Validation of a multi-analyte panel with cell-bound complement activation products for systemic lupus erythematosus

Background: We describe the analytical validation of an assay panel intended to assist clinicians with the diagnosis of systemic lupus erythematosus (SLE). The multi-analyte panel includes quantitative assessment of complement activation and measurement of autoantibodies. Methods: The levels of the complement split product C4d bound to erythrocytes (EC4d) and B-lymphocytes (BC4d) (expressed as mean fluorescence intensity [MFI]) are measured by quantitative flow cytometry, while autoantibodies (inclusive of antinuclear and anti-double stranded DNA antibodies) are determined by immunoassays. Results of the multi-analyte panel are reported as positive or negative based on a 2-tiered index score. Post-phlebotomy stability of EC4d and BC4d in EDTA-anticoagulated blood is determined using specimens collected from patients with SLE and normal donors. Three-level C4 coated positive beads are run daily as controls. Analytical validity is reported using intra-day and inter-day coefficient of variation (CV). Results: EC4d and BC4d are stable for 2 days at ambient temperature and for 4 days at 4 degrees C post-phlebotomy. Median intra-day and inter-day CV range from 2.9% to 7.8% (n = 30) and 73% to 12.4%(n = 66), respectively. The 2-tiered index score is reproducible over 4 consecutive days upon storage of blood at 4 degrees C. A total of 2,888 three-level quality control data were collected from 6 flow cytometers with an overall failure rate below 3%. Median EC4d level is 6 net MFI (Interquartile [IQ] range 4-9 net MFI) and median BC4d is 18 net MFI (IQ range 13-27 net MFI) among 86,852 specimens submitted for testing. The incidence of 2-tiered positive test results is 13.4%. Conclusion: We have established the analytical validity of a multi-analyte assay panel for SLE. (C) 2017 Elsevier B.V. All rights reserved.