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QUANTA Flash SS-B is a chemiluminescent immunoassay for the semi-quantitative determination of IgG anti-SS-B autoantibodies in human serum. The presence of anti-SS-B autoantibodies, in conjunction with clinical findings and other laboratory tests is an aid in the diagnosis of Sjögren's Syndrome and Systemic Lupus Erythematosus.

QUANTA Flash SS-B Calibrators are intended for use with the QUANTA Flash SS-B Reagents for the determination of IgG anti-SS-B autoantibodies in human serum. Each calibrator establishes a point of reference for the working curve that is used to calculate unit values.

QUANTA Flash SS-B Controls are intended for use with the QUANTA Flash SS-B reagents for quality control in the determination of IgG anti-SS-B autoantibodies in human serum.

Device Description

The QUANTA Flash SS-B 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 SS-B assay utilizes a reagent cartridge format, which is compatible with the BIO-FLASH instrument.

Purified recombinant SS-B 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-SS-B antibodies bound to the corresponding beads.

For quantitation, the QUANTA Flash SS-B 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 SS-B 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 SS-B kit contains the following materials:

One (1) QUANTA Flash SS-B Reagent Cartridge One (1) vial of Resuspension buffer One (1) Transfer pipette

The QUANTA Flash SS-B reagent cartridge contains the following reagents for 50 determinations:

a. SS-B antigen coated paramagnetic beads, lyophilized.
Assay buffer colored pink, containing Tris-buffered saline, Tween 20, protein b. stabilizers and preservatives.
C. Tracer IgG - Isoluminol labeled anti-human IgG antibodies in buffer, containing protein stabilizers and preservative.

The QUANTA Flash SS-B Calibrators kit contains two vials of Calibrator 1 and two vials of Calibrator 2:

QUANTA Flash SS-B Calibrators:

। QUANTA Flash SS-B Calibrator 1: Two (2) barcode labeled tubes containing 0.3 mL prediluted, ready to use reagent. Calibrators contain human antibodies to SS-B in stabilizers and preservatives.
QUANTA Flash SS-B Calibrator 2: Two (2) barcode labeled tubes containing 0.3 mL - prediluted, ready to use reagent. Calibrators contain human antibodies to SS-B in stabilizers and preservatives.

The QUANTA Flash SS-B Controls kit contains two vials of Negative Control and two vials of Positive Control:

QUANTA Flash SS-B Controls:

। QUANTA Flash SS-B Negative Control: Two (2) barcode labeled tubes containing 0.5 mL, ready to use reagent. Controls contain human antibodies to SS-B in stabilizers and preservatives.
QUANTA Flash SS-B Positive Control: Two (2) barcode labeled tubes containing 0.5 mL, ready to use reagent. Controls contain human antibodies to SS-B in stabilizers and preservatives.

AI/ML Overview

The document describes the QUANTA Flash® SS-B assay and its performance characteristics. This device is a chemiluminescent immunoassay for the semi-quantitative determination of IgG anti-SS-B autoantibodies in human serum, used as an aid in diagnosing Sjögren's Syndrome and Systemic Lupus Erythematosus.

Here's an analysis of the acceptance criteria and study data:

1. Table of Acceptance Criteria and Reported Device Performance

This section focuses on the analytical performance characteristics and clinical performance.

Test Category	Acceptance Criteria	Reported Device Performance
Precision	Total %CV: < 10%	Achieved. For 10 samples with various concentrations, total CV ranged from 5.1% to 8.9% (for samples 110684-04 and 110689-25 respectively), which is within the acceptance range. The majority of samples showed total CV below 8%.
Reproducibility	Total %CV: < 10%	Achieved. For 7 samples tested across different reagent lots, calibrator lots, and operators, total CV ranged from 3.4% to 7.9% (for Sample 2 and Sample A respectively), well within the 10% limit.
Limit of Blank (LoB)	LoB determined at 95th percentile, using parametric method if data is normal. Consistent with CLSI EP17-A2 guideline.	Achieved. LoB for lot 131009 was 269 RLU, and for lot 141010 was 294 RLU. The final LoB value is 294 RLU. These values are below the analytical measuring range.
Limit of Detection (LoD)	LoD based on proportions of false positives (alpha) < 5% and false negatives (beta) < 5%, consistent with CLSI EP17-A2 guideline.	Achieved. LoD for lot 131009 was 360 RLU, and for lot 141010 was 398 RLU. The final LoD value is 398 RLU. These values are below the analytical measuring range.
Linearity	Recovery between 80-120%, or ± 4 CU, whichever is greater. For linear regression, slope is between 0.9-1.1, and R² is ≥ 0.95.	Achieved. All four specimens showed dilution linearity individually, with slopes ranging from 0.95 to 1.04 and R² values of 0.99 for all. The combined data yielded a slope of 0.98 and R² of 0.99. The upper limit of the analytical measuring range was 1550 CU.
Interference	85% - 115% recovery, or ± 4 CU difference, whichever is greater.	Achieved. No interference detected with bilirubin (95.0% to 108.4% recovery), hemoglobin (96.0% to 105.8%), triglycerides (96.0% to 104.8%), cholesterol (96.0% to 104.8%), and RF IgM (90.7% to 111.5%) within tested concentrations.
Cross-reactivity	No specific numerical acceptance criteria mentioned, but implied expectation is minimal cross-reactivity with other autoantibodies or infection-induced antibodies.	Achieved. Out of 273 control samples from various autoimmune diseases and infectious conditions, only 7 (2.6%) showed anti-SS-B positivity, suggesting minimal cross-reactivity. This includes zero positivity in Graves' Disease, Hashimoto Thyroiditis, HCV, HBV, HIV, Syphilis, Primary Antiphospholipid Syndrome, Vasculitis, and Autoimmune myositis cohorts.
Lot to Lot Comparison	Weighted r: ≥0.975 for linear regression. Intercept of the regression line (constant bias): ± 15% of cut-off (3 CU). Slope of the regression line (proportional bias): 0.9-1.1. Weighted Sy/x: ≤ 0.5. Predicted bias (difference) at cut-off: ±15% (3 CU).	Achieved. All pair-wise comparisons between three reagent lots (131009 vs 14010, 131009 vs 14011, 141010 vs 141011) met the criteria. Weighted r values were 0.99-1.00, intercept biases were within ±15% of cutoff, and slopes were 1.0. Weighted Sy/x was 0.06-0.11. Predicted bias at cutoff was within limits.
Shelf Life Stability	Beads: Lower 95% CI of regression line > 85% at 2 weeks, no individual data point ≤ 75% recovery at 2 weeks. Controls/Calibrators: Lower 95% CI of regression line ≥ 90% at 2 weeks, no individual data point ≤ 80% recovery at 2 weeks.	Achieved. All three lots of beads retained > 85% reactivity. All calibrators and controls maintained > 90% reactivity.
In-use (onboard) Stability	Calibrators: 5 successful calibrations over 8.5 hours, average RLU recovery 90-110%, all controls/patient samples within expected range. Controls: All replicates within established range, linear regression line of %recovery 85-115% at run 15. Reagent Cartridge: Stability claim established when 95% CI of regression line reaches 85% or 115% recovery, or when 2 data points or ≥2% of recovery data is ≤ 75% or ≥ 125%.	Achieved. Calibrators: 5 successful calibrations over 8.5 hours, RLU values within 90-110%, controls/patient panel within expected range. Controls: All controls ran within acceptable ranges, regression line remained between 85% and 115% at run 15. Reagent Cartridge: In-use stability for reagent cartridge was set at 57 days based on the criteria.
Real Time Stability	Controls: Results fall within acceptable ranges. Calibrators: % recovery of average triplicates between 85-115%, %CV < 10%. Reagent Cartridge: QC panel results fall within respective QC ranges.	Achieved for 3, 6, 9, and 12 months. All results were within the acceptance limits for controls, calibrators, and reagent cartridges, supporting a one-year expiration.
Clinical Sensitivity (Sjögren's Syndrome)	Not explicitly stated as acceptance criteria, but reported.	35.0% (95% CI: 20.6-51.7%)
Clinical Specificity (Sjögren's Syndrome)	Not explicitly stated as acceptance criteria, but reported.	97.8% (95% CI: 95.9-99.0%)
Clinical Sensitivity (SLE)	Not explicitly stated as acceptance criteria, but reported.	13.1% (95% CI: 9.4-17.5%)
Clinical Specificity (SLE)	Not explicitly stated as acceptance criteria, but reported.	98.0% (95% CI: 96.0-99.1%)
Agreement with Predicate Device (All Samples)	Not explicitly stated as acceptance criteria, but reported agreement.	Positive Agreement = 81.4% (69.1 – 90.3%), Negative Agreement = 98.8% (97.5 – 99.5%), Total Agreement = 97.2% (95.6 – 98.3%).

Study Details:

This document primarily describes the analytical and clinical validation studies for the QUANTA Flash® SS-B assay, Calibrators, and Controls. There is no information about an AI component or expert readers in this specific submission. The device is an immunoassay, not an AI-driven diagnostic. Therefore, some requested information related to AI and human readers is not applicable.

2. Sample size used for the test set and the data provenance:

Precision Test Set: 10 samples (quantities for each are not specified beyond the number of replicates) for precision, and 7 samples for reproducibility.
LoB/LoD Test Set: 4 blank samples (System Rinse) and 4 low-level samples for LoB/LoD determination. 60 data points were generated for each lot tested.
Linearity Test Set: 5 serum samples with various SS-B antibody concentrations.
Interference Test Set: 3 specimens (near-cutoff negative, weak positive, high positive).
Cross-reactivity Test Set: 273 control samples from patients with various autoimmune diseases and infectious conditions.
Clinical Validation Set: A total of 761 characterized samples.
- Sub-cohort for Clinical Sensitivity/Specificity: Sjögren's Syndrome (n=40), SLE (n=290), Controls (n=431 including various autoimmune/infectious diseases and healthy donors).
- Sub-cohort for Expected Values: 138 apparently healthy blood donors (118 F, 20 M; ages 17-60).
- Sub-cohort for Comparison with Predicate Device: 639 samples (from the 761 clinical validation samples) that had predicate ELISA results available. This cohort included Sjögren's syndrome (n=40), SLE (n=240), and relevant disease controls (n=359). No healthy controls were included in this specific comparison cohort.
Data Provenance: The document does not explicitly state the country of origin but implies the samples are from human patients/donors. The studies are prospective in the sense that the device was tested on these cohorts to establish performance, however, the samples themselves were "characterized samples" meaning they were retrospectively collected with known diagnoses.

3. Number of experts used to establish the ground truth for the test set and the qualifications of those experts:

Not applicable as the device is an immunoassay. The ground truth (diagnosis of Sjögren's Syndrome or SLE, or specific autoimmune/infectious conditions for control samples) was established clinically rather than by expert review of imaging or similar data. The characterization of the samples is the ground truth.

4. Adjudication method for the test set:

Not applicable as the device is an immunoassay measuring anti-SS-B autoantibodies. The "ground truth" for clinical samples was their established clinical diagnosis, not a subjective interpretation requiring adjudication. For analytical studies, internal reference standards and external guidelines (CLSI) were used.

5. If a multi reader multi case (MRMC) comparative effectiveness study was done, If so, what was the effect size of how much human readers improve with AI vs without AI assistance:

Not applicable. This is not an AI-driven device or an imaging diagnostic that involves human readers.

6. If a standalone (i.e. algorithm only without human-in-the loop performance) was done:

Yes, implicitly. The QUANTA Flash SS-B assay is a diagnostic device that performs the tests and yields results semi-quantitatively. Its performance characteristics (precision, linearity, sensitivity, specificity, etc.) are determined in a standalone mode. Human involvement is in operating the instrument, collecting samples, and interpreting the numerical results in a clinical context, but not in directly influencing the assay's biochemical reaction or signal generation.

7. The type of ground truth used (expert consensus, pathology, outcomes data, etc):

Clinical Ground Truth for Clinical Validation: Established clinical diagnoses of Sjögren's Syndrome, Systemic Lupus Erythematosus (SLE), and various other autoimmune/infectious diseases for the control groups. For the reference range and expected values, apparently healthy blood donors were used.
Analytical Ground Truth: For linearity, LoB/LoD, precision, and interference studies, the ground truth was based on known concentrations, dilutions, or characteristics of the samples, derived from accepted laboratory methods and standards (e.g., CDC ANA reference sera, in-house standards).

8. The sample size for the training set:

The document mentions that the cut-off value was initially established using 187 subjects (162 apparently healthy blood donors, 10 viral hepatitis, 5 HIV, 5 Syphilis, 5 Rheumatoid arthritis patients). Then, "32 samples characterized as positive for SS-B antibodies by IIF, FIA, and ELISA" were used to "aid in the determination of the cutoff." It also states "One reference sample tested positive at this threshold."
For the Calibrators and Master Curve Standards, the "target CU is achieved through trial dilutions on small scale" and "Calibrator and Control values are directly traceable to in-house Standards that are used to create the Master Curves for the QUANTA Flash SS-B assay." No specific 'training set' sample size is explicitly defined beyond these descriptions, as the "training" here refers to the development and calibration of the assay itself, rather than training a machine learning model.

9. How the ground truth for the training set was established:

For Cut-off Establishment:
- The "reference population for establishing the reference interval" (187 subjects) was characterized clinically. The distribution of results from these subjects, particularly the 99th percentile (4097 RLU), informed the initial cut-off.
- Additionally, 32 samples "characterized as positive for SS-B antibodies by IIF, FIA, and ELISA" were used. This indicates that these samples had prior positive results from other established methods, forming a reference "positive" ground truth. The cut-off was adjusted to 12,000 RLU (corresponding to 20 CU) to optimize differentiation based on these known positive samples.
For Calibrators and Master Curves: The ground truth for these quantitative assignments is based on "in-house Standards," which are the fundamental reference materials used by the manufacturer to ensure consistency and accuracy of the assay. These standards are likely developed and characterized using highly controlled and validated methods, ensuring traceability where possible (though an international standard for SS-B antibodies is noted as not available). The process involves "trial dilutions," testing on instruments, and "final value assignment."

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