Changes in Testing Algorithm and Reporting Practices for Antinuclear Antibodies and Immunohistochemical Patterns by IFA

Autoimmune diseases, including rheumatoid arthritis, polymyositis, Sjogren’s syndrome, systemic lupus erythematosus, systemic sclerosis, and others, are notoriously difficult to diagnose and the laboratory work-up for these patients is crucial to the process. Anti-nuclear antibodies (ANA) in particular are hallmarks of many autoimmune diseases.
Indirect immunofluorescence assay (IFA) is considered the gold standard for ANA screening by the American College of Rheumatology due to its high sensitivity. However, low specificity for the detection of rheumatic diseases and a high false positive rate are among the limitations of IFA. In an effort to compensate for these drawbacks and provide our clients with the most accurate and timely patient results, Mid America Clinical Laboratories has transitioned to a new platform for ANA Screen by IFA. The substrate will change from HEp-2000 to HEp-2 (human epithelial cell). The new technology offers significant improvement in both turnaround time and slide quality. It incorporates a more purified (Fc specific) anti-human IgG conjugate which ensures detection of only pathogenic antibodies and significantly decreases the false positivity rate of this assay.
To further aid our clients, we are expanding our testing menu to offer a more comprehensive rheumatic disease-targeted diagnostic algorithm. In addition to IFA, Mid America Clinical Laboratories will offer ANA screening by a multiplex platform – ANAChoice. This is a high-throughput multiplex bead immunoassay, which allows for a simultaneous analysis of the most clinically relevant 13 autoimmune analytes (SSA, SSB, Sm, Sm/RNP, RNP, Scl70, centromere B, dsDNA, chromatin, Jo1, and ribosomal P proteins). The ANAChoice screen will be reported as negative if autoantibodies to any of the above 13 antigens are not detected. If they are present, a cascade of reflex testing will be initiated. 
Multiplex immunoassays offer a great improvement in the diagnostic power of ANA testing by providing rapid and more specific results. However, relying on a limited number of antigens can lead to false-negative results. Thus, to better facilitate our clients, we will offer the following three first-line test codes to aid in diagnosing autoimmune diseases:
First-line test codes to aid in diagnosing autoimmune diseases:
        Test Code 249: ANA Screen, IFA with Reflex to Titer and Pattern, IFA
        Test Code 16814: ANA Screen, IFA with Reflex to Titer/Pattern/Specific Antibodies Cascading Reflex
        Test Code 19946:  ANAChoice Screen with Specific Antibodies Cascading Reflex
Addition of new IFA patterns
Two new patterns regarding cytoplasmic fluorescence added. Cytoplasmic fluorescence will be reported if observed on the HEp-2 Slides, even in the case of negative nuclear staining. Cytoplasmic staining observed by IFA has important clinical significance as it is indicative of primary biliary cirrhosis. The following two patterns will be implemented:
1.      “Cytoplasmic pattern detected, ANA positive”
2.      “Cytoplasmic pattern detected, ANA negative”
Seven new subcategories will be added under an “Atypical Pattern” replacing the existing “Atypical Speckled Pattern.” Most recent published guidelines3 and vendor recommendations suggest that the clinical significance of rare patterns (other than the most commonly observed: Homogeneous, Speckled, Centromere and Nucleolar) should not be ignored. The previously available “Atypical Speckled Pattern” category did not correspond to a single particular pattern, but included many rarely seen patterns, which current publications associate with different clinical indications. Thus, this pattern is being replaced by a new umbrella “Atypical Pattern” encompassing seven new rare patterns:
1.      “Possible PCNA pattern detected” – diagnostic of SLE1
2.      “Possible Multiple Nuclear Dots detected” – seen in 27-44% of patients with primary biliary cirrhosis1
3.      “Possible Midbody detected”-recognized in select patients with systemic sclerosis.1,2
4.      “Possible NSpII (CENP F) detected” – associated with malignancies1
5.      “Possible NuMA detected” – linked to Sjogren’s syndrome and primary biliary cirrhosis1
6.       “Possible Nuclear Membrane pattern detected”- associated with autoimmune hepatitis, SLE and RA1
1.      Immuno Concepts. Fluorescent Patterns Associated with Antinuclear Antibody Detection. 2005
2.      Givens T. Anti-Nuclear Antibody with Reflex Titer & Pattern by Zeus Scientific, Inc. ANA/HEp-2 Test System. Quest Diagnostic Incorporated. QDSE817 1.1 (Dec 2009)
3.      Agmon-Levin N, Damoiseaux J, Kallenberg C, et al. International Recommendations for the Assessment of Autoantibodies to Cellular Antigens Referred to as Anti-Nuclear Antibodies. Ann Rheum Dis 10.1136 (Oct 2013).
4.      Kavanaugh A, Tomar R, Reveille J, Solomon D, Homburger H, et al. Guidelines for Clinical Use of the Antinuclear Antibody Test and Tests for Specific Autoantibodies to Nuclear Antigens. Arch Pathol Lab Med 124, 71-81 (Jan 2000).
5.      Solomon D, Kavanaugh A, Schur P. Evidence-Based Guidelines for the Use of Immunologic Tests: Antinuclear Antibody Testing. Arthritis Rheum 47, 4: 434-444 (Aug 2002).

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