Autoimmunity involves the loss of normal immune homeostasis such that the organism produces an abnormal response to its own self tissue. The hallmark of autoimmune diseases generally involves the presence of self-reactive T cells, autoantibodies and inflammation. An area of intense research is determining why the immune system turns against its host. Over the past decade, research has greatly advanced our understanding of autoimmunity and the scientific findings from these investigations are translating new clinical laboratory studies of patients to aid in diagnoses.
Examining patients for potential autoimmune diseases is fraught with difficulty because not one laboratory test establishes such a diagnosis. Typically, multiple laboratory tests are needed and include basic studies like a complete blood count, comprehensive metabolic panel, acute phase reactants, immunologic studies, serologies, flow cytometry, cytokine analysis, and HLA typing. Although some tests may be non-specific, such as the erythrocyte sedimentation rate (ESR), they are useful to assess disease activity. These tests can be useful in the diagnosis and management of patients with autoimmune diseases and help in providing a prognosis, or indicate the severity of organ involvement or damage.
1. Initial laboratory evaluation
Inflammatory diseases will cause abnormalities in routine laboratory studies. Characteristic findings can include a normochromic, normocytic anemia indicating the chronicity or severity of disease. Common hematologic parameters also include an elevated or decreased platelet count and/or white blood cell count. Leukopenia and thrombocytopenia are common in patients with systemic lupus erythematosus (SLE).
Testing will find aberrations in serum levels of specific organ enzymes or abnormalities in metabolic processes that are reflected in the comprehensive metabolic panel. For example, autoimmune hepatitis can be manifested by elevations of transaminases, bilirubin, and serum proteins. One should be aware that these abnormalities can also be associated with drug toxicity.
Coagulation studies such as a prolongation of the activated partial thromboplastin time (aPTT) and/or the prothrombin time (PT) that does not correct with mixing studies suggests an inhibitor of the clotting process is present as seen in the antiphospholipid syndrome. Hypercalcemia can be observed in approximately 30% of patients with sarcoidosis. An increase in muscle enzymes, [creatinine kinase, alanine transaminase (ALT), and aspartate aminotransferase (AST)] can be seen in autoimmune inflammatory myopathies (dermatomyositis, polymyositis, and inclusion body myositis). Serum protein levels are helpful to screen for abnormal elevations of immunoglobulin.
The urinalysis is commonly used to assess renal injury (glomerulonephritis, interstitial nephritis) and will show proteinuria, hematuria or active sediment (white blood cell casts or red blood cell casts). Many other illnesses such as diabetic nephropathy, poorly controlled hypertension, or infections will test similarly but when autoimmune disease is suspect, the common laboratory evaluation will serve as an initial red flag to pursue further testing.1
2. Inflammatory markers
Serum proteins that are produced in response to inflammation can be referred to as inflammatory markers. These proteins are mainly produced by the liver in response to stress and can also be called acute phase reactants. Pro-inflammatory cytokines such as IL-1, IL-6, and TNF-alpha induce synthesis of some acute phase reactants that include CRP, fibrinogen and haptoglobin. Other proteins, like albumin, are not sensitive to inflammatory cytokines for increased synthesis; instead chronic stress (inflammation) results in a lower synthesis rate with resultant decreased serum concentrations. The inflammatory markers are not diagnostic of inflammation, but reflect abnormalities that are seen in autoimmune diseases, infections, malignancies and other illnesses.
Erythrocyte sedimentation rate (ESR)
The ESR is the measure of the quantity of red blood cells (RBC) that precipitate in a tube in a defined time and is based upon serum protein concentrations and RBC interactions with these proteins. Inflammation causes an increase in the ESR. Multiple factors influence the ESR and include patient's age, gender, RBC morphology, hemoglobin concentration, and serum levels of immunoglobulin. The sample must be handled appropriately and processed within a few hours to assure test accuracy. While the ESR is not a diagnostic test, it can be used to monitor disease activity and treatment response and signal that inflammatory or infectious stress is present. For example, in rheumatoid arthritis, the ESR correlates well with disease activity; however normalization of ESR often lags behind successful treatment that causes resolution of the inflammatory state.2, 3
C-reactive protein (CRP)
C-reactive protein (CRP/CRP-high sensitivity) was discovered and named for its reactivity to the C polysaccharide in the cell wall of S. pneumoniae
. CRP, an innate immune protein, helps opsonize pathogens for phagocytosis and activates the complement system. CRP production is under the control of IL-1, IL-6, and TNF-alpha. Changes in serum CRP concentration change more quickly than ESR and therefore CRP may be a better reflection of current inflammation. Unlike the ESR, CRP is a fairly stable serum protein whose measurement is not time-sensitive and is not affected by other serum components. The magnitude of inflammation directly relates to the concentration of CRP. Levels < 0.2 mg/dl are considered normal, while those >1.0 mg/dL are suggestive off inflammation and/or infection. More recently, the use of high sensitivity CRP has been utilized. This test may better quantify lower levels of inflammation and has been important in evaluating cardiac disease and other inflammatory states.2, 3
Serum ferritin is a storage protein for iron and its synthesis is regulated by intracellular iron, cytokines (TNF-alpha, IL-1, and IL-6), products of oxidative stress, and growth factors. Elevated levels can indicate acute or chronic sepsis, inflammation or malignancy. Diseases such as adult Still's disease, systemic-onset juvenile idiopathic arthritis, hemophagocytic lymphohistiocytosis and iron overload diseases, including hemochromatosis or hemosiderosis, should be considered with elevated ferritin levels.4, 5
Less common indicators of inflammatory states include:
the major copper containing protein in the blood that plays a role in iron metabolism and is increased in acute and chronic inflammatory states, pregnancy, lymphoma, rheumatoid arthritis and Alzheimer's disease.7
a hemostatic coagulation factor produced in response to tissue injury. Fibrinogen synthesis is controlled at the transcription level and is increased in the presence of inflammation and stress that is mediated by IL-6.
is produced in response to tissue injury. Increased levels of haptoglobin can be seen during inflammation, malignancy, surgery, trauma, peptic ulcer disease and ulcerative colitis. Decreased levels may indicate chronic liver disease or anemia.
a serum protein synthesized by the liver that aids body tissues in maintaining oncotic pressure necessary for proper body fluid distribution. The average amount of albumin in the plasma is approximately 300 to 400 grams, and about 15 grams is produced by the liver per day. While the rate of synthesis can double in situations of rapid albumin loss as seen in glomerulonephritis or inflammatory bowel disease, serum levels will decline.
3. Autoantibodies and Immunologic Studies
The presence of an autoantibody in a patient does not assure a diagnosis of an autoimmune disease. Rather, a positive serologic test in the company of appropriate signs and symptoms helps to support a diagnosis. Serologic testing is flawed by the presence of autoantibodies in healthy individuals and other patients with non-autoimmune diseases and imperfect testing systems. Historically, many different methods were used to test for the presence an autoantibody. Today, testing is principally done with enzyme immunosorbent assays (EIA) because of cost saving measures with mechanization.
Enzyme-linked immunosorbent assay (ELISA)
ELISA is an immunometric method for detecting and measuring specific antibodies. The basic components of this laboratory method include a substrate where an antigen is fixed (typically a 96 well micro-well plate), patient's sera, washing solutions and a detection method where an enzyme is linked to an antibody that detects the antigen. In a typical double-antibody sandwich ELISA, an antibody that is attached to the bottom of a well provides both antigen capture and immune specificity, while another antibody linked to an enzyme provides detection and acts as an amplification factor. This allows for accurate and sensitive detection of the antigen of interest. However, performance is largely dependent on antibody quantity, kit manufacturer, and operator skill and experience. ELISA permits measurement of only one antigen at a time for a given aliquot of sample. Furthermore, ELISA has a limited dynamic range (that is, the range over which there is a linear relationship between antigen concentration and absorbance reading) – the range is narrow relative to the range for other technologies such as multiplex assays.8
Rheumatoid factor (RF) and Anti-cyclic citrullinated peptide antibody (CCP)
RF is an autoantibody that reacts to the Fc portion of polyclonal IgG; but they can be any class of immunoglobulin. Most assays detect the IgM rheumatoid factor. RF is helpful when evaluating patients who may have rheumatoid arthritis as the sensitivity is ~70% with a specificity of ~70%. Rheumatoid factor is absent in ~15% of patients with rheumatoid arthritis. However, ~15% of the healthy population may have a low titer RF. Rheumatoid factor positive patients are more likely to have progressive, erosive arthritis with loss of joint mobility and also have extraarticular manifestations including rheumatoid nodules, vasculitis, Felty's syndrome and secondary Sjögren's syndrome. In addition, the presence of RF is seen in other autoimmune disorders including Sjogren's syndrome, SLE, cryoglobulinemia, in pulmonary diseases such as interstitial fibrosis and silicosis and various infectious diseases.
Recently, a new biomarker for RA has been described, autoantibodies to cyclic citrullinated peptide (CCP). Inflammation activates the enzyme peptidylarginine deiminase which incorporates citrulline into certain proteins. In RA, autoantibodies are formed against the citrullinated protein (anti-CCP). The presence of serum anti-CCP antibodies are ~95% specific for the diagnosis of RA, with sensitivity similar to rheumatoid factor. Testing for both anti-CCP and RF is beneficial when excluding the diagnosis of RA rather than testing for either antibody alone. In early undifferentiated disease, anti-CCP positive patients tend to go on to have more severe, erosive and aggressive disease. Anti-CCP can also be present in other disease states such as some children with JIA, psoriatic arthritis, lupus, Sjögren's syndrome, inflammatory myopathies and active tuberculosis.2, 3
Anti-nuclear antibody (ANA)
Autoantibodies to nuclear antigens are a diverse group of antibodies that react against nuclear, nucleolar, or perinuclear antigens. These antigens represent cellular components such as nucleic acid, histone, chromatin, nuclear and ribonuclear proteins. Classically, the ANA hallmarks the serologic diagnosis of SLE, but finding an ANA is common to most other autoimmune diseases. Methods used for detection utilize immunofluorescence testing of the patient's serum, at various dilutions, using a cell substrate. Typically, screening patient's serum for the detection of an ANA with ELISA provides high sensitivity but lacks specificity. Results are reported as either the dilution of serum that tests positive or the degree of positivity measured by the testing procedure. Historically, HEp2 cells (a human laryngeal epithelioma cancer cell line) have been used as the cell substrate because the result offers the advantage of detecting a nuclear fluorescent pattern. The fluorescent patterns (homogenous, diffuse, speckled, peripheral and rim) suggest
clinical associations with certain autoimmune diseases
. However, because of the time and expense for testing with HEp2 cells, the assay procedures are largely done by ELISA methods.2, 3
Immunofluorescence is particularly useful as an initial screening test for those individuals suspected of having an autoimmune disease – SLE, Sjögren's syndrome, RA, mixed connective tissue disease (MCTD), scleroderma, polymyositis/dermatomyositis (PM/DM). However, one must use caution when interpreting ANA as this autoantibody is found in nonrheumatic diseases such as Hashimoto's thyroiditis, Graves' disease, autoimmune hepatitis, primary autoimmune cholangitis, primary pulmonary hypertension, and in various infections and malignancies. Furthermore, the presence of low titer ANA occurs more frequently in elderly populations.
details the sensitivity and specificity of the various antinuclear antibodies in several autoimmune diseases. Values are reported as approximate percentages as seen in several published reviews.
Anti-double stranded DNA (anti-dsDNA)
Autoantibodies to double stranded DNA are an important marker used in the diagnosis and monitoring of SLE. Antibodies to dsDNA are highly specific for SLE. However, some patients with other rheumatic diseases or chronic active hepatitis may have mildly or moderately elevated serum titers. Previously, anti-dsDNA was typically measured using radioimmunoassay (particularly the Farr assay). The more common current tests employ an immunofluorescence assay (IFA) or ELISA. The IFA utilizes a target antigen Crithidia luciliae
, a flagellated protozoa containing a dsDNA-containing small organelle called a kinetoplast. The antibodies to dsDNA are detected semiquantitatively by demonstrating IgG bound to the kinetoplast. In contrast, with ELISA testing, the dsDNA is bound to the solid phase of the microwell plate. The serum is incubated and then the bound IgG is detected.2, 3
Anti-extractable nuclear antigen (anti-ENA)
The extractable nuclear antigens consist of the Smith (Sm) antigen, ribonuclear protein (RNP) or U1RNP, anti-SSA (Ro) and anti-SSB (La). They are called extractable because they are readily soluble or extractable in neutral buffers. The Sm antigen is highly specific for SLE, but it is found only in ~25% of SLE patients. The U1RNP antigen is seen in patients with SLE plus systemic sclerosis and in patients with mixed connective tissue disease. The SSA (Ro) and SSB (La) nuclear antigens are often found together in those patients with Sjögren's syndrome. Anti-SSA and anti-SSB are also seen in some subsets of SLE patients. This group includes those patients with subcutaneous lupus erythematosus (SCLE) (prominent photosensitive rashes and sometimes vasculitis), but without severe renal disease. The presence of anti-SSA and anti-SSB is associated with neonatal lupus, in which transplacental transfer of these antibodies (maternal IgG) can cause transient photosensitive rash and/or congenital heart block.2, 3
Anti-signal recognition particle (anti-SRP), anti-JO-1, anti-Mi2, anti-PM/Scl
Anti-SRP, anti-JO-1, anti-Mi-2 and anti-PM/Scl are termed myositis specific antibodies because of the high specificity to the autoimmune inflammatory myopathies (IIM). Anti-SRP antibodies are directed toward an RNA-protein complex consisting of 6 proteins and a 300-nucleotide RNA molecule (7SL RNA). Patients with this antibody have a distinct type of IIM that is characterized by acute onset of muscle weakness, a muscle biopsy that lacks inflammation and the patient shows a poor response to therapy. Anti-JO-1 autoantibodies are the most common autoantibody found in the group of inflammatory myopathies called the anti-synthetase syndrome. Anti-JO-1 autoantibodies are directed against histidyl-tRNA synthetase (an enzyme that attaches histidine to growing polypeptide chains). Other less well understood antisynthetase autoantibodies include anti-PL12, anti-EJ, anti-OJ, anti-PL7, and anti-KS. These antibodies are reportedly more common in PM than DM and are rare in children. Antisynthetase syndrome patients have disease characteristic that are very different than anti-SRP patients and often present with muscle weakness, interstitial lung disease, arthritis and fevers. The anti-JO-1 response appears to be self-antigen driven, with isotype switching and affinity maturation. Anti-Mi2 antibodies recognize a major protein of a nuclear complex formed by about 7 proteins involved in transcription. Autoantibodies to Mi2 are specific for dermatomyositis and associated with acute onset, a better prognosis and good response to therapy. Anti-PM/Scl is an autoantibody to the nucleolar granular component. This is often seen with myositis with scleroderma overlap. Detection of such autoantibodies is done mostly through commercial blot assays utilizing immunoblotting or dot immunoblotting methodologies, in addition to traditional ELISA.8
Antineutrophil cytoplasmic antibody (ANCA) [myeloperoxidase (MPO), proteinase-3 (PR3)
Antineutrophil cytoplasmic antibodies (ANCA) react with cytoplasmic granules of neutrophils. Initial ANCA testing screens sera for the presence of ANCA and two general immunofluorescent staining patterns are observed – cytoplasmic (cANCA) and perinuclear (pANCA). The immunofluorescence pattern is helpful to distinguish various ANCA associated vasculitis syndromes. cANCA is most often seen in Wegener's Granulomatosis (WG), microscopic polyangiitis (MPA) and Churg-Strauss syndrome. pANCA patterns were initially described in microscopic polyangiitis (MPA), but pANCA has now been observed in a variety of diseases including other types of vasculitis, inflammatory bowel disease (IBD), SLE, RA, juvenile idiopathic arthritis (JIA). Antigenic determinants of the ANCA that are important to detect in vasculitis are proteinase 3 (PR3) and myeloperoxidase (MPO). Vasculitic states that test positive are named PR3-ANCA positive or MPO-ANCA positive. The presence of PR3 or MPO can help the clinician to determine the type of vasculitis and activity of disease.
Antibodies to PR3 or MPO are predictive of the various vasculitis syndromes. cANCA plus PR3 have increased positive predictive value (PPV) for ANCA-associated vasculitis, particularly WG. pANCA plus MPO has an increased PPV for MPA and less often for Churg-Strauss. ANCA plus MPO more than PR3 often leads toward a diagnosis of Churg-Straus. With increased disease activity, there is a greater likelihood that ANCA will be positive. ANCA titers may normalize with treatment although persistent ANCA positivity or rising ANCA does not reliably predict disease exacerbation or flare. Therefore, one should not use ANCA titers to determine treatment efficacy. Furthermore, one should also be wary of ANCA positivity as this can be seen in other disease states that include infection, drug use (eg. thyroid medication, particularly PTU), and other autoimmune disease. If the suspicion for vasculitis remains, tissue biopsy should be considered.2, 3, 4, 9
The complement cascade is a complex, tightly regulated series of proteolytic enzymes, regulatory proteins and cell surface receptors that mediate and augment both complement, humoral and cellular immune response. The classical pathway is initiated by immune complexes binding to C1q and involves C4 and C2. The alternative pathway involves factors B, D, and properdin. The mannose-binding lectin pathway, classical and alternative pathways all involve cleavage of C3. This release product induces the formation of the terminal membrane attack complex (C5-C9).
Individual components, such as C3, C4 and factor B, are measured by nephelometry and ELISA. Plasma total hemolytic complement assay (CH50) utilizes a functional assay to assess the functional integrity of the classical pathway. To measure these values, diluted serum is added to sheep antibody-coated erythrocytes and the subsequent value is the reciprocal of the highest dilution able to lyse 50% of the red blood cells. CH50 is a useful screening tool to detect deficiencies of the classical pathway.
Serum levels of complement components can serve as markers of disease activity. In immune complex deposition disease, serum complement proteins are consumed and serum levels decrease. Immune complex disease results from the deposition of antigen-antibody complexes in involved organ tissues. In immune complex glomerulonephritis in SLE, decreased C3 and C4 indicate increased consumption and indicate disease activity. In contrast, increases of C3 and C4 indicate inflammatory disorders as these proteins are also acute phase reactants. Hypocomplementemia is generally not specific for any disease and may be secondary to nonrheumatic diseases such as subacute bacterial endocarditis or poststreptococal glomerulonephritis. If C4 is low compared to C3, it may indicate the presence of cryoglobulins or the genotype C4 null allele. If CH50 is low or undetectable, it may indicate a deficiency of one or more complement components. Genetic/congenital deficiencies of early complement components (C1 to C4) may increase the risk for development of immune-complex diseases. For example, congenital C1q deficiency, although rare in the general population, is associated with individuals who develop lupus.2, 3, 4, 8
Immunoglobulins (quantitative and qualitative)
Measuring total quantitative immunoglobulin (Ig) levels are a key component to any immunologic evaluation. Ig levels reflect B cell function (humoral production and T cell interaction) and serum Ig levels aid in disease detection. Quantitative measurements of serum immunoglobulins, mainly IgG, IgA and IgM are measured via nephelometry. lists diseases that are associated with increased or decreased serum Ig levels.
Differential diagnosis for immunoglobulin levels
Simple qualitative measurements of serum immunoglobulins reflect an individual's ability to mount a humoral immune response. Titers to tetanus, Haemophilus influenza type B (HiB), and pneumococcus can easily be tested to evaluate the quality of the immune response. These levels assess the function of B cells and also detect defects that may indicate immunodeficiency. To assess antibody production, responses to protein and polysaccharide antigens should be evaluated. B-cell testing is done primarily by in vivo (vaccination) studies. Protein vaccinations, like tetanus toxoid, measure T-cell dependent responses. Polysaccharide vaccines, like Pneumovax, measure T-cell independent responses.
Testing of specific antibody titers (such as to influenza immunization) are reported relative to protective values. These values are based on epidemiologic data regarding protection in larger populations. For randomly acquired antibody levels, an initial comparison to protective values can be used to decide if a proper immune response was achieved. A four-fold increase in titers to protein vaccination indicates a normal response. A two-fold increase in titers to a polysaccharide antigen indicates a normal response. Failure to mount an appropriate response to antigen is a clue to the physician to pursue B and T cell function further.2, 3, 4, 8
Cryoglobulins are immunoglobulins that precipitate reversibly in cold temperatures. In disease states, these antibodies can bind with complement proteins and other peptides to form immune complexes and cause tissue damage. Three types of cryoglobulins exist. Type I cryoglobulins are monoclonal immunoglobulins often of the IgM isotype. Type II cryoglobulins are a mixture of polyclonal IgG and monoclonal IgM. Type III cryoglobulins are a combination of polyclonal IgG and polyclonal IgM.
At phlebotomy, whole blood is obtained in pre-warmed tubes without anti-coagulant and maintained at body temperature until coagulation occurs (about one hour). The sample is then centrifuged and the clot is removed. The remaining serum is placed at 4°C up to several days. The specimen is then examined daily to determine if proteins have precipitated. Once a precipitate is present, the sample is spun again and a cryocrit is measured in a calibrated tube. Confirmation of the cryocrit is seen if the precipitate redissolves when placed in a 37°C water bath.
Cryoglobulins are nonspecific indicators of disease states. Type I monoclonal cryoglobulins are associated with multiple myeloma, Waldenstrom's macroglobulinemia, and lymphoproliferative disorders. Type II and III cryoglobulins can bind complement, unlike type I, and are associated with Hepatitis C and small vessel vasculitis. The presence of multiple immunoglobulin components within the cryoglobulin is known as “mixed cryoglobulin.” Symptoms generally associated with cryoglobulins include purpura, ulcerations, Raynaud's phenomenon, arthralgias, proteinuria and renal failure. Cryoglobulins are rarely found in children.2, 3, 4, 8
Lupus Anticoagulant (LAC)/anti-cardiolipin (aCL)/antiphospholipid autoantibodies (aPL)
In autoimmune disease, serum antibodies that inhibit or prolong in vitro
clinical laboratory coagulation tests are termed anti-phospholipid antibodies (aPL, also called anti-cardiolipin antibodies or lupus anticoagulant) because they are directed against phospholipids and phospholipid-binding proteins. The existence of these antibodies is associated with the anti-phospholipid antibody syndrome (APS). APS is suspected in individuals that have venous and/or arterial thromboses, recurrent fetal loss, or thrombocytopenia. APS can occur independent or with systemic rheumatic diseases. Anti-phospholipid antibodies can also be found in healthy individuals and in patients with various infections that do not have features of anti-phospholipid antibody syndrome. The term anticoagulant is paradoxical in that the presence of aPL are associated with thromboses in patients. The presence of aPL can be directly measured by enzyme linked assays. IgG anticardiolipin has a higher predictive value than IgM or IgA of a thrombosis. Beta-2 glycoprotein I has been identified as one of the major antigenic determinants of antiphospholipid antibodies.11, 12, 13, 14
4. Flow cytometry
Flow cytometry is a technique where particles or tagged cells flow through laser light so that populations of particle/cells can be counted and phenotyped using cell characteristics and surface proteins. Initial applications of flow cytometry pertained to the interest in certain cell populations, for example the numbers of lymphocytes in patients infected with human immunodeficiency virus (HIV). The number of T cells that are CD4 positive is an important gauge of severity of HIV infection. However, the methodology has greatly expanded its role such that cell cycle analysis, quantification of malignant cells and activation status of lymphocyte subpopulations can be determined. When evaluating a patient with a suspected immunodeficiency, flow cytometry is crucial to determine the quantitative number of immune cells (typically T, B and NK (natural killer) cells). Remember, flow cytometry testing reveals numbers of cells and does not indicate cellular function. Testing for cellular functioning involves other laboratory methods, such as quantitative immunoglobulin levels to indicate proper B cell function.15, 16
The markers commonly used to assess lymphocyte subsets by flow cytometry are as listed in .
The flow cytometry device consists of an illumination source, an optical bench, fluidic system, electronic monitoring and computer. Cells that will flow through the cytometer are first prepared by tagging cell surface molecules with fluorescently labeled monoclonal antibodies. Illumination of the cell occurs by air-cooled lasers that provide a monochromatic light source (argon at 488 nm or blue, helium neon at 633 nm or red). The point of illumination occurs within the flow cell. The optical bench contains lenses that shape and focus the illumination beam. Nonfluorescent and fluorescent signals generated by the labeled cell are collected and measured by a detection system consisting of filters linked to a photodetector. Cells are injected into a moving fluid sheath to establish a focused single-file flow of cells that move through the analysis point. Differences in the magnitude of emission signal generated from each cell reflect biologic differences between the cells. Software collects data that analyzes cell subpopulations based on the presence or absence of labeled antibody binding. Data is then presented as fluorescence intensity versus cell number.8
5. Cytokine studies
Cytokines are molecules secreted by a variety of cells that function in cellular communication. Immunologists are keenly interested in cytokines, particularly those that influence immune function and inflammation. Commercial testing laboratories do not routinely assay most serum cytokine levels, as this testing is largely done in research laboratories. Testing is laborious because of the labile nature of these small molecules. After phlebotomy, the serum needs to be quickly removed from the cellular components and frozen as quickly as possible and testing should not be delayed. Laboratory methods commonly used to assay cytokine levels include flow cytometry and ELISA.15, 16
Cytokines that influence inflammation include IL-1, IL-6 and TNF-alpha. There is extensive evidence that these cytokines promote inflammation and therefore have become targets for therapy. Rheumatoid arthritis is the best example of an autoimmune illness where anti-TNF therapy has revolutionized the natural history of the disease. Targeting TNF with proteins (fusion produced or monoclonal antibodies) that antagonize TNF action results in dramatic improvement of disease activity. In fact, rheumatoid arthritis is the prototypic autoimmune disease where the efficacy of anti-cytokine therapy is best demonstrated. Currently, anti-TNF, anti-IL-1 and anti-IL-6 therapies have all proven to be effective in treating rheumatoid arthritis.
6. Major Histocompatibility Complex (MHC) (human leukocyte antigen (HLA))
Human leukocyte antigen (HLA) is synonymous with the major histocompatibility complex (MHC). MHC class I and II genes are the major genetic determinants of susceptibility to many autoimmune diseases. MHC class I molecules include HLA-A, -B, and –C. MHC class II molecules include HLA-DR, HLA-DQ, and HLA-DP. Detection of HLA type can be done routinely and can be assayed using several methods that include gel electrophoresis, polymerase chair reaction (PCR), ELISA, and newer methods employing high-throughput detection of nucleic acid. Many antigens of the MHC, especially of HLA class I and II, have been associated with rheumatic disorders. HLA-B27 is present in approximately 90% - 95% of white patients with ankylosing spondylitis and only 7% to 8% of the general population. HLA-DR1 and HLA-DR4 increase the risk of polyarticular juvenile idiopathic arthritis (JIA) in many populations. HLA-DR3 and HLA-DR2 are associated with lupus in Caucasian populations, while much of the risk attributable to MHC is associated with variation at HLA-DRB1 in patients with rheumatoid arthritis.3, 17, 18