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- Chromosomal Microarray, POC, ClariSure®, Oligo-SNP
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- Chromosome Analysis and AFP with Reflex to AChE, Fetal Hgb, Amniotic Fluid
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Tests for Autoimmune DiseasesTest code(s) 249, 16814, 19946, 94954
Question 1. What are autoimmune diseases?
“Autoimmune disease” refers to a diverse group of disorders that can involve almost every one of the body’s organs and systems. It encompasses diseases of the nervous, gastrointestinal, and endocrine systems, as well as skin and other connective tissues, eyes, blood, and blood vessels. In all of these autoimmune diseases, the underlying problem is “autoimmunity”—the body’s immune system becomes misdirected and attacks the very organs it was designed to protect.
Question 2. Why are autoimmune diseases challenging to diagnose?
Diagnosis is challenging for several reasons:
- Patients initially present with nonspecific symptoms such as fatigue, joint and muscle pain, fever, and/or weight change.
- Symptoms often flare and remit.
- Patients frequently have more than 1 autoimmune disease.
According to a survey by the Autoimmune Diseases Association, it takes up to 4.6 years and nearly 5 doctors for a patient to receive a proper autoimmune disease diagnosis.1
Question 3. How common are autoimmune diseases?
At least 30 million Americans suffer from 1 or more of the 80-plus autoimmune diseases.
On average, autoimmune diseases strike 3 times more women than men2. Certain autoimmune diseases have an even higher female:male ratio. Autoimmune diseases are one of the top 10 leading causes of death among women aged 65 and under2 and represent the fourth-largest cause of disability among women in the United States.3
Autoimmune disease commonly occurs in multiple members of a family, indicating a genetic predisposition. Family members are often affected by various autoimmune disorders rather than a single specific disorder.
Question 4. What is the first test to be considered for a patient suspected of having an autoimmune disease?
When evaluating a patient for autoimmune diseases, an antinuclear antibody (ANA) test is typically performed first. The immunofluorescence assay (IFA) (test code 249) screens for approximately 150 autoantibodies that can occur in various autoimmune diseases. The American College of Rheumatology (ACR) recommends IFA as the gold standard method for ANA testing.4
A negative ANA IFA result suggests that ANA-associated autoimmune diseases are not present, but does not rule out the possibility. Patients with negative results on the ANA IFA usually also have negative results for specific ANA antibodies. However, Jo-1 antibody may be detected in ANA IFA-negative patients with some types of myositis, and SSA antibody may be present in some ANA IFA-negative patients with SLE or Sjögren's syndrome.5
A positive result on the ANA IFA screen suggests the presence of autoimmune disease, and will reflex to titer and pattern. A low ANA titer (1:40 to 1:80) may be associated with preclinical disease or lack of disease. Titers >1:80 are consistent with autoimmune disease. In cases of positive ANA, the staining pattern helps predict the disease type.
Specific antibody testing, if clinically indicated, is often useful for diagnosis of specific autoimmune disorders. Quest Diagnostics offers testing options for early diagnosis and prognosis of autoimmune diseases. Please read questions 6, 7, and 8 for more information.
Question 5. What is the significance of ANA staining patterns?
As noted above, the ANA staining pattern can be helpful in suggesting a diagnosis, but does not provide definitive evidence. The significance of various patterns is summarized below.
Question 6. Can I use a single patient specimen to both screen (IFA) and test for specific autoantibodies?
Yes. Quest Diagnostics offers test code 16814 (ANA Screen, IFA, Reflex Titer/Pattern and Reflex to Multiplex 11 Ab Cascade). This test begins with an ANA screen using IFA technology. A positive result reflexes to titer and pattern and to a 3-tiered, 11-antibody cascade. The first tier includes chromatin, dsDNA, RNP, Sm, and Sm/RNP antibodies. If all 5 antibodies are negative, testing proceeds to the second tier, which includes Jo-1, Scl-70, SS-A, and SS-B antibodies. If all 4 of these antibodies are negative, testing proceeds to the final tier, which includes centromere B and ribosomal P antibodies.
The Figure details the cascade and interpretation of specific antibody results. Note that if the ANA IFA result is positive, but all 11 specific antibody results are negative, an autoimmune disease may still be present. The disease may be associated with an antibody not tested in the cascade. Diseases to be considered include rheumatoid arthritis, autoimmune hepatitis, primary biliary cholangitis, autoimmune thyroiditis, Addison disease, pernicious anemia, autoimmune neuropathies, vasculitis, celiac disease, and bullous disease, among others.
Question 7. Can I use a single patient specimen to screen for ANA (IFA), test for specific autoantibodies, and test for rheumatoid arthritis?
Yes. Quest Diagnostics offers an integrated panel to facilitate timelier diagnosis of rheumatoid arthritis in ANA-negative patients as well as those with comorbid rheumatoid diseases: the ANA Screen, IFA, with Reflex to Titer/Pattern, and Reflex to the Multiplex 11 Ab Cascade with IdentRA® (test code 94954). The IdentRA component of this assay includes tests for rheumatoid factor, cyclic citrullinated peptide antibody, and 14-3-3η protein. For patients with positive ANA IFA results, the reflex pattern and testing for specific autoantibodies is identical to that of test code 16814 (see Question 6). The first tier includes chromatin, dsDNA, RNP, Sm, and Sm/RNP antibodies. If all 5 antibodies are negative, testing proceeds to the second tier, which includes Jo-1, Scl-70, SS-A, and SS-B antibodies. If all 4 of these antibodies are negative, testing proceeds to the final tier, which includes centromere B and ribosomal P antibodies.
In addition, all specimens are tested for 3 markers of RA: rheumatoid factor, cyclic citrullinated peptide antibody, and 14-3-3η protein (IdentRA). IdentRA improves diagnostic sensitivity in the diagnosis of early rheumatoid arthritis relative to testing for rheumatoid factor and cyclic citrullinated peptide antibody alone. American College of Rheumatology guidelines indicate that patients who are diagnosed with early RA and are experiencing active disease at the time of testing should be prescribed disease modifying anti-rheumatic drug therapy.6
Question 8. Can I order specific autoantibody testing without an ANA (IFA) screen?
Yes. Testing for each of the autoantibodies included in the 11-antibody cascade may be ordered separately or by using the ANA Multiplex with Reflex to 11 Antibody Cascade (test code 19946). The test 19946 does not include an ANA screen based on IFA technology. Instead, the 3-tier, 11-antibody reflex cascade is triggered if one of the 11 antibodies included in the multiplex screen is positive. This ANA screen is less sensitive, because it tests for only 11 autoantibodies in comparison to ANA IFA screen which tests for approximately 150 autoantibodies. The 3-tier, 11-antibody reflex cascade is identical to that of test code 16814 (ANA Screen, IFA, Reflex Titer/Pattern and Reflex to Multiplex 11 Ab Cascade). The first tier includes chromatin, dsDNA, RNP, Sm, and Sm/RNP antibodies. If all 5 antibodies are negative, testing proceeds to the second tier, which includes Jo-1, Scl-70, SS-A, and SS-B antibodies. If all 4 of these antibodies are negative, testing proceeds to the final tier, which includes centromere B and ribosomal P antibodies. This test may be used when looking for a tiered approach for diagnosis of autoimmune conditions or the patient had a previous positive result on an ANA IFA screening test.
A negative result on the multiplex screen does not rule out autoimmune disease. In addition, a disease associated with an antibody not tested in the cascade may be present, especially if the patient has previously tested positive on an ANA IFA screen. Diseases to be considered include rheumatoid arthritis, autoimmune hepatitis, primary biliary cholangitis, autoimmune thyroiditis, Addison disease, pernicious anemia, autoimmune neuropathies, vasculitis, celiac disease, bullous disease, and others.
Question 9. How predictive are the specific autoantibodies? What is their sensitivity and specificity in various autoimmune diseases?
The presence of a specific antibody is highly suggestive of the associated autoimmune disease. However, these antibodies are not completely specific for a particular disease; thus, results need to be interpreted in context of the clinical information and considering the following antibody prevalence.
Prevalence of Tier 1 Antibodies7-10
Double-stranded DNA (dsDNA) antibodies are present in 57% to 62% of systemic lupus erythematosus (SLE) cases, 10% to 43% of polymyositis, 11% to 20% of Sjögren syndrome, 8% of systemic sclerosis (scleroderma), and 0% to 8% of mixed connective tissue disease (MCTD).
Chromatin antibody is present in >80% of MCTD cases, 37% to 73% of SLE, 14% of systemic sclerosis, 12% of Sjögren syndrome, and 8% of polymyositis.
Ribonucleoprotein (RNP) antibodies target RNP A and/or RNP 68kD proteins; antibodies to one or both are present in >80% of MCTD cases, 22% to 48% of SLE, 14% of systemic sclerosis, 12% of Sjögren syndrome, and 8% of polymyositis.
Sm/RNP antibodies are directed to epitopes formed in a complex of Sm and RNP; antibodies to the Sm/RNP complex are present in 54% to 94% of MCTD cases, 30% of SLE, 4% of systemic sclerosis, and 9% of Sjögren syndrome and polymyositis.
Sm antibody is present in 20% to 30% of SLE cases, 8% of MCTD, 10% of polymyositis, 0% of systemic sclerosis, and 4% of Sjögren syndrome.
Double-stranded DNA, chromatin, ribonucleoprotein, Sm/RNP complex, and Sm antibodies are also present in <2% of normal blood donors.
Prevalence of Tier 2 Antibodies9,10
SS-A and SS-B antibodies are present in >80% of Sjögren syndrome cases and are considered a diagnostic indicator for this autoimmune disease. However, these antibodies are also present in other autoimmune disorders. SS-A antibodies are seen in 33% to 52% of SLE cases, 42% of polymyositis, 23% of systemic sclerosis (scleroderma), and 13% of MCTD. SS-B antibody is present in 13% to 27% of SLE cases, 5% of systemic sclerosis, <2% of polymyositis, and <2% of MCTD.
Scl-70 antibody is present in 16% of systemic sclerosis cases, 7% of MCTD (especially those with features of systemic sclerosis), 2% to 3% of SLE, <2% of Sjögren syndrome, and <2% of polymyositis.
Jo-1 antibody is present in 17% of polymyositis cases, 7% of MCTD (especially in those with features of muscle inflammation), <2% of SLE, Sjögren syndrome, and systemic sclerosis.
Tier 2 antibodies are also present in <2% of normal blood donors.
Prevalence of Tier 3 Antibodies9-12
Centromere B antibody is present in 27% of systemic sclerosis (scleroderma) cases, 66% of CREST syndrome (calcinosis, Raynaud phenomenon, esophageal dysmotility, sclerodactyly, and telangiectasia), 3% to 12% of SLE, 7% of MCTD (typically with features of polymyositis), and <2% of Sjögren syndrome, polymyositis, and normal blood donors.
Ribosomal P antibody is present in 9% to 30% of SLE cases (often with neurological manifestations), 7% of MCTD, and <2% of Sjögren syndrome, systemic sclerosis, polymyositis, and normal blood donors.
The prevalences listed above may vary with the population studied and methods used.7 Values given are for general guidance.
- Autoimmune disease in women. American Autoimmune Related Diseases Association, Inc. Web site. http://www.aarda.org/autoimmune-information/autoimmune-disease-in-women/. Accessed February 4, 2016.
- Walsh SJ, Rau LM. Autoimmune diseases: a leading cause of death among young and middle-aged women in the United States. Am J Public Health. 2000;90:1463-1466.
- Autoimmune diseases fact sheet. Womenshealth.gov Web site. http://www.womenshealth.gov/publications/our-publications/fact-sheet/autoimmune-diseases.html?from=AtoZ. Updated July 16, 2012. Accessed February 4, 2016.
- American College of Rheumatology Position Statement: Methodology of testing for antinuclear antibodies. http://www.rheumatology.org/Portals/0/Files/Methodology%20of%20Testing%20Antinuclear%20Antibodies%20Position%20Statement.pdf. Published January 2009. Updated August 2015. Accessed February 3, 2016.
- Yazdany J, Schmajuk G, Robbins M, et al. Choosing wisely: the American College of Rheumatology's Top 5 list of things physicians and patients should question. Arthritis Care Res (Hoboken). 2013;65:329-339.
- Singh JA, Saag KG, Bridges SL, Jr, et al. 2015 American College of Rheumatology guideline for the treatment of rheumatoid arthritis. Arthritis Rheumatol. 2016;68:1-26.
- Scholz J, Grossmann K, Knütter I, et al. Second generation analysis of antinuclear antibody (ANA) by combination of screening and confirmatory testing. Clin Chem Lab Med. 2015;53:1991-2002.
- Colglazier CL, Sutej PG. Laboratory testing in the rheumatic diseases: a practical review. South Med J. 2005;98:185-191.
- Moder KG, Wener MH, Weisman MH, et al. Measurement of antinuclear antibodies by multiplex immunoassay: a prospective, multicenter clinical evaluation. J Rheumatol. 2000;34:978-986.
- Binder SR, Genovese MC, Merrill JT, et al. Computer-assisted pattern recognition of autoantibody results. Clin Diagn Lab Immunol. 2005;12:1353-1357.
- Mouthon L, Dunogue B, Guillevin L. Diagnosis and classification of eosinophilic granulomatosis with polyangiitis (formerly named Churg-Strauss syndrome). J Autoimmun. 2014;48-49:99-103.
- Tur BS, Süldür N, Ataman S, et al. Anti-neutrophil cytoplasmic antibodies in patients with rheumatoid arthritis: clinical, biological, and radiological correlations. Joint Bone Spine. 2004;71:198-202.