QUANTA FLASH ¿2GP1-DOMAIN1; CONTROLS, HEMOSIL ACUSTAR ANTI-¿2GPI DOMAIN1; CONTROLS.
Device Facts
| Record ID | K141274 |
|---|---|
| Device Name | QUANTA FLASH ¿2GP1-DOMAIN1; CONTROLS, HEMOSIL ACUSTAR ANTI-¿2GPI DOMAIN1; CONTROLS. |
| Applicant | Inova Diagnostics, Inc. |
| Product Code | MSV · Immunology |
| Decision Date | Feb 13, 2015 |
| Decision | SESE |
| Submission Type | Traditional |
| Regulation | 21 CFR 866.5660 |
| Device Class | Class 2 |
Indications for Use
The QUANTA Flash® ß2GP1-Domain1 is an in vitro chemiluminescent immunoassay (CIA) for the semi-quantitative determination of IgG autoantibodies to ß2GP1-Domain1 in human serum. The presence of anti-B2GP1- Domain1 autoantibodies is used in conjunction with clinical and other laboratory findings as an aid in the diagnosis of antiphospholipid syndrome. The OUANTA Flash® B2GP1-Domain1 is not intended to replace assays for antibodies against the whole ß2GP1 molecule. Testing for antibodies to the whole is required according to the classification criteria for antiphospholipid syndrome. The QUANTA Flash ß2GP1-Domain1 Controls are intended for quality control purposes of the QUANTA Flash ß2GP1-Domain1 chemiluminescent immunoassay (CIA) kit. The HemosIL® AcuStar Anti-S2GPI Domain 1 is an in vitro chemiluminescent immunoassay (CIA) for the semiquantitative determination of IgG autoantibodies to B2GPI Domain 1 in human serum. The presence of anti-R2GPI Domain 1 autoantibodies is used in conjunction with clinical and other laboratory findings as an aid in the diagnosis of antiphospholipid syndrome. The HemosIL® AcuStar Anti-ß2GPI Domain 1 is not intended to replace assays for antibodies against the whole ß2GPI molecule. Testing for antibodies to the whole is required according to the classification criteria for antiphospholipid syndrome. The HemosIL AcuStar Anti-B2GPI Domain 1 Controls are intended for quality control purposes of the HemosIL AcuStar Anti-ß2GPI Domain 1 chemiluminescent immunoassay (CIA) kit.
Device Story
Device is an in vitro chemiluminescent immunoassay (CIA) for detecting IgG autoantibodies to β2GP1-Domain 1. Input: human serum samples. Operation: Recombinant β2GP1-Domain 1 protein coated on paramagnetic beads; samples incubated with beads; washed; isoluminol-conjugated anti-human IgG added; trigger reagents added to induce chemiluminescence. Output: Relative Light Units (RLU) converted to Chemiluminescent Units (CU) via instrument-specific working curve. Used on BIO-FLASH® or ACL AcuStar instruments in clinical laboratories. Automated system handles sample dilution, incubation, washing, and signal detection. Results assist clinicians in diagnosing antiphospholipid syndrome (APS) alongside other clinical/laboratory findings. Benefits: provides automated, semi-quantitative measurement of specific domain-targeted antibodies.
Clinical Evidence
Clinical validation used 1,090 samples (270 APS, 820 non-APS). Sensitivity: 51.1% (95% CI: 45.0-57.2%); Specificity: 99.6% (95% CI: 98.9-99.9%). Analytical performance included precision (total CV ≤ 10-15%), linearity (AMR 3.6–1380.4 CU), and interference testing (no interference from bilirubin, hemoglobin, triglycerides, cholesterol, or RF IgM).
Technological Characteristics
Solid-phase chemiluminescent immunoassay (CIA). Reagents: Recombinant β2GP1-Domain 1 coated paramagnetic beads, isoluminol-labeled anti-human IgG conjugate. Instrumentation: BIO-FLASH® or ACL AcuStar (automated closed system). Calibration: Lot-specific Master Curve with two calibrators. Storage: 2-8°C. Software: Automated processing, curve fitting, and result reporting.
Indications for Use
Indicated for semi-quantitative determination of IgG autoantibodies to β2GP1-Domain 1 in human serum to aid in the diagnosis of antiphospholipid syndrome (APS) in conjunction with clinical and other laboratory findings. Not intended to replace whole β2GP1 molecule assays. Prescription use only.
Regulatory Classification
Identification
A multiple autoantibodies immunological test system is a device that consists of the reagents used to measure by immunochemical techniques the autoantibodies (antibodies produced against the body's own tissues) in serum and other body fluids. Measurement of multiple autoantibodies aids in the diagnosis of autoimmune disorders (disease produced when the body's own tissues are injured by autoantibodies).
Predicate Devices
- QUANTA Lite® β2GPI IgG ELISA (K970551)
Related Devices
- K152875 — QUANTA Flash B2GP1-Domain1, QUANTA Flash B2GP1-Domain1 Controls, HemosIL AcuStar Anti-B2GPI Domain 1, HemosIL AcuStar Anti-B2GPI Domain 1 Controls · Inova Diagnostics, Inc. · Dec 21, 2015
Submission Summary (Full Text)
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# 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION MEMORANDUM
A. 510(k) Number:
k141274
B. Purpose for Submission:
New device
C. Measurand:
Anti-β2GP1-Domain 1 autoantibodies
D. Type of Test:
Semi-quantitative chemiluminescent immunoassay
E. Applicant:
INOVA Diagnostics, Inc.
F. Proprietary and Established Names:
QUANTA Flash® β2GP1-Domain1
QUANTA Flash® β2GP1-Domain1 Controls
HemosIL® Acustar Anti-β2GPI-Domain 1
HemosIL® Acustar Anti-β2GPI-Domain 1 Controls
G. Regulatory Information:
1. Regulation section:
21 CFR §866.5660, Multiple autoantibodies immunological test system
21 CFR § 862.1660, Quality control material (assayed and unassayed)
2. Classification:
Class II (assay)
Class I (controls)
3. Product code:
MSV, System, Test, Antibodies, B2-Glycoprotein I (B2-GPI)
JJX, Analyte Controls (Assayed and Unassayed)
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4. Panel:
Immunology (82)
Clinical Chemistry (75)
H. Intended Use:
1. Intended use(s):
QUANTA Flash® β2GP1-Domain1
The QUANTA Flash® β2GP1-Domain1 is an in vitro chemiluminescent immunoassay (CIA) for the semi-quantitative determination of IgG autoantibodies to β2GP1-Domain1 in human serum. The presence of anti-β2GP1-Domain1 autoantibodies is used in conjunction with clinical and other laboratory findings as an aid in the diagnosis of antiphospholipid syndrome. The QUANTA Flash® β2GP1-Domain1 is not intended to replace assays for antibodies against the whole β2GP1 molecule. Testing for antibodies to the whole β2GP1 molecule is required according to the classification criteria for antiphospholipid syndrome.
QUANTA Flash® β2GP1-Domain1 Controls
The QUANTA Flash® β2GP1-Domain1 Controls are intended for quality control purposes of the QUANTA Flash® β2GP1-Domain1 chemiluminescent immunoassay (CIA) kit.
HemosIL® AcuStar Anti-β2GPI Domain 1
The HemosIL® Acustar Anti-β2GPI Domain 1 is an in vitro chemiluminescent immunoassay (CIA) for the semi-quantitative determination of IgG autoantibodies to β2GP1-Domain 1 in human serum. The presence of anti-β2GP1-Domain 1 autoantibodies is used in conjunction with clinical and other laboratory findings as an aid in the diagnosis of antiphospholipid syndrome. The HemosIL® Acustar Anti-β2GPI Domain 1 is not intended to replace assays for antibodies against the whole β2GP1 molecule. Testing for antibodies to the whole β2GP1 molecule is required according to the classification criteria for antiphospholipid syndrome.
HemosIL® AcuStar Anti-β2GPI Domain 1 Controls
The HemosIL® AcuStar Anti-β2GPI Domain 1 Controls are intended for quality control purposes of the HemosIL® Anti-β2GPI Domain 1 chemiluminescent immunoassay (CIA) kit.
2. Indication(s) for use:
Same as Intended use
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3. Special conditions for use statement(s):
For Prescription use only
4. Special instrument requirements:
BIO-FLASH® or ACL AcuStar instrument (k083518)
I. Device Description:
Since the QUANTA Flash® β2GP1-Domain1 assay and the HemosIL® AcuStar β2GP1-Domain 1 assay are identical, device description for the QUANTA Flash® β2GP1-Domain1 assay only is shown below.
The QUANTA Flash® β2GP1-Domain1 kit contains the following materials:
1. QUANTA Flash β2GP1-Domain1 Reagent Cartridge, contains the following reagents for 50 determinations:
a. β2GP1-Domain1 coated paramagnetic beads, preserved prior to first time use.
b. Assay Buffer – colored pink, containing Tris-buffered saline, Tween 20, protein stabilizers, and preservatives.
c. Tracer IgG – Isoluminol labeled anti-human IgG antibodies, containing buffer, protein stabilizers and preservative.
d. Sample diluent – containing Tris-buffered saline, Tween 20, protein stabilizers and preservatives.
2. Resuspension buffer: 1 vial – colorless, containing buffer, protein stabilizers and preservatives.
3. QUANTA Flash β2GP1-Domain1 Calibrator 1, containing: 1 x 1 mL barcoded tube of a solution with human antibodies to β2GP1-Domain1 in buffer, stabilizers, and preservatives.
4. QUANTA Flash β2GP1-Domain1 Calibrator 2, containing: 1 x 1 mL barcoded tube of a solution with human antibodies to β2GP1-Domain1 in buffer, stabilizers, and preservatives.
The QUANTA Flash® β2GP1-Domain1 Controls kit contains two vials of Negative Control and two vials of Positive Control:
1. QUANTA Flash β2GP1-Domain1 Low Control: Three (3) barcode labeled tubes containing 1.0 mL, ready to use reagent. Controls contain human antibodies to β2GP1-Domain1 in stabilizers and preservatives.
2. QUANTA Flash β2GP1-Domain1 High Control: Three (3) barcode labeled tubes containing 1.0 mL, ready to use reagent. Controls contain human antibodies to β2GP1-Domain1 in stabilizers and preservatives.
The QUANTA Flash® β2GP1-Domain1 assay is designed to run on the BIO-FLASH®
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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® $\beta 2\mathrm{GP1}$ -Domain1 assay utilizes a reagent cartridge format, which is compatible with the BIO-FLASH instrument.
# J. Substantial Equivalence Information:
1. Predicate device name(s):
QUANTA Lite® β2GP1 ELISA
2. Predicate 510(k) number(s):
k970551
3. Comparison with predicate:
QUANTA Flash $\beta 2\mathrm{GP1}$ -Domain1 Assay and Calibrators/ HemosIL AcuStar $\beta 2\mathrm{GPI}$ Domain 1 Assay and Calibrators:
| Similarities | | |
| --- | --- | --- |
| Item | Device | Predicate |
| | QUANTA Flash® β2GP1-Domain1 | QUANTA Lite® β2 GP1 IgG |
| Assay methodology | Solid phase (heterogeneous) immunoassay | Solid phase (heterogeneous) immunoassay |
| Assay and Calibrator Shelf life | One year | One year |
| Assay and Calibrator Storage | 2-8 °C | 2-8 °C |
| Sample type | Serum | Serum |
<table><tr><td colspan="3">Differences</td></tr><tr><td>Item</td><td>Device</td><td>Predicate</td></tr><tr><td></td><td>QUANTA Flash®
β2GP1-Domain1</td><td>QUANTA Lite®
β2 GP1 IgG</td></tr><tr><td>Intended use</td><td>The QUANTA Flash® β2GP1-Domain1 and the HemosIL®
Acustar Anti-β2GPI Domain 1
are in vitro chemiluminescent
immunoassays (CIA) for the
semi-quantitative determination
of IgG autoantibodies to
β2GP1-Domain1 in human
serum or citrated plasma. The
presence of anti-β2GP1-
Domain1 autoantibodies is used
in conjunction with clinical and</td><td>QUANTA Lite® β2 GP1 IgG is
an enzyme linked immunoassay
(ELISA) for the semi-
quantitative detection of β2 GP1
IgG antibodies in human serum.
The presence of β2Gp1 IgG
antibodies can be used in
conjunction with clinical
findings and other laboratory
tests to aid in the diagnosis of
certain autoimmune disease
thrombotic disorders such as</td></tr><tr><td></td><td>anti-β2GP1-Domain1 and anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti
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| Differences | | |
| --- | --- | --- |
| Item | Device | Predicate |
| | other laboratory findings as an aid in the diagnosis of antiphospholipid syndrome. The QUANTA Flash® β2GP1-Domain1 and the HemosIL® Acustar Anti-β2GPI Domain 1 are not intended to replace assays for antibodies against the whole β2GP1 molecule. Testing for antibodies to the whole β2GP1 molecule is required according to the classification criteria for antiphospholipid syndrome. | those secondary to systemic lupus erythematosus (SLE) or other lupus-like thrombotic diseases. |
| Detection/Operating principle | Chemiluminescent immunoassay | Enzyme-linked immunosorbent assay |
| Solid phase | Paramagnetic microparticles (beads | 96-well plate |
| Instrumentation | Automated on BIO-FLASH® instrument | Manual |
| Antigen | Recombinant domain1 of β2-Glycoprotein1 | Purified β2-Glycoprotein1 |
| Conjugate | Isoluminol conjugated anti-human IgG | HRP conjugated anti-human IgG |
| Signal Detected | Luminescence (visible light) | Absorbance at 450 nm |
| Calibration | Lot specific Master Curve + two calibrators (included in kit) | Five lot specific calibrators (included in kit) |
| Units | < 20 CU: Negative result ≥ 20 CU: Positive result | < 20 SGU: Negative result ≥ 20 SGU: Positive result |
| Analytical Measuring Range | 3.6 – 1380.4 CU | 9.4 – 150.0 SGU |
QUANTA Flash β2GP1-Domain1 Controls/ HemosIL AcuStar β2GPI-Domain 1 Controls:
| Similarities | | |
| --- | --- | --- |
| Item | QUANTA Flash β2GP1-Domain1 Controls | Predicate Device |
| Matrix | Human serum, stabilizers, and preservative | Human serum, stabilizers, and preservative |
| Physico-chemical characteristics | Liquid, ready to use | Liquid, pre-diluted, ready to use |
| Storage | 2-8 °C | 2-8 °C |
| Shelf life | One year | One year |
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| Differences | | |
| --- | --- | --- |
| Item | QUANTA Flash β2GP1-Domain1 Controls | Predicate Device |
| Intended use | The QUANTA Flash β2GP1-Domain1 Controls are intended for quality control purposes of the QUANTA Flash β2GP1-Domain1 chemiluminescent immunoassay (CIA) kit. | No separate intended use; controls are part of the QUANTA Lite® β2GP1 kit. |
| Analyte | Anti-β2GP1-Domain 1 antibodies | Anti-β2GP1 antibodies |
| Method | QUANTA Flash® β2GP1-Domain1 Chemiluminescent immunoassay | QUANTA Lite® β2GP1 ELISA |
| Unit | QUANTA Flash (CU) Arbitrary Units (U/mL) | SGU |
| Levels | 2 (low and high) | 2 (negative and positive) |
## K. Standard/Guidance Document Referenced (if applicable):
CLSI EP05-A2, Evaluation of Precision Performance of Quantitative Measurement Methods; Approved Guideline, Second Edition
CLSI EP17-A, Protocols for determination of limits of detection and limits of quantitation
CLSI EP06-A, Evaluation of the Linearity of Quantitative Measurement Procedures: A Statistical Approach; Approved Guideline.
CLSI EP09-A2-IR, Method Comparison and Bias Estimation Using Patient Samples; Approved Guideline - Second Edition (Interim Revision)
CLSI EP07-A, Interference Testing in Clinical Chemistry; Approved Guideline
## L. Test Principle:
Recombinant β2GP1-Domain1 protein is coated onto paramagnetic beads, which are stored lyophilized in the reagent cartridge. The reagent pack is prepared for use in the BIO-FLASH® system by pressing down on the grey lid of the reagent pack to pierce the induction seals on the reagent tubes. Once the seals are broken, the beads are rehydrated by adding the entire contents of the vial of resuspension buffer to the bead reagent tube using the transfer pipette supplied with the kit. Only the hole above the bead reagent tube is accessible at this point. The beads are then mixed with the resuspension buffer by pipetting up and down 30 times. This amount of mixing ensures complete resuspension of the beads. The label covering the remaining three reagent holes is now removed, and the reagent cartridge is then loaded onto the BIO-FLASH instrument. Samples are also loaded onto the instrument in sample racks. A patient serum sample is pre-diluted 1:10 by the BIO-FLASH with system rinse in a small disposable plastic cuvette. Small amounts of the diluted patient serum, the
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beads, and assay buffer are all combined into a second cuvette, and mixed. This cuvette is then incubated at $37^{\circ}\mathrm{C}$ . The beads are magnetized and washed several times. Isoluminol conjugated anti-human IgG antibodies are then added to the cuvette, and again incubated at $37^{\circ}\mathrm{C}$ . The beads are magnetized and washed repeatedly. The isoluminol conjugate is oxidized when Trigger 1 and Trigger 2 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- $\beta 2\mathrm{GP1}$ -Domain1 antibodies bound to the corresponding $\beta 2\mathrm{GP1}$ -Domain1 on the beads.
The QUANTA Flash® $\beta 2\mathrm{GP1}$ -Domain1 assay utilizes a pre-defined 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® $\beta 2\mathrm{GP1}$ -Domain1 Calibrators. Based on the results obtained with the two Calibrators included in the reagent kit, an instrument specific Working Curve is created, which is used to calculate chemiluminescent units (CU) from the instrument signal (RLU) obtained for each sample.
# M. Performance Characteristics (if/when applicable):
# 1. Analytical performance:
Since the QUANTA Flash $\beta 2\mathrm{GP1}$ -Domain1 assay and the HemosIL AcuStar $\beta 2\mathrm{GP1}$ -Domain 1 assay are identical, performance data for only the QUANTA Flash $\beta 2\mathrm{GP1}$ -Domain1 assay is shown below.
# a. Precision/Reproducibility:
Precision of the QUANTA Flash® $\beta 2\mathrm{GP1}$ -Domain1 assay was assessed in accordance with CLSI EP5-A2, by running 8 serum samples that were selected to cover the analytical measuring range of the assay, including samples around the medical decision point. Patients were run using duplicate aliquots, twice a day, for 20 days for a total of 80 replicates. Controls were run as quality control during each run. A working curve was generated using calibrators prior to run 1 on day 1. Total $\% \mathrm{CV}$ values were within the manufacturer's pre-determined acceptance limit of $\leq 10\%$ - $15\%$ . The results were analyzed by Analyse-It.
| | | Within Run | | Between-Run | | Between-Day | | Total | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Sample | Mean (CU) | SD | %CV | SD | %CV | SD | %CV | SD | %CV |
| 1 | 10.4 | 0.9 | 8.3% | 0.0 | 0.0% | 0.5 | 4.3% | 1.0 | 9.4% |
| 2 | 18.4 | 0.9 | 4.6% | 0.5 | 2.9% | 0.5 | 2.6% | 1.1 | 6.0% |
| 3 | 22.8 | 1.4 | 6.1% | 0.9 | 3.8% | 0.0 | 0.0% | 1.6 | 7.2% |
| 4 | 54.3 | 2.5 | 4.6% | 1.4 | 2.5% | 1.2 | 2.2% | 3.1 | 5.6% |
| 5 | 99.6 | 6.4 | 6.5% | 0.0 | 0.0% | 0.0 | 0.0% | 6.4 | 6.5% |
| 6 | 318.3 | 18.6 | 5.9% | 0.0 | 0.0% | 4.9 | 1.6% | 19.3 | 6.1% |
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Lot-to-Lot:
A separate lot-to-lot comparison study was performed using 26 samples from INOVA’s serum bank covering the analytical measuring range of the assay and positive and negative controls. Samples were tested in duplicate with three different reagent lots. All results were within the manufacturer’s pre-defined acceptance.
b. Linearity/assay reportable range:
The linearity of the analytical measuring range (AMR, 3.6 – 1380.4 CU) was evaluated by a study according to CLSI EP6-A. Five serum samples with various β2GP1-Domain1 antibody concentrations were serially diluted to obtain 10 dilutions of each sample. Percent recovery of obtained results was calculated compared to the expected results. Obtained values were plotted against expected values, and linear regression analysis was performed using Analyse-It software. The slope and intercept of the regression line were calculated with 95% CI as well as the R² values. All samples met the manufacturer’s pre-defined acceptance criteria. Results of the study are summarized below:
| Sample | Test Range (CU) | Slope (95% CI) | y-intercept (95% CI) | R² | Recovery Range |
| --- | --- | --- | --- | --- | --- |
| 1 | 164.1 – 1640.7 | 1.04 (0.98 – 1.09) | -36.8 (-93.8 to-0.2) | 0.99 | 95.1 – 106.2% |
| 2 | 45.5 – 454.7 | 0.99 (0.96 – 1.03) | 0.2 (-10.3 to 10.6) | 0.99 | 95.5 – 104.1% |
| 3 | 12.5 – 124.9 | 1.03 (1.00 – 1.05) | -1.9 (-3.9 to 0.1) | 1.00 | 94.3 – 103.3% |
| 4 | 8.1 – 81.1 | 1.01 (0.95 – 1.08) | -2.1 (-5.4 to 1.3) | 0.98 | 83.9 – 106.7 % |
| 5 | 1.0 – 10.3 | 0.96 (0.91 – 1.01) | 0.2 (-0.1 to 0.5) | 0.99 | 90.9 – 113.9 % |
The AMR is defined by the values of the lowest and highest Master Curve Standards. The QUANTA Flash® β2GP1-Domain1 AMR is 3.6 CU to 1380.4 CU.
High dose hook effect:
To assess hook effect, the measurement signal (relative light units, RLU) was examined for four high positive specimens, 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 values, thereby confirming that high positive specimens above the analytical measuring range do not show hook effect up to 435,000 RLU.
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c. Traceability, Stability, Expected values (controls, calibrators, or methods):
Traceability:
There are currently no international reference standards for anti-β2GP1-Domain1 antibodies. The assay is calibrated in relative arbitrary units (CU or U/mL). The QUANTA Flash® β2GP1-Domain1 Calibrators and Controls are manufactured by diluting human serum containing high titers of anti-β2GP1-Domain1 antibodies with antibody stabilizer buffer. The human serum is obtained from commercial sources and is tested for markers of infectious substances.
Value Assignment:
The QUANTA Flash® β2GP1-Domain1 assay utilizes a pre-defined lot specific Master Curve that is stored in the reagent pack barcode. 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. 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.
Stability:
Stability studies have been performed and the results support the following claims:
Sample Stability:
Samples were shown to be stable (defined by the manufacturer as recovery of ≤ ± 15%) for up to 48 hours at room temperature, 21 days at 2-8°C, and can undergo freeze/thaw up to 3 times.
Reagent Pack Stability:
Shelf Life: All components tested by accelerated stability testing (4 weeks at 37°C) fulfilled the manufacturer's pre-defined acceptance criteria, therefore one year expiration dating was assigned to each component stored, unopened, at 2-8°C. Real time stability is ongoing and currently supports a claim of 12 months.
Open/In use (onboard) Stability:
Opened reagent packs must be stored on-board the instrument. The in-use (on-board) stability of the reagent cartridge was set at 60 days.
Calibration Stability:
Shelf Life: Calibrators tested by accelerated stability testing (4 weeks at 37°C) fulfilled the manufacturer's pre-defined acceptance criteria, supporting assignment of a one year expiration dating to each component at 2-8°C. Real time stability is ongoing and currently supports a claim of 12 months.
In use (onboard) Stability:
Calibrators can be used for up to 4 calibrations over an 8 hour period onboard the instrument.
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Control Stability:
Shelf Life: Controls tested by accelerated stability testing (4 weeks at 37°C) fulfilled the manufacturer's acceptance criteria, so one year expiration dating was assigned to each component at 2-8°C. Real time stability is ongoing and currently supports a claim of 12 months.
Open/In use (onboard) Stability: The controls were given a maximum of 15 uses with a maximum of 10 minutes onboard the instrument per use. The total time the control tubes can be used is 2.5 hours or 10 minutes per use.
d. Detection limit:
Limit of Blank (LoB):
For the QUANTA Flash β2GP1-Domain1 assay, the LoB was determined according to CLSI EP17-A guideline by running 58 measurements on blank samples.
For the QUANTA Flash β2GP1-Domain1 assay the LoB was determined to 425.7 RLU. The LoB value is below the bottom limit of the curve that the instrument uses to calculate chemiluminescent units (CU), and therefore cannot be converted into CU.
Limit of Detection (LoD):
The LoD of the QUANTA Flash® β2GP1-Domain1 assay is 1296 RLU, which is below the analytical measuring range of the assay. LoD was determined using the CLSI EP17-A guideline with proportions of false positives (alpha) less than 5% and false negatives (beta) less than 5%; based on 264 determinations, with 144 measurements on blank samples and 120 measurements of low level samples. The LoD value is below the bottom limit of the curve that the instrument uses to calculate chemiluminescent units (CU), and therefore cannot be converted into CU. The LoD is below the lower limit of the reportable range, e.g. < 3.6 CU.
e. Analytical specificity:
Interference:
The interference study was performed according to CLSI EP07-A2. Three specimens were tested (negative: 10.7 CU; positive: 55.9 CU; high positive: 446.6 CU). Each interfering substance was spiked into each specimen at three different concentrations per CLSI EP07-A2 and the resulting samples were assessed in triplicate. Recovery of the unit values was calculated compared to control samples spiked with the same volume of diluents (10% of total).
No interference (defined by the manufacturer as ≤ ±15%, or ±4 CU difference, whichever is greater) was detected with bilirubin up to 100 mg/dL (recovery: 92.9% to 97.6%), hemoglobin up to 200 mg/dL (recovery: 95.6% to 97.2%), triglycerides up to 1000 mg/dL (recovery: 92.5% to 100%), cholesterol up to 224.3 mg/dL (recovery: 92.5% to 100%), and RF IgM up to 500 IU/mL (recovery: 86.2% to 98.3%).
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A statement indicating that grossly hemolyzed or icteric serum should not be used is included in the direction insert.
## Cross-Reactivity:
A potential cross-reactivity of the QUANTA Flash® β2GP1 Domain1 CIA with other autoantibodies was evaluated with 111 clinical samples with unknown disease states, most having high levels of various other autoantibodies. These samples were known to be positive for various other autoantibodies including: Sm, RNP, SS-A/Ro60, Ro52, SS-B, Jo-1, Scl-70, CENP, Ribosomal P, and DFS70. These are autoantibodies found in individuals with autoimmune diseases such as Systemic Lupus Erythematous (SLE), Sjögren’s Syndrome, scleroderma, and polymyositis patients. All samples, except for two, scleroderma samples, were negative for QUANTA Flash® β2GP1 Domain1 CIA.
## f. Assay cut-off:
The reference population for establishing the reference interval for the β2GP1-Domain1 assay consisted of 30 subjects with various conditions including infectious diseases and SLE without a history of thrombotic events.
All specimens were the same matrix as specified in the Intended Use. All specimens were unaltered. The cut-off was established Using Analyse-it for Excel, to ensure optimal differentiation between negatives and positives, and found at 7880 RLU. This point was defined as 20 CU. A result below 20 CU is considered negative and greater than 20 CU is considered positive.
## 2. Comparison studies:
### a. Method comparison with predicate device:
Samples for method comparison analysis included those samples from the clinical validation study (below) that were within the reportable range of the assay. For the first table for the analyte, only samples within the measuring ranges defined by the lowest and highest calibrators of the QUANTA Flash β2GP1-Domain1 and predicate assay is included in the method comparison. The 238 samples in this cohort included 183 APS samples and eight contrived samples within ± 25% of the assay cut-off. Twenty-two samples (9.2%) were within ± 25% of the assay cut-off.
| | Predicate ELISA | | | |
| --- | --- | --- | --- | --- |
| | | Positive | Negative | Total |
| QUANTA Flash β2GP1- Domain1 CIA | Positive | 101 | 28 | 129 |
| | Negative | 10 | 99 | 109 |
| | Total | 111 | 127 | 238 |
Positive Percent Agreement (101/111): 91.0% (95% CI: 84.1-95.6%)
Negative Percent Agreement (99/127): 78.0% (95% CI: 69.7-84.8%)
Overall Percent Agreement (200/238): 84.0% (95%CI: 78.7-88.4%)
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b. Matrix comparison:
Not applicable
# 3. Clinical studies:
# a. Clinical Sensitivity and Clinical specificity:
A set of 1090 samples, none of which were used in establishing the reference range, was used to validate the clinical performance of the QUANTA Flash $\beta 2\mathrm{GP1}$ -Domain1 CIA. The validation set consists of a set of clinically characterized sera from Primary APS, Secondary APS, and non-APS diseased controls. The results of the QUANTA Flash $\beta 2\mathrm{GP1}$ -Domain1 CIA device in each disease category are shown below:
| APS Sub-Diagnosis | N | QUANTA Flash β2GP1-Domain1 % Positive (N) |
| --- | --- | --- |
| Primary APS (pAPS) | 180 | 56.6%(102) |
| Secondary APS (sAPS)* | 90 | 40.0% (36) |
| Total APS | 270 | 51.1% (138) |
* All patients with sAPS were diagnosed with SLE
| Non-APS Diagnosis | N | QUANTA Flash β2GP1-Domain1 % Positive (N) |
| --- | --- | --- |
| Hepatitis B virus (HBV) | 21 | 0.0% (0.0) |
| Hepatitis C virus (HCV) | 10 | 0.0% (0.0) |
| Syphilis | 40 | 0.0% (0.0) |
| Crohn's Disease | 104 | 1.0% (1) |
| Ulcerative Colitis | 94 | 0.0% (0.0) |
| Rheumatoid Arthritis | 168 | 0.0% (0.0) |
| Osteoarthritis | 49 | 0.0% (0.0) |
| Scleroderma | 127 | 1.6% (2) |
| Others | 24 | 0.0% (0.0) |
| Pre-eclampsia/eclampsia | 34 | 0.0% (0.0) |
| Fetal loss no APS | 45 | 0.0% (0.0) |
| SLE no APS | 37 | 0.0% (0.0) |
| Thrombosis no APS | 41 | 0.0% (0.0) |
| Atopic Dermatitis | 26 | 0.0% (0.0) |
| Total | 820 | 0.5% (3) |
{12}
| | Clinical Diagnosis | | | |
| --- | --- | --- | --- | --- |
| | | APS | Not APS | Total |
| QUANTA Flash β2GP1-Domain1 | Positive | 138 | 3 | 141 |
| | Negative | 132 | 817 | 949 |
| | Total | 270 | 820 | 1090 |
Sensitivity = 51.1% (95% CI: 45.0-57.2%)
Specificity = 99.6% (95% CI: 98.9-99.9%)
While antibodies to Domain 1 of β2GP1 may be important in the pathology of APS, the test should be used in conjunction with whole β2GP1 molecule testing to aid in establishing a diagnosis of APS per the current practice guidelines outlined in the APS Classification Criteria¹ (Sydney 2006).
b. Other clinical supportive data (when a. and b. are not applicable):
Not Applicable
4. Clinical cut-off:
Not applicable
5. Expected values/Reference range:
The expected value in the normal population is "negative". Anti-β2GP1-Domain1 antibody levels were analyzed in a cohort of 400 apparently healthy blood donors (191 females, ages 17 to 60 years, average age 32.2 years, and 209 males ages 17 to 60 years, average age 34.7 years) using the QUANTA Flash® β2GP1-Domain1. This patient population was different from the one that was used to establish the cut-off. With a cut-off of 20 CU, one sample (0.2%) was positive (34.2 CU) on the QUANTA Flash β2GP1-Domain1. The mean concentration was 3.8 CU, the median concentration 3.6 CU, and the values ranged from <3.6 to 34.2 CU.
N. Proposed Labeling:
The labeling is sufficient and it satisfies the requirements of 21 CFR Part 809.10.
O. Conclusion:
The submitted information in this premarket notification is complete and supports a substantial equivalence decision.
¹ Miyakis S. et. al. International consensus statement on an update of the classification criteria for definite antiphospholipid syndrome (APS). J Thromb Haemost 2006, 4:295 – 306.
