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Methods for analysis of cell mediated immunity
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Methods
• 1.Flow cytometry
• 2.Functional tests of lymphocytes
• 3.Functional tests of phagocyting cells
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What is flow cytometry/ FACS ?
• Flow ~ cells in motion,
Cyto ~ cell, Metry ~ measure
• FACS (fluorescent analysed cell sorting)
• measuring various properties of cells or particles (e.g. synthetic beads with surface bound antibody to detect
cytokines) while in a fluid
stream (biological, chemical,
physical)
(pH, membrane potential, size,
granularity, viability etc.)
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Flow cytometry• Measurement of several parameters at the same time:
- number of cells
- size (FSC)
- granularity (SSC) of cells
- fluorescent signal (FL) (2 or
multiple depending on number of lasers)
• staining of cells with mononuclear antibodies against:
cell surface molecules
cytoplasmatic molecules
nuclear molecules
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• Material: whole blood, bioptic samples of bone
marrow, separated cell subpopulations,
or other cell suspensions obtained by tissue desintegration
• Immunofluorescent staining of cells:
Direct or indirect
Flow cytometry
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Cell staining in flow cytometry
excitation (laser)
emission fluorescenc
e
emission fluorescen
ce
Cell surface marker
Fluorochrome- labelled mAb
Direct staining
Indirect staining
purified/
biotinylated mAb
Fluorochrome-labelled secundar Ab/streptavidin
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List of common fluorochromes used in flow cytometry
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Principle of flow cytometry• One-by-one stream of cells moves rapidly through
the flow cytometer
• Cells pass through a focused beam of light from a laser– Photons scattered in all directions
• Photodetectors capture scattered light and generate digital signals to define cellular characteristics– Size, internal complexity, antigen makeup
• Information stored and analyzed by computer
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LASER
stained cells
Detectors of fluorescence APC
Optical system in flow
cytometer
PE
FITC
PE-Cy7
SSC
FSC
PC analysis
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Typical Dot plot of cell subpopulations in blood samples and double staining
Lympho gate
Dot plot graphs1 dot/event= 1 cell
Single parameter histogram
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Phenotypisation of lymphocytes
• CD3+ T lymphocytes (50-75%)
• CD3+/CD4+ Th lymphocytes (52-69%)
• CD3+/CD8+ cytotoxic T lymphocytes (27-46%)
• CD3+/CD16+/CD56+ NKT lymphocytes ( only
0.2%)
• CD16+/CD56+ NK cells (4-18%)
• CD19+ B lymphocytes (7-18%)
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Application of cytometric analysis
• phenotypisation of cells (diagnostic of primar immunodeficiency, autoimmune diseases, leukemie and lymphoms, etc.)
• functional tests of leukocytes and thrombocytes (proliferating
activity – measurement of DNA content)
• Evaluation of spermatogenesis, detection of viruses,
bacteries and parasites, analysis of chromosomes,
assessment of enzymatic activity, measurement of
intracellular calcium
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Functional tests of lymphocytes
• Proliferation
• Expression of activated markers
• Cytotoxicity
• Cytokine secretion
• Production of antibodies
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Isolation of lymphocytes from blood
• gradient centrifugation (cell separation
according to differences in their density)
Diluted plasma
Separative solution
Lymphocytes
ErythrocytesGranulocytes
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• Important for the process of immune reactions
• Diagnostics of innate immunodeficiency
• Activation of T cell receptor (TCR) → activation of intracellular signal cascade → signal transduction into nucleus → transcription of genes for proliferation
Proliferation of lymphocytes
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Blastic transformation test• Ability of T lymphocytes to response on polyclonal stimuli
3H- tymidin test
• Cultivation of isolated lymphocytes in medium with
stimulators (3-7 days/37°C) incubation with radioactive
stained tymidin (3H) incorporation of 3H- tymidin into DNA of
proliferative lymphocytes detection of -radiation (-
counter)
• more proliferation, more measuring radioactivity
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Activation of lymphocytes
CD69
CD4
Assessment of expression of specific activated markers
Early activated markers(CD69)
Late activated markers (CD25, CD71 )
→ measured by flow cytometry
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Intensity of fluorescence
(DNA content)
• DNA ploidity
• Analysis of cell cycle
• DNA fluorochrome binding
(Propidiumiodid, akridin
orange)
• Intensity of fluorescence
directly proportional to DNA
content in cell
• G2/M phase- proliferative
phase,i.e. % of prolife.cells
Cytometric DNA analysis
Count ofcells
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Cytotoxicity
→ Ability of T cytotoxic lymphocytes and NK cells to kill transformed/tumor or virus infected cells
Different mechanisms:
Fas-FasL
Perforines, granzines
TNF- TNFR
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Cytotoxic tests
• test based on release of radioactive labelled
chrom (51Cr) from tumor cells
• Vital staining of tumor cells microscopic or
cytometric analysis
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Performing of test with 51Cr
+
isoloted lymphocytes
Tumor cells incubated with 51Cr
incubation (37°C/3,5h)
Detection of -radiation in supernatant
51Cr
Calculation of % cytotoxic activity
100x (activity of sample-SPON)/(MAX-SPON)
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Measurement of cytokine secretion
• ELISA
• intracelullar assessement by flow cytometry
• ELISPOT
detection and quantification of T lymphocytes
reacting on antigen stimulus by secretion of specific
cytokines
Number of spots in well = number of
cells secreting cytokines
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ELISPOTprimar Ab against cytokine
T lycytokine
secundar Ab labelled by biotin
streptavidin-enzym
substrate
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Application of functional tests of lymphocytes
• Diagnostic of SCID
• Diagnostic and prognostic of malignant tumours
• Monitoring of cellular immunity (secundar immunodeficiency, sepsi, post-operative state)
• Monitoring of development of graft after transplantation of bone marrow
• Testing of new drugs (pharmacology, anti-cancer immunotherapy)
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Functional tests of phagocytic cells
• Phagocytosis
• Testing of oxidative burst
• Determination of adhesive molecules expression
• Testing of chemotaxis
• Bactericidal test
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Phagocytosis• Phagocytic cells: Neutrophils, monocytes/macrophages• Target for phagocytosis: bacteria, cellular debris• Phases of phagocytosis:
Diapedesis- chemotaxis- recognition- ingestion- killing and destruction of target particles- antigen presentation
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Examination of ingestion phase (engulfment of microorganism)
• substrate Saccharomyces cerevisiae, Candida
albicans, polymer particules
• Suspension of particules or yeasts + blood
(incubation 37°C/1h) making of blood smear
fixation and staining reading in light microscope
• Positive cells- 3 and more engulfed
particles
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• Phagocyting activity-% phagocytes with
absorbed particles from all phagocyting cells
• Another kind of examination: flow cytometry- fluorescent labelled particles
Examination of ingestion phase (Engulfment of microorganism)
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Testing of oxidative burst
• Examination of phagocyte`s ability to build 02 radicals
(activation of NADPH oxidase)
Measurement by flow cytometry
• DHR-123 test: Full blood + phorbol esters +
dihydrorhodamin rhodamin (effect of 02 radicales
measurement of fluorescenct intensity
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• NBT (nitroblue-tetrazolium chlorid) and INT (iod-
nitroblue tetrazolium chlorid) tests• Ability to reduce colourless tetrazolium
salts (result of 02 radicals)
• Full blood + amyloid grains + colourless liquid of NBT or INT colourful formazan (02 radicals) microscopic or spectrophotometric analysis
Testing of oxidative burst
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Bactericidal test
• Assessment of phase of killing the engulfed particle
• substrate Staphylococus aureus, Candida albicans
• Incubation of blood together with microorganism (37°C/1hod)
Analysis:
• microscopic (vital staining with trypan blue)
• cytometric (vital staining with PI, Hoechst)
• Inoculation on plates (counting of colonies)
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Activation of basophils by alergens• Anafylactic degranulation of basophils – fast morphological changes,
exocytosis of IC granules and release of modified mediators• „Piecemeal“ degranulation – slow morphological changes without
degranulation• CD63, CD203c, CRTH2-FITC /CD203c-PE/CD3-PC7• CD69, Cd107a, CD123, CD 164, basogranulin and etc.• Detection of mediators and enzymes
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Baso study in insect allergy
• Diagnostic– Examination of double positivity (CCD)
• Biomarker of anaphylaxis– ↑ expression of CD69 –exposition in vitro and
in vivo
• Monitoring of immunotherapy– Change in reactivity– Prediction of undesirable effects
• Control of effect – exposure test, persistence of treatment effectivity
• Research of alergens
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• Diagnostic of primar immunodeficiency
(LAD-1, LAD-2, chronic granulomatosis)
• Testing of new drugs
• Anticancer immunotherapy (test of DC maturity)
Application of functional tests of phagocytic cells