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THU0337 Evaluation of High-Throughput Platforms for Digital Antinuclear Antibody (ANA) Indirect Immunofluorescence (IFA) Screening
  1. F. Zarabpouri1,
  2. M.-L. V. Zambales1,
  3. V. O. Miralles1,
  4. T. T. Pizarro1,
  5. J. M. Popov1,
  6. S. J. Naides1
  1. 1Immunology, Quest Diagnostics Nichols Institute, San Juan Capistrano, United States


Background ANA screening is a gateway test in the laboratory evaluation of autoimmunity. High-throughput enzyme immunoassays (EIAs) have largely replaced traditional IFA methods but have raised concerns about the limited number of antigen specificities included on immunobeads and about batch variation of antigen content in cell lysate based EIAs. A return to traditional, manual ANA IFA is impeded by labor intensity, lack of standardization, interobserver variation, and the need for highly-trained technical staff.

Objectives A high-throughput IFA method with observer consistency and ease of quality control and monitoring is desirable. To find such a method, we evaluated the performance of 3 commercial platforms for digital ANA IFA image acquisition and analysis.

Methods The 3 digital ANA IFA platforms each employed proprietary HEp-2 slides and antihuman IgG fluorescein conjugates. Results were compared with those obtained using manual IFA on a proprietary HEp-2 cell substrate that used an antihuman polyvalent immunoglobulin conjugate. Results were interpreted by a single senior laboratory scientist.

Results Normal donor (n=120) and ≥153 patient samples submitted for testing were evaluated by manual ANA IFA and on each digital platform. Initial platform screening using the original factory settings indicated the need to adjust the instrument threshold of positivity to approximate the performance of the manual method, which we used as the gold standard. For platform 1, fluorescence intensity positive threshold values of 66, 147, 180, 219, and 280 Units yielded sensitivities of 98%, 90%, 90%, 86%, and 83%, and specificities of 33%, 71%, 78%, 86%, and 92%, respectively. Digital platform specificity was low before we adjusted the threshold for positivity. After detection thresholds were optimized, all 3 platforms were evaluated against the manual IFA (Table). Aligning the platform threshold for positivity with that of the manual method improved specificity without significantly altering sensitivity. Discordant samples tended to have low titer by digital or manual methods. Samples negative by the digital methods but positive by the manual method tended to have nucleolar ANA patterns, several with moderate titers.

Conclusions Thresholds based on total field intensity units may fail to recognize more discrete patterns such as nucleolar or nuclear dots. Technologists should review all images generated on digital platforms to confirm positivity versus negativity and identify patterns that may be overlooked by digital analysis algorithms.

Disclosure of Interest F. Zarabpouri Employee of: Quest Diagnostics Nichols Institute, M.-L. Zambales Employee of: Quest Diagnostics Nichols Institute, V. Miralles Employee of: Quest Diagnostics Nichols Institute, T. Pizarro Employee of: Quest Diagnostics Nichols Institute, J. Popov Employee of: Quest Diagnostics Nichols Institute, S. Naides Employee of: Quest Diagnostics Nichols Institute

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