Predicate Devices

K152013, K123593, K954830, K141655, K141328, K141210, K152635, K151429, K123880, K981237

Reference Devices

Not Found

AI/ML (Artificial Intelligence / Machine Learning)

No
The description details a standard immunoassay process with automated liquid handling, optical imaging, and calculation based on predefined master curves and calibration. There is no mention of AI or ML algorithms being used for data analysis, interpretation, or decision-making.

Therapeutic

No
The device is described as an in vitro diagnostic tool used for the quantitative determination of IgG autoantibodies to aid in the diagnosis of certain diseases, not to treat them.

Diagnostic

Yes

The "Intended Use / Indications for Use" section explicitly states that the device aids in the diagnosis of various autoimmune diseases, such as systemic lupus erythematosus, mixed connective tissue disease, and systemic sclerosis, by quantitatively or semi-quantitatively determining the presence of specific IgG autoantibodies.

SaMD (Software as a Medical Device)

No

The device description clearly outlines a system that includes hardware components such as liquid handling hardware, an optical module, and an integrated computer with a touch screen user interface, in addition to the proprietary software. The device is a complete in vitro diagnostic system, not a software-only medical device.

IVD (In Vitro Diagnostic)

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

Here's why:

Intended Use: The "Intended Use / Indications for Use" section explicitly states that the device is for the "quantitative determination of IgG autoantibodies... in human serum" and that the results are an "aid in the diagnosis" of various autoimmune diseases. This clearly indicates that the device is intended to be used in vitro (outside the body) to examine specimens (human serum) for the purpose of providing information for diagnosis.
Device Description: The description details a laboratory instrument and reagents used to perform tests on patient samples. This aligns with the nature of an IVD.
Intended User / Care Setting: It states "Professional in vitro diagnostic use only," which is a common labeling for IVD devices.

PCCP (Predetermined Change Control Plan) Authorized

N/A

Overview

Intended Use / Indications for Use

The Aptiva CTD Essential Reagent consists of 10 multiplexed immunoassays utilizing particle-based multi-analyte technology for the quantitative determination of IgG autoantibodies against dsDNA, and semi-quantitative determination of IgG autoantibodies against RNP, Sm, Ro52, Ro60, SS-B, Scl-70, Jo-1, centromere, and Ribo-P in human serum:

· The presence of dsDNA antibodies, in conjunction with clinical findings and other laboratory tests, is an aid in the diagnosis of systemic lupus erythematosus.

· The presence of RNP antibodies, in conjunction with clinical findings and other laboratory tests, is an aid in the diagnosis of mixed connective tissue disease and systemic lupus erythematosus.

· The presence of Sm antibodies, in conjunction with clinical findings and other laboratory tests, is an aid in the diagnosis of systemic lupus erythematosus.

· The presence of Ro52 antibodies, in conjunction with clinical findings and other laboratory tests, is an aid in the diagnosis of systemic lupus erythematosus, Sjögren's syndrome, systemic sclerosis, and idiopathic inflammatory myositis.

· The presence of Ro60 antibodies, in conjunction with clinical findings and other laboratory tests, is an aid in the diagnosis of systemic lupus erythematosus and Sjögren's syndrome.

· The presence of SS-B antibodies, in conjunction with clinical findings and other laboratory tests, is an aid in the diagnosis of systemic lupus erythematosus and Sjögren's syndrome.

· The presence of Scl-70 antibodies, in conjunction with clinical findings and other laboratory tests, is an aid in the diagnosis of systemic sclerosis.

· The presence of Jo-1 antibodies, in conjunction with clinical findings and other laboratory tests, is an aid in the diagnosis of idiopathic inflammatory myositis.

· The presence of centromere antibodies, in conjunction with clinical findings and other laboratory tests, is an aid in the diagnosis of systemic sclerosis.

· The presence of Ribo-P antibodies, in conjunction with clinical findings and other laboratory tests, is an aid in the diagnosis of systemic lupus erythematosus.

The individual assays included in the Aptiva CTD Essential Reagent are intended for use with the Inova Diagnostics Aptiva System.

Product codes (comma separated list FDA assigned to the subject device)

LLL, LJM, LKP, LKO, LSW, MQA, OBE

Device Description

The Aptiva CTD Essential reagent utilizes particle based multi-analyte technology (PMAT) in a cartridge format. Each analyte (dsDNA, RNP, Sm, Ro60, Ro52, SS-B, Scl-70, Jo-1, Centromere and Ribo-P) in the Aptiva CTD Essential reagent is a solid phase immunoassay utilizing fluorescent microparticles. This technology allows each of the ten analytes, along with a human anti-lgG capture antibody (IgG Control Microparticle), to be coated onto eleven uniquely recognizable paramagnetic microparticles, which are combined into one tube.

The Aptiva Multi-Analyte Instrument is a fully automated, random-access analyzer. This platform is a closed system with continuous load and random-access capabilities that processes the samples, runs the reagent, and reports results. It includes liquid handling hardware, optical module (OM), and integrated computer with proprietary software and touch screen user interface.

The ten unique populations of microparticles coated with dsDNA, RNP, Sm, Ro60, Ro52, SS-B, Scl-70, Jo-1, Centromere and Ribo-P, along with the one for the control microparticle, are stored in the reagent cartridge under conditions that preserve the autoantigens in their reactive states. When the assay cartridge is ready to be used for the first time, the reagent tube seals are pierced using the cartridge lid. The reagent cartridge is then loaded onto the Aptiva Multi-Analyte Instrument, where the microparticles are automatically rehydrated using buffer located within the cartridge.

A patient's serum is diluted 1:44.4 fold with Aptiva system rinse by the instrument in a disposable cuvette. A small amount of the diluted sample is combined with assay buffer and the microparticle suspension in a second cuvette, and mixed (final serum dilution: 1:230). This reaction cuvette is incubated for 9 ½ minutes at 37°C. The cuvette is then exposed to a magnet that retains the microparticles in place. The liquid is aspirated, and the microparticles are resuspended as system rinse is added to the cuvette and the magnet is removed. This wash cycle is repeated. During the third wash, no system rinse is added after the aspiration step. After the third wash, phycoerythrin conjugated polyclonal anti-human lgG (known as PE Tracer IgG) is added to the cuvette with microparticles, and mixed. Again, the cuvette is incubated for 9 ½ minutes at 37°C. Three wash steps, as described above, are performed on the microparticles. Following the wash steps, the microparticles are transferred to the optical module of the instrument, where a charge coupled device (CCD) camera takes multiple images to identify and count the twelve unique microparticle regions, as well as determine the amount of conjugate on the microparticles. A twelfth particle, coated with goat anti-human IgG, is present in the reagent as a control to flag low concentrations of IgG in the patient serum sample as an assay verification step. The median fluorescent intensity (MFI) is proportional to the amount of PE Tracer that is bound to the human IgG, which is proportional to the amount of IgG antibodies bound to the corresponding microparticle regions.

For quantitation, the ten assays (together as part of the Aptiva CTD Essential Reagent) each utilizes a predefined lot specific Master Curve that is uploaded onto the instrument through the RFID tag on the reagent cartridge. The first time a reagent cartridge of a new lot of Aptiva CTD Essential is placed in the instrument, it must be calibrated. The Aptiva CTD Essential Calibrators are sold separately. The calibration process utilizes the 6 Calibrators that are included in the Calibrators kit to adjust the predefined lot specific dsDNA, RNP, Sm, Ro60, Ro52, SS-B, Scl-70, Jo-1, Centromere and Ribo-P Master Curves into instrument specific Working Curves. These Working Curves are used to calculate FLU (or IU/mL for dsDNA) values from the measured MFI. The Working Curves are lot and instrument specific and stored in the system for use with any reagent cartridge from that lot. The lot specific calibration expires 6 months from the last time the calibration was performed, and re-calibration is required.

Aptiva CTD Essential Calibrators and Aptiva CTD Essential Controls are sold separately.

The Aptiva CTD Essential Reagent kit contains the following materials:

One (1) Aptiva CTD Essential Reagent Cartridge contains the following materials to process 250 determinations:

a. dsDNA, RNP, Sm, Ro60, Ro52, SS-B, Scl-70, Jo-1, Centromere and Ribo-P, and Control paramagnetic particles, preserved.
b. Assay Buffer clear liquid, containing protein stabilizers and preservatives.
c. PE Tracer IgG PE labeled anti-human IgG antibody, containing buffer, protein stabilizers and preservative.
d. Rehydration Buffer containing protein stabilizers and preservatives.

Mentions image processing

Yes, "a charge coupled device (CCD) camera takes multiple images to identify and count the twelve unique microparticle regions"

Mentions AI, DNN, or ML

Not Found

Input Imaging Modality

Not Found

Anatomical Site

human serum

Indicated Patient Age Range

Not Found

Intended User / Care Setting

Not Found

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

Not Found

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

A cohort of characterized samples, none of which were used for establishing the reference range, was used to validate the clinical performance of the Aptiva CTD Essential Reagent. The clinical validation study included 1269 samples from patients with Sjögren's syndrome (SjS, n=141), systemic lupus erythematosus (SLE, n=230), systemic sclerosis (SSc, n=217), mixed connective tissue disease (MCTD, n=91), idiopathic inflammatory myopathy (IIM, n=200) and control samples (n=390) from patients with various types of autoimmune and infectious diseases.

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

Precision Study:
- Study type: Evaluation of Quantitative Measurement Procedures in accordance with CLSI EPO5-A3.
- Sample size: 9 samples for dsDNA, 6 samples for RNP, 7 samples for Sm, 8 samples for Ro60, 9 samples for Ro52, 5 samples for SS-B, 6 samples for Scl-70, 5 samples for Jo-1, 5 samples for Centromere and 6 samples for Ribo-P, containing various concentrations of antibodies. Samples were run in duplicates, twice a day, for 20 days.
- Key results: All samples met the acceptance criteria of Total %CV: 0.95) were fulfilled for all analytes, demonstrating linearity across the entire AMR.
High concentration hook effect Study:
- Study type: Assessment of hook effect.
- Sample size: 2 samples for each analyte (dsDNA, RNP, Sm, Ro52, Ro60, SS-B, Scl-70, Jo-1, Centromere, and Ribo-P).
- Key results: No hook effect was observed for high positive specimens above the AMR.
Interference Study:
- Study type: Interference Testing in Clinical Chemistry in accordance with CLSI EPO7-A2.
- Sample size: Three human serum specimens (one positive, one near cut-off, one negative).
- Key results: The Aptiva CTD Essential Reagent did not show interference with various endogenous and exogenous substances at specified concentrations (e.g., bilirubin up to 1 mg/mL, hemoglobin up to 2 mg/mL, triglycerides up to 1000 mg/dL, ibuprofen up to 21.9 mg/dL).
Sample Stability and Handling Study:
- Study type: Stability assessment.
- Sample size: Three samples for each analyte, tested in duplicates.
- Key results: Samples were stable for up to 24 hours at room temperature, up to 14 days at 2-8°C, and up to 4 freeze/thaw cycles.
Reagent Stability Studies (Shelf life, In-use (onboard) stability, Real time stability):
- Shelf life: Accelerated stability studies performed for 5 weeks at 37°C ± 3°C, equivalent to six months at 5 ± 3°C, using 3 lots of microparticles and 3 lots of rehydration buffer. Acceptance criteria was 95% CI of regression line between 80% and 120% recovery at day 28 (week 4).
- In-use (onboard) stability: One lot of reagent cartridge tested periodically for 45 days.
- Real time stability: Scheduled to be performed every three or six months.
- Key results: All components fulfilled the acceptance criteria for initial shelf life claim (two-year expiration dating). In-use stability was set at 36 days with an 18-day recalibration.
Clinical Sensitivity and Specificity Study:
- Study type: Clinical validation study.
- Sample size: 1269 samples (141 SjS, 230 SLE, 217 SSc, 91 MCTD, 200 IIM, 390 controls from various autoimmune/infectious diseases).
- Key results:
  - dsDNA: SLE Sensitivity (indeterminate as positive) = 50.4% (44.0 - 56.8%), Specificity = 90.1% (88.1 - 91.8%). SLE Sensitivity (indeterminate as negative) = 46.1% (39.8 – 52.5%), Specificity = 92.9% (91.2 – 94.3%).
  - RNP: SLE Sensitivity = 37.4% (31.4 - 43.8%), Specificity = 94.8% (93.2 - 96.1%). MCTD Sensitivity = 68.1% (58.0 – 76.8%), Specificity = 94.8% (93.2 – 96.1%).
  - Sm: SLE Sensitivity = 10.4% (7.1 – 15.1%), Specificity = 99.6% (99.0 – 99.9%).
  - Ro52: SLE Sensitivity = 24.3% (19.3 – 30.3%), Specificity = 93.6% (91.0 - 95.4%). SjS Sensitivity = 60.3% (52.0 - 68.0%), Specificity = 93.6% (91.0 - 95.4%). SSc Sensitivity = 15.2% (11.0 – 20.6%), Specificity = 93.6% (91.0-95.4%). IIM Sensitivity = 19.0% (14.2 – 25.0%), Specificity = 93.6% (91.0 - 95.4%).
  - Ro60: SLE Sensitivity = 52.2% (45.7 – 58.5%), Specificity = 88.8% (86.5 - 90.7%). SjS Sensitivity = 66.7% (58.5 - 73.9%), Specificity = 88.8% (86.5 - 90.7%).
  - SS-B: SLE Sensitivity = 15.7% (11.5 – 20.9%), Specificity = 96.5% (95.1 - 97.6%). SjS Sensitivity = 46.8% (38.8 – 55.0%), Specificity = 96.5% (95.1 – 97.6%).
  - Scl-70: SSc Sensitivity = 30.4% (24.7 – 36.8%), Specificity = 94.3% (92.7 – 95.5%).
  - Jo-1: IIM Sensitivity = 11.5% (7.8 – 16.7%), Specificity = 99.3% (98.7–99.7%).
  - Centromere: SSc Sensitivity = 47.0% (40.5 - 53.6%), Specificity = 97.0% (95.7 - 97.8%).
  - Ribo-P: SLE Sensitivity = 11.7% (8.2 - 16.5%), Specificity = 99.7% (99.2 - 99.9%).
Method Comparison with Predicate Device Study:
- Study type: Comparison of Aptiva CTD Essential Reagent with predicate devices.
- Sample size:
  - dsDNA: N=428
  - RNP: N=480
  - Sm: N=418
  - Ro52: N=1028
  - Ro60: N=551
  - SS-B: N=550
  - Scl-70: N=435
  - Jo-1: N=416
  - Centromere: N=449
  - Ribo-P: N=387
- Key results (Percent Agreement values):
  - dsDNA:
    - With Indeterminate as Positive: NPA = 91.8% (88.2 - 94.4%), PPA = 78.9% (70.8 - 85.1%), TPA = 88.1% (84.7 - 90.8%)
    - With Indeterminate as Negative: NPA = 89.2% (85.0 - 92.3%), PPA = 75.3% (67.9 – 81.5%), TPA = 84.3% (80.6 – 87.5%)
  - RNP: NPA= 90.6% (87.3 – 93.2%), PPA= 97.9% (92.6 – 99.4%), TPA= 92.1% (89.3 - 94.2%)
  - Sm: NPA: 98.2% (96.3-99.1%), PPA: 85.7% (70.6 – 93.7%), TPA: 97.1% (95.0 – 98.4%)
  - Ro52: NPA = 97.8% (96.6 - 98.6%), PPA = 97.6% (94.5 - 99.0%), TPA = 97.8% (96.7 - 98.5%)
  - Ro60: NPA = 91.8% (88.5 – 94.3%), PPA = 98.5% (95.6-99.5%), TPA = 94.2% (91.9 – 95.9%)
  - SS-B: NPA = 96.3% (94.1 – 97.7%), PPA = 96.7% (90.8 – 98.9%), TPA = 96.4% (94.5 - 97.6%)
  - Scl-70: NPA = 97.6% (95.4 – 98.7%), PPA = 95.5% (87.6 - 98.5%), TPA = 97.2% (95.2 – 98.4%)
  - Jo-1: NPA = 99.7% (98.6 – 100.0%), PPA = 96.0% (80.5 – 99.3%), TPA = 99.5% (98.3 – 99.9%)
  - Centromere: NPA = 98.3% (96.2 – 99.2%), PPA = 96.2% (90.6 - 98.5%), TPA = 97.8% (95.9 – 98.8%)
  - Ribo-P: NPA = 99.7% (98.5 – 100.0%), PPA = 100.0% (85.7 – 100.0%), TPA = 99.7% (98.6 – 100.0%)

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

See "Summary of Performance Studies" for Sensitivity and Specificity.
NPA: Negative Percent Agreement; PPA: Positive Percent; TPA: Total Percent Agreement.

Predicate Device(s): If the device was cleared using the 510(k) pathway, identify the Predicate Device(s) K/DEN number used to claim substantial equivalence and list them here in a comma separated list exactly as they appear in the text. List the primary predicate first in the list.

K152013, K123593, K954830, K141655, K141328, K141210, K152635, K151429, K123880, K981237

Reference Device(s): Identify the Reference Device(s) K/DEN number and list them here in a comma separated list exactly as they appear in the text.

Not Found

Predetermined Change Control Plan (PCCP) - All Relevant Information for the subject device only (e.g. presence / absence, what scope was granted / cleared under the PCCP, any restrictions, etc).

Not Found

Regulation Number and Section

§ 866.5100 Antinuclear antibody immunological test system.

(a)
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).(b)
Classification. Class II (performance standards).

Summary

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Image /page/0/Picture/0 description: The image shows the logo of the U.S. Food and Drug Administration (FDA). On the left is the Department of Health & Human Services logo. To the right of that is the FDA logo, with the letters "FDA" in a blue square. To the right of the blue square is the text "U.S. FOOD & DRUG ADMINISTRATION" in blue.

September 29, 2023

Inova Diagnostics, Inc. Andrea Seaman Manager, Research and Development 9900 Old Grove Road San Diego, California 92131

Re: K213403

Trade/Device Name: Aptiva CTD Essential Reagent Regulation Number: 21 CFR 866.5100 Regulation Name: Antinuclear Antibody Immunological Test System Regulatory Class: Class II Product Code: LLL, LJM, LKP, LKO, LSW, MQA, OBE, Dated: February 24, 2023 Received: February 24, 2023

Dear Andrea Seaman:

We have reviewed your Section 510(k) premarket notification of intent to market the device referenced above and have determined the device is substantially equivalent (for the indications for use stated in the enclosure) to legally marketed predicate devices marketed in interstate commerce prior to May 28, 1976, the enactment date of the Medical Device Amendments, or to devices that have been reclassified in accordance with the provisions of the Federal Food, Drug, and Cosmetic Act (Act) that do not require approval of a premarket approval application (PMA). You may, therefore, market the device, subject to the general controls provisions of the Act. Although this letter refers to your product as a device, please be aware that some cleared products may instead be combination products. The 510(k) Premarket Notification Database located at https://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfpmn/pmn.cfm identifies combination product submissions. The general controls provisions of the Act include requirements for annual registration, listing of devices, good manufacturing practice, labeling, and prohibitions against misbranding and adulteration. Please note: CDRH does not evaluate information related to contract liability warranties. We remind you, however, that device labeling must be truthful and not misleading.

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

Please be advised that FDA's issuance of a substantial equivalence determination does not mean that FDA has made a determination that your device complies with other requirements of the Act or any Federal statutes and regulations administered by other Federal agencies. You must comply with all the Act's

1

requirements, including, but not limited to: registration and listing (21 CFR Part 807); labeling (21 CFR Part 801 and Part 809); medical device reporting of medical device-related adverse events) (21 CFR 803) for devices or postmarketing safety reporting (21 CFR 4, Subpart B) for combination products (see https://www.fda.gov/combination-products/guidance-regulatory-information/postmarketing-safety-reportingcombination-products); good manufacturing practice requirements as set forth in the quality systems (OS)

regulation (21 CFR Part 820) for devices or current good manufacturing practices (21 CFR 4, Subpart A) for combination products; and, if applicable, the electronic product radiation control provisions (Sections 531-542 of the Act); 21 CFR 1000-1050.

Also, please note the regulation entitled, "Misbranding by reference to premarket notification" (21 CFR Part 807.97). For questions regarding the reporting of adverse events under the MDR regulation (21 CFR Part 803), please go to https://www.fda.gov/medical-device-safety/medical-device-reportingmdr-how-report-medical-device-problems.

For comprehensive regulatory information about mediation-emitting products, including information about labeling regulations, please see Device Advice (https://www.fda.gov/medicaldevices/device-advice-comprehensive-regulatory-assistance) and CDRH Learn (https://www.fda.gov/training-and-continuing-education/cdrh-learn). Additionally, you may contact the Division of Industry and Consumer Education (DICE) to ask a question about a specific regulatory topic. See the DICE website (https://www.fda.gov/medical-device-advice-comprehensive-regulatoryassistance/contact-us-division-industry-and-consumer-education-dice) for more information or contact DICE by email (DICE@fda.hhs.gov) or phone (1-800-638-2041 or 301-796-7100).

Sincerely,

Ying Mao -S

Ying Mao, Ph.D. Branch Chief Division of Immunology and Hematology Devices OHT7: Office of In Vitro Diagnostics Office of Product Evaluation and Quality Center for Devices and Radiological Health

Enclosure

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Indications for Use

510(k) Number (if known) K213403

Device Name Aptiva CTD Essential Reagents

Indications for Use (Describe)

The Aptiva CTD Essential Reagent consists of 10 multiplexed immunoassays utilizing particle-based multi-analyte technology for the quantitative determination of IgG autoantibodies against dsDNA, and semi-quantitative determination of IgG autoantibodies against RNP, Sm, Ro52, Ro60, SS-B, Scl-70, Jo-1, centromere, and Ribo-P in human serum:

· The presence of dsDNA antibodies, in conjunction with clinical findings and other laboratory tests, is an aid in the diagnosis of systemic lupus erythematosus.

· The presence of RNP antibodies, in conjunction with clinical findings and other laboratory tests, is an aid in the diagnosis of mixed connective tissue disease and systemic lupus erythematosus.

· The presence of Sm antibodies, in conjunction with clinical findings and other laboratory tests, is an ad in the diagnosis of systemic lupus erythematosus.

· The presence of Ro52 antibodies, in conjunction with clinical findings and other laboratory tests, is an aid in the

diagnosis of systemic lupus erythematosus, Sjögren's systemic scleross, and idiopathic inflammatory myositis. · The presence of Ro60 antibodies, in conjunction with clinical findings and other laboratory tests, is an aid in the

diagnosis of systemic lupus erythematosus and Sjögren's syndrome.

· The presence of SS-B antibodies, in conjunction with clinical findings and other laboratory tests, is an aid in the diagnosis of systemic lupus erythematosus and Sjögren's syndrome.

· The presence of Scl-70 antibodies, in conjunction with clinical findings and other laboratory tests, is an aid in the diagnosis of systemic sclerosis.

· The presence of Jo-1 antibodies, in conjunction with clinical findings and other laboratory tests, is an aid in the diagnosis of idiopathic inflammatory myositis.

· The presence of centromere antibodies, in conjunction with clinical findings and other laboratory tests, is an aid in the diagnosis of systemic sclerosis.

· The presence of Ribo-P antibodies, in conjunction with clinical findings and other laboratory tests, is an aid in the diagnosis of systemic lupus erythematosus.

The individual assays included in the Aptiva CTD Essential Reagent are intended for use with the Inova Diagnostics Aptiva System.

Type of Use (Select one or both, as applicable)

X Prescription Use (Part 21 CFR 801 Subpart D)

Over-The-Counter Use (21 CFR 801 Subpart C)

CONTINUE ON A SEPARATE PAGE IF NEEDED.

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510(k) Summary

Aptiva CTD Essential Reagent

Table of Contents
Administrative data.	2
Predicate device.	3
Device description.	3
Intended use(s)	4
Indications for use.	4
Substantial equivalence	5
Comparison to predicate device	5
Analytical performance characteristics	19
Quantitation and units of measure.	19
Precision	21
Reproducibility Studies.	25
Limit of Blank (LoB), Limit of Detection (LoD), and Limit of Quantitation (LoQ).	30
Analytical Measuring Range (AMR)	32
Auto-rerun function and reportable results	32
High concentration hook effect.	32
Linearity	33
Interference.	36
Sample Stability and Handling	36
Reagent Stability.	37
Cut-off, reference range	38
Clinical performance characteristics.	40
Clinical sensitivity, specificity	40
Expected values.	51
Comparison with predicate device	51

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Page 2 of 53

This summary of the 510(k) safety and effectiveness information is being submitted in accordance with the requirements of SMDA 1990 and 21 CFR 807.92.

Administrative data
Submitter:	Inova Diagnostics, Inc
9900 Old Grove Road,
San Diego, CA, 92131
Purpose of submission:	New device
Device in the submission:	Aptiva CTD Essential Reagent
Scientific contact:	Andrea Seaman, Associate Director, Research and Development
Inova Diagnostics, Inc.
9900 Old Grove Road, San Diego, CA, 92131
Phone: 858-586-9900 x77395
Fax: 858-863-0025
Email: aseaman@werfen.com
Quality Systems contact:	Constance Bridges, VP, Quality Systems and RA
Inova Diagnostics, Inc
9900 Old Grove Road, San Diego, CA, 92131
Phone: 858-586-9900 x77212
Fax: 858-863-0025
Email: cbridges@werfen.com
Device name (kit):	Proprietary name: Aptiva CTD Essential Reagent
Common name: anti-nuclear antibody tests
Classification name: Anti-nuclear antibody immunological test system
Regulation Medical Specialty	Immunology
Review Panel	Immunology
Product Code	LLL, Extractable Antinuclear antibody
OBE, Anti-SS-A 52 Autoantibodies
LKP, Anti-Sm antibody
LKO, Anti-RNP Antibody
LJM, Antinuclear Antibody
MQA, Anti-Ribosomal P Antibodies
LSW, Anti-DNA Antibody
Regulation Number	866.5100
Device Class	2

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Predicate device

QUANTA Flash® dsDNA IgG, 510(k) number: K152013. Date declared: April 11, 2016. QUANTA Flash® RNP, 510(k) number: K123593. Date declared: April 17, 2013. Orgentec Sm ELISA, 510(k) number: K954830. Date declared: May 8, 1996. QUANTA Flash® Ro52, 510(k) number: K141655. Date declared: March 5, 2015. QUANTA Flash® Ro60, 510(k) number: K141328. Date declared: February 12, 2015. QUANTA Flash® SS-B, 510(k) number: K141210. Date declared: January 29, 2015. QUANTA Flash® Scl-70, 510(k) number: K152635. Date declared: June 1, 2016. QUANTA Flash® Jo-1, 510(k) number: K151429. Date declared: February 12, 2016. QUANTA Flash® Centromere, 510(k) number: K123880. Date declared: February 7, 2014. QUANTA Lite® Ribo-P, 510(k) number: K981237. Date declared: June 5, 1998.

Device description

The Aptiva CTD Essential reagent utilizes particle based multi-analyte technology (PMAT) in a cartridge format. Each analyte (dsDNA, RNP, Sm, Ro60, Ro52, SS-B, Scl-70, Jo-1, Centromere and Ribo-P) in the Aptiva CTD Essential reagent is a solid phase immunoassay utilizing fluorescent microparticles. This technology allows each of the ten analytes, along with a human anti-lgG capture antibody (IgG Control Microparticle), to be coated onto eleven uniquely recognizable paramagnetic microparticles, which are combined into one tube.

The Aptiva Multi-Analyte Instrument is a fully automated, random-access analyzer. This platform is a closed system with continuous load and random-access capabilities that processes the samples, runs the reagent, and reports results. It includes liquid handling hardware, optical module (OM), and integrated computer with proprietary software and touch screen user interface.

The ten unique populations of microparticles coated with dsDNA, RNP, Sm, Ro60, Ro52, SS-B, Scl-70, Jo-1, Centromere and Ribo-P, along with the one for the control microparticle, are stored in the reagent cartridge under conditions that preserve the autoantigens in their reactive states. When the assay cartridge is ready to be used for the first time, the reagent tube seals are pierced using the cartridge lid. The reagent cartridge is then loaded onto the Aptiva Multi-Analyte Instrument, where the microparticles are automatically rehydrated using buffer located within the cartridge.

A patient's serum is diluted 1:44.4 fold with Aptiva system rinse by the instrument in a disposable cuvette. A small amount of the diluted sample is combined with assay buffer and the microparticle suspension in a second cuvette, and mixed (final serum dilution: 1:230). This reaction cuvette is incubated for 9 ½ minutes at 37°C. The cuvette is then exposed to a magnet that retains the microparticles in place. The liquid is aspirated, and the microparticles are resuspended as system rinse is added to the cuvette and the magnet is removed. This wash cycle is repeated. During the third wash, no system rinse is added after the aspiration step. After the third wash, phycoerythrin conjugated polyclonal anti-human lgG (known as PE Tracer IgG) is added to the cuvette with microparticles, and mixed. Again, the cuvette is incubated for 9 ½ minutes at 37°C. Three wash steps, as described above, are performed on the microparticles. Following the wash steps, the microparticles are transferred to the optical module of the instrument, where a charge coupled device (CCD) camera takes multiple images to identify and count the twelve unique microparticle regions, as well as determine the amount of conjugate on the microparticles. A twelfth particle, coated with goat anti-human IgG, is present in the reagent as a control to flag low concentrations of IgG in the patient serum sample as an assay verification step. The median fluorescent

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intensity (MFI) is proportional to the amount of PE Tracer that is bound to the human IgG, which is proportional to the amount of IgG antibodies bound to the corresponding microparticle regions.

For quantitation, the ten assays (together as part of the Aptiva CTD Essential Reagent) each utilizes a predefined lot specific Master Curve that is uploaded onto the instrument through the RFID tag on the reagent cartridge. The first time a reagent cartridge of a new lot of Aptiva CTD Essential is placed in the instrument, it must be calibrated. The Aptiva CTD Essential Calibrators are sold separately. The calibration process utilizes the 6 Calibrators that are included in the Calibrators kit to adjust the predefined lot specific dsDNA, RNP, Sm, Ro60, Ro52, SS-B, Scl-70, Jo-1, Centromere and Ribo-P Master Curves into instrument specific Working Curves. These Working Curves are used to calculate FLU (or IU/mL for dsDNA) values from the measured MFI. The Working Curves are lot and instrument specific and stored in the system for use with any reagent cartridge from that lot. The lot specific calibration expires 6 months from the last time the calibration was performed, and re-calibration is required.

Aptiva CTD Essential Calibrators and Aptiva CTD Essential Controls are sold separately.

The Aptiva CTD Essential Reagent kit contains the following materials:

One (1) Aptiva CTD Essential Reagent Cartridge contains the following materials to process 250 determinations:

a. dsDNA, RNP, Sm, Ro60, Ro52, SS-B, Scl-70, Jo-1, Centromere and Ribo-P, and Control paramagnetic particles, preserved.
b. Assay Buffer clear liquid, containing protein stabilizers and preservatives.
c. PE Tracer IgG PE labeled anti-human IgG antibody, containing buffer, protein stabilizers and preservative.
d. Rehydration Buffer containing protein stabilizers and preservatives.

Intended use(s)

The Aptiva CTD Essential Reagent consists of 10 multiplexed immunoassays utilizing particle-based multianalyte technology for the quantitative determination of IgG autoantibodies against dsDNA, and semiquantitative determination of IgG autoantibodies against RNP, Sm, Ro52, Ro60, SS-B, Scl-70, Jo-1, centromere, and Ribo-P in human serum:

The presence of dsDNA antibodies, in conjunction with clinical findings and other laboratory tests, is an aid in the diagnosis of systemic lupus erythematosus.
The presence of RNP antibodies, in conjunction with clinical findings and other laboratory tests, is an aid in the diagnosis of mixed connective tissue disease and systemic lupus erythematosus.
The presence of Sm antibodies, in conjunction with clinical findings and other laboratory tests, is an aid in the diagnosis of systemic lupus erythematosus.
. The presence of Ro52 antibodies, in conjunction with clinical findings and other laboratory tests, is an aid in the diagnosis of systemic lupus erythematosus, Sjögren's syndrome, systemic sclerosis, and idiopathic inflammatory myositis.
. The presence of Ro60 antibodies, in conjunction with clinical findings and other laboratory tests, is an aid in the diagnosis of systemic lupus erythematosus and Sjögren's syndrome.
. The presence of SS-B antibodies, in conjunction with clinical findings and other laboratory tests, is an aid in the diagnosis of systemic lupus erythematosus and Sjögren's syndrome.

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. The presence of Scl-70 antibodies, in conjunction with clinical findings and other laboratory tests, is an aid in the diagnosis of systemic sclerosis.
The presence of Jo-1 antibodies, in conjunction with clinical findings and other laboratory tests, is an aid in the diagnosis of idiopathic inflammatory myositis.
The presence of centromere antibodies, in conjunction with clinical findings and other laboratory tests, is an aid in the diagnosis of systemic sclerosis.
The presence of Ribo-P antibodies, in conjunction with clinical findings and other laboratory tests, is an aid in the diagnosis of systemic lupus erythematosus.

The individual assays included in the Aptiva CTD Essential Reagent are intended for use with the Inova Diagnostics Aptiva System.

Indications for use

Same as intended use.

Substantial equivalence

The Aptiva CTD Essential Reagent has the same intended use and assay principle as the predicate devices. Comparison to predicate device

Similarities
Item	Aptiva CTD Essential Reagent
(dsDNA)	QUANTA Flash dsDNA
Intended Use	The Aptiva CTD Essential Reagent
consists of 10 multiplexed
immunoassays utilizing particle-
based multi-analyte technology
for the quantitative determination
of IgG autoantibodies against
dsDNA, and semi-quantitative
determination of IgG
autoantibodies against RNP, Sm,
Ro52, Ro60, SS-B, Scl-70, Jo-1,
centromere, and Ribo-P in human
serum:

The presence of dsDNA
antibodies, in conjunction with
clinical findings and other
laboratory tests, is an aid in the | QUANTA Flash dsDNA is a
chemiluminescent immunoassay
for the quantitative
determination of IgG anti-double
stranded deoxyribonucleic acid
(dsDNA) antibodies in human
serum. The presence of anti-
dsDNA antibodies, in
conjunction with clinical findings
and other laboratory tests, is an
aid in the diagnosis of Systemic
Lupus Erythematosus. |

Aptiva CTD Essential Reagent - dsDNA Assay

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Similarities
Item	Aptiva CTD Essential Reagent (dsDNA)	QUANTA Flash dsDNA
	diagnosis of systemic lupus
erythematosus.

Aptiva CTD Essential Reagent – RNP Assay

Similarities
Item	Aptiva CTD Essential Reagent (RNP)	QUANTA Flash RNP
Intended Use	The Aptiva CTD Essential Reagent consists of 10 multiplexed immunoassays utilizing particle-based multi-analyte technology for the quantitative determination of IgG autoantibodies against dsDNA, and semi-quantitative determination of IgG	The QUANTA Flash RNP is a chemiluminescent immunoassay for the semi-quantitative determination of IgG anti-ribonucleoprotein (RNP) antibodies in human serum. The presence of anti-RNP antibodies, in conjunction with clinical
Similarities
Item	Aptiva CTD Essential Reagent (RNP)	QUANTA Flash RNP
	autoantibodies against RNP, Sm, Ro52, Ro60, SS-B, Scl-70, Jo-1, centromere, and Ribo-P in human serum:

The presence of RNP antibodies, in conjunction with clinical findings and other laboratory tests, is an aid in the diagnosis of mixed connective tissue disease and systemic lupus erythematosus.

10

11

Similarities
Item	Aptiva CTD Essential Reagent (Sm)	Orgentec anti-Sm
Intended Use	The Aptiva CTD Essential Reagent consists of 10 multiplexed immunoassays utilizing particle-based multi-analyte technology for the quantitative determination of IgG autoantibodies against dsDNA, and semi-quantitative determination of IgG autoantibodies against RNP, Sm, Ro52, Ro60, SS-B, Scl-70, Jo-1, centromere, and Ribo-P in human serum:

The presence of Sm antibodies, in conjunction with clinical findings and other laboratory tests, is an aid in the diagnosis of systemic lupus erythematosus.

The Aptiva CTD Essential Reagent is intended for use with the Inova Diagnostics Aptiva System. | Anti-Sm is an ELISA test system for the quantitative measurement of IgG class autoantibodies against Sm in human serum or plasma. This product is intended for professional in vitro diagnostic use only.

The detection of autoantibodies against Sm proteins is a component of the multi-parametric ACR criteria for the diagnosis of systemic lupus erythematosus (SLE). The detection of Sm antibodies serves as a prognostic marker for SLE, there is a relationship between the appearance of Sm antibodies and severe organ manifestations of the disease. Evaluation of a test result should always take into account all clinical and laboratory diagnostic findings. |
| Assay Methodology | solid phase immunoassay | solid phase immunoassay |

Aptiva CTD Essential Reagent – Sm Assay

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Differences
Item	Aptiva CTD Essential Reagent (Sm)	Orgentec anti-Sm
Detection/Operating Principle	fluorescent immunoassay	Chromogenic immunoassay
Conjugate	phycoerythrin conjugated polyclonal anti-human IgG antibody	HRP conjugated anti-human IgG antibody
Antigen	Synthetic Sm peptide	Synthetic Sm peptide
Sample Type	human serum	human serum or plasma
Solid Phase	paramagnetic microparticles	ELISA
Units	fluorescent light units (FLU)	Units (U/mL)
Cut-off	5.00 FLU	25 U/mL
Analytical Measuring Range	0.25 FLU – 256.00 FLU	1 U/mL – 200.0 U/mL
Calibration	Lot specific Master Curve + 6 calibrators (sold separately)	Calibration Curve using 6 calibrators (included)

Aptiva CTD Essential Reagent – Ro52 Assay

Similarities
Item	Aptiva CTD Essential Reagent (Ro52)	QUANTA Flash Ro52
Intended Use	The Aptiva CTD Essential Reagent consists of 10 multiplexed immunoassays utilizing particle-based multi-analyte technology for the quantitative determination of IgG autoantibodies against dsDNA, and semi-quantitative determination of IgG autoantibodies against RNP, Sm, Ro52, Ro60, SS-B, Scl-70, Jo-1, centromere, and Ribo-P in human serum:

The presence of Ro52 antibodies, in conjunction with clinical findings and other laboratory tests, is an aid in the diagnosis of systemic lupus erythematosus, | QUANTA Flash Ro52 is a chemiluminescent immunoassay for the semi-quantitative determination of IgG anti-Ro52 autoantibodies in human serum. The presence of anti-Ro52 autoantibodies, in conjunction with clinical findings and other laboratory tests, is an aid in the diagnosis of Systemic Lupus Erythematosus, Sjögren's Syndrome, Systemic Sclerosis, Idiopathic Inflammatory Myopathies. |

13

Similarities
Item	Aptiva CTD Essential Reagent (Ro52)	QUANTA Flash Ro52
	Sjögren's syndrome, systemic
sclerosis, and idiopathic
inflammatory myositis.

14

Similarities
Item	Aptiva CTD Essential Reagent (Ro60)	QUANTA Flash Ro60
Intended Use	The Aptiva CTD Essential Reagent consists of 10 multiplexed immunoassays utilizing particle-based multi-analyte technology for the quantitative determination of IgG autoantibodies against dsDNA, and semi-quantitative determination of IgG autoantibodies against RNP, Sm, Ro52, Ro60, SS-B, Scl-70, Jo-1, centromere, and Ribo-P in human serum:

The presence of Ro60 antibodies, in conjunction with clinical findings and other laboratory tests, is an aid in the diagnosis of systemic lupus erythematosus and Sjögren's syndrome.

The Aptiva CTD Essential Reagent is intended for use with the Inova Diagnostics Aptiva System. | QUANTA Flash Ro60 is a chemiluminescent immunoassay for the semi-quantitative determination of IgG anti-Ro60 autoantibodies in human serum. The presence of anti-Ro60 autoantibodies, in conjunction with clinical findings and other laboratory tests, aids in the diagnosis of Systemic Lupus Erythematosus and Sjögren's Syndrome. |
| Assay Methodology | solid phase immunoassay | solid phase immunoassay |
| Antigen | Recombinant Ro60 | Recombinant Ro60 |
| Sample Type | human serum | human serum |
| Solid Phase | paramagnetic microparticles | paramagnetic microparticles |

Aptiva CTD Essential Reagent – Ro60 Assay

Differences
Item	Aptiva CTD Essential Reagent
(Ro60)	QUANTA Flash Ro60
	Detection/Operating
Principle

15

| | phycoerythrin conjugated
polyclonal anti-human IgG
antibody | Isoluminol conjugated
monoclonal anti-human IgG
antibody |
|-------------------------------|-------------------------------------------------------------------|----------------------------------------------------------------|
| Conjugate | | |
| Units | fluorescent light units (FLU) | chemiluminescent units (CU) |
| Cut-off | 5.00 FLU | 20.0 CU |
| Analytical Measuring
Range | 0.50 FLU – 583.72 FLU | 4.9 CU – 1374.8 CU |
| Calibration | Lot specific Master Curve + 6
calibrators (sold separately) | Lot specific Master Curve + 2
calibrators (sold separately) |

Aptiva CTD Essential Reagent – SS-B Assay

Similarities
Item	Aptiva CTD Essential Reagent (SS-B)	QUANTA Flash SS-B
Intended Use	The Aptiva CTD Essential Reagent consists of 10 multiplexed immunoassays utilizing particle-based multi-analyte technology for the quantitative determination of IgG autoantibodies against dsDNA, and semi-quantitative determination of IgG autoantibodies against RNP, Sm, Ro52, Ro60, SS-B, Scl-70, Jo-1, centromere, and Ribo-P in human serum:

The presence of SS-B antibodies, in conjunction with clinical findings and other laboratory tests, is an aid in the diagnosis of systemic lupus erythematosus and Sjögren's syndrome.

The Aptiva CTD Essential Reagent is intended for use with the Inova Diagnostics Aptiva System. | QUANTA Flash SS-B is a chemiluminescent immunoassay for the semi-quantitative determination of IgG anti-SS-B autoantibodies in human serum. The presence of anti-SS-B autoantibodies, in conjunction with clinical findings and other laboratory tests is an aid in the diagnosis of Sjögren's Syndrome and Systemic Lupus Erythematosus. |
| Assay Methodology | solid phase immunoassay | solid phase immunoassay |
| Antigen | Recombinant SS-B | Recombinant SS-B |
| Sample Type | human serum | human serum |

16

Similarities
Item	Aptiva CTD Essential Reagent (SS-
B)	QUANTA Flash SS-B
Solid Phase	paramagnetic microparticles	paramagnetic microparticles

Differences
Item	Aptiva CTD Essential Reagent (SS-B)	QUANTA Flash SS-B
Detection/Operating Principle	fluorescent immunoassay	chemiluminescent immunoassay
Conjugate	phycoerythrin conjugated
polyclonal anti-human IgG
antibody	Isoluminol conjugated
monoclonal anti-human IgG
antibody
Units	fluorescent light units (FLU)	chemiluminescent units (CU)
Cut-off	5.00 FLU	20.0 CU
Analytical Measuring Range	0.40 FLU – 195.84 FLU	3.3 CU – 1550.0 CU
Calibration	Lot specific Master Curve + 6
calibrators (sold separately)	Lot specific Master Curve + 2
calibrators (sold separately)

Aptiva CTD Essential Reagent – Scl-70 Assay

Similarities
Item	Aptiva CTD Essential Reagent (Scl-70)	QUANTA Flash Scl-70
Intended Use	The Aptiva CTD Essential Reagent
consists of 10 multiplexed
immunoassays utilizing particle-
based multi-analyte technology
for the quantitative determination
of IgG autoantibodies against
dsDNA, and semi-quantitative
determination of IgG
autoantibodies against RNP, Sm,
Ro52, Ro60, SS-B, Scl-70, Jo-1,
centromere, and Ribo-P in human
serum:

The presence of Scl-70 antibodies,
in conjunction with clinical | QUANTA Flash Scl-70 is a
chemiluminescent immunoassay
for the semi-quantitative
determination of IgG anti-Scl-70
autoantibodies in human serum.
The presence of anti-Scl-70
autoantibodies, in conjunction
with clinical findings and other
laboratory tests, aids in the
diagnosis of systemic sclerosis. |

17

Similarities
Item	Aptiva CTD Essential Reagent (Scl-70)	QUANTA Flash Scl-70
	findings and other laboratory
tests, is an aid in the diagnosis of
systemic sclerosis.

Differences
Item	Aptiva CTD Essential Reagent (Scl-70)	QUANTA Flash Scl-70
Detection/Operating
Principle	fluorescent immunoassay	chemiluminescent immunoassay
Conjugate	phycoerythrin conjugated
polyclonal anti-human IgG
antibody	Isoluminol conjugated
monoclonal anti-human IgG
antibody
Units	fluorescent light units (FLU)	chemiluminescent units (CU)
Cut-off	5.00 FLU	20.0 CU
Analytical Measuring
Range	0.50 FLU - 371.24 FLU	1.2 CU - 786.3 CU
Calibration	Lot specific Master Curve + 6
calibrators (sold separately)	Lot specific Master Curve + 2
calibrators (sold separately)

18

Similarities
Item	Aptiva CTD Essential Reagent (Jo-1)	QUANTA Flash Jo-1
Intended Use	The Aptiva CTD Essential Reagent
consists of 10 multiplexed
immunoassays utilizing particle-
based multi-analyte technology
for the quantitative determination
of IgG autoantibodies against
dsDNA, and semi-quantitative
determination of IgG
autoantibodies against RNP, Sm,
Ro52, Ro60, SS-B, Scl-70, Jo-1,
centromere, and Ribo-P in human
serum:

The presence of Jo-1 antibodies, in
conjunction with clinical findings
and other laboratory tests, is an
aid in the diagnosis of idiopathic
inflammatory myositis.

Aptiva CTD Essential Reagent – Jo-1 Assay

19

Aptiva CTD Essential Reagent – Centromere Assay

Similarities
Item	Aptiva CTD Essential Reagent (Centromere)	QUANTA Flash Centromere
Intended Use	The Aptiva CTD Essential Reagent consists of 10 multiplexed immunoassays utilizing particle-based multi-analyte technology for the quantitative determination of IgG autoantibodies against dsDNA, and semi-quantitative determination of IgG autoantibodies against RNP, Sm, Ro52, R060, SS-B, Scl-70, Jo-1, centromere, and Ribo-P in human serum:

The presence of centromere antibodies, in conjunction with clinical findings and other laboratory tests, is an aid in the diagnosis of systemic sclerosis.

The Aptiva CTD Essential Reagent is intended for use with the Inova Diagnostics Aptiva System. | QUANTA Flash Centromere is a chemiluminescent immunoassay for the semi-quantitative determination of IgG anti-centromere protein B autoantibodies in human serum. The presence of anti-centromere protein B autoantibodies is used as an aid in the diagnosis of systemic sclerosis, in conjunction with clinical finding and other laboratory tests. |
| Assay Methodology | solid phase immunoassay | solid phase immunoassay |
| Antigen | Recombinant Centromere | Recombinant Centromere |
| Sample Type | human serum | human serum |
| Solid Phase | paramagnetic microparticles | paramagnetic microparticles |

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Differences
Item	Aptiva CTD Essential Reagent (Centromere)	QUANTA Flash Centromere
Detection/Operating Principle	fluorescent immunoassay	chemiluminescent immunoassay
Conjugate	phycoerythrin conjugated
polyclonal anti-human IgG
antibody	Isoluminol conjugated
monoclonal anti-human IgG
antibody
Units	fluorescent light units (FLU)	chemiluminescent units (CU)
Cut-off	5.00 FLU	20.0 CU
Analytical Measuring Range	0.50 FLU – 187.69 FLU	3.4 CU – 708.9 CU
Calibration	Lot specific Master Curve + 6
calibrators (sold separately)	Lot specific Master Curve + 2
calibrators (sold separately)

Aptiva CTD Essential Reagent – Ribo-P Assay

Similarities
Item	Aptiva CTD Essential Reagent (Ribo-P)	QUANTA Lite Ribosomal P
Intended Use	The Aptiva CTD Essential Reagent consists of 10 multiplexed immunoassays utilizing particle-based multi-analyte technology for the quantitative determination of IgG autoantibodies against dsDNA, and semi-quantitative determination of IgG autoantibodies against RNP, Sm, Ro52, Ro60, SS-B, Scl-70, Jo-1, centromere, and Ribo-P in human serum:
The presence of Ribo-P antibodies, in conjunction with clinical findings and other laboratory tests, is an aid in the diagnosis of systemic lupus erythematosus.	QUANTA Lite Ribosome P is an enzyme-linked immunosorbent assay (ELISA) for the semi-quantitative detection of Ribosome P antibodies in human serum. The presence of Ribosome P antibodies can be used in conjunction with clinical findings and other laboratory tests to aid in the diagnosis of Systemic Lupus Erythematosus (SLE) and other related connective tissue diseases.

21

Similarities
Item	Aptiva CTD Essential Reagent (Ribo-P)	QUANTA Lite Ribosomal P
	The Aptiva CTD Essential Reagent is intended for use with the Inova Diagnostics Aptiva System.
Assay Methodology	solid phase immunoassay	solid phase immunoassay
Antigen	Synthetic Ribosomal P peptide	Synthetic Ribosomal P peptide
Sample Type	human serum	human serum
Differences
Item	Aptiva CTD Essential Reagent (Ribo-P)	QUANTA Lite Ribosomal P
Solid Phase	paramagnetic microparticles	ELISA
Detection/Operating Principle	fluorescent immunoassay	Chromogenic immunoassay
Conjugate	phycoerythrin conjugated polyclonal anti-human IgG antibody	HRP conjugated anti-human IgG antibody
Units	fluorescent light units (FLU)	Units (U)
Cut-off	5.00 FLU	20.0 U
Analytical Measuring Range	0.25 FLU - 86.86 FLU	N/A
Calibration	Lot specific Master Curve + 6 calibrators (sold separately)	Single point calibration

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Analytical performance characteristics

Quantitation and units of measure

For quantitation, the Aptiva CTD Essential reagent utilizes predefined lot specific Master Curves per each analyte, that is uploaded onto the instrument through the reagent cartridge RFID. The analyte specific Master Curves are generated at Inova for each reagent lot, where in-house Master Curve Standards with assigned FLU (or IU/mL for dsDNA) values are run multiple times. The resulting MFI values generated are used to create a unique 4 parameter logistic (4PL) curve for each of the ten analytes. The IgG control microparticle will flag low concentrations of lgG in the patient serum sample as an assay verification step. This microparticle also has an in-house standard which is run each time a new reagent lot is manufactured. The MFI produced by this standard is used as the cut-off threshold for the IgG control microparticle for that reagent lot. These four parameters of the analyte curves, as well as the MFI cut-off for the IgG control microparticle are embedded in the reagent cartridge RFID.

Material	Assigned Value (IU/mL)
Aptiva CTD Essential Master Curve Standard 1	0.00
Aptiva CTD Essential Master Curve Standard 2	3.15
Aptiva CTD Essential Master Curve Standard 3	12.74
Aptiva CTD Essential Master Curve Standard 4	50.89
Aptiva CTD Essential Master Curve Standard 5	203.49
Aptiva CTD Essential Master Curve Standard 6	814.10

List of Aptiva CTD Essential Master Curve Standards – dsDNA:

List of Aptiva CTD Essential Master Curve Standards - RNP:

Material	Assigned Value (FLU)
Aptiva CTD Essential Master Curve Standard 1	0.00
Aptiva CTD Essential Master Curve Standard 2	0.71
Aptiva CTD Essential Master Curve Standard 3	2.84
Aptiva CTD Essential Master Curve Standard 4	11.37
Aptiva CTD Essential Master Curve Standard 5	45.50
Aptiva CTD Essential Master Curve Standard 6	181.99

List of Aptiva CTD Essential Master Curve Standards – Sm:

Material	Assigned Value (FLU)
Aptiva CTD Essential Master Curve Standard 1	0.00
Aptiva CTD Essential Master Curve Standard 7	1.00
Aptiva CTD Essential Master Curve Standard 8	4.00
Aptiva CTD Essential Master Curve Standard 9	16.00
Aptiva CTD Essential Master Curve Standard 10	64.00
Aptiva CTD Essential Master Curve Standard 11	256.00

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Material	Assigned Value (FLU)
Aptiva CTD Essential Master Curve Standard 1	0.00
Aptiva CTD Essential Master Curve Standard 7	0.77
Aptiva CTD Essential Master Curve Standard 8	3.06
Aptiva CTD Essential Master Curve Standard 9	12.24
Aptiva CTD Essential Master Curve Standard 10	48.96
Aptiva CTD Essential Master Curve Standard 11	195.84

List of Aptiva CTD Essential Master Curve Standards – SS-B:

List of Aptiva CTD Essential Master Curve Standards – Ro52:

Material	Assigned Value (FLU)
Aptiva CTD Essential Master Curve Standard 1	0.00
Aptiva CTD Essential Master Curve Standard 2	0.77
Aptiva CTD Essential Master Curve Standard 3	3.07
Aptiva CTD Essential Master Curve Standard 4	12.27
Aptiva CTD Essential Master Curve Standard 5	49.07
Aptiva CTD Essential Master Curve Standard 6	196.25

List of Aptiva CTD Essential Master Curve Standards – Ro60:

Material	Assigned Value (FLU)
Aptiva CTD Essential Master Curve Standard 1	0.00
Aptiva CTD Essential Master Curve Standard 2	2.28
Aptiva CTD Essential Master Curve Standard 3	9.12
Aptiva CTD Essential Master Curve Standard 4	36.48
Aptiva CTD Essential Master Curve Standard 5	145.93
Aptiva CTD Essential Master Curve Standard 6	583.72

List of Aptiva CTD Essential Master Curve Standards – Jo-1:

Material	Assigned Value (FLU)
Aptiva CTD Essential Master Curve Standard 1	0.00
Aptiva CTD Essential Master Curve Standard 2	0.60
Aptiva CTD Essential Master Curve Standard 3	2.40
Aptiva CTD Essential Master Curve Standard 4	9.60
Aptiva CTD Essential Master Curve Standard 5	38.40
Aptiva CTD Essential Master Curve Standard 6	153.60

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Material	Assigned Value (FLU)
Aptiva CTD Essential Master Curve Standard 1	0.00
Aptiva CTD Essential Master Curve Standard 7	1.45
Aptiva CTD Essential Master Curve Standard 8	5.80
Aptiva CTD Essential Master Curve Standard 9	23.20
Aptiva CTD Essential Master Curve Standard 10	92.81
Aptiva CTD Essential Master Curve Standard 11	371.24

List of Aptiva CTD Essential Master Curve Standards - Scl-70:

List of Aptiva CTD Essential Master Curve Standards - Centromere:

Material	Assigned Value (FLU)
Aptiva CTD Essential Master Curve Standard 1	0.00
Aptiva CTD Essential Master Curve Standard 7	0.73
Aptiva CTD Essential Master Curve Standard 8	2.93
Aptiva CTD Essential Master Curve Standard 9	11.73
Aptiva CTD Essential Master Curve Standard 10	46.92
Aptiva CTD Essential Master Curve Standard 11	187.69

List of Aptiva CTD Essential Master Curve Standards – Ribo-P:

Material	Assigned Value (FLU)
Aptiva CTD Essential Master Curve Standard 1	0.00
Aptiva CTD Essential Master Curve Standard 7	0.34
Aptiva CTD Essential Master Curve Standard 8	1.36
Aptiva CTD Essential Master Curve Standard 9	5.43
Aptiva CTD Essential Master Curve Standard 10	21.71
Aptiva CTD Essential Master Curve Standard 11	86.86

IgG Control Microparticle Standard: 1 mg/dL human IgG

Precision

The precision of the Aptiva CTD Essential Reagent was evaluated on 9 samples for dsDNA, 6 samples for RNP, 7 samples for Sm, 8 samples for Ro60, 9 samples for SS-B, 6 samples for SS-B, 6 samples for Scl-70, 5 samples for Jo-1, 5 samples for Centromere and 6 samples for Ribo-P, containing various concentrations of antibodies in accordance with CLSI EPO5-A3, Evaluation of Quantitative Measurement Procedures; Approved Guideline. Samples were run in duplicates, twice a day, for 20 days.

Data were analyzed with the Analyse-it for Excel method evaluation software, and repeatability (withinrun), between run, between day and within-laboratory precision) were calculated. Results are summarized in the two tables below.

Acceptance criteria: Total %CV: 181.99 FLU for RNP, >256.00 FLU for Sm, >583.72 FLU for Ro60, >196.27 FLU for Ro52, >195.84 FLU for SS-B, >371.24 FLU for Scl-70, >153.60 FLU for Jo-1, >86.86 FLU for Ribo-P, >187.69 FLU for Centromere, or >814.10 IU/mL for dsDNA after performing an additional 10-fold dilution, thereby bringing the measured value within the AMR. The reported result will be calculated by the software factoring the additional dilution. The highest value that can be reported for each measurand is listed in the table below.

Assay	Auto rerun highest value
dsDNA	8141.00 IU/mL
RNP	1819.90 FLU
Sm	2560.00 FLU
Ro52	1962.70 FLU
Ro60	5837.20 FLU
SS-B	1958.40 FLU
Scl-70	3712.40 FLU
Jo-1	1536.00 FLU
Centromere	1876.90 FLU
Ribo-P	868.60 FLU

High concentration hook effect

To assess hook effect, 2 samples for dsDNA, RNP, Sm, Ro52, Ro60, SS-B, Scl-70, Jo-1, Centromere, and Ribo-P were tested at three increasing 2-fold serial dilutions from the standard 1:44.4 dilution used by the Aptiva CTD Essential Reagent. All FLU values above the analytical measuring ranges of the ten assays are theoretical and were mathematically calculated using the 4 parameters of their respective calibration curves. All samples showed increase in FLU values as dilution factor became more concentrated, thereby

36

confirming that high positive specimens above the AMR do not show hook effect up to for the for dsDNA, RNP, Sm, Ro52, Ro60, SS-B, Scl-70, Jo-1, Centromere, and Ribo-P in the Aptiva CTD Essential Reagent.

Linearity

The linearity of the AMR was calculated separately for all analytes (dsDNA, RNP, Sm, Ro60, Ro52, SS-B, Scl-70, Jo-1, Ribo-P and Centromere) as part of the Aptiva CTD Essential Reagent.

The linearity for all analytes was evaluated by a study according to CLSI EP06-Ed2, Evaluation of the Linearity of Quantitative Measurement Procedures: 200 Edition. The study used three human serum samples for the dsDNA assay, four human serum samples for the RNP, Sm, Ro52, SS-B, Scl-70, Jo-1, Centromere and Ribo-P assays and 5 samples for the Ro60 assay with various antibody concentrations in 10% increments (from 0% to 90% of sample) to obtain values that cover the entire AMR of each analyte. The dilutions were assayed in duplicates. Results were analyzed according to the guideline performing regression analysis and identifying the best fitting polynomial.

Acceptance criteria:

Allowable deviation from linearity: +/- 15% or +/- 0.75 FLU (+/- 5.25 IU/mL for dsDNA)
Slope: 0.9-1.1
R2: > 0.95

For dsDNA, all acceptance criteria were fulfilled. Results included in the table below:

dsDNA
Serum Sample	Test Range
(IU/mL)	Slope
(95% CI)	R²	Range of Linearity
Deviations
1	120.85 - 1208.48	1.02 (0.98 - 1.05)	0.99	-9.2% to 6.4%
2	13.09 - 130.89	0.98 (0.96 - 0.99)	1.00	-4.5% to 4.1%
3	1.99 - 19.89	0.98 (0.94 - 1.02)	0.99	-5.0% to 5.3%
and
-1.12 to 0.08 IU/mL

For RNP, all acceptance criteria were fulfilled. Results included in the table below:

RNP
Serum Sample	Test Range
(FLU)	Slope
(95% CI)	R²	Range of Linearity
Deviations
1	20.36 - 203.59	0.96 (0.93 to 1.00)	0.99	-9.6% to 5.8%
2	4.55 - 45.46	1.00 (0.97 to 1.03)	0.99	-11.6% to 6.9%
3	0.71 - 7.09	0.93 (0.88 to 0.99)	0.98	-13.9% to 7.0%
and
-0.11 FLU
4	0.14 - 1.43	0.99 (0.94 to 1.04)	0.99	-0.07 to -0.08 FLU

37

Sm
Serum Sample	Test Range
(FLU)	Slope
(95% Cl)	R²	Range of Linearity
Deviations
1	33.02 - 330.25	0.92 (0.89 to 0.96)	0.98	-12.8% to 8.3%
2	5.87 - 58.72	1.02 (1.00 to 1.04)	1.00	-2.3% to 10.9%
3	0.73 - 7.31	0.96 (0.93 to 0.99)	0.99	-9.0% to 4.2%
and
-0.30 to -0.21 FLU
4	0.13 - 1.33	1.01 (0.97 to 1.04)	0.99	-0.06 to 0.04 FLU

For Sm, all acceptance criteria were fulfilled. Results included in the table below:

For Ro52, all acceptance criteria were fulfilled. Results included in the table below:

Ro52
Serum Sample	Test Range
(FLU)	Slope
(95% CI)	R2	Range of Linearity
Deviations
1	20.07 – 200.73	0.98 (0.95 to 1.01)	0.99	-8.8% to 4.5%
2	4.81 - 48.10	1.02 (1.00 to 1.03)	1.00	-9.8% to 3.5%
3	0.96 - 9.58	1.01 (0.96 to 1.06)	0.98	-8.8% to 7.0%
and
-0.60 to 0.27 FLU
4	0.12 - 1.25	0.97 (0.94 to 0.99)	0.99	-0.04 to 0.04 FLU

For Ro60, all acceptance criteria were fulfilled. Results included in the table below:

Ro60
Serum Sample	Test Range
(FLU)	Slope
(95% Cl)	R²	Range of Linearity
Deviations
1	71.74 - 717.36	0.93 (0.89 to 0.98)	0.98	-8.1% to 7.1%
2	17.17 - 85.85	1.04 (1.01 to 1.07)	0.99	-3.6% to 5.4%
3	5.94 - 59.41	1.02 (0.98 to 1.06)	0.99	-7.0% to 14.4%
4	1.48 - 7.41	1.02 (0.98 to 1.05)	0.99	-5.9% to 11.3%
5	0.30 - 2.96	0.91 (0.84 to 0.97)	0.95	-0.38 to 0.28 FLU

For SS-B, all acceptance criteria were fulfilled. Results included in the table below:

SS-B
Serum Sample	Test Range
(FLU)	Slope
(95% CI)	R²	Range of Linearity
Deviations
1	20.55 - 205.54	0.98 (0.94 to 1.01)	0.99	-13.2% to 7.1%
2	8.89 - 88.85	0.94 (0.92 to 1.07)	0.99	-13.5% to 5.9%
3	1.15 - 11.51	0.88 (0.82 to 0.94)	0.98	-10.3% to 13.7%
and
-0.46 to -0.27 FLU
4	0.16 - 1.55	0.99 (0.96 to 1.02)	0.99	-0.09 to 0.06 FLU

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Scl-70
Serum Sample	Test Range
(FLU)	Slope
(95% CI)	R²	Range of Linearity
Deviations
1	41.46 - 414.62	1.01 (0.99 to 1.03)	0.99	-4.7% to 6.9%
2	5.30 - 53.05	0.98 (0.96 to 0.99)	1.00	-12.9% to 2.5%
3	0.81 - 8.13	0.97 (0.92 to 1.01)	0.99	-14.5% to 4.9%
and
-0.43 to -0.25 FLU
4	0.16 - 1.57	0.94 (0.89 to 0.98)	0.98	-0.09 to 0.10 FLU

For Scl-70, all acceptance criteria were fulfilled. Results included in the table below:

For Jo-1, all acceptance criteria were fulfilled. Results included in the table below:

Jo-1
Serum Sample	Test Range
(FLU)	Slope
(95% CI)	R²	Range of Linearity
Deviations
1	20.60 - 206.02	0.95 (0.92 to 0.98)	0.99	-12.7% to 5.3%
2	5.78 - 57.76	0.96 (0.93 to 0.99)	0.99	-4.3% to 8.6%
3	0.76 - 7.63	1.07 (1.02 to 1.11)	0.98	-10.7% to 11.4%
4	0.14 - 1.38	0.96 (0.94 to 0.98)	0.99	-0.03 to 0.05 FLU

For Centromere, all acceptance criteria were fulfilled. Results included in the table below:

Centromere
Serum Sample	Test Range
(FLU)	Slope
(95% CI)	R²	Range of Linearity
Deviations
1	40.48 - 202.38	1.01 (0.98 to 1.04)	0.99	-12.7% to 5.9%
2	5.96 - 59.56	1.00 (0.99 to 1.01)	1.00	-5.8% to 2.1%
3	0.91 - 9.08	1.01 (0.99 to 1.02)	1.00	-9.9% to 1.9%
4	0.19 - 1.86	0.96 (0.89 to 1.03)	0.97	-0.19 to 0.10 FLU

For Ribo-P, all acceptance criteria were fulfilled. Results included in the table below:

Ribo-P
Serum Sample	Test Range
(FLU)	Slope
(95% CI)	R2	Range of Linearity
Deviations
1	13.91 - 139.12	0.97 (0.95 to 1.00)	0.99	-3.7% to 6.2%
2	2.78 - 27.83	1.00 (0.95 to 1.04)	0.98	-11.9% to 7.2%
3	0.58 - 5.76	0.99 (0.93 to 1.05)	0.99	-7.8% to 6.4%
and
-0.48 to -0.27 FLU
4	0.09 - 0.95	1.05 (0.97 to 1.12)	0.95	-0.10 to 0.11 FLU

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These data demonstrate that the entire analytical measuring range for dsDNA (AMR: 2.30 – 814.10 IU/mL), of RNP (AMR: 0.50 – 181.99 FLU), Sm (AMR: 0.25 – 256.00 FLU) Ro52 (AMR: 0.25 – 196.27 FLU), Ro60 (AMR: 0.50 – 583.72 FLU), SS-B (0.40 – 195.84 FLU Sd-70 (0.50 – 371.24 FLU), Jo-1 (0.25 – 153.60 FLU) Centromere (AMR: 0.50 – 187.69 FLU) and Ribo-P (AMR: 0.25 – 86.86 FLU) have proven to be linear.

Interference

The interference study was performed according to CLSI EPO7-A2, Interference Testing in Clinical Chemistry; Approved Guideline - Second Edition. A set of three human serum specimens, one positive, one near the cutoff and one negative sample were tested using endogenous interfering substances (bilirubin, hemoglobin, triglycerides, cholesterol, rheumatoid factor IgG) and exogenous interfering substances (ibuprofen, acetaminophen, prednisone, warfarin, diltiazem, azathioprine, sildenafil, cyclophosphamide, mycophenolate mofetil and heparin). All interferents were spiked into every serum specimen and the resulting samples were assessed in triplicates with the Aptiva CTD Essential assays (dsDNA, RNP, Sm, Ro52, Ro60, SS-B, Scl-70, Jo-1, Centromere and Ribo-P). Recovery of the unit values was calculated compared to control samples.

Acceptance criteria for the interference studies were 85% - 115% recovery, or ± 15% of the cut-off (±0.75 FLU or ±4.05 IU/mL for dsDNA) difference, whichever is greater.

The Aptiva CTD Essential Reagent did not show interference with bilirubin up to 1 mg/mL, hemoglobin up to 2 mg/mL, triglyceride up to 1000 mg/dL, cholesterol up to 332.5 mg/dL, RF IgM up to 153.4 IU/mL and human IgG up to 35 mg/mL. The Aptiva CTD Essential Reagent did not show interference up to 21.9 mg/dL ibuprofen, 15.6 mg/dL acetaminophen, 0.0099 mg/dL prednisone, 7.5 mg/dL warfarin, 0.09 mg/dL diltiazem, 0.258 mg/dL azathioprine, 0.271 mg/dL sildenafil, 54.9 mg/dL cyclophosphamide, 1.125 mg/mL mycophenolate mofetil and 330 units/dL heparin.

Note: Rituximab was not evaluated for interference with the Aptiva CTD Essential Reagent.

Sample Stability and Handling

For the all analytes included in the Aptiva CTD Essential Reagent (dsDNA, RNP, Sm, Ro52, Ro60, SS-B, Scl-70, Jo-1, Centromere and Ribo-P) three samples were tested. The samples used for this study were achieved by combining high and low antibody level to yield their desired reactivity. All samples were tested in duplicates for up to 21 days while stored at 2-8℃, up to 48 hours while stored at room temperature, and after repeated freeze/thaw cycles up to 5 cycles. Results were compared to those obtained on control samples (time zero / zero cycles).

Acceptance criteria: percent recovery is between 85-115% for positive samples, and between 80-120% for negative samples (