AESKUSLIDES nDNA (Crithidia luciliae), AESKUSLIDES nDNA (Crithidia luciliae) Demo Kit, AESKUSLIDES nDNA (Crithidia luciliae) Bulk kit x5, AESKUSLIDES nDNA (Crithidia luciliae) Bulk kit x10

{0} 1 # 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION SUMMARY A. 510(k) Number: K172348 B. Purpose for Submission: New Device C. Measurand: Anti-DNA Antibodies, IgG D. Type of Test: Indirect Immunofluorescence (IFA) E. Applicant: Aesku.Diagnostics GmbH & Co. KG F. Proprietary and Established Names: AESKUSLIDES nDNA (Crithidia luciliae) G. Regulatory Information: 1. Regulation section: 21 CFR 866.5100 – Antinuclear antibody immunological test system 2. Classification: Class II 3. Product codes: LSW, Anti-DNA Antibody, Antigen and Control PIV, automated indirect immunofluorescence microscope and software-assisted system for clinical use {1} 4. Panel: Immunology (82) H. Intended Use: 1. Intended use: AESKUSLIDES nDNA (Crithidia luciliae) is an indirect immunofluorescence assay utilizing Crithidia luciliae coated slides as a substrate for the qualitative and/or semi-quantitative determination of antibodies to native double stranded DNA (dsDNA) in human serum. This in vitro diagnostic assay is used as an aid for the diagnosis of Systemic Lupus Erythematosus (SLE) in conjunction with other clinical and laboratory findings. The assay can be processed manually and analyzed at the microscope or processed and analyzed with HELIOS AUTOMATED IFA SYSTEM. All suggested results obtained with the HELIOS AUTOMATED IFA SYSTEM must be confirmed by trained personnel. 2. Indication for use: Same as the intended use. 3. Special conditions for use statements: - For prescription use only - The HELIOS AUTOMATED IFA SYSTEM is only for use with reagents that are indicated for use with that system - This device is for use by a trained operator in a clinical laboratory setting - All software-aided results must be confirmed by the trained operator - For use only by manual microscopy or with HELIOS AUTOMATED IFA SYSTEM 4. Special instrument requirements: Not applicable I. Device Description: AESKUSLIDES nDNA (Crithidia luciliae) is an indirect immunofluorescence assay utilizing Crithidia luciliae coated slides as a substrate for the qualitative and/or semi-quantitative determination of antibodies to native double stranded DNA (dsDNA) in human serum. Each kit contains (Quantity depends on product variant): - Slides, each containing 10 wells coated with Crithidia Luciliae cells - 4.0 ml vial containing Fluorescein (FITC) labelled Anti-human Antibody IgG conjugate in a solution of BSA, ready for use 2 {2} - 0.5 ml vial of positive control containing human serum (diluted), ready for use - 0.5 ml vial of negative control containing diluted human serum, ready for use - 8.0 ml vial of mounting medium containing a solution of glycerol and PBS, ready for use - 70 ml bottle of sample buffer, containing BSA, PBS and ready for use - 100 ml bottle of wash buffer, concentrated buffer 1:10 in distilled water, containing BSA, PBS J. Substantial Equivalence Information: 1. Predicate device name: NOVA Lite dsDNA Crithidia luciliae 2. Predicate 510(k) number: K880742 3. Comparison with predicate: | Similarities | | | | --- | --- | --- | | Item | Device | Predicate | | Intended Use | AESKUSLIDES nDNA (Crithidia luciliae) is an indirect immunofluorescence assay utilizing Crithidia luciliae coated slides as a substrate for the qualitative and/or semi-quantitative determination of antibodies to native double stranded DNA (dsDNA) in human serum. This in vitro diagnostic assay is used as an aid for the diagnosis of Systemic Lupus Erythematosus (SLE) in conjunction with other clinical and laboratory findings. The assay can be processed manually and analyzed at the microscope or processed and analyzed with HELIOS® AUTOMATED IFA SYSTEM. All suggested results obtained with the HELIOS® AUTOMATED IFA SYSTEM must be confirmed by trained personnel. | NOVA Lite dsDNA Crithidia luciliae is an indirect immunofluorescence assay for the screening and semi-quantitative determination of anti-double stranded DNA (dsDNA) in human serum. The presence of anti-double stranded DNA can be used in conjunction with other serological tests and clinical findings to aid in the diagnosis of systemic lupus erythematosus (SLE) | {3} | Similarities | | | | --- | --- | --- | | Item | Device | Predicate | | Methodology | Indirect Immunofluorescence assay (IFA) | Same | | Results | Qualitative and semi-quantitative titer | Same | | Sample Matrix | Serum | Same | | Fluorescence Marker | FITC | Same | | Controls | One positive control, one negative control | Same | | Screening dilution | 1:10 | Same | | Shelf life | 24 months (2–8°C) | Same | | Differences | | | | --- | --- | --- | | Item | Device | Predicate | | Manual Interpretation of result | Manual fluorescence microscopy or with HELIOS w/ trained operator verification | Manual fluorescence microscopy | | Automated interpretation of results | HELIOS w/ trained operator verification | Not Applicable | | Slides | 12 wells coated with antigen | 10 wells coated with antigen | # K. Standard/Guidance Document Referenced: CLSI EP07-A2: Interference Testing in Clinical Chemistry CLSI EP17-A2: Evaluation of Detection Capability for Clinical Laboratory Measurement Procedures CLSI EP25-A: Evaluation of Stability of In Vitro Diagnostic Reagents CLSI EP28-A3c: Defining, Establishing and Verifying Reference Intervals in the Clinical Laboratory CLSI EP6-A: Evaluation of the Linearity of Quantitative Measurement Procedures A Statistical Approach GP44-A4: Procedures for the Handling and Processing of Blood Specimens for Common Laboratory Tests; Approved Guideline-Fourth Edition Guidance for Industry and FDA Staff: Recommendations for Anti-Nuclear Antibody (ANA) Test System Premarket (510k) Submissions Guidance for Industry and Staff: Factors to Consider Regarding Benefit-Risk in Medical {4} Device Product Availability, Compliance, and Enforcement Decisions Guidance for Industry and Food and Drug Administration Staff: Applying Human Factors and Usability Engineering to Medical Devices # L. Test Principle: The AESKUSLIDES nDNA assay utilizes indirect immunofluorescent antibody assay techniques. Patient sera are diluted in wash/sample buffer at a recommend screening dilution of 1:10 and applied to a well on the slide. nDNA antibodies, if present, will bind to antigens coated on the slide. After washing with wash/sample buffer, a conjugate specific for human IgG is applied which binds to the nDNA antibodies immobilized on the slide surface. After a final wash to remove excess conjugate, the slide is mounted and read as soon as possible using a fluorescence microscope for manual microscopy or HELIOS AUTOMATED IFA SYSTEM. The HELIOS AUTOMATED IFA SYSTEM will dilute samples, process slides, scans the slides, generates the image, and provides a positive/negative pre-classification suggestion that must be confirmed by trained personnel. # Interpretation of results # Manual interpretation of tests results AESKU recommends a screening dilution of 1:10, followed by serial dilution for semiquantitative determinations and suggests each laboratory establish its own screening dilution and titration scheme based on its population and instrumentation. The fluorescence intensity level is the intensity of the specific fluorescence expressed as a numeric value. These values, if present, are reported as a number between "0" (no specific fluorescence) and "4+" (very strong visible reaction). # Qualitative evaluation A serum dilution is considered negative for nDNA antibodies if the cells exhibit $< 1+$ fluorescence of the kinetoplast. A sample is considered positive for nDNA antibodies if it exhibits exhibit $\geq 1+$ fluorescence of the kinetoplast at a sample dilution of 1:10 or greater. Operators should report all titers and specific fluorescence staining seen. # Semi-quantitative evaluation The endpoint titer is defined as the highest sample dilution factor for which a specific pattern is identifiable. The titers are classified as in the table below: | Dilution | Titer | | --- | --- | | 1:10 | Low | | 1:20 and 1:40 | Medium | | 1:80 and greater | High | {5} Automated instrument interpretation of results The HELIOS DEVICE SOFTWARE examines the fluorescence intensity and uses an analysis algorithm which takes exposure and pixel frequency into account. It examines relevant regions of the captured images, such as the fluorescence cell area and subtracts the background, to provide an assessment of positive or negative results. Trained operators must confirm all assessments made by the HELIOS DEVICE SOFTWARE. Intensity and End Point Titers are identified by the HELIOS DEVICE SOFTWARE and pattern assessmentst are made by the HELIOS PATTERN RECOGNITION tool. | Intensity | Interpretation | | --- | --- | | + | nDNA positive | | - | nDNA negative | AESKU recommends a screening dilution of 1:10, followed by serial dilution for semiquantitative determinations and suggests each laboratory establish its own screening dilution and titration scheme based on its population. # M. Performance Characteristics: Nomenclature and acronyms used in the studies: The HELIOS Microscope and Software system includes a slide processing module (the HELMED). The system allows for automated imaging or manual imaging by the microscope. All studies were evaluated by comparing the four possible reading methods (Method A, B, C and D) as shown in the table below; these modes are consistent throughout this document. All results generated by the HELIOS software must be confirmed by a trained operator. Modes of operations that were evaluated in the study: | Method | Sample Processing | Imaging | Reading/Evaluation of slide | Alternate name of Method | | --- | --- | --- | --- | --- | | A | Automated | Automated | Automated (software interpretation) | HELIOS | | B | Automated | Automated | Manual (read of digital image) | HELIOS User Evaluation | | C | Manual | Manual | Manual (read of microscope field) | Manual AESKUSLIDES nDNA | | D | Manual | Manual | Manual (read of microscope field) | Manual NOVA Lite dsDNA (Predicate) | {6} Fluorescence Interpretation for Manual Processing, Imaging, and Reading of slide (Method C): | Intensity | Interpretation | | | --- | --- | --- | | 4+ | high positive | maximal fluorescence, very strong visible reaction; brilliant yellow-green | | 3+ | positive | strong visible reaction; less brilliant as 4+; yellow-green fluorescence | | 2+ | positive | moderate visible reaction; definite but dull yellow-green fluorescence | | 1+ | positive | weak visible reaction, very dim subdued fluorescence | | 0 | negative | no specific fluorescence | Names of diseases present in samples and their abbreviations: | Disease name | Abbreviations | | --- | --- | | Systemic lupus erythematosus | SLE | | Antiphospholipid Syndrome | APS | | Mixed Connective Tissue Disease | MCTD | | Undifferentiated connective tissue disease | UCTD | | Systemic sclerosis | SSc | | Polymyositis | PM | | Dermatomyositis | DM | | Rheumatoid Arthritis | RA | | Autoimmune Hepatitis | AIH | | Primary biliary cholangitis | PBC | | Primary sclerosing cholangitis | PSC | | ANCA-associated vasculitides | AAV | | Hepatitis C Virus | HCV | | Hepatitis B Virus | HBV | | Epstein–Barr virus | EBV | 1. Analytical performance: All results met the Manufacturer’s pre-determined acceptance criteria. a. Precision/Reproducibility: **Repeatibility** Eleven samples were assayed on five days, two runs per day, and three replicates per sample per run, resulting in 30 data points for each sample for Method A. The same samples were assayed using the same protocol for Method B and C and the results were analyzed by two independent readers, for a total of 60 replicates per sample. All runs have been performed according to the respective instructions for use. One positive and one negative control (kit controls) were included in each run. Samples were tested at 1:10 dilution. The samples and results are summarized in the following table. {7} Samples used for repeatability: | Sample ID | AESKUSLIDES nDNA | | | --- | --- | --- | | | Result | Grading | | 1 | Pos | borderline | | 2 | Pos | low positive | | 3 | Pos | low positive | | 4 | Pos | medium positive | | 5 | Pos | medium positive | | 6 | Pos | medium positive | | 7 | Pos | high positive | | 8 | Pos | high positive | | 9 | Pos | high positive | | 10 | Neg | negative | | 11 | Neg | negative | Results for repeatability: | Sample ID | N=30 | | N=60 | | | | | --- | --- | --- | --- | --- | --- | --- | | | Method A | | Method B | | Method C | | | | % Negative | % Positive | % Negative | % Positive | % Negative | % Positive | | 1 | 100 | 0 | 60 | 40 | 15 | 85 | | 2 | 33.3 | 66.7 | 0 | 100 | 0 | 100 | | 3 | 56.7 | 43.3 | 5 | 95 | 0 | 100 | | 4 | 46.7 | 53.3 | 8.3 | 91.7 | 0 | 100 | | 5 | 0 | 100 | 0 | 100 | 0 | 100 | | 6 | 6.7 | 93.3 | 0 | 100 | 0 | 100 | | 7 | 0 | 100 | 0 | 100 | 0 | 100 | | 8 | 0 | 100 | 0 | 100 | 0 | 100 | | 9 | 3.3 | 96.7 | 0 | 100 | 0 | 100 | | 10 | 83.3 | 16.7 | 100 | 0 | 98.3 | 1.7 | | 11 | 70 | 30 | 100 | 0 | 100 | 0 | ## Reproducibility ## Between-Lab Eleven samples were assayed over five days, two runs per day, three replicates per sample per run, at three different study sites. Two study sites were in the U.S., one study site was in Germany, with one HELIOS instrument at each study site. Results were analyzed by two different readers per study site, resulting in a total of 90 data {8} points per sample per reader. Positive/negative classification was recorded for each sample in each run and for each Method. Positive agreement was calculated as, across all positive samples, the number of correctly found samples divided by the number of total positive samples. Negative agreement was calculated as, across all negative samples, the number of correctly found samples divided by the number of total negative samples. Overall agreement was calculated as, across both positive and negative samples, the number of correctly found samples divided by the number of total samples. Fluorescence intensity (FI) was recorded for samples prepared and processed using Method C. Fluorescence intensity agreement was calculated as, across all samples processed by Method C, the number of correctly found samples divided by the number of total samples. The results are summarized in the following tables: Method A | Agreement (95% CI) | Site 1 vs Site 2 | Site 2 vs Site 3 | Site 1 vs Site 3 | All Sites | | --- | --- | --- | --- | --- | | Positive Agreement | 63.1 (59.0–67.1) | 52.2 (48.0–56.4) | 59.4 (55.3–63.5) | 58.3 (54.8–61.6) | | Negative Agreement | 87.5 (80.4–92.3) | 95.0 (89.5–97.7) | 87.5 (80.4–92.3) | 90.0 (84.7–93.6) | | Overall Agreement | 67.6 (63.9–71.0) | 60.0 (56.2 – 63.7) | 64.5 (60.8 – 68.1) | 64.0 (61.0–67.0) | | FI Agreement | N/A | N/A | N/A | N/A | Method B | Agreement (95% CI) | Site 1 vs Site 2 | Site 2 vs Site 3 | Site 1 vs Site 3 | All Sites | | --- | --- | --- | --- | --- | | Positive Agreement | 92.1 (90.4–93.6) | 84.4 (82.2–86.5) | 84.2 (81.9–86.2) | 86.9 (85.2–88.85) | | Negative Agreement | 99.2 (97.0–99.8) | 99.2 (97.0–99.8) | 100 (98.4–100) | 99.4 (98.0–99.8) | | Overall Agreement | 93.4 (91.9–94.6) | 87.1 (85.2–88.8) | 87 (85.1–88.8) | 89.2 (87.7–90.5) | | FI Agreement | N/A | N/A | N/A | N/A | {9} 10 Method C | Agreement (95% CI) | Site 1 vs Site 2 | Site 2 vs Site3 | Site 1 vs Site 3 | All Sites | | --- | --- | --- | --- | --- | | Positive Agreement | 99.2 (98.4–99.6) | 99.0 (98.2–99.4) | 98.1 (97.2–98.8) | 98.8 (98.1–99.2) | | Negative Agreement | 99.6 (97.7–99.9) | 100 (98.4–100) | 99.6 (97.7–99.9) | 99.7 (98.4–100) | | Overall Agreement | 99.2 (98.6–99.6) | 99.2 (98.5–99.5) | 98.4 (97.6–99.0) | 98.9 (98.4–99.3) | | FI Agreement | 98.7 (97.9–99.2) | 97.7 (96.7–98.3) | 97.4 (96.4–98.2) | 97.9 (97.2–98.5) | Between-Operator Agreement within a site was calculated by determining the total number of samples identified correctly, either positive or negative, divided by the expected number of samples that were pre-determined to be classified as positive or negative. Method B | Agreement (95% CI) | Site 1 | Site 2 | Site 3 | | --- | --- | --- | --- | | Positive Agreement | 91.9 (89.2–93.9) | 92.4 (89.9–94.4) | 76.5 (72.7–79.9) | | Negative Agreement | 100 (96.9–100) | 98.3 (94.1–99.5) | 100 (96.9–100) | | Overall Agreement | 93.3 (91.2–95.0) | 93.5 (91.3–95.1) | 80.8 (77.6–83.6) | | FI Agreement | N/A | N/A | N/A | Method C | Agreement (95% CI) | Site 1 | Site 2 | Site 3 | | --- | --- | --- | --- | | Positive Agreement | 98.3 (96.9–99.1) | 100 (99.3–100) | 98 (96.4–98.9) | | Negative Agreement | 99.2 (95.4–99.9) | 100 (96.9 – 100) | 100 (96.9–100) | | Overall Agreement | 98.5 (97.2–99.2) | 100 (99.4–100) | 98.3 (97–99.1) | | FI Agreement | 98.5 (97.2–99.2) | 98.9 (97.8–99.5) | 96.4 (94.6–97.5) | {10} # Single Operator Assay Methods B and C were performed by two trained readers at three different study sites. The assay was performed according to the instructions for use. Agreement for each method was calculated as the number of samples classified correctly divided by the expected number of positive or negative samples, which were determined prior to the study. Method B | Agreement (95% CI) | Site 1 | | Site 2 | | Site 3 | | | --- | --- | --- | --- | --- | --- | --- | | | Reader 1 | Reader 2 | Reader 3 | Reader 4 | Reader 5 | Reader 6 | | Positive Agreement | 87.4 (82.9–90.8) | 96.3 (93.3–98.0) | 95.2 (91.9–97.2) | 89.6 (85.4–92.7) | 77.4 (72.1–82.0) | 75.6 (70.1–80.3) | | Negative Agreement | 100 (94.0–100) | 100 (94.0–100) | 100 (94.0–100) | 96.7 (88.6–99.1) | 100 (94.0–100) | 100 (94.0–100) | | Overall Agreement | 89.7 (85.9–92.5) | 97.0 (94.5–98.3) | 96.1 (93.4–97.7) | 90.9 (87.3–93.6) | 81.5 (77.0–85.3) | 80.0 (75.4–84.0) | | FI Agreement | N/A | N/A | N/A | N/A | N/A | N/A | Method C | Agreement (95% CI) | Site 1 | | Site 2 | | Site 3 | | | --- | --- | --- | --- | --- | --- | --- | | | Reader 1 | Reader 2 | Reader 3 | Reader 4 | Reader 5 | Reader 6 | | Positive Agreement | 98.5 (96.3–99.4) | 98.1 (95.7–99.2) | 100 (98.6–100) | 100 (98.6–100) | 98.5 (96.3–99.4) | 97.4 (94.7–98.7) | | Negative Agreement | 98.3 (91.1–99.7) | 100 (94.0–100) | 100 (94.0–100) | 100 (94.0–100) | 100 (94.0–100) | 100 (94.0–100) | | Overall Agreement | 98.5 (96.5–99.4) | 98.5 (96.5–99.4) | 100 (98.8–100) | 100 (98.8–100) | 98.8 (96.9–99.5) | 97.9 (95.7–99) | | FI Agreement | 99.7 (98.3–99.9) | 97.3 (94.9–98.6) | 99.7 (98.3–99.9) | 98.2 (96.1–99.2) | 97.6 (95.3–98.8) | 95.2 (92.3–97.0) | # Between-Instrument Imprecision due to instrument for Method A was evaluated at three different study sites. The assay was performed according to the instructions for use. Agreement was calculated as the number of positive or negative samples found correctly by the automated reader, i.e., HELIOS, divided by the expected number of positive or negative samples. {11} 12 Method A | % Agreement (95% CI) | Site 1 | Site 2 | Site 3 | | --- | --- | --- | --- | | Postive Agreement | 70.4 (64.7–75.5) | 55.9 (50–61.7) | 48.5 (42.6–54.5) | | Negative Agreement | 80.0 (68.2–88.2) | 95.0 (86.3–98.3) | 95.0 (86.3–98.3) | | Overall Agreement | 72.1 (67.0–76.7) | 63.0 (57.7–68.1) | 57.0 (51.6–62.2) | | FI Agreement | N/A | N/A | N/A | Between-Lot Imprecision due to reagent lot was performed using three reagent lots each for AESKUSLIDES nDNA. Eleven serum samples were assayed 10 times on each reagent lot to give a total of 30 replicates per serum sample. Slides were processed manually according to the instructions for use and subsequently analyzed at the microscope by two independent readers. The results demonstrated one hundred percent agreement (i.e., positive, negative, overall, and FI agreement) between for all three reagent lots tested. Endpoint titer determination Within-Lab Precision The imprecision associated with reporting endpoint titer was performed at three different sites. Five serum samples with endpoint titers between 1:10 and 1:320 (10, 20,40, 160, 320) were diluted serially, ranging from 1:10 up to 1:1280. For each sample, a minimum of four different dilutions around the expected endpoint titer were made. Serum dilutions were assayed on AESKUSLIDES nDNA and analyzed on HELIOS (Method B) and manually at the microscope (Method C) by two independent readers. For each of the two methods, each serum dilution was assayed on five days, with two runs per day and three replicates per run, resulting in 30 repetitions per serum dilution per reader. For each replicate of the dilution series the endpoint titer was reported as the reciprocal of the highest dilution at which the sample was classified as positive. The percentage of samples that differ a maximum +/-1 titer level from each other and percentage of samples that are > +/-1 titer level away from each other were calculated. The following table represents the samples used in the study: The results of within-lab precision for Method B and Method C are summarized in the following tables: {12} Method B | AESKUSLIDES nDNA | | N | Percentage of samples within +/- 1 titer level | Percentage of samples > +/- 1 titer level | | --- | --- | --- | --- | --- | | Site 1 | Reader 1 | 150 | 92.0 | 8.0 | | | Reader 2 | 150 | 98.0 | 2.0 | | Site 2 | Reader 1 | 150 | 91.0 | 9.0 | | | Reader 2 | 150 | 91.0 | 9.0 | | Site 3 | Reader 1 | 150 | 95.0 | 5.0 | | | Reader 2 | 150 | 90.0 | 10.0 | | All readers combined | | 900 | 93.0 | 7.0 | Method C | AESKUSLIDES nDNA | | N | Percentage of samples within +/- 1 titer level | Percentage of samples > +/- 1 titer level | | --- | --- | --- | --- | --- | | Site 1 | Reader 1 | 150 | 99.0 | 1.0 | | | Reader 2 | 150 | 94.0 | 6.0 | | Site 2 | Reader 1 | 150 | 93.0 | 7.0 | | | Reader 2 | 150 | 93.0 | 7.0 | | Site 3 | Reader 1 | 150 | 93.0 | 7.0 | | | Reader 2 | 150 | 92.0 | 8.0 | | All readers combined | | 900 | 94.0 | 8.0 | Between-Site Imprecision The imprecision associated with endpoint titer determination between sites was evaluated by calculating agreement between titers determined at three sites (site 1 vs site 2, site 2 vs site 3, site 1 vs site 3) for each Methods B and C. | % Titer Agreement (95% CI) | Site 1 vs. Site 2 | Site 2 vs. Site 3 | Site 2 vs. Site 3 | | --- | --- | --- | --- | | Method B | 88.7 (84.6–91.8) | 80.3 (75.5–84.4) | 85.7 (81.2–89.2) | | Method C | 94.3 (91.1–96.4) | 83.0 (78.3–86.8) | 86.7 (82.4–90.1) | b. Linearity/assay reportable range: Five serum samples were assayed in duplicates and analyzed on HELIOS (Method B, Reader Confirmation) and manually at the microscope (Method C) by two independent readers. The reciprocal of the highest dilution in which both replicates were reported as positive was defined as the endpoint titer of the sample. {13} | Sample ID | AESUSLIDES nDNA | | | | | | | --- | --- | --- | --- | --- | --- | --- | | | Pos/Neg | Expected Endpoint Titer | Method B | | Method C | | | | | | Reader 1 | Reader 2 | Reader 1 | Reader 2 | | 1 | Pos | 1280 | 1280 | 1280 | 1280 | 1280 | | 2 | Pos | 640 | 640 | 1280 | 1280 | 1280 | | 3 | Pos | 40 | 80 | 80 | 40 | 40 | | 4 | Pos | 320 | 160 | 160 | 320 | 320 | | 5 | Pos | 80 | 80 | 80 | 40 | 80 | c. Traceability, Stability, Expected values (controls, calibrators, or methods): Traceability: The Positive Control is manufactured by diluting human serum that contains high titter of anti-dsDNA antibodies with protein-based diluent solution, containing preservative. The positive control is a serum from a certified supplier. Stability: Shelf-life Stability: Real-time shelf-life stability testing is ongoing. At the time of submission, stability of three different reagent lots of AESKUSLIDES nDNA stored at $2 - 8^{\circ}\mathrm{C}$ was demonstrated to be three months. Accelerated Stability: Three lots of complete kits (including slides, controls, conjugate, & sample buffer) of AESKUSLIDES nDNA were stored at $37^{\circ}\mathrm{C}$ for 6 weeks. At $t = 0$ , 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, and 6 weeks, a kit was manually tested on a set of eight samples that included negative, low, medium, and high titers. Slides were processed manually according to the instructions for use and subsequently analyzed at the microscope by two independent readers. The data supported a shelf life of 24 months at $2 - 8^{\circ}\mathrm{C}$ . Sample Freeze-Thaw Stability: Serum stability following multiple freeze-thaw intervals was demonstrated using eight serum samples (seven positive, one negative). Low positive, medium positive, and high positive samples were tested. For each serum, one aliquot was prepared to serve as control (no freeze-thawing). A second aliquot for each serum was prepared that underwent four freeze-thaw cycles. All samples and controls were analyzed on AESKUSLIDES nDNA according to the instructions for use. Slides were read by two independent readers. Each freeze/thaw sample was compared to the respective control {14} sample. Freezing and thawing up to four times did not affect serum stability. Long-Term stability Seven serum samples (four positive and three negative) were aliquoted and stored at $-20^{\circ}\mathrm{C}$ . Frozen aliquots of the different sera were thawed and assayed on AESKUSLIDES nDNA at 4, 8, 10, 12, and 14 months. All studies were performed using Method C according to the device's instruction for use. Stability was maintained over the entire 14 months for all samples tested. d. Detection limit: Not applicable e. Analytical specificity: Interference The effect of interfering substances was evaluated by spiking eight different substances, listed in the table below, at two different concentrations into eight different serum samples. Controls were prepared for each serum sample by spiking in only the respective amount of diluent without interfering substances. Spiked serum samples and controls were tested in triplicates for each concentration of interferent with AESKUSLIDES nDNA. All tests were performed manually according to the instruction for use. Results were analyzed by two independent readers at the microscope. No significant interference was observed for samples containing each interference up to the following concentration: | Interferring Substance | Highest Concentration Tested | | --- | --- | | Bilirubin conjugated | 0.4 mg/ml | | Bilirubin unconjugated | 0.4 mg/ml | | Hemoglobin | 5.0 mg/mL | | Triglycerides | 20 mg/mL | | RF IgM | 400 U/mL | | Rituximab | up to 2.0 mg/mL | | Methylprednisolone | 0.8 mg/mL | | Cyclophosphamide | 4.0 mg/mL | | Methotrexate | 0.1 mg/mL | | Azathioprine | 0.03 mg/mL | | Belimumab | 8 mg/ml | | Hydroxychloroquine | 0.024 mg/mL | | Mycophenolat | 0.048 mg/mL | | Ibuprofen | 2 mg/mL | | Naproxen | 0.2 mg/mL | {15} 16 Carry-over A study was performed to demonstrate that the HELIOS instrument does not carry over a positive sample to a negative well. Three high positive samples were run on HELIOS in alternate with a negative serum sample. Tests were performed in accordance with the respective instructions for use. Results were confirmed by a reader. No carry over was observed from well to well. f. Assay cut-off: The manufacturer recommends a screening dilution of 1:10 followed by serial dilutions for semi-quantitative determinations. Each laboratory should establish its own screening dilution and titration scheme based on its patient population. 2. Comparison studies: a. Method comparison with predicate device: Method comparison with the predicate device was performed using the same 776 serum samples (comprising 297 serum samples from patients with SLE and 479 samples from patients with other diseases) tested in the clinical study (see table below). Method comparison was performed with AESKUSLIDES nDNA and the predicate assay (K880742). For each assay, the sample set was processed manually and analyzed (positive/negative classification) at the microscope (Methods C and D). All assays were performed according to the respective instructions for use. Samples were screened at a dilution of 1:10. {16} Samples used for method comparison and clinical sensitivity and specificity: | Diagnosis | | | N | | --- | --- | --- | --- | | Target Disease | | Systemic Lupus Erythematosus | 297 | | Control Diseases | Other Rhuematic Diseases | Antiphospholipid Syndrome | 12 | | | | Sjoegren's Syndrome | 30 | | | | Systemic Sclerosis | 44 | | | | Dermatomyositis/Polymyositis | 31 | | | | Rheumatoid Arthritis | 58 | | | | Mixed Connective Tissue Diseases | 24 | | | | Undifferentiated Connective Tissue Diseases | 21 | | | Autoimmune Liver Diseases | Autoimmune Hepatitis | 21 | | | | Primary Biliary Cirrhosis | 20 | | | | Primary Sclerosing Cholangitis | 10 | | | Vasculitis | ANCA associated Vasculitis | 44 | | | Infectious Diseases | Epstein-Barr Virus Infection | 25 | | | | Hepatitis B Virus Infection | 34 | | | | Hepatitis C Virus Infection | 44 | | | Leukemia | Lymphoma | 31 | | | Other | Fibromyalgia | 30 | | Total | | | 776 | From the results of both assays, the positive, negative and overall agreements were calculated and are presented in the tables below: | Method Comparison | Predicate | | | | | --- | --- | --- | --- | --- | | | | Pos | Neg | Total | | AESKUSLIDES nDNA | Pos | 80 | 13 | 93 | | | Neg | 98 | 585 | 683 | | | Total | 178 | 598 | 776 | | Predicate vs. AESKUSLIDES nDNA | % Agreement (95% CI) | | --- | --- | | Positive Agreement | 44.9 (37.8–52.3) | | Negative Agreement | 97.8 (96.3–98.7) | | Overall Agreement | 85.7 (83.1–88.0) | {17} Comparison of method A, B, and C: Comparison of methods was performed using the same 776 serum samples at 3 different study sites. All assays were performed according to the instructions for uses. Method comparison at each site. | % Agreement (95% CI) | | Method C vs B | Method B vs. A | Method C vs A | | --- | --- | --- | --- | --- | | Site 1 | Positive Agreement | 85.3 (79.0–89.9) | 51.2 (43.7–58.7) | 49.7 (42.1–57.3) | | | Negative Agreement | 98.1 (97.2–98.7) | 84.2 (82.2–86.0) | 83.9 (81.9–85.8) | | | Total Agreement | 96.7 (95.7–97.5) | 80.7 (78.6–82.6) | 80.3 (78.3–82.2) | | Site 2 | Positive Agreement | 91.0 (85.4–94.5) | 55.4 (47.8–62.8) | 54.2 (46.3–61.8) | | | Negative Agreement | 98.2 (97.4–98.8) | 86.3 (84.4–88.0) | 85.8 (83.9–87.6) | | | Total Agreement | 97.5 (96.6–98.2) | 83.0 (81.0–84.8) | 82.7 (80.7–84.5) | | Site 3 | Positive Agreement | 86.2 (79.7–90.9) | 47.7 (39.8–55.6) | 44.8 (37.0–53.0) | | | Negative Agreement | 98.3 (97.5–98.9) | 90.5 (88.9–91.9) | 90.1 (88.5–91.6) | | | Total Agreement | 97.2 (96.2–97.9) | 86.4 (84.6–88.0) | 85.9 (84.1–87.5) | b. Matrix comparison: Not applicable 3. Clinical studies: a. Clinical Sensitivity and Specificity: A clinical study using 776 serum samples was performed with AESKUSLIDES nDNA (Method C) and the predicate assay (Method D), comparing the result of each device to the clinical diagnosis. The samples were the same as those used in the method comparison (see table above). The study was performed at two U.S. sites and one German site. The results are presented in the tables below: {18} 19 Method C | | Diagnosis | | | | | --- | --- | --- | --- | --- | | | | Pos | Neg | Total | | AESKUSLIDES nDNA | Pos | 73 | 25 | 98 | | | Neg | 224 | 454 | 678 | | | Total | 297 | 479 | 776 | Method D | | Diagnosis | | | | | --- | --- | --- | --- | --- | | | | Pos | Neg | Total | | Predicate Assay | Pos | 110 | 68 | 178 | | | Neg | 187 | 411 | 598 | | | Total | 297 | 479 | 776 | | | % Sensitivity (95% CI) | % Specificity (95% CI) | PPV | NPV | | --- | --- | --- | --- | --- | | | SLE (n=297) | Other Diseases (n=479) | | | | AESKUSLIDES nDNA | 24.6 (20.0–29.8) | 94.8 (92.4–96.4) | 74.5 | 67.0 | | Predicate Assay | 37.0 (31.7–42.7) | 85.8 (82.4–88.6) | 61.8 | 68.7 | {19} Clinical Sensitivity and Specificity by Site and Method | % Diagnostic Sensitivity & Specificity | % Sensitivity (95% CI) | Specificity (95% CI) | | % Sensitivity (95% CI) | % Specificity (95% CI) | | | | --- | --- | --- | --- | --- | --- | --- | --- | | | | | | SLE (n=297) | Other Diseases (n=479) | SLE (n=297) | Other Diseases (n=479) | | Site 1 | Manual | Reader 1 | 20.9 (16.6–25.9) | 94.8 (92.4–96.4) | Combined Readers | 20.4 (17.3–23.8) | 95.6 (94.1–96.7) | | | | Reader 2 | 19.9 (15.7–24.8) | 96.5 (94.4–97.8) | | | | | | Reader Confirmation | Reader 1 | 21.2 (16.9–26.2) | 95.4 (93.1–96.9) | Combined Readers | 20.7 (17.6–24.1) | 95.5 (94.0–96.7) | | | | Reader 2 | 20.2 (16.0–25.1) | 95.6 (93.4–97.1) | | | | | | HELIOS | | 20.5 (16.3–25.5) | 81.0 (77.2–84.3) | | | | | Site 2 | Manual | Reader 3 | 22.6 (18.2–27.6) | 97.3 (95.4–98.4) | Combined Readers | 21.7 (18.6–25.2) | 97.3 (96.1–98.1) | | | | Reader 4 | 20.9 (16.6–25.9) | 97.3 (95.4–98.4) | | | | | | Reader Confirmation | Reader 3 | 21.2 (16.9–26.2) | 95.4 (93.1–96.9) | Combined Readers | 21.9 (18.7–25.4) | 96.1 (94.7–97.2) | | | | Reader 4 | 22.6 (18.2–27.6) | 96.9 (94.9–98.1) | | | | | | HELIOS | | 21.9 (17.6–26.9) | 84.1 (80.6–87.1) | | | | | Site 3 | Manual | Reader 5 | 21.9 (17.6–26.9) | 97.7 (95.9–98.7) | Combined Readers | 20.4 (17.3–23.8) | 97.5 (96.3–98.3) | | | | Reader 6 | 18.9 (14.8–23.7) | 97.3 (95.4–98.4) | | | | | | Reader Confirmation | Reader 5 | 19.2 (15.1–24.1) | 97.3 (95.4–98.4) | Combined Readers | 19.2 (16.2–22.6) | 96.9 (95.6–97.8) | | | | Reader 6 | 19.2 (15.1–24.1) | 96.5 (94.4–97.8) | | | | | | HELIOS | | 15.2 (11.5–19.7) | 88.1 (84.9–90.7) | | | | {20} Cross Reactivity: Site 1 | Cross Reactivity | N | Method C | | Method B | | Method A | | --- | --- | --- | --- | --- | --- | --- | | | | Reader 1 | Reader 2 | Reader 1 | Reader 2 | | | | | % Pos | % Pos | % Pos | % Pos | % Pos | | Systemic Lupus Erythematosus | 297 | 20.9 | 19.9 | 21.2 | 20.2 | 20.5 | | Antiphospholipid Syndrome | 12 | 0.0 | 0.0 | 8.3 | 0.0 | 41.7 | | Sjoegren’s Syndrome | 30 | 0.0 | 0.0 | 3.3 | 3.3 | 33.3 | | Systemic Sclerosis | 44 | 2.3 | 0.0 | 0.0 | 0.0 | 13.6 | | Dermatomyositis/Polymyositis | 31 | 3.2 | 3.2 | 3.2 | 3.2 | 19.4 | | Rheumatoid Arthritis | 58 | 13.8 | 12.1 | 12.1 | 13.8 | 25.9 | | Mixed Connective Tissue Diseases | 24 | 8.3 | 8.3 | 12.5 | 8.3 | 41.7 | | Undifferentiated Connective Tissue Diseases | 21 | 0.0 | 0.0 | 0.0 | 0.0 | 9.5 | | Autoimmune Hepatitis | 21 | 9.5 | 9.5 | 9.5 | 9.5 | 23.8 | | Primary Biliary Cirrhosis | 20 | 27.3 | 0.0 | 38.4 | 27.3 | 58.6 | | Primary Sclerosing Cholangitis | 10 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | | ANCA associated Vasculitis | 44 | 11.4 | 11.4 | 4.5 | 4.5 | 25.0 | | Epstein-Barr Virus Infection | 25 | 0.0 | 0.0 | 0.0 | 0.0 | 12.0 | | Hepatitis B Virus Infection | 34 | 5.9 | 0.0 | 2.9 | 2.9 | 2.9 | | Hepatitis C Virus Infection | 44 | 0.0 | 0.0 | 0.0 | 0.0 | 6.8 | | Lymphoma | 31 | 3.2 | 0.0 | 0.0 | 3.2 | 16.1 | | Fibromyalgia | 30 | 0.0 | 0.0 | 0.0 | 0.0 | 10.0 | | Total | 776 | | | | | | Site 2 | Cross Reactivity | N | Manual | | Reader Confirmation | | HELIOS | | --- | --- | --- | --- | --- | --- | --- | | | | Reader 1 | Reader 2 | Reader 1 | Reader 2 | | | | | % Pos | % Pos | % Pos | % Pos | % Pos | | Systemic Lupus Erythematosus | 297 | 22.6 | 20.9 | 21.2 | 22.6 | 21.9 | | Antiphospholipid Syndrome | 12 | 0.0 | 0.0 | 8.3 | 0.0 | 41.7 | | Sjoegren’s Syndrome | 30 | 0.0 | 0.0 | 3.3 | 3.3 | 33.3 | | Systemic Sclerosis | 44 | 0.0 | 0.0 | 3.3 | 0.0 | 13.3 | | Dermatomyositis/Polymyositis | 31 | 3.2 | 3.2 | 6.5 | 12.9 | 15.9 | | Rheumatoid Arthritis | 58 | 10.3 | 10.3 | 12.1 | 10.3 | 20.7 | | Mixed Connective Tissue Diseases | 24 | 8.3 | 8.3 | 12.5 | 8.3 | 33.3 | | Undifferentiated Connective Tissue Diseases | 21 | 0.0 | 0.0 | 0.0 | 0.0 | 14.3 | | Autoimmune Hepatitis | 21 | 9.5 | 9.5 | 9.5 | 4.8 | 14.3 | | Primary Biliary Cirrhosis | 20 | 0.0 | 0.0 | 38.4 | 20.2 | 47.5 | | Primary Sclerosing Cholangitis | 10 | 0.0 | 0.0 | 0.0 | 0.0 | 30.0 | {21} | Cross Reactivity | N | Manual | | Reader Confirmation | | HELIOS | | --- | --- | --- | --- | --- | --- | --- | | | | Reader 1 | Reader 2 | Reader 1 | Reader 2 | | | | | % Pos | % Pos | % Pos | % Pos | % Pos | | ANCA associated Vasculitis | 44 | 2.3 | 2.3 | 4.5 | 2.3 | 9.1 | | Epstein-Barr Virus Infection | 25 | 0.0 | 0.0 | 0.0 | 0.0 | 4.0 | | Hepatitis B Virus Infection | 34 | 2.9 | 2.9 | 2.9 | 2.9 | 11.8 | | Hepatitis C Virus Infection | 44 | 0.0 | 0.0 | 0.0 | 0.0 | 11.4 | | Lymphoma | 31 | 0.0 | 0.0 | 0.0 | 0.0 | 25.8 | | Fibromyalgia | 30 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | | Total | 776 | | | | | | Site 3 | Cross Reactivity | N | Manual | | Reader Confirmation | | HELIOS | | --- | --- | --- | --- | --- | --- | --- | | | | Reader 1 | Reader 2 | Reader 1 | Reader 2 | | | | | % Pos | % Pos | % Pos | % Pos | % Pos | | Systemic Lupus Erythematosus | 297 | 21.9 | 18.9 | 19.2 | 19.2 | 15.2 | | Antiphospholipid Syndrome | 12 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | | Sjoegren's Syndrome | 30 | 0.0 | 0.0 | 3.3 | 3.3 | 13.3 | | Systemic Sclerosis | 44 | 0.0 | 0.0 | 2.2 | 2.2 | 15.9 | | Dermatomyositis/Polymyositis | 31 | 0.0 | 0.0 | 0.0 | 0.0 | 3.2 | | Rheumatoid Arthritis | 58 | 8.6 | 13.8 | 12.1 | 10.3 | 12.1 | | Mixed Connective Tissue Diseases | 24 | 8.3 | 4.2 | 8.3 | 8.3 | 16.7 | | Undifferentiated Connective Tissue Diseases | 21 | 0.0 | 0.0 | 0.0 | 0.0 | 9.5 | | Autoimmune Hepatitis | 21 | 4.8 | 0.0 | 9.5 | 9.5 | 14.3 | | Primary Biliary Cirrhosis | 20 | 0.0 | 11.1 | 0.0 | 11.1 | 20.2 | | Primary Sclerosing Cholangitis | 10 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | | ANCA associated Vasculitis | 44 | 4.5 | 2.3 | 4.5 | 4.5 | 13.6 | | Epstein-Barr Virus Infection | 25 | 0.0 | 0.0 | 0.0 | 0.0 | 12.0 | | Hepatitis B Virus Infection | 34 | 2.9 | 0.0 | 5.9 | 2.9 | 14.7 | | Hepatitis C Virus Infection | 44 | 0.0 | 0.0 | 0.0 | 0.0 | 4.5 | | Lymphoma | 31 | 0.0 | 3.2 | 3.2 | 3.2 | 22.6 | | Fibromyalgia | 30 | 0.0 | 0.0 | 0.0 | 0.0 | 13.3 | | Total | 776 | | | | | | b. Other clinical supportive data (when a. and b. are not applicable): Not applicable {22} 4. Clinical cut-off: Please see assay cut-off for this information. 5. Expected values/Reference range: Expected values for AESKUSLIDES nDNA (Crithidia luciliae) were analyzed with a panel of 164 sera from healthy donors from two different sites: 114 from site 1 (63 females, 51 males, mean age 32 years, range of 18–58 years) and 50 from site 2 (17 females, 33 males, mean age 34 years, range of 19–62 years). Slides were processed manually according to the device instructions for use and analyzed at the microscope by two independent readers. | | Positive (%) | Negative (%) | Total | | --- | --- | --- | --- | | Reader 1 | 1 (0.6) | 163 (99.4) | 164 | | Reader 2 | 0 (0) | 164 (100) | 164 | N. Proposed Labeling: The labeling is sufficient and it satisfies the requirements of 21 CFR Part 809.10. O. Conclusion: The submitted information in this premarket notification is complete and supports a substantial equivalence decision