Predicate Devices

Reference Devices

AI/ML (Artificial Intelligence / Machine Learning)

No
The description details a standard immunoassay process and automated instrument operation, with no mention of AI or ML algorithms for data analysis, interpretation, or decision-making. The results are calculated based on predefined master and working curves.

Therapeutic

No

Explanation: The device is an immunoassay intended for the semiquantitative determination of specific autoantibodies in human serum, which aids in the diagnosis of celiac disease. It is a diagnostic tool, not a therapeutic one that treats or prevents a disease.

Diagnostic

Yes.

The device is an aid in the diagnosis of celiac disease and dermatitis herpetiformis by detecting specific autoantibodies in human serum.

SaMD (Software as a Medical Device)

No

The device description clearly outlines hardware components including liquid handling hardware, optical module, integrated computer, and touch screen user interface, in addition to the software.

IVD (In Vitro Diagnostic)

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

Here's why:

Intended Use: The "Intended Use / Indications for Use" section explicitly states that the device is for the "semiquantitative determination of anti-tissue transglutaminase IgG autoantibodies and anti-deamidated gliadin peptide IgG autoantibodies in human serum." It also states that the presence of these antibodies is "an aid in the diagnosis of celiac disease and dermatitis herpetiformis." This clearly indicates that the device is intended for use in examining specimens derived from the human body (serum) to provide information for the diagnosis of a disease.
Device Description: The description details a laboratory test that analyzes components of human serum using an immunoassay.
Performance Studies: The document includes performance studies evaluating the device's ability to detect these antibodies in human serum samples and its clinical performance in aiding the diagnosis of celiac disease and dermatitis herpetiformis.
Predicate Device: The mention of predicate devices (QUANTA Flash® DGP IgG and QUANTA Flash® h-tTG IgG) which are also IVDs, further supports that this device falls under the IVD category.

All these points align with the definition of an In Vitro Diagnostic device, which is a medical device intended for use in vitro for the examination of specimens derived from the human body solely or principally for the purpose of providing information concerning a physiological or pathological state, or a congenital abnormality, or to determine the safety or suitability of a transfusion or transplantation, or to monitor therapeutic measures.

PCCP (Predetermined Change Control Plan) Authorized

N/A

Overview

Intended Use / Indications for Use

The Aptiva Celiac Disease IgG Reagent is an immunoassay utilizing particle-based multi-analyte technology for the semiquantitative determination of anti-tissue transglutaminase IgG autoantibodies and anti-deamidated gliadin peptide IgG autoantibodies in human serum. The presence of these antibodies, in conjunction with clinical findings and other laboratory tests, is an aid in the diagnosis of celiac disease and dermatitis herpetiformis, particularly in patients with selective IgA deficiency.

The Aptiva Celiac Disease IgG Reagent is intended for use with the Inova Diagnostics Aptiva System.

Product codes

MVM, MST

Device Description

The Aptiva Celiac Disease IgG reagent utilizes particle based multi-analyte technology (PMAT) in a cartridge format. Each analyte (tissue transglutaminase [tTG] and deamidated gliadin peptide [DGP]) in the Aptiva Celiac Disease IgG reagent is a solid phase immunoassay utilizing fluorescent microparticles. This technology allows each of the two analytes, along with a human IgG capture antibody (IgG Control Microparticle), to be coated onto three uniquely recognizable paramagnetic microparticles, which are combined into one tube.

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

The two analyte microparticles, along with the control microparticle, are stored in the reagent cartridge under conditions that preserve the proteins in their reactive states. When the assay cartridge is ready to be used for the first time, the reagent tube seals are pierced using the cartridge lid. The reagent cartridge is then loaded onto the Aptiva instrument, where the microparticles are automatically rehydrated using buffer located within the cartridge.

A patient's serum is diluted 1:23 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 small magnet that holds 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 one more time. 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 microparticles in the cuvette, and mixed. Again, the cuvette is incubated for 9 ½ minutes at 37℃. Three wash steps, as described above, are performed on the microparticles. Following the wash steps, the microparticles are transferred to the of the instrument, where a charge coupled device (CCD) camera takes multiple images in order to identify and count the three unique microparticle regions, as well as determine the amount of conjugate on the microparticles. The control microparticle, a third particle, coated with goat anti-human IgG, is included in the reagent in as a control to flag low concentrations of IgG 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.

Mentions image processing

Yes

Mentions AI, DNN, or ML

Not Found

Input Imaging Modality

Not Found

Anatomical Site

Not Found

Indicated Patient Age Range

Not Found

Intended User / Care Setting

Prescription Use (Part 21 CFR 801 Subpart D)

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

Not Found

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

A cohort of characterized samples, none of which were used for establishing the reference range, was used to validate the clinical performance of the Aptiva Celiac Disease IgG Reagent. A total of 515 characterized samples were included in this Validation Set, including 171 samples from celiac disease patients, 20 samples from patients with IgA deficient celiac disease, 34 dermatitis herpetiformis patients and 290 control samples from patients with various types of autoimmune and infectious diseases. All samples were run on the Aptiva Celiac Disease IgG Reagent.

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

Precision Study:

Study Type: Precision evaluation in accordance with CLSI EPO5-A3.
Sample Size: 8 samples for DGP IgG and tTG IgG.
Results: Data analyzed with Analyse-it for Excel. Repeatability (within-run), between run, between day and within-laboratory precision were calculated.
- DGP IgG Precision:
  - Sample 1 (1.73 FLU): Total SD 0.15 FLU, CV 8.9%
  - Sample 2 (4.29 FLU): Total SD 0.27 FLU, CV 6.2%
  - Sample 3 (4.47 FLU): Total SD 0.29 FLU, CV 6.5%
  - Sample 4 (5.42 FLU): Total SD 0.44 FLU, CV 8.1%
  - Sample 5 (16.17 FLU): Total SD 1.01 FLU, CV 6.3%
  - Sample 6 (31.27 FLU): Total SD 1.57 FLU, CV 5.0%
  - Sample 7 (131.92 FLU): Total SD 7.16 FLU, CV 5.4%
  - Sample 8 (216.46 FLU): Total SD 17.19 FLU, CV 7.9%
- tTG IgG Precision:
  - Sample 1 (1.88 FLU): Total SD 0.14 FLU, CV 7.7%
  - Sample 2 (2.43 FLU): Total SD 0.20 FLU, CV 8.2%
  - Sample 3 (4.53 FLU): Total SD 0.30 FLU, CV 6.5%
  - Sample 4 (5.29 FLU): Total SD 0.31 FLU, CV 5.8%
  - Sample 5 (11.49 FLU): Total SD 0.62 FLU, CV 5.4%
  - Sample 6 (46.27 FLU): Total SD 2.02 FLU, CV 4.4%
  - Sample 7 (117.59 FLU): Total SD 7.49 FLU, CV 6.4%
  - Sample 8 (210.59 FLU): Total SD 17.38 FLU, CV 8.3%
- Acceptance criteria: Total %CV:

Regulation Number and Section

§ 866.5660 Multiple autoantibodies immunological test system.

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

Summary

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August 26, 2021

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

Re: K200230

Trade/Device Name: Aptiva Celiac Disease IgG Reagent Regulation Number: 21 CFR 866.5660 Regulation Name: Multiple Autoantibodies Immunological Test System Regulatory Class: Class II Product Code: MVM, MST Dated: January 27, 2020 Received: January 30, 2020

Dear Andrea Seaman:

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

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

Please be advised that FDA's issuance of a substantial equivalence determination does not mean that FDA has made a determination that your device complies with other requirements of the Act or any Federal statutes and regulations administered by other Federal agencies. You must comply with all the Act's requirements, including, but not limited to: registration and listing (21 CFR Part 807); labeling (21 CFR Part

1

801 and Part 809); medical device reporting of medical device-related adverse events) (21 CFR 803) for devices or postmarketing safety reporting (21 CFR 4, Subpart B) for combination products (see https://www.fda.gov/combination-products/guidance-regulatory-information/postmarketing-safety-reportingcombination-products); good manufacturing practice requirements as set forth in the quality systems (QS) regulation (21 CFR Part 820) for devices or current good manufacturing practices (21 CFR 4, Subpart A) for combination products; and, if applicable, the electronic product radiation control provisions (Sections 531-542 of the Act); 21 CFR 1000-1050.

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

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

Sincerely,

For

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

Enclosure

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Indications for Use

510(k) Number (if known) K200230

Device Name Aptiva Celiac Disease IgG Reagents

Indications for Use (Describe)

The Aptiva Celiac Disease IgG Reagent is an immunoassay utilizing particle-based multi-analyte technology for the semiquantitative determination of anti-tissue transglutaminase IgG autoantibodies and anti-deamidated gliadin peptide IgG autoantibodies in human serum. The presence of these antibodies, in conjunction with clinical findings and other laboratory tests, is an aid in the diagnosis of celiac disease and dermatitis herpetiformis, particularly in patients with selective IgA deficiency.

The Aptiva Celiac Disease IgG Reagent is intended for use with the Inova Diagnostics Aptiva System.

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

X Prescription Use (Part 21 CFR 801 Subpart D)

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

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510(k) Summary

Aptiva Celiac Disease IgG Reagent

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

4

This summary of the 510(k) safety and effectiveness information is being submitted in accordance with the requirements of SMDA 1990 and 21 CFR 807.92. The assigned 510K number is: K200230

Administrative data

| Submitter: | Inova Diagnostics, Inc
9900 Old Grove Road,
San Diego, CA, 92131 | | |
|------------------------------|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-----------------------------------------------------------------------------------------------------------------------------------|--|
| Purpose of submission: | New device | | |
| Device in the submission: | Aptiva Celiac Disease IgG Reagent | | |
| Revision Date: | August 23, 2021 | | |
| Scientific contact: | Andrea Seaman, Manager, Research and Development
Inova Diagnostics, Inc.
9900 Old Grove Road, San Diego, CA, 92131
Phone: 858-586-9900 x1395
Fax: 858-863-0025
Email: aseaman@inovadx.com | | |
| Quality Systems contact: | Ronda Elliott, VP, Quality Systems and RA
Inova Diagnostics, Inc
9900 Old Grove Road, San Diego, CA, 92131
Phone: 858-586-9900 x1381
Fax: 858-863-0025
Email: relliott@inovadx.com | | |
| Device name (kit): | Proprietary name: | Aptiva Celiac Disease IgG Reagent | |
| | Common name: | anti-deamidated gliadin peptide antibody
immunoassay, anti-tissue transglutaminase
antibody immunoassay | |
| | Classification name: | DGP IgG: Radioallergosorbent (RAST)
immunological test system
tTG IgG: Multiple autoantibodies immunological
test system | |
| Regulation Medical Specialty | Immunology | | |
| Review Panel | Immunology | | |
| Product Code | DGP IgG: MST | | |

5

	tTG IgG: MVM
Regulation Number	866.5750, 866.5660
Device Class	2

Predicate device

QUANTA Flash® DGP IgG, 510(k) number: K113863. Date declared: October 23, 2012. QUANTA Flash® h-tTG IgG, 510(k) number: K101644. Date declared: March 23, 2011.

Device description

The Aptiva Celiac Disease IgG reagent utilizes particle based multi-analyte technology (PMAT) in a cartridge format. Each analyte (tissue transglutaminase [tTG] and deamidated gliadin peptide [DGP]) in the Aptiva Celiac Disease IgG reagent is a solid phase immunoassay utilizing fluorescent microparticles. This technology allows each of the two analytes, along with a human IgG capture antibody (IgG Control Microparticle), to be coated onto three uniquely recognizable paramagnetic microparticles, which are combined into one tube.

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

The two analyte microparticles, along with the control microparticle, are stored in the reagent cartridge under conditions that preserve the proteins in their reactive states. When the assay cartridge is ready to be used for the first time, the reagent tube seals are pierced using the cartridge lid. The reagent cartridge is then loaded onto the Aptiva instrument, where the microparticles are automatically rehydrated using buffer located within the cartridge.

A patient's serum is diluted 1:23 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 small magnet that holds 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 one more time. 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 microparticles in the cuvette, and mixed. Again, the cuvette is incubated for 9 ½ minutes at 37℃. Three wash steps, as described above, are performed on the microparticles. Following the wash steps, the microparticles are transferred to the of the instrument, where a charge coupled device (CCD) camera takes multiple images in order to identify and count the three unique microparticle regions, as well as determine the amount of conjugate on the microparticles. The control microparticle, a third particle, coated with goat anti-human IgG, is included in the reagent in as a control to flag low concentrations of IgG 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.

6

For quantitation, the DGP IgG and tTG IgG assays (together as part of the Aptiva Celiac Disease IgG Reagent) each utilizes a predefined lot specific Master Curve that is uploaded onto the instrument through the reagent cartridge RFID tag. Every new lot of reagent cartridge must be calibrated before first use with the reagent specific calibrators. Based on the results obtained with the calibrators included in the Aptiva Celiac Disease IgG Calibrator kit (sold separately), an instrument specific Working Curve is created for each assay, which is used to calculate reported fluorescent light units (FLU) from the median fluorescent intensity (MFI) instrument signal obtained for each sample, on each of the two assays within the reagent.

Aptiva Celiac Disease IgG Calibrators and Aptiva Celiac Disease IgG Controls are sold separately.

The Aptiva Celiac Disease IgG Reagent kit contains the following materials:

One (1) Aptiva Celiac Disease IgG Reagent Cartridge, containing the following reagents for 200 determinations:

a. Aptiva Celiac Disease IgG microparticle containing 3 unique microparticle regions coated with recombinant tissue transglutaminase, deamidated gliadin peptide, or goat antihuman IgG antibody.
b. Assay buffer - colored pink, containing protein stabilizers and preservatives.
C. PE Tracer IgG - phycoerythrin (PE) labeled anti-human IgG antibody, containing buffer, protein stabilizers and preservative.
ರ. Rehydration Buffer - containing protein stabilizers and preservatives.

Intended use(s)

The Aptiva Celiac Disease IgG Reagent is an immunoassay utilizing particle-based multi-analyte technology for the semi-quantitative determination of anti-tissue transglutaminase lgG autoantibodies and anti-deamidated gliadin peptide IgG autoantibodies in human serum. The presence of these antibodies, in conjunction with clinical findings and other laboratory tests, is an aid in the diagnosis of celiac disease and dermatitis herpetiformis, particularly in patients with selective IgA deficiency.

The Aptiva Celiac Disease IgG Reagent is intended for use with the Inova Diagnostics Aptiva System.

Indications for use

Same as intended use.

Substantial equivalence

The Aptiva Celiac Disease IgG Reagent has the same intended use and assay principle as the predicate devices.

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Page 5 of 19

Comparison to predicate device

Aptiva Celiac Disease IgG Reagent – DGP IgG Assay

Similarities
Item	Aptiva Celiac Disease IgG Reagent (DGP IgG)	QUANTA Flash DGP IgG
Intended Use	The Aptiva Celiac Disease IgG Reagent is an immunoassay utilizing particle-based multi-analyte technology for the semi-quantitative determination of anti-tissue transglutaminase IgG autoantibodies and anti-deamidated gliadin peptide IgG autoantibodies in human serum. The presence of these antibodies, in conjunction with clinical findings and other laboratory tests, is an aid in the diagnosis of celiac disease and dermatitis herpetiformis, particularly in patients with selective IgA deficiency.

8

Aptiva Celiac Disease IgG Reagent – tTG IgG Assay

Similarities
Item	Aptiva Celiac Disease IgG Reagent (tTG IgG)	QUANTA Flash tTG IgG
Intended Use	The Aptiva Celiac Disease IgG Reagent is an immunoassay utilizing particle-based multi-analyte technology for the semi-quantitative determination of anti-tissue transglutaminase IgG autoantibodies and anti-deamidated gliadin peptide IgG autoantibodies in human serum. The presence of these antibodies, in conjunction with clinical findings and other laboratory tests, is an aid in the diagnosis of celiac disease and dermatitis herpetiformis, particularly in patients with selective IgA deficiency.
The Aptiva Celiac Disease IgG Reagent is intended for use with the Inova Diagnostics Aptiva System.	The QUANTA Flash h-tTG IgG is a chemiluminescent immunoassay (CIA) for the semi-quantitative detection of IgG anti-human tissue transglutaminase (h-tTG) antibodies in human serum. The presence of IgG anti-h-tTG antibodies, in conjunction with clinical findings and other laboratory tests, can aid in the diagnosis of the gluten sensitive enteropathy celiac disease, particularly in patients with selective IgA deficiency.
Assay Methodology	solid phase (heterogeneous) immunoassay	solid phase (heterogeneous) immunoassay
Antigen	recombinant tissue transglutaminase	recombinant tissue transglutaminase
Sample Type	human serum	human serum
Solid Phase	paramagnetic microparticles	paramagnetic microparticles

Page 6 of 19

9

| Item | Aptiva Celiac Disease IgG Reagent
(tTG IgG) | QUANTA Flash tTG IgG |
|----------------------------------|--------------------------------------------------------------------------------------------------------------------------------------|-----------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Detection/Operating
Principle | fluorescent immunoassay | chemiluminescent immunoassay |
| Conjugate | Phycoerythrin conjugated
polyclonal anti-human IgG
antibody | Isoluminol conjugated
monoclonal anti-human IgG
antibody |
| Units | fluorescent light units (FLU) | chemiluminescent units (CU) |
| Cut-off | 5.00 FLU | 20.0 CU |
| Analytical Measuring
Range | 0.82 FLU - 250.00 FLU | 3.75 CU - 2560.0 CU |
| Control | Controls have lot specific values
assigned.
Control 1
tTG IgA – 8.18 FLU
Control 2
tTG IgA - 16.39 FLU (9.83 – 22.95) | Controls have lot specific values
assigned.
Negative Control
h-tTG IgA - 11.4 CU (6.8 – 16.0)
Positive Control
h-tTG IgA - 50.4 CU (30.2 – 70.6) |
| Calibration | Lot specific Master Curve + 3
calibrators (sold separately) | Lot specific Master Curve + 2
calibrators (sold separately) |

Analytical performance characteristics

Quantitation and units of measure

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

Material	Assigned Value (FLU)
Celiac IgG Master Curve Standard 1	0.00
Celiac IgG Master Curve Standard 2	3.81
Celiac IgG Master Curve Standard 3	8.97
Celiac IgG Master Curve Standard 4	47.37
Celiac IgG Master Curve Standard 5	250.07

List of Aptiva Celiac Disease IgG Master Curve Standards - DGP IgG:

List of Aptiva Celiac Disease IgG Master Curve Standards – tTG IgG

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Material	Assigned Value (FLU)
Celiac IgG Master Curve Standard 1	0.00
Celiac IgG Master Curve Standard 2	2.62
Celiac IgG Master Curve Standard 3	31.14
Celiac IgG Master Curve Standard 4	73.23
Celiac IgG Master Curve Standard 5	386.85

Precision

The precision of the Aptiva Celiac Disease lgG reagent was evaluated on 8 samples for DGP IgG and tTG lgG, containing various concentrations of antibodies in accordance with CLSI EPO5-A3, Evaluation of Precision of Quantitative Measurement Procedures; Approved Guideline. Samples were run in duplicates, twice a day, for 20 days.

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

DGP IgG Precision			Repeatability		Between Run		Between Day		Within Laboratory
Sample	Replicates
(N)	Mean
(FLU)	SD
(FLU)	CV	SD
(FLU)	CV	SD
(FLU)	CV	SD
(FLU)	CV
1	80	1.73	0.1	6.1%	0.07	4.0%	0.09	5.1%	0.15	8.9%
2	80	4.29	0.17	3.9%	0.07	1.6%	0.19	4.5%	0.27	6.2%
3	80	4.47	0.17	3.7%	0.19	4.3%	0.14	3.1%	0.29	6.5%
4	80	5.42	0.23	4.3%	0.30	5.5%	0.22	4.0%	0.44	8.1%
5	80	16.17	0.38	2.3%	0.48	2.9%	0.81	5.0%	1.01	6.3%
6	80	31.27	0.81	2.6%	1.00	3.2%	0.90	2.9%	1.57	5.0%
7	80	131.92	3.25	2.5%	6.32	4.8%	0.94	0.7%	7.16	5.4%
8	80	216.46	9.76	4.5%	7.11	3.3%	12.23	5.6%	17.19	7.9%

Acceptance criteria: Total %CV: 250.00 FLU for DGP IgG or tTG IgG after performing an additional 10-fold dilution, thereby bringing the measured value within the AMR. The reported result will be calculated by the software factoring the additional dilution. As the highest value that can be measured is 2500.00 FLU for DGP IgG or tTG IgG.

14

High concentration hook effect

To assess hook effect, 3 samples for DGP lgG and 3 samples for tTG IgG were tested at two increasing 2fold serial dilutions from the standard 1:23 dilution used by the Aptiva Celiac Disease IgG Reagent. All FLU values above the analytical measuring ranges of the two assays are theoretical and were mathematically calculated using the 4 parameters of their respective calibration curves. All samples showed increase in FLU values as dilution factor became more concentrated, thereby confirming that high positive specimens above the AMR do not show hook effect up to 723.10 FLU for the DGP IgG assay and 465.52 FLU for the tTG IgG assay (theoretical values calculated) in the Aptiva Celiac Disease IgG Reagent.

Linearity

The Linearity of the AMR was calculated separately for DGP IgG as part of the Aptiva Celiac Disease IgG Reagent.

The linearity of the AMR of DGP IgG and tTG IgG was evaluated by a study according to CLSI EPO6-A, Evaluation of the Linearity of Quantitative Measurement Procedures: A Statistical Approved Guideline. Four human serum samples for DGP IgG and three human serum samples for tTG IgG with various antibody concentrations were serially diluted to obtain values that cover the entire AMR. The dilutions were assayed in duplicates. Results were analyzed according to the guideline performing regression analysis and identifying the best fitting polynomial.

Acceptance criteria:

Best fitting polynomial is a linear one, otherwise, the difference between the best-fitting nonlinear and linear polynomial is less than 15% or ±0.75 FLU for low level samples (allowable nonlinearity).

| Sample | Test Range
(FLU) | Slope
(95% CI) | Y-Intercept
(95% CI) | R² | Average % Recovery |
|----------|---------------------|------------------------|----------------------------|------|--------------------|
| 1 | 54.85 - 274.25 | 1.04
(1.00 to 1.09) | -8.73
(-16.69 to -0.77) | 0.99 | 98.0% |
| 2 | 7.13 - 71.33 | 1.04
(1.02 to 1.06) | -2.29
(-3.27 to -1.31) | 1.00 | 95.1% |
| 3 | 4.82 - 48.20 | 1.04
(1.00 to 1.08) | -0.16
(-1.26 to 0.94) | 0.99 | 101.8% |
| 4 | 0.52 - 5.19 | 1.02
(0.99 to 1.05) | -0.15
(-0.24 to -0.05) | 1.00 | 95.4% |
| Combined | 0.52 - 274.25 | 1.00
(0.99 to 1.01) | -0.50
(-1.49 to 0.50) | 1.00 | 97.6% |

For DGP IgG, the best fitting polynomial found for samples 1, 2 and 4 was a linear one, while third order polynomial was found to provide the best fit for sample 3. All acceptance criteria were fulfilled.

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| Sample | Test Range
(FLU) | Slope
(95% CI) | Y-Intercept
(95% CI) | R² | Average % Recovery |
|----------|---------------------|------------------------|---------------------------|------|--------------------|
| 1 | 32.78 - 327.80 | 0.93
(0.88 to 0.99) | 13.20
(1.59 to 24.82) | 0.99 | 103.5% |
| 2 | 7.48 - 74.77 | 1.00
(0.96 to 1.05) | 1.40
(-0.72 to 3.53) | 0.99 | 104.8% |
| 3 | 0.99 - 9.91 | 1.03
(1.00 to 1.06) | -0.22
(-0.42 to -0.02) | 1.00 | 96.3% |
| Combined | 0.99 - 327.80 | 0.98
(0.96 to 1.00) | 2.32
(-0.08 to 4.73) | 0.99 | 101.6% |

For tTG IgG, the best fitting polynomial found for samples 1, 2 and 3 was a linear one. All acceptance criteria were fulfilled.

These data demonstrate the linearity of the analytical measuring range (0.56 FLU – 250.00 FLU) of the DGP lgG assay and the analytical measuring range 0.82 FLU - 250.00 FLU) of the tTG IgG assay, both as part of the Aptiva Celiac Disease IgG Reagent.

Interference

The interference study was performed according to CLSI EPO7-A2, Interference Testing in Clinical Chemistry; Approved Guideline - Second Edition. A set of three human serum specimens, one positive, one near the cutoff and one negative sample were tested using the following interfering substances (bilirubin, hemoglobin, triglycerides, cholesterol, rheumatoid factor IgM and human IgG). All interferents were spiked into every serum specimen and the resulting samples were assessed in triplicates with the Aptiva Celiac Disease IgG assays. Recovery of the unit values was calculated compared to control samples. Acceptance criteria for the interference studies were 85% - 115% recovery, or ± 20% of the cut-off (±1.0 FLU) difference, whichever is greater.

Less than 15% of interference was observed for DGP or tTG IgG with the following interferents: bilirubin up to 1 mg/mL (recovery: from 96.0%-101.3% and from 97.7% to 102.5% for DGP lgG and tTG IgG respectively), hemoglobin up to 2 mg/mL (recovery: from 97.6% to 104.2% and from 96.9% to 100.8% for DGP IgG and tTG IgG respectively), triglycerides up to 1000 mg/dL (recovery: from 93.8% to 102.5% and from 94.5% to 100.8% for DGP IgG and tTG IgG respectively), cholesterol up to 332.5 mg/dL (recovery: from 85.4% to 98.7% and from 89.2% to 100.4% for DGP IgG and tTG IgG respectively), RF IgM up to 250 IU/mL (recovery: 95.4% to 97.6 and 92.1% to 97.3% for DGP IgG and tTG IgG, respectively) and human lgG up to 35 mg/mL (recovery: from 105.0% to 114.1% or -0.80 FLU and from 101.7 to 110.8% or 0.47 FLU for DGP IgG and tTG IgG, respectively).

Sample Stability and Handling

For the DGP IgG assay, six test samples were tested and for tTG IgG assay seven test samples were tested. The samples used for this study were achieved by combining high and low antibody level to yield their desired reactivity. Test samples covered the analytical measuring ranges of each analyte. All samples were tested in duplicates for up to 21 days while stored at 2-8℃, up to 48 hours while stored at room

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temperature, and after repeated freeze/thaw cycles up to 6 cycles. Results were compared to those obtained on control samples (time zero / zero cycles).

Acceptance criteria: percent recovery is between 85-115% for positive samples, and between 80-120% for negative samples ( Negative 5.00 FLU

The reference population for establishing the reference interval for the DGP JgG assays, within the Aptiva Celiac Disease IgG Reagent, consisted of 192 subjects:

Sample Group	N
Crohn's Disease	15
Autoimmune Thyroid Disease	29
Infectious Disease	30
Primary Biliary Cholangitis	30
Rheumatoid Arthritis	40

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Systemic Lupus Erythematosus	13
Systemic Sclerosis	20
Ulcerative Colitis	15

Additionally, 11 diagnosed celiac disease (CD) patient specimens were assayed to aid in the determination of the cutoff values.

All specimens were the same matrix (human serum) as specified in the Intended Use. All specimens were unaltered. The cut-off values were established in accordance to CLSI EP28-A3c: Defining, Establishing, and Verifying Reference Intervals in the Clinical Laboratory; Approved Guideline - Third Edition. The Analyseit for Excel software was used to make the calculations. The results was non-normal (Shapiro-Wilk p