The immuassay handbook parte86

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The immuassay handbook parte86

  1. 1. 869© 2013 David G. Wild. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/B978-0-08-097037-0.00069-5 The term “autoimmune disease” implies the presence of a well-defined entity where an aberrant reaction of the immune system with a specific organ or tissues has resulted in the development of a disease. This is rarely true. In real- ity, the clinical conditions of individuals with autoimmune diseases are extremely heterogeneous. Although they do get lumped together in chapters such as this, they have little in common except that one is able to demonstrate some sort of autoreactive phenomenon—usually autoantibodies in their serum. Indeed, a clear-cut causal relationship between the autoreactive phenomenon and the pathogenesis of the dis- ease is uncommon. Such a relationship is found in some con- ditions such as Goodpasture’s syndrome, where an autoantibody directed to the glomerular basement mem- brane is logically related to the glomerular disease and per- haps to the respiratory disease that these patients experience. However, in the vast majority of autoimmune diseases, the autoantibodies demonstrated in serum are, indirectly, or not at all related to the pathological lesions. A good example is primary biliary cirrhosis, where patients suffer progressive damage to their bile ducts that eventuate in liver failure. Yet, their serum does not contain antibody or cellular reactivity against bile ducts. It contains antibodies that react with pyruvate dehydrogenase complex (PDC-EW) that is present in mitochondria. Because the test was originally described as an immunofluorescence assay, the term used in the literature remains antimitochondrial antibody (AMA). However, these antibodies are not specific for mitochondria in bile ducts. Since mitochondria are not on the cell surface, they are resistant to exposure to these antibodies. Thus, although the presence of AMA is useful to the clinician in confirming the diagnosis of primary biliary cirrhosis, it does not provide information about the pathogenesis of the disease and serves as an example about the assumptions that are inherent in the use of the term “autoimmune disease”. Is primary biliary cir- rhosis caused by the immune system reacting with the bile ducts? It is not known. Yet, because of the presence of AMA and other autoantibodies (see later) in the serum, primary biliary cirrhosis is classified as an autoimmune disease. Because one does not expect the body to develop immune reactivity to its own components, a wide variety of theories and models have been developed to explain how this may come about. This is not a new concept. Indeed, Paul Erlich referred to the ability of the immune system to react against an individual’s own tissues as “horror autotoxicus”. With our more recent information about the subtypes of cells within the immune system, a better understanding has evolved about how such self-reactivity may occur. The immune system has built in checks and balances between the T and B lymphocytes by which the body develops tol- erance to its own antigens. Some of the T cells are suppres- sor cells that inhibit the formation of immune reactivity against a variety of antigens, including self-antigens. The concepts of how the body’s immune system is able to escape from this “tolerance” to self-antigens and produce antibod- ies and/or cell-mediated immunity against its own cells are wide ranging and beyond the scope of this chapter. Some recent review articles are recommended for those inter- ested in more details (Bonilla et al. 2007; Grossman et al. 2011; Hanley et al. 2011; Lopez-Hayes et al. 2007; Maguire et al. 2009; Meroni et al. 2010; Sjowall et al. 2008). Thankfully, for the purposes of using the presence of autoantibodies for clinical diagnosis, it is not necessary to know why they are formed nor does one need to assume that they are involved in the pathogenesis of the disease. They are serving as markers of the diseases described in this chapter. But even as markers of disease, there is con- siderable variation in the utility of autoantibody assays. This is because a certain amount (albeit very low levels) of autoreactivity exists in all of us. Some have suggested that antibodies against nuclear antigens, for example, are a nor- mal part of the tissue repair, aging, cell renewal, or a reac- tion to microbial DNA. Others hypothesize that they are part of our mechanism for dealing with development of neoplastic cells or as a method to heighten the response to infections. As more sensitive assays have been developed, we detect the presence of low levels of many types of auto- antibodies in clinically well individuals. By requiring a rela- tively high level of antinuclear antibodies (ANA) reactivity, such as a screening cutoff of 1:160, one can screen asymp- tomatic individuals while detecting most cases of clinically significant autoimmune diseases such as systemic lupus erythematosus (SLE). However, this will exclude individu- als with more subtle conditions (Kavanaugh et al., 2000). In the past few years, controversies have arisen about the use of older “classical” tests, such as the indirect immuno- fluorescence (IIF) test for ANA versus newer assays that employ specific antigens found in these cells (Meroni and Schur 2010; Maguire et al., 2009). Such controversies often center on whether the testing is to be used as a general screening test for a variety of autoimmune conditions or in detecting a few specific conditions (see below). When reviewing the various tests detailed below, one should focus on the specific antigen preparation used rather than the name of the test given by the manufacturer. For instance, although one may glibly speak about ANA reac- tivity, what does it really mean? There are a plethora of proteins and nucleic acids in the nucleus that can serve as antigens in the ANA test. Therefore, a positive ANA means reactivity against something in the nucleus but does not tell you the antigen specificity of the autoantibody. For this reason, the ANA test as a positive or negative is often viewed as a general screen. When the assay is employed on symptomatic patients and performed with an appropriate screening dilution of serum (1:160 in our laboratory), a negative result makes it highly unlikely that the patient has Autoimmune Disease David F. Keren (dkeren@umich.edu) C H A P T E R 9.15
  2. 2. 870 The Immunoassay Handbook SLE. Therefore, under those conditions, the test retains reasonable sensitivity and the relatively high cutoff of 1:160 improves its positive predictive value. But when used on all patients with minor aches and pains or with serum that is diluted only 1:20, a large number of normal people will have a positive ANA and this compromises its positive predictive value. A lack of understanding about the limita- tions of tests such as ANA leads to misunderstanding about its utility and a global mistrust of the laboratory. Lastly, all ANA screening tests are not identical. When frozen sections of animal tissue substrates are used, different results can be obtained than when tissue culture monolayers are used for these IIF assays. Further, the type of fixative used affects the results. Newer technologies include immu- nometric assays (enzyme immunoassays and chemilumines- cence assays) and multiplex assays such as line probe assays and multiplex bead analysis using a flow cytometer. Because different antigen preparations are used for these assays, it should not be surprising that data obtained from IIF assays may differ from those obtained with the immunoassays that employ different mixtures of nuclear antigens. Unfortunately, standardization of these diverse tech- nologies is still lacking. Results obtained from one manu- facturer’s ANA screen will differ from those of another manufacturer who uses a different mixture of antigens. Yet, no matter which method is used, the final result usu- ally will appear on the patient’s chart as “ANA positive” or “ANA negative”. It is very difficult for clinicians to be knowledgeable about the nuances of the above testing variations. Therefore, a laboratory must be prepared to advise clinicians about appropriate screening tests and to answer questions about possible limitations and helpful further testing. We keep a knowledgeable pathologist available daily to help the clinician interpret this informa- tion and to guide further testing. Analytes ANTINUCLEAR ANTIBODIES ANA are extremely heterogeneous antibodies that are directed against components of the nucleus and despite the test’s name, cytoplasm. Detection of these antibodies is used as a screen for SLE and other connective tissue (rheu- matoid) diseases. Reference Interval The reference interval varies widely depending on the type of assay one is using and the manufacturer of that assay. For an IIF autoantibody test, the ideal reference interval is no detectable nuclear reactivity at the titer chosen by the individual laboratory as its cutoff. That cutoff will vary from laboratory-to-laboratory and from one substrate to another. Some laboratories use the substrate manufactur- er’s suggested cutoff that can be as low as 1:40. However, a standard cutoff for one kit does not take into consideration the variables inherent to each laboratory. For instance, the type of microscope is key; its light source, optics, and con- denser all play a role in the detection of ANA. Other labo- ratoriesestablishtheircutoffbystudyingnormalindividuals of appropriate age and gender for the population tested. A 5–10% positive rate among control individuals is chosen as the typical cutoff. In our laboratory, for instance, we have established a cutoff of 1:160. The disparity from one laboratory to another has made comparisons of samples from the same patient performed at different laboratories difficult and laboratorians need to be aware of it. For the immunometric ANA screening methods, the reference interval is less than a defined optical density or chemiluminescence intensity for a particular lot of a spe- cific company’s kit. Although some studies have found results from immunometric kits compare well with stan- dard IIF ANA kits, the lack of a common standard for the concentration of antigen preparation used in immunomet- ric assays and indeed, the contents of the assays vary mark- edly (Table 1). Not surprisingly, the reaction of some cases varies from laboratory-to-laboratory and kit-to-kit. Fur- ther, little independent information is available in the lit- erature about lot-to-lot variation. The ease in automation of immunometric techniques has led to relatively wide- spread use. Hopefully such broad utilization will encour- age manufacturers to standardize the reactivity expected from the antigen preparations used. Clinical Applications In the past 5 years, a considerable controversy has flared regarding which ANA test is the most suitable screen. A brief glance at Table 1 demonstrates the heterogeneity of antigens included in the testing kits. However, the varia- tion is even greater than listed. The table just shows which antigen preparations are included. It says nothing about how much of each antigen is included. Further, when one is dealing with antibody–antigen interaction, the three- dimensional aspect of the antigen presentation is relevant. Therefore, how it is attached to a microtiter plate or bead is another factor. Solid evidence-based medicine is lacking at the present to allow a clear recommendation of one technique versus another. Because of this, the American College of Rheu- matology presented a position statement stating their pref- erence of the classic IIF HEp-2 cell assay as a screening test for detecting ANA antibodies (Meroni and Schur, 2010). Their recommendations are: G The immunofluorescence ANA test should remain the gold standard for ANA testing. G Hospital and commercial laboratories using bead-based multiplex platforms or other solid-phase assays for detecting ANAs must provide data to ordering physi- cians on request that their assay has the same or improved sensitivity and specificity compared to the IF ANA. G In-house assays for detecting ANA as well as anti-DNA, anti-Sm, anti-ribonucleoprotein (RNP), anti-Ro/SS-A, anti-La/SS-B, etc. should be standardized according to national (e.g., CDC) and/or international (e.g., WHO, IUIS) standards. G Laboratories should specify the methods utilized for detecting ANAs when reporting their results. These recommendations are helpful in many ways but may underestimate the effect of subjectivity and a relatively high false positive rate of the traditional ANA assay by IIF.
  3. 3. 871CHAPTER 9.15 Autoimmune Disease It is certainly useful for laboratories to disclose the type of testing they are performing. However, it is doubtful that most users will understand the variability involved in ANA testing discussed above. Further, the claim of the IIF ANA being the “gold standard” is a product of relatively weak anecdotal case series as evidenced by the information used by Meroni and Schur to support the claim of superiority of the IIF ANA assay (Table 2). Unfortunately, these claims do not take into consider- ation the false positive rate of the assays used. In Kavanaugh et al.’s guidelines of ANA testing, the problem of false positive assays is emphasized. There is no doubt that in some cases of autoimmune disease one method may detect an antibody prior to another. However, the claim that greater than 90% of ANA samples should be positive at the time of diagnosis of conditions such as SLE is not consis- tent with more recent knowledge of the history of the development of autoimmune serology in these patients. A definitive long-term cohort study by Arbuckle looked at the results of 30 million Department of Defense recruits and identified 130 who fulfilled the clinical criteria for SLE. By using the HEp2 ANA test at 1:120, they found that about 80% had a positive ANA test at the time of their first manifestation of SLE (Fig. 1) (Arbuckle et al., 2003). Interestingly, the specific tests for SSA and SSB were pres- ent sooner and also more prevalent at the first manifesta- tions of SLE. Such data suggest that antigen-specific assays, whether immunometric or multiplex bead assays with flow cytometry, will continue to improve and will soon (if they have not already) overtake the subjective IIF assays for ANA screening. Typically, the ANA test is used to screen for the pres- ence of SLE, but antibodies that react with the nucleus are present in a wide variety of other conditions including: mixed connective tissue disease (MCTD), scleroderma, Sjögren’s syndrome (SS), drug-induced lupus, polymyosi- tis, dermatomyositis, Raynaud’s phenomenon, primary biliary cirrhosis, and in as many as 10% of normal indi- viduals. The absence of ANA is useful in ruling out SLE because it is found in 95–98% of patients at some point during their struggle with that disease. However, its pres- ence cannot by itself establish that diagnosis. Multiple spe- cific clinical features must be present along with specific antibodies to define the presence of SLE in an individual. Antibodies to many of the different antigenic components of the nucleus can now be distinguished with specific tests. Some of them, such as antibodies against Sm and DNA, are highly specific for SLE. When the IIF test for ANA is used, a laboratory usually reports a pattern of reactivity in addition to its titer (Table 3). Before the advent of specific antigenic testing, the pattern of reactivity was used as a determinant of subsequent testing. Now the presence of specific antibody tests is the focus of the serologic evalua- tion of patients with positive ANA testing. TABLE 1 Antigens Used in Commercial Multiplexed ANA Screening Tests Antigen Manufacturer and Product* 1* 2 3 4 5 6 7 8 9 Centromere B X X X X X X X X Chromatin X X X ds DNA X X X X X Histones X X X X X Jo-1 X X X X X X X X X Mitochondrial M2 X PCNA X X PM-SCL X Ribosomal P X X X X X X RNP X X X X RNP-68 X X X RNP-A X X X RNP C X X Scl-70 X X X X X X X X X Sm X X X X X X Sm B X X Sm D X X X SmRNP X X X X SSA X X X X SSA-52 X X X X X SSA-60 X X X X X SSB X X X X X X X X X *1. Immunogenetics INNO-LIA™, 2. IMTEC-ANA LIA™, 3. Hikrogen recombLine ANA/EIA, 4. Euroimmun Euroline™ ANA Profile, 5. ZEUS Scientific AtheNA™, 6. Inova QUANTA Plex™ ENA Profile 6, 7. BMD Fidis™ Connective 9, 8. Bio-Rad BioPlex® 2200 ANA Screen, and 9. SmartBead Technologies UltraPlex™ ANA Profile. Source: From Binder (2006).
  4. 4. 872 The Immunoassay Handbook Limitations Although ANA testing is quite sensitive for detecting SLE, it is highly nonspecific. Despite some of the con- cerns expressed above, there does not seem to be any sig- nificant difference in specificity between the IIF method and solid-phase immunoassays. Many normal individuals will have a positive ANA. ANA reactivity has also been found in individuals with nonspecific rheumatologic com- plaints. Sometimes, the titer of unaffected individuals can be relatively high (such as 1:640). Therefore, patient –5 –4 –3 –2 –1 0 1 2 3 4 5 Time (yr) 100 80 60 40 20 0 ANA Anti-Ro Anti-La Anti-Sm Anti-dsDNA Anti-nRNP APL First manifestation of SLE PatientswithPositiveTest FIGURE 1 Temporal sequence of development of autoantibodies prior to and after development of initial clinical symptoms in patients with SLE. (From Arbuckle et al. (2003) used with permission). (The color version of this figure may be viewed at www.immunoassayhandbook.com). TABLE 3 Reactivity of ANA on HEp-2 Substrate Pattern Specificities Homogeneous/Peripheral dsDNA, ssDNA, Histone, DNP Speckled Sm, RNP, SSA/Ro, SSB/La, Scl-70, Centromere Centromere Nucleolar Nucleolar RNA TABLE 2 Comparison of Indirect Immunofluorescence with Solid-phase Immunoassays in Detection of Systemic Lupus Erythematosus Source No. of SLE Patients IFA (% SLE Patients Pos) Solid-Phase Assay (% of SLE Patients Pos) Solid-Phase Assay Method 1* 55 91% (1:80)* 87% 89% 78% Radim SpA EIA Zeus EIA VarElisa ReCombi 2 53 91% (>1:50) 49% Athena Multilyte 3 71 (SLE, DLE, drug-induced) 98% (1:40) 91% RADIAS (Bio-Rad) 4 34 76% (>1:160) 62% ELIA Pharmacia 5 202 87% 75% VarElisa ELISA 6 50 84% at 1:50 80% at 1:100 76% at 1:200 75% 40% 56% VarELisa AntiNucleosomes GmBH QUANTA Lite (INOVA) 7 38 92% 79% QUANTA Lite (INOVA) 8 192 99% (81%)+ 75.5% Bioplex 9 35 97% (>1:160) 100% 94% 100% 60% 62% Quanta Life Bio-Rad Relisa VarElisa UniCap +Two different percentages are reported in the paper. *References: 1. Bernardini et al., 2004; 2. Bonilla et al., 2007; 3. Gniewek et al., 1997; 4. Gonzalez et al., 2005; 5. Lopez-Hoyos et al., 2007; 6. Sjowall et al., 2008; 7. Ulvestad, 2001; 8. Hanly et al., 2010; 9. Fenger et al., 2004. Source: From Meroni and Schur (2010).
  5. 5. 873CHAPTER 9.15 Autoimmune Disease selection prior to testing (i.e., symptomatic not general screening) and subsequent specific antibody testing are critical to its effective use. Assay Technology Most laboratories still use an IIF assay, however, immuno- metric and multiplex assays are now available and will likely replace IIF in the future. Indirect immunofluorescence IIF involves incubation of the patient’s serum on a sub- strate that contains nuclei. At the present time, tissue cul- ture substrates (especially the HEp-2 cell line) have virtually replaced the older frozen section substrates. HEp-2 cells are much larger than the frozen sections of rodent liver and kidney and easier to read. Further, the fro- zen section substrates neither represent centromere anti- gens to advantage nor do they preserve the important SSA/ Ro antigen as well as HEp-2 substrates. HEp-2 cells possess well over 100 antigens in the nucleus and cytoplasm that may have relevance for patients with autoimmune disease. The large number of antigens expressed is one reason that the American College of Rheumatology recommends this technique. However, this also increases the false positive rate and nonspecificity in unaffected individuals. For the technique, the substrate is incubated with the patient’s serum for about 20–30 min (Fig. 2). Following incubation with the patient’s serum, the substrate is washed thoroughly with buffer and then covered with fluorescein- conjugated antibody against human IgG. This binds with any immunoglobulins from the patient’s serum that are attached to the nuclear antigens. After this incubation, the substrate is then washed with buffer, a coverslip is applied to the substrate, and the slide is examined by fluorescence microscopy. The presence of reactivity against the nuclear (or some cytoplasmic) antigens is recorded along with the pattern of fluorescence and the strength of the reaction (indicated by the reciprocal of the highest dilution of the patient’s serum that gives reactivity). One variation of this technique employs an enzyme-conjugated anti-human immunoglobulin that gives a colored reaction product when the chromogenic substrate is added to the enzyme. While this obviates the need for a fluorescence micro- scope, it requires an additional substrate step that slows the overall process. Since most laboratories that perform the ANA test have fluorescence microscopes for other assays and are familiar with its use, the enzyme immuno- histochemical technique is not widely used. The pattern of nuclear fluorescence has traditionally been used to indicate the specificity of autoantibodies present. Homogeneous and peripheral reactivity with the nuclei are the patterns most strongly associated with SLE because they can be a result of antibody directed against double-stranded (ds) DNA. Unfortunately, the specificity of this pattern is often overrated. Antibodies against other nuclear antigens, such as histone, can produce this pattern. Since anti-histone may be associated with drug-induced lupus, a transient phenomenon, this pattern alone is not specific for SLE. The nucleolar pattern has been associated with sclero- derma, but there are much better specific tests such as Scl-70 and centromere antibodies for this condition (see below). Centromere patterns are readily detected on HEp-2 cells. Each centromere is the same size and they line up nicely in mitotic cells. The detection of this pattern is used to define a relatively mild form of scleroderma. The other major reaction seen is the speckled pattern. Attempts to further subdivide the speckled pattern into fine speckles, coarse speckles, or other such descriptions have been difficult to reproduce due to interpretive subjec- tivity. Highly expert technologists can distinguish patterns that may be useful in suggesting conditions such as pri- mary biliary cirrhosis. However, subdividing speckled pat- terns has lost some of its luster now that specific antibody tests are available to identify the most clinically significant autoantibodies. Immunometric assays As noted above, immunometric assays are relatively new techniques that can provide an efficient, automated screen for ANA. However, as shown in Table 1, a wide variety of antigen preparations are used by commercial suppliers and the laboratory needs to be aware of which autoantibody the method can and cannot detect. A typical antigen prepara- tion that includes clinically relevant nuclear components such as chromatin, DNA, Sm, RNP, SSA/Ro, SSB/La, Jo-1, and Scl-70 among others is used to coat a solid sur- face (usually a microtiter well). The immunometric proce- dure is a standard, easily automated serology technique. Serum diluted in buffer reacts with the antigen-coated solid surface (microtiter well or coated bead) followed by a wash step. Several different probes are used to follow the reaction. For immunometric techniques either enzyme or chemiluminescent-conjugated anti-human immunoglobu- lin is used to detect the autoantibodies that have bound to the nuclear antigens on the solid surface (Fig. 3). Follow- ing another wash step, either substrate (for the enzyme) or a catalyst (for chemiluminescence) is added and the result- ing color or light produced is compared to that of standard positive and negative solutions provided. Multiplex assays Although current immunometric assays can detect ANA reactivity in general or specific antibodies in separate reac- tion wells, multiplex assays allow simultaneous detection of autoantibody reactivity to several autoantigens in a single reaction. There are two forms of multiplex assays in current use: the line probe assay and the multiplex bead-based assay. FIGURE 2 IIF technique. A substrate containing the antigen of interest is placed on a glass slide. The patient’s antibodies react with the antigen. After a wash step, a reagent (often mouse or rabbit) anti-human IgG that is labeled with fluorescein is added. Following another wash step, a coverslip is applied to the substrate and the slide is examined with a microscope equipped for darkfield fluorescence.
  6. 6. 874 The Immunoassay Handbook Line probe assay This is an immunoblotting assay similar to the immunob- lot assay used as serologic confirmation for Hepatitis C infection. The LINNO-LIA™ ANA assay (INNOGE- NETICS, Belgium) coats relevant antigen on a nylon membrane with a plastic backing. The system uses several recombinant antigens (SmB, RNP-70k, RNP-A, RNPC, Ro52, SSB/La, Cenp-B, Topo-I [Scl-70], Jo-a), synthetic peptides (SmD and ribosomal P), and natural proteins (Ro60 and histones). FIGURE 3 Immunometric assay using enzyme-labeled antibody as example. Antigen of interest is coated to a solid surface (microtiter well or bead). The patient’s antibodies react with the antigen. After a wash step, a reagent (often mouse) anti-human IgG that is labeled with an enzyme is added. Following another wash step, the substrate for the enzyme produces a color reaction.
  7. 7. 875CHAPTER 9.15 Autoimmune Disease For the procedure, the patient’s serum is incubated in a trough with the antigen-coated nylon strip. After the incu- bation, the strips are washed and a goat anti-human IgG labeled with an enzyme is incubated. Following another wash step, the substrate is added and the resulting bands are compared to that of standard positive and negative solutions provided (Fig. 4) (Maclachlan et al., 2002). Multiplex bead assays The multiplex bead assays merge the advantages of solid- phase antigen presentation together with the discrimina- tory powers of flow cytometry to simultaneously examine several autoantigens. The BioPlex 2200 ANA assay uses microsphere beads that have been dyed with various con- centrations of two fluorochromes. Then each bead of a unique color is coated with one of the following antigens: dsDNA, chromatin, RNP-A, SSB, SSA-52, Scl-70, Sm, Cent B, SmRNP, Ribo P, RNP-68, SSA-60, and Jo-1. As with immunometric techniques, the antigen-coated beads are incubated with the patient’s serum allowing any autoantibodies present to attach to the beads. After a wash step, the beads are exposed to a fluorochrome-conjugated antibody against human immunoglobulin. Following a final wash to remove unbound conjugated antibody, the beads are directed by lamellar flow into single file through the flow cytometer where they are interrogated by two laser beams. The red laser identifies the particular antigen on the bead by their internal dye, the green laser verifies that the bead has a specific autoantibody attached. The result is a detailed account of specific antibodies against a wide variety of antigens in the patient’s serum. While some differences exist between the currently available multiplex systems, current studies of multiplex bead assays have shown a high degree of reliability in the detection of specific nuclear antigens (Grossmann et al., 2011). The greatest challenge to this methodology remains standard- ization. Lack of ribosomal components is a cause of falsely negative studies in patients with scleroderma (Shanmugam et al., 2011). Type of Sample Serum or plasma. ANTI-DSDNA Anti-dsDNA antibodies are found mainly in patients with SLE. They usually produce a peripheral or homogeneous pattern on HEp-2 cell line preparations used in the IIF assay. However, that assay is not specific for anti-dsDNA. Reference Interval Anti-dsDNA should be less than the cutoff. With the IIF test, it will be undetectable at the titer used for the Cri- thidia luciliae test. With the immunometric or multiplex assays, anti-dsDNA will give a reading less than that of the cutoff established by the kit manufacturer. Clinical Applications Anti-dsDNA is one of the most specific laboratory tests for SLE. It correlates with the presence of renal disease in affected patients, one of the most common causes of death in these patients. The glomeruli of patients with SLE who suffer from renal disease usually contain complexes of DNA with anti-DNA antibodies and complement. The titer of anti-dsDNA may correlate with disease activity. Anti- single-stranded (ss) DNA is also present in patients with SLE as well as in patients with drug-induced lupus. It does Control line SmB SmD RNP-70k RNP-A RNP-C SSA/Ro52 SSA/R060 SSB/La Cenp=B Topo=1/Scl--70 Jo-1/HRS Ribosomal P Histones 1. Add serum 2. Wash 3. Add anti-IgG-enzyme 4. Wash 5. Add substrate FIGURE 4 Line probe immunoblot assay. Strips of nylon coated with the antigens indicated (left) are incubated with patient’s serum. The patient’s antibodies react with the antigen. After a wash step, anti-human IgG that is labeled with an enzyme is added. Following another wash step, the substrate for the enzyme produces a color reaction. The intensity of the reactivity is compared to that of controls internal to each strip (right).
  8. 8. 876 The Immunoassay Handbook not have the same specificity as anti-dsDNA for renal dis- ease or disease activity. Anti-ssDNA is not a useful test in the differential diagnosis of SLE (Tran and Pisetsky, 2006). Limitations Anti-dsDNA is not an effective screening test, because patients with SLE may lack them at the onset of symptoms while other antibodies such as SSA/Ro are present (Fig. 1). Thus, the absence of these antibodies does not exclude SLE. However, the presence of anti-dsDNA is strong evi- dence supporting the diagnosis of SLE. Although the assays claim to be specific for anti-dsDNA, many of these patients have antibodies that cross-react with either ss or dsDNA. Anti-ssDNA has less specificity for SLE but is often present in the serum from these patients. Some patients with drug-induced lupus may have anti-ssDNA. Assay Technology Most assays for anti-dsDNA use immunometric techniques as described above. For the assay, preparations of dsDNA are coated onto a solid surface (a microtiter well or a poly- styrene bead). Serum is incubated with these coated sur- faces. Following a wash step with buffer, an antibody labeled with a fluorochrome, enzyme or chemiluminescence probe (depending on the system used) conjugated to anti-human immunoglobulin is incubated with the well. With enzyme labels, an additional substrate reaction step is performed. Finally, the reaction product signal is compared to known reactivity in positive and negative samples provided by the kit manufacturer. The older Farr assay is rarely used today. It was a radioimmunoassay (RIA) wherein radiolabeled DNA was allowed to react with anti-dsDNA in serum. The complexes were precipitated from solution by either spe- cific antibodies or an ammonium sulfate solution. IIF is another method used to identify anti-dsDNA. The substrate is the hemoflagellate C. luciliae. This test takes advantage of the fact that the kinetoplast of these microorganisms contains dsDNA with no ssDNA. It should be noted that the kinetoplast also contains a small amount of histone which may result in a false positive response with strong anti-histone antibodies in some patients with drug-induced lupus. Type of Sample Serum or plasma. ANTI-SM Anti-Sm (short for Smith—the name of the first person described with this reactivity) results in a speckled pattern by IIF ANA assays. Sm is a 95kDa protein that exists as a nuclear protein-RNA complex as part of the RNP antigen. Both Sm and RNP are part of the ribonucleoproteins that function to splice transcriptional messenger RNA. The antibodies against Sm react with several U1RNA antigens: B, B′, D, and E. Reference Interval Most laboratories use an immunometric assay to detect anti-Sm. The reference interval is reactivity below the cut- off established by the manufacturer. Clinical Applications Anti-Sm reactivity is present only in 20–30% of patients with SLE. However, its presence is strong evidence for the pres- ence of SLE. Unlike the ANA test which is very sensitive, but not specific, the anti-Sm test is very specific for SLE. Limitations Only 20–30% of patients with SLE will have anti-Sm. Because of cross-reactivity with RNP antibodies, stringent conditions must be used to avoid false positives with that antibody. Assay Technology Immunometrictechnologyandmultiplexsystemsasdescribed above use antigen preparations that contain Sm. After reac- tion with the patient’s serum, the strength of the reactivity is compared to known positive and negative controls. Immunoblotting analysis may also be used to determine the presence of anti-Sm reactivity. However, this tech- nique is not readily automated and is beyond the degree of complexity offered by most clinical laboratories. Type of Sample Serum or plasma. ANTI-RNP Anti-RNP results in a speckled pattern on IIF ANA screen- ing tests. Anti-RNP reacts with a 70kDa nuclear matrix antigen and epitopes A and C on U1 RNP. It is present in high titers in patients with MCTD. Reference Interval Most laboratories use an immunometric assay to detect anti-RNP. The reference interval is reactivity below the cutoff established by the manufacturer. In the immunoblot assay, lack of band reactivity is the reference interval. Clinical Applications Anti-RNP is present in low titer in many autoimmune dis- eases, including SLE—where it constitutes about 25% of cases. In mixed connective tissue disease (MCTD), anti-RNP is often the only or major reactivity present. About 95% of patients with MCTD will have anti-RNP in their serum. Identification of patients with MCTD is useful because it is usually a milder form of autoimmune disease than SLE. Patients with MCTD often present with Raynaud’s phenom- enon but lack involvement of the kidneys, lungs, or heart. Limitations Although this test may be useful to distinguish patients with SLE from those with MCTD, about a third of patients with SLE also have anti-RNP. Assay Technology Immunometric technology and multiplex systems as described above use antigen preparations that contain
  9. 9. 877CHAPTER 9.15 Autoimmune Disease RNP. After reaction with the patient’s serum, the strength of the reactivity is compared to known positive and nega- tive controls. Immunoblotting analysis may also be used to determine the presence of anti-RNP reactivity. However, this tech- nique is not readily automated and is beyond the degree of complexity offered by most clinical laboratories. Type of Sample Serum or plasma. ANTI-SSA/RO Anti-SSA/Ro produces a speckled pattern on most IIF ANA screening tests. These antibodies were first discov- ered in patients with SS but are also present in patients with SLE and occasionally in patients with rheumatoid arthritis (RA). The antibody recognizes a 52kDa and a 60kDa nuclear protein. However, this antigen is easily extracted by the buffer wash steps in IIF techniques and, therefore, may be missed unless the manufacturer has used special measures to fix it on the substrate. Indeed, prior to widespread use of newer HEp-2 cell lines, SLE patients with mainly or only anti-SSA/Ro reactivity produced neg- ative ANA screening tests. Reference Interval Most laboratories use an immunometric assay to detect anti-SSA/Ro. The reference interval is reactivity below the cutoff established by the manufacturer. In the immunoblot assay, lack of band reactivity is the reference interval. Clinical Applications About 75% of patients with primary SS (protean symp- toms include: dry eyes, dry mouth, and arthritis) will have anti-SSA/Ro. A smaller percentage (10–25%) of patients with secondary SS (the same symptoms together with another autoimmune disease) will have these antibodies. Anti-SSA/Ro is also present in about 25% of patients with SLE. These antibodies are strongly associated with a sub- group of SLE patients who present with photosensitive skin rash and SS. Some patients with drug-induced lupus (especially those associated with toxicity to D-penicilla- mine) have this antibody, as well. SLE and neonatal lupus will often have anti-SSA/Ro activity in the serum. Indeed anti-SSA/Ro is a key marker antibody for neonatal lupus. Quantitative information about the anti-SSA/Ro level in the mother’s serum is an important prognostic marker for the development of cardiac complications in the neonate. Often, it is the only autoantibody present in the serum of these babies (Wisuthsarewong et al., 2011). Limitations When the tissue section immunofluorescence test is used as a screen for ANA, the SSA/Ro antigen may be removed during the wash steps because it is easily eluted from the nuclei in the section. Using these older substrates, about 60% of ANA negative cases who had SLE were found to have anti-SSA/Ro in their serum. Fortunately, HEp-2 substrates are now the standard and manufacturers have improved the ability to detect this important antigen. The level of anti-SSA/Ro provides more prognostic informa- tion than the ANA screening test. Assay Technology Immunometric technology and multiplex systems as described above use antigen preparations that contain SSA/Ro. After reaction with the patient’s serum, the strength of the reactivity is compared to known positive and negative controls. Immunoblotting analysis may also be used to determine the presence of anti-SSA/Ro reactivity. However, this technique is not readily automated and is beyond the degree of complexity offered by most clinical laboratories. Type of Sample Serum or plasma. ANTI-SSB/LA Anti-SSB/La produces a speckled pattern on most IIF ANA screening tests. These antibodies were first discov- ered in patients with SS. SSB/La antigen is a 47kDa pro- tein that associates with RNA polymerase III transcripts. SSB/La is present in much greater concentration than SSA/Ro in substrates for IIF ANA screening. Reference Interval Most laboratories use an immunometric assay to detect anti-SSB/La. The reference interval is reactivity below the cutoff established by the manufacturer. In the immunoblot assay, lack of band reactivity is the reference interval. Clinical Applications About 40% of patients with primary SS have anti-SSB/La. Anti-SSB/La is also found in SLE, but unlike SSA/Ro is neither a marker for neonatal lupus nor subacute cutane- ous lupus erythematosus. Limitations Although anti-SSB/La is found in many individuals with both SLE and SS, it has not proven to be a particularly useful marker of any specific subset of these diseases. Assay Technology The assay technology is identical to that of SSA/Ro (see above). Type of Sample Serum or plasma. ANTI-HISTONE Antibodies against the basic nuclear proteins, histones, usually give a homogeneous pattern on the immunofluo- rescence ANA screen. These antibodies may be present in patients with SLE or in patients with drug-induced
  10. 10. 878 The Immunoassay Handbook lupus. Based on immunologic studies, histones are subdi- vided into five groups: H1, H2, H2B, H3, and H4. Reference Interval Anti-histone may be detected by immunometric or multi- plex assays. The reference interval is absence of detectable antibody. Clinical Applications It is common for patients with SLE to have anti-histone antibodies along with more specific antibodies such as anti-Sm or anti-dsDNA (see above). Because they are not specific for this disease, there is no need to test for these antibodies unless one suspects drug-induced lupus. In patients with SLE, the anti-histone antibodies are usually directed against H1 and H2B. Anti-histone antibodies are also present in about 10% of patients with RA. Testing for these antibodies in patients with RA does not seem to add to the useful information. The vast majority of patients with drug-induced lupus have anti-histone antibodies. Patients with procainamide-induced lupus typically develop antibodies that react with H2A-H2B complex, whereas patients with hydralazine-induced lupus develop antibodies against H3 and H4 (Borchers et al., 2007). Limitations The presence of anti-histone does not mean that the patient has drug-induced lupus. The antibodies are often present in asymptomatic individuals receiving one of the above medications. Further, in patients who do have symp- toms of drug-induced lupus, cessation of the drug usually results in a reversal of the symptoms in weeks although the antibodies may persist for months or years, recall that the half-life of IgG is about 21 days. Assay Technology Immunometric and immunoblot technology are similar to those described above. IIF has been used in the past to detect these antibodies. However, IIF involves extracting and then reconstituting histones in the tissues which is clumsy and insensitive. Therefore, it is not recommended. Type of Sample Serum or plasma. ANTI-CENTROMERE This antibody directed against centromere proteins is use- ful in the diagnosis of the CREST syndrome (see below). On IIF, anti-centromere gives a characteristic staining pattern (Table 4). Uniform-sized speckles are present in resting cells and in dividing cells; speckles conform to and are present on chromosomes. Reference Interval Anti-centromere may be detected by IIF, immunometric, or multiplex flow immunoassay. The reference interval is absence of detectable antibody. Clinical Applications Most patients with CREST syndrome have autoantibod- ies that react with chromosomal centromeres and lack antibodies that react with Scl-70 (topoisomerase I—see below). CREST stands for autoimmune disease with the following manifestations: calcinosis (i.e., rock-like calcifi- cation within soft tissues), Raynaud’s phenomenon (cuta- neous cyanosis), esophageal dysmotility (difficulty swallowing), and telangiectasias (spider-like red patches on skin or mucosal surfaces). It is an important subset of scleroderma, because these individuals usually present with cutaneous scleroderma and lack deep organ involve- ment, and therefore have a much better prognosis (Frit- zler et al., 2011). Limitations Although anti-centromere is present in about 95% of patients with CREST syndrome, it is present in a small number (about 10%) of patients with diffuse scleroderma. Further, it is present in about 25% of patients with isolated Raynaud’s phenomenon who lack any other features of CREST. Assay Technology Immunometric and multiplex flow immunoassay technol- ogy is similar to those described above. IIF on HEp-2 or other tissue culture substrate is an excellent technique to detect these antibodies. They are not definable, however, on frozen section substrates because the nuclei of these normal rat livers or kidneys lack large numbers of dividing cells that produce and concen- trate centromere proteins. In addition, a negative result by IIF on HEp-2 does not rule out some of the more subtle anti-CENP-A and CENP-B that can be detected by the immunometric and multiplex flow immunoassays (Mahler et al., 2011). Type of Sample Serum or plasma. ANTI-SCL-70 (ANTI-DNA TOPOISOMERASE I) Anti-Scl-70, also called the scleroderma antibody or Scl-1, is an antibody against a 70kDa antigen present in DNA topoisomerase I. On IIF, anti-Scl-70 gives a relatively spe- cific pattern in IIF. It stains five areas: the nucleus, nucleo- lus, chromosomes in mitotic cells, the nucleolar organizing TABLE 4 Pattern of ANA in Progressive Systemic Sclerosis Pattern Antigen Significance Nucleolar RNA 60% of PSS* Centromere Centromere protein 80% of CREST patients Fine Speckled Topoisomerase (Scl-70) 20% of PSS Coarse Speckled Unknown Unknown *PSS: progressive systemic sclerosis (scleroderma).
  11. 11. 879CHAPTER 9.15 Autoimmune Disease region, and the cytoplasm in interphase cells (Dellavance et al., 2009). Reference Interval Anti-Scl-70 may be confirmed by immunometric or multi- plex flow immunoassay. The reference interval is absence of detectable antibody. Clinical Applications Anti-Scl-70 identifies about 75% of patients with progres- sive systemic sclerosis (PSS or scleroderma). It is extremely uncommon for this antibody to be present in cases of CREST syndrome subset of PSS. There has been an asso- ciation between the subsequent developments of cancer in scleroderma patients with the presence of anti-Scl-70. Limitations Anti-Scl-70 is also present in about 40% of patients with acrosclerosis, 10% of patients with primary Raynaud’s phenomenon, and about 5% of patients with SS. Assay Technology Immunometric and multiplex flow immunoassays are similar to those described above. Type of Sample Serum or plasma. IgG ANTI-RNA POLYMERASE III (ARA) ARA is an autoantibody that reacts with a major epitope on RNA polymerase III. Reference Interval The reference interval for ARA is reactivity below the cutoff value provided by the manufacturer’s kit. Clinical Applications ARA are present in about 20% of patients with progressive systemic sclerosis (PSS or scleroderma). Its presence in the disease varies from 4-25%. However, its presence is highly specific for PSS and it is most likely to be found in indi- viduals with diffuse cutaneous scleroderma (dcSSc) (Parker et al., 2008). Limitations ARA antibodies have been reported in 2% of controls with no evidence of scleroderm. So, while the test under those conditions had a 98% specificity, the test should not be used alone to establish the diagnosis of scleroderma. Assay Technology Immunometrica assays used are similar to those described above. Type of Sample Serum or plasma. RHEUMATOID FACTOR Rheumatoid factor is an antibody (usually IgM) directed against the Fc portion of human IgG. Reference Interval The reference interval for rheumatoid factor is undetect- able or detectable below a cutoff value provided by the manufacturer’s kit. Clinical Applications More than 90% of patients with RA have rheumatoid fac- tor present in high titer in their serum. These antibodies may precede development of RA by several years. When used in combination with anti-cyclic citrullinated peptide (CCP) assays (see below) their positive predictive value approaches 100% (Taylor et al., 2011). Limitations Most normal individuals develop rheumatoid factor during acute infectious diseases. Thus, the presence of rheuma- toid factor may be confusing. Because of this, the specific- ity of rheumatoid factor is only about 72% in recent studies. The combined use of anti-CCP has been reported to be beneficial in improving the specificity of a positive rheumatoid factor assay. Since the early stages of rheuma- toid arthritis may present with systemic manifestations and fever, repeat testing when the acute period has ended helps to confirm the diagnosis of RA. Of course a negative result does not rule out the disease. The titer of rheumatoid fac- tor will fluctuate, and repeat testing is warranted in cases with strong clinical suspicion despite the absence of detect- able rheumatoid factor on a single sample, especially early in the disease. This suggests that rheumatoid factor is not the etiologic agent of RA but only a by-product of the inflammatory process that is useful for diagnosis. Assay Technology The agglutination and precipitation tests that employed erythrocytes or latex beads coated with immunoglobulin have been being replaced by more reproducible nephelo- metric and turbidometric assays. The older procedures are subjective and suffer from poor reproducibility. In the past 10 years, nephelometric and turbidometric assays with objective rate units have become the mainstay to detect rheumatoid factor. Type of Sample Serum or plasma. ANTI-CYCLIC CITRULLINATED PEPTIDE Anti-CCP is an antibody that recognizes citrullinated forms of native proteins and which is directed against an antigenic determinant containing the deimidated form of arginine (called citrulline). Reference Interval The reference interval for anti-CCP is undetectable or detect- able below a cutoff value provided by the manufacturer’s kit.
  12. 12. 880 The Immunoassay Handbook Clinical Applications The presence of anti-CCP antibodies has a specificity of >90% for RA. When used in combination with the rheu- matoidfactorassay,thepositivepredictivevalueapproaches 100%. They may be detected years before the onset of clinical symptoms of RA. Its presence is also associated with the severity of the disease at the time of diagnosis. Since the presence of anti-CCP early in the disease is asso- ciated with an increased risk of joint erosion, more aggres- sive therapy may be indicated than in anti-CCP negative cases. The RA-7 revised criteria for the diagnosis of RA by the American College of Rheumatology improved the sen- sitivity without decreasing specificity by replacing the requirement for rheumatoid nodules with a positive anti- CCP test result (Zhao et al., 2010; Banal et al., 2009). Limitations While anti-CCP when used alone is more specific than the rheumatoid factor assay (above) for distinguishing patients with RA from controls, the sensitivity of anti- CCP is less than that of the rheumatoid factor test. Because of this, the combination of anti-CCP EIA with IgM anti- rheumatoid factor significantly improves the sensitivity of detection of RA. Assay Technology Immunometric technology is similar to those described above. Type of Sample Serum. ANTI-NEUTROPHIL CYTOPLASMIC ANTIBODIES (C-ANCA, P-ANCA) Cytoplasmic (C) and perinuclear (P) ANCA are found in patients with Wegener’s granulomatosis and microscopic polyangiitis, respectively. They are usually, but not always, directed against serine protease 3 (C-ANCA) and myelo- peroxidase (P-ANCA) in the neutrophil granules. The P-ANCA pattern is also seen in cases of Churg–Strauss syndrome and will usually have anti-myeloperoxidase specificity. The ANCA test is one of the most specific of all autoimmune tests and one of the few that may be consid- ered for a STAT assay in a patient suspected of having rapidly progressive glomerulonephritis. Treatment before the creatinine is markedly elevated can prevent chronic renal failure and may reverse lung damage (Wilde et al., 2010; Wiik, 2010). Reference Interval C-ANCA and P-ANCA may be detected by IIF and/or by immunometric assays. Reference interval is absence of antibody reactivity. Clinical Applications Wegener’s granulomatosis and microscopic polyangi- itis are uncommon conditions wherein small blood vessels in the kidney, lungs, and upper respiratory tract are damaged by necrotizing inflammation. Because the clinical course of these conditions is highly variable and the defini- tive evidence requires kidney and/or lung biopsy, the development of a highly specific and highly sensitive auto- antibody test is of great help. Since C-ANCA is present in the majority of patients with Wegener’s granulomatosis but rarely in patients with other autoimmune disease or controls, it is the test of choice for diagnosing this condi- tion. The presence of this antibody in an appropriate clini- cal setting will result in chemotherapy being administered that may restore renal or pulmonary function. P-ANCA is useful in supporting the diagnosis of micro- scopic polyangiitis and Churg–Strauss syndrome. Limitations Although C-ANCA is highly specific for Wegener’s granu- lomatosis, its absence cannot exclude the disease. The titer of reactivity waxes and wanes with the course of the disease and in some patients it may not be detectable. The presence of granular cytoplasmic staining on the ethanol-fixed neu- trophils (i.e., the hallmark of C-ANCA) must be confirmed by repeating the study on formalin-fixed neutrophils. With true anti-serine protease 3, this will also produce a granular cytoplasmic pattern, typically stronger in reactivity than the ethanol-fixed slides. We also recommend performing a final identification step using an immunometric kit (with the purified protease 3 antigen). The immunometric kits for anti-serine protease 3 have not been standardized with each other and our laboratory has found disparate results between different kits. This antigen deteriorates with time, and reactivity must be verified on each run. P-ANCA is much less specific than C-ANCA. If IIF on ethanol-fixed neutrophils is used, one must be aware that ANA will give a false positive reactivity. We always also react the antisera on formalin-fixed neutrophils where the ANA reactivity usually disappears and myelo- peroxidase (MPO) activity gives a cytoplasmic rather than perinuclear staining pattern. Further, the international consensus recommends that C-ANCA and P-ANCA reactivity be confirmed on EIA kits with the specific antigen. If discrepant reactivity is seen between the two types of assays (IIF and EIA), the clinician should be informed of the atypical reaction. Since the kits are not well standardized, the results may reflect a particular kit’s antigen solution. In addition, other cyto- plasmic antigens such as cathepsin and lactoferrin have been described that also give ANCA patterns in patients with microscopic vasculitis. Assay Technology IIF is the usual screening procedure. Neutrophils from a control individual are cytocentrifuged onto glass slides. Some of the slides are fixed with ethanol, others with for- malin. Serum (usually diluted 1:20 in buffer) is allowed to react with each fixed substrate for about 30min. Following a triple wash in buffer, fluorescein-conjugated antibody against human immunoglobulin is layered onto the slides. After another 30min incubation and wash, the slides are examined under a fluorescence microscope. The presence of granular cytoplasmic fluorescence on both the ethanol- fixed and formalin-fixed slides indicates the presence of
  13. 13. 881CHAPTER 9.15 Autoimmune Disease C-ANCA. The presence of perinuclear staining on the ethanol-fixed slides and cytoplasmic granular staining on the formalin-fixed slides indicates the presence of P-ANCA. Immunometric assays are also used in some centers for the initial screen. More often, they are performed to con- firm the IIF pattern specificity. In the uncommon instances where they do not correlate, we do not dismiss the results. The clinician is informed of the discrepancy. Other anti- gens such as lactoferrin and cathepsin can give reactivity and such antibody reactivity has been described in patients with microscopic vasculitis. P-ANCA reactivity has also been described in patients with inflammatory bowel disease, especially ulcerative coli- tis, and in patients with primary sclerosing cholangitis. The relatives of patients who have inflammatory bowel disease may also demonstrate P-ANCA. This implies the presence of a genetic predisposition for forming such antibodies. Type of Sample Serum or plasma. ANTI-GLOMERULAR BASEMENT MEMBRANE Anti-glomerular basement membrane (GBM) is an anti- body that reacts with glomerular basement membranes and which may cross-react with alveolar basement membranes. It is useful in the diagnosis of Goodpasture’s syndrome. Reference Interval Anti-GBM may be detected by IIF, direct immunofluores- cence, immunometric assays, multiplex flow immunoassay or western blot. The reference interval is absence of detectable antibody. Clinical Applications Patients with Goodpasture’s syndrome suffer from a necrotizing crescentic glomerulonephritis and pulmonary hemorrhage. These clinical features are associated with autoantibodies directed against their glomerular basement membranes. The presence of these antibodies is highly specific for the diagnosis of Goodpasture’s syndrome when found in the appropriate clinical setting. These antibodies react with type IV collagen, a key component in the base- ment membrane. Limitations Most laboratories perform either immunometric immuno- assays or multiplex flow immunoassays. Western blot tech- nology is highly specific, but it is not available in kit form for general laboratory use. The indirect and direct immu- nofluorescence tests require experience to prevent over interpretation. Sinico et al. have reported that there is con- siderable variation from one methodology to another (Sinico et al., 2006). Assay Technology Immunometric kits and multiplex flow immunoassays use a subunit of type IV GBM conjugated either to microtiter wells or polystyrene beads and are performed as described above. Western blotting is available only through some commer- cial or research laboratories that have developed that test. Renal biopsies from a patient may be stained with fluo- rescein-conjugated anti-human IgG to demonstrate the linear fluorescence characteristic of anti-GBM (i.e., direct immunofluorescence). Serum followed by fluorescein-conjugated anti-human IgG may be used to stain glomeruli from kits that use (mainly) monkey kidney sections (i.e., IIF). This technique is subjective and may not be as sensitive as the Western blot methodology. Type of Sample Serum or plasma. ANTI-JO-1 Anti-Jo-1 is an antibody that reacts with the enzyme histidyl- tRNA synthetase located mainly in the cytoplasm and has been used as a marker for myositis, an idiopathic immune or inflammatory disease of skeletal muscle (Targoff, 2008). Reference Interval The reference interval is absence of detectable antibody. Clinical Applications Anti-Jo-1 has been used to identify patients with myositis who may benefit from steroid therapy. Limitations Although relatively sensitive for myositis, a negative Jo-1 cannot rule out myositis, polymyositis, or dermatomyosi- tis. Anti-Jo-1 alone may not give a positive ANA. There- fore, patients with symptoms of myositis may benefit from an anti-Jo-1 test even when their ANA screen is negative. Assay Technology Immunometric, line blot assays and multiplex flow immu- noassays are the techniques used to detect anti-Jo-1 (Ghirardello et al., 2011). The methodologies are the same as described above. Type of Sample Serum or plasma. ANTI-MICROSOMAL (THYROID PEROXIDASE) Antibodies against the microsomal fraction of thyroid epi- thelium are present in patients with autoimmune (Hashi- moto’s) thyroiditis and in those with Graves’ disease. The specific reactivity is against thyroid peroxidase (TPO), a thyroid enzyme responsible for catalyzing oxidation of iodide in order to synthesize thyroid hormones (T3 and T4). Therefore, one may see either the term anti-micro- somal or anti-TPO in the literature. They refer to the same antibody. It seems paradoxical that the same
  14. 14. 882 The Immunoassay Handbook autoantibody is associated both with Hashimoto’s thyroid- itis (a condition usually resulting in hypothyroidism) and Graves’ disease (a condition causing hyperthyroidism). Yet, they share the common association of thyroiditis. Reference Interval The reference interval is absence of detectable antibody. Clinical Applications Patients with Graves’ disease (hyperthyroidism) or Hashi- moto’s thyroiditis (usually hypothyroid, although they may be euthyroid early in the course), will both have anti- microsomal antibodies. The presence of these antibodies and the appropriate clinical and laboratory features help to secure the diagnosis. Anti-TPO fixes complement, dam- ages thyroid tissue, and leads to lymphocytic infiltration of its parenchyma. Other autoantibodies are also present in these conditions. Thyroid-stimulating immunoglobulins (TSI) react with the thyroid-stimulating hormone (TSH) receptors on thyroid epithelium and mimic the stimula- tory effect of TSH resulting in Graves’ disease (hyperthy- roidism). Another autoantibody usually found in these cases is anti-thyroglobulin. Since anti-TPO is almost always present in these cases, the presence or absence of anti-thyroglobulin does not add useful information to the diagnosis. However, anti-thyroglobulin is useful when anti-TPO is negative in a patient suspected of having Graves’ disease. Limitations Rarely, anti-TPO antibodies may be absent in a case of Hashimoto’s thyroiditis or Graves’ disease. In those cases, an anti-thyroglobulin or TSI test may be useful. Since the antibodies do not respond to therapy with levothyroxine, there is no need to monitor the anti-TPO levels after the diagnosis. Assay Technology Most laboratories use immunometric assays for TPO to look for microsomal antibodies. The technique is as described above. Latex agglutination techniques that use beads coated with TPO are also available. The IIF test is an older test that uses tissue sections of thyroid glands from rats or mice. These sections are reacted with the patient’s serum as above for the ANA test. Anti-microsomal antibodies react with the cytoplasm of the gland, while anti-thyroglobulin reacts with the colloid. Type of Sample Serum or plasma. THYROID-STIMULATING HORMONE RECEPTOR ANTIBODY Thyroid-stimulating hormone receptor antibodies (TRAbs) are one of the autoantibodies that have a clear relationship with the disease. They were originally described as “long acting thyroid stimulator” (LATS) before they were recognized as being immunoglobulins. It was called LATS because its half-life was much longer than that of TSH. They have specificity for the thyrotro- pin receptor; their binding activates it yielding an unregu- lated thyrotropin-independent production of thyroid hormones. Other species of antibodies that react with the thyrotropin receptor actually have blocking rather than stimulating activity. These are termed TSH blocking anti- bodies. Interestingly, both may occur simultaneously. Reference Interval The reference interval is absence of detectable antibody. Clinical Applications TRAbs are often used along with TPO to confirm the clinical impression of Graves’ disease. These antibodies are often present prior to the development of clinically detectable hyperthyroidism. As noted above, these are functional antibodies, not just merely markers of the dis- ease. Therefore, in pregnant women, one should be aware that IgG TRAb will cross the placenta and result in neo- natal thyrotoxicosis. The TRAb assays have higher sensi- tivity than the TSI bioassays (Giovanella et al., 2001) between Grave’s disease and destructive thyroiditis (see THYROID). The American Association of Clinical Endocri- nologists endorses the use of TRAb for guiding prognosis and therapy. Measuring TRAb helps to predict which patients can be weaned from anti-thyroid drug therapy (Bahn et al., 2011). Limitations Immunometric assays that use streptavidin in their tech- nique may be interfered with if the patient is receiving bio- tin therapy. Assay Technology The bioassay measuring cyclic AMP product following exposure of in vitro hamster ovarian cells that express the TSH receptor has been the gold standard. But due to its expense and variability, it has largely been replaced by immunometric assays. Some use radioisotopes, while oth- ers use chemiluminescence. Type of Sample Serum. ISLET CELL AUTOANTIBODIES Islet cell autoantibodies (ICA) are often present early on in patients with diabetes mellitus type 1. While they are unlikely to be the cause of type 1 diabetes, their presence indicates the underlying damage to the islets and the rec- ognition of some islet cell antigens as foreign by the humoral immune system (Winter and Schatz, 2011). Further, in individuals who do not have diabetes melli- tus, the presence of ICA indicates the need for close fol- low-up for the likely development of this disease. Detection of these antibodies also can be useful to distin- guish type I diabetes from type II. The four major islet
  15. 15. 883CHAPTER 9.15 Autoimmune Disease cell antibodies are ICA, glutamic acid decarboxylase (GAD) autoantibodies, insulinoma 2 (IA-2)-associated autoantibodies (IA-2A), and insulin autoantibodies (IAA) (Table 5). Reference Interval The reference interval is absence of detectable antibody. Clinical Applications At the present time, the ICA can be used to distinguish type I from type II diabetes mellitus. Detection of these autoantibodies in clinically well family members of indi- viduals with type I diabetes mellitus is a strong predictor of future disease. Limitations The IIF test for ICA is subjective due to the variability of expression of ICA on pancreatic islet cells (Bingley et al., 2010). A negative reaction in any of these assays does not rule out the presence of type I diabetes mellitus. Assay Technology ICA is an IIF test that is largely available from research laboratories. Others, such as IA-2A, glutamate decarboxyl- ase (GADA), and IAA, are available from reference labora- tories as an RIA. Type of Sample Serum or plasma. ANTI-ADRENAL CORTICAL ANTIBODIES (ANTI-21-HYDROXYLASE) Antibodies that react with the adrenal cortex are usually present in the serum from patients with autoimmune adrenal insufficiency (Addison’s disease). The main antigen associated with adrenal cortical antibodies is 21-hydroxylase. It is present in the vast majority of patient’s with Addison’s disease as well as in most indi- viduals with type I or type II autoimmune polyglandular syndrome. Reference Interval The reference interval is absence of detectable antibody. Clinical Applications Patients with idiopathic Addison’s disease will usually have anti-adrenal antibodies in their serum. The demonstration of these antibodies is useful to distinguish idiopathic (auto- immune) Addison’s disease from other causes of adrenal insufficiency, such as tuberculosis. As with most other autoimmune disease testing, patient selection prior to test- ing is key to optimizing the utility of the test. Limitations Anti-adrenal cortical antibodies may be found in about 2–3% of healthy blood donors. The significance of these antibodies in normal individuals is unclear. Elevation of adrenocorticotropic hormone (ACTH) is a more effective test to detect impending Addison’s disease than anti-21- hydroxylase. Recent studies indicated considerable varia- tion of the measurement of anti-21-hydroxylase from one laboratory to another and emphasized the need for better standardization (Falomi et al., 2011). Assay Technology The RIA is the main test for anti-21-hydroxylase. Type of Sample Serum or plasma. ANTI-PARIETAL CELL ANTIBODIES AND ANTI-INTRINSIC FACTOR ANTIBODIES Antibodies against the parietal cells (the cells that make hydrochloric acid) in the stomach are present in most patients with pernicious anemia (PA). Patients with PA also develop antibodies against intrinsic factor. The loss of the acid needed to free cobalamin from binding proteins combined with the interference of anti-intrinsic factor antibodies with the uptake of vitamin B12 by the ileal epi- thelium result in severe vitamin B12 deficiency. Reference Interval The reference interval is absence of detectable antibody. Clinical Applications About 90% of patients with PA will have anti-parietal cell antibodies and/or anti-intrinsic factor antibodies in their serum. When present in the context of an older, usually female patient with a macrocytic anemia, fatigue, dyspepsia, and possibly symptoms of neuropathy, it is a useful confir- mation of the presence of this autoimmune disease. The most recent strategy recommends combined testing of anti- parietal cell antibodies and anti-intrinsic factor antibodies to optimize sensitivity and specificity (Lahner et al., 2009). Limitations Positives for anti-parietal cell antibodies also occur in patients with primary biliary cirrhosis. Assay technology Both assays are now performed mainly by immunometric assays. TABLE 5 The Four Major Islet Cell Antibodies in Diabetes Mellitus Antigen Sensitivitya Specificity ICA 70–80% >99% GADA 70–80% 97–98% IA-2A 60% 97–98% IAA 60%b 95% aFrequency in new-onset type I diabetes mellitus patients. bThis value is for children; IAA are uncommon in adults. Source: From Winter and Schatz (2011).
  16. 16. 884 The Immunoassay Handbook Type of Sample Serum or plasma. ANTI-MITOCHONDRIAL ANTIBODIES (AMA OR M2) Antibodies that react with mitochondria have served as a useful marker for primary biliary cirrhosis. Although nine different mitochondrial antigens have been described, the M2 antigens are the most specific for the diagnosis of pri- mary biliary cirrhosis. These antigens are part of the 2-oxo-acid dehydrogenase complex in mitochondria. Reference Interval The reference interval is absence of detectable antibody. Clinical Applications Patients with primary biliary cirrhosis often have the insidi- ous onset of their disease. Early symptoms of fatigue are often gradual and inconsistent. The patient typically seeks medical attention when her/his pruritis becomes a problem or when the sclera become noticeably yellow. The presence of an ele- vation of alkaline phosphatase in the presence of normal ala- nine aminotransferase (ALT) and aspartate aminotransferase (AST) is the usual pattern of serum enzymes in the disease. AMAs are found in about 90% of patients with primary bili- ary cirrhosis. In some of the patients, anti-smooth muscle antibodies (SMA) may be present. This is not usually a prob- lem because serum from patients with autoimmune chronic active hepatitis will give a much different serum enzyme pat- tern than that from patients with primary biliary cirrhosis. The presence of ANA along with AMA in patients with primary biliary cirrhosis is a negative prognostic feature (Invemizzi et al., 2005). Limitations The titer of AMA does not reflect the severity of the dis- ease in an individual. The degree of liver damage is deter- mined by use of clinical information and biopsy. Because some cases of primary biliary cirrhosis fall into the subset called autoimmune cholangitis, patients who are AMA negative should be tested for the presence of ANA and anti-SMA. When using IIF, antibodies against liver–kidney micro- somal (LKM) fraction can mimic AMA. The LKM anti- bodies are associated with autoimmune hepatitis and stain the proximal tubules but not the distal tubules in the kid- ney in IIF techniques. However, LKM do not stain the gastric epithelium that AMA always do. Assay Technology Immunometric assays have largely replaced IIF for detect- ing AMA. Immunometric tests can distinguish between different types of mitochondrial antigens and provide better speci- ficity in the diagnosis of primary biliary cirrhosis than IIF. The IIF test employs tissue sections of kidney encircled by stomach from rats or mice. These sections are reacted with the patient’s serum as above for the ANA test. AMAs give granular reactivity with the cytoplasm of the kidney tubules and cells within the gastric mucosa. Type of Sample Serum or plasma. ANTI-SMA SMA are antibodies that have as their antigen F-actin. Typically, smooth muscle from rat stomach and/or kidney is used to detect these antibodies. Reference Interval The reference interval is absence of detectable antibody. Clinical Applications Although SMA have been used to detect patients with autoimmune hepatitis, they are also present in patients with conditions such as primary biliary cirrhosis and even in normal individuals. Patients with autoimmune hepatitis frequently have other antibodies as well, including ANA and AMA. The autoantibodies and laboratory features are usually sufficient to diagnose the condition without resort- ing to liver biopsy (Bjomsson et al., 2011). Autoimmune hepatitis is found most often in young- to middle-aged women. The autoimmune nature of the condition is spec- ulative, however, the patients usually respond well to ste- roid therapy. Limitations SMA are not specific for autoimmune hepatitis. Indeed, it is important to use standard serologic tests for hepatitis B and C to rule out infectious etiologies for the condition. Other hepatic diseases such as Wilson’s disease and pri- mary biliary cirrhosis also need to be excluded. Usually the history and serum enzyme pattern help to distinguish pri- mary biliary cirrhosis from autoimmune hepatitis. Because of the nonspecificity of SMA, laboratories usually perform the assay using two or three dilutions of the patient’s serum. Only reactivity at the higher dilutions (typically 1:80 or 1:160) has reasonable predictive value. Assay Technology SMA are detected by IIF on rat stomach and/or kidney sections. The staining pattern on the stomach involves both the muscularis propria and the muscularis mucosa. SMA react mainly with the muscular layers of the arteries in the kidney sections. Although immunometric assays are available, the IIF assays have better diagnostic specificity. Type of Sample Serum or plasma. ANTI-LKM Anti-LKM react with cytochrome P450. The major target antigen of LKM antibodies is cytochrome P4502D6 (CYP 2D6).
  17. 17. 885CHAPTER 9.15 Autoimmune Disease Reference Interval The reference interval is absence of detectable antibody. Clinical Applications Anti-LKM is a helpful marker for a subgroup of patients, mainly children with autoimmune hepatitis type 2. In the United States, less than 5% of cases of autoimmune hepatitis characterized by the presence of anti-LKM reac- tivity are seen in adults. In Europe, 20% of such cases occur among adults. These patients typically lack SMA and AMA (Vergani et al., 2009). Limitations These patients are said to be more likely to have a wide variety of autoantibodies than patients with the usual SMA-associated autoimmune hepatitis. Antibodies against thyroid microsomal and parietal cells are common. The latter may cause confusion with AMA if stomach and renal substrates are not used concomitantly. Assay Technology Immunometric assays and IIF are used to detect LKM. The antibodies react with the proximal convoluted tubules in the kidney, but unlike AMA, they do not react with the distal tubules or the loop of Henle. Further, in gastric sub- strates, the gastric parietal cells are usually negative. Some patients unexpectedly will have staining of the gastric pari- etal cells. Type of Sample Serum or plasma. IGA ANTI-ENDOMYSIUM IgA anti-endomysium antibodies react with smooth mus- cle endomysium (cell membrane) in frozen sections of monkey kidney or human umbilical cord. It is present in the vast majority of patients with celiac disease (sprue) and is uncommon in other gastrointestinal conditions that may be in the differential diagnosis of malabsorption. Reference Interval The reference interval is absence of detectable antibody. Clinical Applications IgA anti-endomysium is used to detect patients with celiac disease. These patients have severe malabsorption due to a reaction to gluten in their diet. Detection of IgA anti-endo- mysium may be helpful in selecting patients who would benefit from a biopsy of the small intestine—currently required for definitive diagnosis. By following the titer of IgA anti-endomysium, one may be able to determine if the patient is maintaining a gluten-free diet without a biopsy of the small intestine. Because IgA anti-endomysium persists longer than anti-gliadin, some authors have suggested that anti-gliadin is a better test to follow therapy. IgA anti-endomysium is a more specific antibody test than the older IgG anti-reticulin. The latter has similar diagnostic uses but has largely and justifiably been replaced. Limitations IgA anti-endomysium is occasionally found in individuals who have normal small bowel biopsies. Therefore, one should confirm the diagnosis of celiac disease by biopsy before submitting the patient to a lifelong gluten-free diet (which requires abstinence of all starches except rice). IgA anti-endomysium may be absent in patients with typical celiac disease. Therefore, in the presence of strong clinical suspicion of celiac disease, a negative study does not preclude biopsy. This assay is still used but is being replaced by the more objective IgA anti-tissue transglutaminase (see below). Assay Technology IIF using frozen sections of monkey esophagus or human umbilical cord is the most common technique to detect IgA anti-endomysium. For this technique to be effective, two dilutions of the patient serum are employed: 1:5 and 1:50. Because some of the patients have other autoantibod- ies such as SMA that obscure detection of IgA anti-endo- mysium, the larger dilution is more specific for celiac disease. The more concentrated serum, however, provides better sensitivity in the majority of sera lacking interfering antibodies. Type of Sample Serum or plasma. IGA ANTI-TISSUE TRANSGLUTAMINASE IgA anti-tissue transglutaminase (tTG) antibodies react with the major autoantigen in smooth muscle endomysium that results in the positive staining on frozen sections of monkey kidney or human umbilical cord. Not surpris- ingly, as with IgA anti-endomysium, IgA anti-tTG is pres- ent in the vast majority of patients with celiac disease (sprue) and is uncommonly present in other gastrointesti- nal conditions that may be involved in the differential diagnosis of malabsorption. Reference Interval The reference interval is absence of detectable antibody. Clinical Applications IgA anti-tTG is used to detect patients with celiac disease. It has been used instead of the IgA anti-endomysium to select patients who would benefit from a biopsy of the small intestine. One function of tTG is to deamidate glu- tamine into glutamic acid which changes the charge on gluten peptides. This action of tTG on gluten results in a heightened immunoreactivity of T cells for gluten in indi- viduals with a genetic proclivity to develop celiac disease. IgG anti-tTG is a useful additional test in individuals who are IgA deficient. Limitations IgA anti-tTG is occasionally found in individuals who have normal small bowel biopsies. Therefore, one should con- firm the diagnosis of celiac disease by biopsy before sub- mitting the patient to a lifelong gluten-free diet (which
  18. 18. 886 The Immunoassay Handbook requires abstinence of all starches except rice). Uncom- monly, IgA anti-tTG may be absent in patients with typi- cal celiac disease. Of course, no individual who is IgA deficient will be able to manufacture IgA anti-tTG. For those individuals, the finding of a positive IgG anti-gliadin (see below) in an appropriate clinical setting would encour- age biopsy proof of celiac disease. Assay Technology Immunometric tests are the current standard for this assay. Type of Sample Serum or plasma. IGG AND IGA ANTI-DEAMIDATED GLIADIN These antibodies react with the alcohol soluble fraction of gluten that is responsible for celiac disease. Reference Interval The reference interval is absence of detectable antibody. Clinical applications IgG and IgA anti-deamidated gliadin are elevated in most patients with celiac disease. Although the IgA anti-gliadin seems to be more specific for celiac disease than IgG anti- gliadin, it is a less sensitive test. The original anti-gliadin tests used intact gliadin and were much less specific than current tests. Once it was recognized that it is the deami- dated gliadin, not intact native gliadin, which binds to the HLA-DQ2 or -DQ8 cells, manufacturers changed the anti- gen used in the test with a marked improvement in both sensitivity and specificity. Because celiac disease occurs with increased frequency among children who are deficient in production of IgA, redundant testing with IgG anti-gliadin may be helpful. IgG anti-gliadin has been useful to follow patients’ adherence to their unpalatable gluten-free diets. Limitations While most manufacturers have switched to deamidated gliadin in their product, it is not clear that all intact gliadin products are no longer being use. Checking on this point with the manufacturer would be prudent. Assay Technology Immunometric kits are available for detection of IgG and IgA anti-deamidated gliadin. Most are just called anti-gli- adin because the deamidation has become the industry standard. Type of Sample Serum or plasma. ANTI-ACETYLCHOLINE RECEPTOR Anti-acetylcholine receptor (A-ACHR) reacts with the acetylcholine receptor at the neuromuscular postsynaptic junction. They are an example of autoantibodies whose specificity is directly related to the clinical symptoms involved with their associated neuromuscular disease— myasthenia gravis. Reference Interval The reference interval is absence of detectable antibody. Clinical Applications In patients who suffer from acquired myasthenia gravis, the normal transmission of acetylcholine across the tiny gap present between the nerve and muscle is blocked by antibodies that react with or near that receptor. This results in weakness in the muscles that are used most fre- quently. Typically this involves the eyelids (ptosis), ocular muscles (diplopia), and when the respiratory muscles are involved it leads to difficulty breathing. As with many autoimmune diseases, women are affected twice as often as men. Different fractions of antibodies that bind to this receptor have been described: binding antibodies, block- ing antibodies, and modulating antibodies. One or more of these antibodies are detectable in about 90% of patients with generalized myasthenia gravis. In addition to the neuromuscular problem, about half of the cases in adolescents or young adults have an associated thymic hyperplasia, and about 10% of cases in middle- aged or older adults have a thymoma. Limitations The absence of A-ACHR does not rule out myasthenia gravis. The presence or absence of the antibodies does not identify whether a patient has a thymoma or thymic hyper- plasia. Patients with congenital myasthenia gravis do not have A-ACHR. Assay Technology Three types of A-ACHR can be detected. A-ACHR binding antibodies are the most common. A-ACHR mod- ulaiting antibodies are somewhat less common and dam- age the receptor itself, while ACHR-blocking antibodies are ones that correlate best with the disease activity. A complex RIA is used mainly in research or commercial laboratories to detect the antibodies associated with acetyl- choline receptors. The technique for ACHR binding anti- bodies takes advantage of the binding of the snake venom, alpha-bungarotoxin, to acetylcholine receptors. For the assay, alpha-bungarotoxin is labeled with 125I. This then reacts with and binds irreversibly to preparations of human acetylcholine receptors prepared from human neural tis- sues. The patient’s serum is allowed to react with prepara- tions of the human acetylcholine receptor complexes with labelled alpha-bungarotoxin. If the serum contains A-ACHR, it binds to this labeled complex and precipitates. The amount of radioactivity, in the precipitate, correlates with the concentration of A-ACHR. The modulating antibody is detected by the binding of antibodies to the surface of cultured muscle cells also using the radiolabeled alpha-bungarotoxin. Modulating anti- bodies can be detected by the amount that they reduce the binding sites available to the labeled snake venom. A-ACHR blocking antibodies are antibodies that bind near the receptor. They are detected by the inhibition of
  19. 19. 887CHAPTER 9.15 Autoimmune Disease binding of a known positive A-ACHR sample to the labeled receptors. Type of Sample Serum or plasma. STRIATIONAL ANTIBODIES Striational antibodies react with many skeletal muscle antigens including actin, myosin, and ryanodine receptor. Reference Interval The reference interval is absence of detectable antibody. Clinical Applications A small number of patients who have symptoms of myas- thenia gravis lack antibodies to ACHR, ACHR modulat- ing antibodies, and ACHR blocking antibodies. This is especially true in older individuals. Some of these patients have striational antibodies. The titer of these antibodies has been used to monitor immunosuppressive therapy. Limitations Because most patients with myasthenia gravis have ACHR antibodies, the striational antibody assay is not the first screening test of choice. Striational antibodies are usually not present in adolescents and children with myasthenia gravis. These antibodies are also found in about 5% of patients with the clinically related Eaton–Lambert syn- drome. Striational antibodies are also present in about 5% of patients with lung cancer and in many patients with autoimmune hepatitis. Assay Technology An immunometric assay using antigen preparations from homogenates of skeletal muscle is the technique used in research and commercial laboratories for detecting stria- tional antibodies. Type of Sample Serum or plasma. CALCIUM CHANNEL ANTIBODIES Calcium channel antibodies react with plasma membrane proteins that are involved with initiating release of acetyl- choline and neurotransmission. Interference with the function of the calcium channels by these antibodies can produce the Lambert–Eaton syndrome. Reference Interval The reference interval is absence of detectable antibody. Clinical Applications Lambert-Eaton syndrome is an autoimmune condition which presents with weakness of the legs and arms due to antibodies formed to neuromuscular junction voltage- gated calcium channels. Because most of these patients have co-existing lung cancer (small cell), the phenomenon has been referred to as a paraneoplastic syndrome. Patients with Lambert–Eaton syndrome can have symptoms that mimic myasthenia gravis. In addition to the weakness, however, they also suffer from xerostomia, xerophthalmia, and other autonomic nervous system impairments. More than 90% of these patients will have antibodies that react with calcium channel peptide antigens, which makes this a sensitive assay for this unusual condition. Limitations As with many autoantibody tests described above, one can- not exclude the diagnosis of Lambert–Eaton syndrome with a negative result. Positive results are present in a small percentage of controls (less than 5%) but are found more frequently in patients with neuropathies associated with epithelial neoplasms including breast, ovary, or lung origin. Assay Technology Antibodies to the P/Q type neuronal calcium channels are the most commonly found in patients with Lambert-Eaton syndrome whether or not they have a diagnosis of cancer. Antibodies to the N type neuronal calcium channels are found in about half of the patients with Lambert-Eaton syn- drome. The assay for these antibodies is a research RIA pro- cedure using antigen from human brain. Radiolabeled calcium channel peptides are used in a radioimmunoprecipi- tation procedure similar to that of ACHR binding antibod- ies. The radiolabeled synthetic calcium channel peptides are added to preparations of human brain. This is added to the patient’s serum. If antibodies are present that bind to the labeled calcium channel peptides (that are attached to high-affinity calcium channel receptors in the human brain), they will react with the brain antigen preparation. Then, anti-human immunoglobulin is used to precipitate the patient’s immunoglobulins. If it has bound to the radiola- beled complexes, radioactivity will be detectable in the immunoprecipitate in proportion to the immune reactivity. Type of Sample Serum or plasma. ANTI-CARDIOLIPIN ANTIBODIES AND BETA-2 GLYCOPROTEIN I ANTIBODIES Antibodies against cardiolipin may actually react with beta-2 glycoprotein I (Beta-2 GPI) that in turn binds to cardiolipin. Beta-2 GPI interacts with anionic phospho- lipid membranes. High levels of Beta-2 GPI antibodies are associated with the phospholipid antibody syndrome (Tebo et al., 2008). Reference Interval The reference interval is absence of detectable antibody. Clinical Applications Cardiolipin and Beta-2 GPI antibodies of the IgG and IgM class have been associated with the phospholipid
  20. 20. 888 The Immunoassay Handbook antibody syndrome. Patients with the phospholipid antibody syndrome have significant problems with both arterial and venous thromboses, thrombocytopenia, and recurrent fetal loss. The vascular thromboses may lead to life-threatening cerebrovascular accidents. In addi- tion, other patients suffer from myocardial infarcts, endocarditis, pulmonary hypertension, and pulmonary infarcts. There is a moderate concordance between the cardiolipin immunoassays and the lupus anticoagulant functional hematologic assay. Patients with the phos- pholipid syndrome may have any or none of these antibodies. Limitations Many individuals will develop antibodies against cardio- lipin transiently during infectious diseases. Cardiolipin is a component of the Venereal Disease Research Labo- ratory (VDRL) test for syphilis. Therefore, some indi- viduals with syphilis will display positive cardiolipin antibody results. To avoid false positives it is recom- mended that a positive test for cardiolipin or Beta-2 GPI antibodies be repeated after about 3 months to allow the response to a transient process, such as an infection, to decline. Assay Technology Immunometric assays for cardiolipin and Beta-2 GPI are the current standard. The reagents should be able to react with IgG and IgM. Some studies suggest that it may be useful to look at IgA anti-cardiolipin, but the data indicate that individuals with only IgA against cardiolipin are van- ishingly rare. Type of Sample Serum or plasma. NEUROPATHY-ASSOCIATED ANTIBODIES: ANTI-MYELIN-ASSOCIATED GLYCOPROTEIN, ANTI-HU, ANTI-RI, AND PURKINJE CELL CYTOPLASMIC ANTIBODIES Anti-myelin-associated glycoprotein (MAG) has been associated with sensory and motor peripheral neuropa- thies. They may be first detected by serum protein electro- phoresis or immunofixation as small IgM monoclonal proteins. Other autoantibodies that affect the nervous system are associated with the paraneoplastic syndrome (Grant and Graus, 2009; Pittock, 2003; Vernino et al., 2002 and King et al., 1999). Anti-Hu (ANNA-1) has been associated with sensory neuropathy and encephalitis in individuals who have underlying neoplasms (often small cell carcinoma) that (usually) are not evident at the time the neurologic symp- toms occur (paraneoplastic syndrome). Anti-Ri (ANNA-2), similar to anti-Hu, is associated with paraneoplastic syndrome; however, in these cases, cerebellar symptoms such as ataxia and midbrain encepha- litis are more often seen. Purkinje cell cytoplasmic antibodies are typically found in patients with cerebellar degeneration accompanied by the paraneoplastic syndrome. Reference Interval The reference interval is absence of detectable antibody. Clinical Applications Sensory and motor neuropathies may be detected as part of the paraneoplastic syndrome. Limitations These antibodies may be detected prior to demonstration of a neoplasm. Assay Technology Immunometric assays are available for MAG. The other assays use IIF as a screen with Western blotting to confirm the screen. Type of Sample Serum. References and Further Reading Arbuckle, M.R., McClain, M.T., Rubertone, M.V., Scofield, R.H., Dennis, G.J., et al. Development of autoantibodies before the clinical onset of systemic lupus erythematosus. N. Engl. J. Med. 349, 1526–1533 (2003). Bahn, R.S., Burch, H.B., Cooper, D.S., Garber, J.R., McDougall, I.R., et al. ATA/ AACE guidelines: hyperthyroidism and other causes of thyrotoxicosis: manage- ment guidelines of the American Thyroid Association and American Association of Clinical Endocrinologists. Endocr. Pract. 17, e1–e56 (2011). Baker, P.R., Nanduri, P., Gottlieb, P.A., Yu, L., Klingensmith, G.J., et al. Predicting the onset of Addison’s disease, ACTH, renin, cortisol, and 21-hydroxylast autoantibodies, Clin. Endocrinol. (2011 Nov 8). doi: 10.1111/j.1365–2265.2011.04276.x. [Epub ahead of print]. Banal, F., Dougados, M., Combescure, C. and Gossec, L. Sensitivity and specificity of the American College of Rheumatology 1987 criteria for the diagnosis of rheumatoid arthritis according to disease duration: a systemic literature review and meta-analysis. Ann. Rheum. Dis. 68, 1184–1191 (2009). Basso, D., Gallo, N., Guariso, G., Pittoni, M., Piva, M.G. and Plebani, M. Role of anti-transglutaminase (anti-tTG), anti-gliadin, and anti-endomysium serum antibodies in diagnosing celiac disease: a comparison of four different commercial kits for anti-tTG determination. J. Clin. Lab. Anal. 15, 112–115 (2001). Bernardini, S., Infantino, M., Bellincampi, L., et al. Screening of antinuclear anti- bodies: comparison between enzyme immunoassay based on nuclear homoge- nates, purified or recombinant antigens and immunofluorescence assay. Clin. Chem. Lab. Med. 42, 1155–1160 (2004). Binder, S. Autoantibody detection using multiplex technologies. Lupus 15, 412–421 (2006). Bingley, P.J., Williams, A.J., Colman, P.G., Gellert, S.A., Eisenbarth, G., et al. Measurement of islet cell antibodies in the type 1 diabetes genetics consortium: efforts to harmonize procedures among the laboratories. Clin. Trials 7(Suppl 1), S56–S64 (2010). Bjomsson, E., Talwalkar, J., Treeprasertusk, S., Neuhauser, M. and Lindor, K. Patients with typical laboratory features of autoimmune hepatitis rarely need a liver biopsy for diagnosis. Clin. Gastroenterol. Hepatol. 9, 57–63 (2011). Bodil, E., Roth, K.S. and Stenberg, P. Biochemical and immuno-pathological aspects of tissue transglutaminase in celiac disease. Autoimmunity 36, 221–226 (2003). Bonilla, E., Francis, L., Allam, F., et al. Immunofluorescence microscopy is superior to fluorescent beads for detection of antinuclear antibody reactivity in systemic lupus erythematosus patients. Clin. Immunol. 24, 18–21 (2007). Borchers, A.T., Keen, C.L. and Gershwin, M.E. Drug-induced lupus. Ann. NY Acad. Sci. 1108, 166–182 (2007).

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