Comparison of Common Tests

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The most commonly performed test to measure autoantibodies in clinical laboratories is IIF to screen for the presence of ANAs [1] (Fig. 8.3). Almost all patients with systemic lupus erythematosus (SLE) are ANA positive, so a negative result on this test virtually rules out SLE [13]. Patients with many other systemic autoimmune diseases, as well as some healthy individuals, are also positive for ANAs [14]. Thus, a positive result is suggestive that the person has an autoimmune disease, but it is not diagnostic. Originally, the ANA IIF test was performed on thin sections of tissue such as mouse kidney. Because of the relatively small size and random orientation of the cells, only a few staining patterns could be observed on this substrate. Also, antibodies to one of the more common autoantigens, SS-A/Ro, were not detected on mouse kidney slides [15]. A significant improvement to screening for ANAs occurred when cells that grow in a monolayer in culture, like HEp-2 cells, were used as the substrate instead of mouse kidney sections. HEp-2 is a human cell line that grows on the surface of the slide and has a relatively large nucleus. The cells are present in all stages of the cell cycle and contain antigens not present in rodent cells, including SS-A/Ro, proliferating cell nuclear antigen that is present at the S phase of the cell cycle [16], and other cell cycle-related antigens. In addition, certain autoantibody specificities, such as anti-centromere, yield clearly identifiable patterns using IIF on HEp-2 cells because of the pattern seen in mitotic cells [17]. While centromere staining is detected in rodent tissues, it is not specifically identifiable, as it requires mitotic-phase cells for confirmation. Other fluorescent patterns in the nucleus include homogeneous, fine-speckled, coarse-speckled, and nucleolar. Several different antibody specificities can yield the same pattern on HEp-2 cells; therefore, it is necessary to perform follow-up testing in order to identify the specificity of the antibody (see below). There are also several structures in the cytoplasm of the cell that react with autoantibodies, particularly mitochondria, Golgi, tRNA synthetases, ribosomes, and GW bodies. Some of these autoantibodies are useful for diagnosing autoimmune diseases [18].

Fig. 8.3 Patterns of immunofluorescence on HEp-2 cells. (A) Antibodies to fibrillarin stain the nucleolus of most cells and decorate the mitotic chromosomes in cells undergoing mitosis. (B) Antibodies to PCNA show various levels of staining in cells that are in S

phase but do not stain cells in other phases of the cell cycle. (C) Antibodies to SS-A/Ro yield a fine-speckled pattern. (D) Antibodies to centromere proteins yield small dots in all cells. In cells that are in mitosis, the dots are aligned with the mitotic chromosomes.

The IIF test for ANA on HEp-2 cells is powerful because it can detect any au-toantibody that binds to structures inside the cell. Antibodies with both known and unknown specificities, giving a wide variety of patterns, are seen. However, as mentioned above, there are a number of problems associated with this test in the clinical lab. It requires a trained technologist to read the slide and thus is labor intensive. There is large lab-to-lab variation in reporting results of IIF on HEp-2 cells due to differences in microscopes such as the power of the objectives and the strength of the fluorescent light, differences in technicians' interpretation of the IIF patterns, differences in the conjugates used to detect the bound autoantibodies (IgG-specific compared to IgG-, IgA-, IgM-, or polyspeci-fic), and differences of the starting dilution of serum (1:40, 1:80, 1:160) among laboratories [14]. More subtle variation is caused by differences in the ways that HEp-2 cells are fixed by various manufacturers [19]. Nonetheless, IIF on HEp-2 cells is the gold standard for screening for ANAs. International standards for identifying many patterns and determining the cutoff between negative and positive are available for ANA screening by IIF on HEp-2 cells [20]. Clinical laboratories could use these standards so that more uniform results can be obtained among laboratories.

Recently, several ELISAs to screen for ANAs were developed [21, 22]. They have gained some popularity because they are easy to automate to reduce labor, the results are objective rather than subjective, and there is not much variation among results performed on ANA ELISAs from the same manufacturer in different laboratories. Notwithstanding these improvements over IIF on HEp-2 cells, the ANA ELISA has many drawbacks. The differences between the ANA ELISAs produced by different manufacturers are much greater than the differences between various HEp-2 cell preparations. Additionally, no ANA ELISA detects all of the autoantibodies detected by HEp-2 cells or all the autoantibodies made by people with diagnosed disease. Thus, some SLE patients are negative on an ANA ELISA but positive by IIF on HEp-2 cells. This defeats the main purpose of the traditional ANA screening test, because a negative result on the ANA ELISA does not rule out SLE. Thus, a negative result on an ANA ELISA is not as useful diagnostically as a negative HEp-2 reading. However, since the best ANA ELISAs will detect the majority of diagnostically important autoantibodies but not many autoantibodies of unknown clinical relevance, a positive result by an ANA ELISA may be more indicative that a person has an autoimmune disease than a positive result by IIF on HEp-2 cells. To a large extent this depends on the cutoff used between positive and negative, since a low cutoff can yield many positive results on an ANA ELISA even in the normal population. The rheumatologist should know what type of ANA screen is used in the laboratory where he sends his patients for testing.

A large number of autoantibodies besides ANAs are detected by IIF on various substrates. Three of the most common and diagnostically important IIF tests are anti-DNA on Crithidia luciliae substrate, ANCA on neutrophils, and anti-endomysial antibody on primate esophagus. These same autoantibodies are identified by other techniques as well. Certain advantages and pitfalls in measuring these antibodies are common to all IIF tests, while others are specific for each type of test.

Because the presence of anti-native DNA antibodies is one of the criteria for diagnosing SLE [13], these autoantibodies have more clinical utility than most. The technique for measuring anti-DNA autoantibodies that has the greatest clinical utility is immunoprecipitation of radiolabeled DNA, commonly called the Farr assay [23]. Patient serum is mixed with radiolabeled DNA, and immune complexes are precipitated with ammonium sulfate. The amount of precipitated radioactivity compared to the radioactivity left in solution determines the amount of anti-DNA autoantibodies present. This technique is quite sensitive and specific, and for many years it was the most common method used in laboratories to measure anti-DNA antibodies. However, it is labor-intensive and uses radioactivity, so it is not as commonly performed today. Either anti-DNA ELISAs [24] or IIF on Crithidia luciliae [25] has largely replaced it. Crithidia luciliae has DNA in both the nucleus and the kinetoplast. Depending on the method of fixation and the fine specificity of the anti-DNA autoantibodies, an anti-DNA-positive sample stains both the nucleus and the kinetoplast or just the kinetoplast.

Numerous studies have compared these three different types of assays [26, 27]. Virtually all of the studies show that the Farr assay has the best clinical sensitivity and specificity to help diagnose patients with SLE. The DNA ELISA is also sensitive, but it is not as specific for SLE patients as the Farr assay. In addition, DNA ELISAs made by different manufacturers vary widely from each other. This is mostly caused by the way that the DNA is bound to the ELISA plate, but is also influenced by the cutoff between positive and negative supplied with the kit and the isotype specificity of the detecting reagent (IgG- or polyspecific). IIF on Crithidia luciliae is the least sensitive of these methods. The general interpretation that ELISA measures both high- and low-affinity antibodies while the Farr assay measures only high-affinity antibodies and IIF measures a subset of anti-DNA antibodies cannot be correct. If this interpretation were right, the ELISA would measure all anti-DNA antibodies, the Farr assay would detect most of those detected by ELISA but not others, and IIF would detect just some of those. This is not what is found. There is usually an 80-90% overlap of reactivity between the techniques, with each technique measuring antibodies that are not detected by the other techniques and not detecting some that are measured by one or both of the other techniques [26, 27]. Thus, there must be complicated interactions between anti-DNA antibodies and the various forms of DNA used to detect the antibodies. Briefly, the Farr assay measures all classes of antibodies that can bind to soluble DNA in high salt and may also measure histone-containing immune complexes that can bind to DNA and cause it to precipitate in high salt [28]. High salt increases the strength of hydrophobic interactions, decreases the strength of ionic interactions, and does not affect van der Waals interactions, the three main forces used in antibody-antigen binding. The DNA in both ELISA and Crithidia luciliae is not soluble but is bound to a solid phase and is thus constrained compared to the DNA in the Farr assay. The kinetoplast DNA in Crithidia luciliae is thought to be super-coiled, while the DNA in most anti-DNA ELISAs is not. Furthermore, even subtle differences in the positively charged substrate used to bind DNA to the ELISA plate can cause large differences in the anti-DNA antibodies detected. The amount of single-stranded DNA in the double-stranded DNA preparation is also important because many people without SLE make anti-single-stranded DNA antibodies. Finally, the specificity of the conjugate (IgG- or polyreactive) that is used to detect the anti-DNA autoantibodies in the ELISA and Crithidia luciliae assay is a strong variable. The subtle differences in the results from these three techniques are not as clinically important as might be expected because the diagnosis of SLE requires that a number of symptoms and laboratory results be positive in a given patient [13, 26].

Positive ANCA results by IIF on ethanol-fixed neutrophils aid in the diagnosis of Wegener s granulomatosis - a rare, life-threatening inflammation of the arteries - and some other types of small-vessel vasculitis. Three specific patterns on ANCA IIF tests are diagnostically important. The c-ANCA pattern, which has a coarse-speckled cytoplasmic stain with interlobular accentuation, indicates that the patient has Wegener's granulomatosis. A perinuclear pattern called p-ANCA is sometimes found in patients with Wegener's granulomatosis but is more common in people with microscopic polyangiitis. [29]. The p-ANCA can be confirmed on formalin-fixed neutrophils by the conversion of the perinuclear pattern to a c-ANCA. Follow-up testing by ELISA for two specific autoantibodies, anti-myeloperoxidase (MPO) for p-ANCA and anti-proteinase-3for c-ANCA, can confirm the IIF results [30]. Patients with anti-PR3 reactivity often have more severe disease than those with anti-MPO antibodies [31]. A few patients with Wegener's granulomatosis are positive on ANCA IIF but not on anti-MPO or PR3 ELISAs, so the ANCA IIF should not be replaced with the ELISAs.

The third important pattern is called X-ANCA or atypical ANCA. This is a perinuclear pattern that is different from p-ANCA and is found primarily in patients with inflammatory bowel disease [32]. The atypical ANCA on ethanol-fixed neutrophils becomes negative on formalin-fixed neutrophils. Any pattern other than the three described above should be called "negative" or "indeterminate." Sometimes the presence of another antibody can mask a c-ANCA or p-ANCA pattern. For example, a strong homogeneous pattern of the nucleus of the neutrophil is not a positive ANCA result because it is not perinuclear. Obviously, it requires a well-trained technician to correctly read ANCA slides.

For many years the gold standard to diagnose people with celiac disease was a characteristic finding on biopsy of the small intestine. Additionally, virtually all of these patients showed an endomysial pattern by IIF on primate esophagus that was from IgA autoantibodies. Recently it was found that autoantibodies to tTG cause the IIF pattern typical of patients with celiac disease [4]. Originally, guinea pig tTG was used as the substrate in ELISA, but it was less sensitive and specific than IIF on primate esophagus. Now that human rather than guinea pig antigen is used, the ELISAs are as good as or better than IIF [33], because antibodies that interfere with IIF do not affect ELISA results. Celiac disease is caused by an immunologic reaction to gluten in wheat and other grains and is typically diagnosed in children who have a failure to thrive and in some other people with stomach ailments. The cure is for the patient to go on a gluten-free diet, which is safe and simple but not easy because wheat is present in so many foods.

With the availability of the tTG ELISA, more immunologic screening for ce-liac disease has been performed in the last few years than ever before. The disease has been found to be more prevalent than previously expected. When an adolescent population in Switzerland was screened for anti-tTG autoantibodies, this reactivity was found in about 1 in 150 people [34]. Because some people with subclinical celiac disease have symptoms that are not usually associated with the disease, such as headache, muscle ache, or general fatigue, the ability to perform widespread screening will be very useful [35]. The symptoms of many people with subclinical celiac disease improve on a gluten-free diet. This is an example where an improvement in technology allowed wider testing of the general population and new insights into the frequency and manifestations of a disease. There is also some discussion among gastroenterologists that a biopsy does not need to be performed to diagnose celiac disease when a person has a positive anti-tTG test, in conjunction with improvement on a gluten-free diet. If this becomes standard, a serologic test will replace an invasive diagnostic procedure.

There are many more clinically useful IIF tests to detect autoantibodies found in both systemic and organ-specific autoimmune diseases. All IIF tests require a large amount of skilled labor to read and interpret the slide. For a number of autoantibodies, alternative tests are available in a format like ELISA that is less labor-intensive and less subjective. Sometimes the ELISA performs as well as or better than the IIF test, while in other cases the ELISA is different enough from the IIF test that it yields different clinical sensitivity and specificity. Each auto-antibody-antigen system needs to be examined individually.

The first three autoantibodies detected were rheumatoid factor (RF), the false-positive VDRL (Venereal Disease Research Laboratory) result, and lupus erythematosus (LE) cell factor. Interestingly, at the time these tests were developed no one knew that they were measuring an autoantibody-antigen reaction. Agglutination of sheep red blood cells coated with rabbit IgG was used to detect RF, usually found in patients with RA [36]. Flocculation seen under a microscope of the reagent from the VDRL was used to detect reagin, which is found in people with syphilis and in some people with SLE. The SLE patients were usually negative on a separate test to detect anti-syphilis reactivity and thus were considered false positive on the VDRL test [37]. Phagocytosis of nuclei by segmented neutrophils seen in a stained blood smear was used to detect LE cell factor [38]. In one form or another, these autoantibodies are still commonly measured today and the techniques used to detect them have evolved over the years.

RFs are autoantibodies that recognize the Fc portion of IgG. Most RFs are IgM class, but may also be IgG or IgA. RFs are found in 50-90% of rheumatoid arthritis patients. RFs are sometimes present even before symptoms of disease develop but become positive in a higher percentage of RA patients as their disease progresses [39]. Even with the large technological advances over the last 50 years, RFs are still often measured by agglutination tests. Some labs still use IgG-coated red blood cells or latex beads, but more often the test is performed in a nephelometer or related machine so that the test is completely automated. Because they are pentamers, IgM antibodies are detected with approximately 10 times more sensitivity than IgG antibodies in agglutination tests. Therefore, any agglutination test is biased to detect IgM isotypes.

ELISAs that detect RFs use IgG-, IgM-, and IgA-specific detecting reagents to detect RFs of each immunoglobulin class. RFs are increased in people with acute infections, some chronic infections such as hepatitis C virus, certain autoimmune diseases such as Sjogren's syndrome, and rarely in healthy individuals [40]. Thus, they are not specific for rheumatoid arthritis. A new diagnostic test to help identify people with rheumatoid arthritis has recently been discovered. The antigen in two IIF tests that were used to help diagnose RA, the anti-keratin test on rat stomach and the perinuclear factor test on buccal cells, was identified as citrullinated filaggrin [5]. An ELISA that contains a peptide with the modified amino acid citrulline was developed [41]. At this time the best-accepted test is to a cyclic citrullinated peptide (CCP) that mimics a citrullinated epitope on filaggrin. Anti-CCP antibodies are found in about 65% of RA patients and are rarely found in people with infections or other autoimmune diseases [42]. Anti-CCP antibodies are found early in the course of disease, often when RFs are not present. About 80% of RF-positive RA patients are also positive for anti-CCP antibodies. Importantly, about 40% of RF-negative RA patients are positive for anti-CCP. There is clinical utility in measuring both RF and anti-CCP. Someone who is positive for both autoantibodies is very likely to have RA. Because some RA patients are positive for only one or the other autoantibody, measuring both autoantibodies detects a greater percentage of RA patients than does measuring one autoantibody alone.

Flocculation of VDRL was generally used as a test for syphilis. When more specific tests for anti-treponema antibodies were developed, it was shown that some people with SLE yielded false-positive results on the VDRL test. Today, the VDRL reagent has been replaced by tests that are more specific for anti-trepone-ma antibodies. An anti-cardiolipin ELISA is used to screen for the type of auto-antibodies that yielded the false-positive VDRL results in patients with SLE. The false-positive VDRL test is one of the criteria to diagnose SLE [13]. Recently, it was suggested that this criterion be changed to a positive anti-cardiolipin result [43]. However, an anti-cardiolipin test and a false-positive VDRL test do not measure the same antibodies [44].

Besides people with SLE, anti-cardiolipin autoantibodies are found in people with antiphospholipid syndrome, a condition in which the chances of thrombosis, stroke, and recurrent fetal loss are increased. Patients with syphilis make true anti-cardiolipin antibodies. However, the majority of diagnostically important autoantibodies measured by the anti-cardiolipin ELISA actually react with beta 2-glycoprotein 1 (S 2-GP1), a positively charged serum protein that binds to the negatively charged cardiolipin on the ELISA plate [45, 46]. The S2-GP1 originates in bovine serum added to the blocking solution for the cardiolipin ELISA plate or the sample diluent or from the patients serum itself. Once S2-GP1 binds to cardiolipin, it becomes reactive with the autoantibodies in sera. S2-GP1 can also bind directly onto an ELISA plate in an immunologically active form. The titers of anti-cardiolipin and anti-S 2-GP1 antibodies have a relatively strong correlation with each other. The data so far suggest that some "anti-cardi-olipin" autoantibodies bind epitopes on S2-GP1 alone and that some bind an epitope comprised of both cardiolipin and S2-GP1, but virtually none of them bind to cardiolipin by itself. Because of historic precedence, the terms "antipho-spholipid syndrome" and "anti-cardiolipin" antibodies are still used, even though they are technically incorrect.

Another assay that measures autoantibodies that are correlated to an autoimmune coagulation disorder is the lupus anticoagulant test [47]. This test is performed on plasma that has been treated with calcium and phospholipid. A positive result is a prolonged clotting time, which is ironic because people with this activity are at risk for increased clotting in vivo. There is only a modest correlation between anti-cardiolipin, anti-S 2-GP1, and the presence of lupus anticoagu lant. Finding an ELISA that matches the lupus anticoagulant test is an active area of research because the lupus anticoagulant test is clinically correlated with clotting problems in SLE patients, some of whom are anti-/52-GP1 negative. Because the lupus anticoagulant has not been identified, it is possible that it is not an autoantibody, while it also might be a set of autoantibodies.

For many years the LE cell test to help diagnose people with SLE was routinely performed in many laboratories throughout the world. In its most typical form, clotted blood was passed through a strainer to break open some lymphocytes, and allowed to incubate for several hours. A drop was smeared on a slide, stained, and examined under a microscope. LE cells were formed when a segmented neutrophil engulfed nuclear material [38]. This occurred in the presence of three things: autoantibodies that bound the nuclear material, active complement, and viable cells. LE cell reactivity was found predominantly in people with SLE, but also in people with drug-induced lupus and lupoid hepatitis. Numerous studies in the 1950s through the 1970s found that adsorption with chro-matin (called deoxyribonucleoprotein at that time), but not its individual components, i.e., DNA-free histone or histone-free DNA, could remove LE cell reactivity from sera [48, 49]. Thus, it was concluded that anti-chromatin, but not anti-DNA or anti-histone, autoantibodies accounted for LE cell reactivity. Recently, some papers have suggested that antibodies to histone H1 account for the LE cell reactivity [50]. There is no explanation for many other researchers finding the opposite result.

The LE cell test is very labor-intensive, is difficult to reproduce, and requires fresh blood. It is rarely performed in the U.S. today but is still performed in other countries. It has largely been replaced or supplemented by anti-DNA testing for these technical reasons. However, the LE cell factor was generally found in a higher percentage of SLE patients than were anti-DNA autoantibodies, so it would be clinically useful if there were an ELISA to replace it [13]. The anti-chromatin ELISA has many of the same properties as the LE cell test. Anti-chromatin autoantibodies are more common than anti-DNA in SLE patients, and they are found in people with SLE, drug-induced lupus, and lupoid hepatitis but not other diseases [51]. Additionally, numerous studies from labs around the world found that anti-chromatin autoantibodies are a sensitive and specific marker for SLE and correlate with kidney disorders or active disease [52]. These are similar to correlations with the LE cell assay.

Autoantibodies to ENAs such as SS-A/Ro, SS-B/La, Sm, RNP, and Scl-70 are common in people with systemic connective tissue diseases such as SLE, Sjogren's syndrome (SS), and systemic sclerosis (SSc) [53]. Jo-1 is an extractable cy-toplasmic antigen and antibodies to it are helpful in diagnosing people with polymyositis or dermatomyositis (PM/DM) [54]. Originally, all these antibodies were detected by the Ouchterlony immunodiffusion technique. This is still the technique used in hundreds of small laboratories and some large ones. Once the above antigens were purified, these tests could all be performed by ELISA. This was the method of choice for large laboratories for several years. Because autoantibodies to the ENAs are typically measured in all people suspected of having one of the autoimmune connective tissue diseases, they were the first autoantibodies detected by the three multiplex technologies described in the last section.

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  • Isembard
    Is it common to check a vdrl for arthritis?
    7 months ago

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