Chapter 19 Immune Function Assessment
Assessment of the Immune System
Measurement of Complement and High-Sensitivity C-Reactive Protein
Assessment of the Immune System
Immune physiology involves complex interactions between cells and proteins to mediate an effective response to infectious disease. A healthy immune system eliminates pathogenic microbes while simultaneously creating a biodynamic relationship with normal microflora. Because the immune system must also effectively distinguish between dangerous foreign invaders and self-antigens, immune dysfunction can result in autoimmunity, cancer, allergies, and asthma, in addition to infection. Healthy immune function is affected by age, gender, sleep, nutrition, and exercise. Natural therapies such as herbs, meditation, hydrotherapy, and supplements can also enhance and manipulate immune reactions.
A variety of tests can be used to evaluate the immune system. To determine what type of immune assessment to use for each clinical diagnosis, it is important to understand how underlying immune physiology contributes to a clinical picture. Clinicians can assess qualitative or functional activity of immune cells. Qualitative data may include things like types, number, and activation states of cells. Cells of the immune system express different proteins on their surface. These proteins, called biomarkers, allow us to identify different cells as well as their activation state. To assess immunodeficiency and lymphoproliferative diseases, the number of T cells and B cells must be quantified. Functional activity of immune cells involves putting the cells into an in vitro assay system, adding antigen or mitogen, and measuring a function such as cytotoxicity, cytokine production, or antibody secretion. Levels of proteins that are produced by lymphocytes and monocytes, such as cytokines and antibodies, can be analyzed as well. Tests of genetic predisposition to disease can be evaluated with genetic tests. An overview of the assays used to measure cells is found in Table 19-1. A description of these tests is found in Box 19-1.
|TYPE OF IMMUNITY||ASSAYS|
|Humoral immunity||Antibody production: ELISA, cytometric bead array|
|Cellular immunity||Cytokines: ELISA, ELISPOT, cytometric bead array, RIA, intracellular cytokine staining|
|Cytotoxicity: 51 chromium release assay|
|Proliferation: CFSE, lymphocyte proliferation assay|
|Genetics||SNPs genetic microarray|
CFSE, carboxyfluorescein succinimidyl ester; ELISA, enzyme-linked immunosorbent assay; ELISPOT, enzyme-linked immunosorbent spot; RIA, radioimmunoassay; SNP, single nucleotide polymorphisms.
BOX 19-1 Testing Methods
Enzyme-linked immunosorbent assay (ELISA) is an assay used to measure proteins of the immune system. In ELISA, an antigen is fixed to the surface of a 96-well plate, then a specific antibody is incubated with the antigen so that it forms an immunocomplex. This antibody is linked to an enzyme that allows the antigen to be converted into a detectable signal, most often a change in color. The more antigen that is present, the darker the color.
A variation of the ELISA is the enzyme-linked immunosorbent assay (ELISPOT). Peripheral blood mononuclear cells (PBMCs) are plated on a filter-bottom 96-well plate coated with anticytokine antibody. The plate is cultured 24 to 48 hours to allow cytokine secretion and capture on the plate. Cells are washed off and detector antibody is added, followed by enzyme substrate. Cytokine-secreting cells are identified as spots of secreted cytokine.
Cytometric Bead Array
A cytometric bead array uses multiplexed beads labeled with capture antibodies for specific analytes, such as cytokines or other serum proteins. Serum is added together with Phycoerythrin (PE) PE-labeled detector antibody. The antibody–antigen complex is run through the flow cytometer, and software calculates the level of each analyte based on PE fluorescence of each bead population relative to a standard curve.
Intracelluar Cytokine Staining
This technique uses the flow cytometer to ensure production of cytokines in short-term stimulated whole blood or PBMCs before the cytokines are secreted from the cells. One advantage of this assay is that multiple cell surface and intracellular markers can be analyzed in combination using multiparameter flow cytometry.
Chromium-51 Release Assay
To test the ability of CD8 T cells or natural killers (NKs) to kill, target cells are incubated with radioactive isotope of chromium-51, which can be released when the cell dies. The CD8 T cells or NKs are then placed in serial dilution with the antigen presenting cells. Antigen is added in the case of the CD8 T cells. When the target cells are killed by the CD8s, the amount of radioactivity or enzyme can be analyzed.
Lymphocyte Proliferation Assay
Proliferation capability of T cells can be measured by isolating PBMCs, separating the T cells, and incubating with tritiated thymidine (3H). A mitogen such as lipopolysaccharide is added to the cell culture. This mitogen may induce cell proliferation. As the cells divide, they incorporate the 3H into the DNA, and analysis of radioactivity can determine how many cell divisions occurred. An ELISA may also be performed to measure cytokine production by the CD4 T cell.
Carboxyfluorescein succinimidyl ester
Carboxyfluorescein succinimidyl ester (CFSE) can be used to measure cell division. In this assay, the parent cell is incubated with CFSE, which is incorporated into DNA during cell division. As the cell divides, each of the daughter cells contains half of the CFSE stain. Every subsequent cell division halves the amount of CFSE in the daughter cells. When these cells are run through a flow cytometer, a characteristic pattern is seen (see Figure 19-2).
Measurement of Antibodies
Antibody (also called immunoglobulin) is a protein made by B cells in response to antigen. Each B cell has specificity for one antigen. This specificity is determined by gene rearrangement long before a B cell ever encounters antigen. The antibody is found both on the surface of B cells and is secreted. B cells are primarily found in the lymph nodes, Peyer’s patches of the gut, and spleen (80%); however, antibody is readily detectable in the blood and other bodily fluids.
Antibodies look like the letter “Y”. Each part of the antibody has a different function: the arms bind specifically to antigen, whereas the base (the Fc region) allows the antibody to bind to receptors. This base region is called the “isotype.” Antibodies come in multiple isotypes based on the constant region of their heavy chain. These different isotypes have functional importance. Some isotypes are better than others at triggering the complement cascade. The Fc receptors of other isotypes allow them to activate specific cells, such as mast cells and eosinophils.
Antibodies are essential to measuring immune function because they can act both as an indicator of disease and as a tool to measure the levels of other proteins and cells. Monoclonal antibodies are a powerful tool (see Box 19-2).
BOX 19-2 Monoclonal Antibodies
One of the primary tools used in many of the assays for immune function is the monoclonal antibody. Monoclonal antibodies are the product of a single B cell. To generate a monoclonal antibody, an antigen is injected into an animal, and the resultant B cells are isolated. These B cells are diluted, and a single B cell is fused with a nonsecreting myeloma cell line to form hybrids. These hybrids then produce the monoclonal antibodies highly specific for the injected antigen. In contrast, when any animal is injected with an antigen, polyclonal antibodies are generated. Many different B cells specific for that antigen release antibody, which is harvested and purified. This collection of antibodies is called polyclonal because, unlike monoclonal antibodies, it has many specificities to the same antigen. Polyclonal and monoclonal antibodies can be tagged with enzymes for fluorescence to detect the original protein antigens for which they are specific.1
Many proteins have monoclonal antibodies specific for them, including cell surface markers, cytokines, and even other antibodies. The quality of the monoclonal antibody can determine the sensitivity and specificity of the test used.
Measuring serum antibody is essential in patients who have repeated or severe infections. Low antibody levels in these patients may indicate that they have an immunodeficiency. Patients with myelomas and lymphoproliferative disorders may have high antibody levels. For example, patients with alcoholic liver disease often have a polyclonal expansion of immunoglobulin-A (IgA), whereas patients with systemic lupus erythematosus (SLE) and Sjögren’s syndrome have polyclonal IgG expansion.1
The type of isotype made by B cells can provide clues as to immunologic status of the patient (see Table 19-2).
The route and duration of exposure, type of antigen, and genetic background may all affect the isotype of antibody that is produced, as well as the subclass of antibody produced. Upon immediate exposure to a novel infectious organism, the body produces IgM. After the B cell makes IgM, it class switches to another isotype, as determined by a combination of activation proteins and cytokines. If the patient has been exposed to an infectious agent, the antibody usually will class switch to IgG.
IgG is the major antibody found in blood, and it consists of four subclasses, originally described in the 1980s according to their abundance in serum. IgG1 and IgG2 are present in much higher concentrations than IgG3 and IgG4. All subclasses of IgG are low in pediatric populations. Deficiency in some IgG subclasses results in increased susceptibility to bacterial infections.2 IgG subtypes vary in their ability to activate complement and Fc receptor binding. IgG3 has a strong affinity for Fc receptors and has the greatest ability to activate complement. The type of antigen can influence the subclass of antibody response. For example, IgG2 antibodies appear to influence polysaccharide antigens, whereas protein antigens and whole bacteria preferentially elicit IgG1.3–5
IgG4 antibodies, found at lowest concentration in the serum, are generated to repeatedly presented antigens. IgG4 deficient individuals may be more susceptible to pyogenic infections of the respiratory tract. Antibodies against dietary antigens are frequently IgG4. In a community survey of 40 individuals, anti-food antibodies to milk, egg, and fish of the IgG4 subclass were found in a significant proportion of a healthy population, indicating that IgG4 antibodies against food antigens cannot serve as markers of atopic disease. Because IgG4 cannot activate complement, it has been hypothesized that it may serve for protective clearance mechanisms and may be a desirable response to dietary antigens.6 IgG1 and IgG2 have also been found for dietary antigens, but the immunologic outcome of these subclasses may be food intolerance that is not caused by IgE mediated hypersensitivity.7 A larger discussion of the immune response to food can be found in Chapter 15.
Antibodies of each of the IgG subclasses can be detected by a variety of techniques, including radioactive iodine labeling, antigen coated red cells, immunofluorescence with antigen coated sepharose, radioimmunoassay (RIA), and enzyme-linked immunosorbent assay (ELISA) with monoclonal antibodies (Figure 19-1).8
FIGURE 19-1 ELISA. A capture antibody specific for an antigen in a sample is attached to the bottom of a plate. The sample (blood, saliva, etc.) is added and excess sample is washed from the plate. A secondary antibody also specific for the antigen is added to the plate. This secondary antibody is labeled with an enzyme. When the substrate for the enzyme is added to the plate, it elicits a color change. The intensity of the color, as measured by spectrophotometry, indicates the amount of antigen that is present.
Courtesy Heather Schiffke.
IgA can also be made to infectious agents and occurs in two forms, IgA1 and IgA2. IgA in serum predominantly exists in the monomeric form, IgA1. The ratio of IgA1:IgA2 in serum is about 9:1. IgA found in secretions, termed secretory IgA, occurs as dimers. The ratio of IgA1:IgA2 in secretions varies, but is approximately 6:4 in saliva.9 Because IgA is found at mucosal surfaces and lines the mucosal surface of the gut, it is not uncommon to find IgA antibody specific for food antigens. Because IgA is at high levels in secretions, it can be found in saliva in addition to serum, and salivary IgA tests are readily available.10,11
An immune response to parasites, specifically worms, triggers an IgE response.12 IgE elicits an immune response by binding to Fc receptors on mast cells, eosinophils, and basophils, causing degranulation and cytokine release. In atopic individuals, IgE is also made to allergens. IgE is at low levels in the blood. Measurement of total serum IgE is useful in patients in whom parasitic infection is suspected, but is not valuable for measuring allergies. Thus, the most common method for allergy testing is the skin prick test.13 In this test, a small amount of the suspected allergen is placed on the skin. Then the skin is pricked so that the allergen goes under the skin’s surface. If the patient is allergic to the allergen, swelling and redness will appear within 15 to 20 minutes.
Measuring total antibody in blood involves protein electrophoresis or ELISA, and is valuable. However, quantifying the total amount of antibody specific for an antigen is even more desirable. Antibody titers for common antigens, such as those found in vaccines, can be ordered. Each laboratory has its own reference levels for titers, because the sensitivity of the test is related to the specificity and affinity of the reagents they are using.
Specific antibodies, rather than total levels, are also measured for circulating autoantibodies. These antibodies can be detected with immunofluorescence, RIA, and ELISA.14 Immunofluorescence involves examining the tissue directly with microscopy and is the least sensitive of these tests. The patient’s tissue is frozen and sections cut on a cryostat. This tissue is incubated with patient serum, such that autoantibodies in the serum can bind. A fluorescently tagged secondary antibody specific for human immunoglobulin is then used to detect the autoantibodies. Immunofluorescence examination of biopsy specimens of damaged or normal tissue may reveal deposits of immunoglobulins caused by antibodies reacting with an organ or tissue specific antigens. This approach is especially important in the diagnosis of antiglomerular basement antibody disease and bullous skin disorders, but may show false positives when used for SLE. (See Table 19-3 for a listing of common autoantibodies.)
|Antinuclear antibody||Systemic rheumatic diseases|
|Smooth-muscle antibody||Nonspecific liver damage; chronic hepatitis|
|Antimitochondrial antibody||Primary biliary cirrhosis|
|Endomysial antibody||Celiac disease; dermatitis; psoriasis|
|Antineutrophil cytoplasmic antibody||Vasculitis|
|Gastric parietal cell antibody||Pernicious anemia|
|Adrenal antibody||Idiopathic Addison’s disease|
|Pancreatic islet cell antibody||Insulin-dependent diabetes mellitus|
|Skin antibodies||Pemphigus vulgaris; Bullous pemphigoid|
|Antiacetylcholine receptor||Myasthenia gravis|
|Antimyelin basic protein||Multiple sclerosis|
|Double-stranded DNA autoantibody||Systemic lupus erythmatosus|
|Thyroid stimulating hormone antibody||Graves’ disease|
RIA is a far more sensitive test used for measuring antibodies that are in low concentrations. In RIA, an antibody specific for human antibody is tagged with a radioactive isotope. When this antibody is incubated with an autoantibody, the contact detects even low levels of autoantibody. ELISA is very similar to RIA; however, the enzymatic substrate is linked to the detection antibody.
When an antibody is made, the antibody light chains, κ and λ, are made in excess. These are present as free forms in serum and urine. Although small amounts of light chains are found in everyone, people with renal damage excrete higher levels of light chains in their urine. Free light chains are associated with malignant plasma dyscrasia and other lymphocyte-related immunoproliferative disorders. Intact immunoglobulin can also be found in urine.15