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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