Snmek 1

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CELL MEDIATED IMMUNITY LABORATORY EVALUATION
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DEFICIENCIES - PRIMARY (inherited) -
SECONDARY (acquired) - infections
(HIV) - malnutrition
- trauma - cancer
- therapy (radiation, cytostatics, steroids)
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PRESENTATION repeated severe infections of lung,
skin, GIT, etc. caused by viral
agents intracellular bacteria
(Mycobacterium) opportunistic
infections (Candida
sp., Pneumocystis carinii)
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DIAGNOSTIC PROCEDURES are complex
patients and family history
clinical evaluation
laboratory tests
1st level leukocytes, differential cell count
2nd level
immunological testing
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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)
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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
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EX VIVO LABORATORY TESTS
enumeration of various
populations of immune
cells functional
tests
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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
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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)
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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
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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
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• 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.

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• 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

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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
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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)
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CELL SCATTERGRAM OF PERIPHERAL BLOOD
granulocytes
FS
monocytes
lymphocytes
debris
SS
CD3-FITC
CD3-FITC
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DETERMINATION OF CD8 T CELLS
DETERMINATION OF CD4 T CELLS
CD8-PE
CD4-PE
CD3-FITC
CD3-FITC
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SARCOIDOSIS - BALF
55 lymphocytes
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92 CD3 T-lymphocytes
SARCOIDOSIS - BALF
8 CD8 T-lymphocytes
85 CD4 T-lymphocytes
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PNEUMONIA - BALF
98 neutrophils
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PNEUMONIA - BALF
4 CD19 lymphocytes
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• 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

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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)
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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.
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LYMPHOCYTE PROLIFERATION IN VITRO
lymphocytes in complete medium
mitogen (phytohem- agglutinin)
3H ( )- thymidine
3H - thymidine uptake
96-wells panel
well
incubation 18h/37C CO2 atmosphere
incubation 72h/37C CO2 atmosphere
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CELL HARVEST (INCORPORATED RADIOACTIVITY)
DNA RADIOACTIVITY MEASUREMENT (cpm)
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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)
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• 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

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• EVALUATION OF PHAGOCYTOSIS
• absolute number of granulocytes (PMNL)
• is limiting for succesful phagocytosis
• four steps priming, activation, adhesion
• chemotaxis (oriented
  movement)
• ingestion
• killing, degradation

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PRIMING, ACTIVATION, ADHESION OF
GRANULOCYTES
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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
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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
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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
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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

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• CHEMOTAXIS
• oriented movement of granulocytes in the
  gradient of chemoattractants
• LABORATORY TEST
• migration of granulocytes under agarose layer
• migration of granulocytes in Boydens chamber
• DEFECTS OF CHEMOTAXIS
• primary defects lazy leukocyte syndrome
• - secondary defects diabetes, juvenile
  periodontitis

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• 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
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• INTRACELLULAR KILLING
• O2 - independent mechanisms
• immunochemical measurement of defensins
• determinantion of lysosyme
• INHERITED DEFECTS
• deficiency of specific granules

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• 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