The antinuclear antibody (ANA) test is a standard screening assay for detecting multiple antibodies that may be produced by a patient with an autoimmune or ANA associated rheumatic disease (AARD). Although there are several methodologies available to screen ANA, the American College of Rheumatology (ACR) issued a statement declaring HEp-2 indirect immunofluorescence (IIF) as the preferred method for ANA screening [
Performing IIF test is labor intensive, subjective, and prone to reader bias [
Autoimmune laboratories have made strides in automation over the past decade but are still far behind other areas of the laboratory, such as chemistry with their fully automated instrumentation. Automated pattern interpretation of HEp-2 ANA was first described in 2002 by Perner et al. [
Based on the recommendation of the ACR for the use of HEp-2 IIF to test for ANA, we aimed to compare the agreement of the NOVA View archived images to the interpretation of the same samples on a manual fluorescent microscope interpreted by a certified medical technologist, with emphasis on agreement of end point titer. In addition, the data were used to calculate ANA titers and positivity rate in various AARD.
Clinically defined serum samples from patients suffering from SLE (
International reference serum panel (CDC ANA #1–12) was obtained from the Centers of Disease Control and Prevention (CDC) (
Lastly, 100 consecutive samples from an individual client, sent to ARUP Laboratories for ANA IIF testing, were reviewed. All patient samples included in the study were deidentified according to the University of Utah Institutional Review Board-approved protocol number 7275 to meet the Health Information Portability and Accountability Act Patient Confidentiality Guidelines.
NOVA Lite HEp-2 IgG ANA with DAPI kit and the NOVA View instrument with 1.0.2 software containing a cut-off value of 100 LIU for positive results (INOVA Diagnostics, San Diego, CA). The conjugate used in this assay contains the usual FITC fluorophor along with diamidino-2-phenylindole (DAPI), a blue nuclear stain that selectively binds to double stranded DNA. DAPI allows the instrument to “find” the cells at a 400 nm wavelength. If the cell density is insufficient or there are no cells in the well, the instrument will not switch to FITC but will produce an “
For the manual reading, a Nikon Eclipse 400 with an LED light source (ARUP Laboratories, Salt Lake City, Utah) was used.
All samples were processed manually and read on both the Nikon microscope and NOVA View, archived images with software version 1.0.2 by a board certified medical technologist. The technologist was blinded to sample classification and has 5 years of reading IIF daily at ARUP laboratories. Intensive training and continuous reading are needed for a technologist to accurately interpret HEp-2 ANA. At ARUP Laboratories, and other facilities, people who interpret HEp-2 ANA on clinical sera must be board certified. In order to read ANA IIF accurately and consistently they read daily and are challenged by internal and external surveys. Patterns recorded at ARUP include speckled, homogeneous, centromere, nucleolar, and nuclear dots, PCNA, and NuMA along with comments on cytoplasmic fluorescence observed.
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 (Tables
NOVA View agreement with manual IIF: 1 : 40 screening dilution.
All patients ( |
Manual IIF | Percent agreement (95% confidence) | ||
---|---|---|---|---|
Positive | Negative | Total | ||
NOVA View | ||||
Positive | 128 | 6 | 134 | Positive agreement = 94.8% (89.6–97.9%) |
Negative | 7 | 228 | 235 | Negative agreement = 97.4% (94.5–99.1%) |
Total | 135 | 234 | 369 | Total agreement = 96.5% (94.1–98.1%) |
|
Manual results were read on a Nikon Eclipse 400 fluorescent microscope with an LED light source.
NOVA View confirmed results = archived images reviewed and confirmed by the operator.
NOVA View agreement with manual IIF: 1 : 80 screening dilution.
All patients ( |
Manual IIF | Percent agreement (95% confidence) | ||
---|---|---|---|---|
Positive | Negative | Total | ||
NOVA View | ||||
Positive | 92 | 7 | 99 | Positive agreement = 92.9% (86.0–97.1%) |
Negative | 7 | 263 | 270 | Negative agreement = 97.4% (94.7–99.0%) |
Total | 99 | 270 | 369 | Total agreement = 96.2% (93.7–97.9%) |
|
Manual results were read on a Nikon Eclipse 400 fluorescent microscope with an LED light source.
NOVA View confirmed results = images reviewed and confirmed by the operator.
Of the 100 samples sent to ARUP Laboratories for routine ANA testing, 63% were negative and 37% were positive. Titers were within plus or minus a doubling dilution between the manual and the NOVA View archived image results, and ranged from 1 : 40 to 1 : 2560 (Figure
Antinuclear antibodies in different patient cohorts. (a) 27 positive systemic lupus erythematosus (SLE) patient sera titered at 1 : 40 through 1 : 2560. (b) Thirteen positive Sjögren’s syndrome (SjS) patient sera titered at 1 : 40 through 1 : 1280. (c) 26 positive scleroderma patient sera titered at 1 : 40 through 1 : 2560. The gray bar represents the end point titer read on the NOVA View archived image. The black bar represents the titer read on a traditional manual microscope. All titers were read by the same technologist.
Good agreement and correlation between manual and NOVA View archived based reading were found. Results of the manual IIF interpretation were grouped into positive and negative. Subsequently, the titers obtained from the NOVA View archived image based interpretation were used to generate a receiver operating characteristic (ROC) curve showing very good agreement (Figure
Agreement and correlation between manual and NOVA View archived image based reading. (a) Receiver operating characteristic (ROC) analysis. Results of the manual indirect immunofluorescence interpretation were grouped into positive and negative. Subsequently, the titers obtained from the NOVA View interpretation were used to generate a ROC curve showing very good agreement (
The 12 CDC samples produced excellent correlation for pattern and titer (data not shown). All samples with ANA were positive with titers ranging from 1 : 40 to 1 : 320. The patterns match their original description of the antibody specificity.
The ROC analyses revealed similar discrimination between AARD and controls using the manual and the NOVA View archive reading (Table
Clinical sensitivity and specificity.
Assay | Sensitivity % (95% CI) | Specificity % (95% CI) | Excluding RA samples specificity % (95% CI) |
---|---|---|---|
Manual IIF, 1 : 40 dilution | 60.5 (50.9–69.6) | 89.5 (83.3–94.0) | 93.9 (87.3–97.7) |
NOVA View, 1 : 40 dilution | 61.4 (51.8–70.4) | 90.9 (85.0–95.1) | 94.9 (88.6–98.3) |
Manual IIF, 1 : 80 dilution | 49.1 (39.6–58.7) | 94.4 (89.3–97.6) | 98.0 (92.9–99.8) |
NOVA View, 1 : 80 dilution | 50.9 (41.3–60.4) | 94.4 (89.3–97.6) | 97.0 (91.4–99.4) |
Comparative receiver operating characteristic (ROC) analyses. ROC for manual indirect immunofluorescence (IIF) manual interpretation and NOVA View results for ANA related autoimmune rheumatic disease (AARD) versus healthy controls and rheumatoid arthritis (RA). The ROC curves were similar for manual IIF and NOVA View, and as expected, specificity improves for both methods when RA patients are removed from analysis.
Antinuclear antibody titer and prevalence among ANA associated rheumatic diseases (AARD,
Although not the focus of our study, we investigated the impact of the NOVA View instrument in the laboratory workflow. We found that the system is a walk-away platform, with user friendly software, and the ability to interface with bar coded slides for positive patient identification.
The recommendation of the ACR to use IIF as the preferred method has triggered the development and validation of automated systems for ANA determination. Although the ANA IIF test is the recommended method for ANA testing, the method has significant limitations, including a high degree of subjectivity [
The NOVA View has a walk-away platform, user friendly software, and the ability to interface with bar coded slides for positive patient identification. In addition, the patient images are stored for later viewing without fluorescent burnout. However, the impact on the workflow might vary from laboratory to laboratory and needs to be quantified in further studies. The good agreement between interpretation using a microscope and using archived images on a screen holds promise to avoid the dark room, which is a source of transcription errors of results.
The specificity against healthy individuals in this study was in keeping with recent recommendations for the determination of anticellular antibodies [
Since we did not analyze the performance of the NOVA View in terms of positive/negative discrimination and pattern recognition, further studies are needed. In recent years, several of those studies have already been performed [
Since this study, NOVA View has had two software updates. The current version, 1.0.3.1, contains a Single Well Titer (SWT) application that utilizes the LIU and assigns pattern to produce a calculated titer from one well. A recent study by Schouwers et al. concluding the estimation of fluorescent intensity offers clinically useful information and value added reporting [
Further studies are desired to underline the clinical utility of the NOVA View system in diagnostic specimens.
The authors declare that there is no conflict of interests regarding the publication of this paper.
This study was supported by funding from the ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, Utah. All kits used in this study were kindly provided free of charge by the manufacturer who had no role in the collection, analysis, and interpretation of the data. The authors thank Marvin Fritzler, PhD, MD, Alberta Research & Innovation Authority, and Rufus Burlingame, VP of Diagnostic Assay Development, Genalyte Inc., for the use of their well-documented disease serum samples.