostoc mesenteroides
Neisseria gonorrhoea
Micrococcus luteus
Propionibacterium acnes

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Blue-green 'algae' (Cyanophyceae)


Anabaena azollae
Anabaena solitaria
Oscillatoria redekei
Mycoplasma
Mycoplasma gallisepticum
M synoviae
Eukaryotes
Fungi
Yeast
Apiotrichum curvature
Candida albicans
Canclida parapsilosis
Candida pseudotropicalis
Candida tropicalis
Hansenula polymorpha
Phaffia rhodozyma
Miscellaneous funGI
Phytophthora cinnatnomi
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DYE

LIGAND/
SUBSTRATE

APPLICATIONS

TOTO - 3

DNA,

RNA

DNA quatification
Cell cycle studies

SYTOX GREEN

DNA,

RNA

Viability
DNA quantification

PI

DNA,

RNA

Viability
DNA quantification

ETHIDIUM
BROMIDE

DNA,

RNA

DNA quantification
Cell cycle studies

HOECHST
33258/33342

DNA

GC PAIRS

Cell cycle studies

SYTO 13

DNA,

RNA

Viability,DNA
quantification
Cell cycle studies

MITHRAMYCIN

DNA

Cell cycle studies

PYRONINE Y

RNA

RNA quantification

FITC

PROTEIN

Microbe detection

TEXAS RED

PROTEIN

Microbe detection 51

CLASSIFICATION OF
FLUOROCHROMES
whose fluorescence increases with binding to specific
cell compounds such as proteins (fluorescein
isothiocyanate (FITC), nucleic acids (propidium iodide
[PI]) and lipids(nile red)
whose fluorescence depends on cellular physiological
parameters (pH, membrane potential etc.)
whose fluorescence depends on enzymatic activity
(fluorogenic substrates) such as esterases, peroxidases
and peptidases
who can be conjugated to antibodies or nucleotide
probes to directly detect microbial antigens or DNA and
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RNA sequences

MEASURABLE
PARAMETERS

size and morphological complexity of cells


cell pigments
total DNA content
total RNA content
chromosome analysis and sorting
cell surface antigens
Intracellular antigens (various cytokines, secondary mediators, etc.)
nuclear antigens
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
oxidative burst
characterising multidrug resistance (MDR) in cancer cells

53

THE ELECTRONIC
SYSTEM
Incident light
Electronic pulses
Distibuted electronically displaying
histogram
Analytical software
54

Detection of viral antigens on cell surfaces by


flow cytometry
Virus

Antigen(s) detected

HSV
Bovine HSV
Duck hepatitis B
Japanese encephalitis
Mouse mammary tumor
Varicella-zoster
Vesicular stomatitis
Rabies
HCMV
HIV
Influenza
Parainfluenza
Feline immunodeficiency
Human herpesvirus 6
Measles
FLV

gB, gC, gD, gE


gll; gIV
Precore protein
E protein
gp52
gpI
G protein
G protein
gB
gpl60/120
HA
F
gpl2O
gpllO/60
H and F
env
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Detection of cell-bound virus by flow cytometry


Virus
receptor
EBV
EBV
SV40
classI
SV40
LCMV
HTLV
Echovirus
Coxsackievirus
Murine leukemia
Measles
CD46
Poliovirus
Sindbis
HIV

Cells

B lymphocyte
Human epithelial
CV-1
Monkey kidney
MC57, BHK, Vero, hela
T and B
BK, Vero, P2002
A9 BK, Vero, P2002
NIH 3T3, HeLa 94
Jurkat
Human mononuclear
Mouse neuronal
MT-4

CR2
CR2
MHC
ND
ND
ND
ND
ND

CD14
ND
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CD4

Analysis of the effect of virus


infection on cell surface proteins
Pseudorabies
decreased

L-929; Neuro 2A

MHC class I expression

HSV
Mouse embryo fibroblasts
decreased

MHC class I expression

CMV
Human foreskin fibroblasts
decreased

MHC class I expression

HSV

Endothelial

GMP140 expression increased

HIV

T cells

CD4 expression decreased

EBV

B cells

Bcl-2 expression decreased


57

Virus-cell interactions leading to


apoptosis
Virus
HIV
lymphoblastoid
HIV
progenitor
HIV
HIV
HIV
HIV
Simian immuno-deficiency
EBV, varicella-zoster
Influenza
LCMV

Apoptotic cells
EBV transformed,
CD34+ bone marrow,
CD8+ T, CD19+ B
gpl60 expressing CD4+ cells
PBMC
CD4+
PBMC
CD 45+
HeLa
Murine T
58

OREGON GREEN
ISOTHIOCYANATE

proteins

Microbe detection

INDO- 1

Ca 2+

Calcium mobilization

FURA- 2

Ca 2+

Calcium mobilization

FLUORO- 3

Ca 2+

Calcium mobilization

BEECF

pH

Metabolic variations

SNARF-1

PH

Metabolic variations

DiOC 6

Membrane potential

Antibiotic senstivity

OXONOL

Membrane potential

Antibiotic senstivity

RHODAMINE 123

Membrane potential

Antibiotic senstivity

FUN - 1

Yeast vacuoler
enzyme activity

Yeast metabolic
states
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VIROLOGY
Virus-Induced Programmed Cell Death
Effects of Virus Infection on Cellular
Proteins Binding of Virus to Cells
Detection of Viral Antigens on the Cell
Surface
Detection of Intracellular Viral
Antigens
Detection of viral antibodies.
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3.Leucocyte analysis
Leukemias
Lymphomas

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2.Erythrocyte analysis
Detection and quantitation of RBC-bound
proteins
Quantitation of RBC-bound immunoglobulins
Detection and quantitation of RBC antigens
and antibodies
Detection and quantitation of minor RBC
populations, including the detection and
quantitation of transfused RBCs and the
detection and quantitation of fetal RBCs in
maternal blood
62

LECTINS

Membrane
oligosaccharides

Cell wall
composition
Microbe detection

FLUORESCENTLY
LABELLED OLIGO
NUCLEOTIDES

Nucleotide sequences

Microbe identification

CALCOFLUOR WHITE

Chitin & other


carbohydrate
polymers

Fungal detection

SUBSTRATES LINKED
TO FLUOROCHROMES

Enzyme activities

Metabolic activities

ANTIBODIES LINKED
TO FLUOROCHROMES

antigens

Microbe detction

63

Protozoa
Flagellakcs
Leishmania chagasi
Tritrichomonas foetus
Trypanosoma congolense
Amoebae
Entamoeba histolytica
Ciliates
Tetrahymena thermophila
Sporozoans
Eimeria tenella
Algae
Diatoms (Bacillariophyeea
Phaeodactylum tricornotum
Dinoflagella
Gonyaulax polyedra
Viruses
Eseherichia coli bacteriophage T4
Pox virus
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I am indebted to my father for living,


but to my teacher for living well. Life
is a journey and your words have
been a guiding light throughout.
Happy Teachers Day!

65

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Data Representation

CD45
CD8

CD4
CD8
leu11a

Mo1

CD20

FITC Fluorescence

67