Immunological laboratory
investigation
CELLULAR METHODS
• Course no. 2
DIFFERENTIAL BLOOD COUNT
Differential blood count gives relative percentage of each type of white blood cell and also
helps reveal abnormal white blood cell populations
infant childs adults
LEUKOCYTES 9 – 15 x 109/l 8 – 12 x 109/l 4 – 9 x 109/l
GRANULOCYTES % % %
neutrophil granulocyte 25 - 65 35 - 70 55 - 70
• segmented cells („segs“) 22 - 65 25 - 65 50 - 70
• bands 0 - 10 0 - 10 3 - 5
eosinophil granulocyte 1 - 7 1 - 5 2 - 4
basophil granulocyte 0 - 2 0 - 1 0 - 1
MONONUCLEAR LEUKOCYTES % % %
lymphocytes 20 - 70 25 - 50 25 – 40
monocytes 7 - 20 1 - 6 2 - 6
proliferation tests
cytotoxicity tests
flow cytometry
ELISPOT
U s i n g o f s e p a r a t e d
PBMCs
CD classification system
(Paris 1982)
CD markers (CD = cluster of differentiation or
cluster of designation)
• is a protocol used for the identification and investigation of cell
surface molecules providing targets for immunophenotyping of cells
• CD for humans is numbered up to 371 (April 2016)
(9th International Conference on Human Leukocyte Differentiation Antigens (HLDA9),
March 2010)
• Usage in clinical practice: investigation of absolute number and
percentage of cell subpopulations by flow cytometry (T cells, B cells
and their subpopulation, NK cells)
• Blood sample: anticoagulant-treated blood (EDTA)
LYMPHOCYTE
SUBPOPULATIONS
CD MARKERS
PERCENTAGE FROM
LYMPHOCYTES
T lymphocytes CD3+ 58 – 85 %
Th lymfocyty CD3+CD4+ 30 – 60 % /CD3+
Tc lymfocyty CD3+CD8+ 15 – 35 % /CD3+
B lymphocytes CD19+ 7 – 23 %
NK cells CD16+/56+ 6 – 20 %
SURVEY OF CELLULAR
IMMUNOLOGICAL INVESTIGATION
We can performed following immunological
cellular investigation:
• percentage and absolute counts of cells of
immune system
• The first step is to find out number of leukocytes and differential
blood counts (percentage and absolute counts of lymphocytes,
monocytes and granulocytes)
• immune cell function
Investigation of percentage and
absolute counts of lymphocyte
subpopulation
FLOW CYTOMETRY
• principle: direct immunofluorescence
• Cells are incubated with secondary antibody
conjugated with fluorescent dye and they are
directed against CD markers of cell surface
• suspending cells in a stream of fluid and passing
them through an electronic detection apparatus
FLOW CYTOMETRY
• cells are differentiated according to size and granularity:
o LYMPHOCYTE, MONOCYTES and GRANULOCYTES
• Cells are differentiate according to cell surface markers:
o Subpopulations of T-LYMPHOCYTES, BLYMPHOCYTES
and NK CELLS
Routinely usage of flow cytometry:
• diagnostic of primary and secondary immunodeficiencies
• diagnostic of hematological malignancies
Investigation of percentage and
absolute counts of lymphocyte
subpopulation
GRANULARITY(SS)
SIZE (FS)
LYMPHOCYTES
MONOCYTES
GRANULOCYTES
principle of the investigation
• PRESTIMULATION by IL-3
• ACTIVATION
– positive control – fMLP
– negative control – negative control from prick tests
– investigated sample – investigated allergen
• STOP OF DEGRANULATION on ice
• LABELING by anti-CD45, anti-IgE, anti-CD63, anti-CD203c
• LYSIS
• ANALYSIS by flow cytometry
BASOPHIL ACTIVATION TEST
CD45
CD45
panleukocyte marker
FcεRI
high-affinity IgE receptor
CD 203c
Cell surface antigen of human
basophils E-NPP3
CD 63
glykoprotein on cell surface of
lysozyme (gp 53)
cell culture is the process by which cells are grown
under controlled conditions, generally outside their
natural environment
the stimulation of lymphocytes by antigens or
mitogens, rendering them metabolically active
and causing them to differentiate into effector cells
evaluation of function of the cells
Lymphocyte cultivation in vitro
Functional investigation of lymphocyte
proliferation
PROLIFERATION OF LYMPHOCYTES
physiological proces during cell activation
• Lymphocytes activated by
• polyclonal mitogens (non-specific stimuli)
• for B-lymphocytes
• pokeweed mitogen (PWM)
• for T-lymphocytes
• phytohemagglutinin (PHA)
• concanavalin A (ConA)
• Antigens (specific stimuli)
• tuberculin
• tetanic toxoid
• widely used proliferation assay is the 3H
thymidine uptake assay
• cells incorporate the radiolabeled nucleotide into
newly synthesized DNA
• in this way, the level of radioactivity as measured
by liquid scintillation gives a relative measure of
cellular proliferation
3H Thymidine Proliferation Assay
Functional investigation of lymphocyte
proliferation in vitro
PROLIFERATION OF LYMPHOCYTES
one of the physiological signs of cellular activation
• isolation of lymphocytes from peripheral blood
• cultivation of lymphocytes with mitogens
tetanic toxoid – 7 days
PHA, PWM – 3 days
• Adding of 3H-thimidin day before end of proliferation
(2nd day or 6th day) – incorporation of thymidine into DNA
of proliferating cells)
• stop of proliferation by freezing
• detection of radioactivity by beta-counter
• result – stimulation index (SI)
• cpm (counts per minute) experimental/ cpm background
unstimulated
• Positivity: SI  5 for antigens, SI  100 for mitogens
Functional investigation of lymphocyte
proliferation in vitro
PROLIFERATION OF LYMPHOCYTES
one of the physiological signs of cellular activation
When investigate proliferation of lymphocytes?
diagnostic of severe immunodeficiencies
(SCID)
Functional investigation of lymphocyte
subpopulations in vitro
further specialized functional investigation
• measurement of production and release of important
cytokines and therefore functional investigation od T-cell
subpopulations (Th1 and Th2)
• ELISA, ELISPOT, PCR
• measurement of cell surface molecule expression, which are
necessary for cell synapsis, or activation markers of cells
• Flow cytometry
• measurement of B lymphocyte immunoglobulin production
• ELISA, ELISPOT
Functional investigation of T cells in vivo
A TUBERCULIN SKIN TEST
is done to see if you have ever been exposed to tuberculosis
(IV. type of hypersenzitivity reactions)
it is called also Mantoux tuberculin test
• the test is done by putting a small amount of TB
protein (antigens) under the top layer of skin on
your inner forearm
• if the patient has ever been exposed to the TB
bacteria (Mycobacterium tuberculosis), his skin
will react to the antigens by developing a firm red
bump at the site within 2 days (24–48 hours)
Functional investigation of T cells in vivo
A TUBERCULIN SKIN TEST
is done to see if you have ever been exposed to tuberculosis
(IV. type of hypersenzitivity reactions)
it is called also Mantoux tuberculin test
• a measured amount of PPD in a shot is put under
the top layer of skin on forearm
• this is a good test for finding a TB infection
• it is often used when symptoms, screening, or
testing, such as a chest X-ray, show that a
person may have TB
A tuberculin skin test cannot tell how long you have been
infected with TB. It also cannot tell if the infection is latent
(inactive) or is active and can be passed to others.
Functional investigation of T cells in vivo
A TUBERCULIN SKIN TEST
is done to see if you have ever been exposed to tuberculosis
(IV. type of hypersenzitivity reactions)
it is called also Mantoux tuberculin test
Test is positive
induration 6–15 mm
• normal response in sensitized person
induration > 15 mm (in children < 5 years < 10 mm)
• indication to chest X-ray
Test is negative
Induration < 6 mm
• patient was not sensitized before
• impaired responsiveness of patient T cells
Functional investigation of T cells in vivo
A TUBERCULIN SKIN TEST
Why is it done?
A tuberculin skin test is done to find people who have
tuberculosis (TB), including:
• people who have been in close contact with someone known
to have TB
• health care workers who are likely to be exposed to TB
• people with TB symptoms, such as an ongoing cough, night
sweats, and unexplained weight loss
• people who have had an abnormal chest X-ray
• people who have had a recent organ transplant or have an
impaired immune system, such as those with human
immunodeficiency virus (HIV).
Functional investigation of T cells in
vivo
CELL MEDIATED IMMUNITY TEST
(CMI test)
• Intradermal application of anamnestic antigens
(tuberculin, candidin, toxoplasmin, tetanus toxoid,
antigens of staphylococci, streptococci, etc.)
• No induration after application of antigen
after 48 hours  impaired T-lymphocyte
responsiveness (patient is anergic)
Determination of number of cells capable of phagocytosis
• number of neutrophil granulocytes
differential blood count
• determination of specific cell surface markers of
granulocytes and monocytes
CD15 for neutrophil granulocytes
CD14 for monocytes
markedly or repeated descrease is indication to investigate phagocyte
functions
number of phagocyte cells
Investigation of phagocyte functions
NBT test
chemiluminiscence
burst test
defects in chronic granulomatous disease
transient defects in infections, traumas and malnutrition
Investigation of phagocyte functions
INVESTIGATION OF RESPIRATORY BURST
NBT test
(nitro blue tetrazolium chloride test)
nitro blue tetrazolium is a chemical compound composed of
two tetrazole moieties. It is used in immunology for sensitive detection
of alkaline phosphatase (with BCIP). NBT serves as the oxidant and BCIP is the
AP-substrate (and gives also dark blue dye)
reduction of colorless nitro blue tetrazolium into colorful
formazan
Investigation of phagocyte functions
INVESTIGATION OF RESPIRATORY BURST
BURST TEST
• heparinized whole blood is incubated at 37 °C with phorbol myristate
acetate (PMA), a compound known to stimulate oxidative burst aktivity,
each flow cytometry pattern is referenced to the patients non-stimulated
cells; in addition, a control blood is included in each run
• upon stimulation, granulocytes and monocytes produce reactive oxygen
metabolites (superoxide anion, hydrogen peroxide, hypochlorous acid)
which destroy bacteria inside the phagosome
formation of the reactive oxidants during the oxidative burst can be
monitored by the addition and enzymatic oxidation of
a fluorogenic substrate, DHR 123
the level of reactive oxygen radicals is determined by flow cytometry
Investigation of phagocyte functions
INVESTIGATION OF RESPIRATORY BURST