ELISA and HEp-2 testing assays yielded 40 (45%) and 72 (81%) positive test results, respectively, demonstrating lack of concordance between test methods. ELISA method did not detect several ANA with nucleolar, homogeneous and speckled immunofluorescence patterns. None of these ELISANEG AN-3485 HEp-2POS ANA were reactive with a panel of six extractable nuclear antigens or with double-stranded DNA. Nonetheless, 13 of these samples (15%) originated from patients with acknowledged ANA-associated disease (n?=?7) or Raynaud’s phenomenon (n?=?6). We conclude that ELISA screening may fail to detect clinically relevant ANA that lack defined specificity for antigen. Keywords: anti-nuclear antibody, autoantibody, autoimmune screening, autoimmunity, audit Introduction Anti-nuclear antibodies (ANA) are extremely useful for the diagnosis of systemic lupus erythematosus (SLE) and systemic sclerosis (SSc), and are somewhat useful for diagnosis of Sj?gren’s syndrome and inflammatory myositis (IM; polymyositis and dermatomyositis) 1C3. These autoantibodies are also found in autoimmune hepatitis type 1 4, and are associated with increased risk of uveitis in patients with juvenile idiopathic arthritis (JIA) 5. Detection of ANA may precede the diagnosis of overt autoimmune disease, sometimes by several years 6. However, ANA may be detected in up to 30% of healthy individuals 7. Such false positive results are a major limitation to diagnostic power 8 and result generally from reactivity to dense fine speckled (DFS) 70 antigen 9. In a recent position statement, the American College of Rheumatology has indicated that immunofluorescence-based screening remains the platinum standard approach to ANA screening 10,11. Furthermore, they state that option diagnostic AN-3485 methods should demonstrate comparable performance to this standard. The status of immunofluorescence-based ANA screening as a reference method has been re-emphasized in a AN-3485 recent international consensus recommendation paper 12. Most commonly, such testing is usually conducted using fixed human epidermoid laryngeal carcinoma (HEp-2) cells, allowing the detection of up to 150 nuclear and cytoplasmic Rabbit Polyclonal to PKC alpha (phospho-Tyr657) autoantibody types. However, HEp-2-based immunofluorescence ANA screening is slow, subjective and requires a high degree of interpretative skill. Moreover, HEp-2 screening is not amenable to standardization and may lack sensitivity for anti-Ro and ribosomal P autoantibodies 2. Owing to these limitations, combined with increasing workloads and budgetary restrictions, many laboratories have relocated to ANA screening using solid-phase assay techniques, notably enzyme-linked immunosorbent assay (ELISA) and fluorescence-based enzyme immunoassays 13. Nonetheless, concerns remain about the level of clinical validation and surveillance that is required in respect of such high-throughput option methods 14, both prior to and after their introduction into clinical practice. At Barnet hospital, we receive approximately 15?000 ANA requests annually. To maintain satisfactory turnaround, screening had been conducted using an ELISA system. However, colleagues in the Department of Rheumatology expressed their issues about the ability of this assay to detect a sufficient proportion of clinically relevant ANA. Consequently, we audited the analytical overall performance of this assay in the field, making comparison with the platinum AN-3485 standard HEp-2-based diagnostic test. While the relative sensitivity of both methods agreed with previous reports, we found that only HEp-2 screening detected a number of clinically relevant ANA that lacked defined target antigen specificity. This failure of ELISA screening to meet the audit standard resulted in the reinstatement of HEp-2 screening into our support. Materials and methods Study design Eighty-nine unselected requests for ANA screening from rheumatology consultants were analysed in parallel using an ANA ELISA [ORG 600 ANA Detect (Orgentec Diagnostika GmbH, Mainz, Germany), comprising 26 human recombinant and highly purified native antigens, including all ANA of acknowledged clinical significance] and a HEp-2 indirect immunofluorescence assay (Nova Lite; Inova Diagnostics, San Diego, CA, USA). Samples were collected prospectively over a 10-month period from January 2013. Screening was also undertaken of ANA requirements [IS2072 (homogeneous)/IS2100 (nucleolar)/IS2134 (centromere)], which were a kind gift of the Center for Disease Control (CDC) Biological Reference Reagents (Atlanta, GA, USA). Diagnosis was subsequently assigned from review of the electronic patient record and medical center letters. Assay performance Samples were analysed by UK state-registered biomedical scientists who were unaware of clinical details and results of other screening. Tests were performed according to the manufacturer’s instructions, except as specified below. The cut-off for the ELISA test had been decided earlier in-house as an optical density (OD) ratio of 08 with respect to the kit control. This locally established threshold provides greater assay sensitivity than the manufacturer’s recommended cut-off, which is usually 12 (borderline significance assigned to indices of 10C12). For HEp-2 screening, sera were screened at a dilution of 1/40 and, if positive, samples were diluted to obtain end-point titre (excluding nucleolar/centromere subtypes). Samples that yielded discordant results in the screening ANA tests were tested for antibodies directed against extractable nuclear antigens [ENA (EliA Symphony, Freiburg, Germany), which detects antibodies to Sm, Ro52/60, La, U1 RNP, Scl-70, CENP-B and Jo-1].