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CELL MEDIATED IMMUNITY LABORATORY EVALUATION. DEFICIENCIES: - PRIMARY (inherited) - SECONDARY (acquired) - infections (HIV) - malnutrition - trauma - cancer

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CELL

MEDIATED

IMMUNITY

LABORATORY EVALUATION


DEFICIENCIES:

- PRIMARY(inherited)

- SECONDARY(acquired)

- infections (HIV)

- malnutrition

- trauma

- cancer

- therapy (radiation, cytostatics, steroids)


PRESENTATION:

repeated severe infections of lung, skin, GIT, etc.

caused by

viral agents

intracellular bacteria (Mycobacterium)

opportunistic infections

(Candida sp., Pneumocystis carinii)


DIAGNOSTIC PROCEDURES:

are complex:

patient’s and family history

clinical evaluation

laboratory tests:

1st level: leukocytes, differential cell count

2nd level: immunological testing


SKIN TEST IN VIVO:

intradermal administration of anamnestic

T-dependent antigens of microbial origin

anamnestic Ag: previous contact is supposed

natural exposition: candidin, toxoplasmin

arteficial exposition: active immunisation

(tetanus toxoid, PPD-purified proteine

derivative of tuberculin, Mantoux reaction)


RESULTS:

induration after 24 and 48 hours is read

(DTH reaction)

CD4+ T cells and macrophages infiltration

(interferon )

INTERPRETATION:

- positive: intact T cell immunity

- negative: anergy


EX VIVO LABORATORY TESTS

enumeration of various populations

of immune cells

functional tests


ENUMERATION OF IMMUNE CELLS

cytomorphological criteria are not sufficient

identification distinct cell population by

the detection of specific surface molecules

detection is based on specific immunochemical

reactions between monoclonal antibodies and their

targets (antigens)

CD molecules: fully characterized

leukocyte molecule


IMMUNOFLUORESCENCE ANALYSIS (IFA)

antibody is labeled by fluorochromes

Fluorochromes: - FITC :

emission at 450 nm (green)

- PE (phycoerythrin):

emission at 580 nm (red)

UV

ABSORPTION  FLUOROCHROME  EMISSION

laser (excitation) (light)


IMMUNOFLUORESCENCE ANALYSIS

DIRECT IMMUNOFLUORESCENCE ANALYSIS:

- primary antibody is labeled

- whole blood

- flow cytometry

INDIRECT IMMUNOFLUORESCENCE ANALYSIS:

- primary antibody is unlabeled

- isolated cells, tissues

- UV microscopy


DIRECT DOUBLE IMMUNOFLUORESCENCE

TO DETECT CD3+ CD4+ T CELLS

INDIRECT IMMUNOFLUORESCENCE

TO DETECT CD3+ T CELLS

Y

Y

ANTI CD3 FITC Mo Ab

Y

ANTI CD4 PE Mo Ab

MOUSE ANTI CD3 Mo Ab

INCUBATION

RED CELLS LYSIS

WASHING

FLOW CYTOMETRY

Y

secondary Ab RaAMIgFITC

INCUBATION

INCUBATION

RED CELLS LYSIS

WASHING

FITC

WASHING

Y

Y

FITC

Y

CD3

FITC

Y

Y

Y

Y

TS/C

CD3

CD3

FITC

CD3

CD3

TS/C

Y

TH

TH

TH

CD8

CD3

CD8

TS/C

CD4

Y

B

B

CD4

CD4

CD8

CD19

CD19

PE

B

CD19

FLOW CYTOMETRY


separation

fluid

(1,076 g/ml)


plasma

LYMPHOCYTES

separation

fluid

ERYTHROCYTES

GRANULOCYTES


FLOW CYTOMETRY

the most modern approach of cell analysis

cells are directed in sheath flow of fluid

CYTOMETRY DETERMINES:

- number of cells

- size of cells (FS parameter)

- morphology of cells (SS)

- the presence of fluorescence signal (FL)

data are processed in PC

MATERIAL EXAMINED:

- whole blood, bone marrow

- isolated cell suspensions

- other body fluids (liquor, ascites, exudates)

- cell suspension obtained by tissue desintegration

CELLS ARE LABELED BY IMMUNOFLUORESCENCE

(either direct or indirect)

monoclonal antibodies are directed against

- surface molecules

- cytoplasmatic molecules

- nuclear molecules


  • IMMUNOFLUORESCENCE ANALYSIS

  • OF THE WHOLE BLOOD

  • cell separation is ommited

  • direct immunofluorescence

  • heparinized blood is incubated with fluorochrome

  • labeled monoclonal antibody (FITC, PE)

  • hypotonic lysis of erythrocytes

  • removal of unbound monoclonal antibodies by washing

  • measurement by flow cytometry

  • Simultaneous determinantion of two (three) different molecules

  • is allowed by the application of two different monoclonals labeled

  • with different fluorochromes.


  • APPLICATION OF FLOW CYTOMETRY

  • IN CLINICAL MEDICINE

  • determination of different populations of leukocytes (other cell types)

  • immunophenotyping analysis of blood malignancies

  • determination of proliferating activity of cells:

  • - DNA content is estimated

  • - DNA is stained by intercalar chemicals which are fluorescent

  • determination of O2- dependent mechanisms of killing of phagocytes:

  • - granulocytes are incubated with chemical compound which is non

  • fluorescent

  • - oxygen species which are raised by NADPH oxidase change this

  • compound into fluorescent

  • cell ploidy determination

  • determination of intracellular biologically active molecules:

  • - cytokines (TH1, TH2 subsets)

  • - molecules regulating cell cycle

  • - molecules regulating cell apoptosis

  • - molecules encoded by oncogenes and antioncogenes


THE MOST IMPORTANT POPULATIONS

OF PERIPHERAL BLOOD

CD3+ T cells: population of mature T cells

50 - 75%

CD4+ T cells: subpopulation of helper inducer T cells

30 - 60%

CD8+ T cells: subpopulation of suppressor cytotoxic T cells

15 - 30%

CD25+ T cells: activated mature T cells

1 - 5%

CD19+ B cells: population of mature B cells

5 - 15%

CD56+ NK cells: natural killers

5 - 15%

CD14+ cells: monocytes

CD15+ cells: granulocytes

CD38+ cells: plasma cells


PRINCIPLES OF FLOW CYTOMETRY

LABELED CELLS

SENSORS DETECTING:

NUMBER OF CELLS

CELL SIZE (FS)

CELL „ MORPHOLOGY“(SS)

FL1 – GREEN EMISSION (FITC)

FL2 – RED EMISSION (PE)


CELL SCATTERGRAM OF PERIPHERAL BLOOD

granulocytes

FS

monocytes

lymphocytes

debris

SS

CD3-FITC

CD3-FITC


DETERMINATION OF

CD8+ T CELLS

DETERMINATION OF

CD4+ T CELLS

CD8-PE

CD4-PE

CD3-FITC

CD3-FITC


SARCOIDOSIS - BALF

55%

lymphocytes


SARCOIDOSIS - BALF

92% CD3+

T-lymphocytes

8% CD8+

T-lymphocytes

85% CD4+

T-lymphocytes


1%

lymphocytes

PNEUMONIA - BALF

98%

neutrophils


76% CD15

granulocytes

22% macrophages

PNEUMONIA - BALF

4% CD19+

lymphocytes


  • FUNCTIONS OF LYMPHOCYTES IN VITRO

  • Functional capacity is tested as:

  • capacity of lymphocytes to proliferate

  • production of cytokines

  • Proliferation test

  • in vitro cultivation

  • cultivating media: supplemented by thymic factors

  • by antibiotics

  • buffer system

  • CO2 atmosphere


STIMULATION IS NECESSARY

FOR LYMPHOCYTES PROLIFERATION

ACTIVATORS:

mitogens: - lectins from plants

- polyclonal activators

- specific binding to cell surface sugars

- non-physiological proliferating stimulus

- phytohemaglutinin (PHA)

antigens: - specific stimulation of the single clone

of T cell through TcR

activators of cell kinases (phorbol esters)


CONCLUSION

Cell proliferation is measured by the incorporation

of isotope labeled nucleotides

(3H thymidine) into newly formed DNA.

Functional tests of T cell system

are COMPLEMENTARY to the enumeration of cells.


LYMPHOCYTE PROLIFERATION IN VITRO

lymphocytes

in complete

medium

mitogen

(phytohem-

agglutinin)

3H ( )- thymidine

3H - thymidine

uptake

96-wells panel

well

incubation

18h/37°C

CO2

atmosphere

incubation

72h/37°C

CO2

atmosphere

division

DNA RADIOACTIVITY

MEASUREMENT

(cpm)

CELL HARVEST

(INCORPORATED

RADIOACTIVITY)


DETERMINATION

OF NATURAL CYTOTOXIC ACTIVITY

NATURAL CYTOTOXIC CELLS:

heterogenous population

killing of viral infected and malignant cells

the most important population are NK cells

NK CELLS PHENOTYPE :

CD56+ (NCAM-1)


  • NK CELL ACTIVITY:

  • the lytic activity of NK cells is tested

  • target cell line is labeled by radioactive Cr

  • target cells are mixed with isolated lymphoid cells

  • (NK cells are included)

  • during incubation target cells are lyzed

  • and released

  • Cr is measured

  • radioactivity release is the function

  • of NK cells activity


  • EVALUATION OF PHAGOCYTOSIS

  • absolute number of granulocytes (PMNL)

  • is limiting for succesful phagocytosis

  • four steps: priming, activation, adhesion

  • chemotaxis (oriented movement)

  • ingestion

  • killing, degradation


PRIMING,

ACTIVATION,

ADHESION

OF GRANULOCYTES


IST STEP OF ADHESION, SELECTINS-MEDIATED

collagens

IMMUNOPATHOLOGY

a1 b1

fibronectin

a1 b1

INFECTION

proinflammatory

proadhesive

signals

a3 b1

MALIGNANCY

MACROPHAGE

CD62E

IL-1

TNF

CD34

CD62P

E-CADHERIN

CHEMOATTRACTANTS

CD15

CD62L

PECAM-1

PSGL-1

ENDOTHEL

GRANULOCYTE

rolling


2ND STEP OF ADHESION

firm adhesion

interaction between LFA-1 and ICAM-1

signaling: outside-in

inside-out

cell spreading

3RD STEP OF ADHESION

diapedesis into tissues


4 b1

 b2

II ND STEP OF ADHESION

A C T I V A TION

C-X-C CHEMOKINES

ENDOTHELIA

MACROPHAGES

C-C CHEMOKINES

ICAM-1

VCAM-1

ICAM-1

VLA-4

LFA-1

DIAPEDESIS

EOSINOPHIL

LFA-1

 b2

NEUTROPHIL

3CYTOADHESINS

FIBRINOGEN vWF


LABORATORY EVALUATION

  • presence of adhesion molecules is tested by immunofluorescence

  • LAD-II deficiency syndrome:

  • extremly rare

  • abnormal glycosylation of selectine ligands

  • LAD-I deficiency syndrome:

  • - absence of  chain (CD18) of LFA-1 integrin heterodimers


  • CHEMOTAXIS

  • oriented movement of granulocytes in the gradient of chemoattractants

  • LABORATORY TEST:

  • migration of granulocytes under agarose layer

  • migration of granulocytes in Boyden’s chamber

  • DEFECTS OF CHEMOTAXIS:

  • primary defects: lazy leukocyte syndrome

  • - secondary defects: diabetes, juvenile periodontitis


  • INGESTION

  • „opsonins“ are necessary

  • incubation of heparinized blood with heat inactivated yeasts

  • granulocytes with ingested yeasts are read after staining under microscope

lymphocyte

erythrocyte

yeast

granulocyte with ingested yeasts


  • INTRACELLULAR KILLING

  • O2 - independent mechanisms:

  • immunochemical measurement of defensins

  • determinantion of lysosyme

  • INHERITED DEFECTS

  • deficiency of specific granules


  • INTRACELLULAR KILLING

  • O2 - dependent mechanisms:

  • INT (NBT) - test

  • isolated granulocytes are stimulated with starch granules in the presence of iodnitrotetrazolium salt (colorless)

  • generation of oxigen species (O2-., superoxide anion) by NADPH oxidases is the cause for the formation of formazan (red color) from INT

  • PRIMARY DEFECTS

  • CGD: - defects in NADPH oxidase assembly

  • - no formazan formation

  • SECONDARY DEFECTS

  • Associated with inflammatory response


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