Is IIFT good for HR
Rudolf Gruber, Lothar Thomas
25.1.1 Mechanisms of Autoimmunity
A critical function of the immune system is to distinguish the self from the foreign. The tolerance to self-antigen is a highly specifically regulated process and in order to maintain it, the immune system must be able to remove its self-reactive lymphocytes / 1 /. A lack of self-tolerance and a defect in the elimination and control of self-reactive lymphocytes are fundamental criteria of autoimmune diseases. It is generally accepted that autoimmune diseases are the result of a complex interaction between genetic and environmental factors / 2 /.
From the pathogenetic point of view, autoimmune diseases are characterized by the chronic activation of the immune system, which leads to tissue inflammation. Innate immunity activates the adaptive immune system, which in turn is responsible for maintaining an inflammatory process / 3 /.
Important clinical criteria of autoimmune diseases are / 2 /:
- A relatively high prevalence in the younger population.
- The chronic behavior.
- The diseases are very different in the affected organs.
- Some diseases are limited to one organ or tissue, others are systemic in nature.
- Most patients present to the doctor in the propagation phase, which is characterized by progressive inflammation and tissue damage.
- It is often difficult to determine the factors that are responsible for the onset of the disease.
- Patients can have autoantibodies several years before the onset of the disease.
In autoimmune diseases, autoantibodies in the serum are directed against structures and functional components of the cells, e.g. against nucleic acids, receptors of the cell nucleus and components of the cytoplasm. The antibodies play an important role in the diagnosis and differentiation of autoimmune diseases. They are synonymous with autoimmune diseases, but this is not always the case because other disease entities such as cancer and acute tissue damage can also be associated with the antibodies / 3 /.
Important autoantigens and corresponding antibodies are:
- DNA molecules, anti-DNA antibodies are formed.
- Centromere, anti-centromere antibodies are formed.
- H2A and H2B, anti-histone antibodies are formed
- Autoepitopes of the cell nucleus, also known as extractable nuclear antigens, and the corresponding antibodies
- Topoisomerase, against which anti-Scl70 antibodies are directed.
- Sm, snRNPs, SSB- and SSA, the corresponding autoantibodies are detectable in Sjögren's syndrome.
The prevalence of systemic and organ-specific autoimmune diseases is 3–5% in the population. This applies to common diseases such as diabetes mellitus type 1, rheumatoid arthritis, autoimmune diseases of the thyroid, but also rare diseases such as diseases of the connective tissue and immune-mediated inflammatory diseases of the gastrointestinal tract (atrophic gastritis, celiac disease, Crohn's disease, ulcerative colitis) or diseases of the central nervous system, e.g. autoimmune neuropathies.
There are associations between the MHC equipment of a person, i.e. the HLA genes and various autoimmune diseases (Tab. 25.1-1 - HLA associations with autoimmune diseases), i.e. some HLA alleles significantly increase the risk of suffering a certain autoimmune disease. HLA-B27 positive individuals have the highest relative risk for the Bechterew disease. These people are 80 times more likely to develop Bechterew disease than HLA-B27 negative people. About 95% of Bechterew's disease patients are HLA-B27 positive. Although 8% of the German population have the HLA-B27 allele, only a small proportion then also develop Bechterew's disease.
Autoimmunity and Primary Immune Deficiency
There are approximately 200-300 primary autoantibody-associated diseases, all of which can lead to abnormal function of the immune system and can be a basis for single or frequent, severe, atypical and chronic infections. Primary immune deficiencies are often associated with autoimmune diseases due to a dysregulation affecting the entire immune system / 4 /.
Some primary immune deficiency diseases have autoimmunity as an essential characteristic / 4 /. See Tab. 25.1-2 - Primary immune deficiency diseases with autoimmunity as an essential characteristic.
Autoinflammation and autoimmunity
The immune system is divided into an innate and an acquired one (adaptive). Immunity disorders can be due to decreased function or hyperactivity of the immune system. Disorders of innate immunity without the involvement of T cells or B cells are called auto-inflammatory syndromes and are characterized by repeated episodes of fever and systemic inflammation. A strong activation of the adaptive immune system results in the formation of reactive lymphocytes against self, an auto-inflammation and the high-titer formation of antibodies, which are typical for an autoimmune disease / 8 /.
In auto-inflammation, the innate immune system directly causes the inflammation of the tissues, while in autoimmune disease, the innate immune system activates the adaptive immune system, which is now responsible for the inflammatory process. Auto-inflammatory diseases are a group of rare hereditary, recurrent and unprovoked diseases that occur without an infection being present. Patients with these diseases do not have autoantibodies or auto-reactive antigen-specific T cells that trigger the disease process.
See also Tab. 25.1-3 - Classification of autoinflammatory and autoimmune diseases.
25.1.2 Clinic of Autoimmunity
Clinically, autoimmune diseases are classified based on the organs or organ systems affected. At one end of the spectrum are systemic autoimmune diseases such as SLE, in which several organ systems are primarily affected, and at the other end autoimmune diseases that are restricted to one organ, such as Hashimoto's thyroiditis, in which only the thyroid is affected.
The degree of manifestation of most autoimmune diseases lies between these two extremes, i.e. they manifest themselves primarily in one organ system, but can also affect others (Table 25.1-4 - spectrum of autoimmune diseases).
In addition to the relatively clearly defined autoimmune diseases, there are inflammatory diseases, infections and also neoplasms that are associated with the phenomenon of autoimmunity and with autoantibodies. Celiacia is an interesting model disease. Here, a foreign antigen is assumed to be gliadin or gluten as the trigger for a pathological immune reaction; so actually in the sense of an allergy. Specific autoantibodies against the enzyme that deaminates gliadin (tissue transglutaminase) develop regularly in the sense of a classic autoimmunity. Interestingly, immune reactions against tissue transglutaminase in the skin lead to the development of dermatitis herpetiformis Duhring, possibly in the sense of self-tolerance.
25.1.3 Antibody determination in autoimmune diseases
Laboratory diagnostics include examinations to demonstrate the activity, organ involvement and side effects of drugs. Depending on the manifestation, different causes, especially infections, come into question in differential diagnosis, so that in some cases extensive laboratory diagnosis is necessary. See Tab. 25.1-5 - Laboratory diagnostics if systemic autoimmune diseases are suspected
The titers of the disease-specific autoantibodies only rarely correlate, e.g. in the case of the anti-ds-DNA antibodies in SLE or the anti-PR3 antibodies in Wegener's granulomatosis, with the prognosis, severity and response to therapy in autoimmune diseases. Inflammation markers are therefore primarily used to assess the course and for therapeutic monitoring.
In addition, markers such as procalcitonin or cytokines such as IL-6 are also determined in certain cases to better differentiate a bacterial superinfection from a disease flare-up.
In principle, all larger molecules can appear as autoantigens in the organism / 7 /:
- Nucleic acids (DNA, RNA).
- Proteins (structural proteins, receptors, intracellular enzymes).
- Glycoproteins (β2-Glycoprotein I).
- Phospholipids (cardiolipin)
- Glycosphingolipids (gangliosides).
The specific autoantibodies that are directed against these antigens can be detected in serum, body fluids and tissues. Antibodies of the IgG class and occasionally of the IgA class are diagnostically relevant in the serum. With a few exceptions, autoantibodies of the IgM class are usually not very meaningful because they are very unspecific and are often detected as physiological auto-reactivity in healthy individuals.
- Suspected systemic autoimmune disease, e.g. if there is an inflammatory process that cannot be explained by an infection.
- Differential diagnosis of systemic diseases, especially in the differentiation from drug-allergic or paraneoplastic diseases.
- Diagnosis and differential diagnosis of inflammatory organ diseases, e.g. stomach, intestines, liver, muscles, bullous skin diseases, diseases of the endocrine system and the central and peripheral nervous system.
- Prognosis of autoimmune diseases, e.g. with collagenosis and myositis.
Important autoantibodies in predominantly systemic autoimmune diseases are shown in Table 25.1-6 - Autoantibodies in systemic autoimmune diseases.
Antibodies in organ-specific autoimmune diseases are listed in Tab. 25.1-7 - Autoantibodies in organ-specific autoimmune diseases.
184.108.40.206 Determination method
Screening for autoantibodies is often done using an indirect immunofluorescence test (IIFT). The disadvantage of the IIFT is its low specificity, i.e. there are frequent false positive results without an associated clinical picture.
In the event of a positive reaction to clarify the specificity of the autoantibodies, immunoassays are used:
- Enzyme immunoassay (ELISA, EIA).
- Luminescence, chemiluminescence or electrochemiluminescence assay.
- Immunoblot (western blot)
- Dot / line immunoblot
- Array techniques (laser / bead array).
- Immunoelectrophoresis (hardly used any more).
- RIA (FARR assay for anti-dsDNA, otherwise hardly used any more).
If an immunoassay using a defined panel of autoantigens is used as the screening test, the positive predictive value is higher due to the better specificity, since only the specific autoantibodies against the antigens used in the test are recorded. The negative predictive value is often lower than with the IIFT, since rare autoantigens are not included in the immunoassays and therefore cannot be recorded. In order to rule out false negative results, several methods should be used in parallel for clinically critical diagnoses such as Goodpasture's syndrome or ANCA-associated vasculitis / 9 /. The autoantibodies most frequently requested in diagnostics are described in separate articles:
220.127.116.11 Examination material
Serum: 1 ml
18.104.22.168 Reference range
Limit values for the individual autoantibodies are applied.
The systemic autoimmune diseases are classified into:
- Rheumatoid arthritis.
- SLE (systemic lupus erythematosus).
- Systemic scleroderma.
- Sjogren's Syndrome.
- Polymyositis / dermatomyositis.
- MCTD (Mixed connective tissue disease, mixed collagenosis, Sharp syndrome).
- Antiphospholipid Syndrome.
- ANCA-associated vasculitis
- Goodpasture syndrome / pulmonary syndrome.
The common pathogenic principle of systemic autoimmune diseases consists in the abolition of the immune system's natural tolerance to the body's own structures. The mechanisms set in motion lead to immune reactive inflammation of the tissues with the impairment of the function of various organs. Systemic autoimmune disease should be considered if inflammatory or ischemic involvement of several organs is present and is associated with pronounced general symptoms.
In the majority of clinical pictures, only unspecific symptoms such as arthralgia, myalgia, weight loss or night sweats are present at the beginning of the disease. The definitive diagnosis can only be made when / 10 / is present:
- Key clinical symptoms, e.g. polyarthritis.
- Characteristic laboratory findings, e.g. autoantibodies.
- Typical lesions using high-resolution imaging methods, e.g. characteristic erosions in the sonography of joints.
22.214.171.124.1 Autoantibodies in organ-specific autoimmune diseases
See Tab. 25.1-7 - Autoantibodies in organ-specific autoimmune diseases.
126.96.36.199.2 Autoantibodies in systemic autoimmune diseases
See Tab. 25.1-8 - Systemic autoimmune diseases.
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2. Rosenblum MD, Remedios Ka, Abbas AK. Mechanisms of human autoimmunity. J Clin Invest 2015; 125: 2228-33.
3. Doria A, Zen M, Bettio S, Gatto M, Bassi N, Nalotto L, et al. Autoinflammation and autoimmunity. Autoimmunity Rev 2012; 12: 22-30.
4. Lehman HK. Autoimmunity and immune dysregulation in primary immune deficiency disorders. Curr Allergy Asthma Rep 2015; 15 DOI 10.1007 / s11882-015-0553-x
5. Husebye ES, Anderson MS, Kämpe O. Autoimmune polyendocrine syndromes. N Engl J Med 2018 378 1132-41.
6. Sneller M, Wang J, Dale JK, Strober W, Middelton LA, Choi Y, et al. Clinical immunologic, and genetic features of an autoimmune lymphoproliferative syndrome associated with abnormal lymphocyte apoptosis. Blood 1997; 89: 1341-8.
7. Wildin RS, Smyk-Pearson S, Filipovich AH. Clinical and molecular features of the immunodysregulation, polyendocrinopathy, eneropathy, X-linked (IPEX) syndrome. J Med Genet 2002; 39: 537-45.
8. Occurede VU, Franchi L. Immunology in clinic review series; focus on autoinflammatory diseases: role of inflammasomes in autoinflammatory syndromes. Clinical and Experimental Imunology 2011; 167: 382-90.
9. Remeny B, ElGuindy A, Smith SC Jr, Yacoub M, Holmes DR JR. Valvular aspects of rheumatic heart disease. Lancet 2016; 387: 1335-46.
10. Hellmich B, Merkel F, Weber M, Gross WL. Early diagnosis of chronic systemic inflammatory disorders. Internist 2005; 46: 421-32.
11. Tozzoli R. The diagnostic role of autoantibodies in the prediction of organ-specific autoimmune diseases. Clin Chem Lab Med 2008; 46: 577-87.
12. Conrad K, Roggenbuck D, Reinhold D, Dorner T. Profiling of rheumatoid arthritis associated autoantibodies. Autoimmune Rev 2010; 9: 431-5.
13. Snow MH, Mikuls TR. Rheumatoid arthritis and cardiovascular disease: the role of systemic inflammation and evolving strategies of prevention. Curr Opin Rheumatol 2005; 17: 234-41.
14. Scott DL, Wolfe F, Huizinga TW. Rheumatoid arthritis. Lancet 2010; 376: 1094-108.
15. Prakken B, Albani S, Martini A. Juvenile idiopathic arthritis. Lancet 2011; 377: 2138-49.
16. Gupta R, Thabah MM, Vaidya B, Gupta S, Lodha R, et al. Anti-cyclic citrullinated peptide antibodies in juvenile idiopathic arthritis. Indian J Pediatr 2010; 77: 41-4.
17. Sawhney S, Magalhaes CS. Pediatric rheumatology - a global perspective. Best Pract Res Clin Rheumatol 2006; 20: 201-21.
18. Bagnari V, Colina M, Ciancio G, Govoni M, Trotta F. Adult-onset Still’s disease. Rheumatol Int 2010; 30: 855-62.
19. Fanouriakis, A, Kostopoulou M, Alunno A, Aringer M, Bajema I, Ioannidis JPA, Boletis JN, et al. 2019 update of the EULAR recommendations for the management of systemic lupus erythematosus. Ann Rheum Dis 2019; 78: 736-45.
20. Bohan A, Peter JB. Polymyositis and dermatomyositis (first of two parts). N Engl J Med 1975; 292: 344-7. Polymyositis and dermatomyositis (second of two parts). N Engl J Med 1975; 292: 403-7.
21. Maddison PJ. Mixed connective tissue disease: overlap syndromes. Baillieres Best Practice Res Clin Rheumatol 2000; 14: 111-24.
22. Vitali C, Bombardieri S, Jonsson R, Moutsopoulos HM, Alexander EL, et al. Classification criteria for Sjogren's syndrome: a revised version of the European criteria proposed by the American-European Consensus Group. Ann Rheum Dis 2002; 61: 554-8.
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24. van der Helm-van Mil AH. Acute rheumatic fever and post streptococcal reactive arthritis reconsidered. Curr Opin Rheumatol 2010; 22: 437-42.
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26. Outhred AC, Kok J, Dwyer DE. Viral arthritis. Expert Rev Anti Infect Ther 2011; 9: 545-54.
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29. Tan EM, Cohen AS, Fries JF, Masi AT, McShane DJ, et al. The 1982 revised criteria for the classification of systemic lupus erythematosus. Arthritis Rheum 1982; 25: 1271-7.
25.2 Antinuclear Antibodies
Rudolf Gruber, Lothar Thomas
Autoantibodies are a hallmark of autoimmunity, with antinuclear antibodies (ANA) being the main laboratory diagnostic indicator. Detection of ANA is essential in diagnosing many autoimmune diseases. Autoantibodies have long been determined using the indirect immunofluorescence test (IIFT) method. Alternative methods have been developed and represent a diagnostic challenge for the IIFT. The alternative methods differ from the IIFT in the antigen profiles and the diagnostic sensitivity and specificity. The term ANA is also no longer technically correct, because it does not include the entire spectrum of relevant autoantibodies directed against cells. In addition to the nuclear pattern of the cells, antigens in the cytoplasm and the mitotic core pattern are important for the diagnosis of autoimmune diseases. It would therefore make sense to change the term ANA to a more appropriate one, i.e. instead of ANA to anti-cellular antibodies / 1 /.
For the detection of extractable nuclear antigens of the cell nucleus (extractable nuclear antigens, ENAs), the samples are first examined with the IIFT and, if positive, the examination for ENAs is then carried out.
Therefore one should expect that the inconsistent terms ANA and ENA will be replaced by the terms anti-cellular antibodies and specific antibodies. But such a change in the nomenclature would not receive the undivided approval of the doctors / 1 /.
ANA and ENA identify a group of autoimmune diseases for which they are an important criterion for diagnosis and differentiation. The titre or the concentration of the ANA and the specific antibodies must be taken into account in the differential diagnosis, because the sensitivity and specificity of the autoantibodies can be very different in the individual autoimmune diseases / 2 /.
Suspected connective tissue disease:
- Systemic lupus erythematosus (SLE).
- Sjogren's Syndrome.
- Scleroderma, including CREST syndrome.
- Mixed collagenosis (MCTD, Sharp syndrome).
- Polymyositis / dermatomyositis.
- Drug-induced lupus erythematosus.
- Therapy with TNF-α blockers.
- Suspected autoimmune hepatitis.
- Juvenile idiopathic arthritis.
25.2.2 Determination method
According to recommendations and guidelines, including those of the American College of Rheumatology (ACR) / 2 /, the ANA is diagnosed as a screening test with the indirect IIFT on HEp2 cells. If the reaction is positive, a differentiation by means of immunoassays is carried out afterwards (Fig. 25.2-1 - Step diagnosis for differentiation of antinuclear antibodies)
188.8.131.52 Indirect Immunofluorescence Test (IIFT)
For the IIFT, HEp2 cells are used on microscope slides, which must have a sufficient number of mitotic cells to show an adequate pattern. HEp2 cells are a cell line that was originally obtained from the epithelial cells of a laryngeal carcinoma. This cell line expresses almost all ANA antigens in such large quantities that most ANA can be detected with a high degree of sensitivity. Only SS-A / Ro is only slightly expressed. For this reason, some manufacturers offer genetically modified cells with the overexpression of SS-A, although the additional diagnostic advantage is not clearly proven. The detection antibody (conjugate) should consist of human antigen-specific IgG antibodies, labeled with fluorescein isothiocyanate (FITC) or a validated fluorochrome of the newer generation. The dilution for screening a serum sample is usually 1: 100 or 1: 160/1 /.
If the IIFT-ANA is positive, it is recommended to state the pattern of fluorescence and the highest positive dilution level in the report. A titration to a dilution of more than 1: 2.560 does not result in any usable expansion of the findings / 1, 12 /.
The IIFT shows whether ANA is contained in the respective sample and, if so, in what titer level (serum dilution). Based on the fluorescence pattern, the respective cell structures and partly also the antigens that bind to the ANA can be deduced. Since some ANA specificities are relatively strongly associated with a certain disease, some fluorescence patterns, e.g. antibodies against centromeres (nuclear dots, few nuclear dots), allow conclusions to be drawn about the underlying disease. Other fluorescence patterns can be described, but do not allow an assignment of the respective target structure, e.g. speckled or homogeneous nuclear fluorescence. Many cytoplasmic target structures such as ribosomes, lysosomes, Golgi apparatus, actin, vimetin, Jo-1 can be suspected on the basis of the fluorescence pattern, but often cannot be clearly assigned. Differentiation should be made depending on the clinical relevance of the suspected autoantibodies.
See Tab. 25.2-1 - Immunofluorescence patterns on HEp2 cells, associated antigens and diseases.
184.108.40.206 Specific antibody determination
A differentiating investigation of the ANA specificities is carried out with an increased ANA titer with defined antigens (Tab. 25.2-2 - autoantibodies and target antigens in systemic autoimmune diseases, especially collagenoses).
Most screening ENAs use defined cocktails of antigens that are effective and inexpensive. The spectrum often enables a focused statement on the autoimmune disease / 3 /. Using the original method of this differentiation, a subgroup of antigens obtained from saline extracts of isolated cell nuclei is often defined as extractable nuclear antigens (ENAs). The antigens Sm, RNP, SS-A and SS-B are essentially present in this coarse cell nucleus extract / 4 /. In the IIFT, antibodies to these antigens create a mottled pattern.
The immunoassays are based on purified native antigens or on recombinant antigens produced by genetic engineering. The advantage of recombinant antigens is better standardization and their constant availability. However, some autoantibodies do not recognize conformational epitopes or post-translationally modified epitopes, which are not always optimally reproduced by recombinantly produced antigens, especially when bacterial expression systems are used.
Methods for the detection of specific autoantibodies are:
- Enzyme immunoassay (ELISA, EIA).
- Luminescence, chemiluminescence or electrochemiluminescence assay.
- Immunoblot (western blot).
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