K Number
K180971
Device Name
QUANTA Flash Calprotectin and Fecal Extraction Device
Date Cleared
2018-10-16

(186 days)

Product Code
Regulation Number
866.5180
AI/MLSaMDIVD (In Vitro Diagnostic)TherapeuticDiagnosticis PCCP Authorized
Intended Use
The Fecal Extraction Device is a single use tube containing extraction buffer intended for sampling and extracting human stool specimens and subsequent analysis with the QUANTA Flash Calprotectin assay.
Device Description
The Fecal Extraction Device acquires the amount of stool necessary to perform the QUANTA Flash Calprotectin assay directly from the primary specimen container instead of weighing the sample. The device consists of a tube, containing 2.8 mL of extraction buffer, and a stick shaped with seven grooves for collecting the sample. The upper end of the device is made up of two parts which can be removed with two separate rotations: the white screw cap (connected to the plastic stick with grooves) is removed by twisting counter-clockwise. The red lower part (for retaining the excess material) is removed by twisting clockwise. Once having completed the extraction procedure, remove both the white and red upper parts. The tube containing the extracted sample can be placed directly into the BIO-FLASH instrument sample rack.
More Information

Not Found

No
The description details a manual extraction device and an automated immunoassay system. While the system has software for calculations and data processing, there is no mention of AI or ML being used for analysis, interpretation, or decision-making. The calculations are based on predefined curves and standard formulas.

No.
The device is intended for sampling and extracting human stool specimens for subsequent analysis, specifically with the QUANTA Flash Calprotectin assay, which is an in vitro diagnostic test. It does not provide any therapeutic benefit or treatment.

No

The Fecal Extraction Device is a sample collection and extraction device used to prepare human stool specimens for subsequent analysis. It does not perform the diagnostic analysis itself, but rather is a component used with a diagnostic assay (QUANTA Flash Calprotectin assay) run on an instrument (BIO-FLASH® instrument). The diagnostic function is performed by the overall system, not by this specific device on its own.

No

The device is a physical tube with extraction buffer and a stick for collecting stool samples. While it is used in conjunction with an automated instrument that includes software, the device itself is a hardware component for sample preparation.

Yes, this device is an IVD (In Vitro Diagnostic).

Here's why:

  • Intended Use: The intended use explicitly states it's for "sampling and extracting human stool specimens and subsequent analysis with the QUANTA Flash Calprotectin assay." This indicates it's used to prepare a biological specimen for an in vitro diagnostic test.
  • Device Description: The device is designed to collect and process a human biological sample (stool) for analysis.
  • Relationship to an IVD Assay: The device is specifically designed to be used with the QUANTA Flash Calprotectin assay, which is described as a "chemiluminescent microparticle immunoassay" run on an automated instrument. This assay is clearly an in vitro diagnostic test.
  • Performance Studies: The document includes performance studies demonstrating the device's ability to properly extract the sample for the subsequent assay, comparing it to a manual extraction method used with a predicate IVD.

The Fecal Extraction Device is a component or accessory used in conjunction with an in vitro diagnostic assay (the QUANTA Flash Calprotectin assay) to prepare the sample for testing. Therefore, it falls under the definition of an IVD device.

N/A

Intended Use / Indications for Use

The Fecal Extraction Device is a single use tube containing extraction buffer intended for sampling and extracting human stool specimens and subsequent analysis with the QUANTA Flash Calprotectin assay.

Product codes

NXO

Device Description

The Fecal Extraction Device acquires the amount of stool necessary to perform the QUANTA Flash Calprotectin assay directly from the primary specimen container instead of weighing the sample. The device consists of a tube, containing 2.8 mL of extraction buffer, and a stick shaped with seven grooves for collecting the sample. The upper end of the device is made up of two parts which can be removed with two separate rotations: the white screw cap (connected to the plastic stick with grooves) is removed by twisting counter-clockwise. The red lower part (for retaining the excess material) is removed by twisting clockwise. Once having completed the extraction procedure, remove both the white and red upper parts. The tube containing the extracted sample can be placed directly into the BIO-FLASH instrument sample rack.

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.

The Fecal Extraction Device kit contains the following materials:
One hundred (100) Fecal Extraction Devices

The Fecal Extraction Device tube contains the following materials for a single stool extraction:
a. 2,8 mL of extraction buffer, colorless, ready to use.

Mentions image processing

Not Found

Mentions AI, DNN, or ML

Not Found

Input Imaging Modality

Not Found

Anatomical Site

human stool specimens

Indicated Patient Age Range

Not Found

Intended User / Care Setting

Not Found

Description of the training set, sample size, data source, and annotation protocol

Not Found

Description of the test set, sample size, data source, and annotation protocol

Not Found

Summary of Performance Studies (study type, sample size, AUC, MRMC, standalone performance, key results)

Stool Extraction method comparison: Fecal Extraction Device vs. Manual weighing method
Study Type: Method Comparison Study
Sample Size: Ninety seven human stool samples
Key Results: All acceptance criteria were met.

  • Intercept of regression line: -1.2 (95% CI: -9.7 to 6.4)
  • Slope of regression line: 0.93 (95% CI: 0.82 to 1.06)
  • Predicted Bias at 120mg/kg: -7.8 % (95% CI: -16.0% to 0.6%)
  • Correlation r: 0.975

Device Validation
Study Type: Performance Measurement Study
Sample Size: Five different human stool samples, collected in replicates of five by three independent operators.
Key Results: All acceptance criteria were met.

  • Mean Sample Weight (95% CI): 56 (55 - 57) mg
  • Median Sample Weight: 56 mg
  • Range: 50 – 62 mg
  • Standard Deviation: ± 3.8 mg
  • % Coefficient of Variation: 6.9%

Extraction Reproducibility
Study Type: Reproducibility Study
Sample Size: Eight samples, tested in replicates of 5, once a day, for 5 days, to generate 25 data points per sample per operator. Total of 75 data points per sample.
Key Results: All acceptance criteria were met.

  • Within Laboratory (Total Imprecision) %CV: ≤ 15% or SD: ≤ 7.5 mg/kg for negative samples.

Sample Stability and Handling
Study Type: Stability Study
Sample Size: Seven human stool samples
Key Results: All samples fulfilled the acceptance criteria (80-120% average recovery) at each time point for each condition.
Recommendations: Extracted samples using the Fecal Extraction Device are stored up to 72 hours at room temperature, up to 14 days at 2-8°C and can be subjected to up to 4 freeze/thaw cycles.

Shelf life (Real Time Stability)
Study Type: Real-Time Stability Study
Sample Size: Three different lots of FED
Key Results: pH remained between 7.70 and 7.90 for up to 26 months. All samples fulfilled the acceptance criteria (70-130% average recovery) for functionality at expiration date.
Conclusion: The Fecal Extraction Device will be given an expiration date of 2 years.

Stability at Room Temperature
Study Type: Room Temperature Stability Study
Sample Size: Three independent lots of Fecal Extraction Device, six human stool samples.
Key Results: All results obtained using the three different lots of Fecal Extraction Device fulfilled the acceptance criteria (80-120% average recovery).
Conclusion: The Fecal Extraction Device can be kept at room temperature up to 72 hours.

Key Metrics (Sensitivity, Specificity, PPV, NPV, etc.)

Stool Extraction method comparison: Fecal Extraction Device vs. Manual weighing method
Regression Analysis:

  • Y-Intercept (95% CI): -1.2 (-9.7 to 6.4)
  • Slope (95% CI): 0.93 (0.82 to 1.06)
  • Bias at 120mg/kg (95% CI): -7.8 % (-16.0% to 0.6%)
  • Correlation r: 0.975

Percent Agreement Analysis (treating Indeterminate as positive):

  • Positive agreement (95%CI): 93.2% (85.1 - 97.1%)
  • Negative agreement (95%CI): 87.0 % (67.9 - 95.5%)
  • Total agreement (95%CI): 91.8% (84.6 - 95.8%)

Percent Agreement Analysis (treating Indeterminate as negative):

  • Positive agreement (95%CI): 93.5% (82.5 - 97.8%)
  • Negative agreement (95%CI): 94.1% (84.1 - 98.0%)
  • Total agreement (95%CI): 93.8% (87.2 - 97.1%)

Device Validation

  • Mean Sample Weight (95% CI): 56 (55 - 57) mg
  • Median Sample Weight: 56 mg
  • Range: 50 – 62 mg
  • Standard Deviation: ± 3.8 mg
  • % Coefficient of Variation: 6.9%

Extraction Reproducibility
(Mean, SD, and CV across various samples and categories of imprecision)
Example:
Sample 1 (N=75, Mean=29.5 mg/kg):

  • Repeatability: SD=2.2 mg/kg, CV=7.3%
  • Between-Day: SD=7.2 mg/kg, CV=24.4%
  • Within Operator: SD=7.5 mg/kg, CV=25.4%
  • Between Operator: SD=0.0 mg/kg, CV=0.0%
  • Within Laboratory: SD=7.5 mg/kg, CV=25.4%

Sample 8 (N=75, Mean=1560.0 mg/kg):

  • Repeatability: SD=63.9 mg/kg, CV=4.1%
  • Between-Day: SD=197.1 mg/kg, CV=12.6%
  • Within Operator: SD=207.2 mg/kg, CV=13.3%
  • Between Operator: SD=0.0 mg/kg, CV=0.0%
  • Within Laboratory: SD=207.2 mg/kg, CV=13.3%

Predicate Device(s)

K170993

Reference Device(s)

Not Found

Predetermined Change Control Plan (PCCP) - All Relevant Information

Not Found

§ 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).

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October 16, 2018

Inova Diagnostics, Inc. Roger Albesa Manager 9900 Old Grove Road San Diego, California 92131-1638

Re: K180971

Trade/Device Name: Fecal Extraction Device Regulation Number: 21 CFR 866.5180 Regulation Name: Fecal calprotectin immunological test system Regulatory Class: Class II Product Code: NXO Dated: April 11, 2018 Received: April 13, 2018

Dear Roger Albesa:

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

1

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/CombinationProducts/GuidanceRegulatoryInformation/ucm597488.html; 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 http://www.fda.gov/MedicalDevices/Safety/ReportaProblem/default.htm.

For comprehensive regulatory information about mediation-emitting products, including information about labeling regulations, please see Device Advice (https://www.fda.gov/MedicalDevices/DeviceRegulationandGuidance/) and CDRH Learn (http://www.fda.gov/Training/CDRHLearn). 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 (http://www.fda.gov/DICE) for more information or contact DICE by email (DICE@fda.hhs.gov) or phone (1-800-638-2041 or 301-796-7100).

Sincerely,

Leonthena R. Carrington -S

Lea Carrington Director Division of Immunology and Hematology Devices Office of In Vitro Diagnostics and Radiological Health Center for Devices and Radiological Health

Enclosure

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Indications for Use

510(k) Number (if known)

Device Name Fecal Extraction Device

Indications for Use (Describe)

The Fecal Extraction Device is a single use tube containing extraction buffer intended for sampling and extracting human stool specimens and subsequent analysis with the QUANTA Flash Calprotectin assay.

Type of Use (Select one or both, as applicable)

X Prescription Use (Part 21 CFR 801 Subpart D)

| Over-The-Counter Use (21 CFR 801 Subpart C)

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510(k) Summary

Fecal Extraction Device

Page 1 of 9

Table of Contents
Administrative data2
Predicate device3
Device description3
Intended use(s)4
Indications for use4
Substantial equivalence4
Comparison to predicate device5
Analytical performance characteristics6
Stool Extraction method comparison: Fecal Extraction Device vs. Manual weighing method6
Device Validation7
Reproducibility Studies7
Sample Stability and Handling8
Fecal Extraction Device Stability8
Clinical performance characteristics9

This summary of the 510(k) safety and effectiveness information is being submitted in accordance with the requirements of SMDA 1990 and 21 CFR 807.92.

4

Administrative data

| Submitter: | Inova Diagnostics, Inc
9900 Old Grove Road,
San Diego, CA, 92131 | | |
|------------------------------|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|----------------------------------------------------------------------------|--|
| Purpose of submission: | The reason for submission is the addition of an optional new device to
aid in the stool extraction process, the Fecal Extraction Device, to work
with the QUANTA Flash Calprotectin assay. | | |
| Devices in the submission: | Fecal Extraction Device | | |
| Scientific contact: | Roger Albesa, Manager, Research and Development
Inova Diagnostics, Inc.
9900 Old Grove Road, San Diego, CA, 92131
Phone: 858-586-9900 x1391
Fax: 858-863-0025
Email: ralbesa@inovadx.com | | |
| Quality Systems contact: | Ronda Elliott, VP, Quality Systems and RA
Inova Diagnostics, Inc
9900 Old Grove Road, San Diego, CA, 92131
Phone: 858-586-9900/1381
Fax: 858-863-0025
Email: relliot@inovadx.com | | |
| Device name: | Proprietary name: | Fecal Extraction Device | |
| | Common name: | Chemiluminescence Fecal Calprotectin assay
with Fecal Extraction Device | |
| | Classification name: | Calprotectin, Fecal | |
| Regulation Description | Fecal Calprotectin Immunological Test System | | |
| Regulation Medical Specialty | Immunology | | |
| Review Panel | Immunology | | |
| Product Code | NXO | | |
| Regulation Number | 866.5180 | | |
| Device Class | 2 | | |

5

Predicate device

QUANTA Flash Calprotectin (using manual extraction), 510(k) number: K170993. Date declared: December 22nd, 2017.

Device description

The Fecal Extraction Device acquires the amount of stool necessary to perform the QUANTA Flash Calprotectin assay directly from the primary specimen container instead of weighing the sample. The device consists of a tube, containing 2.8 mL of extraction buffer, and a stick shaped with seven grooves for collecting the sample. The upper end of the device is made up of two parts which can be removed with two separate rotations: the white screw cap (connected to the plastic stick with grooves) is removed by twisting counter-clockwise. The red lower part (for retaining the excess material) is removed by twisting clockwise. Once having completed the extraction procedure, remove both the white and red upper parts. The tube containing the extracted sample can be placed directly into the BIO-FLASH instrument sample rack.

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

6

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.

The Fecal Extraction Device kit contains the following materials:

One hundred (100) Fecal Extraction Devices

The Fecal Extraction Device tube contains the following materials for a single stool extraction:

  • a. 2,8 mL of extraction buffer, colorless, ready to use.

Intended use(s)

The Fecal Extraction Device is a single use tube containing extraction buffer intended for sampling and extracting human stool specimens and subsequent analysis with the QUANTA Flash Calprotectin assay.

Indications for use

Same as Intended use.

Substantial equivalence

The QUANTA Flash Calprotectin assay system using the Fecal Extraction Device performs equivalently as the predicate device.

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Comparison to predicate device

QUANTA Flash Calprotectin Reagents

Similarities
ItemQUANTA Flash Calprotectin with Fecal Extraction Device.QUANTA Flash Calprotectin
Intended useQUANTA 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).
The Fecal Extraction Device is a single use tube containing extraction buffer intended for sampling stool specimens and subsequent analysis with the QUANTA Flash Calprotectin assay.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).
Assay methodologySolid phase (heterogeneous) immunoassaySame
AntigenRabbit polyclonal anti-calprotectin antibodySame
Shelf lifeOne yearSame
Sample typeExtracted Human StoolSame
Unitsmg/kg (milligram of calprotectin per kilogram of stool)Same
Detection/ Operating principleChemiluminescent immunoassaySame
Solid phaseParamagnetic microparticles (beads)Same
ConjugateIsoluminol conjugated monoclonal anti-calprotectin antibodySame
Analytical Measuring Range$16.1 - 3,500.0$ mg/kgSame
CalibrationLot specific Master Curve + three calibrators (sold separately)Same

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Analytical performance characteristics

Same as approved in 510k submission #K170993 plus the following additional data.

Stool Extraction method comparison: Fecal Extraction Device vs. Manual weighing method

Ninety seven human stool samples containing different levels of calprotectin were extracted in parallel using the manual weighting method and the Fecal Extraction Device. All resulting stool extracts were tested in singleton for calprotectin concentration levels using the QUANTA Flash Calprotectin assay. A scatter plot with a linear fit was created by plotting the mean values obtained with the manual weighting method against those obtained with the Fecal Extraction Device, using the Passing-Bablok fit function of Analyse-it for Excel software. This study has been conducted according to CLSI EPO9-A3 Method Comparison and Bias Estimation Using Patient Samples: Approved Guideline.

Acceptance criteria:

  • Intercept of regression line ± 15% of cut-off (18 mg/kg)
  • Slope of regression line between 0.90 and 1.10
  • Predicted Bias at cut-off ≤ 15%
  • . 95% CI of the bias: does not exceed medically significant difference, 20% of cut-off
  • Correlation r > 0.95

Results are summarized in the Table below:

| Passing-Bablok fit | Y-Intercept
(95% CI) | Slope
(95% CI) | Bias at 120mg/kg
(95% CI) | Correlation r |
|--------------------------------------------------------------------|-------------------------|------------------------|------------------------------|---------------|
| Fecal Collection Device
vs Manual Extraction
comparison n=97 | -1.2
(-9.7 to 6.4) | 0.93
(0.82 to 1.06) | -7.8 %
(-16.0% to 0.6%) | 0.975 |

All acceptance criteria were met.

Additionally, a percent agreement analysis has been performed between the two methods of extraction using the data generated obtaining the following results:

Manual Extraction
PositiveIndeterminateNegativeTotal
Positive433046
Fecal ExtractionIndeterminate320326
DeviceNegative052025
Total46282397
Indeterminate as positive
Positive agreement (95%CI):93.2% (85.1 - 97.1%)
Negative agreement (95%CI):87.0 % (67.9 - 95.5%)
Total agreement (95%CI):91.8% (84.6 - 95.8%)
Indeterminate as negative
Positive agreement (95%CI):93.5% (82.5 - 97.8%)
Negative agreement (95%CI):94.1% (84.1 - 98.0%)
Total agreement (95%CI):93.8% (87.2 - 97.1%)

9

Device Validation

To validate the amount of fecal material that is collected by the Fecal Extraction Device five different human stool samples, encompassing different stool consistencies, were collected using the Fecal Extraction Device in replicates of five by three independent operators. Data were analyzed with the Analyse-it for Excel method evaluation software to calculate weight distribution.

Acceptance criteria: weight collected by the FED is 56 mg ±10% (between 50 and 62 mg). Results of the study are summarized in the Table below:

Sample collection performance of FED
Mean Sample Weight (95% CI)56 (55 - 57) mg
Median Sample Weight56 mg
Range50 – 62 mg
Standard Deviation$\pm$ 3.8 mg
% Coefficient of Variation6.9%

All acceptance criteria were met.

Reproducibility Studies

Extraction Reproducibility

Eight samples were extracted and tested, according to CLSI EP05-A3 Evaluation of Precision of Quantitative Measurement Procedures, in replicates of 5, once a day, for 5 days, to generate 25 data points per sample per operator, for a total of 75 data points per sample. The extractions were performed daily by three independent operators using the Fecal Extraction Device. Data were analyzed with the Analyse-it for Excel method evaluation software to calculate between operator precision.

Acceptance criteria: Within Laboratory (Total Imprecision) %CV: ≤ 15% or SD: ≤ 7.5 mg/kg for negative samples.

Results are summarized in the Table below.

RepeatabilityBetween-DayWithin OperatorBetween OperatorWithin Laboratory
Sample IDNMean
(mg/kg)SD
(mg/kg)CV
(%)SD
(mg/kg)CV
(%)SD
(mg/kg)CV
(%)SD
(mg/kg)CV
(%)SD
(mg/kg)CV
(%)
Sample 17529.52.27.3%7.224.4%7.525.4%0.00.0%7.525.4%
Sample 27540.02.35.7%6.015.0%6.416.1%2.25.5%6.817.0%
Sample 37553.22.13.9%5.410.2%5.810.9%4.89.0%7.514.1%
Sample 475108.24.34.0%8.07.4%9.18.4%7.36.8%11.710.8%
Sample 575222.97.33.3%20.99.4%22.19.9%5.42.4%22.810.2%
Sample 675535.415.72.9%41.27.7%44.18.2%21.94.1%49.29.2%
Sample 7751278.345.03.5%114.69.0%123.29.6%8.90.7%123.59.7%
Sample 8751560.063.94.1%197.112.6%207.213.3%0.00.0%207.213.3%

All acceptance criteria were met.

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Sample Stability and Handling

Seven human stool samples, encompassing indeterminate range (n=1), around the cut-off (n=2), and positive samples (n=4) were extracted using the Fecal Extraction Device and tested in duplicates using the QUANTA Flash Calprotectin assay for up to 21 days while stored at 2-8°C, up to 73 hours while stored at room temperature, and after repeated freeze/thaw cycles up to 5 cycles (thaw samples were homogenized and clarified before its testing by vortex the samples for 30 seconds and centrifygation for 10 min at 3,000 x g)

Results were compared to those obtained on control samples (time zero / zero cycles).

Acceptance criteria: 80-120% average recovery.

All samples fulfilled the acceptance criteria at each time point for each condition. Based on these result, we recommend that extracted samples using the Fecal Extraction Device are stored up to 72 hours at room temperature, up to 14 days at 2-8°C and can be subjected to up to 4 freeze/thaw cycles.

Fecal Extraction Device Stability

Shelf life (Real Time Stability)

Since calprotectin is not stable in human stool for long periods of time stability studies for the Fecal Extraction Device (FED) were conducted by analyzing the pH of the extraction buffer (located inside the device) of three different lots of FED every 3 to 6 months for 24 to 26 months.

Acceptance criteria: pH between 7.70 and 7.90.

Results are summarized in the table below:

| pH testing
Acceptance Criteria
(7.70 - 790) | Reference
mg/kg | 1
mg/kg | 2
mg/kg | 3
mg/kg |
|---------------------------------------------------|--------------------|------------|------------|------------|
| time = 0 months | 7.79 | 7.82 | 7.81 | 7.81 |
| time = 3 months | 7.78 | 7.81 | 7.81 | 7.80 |
| time = 6 months | - | 7.80 | 7.80 | 7.80 |
| time = 9 months | - | 7.81 | 7.79 | 7.78 |
| time = 12 months | - | 7.80 | 7.78 | 7.78 |
| time = 18 months | - | 7.79 | 7.78 | 7.78 |
| time = 24 months | - | 7.79 | 7.78 | 7.78 |
| time = 26 months | - | - | - | 7.77 |

Additionally, in order to test the functionality of the FED at expiration date, three lots of FED, two at expiration date (24 months) and one freshly produced, were used to perform extractions on 15 human stool samples, encompassing negative (n=11), indeterminate (n=2), around the cut-off (n=1) and a positive (n=1) samples. The calprotectin concentration of each stool extract was determined.

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The results of the two lots of FED at expiration date were compared to those obtained with the freshly produced lot. Percent recovery results were calculated where possible.

Acceptance criteria: 70-130% average recovery. This acceptance criterion takes into consideration the variability between lots of FED, the extraction reproducibility (±15%) and the expected recovery on a traditional real time study (±20%).

All samples fulfilled the acceptance criteria. In conclusion, the Fecal Extraction Device will be given an expiration date of 2 years.

Stability at Room Temperature

Three independent lots of Fecal Extraction Device have been evaluated for room temperature stability. Six human stool samples, encompassing indeterminate (n=1 for lots 2 and 3), around the cut-off (n=1) and positive samples (n=4 for lot 1, n=3 for lots 2 and 3) were extracted using the Fecal Extraction Device in two different conditions: using devices kept at 2-8°C and using devices kept at room temperature for at least 73 hours.

All resulting stool extracts were tested for calprotectin concentration levels using the QUANTA Flash Calprotectin assay. Results were compared to those obtained on control devices (2-8°C).

Acceptance criteria: 80-120% average recovery.

All results obtained using the three different lots of Fecal Extraction Device fulfilled the acceptance criteria. In conclusion, the Fecal Extraction Device can be kept at room temperature up to 72 hours.

Clinical performance characteristics

Same as approved in 510k submission #K170993.