The Aptiva APS IgG Reagent is an immunoassay utilizing particle-based multi-analyte technology for the semiquantitative determination of anti-cardiolipin (aCL) and anti-beta 2 glycoprotein 1 (all2GPI) IgG autoantibodies in human serum as an aid in the diagnosis of primary antiphospholipid syndrome (APS), when used in conjunction with other laboratory findings.

The Aptiva APS IgG Reagent is intended for use with the Aptiva System.

The Aptiva APS IgM Reagent is an immunoassay utilizing particle-based multi-analyte technology for the semiquantitative determination of anti-cardiolipin (aCL) and anti-beta 2 glycoprotein 1 (aß2GPI) IgM autoantibodies in human serum as an aid in the diagnosis of primary and secondary antiphospholipid syndrome (APS), when used in conjunction with other laboratory findings.

The Aptiva APS IgM Reagent is intended for use with the Aptiva System.

Device Description

The Aptiva APS IgG and Aptiva APS IgM reagent utilize particle based multi-analyte technology (PMAT) in a cartridge format. Each analyte (anti-cardiolipin [aCL] and anti-B2-Glycoprotein I [aB2GPI]) in the Aptiva APS IgG and Aptiva APS IgM reagent is a solid phase immunoassay utilizing fluorescent microparticles. This technology allows each of the two analytes, along with a human IgG or human IgM capture antibody (IgG or IgM Control Microparticle), to be coated onto three uniquely recognizable paramagnetic microparticles, which are combined into one tube.

The Aptiva 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 two analyte microparticles, along with the control microparticle, are stored in the reagent cartridge under conditions that proteins 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 instrument, where the microparticles are automatically rehydrated using a buffer located within the cartridge.

The Aptiva System dilutes the sample 1:8, then combines an aliquot of diluted sample, and reagent into a cuyette. The mixture is incubated at 37°C. After a wash cvcle, conjugated antihuman IgG or IdM antibodies are added to the particles and this mixture is incubated at 37°C. Excess conjugate is removed in another wash cycle, and the particles are re-suspended in system fluid.

Multiple images are generated by the system to identify and count the two (2) unique analyte particles, as well as determine the amount of coniugate on each particle. Coated with goat anti-human lgG or IdM antibodies, is present as a control to flaq low concentrations of IgG or IgM in the sample as an assay verification step. The median fluorescent intensity (MFI) for each analyte is proportional to the concentration of conjugate bound to human IgG or IgM, which is proportional to the concentration of IgG or IgM antibodies bound to the corresponding particle population. The system uses the MFI from at least 50 particles of each population. The identity of the particles is determined by the unique signature of the particles.

Each analyte in the Aptiva APS IgG Reagent and the Aptiva APS IgM Reagent is assigned a predefined lot specific master curve. The analyte specific master curve is stored on the reagent cartridge RFID label. Based on results obtained by running calibrators (supplied separately), the system creates individual working curves. Working curves are used by the software to calculate Fluorescent Light Units (FLU) for each analyte from the MFI values obtained for each sample.

Aptiva APS IgG and Aptiva APS IgM Calibrators and Aptiva APS IgG and Aptiva APS IgM Controls are sold separately.

AI/ML Overview

The provided text describes the analytical and clinical performance characteristics of the Aptiva APS IgG and Aptiva APS IgM Reagents, which are immunoassays for the semi-quantitative determination of anti-cardiolipin (aCL) and anti-beta 2 glycoprotein 1 (aβ2GPI) IgG/IgM autoantibodies. This information is presented in the context of a 510(k) premarket notification for FDA clearance.

Here's a breakdown of the requested information based on the provided text:

1. A table of acceptance criteria and the reported device performance

The document does not explicitly state "acceptance criteria" as a separate, quantified set of thresholds for each performance metric. Instead, it presents the results of various analytical and clinical studies, implying that these results met the internal criteria for substantial equivalence to predicate devices and overall performance claims for an in vitro diagnostic (IVD) device.

However, we can infer performance targets based on the presented data and the overall context of an FDA submission for an IVD. The primary performance metrics presented are related to precision, detection limits, linearity, interference, and clinical sensitivity/specificity.

Inferred Acceptance Criteria and Reported Device Performance (Summary)

Performance Characteristic	Inferred Acceptance Criteria (General IVD Expectations)	Reported Device Performance (Aptiva APS Reagents)
Precision	CV% to be within acceptable ranges for IVD assays, typically lower for higher concentrations and clinically critical ranges.	Within-Laboratory (Total Precision) CV%:- aCL IgG: 5.6% - 9.5% (generally decreasing with higher FLU)- aβ2GPI IgG: 6.9% - 11.7% (generally decreasing with higher FLU)- aCL IgM: 4.3% - 9.7% (generally decreasing with higher FLU)- aβ2GPI IgM: 5.5% - 10.2% (generally decreasing with higher FLU)Between-Site Reproducibility CV% (3 sites):- aCL IgG: 5.2% - 9.3%- aβ2GPI IgG: 6.4% - 10.0%- aCL IgM: 5.4% - 10.0%- aβ2GPI IgM: 5.9% - 10.5%Between-Lot Reproducibility CV% (3 lots):- aCL IgG: 6.6% - 13.3%- aβ2GPI IgG: 8.5% - 12.1%- aCL IgM: 6.1% - 11.4%- aβ2GPI IgM: 6.0% - 10.5%
Limit of Blank (LoB)	Very low, close to zero, ensuring no signal from blank samples.	aCL IgG: 0.00 FLUaβ2GPI IgG: 0.02 FLUaCL IgM: 0.01 FLUaβ2GPI IgM: 0.03 FLU
Limit of Detection (LoD)	Low, indicating ability to detect small amounts of analyte.	aCL IgG: 0.07 FLUaβ2GPI IgG: 0.09 FLUaCL IgM: 0.04 FLUaβ2GPI IgM: 0.06 FLU
Limit of Quantitation (LoQ)	Low, defining the lowest concentration that can be reliably quantified.	aCL IgG: 0.29 FLUaβ2GPI IgG: 0.21 FLUaCL IgM: 0.06 FLU (set to 0.10 FLU for AMR lower limit)aβ2GPI IgM: 0.09 FLU (set to 0.10 FLU for AMR lower limit)
Analytical Measuring Range (AMR)	Wide enough to cover relevant clinical concentrations, with demonstrated linearity.	aCL IgG: 0.29 - 328.94 FLUaβ2GPI IgG: 0.21 - 256.70 FLUaCL IgM: 0.10 – 114.68 FLUaβ2GPI IgM: 0.10 – 95.86 FLULinearity demonstrated across these ranges with R2 values mostly ≥ 0.98.
High Concentration Hook Effect	No hook effect within or above the AMR.	Confirmed no hook effect up to theoretically calculated values: aCL IgG: 2645.36 FLU, aβ2GPI IgG: 1790.48 FLU, aCL IgM: 167.25 FLU, aβ2GPI IgM: 126.13 FLU.
Interference	No significant interference from common endogenous or exogenous substances at specified concentrations.	No interference detected for aCL IgG, aβ2GPI IgG, aCL IgM, and aβ2GPI IgM with tested interferents (bilirubin, hemoglobin, triglycerides, cholesterol, RF IgM, human IgG, ibuprofen, warfarin, prednisone, acetaminophen, aspirin, hydroxychloroquine, omeprazole, simvastatin, heparin) at their respective tested concentrations. Percent recoveries or FLU differences were within acceptable ranges (generally close to 100% recovery for spiked samples, or low FLU difference for negative samples).
Sample Stability	Samples should be stable for specific storage conditions and freeze/thaw cycles.	Samples stable up to 48 hours at room temperature, up to 14 days at 2-8°C, and for up to 5 freeze/thaw cycles.
Reagent Stability	Reagent shelf-life and in-use stability should be established.	Shelf-life: 9 months for Aptiva APS IgG Reagent, 7 months for Aptiva APS IgM Reagent (based on accelerated stability, verified by ongoing real-time studies).In-use (onboard) stability: 28 days for both, with 14-day recalibration.
Clinical Sensitivity & Specificity	High sensitivity to detect disease (APS) and high specificity to correctly identify non-disease states (controls/non-APS).	Aptiva APS IgG:- aCL IgG: Sensitivity 54.1% (95% CI: 45.3–62.7%), Specificity 99.5% (95% CI: 98.2–99.9%)- aβ2GPI IgG: Sensitivity 53.3% (95% CI: 44.5-61.9%), Specificity 99.0% (95% CI: 97.5-99.6%)Aptiva APS IgM:- aCL IgM: Sensitivity 27.5% (95% CI: 22.7–32.9%), Specificity 97.5% (95% CI: 95.4–98.6%)- aβ2GPI IgM: Sensitivity 24.7% (95% CI: 20.1–30.0%), Specificity 98.5% (95% CI: 96.8–99.3%)
Predicate Method Comparison (Percent Agreement)	High agreement with legally marketed predicate devices.	Aptiva APS IgG (aCL IgG) vs. QUANTA Flash aCL IgG: PPA: 81.6%, NPA: 95.7%, TPA: 93.1% (N=202)Aptiva APS IgG (aβ2GPI IgG) vs. QUANTA Lite Beta 2GP1 IgG ELISA: PPA: 88.0%, NPA: 89.7%, TPA: 88.9% (N=108)Aptiva APS IgM (aCL IgM) vs. QUANTA Flash aCL IgM: PPA: 87.0%, NPA: 90.2%, TPA: 89.8% (N=422)Aptiva APS IgM (aβ2GPI IgM) vs. QUANTA Flash β2GPI IgM: PPA: 88.9%, NPA: 84.3%, TPA: 84.8% (N=244)

2. Sample size used for the test set and the data provenance (e.g. country of origin of the data, retrospective or prospective)

Clinical Performance Test Set Sample Sizes:
- Aptiva APS IgG (aCL IgG & aβ2GPI IgG): N=526 (122 APS combined, 404 controls/non-APS)
- Aptiva APS IgM (aCL IgM & aβ2GPI IgM): N=689 (291 APS combined, 398 controls/non-APS)
- Normal Population for Expected Values: N=200 apparently healthy blood donors.
Method Comparison Test Set Sample Sizes:
- Aptiva APS IgG (aCL IgG vs. QUANTA Flash aCL IgG): N=202
- Aptiva APS IgG (aβ2GPI IgG vs. QUANTA Lite Beta 2GP1 IgG ELISA): N=108
- Aptiva APS IgM (aCL IgM vs. QUANTA Flash aCL IgM): N=422
- Aptiva APS IgM (aβ2GPI IgM vs. QUANTA Flash β2GPI IgM): N=244
Analytical Performance Test Set Sample Sizes:
- Precision: 7 samples for IgG, 7 samples for IgM (80 replicates each for within-lab; 75 replicates each for between-site/lot reproducibility from multiple sites/lots).
- LoB/LoD/LoQ: Blanks (LoB: 4 samples, 60 data points/lot); Low-level samples (LoD/LoQ: 4 samples, 120 data points/assay/lot).
- Interference: 6 human specimens (negative, cutoff, positive) for each analyte, spiked with various interferents and tested in 5 replicates.
- Sample Stability: 5 serum samples (IgG), 5 serum samples (IgM) tested in duplicates over time/cycles.
- In-use Stability: 11 samples (IgG), 7 samples (IgM) tested periodically.
Data Provenance: The document states that a "cohort of characterized samples, none of which were used for establishing the reference range, was used to validate the clinical performance." It does not explicitly state the country of origin of the data or whether the studies were retrospective or prospective. However, for a 510(k) submission, clinical validation studies typically involve retrospective or prospectively collected clinical samples, but the exact nature (e.g., specific clinical sites, patient populations beyond disease groups) and geographic origin are not detailed here. The studies were likely conducted within a controlled laboratory setting by the manufacturer (Inova Diagnostics, Inc. in San Diego, CA) or its affiliates, using sourced human serum samples.

3. Number of experts used to establish the ground truth for the test set and the qualifications of those experts (e.g. radiologist with 10 years of experience)

The establishment of "ground truth" for IVD devices like these typically relies on well-characterized clinical samples and established diagnostic criteria for the disease (Antiphospholipid Syndrome - APS).

The document states that the clinical performance validation was performed using "a cohort of characterized samples." The characterization of these samples (i.e., whether they definitively represent APS or control) would serve as the ground truth.
However, the document does not specify the number of experts or their qualifications (e.g., rheumatologists, clinical immunologists/pathologists) who established the diagnostic status (ground truth) of the clinical samples (APS vs. control) used in the clinical sensitivity and specificity studies. It is implied that these were "characterized samples," meaning their disease status was determined by established clinical and laboratory criteria, likely involving clinical consensus or previous diagnoses.
For cut-off establishment, the reference population included "apparently healthy subjects," and the "internal APS samples (data not provided)" and "distribution of result values of healthy controls" were used. This suggests clinical characterization of these samples.

4. Adjudication method (e.g. 2+1, 3+1, none) for the test set

The concept of "adjudication method" (like 2+1 or 3+1) is typically relevant for interpretative tasks, such as reading medical images, where multiple human readers interpret the same data and their interpretations need to be reconciled to establish a ground truth.

For these types of IVD assays, ground truth for clinical performance is established based on the clinical diagnosis of the patient from whom the sample was collected. This diagnosis is usually a culmination of clinical findings, established criteria (e.g., the revised Sapporo criteria for APS), and other laboratory tests, rather than an "adjudication" of multiple independent interpretations of the test results themselves.

Therefore, the document does not mention any adjudication method in the context of establishing ground truth for the test samples, as it's not applicable in the same way it would be for an AI-medical imaging device. The "ground truth" for the samples (APS vs. non-APS) is assumed to be pre-established clinical diagnosis.

5. If a multi reader multi case (MRMC) comparative effectiveness study was done, If so, what was the effect size of how much human readers improve with AI vs without AI assistance

No MRMC study was conducted or is applicable here.

This device is an in vitro diagnostic (IVD) immunoassay, not an AI-powered image analysis or diagnostic aid that assists human readers (e.g., radiologists interpreting images). The device directly measures biomarker levels in a sample, and its output is a quantitative value (FLU) which then determines a semi-quantitative result (Positive/Negative/Indeterminate based on cut-offs). Human "readers" (laboratory personnel) operate the instrument and interpret the final quantitative results based on predefined cut-offs, but they are not subjectively interpreting complex data that AI would assist with, in the sense of an MRMC study.

Therefore, an MRMC comparative effectiveness study, and an effect size related to human reader improvement with AI assistance, are not relevant for this type of device and are not mentioned in the document.

6. If a standalone (i.e. algorithm only without human-in-the-loop performance) was done

This is a "standalone" device in terms of its core functionality, but the term "algorithm only" or "human-in-the-loop" isn't directly analogous.

The Aptiva System is a fully automated, random-access analyzer (page 6). This means the instrument itself, with its integrated software and optical module, processes the samples, runs the reagents, and reports results independently after the sample is loaded and the assay initiated.
The "performance" described here (sensitivity, specificity, precision, linearity, etc.) is the device's performance (including its internal algorithms and mechanics) in generating quantitative results. There isn't a separate "algorithm only" performance that needs to be differentiated from a "human-in-the-loop" performance, because the device is the automated system determining the FLU values. The human interaction is primarily in sample loading, reagent handling, and result review/reporting, not in interpreting raw data that the device itself would also interpret in an unassisted mode.

7. The type of ground truth used (expert consensus, pathology, outcomes data, etc)

The ground truth for the clinical performance studies (sensitivity and specificity) was established based on "characterized samples" representing patients with Antiphospholipid Syndrome (APS) and various control groups (patients with other autoimmune/infectious diseases, apparently healthy subjects).

While the document doesn't explicitly state "expert consensus," it's highly implied that the "characterization" of these samples as APS or control would be based on:

Clinical findings: Presenting symptoms, medical history.
Other laboratory findings: Beyond the novel antibodies, other relevant diagnostic tests.
Established diagnostic criteria: For APS, this would typically be the revised Sapporo classification criteria, which combine clinical and laboratory criteria.

So, it's a combination of established clinical diagnoses and potentially other laboratory data, which implicitly would involve the consensus or findings of medical experts involved in patient diagnosis. It is not based on pathology (e.g., tissue biopsy) or outcomes data (e.g., long-term disease progression as the sole ground truth).

8. The sample size for the training set

The document describes the submission of a "new device" and its performance characteristics. It does not explicitly mention or quantify a "training set" in the context of machine learning.

For IVD devices, a "training set" isn't a standard concept unless the device incorporates adaptive algorithms or AI that learns from data. In this case, the device is an immunoassay with predefined master curves and calibrated reagents. The master curves are generated "at Inova for each reagent lot, where in-house Master Curve Standards with assigned FLU values are run multiple times." These "in-house Master Curve Standards" could be considered analogous to a "training" or calibration process, but it's not a dataset for training a generalized AI model but rather for calibrating each reagent lot of a classical assay.

The sample sizes provided in the document are for:

Analytical performance (precision, LoB/LoD/LoQ, linearity, interference, stability).
Clinical validation (sensitivity/specificity studies).
Method comparison studies.
Reference range establishment.

None of these are explicitly labeled as a "training set."

9. How the ground truth for the training set was established

As there is no explicitly defined "training set" for a machine learning model, the concept of establishing ground truth for such a set is not applicable here.

However, if we consider the "Master Curve Standards" as analogous to calibration/training data for the assay, their "ground truth" (assigned FLU values) would be established through a rigorous internal process by the manufacturer (Inova Diagnostics) based on:

Carefully prepared and characterized aliquots (standards) with known or assigned concentrations of the target antibodies.
Repeat measurements and statistical analysis for consistent and accurate assignment of FLU values.
This is a standard practice for calibrating quantitative IVD assays, ensuring the device outputs accurate and traceable results.

Summary

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December 17, 2024

Inova Diagnostics, Inc. Constance Bridges VP, Quality & Regulatory Affairs 9900 Old Grove Road San Diego, California 92131

Re: K223093

Trade/Device Name: Aptiva APS IgG Reagent Aptiva APS IgM Reagent Regulation Number: 21 CFR 866.5660 Regulation Name: Multiple Autoantibodies Immunological Test System Regulatory Class: Class II Product Code: MID, MSV Dated: December 14, 2023 Received: December 15, 2023

Dear Constance Bridges:

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 (the 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 available 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.

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Additional information about changes that may require a new premarket notification are provided in the FDA guidance documents entitled "Deciding When to Submit a 510(k) for a Change to an Existing Device" (https://www.fda.gov/media/99812/download) and "Deciding When to Submit a 510(k) for a Software Change to an Existing Device" (https://www.fda.gov/media/99785/download).

Your device is also subject to, among other requirements, the Quality System (QS) regulation (21 CFR Part 820), which includes, but is not limited to, 21 CFR 820.30. Design controls; 21 CFR 820.90. Nonconforming product; and 21 CFR 820.100, Corrective and preventive action. Please note that regardless of whether a change requires premarket review, the QS regulation requires device manufacturers to review and approve changes to device design and production (21 CFR 820.30 and 21 CFR 820.70) and document changes and approvals in the device master record (21 CFR 820.181).

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 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 Part 803) for devices or postmarketing safety reporting (21 CFR Part 4, Subpart B) for combination products (see https://www.fda.gov/combination-products/guidance-regulatory-information/postmarketing-safetyreporting-combination-products); good manufacturing practice requirements as set forth in the quality systems (QS) regulation (21 CFR Part 820) for devices or current good manufacturing practices (21 CFR Part 4, Subpart A) for combination products; and, if applicable, the electronic product radiation control provisions (Sections 531-542 of the Act); 21 CFR Parts 1000-1050.

All medical devices, including Class I and unclassified devices and combination product device constituent parts are required to be in compliance with the final Unique Device Identification System rule ("UDI Rule"). The UDI Rule requires, among other things, that a device bear a unique device identifier (UDI) on its label and package (21 CFR 801.20(a)) unless an exception or alternative applies (21 CFR 801.20(b)) and that the dates on the device label be formatted in accordance with 21 CFR 801.18. The UDI Rule (21 CFR 830.300(a) and 830.320(b)) also requires that certain information be submitted to the Global Unique Device Identification Database (GUDID) (21 CFR Part 830 Subpart E). For additional information on these requirements, please see the UDI System webpage at https://www.fda.gov/medical-device-advicecomprehensive-regulatory-assistance/unique-device-identification-system-udi-system.

Also, please note the regulation entitled, "Misbranding by reference to premarket notification" (21 CFR 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 medical devices and radiation-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-regulatory

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assistance/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) K223093

Device Name Aptiva APS IgG Reagent Aptiva APS IgM Reagent

Indications for Use (Describe)

The Aptiva APS IgG Reagent is intended for use with the Aptiva System.

The Aptiva APS IgM Reagent is intended for use with the 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)
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510(k) Summary

Aptiva APS IgG and IgM Reagent

Table of Contents Adminietrotiv dott

Administrative data
Predicate device
Device description
Intended use(s)
lndications for use
Substantial equivalence
Comparison to predicate device
Analytical performance characteristics
Quantitation and units of measure
Reproducibility Studies
Limit of Blank (LoB), Limit of Detection (LoD), and Limit of Quantitation (LoQ)
Analytical Measuring Range (AMR)
High concentration hook effect
Linearity
Interference
Sample Stability and Handling
Reagent Stability
Cut-off, reference range
Clinical performance characteristics
Clinical sensitivity, specificity
Expected values
Comparison with predicate device

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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, Inc9900 Old Grove Road,San Diego, CA, 92131
Purpose of submission:	New device
Device in the submission:	Aptiva APS IgG ReagentAptiva APS IgM Reagent
Scientific contact:	Andrea Seaman, Associate Director, Research and DevelopmentInova Diagnostics, Inc.9900 Old Grove Road, San Diego, CA, 92131Phone: 858-586-9900 x77395Fax: 858-863-0025Email: aseaman@werfen.com
Quality Systems contact:	Constance Bridges, VP, Quality and RegulatoryInova Diagnostics, Inc9900 Old Grove Road, San Diego, CA, 92131Phone: 858-586-9900 x77212Fax: 858-863-0025Email: cbridges@werfen.com
Device name (kit):	Proprietary name: Aptiva APS IgG ReagentAptiva APS IgM ReagentCommon name: anti-cardiolipin antibody immunoassay, anti-beta2-glycprotein1 immunoassayClassification name: System, Test, Anticardiolipin ImmunologicalSystem, Test, Beta2 Glycoprotein1Immunological
Regulation Description	Multiple autoantibodies immunological test system
Regulation MedicalSpecialty	Immunology
Review Panel	Immunology
Product Code	Anticardiolipin: MIDB2 - Glycoprotein I: MSV
Regulation Number	866.5660
Device Class	2

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Predicate device

HemosIL™ AcuStar Anti-Cardiolipin IgG, 510(k) number: K092181 QUANTA Lite™ Beta 2GP1 IgG ELISA, 510(k) number: K970551 HemosIL™ AcuStar Anti-Cardiolipin IgM, 510(k) number: K092181 HemosIL™ AcuStar Anti-ß2 Glycoprotein-I IgM, 510(k) number: K091556

Device description

Aptiva APS IgG and Aptiva APS IgM Calibrators and Aptiva APS IgG and Aptiva APS IgM Controls are sold separately.

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Page 4 of 28

Contents	Active Ingredient	Quantity	Symbol
1. Aptiva APS IgG ReagentCartridge	-	1 each	RC
- APS IgG Beads	- Paramagnetic beads coated with:- Native Cardiolipin (CL) plus β2GPI antigens (<0.01%)- Native β2GPI antigen (<0.5%)- AffiniPure Goat polyclonal anti-human IgG antigen (<0.01%)- Bovine protein stabilizer (<2.4%)	1 x 0.5mL	-
- Assay Buffer	- Bovine/porcine protein stabilizer (<0.5%)- Bovine protein stabilizer (<0.3%)- Sodium azide (<0.1%)	1 x 17mL	-
- PE Tracer IgG	- PE IgG Conjugate, Goat anti-human IgG antibody (<0.30%)- Bovine protein stabilizer (<1.2%)- Sodium azide (<0.1%)	1 x 17mL	-
- Rehydration Buffer	- Bovine protein stabilizer (<0.03%)- Sodium azide (<0.1%)	1 x 6.5mL	-

The Aptiva APS IgG Reagent kit contains the following materials:

The Aptiva APS IgM Reagent kit contains the following materials:

	Contents	Active Ingredient	Quantity	Symbol
1.	Aptiva APS IgM Reagent Cartridge	-	1 each	RC
	- APS IgM Beads	Paramagnetic beads coated with:- Native Cardiolipin (CL) plus β2GPI antigens (<0.01%)- Native β2GPI antigen (<0.04%)- AffiniPure Goat polyclonal anti-human IgM antigen (<0.01%)- Bovine protein stabilizer (<2.4%)	1 x 0.5mL
	- Assay Buffer	Bovine/porcine protein stabilizer (<0.42%)- Bovine protein stabilizer (<0.27%)- Sodium azide (<0.1%)	1 x 17mL
	- PE Tracer IgM	PE IgG Conjugate, Goat anti-human IgM antibody (<0.15%)- Bovine protein stabilizer (<1.2%)- Sodium azide (<0.1%)	1 x 17mL
	- Rehydration Buffer	Bovine protein stabilizer (< 0.02%)- Sodium azide (<0.1%)	1 x 6.5mL

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Intended use(s)

The Aptiva APS IgG Reagent is an immunoassay utilizing particle-based multi-analyte technology for the semi-quantitative determination of anti-cardiolipin (aCL) and anti-beta 2 qlycoprotein 1 (aß2GPI) IgG autoantibodies in human serum as an aid in the diagnosis of primary and secondary antiphospholipid syndrome (APS), when used in conjunction with other laboratory and clinical findings.

The Aptiva APS IgG Reagent is intended for use with the Aptiva System.

The Aptiva APS IgM Reagent is an immunoassay utilizing particle-based multi-analyte technology for the semi-quantitative determination of anti-cardiolipin (aCL) and anti-beta 2 dlycoprotein 1 (aß2GPI) IgM autoantibodies in human serum as an aid in the diagnosis of primary and secondary antiphospholipid syndrome (APS), when used in conjunction with other laboratory and clinical findings.

The Aptiva APS IgM Reagent is intended for use with the Aptiva System.

Indications for use

Same as intended use.

Substantial equivalence

The Aptiva APS IgG Reagent and the Aptiva APS IgM Reagent have the same intended use and assay principle as the predicate devices.

{9}------------------------------------------------

Comparison to predicate device

Aptiva APS IgG Reagent - aCL IgG Comparison to Predicate Device

This table provides a comparative description of the similarities and differences between the subject device, Aptiva APS IgG Reagent, and its predicate device currently marketed as QUANTA Flash aCL IgG Reagent. The QUANTA Flash aCL IgG Reagent is equivalent to the HemosIL AcuStar Anti-Cardiolipin IgG assay (K092181).

	Subject Device	Predicate Device
Item	Aptiva APS IgG Reagent(aCL IgG)	QUANTA Flash aCL IgG Reagents(aCL IgG)
Trade name	Aptiva APS IgG Reagent	QUANTA Flash aCL IgG Reagents
Intended Use /Indication for Use	The Aptiva APS IgG Reagent is animmunoassay utilizing particle-basedmulti-analyte technology for the semi-quantitative determination of anti-cardiolipin (aCL) and anti-beta 2glycoprotein 1 (aβ2GPI) IgGautoantibodies in human serum as anaid in the diagnosis of primary andsecondary antiphospholipid syndrome(APS), when used in conjunction withother laboratory and clinical findings.The Aptiva APS IgG Reagent isintended for use with the Aptiva Multi-Analyte System.	Fully automated chemiluminescentimmunoassay for the semi-quantitativemeasurement of anti-cardiolipin (aCL)IgG antibodies in human citratedplasma and serum on the BIO-FLASHinstrument as an aid in the diagnosis ofthrombotic disorders related to primaryand secondary antiphospholipidsyndrome (APS), when used inconjunction with other laboratory andclinical findings.
Type of Test	Semi-quantitative	Same
InstrumentPlatform	Aptiva System	BIO-FLASH instrument
Technology	Fluorescent immunoassay	Chemiluminescent immunoassay
Clinical Cut-off	5.00 FLU	20.0 CU* (20 U/mL+)
Calibrator	Three Calibrator Levels	Two Calibrator Levels
Composition	1 cartridge containing 1 vial of magneticparticle suspension coated with bovinecardiolipin and human purified β2GPI, 1vial of assay buffer, 1 vial of tracerconsisting of an anti-human IgGantibody labeled with phycoerythrin,and 1 vial of sample diluent.	1 cartridge containing 1 vial of magneticparticle suspension coated with bovinecardiolipin and human purified β2GPI, 1vial of assay buffer, 1 vial of tracerconsisting of an anti-human IgGantibody labeled with isoluminol, and 1vial of sample diluent.
Sample Type	Serum	Serum or Citrated Plasma
Quality Control	Two Control Levels	Same
Detection Limit	0.07 FLU	2.6 CU* (20 U/mL+)
Linearity	0.29 FLU - 328.94 FLU	2.6 – 2024 U/mL

Applicable to QUANTA Flash aCL IgG Reagent

† Applicable to HemosIL AcuStar Anti-Cardiolipin IgG

{10}------------------------------------------------

Aptiva APS IgG Reagent - β2GPI IgG Comparison to Predicate Device
This table provides a comparative description of the similarities and differences between the subject
device, Aptiva APS IgG Reagent, and its predicate device, the currently marketed QUANTA Lite Beta
2GP1 IgG ELISA (K970551).
Item	Aptiva APS IgG Reagent(ß2GPI IgG)	QUANTA Lite Beta 2GP1 IgG ELISA(aCL IgG)
Trade name	Aptiva APS IgG Reagent	QUANTA Lite Beta 2GP1 IgG ELISA
Intended Use /Indication for Use	The Aptiva APS IgG Reagent is animmunoassay utilizing particle-based multi-analyte technology forthe semi-quantitative determinationof anti-cardiolipin (aCL) and anti-beta 2 glycoprotein 1 (aß2GPI) IgGautoantibodies in human serum asan aid in the diagnosis of primaryand secondary antiphospholipidsyndrome (APS), when used inconjunction with other laboratoryand clinical findings.The Aptiva APS IgG Reagent isintended for use with the AptivaMulti-Analyte System.	QUANTA Lite Beta 2GP1 IgG is anenzyme-linked immunosorbent assay(ELISA) for the semi-quantitativedetection of ß2 GPI IgG antibodies inhuman serum. The presence of ß2GPI IgG antibodies can be used inconjunction with clinical findings andother laboratory tests to aid in thediagnosis of certain autoimmunedisease thrombotic disorders, such asthose secondary to systemic lupuserythematosus (SLE) or other lupus-like thrombotic diseases.
Type of Test	Semi-quantitative	Same
Instrument Platform	Aptiva Multi-Analyte System	N/A - manually run assay
Technology	Fluorescent immunoassay	Enzyme-linked immunosorbent assay
Clinical Cut-off	5.00 FLU	20.0 SGU
Calibrator	Three Calibrator Levels	Five Calibrator Levels
Composition	1 cartridge containing 1 vial ofmagnetic particle suspensioncoated with bovine cardiolipin andhuman purified ß2GPI, 1 vial ofassay buffer, 1 vial of tracerconsisting of an anti-human IgGantibody labeled with phycoerythrin,and 1 vial of sample diluent.	Kit contains 1 β2 GPI ELISA Plate(12-1 x 8 wells), 1 vial predilutedELISA Negative Control, 1 vialprediluted ß2 GPI IgG ELISA Control,5 vials prediluted β2 GPI IgG ELISACalibrators, 1 vial HRP SampleDiluent, 1 vial HRP WashConcentrate (40x), 1 vial HRP IgGConjugate, (goat), anti-human IgG, 1vial 10mL TMB Chromogen, and 1vial HRP Stop Solution
Sample Type	Serum	Same
Quality Control	Two Control Levels	Two Controls - one positive and onenegative
Linearity	0.21 - 256.70 FLU	9.4 - 150.0 SGU

{11}------------------------------------------------

Aptiva APS IgM Reagent - aCL IgM Comparison to Predicate Device
This table provides a comparative description of the similarities and differences between the subject device, Aptiva APS IgM Reagent, and its predicate device currently marketed QUANTA Flash aCL IgM Reagent. The QUANTA Flash aCL IgM Reagent is equivalent to the HemosIL AcuStar Anti-Cardiolipin IgM assay (K092181).
Item	Aptiva APS IgM Reagent (aCL IgM)	QUANTA Flash aCL IgG Reagents (aCL IgM)
Trade name	Aptiva APS IgM Reagent	QUANTA Flash aCL IgM Reagents
Intended Use /Indication for Use	The Aptiva APS IgM Reagent is an immunoassay utilizing particle-based multi-analyte technology for the semi-quantitative determination of anti-cardiolipin (aCL) and anti-beta 2 glycoprotein 1 (aβ2GPI) IgM autoantibodies in human serum as an aid in the diagnosis of primary and secondary antiphospholipid syndrome (APS), when used in conjunction with other laboratory and clinical findings.The Aptiva APS IgM Reagent is intended for use with the Aptiva Multi-Analyte System.	Fully automated chemiluminescent immunoassay for the semi-quantitative measurement of anti-cardiolipin (aCL) IgG antibodies in human citrated plasma and serum on the BIO-FLASH instrument as an aid in the diagnosis of thrombotic disorders related to primary and secondary antiphospholipid syndrome (APS), when used in conjunction with other laboratory and clinical findings.
Type of Test	Semi-quantitative	Same
InstrumentPlatform	Aptiva Multi-Analyte System	BIO-FLASH instrument
Technology	Fluorescent immunoassay	Chemiluminescent immunoassay
Clinical Cut-off	5.00 FLU	20.0 CU* (20U/mL†)
Calibrator	Three Calibrator Levels	Two Calibrator Levels
Composition	1 cartridge containing 1 vial of magnetic particle suspension coated with bovine cardiolipin and human purified β2GPI, 1 vial of assay buffer, 1 vial of tracer consisting of an anti-human IgM antibody labeled with phycoerythrin, and 1 vial of sample diluent.	1 cartridge containing 1 vial of magnetic particle suspension coated with bovine cardiolipin and human purified β2GPI, 1 vial of assay buffer, 1 vial of tracer consisting of an anti-human IgM antibody labeled with isoluminol, and 1 vial of sample diluent.
Sample Type	Serum	Serum or Citrated Plasma
Quality Control	Two Control Levels	Two Control Levels I
Detection Limit	0.04 FLU	1.0 CU* (1 U/mL+)
Linearity	0.10 FLU - 114.68 FLU	1.0 - 774 CU* (1.0 - 774 U/mL+)
Item	Aptiva APS IgM Reagent(β2GPI IgM)	QUANTA Flash β2GPI IgM Reagents(β2GPI IgM)
Trade name	Aptiva APS IgM Reagent	QUANTA Flash β2GPI IgM Reagents
Intended Use /Indication for Use	The Aptiva APS IgM Reagent is animmunoassay utilizing particle-basedmulti-analyte technology for the semi-quantitative determination of anti-cardiolipin (ACL) and anti-beta 2glycoprotein 1 (αβ2GPI) IgMautoantibodies in human serum as anaid in the diagnosis of primary andsecondary antiphospholipid syndrome(APS), when used in conjunction withother laboratory and clinical findings.The Aptiva APS IgM Reagent isintended for use with the Aptiva Multi-Analyte System.	Fully automated chemiluminescentimmunoassay for the semi-quantitativemeasurement of anti-β2 glycoprotein-1(β2GP1) IgM antibodies in humancitrated plasma and serum on the BIO-FLASH® instrument as an aid in thediagnosis of thrombotic disordersrelated to primary and secondaryantiphospholipid syndrome (APS), whenused in conjunction with otherlaboratory and clinical findings.
Type of Test	Semi-quantitative	Same
InstrumentPlatform	Aptiva Multi-Analyte System	BIO-FLASH instrument
Technology	Fluorescent immunoassay	Chemiluminescent immunoassay
Clinical Cut-off	5.00 FLU	20.0 CU* (20 U/mL⁺)
Calibrator	Three Calibrator Levels	Two Calibrator Levels
Composition	1 cartridge containing 1 vial of magneticparticle suspension coated with bovinecardiolipin and human purified β2GPI, 1vial of assay buffer, 1 vial of tracerconsisting of an anti-human IgMantibody labeled with phycoerythrin,and 1 vial of sample diluent.	1 cartridge containing 1 vial of magneticparticle suspension coated with bovinecardiolipin and human purified β2GPI, 1vial of assay buffer, 1 vial of tracerconsisting of an anti-human IgMantibody labeled with isoluminol, and 1vial of sample diluent.
Sample Type	Serum	Serum or Citrated Plasma
Quality Control	Two Control Levels	Two levels at or near cut-off and atabnormal level
Detection Limit	0.04 FLU	1.1 CU* (1.1 U/mL⁺)
Linearity	0.10 FLU - 95.86 FLU	1.0 - 841 CU* (1.0 - 841 U/mL⁺)

Applicable to QUANTA Flash aCL IgM Reagent

† Applicable to HemosIL AcuStar Anti-Cardiolipin IgM

{12}------------------------------------------------

Aptiva APS IgM Reagent - β2GPI IgM Comparison to Predicate Device

This table provides a comparative description of the similarities and differences between the subject device, Aptiva APS IgM Reagent, and its predicate device, the currently marketed QUANTA Flash aCL IgM Reagent. The QUANTA Flash β2GPI IgM Reagent is equivalent to the HemosIL AcuStar Anti-β2 Glycoprotein-I IgM assay (K091556).

Applicable to QUANTA Flash β2GPI IgM Reagent

† Applicable to HemosIL AcuStar Anti-ß2 Glycoprotein-I IgM

{13}------------------------------------------------

Analytical performance characteristics

Quantitation and units of measure

For quantitation, the Aptiva APS IgG and Aptiva APS IgM reagents utilize predefined lot specific Master Curves, one for each analyte (aCL IgG, a82GP1 IgG, aCL IgM and aß2GPI IgM) that are 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 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 two analytes. The IgG and IgM control bead will flag low concentrations of IgG or IgM antibodies in the 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 or IgM control microparticle for that reagent lot. These four parameters of the analyte curves, as well as the MFI cut-off for the IgG or IgM control microparticle are embedded in the reagent cartridge RFID.

Material	aCL IgG - FLU	aβ2GPI IgG - FLU
APS IgG Master Curve Standard 1	0.00	0.00
APS IgG Master Curve Standard 2	1.28	1.00
APS IgG Master Curve Standard 3	5.14	4.01
APS IgG Master Curve Standard 4	20.56	16.04
APS IgG Master Curve Standard 5	82.24	64.17
APS IgG Master Curve Standard 6	328.94	256.70

List of Aptiva APS IgG Master Curve Standards – Assigned Value:

lgG Control Microparticle Standard: 1 mg/dL human lgG

List of Aptiva APS IgM Master Curve Standards - Assigned Value:

Material	aCL IgM - FLU	aβ2GPI IgM- FLU
APS IgM Master Curve Standard 1	0.00	0.00
APS IgM Master Curve Standard 2	1.42	1.18
APS IgM Master Curve Standard 3	4.25	3.55
APS IgM Master Curve Standard 4	12.74	10.65
APS IgM Master Curve Standard 5	38.23	31.95
APS IgM Master Curve Standard 6	114.68	95.86

IgM Control Microparticle Standard: 1.5 mg/dL human IgM

Precision

The precision of the Aptiva APS IgG and Aptiva APS IgM reagents was evaluated on seven samples for aCL IgG, aB2GPI IgG, aCL IgM and a82GPI IgM, containing various concentrations of antibodies in accordance with CLSI EP05-A3, Evaluation of Quantitative Measurement Procedures; Approved Guideline. Samples were run in duplicates, twice a day, for 20 days.

{14}------------------------------------------------

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

aCL IgG Precision		Repeatability		Between Run		Between Day		WithinLaboratory
Sample	Replicates(N)	Mean(FLU)	SD(FLU)	CV	SD(FLU)	CV	SD(FLU)	CV	SD(FLU)	CV
aCL Sample 1	80	2.33	0.13	5.5%	0.08	3.3%	0.10	4.3%	0.18	7.8%
aCL Sample 2	80	5.64	0.29	5.1%	0.10	1.8%	0.28	5.0%	0.42	7.4%
aCL Sample 3	80	8.77	0.37	4.2%	0.32	3.7%	0.00	0.0%	0.49	5.6%
aCL Sample 4	80	26.15	1.41	5.4%	0.00	0.0%	1.42	5.4%	2.00	7.6%
aCL Sample 5	80	66.29	2.53	3.8%	2.22	3.4%	2.71	4.1%	4.32	6.5%
aCL Sample 6	80	201.45	7.94	3.9%	5.58	2.8%	10.65	5.3%	14.40	7.2%
aCL Sample 7	80	265.15	11.06	4.2%	10.94	4.1%	19.96	7.5%	25.30	9.5%

	aβ2GPI IgG Precision			Repeatability		Between Run		Between Day		WithinLaboratory
Sample	Replicates(N)	Mean(FLU)	SD(FLU)	CV	SD(FLU)	CV	SD(FLU)	CV	SD(FLU)	CV
aβ2GPI Sample 1	80	2.04	0.15	7.3%	0.04	1.9%	0.18	9.0%	0.24	11.7%
aβ2GPI Sample 2	80	5.12	0.29	5.7%	0.22	4.4%	0.00	0.0%	0.37	7.1%
aβ2GPI Sample 3	80	9.39	0.50	5.3%	0.29	3.1%	0.89	9.5%	1.06	11.3%
aβ2GPI Sample 4	80	24.55	1.22	5.0%	0.22	0.9%	1.69	6.9%	2.10	8.5%
aβ2GPI Sample 5	80	53.51	2.11	3.9%	2.08	3.9%	2.22	4.1%	3.70	6.9%
aβ2GPI Sample 6	80	170.02	6.40	3.8%	5.05	3.0%	7.83	4.6%	11.30	6.6%
aβ2GPI Sample 7	80	212.10	8.19	3.9%	6.60	3.1%	14.44	6.8%	17.86	8.4%

aCL IgM Precision			Repeatability		Between Run		Between Day		WithinLaboratory
Sample	Replicates(N)	Mean(FLU)	SD(FLU)	CV	SD(FLU)	CV	SD(FLU)	CV	SD(FLU)	CV
aCL Sample 1	80	2.93	0.12	4.1%	0.06	2.1%	0.25	8.5%	0.28	9.7%
aCL Sample 2	80	5.54	0.15	2.8%	0.10	1.9%	0.20	3.6%	0.27	4.9%
aCL Sample 3	80	10.67	0.34	3.2%	0.14	1.3%	0.50	4.7%	0.62	5.8%
aCL Sample 4	80	21.94	0.59	2.7%	0.55	2.5%	0.51	2.3%	0.95	4.3%
aCL Sample 5	80	56.16	1.30	2.3%	0.47	0.8%	3.07	5.5%	3.36	6.0%
aCL Sample 6	80	73.51	2.25	3.1%	1.23	1.7%	3.06	4.2%	3.99	5.4%
aCL Sample 7	80	116.09	2.62	2.3%	2.55	2.2%	5.90	5.1%	6.94	6.0%

{15}------------------------------------------------

aβ2GPI IgM Precision			Repeatability		Between Run		Between Day		Within Laboratory
Sample	Replicates(N)	Mean(FLU)	SD(FLU)	CV	SD(FLU)	CV	SD(FLU)	CV	SD(FLU)	CV
aβ2GPI Sample 1	80	2.47	0.10	3.9%	0.07	2.9%	0.22	9.0%	0.25	10.2%
aβ2GPI Sample 2	80	5.39	0.13	2.5%	0.27	5.0%	0.22	4.1%	0.38	7.0%
aβ2GPI Sample 3	80	8.75	0.28	3.2%	0.19	2.1%	0.38	4.4%	0.51	5.8%
aβ2GPI Sample 4	80	27.26	1.23	4.5%	0.51	1.9%	1.37	5.0%	1.91	7.0%
aβ2GPI Sample 5	80	46.00	1.22	2.7%	0.74	1.6%	2.29	5.0%	2.70	5.9%
aβ2GPI Sample 6	80	63.00	2.10	3.3%	0.63	1.0%	2.70	4.3%	3.48	5.5%
aβ2GPI Sample 7	80	93.18	2.97	3.2%	2.03	2.2%	4.59	4.9%	5.83	6.3%

Reproducibility Studies

Reproducibility between sites (instruments)

Seven samples for aCL IgG and aß2GPI IgG and six samples for aCL IgM and aβ2GPI IgM were tested according to CLSI EP05-A3 Evaluation of Quantitative Measurement Procedures; Approved Guideline, at three different sites. Samples were run in replicates of five, once a day, for five days, to generate 25 data points per site. Data were analyzed with the Analyse-it for Excel method evaluation software to calculate between site precision. Results are summarized in the tables below.

aCL IgG			Repeatability		Between Day		Between-Site		Reproducibility
Sample	Replicates(N)	Mean(FLU)	SD(FLU)	CV%	SD(FLU)	CV%	SD(FLU)	CV%	SD(FLU)	CV%
1	75	1.97	0.10	5.2%	0.04	2.3%	0.12	6.0%	0.16	8.3%
2	75	5.60	0.33	5.9%	0.20	3.6%	0.13	2.3%	0.41	7.2%
3	75	25.24	1.35	5.3%	1.06	4.2%	0.49	1.9%	1.78	7.1%
4	75	53.83	2.46	4.6%	1.48	2.7%	1.40	2.6%	3.19	5.9%
5	75	123.94	6.00	4.8%	2.43	1.0%	0.00	0.0%	6.48	5.2%
6	75	164.49	9.10	5.5%	5.26	3.2%	2.78	1.7%	10.87	6.6%
7	75	279.39	14.03	5.0%	5.79	2.1%	20.98	7.5%	25.89	9.3%

Sample	aẞ2GPI IgG		Replicates(N)	Repeatability		Between Day		Between-Site		Reproducibility
	Mean (FLU)	SD(FLU)		SD(FLU)	CV%	SD(FLU)	CV%	SD(FLU)	CV%	SD(FLU)	CV%
1	1.99	0.11	75	5.7%	0.08	4.0%	0.14	7.1%	0.20	10.0%
2	5.26	0.24	75	4.6%	0.20	3.8%	0.19	3.6%	0.36	6.9%
3	23.58	1.08	75	4.6%	1.18	5.0%	0.96	4.1%	1.87	7.9%
4	52.83	2.49	75	4.7%	1.76	3.3%	1.47	2.8%	3.38	6.4%
5	138.12	8.47	75	6.1%	4.03	2.9%	7.36	5.3%	11.92	8.6%
6	161.78	6.16	75	3.8%	4.09	2.5%	10.78	6.7%	13.07	8.1%
7	217.27	9.54	75	4.4%	4.86	2.2%	12.35	5.7%	16.35	7.5%

{16}------------------------------------------------

aCL IgM			Repeatability		Between Day		Between-Site		Reproducibility
Sample	Replicates(N)	Mean(FLU)	SD(FLU)	CV%	SD(FLU)	CV%	SD(FLU)	CV%	SD(FLU)	CV%
1	75	2.16	0.10	4.6%	0.09	4.3%	0.05	2.2%	0.14	6.7%
2	75	4.56	0.16	3.4%	0.19	4.1%	0.04	0.8%	0.24	5.4%
3	75	6.68	0.28	4.1%	0.19	2.8%	0.33	5.0%	0.47	7.1%
4	75	40.83	1.47	3.6%	1.33	3.3%	3.56	8.7%	4.07	10.0%
5	75	57.09	2.30	4.0%	1.61	2.8%	4.83	8.5%	5.59	9.8%
6	75	92.71	4.27	4.6%	2.36	2.5%	8.24	8.9%	9.58	10.3%

aẞ2GPI IgM			Repeatability		Between Day		Between-Site		Reproducibility
Sample	Replicates(N)	Mean(FLU)	SD(FLU)	CV%	SD(FLU)	CV%	SD(FLU)	CV%	SD(FLU)	CV%
1	75	1.56	0.06	3.7%	0.06	3.9%	0.04	2.3%	0.09	5.9%
2	75	4.63	0.17	3.7%	0.14	3.0%	0.18	3.9%	0.28	6.2%
3	75	6.11	0.24	3.9%	0.20	3.3%	0.36	5.9%	0.47	7.8%
4	75	35.89	1.25	3.5%	1.46	4.1%	3.26	9.1%	3.78	10.5%
5	75	55.66	2.28	4.1%	1.92	3.5%	4.78	8.6%	5.64	10.1%
6	75	82.78	3.71	4.5%	2.20	2.7%	7.25	8.8%	8.44	10.2%

Reproducibility between lots

Seven samples for aCL IgG and aß2GPI IgG and six samples for aCL IgM and aß2GPI IgM were tested according to CLSI EP05-A3 Evaluation of Quantitative Measurement Procedures; Approved Guideline, using three different lots. Samples were run in replicates of 5, once a day, for 5 days, to generate 25 data points per sample, per lot, 75 data points total for each sample. Data were analyzed with the Analyse-it for Excel method evaluation software to calculate between lot precision. Results are summarized in the tables below.

aCL IgG			Repeatability		Between Day		Between-Lot		Reproducibility
Sample	Replicates(N)	Mean(FLU)	SD(FLU)	CV%	SD(FLU)	CV%	SD(FLU)	CV%	SD(FLU)	CV%
1	75	1.61	0.08	5.2%	0.07	4.4%	0.12	7.2%	0.16	9.9%
2	75	4.16	0.21	5.0%	0.18	4.3%	0.19	4.6%	0.34	8.1%
3	75	19.71	0.98	5.0%	0.99	5.0%	2.17	11.0%	2.58	13.1%
4	75	51.11	2.67	5.2%	1.63	3.2%	6.06	11.9%	6.82	13.3%
5	75	124.49	5.93	4.8%	5.92	4.8%	0.00	0.0%	8.37	6.7%
6	75	168.77	7.11	4.2%	8.61	5.1%	0.00	0.0%	11.17	6.6%
7	75	285.27	10.73	3.8%	5.73	2.0%	11.38	4.0%	16.66	5.8%

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Page 14 of 28

aẞ2GPI IgG			Repeatability		Between Day		Between-Lot		Reproducibility
Sample	Replicates(N)	Mean (FLU)	SD(FLU)	CV%	SD(FLU)	CV%	SD(FLU)	CV%	SD(FLU)	CV%
1	75	1.97	0.10	5.0%	0.13	6.7%	0.03	1.7%	0.17	8.5%
2	75	4.94	0.26	5.3%	0.35	7.2%	0.40	8.2%	0.60	12.1%
3	75	23.39	1.08	4.6%	1.24	5.3%	1.10	4.7%	1.98	8.5%
4	75	58.42	3.18	5.5%	2.47	4.2%	3.87	6.6%	5.59	9.6%
5	75	100.56	5.55	5.5%	6.40	6.4%	5.21	5.2%	9.94	9.9%
6	75	144.20	6.89	4.8%	9.57	6.6%	10.69	7.4%	15.92	11.0%
7	75	213.10	8.78	4.1%	5.46	2.6%	21.18	9.9%	23.57	11.1%

aCL IgM			Repeatability		Between Day		Between-Site		Reproducibility
Sample	Replicates(N)	Mean(FLU)	SD(FLU)	CV%	SD(FLU)	CV%	SD(FLU)	CV%	SD(FLU)	CV%
1	75	2.07	0.07	3.3%	0.10	4.9%	0.03	1.7%	0.13	6.1%
2	75	4.39	0.12	2.8%	0.19	4.3%	0.20	4.6%	0.30	6.9%
3	75	5.25	0.15	2.9%	0.24	4.5%	0.24	4.5%	0.36	6.9%
4	75	31.61	0.83	2.6%	0.85	2.7%	2.16	6.8%	2.46	7.8%
5	75	50.07	1.54	3.1%	1.89	3.8%	5.18	10.3%	5.72	11.4%
6	75	88.20	3.24	3.7%	3.80	4.3%	8.10	9.2%	9.51	10.8%

aẞ2GPI IgM			Repeatability		Between Day		Between-Site		Reproducibility
Sample	Replicates(N)	Mean(FLU)	SD(FLU)	CV%	SD(FLU)	CV%	SD(FLU)	CV%	SD(FLU)	CV%
1	75	1.57	0.05	3.3%	0.09	5.6%	0.06	4.0%	0.12	7.6%
2	75	4.62	0.15	3.2%	0.25	5.4%	0.15	3.3%	0.33	7.0%
3	75	5.73	0.20	3.5%	0.28	4.8%	0.00	0.0%	0.34	6.0%
4	75	24.76	0.61	2.5%	0.68	2.7%	1.86	7.5%	2.07	8.4%
5	75	54.40	1.63	3.0%	2.45	4.5%	4.54	8.3%	5.41	9.9%
6	75	80.53	2.52	3.1%	3.27	4.1%	7.35	9.1%	8.43	10.5%

Limit of Blank (LoB), Limit of Detection (LoD), and Limit of Quantitation (LoQ)

The LoB, LoD, and LoQ of the aCL IgG, aß2GPI IgG, aCL IgM and aß2GPI IgM assays in the Aptiva APS IgG and Aptiva APS IgM Reagent were calculated separately by a study according to CLSI EP17-A2, Evaluation of Detection Capability for Clinical Laboratory Measurement Procedures; Approved Guideline- Second Edition.

Study protocol for LoB:

Four blank samples were run in replicates of five on two reagent lots, once per day, for 3 days, with 60 data points generated on each lot. The LoB was determined for each assay, on each reagent lot separately with the Analyse-it for Excel software's Reference Interval function, at the 95th percentile, using the non-parametric method for aCL IgG, a$2GPI IgG, aCL IgM and aβ2GPI IgM assays (all having a p-value = <0.0001)

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The aCL IgG LoB for both reagent lots was determined as 0.00 FLU. The final LoB value for aCL lgG is 0.00 FLU.

The aß2GPI IgG LoB for one reagent lot was determined as 0.00 FLU, and for the second reagent lot as 0.02 FLU. The final LoB value for aß2GPI IgG is 0.02 FLU.

The aCL IgM LoB for one reagent lot was determined as 0.01 FLU, and for the second reagent lot as 0.00 FLU. The final LoB value for aCL IgM is 0.01 FLU.

The aß2GPI IgM LoB for one reagent lot was determined as 0.03 FLU, and for the second reagent lot as 0.02 FLU. The final LoB value for aßB2GPI IgM is 0.03 FLU.

Study protocol for LoD:

Four low level samples for aCL IgG, ap2GPI IgG, aCL IgM and aβ2GPI IgM assays (prepared by mixing human serum samples with high and low levels of antibodies) were run in replicates of five on two reagent lots, twice per day, for 3 days, with 120 data points generated on each assay, on each reagent lot. The LoD was determined separately for each assay, on each reagent lot.

The aCL IgG limit of detection for one reagent lot was determined as 0.07 FLU, and for the second reagent lot as 0.07 FLU. The final LoD value for aCL IgG is 0.07 FLU.

The aß2GPI IgG limit of detection for one reagent lot was determined as 0.07 FLU, and for the second reagent lot as 0.09 FLU. The final LoD value for ap2GPI IgG is 0.09 FLU.

The aCL IgM limit of detection for one reagent lot was determined as 0.04 FLU, and for the second reagent lot as 0.03 FLU. The final LoD value for aCL IgM is 0.04 FLU.

The aβ2GPI IgM limit of detection for one reagent lot was determined as 0.06 FLU, and for the second reagent lot as 0.06 FLU. The final LoD value for a82GPI IgM is 0.06 FLU.

Study protocol for LoQ:

Four low level samples for aCL IgG, aCL IgM and a82GPI IgM assays (prepared by mixing human serum samples with high and low levels of antibodies) were run in replicates of five on two reagent lots, twice per day, for 3 days, with 120 data points generated on each assay, on each reagent lot. The LoQ was determined separately for each assay, on each reagent lot. The LoQ was determined in each case by calculating the total imprecision of each.

The aCL IgG limit of quantitation for one reagent lot was determined as 0.25 FLU, and for the second reagent lot as 0.29 FLU. The final LoQ value is 0.29 FLU, which has been set as the lower limit of the analytical measuring range of the aCL IgG assay.

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The aß2GPI IgG limit of quantitation for one reagent lot was determined as 0.21 FLU, and for the second reagent lot as 0.21 FLU. The final LoQ value is 0.21 FLU, which has been set as the lower limit of the analytical measuring range of the aβ2GPI IgG assay.

The aCL IgM limit of quantitation for one reagent lot was determined as 0.06 FLU, and for the second reagent lot as 0.04 FLU. The final LoQ value is 0.06 FLU. The lower limit of the analytical measuring range of the aCL IgM assay has been set at 0.10 FLU.

The aβ2GPI IgM limit of quantitation for one reagent lot was determined as 0.09 FLU, and for the second reagent lot as 0.09 FLU. The final LoQ value is 0.09 FLU. The lower limit of the analytical measuring range of the aß2GPI IgM assay has been set at 0.10 FLU.

Analytical Measuring Range (AMR)

Within the Aptiva APG IgG Reagent:
aCL IgG:	0.29 - 328.94 FLU
aβ2GPI IgG:	0.21 - 256.70 FLU

Within the Aptiva APG IgM Reagent:
aCL IgM:	0.10 – 114.68 FLU
aβ2GPI IgM:	0.10 – 95.86 FLU

High concentration hook effect

To assess hook effect, 2 samples aCL IgG, aß2GPI IgG, aCL IgM and aβ2GPI IgM assays were tested at increasing 2-fold serial dilutions from the standard 1:8 dilution used by the Aptiva APS IgG and Aptiva APS IgM Reagents. All FLU values above the analytical measuring ranges of the two 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, confirming that high positive specimens above the AMR do not show hook effect up to 2645.36 FLU for aCL lgG, 1790.48 FLU for a82GPI IgG, 167.25 FLU for aCL IgM and 126.13 FLU for the aβ2GPI IgM (theoretical values calculated) in the Aptiva APS IgG and Aptiva APS IgM Reagents.

Linearity

The Linearity of the AMR was calculated separately for aCL IgG, a$2GPI IgG, aCL IgM and aß2GPI IgM assays as part of the Aptiva APS IgG and Aptiva APS IgM Reagents.

The linearity of the AMR of Aptiva APS IgM and Aptiva APS IgG was evaluated by a study according to CLSI EP06, Evaluation of the Linearity of Quantitative Measurement Procedures: A Statistical Approach; Approved Guideline. Five human serum samples for Aptiva APS IgG and four human serum samples for Aptiva APS IgM with various antibody concentrations were serially diluted to obtain values that cover the entire AMR. The dilutions were assayed in duplicates.

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Results were analyzed according to the guideline performing regression analysis.

Results were analyzed according to the guideline performing regression analysis and identifying the best fitting polynomial.

aCllgG:
-------------

Sample	Test Range in FLU	Slope (95% CI)	R2	Range of Linearity Deviations
1	38.22 – 382.20	1.00 (0.96 to 1.03)	0.99	-11.2% to 7.4%
2	9.35 – 46.74	1.00 (0.97 to 1.04)	0.99	-7.2% to 6.6%
3	1.62 – 16.19	0.99 (0.96 to 1.02)	1.00	-13.1% to 2.6%
4	0.96 – 4.79	0.98 (0.94 to 1.02)	0.99	-12.9% to 5.7% or -0.24 FLU
5	0.11 - 1.09	1.00 (0.95 to 1.04)	0.98	-10.3% to 13.5%

aß2GPI IgG:

Sample	Test Range in FLU	Slope (95% CI)	R2	Range of Linearity Deviations
1	30.12 – 301.21	1.00 (0.96 to 1.04)	0.99	-12.1% to 11.6%
2	7.13 – 35.67	1.00 (0.96 to 1.04)	0.98	-6.9% to 12.3%
3	1.45 – 14.55	0.99 (0.95 to 1.02)	0.99	-11.7% to 13.5% or 0.45 FLU
4	0.39 – 3.87	0.91 (0.83 to 0.99)	0.99	-2.1% to 10.1% or -0.46 to -0.17 FLU
5	0.11 - 1.09	1.00 (0.95 to 1.04)	0.98	-10.3% to 13.5%

These data demonstrate the linearity of the analytical measuring range (0.29 – 328.94 FLU) of the aCL IgG assay and the analytical measuring range (0.21 – 256.70 FLU) of the a$2GPI IgG assay, both as part of the Aptiva APS IgG Reagent.

aCL IgM:

Sample	Test Range in FLU	Slope (95% CI)	R2	Range of Linearity Deviations
1	11.81 – 118.12	1.06 (1.02 to 1.11)	0.99	-6.1% to 11.5%
2	1.80 - 18.00	1.03 (1.01 to 1.05)	1.00	-2.7% to 8.8% or 0.57 FLU
3	0.23 – 2.28	1.01 (0.99 to 1.04)	1.00	-9.7% to 5.8%
4	0.04 - 0.37	1.07 (1.02 to 1.12)	0.99	-6.8% to 14.7% or 0.01 to 0.02 FLU

aß2GPI IgM:

Sample	Test Range in FLU	Slope (95% CI)	R2	Range of Linearity Deviations
1	10.21 – 102.10	1.07 (1.02 to 1.13)	0.99	-7.7% to 13.5%
2	1.50 – 15.04	1.06 (1.03 to 1.09)	0.99	-5.8% to 12.6%
3	0.17 – 1.75	1.01 (0.97 to 1.05)	0.99	-10.8% to 9.9% or -0.06 FLU
4	0.04 - 0.37	1.06 (0.99 to 0.13)	0.99	-5.6% to 9.7% or 0.02 to 0.03 FLU

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These data demonstrate the linearity of the analytical measuring range (0.10 – 114.68 FLU) of the aCL IgM assay and the analytical measuring range (0.10 – 95.86 FLU) of the aβ2GPI IgM assay, both as part of the Aptiva APS IgM Reagent.

Interference

The interference study was performed according to CLSI EP07-A2, Interference Testing in Clinical Chemistry; Approved Guideline - Second Edition. Six human specimens, a set of three human serum samples (one positive, one around the cutoff and one negative sample) were tested. Endogenous interfering substances (bilirubin, hemoglobin, triglycerides, cholesterol, rheumatoid factor IgM and human IgG) and exogenous substances (ibuprofen, warfarin, prednisone, and acetaminophen) were spiked into each specimen and the resulting samples were assessed in five replicates with the aCL IgG, a82GPI IgG, aCL IgM and a82GPI IgM assays as part of the Aptiva APS IgG and Aptiva APS IgM Reagents assays. Recovery of the unit values was calculated compared to control samples.

No interference was detected for aCL IgG and aß2GPI IgG with bilirubin at 1.0 mg/mL, hemoglobin at 10.0 g/L, triglycerides at 1000.0 mg/dL, cholesterol at 332.5 mg/dL, RF IgM at 153.4 IU/mL, human IgG at 70 mg/mL, ibuprofen at 21.9 mg/dL, warfarin at 7.5 mg/dL, prednisone at 0.0099 mg/mL acetaminophen at 15.6 mg/dL, aspirin at 3 mg/dL, hydroxychloroquine at 0.465 mg/dL, omeprazole at 0.840 mg/dL, simvastatin at 0.168 mg/dL and heparin at 330 units/dL.

Aptiva APS IgG (aCL IgG)		Percent Recovery or FLU Difference
Endogenous Interfering substance	Final Conc.	Negative	Cutoff	Positive
Bilirubin, Conjugated	1.0 mg/mL	99.8%	105.4%	104.1%
Hemoglobin	10.00 g/L	98.8%	106.7%	114.0%
Triglyceride	1000.0 mg/dL	99.5%	94.0%	95.2%
Cholesterol	332.5 mg/dL	0.39 FLU	105.3%	102.5%
RF IgM	153.4 IU/mL	103.8%	108.7%	100.9%
Human IgG	20.0 mg/mL	0.41 FLU	108.9%	101.1%
Exogenous Interfering substance	Final Conc.	Negative	Cutoff	Positive
Ibuprofen	21.9 mg/dL	97.7%	105.0%	97.1%
Warfarin	7.5 mg/dL	95.9%	102.3%	98.8%
Prednisone	0.0099 mg/dL	94.4%	96.4%	99.8%
Acetaminophen	15.6 mg/dL	98.7%	107.8%	101.0%
Aspirin	3.00 mg/dL	95.4%	97.8%	100.8%
Hydroxychloroquine	0.465 mg/dL	98.2%	102.8%	96.6%
Omeprazole	0.840 mg/dL	97.8%	86.6%	90.4%
Simvastatin	0.168 mg/dL	106.4%	104.3%	102.1%
Heparin	330 units/dL	101.5%	113.3%	101.9%

Aptiva APS IgG (aß2GPI IgG)		Percent Recovery or FLU Difference
Endogenous Interfering substance	Final Conc.	Negative	Cutoff	Positive
Bilirubin, Conjugated	1.0 mg/mL	99.5%	107.7%	104.3%

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Page 19 of 28

Aptiva APS IgG (aβ2GPI IgG)		Percent Recovery or FLU Difference
Hemoglobin	10.00 g/L	104.9%	106.7%	111.6%
Triglyceride	1000.0 mg/dL	99.3%	92.1%	94.4%
Cholesterol	332.5 mg/dL	0.28 FLU	102.1%	99.7%
RF IgM	153.4 IU/mL	102.1%	108.8%	100.7%
Human IgG	20.0 mg/mL	0.18 FLU	104.7%	97.4%
Exogenous Interfering substance	Final Conc.	Negative	Cutoff	Positive
Ibuprofen	21.9 mg/dL	101.1%	102.5%	99.2%
Warfarin	7.5 mg/dL	100.7%	99.3%	102.8%
Prednisone	0.0099 mg/dL	97.6%	95.2%	103.7%
Acetaminophen	15.6 mg/dL	87.0%	103.8%	106.4%
Aspirin	3.00 mg/dL	98.3%	98.3%	100.6%
Hydroxychloroquine	0.465 mg/dL	88.3%	97.9%	93.7%
Omeprazole	0.840 mg/dL	88.3%	85.7%	88.6%
Simvastatin	0.168 mg/dL	96.1%	99.5%	98.4%
Heparin	330 units/dL	113.8%	102.0%	102.7%

No interference was detected for aCL IgM and aβ2GPI IgM with bilirubin at 1.0 mg/mL, hemoglobin at 10.0 mg/mL, triglycerides at 1000.0 mg/dL, cholesterol at 332.5 mg/dL, RF IgM at 153.4 IU/mL, human IgG at 70 mg/mL, ibuprofen at 21.9 mg/dL, warfarin at 7.5 mg/dL, prednisone at 0.0099 mg/mL acetaminophen at 15.6 mg/dL, aspirin at 3 mg/dL, hydroxychloroquine at 0.465 mg/dL, omeprazole at 0.840 mg/dL, simvastatin at 0.168 mg/dL and heparin at 330 units/dL.

Aptiva APS IgM (aCL IgM)		Percent Recovery or FLU Difference
Endogenous Interfering substance	Final Conc.	Negative	Cutoff	Positive
Bilirubin, Conjugated	1.0 mg/mL	105.8%	101.7%	102.3%
Hemoglobin	10.00 g/L	109.4%	105.9%	98.0%
Triglyceride	1000.0 mg/dL	97.6%	99.2%	100.9%
Cholesterol	332.5 mg/dL	106.0%	104.4%	101.2%
RF IgM	153.4 IU/mL	98.3%	100.1%	96.5%
Human IgG	70.0 mg/mL	96.0%	98.2%	96.6%
Exogenous Interfering substance	Final Conc.	Negative	Cutoff	Positive
lbuprofen	21.9 mg/dL	99.5%	100.1%	99.7%
Warfarin	7.5 mg/dL	99.4%	97.1%	100.9%
Prednisone	0.0099 mg/dL	99.7%	102.0%	97.3%
Acetaminophen	15.6 mg/dL	98.9%	97.5%	100.5%
Aspirin	3.00 mg/dL	98.5%	103.9%	102.5%
Hydroxychloroquine	0.465 mg/dL	94.1%	98.2%	101.9%
Omeprazole	0.840 mg/dL	118.4%	90.2%	93.3%
Simvastatin	0.168 mg/dL	84.4%	89.7%	98.6%
Heparin	330 units/dL	108.1%	102.6%	114.4%

{23}------------------------------------------------

Aptiva APS IgM (aβ2GPI IgM)		Percent Recovery or FLU Difference
Endogenous Interfering substance	Final Conc.	Negative	Cutoff	Positive
Bilirubin, Conjugated	1.0 mg/mL	104.2%	99.7%	102.6%
Hemoglobin	10.00 g/L	108.8%	108.1%	98.1%
Triglyceride	1000.0 mg/dL	95.4%	103.0%	102.3%
Cholesterol	332.5 mg/dL	102.1%	101.3%	102.7%
RF IgM	153.4 IU/mL	101.9%	97.7%	97.7%
Human IgG	70.0 mg/mL	97.5%	98.8%	97.9%
Exogenous Interfering substance	Final Conc.	Negative	Cutoff	Positive
Ibuprofen	21.9 mg/dL	98.5%	100.0%	100.7%
Warfarin	7.5 mg/dL	95.5%	99.6%	99.7%
Prednisone	0.0099 mg/dL	98.4%	106.0%	96.9%
Acetaminophen	15.6 mg/dL	100.1%	102.3%	101.1%
Aspirin	3.00 mg/dL	96.9%	102.2%	103.6%
Hydroxychloroquine	0.465 mg/dL	95.6%	96.3%	102.4%
Omeprazole	0.840 mg/dL	0.27 FLU	91.8%	97.2%
Simvastatin	0.168 mg/dL	86.1%	93.4%	102.6%
Heparin	330 units/dL	111.5%	109.0%	112.6%

Sample Stability and Handling

For the aCL IgG, aB2GPI IgG, aCL IgM and a82GPI IgM assays, five serum samples were tested. The samples used for this study were achieved by combining high and low antibody levels to yield their desired reactivity. All samples were tested in duplicates for up to 21 days while stored at 2-8°C, up to 49 hours while stored at room temperature (20-26°C), and after repeated freeze/thaw cycles up to 6 cycles. Results were compared to those obtained on control samples (time zero / zero cycles).

All samples fulfilled the acceptance criteria at each time point for each condition. Based on these results, we recommend that samples may be stored up to 48 hours at room temperature, up to 14 days at 2-8°C and can be subjected to up to 5 freeze/thaw cycles.

Reagent Stability

Shelf life

Real-time stability (on-going) and accelerated stability studies were performed to establish the initial claim for shelf life for the Aptiva APS IgG and the Aptiva APS IgM Reagents, accelerated stability studies were performed on three lots of reagents for 5 weeks at 37°C ± 3°C, where one week is equal to six months at 5 ± 3℃.

Each week a new sealed reagent was placed in the incubator, and all reagents were tested at the end of the experiment together with the one that was stored at 5 ± 3℃. The recovery of the measured values was calculated for each time point (compared to those obtained with 5 ± 3℃ stored reagent). All calculations were performed by comparing results of sealed components stored at 5 ± 3℃ (control) to those stored at 37 ± 3℃ (test) for 1, 2, 3, 4, and 5 weeks, where one

{24}------------------------------------------------

week is equal to six months at 5 ± 3℃. Linear regression analysis was performed between recovery values and the number of days.

A shelf-life of nine months was assigned to the Aptiva APS IgG Reagent and seven months was assigned to the Aptiva APS IgM Reagent.

Real time stability

Real-time stability testing has been scheduled on the Aptiva APS IgA Reagent and Aptiva APS IgM Regents, to verify the assigned expiration dating based on accelerated stability studies. Realtime test samples consisted of the following: low negative, high negative, high negative (near the assay cutoff), low positive (near assay cutoff), mid positive, and high positive.

In-use (onboard) stability

Reagent Cartridge

To establish the in-use stability of the Aptiva APS IgG and Aptiva APS IgM reagents, one lot of reagents was tested using 11 samples (with different reactivity levels) for IgG and seven samples (with different reactivity levels) for IgM. The specimens were tested periodically for 33 days for lgG and 37 days for IgM. On day 14 the reagent was recalibrated, and a specific Working Curve was generated. Percent recoveries were calculated compared to the day zero average values, and linear regression analysis was performed by plotting percent recovery against the number of days. The in-use (onboard) stability of the Aptiva APS IgG and Aptiva APS IgM Reagents was set at 28 days, with a 14-day recalibration.

Cut-off, reference range

The following cut-off is used for the aCL IgG, aCL IgM and aβ2GPI IgM assays in the Aptiva APS IgG and Aptiva APS IgM Reagents:

Negative -<5.00 FLU Positive 25.00 FLU

The reference population for establishing the cutoff values for the aCL IgG and aß2GPI IgG assays in the Aptiva APS IgG Reagent consisted of 52 apparently healthy subjects and for the aCL IgM and aß2GPI IgM assays in the Aptiva APS IgM Reagent 54 apparently healthy subjects.

The cut-off values were established in accordance with CLSI EP28-A3c: Defining, Establishing, and Verifying Reference Intervals in the Clinical Laboratory; Approved Guideline - Third Edition. The Analyse-it for Excel software was used to make the calculations. The results was non-normal (Shapiro-Wilk p<0.0001); therefore, the non-parametric percentile method was used.

{25}------------------------------------------------

The cut-off was established based on greater than the 99th percentile of the results obtained on the reference healthy population.

For the Aptiva APS IgG Reagent (including the aCL and aß2GPI assays) based on the distribution of result values of healthy controls and internal APS samples (data not provided), the cutoff was established at 144 MFI for aCL IgG and 256 MFI for a82GPI IgG and was assigned a value of 5.00 FLU. With this cutoff, we ensure that the cutoff value is greater than the 99th percentile on reference healthy population (105 MFI and 71 MFI for aCL IgG and for a82GPI IgG, respectively).

For the Aptiva APS IgM Reagent (including the aCL and aβ2GPI assays) based on the distribution of result values of healthy controls and internal APS samples (data not provided), the cutoff was established at 564 MFI for aCL IgM and 757 MFI for aß2GPI IgM and was assigned a value of 5.00 FLU. With this cutoff, we ensure that the cutoff value is greater than the 99th percentile on reference healthy population (195 MFI for aCL IgM and 233 MFI for aβ2GPI IgM).

Clinical performance characteristics

Clinical sensitivity, specificity

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 APS IgG and Aptiva APS IgM Reagents.

For Aptiva APS IgG, a total of 526 characterized samples were included in this validation set, including 60 patients with primary antiphospholipid syndrome and 62 patients with secondary antiphospholipid syndrome (pAPS and sAPS) and 404 control samples from patients with various types of autoimmune and infectious diseases. All samples were run on the Aptiva APS IgG Reagent. The distribution of the cohort

Patient Group	N=526	aCL IgG No. Positive	aCL IgG % Positive
APS combined	122	66	54.1%
pAPS	60	33	55.0%
sAPS	62	33	53.2%
Controls	404	2	0.5%
Infectious Disease	58	0	0.0%
PREPI	37	1	2.7%
SLE no APS	27	0	0.0%
Systemic sclerosis	12	1	8.3%
Crohn's Disease	29	0	0.0%
Ulcerative Colitis (UC)	27	0	0.0%
Rheumatoid Arthritis	21	0	0.0%
Fetal Loss no APS	15	0	0.0%
Thrombosis no APS	4	0	0.0%
ANCA-associated vasculitis (AAV)	15	0	0.0%
Autoimmune Thyroid	30	0	0.0%
Celiac Disease (CD)	30	0	0.0%

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Page 23 of 28

Patient Group	N=526	aCL IgG% Positive
COVID-19 related thrombosis	20	0.0%
Hematologic malignancies	16	0.0%
Idiopathic thrombocytopenic purpura (ITP)	17	0.0%
Solid tumor malignancies	16	0.0%
Deep vein thrombosis	30	0.0%

Patient Group	N=526	aβ2GPI IgGNo. Positive	aβ2GPI IgG% Positive
APS combined	122	65	53.3%
pAPS	60	32	53.3%
sAPS	62	33	53.2%
Controls	404	4	1.0%
Infectious Disease	58	0	0.0%
PREPI	37	1	2.7%
SLE no APS	27	0	0.0%
Systemic sclerosis	12	1	8.3%
Crohn's Disease	29	0	0.0%
Ulcerative Colitis (UC)	27	0	0.0%
Rheumatoid Arthritis	21	1	4.8%
Fetal Loss no APS	15	0	0.0%
Thrombosis no APS	4	0	0.0%
ANCA-associated vasculitis (AAV)	15	0	0.0%
Autoimmune Thyroid	30	0	0.0%
Celiac Disease (CD)	30	0	0.0%
COVID-19 related thrombosis	20	1	5.0%
Hematologic malignancies	16	0	0.0%
Idiopathic thrombocytopenic purpura (ITP)	17	0	0.0%
Solid tumor malignancies	16	0	0.0%
Deep vein thrombosis	30	0	0.0%

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Clinical sensitivity and specificity for the Aptiva APS IgG (aCL IgG) were analyzed in the table below:

Clinical Analysis (N=526)		Diagnosis
		APS	Controls orNon-APS	Total
Aptiva APSIgG (aCLIgG)	Positive ≥ 5.00	66	2	68
	Negative < 5.00	56	402	458
	Total	122	404	526

Sensitivity	54.1%	(45.3 – 62.7%)
Specificity	99.5%	(98.2 – 99.9%)

Clinical sensitivity and specificity for the Aptiva APS IgG (a82GPI IgG) were analyzed in the table below:

Clinical Analysis (N=526)			Diagnosis
		APS	Controls orNon-APS	Total
Aptiva APS	Positive ≥ 5.00	65	4	69
IgG (aβ2GPI	Negative < 5.00	57	400	457
IgG)	Total	122	404	526

Sensitivity	53.3 %	(44.5-61.9%)
Specificity	99.0%	(97.5-99.6%)

For Aptiva APS IgM, a total of 689 characterized samples were included in this validation set, including 291 samples from APS patients and 398 control samples from patients with various types of autoimmune and infectious diseases. All samples were run on the Aptiva APS IgM Reagent. The distribution of the cohort and the aCL IgM and aβ2GPI IgM positivity rate is in the tables below:

Patient Group	N=689	aCL IgMNo. Positive	aCL IgM% Positive
APS combined	291	80	27.5%
pAPS	219	60	27.4%
sAPS	72	20	27.8%
Controls	398	10	2.5%
Infectious Disease	69	0	0.0%

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Patient Group	N=689	aCL IgMNo. Positive	aCL IgM% Positive
Myositis	20	1	5.0%
Autoimmune Thyroiditis	35	3	8.6%
Celiac Disease (CD)	50	0	0.0%
Ulcerative Colitis (UC)	19	1	5.3%
Rheumatoid Arthritis	13	1	7.7%
Atopic Dermatitis	11	0	0.0%
Fetal Loss no APS	27	0	0.0%
Pre-Eclampsia	17	0	0.0%
Thrombosis no APS	7	1	14.3%
ANCA-associated vasculities	15	0	0.0%
COVID-19 related thrombosis	20	1	5.0%
Hematologic malignancies	16	0	0.0%
Solid tumor malignancies	16	1	6.3%
Idiopathic thrombocytopenic purpura (ITP)	17	0	0.0%
Deep vein thrombosis	30	1	3.3%
Other disease controls	16	0	0.0%

		aẞ2GPI IgM	aẞ2GPI IgM
Patient Group	N=689	No. Positive	% Positive
APS combined	291	72	24.7%
pAPS	219	52	23.7%
sAPS	72	20	27.8%
Controls	398	6	1.5%
Infectious Disease	69	0	0.0%
Myositis	20	0	0.0%
Autoimmune Thyroiditis	35	2	5.7%
Celiac Disease (CD)	50	0	0.0%
Ulcerative Colitis (UC)	19	1	5.3%
Rheumatoid Arthritis	13	1	7.7%
Atopic Dermatitis	11	0	0.0%
Fetal Loss no APS	27	0	0.0%
Pre-Eclampsia	17	0	0.0%
Thrombosis no APS	7	1	14.3%
ANCA-associated vasculities	15	0	0.0%
COVID-19 related thrombosis	20	0	0.0%
Hematologic malignancies	16	0	0.0%
Solid tumor malignancies	16	1	6.3%
Idiopathic thrombocytopenic purpura (ITP)	17	0	0.0%
Deep vein thrombosis	30	0	0.0%
Other disease controls	16	0	0.0%

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Clinical sensitivity and specificity for the Aptiva APS IgM (aCL IgM) were analyzed in the table below:

Clinical Analysis (N=689)		Diagnosis
		APS	Controls orNon-APS	Total
Aptiva APSIgM (aCL IgM)	Positive ≥ 5.00	80	10	90
	Negative < 5.00	211	388	599
	Total	291	398	689

Sensitivity	27.5% (22.7 – 32.9%)
Specificity	97.5% (95.4 – 98.6%)

Clinical sensitivity and specificity for the Aptiva APS IgM (a82GPI IgM) a were analyzed in the table below:

Clinical Analysis (N=689)		Diagnosis
	APS	Controls orNon-APS	Total
Aptiva APSIgM (aβ2GPIIgM)	Positive ≥ 5.00	72	6	78
	Negative < 5.00	219	392	611
	Total	291	398	689

Sensitivity	24.7%	(20.1 – 30.0%)
Specificity	98.5%	(96.8 – 99.3%)

Expected values

The expected value in the normal population is "negative". A panel of 200 apparently healthy blood donors (106 females/94 males, ages 18 to 70 years, with an average and median age of 37) were tested on the Aptiva APS IgG and Aptiva APS IgM Reagent.

For Aptiva APS IgG, the aCL IgG with a cut-off of 5.00 FLU, no samples were positive, with a mean concentration of 0.29 FLU, and values ranging from 0.29 to 0.42 FLU. For aβ2GPI IgG, with a cut-off of 5.00 FLU, no samples were positive, with a mean concentration of 0.21 FLU, and values ranging from 0.21 to 0.47 FLU.

For Aptiva APS IgM, the aCL IgM with a cut-off of 5.00 FLU, no samples were positive, with a mean concentration of 0.14 FLU, and values ranging from 0.10 to 2.54 FLU. For aβ2GPI IgM, with a cut-off of 5.00 FLU, no samples were positive, with a mean concentration of 0.15 FLU, and values ranging from 0.10 to 3.11 FLU.

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Comparison with predicate device

For Aptiva APS IqG, samples for method comparison analysis included all samples analysis included all samples from the clinical validation study that display results within the analytical measuring range of the assay and its predicate device.

QUANTA Flash aCL IgG Percent Agreement Method Comparison (N=202) Positive Negative Total (95% Confidence) Positive ≥ 5.00 31 7 38 PPA: 81.6% (66.6-90.8%) Aptiva Negative < 5.00 7 157 164 NPA: 95.7% (91.5-97.9%) APS IgG (aCL IgG) Total 38 164 202 TPA: 93.1% (88.7-95.8%)

Method comparison of the Aptiva APS IgG (aCL IgG) with the predicate device:

NPA: Negative Percent Agreement; PPA: Positive Percent Agreement; TPA: Total Percent Agreement

Method comparison of the Aptiva APS IgG (aß2GPI IgG) with the predicate device:

Method Comparison (N=108)		QUANTA LiteBeta 2GP1 IgG ELISA			Percent Agreement
		Positive	Negative	Total	(95% Confidence)
AptivaAPS IgG(aβ2GPIIgG)	Positive ≥ 5.00	44	6	50	PPA: 88.0% (76.2-94.4%)
	Negative < 5.00	6	52	58	NPA: 89.7% (79.2-95.2%)
	Total	50	58	108	TPA: 88.9% (81.6-93.5%)

NPA: Negative Percent Agreement; PPA: Positive Percent Agreement; TPA: Total Percent Agreement

For Aptiva APS IgM, samples for method comparison analysis included all samples from the clinical validation study that display results within the analytical measuring range of the assay and its predicate device These samples were tested on both the Aptiva APS IgM Reagent and on their predicate QUANTA Flash aCL IgM and QUANTA Flash β2GP1 IgM Reagents.

Method comparison of the Aptiva APS IgM (aCL IgM) with the predicate device:

Method Comparison (N=422)		QUANTA Flash aCL IgM			Percent Agreement
		Positive	Negative	Total	(95% Confidence)
AptivaAPS IgM(aCL IgM)	Positive ≥ 5.00	40	37	77	PPA: 87.0% (74.3-93.9%)
	Negative < 5.00	6	339	345	NPA: 90.2% (86.7-92.8%)
	Total	46	376	422	TPA: 89.8% (86.6-92.3%)

NPA: Negative Percent Agreement; PPA: Positive Percent Agreement; TPA: Total Percent Agreement

Method comparison of the Aptiva APS IgM (aß2GPI IgM) with the predicate device:

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Method Comparison (N=244)		QUANTA Flash β2GPI IgM			Percent Agreement
		Positive	Negative	Total	(95% Confidence)
AptivaAPS IgM(aβ2GPIIgM)	Positive ≥ 5.00	24	34	58	PPA: 88.9% (71.9-96.1%)
	Negative < 5.00	3	183	186	NPA: 84.3% (78.9-88.6%)
	Total	27	217	244	TPA: 84.8% (79.8-88.8%)

NPA: Negative Percent Agreement; PPA: Positive Percent Agreement; TPA: Total Percent Agreement

Regulation Number and Section

§ 866.5660 Multiple autoantibodies immunological test system.

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