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LABORATORY DIAGNOSTIC
Subjects: Immunology
Study programme: General Medicine
Academic year: 2015/2016
RNDr. Mira Horváthová, PhD.
Department of Clinical Immunology and Allergology
Faculty of Medicine SMU in Bratislava
Department of Immunolgy
Laboratory of Cellular Immunology
Flow Cytometry Laboratory
Immunochemistry Laboratory
Tissue Culture Laboratory
Advances in diagnostic immunology
are largely driven by instrumentation, automation,
and the implementation of less complex and more
standardized procedures.
Miniaturization
Amplified immunoassays
Flow cytometry
Molecular methods
Assumptions of rational diagnostics
• The broad spectrum of immunoassay methods
• Knowledge of the clinical value of immunological tests
• Rational indication of immunological tests
• A comprehensive assessment of results with clinical
conditions
Determination of Immunological Parameters
• Acute stage of the disease
• Screening
• Treatment monitoring
• Autoimmunity
• Normal function
• Inflammation
• Deficiency or functional
• Hypersensitivity
• Allergy
insufficiency
• Hyperactivity or
inappropriate activity
Examination of cellcell-mediated immunity
• Analysis of living cells
• Correct collection to tube with
anticoagulants - EDTA, heparin
• Sample storage at room
temperature
• Fast transport of material to the
laboratory
• Blood
• BAL
• Cerebrospinal fluid
• Synovial fluid
Examination of humoral immunity
• Serological assays
• Sample storage at
−80°C, −20°C
• Serum
• Plasma
Cellular immune mechanisms
• Leukocyte count and
differential leukogram
• The functional activity of
T and B cells, NK cells
• Examination of
lymphocyte
subpopulations
• Neutrophil function,
basophils, eosinophils,
monocytes / macrophages
• Expression of adhesion
molecules
Humoral immune mechanism
• Detection of plasma proteins
• Immunoglobulins IgG, IgM, IgA, IgE
• Acute-phase proteins (CRP, alfa-1-antitrypsin, alfa-2macroglobulin, orosomucoid, haptoglobin, ceruloplasmin)
• Complement system
• Cryoglobulins
• Immune complexes
• Specific antibodies IgE, IgA, IgG
• Autoantibodies (ANA, anti-ds-DNA, anti-DNP, ANCA, AMA,...)
Laboratory Information System
A laboratory information system (LIS) is a software system that
records, manages, and stores data for clinical laboratories.
Functions of LIS
patient management, including admission date, admitting
physician, ordering department, specimen type, etc.
patient data tracking
test ordering
quality assurance
workload and management reporting
workflow management
billing
Principles of immunological assays
- serological and cellular methods
ANTIGEN – ANTIBODY
INTERACTION
Laboratory Methods of Clinical Immunology
White Blood Cell Counts
and Differential
Microscopy – Hematological
Blood smear
analyzer
Flow
cytometry
HEMATOLOGICAL ANALYSIS
COMPLETE BLOOD COUNT (CBC)
WHITE BLOOD CELL (WBC) DIFFERENTIAL
HEMATOLOGICAL ANALYSIS
HAEMATOLOGICAL SYSTEM
PARAMETERS
Laboratory Methods of Clinical Immunology
Lymphocyte Subpopulations
E-rosette test
Isolation of
Lymphocytes
Fluorscence
Microscopy
Flow
Cytometry
Isolation of
Lymphocytes
FLOW CYTOMETRY
FLOW CYTOMETRY
Hydrodynamic focusing
Identification of immune cell subsets
CD45+
Leukocytes
CD3+
CD3+CD4+ (Th)
CD3+CD8+ (Tc)
T-lymphocytes
CD19+
B-lymphocytes
CD14
monocytes/macrophages
CD3+HLADR+
activated T-Ly
CD3-CD(56+16)+
NK cells
Surface adhesion molecules
Immunoglobulin superfamilly
ICAM-1/CD54
ICAM-2/CD102
ICAM-3/CD50
VCAM-1/CD106
Selectins
E-selectin/CD62E
P-selectin/CD62P
L-selectin/CD62L
Integrins
LFA-1...CD11a/CD18
VLA-4...CD49d/CD29
Importance of Immunophenotyping
• Diagnosis and classification of primary and secondary
immunodeficiency
• Classification of leukemias and lymphomas
• Monitoring of immunotherapy and chemotherapy in
immunodeficient conditions and malignant diseases
• Diagnosis and monitoring of autoimmune diseases
• Monitoring of immunity after organ transplantation
• Assessment of immune status in HIV infection
Laboratory Methods of Clinical Immunology
Function tests of Lymphocytes and NK cells
T lymphocyte
activation
B lymphocyte
function
Lymphocyte
Transformation Testing
- LTT
Secretion of
Immunoglobulins
NK cell function
Cytotoxicity Assay
The importance of lymphocyte function tests
• Diagnostic and monitoring of the immunological profile of
primary and secondary immunodeficiency
• Determination of lymphocyte immunocompetence and
lymphocyte subpopulations disorders
• Monitoring of T lymphocytes immunoregulatory role
• Immunomodulatory therapy monitoring and study of the drugs
influence
• The detection of hypersensitivity to a specific antigen (LTT –
drugs – type IV allergy)
Laboratory Methods of Clinical Immunology
Function tests of Granulocytes
Phagocytic
activity %
Phagocytic
index
Microscopy
Flow Cytometry
Oxidative burst
Chemiluminiscence
Flow Cytometry
Cytotoxic
activity
Microscopy
Flow Cytometry
DETECTION OF PHAGOCYTOSIS
Phagocytosis – tests in vitro – measurement of ingested
material by phagocytic cell (Mo/Ma, Ne, Eo)
Substrate: erythrocytes, E.coli, zymozan (yeast), various
particles
Quantification: colorimetric ELISA method or by
microscopy or flow cytometry
The importance of granulocyte function tests
Diagnostics of primary / secondary deficiencies in phagocytosis
Monitoring of functional and metabolic disorders of phagocytes
TISSUE CULTURE
• Neutralization tests (e.g. viruses)
• Cytotoxicity assays (Tc, NK)
Cytokines secretion by T cells - ELISPOT
Cross--match
Cross
TEST determine compatibility between a donor and recipient in organ
transplantation - recipient serum is tested against donor cells
Immunochemical methods
Antigen-antibodies reactions,
Serological methods, Precipitation methods,
Enzyme Immunomethods, Immunoanalysis
with various particles, Electrophoresis methods,
Immunoafinity methods,...
Serological reaction
Ag + Ab
Specific
Non-specific
Qualitative
Quantitative
Methods for AgAg-Ab detection
PRECIPITATION REACTIONS
GEL ELECTROPHORESIS
The separation of DNA, RNA or proteins
mixtures according to molecular size
ELECTROPHORESIS
Vertical
Electrophoresis
System
Horizontal
Electrophoresis
System
Separácia proteínov na polyakrylovom géle
Autoradiographic
detection
WESTERN BLOT
Fluorescent
detection
Transfer of proteins onto nitrocellulose
COMPLEMENT FIXATION TEST
EXAMINATION OF COMPLEMENT HAEMOLYTIC
ACTIVITY
Functional test of the classical or alternative complement pathway
ELISA
Equipment for ELISA methods
FLUORESCENT TISSUE STAINING
Detection of Autoantibodies
Fluorescence microscope
free antigen
bound antigen
NEPHELOMETRY
immunoassay system for the
quantitative determination of human
proteins in serum
disease state diagnosis and
monitoring
Determination of basic protein in serum, immunoglobulins IgG,
IgA, IgM, IgE, and IgG subclasses, complement components C3
and C4, the acute phase protein (e.g. C-reactive protein,
transferrin, alpha-1-macroglobulin, albumin).
DIAGNOSTIC TESTS
FOR type IV ALLERGY
Mitogen- and Antigen-induced
Lymphocyte Proliferation (LTT)
Clinical Significance
Measurement of human lymphocytes' proliferative responses to
various stimuli is a fundamental technique used to assess their
biological status and functions.
Mitogens, such as plant lectins phytohemagglutinin (PHA),
concanavalin A (Con A) and pokeweed mitogen (PWM), are
able to nonspecifically stimulate lymphocyte proliferation and
used to evaluate patient immune responsiveness.
DETERMINATION OF sIgE
ANTIBODIES AGAINST ALLERGEN
Basic principle - Enzyme Immunoassay
A variety of the antigen (allegen) binding methods
and detection systems
DETERMINATION OF sIgE
Fully automatic system
HYTEC Specific IgE Enzyme Immunoassay
Allergen disc
Allergen-specific IgE from the patient’s serum
Enzyme labelled anti-IgE
Substrate solution
Spectrophotometric measurement
Principle of sIgE detection - chemiluminiscence
BASOPHILS ACTIVATION TESTS
DETECTION OF THE MEDIATORS
PROTEIN MICROARRAY
Microarray technology
The Immunoassay
Handbook
Theory and applications of
ligand bunding, ELISA and
related techniques,
D. Wild, 2013
Clinical Laboratory
Immunology,
C. R. Mahon and D. Tice,
2006
Clinical Immunology,
Principles and Laboratory
Diagnosis,
C. Sheehan, 1996