Aptiva CTD Essential Reagent

K213403 · Inova Diagnostics, Inc. · LLL · Sep 29, 2023 · Immunology

Device Facts

• Record ID: K213403
• Device Name: Aptiva CTD Essential Reagent
• Applicant: Inova Diagnostics, Inc.
• Product Code: LLL · Immunology
• Decision Date: Sep 29, 2023
• Decision: SESE
• Submission Type: Traditional
• Regulation: 21 CFR 866.5100
• Device Class: Class 2

AI Performance

Output	Algorithm	Acceptance	Observed	Dev DS	Dev Readers	Test DS	Test Readers
dsDNA IgG autoantibodies	—	Total %CV: < 12%	Within-laboratory precision CV: 7.7% - 11.8%	—	—	Clinical validation study: 1269 samples	—
RNP IgG autoantibodies	—	Total %CV: < 12%	Within-laboratory precision CV: 7.8% - 11.6%	—	—	Clinical validation study: 1178 samples (SLE vs Controls) and 1039 samples (MCTD vs Controls)	—
Sm IgG autoantibodies	—	Total %CV: < 12%	Within-laboratory precision CV: 6.7% - 11.6%	—	—	Clinical validation study: 1269 samples	—
Ro52 IgG autoantibodies	—	Total %CV: < 12%	Within-laboratory precision CV: 6.4% - 10.8%	—	—	Clinical validation study: 788 samples	—
Ro60 IgG autoantibodies	—	Total %CV: < 12%	Within-laboratory precision CV: 6.9% - 10.7%	—	—	Clinical validation study: 1128 samples (SLE vs Controls) and 1039 samples (SjS vs Controls)	—
SS-B IgG autoantibodies	—	Total %CV: < 12%	Within-laboratory precision CV: 7.0% - 11.7%	—	—	Clinical validation study: 1128 samples (SLE vs Controls) and 1039 samples (SjS vs Controls)	—
Scl-70 IgG autoantibodies	—	Total %CV: < 12%	Within-laboratory precision CV: 6.7% - 11.5%	—	—	Clinical validation study: 1269 samples	—
Jo-1 IgG autoantibodies	—	Total %CV: < 12%	Within-laboratory precision CV: 8.1% - 10.3%	—	—	Clinical validation study: 1269 samples	—
Centromere IgG autoantibodies	—	Total %CV: < 12%	Within-laboratory precision CV: 7.4% - 11.1%	—	—	Clinical validation study: 1269 samples	—
Ribo-P IgG autoantibodies	—	Total %CV: < 12%	Within-laboratory precision CV: 8.1% - 11.1%	—	—	Clinical validation study: 1269 samples	—

Indications for Use

The Aptiva CTD Essential Reagent consists of 10 multiplexed immunoassays utilizing particle-based multi-analyte technology for the quantitative determination of IgG autoantibodies against dsDNA, and semi-quantitative determination of IgG autoantibodies against RNP, Sm, Ro52, Ro60, SS-B, Scl-70, Jo-1, centromere, and Ribo-P in human serum: The presence of dsDNA antibodies, in conjunction with clinical findings and other laboratory tests, is an aid in the diagnosis of systemic lupus erythematosus. The presence of RNP antibodies, in conjunction with clinical findings and other laboratory tests, is an aid in the diagnosis of mixed connective tissue disease and systemic lupus erythematosus. The presence of Sm antibodies, in conjunction with clinical findings and other laboratory tests, is an 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 Story

Aptiva CTD Essential Reagent is a multiplexed immunoassay kit for the Inova Diagnostics Aptiva System; uses particle-based multi-analyte technology (PMAT) to detect 10 specific IgG autoantibodies in human serum. Patient samples are diluted and incubated with microparticles coated with specific autoantigens; PE-conjugated anti-human IgG tracer is added. The system uses a CCD camera to image microparticles, measuring median fluorescent intensity (MFI) proportional to bound IgG. Results are calculated via lot-specific master curves calibrated by the instrument. Output is quantitative (IU/mL) for dsDNA and semi-quantitative (FLU) for others. Used in clinical laboratories to aid in diagnosing connective tissue diseases; results are interpreted by clinicians alongside clinical findings.

Clinical Evidence

Clinical validation study included 1269 samples from patients with Sjögren's syndrome, SLE, systemic sclerosis, MCTD, IIM, and various controls. Sensitivity and specificity were calculated for each analyte. For example, dsDNA sensitivity 50.4%, specificity 90.1%; Sm sensitivity 10.4%, specificity 99.6%; Centromere sensitivity 47.0%, specificity 97.0%. Performance metrics demonstrate clinical utility as an aid in diagnosis.

Technological Characteristics

Multiplexed particle-based multi-analyte technology (PMAT). Uses 11 paramagnetic microparticle populations (10 analytes + 1 IgG control). Detection via phycoerythrin (PE) tracer and CCD camera imaging. Automated system with RFID-based lot-specific calibration. Software uses 4-parameter logistic (4PL) regression for quantitation.

Indications for Use

Indicated for quantitative (dsDNA) or semi-quantitative (RNP, Sm, Ro52, Ro60, SS-B, Scl-70, Jo-1, centromere, Ribo-P) detection of IgG autoantibodies in human serum to aid in the diagnosis of systemic lupus erythematosus, mixed connective tissue disease, Sjögren's syndrome, systemic sclerosis, and idiopathic inflammatory myositis. For prescription use only.

Regulatory Classification

Identification

An antinuclear antibody immunological test system is a device that consists of the reagents used to measure by immunochemical techniques the autoimmune antibodies in serum, other body fluids, and tissues that react with cellular nuclear constituents (molecules present in the nucleus of a cell, such as ribonucleic acid, deoxyribonucleic acid, or nuclear proteins). The measurements aid in the diagnosis of systemic lupus erythematosus (a multisystem autoimmune disease in which antibodies attack the victim's own tissues), hepatitis (a liver disease), rheumatoid arthritis, Sjögren's syndrome (arthritis with inflammation of the eye, eyelid, and salivary glands), and systemic sclerosis (chronic hardening and shrinking of many body tissues).

Predicate Devices

• QUANTA Flash® dsDNA IgG (K152013)
• QUANTA Flash® RNP (K123593)
• Orgentec Sm ELISA (K954830)
• QUANTA Flash® Ro52 (K141655)
• QUANTA Flash® Ro60 (K141328)
• QUANTA Flash® SS-B (K141210)
• QUANTA Flash® Scl-70 (K152635)
• QUANTA Flash® Jo-1 (K151429)
• QUANTA Flash® Centromere (K123880)
• QUANTA Lite® Ribo-P (K981237)

Submission Summary (Full Text)

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FDA

U.S. FOOD & DRUG

ADMINISTRATION

### 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION
DECISION SUMMARY

ASSAY ONLY

### I Background Information:

A 510(k) Number

K213403

B Applicant

Inova Diagnostics, Inc.

C Proprietary and Established Names

Aptiva CTD Essential Reagent

D Regulatory Information

| Product Code(s) | Classification | Regulation Section | Panel |
| --- | --- | --- | --- |
| LJM, LKO, LKP, LLL, LSW, MQA, OBE | Class II | 21 CFR 866.5100 - Antinuclear antibody immunological test system | IM - Immunology |

### II Submission/Device Overview:

A Purpose for Submission:

New Device

B Measurand:

Human anti-dsDNA, anti-RNP, anti-Sm, anti-Ro60, anti-Ro52, anti-SS-B, anti-Scl-70, anti-Jo-1, anti-Centromere, and anti-Ribo-P IgG autoantibodies.

For ease of reference, the aforementioned autoantibody measurands are referred to in this decision summary by their cognate autoantigens.

Food and Drug Administration

10903 New Hampshire Avenue

Silver Spring, MD 20993-0002

www.fda.gov

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| Analyte | Description | Abbreviated Term | Other Names |
| --- | --- | --- | --- |
| Anti-dsDNA | Anti-double-stranded deoxyribonucleic acid IgG autoantibodies | dsDNA | |
| Anti-RNP | Anti-ribonucleoprotein IgG autoantibodies | RNP | Anti-U1RNP |
| Anti-Sm | Anti-Smith antigen IgG autoantibodies | Sm | Anti-Smith |
| Anti-Ro60 | Anti-Ro 60kDa subunit autoantibodies | Ro60 | Anti-SS-A2 Anti-SSA (Anti-Sjögren's Syndrome A) |
| Anti-Ro52 | Anti-Ro 52kDa subunit autoantibodies | Ro52 | Anti-SSA (Anti-Sjögren's Syndrome A) |
| Anti-SS-B | Anti-SS-B (Anti-Sjögren's Syndrome B) autoantibodies | SS-B | Anti-La |
| Anti-Scl-70 | Anti-Scl 70 Antigen autoantibodies | Scl-70 | Anti-DNA Topoisomerase 1 |
| Anti-Jo-1 | Anti-Jo 1 Antigen autoantibodies | Jo-1 | Anti-Histidyl-tRNA Synthetase |
| Anti-Centromere | Anti-Centromere autoantibodies | Centromere | |
| Anti-Ribo-P | Anti-ribosomal P body IgG autoantibodies | Ribo-P | |

### C Type of Test:

Quantitative, Semi-quantitative, Particle-based multi-analyte assay

### III Intended Use/Indications for Use:

### A Intended Use(s):

See Indications for Use below.

### B Indication(s) for Use:

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

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

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- The presence of RNP antibodies, in conjunction with clinical findings and other laboratory tests, is an aid in the diagnosis of mixed connective tissue disease and systemic lupus erythematosus.
- The presence of Sm antibodies, in conjunction with clinical findings and other laboratory tests, is an aid in the diagnosis of systemic lupus erythematosus.
- The presence of Ro52 antibodies, in conjunction with clinical findings and other laboratory tests, is an aid in the diagnosis of systemic lupus erythematosus, Sjögren's syndrome, systemic sclerosis, and idiopathic inflammatory myositis.
- The presence of Ro60 antibodies, in conjunction with clinical findings and other laboratory tests, is an aid in the diagnosis of systemic lupus erythematosus and Sjögren's syndrome.
- The presence of SS-B antibodies, in conjunction with clinical findings and other laboratory tests, is an aid in the diagnosis of systemic lupus erythematosus and Sjögren's syndrome.
- The presence of Scl-70 antibodies, in conjunction with clinical findings and other laboratory tests, is an aid in the diagnosis of systemic sclerosis.
- The presence of Jo-1 antibodies, in conjunction with clinical findings and other laboratory tests, is an aid in the diagnosis of idiopathic inflammatory myositis.
- The presence of centromere antibodies, in conjunction with clinical findings and other laboratory tests, is an aid in the diagnosis of systemic sclerosis.
- The presence of Ribo-P antibodies, in conjunction with clinical findings and other laboratory tests, is an aid in the diagnosis of systemic lupus erythematosus.

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

# C Special Conditions for Use Statement(s):

Rx - For Prescription Use Only

# D Special Instrument Requirements:

Inova Diagnostics Aptiva System

# IV Device/System Characteristics:

# A Device Description:

The Aptiva CTD Essential Reagent consists of 10 multiplexed immunoassays that utilize particle-based multi-analyte technology (PMAT) in a cartridge format. Each Aptiva CTD Essential reagent kit contains the following reagents for 250 determinations:

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

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

- The Aptiva CTD Essential Calibrators includes six calibrators. The calibrators contain human antibodies to dsDNA, RNP, Sm, Ro60, Ro52, SS-B, Scl-70, Jo-1, Centromere, and Ribo-P in stabilizers and preservatives.
- The Aptiva CTD Essential Controls includes two controls. The controls contain human antibodies to dsDNA, RNP, Sm, Ro60, Ro52, SS-B, Scl-70, Jo-1, Centromere, and Ribo-P in stabilizers and preservatives.

### B Principle of Operation:

The Aptiva CTD Essential reagent utilizes particle based multi-analyte technology (PMAT). Ten unique populations of microparticles coated with dsDNA, RNP, Sm, Ro60, Ro52, SS-B, Scl-70, Jo-1, Centromere or Ribo-P autoantigens, along with 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, the reagent tube seals are pierced, and the reagent cartridge is then loaded onto the Aptiva Multi-Analyte Instrument.

The Aptiva System dilutes the patient sample 1:44.4 fold with Aptiva system rinse by the instrument in a disposable cuvette and a small amount of the diluted sample is combined with reagent into a second cuvette. The mixture is incubated at 37°C. After a series of wash cycles, phycoerythrin-conjugated polyclonal anti-human IgG (known as PE Tracer IgG) is 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 Aptiva system fluid.

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 microparticle regions and determine the amount of conjugate on the microparticles. A control microparticle 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, each of the 10 assays in the Aptiva CTD Essential Reagent 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 calibration process utilizes the six 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 Fluorescent Light Units (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.

Based on the defined cut-off value for each analyte, the test results are reported for each sample as “positive” or “negative” with a test value in FLU for the semi-quantitative assays or in IU/mL for quantitative dsDNA assay.

### V Substantial Equivalence Information:

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# A Predicate Device Names and 510k Numbers

| Predicate Name | 510(k) Number |
| --- | --- |
| QUANTA Flash dsDNA | K152013 |
| QUANTA Flash RNP | K123593 |
| Orgentec Sm ELISA | K954830 |
| QUANTA Flash Ro52 | K141655 |
| QUANTA Flash Ro60 | K141328 |
| QUANTA Flash SS-B | K141210 |
| QUANTA Flash Scl-70 | K152635 |
| QUANTA Flash Jo-1 | K151429 |
| QUANTA Flash Centromere | K123880 |
| QUANTA Lite Ribo-P | K981237 |

# B Comparison with Predicate(s):

| Device & Predicate Device(s): | K213403 (Device) | K152013 (Predicate) |
| --- | --- | --- |
| Device Trade Name | Aptiva CTD Essential Reagent (dsDNA) | QUANTA Flash dsDNA |
| General Device Characteristic Similarities | | |
| Intended Use/ Indications For Use | The Aptiva CTD Essential Reagent consists of 10 multiplexed immunoassays utilizing particle-based multi-analyte technology for the quantitative determination of IgG autoantibodies against dsDNA, and semi-quantitative determination of IgG autoantibodies against RNP, Sm, Ro52, Ro60, SS-B, Scl-70, Jo-1, centromere, and Ribo-P in human serum: ... • The presence of dsDNA antibodies, in conjunction with clinical findings and other laboratory tests, is an aid in the diagnosis of systemic lupus erythematosus ... The individual assays included in the Aptiva CTD Essential Reagent are intended for use with the Inova Diagnostics Aptiva System. | QUANTA Flash dsDNA is a chemiluminescent immunoassay for the quantitative determination of IgG anti-double stranded deoxyribonucleic acid (dsDNA) antibodies in human serum. The presence of anti-dsDNA antibodies, in conjunction with clinical findings and other laboratory tests, is an aid in the diagnosis of Systemic Lupus Erythematosus. |
| Assay Methodology | Solid phase immunoassay | Same |

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| Device & Predicate Device(s): | K213403 (Device) | K152013 (Predicate) |
| --- | --- | --- |
| Antigen | Synthetic DNA | Same |
| Sample Matrix | Human serum | Same |
| Solid Phase | Paramagnetic microparticles | Same |
| Units | International Units (IU/mL) | Same |
| Cut-off | 27.00 IU/mL – 35.00 IU/mL | Same |
| Controls | Two dsDNA controls with lot specific values assigned. | Same |
| **General Device Characteristic Differences** | | |
| Detection Principle | Fluorescent immunoassay | Chemiluminescent immunoassay |
| Conjugate | Phycoerythrin conjugated polyclonal anti-human IgG antibody | Isoluminol conjugated monoclonal anti-human IgG antibody |
| Analytical Measuring Range | 2.30 IU/mL – 814.10 IU/mL | 9.8 IU/mL – 666.9 IU/mL |
| Calibration | Lot specific Master Curve + 6 calibrators (sold separately) | Lot specific Master Curve + 2 calibrators (sold separately) |

| Device & Predicate Device(s): | K213403 (Device) | K123593 (Predicate) |
| --- | --- | --- |
| Device Trade Name | Aptiva CTD Essential Reagent (RNP) | QUANTA Flash RNP |
| **General Device Characteristic Similarities** | | |
| Intended Use/ Indications For Use | The Aptiva CTD Essential Reagent consists of 10 multiplexed immunoassays utilizing particle-based multi-analyte technology for the quantitative determination of IgG autoantibodies against dsDNA, and semi-quantitative determination of IgG autoantibodies against RNP, Sm, Ro52, Ro60, SS-B, Scl-70, Jo-1, centromere, and Ribo-P in human serum: ... • The presence of 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 individual assays included in the Aptiva CTD Essential Reagent | The QUANTA Flash RNP is a chemiluminescent immunoassay for the semi-quantitative determination of IgG anti-ribonucleoprotein (RNP) antibodies in human serum. The presence of anti-RNP antibodies, in conjunction with clinical findings and other laboratory tests, can aid in the diagnosis of Systemic Lupus Erythematosus (SLE) and Mixed Connective Tissue Disease (MCTD). |

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| **Device & Predicate Device(s):** | **K213403 (Device)** | **K123593 (Predicate)** |
| --- | --- | --- |
| | are intended for use with the Inova Diagnostics Aptiva System. | |
| Assay Methodology | Solid phase immunoassay | Same |
| Antigen | Native RNP | Same |
| Sample Matrix | Human serum | Same |
| Solid Phase | Paramagnetic microparticles | Same |
| Controls | Two RNP controls with lot specific values assigned. | Same |
| **General Device Characteristic Differences** | | |
| Detection Principle | Fluorescent immunoassay | Chemiluminescent immunoassay |
| Conjugate | Phycoerythrin conjugated polyclonal anti-human IgG antibody | Isoluminol conjugated monoclonal anti-human IgG antibody |
| Units | Fluorescent light units (FLU) | Chemiluminescent units (CU) |
| Cut-off | 5.00 FLU | 20.00 CU |
| Analytical Measuring Range | 0.50 FLU – 181.99 FLU | 3.5 CU – 643.8 CU |
| Calibration | Lot specific Master Curve + 6 calibrators (sold separately) | Lot specific Master Curve + 2 calibrators (sold separately) |

| **Device & Predicate Device(s):** | **K213403 (Device)** | **K954830 (Predicate)** |
| --- | --- | --- |
| Device Trade Name | Aptiva CTD Essential Reagent (Sm) | Orgentec anti-Sm |
| **General Device Characteristic Similarities** | | |
| Intended Use/ Indications For Use | The Aptiva CTD Essential Reagent consists of 10 multiplexed immunoassays utilizing particle-based multi-analyte technology for the quantitative determination of IgG autoantibodies against dsDNA, and semi-quantitative determination of IgG autoantibodies against RNP, Sm, Ro52, Ro60, SS-B, Scl-70, Jo-1, centromere, and Ribo-P in human serum: ... • The presence of Sm 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 | Anti-Sm is an ELISA test system for the quantitative measurement of IgG class autoantibodies against Sm in human serum or plasma. This product is intended for professional in vitro diagnostic use only. The detection of autoantibodies against Sm proteins is a component of the multi-parametric ACR criteria for the diagnosis of systemic lupus erythematosus (SLE). The detection of Sm antibodies serves as a prognostic marker for SLE, there is a relationship between the appearance of Sm antibodies and severe organ manifestations of the disease. Evaluation of a test result should always take into account all clinical and |

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| **Device & Predicate Device(s):** | **K213403 (Device)** | **K954830 (Predicate)** |
| --- | --- | --- |
| | Diagnostics Aptiva System. | laboratory diagnostic findings. |
| Assay Methodology | Solid phase immunoassay | Same |
| Antigen | Synthetic Sm peptide | Same |
| Controls | Two Sm controls with lot specific values assigned. | Same |
| **General Device Characteristic Differences** | | |
| Detection Principle | Fluorescent immunoassay | Chromogenic immunoassay |
| Conjugate | Phycoerythrin conjugated polyclonal anti-human IgG antibody | HRP conjugated anti-human IgG antibody |
| Sample Matrix | Human serum | Human Serum or Plasma |
| Solid Phase | Paramagnetic microparticles | ELISA |
| Units | Fluorescent light units (FLU) | Units (U/mL) |
| Cut-off | 5.00 FLU | 25.00 U/mL |
| Analytical Measuring Range | 0.25 FLU – 256.00 FLU | 1 U/mL – 200.0 U/mL |
| Calibration | Lot specific Master Curve + 6 calibrators (sold separately) | Calibration curve using 6 calibrators (included) |

| **Device & Predicate Device(s):** | **K213403 (Device)** | **K141655 (Predicate)** |
| --- | --- | --- |
| Device Trade Name | Aptiva CTD Essential Reagent (Ro52) | QUANTA Flash Ro52 |
| **General Device Characteristic Similarities** | | |
| Intended Use/ Indications For Use | The Aptiva CTD Essential Reagent consists of 10 multiplexed immunoassays utilizing particle-based multi-analyte technology for the quantitative determination of IgG autoantibodies against dsDNA, and semi-quantitative determination of IgG autoantibodies against RNP, Sm, Ro52, Ro60, SS-B, Scl-70, Jo-1, centromere, and Ribo-P in human serum: ... • The presence of Ro52 antibodies, in conjunction with clinical findings and other laboratory tests, is an aid in the diagnosis of systemic lupus erythematosus, Sjögren's syndrome, systemic sclerosis, and idiopathic inflammatory myositis. ... The individual assays included in the Aptiva CTD Essential Reagent | QUANTA Flash Ro52 is a chemiluminescent immunoassay for the semi-quantitative determination of IgG anti-Ro52 autoantibodies in human serum. The presence of anti-Ro52 autoantibodies, in conjunction with clinical findings and other laboratory tests, is an aid in the diagnosis of Systemic Lupus Erythematosus, Sjögren's Syndrome, Systemic Sclerosis, Idiopathic Inflammatory Myopathies. |

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| **Device & Predicate Device(s):** | **K213403 (Device)** | **K141655 (Predicate)** |
| --- | --- | --- |
| | are intended for use with the Inova Diagnostics Aptiva System. | |
| Assay Methodology | Solid phase immunoassay | Same |
| Antigen | Recombinant Ro52 | Same |
| Sample Matrix | Human serum | Same |
| Solid Phase | Paramagnetic microparticles | Same |
| Controls | Two Ro52 controls with lot specific values assigned. | Same |
| **General Device Characteristic Differences** | | |
| Detection Principle | Fluorescent immunoassay | Chemiluminescent immunoassay |
| Conjugate | Phycoerythrin conjugated polyclonal anti-human IgG antibody | Isoluminol conjugated monoclonal anti-human IgG antibody |
| Units | Fluorescent light units (FLU) | Chemiluminescent units (CU) |
| Cut-off | 5.00 FLU | 20.00 CU |
| Analytical Measuring Range | 0.25 FLU – 196.27 FLU | 2.3 CU – 1685.3 CU |
| Calibration | Lot specific Master Curve + 6 calibrators (sold separately) | Lot specific Master Curve + 2 calibrators (sold separately) |

| **Device & Predicate Device(s):** | **K213403 (Device)** | **K141328 (Predicate)** |
| --- | --- | --- |
| Device Trade Name | Aptiva CTD Essential Reagent (Ro60) | QUANTA Flash Ro60 |
| **General Device Characteristic Similarities** | | |
| Intended Use/ Indications For Use | The Aptiva CTD Essential Reagent consists of 10 multiplexed immunoassays utilizing particle-based multi-analyte technology for the quantitative determination of IgG autoantibodies against dsDNA, and semi-quantitative determination of IgG autoantibodies against RNP, Sm, Ro52, Ro60, SS-B, Scl-70, Jo-1, centromere, and Ribo-P in human serum: ... • The presence of 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. ... | QUANTA Flash Ro60 is a chemiluminescent immunoassay for the semi-quantitative determination of IgG anti-Ro60 autoantibodies in human serum. The presence of anti-Ro60 autoantibodies, in conjunction with clinical findings and other laboratory tests, aids in the diagnosis of Systemic Lupus Erythematosus and Sjögren's Syndrome. |

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| **Device & Predicate Device(s):** | **K213403 (Device)** | **K141328 (Predicate)** |
| --- | --- | --- |
| | The individual assays included in the Aptiva CTD Essential Reagent are intended for use with the Inova Diagnostics Aptiva System. | |
| Assay Methodology | Solid phase immunoassay | Same |
| Antigen | Recombinant Ro60 | Same |
| Sample Matrix | Human serum | Same |
| Solid Phase | Paramagnetic microparticles | Same |
| Controls | Two Ro60 controls with lot specific values assigned. | Same |
| **General Device Characteristic Differences** | | |
| Detection Principle | Fluorescent immunoassay | Chemiluminescent immunoassay |
| Conjugate | Phycoerythrin conjugated polyclonal anti-human IgG antibody | Isoluminol conjugated monoclonal anti-human IgG antibody |
| Units | Fluorescent light units (FLU) | Chemiluminescent units (CU) |
| Cut-off | 5.00 FLU | 20.00 CU |
| Analytical Measuring Range | 0.5 FLU – 583.72 FLU | 4.9 CU – 1374.8 CU |
| Calibration | Lot specific Master Curve + 6 calibrators (sold separately) | Lot specific Master Curve + 2 calibrators (sold separately) |

| **Device & Predicate Device(s):** | **K213403 (Device)** | **K141210 (Predicate)** |
| --- | --- | --- |
| Device Trade Name | Aptiva CTD Essential Reagent (SS-B) | QUANTA Flash SS-B |
| **General Device Characteristic Similarities** | | |
| Intended Use/ Indications For Use | The Aptiva CTD Essential Reagent consists of 10 multiplexed immunoassays utilizing particle-based multi-analyte technology for the quantitative determination of IgG autoantibodies against dsDNA, and semi-quantitative determination of IgG autoantibodies against RNP, Sm, Ro52, Ro60, SS-B, Scl-70, Jo-1, Centromere, and Ribo-P in human serum: ... • The presence of 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 | QUANTA Flash SS-B is a chemiluminescent immunoassay for the semi-quantitative determination of IgG anti-SS-B autoantibodies in human serum. The presence of anti-SS-B autoantibodies, in conjunction with clinical findings and other laboratory tests is an aid in the diagnosis of Sjögren's Syndrome and Systemic Lupus Erythematosus. |

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| **Device & Predicate Device(s):** | **K213403 (Device)** | **K141210 (Predicate)** |
| --- | --- | --- |
| | syndrome. ... The individual assays included in the Aptiva CTD Essential Reagent are intended for use with the Inova Diagnostics Aptiva System. | |
| Assay Methodology | Solid phase immunoassay | Same |
| Antigen | Recombinant SS-B | Same |
| Sample Matrix | Human serum | Same |
| Solid Phase | Paramagnetic microparticles | Same |
| Controls | Two SS-B controls with lot specific values assigned. | Same |
| **General Device Characteristic Differences** | | |
| Detection Principle | Fluorescent immunoassay | Chemiluminescent immunoassay |
| Conjugate | Phycoerythrin conjugated polyclonal anti-human IgG antibody | Isoluminol conjugated monoclonal anti-human IgG antibody |
| Units | Fluorescent light units (FLU) | Chemiluminescent units (CU) |
| Cut-off | 5.00 FLU | 20.00 CU |
| Analytical Measuring Range | 0.40 FLU – 195.84 FLU | 3.3 CU – 1550.0 CU |
| Calibration | Lot specific Master Curve + 6 calibrators (sold separately) | Lot specific Master Curve + 2 calibrators (sold separately) |

| **Device & Predicate Device(s):** | **K213403 (Device)** | **K152635 (Predicate)** |
| --- | --- | --- |
| Device Trade Name | Aptiva CTD Essential Reagent (Scl-70) | QUANTA Flash Scl-70 |
| **General Device Characteristic Similarities** | | |
| Intended Use/ Indications For Use | The Aptiva CTD Essential Reagent consists of 10 multiplexed immunoassays utilizing particle-based multi-analyte technology for the quantitative determination of IgG autoantibodies against dsDNA, and semi-quantitative determination of IgG autoantibodies against RNP, Sm, Ro52, Ro60, SS-B, Scl-70, Jo-1, centromere, and Ribo-P in human serum: ... • The presence of Scl-70 antibodies, in conjunction with clinical findings and other laboratory tests, is an aid in the diagnosis of systemic | QUANTA Flash Scl-70 is a chemiluminescent immunoassay for the semi-quantitative determination of IgG anti-Scl-70 autoantibodies in human serum. The presence of anti-Scl-70 autoantibodies, in conjunction with clinical findings and other laboratory tests, aids in the diagnosis of systemic sclerosis. |

K213403 - Page 11 of 46

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| Device & Predicate Device(s): | K213403 (Device) | K152635 (Predicate) |
| --- | --- | --- |
| | sclerosis. ... The individual assays included in the Aptiva CTD Essential Reagent are intended for use with the Inova Diagnostics Aptiva System. | |
| Assay Methodology | Solid phase immunoassay | Same |
| Antigen | Recombinant Scl-70 | Same |
| Sample Matrix | Human serum | Same |
| Solid Phase | Paramagnetic microparticles | Same |
| Controls | Two Scl-70 controls with lot specific values assigned. | Same |

# General Device Characteristic Differences

| Detection Principle | Fluorescent immunoassay | Chemiluminescent immunoassay |
| --- | --- | --- |
| Conjugate | Phycoerythrin conjugated polyclonal anti-human IgG antibody | Isoluminol conjugated monoclonal anti-human IgG antibody |
| Units | Fluorescent light units (FLU) | Chemiluminescent units (CU) |
| Cut-off | 5.00 FLU | 20.00 CU |
| Analytical Measuring Range | 0.50 FLU – 371.24 FLU | 1.2 CU – 786.3 CU |
| Calibration | Lot specific Master Curve + 6 calibrators (sold separately) | Lot specific Master Curve + 2 calibrators (sold separately) |

| Device & Predicate Device(s): | K213403 (Device) | K151429 (Predicate) |
| --- | --- | --- |
| Device Trade Name | Aptiva CTD Essential Reagent (Jo-1) | QUANTA Flash Jo-1 |

# General Device Characteristic Similarities

| Intended Use/ Indications For Use | The Aptiva CTD Essential Reagent consists of 10 multiplexed immunoassays utilizing particle-based multi-analyte technology for the quantitative determination of IgG autoantibodies against dsDNA, and semi-quantitative determination of IgG autoantibodies against RNP, Sm, Ro52, Ro60, SS-B, Scl-70, Jo-1, centromere, and Ribo-P in human serum: ... • The presence of Jo-1 antibodies, in conjunction with clinical findings and other laboratory tests, is an aid in the diagnosis of idiopathic | QUANTA Flash Jo-1 is a chemiluminescent immunoassay for the semi-quantitative determination of IgG anti-Jo-1 antibodies in human serum. The presence of anti-Jo-1 antibodies, in conjunction with clinical findings and other laboratory tests, is an aid in the diagnosis of idiopathic inflammatory myopathy. |
| --- | --- | --- |

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| Device & Predicate Device(s): | K213403 (Device) | K151429 (Predicate) |
| --- | --- | --- |
| | inflammatory myositis. ... The individual assays included in the Aptiva CTD Essential Reagent are intended for use with the Inova Diagnostics Aptiva System. | |
| Assay Methodology | Solid phase immunoassay | Same |
| Antigen | Recombinant Jo-1 | Same |
| Sample Matrix | Human serum | Same |
| Solid Phase | Paramagnetic microparticles | Same |
| Controls | Two Jo-1 controls with lot specific values assigned. | Same |

# General Device Characteristic Differences

| Detection Principle | Fluorescent immunoassay | Chemiluminescent immunoassay |
| --- | --- | --- |
| Conjugate | Phycoerythrin conjugated polyclonal anti-human IgG antibody | Isoluminol conjugated monoclonal anti-human IgG antibody |
| Units | Fluorescent light units (FLU) | Chemiluminescent units (CU) |
| Cut-off | 5.00 FLU | 20.00 CU |
| Analytical Measuring Range | 0.25 FLU – 153.60 FLU | 2.2 CU – 1147.2 CU |
| Calibration | Lot specific Master Curve + 6 calibrators (sold separately) | Lot specific Master Curve + 2 calibrators (sold separately) |

| Device & Predicate Device(s): | K213403 (Device) | K123880 (Predicate) |
| --- | --- | --- |
| Device Trade Name | Aptiva CTD Essential Reagent (Centromere) | QUANTA Flash Centromere |

# General Device Characteristic Similarities

| Intended Use/ Indications For Use | The Aptiva CTD Essential Reagent consists of 10 multiplexed immunoassays utilizing particle-based multi-analyte technology for the quantitative determination of IgG autoantibodies against dsDNA, and semi-quantitative determination of IgG autoantibodies against RNP, Sm, Ro52, Ro60, SS-B, Scl-70, Jo-1, centromere, and Ribo-P in human serum: ... • The presence of centromere antibodies, in conjunction with clinical findings and other laboratory tests, is an aid in the | QUANTA Flash Centromere is a chemiluminescent immunoassay for the semi-quantitative determination of IgG anti-centromere protein B autoantibodies in human serum. The presence of anti-centromere protein B autoantibodies is used as an aid in the diagnosis of systemic sclerosis, in conjunction with clinical finding and other laboratory tests. |
| --- | --- | --- |

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| **Device & Predicate Device(s):** | **K213403 (Device)** | **K123880 (Predicate)** |
| --- | --- | --- |
| | diagnosis of systemic sclerosis. ... The individual assays included in the Aptiva CTD Essential Reagent are intended for use with the Inova Diagnostics Aptiva System. | |
| Assay Methodology | Solid phase immunoassay | Same |
| Antigen | Recombinant centromere | Same |
| Sample Matrix | Human serum | Same |
| Solid Phase | Paramagnetic microparticles | Same |
| Controls | Two Centromere controls with lot specific values assigned. | Same |
| **General Device Characteristic Differences** | | |
| Detection Principle | Fluorescent immunoassay | Chemiluminescent immunoassay |
| Conjugate | Phycoerythrin conjugated polyclonal anti-human IgG antibody | Isoluminol conjugated monoclonal anti-human IgG antibody |
| Units | Fluorescent light units (FLU) | Chemiluminescent units (CU) |
| Cut-off | 5.00 FLU | 20.00 CU |
| Analytical Measuring Range | 0.50 FLU – 187.69 FLU | 3.4 CU – 708.9 CU |
| Calibration | Lot specific Master Curve + 6 calibrators (sold separately) | Lot specific Master Curve + 2 calibrators (sold separately) |

| **Device & Predicate Device(s):** | **K213403 (Device)** | **K981237 (Predicate)** |
| --- | --- | --- |
| Device Trade Name | Aptiva CTD Essential Reagent (Ribo-P) | QUANTA Lite Ribosomal P |
| **General Device Characteristic Similarities** | | |
| Intended Use/ Indications For Use | The Aptiva CTD Essential Reagent consists of 10 multiplexed immunoassays utilizing particle-based multi-analyte technology for the quantitative determination of IgG autoantibodies against dsDNA, and semi-quantitative determination of IgG autoantibodies against RNP, Sm, Ro52, Ro60, SS-B, Scl-70, Jo-1, centromere, and Ribo-P in human serum: ... • The presence of Ribo-P antibodies, in conjunction with clinical findings and other laboratory tests, is an aid | QUANTA Lite Ribosome P is an enzyme-linked immunosorbent assay (ELISA) for the semi-quantitative detection of Ribosome P antibodies in human serum. The presence of Ribosome P antibodies can be used in conjunction with clinical findings and other laboratory tests to aid in the diagnosis of Systemic Lupus Erythematosus (SLE) and other related connective tissue diseases. |

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| Device & Predicate Device(s): | K213403 (Device) | K981237 (Predicate) |
| --- | --- | --- |
| | 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. | |
| Assay Methodology | Solid phase immunoassay | Same |
| Antigen | Synthetic Ribosomal P peptide | Same |
| Sample Matrix | Human serum | Same |
| Controls | Two Ribo-P controls with lot specific values assigned. | Same |
| **General Device Characteristic Differences** | | |
| Solid Phase | Paramagnetic microparticles | ELISA |
| Detection Principle | Fluorescent immunoassay | Chromogenic immunoassay |
| Conjugate | Phycoerythrin conjugated polyclonal anti-human IgG antibody | HRP conjugated anti-human IgG antibody |
| Units | Fluorescent light units (FLU) | Units (U) |
| Cut-off | 5.00 FLU | 20.00 CU |
| Analytical Measuring Range | 0.25 FLU – 86.86 FLU | N/A |
| Calibration | Lot specific Master Curve + 6 calibrators (sold separately) | Single point calibration |

### VI Standards/Guidance Documents Referenced:

The following Clinical and Laboratory Standards Institute (CLSI) guidelines were used:

- CLSI EP05-A3: Evaluation of Precision of Quantitative Measurement Procedures, Approved Guideline – Third Edition. Reaffirmed September 2019.
- CLSI EP06-Ed2: Evaluation of the Linearity of Quantitative Measurement Procedures – 2nd Edition.
- CLSI EP07-A2: Interference Testing in Clinical Chemistry, Approved Guideline – Second Edition.
- CLSI EP17-A2: Evaluation of Detection Capability for Clinical Laboratory Measurement Procedures, Approved Guideline – Second Edition.
- CLSI EP28-A3c: Defining, Establishing, and Verifying Reference Intervals in the Clinical Laboratory, Approved Guideline – Third Edition.

### VII Performance Characteristics (if/when applicable):

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# A Analytical Performance:

# 1. Precision/Reproducibility:

Precision and reproducibility of the Aptiva CTD Essential Reagent were evaluated in accordance with the CLSI guideline EP05-A3.

# Within-Laboratory Precision:

To evaluate within-laboratory precision of the Aptiva CTD Essential Reagent, a variable number of samples containing various concentrations of antibodies were assayed in duplicate, twice a day, for 20 days, for a total of 80 measurements per sample, using one reagent lot at one laboratory site, by one operator.

The resulting data was analyzed using Analyze-it for Excel software by analysis of variance (ANOVA) methods and the within-run (repeatability), between-run, between-day, and within-laboratory precision were determined. As the number of samples and the values of measurand assayed varies by analyte, the study results are summarized on a per-analyte basis below:

| dsDNA Within-Laboratory Precision | | | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | Repeatability | | Between-Run | | Between-Day | | Within-Laboratory | |
| Sample | Mean (IU/mL) | SD (IU/mL) | %CV | SD (IU/mL) | %CV | SD (IU/mL) | %CV | SD (IU/mL) | %CV |
| 1 | 16.19 | 1.13 | 7.0 | 0.64 | 4.0 | 1.39 | 8.6 | 1.90 | 11.8 |
| 2 | 25.81 | 1.97 | 7.6 | 1.65 | 6.4 | 1.30 | 5.0 | 2.88 | 11.1 |
| 3 | 33.39 | 2.08 | 6.2 | 1.99 | 6.0 | 1.03 | 3.1 | 3.06 | 9.2 |
| 4 | 47.86 | 3.03 | 6.3 | 2.07 | 4.3 | 3.34 | 7.0 | 4.96 | 10.4 |
| 5 | 67.19 | 4.53 | 6.7 | 2.86 | 4.3 | 3.28 | 4.9 | 6.28 | 9.3 |
| 6 | 98.34 | 5.16 | 5.2 | 0.7 | 0.7 | 5.7 | 5.8 | 7.72 | 7.9 |
| 7 | 208.89 | 13.01 | 6.2 | 0.00 | 0.0 | 9.46 | 4.5 | 16.09 | 7.7 |
| 8 | 425.21 | 31.59 | 7.4 | 28.14 | 6.6 | 14.65 | 3.4 | 44.77 | 10.5 |
| 9 | 600.13 | 35.66 | 5.9 | 53.43 | 8.9 | 0.00 | 0.0 | 64.24 | 10.7 |

| RNP Within-Laboratory Precision | | | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | Repeatability | | Between-Run | | Between-Day | | Within-Laboratory | |
| Sample | Mean (FLU) | SD (FLU) | %CV | SD (FLU) | %CV | SD (FLU) | %CV | SD (FLU) | %CV |
| 1 | 1.96 | 0.09 | 4.8 | 0.09 | 4.8 | 0.15 | 7.5 | 0.20 | 10.1 |
| 2 | 2.93 | 0.14 | 4.7 | 0.13 | 4.3 | 0.28 | 9.7 | 0.34 | 11.6 |
| 3 | 5.26 | 0.31 | 5.9 | 0.24 | 4.5 | 0.42 | 8.0 | 0.57 | 10.9 |
| 4 | 14.59 | 0.53 | 3.6 | 0.57 | 3.9 | 0.82 | 5.6 | 1.13 | 7.8 |
| 5 | 45.76 | 2.71 | 5.9 | 1.47 | 3.2 | 2.85 | 6.2 | 4.20 | 9.2 |
| 6 | 131.34 | 8.27 | 6.3 | 2.20 | 1.7 | 9.41 | 7.2 | 12.72 | 9.7 |

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| Sm Within-Laboratory Precision | | | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Sample | Mean (FLU) | Repeatability | | Between-Run | | Between-Day | | Within-Laboratory | |
| | | SD (FLU) | %CV | SD (FLU) | %CV | SD (FLU) | %CV | SD (FLU) | %CV |
| 1 | 3.98 | 0.18 | 4.5 | 0.17 | 4.2 | 0.39 | 9.8 | 0.46 | 11.6 |
| 2 | 5.17 | 0.22 | 4.3 | 0.18 | 3.5 | 0.40 | 7.8 | 0.50 | 9.6 |
| 3 | 9.53 | 0.29 | 3.0 | 0.38 | 4.0 | 0.43 | 4.5 | 0.64 | 6.7 |
| 4 | 51.21 | 2.62 | 5.1 | 0.00 | 0.0 | 3.19 | 6.2 | 4.13 | 8.1 |
| 5 | 122.68 | 5.04 | 4.1 | 6.59 | 5.4 | 8.95 | 7.3 | 12.20 | 9.9 |
| 6 | 156.91 | 9.72 | 6.2 | 10.53 | 6.7 | 9.42 | 6.0 | 17.15 | 10.9 |
| 7 | 188.62 | 13.87 | 7.4 | 12.55 | 6.7 | 6.05 | 3.2 | 19.66 | 10.4 |

| Ro52 Within-Laboratory Precision | | | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Sample | Mean (FLU) | Repeatability | | Between-Run | | Between-Day | | Within-Laboratory | |
| | | SD (FLU) | %CV | SD (FLU) | %CV | SD (FLU) | %CV | SD (FLU) | %CV |
| 1 | 1.16 | 0.06 | 5.3 | 0.05 | 3.9 | 0.06 | 5.0 | 0.10 | 8.3 |
| 2 | 2.57 | 0.16 | 6.4 | 0.14 | 5.3 | 0.17 | 6.8 | 0.28 | 10.8 |
| 3 | 5.59 | 0.32 | 5.7 | 0.17 | 3.1 | 0.40 | 7.1 | 0.54 | 9.6 |
| 4 | 8.14 | 0.28 | 3.4 | 0.23 | 2.8 | 0.46 | 5.6 | 0.58 | 7.1 |
| 5 | 44.31 | 1.33 | 3.0 | 1.48 | 3.3 | 3.17 | 7.2 | 3.75 | 8.5 |
| 6 | 73.45 | 1.80 | 2.4 | 2.45 | 3.3 | 3.56 | 4.9 | 4.68 | 6.4 |
| 7 | 118.51 | 6.22 | 5.2 | 4.90 | 4.1 | 5.49 | 4.6 | 9.63 | 8.1 |
| 8 | 150.71 | 7.42 | 4.9 | 2.14 | 1.4 | 7.04 | 4.7 | 10.45 | 6.9 |
| 9 | 186.21 | 9.86 | 5.3 | 6.60 | 3.5 | 8.88 | 4.8 | 14.82 | 8.0 |

| Ro60 Within-Laboratory Precision | | | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Sample | Mean (FLU) | Repeatability | | Between-Run | | Between-Day | | Within-Laboratory | |
| | | SD | %CV | SD | %CV | SD | %CV | SD | %CV |
| 1 | 2.96 | 0.19 | 6.5 | 0.25 | 8.5 | 0.07 | 2.2 | 0.32 | 10.9 |
| 2 | 5.53 | 0.32 | 5.7 | 0.26 | 4.7 | 0.38 | 6.9 | 0.56 | 10.1 |
| 3 | 8.48 | 0.55 | 6.5 | 0.39 | 4.6 | 0.62 | 7.3 | 0.91 | 10.8 |
| 4 | 19.73 | 0.81 | 4.1 | 0.41 | 2.1 | 1.01 | 5.1 | 1.36 | 6.9 |
| 5 | 58.82 | 2.59 | 4.4 | 1.92 | 3.3 | 3.47 | 5.9 | 4.74 | 8.1 |
| 6 | 80.96 | 3.98 | 4.9 | 4.99 | 6.2 | 4.51 | 5.6 | 7.82 | 9.7 |
| 7 | 226.76 | 14.37 | 6.3 | 16.15 | 7.1 | 9.72 | 4.3 | 23.70 | 10.5 |
| 8 | 400.89 | 24.72 | 6.2 | 27.87 | 7.0 | 20.96 | 5.2 | 42.75 | 10.7 |

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| SS-B Within-Laboratory Precision | | | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Sample | Mean (FLU) | Repeatability | | Between-Run | | Between-Day | | Within-Laboratory | |
| | | SD (FLU) | %CV | SD (FLU) | %CV | SD (FLU) | %CV | SD (FLU) | %CV |
| 1 | 1.33 | 0.05 | 4.0 | 0.06 | 4.2 | 0.12 | 9.0 | 0.14 | 10.7 |
| 2 | 4.69 | 0.16 | 3.3 | 0.18 | 3.8 | 0.49 | 10.5 | 0.55 | 11.7 |
| 3 | 64.34 | 1.87 | 2.9 | 1.60 | 2.5 | 3.74 | 5.8 | 4.48 | 7.0 |
| 4 | 138.52 | 5.76 | 4.2 | 2.62 | 1.9 | 7.95 | 5.7 | 10.16 | 7.3 |
| 5 | 157.86 | 6.60 | 4.2 | 5.43 | 3.4 | 10.42 | 6.6 | 13.48 | 8.5 |

| Scl-70 Within-Laboratory Precision | | | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Sample | Mean (FLU) | Repeatability | | Between-Run | | Between-Day | | Within-Laboratory | |
| | | SD (FLU) | %CV | SD (FLU) | %CV | SD (FLU) | %CV | SD (FLU) | %CV |
| 1 | 3.21 | 0.11 | 3.3 | 0.18 | 5.5 | 0.28 | 8.6 | 0.34 | 10.7 |
| 2 | 5.38 | 0.25 | 4.7 | 0.23 | 4.3 | 0.51 | 9.5 | 0.62 | 11.5 |
| 3 | 11.49 | 0.33 | 2.8 | 0.32 | 2.8 | 0.62 | 5.4 | 0.77 | 6.7 |
| 4 | 62.11 | 2.48 | 4.0 | 0.59 | 0.9 | 3.89 | 6.3 | 4.65 | 7.5 |
| 5 | 109.42 | 3.49 | 3.2 | 1.97 | 1.8 | 6.83 | 6.2 | 7.92 | 7.2 |
| 6 | 307.35 | 18.93 | 6.2 | 25.47 | 8.3 | 9.60 | 3.1 | 33.16 | 10.8 |

| Jo-1 Within-Laboratory Precision | | | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Sample | Mean (FLU) | Repeatability | | Between-Run | | Between-Day | | Within-Laboratory | |
| | | SD (FLU) | %CV | SD (FLU) | %CV | SD (FLU) | %CV | SD (FLU) | %CV |
| 1 | 2.07 | 0.10 | 4.8 | 0.07 | 3.4 | 0.14 | 6.8 | 0.19 | 9.0 |
| 2 | 5.15 | 0.27 | 5.2 | 0.22 | 4.3 | 0.24 | 4.6 | 0.42 | 8.1 |
| 3 | 18.54 | 0.75 | 4.0 | 0.93 | 5.0 | 1.41 | 7.6 | 1.85 | 10.0 |
| 4 | 81.42 | 4.76 | 5.8 | 5.37 | 6.6 | 4.28 | 5.3 | 8.36 | 10.3 |
| 5 | 107.75 | 7.29 | 6.8 | 2.98 | 2.8 | 6.19 | 5.7 | 10.01 | 9.3 |

| Centromere Within-Laboratory Precision | | | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Sample | Mean (FLU) | Repeatability | | Between-Run | | Between-Day | | Within-Laboratory | |
| | | SD (FLU) | %CV | SD (FLU) | %CV | SD (FLU) | %CV | SD (FLU) | %CV |
| 1 | 4.05 | 0.16 | 4.0 | 0.18 | 4.4 | 0.38 | 9.4 | 0.45 | 11.1 |
| 2 | 5.00 | 0.19 | 3.7 | 0.23 | 4.6 | 0.38 | 7.6 | 0.48 | 9.6 |
| 3 | 7.41 | 0.26 | 3.5 | 0.23 | 3.1 | 0.43 | 5.7 | 0.55 | 7.4 |
| 4 | 30.72 | 1.11 | 3.6 | 1.71 | 5.6 | 2.23 | 7.3 | 3.02 | 9.8 |
| 5 | 134.91 | 9.40 | 7.0 | 8.44 | 6.3 | 5.78 | 4.3 | 13.89 | 10.3 |

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| **Ribo-P Within-Laboratory Precision** | | | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Sample | Mean (FLU) | Repeatability | | Between-Run | | Between-Day | | Within-Laboratory | |
| | | SD (FLU) | %CV | SD (FLU) | %CV | SD (FLU) | %CV | SD (FLU) | %CV |
| 1 | 1.42 | 0.09 | 6.5 | 0.00 | 0.0 | 0.10 | 7.1 | 0.14 | 9.6 |
| 2 | 3.39 | 0.16 | 4.8 | 0.12 | 3.4 | 0.21 | 6.1 | 0.29 | 8.4 |
| 3 | 4.85 | 0.27 | 5.5 | 0.25 | 5.2 | 0.39 | 8.1 | 0.54 | 11.1 |
| 4 | 21.77 | 1.23 | 5.6 | 1.07 | 4.9 | 0.71 | 3.3 | 1.77 | 8.1 |
| 5 | 38.98 | 2.76 | 7.1 | 0.00 | 0.0 | 2.63 | 6.7 | 3.81 | 9.8 |
| 6 | 66.90 | 4.89 | 7.3 | 2.54 | 3.8 | 2.95 | 4.4 | 6.25 | 9.3 |

### Reproducibility:

The reproducibility of the Aptiva CTD Essential Reagent was conducted at three sites using a variable number of samples containing various concentrations of antibodies. Samples were assayed in quintuplicate, once a day, for five days to generate 25 data points per sample per site using one reagent lot and a total of 75 replicates per sample. Instrument and operator variables were nested within the multiple site component – i.e., a different operator and instrument was used at each of the three sites.

The resulting data was analyzed using Analyze-it for Excel software by ANOVA methods and precision between sites was determined. As the number of samples assayed varies by analyte, the results are summarized on a per analyte basis below.

| **dsDNA Multi-Site Reproducibility** | | | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Sample | Mean (IU/mL) | Repeatability | | Between-Run | | Between-Site/Instrument | | Reproducibility | |
| | | SD (IU/mL) | %CV | SD (IU/mL) | %CV | SD (IU/mL) | %CV | SD (IU/mL) | %CV |
| 1 | 24.31 | 1.48 | 6.1 | 1.15 | 4.7 | 2 | 8.2 | 2.74 | 11.3 |
| 2 | 36.29 | 2.32 | 6.4 | 1.64 | 4.5 | 2.9 | 8.0 | 4.06 | 11.2 |
| 3 | 120.5 | 7.07 | 5.9 | 2.57 | 2.1 | 6.74 | 5.6 | 10.1 | 8.4 |
| 4 | 248.98 | 11.90 | 4.8 | 8.06 | 3.2 | 4.79 | 1.9 | 15.15 | 6.1 |
| 5 | 554.77 | 34.39 | 6.2 | 22.84 | 4.1 | 62.44 | 11.3 | 74.85 | 13.5 |

| **RNP Multi-Site Reproducibility** | | | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Sample | Mean (FLU) | Repeatability | | Between-Run | | Between-Site/Instrument | | Reproducibility | |
| | | SD (FLU) | %CV | SD (FLU) | %CV | SD (FLU) | %CV | SD (FLU) | %CV |
| 1 | 2.09 | 0.09 | 4.5 | 0.13 | 6.2 | 0.23 | 11.2 | 0.28 | 13.6 |
| 2 | 5.64 | 0.30 | 5.3 | 0.24 | 4.3 | 0.61 | 10.9 | 0.72 | 12.8 |
| 3 | 14.59 | 0.54 | 3.7 | 0.37 | 2.6 | 1.32 | 9.1 | 1.48 | 10.1 |
| 4 | 45.09 | 1.93 | 4.3 | 2.20 | 4.9 | 3.98 | 8.8 | 4.94 | 11.0 |
| 5 | 130.87 | 5.91 | 4.5 | 3.15 | 2.4 | 8.29 | 6.3 | 10.66 | 8.1 |

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| Sm Multi-Site Reproducibility | | | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Sample | Mean (FLU) | Repeatability | | Between-Run | | Between-Site/Instrument | | Reproducibility | |
| | | SD (FLU) | %CV | SD (FLU) | %CV | SD (FLU) | %CV | SD (FLU) | %CV |
| 1 | 2.93 | 0.11 | 3.8 | 0.17 | 5.6 | 0.29 | 9.9 | 0.35 | 12.0 |
| 2 | 5.01 | 0.20 | 4.0 | 0.19 | 3.8 | 0.46 | 9.2 | 0.54 | 10.7 |
| 3 | 7.13 | 0.30 | 4.2 | 0.38 | 5.3 | 0.69 | 9.7 | 0.84 | 11.8 |
| 4 | 50.85 | 2.35 | 4.6 | 2.47 | 4.9 | 4.19 | 8.2 | 5.40 | 10.6 |
| 5 | 84.84 | 3.19 | 3.8 | 1.72 | 2.0 | 7.73 | 9.1 | 8.54 | 10.1 |
| 6 | 142.01 | 6.59 | 4.6 | 6.89 | 4.9 | 16.28 | 11.5 | 18.87 | 13.3 |

| Ro52 Multi-Site Reproducibility | | | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Sample | Mean (FLU) | Repeatability | | Between-Run | | Between-Site/Instrument | | Reproducibility | |
| | | SD (FLU) | %CV | SD (FLU) | %CV | SD (FLU) | %CV | SD (FLU) | %CV |
| 1 | 1.80 | 0.06 | 3.3 | 0.05 | 2.9 | 0.18 | 9.8 | 0.19 | 10.7 |
| 2 | 4.62 | 0.16 | 3.5 | 0.15 | 3.2 | 0.37 | 8.1 | 0.43 | 9.4 |
| 3 | 7.90 | 0.28 | 3.5 | 0.14 | 1.8 | 0.65 | 8.2 | 0.72 | 9.1 |
| 4 | 43.21 | 0.93 | 2.1 | 0.96 | 2.2 | 2.88 | 6.7 | 3.17 | 7.3 |
| 5 | 77.74 | 2.86 | 3.7 | 2.50 | 3.2 | 9.01 | 11.6 | 9.78 | 12.6 |
| 6 | 130.60 | 5.80 | 4.4 | 3.76 | 2.9 | 15.07 | 11.5 | 16.58 | 12.7 |

| Ro60 Multi-Site Reproducibility | | | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Sample | Mean (FLU) | Repeatability | | Between-Run | | Between-Site/Instrument | | Reproducibility | |
| | | SD (FLU) | %CV | SD (FLU) | %CV | SD (FLU) | %CV | SD (FLU) | %CV |
| 1 | 2.69 | 0.17 | 6.5 | 0.08 | 2.9 | 0.19 | 7.1 | 0.27 | 10.0 |
| 2 | 5.99 | 0.34 | 5.6 | 0.21 | 3.6 | 0.70 | 11.6 | 0.80 | 13.4 |
| 3 | 29.52 | 1.29 | 4.4 | 1.10 | 3.7 | 2.07 | 7.0 | 2.68 | 9.1 |
| 4 | 79.82 | 3.90 | 4.9 | 1.02 | 1.3 | 5.12 | 6.4 | 6.52 | 8.2 |
| 5 | 212.42 | 10.48 | 4.9 | 7.77 | 3.7 | 7.80 | 3.7 | 15.20 | 7.2 |
| 6 | 325.49 | 25.27 | 7.8 | 19.39 | 6.0 | 25.95 | 8.0 | 41.09 | 12.6 |

| SS-B Multi-Site Reproducibility | | | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Sample | Mean (FLU) | Repeatability | | Between-Run | | Between-Site/Instrument | | Reproducibility | |
| | | SD (FLU) | %CV | SD (FLU) | %CV | SD (FLU) | %CV | SD (FLU) | %CV |
| 1 | 1.27 | 0.06 | 4.5 | 0.05 | 4.3 | 0.14 | 10.7 | 0.16 | 12.4 |
| 2 | 4.17 | 0.16 | 3.8 | 0.15 | 3.6 | 0.29 | 6.9 | 0.36 | 8.6 |
| 3 | 22.80 | 0.88 | 3.9 | 1.20 | 5.2 | 1.71 | 7.5 | 2.27 | 10.0 |

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| **SS-B Multi-Site Reproducibility** | | | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Sample | Mean (FLU) | Repeatability | | Between-Run | | Between-Site/Instrument | | Reproducibility | |
| | | SD (FLU) | %CV | SD (FLU) | %CV | SD (FLU) | %CV | SD (FLU) | %CV |
| 4 | 61.69 | 1.80 | 2.9 | 2.21 | 3.6 | 3.31 | 5.4 | 4.37 | 7.1 |
| 5 | 136.07 | 5.76 | 4.2 | 3.91 | 2.9 | 10.08 | 7.4 | 12.25 | 9.0 |
| 6 | 149.63 | 6.64 | 4.4 | 4.44 | 3.0 | 10.67 | 7.1 | 13.33 | 8.9 |

| **Scl-70 Multi-Site Reproducibility** | | | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Sample | Mean (FLU) | Repeatability | | Between-Run | | Between-Site/Instrument | | Reproducibility | |
| | | SD (FLU) | %CV | SD (FLU) | %CV | SD (FLU) | %CV | SD (FLU) | %CV |
| 1 | 3.39 | 0.20 | 6.0 | 0.23 | 6.8 | 0.26 | 7.7 | 0.40 | 11.9 |
| 2 | 5.93 | 0.26 | 4.4 | 0.17 | 2.9 | 0.47 | 8.0 | 0.57 | 9.6 |
| 3 | 12.51 | 0.41 | 3.2 | 0.37 | 2.9 | 0.60 | 4.8 | 0.81 | 6.5 |
| 4 | 70.19 | 2.23 | 3.2 | 3.19 | 4.5 | 2.37 | 3.4 | 4.55 | 6.5 |
| 5 | 123.54 | 3.76 | 3.0 | 5.14 | 4.2 | 3.50 | 2.8 | 7.27 | 5.9 |
| 6 | 220.48 | 8.57 | 3.9 | 7.79 | 3.5 | 26.09 | 11.8 | 28.54 | 12.9 |

| **Jo-1 Multi-Site Reproducibility** | | | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Sample | Mean (FLU) | Repeatability | | Between-Run | | Between-Site/Instrument | | Reproducibility | |
| | | SD (FLU) | %CV | SD (FLU) | %CV | SD (FLU) | %CV | SD (FLU) | %CV |
| 1 | 3.23 | 0.17 | 5.1 | 0.12 | 3.6 | 0.24 | 7.3 | 0.31 | 9.7 |
| 2 | 5.41 | 0.22 | 4.1 | 0.20 | 3.7 | 0.43 | 7.9 | 0.52 | 9.6 |
| 3 | 17.49 | 0.60 | 3.5 | 0.55 | 3.2 | 1.03 | 5.9 | 1.32 | 7.5 |
| 4 | 79.52 | 3.72 | 4.7 | 4.09 | 5.1 | 9.30 | 11.7 | 10.82 | 13.6 |
| 5 | 108.64 | 7.01 | 6.5 | 2.60 | 2.4 | 13.06 | 12.0 | 15.05 | 13.9 |

| **Centromere Multi-Site Reproducibility** | | | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Sample | Mean (FLU) | Repeatability | | Between-Run | | Between-Site/Instrument | | Reproducibility | |
| | | SD (FLU) | %CV | SD (FLU) | %CV | SD (FLU) | %CV | SD (FLU) | %CV |
| 1 | 2.28 | 0.13 | 5.6 | 0.15 | 6.8 | 0.15 | 6.4 | 0.25 | 10.9 |
| 2 | 4.34 | 0.14 | 3.2 | 0.16 | 3.7 | 0.37 | 8.6 | 0.43 | 9.9 |
| 3 | 6.79 | 0.22 | 3.2 | 0.21 | 3.1 | 0.66 | 9.7 | 0.73 | 10.7 |
| 4 | 28.61 | 1.04 | 3.6 | 1.20 | 4.2 | 1.32 | 4.6 | 2.07 | 7.2 |
| 5 | 43.74 | 1.75 | 4.0 | 1.88 | 4.3 | 5.01 | 11.5 | 5.63 | 12.9 |
| 6 | 112.03 | 6.74 | 6.0 | 6.21 | 5.5 | 8.78 | 7.8 | 12.69 | 11.3 |

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| **Ribo-P Multi-Site Reproducibility** | | | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Sample | Mean (FLU) | Repeatability | | Between-Run | | Between-Site/Instrument | | Reproducibility | |
| | | SD (FLU) | %CV | SD (FLU) | %CV | SD (FLU) | %CV | SD (FLU) | %CV |
| 1 | 1.56 | 0.07 | 4.4 | 0.04 | 2.5 | 0.18 | 11.7 | 0.20 | 12.8 |
| 2 | 4.78 | 0.25 | 5.2 | 0.27 | 5.7 | 0.31 | 6.6 | 0.48 | 10.1 |
| 3 | 21.96 | 1.21 | 5.5 | 0.26 | 1.2 | 2.17 | 9.9 | 2.50 | 11.4 |
| 4 | 38.20 | 1.76 | 4.6 | 1.40 | 3.7 | 3.17 | 8.3 | 3.89 | 10.2 |
| 5 | 61.32 | 3.38 | 5.5 | 1.15 | 1.9 | 5.46 | 8.9 | 6.53 | 10.6 |

#### Lot-to-lot imprecision:

To evaluate the between-lot imprecision of the Aptiva CTD Essential Reagent, a variable number of samples containing various concentrations of antibodies were assayed in quintuplicate, once a day, for 5 days, using three reagent lots, using one instrument, for a total of 75 replicates per sample.

The resulting data was analyzed using Analyze-it for Excel software by ANOVA methods and precision between lots was determined. As the number of samples assayed varies by analyte, the results are summarized on a per analyte basis below:

| **dsDNA Multi-Lot Imprecision** | | | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Sample | Mean (IU/mL) | Repeatability | | Between-Day | | Between-Lot | | Total | |
| | | SD (IU/mL) | %CV | SD (IU/mL) | %CV | SD (IU/mL) | %CV | SD (IU/mL) | %CV |
| 1 | 14.26 | 1.04 | 7.3 | 0.59 | 4.2 | 0.97 | 6.8 | 1.54 | 10.8 |
| 2 | 26.41 | 2.30 | 8.7 | 1.92 | 7.3 | 1.75 | 6.6 | 3.47 | 13.1 |
| 3 | 87.69 | 6.04 | 6.9 | 4.73 | 5.4 | 3.64 | 4.2 | 8.49 | 9.7 |
| 4 | 191.71 | 10.49 | 5.5 | 9.92 | 5.2 | 1.98 | 1.0 | 14.57 | 7.6 |
| 5 | 402.15 | 32.20 | 8.0 | 5.28 | 1.3 | 33.19 | 8.3 | 46.54 | 11.6 |
| 6 | 557.44 | 30.79 | 5.5 | 18.13 | 3.3 | 61.67 | 11.1 | 71.27 | 12.8 |

| **RNP Multi-Lot Imprecision** | | | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Sample | Mean (FLU) | Repeatability | | Between-Day | | Between-Lot | | Total | |
| | | SD (FLU) | %CV | SD (FLU) | %CV | SD (FLU) | %CV | SD (FLU) | %CV |
| 1 | 1.72 | 0.16 | 9.4 | 0.10 | 5.9 | 0.04 | 2.3 | 0.19 | 11.3 |
| 2 | 4.36 | 0.30 | 6.9 | 0.19 | 4.4 | 0.25 | 5.8 | 0.44 | 10.0 |
| 3 | 21.30 | 1.48 | 7.0 | 0.65 | 3.0 | 1.33 | 6.2 | 2.09 | 9.8 |
| 4 | 114.31 | 7.08 | 6.2 | 2.24 | 2.0 | 4.21 | 3.7 | 8.54 | 7.5 |

| **Sm Multi-Lot Imprecision** | | | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Sample | Mean (FLU) | Repeatability | | Between-Day | | Between-Lot | | Total | |
| | | SD (FLU) | %CV | SD (FLU) | %CV | SD (FLU) | %CV | SD (FLU) | %CV |
| 1 | 2.90 | 0.13 | 4.6 | 0.24 | 8.3 | 0.35 | 12.0 | 0.44 | 15.3 |

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# **Sm Multi-Lot Imprecision**

| Sample | Mean (FLU) | Repeatability | | Between-Day | | Between-Lot | | Total | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | SD (FLU) | %CV | SD (FLU) | %CV | SD (FLU) | %CV | SD (FLU) | %CV |
| 2 | 3.90 | 0.26 | 6.8 | 0.23 | 6.0 | 0.34 | 8.8 | 0.49 | 12.6 |
| 3 | 47.73 | 1.71 | 3.6 | 3.35 | 7.0 | 3.25 | 6.8 | 4.97 | 10.4 |
| 4 | 115.48 | 4.70 | 4.1 | 9.17 | 7.9 | 5.14 | 4.5 | 11.51 | 10.0 |
| 5 | 163.02 | 6.28 | 3.9 | 10.02 | 6.1 | 15.23 | 9.3 | 19.28 | 11.8 |

# **Ro52 Multi-Lot Imprecision**

| Sample | Mean (FLU) | Repeatability | | Between-Day | | Between-Lot | | Total | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | SD (FLU) | %CV | SD (FLU) | %CV | SD (FLU) | %CV | SD (FLU) | %CV |
| 1 | 1.77 | 0.07 | 4.2 | 0.10 | 5.8 | 0.17 | 9.4 | 0.21 | 11.9 |
| 2 | 4.11 | 0.16 | 3.9 | 0.23 | 5.6 | 0.11 | 2.7 | 0.30 | 7.3 |
| 3 | 7.50 | 0.33 | 4.4 | 0.45 | 6.0 | 0.52 | 7.0 | 0.77 | 10.2 |
| 4 | 39.74 | 2.11 | 5.3 | 2.79 | 7.0 | 1.38 | 3.5 | 3.76 | 9.5 |
| 5 | 71.05 | 2.15 | 3.0 | 6.03 | 8.5 | 6.23 | 8.8 | 8.93 | 12.6 |
| 6 | 118.01 | 7.98 | 6.8 | 5.99 | 5.1 | 10.71 | 9.1 | 14.64 | 12.4 |

# **Ro60 Multi-Lot Imprecision**

| Sample | Mean (FLU) | Repeatability | | Between-Day | | Between-Lot | | Total | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | SD (FLU) | %CV | SD (FLU) | %CV | SD (FLU) | %CV | SD (FLU) | %CV |
| 1 | 3.06 | 0.44 | 14.5 | 0.20 | 6.6 | 0.14 | 4.7 | 0.51 | 16.6 |
| 2 | 4.72 | 0.37 | 7.9 | 0.20 | 4.2 | 0.09 | 1.9 | 0.43 | 9.1 |
| 3 | 72.00 | 4.61 | 6.4 | 2.08 | 2.9 | 6.00 | 8.3 | 7.84 | 10.9 |
| 4 | 251.18 | 12.67 | 5.0 | 11.98 | 4.8 | 19.10 | 7.6 | 25.86 | 10.3 |
| 5 | 359.05 | 19.11 | 5.3 | 9.87 | 2.7 | 22.33 | 6.2 | 31.00 | 8.6 |

# **SS-B Multi-Lot Imprecision**

| Sample | Mean (FLU) | Repeatability | | Between-Day | | Between-Lot | | Total | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | SD (FLU) | %CV | SD (FLU) | %CV | SD (FLU) | %CV | SD (FLU) | %CV |
| 1 | 1.31 | 0.07 | 5.2 | 0.07 | 5.7 | 0.12 | 8.9 | 0.15 | 11.8 |
| 2 | 4.66 | 0.19 | 4.0 | 0.29 | 6.2 | 0.50 | 10.8 | 0.61 | 13.1 |
| 3 | 65.82 | 1.81 | 2.7 | 3.54 | 5.4 | 5.86 | 8.9 | 7.08 | 10.8 |
| 4 | 138.38 | 5.93 | 4.3 | 7.58 | 5.5 | 5.30 | 3.8 | 10.99 | 7.9 |

# **Scl-70 Multi-Lot Imprecision**

| Sample | Mean (FLU) | Repeatability | | Between-Day | | Between-Lot | | Total | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | SD (FLU) | %CV | SD (FLU) | %CV | SD (FLU) | %CV | SD (FLU) | %CV |
| 1 | 2.23 | 0.16 | 7.3 | 0.10 | 4.6 | 0.25 | 11.1 | 0.31 | 14.1 |
| 2 | 4.58 | 0.20 | 4.5 | 0.26 | 5.6 | 0.49 | 10.8 | 0.59 | 12.9 |
| 3 | 85.97 | 3.19 | 3.7 | 7.35 | 8.6 | 3.27 | 3.8 | 8.66 | 10.1 |
| 4 | 287.33 | 13.38 | 4.7 | 17.36 | 6.0 | 19.27 | 6.7 | 29.19 | 10.2 |

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| **Jo-1 Multi-Lot Imprecision** | | | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Sample | Mean (FLU) | Repeatability | | Between-Day | | Between-Lot | | Total | |
| | | SD (FLU) | %CV | SD (FLU) | %CV | SD (FLU) | %CV | SD (FLU) | %CV |
| 1 | 3.05 | 0.13 | 4.4 | 0.20 | 6.6 | 0.07 | 2.2 | 0.25 | 8.2 |
| 2 | 4.96 | 0.30 | 6.1 | 0.27 | 5.5 | 0.10 | 2.0 | 0.42 | 8.4 |
| 3 | 16.14 | 0.89 | 5.5 | 0.82 | 5.1 | 1.01 | 6.2 | 1.58 | 9.8 |
| 4 | 69.67 | 5.20 | 7.5 | 2.21 | 3.2 | 6.10 | 8.8 | 8.31 | 11.9 |
| 5 | 97.37 | 7.35 | 7.6 | 2.02 | 2.1 | 9.19 | 9.4 | 11.94 | 12.3 |

| **Centromere Multi-Lot Imprecision** | | | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Sample | Mean (FLU) | Repeatability | | Between-Day | | Between-Lot | | Total | |
| | | SD (FLU) | %CV | SD (FLU) | %CV | SD (FLU) | %CV | SD (FLU) | %CV |
| 1 | 1.69 | 0.25 | 14.9 | 0.17 | 9.9 | 0.09 | 5.1 | 0.31 | 18.6 |
| 2 | 4.36 | 0.18 | 4.2 | 0.35 | 8.1 | 0.16 | 3.6 | 0.43 | 9.8 |
| 3 | 28.59 | 1.16 | 4.1 | 2.66 | 9.3 | 1.58 | 5.5 | 3.30 | 11.5 |
| 4 | 128.28 | 6.57 | 5.1 | 6.06 | 4.7 | 13.72 | 10.7 | 16.38 | 12.8 |

| **Ribo-P Multi-Lot Imprecision** | | | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Sample | Mean (FLU) | Repeatability | | Between-Day | | Between-Lot | | Total | |
| | | SD (FLU) | %CV | SD (FLU) | %CV | SD (FLU) | %CV | SD (FLU) | %CV |
| 1 | 1.33 | 0.08 | 5.8 | 0.09 | 6.7 | 0.00 | 0.0 | 0.12 | 8.9 |
| 2 | 4.81 | 0.32 | 6.7 | 0.16 | 3.3 | 0.34 | 7.0 | 0.49 | 10.2 |
| 3 | 23.81 | 0.99 | 4.1 | 0.87 | 3.6 | 0.91 | 3.8 | 1.60 | 6.7 |
| 4 | 62.64 | 2.54 | 4.0 | 2.89 | 4.6 | 3.49 | 5.6 | 5.19 | 8.3 |

## 2. Linearity:

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

The linearity for all analytes was evaluated by a study following CLSI EP06-Ed2. Various samples were prepared as overlapping dilution series by mixing human serum samples with high antibody concentrations and samples with low antibody concentrations to cover portions of the analytical measuring interval (AMI) of each analyte. Each sample was tested in duplicate and the results from each sample were analyzed separately using a weighted least squares regression analysis.

The percent deviation from the weighted least squares regression analysis was used to assess the fit of the regression for each sample and each analyte. For values on the low end of the measuring interval, absolute deviations were used in lieu of the percentage. The results are summarized on a per analyte basis below.

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

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

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

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

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

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

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

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

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

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

#### Auto-rerun and reportable results

The Aptiva software has an auto-rerun option available. If the option is selected, the instrument will automatically rerun any sample that has a result above the upper limit of the

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analytical measuring range by performing a 10-fold dilution to bring the measured value to within the AMI, followed by a software calculation to account for the additional 10-fold dilution. To validate the auto-rerun function with 1:10 dilutions, two positive samples for each analyte, with concentrations well above the assay measuring range, were run with the auto-rerun function enabled on the Aptiva Instrument. The same set of samples were manually diluted 1:10, tested, and used as reference values. A percent recovery comparison of the values obtained by the auto-rerun and the manual dilution was conducted to determine the highest Auto-rerun values.

The highest value that is $\leq 15\%$ of the manual concentration for each measurand is listed in the table below:

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

#### High concentration hook effect:

To assess the Aptiva CTD Essential Reagent for hook effects, two samples for dsDNA, RNP, Sm, Ro52, Ro60, SS-B, Scl-70, Jo-1, Centromere, and Ribo-P were tested at three increasing two-fold serial dilutions from the standard 1:44.4 dilution used by the Aptiva CTD Essential Reagent.

All samples showed an increase in FLU values as the dilution factor became more concentrated, demonstrating that high positive specimens above the AMR do not demonstrate a hook effect up to the following concentrations:

| Assay | Highest concentrations tested |
| --- | --- |
| dsDNA | 4314.85 IU/mL |
| RNP | 25878.44 FLU |
| Sm | 1864.64 FLU |
| Ro52 | 2962.51 FLU |
| Ro60 | 3653.43 FLU |
| SS-B | 4562.22 FLU |
| Scl-70 | 10562.70 FLU |
| Jo-1 | 1932.57 FLU |
| Centromere | 1523.35 FLU |
| Ribo-P | 1144.80 FLU |

### 3. Analytical Specificity/Interference:

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# Interference:

An interference study was performed based on the recommendations contained in CLSI EP07-A2. A set of three human serum specimens – one positive specimen, one specimen near the cutoff, and one negative specimen, were assessed as vehicle-control or interferent-spiked contrived specimens in triplicate using the Aptiva CTD Essential Reagent assays (dsDNA, RNP, Sm, Ro52, Ro60, SS-B, Scl-70, Jo-1, Centromere and Ribo-P). The percent recovery for each sample that was spiked with the potential interfering substance was calculated by comparing its result to that of the corresponding control sample without the interfering substance.

# Endogenous:

The following endogenous interferents were tested: bilirubin, hemoglobin, triglycerides, cholesterol, rheumatoid factor IgM and human IgG. The Aptiva CTD Essential Reagent showed <±15% recovery with bilirubin up to 1 mg/mL, hemoglobin up to 2 mg/mL, triglyceride up to 1000 mg/dL, cholesterol up to 332.5 mg/dL, RF IgM up to 153.4 IU/mL and human IgG up to 35 mg/mL.

# Exogenous:

The following exogenous interferents were tested: ibuprofen, acetaminophen, prednisone, warfarin, diltiazem, azathioprine, sildenafil, cyclophosphamide, mycophenolate mofetil and heparin. The Aptiva CTD Essential Reagent showed <±15% recovery up to 21.9 mg/dL ibuprofen, 15.6 mg/dL acetaminophen, 0.0099 mg/dL prednisone, 7.5 mg/dL warfarin, 0.09 mg/dL diltiazem, 0.258 mg/dL azathioprine, 0.271 mg/dL sildenafil, 54.9 mg/dL cyclophosphamide, 1.125 mg/mL mycophenolate mofetil and 330 units/dL heparin. Rituximab was not evaluated for interference with the Aptiva CTD Essential Reagent.

# Analytical Specificity:

CDC (Center for Disease Controls and Prevention) ANA Reference Panel samples 1–12 were tested using one lot of Aptiva CTD Essential Reagent to demonstrate the analytical specificity of the assays. The results have been outlined below.

| CDC ID | Sample ID | CDC Description | Aptiva Result |
| --- | --- | --- | --- |
| IS2072 | CDC ANA 01 | ANA Homogeneous Positive/Anti-native DNA | dsDNA Positive |
| IS2073 | CDC ANA 02 | ANA Speckled Positive/Anti-SS-B Positive | SS-B, Ro52, Ro60 Positive |
| IS2074 | CDC ANA 03 | ANA Speckled Positive | RNP, Ro60, SS-B Positive |
| IS2075 | CDC ANA 04 | ANA-U1 RNP Positive | RNP Positive |
| IS2076 | CDC ANA 05 | Anti-Sm Positive | RNP and Sm Positive |
| IS2105 | CDC ANA 07 | Anti-SS-A Positive | Ro52 and Ro60 Positive |
| IS2134 | CDC ANA 08 | ANA Centromere Positive | Centromere Positive |
| IS2135 | CDC ANA 09 | Anti-Scl-70 Positive | Scl-70 Positive |
| IS2187 | CDC ANA 10 | Anti-Jo-1 Positive | Jo-1 and Ro52 Positive |
| IS2706 | CDC ANA 12 | Anti-Ribosomal P Positive | Ribo-P Positive |

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# 4. Assay Reportable Range:

The analytical measuring range (AMR) of the dsDNA, RNP, Sm, Ro60, Ro52, SS-B, Scl-70, Jo-1, Centromere and Ribo-P assays are outlined in the table below:

| Assay | Analytical Measuring Interval (AMI) |
| --- | --- |
| dsDNA | 2.3 – 814.10 IU/mL |
| RNP | 0.50 – 181.99 FLU |
| Sm | 0.25 – 256.00 FLU |
| Ro52 | 0.25 – 196.27 FLU |
| Ro60 | 0.50 – 583.72 FLU |
| SS-B | 0.40 – 195.84 FLU |
| Scl-70 | 0.50 – 371.24 FLU |
| Jo-1 | 0.25 – 153.60 FLU |
| Centromere | 0.50 – 187.69 FLU |
| Ribo-P | 0.25 – 86.86 FLU |

# 5. Traceability, Stability, Expected Values (Controls, Calibrators, or Methods):

# Traceability

For Aptiva dsDNA, the Master Curve Standards are traceable to the First International Standard Preparation (WHO code: Wo/80). Based upon this standardization, results are reported in International Units (IU)/mL. There are no international reference standards for the other analytes that allow the standardization of the assays. Calibrator and control values are directly traceable to in-house reference materials that are used to create the master curves for the Aptiva CTD Essential Reagent.

No international reference serum panel for anti-ANA antibodies is available that allows for the standardization of anti-ANA antibody assays.

# Sample Stability

To determine the stability of patient samples, three samples were tested for each analyte in the Aptiva CTD Essential Reagent assay. The samples used in the study were contrived by combining native high and low measurand (auto-antibody) serum samples to yield a desired reactivity. The samples were assayed in duplicate for up to 21 days while stored at 2-8°C, up to 48 hours while stored at room temperature (e.g., 20–25°C), and after repeated freeze/thaw cycles up to 5 cycles. The results were compared to the result obtained from control samples and conditions (i.e., timepoint zero / zero cycles).

Based on these results, serum samples may be stored up to 48 hours at room temperature, up to 14 days at 2–8°C, and may be frozen/thawed for up to 5 cycles when stored at or below -20°C.

# Reagent Stability

# Shelf life:

A Real Time stability study was conducted at 3-month intervals using Aptiva CTD Essential Reagent stored at 2–8°C. At each timepoint, a low-negative sample, a mid-range positive

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sample, and a high-range positive sample were tested at time 0, 6, 9, and 12-months with additional testing scheduled to be performed up to 24 months. The currently available results support a 12-month stability claim.

An accelerated stability study was performed for 5 weeks at 37°C ± 3°C to simulate 30-months at 5 ± 3°C. The recovery of the measured values relative to the control stored at 5 ± 3°C was determined and a linear regression analysis was performed between recovery values and the number of days for each bead component of the Aptiva CTD Essential Reagent. At each timepoint, a minimum of six samples were tested, including low-negative sample, mid-range positive sample, and high-range positive samples. The accelerated stability testing results support that the Aptiva CTD Essential Reagent may be stable for up to 24-months.

# In use (onboard) stability:

To establish the in-use stability of the Aptiva CTD Essential reagent cartridge onboard the Aptiva instrument, one lot of reagent cartridge was tested using human serum samples for all analytes. The specimens were tested periodically for 45 days. At day 21, the reagent cartridge was recalibrated, and a new cartridge specific working curve was generated. Percent recoveries were calculated compared to the day zero average values, and a linear regression analysis was performed by plotting percent recovery against time.

The test results support an in-use (onboard) stability for the Aptiva CTD Essential Reagent as 36 days with an 18-day recalibration.

# 6. Detection Limit:

Limit of Blank (LoB), Limit of Detection (LoD), and Limit of Quantitation (LoQ) were assessed for each analyte in the Aptiva CTD Essential Reagent and were calculated separately based on the recommendations contained in CLSI EP17-A2.

# LoB:

To determine the Limit of Blank, four blank samples were prepared using three lots of Aptiva system rinse, and were run in quintuplicate, once a day, for three days using two reagent lots for a total of 60 data points per lot. The LoB was determined for each assay, on each reagent lot separately using Analyse-it for Excel, at the 95th percentile, using the non-parametric method for all analyses. The higher LoB result between the two lots was selected for the final LoB value.

# LoD:

To determine the LoD for each analyte, four low level samples were contrived for each analyte by mixing human serum samples with high and low levels of antibodies, and were assayed in quintuplicate on two reagent lots, twice per day, for three days, for a total of 120 data points for each assay on each reagent lot. The LoD was determined separately for each assay on each reagent lot and the highest LoD result was selected for the final LoD value.

# LoQ:

To determine the LoQ for each analyte, four low level samples were contrived by mixing human serum samples with high and low levels of antibodies, which were run in quintuplicate, using two reagent lots, twice per day for three days, to generate 120 data points for each assay on each reagent lot. The LoQ was determined separately for each assay on

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each reagent lot, by calculating the total imprecision of each sample. The LoQ was defined to be the lowest concentration level that meets the within laboratory imprecision of <20% for each lot. The LoQ for each assay was determined as the greatest LoQ across the two lots and set as the lower limit of the AMR.

The LoB, LoD, and LoQ for each measurand is summarized as follows:

| Analyte | LoB | LoD | LoQ |
| --- | --- | --- | --- |
| dsDNA | 0.11 IU/mL | 1.64 IU/mL | 2.30 IU/mL |
| RNP | 0.03 FLU | 0.12 FLU | 0.15 FLU |
| Sm | 0.01 FLU | 0.13 FLU | 0.18 FLU |
| Ro52 | 0.00 FLU | 0.10 FLU | 0.23 FLU |
| Ro60 | 0.01 FLU | 0.06 FLU | 0.14 FLU |
| SS-B | 0.00 FLU | 0.14 FLU | 0.33 FLU |
| Scl-70 | 0.02 FLU | 0.12 FLU | 0.16 FLU |
| Jo-1 | 0.01 FLU | 0.03 FLU | 0.07 FLU |
| Centromere | 0.00 FLU | 0.06 FLU | 0.21 FLU |
| Ribo-P | 0.02 FLU | 0.16 FLU | 0.16 FLU |

# 7. Assay Cut-Off:

A cut-off study was performed using clinical samples. Please refer to the Clinical Cut-off (section §VII.D) below.

# B Comparison Studies:

# 1. Method Comparison with Predicate Device:

Samples for the method comparison analysis included the samples from the clinical validation study (see VII.C below). Samples were tested on both the Aptiva CTD Essential Reagent and their respective predicate devices. Positive percent agreement (PPA) and negative percent agreement (NPA), with 95% confidence intervals were calculated for each analyte comparison, excluding values that were outside of the measuring ranges of either assay. The following tables summarize these method comparison results:

| Method comparison of the Aptiva dsDNA with the predicate device | | | | | | |
| --- | --- | --- | --- | --- | --- | --- |
| | Predicate | | | | | |
| | | | Positive | Indeterminate | Negative | Total |
| Aptiva dsDNA | Positive | >35.0 | 97 | 8 | 17 | 122 |
| | Indeterminate | 27.0–35.0 | 5 | 3 | 13 | 21 |
| | Negative | <27.0 | 21 | 16 | 248 | 285 |
| | Total | | 123 | 27 | 278 | 428 |

| Indeterminate as positive results | PPA | 75.3% | (67.9–81.5%) |
| --- | --- | --- | --- |
| | NPA | 89.2% | (85.0–92.3%) |
| Indeterminate as negative results | PPA | 78.9% | (70.8–85.1%) |
| | NPA | 91.8% | (88.2–94.4%) |

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| Method comparison of the Aptiva RNP with the predicate device | | | | | |
| --- | --- | --- | --- | --- | --- |
| | | | Predicate | | |
| | | | Positive | Negative | Total |
| **Aptiva RNP** | Positive | ≥5.0 | 93 | 36 | 129 |
| | Negative | <5.0 | 2 | 349 | 351 |
| | Total | | 95 | 385 | 480 |

| PPA | 97.9% | (92.6–99.4%) |
| --- | --- | --- |
| NPA | 90.6% | (87.3–93.2%) |

| Method comparison of the Aptiva Sm with the predicate device | | | | | |
| --- | --- | --- | --- | --- | --- |
| | | | Predicate | | |
| | | | Positive | Negative | Total |
| **Aptiva Sm** | Positive | ≥5.0 | 30 | 7 | 37 |
| | Negative | <5.0 | 5 | 376 | 381 |
| | Total | | 35 | 383 | 418 |

| PPA | 85.7% | (70.6–93.7%) |
| --- | --- | --- |
| NPA | 98.2% | (96.3–99.1%) |

| Method comparison of the Aptiva Ro52 with the predicate device | | | | | |
| --- | --- | --- | --- | --- | --- |
| | | | Predicate | | |
| | | | Positive | Negative | Total |
| **Aptiva Ro52** | Positive | ≥5.0 | 203 | 18 | 221 |
| | Negative | <5.0 | 5 | 802 | 807 |
| | Total | | 208 | 820 | 1028 |

| PPA | 97.6% | (94.5–99.0%) |
| --- | --- | --- |
| NPA | 97.8% | (96.6–98.6%) |

| Method comparison of the Aptiva Ro60 with the predicate device | | | | | |
| --- | --- | --- | --- | --- | --- |
| | | | Predicate | | |
| | | | Positive | Negative | Total |
| **Aptiva Ro60** | Positive | ≥5.0 | 193 | 29 | 222 |
| | Negative | <5.0 | 3 | 326 | 329 |
| | Total | | 196 | 355 | 551 |

| PPA | 98.5% | (95.6–99.5%) |
| --- | --- | --- |
| NPA | 91.7% | (88.5–94.3%) |

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| Method comparison of the Aptiva SS-B with the predicate device | | | | | |
| --- | --- | --- | --- | --- | --- |
| | | | Predicate | | |
| | | | Positive | Negative | Total |
| **Aptiva SS-B** | Positive | ≥5.0 | 88 | 17 | 105 |
| | Negative | <5.0 | 3 | 442 | 445 |
| | Total | | 91 | 459 | 550 |

| PPA | 96.7% | (90.8–98.9%) |
| --- | --- | --- |
| NPA | 96.3% | (94.1–97.7%) |

| Method comparison of the Aptiva Scl-70 with the predicate device | | | | | |
| --- | --- | --- | --- | --- | --- |
| | | | Predicate | | |
| | | | Positive | Negative | Total |
| **Aptiva Scl-70** | Positive | ≥5.0 | 64 | 9 | 73 |
| | Negative | <5.0 | 3 | 359 | 362 |
| | Total | | 67 | 368 | 435 |

| PPA | 95.5% | (87.6–98.5%) |
| --- | --- | --- |
| NPA | 97.6% | (95.4–98.7%) |

| Method comparison of the Aptiva Jo-1 with the predicate device | | | | | |
| --- | --- | --- | --- | --- | --- |
| | | | Predicate | | |
| | | | Positive | Negative | Total |
| **Aptiva Jo-1** | Positive | ≥5.0 | 24 | 1 | 25 |
| | Negative | <5.0 | 1 | 390 | 391 |
| | Total | | 25 | 391 | 416 |

| PPA | 96.0% | (80.5–99.3%) |
| --- | --- | --- |
| NPA | 99.7% | (98.6–100%) |

| Method comparison of the Aptiva Centromere with the predicate device | | | | | |
| --- | --- | --- | --- | --- | --- |
| | | | Predicate | | |
| | | | Positive | Negative | Total |
| **Aptiva Centromere** | Positive | ≥5.0 | 101 | 6 | 107 |
| | Negative | <5.0 | 4 | 338 | 342 |
| | Total | | 105 | 344 | 449 |

| PPA | 9…