Anti-Neutrophil Cytoplasmic Antibodies (Ethanol-Fixed); Anti-Neutrophil Cytoplasmic Antibodies (Formalin-Fixed)

{0} FDA U.S. FOOD & DRUG ADMINISTRATION # 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION SUMMARY ASSAY ## I Background Information: A 510(k) Number K243776 B Applicant ZEUS Scientific C Proprietary and Established Names Anti-Neutrophil Cytoplasmic Antibodies (Ethanol-fixed) Anti-Neutrophil Cytoplasmic Antibodies (Formalin-fixed) D Regulatory Information | Product Code(s) | Classification | Regulation Section | Panel | | --- | --- | --- | --- | | MOB | Class II | 21 CFR 866.5660 - Multiple Autoantibodies Immunological Test System | IM - Immunology | | PIV | Class II | 21 CFR 866.4750 - Automated Indirect Immunofluorescence Microscope and Software-Assisted System | IM - Immunology | ## II Submission/Device Overview: A Purpose for Submission: New assay on a previously cleared instrument B Measurand: Anti-Neutrophil Cytoplasmic Antibodies (ANCA) C Type of Test: Qualitative and/or semi-quantitative indirect immunofluorescence assay (IFA); manual or semi-automated Food and Drug Administration 10903 New Hampshire Avenue Silver Spring, MD 20993-0002 www.fda.gov {1} K243776 - Page 2 of 28 ## III Intended Use/Indications for Use: ### A Intended Use(s): See Indications for Use below. ### B Indication(s) for Use: The Anti-Neutrophil Cytoplasmic Antibodies (Ethanol-fixed) test system is an indirect immunofluorescence assay (IFA) for the qualitative and semi-quantitative determination of anti-neutrophil cytoplasmic antibodies (ANCA) of the IgG isotype in human serum by manual fluorescence microscopy or with dIFine. The presence of ANCA in conjunction with other clinical and laboratory findings can be used to aid in the diagnosis of ANCA associated vasculitis (AAV). All suggested results obtained with dIFine must be confirmed by a trained operator. The Anti-Neutrophil Cytoplasmic Antibodies (Formalin-fixed) test system is an indirect immunofluorescence assay (IFA) for the qualitative and semi-quantitative determination of anti-neutrophil cytoplasmic antibodies (ANCA) of the IgG isotype in human serum by manual fluorescence microscopy or with dIFine. The presence of ANCA in conjunction with other clinical and laboratory findings can be used to aid in the diagnosis of ANCA associated vasculitis (AAV). All suggested results obtained with dIFine must be confirmed by a trained operator. ### C Special Conditions for Use Statement(s): Rx - For Prescription Use Only - For In Vitro diagnostic use. - The device is for use by a trained operator in a clinical laboratory setting. - All software-aided results must be confirmed by the trained operator. ### D Special Instrument Requirements: Zeus dIFine or manual fluorescence microscope ## IV Device/System Characteristics: ### A Device Description: The Anti-Neutrophil Cytoplasmic Antibodies (Ethanol-fixed) and the Anti-Neutrophil Cytoplasmic Antibodies (Formalin-fixed) test systems each contain the following components: - ANCA Substrate Slides: Ten, 10-well Slides with absorbent blotter and desiccant pouch - Conjugate: 2 x 3 mL, Goat anti-human IgG labeled with FITC in phosphate buffer with BSA and counterstain {2} - p-ANCA Positive Control: 1 x 0.5 mL, p-ANCA positive human serum with preservatives. - c-ANCA Positive Control: 1 x 0.5 mL, c-ANCA positive human serum with preservatives. - Negative Control: 1 x 0.5 mL, human serum negative for ANCA with preservatives. 0.5 mL - Cover Glass: 24 x 60 mm, Thickness #1 - Phosphate-buffered-saline (PBS): 2 packets, each buffer packet for one liter of distilled or deionized water, pH 7.2 ± 0.2 - Mounting Media (Buffered Glycerol): 1 x 12 mL (in 15 mL bottle), ready to use Materials that are required but not provided with the kit: - dIFine instrument or manual fluorescence microscope - General laboratory materials (e.g., pipettors, disposable pipette tips, test tubes, dilutions plates, slide washer, distilled or deionized water, etc.) ## B Principle of Operation: The ANCA IFA test systems are designed to detect the presence of ANCA in human serum. Each test system employs either ethanol-fixed or formalin-fixed human granulocyte substrate, optimized goat anti-human IgG fluorescein isothiocyanate (FITC) conjugate solution, and wash solution. For either product, the reaction occurs as follows: 1. Step one is the sample incubation where ANCA present in the patient sample may bind to the cell substrate, forming an antigen-antibody complex. Other serum components are subsequently washed away. 2. Step two is the Conjugate incubation where the anti-human IgG FITC conjugate reacts with any human IgG bound to the substrate during the sample incubation. This will form a stable antigen-antibody-Conjugate complex at the location where the initial patient antibody bound to the cell substrate. Excess Conjugate is subsequently washed away. 3. The results of the assay can be visualized using a properly equipped fluorescent microscope or dIFine. Any positive reactions will appear as apple-green, fluorescent staining within the cell. If the sample did not have ANCA present, there will be no distinct nuclear or cytoplasmic staining of the cell. ## Manual Interpretation: The interpretation of the results depends on the pattern observed as well as the titer of the autoantibody present in the specimen. A positive reaction is the presence of any pattern of nuclear apple-green staining observed at a 1:20 dilution based on a 1+ to 4+ scale of staining intensity where 1+ is considered a weak reaction and 4+ a strong reaction. Zeus recommends sera positive at 1:20 should be titered to endpoint dilution by making 1:40, 1:80, etc. serial dilutions. The endpoint titer is the highest dilution that produces a 1+ positive reaction. ## dIFine Interpretation: When slides are analyzed by dIFine, digital images of representative fields of view of the well are captured. The default scanning area is composed of 12 fields with each field approximately 610 µm x 510 µm using the 20X objective. All images are taken through a fluorescein isothiocyanate (FITC) filter. The dIFine System reads the slides and measures the FITC light K243776 - Page 3 of 28 {3} intensity of the cell substrate that are included in the region. Then, the dIFine System reports the measured average fluorescence intensity as an index percentage and recommends a qualitative result. To facilitate the interpretation, the dIFine System presents acquired digital images of the slide and the following information for human analysis: Negative, Positive or Uncertain classification as well as a suggested pattern (c-ANCA, p-ANCA, or Others). Trained operators then review the images taken by dIFine System. During the review process, further options include: - Navigation of digitized well using virtual microscope tools (zooming/browsing images at different magnifications). - Enlargement of images to examine detail. - Image Atlas to assist with identification of patterns. The trained operator can confirm the results by clicking the "Validate" button on the screen and accepting the classification (negative/positive and pattern) suggested by the dIFine System, or they can revise the suggested dIFine classification (negative to positive and vice versa or from Uncertain to either positive or negative), add comments (if any), change the pattern (if necessary) and eventually click the "Validate" button on the screen. ## V Substantial Equivalence Information: ### A Predicate Device Name(s): NOVA Lite DAPI ANCA Ethanol Kit NOVA Lite DAPI ANCA Formalin Kit ### B Predicate 510(k) Number(s): K161258 ### C Comparison with Predicate(s): K243776 - Page 4 of 28 {4} K243776 - Page 5 of 28 | Device & Predicate Device(s): | K243776 (Candidate) | K161258 (Predicate) | | --- | --- | --- | | Device Trade Name | Anti-Neutrophil Cytoplasmic Antibodies (Ethanol-fixed) Anti-Neutrophil Cytoplasmic Antibodies (Formalin-fixed) | NOVA Lite DAPI ANCA (Ethanol) NOVA Lite DAPI ANCA (Formalin) | | **General Device Characteristic Similarities** | | | | Intended Use/Indications For Use | The Anti-Neutrophil Cytoplasmic Antibodies (Ethanol-fixed) and Anti-Neutrophil Cytoplasmic Antibodies (Formalin-fixed) test systems are indirect immunofluorescence assays (IFA) for the qualitative and semi-quantitative determination of anti-neutrophil cytoplasmic antibodies (ANCA) of the IgG isotype in human serum by manual fluorescence microscopy or with dIFine. The presence of ANCA in conjunction with other clinical and laboratory findings can be used to aid in the diagnosis of ANCA associated vasculitis (AAV). All suggested results obtained with dIFine must be confirmed by a trained operator. | NOVA Lite DAPI ANCA (Ethanol) Kit or NOVA Lite DAPI ANCA (Formalin) Kit is indirect immunofluorescence assay for the qualitative detection and semi-quantitative determination of anti-neutrophil cytoplasmic antibodies (ANCA) of IgG isotypes in human serum by manual fluorescence microscopy or with NOVA View Automated Fluorescence Microscope. The presence of ANCA, in conjunction with other serological, radiological, histological, and clinical findings, aids in the diagnosis of ANCA associated vasculitis. A trained operator must confirm results when generated with the NOVA View device. | | Solid Phase (Substrate) | Ethanol-fixed or Formalin-fixed human neutrophils | Same | | Technology | Indirect Immunofluorescence Assay | Same | | Sample | Human Serum | Same | | Positive Control | Positive pANCA human control Positive cANCA human control | Same | | Negative control | Human Serum with no ANCA activity | Same | | Sample Diluent | PBS Buffer | Same | | Wash Solution | PBS Buffer | Same | | Screening Dilution | 1:20 dilution in PBS | Same | | Staining Pattern Observed | pANCA, cANCA, Other, Uncertain | Same | | **General Device Characteristic Differences** | | | | Conjugate | Anti-human IgG labeled with FITC | FITC-labeled anti-human IgG (Fc) Conjugate with DAPI | | Mounting Media | Buffered Glycerol | Unknown | | Interpretation Method | Manual microscope or dIFine automated microscope | Manual microscope or NOVA View automated microscope | {5} VI Standards/Guidance Documents Referenced: The following Clinical and Laboratory Standards Institute (CLSI) guidelines were used: - CLSI EP05-A3 Evaluation of Precision Performance of Quantitative Measurement Methods, Approved Guideline - 3rd Edition - CLSI EP06, 2nd ed. Evaluation of the Linearity of Quantitative Measurement Procedures – Second Edition - CLSI EP07-A2 Interference Testing in Clinical Chemistry, 2nd Edition - CLSI EP17-A2 Evaluation of Detection Capability for Clinical Laboratory Measurement Procedures, 2nd Edition - CLSI EP28-A3c Defining, Establishing and Verifying Reference Intervals in the Clinical Laboratory VII Performance Characteristics (if/when applicable): A Analytical Performance: When establishing performance characteristics of the ANCA Ethanol-fixed IFA test system or ANCA Formalin-fixed IFA test system, slides were interpreted using three different methods as outlined below: | Method | Processing | Imaging | Reading/Evaluation of Slides | | --- | --- | --- | --- | | A | Manual | Manual | Manual (read of microscope field) | | B | Automated | Automated | Manual (read of digital image) | | C | Automated | Automated | Automated (software interpretation) | Data analysis was performed to determine the agreement based on the qualitative results and the staining pattern results: - The qualitative agreement is the output read by each interpretation method to conclude if a sample is positive or negative. - The staining pattern agreement defines if the positive sample belongs to c-ANCA (cytoplasmic ANCA) pattern or p-ANCA (perinuclear ANCA) pattern. 1. Precision/Reproducibility: a. Repeatability: 20-day Within-Laboratory Precision Study A panel of samples including: two low positive samples (~1:20-1:40 endpoint) with one anti-PR3 (c-ANCA) and one anti-MPO (p-ANCA), two mid positive samples (~1:80-1:160 endpoint) with one anti-PR3 (c-ANCA) and one anti-MPO (p-ANCA), and two high positive samples (> 1:160 endpoint) with one anti-PR3 (c-ANCA) and one anti-MPO (p-ANCA), and two negative samples, were assayed in triplicate, at a 1:20 K243776 - Page 6 of 28 {6} screening dilution, once per day, on 20 different days, resulting a total of 60 results per sample for each of the Anti-Neutrophil Cytoplasmic Antibodies (Ethanol-fixed) and the Anti-Neutrophil Cytoplasmic Antibodies (Formalin-fixed) test systems. Qualitative results and pattern results were interpreted by two technicians for Methods A and B, and by a single dIFine instrument for Method C. For each test system, the summary results for within-method and between method agreements (for both qualitative and patten) with combined results from two technicians (a total of 120 results per sample) are presented in the tables below: # Anti-Neutrophil Cytoplasmic Antibodies (Ethanol-Fixed) Within-Method Agreement - Qualitative Result | Sample | Agreement% (95% CI) | | | | --- | --- | --- | --- | | | Method A | Method B | Method C | | Low Positive – c-ANCA | 100% (96.9–100.0%) | 100% (96.9–100.0%) | 100% (96.9–100.0%) | | Low Positive – p-ANCA | 100% (96.9–100.0%) | 100% (96.9–100.0%) | 100% (96.9–100.0%) | | Mid Positive – c-ANCA | 100% (96.9–100.0%) | 100% (96.9–100.0%) | 100% (96.9–100.0%) | | Mid Positive – p-ANCA | 100% (96.9–100.0%) | 100% (96.9–100.0%) | 100% (96.9–100.0%) | | High Positive – c-ANCA | 100% (96.9–100.0%) | 100% (96.9–100.0%) | 100% (96.9–100.0%) | | High Positive – p-ANCA | 100% (96.9–100.0%) | 100% (96.9–100.0%) | 100% (96.9–100.0%) | | Negative 1 | 100% (96.9–100.0%) | 100% (96.9–100.0%) | 100% (96.9–100.0%) | | Negative 2 | 100% (96.9–100.0%) | 100% (96.9–100.0%) | 100% (96.9–100.0%) | Within-Method Agreement - Pattern Result | Sample | Agreement% (95% CI) | | | | --- | --- | --- | --- | | | Method A | Method B | Method C | | Low Positive – c-ANCA | 100% (96.9–100.0%) | 100% (96.9–100.0%) | 98.3% (94.1–99.5%) | | Low Positive – p-ANCA | 100% (96.9–100.0%) | 100% (96.9–100.0%) | 96.7% (91.7–98.7%) | | Mid Positive – c-ANCA | 100% (96.9–100.0%) | 100% (96.9–100.0%) | 100% (96.9–100.0%) | | Mid Positive – p-ANCA | 100% (96.9–100.0%) | 100% (96.9–100.0%) | 100% (96.9–100.0%) | | High Positive – c-ANCA | 100% (96.9–100.0%) | 100% (96.9–100.0%) | 96.7% (91.7–98.7%) | | High Positive – p-ANCA | 100% (96.9–100.0%) | 100% (96.9–100.0%) | 100% (96.9–100.0%) | Between-Method Agreement - Qualitative Result | Sample | Agreement% (95% CI) | | | | --- | --- | --- | --- | | | Method A | Method B | Method C | | Low Positive – c-ANCA | 100% (96.9–100.0%) | 100% (96.9–100.0%) | 100% (96.9–100.0%) | | Low Positive – p-ANCA | 100% (96.9–100.0%) | 100% (96.9–100.0%) | 100% (96.9–100.0%) | | Mid Positive – c-ANCA | 100% (96.9–100.0%) | 100% (96.9–100.0%) | 100% (96.9–100.0%) | | Mid Positive – p-ANCA | 100% (96.9–100.0%) | 100% (96.9–100.0%) | 100% (96.9–100.0%) | | High Positive – c-ANCA | 100% (96.9–100.0%) | 100% (96.9–100.0%) | 100% (96.9–100.0%) | | High Positive – p-ANCA | 100% (96.9–100.0%) | 100% (96.9–100.0%) | 100% (96.9–100.0%) | | Negative 1 | 100% (96.9–100.0%) | 100% (96.9–100.0%) | 100% (96.9–100.0%) | | Negative 2 | 100% (96.9–100.0%) | 100% (96.9–100.0%) | 100% (96.9–100.0%) | K243776 - Page 7 of 28 {7} Between-Method Agreement - Pattern Result | Sample | Agreement% (95% CI) | | | | --- | --- | --- | --- | | | Method A | Method B | Method C | | Low Positive – c-ANCA | 100% (96.9–100.0%) | 98.3% (94.1–99.5%) | 98.3% (94.1–99.5%) | | Low Positive – p-ANCA | 100% (96.9–100.0%) | 96.7% (91.7–98.7%) | 96.7% (91.7–98.7%) | | Mid Positive – c-ANCA | 100% (96.9–100.0%) | 100% (96.9–100.0%) | 100% (96.9–100.0%) | | Mid Positive – p-ANCA | 100% (96.9–100.0%) | 100% (96.9–100.0%) | 100% (96.9–100.0%) | | High Positive – c-ANCA | 100% (96.9–100.0%) | 96.7% (91.7–98.7%) | 96.7% (91.7–98.7%) | | High Positive – p-ANCA | 100% (96.9–100.0%) | 100% (96.9–100.0%) | 100% (96.9–100.0%) | ## Anti-Neutrophil Cytoplasmic Antibodies (Formalin-Fixed) Within-Method Agreement - Qualitative Result | Sample | Agreement% (95% CI) | | | | --- | --- | --- | --- | | | Method A | Method B | Method C | | Low Positive – c-ANCA | 100% (96.9–100.0%) | 100% (96.9–100.0%) | 100% (96.9–100.0%) | | Low Positive – p-ANCA | 96.7% (91.7–98.7%) | 96.7% (91.7–98.7%) | 88.3% (81.4–92.9%) | | Mid Positive – c-ANCA | 100% (96.9–100.0%) | 100% (96.9–100.0%) | 100% (96.9–100.0%) | | Mid Positive – p-ANCA | 98.3% (94.1–99.5%) | 98.3% (94.1–99.5%) | 91.7% (85.3–95.4%) | | High Positive – c-ANCA | 98.3% (94.1–99.5%) | 98.3% (94.1–99.5%) | 98.3% (94.1–99.5%) | | High Positive – p-ANCA | 100% (96.9–100.0%) | 100% (96.9–100.0%) | 96.7% (91.7–98.7%) | | Negative 1 | 100% (96.9–100.0%) | 100% (96.9–100.0%) | 100% (96.9–100.0%) | | Negative 2 | 100% (96.9–100.0%) | 100% (96.9–100.0%) | 100% (96.9–100.0%) | Within-Method Agreement - Pattern Result | Sample | Agreement% (95% CI) | | | | --- | --- | --- | --- | | | Method A | Method B | Method C | | Low Positive – c-ANCA | 100% (96.9–100.0%) | 98.3% (94.1–99.5%) | 98.3% (94.1–99.5%) | | Low Positive – p-ANCA | 100% (96.9–100.0%) | 100% (96.9–100.0%) | 96.7% (91.7–98.7%) | | Mid Positive – c-ANCA | 100% (96.9–100.0%) | 100% (96.9–100.0%) | 100% (96.9–100.0%) | | Mid Positive – p-ANCA | 100% (96.9–100.0%) | 100% (96.9–100.0%) | 100% (96.9–100.0%) | | High Positive – c-ANCA | 100% (96.9–100.0%) | 100% (96.9–100.0%) | 98.3% (94.1–99.5%) | | High Positive – p-ANCA | 100% (96.9–100.0%) | 100% (96.9–100.0%) | 94.9% (89.4–97.7%) | Between-Method Agreement - Qualitative Result | Sample | Agreement% (95% CI) | | | | --- | --- | --- | --- | | | Method A | Method B | Method C | | Low Positive – c-ANCA | 100% (96.9–100.0%) | 100% (96.9–100.0%) | 100% (96.9–100.0%) | | Low Positive – p-ANCA | 100% (96.9–100.0%) | 91.7% (85.3–95.4%) | 91.7% (85.3–95.4%) | | Mid Positive – c-ANCA | 100% (96.9–100.0%) | 100% (96.9–100.0%) | 100% (96.9–100.0%) | | Mid Positive – p-ANCA | 100% (96.9–100.0%) | 93.3% (87.4–96.6%) | 93.3% (87.4–96.6%) | | High Positive – c-ANCA | 100% (96.9–100.0%) | 100% (96.9–100.0%) | 100% (96.9–100.0%) | | High Positive – p-ANCA | 100% (96.9–100.0%) | 96.7% (91.7–98.7%) | 96.7% (91.7–98.7%) | | Negative 1 | 100% (96.9–100.0%) | 100% (96.9–100.0%) | 100% (96.9–100.0%) | | Negative 2 | 100% (96.9–100.0%) | 100% (96.9–100.0%) | 100% (96.9–100.0%) | K243776 - Page 8 of 28 {8} Between-Method Agreement - Pattern Result | Sample | Agreement% (95% CI) | | | | --- | --- | --- | --- | | | Method A | Method B | Method C | | Low Positive – c-ANCA | 100% (96.9–100.0%) | 98.3% (94.1–99.5%) | 98.3% (94.1–99.5%) | | Low Positive – p-ANCA | 100% (96.9–100.0%) | 100% (96.9–100.0%) | 100% (96.9–100.0%) | | Mid Positive – c-ANCA | 100% (96.9–100.0%) | 100% (96.9–100.0%) | 100% (96.9–100.0%) | | Mid Positive – p-ANCA | 100% (96.9–100.0%) | 100% (96.9–100.0%) | 100% (96.9–100.0%) | | High Positive – c-ANCA | 100% (96.9–100.0%) | 98.3% (94.1–99.5%) | 98.3% (94.1–99.5%) | | High Positive – p-ANCA | 100% (96.9–100.0%) | 94.9% (89.4–97.7%) | 94.9% (89.4–97.7%) | b. Lot-to-Lot Reproducibility A panel of 10 samples includes: two low positive samples (~1:20-1:40 endpoint) with one anti-PR3 (c-ANCA) and one anti-MPO (p-ANCA), two mid positive samples (~1:80-1:160 endpoint) with one anti-PR3 (c-ANCA) and one anti-MPO (p-ANCA), two high positive samples (> 1:160 endpoint) with one anti-PR3 (c-ANCA) and one anti-MPO (p-ANCA), and four negative samples, were each assayed at a 1:20 screening dilution using three different lots of the Anti-Neutrophil Cytoplasmic Antibodies (Ethanol-fixed) test system or the Anti-Neutrophil Cytoplasmic Antibodies (Formalin-fixed) test system. The six positive samples were also serially diluted and assayed using the same three lots of each test system. Results were interpreted by all three methods noted above. A total of 44 results per lot from the diluted positive samples and the 4 negative samples were analyzed for qualitative agreement and pattern agreement analyses. Endpoint dilutions were also determined for the serially diluted samples. The results are summarized in the tables below: Anti-Neutrophil Cytoplasmic Antibodies (Ethanol-Fixed) Within-Lot Agreement - Qualitative Results between Three Methods | Interpretation Methods | Agreement% (95% CI) | | | | --- | --- | --- | --- | | | Lot 1 | Lot 2 | Lot 3 | | A vs B | 100.0% (92.0–100.0%) | 97.7% (88.2–99.6%) | 95.5% (84.9–98.7%) | | A vs C | 86.4% (73.3–93.6%) | 79.6% (65.5–88.9%) | 77.3% (63.0–87.1%) | | B vs C | 90.9% (78.8–96.4%) | 81.8% (68.0–90.5%) | 81.8% (68.0–90.5%) | Within-Lot Agreement - Pattern Results between Three Methods | Interpretation Methods | Agreement% (95% CI) | | | | --- | --- | --- | --- | | | Lot 1 | Lot 2 | Lot 3 | | A vs B | 100.0% (85.7–100.0%) | 100.0% (85.1–100.0%) | 100.0% (85.7–100.0%) | | A vs C | 100.0% (85.7–100.0%) | 86.4% (66.7–95.3%) | 82.6% (62.9–93.0%) | | B vs C | 96.0% (80.5–99.3%) | 86.4% (66.7–95.3%) | 84.0% (65.4–93.6%) | K243776 - Page 9 of 28 {9} Between Lot Agreement - Qualitative Results between Three Methods | Interpretation Methods | Agreement% (95% CI) | | | | --- | --- | --- | --- | | | Lot 1 vs. Lot 2 | Lot 1 vs. Lot 3 | Lot 2 vs. Lot 3 | | A vs A | 97.7% (88.2–99.6%) | 95.5% (84.9–98.7%) | 97.7% (88.2–99.6%) | | B vs B | 95.5% (84.9–98.7%) | 100.0% (92.0–100.0%) | 95.5% (84.9–98.7%) | | C vs C | 84.1% (70.6–92.1%) | 86.4% (73.3–93.6%) | 79.6% (65.5–88.9%) | | A vs B | 95.5% (84.9–98.7%) | 95.5% (84.9–98.7%) | 93.2% (81.8–97.7%) | | A vs C | 77.3% (63.0–87.2%) | 77.3% (63.0–87.2%) | 75.0% (60.6–85.4%) | | B vs C | 79.6% (65.5–88.9%) | 81.8% (68.0–90.5%) | 77.3% (63.0 - 87.2%) | | B vs A | 93.2% (81.8–97.7%) | 95.5% (84.9–98.7%) | 95.5% (84.9–98.7%) | | C vs A | 84.1% (70.6–92.1%) | 86.4% (73.3–93.6%) | 79.6% (65.5–88.9%) | | C vs B | 86.4% (73.3–93.6%) | 90.9% (78.8–96.4%) | 79.6% (65.5–88.9%) | Between Lot Agreement - Pattern Results between Three Methods | Interpretation Methods | Agreement% (95% CI) | | | | --- | --- | --- | --- | | | Lot 1 vs. Lot 2 | Lot 1 vs. Lot 3 | Lot 2 vs. Lot 3 | | A vs A | 100.0% (85.1–100.0%) | 100.0% (85.1–100.0%) | 100.0% (85.1–100.0%) | | B vs B | 100.0% (85.1–100.0%) | 100.0% (86.7–100.0%) | 100.0% (85.7–100.0%) | | C vs C | 83.3% (64.2–93.3%) | 80.8% (62.1–91.5%) | 80.0% (60.9–91.1%) | | A vs B | 100.0% (85.1–100.0%) | 100.0% (85.7–100.0%) | 100.0% (85.1–100.0%) | | A vs C | 86.4% (66.7–95.3%) | 82.6% (62.9–93.0%) | 86.4% (66.7–95.3%) | | B vs C | 82.6% (62.9- 93.0%) | 84.0% (65.4–93.6%) | 87.0% (67.9–95.5%) | | B vs A | 100.0% (85.1–100.0%) | 100.0% (85.7–100.0%) | 100.0% (85.1–100.0%) | | C vs A | 100.0% (85.1–100.0%) | 95.7% (79.0–99.2%) | 86.4% (66.7–95.3%) | | C vs B | 100.0% (85.1–100.0%) | 96.0% (80.5–99.3%) | 87.0% (67.9–95.5%) | Endpoint Titers Agreement: | Sample | Method A | Method B | Method C | | | | --- | --- | --- | --- | --- | --- | | | | | UNC as (-) | UNC as (+) | | | Low Positive - c-ANCA | Lot 1 | 1:40 | 1:40 | 1:40 | 1:40 | | | Lot 2 | 1:40 | 1:40 | 1:160 | 1:320 | | | Lot 3 | 1:40 | 1:40 | 1:80 | 1:160 | | Low Positive - p-ANCA | Lot 1 | 1:40 | 1:40 | 1:80 | 1:160 | | | Lot 2 | 1:40 | 1:40 | 1:80 | 1:160 | | | Lot 3 | 1:40 | 1:40 | 1:80 | 1:80 | | Mid Positive - c-ANCA | Lot 1 | 1:80 | 1:80 | 1:80 | 1:80 | | | Lot 2 | 1:80 | 1:80 | 1:80 | 1:160 | | | Lot 3 | 1:80 | 1:80 | 1:160 | 1:320 | | Mid Positive - p-ANCA | Lot 1 | 1:80 | 1:160 | 1:160 | 1:320 | | | Lot 2 | 1:80 | 1:160 | 1:160 | 1:320 | | | Lot 3 | 1:80 | 1:160 | 1:160 | 1:320 | | High Positive - c-ANCA | Lot 1 | 1:1280 | 1:2560 | 1:2560 | 1:5120 | | | Lot 2 | 1:1280 | 1:1280 | 1:1280 | 1:2560 | | | Lot 3 | 1:2560 | 1:2560 | 1:2560 | 1:5120 | | High Positive - p-ANCA | Lot 1 | 1:640 | 1:640 | 1:640 | 1:640 | | | Lot 2 | 1:320 | 1:320 | 1:320 | 1:320 | | | Lot 3 | 1:320 | 1:640 | 1:640 | 1:1280 | K243776 - Page 10 of 28 {10} Anti-Neutrophil Cytoplasmic Antibodies (Formalin-Fixed) Within-Lot Agreement - Qualitative Results between Three Methods | Interpretation Methods | Agreement% (95% CI) | | | | --- | --- | --- | --- | | | Lot 1 | Lot 2 | Lot 3 | | A vs B | 100.0% (92.0–100.0%) | 100.0% (92.0–100.0%) | 100.0% (92.0–100.0%) | | A vs C | 88.6% (76.0–95.1%) | 86.4% (73.3–93.6%) | 81.8% (68.0–90.5%) | | B vs C | 88.6% (76.0–95.1%) | 86.4% (73.3–93.6%) | 81.8% (68.0–90.5%) | Within-Lot Agreement – Pattern Results between Three Methods | Interpretation Methods | Agreement% (95% CI) | | | | --- | --- | --- | --- | | | Lot 1 | Lot 2 | Lot 3 | | A vs B | 100.0% (86.2–100.0%) | 100.0% (85.7–100.0%) | 100.0% (85.7–100.0%) | | A vs C | 65.0% (43.3–81.9%) | 79.0% (56.7–91.5%) | 100.0% (83.9–100.0%) | | B vs C | 65.0% (43.3–81.9%) | 79.0% (56.7–91.5%) | 100.0% (83.9–100.0%) | Between Lot Agreement - Qualitative Results between Three Methods | Interpretation Methods | Agreement% (95% CI) | | | | --- | --- | --- | --- | | | Lot 1 vs. Lot 2 | Lot 1 vs. Lot 3 | Lot 2 vs. Lot 3 | | A vs A | 97.7% (88.2–99.6%) | 97.7% (88.2–99.6%) | 100.0% (92.0–100.0%) | | B vs B | 97.7% (88.2–99.6%) | 97.7% (88.2–99.6%) | 100.0% (92.0–100.0%) | | C vs C | 79.6% (65.5–88.9%) | 79.6% (65.5–88.9%) | 79.6% (65.5–88.9%) | | A vs B | 97.7% (88.2–99.6%) | 97.7% (88.2–99.6%) | 100.0% (92.0–100.0%) | | A vs C | 84.1% (70.6–92.1%) | 79.6% (65.5–88.9%) | 81.8% (68.0–90.5%) | | B vs C | 84.1% (70.6–92.1%) | 79.6% (65.5–88.9%) | 81.8% (68.0–90.5%) | | B vs A | 97.7% (88.2–99.6%) | 97.7% (88.2–99.6%) | 100.0% (92.0–100.0%) | | C vs A | 88.6% (76.0–95.1%) | 88.6% (76.0–95.1%) | 86.4% (73.3–93.6%) | | C vs B | 88.6% (76.0–95.1%) | 88.6% (76.0–95.1%) | 86.4% (73.3–93.6%) | Between Lot Agreement - Pattern Results between Three Methods | Interpretation Methods | Agreement% (95% CI) | | | | --- | --- | --- | --- | | | Lot 1 vs. Lot 2 | Lot 1 vs. Lot 3 | Lot 2 vs. Lot 3 | | A vs A | 100.0% (85.7–100.0%) | 100.0% (85.7–100.0%) | 100.0% (85.7–100.0%) | | B vs B | 100.0% (85.7–100.0%) | 100.0% (85.7–100.0%) | 100.0% (85.7–100.0%) | | C vs C | 70.6% (46.9–86.7%) | 66.7% (43.8–83.7%) | 82.4% (59.0–93.8%) | | A vs B | 100.0% (85.7–100.0%) | 100.0% (85.7–100.0%) | 100.0% (85.7–100.0%) | | A vs C | 79.0% (56.7–91.5%) | 100.0% (83.9–100.0%) | 100.0% (83.9–100.0%) | | B vs C | 79.0% (56.7–91.5%) | 100.0% (83.9–100.0%) | 100.0% (83.9–100.0%) | | B vs A | 100.0% (85.7–100.0%) | 100.0% (85.7–100.0%) | 100.0% (85.7–100.0%) | | C vs A | 65.0% (43.3–81.9%) | 65.0% (43.3–81.9%) | 79.0% (56.7–91.5%) | | C vs B | 65.0% (43.3–81.9%) | 65.0% (43.3–81.9%) | 79.0% (56.7–91.5%) | K243776 - Page 11 of 28 {11} Endpoint Titers Agreement: | Sample | Method A | Method B | Method C | | | | --- | --- | --- | --- | --- | --- | | | | | UNC as (-) | UNC as (+) | | | Low Positive - c-ANCA | Lot 1 | 1:40 | 1:40 | 1:40 | 1:80 | | | Lot 2 | 1:40 | 1:40 | 1:20 | 1:80 | | | Lot 3 | 1:40 | 1:40 | 1:40 | 1:80 | | Low Positive - p-ANCA | Lot 1 | 1:40 | 1:40 | 1:40 | 1:40 | | | Lot 2 | 1:40 | 1:40 | 1:40 | 1:80 | | | Lot 3 | 1:40 | 1:40 | 1:80 | 1:160 | | Mid Positive - c-ANCA | Lot 1 | 1:80 | 1:80 | 1:80 | 1:80 | | | Lot 2 | 1:80 | 1:80 | 1:80 | 1:160 | | | Lot 3 | 1:80 | 1:80 | 1:40 | 1:160 | | Mid Positive - p-ANCA | Lot 1 | 1:160 | 1:160 | 1:80 | 1:160 | | | Lot 2 | 1:160 | 1:160 | 1:80 | 1:160 | | | Lot 3 | 1:160 | 1:160 | 1:160 | 1:320 | | High Positive - c-ANCA | Lot 1 | 1:1280 | 1:1280 | 1:640 | 1:1280 | | | Lot 2 | 1:640 | 1:640 | 1:160 | 1:640 | | | Lot 3 | 1:640 | 1:640 | 1:320 | 1:640 | | High Positive - p-ANCA | Lot 1 | 1:640 | 1:640 | 1:160 | 1:640 | | | Lot 2 | 1:640 | 1:640 | 1:640 | 1:640 | | | Lot 3 | 1:640 | 1:640 | 1:320 | 1:640 | c. Multi-Site Reproducibility Study To assess the multi-site reproducibility of the Anti-Neutrophil Cytoplasmic Antibodies (Ethanol-fixed) and the Anti-Neutrophil Cytoplasmic Antibodies (Formalin-fixed), a panel of 8 samples including two low positive samples (~1:20-1:40 endpoint) with one anti-PR3 (c-ANCA) and one anti-MPO (p-ANCA), two mid positive samples (~1:80-1:160 endpoint) with one anti-PR3 (c-ANCA) and one anti-MPO (p-ANCA), two high positive samples (>1:160 endpoint) with one anti-PR3 (c-ANCA) and one anti-MPO (p-ANCA), and two negative samples were each assayed in triplicate at a 1:20 screening dilution twice per day, on five different days, by three different technicians at three different laboratories using each test system. Qualitative and pattern results were interpreted by two technicians for Methods A and B, and by a single dIFine instrument for Method C (six total technicians and three total dIFine instruments). The results (30 results per sample per method per technician) were analyzed for each site and for combined. The combined results (a total of 240 results for each method per technician for qualitative analysis and a total of 180 results for each method per technician for pattern analysis) are summarized in the tables below: K243776 - Page 12 of 28 {12} # Anti-Neutrophil Cytoplasmic Antibodies (Ethanol-Fixed) Site-to-Site and Technician-to-Technician Agreement – Qualitative Results | Method A | Site 1 | | Site 2 | | Site 3 | | | | --- | --- | --- | --- | --- | --- | --- | --- | | | | Technician 1 | Technician 2 | Technician 1 | Technician 2 | Technician 1 | Technician 2 | | Site 1 | Technician 1 | | 100% (98.4–100.0%) | 100% (98.4–100.0%) | 100% (98.4–100.0%) | 100% (98.4–100.0%) | 100% (98.4–100.0%) | | | Technician 2 | | | 100% (98.4–100.0%) | 100% (98.4–100.0%) | 100% (98.4–100.0%) | 100% (98.4–100.0%) | | Site 2 | Technician 1 | | | | 100% (98.4–100.0%) | 100% (98.4–100.0%) | 100% (98.4–100.0%) | | | Technician 2 | | | | | 100% (98.4–100.0%) | 100% (98.4–100.0%) | | Site 3 | Technician 1 | | | | | | 100% (98.4–100.0%) | | | Technician 2 | | | | | | | | Method B | Site 1 | | Site 2 | | Site 3 | | | | --- | --- | --- | --- | --- | --- | --- | --- | | | | Technician 1 | Technician 2 | Technician 1 | Technician 2 | Technician 1 | Technician 2 | | Site 1 | Technician 1 | | 100% (98.4–100.0%) | 100% (98.4–100.0%) | 100% (98.4–100.0%) | 100% (98.4–100.0%) | 100% (98.4–100.0%) | | | Technician 2 | | | 100% (98.4–100.0%) | 100% (98.4–100.0%) | 100% (98.4–100.0%) | 100% (98.4–100.0%) | | Site 2 | Technician 1 | | | | 100% (98.4–100.0%) | 100% (98.4–100.0%) | 100% (98.4–100.0%) | | | Technician 2 | | | | | 100% (98.4–100.0%) | 100% (98.4–100.0%) | | Site 3 | Technician 1 | | | | | | 100% (98.4–100.0%) | | | Technician 2 | | | | | | | | Method C | Site 1 | Site 2 | Site 3 | | --- | --- | --- | --- | | Site 1 | | 99.6% (97.7–99.9%) | 99.6% (97.7–99.9%) | | Site 2 | | | 99.2% (97.0–99.8%) | | Site 3 | | | | There was 100% qualitative pattern agreement between sites and between technicians for Method A and Method B. While there was high qualitative agreement for Method C between sites dIFine was not able to identify low positive p-ANCA sample at Site 2 for one of the replicates across days and runs and low positive p-ANCA sample at Site 3 for couple of replicates across days. Additionally, dIFine did not correctly recognize a negative sample at Site 3 for one of the replicates across days and runs. K243776 - Page 13 of 28 {13} Site-to-Site and Technician-to-Technician Agreement – Pattern Results | Method A | Site 1 | | Site 2 | | Site 3 | | | | --- | --- | --- | --- | --- | --- | --- | --- | | | | Technician 1 | Technician 2 | Technician 1 | Technician 2 | Technician 1 | Technician 2 | | Site 1 | Technician 1 | | 100% (98.4–100.0%) | 100% (98.4–100.0%) | 100% (98.4–100.0%) | 100% (98.4–100.0%) | 100% (98.4–100.0%) | | | Technician 2 | | | 100% (98.4–100.0%) | 100% (98.4–100.0%) | 100% (98.4–100.0%) | 100% (98.4–100.0%) | | Site 2 | Technician 1 | | | | 100% (98.4–100.0%) | 100% (98.4–100.0%) | 100% (98.4–100.0%) | | | Technician 2 | | | | | 100% (98.4–100.0%) | 100% (98.4–100.0%) | | Site 3 | Technician 1 | | | | | | 100% (98.4–100.0%) | | | Technician 2 | | | | | | | | Method B | Site 1 | | Site 2 | | Site 3 | | | | --- | --- | --- | --- | --- | --- | --- | --- | | | | Technician 1 | Technician 2 | Technician 1 | Technician 2 | Technician 1 | Technician 2 | | Site 1 | Technician 1 | | 100% (98.4–100.0%) | 100% (98.4–100.0%) | 100% (98.4–100.0%) | 100% (98.4–100.0%) | 100% (98.4–100.0%) | | | Technician 2 | | | 100% (98.4–100.0%) | 100% (98.4–100.0%) | 100% (98.4–100.0%) | 100% (98.4–100.0%) | | Site 2 | Technician 1 | | | | 100% (98.4–100.0%) | 100% (98.4–100.0%) | 100% (98.4–100.0%) | | | Technician 2 | | | | | 100% (98.4–100.0%) | 100% (98.4–100.0%) | | Site 3 | Technician 1 | | | | | | 100% (98.4–100.0%) | | | Technician 2 | | | | | | | | Method C | Site 1 | Site 2 | Site 3 | | --- | --- | --- | --- | | Site 1 | | 92.7% (88.0–95.7%) | 94.4% (90.1–97.0%) | | Site 2 | | | 91.6% (86.6–94.9%) | | Site 3 | | | | There was 100% pattern agreement between sites and technicians for Method A and Method B. While there was high pattern agreement for Method C between sites dIFine was not able to identify low positive p-ANCA sample and mid positive c-ANCA sample at Site 1 across days and runs. Additionally, at Site 2 dIFine did not correctly recognize pattern for low positive p-ANCA sample, low positive c-ANCA sample and mid positive c-ANCA sample for few of the replicates across days and runs. At site 3, dIFine did not accurately identify patterns for low positive and mid positive p-ANCA samples at few replicates across days and runs. K243776 - Page 14 of 28 {14} # Anti-Neutrophil Cytoplasmic Antibodies (Formalin-Fixed) Site-to-Site and Technician-to-Technician Agreement – Qualitative Results | Method A | Site 1 | | Site 2 | | Site 3 | | | | --- | --- | --- | --- | --- | --- | --- | --- | | | | Technician 1 | Technician 2 | Technician 1 | Technician 2 | Technician 1 | Technician 2 | | Site 1 | Technician 1 | | 100% (98.4–100.0%) | 100% (98.4–100.0%) | 100% (98.4–100.0%) | 100% (98.4–100.0%) | 100% (98.4–100.0%) | | | Technician 2 | | | 100% (98.4–100.0%) | 100% (98.4–100.0%) | 100% (98.4–100.0%) | 100% (98.4–100.0%) | | Site 2 | Technician 1 | | | | 100% (98.4–100.0%) | 100% (98.4–100.0%) | 100% (98.4–100.0%) | | | Technician 2 | | | | | 100% (98.4–100.0%) | 100% (98.4–100.0%) | | Site 3 | Technician 1 | | | | | | 100% (98.4–100.0%) | | | Technician 2 | | | | | | | | Method B | Site 1 | | Site 2 | | Site 3 | | | | --- | --- | --- | --- | --- | --- | --- | --- | | | | Technician 1 | Technician 2 | Technician 1 | Technician 2 | Technician 1 | Technician 2 | | Site 1 | Technician 1 | | 100% (98.4–100.0%) | 100% (98.4–100.0%) | 100% (98.4–100.0%) | 100% (98.4–100.0%) | 100% (98.4–100.0%) | | | Technician 2 | | | 100% (98.4–100.0%) | 100% (98.4–100.0%) | 100% (98.4–100.0%) | 100% (98.4–100.0%) | | Site 2 | Technician 1 | | | | 100% (98.4–100.0%) | 100% (98.4–100.0%) | 100% (98.4–100.0%) | | | Technician 2 | | | | | 100% (98.4–100.0%) | 100% (98.4–100.0%) | | Site 3 | Technician 1 | | | | | | 100% (98.4–100.0%) | | | Technician 2 | | | | | | | | Method C | Site 1 | Site 2 | Site 3 | | --- | --- | --- | --- | | Site 1 | | 97.5% (94.7–98.9%) | 99.2% (97.0–99.8%) | | Site 2 | | | 97.9% (95.2–99.1%) | | Site 3 | | | | There was 100% qualitative result agreement in all cases across sites and technicians for Method A and Method B. While there was high qualitative agreement for Method C between sites, dIFine was not able to identify low positive c-ANCA sample at Sites 1 and 2 and low positive p-ANCA sample at Site 3 for couple of replicates across days. K243776 - Page 15 of 28 {15} Site-to-Site and Technician-to-Technician Agreement – Pattern Results | Method A | Site 1 | | Site 2 | | Site 3 | | | | --- | --- | --- | --- | --- | --- | --- | --- | | | | Technician 1 | Technician 2 | Technician 1 | Technician 2 | Technician 1 | Technician 2 | | Site 1 | Technician 1 | | 99.4% (96.9–99.9%) | 100% (97.9–100.0%) | 100% (97.9–100.0%) | 100% (97.9–100.0%) | 100% (97.9–100.0%) | | | Technician 2 | | | 99.4% (96.9–99.9%) | 99.4% (96.9–99.9%) | 99.4% (96.9–99.9%) | 99.4% (96.9–99.9%) | | Site 2 | Technician 1 | | | | 100% (97.9–100.0%) | 100% (97.9–100.0%) | 100% (97.9–100.0%) | | | Technician 2 | | | | | 100% (97.9–100.0%) | 100% (97.9–100.0%) | | Site 3 | Technician 1 | | | | | | 100% (97.9–100.0%) | | | Technician 2 | | | | | | | | Method B | Site 1 | | Site 2 | | Site 3 | | | | --- | --- | --- | --- | --- | --- | --- | --- | | | | Technician 1 | Technician 2 | Technician 1 | Technician 2 | Technician 1 | Technician 2 | | Site 1 | Technician 1 | | 99.4% (96.9–99.9%) | 100% (97.9–100.0%) | 100% (97.9–100.0%) | 100% (97.9–100.0%) | 100% (97.9–100.0%) | | | Technician 2 | | | 99.4% (96.9–99.9%) | 99.4% (96.9–99.9%) | 99.4% (96.9–99.9%) | 99.4% (96.9–99.9%) | | Site 2 | Technician 1 | | | | 100% (97.9–100.0%) | 100% (97.9–100.0%) | 100% (97.9–100.0%) | | | Technician 2 | | | | | 100% (97.9–100.0%) | 100% (97.9–100.0%) | | Site 3 | Technician 1 | | | | | | 100% (97.9–100.0%) | | | Technician 2 | | | | | | | | Method C | Site 1 | Site 2 | Site 3 | | --- | --- | --- | --- | | Site 1 | | 98.3% (95.0–99.4%) | 98.3% (95.2–99.4%) | | Site 2 | | | 98.9% (95.9–99.7%) | | Site 3 | | | | While there was 100% pattern agreement in most cases across sites and technicians for Method A and Method B, there was one discrepant result for mid-positive p-ANCA sample for Technician 2 where the pattern was wrongly identified for one of the three replicates at Site 1. While there was high pattern agreement for Method C between sites dIFine was not able to recognize pattern for low positive c-ANCA sample at Site 2 for few of the replicates across days. K243776 - Page 16 of 28 {16} 2. Linearity: Six samples including: two low positive samples (~1:20-1:40 endpoint) with one anti-PR3 (c-ANCA) and one anti-MPO (p-ANCA), two mid positive samples (~1:80-1:160 endpoint) with one anti-PR3 (c-ANCA) and one anti-MPO (p-ANCA), and two high positive samples (> 1:160 endpoint) with one anti-PR3 (c-ANCA) and one anti-MPO (p-ANCA), were each assayed at a 1:20 screening dilution, as well as at various corresponding serial dilutions. For Methods A and B, the fluorescence intensity was recorded at each dilution using a scale of "4" (very intense) and "0" (no fluorescence). Qualitative results and pattern results were interpreted by all three methods. For each the Anti-Neutrophil Cytoplasmic Antibodies (Ethanol-fixed) and the Anti-Neutrophil Cytoplasmic Antibodies (Formalin-fixed) test system, the endpoints for each sample and each method, and the sample dilutions and the associated fluorescence intensities for Method A and Method B (Method C cannot provide an intensity grade, only a positive, negative, or uncertain call) are summarized in the tables below: Anti-Neutrophil Cytoplasmic Antibodies (Ethanol-Fixed) | Endpoint Titers across Methods | | | | | | --- | --- | --- | --- | --- | | Sample | Method A | Method B | Method C | | | | | | UNC as (-) | UNC as (+) | | Low Positive PR3 (c-ANCA) | 1:40 | 1:40 | 1:40 | 1:40 | | Low Positive MPO (p-ANCA) | 1:40 | 1:40 | 1:80 | 1:160 | | Medium Positive PR3 (c-ANCA) | 1:80 | 1:80 | 1:80 | 1:80 | | Medium Positive MPO (p-ANCA) | 1:80 | 1:160 | 1:160 | 1:320 | | High Positive PR3 (c-ANCA) | 1:1280 | 1:2560 | 1:2560 | 1:5120 | | High Positive MPO (p-ANCA) | 1:640 | 1:640 | 1:640 | 1:640 | The sample dilutions and associated intensity grade results | Sample | | 1:20 | 1:40 | 1:80 | 1:160 | 1:320 | 1:640 | 1:1280 | 1:2560 | 1:5120 | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | Low Positive c-ANCA | Method A | 2+ | 1+ | 0 | 0 | 0 | | | | | | | Method B | 2+ | 1+ | 0 | 0 | 0 | | | | | | Low Positive p-ANCA | Method A | 2+ | 1+ | 0 | 0 | 0 | | | | | | | Method B | 2+ | 1+ | 0 | 0 | 0 | | | | | | Mid Positive c-ANCA | Method A | 3+ | 2+ | 1+ | 0 | 0 | | | | | | | Method B | 3+ | 2+ | 1+ | 0 | 0 | | | | | | Mid Positive p-ANCA | Method A | 3+ | 2+ | 1+ | 0 | 0 | | | | | | | Method B | 4+ | 3+ | 2+ | 1+ | 0 | | | | | | High Positive c-ANCA | Method A | 4+ | 4+ | 4+ | 4+ | 3+ | 2+ | 1+ | 0 | 0 | | | Method B | 4+ | 4+ | 4+ | 4+ | 4+ | 3+ | 2+ | 1+ | 0 | | High Positive p-ANCA | Method A | 4+ | 4+ | 4+ | 3+ | 2+ | 1+ | 0 | 0 | 0 | | | Method B | 4+ | 4+ | 4+ | 3+ | 2+ | 1+ | 0 | 0 | 0 | K243776 - Page 17 of 28 {17} Anti-Neutrophil Cytoplasmic Antibodies (Formalin-Fixed) | Endpoint Titers across Methods | | | | | | --- | --- | --- | --- | --- | | Sample | Method A | Method B | Method C | | | | | | UNC as (-) | UNC as (+) | | Low Positive PR3 (c-ANCA) | 1:40 | 1:40 | 1:40 | 1:80 | | Low Positive MPO (p-ANCA) | 1:40 | 1:40 | 1:40 | 1:40 | | Medium Positive PR3 (c-ANCA) | 1:80 | 1:80 | 1:80 | 1:80 | | Medium Positive MPO (p-ANCA) | 1:160 | 1:160 | 1:80 | 1:160 | | High Positive PR3 (c-ANCA) | 1:1280 | 1:1280 | 1:640 | 1:1280 | | High Positive MPO (p-ANCA) | 1:640 | 1:640 | 1:160 | 1:640 | The sample dilutions and associated intensity grade results | Sample | | 1:20 | 1:40 | 1:80 | 1:160 | 1:320 | 1:640 | 1:1280 | 1:2560 | 1:5120 | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | Low Positive c-ANCA | Method A | 2+ | 1+ | 0 | 0 | 0 | | | | | | | Method B | 2+ | 1+ | 0 | 0 | 0 | | | | | | Low Positive p-ANCA | Method A | 2+ | 1+ | 0 | 0 | 0 | | | | | | | Method B | 2+ | 1+ | 0 | 0 | 0 | | | | | | Mid Positive c-ANCA | Method A | 3+ | 2+ | 1+ | 0 | 0 | | | | | | | Method B | 3+ | 2+ | 1+ | 0 | 0 | | | | | | Mid Positive p-ANCA | Method A | 4+ | 3+ | 2+ | 1+ | 0 | | | | | | | Method B | 4+ | 3+ | 2+ | 1+ | 0 | | | | | | High Positive c-ANCA | Method A | 4+ | 4+ | 4+ | 4+ | 3+ | 2+ | 1+ | 0 | 0 | | | Method B | 4+ | 4+ | 4+ | 4+ | 3+ | 2+ | 1+ | 0 | 0 | | High Positive p-ANCA | Method A | 4+ | 4+ | 4+ | 3+ | 2+ | 1+ | 0 | 0 | 0 | | | Method B | 4+ | 4+ | 4+ | 3+ | 2+ | 1+ | 0 | 0 | 0 | # 3. Analytical Specificity/Interference: The panel of samples include: two low positive samples ( $\sim 1:20-1:40$ endpoint) with one anti-PR3 (c-ANCA) and one anti-MPO (p-ANCA); two mid positive samples ( $\sim 1:80-1:160$ endpoint) with one anti-PR3 (c-ANCA) and one anti-MPO (p-ANCA); two high positive samples ( $>1:160$ endpoint) with one anti-PR3 (c-ANCA) and one anti-MPO (p-ANCA); and two negative samples, were spiked with two different concentrations (Low Concentration and High Concentration) of 19 different interferents as outlined in the table below. All specimens were assayed in triplicate by each of the Anti-Neutrophil Cytoplasmic Antibodies (Ethanol-fixed) and the Anti-Neutrophil Cytoplasmic Antibodies (Formalin-fixed) at a 1:20 dilution and interpreted by all three methods. Qualitative results and pattern results were interpreted by two technicians for Methods A and B, and by a single dIFine instrument for Method C. | Endogenous Substances | | | | --- | --- | --- | | Substance | Low Concentration | High Concentration | | Bilirubin (Unconjugated) | 0.02 mg/mL | 0.15 mg/mL | | Cholesterol (total) | 1.5 mg/mL | 2.2 mg/mL | | Triglycerides (total) | 1.0 mg/mL | 2.5 mg/mL | | Albumin | 35.0 mg/mL | 52.0 mg/mL | | Hemoglobin | 100.0 mg/mL | 200.0 mg/mL | | RF | 200.0 U/mL | 400.0 U/mL | K243776 - Page 18 of 28 {18} | Exogenous Substances | | | | --- | --- | --- | | Substance | Low Concentration | High Concentration | | Intralipids | 2.0 mg/mL | 20.0 mg/mL | | Cyclophosphamide | 0.183 mg/mL | 0.549mg/mL | | Ibuprofen | 0.073 mg/mL | 0.219 mg/mL | | Hydroxychloroquine | 0.006 mg/mL | 0.024 mg/mL | | Simvastatin | 0.0000277 mg/mL | 0.000083 mg/mL | | Prednisone | 0.000033 mg/mL | 0.000099 mg/mL | | Azathioprine | 0.00086 mg/mL | 0.00258 mg/mL | | Diltiazem | 0.0003 mg/mL | 0.0009 mg/mL | | Mycophenolate mofetil | 0.012 mg/mL | 0.048 mg/mL | | Rituximab | 0.5 mg/mL | 2.0 mg/mL | | Belimumab | 2.0 mg/mL | 8.0 mg/mL | | Methotrexate | 0.454 mg/mL | 1.36 mg/mL | | Naproxen | 0.12 mg/mL | 0.36 mg/mL | For both Anti-Neutrophil Cytoplasmic Antibodies (Ethanol-fixed) and the Anti-Neutrophil Cytoplasmic Antibodies (Formalin-fixed) test systems, none of the interferents affected the expected qualitative results for any samples when read by Methods A and B. Method C yielded qualitatively correct results for the Anti-Neutrophil Cytoplasmic Antibodies (Formalin-fixed) test systems, however there were few instances of discrepant results for Anti-Neutrophil Cytoplasmic Antibodies (Ethanol-fixed) as noted below: - The low positive PR3 (c-ANCA) sample showed effect of interference for Diltiazem spiked at a low concentration. - The low positive PR3 (c-ANCA) sample showed effect of interference for cholesterol spiked at a low concentration. - The low positive PR3 (c-ANCA) sample showed effect of interference for Prednisone spiked at a low concentration. - The low positive MPO (p-ANCA) sample showed effect of interference for hemoglobin spiked at a high concentration. 4. Assay Reportable Range: Not Applicable. 5. Traceability, Stability, Expected Values (Controls, Calibrators, or Methods): a. Traceability: A recognized standard for anti-neutrophil cytoplasmic antibodies is not available. b. Stability Sample Stability: Three serum samples including one negative sample, one low positive c-ANCA sample and one low positive p-ANCA sample were assayed using the ZEUS ANCA kits (Ethanol-fixed and Formalin-fixed) to assess the stability of ANCAs in serum samples K243776 - Page 19 of 28 {19} stored for various conditions. Multiple aliquots of the three serum samples were prepared and one set of aliquot served as the baseline or time T₀ time-point. The remaining aliquots were placed at room temperature and -20°C. An aliquot from each storage temperature was assayed in triplicate, at a 1:20 screening dilution by one technician using Methods A and B and one dIFine instrument for Method C, at the following time points: - Room temperature and 2-8°C storage: 1 day, 3 days, 7 days, and 14 days - -20°C storage (frozen): 7 days, 14 days, and 21 days For the samples stored at room temperature, there was no change in qualitative outcome or pattern over the 14-day testing period, for Methods A and B on both the Ethanol-fixed and Formalin-fixed slides. Likewise, for the frozen samples, there was no change in the qualitative outcome, or the pattern observed over the 21-day period, regardless of the method interpretation on both the Ethanol-fixed and Formalin-fixed slides. This study demonstrates that the ANCA antibody in the human serum samples are stable for a minimum of 7 days at room temperature and 14 days when frozen. ## Reagent Stability: The reagent stability was assessed based on the real-time stability study in which a panel of samples includes: two low positive samples (~1:20-1:40 endpoint) with one anti-PR3 (c-ANCA) and one anti-MPO (p-ANCA), two mid positive samples (~1:80-1:160 endpoint) with one anti-PR3 (c-ANCA) and one anti-MPO (p-ANCA), two high positive samples (> 1:160 endpoint) with one anti-PR3 (c-ANCA) and one anti-MPO (p-ANCA), and two negative samples, were assayed in singlicate at a 1:20 screening dilution to represent the baseline or time-point T₀. Different lots of ANCA Ethanol-fixed or ANCA Formalin-fixed reagent kits stored at 2-8°C were tested at different time-points. The data were analyzed for the qualitative results and patterns agreement results comparing to the baseline. The results support that the reagent kits of the Anti-Neutrophil Cytoplasmic Antibodies (Ethanol-Fixed) and Anti-Neutrophil Cytoplasmic Antibodies (Ethanol-Fixed) are stable up to 7 months when stored at 2-8°C. ## 6. Detection Limit: Not Applicable. ## 7. Assay Cut-Off: It is recommended to perform the test using a screening dilution of 1:20 followed by serial dilutions for semi-quantitative determinations. ## B Comparison Studies: ### 1. Method Comparison with Predicate Device: In order to show comparable performance between the ANCA Ethanol-fixed or Formalin-fixed IFA test system and the predicate ANCA Ethanol-Fixed or Formalin-fixed IFA test system, 583 clinically characterized serum specimens that included 223 serum specimens with ANCA Associated Vasculitis (AAV) and 360 serum specimens with control diseases were tested. These 583 clinically characterized specimens included in the study are outlined in the table below. K243776 - Page 20 of 28 {20} | Diseases | | N | | --- | --- | --- | | ANCA Associated Vasculitis | Microscopic Polyangiitis (MPA) | 123 | | | Granulomatosis with Polyangiitis (GPA) | 92 | | | Eosinophilic Granulomatosis with Polyangiitis (eGPA) | 8 | | Control Diseases | Non-ANCA Associated Vasculitis | 30 | | | Autoimmune Thyroid | 30 | | | Rheumatoid Arthritis | 30 | | | Chronic Kidney Disease | 30 | | | Systemic Sclerosis | 30 | | | Systemic Lupus Erythematosus | 30 | | | Dermatomyositis/Polymyositis | 30 | | | Inflammatory Bowel Disease | 30 | | | Sinusitis/Allergic Rhinitis | 30 | | | Infectious Disease | 30 | | | Asthma/COPD | 30 | | | Cancer | 30 | | Total | | 583 | All 583 samples were tested by three different technicians at a 1:20 screening dilution using the candidate device, i.e., the Anti-Neutrophil Cytoplasmic Antibodies (Ethanol-fixed) or Anti-Neutrophil Cytoplasmic Antibodies (Formalin-fixed) test system. The samples were also tested by a single technician using the predicate device, i.e., NOVA Lite DAPI ANCA (Ethanol) or NOVA Lite DAPI ANCA (Formalin). Qualitative and pattern results for the candidate assay were interpreted by two technicians at three different sites for Methods A and B, and by a single dIFine instrument at each laboratory for Method C (6 total technicians and 3 total dIFine instruments) while qualitative and pattern results for the predicate were interpreted at a single laboratory for Method A. The results are summarized in the tables below. ## Anti-Neutrophil Cytoplasmic Antibodies (Ethanol-Fixed) ANCA (Ethanol-fixed) vs NOVA Lite DAPI ANCA (Ethanol) – Qualitative Results | | % Positive Agreement (95% CI) | % Negative Agreement (95% CI) | % Total Agreement (95% CI) | | --- | --- | --- | --- | | Method A vs Predicate | 92.3% (91.1-93.4%) | 88.5% (86.8-90.1%) | 90.8% (89.8-91.7%) | | Method B vs Predicate | 93.0% (91.8-94.0%) | 87.7% (85.9-89.3%) | 90.8% (89.8-91.8%) | | Method C vs Predicate (UNC as positive) | 94.4% (92.8-95.6%) | 83.5% (80.6-96.1%) | 90.0% (88.5-91.3%) | | Method C vs Predicate (UNC as negative) | 91.8% (90.0-93.3%) | 90.0% (87.6-92.0%) | 91.1% (89.7-92.3%) | ANCA (Ethanol-fixed) vs NOVA Lite DAPI ANCA (Ethanol) – Pattern Results | | % Agreement (95% CI) | | --- | --- | | Method A vs Predicate | 79.7% (78.4-81.1%) | | Method B vs Predicate | 79.6% (78.2-80.9%) | | Method C vs Predicate | 78.4% (76.4-80.2%) | K243776 - Page 21 of 28 {21} Between-Method Agreement of the ANCA (Ethanol-fixed) - Qualitative Results | | % Positive Agreement (95% CI) | % Negative Agreement (95% CI) | % Total Agreement (95% CI) | | --- | --- | --- | --- | | Method A vs Method B | 99.4% (98.9-99.6%) | 97.3% (86.8-90.1%) | 98.5% (98.1-98.9%) | | Method A vs Method C (UNC as positive) | 99.3% (98.9-99.6%) | 91.0% (89.4-92.4%) | 96.0% (95.2-96.6%) | | Method A vs Method C (UNC as negative) | 96.7% (95.8-97.4%) | 97.4% (96.4-98.1%) | 97.0% (96.4-97.5%) | | Method B vs Method C (UNC as positive) | 99.5% (99.1-99.7%) | 93.0% (91.4-94.1%) | 96.9% (96.2-97.4%) | | Method B vs Method C (UNC as negative) | 96.1% (95.1-96.8%) | 98.2% (97.4-98.8%) | 96.9% (96.3-97.4%) | Between-Methods Agreement of the ANCA (Ethanol-fixed) - Pattern Results | | % Agreement (95% CI) | | --- | --- | | Method A vs Method B | 95.5% (94.5-96.3%) | | Method A vs Method C | 85.5% (83.9-87.0%) | | Method B vs Method C | 86.5% (85.0-87.9%) | # Anti-Neutrophil Cytoplasmic Antibodies (Formalin-Fixed) ANCA (Formalin-fixed) vs NOVA Lite DAPI ANCA (Formalin) - Qualitative Results | | % Positive Agreement (95% CI) | % Negative Agreement (95% CI) | % Total Agreement (95% CI) | | --- | --- | --- | --- | | Method A vs Predicate | 92.0% (90.5-93.3%) | 95.6% (94.6-96.4%) | 94.8% (92.6-96.2%) | | Method B vs Predicate | 91.9% (90.3-93.2%) | 95.5% (94.5-96.3%) | 94.0% (93.2-94.8%) | | Method C vs Predicate (UNC as positive) | 88.9% (86.4-91.1%) | 97.7% (96.6-98.5%) | 94.2% (93.0-95.2%) | | Method C vs Predicate (UNC as negative) | 84.9% (82.1-87.4%) | 98.0% (97.0-98.7%) | 92.8% (91.5-93.9%) | ANCA (Formalin-fixed) vs NOVA Lite DAPI ANCA (Formalin) - Qualitative Results | | % Agreement (95% CI) | | --- | --- | | Method A vs Predicate | 93.2% (92.3-94.0%) | | Method B vs Predicate | 93.3% (92.4-94.1%) | | Method C vs Predicate | 91.5% (90.1-92.7%) | Between-Method of the ANCA (Formalin-fixed) - Qualitative Results | | % Positive Agreement (95% CI) | % Negative Agreement (95% CI) | % Total Agreement (95% CI) | | --- | --- | --- | --- | | Method A vs Method B | 99.2% (98.6-99.6%) | 99.5% (99.1-99.7%) | 99.4% (99.1-99.6%) | | Method A vs Method C (UNC as positive) | 92.9% (91.4-94.1%) | 99.5% (99.1-99.7%) | 96.9% (96.3-97.4%) | | Method A vs Method C (UNC as negative) | 88.8% (87.0-90.4%) | 99.8% (99.5-99.9%) | 95.5% (94.7-96.1%) | | Method B vs Method C (UNC as positive) | 93.1% (91.6-94.3%) | 99.7% (99.3-99.8%) | 97.1% (96.5-97.6%) | | Method B vs Method C (UNC as negative) | 88.9% (87.2-90.5%) | 99.9% (99.7-100.0%) | 95.6% (94.9-96.2%) | K243776 - Page 22 of 28 {22} Between-Methods of the ANCA (Ethanol-fixed) – Pattern Results | | % Agreement (95% CI) | | --- | --- | | Method A vs Method B | 95.5% (94.5-96.3%) | | Method A vs Method C | 85.5% (83.9–87.0%) | | Method B vs Method C | 86.5% (85.0-87.9%) | 2. Matrix Comparison: Not Applicable. C Clinical Studies: 1. Clinical Sensitivity and Clinical Specificity: The samples tested in the Method Comparison study (see Section above) were evaluated for clinical performance of each of the Anti-Neutrophil Cytoplasmic Antibodies (Ethanol-fixed) and the Anti-Neutrophil Cytoplasmic Antibodies (Formalin-fixed) test systems. A total of 583 samples including 223 AAV samples and 360 control disease samples were tested with each test system at three sites. The clinical performance of the candidate devices is summarized in the tables below: Anti-Neutrophil Cytoplasmic Antibodies (Ethanol-Fixed) Percent positives at each site and each technician by different interpretation methods are presented in the tables below: | Site 1 | | | | | | | | | --- | --- | --- | --- | --- | --- | --- | --- | | ANCA (Ethanol-Fixed) | Method A | | Method B | | Method C | | | | | | Tech A | Tech B | Tech A | Tech B | UNC (-) | UNC (+) | | Diagnosis | N | % Pos | % Pos | % Pos | % Pos | % Pos | % Pos | | Target Disease (ANCA Associated Vasculitis) | | | | | | | | | MPA | 123 | 97.6% | 98.4% | 98.4% | 98.4% | 98.4% | 99.2% | | GPA | 92 | 92.4% | 92.4% | 92.4% | 92.4% | 89.1% | 92.4% | | eGPA | 8 | 87.5% | 100.0% | 87.5% | 87.5% | 87.5% | 100.0% | | Control Disease | | | | | | | | | Non-AAV* | 30 | 40.0% | 40.0% | 40.0% | 40.0% | 43.3% | 50.0% | | Autoimmune Thyroid | 30 | 36.7% | 36.7% | 40.0% | 36.7% | 40.0% | 46.7% | | Rheumatoid Arthritis | 30 | 53.3% | 53.3% | 53.3% | 53.3% | 50.0% | 56.7% | | Chronic Kidney Disease | 30 | 46.7% | 43.3% | 46.7% | 43.3% | 36.7% | 50.0% | | Systemic Sclerosis | 30 | 56.7% | 56.7% | 56.7% | 56.7% | 56.7% | 56.7% | | SLE* | 30 | 83.3% | 83.3% | 83.3% | 83.3% | 86.7% | 90.0% | | DM/PM* | 30 | 26.7% | 26.7% | 26.7% | 26.7% | 23.3% | 30.0% | | IBD* | 30 | 40.0% | 40.0% | 40.0% | 40.0% | 40.0% | 40.0% | | Sinusitis/Allergic Rhinitis | 30 | 13.3% | 13.3% | 13.3% | 13.3% | 20.0% | 20.0% | | Infectious Disease | 30 | 26.7% | 30.0% | 26.7% | 26.7% | 26.7% | 46.7% | | Asthma/COPD | 30 | 13.3% | 13.3% | 13.3% | 13.3% | 13.3% | 13.3% | | Cancer | 30 | 16.7% | 13.3% | 16.7% | 16.75 | 13.3% | 23.3% | | Total | 583 | | | | | | | * Non-AAV: Non-ANCA Associated Vasculitis; SLE: Systemic Lupus Erythematosus; DM/PM: Dermatomyositis/Polymyositis; IBD: Inflammatory Bowel Disease K243776 - Page 23 of 28 {23} | Site 2 | | | | | | | | | --- | --- | --- | --- | --- | --- | --- | --- | | ANCA (Ethanol-Fixed) | Method A | | Method B | | Method C | | | | | | Tech A | Tech B | Tech A | Tech B | UNC (-) | UNC (+) | | Diagnosis | N | % Pos | % Pos | % Pos | % Pos | % Pos | % Pos | | Target Disease (ANCA Associated Vasculitis) | | | | | | | | | MPA | 123 | 98.4% | 98.4% | 98.4% | 98.4% | 98.4% | 99.2% | | GPA | 92 | 90.2% | 89.1% | 90.2% | 89.1% | 89.1% | 90.2% | | eGPA | 8 | 100.0% | 100.0% | 100.0% | 100.0% | 100.0% | 100.0% | | Control Disease | | | | | | | | | Non-AAV* | 30 | 40.0% | 40.0% | 40.0% | 43.3% | 36.7% | 43.3% | | Autoimmune Thyroid | 30 | 26.7% | 30.0% | 33.3% | 26.7% | 26.7% | 36.7% | | Rheumatoid Arthritis | 30 | 50.0% | 50.0% | 50.0% | 50.0% | 46.7% | 50.0% | | Chronic Kidney Disease | 30 | 36.7% | 40.0% | 40.0% | 40.0% | 36.7% | 43.3% | | Systemic Sclerosis | 30 | 53.3% | 56.7% | 56.7% | 53.3% | 53.3% | 60.0% | | SLE* | 30 | 80.0% | 80.0% | 80.0% | 80.0% | 76.7% | 83.3% | | DM/PM* | 30 | 13.3% | 13.3% | 16.7% | 13.3% | 16.7% | 20.0% | | IBD* | 30 | 36.7% | 36.7% | 36.7% | 36.7% | 40.0% | 43.3% | | Sinusitis/Allergic Rhinitis | 30 | 6.7% | 6.7% | 6.7% | 6.7% | 6.7% | 6.7% | | Infectious Disease | 30 | 20.0% | 20.0% | 20.0% | 20.0% | 20.0% | 26.7 | | Asthma/COPD | 30 | 10.0% | 10.0% | 10.0% | 10.0% | 10.0% | 10.0% | | Cancer | 30 | 16.7% | 16.7% | 16.7% | 16.7% | 13.3% | 16.7% | | Total | 583 | | | | | | | * Non-AAV: Non-ANCA Associated Vasculitis; SLE: Systemic Lupus Erythematosus; DM/PM: Dermatomyositis/Polymyositis; IBD: Inflammatory Bowel Disease | Site 3 | | | | | | | | | --- | --- | --- | --- | --- | --- | --- | --- | | ANCA (Ethanol-Fixed) | Method A | | Method B | | Method C | | | | | | Tech A | Tech B | Tech A | Tech B | UNC (-) | UNC (+) | | Diagnosis | N | % Pos | % Pos | % Pos | % Pos | % Pos | % Pos | | Target Disease (ANCA Associated Vasculitis) | | | | | | | | | MPA | 123 | 99.2% | 99.2% | 99.2% | 99.2% | 99.2% | 99.2% | | GPA | 92 | 95.7% | 95.7% | 95.7% | 94.6% | 91.3% | 93.5% | | eGPA | 8 | 100.0% | 100.0% | 100.0% | 100.0% | 100.0% | 100.0% | | Control Disease | | | | | | | | | Non-AAV* | 30 | 40.0% | 40.0% | 40.0% | 43.3% | 36.7% | 43.3% | | Autoimmune Thyroid | 30 | 26.7% | 30.0% | 33.3% | 26.7% | 26.7% | 36.7% | | Rheumatoid Arthritis | 30 | 50.0% | 50.0% | 50.0% | 50.0% | 46.7% | 50.0% | | Chronic Kidney Disease | 30 | 36.7% | 40.0% | 40.0% | 40.0% | 36.7% | 43.3% | | Systemic Sclerosis | 30 | 53.3% | 56.7% | 56.7% | 53.3% | 53.3% | 60.0% | | SLE* | 30 | 80.0% | 80.0% | 80.0% | 80.0% | 76.7% | 83.3% | | DM/PM* | 30 | 13.3% | 13.3% | 16.7% | 13.3% | 16.7% | 20.0% | | IBD* | 30 | 36.7% | 36.7% | 36.7% | 36.7% | 40.0% | 43.3% | | Sinusitis/Allergic Rhinitis | 30 | 6.7% | 6.7% | 6.7% | 6.7% | 6.7% | 6.7% | | Infectious Disease | 30 | 20.0% | 20.0% | 20.0% | 20.0% | 20.0% | 26.7% | | Asthma/COPD | 30 | 10.0% | 10.0% | 10.0% | 10.0% | 10.0% | 10.0% | | Cancer | 30 | 16.7% | 16.7% | 16.7% | 16.7% | 13.3% | 16.7% | | Total | 583 | | | | | | | * Non-AAV: Non-ANCA Associated Vasculitis; SLE: Systemic Lupus Erythematosus; DM/PM: Dermatomyositis/Polymyositis; IBD: Inflammatory Bowel Disease K243776 - Page 24 of 28 {24} When the same samples were also run on the predicate device, there is a high correlation for % positive samples in the AAV and the control disease group between the candidate device across sites and technicians when compared with the predicate device. Clinical Performance Summary for ANCA Ethanol-Fixed | Site | Tech | Interpretation | AAV* (n = 223) | CD** (n = 360) | | --- | --- | --- | --- | --- | | | | | % Sensitivity (95% CI) | % Specificity 95% CI | | 1 | A | Method A | 95.1% (91.4-97.2%) | 62.2% (57.1-67.1%) | | | | Method B | 95.5% (91.9-97.6%) | 61.9% (56.8-66.8%) | | | B | Method A | 96.0% (92.5-97.9%) | 62.5% (57.4-67.3%) | | | | Method B | 95.5% (91.9-97.6%) | 62.5% (57.4-67.3%) | | | dIFine | Method C - UNC as (-) | 94.2% (90.3-96.6%) | 62.5% (57.4-67.3%) | | | | Method C - UNC as (+) | 96.4% (93.1-98.2%) | 56.7% (51.5-61.7%) | | 2 | A | Method A | 95.1% (91.4-97.2%) | 67.5% (62.5-72.1%) | | | | Method B | 95.1% (91.4-97.2%) | 66.1% (61.1-70.8%) | | | B | Method A | 94.6% (90.8-96.9%) | 66.7% (61.6-71.3%) | | | | Method B | 94.6% (90.8-96.9%) | 66.9% (61.9-71.6%) | | | dIFine | Method C - UNC as (-) | 94.6% (90.8-96.9%) | 68.1% (63.1-72.7%) | | | | Method C - UNC as (+) | 95.5% (91.9-97.6%) | 63.3% (58.2-68.2%) | | 3 | A | Method A | 97.8% (94.9-99.0%) | 60.0% (54.9-64.9%) | | | | Method B | 97.8% (94.9-99.0%) | 57.2 (52.1-62.2%) | | | B | Method A | 97.8% (94.9-99.0%) | 60.3 (55.1-65.2%) | | | | Method B | 97.3% (94.3-98.8%) | 56.9 (51.8-62.0%) | | | dIFine | Method C - UNC as (-) | 96.0% (92.5-97.9%) | 61.4 (56.3-66.3%) | | | | Method C - UNC as (+) | 96.9% (93.7-98.5%) | 54.2(49.0-59.2%) | Samples were run on predicate manual interpretation method at Site 1 and the clinical performance was evaluated. The clinical performance between the candidate device and the predicate device at the same site using the same technician showed the comparable clinical sensitivity and specificity as shown below. | Interpretation (Site 1, Tech A, Method A) | AAV (n = 223) | CD (n = 360) | | --- | --- | --- | | | Sensitivity% (95% CI) | Specificity% (95% CI) | | ANCA (Ethanol-Fixed) | 95.1% (91.4-97.2%) | 62.2% (57.1-67.1%) | | INOVA ANCA (Ethanol) | 92.8% (88.7-95.5%) | 61.4% (56.3-66.3%) | # Anti-Neutrophil Cytoplasmic Antibodies (Formalin-Fixed) Percent positives at each site and each technician by different interpretation methods are presented in the tables below. K243776 - Page 25 of 28 {25} | Site 1 | | | | | | | | | --- | --- | --- | --- | --- | --- | --- | --- | | ANCA (Formalin-Fixed) | Method A | | Method B | | Method C | | | | | | Tech A | Tech B | Tech A | Tech B | UNC (-) | UNC (+) | | Diagnosis | N | % Pos | % Pos | % Pos | % Pos | % Pos | % Pos | | Target Disease (ANCA Associated Vasculitis) | | | | | | | | | MPA | 123 | 89.4% | 90.2% | 91.1% | 90.2% | 87.8% | 88.6% | | GPA | 92 | 83.7% | 83.7% | 83.7% | 82.6% | 80.4% | 83.7% | | eGPA | 8 | 87.5% | 87.5% | 87.5% | 87.5% | 87.5% | 87.5% | | Control Disease | | | | | | | | | Non-AAV* | 30 | 3.3% | 3.3% | 3.3% | 3.3% | 0.0% | 3.3% | | Autoimmune Thyroid | 30 | 6.6% | 6.7% | 6.7% | 6.7% | 3.3% | 3.3% | | Rheumatoid Arthritis | 30 | 13.3% | 13.3% | 13.3% | 13.3% | 13.3% | 13.3% | | Chronic Kidney Disease | 30 | 6.7% | 6.7% | 6.7% | 6.7% | 0.0% | 3.3% | | Systemic Sclerosis | 30 | 13.3% | 13.3% | 13.3% | 13.3% | 6.7% | 10.0% | | SLE* | 30 | 43.3% | 43.3% | 36.7% | 36.7% | 40.0% | 40.0% | | DM/PM* | 30 | 0.0% | 0.0% | 0.0% | 0.0% | 0.0% | 0.0% | | IBD* | 30 | 0.0% | 0.0% | 0.0% | 0.0% | 0.0% | 0.0% | | Sinusitis/Allergic Rhinitis | 30 | 0.0% | 0.0% | 0.0% | 0.0% | 0.0% | 0.0% | | Infectious Disease | 30 | 0.0% | 0.0% | 0.0% | 0.0% | 0.0% | 0.0% | | Asthma/COPD | 30 | 0.0% | 0.0% | 0.0% | 0.0% | 0.0% | 0.0% | | Cancer | 30 | 3.3% | 3.3% | 3.3% | 3.3% | 3.3% | 3.3% | | Total | 583 | | | | | | | * Non-AAV: Non-ANCA Associated Vasculitis; SLE: Systemic Lupus Erythematosus; DM/PM: Dermatomyositis/Polymyositis; IBD: Inflammatory Bowel Disease | Site 2 | | | | | | | | | --- | --- | --- | --- | --- | --- | --- | --- | | ANCA (Formalin-Fixed) | Method A | | Method B | | Method C | | | | | | Tech A | Tech B | Tech A | Tech B | UNC (-) | UNC (+) | | Diagnosis | N | % Pos | % Pos | % Pos | % Pos | % Pos | % Pos | | Target Disease (ANCA Associated Vasculitis) | | | | | | | | | MPA | 123 | 87.0% | 84.6% | 87.0% | 84.6% | 82.9% | 85.4% | | GPA | 92 | 85.9% | 82.6% | 85.9% | 80.4% | 73.9% | 78.3% | | eGPA | 8 | 100.0% | 100.0% | 100.0% | 100.0% | 100.0% | 100.0% | | Control Disease | | | | | | | | | Non-AAV* | 30 | 3.3% | 3.3% | 3.3% | 3.3% | 3.3% | 3.3% | | Autoimmune Thyroid | 30 | 6.7% | 6.7% | 6.7% | 6.7% | 6.7% | 6.7% | | Rheumatoid Arthritis | 30 | 10.0% | 10.0% | 10.0% | 10.0% | 6.7 | 10.0% | | Chronic Kidney Disease | 30 | 0.0% | 0.0% | 0.0% | 0.0% | 0.0% | 0.0% | | Systemic Sclerosis | 30 | 10.0% | 10.0% | 10.0% | 10.0% | 10.0% | 10.0% | | SLE* | 30 | 40.0% | 30.0% | 36.7% | 40.0% | 33.3% | 43.3% | | DM/PM* | 30 | 0.0% | 0.0% | 0.0% | 0.0% | 0.0% | 0.0% | | IBD* | 30 | 0.0% | 0.0% | 0.0% | 0.0% | 0.0% | 0.0% | | Sinusitis/Allergic Rhinitis | 30 | 0.0% | 0.0% | 0.0% | 0.0% | 0.0% | 0.0% | | Infectious Disease | 30 | 0.0% | 0.0% | 0.0% | 0.0% | 0.0% | 0.0% | | Asthma/COPD | 30 | 0.0% | 0.0% | 0.0% | 0.0% | 0.0% | 0.0% | | Cancer | 30 | 3.3% | 0.0% | 3.3% | 0.0% | 0.0% | 0.0% | | Total | 583 | | | | | | | * Non-AAV: Non-ANCA Associated Vasculitis; SLE: Systemic Lupus Erythematosus. DM/PM: Dermatomyositis/Polymyositis; IBD: Inflammatory Bowel Disease K243776 - Page 26 of 28 {26} | Site 3 | | | | | | | | | --- | --- | --- | --- | --- | --- | --- | --- | | ANCA (Formalin-Fixed) | Method A | | Method B | | Method C | | | | | | Tech A | Tech B | Tech A | Tech B | UNC (-) | UNC (+) | | Diagnosis | N | % Pos | % Pos | % Pos | % Pos | % Pos | % Pos | | Target Disease (ANCA Associated Vasculitis) | | | | | | | | | MPA | 123 | 93.5% | 95.1% | 94.3% | 95.1% | 83.7% | 89.4% | | GPA | 92 | 92.4% | 92.4% | 93.5% | 93.5% | 82.6% | 84.8% | | eGPA | 8 | 100.0% | 100.0% | 100.0% | 100.0% | 87.5% | 87.5% | | Control Disease | | | | | | | | | Non-AAV* | 30 | 3.3% | 0.0% | 3.3% | 3.3% | 0.0% | 0.0% | | Autoimmune Thyroid | 30 | 13.3% | 10.0% | 13.3% | 13.3% | 3.3% | 6.7% | | Rheumatoid Arthritis | 30 | 23.3% | 23.3% | 23.3% | 23.3% | 13.3% | 16.7% | | Chronic Kidney Disease | 30 | 6.7% | 6.7% | 6.7% | 6.7% | 0.0% | 0.0% | | Systemic Sclerosis | 30 | 20.0% | 20.0% | 20.0% | 20.0% | 10.0% | 13.3% | | SLE* | 30 | 53.3% | 53.3% | 53.3% | 53.3% | 40.0% | 43.3% | | DM/PM* | 30 | 6.7% | 10.0% | 3.3% | 6.7% | 0.0% | 0.0% | | IBD* | 30 | 6.7% | 6.7% | 6.7% | 6.7% | 0.0% | 0.0% | | Sinusitis/Allergic Rhinitis | 30 | 0.0% | 0.0% | 0.0% | 0.0% | 0.0% | 0.0% | | Infectious Disease | 30 | 6.7% | 6.7% | 6.7% | 6.7% | 0.0% | 0.0% | | Asthma/COPD | 30 | 6.7% | 6.7% | 6.7% | 6.7% | 0.0% | 0.0% | | Cancer | 30 | 6.7% | 6.7% | 6.7% | 6.7% | 3.3% | 3.3% | | Total | 583 | | | | | | | * Non-AAV: Non-ANCA Associated Vasculitis; SLE: Systemic Lupus Erythematosus. DM/PM: Dermatomyositis/Polymyositis; IBD: Inflammatory Bowel Disease When the same samples were run on the predicate device, there is a high correlation for % positive samples in the AAV and the control disease group between the candidate device across sites and technicians when compared with the predicate device. Clinical Performance for the Anti-Neutrophil Cytoplasmic Antibodies (Formalin-Fixed) | Site | Tech | Interpretation | AAV (n = 223) | CD (n = 360) | | --- | --- | --- | --- | --- | | | | | Sensitivity% (95% CI) | Specificity% (95% CI) | | 1 | A | Method A | 87.0% (82.0-91.0%) | 92.5% (89.3-94.8%) | | | | Method B | 87.9% (83.0-91.5%) | 93.1% (90.0-95.3%) | | | B | Method A | 87.4% (82.5-91.2%) | 92.5% (89.3-94.8%) | | | | Method B | 87.0% (82.0-91.0%) | 93.1% (90.0-95.3%) | | | dIFine | Method C - UNC as (-) | 84.8% (79.5-88.9%) | 94.4% (91.6-96.4%) | | | | Method C - UNC as (+) | 86.5% (81.4-90.4%) | 93.6% (90.6-95.7%) | | 2 | A | Method A | 87.0% (82.0-90.8%) | 93.9% (90.9-95.9%) | | | | Method B | 87.0% (82.0-90.8%) | 94.2% (91.3-96.2%) | | | B | Method A | 84.3% (79.0-88.5%) | 95.0% (92.2-96.8%) | | | | Method B | 83.4% (78.0-87.7%) | 94.2% (91.3-96.2%) | | | dIFine | Method C - UNC as (-) | 79.8% (74.0-84.6%) | 95.0% (92.2-96.8%) | | | | Method C - UNC as (+) | 83.0% (77.5-87.3%) | 93.9% (90.9-95.9%) | | 3 | A | Method A | 93.3% (89.2-95.9%) | 87.2% (83.4-90.3%) | | | | Method B | 94.2% (90.3-96.6%) | 87.5% (83.7-90.5%) | | | B | Method A | 94.2% (90.3-96.6%) | 87.5% (83.7-90.5%) | | | | Method B | 94.6% (90.8-96.9%) | 87.2% (83.4-90.3%) | | | dIFine | Method C - UNC as (-) | 83.4% (78.0-87.7%) | 94.2% (91.3-96.2%) | | | | Method C - UNC as (+) | 87.4% (82.5-91.2%) | 93.1% (90.0-95.3%) | K243776 - Page 27 of 28 {27} Samples were analyzed using the predicate manual interpretation method at Site 1 and the clinical performance was evaluated. The clinical performance between the candidate device and the predicate device at the same site using the same technician showed comparable clinical sensitivity and specificity as shown below. | Interpretation (Site 1, Tech A, Method A) | AAV (n = 223) | CD (n = 360) | | --- | --- | --- | | | Sensitivity% (95% CI) | Specificity% (95% CI) | | ANCA (Formalin-Fixed) | 88.8% (84.0-92.3%) | 90.6% (87.1-93.2%) | | INOVA ANCA (Formalin) | 87.0% (82.0-91.0%) | 92.5% (89.3-94.8%) | ## D Clinical Cut-Off: Not applicable ## E Expected Values/Reference Range: One hundred and fifty serum samples were acquired from apparently healthy donors within the United States. Each sample was tested at the screening dilution of 1:20, then scanned by ZEUS dIFine and the qualitative results (i.e., Positive, Negative, Uncertain) were determined via Methods A, B, and C. The percent positivity was determined for the 1:20 screening data and the results are summarized below in the table: Anti-Neutrophil Cytoplasmic Antibodies (Ethanol-Fixed) | Method | N | Number of Positives | % Positives | Number of Negatives | % Negatives | Number of Uncertain | % Uncertain | | --- | --- | --- | --- | --- | --- | --- | --- | | A | 150 | 16* | 10.7 | 134 | 89.3 | NA | NA | | B | 150 | 16** | 10.7 | 134 | 89.3 | NA | NA | | C | 150 | 19*** | 12.7 | 125 | 83.3 | 6*** | 4.0 | *6 of 16 ANA positive by HEp-2 IFA; **7 of 19 ANA positive by HEp-2 IFA; ***1 of 6 ANA positive by HEp-2 IFA Anti-Neutrophil Cytoplasmic Antibodies (Formalin-Fixed) | Method | N | Number of Positives | % Positives | Number of Negatives | % Negatives | Number of Uncertain | % Uncertain | | --- | --- | --- | --- | --- | --- | --- | --- | | A | 150 | 2* | 1.3 | 148 | 98.7 | NA | NA | | B | 150 | 2** | 1.3 | 148 | 98.7 | NA | NA | | C | 150 | 1 | 0.7 | 148 | 98.7 | 1** | 0.7 | *1 of 2 ANA positive by HEp-2 IFA; ** 1 of 1 ANA positive by HEp-2 IFA ## VIII Proposed Labeling: The labeling supports the finding of substantial equivalence for this device. ## IX Conclusion: The submitted information in this premarket notification is complete and supports a substantial equivalence decision. K243776 - Page 28 of 28