Contributor
Diane Davis, PhD
MLS Immuno 11
Immunology
Medical Laboratory Scientist
I. Basic Concepts
A. Define (Level 1)
1. Natural (innate) immunity
2. Acquired (adaptive) immunity
a) Active immunity
b) Passive immunity
B. State the major components of the natural/innate immune system (Level 1)
1. Physical barriers such as skin, normal flora and mucosal membranes
2. Phagocytic cells and NK cells
3. Chemical barriers such as lytic enzymes and pH
4. Soluble immune mediators such as complement, acute phase reactants, cytokines (especially IL-1)
5. Inflammatory response
a) “Acute phase reaction” changes in serum composition
b) Positive acute phase reactants used as inflammatory markers
(1) C-reactive protein
(2) Procalcitonin
(3) Others
c) Erythrocyte sedimentation rate
C. Describe the components of the acquired/adaptive immune system (Level 1)
1. Lymphoid Tissues
2. Bridge between innate and adaptive: antigen presenting cells (APC)
3. Gene rearrangement in T and B cell development to create antigen receptors
4. Anamnestic, memory, or secondary immune response
D. Describe the origin of immune cells in cell-mediated immunity (Level 1)
1. Lymphocytes
a) Precursor cells in bone marrow and thymus
b) T cells
MLS Immuno 11
(1) CD4+ (T helper or TH/regulatory T or Treg)
(2) CD8+ (T cytotoxic or TC)
c) B cells
2. Macrophages/monocytes
3. Dendritic cells
E. Differentiate cell-mediated immunity from humoral immunity in general terms of mechanisms and efficacy against particular targets, e.g., T cells better against fungi
1. Mediation by CD4+ cells
a) Class II major histocompatibility complex (MHC) restriction
b) Interaction with antigen presenting cells
c) Cytokine activation (especially IL-1, IL-6, TNF-alpha)
2. Effector function of CD8+ cells
a) Class I MHC restriction
b) Stimulation by T helper cells
c) Stimulation by infected/abnormal host cells
d) Cytokine activation
e) Direct cytotoxicity
f) Immune manifestations, i.e., delayed type hypersensitivity, graft rejection
F. Describe antibody-mediated (humoral) immunity (Level 1)
1. B cell development--gene rearrangement to create immunoglobulin
2. Mature B cell
a) Surface immunoglobulin IgM and IgD on naïve mature B cells
b) Isotype switching to IgG, IgA or IgE following activation and T cell help
3. Plasma cell antibody secretion
4. Stimulatory mechanisms through APC and T helper lymphocyte
5. Immunoglobulins
a) Structure
b) Function
G. Discuss features of the antigen molecule that determines immunogenicity (Level 2)
1. Foreignness
2. Molecular size
3. Chemical-structural complexity
4. Epitope
5. Dosage
6. Route and timing of exposure
H. Describe the regulatory functions (Level 2)
1. TH, Treg, and Tc cells
a) Antibody production
b) Cell-mediated immunity
2. B cells
a) Activation
b) Antibody production
I. Discuss monocyte/macrophage functions (Level 1)
1. Phagocytosis
2. Antigen presentation
3. Cytokine production
J. List and define the cytokines and discuss their general functions (Level 1)
1. Interleukins
2. Colony stimulating factors
3. Interferons
4. Tumor necrosis factors
K. Describe immunoglobulin structure and how it relates to function (Level 1)
1. Polypeptide chain
a) Light
(1) Kappa
(2) Lambda
b) Heavy
(1) Alpha
(2) Mu
(3) Delta
(4) Epsilon
(5) Gamma
2. Regions
a) Constant
b) Variable
c) Hypervariable
d) Hinge region and disulfide bonds
3. Fragments
a) Fab
b) Fc
L. List the properties of the immunoglobulin classes, including serum concentration, relative size, valence, ability to cross the placenta, complement activation, affinity/avidity (Level 1)
1. IgG
2. IgA
3. IgM
4. IgD
5. IgE
M. Define (Level 1)
1. Isotype
2. Allotype
3. Idiotype
4. Affinity/avidity
5. Prozone/postzone
6. Titer
7. Polyclonal
8. Monoclonal
N. Discuss the complement system (Level 1)
1. Activation
a) Classic pathway
b) Alternative pathway
c) Lectin pathway
2. Regulation
3. Biologic effects
a) Opsonization
b) Chemotaxis
c) Anaphylatoxin
d) Cytolysis
II. Major histocompatibility complex (MHC)/human leukocyte antigen (HLA)
A. Describe the genetic variability, mechanisms and functions for determining self from non-self.
1. Class I antigens
a) HLA-A
b) HLA-B
c) HLA-C
2. Class II antigens
a) HLA-DR
b) HLA-DP
c) HLA-DQ
B. Discuss the clinical importance for identifying or matching HLA antigens (Level 1)
1. Disease association
2. Paternity testing
3. Transplantation
4. Platelet transfusion
C. Describe the principles for antibody screening and crossmatching procedures for human leukocyte antigens (Level 1)
III. Immunologic techniques used in the clinical immunology laboratory
A. Discuss and describe basic immunoassay principles (Level 1)
1. Precipitation
a) Nephelometric
b) Gel, i.e., radial immunodiffusion
2. Electrophoresis
a) Immunofixation
b) Protein electrophoresis
3. Agglutination
a) Direct
b) Passive
c) Anti-globulin techniques
d) Absorption techniques, e.g., heterophile antibody
e) Agglutination inhibition
4. Flocculation
5. Competitive binding
a) Labels used to detect antigen/antibody reaction
(1) Radioisotopes
(2) Fluorophors
(3) Chemiluminescent molecules
(4) Enzymes
(5) Avidin/biotin
b) Techniques for interpretation
(1) Dose/response curve construction
(2) Cut-off value calculation formats
(a) Homogeneous
(b) Heterogeneous
6. Noncompetitive binding
a) Labels used to detect antigen/antibody reaction
(1) Radioisotopes
(2) Fluorophors
(3) Chemiluminescent molecules
(4) Enzymes
(5) Avidin/Biotin
b) Techniques
(1) Direct and indirect “sandwich” assays
(2) Immunoblotting/Western blotting
(3) Specific IgE, i.e., sIgE, formerly RAST
(4) Antibody capture
c) Dose/response curve construction technique for interpretation
7. Rapid assay/point of care devices
8. Complement fixation
9. Cold agglutinin testing
10. Anti-nuclear antibody testing—microscopic and automated
11. Cytokine detection, such as in vitro T cell reaction to Mycobacterium tuberculosis
12. Calculations
a) Dilutions
b) Titers
c) Cell counts (in flow cytometry)
B. Specify and perform appropriate specimen collection handling (Level 2)
C. Assemble and prepare appropriate materials and equipment for the performance of test procedures above (Level 2)
D. Perform test procedures according to established laboratory protocol (Level 2) and interpret results above (Level 3)
E. Determine acceptability of results (Level 3)
F. Report results according to laboratory protocol (Level 2)
G. Identify and correct sources of error in test procedures according to laboratory protocol (Level 3)
1. Prozone/postzone
2. Inappropriate specimen
3. Specimen interferences
4. Instrument malfunction
H. Perform, document, and evaluate quality control (Level 3)
I. Perform and document routine preventive maintenance (Level 2)
1. Develop a maintenance procedure
2. Develop a documentation tool and procedure
J. Correlate test results with other laboratory tests and patient diagnoses (Level 3)
IV. Immunologic techniques used in flow cytometry
A. Identify basic concepts of flow cytometry (Level 1)
1. Fluid system
2. Optical system
3. Signal detector system
a) Forward/side scatter
b) Multi-color fluorescence
4. Data management system
5. Cell sorter
B. Discuss the operation of a flow cell cytometry instrument (Level 1)
1. Sample preparation and evaluation
a) Collection and storage to insure cell viability
b) Evaluation of cell viability prior to analysis
c) Sample preparation appropriate for desired analysis
2. Light scatter
a) Forward-angle (FALS)
b) Side (SS)
3. Gating
4. Cell sorting
C. Discuss the role of monoclonal antibodies in flow cell cytometry (Level 1)
1. Production
2. Cluster designations
3. Fluorescent labeling
D. Discuss the clinical application of flow cell cytometry (Level 1)
1. Lymphocyte immunophenotyping
2. Leukemia/lymphoma immunophenotyping
3. DNA ploidy analysis
4. Reticulocyte enumeration
5. Tumor specific antigens
6. Cell counts, e.g., CD4+ cells in AIDS
7. Other
E. Perform basic scatterplot interpretation, including calculations related to cell counts
V. Disease states
A. Define tolerance (Level 1)
B. Describe proposed mechanisms for autoimmunity (Level 1)
1. Release of sequestered antigens
2. Escape of tolerance at the T cell level
3. Diminished regulatory T cell function
4. Genetic pre-disposition
5. Mimicry/cell damage
C. Describe the clinical symptoms and laboratory findings for classic autoimmune diseases (Level 1)
1. Addison’s disease
2. Hyperthyroidism (Graves’ disease)
3. Hypothyroidism (Hashimoto’s thyroiditis)
4. Idiopathic thrombocytopenic purpura
5. Type I diabetes mellitus
6. Pernicious anemia
7. Multiple sclerosis
8. Rheumatoid arthritis
9. Systemic lupus erythematosus
10. Progressive systemic sclerosis (scleroderma)
11. Myasthenia gravis
D. Describe the surveillance function of the immune system (Level 1)
E. List and describe antigens that are associated with human tumors (Level 1)
1. Alpha-fetoprotein (AFP)
2. Beta-2-microglobulin
3. Carcinoembryonic antigen (CEA)
4. Prostate-specific antigen (PSA)
5. Human chorionic gonadotropin
6. Others
F. Define immunodeficiency (Level 1)
G. Differentiate between primary and acquired immunodeficiency disorders (Level 1)
H. Discuss the congenital lymphocyte immunodeficiencies (Level 2)
1. B cell deficiencies
a) X-linked hypogammaglobulinemia (Bruton’s agammaglobulinemia)
b) Selective immunoglobulin deficiencies
2. T cell deficiencies--thymic aplasia (DiGeorge’s syndrome)
3. Combined B cell and T cell deficiencies--severe combined immunodeficiency disease (SCID)
I. Discuss the etiology of complement deficiencies (Level 1)
1. Genetic deficiency of complement components
2. Genetic deficiency of complement regulatory components
a) Hereditary angioedema
b) PNH
3. Complement consumption by disease- infection, autoimmunity
J. Discuss phagocyte deficiencies (Level 1)
1. Etiology and clinical findings
a) Chronic granulomatous disease (CGD)
b) Leukocyte adhesion defect (LAD)
2. Laboratory techniques to evaluate phagocytes
K. Describe causes of acquired immunodeficiencies--viral, chemical, pharmaceutical (Level 1)
L. Describe the clinical and laboratory findings in a patient with acquired immunodeficiency syndrome (AIDS) (Level 1)
M. Describe the clinical and laboratory findings for viral infections (Level 2)
1. Epstein Barr Virus
2. Cytomegalovirus
3. Hepatitis A
4. Hepatitis B
5. Hepatitis C
6. Hepatitis D
7. Hepatitis E
8. HIV
9. Rubella
10. Other
N. List the types of hypersensitivity (Level 1)
1. Type I
2. Type II
3. Type III
4. Type IV
O. Discuss the immunologic mechanisms unique to each type of hypersensitivity (Level 1)
P. Correlate hypersensitivity reactions with autoimmune diseases (Level 3)
Q. Discuss the events leading to and the symptoms in anaphylaxis (Level 1)
R. Describe laboratory findings associated with each type of hypersensitivity (Level 1)
S. Describe the clinical and laboratory findings of bacterial diseases (Level 2)
1. Lyme disease
2. Streptococcal
3. Syphilis
4. Mycoplasma
5. Systemic inflammatory response syndrome (SIRS)/sepsis
MLS Immuno 11