(267 days)
QUANTA Flash Ro60 is a chemiluminescent immunoassay for the semi-quantitative determination of IgG anti-Ro60 autoantibodies in human serum. The presence of anti-Ro60 autoantibodies, in conjunction with clinical findings and other laboratory tests, is an aid in the diagnosis of Systemic Lupus Erythematosus and Sjögren's Syndrome.
QUANTA Flash Ro60 Calibrators are intended for use with the QUANTA Flash Ro60 Reagents for the determination of Ig G anti-Ro60 autoantibodies in human serum. Each calibrator establishes a point of reference for the working curve that is used to calculate unit values.
QUANTA Flash Ro60 Controls are intended for use with the QUANTA Flash Ro60 reagents for quality control in the determination of IgG anti-Ro60 autoantibodies in human serum.
The QUANTA Flash Ro60 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 Ro60 assay utilizes a reagent cartridge format, which is compatible with the BIO-FLASH instrument.
Purified recombinant Ro60 antigen 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 prediluted 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 all 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 cuvette, and 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-Ro60 antibodies bound to the corresponding beads.
For quantitation, the QUANTA Flash Ro60 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 Ro60 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 Ro60 kit contains the following materials:
One (1) QUANTA Flash Ro60 Reagent Cartridge
One (1) vial of Resuspension buffer
One (1) Transfer pipette
The QUANTA Flash Ro60 reagent cartridge contains the following reagents for 50 determinations:
- a. Ro60 antigen 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 Ro60 Calibrators kit contains two vials of Calibrator 1 and two vials of Calibrator 2:
QUANTA Flash Ro60 Calibrators:
- QUANTA Flash Ro60 Calibrator 1: Two (2) barcode labeled tubes containing 0.3 mL ı prediluted, ready to use reagent. Calibrators contain human antibodies to Ro60 in stabilizers and preservatives.
- -QUANTA Flash Ro60 Calibrator 2: Two (2) barcode labeled tubes containing 0.3 mL prediluted, ready to use reagent. Calibrators contain human antibodies to Ro60 in stabilizers and preservatives.
The QUANTA Flash Ro60 Controls kit contains two vials of Negative Control and two vials of Positive Control:
QUANTA Flash Ro60 Controls:
- । QUANTA Flash Ro60 Negative Control: Two (2) barcode labeled tubes containing 0.5 mL, ready to use reagent. Controls contain human antibodies to Ro60 in stabilizers and preservatives.
- i QUANTA Flash Ro60 Positive Control: Two (2) barcode labeled tubes containing 0.5 mL, ready to use reagent. Controls contain human antibodies to Ro60 in stabilizers and preservatives.
The document describes the analytical and clinical performance of the QUANTA Flash® Ro60 chemiluminescent immunoassay for the semi-quantitative determination of IgG anti-Ro60 autoantibodies in human serum.
Here's a breakdown of the requested information:
1. Table of Acceptance Criteria and Reported Device Performance:
| Performance Metric | Acceptance Criteria (QUANTA Flash® Ro60) | Reported Device Performance (QUANTA Flash® Ro60) |
|---|---|---|
| Precision (Total %CV) | < 10% | Range: 4.4% to 8.4% (for 10 samples with various concentrations) |
| Reproducibility (Total %CV) | < 10% | Range: 5.3% to 5.4% (for 3 samples) |
| Linearity (% Recovery) | 80-120%, or ± 4 CU, whichever is greater | All three specimens showed dilution linearity individually and combined data showed excellent linearity (Slope 1.07, R2 1.00 for range 7.5 to 1372.2 CU) |
| Linearity (Slope for regression) | 0.9-1.1 | Range: 1.00 to 1.05 for individual samples, 1.07 for combined data |
| Linearity (R2 for regression) | ≥ 0.95 | Range: 0.99 to 1.00 for individual samples, 1.00 for combined data |
| Interference (% Recovery) | 85% - 115%, or ± 4 CU difference, whichever is greater | No interference detected with bilirubin (99-109%), hemoglobin (100-108%), triglycerides (89-109%), cholesterol (89-109%), and RF IgM (90-107%). |
| Lot to Lot Comparison (Weighted R) | ≥ 0.975 | Range: 0.994 to 0.996 |
| Lot to Lot Comparison (Slope) | 0.9-1.1 | 1.0 for all comparisons |
| Lot to Lot Comparison (Intercept) | ± 15% of cut-off (3 CU) | Range: -1.2 to 1.0 |
| Lot to Lot Comparison (Predicted bias at cut-off) | ± 15% (3 CU) | Range: -1.1 to 1.3 |
| Sample Stability (% Recovery) | 85-115% average recovery | Supported claims for up to 48 hours at RT, up to 14 days at 2-8°C, and up to 3 freeze/thaw cycles. |
| Shelf Life (Beads) (Lower 95% CI) | ≥ 85% at 2 weeks (accelerated testing) | All three lots of beads retained > 85% reactivity after two weeks at 37 ± 3 ℃. |
| Shelf Life (Beads) (Individual data point recovery) | No individual data point ≤ 75% recovery at 2 weeks | Not explicitly stated, but "pass the acceptance criteria" implies this was met. |
| Shelf Life (Calibrators & Controls) (Lower 95% CI) | ≥ 90% at 2 weeks (accelerated testing) | All Calibrators and Controls maintained > 90% reactivity when stored at 37 ± 3°C for 2 weeks. |
| Shelf Life (Calibrators & Controls) (Individual data point recovery) | No individual data point ≤ 80% recovery at 2 weeks | Not explicitly stated, but "pass the acceptance criteria" implies this was met. |
| Onboard Stability (Calibrators) (RLU recovery) | 90-110% compared to first use | Calibrator RLU values remained within the 90-110% range over 8.5 hours. |
| Onboard Stability (Controls) (Regression line) | Between 85% and 115% at run 15 | Regression line remained between 85% and 115% at run 15 for both Controls. |
| Real-time Stability (Controls) | Results within established acceptable ranges | All results were within the acceptance limits. |
| Real-time Stability (Calibrators) (% Recovery) | 85-115% | All results were within the acceptance limits. |
| Real-time Stability (Calibrators) (%CV) | < 10% | All results were within the acceptance limits. |
| Real-time Stability (Reagent Cartridge) | Results within respective QC ranges | All results were within the acceptance limits. |
| Sjögren's Syndrome Clinical Sens. | N/A (reported as part of clinical validation) | 66.7% (95% CI: 49.8-80.9%) |
| Sjögren's Syndrome Clinical Spec. | N/A (reported as part of clinical validation) | 97.4% (95% CI: 94.8-99.0%) |
| SLE Clinical Sensitivity | N/A (reported as part of clinical validation) | 24.0% (95% CI: 17.4-31.6%) |
| SLE Clinical Specificity | N/A (reported as part of clinical validation) | 97.4% (95% CI: 94.8-99.0%) |
| SLE+SS Clinical Sensitivity | N/A (reported as part of clinical validation) | 32.8% (95% CI: 26.2-40.0%) |
| SLE+SS Clinical Specificity | N/A (reported as part of clinical validation) | 97.4% (95% CI: 94.8-99.0%) |
| Method Comparison (predicate) Pos. Agree (ELISA equivocal as negative) | N/A (reported as part of predicate comparison) | 93.5% (82.1 – 98.6%) |
| Method Comparison (predicate) Neg. Agree (ELISA equivocal as negative) | N/A (reported as part of predicate comparison) | 95.9% (89.8 – 98.9%) |
| Method Comparison (predicate) Total Agree (ELISA equivocal as negative) | N/A (reported as part of predicate comparison) | 95.1% (90.2 – 98.0%) |
| Method Comparison (predicate) Pos. Agree (ELISA equivocal as positive) | N/A (reported as part of predicate comparison) | 82.7% (69.7 – 91.8%) |
| Method Comparison (predicate) Neg. Agree (ELISA equivocal as positive) | N/A (reported as part of predicate comparison) | 95.6% (89.1-98.8%) |
| Method Comparison (predicate) Total Agree (ELISA equivocal as positive) | N/A (reported as part of predicate comparison) | 90.9% (85.0 - 95.1%) |
2. Sample Size Used for the Test Set and Data Provenance:
- Precision Study: 10 samples (run in duplicates, twice a day, for 21 days), 84 replicates each.
- Reproducibility Study: 3 samples (run in quadruplicates, two times a day, for 10 days), 80 data points per sample.
- Limit of Detection (LoD) Study: 120 determinations (60 blank, 60 low-level samples) per reagent lot. 4 low-level samples (diluted anti-Ro60 positive samples) for LoD determination.
- Limit of Blank (LoB) Study: 4 blank samples (System Rinse) from two different lots, run in replicates of five per day for 3 days. 60 data points per lot.
- Linearity Study: 3 serum samples (diluted in 10% increments).
- Interference Study: 3 specimens (one near-cutoff negative, one weak positive, one high positive), spiked with interfering substances at three concentrations.
- Cross-reactivity Study: 213 "control" clinical samples from patients with various autoimmune diseases or infectious disease serology.
- Lot to Lot Comparison Study: 21 unique samples and 2 Positive Controls (23 specimens in total).
- Sample Stability Study: 6 samples (negative, around cut-off, positive).
- Accelerated Stability Studies: 3 lots of beads and 3 lots of Calibrators and Controls.
- Onboard Stability (Calibrators): Not explicitly stated, but involved running Calibrators 5 times over 8.5 hours with Controls and a panel of characterized patient specimens.
- Onboard Stability (Controls): 2 vials of each Control, assayed twice a day for a total of 20 runs.
- Reagent Cartridge Onboard Stability: Three lots of cartridges, tested with up to 6 serum specimens and controls.
- Real-time Stability: Not explicitly stated, but involved testing at various time points (3, 6, 9, 12 months for Calibrators/Controls; 2, 5, 8, 12 months for reagent cartridge).
- Reference Range Establishment: 156 subjects (apparently healthy blood donors, viral hepatitis, HIV, syphilis, rheumatoid arthritis patients). Data provenance not specified beyond "commercial sources" for certain materials.
- Clinical Performance (Sensitivity/Specificity): 475 characterized samples for the Validation Set (from Sjögren's Syndrome, SLE, and various control groups). Data provenance not specified, but likely from a clinical setting.
- Method Comparison: 143 samples from SS and SLE patients and disease controls.
The data provenance for most studies is not explicitly stated beyond general descriptions ("human serum," "commercial sources," "characterized samples"). It is not specified whether the studies were retrospective or prospective, or the country of origin for the patient/sample cohorts.
3. Number of Experts Used to Establish the Ground Truth for the Test Set and Qualifications:
The document does not mention the use of experts to establish ground truth for the test set. For the clinical performance, samples were "characterized" and grouped into patient categories (e.g., Sjögren's Syndrome, SLE), but the method of this characterization (e.g., clinical diagnosis by a panel of rheumatologists, specific diagnostic criteria) and the number/qualifications of experts involved are not provided.
4. Adjudication Method:
The document does not describe any adjudication method (e.g., 2+1, 3+1) for the test set.
5. Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study:
No MRMC comparative effectiveness study is mentioned. This device is an immunoassay, typically read by an instrument rather than human readers, so such a study would not be applicable.
6. Standalone Performance:
Yes, the entire document details the standalone performance of the QUANTA Flash® Ro60 immunoassay. All the analytical and clinical performance characteristics (precision, reproducibility, linearity, interference, stability, sensitivity, specificity, method comparison) are based on the algorithm's (instrument's) output without human intervention in the result interpretation beyond setting the cut-off. The device is described as "a fully automated closed system with continuous load and random access capabilities that automatically processes the samples, runs the assay and reports the results."
7. Type of Ground Truth Used:
- Analytical Studies (Precision, Reproducibility, Linearity, Interference, LoD, LoB, Lot-to-Lot, Sample Stability, Instrument Stability): Ground truth is typically based on pre-defined concentrations, spiked samples, or reference materials. For example, for linearity, expected values were based on dilution factors. For LoD/LoB, the truth was whether a sample was blank or low-level.
- Clinical Performance (Sensitivity, Specificity) and Cross-reactivity: Ground truth was based on clinical diagnoses (e.g., Sjögren's Syndrome, SLE, Graves' Disease, etc.) for the characterized samples. For cross-reactivity, it also included the presence of specific autoantibodies or infectious disease serology.
- Cut-off Establishment: Used a reference population of 156 subjects (apparently healthy, viral hepatitis, HIV, syphilis, rheumatoid arthritis) and also included 12 proficiency testing samples from CAP and UKNEQAS with known consensus/target results.
- Method Comparison: Ground truth was the result from a predicate device (Hycor AUTOSTAT™ II Anti-SS-A Ro ELISA).
8. Sample Size for the Training Set:
The document does not specify a separate "training set" in the context of machine learning. For an immunoassay, the "training" equivalent would be the data used to establish master curves, calibrator values, and cut-offs.
- Master Curve/Calibrator Values: Based on "in-house Standards" and trial dilutions (sample size for these not specified, but implied to be robust enough for value assignment).
- Cut-off Establishment: 156 subjects (reference population) and 12 proficiency testing samples were used to establish the cut-off. This could be considered part of the "training" for the interpretation of results into positive/negative.
9. How Ground Truth for the Training Set Was Established:
As explained in point 8, if we consider cut-off establishment and master curve definition as part of "training":
- Master Curves and Calibrator/Control Values: Established internally using "in-house Standards" and extensive testing on multiple instruments and reagent lots.
- Cut-off for Classification: Established by analyzing the results from a reference population of 156 subjects using statistical methods (99th percentile, non-parametric method) and further informed by 12 proficiency testing samples with known consensus/target results from external quality assessment schemes (CAP, UKNEQAS).
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Food and Drug Administration 10903 New Hampshire Avenue Document Control Center - WO66-G609 Silver Spring, MD 20993-0002
February 12, 2015
INOVA DIAGNOSTICS, INC. GABRIELLA LAKOS, MD, PhD, D (ABMLI) DIRECTOR, RHEUMATOLOGY RESEARCH 9900 OLD GROVE ROAD SAN DIEGO CA 92131-1638
Re: K141328
Trade/Device Name: Quanta Flash® Ro60 Quanta Flash® Ro60 Calibrators Ouanta Flash® Ro60 Controls Regulation Number: 21 CFR 866.5100 Regulation Name: Antinuclear antibody immunological test system Regulatory Class: Class II Product Code: LLL, JIT, JJX Dated: January 8, 2015 Received: January 9, 2015
Dear Dr. Lakos:
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 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
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as set forth in the quality systems (OS) 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 regulations (21 CFR Parts 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/ResourcesforYou/Industry/default.htm. 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
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 -A
Leonthena R. Carrington, MS, MBA, MT(ASCP) Director (Acting) 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) K141328
Device Name QUANTA Flash® Ro60 QUANTA Flash® Ro60 Calibrators QUANTA Flash® Ro60 Controls
Indications for Use (Describe)
QUANTA Flash Ro60 is a chemiluminescent immunoassay for the semi-quantitative determination of IgG anti-Ro60 autoantibodies in human serum. The presence of anti-Ro60 autoantibodies, in conjunction with clinical findings and other laboratory tests, is an aid in the diagnosis of Systemic Lupus Erythematosus and Sjögren's Syndrome.
QUANTA Flash Ro60 Calibrators are intended for use with the QUANTA Flash Ro60 Reagents for the determination of Ig G anti-Ro60 autoantibodies in human serum. Each calibrator establishes a point of reference for the working curve that is used to calculate unit values.
QUANTA Flash Ro60 Controls are intended for use with the QUANTA Flash Ro60 reagents for quality control in the determination of IgG anti-Ro60 autoantibodies 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® Ro60 QUANTA Flash® Ro60 Calibrators QUANTA Flash® Ro60 Controls
Table of Contents
| Administrative data | |
|---|---|
| Device description | |
| Intended use(s) | |
| Substantial equivalence | |
| Comparison to predicate device | |
| Analytical performance characteristics | |
| 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 | |
| Sample stability | |
| Stability | |
| Cut-off, reference range | |
| Clinical performance characteristics | |
| Clinical sensitivity, specificity | |
| Expected values | |
| Comparison with predicate device |
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This summary of the 510(k) safety and effectiveness information is being submitted in accordance with the requirements of SMDA 1990 and 21 CFR 807.92.
Administrative data
| Submitter: | Inova Diagnostics, Inc9900 Old Grove Road,San Diego, CA, 92131 | |
|---|---|---|
| Purpose of submission: | New device(s) | |
| Devices in the submission: | QUANTA Flash® Ro60QUANTA Flash® Ro60 CalibratorsQUANTA Flash® Ro60 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 | |
| Preparation date: | 05/19/2014 | |
| Device name (assay kit): | Proprietary name: | QUANTA Flash® Ro60 |
| Common name: | Anti-Ro60 Chemiluminescent Immunoassay | |
| Classification name: | anti-Ro60 antibody, antigen and control | |
| Regulation Description | Antinuclear antibody immunological test system | |
| Regulation Medical Specialty | Immunology | |
| Review Panel | Immunology | |
| Product Code | LLL. Extractable Antinuclear Antibody. Antigen And Control |
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510(k) Summary QUANTA Flash® Ro60
| Regulation Number | 866.5100 | |
|---|---|---|
| Device Class | 2 | |
| Device name (Calibrators): | Proprietary name:Common name:Classification name: | QUANTA Flash® Ro60 CalibratorsRo60 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® Ro60 ControlsRo60 Controlssingle (specified) analyte controls (assayed and unassayed) |
| 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: | Hycor AUTOSTAT™ II Anti-SS-A Ro ELISA, 510(k) number: K962719 | |
| Device description |
The QUANTA Flash Ro60 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 Ro60 assay utilizes a reagent cartridge format, which is compatible with the BIO-FLASH instrument.
Purified recombinant Ro60 antigen 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
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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 prediluted 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 all 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 cuvette, and 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-Ro60 antibodies bound to the corresponding beads.
For quantitation, the QUANTA Flash Ro60 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 Ro60 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 Ro60 kit contains the following materials:
One (1) QUANTA Flash Ro60 Reagent Cartridge
One (1) vial of Resuspension buffer
One (1) Transfer pipette
The QUANTA Flash Ro60 reagent cartridge contains the following reagents for 50 determinations:
- a. Ro60 antigen 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 Ro60 Calibrators kit contains two vials of Calibrator 1 and two vials of Calibrator 2:
QUANTA Flash Ro60 Calibrators:
- QUANTA Flash Ro60 Calibrator 1: Two (2) barcode labeled tubes containing 0.3 mL ı prediluted, ready to use reagent. Calibrators contain human antibodies to Ro60 in stabilizers and preservatives.
- -QUANTA Flash Ro60 Calibrator 2: Two (2) barcode labeled tubes containing 0.3 mL
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prediluted, ready to use reagent. Calibrators contain human antibodies to Ro60 in stabilizers and preservatives.
The QUANTA Flash Ro60 Controls kit contains two vials of Negative Control and two vials of Positive Control:
QUANTA Flash Ro60 Controls:
- । QUANTA Flash Ro60 Negative Control: Two (2) barcode labeled tubes containing 0.5 mL, ready to use reagent. Controls contain human antibodies to Ro60 in stabilizers and preservatives.
- i QUANTA Flash Ro60 Positive Control: Two (2) barcode labeled tubes containing 0.5 mL, ready to use reagent. Controls contain human antibodies to Ro60 in stabilizers and preservatives.
Intended use(s)
QUANTA Flash Ro60 is a chemiluminescent immunoassay for the semi-quantitative determination of IgG anti-Ro60 autoantibodies in human serum. The presence of anti-Ro60 autoantibodies, in conjunction with clinical findings and other laboratory tests, is an aid in the diagnosis of Systemic Lupus Erythematosus and Sjögren's Syndrome.
QUANTA Flash Ro60 Calibrators are intended for use with the QUANTA Flash Ro60 Reagents for the determination of IgG anti-Ro60 autoantibodies in human serum. Each calibrator establishes a point of reference for the working curve that is used to calculate unit values.
QUANTA Flash Ro60 Controls are intended for use with the QUANTA Flash Ro60 Reagents for quality control in the determination of IgG anti-Ro60 autoantibodies in human serum.
Substantial equivalence
The QUANTA Flash Ro60 Reagent, the QUANTA Flash Ro60 Calibrators and the QUANTA Flash Ro60 Controls have the same intended use and assay principle as the predicate device.
Comparison to predicate device
| Similarities | ||
|---|---|---|
| Item | QUANTA Flash Ro60 | Predicate Device |
| Intended use | Semi-quantitative determination of anti-Ro60 antibodies in human serum | Semi-quantitative detection of anti-Ro60 antibodies in human serum |
QUANTA Flash Ro60 reagent kit
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| Similarities | ||
|---|---|---|
| Item | QUANTA Flash Ro60 | Predicate Device |
| Assay methodology | Solid phase (heterogeneous)immunoassay | Solid phase (heterogeneous)immunoassay |
| Traceability | International Reference Preparationis not availableResults are traceable to in-houseStandards | International Reference Preparation isnot available |
| Sample type | Serum | Serum |
| Shelf life | One year | One year |
| Differences | ||
|---|---|---|
| Item | QUANTA Flash Ro60 | Predicate Device |
| Detection/Operating principle | Chemiluminescent immunoassay | Enzyme-linked immunosorbent assay |
| Solid phase | Paramagnetic microparticles (beads) | 96-well plate |
| Antigen | Purified recombinant Ro60 antigen | Native Ro60 antigen, purified from bovine sources |
| Conjugate | Isoluminol conjugated anti-human IgG | HRP conjugated anti-human IgG |
| Calibration | Lot specific Master Curve + two calibrators (sold separately) | Single standard(Included in the kit) |
QUANTA Flash Ro60 Calibrators
| Item | QUANTA Flash Ro60 Calibrators | Predicate Device |
|---|---|---|
| Intended use | QUANTA Flash Ro60 Calibrators are intended for use with the QUANTA Flash Ro60 Reagents for the determination of IgG anti-Ro60 autoantibodies in human serum. Each calibrator establishes a point of reference for the working curve that is used to calculate unit values. | No separate intended use; calibrator is part of the kit. |
| Analyte | Anti-Ro60 antibodies | Anti-Ro60 antibodies |
| Method | QUANTA Flash Ro60 chemiluminescent immunoassay | HYCOR Anti-SS-A/Ro ELISA |
| Matrix | Human serum, stabilizers, and preservative | Human serum with preservative |
| Unit | CU (Chemiluminescent units) | U/mL (arbitrary) |
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| Item | QUANTA Flash Ro60 Calibrators | Predicate Device |
|---|---|---|
| (arbitrary) | ||
| Physico-chemicalcharacteristics | Liquid, prediluted, ready to use | Liquid, prediluted, ready to use |
| Storage | 2-8 °C | 2-8 °C |
| Shelf life | One year | One year |
QUANTA Flash Ro60 Controls
| Item | QUANTA Flash Ro60 Controls | Predicate Device |
|---|---|---|
| Intended use | QUANTA Flash Ro60 Controls are intended for use with the QUANTA Flash Ro60 reagents for quality control in the determination of IgG anti-Ro60 autoantibodies in human serum. | No separate intended use; controls are part of the kit. |
| Analyte | Anti-Ro60 antibodies | Anti-Ro60 antibodies |
| Method | QUANTA Flash Ro60 chemiluminescent immunoassay | HYCOR Anti-SS-A/Ro ELISA |
| Matrix | Human serum, stabilizers, and preservative | Human serum with preservative |
| Unit | CU (Chemiluminescent units) (arbitrary) | U/mL (arbitrary) |
| Physico-chemical characteristics | Liquid, ready to use | Liquid, prediluted, ready to use |
| Levels | 2 (negative and positive) | 2 (negative and positive) |
| Storage | 2-8 °C | 2-8 °C |
| Shelf life | One year | One year |
Analytical performance characteristics
Value assignment and traceability of Calibrators and Controls
There is currently no recognized international standard for the measurement of Ro60 antibodies. The CDC ANA reference sera #7 (REFERENCE SERUM FOR HUMAN ANTIBODIES TO SSA-60/Ro) and #3 (REFERENCE SERUM, FLUORESCENCE ANTINUCLEAR ANTIBODY, SPECKLED PATTERN) were tested for Ro60 and produced the following results: CDC ANA #7: >1374.8 CU CDC ANA #3: 920.0 CU
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The QUANTA Flash Ro60 Calibrators and Controls are manufactured by diluting human serum that contains high titer of antibodies with commercial antibody stabilizer, containing 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 in-house Standards that are used to create the Master Curves for the QUANTA Flash Ro60 assay.
| Material | Assigned Value |
|---|---|
| Ro60 Master Curve Standard 1 | 4.9 CU |
| Ro60 Master Curve Standard 2 | 13.0 CU |
| Ro60 Master Curve Standard 3 | 64.1 CU |
| Ro60 Master Curve Standard 4 | 145.5 CU |
| Ro60 Master Curve Standard 5 | 472.1 CU |
| Ro60 Master Curve Standard 6 | 1374.8 CU |
List of Ro60 Standards, Calibrators and Controls:
| Material | ManufacturingTarget Value | ManufacturingTarget Range |
|---|---|---|
| Ro60 Calibrator 1 | 12 CU | 10-14 CU |
| Ro60 Calibrator 2 | 360 CU | 324 - 396 CU |
| Ro60 Negative Control | 10 CU | 8 - 12 CU |
| Ro60 Positive Control | 50 CU | 40-60 CU |
Precision
The precision of the QUANTA Flash Ro60 assay was evaluated on 10 samples containing various concentrations of Ro60 antibodies in accordance with CLSI EP5-A2, Evaluation of Precision Performance of Quantitative Measurement Procedures - Approved Guideline: samples were run in duplicates, twice a day, for 21 days. Production reagent lots 131007 and 141008 were used for the studies.
Data were analyzed with the Analyse-it for Excel method evaluation software, and within run, between run, between day and total precision were calculated.
Acceptance criteria: Total %CV: < 10%
Results are summarized in the Table below.
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| QUANTA Flash Ro60 | Within Run | Between Runs | Between Days | Total | ||||||
|---|---|---|---|---|---|---|---|---|---|---|
| Sample ID | Number ofreplicates | Mean(CU) | SD(CU) | CV(%) | SD(CU) | CV(%) | SD(CU) | CV(%) | SD(CU) | CV(%) |
| 110685-1800 | 84 | 11.5 | 0.6 | 5.0 | 0.0 | 0.0 | 0.5 | 4.5 | 0.8 | 6.8 |
| 000674-800 | 84 | 16.7 | 0.9 | 5.4 | 0.8 | 4.8 | 0.7 | 4.4 | 1.4 | 8.4 |
| 110688-1000 | 84 | 20.7 | 1.4 | 6.9 | 0.0 | 0.0 | 0.9 | 4.3 | 1.7 | 8.1 |
| 110689-950 | 84 | 25.7 | 0.9 | 3.4 | 0.8 | 3.3 | 1.0 | 4.0 | 1.6 | 6.2 |
| 110687-1500 | 84 | 26.4 | 0.8 | 3.1 | 0.6 | 2.4 | 0.5 | 1.9 | 1.1 | 4.4 |
| 110687-200 | 84 | 142.1 | 7.7 | 5.4 | 4.5 | 3.1 | 6.2 | 4.3 | 10.8 | 7.6 |
| 110688-85 | 84 | 406.3 | 26.6 | 6.6 | 0.0 | 0.0 | 21.2 | 5.2 | 34.0 | 8.4 |
| 110684-28 | 84 | 807.5 | 25.9 | 3.2 | 20.3 | 2.5 | 18.6 | 2.3 | 37.8 | 4.7 |
| 000674-5.83 | 84 | 1181.0 | 45.3 | 3.8 | 49.2 | 4.2 | 30.6 | 2.6 | 73.6 | 6.2 |
| 110686-55 | 84 | 1246.3 | 67.9 | 5.4 | 35.3 | 2.8 | 60.8 | 4.9 | 97.7 | 7.8 |
Reproducibility
Three samples were tested on two different reagent lots, using two different lots of Calibrators, by two operators. Samples were run in quadruplicates, two times a day, for 10 days, to generate 80 data points per sample. Production reagent lots 131007 and Calibrator and Control lots 131005 and 131006 were used for the studies. Data were analyzed with the Analyse-it for Excel method evaluation software, and within run, between reagent lots, between calibrator lots, between operators and total precision were calculated.
Acceptance criteria: Total %CV: < 10%
Results are summarized in the Table below.
| QUANTA Flash Ro60 | ||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Sample | Number ofreplicates | Mean(CU) | Within RunSD(CU) | Within RunCV(%) | BetweenReagent LotsSD(CU) | BetweenReagent LotsCV(%) | BetweenCalibrator LotsSD(CU) | BetweenCalibrator LotsCV(%) | BetweenOperatorsSD(CU) | BetweenOperatorsCV(%) | TotalSD(CU) | TotalCV(%) |
| 1 | 80 | 11.2 | 0.2 | 1.9 | 0.6 | 5.8 | 0.6 | 5.4 | 0.5 | 4.3 | 0.6 | 5.3 |
| 2 | 80 | 21.0 | 0.5 | 2.5 | 1.6 | 7.4 | 0.8 | 3.9 | 0.7 | 3.1 | 1.1 | 5.4 |
| 3 | 80 | 100.0 | 2.7 | 2.7 | 5.0 | 5.0 | 5.3 | 5.3 | 4.2 | 4.2 | 5.1 | 5.1 |
Limit of Blank (LoB) and Limit of Detection (LoD)
The LoD of the QUANTA Flash Ro60 assay is 519 RLU, which is below the analytical measuring range of the assay. It was determined 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.
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For determining the LoB, 4 blank samples (System Rinse) from two different lots were run in replicates of five on two reagent lots, once per day, for 3 days. Production reagent lots 131007 and 141008 were used for the studies. Sixty data points were generated on each lot.
The LoB was determined on each lot separately with the Analyse-it for Excel software's Reference Interval function, at the 95" percentile, using the non-parametric method, as the dataset showed nonnormal distribution (p values < 0.0001). The LoB for lot 131007 was determined as 398 RLU, and for lot 141008 as 452 RLU. The final LoB value is 452 RLU.
For determining the LoD, 4 low level samples (prepared by diluting anti-Ro60 positive samples with System Rinse) were run in replicates of five on two reagent lots, once per day, for 3 days. Production reagent lots 131007 and 141008 were used for the studies. Sixty data points were generated on each lot.
The LoD was determined separately on each lot according to CLSI EP17-A2 guideline. The limit of detection for lot 131007 was determined as 519 RLU, and for lot 141010 as 502 RLU. The final LoD value is 519 RLU.
These values are below the value of the lowest QUANTA Flash Ro60 Master Curve standard, i.e. below the Analytical Measuring Range.
Analytical Measuring Range (AMR)
4.9 CU - 1374.8 CU QUANTA Flash Ro60: 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 >1374.8 by 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 measured is 1374.8 CU, the highest value that can be reported is 27496 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 19,707 CU in the Ro60 assay (the highest concentration that was tested).
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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 Approach; Approved Guideline. Production reagent lot 131007 was used for the study. Three serum samples with various Ro60 antibody concentrations were diluted in 10% increments (from 0% to 90% diluent) to obtain values that cover the AMR. The dilutions were assayed in duplicates. Percent recovery of obtained results was calculated compared to the expected 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 three specimens showed dilution linearity individually.
| Sample | Test Range (CU) | Slope (95% CI) | R2 |
|---|---|---|---|
| 1 | 148.3 to 1372.2 | 1.05 (1.02 to 1.08) | 1.00 |
| 2 | 15.3 to 178.6 | 1.00 (0.96 to 1.04) | 0.99 |
| 3 | 7.5 to 45.6 | 1.04 (1.00 to 1.08) | 0.99 |
The combined data yielded the following results with linear regression:
| Sample | Test Range (CU)* | Slope (95% CI) | R2 |
|---|---|---|---|
| All | 7.5 to 1372.2 | 1.07 (1.06 to 1.08) | 1.00 |
*The analytical measuring range of the assay is 4.9 CU to 1374.8 CU.
Interference
The interference study was performed according to CLSI EPO7-A2, Interference Testing in Clinical Chemistry; Approved Guideline - Second Edition. Three specimens were tested (near-the-cutoff negative: 15.2 CU; weak positive: 40.3 CU; high positive: 161.4 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 Ro60 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: 99% to 109%), hemoglobin up to 200 mg/dL (recovery: 100% to 108%), triglycerides up to 1000 mg/dL (recovery: 89% to 109%),
{14}------------------------------------------------
cholesterol up to 224.3 mg/dL (recovery: 89% to 109%), and RF IgM up to 500 IU/mL (recovery: 90% to 107%).
Cross-reactivity
To test potential cross-reactivity with autoantibodies and infection-induced antibodies, results obtained on 213 of the total 286 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-Ro60 positivity rate is shown in the Table below:
| Diagnosis | Number of samples | # pos | % pos |
|---|---|---|---|
| Graves' Disease | 10 | 0 | 0.0% |
| Hashimoto Thyroiditis | 10 | 0 | 0.0% |
| Celiac Disease | 11 | 0 | 0.0% |
| Crohn's Disease | 20 | 1 | 5.0% |
| Ulcerative Colitis | 20 | 0 | 0.0% |
| HCV | 9 | 0 | 0.0% |
| HBV | 9 | 0 | 0.0% |
| HIV | 5 | 0 | 0.0% |
| Syphilis | 10 | 0 | 0.0% |
| Primary Antiphospholipid Syndrome | 15 | 1 | 6.7% |
| Vasculitis | 1 | 0 | 0.0% |
| Systemic sclerosis | 48 | 1 | 2.1% |
| Autoimmune myositis | 1 | 0 | 0.0% |
| Rheumatoid arthritis | 20 | 1 | 5.0% |
| Autoimmune liver disease* | 24 | 1 | 4.2% |
| Total controls | 213 | 5 | 2.3% |
Based on the results, the QUANTA Flash Ro60 assay does not show cross-reactivity with autoantibodies that are present in various autoimmune diseases, or antibodies against infectious agents.
Lot to lot comparison
Twenty-one unique samples and the Positive Controls (altogether 23 specimens) with various reactivity levels were tested in triplicates with three different reagent lots: 131007 and 141008. The samples covered the total analytical measuring range of the assay. Results were processed
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510(k) Summary QUANTA Flash® Ro60
by linear regression analysis and bias calculation according to CLSI EPO9-A2, Method Comparison and Bias Calculation Using Patient Samples; Approved Guideline - Second Edition.
Pair-wise comparisons were performed between lot 131007, lot 131006 vs 14008 and lot 131007 vs 141008, considering individual replicates instead of the mean of replicates.
Acceptance criteria and results are in the Table below. All results were within the acceptance limits.
| 131006 vs131007 | 131006 vs14008 | 131007 vs141008 | |
|---|---|---|---|
| Acceptance criteria | |||
| Weighted r: ≥0.975 for linear regression | 0.994 | 0.996 | 0.996 |
| Intercept of the regression line (constant bias):± 15% of cut-off (3 CU) | -1.2 | -0.3 | 1.0 |
| Slope of the regression line (proportional bias): 0.9-1.1 | 1.0 | 1.0 | 1.0 |
| Weighted S y/x: ≤ 0.5 | 0.095 | 0.076 | 0.075 |
| Predicted bias (difference) at cut-off: ±15% (3 CU) | -1.1 | 0.0 | 1.3 |
Sample stability
Six samples, encompassing negative, around the cut-off, and positive samples were tested for up to 21 days at 2-8℃, up to 48 hours at room temperature, moreover, after repeated freeze/thaw cycles up to 3 cycles. One sample had insufficient volume, and could not be tested on days 14 and 21 of the 2-8°C storage study.
Acceptance criteria: 85-115% average recovery.
Claim: up to 48 hours of storage at room temperature, up to 14 days of storage at 2-8°C, and up to 3 freeze/thaw cycles when samples are stored at or below -20°C.
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 Ro60 to establish initial stability claim: the beads, 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℃. The recovery of the measured values was calculated for each time point (compared to those obtained with 5 ± 3°C stored reagent). All calculations were performed by comparing results of sealed components stored at 5 ± 3℃ (control) to those stored at 37 ± 3℃ (test) for 1, 2, 3, and 4 weeks, where one week is equal to six months at 5 ± 3℃. Linear regression analysis was performed between recovery values and the number of days.
Acceptance criteria for one year preliminary expiration dating:
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- Beads:
With regression analysis, the lower 95% Cl interval of the regression line is ≥ 85% at 2 weeks, and no individual data point has ≤ 75% recovery at 2 weeks.
- Controls and Calibrators:
With regression analysis, the lower 95% Cl interval of the regression line is ≥ 90% at 2 weeks, and no individual data point has ≤ 80% recovery at 2 weeks.
Beads
Testing was performed on three lots of Ro60 coupled beads using up to 6 characterized samples with various reactivity levels.
All three lots of beads retained > 85% 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 Ro60 Calibrators and Controls. All Calibrators and Controls maintained > 90% reactivity (considering the 95% Cl) when sored at 37 ± 3°C 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 20 runs. The first run (each vial run in duplicate) was used to establish baseline value, and then additional 19 runs (each vial run in singleton) were performed. 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 against the mean of duplicates of each vial from the first run. Controls are considered stable when all values run within their established range, and the linear
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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.
Reagent Cartridge
To establish the in-use stability of the QUANTA Flash Ro60 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.
Using these criteria, the in-use (onboard) stability of Ro60 reagent cartridge was set at 49 days.
Real time stability
Real time stability testing was performed at 3, 6, 9 and 12 months on Calibrators and Controls, while testing was performed at 2, 5, 8, and 12 months on the reagent cartridge to support the one year expiration.
For Controls, each control was tested in triplicates at each time point.
- Acceptance criteria: results should fall within their acceptable ranges as it was established at the release of the controls.
Calibrators were tested in triplicates at each time point as it is done during calibration. 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%
For reagent cartridge, QC panel samples were tested at each time point. This QC panel is used by the QC Department for reagent release and QC.
- Acceptance criteria: results should fall within their respective QC ranges.
All results were within the acceptance limits.
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Cut-off, reference range
| QUANTA Flash Ro60: | Negative | <20 CU |
|---|---|---|
| Positive | $\geq$ 20 CU |
The reference population for establishing the reference interval for the Ro60 assay consisted of 156 subjects:
| Sample Group | N |
|---|---|
| Apparently healthy blood donors | 115 |
| Viral hepatitis positive samples | 8 |
| HIV positive samples | 5 |
| Syphilis positive samples | 5 |
| Rheumatoid arthritis patients | 23 |
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 C28-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. The 99th percentile of the obtained values calculated as 8193.6 RLU.
Additionally, 12 proficiency testing samples from the College of American Pathologists (CAP) and United Kingdom National External Quality Assessment Service (UKNEQAS) with known consensus/target results were tested to aid in the determination of the cutoff. Taking into account the target results of the proficiency testing samples, the cutoff was increased to 12,000 RLU to ensure optimal differentiation between negatives and positives, and a 20 CU value was assigned to this RLU value. No reference sample tested positive at this cutoff level.
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 Ro60. A total of 475 characterized samples were included in the Validation Set for the QUANTA Flash Ro60. All samples were run on the QUANTA Flash Ro60. The distribution of the Ro60 positivity rate is in the Table below:
| Patient group | N | Number positive | % positive |
|---|---|---|---|
| Graves' Disease | 10 | 0 | 0.0% |
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510(k) Summary QUANTA Flash® Ro60
| Patient group | N | Number positive | % positive |
|---|---|---|---|
| Hashimoto Thyroiditis | 10 | 0 | 0.0% |
| Celiac Disease | 11 | 0 | 0.0% |
| Crohn's Disease | 20 | 1 | 5.0% |
| Ulcerative Colitis | 20 | 0 | 0.0% |
| HCV | 9 | 0 | 0.0% |
| HBV | 9 | 0 | 0.0% |
| HIV | 5 | 0 | 0.0% |
| Syphilis | 10 | 0 | 0.0% |
| Osteoarthritis | 20 | 1 | 5.0% |
| Primary Antiphospholipid Syndrome | 15 | 1 | 6.7% |
| Secondary Antiphospholipid Syndrome* | 15 | 4 | 26.7% |
| Other rheumatic diseases | 38 | 1 | 2.6% |
| Vasculitis | 1 | 0 | 0.0% |
| Systemic sclerosis | 48 | 1 | 2.1% |
| Autoimmune myositis | 1 | 0 | 0.0% |
| Rheumatoid arthritis | 20 | 1 | 5.0% |
| Autoimmune liver disease** | 24 | 1 | 4.2% |
| Total controls | 286 | 11 | 3.8% |
| Sjögren's Syndrome | 39 | 26 | 66.7% |
| SLE | 150 | 36 | 24.0% |
| Total | 475 | ||
| * Patients may have SLE | |||
| ** Samples contain autoimmune liver disease specific antibodies (SLA, F-actin, M2) |
The results were analyzed to calculate sensitivity and specificity for SLE (n=150) and Sjögren's syndrome (SS) (n=39) separately, and SLE and SS combined. The secondary APS group was excluded from all calculations.
Clinical sensitivity and specificity of the QUANTA Flash Ro60 in Sjögren's syndrome
| n=310 | Diagnosis | Analysis(95% confidence) | |||
|---|---|---|---|---|---|
| SS | Controls(excludingSLE) | Total | |||
| QUANTA FlashRo60 | Positive | 26 | 7 | 33 | Sensitivity = 66.7% (49.8-80.9%) |
| Negative | 13 | 264 | 277 | Specificity = 97.4% (94.8-99.0%) | |
| Total | 39 | 271 | 310 |
Clinical sensitivity and specificity of the QUANTA Flash Ro60 in SLE
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| n=421 | Diagnosis | ||||
|---|---|---|---|---|---|
| SLE | Controls(excludingSjögren's) | Total | Analysis(95% confidence) | ||
| QUANTA FlashRo60 | Positive | 36 | 7 | 43 | Sensitivity = 24.0% (17.4-31.6%) |
| Negative | 114 | 264 | 378 | Specificity = 97.4% (94.8-99.0%) | |
| Total | 150 | 271 | 421 |
Clinical sensitivity and specificity of the QUANTA Flash Ro60 in SLE+SS
| Diagnosis | Analysis(95% confidence) | ||||
|---|---|---|---|---|---|
| n=460 | SS or SLE | Controls | Total | ||
| QUANTA FlashRo60 | Positive | 62 | 7 | 69 | Sensitivity = 32.8% (26.2-40.0%) |
| Negative | 127 | 264 | 391 | Specificity = 97.4% (94.8-99.0%) | |
| Total | 189 | 271 | 460 |
Expected values
The expected value in the normal population is "negative". Anti-Ro60 autoantibody levels were analyzed in a cohort of 98 apparently healthy blood donors (52 females and 46 males, ages 17 to 60 years, with an average age of 33.2 years and median age of 29 years) using the QUANTA Flash Ro60. 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, 2 (2 %) of the samples were positive on the QUANTA Flash Ro60. The mean concentration was 6 CU, and the values ranged from <4.9 to 84.8 CU.
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Comparison with predicate device
Samples for method comparison analysis included a total of 143 samples from SS and SLE patients and disease controls, with results for 63 samples being within the reportable range of the assy. These samples were tested on both the QUANTA Flash Ro60 and on the predicate ELISA. has an equivocal range. The data are presented in two ways; with ELISA equivocal samples as negative in the first table, then as positive in the following table:
Method Comparison, all samples:
| Method Comparison (N=143)ELISA equivocal as negative | Ro60 ELISA | Percent Agreement(95% Confidence) | |||
|---|---|---|---|---|---|
| QUANTA Flash®Ro60 CIA | Negative | 93 | 3 | 96 | Pos. Agree = 93.5% (82.1 – 98.6%) |
| Positive | 4 | 43 | 47 | Neg. Agree = 95.9% (89.8 – 98.9%) | |
| Total | 97 | 46 | 143 | Total Agree = 95.1% (90.2 – 98.0%) |
| Method Comparison (N=143)ELISA equivocal as positive | Ro60 ELISA | Percent Agreement(95% Confidence) | |||
|---|---|---|---|---|---|
| Negative | Positive | Total | |||
| QUANTA Flash®Ro60 CIA | Negative | 87 | 9 | 96 | Pos. Agree = 82.7% (69.7 – 91.8%) |
| Positive | 4 | 43 | 47 | Neg. Agree = 95.6% (89.1-98.8%) | |
| Total | 91 | 52 | 143 | Total Agree = 90.9% (85.0 - 95.1%) |
Method Comparison, samples within reportable range:
| Method Comparison (N=63)ELISA equivocal as negative | Ro60 ELISA | Percent Agreement(95% Confidence) | |||
|---|---|---|---|---|---|
| Negative | Positive | Total | |||
| QUANTA Flash®Ro60 CIA | Negative | 16 | 3 | 19 | Pos. Agree = 93.0% (80.9 – 98.5%) |
| Positive | 4 | 40 | 44 | Neg. Agree = 80.0% (56.3 – 94.3%) | |
| Total | 20 | 43 | 63 | Total Agree = 88.9% (78.4 – 95.4%) |
| Method Comparison (N=63)ELISA equivocal as positive | Ro60 ELISA | Percent Agreement(95% Confidence) | |||
|---|---|---|---|---|---|
| QUANTA Flash®Ro60 CIA | Negative | Positive | Total | ||
| Negative | 13 | 6 | 19 | Pos. Agree = 87.0% (73.7 – 95.1%) | |
| Positive | 4 | 40 | 44 | Neg. Agree = 76.5% (50.1 - 93.2%) | |
| Total | 17 | 46 | 63 | Total Agree = 84.1% (72.7 – 92.1%) |
§ 866.5100 Antinuclear antibody immunological test system.
(a)
Identification. An antinuclear antibody immunological test system is a device that consists of the reagents used to measure by immunochemical techniques the autoimmune antibodies in serum, other body fluids, and tissues that react with cellular nuclear constituents (molecules present in the nucleus of a cell, such as ribonucleic acid, deoxyribonucleic acid, or nuclear proteins). The measurements aid in the diagnosis of systemic lupus erythematosus (a multisystem autoimmune disease in which antibodies attack the victim's own tissues), hepatitis (a liver disease), rheumatoid arthritis, Sjögren's syndrome (arthritis with inflammation of the eye, eyelid, and salivary glands), and systemic sclerosis (chronic hardening and shrinking of many body tissues).(b)
Classification. Class II (performance standards).