Abstract
Background
The indirect immunofluorescence assay (IIFA) for the detection of antinuclear antibodies (ANA) was firstly described in 1958 and is still considered the reference method for ANA screening. Currently, an automated processing and recognition system for standardized and efficient ANA interpretation by human epithelial (HEp‐2) cell‐based immunofluorescence (IIF; EUROPattern Suite, Euroimmun) is available in China.
Methods
In this study, the performance of this novel system for positive/negative classification, pattern recognition (including homogenous, speckled, nucleolar, nuclear dots, cytoplasmic, and centromeres patterns) and titers evaluation was evaluated by comparing to visual interpretation.
Results
Referring to the total of 3681 collected samples, there was an agreement of 98.7% (κ = 0.973) between the visual and automated examination regarding positive/negative discrimination. In sera with single pattern, correct pattern recognition was observed in 94.6% of the samples. The efficiency of automated recognition for single pattern varied for the different patterns. The automatically determined patterns were correct and complete in 1071 of 1620 cases and correct and meaningful but not complete (“main pattern”) in another 405 cases, enabling main pattern recognition in 91.1% of all cases. Referring to the titers evaluation, the results within the next titer were considered to be consistent. In 1603 positive sera both by visual and automated evaluation, titers of 1514 sample were consistent, accounting for 94.4%.
Conclusion
Attributed to the performance characteristics, EUROPattern system is suitable for clinical use as its high degree of automation and result reliability, and may help clinical laboratories to standardize of IIF evaluation.
Keywords: antinuclear antibodies, automated interpretation, immunofluorescence pattern, indirect immunofluorescence, visual interpretation
1. INTRODUCTION
Antinuclear antibodies (ANA) are immunoglobulin directed against autologous cell nuclear and cytoplasmic components. ANAs are found in patients with a number of autoimmune diseases, such as systemic lupus erythematosus, Sj¨ogren's syndrome, rheumatoid arthritis, polymyositis, scleroderma, and chronic active autoimmune hepatitis.1, 2 ANAs can also be found in patients with chronic infections and cancer.
The fluorescent antinuclear antibody test was designed by George Friou, MD in 1957.3 It is a sensitive screening test used to diagnosis of autoimmune diseases. Since the 1970s, human epithelial (HEp‐2) cells have been increasingly used and were eventually adopted as the universal standard substrate in practically all commercially available ANA assay kits. At present, indirect immunofluorescence (IIF) on HEp‐2 cells is still considered as the preferred method for ANA screening.4, 5 Using HEp‐2 cells as the substrate, the IIF allows detection of more than 50 autoantibodies to 30 different nuclear and cytoplasmic antigens.6 However, standardization of this assay is difficult due to inter‐manufacturer variations in the substrate and the fixation process, characteristics of the secondary antibody used.7 Inter‐laboratory variations in microscopy apparatus, and, especially, the subjective interpretation of the results.8, 9 Although there are already some commercial automation instruments for IIF processing, the automation of interpretation of ANA is at starting stage still.
During the last few years, automated hardware and software‐based pattern recognition platforms have become available.10, 11 Currently, an automated processing and recognition system for standardized and ANA interpretation by HEp‐2 cell‐based IIF (EUROPattern Suite; Euroimmun, Lubeck, Germany) is available in China. The purpose of this study was to evaluate the performance of this novel system for positive/negative classification, pattern recognition and titers evaluation by comparing to Conventional visual interpretation.
2. MATERIALS AND METHODS
2.1. Samples
All serum samples submitted for routine ANA testing were continuously enrolled in this study, including samples from the various clinical departments. There were 1620 antibody‐positive samples and 2061 visually negative samples collected in the final. Specimens were stored at −20°C until testing and then stored at 2‐8°C. The local ethics committee approved the study.
2.2. Reagents and apparatus
All sera prospectively included, were tested for ANA by IIF using HEp‐2 cells and primary liver (Euroimmun). The assay was performed according to the manufacturer's instructions, using a screening serum dilution of 1:100. The slides were evaluated in two ways: (a) automated recognition by the EUROPattern System (Euroimmun) and (b) visual interpretation by two experienced technicians who worked independently without reference to the other's. Sera with an antibody titer equal to or >1:100 were considered as positive. In each batch of tests, a negative and a weak positive control were tested simultaneously with clinical samples.
Immunofluorescence Patterns, such as homogenous, speckled, nucleolar, nuclear dots, cytoplasmic and centromeres patterns, and titers were also reported for positive samples.
2.3. Statistical analysis
Agreement between visual and automated interpretation was assessed using the percentage of concordance and kappa coefficients. Kappa (κ) Values of 0.41‐0.60 indicate moderate agreement, 0.61‐0.80 good and 0.81‐1.00 an lmost perfect agreement.12 Statistical calculation was performed using MedCalc version 6 (Medcalc software, Mariakerke, Belgium).
3. RESULTS
3.1. Distinguish between positive and negative
A total of 3681 patients submitted for routine ANA testing were included. There were 1603 sera classified as ANA positive and 2029 as negative both by visual and automated interpretation. In total, 32 discrepant sera were reported negative by artificial interpretation, but weakly positive by automated interpretation. Of 1620 sera tested positive by visual examination, 17 sera were negative in EUROPattern.
As for the total of 3681 sera, there was an agreement of 98.7% (κ = 0.973) between the artificial and automated interpretation for positive/negative classification. The analytical sensitivity and specificity of automated interpretation were 98.95% and 98.4% respectively. The positive and negative predictive values of EUROPattern were 98.0% and 99.2% respectively.
3.2. Immunofluorescence pattern interpretation
The ability of EUROPattern for recognition of homogenous, speckled, nucleolar, centromere, nuclear dotted, and cytoplasmic patterns were analyzed. In1620 positive sera by visual interpretation, 608 sera were assessed as single immunofluorescence pattern 1012 as mixed patterns.
In sera with single pattern, correct pattern recognition was observed in 94.6% of the sera. The efficiency of automated recognition for single pattern varied for the different patterns: cytoplasmic pattern, nucleolar pattern (100%) > speckled pattern (97.2%) > homogenous pattern (91.6%) > nuclear dots pattern (75%) > centromeres pattern (60.7%; Table 1).
Table 1.
Single pattern recognition by EUROPattern
| ANA pattern by visual interpretation | Automated recognition (n = 608) | |
|---|---|---|
| n | Pattern recognized line feeds: (n, %) | |
| Homogenous | 143 | 131, 91.6 |
| Speckled | 213 | 207, 97.2 |
| Nucleolar | 45 | 45, 100 |
| Nuclear dots | 16 | 12, 75 |
| Centromeres | 28 | 17, 60.7 |
| Cytoplasmic | 146 | 146, 100 |
ANA, antinuclear antibodies.
As for the 1012 sera with mixed patterns, the automatically reported results were exactly correct in 49% of sera, only main pattern correct in 40% of the samples, and only secondary pattern correct in 6% of the samples. In 51 of 1012 (5.0%) sera, the pattern was improperly recognized. The efficiency of automated recognition for multiple patterns was shown in Table 2.
Table 2.
Multiple patterns recognition by EUROPattern
| ANA pattern by visual interpretation | Automated recognition (n = 608) | |
|---|---|---|
| n | Pattern recognized (n, %) | |
| Homogenous | 462 | 424, 91.8 |
| Speckled | 708 | 656, 92.7 |
| Nucleolar | 174 | 153, 87.9 |
| Nuclear dots | 65 | 49, 75.4 |
| Centromeres | 119 | 72, 60.5 |
| Cytoplasmic | 496 | 486, 97.98 |
ANA, antinuclear antibodies.
In EUROPattern, the automatically reported patterns were correct and complete in 1071 of 1620 cases and correct and meaningful but not complete (“main pattern”) in another 405 cases, enabling main pattern recognition in 91.1% of all cases.
3.3. Interpretation of ANA titers
Referring to the titers evaluation, the results within the next titer were considered to be consistent. In 1603 positive sera both by visual and automated evaluation, titers of 1514 samples were consistent, accounting for 94.4%. The main nonconformity was that automatic interpretation would underestimate the strong positive results.
4. DISCUSSION
Human epithelial‐2‐cell‐based IIF is the gold standard for ANA screening. Although there are some automation solutions for IIF incubation, visual evaluation by laboratory technicians is still carried out in most laboratories in China. Visual interpretation is time consuming, subjective and difficult to standardize.13 Automatic operation and interpretation may help reduce subjective errors and advance the ANA standardization process.14
As automated recognition system, EUROPattern can carry out the microscopic process and obtain high‐resolution immunofluorescence images, recognize most of the meaningful patterns and report the corresponding titers. In this study, the results of classical visual reading and automated pattern recognition by EUROPattern were compared in 3681 sera. Overall, automated recognition shew a 98.7% agreement with visual interpretation, and also methodological comparison of consistency tests showed better consistency (κ = 0.973). This κ value was higher than the result between HELIOS IIF processor and visual assessment.15 The analytical sensitivity and specificity of EUROPattern amounted to 98.95% and 98.4% respectively. So, EUROPattern proved to have the ability to distinguish between positive and negative results. Of a total of 3681 sera, 17 of 1620 positive sera and 32 of 2061 negatives were misinterpreted. So, in clinical practice, a laboratory technician should browse images obtained by EUROPattern and validate the final results.
In EUROPattern, the automatically reported patterns were correct in 94.6% of sera with single pattern, and 91.1% of all positive cases. The lowest performance in pattern recognition was found for the anticentromere and antinuclear dot pattern. This finding may be due to the fact that this system was poor for small dot‐like fluorescence recognition. By comparing EUROPattern, AKLIDES, Nova View, HELIOS, Zenit G Sight and Image Navigator for ANA IIF interpretation, the classic nuclear (homogeneous, speckled, and centromeric) and nucleolar patterns are identified in 70%‐85% of the cases, the rarer patterns (multiple nuclear dots, nuclear rim, midbody, PCNA, and nuclear matrix) are found at a significantly lower rate of 25%‐50%.16
It is vital to identify mixed patterns correctly for ANA interpretation. However, discrimination of patterns with two or more autoantibodies is difficult, depending on their targets and titers.17, 18 In this study, the automatically determined patterns by EUROPattern were correct and complete in 49% and only main pattern correct in 40% of all samples with multiple patterns. This rate was fairly good given the complexity.
Referring to the titers evaluation, EUROPattern automated evaluation shew a 94.4% agreement with visual interpretation. The main nonconformity was that automatic interpretation would underestimate the strong positive results. So, it was necessary for laboratory personnel to validate positive results patient by patient in EUROPattern system.
Our study has several limitations. Patterns of ANA by immunofluorescence assay (IIFA) reflect the topographic distribution of target autoantigens and may convey significant information about antibody specificity.19, 20 Only six patterns which are the most commonly recognized in clinical practice was evaluated by EUROPattern automated evaluation and visual interpretation. Although this study proved that EUROPattern automated evaluation had a good agreement with visual interpretation, the associations between positive results by automated evaluation and clinical diagnosis of an autoimmune disease need additional exploration in future studies.
5. CONCLUSION
The results of EUROPattern automatic Immunofluorescence system and conventional visual interpretation are in good consistency. EUROPattern also proved to be very effective in listing negative results and pattern identification. So, the system is suitable for clinical use as its high degree of automation and result reliability, and may help clinical laboratories to improve standardization of IIF evaluation.
Li ZY, Han RL, Yan ZL, Li LJ, Feng ZR. Antinuclear antibodies detection: A comparative study between automated recognition and conventional visual interpretation. J Clin Lab Anal. 2019;33:e22619 10.1002/jcla.22619
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