25

Autoimmunity and antibody testing

25

Autoimmunity and antibody testing

25

Autoimmunity and antibody testing

25

Autoimmunity and antibody testing

25.1 Autoimmunity

Rudolf Gruber, Lothar Thomas

25.1.1 Mechanisms of autoimmunity

The critical function of the immune system is to discriminate self from non self. Tolerance against self-antigens is a highly regulated process and, in order to maintain it, the immune system must be able to distinguish self-reactive lymphocytes as they develop /1/.

A dynamic process of self-nonself discrimination is the basis of immune tolerance.Two distinctive types of immune cells, T and B cells are the hallmark of antigen-specific adaptive immunity. T cells orchestrate immune responses /30/:

  • directly, by killing foreign and infected tissues through cellular and soluble mediators.
  • indirectly by providing soluble and and membrane-associated signals that promote the survival, expansion, and differentiation of B cells.

In a parallel system regulatory Tcells (Tregs) recognize self-peptides, and when activated, control self-reactive pathogenic T cells. This complex, dynamic process of self-nonself discrimination is the basis of immune tolerance.

T cells recognize foreign antigens through a unique, highly diverse set of T cell receptors designed to mediate immunity without collateral damage of destroying native tissues.

Failure of self-tolerance and defective elimination and control of self-reactive lymphocytes are the fundamental abnormalities in autoimmune diseases. However, it is generally accepted that autoimmune diseases are the result of a complex interaction between genetic and environmental factors, most of which have not been identified /2/.

Autoimmune diseases

From a pathogenetic point of view autoimmune diseases are characterized by a chronic activation of the immune system, which leads to tissue inflammation. The innate immune system activates the adaptive immune response which, in turn, is responsible for the inflammatory process /3/.

A significant clinical problem of autoimmune diseases are /2/:

  • Their prevalence in the younger population
  • The chronic nature
  • Diseases vary greatly in the organs they affect and in their clinical manifestation
  • Some being limited to particular tissues and others being systemic or disseminated
  • Most patients present with clinical disease during the propagation phase, which is characterized by progressive inflammation and tissue damage
  • It is often difficult to evaluate the factors responsible for the initiation of disease
  • Patients may carry antibodies many years before they manifest clinical symptoms

In autoimmune diseases a large number of antibodies in serum are directed against structures and functional components of the cell e.g., nucleic acid, nuclear molecular receptors and components of the cytoplasm can be detected. The antibodies play an important role in the diagnosis and differentiation of the autoimmune disorders. They were considered synonymous of autoimmune disease, but that is not always the case, because other conditions are associated with their presence e.g., acute tissue damage and cancer /3/.

Identified auto antigens are /3/:

  • DNA molecules and the bound histones
  • Anti histone antibodies directed against H2A and H2B
  • Anti-centromere (expressed or activated during specific phases of the cell cycle)
  • Auto epitopes of the nucleus , called extractable nuclear antigens (ENA)
  • Anti Scl70 antibodies directed against topoisomerase
  • Anti-Sm, snRNPs, SSB and SSA antibodies found in Sjögren’s syndrome.

The prevalence of systemic and organ specific autoimmune diseases in the population is 3–5%. This includes common diseases such as diabetes mellitus type I, rheumatoid arthritis and autoimmune thyroid disease, as well as rare diseases such as connective tissue diseases, immune mediated inflammatory diseases of the gastrointestinal tract (atrophic gastritis, celiac disease, Crohn’s diseases, and ulcerative colitis) or diseases of the central and peripheral nervous system (e.g., autoimmune neuropathies).

There are associations between MHC alleles (HLA genes) of an individual and various autoimmune diseases (Tab. 25.1-1 – HLA associations with autoimmune diseases). Some HLA alleles significantly increase the risk of contracting a specific autoimmune disease. The highest relative risk for ankylosing spondylitis (Bekhterev’s disease) is seen in HLA-B27 positive individuals. These persons are 80 times more likely to develop ankylosing spondylitis than HLA-B27 negative individuals. Approximately 95% of patients with ankylosing spondylitis are HLA-B27 positive. Although 8% of the German population have the HLA-B27 allele, only a small proportion of these individuals develop ankylosing spondylitis.

Autoimmunity and primary immune deficiency disorders

Primary immune deficiency diseases consist of a group of over 200 genetic defects, all of which can ultimately lead to an ab errantly functioning immune system and predispose individual to recurrent, chronic, atypical, or severe infections. Primary immune deficiencies are often associated with autoimmune diseases due to deregulation of the immune system as a whole /4/.

Some primary immune deficiencies have autoimmunity as a defining feature /4/. Refer to Tab. 25.1-2 – Primary immune deficiencies with autoimmunity as a defining feature.

Auto inflammation and autoimmunity

The immune system is categorized broadly as innate and adaptive. Disorders of the immune system may be due to impaired activation or hyper activation of either the innate or the adaptive immune system. Disorders of the innate immune system with no or little involvement of T and B cells are called auto inflammatory syndromes and are characterized by recurrent episodes of fever and systemic inflammation. An ex aggregated activation of the adaptive immune system results in the generation of self-reactive lymphocytes, auto inflammation and high-titre antibodies that are typical features of autoimmune diseases /8/. In auto inflammation the innate immune system directly causes tissue inflammation, whereas in autoimmune disease the innate immune system activates the adaptive immune system which, in turn, is responsible for the inflammatory process. Auto inflammatory diseases refer to a group of rare hereditary recurrent, unprovoked inflammatory disorders which occur in the absence of infection. Patients affected with auto inflammation do not have autoantibodies or auto reactive antigen specific T cells driving the disease process /3/.

Refer to Tab. 25.1-3 – Classification of auto inflammatory and systemic autoimmune diseases.

25.1.2 Clinical characteristics of autoimmunity

Clinically, autoimmunopathies are categorized on the basis of organs or organ systems affected. At one end of the spectrum are systemic autoimmune diseases, such as systemic lupus erythematosus (SLE), in which several organ systems are affected primarily, while the other end represents autoimmune diseases that remain restricted to a single organ, such as Hashimoto’s disease, in which only the thyroid is affected. In most autoimmune diseases the degree of manifestation is between these two extremes (i.e., the disease predominantly manifests in a single organ system, but also affects other organs).

Refer to Tab. 25.1-4 – Spectrum of autoimmune diseases.

25.1.3 Antibody testing of autoimmune diseases

General laboratory testing comprises for determining activity, organ involvement and drug side effects (Tab. 25.1-5 – Laboratory workup for suspected systemic autoimmune diseases). Depending on the manifestation, various causes, above all infections, must be considered when making a differential diagnosis. This may required extensive laboratory testing.

The titers of disease specific autoantibodies rarely (e.g., in the case of anti-dsDNA antibodies in SLE or anti-PR3 antibodies in Wegener’s granulomatosis) correlate with the prognosis, the severity and the treatment response in autoimmune diseases. Therefore, disease and treatment monitoring focuses mainly on measuring inflammatory parameters.

In addition, in some cases inflammatory markers are measured e.g., cytokines such as IL-6 (especially in pediatrics), or procalcitonin to help differentiate a bacterial superinfection from a flare-up.

In principle, all large molecule structures in the body may act as auto antigens /9/. The spectrum of autoantibodies includes:

  • Nucleic acids (DNA, RNA)
  • Proteins (structural proteins, receptors, intracellular enzymes)
  • Glycoproteins (β2-glycoprotein I)
  • Phospholipids (cardiolipin)
  • Glycosphingolipids (gangliosides).

The autoantibodies specifically directed against these antigens can be detected in serum, body fluids and tissues. Serum antibodies of diagnostic relevance are IgG and, sometimes, IgA class antibodies. IgM class autoantibodies are usually of little diagnostic significance, since they are very nonspecific and can often also be found in healthy individuals, where they represent physiological auto reactivity.

25.1.3.1 Indication

  • Suspected systemic autoimmune disease (e.g., when there is an inflammatory reaction that cannot be explained by an infection)
  • Differential diagnostic workup of systemic diseases, especially for differentiating such diseases from drug allergy or para neoplastic disease
  • Diagnostic and differential diagnostic workup of inflammatory organ diseases such as stomach, bowel, liver, muscle, bullous skin diseases, and diseases of the endocrine system and the central and peripheral nervous system
  • Prognosis of autoimmune diseases (e.g., connective tissue disease and myositis).

Important autoantibodies in predominantly systemic autoimmune diseases are listed in Tab. 25.1-6 – Autoantibodies in systemic autoimmune diseases.

Autoantibodies in organ specific diseases are listed in Tab. 25.1-7 – Autoantibodies in organ specific autoimmune diseases.

25.1.3.2 Method of determination

Screening for autoantibodies is often performed by indirect immunofluorescence test (IIFT). If autoantibodies are detected, their specificities are determined with immunoassays:

  • Enzyme immunoassay (ELISA, EIA)
  • Luminescence, chemoluminescence or electrochemoluminescence assay
  • Immunoblot (Western blot)
  • Dot/line immunoblot
  • Array techniques (laser/bead array)
  • Immune electrophoresis, rarely used today
  • RIA (FARR assay for anti-dsDNA, otherwise rarely used).

Note: the disadvantage of IIFT is its low specificity. The test often produces false positive results in the absence of a corresponding clinical picture.

If an immunoassay with a specific panel of auto antigens is used as a screening test, the positive predictive value will be higher due to better diagnostic specificity, since only the antibodies specifically directed against the antigens employed in the assay are measured. The negative predictive value is often lower than in the IIFT, since rare auto antigens are not included and thus not detectable in the immunoassays. To exclude false negatives, several methods should be used in parallel for clinically critical diagnoses such as Goodpasture syndrome or ANCA-associated vasculitides /9/.

The most commonly requested diagnostic autoantibody tests are:

  • Antinuclear antibodies
  • Anti phospholipid antibodies
  • Rheumatoid factors
  • Anti cyclic citrullinated protein/peptide antibodies (ACPA, anti-CCP).

25.1.3.3 Specimen

Serum: 1 mL

25.1.3.4 Reference interval

Depending on the assay and the specific antibody.

25.1.3.5 Clinical assessment

Systemic autoimmune diseases are classified into two main groups:

Autoimmune arthritis

  • Rheumatoid arthritis
  • Spondyloarthropathy

Connective tissue diseases

  • Systemic lupus erythematosus
  • Systemic scleroderma
  • Sjögren’s syndrome
  • Polymyositis/dermatomyositis
  • Mixed connective tissue disease, Sharp’s syndrome
  • Anti-phospholipid syndrome
  • ANCA associated vasculitis
  • Goodpasture syndrome/pulmonary-renal syndrome.

The common pathogenic principle in systemic autoimmune disease is the loss of natural immunological tolerance to self structures. Activation of the relevant mechanisms results in immunoreactive inflammation of tissues with impairment of various organ functions. Systemic autoimmune disease should be considered if there is inflammatory or ischemic involvement of several organs along with obvious general symptoms.

In most cases the disease begins with nonspecific symptoms such as arthralgia, myalgia, weight loss or nocturnal hyperhidrosis. For a definite diagnosis to be made, the following must be present /10/:

  • Cardinal clinical symptoms such as polyarthritis
  • Characteristic laboratory findings such as autoantibodies
  • Typical lesions on high resolution imaging studies (e.g., characteristic erosions on joint sonography).
25.1.3.5.1 Autoantibodies in organ specific autoimmune diseases

Refer to Tab. 25.1-7 – Autoantibodies in organ specific autoimmune diseases.

25.1.3.5.2 Systemic autoimmune diseases

Refer to:

References

1. Lieo A, Invernizzi P, Gao B, Podda M, Gershwin ME. Definition of human autoimmunity-autoantibodies versus autoimmune disease. Autoimmunity Rev 2010; 9: A259-A266.

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. https://doi.org/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. Okzurede 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. Autoimmun 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. Paediatric 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.

21. Maddison PJ. Mixed connective tissue disease: overlap syndromes. Baillieres Best Pract Res Clin Rheumatol 2000; 14: 111–24.

22. Vitali C, Bombardieri S, Jonsson R, Moutsopoulos HM, Alexander EL, et al. Classification criteria for Sjögren’s syndrome: a revised version of the European criteria proposed by the American-European Consensus Group. Ann Rheum Dis 2002; 61: 554–8.

23. Dougados M, Baeten D. Spondyloarthritis. Lancet 2011; 377: 2127–37.

24. van der Helm-van Mil AH. Acute rheumatic fever and post streptococcal reactive arthritis reconsidered. Curr Opin Rheumatol 2010; 22: 437–42.

25. Ross JJ, Saltzman CL, Carling P, Shapiro DS. Pneumococcal septic arthritis: review of 190 cases. Clin Infect Dis 2003; 36: 319–27.

26. Outhred AC, Kok J, Dwyer DE. Viral arthritides. Expert Rev Anti Infect Ther 2011; 9: 545–54.

27. Petri M, Orbai AM, Alarcón GS, Gordon C, Merrill JT, Fortin PR, et al. Derivation and validation of the Systemic Lupus International Collaborating Clinics classification criteria for systemic lupus erythematosus. Arthritis Rheum 2012; 64: 2677–86.

28. Aletaha D, Neogi T, Silman AJ, Funovits J, Felson DT, Bingham CO, Birnbaum NS, et al. 2010 Rheumatoid arthritis classification criteria. Arthritis & Rheumatism 2010; 62: 2569–81.

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.

30. Bluestone JA, Anderson M. Tolerance in the age of immunotherapy. N Engl J Med 2020; 383: 1156–66.

25.2 Antinuclear antibodies

Rudolf Gruber, Lothar Thomas

Autoantibodies are the hallmark of autoimmunity, of which antinuclear antibodies (ANAs) are the centre stage. Detection of the ANA family is pivotal to the diagnosis of many autoimmune diseases. ANA have been determined by indirect immunofluorescence test (IIFT) for decades. Alternative techniques were developed challenging the classic IIFT. These alternative techniques differ in their antigen profiles, sensitivity and specificity. Therefore the term ANA is no longer technically correct and does not cover the entire spectrum of relevant autoantibodies. Because for the diagnosis of autoimmune diseases besides nuclear patterns of cells also cytoplasmic and mitotic apparatus patterns are relevant. Therefore, the term ANA may be changed to an appropriate one such as anti cellular antibodies /1/.

For detection of extractable nuclear antigens (ENAs) samples frequently are screened with IIFT; further determination of anti-ENA antibodies is performed only when the result of ENA is positive.

Therefore one may suggest changing the outdated terms ANA and ENA to such as anti-cellular antibodies and specific antibodies, respectively. However such a change in nomenclature would not receive broad agreement within the medical community /1/.

ANA and ENA identify a group of autoimmune diseases for which they are important diagnostic and classification criteria. The titer, or concentration, of ANA and their different specificities (target structures) must be taken into account when making a differential diagnosis. The diagnostic sensitivity and specificity of these antibodies for different diseases can vary significantly /2/.

25.2.1 Indication

Suspected connective tissue disease:

  • Systemic lupus erythematosus (SLE)
  • Sjögren’s syndrome
  • Scleroderma, including CREST syndrome
  • Mixed connective tissue disease (MCTD, Sharp’s syndrome)
  • Polymyositis/dermatomyositis
  • Drug induced lupus erythematosus
  • Treatment with TNF-α blockers
  • Suspected autoimmune hepatitis
  • Juvenile idiopathic arthritis.

25.2.2 Method of determination

According to recommendations and guidelines (e.g., of the American College of Rheumatology; ACR) /2/, ANA testing should be performed as a screening test by indirect immunofluorescence test (IIFT) on HEp-2 cells. Positive results should be followed by an immunoassay for differentiation (Fig. 25.2-1 – Assessment of a negative and positive antinuclear antibody result).

25.2.2.1 Indirect immunofluorescence test (IIFT)

The IIFT uses HEp-2 cells fixed on slides. The cells should contain a sufficient number of mitotic cells to enable adequate pattern recognition. HEp-2 is a cell line originally derived from epithelial cells from a larynx carcinoma. This cell line expresses almost all ANA antigens at levels that allow most ANA to be detected with high sensitivity. Only SSA/Ro antigens are expressed at low levels. Some manufacturers therefore offer genetically modified cells with over expression of SSA, although there is no clear evidence that this is of additional diagnostic advantage. The detection antibody (conjugate) should consist of fluorochrome labelled anti-human IgG-specific antiserum, either FITC or another validated new-generation fluorochrome. The optimal screening dilution of 1 : 100 or 1 : 160 is often found to be suitable /1/.

In the case of a positive ANA test it is recommended that the pattern and the highest dilution to demonstrate reactivity be reported /1/.

The IIFT shows whether the relevant patient serum contains ANA and, if so, at what titer levels (serum dilution). Based on the fluorescence pattern, the relevant cell structures and, to some extent, the antigen to which the ANA bind can be derived. Since some ANA specificities are relatively highly associated with a certain disease, some fluorescence patterns (e.g., antibodies to centromeres, nuclear dots and few nuclear dots) may provide clues to the underlying disease process. Other fluorescence patterns can be described (e.g. speckled or homogeneous nuclear fluorescence), but the relevant target structure cannot be assigned. Many cytoplasmic target structures, such as ribosomes, lysosomes, Golgi apparatus, actin, vimetin and Jo-1, can be assumed based on the fluorescence pattern, but often cannot be clearly assigned. Depending on the clinical relevance of the suspected autoantibodies, a differentiation should be made. Refer to Tab. 25.2-1 – Immunofluorescent patterns on HEp-2 cells, associated antigens and diseases.

25.2.2.2 Determination of specific antibodies

When the ANA titer is elevated, a differentiating analysis of ANA specificity is performed with specific antigens (Tab. 25.2-2 – Autoantibodies and target antigens in systemic autoimmune diseases, especially connective tissue diseases).

Most screening ENA tests utilize the single-well antigen cocktail approach, emphasizing efficiency and cost containment. The profile approach allows a more focused evaluation for defined clinically relevant autoantibodies /3/. Based on the original methodology of this differentiation, a subgroup of antigens that are derived from saline extracts of isolated nuclei are often defined as extractable nuclear antigens (ENAs) /4/. This crude nuclear extract contains mainly the Sm, RNP, SSA and SSB antigens. These antigens produce a speckled pattern in the IIFT.

The assays are based on highly purified native antigens or genetically produced recombinant antigens. The advantage of recombinant antigens is easy availability and better standardization.

The following immunoassays are used for detecting specific autoantibodies:

  • Enzyme immunoassay (ELISA, EIA)
  • Luminescence, chemiluminescence or electrochemiluminescence assay
  • Immunoblot (Western blot)
  • Dot/line immunoblot
  • Array techniques (laser/bead array)
  • RIA (FARR assay for anti-dsDNA, otherwise rarely used).

Both IIFT screening tests and immunoassays routinely detect IgG autoantibodies. There is insufficient evidence of the diagnostic value of IgM and IgA ANA/ENA.

25.2.3 Specimen

Serum, plasma: 1 mL

25.2.4 Thresholds

Indirect immunofluorescence test (IIFT)

When using HEp-2 cells, the titration of the serum is started with a dilution of 1 : 80 or 1 : 100 or, for children, with a dilution of 1 : 40. When interpreting the titer, the patient’s age and sex and well as the fluorescence pattern should be taken into account. An ANA titer ≥ 1 : 320 is generally interpreted as positive. However, in young adults, titers as low as 1 : 80 or 1 : 100 must be interpreted as weakly positive, and in children even titers as low as 1 : 40 can indicate an autoimmune disease. Older people (> 60 years) frequently have titers ≥ 1 : 320 without a disease correlate.

Immunoassay

The thresholds are specified by the manufacturer and are not standardized and thus cannot be compared between assays. There is a quantitative standard published by the WHO only for anti-dsDNA antibodies.

25.2.5 Clinical significance

Besides autoimmune diseases for which ANA testing is indicated, ANA can be detected in patients with malignant or infectious diseases as well as in healthy subjects. In the general population some individuals with a positive ANA test by IIFT do not have an autoimmune disease and are unlikely to develop one. Various drugs can induce the production of ANA (drug induced LE), usually transiently and without clinical manifestations of LE. High titers of ANA are seen in up to 30% of patients receiving TNF-α inhibitors, often without a demonstrable reaction to a specific antigen.

The prevalence of positive ANA /5/in various diseases is shown in Tab. 25.2-3 – Prevalence of ANA and association with disease.

The ANA assay is not a screening test for autoimmune diseases, as is often incorrectly assumed. ANA should be ordered only if there is clinical suspicion for one of the autoimmune diseases listed in

The workup of an incidentally detected elevated ANA titer is stressful for the patient and requires the exclusion of a rheumatoid disease. The positive predictive value of an incidentally detected elevated ANA titer is less than 5%. However, in some cases ANA may precede the clinical onset of disease for many years. A positive ANA result should therefore not be ignored, but should be monitored.

25.2.5.1 Assessment of positive ANA result

ANA are of particular diagnostic relevance if they are high titer, high avidity IgG type antibodies of defined specificity.

Significantly elevated titers (≥ 1 : 1,000) are mainly found in systemic autoimmune diseases such as connective tissue disease, autoimmune hepatitis, and juvenile idiopathic oligoarthritis, a subgroup of juvenile idiopathic arthritis /6/, or after treatment with TNF-α inhibitors. The higher the titer or antibody concentration, the higher the likelihood of autoimmune disease being present. Depending on the stated criteria, every positive ANA finding, even if detected incidentally, should thus be followed by a thorough check of the patient’s medical history, a clinical investigation, and further diagnostic and follow up tests.

In healthy individuals, the prevalence of ANA varies between 3% and 30%, depending on age, sex and titer. The fluorescence patterns are mainly homogeneous or speckled. The titers usually remain constant during the monitoring period. For example, individuals with an initially high ANA titer still showed the same high titer result 4 years later, and individuals with an initially low ANA titer (1 : 80 or 1 : 100) showed a decline, sometimes to undetectable levels, during the same period /7/. Various publications point out that irrelevant ANA may be differentiated based on the pattern and the reactivity to certain antigens, especially against DSF70 (dense fine speckled) /8/.

Assessment of IIFT titers ≥ 1 : 320, a diagnostic specificity of 95% can be achieved.

Diagnostic sensitivities of the IIFT /9/:

  • 95% for SLE
  • 85% for scleroderma (systemic sclerosis)
  • 75% for Sjögren’s syndrome.

Certain immunofluorescent patterns can be due to defined antibody specificities and can thus help narrow down the differential diagnosis (Tab. 25.2-1 – Immunofluorescent patterns on HEp-2 cells, associated antigens and diseases).

Antibody specificities should be differentiated when the ANA titer is ≥ 1 : 320 or, if there is a high clinical suspicion, even when the titer is as low as 1 : 100 or the ANA screen is negative. Knowing the fluorescence pattern does not replace testing of specific antibodies. In connective tissue diseases, the immune response is often directed against several antigens rather than just a single one.

This is particularly apparent in SLE (Tab. 25.2-4 – Prevalence of antibody findings in systemic autoimmune diseases).

25.2.5.2 Assessment of negative ANA results

A negative ANA result excludes the diagnosis of SLE with a diagnostic specificity of more than 95%. The previous concept of ANA negative SLE was based on ANA testing on rat liver sections. However, if the ANA test is negative but SLE continues to be strongly suspected, specific antibody testing by immunoassay must be performed.

Treatment with immunosuppressants and glucocorticoids has little influence on the ANA titer, although false negatives are possible in isolated cases. In addition, no plasmapheresis or treatment with immunoglobulin should have taken place prior to blood collection.

Some ENAs can be missed on IIFT, such as anti-SSA/Ro and anti-SSB/la antibodies. In addition, anti-Jo1 antibodies are not excluded in the case of negative IIFT because Jo1 is a cytoplasmic antigen. In a study /10/of 291 samples negative on IIFT (serum diluted 1 : 40) 12 showed anti-ENA reactivity on luminescence immunoassay. The authors argue that when clinical suspicion for rheumatic connective tissue disease is present, testing for ENAs should be performed.

If ANA-IIFT is negative one should not proceed to defining anti-dsDNA antibodies. On the other hand, certain IIFT patterns (e.g., homogenous) are suggestive of anti-dsDNA antibody /112/.

25.2.6 Comments and problems

Indirect immunofluorescence test (IIFT)

Many antibody specificities lead to nuclear, nucleolar and cytoplasmic fluorescence patterns, but only few are strictly antigen specific. If HEp-2 cells are used as a substrate, they should show sufficient mitotic stages. During the metaphase the chromosomes are more accessible to the autoantibodies.

Patients often have autoantibodies to several antigens. Some antigens are masked. For example, a speckled fluorescence pattern can be masked by homogeneous fluorescence at low serum dilution and only become prominent at a higher dilution.

The ANA titer is generally interpreted to be higher when the fluorescence pattern is non homogeneous (e.g., speckled). This is due to the fact that non homogeneous fluorescent staining patterns are of higher contrast, which can be misinterpreted as higher brightness.

Because ANA IIFT is not standardized, the ANA titers determined by different laboratories are only moderately comparable. To achieve comparability, there are various initiatives that seek to establish general standards /2/. When monitoring titers, physicians should be aware that a variation of one titer level, including 1 : 100 and below, is of no significance and may merely be due to inaccurate reading.

Immunoassays as screening for ANA

Immunoassays used as screening tests often employ nuclear lysates of HEp-2 cells or a mixture of recombinant antigen specificities. The threshold of various manufacturers’ immunoassays that use nuclear lysates corresponds approximately to the detection limit of IIFTs at a titer of 1 : 100. Tests using nuclear lysates offer the advantage of a high degree of automation, but lack specificity /11/.

If assays with defined mixtures of recombinant antigens are used for screening, only autoantibodies directed against the antigens contained in the mixture can be detected. Often, antigens of rare antibodies (e.g. PM-Scl) are not included in the test and can therefore not be detected. However, the number of false positive results is lower than that produced by test systems using nuclear lysates or by the IIFT.

Specimen

Analysis from synovial fluid or cerebrospinal fluid is of no significance in routine diagnosis.

Immunoassays, especially multiplex assays, often require no more than 10–20 μL of sample to identify more than 10, and in the future several hundred, antibody specificities. This is of particular interest in cases where only very small volumes of sample can be collected (e.g., in pediatrics and small animal medicine).

Stability

1 day at room temperature, 10 days at 4–8 °C, several years at –20 °C. Because antibodies and thus autoantibodies are very stable proteins, the time periods stated can usually be exceeded multiple times without loss of reactivity, although this has generally not been validated.

References

1. Agmon-Levin N, Damoiseaux J, Kallenberg C, Sack U, Witte T, Herold M, et al. International recommendations for the assessment of autoantibodies to cellular antigens referred to as anti-nuclear antibodies. Ann Rheum Dis 2014; 73: 17–23.

2. Kavanaugh A, Tomar R, Reveille J, Solomon DH, Homburger HA. Guidelines for clinical use of the antinuclear antibody test and tests for specific autoantibodies to nuclear antigens. American College of Pathologists. Arch Pathol Lab Med 2000.

3. Carey JL. Enzyme immunoassays for antinuclear antibodies. Clin Lab Med 1997; 355–65.

4. Dorsch CA, White GM, Berzofsky RN. The measurement of antibodies to extractable nuclear antigen. Am J Clin Pathol 1979; 71: 333–7.

5. Solomon DH, Kavanaugh AJ, Schur PH. Evidence-based guidelines for the use of immunologic tests: Antinuclear antibody testing. Arthritis Rheum 2002; 47: 434–44.

6. Malleson PN, Mackinnon MJ, Sailer-Hoeck M, Spencer CH. Review for the generalist: The antinuclear antibody test in children. When to use it and what to do with a positive titer. Pediatr Rheumatol Online J 2010; 8: 27.

7. Xavier RM, Yamauchi Y, Nakamura M, Tanigawa Y, Ishikura H, et al. Antinuclear antibodies in healthy aging people: a prospective study. Mech Ageing Dev 1995; 78: 145–54.

8. Seelig CA, Bauer O, Seelig HP. Autoantibodies against DFS70/LEDGF exclusion markers for systemic autoimmune rheumatic diseases. Clin Lab 2016; 62: 499–517.

9. Tan EM, Feltkamp TE, Smolen JS, Butcher B, Dawkins R, et al. Range of antinuclear antibodies in “healthy” individuals. Arthritis Rheum 1997; 40: 1601–11.

10. Hoffman IEA, Peene I, Veys EM, de Keyser F. Detection of specific antinuclear reactivities in patients with negative antinuclear antibody immunofluorescence screening tests. Clin Chem 2002; 48: 2171–6.

11. Meroni PL, Schur PH. ANA screening: an old test with new recommendations. Ann Rheum Dis 2010; 69: 1420–2.

12. Herold M, Klotz W, Demel U, Endler G, Forster E, Griesmacher A, et al. Internationaler Konsensus zur ANA-Bestimmung – was ändert sich im deutschen Sprachraum? J Lab Med 2015; 39: 145–52.

13. Röther E, Peter HH. Antinukleäre Antikörper. Internist 1995; 36: 277–81.

14. Kavanaugh AF, Solomon DH. Guidelines for immunologic laboratory testing in the rheumatic diseases: anti-DNA antibody tests. Arthritis Rheum 2002; 47: 546–55.

15. Ghillani P, Andre C, Toly C, Rouquette AM, Bengoufa D, et al. Clinical significance of anti-Ro52 (TRIM21) antibodies non-associated with anti-SSA 60kDa antibodies: results of a multicentric study. Autoimmun Rev 2011; 10: 509–13.

16. Hamaguchi Y. Autoantibody profiles in systemic sclerosis: predictive value for clinical evaluation and prognosis. J Dermatol 2010; 37: 42–53.

17. Gunawardena H, Betteridge ZE, McHugh NJ. Myositis-specific autoantibodies: their clinical and pathogenic significance in disease expression. Rheumatology (Oxford) 2009; 48: 607–12.

18. Koenig M, Fritzler MJ, Targoff IN, Troyanov Y, Senecal JL. Heterogeneity of autoantibodies in 100 patients with autoimmune myositis: insights into clinical features and outcomes. Arthritis Res Ther 2007; 9: R78.

19. Tuteja R, Tuteja N. Ku autoantigen: a multifunctional DNA-binding protein. Crit Rev Biochem Mol Biol 2000; 35: 1–33.

20. Hahn BH. Antibodies to DNA. N Engl J Med 1998; 338: 1359–68.

21. Pisetsky DS. Anti-DNA antibodies in systemic lupus erythematosus. Rheum Dis Clin North Am 1992; 18: 437–54.

22. Reichlin M. Systemic lupus erythematosus. Antibodies to ribonuclear proteins. Rheum Dis Clin North Am 1994; 20: 29–43.

23. Op De Beeck K, Vermeersch P, Verschueren P, Westhovens R, Marien G, et al. Detection of anti-nuclear antibodies by indirect immunofluorescence and by solid phase assay. Autoimmun Rev 2011; 10: 801–10.

25.3 Rheumatoid factors in rheumatoid arthritis

Rudolf Gruber, Lothar Thomas

In rheumatoid arthritis (RA) diagnosis and disease activity cannot be measured by a single variable. In clinical practice an opinion is formed from a combination of clinical, radiological and laboratory findings. Rheumatoid factors (RFs) are important laboratory markers. RFs are autoantibodies that bind to the Fc portion of IgG molecules /1/. The classic RF is an IgM isotype and most commonly seen in patients with RA. RFs were, in fact, so important that clinically diagnosed RA without evidence of RF was referred to as seronegative RA, and RFs were the only laboratory parameter to be listed as a main criterion in the RA classification criteria set up by the American College of Rheumatology (ACR criteria). With the advent of anti-citrullinated protein/peptide antibodies, which have considerably more diagnostic specificity at similar diagnostic sensitivity and have been included in the latest version of the ACR criteria, RF has become less important /23/.

25.3.1 Indication

Main indications:

  • Rheumatoid arthritis
  • Mixed cryoglobulinemia (type II).

Further indications:

  • Nonspecific arthritis
  • Vasculitis
  • Serositis
  • Connective tissue disease (suspected Sjögren’s syndrome).

25.3.2 Method of determination

Immunonephelometry and immunoturbidimetry

Principle: see Section 52.1.6 – Indirect measure of antigens and antibodies.

Enzyme immunoassay,luminescence immunoassay

Principle: see Section 52.1.8 – Assays with labeling of a reactant.

Latex agglutination test

Principle: human IgG molecules bound to latex particles are used as an antigen. See Fig. 25.3-1 – Principle of rheumatoid factor determination by latex enhanced immunoassay.

Waaler-Rose test

Principle: direct hemagglutination of erythrocytes coated with animal (usually rabbit) anti-IgG by rheumatoid factors in the sample. Principle: see Section 52.1.6.

25.3.3 Specimen

Serum, plasma, synovial fluid: 1 mL

25.3.4 Threshold value

Depending on the reagent and assay manufacturer. The threshold is usually 10–20 kU/L related to the international reference preparation /4/. The test used for the measurement must have a diagnostic specificity of more than 95% /5/.

25.3.5 Clinical significance

RFs occur transiently in infectious diseases, in particular in viral infections and viral hepatitides. Similar to ANA, RF increases in prevalence with age, without any clinical significance. Low levels of RF can be found in rheumatoid and non rheumatoid diseases (Tab. 25.3-1 – Prevalence of rheumatoid factors in rheumatic diseases).

25.3.5.1 Rheumatoid arthritis (RA)

The signs and symptoms of RA typically result from synovitis, the inflammation of the synovial membrane within joints. A complex, interactive network of cells and cytokines are involved in the pathogenesis of RA, particularly in the recruitment, activation, and effector functions of immune cells. The synovial layer becomes neovascularized and infiltrated with macrophages and fibroblasts, in addition to an influx of B and T lymphocytes, plasma cells, mast cells, dendritic cells and neutrophils /6/.

RA is the most frequent inflammatory rheumatic disease with a prevalence of 0.5 to 0.85% in the adult population. The current guidelines demand /7/:

  • Disease modifying treatment in the first 3 months
  • Continuous monitoring of disease activity
  • Remission as treatment goal
  • Management by a multidisciplinary team

There is an association with MHC class II antigens. RFs can be found in 70–90% of patients with RA. RF is often detected years before clinical symptoms develop, and RF positive healthy individuals have a 5–40-fold higher risk of developing RA than RF negative individuals /8/. High titers are common in patients with rapidly progressing joint destruction and in those with extra-articular manifestations such as rheumatoid nodules, polyneuropathy, vasculitis, serositis or Sicca syndrome.

Although the RF titer usually decreases following initiation of anti-inflammatory treatment, there is no strict correlation with disease activity.

The RF test is useful in the diagnosis of rheumatoid diseases, since patients with rheumatoid diseases relevant for differential diagnosis, such as psoriatic arthritis, ankylosing spondylitis, gout, reactive arthritides, polymyalgia rheumatica and arthroses do not show an elevated prevalence of RF compared to the healthy population /9/. RF is an important criterion of the ACR criteria and therefore must be assayed at least in all RA patients involved in treatment studies.

According to the 2010 rheumatoid arthritis classification criteria the classification as definite RA is based on /3/:

  • The confirmed presence of synovitis in at least one joint
  • Absence of an alternative diagnosis better explaining the synovitis
  • Achievement of a total score of 6 or greater (of a possible 10) from the individual scored in four domains: number and site of involved joints (range 0–5), serological abnormality (range 0–3), elevated acute-phase response (range 0–1) and symptom duration (range 0–1).

Refer to Tab. 25.1-10 – The 2010 rheumatoid classification criteria.

25.3.5.2 Mixed cryoglobulinemia type II and type III

Approximately 40–60% of patients with hepatitis C infection develop cryoglobulinemia, women more often than men (Section 18.11 – Cryoglobulins and cryofibrinogen). In all cases there is RF activity, which is almost always due to a monoclonal IgM κ-type and precipitates readily at 4 °C /1/. The IgM RF is complexed with polyclonal IgG in the cryoprecipitate from the serum of these patients. Since monoclonal RF do not always have the same specificity as the RF typical of RA, there may not always be a detectable reaction with rabbit IgG (Waaler-Rose test).

25.3.5.3 IgG and IgA type rheumatoid factors

There is no consensus on the diagnostic value of class-specific RF tests. IgA RF are more specific than IgM RF, comparable to the value of anti-CCP antibodies, but have a diagnostic sensitivity of only 50–60%. Even when measuring all RF classes, approximately 10% of RA patients remain seronegative. A positive result for all three RF classes is considered specific for RA. In isolated cases, elevated IgA RF and IgG RF have been ascribed prognostic value with regard to the progression of joint erosions in rheumatoid patients. Extra articular manifestations of RA are thought to be associated with IgA RF /910/.

25.3.6 Comments and problems

Method of determination

Immunonephelometry, immunoturbidimetry /11/: RF is measured by latex enhanced assays, usually with latex particles coated with human IgG. Most assays detect only IgM RFs, because their pentameric shape allows them to cause sufficient agglutination of the latex particles. Turbidimetric methods are used on the clinical chemistry analyzers. It must be kept in mind that, compared to ELISA, this method is more susceptible to nonspecific reactions. Since the turbidity is proportional to the concentration of RF, a quantitative result is generated (in kU/L).

ELISA assays are predominantly used to measure RF of the IgG and IgA isotypes.

Determination of RF in synovial fluid

In RA, the results correspond to those obtained in serum in almost all cases /12/. No valid statement can be made on a positive synovial fluid test with a negative result in serum, since a false positive reaction due to the matrix effects in the synovial fluid is more likely than a false negative result in serum. RF in synovial fluid should therefore not be part of routine diagnostic testing.

Interference

RFs pose a technical problem in the laboratory, as they can interfere with specific antibody immunoassays (Tab. 52.1-4 – Factors interfering with immunoassays).

References

1. Newkirk M. Rheumatoid factors: host resistance to autoimmunity? Clin Immunol 2002; 104: 1–13.

2. Dörner T, Egerer K, Feist E, Burmester GR. Rheumatoid factor revisited. Curr Opin Rheumatol 2004; 16: 246–53.

3. Aletaha D, Neogi T, Silman AJ, Funovits J, Felson DT, et al. 2010 Rheumatoid arthritis classification criteria: an American College of Rheumatology/European League Against Rheumatism collaborative initiative. Arthritis Rheum 2010; 62: 2569–81.

4. Anderson SG, Bentzon MW, Houba V, Krag P. International reference preparation of rheumatoid arthritis serum. Bull World Health Organ 1970; 42: 311–18.

5. Carson DA. Rheumatoid factor. In: Kelley WN, Harris ED, Ruddy S, Sledge CB, eds. Textbook of rheumatology. Philadelphia: Saunders, 1993: 155–63.

6. Siebert S, Tsoukas A, Robertson J, McInnes I. Cytokines as therapeutic targsts in rheumatoid arthritis and other inflammatory diseases. Pharmacol Rev 2015; 67: 280–309.

7. Schneider M, Krüger K. Rheumatoid arthritis – early diagnosis and disease management. Dtsch Arztebl Int 2013; 110: 477–84

8. Shmerling RH, Delbanco TL. The rheumatoid factor: an analysis of clinical utility. Am J Med 1991; 91: 528–34.

9. Conrad K, Roggenbuck D, Reinhold D, Dorner T. Profiling of rheumatoid arthritis associated autoantibodies. Autoimmun Rev 2010; 9: 431–5.

10. Mierau R, Gause A, Küppers R, Michels M, Mageed RA, Jefferis R, et al. A human monoclonal IgA rheumatoid factor using the VkIV light chain gene. Rheumatol Int 1992; 31: 23–31.

11. Roberts-Thomson PJ, Wernick RM, Ziff M. Quantitation of rheumatoid factor by laser nephelometry. Rheumatol Int 1982; 2: 17–20.

12. Mierau R, Wohltmann D, Heinrichs R, Genth E. Nephelometrische Bestimmung von IgM-Rheumafaktoren. Med Welt 1985; 36: 335–43.

25.4 Anti-citrullinated peptide/protein antibodies in rheumatoid arthritis

Rudolf Gruber, Lothar Thomas

Rheumatoid arthritis (RA) is the most common autoimmune arthritis worldwide with a prevalence of 0.5 to 0.8%. Citrullinated peptides (CCP) are peptides and proteins in which arginine residues have been converted to citrulline residues. Testing for anti-CCP antibodies in undifferentiated arthritis allows prediction of approximately 80% of patients who will fulfill the criteria for RA /12/.

25.4.1 Indication

Suspicion of /3/:

  • Rheumatoid arthritis
  • Seronegative (RF negative) rheumatoid arthritis
  • Undifferentiated arthritis
  • Early RA, to determine indications for RA specific treatment (e.g., TNF-α inhibitors).

In addition:

  • Differentiation of nonspecific arthritides with demonstrable rheumatoid factor
  • Marker for the prognostic evaluation of RA in combination with other criteria
  • Prognostic parameter in juvenile idiopathic arthritis (group of RF positive polyarthritides).

25.4.2 Method of determination

Immunoassays using anti-cyclic citrullinated peptides (anti-CCP) as antigens /4/ e.g., an immunoassay based on the biotin streptavidin technology employing an IgG capture assays format. The format uses biotinylated peptides that are recognized by anti-CCP antibodies from patient samples and a ruthenium labeled monoclonal antibody that recognizes aggregated human IgG. The resulting immune complexes immobilize at the streptavidin coated magnetic microbeads. The measured electrochemiluminescence signal is proportional to the anti-CCP-IgG antibody concentration in the sample.

25.4.3 Specimen

Serum, plasma: 1 mL

25.4.4 Reference interval

Depending on the manufacturer of the commercial assay. Some manufacturers use the cut-off 17 U/mL, a few others use 25 U/mL.

25.4.5 Clinical significance

The 2010 American College of Rheumatology/European League Against Rheumatism classification criteria for RA validate rheumatic factor (RF) and anti-CCP with identical score in patients with synovitis /1/. Refer to Tab. 25.1-10 – The 2010 rheumatoid classification criteria.

According to a systematic review of 151 studies investigating the accuracy of anti-CCP for diagnosing RA the sensitivity range was 12–93% and specificity range 63–100%. In a cohort study that investigated anti-CCP antibodies in patients with early rheumatoid arthritis (< 2 years), summary sensitivity and specificity were 57% (95% CI 51 to 63%) and 96% (95% CI 93–97%), respectively. Case-control and cross-sectional studies and studies of patients with established RA all overestimated sensitivity. Anti-CCP had greater specificity than RF (96% vs. 86%) with similar sensitivity. Evidence was insufficient to ascertain whether the combination of anti-CCP and RF provides additional benefit over anti-CCP alone /5/.

Like RF positivity, the presence of anti-CCP is associated with severe disease and thus a significant prognostic factor /6/. Tab. 25.4-1 – Prevalence of anti-CCP antibodies in diseases shows the prevalence of ACPA in different diseases /7/.

Combining ACPA and IgM-RF tests allows a better accuracy in the diagnosis of RA. In a study population at risk for RA, the positive predictive value (PPV) of anti-CCP and IgM RF for RA was 61% and the negative predictive value (NPV) 92% in each case. When both biomarkers were positive, the PPV increased to 98%; when both were negative, the NPV was 92.5%. Combining the two tests therefore markedly increases the PPV. A higher diagnostic specificity (increase from 82–90% to 98%) was also achieved by combining anti-CCP and IgA-RF /89/.

Anti-CCP antibodies can be detected with a diagnostic sensitivity of 40–70% in the early stages of RA, sometimes even up to 10 years before the onset of clinical symptoms /10/. Due to the high diagnostic specificity of anti-CCP antibodies , all positive patients without corresponding clinical symptoms must be counseled and monitored. Although some studies have reported a decrease in anti-CCP antibodies titers in patients receiving immunosuppressive treatment, there is no clear evidence to use antibody titers for monitoring of the disease activity.

Anti-CCP antibody positive patients have a higher risk of developing progression of joint damage within 5 years than Anti-CCP antibody negative patients, when evaluating radiographic joint damage using Sharp scores /11/.

25.4.6 Comments and problems

Method of determination

ACPA can be assayed using the standardized first commercial ELISA (anti-CCP-1 i.e., first generation). A lot of data was obtained with anti-CCP-1 assays. Using the anti-CCP-2 assay increased the diagnostic sensitivity at the same specificity. Although all commercially available anti-CCP assays contain the same highly standardized peptides, various studies showed intra assay variations of 0.5–19% and inter assay variations of 0.4–22%. At a fixed diagnostic specificity of 98.5%, the sensitivities of different assays for RA ranged from 41% to 74%. The most false positives are seen in viral infections /12/. A diagnostic advantage of other ACPA tests (e.g., anti-MCV) over anti-CCP-2 tests could not be demonstrated, as the results from comparative studies vary /13/.

Specimen

The test can be performed with sample volumes as low as 100 μL, allowing it to be used even in cases where only low test volumes are available (e.g., in pediatrics).

Assaying anti-CCP antibodies in synovial fluid barely increases the diagnostic sensitivity for RA and should not be part of routine diagnostic testing due to the technical issues (tests not validated, matrix effects).

Stability

10 days at room temperature, four weeks at 4–8 °C, several years at –20 °C.

25.4.7 Pathophysiology

Antibodies to citrullinated protein/peptide antigens (anti-CCP antibodies) are directed against an individual’s own citrullinated proteins and peptides. For the measurement of anti-CCP antibodies cyclic citrullinated peptides (CCP) are used in immunoassays.

Citrulline is a modified amino acid. It is produced as a by product of the enzymatic deamination of the essential amino acid arginine (i.e., positively charged amino groups are hydrolyzed to a neutral oxygen group) (Fig. 25.4-1 – Removal of the arginine residue of a peptide by the enzyme peptidylarginine deiminase). The deamination of arginine in peptides and proteins is catalyzed by four known peptidylarginine deiminases (PAD 1–4), which exhibit a tissue specific distribution. PADs are also referred to as PADIs.

Physiologically, only few proteins are citrullinated by the enzyme PAD (e.g., vimentin, filaggrin, fibrin, α-enolase). Citrullination may be enhanced in certain situations. For example, the protein filaggrin is citrullinated during the late phase of differentiation of epidermal cells, and fibrin is citrullinated during joint inflammation in RA. In addition, antibodies to CCP are produced in the inflamed synovia, and the concentration of IgG antibodies to CCP in the pannus is multiple times higher than in synovial fluid or serum /14/. It is well established that antigen stimulated B cells produce IgG antibodies to CCP in the joints of RA patients and that this occurs locally in the inflamed joint rather than as a systemic process.

There is evidence to suggest that abnormal citrullination may play role in RA. For example, single nucleotide polymorphisms (SNPs) in the PADI 4 gene which may lead to a more stable mRNA and thus higher activity of the enzyme have been associated with RA /15/. The altered self hypothesis is also being discussed in relation to the formation of anti-CCP antibodies. In RA, autoantibodies to the enzyme PAD 4 were found. Other recently described autoantibodies with a potential for diagnostic use is also directed against “altered self”. They are directed against carbamylated proteins and are called anti-CarbP /16/.

Similar to celiac disease, here, too, the association with certain HLA alleles is a major factor in the exaggerated immune response. In RA there is a high association with the shared epitope, a peptide sequence in the HLA-DR that occurs in the alleles previously called HLA-DR4 and now known as HLA-DR*0401, 0404, as well as in HLA-DR*0101. Citrullinated peptides have been shown to induce a particularly strong T cell response when presented by cells with the shared epitope /17/. The abnormal response is triggered or at least amplified by external triggers. Smoking is a major risk factor for RA. Carriers of the shared epitope have a higher risk of developing RA, and smoking increases this risk dramatically /18/.

References

1. Aletaha D, Neogi T, Silman AJ, Funovits J, Felson DT, et al. Rheumatoid arthritis classification criteria: an American College of Rheumatology/European League Against Rheumatism collaborative initiative. Arthritis Rheum 2010; 62: 2569–81.

2. Schellekens GA, de Jong BA, van den Hoogen FH, van de Putte LB, van Venrooij WJ. Citrulline is an essential constituent of antigenic determinants recognized by rheumatoid arthritis-specific autoantibodies. J Clin Invest 1998; 101: 273–81.

3. Kamoun M. Diagnostic performance and predictive value of anti-citrullinated peptide antibodies for diagnosis of rheumatoid arthritis: toward more accurate detection? Clin Chem 2005; 51: 12–3.

4. van Venrooij WJ, Hazes JM, Visser H. Anticitrullinated protein/peptide antibody and its role in the diagnosis and prognosis of early rheumatoid arthritis. Neth J Med 2002; 60: 383–8.

5. Whiting PF, Smidt N, Sterne JAC, Harbord R, Burton A, Burke M, et al. Systematic review: accuracy of anti-citrullinated peptide antibodies for diagnosing rheumatoid arthritis. Ann Intern Med 2010; 152: 456–64.

6. Dekkers J, Toes RE, Huinzinga TW, van der Woude D. The role of anti-citrullinated protein antibodies in the early stages of rheumatoid arthritis. Curr Opin Rheumatol 2016; PMID:26945334.

7. Mierau R, Genth E. Diagnosis and prognosis of early rheumatoid arthritis, with special emphasis on laboratory analysis. Clin Chem Lab Med 2006; 44: 138–43.

8. Bas S, Genevay S, Meyer O, Gabay C. Anti-cyclic citrullinated peptide antibodies, IgM and IgA rheumatoid factors in the diagnosis and prognosis of rheumatoid arthritis. Rheumatology (Oxford) 2003; 42: 677–80.

9. Hoffman IE, Peene I, Pottel H, Union A, Hulstaert F, et al. Diagnostic performance and predictive value of rheumatoid factor, anti-citrullinated peptide antibodies, and the HLA shared epitope for diagnosis of rheumatoid arthritis. Clin Chem 2005; 51: 261–3.

10. Vallbracht I, Rieber J, Oppermann M, Forger F, Siebert U, et al. Diagnostic and clinical value of anti-cyclic citrullinated peptide antibodies compared with rheumatoid factor isotypes in rheumatoid arthritis. Ann Rheum Dis 2004; 63: 1079–84.

11. Meyer O, Labarre C, Dougados M, Goupille P, Cantagrel A, et al. Anti-citrullinated protein/peptide antibody assays in early rheumatoid arthritis for predicting five year radiographic damage. Ann Rheum Dis 2003; 62: 120–6.

12. Bizzaro N. Antibodies to citrullinated peptides: a significant step forward in the early diagnosis of rheumatoid arthritis. Clin Chem Lab Med 2007; 45: 150–7.

13. Wiik AS, van Venrooij WJ, Pruijn GJ. All you wanted to know about anti-CCP but were afraid to ask. Autoimmun Rev 2010; 10: 90–3.

14. Masson-Bessiere C, Sebbag M, Durieux JJ, Nogueira L, Vincent C, et al. In the rheumatoid pannus, anti-filaggrin autoantibodies are produced by local plasma cells and constitute a higher proportion of IgG than in synovial fluid and serum. Clin Exp Immunol 2000; 119: 544–52.

15. Suzuki A, Yamada R, Chang X, Tokuhiro S, Sawada T, et al. Functional haplotypes of PADI4, encoding citrullinating enzyme peptidylarginine deiminase 4, are associated with rheumatoid arthritis. Nat Genet 2003; 34: 395–402.

16. Shi J, van Veelen PA, Mahler M, Janssen GMC, et al. Carbamylation and antibodies against carbamyled proteins in autoimmunity and other pathologies. Autoimmun Rev 2014; 13: 225–30.

17. Hill JA, Southwood S, Sette A, Jevnikar AM, Bell DA, et al. Cutting edge: the conversion of arginine to citrulline allows for a high-affinity peptide interaction with the rheumatoid arthritis-associated HLA-DRB1*0401 MHC class II molecule. J Immunol 2003; 171: 538–41.

18. Morgan AW, Thomson W, Martin SG, Carter AM, Erlich HA, et al. Reevaluation of the interaction between HLA-DRB1 shared epitope alleles, PTPN22, and smoking in determining susceptibility to autoantibody-positive and autoantibody-negative rheumatoid arthritis in a large UK Caucasian population. Arthritis Rheum 2009; 60: 2565–76.

25.5 Antibody diagnostics of myopathies

Lothar Thomas

Myopathies are a heterogenous group of disorders. Based on etiopathogenesis one may distinguish among the following myopathies /12/:

  • Hereditary degenerative (e.g., muscular dystrophy, congenital dystrophy, and sarcopenia)
  • Hereditary metabolic (e.g., glycogen storage myopathy, lipid storage myopathy, mitochondrial myopathy)
  • Inflammatory ; autoimmune (polymyositis, dermatomyositis, immune-mediated necrotizing myopathy) and infectious (e.g., viral, bacterial, and fungal genesis)
  • Medicinal-toxic genesis (e.g., statins, steroids, and alcohol)
  • Endocrine (e.g., hypothyroidism)
  • Neoplastic infiltrative (e.g., rhabdomyosarcoma) and para neoplastic (e.g., dermatomyositis)
  • Mechanically (e.g., march rhabdomyolysis)

Hereditary myopathies have a slow course and start early in life, acquired myopathies have a fast course and start later in life.

25.5.1 Myositis-specific and myositis- associated autoantibodies

Myositis-specific autoantibodies (MSA) and myositis-associated autoantibodies (MAA) can help to diagnose idiopathic inflammatory myopathies and define subgroups of patients in terms of clinical or pathological phenotypes, prognosis, and response to treatment.

Patients can be tested for the following antibodies /3/:

  • Anti-histidyl-ARN-t-synthetase (Jo-1)
  • Anti-threonine-ARN-t-synthetase (PL7)
  • Anti-alanine-ARN-t-sythetase (PL12)
  • Anti-complex nucleosome remodeling histone deacetylase (Mi2)
  • Anti-Ku
  • Anti-polymyositis/systemic scleroderma (PMScl)
  • Anti-topoisomerase 1 (SCL70)
  • Anti-signal recognition particle (SRP).

Refer to:

25.5.1.1 Indication

Diagnosis of idiopathic inflammatory myopathy (IMM) and to define subgroups of patients.

25.5.1.2 Method of determination

Most of the commercial kits are line immune assays in which several antigens are coated on a strip. Some ELISA are also developed.

25.5.1.3 Sample

Serum: 1 ml

25.5.1.4 Reference interval

Depending on the manufacturer of the commercial assay.

25.5.1.5 Clinical significance

Based on clinical and pathological descriptions one may distinguish among idiopathic inflammatory myopathies (IMM) a heterogenous group of myopathies in their pathophysiologic features and prognosis: dermatomyositis, polymyositis, overlap myositis, inclusion body myositis, immune-mediated necrotizing myopathy and other nonspecific myositis /4/.

The emergence of myositis specific autoantibodies suggests that subgroups of patients with IMM may be differentiated /34/.

25.5.1.5.1 Idiopathic inflammatory myopathy (IMM)

The IMM are a group of acquired myopathies characterized by muscle inflammation that are associated with motor weakness of varying severity. They are rare autoimmune diseases and heterogeneous in their muscle phenotype and extra muscular manifestations /3/.

The symptoms are muscle weakness and sometimes pain, often but not always symmetrical and proximal, are limited to the trunk, neck, and limbs /4/. These symptoms are accompanied by signs of muscle damage: the enzymes CK, AST and LD are elevated.

Epidemiology

The IIM has a prevalence of about 60 per 1 million and the incidence is 1.9–7.7 per 1 million in the general population of Caucasians /5/. The most common form of IIM is dermatomyositis (DM), followed by inclusion body myositis and Polymyositis (PM). Inclusion body myositis accounts for 10% of IIM cases in general, and for up to 25% of IIM cases in older patients. DM and PM are 2–3 times more common in women than in men; in childhood, both sexes are affected approximately equally. Juvenile DM is the most common presentation, followed by far fewer cases of juvenile PM, overlap myositis, and inclusion body myositis /6/. Myositis overlap syndromes are 5–10 times more common in women than in men.

25.5.1.5.2 Subgroups of idiopathic inflammatory myopathies

The major inflammatory subgroups of idiopathic inflammatory myopathies (IIM) are /4/:

  • Overlap myositis
  • Dermatomyositis
  • Immune mediated necrotizing myopathy
  • Inclusion body myositis.

A new classification system for IIM based on clinical manifestations and myositis specific antibodies defined the following subgroups /3/:

  • Anti synthetase syndrome; presence of anti-Jo-1 antibodies or anti-PL7 antibodies
  • Dematomyositis; presence of anti-Mi2 antibodies, anti-melanoma differentiation-associated protein 5 (MDA5) antibodies, or anti-transcription intermediary factor 1γ (TIF1γ) antibodies
  • Immune mediated necrotizing myopathy; presence of anti-SRP antibodies or anti-3-hydroxy-3-methyl glutaryl-coenzyme A reductase (HMGCR) antibodies
  • Inclusion body myositis; presence of cytosolic 5’ nucleotidase antibodies (anti-cN1A).

Refer to Tab. 25.5-3 – Frequency of autoantibodies in inflammatory idiopathic myopathies.

The IMM subgroups share some common characteristics in terms of phenotype and pathogenesis. The characteristics of the subgroups are shown in Tab. 25.5-4 – Subgroups of idiopathic inflammatory syndromes.

25.5.1.5.3 Laboratory tests

Patients with muscular complaints should primarily undergo the following basic tests:

  • Blood count and differential count
  • Inflammatory markers (e.g., C-reactive protein, ESR)
  • Muscle enzymes (e.g., creatine kinase, serum myoglobin) which have a diagnostic sensitivity of 90%, but low specificity for IIM
  • Anti-CCP or rheumatoid factor to differentiate rheumatoid arthritis
  • Antinuclear antibodies (ANA), since elevated ANA are found in 40–80% of patients, depending on the clinical group of myopathies
  • Organ specific laboratory tests: kidney function (creatinine), liver damage (ALT), focus on organ damage (LD), lactate and TSH to differentiate metabolic and endocrine myopathies.

References

1. Jungbluth H, Voermans NC. Congenital myopathies. Curr Opin Neurol 2016; 29: 642–50.

2. Dimachkie MM, Barohn RJ, Amato A. Idiopathic inflammatory myopathies. Neurol Clin 2014; 32: 595–628

3. Mariampillai K, Granger B, Amelin D, Guiguet M, Hachulla E, Maurier F, et al. Development of a new classification system for idiopathic inflammatory myopathies based on clinical manifestations and myositis-specific antibodies. JAMA Neurol 2018; 75: 1528–37.

4. Benveniste O, Stenzel W, Allenbach Y. Advances in serological diagnostics of inflammatory myopathies. Curent Opinion Neurol 2016; 29: 662–73.

5. Schoser B. Inflammatorische Myopathien. Z Rheumatol 2009; 68: 665–77.

6. Compeyrot-Lacassagne Feldman BM. Inflammatory myopathies in children. Pediatr Clin N Am 2005; 52: 493–520.

7. Dalakas MC. Inflammatory muscle diseases: a critical review on pathogenesis and therapies. Curr Opin Pharmacol 2010; 10: 346–52.

8. Benviste O, Stenzel W, Hilton-Jones D, Sandri M, Boyer O, van Engelem BGM, et al. Amyloid deposits and inflammatory infiltrates in sporadic inclusion body myositis: the inflammatory egg comes before the degenerative chicken. Acta Neuropatol (Berl) 2015; 129: 611–24.

9. Cavagna L, Nuno L, Scire CA, Govoni M, Longo FJ, Franchechini F, et al. Serum Jo-1 antibody and isolated arthritis in the antisynthetase syndrome: Review of the literature and report of the experiences of the AENEAS Collaborative Group. Clin Rev Allergy Immunol 2016; www.ncbi.nlm.nih.gov/pubmed/26782036.

10. Gunawardena H, Betteridge ZE, McHugh NJ. Myositis-specific antibodies: their clinical and pathogenic significance in disease expression. Rheumatology 2009; 68: 665–77.

11. Herbert MK, Stammen-Vogelzang J, Verbeek MM, Rietveld A, Lundberg IE, Chinoy H, et al. Disease specificity of autoantibodies to cytosolic 5’nucleotidase 1A in sporadic inclusion body myositis versus known autoimmune diseases. Ann Rheum Dis 2016; 75: 696–701.

25.6 Neurological syndromes associated with autoantibodies

Lothar Thomas

Neurological syndromes can result from vascular, inflammatory, degenerative, metabolic or genetic causes /12/.

Some of the non-infectious disorders are associated with autoantibodies. The following autoantibodies are differentiated:

  • Autoantibodies targeting neuronal cell-surface antigens. These antibodies suggest a direct pathogenic effect and cause neuronal damage and neuronal death /34/. They have been identified in patients presenting with neurological symptoms resembling para neoplastic syndromes.
  • Autoantibodies targeting intracellular antigens do not appear to be directly pathogenic, but may be used as diagnostic biomarkers of cancer.

Autoantibodies are diagnostically relevant in /5/:

  • Syndromes of the central nervous system
  • Syndromes of the peripheral nervous system
  • Syndromes of the neuromuscular junction and muscle.

Refer to Tab. 25.6-1 – Neurological syndromes.

25.6.1 Paraneoplastic neurological syndroms

Paraneoplastic neurological syndroms (PNS) are a diverse group of disorders that can occur with any type of malignancy and can affect any part of the central nervous system. Many PNS are immune-mediated and are triggered when tumors express proteins that are normally restricted to immune privileged neurons /6/.

The immune responses often manifest as anti neuronal antibodies that can be measured in cerebrospinal fluid (CSF) and serum. Anti neuronal antibodies also referred as onconeural antibodies or para neoplastic antibodies are directed against intracellular neuronal antigens. Onconeural antibodies are commonly associated with small cell lung cancer (SCLC), ovarian cancer, breast cancer, neuroendocrine tumors, thymoma and lymphoma. The antibodies serve as markers of the para neoplastic neurologic syndromes and, in some cases, of the presence of specific types of tumors /6/.

Refer to:

Onconeural antibodies are also seen in cancer patients without PNS or in patients without cancer. Therefore, other neurological causes should be considered in the differential diagnosis, even if an onconeural antibody is present.

Most PNS have a prevalence of far less than 1%, with the exception of the following /6/:

  • Paraneoplastic cerebellar degeneration
  • Limbic encephalitis
  • Paraneoplastic sensory neuropathy
  • Anti-N-methyl-D-aspartate receptor encephalitis

PNS is suspected in the following cases:

  • Signs of classical PNS
  • Sub acute neurological symptoms which cannot be diagnosed with regular neurological investigations or laboratory tests
  • Cancer patients whose symptoms cannot be neurologically explained by either the primary tumor, its localization and metastatic spread, or by cytostatic treatment.

Refer to Fig. 25.6-1 – Diagnostic approach to suspected paraneoplastic neurological syndrome.

The history, symptoms and progression of PNS are generally as follows:

  • Most PNS occur in patients with previously unknown cancer
  • PNS has an acute or subacute onset and progresses over weeks to months before stabilizing.

25.6.1.1 Paraneoplastic antibodies (PNA)

Antibodies that occur in paraneoplastic syndromes have been divided in two categories depending on the location of the antigen. Intracellular antibodies are directed against intracellular neural proteins. Extracellular antibodies are directed against extracellular (surface) proteins. Antibodies to intracellular neural proteins, also called classical para neoplastic antibodies (PNA) or onconeural antibodies are well characterized PNA and their detection almost always indicates the presence of an underlying cancer /1/.

These PNA include:

  • Anti-neuronal antibody type 1 (ANNA-1), anti-Hu
  • Anti-neuronal antibody type 2 (ANNA-2), anti-Ri
  • Anti-amphysin
  • Anti-collapsin response mediator protein type 5 (anti CRMP5)
  • Anti-Purkinje cell cytoplasm antibody type 1 (anti-CA-1), anti-Yo
  • Anti-para neoplastic neuronal antigen Ma2 (anti-Ma2)
  • Anti-recoverin.

PNA are usually of the IgG isotype and rarely found in healthy individuals. In patients with paraneoplastic syndrome, the antibodies are generally found in high titers in the cerebrospinal fluid (CSF). Although the PNA titer does not correlate with the severity of disease, the antibodies are more common in patients with cancer and neurological symptoms than in patients with cancer alone.

Low PNA titers are also seen in cancer patients without neurological symptoms. The presence of PNA without neurological symptoms is thus not necessarily diagnostic of PNS.

Although PNA are sensitive markers of PNS, they are only diagnosed in up to 50% of PNS patients. They are also detected in 16% of cancer patients, even though the patients have no neurological symptoms. Up to 30% of patients with PNS have several paraneoplastic neurological syndromes. PNA can be detected as early as 20 months before the cancer is diagnosed.

Refer to:

25.6.2 Antibodies in syndromes of the central nervous system (encephalitis/cerebellitis)

Encephalitis associated with autoantibodies account for approximately 8% of encephalitis cases. Targets of autoantibodies include /4/:

  • Ionotropic receptors; N-methyl-D-aspartate receptor (NMDAR), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR), γ-amino butyric acid (GABA)A receptor and glycine receptor (GlyR)
  • Metabotropic receptors; GluR1, GluR5, GABABR
  • Proteins belonging to the voltage-gated potassium channel (VGKC) complex, namely Lgi1 and Caspr2.

Refer to:

25.6.3 Antibodies in immune-mediated neuropathies

The evaluation of immune-mediated polyneuropathies can be challenging and considers careful consideration because these neuropathies vary in their spectrum. Symptoms of distal numbness, tingling and pain, and weakness are common complaints. The diagnosis is based on clinical, electrophysiological, and immunological features of the syndrome. One important finding of the antibody associated polyneuropathy are autoantibodies directed against targets of glycolipids or proteins with a receptor or ion channel function /7/.

Immune mediated neuropathies comprise:

Antibodies detectable in immune mediated neuropathies are listed in Tab. 25.6-8 – Antibodies in immune-mediated polyneuropathies. Of diagnostic relevance are anti-ganglioside antibodies, the structure of which is shown in Fig. 25.6-2 – Structure of the gangliosides.

25.6.3.1 Guillain-Barré syndrome (GBS)

GBS is an acute, self-limiting motor dominant polyneuropathy, often preceded by an infection. The annual incidence has been estimated at 1–2 per 100,000 population and is the most common form of polyneuropathy in Europe and in North America, accounting for 85–90% of cases. GBS includes the acute inflammatory demyelinating polyneuropathy (ADIP) and the acute axonal neuropathy (AMAN) /8/. The mortality rate in the acute phase is 3.5–12%, and 20% of patients are left with residual complaints.

GBS represents the prototype of immune-mediated polyneuropathy. In GBS, the body launches an autoimmune attack on nerve roots of the spinal cord in a primarily healthy individual without any other autoimmune disease being present. The inflammation leads to demyelination of nerve segments, causing ascending paralysis which usually begins in the legs. This is sometimes preceded by an infection of the upper respiratory tract or the gastrointestinal tract. The main pathogens are Cytomegalovirus, Epstein-Barr virus, Mycoplasma pneumoniae and Campylobacter jejuni. This bacterium is thought to share certain structurally similar components with the peripheral nervous system.

Besides the classical GBS several variants are differentiated (Tab. 25.6-7 – Neuropathies phenotypically associated with anti-ganglioside antibodies).

Clinical presentation

GBS is difficult to diagnose in the initial stages, since patients usually have vague symptoms such as weakness, neck or back pain before the classic clinical picture is evident /9/. The latter manifests as quadriplegia, possibly with weakness of the respiratory muscles as well as facial and bulbar weakness. By definition, the progression of muscle weakness in two or more extremities to a nadir occurs within 4 weeks. Sensory symptoms are usually mild. The acute phase is followed by a variable plateau phase and then spontaneous remission.

25.6.3.1.1 Anti-ganglioside antibodies

Anti-ganglioside antibodies, mostly IgG type, are present in sera of about 60% of patients in the acute phase of GBS and are a characteristic feature. The serum antibodies in GBS are subdivided into the following types: anti-GM1 (40%), anti-GD1a (20%), anti-GalNAc-GD1a and anti-Asialo-GM-1 (17%), respectively /10/.

Interpretation 

Refer to Tab. 25.6-7 – Neuropathies phenotypically associated with anti-ganglioside antibodies.

Cerebrospinal fluid

The cell count is normal or slightly elevated (up to 50 cells/μL, predominately lymphocytes, monocytes). Total protein is initially normal and later elevated, the QAlb can be up to 50 × 10–3, no oligoclonal bands.

25.6.3.2 Miller Fisher syndrome (MFS)

Acute immune-mediated immunopathies include GBS, MFS, MFS variants, and Bickerstaff’s brainstem encephalitis. MFS is characterized by acute onset of ophthalmoplegia, ataxia and areflexia, and is the most common variant of GBS. The syndrome also includes patients with cranial nerve involvement and can show overlap with GBS. The annual incidence has been estimated at 0.09 per 100,000 population, accounting for about 5–10% of GBS cases. MFS patients have anti-GQ1b IgG antibodies during the acute phase of the illness. Many patients with elevated titers of anti-GQ1b IgG antibodies report about respiratory tract infection preceding the onset of neurological symptoms.

The anti-GQ1b IgG antibodies can be positive in Bickerstaff’s brain stem encephalitis. These patients have opthalmoplegia, areflexia or hyporeflexia and also show impaired consciousness, which is not common in MFS /11/.

25.6.3.3 Acute motor axonal neuropathy (AMAN)

AMAN is a multi focal demyelinating neuropathy characterized by a slowly progressive, predominantly distal, asymmetric weakness. Weakness begins distally in upper extremity in 80% of patients. The diagnosis depends on the presence of persistent, focal motor conduction block in one or more nerves at sites not prone to compression. AMAN involves at least two motor nerves for at least one month. The disease mostly affects the arms; tendon reflexes are weak or absent. Sensory disturbances are absent or mild. There is no cranial nerve involvement. AMAN runs a chronic course, lasting years /1213/.

25.6.3.3.1 Laboratory findings

Mild elevation of total protein in CSF, but never above 1 g/L /13/. High titer serum anti-GM-1 IgM antibodies in 75–80% of patients. Another 10–15% of patients have serum IgM binding to GalNAc-GD1a ganglioside. The IgM antibodies may be polyclonal or monoclonal. On immunofixation electrophoresis 10–20% of AMAN patients have a serum IgM monoclonal protein /7/.

25.6.3.4 Chronic inflammatory demyelinating polyneuropathy (CIDP)

CIDP is a chronic neuropathy characterized by more than 2 months of progressive weakness, sensory loss and decreases or absent tendon reflexes. At clinical presence of neuropathy or in neuromuscular centers, CIDP accounts for approximately 20% of patients with initially undiagnosed neuropathies. The polyneuropathy is characterized by symmetric proximal and distal sensory and motor polyneuro radiculopathy. It is an autoimmune disease that involves the peripheral nerve sheaths. T cell deregulation is thought to play a role in the pathogenesis. CIDP runs a chronic progressive or relapsing remitting course.

A non inflammatory, similar condition occurs in diabetes mellitus and type 1 Charcot-Marie-Tooth disease (CMT1). In the general population the prevalences of CIDP and CMT1 are 5/100,000 and 10/100,000, respectively /14/. Autoantibody testing is of little value in CIDP.

Cerebrospinal fluid

Total protein approximately 450 mg/L and a cell count 10/μL (albumino cytologic dissociation) are seen in 83–95% of patients. Albumino cytologic dissociation can also occur in diabetic neuropathy and in GBS /15/.

25.6.3.5 Paraproteinemic neuropathy (PPN)

IgM paraproteinemic neuropathy

The most common types of PPN are those with demyelinating neuropathy and without non neurological symptoms /16/. The neuropathy is defined as demyelinating if it satisfies electrophysiological criteria for chronic inflammatory demyelinating neuropathy (CDIP). If there are subtle features of demyelination not meeting these criteria, further investigations should be considered to confirm the evidence of immune mediated demyelination. Patients with IgM MGUS often develop PPN. Most patients with IgM PPN have the acquired demyelinating symmetrical clinical phenotype of predominantly distal, chronic (duration over 6 months), slowly progressive, symmetric, predominantly sensory impairment, with ataxia and relatively mild or no weakness, and often tremor /14/.

Patients with IgG and IgA PPN usually have proximal and distal motor and sensory weakness that is clinically and electrophysiologically indistinguishable from CIDP.

If a multiple myeloma is present, the neuropathy can be of heterogeneous etiology (amyloidosis, metabolic and toxic damage, nerve root compression).

The Guidelines of the European Federation of Neurological Societies recommend /14/:

  • Patients with PPN should be investigated for a malignant plasma cell dyscrasia
  • The paraprotein is more likely to be causing the neuropathy if the paraprotein is immunoglobulin (IgM), antibodies are present in serum or on biopsy, or the clinical phenotype is chronic distal neuropathy
  • Patients with IgM PPN usually have predominantly distal and sensory impairment, with prolonged distal motor latencies, and often anti-myelin associated glycoprotein antibodies
  • IgM PPN sometimes responds to immunotherapies
  • IgG and IgA PPN may be indistinguishable from CDIP clinically, electrophysiologically, and in response to treatment
  • For POEMS syndrome, local irradiation or resection of an isolated plasmacytoma, or melphalan with or without corticosteroids, should be considered, with hemato-oncology advice.
25.6.3.5.1 Laboratory findings

Anti-Myelin associated glycoprotein (MAG) antibodies are found in the serum of 50% of patients with IgM PPN in high titers (above 1 : 6,400) more commonly associated with kappa than lambda light chains. Patients with IGM PPN and no anti-MAG antibodies should be tested for IgM antibodies to other neural antigens such as the gangliosides and sulfatides /14/. Cerebrospinal fluid analysis shows elevated total protein in 75–86% of PPN patients /14/.

25.6.3.6 CONAMAD

The syndrome of chronic ataxic neuropathy with opthalmoplegia, IgM monoclonal gammopathy, cold agglutinins and ganglioside IgM antibodies (anti-GD1b and anti-GQ1b) is a rare neuropathy. CONAMAD is similar to the chronic form of Miller Fisher syndrome, with mixed demyelinating and axonal electrophysiology. Ataxia is profound, severely impaired function, but motor strength remains relatively spared /15/.

25.6.3.7 POEMS

The acronym POEMS refers to polyneuropathy, organomegaly, endocrinopathy, M protein and skin changes. Some of these elements my be lacking. The syndrome is also known as osteosclerotic myeloma. Neuropathy is the main feature of this syndrome and often precedes the diagnosis of osteosclerotic myeloma. Patients with POEMS typically have IgG or IgA monoclonal gammopathy of the lambda type /17/, the paraprotein level is usually below 2 g/L. Half the patients have thrombocytosis and polycythemia. The vascular endothelial growth factor level is diagnostically useful.

25.6.3.8 Amyloidosis

Primary amyloidosis can be present in up to 10% of patients with multiple myeloma, predominantly of the IgG class and lambda type. Approximately 20% of these patients have an axonal neuropathy /14/. Refer to Tab. 22-26 – Findings in amyloidosis.

25.6.4 Syndromes of the neuromuscular junction and muscle

Neuromuscular diseases affect the nerves that control the voluntary muscles. Neurons send the messages that control these muscles. Most diseases causing muscle weakness that can lead to twitching, cramps, aches, pains and joint problems result from disorders of neuromuscular transmission and are associated with autoantibodies.

Refer to:

25.6.4.1 Neuromuscular transmission

Each muscle fiber is supplied by only a single axonal branch and has one neuromuscular junction. In the junction the presynaptic nerve terminal and the postsynaptic muscle membrane are separated by a 50 nanometer space. The nerve terminals contains an abundance of synaptic vesicles. Each vesicle contains about 5000–10.000 acetylcholine (ACh) molecules. When an electrical nerve impulse invades the motor nerve terminal, it causes voltage gated calcium channels in the nerve terminal to open, allowing Ca2+ to enter. When ACh is released from the motor nerve terminal, it crosses the synaptic cleft and binds to the acetylcholine receptor (AChR) at the muscle membrane. This induces a rapid increase in permeability in Na+, K+, as well as Ca2+ and Mg2+. The main function of ACh transmission at the neuromuscular junction is to trigger contractions of skeletal muscle.

Voltage gated channels perform a unique role in excitable cells. By gating Ca2+ influx in response to depolarization, voltage gated Ca2+channels couple changes in membrane potential to numerous intracellular mechanisms regulated by Ca2+, including neurotransmitter release, muscle contraction, gene expression and cellular differentiation /16/.

25.6.4.2 Neuromuscular junction disorders

A range of antibody mediated disorders of the neuromuscular junction have been described (Tab. 25.6-9 – Autoantibodies in immune-mediated muscular disease), each associated with an autoantibody to a specific ligand gated receptor, voltage gated ion channel or related protein /17/. Besides autoantibodies directed against postsynaptic AChR (e.g., in myasthenia gravis) and autoantibodies that interfere with acetylcholine release at motor nerve terminals (e.g., in Lambert-Eaton myasthenia syndrome) autoantibodies against voltage gated calcium channels are of importance.

25.6.4.3 Lambert-Eaton myasthenia syndrome (LEMS)

LEMS is a presynaptic disorder of neuromuscular transmission characterized by reduction of ACh which is released from the synaptic vesicles of the nerve terminals in response to a nerve impulse. This process requires the influx of Ca2+ through voltage gated Ca2+channels into the nerve terminals. In LEMS, these channels are blocked by autoantibodies. Whereas at rest there is still sufficient release of acetylcholine into the synaptic cleft, this is not the case during muscle activity resulting in muscle weakness /18/.

LEMS is an autoimmune disease mediated by autoantibodies against P/Q-type α1a voltage gated Ca2+ channel (VGCC) at motor nerve terminals. LEMS is a para neoplastic myasthenic syndrome and usually occurs in association with small cell lung cancer (SCLC). The antigenic stimulus for anti-VGCC autoantibody production with SCLC-LEMS appears to be tumor VGCC. About 40% of patients do not have a malignancy. It is believed that proteins of the SCLC provide the antigenic stimuli for autoantibody synthesis. The trigger for the production of anti-VGCC antibodies in individuals with LEMS without detectable lung cancer is unknown. The SCLC develops from cells that ontogenetically originate from the neural crest and form neuroectodermal peptides. The SCLC cells also have Ca2+ channels that are blocked by autoantibodies to voltage gated Ca2+channels /19/.

Clinical presentation

LEMS accounts for 1% of myasthenic syndromes and develops in 3% of patients with SCLC. A frequent initial symptom is proximal muscle weakness, more commonly affecting the muscles of the lower extremities. Patients complain of feeling increasingly exhausted when walking and especially when climbing stairs. Further symptoms are clumsy movements, horizontal diplopia, ptosis and dysphagia. Autonomic disturbances usually begin with dry mouth.

25.6.4.3.1 Laboratory findings

Anti-VGCC autoantibodies are detected in 85% of patients using an immunoprecipitation assay with 125J-conotoxin-labeled MVIIC-VGCC.

25.6.4.4 Neuromyelitis optica

Neuromyelitis optica (NMO), or Devic’s syndrome, is an inflammatory demyelinating disorder of the CNS with lesions predominantly in the optic nerve and spinal cord /20/. It can cause blindness and/or paralysis. The longitudinally extensive transverse myelitis is more than three vertebral segments in length. The interval between optic neuritis and transverse myelitis can be several years. NMO can be monophasic or relapsing. The latter is more common in women.

25.6.4.4.1 Laboratory findings

The anti-AQP4 antibody test has a diagnostic sensitivity of 99% and a specificity of 90%. The anti-AQP4 antibody concentration increases as the disease progresses and may not be pathological during the initial stage of clinical symptoms /20/. Anti-AQP4 antibodies may also be present in SLE, Sjögren’s syndrome and myasthenia gravis. Cerebrospinal fluid shows lymphocytic or neutrophil pleocytosis.

25.6.4.5 Myasthenia gravis

The nicotinic acetylcholine receptors (AChR) belongs to the super family of neurotransmitter gated ion channels /21/. The transmitter of the neuromuscular interaction is acetylcholine (ACh). This is stored in the synapses of the motor nerve terminals and released into the synaptic cleft upon a nerve impulse. On the muscle membrane, ACh activates the AChR, causing the ion channel to open and the motor end plate to depolarize. The AChR is a glycoprotein with a molecular weight of 300 kDa.

Myasthenia gravis is caused by autoantibodies to the muscle nicotinic P/Q-type AChR /22/. This leads to reduced membrane depolarization and weakened muscle action /21/.

Clinical presentation

The prevalence of myasthenia gravis is 40–50 per 1 million population. The early onset form of the disease is mainly seen in women aged 20–30 years, while the late onset form is more common in men aged over 50 years. Symptoms include skeletal muscle weakness, diplopia due to ocular myasthenia, ptosis, fatigable chewing due to pharyngeal muscle weakness, difficulty lifting arms and climbing stairs due to muscle weakness in limbs /22/. Most patients do not receive a clinical diagnosis until two years after symptoms first appear. Some patients are primarily given a psychiatric diagnosis. Most patients with myasthenia gravis have an abnormal thymus, 15% of them have a thymoma /23/.

There are various forms of myasthenia gravis:

  • Generalized myasthenia
  • Ocular myasthenia
  • Neonatal myasthenia. Due to anti-AChR antibodies crossing the placenta into the fetal circulation, 12–20% of newborns to mothers with myasthenia gravis will have myasthenic symptoms such as difficulty swallowing, but these usually disappear within the first six months as the antibody concentration decreases.
  • Paraneoplastic myasthenia gravis. The disease is usually associated with thymic hyperplasia; in the paraneoplastic form there is a thymoma.
  • Congenital myasthenia; is not of autoimmune origin and therefore autoantibody negative.

Relevant autoantibodies in myasthenia are /23/:

  • Anti-acetylcholine receptor antibodies (anti-AChR)
  • Anti-muscle specific tyrosine kinase antibodies (anti-MuSK)
  • Anti-ryanodine receptor antibodies
  • Anti-titin antibodies.

Refer to:

25.6.4.5.1 Anti-acetylcholine receptor (anti-AChR)

The antibodies are detected in serum using an radioimmunoassay. The upper reference value is 0.25 nmol/L; a concentration above 0.4 nmol/L is indicative of myasthenia gravis /24/. A positive anti-AChR antibody test is diagnostic of myasthenia gravis; a negative test does not exclude the disease, since 10–20% of patients, in particular adolescent patients, are seronegative. However, two thirds of patients with anti-AChR antibodies have autoantibodies to muscle specific tyrosine kinase (MuSK).

25.6.4.5.2 Anti-muscle specific tyrosine kinase antibodies (anti-MuSK)

Autoantibodies to MuSK are detected in serum with an immunoprecipitation assay using 125J MuSK as the antigen /25/. The upper reference interval value is 0.05 nmol/L; patients with anti-AChR negative myasthenia gravis have levels above 1 nmol/L /26/.

25.6.4.5.3 Anti-titin antibodies

Patients with thymoma associated myasthenia gravis often have autoantibodies to the muscle protein titin and the ryanodine receptor in addition to anti-AChR antibodies /27/. The antibody tests are performed when the presence of a thymoma cannot be confirmed by imaging studies.

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51. Jaskowski TD, Prince HE, Greer RW, Litwin CM, Hill HR. Further comparisons of assays for detecting MAG IgM autoantibodies. J Neuroimmunol 2007; 187: 175–8.

52. Mata S, Ambrosini S, Mello T, Lolli F, Miniciacchi D. Anti-myelin associated glycoprotein antibodies recognize HNK-1 epitope of CNS. J Neuroimmunol 2011; 236: 99–105.

53. Lennon VA, Wingerchuk DM, Kryzer TJ, Pittock SJ, Lucchinetti CF, Fuzihara K, et al. A serum antibody marker of neuromyelitis optica: distinction from multiple sclerosis. Lancet 2004; 364: 2106–12.

54. Stergiou C, Lazaridis K, Zouvelou V, Tzardos J, Mategazza R, Antizzi C, et al. Titin antibodie in seronegative myasthenia gravis – a new role for an old antigen. J Neuroimmunol 2016; 292: 108–15.

55. Yamamoto AM, Gajdos P, Eymard B, Tranchant C, Warter JM, Gomez L, et al. Anti-titin antibodies in myasthenia gravis: tight association with thymoma and heterogeneity of nonthymoma patients. Arch Neurol 2001; 58: 885–90.

56. Budhram A, Dubey D, Sechi E, Flanagan EP, Yang L. Bhayaana V, et al. Neural antibody testing in patients with suspected autoimmune encephalitis. Clin Chem 2020; 66 (12): 1496–1509.

25.7 Autoantibodies in liver disease

R. Gruber, S. Borgmann, L. Thomas

The demonstration and characterization of serum autoantibodies in patients with acute and chronic liver disease is important for diagnostic, therapeutic and pathogenetic reasons. Autoimmune liver disease is associated with the presence of liver-specific or/and non-organ specific antibodies /1/.

25.7.1 Autoimmune liver disease

Autoimmune hepatitis (AIH), primary biliary cirrhosis (PBC) and primary sclerosing cholangitis (PSC) are three mayor forms of autoimmune liver disease, which differ according to the focus of autoimmune injury, the pattern of inflammation and the clinical phenotype. All three disorders have a progressive course, that, if untreated, develop into liver failure requiring liver transplantation. AIH, PBC, and PSC represent complex disorders, in that they result from the interaction between genetic and environmental factors /2/.

It is believed that the autoimmune condition of AIH, PBC and PSC is determined by the breakdown of immune-regulatory mechanisms with autoimmune etiology. Among multiple T cell subsets with suppressive function, the regulatory T cells (Tregs), defined by the expression of CD4, IL-2 receptor α chain (CD25) and the transcription factor FOXP3, have emerged as having a central role in maintaining immune-tolerance to auto antigens. A reduced frequency and/or function of Tregs in all three autoimmune liver diseases stand under discussion /3/.

Autoimmune liver diseases are differentiated according to clinical findings and the evaluation of autoantibodies in serum. Refer to Fig. 25.7-1 – Diagnostic algorithm for differentiation of autoantibodies associated with liver disease.

25.7.1.1 Autoimmune hepatitis

Refer to Tab. 25.7-1 – Autoimmune liver diseases.

25.7.1.2 Primary biliary cirrhosis

Refer to Tab. 25.7-1 – Autoimmune liver diseases.

25.7.1.3 Primary biliary cholangitis

Refer to Tab. 25.7-1 – Autoimmune liver diseases.

25.7.1.4 Anti-mitochondrial antibodies (AMA)

Refer to:

Shared serological, immunological and histological patterns exist across the spectrum of AIH, PBC, and PSC. Conditions exhibiting features of two different autoimmune liver diseases are commonly designated overlap syndromes. These are represented by various forms of AIH, in which there are characteristics of both AIH and PBC (AIH/PBC overlap) or AIH and PSC (AIH/PSC overlap). However, there is barely any significant genetic overlap between PBC and PSC /2/.

Approximately 90–95% of patients with PBC have anti-mitochondrial antibodies (AMA), sometimes accompanied by certain ANA such as nuclear dots/SP100, nuclear membrane/gp210, and lamin B receptor (LBR). In rare cases no autoantibodies are found. Some authors refer to AMA negative PBC as autoimmune cholangitis /9/. AMA can be confirmed by enzyme immunoassay or immunoblotting using pyruvate dehydrogenase (PDH)-E2 as antigen (AMA-M2). To improve the detection limit, two additional epitopes, namely branched chain α-keto acid dehydrogenase and oxoglutarate dehydrogenase, are implemented in some assays, partly in the form of recombinant proteins. Overall, however, these proteins are much less commonly used as auto antigens than PDH-E2.The diagnostic sensitivity of the antibody test can be increased further without loss of specificity by including the PBC-specific ANA antigens (gp210 and SP100) in the test.

The following Tables and Figures show data about the differentiation and determination of AMA

Approximately 80% of PSC patients are found to have p-ANCA, but these are not disease specific.

The antibodies described may also develop secondarily, especially in systemic autoimmune diseases. For example, 25–50% of patients with SLE have elevated liver enzymes in the course of their lives. Of these patients, 70% meet the diagnostic criteria for autoimmune liver disease, but only 20% exhibit corresponding histological changes. The prevalence of autoimmune liver disease in SLE patients is 5% /10/.

In addition, there are associations between autoimmune liver diseases and other autoimmune disorders:

  • Autoimmune liver disease with autoimmune thyroiditis
  • PBC with CREST syndrome
  • PSC with ulcerative colitis.

Secondary antibody development is also seen in viral hepatitides. More than 50% of patients with HCV infection have autoantibodies, usually rheumatoid factors with cryoglobulin activity. In 5–10% of cases, these antibodies are present in high titers and often lead to clinically manifest symptoms. Approximately 2–5% of patients infected with HCV have anti-LKM-1 Ab, which are otherwise highly specific for type II AIH; therefore, patients with anti-LKM-1 Ab should be tested for HCV infection /4/.

A negative autoantibody test does not exclude an autoimmune liver disease (e.g., AMA-negative PBC). Conversely, autoantibodies may occur in non autoimmune liver diseases (e.g., in the presence of extrahepatic autoimmune diseases or infections).

Unlike previously thought, the development of anti-LKM-1 Ab triggered by HCV infection does not have a negative impact on the success of treatment with immune stimulating substances such as interferon-α /5/.

References

1. Neuberger J, Bradwell AR. Milestones in liver disease. J Hepatol 2002; 37: 712–6.

2. Carbone M, Neuberger JM. Autoimmune liver disease, autoimmunity and liver transplantation. EASL Journal of Hepatology 2014; 60: 210–23.

3. Liberal R, Grant CR, Longhi MS, Mieli-Vergani G, Vergani D. Regulatory T cells: mechanisms of suppression and impairment in autoimmune liver disease. IUBMB Life 2015; 67: 88–97

4. Gatselis NK, Zachou K, Koukoulis GK, Dalekos GN. Autoimmune hepatitis, one disease with many faces: Etiopathogenetic, clinico-laboratory and histological characteristics. World J Gastroenerol 2015; 21: 60–83.

5. Feld JJ, Heathcote EJ. Epidemiology of autoimmune liver disease. J Gastroenterol Hepatol 2003; 18: 1118–28.

6. Takahashi H, Zeniya M. Acute presentation of autoimmune hepatitis: Does it exist? A published work review. Hepatol Res 2011; 41: 498–504.

7. Hennes EM, Zeniya M, Czaja AJ, Pares A, Dalekos GN, et al. Simplified criteria for the diagnosis of autoimmune hepatitis. Hepatology 2008; 48: 169–76

8. Krawitt EL. Autoimmune hepatitis. N Engl J Med 2006; 354: 54–66.

9. Beuers U. Hepatic overlap syndromes. J Hepatol 2005; 42 Suppl: S93–S99.

10. Efe C, Purnak T, Ozaslan E. Systemic lupus erythematosus and autoimmune hepatitis. Rheumatol Int 2011; 31: 419.

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. Gulamhusein AF, Hirschfield GM. Pathophysiology of primary biliary cholangitis. Best Pract Res Clin Gastroenterol 2018; 34–35: 17–25.

13. Selmi C, Mayo MJ, Bach N, Ishibashi H, Invernizzi P, et al. Primary biliary cirrhosis in monozygotic and dizygotic twins: genetics, epigenetics, and environment. Gastroenterology 2004; 127: 485–92.

14. Hirschfield GM, Siminovitch KA. Toward the molecular dissection of primary biliary cirrhosis. Hepatology 2009; 50: 1347–50.

15. Berg PA, Binder T, Lindner H, Bannaski H, Maas D, et al. Heterogenität mitochondrialer Antikörper. Dtsch Med Wschr 1975; 100: 1123–7.

16. Muratori P, Muratori L, Ferrari R, Cassani F, Bianchi G, et al. haracterization and clinical impact of antinuclear antibodies in primary biliary cirrhosis. Am J Gastroenterol 2003; 98: 431–7.

17. Worman HJ, Courvalin JC. Antinuclear antibodies specific for primary biliary cirrhosis. Autoimmun Rev 2003; 2: 211–7.

18. De Liso F, Matinato C, Ronchi M, Maiavacca R. The diagnostic accuracy of biomarkers for diagnosis of primary biliary cholangitis (PBC) in anti-mitochondrial antibody negative PBC patients: a review of literature. Clin Chem Lab Med 2018; 56: 25–31.

19. Lee YM, Kaplan MM. Primary sclerosing cholangitis. N Engl J Med 1995; 332: 924–33.

20. Khosroshahi A, Stone JH. A clinical overview of IgG4-related systemic disease. Curr Opin Rheumatol 2011; 23: 57–66.

21. Hov JR, Boberg KM, Karlsen TH. Autoantibodies in primary sclerosing cholangitis. World J Gastroenterol 2008; 14: 3781–91.

22. Manns MP, Czaja AJ, Gorham JD, Krawitt EL, Mieli-Vergani G, et al. Diagnosis and management of autoimmune hepatitis. Hepatology 2010; 51: 2193–213.

23. Albayda J, Khan A, Casciola Rosen L, Corse AM, Paik JJ, Christopher-Sine L. Inflammatory myopathy associated with anti-mitochondrial antibodies: a distinct phenotype with cardiac involvement. Semin Arthritis Rheum 2018; 47: 552–6.

24. Konishi H, Fukuzawa K, Mori S, Satomi-Kobayashi S, Kiuchi K, Suzuki A, et al. Anti-mitochondrial M2 antibodies enhance the risk of supraventricular arrhythmias in patients with elevated hepatobiliary enzymes. Intern Med 2017; 56: 1771–9.

25. Nguyen DL, Juran BD, Lazaridis KN. Primary biliary cirrhosis. Best Pract Res Clin Gastroenterol 2010; 24: 647–54.

25.8 ANCA associated vasculitides, Goodpasture syndrome

Stefan Borgmann, Rudolf Gruber

The four ANCA associated diseases, which are of autoimmune genesis and predominantly affect the lungs and kidneys as target organs are:

  • Primary small vessel vasculitides (AVV)
  • Microscopic polyangiitis (MPA)
  • Churg-Strauss syndrome (CSS)
  • Goodpasture syndrome (GPS).

While AVV, MPA and CSS are primary vasculitides, GPS is due to direct damage to the pulmonary and glomerular basement membranes. This grouping is therefore artificial in terms of pathogenic aspects. However, there are systematic overlaps, which could be overlooked if the diseases were described separately.

25.8.1 Vasculitis

Vasculitis is an inflammatory process that destroys or impairs the function of blood vessels. In addition, many types of vasculitides are associated with glomerular damage, which often determines the clinical course of disease.

Vasculitis is characterized by general symptoms such as exhaustion, fever and weight loss. The clinical presentation varies depending on the pattern of vessel involvement. For example, small vessel involvement manifests as palpable purpura, polyneuritis, episcleritis, hemoptysis or micro hematuria. Medium vessel involvement leads to infarction of the heart, kidneys, bowel or extremities, or to cerebral insults. Large vessel involvement may manifest as aortic arch syndrome or thrombotic venous occlusion.

Classification

Vasculitis may occur as a primary disease or secondary to another underlying disease (secondary vasculitis). Secondary vasculitis occurs in connective tissue disease (SLE, Sjögren’s syndrome), rheumatoid arthritis, infections (hepatitis B and C, HIV), and as a result of the use of drugs (cocaine) and medicines.

Vasculitis is classified based on pathogenic as well as histological (granulomatous vasculitis, leukocytoclastic vasculitis, vasculitis with glomerular crescent formation) or immunopathological criteria (immune complex vasculitis vs. pauci immune vasculitis). Unlike immune complex vasculitis, the pauci immune forms are not associated with immune deposits in inflamed areas. Examples of pauci immune vasculitis are Wegener’s granulomatosis, microscopic polyangiitis and Churg-Strauss syndrome. Pauci immune vasculitis is a form of vasculitis that is associated with minimal evidence of hypersensitivity upon immunofluorescent staining for IgG. Immune complex vasculitis is seen in SLE and in Schoenlein-Henoch purpura.

The American College of Rheumatology (ACR) classification provides diagnostic criteria for the presence of rheumatic disease. The criteria are continually reviewed by the ACR in cooperation with the European League against Rheumatism (EULAR) and updated to reflect new knowledge as it becomes available /1/.

Tab. 25.8-1 – ACR criteria for granulomatosis with polyangiitis.

The Chapel Hill Consensus Conference /2/, in contrast, has defined criteria for distinguishing between different types of primary vasculitis /2/. At the heart of this classification is the inclusion of the caliber of inflamed vessels. The main significance of this classification lies in the first time division of classic pan- or polyarteritis nodosa into medium vessel panarteritis nodosa and microscopic polyangiitis (MPA), especially of small vessels. Since p-ANCA only occur in MPA, this division impacts on laboratory diagnostic testing.

Tab. 25.8-2 – Chapel Hill Consensus Conference nomenclature of vasculitis.

Clinical symptoms vary according to the size of the affected vessels /3/:

  • Inflammation of the large vessels mainly results in vein thrombosis and arterial stenosis
  • Medium vessel involvement results in hemorrhage, infarction and arterial stenosis
  • Small vessel inflammation causes episcleritis, hearing loss, hemorrhagic rhinitis, vertigo, hemoptysis, melaena, micro hematuria, neuritis, palpable purpura, and perimyocarditis /3/.

Clinical presentation

The ANCA associated vasculitides AVV, MPA and CSS are pauci immune forms /45/. In these forms of vasculitis there are few or no in situ immune deposits. Therefore, unlike immune complex vasculitis (polyarteritis nodosa) and vasculitis in connective tissue disease (systemic lupus erythematosus), these disorders are not associated with a decline in peripheral complement levels. The full blown clinical picture of the three diseases often includes involvement of the upper respiratory tract, the lungs, and the kidneys. As symptoms are frequently very unspecific at first, an impaired general condition is often misinterpreted as an infection or tumorous process. The clinical presentation of ANCA associated vasculitides is described in Tab. 25.8-3 – ANCA associated vasculitides.

Laboratory tests

Tab. 25.8-4 – Laboratory tests in the diagnosis of vasculitis and pulmonary-renal syndrome lists the laboratory tests related to the diagnosis, activity of vasculitis and/or organ involvement. Depending on the stage of disease, CRP or, if the kidneys are involved, creatinine levels are elevated. The presence of erythrocytes or erythrocyte cylinders in urine sediment is a sign of glomerular damage.

In addition to the laboratory tests, the diagnosis should be confirmed by biopsy histology and immunohistology. The tissue samples must be taken from pathologically abnormal areas.

25.8.2 Anti-neutrophil cytoplasmic antibodies (ANCA)

ANCA, such as those directed towards proteinase 3 (PR3) and myeloperoxidase (MPO), are associated with a distinct form of small-vessel vasculitis, known as ANCA associated vasculitis (AAV), a term that encompasses granulomatosis with polyangiitis (GPA) and microscopic polyangiitis (MPA) /6/. AAV can take a peracute course and lead to dialysis dependence within a day. A significant increase in titer or detection of c-ANCA should immediately be communicated to the physician in charge.

25.8.2.1 Indication

Recommendation for ANCA testing /6/:

  • Glomerulonephritis, especially rapidly progressive glomerulonephritis
  • Pulmonary hemorrhage, especially pulmonary renal syndrome
  • Cutaneous vasculitis with systemic features
  • Multiple lung nodules
  • Chronic destructive disease of the upper airways
  • Long-standing sinusitis or otitis
  • Subglottic tracheal stenosis
  • Mononeuritis multiplex or other peripheral neuropathy
  • Retro-orbital mass
  • Scleritis

25.8.2.2 Method of determination

Target antigens of the immunological methods are myeloperoxidase (MPO) and PR3. The international recommendations suggest that the (IIFT) on ethanol stained human neutrophils should be used for ANCA screening. However, for optimal screening, the combined use of IIFTs and immunoassays for the detection of antibodies to proteinase 3 and myeloperoxidase is recommended /78/. A revised 2017 international consensus /6/ proposes that high-quality immunoassays can be used as the primary screening method for patients suspected as having the ANCA associated vasculitis granulomatosis with polyangiitis (GPA) and microscopic polyangiitis (MPA) without the categorical need of IIFT. A second PR3 and MPO ANCA immunoassay or IIFT can be considered for negative results in patients with a high clinical suspicion (to increase sensitivity) or in case of low antibody levels (to increase specificity) /6/.

Indirect immunofluorescence test (IIFT)

Using ethanol and formalin stained neutrophils in the IIFT, the following different fluorescence patterns can be detected:

  • Typical c-ANCA produce an identical pattern on ethanol and formalin stained neutrophils, which appears in the form of a diffuse coarse granular fluorescent staining of the cytoplasm with interlobular accentuation
  • Atypical c-ANCA usually give a diffuse to fine granular pattern without interlobular accentuation on ethanol stained neutrophils and are usually negative on formalin stained neutrophils
  • Typical p-ANCA show a perinuclear fluorescence pattern on ethanol stained neutrophils and a granular cytoplasmic fluorescence pattern identical to that of typical c-ANCA on formalin stained neutrophils.
  • Atypical p-ANCA produce a perinuclear fluorescence pattern on ethanol stained neutrophils and are usually negative on formalin stained neutrophils.

To be able to interpret the perinuclear fluorescence pattern of p-ANCA on ethanol stained neutrophils, it is important to know that the pattern is due to a fixation artefact caused by translocation of the autoantigens from the azurophilic granules into the perinuclear area. In reality, the antigens of both the c-ANCA and the p-ANCA are contained in the azurophilic granules. The granular cytoplasmic fluorescence pattern found on formalin stained neutrophils thus corresponds to the in situ localization.

The group of atypical p-ANCA are often referred to as a-ANCA (a stands for atypical) or, by some authors, as x-ANCA. Overall, however, the term a-ANCA appears to be more appropriate, since these autoantibodies are not associated with vasculitides and a finding of p-ANCA could mislead the clinician. In view of this fact, the authors, in agreement with various literature, suggest that atypical ANCA (p- or c-ANCA) always be referred to as a-ANCA /9/, allowing clinicians to tell from the nomenclature alone whether or not primary vasculitis is present.

Immunoassay

EIA, ELISA, chemiluminescence tests, bead assays, immunoblot. These assays use the antigens proteinase 3 (PR3) and MPO which have been purified or recombined from granulocyte extracts /6/.

25.8.2.3 Specimen

Serum, plasma: 1 mL

25.8.2.4 Threshold values

IIFT ≥ 1 : 10 positive

Immunoassay: not standardized; specifications from test kit manufacturer.

25.8.2.5 Clinical significance

The ANCA assay is an established method used in cases where ANCA associated vasculitis is suspected. A negative test result does, however, not exclude the presence of ANCA associated vasculitis. ANCA may be negative especially in phases of low disease activity. In a study /10/ of patients with biopsy confirmed granulomatosis with polyangiitis, 96% of patients had active generalized disease, but only 67% of those in the initial stage of disease were ANCA positive. After reaching the remission stage, the proportion of ANCA positive patients decreased by 50%.

High ANCA titers are associated with active phases of the disease. This applies to anti-proteinase 3 antibody rather than anti-MPO antibody. Rises in titers can occur weeks or months before exacerbation of vasculitis (9–106 days, on average 49 days) /11/. Unless there is a concomitant rise in ANCA, a relapse is rare. However, a mere rise in titer without concomitant worsening of symptoms is no reason to intensify immunosuppressive treatment. ANCA negative patients with biopsy confirmed Wegener’s granulomatosis have a better prognosis.

ANCA testing is also suitable for monitoring treatment of primary vasculitis. Successful immunosuppressive treatment with steroids or cyclophosphamide should be followed by a decrease in titer or a negative ANCA test. Terminating immunosuppressive treatment before the patient tests completely negative for ANCA increases the risk of reactivation of the vasculitis within the following 3–6 months. Patients in remission who have persistently or intermittently elevated ANCA titers appear to have a higher risk of relapse /1213/.

For further information refer to:

25.8.2.6 Comments and problems

Besides the typical constellations of vasculitis associated ANCA, there are often p-ANCA findings and atypical c-ANCA that would be better referred to as a-ANCA, as they are not associated with vasculitis.

A-ANCA are commonly seen (up to 80%) in patients with ulcerative colitis, primary sclerosing cholangitis or autoimmune hepatitis, less commonly in Crohn’s disease, rheumatoid arthritis, chronic inflammatory diseases and in connective tissue diseases (lupus erythematosus) /14/. A number of other auto antigens have been described in these diseases, among them the bactericidal/permeability increasing protein, which commonly produces a cytoplasmic staining pattern on ethanol fixed neutrophils in IIFT, as well as neutrophil elastase, azurocidin and cathepsin G. There are published data on specific auto antigens of a-ANCA in autoimmune hepatitis, PSC and ulcerative colitis in association with DNA associated lactoferrin /15/, human β-tubulin isotype 5 and the microbial protein FtsZ /16/.

If p-ANCA and a-ANCA are present together with ANA, IIFT is usually inconclusive due to the fluorescence overlay. In this case, a supplemental immunoassay should be used.

Immunoassays

Anti-PR3 Ab produce a c-ANCA pattern in IIFT in more than 90% of cases, but may also appear as p-ANCA, whereas anti-MPO Ab show a perinuclear pattern (p-ANCA) in more than 90% of cases, but may also give a c-ANCA pattern. Commercial ANCA immunoassays may have high quality /6/.

Stability

1 day at room temperature, 10 days at 4–8 °C. Long-term storage (years) at –20 °C in a polypropylene tube.

25.8.3 Goodpasture syndrome

Goodpasture syndrome (GPS) is a complex of clinical symptoms associated with rapidly progressive glomerulonephritis in combination with progressive pulmonary disease /17/. The latter can persist with relatively mild symptoms (mild dyspnea) for several months, but then exacerbate rapidly, with hemorrhages into the lungs and hemoptyses. Hemorrhages are especially seen in smokers. GPS is characterized by renal and pulmonary dysfunction as a result of the presence of autoantibodies to the glomerular basement membrane /18/. If left untreated, the disease is always fatal, and most patients will have to undergo chronic hemodialysis even under immunosuppressive treatment and plasmapheresis. GPS is relatively rare, with an incidence of 1–5 per 1 million population and year, and has two age peaks (18–30 years and 50–65 years). The median age at diagnosis is 59 years, and 44% of patients are female.

25.8.3.1 Laboratory findings

Serum creatinine and K+ elevated, blood gas analysis (PO2 , PCO2 , pH ), anti-GBM positive. After lung hemorrhage, siderophages can be detected in bronchoalveolar lavage fluid.

Although there is an HLA association, HLA typing is of diagnostic relevance only in exceptional cases. HLA DRB1*1501 is 3.5 times more common in patients with GPS than in the normal population. HLA DRB1*03 and HLA DRB1*04 are also more frequent in GPS patients. By contrast, the HLA DRB1*01 alleles of HLA DRB1*07 are detected less frequently and thus seem to confer some protection against the development of GPS.

25.8.3.2 Anti-glomerular basement membrane antibodies

Anti-glomerular basement membrane antibodies (anti-GBM Ab) can be assayed by both immunoassay and IIFT and immunoassay using cryostat sections of monkey kidney. Due to the IgG deposits commonly found there, the linear fluorescent pattern typical of anti-GBM Ab can be seen even with normal sera /19/.

Due to the sometimes peracute worsening of the clinical situation, positive initial results should always be communicated by telephone to the physician in charge. Initially high antibody titers are reported to be associated with an increased risk of becoming permanently dependent on hemodialysis. Nearly half of all patients with GBS are found to have ANCA, usually p-ANCA directed against myeloperoxidase .

25.8.4 Pathophysiology

ANCA-associated vasculitis

The pathogenesis of ANCA associated vasculitis is unknown. However, a model summarizing the findings can be derived from existing clinical observations and experimental data, most of which relate to granulomatosis associated with polyangiitis.

It is widely accepted that neutrophils are initially activated by cytokines and adhere to endothelial cells. ANCA in plasma bind to PR3 in the cell membrane and activate the neutrophils for the further translocation of PR3 from the azurophilic granules into the cell membrane. The continuous binding of ANCA to membrane-bound PR3 leads to further activation of the neutrophils and finally their respiratory burst and the release of proteolytic enzymes, resulting in damage to the endothelium.

The proportion of neutrophils that express PR3 on their cell membrane (mPR3) varies from individual to individual and is likely determined by genetics. The activation induced translocation of granular PR3 into the cell membrane does not increase this proportion, as only the amount of membrane PR3 in mPR3-positive neutrophils is increased, but not the amount of mPR3-positive neutrophils. The translocation requires a certain amount of HLA antigens plus the glycoprotein NB1 (CD177) /2021/. AVV patients have a higher percentage of mPR3-positive cells of the total neutrophil population than the healthy population.

Although neutrophils cause the vasculitis, cases have been described in which ANCA occurred in association with neutropenia. Since the mediator of the inflammation is limited in its extent in the presence of neutropenia, the resulting vasculitis should be mild. In agreement with this assumption, the vasculitis, if present at all, was limited to the skin. The causes of ANCA triggered vasculitides in the presence of neutropenia includes the use of drugs (propylthiouracil, methimazole, minocycline), misuse of cocaine, or the presence of other autoimmune diseases (Felty’s syndrome, autoimmune hepatitis, Sjögren’s syndrome) /22/.

The initial priming of the neutrophils is likely caused by infections. Nasal S. aureus carrier rates are higher in patients with AVV than in the healthy population. In addition, carriage of S. aureus appears to be associated with a higher rate of disease relapse /23/. Studies /24/ suggest that first there is an infection, which results in the production of antibodies to the microbes. Then, for unknown reasons, (idiotypical) antibodies are produced against the antigen binding regions of the anti microbe antibodies. The idiotypical antibodies finally cross react with PR3, so that the development of PR3-ANCA is ultimately due to an infection. However, PR3-ANCA positive patients showed reduced rather than the expected increased reactivity against complementary PR3 in comparison to healthy controls and MPO-ANCA positive patients, which does little to help improve the understanding of the pathogenesis /25/.

The observation that both patients with florid PR3- and patients with florid MPO-ANCA associated vasculitis have antibodies to the bacterial adhesin FimH suggests that infections caused by Gram negative bacteria may be involved in the pathogenesis. Patients in remission, by contrast, do not have any anti-FimH antibodies. FimH, in turn, is partly identical with LAMP-2, which is present on neutrophils and endothelial cells. Like PR3 and MPO, LAMP-2 is found in neutrophil azurophilic granules, but is also transported from there to the cell membrane and back. By binding to membrane bound LAMP-2, the antibodies can bypass the neutrophils and develop direct cytotoxicity. An association with antibodies to LAMP-2 was not only established for patients with florid vasculitis, but also in animal tests, where injection of anti-LAMP-2 Ab caused pauci-immune vasculitis /26/.

A summary of the pathogenesis is shown in Fig. 25.8-1 – Pathogenesis of ANCA associated vasculitis. One of the first genetic associations of ANCA associated vasculitis was the finding that the PI*Z allele of α1-antitrypsin occurs more frequently in patients with α1-antitrypsin deficiency than in the normal population. α1-antitrypsin is the physiological inhibitor of PR3. About 25% of homozygous PI*Z carriers have liver cirrhosis in childhood and virtually always develop pulmonary emphysema in adulthood. Heterozygous carriers of this deficiency allele are overrepresented among ANCA associated vasculitis patients. It is, however, unlikely that expression of this allele in itself is a risk factor, because although at 5% the frequency of carriers of this allele is 100-fold higher among ANCA associated vasculitis patients than among the general population, as many as 95% of all patients do not have the allele. It is therefore possible that, rather than α1-antitrypsin itself, an unknown protein whose gene is located near the PI*Z gene is involved in the pathogenesis. This hypothesis is supported by the fact that, unlike normal controls, ANCA associated vasculitis patients show a linkage disequilibrium in the serpin gene cluster that includes the α1-antitrypsin gene /27/. However, searching in the serpin gene cluster has not produced any indication of the protein involved /28/. In addition to the PI*Z allele, the S allele is also thought to be involved in the development of ANCA associated vasculitis /29/.

Goodpasture syndrome

Certain MHC class II antigens can promote or reduce the development of GPS. The sine qua non for the diagnosis of this disease is an IgG autoantibody which attacks both the glomerular and the alveolar basement membranes. The basement membrane essentially consists of type IV collagen (Fig. 25.8-2 – Basement membranes consist of collagen type IV), which is made up of a helical collagenous domain and a non collagenous domain (NCl). The NCl in turn is composed of a hexamer of subunits. One subunit each of α3 NCl, α4 NCl and α5 NCl together form an α345 NCl domain, and two of these domains form an NCl hexamer.

Each α345 NCl domain has a sulfilimine bond that links the relevant domain. The GP autoantibody binds to the α3 NCl subunit; in more advanced stages of the disease, antibodies to the α5 NCl subunit may also develop. If the α3 NCl subunit is in a crosslinked hexamer, the GP autoantibody cannot bind to it. In a non crosslinked hexamer, the autoantibody is able to first dissociate the hexamer into subunits and then bind to the α3 NCl subunit. However, if sulfilimine bonds have formed, dissociation cannot be induced and there is no antibody binding /30/. Animal tests indicate that the cell mediated immune system may also be involved in the pathogenesis of GPS and provide therapeutic targets /31/.

Almost half of all GPS patients have ANCA directed against MPO, and some of the patients have lung involvement. Since some authors exclude the presence of microscopic polyangiitis when the respiratory tract is involved, development of ANCA directed against MPO in these patients should rather be seen as a secondary phenomenon to GPS.

References

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5. Jennette JC. Nomenclature and classification of vasculitis: lessons learned from granulomatosis with polyangiitis (Wegener’s granulomatosis). Clin Exp Immunol 2011; 164 Suppl 1: 7–10.

6 Bussuyt X, Tervaert JWC, Arimura Y, Blockmans D, Suarez LFF, Guillevin L, et al. Revised 2017 international consensus on testing of ANCA in granulomatosis with polyangiitis and microscopic polyangiitis. Nature Reviews Rheumatology 2017; 13: 683–92.

7. Savige J, Gillis D, Benson E, Davies D, Esnault V, et al. International Consensus Statement on Testing and Reporting of Antineutrophil Cytoplasmic Antibodies (ANCA). Am J Clin Pathol 1999; 111: 507–13.

8. Savige J, Dimech W, Fritzler M, Goeken J, Hagen EC, et al. Addendum to the International Consensus Statement on testing and reporting of antineutrophil cytoplasmic antibodies. Quality control guidelines, comments, and recommendations for testing in other autoimmune diseases. Am J Clin Pathol 2003; 120: 312–8.

9. Terjung B, Worman HJ, Herzog V, Sauerbruch T, Spengler U. Differentiation of antineutrophil nuclear antibodies in inflammatory bowel and autoimmune liver diseases from antineutrophil cytoplasmic antibodies (p-ANCA) using immunofluorescence microscopy. Clin Exp Immunol 2001; 126: 37–46.

10. Schulte-Pelkum J, Radice A, Norman GL, Hoyos ML, Lakos G, Buchner C, Musset L, et al. Novel clinical and diagnostic aspects of antineutrophil cytoplasmic antibodies. J Immunol Res 2014; article ID 185416; https://dx.doi.org/10.1155/2014/185416.

11. Jayne DR, Gaskin G, Pusey CD, Lockwood CM. ANCA and predicting relapse in systemic vasculitis. QJM 1995; 88: 127–33.

12. Boomsma MM, Stegeman CA, van der Leij MJ, Oost W, Hermans J, et al. Prediction of relapses in Wegener’s granulomatosis by measurement of antineutrophil cytoplasmic antibody levels: a prospective study. Arthritis Rheum 2000; 43: 2025–33.

13. Stegeman CA, Tervaert JW, Sluiter WJ, Manson WL, de Jong PE, et al. Association of chronic nasal carriage of Staphylococcus aureus and higher relapse rates in Wegener granulomatosis. Ann Intern Med 1994; 120: 12–7.

14. Wieslander J. How are antineutrophil cytoplasmic autoantibodies detected? Am J Kidney Dis 1991; 18: 154–8.

15. Teegen B, Niemann S, Probst C, Schlumberger W, Stöcker W, et al. DNA-bound lactoferrin is the major target for antineutrophil perinuclear cytoplasmic antibodies in ulcerative colitis. Ann N Y Acad Sci 2009; 1173: 161–5.

16. Terjung B, Sohne J, Lechtenberg B, Gottwein J, Muennich M, et al. p-ANCAs in autoimmune liver disorders recognise human beta-tubulin isotype 5 and cross-react with microbial protein FtsZ. Gut 2010; 59: 808–16.

17. Greco A, Rizzo ML, De virgilio A, Gallo A, Fusconi M, Pagliuca G, et al. Goodpasture’s syndrome: a clinical update. Autoimmun Rev 2015; 14: 246–53.

18. Pedchenko V, Bondar O, Fogo AB, Vanacore R, Voziyan P, et al. Molecular architecture of the Goodpasture autoantigen in anti-GBM nephritis. N Engl J Med 2010; 363: 343–54.

19. McAdoo SP, Pusey CD. Anti-glomerular basement membrane disease. Clin J Am Soc Nephrol 2017; 12: 1162–72.

20. Hagen EC, Daha MR, Hermans J, Andrassy K, Csernok E, Gaskin G, et al. Diagnostic value of standardized assays for anti-neutrophil cytoplasmic antibodies in idiopathic systemic vasculitis. Kideney Int 1998; 53: 743–53.

21. von Vietinghoff S, Tunnemann G, Eulenberg C, Wellner M, Cristina Cardoso M, et al. NB1 mediates surface expression of the ANCA antigen proteinase 3 on human neutrophils. Blood 2007; 109: 4487–93.

22. Grayson PC, Sloan JM, Niles JL, Monach PA, Merkel PA. Antineutrophil Cytoplasmic antibodies, autoimmune neutropenia, and vasculitis. Semin Arthritis Rheum 2011; 41: 424–33.

23. Zycinska K, Wardyn KA, Zielonka TM, Demkow U, Traburzynski MS. Chronic crusting, nasal carriage of staphylococcus aureus and relapse rate in pulmonary Wegener’s granulomatosis. J Physiol Pharmacol 2008; 59 Suppl 6: 825–31.

24. Pendergraft WF, 3rd, Preston GA, Shah RR, Tropsha A, Carter CW, Jr., et al. Autoimmunity is triggered by cPR-3(105–201), a protein complementary to human autoantigen proteinase-3. Nature Med 2004; 10: 72–9.

25. Tadema H, Kallenberg CG, Stegeman CA, Heeringa P. Reactivity against complementary proteinase-3 is not increased in patients with PR3-ANCA-associated vasculitis. PLoS One 2011; 6: e17972.

26. Kain R, Exner M, Brandes R, Ziebermayr R, Cunningham D, et al. Molecular mimicry in pauci-immune focal necrotizing glomerulonephritis. Nature Med 2008; 14: 1088–96.

27. Borgmann S, Endisch G, Urban S, Sitter T, Fricke H. A linkage disequilibrium between genes at the serine protease inhibitor gene cluster on chromosome 14q32.1 is associated with Wegener’s granulomatosis. Clin Immunol 2001; 98: 244–8.

28. Borgmann S, Haubitz M, Schwab SG. Lack of association of alpha-1 antichymotrypsin gene polymorphism with PR3-ANCA and MPO-ANCA associated vasculitis. Autoimmunity 2002; 35: 435–9.

29. Mahr AD, Edberg JC, Stone JH, Hoffman GS, St Clair EW, et al. Alpha-antitrypsin deficiency-related alleles Z and S and the risk of Wegener’s granulomatosis. Arthritis Rheum 2010; 62: 3760–7.

30. Vanacore R, Pedchenko V, Bhave G, Hudson BG. Sulphilimine cross-links in Goodpasture’s disease. Clin Exp Immunol 2011; 164 Suppl 1: 4–6.

31. Reynolds J. Strain differences and the genetic basis of experimental autoimmune anti-glomerular basement membrane glomerulonephritis. Int J Exp Pathol 2011; 92: 211–7.

25.9 Autoantibodies and type 1 diabetes

Lothar Thomas

Type 1 diabetes (autoimmune diabetes) is a chronic progressive disease characterized by the destruction of insulin secreting β cells within the pancreatic islets. Autoimmune diabetes is a T cell-mediated disease that results from immune dysfunction, with subsequent loss of tolerance to β cell antigens and destructive lymphocytic infiltration of the islets. This, in turn, leads to insulin deficiency, impaired glucose tolerance, and symptomatic hyperglycemia. Monitoring of circulating autoantibodies targeting β cell proteins is the most reliable diagnostic procedure in the prodromal phase of type 1 diabetes, since antibody appearance precedes overt type 1 diabetes for years /12/. This provides a window for therapeutic intervention.

25.9.1 Indication

Prediction of type 1 diabetes /34/:

  • Prodromal phase of type 1 diabetes
  • Acute onset of ketoacidotic diabetes in children or overweight adults
  • Onset of non ketotic diabetes mellitus in lean patients
  • Screening of at risk relatives of T1D patients.

25.9.2 Method of determination

The first evidence for islet autoimmunity was the classic islet cell autoantibody (ICA) immunofluorescence test /5/.

ICA immunofluorescence test

ICA are detected on unfixed frozen sections of human pancreas from blood type 0 donors. Following incubation with the patient serum and a washing step, the sections are incubated with a fluorescein isothiocyanate labeled secondary antibody directed against the Fc component of human IgG and analyzed under the fluorescence microscope. ICA typically stain the cytoplasm of islet cells. To determine the antibody titers, the serum is geometrically diluted in phosphate buffered saline (PBS). To allow the results to be compared, the titer is converted to Juvenile Diabetes Foundation (JDF) units by comparison with a standard curve. The standard curve is generated for each pancreas using an internationally available reference serum.

Determination of autoantibodies targeting islet cell antigens

The major autoantibodies for the detection of type 1 diabetes target the following antigens:

  • Insulin; associated autoantibodies are IAA
  • The 65-kDa isoform of glutamic acid decarboxylase; associated antibodies are GADA
  • Insulinoma antigen 2; associated antibodies are IA-2A
  • Zinc transporter 8 protein; associated antibodies are anti-ZnT8.

The islet cell auto antigens have been specifically identified and cloned (recombinant antigens) to determine IAA, IA-2A and GADA. For RIAs, [35S]-methionine-labeled GAD65 or IA-2 produced by in vitro transcription/translation or biochemically isolated 125J-labeled GAD65, IA-2 or insulin is used. In an enzyme immunoassay, the autoantibodies bind to solid phase bound GAD65, IA-2 or insulin. Alternatively, the autoantibodies bind to biotinylated islet cell antigens or insulin in the liquid phase and subsequently to streptavidin coated micro titer plates. Most assays also include internal positive and negative standard sera. International workshops (Diabetes Antibody Standardization Program) have established reference sera for demonstrating GADA and IA-2A in order to standardize the quantification of antibody titers when using different assays /6/. For detection of anti-ZnT8 an optimized radioimmunoassays was developed /7/.

Refer to Tab. 25.9-1 – Relevant autoantibodies in the diagnosis of type 1 diabetes.

25.9.3 Specimen

Serum: 1 mL

25.9.4 Reference interval

See Tab. 25.9-2 – Thresholds for positive results.

25.9.5 Clinical significance

Type 1 diabetes (T1D) is usually preceded by clinical prodromes in the form of ICA and the biochemical antibodies IAA, GADA, IA-2A and anti-ZnT8.

25.9.5.1 Autoantibodies in the diagnosis of T1D

Autoantibodies are the most important markers for identifying individuals at increased risk for diabetes at an early stage when all the available metabolic tests are still normal. None of the autoantibodies is sufficiently sensitive by itself to predict T1D, since healthy individuals can also be autoantibody positive. Combining anti-ZnT8 with IAA, GADA, and IA2A assays increases the sensitivity of autoimmune detection to 96% with an about 26% reduction in the number of antibody negative results in subjects ranging in the age from 2 to 40 years /8/.

Refer to:

25.9.5.2 Seroconversion and risk of progression to T1D

The data from prospective studies undertaken in the USA (Environmental Determinants of Diabetes in the Young, TEDDY; Diabetes Autoimmunity Study in the Young, DAISY), Finland (T1D Prediction and Prevention, DIPP) and Germany (BABYDIAB and BABYDIET) /9/ were used to determine the rate of progression to T1D in high risk children (HLA-DR/DQ genotype and two or more first degree relatives with T1D). Thousands of children were tested every 1–3 years over periods of 3 and 15 years.

The results are shown in Tab. 25.9-5 – Risk of developing islet cell autoantibodies and type 1 diabetes in children and in gestational diabetes

Approximately 85% of patients with newly diagnosed T1D do not have any first degree relative with T1D and about 20% of these T1D patients have only one islet cell autoantibody. However, a single autoantibody to GAD65, insulin, IA-2 or ZnT8 are found in 1–2% of healthy individuals at low risk of progressing to T1D. Due to the low prevalence of T1D (0.3%) in the population, the positive predictive value for progression to T1D is very low in individuals with only one autoantibody /6/. The prerequisite for progression to T1D in children is a genetic risk and the presence of multiple autoantibodies /9/. This is confirmed by the results of the Diabetes Prevention Trial 1 (Tab. 25.9-6 – Progression to diabetes type 1 in dependence of autoantibodies/10/. Higher ICA titers and higher concentrations of GADA are more powerful predictors of T1D than lower titers and lower concentrations.

Some patients with newly diagnosed T1D have no autoantibodies. For example, in one study /11/, 5.8% of the patients were negative for GADA, IA-2A and IAA, and 72% were positive for two and more autoantibodies. When ZnT8A was added to the panel, the negative rate was as low as 1.8% and the positive rate improved to 82% /12/.

For screening of relatives of type 1 diabetics, the Type 1 Diabetes TrialNet Natural History Study recommends testing for GADA and IA-2A, since both assays can easily be automated. Individuals positive for these autoantibodies are then additionally tested for ICA and IAA /13/.

Autoantibodies can develop as early as in childhood. In the BABYDIAB study /14/, 11% of children of diabetic parents were positive for a single autoantibody at age 2, and 3.5% already had multiple autoantibodies. 50% of the children developed diabetes by age 5 and more than 75% by age 10 /1415/.

To identify children at increased risk for diabetes at an early stage, screening for ICA, GADA and IAA should start as early as at age 2–3 years. If only a single autoantibody is found, follow up tests should be performed annually to detect seroconversion to multiple autoantibodies. A second screening (IA-2A and GADA) is performed at about age 10. In antibody positive cases, the positive predictive value can be further increased by HLA typing and a glucose tolerance test.

25.9.5.3 Autoantibodies in the differential diagnosis of diabetes

Islet autoantibodies are important in differentiating latent autoimmune diabetes in adults (LADA) from T2D (Fig. 25.9-1 – Differentiation of diabetes mellitus in the presence of autoantibodies). Clinically, LADA is an adult onset, non insulin requiring diabetes like T2D, but in laboratory tests it behaves like T1D due to the presence of islet cell autoantibodies. LADA is characterized by positive GADA and/or ICA /16/. The presence of these antibodies indicates a lack of insulin and/or need for insulin (Section 3.1.5.3 – Latent autoimmune diabetes in adults (LADA)).

25.9.6 Comments and problems

ICA

Pancreatic tissue from different donors can vary significantly in the detection limit and specificity for ICA.

GADA

Since the available GAD65A and IA-2A ELISAs have a lower detection limit compared to RIAs, false negative results must be expected /17/. The CV of imprecision of these assays is near 5% /3/.

IAA

Due to high concentration of insulin in the patient’s blood, the competition between unlabeled and labeled insulin in the immunoassay may be impaired, resulting in less labeled insulin binding to the antibodies. A falsely low concentration of IAA is measured. Therefore, a fasting blood sample should be collected for determining IAA. The CV of imprecision of the IAA assays is 20–25% /3/.

Thresholds

The ICA test, positivity is defined as islet fluorescence at a minimum sample dilution of 2. In the biochemical antibody assays, positivity is defined as signals that exceed the 99th percentile for the general population /3/.

25.9.7 Physiology

The peptide insulin undergoes a regulated secretory pathway after synthesis /8/:

After passage through the endoplasmic reticulum and golgi apparatus, it is sorted into secretory granules for proteolytic processing, storage and subsequent release in response to appropriate stimuli

  • IA2 is integrated in the membrane of the secretory granule and plays a pivotal role in insulin secretion. IA-2 is a member of the conserved protein tyrosine phosphatase family, albeit catalytically inactive.
  • ZTn 8 functions to channel zinc into the secretory granule, where it is instrumental in the formation of insulin hexamers for crystallization
  • GAD 65 is a peripheral membrane protein associated with synaptic-like micro vesicles within the β cell and the enzyme responsible for gamma amino butyric acid synthesis.

Refer further to Section 3.7.8 – Pathophysiology of insulin.

References

1. Eisenbarth GS. Type 1 diabetes mellitus. A chronic autoimmune disease. N Engl J Med 1986; 314: 1360–8.

2. Pihoker C, Gilliam I, Hsmpe C, Lenmark A. Autoantibodies in diabetes. Diabetes 2005; 54: S52–61.

3. Winter WE, Schatz DA. Autoimmune markers in diabetes. Clin Chem 2011; 57: 168–75.

4. Calderon B, Sacks DB. Islet autoantibodies and type 1 diabetes: does the evidence support screening? Clin Chem 2014; 60: 438–40.

5. Bottazzo GF, Florin-Christensen A, Doniach D. Islet-cell antibodies in diabetes mellitus with autoimmune polyendocrine deficiencies. Lancet 1974; 2 (7892): 1279–83.

6. Calderon B, Sacks DB. Islet autoantibodies and type 1 diabetes: does the evidence support screening? Clin Chem 2014; 60: 438–40.

7. Wenzlau JM, Juhl K, Yu L, Moua O, Sarkar S, Gottlieb P, et al. The cation efflux transporter ZnT8 (Slc30A8) is a major autoantigen in human type 1 diabetes. Proc Natl Acad Sci USA 2007; 104: 17040–5.

8. Wenzlau JM, Hutton JC. Novel diabetes autoantibodies and prediction of type 1 diabetes. Curr Diab Rep 2013; 13: 608–15.

9. Ziegler AG, Rewers M, Simell O, Lempainen J, Steck A, Winkler C, et al. Seroconversion to multiple islet autoantibodies and risk of progression to diabetes in children. JAMA 2013; 309: 2473–9.

10. Orban T, Sosenko JM, Cuthbertson D, Krischer JP, Skyler JS, Jackson R, et al. Pancreatic islet antibodies as predictors of type 1 diabetes in the Diabetes Prevention Trial-Type 1. Diabetes Care 2009; 32: 2269–74.

11. Scherbaum WA, Mirakian R, Pujol-Borrell R, Dean BM, Bottazzo GF. Immunochemistry in the study and diagnosis of organ-specific autoimmune disease. In: Polak JM, van Noorden S, eds. Immunochemistry. Modern Methods and Applications. Bristol; Wright 1986: 456.

12. Wenzlau JM, Walter M, Gardner TJ, Frisch LM, Yu L, Eisenbarth GS, et al. Kinetics of the post-onset decline in zinc transporter 8 autoantibodies in type 1 diabetic human subjects. J Clin Endocrinol Metab 2010; 95: 4712–9.

13. Mahon JL, Sosenko JM, Rafkin-Mervis L, Krause-Steinrauf H, Lachim JM, Thompson C, et al. The TrialNet Natural History study on the development of type 1 diabetes: objectives, design, and initial results. Pediatr Diabetes 2009; 10: 97–104.

14. Hummel M, Bonifacio E, Schmid S, Walter M, Knopff A, Ziegler AG. Brief communication: early appearance of islet autoantibodies predicts childhood type 1 diabetes in offspring of diabetic parents. Ann Intern Med 2004; 140: 882–6.

15. Kimpimaki T, Kulmala P, Savola K, et al. Disease-associated autoantibodies as surrogate markers of type 1 diabetes in young children at increased genetic risk. Childhood Diabetes in Finland Study Group. J Clin Endocrinol Metab 2000; 85: 1126–32.

16. Leslie RD, Kolb H, Schloot NC, Buzzetti R, Mauricio D, De Leiva A, et al. Diabetes classification: gray zones, sound and smoke: action LADA 1. Diabetes Metab Res Rev 2008; 24: 511–9.

17. Herold KC, Vignali DA, Cook A, Bluestone JA. Type 1 diabetes: translating mechanistic observations into effective clinical outcomes. Nat Rev Immunol 2013; 13: 243–56.

18. Grönholm J, Lenardo MJ. Novel diagnostic and therapeutic approaches for autoimmune diabetes – a prime time to treat insulitis as a disease. Clin Immunol 2015; 156: 109–18.

19. Seissler J, Scherbaum WA. Autoimmune diagnostics in diabetes mellitus. Clin Chem Lab Med 2006; 44: 133–7.

20. Chimienti F, Devergnas S, Pattou F, Schuit F, Garcia-Cuenca R, Vandewalle B, et al. In vivo expression and functional characterization of the zinc transporter ZnT8 in glucose-induced insulin secretion. J Cell Science 2006; 119: 4199–206.

21. Krischer JP, Lynch KF, Schatz DA, Ilonen J, Lenmark A, Hagopian WA, et al. The 6 year incidence of diabetes associated autoantibodies in genetically at-risk children: the Teddy study. Diabetologia 2015; 58: 980–7.

22. Bonifacio E, Beyerlein A, Hippich M, Winkler C, Vehik K, Weedon MN, et al. Genetic scores to stratify risk of developing multiple islet autoantibodies and type 1 diabetes: a prospective study in children. PLOS Medicine 2018; https://doi.org//10.1371/journal.pmed.1002548.

23. Incani M, Baroni MG, Cossu E. Testing for type 1 diabetes autoantibodies in gestational diabetes mellitus (GDM): is it clinically useful? BMC Endocrine Disorders 2019; 19: 44; https://doi.org/10.1186/s12902-019-0373-4.

25.10 Antibodies in gastrointestinal diseases

Wilma Höchtlen-Vollmar, Lothar Thomas, Rudolf Gruber

Autoimmune diseases currently affect up to 9% of the population and occur significantly more often in females. Circulating antibodies are detected in a broad spectrum of gastrointestinal diseases (Tab. 25.10.1 – Diagnostic antibodies in gastrointestinal diseases).

25.10.1 Chronic atrophic gastritis

Chronic atrophic gastritis is a special form of chronic gastric disease and characterized by /1/:

  • The presence of autoantibodies targeting parietal cells and/or intrinsic factor
  • Hypochlorhydria or achlorhydria and hyper gastrinemia
  • Iron deficiency in 20–40% of patients
  • Vitamin B12 deficiency, neurological disorders and macrocytic, hyperchromic anemia in relation to the duration of the deficiency in 15–20% of patients
  • Carcinoid tumors or gastric adenocarcinoma in up to 10% of patients
  • Association with autoimmune endocrinopathies such as type 1 diabetes, Hashimoto’s disease, Graves’ disease. The prevalence of these endocrinopathies is increased 3 to 5-fold.

Helicobacter pylori is the main etiologic factor for chronic gastritis worldwide. The degree of inflammation and the evolution of this form of chronic gastritis can vary largely depending on virulence factors, host susceptibility factors and environmental conditions /2/.

Autoimmune atrophic gastritis is a special form of chronic atrophic gastritis with inflammation of the gastric mucosa, that may progress to pernicious anemia. It is characterized by damage to the gastric corpus and fundus. The mucosa shows lymphocytic infiltration and loss of main cells and parietal cells. Auto immune chronic atrophic gastritis is characterized by the presence of autoantibodies in serum against H+-K+ adenosin triphosphatase (H+-K+-ATPase) the gastric proton pump located on parietal cells. In addition, the parietal cells produce intrinsic factor, which binds vitamin B12, allowing the vitamin to be reabsorbed in the ileum. Refer to Section 13.3 – Vitamin B12.

Autoimmune chronic atrophic gastritis can be found in up to 2% of the population /3/. Due to the frequent asymptomatic nature, the prevalence of autoimmune chronic atrophic gastritis is probably underestimated. In a study /4/ including 9,684 subjects aged 50–74 years the presence of anti-parietal cell antibodies was approximately 20%.

25.10.1.1 Anti-parietal cell antibodies (APCA)

The relatively large parietal cells reside in the gastric mucosa between the main cells. The target for APCA is the gastric H+ pump (H+-K+-ATPase), which is composed of four subunits (two alpha of 100 kDa and two beta of 60–90 kDa transmembrane proteins) that reside in the membrane of the intracellular secretory channels of parietal cells. The H+ pump functions to produce HCl by exchanging cytoplasmic H+ for extracellular K+. APCA target the highly glycosylated β-subunit as well as the α-subunit of the H+-K+-ATPase. The autoimmune reaction mediated mainly by CD4+T cells reactive to the gastric H+ pump and stimulated by ACPA leads to the destruction of parietal cells in the stomach /5/.

25.10.1.1.1 Indication

Screening for autoimmune atrophic gastritis

Megaloblastic anemia

Neurologic disorders (numbness, paresthesia, weakness ataxia)

25.10.1.1.2 Method of determination

Indirect immunofluorescence test (IIFT)

Frozen sections of rat or monkey stomach are used. APCA produce a coarse, reticular cytoplasmic fluorescence pattern of the parietal cells, while anti-mitochondrial antibodies (AMA) produce a more uniform, finer staining pattern. An exact differentiation is possible by preincubation with a glycine urea buffer solution, which weakens the AMA reactivities. Titers > 1 : 10 are pathologic. APCA belong to the IgG class.

Reactivities to rodent stomach (of the rat, less pronounced with mouse stomach), may give false positive reactions for APCA in the presence of AMA.

Enzyme immunoassay

Semi quantitative or quantitative detection of IgG APCA using highly purified H+-K+-ATPase from gastric mucosa from monkeys or recombinant H+-K+-ATPase. ELISAs show a more sensitive detection limit. They should be applied for early detection of APCA /3/.

25.10.1.1.3 Specimen

Serum, plasma: 1 mL

25.10.1.1.4 Reference interval

Threshold value of the test kit manufacturer

25.10.1.1.5 Clinical significance

The APCA prevalence of the population in Germany is 19.5%. ACPA prevalence is strongly associated with chronic atrophic gastritis (CAG), and the association is increasing with increasing severity of CAG. Furthermore, the association between APCA and CAG is even stronger among Helicobacter pylori negative subjects (Odds ratio 11.3) than among Helicobacter pylori positive subjects (Odds ratio 2.6) /4/. ACPA are found in 90% of patients with CAG with or without pernicious anemia, but also in up to 12% of healthy individuals without CAG or pernicious anemia. The prevalence in healthy individuals increases with age, from 2.5% in the 3rd decade to 12% in the 8th decade of life. APCA are also detected in younger patients with autoimmune gastritis, but not as commonly as antibodies to intrinsic factor.

APCA are more prevalent in patients with extra gastric autoimmune diseases. In type 1 diabetes, 10–15% of children and 15–25% of adults have APCA. Approximately 22% of patients with Graves’ disease and 32–40% of patients with Hashimoto’s disease are positive for APCA. The antibodies are also found in 20–30% of relatives of patients with CAG, which indicates a genetic disposition /5/.

The presence of APCA predicts the development of CAG and pernicious anemia. In patients with autoimmune thyroid disease who developed CAG, the APCA levels were found to rise progressively, reach a peak level and then fall again according to the progressive destruction of the parietal cells and disappearance of the target antigen /6/. In a study /7/ it was concluded that approximately one-third of patients with recurrent aphthous stomatitis have positive ACPA. These patients should be examined for pernicious anemia, CAG, and hypothyroidism or hyperthyroidism.

25.10.1.1.6 Comments and problems

Titre

In patients with severe autoimmune gastritis the disappearance of the antigen source due to advanced mucosa atrophy results in decrease of the ACPA titre.

Stability

Approximately 14 days at 4 °C.

25.10.1.2 Intrinsic factor antibodies

Intrinsic factor antibodies (IFA) are considered to be specific antibodies in chronic atrophic gastritis (CAG). Intrinsic factor (IF) is a 57 kDa glycoprotein which is resistant to proteolysis. It binds vitamin B12, and the IF-vitamin B12 complex then migrates to the distal ileum where it is absorbed by specific receptors.

Refer to Fig. 13.3-4 – Transport and cellular uptake of vitamin B12.

Two types of autoantibodies to IF are present in the serum of patients with CAG:

  • Type 1 antibodies bind to the vitamin B12 binding site of IF and prevent the binding of vitamin B12 (blocking antibodies)
  • Type 2 antibodies bind to IF or the vitamin B12-IF complex without displacing or preventing the binding of vitamin B12 (binding antibodies).

However, both antibody types have the same pathogenic effect: they prevent the reabsorption of vitamin B12 in the ileum.

25.10.1.2.1 Indication

Patients with macrocytic anemia being ACPA positive.

25.10.1.2.2 Method of determination

Radioimmunoassay with 57Co-labeled vitamin B12 for determination type 1 IFA

IF is bound to a solid phase (e.g., the wall of a tube). The tube is first incubated with patient serum and then 57Co-labeled vitamin B12 tracer is added. Type 1 IFA prevent the binding of the tracer to the IF, resulting in less radioactivity being bound compared to normal serum. The lower the amount of bound radioactivity, the higher the concentration of type 1 IFA in the patient sample.

Radio immune precipitation with complexes of IF and 57Co-labeled vitamin B12 for demonstrating type 2 IFA

Patient serum is incubated with radioactively labeled IF-B12 complex, leading to the formation of immune complexes. The amount of radioactivity is measured in the precipitate. The higher the type 2 IFA concentration in the patient serum, the more radioactivity is measured in the precipitate.

Immunoassays (especially ELISA)

Quantitative detection of IgG class IFA using highly purified IF from the gastric mucosa of pigs or recombinant human intrinsic factor antigen. Type 1 and type 2 IFA cannot be differentiated /8/.

25.10.1.2.3 Specimen

Serum, plasma: 1 mL

25.10.1.2.4 Reference interval

Reference interval values of the test kit manufacturer.

25.10.1.2.5 Clinical significance

IFA are highly specific for CAG and the associated vitamin B12 deficiency syndromes (Tab. 25.10-2 – Vitamin B12 level and proportion of patients positive for intrinsic factor antibodies).

IFA are detected in 40–70% of CAG cases, with diagnostic sensitivity depending on the assay used. Type 1 IFA can be found in 70% of patients with pernicious anemia, type 2 IFA in 35–40% of cases, often together with anti-type 1 antibodies. IFA and APCA complement each other in terms of their diagnostic sensitivity. Approximately 96% of patients with pernicious anemia are positive for at least one antibody and should therefore always be tested for both autoantibodies /9/.

25.10.1.2.6 Comments and problems

Method of determination

Since radioactive isotopes are no longer routinely used and differentiating between type 1 and type 2 IFA is of little diagnostic value, IFA are usually measured with enzyme immunoassays. Another disadvantage of competitive radio immunoassays is that therapeutically administered vitamin B12 can lead to false positive results.

Stability

Approximately 14 days at 4 °C.

25.10.2 Inflammatory bowel disease

Inflammatory bowel disease (IBD) is a chronic relapsing disorder of the gastrointestinal tract accompanied by abdominal pain, rectal bleeding and malabsorption. IBD manifests between adolescence and the third decade of life with about 10% of cases in persons younger than 18 years. The diagnosis is based upon clinical findings, endoscopic, radiological, histological and laboratory investigations /10/.

IBD comprises the following major entities:

  • Ulcerative colitis
  • Crohn’s disease.

25.10.2.1 Ulcerative Colitis

Ulcerative colitis is a chronic idiopathic inflammatory disease that affects the colon. It is characterized by relapsing and remitting mucosal inflammation, starting in the rectum and extending to proximal segments of the colon. The peak age of disease onset is between 30 and 40 years, no sex predominance exists. The incidence is 24.3 per 100.000 and the prevalence rate 505 per 100.000 in Northern Europe. In North America the prevalence is 214 per 100,000. Approximately 8–14% of patients with ulcerative colitis have a family history of inflammatory bowel disease and first-degree relatives have four times the risk of developing the disease /15/.

Clinical symptoms include urgency, incontinence, fatigue, increased movements, mucus discharge, nocturnal discharge and cramps.

The diagnosis of ulcerative colitis is based on the combination of symptoms, endoscopic findings, histology and the absence of alternative diagnosis.

Laboratory findings

Laboratory investigations are ordered for /15/:

  • Fecal calprotectin, which appears to have the highest sensitivity and specificity for active inflammation
  • Stool culture to exclude Clostridium difficile to rule out enteric superimposed infection
  • Iron deficiency in anemia of chronic disease (ferritin, transferrin saturation, soluble transferrin receptor)
  • Hypoalbuminemia in severe disease
  • CRP and erythrocyte sedimentation rate as markers of inflammation
  • Perinuclear anti-neutrophil cytoplasmic antibodies (p-ANCA). They can be elevated, but are non-specific and have a low sensitivity.

Endoscopy with biopsies is the only way to establish the diagnosis of ulcerative colitis.

25.10.2.2 Crohn’s disease

Crohn’s disease is a chronic, destructive, inflammatory disease of the intestines of unknown cause. In contrast to ulcerative colitis patients with Crohn’s disease have mucosal inflammation affecting both colon and small bowel /16/.

25.10.3 Celiac disease

Celiac disease (CD) is a life-long gluten-sensitive immune mediated systemic disorder /11/. The classical disease includes gastrointestinal-related symptoms such as diarrhea, steatorrhea and weight loss due to malabsorption. The CD is one of the most common chronic diseases affecting 0.5–1% of the population. Patients with diabetes, autoimmune disorders or relatives of CD patients have a higher risk for the development of CD.

About 50% of CD patients present extra intestinal atypical symptoms such as anemia, osteoporosis, dermatitis herpetiformis, neurological problems and dental enamel hypoplasia. Positive anti-tissue trans glutaminase antibody or anti-endomysium antibody helps to confirm the diagnosis.

In patients with CD 95% have HLA-DQ2 and 5–10% have HLA-DQ8 /12/. However, 95–98% of persons who carry alleles that encode either HLA-DQ2 or HLA-DQ8 alleles do not develop CD.

The pathophysiology of CD is complex and involves both environmental (gluten) and genetic factors (human leukocyte antigen; HLA gene). It is suggested that HLA-DQ2 and HLA-DQ8 proteins activate T cells by presenting a peptide fragment derived from gluten. This fragment is created by the enzymatic activity of trans glutaminase 2 /13/. But the ingredients must be incomplete and a missing ingredient could be the intestinal effect of enteral Reovirus infection /14/.

CD usually regresses after a gluten-free diet and is triggered again on exposure to gluten.

CD is associated with histological abnormalities, which require investigation by small bowel biopsy. The histological findings are staged according to the Marsh-Oberhuber classification, where stage III of advanced celiac disease is characterized by crypt hyperplasia and villous atrophy in addition to lymphocytic infiltration of the epithelium. These histological characteristics are not specific to CD. They are also seen in cow milk allergy, advanced lambliasis and tropic sprue. In addition, the distribution pattern of histological abnormalities is inconsistent /33/.

The abnormalities of CD result in a reduced absorptive surface area (malabsorption), secondary disaccharide deficiency (lactose intolerance) and secondary pancreas insufficiency with steatorrhea. Classic sprue, which clinically presents with the typical signs of malabsorption syndrome (e.g., significant weight loss, muscle wasting and protein deficiency edemas) is rare, whereas mono- and oligo symptomatic forms of the disease are common.

In young children, CD mainly presents with abdominal symptoms such as diarrhea as well as failure to thrive and poor growth, while in adults it often manifests not only with abdominal symptoms, but also with anemia (iron, B12 and folic acid deficiencies), osteoporosis (vitamin D and calcium deficiencies) or neuropsychiatric symptoms (thiamine, vitamin B12, calcium and magnesium deficiencies). There are also atypical forms of CD, which are not due to malabsorption. These include dermatitis herpetiformis (Duhring’s disease), gluten ataxia, arthropathies and endocrine disorders (e.g., delayed puberty, amenorrhea, infertility). In addition, it must be taken into account that aminotransferases and pancreatic enzymes may be elevated in CD /34/.

Research based on antibody screening has revealed that CD affects between 0.5 to 1% of the population, whereas the prevalence of clinically diagnosed CD is 0.05% in Germany. The first peak of incidence occurs between the ages of 9 months and 2 years (i.e., 3–6 months after having been on a gluten-containing diet); a second peak of incidence occurs in the fourth decade.

Laboratory testing for CD is recommended not only for the diagnosis of suspected CD, but also for autoimmune and genetic disorders associated with CD as well as for relatives of patients with CD (Tab. 25.10-3 – Diseases with increased prevalence of celiac disease and genetic predisposition).

25.10.4 Autoimmune pancreatitis

Autoimmune pancreatitis (AIP) is a unique disease within the spectrum of inflammatory disorders of the pancreas, frequently associated with other autoimmune diseases, presence of autoantibodies, and the response to steroid treatment. Elevated IgG4 levels are regarded as the most sensitive serum parameter for diagnosing AIP. Circulating antibodies to AIP include autoantibodies against carbo anhydrase II, lactoferrin, ANA and ASMA /17/.

Clinically, patients with AIP suffer from abdominal pain that may be recurrent or last for several months, as well as by signs of obstructive jaundice. Like IgG4 cholangiopathy, AIP is an IgG4 associated sclerosing disease, with the two diseases often occurring together.

Imaging shows narrowing of the pancreatic and bile ducts as well as parenchymal swelling, similar to the features seen in pancreatic ductal adenocarcinoma. AIP responds well to steroid treatment, which helps differentiate it from pancreatic cancer. The main histopathological feature is a lymphoplasmocytic infiltration with IgG4 positive plasma cells /18/.

25.10.5 Antibodies in inflammatory bowel disease

Antibodies related to inflammatory bowel disease encompass antibodies targeting microbial antigens and autoantibodies.

Refer to Tab. 25.10-4 – Serologic markers of inflammatory bowel disease.

The usefulness of antibodies in the investigation of inflammatory bowel disease (IBD) is as follows /5/:

  • Distinguishing IBD from other diseases with groups of non-IBT gastrointestinal disorders (e.g., irritable bowel syndrome, functional disorders, and infectious enteritis)
  • Differentiation of IBD subtypes (e.g., Crohn’s disease from ulcerative colitis and celiac disease)
  • Evaluation of the prognostic value in stratifying disease phenotypes
  • Monitoring disease activity and reflecting the response to therapy.

Although the diagnosis of IBD can be suspected on clinical grounds a positive antibody test result only modestly influences the pretest/post test probability in distinguishing IBD from other intestinal disorders with similar clinical presentation. Although IBD associated antibodies have a diagnostic sensitivity of 75 to 99% caution should be taken in considering the non-IBD gastrointestinal disorders. A negative test result has no clinical value /5/.

Pediatric patients with abdominal complaints are a particular population in whom non-invasive testing is desirable, especially in celiac disease for diagnostic and follow up purposes /19/.

Combined instead of individual testing of antibodies is more useful in differentiation ulcerative colitis versus Crohn’s disease. The profile ASCA positive/pANCA negative increases specificity and positive predictive value for diagnosis of Crohn’s disease in comparison to ASCA positive as an isolated result. The reverse result profile has a higher specificity and positive predictive value for diagnosis of ulcerative colitis than positive pANCA alone. Overall, ASCA have the best combined sensitivity and specificity for Crohn’s disease and pANCA for ulcerative colitis /5/.

There is no use of serial measurement of IBD serological markers in monitoring disease activity /5/.

Circulating autoantibodies in ulcerative colitis and Crohn’s disease include:

  • Pancreatic acinar cell surface antibodies
  • Anti-saccharomyces cerevisiae antibodies (ASCA)
  • Goblet cell antibodies
  • Atypical ANCA

25.10.5.1 Antibodies against exocrine pancreas

Antibodies against exocrine pancreas (PABA) target the exocrine pancreas. The main antigens are zona pellucida like domain containing protein 1 (CUZD 1) and glycoprotein 2 (GP 2). CUZD 1 is a protein that is expressed by the pancreatic epithelium and plays a role in cell-cell interactions. GP 2 is a 78 kDa protein found in the zymogen granules of the pancreatic cells and, following its release from the granules, in the excretory ducts. Patients with Crohn’s disease express CUZD 1 and GP 2 in the enterocytes and Peyer’s plaques /20/.

Method of determination

Indirect immunofluorescence test (IIFT)

The assay uses cryocut sections of monkey pancreas. The following antibodies can be detected:

  • Subtype 1 PABA (anti-CUZD 1), which produce a reticular to granular cytoplasmic fluorescence pattern of the pancreatic acinar cells
  • Subtype 2 PABA (anti-GP 2), which produce a drop like fluorescence pattern in the acinar lumen.

Immunoassay

Determination of anti-GP 2 Ab and anti-CUZD 1 Ab. Recombinant CUZD 1 and GP 2 are used as antigens.

Specimen

Serum, plasma: 1 mL

Clinical significance

PABA are present in 39% of patients with Crohn’s disease and rarely in other diseases (e.g. in 2% of patients with ulcerative colitis). They are usually of the IgG class, sometimes of the IgA class. PABA titers of 1 : 32 and higher are highly specific for Crohn’s disease /21/.

Stability

Approximately 14 days at 4 °C.

25.10.5.2 Anti-Saccharomyces cerevisiae antibodies (ASCA)

Oligomannans are part of Saccharomyces cerevisiae (brewer’s yeast) and other microorganisms. It is therefore not clear whether ASCA are actually directed against Saccharomyces cerevisiae or whether there just is cross reactivity with unknown other microorganisms with similar mannose structures. It is believed that due to increased permeability of the intestinal mucosa in patients with Crohn’s disease, there is excessive antigen exposure to the immune system /22/. Patients with Crohn’s disease are therefore more often positive for antibodies to antigens of microorganisms than healthy individuals. For example, patients with Crohn’s disease were found to have high levels of antibodies to OmpC from E. coli, to I 2 from Pseudomonas aeruginosa and to CBir1 flagellin from commensal bacteria. The number and concentration of antibodies detected correlate with the severity of disease /23/.

Method of determination

Immunoassay

The assay uses purified mannan from the cell wall of Saccharomyces cerevisiae as antigen. IgG and IgA antibodies are determined.

IIFT

Smears of brewer’s yeast are used as an antigen.

Specimen

Serum, plasma 1 mL

Clinical significance

The ASCA tests are not standardized. Since studies were performed with various commercial and in house tests, data on the diagnostic sensitivity and specificity for ASCA in Crohn’s disease vary significantly.

ASCA can be found in 39–72% of patients with Crohn’s disease, in 5–15% of patients with ulcerative colitis, in up to 3% of healthy controls and in up to 11% of patients with other diseases /24/. The diagnostic sensitivity ranges from 39% to 61% with a specificity of 86–95% and a positive predictive value of 54–92% /25/. Determining IgA antibodies is more important than determining IgG antibodies. ASCA are found in 20–25% of first degree relatives of patients with Crohn’s disease and are thought to have predictive value. There is no correlation between the serum level of ASCA and disease activity /5/.

Stability

Approximately 14 days at 4 °C.

25.10.5.3 Goblet cell antibodies

Goblet cell antibodies (GABA) are mucin producing, goblet shaped cells that reside throughout the length of the intestine. Their main localization (colon and rectum) correlates with the site of disease manifestation. The mucins are the target antigens /526/.

Specimen

Serum, plasma: 1 mL

Method of determination

IIF on human fetal intestinal tissue which has not previously been exposed to bacteria or other antigens, or on goblet cells from human enterocyte cultures. Rodent tissue shows non specific fluorescence. GABA produce a drop like and/or cloud like fluorescence pattern of the goblet cells. GABA are IgA and/or IgG, with IgA class antibodies prevailing. Titers range between 1 : 10 and 1 : 100.

Clinical significance

GABA are thought to be specific for ulcerative colitis, although their prevalence is low. They are present in only 15–28% of patients. GABA also occur in first degree relatives of patients with ulcerative colitis.

Stability

Approximately 14 days at 4 °C.

25.10.5.4 Atypical anti neutrophil cytoplasmic antibodies (a-ANCA)

ANCA found in inflammatory bowel disease are usually atypical p-ANCA and less frequently atypical c-ANCA. As described in Section 25.8.2 – ANCA associated vasculitides and pulmonary renal syndromes, atypical ANCA must be distinguished from typical ANCA according to the consensus statement. While typical p-ANCA are characterized by a perinuclear fluorescent staining pattern with nuclear extension on ethanol-fixed neutrophils, atypical p-ANCA give a broad, non homogenous rim like staining of the nuclear periphery. The atypical c-ANCA produce a cytoplasmic staining pattern without interlobular accentuation.

Atypical p-ANCA and c-ANCA do not usually result in cytoplasmic fluorescence on formalin stained neutrophils. Approximately 95% of sera from vasculitis patients, in whom typical p-ANCA are expected, show a granular cytoplasmic fluorescent pattern on formalin stained neutrophils, while 96% of serum samples from patients with ulcerative colitis do not show any reactivity on formalin stained neutrophils /27/.

It was shown that p-ANCA, which are associated with chronic inflammatory bowel diseases (ulcerative colitis 67%, Crohn’s disease 7%, healthy individuals less than 2%) or primary sclerosing cholangitis (70%), target DNA complexed granulocyte antigens, which were identified as lactoferrin and azurocidin /28/. Human β-tubulin isotype 5 and the microbial protein FtsZ have also been described as target antigens of these atypical p-ANCA /29/.

Specimen

Serum, plasma: 1 mL

Method of determination

IIFT on human ethanol and formalin stained neutrophils. Initial dilution 1 : 10, IgG conjugate. Testing of DNA associated ANCA on ethanol-fixed neutrophils treated with 1 mol/L of MgSO4 and reconstituted with lactoferrin.

Clinical significance

Due to ethnic variations and non standardized assays, the reported diagnostic sensitivity for ulcerative colitis varies from 50–67% with a specificity of 65–92% and a positive predictive value of 54–93%. Approximately 6–16% of patients with Crohn’s disease are positive for atypical ANCA (a-ANCA) /24/. The diagnostic specificity of a-ANCA is low, since the antibodies are also present in primary sclerosing cholangitis and autoimmune hepatitis as well as in up to 3% of healthy controls.

Discrepancies must be expected, especially in the evaluation of a-ANCA, depending on the substrate used and the observer’s experience and bias. For example, the inter observer agreement rate for a-ANCA on formalin stained neutrophils was only 74.1% /30/.

Approximately 55% of previously ANCA negative sera from ulcerative colitis patients, 20% of those from PSC patients, but only 1% of those from Crohn’s disease patients tested ANCA positive on ethanol stained neutrophils reconstituted with lactoferrin. This substrate thus increased the diagnostic sensitivity of the a-ANCA from 72% to 87% for ulcerative colitis and from 42% to 54% for PSC /28/.

The immunoassays measuring antibodies to lactoferrin, BPI, elastase and cathepsin G are positive only in a small proportion of patients with inflammatory bowel disease, the prevalence of anti-lactoferrin being 10% vs. 5% and that of anti-BPI 10% vs. 7% in ulcerative colitis and Crohn’s disease, respectively. Some patients with inflammatory bowel disease (IBD), especially ulcerative colitis, also have low concentrations of antibodies to proteinase 3 and myeloperoxidase /28/.

The combination of ASCA and a-ANCA is useful in differentiating ulcerative colitis from Crohn’s disease, especially in patients with indeterminate colitis, which are reported to initially account for 5–23% of IBD cases and have an incidence of 2.4 per 100,000 population per year. Up to 80% of these patients are later classified as having ulcerative colitis or Crohn’s disease.

Studies showed that the combination of /3132/:

  • ANCA positive and ASCA negative predicts ulcerative colitis with a diagnostic sensitivity of 30–56% at a specificity of 94–97% and with a positive predictive value of 77–96%.
  • ANCA negative and ASCA positive predicts Crohn’s disease with a diagnostic sensitivity of 44–58% at a specificity of 81–97% and with a positive predictive value of 75–93%.

In patients with indeterminate colitis who were able to be diagnosed definitely 1 year later /3132/:

  • The combination of ANCA positive and ASCA negative predicts ulcerative colitis in 64% of cases.
  • The combination von ANCA negative and ASCA positive predicts Crohn’s disease in 80% of cases.

However, since 48.5% of patients with indeterminate colitis were negative for ANCA and ASCA, the utility of combined autoantibody testing in these patients is doubtful /32/.

Stability

Approximately 14 days at 4 °C.

25.10.6 Celiac disease specific antibodies

The following antibodies have high diagnostic sensitivity and specificity for celiac disease detection:

  • Tissue trans glutaminase antibodies (tTGA )
  • Endomysial antibodies (EMA)
  • Antibodies to deamidated gliadin and gliadin peptides (DGPA).

25.10.6.1 Tissue trans glutaminase antibodies

Tissue trans glutaminase (tTG) is a Ca2+-dependent cytoplasmic enzyme that is present in all tissues, especially in the intestine, and is released by damaged cells. The main function of tTG is the irreversible cross-linking of glutamine side chains with lysin. In the absence of lysine residues, glutamine side chains are de aminated (i.e., by gliadin) producing glutamic acid. The de amidation of gliadin changes the charge, enabling a more effective presentation via the MHC complex, which results in an increased T cell response in celiac patients /36/. It is also thought that de amidated gluten peptides cross-link with tTG, inducing an autoimmune response to tTG or neoantigens /37/.

Three isoenzymes of tTG with corresponding sequence homologies play a role in the pathogenesis of gluten sensitive disease:

  • Tissue trans glutaminase (tTG1 or tTG2) in the pathogenesis of celiac disease
  • Epidermal trans glutaminase (eTG or eTG3) in the pathogenesis of dermatitis herpetiformis
  • Neuronal trans glutaminase (TG6) in the pathogenesis of gluten ataxia /37/.

Method of determination

Immunoassay using recombinant or purified human tTG as an antigen. IgA- and IgG-class antibodies are determined. Most commercial kits use human tTG as an antigen. Guinea pig tTG should no longer be used, as it leads to lower diagnostic sensitivity and specificity /37/. The result should be quantitative for monitoring. Due to the lack of an international standard, results are reported in relative units.

Specimen

Serum, plasma 1 mL

Clinical significance

In a comparison between 20 laboratories the diagnostic sensitivity of assays detecting anti-tTG antibodies (tTGA) of the IgA type (tTGA IgA) ranged from 96% to 100% /38/. In a review on tTGA IgA, the mean diagnostic sensitivity was 93.8% at a mean specificity of 98.7%. The diagnostic sensitivity tTGA IgA was 93% in children under the age of 2 with celiac disease and 99% in children older than 2 years /39/.

Various authors have reported low tTGA IgA in patients with other autoimmune diseases and transient low tTGA IgA in children with infections (EBV) that were neither associated with celiac disease nor with the presence of anti-endomysial antibodies or HLA-DQ2 and/or HLA-DQ8 positivity /4041/. At high serum IgA levels, unspecific elevations of tTGA IgA with low antibody titers are possible /42/.

High concentrations of tTGA IgA correlate with the severity of histological abnormalities /34/. tTGA IgA levels greater than 10 times the assay manufacturer’s diagnostic threshold are indicative of celiac disease and associated with corresponding characteristic histological abnormalities /42/. If patients adhere to a gluten-free diet, tTGA IgA levels should begin to decrease within 9 to 12 months /43/ and histological abnormalities regress. tTGA IgA have predictive value, as they may be present years before celiac disease manifests clinically.

IgG-class tTGA (tTGA IgG) have a markedly lower diagnostic sensitivity than tTGA IgA in patients without IgA deficiency. The diagnostic relevance of isolated tTGA IgG in patients without IgA deficiency is unclear. tTGA IgG are relevant in patients with IgA deficiency, where their diagnostic sensitivity is 82% /4041/. Refer to:

tTGA IgA and tTGA IgG can also be detected in saliva or stool, but are less sensitive compared to antibodies in blood /43/.

Significant levels of tTGA are also found in dermatitis herpetiformis. There are high sequence homologies between epidermal tTG and tissue tTG.

Stability

Approximately 14 days at 4 °C.

25.10.6.2 Endomysial antibodies (EMA)

The first diagnostic screening test for celiac disease was the detection of antibodies to endomysium with tTG as the target antigen /35/. The endomysium is a thin, delicate layer of connective tissue that surrounds individual muscle fibers. Autoantibodies to endomysium can be visualized with many types of tissues.

Method of determination

IIFT using frozen sections of monkey liver or frozen sections of monkey esophagus or human umbilical cord tissue as a substrate. IgA and IgG class antibodies are determined. Titers > 1 : 10 are pathologic. The frozen sections of monkey liver show fluorescent staining of the filamentous linings of the intralobular sinusoids; on the esophagus sections, the connective tissue layer between the mucosa and the smooth muscle cells of the muscularis mucosae is stained.

Specimen

Serum, plasma. 1 mL

Clinical significance

A meta analysis demonstrated that EMA on monkey esophagus had a diagnostic sensitivity of 93% and specificity of 99% for the diagnosis of celiac disease /37/. In a comparison of 18 studies in which tTGA IgA and EMA IgA were determined simultaneously, tTGA IgA proved to be more sensitive in 8 studies (44%) and EMA IgA in 10 studies; 5 studies showed equal sensitivities and 4 studies equal specificities of the assays /35/. It must be taken into account that the EMA test is an IIFT in which the results are subject to variability dependent on the observer and his/her level of experience. Moreover, as a semi quantitative method, the EMA assay is not equally suited for monitoring. However, the evaluation of the ESPGHAN celiac guidelines has shown that a positive EMA titer improves the positive predictive value of tTGA IgA screen by ELISA /44/.

Like tTGA IgA, EMA IgA correlate well with the extent of villous atrophy of the intestinal mucosa and decline on a gluten free diet. Low titers of EMA can indicate the development of celiac disease, even before the intestinal mucosa shows any histological abnormalities. EMA IgG are less specific than EMA IgA. EMA IgG can be found in 76% of patients with celiac disease and selective IgA deficiency /45/.

EMA are also present in dermatitis herpetiformis. They have a diagnostic sensitivity of 65% to 78%, or even 100% in the setting of severe villous atrophy /46/.

Stability

Approximately 14 days at 4 °C.

25.10.6.3 Antibodies to deamidated gliadins and gliadin peptides (anti-DGPA)

Glutens are storage proteins (cohesins) found in various types of cereals. In wheat they are called gliadins (prolamine fraction). This comprises 50 different proteins with a molecular weight of 16–40 kDa. The gliadins are divided into α-, β-, γ- and ω-gliadins, based on their electrophoretic mobility. Previous assays used to detect antibodies to native gliadin mostly detected antibodies of the IgG class, which also occur in healthy individuals and patients without celiac disease. A critical factor for immunogenicity is the deamidation of the gliadins by trans glutaminase and their presentation via the MHC complex. The assays detect antibodies to deamidated gliadin peptides that are specific to celiac disease.

Method of determination

Enzyme immunoassay with deamidated synthetic gliadin peptides as antigens; IgG and IgA antibodies are detected.

Specimen

Serum, plasma: 1 mL

Clinical significance

DGPA have a higher diagnostic sensitivity and specificity than antibodies to native gliadin. The latter are therefore no longer recommended for the primary detection of gluten sensitive disease. DGPA IgG have a higher specificity than DGPA IgA. In a comparison of four assays from different manufacturers, the diagnostic sensitivity of DPGA IgG varied between 76.7% and 86% and the specificity between 97.3% and 99.2%, with sensitivity correlating inversely with specificity /46/. However, DPGA IgA showed a lower diagnostic sensitivity in comparison to DPGA IgG using assays of three manufacturers /47/. The higher the antibody concentrations of DPGA IgG and DPGA IgA, the more likely the presence of celiac disease. IgA antibodies are less predictive than IgG antibodies /48/.

DGPA IgG have a diagnostic sensitivity of 88.2% in celiac patients with IgA deficiency, which is comparable with the prevalence in patients without IgA deficiency. DGPA IgG should therefore be preferred over tTGA IgG in the diagnostic testing of patients with IgA deficiency /45/. Refer to:

There is general consensus that both DGPA IgG and tTGA IgA can be used for monitoring gluten free diet adherence. While some authors consider celiac specific antibodies of the IgG and IgA class to be equally suited /3949/, others prefer IgA antibodies for monitoring /49/. It must be taken into account that antibody concentrations will slowly decrease, regardless of the assay method. The tests should therefore be repeated at 3 to 6 months intervals.

Many studies have investigated the suitability of different antibodies and antibody combinations for the diagnosis of celiac disease. In particular, the studies compared DGPA IgG and tTGA IgA, as both assays have a high sensitivity and specificity.

Stability

Approximately 14 days at 4 °C.

25.10.6.4 Antibody tests indicated in celiac disease

Most studies have found tTGA IgA to be the antibodies with the highest prevalence in celiac disease (CD), although in children aged under 2 years DGPA IgG are reported to be equally prevalent. In one study, the diagnostic sensitivity of DGPA IgG was 96.4% in children under the age of 2 without IgA deficiency and corresponded to the sensitivity of tTGA IgA, whereas only 62.5% of children over the age of 10 were positive for DGPA IgG and 81.3% were positive for tTGA IgA /41/. Combined tTGA IgA and DGPA IgG testing also detects all patients with IgA deficiency. In addition, various studies report a higher diagnostic sensitivity for this combination, especially in young children /48/.

In a meta analysis of patients with villous atrophy on gluten free diets and biopsy confirmed CD tests for serum tTD IgA and EMA IgA had a diagnostic sensitivity below 50% /50/.

European Society for Paediatric Gastroenterology Hepatology and Nutrition (ESPGHAN) criteria

The ESPGHAN criteria /51/ contain recommendations on the diagnosis of CD in children and adolescents. According to the recommendations no small bowel biopsy is necessary in symptomatic children who have tTGA IgA titers greater than 10 times the upper reference interval value and also fulfill the following criteria: verification of tTGA IgA positivity by endomysial antibodies and HLA-DQ2/8 positivity.

Refer to Fig. 25.10-1 – Recommendations of the ESPGHAN on the diagnosis of celiac disease in children.

HLA analysis is indicated in at-risk groups (e.g., individuals with a familial predisposition or conditions highly associated with CD) /51/. Individuals who are negative for HLA-DQ2/8 do not require further testing for celiac specific antibodies.

Refer to Fig. 25.10-2 – Recommendations of the ESPGHAN on the diagnosis of celiac disease in high risk populations.

Diagnostic approach in symptomatic children

Determination of tTGA IgA and total IgA (alternatively determination of tTGA IgA and DGPA IgG) /4451/:

  • Patients who are negative for tTG IgA and whose serum IgA level is within the reference range are unlikely to have CD
  • Patients with positive tTG IgA levels lower than 10 times the upper reference interval value of the assay should undergo endoscopy with small bowel biopsy
  • Patients with positive tTG IgA levels greater than 10 times the upper reference interval value of the assay should undergo EMA IgA and HLA DQ2/8 testing. If these tests are positive, the option and implications of omitting the biopsy should be discussed with the parents, before the diagnosis of CD is made.

Diagnostic approach in asymptomatic children who belong to a high-risk group

Testing for HLA-DQ2 and DQ8 /4451/:

  • HLA-DQ2/DQ8 negative: no further serological testing
  • HLA-DQ2/DQ8 positive: determination of tTG IgA and serum levels of IgA
  • tTGA IgA and IgA within the reference range: further follow up tests at 2-year intervals
  • tTGA IgA greater than 3 times the upper reference interval and serum levels of IgA within the reference interval: endoscopy with small bowel biopsy
  • tTGA IgA elevated but lower than 3 times the upper reference interval value and serum levels of serum IgA within the reference interval: determination of EMA IgA; if the EMA IgA is positive, then a small bowel biopsy should be performed. If the EMA IgA are negative, follow up tests should be performed at 3 to 6 months intervals.

Diagnostic approach in adults

Biopsy is further considered as essentially for the diagnosis of adult CD. The combination of tTG IgA with DGPA IgG is considered as useful to recognize also these patients who are IgA deficient.

25.10.7 Antibodies in autoimmune pancreatitis

A number of autoantibodies have been identified in autoimmune pancreatitis (AIP), although they are not specific to the disease. Autoantibodies targeting pancreatic enzymes are suggested as pathophysiology of the inflammatory process /17/.

Apart from ANA, ASMA and rheumatoid factors, the following autoantibody prevalences to pancreatic antigens have been described:

  • Pancreatic acinar cell antibodies (10%) /43/,
  • Lactoferrin antibodies (73%) /52/,
  • Type II carbonic anhydrase antibodies (54%) /52/,
  • Pancreatic secretory trypsin inhibitor (PSTI) antibodies (40%) /43/.

Approximately 94% of patients with AIP were reported /53/ to have antibodies to the plasminogen binding protein of Helicobacter pylori, which supports the hypothesis that Helicobacter pylori stimulates the autoimmune process. In addition, there is a homology between carbonic anhydrase and Helicobacter pylori. However, there is currently no commercial assay available for demonstrating antibodies to the plasminogen binding protein of Helicobacter pylori. It is therefore recommended that individuals with suspected AIP be tested for lactoferrin antibodies and type II carbonic anhydrase antibodies in combination with IgG4.

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22. Sendid B, Colombel JF, Jacquinot PM, et al. Specific antibody response to oligomannosidic epitopes in Crohn’s disease. Clinical Diagnosis and Laboratory Immunology 1996; 3: 219–26.

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24. Bossuyt X. Serologic markers in inflammatory bowel disease. Clin Chem 2006; 52: 171–81.

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28. Teegen B, Niemann S, Probst C, Schlumberger W, Stöcker W, Komorowski L. DNA-bound lactoferrin is the major target for antineutrophil perinuclear cytoplasmic antibodies in ulcerative colitis. Ann N Y Acad Sci. 2009; 1173: 161–5.

29. Terjung B, Sohne J, Lechtenberg B, Gottwein J, Muennich M, et al. p-ANCAs in autoimmune liver disorders recognise human beta-tubulin isotype 5 and cross-react with microbial protein FtsZ. Gut 2010; 59: 808–16.

30. Papp M, Altorjay I, Lakos G, Tumpek J, Sipka S, Dinya T, et al. Evaluation of the combined application of ethanol-fixed and formaldehyde-fixed neutrophil substrates for identifying atypical perinuclear antineutrophil cytoplasmic antibodies in inflammatory bowel disease. Clin Vaccine Immunol 2009; 16: 464–70.

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Table 25.1-1 HLA associations with autoimmune diseases (selection)

Disease

HLA and
%Relative
risk RR)(1)

Association with MHC class I (HLA-A,-B,-C)

Ankylosing spondylitis

B27

> 85%

Reiter’s syndrome

B27

40%

Reactive arthritis

B27

20%

Psoriatic arthritis

B27; B38

10%

Acute anterior uveitis

B27

10%

Association with MHC class II (HLA-DR, -DP, -DQ)

Systemic lupus
erythematosus

DR3, B8

5%

Sjögren’s syndrome

DR3

10%

Scleroderma
(Caucasians)

DR3

15%

Pemphigus vulgaris

DR4

14%

Graves’ disease

DR3, B8

3%

Hashimoto’s thyroiditis
(atrophic)

DR4, DR5

3%

Juvenile rheumatoid
arthritis

DR8

10%

Goodpasture syndrome

DR2

15%

Complex association with MHC

Rheumatoid arthritis

Shared epitope(2) (e.g., DRB1*0101, *0102, *0401, *0404; previously subsumed also under DR4)

15%

  • Diabetes mellitus type I

DQA1*0501/DQB1*02 (also simply known as DQ2)

DQA1*0301/DQB1*0302 (DQ8)(previously also described as association with HLA-DR3/4)

  • Celiac disease(3)

DQA1*0501/DQB1*02 (DQ2)

DQA1*0301/DQB1*0302 (DQ8)

(1) Presence of this allele is associated with an X-fold increased risk for the disease compared to individuals without the allele; approximate data (marked differences depending on the study and patient population)

(2) Shared epitope refers to peptide sequences in the HLA molecule that may be present in different HLA subgroups

(3) Besides the common HLA association, patients with celiac disease also have a markedly increased clinical risk of developing diabetes mellitus type I

Table 25.1-2 Primary immune deficiencies with autoimmunity as defining feature /4/

Clinical and laboratory findings

Autoimmune polyendocrinopathy candidiasis and ectodermal dystrophy (APECED)

This syndrome, also referred to as autoimmune polyendocrine syndrome type1 is caused by an autosomal recessive mutation in the AIRE gene /5/. The classical findings are chronic mucocutaneous candidiasis, hypoprathyroidism, and adrenocortical failure.

Autoimmune lymphoproliferative syndrome (ALS)

The ALS is an autosomal dominant disorder caused by abnormalities in Fas-mediated lymphocyte apoptosis. Clinical features are splenomegaly, lymphadenopathy, and various autoimmune manifestations /6/.

Immunodysregulation, polyendocrinopathy, enteropathy, and X-linked (IPEX)

An X-linked recessive disorder caused by a mutation in FOXP3 which is expressed primarily in CD4+CD25+ regulatory T cells playing a key role in regulatory T cell development and function. A severe enteropathy with villous atrophy and infiltration of lymphocytes into the submucosa or lamina propria is found in nearly all patients. Clinical symptoms are failure to thrive, malabsorption and diarrhea. Insulin-dependent diabetes mellitus with onset in infancy is the most common endocrinopathy found in IPEX patients /7/.

IL10/IL-10 receptor deficiencies

Mutations in the genes encoding the regulatory cytokine IL-10 and its receptor have been implicated as causes of very early onset inflammatory bowel disease presenting in infancy.

Table 25.1-3 Classification of inflammatory and systemic autoimmune diseases /3/

Autoinflammatory diseases

Autoimmune diseases

Monogenic

Polygenic

Monogenic

Polygenic

FMF

Still’s disease

APS type1

Rheumatoid arthritis

TRAPS

Crohn’s disease

IPEX

SLE

CAPS, FCAS,
MWS, NOMID

Behcet disease

ALPS

Systemic sclerosis

HIDS

Gout

C1q deficiency

Poly-myositis

Blau’s syndrome,

PAPA syndrome,

CRMO,

DIRA,

Majeed’s syndrome,

IL-10 deficiency

sJLA

Dermato-myositis,

Sjögren syndrome,

UCTD,

MCTD,

Overlap syndromes

FMF, familial Mediterranean fever; TRAPS, TNF receptor associated syndrome; CAPS, cryopyrin associated periodic syndrome; FCAS, familial cold auto inflammatory syndrome; MWS, Muckle-Wells syndrome; NOMID, neonatal onset multisystem inflammatory disease; HIDS, hyper immunoglobulin D syndrome; PAPA, pyogenic arthritis, pyodermia gangrenosum and acne; DIRA, deficiency of the interleukin-1 receptor antagonist syndrome; sJLA, systemic juvenile idiopathic arthritis; CRMO, chronic recurrent multi focal osteomyelits syndrome; APS, autoimmune polyendocrinopathy syndrome; IPEX, immune dysregulation polyendocrinopathy enteropathy X-linked syndrome; ALPS, autoimmune lymphoproliferative syndrome; UCTD, undifferentiated connective tissue disease; MCTD, mixed connective tissue disease

Table 25.1-4 Spectrum of autoimmune diseases (selection)

Disease picture Systemicautoimmune disease Organ specificautoimmune disease Systemic lupus erythematosus (SLE) Mixed connective tissue disease (MCTD) Scleroderma Sjögren syndrom Antiphospholipid syndrome ANCA -associated vasculitis Rheumatoid arthritis Dermatomyositis Ulcerative colitis , Crohn's disease Primary biliary cirrhosis Autoimmune hepatitis Goodpasture syndrom Myasthenia gravis Stiff mans syndrome Guillain-Barré syndrom Multiple sclerosis Type I diabetes mellitus Vulgar pemphigus , bullous pemphigoid Autoimmune atrophic gastritis, pernicious anemia Hashimoto's thyroiditis, M. Basedow M. Addison Immune thrombocytopenia (ITP) Autoimmune hemolytic anemia

Table 25.1-5 Laboratory workup for suspected systemic autoimmune diseases

Specific work-up

Autoantibodies (e.g., ANA, rheumatoid factors, ACPA/anti-CCP, ANCA)

Genetic analysis (e.g., HLA-B27 in ankylosing spondylitis)

Differential workup

Frequently: infections (viral, bacterial)

Metabolic diseases

Chronic inflammatory diseases (fever syndromes)

Immune defects

Paraneoplastic syndromes

Disease activity, course of disease (inflammatory markers)

C-reactive protein

Erythrocyte sedimentation rate

Differential blood count

Serum protein electrophoresis

Procalcitonin

IL-6

Organ involvement effects,

Side effects of medications

Kidney: Creatinine (GFR), cystatin C, urine status, albumin excretion

Liver: Aminotransferases (ALT, AST), γ-GT, ALP, bilirubin

Pancreas: Lipase, amylase

Muscles: Creatine kinase (CK)

Endocrine organs: Hormones (e.g., TSH)

Lungs/heart: Blood gas analysis, NTproBNP

Bone marrow, blood: Blood count, LD, haptoglobin

Coagulation system: Platelets, aPTT, PT

Immune system: Interferon-gamma release assay to exclude tuberculosis prior to administering biological agents

Immune system: B cells under treatment with anti-CD20 antibodies

Comprehensive workup

Genetic analysis (e.g., HLA-DQ2/8 in celiac disease, HLA-B27 in spondylarthropathies, HLA-DR4 in rheumatoid arthritis)

For further workup refer to the sections on the individual systemic autoimmune diseases

Table 25.1-6 Autoantibodies in systemic autoimmune diseases /911/

Disease

Specific auto-
antibodies

Prevalence
of positive
results

Diagnostic
relevance

Autoantibodies
of lower
significance

Rheumatoid
arthritis

ACPA, anti-CCP, rheumatoid factor

60–80%

60–80%

American College of Rheumatology criterion

ANA, SSA, RA 33

Systemic lupus
erythema-
tosus
(SLE)

ANA

dsDNA Ab

Sm Ab

> 95%

40–90%

< 10% Caucasians, up to 30% other ethnic groups

Very sensitive

Highly specific

Highly specific

Nucleosome Ab (80%)

SSA/Ro Ab, 60 kDa; approximately 60%

Histone Ab (30%)

Ribosomal pancreatic Ab (10%)

PCNA (5–10%)

Subacute
cutaneous LE

ANA

> 95%

 

SSB/La Ab (25–35%)

Cardiolipin, lupus anticoagulant (25–50%)

SSA/Ro Ab

75%

 

Neonatal LE

ANA

> 95%

Associated with congenital heart block

SSB/La Ab (40%)

SSA/Ro Ab

> 95%

Drug-induced
LE

ANA

 

Diagnostic criterion

ssDNA, if highly positive

Histone Ab

60–100%

Mixed
connective
tissue disease
(MCTD)

ANA

> 95%

Diagnostic criterion

 

U1-nRNP Ab

100%

Systemic
scleroderma
(SS)

ANA

> 95%

Positive relevance

 

RNA polymerase
Ab

5–22%

Scl-70 Ab

20–40%

Diffuse Sjögren’s syndrome (SS)

zSS (muscle joints)

Overlap
syndrome 25%

PMScl Ab

5%

CREST
syndrome

Centromere Ab

> 80%

Differentiation of SS from primary Raynaud’s

 

Polymyositis
Dermato-
myositis

ANA

< 80%

Nucleolar fluorescence

Mi-2 Ab (28% in D, 9% in P)

Ku Ab

Anti-PM-Scl Ab

t-RNA
synthetases:
Jo-1 Ab

46%

Antisynthetase syndrome

Further t-RNA
synthetases

< 3%

 

Anti-SRP

5%

High specificity of these autoantibodies

Sjögren’s
syndrome

ANA

80%

 

SSA/Ro Ab (52 kDa) 60%

Rheumatoid factors 70%

SSA/Ro (60 kDa)
Ab

80–95%

SSB/La Ab

40–80%

Anti-
phospholipid
syndrome
(APS)

Cardiolipin Ab

> 95%

The autoantibodies define the disease

ANA (secondary APS in SLE)

β2-glycoprotein
I Ab

> 95%

Wegener’s
granulo-
matosis

c-ANCA

80–95%

The autoantibodies define the disease

Rarely p-ANCA or MPO-ANCA

PR3-ANCA

> 85%

Microscopic
polyangiitis

p-ANCA

80–95%

The autoantibodies define the disease

Rarely c-ANCA or PR3-ANCA

MPO-ANCA

> 85%

Churg-Strauss
syndrome

p-ANCA

45%

 

 

MPO-ANCA

45%

Goodpasture
syndrome

Anti-GBM

100%

The autoantibodies define the disease

 

Table 25.1-7 Autoantibodies in organ specific autoimmune diseases /1112/

Disease

Autoantibody

Antigen

Ulcerative colitis

a-ANCA

Polymorphonuclear granulocytes

GAB

Goblet cells

Crohn’s disease

PAB

Exocrine pancreas

ASCA

Saccharomyces cerevisiae

Celiac disease

Gliadin Ab

Deaminated gliadin

EMA

Endomysium of the mucous membrane of the small intestine

htTG Ab

Human tissue transglutaminase

Polyglandular syndrome

Ab to the adrenal cortex, thyroid, pancreas,

ANA, ASMA

See individual organs

Dermatomyositis

Mi-2 Ab

Protein of the helicase/ATPase domain

Dermatitis
herpetiformis

Gliadin Ab

Gliadin

Bullous pemphigoid

BMZ Ab

Basement membrane zone

BPAG1 Ab

Bullous pemphigoid antigen 1 (BPAG1)

BPAG2 Ab

Bullous pemphigoid antigen 2 (BPAG2)

Pemphigus vulgaris

Desmoglein Ab

ICS Ab

Desmosomal antigens of the epidermal intercellular substance (ICS)

Subacute cutaneous
SLE

SSA/Ro60 Ab

Ribonucleoproteins

SSB/La Ab

SSA/Ro-RNP particles

Dilatative
cardiomyopathy

β-adrenoreceptor

Autoimmune
hepatitis

Actin Ab

SMA (ASMA)

Smooth muscle

LKM-1 Ab

Endoplasmic reticulum (cytochrome P450)

SLA Ab

Soluble liver antigen

LC-1 Ab

Liver cytosol antigen (LC-1)

ANA

Nucleus of liver cells

Primary biliary
cirrhosis (PBC)

AMA-M2

Mitochondrial pyruvate dehydrogenase

Sp100 Ab

ANA (nuclear dots, Sp100)

CENP

ANA (anti-centromere antibody, CENP-A,B,D)

Gp210, lamin B receptor

ANA (fluorescence of the nuclear membrane)

Primary sclerosing
cholangitis

a-ANCA

Granulocytes (tubulin 5, DNA-associated lactoferrin)

Pulmonary
syndrome:
Goodpasture
syndrome

GBM Ab

Glomerular/alveolar basement membrane

with ANCA
association

p-ANCA

Myeloperoxidase (MPO) of the granulocytes

Granulomatosis
with polyangiitis
(Wegener’s
granulomatosis)

c-ANCA

Serine proteinase 3 (PR3) of the granulocytes

Chronic atrophic
gastritis, pernicious
anemia, funicular
myelosis

Parietal cell Ab (H+–K+-ATPase)

Parietal cells (IFT)

H+–K+-ATPase (immunoassay)

Intrinsic factor Ab

Intrinsic factor

Myasthenia

AchR Ab

Acetylcholine receptors

Myasthenia with indication of thymoma

Titin Ab

Tyrosine kinase (MuSK)

Lambert-Eaton
myasthenia
syndrome

VGCC Ab

Voltage gated calcium channels (VGCC)

Inflammatory
idiopathic myopathy*

Jo-1 Ab, PL-7 Ab, PL-12 Ab, EJ Ab, OJ Ab

Aminoacyl-tRNA synthetases

SRP Ab

Cytoplasmic anti-signal recognition particle

Dermatomyositis*

Mi-2 Ab

Nuclear antigen

Polymyositis,
dermatomyositis*

PM-Scl Ab

Complex consisting of 16 nucleolar proteins

Myositis-
scleroderma overlap
syndrome*

Ku Ab

DNA-binding proteins

SSB/La Ab

Phosphoprotein antigen

SSA/Ro Ab

Ribonucleoprotein

U1–U3-RNP Ab

Ribonucleoprotein particles of the nucleus

Polyglandular
autoimmune
syndrome

Addison’s disease

Adrenal cortex Ab

(21-hydroxylase Ab)

Adrenal cortex (IFT)

21-hydroxylase (immunoassay)

Goodpasture
syndrome
(anti-GBM nephritis)

GBM Ab

Glomerular basement membrane

RPGM type 3
(caused by vasculitis)

ANCA

Granulocytes

Diabetes
mellitus type 1

ICA

Islet-cell cytoplasm

Glutamate decarboxylase

Islet cell protein IA-2

GADA, GAD65A

IA-2 Ab

Zinc transporter 8

Guillain-Barré
syndrome

Anti-GM1, anti-GD1

Glycolipids (gangliosides)

Miller-Fisher
syndrome

Anti-GQ1b

IgM para
proteinemic
neuropathy

Anti-MAG

Myelin-associated glycoprotein (MAG)

Stiff person
syndrome

GADA, GAD65A

Glutamate decarboxylase (GAD)

Paraneoplastic
neurological
syndrome

Limbic encephalitis

Anti-Hu, anti-Yo, anti-Ri

Anti-VGKC, anti-AMBAR

Onconeural antigens

Surface proteins of the neurons

Antiphospholipid
syndrome

Antiphospholipid, anti-β2GPI Ab

Phospholipids, glycoproteins

Thyroiditis

TPO Ab

Thyroid peroxidase (TPO)

TG Ab

Thyroid globulin (TG)

Graves’ disease

TR Ab

TSH receptor (TR)

Table 25.1-8 Systemic autoimmune diseases

Clinical and laboratory findings

Autoimmune arthritis

Autoimmune forms of arthritis are often classed as systemic autoimmune diseases. They primarily affect the joints and therefore exhibit a certain organ specificity. On the other hand, rheumatoid arthritis (RA) in particular must be seen as a systemic disease, since besides the typical skin involvement manifesting as rheumatoid nodules it can also lead to serositis (e.g. of the pleura) and pulmonary fibrosis. In addition, RA is associated with a markedly increased risk of morbidity and mortality from cardiovascular disease, probably due to the high chronic inflammatory activity.

Spondyloarthropathies are heterogeneous and differ from typical autoimmune disease in that no characteristic autoantibodies, especially none of diagnostic relevance, have yet been identified. The absence of detectable rheumatoid factor led to the original term seronegative spondyloarthropathy

General laboratory testing comprises the tests listed in Tab. 25.1-5 – Laboratory workup for suspected systemic autoimmune diseases for determining activity, organ involvement and drug side effects. For differential diagnosis, a range of other causes of arthritis (Tab. 25.1-9 – Laboratory workup for arthritis spectrum disorders), including connective tissue diseases, must be considered in addition to RA, depending on the manifestation. This may required comprehensive laboratory testing /12/.

One important step in many forms of arthritis with articular effusion is joint aspiration for synovial fluid analysis. The detection of pathogens can be essential in saving the joint, in particular in septic arthritis (refer to Section 49.4.1 – Microbiological evidence of septic arthritis). The presence of granulocytic uric acid crystals is pathognomonic of gout and the presence of calcium pyrophosphate crystals pathognomonic of pseudo gout.

Rheumatoid arthritis (RA)

RA, previously also primary chronic polyarthritis (PCP), is a chronic inflammatory systemic disease which predominantly affects the joints. Systemic vasculitis may be accompanied by severe life limiting diseases of visceral organs. For example, RA patients have a 2-fold higher rate of chronic heart failure, a 40% higher incidence of heart attack and a 2.2-fold increased mortality risk compared to the normal population /13/. About 30% of patients develop bone and cartilage destruction within one year and 70% within 2 years of diagnosis. More than 50% of patients are no longer working within 5 years of diagnosis.

The prevalence of RA in the general population is 0.5–1%, making it the most common inflammatory joint disease. Female individuals are 2–3 times more affected than male. The diagnosis is often made between 40 and 60 years of age. Early diagnosis is difficult because initial symptoms are nonspecific and laboratory results are not always conclusive during the early phase of disease. However, early diagnosis is crucial since early treatment can have a positive influence on the course of this chronic destructive disease /14/.

In two thirds of patients clinical symptoms first manifest in the joints of hands and feet (metacarpophalangeal, proximal interphalangeal and metatarsophalangeal joints) before affecting the large joints. In most cases, the joints are affected symmetrically, although RA can also begin atypically:

Polymyalgic onset; patients are generally older and present with stiffness of the shoulder and pelvic girdle

Palindromic onset; patients have recurring episodes of pain as well as swelling and redness of one or several joints for 1–2 days; later on the pain becomes permanent

Systemic onset; initial symptoms are not focal, but extra articular and of a general nature (e.g., fatigue, loss of weight, depression, fever).

Mono arthritic onset; patients initially suffer from persistent pain in one of the large joints, such as the knee, shoulder, elbow or ankle joint.

RA is classified for treatment studies, based on the criteria of the American College of Rheumatology (Tab. 25.1-10 – The 2010 American College of Rheumatology/European League Against Rheumatism classification criteria for rheumatoid arthritis). In practice these criteria are not always met even if RA is present. This classification is not suitable for diagnosis, especially in the early stages of RA. Patients who do not reach the score required for diagnosis should be retested at a later stage /14/.

Specific testing for RA includes screening for anti-citrullinated protein/peptide antibodies (ACPA, anti-CCP) and RF. HLA typing for the detection of the shared epitope is important for disease prognosis and is performed in many treatment studies. Yet, it has not become established in routine diagnostics. Screening for HLA-B27 is indicated whenever spondyloarthropathy is suspected.

Special forms of rheumatoid arthritis

Juvenile idiopathic arthritis (JIA) /15/

JIA is a heterogeneous group of rheumatoid joint diseases with onset before the age of 16, usually at age 2–3. Early childhood oligoarthritis with involvement of the lower extremities usually interferes with learning to walk. 80% of patients are girls. RF and/or ACPA are positive in 40–50% of children with polyarthritic JIA. ANA can be found in 75–85% of children with an oligoarthritic pattern and are associated with a higher risk of developing uveitis /16/. The prevalence of JIA is 0.07–4.01 per 1,000 children, depending on the geographical region /17/.

Adult rheumatoid arthritis

The disease begins acutely after age 60 with extensive signs of inflammation and rapid progression, with myalgic symptoms during the early stage. Markedly elevated CRP, anemia of chronic disease, RF rarely positive, ACPA more often positive than RF.

Felty’s syndrome

Characteristic triad of arthritis, leukocytopenia and, in some cases, splenomegaly. Affects approximately 1% of RA patients with extra-articular manifestation. High concentration of RF, ANA positive in 75–100% of cases, granulocytopenia below 2 × 109/L, HLA DR4 positive.

Still’s disease /18/

Disease that occurs in episodes, with the following relative prevalences of symptoms: arthralgias (100%), fever (97%), arthritis (94%), sore throat (92%), exanthemas (88%), myalgias (84%), weight loss (74%), lymphadenopathies (63%), splenomegaly (52%) and hepatomegaly (42%).

The prevalences of diagnostic test results are as follows: elevated ESR (99%), granulocytosis (92%), negative rheumatism serology (92%), serum albumin below 3.5 g/dL (81%), elevated aminotransferases (73%), hemoglobin below 100 g/L (68%) and high ferritin, usually above 1,000 μg/L.

The main criteria are: fever above 39 °C, arthralgias or arthritis, RF negative, ANA negative.

The differential diagnosis includes other arthritides such as spondylarthropathies, including reactive arthritides, Lyme disease and septic arthritis (Tab. 25.1-9 – Laboratory workup for arthritis spectrum disorders).

Spondylarthropathy

The spondyloarthropathy group of diseases comprises the following disorders /16/:

  • Ankylosing spondylitis (Bechterew’s disease)
  • Reactive arthritides and Reiter’s syndrome
  • Psoriatic arthritis
  • Enteropathic arthritides
  • Undifferentiated SpA.

SpA (Greek for inflammation of spine joints), have a prevalence of 0.5–2% in the population. SpA with isolated involvement of the axial skeleton is characterized by deep-seated lower back and/or gluteal pain indicative of sacroiliitis. Patients suffer from early morning stiffness and spinal motion restriction, which will eventually also affect the thoracic and cervical spine. Approximately one quarter of patients have anterior uveitis as an extra-skeletal manifestation. The disease occurs in episodes and is chronically progressive. Men and women are equally affected. SpA is associated with HLA-B27 at varying degrees.

For further diagnostic tests for spondyloarthropathies and workup of arthritis refer to Tab. 25.1-9 – Laboratory workup for arthritis spectrum disorders.

Systemic lupus erythematosus (SLE)

SLE may differ between individuals in terms of severity and organ manifestations. It has a prevalence of 10–50 per 100,000 population in the Western countries. The most common symptoms are arthralgias or arthritis, with a prevalence of 60–90%, followed by abnormalities of the skin and mucous membranes and involvement of internal organs (kidneys, heart, lungs, muscles) and of the central nervous system. SLE usually occurs between the ages of 15 and 30 years, with women being affected ten times more often than men.

SLE usually takes between 1 and 2 years from the first medical visit for the final diagnosis. The American College of Rheumatology’s (ACR) criteria for the classification of SLE are shown in Tab. 25.1-11 – The 1982 Revised criteria for the classification of systemic lupus erythematosus [American Rheumatism Association (ACR)]. Only approximately 50% of patients meet these criteria during the early phase of the disease while others fail to meet them even after 10 years of disease. The criteria are therefore not a diagnostic tool, but are used for the scientific standardization of a patient population and for the classification into treatment studies /1718/.

Labordiagnostik: Up to 50% of patients have general findings such as mild elevation of ESR and CRP, anemia, lymphopenia and thrombocytopenia.

The diagnosis of SLE is confirmed or excluded by testing for autoantibodies, especially ANA and their specific subtypes ENA and anti-dsDNA. ANA, anti-Sm, anti-dsDNA and anti phospholipid antibodies are part of the ACR criteria. The latter antibodies are indicative of secondary anti phospholipid syndrome. The LE cell test previously commonly used for diagnosis is no longer performed, since the LE cells are difficult to detect systematically and have significantly lower diagnostic sensitivity and specificity compared to ANA. For 2019 update of EULAR recommendations for the management of systemic lupus erythematosus refer to Ref. /19/.

Polymyositis (PM) and dermatomyositis (DM)

PM and DM are rare autoimmune forms of myositis. PM involves only the muscles, while DM also affects the skin. The diagnosis is made based on the following criteria proposed by Bohan and Peter /1920/:

1. Symmetric weakness of the shoulder and pelvic girdle muscles over weeks to months.

2. Histological demonstration of muscle fiber necrosis, phagocytosis and regeneration.

3. Increase in serum enzymes (CK, AST, LD).

4. Characteristic electromyographic pattern.

5. Typical rash of dermatomyositis.

Clinical significance

4 criteria met; definite diagnosis of PM

3 criteria met plus exanthema typical of DM; definite diagnosis of DM.

Special forms

Anti-signal recognition particle (SRP) syndrome. Anti-SRP syndrome is an acute- or subacute onset form of polymyositis with severe muscle necrosis and mild inflammation, characterized by the presence of anti-SRP antibodies

Polymyositis/scleroderma overlap syndrome. Characterized by the symptoms of PM and systemic scleroderma.

Anti-synthetase syndrome (most commonly anti-Jo-1 syndrome): autoimmune form of myositis, which is diagnosed in 25% of patients with adult myositis and associated with interstitial lung disease, usually fibrosing alveolitis.

Laboratory findings: The increase in CK is a sign of damaged skeletal muscles. During the early phases of PM/DM as well as in chronic PM, enzyme levels may not be elevated. Myositis associated and/or myositis specific antibodies occur in approximately 50% of patients with PM/DM. The antibodies detected are considered to be ANA, although some of the corresponding antigens are found in the cytoplasm /21/.The autoantibodies relevant for the diagnosis or exclusion of PM/DM are described in Section 25.2 – Antinuclear antibodies

Sjögren’s syndrome

This disease is characterized by autoimmune inflammation of exocrine glands with reduced secretion. According to the revised European classifications, 4 of the following 6 criteria must be met for a diagnosis of Sjögren’s syndrome to be made /22/:

1. Persistently dry eyes or foreign body sensation in the eyes for at least 3 months.

2. Oral symptoms: daily feeling of dry mouth for more than 3 months. Recurrently or frequently swollen salivary glands.

3. Ocular signs: positive Schirmer I test, positive rose bengal staining test.

4. Histopathological focus score > 1 (e.g., in lip biopsy).

5. Objective evidence of salivary gland involvement defined as abnormal parotid sialography, abnormal salivary scintigraphy or non stimulated salivary flow (less than 1.5 mL in 15 min.).

6. Presence of serum anti-SSA and/or anti-SSB autoantibodies.

In patients with another autoimmune disease, the presence of symptoms of groups 1 and 2 plus two findings of groups 3–5 are indicative of secondary Sjögren’s syndrome, provided that the symptoms cannot be attributed to other diseases such as non Hodgkin lymphoma, AIDS, sarcoidosis, or bone marrow transplantation.

The ANA IIFT is positive in 95% of cases and shows a speckled pattern. For autoantibody specificities refer to Section 25.2 – Antinuclear antibodies.

Systemic scleroderma

Systemic scleroderma, also known as sclerosis, is an inflammatory, fibrosing, multi organ disease that affects the skin, but may also involve other organs and, less commonly, the locomotor system, heart and kidneys.

The main criteria are /21/:

  • ANA positivity
  • Sclerodactyly in more than 95% of patients
  • Raynaud’s phenomenon in more than 90% of patients.

Patients who fail to meet any of these three criteria probably suffer from a different disease (eosinophilic fasciitis, eosinophilic myalgia syndrome). The most common clinical manifestations are dermatofibrosis and circulation problems in the fingers and toes.

Special forms of systemic sclerosis include:

Systemic scleroderma with diffuse skin involvement (inclusive trunk and proximal to the elbow joints)

Limited scleroderma with involvement of the face and the regions distal to the elbow and knee joints. This form corresponds to CREST syndrome (C, cutaneous calcinosis; R, Raynaud’s phenomenon; E, esophageal dysmotility; S, sclerodactyly; T, teleangiectasia).

Sclerosis sine scleroderma. Monosymptomatic or oligosymptomatic systemic sclerosis without skin involvement, but with ANA positivity.

Mixed connective tissue disease (MCTD)

MCTD is a term used to describe anti-U1 nRNP positive connective tissue disease with clinical characteristics of SLE, of systemic scleroderma and of DM/PM /19/. It is also known as Sharp’s syndrome. MCTD may also include overlap syndromes of polymyositis and systemic scleroderma. These are characterized by symptoms and/or antibodies of systemic scleroderma and PM/DM.

One important characteristic feature of MCTD is general proliferative vasculopathy with proliferation of the vascular intima and media, which leads to vasoconstriction.

Criteria of MCTD as proposed by Sharp:

  • Presence of anti-U1 nRNP plus at least two of the following disorders: SLE, systemic scleroderma, myositis, rheumatoid arthritis
  • At least three of the following symptoms: Raynaud’s phenomenon, swollen hands, scleroderma, proximal muscle weakness, synovitis.

The IIFT usually shows high titers of ANA in MCTD. The diagnosis is based on the presence of high titers of autoantibodies to U1-RNP. A negative result therefore excludes MCTD. Production of additional SLE or systemic scleroderma autoantibodies during the course of a MCTD is unlikely. During remission of the disease, anti-U1 nRNP antibody levels may decline significantly.

Table 25.1-9 Laboratory workup for arthritis spectrum disorders

Clinical presentation

Laboratory investigations

Rheumatoid arthritis (RA)

See Section 25.3.5.1 – Rheumatoid arthritis (RA)

ACPA/anti-CCP and RF

Spondyloarthropathy (SpA) /23/

  • Ankylosing spondylitis (Bechterew’s disease)
  • Reactive arthritides and Reiter’s syndrome
  • Enteropathic arthritides
  • Psoriatic arthritis
  • Undifferentiated spondyloarthropathy
  • Juvenile spondyloarthropathies (subgroup of juvenile idiopathic arthritis)

Inflammatory markers: ESR and CRP

HLA-B27: SpA have variable associations with HLA-B27

Ankylosing spondylitis (Bechterew’s disease)

The main symptom is inflammatory back pain in the loin and gluteal regions, accompanied by morning stiffness; onset is usually in late adolescence or early adulthood. Approximately 25–35% of patients have arthritis of the shoulder and hip as well as sacroiliitis. 30% of patients have (frequently asymmetric) arthritis of other joints. Ankylosing spondylitis can also manifest as enthesopathies, particularly affecting the Achilles tendon and femoral and pelvic entheses (trochanters, ischium, iliac crest). In addition, patients may experience limited spine mobility due to the formation of syndesmophytes, ossifications which bridge the intervertebral disc spaces. The disease runs a very variable course, from mild stiffness to complete fusion of the vertebrae accompanied by restricted mobility of the upper body.

The most common complication outside the joints is acute unilateral anterior uveitis. 5–10% of patients also have chronic inflammatory bowel diseases. In rare cases, ankylosing spondylitis may cause damage to the lungs, aorta or heart.

HLA-B27 frequency: 95%

Acute inflammatory markers (CRP, ESR), usually markedly elevated especially during flare-ups

Enteropathic arthritis

Approximately 30% of patients with Crohn’s disease and ulcerative colitis have joint involvement. One in five patients have asymptomatic sacroiliitis following long standing disease, and 10% have concomitant spondyloarthritis. However, peripheral arthritis rarely leads to deformation of the affected joints.

HLA-B27 (high association)

Psoriatic arthritis

Approximately 10–20% of psoriatrics are affected with arthritis. Psoriasis of the skin usually precedes joint involvement, but may also be absent or occur years after joint manifestation. The disease mainly involves the peripheral joints; in 20–40% of cases the sacroiliac joints are also affected. The spondylitis manifests as syndesmophytes (bony growth originating inside a ligament) and para syndesmophytes in all sections of the spine.

HLA-B27 frequency: 60–70%

Undifferentiated spondyloarthropathy

The most commonly diagnosed type of spondyloarthropathy. Patients present with inflammatory back pain with or without oligoarthritis. History of restricted spine mobility or restricted respiratory excursion, often the only clinical symptoms.

HLA-B27 frequency: 70%

Juvenile enthesitis (spondyloarthropathy)

This subgroup of juvenile idiopathic arthritis is a form of undifferentiated spondyloarthropathy. In 30–40% of patients the disease progresses to sacroiliitis.

HLA-B27 frequency: 80%

Reactive arthritis, Reiter’s syndrome (HLA-B27 associated)

Common pathogens:

  • Chlamydia trachomatis
  • Mycoplasma, Ureaplasma
  • Enteropathogenic pathogens, Campylobacter, Yersinia
  • Rarely Salmonella and Shigella

Reactive arthritis is most commonly and asymmetric oligoarthritis involving less than six joints. The arthritis has a predilection for joints of the lower extremity, in particular the knee and ankle joints. Affected patients have more severe and longer lasting clinical symptoms.

Reiter’s syndrome: triad of arthritis, urethritis and conjunctivitis. The latter is present in 30–60% of cases, affects both eyes and manifests earlier than the arthritis.

Reactive arthritis typically begins within 4 weeks after infection. The incidence of reactive arthritis is 5–14 per 100,000 general population. Since gastrointestinal symptoms are often mild, they are usually denied by patients and not mentioned when their history is taken. Approximately 75% of reactive arthritis patients have musculoskeletal pain for more than 1 year, and 30% of them still suffer from pain after 6 years. The joint problems usually only last 3–5 months; about 15% of patients develop chronic symptoms. One common characteristic of all pathogens of enteropathogenic reactive arthritis is that they also propagate within cells and may persist for months in cells of the intestinal mucosa, lymph nodes, liver, spleen or synovial fibroblasts.

Reactive arthritis, Reiter’s syndrome: HLA-B27 frequency 40–50%

Serology: high titers of specific IgA antibodies indicate recent infection

CRP, ESR elevated, neutrophilia

Synovial fluid: inflammatory synovitis, but no pathogens detectable.

The usefulness of stool screening is doubtful, since the pathogen is usually no longer detectable.

Cervical smear/urethral smear: 1. First-void urine to test for Chlamydia and/or Mycoplasma/Ureaplasma (culture, better: PCR)

Other reactive arthritides caused by enteropathogenic pathogens

Inflammatory arthritis may occur after bowel infection with Clostridium difficile, Brucella, Giardia lamblia and Tropheryma whippeli.

Clostridium difficile causes pseudomembranous colitis, but is also responsible for 20% of antibiotic associated diarrheas without colitis. It is believed that Clostridium difficile associated arthritides are responsible for a considerable portion of undifferentiated oligoarthritides.

Giardia lamblia causes enteric infections worldwide that can result in arthritides, especially in children.

Tropheryma whippeli is detected by molecular diagnostic testing in the saliva of 35% of healthy individuals, but the pathogen does not routinely colonize the small intestine. The pathogen causes Whipple´s disease, a diarrheal disorder with arthritis, which primarily affects men aged 45–50 years.

Low or no association with HLA-B27

Pathogen detection in stool (C. difficile, G. lamblia) or duodenal juice (T. Whippeli)

Serological detection of specific antibodies to Brucella.

Other bacteria as rare causes of reactive arthritis

Treponema pallidum, and Brucella have been described as pathogens of reactive arthritides.

N. gonorrhoeae and Mycobacterium can cause infectious arthritis as well as reactive arthritis. A rare but severe complication is reactive arthritis after BCG immunotherapy for bladder cancer.

Serology; pathogen detection is usually not possible; possibly blood culture for the detection of Brucella.

Streptococcal arthritis /24/ – Rheumatic fever and reactive arthritis

Due to the differences in the course of disease, post streptococcal arthritides can be divided into two entities: acute rheumatic fever and post streptococcal reactive arthritis. It has been proposed that both diseases arise from molecular mimicry with cross reactivity of antibodies to the M protein of streptococci with cardiac valve endothelium and synovial cells.

Antistreptolysin O (ASL, ASO), anti-DNAse B: 80% sensitivity with low specificity. A very high titer or marked increase indicate an acute infection. A throat culture test should be performed for the detection of Streptococcus type A.

Lyme arthritis

Lyme disease is caused by the tick transmitted spirochete Borrelia burgdorferi. Multi systemic disease causing symptoms predominantly in the skin, nervous system, muscles and joints results only when pathogens disseminate. Lyme arthritis is a rare (definitely not common) manifestation of Lyme disease. The disease is often described to proceed in three clinical stages, which may overlap considerably. In addition, patients can go through one or more of these stages without showing any symptoms, so that the disease may only become clinically apparent at an advanced stage. Only about one third of all patients with Lyme arthritis remember being bitten by a tick. Stage I presents as erythema migrans. The early rheumatic symptoms during this stage (stage II) are typically fleeting and manifest as migratory, sometimes severe arthralgia and myalgia that can last from a few hours to several days. Joint swelling is rarely seen in this stage.

Classic Lyme arthritis corresponds to stage III. The arthritis occurs on average 6 months after infection. Lyme arthritis is typically a recurrent mono arthritis or oligoarthritis, mainly of the large joints in the region of the lower extremities. In most patients, one knee joint becomes affected during the course of the disease. However, similar to stage II, patients may only have severe arthralgias without apparent synovitis. Symmetric involvement of small vessels as seen in rheumatoid arthritis is rare.

Lyme arthritis is diagnosed by detecting IgG antibodies to B. burgdorferi in the ELISA, confirmed by immunoblot. The blot shows a clear positive reaction in more than two bands. The IgM test is usually negative again due to the advanced stage.

The inflammatory parameters (e.g., ESR and CRP) are often only slightly or not at all elevated.

Septic arthritis (monoarthritis)

Septic arthritis is often caused by occult bacteremia. The synovia is very susceptible to bacterial colonization. Septic arthritis is usually caused by Gram positive bacteria; only approximately 10% of septic arthritides are due to Gram negative bacteria. The pathogens originate from the gastrointestinal or urogenital tract. Main pathogens (USA, 2407 cases /25/): Staphylococcus aureus (44.3%), Streptococcus pyogenes (7.6%), Streptococcus pneumoniae (6.5%), H. influenzae (4.2%), and E. coli (3,8%).

The most important risk factor for septic arthritis is preexisting joint disease, with up to 47% of septic arthritis patients having previous joint problems. Usually only one joint, in particular the knee joint, is affected. Patients will present with fever and a warm, swollen and painful joint.

Whenever there is any suspicion of septic arthritis, joint aspiration must be performed.

Detection of the causative pathogen from the synovial fluid culture or blood culture (approximately 15% of pathogens are identifiable only from the blood culture). Gram staining of the synovial fluid has low sensitivity (positive in approx. 70% of Gram positive and 40–50% of Gram negative septic arthritides). Leukocyte count > 50 × 109/L, > 95% granulocytes

Gonococcal arthralgia

Neisseria gonorrhoeae causes disseminated infection, resulting in asymmetric polyarthralgia within days of infection, and in a skin rash. The disease runs in two phases: the bacteremic phase and the joint phase, with suppurative arthritis; many patients go through both phases. During the bacteremic phase, patients typically have high fever, sometimes with convulsions, skin rash and polyarthralgia, sometimes in combination with tenosynovitis. There is usually pain in the knee, elbow and distal joints; the axial skeleton is generally not affected. Up to 40% of patients do not experience fever. The incidence of gonococcal arthritis varies significantly according to region and age group. In Great Britain it is 0.06% and in France 1.6%, but in the USA it is the most common etiology of septic arthritis in otherwise healthy young adults.

Leukocyte count in synovial fluid (SF) is above 40 × 109/L with over 80% granulocytes. Gram stain shows less than 40% intra- and extracellular diplococci in culture positive SF. PCR has a diagnostic sensitivity of approximately 80%. Although most patients deny local urogenital, rectal or pharyngeal symptoms, 70–80% of smears from patients or their sexual partners test culture- or PCR-positive. 30% of patients positive for Neisseria gonorrhoeae have Chlamydia in addition.

Arthritis caused by mycobacteria

The pathogen of mycobacterial osteomyelitis and arthritis is M. tuberculosis colonized (e.g., in the lungs and bone). The main sites of infection in bone and joint tuberculosis are the spine (40%), followed by the hip and knee joints. The most common symptoms are pain and local swelling, while fever and weight loss occur much more rarely.

A histological examination of the aspirate should be performed (94% diagnostic sensitivity). The synovial fluid shows mild to moderate leukocytosis with neutrophil dominance.

To determine whether a person has developed an immune response to M. tuberculosis, an interferon-gamma release assay may be performed (refer to Section 42.12.4.2 – Interferon gamma release assay (IGRA)). The diagnostic sensitivity is high, but the specificity for new/active infection is low, since individuals with latent infection will usually also test positive.

Viral arthritides – Generally /26/

Arthritogenic viruses can cause acute or chronic arthritides. They can cause direct infection of the joint, or the involvement of the joint can lead to inflammation and/or damage to the joint through a reactive or autoimmune response. Viral arthritides are generally self limiting and do not lead to permanent joint damage. Nevertheless, a definite diagnosis should be made whenever possible, if for no other reason than to exclude with more certainty any causes of arthritis that can lead to the destruction of joints.

The viruses can be divided into arthritogenic Arbovirus, which is transmitted through vectors, usually mosquitoes, and is relevant in Germany in the context of travel diseases, and endemic viruses, which are transmitted via body fluids.

The diagnosis is usually made by serological testing for specific antibodies, rarely by PCR or direct viral culture, with the latter generally being used only in scientific or specific epidemiological investigations.

– Parvovirus B19

The most common presentation of Parvovirus B19 related arthropathy is the acute clinical onset of symmetric peripheral joint involvement, transient rash, myalgia, and fever. The arthralgia mainly involves the wrists, knee and ankle joints and may persist for a long time in some patients.

Serology, especially testing for specific anti-IgM antibodies.

– HCV

HCV associated arthropathy is a common extra hepatic manifestation of hepatitis C which affects up to 20% of those infected with HCV. The disease can proceed in two forms: symmetric involvement of many small joints, similar to rheumatoid arthritis, but non erosive, or cryoglobulinemia related mono-/oligoarthritis.

HCV antibodies, HCV RNA. Only half the patients have elevated CRP levels and an increased ESR. The RF may be positive, especially in the presence of cryoglobulins.

– HBV

The arthritis in HBV infection often precedes the onset of hepatitis with jaundice by several weeks and may be the only manifestation of acute HBV infection. It presents as polyarthritis of the small joints, caused by immune complex deposits.

Specific serology; measurement of HBsAg and HBVDNA.

– Rubella

Approximately half of all patients with acute rubella infection have arthralgia or arthritis during the week of the rash. The arthritis is symmetric and affects the finger, elbow and knee joints and wrists. The joint symptoms usually resolve within a week, but may also persist for up to 1 year.

Serology (rubella IgM Ab).

CRP and ESR are usually not elevated.

– Retroviruses (HIV, HTLV-1)

Arthralgias are common in acute HIV infection and may occur during any stage of the infection, usually in the form of non erosive oligoarthritis of the lower extremities.

Human T cell lymphotropic virus type-I infection is usually associated with arthropathy, which is clinically very similar to rheumatoid arthritis. Patients are usually older women who live or have lived in regions with endemic HTLV-1 infection. Onset is acute, with symptoms in the large joints such as knee and shoulder joints and wrists.

Serology (HIV/HTLV-1 antibodies). Virus detection (PCR).

– Chikungunya (Arbovirus)

Occurs mainly in Africa, South and South East Asia. 6–12 days incubation time, arthritis and lymphadenopathy.

Serology, RT-PCR

Viral arthritides – Ross River virus (Arbovirus)

Australia; 5–15 days incubation time; causes epidemic polyarthritis, chronic arthritis, and glomerulonephritis.

Serology

– Dengue virus (Arbovirus)

Asia, Central and South America; 4–7 days incubation time; often associated with severe joint pain (break bone fever); known to cause Dengue hemorrhagic fever.

Serology, RT-PCR

– Other viruses as rare causes of arthritides

ECHO virus, Coxsackie virus, Adeno virus, Cytomegalo virus, Epstein Barr virus, Herpes simplex virus 1+2, Varicella zoster virus

Serology

Other etiologies/differential diagnoses of arthritides – Generally

Arthritides must be included in the differential diagnosis of gout, pseudo gout, hemochromatosis and sarcoidosis.

 

– Gout

Gout is a disorder of purine metabolism which leads to elevated uric acid levels in the blood and deposition of urate crystals in peripheral joints and tissues. Acute symptoms include sudden severe pain and clear signs of inflammation, usually in a single joint. Often the big toe joint is involved (podagra). Refer to Section 5.4 – Uric acid.

Diagnosis relies on the detection of specific crystals in the synovial fluid (uric acid crystals in gout, calcium pyrophosphate in pseudo gout). In addition, synovial fluid analysis may show elevated inflammatory markers. Refer to Chapter 49 – Synovial fluid.

In the blood (CRP, ESR elevated).

Serum and synovial fluid uric acid are not necessarily elevated during a gout attack.

– Pseudogout

Pseudo gout (chondrocalcinosis) is a disease of the joints that is similar to gout but has a fundamentally different pathomechanism. While gout results from the deposition of urate crystals, in pseudo gout the degeneration of cartilage is due to calcium pyrophosphate deposition. The disease predominantly affects large joints (often first involving the knee).

– Hemochromatosis

Hereditary hemochromatosis (HH) is a genetic disorder of iron overload that affects the pancreas, liver (cirrhosis), heart, and skin (bronze diabetes).

Joint involvement has been described in 50–75% of patients with HH. The cardinal symptom of HH are arthralgias, which occur early in the course of the disease. Joint involvement has been described as initial manifestation in 30% of 35–65 year olds.

Two different forms can be characterized: the first form manifests as chronic degenerative joint changes with the clinical picture of exercise related arthralgias that is typical of arthrosis, the second form as chondrocalcinosis, which accounts for 5–30% of patients with HH associated arthropathy and can cause acute inflammatory pseudo gout attacks.

Elevated ferritin and high transferrin saturation.

Definite diagnosis by screening for gene mutations (see Section 7.1.5.1 – Hereditary hemochromatosis (HH)).

– Acute sarcoidosis, Löfgren’s syndrome

Löfgren’s syndrome is characterized by the triad of (1) arthritis, predominantly involving the ankle, but possibly also knee and elbow joints, (2) erythema nodosum and (3) bilateral hilar lymphadenopathy of the lungs. In addition, there are nonspecific general symptoms such as fever, fatigue, myalgias, and cough.

CRP, ESR and angiotensin converting enzyme are usually elevated, although the elevation is nonspecific. Elevation of sIL2 receptor and neopterin (markers of T cell and monocyte activation).

Table 25.1-10 The 2010 American College of Rheumatology/European League Against Rheumatism classification criteria for rheumatoid arthritis

Target population (who should be tested?) Patientes who

1) have at least 1 joint with definite clinical synovitis (swelling)

2) with the synovitis not better explained by another disease

Classification criteria for RA (score based algorithm: add score of categories A-D; a score of 6/10 is needed for classification of a patient as having definitive RA

A. Joint involvement

Score

1 large joint

0

2–10 large joints

1

1–3 small joints (with or without involvement of large joints)

2

4–10 small joints (with or without involvement of large joints)

3

> 10 joints (at least 1 small joint)

5

B.Serology (at least 1 test result is needed for classification)

Negative RF and negative ACPA

0

Low-positive RF or low positive ACPA

2

High-positive RF or high positive ACPA

3

C. Acute-phase reactants (at least 1 test result is needed for classification)

Normal CRP and normal ESR 0

0

Abnormal CRP or normal ESR1

1

D. Duration of symptoms

Below 6 weeks

0

More than or equal to 6 weeks

1

Normal/abnormal is determined by local laboratory reference intervals; ESR erythrocyte sedimentation rate; ACPA, anti-citrullinated protein antibody. ACPA and IgM RF levels are usually reported in IU. Based on the upper level of normal (ULN) the following decisions can be made: negative=less than or equal to the ULN; low-positive=less than or equal 3 times the ULN; high-level positive more than 3 times the ULN. When RF information is available only qualitatively or as a level, and thus positive or negative, patients with a positive level should be scored as low-level positive for RF.

Table 25.1-11 The 1982 Revised criteria for the classification of systemic lupus erythematosus [American Rheumatism Association (ACR)*] /29/

1.

Malar rash (fixed erythema over the malar eminences)

2.

Discoid rash (erythematous raised patches)

3.

Photosensitivity (skin rash as a result of unusual reaction to sunlight)

4.

Oral ulcers (oral or nasopharyngeal ulceration)

5.

Arthritis (non erosive arthritis involving 2 or more peripheral joints)

6.

Serositis (pleuritis or pericarditis)

7.

Renal disorder (proteinuria ≥ 0.5 g/24 h or cellular casts; red cell, hemoglobin, granular, mixed)

8.

Neurologic disorder (seizures or psychosis)

9.

Hematologic disorder [hemolytic anemia with reticulocytosis or leukopenia < 4 × 109/L on 2 or more occasions, or lymphopenia < 1.5 × 109/L on 2 or more occasions or thrombocytopenia < 100 × 109/L in the absence of offending drugs

10.

Immunologic disorder (specific autoantibodies)

11.

Antinuclear antibody (abnormal titer)

* The proposed classification is based on 11 criteria. For the purpose of identifying patients in clinical studies, a person shall be said to have systemic lupus erythematosus if any 4 or more of the 11 criteria are present, serially or simultaneously, during any interval of observation.

Table 25.2-1 Immunofluorescent patterns on HEp-2 cells, associated antigens and diseases /13/

Immuno-
fluorescent
pattern

Antigens

Disease
association

Nuclear pattern (fluorescent pattern of the nucleus)

Homogeneous nuclear
fluorescence

dsDNA

SLE; rarely other autoimmune diseases such as autoimmune hepatitis, primary Sjögren’s syndrome

ssDNA

Drug-induced SLE (high-titer), SLE, various other diseases; non disease specific

Nucleosomes (chromatin)

SLE (highly specific)

Histones H1, H2A, H2B, H3, H4

DNA-histone complexes

Drug-induced SLE (high titer), SLE and others

Fine speckled nuclear
fluorescence

SSA/Ro

Primary Sjögren’s syndrome, SLE, rheumatoid arthritis, mixed connective tissue disease (MCTD), neonatal lupus

SSB/La

Primary Sjögren’s syndrome (pSS), SLE, rarely other diseases (e.g., autoimmune hepatitis)

Sm

SLE (highly specific)

Mi-2

Dermatomyositis

Ku

Overlap syndrome (scleroderma/dermatomyositis), SLE, pSS

Coarse speckled nuclear
fluorescence

U1-RNP

MCTD, rarely SLE, systemic scleroderma

Sm

SLE (highly specific)

Scl-70
(DNA topoisomerase I)

Scleroderma

Nucleolar circular

Scl-70
(DNA topoisomerase I)

Scleroderma

Nucleolar homogeneous

Complex of nucleoproteins such as PM-Scl, To/Th

Myositis/scleroderma overlap syndrome, scleroderma, polymyositis, dermatomyositis

Nucleolar clumpy

Fibrillarin
(U3-RNP-associated protein)

Scleroderma

Nucleolar granular

RNA polymerase I, II, III

Scleroderma (diffuse form)

Nucleolar dots

NOR-90

Scleroderma

Heterogeneous nuclear
staining in different phases
of mitosis

PCNA (proliferating cell nuclear antigen)

SLE (highly specific)

PCNA like (antigen unknown)

Association with malignant disease has been described

Few nuclear dots(1)

P80 coilin

Primary biliary cirrhosis (PBC), rarely scleroderma

Nuclear dots(1)

SP100 protein

Primary biliary cirrhosis (PBC), rarely scleroderma

Centromeres(1)

CENP-A, -B, -C

CREST syndrome, PBC

CENP-F (mitosin)

Association with malignant disease has been described

Membranous(1)

Lamin B receptor, gp210

Primary biliary cirrhosis (highly specific)

Cytoplasmic patterns

Fine speckled
(fine granular)

Aminoacyl-tRNA synthetase, e.g. Jo-1 (histidyl-tRNA synthetase), PL-7, PL-12

Polymyositis, Jo-1 syndrome

Coarse speckled

Mitochondria

Primary biliary cirrhosis

Perinuclear

Golgi apparatus

SLE, rheumatoid arthritis, primary Sjögren’s syndrome

Filamentous

Cytoskeleton (keratin, vimentin, desmin, neurofilaments)

No specific disease association described

Actin (especially F-actin)

Autoimmune hepatitis

(1) Immunofluorescence patterns on HEp-2 cells are generally very typical, thus often eliminating the need for differentiation by immunoassay.

Table 25.2-2 Autoantibodies and target antigens in systemic autoimmune diseases, especially connective tissue diseases

Autoantibodies, Association

Clinical and laboratory findings

ANA

Connective tissue disease (inclusive myositis,drug-induced SLE), autoimmune hepatitis, juvenile idiopathic arthritis

ANA: general term used to describe the group of non organ specific antibodies that are directed against components of the cell nucleus. In a broader sense, it also includes antibodies directed against cytoplasmic antigens. The antibodies are detected by IIFT on HEp-2 cells or by immunoassays using cell lysate or a combination of defined nuclear antigens as a substrate. If the reaction is positive, differentiation using specific antigens is recommended.

ENA

Connective tissue disease (inclusive myositis, drug-induced SLE)

ENA: umbrella term for certain ANA whose antigens are obtained from cell lysate by saline extraction. More specifically, these are SS-A/Ro, SS-B/La, U1-RNP, Sm, and Scl70, although often further antigens are included (e.g., PM/Scl, Jo-1, CENP). Anti-dsDNA antibodies were originally not included, because DNA does not dissolve in the saline extract. These purification processes are increasingly being replaced by the use of recombinant proteins/DNA. A laboratory order for ENA testing often means the determination of autoantibodies to specific single antigens for the purpose of ANA differentiation or ANA profiling including anti-dsDNA antibodies.

Autoantibodies relevant for differential diagnosis

dsDNA /14/

Systemic lupus erythematosus (SLE)

Antibodies to native double stranded DNA of the nucleus are directed against the phosphoribosyl backbone of the native double-stranded DNA helix. They are one of 11 criteria for the classification of SLE.

Method of determination: antibody screening and monitoring is usually performed with an enzyme immunoassay. These assays often also detect low avidity antibodies. Older ELISAs already were very sensitive, but not very specific. Newer ELISAs, in contrast, have become significantly more specific. However, radio immunoassays (Farr test) are still the gold standard. The Crithidia luciliae IIFT detects only high avidity, high titer dsDNA antibodies and thus has low diagnostic sensitivity with high specificity for SLE. The assay is semi quantitative and, like all IIFTs, subject to subjective evaluation, which limits its suitability for monitoring.

Clinical significance: at an activity of greater than 70 U/L, the diagnostic specificity of the Farr assay for SLE is 95%. The diagnostic sensitivity is 90% for active SLE with kidney involvement and 60% for inactive SLE. Some enzyme immunoassays also detect low avidity antibodies and may also be positive in Sjögren’s syndrome, systemic sclerosis and autoimmune hepatitis. Rising titers (concentrations) correlate with the activity of SLE and its organ manifestations, such as glomerulonephritis.

Nucleosome Ab (chromatin Ab)

Systemic lupus erythematosus

Nucleosomes are the structural subunits of chromatin. Autoantibodies to nucleosomes mainly target dsDNA and histone proteins.

Method of determination: in the ANA IIFT, anti nucleosome antibodies produce a homogeneous fluorescent pattern. For specific detection, immunoassays are used.

Clinical significance: approximately 70% of SLE patients have autoantibodies to nucleosomes. The significance of the reactivity of antibodies to isolated nucleosomes in the ELISA is controversial. Anti-nucleosome antibodies may be detectable before anti-dsDNA antibodies and are thus a potential marker for the very early diagnosis of SLE.

SSA/Ro

These autoantibodies are usually referred to as SSA. Ro is an older term. In the antigen preparation from cell extracts, the SSA complex consists of a 60 kDa and a 52 kDa protein. By using recombinant antigens it has been shown that the two proteins belong to different protein families.

Together with a low molecular weight RNA, SSA60 forms den hY ribonucleoprotein particle (h, human; Y, cytoplasmic). Ro52 has been identified as TRIM21 [RING/Bbox/coiled-coil (RBCC) tripartite motif (TRIM)] protein with the function of a ubiquitin ligase /15/. Since the two antibodies also differ in their clinical associations, some authors no longer consider Ro52 a subunit of SSA.

SSA60

  • Sjögren’s syndrome,
  • SLE,
  • subacute cutaneous LE,
  • neonatal LE,
  • SLE/SS overlap,
  • ANA-negative SLE

Method of determination: in the ANA IIFT, SSA antibodies produce a fine speckled nuclear pattern. SSA60 is species specific, and 10% of anti-SSA/Ro-positive sera do not react with extracts from rodent organs, which is one explanation for the ANA-IFA-negative SLE described previously when rat liver sections were used for the detection of ANA. For specific detection, immunoassays with recombinant antigen are used.

Clinical significance for Sjögren’s syndrome: anti-SSA are present in 95% of patients.

Clinical significance for SLE: high association with congenital heart block in neonatal LE. The latter is likely caused by anti-SSA and anti-SSB crossing the placenta. Skin disorders (annular and periorbital edemas, discoid skin lesions) as well as splenomegaly, thrombocytopenia and hemolytic anemia occur during the first few weeks of life and disappear by 6 months of age, when the autoantibodies acquired from the mother have largely been eliminated. The incidence of congenital complete heart block is 1 per 20,000 newborns. Anti-SSA60 is detected in > 90% of mothers. There is an association with homozygous C2 complement deficiency and a clinical picture similar to that of LE, in particular subacute cutaneous LE and the detection of anti-SSA60. Older people with SLE are more frequently positive for anti-SSA60, especially if they are also found to have anti-SSB autoantibodies. The SLE is usually mild.

Ro52,TRIM21

Myositis, Sjögren’s syndrome, SLE, interstitial lung disease/fibrosis, rarely autoimmune hepatitis, RA, other connective tissue diseases without clinical association

Ro52 most likely represents one of the most immunogenic human proteins. Antibodies to Ro52 are frequently associated with other, more specific autoantibodies. The significance of isolated anti-Ro52 autoantibodies is controversial, although newer studies performed with improved assays do seem to show a clear association with various immune diseases /15/. Nevertheless they are associated with congenital heart block and must be reported especially in pregnant women.

SSB/La

Sjögren’s syndrome (SS), rarely SLE

SSB/La autoantibodies are usually referred to as (anti-)SSB. La is an older term. SSB is a phosphoprotein (MW 48 kDa). It acts as a transcription termination factor of RNA polymerase III and is localized in the nucleus and in the cytoplasm. SSB antibodies almost always occur together with SSA60 antibodies. In the ANA IIFT on HEp-2 cells, anti-SSB antibodies produce a fine speckled nuclear pattern. For specific detection, immunoassays with recombinant protein are used.

Anti-SSB is present in 40–70% of patients with Sjögren’s syndrome, rarely in patients with SLE.

U1-nRNP

Mixed connective tissue disease (MCTD; Sharp’s syndrome), rarely in SLE, systemic scleroderma (with joint and muscle involvement)

The complex to which U1 nRNP antibodies bind consists of an RNA component known as U1 (rich in uridine), which is complexed with seven or more D proteins (MW 12–68 kDa).

Method of determination: in the ANA IIFT, U1-RNP antibodies produce a coarse speckled nuclear pattern. For specific detection, immunoassays with the recombinant protein are used.

Clinical significance: patients with MCTD are always anti-U1 nRNP positive, since this marker is part of the disease definition.

Sm

Systemic lupus erythematosus

Anti-Sm react mostly with the D proteins D1, D2, D3, rarely with the E, F or G proteins of the U1 nRNP particles in the spliceosomes. For this reason, anti-Sm antibodies usually also react to native U1 nRNP preparations.

Method of determination: in the ANA IIFT, anti-U1 nRNP and anti-Sm antibodies produce a coarse speckled nuclear pattern. Anti-Sm may also give a fine speckled pattern. For specific detection, immunoassays with native antigen are used.

Clinical significance: although anti-Sm antibodies are highly specific for SLE (> 95%), they only occur in 10–30% of SLE patients.

PCNA

Systemic lupus erythematosus

Proliferating cell nuclear antigen (PCNA) is a protein with a molecular weight of 35 kDa. PCNA is also known as cyclin. During the S phase of mitosis it is part of the DNA synthetase complex.

Method of determination: in the ANA IIFT, PCNA antibodies produce a characteristic pattern in which the cells are stained very in homogeneously depending on the mitotic phase. Some cells show almost negative staining, others a clearly positive fine granular staining pattern, and yet others a coarse granulated pattern. It is sometimes difficult to distinguish these patterns from the PCNA like pattern that is produced by a not well characterized autoantibody and may be associated with tumors. For specific detection of PCNA antibodies, immunoassays are used.

Clinical significance: although PCNA antibodies are rare in SLE (2–5%), they are highly specific (> 95%).

Scleroderma associated autoantibodies /16/

Scl-70

Diffuse systemic scleroderma

Scl-70 is identical to DNA topoisomerase I. It is a nuclear enzyme that introduces or removes super helical turns from the DNA strands by breaking and rejoining a singular DNA strand within the DNA double helix.

Method of determination: in the ANA IIFT, anti-Scl-70 antibodies produce a mixed coarse speckled nuclear and, in some cells, nucleolar pattern, usually also cytoplasmic fluorescence. For specific detection, immunoassays are used.

Clinical significance: anti-Scl-70 antibodies can be found in diffuse systemic scleroderma with early involvement of internal organs and pulmonary fibrosis. They are present in 20–40% of Caucasians, 37% of Blacks in the USA, and in 76% of Asians.

Centromere protein (CENP)

CREST syndrome, PBC

The antibodies to centromere proteins react with several of the following proteins, but most commonly with CENP-B:

  • CENP-A, a protein related to histone 3 with a MW of 17 kDa
  • CENP-B, a DNA-binding protein (MW 80 kDa)
  • CENP-C, a component of the chromosomes which plays a role in kinetochore assembly; MW 140 kDa
  • CENP-D, a kinesin like motor protein; MW 50 kDa
  • CENP-E, a kinesin like motor protein; MW 312 kDa
  • CENP-F, mitosin (matrix protein of the chromosomes).

Method of determination: in the ANA IIFT on HEp-2 cells, CENP antibodies produce a centromeric pattern. For specific detection, immunoassays are used.

Clinical significance: anti-centromere antibodies occur specifically in systemic scleroderma and primary biliary cirrhosis (PBC). They are most commonly found in CREST syndrome (55–80%), rarely in the diffuse (3–12%) and systemic (10–40%) forms. CENP-F antibodies have been described in association with malignant disease.

RNA polymerase III (RNAP)

Usually diffuse scleroderma, often involvement of the kidneys and heart

RNAPs are multi protein complexes of 8–14 proteins with a MW between 10 and 220 kDa. There are 3 classes of RNAPs which, due to their different locations in the cell, can cause mixed patterns of nucleolar and cytoplasmic fluorescence on Hep-2 cells. Anti-RNAP III are found in 5–22% of scleroderma patients and are highly specific. For specific detection, radio immune precipitation, immunoblot or ELISA with recombinant RNAP-III fragment are used.

Fibrillarin U3 RNP = Scl34

Usually diffuse scleroderma, often involvement of the kidneys and heart

Fibrillarin has a MW of 34 kDa and is the main component of the nucleolar U3-RNP complexes. Autoantibodies to fibrillarin produce a granular pattern of the nucleoli on HEp-2 cells. For specific routine detection of the autoantibodies, a line blot with recombinant fibrillarin is used. Fibrillarin antibodies are found in 2–5% of scleroderma patients and are highly specific.

To (Th), PM-Scl, Ku, U1-RNP

See myositis associated autoantibodies in this table.

Myositis specific autoantibodies (> 98% specificity) /17/

Aminoacyl-tRNA synthetases

Polymyositis/ dermato­myositis, anti-synthetase syndrome, Jo-1 syndrome

Histidyl-tRNA synthetase (Jo-1)

Prevalence 20–30%

Threonine-tRNA synthetase (PL-7)

Prevalence 2–3%

Alanine-tRNA synthetase (PL-12)

Prevalence 2–3%

Glycine-tRNA synthetase (EJ)

Prevalence 1–2%

Isoleucine-tRNA synthetase (OJ)

Prevalence 1–2%

These are antibodies to cytoplasmic aminoacyl-tRNA synthetases, a group of 20 different enzymes. The latter catalyze the loading of the tRNA at their 3’ terminal ends with a specific amino acid. For each amino acid there are one or several aminoacyl-tRNA synthetases.

Jo-1 antibodies are the most frequent of this group (directed against histidyl-tRNA synthetase). There are also autoantibodies to threonine-tRNA synthetase (PL-7) and alanine-tRNA synthetase (PL-12).

Method of determination: in the ANA IIFT on HEp-2 cells, these antibodies produce a cytoplasmic fine speckled pattern (ribosomal pattern). For specific detection, immunoassays are used.

Clinical significance: Jo-1 antibodies can be found in 15–45% of patients with autoimmune myositis and especially polymyositis. Jo-1 antibodies are always present in Jo-1 syndrome, whereas other myositis associated autoantibodies (anti-U1RNP, anti-Mi-2) are rare. PL-7 and PL-12 antibodies are considerably less common in polymyositis, each occurring with a prevalence of about 2–3%.

Anti-SRP (signal recognition article)

Prevalence 4%

Clinical significance: severe acute polymyositis with necrotizing myopathy, no involvement of joints, lungs and skin.

Mi-2

Dermatomyositis, rarely polymyositis

Anti-Mi-2 are directed against a group of at least 6 nuclear proteins, including a 240 kDa helicase.

Method of determination: in the ANA IIFT, anti-Mi-2 produce a fine speckled nuclear pattern. For specific detection, immunoassays are used.

Clinical significance: anti-Mi-2 can be found in 5–31% of adults and in 10–15% of children with dermatomyositis with a specificity of more than 95%. Anti-Mi-2 are detectable once clinical symptoms appear. Patients with anti-Mi-2 dermatomyositis have a better prognosis than those with anti-Jo-1. Anti-Mi-2 are rarely seen in polymyositis.

Myositis associated autoantibodies /18/

Ku /19/

Polymyositis/dermatomyositis overlap syndrome, Sjögren’s syndrome

1–7%

Ku is a heterodimeric protein composed of one 70 kDa and one 80 kDa subunit. It has a high binding affinity for damaged DNA. Ku is the first name of the Japanese patient in whom the protein was first identified. Ku is localized in the nucleus and the nucleoli.

Method of determination: in the ANA IIFT on HEp-2 cells, anti-Ku antibodies produce a reticular granular cytoplasmic pattern. For specific detection, immunoassays are used.

Clinical significance: Ku antibodies can be found in 20% of patients with the above autoimmune diseases.

PM-Scl

Polymyositis/scleroderma overlap syndrome, systemic sclerosis

Prevalence 8–12%

The PM-Scl complex consists of up to 16 nucleolar proteins.

Method of determination: in the ANA IIFT, PM-Scl antibodies produce a nucleolar pattern. For specific detection, immunoassays are used.

Clinical significance: PM-Scl antibodies can be found in approximately 25% of patients with polymyositis/scleroderma overlap syndrome.

U1-snRNP

Prevalence 4–17%

Overlap syndrome with mixed connective tissue disease

SSA/Ro60

Prevalence 5–10%

Overlap syndrome with Sjögren’s syndrome

SSA/Ro52

Prevalence 25%

Often in myositis in association with other myositis antibodies

Antigens of lesser relevance

ssDNA /20/

Positive ANA without a clear clinical association and negative ENA, dsDNA

Method of determination: IIFT, homogeneous pattern, differentiation by immunoassay.

Clinical significance: high titer anti-ssDNA antibodies can mainly be found in drug induced LE. They are of little diagnostic significance due to their low specificity. ssDNA antibodies are seen in many disorders, not only rheumatic diseases. They may also occur transiently due to an infection as well as in healthy individuals.

Histone /21/

Drug induced SLE, rheumatoid arthritis, SLE, Felty’s syndrome

Histones are a group of basic proteins named H1, H2A, H2B, H3 and H4 according to their chromatographic behavior. Together with the dsDNA they form the nucleosome.

Method of determination: IIFT, homogeneous pattern, differentiation by immunoassay.

Clinical significance: high titer anti-histone antibodies can be found especially in drug induced LE. Approximately 85% of patients with Felty’s syndrome are positive for histone antibodies. The antibodies are detectable even before the onset of clinical symptoms and may persist for years after therapy. They are of little diagnostic significance due to their low specificity. Anti-histone antibodies are seen in many disorders, not only rheumatic diseases. They may also occur transiently due to an infection as well as in healthy individuals.

To/Th

SLE, Raynaud’s syndrome

To and Th antigens are endoribonucleases in RNAse particles. To/Th antibodies produce a homogeneous nucleolar pattern in the IIFT. For specific detection, an immunoblot with recombinant Th40/Rpp38 protein is used. No commercial assay is available.

Cytoplasmic antigens

Ribosomal P proteins /22/

SLE

Ribosomal P proteins (RPP) are phosphoproteins of the 60 S subunit of ribosomal complexes. A distinction is made between the P0 (38 kDa), P1 (19 kDa) and P2 (17 kDa) proteins. RPP antibodies are detected by immunoblot analysis. They are present in approximately 20% of SLE patients.

Jo-1

Polymyositis, .Jo-1 syndrome

See aminoacyl-tRNA synthetases.

Mitochondria

Primary biliary cirrhosis

See Section 25-8 –ANCA associated liver diseases.

Golgi apparatus

Non specific

No diagnostic relevance, present in SLE, rheumatoid arthritis, primary Sjögren’s syndrome.

Cytoskeleton

No specific disease association

Antibodies to keratin, vimentin, desmin and neurofilaments have no diagnostic relevance.

Cytoskeleton

Autoimmune hepatitis

Anti-actin: see Section 25-8 – ANCA associated liver diseases.

Table 25.2-3 Prevalence of ANA and association with disease /5/

Disease

Prevalence (%)

Indication for ANA testing

  • SLE

95–100 (total)

80–100 (inactive)

100 (active)

  • Drug induced LE

10–50

  • Discoid LE

20–80

  • Subacute cutaneous LE

100

  • Mixed connective tissue disease
  • (Sharp’s syndrome)

85–98

  • Progressive systemic sclerosis

95–100

  • CREST syndrome

95–100

  • Polymyositis/dermatomyositis

63–78

  • Sjögren’s syndrome

50–70

  • Juvenile idiopathic arthritis

About 15

  • Autoimmune chronic active hepatitis

25–33

ANA as a minor finding

  • Panarteritis nodosa

18–25

  • Rheumatoid arthritis

15–40

  • Felty’s syndrome

30–60

  • Psoriatic arthritis

24–67

  • Other rheumatic diseases

< 5

  • Fibrosing alveolitis

About 33

  • Myasthenia gravis

40–60

  • Healthy blood donors by comparison:

0–2 (< 40 years)

0–5 (41–60 years)

5–20 (< 60 years)

Table 25.2-4 Prevalence (%) of antibody findings in systemic autoimmune diseases /523/

Auto-
antibody

SLE

Sjögren’s
syndrome

Sclero-
derma

MCD

ANA

95

85

95

100

dsDNA

50

< 5

5

SSA/Ro

50

85

5

SSB/La

15

60

Sm

10

U1-nRNP

15

100

Scl-70

30

PCNA

5

Centromere

35

SLE, systemic lupus erythematosus; MCD, mixed connective tissue disease

Table 25.3-1 Prevalence of rheumatoid factors in rheumatic diseases /56/

Disease

Prevalence (%)

Rheumatoid arthritis

70– 90

Systemic lupus erythematosus

15– 35

Sjögren’s syndrome

75– 95

Mixed connective tissue disease

50– 60

Systemic sclerosis

20– 30

Essential mixed cryoglobulinemia type II*

100*

Systemic vasculitides (e.g., panarteritis nodosa, Wegener’s granulomatosis)

5– 20

Chronic sarcoidosis

5– 30

Chronic liver diseases (e.g., chronic active hepatitis, primary biliary cirrhosis)

15– 70

Chronic inflammatory lung diseases (e.g., pulmonary fibrosis, silicosis, asbestosis)

10– 50

Subacute bacterial endocarditis

25– 65

Other bacterial infections (e.g., mycobacteria, spirochetes, brucellae, salmonellae)

5– 60

Parasitic infections (e.g., trypanosomes, plasmodium, schistosomes, trichinae)

20– 90

Viral infections and vaccinations (e.g., EBV, CMV, HIV, hepatitis, influenza, rubella)

15– 65

Neoplasias, especially following irradiation or chemotherapy

5– 25

Healthy individuals < 50 yearsS

< 5

Healthy individuals > 70 years

10– 25

* Monoclonal IgM rheumatoid factors

Table 25.4-1 Prevalence of anti-CCP antibodies (anti-CCP-2) in diseases according to Ref. /7/

Disease

Number
of cases

Prevalence
(%)

Rheumatoid arthritis

2958

71

  • Early RA (< 1 year)

2126

57

  • Juvenile chronic arthritis

696

6

Systemic LE

817

6

Sjögren’s syndrome

625

5

Poly-/Dermatomyositis

195

2

Systemic vasculitis

74

4

Systemic sclerosis

317

6

Psoriatic arthritis

381

8

Other types of spondyloarthritis

161

2

Lyme disease

86

5

Reactive and viral types of arthritis

109

4

Polymyalgia rheumatica

109

2

Osteoarthritis and other non inflammatory rheum. diseases

178

5

Infectious diseases without arthritis

679

2

Healthy individuals (including elderly people)

3949

0.5

Table 25.5-1 Idiopathic inflammatory myopathy (IIM): muscle-specific autoantibodies /7/

Clinical and laboratory findings

Anti-synthetase autoantibodies

Anti-synthetase antibodies (ASA) are directed against aminoacyl-tRNA synthetase enzymes. Every synthetase catalyzes attachment of a particular amino acid to its cognate tRNA. Synthetases differ greatly in size and primary structure and are immunologically distinct. Autoantibodies to 8 of the 20 aminoacyl-tRNA synthetase enzymes have been described. Patients with idiopathic inflammatory myositis and ASAs make the anti synthetase syndrome. However, ASAs are non-specific and can be detected in many situations (e.g., myositis, interstitial lung disease or apparently isolated polyarthritis) /4/. The phenotype and prognosis of the different subtypes of IMM and the correspondent ASAs are different. Anti-histidyl-t-RNA synthetase (anti-Jo-1) is found in about 30%, anti-PL7 or anti-PL12 in 3–4% and other ASAs in less than 2% of patients with IMM. Although the correlation between anti-Jo-1 levels and CK elevation is only moderate at diagnosis, in longitudinal analysis the anti-Jo-1 antibody titers are very well correlated with the CK level /4/.

Anti-Jo-1 antibodies are found in up to 95% of patients with anti-synthetase syndrome and idiopathic interstitial pneumonia. Patients who are negative for anti-Jo-1, but positive for anti-PL-12, anti-KS or anti-OJ also frequently have idiopathic interstitial pneumonia. Patients with anti-PL-7 also have interstitial pneumonia, but milder muscle symptoms and lower CK levels compared to those with anti-Jo-1. In children with juvenile myositis, the prevalence of anti-synthetase antibodies is 2.6%, which is markedly lower than in adults.

Individual patients generally have autoantibodies to only one synthetase /2/. Anti-Jo-1 is found in about 20% of patients with PM or DM. The frequency of other anti synthetases among myositis patients depends on the area of a country /5/.

According to a study /3/ anti-synthetase syndrome is a subgroup of IMM.

Anti-signal recognition particle (SRP) autoantibodies

The SRP is a ribonucleoprotein complex and contains six proteins with molecular weights of 9, 14, 19, 54, 68 and 72 kDa. The cytoplasmic SRP recognizes secretory or cytoplasmic proteins and regulates their translocation through the endoplasmic reticulum.

Approximately 5% of patients with IIM are positive for anti-SRP and belong to the immune-mediated necrotizing myopathies (IMNM) of the IMM subgroup. The phenotype is an isolated myopathy associated with up to one third of cases with cardiomyopathy. The patients usually have many necrotic muscle fibers as the predominant abnormal feature. Inflammatory cells are sparse or only slight perivascular; perimysial infiltrate is not evident /4/. The disease progression might be very slow notably in white patients, mimicking then a limb girdle muscular dystrophy. Patients often present with acute severe myopathy and dysphagia and have markedly elevated CK. There is a good correlation between anti-SRP titers, CK levels and muscle weekness.

If anti-SRP are detected in children with myopathy, there is often no extra muscular involvement /8/.

The anti-SRP antibody reacts with SRP, which is an RNA protein complex that binds newly synthesized proteins and directs them to the endoplasmic reticulum for translocation. Anti-SRP autoantibodies precipitate the 7SL RNA that is specific for the antigen. Almost all patients have PM rather than DM. The myositis of anti-SRP patients is more likely to be acute in onset, severe, and resistant to therapy /5/.

Anti-Mi2 autoantibodies

Mi-2 is a nuclear helicase protein and part of a nucleosome remodelling de acetylase (NuRD) complex which plays a role in the transcription of genes.

Anti-Mi2 antibodies (anti-complex nucleosome remodelling histone deacetylase) are positive in 9% of patients with IIM and in 20% of patients with DM rash. The target of this autoantibody is a nuclear antigen. The antibody is present almost exclusively in patients with DM. Anti-Mi2 are also present in 4–10% in cases with juvenile DM or juvenile myositis overlap syndrome. Anti-Mi2 are predominantly positive in DM patients with significant skin lesions. Patients with anti-Mi2 have mild muscle involvement and a low risk of idiopathic interstitial pneumonia.

According to a study /3/ patients who have anti-Mi2, anti-melanoma differentiation-associated protein 5 (MDA5), or anti-transcription intermediary factor 1γ (TIF1γ) antibodies mainly correspond with patients who have dermatomyositis with rash.

Anti-MAD5

The antigen of these antibodies is melanoma differentiation associated gene 5 (MAD 5). MAD5 is involved in innate immune defense against viruses. Patients with anti-MAD5 antibodies do not harbor the classical feature of dermatomyositis and do not meet pathological criteria for diagnosis of definitive or possible dermatomyositis /4/. They have the cutaneous symptoms of DM, but no clinically significant muscle involvement. They are, however, at risk of developing idiopathic interstitial lung disease.

Anti cytosolic 5’nucleotidase antibodies (anti-cN1A) are present in inclusion body myositis. The disease is first an inflammatory condition with a second aggravating vacuolar myopathy by lysosome/proteasome dysfunctions /8/.

Table 25.5-2 Muscle-associated autoantibodies in idiopathic inflammatory myopathy (IIM) /1/

Antibodies

Clinical and laboratory findings

Anti-Ku

Patients with these autoantibodies often have an overlap of symptoms of IIM and systemic sclerosis (30–55%).

Anti-U1-RNP

Approximately 48–73% of patients with high antibody titers have overlap symptoms of the mixed connective tissue disease type (MCTD, Sharp’s syndrome). About 25% of SLE patients have low titers of these autoantibodies. Patients with a high titer often have scleroderma and/or myositis.

Anti-PmScl

Approximately half of all patients with this antibody have myositis. Approximately 80% of patients develop skin changes typical of scleroderma as well as Raynaud’s symptoms during the course of disease.

Table 25.5-3 Frequency of autoantibodies in inflammatory idiopathic myopathies

Name

Antigen

Autoantibody
frequency (%)

Myositis-specific autoantibodies

Jo-1

Histidyl-tRNA synthetase

25–30%

PL-7

Threonyl-tRNA synthetase

< 3

PL-12

Alanyl-tRNA synthetase

< 3

EJ

Isoleucyl-tRNA synthetase

< 2

OJ

Glycyl-tRNA synthetase

< 2

KS

Asparaginyl synthetase

< 2

Ha

Tyrosyl synthetase

< 2

ZO

Phenylalanyl synthetase

< 2

Total

Aminoacyl-tRNA synthetases

30–40

SRP

Signal recognition particle

4–5

Mi-2

Helicase of the SNF2 family

8–12

Myositis associated autoantibodies

PmScl

Exonuclease complex

8–15

U1-RNP

U1 small nuclear ribonucleoprotein

12

U2-RNP

U2 small nuclear ribonucleoprotein

< 3

Ku

Component of the DNA dependent protein kinase

1–7

Table 25.5-4 Subgroups of idiopathic inflammatory myopathies (IIM) /4910/

Clinical and laboratory findings

Dermatomyositis (DM)

Clinical presentation: DM is characterized by a skin rush that accompanies or precedes proximal symmetrical muscle weakness. Patients of 5–15 years and 45–65 years are affected. DM is an acute severe illness characterized by heliotrope colored erythema (Lilac disease) on the face, trunk and extremities. More specifically, the erythema occurs around the eyelids, on the cheeks and the anterior cervical triangle. Subcutaneous calcifications may develop, especially in childhood and juvenile DM. In some cases there is involvement of organs such as the pharynx and lower esophagus.

Pathophysiology /10/: an early event in the disease is the damage to the endothelial cells of endomysial capillaries mediated by complement activation and formation of the membrane attack complex (MAC), which causes lysis of the endothelial cells, destruction of capillaries, and muscle ischemia. As a result, the number of capillaries is reduced, while the lumen of the remaining ones is dilated to compensate for the ischemic process. The complement activation triggers pro- inflammatory cytokines and thus activates the immune system. The histological picture is that of perifascicular interstitial inflammation with infiltration of inflammatory cells.

Laboratory findings: during the acute phase, CK rises up to 50-fold, in rare cases normal activities may be found. Anti- Mi2 antibodies are found in 20% of patients with DM rash.

Polymyositis (PM)

PM develops subacutely, proximal symmetrical skeletal muscles become atrophic, and almost exclusively adults. There are no inflammatory skin conditions. The main manifestation is weakness of the proximal arm and leg muscles. The muscle atrophies are significant, but often asymmetric in nature. Progressive muscle weakness leads to difficulty in swallowing, speaking, rising from sitting position and climbing stairs. Polymyositis is an over diagnosed entity most of these patients can be reclassified among dermatomyositis (sine dermatitis), overlap myositits, immune-mediated necrotizing myopathy, inclusion body myositis or inherited myopathies with inflammation /4/.

Laboratory findings: during the acute phase, CK rises up to 50-fold, in rare cases normal activities may be found. Anti- Mi2 antibodies are found in 20% of patients with rash indicating dermatomyositis.

Overlap syndrome

One third of patients with IIM have overlap syndrome. This occurs predominantly in conjunction with DM. Overlap syndromes include progressive systemic sclerosis and Sharp’s syndrome. Besides the symptoms of DM or PM, the syndrome may also clinically manifest as SLE, systemic sclerosis or Sjögren’s syndrome. One clinical entity of overlap syndrome is anti-synthetase disease, within which Jo-1 syndrome is a distinct entity.

Anti-synthetase disease (ASSD): the ASSD is an autoimmune disease characterized by arthritis, myositis, and interstitial lung disease. Patients have anti-synthetase antibodies and clinical multi-organ disease with polymyositis, polysynovitis (arthralgias, arthritis, tenosynovitis) and fibrosing alveolitis. Rhagades and keratoses on the hands (mechanics’ hands) are common. Onset of the disease usually occurs in spring, with fever, leukocytosis and diffuse hand and feet edemas. Caucasian patients with anti-synthetase syndrome often have the HLA alleles DRB1*0301, DQA1*0501. According to a review 24% of patients have isolated arthritis as the present feature /9/. Anti-Jo-1 is the most frequently detectable antibody in the ASSD.

Jo-1 syndrome: patients with anti-Jo 1 differ from those with other anti-synthetase antibodies. The disease usually has a subacute onset. Patients typically suffer from proximal muscle weakness in combination with exertional dyspnea and often have dry cough, dysphagia, arthritis, sicca syndrome and Raynaud’s phenomenon. The disease prognosis is determined by the degree of lung involvement.

Laboratory findings: anti-PmScl, anti-Ku or anti-U1-RNP antibodies, anti-Jo-1 or other anti-synthetase antibodies, or anti-Mi2 or anti-SRP are positive. Furthermore, CK and CRP are elevated and there is leukocytosis.

Inclusion body myositis (IBM)

Clinical presentation: IBM differs from DM and PM in that it develops slowly and progressively from age 60, affecting both proximal and distal muscles, sometimes asymmetrically. Falling and tripping are usually the first noticeable symptoms of IBM. Patients have no inflammatory skin disease, but exhibit relative resistance to corticosteroid therapy and other immunosuppressive therapies.

Pathophysiology: IBM is a separate form of chronic inflammatory myopathies in terms of pathophysiology. Histopathologically, the muscle cells have characteristically altered vacuoles of ubiquitin or β-amyloid, and electron microscopy shows microtubular filaments in cytoplasmic or nuclear inclusions. The inflammatory infiltrates contain a high percentage of CD8+T cells. There is significant correlation with HLA-DR3.

Laboratory findings: slightly elevated CK. Identification of anti-cN1A antibodies in about 34% of patients. The specificity of the antibodies is disappointing as 36% of Sjögren’s syndrome sera and 20% of systemic lupus erythematosus sera are also positive /11/.

Necrotizing myositis (NM) /7/

During the acute or subacute onset of disease, patients have moderate to severe muscle weakness as a result of muscle fiber necrosis caused by macrophages. There are no T cell infiltrates or other histological findings such as are seen in DM and PM. The disease is multifactorial. Some patients have cancer, others a viral infection (HIV) and yet others are under statin treatment. Statins can cause both toxic and autoimmune myopathy (Tab. 1.8-5 – CK and CK-MB activities in acute skeletal muscle damage).

Laboratory findings: significant elevation of CK, some patients have antibodies to the signal recognition particle (SRP).

Table 25.6-1 Paraneoplastic neurological syndromes /1/

Syndromes of central nervous system

Encephalomyelitis

Limbic encephalitis

Brainstem encephalitis

Subacute cerebellar degeneration

Opsoclonus-myoclonus

Optic neuritis

Cancer associated myopathy

Melanoma associated retinopathy

Stiff person syndrome

Necrotising myelopathy

Motor neuron disease

Syndromes of peripheral nervous system

Subacute sensory neuronopathy

Acute sensimotor neuropathy

  • Guillain-Barre syndrome
  • Brachial neuritis

Subacute/chronic sensimotor neuropathies

Neuropathy and paraproteinemia

Neuropathy with vasculitis

Autonomic neuropathies

  • Chronic gastrointestinal pseudoobstruction
  • Acute pandysautonomia

Syndromes of the neuromuscular junction and muscle

Myasthenia gravis

Lambert-Eaton myasthenic syndrome

Acquired neuromyotonia

Dermatomyositis

Acute necrotising myopathy

Italics; classical paraneoplastic neurological syndromes

Table 25.6-2 Diagnostic criteria for paraneoplastic neurological syndromes (PNS) /6/

Definite PNS

1. A classical syndrome and cancer that develops within 5 years of the diagnosis of the neurological disorder.

2. A non classical syndrome that resolves or significantly improves after cancer treatment without concomitant immunotherapy, provided that the syndrome is not susceptible to spontaneous remission.

3. A non classical syndrome with onconeural antibodies (well characterized or not) and cancer that develops within 5 years of the diagnosis of the neurological disorder.

4. A neurological syndrome (classical or not) with well characterized onconeural antibodies (anti-Hu, anti-Yo, anti-CV2, anti-Ri, anti-Ma2, or anti-amphiphysin), and no cancer.

Possible PNS

1. A classical syndrome, no onconeural antibodies, no cancer but a high risk to have an underlying tumor.

2. A neurological syndrome (classical or not) with partially characterized onconeural antibodies and no cancer.

3. A non-classical syndrome, no onconeural antibodies, and cancer present within 2 years of diagnosis.

Table 25.6-3 Paraneoplastic antibodies (PNA) against neuronal intracellular antigens /1628/

Antibodies

Anti-Hu Ab (ANNA-1)

Anti-Hu antibodies target nuclear material of neurons of the central and peripheral nervous systems, of the retina, the adrenal cortex, and tumor cells. CNS syndromes associated with anti-Hu antibodies include para neoplastic encephalomyelitis (PEM), para neoplastic cerebellar degeneration (PCD), limbic encephalitis (LE), and brain stem encephalitis. Patients with LE are typically over 40 years old and smokers.

Laboratory findings: if detected, the antibody is highly sensitive for para neoplastic syndrome (PNS) and the clinical presentation of peripheral neuropathy. Approximately 70% of patients with LE and anti-Hu antibodies have small cell lung cancer (SCLC). Only 50% of patients with LE and PNS have detectable titers of anti-Hu antibodies. The proportion of anti-Hu positive patients without cancer is 2%. The prevalence of anti-Hu antibodies in cancer patients without PNS is 16%. However, anti-Hu titers are low in these patients, whereas patients with PNS have markedly higher titers, unless they are receiving cytostatic treatment, in which case titers may be low despite the presence of PNS. Anti-Hu antibodies often coexist with anti-amphiphysin, anti-CRMP-5 and anti-Ri.

Anti-Ri Ab (ANNA-2)

These antibodies target a 55 kDa and an 80 kDa antigen of the nuclei of peripheral neurons. The antibodies are typically seen in patients with opsoclonus myoclonus syndrome and ataxia in SCLC and breast cancer, but rarely found in LE. The proportion of anti-Ri positive patients without cancer is 3%. The prevalence of anti-amphiphysin antibodies in cancer patients without PNS is 4%. Anti-Ri antibodies often coexist with anti-amphiphysin, anti-CRMP-5 and anti-Hu.

ANNA-3

This antibody is rare and has been described in association with SCLC in few families. Immunofluorescence shows intense nuclear staining of Purkinje cells and podocytes.

Anti-CV2/CRMP-5 Ab

The antigen CRMP-5 (collapsin response mediator protein type 5), also known as CV2, is a 66 kDa phosphoprotein in the cytoplasm of oligodendrocytes, certain sensory peripheral neurons, Schwann cells and SCLC cells. Anti-CRMP-5 antibodies are the second most commonly diagnosed para neoplastic antibodies. They are similar to ANNA-1 in regards to the clinical picture and tumor diseases and are associated with SCLC in 80% of cases. Peripheral neuropathies are common, as well as hyperkinetic disorders, in particular chorea. The proportion of anti-CRMP-5 positive patients without cancer is 4%. The prevalence of anti-CRMP-5 antibodies in cancer patients without PNS is 9%. Patients with PNS and anti-CRMP-5 survive longer than those with ANNA-1. PNS patients with both antibodies have a median survival time of 18 months.

Purkinje cell antibodies, PCA-1 (YO)

Anti-PCA-1 antibodies target a 34 kDa and a 62 kDa antigen in the cytoplasm of Purkinje cells, where they bind to ribosomes, the endoplasmic reticulum and to vesicles of the Golgi apparatus. The antibodies are of high homogeneity and correlate strongly with gender and the type of cancer. Anti-PCA-1 occur almost exclusively in women with para neoplastic cerebellar degeneration and ovarian cancer or breast cancer. The proportion of anti-PCA-1 positive patients without cancer is 2%. The prevalence of anti-PCA-1 in cancer patients without PNS is 1%. Testing should be performed in cerebrospinal fluid, where the antibody concentration is higher than in serum due to intrathecal synthesis. In contrast to ANNA-1 and ANNA-2, anti-PCA-1 antibodies occur in isolation.

Anti-Tr Ab

These antibodies occur almost exclusively in men with para neoplastic cerebellar degeneration and Hodgkin’s disease. The proximal dendrites of Purkinje cells produce a fine speckled pattern on immunofluorescence.

Anti-amphiphysin Ab

Anti-amphiphysin Ab target a 128 kDa antigen which is located predominantly in presynaptic terminals in the granular layer. The granular layer of the cerebellum has many such terminals. In 74% of patients, anti-amphiphysin antibodies coexist with other para neoplastic antibodies such as ANNA-1, ANNA-2, anti-Purkinje cell Ab or anti-CRMP-5. The most common malignancy in men with para neoplastic syndrome and anti-amphiphysin Ab is SCLC (86%). The most common neurological manifestations are neuropathies, followed by encephalopathies. Anti-amphiphysin Ab are also found in stiff person syndrome, myoclonus and encephalomyelitis.

Anti-amphiphysin Ab are often also seen in cancer patients without para neoplastic syndrome, less frequently in patients without a tumor. The proportion of anti-amphiphysin Ab positive patients without cancer is 5%. The prevalence of anti-amphiphysin Ab in cancer patients without PNS is 1%.

Anti-Ma2 Ab (also known as anti-Ta)

Anti-Ma antibodies react with the Ma1, Ma2 and Ma3 proteins and cross react with a number of tumor antigens. Most commonly there is a para neoplastic association with anti-Ma2 Ab. The target antigen of the antibodies, a 41 kDa antigen, is localized in the neuronal nucleoli, less commonly in the nucleolus or the cytoplasm. Anti-Ma2 Ab are identified by immunofluorescence staining as a single dot in the nucleoli of cerebellar Purkinje cells.

Patients are predominantly male and under 40 years of age. Cancers associated with anti-Ma2 Ab include seminoma, testicular germ cell tumors and breast cancer. The proportion of anti-Ma2 positive patients without cancer is 4%. Anti-Ma2 Ab are not known to occur in cancer patients without para neoplastic syndrome. In such cases the patients have LE and common symptoms include hallucinations, loss of memory and seizures.

Anti-glial nuclear antibodies (SOX-1 Ab)

Anti-glial nuclear antibodies (AGNA) target the sex determining region Y-Box1 (SOX1). The antigen is localized in the nuclei of the cerebellar Bergmann glia cells. SOX1 belongs to the family of DNA binding transcription factors. The gene encoding SOX-1 is differentiated into subgroups A to H. SOX-1 antibodies occur in isolation either in neurological syndromes or in malignant tumors. SOX-1 Ab are no para neoplastic antibodies, but their presence together with VGCC Ab is highly indicative of SCLC. The frequency of SOX-1 Ab is significantly higher in patients with para neoplastic Lambert-Eaton myasthenia syndrome (LEMS) caused by VGCC (64%) than in those with idiopathic LEMS or SCLC alone (22%). SOX-1 Ab are thus important in distinguishing idiopathic LEMS from neoplastic LEMS, because the two entities cannot clinically be differentiated from one another.

Antigens are the zinc finger proteins Zic 1 to Zic 5. ZIC 4 genes are expressed in the cerebellum. Of clinical importance are anti-Zic 4 Ab. These react with the nuclei of the granular region of the cerebellum. The antibodies rarely occur in isolation. Over 80% of patients with anti-Zic4 Ab have other coexisting SCLC associated PNA (ANNA-1, CRMP-5). Approximately 92% of patients with isolated anti-Zic 4 Ab have SCLC. The most common clinical manifestations are disorders of the cerebellum.

Table 25.6-4 Non paraneoplastic antibodies against intracellular antigens /1628/

Antibodies

Anti-GAD Ab

Glutamic acid decarboxylase (GAD): the cytoplasmic enzyme GAD catalyzes the decarboxylation of glutamate to γ-aminobutyric acid, the main inhibitory transmitter of the CNS. GAD is synthesized mainly by GABAergic neurons of the CNS and in β cells of the pancreas /29/. The enzyme has two isoforms, the membrane associated GAD 65 and the soluble GAD 67, which differ in their amino-terminal regions. GAD 65 is attached to the inner surface of the synaptic vesicles of GABAergic neurons.

Anti-GAD65 Ab: these antibodies are found in 1% of the normal population and in 5% of patients with neurological disorders. There is a difference in the antigen spectrum between anti-GAD65 Ab in disorders of the CNS and type 1 diabetes, because anti-GAD65 Ab from type 1 diabetics do not react with GABAergic neurons from brain sections.

Indication: stiff person syndrome (SPS), where the antibodies are present in the sera of 80% and in the cerebrospinal fluid of 75% of SPS patients. They are also found in cerebellar ataxia, temporal lobe epilepsy, myoclonus, and non para neoplastic limbic encephalitis (LE). In patients with LE, often younger women, the clinical picture is dominated by temporal lobe epilepsy. They do not have a malignant tumor. Although anti-GAD65 Ab are not markers of para neoplastic disease, patients who test positive for these antibodies should be screened for tumors, since the antibodies have increasingly been observed in cancers of the lung, kidney, pancreas or thymus.

Laboratory findings: anti-GAD65 Ab are detected by RIA and ELISA. The upper reference interval value is 100 to 150 U/L. Levels above 1,000 U/L are measured in stiff person syndrome, GAD associated LE, cerebellar ataxia, and epilepsy. In non selected patients with inflammatory/autoimmune disorders of the nervous system, the prevalence of anti-GAD65 Ab is 0.48% /30/.

Anti-adenylate kinase 5 Ab

The antibodies targeting this enzyme can be detected in Lambert-Eaton myasthenie syndrome in the absence of a malignant tumor.

Anti-MOG Ab

Myelin oligodentrocyte glycoprotein (MOG) is expressed by the outer lamellae of the myelin sheath of the CNS. Anti-MOG Ab cause demyelination and, at the onset of acute disseminated encephalomyelitis (ADEM), are present in titers that do not correlate with the severity of disease and will decline again during the course of the disease. The highest titers are seen in children at the onset of ADEM /31/.

Table 25.6-5 Antibodies targeting extracellular cell-surface/synaptic neural antigens /1432, 3356/

Antibodies

Anti-glutamate receptor antibodies

Glutamate belongs to the group of excitatory amino acid transmitters of the CNS. It is involved in many functions of the brain, such as perception, learning, movement, and development. As a neurotransmitter, glutamate binds to its postsynaptic receptor (GluR), mediating a nerve pulse via the synapse between two nerve endings. Approximately 50% of the synaptic transmissions in the CNS are mediated via glutamate, which acts through inotropic receptors (iGluR) and metabotropic receptors (mGluR) localized on pre- and postsynaptic membranes. The iGluR has two subtypes, which are named according to their affinity to N-methyl-D aspartate (NMDA) or α-amino-3-hydroxy-5-methyl-4-isoxazolpropionate (AMPA). Both receptors are ion channels which, upon stimulation by glutamate, allow Na+ flow and membrane depolarization. Changes in the receptor activity, in particular hyperactivity, can lead to neurotoxicity with severe neuronal damage. Antibodies to the NMDA- and AMPA-sensitive iGluR can cause such damage.

Anti-NMDAR

N-methy-D-aspartate receptor (NMDAR): the receptor is named after the selective agonist N-methyl-D aspartate (NMDA). The receptor consist of four subunits, two obligatory GluN1 subunits, and GluN2 or GluN3 subunits, which form the glutamate-gated ion channel. The two GluNR1 units make up the binding site for glycine; the two NR2 units bind glutamate. The activation of NMDAR increases the content of Ca2+ within the cell, which triggers a cascade of events.

Anti-NMDAR: the antibodies of patients with anti-NMDAR encephalitis target the obligatory subunit GluN1, inhibit the receptor at the presynaptic GABAergic inter- neurons and reduce the release of GABA and thus the inhibition of postsynaptic glutamate transmission. As a result there is excessive release of glutamate in various parts of the brain (hippocampus and cerebellum) with enormous excitatory effect.

Anti-AMPAR

The AMPA ((amino-3-hydroxy-5-methylisoxazole-4-propionic acid-) subtype excitatory amino acid receptor is the principal receptor mediating fast synaptic transmission at most CNS excitatory synapses /37/. The AMPA receptor consists of subunits called GluR1, GluR2 and GluR3. The anti-AMBAR induce pathological effects: they activate the AMPAR, kill neurons by exocytosis, and/or by complement activation, and cause multiple brain damage /38/. Antibodies to GLuR1/2 associated with LE are often para neoplastic /39/.

Anti-GABAAR

GABAAR belongs to the family of heteropentameric ligand-gated ion channels, which also includes the glycine receptor. Synaptic GABAARs are composed of three α subunits (α1–3) and two β subunits (β2 and β3) and a single γ2 unit.

Two subgroups of patients can be differentiated. The first group presents with encephalitis, refractory seizures and high titers of serum and CSF anti-GABAAR, the second group presents with diverse clinical features (encephalitis with seizures, stiff-person syndrome and opsoclonus-myoclonus).

Anti-VGKC

The voltage-gated potassium channels (VGKCs) are transmembrane K+ sensitive channels of the cell membrane. The channels are assemblies of four identical transmembrane subunits surrounding a central pore. VGKC respond sensitively to changes in the cell membrane potential. Opening of a Na+ channel leads to an influx of Na+ and Ca2+ into the cell. As a result, an excess of positive charge builds up inside the cell, generating an action potential. This activates the VGKC, K+ leave the cell, reduce the positive charge and end the action potential.

Anti-VGKC Ab: the target of the autoantibodies are proteins interacting with the VGKC complex, such as leucine rich glioma inactivated 1 (LGI 1) and contactin associated protein like 2 (CASPR 2).

Antibodies are determined against:

  • The VGKC complex consisting of LGI 1 and CASPR.
  • LGI 1 (lethal giant larvae homolog 1): this glycoprotein is secreted by presynaptic terminals and constitutes the main component of the complex, accounting for 70% of it. Receptors for LGI 1 are the postsynaptically secreted disintegrin as well as proteins 22 and 23 of the metalloprotease domain (ADAM 22/23).
  • CASPR (contactin-associated protein-like 2): this membrane protein has a large extracellular domain. It is a cell adhesion molecule which is important for the localization of the VGKC complex. CASPR accounts for approximately 20% of the VGKC complex.

Anti-GABABR

The synthesis of the neurotransmitter γ-aminobutyric acid (GABA) is catalyzed by the enzyme glutamate decarboxylase (GAD). The GABAergic system plays an important role in the regulation of neuronal activity in the brain and spinal cord. GABA acts via ionotropic GABAA and metabotropic GABAB receptors. On the cellular level, GABA acts in an inhibitory way via GABAB receptors by hyperpolarization of the membrane. The GABAB receptor is a postsynaptic ion channel for K+ that can be inhibited by γ-amino butyric acid. GABAB receptors are functional only as heterodimers and consist of a GABR1 and a GABR2 subunit.

Anti-GlyR

Glycine is an important inhibitory neurotransmitter in the brain stem and spinal cord. Glycine receptors (GlyRs) are pentameric chloride channels composed of a variable arrangement of α and β subunits. The binding of glycine to its corresponding receptor causes a significant increase in the transport of Cl and leads to hyperpolarization of the cell membrane. GlyR antibodies are predominatly of the IgG1subclass, intrathecally synthesized and directed against an extracellular epitope on the GlyR subunit. Mutations of the receptor cause hereditary hyperekplexia, a condition with increased jumpiness and increased muscle tone in children.

Clinical presentation: the most clinical presentations are progressive encephalomyelitis with myoclonus. Common cancer association: infrequent thymomoma or lymphoma.

Laboratory findings: GlyR antibody titers in serum and CSF correlate well with the clinical course.

Anti-DPPX

Dipeptidyl-peptidase-like protein X (DPPX) is an accessory subunit of Kv4.2 potassium channels.

Clinical presentation: patients with anti-DPPC present with hallucinations and confusion associated with symptoms reflecting CNS hyperexcitability. Half of the patients have gastrointestinal symptoms, mainly diarrhea.

Anti-GluR5

Clinical presentation: Limbic encephalitis. Common cancer association Hodgkin’s lymphoma.

Anti-amphysin

Amphysin is an intracellular synaptic vesicle protein involved in clathrin-mediated endocytosis.

Amphysin antibodies are described in para neoplastic stiff-person syndrome. They can also be found in limbic encephalitis and para neoplastic cerebellar degeneration. Classically, patients with stiff-person syndrome and anti-amphysin antibodies are female with a median age at symptom onset around 60 years and associated breast cancer.

Anti-GAD

Glutamate decarboxylase (GAD) is the rate-limiting enzyme involved in the synthesis of gamma amino butyric acid (GABA), which is the major inhibitory neurotransmitter in the CNS. Two isoforms GAD 65 and GAD 67 are expressed in the brain. GAD 65 is found in presynaptic terminals and is the dominant target of autoantibodies.

Anti-GAD antibodies may have a specific role in the pathogenesis of limbic encephalitis, cerebellar ataxia, and Stiff-person syndrome. Common cancer associations are neuroendocrine tumors. Risk of cancer increases with age, male sex, presence of concurrent neuronal surface antibodies, and limbic encephalitis.

Anti-VGCC

The P/Q type voltage-gated calcium channel (VGCC) consist of α1- and β-subunits; the α1-subunit has three, the β-subunit four subtypes. While the α1-subunit determines the type of ion flux, the β-subunit has auxiliary functions. The α1-subunit directs the ions into the cell membrane and determines the kinetics of ion flux.

Autoantibodies to the presynaptic P/Q type of the VGCC are responsible for reduced release of acetylcholine, which leads to muscle weakness and autonomous symptoms of neuromuscular disease, also known as Lambert-Eaton myasthenia syndrome (LEMS). Anti-VGCC are IgG type autoantibodies that bind to calcium channels of the nerve cell or muscle cell membrane. Depolarization of the presynaptic nerve cell membrane leads to an influx of Ca2+, which causes the release of acetylcholine into the synaptic cleft. The inhibition of the VGCC by the binding of antibodies leads to a structural change of the VGCC resulting in internalization into the cell and degradation. Diagnostically relevant antibodies target the α1-subunit, the neurotransmitter releasing channel type of the motor end plate (anti-P/Q antibodies) and the neurotransmitter releasing channel type at the synapses of some autonomous nerves (anti-N antibodies). In Patients with anti-VGCC there is an early and diffuse loss of Purkinje cells.

Clinical presentation: Anti-VGCC are associated with Lambert-Eaton myasthenic syndrome (LEMS), cerebellar ataxia or both. Cerebellar ataxia associated with anti-VGCC is essentially para neoplastic, associated with small cell lung carcinoma (SCLC) expressing functional VGCC. Cerebellar ataxia is usually subacute with symmetrical gait and limb ataxia.

Anti-GluR1

GLuR1 is a metabotropic glutamate receptor localized postsynaptically in somatodentric domains and strongly expressed in hypo campus and cerebellum.

Clinical presentation: para neoplastic cerebellar degeneration. All patients experience severe cerebellar ataxia with balance and gait disturbances, dysarthria and nystagmus. Common cancer association: Hodgkin’s lymphoma.

Anti-Tr/DNER

Anti-Tr/Delta/notch-like epidermal growth factor-related receptor (DNER) antibodies are associated with para neoplastic cerebellar degeneration and Hodgkin’s lymphoma.

Anti-AQP4

The IgG antibodies target the astrocytic water channel and are found in neuromyelitis optica, a syndrome consisting of optic nerve and/or spinal cord inflammation.

Table 25.6-6 Encephalitis and cerebellitis associated neurological diseases

Clinical and laboratory findings

Limbic encephalitis (LE)

The clinical findings of LE are related to inflammation of the medial temporal lobes. LE is classified according to the presence of autoantibodies in two broad categories, one associated with antibodies to intracellular neuronal antigens and the other to cell surface antigens /42/.

The clinical picture of LE is also seen not associated with autoantibodies and para neoplastic diseases in other disorders such as viral encephalitis (HSV-1, EBV), syphilis, Wernicke’s encephalopathy, local tumors, gliomas and in organ transplanted patients.

Anti-NMDAR encephalitis /35/

Autoimmune encephalitis associated with antibodies against N-methyl D-aspartate (NMDA) receptor affects many more brain regions than purely the limbic system, and therefore is not classified as a limbic encephalitis. The NMDA receptor is a protein in the brain that helps control the electrical activity of nerves and therefore antibodies against these receptors are likely to have an important role in directly causing the disease /36/. The early phase is characterized by psychosis, amnesia and confusion, followed by motor disturbances, autonomous instability, hypo ventilation and impaired consciousness. Some patients have a malignant tumor. The UK encephalitis study showed that 4% of patients had anti-NMDAR encephalitis. The encephalitis is associated with tumors, mostly teratomas of the ovaries, however a substantial proportion of patients have no detectable tumor. Anti-NMDAR Ab that target receptors in the brain are produced by cross reactivity with NMDA receptors in the teratoma.

Laboratory findings: moderate lymphocytic pleocytosis (median 32 lymphocytes/μL, range of 5–480/μL). The anti-NMDAR Ab level is higher in serum (approximately 10-fold) than in cerebrospinal fluid. When total IgG in CSF is normalized intrathecal synthesis of anti-NMDAR Ab is detected

Autoimmune limbic encephalitis

Limbic encephalitis with prominent psychiatric features. In comparison to patients with anti-NMDAR the presence of anti-AMPAR encephalitis is more variable. The glutamate receptor antibodies anti-AMPAR are present in 25–30% of patients with different types of epilepsy. When these or other types of autoimmune antibodies are found in epilepsy patients, and when these autoimmune antibodies are suspected to induce or aggravate the seizures and/or the cognitive/psychiatric/behavioral impairments that sometimes accompany the seizures, the epilepsy is called autoimmune epilepsy. Cancer association: approximately 50% of cases small cell lung cancer (SCLC), approximately 70% of cases SCLC, thymoma or breast cancer. AMPAR encephalitis responds well to treatment.

Morvan syndrome /40/

Morvan syndrome is a rare disease characterized by widespread neurological symptoms involving the peripheral nervous system (neuromyotonia), autonomic system (hyperhidrosis, severe constipation, urinary incontinence, and cardiac arrhythmia) and the CNS (severe insomnia, hallucinations, impairment of short term memory and epilepsy). Many patient have an underlying tumor, for example thymoma, lung cancer, testicular cancer and lymphoma; this indicates the para neoplastic nature of the disease.

Laboratory findings: serum CK elevated, anti-VGKC above 100 pmol/L, some patients show oligoclonal bands in the cerebrospinal fluid.

Anti-GABAR limbic encephalitis

Patients have the clinical picture of LE, but present with early onset seizures, which may be the dominating symptom. Anti-GABABR are the most common antibodies found in LE associated with SCLC. In patients with anti-GAD Ab, the frequency of anti-GABABR is low, and the antibodies are only seen in the context of malignant diseases /45/.

Laboratory findings: a subset of patients have low titres of anti-GABABR.

Autoimmune limbic encephalitis associated with GAD antibodies /41/

New onset temporal lobe epilepsy, particularly when intractable and associated with memory loss, behavioral, or psychiatric features should raise suspicion of autoimmune LE. Features of increased signal on MRT-FLAIR in the medical temporal lobes, hyponatremia, dysthyroidism, and/or neurologic findings associated with cerebellar dysfunction mandate anti-neuronal antibody screening for autoimmune LE that includes glutamic acid decarboxylase antibodies (anti-GAD).

Laboratory findings: the serum concentration of anti-GAD is usually above 1,000 U/L. Due to intrathecal synthesis, anti-GAD Ab can also be detected in cerebrospinal fluid, as can oligoclonal bands.

Anti-VGKC limbic encephalitis

Limbic encephalitis in association with anti-voltage-gated potassium channel-complex (anti-VGKC) is regularly diagnosed in Europe, in North America and in Australia. The incidence of high antibody levels in Great Britain is 1–2 per 1 million population per year. Of these, 67% have LE, 11% neuromyotonia, 5% Morvan syndrome, 4% epilepsy, and 11% cannot be categorized /42/. Patients present with an acute or subacute clinical picture with loss of memory, confusion, temporal lobe related seizures and psychiatric symptoms. Patients are over 40 years of age, the ratio of men to women is 2 : 1. According to a British study, 3% of patients with encephalitis have antibodies to the VGKC complex /43/. Children with encephalitis and anti-VGKC Ab have status epilepticus and focal epilepsy. Anti-VGKC Ab are not directed against the antigens LGI 1 and CASPR2 antigens /44/.

High levels of anti-VGKC Ab are also seen in 9% of patients with seizures and resistance to anti epileptic drugs and patients with faciobrachial dystonic seizures.

Laboratory findings: anti-VGKC are most commonly detected by immune precipitation. VGKC, extracted from rabbit brain, is labeled with 125J α-dendrotoxin, binds the anti-VGKC antibodies and is precipitated as immune complex. The VGKC complex can also be identified by radioimmunoassay. Antibodies to LGl 1 and CASPR 2 are also assayed using cell based methods. Anti-VGKC antibodies are not always detectable in cerebrospinal fluid (CSF) and their levels are higher in serum than in CSF . VGKC antibodies are found in 90% of men with limbic encephalitis and Morvan syndrome.

Neuromyotonia /46/

Neuromyotonia, also called Isaac’s syndrome, is a peripheral nerve hyperexcitability syndrome that presents motor activity. Clinical findings include cramps, fasciculations, and myokymia. Etiopathogenesis involves the interaction of genetic, autoimmune, and para neoplastic factors. The disease may occur in combination with autoimmune diseases such as myasthenia gravis or malignant tumors such as SCLC, malignant lymphoma and thymoma.

Laboratory findings: anti-VGKC 100–400 pmol/L.

Stiff person syndrome (SPS) /47/

SPS is a rare form of chronic encephalomyelitis (prevalence of 1–2 per 1 million population) characterized by progressive muscle stiffness, rigidity and spasms. It also affects the axillary muscles, restricting arm swinging, which leads to a stiff posture. Women are affected more often than men. Clinical symptoms occur between the 3rd and 7th decade of life. SPS is frequently associated with autoimmune polyglandular syndrome (thyroiditis, diabetes type 1) and other diseases (psoriasis, pernicious anemia, vitiligo, connective tissue disease, myasthenia gravis). A small minority of patients have the para neoplastic form of SPS. Based on the clinical and laboratory findings, SPS is categorized into three groups:

  • Autoimmune group; 55–60% of patients, possible autoimmune diseases are diabetes type 1, Graves’ disease or hypothyroidism, pernicious anemia, epilepsy; findings of GAD, islet cell antibodies, parietal cell antibodies or thyroid peroxidase antibodies.
  • Para neoplastic group; 0–5% of patients. Possible associations: breast cancer, Hodgkin’s lymphoma, colon cancer, lung cancer; anti-GAD antibodies are negative, but the following autoantibodies may be positive: anti-amphiphysin Ab, anti-smooth muscle Ab and antinuclear Ab.
  • Idiopathic; no clinical association, no autoantibodies.

Laboratory findings: autoantibodies to glutamic acid decarboxylase (GAD65) are in the serum and cerebrospinal fluid of 60–80% of SPS patients, anti-GABAR Ab in 70% of SPS patients with anti-GAD Ab; detection of anti-amphiphysin Ab. The main target antigens are the inhibitory synapses of the CNS. The titers of anti-GAD Ab in the CSF are 50-fold lower than in the serum.

Progressive encephalomyelitis (PERM)

PERM with rigidity and myoclonus is a disease that belongs to the spectrum of stiff person syndrome. Clinical features include muscle stiffness and rigidity, violent jerks triggered by various stimuli, and brainstem impairment with oculomotor dysfunction /48/.

Laboratory findings: anti-GlyR antibodies /4/.

Table 25.6-7 Neuropathies phenotypically associated with anti-ganglioside antibodies /8/

Disease

Associations

Anti-gangliosides

Acute motor axonal neuropathy (AMAN)

Guillain-Barre syndrome (GBS) with cranial nerve involvement, pharyngeal-cervical-brachial variant (PCB), polyneuritis cranialis

Anti-GD1a

AMAN

Pure motor GBS, GBS without cranial nerve involvement

Anti-GM1

Miller Fisher syndrome (MFS), Bickerstaff brainstem encephalitis (BBE)

MFS-PCB, MFS-GBS, MFS-BBE, polyneuritis cranialis, acute bulbar palsy, extraocular muscle weakness

Anti-GQ1b

Pharyngeal-cervical-brachial variant (PCB)

GBS with bulbar weakness, acute bulbar palsy, AMAN

Anti-GT1a

Acute inflammatory demyelinating neuropathy

Limited cranial nerve palsies

Anti-GM2

Sensory GBS with bulbar weakness

Anti-GD1b

Pure motor GBS

Anti-GalNAc-GD1a

Table 25.6-8 Antibodies in immune mediated polyneuropathies

Antibodies

Anti-ganglioside IgG antibodies /910/

Gangliosides: gangliosides are glycosphingolipids which are composed of a ceramide (N-acetylated sphingosine) attached to one or more hexoses. The hydrophobic ceramide is immersed in the lipid membrane and the hydrophilic carbohydrate structure is exposed extracellularly, as is the case in plasma membranes, it is capable as acting as an autoantibody target. All known neuropathy associated antibodies target this extracellular carbohydrate structure. The term gangliosides refers to the group of glycosphingolipids that contain sialic acid linked to the oligosaccharide core, synthesized through addition of monosaccharides in a stepwise fashion by glycosyltransferases and sialyltransferases) /11/. Within the plasma membrane, gangliosides interact with transmembrane receptors and signal transduction molecules. The action of anti-ganglioside IgG antibodies lead to the formation of ganglioside complexes in the axons and Schwann cells of the nervous system and thus may cause dysfunction.

The G1 series of gangliosides comprises GM1, GD1a, GD1b, and GT1b. These gangliosides differ with regard to the number and position of their sialic acids where M, D and T stand for mono-, di-, and tri-sialosyl groups. Thus there are two disialosylgangliosides, GD1a and GD1b. The term GM1b is used for the monosialosylgangliotetraos ceramide in which the sialosyl group is attached to the terminal galactose, in contrast to GM1a. GQ1b corresponds to GM1 but, unlike the latter, has two N-acetyl neuraminic acid molecules bound to each of the two galactose molecules. The term LM1 is used for the sialosylneolactotetraos ceramide, which is also known as sialosyl paragloboside. Sialosyl lactosaminyl paragloboside (SGPG) and its higher homologue SGLPG have structures similar to that of LM1 /11/. Cells of the ventral horn of the spinal cord have a relatively high content of GM1 and GD1a compared to the sensory posterior horn cells. Motor nerves supplying the eye muscles are rich in GQ1b.

Anti-ganglioside: these antibodies belong to the IgG class and are found in 60% of patients with acute immune mediated polyneuropathy, also known as Guillain-Barré syndrome (GBS). The antibodies are elevated especially during the acute phase of the disease and can therefore be used as a marker of GBS. Anti-GQ1b are specific for Miller-Fisher syndrome, a variant of GBS. The sera of patients with GBS also react with ganglioside complexes that consist of two different gangliosides, but not with the individual gangliosides of these complexes, since the interaction of two gangliosides creates a new epitope.

Serum anti-ganglioside Ab were subdivided into three groups /49/:

  • Antibodies specific to GQ1b and/or GT1a without anti-ganglioside complex (GC) reactivity
  • Antibodies hat recognize a combination of [Galβ1-3GalNAc] and [NeuAcα2-8 NeuAcα2-3Galβ1-3GalNAc] in the terminal residues of ganglio-N-tetraose structures such as antibodies to GCs (e.g., GQ1b/GM1, GQ1b/GD1b, GT1a/GM1, GT1a/GD1b)
  • Antibodies that recognize a combination of [NeuAcα2-3Galβ1-3GalNAc] and [NeuAcα2-8 NeuAcα2-3Galβ1-3GalNAc] in the terminal residues, such as antibodies to GQ1b/GD1a, GT1a/GD1a, GQ1b/GT1b, GT1a/GT1b.

Laboratory findings: anti-ganglioside IgG antibodies are usually demonstrated with solid phase enzyme immunoassays based on the ELISA method. The determination is quantitative. The ganglioside content of the patient sample is determined relative to a healthy control population. A semi quantitative determination can also be performed by immunoblot.

Anti-ganglioside IgM antibodies /50/

Anti-ganglioside IgM (anti-GM1, anti-GM2, anti-GD1a, anti-GD1b, anti-GQ1b) and anti-sulfatide Ab are associated with multi focal motor neuropathy (MMN).

Laboratory findings: in most cases, solid phase enzyme immunoassays are used. The determination is quantitative. The ganglioside content of the patient sample is determined relative to a healthy population. Semi quantitative determination can be performed by immunoblot using antibodies against GM1.

Anti-myelin-associated glycoprotein antibodies (Anti-MAG)

Myelin-associated glycoprotein (MAG): the protein has a molecular weight of 110 kDa and belongs to the SIGLEC (sialic-acid-binding immunoglobulin-like lectins) family of proteins. MAG is part of peripheral nerve myelin and contains approximately 30% carbohydrates. The MAG antigen is a sulfated glucuronic acid which also occurs in other glycoconjugates such as sulfate-3-glucuronyl paragloboside (SGPG) and the myelin proteins P0 and P22.

Anti-MAG Ab: the IgM class antibodies are present in demyelinating sensorimotor polyneuropathy and IgM paraproteinemia. Anti-MAG antibodies bind to MAG in 50% of patients with IgM monoclonal gammopathy of undetermined significance (IgM-MGUS). Their binding results in changes in the myelin of the peripheral nerves, with widening of the myelin lamellae and consecutive demyelinating neuropathy. Although anti-MAG Ab most commonly occur in IgM-MGUS, they are also seen in Waldenstroem’s macroglobulinemia and other B cell lymphomas.

Laboratory findings: the antibodies are determined with the following tests /51/:

  • Immunoblot (antigen: MAG from human brain): diagnostic sensitivity 72.5%, specificity 100%
  • MAG-EIA (antigen: MAG from human brain): diagnostic sensitivity 97.5% specificity 80.0%
  • IIFT (antigen; monkey peripheral nerve): diagnostic sensitivity 92.5%, specificity 94.3%.

Patients with low EIA titers but a negative immunoblot result are likely to have other autoimmune neuropathies without demyelination.

Use of the HNK-1 carbohydrate epitope of MAG is also said to be effective /52/. Normal results are a titer less than 1 : 640 in the IFA and a titer up to 1,000 U/mL in the ELISA /50/. However, many laboratories use higher thresholds for a significant finding (e.g., 1 : 5000 for the IFA and 1 : 3200 for the ELISA /52/.

Cryoglobulins

Cryoglobulin analysis is described in Section 18.11 – Cryoglobulins and cryofibrinogen. Up to 86% of mixed cryoglobulinemias are associated with polyneuropathy.

Table 25.6-9 Autoantibodies in immune mediated muscular disease:

Antibodies

Anti-AQP4/NMO antibodies

Aquaporin channels: they play a prominent role in water homeostasis and are responsible for a rapid response to changes in the cell volume when the osmolality in the extracellular space changes (Fig. 8.6-6). More than 10 isoforms of AQP4 are known, of which 7 are localized in various segments of the nephron. Aquaporin isoform 4 (AQP4) is prevalent in the CNS and primarily found in the plasma membranes of astrocytes.

Laboratory findings: in neuromyelitis optica (NMO), also known as Devic’s syndrome, IgG antibodies to AQP4/NMO are detected with a diagnostic sensitivity of 73% and a specificity of 91% /53/. Anti-NMO Ab help distinguish between NMO and multiple sclerosis, especially in early stages of the diseases.

Anti-AChR antibodies

Acetylcholine receptor (AChR): the nicotinic AChR is the prototype of ligand gated ion channels. It consists of a pentamer of protein subunits with two binding sites for acetylcholine (ACh). When ACh binds to these sites, the receptor undergoes a conformational change and a pore opens, which allows Na+ to move into the cell along a gradient. When sufficient pores are opened, the intracellular Na+ concentration increases, and the influx of positive charge into the cell leads to depolarization of the membrane and consequent generation of an action potential.

Anti-AChR Ab: anti-AChR antibody levels are graded as follows (nmol/L): 1–10 as low, 10–100 as intermediate, above 100 as high /54/. There is no inter individual correlation between the anti-AChR Ab level and the severity of clinical symptoms. Intraindividual monitoring, however, does reveal a correlation. This can be seen especially in plasmapheresis therapy. During myasthenic crises the anti-AChR Ab level is above 1,000 nmol/L. Plasmapheresis and high-dose immune globulin therapy lead to a rapid decline.

By monitoring the anti-AChR Ab level, effective long-term immunosuppressive therapy can be achieved. The decline in the anti-AChR Ab level corresponds to that of IgG with a half life of about 20 days. A change in the antibody level by 20% or more can be expected to result in a change in Osserman score /18/. Osserman score: I, ocular myasthenia; IIa, mild generalized myasthenia; IIb, moderately severe generalized myasthenia; III, with bulbar manifestation; IV, ventilation required. While during younger years IIa and IIb prevail, the purely ocular form is characteristic of older ages.

Anti-MuSK antibodies /26/

Muscle-specific tyrosine kinase (MuSK): MuSK is a protein of the agrin receptor at the motor end-plate. Upon activation by agrin via a signal transduction path, MuSK is crucial for the AChR assembly during end-plate development. However, it has been shown that inhibition of MuSK synthesis leads to AChR dispersion and end-plate disruption also in mature muscle.

Ten to 15% of patients with generalized myasthenia gravis do not have antibodies to the AChR. In a proportion of these persons anti-MuSK IgG antibodies has been identified. The detection of anti-MuSK represents a valid diagnostic tool, as they proved to be fairly specific for anti-ChR-negative myasthenia gravis. In most patients with anti-MuSK in different phases of the disease a correspondence between variations in clinical symptoms and antibody concentrations was found. Immunosuppression therapy caused a sharp decrease of anti-MuSK IgG, in the general population, the difference between anti-MuSK concentrations in samples from treated and untreated patients was not significant /26/.

Anti-titin antibodies /27/

Titin: titin is a filamentous muscle protein. Depending on the respective musclar type the isoforms varies in molecular weight between 3000 and 4200 kDa. Sera from most anti-titin positive patients recognize a specific 30 kDa segment corresponding to 1% of titin’s mass. Titin represents 10% of the total protein of striated muscle.

Myasthenia gravis is an autoimmune disease caused by antibodies targeting the neuromuscular junction of skeletal muscles. The majority of patients with generalized myasthenia gravis have antibodies directed against the AChR, 6% have antibodies against MuSK and 2% have antibodies against the low-density lipoprotein receptor-related protein 4 (LRP4). Titin antibodies have not been found in myastenia gravis patients negative for AChR antibodies.

Approximately 10% of myastenia gravis patients do not have detectable antibodies. Overall 20–40% of myastenia gravis patients are positive for titin antibodies. The presence of titin antibodies is age-related, the prevalence is about 6% in early onset myasthenia gravis, and rises to 50–80% in non-thymomatous patients with late-onset myasthenia gravis. In patients with early onset myasthenia gravis titin antibodies are a strong indication of thymoma (50–95%) but only in few non-thymoma early onset myasthenia gravis patients. The presence of titin antibodies in all age groups is related to a more severe symptom manifestation /54/.

Anti-titin antibodies are a sensitive marker of thymoma associated with myasthenia gravis in patients 60 years and younger, justifying the insistent search for a thymoma in myasthenia gravis patients of this age group who have these antibodies /55/.

Table 25.6-10 Prevalence (%) of antibodies in myasthenia gravis

M. gravis

AChR Ab

MuSK Ab

Titin Ab

RR Ab

Generalized

80–90

 

60–80(1

15

Ocular

50

 

90(2

 

Thymoma associated

80–90

 

75–95

50–70

Seronegative

 

70

 

15(3

RR antibodies, ryanodine receptor antibodies; 1) Age ≥ 40 years; 2) Age ≥ 60 years, 3) Age ≥ 50 years.

Table 25.7-1 Autoimmune liver diseases

Clinical and laboratory findings

Autoimmune Hepatitis (AIH) /4/

AIH is a non resolving progressive liver disease of unknown etiology characterized by interface hepatitis, hyper gamma globulinemia, and autoantibodies.

In Europe, AIH has an incidence of 0.1–1.9 per 100,000 population/year and a prevalence of 2.2–17 per 100,000 population /8/. Approximately 80% of AIH patients are women. The disease usually has an insidious onset and is only diagnosed in the chronic phase; hence the name chronic autoimmune hepatitis. In 10–25% of cases, AIH presents as acute hepatitis, rarely with a fulminant course /6/. The endoscopic retrograde cholangiopancreatogram (ERCP) finding is uncharacteristic and is used to exclude other liver diseases such as PSC. AIH often has a characteristic histological finding with piecemeal necroses as well as portal and lobular infiltration with inflammatory cells.

The infiltration provides clues to the degree of inflammation. Together with the degree of fibrosis or cirrhosis as well as laboratory and clinical parameters, a hepatitis score can be built /7/.

AIH is classed into type 1 and type 2, depending on the target antigen of the autoantibodies detected. Both types predominate in women /8/.

Type 1 AIH shows the following pattern of antibodies: ANA and/or ASMA and anti-ALA/LP Ab positive. This most common type (80%) usually occurs in young women. The disease has an early onset, runs a severe course and responds well to immunosuppression. In approximately 20% of patients, the onset is acute, similar to viral hepatitis.

Type 2 AIH is anti-LKM Ab positive. The disease often begins as early as in childhood, with a second incidence peak occurring between the ages of 35 and 65 years. Type 1 has a worse course, since half of the patients already have liver cirrhosis at the time of diagnosis. Type 2 is more often associated with extrahepatic autoimmune syndromes such as thyroiditis, arthritis, neuropathy and pernicious anemia than type 1.

Some authors defined a third type characterized by the presence of anti-SLA Ab. However, since this was the only major difference compared to type 1, this differentiation did not become established and was therefore abandoned.

Primary biliary cholangitis (PBC), also known as primary biliary cirrhosis

The differential diagnosis in patients with prolonged jaundice of obstructive type is difficult. PBC, the chronic idiopathic obstructive jaundice occurring chiefly in middle-aged women may be extremely difficult to differentiate from obstruction to main bile ducts which requires surgical relief /1/. The destruction of the bile capillaries and the consecutive cholestasis with accumulation of bile acids leads to continuous necrosis of parenchymal cells in PBC. The subsequent fascial reconstruction of the hepatic architecture ultimately results in the development of liver cirrhosis.

A consensus of experts recommends that PBC should be diagnosed when two of the following criteria are fulfilled:

  • Elevated cholestasis parameters (ALP elevated for over 6 months)
  • AMA positivity
  • Characteristic histology.

Since AMA may be detectable years to decades before clinical manifestation of the disease /11/, more than 60% of PBC cases are identified as early as during the asymptomatic stage. By contrast, only 4% of patients of a cohort had no clinical symptoms at initial diagnosis. For pathophysiology of PBC refer to Ref. /12/.

Clinical findings:

  • Asymptomatic phase, which can last up to 20 years. During this phase, ALP and GGT are elevated with normal or only slightly increased aminotransferase activities
  • Symptomatic phase. The main symptoms are exhaustion, fatigue and itching, especially at night, without elevated bilirubin for a long time. The cause of the itching is unknown. The serum concentration of bile acids is several times higher than normal, and it is believed that other pruritogenic substances, which are normally eliminated via bile, accumulate in the skin.
  • Incidence/prevalence: in Great Britain, PBC has an incidence of 3.1 per 100,000 population/year and a prevalence of 25.1 per 100,000 population. In North America, the disease has an incidence of 2.7 and a prevalence of 42.1. In Asia, the incidence is low at approximately 0.4, and the prevalence is 2 per 100,000 population.
  • The female/male ratio ranges from 6/1 to 22/1 depending on the study group. A study conducted over a period of 20 years reports an annual incidence of 4.5 for women and 0.7 for men related to a population of 100,000 /5/. The disease is typically diagnosed between the ages of 45 and 65 years. The youngest female patient was 15 years old.
  • Compared to the general population, a clear predisposition to disease was found in monozygotic twins > dizygotic twins > first-degree relatives /13/.

A large genome wide association study (GWAS) on PBC was not only able to confirm the known HLA association but also found considerable associations with certain SNPs (single nucleotide polymorphisms) and certain alleles of several gene loci /14/.

Anti-mitochondrial antibodies: nine different AMA subtypes (M1–M9) have been described, of which only the highly specific AMA-M2 are of relevance in the diagnostic testing for PBC. AMA-M2 can be directed against three main antigens: pyruvate dehydrogenase (PDH); branched chain α-keto acid dehydrogenase (BCKD); oxoglutarate dehydrogenase (OCKD). AMA-M4, AMA-M8 and AMA-M9 are probably not of any additional diagnostic value /15/.

Besides AMA, certain ANA can also indicate the presence of PBC. This includes ANA directed against the SP100 antigen, which produce a nuclear dot pattern in the IIFT, as well as anti-gp210 antibody, which cause staining of the nuclear membrane in the IIFT. These ANA may occur as isolated autoantibodies or in combination with AMA.

The diagnostic sensitivity and specificity of AMA for PBC are both > 90–95%. PBC specific ANA can be detected in approximately 50% of AMA negative patients, leaving only 2–5% of PBC patients that cannot be clearly diagnosed based on the autoantibodies /1617/. The clinical picture of AMA-negative PBC is referred to as autoimmune cholangitis (AIC). The diagnostic sensitivity to identity AIC using biomarkers (kelch-like 12, hexokinase-1) is poor /18/.

Primary sclerosing cholangitis (PSC)

Primary sclerosing cholangitis (PSC) is characterized by a fibrosing and sclerosing inflammation of the intrahepatic and extrahepatic bile ducts /19/. Over time the bile ducts become narrow and obliterated and, as a result, the small bile ducts eventually disappear completely. Focal dilations of the bile ducts proximal to the strictures have a characteristic “string of beads” appearance on ERCP.

Clinical findings

  • Incidence/prevalence: in Norway, an incidence of 1.3 per 100,000 population/year and a prevalence of 8.5 per 100,000 population have been described. Approximately 70% of those affected are men. The average age at diagnosis is 39 years. However, PSC is increasingly diagnosed in children as the cause of chronic liver disease, often as an overlap syndrome with AIH. About 75% of patients with PSC have concomitant inflammatory bowel disease and 87% have ulcerative colitis, while Crohn’s disease is rare in PSC patients.
  • The clinical picture is heterogeneous. Patients are initially asymptomatic but, as the disease progresses, may experience exhaustion, weight loss, pruritus and recurring episodes of fever due to acute cholangitis.
  • When making a differential diagnosis, PSC must be distinguished from IgG4 associated cholangitis. IgG4 associated PSC may appear clinically and histologically identical to PSC. IgG4 associated cholangitis is one of the IgG4 associated autoimmune diseases that were not described until 2003. The dominant clinical picture is usually one of autoimmune pancreatitis, which in 70–100% of cases is accompanied by cholangitis. Serum IgG4 is elevated in approximately 75% of patients. The final diagnosis is made histologically based on the characteristic infiltrate of IgG4 positive plasma cells. Differentiating the two diseases is important because, unlike classical PSC, IgG4 associated PSC responds to steroid therapy /20/.

Laboratory findings

In the early, asymptomatic phase, only ALP and GGT are increased and aminotransferases may be mildly elevated. As the disease progresses, ALP levels may increase to up to 20-fold and aminotransferases to up to 5-fold the upper reference interval value. Bilirubin is mildly elevated at diagnosis in about 50% of patients before increasing continuously with fluctuating levels. The bile acid concentration is several times higher than the upper reference interval value (Tab. 47.4-1 – Reference intervals for bile analytes).

All forms of cholestasis must be considered in a differential diagnosis. In contrast to AIH and PBC, the specific diagnosis of PSC is made based on the characteristic image obtained with ERCP. Perinuclear anti-neutrophil cytoplasmic antibodies (ANCA) testing is useful.

Like in ulcerative colitis, ANCA can be found in up to 80% of patients with PSC. Unlike vasculitis associated p-ANCA, the ANCA found in PSC do not produce a granular cytoplasmic staining pattern on ethanol-fixed neutrophil granulocytes, but an uncharacteristic streaky fluorescence pattern or no fluorescence at all. These ANCA should therefore be named a-ANCA (atypical ANCA) /21/.

Table 25.7-2 Autoantibodies of high relevance in autoimmune liver disease /822/

Autoantibodies

Antinuclear antibodies (ANA) in AIH (type 1)

ANA are usually detected on microscopic slides with cultivated human epithelial cells (HEp-2 cells). Titers ≥ 1 : 100 are generally interpreted as a positive, and titers > 1 : 320 as a clearly positive test result. This interpretation can be normalized by the patient’s age, since the physiological production of ANA increases with age. Healthy individuals aged ≥ 50 may have ANA titers of up to 1 : 320 without any pathological significance. In children, however, even ANA titers as low as 1 : 32 must be interpreted as pathologic. Especially children with juvenile chronic polyarthritis usually test positive for ANA.

ANA are seen in 40–50% of patients with AIH and thus represent the most characteristic serologic marker of type 1 AIH. The fluorescent staining pattern may vary; it is usually homogenous or speckled. A nucleolar pattern is rare. Specific other patterns, in contrast, are associated with PBC and can be helpful in excluding AIH.

Since the ANA found in AIH can only rarely be associated with a known antigen, additional testing with ELISA or immunoblot is unlikely to provide any useful information. In the rare case that the ANA in AIH are directed against a known antigen, this is usually SSA. Differential diagnosis is complicated by the fact that up to 50% of SLE patients have transiently elevated liver enzymes /4/, so that a positive ANA screen would be consistent with both AIH and SLE. A positive result for known extractable nuclear antigen (ENA) Ab and dsDNA Ab would then suggest the presence of a connective tissue disease (e.g. SLE) and contradict a diagnosis of AIH, so that in this case further testing with ELISA or immunoblot would in fact be helpful for differential diagnosis. Interpreting an ANA test result is further complicated by the fact that ANA are also found in 5–10% of patients with hepatitis C, where the antibodies are produced as a result of the infection and are therefore not the cause of the elevated liver enzymes, and in usually low titers in 20–50% of PSC patients.

Antinuclear antibodies (ANA) in PBC

While the most specific antibodies for PBC are AMA-M2, up to 50% of PBC patients are also positive for ANA. The fluorescent staining pattern allows discriminating between ANA produced during PBC and ANA that are indicative of an overlap syndrome with features of PBC and another autoimmune disease.

PBC specific ANA produce patterns of nuclear dots, few nuclear dots, nuclear membrane staining and, to some extent, centromeric staining. These staining patterns are generally created by autoantibodies to sp100 (nuclear dots), to coilin (few nuclear dots) and to the lamin B receptor or gp210 (nuclear membrane). However, since there is no 100% correspondence between the staining pattern and the target antigen, further testing with ELISA or immunoblot should be performed if there are any doubts.

A further complication is that some antibodies, in particular those directed against centromeres, are found not only in PBC but also in CREST syndrome, a subtype of scleroderma. Some scleroderma patients may also have antibodies to sp100 and coilin p80. So, although the staining patterns mentioned are caused by PBC specific ANA, the presence of these patterns may also be indicative of PBC/scleroderma overlap syndrome.

Antibodies to coilin p80 are also seen in individuals with neither PBC nor scleroderma. The few nuclear dots pattern is therefore less specific by comparison.

Conversely, there are cases in which all clinical symptoms and biochemical markers support the diagnosis of PBC even though no AMA can be detected. The majority of these patients have the above mentioned PBC specific ANA. In these cases the presence of PBC can be confirmed serologically on the basis of positive PBC specific ANA.

Patients with nuclear staining patterns other than the above also may have AIH/PBC overlap syndrome. It is also possible that a non PBC specific ANA pattern is produced, although the clinical and/or histological findings are suggestive of PBC but no AMA are detected. In this case the patient may have an overlap syndrome of AIH and autoimmune cholangitis.

Anti-smooth muscle antibodies (ASMA) (type 1 AIH)

ASMA react with actin of the micro filaments of muscle cells. In the IIFT, ASMA are seen predominantly on rat stomach sections, but they also bind smooth muscle cells in hepatic and renal vessels as well as HEp-2 cells. To date, no satisfactory ELISA is available.

ASMA have low specificity per se, because they are present in low titers (1 : 100) in different liver diseases (e.g., in 10–15% of hepatitis C patients). High titers of ASMA (> 1 : 320) against F-actin (polymerized actin) or the simultaneous presence of ASMA, ANA and elevated liver enzymes, in contrast, are very specific for type 1 AIH. High titers of ASMA alone, without any detectable ANA or anti-SLA Ab, are rare in type 1 AIH.

Anti-soluble liver antigen/liver-pancreas antigen antibodies (anti-SLA/LP Ab)

Anti-SLA/LP Ab are highly specific for type 1 AIH. Anti-SLA/LP Ab are directed against the enzyme O-phosphoseryl-tRNA:selenocysteinyl-tRNA synthase (SepSecS), which converts phosphoseryl-tRNA(Sec) to selenocysteinyl-tRNA(Sec). The enzyme consists of 422 amino acids, of which amino acids 371–409 (catalytically active center) are bound by the autoantibodies. Testing for these antibodies is performed with immunoassays or immunoblot methods. Anti-SLA/LP are also seen in ANA negative or ASMA negative patients. Simultaneous occurrence of anti-SLA/LP Ab and anti-LKM Ab has not been described. Detection of this autoantibody clearly differentiates AIH from viral hepatitis /56/.

Anti-liver/kidney microsomal antibodies (anti-LKM Ab)

Anti-LKM Ab are specific for type 2 AIH. Anti-LKM Ab bind to microsomal proteins. In the IIFT, the antibodies produce a diffuse cytoplasmic fluorescent staining of the hepatocytes; on kidney sections they produce extensive cellular fluorescent staining, which only spares the glomeruli and distal tubules. In total, there are three different anti-LKM Ab, which can hardly be distinguished by IIFT and of which only the anti-LKM-1 Ab are specific for type 2 AIH. A positive IIFT result should therefore be specified. This can be done with immunoblot or ELISA which contains cytochrome p450 isoenzyme 2D6, the antigen of the anti-LKM-1 Ab. Anti-LKM-1 Ab are seen in 4% of adults and 20% of children with AIH, but also in approximately 2–5% of all hepatitis C patients.

Anti-LKM-2 Ab are found in drug-induced hepatitis caused by the diuretic ticrynafen. The target antigen is the enzyme cytochrome P4502C9, which metabolizes the diuretic. Ticrynafen was approved only temporarily (e.g. in France and in the USA) before being withdrawn in 1982 after case reports indicated a link between the use of the drug and hepatitis. Therefore, it is expected that there will be no more anti-LKM-2 positive patients. Anti-LKM-3 Ab occur in 10–20% of patients with chronic hepatitis D.

Anti-LC-1 antibodies (type 2 AIH)

Anti-LC-1 Ab are specific for type 2 AIH. They were identified by their characteristic fluorescent pattern on rat liver sections. Formiminotransferase cyclodeaminase (FTCD) was defined as the target antigen, and specific immunoassays (ELISA, immunoblot) are now available. Anti-LC-1 Ab usually occur in association with anti-LKM-1 Ab, although, especially in children, cases of type 2 AIH have been described in which anti-LC-1 Ab is the only autoantibody /8/.

Anti-mitochondrial antibodies (AMA)

In the IIFT, AMA produce a coarse granular cytoplasmic fluorescent pattern. IIFT detectable AMA may target different auto antigens of the inner (M1, M2, M5a and M7) and outer (M3, M4, M5b, M6, M8, M9) mitochondrial membrane, but only AMA-M2 are specific for PBC.

AMA-M2 are directed against three proteins of the oxo(-keto-)acid dehydrogenase complex, which are localized in the inner mitochondrial membrane: pyruvate dehydrogenase (PDH), ketoglutarate dehydrogenase (oxoacid dehydrogenase, OADC), and branched chain oxoacid dehydrogenase (BCKD). Each of these target antigens is divided into subunits. AMA-M2 recognize the E2 subunit of all three antigens. AMA-M2 have the highest affinity for the PDH E2 subunit, to which 95% of PBC patients have autoantibodies. Approximately 40–80% of PBC patients have autoantibodies to the OADC-E2 and BCKD-E2 antigens. Thus, the majority of patients have autoantibodies to several components of the oxo(-keto-)acid dehydrogenase complex.

The antigens PDH-E2, OADC-E2 and BCKD-E2 correspond to the so called M2 autoantibody epitopes in the classification according to Berg /22/. A positive AMA IIFT should always be specified by ELISA or immunoblot tests.

The AMA IIFT can also be positive in syphilis, iproniazid induced hepatitis and venocuran induced pseudolupus. Moreover, the AMA IIFT has relatively low diagnostic sensitivity. Approximately 10% to > 30% of PBC patients who tested negative for AMA in the IIFT (large variations between studies) had a positive immunoblot result /5/. If there is sufficient clinical suspicion, an immunoassay for AMA-M2 should be performed even if the patient tested negative in the AMA IIFT.

AMA identify a distinct inflammatory myopathy phenotype that is frequently associated with chronic skeletal muscle disease and severe cardiac involvement /23/. AMA-M2 enhance the risk of supraventicular arrhythmias in patients with elevated hepatobiliary enzyme levels /24/.

Anti-neutrophil cytoplasmic antibodies (ANCA)

ANCA are detected by IIFT (the substrate primarily consists of ethanol fixed, usually additionally formalin fixed neutrophil preparations). ANCA can be distinguished by their cytoplasmic (c-ANCA) and perinuclear (p-ANCA) fluorescent staining patterns. Over 90% of c-ANCA is directed against serine proteinase 3. C-ANCA are highly specific for Wegener’s granulomatosis. The vasculitis associated p-ANCA target myeloperoxidase and, in rare cases, serine proteinase 3.

In contrast to the vasculitis associated p-ANCA, the PSC associated ANCA do not produce a granular, cytoplasmic fluorescent staining pattern, but only an uncharacteristic streaky fluorescent pattern or no fluorescence at all on formalin fixed neutrophils. These ANCA are therefore referred to as a-ANCA (atypical ANCA) or, by some authors, as x-ANCA. A-ANCA can be found in approximately 80% of patients with PSC, in 75% of patients with ulcerative colitis (80% of patients with PSC have inflammatory bowel disease, usually ulcerative colitis) and markedly less frequently in patients with Crohn’s disease. Various studies found high prevalences of a-ANCA in AIH /8/.

Table 25.7-3 Autoantibodies of low relevance in autoimmune liver diseases /822/

Antibodies

Asialoglycoprotein receptor (ASGPR) Ab

The ASGPR is likely a component of the liver specific protein. The ASGPR regulates the uptake of glycosylated proteins into the hepatocyte. Anti-ASGPR Ab were classified as diagnostically relevant in some early publications. These results could not be confirmed. Anti-ASGPR Ab do not have sufficient diagnostic specificity and sensitivity to be of additional use and are therefore no longer routinely assayed. The antibodies are demonstrated by RIA or ELISA.

Liver specific protein (LSP) Ab

Anti-LSP Ab are detected by IIFT on liver sections. Some of the reactivity can likely be explained by antibodies to ASGPR. It is difficult to attribute with certainty, and the relevance is low.

Liver membrane antigen (LMA) Ab

IIFT sometimes shows marked linear staining of the hepatocyte membrane. Here, too, the reactivity is difficult to attribute with certainty, and the relevance is low.

Liver microsome (LM) Ab

Drug induced hepatitis due to dihydralazine may cause anti-LM Ab by IIFT on rat liver sections. Staining only in the liver; hepatocytes near the central lobular area. The antigen is not defined; the reactivity is difficult to attribute with certainty, and the relevance is low.

Table 25.7-4 Diagnostic findings in autoimmune liver diseases /822/

Criterion

AIH

PBC

PSC

Antibodies
(required for
diagnosing
autoimmune
hepatitis
and PBC)

Type 1 (SLA/LP Ab, ANA, ASMA)

Type 2 (anti-LKM Ab)

AMA (IFT), AMA-M2, PBC-specific ANA (nuclear dots/sp100, nuclear membrane/gp210, centromere/CENP)

p-ANCA; sometimes also referred to as atypical ANCA (a-ANCA)

Special
forms

AMA negative PBC (autoimmune cholangitis), PBC specific ANA usually positive

IgG4 associated cholangitis; small bile duct PSC (normal cholangiography, typical histological abnormalities in the small bile ducts)

ERCP
finding

Non specific

Non specific

Characteristic

Liver
histology

Periportal hepatitis +/– fibrosis

Non suppurative cholangitis and fibrosis

Cholangitis, missing bile ducts

γ-globulins
elevated

Yes

No

No

Ig elevation

IgG

IgM

No (IgG4, see above)

HLA
association

B8, DR3, DR4

DR8

B8, DR3

Table 25.7-5 Prevalence (%) of autoantibodies in chronic liver diseases /822/

Diagnosis

ANA

ASMA

SLA/LP
Ab

LKM
Ab

ANCA

AMA

AIH

50

50

25

10

50

10*

PBC

50

10

< 5

95

PSC

25

15

80

Toxic hepatitis

10

10

< 5

Hepatitis B

5

10

< 5

Hepatitis C

10

15

< 5

< 5

Hepatitis D

5

10

10

< 5

Hereditary
hemachro-
matosis

< 5

Wilson’s
disease

α1-AT
deficiency

Some percentages vary significantly between studies and should be seen only as an indication of scale; * AIH/PBC overlap syndrome; AIH, autoimmune hepatitis; PBC, primary biliary cirrhosis; PSC, primary sclerosing cholangitis.

Table 25.7-6 Assessment of AMA types and ANA in primary biliary cirrhosis /1151725/

Criterion

New/(old)
nomen-
clature

MW

(kDa)

Pre-
valence

Significance

Confirmed
relevant
autoantibodies

PDH-E2
(M2a)

74

95%

AMA-M2, highest diagnostic sensitivity and specificity

PDH-E1α
(M2d)

41

41–66%

 

PDH-E1β
(M2e)

36

5%

 

Protein X
(M2c)

52

95%

 

BCKD-E2

50

53–55%

AMA-M2, highest diagnostic sensitivity and specificity

BCKD-E1α

46

 

BCKD-E1β

38

 

OADC-E2

48

39–88%

AMA-M2, highest diagnostic sensitivity and specificity

OADC-E1

110

< 5%

 

OADC-E3

55

38%

 

Formerly
described
AMA subtypes
(classification
has little
relevance today)

(M1)

 

< 5%

Anti-cardiolipin Ab; non specific (e.g., 100% positive in active syphilis)

(M2)

AMA-M2 (see above)

(M3)

 

< 5%

Pseudo lupus

(M4)

 

50%

PBC, little additional value

(M5a/b)

 

< 5%

Connective tissue diseases

(M6)

 

< 5%

Hepatitis

(M7)

 

< 5%

Myocarditis

(M8)

 

50%

PBC, little additional value

(M9)

 

80%

PBC, little additional value

PBC
specific
ANA

Immuno-
assay/
IIFT

MW (kDa)

Pre-valence

Significance

 

Sp100

(Nuclear
dots)

100

30%

High specificity for PBC, also prevalent in scleroderma

Coilin p80

(Few
nuclear
dots)

80

15%

Prevalent in PBC and sclerodermas, but also without specific clinical association

Gp210

(Nuclear
membrane)

210

25%

High specificity for PBC

Lamin B
receptor

 

< 5%

CENP
(Centro-
mere)

 

30%

High specificity for CREST and PBC

Table 25.8-1 ACR criteria for granulomatosis with polyangiitis /1/

Disease

Criteria

Granulomatosis with polyangiitis

The diagnosis of GMP is supported by the presence of at least two of the four criteria. The presence of two or more criteria yields a diagnostic sensitivity of 88.2% and a specificity of 92.0%. For the nomenclature of the disease refer to the text.

1. Nasal or oral inflammations: painful or painless oral ulcerations or nasal discharge, purulent or bloody

2. Abnormal findings on chest radiograph: presence of nodular infiltrates or cavities

3. Urinary sediment: micro hematuria (> 5 red blood cells per high power field) or red cell casts

4. Granulomatous inflammation on biopsy: histological abnormalities with granulomatous inflammation in the arterial wall or in the perivascular or extravascular region (artery or arteriole).

Churg-Strauss syndrome

The diagnosis of CSS is supported by the presence of 4–6 criteria. The presence of 4 or more criteria yields a diagnostic sensitivity of 85% and a specificity of 99.7%.

1. Asthma

2. Eosinophilia > 10%

3. Mono- or polyneuropathy

4. Pulmonary infiltrates (non fixed)

5. Paranasal sinus abnormalities

6. Extravascular eosinophilia

Table 25.8-2 Chapel Hill Consensus Conference definition of vasculitides /2/

Vasculitis

Disease definition

Large vessels

Giant cell arteritis (temporal arteritis): granulomatous arteritis of the aorta and its major branches

Takayasu arteritis: granulomatous inflammation of the aorta and its major branches

Medium vessels

Polyarteritis nodosa (classic polyarteritis nodosa): necrotizing inflammation of medium-sized or small arteries without glomerulonephritis or vasculitis in arterioles, capillaries, or venules

Kawasaki disease: arteritis involving large, medium-sized, and small arteries, and associated with mucocutaneous lymph node syndrome

Small vessels

Wegners granulomatosis: granulomatous inflammation involving the respiratory tract

Churg-Strauss syndrome: eosinophil-rich and granulomatous inflammation involving the respiratory tract

Microscopic polyarteritis: necrotizing vasculitis, with few or no immune deposits, affecting small vessels

Schoenlein-Henoch purpura: vasculitis, with IgA-dominant immune deposits

Essential cryoglobulinemic vasculitis: vasculitis, with cryoglobulin immune deposits

Cutaneous leukocytoclastic angiitis: isolated cutaneous leukoclastic angiitis without systemic vasculitis or glomerulonephritis

Table 25.8-3 ANCA associated vasculitides

Clinical and laboratory findings

Granulomatosis with polyangiitis (Wegener’s granulomatosis; WG)

The disease is characterized by Wegener’s triad. It comprises necrotizing granulomatous processes of the upper or lower respiratory tract, a focal segmental glomerulonephritis and, in some cases, the development of granulomas on the glomeruli as well as disseminated, focal necrotizing vasculitis of the arteries or veins. WG was defined by the Chapel Hill Consensus Conference as granulomatous inflammation involving the respiratory tract, and necrotizing vasculitis affecting small to medium sized vessels /2/. The disease can be divided into two phases: the initial phase and the generalized phase /4/. In the initial phase, the disease is limited to granulomatous inflammation of the eyes or the ENT area. This may persist for variable amounts of time before progressing to the vasculitic generalized phase with involvement of various organs, where the prognosis is often determined by the degree of renal involvement. Wegener’s granulomatosis has recently been renamed granulomatosis with polyangiitis /5/.

Microscopic polyangiitis (MPA)

According to the 1992 Chapel Hill Consensus Conference, MPA is a form of vasculitis primarily involving small vessels. It differs from GMP by the absence of biopsy detectable granulomas and from polyarteritis nodosa by the absence of detectable immune complexes. Due to their strong similarity in clinical symptoms, it may be difficult or impossible to differentiate between MPA and GMP /6/.

MPA is primarily characterized by renal involvement, usually presenting as focal necrotizing glomerulonephritis, which determines the prognosis. In contrast to WG, MPA often affects only the kidneys. In addition, patients frequently suffer from nonspecific rheumatic symptoms, peripheral neuropathy and pulmonary renal syndrome.

Churg-Strauss syndrome (CSS)

CSS is an eosinophilic, granulomatous necrotizing vasculitis of the small and medium sized vessels /2/. This disease, which is usually associated with bronchial asthma, is characterized by eosinophilia of more than 10% in peripheral blood. CSS has three phases. During the prodromal phase, which may persist for years, patients often suffer from allergic reactions of the ENT tract (allergic rhinitis, nasal polyposis, bronchial asthma) and occasionally from cutaneous allergies. This is followed by a second phase with blood and tissue eosinophilia as well as elevated serum IgE. The third, generalized phase of CSS is marked by renal involvement, neuropathies with possible progression to tetra pareses, and involvement of the lungs in the form of non cavitating nodules. The prognosis is generally determined by the extent of cardiac involvement. ANCA, generally p-ANCA targeting MPO, can only be found in half of all CSS patients.

Table 25.8-4 Laboratory tests in the diagnosis of vasculitis and pulmonary-renal syndrome

Test

Disease/Condition

Etiology

  • ANCA

ANCA associated vasculitis

  • Anti-GBM antibodies

Goodpasture syndrome

Primary vs. secondary vasculitis/differentiation

  • Complement reduction

Complement defect

  • Hepatitis B antigen (HBV RNA)

Classic polyarteritis

  • Hepatitis C antigen (HCV RNA)

Mixed cryoglobulinemia

  • Cryoglobulins

Cryoglobulinemia (often in HCV infection)

  • Antiphospholipid antibodies

Antiphospholipid syndrome

  • Eosinophils, elevated IgE

Churg-Strauss syndrome

Activity parameters

  • ESR, C-reactive protein

Vasculitides in general

  • Leukocytosis, thrombocytosis

Vasculitides in general

  • Complement decline

Immune complex vasculitis (e.g., SLE)

  • ANCA titer change (especially in the immunoassay)

Especially in PR3 positive ANCA associated vasculitis

Organ involvement

  • Hematuria

Glomerulonephritis

  • Proteinuria

Glomerulonephritis

  • Elevated creatinine

Glomerulonephritis

Table 25.8-5 Frequency (%) of anti-proteinase 3-Ab and anti-myeloperoxidase Ab /20/

Disease

cANCA
(IIFT)

Protei-
nase
3-Ab (IA)

pANCA
(IIFT)

MPO-Ab
(IA)

1. Wegeners’
granulomatosis

64

67

21

24

2. Mikroscopic
polyangiitis

23

27

58

58

3. Churg-Strauss
syndrome

33

33

33

33

Patients with
diseases 1–3

5

11

19

9

Healthy controls

2

1

4

4

IA, Immunoassay; Ab, antibodies; MPO, myeloperoxidase; IA, immunoassay; IIFT indirect immune fluorescence test

Table 25.8-6 Interpretation of ANCA based on IIFT patterns and immunoassay results /78/

Antibodies

Ethanol-fixed neutrophils(1)

Formalin-fixed neutrophils

HEp-2
cells

ELISA result(2)

c-ANCA

Cytoplasmic granular

Cytoplasmic granular

Irrelevant

PR3 (90%) or MPO (10%) clearly positive

Interpretation: High serological suspicion of granulomatosis with polyangiitis or, in rare cases, microscopic polyangiitis or Churg-Strauss syndrome.

p-ANCA

Nuclear/ perinuclear

Cytoplasmic granular

Negative

MPO (90%) or PR3 (10%) clearly positive

Interpretation: High serological suspicion of microscopic polyangiitis, Churg-Strauss syndrome or, in rare cases, granulomatosis with polyangiitis.

a-ANCA

Cytoplasmic diffuse

Negative

Negative

MPO and PR3 negative

Interpretation: Not indicative of ANCA-associated vasculitis (microscopic polyangiitis, Churg-Strauss syndrome or granulomatosis with polyangiitis).

Not specifically indicative of a specific disease; may be present in chronic inflammatory bowel diseases, PSC, AIH, as well as in other autoimmune diseases or chronic infection or without disease association.

c-ANCA

Cytoplasmic granular

Cytoplasmic granular

Irrelevant

PR3 and MPO negative or borderline reactive

Interpretation: May be present in treated, inactive or relapsing granulomatosis with polyangiitis, microscopic polyangiitis or Churg-Strauss syndrome; differential diagnosis must consider chronic infection, inflammatory bowel disease or other autoimmune diseases.

c-ANCA (atypical)

Cytoplasmic fine granular without interlobular accentuation

Negative

Negative or cytoplasmic fluorescence

PR3 and MPO negative or borderline reactive

Interpretation: Not indicative of ANCA-associated vasculitis. Not specifically indicative of a specific disease; may be present in chronic inflammatory bowel diseases, PSC, AIH, as well as in other autoimmune diseases or chronic infection or without disease association.

p-ANCA

Nuclear/ perinuclear

Cytoplasmic granular

Negative or cytoplasmic fluorescence

PR3 and MPO negative or borderline reactive

Interpretation: May be present in treated, inactive or relapsing microscopic polyangiitis or Churg-Strauss syndrome or, rarely, granulomatosis with polyangiitis; differential diagnosis must consider chronic infection, inflammatory bowel disease or other autoimmune diseases.

Anti-PR3 or anti-MPO Ab

Negative

Negative

Irrelevant

PR3 or MPO clearly positive

Interpretation: Indicative of vasculitis (further investigation by biopsy or follow-up tests)

Suspicion for p-ANCA

Nuclear/ perinuclear

Negative

Nuclear

PR3 and MPO negative

Interpretation: ANA or ANA and a-ANCA (if necessary, further investigation by differentiation of ANA)

Suspicion for p-ANCA

Nuclear/ perinuclear

Cytoplasmic granular

Nuclear

MPO (or rarely PR3) positive

Interpretation: ANA and p-ANCA (suspicion for vasculitis)

ANCA, anti-neutrophil cytoplasmic antibodies; p, perinuclear; c, cytoplasmic; a, atypical; PR3, proteinase 3; MPO, myeloperoxidase.

The International Consensus Statement /78/ recommends:

(1) The use of ethanol-fixed neutrophils; additional testing on formalin-fixed neutrophils for differentiating MPO-specific p-ANCA is not required.

(2) Determination of anti-PR3 and anti-MPO antibodies by ELISA

Table 25.9-1 Relevant autoantibodies in the diagnosis type 1 diabetes /3/

Autoantibodies in type 1 diabetes

Glutamic acid decarboxylase autoantibodies (GADA) /5/

The enzyme GAD catalyzes the conversion of glutamic acid to the inhibitory neurotransmitter γ-amino butyric acid (GABA). The highest concentrations of GAD are found in the central nervous system. GAD is an intracellular enzyme. Two isoforms of GAD are known in humans:

  • GAD65 ( molecular weight 65 kDa); the autoimmune response is directed against this protein
  • GAD67 with a molecular weight of 67 kDa. The percentage of cross-reaction with GAD65 is low. Only the detection of GAD65A is relevant in routine diagnostic testing for T1D.

Following the clinical diagnosis of T1D, GADA are more persistent than ICA. For this reason, GADA testing is preferred over ICA testing when latent autoimmune diabetes of adulthood (LADA) is suspected. In addition to T1D, GADA are also positive in stiff person syndrome.

Insulinoma-2-associated autoantibodies (IA-2A)

IA-2A were detected by screening an insulinoma expression library for reactivity with sera from type 1 diabetes patients. IA-2 is a member of the tyrosine kinase family and is a transmembrane protein. The auto reactive epitopes are located in the C-terminal region and thus oriented intracellularly. Auto reactivity to the C-terminal construct of IA-2A (ICA 512) is known as ICA512 autoantibody (ICA512A). IA-2A are less common at the onset of T1D than either ICA or GADA.

Insulin autoantibodies (IAA)

Most auto antigens, except insulin, are not specific to β cells. Insulin has been proposed as the primary target antigen in T1D. Loss of tolerance to insulin or failure to develop tolerance to insulin is believed to be the trigger to β cell autoimmunity and eventual β cell destruction. Insufficient thymic expression of insulin theoretically allows insulin-auto reactive clones of CD4+T cells and CD8+T cells to exit the thymus and to react with the insulin of pre damaged β cells.

IAA are more common in younger children with new-onset T1D than in adults. Once insulin is exogenously administered for more than 10 days, the determination of IAA is no longer useful because exogenous insulin injection can trigger the formation of polyclonal antibodies that cannot be distinguished from the IAA. However, the concentration of antibodies is usually higher if the antibodies are IAA.

Cytoplasmic islet cell antibodies (ICA) /5/

ICA are IgG-type autoantibodies that react with islet cells. They are directed against sialoglycoconjugates, an insulinoma-associated auto antigen, and against GAD. The advantage of the ICA assay is that it simultaneously detects autoantibodies to several antigens. The assay must be performed using blood type 0 human pancreatic tissue, since the test sensitivity and specificity are significantly lower with tissues of other species. The drawback of the ICA assay is that it gives only semi quantitative results.

Zinc transporter 8 (ZnT8) autoantibodies

Insulin is stored in pancreatic islet cell secretory vesicles, in which six insulin molecules form solid hexamers with two Zn2+. This crystalline structure is osmotically stable at pH 5.5 until secretion. Zn2+ is transported by the ZnT8 transporter (see Section 10.10 – Zinc) , a member of the SLC30 family. The ZnT8 transporter is present only in the human pancreas and is a key protein for insulin secretion by the islet cells /20/. ZnT8A can predict T1D.

Table 25.9-2 Threshold values for positive results

Autoantibodies

Positive result

ICA

≥ 10 JDF units /18/

GADA

> 1 GAD unit /19/

IA-2A

> 1.0 kU/L /19/

IAA

≥ 0.4 insulin binding units /10/

ZnT8A

Positive in the radio-precipitation assay

Table 25.9-3 Sensitivity and specificity of autoantibodies for the diagnosis of type 1 diabetes

Auto-
antibody

Sens.
(%)

Spec.
(%)

Clinical and laboratory findings

ICA

70–
100

> 99

ICA are found in 70–100% of patients with new onset T1D. ICA positivity declines following diagnosis of TD1, and only about 5% of T1D patients remain ICA positive after 10 years. Nondiabetic ICA positive relatives of type 1 diabetics who showed a reduced first phase insulin response had a 5-year risk of 60% and a 10-year-risk of 90% for the development of T1D, as demonstrated by data from the Diabetes Prevention Trial 1 /10/.

GADA

70–
80

97–
98

GADA occur with approximately the same frequency as ICA in patients with new onset T1D. However, since GADA persist longer than ICA, testing for GADA is preferred to ICA when LADA is suspected in adults with long standing diabetes.

IA-2A

60

97–
98

IA-2A are found less frequently than ICA or GADA in patients with new onset T1D. GADA are therefore the preferred markers for the diagnosis of T1D.

IAA

60

95

IAA are more frequently found in younger children with T1D than in older children and adults. The diagnostic sensitivity of IAA decreases with increasing age of the patient.

ZnT8A

63

 

Within a group of young patients with new onset T1D, 63% were positive for ZnT8A, 72% for GADA, 68% for IA-2A and 55% for IAA /12/. Within a group of T1D patients with negative ICA and biochemical islet cell antibodies, 26% were positive for ZnT8A /17/. After the onset of T1D a rapid decline of ZnT8A occurs. In the TrialNet NHS /13/ in relatives with one standard biochemical autoantibody, ZnT8A identified a subset at higher diabetes risk. ZnT8A predicted diabetes independently of ICA, age, and HLA type. ZnT8A should be included in T1D prediction and prevention studies. The ZNT8 antibody can add 3–4% to the antibody sensitivity forT1D. This is because ZnT8 antibody is positive in 3–4% of patients who are negative for GAD 65, IA-2 and insulin antibodies. Use of these 4 antibodies results in 93–98% diagnostic sensitivity.

Sens, diagnostic sensitivity; Spec, diagnostic specificity

Table 25.9-4 Prevalence of autoantibodies in non-diabetic children with (left column) and without (right column) familial diabetes predisposition /15/

Auto-
antibody

Prevalence of autoantibodies (%)

First degree
relatives

School-
children

GADA

4.6–7.0

0.3–3.0

IA-2A

2.1–5.3

0.1–2.4

IAA

2.5–3.7

1.5–3.9

At least 1 AAb

10.7–12.5

1.0–9.4

2 or 3 AAb

2.0–6.2

0.1–0.8

AAb, autoantibody/ies

Table 25.9-5 Risk of developing islet cell autoantibodies and type 1 diabetes in children and in gestational diabetes mellitus

Clinical and laboratory findings

TEDDY STUDY AND GENETIC SCORE

Around 0.3% of newborns will develop autoimmunity to pancreatic beta cells in childhood and subsequently develop type 1 diabetes before adulthood.The environmental Determinants of Diabetes in the Young (TEDDY) study followed the genetically at-risk children at 3- to 6-monthly intervals from birth for the development of islet autoantibodies and type 1 diabetes. The risk for developing multiple islet autoantibodies (pre-symptomatic type 1 diabetes) and type 1 diabetes in children who had no first-degree relatives with type 1 diabetes and either high risk genotypes (e.g., heterozygous HLADR 3 and DR4-DQ8 risk genotype or a homozygous DR4-DQ8 genotype). Risk was 5% in children with the two highest-risk HLA genotypes. In children with one of the 2 HLA risk genotypes, risk for developing multiple islet cell antibodies was 5.8% by age 6 years and 7.6% by age 10 years, respectively /21/. Children without a family history of type 1 diabetes and a merged genetic score (> 14.4) had a nearly 2 fold higher risk than children identified by high-risk genotypes alone /22/.

DAISY, BABABYDIAB, BABYDIET and DIPP STUDIES /917/

The time interval from seroconversion to onset of T1D varied from weeks to 18 years.

Only 7.9% of the study population developed autoantibodies to islet cells over 15 years. 55% of these individuals expressed multiple autoantibodies.

The number of autoantibodies predicted development of T1D, ranging from 10% in children with 1 autoantibody to almost 100% in children with multiple autoantibodies [hazard ratios for progression to diabetes (compared to children with no antibodies) were 395.6 and 52.7 for multiple and single autoantibodies, respectively].

The combination of IAA with IA-2 had a greater risk of progression to T1D than the combination of either IAA/GADA or IA-2/GADA.

Approximately 0.2% of the children who progressed to T1D were negative for autoantibodies to islet cells.

Faster progression to T1D after seroconversion was associated with younger age at seroconversion.

Gestational Diabetes mellitus (GDM)

GDM is the most common metabolic disorder in pregnancy with a prevalence of 2–17%. Normally the β cell pool adapts to physiological needs and increased functional demands. A subgroup of women of all GDM cases have a high risk to type 1 diabetes and/or latent autoimmune diabetes of adulthood (LADA) after pregnancy. Islet-cell autoantibodies, are present in sera from women with GDM in usually 10%. Titres for all autoantibodies are lower in GDM patients than in cases with newly diagnosed type 1 diabetes or in first degree relatives of patients with type 1 diabetes. The titres are similar to those observed in LADA patients, and are considered indicative of a slow developing autoimmune process. GADA are the most common autoantibody compared to the other autoantibodies, but autoantibodies show similar frequencies in GDM and women without GDM, suggesting that there is no strong correlation between autoantibody positivity and β cell impairment. Unless a cluster of clinical features is strongly suggestive of a diabetes type 1 like form of GDM (young age, low body mass index, early insulin therapy, presence of ketones) is present, autoantibody screening may not be needed /23/.

Table 25.9-6 Progression to type 1 diabetes in dependence of autoantibodies /10/

Diabetes associated
antibody

Positive
predictivity (%)

ICA positive

2.8

ICA and an additional
antibody positive

17.9

GADA positive

2.3

GADA and an additional
antibody positive

13.9

IAA

No predictivity

4 antibodies positive

50.0

3 antibodies positive

40.3

2 antibodies positive

16.1

1 antibody positive

3.1

No antibody positive

0.5

Table 25.10-1 Diagnostic antibodies in gastrointestinal diseases

Disease

Antibodies

Chronic atrophic
gastritis

Parietal cell antibodies (APCA)

Intrinsic factor antibodies (IFA)

Ulcerative colitis,
Crohn’s disease

Anti-pancreatic acinar cell antibodies (APAB)

Anti-Saccharomyces cerevisiae antibodies (ASCA)

Goblet cell antibodies (GAB)

Atypical anti-neutrophil cytoplasmic antibodies (atypical p-ANCA and atypical c-ANCA)

Celiac disease

Tissue transglutaminase antibodies (tTGA)

Endomysial antibodies (EMA)

Deamidated gliadin (peptide) antibodies (DG(P)A)

Autoimmune
pancreatitis

IgG4

Anti-carbonic anhydrase II antibodies

Anti-lactoferrin antibodies

Table 25.10-2 Vitamin B12 level and proportion of patients positive for intrinsic factor antibodies /8/

Vitamin B12
(pmol/L)

Proportion

%

201–600

2 of 254

0.82

151–200

4 of 65

6.2

100–150

4 of 23

17.4

< 100

9 of 13

69.2

Table 25.10-3 Diseases with increased prevalence of celiac disease and genetic predisposition /3334/

Disease

Prevalence (%)

Autoimmune diseases

Diabetes mellitus type 1

4–10

Autoimmune thyroiditis

4–5

Sjögren’s syndrome

4–12

Autoimmune hepatitis

3–6

Addison’s disease

5

Genetic syndromes

Down syndrome

5–10

Ullrich-Turner syndrome

8

Williams syndrome

8

Selective IgA deficiency

7–10

Relatives of celiac patients

Monozygotic twins

75

First degree relatives

5

Table 25.10-4 Serological markers of inflammatory bowel disease /5/

Antibodies to microbial antigens

Anti-glycan antibodies: anti-saccharomyces cerevisiae antibodies (ASCA); anti-chitibioside carbohydrate antibodies (ACCA); anti-laminaribioside carbohydrate antibodies (ACLA); anti-mannobioside carbohydrate antibodies (AMCA); anti-laminin antibodies (anti-L); anti-chitin antibodies (anti-C)

These antibodies target cell wall carbohydrate epitopes found in microorganisms like bacteria and yeasts. ASCA are the most prominent member. The glycan antibodies target carbohydrates. Similarly to ASCA the other carbohydrate IgG antibodies are specific to ulcerative colitis and Crohn’s disease, though with lower sensitivity.

Antibody to outer membrane porin (anti-OmpC): OmpC is an outer membrane protein of Escherichia coli. Patients with ulcerative colitis and Crohn’s disease have IgA type anti-OmpC.

Antibody to Pseudomonas fluorescens-associated sequence 12 (anti-12): the gene product has been shown to derive from Pseudomonas fluorescens. Patients with ulcerative colitis and Crohn’s disease have IgA type anti-12.

Antibody to bacterial flagellin (anti-Cbir1): patients with ulcerative colitis and Crohn’s disease have elevated anti-flagellin IgG2 response.

Autoantibodies

Atypical anti-neutrophil cytoplasmic antibodies (p-ANCA): the antibodies are present in serum of patients with ulcerative colitis and Crohn’s disease.

Antibodies against exocrine pancreas (PAB): the antibodies are present exclusively in serum of patients with ulcerative colitis and Crohn’s disease. The diagnostic sensitivity is low.

Antibodies to goblet cells (GAB): goblet cells are specialized intestinal epithelial cells. They regulate the production of mucus and factors that contribute to epithelial repair and regulation of inflammation. The antibodies are present in serum of patients with ulcerative colitis and Crohn’s disease

Endomysial antibodies (EMAs). The endomysium is the perivascular connective tissue which lines smooth muscle bundles. The target antigen in endomysium is tissue trans glutaminase. The enzyme is calcium dependent and cross links proteins. When it reacts with gliadin, neoepitopes are formed. The immunological response to these neoepitopes may initiate the mucosal damage in celiac disease /56/.

Table 25.10-5 Multicenter study on the sensitivity and specificity of anti-gliadin antibodies /50/

ELISA

Sens.
(%)

Spec.
(%)

Sens.
at 95%
spec.

IgA

Gliadin Ab (native)

79

80 

54 

Gliadin (deaminated)*Ab

88

89

83 

Transglutaminase Ab

96 

96

96 

IgG

Gliadin (native) Ab

11

79

31

Giadin (deaminated)*Ab

95

95 

94

Transglutaminase Ab

82

87

62

Test evaluation based on 181 sera from patients with biopsy confirmed gluten sensitive disease, 220 sera from controls with biopsy confirmed disease /56/. * “Anti-GAF-3X” antibodies, where GAF stands for gliadin analogous fusion protein; Sens, diagnostic sensitivity; Spec, diagnostic specificity

Table 25.10-6 Antibodies for the diagnosis of celiac disease in children below 6 years /55/

ELISA

Sens.
(%)

Spec.
(%)

PPV
(%)

NPV
(%)

IgA

Gliadin
(native) Ab

45.3

96.4

95.6

50.5

Gliadin
(deaminated) Ab

45.3

100

100

51.4

Endomysial Ab

77.9

100

100

72.4

Transglutaminase
Ab

77.9

100

100

72.4

Actin Ab

77.9

83.6

89.2

68.7

IgG

Gliadin
(native) Ab

50.5

74.6

77.4

46.6

Gliadin
(deaminated) Ab

60.0

100

100

59.1

Transglutaminase
Ab

54.7

100

100

56.1

PPV, positive predictive value; NPV, negative predictive value; Sens, diagnostic sensitivity; Spec, diagnostic specificity

Figure 25.2-1 Assessment of a negative and positive antinuclear antibody result. 1) Immunofluorescence screening on HEp2 cells and further steps depending on the reaction and fluorescent pattern.

ANA screening on HEp2 cells Negative Positive No further ANA differentiation (1) (1) Only at urgent clinical suspicion on collagenosis Pattern: Homogenous Fine/course speckled Nucleolar Characteristic patterns: Centromer Nuclear dots Few nuclear dots PCNA ANA differentiation with specific immunoassays on following antigens (usually parallel specifications): ds-DNA, SS-A/Ro, SS-B/La, Sm, U1-nRNP, Scl70and depending on the clinic of additional tests (e.g. myositis or scleroderma specific or associated antibodies) Differentiation with specific immunoassays: Centromere → CENP-B (A, D, F) Nuclear dots → Sp100 Few nuclear dots → Coilin-p80 PCNA → Cyclin Nuclear membrane with rim → gp210, Lamin-B receptor

Figure 25.3-1 Principle of rheumatoid factor (RF) determination by latex enhanced immunoassay. RF binds to the Fc portion of a human IgG antibody attached to latex beads. The resulting antigen-antibody reaction leads to formation of large immune complexes. These are quantified with turbidimetric or nephelometric measurements. The turbidity or scattered light measured is proportional to the RF concentration.

IgM Rheumatoid factor (IgM anti-IgG-Fc) Latex bead Human IgG

Figure 25.4-1 Removal of the arginine residue of a peptide by the enzyme peptidylarginine deiminase (PAD).

O N H O NH NH 3 Peptidylarginine-deiminase (PAD) Ca 2+ L-citrulline residue(neutral) N H O NH NH3 NH 4+ L-arginine residue (+-charged)

Figure 25.6-1 Diagnostic approach to suspected paraneoplastic neurological syndrome /4/. PNS, para-neoplastic syndrome.

Classical symptoms Non classical symptoms Neurological syndrome Tumor is present Tumor is present No tumor No tumor No onconeural Ab No onconeural Ab Onconeural Ab present or not Definitely PNS High cancer risk Possible PNS Definitely PNS Definitely PNS Possible PNS Possible PNS Proof of onconeural Ab Improvement after tumor therapy or proof of onconeural Ab Good character- ized onco- neural Ab Lower character- ized onco- neural Ab

Figure 25.6-2 Structure of the gangliosides. They consist of a ceramide body which is glycosidically connected to an oligosaccharide via a hydroxyl group that carries one or more molecules of N-acetyl neuraminic acid (sialic acid). The most simple structure is galactocerebroside (A); sulfatides are galactocerebrosides (B) sulfated at the third C atom of galactose. Gangliosides are a large family in which sialic acid is bound to galactose (C) or lactose (D), as in the LM1 gangliosides. Modified with kind permission from Ref. /11/. Gal, galactose; GalNAc, N-acetyl galactosamine; Glc, glucose; GlcNAc, N-acetyl glucosamine; NeuNAc, N-acetyl neuraminic acid; GlcUA, glucuronic acid; Cer, ceramide.

A GalNeuNAcGalNAcGluGlcNAcGlcUACeramid GalC B GM1 C SGPG SO 3 D LM1

Figure 25.6-3 Frequency of autoantibodies in immune mediated muscular disease. AChR, anti-acetylcholine receptor antibody (Ab); VGCC, anti-voltage-gated Ca2+ channel Ab; AQP4, anti-aquaporin Ab; MuSk, muscle-specific tyrosine kinase Ab; VGKC, anti-voltage-gated K+ channel Ab.

AChR VGCC VGKC MuSK AQP4 0 20 40 80 % 60 100

Figure 25.6-4 Antibody testing for suspected myasthenia gravis.

AChR-Ab Thymoma associated myasthenia positive Myasthenia gravis Titin Ab, if suspicion of thymoma Myasthenia gravis Anti-MuSK positive negative positive negative Myastheniagravisexcluded

Figure 25.7-1 Diagnostic algorithm for differentiation of autoantibodies associated with liver disease. ANA, antinuclear antibody (Ab); ASMA, anti-smooth muscle antibody; SLA/LP, anti-soluble liver antigen/liver pancreas antigen Ab; LKM, anti-liver/kidney microsomal Ab; AMA, anti-mitochondrial Ab; ENA, antibodies against extractable nuclear antigens; AIH, autoimmune hepatitis; PBC, primary biliary cirrhosis; PSC, primary sclerosing cholangitis.

Immunofluorescence test(HEp2 cell, rat liver, kidney, stomach) ANAnuclearfluorescence (HEp2-cell) ASMAF-actinfluorescence(rat stomach,HEp2 cell) LKMdistal tubularfluorescence (kidney)cytoplasmicfluorescence (liver) AMAtubular fl. (kidney)cytoplasmic fl.(liver, HEp2and parietal cells too) Homogenous,speckled,partial nucleolaror mixed Nucleolar dots,membranous,centromere SLA/LP(ELISA,immuno-blot) AIH type 1 AIH type 2 PBC ENA, dsDNAmostly negative Cytochromep450 2D6 AMA-M2 Sp100,gp210