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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.

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.

AI/ML Overview

This document describes the analytical and clinical performance characteristics of the Aptiva CTD Essential Reagent, a multiplexed immunoassay system, and its comparison to predicate devices.

Here's an analysis of the acceptance criteria and study proving device performance:

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

Acceptance Criteria Category	Specific Acceptance Criteria	Reported Device Performance (Summary)
Precision	Total %CV: < 12%	All samples for all analytes met this criterion, with most Total %CV values well below 12%.
Reproducibility (Between-Site)	Reproducibility Between-Site %CV: < 12%	Most samples for all analytes met this, with a few exceptions slightly exceeding (e.g., RNP Sample 1 at 13.6%, Sm Sample 6 at 13.3%, Ro52 Sample 6 at 12.7%, Ro60 Sample 2 at 13.4%, Jo-1 Sample 4 at 13.6%, Jo-1 Sample 5 at 13.9%, Centromere Sample 5 at 12.9%, Ribo-P Sample 1 at 12.8%). However, the majority fell within the acceptance range.
Reproducibility (Between-Lot)	Reproducibility Between-Lot %CV: < 12%	All analytes met this criterion, with a few samples slightly exceeding it (dsDNA Sample 2 at 13.1%, dsDNA Sample 6 at 12.8%, Sm Sample 1 at 15.3%, Sm Sample 2 at 12.6%, Ro52 Sample 1 at 11.9%, Ro52 Sample 6 at 12.4%, Ro60 Sample 1 at 16.6%, SS-B Sample 1 at 11.8%, SS-B Sample 2 at 13.1%, Scl-70 Sample 1 at 14.1%, Scl-70 Sample 2 at 12.9%, Centromere Sample 1 at 18.6%, Centromere Sample 4 at 12.8%). The majority were within limits.
Linearity	Allowable deviation from linearity: +/- 15% or +/- 0.75 FLU (+/- 5.25 IU/mL for dsDNA)	All analytes fulfilled the acceptance criteria for linearity across their respective AMRs.
Linearity	Slope: 0.9-1.1	All analytes fulfilled the acceptance criteria.
Linearity	R²: > 0.95	All analytes fulfilled the acceptance criteria.
Interference	Recovery of unit values: 85% - 115% or ± 15% of the cut-off (±0.75 FLU or ±4.05 IU/mL for dsDNA)	The device did not show interference with various endogenous (bilirubin, hemoglobin, triglycerides, cholesterol, RF IgM, human IgG) and exogenous (ibuprofen, acetaminophen, prednisone, warfarin, diltiazem, azathioprine, sildenafil, cyclophosphamide, mycophenolate mofetil, heparin) substances at tested concentrations.
Sample Stability and Handling	Percent recovery: 85-115% for positive samples, 80-120% for negative samples	All samples fulfilled the acceptance criteria for storage up to 24 hours at room temperature, up to 14 days at 2-8°C, and up to 4 freeze/thaw cycles.
Reagent Shelf Life (Accelerated Stability)	Lower and upper 95% CI interval of the regression line: between 80% and 120% recovery at day 28 (week 4)	All components tested fulfilled the acceptance criteria, leading to an initial two-year expiration dating.
In-use (Onboard) Stability	Stability claim established at actual measurement day preceding 95% CI of regression line reaching 85% or 115% recovery OR 2% of recovery data is <75% or ≥125% recovery (whichever is fulfilled first).	All data obtained fulfilled the acceptance criteria, and the in-use stability was set at 36 days with an 18-day recalibration.
Method Comparison (Agreement vs. predicate devices)	No explicit numerical acceptance criteria for agreement percentages are provided in the excerpt for method comparison, but the reported percentages indicate the level of agreement.	NPA, PPA, and TPA values are consistently high for all analytes when compared to predicate devices (generally in the high 80s and 90s, with some PPA nearing 100%), demonstrating substantial agreement.

2. Sample size used for the test set and the data provenance

Clinical Validation Study (Test Set for Clinical Sensitivity and Specificity):
- Sample Size: 1269 samples in total. This included 141 patients with Sjögren's syndrome (SjS), 230 with systemic lupus erythematosus (SLE), 217 with systemic sclerosis (SSc), 91 with mixed connective tissue disease (MCTD), 200 with idiopathic inflammatory myopathy (IIM), and 390 control samples from patients with various types of autoimmune and infectious diseases.
- Data Provenance: The document does not explicitly state the country of origin. It indicates that the samples were "from patients," implying clinical samples. The study describes them as a "cohort of characterized samples," and it's mentioned that these were "none of which were used for establishing the reference range." This typically suggests retrospective collection for validation purposes.
Method Comparison Study (Test Set for Agreement with Predicate):
- Sample Size: The sample sizes vary by analyte due to comparisons against different predicate devices. Examples include:
  - dsDNA: 428 samples
  - RNP: 480 samples
  - Sm: 418 samples
  - Ro52: 1028 samples
  - Ro60: 551 samples
  - SS-B: 550 samples
  - Scl-70: 435 samples
  - Jo-1: 416 samples
  - Centromere: 449 samples
  - Ribo-P: 387 samples
- Data Provenance: "Samples for the method comparison analysis included the samples from the clinical validation study." Therefore, the provenance is the same as the clinical validation study: clinical samples, likely retrospectively collected, and country of origin not specified.

3. Number of experts used to establish the ground truth for the test set and the qualifications of those experts

The document states that the ground truth for the clinical validation samples (test set) was established using a "cohort of characterized samples" from patients with specific autoimmune diseases (SjS, SLE, SSc, MCTD, IIM) and control samples. It does not specify:

The number of experts
The qualifications of those experts (e.g., radiologist with 10 years of experience)
The method by which the characterization/diagnosis was made.

The characterization is implied to be clinical diagnosis (e.g., "patients with systemic lupus erythematosus"), which generally would involve medical professionals, but specifics are missing.

4. Adjudication method for the test set

The document does not describe any formal adjudication method (like 2+1 or 3+1) for establishing the ground truth of the clinical validation test set. The samples are referred to as "characterized samples," suggesting that their disease status was determined prior to the study by 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 such MRMC comparative effectiveness study is described in the provided text. The device is an immunoassay (a diagnostic test for autoantibodies), not an AI-powered image analysis tool that assists human readers. Therefore, the concept of "human readers improve with AI vs without AI assistance" is not applicable here.

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

Yes, a standalone performance evaluation was done. The entire document focuses on the performance of the Aptiva CTD Essential Reagent (the immunoassay system) itself. The reported sensitivity, specificity, precision, reproducibility, linearity, interference, and stability data are all measures of the device's performance in isolation, without an explicit human-in-the-loop component beyond standard laboratory operation.

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

For the clinical validation study, the ground truth was based on the clinical diagnosis of patients belonging to specific disease groups (e.g., Systemic Lupus Erythematosus, Sjögren's Syndrome, Systemic Sclerosis, etc.) and control groups with other autoimmune or infectious diseases. This implies that the ground truth was established by medical professionals through clinical findings and other laboratory tests, representing a form of "expert diagnosis/characterization" rather than specific pathology or outcomes data.

For establishing the cut-offs, for several analytes (dsDNA, Sm, Ribo-P, RNP, Ro60, SS-B, Scl-70, Centromere, Ro52, Jo-1), the cut-off was established based on the 95th percentile of results from "reference subjects" (presumably healthy or non-diseased controls) and results from patients with the relevant disease (e.g., SLE patients for dsDNA) "to ensure optimal differentiation." This is a statistical approach tied to distinguishing patient populations.

8. The sample size for the training set

The document does not explicitly use the term "training set" in the context of machine learning. However, for establishing the cut-offs and calibrators for the assays:

Reference population for cut-offs: 120 samples from reference subjects (various autoimmune and infectious diseases, but generally considered "controls" for the target diseases), plus specific numbers of diseased patient samples (e.g., 13 SLE samples for dsDNA, 7 SLE and 5 MCTD samples for RNP, etc.). These samples were used to define the diagnostic thresholds.
Master Curves/Calibrators: These were generated "in-house" using "in-house Master Curve Standards" with assigned values. The document states that these standards were run "multiple times" to create the 4-parameter logistic curve for each analyte. The exact number of runs or distinct samples used to define these master curves is not provided with an aggregate number.

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

For the reference population used to establish cut-offs: The ground truth was established by clinical diagnosis of the subjects ("patients with celiac disease," "patients with systemic lupus erythematosus," etc.). These diagnoses serve as the reference standard for defining what constitutes a positive or negative result for the assay.
For the Master Curve Standards (calibrators): These standards have "assigned values" (e.g., IU/mL or FLU), indicating that their "ground truth" (concentration) was determined by an internal, established method at Inova Diagnostics. This is a common practice for calibrators in immunoassay development.

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