(90 days)
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 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.
The BÜHLMANN CALEX Cap is a single use tube intended for the preparation of human stool samples to be used with the BÜHLMANN fCAL turbo.
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.
Sample extracts may be prepared using manual weighing extraction methods or the CALEX® Cap.
The CALEX® Cap is a single use tilled with extraction buffer. The sampling pin houses a dosing tip which is used to obtain sufficient stool sample for the extraction process. The extraction method leads to stool specimen extracts which can be measured directly using the BÜHLMANN fCAL® turbo assay.
This document describes the validation of the BÜHLMANN fCAL® turbo and CALEX® Cap device, an in vitro diagnostic assay for measuring fecal calprotectin.
1. Table of Acceptance Criteria (Implied) and Reported Device Performance
The document doesn't explicitly list "acceptance criteria" in a separate table, but the performance data sections demonstrate that the device meets various analytical and clinical performance standards. The key performance metrics are presented as results from studies.
| Performance Metric | Reported Device Performance |
|---|---|
| Precision | Single-Site Repeatability (Manual Weighing): %CVs ranged from 0.7% to 8.3%. Multi-Site Reproducibility (Manual Weighing): Total Precision %CVs ranged from 3.2% to 9.1%. Lot-to-Lot Precision (Manual Weighing): Total Precision %CVs ranged from 3.6% to 11.3%. Extraction Reproducibility (Manual Weighing): Total Precision %CVs ranged from 4.5% to 13.0%. Extraction Reproducibility (CALEX® Cap): Total Precision %CVs ranged from 8.1% to 19.7%. |
| Linearity | R^2 values: 0.9983 and 0.9984 for two dilution series. Measuring Range: Direct 30 - 2000 µg/g; with automatic dilution 30 - 10,000 µg/g. |
| High Dose Hook Effect | No high dose hook effect observed up to 45,715 µg/g. |
| Accuracy/Recovery | Total recovery ranged from 93.6% to 102.0% across various spiked samples. |
| Analytical Sensitivity | LoB: 16.7 µg/g LoD: 23.7 µg/g LoQ: 30 µg/g |
| Interfering Substances | No interference observed from various common pharmaceuticals, nutritional supplements, and enteropathological microorganisms at tested concentrations. |
| Method Comparison (vs. predicate ELISA) | Correlation (r): 0.972 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%] |
| Extraction Method Comparison (CALEX® Cap vs. Manual) | Correlation (r): 0.921 PPA (lower cutoff): 98.1% [94.7%, 99.6%] NPA (lower cutoff): 89.9% [81.0%, 95.5%] PPA (upper cutoff): 97.6% [93.1%, 99.5%] NPA (upper cutoff): 96.6% [91.4%, 99.1%] |
| Clinical Sensitivity/Specificity (Manual Weighing) | IBD vs. IBS (Borderline Considered Positive): Sensitivity 91.1%, Specificity 76.2% IBD vs. IBS (Borderline Considered Negative): Sensitivity 80.0%, Specificity 87.7% IBD vs. non-IBD (Borderline Considered Positive): Sensitivity 91.1%, Specificity 74.3% IBD vs. non-IBD (Borderline Considered Negative): Sensitivity 80.0%, Specificity 85.1% |
| Expected Values/Reference Range | 75.2% of apparently healthy adults (<80 µg/g), 12.8% (80-160 µg/g), 12.1% (>160 µg/g). |
2. Sample Size and Data Provenance
-
Test Set Sample Sizes:
- Precision Studies: 80 replicates per sample for Single-Site and Extraction Reproducibility, 75 replicates per sample for Multi-Site and Lot-to-Lot precision.
- Linearity: Not explicitly stated as a number of unique patient samples, but involves two dilution series with "various mixing ratios" and 4 replicates per dilution.
- Accuracy/Recovery: 7 samples.
- Interfering Substances: Stool specimen extracts with 4 approximate calprotectin concentrations; specific number of runs not explicitly stated.
- Method Comparison (vs. predicate ELISA): 248 clinical study samples.
- Extraction Method Comparison (CALEX® Cap vs. Manual): 241 clinical study samples.
- Clinical Sensitivity/Specificity: 265 samples for IBD vs. IBS analysis, 337 samples for IBD vs. non-IBD analysis.
- Expected Values/Reference Range: 141 apparently healthy normal adults.
-
Data Provenance: The document does not explicitly state the country of origin for the clinical study data or whether it was retrospective or prospective. Given the submitter's location (Switzerland), it's possible some or all studies were conducted there or internationally. The term "clinical study samples" usually implies prospective collection, but it's not explicitly stated.
3. Number of Experts and Qualifications for Ground Truth
- This document is for an in vitro diagnostic (IVD) assay, specifically an immunological test system for fecal calprotectin. The "ground truth" for such devices often relies on established laboratory methods, clinical diagnoses based on a constellation of evidence (e.g., endoscopy, histology, imaging, other lab findings), or consensus among clinicians.
- No "experts" in the sense of human readers for images are mentioned or used for establishing ground truth. Instead, the device's performance is compared against:
- Itself (for precision, linearity, recovery, sensitivity).
- A legally marketed predicate device (for method comparison).
- External clinical diagnoses (for clinical sensitivity/specificity).
- For clinical diagnosis (e.g., IBD, IBS, non-IBD), the ground truth is implicitly established by clinical findings, which would typically involve qualified physicians (e.g., gastroenterologists) and pathologists, but their specific number or qualifications are not detailed as this is standard for IVD test validation.
4. Adjudication Method for the Test Set
- Adjudication methods (like 2+1, 3+1) are typically used for studies involving human interpretation of complex data (e.g., medical images) where a consensus is needed to define the ground truth for equivocal cases.
- This is an IVD device, not an image-based AI system. Therefore, there is no "adjudication method" as described in the context of human reader studies. The "ground truth" for clinical sensitivity/specificity would be the established clinical diagnosis for each patient, determined by standard medical practice, not by expert adjudication of the test results themselves.
5. Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study
- No MRMC study was done. MRMC studies are designed to compare the diagnostic performance of human readers, often with and without AI assistance, on a set of medical cases.
- This document describes the validation of an in vitro diagnostic (IVD) test kit, which is a laboratory assay. It does not involve human readers interpreting images or any AI assistance for human interpretation.
6. Standalone (Algorithm Only Without Human-in-the-Loop Performance) Study
- Yes, this entire submission effectively represents a standalone performance study of the BÜHLMANN fCAL® turbo and CALEX® Cap device.
- As an IVD assay, its core function is to produce quantitative results based on the chemical reaction. The performance data (precision, linearity, accuracy, analytical sensitivity, method comparison) demonstrate its intrinsic performance independent of human interpretation of its raw output. The subsequent clinical sensitivity and specificity studies evaluate the diagnostic accuracy of the device's numerical output against clinical diagnoses. There is no "human-in-the-loop" in the direct operation or result generation of the assay itself, apart from standard laboratory procedures and subsequent clinical interpretation of the numerical calprotectin values.
7. Type of Ground Truth Used
- Analytical Ground Truths:
- Known concentrations: For linearity, analytical sensitivity (LoB, LoD, LoQ), accuracy/recovery, and interfering substances, the ground truth involves samples with known or spiked concentrations of calprotectin or interferents.
- Reference Methods/Predicate Device: For method comparison, the predicate BÜHLMANN fCAL® ELISA assay serves as a comparative ground truth.
- Clinical Ground Truths:
- Clinical Diagnosis: For clinical sensitivity and specificity, the ground truth for patients was their established clinical diagnosis (e.g., IBD, IBS, non-IBD). This diagnosis is typically based on an aggregation of clinical findings, endoscopic results, histological reports, and other laboratory tests, representing the best available clinical determination.
- Healthy Normal Cohort: For expected values/reference range, the ground truth was a cohort of "apparently healthy normal adults" based on lack of symptoms or signs of gastrointestinal disease, implying a clinical assessment.
8. Sample Size for the Training Set
- This document describes the validation studies for a pre-market submission (510(k)) for an IVD device. For such devices, particularly those based on immunoassay technology, the concept of a "training set" like that used in machine learning (ML) is generally not applicable in the same way. The device's underlying immunoassay methodology is laboratory-based and its parameters are established through extensive analytical development rather than data-driven "training" in the ML sense.
- The document presents performance data from verification and validation studies, which are essentially "test sets" for the final device. There is no mention of a separate "training set" of patient data for model development as one would see with a deep learning algorithm. The analytical parameters and performance characteristics of the immunoassay are developed and optimized by the manufacturer.
9. How the Ground Truth for the Training Set Was Established
- As explained above, the concept of a distinct "training set" and associated ground truth establishment in the context of an ML algorithm is not directly applicable to this immunoassay device's validation as described in the 510(k) summary. The development of such assays relies on biochemical principles, calibration curves established with known standards, and analytical validation experiments performed on various matrices and samples. The "ground truth" for developing the assay itself would be the precisely measured values of known calprotectin standards.
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September 24, 2019
BÜHLMANN Laboratories AG Roshana Ahmed President Quaras, LLC 2101 Camino Rey Fullerton, California 92833
Re: K191718
Trade/Device Name: BÜHLMANN fCAL turbo and CALEX Cap Regulation Number: 21 CFR 866.5180 Regulation Name: Fecal calprotectin immunological test system Regulatory Class: Class II Product Code: NXO Dated: June 26, 2019 Received: June 26, 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.
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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 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 mediation-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,
for
Douglas Jeffery, Ph.D. Branch Chief Division of Immunology and Hematology Devices OHT7: Office of In Vitro Diagnostics and Radiological Health Office of Product Evaluation and Ouality Center for Devices and Radiological Health
Enclosure
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Indications for Use
510(k) Number (if known)
Device Name BÜHLMANN fCAL turbo, CALEX Cap
Indications for Use (Describe)
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 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.
The BÜHLMANN CALEX Cap is a single use tube intended for the preparation of human stool samples to be used with the BÜHLMANN fCAL turbo.
| 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
BUHLMANN Laboratories AG Baselstrasse 55 Schönenbuch CH-4124 Switzerland Phone: +41 61 487 12 50
Contact Person: Laura Zurbrügg Date Prepared: September 11, 2019
II. Device
| Device Proprietary Names: | BÜHLMANN fCAL® turbo, CALEX® Cap |
|---|---|
| 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® turbo, K190784, BÜHLMANN Laboratories AG
Device Description IV.
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.
Sample extracts may be prepared using manual weighing extraction methods or the CALEX® Cap.
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The CALEX® Cap is a single use tilled with extraction buffer. The sampling pin houses a dosing tip which is used to obtain sufficient stool sample for the extraction process. The extraction method leads to stool specimen extracts which can be measured directly using the BÜHLMANN fCAL® turbo assay.
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.
The BÜHLMANN CALEX® Cap is a single use tube intended for the preparation of human stool samples to be used with the BÜHLMANN fCAL® turbo.
VI. Comparison of Technological Characteristics
The subject and predicate device utilize the same exact assay, controls, and calibrators. The only difference between the two products is the provision of the CALEX® Cap as an alternative sample extraction method to manual weighing.
The tables below compare key technological features between the subject and predicate devices.
Technological comparison
Comparison of Assay
| BÜHLMANN fCAL® turbo andCALEX® Cap | BÜHLMANN fCAL® turbo(K190784) | |
|---|---|---|
| Analyte | Human fecal calprotectin(MRP8/14) | Human fecal calprotectin(MRP8/14) |
| Assay format | Quantitative | Quantitative |
| Specimen type | Human stool | Human stool |
| 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 |
| Method | PETIA | PETIA |
| Automation | Automated | Automated |
| Solid phase | Polystyrene nanoparticles (beads) | Polystyrene nanoparticles (beads) |
| Detection method | Automated clinical chemistry analyzer read at 546 nm | Automated clinical chemistry analyzer read at 546 nm |
| Analyte-specificantibodycomponents | Polyclonal antibodies against human calprotectin coated on polystyrene beads | Polyclonal antibodies against human calprotectin coated on polystyrene beads |
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| BÜHLMANN fCAL® turbo andCALEX® Cap | BÜHLMANN fCAL® turbo(K190784) | |
|---|---|---|
| Measuring range | Direct measuring range:30 - 2000 μg/gMeasuring range with automaticdilution: 30 - 10,000 μg/g | Direct measuring range:30 - 2000 μg/gMeasuring range with automaticdilution: 30 – 10,000 μg/g |
| ExtractionMethod | Manual Weighing (1:50 dilution inExtraction Buffer)CALEX® Cap (1:500 dilution inExtraction Buffer) | Manual Weighing (1:50 dilution inExtraction Buffer)N/A |
Comparison of Calibrators
| BÜHLMANN fCAL® turbo | BÜHLMANN fCAL® turbo (K190784) | |
|---|---|---|
| Indications for Use | 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. | 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. |
| Method | BÜHLMANN fCAL® turbo | BÜHLMANN fCAL® turbo |
| Analyte | Native human calprotectinSource: human granulocyte extract | Native human calprotectinSource: human granulocyte extract |
| Calibrators | 6 levels:Target values: 0, 50, 200, 500,1000, 2000 µg/g | 6 levels:Target values: 0, 50, 200, 500,1000, 2000 µg/g |
| Conversion factor | N/A | N/A |
| Value assignment: | Calibrator values assigned using avalue transfer protocol for eachcalibrator lot. Values are indicatedin the QC datasheet. | Calibrator values assigned using avalue transfer protocol for eachcalibrator lot. Values are indicatedin the QC datasheet. |
| Configuration | Available as a separateBÜHLMANN fCAL® turboCalibrator Kit | Available as a separateBÜHLMANN fCAL® turboCalibrator Kit |
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| BÜHLMANN fCAL® turbo | BÜHLMANN fCAL® turbo(K190784) | ||
|---|---|---|---|
| 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. | 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 inextracted stool samples. | |
| Method | BÜHLMANN fCAL® turbo | ||
| Analyte | Native human calprotectin | Native human calprotectin | |
| Source: human granulocyte extract | Source: human granulocyte extract | ||
| 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 | Available as a separateBÜHLMANN fCAL® turboControl Kit |
Comparison of Controls
Discussion
As seen above, the only difference between the subject and predicate devices is the provision of the CALEX® Cap as an alternative sample extraction method. This technological difference does not create new questions of safety and effectiveness and the differences are addressed by the performance studies identified below.
Performance Data VII.
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 |
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B. Precision
| 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% |
Single-Site Repeatability Study Results (Manual Weighing Extraction Method):
| Multi-Site Reproducibility Study Results (Manual Weighing Extraction Method): | ||||
|---|---|---|---|---|
| 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 | TotalPrecision | ||||
|---|---|---|---|---|---|---|---|---|---|---|
| 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 (Manual Weighing Extraction Method):
Extraction Reproducibility (Manual Weighing Extraction Method) Study Results:
| Sample | n | Mean(µg/g) | 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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| Sample | n | Mean(µg/g) | Within-Run(Repeatability) | Between-extraction | Between-day | Between-Lot | Between-operator | TotalPrecision | ||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| SD(µg/g) | %CV | SD(µg/g) | %CV | SD(µg/g) | %CV | SD(µg/g) | %CV | SD(µg/g) | %CV | SD(µg/g) | %CV | |||
| S1 | 72 | 42.7 | 3.2 | 7.5 | 4.6 | 10.8 | 0.0 | 0.0 | 2.7 | 6.3 | 0.0 | 0.0 | 6.2 | 14.5 |
| S2 | 72 | 71.5 | 3.9 | 5.4 | 6.9 | 9.6 | 9.4 | 13.1 | 0.0 | 0.0 | 0.0 | 0.0 | 12.2 | 17.1 |
| S3 | 72 | 111.3 | 3.3 | 2.9 | 14.2 | 12.7 | 0.0 | 0.0 | 6.8 | 6.1 | 7.8 | 7.0 | 17.9 | 16.1 |
| S4 | 72 | 119.8 | 2.9 | 2.4 | 7.2 | 6.0 | 5.8 | 4.8 | 0.0 | 0.0 | 0.0 | 0.0 | 9.7 | 8.1 |
| S5 | 72 | 213.0 | 3.2 | 1.5 | 27.9 | 13.1 | 0.0 | 0.0 | 0.0 | 0.0 | 9.0 | 4.2 | 29.5 | 13.8 |
| S6 | 72 | 297.2 | 3.7 | 1.2 | 24.5 | 8.2 | 13.5 | 4.6 | 18.0 | 6.1 | 12.3 | 4.1 | 35.6 | 12.0 |
| S7 | 72 | 561.2 | 5.5 | 1.0 | 18.6 | 3.3 | 66.1 | 11.8 | 0.0 | 0.0 | 0.0 | 0.0 | 68.9 | 12.3 |
| S8 | 72 | 610.0 | 4.7 | 0.8 | 74.3 | 12.2 | 28.2 | 4.6 | 0.0 | 0.0 | 0.0 | 0.0 | 79.6 | 13.1 |
| S9 | 72 | 940.4 | 12.2 | 1.3 | 152.7 | 16.2 | 34.8 | 3.7 | 0.0 | 0.0 | 97.5 | 10.4 | 184.9 | 19.7 |
| S10 | 72 | 1558.4 | 7.8 | 0.5 | 152.0 | 9.8 | 39.9 | 2.6 | 98.6 | 6.3 | 146.2 | 9.4 | 236.4 | 15.2 |
| S11 | 72 | 2041.6 | 27.2 | 1.3 | 150.3 | 7.4 | 133.8 | 6.6 | 88.9 | 4.4 | 10.5 | 0.5 | 221.9 | 10.9 |
| S12 | 72 | 3440.0 | 48.7 | 1.4 | 177.7 | 5.2 | 321.5 | 9.3 | 0.0 | 0.0 | 0.0 | 0.0 | 370.5 | 10.8 |
Extraction Reproducibility (CALEX® Cap) Study Results:
C. Linearity
Study procedures were performed using two dilution series. For each dilution series, a stool specimen extract with a calprotectin concentration above the anticipated upper limit of the analytical measuring range was combined with a stool specimen extract with a calprotectin concentration below the anticipated lower limit of the analytical measuring range, in various mixing ratios covering the range; each dilution was tested in four (4) replicates. Results of the linear regression analyses are presented in the table below.
| DilutionSeries | 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 µg/g
- Measuring range with automatic dilution: 30 - 10,000 ug/g
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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 µg/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
Interfering Substances G.
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 |
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| 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 |
The following enteropathological microorganisms did not interfere with the BÜHLMANN fCAL® turbo when added to stool extracts at the given cell counts:
H. Method Comparison
A total of 248 clinical study samples, prepared using the manual weighing extraction method, 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(0.990, 1.058) | -4.5(-8.7, 0.3) | -3.1%(-7.2%, 0.5%) | -0.3%(-2.4%, 2.7%) | 0.972 |
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. Extraction Method Comparison
A total of 241 clinical study samples were extracted using the CALEX® Cap and manual weighing extraction methods and tested using the BÜHLMANN fCAL® turbo. Valid results within the linear measuring range were obtained for 202 of these samples using both extraction methods.
Results were analyzed by Passing-Bablok regression analysis.
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| Slope(95% CI) | Intercept (µg/g)(95% CI) | Bias at 80 µg/g(95% CI) | Bias at 160 µg/g(95% CI) | Correlationr |
|---|---|---|---|---|
| 1.149 | -8.3 | 4.6% | 9.7% | 0.921 |
| (1.100, 1.201) | (-17.1, -2.0) | (-4.3%, 9.1%) | (4.2%, 13.8%) |
Frequency counts of BÜHLMANN fCAL® turbo test results using the CALEX® Cap and manual weighing extraction methods within each of the BÜHLMANN fCAL® turbo diagnostic ranges are provided below.
| # of CALEX® Cap results | |||||
|---|---|---|---|---|---|
| < 80 | 80 - 160 | > 160 | Total | ||
| # of manualweighingresults | < 80 | 71 | 8 | 0 | 79 |
| 80 - 160 | 3 | 30 | 4 | 37 | |
| > 160 | 0 | 3 | 122 | 125 | |
| Total | 74 | 41 | 126 | 241 |
Estimates of positive percent agreement (PPA) and negative percent agreement (NPA) between the BÜHLMANN fCAL® turbo results using the CALEX® Cap extraction method and corresponding results using the manual weighing extraction method, using both sets of assay cutoffs are shown in the table below.
| Metric | Estimate | 95% C.I. |
|---|---|---|
| PPA (lower cutoff) | $159/162 = 98.1%$ | [94.7%, 99.6%] |
| NPA (lower cutoff) | $71/79 = 89.9%$ | [81.0%, 95.5%] |
| PPA (upper cutoff) | $122/125 = 97.6%$ | [93.1%, 99.5%] |
| NPA (upper cutoff) | $112/116 = 96.6%$ | [91.4%, 99.1%] |
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Clinical Sensitivity/Specificity (Manual Weighing Extraction Method) J.
| IBD vs. | IBS: |
|---|---|
| --------- | ------ |
| Borderline ValuesConsidered Positive | Clinical Diagnosis | Total | ||
|---|---|---|---|---|
| IBD | IBS | |||
| BÜHLMANNfCAL® turbo | Positive | 123 | 31 | 154 |
| 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%) |
| Borderline ValuesConsidered Negative | Clinical Diagnosis | Total | ||
|---|---|---|---|---|
| IBD | IBS | |||
| BÜHLMANN | Positive | 108 | 16 | 124 |
| fCAL® turbo | 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 ValuesConsidered Positive | Clinical Diagnosis | Total | ||
|---|---|---|---|---|
| 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%) |
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| Borderline ValuesConsidered Negative | Clinical Diagnosis | Total | ||
|---|---|---|---|---|
| BÜHLMANNfCAL® turbo | IBD | IBS | ||
| Positive | 108 | 30 | 138 | |
| Negative | 27 | 172 | 199 | |
| Total | 135 | 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%) |
K. Expected Values/Reference Range:
Stool samples, prepared using the manual weighing extraction method, were obtained from 141 apparently healthy normal adults (> 21 years of age) with no symptoms or signs of gastrointestinal disease and were. 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 and CALEX® Cap are as safe and effective as the predicate device. Verification and validation studies support that the use of the CALEX® Cap to prepare stool sample extracts does not raise any new questions of safety and effectiveness. Therefore, it is concluded that the BÜHLMANN fCAL® turbo and CALEX® Cap are substantially equivalent to the predicate device.
§ 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).