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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.

The QUANTA Flash® ß2GP1-Domain1 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® ß2GP1-Domain1 assay utilizes a reagent cartridge format, which is compatible with the BIO-FLASH instrument.

The assays included in this submission, the QUANTA Flash ß2GP1-Domain1 marketed by Inova Diagnostics Inc. (9900 Old Grove Road, San Diego, CA 92131) and HemoslL AcuStar Anti-ß-GPI Domain 1 marketed by Instrumentation Laboratory (180 Hartwell Road Bedford, MA 01730), are equivalent assays. Therefore all data stated hereafter and referred to as: QUANTA Flash §2GP1-Domain1 data is equivalently also valid for HemosIL "AcuStar Anti-ß₂GPI Domain 1.

Recombinant ß2GP1-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 prediluted 1:10 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(III) coproporphyrin in sodium hydroxide solution) and Trigger 2 (urea-hydrogen 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- ß2GP1-Domain1 antibodies bound to the corresponding ß2GP1-Domain1 on the beads.

The QUANTA Flash® ß2GP1-Domain1 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® ß2GP1-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.

This document describes the analytical and clinical performance characteristics of the QUANTA Flash® β2GP1-Domain1 and HemosIL® AcuStar Anti-β2GPI Domain 1 assays, which are in vitro chemiluminescent immunoassays (CIA) for the semi-quantitative determination of IgG autoantibodies to β2GP1-Domain1 in human serum, used as an aid in diagnosing antiphospholipid syndrome (APS).

1. Table of Acceptance Criteria and Reported Device Performance

Detailed Study Information:

2. Sample Size and Data Provenance

• Test Set (Clinical Performance):

  • Clinical Validation Set: 1090 samples.
    • 270 samples from APS patients.
    • 820 samples from a "control population" (71 infectious disease samples (40 syphilis, 10 HCV, 21 HBV), 104 Crohn's Disease, 94 Ulcerative Colitis, 127 scleroderma, 168 rheumatoid arthritis, 49 osteoarthritis, 24 other diseases, 183 "without APS" conditions like pre-eclampsia/eclampsia, fetal loss, SLE, thrombosis, atopic dermatitis).
  • Expected Values (Normal Population): 400 apparently healthy blood donors (191 females, 209 males). This was used to validate the cutoff and assess the expected value in a normal population, differing from the reference range establishment set.
  • Provenance: Not explicitly stated, but the mention of "human serum from commercial sources" for calibrators and controls implies a general origin rather than specific countries. The description of diverse disease cohorts suggests clinical sites. It is implied these are retrospective samples collected from various patient populations.

• Test Set (Analytical Performance - Precision): 8 quantitative samples were tested. Each sample was run in duplicate, twice a day, for 20 days (total 80 measurements per sample).

• Test Set (Analytical Performance - LoD/LoB): 264 determinations (144 measurements on blank samples and 120 measurements of low level samples).

• Test Set (Analytical Performance - Linearity): Five serum samples with various β2GP1-Domain1 antibody concentrations were diluted.

• Test Set (Analytical Performance - Interference): Unspecified number of specimens, each spiked at three different concentrations and assessed in triplicates.

• Test Set (Analytical Performance - Cross-reactivity): 111 clinical samples with unknown disease states, most having high levels of various other autoantibodies.

• Test Set (Stability):

  • Accelerated Stability: 1 lot of Reagent Kit, 3 lots of β2GP1-Domain1 beads, 3 lots of Resuspension Buffer, 3 lots of Calibrators, 3 lots of Low and High controls.
  • In-use Stability (Calibrators): Not specified by number, but tested over 8.5 hours.
  • In-use Stability (Controls): Low and high controls assayed for a total of 20 runs over 9 days.
  • In-use Stability (Reagent Cartridge): Four serum specimens (with different reactivity levels) along with Low and High Controls tested periodically for 64 days using three lots of reagent cartridge.

3. Number of Experts (Ground Truth for Test Set)

The document does not specify the number or qualifications of experts used to establish the ground truth for the clinical test set. The ground truth (diagnoses of APS, infectious diseases, autoimmune diseases, etc.) appears to be based on pre-existing clinical diagnoses and classifications from the source of the samples.

4. Adjudication Method (Test Set)

The document does not describe any specific adjudication method (e.g., 2+1, 3+1) for establishing the ground truth of the clinical test set. The clinical diagnoses were presumably accepted as the ground truth.

5. Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study

No MRMC comparative effectiveness study was done. This device is an in vitro diagnostic (IVD) assay designed to semi-quantitatively determine autoantibodies in serum, not an imaging or AI-assisted diagnostic tool that involves human readers interpreting results. Therefore, the concept of "human readers improve with AI vs without AI assistance" does not apply here.

6. Standalone Performance

Yes, a standalone performance study was done. The entire document describes the performance of the assay (algorithm and system) itself, without human interpretation as part of the primary diagnostic output. The clinical sensitivity and specificity, as well as analytical performance characteristics, are all measures of the algorithm's standalone performance.

7. Type of Ground Truth Used

• Clinical Performance (APS diagnosis): The ground truth for APS and other disease categories was based on clinical diagnoses of the patients from whom the samples were collected. For example, "270 samples from APS patients" implies these patients had a clinical diagnosis of APS.
• Analytical Performance:
  • Precision, LoD/LoB, Linearity, AMR, Hook Effect: Ground truth established through reference methods and known concentrations/dilutions of samples.
  • Interference/Cross-reactivity: Ground truth established by spiking known interfering substances or by using samples with known high levels of other autoantibodies/diseases.

8. Sample Size for the Training Set

• Cut-off Establishment (Reference Range): 30 subjects were used to establish the cut-off. This can be considered a form of "training set" for the cut-off. These subjects included apparently healthy blood donors (5), viral hepatitis (4), Systemic Lupus erythematosus (without history of thrombotic events) (10), Syphilis (10), and HIV (1).
• The document also implies other internal validation/development data may have been used to define the Master Curve and instrument parameters, but specific sample sizes and details for such internal "training" are not provided.

9. How the Ground Truth for the Training Set was Established

• Cut-off (Reference Range) Ground Truth:
  • The "training set" for establishing the cut-off consisted of 30 subjects with defined conditions (apparently healthy, specific infectious diseases, SLE without thrombotic events).
  • The ground truth was established by the clinical diagnosis or health status of these subjects. The device was expected to differentiate between "negatives" and "positives" based on these known conditions.
  • The cut-off was derived using "Analyse-it for Excel, to ensure optimal differentiation between negatives and positives," which suggests statistical analysis of the results from these 30 subjects to find the optimal threshold (20 CU in this case).

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