(298 days)
The KRONUS Aquaporin-4 Autoantibody (AQP4Ab) ELISA Assay is for the semiquantitative determination of autoantibodies to Aquaporin-4 in human serum. The KRONUS Aquaporin-4 Autoantibody (AQP4Ab) ELISA Assay may be useful as an aid in the diagnosis of Neuromyelitis Optica (NMO) and Neuromyelitis Optica Spectrum Disorders (NMOSD). The KRONUS Aquaporin-4 Autoantibody (AQP4Ab) ELISA Assay is not to be used alone and is to be used in conjunction with other clinical, laboratory, and radiological (e.g., MRI) findings.
The KRONUS Aquaporin-4 Autoantibody (AQP4Ab) ELISA Assay consists of ELISA strip wells coated with human recombinant AQP4 protein (amino acid position: 23-323) (96 wells in total) and supplied as 12 strips of 8 wells in a frame and sealed in a foil pouch with desiccant; ready-to-use 5 levels of calibrators (1.5, 5, 20, 40, and 80 U/mL), 5 x 0.7 mL; a ready-to-use positive (2 x . 7 mL) and a negative control serum (1 x 0.7 mL); AQP4-Biotin (lyophilized), 3 x 1.5 mL (reconstituted); ready-to-use reconstitution buffer for AQP4-Biotin, 1 x 10 mL; Streptavidin Peroxidase (SA-POD dilute before use) 1 x 0.8 mL; ready-to-use diluent for SA-POD, 1 x 15 mL; ready-to-use peroxidase substrate (TMB), 1 x 15 mL; concentrated wash solution (dilute 1:10 with deionized water before use), 1 x 120 mL and ready-to-use stop solution, 1 x 14 mL.
KRONUS Aquaporin-4 Autoantibody (AQP4Ab) ELISA Assay: Acceptance Criteria and Performance Study
This document summarizes the acceptance criteria and the study demonstrating the KRONUS Aquaporin-4 Autoantibody (AQP4Ab) ELISA Assay meets these criteria, based on the provided text.
1. Table of Acceptance Criteria and Reported Device Performance
The provided text focuses on the performance characteristics of the KRONUS Aquaporin-4 Autoantibody (AQP4Ab) ELISA Assay. While explicit "acceptance criteria" are not always presented as a direct numerical threshold in this document, the studies demonstrate that the device's performance metrics were "within the manufacturer's pre-determined acceptance limit" or "deemed appropriate" based on the established cut-off. The table below compiles the reported performance metrics.
| Performance Characteristic | Acceptance Criteria (Implied/Stated) | Reported Device Performance |
|---|---|---|
| Precision | Within manufacturer's pre-determined acceptance limit for %CV | All %CV values within limit. Example Total %CVs: 6.7% - 15.0% |
| Reproducibility (Site-to-site) | R² > 0.99 for correlation between sites | R² > 0.99 for all comparisons (Lab 1 vs. Lab 2, Lab 1 vs. Lab 3, Lab 2 vs. Lab 3) |
| Reproducibility (Lot-to-lot) | %CV for all samples within a specified range | %CV values ranged from 3.22% - 14.13% |
| Linearity Range | Within manufacturer's pre-determined acceptance criteria for recovery | Linear from 1.5 - 79.5 U/mL. Recovery: 80.76% - 118.38% |
| Hook Effect | No hook effect observed up to a certain concentration | No hook effect observed up to 14000 U/mL |
| Limit of Blank (LoB) | Not explicitly stated; determined by testing negative samples | <1.04 U/mL (higher LoB from b(4) lots) |
| Limit of Detection (LoD) | Not explicitly stated; determined by testing low-positive samples | <1.04 U/mL (higher LoD from b(4) lots) |
| Limit of Quantitation (LoQ) | Accuracy goal of 20% CV | <1.04 U/mL (higher LoQ from b(4) lots) |
| Analytical Specificity (Interference) | No interference detected up to specified concentrations | No interference detected for Bilirubin (20 mg/dL), Intralipid (3000 mg/dL), Hemoglobin (150 mg/dL), Rheumatoid factor (50 IU/mL) |
| Assay Cut-off | Appropriate for distinguishing AQP4Ab positive/negative | 3.0 U/mL (based on 97.5 percentile + 2 SD from healthy donors) |
| Clinical Sensitivity (NMO) | Not explicitly stated as a target numerical value, demonstrated with clinical data | 81% (69/85) for NMO |
| Clinical Sensitivity (NMOSD) | Not explicitly stated as a target numerical value, demonstrated with clinical data | 48% (25/52) for NMOSD |
| Clinical Sensitivity (NMO or NMOSD combined) | Not explicitly stated as a target numerical value, demonstrated with clinical data | 68.6% (94/137) for NMO or NMOSD |
| Clinical Specificity | Not explicitly stated as a target numerical value, demonstrated with clinical data | 98.1% (474/483) against Non-Target Diseases |
| Overall Agreement (NMOSD vs. Non-Target) | Not explicitly stated as a target numerical value, demonstrated with clinical data | 93.2% (499/535) |
| Overall Agreement (NMO or NMOSD vs. Non-Target) | Not explicitly stated as a target numerical value, demonstrated with clinical data | 91.6% (568/620) |
2. Sample Size Used for the Test Set and Data Provenance
- Test Set Sample Size:
- Clinical Validation: A total of 620 serum samples were included. This broke down into:
- 85 patients diagnosed with NMO
- 52 patients diagnosed with NMOSD
- 483 samples from patients with multiple sclerosis and other non-target diseases (detailed list provided in Section 7b).
- Assay Cut-off Determination: 509 healthy blood donors in the U.S.
- Reproducibility (Site-to-site): 50 serum samples.
- Lot-to-lot Reproducibility: 15 samples.
- Precision: 10 human sera samples.
- Detection Limit: "b(4) Trade Secret" (number not explicitly stated for publication).
- Interference: 5-11 samples.
- Clinical Validation: A total of 620 serum samples were included. This broke down into:
- Data Provenance:
- The document implies the samples were collected from patients diagnosed with NMO, NMOSD, MS, and various other infectious and autoimmune diseases, as well as healthy blood donors.
- For reproducibility, three different laboratories were used (two in the U.K. and one in the U.S.).
- For assay cut-off, 509 healthy blood donors in the U.S. were used.
- The other studies do not explicitly state the country of origin but refer to "human sera samples" or "patient sera".
- The study appears to be retrospective in nature, utilizing pre-existing serum samples from diagnosed patients or healthy individuals.
3. Number of Experts Used to Establish the Ground Truth for the Test Set and Their Qualifications
The document does not explicitly state the number of experts or their specific qualifications (e.g., "radiologist with 10 years of experience") used to establish the ground truth for the test set. However, it does outline how diagnoses were made:
- NMO and NMOSD diagnoses: "...must be based on clinical findings, laboratory tests (e.g., serological tests), and radiological tests (e.g., Magnetic Resonance Imaging)." Specific criteria for NMO were referenced (Wingerchuk DM et al., 2006).
- Non-Target Disease Group diagnoses: "...must be based on established diagnostic criteria and clinical evaluation." For Multiple Sclerosis, "McDonald Criteria" (Polman CH et al., 2005) were referenced.
This implies that the ground truth for patient diagnosis was established through standard clinical practice by medical professionals, likely involving neurologists, radiologists, and laboratory specialists, following recognized diagnostic criteria.
4. Adjudication Method for the Test Set
The document does not explicitly describe an adjudication method (like 2+1, 3+1) for establishing the ground truth of the test set samples. The diagnoses for NMO, NMOSD, and non-target diseases were stated to be based on established diagnostic criteria and clinical evaluation, which suggests a standard clinical diagnostic process rather than a specific study-defined adjudication panel.
5. Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study
No Multi-Reader Multi-Case (MRMC) comparative effectiveness study was mentioned. This device is an immunoassay (ELISA) for the semi-quantitative determination of autoantibodies, not an imaging device that would typically involve multiple human readers interpreting results with or without AI assistance. Therefore, the concept of human readers improving with AI vs. without AI assistance is not applicable here.
6. Standalone Performance Study
Yes, a standalone performance study was conducted. The entire clinical validation and analytical performance sections describe the performance of the KRONUS Aquaporin-4 Autoantibody (AQP4Ab) ELISA Assay as an algorithm/device only, without human-in-the-loop performance modifications during the testing itself. The results presented (sensitivity, specificity, precision, linearity, etc.) are a direct measure of the device's performance.
7. Type of Ground Truth Used
The ground truth used was primarily expert clinical diagnosis based on established diagnostic criteria, combining clinical findings, laboratory tests (other than the device under evaluation), and radiological tests. This is further elaborated:
- For NMO: Wingerchuk DM, Lennon VA, Pittock SJ, Lucchinetti CF, Weinshenker BG. 2006. Revised diagnostic criteria for neuromyelitis optica. Neurology 66:1485-1489.
- For MS: Polman CH, Reingold SC, Edan G, Filippi M, Hartung HP, Kappos L, Lublin FD, Metz LM, McFarland HF, O'Connor PW, Sandberg-Wollheim M, Thompson AJ, Weinshenker BG, Wolinsky JS, 2005. Diagnostic criteria for multiple sclerosis: 2005 revisions to the "McDonald Criteria". Ann Neurol. 58:840-846.
- For other differential diagnoses: "Diagnosis of diseases or conditions for the differential or nontarget disease groups must be based on established diagnostic criteria and clinical evaluation."
8. Sample Size for the Training Set
The document explicitly states that the KRONUS Aquaporin-4 Autoantibody (AQP4Ab) ELISA Assay is a manual enzyme-linked immunosorbent assay, semi-quantitative. It does not describe an AI or machine learning component that requires a "training set" in the conventional sense (i.e., data used to train a predictive model). The assay's "training" in this context would refer to the development and optimization of the assay itself and its calibrators/controls.
The calibrator and control values are traceable to "a panel of Master Control samples prepared from AQP4Ab positive patient sera that are used to create the linear calibration curves". The size of this "panel of Master Control samples" is not specified but serves a function analogous to a training or reference set for the assay's internal calibration.
9. How the Ground Truth for the Training Set Was Established
As noted above, a "training set" for an AI algorithm is not applicable here. However, for the assay's internal calibration via "Master Control samples" and calibrators, the ground truth was established by:
- "Master Control samples prepared from AQP4Ab positive patient sera". This implies that these were clinically confirmed positive patients.
- "Newly manufactured AQP4Ab calibrators are checked against a previous lot of calibrators (as a reference) and a panel of Master Control samples. Each calibrator and positive control level must fall within their previously established OD range."
This indicates that the ground truth for these internal controls and calibrators is based on a well-characterized set of patient sera with known AQP4Ab positivity and concentration values, likely determined through rigorous analytical methods to ensure consistency and accuracy across different lots and during development. The document does not provide details on how the initial master control samples were specifically characterized (e.g., by an independent gold-standard method), but implies a traceable and established process within the manufacturer's quality system.
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EVALUATION OF AUTOMATIC CLASS III DESIGNATION FOR KRONUS Aquaporin-4 Autoantibody (AQP4Ab) ELISA Assay
DECISION SUMMARY
A. DEN Number:
B. Purpose for Submission:
De Novo request for evaluation of automatic class III designation of the KRONUS Aquaporin-4 Autoantibody (AQP4Ab) ELISA Assay
C. Measurand:
Aquaporin-4 Autoantibody (AQP4Ab)
D. Type of Test:
Manual enzyme-linked immunosorbent assay, semi-quantitative
E. Applicant:
KRONUS, Inc.
F. Proprietary and Established Names:
KRONUS Aquaporin-4 Autoantibody (AQP4Ab) ELISA Assay
G. Regulatory Information:
-
- Regulation section:
21 CFR §866.5665, Aquaporin-4 autoantibody immunological test system
- Regulation section:
-
- Classification:
Class II (special controls)
- Classification:
-
- Product code:
PNI - Aquaporin-4 Autoantibody
- Product code:
-
- Panel: Immunology (82)
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H. Indications for use:
The KRONUS Aquaporin-4 Autoantibody (AQP4Ab) ELISA Assay is for the semiquantitative determination of autoantibodies to Aquaporin-4 in human serum. The KRONUS Aquaporin-4 Autoantibody (AQP4Ab) ELISA Assay may be useful as an aid in the diagnosis of Neuromyelitis Optica (NMO) and Neuromyelitis Optica Spectrum Disorders (NMOSD). The KRONUS Aquaporin-4 Autoantibody (AQP4Ab) ELISA Assay is not to be used alone and is to be used in conjunction with other clinical, laboratory, and radiological (e.g., MRI) findings.
Special conditions for use statement(s):
For prescription use only
Special instrument requirements:
ELISA Plate Reader suitable for 96-well format and capable of measuring at 405 nm and ELISA plate shaker capable of 500 shakes/minute.
I. Device Description:
The KRONUS Aquaporin-4 Autoantibody (AQP4Ab) ELISA Assay consists of ELISA strip wells coated with human recombinant AQP4 protein (amino acid position: 23-323) (96 wells in total) and supplied as 12 strips of 8 wells in a frame and sealed in a foil pouch with desiccant; ready-to-use 5 levels of calibrators (1.5, 5, 20, 40, and 80 U/mL), 5 x 0.7 mL; a ready-to-use positive (2 x . 7 mL) and a negative control serum (1 x 0.7 mL); AQP4-Biotin (lyophilized), 3 x 1.5 mL (reconstituted); ready-to-use reconstitution buffer for AQP4-Biotin, 1 x 10 mL; Streptavidin Peroxidase (SA-POD dilute before use) 1 x 0.8 mL; ready-to-use diluent for SA-POD, 1 x 15 mL; ready-to-use peroxidase substrate (TMB), 1 x 15 mL; concentrated wash solution (dilute 1:10 with deionized water before use), 1 x 120 mL and ready-to-use stop solution, 1 x 14 mL.
J. Standard/Guidance Document Referenced (if applicable):
CLSI EP05-A2: Evaluation of Detection Capability for Clinical Laboratory Measurement Procedures: Approved Guideline - Second Edition
CLSI EP6-A: Evaluation of Linearity of Quantitative Measurement Procedures: A Statistical Approach; Approved Guideline
CSLI EP17-A2: Evaluation of Detection Capability for Clinical Laboratory Measurement Procedures; Approved Guideline- Second Edition
CLSI C28-A3c: Defining, Establishing, and Verifying Reference Intervals in the Clinical Laboratory: Approved Guideline- Third Edition
K. Test Principle:
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The KRONUS Aquaporin-4 Autoantibody (AQP4Ab) ELISA Assay depends on the divalent properties of the AQP4 autoantibodies (AQP4Ab) to form a bridge between AQP4 coated on ELISA plate wells and liquid phase AQP4-biotin. The level of bound AQP4Ab is then quantitated by the sequential addition of streptavidin peroxidase and a chromogenic substrate with reading of final absorbance at (4) nm. The absorbance of each well is directly proportional to the amount of autoantibody present in the patient samples. Calibrator values are plotted on semi-log graph paper and the antibody concentrations of the controls and patient samples are interpolated from the curve.
L. Performance Characteristics (if/when applicable):
1. Analytical performance:
- a. Precision/Reproducibility:
Precision: Precision measurements were conducted according to CLSI EP05-A2. A panel consisting of ten human sera samples with levels of AQP4Ab that cover the measuring range was assayed in duplicate, twice a day, for 20 days with one reagent lot (b(4) Trade Secret ). All %CV values were within the manufacturer's pre-determined acceptance limit. The results are summarized in the table below:
| Precision | ||||||||
|---|---|---|---|---|---|---|---|---|
| Mean value | Within-Run | Between-Run | Between-day | Total | ||||
| (U/mL) | SD | %CV | SD | %CV | SD | %CV | SD | %CV |
| 2.1 | 0.2 | 8.2 | 0.2 | 10.2 | 0.2 | 7.3 | 0.3 | 14.5 |
| 2.8 | 0.2 | 6.1 | 0.2 | 8.2 | 0.2 | 6.1 | 0.3 | 11.8 |
| 3.5 | 0.2 | 6.5 | 0.2 | 4.5 | 0.2 | 6.0 | 0.4 | 9.9 |
| 4.5 | 0.3 | 5.7 | 0.5 | 10.8 | 0.4 | 8.8 | 0.7 | 15.0 |
| 4.4 | 0.2 | 4.5 | 0.4 | 9.5 | 0.4 | 8.2 | 0.6 | 13.4 |
| 7.8 | 0.4 | 5.2 | 0.8 | 10.6 | 0.6 | 8.2 | 1.1 | 14.3 |
| 9.2 | 0.5 | 5.2 | 0.9 | 9.4 | 0.7 | 7.4 | 1.2 | 13.0 |
| 32.0 | 1.1 | 3.4 | 3.5 | 10.9 | 2.5 | 7.8 | 4.4 | 13.8 |
| 70.2 | 2.9 | 4.1 | 4.6 | 6.6 | 3.3 | 4.6 | 6.7 | 9.6 |
| 72.1 | 3.6 | 4.9 | 2.0 | 2.8 | 3.9 | 5.5 | 5.3 | 6.7 |
Reproducibility: A panel of 50 serum samples with AOP4Ab concentration at various levels across the measuring range was tested in duplicate in one run at three different laboratories (two in the U.K. and one located in the U.S.) with one reagent lot to evaluate the site-to-site reproducibility. The correlation between sites was R2 > 0.99. The results are summarized in the table below:
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| Site-to-site reproducibility | ||||
|---|---|---|---|---|
| Sample | Regression | Slope(95% CI) | Y- Intercept(95% CI) | R2 |
| Laboratory 1 vs.Laboratory 2 | y= 0.96x-0.23 | 0.96b(4) Trade | -0.23b(4) Trade | 0.99 |
| Laboratory 1 vs.Laboratory 3 | y= 1.02x-0.39 | 1.02b(4) Trade | 0.39b(4) Trade | 1.00 |
| Laboratory 2 vs.Laboratory 3 | y= 1.02x-0.16 | 1.0188b(4) Trade | -0.16b(4) Trade | 0.99 |
To evaluate lot-to-lot reproducibility, a panel of 15 samples with AQP4Ab concentration at various levels across the measuring range was assayed with b(4) b(4) Trade production lots of the KRONUS Aquaporin-4 Autoantibody (AQF499) ELISA Assay. Mean and %CV for each sample were calculated and %CV values were from 3.22-14.13% for all samples. The results are summarized in the table below:
| Lot-to-lot reproducibility | ||||
|---|---|---|---|---|
| Sample | Numberof lots | Mean(U/mL) | SD(U/mL) | %CV |
| 1 | b(4) TradeSecret | 2.49 | 0.35 | 13.98 |
| 2 | 3.48 | 0.36 | 10.44 | |
| 3 | 4.68 | 0.21 | 4.52 | |
| 4 | 5.60 | 0.54 | 9.70 | |
| 5 | 7.04 | 0.72 | 10.20 | |
| 6 | 13.25 | 1.52 | 11.46 | |
| 7 | 14.89 | 1.45 | 9.72 | |
| 8 | 15.49 | 0.50 | 3.22 | |
| 9 | 27.28 | 2.90 | 10.62 | |
| 10 | 32.29 | 3.65 | 11.29 | |
| 11 | 35.52 | 4.75 | 13.45 | |
| 12 | 53.89 | 5.98 | 11.09 | |
| 13 | 60.35 | 8.08 | 13.38 | |
| 14 | 60.44 | 7.22 | 11.75 | |
| 15 | 63.27 | 8.94 | 14.13 |
b. Linearity/assay reportable range:
Linearity: Linearity and recovery characteristics of the KRONUS Aquaporin-4 Autoantibody (AQP4Ab) ELISA Assay were evaluated according to the CLSI EP6-A. b(4) Trade Secret
1.5-79.5 U/mL. Each dilution was tested in duplicate. The observed values were graphed against the calculated values and a linear regression
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| Samplepool | Test Range(U/mL) | Slope(95% CI) | Y-Intercept(95% CI) | R2 | % Recovery |
|---|---|---|---|---|---|
| 1 | 2.0-10.0 | 1.00b(4) Trade | 0.38b(4) Trade | 0.99 | 101.67-118.38 |
| 2 | 1.5-20.0 | 0.95b(4) Trade | 0.05b(4) Trade | 0.99 | 90.77-106.74 |
| 3 | 8.0-72.0 | 0.97b(4) Trade | -1.42b(4) Trade | 0.99 | 85.31-112.29 |
| 4 | 10.0-79.5 | 0.97b(4) Trade | -3.13b(4) Trade | 0.98 | 80.76-101.40 |
was performed. Results summarized in the table below were within the manufacturer's pre-determined acceptance criteria of recovery.
The assay is linear from 1.5-79.5 U/mL. The Package Insert Calculation Section states: "For samples that result in values greater than the 80 UlmL (greater than the highest calibrator) KRONUS recommends reporting the value as "greater than 80 U/mL".
Hook effect: Three native serum samples having AQP4Ab concentration above assay measuring range (> 80 U/mL) were serially diluted in assay negative control. No hook effect was observed up to 14000 U/mL.
- Traceability, Stability, Expected values (controls, calibrators, or methods): C.
- i) Traceability and value assignment:
There is no recognized standard or reference material for AQP4Ab. The calibrator and control values are directly traceable to a panel of Master Control samples prepared from AQP4Ab positive patient sera that are used to create the linear calibration curves for the KRONUS Aquaporin-4 Autoantibody (AQP4Ab) ELISA Assay.
The calibrators and positive controls are made by b(4) Trade Secret
There are five levels with assigned values from 1.5-80.0 U/mL. Newly manufactured AQP4Ab calibrators are checked against a previous lot of calibrators (as a reference) and a panel of Master Control samples. Each calibrator and positive control level must fall within their previously established OD range.
- ii) Assay stability:
Closed assay stability- An ongoing real-time stability study performed on 2(4) n lots of the KRONUS Aquaporin-4 Autoantibody (AQP4Ab) ELISS Assay supports a shelf-life claim of 4.5 months at 2-8°C.
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Open assay stability- A real-time stability study supports the stability of the ELISA strip wells after first opening of the foil pouch at four months at 2-8°C. The package insert recommends that unused wells be placed in the self-seal plastic bag provided. Opened and diluted SA-POD and Concentrated Wash Solution are stable at 2-8°C for up to 16 weeks and 11 months, respectively. The AQP4-Biotin is single-use and to be reconstituted immediately before use. Opened calibrators and controls are stable for three months at 2-8°C.
-
iii) Sample stability and storage:
The package insert recommends that sera be assayed soon after separation or stored in aliquots at or below -20℃. -
d. Detection limit:
The analytical sensitivity was determined in accordance with CLSI EP17-A2. The limit of blank (LoB) was determined by testing b(4) Trade Secret
. The higher LoB calculated from the b(4) lots i:b(4) JJmL. The limit of detection (LoD) was determined by testing b(4) Trade Secret
higher LoD calculated from theb(4) lots is [4) U/mL. The limit of quantitation (LoQ) was calculated using within-laboratory precision as the acceptance goal. Using an accuracy goal of 20%CV, the higher LoQ calculated
from the b(4) ots is b(4)
- e. Analytical specificity:
- Endogenous interference: i)
Interferences were assessed by testing 5-11 samples with AOP4Ab concentration that covers the entire assay measuring range. Each sample was spiked with known quantities of the interfering substances and analyzed in triplicate, in one assay run, with one assay lot. The recovery was calculated by comparing to control samples spiked with the same volume of diluents. No interference was detected in the samples up to the test concentrations listed in the table below:
| Potential Interfering Substances | Test Concentration |
|---|---|
| Bilirubin | 20 mg/dL |
| Intralipid | 3000 mg/dL |
| Hemoglobin | 150 mg/dL |
| Rheumatoid factor | 50 IU/mL |
The Package Insert 'Specimen Collection and Handling Section' lists rheumatoid factor (at > 50 IU/mL) and hemoglobin (at > 250 mg/dL) as interfering substances and states "Do not use lipemic or hemolysed serum samples".
The
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ii) Cross-reactivity:
Refer to test results for 483 serum samples from patients in the Non-Target Disease Group in the table presented in the section on Clinical studies.
- f. Assay cut-off:
The assay cut-off (greater than or equal to 3.0 U/mL is positive) for the KRONUS Aquaporin-4 Autoantibody (AQP4Ab) ELISA Assay was determined by testing specimens from 509 healthy blood donors in the U.S. Using the 97.50 percentile value plus 2 SD (1.4) for samples > LoD, a cut-off value of 3.0 U/mL was deemed appropriate. Utilizing the 3.0 U/mL cut-off value, 1.86% (9/483) of the samples from patients in the Non-Target Disease Group were positive for AOP4Ab. Given these results and taking into account the analytical sensitivity of the assay, values less than 3 U/mL are considered negative for AQP4Ab, and values greater than or equal to 3 U/mL are considered positive for AQP4Ab.
2. Comparison studies:
-
a. Method comparison with predicate device:
Not applicable -
b. Matrix comparison:
Not applicable. Serum is the only recommended matrix. The Package Insert 'Specimen Collection and Handling Section' states "Do not use plasma in the assay".
3. Clinical studies:
- Clinical sensitivity and specificity: a.
A total of 620 serum samples were included in the clinical validation for the KRONUS Aquaporin-4 Autoantibody (AQP4Ab) ELISA Assay. The validation set of samples includes 85 patients diagnosed with NMO , 52 patients diagnosed with NMOSD1, and 483 samples from patients with multiple sclerosis- and other nontarget diseases listed in the table below.
1Wingerchuk DM, Lennon VA, Pittock SJ, Lucchinetti CF, Weinshenker BG. 2006. Revised diagnostic criteriafor neuromyelitis optica. Neurology 66:1485-1489.
2 Polman CH, Reingold SC, Edan G, Filippi M, Hartung HP, Kappos L, Lublin FD, Metz LM, McFarland HF, O'Connor PW, Sandberg-Wollheim M, Thompson AJ, Weinshenker BG, Wolinsky JS, 2005. Diagnostic criteria for multiple sclerosis: 2005 revisions to the "McDonald Criteria". Ann Neurol. 58:840-846.
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| Non-Target Disease Group | |||
|---|---|---|---|
| n | #Positive | %Positive | |
| Multiple sclerosis (MS) | 75 | 1 | 1.33 |
| Relapsing-Remitting (RRMS) | 53 | 1 | 1.9 |
| Secondary- Progressive (SPMS) | 7 | 0 | 0 |
| Primary-Progressive (PPMS) | 5 | 0 | 0 |
| Progressive-Relapsing (PRMS) | 10 | 0 | 0 |
| Stroke | 21 | 0 | 0 |
| Infectious diseases | 119 | 5 | 4.20 |
| Lyme disease | 30 | 1 | 3.33 |
| Shingles | 10 | 0 | 0 |
| Syphilis | 30 | 4 | 13.33 |
| Human Immunodeficiency Virus (HIV) | 10 | 0 | 0 |
| Hepatitis B | 31 | 0 | 0 |
| Tuberculosis | 8 | 0 | 0 |
| Autoimmune diseases | 268 | 3 | 1.12 |
| Sjogren's Syndrome | 30 | 2 | 6.67 |
| Systemic Lupus Erythematosus (SLE) | 30 | 0 | 0 |
| Systemic Vasculitis | 4 | 0 | 0 |
| Sarcoidosis | 6 | 0 | 0 |
| Graves' disease | 110 | 0 | 0 |
| Type 1 Diabetes | 33 | 0 | 0 |
| Rheumatoid Arthritis | 17 | 1 | 5.9 |
| Hashimoto's Disease | 16 | 0 | 0 |
| Addison's Disease | 12 | 0 | 0 |
| Myasthenia Gravis | 10 | 0 | 0 |
| Combined total non-target diseases | 483 | 9 | 1.86 |
Using a cut-off of 3 U/mL, 69 (81%) of the 85 NMO patient samples and 25 (48%) of the 52 NMOSD tested positive. When combined, 94 (68.6%) of the 137 NMO/NMOSD samples tested positive.
One of 75 MS samples (1.3%) was positive for AQP4Ab. None of the 21 samples from patients with stroke were positive for AQP4Ab. Eight of 387 samples (2.1%) from patients with either infectious or autoimmune diseases tested positive for AQP4 Ab. Four of the 30 samples (13.33%) from patients with syphilis were positive. The package insert states that "a current or past syphilis diagnosis may increase risk of obtaining a false positive result."
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| KRONUS Aquaporin-4Autoantibody (AQP4Ab)ELISA Assay | Diagnosis | ||
|---|---|---|---|
| PositiveNMO | Negative(Non-Target Diseases) | Total | |
| Positive | 69 | 9 | 78 |
| Negative | 16 | 474 | 490 |
| Total | 85 | 483 | 568 |
Clinical sensitivity, specificity and overall agreement in this sample cohort are summarized in the following tables:
| Diagnosis | |||||||
|---|---|---|---|---|---|---|---|
| KRONUS Aquaporin-4 | Positive | Negative | |||||
| Autoantibody (AQP4Ab) | NMOSD | (Non-Target Diseases) | Total | ||||
| ELISA Assay | |||||||
| Positive | 25 | 9 | 34 | ||||
| Negative | 27 | 474 | 501 | ||||
| Total | 52 | 483 | રેડિયા રહેરાં રહ્યું હતું. સંદર્ભ દિવેલી દિવેલી દિવસ દિવસ દિવસ દિવસ દિવસ દિવસ દિવસ દિવસ દિવસ દિવસ દિવસ દિવસ દિવસ દિવસ દિવેલી દિવસ દિવેલી દિવસ દિવસ દિવસ | ||||
| Sensitivity: 48.1% (25/52) 95% CI: 35.1%-61.3% | |||||||
| Specificity: 98.1% (474/483) 95% CI: 96.5%-99.0% | |||||||
| Overall agreement: 93.2% (499/535) 95% CI: 90.8%-95.1% |
| Diagnosis | |||
|---|---|---|---|
| KRONUS Aquaporin-4Autoantibody (AQP4Ab)ELISA Assay | PositiveNMO orNMOSD | Negative(Non-Target Diseases) | Total |
| Positive | 94 | 9 | 103 |
| Negative | 43 | 474 | 517 |
| Total | 137 | 483 | 620 |
| Sensitivity: 68.6% (94/137) 95% CI: 66.4%-75.8% | |||
| Specificity: 98.1% (474/483) 95% CI: 96.5%-99.0% | |||
| Overall agreement: 91.6% (568/620) 95% CI: 89.2%-93.6% |
- b. Other clinical supportive data (when a. is not applicable):
Not applicable.
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4. Clinical cut-off:
Refer to assay cut-off.
-
- Expected values/Reference range:
The expected value from 509 healthy individual blood donors was determined in accordance with CSLI C28-A3c. The AQP4Ab values in normal healthy sera (NHS) ranged from <1.04 - 4.20 U/mL. Two NHS samples (0.4%) were positive for AQP4Ab. The 97.5th percentile value for all samples was 1.55 U/mL. Similar values were obtained when evaluations were based on gender, age and sex. It is the responsibility of each laboratory to establish its own reference ranges for the population of patients it serves, as expected values are affected by many different factors.
- Expected values/Reference range:
| Frequency of NHS AQP4Ab Values by Gender | ||||||||
|---|---|---|---|---|---|---|---|---|
| Gender | <1.04 U/mL | 1.04–1.49 U/mL | 1.50–1.99 U/mL | 2.00–2.49 U/mL | 2.50–2.99 U/mL | 3.00–3.49 U/mL | 3.5–3.99 U/mL | >3.99 U/mL |
| Male | 0.96 | 0.02 | 0.01 | 0 | 0 | 0 | 0 | 0.01 |
| Female | 0.91 | 0.02 | 0.06 | 0.01 | 0 | 0 | 0 | 0 |
| Frequency of NHS AQP4Ab Values by Age | ||||||||
|---|---|---|---|---|---|---|---|---|
| Age | <1.04 U/mL | 1.04–1.49 U/mL | 1.50–1.99 U/mL | 2.00–2.49 U/mL | 2.50–2.99 U/mL | 3.00–3.49 U/mL | 3.5–3.99 U/mL | >3.99 U/mL |
| < 20 | 0.93 | 0.04 | 0.04 | 0 | 0 | 0 | 0 | 0 |
| 20-39 | 0.95 | 0.02 | 0.02 | 0.01 | 0 | 0 | 0 | 0.01 |
| 40-59 | 0.95 | 0.05 | 0.03 | 0 | 0.01 | 0 | 0 | 0 |
| > 59 | 0.95 | 0.03 | 0.03 | 0 | 0 | 0 | 0 | 0 |
| Frequency of NHS AQP4Ab Values by Race | ||||||||
|---|---|---|---|---|---|---|---|---|
| Race | <1.04U/mL | 1.04-1.49U/mL | 1.50-1.99U/mL | 2.00-2.49U/mL | 2.50-2.99U/mL | 3.00-3.49U/mL | 3.5-3.99U/mL | >3.99U/mL |
| Black | 0.98 | 0.01 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 |
| Hispanic | 0.91 | 0.04 | 0.02 | 0.00 | 0.02 | 0.00 | 0.00 | 0.02 |
| Caucasian | 0.91 | 0.04 | 0.04 | 0.01 | 0.00 | 0.00 | 0.00 | 0.01 |
| Unknown | 0.88 | 0.13 | 0 | 0 | 0 | 0 | 0 | 0 |
M. Proposed Labeling:
The labeling is sufficient and it satisfies the requirements of 21 CFR Parts 801 and 809 and the special controls for this device type.
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N. Identified Risks and Required Mitigations:
| Identified Risks to Health | Required Mitigations |
|---|---|
| Inaccurate test results that provide falsepositive or false negative results can lead toimproper patient management | Special controls (1), (2), and (3) |
| Failure to correctly interpret test results canlead to false positive or false negative results | Special controls (1) (iii), (2), and (3) |
O. Benefit/Risk Analysis:
| Summary | |
|---|---|
| Summary ofthe Benefit(s) | This is the first serological test to the aid in the diagnosis of NMO/NMSD tobe commercially available in the U.S., providing a clinically important unmetmedical need. |
| Summary ofthe Benefit(s) | This is of benefit to patients by providing the possibility of earlier diagnosisof NMO/NMOSD and earlier initiation of appropriate therapy. |
| Summary ofthe Benefit(s) | The presence of the antibody is useful in helping to distinguishNMO/NMOSD from MS, which is important because the drug treatment forNMO/NMOSD and MS differ. |
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| Summary | |
|---|---|
| Summary ofthe Risk(s) | The primary risks to patients are related to the consequences of clinicaldecisions based on false negative and false positive results for a patient due toinaccurate test results or failure to correctly interpret test results. For a falsepositive result, the risks could include unnecessary testing or inappropriatetreatment related to an inaccurate result. For a false negative result, the riskcould include a missed or delayed diagnosis. The results from this test wouldbe used with results from other clinical, laboratory, and radiological (e.g.,MRI) findings, which would mitigate these risks.The risk to patients of false negative and false positive results are alsomitigated by statements in the indications for use statement which states thatthe assay is not to be used alone and is to be used in conjunction with otherclinical, laboratory, and radiological (e.g. MRI) findings. The risks are furthermitigated by the special controls established for this device.The test requires that a blood sample be obtained during routine phlebotomy. |
| This is standard procedure in clinical care, and the risk to patients is minimal.The risk to laboratory workers is no greater than that for the routine collectionand handling of blood specimens, given that the test is for use by laboratoryprofessionals in a clinical laboratory setting. | |
| Summary ofOther Factors | None |
| ConclusionsDo theprobablebenefitsoutweigh theprobable risks? | Given the well characterized performance characteristics, statements in theindications for use statement, the combination of required general controlsand the special controls established for this device, the performance data,warnings, and precautions and limitations required in the labeling, theprobable benefits outweigh the probable risks for this device. |
P. Conclusion:
The information provided in this de novo submission is sufficient to classify this device into class II under regulation 21 CFR 866.5665. FDA believes that the stated special controls and applicable general controls, including design controls, provide reasonable assurance of the safety and effectiveness of the device type. The device is classified under the following:
| Product Code: | PNI |
|---|---|
| Device Type: | Aquaporin-4 autoantibody immunological test system |
| Class: | II (special controls) |
| Regulation: | 21 CFR 866.5665 |
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- (a) Identification. An Aquaporin-4 autoantibody immunological test system is a device that consists of reagents used to measure by immunochemical techniques autoantibodies in human serum samples that react with Aquaporin-4 (AOP4Ab). The measurements aid in the diagnosis of Neuromyelitis Optica (NMO) and Neuromyelitis Optica Spectrum Disorders (NMOSD) in conjunction with other clinical, laboratory, and radiological (e.g., MRI) findings.
- (b) Classification. Class II (special controls). An Aquaporin-4 autoantibody immunological test system must comply with the following special controls:
-
- Premarket notification submissions must include the following information:
- i) A detailed device description including:
- A) A detailed description of all components including all required ancillary reagents in the test.
- B) If applicable, a detailed description of instrumentation and equipment, including illustrations or photographs of non-standard equipment or manuals.
- C) If applicable, detailed documentation of the device software, including, but not limited to, standalone software applications and hardware-based devices that incorporate software.
- D) A detailed description of appropriate internal and external quality controls that are recommended or provided. The description must identify those control elements that are incorporated into the specified testing procedures.
- E) Detailed specifications for sample collection, processing, and storage.
- F) A detailed description of methodology and assay procedure.
- G) A description of how the assay cut-off (the medical decision point between positive and negative) was established and validated as well as supporting data.
- H) Detailed specification of the criteria for test results interpretation and reporting.
- ii) Detailed information demonstrating the performance characteristics of the device, including:
- A) Device precision/reproducibility data generated from within-run, between-run, between-day, between-lot, between-site, and total precision for multiple nonconsecutive days, as applicable. A well characterized panel of patient samples or pools from the indicated population that covers the device measuring range must be used.
- B) Device linearity data generated from samples covering the device measuring range, if applicable.
- C) Information on traceability to a reference material and description of value assignment of calibrators and controls, if applicable.
- D) Device analytical sensitivity data, including limit of blank, limit of detection, and limit of quantitation, if applicable.
-
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- E) Device analytical specificity data, including interference by endogenous and exogenous substances, as well as cross-reactivity with samples derived from patients with other autoimmune diseases or conditions.
- F) Device instrument carryover data, when applicable.
- G) Device stability data, including real-time stability under various storage times and temperatures.
- H) Specimen stability data, including stability under various storage times, temperatures, freeze-thaw, and transport conditions, where appropriate.
- Method comparison data generated by comparison of the results obtained I) with the device to those obtained with a legally marketed predicate device with similar indications of use. A well-characterized panel of patient samples from the indicated population covering the device measuring range must be used.
- J) Specimen matrix comparison data, if more than one specimen type or anticoagulant can be tested with the device. Samples used for comparison must be from well-characterized patient samples covering the device measuring range.
- K) Clinical performance must be established by comparing data generated by testing samples from the indicated population and the differential diagnosis or non-target disease groups with the device to the clinical diagnostic standard.
- (1) The diagnosis of NMO and NMOSD must be based on clinical findings, laboratory tests (e.g., serological tests), and radiological tests (e.g., Magnetic Resonance Imaging).
- (2) The differential diagnosis or non-target disease group must include the applicable diseases or conditions, including but not be limited to the following: multiple sclerosis, stroke, lyme disease, shingles, syphilis, human immunodeficiency virus, hepatitis B, tuberculosis, Sjörgen's Syndrome, systemic lupus erythematous, systemic vasculitis, sarcoidosis, Graves' disease, Hashimoto's disease. Type I diabetes, rheumatoid arthritis, Addison's disease, and Myasthenia Gravis.
- (3) Diagnosis of diseases or conditions for the differential or nontarget disease groups must be based on established diagnostic criteria and clinical evaluation.
- (4) For all samples, the diagnostic clinical criteria and the demographic information must be collected and provided.
- (5) The clinical validation results must demonstrate clinical sensitivity and clinical specificity for the test values based on the presence or absence of NMO and NMOSD.
- (6) The data must be summarized in tabular format comparing the interpretation of results to the disease status.
- L) Expected/ reference values generated by testing an adequate number of samples from apparently healthy normal individuals.
- iii) Identification of risk mitigation elements used by the device, including
{14}------------------------------------------------
description of all additional procedures, methods, and practices incorporated into the directions for use that mitigate risks associated with testing.
-
- The device's 21 CFR 809.10(b) compliant labeling must include warnings relevant to the device including:
- i) A warning statement that reads "The device is for use by laboratory professionals in a clinical laboratory setting."
- ii) A warning statement that reads "The device is not to be used as a standalone device but as an adjunct to other clinical information. A diagnosis of Neuromyelitis Optica (NMO) and Neuromyelitis Optica Spectrum Disorders (NMOSD) should not be made on a single test result. The clinical symptoms, results from physical examination, laboratory tests (e.g., serological tests), and radiological tests (e.g. Magnetic Resonance Imaging), when appropriate, should always be taken into account when considering the diagnosis of NMO and NMOSD."
-
- The device's 21 CFR 809.10(b) compliant labeling must include a detailed description of the protocol and performance studies performed in accordance with special control (1)(ii) and a summary of the results.
§ 866.5665 Aquaporin-4 autoantibody immunological test system.
(a)
Identification. An Aquaporin-4 autoantibody immunological test system is a device that consists of reagents used to measure by immunochemical techniques autoantibodies in human serum samples that react with Aquaporin-4 (AQP4Ab). The measurements aid in the diagnosis of neuromyelitis optica (NMO) and neuromyelitis optica spectrum disorders (NMOSD) in conjunction with other clinical, laboratory, and radiological (e.g., magnetic resonance imaging) findings.(b)
Classification. Class II (special controls). The special controls for this device are:(1) Premarket notification submissions must include the following information:
(i) A detailed device description including:
(A) A detailed description of all components including all required ancillary reagents in the test;
(B) If applicable, a detailed description of instrumentation and equipment, including illustrations or photographs of non-standard equipment or manuals;
(C) If applicable, detailed documentation of the device software, including, but not limited to, standalone software applications and hardware-based devices that incorporate software;
(D) A detailed description of appropriate internal and external quality controls that are recommended or provided. The description must identify those control elements that are incorporated into the specified testing procedures;
(E) Detailed specifications for sample collection, processing, and storage;
(F) A detailed description of methodology and assay procedure;
(G) A description of how the assay cutoff (the medical decision point between positive and negative) was established and validated as well as supporting data; and
(H) Detailed specification of the criteria for test results interpretation and reporting.
(ii) Detailed information demonstrating the performance characteristics of the device, including:
(A) Device precision/reproducibility data generated from within-run, between-run, between-day, between-lot, between-site, and total precision for multiple nonconsecutive days, as applicable. A well characterized panel of patient samples or pools from the indicated population that covers the device measuring range must be used.
(B) Device linearity data generated from samples covering the device measuring range, if applicable.
(C) Information on traceability to a reference material and description of value assignment of calibrators and controls, if applicable.
(D) Device analytical sensitivity data, including limit of blank, limit of detection, and limit of quantitation, if applicable.
(E) Device analytical specificity data, including interference by endogenous and exogenous substances, as well as cross-reactivity with samples derived from patients with other autoimmune diseases or conditions.
(F) Device instrument carryover data, when applicable.
(G) Device stability data, including real-time stability under various storage times and temperatures.
(H) Specimen stability data, including stability under various storage times, temperatures, freeze-thaw, and transport conditions, where appropriate.
(I) Method comparison data generated by comparison of the results obtained with the device to those obtained with a legally marketed predicate device with similar indications of use. A well-characterized panel of patient samples from the indicated population covering the device measuring range must be used.
(J) Specimen matrix comparison data, if more than one specimen type or anticoagulant can be tested with the device. Samples used for comparison must be from well-characterized patient samples covering the device measuring range.
(K) Clinical performance must be established by comparing data generated by testing samples from the indicated population and the differential diagnosis or non-target disease groups with the device to the clinical diagnostic standard.
(
1 ) The diagnosis of NMO and NMOSD must be based on clinical findings, laboratory tests (e.g., serological tests), and radiological tests (e.g., magnetic resonance imaging).(
2 ) The differential diagnosis or non-target disease group must include the applicable diseases or conditions, including but not be limited to the following: Multiple sclerosis, stroke, Lyme disease, shingles, syphilis, human immunodeficiency virus, hepatitis B, tuberculosis, Srgen's syndrome, systemic lupus erythematous, systemic vasculitis, sarcoidosis, Graves' disease, Hashimoto's disease, Type I diabetes, rheumatoid arthritis, Addison's disease, and myasthenia gravis.(
3 ) Diagnosis of diseases or conditions for the differential or non-target disease groups must be based on established diagnostic criteria and clinical evaluation.(
4 ) For all samples, the diagnostic clinical criteria and the demographic information must be collected and provided.(
5 ) The clinical validation results must demonstrate clinical sensitivity and clinical specificity for the test values based on the presence or absence of NMO and NMOSD.(
6 ) The data must be summarized in tabular format comparing the interpretation of results to the disease status.(L) Expected/reference values generated by testing an adequate number of samples from apparently healthy normal individuals.
(iii) Identification of risk mitigation elements used by the device, including description of all additional procedures, methods, and practices incorporated into the directions for use that mitigate risks associated with testing.
(2) The device's 21 CFR 809.10(b) compliant labeling must include warnings relevant to the device including:
(i) A warning statement that reads “The device is for use by laboratory professionals in a clinical laboratory setting”; and
(ii) A warning statement that reads “The device is not to be used as a stand-alone device but as an adjunct to other clinical information. A diagnosis of Neuromyelitis Optica (NMO) and Neuromyelitis Optica Spectrum Disorders (NMOSD) should not be made on a single test result. The clinical symptoms, results from physical examination, laboratory tests (
e.g., serological tests), and radiological tests (e.g. Magnetic Resonance Imaging), when appropriate, should always be taken into account when considering the diagnosis of NMO and NMOSD.”(3) The device's 21 CFR 809.10(b) compliant labeling must include a detailed description of the protocol and performance studies performed in accordance with paragraph (b)(1)(ii) of this section and a summary of the results.