Screening for IgG Antinuclear Autoantibodies by HEp-2 Indirect Fluorescent Antibody Assays and the Need for Standardization

Abstract

We evaluated 5 commercially available HEp-2 antinuclear antibody (ANA) indirect fluorescent antibody (IFA) assays using patient serum samples from 45 patients with rheumatoid arthritis, 50 with systemic lupus erythematosus (SLE), 35 with scleroderma, 20 with Sjögren syndrome, 10 with polymyositis, and 100 healthy control subjects. In addition, 12 defined serum samples from the Centers for Disease Control and Prevention and 100 patient serum samples sent to ARUP Laboratories (Salt Lake City, UT) for ANA IFA testing were also examined (n = 372). Standardization among the HEp-2 IFA assays occurred when they exhibited the same titer ± 1 doubling dilution. Agreement of the 5 assays was 78%. Within the specific groups of serum samples, agreement ranged from 44% in scleroderma serum samples to 93% in healthy control subjects, with 72% agreement in the SLE group. Variations in slide and substrate quality were also noted (ie, clarity, consistency of fluorescence, cell size, number and quality of mitotic cells). Along with subjectivity of interpretation, HEp-2 IFA assays are also vulnerable to standardization issues similar to other methods for ANA screening.

Comparative Study of Human and Automated Screening for Antinuclear Antibodies by Immunofluorescence on HEp-2 Cells

“…The best agreements involved specifically the most common IIF patterns, homogenous, speckled, nuclear dots and membranous nuclear. This results corroborates that the study of Copple et al [4], in which 5 HEp-2 kits (Kallestad®, ImmunoConcepts®, Zeus®, Euroimmun® and Inova®) were compared using 372 sera (50 SLE, 45 rheumatoid arthritis, 35 scleroderma, 20 Sjögren syndrome, 10 polymyositis, 12 reference sera from the CDC, 100 healthy blood donors and 100 sera from private laboratories). In this study, three skilled technologists made evaluation independently and blindly to sample classification and each other's reading [4].…”

“…In this study, three skilled technologists made evaluation independently and blindly to sample classification and each other's reading [4]. Concordance between the 3 readers varied from 96% to 99% [4]. Overall, the percentage of agreement for the 5 HEp-2 assays was 78% and varied from 44% in the scleroderma sera to 93% in the healthy blood donor group [4].…”

“…Concordance between the 3 readers varied from 96% to 99% [4]. Overall, the percentage of agreement for the 5 HEp-2 assays was 78% and varied from 44% in the scleroderma sera to 93% in the healthy blood donor group [4]. Furthermore, the authors reported discrepancies in titers' estimation that reached for some specimens from to 1/80 th to 1/1280 th [4].…”

“…Overall, the percentage of agreement for the 5 HEp-2 assays was 78% and varied from 44% in the scleroderma sera to 93% in the healthy blood donor group [4]. Furthermore, the authors reported discrepancies in titers' estimation that reached for some specimens from to 1/80 th to 1/1280 th [4]. It has been proposed that the use of a polyconjugate versus an IgG-specific conjugate results in variability among the 5 tested kits.…”

Prevalence and Clinical Significance of Anti-DFS70 in Antinuclear Antibody (ANA)–positive Patients Undergoing Routine ANA Testing in a New Zealand Public Hospital

“…In our laboratory, ANA screening slides are interpreted by a NOVA View automated IIF slide reader (INOVA Diagnostics Inc.), which incorporates a digital analysis image system, pattern recognition algorithms, and preset cutoff values. Problems still exist for the laboratory community to determine whether this system efficiently identifies antigens of clinical significance and whether the different automated systems have an appropriate level of pattern recognition agreement 4 .…”