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QUANTA Flash Calprotectin is a chemiluminescent immunoassay for the quantitative determination of fecal calprotectin in extracted human stool samples. Elevated levels of fecal calprotectin, in conjunction with clinical findings and other laboratory tests, can aid in the diagnosis of inflammatory bowel disease (IBD) (ulcerative colitis and Crohn's disease), and in the differentiation of IBD from irritable bowel syndrome (IBS).

QUANTA Flash Calprotectin Calibrators are intended for use with the QUANTA Flash Calprotectin Reagents for the determination of fecal calprotectin levels in extracted stool samples. Each calibrator establishes a point of reference for the working curve that is used to calculate unit values.

QUANTA Flash Calprotectin Controls are intended for use with the QUANTA Flash Calprotectin Reagents for quality control in the determination of fecal calprotectin levels in extracted stool samples.

QUANTA Flash Calprotectin Extraction Buffer is intended for use with the QUANTA Flash Calprotectin Reagents as sample extraction solution.

Device Description

The principle of the assay is chemiluminescent microparticle immunoassay, a variation of solid phase immunoassay. The QUANTA Flash® Calprotectin 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® Calprotectin assay utilizes a reagent cartridge format, which is compatible with the BIO-FLASH® instrument.

Calprotectin-specific capture antibodies are coated on to paramagnetic beads, which are stored in the reagent cartridge under conditions that preserve the antibody in its reactive state. Prior to use in the BIO-FLASH® system, the reagent pack containing all the necessary assay reagents is mixed thoroughly by being inverted several times. The sealed reagent tubes are pierced with the reagent cartridge lid, and the reagent cartridge is loaded onto the instrument. Reagents are calibrated when the lot is first used. A patient extracted stool sample is prediluted by the BIO-FLASH® with sample buffer in a disposable plastic cuvette. Small amounts of the diluted patient extracted stool, the beads, and the 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 monoclonal 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 (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 measured RLU is proportional to the amount of bound isoluminol conjugate, which is in turn proportional to the amount of calprotectin antigen captured by the antibodies (anti-calprotectin polyclonal antibodies in this case) on the beads. For quantitation, the QUANTA Flash® Calprotectin will utilize a predefined lot specific Master Curve that is uploaded onto the instrument through the reagent cartridge barcode. The Master Curve is generated by Inova Diagnostics for each reagent pack lot with in-house Standards with assigned unit values (ng/mL). The RLU and assigned ng/mL values of the Standards are used to create a 4 parameter logistic curve. These four parameters are embedded in the reagent pack barcode. When the lot is used the first time, the Calibrators are run, and based on the results obtained on the Calibrators, an instrument specific Working Curve is created; The Working Curve is used to calculate units (ng/mL) based on RLU values obtained on each sample. The obtained ng/mL values will be converted to mg/kg by a calculation that takes into account the dilution of the samples. This unit conversion is calculated automatically by the software.

AI/ML Overview

Here's an analysis of the provided text, extracting the requested information about acceptance criteria and the study proving device performance:

1. Table of Acceptance Criteria and Reported Device Performance

The device is an in vitro diagnostic (IVD) test system, so performance metrics like sensitivity and specificity are evaluated, alongside analytical performance criteria common for laboratory assays.

Performance Characteristic	Acceptance Criteria	Reported Device Performance
Precision (Within-Laboratory)	Total %CV: < 12%	Ranged from 3.1% to 6.2% for various samples. All met the acceptance criteria.
Reproducibility (Between Sites)	Total %CV: < 15%	Ranged from 3.2% to 10.8% for various samples. All met the acceptance criteria.
Reproducibility (Between Lots)	Total %CV: < 15%	Ranged from 4.7% to 12.9% for various samples. All met the acceptance criteria.
Reproducibility (Extraction)	Total %CV: < 15%	Ranged from 4.5% to 9.8% for various samples. All met the acceptance criteria.
Limit of Quantitation (LoQ)	Total imprecision CV% < 20%	LoQ was determined at 14.1 mg/kg, with CV% of 14.7% for Sample 2 (and 12.6% for Sample 1). Meets criteria.
Analytical Measuring Range (AMR)	Not explicitly stated as acceptance criteria, but defined.	16.1 mg/kg - 3,500.0 mg/kg. The highest reportable value is 35,000.0 mg/kg with auto-rerun.
High Concentration Hook Effect	No hook effect up to a certain high concentration.	RLU values increased with increasing antibody concentrations above the AMR, confirming no hook effect up to 21,753.6 mg/kg.
Linearity	Best fitting polynomial is linear, or difference between best-fitting nonlinear and linear is < 15%.	For stool samples and most recombinant antigen (rAg) samples, the best fit was linear. For one rAg sample, a second-order polynomial was best, but non-linearity ranged from -13.0% to 2.1%, fulfilling the acceptance criteria. Spearman's rs: 0.983 (95% Cl, 0.973 - 0.989) on the method comparison study.
Recovery	Percent Recovery between 88% and 112%.	Ranged from 94.7% to 109.8%. All fulfilled the acceptance criteria.
Interference	85-115% recovery, or ± 15% of low indeterminate range (±7.5 mg/kg) difference, whichever is greater.	No interference detected from various substances (drugs, nutrients, bacterial cultures) at the concentrations tested.
Sample Stability (Extracted)	80-120% average recovery.	All samples fulfilled acceptance criteria up to 72 hours at room temperature, up to 21 days at 2-8°C, and up to 3 months frozen at -20±5 °C, and up to 4 freeze/thaw cycles.
Reagent Shelf Life (Accelerated)	95% Cl of regression line between 80% and 120% recovery at day 14 (equating to 1 year).	All components (beads, tracer, calibrators, controls, extraction buffer, special wash) fulfilled the criteria. One-year expiration dating was assigned.
Calibrators Onboard Stability	All 4 calibrations successful within 8 hours; mean RLU recovery 90-110%; control/patient panel recovery 85-115%.	Four calibrations within 8 hours were valid. Average RLU recovery: 100.0% to 107.1%. Control/patient panel recovery: 89.1% to 105.2%. Supports claim for 4 calibrations over 8 hours.
Controls Onboard Stability	All values within established range; linear regression line of percent recovery between 85% and 115% at run 15.	All controls ran within acceptable ranges for all runs. Regression line remained between 85% and 115% at run 15. Supports claim for 15 uses (10 min/use).
Reagent Cartridge In-Use Stability	Regression line 95% CI reaches 85% or 115% recovery, OR ≥2% of recovery data (<75% or ≥125%).	One lot was stable for 97 days, supporting a 90-day in-use (onboard) stability.
Extraction Buffer In-Use Stability	r≥0.975; intercept ±15% of cut-off; slope 0.9-1.1; weighted S y/x ≤0.5; predicted bias at cut-off ≤15%; 95% Cl of bias does not exceed 20% of cut-off.	All criteria met at 91 days (weighted r=0.999, intercept=3.87 mg/kg, slope=0.9877, weighted S y/x=0.10, predicted bias=2.39 mg/kg, 95% Cl of bias=-2.81-7.59 mg/kg). Stable for 90 days at 2-8°C.
Special Wash Onboard Stability	Regression line 95% CI reaches 85% or 115% recovery, OR ≥2% of recovery data (≤75% or ≥125%).	All criteria met at 91 days. 95% CI of regression line between 97.9% and 105.5%. Supports 30 days uncapped continuous use, or 720 hours distributed over 90 days onboard.
Real Time Stability	Results should fall within their respective ranges (reagent cartridge specimens), 85-115% recovery & %CV<10% (calibrators), within acceptable ranges (controls).	All results to date (up to 6 months at the time of submission) were within the acceptance limits for reagent cartridge, calibrators, and controls.
Clinical Sensitivity (IBD vs Controls)	Not explicitly stated as acceptance criteria, but calculated.	Indeterminate = Negative: 89.5% (78.9 - 95.1%)Indeterminate = Positive: 96.5% (88.1 - 99.0%)
Clinical Specificity (IBD vs Controls)	Not explicitly stated as acceptance criteria, but calculated.	Indeterminate = Negative: 90.9% (83.1 - 95.3%)Indeterminate = Positive: 78.4% (68.7 - 85.7%)
Positive Percent Agreement (PPA)	No explicit acceptance criterion given, but reported from predicate device comparison.	On all samples (N=137), Indeterminate = Negative: 98.1% (90.2 – 99.7%)On all samples (N=137), Indeterminate = Positive: 98.5% (91.9 – 99.7%)Within AMR (N=77), Indeterminate = Negative: 98.1% (89.9 – 99.7%)Within AMR (N=77), Indeterminate = Positive: 98.4% (91.7 – 99.7%)
Negative Percent Agreement (NPA)	No explicit acceptance criterion given, but reported from predicate device comparison.	On all samples (N=137), Indeterminate = Negative: 97.6% (91.6 – 99.3%)On all samples (N=137), Indeterminate = Positive: 94.4% (86.4 - 97.8%)Within AMR (N=77), Indeterminate = Negative: 92.0% (75.0 – 97.8%)Within AMR (N=77), Indeterminate = Positive: 69.2% (42.4 – 87.3%)
Total Percent Agreement (TPA)	No explicit acceptance criterion given, but reported from predicate device comparison.	On all samples (N=137), Indeterminate = Negative: 97.8% (93.8 – 99.3%)On all samples (N=137), Indeterminate = Positive: 96.4% (91.4 - 98.4%)Within AMR (N=77), Indeterminate = Negative: 96.1% (89.2 – 98.7%)Within AMR (N=77), Indeterminate = Positive: 93.5% (85.7 -97.2%)
Spearman's correlation with predicate	No explicit acceptance criterion given, but reported.	0.983 (95% Cl, 0.973 - 0.989) for 77 samples within AMR.
Linear regression with predicate	No explicit acceptance criterion given, but reported.	Slope: 1.10 (1.01 - 1.18), Intercept: 0.52 (-9.47 - 14.0), Correlation Coefficient: 0.956 for 77 samples within AMR.

2. Sample Size Used for the Test Set and Data Provenance

Clinical Performance Test Set:
- Sample Size: A total of 165 characterized samples were included in the validation set.
- Data Provenance: Samples came from studies performed at two different sites (Site A and Site B) and a commercial source.
  - Site A: Samples 1 to 107. Inclusion criteria for IBD patients included suspicion of IBD, a calprotectin test request, and underwent ileocolonoscopy. Patients diagnosed with IBD or without ileocolonoscopy were excluded.
  - Site B: Samples 108 to 175. Sixty-eight consecutive samples requested for a calprotectin determination test by a gastroenterologist. Patients with excessive mucous, unclear diagnosis, or without colonoscopy were excluded.
- Retrospective/Prospective: The description implies a retrospective collection of "characterized samples" from previously conducted studies or existing cohorts. For Site A, samples were "recruited over a period of 6 months," suggesting prospective collection during that period, but then used retrospectively for this validation. For Site B, "consecutive samples requested" also suggests a retrospective collection from a past clinical workflow.
Method Comparison Test Set:
- Sample Size: 137 samples out of the 165 clinical validation study samples. Of these, 77 samples fell within the AMR of both assays.
- Data Provenance: Same as the clinical performance test set (two sites and commercial source).
Precision and Reproducibility Studies: Sample numbers are specified per study (e.g., 8 samples for within-laboratory precision, 8 samples for between-sites, 8 samples for between-lots, 5 samples for extraction reproducibility). These are generally manufactured or spiked samples, not from patients.
LoD/LoQ: 2 low-level samples for LoQ, 60 blank samples and 60 low-level samples (per lot) for LoD.
Linearity: Three extracted stool samples and three recombinant antigen samples.
Recovery: Seven extracted stool samples.
Interference: Six human stool specimens (one high positive, one moderately positive, one low positive, one near cut-off, one indeterminate, one negative).
Sample Stability: Eight extracted human stool samples (negative, indeterminate, around cut-off, positive) and three extracted stool samples (indeterminate, around cut-off, high positive) for frozen stability.
Reference Range Establishment: 61 subjects (presumably healthy donors and some with specific benign conditions).
Expected Values (Normal Population): 164 apparently healthy stool donors.

3. Number of Experts Used to Establish the Ground Truth for the Test Set and Qualifications of Those Experts

Clinical Performance Ground Truth:
- For IBD diagnosis at Site A, "Senior gastroenterologists performed all endoscopies and findings were documented in a computer-based database. The final diagnosis of IBD (i.e. CD and UC) was independently made by a pathologist or gastroenterologist who was blinded for calprotectin results." This indicates multiple Senior Gastroenterologists and Pathologists/Gastroenterologists were involved. Specific years of experience are not mentioned, but "Senior" implies significant experience.
- For IBD diagnosis at Site B, "Patients were diagnosed after exclusion of organic pathology on the basis of routine blood tests, thyroid function tests, serological screening for coeliac disease, stool examination for bacteria and parasites, ultrasound examination, and eventually colonoscopy using the ROME III criteria. The diagnosis of IBD was made upon clinical, endoscopic and histological findings as described in J Crohn Colitis 2012:6: 965-990 (ulcerative colitis) and J Crohn Colitis 2010:4:7-27 (Crohn's disease)." This implies multiple Gastroenterologists and Pathologists following established diagnostic guidelines.
Reference Range Establishment: For the 61 subjects used to establish the cut-off, the description indicates they were "apparently healthy donors" or had specific benign conditions (e.g., Squamous Cell Carcinoma, Glandular Polyp, Hyperplastic Polyp, Adenoma). The diagnosis here would be based on standard clinical and pathological evaluations, though specific experts for this ground truth are not detailed.

4. Adjudication Method for the Test Set

For the IBD diagnosis at Site A, the final diagnosis was "independently made by a pathologist or gastroenterologist who was blinded for calprotectin results." This suggests a consensus-based approach or review by independent experts, but a specific (e.g., 2+1, 3+1) method is not explicitly stated. The term "independently made" implies that the diagnosis of IBD (considered the ground truth) was confirmed by one or more experts who were shielded from the calprotectin results.

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

No, an MRMC comparative effectiveness study involving human readers with and without AI assistance was not done. This device is an automated in vitro diagnostic test (chemiluminescent immunoassay) for measuring fecal calprotectin, not an AI-powered image analysis or decision support system that directly assists human readers in real-time interpretation. The studies focus on the analytical and clinical performance of the assay itself compared to a predicate device or clinical diagnosis.

6. Standalone (Algorithm Only Without Human-in-the-Loop) Performance

Yes, performance metrics like precision, reproducibility, LoD, LoQ, linearity, recovery, interference, and stability are all "standalone" performance measures of the device itself (its reagents and instrument) without human-in-the-loop directly influencing the measurement result.
The "Clinical Performance Characteristics" (sensitivity, specificity) are also standalone performance data for the device's ability to differentiate IBD from controls, given a clinical ground truth.
The "Comparison with predicate device" also serves as a standalone comparison of the new device against an existing, legally marketed device.

7. Type of Ground Truth Used

Clinical Performance / Clinical Validation: The ground truth for IBD diagnosis was established through comprehensive medical work-up, including "clinical findings and other laboratory tests," "endoscopic and histologic analysis, radiologic work-up and laboratory tests including ileocolonoscopy" (Site A), and "exclusion of organic pathology on the basis of routine blood tests, thyroid function tests, serological screening for coeliac disease, stool examination for bacteria and parasites, ultrasound examination, and eventually colonoscopy using the ROME III criteria" (Site B). This is robust clinical diagnosis/outcomes data, supported by pathology and endoscopy.
Analytical Performance (e.g., LoD, LoQ, Linearity, Recovery, Interference, Stability): The ground truth for these studies involves pre-defined concentrations of calprotectin (e.g., recombinant calprotectin antigen, spiked samples) or known conditions (e.g., specific concentrations of interferents, different storage temperatures/durations). This is essentially known concentrations/states as ground truth.
Reference Range / Cut-off: The cut-off was established based on a reference population (61 subjects) and further informed by 31 diagnosed IBD patients. The definition of "negative," "indeterminate," and "positive" ranges are based on statistical analysis of these populations and clinical considerations.

8. Sample Size for the Training Set

The document does not explicitly describe a "training set" in the context of an AI/machine learning model. The device is a chemiluminescent immunoassay run on an automated instrument.

However, the "Master Curve" for the assay is established during manufacturing using "in-house Standards with assigned unit values". This master curve can be considered analogous to a foundational calibration or "training" for the assay's quantitative functionality.

Master Curve Standards: Seven calprotectin standards (0.0 ng/mL to 3478.3 ng/mL) are used to create the lot-specific Master Curve. The number of samples for generating this master curve is not explicitly stated beyond "7 Standards", but it is a manufacturing process.
Calibrators and Controls Value Assignment: Calibrators and controls are assigned values by testing on at least two instruments, on at least two lots of reagent cartridge, in replicates of 5 to obtain a minimum of 10 data points. This process of value assignment, while not for an AI training set, is how the reference values are established for the functional units of the assay.

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

As noted above, there isn't a traditional "training set" for an AI model. For the functional calibration of the assay:

Master Curve Ground Truth: The "Master Curve Standards" are "in-house Standards with assigned unit values (ng/mL)." This effectively means the ground truth for the Master Curve is based on traceable, pre-assigned concentrations of calprotectin.
Calibrators and Controls Ground Truth: The "Calibrator and Control values are directly traceable to the in-house Standards that are used to create the Master Curves for the QUANTA Flash Calprotectin assay." This means their ground truth is rooted in the same pre-assigned values of the in-house standards.

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