CalprestNG is a quantitative ELISA for detecting concentration of fecal calprotectin. CalprestNG can in vitro diagnostic to aid in the diagnosis of Inflammatory Bowel Diseases (IBD, Crohn's disease and ulcerative colitis) and to differentiate IBD from Irritable Bowel Syndrome (IBS) in conjunction with other clinical and laboratory findings.

Device Description

Calprest®NG is an enzyme-linked immunosorbent assay (ELISA) system with colorimetric detection based on the use of antibodies against calprotectin. Calprotectin present in the diluted sample is bound by the antibody adsorbed to the surface of the plastic well. The enzyme conjugated antibody binds to the captured antigen and subsequently the enzyme catalyzes the conversion of the substrate to a colored product. The intensity of the color is proportional to the amount of conjugate bound, and thus to the amount of captured calprotectin. Concentration of calprotectin in the samples is calculated using the provided standards.

EasyCal is a single-use device for stool sample pre-analytical processing that allows the extraction of calprotectin from the specific amount of collected fecal sample required to perform Eurospital's Calprest® and Calprest®NG assays.

The device consists of a tube, containing 2.8 ml of extraction solution, a stick shaped with seven grooves for collecting the sample. The upper end is made up by two components which can be removed by opposite rotations. The screw cap (white) connected to the shaped stick traps the sample excess and can be then removed by counter-clockwise rotation. Once the extraction procedure has been completed, the sample can be transferred to an automated ELISA instrumentation, placing it directly into the sample rack. EasyCal allows an easy, reliable and reproducible way to sample from primary containers and analyze the extract directly from the device, without the need to weight the stool sample.

AI/ML Overview

The document describes the CalprestNG device, an ELISA (enzyme-linked immunosorbent assay) for detecting fecal calprotectin, and its new pre-analytical processing accessory, EasyCal. The submission is for K191592, establishing substantial equivalence to the predicate device CalprestNG (K160447), which used manual extraction. The core of the study is to demonstrate that using EasyCal for sample extraction yields comparable results to the manual extraction method.

Here's an analysis of the acceptance criteria and the study proving the device meets them:

1. A table of acceptance criteria and the reported device performance:

The document primarily focuses on demonstrating the comparability of CalprestNG with EasyCal to CalprestNG with manual extraction, rather than predefined quantitative acceptance criteria with specific ranges. Instead, the "acceptance criteria" are implied by showing the comparability of the new method to the predicate method.

However, we can infer the performance metrics tested and their results, which serve as evidence of acceptance.

Table 1. Method Comparison and Performance

Performance Metric	Acceptance Criteria (Implied/Study Goal)	Reported Device Performance (CalprestNG with EasyCal vs. CalprestNG Manual Extraction)
Method Comparison (Quantitative)	Slope and Y-intercept close to 1 and 0 respectively; high correlation (r); low bias at the clinical cut-off (120 mg/kg).	Passing-Bablok Regression:- Slope (95% CI): 0.9778 (0.901 to 1.003) - Y-intercept (95% CI): 0.8495 (-0.8360 to 3.978) - Correlation (r): 0.954 - Bias at 120 mg/kg (95% CI): -1.5% (-9.0% to 1.5%)
Qualitative Agreement	High agreement (Negative, Positive, and Total Agreement) when classifying samples based on a clinical cut-off.	Referencing 120 mg/kg cut-off (borderline as positive/negative based on interpretation):- Borderline as positive: - Negative Agreement (95% CI): 96.2% (81.1 to 99.3%) - Positive Agreement (95% CI): 100.0% (95.1 to 100%) - Total Agreement (95% CI): 99.0% (94.6 to 99.8%) - Borderline as negative: - Negative Agreement (95% CI): 100.0% (90.1 to 100%) - Positive Agreement (95% CI): 95.4% (87.3 to 98.4%) - Total Agreement (95% CI): 97.0% (91.5 to 99.0%)
Stool Sample Collection Performance	Maintain consistent and appropriate sample weight.	Mean weight collected by EasyCal: 56 mg. (This is a specific internal metric, the acceptance criteria implicitly being that this weight is consistent and adequate for the assay).
Extraction Reproducibility	Low coefficient of variation (CV%) for within-run, between-day, within-operator, between-operator, and within-laboratory precision.	CV% for 8 samples across quantification range (average or example values presented): Representative CVs range from 3.8% to 6.9% for repeatability, and overall within-laboratory CVs up to 18.3%. (Specific ranges were not given as 'criteria', but the detailed table data demonstrates the measured precision). The study concludes: "Reproducibility of results obtained with Calprest® with EasyCal is confirmed."
Extracted Sample Stability	Extracted samples should remain stable at specific temperatures and after freeze/thaw cycles for defined durations.	Samples met acceptance criteria for: - 2-8 °C up to 21 days (recommendation: up to 14 days) - Room temperature up to 73 hours (recommendation: up to 72 hours) - Up to 5 freeze/thaw cycles (recommendation: not to exceed 4 cycles)
EasyCal Device Stability (Shelf Life)	pH and volume stability over time; no adverse effect on CalprestNG performance.	pH and Volume: Between 7.77 and 7.84 (mean 7.80) for pH; 99.4% to 100.6% (mean 100.0%) for volume variation up to 25 months. All met acceptance criteria. CalprestNG performance: All acceptance criteria met at 25 months. Device stable for 24 months at 4 °C and does not affect CalprestNG performance.
EasyCal Device Stability (Room Temp.)	Maintain performance when stored at room temperature for specified duration.	All acceptance criteria were met. Device stable for 72 hours at room temperature and doesn't affect CalprestNG performances.

2. Sample sizes used for the test set and the data provenance

Method Comparison (Stool extraction method comparison: EasyCal vs manual extraction procedure):
- Sample Size: One hundred (100) stool samples.
- Data Provenance: Not explicitly stated, but clinical studies for such devices typically involve prospective collection or use of banked samples from clinical sites. The document implies these are "patient samples" (from CLSI EP09c reference). Country of origin is not specified.
- Retrospective/Prospective: Not explicitly stated for this particular sample set, but device validation studies often use a mix. Given the "CLSI EP09c" reference, it points to a formal comparison study, which could be prospective collection for the purpose of the study.
Reproducibility study: Extraction Reproducibility:
- Sample Size: Eight (8) stool samples (tested in replicate of five, once a day, for five days, by three different operators). This results in 8 samples * 5 replicates * 5 days * 3 operators = 600 total test points.
- Data Provenance: Not specified.
- Retrospective/Prospective: Not specified.
Stool sample collection performance of EasyCal:
- Sample Size: Five (5) different human stool samples. Each collected in replicates of five by three independent operators (5 samples * 5 replicates * 3 operators = 75 total weight measurements).
- Data Provenance: Not specified.
- Retrospective/Prospective: Not specified.
Samples stability and handling:
- Sample Size: Eight (8) stool samples.
- Data Provenance: Not specified.
- Retrospective/Prospective: Not specified.
EasyCal device stability (Shelf life and Room Temperature):
- Sample Size: Three lots of EasyCal devices were used for pH/volume tests. Twelve (12) samples were extracted for performance validation with aged vs. fresh devices. Eight (8) stool samples were used for room temperature storage validation.
- Data Provenance: Not specified.
- Retrospective/Prospective: Real-time stability studies are inherently prospective observations over time.

3. Number of experts used to establish the ground truth for the test set and the qualifications of those experts (e.g. radiologist with 10 years of experience)

This device is an in vitro diagnostic (IVD) quantitative assay for fecal calprotectin. The "ground truth" for such devices is established through:

Referenced methods: The manual extraction method of the predicate device (K160447) served as the reference or comparative "truth" for the EasyCal method. The study aimed to show equivalence to this established method.
Known concentrations: For reproducibility and stability studies, often spiked samples or samples with predetermined concentrations are used, verified by validated analytical methods.
Clinical correlation (indirect): The output of the assay (Calprotectin concentration) is correlated with clinical findings (IBD vs. IBS diagnosis). However, the direct clinical diagnostic "ground truth" (e.g., patient biopsy, endoscopy results) is not used within this specific analytical validation section to establish the "truth" for the quantitative assay itself. The clinical performance characteristics are stated to be "Same as approved in 510(k) submission # K160447," implying that clinical utility was established previously, and this submission focuses on analytical equivalence.

Therefore, human experts in the context of "ground truth establishment" for an image analysis or diagnostic interpretation task (like radiologists for imaging) are not directly applicable here. The "ground truth" is the precise measurement of calprotectin or the established manual extraction benchmark.

4. Adjudication method (e.g. 2+1, 3+1, none) for the test set

Adjudication methods like 2+1 or 3+1 are typically used in studies where human readers/experts independently interpret complex data (e.g., medical images), and a consensus or adjudication process is needed to resolve discrepancies and establish a robust ground truth for classification tasks.

This study is an analytical performance study of a quantitative in vitro diagnostic (IVD) device. The "truth" is either the measured concentration by a reference method or the established manual extraction procedure. Therefore, no adjudication method involving multiple human readers/experts is pertinent or mentioned for establishing the "ground truth" of the test set in this context.

5. If a multi reader multi case (MRMC) comparative effectiveness study was done, If so, what was the effect size of how much human readers improve with AI vs without AI assistance

No MRMC comparative effectiveness study was done or is relevant here. This is a submission for an IVD device measuring a biomarker (fecal calprotectin), not an AI-assisted diagnostic imaging or interpretation tool for human readers. There are no "human readers" interpreting the output in a way that would be "improved with AI assistance." The technology is a laboratory assay.

6. If a standalone (i.e. algorithm only without human-in-the-loop performance) was done

This is an analytical device, and its performance is inherently "standalone" in terms of its measurement capabilities. The CalprestNG ELISA, whether with manual or EasyCal extraction, provides a quantitative result for fecal calprotectin. There isn't an "algorithm" in the sense of AI or machine learning that processes images or complex data for interpretation. The "algorithm" is the biochemical assay itself, which delivers a numerical value.

The performance studies (method comparison, reproducibility) directly evaluate this standalone performance. The "human-in-the-loop" aspect relates to standard laboratory practices (performing the assay, pipetting, reading results from the instrument), but not an assistive AI.

7. The type of ground truth used (expert consensus, pathology, outcomes data, etc)

The "ground truth" for the test set in this analytical validation is established by:

Reference Method Comparison: The results obtained via the CalprestNG assay with the manual extraction procedure (K160447 predicate device) served as the primary reference method to which the new EasyCal extraction method was compared for equivalence.
Known samples/standards: For reproducibility and stability, samples with known or well-characterized concentrations of calprotectin (likely established using a highly accurate and precise method) are typically used.
Internal standards and controls: The assay uses calibrators (0, 2.5, 12.5, 25, 50, 150 ng/ml) to quantify calprotectin concentration, which act as internal "ground truth" for the assay's operational range.

8. The sample size for the training set

This document describes the validation of an in vitro diagnostic assay kit, not a machine learning or AI model that requires a "training set" to learn from data. Therefore, the concept of a "training set" in the AI/ML sense is not applicable here. The assay is based on established biochemical principles (ELISA) and does not "learn" from data in the way an algorithm does.

9. How the ground truth for the training set was established

As explained above, there is no "training set" in the context of AI/ML for this device. The "ground truth" for the assay's core function is inherent in its biochemical design, the use of validated reagents, and the calibration curves established with known standards. The validation studies described in the document (method comparison, reproducibility, stability) assess the performance of the final manufactured product against predefined analytical metrics and comparison to a predicate device.

Summary

{0}------------------------------------------------

Image /page/0/Picture/0 description: The image shows the logo for the U.S. Food & Drug Administration (FDA). The logo consists of two parts: a symbol on the left and the FDA name on the right. The symbol is a stylized representation of a human figure, while the FDA name is written in blue and includes the words "U.S. Food & Drug Administration".

July 10, 2019

Eurospital S.p.A. Chiara Pelillo Regulatory Affairs Assistant Via Flavia 122 Trieste. 34147 Italy

Re: K191592

Trade/Device Name: CalprestNG, EasyCal Regulation Number: 21 CFR 866.5180 Regulation Name: Fecal Calprotectin Immunological Test System Regulatory Class: Class II Product Code: NXO Dated: June 11, 2019 Received: June 14, 2019

Dear Chiara Pelillo:

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/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. Branch Chief Immunology and Flow Cytometry Branch 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

{2}------------------------------------------------

Indications for Use

510(k) Number (if known) K191592

Device Name CalprestNG

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)

CONTINUE ON A SEPARATE PAGE IF NEEDED.

This section applies only to requirements of the Paperwork Reduction Act of 1995.

DO NOT SEND YOUR COMPLETED FORM TO THE PRA STAFF EMAIL ADDRESS BELOW.

The burden time for this collection of information is estimated to average 79 hours per response, including the time to review instructions, search existing data sources, gather and maintain the data needed and complete and review the collection of information. Send comments regarding this burden estimate or any other aspect of this information collection, including suggestions for reducing this burden, to:

Department of Health and Human Services Food and Drug Administration Office of Chief Information Officer Paperwork Reduction Act (PRA) Staff PRAStaff@fda.hhs.gov

"An agency may not conduct or sponsor, and a person is not required to respond to, a collection of information unless it displays a currently valid OMB number."

{3}------------------------------------------------

Image /page/3/Picture/1 description: The image shows the word "Eurospital" in a blue, sans-serif font. To the right of the word is a graphic of several curved lines in shades of blue and green. The lines appear to be intersecting and overlapping each other, creating a sense of movement and flow.

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

1. Administrative data

Submitter:	Eurospital S.p.A.
Submitter'sAddress:	Via Flavia, 12234147 TriesteItaly
Submitter'scontact:	Chiara PelilloRegulatory Affairs SpecialistPhone: 0039 0408997266Fax: 0039 040280944Email: cpelillo@eurospital.it
Device name:	Proprietary name: Calprest®NGCommon name: Fecal Calprotectin enzyme-linked immunosorbent assay with EasyCal
RegulationDescription:	Fecal Calprotectin Immunological TestSystem
Regulation MedicalSpecialty	Immunology
Review Panel	Immunology
Product Code	NXO
Regulation Number	21 CFR 866.5180
Device Class	2
Date of 510(k)Preparation:	June 11th, 2019

{4}------------------------------------------------

Image /page/4/Picture/1 description: The image shows the Eurospital logo. The word "Eurospital" is written in a blue sans-serif font. To the right of the word is a graphic of curved lines in shades of blue and green. The lines appear to be flowing or radiating outward.

2. Predicate device

Calprest®NG (using manual extraction), 510 (k) number K160447. Date of clearance: November 10th, 2016.

3. Device description

The EasyCal kit provides the materials listed below:

One hundred (100) EasyCal kits. ●
Each tube of EasyCal contains:
2,8 mL of extraction buffer, colorless, ready to use.

{5}------------------------------------------------

Image /page/5/Picture/1 description: The image shows the Eurospital logo. The word "Eurospital" is written in a blue, sans-serif font. To the right of the word is a graphic of curved lines in blue and green. The lines appear to be intersecting.

4. Intended use and Indications for use

Calprest®NG is a quantitative ELISA for detecting concentration of fecal calprotectin. Calprest®NG can be used as an in vitro diagnostic to aid in the diagnosis of Inflammatory Bowel Diseases (IBD, Crohn's disease and ulcerative colitis) and to differentiate IBD from Irritable Bowel Syndrome (IBS) in conjunction with other clinical and laboratory findings.

5. Pre-Analytical Processing

The device object of the present submission is the same in terms of chemical composition, formulation, principle of operations to the device cleared in 2016 (K160447). The change is due to the introduction of the EasyCal, which is an optional single-use device for stool sample pre-analytical processing sold separately from Calprest®NG. It allows the extraction of calprotectin from the specific amount of collected fecal sample required to perform Eurospital's Calprest® and Calprest®NG calprotectin determination assays.

Substantial equivalence 6.

The new device and the predicate Device (K160447) have the same Intended Use/Indications for Use, same fundamental technological characteristics, principles of operation and comparable performances characteristics. The modifications consist in the addition of an optional accessory, the EasyCal device, which is a single-use device for stool sample pre-analytical processing as alternative to the manual extraction procedure. It allows the extraction of calprotectin from the specific amount of collected fecal sample required to perform Eurospital's Calprest® and Calprest®NG calprotectin determination assays.

As evidenced by Risk Assessment and Validation Studies, no new questions were raised regarding the Safety, Effectiveness, Performance, Indication for Use, Technology and the Principles of Operation. Therefore, Calprest®NG in combination with EasyCal as stool extraction device performs equivalently as the predicate device, Calprest®NG in combination with manual extraction procedure Device.

{6}------------------------------------------------

Image /page/6/Picture/1 description: The image shows the Eurospital logo. The logo is in blue and green. The word "Eurospital" is in blue, and there are blue and green lines to the right of the word.

7. Comparison to predicate device

7. Comparison to predicate deviceSimilarities
Item	New device	Predicate (K160447)
	Calprest®NG with EasyCal	Calprest®NG
Intended use	Calprest®NG is a quantitative ELISA for detecting concentration of fecal calprotectin. Calprest®NG can be used as an in vitro diagnostic to aid in the diagnosis of Inflammatory Bowel Diseases (IBD, Crohn's disease and ulcerative colitis) and to differentiate IBD from Irritable Bowel Syndrome (IBS) in conjunction with other clinical and laboratory findings.	Calprest®NG is a quantitative ELISA for detecting concentration of fecal calprotectin. Calprest®NG can be used as an in vitro diagnostic to aid in the diagnosis of Inflammatory Bowel Diseases (IBD, Crohn's disease and ulcerative colitis) and to differentiate IBD from Irritable Bowel Syndrome (IBS) in conjunction with other clinical and laboratory findings.
Assay methodology	ELISA	Same
Antigen	Calprotectin	Same
Conjugate	Horseradish peroxidase	Same
Shelf life	18 months	Same
Sample type	Extracted human stool	Same
Sample dilution	1:20000	Same
Sample units	mg/kg (mg of calprotectinper kg of stool)	Same
Detection/ operating principle	Colorimetric assay	Same
Analytical Measuring Range	27.1 - 3000 mg/kg	Same
Calibration	6 calibrators: 0, 2.5, 12.5, 25, 50, 150 ng/ml	Same
Differences
Item	New device	Predicate (K160447)
	Calprest®NG with EasyCal	Calprest®NG
Pre-analyticalprocessingprocedure	Manual extraction procedureorStool extraction procedureusing EasyCal, a tubecontaining 2.8 ml of extractionsolution and a stick shaped withseven grooves for collecting thesample.	Manual extractionprocedure.

{7}------------------------------------------------

Image /page/7/Picture/1 description: The image shows the logo for Eurospital. The word "Eurospital" is written in a sans-serif font in blue. To the right of the word is a graphic of curved lines in blue and green. The lines appear to be moving upward and outward.

{8}------------------------------------------------

Image /page/8/Picture/1 description: The image shows the word "Eurospital" in a bold, sans-serif font, colored in a light blue. To the right of the word is an abstract graphic consisting of curved lines in shades of blue and green. The lines appear to be layered and overlapping, creating a sense of depth and movement.

8. Analytical performance characteristics

Same as approved in 510(k) submission #K160447 plus the following additional data.

8.1. Stool extraction method comparison: EasyCal vs manual extraction procedure

One hundred (100) stool samples containing different levels of calprotectin evenly distributed and covering the quantification range of Calprest®NG, were extracted in parallel with the EasyCal and using the manual extraction procedure. All the extracted stool samples were tested in duplicate for calprotectin concentration using the Calprest®NG assay according to the package insert. The following parameters were analyzed with Passing-Bablok regression and Analyse-it software: intercept, slope, predicted bias at cut-off, 95% CI, correlation index (r). The study was conducted according to the CLSI EP09c "Measurement Procedure Comparison and Bias Estimation Using Patient Samples, 3rd Edition" (June 2018).

The results are summarized in the table below:

Table 1. Method comparison Calprest®NG with EasyCal (y) versus Calprest®NG with manual extraction procedure (x)

Passing-Bablok regression	Calprest®NG with EasyCal = 0.8495 +0.9778 Calprest®NG manual weight
Slope (95% CI)	0.9778	(0.901 to 1.003)
Y-intercept (95% CI)	0.8495	(-0.8360 to 3.978)
Correlation - r	0.954
Bias at 120 mg/kg (95% CI)	-1.5%	(-9.0% to 1.5%)

Qualitative agreement Calprest NG with EasyCal versus Calprest NG withmanual weight
Calprest NG with EasyCal
Borderline	Negative	Positive	Total
9	0	0	9
1	25	0	26
3	0	62	65
13	25	62	100
Borderline as positive
Negative Agreement (95% CI)		96.2% (81.1 to 99.3%)
Positive Agreement (95% CI)		100.0% (95.1 to 100%)
Total Agreement (95% CI)		99.0% (94.6 to 99.8%)
Borderline as negative
Negative Agreement (95% CI)		100.0% (90.1 to 100%)
Positive Agreement (95% CI)		95.4% 87.3 to 98.4%)
Total Agreement (95% CI)		97.0% (91.5 to 99.0%)

The results demonstrate that the performances of Calprest®NG with EasyCal is comparable to Calprest®NG with manual extraction procedure.

{9}------------------------------------------------

Image /page/9/Picture/1 description: The image shows the Eurospital logo. The word "Eurospital" is written in a blue, sans-serif font. To the right of the word is a graphic of curved lines in shades of blue and green. The lines appear to be stylized representations of waves or wind.

Stool sample collection performance of EasyCal 8.2.

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

The results obtained confirm that the stool sample mean weight collected by EasyCal is 56 mg.

Reproducibility study: Extraction Reproducibility 8.3.

Eight (8) stool samples covering the quantification range of Calprest NG assay were extracted and tested in replicate of five, once a day, for five days, by three different operators (scheme 5x5x3).

CV(%) and standard deviation were calculated for each set of analysis performed with Analyse-it software.

Faecal extracts tested with Calprest® NG
			Repeatability		Between-Day		Within Operator		BetweenOperator		WithinLaboratory
Sample ID	N	Mean(mg/kg)	SD(mg/kg)	CV(%)	SD(mg/kg)	CV (%)	SD(mg/kg)	CV (%)	SD(mg/kg)	CV (%)	SD(mg/kg)	CV (%)
Sample1	75	32.61	2.03	6.2%	0.76	2.3%	2.16	6.6%	0.69	2.1%	2.27	7.0%
Sample2	75	57.77	2.22	3.8%	5.10	8.8%	5.56	9.6%	0.51	0.9%	5.59	9.7%
Sample3	75	232.86	6.40	2.7%	26.02	11.2%	26.79	11.5%	9.61	4.1%	28.47	12.2%
Sample4	75	335.40	10.48	3.1%	47.95	14.3%	49.08	14.6%	27.56	8.2%	56.29	16.8%
Sample5	75	529.88	18.46	3.5%	95.44	18.0%	97.21	18.3%	0.00	0.0%	97.21	18.3%
Sample6	75	871.67	28.92	3.3%	44.80	5.1%	53.32	6.1%	50.52	5.8%	73.46	8.4%
Sample7	75	1284.61	70.34	5.5%	87.01	6.8%	111.89	8.7%	0.00	0.0%	111.89	8.7%
Sample8	75	2539.14	174.85	6.9%	75.74	3.0%	190.55	7.5%	17.25	0.7%	191.33	7.5%

The study was conducted according to the CLSI EP05A3 "Evaluation of Precision of Quantitative Measurement Procedures, 3rd Edition" (October 2014)

Reproducibility of results obtained with Calprest® with EasyCal is confirmed.

8.4. Samples stability and handling

Eight (8) stool samples covering the quantification range of Calprest NG assay were extracted with EasyCal and tested, using Calprest®NG assay, for stability of the extracted stool sample at 2-8 °C up to 21 days (day 0, day 1, day 3, day 7, day 14 and day 21), at room temperature up to 73h (0h, 16h, 24h, 72h and 73h) and after up to 5 freeze/thaw cycles (0 cycle, 1 cycle, 2 cycles, 4 cycles and 5 cycles). Data related to the extracted stool samples were analyzed compared to those obtained at time zero/cycle zero and the percentage of recovery was calculated.

{10}------------------------------------------------

Image /page/10/Picture/1 description: The image shows the Eurospital logo. The word "Eurospital" is written in a sans-serif font in blue. To the right of the word is a graphic of curved lines in shades of blue and green. The lines are arranged in a fan-like shape.

All samples met the acceptance criteria at 21 days, 73 hours and after five cycle of freeze-thaw, therefore, the extracted stool samples in the EasyCal can be stored: up to 14 days at 2-8 °C and up to 72h at RT. Even though after five freeze-thaw cycles all samples met the acceptance criteria, we recommend not to exceed 4 freeze-thaw cycles to remain in a safety stability condition.

8.5. EasyCal device stability

8.5.1. Shelf life (Real time stability study)

To evaluate the real time stability of EasyCal, pH values for the extraction buffer were assessed and variation of the volume (evaporation) was verified by weighting the devices at time zero and up to 25 months (0; 7; 13; 19; 24 and 25 months) on three lots. Data were compared to those obtained at time zero.

The pH measures were between 7.77 and 7.84 (mean 7.80) and volume variation expressed in percentage was between 99.4% and 100.6% (mean 100.0%); all measures met the acceptance criteria up to 25 months.

Additionally, to confirm that EasyCal stability guarantees no changes in Calprest®NG's performances, twelve (12) samples were extracted with three lots of EasyCal stored at 2-8 °C up to 25 months (24 and 25 months) and with a freshly produced one for comparison. The extracted stool samples were tested using Calprest®NG assay.

All acceptance criteria were met at 25 months, therefore the EasyCal device is stable for 24 months when kept at 4 °C and does not affect Calprest®NG's performances.

8.5.2. Calprest®NG performances with EasyCal stored at room temperature

Eight (8) stool samples, covering the quantification range of Calprest®NG assay, were extracted with three different lots of EasyCal stored at 2-8 °C or at room temperature up to 73h and tested using Calprest®NG assay. The results obtained with the EasyCal stored at room temperature were compared to those obtained with the EasyCal stored at 2-8 °C and the percentage of recovery calculated.

All acceptance criteria were met, therefore, the device is stable for 72h at room temperature and doesn't affect Calprest®NG performances.

9. Clinical performance characteristics

Same as approved in 510(k) submission # K160447

10. Conclusions

The Calprest®NG, with EasyCal is a modification of existing and legally marketed IVD device Calprest®NG. The modification consist in the introduction of a stand-alone optional accessory, the EasyCal, which is a single-use device for stool sample preanalytical processing. It allows the extraction of calprotectin from the specific amount of collected fecal sample required to perform Eurospital's Calprest®NG calprotectin determination assays. This modification does not affect their intended use either the technological characteristics of the cleared Calprest®NG assay (K160447).

{11}------------------------------------------------

Image /page/11/Picture/1 description: The image shows the logo for Eurospital. The word "Eurospital" is written in a sans-serif font in blue. To the right of the word is a graphic of curved lines in blue and green. The lines appear to be intersecting each other.

The design validation of Calprest®NG plus EasyCal has been performed following same standards and verification-validation testing already approved for Calprest®NG.

The IVD medical device design validation revealed compliant and without affecting the requirements of safety and effectiveness of Calprest®NG.

Therefore, the Calprest®NG plus EasyCal is substantially equivalent to the cleared Calprest®NG (K160447).

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).