Assessment of antinuclear antibodies by indirect immunofluorescence assay: report from a survey by the American Association of Medical Laboratory Immunologists.

Background: The indirect immunofluorescence assay (IFA) using HEp-2 cell substrates is the preferred method by some for detecting antinuclear antibodies (ANA) as it demonstrates a number of characteristic staining patterns that reflect the cellular components bound as well as semi-quantitative results. Lack of harmonized nomenclature for HEp-2 IFA patterns, subjectivity in interpretation and variability in the number of patterns reported by different laboratories pose significant harmonization challenges. The main objectives of this study were to assess current practice in laboratory assessment of HEp-2 IFA, identify gaps and define strategies to improve reading, interpretation and reporting. Methods: We developed and administered a 24-item survey based on four domains: educational and professional background of participants, current practice of HEp-2 IFA testing and training, gap assessment and the perceived value of International Consensus on Antinuclear Antibody Patterns (ICAP) and other factors in HEp-2 IFA assessment. The Association of Medical Laboratory Immunologists (AMLI) and American Society for Clinical Pathology administered the survey from April 1 to June 30, 2018, to members involved in ANA testing. This report summarizes the survey results and discussion from a dry workshop held during the 2019 AMLI annual meeting. Results: One hundred and seventy-nine (n = 179) responses were obtained where a significant number were clinical laboratory scientists (46%), laboratory directors (24%), supervisors (13%) or others (17%). A majority of respondents agreed on the need to standardize nomenclature and reporting of HEp-2 IFA results. About 55% were aware of the ICAP initiative; however, among those aware, a significant majority thought its guidance on HEp-2 IFA nomenclature and reporting is of value to clinical laboratories. To improve ICAP awareness and further enhance HEp-2 IFA assessment, increased collaboration between ICAP and the clinical laboratory community was suggested with emphasis on education and availability of reference materials. Conclusions: Based on these suggestions, future efforts to optimize HEp-2 IFA reading, interpretation and reporting would benefit from more hands-on training of laboratory personnel as well as continuous collaboration between professional organizations, in vitro diagnostic manufacturers and clinical laboratories.

Interpretation of ANA indirect immunofluorescence test outside the darkroom using NOVA view compared to manual microscopy.

OBJECTIVE: To evaluate NOVA View with focus on reading archived images versus microscope based manual interpretation of ANA HEp-2 slides by an experienced, certified medical technologist. METHODS: 369 well defined sera from: 44 rheumatoid arthritis, 50 systemic lupus erythematosus, 35 scleroderma, 19 Sjögren's syndrome, and 10 polymyositis patients as well as 99 healthy controls were examined. In addition, 12 defined sera from the Centers for Disease Control and 100 random patient sera sent to ARUP Laboratories for ANA HEp-2 IIF testing were included. Samples were read using the archived images on NOVA View and compared to results obtained from manual reading. RESULTS: At a 1 : 40/1 : 80 dilution the resulting comparison demonstrated 94.8%/92.9% positive, 97.4%/97.4% negative, and 96.5%/96.2% total agreements between manual IIF and NOVA View archived images. Agreement of identifiable patterns between methods was 97%, with PCNA and mixed patterns undetermined. CONCLUSION: Excellent agreements were obtained between reading archived images on NOVA View and manually on a fluorescent microscope. In addition, workflow benefits were observed which need to be analyzed in future studies.

Anti-NMDA-receptor antibody encephalitis: performance evaluation and laboratory experience with the anti-NMDA-receptor IgG assay.

BACKGROUND: Antibodies targeting the NR1 subunit of the N-methyl-d-aspartate-receptor (NMDAR) are considered diagnostic for a novel form of autoimmune encephalitis. We report the validation of a qualitative indirect immunofluorescence antibody (IFA) test for the detection of anti-NMDAR IgG and describe the attributes of antibody-positive patients. METHODS: The anti-NMDAR IgG assay (Euroimmun Diagnosika, Lübeck, Germany) was validated with serum and cerebrospinal fluid (CSF) specimens from 30 healthy and 50 disease controls as well as 5 anti-NMDAR IgG-positive individuals. Consecutive specimens (n=1671) for anti-NMDAR IgG antibodies were evaluated and positive specimens titrated to end-point [starting dilutions: CSF; 1:1 and serum; 1:10]. In a subset of antibody-positive patients, we sought clinical information for correlation with diagnostic and treatment outcomes. RESULTS: The assay demonstrated excellent performance characteristics in all groups evaluated. Of the 1671 specimens tested, 1389 were unique cases with a positivity rate of 9.0% (n=123). For the antibody-positive samples, the female to male ratio was 2:1 with a prevalence of 46% in the pediatric population (≤17 years). Antibody titers were titrated to end-point for 106/123 specimens [45 CSF, 41 sera, and 20 CSF and serum pairs] with more than 75% having titers greater than 1:10 (CSF) and 1:20 (serum). Overall, high levels of these antibodies showed correlation to disease severity with variable response to treatment in the subset of patients evaluated. CONCLUSION: Our data suggests a high prevalence for anti-NMDAR antibody encephalitis irrespective of age and gender in our unselected disease cohort with support for measuring antibody titers in the evaluation of these patients.

Screening for IgG antinuclear autoantibodies by HEp-2 indirect fluorescent antibody assays and the need for standardization.

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.

Enzyme-linked immunosorbent assay screening then indirect immunofluorescence confirmation of antinuclear antibodies: a statistical analysis.

The purpose of this study was to analyze antinuclear antibody (ANA) screening by enzyme-linked immunosorbent assay (ELISA) followed by indirect fluorescent antibody (IFA) testing to confirm and characterize the pattern and titer of the antibody. We evaluated 4 ANA ELISAs and 1 HEp-2 IFA substrate in 224 clinically defined serum samples consisting of 30 from systemic lupus erythematosus (SLE) cases, 94 from rheumatoid arthritis cases, and 100 from healthy donors plus 495 serum samples submitted for routine ANA testing and 12 reference serum samples distributed by the Centers for Disease Control and Prevention. IFA tests were read independently by 2 certified medical technologists. ELISA sensitivities ranged from 90% to 97% compared with 80% by IFA in the SLE serum samples. The ELISAs had specificities of 36% to 94%, whereas the IFA had 99% specificity. Overall, ELISAs for ANA assays demonstrated better sensitivity and good specificity, suggesting ELISA is a more cost-effective alternative to IFA testing for initial ANA screening. Samples positive by ANA ELISA should be tested on HEp-2 to determine the titer and pattern.

Elimination of false-positive results in a luminex assay for pneumococcal antibodies.

In our 14-valent Luminex assay for pneumococcal antibodies, we identified two groups of sera that caused false-positive results. The first group bound nonspecifically to the Luminex microspheres. The second group reacted specifically with bovine serum albumin (BSA). We describe here methods that eliminated the false-positive reactivity of both groups.

Comparison of three multiplex immunoassays for detection of antibodies to extractable nuclear antibodies using clinically defined sera.

We set out to determine the agreement of three multiplex immunoassays for the detection of autoantibodies involved in connective tissue disease using clinically defined sera. Usefulness of the immunoassays will be defined by correlation to disease state. Using the immunoassays from Inova Diagnostics, Biomedical Diagnostics (BMD), and AtheNA, reactivity, to Smith (Sm), ribonucleic protein (RNP), SSA (Ro), SSB (La), Scl-70, and dsDNA or chromatin, we tested 273 clinically defined sera consisting of 57 systemic lupus erythrematosus (SLE) sera, 69 rheumatoid arthritis (RA) sera, 47 sera defined as various other connective tissue diseases, and 100 normal donor sera. Samples were also tested for anti-nuclear antibody (ANA) oN HEp-2 cells by IFA for analysis of discrepant results. Inova, BMD, and AtheNA assays demonstrated 57%, 89%, and 80% concordance, respectively, in the 47 connective tissue disease sera. The BMD assay was the most sensitive in detecting Scl-70. The immunoassays did not correlate well in the 57 SLE-defined sera; however, each had a variety of antibodies positive for each serum. The AtheNA assay demonstrated the highest degree of nonspecificity. Inova, BMD, AtheNA, and the Inova ANA HEp-2 IFA demonstrated 97%, 98%, 97%, and 99% specificity, respectively, using normal sera. Thus, all three assays showed a 97% or better negative predictive value. Positive correlation varied from 83% to 98%. Antibodies in SLE sera did not compare well among the three immunoassays. Significant variation in specificity and sensitivity due to individual characteristics of each assay was demonstrated, with the BMD assay showing the highest correlation with clinical diagnosis.