← Product Code [LSW](/submissions/IM/subpart-f%E2%80%94immunological-test-systems/LSW) · K172348 # AESKUSLIDES nDNA (Crithidia luciliae), AESKUSLIDES nDNA (Crithidia luciliae) Demo Kit, AESKUSLIDES nDNA (Crithidia luciliae) Bulk kit x5, AESKUSLIDES nDNA (Crithidia luciliae) Bulk kit x10 (K172348) _Aesku.Diagnostics GmbH & Co. KG · LSW · Feb 16, 2018 · Immunology · SESE_ **Canonical URL:** https://fda.innolitics.com/submissions/IM/subpart-f%E2%80%94immunological-test-systems/LSW/K172348 ## Device Facts - **Applicant:** Aesku.Diagnostics GmbH & Co. KG - **Product Code:** [LSW](/submissions/IM/subpart-f%E2%80%94immunological-test-systems/LSW.md) - **Decision Date:** Feb 16, 2018 - **Decision:** SESE - **Submission Type:** Traditional - **Regulation:** 21 CFR 866.5100 - **Device Class:** Class 2 - **Review Panel:** Immunology ## Intended Use AESKUSLIDES® nDNA (Crithidia luciliae) is an indirect immunofluorescence assay utilizing Crithidia luciliae coated slides as a substrate for the qualitative and/or semi-quantitative determination of antibodies to native double stranded DNA (dsDNA) in human serum. This in vitro diagnostic assay is used as an aid for the diagnosis of Systemic Lupus Erythematosus (SLE) in conjunction with other clinical and laboratory findings. The assay can be processed manually and analyzed at the microscope or processed and analyzed with HELIOS® AUTOMATED IFA SYSTEM. All suggested results obtained with the HELIOS® AUTOMATED IFA SYSTEM must be confirmed by trained personnel. ## Device Story AESKUSLIDES® nDNA is an indirect immunofluorescence (IFA) assay using Crithidia luciliae-coated slides to detect anti-dsDNA antibodies in human serum. The assay involves incubating patient serum on slides, followed by a FITC-labeled anti-human IgG conjugate. The resulting fluorescence is analyzed either manually via fluorescence microscopy or automatically using the HELIOS® AUTOMATED IFA SYSTEM. In the automated workflow, the HELIOS system captures images, and the HELIOS DEVICE SOFTWARE (using the DNA Pattern Plus tool) performs image analysis to suggest positive/negative results based on fluorescence intensity and cell area. All automated suggestions require confirmation by a trained operator. The device aids in SLE diagnosis by identifying specific kinetoplast fluorescence. Benefits include standardized processing and automated image analysis, potentially improving laboratory efficiency while maintaining clinical accuracy through mandatory human verification. ## Clinical Evidence Clinical study evaluated 776 serum samples (297 SLE, 479 other diseases). AESKUSLIDES nDNA showed 24.6% sensitivity and 94.8% specificity, compared to 37% sensitivity and 85.8% specificity for the predicate. The higher specificity of the subject device resulted in a higher PPV (74.5% vs 61.8%). Precision studies (within-lab, between-lab, between-operator, single-operator) confirmed that all methods (manual and automated) met acceptance criteria, with overall agreements >85% for manual and reader-confirmed automated methods. ## Technological Characteristics Indirect immunofluorescence assay using Crithidia luciliae-coated slides. Components include FITC-labeled anti-human IgG conjugate, positive/negative controls, mounting medium, and buffers. Connectivity: Standalone or integrated with HELIOS AUTOMATED IFA SYSTEM. Software: DNA Pattern Plus tool for automated image analysis. Sterilization: Not applicable (reagents). ## Regulatory Identification An antinuclear antibody immunological test system is a device that consists of the reagents used to measure by immunochemical techniques the autoimmune antibodies in serum, other body fluids, and tissues that react with cellular nuclear constituents (molecules present in the nucleus of a cell, such as ribonucleic acid, deoxyribonucleic acid, or nuclear proteins). The measurements aid in the diagnosis of systemic lupus erythematosus (a multisystem autoimmune disease in which antibodies attack the victim's own tissues), hepatitis (a liver disease), rheumatoid arthritis, Sjögren's syndrome (arthritis with inflammation of the eye, eyelid, and salivary glands), and systemic sclerosis (chronic hardening and shrinking of many body tissues). ## Predicate Devices - NOVA Lite dsDNA Crithidia luciliae ([K880742](/device/K880742.md)) ## Submission Summary (Full Text) > This content was OCRed from public FDA records by [Innolitics](https://innolitics.com). If you use, quote, summarize, crawl, or train on this content, cite Innolitics at https://innolitics.com. > > Innolitics is a medical-device software consultancy. We help companies design, build, and clear FDA-regulated software and AI/ML devices, including [a 510(k)](https://innolitics.com/services/510ks/), [a De Novo](https://innolitics.com/services/regulatory/), [a SaMD](https://innolitics.com/services/end-to-end-samd/), [an AI/ML medical device](https://innolitics.com/services/medical-imaging-ai-development/), or [an FDA regulatory strategy](https://innolitics.com/services/regulatory/). {0}------------------------------------------------ # Please wait... If this message is not eventually replaced by the proper contents of the document, your PDF viewer may not be able to display this type of document. You can upgrade to the latest version of Adobe Reader for Windows®, Mac, or Linux® by visiting http://www.adobe.com/go/reader_download. For more assistance with Adobe Reader visit http://www.adobe.com/go/acrreader. Windows is either a registered trademark of Microsoft Corporation in the United States and/or other countries. Mac is a trademark of Apple Inc., registered in the United States and other countries. Linux is the registered trademark of Linus Torvalds in the U.S. and other countries. {1}------------------------------------------------ ### 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION MEMORANDUM ASSAY AND INSTRUMENT COMBINATION TEMPLATE - A. 510(k) Number: K172348 - B. Purpose for Submission: New Device - C. Measurand: native double helix DNA (dsDNA) - D. Type of Test: Qualitative and/or semi-quantitative, indirect immunofluorescence - E. Applicant: AESKU.Diagnostics GmbH & Co. KG - F. Proprietary and Established Names: AESKUSLIDES® nDNA (Crithidia luciliae) ### G. Regulatory Information: 1. Regulation section: §CFR 866.5100 - Antinuclear antibody immunological test system 2. Classification: Class II 3. Product code: LSW, Anti-DNA Antibody, Antigen and Control 4. Clinical use Immunology (82) {2}------------------------------------------------ ### H. Intended Use: - 1. Intended use(s): AESKUSLIDES® nDNA (Crithidia luciliae) is an indirect immunofluorescence assay utilizing Crithidia luciliae coated slides as a substrate for the qualitative and/or semi-quantitative determination of antibodies to native double stranded DNA (dsDNA) in human serum. This in vitro diagnostic assay is used as an aid for the diagnosis of Systemic Lupus Erythematosus (SLE) in conjunction with other clinical and laboratory findings. The assay can be processed manually and analyzed at the microscope or processed and analyzed with HELIOS® AUTOMATED IFA SYSTEM. All suggested results obtained with the HELIOS® AUTOMATED IFA SYSTEM must be confirmed by trained personnel. - 2. Indication(s) for use: Same as intended use - 3. Special conditions for use statement(s): - 1. For prescription use only 2. This device is only for use with reagents that are indicated for use with the device. 3. The device is for use by a trained operator in a clinical laboratory setting. 4. 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® nDNA (Crithidia luciliae) is an indirect immunofluorescence assay utilizing Crithidia luciliae coated slides as a substrate for the qualitative and/or semiquantitative determination of antibodies to native double stranded DNA (dsDNA) in human serum. Each kit contains (Quantity depends on product variant): - -Slides, each containing 10 wells coated with Crithidia Luciliae cells - 4.0 ml vial containing Fluorescein (FITC) labelled Anti-human Antibody lgG 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 BSA, PBS. {3}------------------------------------------------ | Standard Ref. | Description | Tests | |------------------|------------------------------------------------------|-------| | 53.100.US | nDNA (Crithidia luciliae) (10
wells) | 100 | | 53.100.US.Demo | nDNA (Crithidia luciliae) (10
wells) Demo kit | 20 | | 53.100.US.Bulk5 | nDNA (Crithidia luciliae) (10
wells) bulk kit x5 | 500 | | 53.100.US.Bulk10 | nDNA (Crithidia luciliae) (10
wells) bulk kit x10 | 1000 | ### 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 dsDNA Crithidia luciliae - 2. Predicate 510(k) number(s): K880742 {4}------------------------------------------------ July 28, 2017 ### 3. Comparison with predicate device: | | Item | Predicate
NOVA Lite dsDNA Crithidia luciliae | AESKUSLIDES® nDNA (Crithidia luciliae) | |--------------|-------------------------------------------|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------| | Similarities | Intended Use | NOVA Lite® dsDNA Crithidia luciliae is an
indirect immunofluorescent assay for the
screening and semi-quantitative
determination of anti-double stranded
DNA (dsDNA) in human serum. The
presence of anti-double stranded DNA can
be used in conjunction with other
serological tests and clinical findings to aid
in the diagnosis of systemic lupus
erythematosus (SLE) | AESKUSLIDES® nDNA (Crithidia luciliae) is an
indirect immunofluorescence assay utilizing Crithidia
luciliae coated slides as a substrate for the qualitative
and/or semi-quantitative determination of antibodies to
native double stranded DNA (dsDNA) in human serum.
This in vitro diagnostic assay is used as an aid for the
diagnosis of Systemic Lupus Erythematosus (SLE) in
conjunction with other clinical and laboratory findings.
The assay can be processed manually and analyzed at
the microscope or processed and analyzed with
HELIOS® AUTOMATED IFA SYSTEM. All suggested
results obtained with the HELIOS® AUTOMATED IFA
SYSTEM must be confirmed by trained personnel. | | | Methology | Immunofluoreszenz assay (IFA) | same as Predicate | | | Procedure | Standard IFA technique | same as Predicate | | | Results | qualitative and semi-quantitative titer | same as Predicate | | | Samples
Matrix | Serum | same as Predicate | | | Analyte | dsDNA | same as Predicate | | | Antigen | dsDNA Crithidia luciliae cells | same as Predicate | | | Fluorescence
Marker | FITC | same as Predicate | | | Controls | one positive control, one negative control | same as Predicate | | | conjugate
Screening | anti-human IgG | same as Predicate | | Differences | Screening
dilution | 1:10 | same as Predicate | | | Storage | 2 - 8 °C | same as Predicate | | | Shelf life | 24 months | same as Predicate | | | Slides | 12 wells coated with antigen | 10 wells coated with antigen | | | Manual
Interpretation
of result | Manual fluorescense microscopy | Manual fluorescense microscopy or with HELIOS w/
trained operator verification | | | Automated
interpretation
of results | N/A | HELIOS w/ trained operator verification | Table 6: comparison with predicate device {5}------------------------------------------------ ### K. Standard/ Guidance Document Referenced (if applicable): Table 7: 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 The AESKUSLIDES nDNA assay utilizes indirect immunofluorescent antibody assay techniques. Patient sera are diluted in wash/sample buffer and applied to a well on the slide. nDNA antibodies, if present, will bind to antigens coated on the slide. After washing with wash/sample buffer, a conjugate specific for human IgG is applied which binds to the nDNA antibodies immobilized on the slide surface. After a final wash to remove excess conjugate, the slide is mounted and read as soon as possible using a fluorescence microscope for manual microscopy or HELIOS AUTOMATED IFA SYSTEM. ### Manual interpretation of test results 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 "O" (no specific flurescence) and "4+" (very strong visible reaction). {6}------------------------------------------------ July 28, 2017 | 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 1:10, followed by serial dilution for semiquantitative determinations and suggests each laboratory establish its own screening dilution and titration scheme based on its population and instrumentation. ### Qualitative evaluation A serum dilution is considered negative for nDNA antibodies if the cells exhibit < 1+ fluorescence of the kinetoplast. Likewise, a serum dilution is considered positive for nDNA antibodies if the cells exhibit ≥ 1+ fluorescence of the kinetoplast. A sample is considered positive for nDNA antibodies if it exhibits exhibit ≥ 1+ fluorescence of the kinetoplast at a sample dilution of 1:10 or greater. Operators should report all titers and specific fluorescence staining seen. ### Semi-quantitative evaluation The endpoint titer is defined as the highest sample dilution factor for which specific fluorescence of the kinetoplast is identifiable. The titers are classified as: - 1:10 are considered low titers - 1:20 and 1:40 are considered medium titers - 1:80 and greater are considered high titers {7}------------------------------------------------ ### Automated instrument interpretation of test results The HELIOS DEVICE SOFTWARE examines the fluorescence intensity and uses an analysis algorithm which takes exposure and pixel frequency into account. It examines relevant regions of the captured images, such as the fluorescence cell area and subtracts the background, to provide an assessment of positive or negative results. | Intensity | Interpretation | |-----------|----------------| | + | nDNA positive | | - | nDNA negative | Trained operators must confirm all suggestions. Intensity and End Point Titers are identified by the HELIOS DEVICE SOFTWARE and pattern suggestions are made by the HELIOS PATTERN RECOGNITION tool. AESKU recommends a screening dilution of 1:10, followed by serial dilution for semiquantitative determinations and suggests each laboratory establish its own screening dilution and titration scheme based on its population. #### M. Performance Characteristics: ### 1. Analytical performance During the study 4 different methods have been applied: | Method | Processing | Imaging | Reading/Evaluation od
Slide | Alternate Name of
Method | |--------|------------|-----------|----------------------------------------|---------------------------------------| | A | Automated | Automated | Automated (Software
Interpretation) | HELIOS | | B | Automated | Automated | Manual (read of digital
image) | HELIOS User
Evaluation | | C | Manual | Manual | Manual (read of
microscope field) | Manual
AESKUSLIDES
nDNA | | D | Manual | Manual | Manual (read of
microscope field) | Manual - Predicate
NOVA Lite dsDNA | Method D is the predicate for method C In the clinical study the method comparison was: Method D vs Method C Method C vs. Method A, B Method B vs Method A Nomenclature and acronyms used in studies {8}------------------------------------------------ July 28, 2017 | Nomenclature | Description/ Also known as | |-------------------------------------|-----------------------------------------------------------------------------------------------------------------------| | AESKUSLIDES nDNA | nDNA | | HELIOS AUTOMATED IFA SYSTEM | HELIOS | | Manual or Manual Reading | Results obtained by the operator after reading and interpreting the slides with a traditional fluorescence microscope | | Method A | HELIOS + AESKUSLIDES nDNA w/slides read by HELIOS | | Method B | HELIOS + AESKUSLIDES nDNA w/slides read by User | | Method C | Manual AESKUSLIDES nDNA w/slides read by User | | Method D | Manual Nova Lite dsDNA w/slides read by User | | Negative IFA score/result, negative | neg, negative | | Positive IFA score/result | pos, psoitive | | Diseases Abbreviation | Diseases Name | | SLE | Systemic lupus erythematosus | | APS | Antiphospholipid Syndrome | | MCTD | Mixed Connective Tissue Disease | | UCTD | Undifferentiated connective tissue disease | | SSc | Systemic sclerosis | | PM | Polymyositis | | DM | Dermatomyositis | | RA | Rheumatoid Arthritis | | AIH | Autoimmune Hepatitis | | PBC | Primary biliary cholangitis | | PSC | Primary sclerosing cholangitis | | AAV | ANCA-associated vasculitides | | HCV | Hepatitis C Virus | | HBV | Hepatitis B Virus | # Table 8: Nomenclature and acronyms {9}------------------------------------------------ ### Description of study sample set: 776 Clinical samples have been used for the Clinical Evaluation and Method Comparisons studies described herein. An exhaustive search for US Clinical Samples from 10 BioBanks (BioChain, BioReclamationIVT, Bioserve, ConversantBio, Cureline, DiscoveryLifeSciences, iSpecimen, Precision for Medicine, ProMedDx, and Vitrologic) has been performed and 746 Clinical Samples have been found. The samples have been selected according to their diagnosis, to reflect all important diagnosis for this study (see table). The sample set has been completed with 30 serum samples from a German University Hospital to complement some rare diagnosis (Vasculitis). | Study Sample Set | | | | |---------------------------|------------------------------|---------------------------------------------------|-----| | Sample Definition | | Diagnosis | n | | Target
Diagnosis | SLE | SLE | 277 | | Differential
Diagnosis | APS | APS | 19 | | | Other rheumatic
diseases | Mixed connective tissue disease (MCTD) | 31 | | | | Undifferentiated connective tissue disease (UCTD) | 21 | | | | Systemic sclerosis (SSc) | 44 | | | | Sjögren's syndrome | 30 | | | | Dermatomyositis (DM) and polymyositis (PM) | 37 | | | | Rheumatoid arthritis (RA) | 58 | | | Autoimmune Liver
Diseases | Autoimmune Hepatitis | 21 | | | | Primary biliary cholangitis (PBC) | 20 | | | | Primary sclerosing cholangitis (PSC) | 10 | | | Vasculitis | AAVs | 44* | | | Fibromyalgia | Fibromyalgia | 30 | | | Infections | HCV | 44 | | | | HBV | 34 | | | | EBV | 25 | | | Leukemia | Lymphoma, Myeloma | 31 | | Sum | | | 776 | Table: 9 Serum samples set for clinical and method comparison studies *30samples from Germany The diagnosis criteria of the different samples have been made in agreement with diagnostic standards used in the U.S and Germany. A written statement from different serum suppliers is available on request. Standard criteria are for example ACR criteria. The US sample set was also selected to contain different ethnic groups (e.g. White, Black/Black African, Asian, Hispanic) to reflect the ethnic composition of the US population as good as possible. {10}------------------------------------------------ The samples have been checked for purity and volume by visual inspection and for further contaminations by an in house prescreening before inclusion into the study. All 776 sera passed and were determined suitable for the study. All serum samples which have been used for this studies have been shipped and stored at -20°C. Upon arrival from the Biobanks they were thawed once to aliquot them for the different studies. Subsequently, samples were frozen again. Therefore, samples underwent 2-3 freezing-thawing cycles before they were used in the studies. To confirm that repeated thawing and freezing and also long term storage at -20°C has no influence on the samples we have performed the following two studies: ### Serum Stability AESKUSLIDES nDNA (Crithidia luciliae) #### Procedure: Serum Stability was shown by testing 8 serum samples (7 positive, 1 negative). For each serum, one aliquot has been prepared that serves as control (no freezethawing). A second aliquot for each serum has been prepared that underwent 4 freeze-thaw cycles. Then, all samples and controls have been analyzed on AESKUSLIDES nDNA according to the IFU. Slides were read by two independent readers. Each freeze/thaw sample was compared to the respective control sample. | Sample
ID | AESKUSLIDES nDNA
Result | Grading | |--------------|----------------------------|-----------------| | 1 | Pos | low positive | | 2 | Pos | low positive | | 3 | Pos | low positive | | 4 | Pos | medium positive | | 5 | Pos | medium positive | | 6 | Pos | high positive | | 7 | Pos | high positive | | 8 | Neg | Neg | {11}------------------------------------------------ ### Acceptance criteria: - -Pos/Neg/Overall Agreement: All positive sera have to be found positive and all negative sera have to be found negative. - -Overall 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 AESKUSLIDES nDNA. All positive samples have been found positive, all negative samples have been found negative. - FI agreement was 100% for AESKUSLIDES nDNA. No deviations of fluorescence intensities greater than +/-1 from the initial value have been observed. ### Raw data are in Attachment 10_Section 12_Raw data serum stability Conclusions All criteria are fulfilled in each test. The results show that multiple freeze-thaw cycles have no effect on test results. ### Long Term Serum Stability AESKUSLIDES nDNA (Crithidia luciliae) To show that long term storage of serum samples at -20°C has no effect on performance and results of AESKUSLIDES nDNA. ### Procedure: Seven different serum samples (4 positive and 3 negative) have been aliquoted and stored at -20°C for a time period of at least 14 months. Frozen aliquots of the different sera have been thawed and assayed on AESKUSLIDES nDNA at the indicated time points. All tests have been performed manually according to the IFU and subsequently analyzed at the microscope | Sample
ID | Expected Result | | |--------------|-----------------|----| | | Pos/Neg | FI | | Sample 1 | Pos | 3 | | Sample 2 | Pos | 2 | | Sample 3 | Pos | 4 | | Sample 4 | Neg | 0 | | Sample 5 | Neg | 0 | | Sample 6 | Neg | 0 | | Sample 7 | Pos | 2 | {12}------------------------------------------------ ### Acceptance Criteria: Positive sera have to be found positive and negative sera have to be found negative throughout the whole testing period. 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 at all test time points. FI did not differ more than +/- 1 level from the expected values. | Sample ID | month 0 | | month 4 | | month 8 | | month 10 | | month 12 | | month 14 | | |-----------|---------|----|---------|----|---------|----|----------|----|----------|----|----------|----| | | Pos/Neg | FI | Pos/Neg | FI | Pos/Neg | FI | Pos/Neg | FI | Pos/Neg | FI | Pos/Neg | FI | | Sample 1 | Pos | 3 | Pos | 3 | Pos | 4 | Pos | 3 | Pos | 3 | Pos | 3 | | Sample 2 | Pos | 2 | Pos | 2 | Pos | 2 | Pos | 2 | Pos | 1 | Pos | 2 | | Sample 3 | Pos | 4 | Pos | 3 | Pos | 4 | Pos | 3 | Pos | 4 | Pos | 3 | | Sample 4 | Neg | 0 | Neg | 0 | Neg | 0 | Neg | 0 | Neg | 0 | Neg | 0 | | Sample 5 | Neg | 0 | Neg | 0 | Neg | 0 | Neg | 0 | Neg | 0 | Neg | 0 | | Sample 6 | Neg | 0 | Neg | 0 | Neg | 0 | Neg | 0 | Neg | 0 | Neg | 0 | | Sample 7 | Pos | 2 | Pos | 2 | Pos | 2 | Pos | 2 | Pos | 2 | Pos | 2 | ### Table 10: Results long term serum stability ### 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 14 months has no effect on performance and test results of AESKUSLIDES nDNA. ## a) Precision/Reproducibilty ### Within-Lab Precision AESKUSLIDES nDNA (Crithida luciliae) To assess Within-Lab Precision of AESKUSLIDES nDNA (Crithidia luciliae) 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 11 samples were assayed on AESKUSLIDES nDNA. The sample set includes borderline, low, medium, high positive, and negative samples. The samples are characterized in the following table: | Sample ID | Result | Grading | |-----------|--------|-----------------| | 1 | Pos | borderline | | 2 | Pos | low positive | | 3 | Pos | low positive | | 4 | Pos | medium positive | | 5 | Pos | medium positive | | 6 | Pos | medium positive | Table 11: Sample table for Within-Lab Precision {13}------------------------------------------------ July 28, 2017 | 7 | Pos | high positive | |----|-----|---------------| | 8 | Pos | high positive | | 9 | Pos | high positive | | 10 | Neg | negative | | 11 | Neg | negative | ### Procedure 11 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 1:10 dilution. Results were analyzed by two independent readers. ### Data Analysis Positive/Negative Classification was recorded for each sample in each run and for each Method. Pos/Neg Classification in Method A (HELIOS) is provided by the nDNA 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 neqative 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) 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 {14}------------------------------------------------ * 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 The results are presented in % Positive and % Negative tables 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). ### Raw Data can be found in 2 Report Within-Lab Precision re-evaluation nDNA Table 12: Results - Within Lab Precision AESKUSLIDES nDNA - Method A, B, and C number of positive and negative results | Sample ID | N | Method A | | Method B | | | | Method C | | | | |-----------|----|----------|-------|----------|----------|----------|----------|----------|----------|----------|----------| | | | 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 | 30 | 0 | 30 | 6 | 0 | 24 | 4 | 5 | 26 | 25 | | S2 | 30 | 10 | 20 | 0 | 0 | 30 | 30 | 0 | 0 | 30 | 30 | | S3 | 30 | 17 | 13 | 0 | 3 | 30 | 27 | 0 | 0 | 30 | 30 | | S4 | 30 | 14 | 16 | 4 | 1 | 26 | 29 | 0 | 0 | 30 | 30 | | S5 | 30 | 0 | 30 | 0 | 0 | 30 | 30 | 0 | 0 | 30 | 30 | | S6 | 30 | 2 | 28 | 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 | 1 | 29 | 0 | 0 | 30 | 30 | 0 | 0 | 30 | 30 | | S10 | 30 | 25 | 5 | 30 | 30 | 0 | 0 | 29 | 30 | 1 | 0 | | S11 | 30 | 21 | 9 | 30 | 30 | 0 | 0 | 30 | 30 | 0 | 0 | {15}------------------------------------------------ | Sample ID | N | Method A | | Method B | | | | Method C | | | | |-----------|----|------------|------------|------------|----------|------------|----------|------------|----------|------------|----------| | | | % Negative | % Positive | % Negative | | % Positive | | % Negative | | % Positive | | | | | | | Reader 1 | Reader 2 | Reader 1 | Reader 2 | Reader 1 | Reader 2 | Reader 1 | Reader 2 | | S1 | 30 | 100 | 0 | 100 | 20.0 | 0 | 80.0 | 13.3 | 16.7 | 86.7 | 83.3 | | S2 | 30 | 33.3 | 66.7 | 0 | 0 | 100 | 100 | 0 | 0 | 100 | 100 | | S3 | 30 | 56.7 | 43.3 | 0 | 10 | 100 | 90 | 0 | 0 | 100 | 100 | | S4 | 30 | 46.7 | 53.3 | 13.3 | 3.3 | 86.7 | 96.7 | 0 | 0 | 100 | 100 | | S5 | 30 | 0 | 100 | 0 | 0 | 100 | 100 | 0 | 0 | 100 | 100 | | S6 | 30 | 6.7 | 93.3 | 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 | 3.3 | 96.7 | 0 | 0 | 100 | 100 | 0 | 0 | 100 | 100 | | S10 | 30 | 83.3 | 16.7 | 100 | 100 | 0 | 0 | 96.7 | 100 | 3.3 | 0 | | S11 | 30 | 70.0 | 30.0 | 100 | 100 | 0 | 0 | 100 | 100 | 0 | 0 | Table 13: Results – Within Lab Precision AESKUSLIDES nDNA – Method A, B and C – percentages of positive and negative results Table 14: Results – Within Lab Precision AESKUSLIDES nDNA – Method A, B and C – percentages of positive and negative results for both readers combined | Sample ID | N
(Method A) | Method A | | N
(Method B/C) | Method B | | Method C | | |-----------|-----------------|---------------|---------------|-------------------|---------------|---------------|---------------|---------------| | | | %
Negative | %
Positive | | %
Negative | %
Positive | %
Negative | %
Positive | | S1 | 30 | 100 | 0 | 60 | 60.0 | 40.0 | 15.0 | 85.0 | | S2 | 30 | 33.3 | 66.7 | 60 | 0 | 100 | 0 | 100 | | S3 | 30 | 56.7 | 43.3 | 60 | 5.0 | 95.0 | 0 | 100 | | S4 | 30 | 46.7 | 53.3 | 60 | 8.3 | 91.7 | 0 | 100 | | S5 | 30 | 0 | 100 | 60 | 0 | 100 | 0 | 100 | | S6 | 30 | 6.7 | 93.3 | 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 | 3.3 | 96.7 | 60 | 0 | 100 | 0 | 100 | | S10 | 30 | 83.3 | 16.7 | 60 | 100 | 0 | 98.3 | 1.7 | | S11 | 30 | 70.0 | 30.0 | 60 | 100 | 0 | 100 | 0 | For manual testing (Method C) fluorescence intensities differed at maximum ±1 level within one run and between the runs. ### Conclusion AESKUSLIDES nDNA (Crithidia luciliae) 510(k) Traditional Submission All pre-determined acceptance criteria were met. {16}------------------------------------------------ ### Between-Lab Precision AESKUSLIDES nDNA (Crithida luciliae) To assess Between-Lab Precision of AESKUSLIDES nDNA (Crithidia luciliae) processed on HELIOS (Method A - HELIOS suggestions and Method B - Reader Confirmation of HELIOS images) and processed manually (Method C). ### Sample Set 11 samples were assayed on AESKUSLIDES nDNA. The sample set includes borderline, low, medium, high positive, and negative samples. The samples are characterized in the following table: | Sample ID | AESKUSLIDES nDNA | | | |-----------|------------------|---------------------|-----------------------------| | | Result | Grading | Fluorescence Intensity (FI) | | 1 | Pos | borderline positive | 1 | | 2 | Pos | low positive | 2 | | 3 | Pos | borderline positive | 1 | | 4 | Pos | medium positive | 3 | | 5 | Pos | medium positive | 3 | | 6 | Pos | medium positive | 3 | | 7 | Pos | high positive | 4 | | 8 | Pos | high positive | 4 | | 9 | Pos | high positive | 4 | | 10 | Neg | negative | 0 | | 11 | Neg | negative | 0 | | | | | Table 15: Sample table for Precision study | | |--|--|--|--------------------------------------------|--| ### Procedure 11 samples have been tested on five days, two runs per day, three replicates per sample per run, on 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 | 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 1:10 dilution. Results were analyzed by two independent readers. {17}------------------------------------------------ ### Data Analysis Positive/Negative Classification was recorded for each sample in each run and for each Method. Pos/Neg Classification in Method A (HELIOS) is provided by the nDNA Pattern Plus software tool. For Method C (Manual performance) fluorescence intensity was also reported. The following % Agreements including 95% CI (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) - -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. - Reported FI is allowed to differ max. ± 1 level from the expected value. - - -Agreements should meet the following criteria: Table 16: Acceptance criteria for between-lab precision study | 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% | | 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. 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. From the results of those study, the following will be calculated: 1. Between-Lab Precisions, 2. Between-Operator Precisions, 3. Single-Operator Precisions, and 4. Instrument Precision "The e-Copy is an exact duplicate of the paper copy" {18}------------------------------------------------ ### Results Results are presented in the tables below. Agreements were calculated 1. including the results of the two borderline positive samples, 2. excluding the results of those two samples. All predetermined acceptance criteria were met. ### Between-Site Agreement Manual (Method C) Between-Site Agreement (Calculation including borderline samples): Table 17: Between-Site Agreement (Calculation including borderline samples) Number of Correctly/Incorrectly found samples Method C | 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 | 1071 | 9 | 1080 | 1069 | 11 | 1080 | 1060 | 20 | 1080 | 1600 | 20 | 1620 | | Negative
Agreement | 239 | 1 | 240 | 240 | 0 | 240 | 239 | 1 | 240 | 359 | 1 | 360 | | Overall
Agreement | 1310 | 10 | 1320 | 1309 | 11 | 1320 | 1299 | 21 | 1320 | 1959 | 21 | 1980 | | FI Agreement | 1303 | 17 | 1320 | 1289 | 31 | 1320 | 1286 | 34 | 1320 | 1939 | 41 | 1980 | Table 18: Between-Site Agreement (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 | 99.2
(98.4 - 99.6) | 99
(98.2 - 99.4) | 98.1
(97.2 - 98.8) | 98.8
(98.1 - 99.2) | | Negative
Agreement | 99.6
(97.7 - 99.9) | 100
(98.4 - 100) | 99.6
(97.7 - 99.9) | 99.7
(98.4 - 100) | | Overall
Agreement | 99.2
(98.6 - 99.6) | 99.2
(98.5 - 99.5) | 98.4
(97.6 - 99) | 98.9
(98.4 - 99.3) | | FI Agreement | 98.7
(97.9 - 99.2) | 97.7
(96.7 - 98.3) | 97.4
(96.4 - 98.2) | 97.9
(97.2 - 98.5) | Manual (Method C) Between-Site Agreement (Calculation excluding borderline samples): {19}------------------------------------------------ --- #### Table 19: Between-Site Agreement (Calculation excluding borderline samples) Number of Correctly/Incorrectly found samples Method C | 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 | 840 | 0 | 840 | 834 | 6 | 840 | 834 | 6 | 840 | 1254 | 6 | 1260 | | Negative
Agreement | 239 | 1 | 240 | 240 | 0 | 240 | 239 | 1 | 240 | 359 | 1 | 360 | | Overall
Agreement | 1079 | 1 | 1080 | 1074 | 6 | 1080 | 1073 | 7 | 1080 | 1613 | 7 | 1620 | | FI Agreement | 1063 | 17 | 1080 | 1049 | 31 | 1080 | 1046 | 34 | 1080 | 1579 | 41 | 1620 | Table 20: Between-Site Agreement (Calculation excluding borderline samples) % Agreements (95% Cl) 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
(99.5 - 100) | 99.3
(98.5 - 99.7) | 99.3
(98.5 - 99.7) | 99.5
(99 - 99.8) | | Negative
Agreement | 99.6
(97.7 - 99.9) | 100
(98.4 - 100) | 99.6
(97.7 - 99.9) | 99.7
(98.4 - 100) | | Overall
Agreement | 99.9
(99.5 - 100) | 99.4
(98.8 - 99.7) | 99.4
(98.7 - 99.7) | 99.6
(99.1 - 99.8) | | FI Agreement | 98.4
(97.5 - 99) | 97.1
(96 - 98) | 96.9
(95.6 - 97.7) | 97.5
(96.6 - 98.1) | {20}------------------------------------------------ July 26, 201 Reader Confirmation (Method B) Between-Site Agreement (Calculation including borderline samples): Table 21: Between-Site Agreement (Calculation including 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 | | Positive
Agreement | 995 | 85 | 1080 | 912 | 168 | 1080 | 909 | 171 | 1080 | 1408 | 212 | 1620 | | Negative
Agreement | 238 | 2 | 240 | 238 | 2 | 240 | 240 | 0 | 240 | 358 | 2 | 360 | | Overall
Agreement | 1233 | 87 | 1320 | 1150 | 170 | 1320 | 1149 | 171 | 1320 | 1766 | 214 | 1980 | | FI Agreement | na | na | na | na | na | na | na | na | na | na | na | | #### Table 22: Between-Site Agreement (Calculation including borderline samples) % Agreements (95% Cl) Method B | % Agreement
(95% CI) | Site 1 vs Site 2 | Site 2 vs Site 3 | Site 1 vs Site 3 | All Sites | |-------------------------|-----------------------|-----------------------|-----------------------|-----------------------| | Positive
Agreement | 92.1
(90.4 - 93.6) | 84.4
(82.2 - 86.5) | 84.2
(81.9 - 86.2) | 86.9
(85.2 - 88.5) | | Negative
Agreement | 99.2
(97 - 99.8) | 99.2
(97 - 99.8) | 100
(98.4 - 100) | 99.4
(98 - 99.8) | | Overall
Agreement | 93.4
(91.9 - 94.6) | 87.1
(85.2 - 88.8) | 87
(85.1 - 88.8) | 89.2
(87.7 - 90.5) | | FI Agreement | na | na | na | na | {21}------------------------------------------------ > Reader Confirmation (Method B) Between-Site Agreement (Calculation excluding borderline samples): Table 23: Between-Site Agreement (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 | | Positive
Agreement | 833 | 7 | 840 | 818 | 22 | 840 | 815 | 25 | 840 | 1233 | 27 | 1260 | | Negative
Agreement | 238 | 2 | 240 | 238 | 2 | 240 | 240 | 0 | 240 | 358 | 2 | 360 | | Overall
Agreement | 1071 | 9 | 1080 | 1056 | 24 | 1080 | 1055 | 25 | 1080 | 1591 | 29 | 1620 | | FI Agreement | na | na | na | na | na | na | na | na | na | na | na | na | #### Table 24: Between-Site Agreement (Calculation excluding borderline samples) % Agreements (95% Cl) Method B | % Agreement
(95% CI) | Site 1 vs Site 2 | Site 2 vs Site 3 | Site 1 vs Site 3 | All Sites | |-------------------------|-----------------------|-----------------------|-----------------------|-----------------------| | Positive
Agreement | 99.2
(98.3 - 99.6) | 97.4
(96.1 - 98.3) | 97
(95.6 - 98) | 97.9
(96.9 - 98.5) | | Negative
Agreement | 99.2
(97 - 99.8) | 99.2
(97 - 99.8) | 100
(98.4 - 100) | 99.4
(98 - 99.8) | | Overall
Agreement | 99.2
(98.4 - 99.6) | 97.8
(96.7 - 98.5) | 97.7
(96.6 - 98.4) | 98.2
(97.4 - 98.8) | | FI Agreement | na | na | na | na | {22}------------------------------------------------ July 28, 2017 ### HELIOS (Method A) Between-Site Agreement (Calclation including borderline samples): Table 25: Between-Site Agreement (Calculation including borderline 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 | | Positive
Agreement | 341 | 199 | 540 | 282 | 258 | 540 | 321 | 219 | 540 | 472 | 338 | 810 | | Negative
Agreement | 105 | 15 | 120 | 114 | 6 | 120 | 105 | 15 | 120 | 162 | 18 | 180 | | Overall
Agreement | 446 | 214 | 660 | 396 | 264 | 660 | 426 | 234 | 660 | 634 | 356 | 990 | | FI Agreement | na | na | na | na | na | na | na | na | na | na | na | | Table 26: Between-Site Agreement (Calculation including borderline samples) % 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 | 63.1
(59 - 67.1) | 52.2
(48 - 56.4) | 59.4
(55.3 - 63.5) | 58.3
(54.8 - 61.6) | | Negative
Agreement | 87.5
(80.4 - 92.3) | 95
(89.5 - 97.7) | 87.5
(80.4 - 92.3) | 90
(84.7 - 93.6) | | Overall
Agreement | 67.6
(63.9 - 71) | 60
(56.2 - 63.7) | 64.5
(60.8 - 68.1) | 64
(61 - 67) | | FI Agreement | na | na | na | na | {23}------------------------------------------------ HELIOS (Method A) Between-Site Agreement (Calclati… --- **Source:** [https://fda.innolitics.com/submissions/IM/subpart-f%E2%80%94immunological-test-systems/LSW/K172348](https://fda.innolitics.com/submissions/IM/subpart-f%E2%80%94immunological-test-systems/LSW/K172348) **Published by [Innolitics](https://innolitics.com)** — a medical-device software consultancy. We help companies design, build, and clear FDA-regulated software and AI/ML devices. If you're preparing [a 510(k)](https://innolitics.com/services/510ks/), [a De Novo](https://innolitics.com/services/regulatory/), [a SaMD](https://innolitics.com/services/end-to-end-samd/), [an AI/ML medical device](https://innolitics.com/services/medical-imaging-ai-development/), or [an FDA regulatory strategy](https://innolitics.com/services/regulatory/), [get in touch](https://innolitics.com/contact). **Cite:** Innolitics at https://innolitics.com