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FLOW
CYTOMETRY
NATASHA
SAWHNEY
CYTO - CELLS
METRY MEASUREMENT
FLOW CYTOMETRY
MEASUREMENT OF CELLS IN
FLOW
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)
5
WOLFGANG
GOHDE.19
68
FLUIDIC SYSTEM..
Which presents sample to the
interrogation point
HYDRO DYNAMIC
FOCUSSING
Fluid dynamics.
Lamella in the
middle of the
channel takes
smaller
crossection
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
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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pH
membrane integrity
SNARF 1
bromide
Ethidium
propidium iodide
sytox
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FLOW CYTOMETRY
&
MICROBIOLOGY
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DETECTION OF MICROBES
IDENTIFICATION OF SPECIFIC
MICRO ORGANISMS
GRAM VARIABILITY
CELL VIABILITY
ANTIMICROBIAL AGENTS
19
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
20
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.
23
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.
24
IMMUNOFLUORESCEN
CE APPROACH
FLUOROPHORE labelled antibody
Added to cell
Fluorescent signal
emitted
Detected by
detectors
25
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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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.
30
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.
32
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
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
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
34
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
35
VIROLOGY
HIV
FARMER et al..1954
VZV
INFLUENZA
PARAINFLUENZA
RABIES
SV 40
HORAN M et al.1975
HCMV
ELMENDORF et al.1988
EBV
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
Platelet analysis
40
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
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Analyser a
new milestone
in flow
cytometry
Powerful: Multisample
& multiparameter cell
analysis
Capable: Fast and
sensitive rare cell
analysis
Accurate: Volumetric
absolute cell counting
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FLOW CYTOMETRY
&
HEMATOLOGY
48
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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
SYTO 13
DNA,
RNA
Viability,DNA
quantification
Cell cycle studies
MITHRAMYCIN
DNA
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
52
RNA sequences
MEASURABLE
PARAMETERS
53
THE ELECTRONIC
SYSTEM
Incident light
Electronic pulses
Distibuted electronically displaying
histogram
Analytical software
54
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
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
56
CD4
L-929; Neuro 2A
HSV
Mouse embryo fibroblasts
decreased
CMV
Human foreskin fibroblasts
decreased
HSV
Endothelial
HIV
T cells
EBV
B cells
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
59
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.
60
3.Leucocyte analysis
Leukemias
Lymphomas
61
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
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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Data Representation
CD45
CD8
CD4
CD8
leu11a
Mo1
CD20
FITC Fluorescence
67