Exciting discoveries in molecular biology, recombinant DNA and proteins, cytokine biology, and human genetics have enhanced our understanding of immune-mediated diseases. With these advancements, clinical laboratory immunology has matured, and its applications have increased extensively. Hence, the scope of the clinical immunology laboratory now extends to a wide variety of disciplines, such as trans plantation, rheumatology, oncology, dermatology, infectious disease, allergy, and immunodeficiencies. The goal of the clinical immunology laboratory is to provide laboratory testing to support the diagnosis and monitoring of patients with immune disorders. A variety of technologies are used to evaluate both the antibody and cellular components of the immune response. For a comprehensive review of current test systems used the clinical immunology hospital setting, see Detrick et al (2015). Selected assays are highlighted.

Antibody Evaluation Assays

 A. Enzyme-Linked Immunosorbent Assay

 The enzyme immunoassay (EIA) testing system is one of the most popular tests used in the clinical laboratory to monitor a variety of antibody specificities. This method depends on the conjugation of an enzyme to an antibody. The enzyme is detected by assaying for enzyme activity with its substrate. To measure antibody concentration, known antigens are bound to a solid phase (eg, plastic microtiter plate), incubated with test antibody dilutions, washed, and reincubated with an anti-immunoglobulin labeled with an enzyme (eg, horseradish peroxidase). The enzyme conjugated to the detection moiety produces a color when the specific substrate is added. The more antigen that binds to antibody results in higher concentrations of enzyme that leads to stronger color development. Thus, the intensity of the color developed is a direct function of the concentration of antibody bound. This serologic test is used to detect antibodies to a number of infectious diseases, such as antibodies to HIV proteins in blood samples or antibodies to the syphilis organism, Treponema pallidum. This assay is also widely used to detect autoantibodies present in the circulation of patients with systemic and organ-specific autoimmune diseases (eg, antibodies in systemic lupus erythematosus, scleroderma, or Sjögren syndrome). Variations of the traditional enzyme-linked immunosorbent assay include some of the newer technologies, such as chemiluminescence assay (CIA) and multiplex particle-based assays.

B. Immunofluorescence

When an antibody is labeled with a fluorescent dye (eg, fluorescein, rhodamine), the presence of the antibody can be observed using an ultraviolet light source in a fluorescence microscope. This assay system can be applied in two ways: a direct immunofluorescence assay or an indirect immunofluorescence assay. In the direct immunofluorescence assay, a known specific antibody is labeled with a fluorescent dye. A specimen with unknown organisms is added to a slide and the slide is incubated with the fluorescein-labeled specific antibody (eg, antistreptococcal antibody). The slide is washed and evaluated under a fluorescence microscope. If the unknown specimen contains streptococcus organisms, they will appear green. In the indirect immunofluorescence assay, a two-step procedure is used to detect the presence of organism-specific antibodies (such as treponemal antibodies) in a serum specimen. First, a known antigen (treponema) is attached to a slide. A serum sample is incubated with the slide, the sample is removed, the slide is washed, and a second fluorescein-labeled anti-immunoglobulin is added. The slide is washed and examined under a fluorescence microscope. If the patient’s serum contained antitreponemal antibodies, the organism will appear green under the fluorescence microscope. Historically this assay has been used to detect antibodies to certain microorganisms (eg, T pallidum) and is the standard procedure for the detection of autoantibodies in autoimmune diseases (eg, antinuclear antibodies).

C. Immunoblot

Immunoblot or Western blot is used to identify an antigen in a complex mixture of proteins. The complex mixture of proteins (eg, microorganism) is subjected to sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis (PAGE). This separates the proteins according to charge and molecular size. The gel is then covered with a membrane (such as nitrocellulose), and the proteins are transferred to the membrane. The nitrocellulose membrane (blot) now contains the separated proteins. The membrane is incubated with a serum sample. If the serum contains specific antibody that reacts with a protein on the membrane, the antibody will remain on the membrane. The membrane is now incubated with an enzyme labeled anti-immunoglobulin. The membrane is washed and incubated with the enzyme substrate. The enzyme and enzyme substrate mixture allows for colorimetric detection.

The antigen-antibody complex is visible as a separate band. This method is widely used as a secondary test for HCV and Lyme disease. More recently, this technology is being applied to identification of autoantibodies in selected autoimmune diseases (eg, polymyositis). Variations of the immunoblot techniques include dot or slot blot assays, both of which use purified antigens. In these techniques, purified antigens are attached to the nitrocellulose membrane.

 D. Other Laboratory Assays

Other technologies often available in the clinical immunology laboratory include protein electrophoresis and immunofixation electrophoresis, which are essential tests used to identify abnormal immunoglobulin production in the serum or urine of patients with myeloma. Nephelometry is another laboratory test that quantifies a wide variety of analytes in serum or plasma. This is the method of choice for quantitating complement components, immunoglobulins, and other serum analytes. These assays can also be used to evaluate other abnormalities associated with these selected infectious diseases (eg, HCV can be associated with a monoclonal protein and the presence of cryoglobulins).

Evaluation of Cellular Responses

 A. Flow Cytometry

 Flow cytometry is a laser-based method used for the analysis of cells and selected cell components. One of the most popular applications of flow cytometry is immunophenotyping of cell populations. In this method, single-cell suspensions are stream through a flow cell in which the cells pass through a laser beam for sensing. As the cells pass through the laser, they scatter light. If cells also contain fluorescent molecules, this will be detected. Both scattered light and fluorescent light information is recorded and analyzed to identify subpopulations within the sample. It is relatively easy to separate the cells into major classes, such as small lymphocytes separated from granulocytes that are larger and contain more granules (scatter more light).

A second way to analyze these cells is to evaluate cell sur face molecules that can be labeled with a fluorescent dye. The cluster of differentiation (CD) nomenclature is used for the identification of cell surface molecules. Presently there are over 300 CD molecules that have been identified. Monoclonal antibodies directed against the CD molecules have been generated and can be tagged with fluorescent labels. Incubation of the cells with a variety of different CD labeled antibodies allows the flow cytometric analysis of distinct populations of cells in the mixture. Using this method, one can identify CD4-positive cells, CD8-positive cells, B cells, macrophages, and cells expressing a variety of cytokines. This technology is widely used both in clinical laboratory and in biomedical research (eg, to enumerate CD4 T cells in HIV-positive patients or to distinguish tumor cells from normal white blood cells).

B. Functional Cellular Assays

In order to measure T cell function in vitro, the cells’ ability to proliferate or produce specific cytokines, such as IFN-γ, is analyzed. This assay is the in vitro counterpart of type IV hypersensitivity reactions, with TB skin test as a model. In the skin, the administered TB antigen interacts with specific T cells to proliferate, produce IFN-γ, and give a positive skin reaction. In this in vitro assay, peripheral blood leukocytes (PBLs) are incubated with a specific antigen for 24–72 hours. When specifically sensitized T cells in the PBLs interact with their specific antigen (eg, TB antigen), the cells will proliferate and produce IFN-γ. Proliferation can be measured by H3 thymidine incorporation, or IFN-γ production can be monitored by EIA or flow cytometry. This assay can be used to assess the immune status of an individual, particularly patients who are immunocompromised because of an infectious disease, malignancy, or drug therapy.

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مواضيع ذات صلة

phosphate (PO4 , Phosphorus [P])

pheochromocytoma suppression and provocative testing (Clonidine suppression test [CST], Glucagon stimulation test)

parvovirus B19 antibody

Applications of Molecular Biology to Erythrocyte Immunohematology

Direct Antiglobulin Test (Direct Coombs Test)

Indirect Antiglobulin Test (Indirect Coombs Test)

Partial thromboplastin time, activated (APTT, Partial thromboplastin time [PTT])

parathyroid scan (Parathyroid scintigraphy)

parathyroid hormone (PTH, Parathormone)

Paracentesis (Peritoneal fluid analysis, Ascitic fluid cytology, Peritoneal tap)

Determination of Type D and Rh Phenotype

Determination of the ABO Group