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Life is a journey and your words have been a guiding light throughout.

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FLOW CYTOMETRY

NATASHA SAWHNEY

Supervised by-DR. VARSHA A.SINGH

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CYTO - CELLS

METRY – MEASUREMENT

FLOW CYTOMETRY – MEASUREMENT OF CELLS IN FLOW

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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.

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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….1968

• 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

• 1978…Conference of the American Engineering Foundation in Pensacola, Florida – FLOW CYTOMETER

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FLUIDIC SYSTEM…..Which presents sample to the interrogation point

THE ILLUMINATION SYSTEM…Which are the light source for scatter and fluorescence…LASER 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.

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1. ONLY SAMPLE IS USED

HYDRO DYNAMIC FOCUSSING

Fluid dynamics….“Lamella in the

middle of the channel takes smaller crossection”

Flow Cell

Image from SonyInsider

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FORWARD SCATTERThe amount of 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 SCATTERThe amount of 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

FSC

SSC

Lymphocytes

Monocytes

Granulocytes

RBCs, Debris,Dead Cells

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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 NUCLEIC ACIDS LIPIDS

FITC ETHIDIUM BROMIDE NILE RED

TEXAS RED YOYO 1 HOECHST 3325

CHROMOMYCIN A3

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2. Whose fluorescence depend on physiological parameters

pH membrane integrity

SNARF 1 Ethidium bromide

propidium iodide

sytox green

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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

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 rodsCocciSpirillasSpirochaetesVibrios• 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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IMMUNOFLUORESCENCE 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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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 VIABILITYMEMBRANE INTEGRITY-• Ethidium bromide• Propidium iodide• Sytox greenPassively 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/α 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 .ColiPs. aeruginosaS .AureusStr. pyogenes

amikacin Cohen and sahar et al….1989

Ethidium bromide & mithramycin

e. coli CeftazidineCiprofloxacingentamicin

Walburg et al 1997

Acridine orange

e. coli Gentamicin Mason & lloyd 1997

Propidium iodide

e. coli GentamicinCefotaximeAmpicillinciprofloxacin

Gant et al 1993

Sytox green e. Colis. Aureusb. cereus

AmpicilinAmoxicillinPenicillinvancomycin

Roth et al 1997

Rhodamine 123

e. coli vancomycin Perter et al 1995

FITC e. Colis. aureus

Ceftazidimevancomycin

Suller & lloyd 1999

Di BAC 4 e. coli Azithromycinciprofloxacin

Jepras et al 1997

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PARASITOLOGYNAEGLERIA 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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MYCOLOGYCANDIDA ALBICANS GROSHEN et al….1983

CHAFFIN et al….1998HAN 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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VIROLOGYHIV 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 MICROBESLight scatter

measurements+

Nucleic acid dyes

ID. OF MICROORGANISMSLight scatter

measurements +

Dna specific dye+

Immunofluorescencre approach

GRAM STAININGCELL VIABILITY

Membrane integrity specific

dyes

ANTIMICROBIAL SUSCEPTIBILITY

Membrane integrity

Specific dyes

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DNA content analysis By using fluorescent dyes……DNA

aneuploidy• Rhabdomyosarcoma• Neuroblastoma• Multiple myeloma• Acute lymphoblastic leukemia• Myelodysplastic syndromes

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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 matchPost 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

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DISADVANTAGES• EXPENSIVE• EXPERTISE ASSISTANCE AND

TECHNICAL SUPPORT REQUIRED• INSTRUMENT MAINTENENCE

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The MACSQuant 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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FLOW CYTOMETRY

&

HEMATOLOGY

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New microbial species analysed by flow cytometry ProkaryotesBacteriaGram-negative bacteriaAzotobacter 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 lipoferunGram-positive bacteriaLeuconostoc mesenteroides Neisseria gonorrhoea Micrococcus luteus Propionibacterium acnes

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Blue-green 'algae' (Cyanophyceae)Anabaena azollae Anabaena solitaria Oscillatoria redekei MycoplasmaMycoplasma gallisepticum M synoviae EukaryotesFungiYeastApiotrichum curvature Candida albicans Canclida parapsilosis Candida pseudotropicalis Candida tropicalis Hansenula polymorpha Phaffia rhodozyma Miscellaneous funGIPhytophthora cinnatnomi

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DYE LIGAND/ SUBSTRATE

APPLICATIONS

TOTO - 3 DNA, RNA DNA quatificationCell cycle studies

SYTOX GREEN DNA, RNA ViabilityDNA quantification

PI DNA, RNA ViabilityDNA quantification

ETHIDIUM BROMIDE

DNA, RNA DNA quantificationCell cycle studies

HOECHST 33258/33342

DNA GC PAIRS Cell cycle studies

SYTO 13 DNA, RNA Viability,DNA quantificationCell cycle studies

MITHRAMYCIN DNA Cell cycle studies

PYRONINE Y RNA RNA quantification

FITC PROTEIN Microbe detection

TEXAS RED PROTEIN Microbe detection

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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 RNA sequences

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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

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THE ELECTRONIC SYSTEM

Incident light Electronic pulses

Distibuted electronically displaying histogram

Analytical software

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Detection of viral antigens on cell surfaces byflow cytometry

Virus Antigen(s) detected

HSV gB, gC, gD, gE Bovine HSV gll; gIV Duck hepatitis B Precore protein Japanese encephalitis E protein Mouse mammary tumor gp52 Varicella-zoster gpI Vesicular stomatitis G protein Rabies G protein HCMV gB HIV gpl60/120 Influenza HA Parainfluenza F Feline immunodeficiency gpl2O Human herpesvirus 6 gpllO/60 Measles H and F FLV env

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Detection of cell-bound virus by flow cytometryVirus Cells receptor

EBV B lymphocyte CR2 EBV Human epithelial CR2 SV40 CV-1 MHC classI SV40 Monkey kidney ND LCMV MC57, BHK, Vero, hela ND HTLV T and B ND Echovirus BK, Vero, P2002 ND Coxsackievirus A9 BK, Vero, P2002 ND Murine leukemia NIH 3T3, HeLa 94Measles Jurkat CD46Poliovirus Human mononuclear CD14 Sindbis Mouse neuronal ND HIV MT-4 CD4

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Analysis of the effect of virus infection on cell surface proteins

Pseudorabies L-929; Neuro 2A MHC class I expression decreased

HSV Mouse embryo fibroblasts MHC class I expression decreased

CMV Human foreskin fibroblasts MHC class I expression decreased

HSV Endothelial GMP140 expression increased

HIV T cells CD4 expression decreased

EBV B cells Bcl-2 expression decreased

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Virus-cell interactions leading to apoptosis

Virus Apoptotic cells

HIV EBV transformed, lymphoblastoid HIV CD34+ bone marrow, progenitor HIV CD8+ T, CD19+ B HIV gpl60 expressing CD4+ cells HIV PBMC HIV CD4+ Simian immuno-deficiency PBMCEBV, varicella-zoster CD 45+Influenza HeLa LCMV Murine T

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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

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LECTINS Membrane oligosaccharides

Cell wall compositionMicrobe 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

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ProtozoaFlagellakcsLeishmania chagasi Tritrichomonas foetus Trypanosoma congolense AmoebaeEntamoeba histolytica CiliatesTetrahymena thermophila SporozoansEimeria tenellaAlgaeDiatoms (BacillariophyeeaPhaeodactylum tricornotum DinoflagellaGonyaulax polyedra VirusesEseherichia 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!

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67FITC Fluorescence

Mo1

CD4 CD8

CD8

CD45

leu11a

CD20 Tube

ID