Predicate Devices

Reference Devices

AI/ML (Artificial Intelligence / Machine Learning)

Yes
The document explicitly states that the HELIOS Pattern Recognition software uses SVM (Support Vector Machine) technology, which is a type of machine learning algorithm.

Therapeutic

No.
The device is an in vitro diagnostic assay used as an aid for diagnosis, not for treating a condition.

Diagnostic

Yes

The device is an "in vitro diagnostic assay" used as an "aid for the diagnosis of ANCA-associated vasculitides (AAV)".

SaMD (Software as a Medical Device)

No

The device description explicitly states it is an "indirect immunofluorescence assay utilizing human neutrophil granulocyte coated slides" and is used with "human serum". It also mentions the "HELIOS® AUTOMATED IFA SYSTEM" which is a hardware system. While it includes software for image processing and pattern recognition, it is not solely software.

IVD (In Vitro Diagnostic)

Yes, this device is an IVD (In Vitro Diagnostic).

Here's why:

Explicitly Stated: The "Intended Use / Indications for Use" and "Device Description" sections both explicitly state that this is an "in vitro diagnostic assay".
Purpose: The device is designed to determine the presence of anti-neutrophil cytoplasmic autoantibodies (ANCA) in human serum, which is a biological sample taken from the body.
Diagnostic Aid: The intended use is "as an aid for the diagnosis of ANCA-associated vasculitides (AAV) in conjunction with other clinical and laboratory findings." This clearly indicates its role in the diagnostic process.
Laboratory Use: The intended user is a "trained operator in a clinical laboratory setting," which is typical for IVD devices.

PCCP (Predetermined Change Control Plan) Authorized

No
The letter does not state that the FDA has reviewed and approved or cleared a Predetermined Change Control Plan (PCCP) for this specific device.

Overview

Intended Use / Indications for Use

AESKUSLIDES® ANCA is an indirect immunofluorescence assay utilizing human neutrophil granulocyte coated slides, fixed with Ethanol or Formalin, as a substrate for the qualitative and semi-quantitative determination of anti-neutrophil cytoplasmic autoantibodies (ANCA) in human serum by manual microscopy or with the HELIOS® AUTOMATED IFA SYSTEM.

This in vitro diagnostic assay is used as an aid for the diagnosis of ANCA-associated vasculitides (AAV) in conjunction with other clinical and laboratory findings. All suggested results obtained with the HELIOS AUTOMATED IFA SYSTEM must be confirmed by trained personnel.

Product codes

MOB

Device Description

AESKUSLIDES ANCA is an indirect immunofluorescence assay utilizing human neutrophil granulocyte coated slides, fixed with Ethanol or Formalin, as a substrate for the qualitative and semi-quantitative determination of anti-neutrophil cytoplasmic autoantibodies (ANCA) in human serum by manual microscopy or with the HELIOS® AUTOMATED IFA SYSTEM.

This in vitro diagnostic assay is used as an aid for the diagnosis of ANCA-associated vasculitides (AAV) in coniunction with other clinical and laboratory findings. All suggested results obtained with the HELIOS AUTOMATED IFA SYSTEM must be confirmed by trained personnel.

Slides coated with human neutrophil granulocytes for autoantibody detection are fixated by two different methods: ethanol (EtOH) fixation or formalin fixation. Ethanol fixation allows cell components to move through the cells after the fixation process. Formalin fixation causes cellular components to cross-link (a movement of cellular components is abrogated and the patterns are distinct). By processing serum on both Ethanol and Formalin-fixed slides, the user can confirm if the pattern is C-, P-, or A-ANCA, according to the table below.

ANCA Formalin test is not intended to be used by itself, but in conjunction with ANCA Ethanol test.

The HELIOS Vasculitis Pattern Plus software is able to detect both C- and P-ANCA. A-ANCA are reported as undefined positive. All suggested results obtained with the HELIOS AUTOMATED IFA SYSTEM must be confirmed by trained personnel.

Each kit of ANCA Ethanol and ANCA Formalin contains (Quantity depends on product variant):

Slides, each containing 6 or 12 wells coated with human neutrophils (ethanol fixation)/ human neutrophils (formalin fixation) cells
-2.0 ml/ 4.0 ml vial containing Fluorescein (FITC) labelled Anti-human Antibody IgG conjugate in a solution of BSA, ready for use
-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 PBS

Mentions image processing

Yes

Mentions AI, DNN, or ML

Yes

Input Imaging Modality

Indirect immunofluorescence microscopy
Digital images from HELIOS AUTOMATED IFA SYSTEM

Anatomical Site

Not Found

Indicated Patient Age Range

Not Found

Intended User / Care Setting

Trained operator in a clinical laboratory setting.

Description of the training set, sample size, data source, and annotation protocol

Not Found

Description of the test set, sample size, data source, and annotation protocol

Sample set for Clinical Evaluation and Method Comparison studies:

Sample Size: 630 clinical samples.
Data Source: 510 clinical samples from 10 US BioBanks (BioChain, BioReclamationIVT, Bioserve, ConversantBio, Cureline, DiscoveryLifeSciences, iSpecimen, Precision for Medicine, ProMedDx, and Vitrologic). 120 serum samples from a German University Hospital.
Sample Selection Criteria: Selected according to diagnosis to reflect important diagnoses for the study, including specific ANCA-associated vasculitides (AAV) and various control diagnoses (Autoimmune Liver Diseases, Inflammatory Bowel Diseases, Other Rheumatic Diseases, Infections, Non-ANCA associated Vasculitides, Purpura, Leukemia, Other diagnoses like Celiac disease, Chronic kidney disease, Sinusitis, Asthma). The US sample set aimed to reflect ethnic composition (White, Black/Black African, Asian, Hispanic).
Diagnosis Criteria: Made in agreement with diagnostic standards used in the U.S. and Germany (e.g., ACR criteria). Written statement from serum suppliers available.
Sample Purity and Volume: Checked by visual inspection.
Contaminations: In-house pre-screening performed before inclusion into the study. All 630 sera passed and were found suitable.
Storage and Handling: Shipped and stored at -20°C. Thawed once upon arrival to aliquot, then re-frozen. Samples underwent 2-3 freezing-thawing cycles. Serum stability studies confirmed no influence of repeated thawing/freezing or long-term storage at -20°C on results.
Annotation Protocol: Not explicitly stated as an annotation protocol, but procedures for processing and analysis are described for different methods (manual, HELIOS automated, HELIOS user evaluation).

Endpoint Titer Comparison (for Endpoint Titer Determination):

Sample Size: Five serum samples.
Sample Selection Criteria: Samples with expected endpoint titers between 1:10 and 1:320.
Dilution Protocol: Serially diluted from 1:10 up to 1:1280, with at least 4 different dilutions around the expected endpoint titer.
Testing Protocol: For each of two methods (HELIOS Reader Confirmation and Manual), 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. Performed at three different study sites.
Annotation Protocol: Endpoint titer was reported as the reciprocal of the highest dilution at which the sample was classified as positive. Positive/Negative classification and pattern recorded for each sample dilution.

Normal Range Study:

Sample Size: 150 sera samples.
Data Source: 100 samples from Germany (56 females, 44 males, mean age 31 years, range 18-58 years, Ethnicity: German). 50 samples from the US (17 females, 33 males, mean age 34 years, range 19-62 years, Ethnicity: 30 Black, 10 Hispanic, 10 White).
Annotation Protocol: Slides processed manually according to IFU and analyzed at the microscope by two independent readers. Positive/Negative classification, ANCA pattern, and fluorescence intensity (FI) recorded.

Summary of Performance Studies (study type, sample size, AUC, MRMC, standalone performance, key results)

1. Analytical Performance Studies:

Serum Stability AESKUSLIDES ANCA:
- Study Type: Freeze-thaw stability.
- Sample Size: 10 serum samples (7 positive, 3 negative).
- Key Results: 100% positive/negative/overall agreement and pattern agreement for both ANCA Ethanol and Formalin. FI agreement was 100%, with no deviations greater than +/-1. Multiple freeze-thaw cycles had no effect.
Long Term Serum Stability AESKUSLIDES ANCA:
- Study Type: Long-term storage stability at -20°C.
- Sample Size: 9 different serum samples.
- Key Results: All positive samples found positive, negative samples negative, and patterns found correctly throughout the 14-month testing period. FI did not differ more than +/-1 level.
Precision (Within-Lab/Repeatability Between-Lab/Reproducibility):
- Study Type: Multi-site precision study (Within-lab, Between-lab, Between-Operator, Single-Operator, Instrument).
- Sample Size: 10 samples (borderline, low, medium, high positive, and negative).
- Key Results (ANCA Ethanol):
  - Within-Lab: All predetermined acceptance criteria met. % Positives/Negatives met criteria. Pattern determination consistent >95% for Method B/C, >85% for Method A for positive samples.
  - Between-Lab: All acceptance criteria met. Overall-Between-Lab agreements ranged from 92.5% to 96.5% for Method A (HELIOS), 98.5% to 100% for Method B (Reader Confirmation), and 100% for Method C (Manual).
  - Between-Operator: All acceptance criteria met. Overall-Between-Operator agreements ranged from 97.1% to 100% for Method B, and 100% for Method C.
  - Single-Operator: All acceptance criteria met. Overall-Single-Operator agreements ranged from 96.7% to 100% for Method B, and 100% for Method C.
  - Instrument Precision: All acceptance criteria met. Overall-Instrument to Instrument agreements ranged from 91.3% to 99.2%.
- Key Results (ANCA Formalin):
  - Within-Lab: All acceptance criteria met (except one reader for Method C, slightly below for FI, but tolerated). Percentage of samples within +/-1 titer level met criteria.
  - Between-Lab: All acceptance criteria met. Overall-Between-Lab agreements ranged from 89.8% to 94.7% for Method A (HELIOS), 95.8% to 98.6% for Method B (Reader Confirmation), and 98.2% to 99.4% for Method C (Manual).
  - Between-Operator: All acceptance criteria met. Overall-Between-Operator agreements ranged from 93.5% to 99% for Method B, and 97.5% to 100% for Method C.
  - Single-Operator: All acceptance criteria met. Overall-Single-Operator agreements ranged from 92.7% to 99% for Method B, and 97% to 100% for Method C.
  - Instrument Precision: All acceptance criteria met, except for negative agreement at site 3 (61.7%), due to HELIOS misinterpreting unspecific particles as positive. The report emphasizes human confirmation. Overall-Instrument to Instrument agreements ranged from 89% to 90.7%.
Lot to Lot Precision/Reproducibility Study AESKUSLIDES ANCA:
- Study Type: Reagent lot variability.
- Sample Size: 12 sera tested 10 times on each of 3 reagent lots (30 replicates per serum sample).
- Key Results (ANCA Ethanol): All agreements fulfilled criteria, ranging from 96.3% to 100% for positive, 100% for negative, 96.9% to 100% overall, 100% for pattern, and 97.8% to 100% for FI.
- Key Results (ANCA Formalin): All agreements fulfilled criteria, ranging from 99.5% to 100% for positive, 96.2% to 99.0% for negative, 98.4% to 99.7% overall, 100% for pattern, and 97.5% to 99.1% for FI.
Carry over:
- Study Type: Evaluate sample carry-over on HELIOS.
- Sample Size: Three high positive, one negative serum.
- Key Results: No carry-over observed. All samples identified correctly with expected patterns and FI.
Time Extension Study:
- Study Type: Evaluate stability of performance with varying incubation times.
- Sample Size: 10 samples (positive/negative).
- Key Results: All acceptance criteria met. Stability shown for tested incubation times (15, 30, 45 min) for both ANCA Ethanol and Formalin. FI did not deviate more than +/-1 level.
Analytical specificity (Interfering substances):
- Study Type: Evaluate interference from various substances.
- Sample Size: 14 serum samples.
- Key Results: No significant interference detected up to maximal tested concentrations of bilirubin, hemoglobin, triglycerides, RF IgM, Rituximab, Methylprednisolone, Cyclophosphamide, Methotrexate, and Azathioprine. All acceptance criteria (Pos/Neg/Overall agreement >90%, Pattern agreement >90%, FI agreement >90%) were met.
Stability Studies (Accelerated, Real Time, In Use, Transport):
- Accelerated Stability Report:
  - Study Type: Product shelf life estimation at elevated temperature (37°C).
  - Sample Size: 3 lots of complete kits tested with 8 samples.
  - Key Results (ANCA Ethanol): All acceptance criteria (all agreements >85%) met for 6 weeks at 37°C. Claimed shelf life of at least 24 months (24+3 months) at 2-8°C based on Arrhenius equation.
  - Key Results (ANCA Formalin): All acceptance criteria (all agreements >85%) met for 4 weeks at 37°C (excluding borderline samples). Claimed shelf life of at least 18 months at 2-8°C.
- Real Time Stability Report:
  - Study Type: Ongoing long-term stability at 2-8°C to confirm accelerated stability.
  - Sample Size: 3 different reagent lots with 8 serum samples.
  - Key Results: Data available for 3 months currently. All agreements met criteria (>85%). Supports stability for at least 3 months at 2-8°C.
- In Use Stability Report:
  - Study Type: Stability of opened kits.
  - Sample Size: 3 different reagent lots with 8 serum samples.
  - Key Results (ANCA Ethanol): All acceptance criteria (>85%) met for 6 weeks at 2-8°C. Claimed In Use Stability of 6 weeks.
  - Key Results (ANCA Formalin): All acceptance criteria (>85%) met for 6 weeks at 2-8°C (excluding borderline samples). Claimed In Use Stability of 6 weeks.
- Transport Stability Report:
  - Study Type: Based on accelerated stability study results.
  - Key Results: Kit can resist 37°C for at least 2 weeks. All acceptance criteria (>85%) were met from the accelerated stability study, which simulates worst-case transport.

2. Comparison Studies:

Method comparison with predicate device:
- Study Type: Comparative study with NOVA Lite ANCA (K961340).
- Sample Size: 507 serum samples (132 AAV, 375 other diseases).
- Key Results (ANCA Ethanol):
  - Diagnostic Sensitivity/Specificity: AESKUSLIDES ANCA Ethanol showed higher sensitivity (48.5% vs 36.4%) and specificity (69.3% vs 55.2%) compared to the predicate. Higher PPV (35.8% vs 22.2%) and NPV (79.3% vs 71.1%).
  - Relative Sensitivity/Specificity (Agreement with Predicate): Positive Agreement 67.1%, Negative Agreement 88.3%, Overall Agreement 79.3%.
  - Conclusion: AESKU assay is comparable or better than the predicate.
- Key Results (ANCA Formalin):
  - Diagnostic Sensitivity/Specificity: AESKUSLIDES ANCA Formalin showed higher sensitivity (50.0% vs 37.9%) and comparable specificity (90.7% vs 91.5%). PPV/NPV were comparable (65.3% vs 61.0%, 83.7% vs 80.7%).
  - Relative Sensitivity/Specificity (Agreement with Predicate): Positive Agreement 80.5%, Negative Agreement 91.8%, Overall Agreement 89.9%.
  - Conclusion: AESKU assay is comparable or better than the predicate.
Method Comparison of Method A, B, and C and clinical study:
- Study Type: Multi-site clinical comparison of manual microscopy (Method C), HELIOS with reader confirmation (Method B), and HELIOS automated classification (Method A).
- Sample Size: 630 serum samples (135 AAV, 120 characterized MPO/PR3/ANCA positive, 375 other diseases).
- Key Results (ANCA Ethanol):
  - Method C vs B: Positive Agreement 85.1-92.8%, Negative Agreement 91.3-99.8%, Overall Agreement 89.4-95.7%, Pattern Agreement 82.5-89.2%. All acceptance criteria (>80%) met.
  - Method B vs A: Positive Agreement 79.0-92.6%, Negative Agreement 98.3-99.3%, Overall Agreement 92.2-96.8%, Pattern Agreement 81.0-85.6%. Acceptance criteria met.
  - Method C vs A: Positive Agreement 70.7-83.2%, Negative Agreement 94.5-99.1%, Overall Agreement 89.7-93.0%, Pattern Agreement 77.8-81.8%. Acceptance criteria met.
  - Diagnostic Sensitivity/Specificity: Varied by site and method (e.g., Site 1 Manual Reader 1: Sens 24.4%, Spec 73.9%; Site 3 HELIOS: Sens 28.9%, Spec 80.8%).
- Key Results (ANCA Formalin):
  - Method C vs B: Positive Agreement 85.7-89.6%, Negative Agreement 86.3-97.6%, Overall Agreement 87.0-95.4%, Pattern Agreement 82.1-87.5%. All acceptance criteria (>80%) met.
  - Method B vs A: Positive Agreement 73.6-99.4%, Negative Agreement 72.2-99.2%, Overall Agreement 79.2-95.9%, Pattern Agreement 76.1-90.8%. All acceptance criteria met.
  - Method C vs A: Positive Agreement 71.0-98.7%, Negative Agreement 70.7-95.2%, Overall Agreement 77.6-90.5%, Pattern Agreement 69.7-79.6%. Pattern Agreement was slightly below criterion (69.7%), but tolerated.
  - Diagnostic Sensitivity/Specificity: Varied by site and method (e.g., Site 1 Manual Reader 1: Sens 40%, Spec 93.1%; Site 3 HELIOS: Sens 63.7%, Spec 71.7%).
Endpoint Titer comparison:
- Study Type: Repeatability and reproducibility of endpoint titer determination.
- Sample Size: 5 serum samples with varying endpoint titers.
- Key Results (ANCA Ethanol):
  - Within-Lab: % samples within +/-1 titer level: 95.1% for Method B, 95.4% for Method C (combined readers). All readers met >90% criteria.
  - Between-Lab: Titer agreements 82.3-93.3% for Method B, 87.0-98.3% for Method C. All acceptance criteria (>80%) met.
- Key Results (ANCA Formalin):
  - Within-Lab: % samples within +/-1 titer level: 94.7% for Method B, 96.1% for Method C (combined readers). One reader for Method C was slightly below 90% (89.3%), tolerated due to subjectivity.
  - Between-Lab: Titer agreements 79.0-86.7% for Method B, 80.3-93.7% for Method C. One comparison for Method B (site 1 vs 3) was slightly below 80% (79.0%), tolerated due to site differences.
- Conclusion: Good/acceptable repeatability and reproducibility for endpoint titer determination.

3. Reference Range / Expected Values:

Study Type: Normal Range Study.
Sample Size: 150 healthy donor sera.
Key Results:
- ANCA Ethanol: Reader 1 found 6 (4%) positive, Reader 2 found 3 (2%) positive.
- ANCA Formalin: Reader 1 found 6 (4%) positive, Reader 2 found 4 (2.7%) positive.
- Low number of positive samples found correlates with literature.

Key Metrics (Sensitivity, Specificity, PPV, NPV, etc.)

Method comparison with predicate device ANCA Ethanol:

% Sensitivity (95% CI): 48.5 (40.1 - 56.9)
% Specificity (95% CI): 69.3 (64.5 - 73.8)
Predictive Value of a Positive Test Result (PPV): 35.8%
Predictive Value of a Negative Test Result (NPV): 79.3%
Positive Agreement (Method Comparison Predicate vs AESKU): 67.1 (60.6 - 73)
Negative Agreement (Method Comparison Predicate vs AESKU): 88.3 (84.1 - 91.5)
Overall Agreement (Method Comparison Predicate vs AESKU): 79.3 (75.5 - 82.6)

Method comparison with predicate device ANCA Formalin:

% Sensitivity (95% CI): 50 (41.6 - 58.4)
% Specificity (95% CI): 90.7 (87.3 - 93.2)
Predictive Value of a Positive Test Result (PPV): 65.3%
Predictive Value of a Negative Test Result (NPV): 83.7%
Positive Agreement (Method Comparison Predicate vs AESKU): 80.5 (70.6 - 87.6)
Negative Agreement (Method Comparison Predicate vs AESKU): 91.8 (88.8 - 94)
Overall Agreement (Method Comparison Predicate vs AESKU): 89.9 (87 - 92.3)

Method Comparison of Method A, B and C_ANCA Ethanol_Combined Reader Agreements:

Site 1 C vs B: Positive Agreement 86.2 (82.3 - 89.3), Negative Agreement 99.5 (98.8 - 99.8), Total Agreement 95.5 (94.2 - 96.5)
Site 1 B vs A: Positive Agreement 79 (74.4 - 83.1), Negative Agreement 98.3 (97.2 - 98.9), Total Agreement 93.2 (91.6 - 94.4)
Site 1 C vs A: Positive Agreement 70.8 (66 - 75.1), Negative Agreement 99 (98.1 - 99.5), Total Agreement 90.4 (88.6 - 91.9)
Site 2 C vs B: Positive Agreement 90.6 (87.4 - 93), Negative Agreement 97.6 (96.4 - 98.5), Total Agreement 95.3 (94 - 96.4)
Site 2 B vs A: Positive Agreement 89.6 (86.3 - 92.3), Negative Agreement 99 (98 - 99.5), Total Agreement 96 (94.8 - 97)
Site 2 C vs A: Positive Agreement 82.4 (78.5 - 85.8), Negative Agreement 97.4 (96.1 - 98.3), Total Agreement 92.5 (90.9 - 93.8)
Site 3 C vs B: Positive Agreement 87.9 (84.4 - 90.6), Negative Agreement 93.6 (91.7 - 95), Total Agreement 91.7 (90 - 93.1)
Site 3 B vs A: Positive Agreement 81.6 (77.6 - 85), Negative Agreement 98.7 (97.7 - 99.3), Total Agreement 92.9 (91.4 - 94.2)
Site 3 C vs A: Positive Agreement 77.4 (73.1 - 81.1), Negative Agreement 96.3 (94.8 - 97.4), Total Agreement 90 (88.2 - 91.5)

Method Comparison of Method A, B and C_ANCA Formalin_Combined Reader Agreements:

Site 1 C vs B: Positive Agreement 86.6 (82.1 - 90.1), Negative Agreement 95.2 (93.7 - 96.4), Total Agreement 93.3 (91.7 - 94.5)
Site 1 B vs A: Positive Agreement 83.6 (78.9 - 87.4), Negative Agreement 98.8 (97.8 - 99.3), Total Agreement 95.2 (93.9 - 96.3)
Site 1 C vs A: Positive Agreement 73.1 (67.7 - 78), Negative Agreement 95 (93.4 - 96.2), Total Agreement 90.1 (88.3 - 91.6)
Site 2 C vs B: Positive Agreement 86.9 (82.6 - 90.3), Negative Agreement 94.9 (93.4 - 96.1), Total Agreement 93.1 (91.5 - 94.4)
Site 2 B vs A: Positive Agreement 79.8 (74.9 - 83.9), Negative Agreement 95.9 (94.5 - 97), Total Agreement 92.1 (90.4 - 93.4)
Site 2 C vs A: Positive Agreement 74.9 (69.6 - 79.5), Negative Agreement 93.6 (91.9 - 95), Total Agreement 89.3 (87.4 - 90.9)
Site 3 C vs B: Positive Agreement 89.1 (85.3 - 92), Negative Agreement 90.8 (88.7 - 92.5), Total Agreement 90.3 (88.5 - 91.8)
Site 3 B vs A: Positive Agreement 99 (97.4 - 99.6), Negative Agreement 77.1 (74.2 - 79.7), Total Agreement 83.8 (81.6 - 85.7)
Site 3 C vs A: Positive Agreement 95.6 (92.8 - 97.3), Negative Agreement 71.8 (68.9 - 74.7), Total Agreement 78.2 (75.9 - 80.4)

Predicate Device(s)

K961340

Reference Device(s)

K153117

Predetermined Change Control Plan (PCCP) - All Relevant Information

Not Found

Regulation Number and Section

§ 866.5660 Multiple autoantibodies immunological test system.

(a)
Identification. A multiple autoantibodies immunological test system is a device that consists of the reagents used to measure by immunochemical techniques the autoantibodies (antibodies produced against the body's own tissues) in serum and other body fluids. Measurement of multiple autoantibodies aids in the diagnosis of autoimmune disorders (disease produced when the body's own tissues are injured by autoantibodies).(b)
Classification. Class II (performance standards).

Summary

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March 23, 2018

Aesku.Diagnostics GmbH & Co. KG Sandra Reuter Regulatory Affairs Manager Mikro-Forum-Ring 2 Wendelsheim, Rheinland-Pfalz55234 Germany

Re: K172461

Trade/Device Name: AESKUSLIDES® ANCA Ethanol AESKUSLIDES® ANCA Formalin

Regulation Number: 21 CFR 866.5660 Regulation Name: Multiple autoantibodies immunological test system Regulatory Class: Class II Product Code: MOB Dated: July 31, 2017 Received: August 14, 2017

Dear Sandra Reuter:

We have reviewed your Section 510(k) premarket notification of intent to market the device referenced above and have determined the device is substantially equivalent (for the indications for use stated in the enclosure) to legally marketed predicate devices marketed in interstate commerce prior to May 28, 1976, the enactment date of the Medical Device Amendments, or to devices that have been reclassified in accordance with the provisions of the Federal Food, Drug, and Cosmetic Act (Act) that do not require approval of a premarket approval application (PMA). You may, therefore, market the device, subject to the general controls provisions of the Act. The general controls provisions of the Act include requirements for annual registration. listing of devices, good manufacturing practice, labeling, and prohibitions against misbranding and adulteration. Please note: CDRH does not evaluate information related to contract liability warranties. We remind you, however, that device labeling must be truthful and not misleading.

If your device is classified (see above) into either class II (Special Controls) or class III (PMA), it may be subject to additional controls. Existing major regulations affecting your device can be found in the Code of Federal Regulations, Title 21, Parts 800 to 898. In addition, FDA may publish further announcements concerning your device in the Federal Register.

Please be advised that FDA's issuance of a substantial equivalence determination does not mean that FDA has made a determination that your device complies with other requirements of the Act or any Federal

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statutes and regulations administered by other Federal agencies. You must comply with all the Act's requirements, including, but not limited to: registration and listing (21 CFR Part 807); labeling (21 CFR Part 801 and Part 809); medical device reporting of medical device-related adverse events) (21 CFR 803); good manufacturing practice requirements as set forth in the quality systems (QS) regulation (21 CFR Part 820); and if applicable, the electronic product radiation control provisions (Sections 531-542 of the Act); 21 CFR 1000-1050.

Also, please note the regulation entitled, "Misbranding by reference to premarket notification" (21 CFR Part 807.97). For questions regarding the reporting of adverse events under the MDR regulation (21 CFR Part 803), please go to http://www.fda.gov/MedicalDevices/Safety/ReportaProblem/default.htm for the CDRH's Office of Surveillance and Biometrics/Division of Postmarket Surveillance.

For comprehensive regulatory information about medical devices and radiation-emitting products, including information about labeling regulations, please see Device Advice (https://www.fda.gov/MedicalDevices/DeviceRegulationandGuidance/) and CDRH Learn (http://www.fda.gov/Training/CDRHLearn). Additionally, you may contact the Division of Industry and Consumer Education (DICE) to ask a question about a specific regulatory topic. See the DICE website (http://www.fda.gov/DICE) for more information or contact DICE by email (DICE@fda.hhs.gov) or phone (1-800-638-2041 or 301-796-7100).

Sincerely.

Lea Carrington Director Division of Immunology and Hematology Devices Office of In Vitro Diagnostics and Radiological Health Center for Devices and Radiological Health

Enclosure

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510(k) Summary

A. 510(k) Number: K172461
B. Purpose for Submission: New Device

C. Measurand:

Anti-neutrophil cytoplasmic autoantibodies (ANCA)

D. Type of Test:

Qualitative and semi-quantitative, indirect immunofluorescence

E. Applicant:

AESKU.Diagnostics GmbH & Co.KG Mikro-Forum-Ring 2 55234 Wendelsheim Germany

Contact Person: Dr. Sandra Reuter Regulatory Affairs registrations@aesku.com p: +49 6734 9622 0

prepared on March 23rd, 2018

F. Proprietary and Established Names:

AESKUSLIDES® ANCA Ethanol AESKUSLIDES® ANCA Formalin

G. Regulatory Information:

Regulation section: §CFR 866.5660 - Multiple autoantibodies immunological test system
Classification: Class II
Product code: MOB - Anti-neutrophil cytoplasmic antibodies (ANCA)
Clinical use Immunology (82)

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H. Intended Use:

1. Intended use(s):
  AESKUSLIDES® ANCA is an indirect immunofluorescence assay utilizing human neutrophil granulocyte coated slides, fixed with Ethanol or Formalin, as a substrate for the qualitative and semi-quantitative determination of anti-neutrophil cytoplasmic autoantibodies (ANCA) in human serum by manual microscopy or with the HELIOS® AUTOMATED IFA SYSTEM.

This in vitro diagnostic assay is used as an aid for the diagnosis of ANCA-associated vasculitides (AAV) in conjunction with other clinical and laboratory findings. All suggested results obtained with the HELIOS AUTOMATED IFA SYSTEM must be confirmed by trained personnel.

1. Indication(s) for use: Same as intended use
1. Special conditions for use statement(s):
- 1. For prescription use only

This device is only for use with reagents that are indicated for use with the device.
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.

5.For use only by manual microscopy or with HELIOS® AUTOMATED IFA SYSTEM.

l. Device Description:

AESKUSLIDES ANCA is an indirect immunofluorescence assay utilizing human neutrophil granulocyte coated slides, fixed with Ethanol or Formalin, as a substrate for the qualitative and semi-quantitative determination of anti-neutrophil cytoplasmic autoantibodies (ANCA) in human serum by manual microscopy or with the HELIOS® AUTOMATED IFA SYSTEM.

This in vitro diagnostic assay is used as an aid for the diagnosis of ANCA-associated vasculitides (AAV) in coniunction with other clinical and laboratory findings. All suggested results obtained with the HELIOS AUTOMATED IFA SYSTEM must be confirmed by trained personnel.

Slides coated with human neutrophil granulocytes for autoantibody detection are fixated by two different methods: ethanol (EtOH) fixation or formalin fixation. Ethanol fixation allows cell components to move through the cells after the fixation process. Formalin fixation causes cellular components to cross-link (a movement of cellular components is abrogated and the patterns are distinct). By processing serum on both Ethanol and Formalin-fixed slides, the user can confirm if the pattern is C-, P-, or A-ANCA, according to the table below.

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Ethanol-fixed Result	Formalin-Fixed Result	Pattern
Cytoplasmic (C-ANCA)	Cytoplasmic (C-ANCA)	C-ANCA
Perinuclear (P-ANCA)	Cytoplasmic (C-ANCA)	P-ANCA
Perinuclear (P-ANCA)	Negative / unclear	Confirm with ANA
Mixed (P-ANCA + C-ANCA)	Negative / weak positive	A-ANCA
Very Perinuclear	A-ANCA	A-ANCA

ANCA Formalin test is not intended to be used by itself, but in conjunction with ANCA Ethanol test.

The HELIOS Vasculitis Pattern Plus software is able to detect both C- and P-ANCA. A-ANCA are reported as undefined positive. All suggested results obtained with the HELIOS AUTOMATED IFA SYSTEM must be confirmed by trained personnel.

Each kit of ANCA Ethanol and ANCA Formalin contains (Quantity depends on product variant):

Slides, each containing 6 or 12 wells coated with human neutrophils (ethanol fixation)/ human neutrophils (formalin fixation) cells
-2.0 ml/ 4.0 ml vial containing Fluorescein (FITC) labelled Anti-human Antibody IgG conjugate in a solution of BSA, ready for use
-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 PBS

Standard Ref.	Description	Tests
54.100.US	ANCA Ethanol (12 wells)	120
54.101.US	ANCA Formalin (12 wells)	120
54.050.US	ANCA Ethanol (6 wells)	60
54.051.US	ANCA Formalin (6 wells)	60
54.100.US.Demo	ANCA Ethanol (12 wells)	24
54.100.US.Bulk5	ANCA Ethanol (12 wells)	600
54.100.US.Bulk10	ANCA Ethanol (12 wells)	1200
54.101.US.Demo	ANCA Formalin (12 wells)	24
54.101.US.Bulk5	ANCA Formalin (12 wells)	600
54.101.US.Bulk10	ANCA Formalin (12 wells)	1200

6

Not provided in the kit:

| Ref. | Reagent | | Quantity
/ Volume | Description | Ready
to use |
|-------|-----------------------|----|----------------------|--------------------------------------------------------------------------------------------------------------------------------|-----------------|
| EBIFA | Evans
Blue
0.2% | 1x | 3ml | Capped white: Blue coloured
solution
Containing: PBS, Evans
Blue.
Dilute the Evans Blue 0.2%
1:3000 in 1x WBIFA | NO |

HELIOS AUTOMATED IFA SYSTEM (K153117) or equivalent manual microscope.

J. Substantial Equivalence Information:

1. Predicate device name(s):
  NOVA Lite ANCA
1. Predicate 510(k) number(s): K961340
1. Comparison with predicate device:

Table 7: comparison table with predicate device

| | Item | Predicate
NOVA Lite ANCA | AESKUSLIDES® ANCA |
|--------------|------------------------|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Similarities | Intended Use | NOVA Lite® ANCA is an indirect
immunofluorescent assay for the
screening and semi-quantitative
determination of anti-neutrophil
cytoplasmic antibodies (ANCA) in
human serum. The presence of
antineutrophil cytoplasmic
antibodies can be used in
conjunction with other serological
tests and clinical findings aids in
the assessment of various
systemic vasculitides. | AESKUSLIDES ANCA is an indirect
immunofluorescence assay utilizing human
neutrophil granulocyte coated slides, fixed with
Ethanol or Formalin, as a substrate for the
qualitative and semi-quantitative determination of
anti-neutrophil cytoplasmic autoantibodies (ANCA)
in human serum by manual microscopy or with the
HELIOS® AUTOMATED IFA SYSTEM.
This in vitro diagnostic assay is used as an aid for
the diagnosis of ANCA-associated vasculitides
(AAV) in conjunction with other clinical and
laboratory findings.
All suggested results obtained with the HELIOS
AUTOMATED IFA SYSTEM must be
confirmed by trained personnel. |
| | Methology | Immunofluorescence assay (IFA) | same as Predicate |
| | Procedure | Standard IFA technique
qualitative and semi-quantitative
titer | same as Predicate |
| | Results | Serum | same as Predicate |
| | Samples Matrix | anti-neutrophil cytoplasmic
autoantibodies (ANCA) | same as Predicate |
| | Analyte | ethanol-fixed human neutrophil /
formalin-fixed human neutrophil | same as Predicate |
| | Antigen | FITC | same as Predicate |
| | Fluorescence
Marker | cANCA Positive, pANCA Positive
and one negative control | same as Predicate |
| | Controls | Anti-Human IgG Conjugate | same as Predicate |
| | conjugate | 1:20 | same as Predicate |
| | Screening
dilution | 2 - 8 °C | same as Predicate |
| | Storage | | |

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	Slides	6 or 12 wells coated with antigen	same as Predicate
Differences	Shelf life	18 months	24 months for ANCA Ethanol
18 months for ANCA Formalin
	Manual
Interpretation of
result	Manual fluorescence microscopy	Manual fluorescence microscopy or with HELIOS
w/ trained operator verification
	Automated
interpretation of
results	N/A	HELIOS w/ trained operator verification

K. Standard/ Guidance Document Referenced (if applicable):

Table 8: List of Standards / Guidance Documents

#	Standards Title	Version
1	ISO 14971 - Medical Devices - Application of risk management to medical
devices	Second
Edition
2	IEC 62366 - Medical Devices - Part 1: Application Of Usability Engineering To
Medical Devices [Including CORRIGENDUM 1 (2016)]	Edition 1.0
3	15223-1 - Medical Devices - Symbols To Be Used With Medical Device Labels,
Labelling, and Information to be supplied - Part 1: General Requirements	Second
Edition
4	Interference Testing in Clinical Chemistry	EP07-A2
5	Evaluation of Detection Capability for Clinical Laboratory Measurement
Procedures	EP17-A2
6	Evaluation of Stability of In Vitro Diagnostic Reagents	EP25-A
7	Defining, Establishing and Verifying Reference Intervals in the Clinical
Laboratory	EP28-A3c
8	Evaluation of the Linearity of Quantitative Measurement Procedures A
Statistical Approach	EP06-A
9	Procedures for the Handling and Processing of Blood Specimens for Common
Laboratory Tests; Approved Guideline-Fourth Edition	GP44-A4
10	Guidance for Industry and FDA Staff: Recommendations for Anti-Nuclear
Antibody (ANA) Test System Premarket (510k) Submissions	-
11	Factors to Consider Regarding Benefit-Risk in Medical Device Product
Availability, Compliance, and Enforcement Decisions	-
12	Applying Human Factors and Usability Engineering to Medical Devices	-

L. Test Principle

AESKUSLIDES® ANCA is an indirect fluorescent antibody assay utilizing human neutrophil granulocyte coated slides, fixed with Ethanol or Formalin, as a substrate for the qualitative and semi-quantitative determination of anti-neutrophil cytoplasmic autoantibodies (ANCA) in human serum by manual microscopy or with HELIOS® AUTOMATED IFA SYSTEM. This in vitro diagnostic assay is used as an aid for the diagnosis of ANCA-associated vascultitides (AAV) in conjunction with other clinical and laboratory findings. All suggested results obtained with the HELIOS® AUTOMATED IFA SYSTEM must be confirmed by trained personnel.

Manual interpretation of AESKUSLIDES® ANCA Ethanol:

The two main patterns seen on an ethanol-fixed substrate are perinuclear (P-ANCA) and cytoplasmic (C-ANCA):

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Image /page/8/Picture/1 description: The image is a logo for AESKU.DIAGNOSTICS. The logo features a green square with a white image of a medical symbol on the left. To the right of the square is the text "AESKU.DIAGNOSTICS" in a gray sans-serif font. Below the text is the tagline "THE DIAGNOSTIC TOOL THAT WORKS" in a smaller, lighter gray font.

-C-ANCA presents as coarse speckled cytoplasmic fluorescence, often with accentuated staining between the nuclear lobes. This pattern is characteristic for antibodies reacting with PR3.
-P-ANCA presents as perinuclear staining with or without nuclear extension. This pattern is usually characteristic for antibodies reacting with MPO. Note that anti-nuclear antibody (ANA) positive samples (containing anti-DNA/histones) may react with the nuclei of ethanol-fixed neutrophils, causing nuclear staining, and may mask or mimic the P-ANCA pattern(s).

A third pattern, less commonly seen, is called atypical ANCA (A-ANCA or X-ANCA):

। A-ANCA presents as a cytoplasmic and perinuclear or very perinuclear staining on ethanol-fixed neutrophil substrate and usually becomes negative on formalin fixed substrate.

Manual interpretation of AESKUSLIDES® ANCA Formalin:

On formalin fixed substrate, both MPO and PR3 antibodies appear as coarse cytoplasmic granular staining with interlobular accentuation.

Results from both AESKUSLIDES® ANCA Ethanol and AESKUSLIDES® ANCA Formalin provide further information on the antibodies present in the serum.

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).

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

AESKU recommends a screening dilution of 1:20, followed by serial dilutions for semiquantitative determinations and suggests each laboratory establish its own screening dilution and titration scheme based on its population and instrumentation.

Qualitative evaluation

A serum dilution is considered negative for ANCA antibodies if the cells exhibit 85%.

-All patterns have to be found correctly.
-Pattern Agreement: > 85%
-FI is allowed to differ maximum + or - 1 from the expected value. FI Agreement: > 85%.

Results

All samples fulfilled the above criteria. No differences were observed between sera thawed a single time and sera that had undergone 4 cycles of freeze/thawing:

-Positive/Negative/Overall Agreement was 100% for both AESKUSLIDES® ANCA Ethanol and Formalin. All positive samples have been found positive, all negative samples have been found negative.
-Pattern Agreement was 100% for both AESKUSLIDES® ANCA Ethanol and Formalin. All patterns have been found as expected.
FI agreement was 100% for both AESKUSLIDES® ANCA Ethanol and Formalin. -No deviations of fluorescence intensities greater than +/-1 from the initial value have been observed.

Conclusions

All criteria are fulfilled in each test. The results show that multiple freeze-thaw cycles have no effect on test results.

14

Long Term Serum Stability AESKUSLIDES® ANCA

To show that long term storage of serum samples at -20°C has no effect on performance and results of AESKUSLIDES® ANCA Ethanol and ANCA Formalin.

Procedure:

Nine different serum samples have been aliquoted and stored at -20°C for a time period of at least 12 months. Frozen aliquots of the different sera have been thawed and assayed on AESKUSLIDES® ANCA at the indicated time points. All tests have been performed manually according to the IFU and subsequently analyzed at the microscope.

| Sample
ID | AESKUSLIDES ANCA Ethanol
Expected Result | | | AESKUSLIDES ANCA Formalin
Expected Result | | |
|--------------|---------------------------------------------|---------|----|----------------------------------------------|---------|----|
| | Pos/Neg | Pattern | FI | Pos/Neg | Pattern | FI |
| 1 | Pos | P | 3 | Pos | C | 3 |
| 2 | Pos | C | 3 | Pos | C | 3 |
| 3 | Pos | A | 1 | Neg | Neg | 0 |
| 4 | Pos | C | 1 | Pos | C | 1 |
| 5 | Neg | 0 | 0 | Neg | 0 | 0 |
| 6 | Neg | 0 | 0 | Neg | 0 | 0 |
| 7 | Neg | 0 | 0 | Neg | 0 | 0 |
| 8 | Pos | P | 3 | Pos | P | 2 |
| 9 | Pos | P | 3 | Pos | C | 3 |

Table 12: Serum sample set for long term serum stability

Acceptance Criteria:

Positive sera have to be found positive and negative sera have to be found negative throughout the whole testing period. Correct patterns have to be found. The fluorescence intensity (FI) is allowed to differ maximum +/- 1 level from the expected value at each test time point.

Results:

Positive samples have been found positive, negative samples have been found negative, and all patterns have been found correctly at all test time points. FI did not differ more than +/- 1 level from the expected values.

AESKUSLIDES ANCA Ethanol
Sample
ID	month 0		month 3		month 6		month 8		month 10		month 14
	Pos/Neg	FI	Pos/Neg	FI	Pos/Neg	FI	Pos/Neg	FI	Pos/Neg	FI	Pos/Neg	FI
1	Pos	3	Pos	3	Pos	3	Pos	3	Pos	3	Pos	3
2	Pos	3	Pos	3	Pos	3	Pos	3	Pos	3	Pos	3
3	Pos	1	Pos	1	Pos	1	Pos	1	Pos	1	Pos	1
4	Pos	1	Pos	1	Pos	1	Pos	1	Pos	1	Pos	1
5	Neg	0	Neg	0	Neg	0	Neg	0	Neg	0	Neg	0
6	Neg	0	Neg	0	Neg	0	Neg	0	Neg	0	Neg	0
7	Neg	0	Neg	0	Neg	0	Neg	0	Neg	0	Neg	0
8	Pos	3	Pos	3	Pos	3	Pos	3	Pos	3	Pos	3
9	Pos	3	Pos	3	Pos	3	Pos	3	Pos	3	Pos	3

Table 13: Long Term Serum Stability AESKUSLIDES ANCA Ethanol

15

AESKUSLIDES ANCA Formalin
Sample
ID	month 0		month 2		month 5		month 8		month 10		month 12
	Pos/Neg	FI	Pos/Neg	FI	Pos/Neg	FI	Pos/Neg	FI	Pos/Neg	FI	Pos/Neg	FI
1	Pos	3	Pos	3	Pos	3	Pos	2	Pos	2	Pos	2
2	Pos	3	Pos	3	Pos	3	Pos	3	Pos	3	Pos	3
3	Neg	0	Neg	0	Neg	0	Neg	0	Neg	0	Neg	0
4	Pos	1	Pos	1	Pos	1	Pos	1	Pos	1	Pos	1
5	Neg	0	Neg	0	Neg	0	Neg	0	Neg	0	Neg	0
6	Neg	0	Neg	0	Neg	0	Neg	0	Neg	0	Neg	0
7	Neg	0	Neg	0	Neg	0	Neg	0	Neg	0	Neg	0
8	Pos	1	na	na	Pos	2	na	na	Pos	1	Pos	2
9	Pos	3	Pos	3	Pos	3	Pos	3	Pos	3	Pos	3

Table 14: Long Term Serum Stability AESKUSLIDES® ANCA Formalin

Conclusion:

All acceptance criteria have been fulfilled for the tested sera. The results show that long term storage of sera at -20°C over a time period of at least 12 months has no effect on performance and test results of AESKUSLIDES® ANCA.

Precision

a) Within-Lab Precision/Repeatability AESKUSLIDES® ANCA

Within-Lab Precision of AESKUSLIDES® ANCA Ethanol and ANCA Formalin processed on HELIOS® (Method A - HELIOS suggestions and Method B - Reader Confirmation of HELIOS images) and processed manually (Method C), based on CLSI Guideline EP12-A2.

Sample Set

10 samples were assayed on AESKUSLIDES® ANCA Ethanol and Formalin. The sample set includes borderline, low, medium, high positive, and negative samples. The samples are characterized in the following table:

| Sample

ID	Result	Pattern	Grading
1	Pos	P	high positive
2	Pos	P	high positive
3	Pos	P	medium positive
4	Pos	P	medium positive
5	Pos	C	borderline positive
6	Pos	C	high positive
7	Pos	C	borderline positive
8	Pos	C	medium positive
9	Neg	negative	negative
10	Neg	negative	negative

Table 15: Sample table for Precision study_ANCA Ethanol_Within-Lab Samnle AESKUSLIDES ANCA Ethanol

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Sample ID	AESKUSLIDES ANCA Formalin
	Result	Pattern	Grading
1	Pos	C	medium positive
2	Pos	C	high positive
3	Pos	C	medium positive
4	Pos	C	high positive
5	Pos	C	low positive
6	Pos	C	high positive
7	Pos	C	medium positive
8	Pos	C	high positive
9	Neg	negative	negative
10	Neg	negative	negative

Table 16: Sample table for Precision study ANCA Formalin Within-Lab

Procedure

10 samples have been tested on five days, two runs per day, three replicates per sample per run, resulting in 30 data points for each sample.

This Within-lab precision study was done for three different Methods:

| Method A (HELIOS) | Processing of slides and image recording at HELIOS +
HELIOS result suggestion |
|-----------------------------------|-------------------------------------------------------------------------------------------------|
| Method B (Reader
Confirmation) | HELIOS images recorded in Method A + result analysis
by two independent readers |
| Method C (Manual) | Manual processing of slides and result analysis at the
microscope by two independent readers |

All runs have been performed according to the respective IFUs. 1 positive and 1 negative control (kit controls) were included in each run. Samples were tested at 1:20 dilution. Results were analyzed by two independent readers.

Data Analysis

Positive/Negative Classification and Pattern was recorded for each sample in each run and for each Method. Pattern suggestion in Method A (HELIOS) is provided by the Vasculitis Pattern Plus software tool.

For Method C (Manual performance) fluorescence intensity was also reported.

As an estimate of the imprecision of the three methods A, B and C the percentages of positive and negative results will be calculated for each sample:

% Positive (number of positive calls divided by the total number of data points for each sample)
% Negative (number of negative calls divided by the total number of data points per sample)

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Image /page/17/Picture/0 description: The image contains the logo for AESKU.DIAGNOSTICS. The logo consists of a green square with a white stylized image of a zipper inside. To the right of the square is the text "AESKU.DIAGNOSTICS" in a gray sans-serif font, with the word "AESKU" being larger and bolder than "DIAGNOSTICS". Below the text is the phrase "THE DIAGNOSTIC TOOL THAT WORKS" in a smaller, lighter gray font.

Results of the two readers (Method B/C) will be calculated and displayed separately as well as combined.

Acceptance Criteria

Positive sera have to be found positive, and negative sera have to be found negative*.

Reported FI is allowed to differ max. ± 1 level within the study ●
Calculations should meet the following criteria:

| Estimate of imprecision | HELIOS | Reader
Confirmation | Manual |
|-------------------------------------------------------------------|--------|------------------------|--------|
| % Positives of positive samples (excluding borderline
samples) | > 80% | > 90% | > 90% |
| % Negatives of negative samples | > 80% | > 90% | > 90% |

borderline samples are samples with analyte concentration near the cutoff. These samples are very low positive. They can be evaluated also negative in certain cases, depending on the reader, the subjective manner of result analysis and normal test variances.

For Method A we accept lower percentage of positive/negative results for positive and negative samples, respectively, because we state in the IFU that all results generated by the HELIOS (Method A) must be confirmed by trained personnel.

Results

Results are presented as % positive and % negative results of each sample. Percentages were calculated for the two readers separately (30 replicates for each sample) and for both readers combined (60 data points per sample). All predetermined acceptance criteria were met.

| Sample

ID		HELIOS		User Confirmation				Manual
	N	n Neg	n Pos	n Neg		n Pos		n Neg		n Pos
				Reader
1	Reader
2	Reader
1	Reader
2	Reader
1	Reader
2	Reader
1	Reader
2
S1	30	0	30	0	0	30	30	0	0	30	30
S2	30	4	26	0	0	30	30	0	0	30	30
S3	30	1	29	0	0	30	30	0	0	30	30
S4	30	2	28	0	0	30	30	0	0	30	30
S5	30	23	7	27	19	3	11	27	19	3	11
S6	30	3	27	1	1	29	29	0	0	30	30
S7	30	28	2	19	10	11	20	3	17	27	13
S8	30	2	28	3	3	27	27	0	0	30	30
S9	30	26	4	26	28	4	2	30	30	0	0
S10	30	25	5	30	30	0	0	30	30	0	0

Table 17: Within-Lab Precision AESKUSLIDES ANCA Ethanol number of positive and negative results

18

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Table 18: Within-Lab Precision AESKUSLIDES ANCA Ethanol percentages of positive and negative results

| Sample

ID	N	HELIOS		User Confirmation				Manual
		% Negative	% Positive	% Negative		% Positive		% Negative		% Positive
				Reader
1	Reader
2	Reader
1	Reader
2	Reader
1	Reader
2	Reader
1	Reader 2
S1	30	0	100	0	0	100	100	0	0	100	100
S2	30	13.3	86.7	0	0	100	100	0	0	100	100
S3	30	3.3	96.7	0	0	100	100	0	0	100	100
S4	30	6.7	93.3	0	0	100	100	0	0	100	100
S5	30	76.7	23.3	90.0	63.3	10.0	36.7	90.0	63.3	10.0	36.7
S6	30	10.0	90.0	3.3	3.3	96.7	96.7	0	0	100	100
S7	30	93.3	6.7	63.3	33.3	36.7	66.7	10	56.7	90.0	43.3
S8	30	6.7	93.3	10.0	10.0	90.0	90.0	0	0	100	100
S9	30	86.7	13.3	86.7	93.3	13.3	6.7	100	100	0	0
S10	30	83.3	16.7	100	100	0	0	100	100	0	0

Table 19: Within-Lab Precision AESKUSLIDES ANCA Ethanol percentages of positive and negative results for both readers combined

| Sample
ID | N
(Method A) | HELIOS | | N
(Method B/C) | User Confirmation | | Manual | |
|--------------|-----------------|------------|------------|-------------------|-------------------|------------|------------|------------|
| | | % Negative | % Positive | | % Negative | % Positive | % Negative | % Positive |
| S1 | 30 | 0 | 100 | 60 | 0 | 100 | 0 | 100 |
| S2 | 30 | 13.3 | 86.7 | 60 | 0 | 100 | 0 | 100 |
| S3 | 30 | 3.3 | 96.7 | 60 | 0 | 100 | 0 | 100 |
| S4 | 30 | 6.7 | 93.3 | 60 | 0 | 100 | 0 | 100 |
| S5 | 30 | 77 | 23 | 60 | 76.7 | 23.3 | 76.7 | 23.3 |
| S6 | 30 | 10.0 | 90.0 | 60 | 3.3 | 96.7 | 0 | 100 |
| S7 | 30 | 93 | 7 | 60 | 48.3 | 51.7 | 33.3 | 66.7 |
| S8 | 30 | 7 | 93 | 60 | 10.0 | 90.0 | 0 | 100 |
| S9 | 30 | 86.7 | 13.3 | 60 | 90.0 | 10.0 | 100 | 0 |
| S10 | 30 | 83.3 | 16.7 | 60 | 100 | 0 | 100 | 0 |

Table 20: Within-Lab Precision AESKUSLIDES ANCA Formalin number of positive and negative results

	N	HELIOS		User Confirmation				Manual
Sample
ID		n Neg		n Neg
n Pos				n Neg	n Pos
			n Pos	Reader
1	Reader
2	Reader
1	Reader
2	Reader
1	Reader
2	Reader
1	Reader
2
S1	30	0	30	0	0	30	30	0	0	30	30
S2	30	0	30	0	0	30	30	0	0	30	30
S3	30	0	30	0	0	30	30	0	0	30	30
S4	30	0	30	0	0	30	30	0	0	30	30
S5	30	0	30	0	0	30	30	0	0	30	30
S6	30	0	30	0	0	30	30	0	0	30	30
S7	30	0	30	0	0	30	30	0	0	30	30
S8	30	0	30	0	0	30	30	0	0	30	30
S9	30	29	1	27	30	3	0	29	26	1	4
S10	30	27	3	30	27	0	3	30	28	0	2

19

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Table 21: Within-Lab Precision AESKUSLIDES ANCA Formalin percentages of positive and negative results

| Sample

ID	N	HELIOS		User Confirmation				Manual
		%
Negative	%
Positive	% Negative
Reader
1	% Negative
Reader
2	% Positive
Reader
1	% Positive
Reader
2	% Negative
Reader
1	% Negative
Reader
2	% Positive
Reader
1	% Positive
Reader
2
S1	30	0	100	0	0	100	100	0	0	100	100
S2	30	0	100	0	0	100	100	0	0	100	100
S3	30	0	100	0	0	100	100	0	0	100	100
S4	30	0	100	0	0	100	100	0	0	100	100
S5	30	0	100	0	0	100	100	0	0	100	100
S6	30	0	100	0	0	100	100	0	0	100	100
S7	30	0	100	0	0	100	100	0	0	100	100
S8	30	0	100	0	0	100	100	0	0	100	100
S9	30	96.7	3.3	90.0	100	10.0	0	97	86.7	3	13.3
S10	30	90.0	10.0	100	90.0	0	10.0	100	93.3	0	6.7

Table 22: Within-Lab Precision AESKUSLIDES ANCA Formalin percentages of positive and negative results for both readers combined

| Sample
ID | N
(Method A) | HELIOS | | N
(Method B/C) | User Confirmation | | Manual | |
|--------------|-----------------|---------------|------------|-------------------|-------------------|------------|---------------|------------|
| | | %
Negative | % Positive | | %
Negative | % Positive | %
Negative | % Positive |
| S1 | 30 | 0 | 100 | 60 | 0 | 100 | 0 | 100 |
| S2 | 30 | 0 | 100 | 60 | 0 | 100 | 0 | 100 |
| S3 | 30 | 0 | 100 | 60 | 0 | 100 | 0 | 100 |
| S4 | 30 | 0 | 100 | 60 | 0 | 100 | 0 | 100 |
| S5 | 30 | 0 | 100 | 60 | 0 | 100 | 0 | 100 |
| S6 | 30 | 0 | 100 | 60 | 0 | 100 | 0 | 100 |
| S7 | 30 | 0 | 100 | 60 | 0 | 100 | 0 | 100 |
| S8 | 30 | 0 | 100 | 60 | 0 | 100 | 0 | 100 |
| S9 | 30 | 96.7 | 3.3 | 60 | 95.0 | 5.0 | 91.7 | 8.3 |
| S10 | 30 | 90.0 | 10.0 | 60 | 95.0 | 5.0 | 96.7 | 3.3 |

For manual testing (Method C) of AESKUSLIDES ANCA Ethanol and Formalin fluorescence intensities were at maximum ±1 level within one run (triplicates) and between the runs. For samples found to be positive, pattern determination was consistent for more than 95% of the replicates, using Method B and C, whereas for Method A more than 85% of the determined patterns correspond to each other.

Conclusion

All pre-determined acceptance criteria were met.

b) Between-Lab Precision/Reproducibility AESKUSLIDES ANCA

To assess Between-Lab Precision of AESKUSLIDES ANCA Ethanol and ANCA Formalin processed on HELIOS (Method A - HELIOS suggestions and Method B -Reader Confirmation of HELIOS images) and processed manually (Method C).

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Image /page/20/Picture/0 description: The image is a logo for AESKU.DIAGNOSTICS. The logo features a green square with a white image inside. To the right of the square is the text "AESKU.DIAGNOSTICS" in gray, with the word "AESKU" being larger and bolder than "DIAGNOSTICS". Below the text is the phrase "THE DIAGNOSTIC TOOL THAT WORKS" in a smaller, lighter gray font.

Sample Set

10 samples were assayed on AESKUSLIDES ANCA Ethanol and ANCA Formalin. The sample set includes borderline, low, medium, high positive, and negative samples. The samples are characterized in the following table:

| Sample

ID	AESKUSLIDES ANCA Ethanol
1	Pos	P	high positive
2	Pos	P	high positive
3	Pos	P	medium positive
4	Pos	P	medium positive
5	Pos	C	borderline positive
6	Pos	C	high positive
7	Pos	C	borderline positive
8	Pos	C	medium positive
9	Neg	negative	negative
10	Neg	negative	negative

Table 23: Sample table for Precision study_ANCA Ethanol_Between-Lab

Sample ID	AESKUSLIDES ANCA Formalin
	Result	Pattern	Grading
1	Pos	C	medium positive
2	Pos	C	high positive
3	Pos	C	medium positive
4	Pos	C	high positive
5	Pos	C	low positive
6	Pos	C	high positive
7	Pos	C	medium positive
8	Pos	C	high positive
9	Neg	negative	negative
10	Neg	negative	negative

Procedure

10 samples have been tested on five days, two runs per day, three replicates per sample per run, at three different study sites. Two study sites were in the US, one study site was in Germany, with one HELIOS device per study site. Results were analyzed by two different readers per study site, resulting in a total of 90 data points per sample per reader.

This Between-Lab Precision study was done for three different Methods:

| Method A (HELIOS) | Processing of slides and image recording at HELIOS +
HELIOS result suggestion |
|-----------------------------------|-------------------------------------------------------------------------------------------------|
| Method B (Reader
Confirmation) | HELIOS images recorded in Method A + result analysis
by two independent readers |
| Method C (Manual) | Manual processing of slides and result analysis at the
microscope by two independent readers |

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Image /page/21/Picture/0 description: The image shows the logo for AESKU.DIAGNOSTICS. The logo consists of a green square with a white symbol inside, followed by the text "AESKU.DIAGNOSTICS" in gray. Below the text, there is a tagline that reads "THE DIAGNOSTIC TOOL THAT WORKS" in a smaller, lighter gray font. The symbol inside the green square appears to be a stylized representation of a medical or diagnostic tool.

All runs have been performed according to the respective IFUs. 1 positive and 1 negative control (kit controls) were included in each run. Samples were tested at 1:20 dilution. Results were analyzed by two independent readers.

Data Analysis

Positive/negative classification and pattern was recorded for each sample in each run and for each method. Pattern suggestion in Method A (HELIOS) is provided by the Vasculitis Pattern Plus software tool (ANCA).

For Method C (Manual performance) fluorescence intensity was also reported.

The following % agreements including 95% Cl (confidence intervals) will be calculated for each Method A, B and C:

-Positive % agreement (across all positive samples: number of correctly found samples divided through number of total positive samples)
-Negative % agreement (across all negative samples: number of correctly found samples divided through number of total negative samples)
-Pattern % agreement (across all across all positive samples: number of correctly found samples divided through number of total positive samples)
-Overall % agreement (across all positive and negative samples: number of correctly found samples divided through number of total samples)
-Fluorescence Intensity % agreement (across all samples, only for Method C: number of correctly found samples divided through number of total samples)

Results of the two readers will be calculated separately as well as combined.

Acceptance Criteria

Positive sera have to be found positive, and negative sera have to be found negative.

Patterns have to be found correctly.

Reported FI is allowed to differ max. ± 1 level from the expected value.

Agreements should meet the following criteria:

Type of Agreement (borderline positive samples excluded*)	Method A	Method B	Method C
Positive Agreement	≥ 70%	≥ 90%	≥ 90%
Negative Agreement	≥ 70%	≥ 90%	≥ 90%
Overall Agreement	≥ 70%	≥ 90%	≥ 90%
Pattern Agreement	≥ 70%	≥ 90%	≥ 90%
Fluorescence Intensity Agreement	---	---	≥ 90%

borderline samples are very low positive samples that can be evaluated also negative in certain cases, depending on the reader, the subjective manner of result analysis and normal test variances. Therefore, they may distort positive and pattern agreement, resulting in lower agreements. Therefore, agreements are calculated once including those borderline samples and once excluding them.

For Method A we accept a lower agreement (>70%), because we state in the IFU that all results generated by the HELIOS (Method A) must be confirmed by trained personnel.

22

From the results of those study, the following will be calculated:

Between-Lab Precisions, 2. Between-Operator Precisions, 3. Single-Operator Precisions, and 4. Instrument Precision

Results

Results are presented in the tables below. Agreements for AESKUSLIDES® ANCA Ethanol were calculated 1. including the results of the two borderline positive samples, 2. excluding the results of those two samples. For AESKUSLIDES ANCA Formalin results were only calculated once for all tested sera, since no borderline sera were included.

All predetermined acceptance criteria were met.

Between-Site Agreement ANCA Ethanol

Manual (Method C) Between-Site Agreement (Calculation including borderline samples):

Number of samples	Site 1 vs Site 2			Site 2 vs Site 3			Site 1 vs Site 3			All Sites
	Found Correctly	Found Incorrectly	Expected Numbers	Found Correctly	Found Incorrectly	Expected Numbers	Found Correctly	Found Incorrectly	Expected Numbers	Found Correctly	Found Incorrectly	Expected Numbers
Positive Agreement	894	66	960	948	12	960	948	12	960	1362	78	1440
Negative Agreement	240	0	240	240	0	240	240	0	240	360	0	360
Overall Agreement	1134	66	1200	1188	12	1200	1188	12	1200	1722	78	1800
Pattern Agreement	894	66	960	945	15	960	945	15	960	1359	81	1440
FI Agreement	1427	13	1440	1416	24	1440	1416	24	1440	2136	24	2160

Table 25: Between-Site Agreement_ANCA Ethanol_(Calculation including borderline samples). Number of correctly/incorrectly found samples Method C

Table 26: Between-Site Agreement_ANCA Ethanol_(Calculation including borderline samples). % Agreements (95% CI) Method C

% Agreement (95% CI)	Site 1 vs Site 2	Site 2 vs Site 3	Site 1 vs Site 3	All Sites
Positive Agreement	93.1	91.9	98.8	94.6
	(91.3 - 94.6)	(90 - 93.4)	(97.8 - 99.3)	(93.3 - 95.6)
Negative Agreement	100	100	100	100
	(98.4 - 100)	(98.4 - 100)	(98.4 - 100)	(98.9 - 100)
Overall Agreement	94.5	93.5	99	95.7
	(93.1 - 95.7)	(92 - 94.8)	(98.3 - 99.4)	(94.6 - 96.5)
Pattern Agreement	93.1	91.6	98.4	94.4
	(91.3 - 94.6)	(89.6 - 93.2)	(97.4 - 99.1)	(93.1 - 95.5)
FI Agreement	99.1	99.2	98.3	98.9
	(98.5 - 99.5)	(98.6 - 99.6)	(97.5 - 98.9)	(98.4 - 99.3)

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Manual (Method C) Between-Site Agreement (Calculation excluding borderline samples):

Number of samples	Site 1 vs Site 2			Site 2 vs Site 3			Site 1 vs Site 3			All Sites
	Found Correctly	Found Incorrectly	Expected Numbers	Found Correctly	Found Incorrectly	Expected Numbers	Found Correctly	Found Incorrectly	Expected Numbers	Found Correctly	Found Incorrectly	Expected Numbers
Positive Agreement	720	0	720	720	0	720	720	0	720	1080	0	1080
Negative Agreement	240	0	240	240	0	240	240	0	240	360	0	360
Overall Agreement	960	0	960	960	0	960	960	0	960	1440	0	1440
Pattern Agreement	720	0	720	717	3	720	717	3	720	1077	3	1080
FI Agreement	949	11	960	938	22	960	938	22	960	1418	22	1440

Table 27: Between-Site Agreement_ANCA Ethanol_(Calculation excluding borderline

Table 28: Between-Site Agreement_ANCA Ethanol_(Calculation excluding borderline samples). % Agreements (95% CI) Method C

% Agreement (95% CI)	Site 1 vs Site 2	Site 2 vs Site 3	Site 1 vs Site 3	All Sites
Positive Agreement	100	100	100	100
	(99.5 - 100)	(99.5 - 100)	(99.5 - 100)	(99.6 - 100)
Negative Agreement	100	100	100	100
	(98.4 - 100)	(98.4 - 100)	(98.4 - 100)	(98.9 - 100)
Overall Agreement	100	100	100	100
	(99.6 - 100)	(99.6 - 100)	(99.6 - 100)	(99.7 - 100)
Pattern Agreement	100	99.6	99.6	99.7
	(99.5 - 100)	(98.8 - 99.9)	(98.8 - 99.9)	(99.2 - 99.9)
FI Agreement	na	na	na	na

Reader Confirmation (Method B) Between-Site Agreement (Calculation including borderline samples):

Table 29: Between-Site Agreement_ANCA Ethanol_(Calculation including borderline samples). Number of correctly/incorrectly found samples Method B

		Site 1 vs Site 2			Site 2 vs Site 3			Site 1 vs Site 3			All Sites
Number of
Samples		Found
Correctly	Found
Incorrectly	Expected
Numbers	Found
Correctly	Found
Incorrectly	Expected
Numbers	Found
Correctly	Found
Incorrectly	Expected
Numbers	Found
Correctly	Found
Incorrectly	Expected
Numbers
Pos		869	91	960	952	8	960	877	83	960	1349	91	1440
Neg		234	6	240	240	0	240	234	6	240	354	6	360
Overall		1103	97	1200	1192	8	1200	1111	89	1200	1703	97	1800
Total Pattern		837	123	960	897	63	960	850	110	960	1292	148	1440
FI		na	na	na	na	na	na	na	na	na	na	na	na

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Image /page/24/Picture/0 description: The image is a logo for AESKU.DIAGNOSTICS. The logo features a green square with a white abstract design on the left. To the right of the square is the company name, "AESKU.DIAGNOSTICS," in gray text. Below the company name is the tagline "THE DIAGNOSTIC TOOL THAT WORKS" in a smaller, lighter gray font.

Table 30: Between-Site Agreement_ANCA Ethanol_(Calculation including borderline samples). % Agreements (95% CI) Method B

% Agreement (95% CI)	Site 1 vs Site 2	Site 2 vs Site 3	Site 1 vs Site 3	All Sites
Positive Agreement	90.5
(88.5 - 92.2)	99.2
(98.4 - 99.6)	91.4
(89.4 - 93)	93.7
(92.3 - 94.8)
Negative Agreement	97.5
(94.7 - 98.8)	100
(98.4 - 100)	97.5
(94.7 - 98.8)	98.3
(96.4 - 99.2)
Overall Agreement	91.9
(90.2 - 93.3)	99.3
(98.7 - 99.7)	92.6
(91 - 93.9)	94.6
(93.5 - 95.6)
Total Pattern Agreement	87.2
(84.9 - 89.2)	93.4
(91.7 - 94.8)	88.5
(86.4 - 90.4)	89.7
(88 - 91.2)
FI Agreement	na	na	na	na

Reader Confirmation (Method B) Between-Site Agreement (Calculation excluding borderline samples):

Table 31: Between-Site Agreement_ANCA Ethanol_(Calculation excluding borderline samples). Number of correctly/incorrectly found samples Method B

| Number of

Samples	Site 1 vs Site 2			Site 2 vs Site 3			Site 1 vs Site 3			All Sites
	Found
Correctly	Found
Incorrectly	Expected
Numbers	Found
Correctly	Found
Incorrectly	Expected
Numbers	Found
Correctly	Found
Incorrectly	Expected
Numbers	Found
Correctly	Found
Incorrectly	Expected
Numbers
Pos	712	8	720	720	0	720	712	8	720	1072	8	1080
Neg	234	6	240	240	0	240	234	6	240	354	6	360
Overall	946	14	960	960	0	960	946	14	960	1426	14	1440
Total Pattern	680	40	720	665	55	720	685	35	720	1015	65	1080
FI	na	na	na	na	na	na	na	na	na	na	na

Table 32: Between-Site Agreement_ANCA Ethanol_(Calculation excluding borderline samples). % Agreements (95% CI) Method B

% Agreement (95% CI)	Site 1 vs Site 2	Site 2 vs Site 3	Site 1 vs Site 3	All Sites
Positive Agreement	98.9
(97.8 - 99.4)	100
(99.5 - 100)	98.9
(97.8 - 99.4)	99.3
(98.5 - 99.6)
Negative Agreement	97.5
(94.7 - 98.8)	100
(98.4 - 100)	97.5
(94.7 - 98.8)	98.3
(96.4 - 99.2)
Overall Agreement	98.5
(97.6 - 99.1)	100
(99.6 - 100)	98.5
(97.6 - 99.1)	99
(98.4 - 99.4)
Total Pattern Agreement	94.4
(92.5 - 95.9)	92.4
(90.2 - 94.1)	95.1
(93.3 - 96.5)	94
(92.4 - 95.3)
FI Agreement	na	na	na	na

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HELIOS (Method A) Between-Site Agreement (Calclation including borderline samples):

samples). Number of correctly/incorrectly found samples Method A
Number of
Samples	Site 1 vs Site 2			Site 2 vs Site 3			Site 1 vs Site 3			All Sites
	Found
Correctly	Found
Incorrectly	Expected
Numbers	Found
Correctly	Found
Incorrectly	Expected
Numbers	Found
Correctly	Found
Incorrectly	Expected
Numbers	Found
Correctly	Found
Incorrectly	Expected
Numbers
Pos	411	69	480	472	8	480	415	65	480	649	71	720
Neg	102	18	120	111	9	120	111	9	120	162	18	180
Overall	513	87	600	583	17	600	526	74	600	811	89	900
Total Pattern	298	182	480	345	135	480	309	171	480	476	244	720
FI	na	na	na	na	na	na	na	na	na	na	na	na

Table 33: Between-Site Agreement_ANCA Ethanol_(Calculation including borderline

Table 34: Between-Site Agreement_ANCA Ethanol_(Calculation including borderline samples). % Agreements (95% CI) Method A

% Agreement (95% CI)	Site 1 vs Site 2	Site 2 vs Site 3	Site 1 vs Site 3	All Sites
Positive Agreement	85.6
(82.2 - 88.5)	98.3
(96.7 - 99.2)	86.5
(83.1 - 89.2)	90.1
(87.7 - 92.1)
Negative Agreement	85
(77.5 - 90.3)	92.5
(86.4 - 96)	92.5
(86.4 - 96)	90
(84.7 - 93.6)
Overall Agreement	85.5
(82.5 - 88.1)	97.2
(95.5 - 98.2)	87.7
(84.8 - 90.1)	90.1
(88 - 91.9)
Total Pattern Agreement	62.1
(57.7 - 66.3)	71.9
(67.7 - 75.7)	64.4
(60 - 68.5)	66.1
(62.6 - 69.5)
FI Agreement	na	na	na	na

HELIOS (Method A) Between-Site Agreement (Calclation excluding borderline samples):

| Table 35: Between-Site Agreement_ANCA Ethanol_(Calculation excluding borderline

samples). Number of correctly/incorrectly found samples Method A
	Site 1 vs Site 2			Site 2 vs Site 3			Site 1 vs Site 3			All Sites
Number of
Samples	Found
Correctly	Found
Incorrectly	Expected
Numbers	Found
Correctly	Found
Incorrectly	Expected
Numbers	Found
Correctly	Found
Incorrectly	Expected
Numbers	Found
Correctly	Found
Incorrectly	Expected
Numbers
Pos	342	18	360	352	8	360	346	14	360	520	20	540
Neg	102	18	120	111	9	120	111	9	120	162	18	180
Overall	444	36	480	463	17	480	457	23	480	682	38	720
Total Pattern	278	82	360	308	52	360	292	68	360	439	101	540
FI	na	na	na	na	na	na	na	na	na	na	na	na

Table 35: Between-Site Agreement ANCA Ethanol (Calculation excluding borderline

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Image /page/26/Picture/0 description: The image is a logo for AESKU.DIAGNOSTICS. The logo has a green square on the left with a white image inside. The text "AESKU.DIAGNOSTICS" is in gray, with the word "AESKU" being larger than the word "DIAGNOSTICS". Below the text is the phrase "THE DIAGNOSTIC TOOL THAT WORKS" in a smaller, lighter gray font.

Table 36: Between-Site Agreement ANCA Ethanol (Calculation excluding borderline samples). % Agreements (95% CI) Method A

% Agreement (95% CI)	Site 1 vs Site 2	Site 2 vs Site 3	Site 1 vs Site 3	All Sites
Positive Agreement	95
(92.2 - 96.8)	97.8
(95.7 - 98.9)	96.1
(93.6 - 97.7)	96.3
(94.3 - 97.6)
Negative Agreement	85
(77.5 - 90.3)	92.5
(86.4 - 96)	92.5
(86.4 - 96)	90
(84.7 - 93.6)
Overall Agreement	92.5
(89.8 - 94.5)	96.5
(94.4 - 97.8)	95.2
(92.9 - 96.8)	94.7
(92.8 - 96.1)
Total Pattern Agreement	77.2
(72.6 - 81.3)	85.6
(81.5 - 88.8)	81.1
(76.7 - 84.8)	81.3
(77.8 - 84.4)
FI Agreement	na	na	na	na

For Between-site Agreement of ANCA Ethanol, all acceptance critieria were met. All agreements were >90% for Method C (Manual) and Method B (Reader Confirmation) for all study sites. All agreements were > 70% for Method A (HELIOS suggestion) for all study sites.

Overall-Between-Lab agreements ranged from 92.5% to 96.5% for Method A (HELIOS), from 98.5% to 100% for Method B (Reader Confirmation), and was 100% for Method C (Manual).

Between-Operator Agreement ANCA Ethanol

Manual (Method C) Between-Operator Agreement (Calculation including borderline samples):

Number of Samples	Site 1			Site 2			Site 3
	Found Correctly	Found Incorrectly	Expected Numbers	Found Correctly	Found Incorrectly	Expected Numbers	Found Correctly	Found Incorrectly	Expected Numbers
Positive Agreement	414	66	480	480	0	480	468	12	480
Negative Agreement	120	0	120	120	0	120	120	0	120
Overall Agreement	534	66	600	600	0	600	588	12	600
Pattern Agreement	414	66	480	480	0	480	465	15	480
FI Agreement	720	0	720	707	13	720	709	11	720

Table 37: Between-Operator Agreement_ANCA Ethanol_(Calculation including borderline
samples). Number of correctly/incorrectly found samples Method C

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Image /page/27/Picture/0 description: The image is a logo for AESKU.DIAGNOSTICS. The logo features a green square with a white image inside, followed by the text "AESKU.DIAGNOSTICS" in gray. Below the text is the phrase "THE DIAGNOSTIC TOOL THAT WORKS" in a smaller, lighter gray font.

Table 38: Between-Operator Agreement_ANCA Ethanol_(Calculation including borderline samples). % Agreements (95% CI) Method C

% Agreement (95% CI)	Site 1	Site 2	Site 3
Positive Agreement	86.3
(82.9 - 89)	100
(99.2 - 100)	97.5
(95.7 - 98.6)
Negative Agreement	100
(96.9 - 100)	100
(96.9 - 100)	100
(96.9 - 100)
Overall Agreement	89
(86.2 - 91.3)	100
(99.4 - 100)	98
(96.5 - 98.9)
Pattern Agreement	86.3
(82.9 - 89)	100
(99.2 - 100)	96.9
(94.9 - 98.1)
FI Agreement	100
(99.5 - 100)	98.2
(96.9 - 98.9)	98.5
(97.3 - 99.1)

Manual (Method C) Between-Operator Agreement (Calculation excluding borderline samples):

Table 39: Between-Operator Agreement_ANCA Ethanol_(Calculation excluding borderline samples). Number of correctly/incorrectly found samples Method C

	Site 1			Site 2			Site 3
Number of Samples	Found
Correctly	Found
Incorrectly	Expected
Numbers	Found
Correctly	Found
Incorrectly	Expected
Numbers	Found
Correctly	Found
Incorrectly	Expected
Numbers
Positive Agreement	360	0	360	360	0	360	360	0	360
Negative Agreement	120	0	120	120	0	120	120	0	120
Overall Agreement	480	0	480	480	0	480	480	0	480
Pattern Agreement	360	0	360	360	0	360	357	3	360
FI Agreement	480	0	480	469	11	480	469	11	480

Table 40: Between-Operator Agreement_ANCA Ethanol_(Calculation excluding borderline samples). % Agreements (95% CI) Method C

% Agreement (95% CI)	Site 1	Site 2	Site 3
Positive Agreement	100
(98.9 - 100)	100
(98.9 - 100)	100
(98.9 - 100)
Negative Agreement	100
(96.9 - 100)	100
(96.9 - 100)	100
(96.9 - 100)
Overall Agreement	100
(99.2 - 100)	100
(99.2 - 100)	100
(99.2 - 100)
Pattern Agreement	100
(98.9 - 100)	100
(98.9 - 100)	99.2
(97.6 - 99.7)
FI Agreement	100
(99.2 - 100)	97.7
(95.9 - 98.7)	97.7
(95.9 - 98.7)

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Image /page/28/Picture/0 description: The image is a logo for AESKU.DIAGNOSTICS. The logo consists of a green square with a white stylized image of a molecule or antibody inside. To the right of the square is the text "AESKU.DIAGNOSTICS" in a dark gray, sans-serif font. Below the company name is the tagline "THE DIAGNOSTIC TOOL THAT WORKS" in a lighter gray font.

Reader Confirmation (Method B) Between-Operator Agreement (Calculation including borderline samples):

Table 41: Between-Operator Agreement_ANCA Ethanol_(Calculation including borderline samples). Number of correctly/incorrectly found samples Method B

Number of Samples	Site 1			Site 2			Site 3
	Found Correctly	Found Incorrectly	Expected Numbers	Found Correctly	Found Incorrectly	Expected Numbers	Found Correctly	Found Incorrectly	Expected Numbers
Pos	397	83	480	472	8	480	480	0	480
Neg	114	6	120	120	0	120	120	0	120
Overall	511	89	600	592	8	600	600	0	600
Total Pattern	395	85	480	442	38	480	455	25	480
FI	na	na	na	na	na	na	na	na	na

Table 42: Between-Operator Agreement_ANCA Ethanol_(Calculation including borderline samples). % Agreements (95% CI) Method B

% Agreement (95% CI)	Site 1	Site 2	Site 3
Positive Agreement	82.7
(79.1 - 85.8)	98.3
(96.7 - 99.2)	100
(99.2 - 100)
Negative Agreement	95
(89.5 - 97.7)	100
(96.9 - 100)	100
(96.9 - 100)
Overall Agreement	85.2
(82.1 - 87.8)	98.7
(97.4 - 99.3)	100
(99.4 - 100)
Total Pattern Agreement	82.3
(78.6 - 85.4)	92.1
(89.3 - 94.2)	94.8
(92.4 - 96.4)
FI Agreement	na	na	na

Reader Confirmation (Method B) Between-Operator Agreement (Calculation excluding borderline samples):

Table 43: Between-Operator Agreement_ANCA Ethanol_(Calculation excluding borderline samples). Number of correctly/incorrectly found samples Method B

Number of Samples	Site 1			Site 2			Site 3
	Found
Correctly	Found
Incorrectly	Expected
Numbers	Found
Correctly	Found
Incorrectly	Expected
Numbers	Found
Correctly	Found
Incorrectly	Expected
Numbers
Pos	352	8	360	360	0	360	360	0	360
Neg	114	6	120	120	0	120	120	0	120
Overall	466	14	480	480	0	480	480	0	480
Total Pattern	350	10	360	330	30	360	335	25	360
FI	na	na	na	na	na	na	na	na	na

29

Image /page/29/Picture/0 description: The image is a logo for AESKU.DIAGNOSTICS. The logo consists of a green square with a white symbol inside, followed by the text "AESKU.DIAGNOSTICS" in gray. Below the text, there is a tagline that reads "THE DIAGNOSTIC TOOL THAT WORKS" in a smaller, lighter gray font. The symbol in the green square appears to be a stylized representation of a medical or diagnostic tool.

Table 44: Between-Operator Agreement_ANCA Ethanol_(Calculation excluding borderline samples). % Agreements (95% CI) Method B

% Agreement (95% CI)	Site 1	Site 2	Site 3
Positive Agreement	97.8
(95.7 - 98.9)	100
(98.9 - 100)	100
(98.9 - 100)
Negative Agreement	95
(89.5 - 97.7)	100
(96.9 - 100)	100
(96.9 - 100)
Overall Agreement	97.1
(95.2 - 98.3)	100
(99.2 - 100)	100
(99.2 - 100)
Total Pattern Agreement	97.2
(95 - 98.5)	91.7
(88.4 - 94.1)	93.1
(89.9 - 95.3)
FI Agreement	na	na	na

For Between-Operator Agreement of ANCA Ethanol, all acceptance criteria were met. All agreements were >90% for Method C (Manual) and Method B (Reader Confirmation) for all study sites.

Overall- Between-Operator agreements ranged from 97.1% to 100% for Method B (Reader Confirmation), and was 100% for Method C (Manual).

Single-Operator Agreement ANCA Ethanol

Manual (Method C) Single-Operator Agreement (Calculation including borderline samples):

Table 45: Single-Operator Agreement_ANCA Ethanol_(Calculation including borderline
samples). Number of correctly/incorrectly found samples Method C

| Number of

Samples	Site 1			Site 2			Site 3
	Reader 1	Reader 2	Reader 3	Reader 4	Reader 5	Reader 6
	Found
Correctly	Found
Incorrectl	Expected
Numbers	Found
Correctly	Found
Incorrectl	Expected
Numbers	Found
Correctly	Found
Incorrectl	Expected
Numbers
Positive
Agreement	210	30	240	204	36	240	240	0	240	228	12	240	240	0	240
Negative
Agreement	60	0	60	60	0	60	60	0	60	60	0	60	60	0	60
Overall
Agreement	270	30	300	264	36	300	300	0	300	288	12	300	300	0	300
Pattern
Agreement	210	30	240	204	36	240	240	0	240	228	12	240	237	3	240
FI Agreement	360	0	360	360	0	360	351	9	360	356	4	360	353	7	360

30

Image /page/30/Picture/0 description: The image shows the logo for AESKU.DIAGNOSTICS. The logo consists of a green square with a white medical symbol inside, followed by the text "AESKU.DIAGNOSTICS" in gray. Below the text is the phrase "THE DIAGNOSTIC TOOL THAT WORKS" in a smaller, lighter gray font.

Table 46: Single-Operator Agreement_ANCA Ethanol_(Calculation including borderline
samples). % Agreements (95% CI) 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	87.5
(82.7 - 91.1)	85
(79.9 - 89)	100
(98.4 - 100)	100
(98.4 - 100)	95
(91.5 - 97.1)	100
(98.4 - 100)
Negative Agreement	100
(94 - 100)	100
(94 - 100)	100
(94 - 100)	100
(94 - 100)	100
(94 - 100)	100
(94 - 100)
Overall Agreement	90
(86.1 - 92.9)	88
(83.8 - 91.2)	100
(98.7 - 100)	100
(98.7 - 100)	96
(93.1 - 97.7)	100
(98.7 - 100)
Pattern Agreement	87.5
(82.7 - 91.1)	85
(79.9 - 89)	100
(98.4 - 100)	100
(98.4 - 100)	95
(91.5 - 97.1)	98.8
(96.4 - 99.6)
FI Agreement	100
(98.9 - 100)	100
(98.9 - 100)	97.5
(95.3 - 98.7)	98.9
(97.2 - 99.6)	98.9
(97.2 - 99.6)	98.1
(96 - 99.1)

Manual (Method C) Single-Operator Agreement (Calculation excluding borderline samples):

Number of Samples	Site 1						Site 2						Site 3
	Reader 1			Reader 2			Reader 3			Reader 4			Reader 5			Reader 6
	Found Correctly	Found Incorrectly	Expected Numbers	Found Correctly	Found Incorrectly	Expected Numbers	Found Correctly	Found Incorrectly	Expected Numbers	Found Correctly	Found Incorrectly	Expected Numbers	Found Correctly	Found Incorrectly	Expected Numbers	Found Correctly	Found Incorrectly	Expected Numbers
Positive Agreement	180	0	180	180	0	180	180	0	180	180	0	180	180	0	180	180	0	180
Negative Agreement	60	0	60	60	0	60	60	0	60	60	0	60	60	0	60	60	0	60
Overall Agreement	240	0	240	240	0	240	240	0	240	240	0	240	240	0	240	240	0	240
Pattern Agreement	180	0	180	180	0	180	180	0	180	180	0	180	180	0	180	177	3	180
FI Agreement	240	0	240	240	0	240	231	9	240	238	2	240	236	4	240	233	7	240

Table 47: Single-Operator Agreement_ANCA Ethanol_(Calculation excluding borderline than C

Table 48: Single-Operator Agreement_ANCA Ethanol_(Calculation excluding borderline samples). % Agreements (95% CI) Method C

	Site 1		Site 2		Site 3
% Agreement (95%
CI)	Reader 1	Reader 2	Reader 3	Reader 4	Reader 5	Reader 6
Positive Agreement	100
(97.9 - 100)	100
(97.9 - 100)	100
(97.9 - 100)	100
(97.9 - 100)	100
(97.9 - 100)	100
(97.9 - 100)
Negative Agreement	100
(94 - 100)	100
(94 - 100)	100
(94 - 100)	100
(94 - 100)	100
(94 - 100)	100
(94 - 100)
Overall Agreement	100
(98.4 - 100)	100
(98.4 - 100)	100
(98.4 - 100)	100
(98.4 - 100)	100
(98.4 - 100)	100
(98.4 - 100)
Total Pattern
Agreement	100
(97.9 - 100)	100
(97.9 - 100)	100
(97.9 - 100)	100
(97.9 - 100)	100
(97.9 - 100)	98.3
(95.2 - 99.4)
FI Agreement	100
(98.4 - 100)	100
(98.4 - 100)	96.3
(93 - 98)	99.2
(97 - 99.8)	98.3
(95.8 - 99.4)	97.1
(94.1 - 98.6)

31

Image /page/31/Picture/0 description: The image is a logo for AESKU.DIAGNOSTICS. The logo features a green square with a white symbol inside, followed by the text "AESKU.DIAGNOSTICS" in gray. Below the text, there is a tagline that reads "THE DIAGNOSTIC TOOL THAT WORKS" in a smaller, lighter gray font.

Reader Confirmation (Method B) Single-Operator Agreement (Calculation including borderline samples):

Table 49: Single-Operator Agreement_ANCA Ethanol_(Calculation including borderline samples). Number of correctly/incorrectly found samples Method B

| Number of

Samples	Site 1			Site 2			Site 3
	Reader 1
Found
Correctly	Reader 1
Found
Incorrectly	Reader 1
Expected
Numbers	Reader 2
Found
Correctly	Reader 2
Found
Incorrectly	Reader 2
Expected
Numbers	Reader 3
Found
Correctly	Reader 3
Found
Incorrectly	Reader 3
Expected
Numbers	Reader 4
Found
Correctly	Reader 4
Found
Incorrectly	Reader 4
Expected
Numbers	Reader 5
Found
Correctly	Reader 5
Found
Incorrectly	Reader 5
Expected
Numbers	Reader 6
Found
Correctly	Reader 6
Found
Incorrectly	Reader 6
Expected
Numbers
Positive
Agreement	190	50	240	207	33	240	236	4	240	236	4	240	240	0	240	240	0	240
Negative
Agreement	56	4	60	58	2	60	60	0	60	60	0	60	60	0	60	60	0	60
Overall
Agreement	246	54	300	265	35	300	296	4	300	296	4	300	300	0	300	300	0	300
Pattern
Agreement	190	50	240	205	35	240	221	19	240	221	19	240	228	12	240	227	13	240
FI Agreement	na	na	na	na	na	na	na	na	na	na	na	na	na	na	na	na	na	na

Table 50: Single-Operator Agreement_ANCA Ethanol_(Calculation including borderline samples). % Agreements (95% CI) 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	79.2
(73.6 - 83.8)	86.3
(81.3 - 90)	98.3
(95.8 - 99.4)	98.3
(95.8 - 99.4)	100
(98.4 - 100)	100
(98.4 - 100)
Negative Agreement	93.3
(84.1 - 97.4)	96.7
(88.6 - 99.1)	100
(94 - 100)	100
(94 - 100)	100
(94 - 100)	100
(94 - 100)
Overall Agreement	82
(77.3 - 85.9)	88.3
(84.2 - 91.5)	98.7
(96.6 - 99.5)	98.7
(96.6 - 99.5)	100
(98.7 - 100)	100
(98.7 - 100)
Total Pattern
Agreement	79.2
(73.6 - 83.8)	85.4
(80.4 - 89.3)	92.1
(88 - 94.9)	92.1
(88 - 94.9)	95
(91.5 - 97.1)	94.6
(91 - 96.8)
FI Agreement	na	na	na	na	na	na

32

Image /page/32/Picture/0 description: The image is a logo for AESKU.DIAGNOSTICS. The logo features a green square with a white image of a zipper inside. To the right of the square is the text "AESKU.DIAGNOSTICS" in gray, with the word "AESKU" being larger than the word "DIAGNOSTICS". Below the text is the phrase "THE DIAGNOSTIC TOOL THAT WORKS" in a smaller, lighter gray font.

Reader Confirmation (Method B) Single-Operator Agreement (Calculation excluding borderline samples):

Table 51: Single-Operator Agreement_ANCA Ethanol_(Calculation excluding borderline samples). Number of correctly/incorrectly found samples Method B

Number of Samples	Site 1			Site 2			Site 3
	Reader 1
Found Correctly	Reader 1
Found Incorrectly	Reader 1
Expected Numbers	Reader 2
Found Correctly	Reader 2
Found Incorrectly	Reader 2
Expected Numbers	Reader 3
Found Correctly	Reader 3
Found Incorrectly	Reader 3
Expected Numbers	Reader 4
Found Correctly	Reader 4
Found Incorrectly	Reader 4
Expected Numbers	Reader 5
Found Correctly	Reader 5
Found Incorrectly	Reader 5
Expected Numbers	Reader 6
Found Correctly	Reader 6
Found Incorrectly	Reader 6
Expected Numbers
Positive Agreement	176	4	180	176	4	180	180	0	180	180	0	180	180	0	180	180	0	180
Negative Agreement	56	4	60	58	2	60	60	0	60	60	0	60	60	0	60	60	0	60
Overall Agreement	232	8	240	234	6	240	240	0	240	240	0	240	240	0	240	240	0	240
Pattern Agreement	176	4	180	174	6	180	165	15	180	165	15	180	168	12	180	167	13	180
FI Agreement	na	na	na	na	na	na	na	na	na	na	na	na	na	na	na	na	na	na

Table 52: Single-Operator Agreement_ANCA Ethanol_(Calculation excluding borderline samples). % Agreements (95% CI) 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	97.8
(94.4 - 99.1)	97.8
(94.4 - 99.1)	100
(97.9 - 100)	100
(97.9 - 100)	100
(97.9 - 100)	100
(97.9 - 100)
Negative Agreement	93.3
(84.1 - 97.4)	96.7
(88.6 - 99.1)	100
(94 - 100)	100
(94 - 100)	100
(94 - 100)	100
(94 - 100)
Overall Agreement	96.7
(93.6 - 98.3)	97.5
(94.7 - 98.8)	100
(98.4 - 100)	100
(98.4 - 100)	100
(98.4 - 100)	100
(98.4 - 100)
Total Pattern
Agreement	97.8
(94.4 - 99.1)	96.7
(92.9 - 98.5)	91.7
(86.7 - 94.9)	91.7
(86.7 - 94.9)	93.3
(88.7 - 96.1)	92.8
(88 - 95.7)
FI Agreement	na	na	na	na	na	na

For Single-Operator Agreement of ANCA Ethanol, all acceptance criteria were met. All agreements were >90% for Method C (Manual) and Method B (Reader Confirmation) for all study sites.

Overall- Single-Operator agreements ranged from 96.7% to 100% for Method B (Reader Confirmation), and was 100% for Method C (Manual).

33

Instrument Precision ANCA Ethanol

HELIOS (Method A) Instrument Agreement (Calculation including borderline samples):

Table 53: Instrument Agreement_ANCA Ethanol_(Calculation including borderline samples). Number of correctly/incorrectly found samples Method A

| Number of
Samples | Site 1
HELIOS 1 | | | Site 2
HELIOS 2 | | | Site 3
HELIOS 3 | | |
|-----------------------|--------------------|-------------------|------------------|--------------------|-------------------|------------------|--------------------|-------------------|------------------|
| | Found Correctly | Found Incorrectly | Expected Numbers | Found Correctly | Found Incorrectly | Expected Numbers | Found Correctly | Found Incorrectly | Expected Numbers |
| Positive
Agreement | 177 | 63 | 240 | 234 | 6 | 240 | 238 | 2 | 240 |
| Negative
Agreement | 51 | 9 | 60 | 51 | 9 | 60 | 60 | 0 | 60 |
| Overall
Agreement | 228 | 72 | 300 | 285 | 15 | 300 | 298 | 2 | 300 |
| Pattern
Agreement | 131 | 109 | 240 | 167 | 73 | 240 | 178 | 62 | 240 |
| FI Agreement | na | na | na | na | na | na | na | na | na |

Table 54: Instrument Agreement_ANCA Ethanol_(Calculation including borderline samples). % Agreements (95% CI) Method A

% Agreement (95% CI)	Site 1	Site 2	Site 3
	HELIOS 1	HELIOS 2	HELIOS 3
Positive Agreement	73.8
(67.8 - 78.9)	97.5
(94.7 - 98.8)	99.2
(97 - 99.8)
Negative Agreement	85
(73.9 - 91.9)	85
(73.9 - 91.9)	100
(94 - 100)
Overall Agreement	76
(70.9 - 80.5)	95
(91.9 - 96.9)	99.3
(97.6 - 99.8)
Pattern Agreement	54.6
(48.3 - 60.8)	69.6
(63.5 - 75.1)	74.2
(68.3 - 79.3)
FI Agreement	na	na	na

34

Image /page/34/Picture/0 description: The image shows the logo for AESKU.DIAGNOSTICS. The logo consists of a green square with a white stylized image of a molecule inside. To the right of the square is the text "AESKU.DIAGNOSTICS" in gray, with the word "AESKU" being larger and bolder than the word "DIAGNOSTICS". Below the text is the phrase "THE DIAGNOSTIC TOOL THAT WORKS" in a smaller, lighter gray font.

HELIOS (Method A) Instrument Agreement (Calculation excluding borderline samples):

| Number of
Samples | Site 1
HELIOS 1 | | | Site 2
HELIOS 2 | | | Site 3
HELIOS 3 | | |
|-----------------------|--------------------|----------------------|---------------------|--------------------|----------------------|---------------------|--------------------|----------------------|---------------------|
| | Found
Correctly | Found
Incorrectly | Expected
Numbers | Found
Correctly | Found
Incorrectly | Expected
Numbers | Found
Correctly | Found
Incorrectly | Expected
Numbers |
| Positive
Agreement | 168 | 12 | 180 | 174 | 6 | 180 | 178 | 2 | 180 |
| Negative
Agreement | 51 | 9 | 60 | 51 | 9 | 60 | 60 | 0 | 60 |
| Overall
Agreement | 219 | 21 | 240 | 225 | 15 | 240 | 238 | 2 | 240 |
| Pattern
Agreement | 131 | 49 | 180 | 147 | 33 | 180 | 161 | 19 | 180 |
| FI Agreement | na | na | na | na | na | na | na | na | na |

Table 55: Instrument Agreement_ANCA Ethanol_(Calculation excluding borderline samples). Number of correctly/incorrectly found samples Method A

Table 56: Instrument Agreement_ANCA Ethanol_(Calculation excluding borderline samples). % Agreements (95% CI) Method A

| % Agreement (95% CI) | Site 1
HELIOS 1 | Site 2
HELIOS 2 | Site 3
HELIOS 3 |
|----------------------|-----------------------|-----------------------|-----------------------|
| Positive Agreement | 93.3
(88.7 - 96.1) | 96.7
(92.9 - 98.5) | 98.9
(96 - 99.7) |
| Negative Agreement | 85
(73.9 - 91.9) | 85
(73.9 - 91.9) | 100
(94 - 100) |
| Overall Agreement | 91.3
(87 - 94.2) | 93.8
(89.9 - 96.2) | 99.2
(97 - 99.8) |
| Pattern Agreement | 72.8
(65.9 - 78.8) | 81.7
(75.4 - 86.6) | 89.4
(84.1 - 93.1) |
| FI Agreement | na | na | na |

For Instrument to Instrument Agreement with ANCA Ethanol, all acceptance criteria were met. All agreements were >70% for all study sites.

Overall- Instrument to Instrument agreements ranged from 91.3% to 99.2%.

We clearly state that all results suggested by HELIOS have to be confirmed by a trained reader.

35

Between-Site Agreement ANCA Formalin

Manual (Method C) Between-Site Agreement:

Table 57: Between-Site Agreement_ANCA Formalin_Number of correctly/incorrectly found samples Method C

| | AESKUSLIDES ANCA Formalin Manual Performance (Method C)
Site 1 vs Site 2 | | | Site 2 vs Site 3 | | | Site 1 vs Site 3 | | | All Sites | | |
|-----------------------|-----------------------------------------------------------------------------|----------------------|---------------------|--------------------|----------------------|---------------------|--------------------|----------------------|---------------------|--------------------|----------------------|---------------------|
| Number of
Samples | Found
Correctly | Found
Incorrectly | Expected
Numbers | Found
Correctly | Found
Incorrectly | Expected
Numbers | Found
Correctly | Found
Incorrectly | Expected
Numbers | Found
Correctly | Found
Incorrectly | Expected
Numbers |
| Positive
Agreement | 960 | 0 | 960 | 945 | 15 | 960 | 945 | 15 | 960 | 1425 | 15 | 1440 |
| Negative
Agreement | 233 | 7 | 240 | 240 | 0 | 240 | 233 | 7 | 240 | 353 | 7 | 360 |
| Overall
Agreement | 1193 | 7 | 1200 | 1185 | 15 | 1200 | 1178 | 22 | 1200 | 1778 | 22 | 1800 |
| Pattern
Agreement | 960 | 0 | 960 | 945 | 15 | 960 | 945 | 15 | 960 | 1425 | 15 | 1440 |
| FI Agreement | 897 | 3 | 900 | 1166 | 34 | 1200 | 869 | 31 | 900 | 1466 | 34 | 1500 |

Table 58: Between-Site Agreement_ANCA Formalin % Agreements (95% CI) Method C

% Agreement (95% CI)	Site 1 vs Site 2	Site 2 vs Site 3	Site 1 vs Site 3	All Sites
Positive Agreement	100	98.4	98.4	99
	(99.6 - 100)	(97.4 - 99.1)	(97.4 - 99.1)	(98.3 - 99.4)
Negative Agreement	97.1	100	97.1	98.1
	(94.1 - 98.6)	(98.4 - 100)	(94.1 - 98.6)	(96 - 99.1)
Overall Agreement	99.4	98.8	98.2	98.8
	(98.8 - 99.7)	(97.9 - 99.2)	(97.2 - 98.8)	(98.2 - 99.2)
Pattern Agreement	100	98.4	98.4	99
	(99.6 - 100)	(97.4 - 99.1)	(97.4 - 99.1)	(98.3 - 99.4)
FI Agreement	99.7	97.2	96.6	97.7
	(99 - 99.9)	(96.1 - 98)	(95.2 - 97.6)	(96.8 - 98.4)

Reader Confirmation (Method B) Between-Site Agreement:

Table 59: Between-Site Agreement_ANCA Formalin_Number of correctly/incorrectly found samples Method B

Number of Samples	Site 1 vs Site 2			Site 2 vs Site 3			Site 1 vs Site 3			All Sites
	Found Correctly	Found Incorrectly	Expected Numbers	Found Correctly	Found Incorrectly	Expected Numbers	Found Correctly	Found Incorrectly	Expected Numbers	Found Correctly	Found Incorrectly	Expected Numbers
Positive Agreement	931	29	960	924	36	960	953	7	960	1404	36	1440
Negative Agreement	224	16	240	226	14	240	230	10	240	340	20	360
Overall Agreement	1155	45	1200	1150	50	1200	1183	17	1200	1744	56	1800
Pattern Agreement	914	46	960	898	62	960	944	16	960	1378	62	1440
FI Agreement	na	na	na	na	na	na	na	na	na	na	na

36

Image /page/36/Picture/0 description: The image is a logo for AESKU.DIAGNOSTICS. The logo consists of a green square with a white stylized image of a zipper in the left side of the logo. To the right of the square is the text "AESKU.DIAGNOSTICS" in gray, with the word "AESKU" being larger and bolder than the word "DIAGNOSTICS". Below the text is the phrase "THE DIAGNOSTIC TOOL THAT WORKS" in a smaller, lighter gray font.

% Agreement (95% CI)	Site 1 vs Site 2	Site 2 vs Site 3	Site 1 vs Site 3	All Sites
Positive Agreement	97
(95.7 - 97.9)	96.3
(94.9 - 97.3)	99.3
(98.5 - 99.6)	97.5
(96.6 - 98.2)
Negative Agreement	93.3
(89.4 - 95.9)	94.2
(90.4 - 96.5)	95.8
(92.5 - 97.7)	94.4
(91.6 - 96.4)
Overall Agreement	96.3
(95 - 97.2)	95.8
(94.5 - 96.8)	98.6
(97.7 - 99.1)	96.9
(96 - 97.6)
Pattern Agreement	95.2
(93.7 - 96.4)	93.5
(91.8 - 94.9)	98.3
(97.3 - 99)	95.7
(94.5 - 96.6)
FI Agreement	na	na	na	na

Table 60: Between-Site Agreement ANCA Formalin % Agreements (95% Cl) Method B

HELIOS (Method A) Between-Site Agreement (Calclation including borderline samples):

Table 61: Between-Site Agreement_ANCA Formalin_Number of correctly/incorrectly found samples Method A

	Site 1 vs Site 2			Site 2 vs Site 3			Site 1 vs Site 3			All Sites
Number of Samples	Found Correctly	Found Incorrectly	Expected Numbers	Found Correctly	Found Incorrectly	Expected Numbers	Found Correctly	Found Incorrectly	Expected Numbers	Found Correctly	Found Incorrectly	Expected Numbers
Positive Agreement	461	19	480	456	24	480	475	5	480	696	24	720
Negative Agreement	102	18	120	83	37	120	93	27	120	139	41	180
Overall Agreement	563	37	600	539	61	600	568	32	600	835	65	900
Pattern Agreement	422	58	480	404	76	480	440	40	480	633	87	720
FI Agreement	na	na	na	na	na	na	na	na	na	na	na

Table 62: Between-Site Agreement ANCA Formalin. % Agreements (95% Cl) Method A

% Agreement (95% CI)	Site 1 vs Site 2	Site 2 vs Site 3	Site 1 vs Site 3	All Sites
Positive Agreement	96
(93.9 - 97.5)	95
(92.7 - 96.6)	99
(97.6 - 99.6)	96.7
(95.1 - 97.7)
Negative Agreement	85
(77.5 - 90.3)	69.2
(60.4 - 76.7)	77.5
(69.2 - 84.1)	77.2
(70.6 - 82.7)
Overall Agreement	93.8
(91.6 - 95.5)	89.8
(87.2 - 92)	94.7
(92.6 - 96.2)	92.8
(90.9 - 94.3)
Pattern Agreement	87.9
(84.7 - 90.5)	84.2
(80.6 - 87.2)	91.7
(88.9 - 93.8)	87.9
(85.3 - 90.1)
FI Agreement	na	na	na	na

For Between-site Agreement of ANCA Formalin, all acceptance criteria were met. All agreements were >90% for Method C (Manual) and Method B (Reader Confirmation) for all study sites. All agreements were > 70% for Method A (HELIOS suggestion) for all study sites.

Overall-Between-Lab agreements ranged from 89.8% to 94.7% for Method A (HELIOS), from 95.8% to 98.6% for Method B (Reader Confirmation), and from 98.2% to 99.4% for Method C (Manual).

37

Between-Operator Agreement ANCA Formalin

Manual (Method C) Between-Operator Agreement (Calculation including borderline samples):

Table 63: Between-Operator Agreement_ANCA Formalin_Number of correctly/incorrectly found samples Method C

Number of Samples	Site 1			Site 2			Site 3
	Found
Correctly	Found
Incorrectly	Expected
Numbers	Found
Correctly	Found
Incorrectly	Expected
Numbers	Found
Correctly	Found
Incorrectly	Expected
Numbers
Pos	480	0	480	480	0	480	465	15	480
Neg	113	7	120	120	0	120	120	0	120
Overall	593	7	600	600	0	600	585	15	600
Single Pattern C-ANCA	480	0	480	480	0	480	465	15	480
FI	300	0	300	597	3	600	569	31	600

Table 64: Between-Operator Agreement_ANCA Formalin. % Agreements (95% CI) Method C

% Agreement (95% CI)	Site 1	Site 2	Site 3
Positive Agreement	100
(99.2 - 100)	100
(99.2 - 100)	96.9
(94.9 - 98.1)
Negative Agreement	94.2
(88.4 - 97.1)	100
(96.9 - 100)	100
(96.9 - 100)
Overall Agreement	98.8
(97.6 - 99.4)	100
(99.4 - 100)	97.5
(95.9 - 98.5)
Single Pattern C-ANCA Agreement	100
(99.2 - 100)	100
(99.2 - 100)	96.9
(94.9 - 98.1)
FI Agreement	100
(98.7 - 100)	99.5
(98.5 - 99.8)	94.8
(92.8 - 96.3)

38

Reader Confirmation (Method B) Between-Operator Agreement:

Table 65: Between-Operator Agreement_ANCA Formalin_Number of correctly/incorrectly found samples Method B

	Site 1			Site 2			Site 3
Number of Samples	Found
Correctly	Found
Incorrectly	Expected
Numbers	Found
Correctly	Found
Incorrectly	Expected
Numbers	Found
Correctly	Found
Incorrectly	Expected
Numbers
Positive Agreement	480	0	480	451	29	480	473	7	480
Negative Agreement	114	6	120	110	10	120	116	4	120
Overall Agreement	594	6	600	561	39	600	589	11	600
Pattern Agreement	480	0	480	434	46	480	464	16	480
FI Agreement	na	na	na	na	na	na	na	na	na

Table 66: Between-Operator Agreement_ANCA Formalin. % Agreements (95% CI) Method B

% Agreement (95% CI)	Site 1	Site 2	Site 3
Positive Agreement	100
(99.2 - 100)	94
(91.5 - 95.8)	98.5
(97 - 99.3)
Negative Agreement	95
(89.5 - 97.7)	91.7
(85.3 - 95.4)	96.7
(91.7 - 98.7)
Overall Agreement	99
(97.8 - 99.5)	93.5
(91.2 - 95.2)	98.2
(96.7 - 99)
Pattern Agreement	100
(99.2 - 100)	90.4
(87.5 - 92.7)	96.7
(94.7 - 97.9)
FI Agreement	na	na	na

For Between-Operator Agreement of ANCA Formalin, all acceptance critieria were met. All agreements were >90% for Method C (Manual) and Method B (Reader Confirmation) for all study sites.

Overall- Between-Operator agreements ranged from 93.5% to 99% for Method B (Reader Confirmation), and from 97.5% to 100% for Method C (Manual).

39

Single-Operator Agreement ANCA Formalin

Manual (Method C) Single-Operator Agreement:

Table 67: Single-Operator Agreement_ANCA Formalin_Number of correctly/incorrectly found samples Method C

			Site 2			Site 3
Number of Samples	Reader 1	Reader 2	Reader 3	Reader 4	Reader 5	Reader 6
	Found Correctly	Found Incorrectl	Expected Numbers	Found Correctly	Found Incorrectl	Expected Numbers	Found Correctly	Found Incorrectl	Expected Numbers	Found Correctly	Found Incorrectl	Expected Numbers	Found Correctly	Found Incorrectl	Expected Numbers	Found Correctly	Found Incorrectl	Expected Numbers
Positive Agreement	240	0	240	240	0	240	240	0	240	240	0	240	234	6	240	231	9	240
Negative Agreement	59	1	60	54	6	60	60	0	60	60	0	60	60	0	60	60	0	60
Overall Agreement	299	1	300	294	6	300	300	0	300	300	0	300	294	6	300	291	9	300
Pattern Agreement	240	0	240	240	0	240	240	0	240	240	0	240	234	6	240	231	9	240
FI Agreement	300	0	300	300	0	300	300	0	300	297	3	300	293	7	300	276	24	300

Table 68: Single-Operator Agreement_ANCA Formalin. % Agreements (95% CI) 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	100
(98.4 - 100)	100
(98.4 - 100)	100
(98.4 - 100)	100
(98.4 - 100)	97.5
(94.7 - 98.8)	96.3
(93 - 98)
Negative Agreement	98.3
(91.1 - 99.7)	90
(79.9 - 95.3)	100
(94 - 100)	100
(94 - 100)	100
(94 - 100)	100
(94 - 100)
Overall Agreement	99.7
(98.1 - 99.9)	98
(95.7 - 99.1)	100
(98.7 - 100)	100
(98.7 - 100)	98
(95.7 - 99.1)	97
(94.4 - 98.4)
Single Pattern C-ANCA
Agreement	100
(98.4 - 100)	100
(98.4 - 100)	100
(98.4 - 100)	100
(98.4 - 100)	97.5
(94.7 - 98.8)	96.3
(93 - 98)
FI Agreement	100
(98.7 - 100)	100
(98.7 - 100)	100
(98.7 - 100)	99
(97.1 - 99.7)	97.7
(95.3 - 98.9)	92
(88.4 - 94.6)

40

Reader Confirmation (Method B) Single-Operator Agreement (Calculation including borderline samples):

Table 69: Single-Operator Agreement ANCA Formalin Number of correctly/incorrectly found
samples Method B

| Number of

Samples	Site 1						Site 2						Site 3
	Reader 1			Reader 2			Reader 3			Reader 4			Reader 5			Reader 6
	Found	Correctly	Found
Incorrectly	Expected
Numbers	Found	Correctly	Found
Incorrectly	Expected
Numbers	Found	Correctly	Found
Incorrectly	Expected
Numbers	Found	Correctly	Found
Incorrectly	Expected
Numbers	Found	Correctly	Found
Incorrectly	Expected
Numbers	Found	Correctly	Found
Incorrectly	Expected
Numbers
Positive
Agreement	240	0	240	240	0	240	223	17	240	228	12	240	235	5	240	238	2	240
Negative
Agreement	57	3	60	57	3	60	55	5	60	55	5	60	59	1	60	57	3	60
Overall
Agreement	297	3	300	297	3	300	278	22	300	283	17	300	294	6	300	295	5	300
Pattern
Agreement	240	0	240	240	0	240	218	22	240	216	24	240	232	8	240	232	8	240
FI Agreement	na	na	na	na	na	na	na	na	na	na	na	na	na	na	na	na	na

Table 70: Single-Operator Agreement_ANCA Formalin. % Agreements (95% CI) 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	100
(98.4 - 100)	100
(98.4 - 100)	92.9
(89 - 95.5)	95
(91.5 - 97.1)	97.9
(95.2 - 99.1)	99.2
(97 - 99.8)
Negative Agreement	95
(86.3 - 98.3)	95
(86.3 - 98.3)	91.7
(81.9 - 96.4)	91.7
(81.9 - 96.4)	98.3
(91.1 - 99.7)	95
(86.3 - 98.3)
Overall Agreement	99
(97.1 - 99.7)	99
(97.1 - 99.7)	92.7
(89.1 - 95.1)	94.3
(91.1 - 96.4)	98
(95.7 - 99.1)	98.3
(96.2 - 99.3)
Pattern Agreement	100
(98.4 - 100)	100
(98.4 - 100)	90.8
(86.5 - 93.9)	90
(85.6 - 93.2)	96.7
(93.6 - 98.3)	96.7
(93.6 - 98.3)
FI Agreement	na	na	na	na	na	na

For Single-Operator Agreement of ANCA Formalin, all acceptance critieria were met. All agreements were >90% for Method C (Manual) and Method B (Reader Confirmation) for all study sites.

Overall- Single-Operator agreements ranged from 92.7% to 99% for Method B (Reader Confirmation), and from 97% to 100% for Method C (Manual).

41

Instrument Precision ANCA Formalin

HELIOS (Method A) Instrument Agreement:

Table 71: Instrument Agreement_ANCA Formalin_Number of correctly/incorrectly found samples Method A

Number of Samples	Site 1			Site 2			Site 3
	HELIOS 1			HELIOS 2			HELIOS 3
Positive Agreement	240	0	240	221	19	240	235	5	240
Negative Agreement	56	4	60	46	14	60	37	23	60
Overall Agreement	296	4	300	267	33	300	272	28	300
Pattern Agreement	229	11	240	193	47	240	211	29	240
FI Agreement	na	na	na	0	0	0	0	0	0

Table 72: Instrument Agreement_ANCA Formalin. % Agreements (95% CI) Method A

% Agreement (95% CI)	Site 1	Site 2	Site 3
	HELIOS 1	HELIOS 2	HELIOS 3
Positive Agreement	100
(98.4 - 100)	92.1
(88 - 94.9)	97.9
(95.2 - 99.1)
Negative Agreement	93.3
(84.1 - 97.4)	76.7
(64.6 - 85.6)	61.7
(49 - 72.9)
Overall Agreement	98.7
(96.6 - 99.5)	89
(85 - 92.1)	90.7
(86.8 - 93.5)
Pattern Agreement	95.4
(92 - 97.4)	80.4
(74.9 - 84.9)	87.9
(83.2 - 91.5)
FI Agreement	na	0	0

For Instrument to Instrument Agreement with ANCA Formalin, all acceptance criteria were met, except for negative agreement at site 3. There, negative agreement was 61.7% whereas negative agreement at site 1 and was 93.3% and 76.7%. Sometimes it may occur, that HELIOS focusses falsly on unspecific, fluorescing particles, thereby reporting the sample as positive, while the reader can still see from the images that the cells are negative. Therefore, we clearly state that all results suggested by HELIOS have to be confirmed by a trained reader.

Overall- Instrument to Instrument agreements ranged from 89% to 90.7%.

42

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Conclusions

ANCA Ethanol

Overall-Between-Lab agreements ranged from 92.5% to 96.5% for Method A (HELIOS), from 98.5% to 100% for Method B (Reader Confirmation), and was 100% for Method C (Manual).

Overall- Between-Operator agreements ranged from 97.1% to 100% for Method B (Reader Confirmation), and was 100% for Method C (Manual).

Overall- Single-Operator agreements ranged from 96.7% to 100% for Method B (Reader Confirmation), and was 100% for Method C (Manual).

Overall- Instrument to Instrument agreements ranged from 91.3% to 99.2%.

ANCA Formalin

Overall-Between-Lab agreements ranged from 89.8% to 94.7% for Method A (HELIOS), from 95.8% to 98.6% for Method B (Reader Confirmation), and from 98.2% to 99.4% for Method C (Manual).

Overall- Between-Operator agreements ranged from 93.5% to 99% for Method B (Reader Confirmation), and from 97.5% to 100% for Method C (Manual).

Overall- Single-Operator agreements ranged from 92.7% to 99% for Method B (Reader Confirmation), and from 97% to 100% for Method C (Manual).

Overall- Instrument to Instrument agreements ranged from 89% to 90.7%.

c) Lot to Lot Precision/Reproducibility Study AESKUSLIDES® ANCA A lot to lot reproducibility study was performed using three reagent lots each for AESKUSLIDES® ANCA Ethanol and ANCA Formalin. 12 sera were tested:

| Sample
ID | Result | Pattern ANCA
Ethanol | Grading | Result | Pattern ANCA
Formalin | Grading |
|--------------|--------|-------------------------|-----------------|--------|--------------------------|-----------------|
| 1 | Pos | P-ANCA | high positive | Pos | C-ANCA | high positive |
| 2 | Pos | P-ANCA | medium positive | Pos | C-ANCA | medium positive |
| 3 | Pos | P-ANCA | high positive | Pos | C-ANCA | high positive |
| 4 | Pos | P-ANCA | medium positive | Pos | C-ANCA | medium positive |
| 5 | Pos | C-ANCA | high positive | Pos | C-ANCA | high positive |
| 6 | Pos | C-ANCA | medium positive | Pos | C-ANCA | medium positive |
| 7 | Pos | C-ANCA | high positive | Pos | C-ANCA | high positive |
| 8 | Pos | C-ANCA | low positive | Pos | C-ANCA | low positive |
| 9 | Pos | A-ANCA | high positive | Neg | Neg | negative |
| 10 | Pos | A-ANCA | medium positive | Neg | Neg | negative |
| 11 | Neg | Neg | negative | Neg | Neg | negative |
| 12 | Neg | Neg | negative | Neg | Neg | negative |

Table 73: Serum sample set for Lot to Lot Precision Study

43

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Each of the 12 serum samples was assayed 10 times on each reagent lot to give a total of 30 replicates per serum sample.

Slides were processed manually according to the IFU and subsequently analyzed at the microscope by two independent readers.

Agreements were calculated between the three reagent lots (lot 1 vs lo2 , lot 2 vs lot 3, lot 1 vs lot 3), for individual as well as for both readers combined.

Acceptance Criteria:

Positive sera have to be found positive, and negative sera have to be found negative.
-Correct pattern has to be found.
-Reported FI is allowed to differ max. ± 1 level from the expected value.
-Agreements should meet the following criteria:

Type of Agreement	% Agreement
Positive Agreement	> 85%
Negative Agreement	> 85%
Overall Agreement	> 85%
Total Pattern Agreement	> 85%
Single Pattern Agreement P-ANCA	> 85%
Single Pattern Agreement C-ANCA	> 85%
Single Pattern % Agreement A-ANCA	> 85%
Fluorescence Intensity Agreement	> 85%

Results:

Table 74a: Lot to Lot Precision AESKUSLIDES ANCA Ethanol, Lot to lot agreements – Lot 1 vs Lot 2:

| % Agreement (95% CI)
Lot 1 vs Lot 2 | Reader 1 | Reader 2 | Combined
Readers |
|----------------------------------------------------|-----------------------|-----------------------|-----------------------|
| Positive agreement
(97.1 - 100) | 100
(97.1 - 100) | 92.9
(87.4 - 96.1) | 96.3
(93.3 - 98) |
| Negative agreement
(88.6 - 100) | 100
(88.6 - 100) | 100
(83.9 - 100) | 100
(92.9 - 100) |
| Overall agreement
(97.7 - 100) | 100
(97.7 - 100) | 93.8
(88.9 - 96.6) | 96.9
(94.3 - 98.3) |
| Single Pattern C-ANCA
agreement
(92.9 - 100) | 100
(92.9 - 100) | 100
(92.9 - 100) | 100
(96.3 - 100) |
| Single Pattern P-ANCA
agreement
(94 - 100) | 100
(94 - 100) | 100
(94 - 100) | 100
(96.9 - 100) |
| Single Pattern A-ANCA
agreement
(83.9 - 100) | 100
(83.9 - 100) | 100
(83.9 - 100) | 100
(91.2 - 100) |
| Total Pattern agreement
(97.1 - 100) | 100
(97.1 - 100) | 100
(97.1 - 100) | 100
(98.5 - 100) |
| Fl agreement
(96.5 - 99.9) | 99.4
(96.5 - 99.9) | 98.8
(95.6 - 99.7) | 99.1
(97.3 - 99.7) |

44

Table 74b: Lot to Lot Precision AESKUSLIDES ANCA Ethanol, Lot to lot agreements – Lot 2 vs Lot 3:

| % Agreement (95% CI)
Lot 2 vs Lot 3 | Reader 1 | Reader 2 | Combined
Readers |
|----------------------------------------|------------------------------------|-----------------------|---------------------|
| Positive agreement | 100
(97.1 - 100) | 100
(97.1 - 100) | 100
(98.5 - 100) |
| | Negative agreement | 100
(88.6 - 100) | 100
(88.6 - 100) |
| Overall agreement | | 100
(97.7 - 100) | 100
(97.7 - 100) |
| | Single Pattern C-ANCA
agreement | 100
(92.9 - 100) | 100
(92.9 - 100) |
| Single Pattern P-ANCA
agreement | | 100
(94 - 100) | 100
(94 - 100) |
| | Single Pattern A-ANCA
agreement | 100
(83.9 - 100) | 100
(83.9 - 100) |
| Total Pattern agreement | | 100
(97.1 - 100) | 100
(97.1 - 100) |
| | Fl agreement | 98.1
(94.6 - 99.4) | 97.5
(93.7 - 99) |

FI = Fluorescence Intensity

Table 74c: Lot to Lot Precision AESKUSLIDES ANCA Ethanol, Lot to lot agreements – Lot 1 vs Lot 3:

| % Agreement (95% CI)
Lot 1 vs Lot 3 | Reader 1 | Reader 2 | Combined
Readers |
|----------------------------------------|---------------------|--------------------------|-----------------------|
| Positive agreement | 100
(97.1 - 100) | 92.9
(87.4 -
96.1) | 96.3
(93.3 - 98) |
| Negative agreement | 100
(88.6 - 100) | 100
(83.9 - 100) | 100
(92.9 - 100) |
| Overall agreement | 100
(97.7 - 100) | 93.8
(88.9 -
96.6) | 96.9
(94.3 - 98.3) |
| Single Pattern C-ANCA
agreement | 100
(92.9 - 100) | 100
(92.9 - 100) | 100
(96.3 - 100) |
| Single Pattern P-ANCA
agreement | 100
(94 - 100) | 100
(94 - 100) | 100
(96.9 - 100) |
| Single Pattern A-ANCA
agreement | 100
(83.9 - 100) | 100
(83.9 - 100) | 100
(91.2 - 100) |
| Total Pattern agreement | 100
(97.1 - 100) | 100
(97.1 - 100) | 100
(98.5 - 100) |
| Fl agreement | 100
(97.7 - 100) | 100
(97.7 - 100) | 100
(98.8 - 100) |

45

% Agreement (95% CI)	Lot 1 vs Lot 2	Lot 2 vs Lot 3	Lot 1 vs Lot 3
Positive agreement	96.3
(93.3 - 98)	100
(98.5 - 100)	96.3
(93.3 - 98)
Negative agreement	100
(92.9 - 100)	100
(94 - 100)	100
(92.9 - 100)
Overall agreement	96.9
(94.3 - 98.3)	100
(98.8 - 100)	96.9
(94.3 - 98.3)
Single Pattern C-ANCA
agreement	100
(96.3 - 100)	100
(96.3 - 100)	100
(96.3 - 100)
Single Pattern P-ANCA
agreement	100
(96.9 - 100)	100
(96.9 - 100)	100
(96.9 - 100)
Single Pattern A-ANCA
agreement	100
(91.2 - 100)	100
(91.2 - 100)	100
(91.2 - 100)
Total Pattern agreement	100
(98.5 - 100)	100
(98.5 - 100)	100
(98.5 - 100)
FI agreement	99.1
(97.3 - 99.7)	97.8
(95.6 - 98.9)	100
(98.8 - 100)

Table 75: Overview Lot to lot agreements (combined readers):

FI = Fluorescence Intensity

All agreements fulfilled the above criteria.

Positive agreement ranged from 96.3% to 100% for AESKUSLIDES ANCA Ethanol for both readers.

Negative agreement for ANCA Ethanol was 100% for both readers.

Overall agreement for ANCA Ethanol ranged from 96.9% to 100% for combined readers.

Pattern agreement for ANCA Ethanol was 100% for both readers, for all single pattern and total pattern agreement.

FI agreement for ANCA Ethanol ranged from 97.8% to 100% for combined readers.

46

Image /page/46/Picture/0 description: The image shows the logo for AESKU.DIAGNOSTICS. The logo consists of a green square with a white symbol inside, followed by the text "AESKU.DIAGNOSTICS" in gray. Below the text, there is a tagline that reads "THE DIAGNOSTIC TOOL THAT WORKS" in a smaller, lighter gray font. The symbol inside the green square appears to be a stylized representation of a medical or diagnostic tool.

Table 76a: Lot to Lot Precision AESKUSLIDES ANCA Formalin, Lot to lot agreements – Lot 1 vs Lot 2

| % Agreement (95% CI)
Lot 1 vs Lot 2 | Reader 1 | Reader 2 | Combined
Readers |
|----------------------------------------|-----------------------|-----------------------|-----------------------|
| Positive agreement | 100
(96.6 - 100) | 99.1
(94.9 - 99.8) | 99.5
(97.4 - 99.9) |
| Negative agreement | 98
(89.7 - 99.7) | 94.3
(84.6 - 98.1) | 96.2
(90.5 - 98.5) |
| Overall agreement | 99.4
(96.5 - 99.9) | 97.5
(93.7 - 99) | 98.4
(96.4 - 99.3) |
| Single Pattern C-ANCA agreement | 100
(96.3 - 100) | 100
(96.2 - 100) | 100
(98.1 - 100) |
| Total Pattern agreement | 100
(96.6 - 100) | 100
(96.5 - 100) | 100
(98.2 - 100) |
| Fl agreement | 98.8
(95.6 - 99.7) | 96.9
(92.9 - 98.7) | 97.8
(95.6 - 98.9) |

FI = Fluorescence Intensity

Table 76b: Lot to Lot Precision AESKUSLIDES ANCA Formalin, Lot to lot agreements – Lot 2 vs Lot 3:

| % Agreement (95% CI)
Lot 2 vs Lot 3 | Reader 1 | Reader 2 | Combined
Readers |
|----------------------------------------|-----------------------|-----------------------|-----------------------|
| Positive agreement | 100
(96.6 - 100) | 100
(96.6 - 100) | 100
(98.3 - 100) |
| Negative agreement | 100
(92.9 - 100) | 98
(89.7 - 99.7) | 99
(94.6 - 99.8) |
| Overall agreement | 100
(97.7 - 100) | 99.4
(96.5 - 99.9) | 99.7
(98.3 - 99.9) |
| Single Pattern C-ANCA
agreement | 100
(96.3 - 100) | 100
(96.3 - 100) | 100
(98.1 - 100) |
| Total Pattern agreement | 100
(96.6 - 100) | 100
(96.6 - 100) | 100
(98.3 - 100) |
| Fl agreement | 99.4
(96.5 - 99.9) | 98.8
(95.6 - 99.7) | 99.1
(97.3 - 99.7) |

47

Table 76a: Lot to Lot Precision AESKUSLIDES ANCA Formalin, Lot to lot agreements – Lot 1 vs Lot 3:

| % Agreement (95% CI)
Lot 1 vs Lot 3 | Reader 1 | Reader 2 | Combined
Readers |
|----------------------------------------|-----------------------|-----------------------|-----------------------|
| Positive agreement | 100
(96.6 - 100) | 100
(96.5 - 100) | 100
(98.3 - 100) |
| Negative agreement | 98
(89.7 - 99.7) | 94.3
(84.6 - 98.1) | 96.2
(90.5 - 98.5) |
| Overall agreement | 99.4
(96.5 - 99.9) | 98.1
(94.6 - 99.4) | 98.8
(96.8 - 99.5) |
| Single Pattern C-ANCA
agreement | 100
(96.3 - 100) | 100
(96.2 - 100) | 100
(98.1 - 100) |
| Total Pattern agreement | 100
(96.6 - 100) | 100
(96.5 - 100) | 100
(98.3 - 100) |
| Fl agreement | 98.1
(94.6 - 99.4) | 96.9
(92.9 - 98.7) | 97.5
(95.1 - 98.7) |

FI = Fluorescence Intensity

Table 77: Overview Lot to Lot agreements (combined readers):

% Agreement (95% CI)	Lot 1 vs Lot 2	Lot 2 vs Lot 3	Lot 1 vs Lot 3
Positive agreement	99.5
(97.4 - 99.9)	100
(98.3 - 100)	100
(98.3 - 100)
Negative agreement	96.2
(90.5 - 98.5)	99
(94.6 - 99.8)	96.2
(90.5 - 98.5)
Overall agreement	98.4
(96.4 - 99.3)	99.7
(98.3 - 99.9)	98.8
(96.8 - 99.5)
Single Pattern C-ANCA agreement	100
(98.1 - 100)	100
(98.1 - 100)	100
(98.1 - 100)
Total Pattern agreement	100
(98.2 - 100)	100
(98.3 - 100)	100
(98.3 - 100)
FI agreement	97.8
(95.6 - 98.9)	99.1
(97.3 - 99.7)	97.5
(95.1 - 98.7)

FI = Fluorescence Intensity

All agreements fulfilled the above criteria.

Positive agreement for AESKUSLIDES ANCA Formalin ranged from 99.5% to 100% for combined readers.

Negative agreement for ANCA Formalin ranged from 96.2% to 99.0% for both readers.

Overall agreement for ANCA Formalin ranged from 98.4% to 99.7% for combined readers.

Pattern agreement for ANCA Formalin was 100% for both readers, for all single pattern and total pattern agreement.

FI agreement for ANCA Formalin ranged from 97.8% to 99.1%% for combined readers.

48

Image /page/48/Picture/0 description: The image is a logo for AESKU.DIAGNOSTICS. The logo features a green symbol on the left, followed by the text "AESKU.DIAGNOSTICS" in gray. Below the text is the phrase "THE DIAGNOSTIC TOOL THAT WORKS" in a smaller, lighter gray font.

Conclusion:

Overall agreement for AESKUSLIDES ANCA Ethanol ranged from 96.9% to 100% and was acceptable. Overall agreement for AESKUSLIDES ANCA Formalin ranged from 98.4% to 99.7% and was acceptable.

Carry over:

To investigate and evaluate the carry over of samples from one reaction well to the following reaction well when slides are processed by HELIOS.

Three high positive samples were run on HELIOS in alternate with a negative serum sample. Results were confirmed by a reader. Tests were performed in accordance with the respective IFUs.

Sample ID	Pos/Neg	AESKUSLIDES ANCA Ethanol		AESKUSLIDES ANCA Formalin
		Pattern ANCA Ethanol	Grading	Pos/Neg	Pattern ANCA Formalin	Grading
1	Pos	P-ANCA	high positive	Pos	C-ANCA	high positive
2	Pos	C-ANCA	high positive	Pos	C-ANCA	high positive
3	Pos	P-ANCA	high positive	Pos	C-ANCA	high positive
4	Neg	Neg	negative	Neg	Neg	negative

Table 78: Serum sample set for Carry Over study

Acceptance criteria:

-Pos/Neg/Overall Agreement: All positive sera have to be found positive and all negative sera have to be found negative.
-All patterns have to be found correctly.
-FI is allowed to differ maximum + or - 1 from the expected value.

Results:

Table 79: Results Carry over for AESKUSLIDES ANCA

| Sample

ID	AESKUSLIDES ANCA Ethanol			AESKUSLIDES ANCA Formalin
	Result	Pattern	FI	Result	Pattern	FI
1	Pos	P	3	Pos	C	3
4	Neg	Neg	0	Neg	Neg	0
4	Neg	Neg	0	Neg	Neg	0
2	Pos	C	3	Pos	C	3
4	Neg	Neg	0	Neg	Neg	0
4	Neg	Neg	0	Neg	Neg	0
3	Pos	P	3	Pos	C	3
4	Neg	Neg	0	Neg	Neg	0
4	Neg	Neg	0	Neg	Neg	0

All samples fulfilled the above criteria. No carry over has been observed from well to well. All positive samples were identified as positive. All negative samples were identified as negative. All patterns have been identified correctly.

49

Image /page/49/Picture/0 description: The image contains the logo for AESKU.DIAGNOSTICS. The logo features a green square with a white symbol inside, followed by the text "AESKU.DIAGNOSTICS" in gray. Below the text, there is a tagline that reads "THE DIAGNOSTIC TOOL THAT WORKS" in a lighter shade of gray.

Conclusions

No carry over could be observed.

Time Extension Study

To investigate the stability of AESKUSLIDES ANCA Ethanol and ANCA Formalin performance upon shortening and elongation of the recommended incubation times for samples and conjugate.

Procedure:

AESKUSLIDES ANCA Ethanol and ANCA Formalin was assayed with 10 samples on HELIOS with the following incubation times:

1. 15 min sera incubation + 15min incubation of conjugate
1. 30 min sera incubation + 30min incubation of conjugate
1. 45 min sera incubation + 45min incubation of conjugate

Tabel 80: Samples used for Time Extension

| Sample

ID	Pos/Neg	AESKUSLIDES ANCA Ethanol		AESKUSLIDES ANCA Formalin
		Pattern
ANCA
Ethanol	Grading	Pos/Neg	Pattern
ANCA
Formalin	Grading
1	Pos	P-ANCA	high positive	Pos	C-ANCA	high positive
2	Pos	P-ANCA	medium positive	Pos	C-ANCA	medium positive
3	Pos	P-ANCA	high positive	Pos	C-ANCA	high positive
4	Pos	P-ANCA	medium positive	Pos	C-ANCA	medium positive
5	Pos	P-ANCA	medium positive	Pos	C-ANCA	medium positive
6	Pos	C-ANCA	high positive	Pos	C-ANCA	high positive
7	Pos	C-ANCA	medium positive	Pos	C-ANCA	medium positive
8	Pos	C-ANCA	medium positive	Pos	C-ANCA	medium positive
9	Neg	Neg	negative	Neg	Neg	negative
10	Neg	Neg	negative	Neg	Neg	negative

Tests were performed in accordance with the respective IFUs. Results have been confirmed by a trained reader.

Results of the 30 min protocol will be compared to the results of the 15 min and 45 min protocol.

Acceptance criteria:

-Pos/Neg/Overall Agreement: All positive sera have to be found positive and all negative sera have to be found negative.
-FI is allowed to differ maximum + or - 1 from the expected value.

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AESKUSLIDES ANCA 510(k) Traditional Submission

Results

| Incubation

Times	2 x 15 Minutes			2 x 30 Minutes			2 x 45 Minutes
Sample ID	Result	Pattern	FI	Result	Pattern	FI	Result	Pattern	FI
PC-C	Pos	C	3	Pos	C	3	Pos	C	3
PC-P	Pos	P	3	Pos	P	3	Pos	P	3
NC	Neg	Neg	Neg	Neg	Neg	Neg	Neg	Neg	Neg
Sample 1	Pos	P	3	Pos	P	3	Pos	P	3
Sample 2	Pos	P	2	Pos	P	3	Pos	P	2
Sample 3	Pos	P	3	Pos	P	3	Pos	P	3
Sample 4	Pos	P	3	Pos	P	3	Pos	P	3
Sample 5	Pos	P	2	Pos	P	3	Pos	P	3
Sample 6	Pos	C	3	Pos	C	3	Pos	C	3
Sample 7	Pos	C	2	Pos	C	2	Pos	C	2
Sample 8	Pos	C	3	Pos	C	3	Pos	C	3
Sample 9	Neg	Neg	Neg	Neg	Neg	Neg	Neg	Neg	Neg
Sample 10	Neg	Neg	Neg	Neg	Neg	Neg	Neg	Neg	Neg

Table 81: Results Time extension study AESKUSLIDES ANCA Ethanol

FI = Fluorescence Intensity

| Incubation

Times	2 x 15 Minutes			2 x 30 Minutes			2 x 45 Minutes
Sample ID	Result	Pattern	FI	Result	Pattern	FI	Result	Pattern	FI
PC-C	Pos	C	3	Pos	C	3	Pos	C	3
PC-P	Pos	C	3	Pos	C	3	Pos	C	3
NC	Neg	Neg	Neg	Neg	Neg	Neg	Neg	Neg	Neg
Sample 1	Pos	C	3	Pos	C	3	Pos	C	3
Sample 2	Pos	C	2	Pos	C	3	Pos	C	2
Sample 3	Pos	C	3	Pos	C	2	Pos	C	3
Sample 4	Pos	C	3	Pos	C	3	Pos	C	3
Sample 5	Pos	C	2	Pos	C	3	Pos	C	2
Sample 6	Pos	C	3	Pos	C	3	Pos	C	3
Sample 7	Pos	C	3	Pos	C	3	Pos	C	3
Sample 8	Pos	C	3	Pos	C	3	Pos	C	3
Sample 9	Neg	Neg	Neg	Neg	Neg	Neg	Neg	Neg	Neg
Sample 10	Neg	Neg	Neg	Neg	Neg	Neg	Neg	Neg	Neg

Table 82: Results Time extension study AESKUSLIDES ANCA Formalin

FI = Fluorescence Intensity

All acceptance criteria were fulfilled in each assay. All positive samples were identified as positive. All negative samples were identified as negative. All patterns have been found as expected. Fluorescence intensities did not deviate more than +/-1 level.

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Conclusions

The results demonstrate stability of the AESKUSLIDES ANCA Ethanol, as well as AESKUSLIDES ANCA Formalin during the tested incubation times of sample and conjugate.

Detection limit

Not applicable

Analytical specificity

Interfering substances:

The interference study was performed according to CLSI EP07-A2, Interference Testing in Clinical Chemistry; Approved Guideline - Second Edition. Interference by 10 different substances was assessed by testing 14 serum samples.

Table 83: Samples used for Interfering substances study

AESKUSLIDES ANCA Ethanol				AESKUSLIDES ANCA Formalin
Sample
ID	Pos/Neg	Pattern
ANCA
Ethanol	Grading	Pos/Neg	Pattern ANCA
Formalin	Grading
1	Pos	P-ANCA	medium positive	Pos	C-ANCA	medium positive
2	Pos	P-ANCA	high positive	Pos	C-ANCA	high positive
3	Pos	C-ANCA	medium positive	Pos	C-ANCA	medium positive
4	Pos	C-ANCA	low positive	Pos	C-ANCA	high positive
5	Pos	A-ANCA	medium positive	Neg	Neg	negative
6	Pos	A-ANCA	low positive	Neg	Neg	negative
7	Neg	Neg	negative	Neg	Neg	negative
8	Neg	Neg	negative	Neg	Neg	negative
9	Pos	P-ANCA	low positive	Pos	C-ANCA	low positive
10	Pos	P-ANCA	low positive	Pos	C-ANCA	low positive
11	Pos	P-ANCA	low positive	Pos	C-ANCA	low positive
12	Pos	C-ANCA	high positive	Pos	C-ANCA	high positive
13	Pos	C-ANCA	low positive	Pos	C-ANCA	low positive
14	Pos	C-ANCA	low positive	Pos	C-ANCA	low positive

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The following substances have been tested with the indicated concentrations:

Table 84: Concentrations of Interfering substances
Interfering Substance	Minimum final
Concentration
tested	Maximum final
Concentration
tested
Bilirubin conjugated	0.1 mg/mL	0.4 mg/mL
Bilirubin unconjugated	0.1 mg/mL	0.4 mg/mL
Hemoglobin	2.5 mg/mL	5.0 mg/mL
Triglycerides	5 mg/mL	20 mg/mL
RF IgM	200 IU/mL	400 IU/mL
Rituximab	0.5 mg/mL	2.0 mg/mL
Methylprednisolone	0.2 mg/mL	0.8 mg/mL
Cyclophosphamide	1.0 mg/mL	4.0 mg/mL
Methotrexate	0.025 mg/mL	0.1 mg/mL
Azathioprine	0.0075 mg/mL	0.03 mg/mL

Table 84: Concentrations of Interfering substances

Interfering substances were spiked in each of the 14 samples in two different concentrations. Controls were prepared for each serum sample by spiking in only the respective amount of diluent without interfering substances. Spiked serum samples and their controls have been tested in triplicates for each concentration of interferent with AESKUSLIDES ANCA Ethanol and ANCA Formalin. All tests have been performed manually according to the IFUs. Results have been analyzed by two independent readers at the microscope.

The results (positive/negative classification, pattern, fluorescence intensity (FI)) of the spiked samples were compared to the results of the respective controls. Agreements have been calculated by dividing the number of correctly found results through the number of total results for spiked samples.

Acceptance criteria:

-Pos/Neq/Overall Agreement: All positive sera have to be found positive and all negative sera have to be found negative.
-Overall Agreement: > 90%.
-All patterns have to be found correctly. Pattern Agreement: > 90%
-FI is allowed to differ maximum + or - 1 from the expected value. FI Agreement: > 90%.

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Results

All samples fulfilled the above criteria. No significant differences were observed between sera spiked with different concentrations of the interferents and their controls:

ANCA Ethanol:

Positive Agreement ranged from 97% to 100% for all tested interferents and ● concentrations. It was 100% for all tested substances and concentrations, except for 5 mg/ml
Triglycerides with Reader 2 (97%), 200 IU/ml RF with both readers (99%), and 1 mg/ml Cyclophosphamide with both readers (99%).
Negative Agreement was 100% for all tested interferents and concentrations.
Overall Agreement ranged from 98% to 100% for all tested interferents and concentrations. It was 100% for all tested substances and concentrations, except for 5 mg/ml Triglycerides with Reader 2 (98%), 200 IU/ml RF with both readers (99%), and 1 mg/ml Cyclophosphamide with both readers (99%).
. Pattern Agreement ranged from 97% to 100% for all tested interferents and concentrations. It was 100% for all tested substances and concentrations, except for 5 mg/ml Triglycerides with Reader 2 (97%), 200 IU/ml RF with both readers (99%), 1 mq/ml Cyclophosphamide with both readers (99%), and 0.0075 mg/ml Azathioprine with both readers (99%).
. FI agreement ranged from 98% to 100% for all tested interferents and concentrations. It was 100% for all tested substances and concentrations, except for 5 mg/ml Triglycerides with Reader 2 (98%), and 1 mq/ml Cyclophosphamide with both readers (99%).

ANCA Formalin:

. Positive Agreement was 100% for all tested interferents and concentrations.
Negative Agreement ranged from 92% to 100% for all tested interferents and . concentrations. It was 100% for all tested substances and concentrations, except for 0.025 mq/ml Methotrexate with Reader 1 (92%) and with Reader 2 (96%).
Overall Agreement ranged from 98% to 100% for all tested interferents and . concentrations. It was 100% for all tested substances and concentrations, except for 0.025 mg/ml Methotrexate with Reader 1 (98%) and with Reader 2 (99%).
Pattern Agreement was 100% for all tested interferents and concentrations. ●

54

FI Agreement was 100% for all tested interferents and concentrations. ●

Conclusions

All criteria are fulfilled in each test. No interference was detected with the tested substances, up to the maximal concentrations indicated above.

Assay cut-off:

AESKU recommends a screening dilution of 1:20. followed by serial dilutions for semiquantitative determinations. but suggests each laboratory establish its own screening dilution and titration scheme based on its population. The titers of 1:10 and 1:20 are considered low titers. 1:40 and 1:80 are considered medium titers. and 1:160 and greater are considered high titers.

Stability studies

Accelerated Stability Report

3 lots of complete kits (including slides, controls, conjugate, & sample buffer) of AESKUSLIDES ANCA Ethanol and Formalin were stored at 37°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 the same set of 8 samples:

Sample ID	AESKUSLIDES ANCA Ethanol			AESKUSLIDES ANCA Formalin
	Pos/Neg	Pattern ANCA Ethanol	Grading	Pos/Neg	Pattern ANCA Formalin	Grading
1	Pos	C-ANCA	high positive	Pos	C-ANCA	high positive
2	Pos	C-ANCA	medium positive	Pos	C-ANCA	medium positive
3	Pos	C-ANCA	low positive	Pos	C-ANCA	low positive
4	Pos	P-ANCA	high positive	Pos	C-ANCA	medium positive
5	Pos	P-ANCA	medium positive	Pos	C-ANCA	borderline positive
6	Pos	P-ANCA	low positive	Pos	Neg	Neg
7	Pos	A-ANCA	medium positive	Neg	Neg	Neg
8	Neg	Neg	Neg	Neg	Neg	Neg

Slides were processed manually according to the IFU and subsequently analyzed at the microscope by two independent readers.

The stability data is covering the stability of all components of the kit. Agreements were calculated for individual and combined readers. Results of the kit controls were not included in the agreement calculations.

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Acceptance Criteria:

. Positive sera have to be found positive, and negative sera have to be found negative.
Correct pattern has to be found. ●
Reported FI is allowed to differ max. ± 1 level from the expected value. .
Agreements should meet the following criteria: ●

Type of Agreement	% Agreement
Positive Agreement	> 85%
Negative Agreement	> 85%
Overall Agreement	> 85%
Total Pattern Agreement	> 85%
Fluorescence Intensity Agreement	> 85%

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AESKUSLIDES ANCA 510(k) Traditional Submission

Results:

Table 85: Accelerated Stability AESKUSLIDES ANCA Ethanol – Number of Samples:

| No of samples

(Incorrect/total)	Lot 1			Lot 2			Lot 3
	Reader 1	Reader 2	both
Readers	Reader 1	Reader 2	both
Readers	Reader 1	Reader 2	both
Readers
Positive Agreement	3 / 98	5 / 98	8 / 196	9 / 98	12 / 98	21 / 196	2 / 98	5 / 98	7 / 196
Negative Agreement	1 / 14	1 / 14	2 / 28	0 / 14	0 / 14	0 / 28	0 / 14	0 / 14	0 / 28
Overall Agreement	4 / 112	6 / 112	10 / 224	9 / 112	12 / 112	21 / 224	2 / 112	5 / 112	7 / 224
Pattern Agreement	3 / 98	5 / 98	8 / 196	11 / 98	15 / 98	26 / 196	2 / 98	8 / 98	10 / 196
FI Agreement	3 / 112	4 / 112	7 / 224	4 / 112	6 / 112	10 / 224	2 / 112	2 / 112	4 / 224

FI = Fluorescence Intensity

Table 86: Accelerated Stability AESKUSLIDES ANCA Ethanol – % Agreement:

	Lot 1			Lot 2			Lot 3
% Agreement
(95% CI)	Reader 1	Reader 2	both Readers	Reader 1	Reader 2	both Readers	Reader 1	Reader 2	both Readers
Positive Agreement	96.9
(91.4 - 99)	94.9
(88.6 - 97.8)	95.9
(92.2 - 97.9)	90.8
(83.5 - 95.1)	87.8
(79.8 - 92.9)	89.3
(84.2 - 92.9)	98
(92.9 - 99.4)	94.9
(88.6 - 97.8)	96.4
(92.8 - 98.3)
Negative Agreement	92.9
(68.5 - 98.7)	92.9
(68.5 - 98.7)	92.9
(77.4 - 98)	100
(78.5 - 100)	100
(78.5 - 100)	100
(87.9 - 100)	100
(78.5 - 100)	100
(78.5 - 100)	100
(87.9 - 100)
Overall Agreement	96.4
(91.2 - 98.6)	94.6
(88.8 - 97.5)	95.5
(92 - 97.6)	92
(85.4 - 95.7)	89.3
(82.2 - 93.8)	90.6
(86.1 - 93.8)	98.2
(93.7 - 99.5)	95.5
(90 - 98.1)	96.9
(93.7 - 98.5)
Pattern Agreement	96.9
(91.4 - 99)	94.9
(88.6 - 97.8)	95.9
(92.2 - 97.9)	88.8
(81 - 93.6)	84.7
(76.3 - 90.5)	86.7
(81.3 - 90.8)	98
(92.9 - 99.4)	91.8
(84.7 - 95.8)	94.9
(90.9 - 97.2)
FI Agreement	97.3
(92.4 - 99.1)	96.4
(91.2 - 98.6)	96.9
(93.7 - 98.5)	96.4
(91.2 - 98.6)	94.6
(88.8 - 97.5)	95.5
(92 - 97.6)	98.2
(93.7 - 99.5)	98.2
(93.7 - 99.5)	98.2
(95.5 - 99.3)

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Agreement calculations for AESKUSLIDES ANCA Ethanol included all data points of the whole test time span of 6 weeks at 37°C:

All agreements fulfilled the above criteria.

Positive agreement for AESKUSLIDES ANCA Ethanol for both readers ranged from 89.3% to 96.4% for all 3 reagent lots.

Negative agreement for ANCA Ethanol for both readers ranged from 92.9% to 100% for all 3 reagent lots.

Overall agreement for ANCA Ethanol for both readers ranged from 90.6% to 96.9% for all 3 reagent lots.

Pattern agreement for ANCA Ethanol for both readers ranged from 86.7% to 95.9% for all 3 reagent lots.

FI agreement for ANCA Ethanol for both readers ranged from 95.5% to 98.2% for all 3 reagent lots.

No deviations of fluorescence intensities greater than +/-1 from the initial value have been observed.

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AESKUSLIDES ANCA 510(k) Traditional Submission

Table 87: Accelerated Stability AESKUSLIDES ANCA Formalin - Number of Samples:

| No of samples

(Incorrect/total)	Lot 1			Lot 2			Lot 3
	Reader 1	Reader 2	both
Readers	Reader 1	Reader 2	both
Readers	Reader 1	Reader 2	both
Readers
Positive Agreement	4 / 50	14 / 50	18 / 100	4 / 50	12 / 50	16 / 100	5 / 50	16 / 50	21 / 100
Negative Agreement	0 / 30	0 / 30	0 / 60	0 / 30	0 / 30	0 / 60	0 / 30	0 / 30	0 / 60
Overall Agreement	4 / 80	14 / 80	18 / 160	4 / 80	12 / 80	16 / 160	5 / 80	16 / 80	21 / 160
Pattern Agreement	4 / 50	14 / 50	18 / 100	4 / 50	12 / 50	16 / 100	5 / 50	16 / 50	21 / 100
FI Agreement	0 / 80	3 / 80	3 / 160	0 / 80	2 / 80	2 / 160	1 / 80	4 / 80	5 / 160

FI = Fluorescence Intensity

Table 88: Accelerated Stability AESKUSLIDES ANCA Formalin – % Agreement:

| % Agreement

(95% CI)	Lot 1
	Reader 1	Reader 2	both
Readers	Reader 1	Reader 2	both
Readers	Reader 1	Reader 2	both
Readers
Positive Agreement	92
(81.2 - 96.8)	72
(58.3 - 82.5)	82
(73.3 - 88.3)	92
(81.2 - 96.8)	76
(62.6 - 85.7)	84
(75.6 - 89.9)	90
(78.6 - 95.7)	68
(54.2 - 79.2)	79
(70 - 85.8)
Negative Agreement	100
(88.6 - 100)	100
(88.6 - 100)	100
(94 - 100)	100
(88.6 - 100)	100
(88.6 - 100)	100
(94 - 100)	100
(88.6 - 100)	100
(88.6 - 100)	100
(94 - 100)
Overall Agreement	95
(87.8 - 98)	82.5
(72.7 - 89.3)	88.8
(82.9 - 92.8)	95
(87.8 - 98)	85
(75.6 - 91.2)	90
(84.4 - 93.8)	93.8
(86.2 - 97.3)	80
(70 - 87.3)	86.9
(80.8 - 91.3)
Pattern Agreement	92
(81.2 - 96.8)	72
(58.3 - 82.5)	82
(73.3 - 88.3)	92
(81.2 - 96.8)	76
(62.6 - 85.7)	84
(75.6 - 89.9)	90
(78.6 - 95.7)	68
(54.2 - 79.2)	79
(70 - 85.8)
FI Agreement	100
(95.4 - 100)	96.3
(89.5 - 98.7)	98.1
(94.6 - 99.4)	100
(95.4 - 100)	97.5
(91.3 - 99.3)	98.8
(95.6 - 99.7)	98.8
(93.3 - 99.8)	95
(87.8 - 98)	96.9
(92.9 - 98.7)

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Image /page/59/Picture/0 description: The image contains a logo for AESKU.DIAGNOSTICS. The logo features a green symbol on the left, followed by the text "AESKU.DIAGNOSTICS" in gray. Below the text, there is a tagline that reads "THE DIAGNOSTIC TOOL THAT WORKS" in a smaller, lighter gray font. The overall design is clean and professional, suggesting a company in the medical or diagnostic field.

AESKUSLIDES ANCA 510(k) Traditional Submission

Table 89: Accelerated Stability AESKUSLIDES ANCA Formalin – Number of Samples, borderline positive samples excluded:

| No of samples

(Incorrect/total)	Lot 1			Lot 2			Lot 3
	Reader 1	Reader 2	both
Readers	Reader 1	Reader 2	both
Readers	Reader 1	Reader 2	both
Readers
Positive Agreement	0 / 40	5 / 40	5 / 80	0 / 40	2 / 40	2 / 80	1 / 40	6 / 40	7 / 80
Negative Agreement	0 / 30	0 / 30	0 / 60	0 / 30	0 / 30	0 / 60	0 / 30	0 / 30	0 / 60
Overall Agreement	0 / 70	5 / 70	5 / 140	0 / 70	2 / 70	2 / 140	1 / 70	6 / 70	7 / 140
Pattern Agreement	0 / 40	5 / 40	5 / 80	0 / 40	2 / 40	2 / 80	1 / 40	6 / 40	7 / 80
FI Agreement	0 / 70	3 / 70	3 / 140	0 / 70	2 / 70	2 / 140	1 / 70	4 / 70	5 / 140

FI = Fluorescence Intensity

Table 90: Accelerated Stability AESKUSLIDES ANCA Formalin – % Agreement, borderline positive samples excluded:

| % Agreement

(95% CI)	Lot 1
	Reader 1	Reader 2	both
Readers	Reader 1	Reader 2	both
Readers	Reader 1	Reader 2	both
Readers
Positive Agreement	100
(91.2 - 100)	87.5
(73.9 - 94.5)	93.8
(86.2 - 97.3)	100
(91.2 - 100)	95
(83.5 - 98.6)	97.5
(91.3 - 99.3)	97.5
(87.1 - 99.6)	85
(70.9 - 92.9)	91.3
(83 - 95.7)
Negative Agreement	100
(88.6 - 100)	100
(88.6 - 100)	100
(94 - 100)	100
(88.6 - 100)	100
(88.6 - 100)	100
(94 - 100)	100
(88.6 - 100)	100
(88.6 - 100)	100
(94 - 100)
Overall Agreement	100
(94.8 - 100)	92.9
(84.3 - 96.9)	96.4
(91.9 - 98.5)	100
(94.8 - 100)	97.1
(90.2 - 99.2)	98.6
(94.9 - 99.6)	98.6
(92.3 - 99.7)	91.4
(82.5 - 96)	95
(90 - 97.6)
Pattern Agreement	100
(91.2 - 100)	87.5
(73.9 - 94.5)	93.8
(86.2 - 97.3)	100
(91.2 - 100)	95
(83.5 - 98.6)	97.5
(91.3 - 99.3)	97.5
(87.1 - 99.6)	85
(70.9 - 92.9)	91.3
(83 - 95.7)
FI Agreement	100
(95.4 - 100)	96.3
(89.5 - 98.7)	97.9
(93.9 - 99.3)	100
(95.4 - 100)	97.5
(91.3 - 99.3)	98.6
(94.9 - 99.6)	98.8
(93.3 - 99.8)	95
(87.8 - 98)	96.4
(91.9 - 98.5)

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Agreement calculations for AESKUSLIDES ANCA Formalin included all data points of a test time span of 4 weeks at 37°C:

The sample set for AESKUSLIDES ANCA Formalin included one sample (sample 5) that was borderline positive. For this sample we expect either a low positive result (Fl=1) or a negative result (Fl=0). Therefore, this sample leads to many false negatives and therefore has a big impact on positive agreement and on pattern agreement which is not due to stability issues of the assay.

Therefore, we did an alternative agreement calculation excluding this borderline positive sample. In this calculation all acceptance criteria have been met:

Positive agreement for AESKUSLIDES ANCA Formalin for both readers ranged from 91.3% to 97.5% for all 3 reagent lots.

Negative agreement for ANCA Formalin for both readers was 100% for all 3 reagent lots.

Overall agreement for ANCA Formalin for both readers ranged from 95% to 98.6% for all 3 reagent lots.

Pattern agreement for ANCA Formalin for both readers ranged from 91.3% to 97.5% for all 3 reagent lots.

FI agreement for ANCA Formalin for both readers ranged from 96.4% to 98.6% for all 3 reagent lots.

No deviations of fluorescence intensities greater than +/-1 from the initial value have been observed.

Conclusion:

For AESKUSLIDES ANCA Ethanol all acceptance criteria were met for a time span of 6 weeks at 37°C. Therefore, according to Arrhenius equation, we claim a shelf life of at least 24 months (24+3 months) at 2-8°C.

For AESKUSLIDES ANCA Formalin all acceptance criteria were met for a time span of 4 weeks at 37°C. Therefore, according to Arrhenius equation, we claim a shelf life of at least 18 months at 2-8°C.

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Real Time Stability Report

Shelf life of AESKUSLIDES ANCA Ethanol has been claimed to be at least 24 months at 2-8°C storage temperature based on the results of the Accelerated Stability Study of AESKUSLIDES ANCA Ethanol. Shelf life of AESKUSLIDES ANCA Formalin has been claimed to be at least 18 months at 2-8°C storage temperature based on the results of the Accelerated Stability Study of AESKUSLIDES ANCA Formalin. To confirm this, a real time / long term stability study over a time period of 27 months is conducted.

Procedure:

Three different reagent lots of AESKUSLIDES ANCA Ethanol and ANCA Formalin will be stored at 2-8°C over a time period of 27 months. The same eight sera will be assayed at each of the following time points:

Starting point of the stability study to
3 months after to t
6 months after to t2
12 months after to t3
18 months after to t4
24 months after to t5
27 months after to te

Sample description:

			AESKUSLIDES ANCA Ethanol	AESKUSLIDES ANCA Formalin
Sample ID	Expected
Result	Expected
Pattern	Grading	Expected
Result	Expected
Pattern	Grading
Sample 1	Pos	C	high positive	Pos	C	high positive
Sample 2	Pos	C	medium positive	Pos	C	medium positive
Sample 3	Pos	C	low positve	Pos	C	low posiitve
Sample 4	Pos	P	high positive	Pos	C	medium positive
Sample 5	Pos	P	medium positive	Pos	C	low posiitve
Sample 6	Pos	P	low positve	Neg	Neg	negative
Sample 7	Pos	A	medium positive	Neg	Neg	negative
Sample 8	Neg	Neg	negative	Neg	Neg	negative

Each serum is tested in triplicates in each run.

At time point to three runs will be performed for each of the three different kit lots to give a total of 9 repetitions per serum per lot.

At the following time points (t, to ts) only two runs will be performed for each of the three different kit lots to give a total of 6 repetitions per serum per lot.

All tests are performed manually according to the IFU. Results are analyzed by two independent readers at the microscope.

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The results (pos/neg, pattern, Fl) at each time point and for each sample are compared to the respective results, determined at start of the stability study (to) and will be displayed as % agreements (pos/neq/overall agreement, pattern agreement. Fl agreement).

The results of both readers are combined for agreement calculations. Results of the kit controls are not included in the agreement calculations.

The stability data is covering the stability of all (undiluted) components of the kit. For each test time point only the minimal needed amount of concentrated wash buffer will be removed from the original bottle and diluted to give 1x wash buffer.

Acceptance criteria:

Positive/Neqative /Overall Agreement: All positive sera have to be found positive and all negative sera have to be found negative. Agreement is calculated from the number of correctly and incorrectly found samples, for positive samples (positive agreement), negative samples (negative agreement) and total samples (overall agreement).

Positive/Negative/Overall Agreement > 85%.

Patterns have to be found correctly.

Pattern Agreement > 85%.

FI is allowed to differ maximum + or - 1 from the expected value. Agreement is calculated from the number of correctly and incorrectly found FI values (of all tested samples).

FI Agreement > 85%.

Interpretation for Real time stability:

Acceptance criteria have to be fulfilled over the whole time period.

Results:

At the time of preparation of this report, only the results for time points to and to have been acquired so far, covering a time period of 3 months at 2-8°C.

Real time stability study is still ongoing. Further time points to be tested are 6, 12, 18, 24, and 27 months.

The results of the In Use Stability Study of AESKUSLIDES ANCA Ethanol and ANCA Formalin described elsewhere, can also serve as a real time stability study, covering a time period of 6 weeks at 2-8°C. In this In Use study the same sera have been used as for the herein described real time / long term stability study.

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AESKUSLIDES ANCA 510(k) Traditional Submission

Number of Samples	Lot 1			Lot 2			Lot 3
	Found Correctly	Found Incorrectly	Expected Numbers	Found Correctly	Found Incorrectly	Expected Numbers	Found Correctly	Found Incorrectly	Expected Numbers
Positive Agreement	101	4	105	105	0	105	105	0	105
Negative Agreement	15	0	15	15	0	15	15	0	15
Overall Agreement	116	4	120	120	0	120	120	0	120
Pattern Agreement	101	4	105	105	0	105	104	1	105
FI Agreement	119	1	120	120	0	120	120	0	120

Table 91: Real time Stability AESKUSLIDES ANCA Ethanol (Reader 1) - Number of Samples:

FI = Fluorescence Intensity

Table 92: Real time Stability AESKUSLIDES ANCA Ethanol (Reader 2) – Number of Samples:

Number of Samples	Reader 2
	Lot 1			Lot 2			Lot 3
	Found
Correctly	Found Incorrectly	Expected
Numbers	Found
Correctly	Found
Incorrectly	Expected
Numbers	Found
Correctly	Found
Incorrectly	Expected
Numbers
Positive Agreement	98	7	105	105	0	105	101	4	105
Negative Agreement	14	1	15	15	0	15	15	0	15
Overall Agreement	112	8	120	120	0	120	116	4	120
Pattern Agreement	98	7	105	105	0	105	100	5	105
FI Agreement	119	1	120	120	0	120	120	0	120

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AESKUSLIDES ANCA 510(k) Traditional Submission

Table 93: Real time Stability AESKUSLIDES ANCA Ethanol (Combined Readers) – Number of Samples:

Number of Samples	Combined Readers
	Lot 1			Lot 2			Lot 3
	Found
Correctly	Found
Incorrectly	Expected
Numbers	Found
Correctly	Found
Incorrectly	Expected
Numbers	Found
Correctly	Found
Incorrectly	Expected
Numbers
Positive Agreement	199	11	210	210	0	210	206	4	210
Negative Agreement	29	1	30	30	0	30	30	0	30
Overall Agreement	228	12	240	240	0	240	236	4	240
Pattern Agreement	199	11	210	210	0	210	204	6	210
FI Agreement	238	2	240	240	0	240	240	0	240

65

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| % Agreement

(95% CI)	Lot 1	Lot 2	Lot 3
Positive Agreement	96.2
(90.6 - 98.5)	100
(96.5 - 100)	100
(96.5 - 100)
Negative Agreement	100
(79.6 - 100)	100
(79.6 - 100)	100
(79.6 - 100)
Overall Agreement	96.7
(91.7 - 98.7)	100
(96.9 - 100)	100
(96.9 - 100)
Pattern Agreement	96.2
(90.6 - 98.5)	100
(96.5 - 100)	99
(94.8 - 99.8)
FI Agreement	99.2
(95.4 - 99.9)	100
(96.9 - 100)	100
(96.9 - 100)

Table 94: Real time Stability AESKUSLIDES ANCA Ethanol (Reader 1) – %Agreement:

FI = Fluorescence Intensity

Table 95: Real time Stability AESKUSLIDES ANCA Ethanol (Reader 2) – %Agreement:

| % Agreement

(95% CI)	Reader 2
	Lot 1	Lot 2	Lot 3
Positive Agreement	93.3
(86.9 - 96.7)	100
(96.5 - 100)	96.2
(90.6 - 98.5)
Negative Agreement	93.3
(70.2 - 98.8)	100
(79.6 - 100)	100
(79.6 - 100)
Overall Agreement	93.3
(87.4 - 96.6)	100
(96.9 - 100)	96.7
(91.7 - 98.7)
Pattern Agreement	93.3
(86.9 - 96.7)	100
(96.5 - 100)	95.2
(89.3 - 97.9)
FI Agreement	99.2
(95.4 - 99.9)	100
(96.9 - 100)	100
(96.9 - 100)

FI = Fluorescence Intensity

Table 96: Real time Stability AESKUSLIDES ANCA Ethanol (Combined Readers) – %Agreement:

| % Agreement

(95% CI)	Combined Readers
	Lot 1	Lot 2	Lot 3
Positive Agreement	94.8
(90.9 - 97.1)	100
(98.2 - 100)	98.1
(95.2 - 99.3)
Negative Agreement	96.7
(83.3 - 99.4)	100
(88.6 - 100)	100
(88.6 - 100)
Overall Agreement	95
(91.5 - 97.1)	100
(98.4 - 100)	98.3
(95.8 - 99.4)
Pattern Agreement	94.8
(90.9 - 97.1)	100
(98.2 - 100)	97.1
(93.9 - 98.7)
FI Agreement	99.2
(97 - 99.8)	100
(98.4 - 100)	100
(98.4 - 100)

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Agreement calculations for AESKUSLIDES ANCA Ethanol included all data points of the test over a time period of 3 months at 2-8°C:

All agreements fulfilled the above criteria.

Positive agreement for AESKUSLIDES ANCA Ethanol for both readers ranged from 94.8% to 100% for all 3 reagent lots.

Negative agreement for ANCA Ethanol for both readers ranged from 96.7% to 100% for all 3 reagent lots.

Overall agreement for ANCA Ethanol for both readers ranged from 95.0% to 100% for all 3 reagent lots.

Pattern agreement for ANCA Ethanol for both readers ranged from 94.8% to 100% for all 3 reagent lots.

FI agreement for ANCA Ethanol for both readers ranged from 99.2% to 100% for all 3 reagent lots.

Just one deviation of fluorescence intensity greater than +/-1 from the initial value has been observed for each reader.

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AESKUSLIDES ANCA 510(k) Traditional Submission

Number of Samples	Reader 1
	Lot 1			Lot 2			Lot 3
	Found Correctly	Found Incorrectly	Expected Numbers	Found Correctly	Found Incorrectly	Expected Numbers	Found Correctly	Found Incorrectly	Expected Numbers
Positive Agreement	72	3	75	71	4	75	67	8	75
Negative Agreement	45	0	45	45	0	45	44	1	45
Overall Agreement	117	3	120	116	4	120	111	9	120
Pattern Agreement	72	3	75	71	4	75	67	8	75
FI Agreement	120	0	120	120	0	120	120	0	120

Table 97: Real time Stability AESKUSLIDES ANCA Formalin (Reader 1) – Number of Samples:

FI = Fluorescence Intensity

Table 98: Real time Stability AESKUSLIDES ANCA Formalin (Reader 1) – Number of Samples:

Number of Samples	Reader 2
	Lot 1			Lot 2			Lot 3
	Found
Correctly	Found
Incorrectly	Expected
Numbers	Found
Correctly	Found
Incorrectly	Expected
Numbers	Found
Correctly	Found
Incorrectly	Expected
Numbers
Positive Agreement	64	11	75	64	11	75	65	10	75
Negative Agreement	45	0	45	45	0	45	44	1	45
Overall Agreement	109	11	120	109	11	120	109	11	120
Pattern Agreement	64	11	75	64	11	75	65	10	75
FI Agreement	120	0	120	119	1	120	120	0	120

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AESKUSLIDES ANCA 510(k) Traditional Submission

Table 99: Real time Stability AESKUSLIDES ANCA Formalin (Combined Readers) – Number of Samples:

Number of Samples	Combined Readers
	Lot 1			Lot 2			Lot 3
	Found
Correctly	Found
Incorrectly	Expected
Numbers	Found
Correctly	Found
Incorrectly	Expected
Numbers	Found
Correctly	Found
Incorrectly	Expected
Numbers
Positive Agreement	136	14	150	135	15	150	132	18	150
Negative Agreement	90	0	90	90	0	90	88	2	90
Overall Agreement	226	14	240	225	15	240	220	20	240
Pattern Agreement	136	14	150	135	15	150	132	18	150
FI Agreement	240	0	240	239	1	240	240	0	240

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| % Agreement
(95% CI) | Reader 1
Lot 1 | Lot 2 | Lot 3 |
|-------------------------|-----------------------|-----------------------|-----------------------|
| Positive Agreement | 96
(88.9 - 98.6) | 94.7
(87.1 - 97.9) | 89.3
(80.3 - 94.5) |
| Negative Agreement | 100
(92.1 - 100) | 100
(92.1 - 100) | 97.8
(88.4 - 99.6) |
| Overall Agreement | 97.5
(92.9 - 99.1) | 96.7
(91.7 - 98.7) | 92.5
(86.4 - 96) |
| Pattern Agreement | 96
(88.9 - 98.6) | 94.7
(87.1 - 97.9) | 89.3
(80.3 - 94.5) |
| FI Agreement | 100
(96.9 - 100) | 100
(96.9 - 100) | 100
(96.9 - 100) |

Table 100: Real time Stability AESKUSLIDES ANCA Formalin (Reader 1) – %Agreement:

FI = Fluorescence Intensity

Table 101: Real time Stability AESKUSLIDES ANCA Formalin (Reader 2) – %Agreement:

| % Agreement

(95% CI)	Lot 1	Lot 2	Lot 3
Positive Agreement	85.3
(75.6 - 91.6)	85.3
(75.6 - 91.6)	86.7
(77.2 - 92.6)
Negative Agreement	100
(92.1 - 100)	100
(92.1 - 100)	97.8
(88.4 - 99.6)
Overall Agreement	90.8
(84.3 - 94.8)	90.8
(84.3 - 94.8)	90.8
(84.3 - 94.8)
Pattern Agreement	85.3
(75.6 - 91.6)	85.3
(75.6 - 91.6)	86.7
(77.2 - 92.6)
FI Agreement	100
(96.9 - 100)	99.2
(95.4 - 99.9)	100
(96.9 - 100)

FI = Fluorescence Intensity

Table 102: Real time Stability AESKUSLIDES ANCA Formalin (Combined Readers) – %Agreement:

| % Agreement

(95% CI)	Combined Readers	Lot 1	Lot 2	Lot 3
Positive Agreement	90.7
(84.9 - 94.4)	90
(84.2 - 93.8)	88
(81.8 - 92.3)
Negative Agreement	100
(95.9 - 100)	100
(95.9 - 100)	97.8
(92.3 - 99.4)
Overall Agreement	94.2
(90.4 - 96.5)	93.8
(89.9 - 96.2)	91.7
(87.5 - 94.5)
Pattern Agreement	90.7
(84.9 - 94.4)	90
(84.2 - 93.8)	88
(81.8 - 92.3)
FI Agreement	100
(98.4 - 100)	99.6
(97.7 - 99.9)	100
(98.4 - 100)

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Agreement calculations for AESKUSLIDES ANCA Formalin included all data points of the test over a time period of 3 months at 2-8°C:

All agreements fulfilled the above criteria.

Positive agreement for AESKUSLIDES ANCA Formalin for both readers ranged from 88.0% to 90.7% for all 3 reagent lots.

Negative agreement for ANCA Formalin for both readers ranged from 97.8% to 100% for all 3 reagent lots.

Overall agreement for ANCA Formalin for both readers ranged from 91.7% to 94.2% for all 3 reagent lots.

Pattern agreement for ANCA Formalin for both readers ranged from 88.0% to 90.7% for all 3 reagent lots.

FI agreement for ANCA Formalin for both readers ranged from 99.6% to 100% for all 3 reagent lots.

Just one deviation of fluorescence intensity greater than +/-1 from the initial value was observed for reader 2.

Conclusion:

All acceptance criteria were met. All agreements were found to be > 85%. The presented data support stability for AESKUSLIDES ANCA Ethanol and ANCA Formalin for at least 3 months at 2-8°C, so far. Other test time points will follow to support the claimed shelf life of 24 months (based on the accelerated stability study of AESKUSLIDES ANCA described elsewhere).

In Use Stability Report

In Use stability testing is designed to predict the period of time that a product remains viable once placed into use. Therefore, In Use stability of AESKUSLIDES ANCA was tested over a period of 6 weeks.

Procedure:

Three different reagent lots were stored at 2-8°C over a time period of 6 weeks. Tests have been performed at the following time points: t0, t1, t2, t4, t6 (t1= after 1 week, t2= after 2 weeks, t4= after 4 weeks, t6 after 6 weeks) for each reagent lot. After the first test at time point t0, kits were incubated at the indicated temperature (2-8°C). Each kit component (with the exception of slide foils) had been opened and all buffers (except wash buffer) had been prepared at time point t0. Eight serum samples have been tested at each time point:

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| Sample

ID	AESKUSLIDES ANCA Ethanol			AESKUSLIDES ANCA Formalin
	Pos/
Neg	Pattern ANCA
Ethanol	Grading	Pos/
Neg	Pattern ANCA
Formalin	Grading
1	Pos	C-ANCA	high positive	Pos	C-ANCA	high positive
2	Pos	C-ANCA	medium positive	Pos	C-ANCA	medium positive
3	Pos	C-ANCA	low positive	Pos	C-ANCA	low positive
4	Pos	P-ANCA	high positive	Pos	C-ANCA	medium positive
5	Pos	P-ANCA	medium positive	Pos	C-ANCA	borderline positive
6	Pos	P-ANCA	low positive	Pos	C-ANCA	borderline positive
7	Pos	A-ANCA	high positive	Neg	Neg	negative
8	Neg	Neg	negative	Neg	Neg	negative

Slides were processed manually according to the IFU and subsequently analyzed at the microscope by two independent readers.

The stability data is covering the stability of all components of the kit, except for diluted wash buffer and opened slides. Diluted wash buffer is stable for 1 week at 2-8°C, as has been shown in a wash buffer stability study. This is explicitly written in the IFU. Also noted in the IFU is the recommendation to process slides immediately upon opening the silde foil.

Agreements were calculated for individual and combined readers. Results of the kit controls were not included in the agreement calculations.

Acceptance Criteria:

Positive sera have to be found positive, and negative sera have to be found negative. ●
. Correct pattern has to be found.
Reported FI is allowed to differ max. ± 1 level from the expected value.
. Agreements should meet the following criteria:

Type of Agreement	% Agreement
Positive Agreement	> 85%
Negative Agreement	> 85%
Overall Agreement	> 85%
Total Pattern Agreement	> 85%
Fluorescence Intensity Agreement	> 85%

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Image /page/72/Picture/0 description: The image is a logo for AESKU.DIAGNOSTICS. The logo features a green square with a white symbol inside, followed by the text "AESKU.DIAGNOSTICS" in gray. Below the text, there is a tagline that reads "THE DIAGNOSTIC TOOL THAT WORKS" in a smaller, lighter gray font. The logo is clean and professional, with a focus on the company's name and its diagnostic tools.

AESKUSLIDES ANCA 510(k) Traditional Submission

Results:

Table 103: In Use Stability AESKUSLIDES ANCA Ethanol – Number of Samples:

| No of samples

(Incorrect/total)	Lot 1			Lot 2			Lot 3
	Reader 1	Reader 2	both
Readers	Reader 1	Reader 2	both
Readers	Reader 1	Reader 2	both
Readers
Positive Agreement	1 / 70	1 / 70	2 / 140	1 / 70	1 / 70	2 / 140	0 / 70	0 / 70	0 / 140
Negative Agreement	0 / 10	0 / 10	0 / 20	0 / 10	0 / 10	0 / 20	0 / 10	0 / 10	0 / 20
Overall Agreement	1 / 80	1 / 80	2 / 160	1 / 80	1 / 80	2 / 160	0 / 80	0 / 80	0 / 160
Pattern Agreement	1 / 70	1 / 70	2 / 140	4 / 70	4 / 70	8 / 140	0 / 70	1 / 70	1 / 140
FI Agreement	0 / 80	0 / 80	0 / 160	0 / 80	0 / 80	0 / 160	0 / 80	0 / 80	0 / 160

FI = Fluorescence Intensity

Table 104: In Use Stability AESKUSLIDES ANCA Ethanol – % Agreement:

| %
Agreement

(95% CI)	Lot 1			Lot 2			Lot 3
	Reader 1	Reader 2	both Readers	Reader 1	Reader 2	both Readers	Reader 1	Reader 2	both Readers
Positive
Agreement	98.6
(92.3 - 99.7)	98.6
(92.3 - 99.7)	98.6
(94.9 - 99.6)	98.6
(92.3 - 99.7)	98.6
(92.3 - 99.7)	98.6
(94.9 - 99.6)	100
(94.8 - 100)	100
(94.8 - 100)	100
(97.3 - 100)
Negative
Agreement	100
(72.2 - 100)	100
(72.2 - 100)	100
(83.9 - 100)	100
(72.2 - 100)	100
(72.2 - 100)	100
(83.9 - 100)	100
(72.2 - 100)	100
(72.2 - 100)	100
(83.9 - 100)
Overall
Agreement	98.8
(93.3 - 99.8)	98.8
(93.3 - 99.8)	98.8
(95.6 - 99.7)	98.8
(93.3 - 99.8)	98.8
(93.3 - 99.8)	98.8
(95.6 - 99.7)	100
(95.4 - 100)	100
(95.4 - 100)	100
(97.7 - 100)
Pattern
Agreement	98.6
(92.3 - 99.7)	98.6
(92.3 - 99.7)	98.6
(94.9 - 99.6)	94.3
(86.2 - 97.8)	94.3
(86.2 - 97.8)	94.3
(89.1 - 97.1)	100
(94.8 - 100)	98.6
(92.3 - 99.7)	99.3
(96.1 - 99.9)
FI
Agreement	100
(95.4 - 100)	100
(95.4 - 100)	100
(97.7 - 100)	100
(95.4 - 100)	100
(95.4 - 100)	100
(97.7 - 100)	100
(95.4 - 100)	100
(95.4 - 100)	100
(97.7 - 100)

73

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Agreement calculations for AESKUSLIDES ANCA Ethanol included all data points of the whole test time span of 6 weeks at 2-8°C:

All agreements fulfilled the above acceptance criteria.

Positive agreement for AESKUSLIDES ANCA Ethanol for both readers ranged from 98.6% to 100% for all 3 reagent lots.

Negative agreement for ANCA Ethanol for both readers was 100% for all 3 reagent lots.

Overall agreement for ANCA Ethanol for both readers ranged from 98.8% to 100% for all 3 reagent lots.

Pattern agreement for ANCA Ethanol for both readers ranged from 94.3% to 99.3% for all 3 reagent lots.

FI agreement for ANCA Ethanol for both readers was 100% for all 3 reagent lots.

No deviations of fluorescence intensities greater than +/-1 from the initial value have been observed.

74

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AESKUSLIDES ANCA 510(k) Traditional Submission

| No of samples

(Incorrect/total)	Lot 1			Lot 2			Lot 3
	Reader 1	Reader 2	both
Readers	Reader 1	Reader 2	both
Readers	Reader 1	Reader 2	both
Readers
Positive Agreement	13 / 60	21 / 60	34 / 120	11 / 60	16 / 60	27 / 120	11 / 60	19 / 60	30 / 120
Negative Agreement	0 / 20	0 / 20	0 / 40	0 / 20	0 / 20	0 / 40	0 / 20	0 / 20	0 / 40
Overall Agreement	13 / 80	21 / 80	34 / 160	11 / 80	16 / 80	27 / 160	11 / 80	19 / 80	30 / 160
Pattern Agreement	13 / 60	21 / 60	34 / 120	11 / 60	16 / 60	27 / 120	11 / 60	19 / 60	30 / 120
FI Agreement	0 / 80	0 / 80	0 / 160	0 / 80	0 / 80	0 / 160	0 / 80	2 / 80	2 / 160

Table 105: In Use AESKUSLIDES ANCA Formalin – Number of Samples (including borderline samples 5 and 6):

FI = Fluorescence Intensity

Table 106: In Use Stability AESKUSLIDES ANCA Formalin – % Agreement (including borderline samples 5 and 6):

| % Agreement

(95% CI)	Lot 1				Lot 2		Lot 3
	Reader 1	Reader 2	both
Readers	Reader 1	Reader 2	both
Readers	Reader 1	Reader 2	both
Readers
Positive Agreement	78.3	65	71.7	81.7	73.3	77.5	81.7	68.3	75
	(66.4 - 86.9)	(52.4 - 75.8)	(63 - 79)	(70.1 - 89.4)	(61 - 82.9)	(69.2 - 84.1)	(70.1 - 89.4)	(55.8 - 78.7)	(66.6 - 81.9)
Negative Agreement	100	100	100	100	100	100	100	100	100
	(83.9 - 100)	(83.9 - 100)	(91.2 - 100)	(83.9 - 100)	(83.9 - 100)	(91.2 - 100)	(83.9 - 100)	(83.9 - 100)	(91.2 - 100)
Overall Agreement	83.8	73.8	78.8	86.3	80	83.1	86.3	76.3	81.3
	(74.2 - 90.3)	(63.2 - 82.1)	(71.8 - 84.4)	(77 - 92.1)	(70 - 87.3)	(76.6 - 88.1)	(77 - 92.1)	(65.9 - 84.2)	(74.5 - 86.5)
Pattern Agreement	78.3	65	71.7	81.7	73.3	77.5	81.7	68.3	75
	(66.4 - 86.9)	(52.4 - 75.8)	(63 - 79)	(70.1 - 89.4)	(61 - 82.9)	(69.2 - 84.1)	(70.1 - 89.4)	(55.8 - 78.7)	(66.6 - 81.9)
FI Agreement	100	100	100	100	100	100	100	97.5	98.8
	(95.4 - 100)	(95.4 - 100)	(97.7 - 100)	(95.4 - 100)	(95.4 - 100)	(97.7 - 100)	(95.4 - 100)	(91.3 - 99.3)	(95.6 - 99.7)

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AESKUSLIDES ANCA 510(k) Traditional Submission

| No of samples

(Incorrect/total)	Lot 1			Lot 2			Lot 3
	Reader 1	Reader 2	both
Readers	Reader 1	Reader 2	both
Readers	Reader 1	Reader 2	both
Readers
Positive Agreement	0 / 40	2 / 40	2 / 80	0 / 40	1 / 40	1 / 80	0 / 40	2 / 40	2 / 80
Negative Agreement	0 / 20	0 / 20	0 / 40	0 / 20	0 / 20	0 / 40	0 / 20	0 / 20	0 / 40
Overall Agreement	0 / 60	2 / 60	2 / 120	0 / 60	1 / 60	1 / 120	0 / 60	2 / 60	2 / 120
Pattern Agreement	0 / 40	2 / 40	2 / 80	0 / 40	1 / 40	1 / 80	0 / 40	2 / 40	2 / 80
FI Agreement	0 / 60	0 / 60	0 / 120	0 / 60	0 / 60	0 / 120	0 / 60	2 / 60	2 / 120

Table 107: In Use AESKUSLIDES ANCA Formalin - Number of Samples (excluding borderline samples 5 and 6):

FI = Fluorescence Intensity

Table 108: In Use Stability AESKUSLIDES ANCA Formalin – % Agreement (excluding borderline samples 5 and 6):

| % Agreement

(95% CI)	Lot 1			Lot 2			Lot 3
	Reader 1	Reader 2	both
Readers	Reader 1	Reader 2	both
Readers	Reader 1	Reader 2	both
Readers
Positive Agreement	100	95	97.5	100	97.5	98.8	100	95	97.5
	(91.2 - 100)	(83.5 - 98.6)	(91.3 - 99.3)	(91.2 - 100)	(87.1 - 99.6)	(93.3 - 99.8)	(91.2 - 100)	(83.5 - 98.6)	(91.3 - 99.3)
Negative Agreement	100	100	100	100	100	100	100	100	100
	(83.9 - 100)	(83.9 - 100)	(91.2 - 100)	(83.9 - 100)	(83.9 - 100)	(91.2 - 100)	(83.9 - 100)	(83.9 - 100)	(91.2 - 100)
Overall Agreement	100	96.7	98.3	100	98.3	99.2	100	96.7	98.3
	(94 - 100)	(88.6 - 99.1)	(94.1 - 99.5)	(94 - 100)	(91.1 - 99.7)	(95.4 - 99.9)	(94 - 100)	(88.6 - 99.1)	(94.1 - 99.5)
Pattern Agreement	100	95	97.5	100	97.5	98.8	100	95	97.5
	(91.2 - 100)	(83.5 - 98.6)	(91.3 - 99.3)	(91.2 - 100)	(87.1 - 99.6)	(93.3 - 99.8)	(91.2 - 100)	(83.5 - 98.6)	(91.3 - 99.3)
FI Agreement	100	100	100	100	100	100	100	96.7	98.3
	(94 - 100)	(94 - 100)	(96.9 - 100)	(94 - 100)	(94 - 100)	(96.9 - 100)	(94 - 100)	(88.6 - 99.1)	(94.1 - 99.5)

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Agreement calculations for AESKUSLIDES ANCA Formalin included all data points of a test time span of 6 weeks at 2-8°C:

The sample set for AESKUSLIDES ANCA Formalin included two samples (sample 5 and 6) that were borderline positive. For these samples we expect either a low positive result (Fl=1) or a neqative result (Fl=0). Therefore, these samples lead to many "false negatives" when they are defined as "positive" for the calculation (or to many "false positives" when they are defined as "negative" for the calculation). These "false negatives" (or "false positives") have a big impact on positive agreement and on pattern agreement which is due to normal test variances that have a bigger impact on borderline samples than on samples that are clearly negative or clearly positive. Therefore, the lower positive (and pattern) agreements we observed with the agreement calculation for ANCA Formalin are not due to stability issues of the assay.

Therefore, we did an alternative agreement calculation excluding these two borderline positive samples. In this calculation all acceptance criteria have been met:

Positive agreement for AESKUSLIDES ANCA Formalin for both readers ranged from 97.5% to 98.8% for all 3 reagent lots.

Negative agreement for ANCA Formalin for both readers was 100% for all 3 reagent lots.

Overall agreement for ANCA Formalin for both readers ranged from 98.3% to 99.2% for all 3 reagent lots.

Pattern agreement for ANCA Formalin for both readers ranged from 97.5% to 98.8% for all 3 reagent lots.

FI agreement for ANCA Formalin for both readers ranged from 98.3% to 100% for all 3 reagent lots. No deviations of fluorescence intensities greater than +/-1 from the initial value have been observed.

Conclusion:

For AESKUSLIDES ANCA Ethanol all acceptance criteria were met for a time span of 6 weeks at 2-8°C. Therefore, we claim an In Use Stability of 6 weeks at 2-8°C storage temperature.

For AESKUSLIDES ANCA Formalin all acceptance criteria were met for a time span of 6 weeks at 2-8°C. Therefore, we claim an In Use Stability of 6 weeks at 2-8°C storage temperature.

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Transport Stability Report

To determined transport stability of AESKUSLIDES ANCA, data were taken from Accelerated Stability Report, as these conditions used in the study simulate a worst case scenario of transport of the kits.

The kit components were incubated at 37°C over a period of 6 weeks.

Acceptance Criteria:

Positive sera have to be found positive, and negative sera have to be found negative. ●
Correct pattern has to be found.
Reported FI is allowed to differ max. ± 1 level from the expected value. ●
. Agreements should meet the following criteria:

Type of Agreement	% Agreement
Positive Agreement	> 85%
Negative Agreement	> 85%
Overall Agreement	> 85%
Total Pattern Agreement	> 85%
Fluorescence Intensity Agreement	> 85%

Conclusion:

All criteria were fulfilled in each test. Therefore, we claim that the kit can resist a temperature of 37°C for at least 2 weeks.

For more information please see at "Accelerated Stability Report".

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2. Comparison studies

Method comparison with predicate device:

To show comparable performance of AESKUSLIDES ANCA (Ethanol and Formalin) and the predicate assay NOVA Lite ANCA (Ethanol and Formalin) of INOVA.

Procedure:

A method comparison study with 507 serum samples (comprising 132 serum samples from patients with AAV and 375 samples from patients with other diseases) was performed in house. A detailed description of the study sample set can be found on page 12-79, table 110.

Healthy donor samples have been excluded from this study.

For each assay the sample set was processed manually and analyzed (positive/negative classification, pattern classification) at the microscope (Method C).

All assays were performed according to the respective IFUs. Sample were screened at a dilution of 1:20.

From the results of both assays the relative sensitivity, specificity, positive (%), negative (%) and overall agreements (%) were calculated.

Furthermore, diagnostic sensitivity and specificity were calculated for both assays, and the cross reactivities for different diagnoses are shown in a table.

Results:

Results Predicate comparison ANCA Ethanol

Diagnostic Sensitivity and Specificity

Table 109a: Method comparison with predicate device ANCA Ethanol Diagnostic Sensitivity and Specificity Number of positive and negative results:

Diagnostic Sensitivity & Specificity	Diagnosis
	AAVs	Other Diseases	Total
AESKUSLIDES ANCA Ethanol	Pos	64	115	179
	Neg	68	260	328
	Total	132	375	507
Predicate Assay	Pos	48	168	216
	Neg	84	207	291
	Total	132	375	507

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Table 109b: Method comparison with predicate device ANCA Ethanol Diagnostic Sensitivity and Specificity PPV/NPV:

| % Diagnostic
Sensitivity &
Specificity (95% CI) | % Sensitivity
(95% CI)
AAVs (n= 132) | % Specificity
(95% CI)
OD (n=375) | Predictive
Value of a
Positive Test
Result (PPV) | Predictive Value of
a Negative Test
Result (NPV) |
|-------------------------------------------------------|--------------------------------------------|-----------------------------------------|-----------------------------------------------------------|--------------------------------------------------------|
| AESKUSLIDES ANCA
Ethanol | 48.5
(40.1 - 56.9) | 69.3
(64.5 - 73.8) | 35.8% | 79.3% |
| Predicate Assay | 36.4
(28.7 - 44.8) | 55.2
(50.1 - 60.2) | 22.2% | 71.1% |

Sensitivity of AESKUSLIDES ANCA Ethanol is higher (48.5%) than that calculated for the predicate assay (36.4%). Specificity for AESKUSLIDES ANCA Ethanol was 69.3%, whereas for the predicate assay a specificity of 55.2% was calculated. Values reported in literature are variable and spread over a wide range, depending on the serum panel used in the study. The ANCA titer may be associated with disease activity and may also disappear upon treatment. The AAV cohort tested in this study was not selected for disease activity or treatment.

The higher sensitivity and specificity of AESKUSLIDES ANCA Ethanol go along with a higher PPV (35.0% compared to 22.2% for the predicate) and NPV (79.3% vs 71.1%).

The results for both assays are shown in detail in the cross-reactivity table below. With AESKUSLIDES ANCA Ethanol fewer cross-reactivity are seen. Cross-reactivity could be detected in autoimmune liver diseases (up to 50%), inflammatory bowel diseases (up to 50.7%) and also other rheumatic diseases (SLE, RA, Scleroderma: up to 47.4%). The predicate assay detected positives in similar diagnoses but with higher number of positives.

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AESKUSLIDES ANCA

510(k) Traditional Submission

			AESKUSLIDES ANCA Ethanol			Predicate
Diagnosis		n	n Pos	%Pos	n Neg	%Neg	n Pos	%Pos	n Neg	%Neg
Target
Diagnosis	ANCA associated
Vasculitis (AAV)	Wegener's Granulomatosis	78	33	42.3%	45	57.7%	20	25.6%	58	74.4%
		MPA	29	22	75.9%	7	24.1%	20	69.0%	9	31.0%
		Churg-Strauss Syndrome	25	9	36.0%	16	64.0%	8	32.0%	17	68.0%
		Autoimmune Hepatitis (AIH)	8	3	37.5%	5	62.5%	5	62.5%	3	37.5%
	Autoimmune Liver
Diseases	Autoimmune Hepatitis/ Primary biliary
cholangitis	6	3	50.0%	3	50.0%	6	100.0%	0	0.0%
		Primary biliary cholangitis (PBC)	11	2	18.2%	9	81.8%	8	72.7%	3	27.3%
		Primary sclerosing cholangitis (PSC)	10	5	50.0%	5	50.0%	5	50.0%	5	50.0%
		Ulcerative Colitis	71	36	50.7%	35	49.3%	42	59.2%	29	40.8%
	Inflammatory bowel
diseases	Crohn's disease	40	16	40.0%	24	60.0%	26	65.0%	14	35.0%
		Inflammatory bowel disease (IBD)	9	4	44.4%	5	55.6%	5	55.6%	4	44.4%
		Rheumatoid Arthritis (RA)	12	4	33.3%	8	66.7%	9	75.0%	3	25.0%
	Other rheumatic
diseases	Systemic Lupus Erythematosus (SLE)	30	14	46.7%	16	53.3%	22	73.3%	8	26.7%
Control
diagnosis		Scleroderma	19	9	47.4%	10	52.6%	13	68.4%	6	31.6%
		Myositis	1	0	0.0%	1	100.0%	0	0.0%	1	100.0%
		Infections	Hepatitis C Virus (HCV )	16	3	18.8%	13	81.3%	5	31.3%	11
			Hepatitis B Virus (HBV )	8	0	0.0%	8	100.0%	0	0.0%	8
	Vasculitides NOT
associated with ANCA	Polymyalgia rheumatica (PMR)	18	3	16.7%	15	83.3%	5	27.8%	13	72.2%
		Giant cell artertitis (GCA)	4	0	0.0%	4	100.0%	2	50.0%	2	50.0%
		Purpura	1	1	100.0%	0	0.0%	1	100.0%	0	0.0%
	Leukemia	Lymphoma, Myeloma	15	3	20.0%	12	80.0%	4	26.7%	11	73.3%
	Other diagnosis	Celiac disease	10	0	0.0%	10	100.0%	0	0.0%	10	100.0%
		Chronic kidney disease	32	2	6.3%	30	93.8%	3	9.4%	29	90.6%
		Sinusitis	22	4	18.2%	18	81.8%	4	18.2%	18	81.8%
		Asthma	32	3	9.4%	29	90.6%	3	9.4%	29	90.6%
Sum			507	179	35.3%	328	64.7%	216	42.6%	291	57.4%

Table 110: Method comparison with predicate device ANCA Ethanol. Number and positive samples in the different tested diagnosis

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Relative sensitivity and specificity

Table 110a: Method comparison with predicate device ANCA Ethanol Relative Sensitivity and Specificity Number of positive and negative results and % agreements:

Method Comparison		Predicate
		Pos	Neg	Total
AESKU ANCA
Ethanol	Pos	145	34	179
	Neg	71	257	328
	Total	216	291	507

Method Comparison Predicate vs AESKU	% Agreement (95% CI)
Positive Agreement	67.1
(60.6 - 73)
Negative Agreement	88.3
(84.1 - 91.5)
Overall Agreement	79.3
(75.5 - 82.6)

Positive, negative and overall agreement between both assays were acceptable (67.1%, 88.3% and 79.3%). The rather low positive agreement was due to the fact, that the predicate assay detects less positive results.

Conclusions

AESKUSLIDES ANCA Ethanol showed higher diagnostic sensitivity (48.5%) and specificity (69.3%) compared to the predicate assay NOVA Lite provided by INOVA (36.4%, 55.2%). This is due to the fact, that AESKU assay detects more positives in the AAV cohort, and less positives in the other disease groups.

This study shows that the AESKU assay is comparable or better than the predicate device.

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Results Predicate comparison ANCA Formalin

Diagnostic Sensitivity and Specificity

Table 111: Method comparison with predicate device_ANCA Formalin_Diagnostic Sensitivity and Specificity Number of positive and negative results:

| Diagnostic Sensitivity &

Specificity		Diagnosis
		AAVs	Other Diseases	Total
AESKUSLIDES
ANCA Formalin	Pos	66	35	101
	Neg	66	340	406
	Total	132	375	507
Predicate Assay	Pos	50	32	82
	Neg	82	343	425
	Total	132	375	507

Table 112: Method comparison with predicate device_ANCA Formalin_Diagnostic Sensitivity and Specificity PPV/NPV:

| % Diagnostic Sensitivity &
Specificity (95% CI) | % Sensitivity
(95% CI) | % Specificity
(95% CI) | Predictive
Value of a
Positive Test
Result (PPV) | Predictive Value of
a Negative Test
Result (NPV) |
|----------------------------------------------------|---------------------------|---------------------------|-----------------------------------------------------------|--------------------------------------------------------|
| | AAVs (n= 132) | OD (n=375) | | |
| AESKUSLIDES ANCA
Formalin | 50
(41.6 - 58.4) | 90.7
(87.3 - 93.2) | 65.3% | 83.7% |
| Predicate Assay | 37.9
(30.1 - 46.4) | 91.5
(88.2 - 93.9) | 61.0% | 80.7% |

Sensitivity of AESKUSLIDES ANCA Formalin is higher (50.0%) than that calculated for the predicate assay (37.9%) in this study. Specificities for both assays were comparable (90.7% vs 91.5%). PPVs and NPVs of both assays were comparable. However, values for AESKUSLIDES were slightly higher.

The cross-reactivity table shows equivalent results for both assays. Cross-reactivity could be detected in AIH (25.0%), PSC (20.0%), Ulcerative Colitis (16.9% and SLE (20.0%)

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AESKUSLIDES ANCA

510(k) Traditional Submission

Table 113: Method comparison with predicate device_ANCA Formalin. Number and percent positive samples in the different tested diagnosis
		AESKUSLIDES ANCA Formalin				Predicate
	Diagnosis	n	n Pos	%Pos	n Neg	%Neg	n Pos	%Pos	n Neg	%Neg
Target
Diagnosis	ANCA associated
Vasculitis (AAV)	Wegener's Granulomatosis	78	45	57.7%	33	42.3%	37	47.4%	41	52.6%
		MPA	29	15	51.7%	14	48.3%	12	41.4%	17	58.6%
		Churg-Strauss Syndrome	25	6	24.0%	19	76.0%	1	4.0%	24	96.0%
	Autoimmune Liver
Diseases	Autoimmune Hepatitis (AIH)	8	2	25.0%	6	75.0%	1	12.5%	7	87.5%
		Autoimmune Hepatitis/ Primary biliary
cholangitis	6	0	0.0%	6	100.0%	0	0.0%	6	100.0%
		Primary biliary cholangitis (PBC)	11	0	0.0%	11	100.0%	0	0.0%	11	100.0%
		Primary sclerosing cholangitis (PSC)	10	2	20.0%	8	80.0%	0	0.0%	10	100.0%
	Inflammatory
bowel diseases	Ulcerative Colitis	71	12	16.9%	59	83.1%	10	14.1%	61	85.9%
		Crohn's disease	40	1	2.5%	39	97.5%	0	0.0%	40	100.0%
		Inflammatory bowel disease (IBD)	9	1	11.1%	8	88.9%	1	11.1%	8	88.9%
Control
diagnosis	Other rheumatic
diseases	Rheumatoid Arthritis (RA)	12	1	8.3%	11	91.7%	1	8.3%	11	91.7%
		Systemic Lupus Erythematosus (SLE)	30	6	20.0%	24	80.0%	8	26.7%	22	73.3%
		Scleroderma	19	0	0.0%	19	100.0%	2	10.5%	17	89.5%
		Myositis	1	0	0.0%	1	100.0%	0	0.0%	1	100.0%
		Infections	Hepatitis C Virus (HCV )	16	2	12.5%	14	87.5%	3	18.8%	13
			Hepatitis B Virus (HBV )	8	0	0.0%	8	100.0%	0	0.0%	8
		Vasculitides NOT
associated with
ANCA	Polymyalgia rheumatica (PMR)	18	2	11.1%	16	88.9%	1	5.6%	17
			Giant cell artertitis (GCA)	4	0	0.0%	4	100.0%	2	50.0%	2
		Leukemia	Purpura	1	1	100.0%	0	0.0%	1	100.0%	0
			Lymphoma, Myeloma	15	2	13.3%	13	86.7%	0	0.0%	15
		Other diagnosis	Celiac disease	10	0	0.0%	10	100.0%	0	0.0%	10
			Chronic kidney disease	32	2	6.3%	30	93.8%	1	3.1%	31
		Sinusitis	22	1	4.5%	21	95.5%	1	4.5%	21	95.5%
		Asthma	32	0	0.0%	32	100.0%	0	0.0%	32	100.0%
Sum			507	101	19.9%	406	80.1%	82	16.2%	425	83.8%

Table 113: Method comparison with predicate device ANCA Formalin. Number and percent positive samples in the different tested diagnosis

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Relative sensitivity and specificity

Table 114: Method comparison with predicate device ANCA Formalin Relative Sensitivity and Specificity Number of positive and negative results and % agreements:

Method Comparison		Predicate
		Pos	Neg	Total
AESKUSLIDES
ANCA Formalin	Pos	66	35	101
	Neg	16	390	406
	Total	82	425	507

| Method Comparison ANCA
Formalin | % Agreement
(95% CI) |
|------------------------------------|-------------------------|
| Positive Agreement | 80.5
(70.6 - 87.6) |
| Negative Agreement | 91.8
(88.8 - 94) |
| Overall Agreement | 89.9
(87 - 92.3) |

Overall agreement between both assays was good (89.9%). The same holds true for the negative agreement (91.8%). The relatively low positive agreement (80.5%) is due to the fact, that the AESKU assay detects more samples as positive.

Conclusions

AESKUSLIDES ANCA Formalin showed a diagnostic sensitivity (50.0%) compared to the predicate assay NOVA Lite provided by INOVA (37.9%). However, the diagnostic sensitivity was comparable between the two (90.7% vs 91.5%).

This study shows that the AESKU assay is comparable or better than the predicate device.

Method Comparison of Method A, B, and C and clinical study

To show comparable performance of AESKUSLIDES ANCA Ethanol and ANCA Formalin when processed manually and on HELIOS.

Procedure:

A clinical study with the same 630 serum samples (comprising 135 serum samples from patients with AAV, 120 characterized MPO/PR3/ANCA positive sera and 375 samples from patients with other diseases) was performed on 3 different study sites (2 US sites, one German site). A detailed description of the study sample can be found on page 12-87/88, table 118.

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At each study site the sample set was processed

manually and analyzed (positive/negative classification) at the microscope by two independent readers (Method C)
on HELIOS and HELIOS images were analyzed by two independent readers at the PC (Method B)
on HELIOS and positive/negative classification was done by the HELIOS Vasculitis Pattern Plus Software (ANCA) (Method A)

All assays were performed according to the IFU.

For each study site, the results of the single and combined readers with the different Methods were compared to each other and % agreements were calculated:

Method C (Manual) vs Method B (Reader Confirmation)

Method B (Reader Confirmation) vs Method A (HELIOS)

Method C (Manual) vs Method A (HELIOS).

Furthermore, the number of positive samples found for each tested diagnosis for all study sites and all methods will be compared as well as the calculated diagnostic sensitivities and specificities. For the calculation of diagnostic sensitivity and specificity the 120 characterized MPO/PR3/ANCA positive sera have been excluded.

Acceptance Criteria:

Positive, Negative, and Overall Agreements between the different Methods should be >85%.

Positive, Negative, Overall and Pattern Agreements between the different methods were set to be >80%. This is due to the fact that unknown clinical samples often results in mixed patterns, which are not easy to characterize. For example, in general one reader may report a sample with unspecific fluorescence as positive undefined, whereas another evaluates it as negative. Thus, the evaluation of ANCA slides is very subjective and strongly dependent on the reader's experience. Therefore, we expect and accept variances up to 15% in this clinical study.

For Method A we accept a lower agreement (>70%). Comparing Methods B vs A and C vs A. In general, method A (HELIOS Vasculitis Pattern Plus software) detects less positive samples. In addition, positive samples are mostly classified as positive undefined. This can be explained by the complex patterns, which can be distinguished by the readers at the microscope or at the screen, but to limited extent by the software. Because of this, all results generated by the HELIOS (Method A) must be confirmed by trained personnel.

86

Results:

Results Clinical study and method comparison ANCA Ethanol

Table 115: Method Comparison of Method A, B and C_ANCA Ethanol_Single Reader Agreements

| % Agreement

(95% CI)		Method C vs B		Method B vs A		Method C vs A
		Reader 1	Reader 2	Reader 1	Reader 2	Reader 1	Reader 2
Site 1	Positive
Agreement	86.5
(80.9 - 90.6)	85.9
(80.2 - 90.1)	79
(72.3 - 84.5)	79
(72.3 - 84.5)	70.8
(64 - 76.8)	70.7
(63.9 - 76.7)
	Negative
Agreement	99.8
(98.7 - 100)	99.3
(98 - 99.8)	98.3
(96.6 - 99.1)	98.3
(96.6 - 99.1)	99.1
(97.7 - 99.6)	98.9
(97.4 - 99.5)
	Total
Agreement	95.7
(93.8 - 97)	95.2
(93.3 - 96.6)	93.2
(90.9 - 94.9)	93.2
(90.9 - 94.9)	90.5
(87.9 - 92.5)	90.3
(87.8 - 92.4)
Site 2	Positive
Agreement	88.5
(83.4 - 92.1)	92.8
(88.4 - 95.6)	92.6
(88 - 95.6)	86.9
(81.6 - 90.8)	83.2
(77.5 - 87.6)	81.6
(75.8 - 86.3)
	Negative
Agreement	98.6
(96.9 - 99.3)	96.7
(94.5 - 98)	98.6
(97.1 - 99.4)	99.3
(97.9 - 99.8)	97.9
(96 - 98.9)	96.9
(94.8 - 98.2)
	Total
Agreement	95.2
(93.3 - 96.6)	95.4
(93.5 - 96.8)	96.8
(95.1 - 97.9)	95.2
(93.3 - 96.6)	93
(90.8 - 94.8)	91.9
(89.5 - 93.8)
Site 3	Positive
Agreement	90.2
(85.6 - 93.5)	85.1
(79.5 - 89.4)	79.5
(73.6 - 84.3)	83.8
(78.2 - 88.2)	76
(70 - 81.1)	79
(72.7 - 84.1)
	Negative
Agreement	96
(93.7 - 97.6)	91.3
(88.2 - 93.6)	99
(97.5 - 99.6)	98.4
(96.6 - 99.2)	98.3
(96.5 - 99.2)	94.5
(91.9 - 96.3)
	Total
Agreement	94
(91.8 - 95.6)	89.4
(86.7 - 91.5)	92.2
(89.9 - 94.1)	93.7
(91.5 - 95.3)	90.3
(87.8 - 92.4)	89.7
(87.1 - 91.8)

Table 116: Method Comparison of Method A, B and C_ANCA Ethanol_Combined Reader Agreements

	% Agreement (95% CI)	Method C vs B
	Positive Agreement	86.2
(82.3 - 89.3)	79
(74.4 - 83.1)	70.8
(66 - 75.1)
Site 1	Negative Agreement	99.5
(98.8 - 99.8)	98.3
(97.2 - 98.9)	99
(98.1 - 99.5)
	Total Agreement	95.5
(94.2 - 96.5)	93.2
(91.6 - 94.4)	90.4
(88.6 - 91.9)
	Positive Agreement	90.6
(87.4 - 93)	89.6
(86.3 - 92.3)	82.4
(78.5 - 85.8)
Site 2	Negative Agreement	97.6
(96.4 - 98.5)	99
(98 - 99.5)	97.4
(96.1 - 98.3)
	Total Agreement	95.3
(94 - 96.4)	96
(94.8 - 97)	92.5
(90.9 - 93.8)
	Positive Agreement	87.9
(84.4 - 90.6)	81.6
(77.6 - 85)	77.4
(73.1 - 81.1)
Site 3	Negative Agreement	93.6
(91.7 - 95)	98.7
(97.7 - 99.3)	96.3
(94.8 - 97.4)
	Total Agreement	91.7
(90 - 93.1)	92.9
(91.4 - 94.2)	90
(88.2 - 91.5)

87

% Agreement (95% CI)	Method C vs B	Method B vs A	Method C vs A
Site 1	87
(85 - 88.7)	81
(78.8 - 82.9)	77.8
(75.4 - 79.9)
Site 2	89.2
(87.5 - 90.7)	84.7
(82.6 - 86.5)	81.8
(79.7 - 83.8)
Site 3	82.5
(80.5 - 84.2)	85.6
(83.7 - 87.4)	80.3
(78.2 - 82.3)
All sites	86.2
(85.2 - 87.2)	83.8
(82.6 - 84.8)	80
(78.7 - 81.2)

Table 117: Method Comparison of Method A, B and C – Pattern Agreements

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AESKUSLIDES ANCA 510(k) Traditional submission

Table 118: Method comparison. Number and percent positive samples in the different tested diagnosis_ANCA Ethanol

		Site 1										Site 2										Site 3
Cross
Reactivity				Manual Performance				Reader Confirmation at HELIOS				HELIOS
suggestion		Manual Performance				Reader Confirmation at HELIOS				HELIOS
suggestion		Manual Performance				Reader Confirmation at HELIOS				HELIOS
suggestion
Diagnosis	n	Reader 1		Reader 2		Reader 1		Reader 2		n	Reader 3		Reader 4		Reader 3		Reader 4		n	Reader 5			Reader 6		Reader 5		Reader 6
		n
Pos	%
Pos	n
Pos	%
Pos	n
Pos	%
Pos	n
Pos	%
Pos	n	%
Pos	n
Pos	%
Pos	n
Pos	%
Pos	n
Pos	%
Pos	n
Pos	%
Pos	n	%
Pos	n
Pos	%
Pos	n
Pos	%
Pos	n
Pos	%
Pos	n
Pos	%
Pos
Wegener's	80	13	16.3	13	16.3	13	16.3	13	16.3	11	13.8	20	25.0	22	27.5	17	21.2	21	26.2	17	21.2	19	23.7	21	26.2	20	25	22	27.5	17	21.2
MPA	29	18	62.1	18	62.1	16	55.2	16	55.2	14	48.3	19	65.5	18	62.0	20	68.9	20	68.9	20	68.9	19	65.5	19	65.5	19	65.5	19	65.5	18	62.0
Churg-Strauss	26	2	7.7	2	7.7	2	7.7	2	7.7	0	0.0	5	19.2	4	15.3	5	19.2	5	19.2	6	23.0	6	23.0	6	23.0	8	30.7	8	30.7	4	15.3
C-ANCA	20	14	70.0	14	70.0	13	65.0	13	65.0	11	55.0	17	85.0	17	85	16	80	16	80	14	70	14	70	13	65	13	65	14	70	13	65
P-ANCA	50	24	48.0	23	46.0	24	48.0	24	48.0	25	50.0	23	46.0	23	46	23	46	25	50	24	48	25	50	25	50	25	50	26	52	25	50
MPO	25	18	72.0	18	72.0	17	68.0	17	68.0	18	72.0	23	92.0	22	88	19	76	22	88	18	72	22	88	20	80	20	80	19	76	18	72
PR3	25	5	20.0	5	20.0	3	12.0	3	12.0	3	12.0	9	36.0	7	28	8	32	7	28	7	28	15	60	12	48	10	40	10	40	11	44
AIH	8	5	62.5	5	62.5	5	62.5	5	62.5	4	50.0	3	37.5	3	37.5	2	25	3	37.5	2	25	2	25	1	12.5	3	37.5	1	12.5	1	12.5
AIH/PBC	6	4	66.7	4	66.7	4	66.7	4	66.7	3	50.0	3	50.0	3	50	3	50	3	50	2	33.3	1	16.6	4	66.6	3	50	2	33.3
PBC	11	3	27.3	3	27.3	3	27.3	3	27.3	2	18.2	2	18.2	2	18.1	3	27.2	4	36.3	1	9.09	4	36.3	2	18.1	4	36.3	3	27.2	1	9.0
PSC	10	5	50.0	5	50.0	5	50.0	5	50.0	3	30.0	5	50.0	5	50	5	50	5	50	4	40	1	10	4	40	4	40	3	30
Ulcerative Colitis	71	28	39.4	28	39.4	16	22.5	16	22.5	15	21.1	30	42.3	30	42.2	28	39.4	29	40.8	28	39.4	32	45.0	27	38.0	34	47.8	25	35.2	27	38.0
Crohn's	40	10	25.0	9	22.5	8	20.0	8	20.0	2	5.0	13	32.5	11	27.5	9	22.5	7	17.5	6	15	13	32.5	12	30	12	30	10	25	9	22.5
IBD	9	1	11.1	1	11.1	1	11.1	1	11.1	0	0.0	4	44.4	4	44.4	4	44.4	4	44.4	3	33.3	5	55.5	3	33.3	5	55.5	3	33.3	3	33.3
RA	12	1	8.3	1	8.3	1	8.3	1	8.3	1	8.3	1	8.3	2	16.6	0	0	1	8.33	0	0	4	33.3	4	33.3	1	8.3	1	8.3	1	8.3
SLE	30	16	53.3	16	53.3	15	50.0	15	50.0	12	40.0	14	46.7	14	46.6	14	46.6	14	46.6	13	43.3	16	53.3	12	40	14	46.6	12	40	10	33.3
Myositis	1	0	0.0	0	0.0	0	0.0	0	0.0	0	0.0	0	0.0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
Scleroderma	19	10	52.6	10	52.6	10	52.6	10	52.6	6	31.6	5	26.3	8	42.1	4	21.0	9	47.3	5	26.3	8	42.1	0	0	10	52.6	9	47.3	5	26.3
HBV	8	0	0.0	0	0.0	0	0.0	0	0.0	0	0.0	0	0.0	0	0	0	0	0	0	0	0	1	12.5	1	12.5	0	0	0	0	0	0
HCV	16	3	18.8	3	18.8	2	12.5	2	12.5	3	18.8	3	18.8	3	18.7	3	18.7	3	18.7	3	18.7	3	18.7	3	18.7	3	18.7	3	18.7	2	12.5
GCA	4	0	0.0	0	0.0	0	0.0	0	0.0	0	0.0	0	0.0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
PMR	18	4	22.2	4	22.2	3	16.7	3	16.7	3	16.7	2	11.1	2	11.1	0	0	1	5.5	0	0	3	16.6	3	16.6	2	11.1	2	11.1	1	5.5
Purpura	1	1	100	1	100	0	0.0	0	0.0	0	0.0	1	100	1	100	1	100	1	100	1	100	1	100	1	100	1	100	1	100	1	100
Leukemia	15	3	20.0	4	26.7	2	13.3	2	13.3	1	6.7	2	13.3	2	13.3	2	13.3	2	13.3	2	13.3	3	20	2	13.3	3	20	2	13.3	2	13.3
Celiac
disease	10	1	10.0	1	10.0	1	10.0	1	10.0	1	10.0	0	0.0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
CKD	32	1	3.1	1	3.1	1	3.1	1	3.1	1	3.1	1	3.1	1	3.1	1	3.1	1	3.1	0	0	1	3.1	3	9.3	1	3.1	3	9.3	1	3.1
Asthma	32	1	3.1	1	3.1	1	3.1	1	3.1	0	0.0	1	3.1	1	3.1	1	3.1	1	3.1	2	6.2	1	3.1	1	3.1	1	3.1	2	6.2	1	3.1
Sinusitis	22	1	4.5	1	4.5	1	4.5	1	4.5	1	4.5	2	9.1	2	9.0	2	9.0	2	9.0	2	9.0	2	9.0	2	9.0	2	9.0	2	9.0	2	9.0

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AESKUSLIDES ANCA 510(k) Traditional submission

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AESKUSLIDES ANCA 510(k) Traditional submission

| | % Diagnostic Sensitivity & Specificity
(95% CI) | | % Sensitivity
(95% CI) | % Specificity
(95% CI) | | % Sensitivity
(95% CI) | % Specificity
(95% CI) |
|-----------|----------------------------------------------------|----------|---------------------------|---------------------------|---------------------|---------------------------|---------------------------|
| | | | AAV (n= 135) | OD (n=375) | | AAV (n= 135) | OD (n=375) |
| Site
1 | Manual | Reader 1 | 24.4
(18 - 32.3) | 73.9
(69.2 - 78.1) | Combined
Readers | 24.4
(19.7 - 29.9) | 73.9
(70.6 - 76.9) |
| | | Reader 2 | 24.4
(18 - 32.3) | 73.9
(69.2 - 78.1) | | | |
| | Reader
Confirmation
at HELIOS | Reader 1 | 23
(16.7 - 30.7) | 78.9
(74.5 - 82.8) | Combined
Readers | 23
(18.3 - 28.3) | 78.9
(75.9 - 81.7) |
| | | Reader 2 | 23
(16.7 - 30.7) | 78.9
(74.5 - 82.8) | | | |
| | HELIOS | | 18.5
(12.9 - 25.9) | 84.5
(80.5 - 87.8) | | | |
| Site
2 | Manual | Reader 3 | 32.6
(25.3 - 40.9) | 75.5
(70.9 - 79.5) | Combined
Readers | 32.6
(27.3 - 38.4) | 75.2
(72 - 78.2) |
| | | Reader 4 | 32.6
(25.3 - 40.9) | 74.9
(70.3 - 79.1) | | | |
| | Reader
Confirmation
at HELIOS | Reader 3 | 31.1
(23.9 - 39.4) | 78.1
(73.7 - 82) | Combined
Readers | 32.6
(27.3 - 38.4) | 77.1
(73.9 - 79.9) |
| | | Reader 4 | 34.1
(26.6 - 42.4) | 76
(71.4 - 80) | | | |
| | HELIOS | | 31.9
(24.6 - 40.1) | 79.7
(75.4 - 83.5) | | | |
| Site
3 | Manual | Reader 5 | 32.6
(25.3 - 40.9) | 72
(67.3 - 76.3) | Combined
Readers | 33.3
(28 - 39.2) | 75.5
(72.3 - 78.4) |
| | | Reader 6 | 34.1
(26.6 - 42.4) | 78.9
(74.5 - 82.8) | | | |
| | Reader
Confirmation
at HELIOS | Reader 5 | 34.8
(27.3 - 43.2) | 72.3
(67.5 - 76.6) | Combined
Readers | 35.6
(30.1 - 41.4) | 74.7
(71.4 - 77.6) |
| | | Reader 6 | 36.3
(28.7 - 44.7) | 77.1
(72.5 - 81) | | | |
| | HELIOS | | 28.9
(21.9 - 37) | 80.8
(76.5 - 84.5) | | | |

Table 119: Diagnostic Sensitivity and Specificity ANCA Ethanol

Note: The 120 characterized MPO/PR3/ANCA positive sera have been excluded from the calculation of diagnostic sensitivity and specificity.

Summary of Results:

Positive, Negative, Overall and Pattern Agreements for Method Comparison between Method C (Manual) and Method B (Reader Confirmation of HELIOS images) were > 80%. All acceptance criteria were met.

Positive Agreement of Method C vs B ranged from 85.1% to 92.8%.

91

Negative Agreement of Method C vs B ranged 91.3% to 99.8%.

Overall Agreement of Method C vs B ranged from 89.4% to 95.7%.

Pattern Agreement of Method C vs B ranged from 82.5% to 89.2%.

The results show that Methods C (Manual) and B (Reader Confirmation of HELIOS images) give equivalent results. Furthermore, the results of the 3 study site were comparable with each other.

Agreements for Comparison between Method B (Reader Confirmation of HELIOS images) and Method A (HELIOS positive/negative classification by the Vasculitis Pattern Plus software) were as follows:

Positive Agreement of Method B vs A ranged from 79.0% to 92.6%.

Negative Agreement of Method B vs A ranged from 98.3% to 99.3%.

Overall Agreement of Method B vs A ranged from 92.2% to 96.8%.

Pattern Agreement of Method B vs A ranged from 81.0% to 85.6%.

According to this, acceptance criteria for comparison of Method B vs A were met. The same holds true for the comparison of Method C (Manual) vs A (HELIOS suggested results).

Positive Agreement of Method C vs A ranged from 70.7% to 83.2%.

Negative Agreement of Method C vs A ranged from 94.5% to 99.1%.

Overall Agreement of Method C vs A ranged from 89.7% to 93.0%.

Pattern Agreement of Method C vs A ranged from 77.8% to 81.8%.

Overall, the crossreactivity table (table No.96) shows few crossreactivities for Method A, B and C. The most crossreactivities could be seen in the autoimmune liver disease group (AIH, PBC, PSC: up to 66.7%) and in inflammatory bowel diseases (Ulcerative Colitis, Crohn's disease: up to 45.0%). However, these values are in accordance with literature, where it is stated that ANCAs could also be associated with inflammatory bowel disease and autoimmune liver disease.

92

Results Clinical study and method comparison ANCA Formalin

Table 120: Method Comparison of Method A, B and C_ANCA Formalin_Single Reader Agreements

% Agreement (95% CI)		Method C vs B		Method B vs A		Method C vs A
		Reader 1	Reader 2	Reader 1	Reader 2	Reader 1	Reader 2
Site 1	Positive
Agreement	87.5
(80.9 - 92)	85.7
(79.1 - 90.5)	84.9
(78.2 - 89.8)	82.2
(75.2 - 87.5)	75
(67.1 - 81.5)	71.4
(63.7 - 78.1)
	Negative
Agreement	94.5
(92.2 - 96.2)	95.9
(93.7 - 97.3)	99.2
(97.9 - 99.7)	98.3
(96.8 - 99.2)	94.7
(92.4 - 96.4)	95.2
(93 - 96.8)
	Total
Agreement	93
(90.8 - 94.8)	93.5
(91.3 - 95.2)	95.9
(94 - 97.2)	94.6
(92.6 - 96.1)	90.5
(87.9 - 92.5)	89.7
(87.1 - 91.8)
Site 2	Positive
Agreement	86.8
(79.9 - 91.6)	87
(81 - 91.4)	88.7
(81.9 - 93.2)	73.6
(66.7 - 79.5)	79.8
(72.1 - 85.9)	71
(63.6 - 77.4)
	Negative
Agreement	97.6
(95.9 - 98.6)	92.1
(89.3 - 94.2)	94.1
(91.6 - 95.8)	98
(96.3 - 98.9)	92.6
(90 - 94.6)	94.6
(92.2 - 96.3)
	Total
Agreement	95.4
(93.5 - 96.8)	90.8
(88.3 - 92.8)	93
(90.7 - 94.7)	91.1
(88.6 - 93.1)	90
(87.4 - 92.1)	88.6
(85.8 - 90.8)
Site 3	Positive
Agreement	89.6
(83.8 - 93.5)	88.6
(83.2 - 92.4)	99.4
(96.6 - 99.9)	98.7
(96.1 - 99.5)	98.7
(95.4 - 99.6)	92.9
(88.3 - 95.8)
	Negative
Agreement	94.9
(92.6 - 96.6)	86.3
(82.8 - 89.2)	72.2
(67.9 - 76)	82.7
(78.7 - 86.1)	70.7
(66.5 - 74.6)	73
(68.7 - 76.9)
	Total
Agreement	93.6
(91.5 - 95.3)	87
(84.1 - 89.4)	79.2
(75.8 - 82.2)	88.4
(85.7 - 90.7)	77.6
(74.2 - 80.7)	78.9
(75.5 - 81.9)

Table 121: Method Comparison of Method A, B and C_ANCA Formalin_Combined Reader Agreements

| | % Agreement
(95% CI) | Method C vs B | Method B vs A | Method C vs A |
|--------|-------------------------|-----------------------|-----------------------|-----------------------|
| Site 1 | Positive
Agreement | 86.6
(82.1 - 90.1) | 83.6
(78.9 - 87.4) | 73.1
(67.7 - 78) |
| | Negative
Agreement | 95.2
(93.7 - 96.4) | 98.8
(97.8 - 99.3) | 95
(93.4 - 96.2) |
| | Total
Agreement | 93.3
(91.7 - 94.5) | 95.2
(93.9 - 96.3) | 90.1
(88.3 - 91.6) |
| Site 2 | Positive
Agreement | 86.9
(82.6 - 90.3) | 79.8
(74.9 - 83.9) | 74.9
(69.6 - 79.5) |
| | Negative
Agreement | 94.9
(93.4 - 96.1) | 95.9
(94.5 - 97) | 93.6
(91.9 - 95) |
| | Total
Agreement | 93.1
(91.5 - 94.4) | 92.1
(90.4 - 93.4) | 89.3
(87.4 - 90.9) |
| Site 3 | Positive
Agreement | 89.1
(85.3 - 92) | 99
(97.4 - 99.6) | 95.6
(92.8 - 97.3) |
| | Negative
Agreement | 90.8
(88.7 - 92.5) | 77.1
(74.2 - 79.7) | 71.8
(68.9 - 74.7) |
| | Total
Agreement | 90.3
(88.5 - 91.8) | 83.8
(81.6 - 85.7) | 78.2
(75.9 - 80.4) |

93

Table 122: Method Comparison of Method A, B and C_ANCA Formalin_Pattern Agreements

% Agreement (95% CI)	Method C vs B	Method B vs A	Method C vs A
Site 1	82.1
(79.9 - 84.2)	90.8
(89.1 - 92.3)	77.9
(75.5 - 80.1)
Site 2	87.5
(85.5 - 89.2)	84
(81.9 - 86)	79.6
(77.3 - 81.7)
Site 3	82.1
(79.8 - 84.1)	76.1
(73.7 - 78.4)	69.7
(67.1 - 72.2)
All sites	83.9
(82.7 - 85)	83.7
(82.4 - 84.8)	75.7
(74.3 - 77.1)

94

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AESKUSLIDES ANCA 510(k) Traditional submission

Table 123: Method comparison. Number and percent positive samples in the different tested diagnosis_ANCA Formalin

| | Site 2 | | |-----------------------|----|-- | Cross Reactivity | | HELIOS | | HELIOS | HELIOS | | | suggestion | |
| | | Reader 1 Reader 6 | | Diagnosis | n | n Pos Pos | n
Pos | % Pos | n
Pos | %
Pos | n Pos Pos | 0
Pos | %
Pos | n
Pos | 0%
Pos | 0
Pos | %
Pos | n
Pos | %
Pos | n Pos | %
Pos | n Pos | %
Pos | n Pos Pos | %
Pos | n Pos | %
Pos |
| Wegener's | MPA | 29 | 13 | Churg-
Strauss | 26 | 2 | C-ANCA | 20 | 14 | P-ANCA | 50 | 20 | MPO | 25 | 8 | PR3 | 25 | 14 | AlH | 8 | 4 | AIH/PBC | ଚ | 0 | РВС | 11 | O | PSC | 10 | O | Ulcerative
Colitis | 71 | 6 | Crohn`s
Disease | 40 | O | IBD | 9 | 0 | RA | 12 | 1 | SLE | 30 | 5 | Myositis | 1 | O | Scleroderma | 19 | 3 | HBV | 8 | 0 | HCV | 16 | త | GCA | イ | 1 | PMR | 18 | 1 | Purpura | 1 | O | Leukemia | 15 | O | Celiac
disease | 10 | 1 | CKD | 32 | O | Asthma | 32 | 1 | Sinusitis | 22 | O | Site 1
| | | | | | Site 3 | | | | | | | | | | | | | | | | | | | | | |
----------------|--------------------|----------|----------|----------|----------|----------------------------------|----------|------------|----------|----------|--------------------|----------|-----------|----------|------------------------|----------|----------|------------|----------|-------|----------------------|--------------------|-------|----------------------------------|-------|----------|----------|----------------------|----------|
| Manual Performance | | | | | Reader Confirmation at
| | Manual Performance | | | | Reader Confirmation at | HELIOS | | HELIOS | | | | Manual Performance | | Reader Confirmation at
| HELIOS
| | | Reader 2 | | Reader 1 | | Reader 2 | suggestion | | | Reader 3 | | Reader 4 | | Reader 3 | | Reader 4 | suggestion | | | Reader 5
| | Reader 5 | | | Reader 6 | | |
| %
| % Pos | n Pos | %
| % Pos | n Pos | % Pos | n
| 80 | રૂપ | 48.8 | 42 | 52.5 | 45 | 56.3 | 43 | 53.8 | 41 | 51.3 | રૂદિ | 45.0 | રૂત્વ | 48.8 | 31 | 38.8 | 37 | 46.3 | 37 | 46.3 | 38 | 47.5 | 51 | 63.8 | 38 | 47.5 | 50 | 62.5 | 56 | 70.0 |
| 44.8 | 12 | 41.4 | 15 | 51.7 | 14 | 48.3 | 14 | 48.3 | 11 | 37.9 | 16 | 55.2 | 13 | 44.8 | 17 | 58.6 | 14 | 48.3 | 12 | 41.4 | 13 | 44.8 | 13 | 44.8 | 14 | 48.3 | 19 | 65.5 |
| 7.7 | 3 | 11.5 | 7 | 26.9 | 4 | 15.4 | ઉ | 23.1 | | 15.4 | 5 | 19.2 | 5 | 19.2 | | 34.6 | 6 | 23.1 | | 3.8 | 1 | 3.8 | 2 | 7.7 | प | 15.4 | 11 | 42.3 |
| 70.0 | 15 | 75.0 | 14 | 70.0 | 15 | 75.0 | 14 | 70.0 | 15 | 75.0 | 17 | 85.0 | 15 | 75.0 | 15 | 75.0 | 13 | 65.0 | 19 | 95.0 | 19 | 95.0 | 19 | 95.0 | 18 | 95.0 | 20 | 100 |
| 40.0 | 20 | 40.0 | 18 | 36.0 | 19 | 38.0 | 17 | 34.0 | 17 | 34.0 | 18 | 36.0 | 16 | 32.0 | 23 | 46.0 | 20 | 40.0 | 22 | 44.0 | 25 | 50.0 | 20 | 40.0 | 25 | 50.0 | 32 | 64.0 |
| 32.0 | 7 | 28.0 | 8 | 32.0 | 7 | 28.0 | 3 | 12.0 | | 28.0 | 15 | 60.0 | | 28.0 | | 68.0 | 12 | 48.0 | 19 | 76.0 | 19 | 76.0 | 16 | 64.0 | 18 | 76.0 | 24 | 96.0 |
| 56.0 | 16 | 64.0 | 14 | 56.0 | 14 | 56.0 | 15 | 60.0 | ਤ | 52.0 | 16 | 64.0 | 13 | 52.0 | 16 | 64.0 | 17 | 68.0 | 19 | 76.0 | 20 | 80.0 | 15 | 60.0 | 20 | 80.0 | 23 | 92.0 |
| 50.0 | 2 | 25.0 | ব | 50.0 | 2 | 25.0 | 2 | 25.0 | | 37.5 | 3 | 37.5 | | 62.5 | | 50.0 | 3 | 37.5 | 2 | 25.0 | 2 | 25.0 | 3 | 37.5 | | 37.5 | ব | 50.0 |
| 0.0 | 0 | 0.0 | 0 | 0.0 | O | 0.0 | 0 | 0.0 | | 16.7 | | 0.0 | | 16.7 | | 0.0 | 0 | 0.0 | 0 | 0.0 | 0 | 0.0 | 0 | 0.0 | | 0.0 | ব | 66.7 |
| 0.0 | O | 0.0 | 0 | 0.0 | O | 0.0 | 0 | 0.0 | | 18.2 | | 0.0 | | 18.2 | | 0.0 | 0 | 0.0 | 2 | 18.2 | 2 | 18.2 | 2 | 18.2 | | 27.3 | 4 | 36.4 |
| 0.0 | 1 | 10.0 | 0 | 0.0 | 1 | 10.0 | 0 | 0.0 | | 30.0 | ব | 40.0 | | 40.0 | | 30.0 | O | 0.0 | 3 | 30.0 | 3 | 30.0 | 3 | 30.0 | | 70.0 | 8 | 80.0 |
| 8.5 | 8 | 11.3 | ব | 5.6 | 8 | 11.3 | प | 5.6 | 5 | 7.0 | 7 | 0.9 | ব | 5.6 | | 5.6 | 5 | 7.0 | 7 | ರಿ. ಇ | 9 | 12.7 | 14 | 19.7 | 27 | 38.0 | 37 | 52.1 |
| 0.0 | 2 | 5.0 | 0 | 0.0 | വ | 5.0 | 0 | 0.0 | | 2.5 | 2 | 5.0 | 0 | 0.0 | 2 | 5.0 | 0 | 0.0 | O | 0.0 | 3 | 7.5 | 2 | 5.0 | 11 | 27.5 | 13 | 32.5 |
| 0.0 | 1 | 11.1 | 0 | 0.0 | 1 | 11.1 | 0 | 0.0 | | 0.0 | | 11.1 | | 0.0 | | 11.1 | 0 | 0.0 | 0 | 0.0 | | 11.1 | 0 | 0.0 | | 11.1 | 1 | 11.1 |
| 8.3 | 1 | 8.3 | | 8.3 | 1 | 8.3 | 1 | 8.3 | | 0.0 | 0 | 0.0 | () | 0.0 | | 0.0 | | 8.3 | 0 | 0.0 | | 8.3 | O | 0.0 | | 0.0 | 0 | 0.0 |
| 16.7 | র্ব | 13.3 | 5 | 16.7 | 3 | 10.0 | 3 | 10.0 | 5 | 16.7 | | 23.3 | 5 | 16.7 | | 23.3 | 5 | 16.7 | 2 | 6.7 | प | 13.3 | 5 | 16.7 | | 23.3 | 6 | 20.0 |
| 0.0 | O | 0.0 | O | 0.0 | O | 0.0 | 0 | 0.0 | | 0.0 | 0 | 0.0 | O | 0.0 | | 0.0 | O | 0.0 | 0 | 0.0 | 0 | 0.0 | 0 | 0.0 | | 0.0 | 0 | 0.0 |
| 15.8 | 3 | 15.8 | | 21.1 | 2 | 10.5 | 3 | 15.8 | | 0.0 | | 5.3 | | 5.3 | | 21.1 | 2 | 10.5 | | 21.1 | 3 | 15.8 | 4 | 21.1 | 3 | 15.8 | 8 | 42.1 |
| 0.0 | 0 | 0.0 | 0 | 0.0 | O | 0.0 | 0 | 0.0 | | 0.0 | 0 | 0.0 | 0 | 0.0 | | 0.0 | 0 | 0.0 | 0 | 0.0 | 0 | 0.0 | 0 | 0.0 | | 0.0 | 0 | 0.0 |
| 18.8 | 2 | 12.5 | 3 | 18.8 | വ | 12.5 | 1 | 6.3 | | 6.3 | 2 | 12.5 | | 6.3 | 2 | 12.5 | 3 | 18.8 | 2 | 12.5 | ব | 25.0 | 2 | 12.5 | | 25.0 | ട | 37.5 |
| 25.0 | 1 | 25.0 | | 25.0 | 1 | 25.0 | | 25.0 | | 0.0 | 0 | 0.0 | () | 0.0 | | 0.0 | O | 0.0 | 0 | 0.0 | 0 | 0.0 | 0 | 0.0 | | 0.0 | 1 | 25.0 |
| 5.6 | 1 | 5.6 | | 5.6 | 1 | 5.6 | 1 | 5.6 | | 16.7 | 3 | 16.7 | | 5.6 | 2 | 11.1 | | 5.6 | O | 0.0 | 0 | 0.0 | 0 | 0.0 | | 0.0 | 0 | 0.0 |
| 0.0 | O | 0.0 | O | 0.0 | O | 0.0 | 0 | 0.0 | | 0.0 | | 100 | O | 0.0 | | 100 | O | 0.0 | | 100 | | 100 | 1 | 100 | | 100 | 1 | 100 |
| 0.0 | O | 0.0 | 0 | 0.0 | O | 0.0 | 0 | 0.0 | 0 | 0.0 | | 6.7 | O | 0.0 | | 6.7 | | 6.7 | 0 | 0.0 | 2 | 13.3 | 2 | 13.3 | ব | 26.7 | 7 | 46.7 |
| 10.0 | 1 | 10.0 | | 10.0 | 1 | 10.0 | | 10.0 | | 0.0 | | 10.0 | O | 0.0 | | 30.0 | O | 0.0 | O | 0.0 | 0 | 0.0 | O | 0.0 | O | 0.0 | 3 | 30.0 |
| 0.0 | 3 | 9.4 | 0 | 0.0 | 3 | 9.4 | 0 | 0.0 | | 3.1 | 2 | 6.3 | | 0.0 | | 9.4 | 0 | 0.0 | | 3.1 | | 3.1 | O | 0.0 | | 3.1 | 2 | 6.3 |
| 3.1 | 1 | 3.1 | 1 | 3.1 | 1 | 3.1 | 1 | 3.1 | 0 | 0.0 | 0 | 0.0 | O | 0.0 | | 3.1 | O | 0.0 | O | 0.0 | 0 | 0.0 | 1 | 3.1 | 1 | 3.1 | 1 | 3.1 |
| 0.0 | 1 | 4.5 | 0 | 0.0 | 1 | 4.5 | 0 | 0.0 | | 4.5 | | 4.5 | () | 0.0 | 6 | 27.3 | O | 0.0 | O | 0.0 | O | 0.0 | 0 | 0.0 | O | 0.0 | 0 | 0.0 |

95

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AESKUSLIDES ANCA 510(k) Traditional submission

Table 124: Diagnostic Sensitivity and Specificity_ANCA Formalin
	% Diagnostic Sensitivity & Specificity
(95% CI)		% Sensitivity
(95% CI)		% Specificity
(95% CI)
			AAV (n= 135)	OD (n=375)	AAV (n= 135)	OD (n=375)
Site 1	Manual	Reader 1	40
(32.1 - 48.4)	93.1
(90 - 95.2)	Combined
Readers	41.1
(35.4 - 47.1)	92.3
(90.1 - 94)
		Reader 2	42.2
(34.2 - 50.7)	91.5
(88.2 - 93.9)
	Reader
Confirmation
at HELIOS	Reader 1	49.6
(41.3 - 58)	93.3
(90.3 - 95.4)	Combined
Readers	47.4
(41.5 - 53.4)	92.7
(90.6 - 94.3)
		Reader 2	45.2
(37 - 53.6)	92
(88.8 - 94.3)
	HELIOS		45.2
(37 - 53.6)	95.2
(92.5 - 96.9)
Site 2	Manual	Reader 3	37.8
(30 - 46.2)	93.1
(90 - 95.2)	Combined
Readers	41.1
(35.4 - 47.1)	91.7
(89.5 - 93.5)
		Reader 4	44.4
(36.3 - 52.9)	90.4
(87 - 93)
	Reader
Confirmation
at HELIOS	Reader 3	36.3
(28.7 - 44.7)	93.6
(90.7 - 95.7)	Combined
Readers	41.5
(35.8 - 47.4)	90.9
(88.7 - 92.8)
		Reader 4	46.7
(38.5 - 55.1)	88.3
(84.6 - 91.1)
	HELIOS		42.2
(34.2 - 50.7)	94.4
(91.6 - 96.3)
Site 3	Manual	Reader 5	37.8
(30 - 46.2)	93.6
(90.7 - 95.7)	Combined
Readers	43
(37.2 - 48.9)	92
(89.8 - 93.7)
		Reader 6	48.1
(39.9 - 56.5)	90.4
(87 - 93)
	Reader
Confirmation
at HELIOS	Reader 5	39.3
(31.4 - 47.7)	89.6
(86.1 - 92.3)	Combined
Readers	44.8
(39 - 50.8)	85.1
(82.3 - 87.4)
		Reader 6	50.4
(42 - 58.7)	80.5
(76.2 - 84.2)
	HELIOS		63.7
(55.3 - 71.3)	71.7
(67 - 76.1)

Table 124: Diagnostic Sensitivity and Specificity_ANCA Formalin

Note: The 120 characterized MPO/PR3/ANCA positive sera have been excluded from the calculation of
diagnostic sensitivity and specificity.

96

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Summary of Results:

Positive, Negative, Overall and Pattern Agreements for Method Comparison between Method C (Manual) and Method B (Reader Confirmation of HELIOS images) were > 80%. All acceptance criteria were met.

Positive Agreement of Method C vs B ranged from 85.7% to 89.6%.

Negative Agreement of Method C vs B ranged 86.3% to 97.6%.

Overall Agreement of Method C vs B ranged from 87.0% to 95.4%.

Pattern Agreement of Method C vs B ranged from 82.1% to 87.5%.

The results show that Methods C (Manual) and B (Reader Confirmation of HELIOS images) give equivalent results. Furthermore, the results of the 3 study site were comparable with each other.

Agreements for Comparison between Method B (Reader Confirmation of HELIOS images) and Method A (HELIOS positive/negative classification by the Vasculitis Pattern Plus software) were as follows:

Positive Agreement of Method B vs A ranged from 73.6% to 99.4%.

Negative Agreement of Method B vs A ranged from 72.2% to 99.2%.

Overall Agreement of Method B vs A ranged from 79.2% to 95.9%.

Pattern Agreement of Method B vs A ranged from 76.1% to 90.8%.

According to this, acceptance criteria for comparison of Method B vs A were met.

The comparison of Method C (Manual) vs A (HELIOS suggested results) gave good results within the acceptance criteria for positive, negative and overall agreement. In the case of pattern agreement, 69.7% was reached. This is just below the acceptance criteria and we tolerate the deviation since this is a slight and unique occurrence.

Positive Agreement of Method C vs A ranged from 71.0% to 98.7%.

Negative Agreement of Method C vs A ranged from 70.7% to 95.2%.

Overall Agreement of Method C vs A ranged from 77.6% to 90.5%.

Pattern Agreement of Method C vs A ranged from 69.7% to 79.6%.

The Cross Reactivity Table (table no.: 101) shows that there were only few crossreactivities for Method A, B and C, which is also reflected by the high specificity of this methods (90%). The most crossreactivities could be seen in the Autoimmune Liver Disease Group (AIH, PBC, and PCS: up to 62.5%), in the inflammatory bowel disease group (Crohn' Disease, Ulcerative Colitis, IBD: up to 11.3%) and in SLE (up to 23%). These results reflect values reported in the literature. Method A (site 3) has a lower specificity as described and therefore, higher crossreactivities can be seen.

97

Conclusion:

Method C and Method B show substantial equivalence. AESKUSLIDES ANCA Ethanol and Formalin perform substantially equivalent when processed manually and on HELIOS with Reader Confirmation of HELIOS images, as shown by the method comparison study, the calculated diagnostic sensitivities and specificities and the cross reactivity table.

For Method A we found a lower agreement compared to Method B and C. This can be explained by the complex patterns, which can be distinguished by the readers at the microscope or at the screen, but to limited extent by the software.

Our results show that results generated at HELIOS must always be confirmed by trained readers. The Vasculitis Pattern Plus software can support the reader to preselect positive samples, regardless of the detected pattern. According to the international recommendations on ANCA testing and the literature, it is recommended to further characterize ANCA positive IFA results by another method, for example PR3- and/or MPO-ELISA.

Endpoint Titer comparison:

To evaluate Within-Lab Precision and Between-Lab Precision of endpoint titer determination on AESKUSLIDES ANCA Ethanol and ANCA Formalin processed on HELIOS and processed manually, based on CLSI Guideline EP12-A2.

Procedure:

Five serum samples with endpoint titers between 1:10 and 1:320 were diluted serially, ranging from 1:10 up to 1:1280 sample dilution, depending on the expected enpoint titer displayed in the table below. For each sample at least 4 different dilutions around the expected endpoint titer were made. Those serum dilutions were assayed on AESKUSLIDES ANCA Ethanol and Formalin and analyzed on HELIOS (Method B, Reader Confirmation) 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.

| Sample
ID | Pos/Neg | Pattern
ANCA Ethanol | Endpoint titer
ANCA Ethanol | Pattern
ANCA Formalin | Endpoint titer
ANCA
Formalin |
|--------------|---------|-------------------------|--------------------------------|--------------------------|------------------------------------|
| 1 | Pos | C-ANCA | 10 | C-ANCA | 20 |
| 2 | Pos | C-ANCA | 160 | C-ANCA | 160 |
| 3 | Pos | C-ANCA | 160 | C-ANCA | 320 |
| 4 | Pos | P-ANCA | 80 | C-ANCA | 20 |
| 5 | Pos | P-ANCA | 320 | C-ANCA | 40 |

All runs have been performed according to the respective IFUs. 1 positive and 1 negative control (kit controls) were included in each run.

The described study has been conducted at three different study sites to allow the calculation of Between-Lab Precision.

98

Data Analysis

Positive/Negative Classification and Pattern were recorded for each sample dilution in each run and for each Method. For Method C (Manual performance) fluorescence intensity was also reported.

Determination of endpoint titer:

For each replicate of the dilution series endpoint titer was reported as the reciprocal of the highest dilution at which the sample was classified as positive.

After pairwise comparison of the datapoints Within-lab precision was calculated for Methods B and C, for each of the three study sites.

Data will be presented as

percentage of samples that differ maximum ±1 titer level from each . other and
. percentage of samples that are > ±1 titer level away from each other.

For Between-Lab Precision the titer agreements (including 95% CI) between the three study sites were determined for each Method B and C (site 1 vs site 2, site 2 vs site 3, site 1 vs site 3).

Acceptance Criteria

. For each serum reported endpoint titers are allowed to differ maximum ±1 titer level from each other
. Calculations should meet the following criteria:

Calculations	Method B	Method C
Percentage of samples that differ maximum ±1 titer level	≥ 90%	≥ 90%
Titer Agreement	≥ 80%	≥ 80%

Results - Within-Lab Precision of endpoint titer determination

Results of endpoint titer determination for each of the three replicates per sample dilution can be found in the appendix.

For calculation of Within-Lab Precision the percentages of samples that are within and outside +/-1 titer level were calculated for the two readers separately (30 replicates for each sample dilution) and for all readers combined. The results of Within-Lab Precision for study sites 1, 2 and 3 are presented in the tables below.

99

AESKUSLIDES ANCA Ethanol

Table 125a: Number and percentages of samples that are within and outside +/-1 titer level – Method B and C

| AESKUSLIDES
ANCA Ethanol -
Method B | | Number of
samples within
+/-1 titer level | Number of
samples

+/-1 titer level | Total
number | Percentage of
samples that
are +/-1 titer
level | Percentage of
samples
+/-1 titer level |
|-------------------------------------------|----------|-------------------------------------------------|--------------------------------------------|-----------------|----------------------------------------------------------|------------------------------------------------|
| Site 1 | Reader 1 | 147 | 3 | 150 | 98.0% | 2.0% |
| | Reader 2 | 139 | 11 | 150 | 92.7% | 7.3% |
| Site 2 | Reader 3 | 139 | 11 | 150 | 92.7% | 7.3% |
| | Reader 4 | 138 | 12 | 150 | 92.0% | 8.0% |
| Site 3 | Reader 5 | 149 | 1 | 150 | 99.3% | 0.7% |
| | Reader 6 | 144 | 6 | 150 | 96.0% | 4.0% |
| All readers combined | | 856 | 44 | 900 | 95.1% | 4.9% |

| AESKUSLIDES
ANCA Ethanol -
Method C | | Number of
samples within
+/-1 titer level | Number of
samples

+/-1 titer
level | Total
number | Percentage of
samples that
are +/-1 titer
level | Percentage of
samples
+/-1 titer level |
|-------------------------------------------|----------|-------------------------------------------------|-----------------------------------------------|-----------------|----------------------------------------------------------|------------------------------------------------|
| Site 1 | Reader 1 | 143 | 7 | 150 | 95.3% | 4.7% |
| | Reader 2 | 144 | 6 | 150 | 96.0% | 4.0% |
| Site 2 | Reader 3 | 143 | 7 | 150 | 95.3% | 4.7% |
| | Reader 4 | 142 | 8 | 150 | 94.7% | 5.3% |
| Site 3 | Reader 5 | 146 | 4 | 150 | 97.3% | 2.7% |
| | Reader 6 | 141 | 9 | 150 | 94.0% | 6.0% |
| All readers combined | | 859 | 41 | 900 | 95.4% | 4.6% |

Acceptance criteria for Within-Lab Precision of endpoint titer determination on ANCA Ethanol were met. Percentage of samples that were within +/-1 titer level was 95.1% for Method B and 95.4% for Method C, for combined readers from all study sites. Considering all three study sites and readers separately, percentage of samples that were within +/-1 titer level ranged from 92.0% to 99.3% for Method B (Reader Confirmation) and from 94.0% to 97.3% for Method C (Manual). Results for Methods B and C were comparable.

Pos/Neg classification and pattern determination were consistent for 100% of the replicates.

100

AESKUSLIDES ANCA Formalin

Table 125b: Number and percentages of samples that are within and outside +/-1 titer level – Method B and C

| | AESKUSLIDES
ANCA Formalin -
Method B | Number of
samples within
+/-1 titer level | Number of
samples

+/-1 titer level | Total
number | Percentage of
samples that are
+/-1 titer level | Percentage of
samples
+/-1 titer level |
|----------------------|--------------------------------------------|-------------------------------------------------|--------------------------------------------|-----------------|-------------------------------------------------------|------------------------------------------------|
| Site 1 | Reader 1 | 145 | 5 | 150 | 96.7% | 3.3% |
| | Reader 2 | 145 | 5 | 150 | 96.7% | 3.3% |
| Site 2 | Reader 3 | 147 | 3 | 150 | 98.0% | 2.0% |
| | Reader 4 | 135 | 15 | 150 | 90.0% | 10.0% |
| Site 3 | Reader 5 | 145 | 5 | 150 | 96.7% | 3.3% |
| | Reader 6 | 135 | 15 | 150 | 90.0% | 10.0% |
| All readers combined | | 852 | 48 | 900 | 94.7% | 5.3% |

| | AESKUSLIDES
ANCA Formalin -
Method C | Number of
samples within
+/-1 titer level | Number of
samples

+/-1 titer level | Total
number | Percentage of
samples that are
+/-1 titer level | Percentage of
samples
+/-1 titer level |
|--------|--------------------------------------------|-------------------------------------------------|--------------------------------------------|-----------------|-------------------------------------------------------|------------------------------------------------|
| Site 1 | Reader 1 | 145 | 5 | 150 | 96.7% | 3.3% |
| | Reader 2 | 148 | 2 | 150 | 98.7% | 1.3% |
| Site 2 | Reader 3 | 145 | 5 | 150 | 96.7% | 3.3% |
| | Reader 4 | 144 | 6 | 150 | 96.0% | 4.0% |
| Site 3 | Reader 5 | 149 | 1 | 150 | 99.3% | 0.7% |
| | Reader 6 | 134 | 16 | 150 | 89.3% | 10.7% |
| | All readers combined | 865 | 35 | 900 | 96.1% | 3.9% |

Acceptance criteria for Within-Lab Precision of endpoint titer determination on ANCA Formalin were met. Percentage of samples that were within +/-1 titer level was 94.7% for Method B and 96.1% for Method C, for combined readers from all study sites.

Considering all three study sites and readers separately, percentage of samples that were within +/-1 titer level ranged from 90.0% to 98.0% for Method B (Reader Confirmation) and from 89.3% to 99.3 % for Method C (Manual). Only for reader 6 in Method C the percentage of samples was slightly below our acceptance criterion (89.3% instead of 90%). We tolerate this deviation, since this only concerns one reader out of six. However, this result shows that positive classification of samples near its endpoint titer is subjective and highly depends on the experience of the reader.

Results for Methods B and C were comparable.

Pos/Neg classification and pattern determination were consistent for 100% of the replicates.

Results - Between-Lab Precision of endpoint titer determination

In order to evaluate Between-Lab Precision of endpoint titer determination, titer agreements (including 95% Cls) were determined between the three sites (site 1 vs site 2, site 2 vs site 3, site 1 vs site 3) for each Method B and C. Results are displayed in the following tables.

101

AESKUSLIDES ANCA Ethanol

Table 126a: % Titer agreement between three study sites – Method B and C

| % Titer agreement

(95% CI)	Site 1 vs. Site 2	Site 1 vs. Site 3	Site 2 vs. Site 3
Method B
(Reader
Confirmation)	93.3
(89.9 - 95.6)	86.7
(82.4 - 90.1)	82.3
(77.6 - 86.2)
Method C
(Manual)	98.3
(96.2 - 99.3)	88.7
(84.6 - 91.8)	87
(82.7 - 90.3)

For Between-lab Precision of endpoint titer determination on ANCA Ethanol, all acceptance criteria were met. Titer agreements comparing the three study sites ranged from 82.3% to 93.3% for Method B (Reader Confirmation) and from 87.0% to 98.3% for Method C (Manual). With manual reading of the slides (Method C) slightly higher titer agreements have been achieved.

AESKUSLIDES ANCA Formalin

Table 126b: % Titer agreement between three study sites - Method B and C

| % Titer agreement

(95% CI)	Site 1 vs. Site 2	Site 1 vs. Site 3	Site 2 vs. Site 3
Method B
(Reader
Confirmation)	86.7
(82.4 - 90.1)	79
(74 - 83.2)	82.7
(78 - 86.5)
Method C
(Manual)	93.7
(90.3 - 95.9)	80.3
(75.5 - 84.4)	82.7
(78 - 86.5)

Titer agreement between the three study sites ranged from 79.0% to 86.7% for Method B (Reader Confirmation) and from 80.3% to 93.7% for Method C (Manual). For Between-Lab Precision of endpoint titer determination on ANCA Formalin, all acceptance criteria were met, except for Method B site 1 vs. site 3. Here, titer agreement was slightly below the acceptance criterion (79.0% instead of 80%). However, we tolerate this deviation, since at study site 3, consistently higher endpoint titers have been obtained than at study sites 1 and 2. This is also reflected by the generally lower agreements when comparing site 3 to the other study sites. This study shows that the evaluation of sample titer is subjective and can vary from laboratory to laboratory, from microscope to microscope. Thus, we suggest for each laboratory to perform a titer control using the positive control or other well-known samples to balance variations in endpoint titer determination.

Results for Methods B and C were comparable when contrasting Site 1 vs Site 3 and Site 2 vs Site 3. When comparing Site 1 vs Site 2 titer agreements were slightly higher for Method C (Manual).

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Conclusion

Pre-determined acceptance criteria were met. Method B (Reader Confirmation) as well as Method C (Manual) showed a good and acceptable repeatability (Within-Lab Precision) and reproducibility (Between-Lab Precision) for enpoint titer determination.

3. Clinical cut-off:

See assay cut-off

4. Expected values/Reference range

Expected values for ANCA were analyzed with a panel of 150 sera from healthy donors: 100 from Germany (56 females,44 males, mean age 31 years, range of 18-58 years, Ethnicity: German) and 50 from the US (17 females, 33 males, mean age 34 years, range of 19-62 years, Ethnicity: 30 Black, 10 Hispanic, 10 White).

Slides were processed manually according to the IFU and subsequently analyzed at the microscope by two independent readers.

Results:

With AESKUSLIDES ANCA Ethanol, there were 6 positive results for reader 1 and 3 positive results for reader 2, and with AESKUSLIDES ANCA Formalin, there were 6 positive results for reader 1 and 4 positive results for reader 2.

Table 127. Normal Range Study, number and percent positive and negative samples.
Normal Range
Study	ANCA Ethanol			ANCA Formalin
	Pos (%)	Neg (%)	Total	Pos (%)	Neg (%)	Total
Reader 1	6 (4)	144 (96)	150	6 (4)	144 (96)	150
Reader 2	3 (2)	147 (98)	150	4 (2.7)	146 (97.3)	150

Table 127: Normal Range Study, number and percent positive and negative samples:

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	Normal Range Study	ANCA Ethanol			ANCA Formalin
Sample ID	Sample Origin	Reader 1		Reader 2		Reader 1		Reader 2
		Pos/Neg	ANCA Pattern FI	Pos/Neg	ANCA Pattern FI	Pos/Neg	ANCA Pattern FI	Pos/Neg	ANCA Pattern FI
NC 008	USA	Pos	A 2	Pos	A 2	Pos	C 1	Pos	C 1
NC 010	USA	Neg	Neg 0	Neg	Neg 0	Neg	Neg 0	Pos	C 1
NC 028	USA	Pos	P 1	Pos	P 1	Neg	Neg 0	Neg	Neg 0
NC 085	Germany	Pos	A 1	Pos	A 1	Neg	Neg 0	Neg	Neg 0
NC 109	Germany	Neg	Neg 0	Neg	Neg 0	Pos	C 1	Pos	C 1
NC 123	Germany	Pos	C 1	Neg	Neg 0	Pos	C 1	Neg	Neg 0
NC 126	Germany	Neg	Neg 0	Neg	Neg 0	Pos	C 1	Neg	Neg 0
NC 127	Germany	Pos	P 1	Neg	Neg 0	Neg	Neg 0	Neg	Neg 0
NC 139	Germany	Pos	C 1	Neg	Neg 0	Pos	C 1	Neg	Neg 0
NC 144	Germany	Neg	Neg 0	Neg	Neg 0	Pos	C 1	Pos	C 1

Table 128: Normal Range Study, positive samples:

FI = Fluorescence Intensitiy

Only one sample (NC 008) has been found positive in both ANCA Ethanol (FI = 2) and ANCA Formalin (FI = 1) assays by both readers. 2 samples (NC 123, NC 139) have been found positive in both assays (Fl=1) by reader 1 and negative by reader 2. All other samples have been found positive either in the ANCA Ethanol or in the ANCA Formalin assay with an FI of 1.

Conclusion:

The low number of positive samples we found with AESKUSLIDES ANCA Ethanol and ANCA Formalin in the tested healthy population correlate well with numbers reported in the literature.

N. Instrument Name

HELIOS AUTOMATED IFA SYSTEM

O. System Description

The HELIOS AUTOMATED IFA SYSTEM is an automated system including pipetting unit with microscope and software that acquires. stores and displays digital images of stained indirect immunofluorescence slides. The HELIOS DEVICE SOFTWARE is designed to support input of results from the AESKUSLIDES into electronic laboratory data management systems.

The HELIOS AUTOMATED IFA SYSTEM should only be used with AESKUSLIDES assays that are cleared or approved for use on the instrument. All suggested results obtained with the HELIOS DEVICE SOFTWARE must be confirmed by trained personnel.

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Image /page/104/Picture/1 description: The image is a logo for AESKU.DIAGNOSTICS. The logo features a green square with a white image of a vest inside. To the right of the square is the text "AESKU.DIAGNOSTICS" in gray, with the words "THE DIAGNOSTIC TOOL THAT WORKS" in a smaller font below.

1. Modes of Operation:

HELIOS: automated processing with automated imaging and automated reading HELIOS User Eval: automated processing with automated imaging and manual reading

HELMED Mode: automated processing with manual imaging and manual reading Manual Mode: manual processing with manual imaging and manual reading

Does the applicant's device contain the ability to transmit data to a computer, webserver, or mobile device?

Yes

Does the applicant's device transmit data to a computer, webserver, or mobile device using wireless transmission?

No

2. Software:

General

The HELIOS AUTOMATED IFA SYSTEM is an automated system for immunofluorescence processing with an integrated camera with an optic (microscope) and software for routine laboratory use by professional users under controlled environmental conditions.

The HELIOS camera with an optic (microscope) automatically focuses on the areas of biological material on the slides. Pictures are stored and evaluated by the HELIOS DEVICE SOFTWARE. The software performs a positive/negative of the cells. The software suggests the result as a qualitative result (positive. negative). All suggested results obtained with the HELIOS AUTOMATED IFA SYSTEM and the HELIOS DEVICE SOFTWARE must be confirmed by trained personnel before the result is finalized.

Level of Concern

The Level of concern was determined according to the "Guidance for the Content of Premarket Submissions for Software Contained in Medical Devices" dated May 11. 2005. Following the recommendations of this standard. the HELIOS DEVICE SOFTWARE is assigned moderate level of concern.

FDA has reviewed applicant's Hazard Analysis and software development processes for this line of product types.

Specimen Identification: 3. Manual sample identification and/or Barcode
1. Specimen Sampling and Handling: Not applicable

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5. Calibration:

There is no calibration of the instrument by the user.

6. Quality Control:

Positive and negative controls are supplied with the assay reagents (see assay description above).

P. Other Supportive Instrument Performance Characteristics Data Not Covered In The "Performance Characteristics" Section above:

Not applicable