Autoimmune Disease Tests
Autoimmune disease tests are screening procedures used in the diagnosis of immune system disorders and other disease states to detect autoantibodies, which are antibodies produced against the body's own tissues.
Hundreds of different autoantibodies have been described, not all of which are involved in disease. Autoantibodies are classified into two broad catagories, organ-specific, in which the targeted antigen is located in a single organ or cell type, and organ-nonspecific, in which the targeted antigen is widely distributed, usually a ubiquitous component of all cell types. Autoantibodies may be further classified as primary pathogenic antibodies, which directly cause a disorder by blocking a normal
Autoimmune diseases are illnesses in which the immune system produces autoantibodies that attack the body's own cells or tissues as though they are foreign substances. Autoimmune diseases are generally difficult to diagnose, as individuals exhibiting very different symptoms can have the same underlying disease. Like the causative autoantibodies, the diseases are classified as organ-specific and organ-nonspecific (or systemic). In organ-specific autoimmunity, the autoantibodies are produced against a specific target antigen in a specialized cell, tissue, or organ in response to injury, inflammation, or other stimulus. Examples include autoimmune hemolytic anemia, in which anti-erythrocyte antibodies are produced, insulin-dependent or type I diabetes, characterized by T cells and antibodies against beta-cells in the pancreas, and myasthenia gravis, involving antibodies against the acetylcholine receptor. In systemic autoimmune diseases, the tissue injury and inflammation is generally initiated by vascular leakage and deposition in multiple sites of circulating autoimmune complexes formed against ubiquitous soluble cellular antigens, usually nuclear in origin. Systemic lupus erythematosus (SLE), characterized by the production of multiple anti-nuclear and anti-DNA autoantibodies, is the classical example of a systemic autoimmune disease. Autoimmune disease tests can help to identify the causative autoantibody in the blood.
For all autoimmune disease tests, a blood sample is required, usually taken from a vein on the back of the hand or the inside of the elbow. The nurse or phlebotomist collecting the sample should observe universal precautions for the pevention of transmission of blood-borne pathogens. Generally, blood samples that are grossly hemolyzed or lipemic are unacceptable.
The diagnosis of autoimmune diseases is very challenging; it can take months or even years for the health care team to put together the symptoms and laboratory findings to make an accurate diagnosis. New symptoms may appear over time to aid the process of reaching a diagnosis. Knowledge and awareness on the part of the doctor and effective communication between doctor and patient, including a complete and accurate medical history, together with a thorough physical examination and carefully chosen laboratory tests, are essential to the process.
Conditions other than autoimmune diseases can give rise to autoantibodies. In many cases, the tests performed on healthy individuals, especially the elderly, give positive results. It should be kept in mind that transient appearance of autoantibodies is a common feature of some infections, especially Epstein-Barr virus, adenovirus, and mycoplasmal pneumonia. Ideally, in the end, each positive result is interpreted in the context of the patient's symptoms and history, and all other diagnostic findings.
Access to a medical laboratory that specializes in autoantibody analysis is an advantage. Clinical immunology is a rapidly changing field: new, more convenient, and more specific test protocols are evolving, and a specialized laboratory is likely to have access to the most modern diagnostic tools.
In immunofluorescence-based tests, the tissues used in the assays must be carefully chosen and handled. It is important to use serum and not plasma, since the fibrinogen in the plasma can give rise to stray fluorescence and false-positive reports. The serum must be adequately diluted to avoid non-specific interactions with the target antigens. The ratio of FTIC-bound to unlabeled immunoglobulin must be sufficiently high to allow detection but not so high as to give rise to non-specific fluorescence. It is essential that control assays—a negative control that contains no antibody, as well as positive controls that contain known amounts of autoantibody at high and low levels—be included to ensure the accuracy of the test results.
In the tests for cryoglobulins and cold agglutinins, it is essential that the syringe used to draw blood and the container used to transport it to the laboratory be prewarmed and maintained at 37°C. The sample in these tests must be brought to the laboratory immediately for processing, and the procedure should be scheduled with the laboratory in advance.
INDIRECT IMMUNOFLUORESCENCE. The standard detection technique for identification of many serum autoantibodies is immunofluorescence. In indirect immunofluorescence testing for the widest range of antibodies, a "multiblock" of commercial test tissues (for example, rat liver, kidney, and stomach) or a single tissue such as mouse kidney with both muscle and blood vessel cells can be used. The tissues are frozen and cut with a cryostat into sections about 4 micrometers thick, and the sections are mounted onto sections (wells) of a glass slide and frozen. Alternatively, other cells or tissues
As an alternative to the fluorescence detection method, instead of FITC, the anti-human immunoglobin antibody is bound to an enzyme, such as horseradish peroxidase, that produces a colored product when treated with the appropriate substrate. The advantage of enzyme-based test is that an ordinary high quality light microscope may be used for detection; however, extra wash and incubation steps are necessary to produce the color change.
In a related technique, direct immunofluorescence, the tissue to be tested is obtained from the patient, frozen, and thin-sectioned using a cryostat. The sections of tissue are mounted onto microscope slides and treated directly with the FITC-linked antibody. This method detects autoantibodies that have attached in vivo to the specific tissue.
ENZYME-LINKED IMMUNOASSAY (EIA), ALSO CALLED ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA). This detection procedure is similar to immunofluorescence, except that, instead of cells, a specific antigen covalently bound to a solid surface (usually a commercially available microwell plate) is allowed to react with the test serum. The plate is then washed and reacted with an enzyme-labeled antihuman immunoglobulin, then washed and allowed to react with a substrate that gives a quantifiable color change. This technique is generally costlier and less information-rich than immunofluorescence on muliblock tissues, but it is often used to confirm a diagnosis based on the immunofluorescence result. Commercial EIA test kits are available for a wide variety of autoantibodies including anticardiolipin (antiphospholipid), and thyroid antibodies such as anti-thyroglobulin and anti-thyroid peroxidase.
PARTICLE AGGLUTINATION. Detection of antibodies by particle agglutination is an old—but reliable and relatively inexpensive—technique. The target antigen is coated onto an inert gelatin or latex particle and treated with the test serum. Agglutination or clumping of the particles indicates a positive response, and the serum is then titered as in the immunofluorescence assay.
IMMUNOPRECIPITATION. The Ouchterlony double diffusion test is the prototype immunoprecipitation technique. The serum to be tested for the presence of an antibody and the antigen are added to separated wells cut into an agar gel slab and allowed to diffuse towards one another. A positive test is indicated when a precipitation line forms in the zone of the gel where the antibody comes into contact with the antigen. The process, which may require up to 72 hours, can be accelerated by using agarose gels with an electrolyte and applying a current, in a technique called immunoelectrophoresis. In a related method called nephelometry, the immunoprecipitation, carried out in a solution containing the antigen, gives rise to turbidity that can be quantified by an instrument that measures light scattering.
ANTINUCLEAR ANTIBODY TEST (ANA), ALSO CALLED FLUORESCENT ANTINUCLEAR ANTIBODY TEST (FANA) AND ANTINUCLEAR ANTIBODY PANEL. The antinuclear antibody (ANA) test, which screens for antibodies to components of the nuclei of all cells, is generally performed first in the evaluation of a person for autoimmune diseases, particularly when systemic lupus erythematosus (SLE) is suspected. Usually HEp-2 cells are tested with 5–10 microliters of the patient's serum. HEp-2 cells are preferred because of their very large nuclei. Recognition of the patterns of immunofluorescence staining of the nucleus is critical in the interpretation of the ANA test. EIA kits for testing ANA IgG and IgM antibodies are also commercially available, but give only titre results without staining pattern information.
ANTIMITOCHONDRIAL ANTIBODY TEST. This test is primarily used when there are symptoms of liver or other organ damage, to confirm a diagnosis, or help to identify possible causes of tissue damage. Antimitochondrial antibodies are helpful in distinguishing primary biliary cirrhosis (PBC) from other extrahepatic obstruction, hepatitis, and alcoholic cirrhosis. Nine discrete subtypes of antimitochondrial antibodies, designated M1–M9, have been described and are associated with different disease
ANTIPHOSPHOLIPID ANTIBODY TEST, ALSO CALLED CARDIOLIPIN ANTIBODY TEST. Antiphospholipid antibodies are detected in combination with circulation disorders (livedo reticularis and Sneddon's syndrome), in SLE, in Sjögren's syndrome, and in women suffering recurrent spontaneous abortions. In these conditions, collectively called antiphospholipid syndrome, the antibodies detected are of the IgG and IgM classes. In more than 75% of the patients with spontaneous abortions, only IgG antibodies are found. EIA detection is generally used to test for antiphospholipid antibodies, and antigens to detect each of the IgG-, IgM-, and IgA-type antibodies are commercially available.
CRYOGLOBULIN TEST. Cryoglobulins are tested in the presence of symptoms induced by cold, such as purpura and Raynaud's phenomenon, and when immune-complex-mediated disease is suspected. Cryoglobulins, often confused with cold agglutinins, are abnormal immunoglobulins that precipitate to varying degrees at temperatures below 37°C and become resolubilized upon rewarming; the precipitates formed can block small blood vessels. Cyroglobulins have been purified and immunochemically characterized, and three major types having distinct clinical features have been described. Type 1 cryoglobulins are associated with single monoclonal immunoglobulins (IgM, IgG, or IgA), Type II with mixed monoclonal immunoglobulins, and Type III with mixed polyclonal immunoglobulins. Cryofibrinogen and C-reactive protein-albumin complexes are nonimmunoglobiulin cryoprecipitable plasma proteins with similar clinical manifestations.
For the test, 10mL of blood is collected with a syringe warmed to 37°C, and 6mL is transferred to a specifically designed glass cryoglobulin tube, also prewarmed to 37°C. The sample should be hand carried to the laboratory and must be kept at 37°C until the blood has clotted and the serum is collected by centrifugation. The serum sample is then stored at 4°C for 24–72 hours and observed for formation of a white precipitate (cryoglobulins). An aliquot of the serum is rewarmed at 37°C for 24 hours to test for reversibility of cryoprecipitation. The amount of cryoglobulin is quantified by measuring the serum protein concentration before and after cryoprecipitation, or by collecting the precipitate from an aliquot of serum, dissolving it in an acidic buffer, and measuring the cryoglobulin protein level. The individual cryoglobulin components may be identified from the isolated cryoprecipitate by immunoelectrophoresis, performed at 37°C to prevent precipitation of the cryoglobulin during the procedure.
COLD AGGLUTININS. Cold agglutinin disease is an acquired autoimmune hemolytic anemia. Cold agglutinins, which are frequently confused with cryoglobulins, are an IgM-type autoantibody directed against an antigen (I or i) on the surface of red blood cells and react only at temperatures lower than 37°C. The antibody reaction occurs in the extremities, which are generally cooler, causing red blood cells to briefly stick together, leading to obstruction of the small blood vessels; Raynaud's syndrome is commonly associated with cold agglutinins, and hemolytic anemia may result when red blood cells under-go lysis upon rewarming. As with the cryoglobulin test, blood for the cold agglutinins test must be collected with a warm syringe and kept at 37°C during transport to the laboratory for analysis. The test is performed by mixing serial dilutions of the patient's serum with group O red blood cells and incubating at 2-8°C overnight. The tubes are examined for clumping (agglutination) and the highest dilution giving agglutination is noted. Following this, the tubes must be incubated at 37°C for 30 minutes to reverse the agglutination that occurred in the cold. The test is positve only when the agglutination is reversed by 37°C incubation.
RHEUMATOID FACTOR TEST, ALSO CALLED RF OR RHF TEST. Rheumatoid factors are associated with many different conditions, including viral and chronic bacterial infections, lymphomas and myelomas, and connective tissue diseases. Standard tests are non-specific, detecting all classes of immunoglobulins that react with the Fc region of other immunoglobulins. Commercially available EIA kits have recently become available that allow testing of individual immunoglobulin classes. The test is used mainly in the diagnosis of rheumatoid arthritis, although it is not very specific unless the disease is progressive with vasculitis. Elevated rheumatoid factor is also found with other rheumatologic diseases, such as SLE. Rheumatoid factors are detected by particle agglutination, nephelometry, or by EIA.
Certain drugs, including antibiotics, oral contraceptives, procainamide, tranquilizers, steroids, thiazide diuretics, and some antihypertensive drugs give a false positive result in the ANA test, and the patient should be advised to stop taking these before the test. For the antimitochondrial antibody and cryoglobulin tests, fasting for six hours or overnight before the test may be required.
After the blood sample is drawn, pressure should be applied to the puncture site until the bleeding stops to reduce bruising, and a bandage may be applied to the site. A warm pack may be applied to the site to relieve discomfort.
Complications associated with blood drawing may be anticipated, but are minimal when the procedure is performed correctly. Multiple punctures to locate suitable veins may be necessary to obtain a blood sample from some patients. The patient may faint or feel dizzy while the blood sample is being drawn. Discomfort or bruising may occur at the puncture site, and there is a slight risk of infection.
With immunofluorescence detection, lower than reference level titers that produce a fluorescent pattern in the test cells is interpreted as a negative result. In EIA, a lower than reference level titer that produces a color change, or a level of color change less than or equal to that found for a reference negative control, is interpreted as a negative result. With particle agglutination, absence of particle clumping, and in immunoprecipitation the lack of a precipitation line or solution turbidity, at titers lower than reference levels is negative.
Several factors should be considered as part of interpreting a positive test, and additional tests are often indicated to aid in identification of the individual causative autoantibodies.
Antinuclear antibodies test
Normal titer ranges for antinuclear antibodies is age-dependent:
- 18 years and younger: < 1:20
- 18–65: < 1:40
- older than 65: < 1:80
The pattern of immunofluorescent staining should be reported along with the titer. Homogeneous staining indicates antibodies to histones and deoxyribonucleoproteins. A speckled pattern is found with antibodies against ENA, a group of saline-soluble antigens that include nonhistone proteins and nuclear ribonucleoprotein (RNP). Peripheral staining indicates double-stranded (ds) DNA, rheumatoid factor, and antiphospholipid. Nucleolar staining is positive for anti-nucleolar RNP, a result specific for the autoimmune skin disorder scleroderma. Specific staining of centromeres is indicative of a systemic sclerosis varient known as CREST syndrome. More than 95% of people with SLE have a positive ANA test. Scleroderma has a 60–71% positive rate; Sjögren's disease, 50–60%, and rheumatoid arthritis, 25–30%. Serum that tests positive for ANA can be further tested by EIA with specific antigens to confirm a diagnosis. Positive tests for anti-double-stranded (ds) DNA or anti-Sm
Antimitochondrial antibody test
The titer in a normal sample is < 1:40. A positive result is typified by granular staining in the cytoplasm of all tissue types in the commercial multiblock. Antimitochondrial antibody tests are positive in about 94% of patients with primary biliary cirrhosis (PBC), with typical titers of > 1:160, and in 25% of patients with chronic hepatic cirrhosis. Positive samples should be screened by EIA for antibodies specific to the M2 antigen to confirm the diagnosis of PBC.
Antiphospholipid antibody test
Reference levels will be dependent on the type of commercial EIA kit used and should be independently established in each laboratory. Antiphospholipid antibodies occur with a prevalence of 40% in patients with SLE. Over 75% of patients presenting with spontaneous abortions were positive for only IgG antibodies, while patients with thromboses had mainly IgM antibodies. IgG and IgM antibodies were found simultaneously in 31% of patients with Sjögren's syndrome.
The percent cryoprecipitation is reported (cryocrit). Although there is considerable overlap, Type I and Type II cyroglobulins, positive in Waldenström's macroglobulinaemia and in chronic lymphocytic leukemia, are often present in relatively high concentrations, with cryocrits usually > 5%, sometimes as high as 20–40%, especially with Type I cyroglobulins. Type III cryoglobulins generally give a cryocrit of < 1% and are associated with autoimmune diseases, including systemic lupus erythematosus (SLE), rheumatoid arthritis, scleroderma, and Sjögren's syndrome.
Cold agglutinins test
A normal titre for cold agglutinins is less than 1:64. Cold agglutinin disease associated with autoimmune hemolytic anemia usually produces titres between 1,000-100,000. Higher titers are alwo seen in about 60% of persons with Mycoplasma pneumoniae infections and other acute infectious diseases such as infectious mononucleosis. In cold autoimmune hemolytic anemia, the peripheral blood smear may show red blood cells present in clumps caused by their reaction with cold agglutinins. Some cases of chronic cold agglutinin disease occur in association with Waldenström's macroglobulinemia.
Rheumatoid factor test
Normal rheumatoid factor titer for adults is < 1:80 (by particle agglutination) or 60 U/ml (by nephelometry). A positive test may result with chronic hepatitis, chronic viral infection, dermatomyositis, infectious mononucleosis, leukemia, rheumatoid arthritis, scleroderma, or SLE. Adult Still's disease, chronic infection, juvenile rheumatoid arthritis, nephrotic syndrome, and Sjögren's syndrome may also give positive results. Reference levels with commercial EIA kits will be dependent on the type of kit used and should be independently established in each laboratory. IgM-type rheumatoid factors are present in 75% of patients with chronic polyarthritis (CP), in 50–65% of patients with interstitial pulmonary fibrosis, in only 30% of patients with mixed connective tissue disease (SLE, Sjögren's syndrome), and with a prevalence of 10–20% in viral hepatitis, liver cirrhosis, sarcoidosis, and tuberculosis. IgA-class rheumatoid factors can be an early finding in severe rheumatic disease and are also found in SLE patients. Specific determination of IgG class rheumatoid factors, mainly present as immune complexes, have been described in both juvenile and chronic polyarthritis.
Health care team roles
A nurse or phlebotomist usually collects the blood sample by venipuncture and sees to the timely and appropriate transport to the laboratory for analysis. A member of the health care team should observe the patient to ensure that bleeding from the puncture site is stopped.
Autoimmunity tests are performed by a clinical laboratory scientist/medical technologist. For immunofluorescence assays the technologist will usually have advanced training in fluorescent microscopy and the interpretation of immunofluorescence patterns.
A physician interprets the autoantibody test results and uses them in combination with a complete medical history and thorough physical examination to reach a diagnosis.
The physicians, nurses, and other caregivers should instruct the patient that diagnosis of an autoimmune disorder is a difficult process that may take considerable time and strive to allay the patient's feelings of frustration and anxiety.
Agglutination—Antibody-induced clumping of cells.
Antibody—Y-shaped protein molecule produced by B cells as part of the primary immune defense; each molecule and its clones have a unique binding site that combines with a complementary site of an antigen, to disable the antigen and signal other immune defenses.
Autoantibody—An antibody produced by an organism against any of its own tissues, cells, or cell components.
Autoimmune disease—Any of a variety of diseases in which the body's immune system attacks its own tissues.
Capillary—A small-bored tube used to collect, store, or test fluid samples.
Cold agglutinins—An autoantibody found on the surface of red blood cells in certain diseases that can cause the cells to clump at temperatures below 37°C and cause red blood cell lysis.
Cryoglobulin—A plasma immunoglobulin that is insoluble when cold and dissolved at body temperature.
Cryoprecipitation—Cold-induced precipitation of a substance from solution.
Deoxyribonucleoproteins—A class of proteins associated with DNA, found in the nucleus of the cell.
Enzyme—A protein that produces specific chemical changes in an organic target molecule by catalytic action.
Fluorescein isothiocyanate (FITC)—An organic molecule that can be covalently bound to other molecules, and that emits visible light when exposed to ultraviolet radiation.
Hemolytic anemia—Anemic condition characterized by the destruction of red blood cells.
Histone—Any of a group of five small alkaline proteins that occur in the nucleus of eukaryotic cells and form molecular complexes with DNA, around which the DNA is wound in the nucleosomes.
Immunofluorescence—Any of various techniques to detect an antigen or antibody in a sample by coupling its target antibody or antigen to a fluorescent compound and observing its reaction with the sample under an ultraviolet-light.
Immunoglobin (Ig)—Any of several classes of structurally related proteins that function as antibodies and are found in plasma and other body fluids and in the membrane of certain cells; classes include IgA, IgD, IgE, IgG, and IgM.
Microwell plate—A rectangular disposable culture dish constructed with an array of small depressions used to carry out reactions.
Mitochondrion—An organelle in the cytoplasm of cells that functions in energy production.
Monoclonal—Pertaining to cells or cellular products that are derived from a single cell; a monoclonal antibody is an immunoglobulin directed against a specific epitope of an antigen, and isolated from a culture of identical cells grown from a single clone.
Nephelometry—A method for studying the density of suspended particles in a liquid sample by measuring the amount of light scattered by the particles.
Nucleolus—One or more densely staining bodies, composed mainly of RNA, within the nucleus of a cell.
Nucleus—A specialized spherical structure, present in most living eukaryotic cells and enclosed in a membrane, that is the center for metabolism of DNA and RNA.
Phospholipid—Any of a group of fatty biomolecules composed of phosphoric esters.
Polyclonal—Pertaining to cells or cellular products that are derived from several lines of clones; a polyclonal antibody is a mixture of immunoglobulins isolated from antisera that is directed against all epitopes of a single antigen.
Raynaud's phenomenon—Sporadic attacks of blood flow interruption to the fingers, toes, ears, or nose caused by exposure to cold or by emotional stimulation, in which the affected area becomes bluish white and numb, then red, warm, and tingly, before returning to normal.
Rheumatoid factor—An autoantibody found in the blood, often associated with rheumatoid arthritis.
Ribonucleoprotein—A class of protein found in the nucleus associated with RNA.
Scleroderma—An autoimmune disease in which the connective tissue becomes hardened and rigid.
Sjögren's disease—An autoimmune disease characterized by fatigue and malaise, dry gritty eyes and dry mouth, and recurrent gingivitis and respiratory infection.
Smith's (Sm) antibodies—A nuclear autoantibody specific for SLE, named for the patient in which first described.
Systemic lupus erythematosus (SLE)—An autoimmune inflammatory disease of the connective tissue that occurs mainly among middle-aged women, characterized by skin eruptions, joint pain, recurrent pleurisy, and kidney disease.
Titer—The concentration of substance in a solution described in terms of the degree of dilution required to achieve a threshold effect.
Vacutainer—A glass tube fitted with a rubber stopper from which air is evacuated to produce a slight vacuum, used for blood specimen collection.
Venipuncture—The puncture of a vein for therapeutic purpose or to collect a specimen.
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Patricia L. Bounds, Ph.D.
Table Of Contents
- Detection techniques
- Representative tests
- Antinuclear antibodies test
- Antimitochondrial antibody test
- Antiphospholipid antibody test
- Cryoglobulin test
- Cold agglutinins test
- Rheumatoid factor test
- Health care team roles
- KEY TERMS