title: Validation of a multi-analyte panel with cell-bound complement activation products for systemic lupus erythematosus.
abstract: 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.
  
  
  
  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).
  
  
  
  EC4d and BC4d are stable for 2days at ambient temperature and for 4days at 4°C post-phlebotomy. Median intra-day and inter-day CV range from 2.9% to 7.8% (n=30) and 7.3% to 12.4% (n=66), respectively. The 2-tiered index score is reproducible over 4 consecutive daysupon storage of blood at 4°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%.
  
  
  
  We have established the analytical validity of a multi-analyte assay panel for SLE.
authors.affiliation: Exagen Diagnostics, Vista, CA, United States. Electronic address: tdervieux@exagen.com.
authors.forename: Thierry
authors.initials: T
authors.lastname: Dervieux
authors.affiliation: Exagen Diagnostics, Vista, CA, United States.
authors.forename: John
authors.initials: J
authors.lastname: Conklin
authors.affiliation: Exagen Diagnostics, Vista, CA, United States.
authors.forename: Jo-Anne
authors.initials: JA
authors.lastname: Ligayon
authors.affiliation: Exagen Diagnostics, Vista, CA, United States.
authors.forename: Leilani
authors.initials: L
authors.lastname: Wolover
authors.affiliation: Exagen Diagnostics, Vista, CA, United States.
authors.forename: Tyler
authors.initials: T
authors.lastname: O'Malley
authors.affiliation: Exagen Diagnostics, Vista, CA, United States.
authors.forename: Roberta Vezza
authors.initials: RV
authors.lastname: Alexander
authors.affiliation: Georgetown University, Washington, DC, United States.
authors.forename: Arthur
authors.initials: A
authors.lastname: Weinstein
authors.affiliation: Exagen Diagnostics, Vista, CA, United States.
authors.forename: Claudia A
authors.initials: CA
authors.lastname: Ibarra
mesh_terms: D000328:Adult; D000974:Antibodies, Antinuclear / Q000097:blood; D001323:Autoantibodies / Q000097:blood* / Q000302:isolation & purification; D015415:Biomarkers / Q000097:blood; D003167:Complement Activation; D015935:Complement C4b / Q000276:immunology / Q000302:isolation & purification; D005260:Female; D005434:Flow Cytometry / Q000379:methods; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D008180:Lupus Erythematosus, Systemic / Q000175:diagnosis* / Q000276:immunology; D008297:Male; D008875:Middle Aged; D010446:Peptide Fragments / Q000276:immunology / Q000302:isolation & purification
chemical_list: D000974:Antibodies, Antinuclear; D001323:Autoantibodies; D015415:Biomarkers; D010446:Peptide Fragments; D015935:Complement C4b; C032261:complement C4d
keywords: Biomarkers; Complement; Diagnostic immunology