(264 days)
QUANTA Flash CCP3 is a chemiluminescent immunoassay for the semi-quantitative determination of IgG anti-CCP3 antibodies in human serum. The presence of anti-CCP3 antibodies, in conjunction with clinical findings and other laboratory tests, is an aid in the diagnosis of rheumatoid arthritis.
QUANTA Flash CCP3 Calibrators are intended for use with the QUANTA Flash CCP3 chemiluminescent immunoassay for the determination of IgG anti-CCP3 antibodies in human serum. Each calibrator establishes a point of reference for the working curve that is used to calculate unit values.
QUANTA Flash CCP3 Controls are intended for use with the QUANTA Flash CCP3 chemiluminescent immunoassay for quality control in the determination of IgG anti-CCP3 antibodies in human serum.
The QUANTA Flash CCP3 assay is designed to run on the BIO-FLASH® instrument. This platform is a fully automated closed system with continuous load and random access capabilities that automatically processes the samples, runs the assay and reports the results. It includes liquid handling hardware, luminometer and computer with software-user interface. The QUANTA Flash CCP3 assay utilizes a reagent cartridge format, which is compatible with the BIO-FLASH instrument.
Synthetic cyclic citrullinated peptide is coated onto paramagnetic beads. The bead suspension is lyophilized and stored in the bead tube. Prior to use in the BIO-FLASH system, the sealed reagent tubes are pierced with the reagent cartridge lid and the beads are rehydrated and resuspended using resuspension buffer by pipetting up and down with a transfer pipette. The reagent cartridge is then loaded onto the BIO-FLASH instrument. Samples are also loaded onto the instrument in sample racks. Serum samples are diluted by the BIO-FLASH with system rinse in a small disposable plastic cuvette. Small amounts of the diluted patient serum, the beads, and assay buffer are combined into a second cuvette, and mixed. This cuvette is then incubated at 37°C. The beads are magnetized and washed several times. Isoluminol conjugated anti-human IgG antibodies are then added to the cuvette, and again incubated at 37°C. The beads are magnetized and washed repeatedly. The isoluminol conjugate is oxidized when Trigger 1 (Fe(II)coproporphyrin in sodium hydroxide solution) and Trigger 2 (ureahydrogen peroxide in sodium chloride solution) are added to the flash of light produced from this reaction is measured as Relative Light Units (RLU) by the BIO-FLASH optical system. The RLU are proportional to the amount of isoluminol conjugate that is bound to the human IgG, which is in turn proportional to the amount of anti-CCP3 antibodies bound to the corresponding beads.
For quantitation, the QUANTA Flash CCP3 assay utilizes a predefined lot specific Master Curve that is uploaded onto the instrument through the reagent cartridge barcode. Every new lot number of reagent cartridge must be calibrated before first use, with the QUANTA Flash CCP3 Calibrators. Based on the results obtained with the two Calibrators included in the Calibrator Set (sold separately), an instrument specific Working Curve is created, which is used to calculate chemiluminescent units (CU) from the instrument signal (RLU) obtained for each sample.
The QUANTA Flash CCP3 kit contains the following materials:
One (1) QUANTA Flash CCP3 Reagent Cartridge One (1) vial of Resuspension buffer One (1) Transfer pipette
The QUANTA Flash CCP3 reagent cartridge contains the following reagents for 100 determinations:
- a. CCP3 coated paramagnetic beads, lyophilized.
- b. Assay buffer - colored pink, containing Tris-buffered saline, Tween 20, protein stabilizers and preservatives.
- C. Tracer IgG - Isoluminol labeled anti-human IgG antibodies in buffer, containing protein stabilizers and preservative.
The QUANTA Flash CCP3 Calibrators kit contains two vials of Calibrator 1 and two vials of Calibrator 2:
QUANTA Flash CCP3 Calibrators:
-
QUANTA Flash CCP3 Calibrator 1: Two (2) barcode labeled tubes containing 0.7 mL prediluted, ready to use reagent. Calibrators contain human antibodies to CCP3 in stabilizer and preservative.
-
QUANTA Flash CCP3 Calibrator 2: Two (2) barcode labeled tubes containing 0.7 mL prediluted, ready to use reagent. Calibrators contain human antibodies to CCP3 in stabilizer and preservative.
The QUANTA Flash CCP3 Controls kit contains two vials of Negative Control and two vials of Positive Control:
QUANTA Flash CCP3 Controls:
- QUANTA Flash CCP3 Negative Control: Two (2) barcode labeled tubes containing 0.5 mL, ready to use reagent. Controls contain human antibodies to CCP3 in stabilizer and preservative.
- QUANTA Flash CCP3 Positive Control: Two (2) barcode labeled tubes containing 0.5 mL, ready to use reagent. Controls contain human antibodies to CCP3 in stabilizer and preservative.
The provided text describes the QUANTA Flash® CCP3 chemiluminescent immunoassay and its associated calibrators and controls. The study focuses on establishing the substantial equivalence of the new device to a predicate device (QUANTA Lite® CCP3 IgG ELISA) and demonstrating its analytical and clinical performance characteristics.
Here's an analysis of the acceptance criteria and study as requested:
1. Table of Acceptance Criteria and Reported Device Performance
The document details numerous acceptance criteria for various analytical and clinical performance characteristics. Below is a summary of some key areas:
| Performance Characteristic | Acceptance Criteria | Reported Device Performance |
|---|---|---|
| Precision | Total %CV: < 10% | All samples met this, with total %CV ranging from 5.0% to 6.8%. |
| Reproducibility | Total %CV: < 10% | All samples met this, with total %CV ranging from 0.0% to 3.2%. |
| Linearity (Recovery) | 80-120% recovery, or ± 4 CU (whichever is greater) | All five specimens showed dilution linearity individually. Specific recovery values are not explicitly stated for each dilution, but the overall conclusion is that all specimens met the criteria. |
| Linearity (Regression) | Slope: 0.9-1.1, R²: ≥ 0.95 | Individual samples slopes ranged from 0.94 to 1.00 (with CIs largely within 0.9-1.1), and R² values were 0.98 or 0.99. Combined data showed a slope of 1.07 (CI 1.05-1.10) and R² of 0.99. |
| Interference | 85-115% recovery, or ± 4 CU (whichever is greater) | No interference detected with bilirubin (101-102% recovery), hemoglobin (97-108% recovery), triglycerides (98-111% recovery), and cholesterol (98-111% recovery). |
| Cross-reactivity | Not explicitly stated as a quantitative criterion, but implied as a low positivity rate in non-RA conditions. | Out of 234 samples from various autoimmune diseases and infectious conditions, only 5 (2.1%) tested positive, indicating low cross-reactivity. |
| Lot to Lot Comparison | Between lot %CV: < 10% | All samples met this, with between lot %CV ranging from 2.8% to 4.0%. |
| Shelf Life (Accelerated Stability - Beads & Resuspension Buffer) | Lower 95% CI of regression line ≥ 85% and upper 95% CI ≤ 115% at day 14; no individual data point ≤ 75% or ≥ 125% at day 14. | All three lots of beads and resuspension buffer met these criteria, retaining 85-115% reactivity. |
| Shelf Life (Accelerated Stability - Calibrators & Controls) | Lower 95% CI of regression line ≥ 90% and upper 95% CI ≤ 110% at day 14; no individual data point ≤ 80% or ≥ 120% at day 14. | All three lots of calibrators and controls met these criteria, maintaining 90-110% reactivity. |
| In-use Stability (Calibrators) | 5 successful calibrations in 8.5 hours; average RLU recovery 90-110% compared to first use. | All 5 successful calibrations and RLU values remained within 90-110%, supporting a claim of up to 4 calibrations over 8 hours. |
| In-use Stability (Controls) | All values run within established range; linear regression line for %recovery 85-115% at run 15. | All controls ran within acceptable ranges and regression line remained between 85-115% at run 15, supporting usage for up to 15 times. |
| In-use Stability (Reagent Cartridge) | Regression line 95% CI reaches 85% or 115% recovery; OR 2 data points or ≥2% of recovery data is ≤ 75% or ≥ 125% recovery. | Onboard stability determined to be 60 days based on individual lot results (63-83 days). |
| Real-time Stability | Results within respective QC ranges; % recovery 85-115% for calibrators, %CV < 10%; results within acceptable ranges for controls. | All results to date (6 months for reagent cartridge, 9 months for calibrators and controls) were within acceptance limits. |
| Clinical Sensitivity for RA | Not explicitly stated as a single numerical acceptance criteria within the document, but is part of the clinical performance evaluation. | 70.7% (95% CI: 65.8-75.2%) |
| Clinical Specificity for RA | Not explicitly stated as a single numerical acceptance criteria within the document, but is part of the clinical performance evaluation. | 96.5% (95% CI: 94.2-98.0%) |
| Method Comparison (Positive Agreement) | Not explicitly stated. | 92.7% (89.0-95.3%) for all samples; 94.2% (90.3-96.6%) for samples within AMR. |
| Method Comparison (Negative Agreement) | Not explicitly stated. | 95.9% (93.7-97.4%) for all samples; 90.3% (85.4-93.7%) for samples within AMR. |
| Method Comparison (Total Agreement) | Not explicitly stated. | 94.8% (92.9-96.2%) for all samples; 92.4% (89.4-94.6%) for samples within AMR. |
2. Sample Size Used for the Test Set and the Data Provenance
-
Test Set for Clinical Performance (Sensitivity/Specificity, Method Comparison):
- Sample Size: 728 characterized samples (352 Rheumatoid Arthritis (RA) samples and 376 control samples, which include various other diseases and healthy individuals).
- Data Provenance: Not explicitly stated regarding country of origin. The study implies these are patient samples ("clinical validation study," "cohort of characterized samples"). It's a retrospective analysis of previously collected and characterized samples.
-
Test Set for Cross-reactivity:
- Sample Size: 234 control samples (subset of the 376 total control samples).
- Data Provenance: Not explicitly stated regarding country of origin, appears to be retrospective.
-
Test Set for Precision: 8 samples.
-
Test Set for Reproducibility: 8 (3+5) samples.
-
Test Set for Linearity: 5 serum samples.
-
Test Set for Interference: 3 specimens.
-
Test Set for Lot-to-Lot Comparison: 5 unique samples.
-
Test Set for Shelf Life (Accelerated, In-Use, Real-time): "up to 9 characterized samples with various reactivity levels," "up to 6 characterized samples," "QC panel samples."
-
Test Set for Reference Range (Cut-off): 210 subjects (170 apparently healthy blood donors, 20 viral hepatitis positive, 20 autoimmune thyroid disease).
-
Test Set for Expected Values: 146 apparently healthy blood donors (different from the cut-off establishment cohort).
3. Number of Experts Used to Establish the Ground Truth for the Test Set and the Qualifications of Those Experts
The document does not explicitly state the number or qualifications of experts used to establish the ground truth for the clinical diagnosis of the test set (e.g., confirming RA diagnosis for the 352 RA patients). It refers to the samples as "characterized samples." This suggests that their disease status was determined by standard diagnostic procedures, likely involving clinicians and laboratory specialists, but the specific details are not provided in this summary.
4. Adjudication Method for the Test Set
The document does not describe a formal adjudication method (e.g., 2+1, 3+1) for the clinical diagnosis of the test set samples. The samples are referred to as "characterized samples," implying their diagnosis was already established prior to their use in this study.
5. If a Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study Was Done, If So, What Was the Effect Size of How Much Human Readers Improve with AI vs Without AI Assistance
This document describes an in vitro diagnostic (IVD) device, specifically a chemiluminescent immunoassay for semi-quantitative determination of antibodies. It is a laboratory test, not an imaging device or AI algorithm intended for interpretation by human readers. Therefore, an MRMC comparative effectiveness study where human readers (e.g., radiologists) improve with AI assistance is not applicable to this type of device and study.
6. If a Standalone (i.e., algorithm only without human-in-the-loop performance) Was Done
Yes, the study primarily demonstrates the standalone performance of the QUANTA Flash® CCP3 assay. It evaluates the assay's ability to accurately detect anti-CCP3 antibodies in human serum, with the BIO-FLASH® instrument performing the automated processing and reporting. The "human-in-the-loop" aspect is limited to laboratory personnel operating the instrument and interpreting the reported numerical results based on established cut-offs, rather than an interactive AI interpretation.
7. The Type of Ground Truth Used (Expert Consensus, Pathology, Outcomes Data, etc.)
The ground truth for the clinical performance characteristics (sensitivity, specificity) relies on the clinical diagnosis of the patient samples:
- Rheumatoid Arthritis (RA) patients: Diagnosed by unspecified clinical criteria, likely rheumatologist diagnosis based on established classification criteria for RA.
- Control samples: Patients with various other diseases or apparently healthy blood donors, where the absence of RA (or other specific autoantibodies/infections for cross-reactivity) serves as the ground truth.
For analytical characteristics (precision, linearity, interference, etc.), the ground truth is often established by reference materials, defined concentrations, or established spiking protocols (e.g., known concentrations of interfering substances).
8. The Sample Size for the Training Set
The document does not explicitly describe a separate "training set" in the context typically used for machine learning algorithms. This is a traditional IVD device, not an AI algorithm that learns from data.
However, the concept of "training" or "calibration" is present in the context of the assay's internal calibration and master curve generation:
- Master Curve Standards: The QUANTA Flash CCP3 assay uses 7 "Master Curve Standards" with assigned CU values (e.g., 4.6 CU to 2776.8 CU) to create a lot-specific Master Curve. This could be considered analogous to a calibration/training data set for the instrument's internal quantitation.
- Calibrators: Two calibrators are used with each new reagent cartridge lot to create an instrument-specific Working Curve.
9. How the Ground Truth for the Training Set Was Established
Given that this is an immunoassay, not an AI algorithm, the "ground truth" for its internal calibration (Master Curve Standards and Calibrators) is established through:
- Assigned Values: The 7 Master Curve Standards have "Assigned Values" in CU (Chemiluminescent units), determined during the manufacturing process.
- Traceability: Calibrator and Control values are "directly traceable to the in-house Standards that are used to create the Master Curves."
- Reference Material: Traceability to an external reference material was also established by testing the IUIS-Centers for Disease Control and Prevention (CDC) reference reagent for ACPA, which has an assigned value of 100 U/mL. This allows for conversion between CU and U/mL.
Essentially, the "ground truth" for the device's internal quantitation is derived from a hierarchy of internal standards, calibrated and assigned specific values during manufacturing, and traceable to external reference materials.
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Food and Drug Administration 10903 New Hampshire Avenue Document Control Center - WO66-G609 Silver Spring, MD 20993-0002
October 23, 2015
INOVA DIAGNOSTICS, INC. DR. GABRIELLA LAKOS DIRECTOR, RESEARCH AND DEVELOPMENT 9900 OLD GROVE ROAD SAN DIEGO, CA 92131-1638
Re: K143754 Trade/Device Name: Quanta Flash® CCP3 Quanta Flash® CCP3 Calibrators Quanta Flash® CCP3 Controls Regulation Number: 21 CFR 866.5775 Regulation Name: Rheumatoid Factor Immunological Test System Regulatory Class: Class II Product Code: NHX, JIT, JIT, JJX Dated: August 19, 2015 Received: August 20, 2015
Dear Dr. Lakos:
This letter corrects our substantially equivalent letter of September 21, 2015.
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. 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
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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 Parts 801 and 809); medical device reporting (reporting of medical device-related adverse events) (21 CFR 803); good manufacturing practice requirements as set forth in the quality systems (QS) regulation (21 CFR Part 820); and if applicable, the electronic product radiation control provisions (Sections 531-542 of the Act); 21 CFR 1000-1050.
If you desire specific advice for your device on our labeling regulation (21 CFR Part 801 and 809), please contact the Division of Industry and Consumer Education at its toll-free number (800) 638-2041 or (301) 796-7100 or at its Internet address
http://www.fda.gov/MedicalDevices/Resourcesfor You/Industry/default.htm. Also, please note the regulation entitled. "Misbranding by reference to premarket notification" (21CFR Part 807.97). For questions regarding the reporting of adverse events under the MDR regulation (21 CFR Part 803), please go to
http://www.fda.gov/MedicalDevices/Safety/ReportaProblem/default.htm for the CDRH's Office of Surveillance and Biometrics/Division of Postmarket Surveillance.
You may obtain other general information on your responsibilities under the Act from the Division of Industry and Consumer Education at its toll-free number (800) 638-2041 or (301) 796-7100 or at its Internet address
http://www.fda.gov/MedicalDevices/ResourcesforYou/Industry/default.htm.
Sincerely yours,
Leonthena R. Carrington -S
Leonthena R. Carrington, MS, MBA, MT(ASCP) Director Division of Immunology and Hematology Devices Office of In Vitro Diagnostics and Radiological Health Center for Devices and Radiological Health
Enclosure
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Indications for Use
510(k) Number (if known) K143754
Device Name
QUANTA Flash® CCP3, QUANTA Flash® CCP3 Calibrators, QUANTA Flash® CCP3 Controls
Indications for Use (Describe)
QUANTA Flash CCP3 is a chemiluminescent immunoassay for the semi-quantitative determination of IgG anti-CCP3 antibodies in human serum. The presence of anti-CCP3 antibodies, in conjunction with clinical findings and other laboratory tests, is an aid in the diagnosis of rheumatoid arthritis.
QUANTA Flash CCP3 Calibrators are intended for use with the QUANTA Flash CCP3 chemiluminescent immunoassay for the determination of IgG anti-CCP3 antibodies in human serum. Each calibrator establishes a point of reference for the working curve that is used to calculate unit values.
QUANTA Flash CCP3 Controls are intended for use with the QUANTA Flash CCP3 chemiluminescent immunoassay for quality control in the determination of IgG anti-CCP3 antibodies in human serum.
| Type of Use (Select one or both, as applicable) | |
|---|---|
| ☒ Prescription Use (Part 21 CFR 801 Subpart D) | |
| ☐ Over-The-Counter Use (21 CFR 801 Subpart C) |
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510(k) Summary
QUANTA Flash® CCP3 QUANTA Flash® CCP3 Calibrators QUANTA Flash® CCP3 Controls
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 | |
| Value assignment and traceability of Calibrators and Controls | |
| Precision | |
| Reproducibility | |
| Limit of Blank (LoB) and Limit of Detection (LoD) | |
| Analytical Measuring Range (AMR) | |
| Auto-rerun function and reportable results | |
| High concentration hook effect | |
| Linearity | |
| Interference | |
| Cross-reactivity | |
| Lot to lot comparison | |
| Stability | |
| Cut-off, reference range | |
| Clinical performance characteristics | |
| Clinical sensitivity, specificity | |
| Expected values |
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Comparison with predicate device ............................................................................................................................................
This summary of the 510(k) safety and effectiveness information is being submitted in accordance with the requirements of SMDA 1990 and 21 CFR 807.92.
Administrative data
| Submitter: | INOVA Diagnostics, Inc9900 Old Grove Road,San Diego, CA, 92131 |
|---|---|
| Purpose of submission: | New device(s) |
| Devices in the submission: | QUANTA Flash® CCP3QUANTA Flash® CCP3 CalibratorsQUANTA Flash® CCP3 Controls |
| Scientific contact: | Gabriella Lakos, Director of Research, RheumatologyInova Diagnostics, Inc.9900 Old Grove Road, San Diego, CA, 92131Phone: 858-586-9900/1393Fax: 858-863-0025email: glakos@inovadx.com |
| Quality Systems contact: | Ronda Elliott, VP, Quality Systems and RAInova Diagnostics, Inc9900 Old Grove Road, San Diego, CA, 92131Phone: 858-586-9900/1381Fax: 858-863-0025email: relliot@inovadx.com |
| Device name (assay kit): | Proprietary name: QUANTA Flash® CCP3Common name: Anti-CCP3 Chemiluminescent ImmunoassayClassification name: antibodies, anti-cyclic citrullinated peptide (ccp) |
| Regulation Description | Rheumatoid factor immunological test system |
| Regulation Medical Specialty | Immunology |
| Review Panel | Immunology |
| Product Code | NHX |
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| Regulation Number | 866.5775 | |
|---|---|---|
| Device Class | 2 | |
| Device name (Calibrators): | Proprietary name:Common name:Classification name: | QUANTA Flash® CCP3 CalibratorsCCP3 CalibratorsCalibrator, secondary |
| Regulation Description | Calibrator | |
| Regulation Medical Specialty | Clinical Chemistry | |
| Product Code | JIT | |
| Regulation Number | 862.1150 | |
| Device Class | 2 | |
| Device name (Controls): | Proprietary name:Common name:Classification name: | QUANTA Flash® CCP3 ControlsCCP3 Controlssingle (specified) analyte controls (assayed andunassayed) |
| Regulation Description | Quality control material (assayed and unassayed) | |
| Regulation Medical Specialty | Clinical Chemistry | |
| Product Code | JJX | |
| Regulation Number | 862.1660 | |
| Device Class | 1 (reserved) |
Predicate device
QUANTA Lite® CCP3 IgG ELISA, 510(k) number: K052264
Device description
The QUANTA Flash CCP3 assay is designed to run on the BIO-FLASH® instrument. This platform is a fully automated closed system with continuous load and random access capabilities that automatically processes the samples, runs the assay and reports the results. It includes liquid handling hardware, luminometer and computer with software-user interface. The QUANTA Flash CCP3 assay utilizes a reagent cartridge format, which is compatible with the BIO-FLASH instrument.
Synthetic cyclic citrullinated peptide is coated onto paramagnetic beads. The bead suspension is
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lyophilized and stored in the bead tube. Prior to use in the BIO-FLASH system, the sealed reagent tubes are pierced with the reagent cartridge lid and the beads are rehydrated and resuspended using resuspension buffer by pipetting up and down with a transfer pipette. The reagent cartridge is then loaded onto the BIO-FLASH instrument. Samples are also loaded onto the instrument in sample racks. Serum samples are diluted by the BIO-FLASH with system rinse in a small disposable plastic cuvette. Small amounts of the diluted patient serum, the beads, and assay buffer are combined into a second cuvette, and mixed. This cuvette is then incubated at 37°C. The beads are magnetized and washed several times. Isoluminol conjugated anti-human IgG antibodies are then added to the cuvette, and again incubated at 37°C. The beads are magnetized and washed repeatedly. The isoluminol conjugate is oxidized when Trigger 1 (Fe(II)coproporphyrin in sodium hydroxide solution) and Trigger 2 (ureahydrogen peroxide in sodium chloride solution) are added to the flash of light produced from this reaction is measured as Relative Light Units (RLU) by the BIO-FLASH optical system. The RLU are proportional to the amount of isoluminol conjugate that is bound to the human IgG, which is in turn proportional to the amount of anti-CCP3 antibodies bound to the corresponding beads.
For quantitation, the QUANTA Flash CCP3 assay utilizes a predefined lot specific Master Curve that is uploaded onto the instrument through the reagent cartridge barcode. Every new lot number of reagent cartridge must be calibrated before first use, with the QUANTA Flash CCP3 Calibrators. Based on the results obtained with the two Calibrators included in the Calibrator Set (sold separately), an instrument specific Working Curve is created, which is used to calculate chemiluminescent units (CU) from the instrument signal (RLU) obtained for each sample.
The QUANTA Flash CCP3 kit contains the following materials:
One (1) QUANTA Flash CCP3 Reagent Cartridge One (1) vial of Resuspension buffer One (1) Transfer pipette
The QUANTA Flash CCP3 reagent cartridge contains the following reagents for 100 determinations:
- a. CCP3 coated paramagnetic beads, lyophilized.
- b. Assay buffer - colored pink, containing Tris-buffered saline, Tween 20, protein stabilizers and preservatives.
- C. Tracer IgG - Isoluminol labeled anti-human IgG antibodies in buffer, containing protein stabilizers and preservative.
The QUANTA Flash CCP3 Calibrators kit contains two vials of Calibrator 1 and two vials of Calibrator 2:
QUANTA Flash CCP3 Calibrators:
- QUANTA Flash CCP3 Calibrator 1: Two (2) barcode labeled tubes containing 0.7 mL prediluted, ready to use reagent. Calibrators contain human antibodies to CCP3 in stabilizer
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and preservative.
- QUANTA Flash CCP3 Calibrator 2: Two (2) barcode labeled tubes containing 0.7 mL । prediluted, ready to use reagent. Calibrators contain human antibodies to CCP3 in stabilizer and preservative.
The QUANTA Flash CCP3 Controls kit contains two vials of Negative Control and two vials of Positive Control:
QUANTA Flash CCP3 Controls:
- QUANTA Flash CCP3 Negative Control: Two (2) barcode labeled tubes containing 0.5 mL, ready to use reagent. Controls contain human antibodies to CCP3 in stabilizer and preservative.
- । QUANTA Flash CCP3 Positive Control: Two (2) barcode labeled tubes containing 0.5 mL, ready to use reagent. Controls contain human antibodies to CCP3 in stabilizer and preservative.
Intended use(s)
QUANTA Flash CCP3 is a chemiluminescent immunoassay for the semi-quantitative determination of IgG anti-CCP3 antibodies in human serum. The presence of antibodies, in conjunction with clinical findings and other laboratory tests, is an aid in the diagnosis of rheumatoid arthritis.
QUANTA Flash CCP3 Calibrators are intended for use with the QUANTA Flash CCP3 chemiluminescent immunoassay for the determination of IgG anti-CCP3 antibodies in human serum. Each calibrator establishes a point of reference for the working curve that is used to calculate unit values.
QUANTA Flash CCP3 Controls are intended for use with the QUANTA Flash CCP3 chemiluminescent immunoassay for quality control in the determination of IgG antibodies in human serum.
Indications for use
Same as Intended use.
Substantial equivalence
The QUANTA Flash CCP3 Reagent, the QUANTA Flash CCP3 Calibrators and the QUANTA Flash CCP3 Controls have the same intended use and assay principle as the predicate device.
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Comparison to predicate device
QUANTA Flash CCP3 reagent kit
| Similarities | ||
|---|---|---|
| Item | QUANTA Flash CCP3 | Predicate Device |
| Intended use | Semi-quantitative determination of anti-CCP3 antibodies in human serum | Semi-quantitative detection of anti-CCP3 antibodies in human serum, citrated and EDTA plasma |
| Assay methodology | Solid phase (heterogeneous) immunoassay | Solid phase (heterogeneous) immunoassay |
| Antigen | Synthetic peptide | Synthetic peptide |
| Shelf life | One year | One year |
| Differences | ||
|---|---|---|
| Item | QUANTA Flash CCP3 | Predicate Device |
| Detection/Operating principle | Chemiluminescent immunoassay | Enzyme-linked immunosorbent assay |
| Sample type | Serum | Serum; citrated or EDTA plasma |
| Solid phase | Paramagnetic microparticles (beads) | 96-well polystyrene plate |
| Conjugate | Isoluminol conjugated anti-humanIgG | HRP conjugated anti-human IgG |
| Calibration | Lot specific Master Curve + twocalibrators (sold separately) | CCP3 ELISA Low Positive, andCalibrators A through E(Included in the kit) |
QUANTA Flash CCP3 Calibrators
| Item | QUANTA Flash CCP3 Calibrators | Predicate Device |
|---|---|---|
| Intended use | For use with the QUANTA Flash CCP3Reagents Each calibrator establishesa point of reference for the workingcurve that is used to calculate unitvalues. | No separate intended use; calibratorsare part of the kit. |
| Analyte | Anti-CCP3 antibodies | Anti-CCP3 antibodies |
| Method | QUANTA Flash CCP3chemiluminescent immunoassay | QUANTA Lite CCP3 IgG ELISA |
| Matrix | Human serum, stabilizer, andpreservative | Human serum, buffer, proteinstabilizer, and preservative |
| Unit | CU (Chemiluminescent units)(arbitrary)U/mL (based on IUIS – CDC referencereagent) | units (arbitrary) |
| Physico-chemical | Liquid, prediluted, ready to use | Liquid, prediluted, ready to use |
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| Item | QUANTA Flash CCP3 Calibrators | Predicate Device |
|---|---|---|
| characteristics | ||
| Storage | 2-8 °C | 2-8 °C |
| Shelf life | One year | One year |
QUANTA Flash CCP3 Controls
| Item | QUANTA Flash CCP3 Controls | Predicate Device |
|---|---|---|
| Intended use | For use with the QUANTA Flash CCP3 reagents for quality control in the determination of IgG anti-CCP3 autoantibodies in human serum. | No separate intended use; controls are part of the kit. |
| Analyte | Anti-CCP3 antibodies | Anti-CCP3 antibodies |
| Method | QUANTA Flash CCP3 chemiluminescent immunoassay | QUANTA Lite CCP3 IgG ELISA |
| Matrix | Human serum, stabilizers, and preservative | Human serum, buffer, protein stabilizer, and preservative |
| Unit | CU (Chemiluminescent units) (arbitrary)U/mL (based on IUIS – CDC reference reagent) | units (arbitrary) |
| Physico-chemical characteristics | Liquid, ready to use | Liquid, prediluted, ready to use |
| Levels | 2 (negative and positive) | 2 (ELISA negative and High positive) |
| Storage | 2-8 °C | 2-8 °C |
| Shelf life | One year | One year |
Analytical performance characteristics
Quantitation and units of measure
For quantitation, the QUANTA Flash CCP3 assay utilizes a lot specific Master Curve that is uploaded onto the instrument through the reagent cartridge barcode. The Master Curve for QUANTA Flash CCP3 consists of 7 Standards. These Master Curve Standards are used to create the lot specific Master Curve during the manufacturing procedure.
List of CCP3 Standards:
| Material | Assigned Value |
|---|---|
| CCP3 Master Curve Standard 1 | 4.6 CU |
| CCP3 Master Curve Standard 2 | 14.6 CU |
| CCP3 Master Curve Standard 3 | 42.2 CU |
| CCP3 Master Curve Standard 4 | 124.3 CU |
| CCP3 Master Curve Standard 5 | 313.7 CU |
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| CCP3 Master Curve Standard 6 | 996.1 CU |
|---|---|
| CCP3 Master Curve Standard 7 | 2776.8 CU |
To establish traceability to a reference material, the IUIS-Centers for Disease Control and Prevention (CDC) reference reagent for anti-citrullinated peptide/protein antibodies (ACPA) (P/N IS2723 L/N 08-0202) was tested, and the value of 379.5 CU was obtained. The reference reagent has an assigned value of 100 U/mL.
Using the 100 U/mL assigned value of the reference material, the correlation between CU and U/mL values was established. Based on the obtained correlation, the BIO-FLASH software is able to automatically convert anti-CCP3 CU values into U/mL values. The 20 CU cutoff value equals 5.3 U/mL.
Value assignment and traceability of Calibrators and Controls
The QUANTA Flash CCP3 Calibrators and Controls are manufactured by diluting human serum that contains high titer of antibodies with stabilizer and preservative. The human serum is obtained from commercial sources and it is tested for markers of infectious substances.
The target CU is achieved through trial dilutions on small scale. Once a dilution is selected, the Calibrators and Control are bulked, tested, and adjusted. Upon completion of the manufacturing process, the Calibrators and Controls are tested on at least two instruments, on at least two lots of reagent cartridge, in replicates of 10 to determine final value assignment.
Calibrator and Control values are directly traceable to the in-house Standards that are used to create the Master Curves for the QUANTA Flash CCP3 assay.
| Material | ManufacturingTarget Value | ManufacturingTarget Range |
|---|---|---|
| CCP3 Calibrator 1 | 14 CU | 10-16 CU |
| CCP3 Calibrator 2 | 300 CU | 270 - 330 CU |
| CCP3 Negative Control | 10 CU | 8-12 CU |
| CCP3 Positive Control | 50 CU | 40-60 CU |
CCP3 Calibrators and Controls with target manufacturing values:
Precision
The precision of the QUANTA Flash CCP3 assay was evaluated on 8 samples containing various concentrations of CCP3 antibodies in accordance with CLSI EP05-A2, Evaluation of Precision Performance of Quantitative Measurement Methods; 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 within run, between run, between day and total precision were calculated.
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| QUANTA Flash CCP3 | Within Run | Between Runs | Between Days | Total | ||||||
|---|---|---|---|---|---|---|---|---|---|---|
| Sample ID | Number of replicates | Mean (CU) | SD (CU) | CV (%) | SD (CU) | CV (%) | SD (CU) | CV (%) | SD (CU) | CV (%) |
| 1 | 80 | 11.7 | 0.7 | 5.8 | 0.0 | 0.0 | 0.4 | 3.5 | 0.8 | 6.8 |
| 2 | 80 | 21.3 | 1.0 | 4.6 | 0.7 | 3.2 | 0.7 | 3.5 | 1.4 | 6.6 |
| 3 | 80 | 21.4 | 0.8 | 3.6 | 0.6 | 2.8 | 0.9 | 4.1 | 1.3 | 6.1 |
| 4 | 80 | 23.0 | 1.0 | 4.4 | 0.0 | 0.0 | 0.6 | 2.8 | 1.2 | 5.2 |
| 5 | 80 | 55.9 | 1.4 | 2.6 | 1.1 | 1.9 | 2.1 | 3.8 | 2.8 | 5.0 |
| 6 | 80 | 195.1 | 8.8 | 4.5 | 6.0 | 3.1 | 1.3 | 0.6 | 10.7 | 5.5 |
| 7 | 80 | 1155.3 | 56.2 | 4.9 | 20.3 | 1.8 | 32.6 | 2.8 | 68.0 | 5.9 |
| 8 | 80 | 2210.3 | 119.9 | 5.4 | 42.3 | 1.9 | 36.6 | 1.7 | 132.3 | 6.0 |
Acceptance criteria: Total %CV: < 10% Results are summarized in the Table below.
Reproducibility
Three samples were tested at three different sites. Samples were run in quadruplicates, two times a day, for 5 days, to generate 40 data points per sample, per site. An additional 5 samples were tested at three different sites. Samples were run in replicates of 5, once a day, for 5 days, to generate 25 data points per sample, per site. Data were analyzed with the Analyse-it for Excel method evaluation software to calculate between site precision.
Acceptance criteria: Total %CV: < 10%
Results are summarized in the Table below.
| QUANTA Flash CCP3 | Between Site Precision (Reproducibility) | |||
|---|---|---|---|---|
| Sample ID | Number of replicates | Mean (CU) | SD (CU) | CV (%) |
| 1 | 120 | 9.3 | 0.0 | 0.0 |
| 2 | 120 | 35.1 | 1.1 | 3.0 |
| 3 | 120 | 207.9 | 4.1 | 2.0 |
| 4 | 75 | 16.0 | 0.5 | 3.2 |
| 5 | 75 | 24.4 | 0.5 | 1.9 |
| 6 | 75 | 767.5 | 11.5 | 1.5 |
| 7 | 75 | 1447.6 | 38.2 | 2.6 |
| 8 | 75 | 2274.4 | 0.0 | 0.0 |
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Limit of Blank (LoB) and Limit of Detection (LoD)
The LoD of the QUANTA Flash CCP3 assay is 478 RLU, which is below the analytical measuring range of the assay. It was determined by using two reagent lots, consistent with CLSI EP17-A2 guideline with proportions of false positives (alpha) less than 5% and false negatives (beta) less than 5%; based on 120 determinations, with 60 measurements on blank samples and 60 measurements of low level samples, per reagent lot. The LoB is 410 RLU.
These values are below the value of the lowest QUANTA Flash CCP3 Master Curve standard, i.e. below the Analytical Measuring Range.
Analytical Measuring Range (AMR)
QUANTA Flash CCP3: 4.6 CU - 2776.8 CU The AMR is defined by the values of the lowest and highest Master Curve Standards.
Auto-rerun function and reportable results
The BIO-FLASH software has an Auto-rerun option available. If this option is selected, the instrument will automatically rerun any sample that has a result of >2776.8 after further diluting it by 20 fold, thereby bringing the measured value within the AMR. The final result will be calculated by the software by taking into account the additional dilution factor. As the highest value that can be directly measured is 2776.8 CU, the highest value that can be reported is 55536 CU.
High concentration hook effect
To assess hook effect, measurement signal (relative light units, RLU) was examined for four high positive samples (results above the AMR) before and after automatic or manual dilution. All sera produced significantly higher RLU values (above the AMR) when used "as is" compared to the manually or automatically diluted ones (that were within the AMR), thereby confirming that high positive specimens above the analytical measuring range do not show hook effect up to 113191 CU in the CCP3 assay (the highest concentration that was tested).
Linearity
The linearity of the AMR was evaluated by a study according to CLSI EPG-A, Evaluation of the Linearity of Quantitative Measurement Procedures: A Statistical Approved Guideline. Five serum samples with various CCP3 antibody concentrations were diluted with negative serum in 10% increments (from
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0% to 90% negative serum) to obtain values that cover the AMR. The dilutions were assayed in duplicates. Percent recovery of obtained mean results was calculated compared to the expected mean results (based on the dilution factor). Moreover, obtained values were plotted against expected values, and linear regression analysis was performed.
Acceptance criteria:
- Recovery is between 80-120%, or ± 4 CU, whichever is greater.
- For linear regression analysis, slope is between 0.9-1.1, and R-is ≥ 0.95.
All five specimens showed dilution linearity individually.
| Sample | Test Range (CU) | Slope (95% CI) | R2 |
|---|---|---|---|
| 1 | 358.3 to 2772.3 | 1.00 (1.00 to 1.00) | 1.00 |
| 2 | 190.7 to 2030.0 | 0.99 (0.96 to 1.03) | 0.99 |
| 3 | 82.5 to 705.4 | 1.00 (0.95 to 1.04) | 0.99 |
| 4 | 14.0 to 134.5 | 0.94 (0.88 to 1.00) | 0.98 |
| 5 | 6.6 to 34.9 | 0.97 (0.93 to 1.02) | 0.99 |
The combined data yielded the following results with linear regression:
| Sample | Test Range (CU) | Slope (95% CI) | D2 |
|---|---|---|---|
| All | 6.6 to 2772.3 | 1.07 (1.05 to 1.10) | 0.99 |
These data demonstrate the linearity of the analytical measuring range (4.6 CU - 2776.8 CU) of the QUANTA Flash CCP3 assay.
Interference
The interference study was performed according to CLSI EPO7-A2, Interference Testing in Clinical Chemistry; Approved Guideline - Second Edition. Three specimens were tested (negative: 10.1 CU; low positive: 40.5 CU; positive: 939.8 CU). Interfering substances were spiked into every specimen at three different concentrations in 10% of total specimen volume, and the resulting samples were assessed in triplicates with the CCP3 assay. Recovery of the unit values was calculated compared to control samples spiked with the same volume of diluents (10% of total). Acceptance criteria for the interference studies were 85% - 115% recovery, or ± 4 CU difference, whichever is greater.
No interference was detected with bilirubin up to 10 mg/dL (recovery: 101% to 102%), hemoglobin up to 200 mg/dL (recovery: 97% to 108%), triglycerides up to 1000 mg/dL (recovery: 98% to 111%), and cholesterol up to 224.3 mg/dL (recovery: 98% to 11%). Interference of rheumatoid factor was not tested.
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Cross-reactivity
To test potential cross-reactivity with autoantibodies and infection-induced antibodies, results obtained on 234 of the total 376 control samples that were included in the clinical validation study were assessed. These samples were from patients with autoimmune diseases that are characterized with disease specific autoantibodies, or from patients with positive infectious disease serology. The composition of the cohort and the anti-CCP3 positivity rate is shown in the Table below:
| Diagnosis | Number of samples | # pos | % pos |
|---|---|---|---|
| Systemic Lupus Erythematosus | 53 | 2 | 3.8% |
| HBV | 22 | 0 | 0.0% |
| HCV | 13 | 0 | 0.0% |
| Limited Systemic Sclerosis | 8 | 0 | 0.0% |
| Mixed Connective Tissue Disease | 7 | 0 | 0.0% |
| Primary Biliary Cirrhosis | 7 | 1 | 14.3% |
| Vasculitis | 6 | 0 | 0.0% |
| Wegener's Granulomatosis | 1 | 0 | 0.0% |
| Parvo virus infection | 12 | 0 | 0.0% |
| Salmonella infection | 10 | 0 | 0.0% |
| Lyme disease | 25 | 0 | 0.0% |
| Ulcerative colitis (UC) | 11 | 0 | 0.0% |
| Autoimmune hepatitis (AIH) | 19 | 1 | 5.3% |
| Celiac disease (CD) | 20 | 1 | 5.0% |
| Sjögren's syndrome | 20 | 0 | 0.0% |
| Total | 234 | 5 | 2.1% |
Based on the results, the QUANTA Flash CCP3 assay does not show cross-reactivity with autoantibodies that are present in various autoimmune diseases, or with antibodies against infectious agents.
Lot to lot comparison
Five unique samples with various reactivity levels were tested with three different reagent lots: 141004, 141005 and 151006. The samples covered the analytical measuring range of the assay. Samples were tested in replicates of 5, once per day, for 5 days according to CLSI EPO5-A3, Measurement Procedure Comparison and Bias Estimation Using Patient Samples; Approved Guideline.
Data were analyzed with the Analyse-it for Excel method evaluation software to calculate between lot precision.
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| QUANTA Flash CCP3 | Between Lot Precision | |||
|---|---|---|---|---|
| Sample ID | Mean (CU) | N | SD (CU) | CV (%) |
| 1 | 16.2 | 75 | 0.6 | 3.9 |
| 2 | 24.9 | 75 | 0.7 | 2.8 |
| 3 | 77.5 | 75 | 2.6 | 3.4 |
| 4 | 341.1 | 75 | 10.2 | 3.0 |
| 5 | 1528.7 | 75 | 61.2 | 4.0 |
Acceptance criteria: Between lot %CV: < 10% Results are summarized in the Table below.
Stability
Shelf life
To establish the initial claim for shelf life, accelerated stability studies were performed for 4 weeks at 37°C ± 3°C, where one week is equal to six months at 5 ± 3°C.
Accelerated stability testing was performed on each of the following sealed components of the QUANTA Flash CCP3 to establish initial stability claim: the beads, the resuspension buffer, the two Calibrators, and the Negative and Positive Controls. Each week a new sealed component was placed in the incubator, and all components were tested at the end of the experiment together with the one that was stored at 5 ± 3°C. The recovery of the measured values was calculated for each time point (compared to those obtained with 5 ± 3℃ stored reagent). All calculations were performed by comparing results of sealed components stored at 5 ± 3°C (control) to those stored at 37 ± 3°C (test) for 1, 2, 3, and 4 weeks, where one week is equal to six months at 5 ± 3°C. Linear regression analysis was performed between recovery values and the number of days.
Acceptance criteria for one year preliminary expiration dating:
- Beads and Resuspension Buffer:
With regression analysis, the lower 95% Cl interval of the regression line is ≥85% and the upper 95% Cl interval is at ≤115% at day 14, and no individual data point has ≤75% or ≥125% recovery at day 14.
- Controls and Calibrators:
With regression analysis, the lower 95% Cl interval of the regression line is ≥90% and the upper 95% Cl interval is ≤110% at day 14, and no individual data point has ≤80% or ≥120% recovery at day 14.
Beads
Testing was performed on three lots of CCP3 coupled beads using up to 9 characterized samples with various reactivity levels, along with the negative and positive controls.
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All three lots of beads retained between 85% and 115% reactivity (considering the 95% Cl) after two weeks at 37 ± 3°C, and therefore pass the acceptance criteria for one year expiration date.
Resuspension Buffer
Testing was performed on three lots of resuspension buffer using up to 6 characterized samples with various reactivity levels, along with the negative and positive controls.
All three lots of resuspension buffer retained between 85% and 115% reactivity (considering the 95% Cl) after two weeks at 37 ± 3℃, and therefore pass the acceptance criteria for one year expiration date.
Calibrators and Controls
Testing was performed on three lots of CCP3 Calibrators and Controls. All Calibrators and Controls maintained between 90% and 110% reactivity (considering the 95% Cl) when sored at 37 ± 3℃ for 2 weeks, and therefore pass the acceptance criteria for one year expiration dating.
In-use (onboard) stability
Calibrators
Onboard stability claim: 4 calibrations, or 8 hours onboard
During assessing on-board stability, Calibrators were placed uncapped, onboard the instrument, and calibration was performed altogether five times over 8.5 hours. Controls and a panel of characterized patient specimens were run on each calibration curve.
Calibrators are considered stable if all five calibrations performed in the 8.5 hour period are successful, and average Calibrator RLU recovery values are between 90% and 110% compared to the first use.
A total of 5 successful calibrations were performed over an 8.5 hour period. Calibrator RLU values remained within the 90-110% range. Moreover, all Controls and patient panel samples ran within their expected range. This supports the claim that calibrators can be used for up to 4 calibrations over an 8 hour period.
Controls
Onboard stability claim: up to 15 uses, at 10 minutes onboard per use
During assessing on-board stability, 2 vials of each Control were assayed twice a day for a total of 21 runs. The first run was used to establish baseline value, by running each vial in duplicate, and then additional 20 runs were performed, by running each vial in singleton. During runs, the Controls were left uncapped, onboard the instrument for 15 minutes per run. When not in use, the controls were capped, and stored at 5 ± 3°C.
Percent recovery of each value was calculated compared to the baseline value. Controls are considered stable when all values run within their established range, and the linear regression line obtained by plotting %recovery values against the number of runs stays between 85% and 115% at run 15.
All controls ran within their respective acceptable ranges for all runs. Moreover, the regression line remained between 85% and 115% at run 15 for both Controls. These results support the claim that controls can be used for up to 15 times, at 10 minutes per use.
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Reagent Cartridge
To establish the in-use stability of the QUANTA Flash CCP3 reagent cartridge, three lots of cartridges were tested with up to 6 serum specimens (with different reactivity levels) along with the Negative and Positive Controls. The specimens were tested periodically up to 90 days. Percent recoveries were calculated compared to the day zero average values, and linear regression analysis was performed by plotting %recovery against the number of days. The claim was established using the following criteria (using the one that is fulfilled first):
-
The stability claim is established at the actual measurement day proceeding the 95% confidence interval of the regression line reaches 85% or 115% recovery, or
-
At the actual measurement day preceding the day when 2 data points or ≥2% of the recovery data (whichever is greater) is ≤ 75% or ≥ 125% recovery.
The onboard stability results of the three lots are as follows:
RP0001: 64 days 131001: 83 days 141002: 63 days Using these criteria, the in-use (onboard) stability of CCP3 reagent cartridge was set at 60 days.
Real time stability
Real time stability testing has been scheduled to be performed every three months on the reagent cartridge, Calibrators and Controls, to verify the one year expiration that was assigned based on accelerated stability studies. At the time of the submission, results were available up to 6 months for reagent cartridge, and up to 9 months on Calibrators and Controls.
For reagent cartridge, QC panel samples were tested in duplicates at each time point. The QC panel is a group of characterized patient samples with target values, used by the QC Department for reagent release and QC.
- Acceptance criteria: results should fall within their respective QC ranges.
Calibrators were tested in triplicates on a calibrated cartridge at each time point. Averages of the triplicates were compared to the value that was assigned to the Calibrators at release.
- Acceptance criteria: % recovery of the average of the triplicates is between 85% and 115%, and %CV of the triplicates is < 10%.
Controls were tested in triplicates on a calibrated cartridge at each time point. Individual values, and averages of the triplicates were compared to the values that were assigned to the Controls at release.
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Page 16 of 19
- Acceptance criteria: results should fall within their acceptable ranges as were established at the release of the Controls.
All results to date were within the acceptance limits.
Cut-off, reference range
| QUANTA Flash CCP3: | Negative | $<$ 20 CU |
|---|---|---|
| Positive | $ \ge $ 20 CU |
The reference population for establishing the reference interval for the CCP3 assay consisted of 210 subjects:
| Sample Group | N |
|---|---|
| Apparently healthy blood donors | 170 |
| Viral hepatitis positive samples | 20 |
| Autoimmune thyroid disease | 20 |
All specimens were the same matrix (serum) as specified in the Intended Use. All specimens were unaltered. The cut-off was 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 distribution of the results was non-normal (Saphiro-Wilk p<0.0001), so the non-parametric percentile method was used. One high positive sample was excluded from the calculations as an outlier. This sample was confirmed to be true positive with the predicate device. The 99th percentile of the remaining obtained values was calculated as 11294 RLU. The cutoff was set to 12000 RLU. One hepatitis C patient tested positive at this cutoff level, along with the one apparently healthy blood donor outlier.
Clinical performance characteristics
Clinical sensitivity, specificity
A cohort of characterized samples, none of which were used for establishing the reference range, was used to validate the clinical performance of the QUANTA Flash CCP3. A total of 728 characterized samples were included in the Validation Set for the QUANTA Flash CCP3. All samples were run on the QUANTA Flash CCP3. The distribution of the cohort and the CCP3 positivity rate is in the Table below:
| Patient group | N | Number positive | % positive |
|---|---|---|---|
| Ankylosing Spondylitis | 13 | 0 | 0.0% |
| Osteoarthritis | 49 | 2 | 4.1% |
| Polymyalgia Rheumatica | 20 | 2 | 10.0% |
| Psoriasis Arthritis | 14 | 1 | 7.1% |
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| Patient group | N | Number positive | % positive |
|---|---|---|---|
| Systemic Lupus Erythematosus | 53 | 2 | 3.8% |
| HBV | 22 | 0 | 0.0% |
| HCV | 13 | 0 | 0.0% |
| Parvo virus infection | 12 | 0 | 0.0% |
| Salmonella infection | 10 | 0 | 0.0% |
| Lyme disease | 25 | 0 | 0.0% |
| Ulcerative colitis (UC) | 11 | 0 | 0.0% |
| Autoimmune hepatitis (AIH) | 19 | 1 | 5.3% |
| Celiac disease (CD) | 20 | 1 | 5.0% |
| Sjögren's syndrome | 20 | 0 | 0.0% |
| Other diseases* | 75 | 4 | 5.3% |
| Total controls | 376 | 13 | 3.5% |
| Rheumatoid arthritis (RA) | 352 | 249 | 70.7% |
| Total | 728 | - | - |
| *List of other diseases | n |
|---|---|
| Arthritis with Colitis ulcerosa | 1 |
| Arthritis with Crohn' disease | 1 |
| Colon Carcinoma | 1 |
| Connective Tissue Disease | 2 |
| Cryoglobulinemia | 1 |
| Degenerative Spine Disease | 6 |
| Fibromyalgia | 6 |
| Fibromyalgia, Connective Tissue Disease | 1 |
| Fibromyalgia, Osteoarthrosis | 1 |
| Gout | 3 |
| Juvenile Rheumatoid Arthritis | 1 |
| Limited Systemic Sclerosis | 8 |
| Mixed Connective Tissue Disease | 7 |
| Monoarthritis | 2 |
| Monoclonal Gammopathy of Unknown Significance (MGUS) | 2 |
| Multiple Sclerosis | 1 |
| Osteoarthrosis, MGUS | 1 |
| Osteoarthrosis, Osteoarthritis | 1 |
| Periarthropathia Humero-Scapularis | 1 |
| Polymyalgia Rheumatica, Arteriitis temporalis | 1 |
| Polymyositis | 2 |
{20}------------------------------------------------
| *List of other diseases | n |
|---|---|
| Primary Biliary Cirrhosis | 7 |
| Raynaud-Syndrome | 2 |
| Rosacea | 1 |
| Sakroiliitis | 1 |
| Scoliosis | 1 |
| Spondylosis | 1 |
| Undifferentiated Connective Tissue Disease | 5 |
| Vasculitis | 6 |
| Wegener's Granulomatosis | 1 |
The results were analyzed to calculate sensitivity and specificity for RA (n=352), using all 376 controls.
| N=728 | Diagnosis | Analysis(95% confidence) | |||
|---|---|---|---|---|---|
| RA | Controls | Total | |||
| QUANTA FlashCCP3 | Positive | 249 | 13 | 262 | Sensitivity = 70.7% (65.8-75.2%) |
| Negative | 103 | 363 | 466 | Specificity = 96.5% (94.2-98.0%) | |
| Total | 352 | 376 | 728 |
Clinical sensitivity and specificity of the QUANTA Flash CCP3 in RA
Expected values
The expected value in the normal population is "negative". Anti-CCP3 antibody levels were analyzed in a cohort of 146 apparently healthy blood donors (66 females and 80 males, ages 17 to 60 years, with an average and median age of 34 years) using the QUANTA Flash CCP3. This patient population was different from the one that was used to establish the cutoff, and was only used to assess expected values. With the cut-off of 20 CU, one sample was positive on the QUANTA Flash CCP3. The mean concentration was 5.5 CU, and the values ranged from <4.6 to 31.1 CU.
Comparison with predicate device
Samples for method comparison analysis included all 728 samples from the clinical validation study. These samples were tested on both the QUANTA Flash CCP3 and on the predicate ELISA. The data are presented in two ways; first using all samples, and next using only samples within the AMR:
| Method Comparison (N=728) | CCP3 ELISA | Percent Agreement(95% Confidence) | |||
|---|---|---|---|---|---|
| Negative | Positive | Total | |||
| QUANTA Flash® | Negative | 447 | 19 | 466 | Pos. Agree = 92.7% (89.0 - 95.3%) |
Method Comparison, all samples:
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| CCP3 CIA | |||||
|---|---|---|---|---|---|
| Positive | 19 | 243 | 262 | Neg. Agree = 95.9% (93.7 – 97.4%) | |
| Total | 466 | 262 | 728 | Total Agree = 94.8% (92.9 -96.2%) |
Method Comparison, samples with the AMR:
| Method Comparison (N=420) | CCP3 ELISA | Percent Agreement(95% Confidence) | |||
|---|---|---|---|---|---|
| Negative | Positive | Total | |||
| QUANTA Flash®CCP3 CIA | Negative | 177 | 13 | 190 | Pos. Agree = 94.2% (90.3 – 96.6%) |
| Positive | 19 | 211 | 230 | Neg. Agree = 90.3% (85.4 – 93.7%) | |
| Total | 196 | 224 | 420 | Total Agree = 92.4% (89.4 – 94.6%) |
§ 866.5775 Rheumatoid factor immunological test system.
(a)
Identification. A rheumatoid factor immunological test system is a device that consists of the reagents used to measure by immunochemical techniques the rheumatoid factor (antibodies to immunoglobulins) in serum, other body fluids, and tissues. Measurement of rheumatoid factor may aid in the diagnosis of rheumatoid arthritis.(b)
Classification. Class II (performance standards).