K Number

Device Name

KRONUS Aquaporin-4 Autoantibody (AQP4Ab) ELISA Assay

Manufacturer

KRONUS, INC.

Date Cleared

2016-04-25

(298 days)

Product Code

PNI

Regulation Number

866.5665

Intended 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.

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.

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AI/ML (Artificial Intelligence / Machine Learning)

No
The summary describes a standard ELISA assay kit and its performance characteristics, with no mention of AI or ML components in the device description, intended use, or performance studies.

Therapeutic

No
This device is an in vitro diagnostic (IVD) assay designed to detect autoantibodies as an aid in diagnosis, not to treat or cure a disease.

Diagnostic

Yes
The "Intended Use / Indications for Use" states that the assay "may be useful as an aid in the diagnosis of Neuromyelitis Optica (NMO) and Neuromyelitis Optica Spectrum Disorders (NMOSD)." This directly indicates its role in aiding diagnosis.

SaMD (Software as a Medical Device)

The device description clearly outlines a physical ELISA assay kit with various reagents and components, indicating it is a hardware-based diagnostic test, not software only.

IVD (In Vitro Diagnostic)

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

Here's why:

Intended Use: The intended use explicitly states it's for the "semiquantitative determination of autoantibodies to Aquaporin-4 in human serum." This involves testing a sample taken from the human body (serum) in vitro (outside the body) to provide information about a medical condition (presence of AQP4 autoantibodies).
Device Description: The description details a kit containing reagents and components designed for performing an ELISA assay on serum samples. This is a typical format for IVD kits.
Intended User / Care Setting: The intended users are "laboratory professionals in a clinical laboratory setting," which is where IVD testing is performed.
Performance Studies: The document includes a summary of performance studies using human serum samples to evaluate the device's clinical sensitivity and specificity in relation to diagnosing NMO and NMOSD. This is a requirement for demonstrating the clinical utility of an IVD.

The core function of the device is to analyze a biological sample in vitro to provide information for medical diagnosis, which is the definition of an In Vitro Diagnostic device.

PCCP (Predetermined Change Control Plan) Authorized

N/A

Overview

Intended Use / Indications for Use

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.

Product codes

PNI

Device Description

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Intended User / Care Setting

"The device is for use by laboratory professionals in a clinical laboratory setting."

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

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

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Summary of Performance Studies

Clinical sensitivity and specificity: 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 NMOSD, and 483 samples from patients with multiple sclerosis and other non-target diseases.

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 expected value from 509 healthy individual blood donors was determined. The AQP4Ab values in normal healthy sera (NHS) ranged from

Regulation Number and Section

§ 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.

Summary

EVALUATION OF AUTOMATIC CLASS III DESIGNATION FOR KRONUS Aquaporin-4 Autoantibody (AQP4Ab) ELISA Assay

DECISION SUMMARY

A. DEN Number:

DEN150030

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:

1. Regulation section:
  21 CFR §866.5665, Aquaporin-4 autoantibody immunological test system
1. Classification:
  Class II (special controls)
1. Product code:
  PNI - Aquaporin-4 Autoantibody
1. Panel: Immunology (82)

H. Indications for use:

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:

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:

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:

Site-to-site reproducibility
Sample	Regression	Slope
(95% CI)	Y- Intercept
(95% CI)	R2
Laboratory 1 vs.
Laboratory 2	y= 0.96x-0.23	0.96
b(4) Trade	-0.23
b(4) Trade	0.99
Laboratory 1 vs.
Laboratory 3	y= 1.02x-0.39	1.02
b(4) Trade	0.39
b(4) Trade	1.00
Laboratory 2 vs.
Laboratory 3	y= 1.02x-0.16	1.0188
b(4) Trade	-0.16
b(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	Number
of lots	Mean
(U/mL)	SD
(U/mL)	%CV
1	b(4) Trade
Secret	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

| Sample
pool | Test Range
(U/mL) | Slope
(95% CI) | Y-Intercept
(95% CI) | R2 | % Recovery |
|----------------|----------------------|--------------------|-------------------------|------|---------------|
| 1 | 2.0-10.0 | 1.00
b(4) Trade | 0.38
b(4) Trade | 0.99 | 101.67-118.38 |
| 2 | 1.5-20.0 | 0.95
b(4) Trade | 0.05
b(4) Trade | 0.99 | 90.77-106.74 |
| 3 | 8.0-72.0 | 0.97
b(4) Trade | -1.42
b(4) Trade | 0.99 | 85.31-112.29 |
| 4 | 10.0-79.5 | 0.97
b(4) Trade | -3.13
b(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.

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

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.

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.

| KRONUS Aquaporin-4
Autoantibody (AQP4Ab)

ELISA Assay	Diagnosis
	Positive
NMO	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-4
Autoantibody (AQP4Ab)
ELISA Assay	Positive
NMO or
NMOSD	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.

4. Clinical cut-off:

Refer to assay cut-off.

1. 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
  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
3.99 U/mL
59	0.95	0.03	0.03	0	0	0	0	0

Frequency of NHS AQP4Ab Values by Race
Race
3.99
U/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.

N. Identified Risks and Required Mitigations:

Identified Risks to Health	Required Mitigations
Inaccurate test results that provide false
positive or false negative results can lead to
improper patient management	Special controls (1), (2), and (3)
Failure to correctly interpret test results can
lead to false positive or false negative results	Special controls (1) (iii), (2), and (3)

O. Benefit/Risk Analysis:

Summary
Summary of
the Benefit(s)	This is the first serological test to the aid in the diagnosis of NMO/NMSD to
be commercially available in the U.S., providing a clinically important unmet
medical need.
Summary of
the Benefit(s)	This is of benefit to patients by providing the possibility of earlier diagnosis
of NMO/NMOSD and earlier initiation of appropriate therapy.
Summary of
the Benefit(s)	The presence of the antibody is useful in helping to distinguish
NMO/NMOSD from MS, which is important because the drug treatment for
NMO/NMOSD and MS differ.

Summary
Summary of
the Risk(s)	The primary risks to patients are related to the consequences of clinical
decisions based on false negative and false positive results for a patient due to
inaccurate test results or failure to correctly interpret test results. For a false
positive result, the risks could include unnecessary testing or inappropriate
treatment related to an inaccurate result. For a false negative result, the risk
could include a missed or delayed diagnosis. The results from this test would
be 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 also
mitigated by statements in the indications for use statement which states that
the 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 risks are further
mitigated 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 collection
and handling of blood specimens, given that the test is for use by laboratory
professionals in a clinical laboratory setting.
Summary of
Other Factors	None
Conclusions
Do the
probable
benefits
outweigh the
probable risks?	Given the well characterized performance characteristics, statements in the
indications for use statement, the combination of required general controls
and the special controls established for this device, the performance data,
warnings, and precautions and limitations required in the labeling, the
probable 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

(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:
- 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 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.

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

description of all additional procedures, methods, and practices incorporated into the directions for use that mitigate risks associated with testing.

1. 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."
1. 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.