Laboratory diagnostics is of great importance for the diagnosis of systemic lupus erythematosus (SLE). Besides hematologic parameters like the sedimentation rate, leukopenia, or lymphopenia, autoantibodies against components of the cell nuclei (anti-nuclear antibodies, ANA) are the parameter of interest. Among ANA, antibodies against double-stranded DNA (anti-dsDNA) are one of the most reliable serological markers for SLE. Accordingly, they were listed as an important immunological feature for SLE diagnosis in the revised classification criteria by the American College of Rheumatology in 1982, and in the updated version from 1997 , . In the most recent revision and validation of the criteria by the Systemic Lupus International Collaborating Clinics (SLICC)  anti-dsDNA antibodies were attributed an even more prominent role in SLE diagnosis. Also, anti-dsDNA antibody levels may reflect disease activity and kidney involvement; titer increases can precede a flareup of disease , .
The prevalence of anti-dsDNA antibodies in SLE is reported to range from 30% to 98% , , . One cause of this variability is the use of different test systems for antibody detection, including radio immunoassay (RIA), Crithidia luciliae indirect immunofluorescence tests (CLIFT), enzyme-linked immunosorbent assays (ELISA) and other solid phase immunoassays. Each assay is supposed to bind a different subset of anti-dsDNA antibodies, e.g. of high or low avidity , . The Farr assay, a certain form of RIA, is often considered as the gold standard for anti-dsDNA antibody detection , , . However, as many clinics and laboratories avoid handling radioactive material, CLIFT and ELISA are today’s preferred methods . ELISA is easy-to-use and cost effective, and therefore commonly used as screening assay. However, ELISA are often less disease specific as they also detect anti-dsDNA antibodies of low avidity, which are less relevant for SLE diagnostics, resulting in a higher number of false positive results. This is also reflected in the SLICC classification criteria which prescribe a clinical relevant antibody level threshold of >2 times the reference range if measured by ELISA . Furthermore, ELISA are prone to a high performance variability between test kits and manufacturers, depending on the antigenic substrate used , , . Even though great improvements have been made in recent years , , , confirmation of positive ELISA results with a second, more specific assay, like CLIFT or Farr assay, is advised . The indirect immunofluorescence (IIF) test for anti-dsDNA antibodies is commonly based on C. luciliae as the substrate, a flagellate with a large mitochondrion, called a kinetoplast, containing a complex network of dsDNA, which is almost free of proteins and histones. This increases the assay’s specificity for SLE, as it is selective to high avide anti-dsDNA antibodies, which are most disease relevant , . More sensitive CLIFT assays also detecting antibodies of low avidity can be achieved using modified buffer compositions.
A general drawback of the IIF test lies in the visual and subjective evaluation of test results which leads to high intra- and interlaboratory variability , , . To reduce such variances and to optimize workflow, automated systems have been developed, which assist the operator in the handling of slides and result evaluation , , , , , , , , . The first computer-aided immunofluorescence microscopy (CAIFM) systems were introduced into the field of ANA diagnostics for IIF on HEp-2 cells, which is regarded as the gold standard , . As intended, automation could contribute to standardization of assay evaluation and fluorescence interpretation , , , , . Especially software performed discrimination of positive and negative results greatly matches with visual evaluations by the operator. Several commercial platforms are on the market, among them the EUROPattern Suite encompassing the automated EUROPattern microscope and different software modules , . The system offers fully automated image acquisition for a high number of different IIF substrates and is capable of automated IIF (pattern) classification for ANA (HEp-2 and HEp-20-10), ANCA (ethanol- and formalin-fixed), and CLIFT assays. Results will be suggested to the operator and verified by him.
Only few CAIFM systems are capable of automated CLIFT analyses , , , , . Data on these systems are mainly focused on either their development or their performance characteristic. Only little is published on their performance in routine diagnostics in a clinical laboratory so far. We aimed to test the EUROPattern Suite performance in the routine setting of a clinical laboratory.
Materials and methods
Serum samples were collected at the Central Diagnostic Laboratory of the Maastricht University Medical Centre over a period of 7 months. All samples requested for anti-dsDNA analyses were considered, 332 serum samples in total. Fifteen samples had to be excluded due to insufficient material and five samples were assessed to be not evaluable by the visual reader leaving 312 samples in the final analysis. Collected serum samples included specimens sent to the laboratory for either diagnostic or follow-up purposes. The 312 serum samples were assigned to 281 patients, of which 256 patients provided one sample, 19 patients two samples and six patients three samples. Analysis was performed blinded. Matching with clinical data, being either SLE or non-SLE, was performed afterwards and only in case of positive results. This study was performed in accordance with the 1997 Declaration of Helsinki of the World Medical Association. For the analyses on patient material serum was obtained for diagnostic or follow-up purposes. As rest-serum was used in a semi-anonymous way, ethical approval was not necessary according to the Dutch guidelines.
Anti-dsDNA antibody detection
Three different CLIFT and evaluation procedures were applied for anti-dsDNA detection: (1) Routine analysis of anti-dsDNA antibodies at the Central Diagnostic Laboratory of Maastricht University Medical Centre was performed using the Fluorescent nDNA Test System (ImmunoConcepts, Sacramento, CA, USA) according to the manufacturer’s instructions . Slides were prepared manually and evaluation was performed visually by trained experts utilizing a fluorescent microscope (Axioskop, Carl Zeiss Microscopy GmbH, Jena, Germany) with LED light source. (2) dsDNA EUROPattern test kit, (Euroimmun AG, Lübeck, Germany) and (3) dsDNA (sensitive) EUROPattern test kit (Euroimmun AG) were conducted at the Central Diagnostic Laboratory of Maastricht University Medical Centre according to the manufacturer’s instructions. Each dsDNA EUROPattern slide contains 10 reaction areas, each providing one biochip (2×2 mm fragment of coated cover slip glued into the reaction fields), coated with the protist. Slides were automatically incubated and washed using the IF Sprinter (Euroimmun AG, Lübeck, Germany). For initial screening, samples were applied at a dilution of 1:10. Fluorescein isothiocyanate (FITC)-labeled goat anti-human IgG was used for green fluorescent staining. Antiserum was supplied with Evans Blue used for red fluorescent counterstaining of the cells. Images of each incubated biochip were automatically acquired by the EUROPattern microscope, evaluation of the images was performed either visually by two trained experts or automatically by the EUROPattern software. Samples reactive at screening dilution were further analyzed at dilutions of 1:100 and 1:1000. Anti-dsDNA antibody titers ≥1:10 were considered positive. CLIFT 3 was only performed in case of discrepant results between CLIFT 1 and CLIFT 2.
The EUROPattern Suite (EUROIMMUN AG, Lübeck, Germany) comprises an automated fluorescence microscope and associated software modules for automated image acquisition and assay evaluation (for details see ). A focused image of each biochip is automatically taken by the EUROPattern microscope. A novel software feature induces acquisition of a second image of the same biochip with reduced exposure time in the case of overexposed images (low-light image). Automated titer estimation was performed by the software based on the original image data. During the classification process confidence values are calculated. They are a measure of how certain the system is about the proposed result (for details see ).
For statistical comparison the Mann-Whitney rank sum test was employed using SigmaPlot 13 (Systat Software, San Jose, USA). Values of p<0.05 were considered significant, whereas values of p<0.01 and p<0.001 were defined as very significant and highly significant, respectively.
Appraisal of dsDNA EUROPattern test kit in the routine environment of the MUMC
Of 312 serum samples, 304 had a clear positive or negative result in the routine CLIFT 1 of the Central Diagnostic Laboratory of the Maastricht University Medical Centre. In 290 cases concordant results (35 positive, 255 negative) were obtained with the EUROPattern CLIFT 2 and subsequent visual evaluation by one expert (Table 1). Of the 14 discrepant samples, one was tested negative in the routine laboratory but positive in CLIFT 2 (diagnosis SLE); 13 were positive in routine testing but negative in CLIFT 2 – of which 12 samples belonged to patients who were finally diagnosed with SLE. These 12 samples were further analyzed with the more sensitive EUROPattern CLIFT 3. To avoid bias due to knowledge of former results, analysis was performed blinded with additional positive and negative samples. Reevaluation with CLIFT 3 revealed 11 positive results. With an overall agreement of 95.4% between CLIFT 1 and 2, and 99.0% between CLIFT 1 and 2/3, respectively, substantial equivalence of the assays can be assumed. The next step was now to assess the performance of the automated assay evaluation by EUROPattern.
Automated CLIFT evaluation compared to visual interpretation
Out of 312 samples tested with CLIFT 2, 32 samples were found positive by visual read-out as well as automated EUROPattern classification (Table 2). Thirty-one samples were identified to be derived from patients diagnosed with SLE; one sample with low anti-dsDNA titer originated from a patient with primary anti-phospholipid syndrome. Concordant negative classifications of samples by the expert and EUROPattern were found in 259 cases, while discrepant results were reported for 21 samples. Thus, visual and automated classification showed an overall concordance of 93.3%. Concerning the estimated titers visual reading by one expert and EUROPattern classification agreed in 81.3% of the cases within the range of ±1 dilution step. EUROPattern determined a two steps higher titer for the remaining 18.8% of samples (Figure 1). However, these values vary with inter-reader differences, as agreement in ±1 titer step between two visual reads was also just 81.3%.
In total, EUROPattern gave apparently false positive results in 19 cases and false negative results for two samples with respect to visual interpretation as reference standard (Table 3), yielding a relative sensitivity of 94.1% and a relative specificity of 93.2%.
More detailed analysis of the discrepant samples revealed that the two being evaluated negative by EUROPattern had a low titer of only 1:10 as determined visually (and this was confirmed by a re-evaluation of the images by a second visual reader) (Table 3; upper part). Confirmed SLE diagnosis of the respective patients supported the correct evaluation by the experts in these cases. Titers given by EUROPattern in the remaining 19 discrepant cases ranged from 1:32 up to 1:1000 (Table 3). In three patients, the software-generated positive results were confirmed by positive routine testing and final SLE diagnosis; in one sample, the routine test result as well as the diagnosis were doubtful. In the other 15 samples, routine test results agreed with the negative classifications by the expert, clinical diagnosis excluded SLE, or only revealed cutaneous LE, in 14 of the respective patients.
Fluorescent images concerning these 15 patients showed either a strong, granular background staining of the cell body or a comma-like accentuation of the kinetoplast. Also, certain samples revealed a very bright background staining of the cells which perturbed the EUROPattern classification software at the default setting. In these cases a low light image, as depicted in Figure 2, was automatically acquired. Therefore, overexposure was not responsible for any misclassification, as all additional low light images were easily evaluable and could be classified as any image with default settings.
Looking at the confidence values revealed that the median confidence value of results matching with the visual read-out was considerably higher than the corresponding value for deviating results. Figure 3 shows the fluorescence images for whom positive and negative classification, respectively, revealed the highest confidence values. The distribution of confidence values for concordantly (true) positive and negative as well as discrepant (false positive and false negative) results are given in Figure 4. However, only the difference between the medians of concordantly positive and false positive results was (highly) significant (p<0.001).
Antibodies directed against dsDNA are highly specific diagnostic markers for SLE , , . A well-established assay with high disease specificity is the CLIFT , , , , , . The sensitivity of CLIFT for SLE is reported to be approximately 30%–60% , , ; prevalence may vary with the used test system and the manufacturer. This may explain the differences between EUROPattern CLIFT 2 results and the results yielded by routine testing (CLIFT 1). Depending on, e.g. sample buffer composition, binding capacities for antibodies may vary. The hypothesis is that special buffer formulations allow binding of only the clinically relevant antibodies of high avidity making an assay less sensitive but more specific. However, as the clinical data in our study were only accessed in the case of any positive test result, no sensitivity and specificity data for the used CLIFT assays can be calculated. But further analysis of the 12 samples which showed discrepant results between routine CLIFT 1 and CLIFT 2 with the CLIFT 3 revealed that 11 samples reacted positive with the more sensitive test kit; highlighting the importance to know the specifications of an assay in use and to assess the preferred weighting of sensitivity and specificity .
Reliable automation and standardization are the future for clinical application of indirect immunofluorescence microscopy to avoid subjective interpretation of immunofluorescence results and to optimize workflow , , , . The EUROPattern Suite is an advanced system for automated image acquisition and classification in indirect immunofluorescence diagnostics including CLIFT. Overall concordance, sensitivity and specificity of the system with respect to classical visual evaluation determined in this study are comparable to those reported in the literature for similar systems , .
Comparing the results of EUROPattern’s automated CLIFT evaluation with visual reading of the acquired fluorescence images by trained and experienced lab technicians, a relative sensitivity of 94.1% was found. In total numbers, only two samples were missed by the software which were classified positive with a titer of 1:10 by visual interpretation. These data are in line with an earlier study demonstrating the very high accuracy of the EUROPattern Suite . Discrepant positive classification in 19 cases results in a relative specificity of 93.2%. Those deviating classifications of single samples can either be explained by a bright and granular background staining of the whole Crithidia’s cell body or a comma-like accentuation of the kinetoplast, which is judged negative by visual reading but positive by the software. Intensive fluorescence of the basal bodies within the analyzed cells, earlier described to cause misclassification, does not seem to influence automated classification in this study, showing that this issue was successfully corrected . As the confidence values calculated by the software are significantly higher for samples which are concordantly positive compared to those that are false positive with respect to visually obtained results, further optimization of the classification process might be realized in the near future. Still, automated classification by the EUROPattern Suite is already very accurate and reliable. Of all negative classifications, 99.2% were correct compared to visual evaluation. Additionally, it is important to stress that in routine operation of the EUROPattern Suite, software results have to be validated by the operator before being included in the final result report. Negative samples can be verified batch-wise, but it is easily possible to further evaluate single samples by removing the check mark for negative. Samples will then be displayed along with the positive sera and verification is performed one by one. All images generated during sample analysis will be integrated into the patient’s data history, making them readily accessible in any case that might demand reevaluation of the patient’s diagnosis.
Additionally to positive-negative discrimination, titer estimation is an essential part of serological diagnostics to evaluate antibody levels in patient samples. Although automated titer calculation cannot be taken for granted in all CAIFM systems for CLIFT analysis , , , , this is an integral feature of the EUROPattern classification software module . Automated titer estimation worked very well compared to visual evaluations with an agreement of 81.3% (expert 1) and 100.0% (expert 2) within the range of ±1 titer step, respectively.
Modern health care demands both, cost effectiveness and high quality results from laboratory diagnostics. Therefore, current methodological improvements in clinical laboratories focus on the standardization of diagnostics and workflow optimization. We show that computer-aided immunofluorescence microscopy with an advanced system like the EUROPattern Suite is able to perform reliable assay analysis and can be of great help in routine laboratories. Automated image acquisition is readily performed and automated image classification gives a reliable result proposal to the human operator. Adjustment of results is only seldom necessary, comprising almost exclusively false positive results, which are verified by the operator one after another, and thus can be easily revised.
We thank Tom Avery, Kathleen Mallet, Veerle Mengelers, and Angelina Misiou for their participation in the analyses of the IIF assays and Johanna Fraune for her assistance in the final figure design and for careful and critical reading of the manuscript.
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About the article
Published Online: 2017-06-23
Published in Print: 2017-11-27
Author contributions: All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission.
Research funding: None declared.
Employment or leadership: W.H., M.H., S.G., K.A., N.H., and K.F. are employees of EUROIMMUN medizinische Labordiagnostika AG, Lübeck, Germany.
Honorarium: None declared.
Competing interests: The funding organization(s) played no role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the report for publication.