Autoimmunity Testing Options

Autoantibodies can be specific markers for autoimmune (AI) diseases and are a key component in the diagnosis of diseases which are often underestimated and difficult to determine.

Criteria and Tests for
Autoimmune Disease Diagnosis

Laboratory tests performed to aid in the diagnosis of autoimmune disorders depend on the particular disorder(s) the healthcare provider suspects a patient has, but usually include blood tests for one or more autoantibodies as well as tests for inflammation such as C-reactive protein levels (CRP) and elevated erythrocyte sedimentation rate (ESR).

Blood testing options

Antiphospholipid Syndrome

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According to international classification criteria, identification of APS requires the presence of vascular thrombosis and/or pregnancy morbidity, along with at least one of the following antiphospholipid antibody tests, which are often performed in parallel:¹

Lupus Anticoagulant (LA)
Cardiolipin IgG and/or IgM antibodies

β2-glycoprotein I (β2-GPI) IgG and/or IgM antibodies¹

Connective Tissue Disease

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Sjögren’s syndrome

According to the 2016 American College of Rheumatology/European League Against Rheumatism classification criteria for primary Sjögren’s syndrome, individuals with signs and/or symptoms suggestive of Sjögren’s syndrome who have a total score of ≥4 for the items below meet the criteria for primary Sjögren’s syndrome:²

Anti-SSA/Ro antibody positivity and focal lymphocytic sialadenitis with a focus score of ≥1 foci/4 mm³, each scoring 3
An abnormal Ocular Staining Score of ≥5 (or van Bijsterveld score of ≥4)
A Schirmer’s test result of ≤5 mm/5 min
An unstimulated salivary flow rate of ≤0.1 mL/min, each scoring 1

Systemic lupus erythematosus (SLE)

According to the 1997 Update of the 1982 American College of Rheumatology Revised Criteria for Diagnosis of SLE, four of 11 following criteria should be fulfilled at the same time or in succession:⁴

Malar rash
Discoid rash
Photosensitivity
Oral ulcers
Non-erosive arthritis
Pleuritis or pericarditis
Renal disorder (proteinuria >0.5 g/day or cellular casts)
Neurologic disorder
Hematologic disorder
Immunologic disorder (anti-DNA/anti-Sm/LE cell/false-positive STS
A Positive ANA: An abnormal titre of antinuclear antibody by immunofluorescence OR an equivalent assay at any point in time and (in the absence of drugs)

Systemic sclerosis

According to the 2013 ACR/EULAR classification criteria for Systemic Sclerosis, patients with a total score of ≥9 are classified as having definite SSc.⁵

Items	Sub-items	Score
Skin thickening of the fingers of both hands extending proximal to the MCP joints (sufficient criteria)	--	9
Skin thickening of the fingers (only count the highest score)	Puffy fingers Sclerodactyly of the fingers	2 4
Fingertip lesions (only count the highest score)	Digital tip ulcers Fingertip pitting scars	2 3
Telangiectasia	--	2
Abnormal nail fold capillaries	--	2
Pulmonary arterial hypertension and/or interstitial lung disease (maximum score is 2)	Pulmonary arterial hypertension Interstitial lung disease	2 2
Raynaud's phenomenon	--	3
SSc-related antibodies	Anti-centromere Anti- Scl-70 Anti-RNA polymerase III	3 -- --

Polymyositis/Dermatomyositis

According to the 2017 EULAR/ACR classification criteria for adult and juvenile IdiopathicInflammatory Myopathies (IMM) and their major subgroups, includingpolymyositis/dermatomyositis, patients with probability above 55 percent, when no better explanation for the symptoms or signs exists, are classified as having IIM.⁶

Items	No Biopsy	Biopsy
Age of onset of first related symptoms 18-40 age ≥40 age	1.3 2.1	1.5 2.2
Muscle weakness Objective symmetric weakness, usually progressive, of proximal upper extremities Objective symmetric weakness, usually progressive, of proximal lower extremities Neck flexors are relatively weaker than neck extensors In the legs, proximal muscles are relatively weaker than distal muscles	0.7 0.8 1.9 0.9	0.7 0.5 1.6 1.2
Skin Manifestations Heliotrope rash Gottron's papules Gottron's sign	3.1 2.1 3.3	3.2 2.7 3.7
Other clinical manifestations Dysphagia or esphageal dysmotility	0.7	0.6
Laboratory measurements Anti-Jo1 autoantibody positivity Elevated serum level (above uppe limit of normal) of creatine kinase or lactate dehydrogenase or aspartate aminotransferase or alanine aminotransferase	3.9 1.3	3.8 1.4
Muscle biopsy features Endomysial infiltration of mononuclear cells surrounding, but not invading, myofibres Perimysial and/or perivascular infiltration of mononuclear cells Perifascicular atrophy Rimmed vacuoles	-- -- -- --	1.7 1.2 1.9 3.1

Items

No Biopsy

Biopsy

Age of onset of first related symptoms

18-40 age ≥40 age

1.3

2.1

1.5

2.2

Muscle weakness

Objective symmetric weakness, usually progressive, of proximal upper extremities
Objective symmetric weakness, usually progressive, of proximal lower extremities
Neck flexors are relatively weaker than neck extensors
In the legs, proximal muscles are relatively weaker than distal muscles

0.7

0.8

1.9

0.9

0.7

0.5

1.6

1.2

Skin Manifestations

Heliotrope rash
Gottron's papules
Gottron's sign

3.1

2.1

3.3

3.2

2.7

3.7

Other clinical manifestations

Dysphagia or esphageal dysmotility

0.7

0.6

Laboratory measurements

Anti-Jo1 autoantibody positivity
Elevated serum level (above uppe limit of normal) of creatine kinase or lactate dehydrogenase or aspartate aminotransferase or alanine aminotransferase

3.9

1.3

3.8

1.4

Muscle biopsy features

Endomysial infiltration of mononuclear cells surrounding, but not invading, myofibres
Perimysial and/or perivascular infiltration of mononuclear cells
Perifascicular atrophy
Rimmed vacuoles

1.7

1.2

1.9

3.1

Mixed connective tissue disease (MCTD)

There is overlapping diagnostic criteria for MCTDs. A comparison study determined that MCTD was best identified with the Alarcón-Segovia criteria and the Kahn criteria.⁷

The Alarcón-Segovia diagnostic criteria:

Serological criteria and at least three clinical criteria.
If the clinical criteria 1, 4, and 5 are present, then 2 or 3 are required as well.

Serological criteria:

A positive anti-RNP at a hemagglutination titer of 1:1600 or higher AND at least three of the following five clinical criteria:

Edema of the hands
Synovitis
Myositis
Raynaud’s phenomenon
Acrosclerosis

The Kahn diagnostic criteria:

Serological criteria and clinical criteria.¹
At least two of the other clinical criteria.

Serological criteria:

A high titer of anti-RNP corresponding to speckled ANA at titer 1:2000 or higher.

Clinical criteria:

Raynaud’s phenomenon
Synovitis
Myositis
Swollen finger

Celiac Disease

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Serological testing can help differentiate this disease and provide an accurate, expedited diagnosis. It may help to consider testing when there is presentation of gastrointestinal (GI) symptoms or a new diagnosis for a condition that increases the risk for having CD.⁸

International classification criteria advocates for serologic testing to help aid in the diagnosis of celiac disease. The European Society for Pediatric Gastroenterology, Hepatology and Nutrition has published guidelines for the diagnosis of celiac disease for children or adolescents with otherwise unexplained symptoms and signs suggestive of CD.⁹It states that children should be tested for:

Immunoglobulin A (IgA) Transglutaminase (tTG) and total IgA, as the first choice
Deamidated Gliadin (DGP-AGA) IgG, tTG IgG, or IgG Endomysial Antibodies (EMA) if IgA is deficient

In the UK, the National Institute for Health and Care Excellence encourages young people and adults to be tested for:¹⁰

tTG IgA and total IgA as the first choice
IgA EMA if tTG IgA is weakly positive
IgG deamidated gliadin peptide, tTG IgG, or IgG EMA if IgA is deficient

The American College of Gastroenterology recommends that adults in whom celiac disease is suspected by testing according to the following algorithms.³

Adapted from Husby, et al 2012, Werkstetter et al., 2017, and World Health Organization, 2015. Please be aware that additional analytical parameters can be necessary.

Crohn’s Disease

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Testing to differentiate Crohn’s Disease (CrD) from Ulcerative Colitis (UC), both main IBD entities, can help you choose the most appropriate therapy for your patient.¹¹ Anti-Saccharomyces Cerevisiae Antibodies (ASCA) testing, in combination with Perinuclear Antineutrophil Cytoplasmic Autoantibodies (pANCA), may be used to aid in differential diagnosis of CrD and UC, especially in IBD-unclassified (IBD-U) patients.^12,13 In many IBD-U patients, pathology makes endoscopic differentiation extremely difficult.^14,15

The combination of positive pANCA with negative ASCA is indicative for UC, whereas the combination of positive ASCA with negative pANCA is indicative for Crohn´s disease.¹⁶

Using ASCA and pANCA to help differentiate Crohn’s disease and ulcerative colitis:¹⁶

Thyroid Diseases

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The following algorithms may be helpful in diagnosing AITDs, i.e., autoimmune thyroiditis or Hashimoto’s thyroiditis and Graves’ disease. Getting these patients the appropriate diagnosis is very important. The presence of AITDs, especially Hashimoto’s thyroiditis, increases the risk for other autoimmune diseases such as Type 1 diabetes mellitus and Addison’s disease.^17,18 Be aware of this link and consider testing your patient for other autoimmune diseases, if appropriate.

Rheumatoid Arthritis

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Numerous international guidelines recommend RF IgM and anti-CCP as first-line tests to aid in the diagnosis of rheumatoid arthritis. CCP antibodies appear in the early stages of rheumatoid disease.^19,20

International guidelines classification criteria also recommend laboratory testing for:

Erythrocyte Sedimentation Rate (ESR)
C-Reactive Protein (CRP)

Many tests measure rheumatoid factor (RF) using nephelometry or turbidometry. However, using an RA panel that can distinguish between the different RF isotypes—RF IgA and RF IgM in particular—can give you important additional diagnostic guidance.^21-23

Vasculitis

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International guidelines recommend positive serology for ANCAs. These diagnostic tools can help you identify the disease and start your patients on the road to treatment for granulomatosis with polyangiitis (GPA), microscopic polyangiitis (MPA), and eosinophilic granulomatosis with polyangiitis (EGPA).²⁴

The revised 2017 international consensus on testing of ANCAs in GPA and MPA proposes that high-quality immunoassays for MPO and PR3 antibodies should be used as the primary screening method for patients suspected of having the ANCA-associated vaculitides GPA and MPA without the need for indirect immunofluorescence (IIF).²⁴

References

Miyakis S, Lockshin MD, Atsumi T, et al. International consensus statement on an update of the classification criteria for definite antiphospholipid syndrome (APS). J Thromb Haemost. 2006;4(2):295-306.
Shiboski CH, Shiboski SC, Seror R, et al. 2016 American College of Rheumatology/European League Against Rheumatism Classification Criteria for Primary Sjogren’s Syndrome. Ann Rheum Dis. 2017;76(1):9-16.
Gaubitz M. Epidemiology of connective tissue disorders. Rheumatology (Oxford). 2006;45(Suppl 3):iii3-4.
American College of Rheumatology. 1997 Update of the 1982 ACR Criteria for the Classification of SLE. http://www.rheumatology.org/Portals/0/Files/1997%20Update%20of%201982%20Revised.pdf. Accessed December 2017.
van den Hoogen F. Classification Criteria for Systemic Sclerosis: An ACR-EULAR Collaborative Initiative Arthritis Rheum. 2013; 65(11):2737-2747.
Bottai M, Tjärnlund A, Santoni G, et al. EULAR/ACR Classification criteria for adult and juvenile idiopathic inflammatory myopathies and their major subgroups: a methodology report. RMD Open 2017;3:e000507.
JM Amigues, A Cantagrel, M Abbal, et al. Comparative study of 4 diagnosis criteria sets for mixed connective tissue disease in patients with anti-RNP antibodies. Autoimmunity Group of the Hospitals of Toulouse. J of Rheum. 1997;(23)2055-62.
Rubio-Tapia A, Hill ID, Kelly CP, et al. ACG Clinical Guidelines: Diagnosis and Management of Celiac Disease. Am J Gastroenterol. 2013;108:656-676.
Husby S, Koletzko S, Korponay-Szabó IR, et al. European Society for Pediatric Gastroenterology, Hepatology, and Nutrition Guidelines for the Diagnosis of Coeliac Disease. J Pediatr Gastroenterol Nutr. 2012;54:136-160.
National Institute for Health and Care Excellence. Coeliac disease: recognition, assessment and management. https://www.nice.org.uk/guidance/ng20/resources/coeliac-disease-recognition-assessment-and-management-pdf-1837325178565. Accessed November 2017.
Kovács M, Müller KE, Papp M, et al. New serological markers in pediatric patients with inflammatory bowel disease. World J Gastroenterol. 2014;20(17):4873-4882.
Levine A, Koletzko S, Turner D, et al. ESPGHAN Revised Porto Criteria for the Diagnosis of Inflammatory Bowel Disease in Children and adolescents. J Pediatr Gastroenterol Nutr. 14;58:795-806.
Joossens S, Reinisch W, Vermeire S, et al. The value of serologic markers in indeterminate colitis: a prospective follow-up study. Gastroenterology. 2002;122:1242-1247.
Burakoff R. Indeterminate colitis: clinical spectrum of disease. J Clin Gastroenterol. 2004;38(5 Suppl 1):S41-S43.
Bousvaros A, Antonioli DA, Colletti RB, et al. Differentiating ulcerative colitis from Crohn disease in children and young adults: report of a working group of the North American Society for Pediatric Gastroenterology, Hepatology, and Nutrition and the Crohn’s and Colitis Foundation of America. J Pediatr Gastroenterol Nutr. 2007;44(5):653-674.
Bossuyt X. Serologic markers in inflammatory bowel disease. Clin Chem. 2006;52:2:171-181.
Iddah MA, Macharia BN. Autoimmune thyroid disorders. ISRN Endocrinol. 2013;2013:509764.
Boelaert K, Newby PR, Simmonds MJ, et al. Prevalence and Relative Risk of Other Autoimmune Diseases in Subjects with Autoimmune Thyroid Disease. Am J Med. 2010;123(2):183.e1-9.
Schellekens GA, Visser H, de Jong BA, et al. The diagnostic properties of rheumatoid arthritis antibodies recognizing a cyclic citrullinated peptide; Arthritis Rheum. 2000;43(1):155-163.
Berglin E, Padyukov L, Sundin U, et al. A combination of autoantibodies to cyclic citrullinated peptide (CCP) and HLA-DRB1 locus antigens is strongly associated with future onset of rheumatoid arthritis. Arthritis Res Ther. 2004;6(4):R303-R308.
Bobbio-Pallavicini F, Caporali R, Alpini C, et al. High IgA rheumatoid factor levels are associated with poor clinical response to tumour necrosis factor α inhibitors in rheumatoid arthritis. Ann Rheum Dis. 2007;66(3): 302-307.
Jónsson T, Valdimarsson H. Is measurement of rheumatoid factor isotypes clinically useful? Ann Rheum Dis. 1993;52(2):161-164.
Shakiba Y, Koopah S, Jamshidi AR, et al. Anti-cyclic citrullinated peptide antibody and rheumatoid factor isotypes in Iranian patients with rheumatoid arthritis: evaluation of clinical value and association with disease activity. Iran J Allergy Asthma Immunol. 2014;13(3):147-156.
Bossuyt X, Cohen Tervaert J-W, Arimura Y, et al. Revised 2017 international consensus on testing of ANCAs in granulomatosis with polyangiitis and microscopic polyangiitis. Nature Rev Rheumatol. 2017;doi: 10.1038/nrrheum.2017.140

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