The BÜHLMANN fCAL turbo is an in vitro diagnostic assay intended for the quantitative measurement of fecal calprotectin, a neutrophilic protein that is a marker of intestinal mucosal inflammation, in human stool. The BÜHLMANN fCAL turbo aids in the diagnosis of inflammatory bowel disease (IBD), specifically Crohn's disease (CD) and ulcerative colitis (UC) and aids in the differentiation of IBD from irritable bowel syndrome (IBS) in conjunction with other laboratory and clinical findings.

Device Description

The BÜHLMANN fCAL® turbo, a particle-enhanced turbidimetric immunoassay (PETIA), is performed using patient stool extracts collected without preservatives. Calprotectin within the sample extract mediates immunoparticle agglutination; sample turbidity is proportional to calprotectin concentration. The detected light absorbance allows quantification of calprotectin concentration via interpolation of an established calibration curve. The assay is validated for use on clinical chemistry analyzers such as the Roche cobas® c501/c502 platforms.

The BÜHLMANN fCAL® turbo Reagent Kit is to be used in conjunction with the BÜHLMANN fCAL® turbo Calibrator Kit and BÜHLMANN fCAL® turbo Control Kit, which are available separately.

AI/ML Overview

The provided document describes the 510(k) premarket notification for the BÜHLMANN fCAL® turbo device, an in vitro diagnostic assay. It details the device's indications for use, its technological characteristics compared to a predicate device, and the performance data collected to demonstrate its safety and effectiveness.

Here's an analysis of the acceptance criteria and the study proving the device meets them, based on the provided text:

Acceptance Criteria and Device Performance

The core of the acceptance criteria for the BÜHLMANN fCAL® turbo, as a new device seeking substantial equivalence to a legally marketed predicate (BÜHLMANN fCAL® ELISA), is demonstrated through various performance studies. The primary goal is to show that, despite technological differences, the new device performs comparably to the predicate and is safe and effective for its stated indications.

The document implicitly defines acceptance criteria through the successful outcomes of its performance studies, demonstrating analytical parity and comparable clinical utility. Explicit numerical thresholds for acceptance are not always stated outright as "acceptance criteria" but are demonstrated by the presented results falling within acceptable ranges or showing strong correlation/agreement with the predicate.

Here's a table summarizing the key performance metrics presented, which serve as the de facto acceptance criteria and the device's reported performance:

Criterion (Implicit Acceptance Threshold)	Reported Device Performance and Study Type
Precision (Low %CV indicating reproducibility and repeatability)	Single-Site Repeatability Study: - 8 samples tested (S01-S08). - Mean values ranged from 42.9 µg/g to 5405.6 µg/g. - Within-run %CV ranged from 0.7% to 8.3%. - Within-laboratory %CV ranged from 1.4% to 9.1%. Multi-Site Reproducibility Study: - 8 samples tested (S01-S08). - Mean values ranged from 47.2 µg/g to 5475.6 µg/g. - Total Precision %CV ranged from 3.2% to 11.3%. Lot-to-Lot Precision Study: - 8 samples tested (S1-S8). - Mean values ranged from 45.2 µg/g to 5303.1 µg/g. - Total Precision %CV ranged from 3.6% to 11.3%. Extraction Reproducibility Study: - 10 samples tested (S1-S10). - Mean values ranged from 47.7 µg/g to 3330.4 µg/g. - Total Precision %CV for extraction ranged from 1.4% to 13.0%.
Linearity/Analytical Measuring Range (R² close to 1, small intercept, slope close to 1)	Linearity Study: - Two dilution series analyzed. - Dilution Series 1: Covered 37.6 – 12,216.0 µg/g. Regression: Intercept=5.7 (95% C.I. 1.6, 16.9), Slope=1.057 (95% C.I. 1.044, 1.075), R²=0.9983. - Dilution Series 2: Covered 33.5 – 13,339.5 µg/g. Regression: Intercept=3.8 (95% C.I. -0.4, 13.3), Slope=1.031 (95% C.I. 1.014, 1.042), R²=0.9984. Claimed Analytical Measuring Range: Direct: 30 - 2000 µg/g. With automatic dilution: 30 - 10,000 µg/g. (Supported by data)
High Dose Hook Effect (No significant effect at high concentrations)	High Dose Hook Effect Study: - No high dose hook effect observed up to 45,715 µg/g.
Accuracy/Recovery (Total Recovery % close to 100%)	Accuracy/Recovery Study: - 7 spiked samples (varied baselines). - Total recovery ranged from 93.6% to 102.0%.
Analytical Sensitivity (Low LoB, LoD, LoQ)	Analytical Sensitivity Studies: - LoB (Limit of Blank) = 16.7 µg/g. - LoD (Limit of Detection) = 23.7 µg/g. - LoQ (Limit of Quantitation) = 30 µg/g. Supports claimed direct measuring range of 30 - 2000 µg/g, and 30 - 10,000 µg/g with automatic dilution.
Interfering Substances (No significant interference)	Interfering Substances Study: - Tested various analytes (e.g., Iron, Prednisone, Mesalamine, Vancomycin, antibiotics, vitamins, hemoglobin) and common enteropathological microorganisms (e.g., E. coli, Salmonella, Klebsiella, Shigella, Yersinia) at specified concentrations. - Confirmed no interference.
Method Comparison with Predicate (Strong correlation, low bias, high PPA/NPA)	Method Comparison Study: - Statistical Comparison (Passing-Bablok): - Slope = 1.025 (95% CI: 0.990, 1.058) (close to 1). - Intercept = -4.5 µg/g (95% CI: -8.7, 0.3) (close to 0). - Bias at 80 µg/g = -3.1% (95% CI: -7.2%, 0.5%). - Bias at 160 µg/g = -0.3% (95% CI: -2.4%, 2.7%). - Correlation (r) = 0.972 (strong positive correlation). - Clinical Agreement (PPA/NPA): - All subjects combined: PPA (lower cutoff) = 93.6% (88.5%, 96.9%), NPA (lower cutoff) = 91.3% (83.6%, 96.2%). - PPA (upper cutoff) = 93.9% (87.1%, 97.7%), NPA (upper cutoff) = 95.3% (90.6%, 98.1%). (High PPA/NPA values indicate good agreement with the predicate across various patient subgroups and diagnostic cutoffs).
Clinical Sensitivity/Specificity (Clinically meaningful diagnostic performance for IBD vs. IBS/non-IBD)	Clinical Sensitivity/Specificity Study: - IBD vs. IBS: - Borderline considered Positive: Sensitivity = 91.1%, Specificity = 76.2%. - Borderline considered Negative: Sensitivity = 80.0%, Specificity = 87.7%. - IBD vs. Non-IBD: - Borderline considered Positive: Sensitivity = 91.1%, Specificity = 74.3%. - Borderline considered Negative: Sensitivity = 80.0%, Specificity = 85.1%. (Demonstrates ability to differentiate IBD from IBS and non-IBD conditions).
Expected Values/Reference Range (Distribution in healthy population)	Expected Values/Reference Range Study: - Tested 141 healthy normal adults. - Distribution: 75.2% < 80 µg/g, 12.8% 80-160 µg/g, 12.1% > 160 µg/g. (Provides population data for interpretation).

Study Details Proving Device Meets Acceptance Criteria

Sample Size and Data Provenance:
- Precision Studies (Single-site, Multi-site, Lot-to-Lot): For each sample (typically 8 different concentration levels), n=80 (Single-site) or n=75 (Multi-site, Lot-to-Lot) measurements were performed. This refers to the number of replicates / runs, not distinct clinical samples. These are analytical performance studies using spiked samples or controls.
- Extraction Reproducibility Study: n=80 for each of the 10 samples (again, replicates/runs).
- Linearity Study: Each dilution was tested in 4 replicates. Number of unique stool specimens used for creating dilution series is not explicitly stated but implied to be at least two (one high, one low).
- Accuracy/Recovery Study: 7 clinical samples were spiked and tested.
- Interfering Substances Study: Stool extracts with 4 different calprotectin concentrations were used for analytes/pharmaceuticals/supplements. Microorganism interference was tested at given cell counts, presumably across various stool extracts. The exact number of distinct stool samples used for interference testing is not specified.
- Method Comparison Study: A total of 248 clinical study samples were tested. 220 had valid results within the linear measuring range for both devices.
- Clinical Sensitivity/Specificity Study: 337 clinical samples were included for IBD vs. non-IBD analysis, and 265 clinical samples for IBD vs. IBS.
- Expected Values/Reference Range Study: 141 apparently healthy normal adults provided stool samples.
- Data Provenance: Not explicitly stated but typically for an FDA 510(k) submission, these studies would be conducted in the country of origin of the manufacturer (Switzerland, in this case) or in clinical sites compliant with GCLP/GCP, often with diverse patient populations to support generalizability (though not explicitly stated as multi-national for every study). The studies are inherently prospective in nature, as they involve newly generated data from specified experimental designs for device validation.
Number of Experts and Qualifications for Ground Truth of Test Set:
- This section is not applicable for this type of in-vitro diagnostic device (IVD). The device measures a biomarker (fecal calprotectin). The "ground truth" for the test set is either:
  - Analytical True Value: For analytical studies (precision, linearity, accuracy, analytical sensitivity, interference), the ground truth is established by the known concentrations of spiked samples, reference materials, or highly accurate reference methods.
  - Clinical Diagnosis: For the method comparison and clinical sensitivity/specificity studies, the ground truth for "IBD," "IBS," and "non-IBD" is based on the clinical diagnosis of the patient from whom the stool sample was collected. This diagnosis is established by healthcare professionals (e.g., gastroenterologists) using a combination of clinical findings, endoscopy, histology, and other laboratory tests, not by a panel of independent "experts" adjudicating images or cases for the purpose of a study (which is common in AI/Imaging studies).
Adjudication Method for the Test Set:
- Not applicable in the context of expert adjudication for diagnostic calls, as this is an IVD measuring a biomarker.
- For the clinical ground truth, it's based on the established clinical diagnosis by treating physicians. There is no mention of a separate adjudication process (e.g., 2+1 or 3+1 reader consensus) for these clinical diagnoses for the purpose of this device study.
Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study:
- No, an MRMC study was not done. This type of study is specifically relevant for image-based diagnostic aids where the output of the AI directly influences a human reader's interpretive performance. The BÜHLMANN fCAL® turbo is a laboratory-based immunoassay that quantifies a biomarker. Its output is a numerical concentration, not an image interpretation requiring multiple readers. The "comparative effectiveness" is demonstrated against the predicate device's performance, not how it assists human readers in interpreting complex visual information.
Standalone (Algorithm Only) Performance:
- Yes, this entire submission effectively describes a standalone performance. The BÜHLMANN fCAL® turbo is an automated analyzer/reagent system that directly measures fecal calprotectin concentration from a stool sample extract. The reported results (e.g., precision, linearity, accuracy, clinical sensitivity/specificity) are the direct output of the device itself, without human-in-the-loop interpretation of its primary numerical output. The device "aids in the diagnosis" in conjunction with other findings, meaning the numerical output is used by clinicians, but the device itself functions as a standalone measurement tool.
Type of Ground Truth Used:
- Analytical Ground Truth: For the analytical performance studies (Precision, Linearity, Accuracy, Analytical Sensitivity, Interfering Substances), the ground truth is established through known concentrations of reference materials, spiked samples, or comparison to established reference methods.
- Clinical Ground Truth: For the Method Comparison and Clinical Sensitivity/Specificity studies, the ground truth for patient classification (IBD, IBS, non-IBD, normal) is based on clinical diagnosis, which would typically include outcomes data, endoscopic findings, histological results, and the overall clinical picture as determined by a treating physician. It is not expert consensus of independent reviewers explicitly for the study, nor is it pathology in the sense of a single definitive pathology report for every case, but rather a comprehensive clinical workup.
Sample Size for the Training Set:
- Not applicable / Not explicitly stated as a separate "training set" in the context of traditional machine learning. This device is an in vitro diagnostic (IVD) immunoassay, not an AI/ML algorithm that is "trained" on a large dataset of patient samples in the same way a deep learning model for image analysis is. The "training" here refers to the internal development and calibration of the assay (e.g., establishing the standard curve, optimizing reagent concentrations), which is part of the product development process, not a distinct "training set" like in AI/ML validation studies. The analytical performance and clinical validation studies presented here are akin to a "test set" and "validation set" in AI/ML terminology, but the concept of a separate "training set" for the device itself (rather than the general scientific knowledge used in developing the immunoassay) is not present.
How Ground Truth for Training Set Was Established:
- As noted above, the concept of a "training set" for this type of IVD device is different from AI/ML models. The ground truth for developing and calibrating the assay (which could be loosely considered "training") involves:
  - Reference materials with known concentrations of calprotectin.
  - Standard curves generated using defined calibrators.
  - Analytical methods and clinical literature informing the selection of antibodies, detection methods, and clinical cut-offs.
  - Internal validation and optimization processes during product development.

In summary, the document thoroughly details the analytical and clinical performance of the BÜHLMANN fCAL® turbo, demonstrating substantial equivalence to its predicate by meeting rigorous criteria in terms of precision, linearity, accuracy, analytical sensitivity, and demonstrating comparable clinical utility in differentiating IBD from other gastrointestinal conditions. The nature of the device (an immunoassay) means certain aspects typically found in AI/ML performance studies (like MRMC or reader adjudication) are not applicable.

Summary

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Image /page/0/Picture/0 description: The image contains the logo of the U.S. Food and Drug Administration (FDA). On the left is the Department of Health & Human Services logo. To the right of that is the FDA logo, which is a blue square with the letters "FDA" in white. To the right of the blue square is the text "U.S. FOOD & DRUG ADMINISTRATION" in blue.

June 25, 2019

Roshana Ahmed President Quaras. LLC 2101 Camino Rey Fullerton, California 92833

Re: K190784

Trade/Device Name: BUHLMANN fCAL turbo Regulation Number: 21 CFR 866.5180 Regulation Name: Fecal calprotectin immunological test system Regulatory Class: Class II Product Code: NXO Dated: March 25, 2019 Received: March 27, 2019

Dear Roshana Ahmed:

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. Although this letter refers to your product as a device, please be aware that some cleared products may instead be combination products. The 510(k) Premarket Notification Database located at https://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfpmn/pmn.cfm identifies combination product submissions. 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 Part

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801 and Part 809); medical device reporting of medical device-related adverse events) (21 CFR 803) for devices or postmarketing safety reporting (21 CFR 4, Subpart B) for combination products (see https://www.fda.gov/combination-products/guidance-regulatory-information/postmarketing-safety-reportingcombination-products); good manufacturing practice requirements as set forth in the quality systems (QS) regulation (21 CFR Part 820) for devices or current good manufacturing practices (21 CFR 4, Subpart A) for combination products; and, if applicable, the electronic product radiation control provisions (Sections 531-542 of the Act); 21 CFR 1000-1050.

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 https://www.fda.gov/medical-device-safety/medical-device-reportingmdr-how-report-medical-device-problems.

For comprehensive regulatory information about medical devices and radiation-emitting products, including information about labeling regulations, please see Device Advice (https://www.fda.gov/medicaldevices/device-advice-comprehensive-regulatory-assistance) and CDRH Learn (https://www.fda.gov/training-and-continuing-education/cdrh-learn). Additionally, you may contact the Division of Industry and Consumer Education (DICE) to ask a question about a specific regulatory topic. See the DICE website (https://www.fda.gov/medical-device-advice-comprehensive-regulatoryassistance/contact-us-division-industry-and-consumer-education-dice) for more information or contact DICE by email (DICE@fda.hhs.gov) or phone (1-800-638-2041 or 301-796-7100).

Sincerely,

Doug Jeffery, Ph.D. Acting Deputy Division Director Division of Immunology and Hematology Devices OHT7: Office of In Vitro Diagnostics and Radiological Health Office of Product Evaluation and Quality Center for Devices and Radiological Health

Enclosure

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Indications for Use

510(k) Number (if known) K190784

Device Name BÜHLMANN fCAL turbo

Indications for Use (Describe)

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

I. Submitter

BÜHLMANN Laboratories AG Baselstrasse 55 Schönenbuch CH-4124 Switzerland Phone: +41 61 487 12 50

Contact Person: Laura Zurbrügg Date Prepared: June 20, 2019

II. Device

Device Proprietary Name:	BUHLMANN fCAL® turbo
Common or Usual Name:	Fecal calprotectin immunological test system
Classification Name:	Calprotectin, Fecal
Regulation Number:	21 CFR 866.5180
Product Code:	NXO
Device Classification:	II

III. Predicate Device

Substantial equivalence is claimed to the following device:

BÜHLMANN fCAL® ELISA, K181012, BÜHLMANN Laboratories AG

IV. Device Description

The BÜHLMANN fCAL® turbo Reagent Kit is to be used in conjunction with the BÜHLMANN fCAL® turbo Calibrator Kit and BÜHLMANN fCAL® turbo Control Kit, which are available separately.

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V. Indications for Use

The BÜHLMANN fCAL® turbo is an in vitro diagnostic assay intended for the quantitative measurement of fecal calprotectin, a neutrophilic protein that is a marker of intestinal mucosal inflammation, in human stool. The BÜHLMANN fCAL® turbo aids in the diagnosis of inflammatory bowel disease (IBD), specifically Crohn's disease (CD) and ulcerative colitis (UC) and aids in the differentiation of IBD from irritable bowel syndrome (IBS) in conjunction with other laboratory and clinical findings.

VI. Comparison of Technological Characteristics

The BÜHLMANN fCAL® turbo and the predicate device share the following characteristics:

measurement of human fecal calprotectin in human stool;
use of a quantitative platform;
use of manual weighing extraction method for stool samples; and ●
clinical decision thresholds. ●

The BÜHLMANN fCAL® turbo is technologically different from the predicate device as follows:

assay method;
use of an alternate, automated, detection method;
use of polyclonal antibodies; and ●
broader measuring range. ●

The tables below compare key technological features between the subject and predicate device.

Technological comparison

Comparison of Assay

Similarities
	BÜHLMANN fCAL® turbo	BÜHLMANN fCAL® ELISA(K181012)
Analyte	Human fecal calprotectin(MRP8/14)	Human fecal calprotectin(MRP8/14)
Assay format	Quantitative	Quantitative
Specimen type	Human stool	Human stool
ExtractionMethod	Manual Weighing (1:50 dilution inExtraction Buffer)	Manual Weighing (1:50 dilution inExtraction Buffer)
Clinical DecisionThresholds	Normal: < 80 µg/gGray-zone/borderline: 80 - 160 µg/gElevated: > 160 µg/g	Normal: < 80 µg/gGray-zone/borderline: 80 - 160 µg/gElevated: > 160 µg/g

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Differences
	BÜHLMANN fCAL® turbo	BÜHLMANN fCAL® ELISA(K181012)
Method	PETIA	ELISA
Automation	Automated	Not automated
Solid phase	Polystyrene nanoparticles (beads)	96-well polystyrene microtiter plate
Detection method	Automated clinical chemistryanalyzer read at 546 nm	Microtiter plate reader read at450 nm
Analyte-specificantibodycomponents	Polyclonal antibodies againsthuman calprotectin coated onpolystyrene beads	Capture antibodies: monoclonalantibodies against humancalprotectin coated on microtiterplates
		Detection antibodies: monoclonalantibodies against humancalprotectin conjugated to HRP
Measuring range	Direct measuring range:30 – 2000 µg/gMeasuring range with automaticdilution: 30 – 10,000 µg/g	30 – 1800 µg/g

Comparison of Calibrators

	BÜHLMANN fCAL® turbo	BÜHLMANN fCAL® ELISA
Indications forUse	The BÜHLMANN fCAL® turboCalibrator Kit is intended for usewith the BÜHLMANN fCAL®turbo Reagent Kit for thedetermination of fecal calprotectinlevels in extracted stool samples.Comprised of six (6) calibrators,each calibrator establishes a pointof reference for the working curvethat is used to calculate test resultsfrom patient samples.	N/A
Method	BÜHLMANN fCAL® turbo	BÜHLMANN fCAL® ELISA
Analyte	Native human calprotectinSource: human granulocyte extract	Native human calprotectinSource: human serum
Calibrators	6 levels:Target values: 0, 50, 200, 500,1000, 2000 µg/g	5 levels:4, 12, 40, 120, and 240 ng/mL
Conversion factor	N/A	7.5
Value assignment:	Calibrator values assigned using avalue transfer protocol for eachcalibrator lot. Values are indicatedin the QC datasheet.	Nominal values:30 µg/g, 90 µg/g, 300 µg/g, 900µg/g, 1800 µg/g

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	BÜHLMANN fCAL® turbo	BÜHLMANN fCAL® ELISA
Configuration	Available as a separateBÜHLMANN fCAL® turboCalibrator Kit.	Included within the BÜHLMANNfCAL® ELISA kit.

Comparison of Controls

	BÜHLMANN fCAL® turbo	BÜHLMANN fCAL® ELISA
Indications forUse	The BÜHLMANN fCAL® turboControl Kit, comprised of a highand low control, is intended for usewith the BÜHLMANN fCAL®turbo Reagent Kit, for qualitycontrol, in the determination offecal calprotectin levels in extractedstool samples.	N/A
Method	BÜHLMANN fCAL® turbo	BÜHLMANN fCAL® ELISA
Analyte	Native human calprotectinSource: human granulocyte extract	Native human calprotectinSource: human serum
Levels	2 (low and high)	2 (low and high)
Physio-chemicalcharacteristics	Ready to use	Ready to use
Configuration	Available as a separateBÜHLMANN fCAL® turbo ControlKit	Included within the BÜHLMANNfCAL® ELISA kit

Discussion

As seen above, differences between the subject and predicate device include the assay and detection methods, antibodies, and reportable measuring range. These technological differences do not create new questions of safety and effectiveness and the differences are addressed by the performance studies identified below.

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VII. Performance Data

A. Clinical Thresholds

Calprotectin Concentration	Interpretation	Follow-Up
< 80 µg/g	Normal	None
80 µg/g ≤ x ≤ 160 µg/g	Gray-zone/Borderline	Retest within 4 – 6 weeks
> 160 µg/g	Elevated	Retest as appropriate

B. Precision

Single-Site Repeatability Study Results:

ID	Mean[µg/g]	n	Within-run(Repeatability)		Between-run		Between-day		Within-laboratory
			SD	%CV	SD	%CV	SD	%CV	SD	%CV
S01	42.9	80	3.6	8.3%	1.2	2.7%	1.1	2.5%	3.9	9.1%
S02	98.4	80	2.5	2.6%	1.8	1.8%	2.2	2.2%	3.7	3.8%
S03	166.5	80	4.3	2.6%	0.8	0.5%	1.9	1.2%	4.8	2.9%
S04	267.6	80	3.9	1.4%	2.5	0.9%	1.8	0.7%	5.0	1.9%
S05	642.0	80	20.1	3.1%	14.9	2.3%	0.0	0.0%	25.1	3.9%
S06	1414.2	80	19.6	1.4%	11.1	0.8%	3.5	0.2%	22.8	1.6%
S07	3251.4	80	40.8	1.3%	21.4	0.7%	19.7	0.6%	50.1	1.5%
S08	5405.6	80	40.2	0.7%	56.6	1.0%	34.5	0.6%	77.5	1.4%

Multi-Site Reproducibility Study Results:

ID	Mean[µg/g]	n	Within-run(Repeatability)		Between-day		Between-site		TotalPrecision
			SD	%CV	SD	%CV	SD	%CV	SD	%CV
S01	47.2	75	3.6	7.6	2.4	5.0	0.0	0.0	4.3	9.1
S02	91.1	75	3.5	3.8	3.5	3.8	2.8	3.1	5.7	6.2
S03	185.4	75	5.1	2.7	2.7	1.4	5.5	3.0	7.9	4.3
S04	276.9	75	6.4	2.3	4.5	1.6	9.7	3.5	12.5	4.5
S05	674.5	75	12.9	1.9	1.2	0.2	22.8	3.4	26.3	3.9
S06	1519.6	75	25.3	1.7	17.8	1.2	62.3	4.1	69.6	4.6
S07	3343.8	75	54.6	1.6	35.6	1.1	100.0	3.0	119.4	3.6
S08	5475.6	75	72.1	1.3	35.8	0.7	154.2	2.8	173.9	3.2

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ID	Mean [µg/g]	n	Within-Run (Repeatability)		Between-Day		Between-Lot		Total Precision
			SD	%CV	SD	%CV	SD	%CV	SD	%CV
S1	45.2	75	3.22	7.1%	1.36	3.0%	3.70	8.2%	5.09	11.3%
S2	86.4	75	3.69	4.3%	1.19	1.4%	5.66	6.6%	6.86	7.9%
S3	175.8	75	5.04	2.9%	0.29	0.2%	9.90	5.6%	11.11	6.3%
S4	263.9	75	7.55	2.9%	0.00	0.0%	9.98	3.8%	12.52	4.7%
S5	647.4	75	15.47	2.4%	0.00	0.0%	15.28	2.4%	21.74	3.4%
S6	1460.7	75	33.66	2.3%	11.64	0.8%	41.01	2.8%	54.32	3.7%
S7	3234.5	75	71.23	2.2%	8.90	0.3%	130.29	4.0%	148.76	4.6%
S8	5303.1	75	97.42	1.8%	11.18	0.2%	163.87	3.1%	190.97	3.6%

Lot-to-Lot Precision Study Results:

Extraction Reproducibility Study Results:

Sample	n	Mean(µg/g)	SD(µg/g)	%CV	Within-Run(Repeatability)		Between-extraction		Between-day		Between-operator		TotalPrecision
					SD(µg/g)	%CV	SD(µg/g)	%CV	SD(µg/g)	%CV	SD(µg/g)	%CV	SD(µg/g)	%CV
S1	80	47.7	2.8	5.9	1.1	2.4	0.7	1.5	1.4	2.9	3.4	7.2
S2	80	72.3	3.8	5.2	3.9	5.4	4.2	5.8	0.0	0.0	6.8	9.5
S3	80	96.1	3.8	3.9	2.2	2.3	1.4	1.4	0.0	0.0	4.6	4.8
S4	80	170.6	4.0	2.4	2.5	1.5	8.7	5.1	0.0	0.0	9.9	5.8
S5	80	277.0	3.7	1.4	27.9	10.1	10.0	3.6	11.0	4.0	31.8	11.5
S6	80	421.1	9.8	2.3	5.9	1.4	15.3	3.6	0.0	0.0	19.1	4.5
S7	80	573.9	5.4	0.9	39.5	6.9	0.0	0.0	0.0	0.0	39.9	6.9
S8	80	1387.4	39.1	2.8	75.1	5.4	159.9	11.5	0.0	0.0	180.9	13.0
S9	80	3264.9	87.2	2.7	236.2	7.2	256.9	7.9	0.0	0.0	359.7	11.0
S10	80	3330.4	89.8	2.7	92.4	2.8	75.7	2.3	0.0	0.0	149.4	4.5

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C. Linearity

Study procedures were performed using two dilution series. For each dilution series, a stool specimen extract with concentration above the anticipated upper limit of the analytical measuring range was combined with a stool specimen extract with concentration below the anticipated lower limit of the analytical measuring range, in various mixing ratios covering the range; each dilution was tested in 4 replicates. Results of the linear regression analyses are presented in the table below.

Best	MeasuringRange [µg/g]	Linear regression parameters
		Intercept(95% C.I.)	Slope(95% C.I.)	R2
1	37.6 – 12,216.0	5.7(1.6, 16.9)	1.057(1.044, 1.075)	0.9983
2	33.5 – 13,339.5	3.8(-0.4, 13.3)	1.031(1.014, 1.042)	0.9984

The data supports the following claims for analytical measuring range:

Direct analytical measuring range: 30 - 2000 ug/g Measuring range with automatic dilution: 30 - 10,000 ug/g

D. High Dose Hook Effect

No high dose hook effect at theoretical concentrations up to 45,715 ug/g.

E. Accuracy/Recovery

Sample No	7226	7228	7238	7236	7244	7234	7246
Baseline result [µg/g]	44.10	65.45	116.43	138.48	230.88	510.78	1076.33
Expected post-spike result [µg/g]	101.04	122.39	173.37	195.42	458.65	738.55	1304.10
Observed post-spike result [µg/g]	94.55	114.53	170.23	186.93	453.10	753.18	1309.28
Total recovery [%]	93.6%	93.6%	98.2%	95.7%	98.8%	102.0%	100.4%

F. Analytical Sensitivity

Results of the analytical sensitivity studies support a claimed direct measuring range of 30 -2000 ug/g and a measuring range of 30 - 10,000 µg/g with automatic dilution.

LoB = 16.7 µg/g LoD = 23.7 µg/g LoQ = 30 µg/g

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G. Interfering Substances

Study procedures were performed using stool specimen extracts with the following approximate calprotectin concentrations: 30 µg/g, 100 µg/g, 300 µg/g, and 550 µg/g. The following analytes, pharmaceuticals, and nutritional supplements did not interfere with the BÜHLMANN fCAL® turbo:

Trade name	Active component	Solvent	Concentrationmg/50 mgstool
gyno-Tardyferon	Iron (II) sulfate	HCl/NaOH	0.11
Prednisone	Prednisone	DMSO	0.31
Imurek	Azathioprine	DMSO	0.19
Salofalk	Mesalamine; 5-ASA	DMSO	5.21
Agopton	Lansoprazole	Dimethylformamide	0.18
Asacol	Mesalamine; 5-ASA	DMSO	2.50
Vancocin	Vancomycin	H2Odd	2.00
Sulfamethoxazole	Sulfamethoxazole	DMSO	1.60
Trimethoprim	Trimethoprim lactate	DMSO/Exbuffer	0.35
Ciproxine	Ciprofloxacin	solvent from manufacturer/H2Odd	1.25
Vitamin E	DL-α Tocopherol Acetate	H2O + Tween	0.30
Bion 3	Multivitamin	HCl/NaOH	1.06
Hemoglobin	Hemoglobin	H2Odd	1.25

The following enteropathological microorganisms did not interfere with the BÜHLMANN fCAL® turbo when added to stool extracts at the given cell counts:

Microorganism	Concentration(cfu/mL)
Escherichia coli	3.3 x 107
Salmonella enterica subsp. enterica	9.0 x 107
Klebsiella pneumoniae subsp. pneumonia	5.3 x 107
Citrobacter freundii	12.9 x 107
Shigella flexneri	5.0 x 107
Yersinia enterocolitica subsp. enterocolitica	9.8 x 107

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H. Method Comparison

A total of 248 clinical study samples were tested using the BÜHLMANN fCAL® turbo and the predicate device (BÜHLMANN fCAL® ELISA assay); valid results within the linear measuring range for both assays were obtained for 220 of these samples. Results were analyzed by Passing-Bablok regression analysis.

Slope(95% CI)	Intercept (µg/g)(95% CI)	Bias at 80 µg/g(95% CI)	Bias at 160 µg/g(95% CI)	Correlationr
1.025	-4.5	-3.1%	-0.3%	0.972
(0.990, 1.058)	(-8.7, 0.3)	(-7.2%, 0.5%)	(-2.4%, 2.7%)

Frequency counts of BÜHLMANN fCAL® turbo test results and corresponding BÜHLMANN fCAL® ELISA assay results within each of the diagnostic ranges of these tests are provided below.

		# in BÜHLMANN fCAL® ELISA assay range (µg/g)
		< 80	80 - 160	> 160	Total
# in fCALturbo range(µg/g)	< 80	84	10	0	94
	80 - 160	8	41	6	55
	> 160	0	7	92	99
Total		92	58	98	248

Estimates of positive percent agreement (PPA) and negative percent agreement (NPA) between the BÜHLMANN fCAL® turbo results and corresponding BÜHLMANN fCAL® ELISA assay results, using both sets of assay cutoffs, with respect to IBD subjects, other GI subjects, normal subjects, and all subjects combined are shown in the table below.

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Subgroup	Metric	Estimate	95% C.I.
IBD	PPA (lower cutoff)	68/70 = 97.1%	[90.1%, 99.7%]
	NPA (lower cutoff)	6/7 = 85.7%	[42.1%, 99.6%]
	PPA (upper cutoff)	52/56 = 92.9%	[82.7%, 98.0%]
	NPA (upper cutoff)	19/21 = 90.5%	[69.6%, 98.8%]
IBS	PPA (lower cutoff)	28/32 = 87.5%	[71.0%, 96.5%]
	NPA (lower cutoff)	31/33 = 93.9%	[79.8%, 99.3%]
	PPA (upper cutoff)	12/13 = 92.3%	[64.0%, 99.8%]
	NPA (upper cutoff)	49/52 = 94.2%	[84.1%, 98.8%]
Other GI	PPA (lower cutoff)	20/21 = 95.2%	[76.2%, 99.9%]
	NPA (lower cutoff)	16/16 = 100%	[79.4%, 100%]
	PPA (upper cutoff)	13/14 = 92.9%	[66.1%, 99.8%]
	NPA (upper cutoff)	23/23 = 100%	[85.2%, 100%]
Normal	PPA (lower cutoff)	30/33 = 90.9%	[75.7%, 98.1%]
	NPA (lower cutoff)	31/36 = 86.1%	[70.5%, 95.3%]
	PPA (upper cutoff)	15/15 = 100%	[78.2%, 100%]
	NPA (upper cutoff)	52/54 = 96.3%	[87.3%, 99.5%]
All subjects	PPA (lower cutoff)	146/156 = 93.6%	[88.5%, 96.9%]
	NPA (lower cutoff)	84/92 = 91.3%	[83.6%, 96.2%]
	PPA (upper cutoff)	92/98 = 93.9%	[87.1%, 97.7%]
	NPA (upper cutoff)	143/150 = 95.3%	[90.6%, 98.1%]

I. Clinical Sensitivity/Specificity

IBD vs. IBS:

Borderline ValuesConsidered Positive		Clinical Diagnosis		Total
BÜHLMANNfCAL® turbo		IBD	IBS
BÜHLMANNfCAL® turbo	Positive	123	31	154
BÜHLMANNfCAL® turbo	Negative	12	99	111
	Total	135	130	265
Sensitivity = 91.1%; 95% C.I. (85.0%, 95.3%)
Specificity = 76.2%; 95% C.I. (67.9%, 83.2%)
PPV = 79.9%; 95% C.I. (72.7%, 85.9%)
NPV = 89.2%; 95% C.I. (81.9%, 94.3%)

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Borderline ValuesConsidered Negative		Clinical Diagnosis		Total
		IBD	IBS
BÜHLMANNfCAL® turbo	Positive	108	16	124
	Negative	27	114	141
	Total	135	130	265
Sensitivity = 80.0%; 95% C.I. (72.3%, 86.4%)
Specificity = 87.7%; 95% C.I. (80.8%, 92.8%)
PPV = 87.1%; 95% C.I. (79.9%, 92.4%)
NPV =80.9%; 95% C.I. (73.4%, 87.0%)

IBD vs. non-IBD:

Borderline Values		Clinical Diagnosis		Total
Considered Positive		IBD	Non-IBD
BÜHLMANN	Positive	123	52	175
fCAL® turbo	Negative	12	150	162
	Total	135	202	337
Sensitivity = 91.1%; 95% C.I. (85.0%, 95.3%)
Specificity = 74.3%; 95% C.I. (67.7%, 80.1%)
PPV = 70.3%; 95% C.I. (62.9%, 76.9%)
NPV = 92.6%; 95% C.I. (87.4%, 96.1%)

Borderline Values		Clinical Diagnosis		Total
Considered Negative		IBD	IBS
BÜHLMANN	Positive	108	30	138
fCAL® turbo	Negative	27	172	199
	Total	ા રેર	202	337
Sensitivity = 80.0%; 95% C.I. (72.3%, 86.4%)
Specificity = 85.1%; 95% C.I. (79.5%, 89.8%)
PPV = 78.3%; 95% C.I. (70.4%, 84.8%)
NPV =86.4%; 95% C.I. (80.9%, 90.9%)

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J. Expected Values/Reference Range:

Stool samples were obtained from 141 apparently healthy normal adults (> 21 years of age) with no symptoms or signs of gastrointestinal disease. The test results were categorized by the assay cut-offs below.

Calprotectin level by BÜHLMANN fCAL turbo
	< 80 μg/g	80 - 160 μg/g	> 160 μg/g	Total
Number ofsubjects (%)	106 (75.2%)	18 (12.8%)	17 (12.1%)	141 (100%)

VIII. Conclusion

The information provided above supports that the BÜHLMANN fCAL® turbo is as safe and effective as the predicate device. Although differences exist between the subject and predicate device, verification and validation studies support that these differences do not raise any new questions of safety and effectiveness. Therefore, it is concluded that the BÜHLMANN CAL® turbo is substantially equivalent to the predicate device.

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Regulation Number and Section

§ 866.5180 Fecal calprotectin immunological test system.

(a)
Identification. A fecal calprotectin immunological test system is anin vitro diagnostic device that consists of reagents used to quantitatively measure, by immunochemical techniques, fecal calprotectin in human stool specimens. The device is intended forin vitro diagnostic use as an aid in the diagnosis of inflammatory bowel diseases (IBD), specifically Crohn's disease and ulcerative colitis, and as an aid in differentiation of IBD from irritable bowel syndrome.(b)
Classification. Class II (special controls). The special control for these devices is FDA's guidance document entitled “Class II Special Controls Guidance Document: Fecal Calprotectin Immunological Test Systems.” For the availability of this guidance document, see § 866.1(e).