Interpretation and Reporting of Antinuclear Antibodies by HEp-2 Indirect Immunofluorescence Assay - Consensus-Guided Multisite Performance Study

Oct 6, 2026

Uploaded by Mary Man

Evaluate interpretation and reporting performance for antinuclear antibodies (ANA) using HEp-2 indirect immunofluorescence assay (IFA) in line with common practice and International Consensus on ANA patterns (ICAP). Sixteen clinical laboratories and eight in vitro diagnostic manufacturers tested, interpreted, and reported HEp-2 IFA results for twelve pre-qualified specimens across nuclear, cytoplasmic, and mitotic categories. Accuracy for nuclear categorical patterns was highest (99%); cytoplasmic was lower (78%) with notable titer variability. Results also compared common versus ICAP nomenclature and assessed how breadth of pattern reporting affected performance, with implications for training competencies.

LanguageEnglish

CategoryHealthcare

FormatPDF

Pages20

Reading Time50 min

AccessFree to download

Study Objective

Participants and Specimens

Reporting Performance Results

Categorical Pattern Accuracy

Common vs ICAP Nomenclature

Titer Variability

Discussion and Clinical Implications

Competency and Training Needs

􀂦 The University of Utah School of Medicine [https://orcid.org/0000-0001-9553-298X](https://orcid.org/0000-0001-9553-298X) The University of Utah Department of Pathology Cleveland Clinic's Robert J Tomsich Pathology and Laboratory Medicine Institute The University of Utah Department of Pathology Department of Oral Biology, University of Florida University of Calgary Cumming School of Medicine University of Washington Anti-nuclear antibodies, Cytoplasmic patterns, Performance Survey, Indirect immuno§uorescence, Mitotic patterns, Nuclear patterns [https://doi.org/10.21203/rs.3.rs-80095/v1](https://doi.org/10.21203/rs.3.rs-80095/v1) 􀁱 􀅏 This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License To evaluate the interpretation and reporting of antinuclear antibodies (ANA) by indirect immuno§uorescence assay (IFA) using HEp-2 substrate based on common practice and guidance by the International Consensus on ANA patterns (ICAP) . Participants included two groups [ 16 clinical laboratories (CL) and 8 in vitro diagnostic manufacturers (IVD)] recruited via an email sent to the Association of Medical Laboratory Immunologists (AMLI) membership. Twelve (n =12) pre-quali¦ed specimens were distributed to participants for testing, interpretation and reporting HEp-2 IFA. Results obtained were analyzed for accuracy with the intended and consensus response for three main categorical patterns (nuclear, cytoplasmic and mitotic), common patterns and ICAP report nomenclatures. The distributions of antibody titers of specimens were also compared. Laboratories differed in the categorical patterns reported; 8 reporting all patterns, 3 reporting only nuclear patterns and 5 reporting nuclear patterns with various combinations of other patterns. For all participants, accuracy with the intended response for the categorical nuclear pattern was excellent at 99%[95% con¦dence interval (CI) : 97-100%] compared to 78%[95% CI: 67-88%] for the cytoplasmic, and 93%[95% CI: 86%-100%] for mitotic patterns. The accuracy was 13% greater for the common nomenclature [87%, 95% CI: 82-90%] compared to the ICAP nomenclature [74%, 95% CI: 68-79%] for all participants. Participants reporting all three main categories demonstrated better performances compared to those reporting 2 or less categorical patterns. The average accuracies varied between participant groups, however, with the lowest and most variable performances for cytoplasmic pattern specimens. The reported titers for all specimens varied, with the least variability for nuclear patterns and most titer variability associated with cytoplasmic patterns. Our study demonstrates signi¦cant accuracy for all participants in identifying the categorical nuclear staining as well as traditional pattern assignments for nuclear patterns. However, there was less consistency in reporting cytoplasmic and mitotic patterns, with implications for assigning competencies and training for clinical laboratory personnel. The presence of antinuclear antibodies (ANA) is a hallmark and classi¦cation criterion for a number of systemic autoimmune rheumatic diseases (SARD) . ANA testing is usually performed as part of the initial diagnostic workup when suspicion of an underlying autoimmune disorder is high. The indirect immuno§uorescence antibody (IFA) technique on HEp-2 substrate has been considered the traditional and preferred method for detecting ANA by some [ 1]. It allows detection of antibody binding to speci¦c intracellular targets, resulting in diverse staining patterns that are usually categorized based on the cellular components recognized and the degree of binding, as re§ected by the §uorescence intensity or titer [2, 3]. As a screening tool, the recognition of a well-de¦ ned HEp-2 IFA staining pattern may be helpful in determining the most likely speci¦c autoantibodies present, as well as suggesting possible clinical associations for known speci¦cities

FAQs

What was the study designed to evaluate for ANA testing?

The study evaluated how well participants interpreted and reported antinuclear antibodies (ANA) using HEp-2 indirect immunofluorescence assay (IFA) based on common practice and ICAP guidance.

How did accuracy differ across nuclear, cytoplasmic, and mitotic categorical patterns?

Accuracy was excellent for the categorical nuclear pattern (99%), lower for cytoplasmic patterns (78%), and high for mitotic patterns (93%).

What comparisons were made regarding nomenclature and its effect on performance?

Accuracy was compared between common nomenclature and ICAP nomenclature across participants, showing higher accuracy for common nomenclature than for ICAP nomenclature.

Oct 6, 2026

Uploaded by Mary Man