LIAISON Calprotectin, LIAISON Calprotectin Control Set, LIAISON Calprotectin Calibration Verifiers, LIAISON Q.S.E.T. Buffer, LIAISON Q.S.E.T. Device

K182698 · DiaSorin, Inc. · NXO · Dec 26, 2018 · Immunology

Device Facts

• Record ID: K182698
• Device Name: LIAISON Calprotectin, LIAISON Calprotectin Control Set, LIAISON Calprotectin Calibration Verifiers, LIAISON Q.S.E.T. Buffer, LIAISON Q.S.E.T. Device
• Applicant: DiaSorin, Inc.
• Product Code: NXO · Immunology
• Decision Date: Dec 26, 2018
• Decision: SESE
• Submission Type: Traditional
• Regulation: 21 CFR 866.5180
• Device Class: Class 2
• Attributes: Pediatric

Indications for Use

The DiaSorin LIAISON® Calprotectin assay is an in vitro diagnostic chemiluminescent immunoassay (CLIA) intended for the quantitative measurement, in human stool, of fecal calprotectin, a neutrophilic protein that is a marker of mucosal inflammation. The LIAISON® Calprotectin assay can be used 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 (IBS). Test results are to be used in conjunction with information obtained from the patients' clinical evaluation and other diagnostic procedures. The test has to be performed on the LIAISON® XL Analyzer. The DiaSorin LIAISON® Calprotectin Control Set is intended for use as assayed quality control samples to monitor the performance of the LIAISON® Calprotectin assay. The performance characteristics of the LIAISON® Calprotectin Control Set have not been established for any other assay or instrument platforms different from the LIAISON® XL Analyzer. The DiaSorin LIAISON® Calprotectin Calibration Verifiers are assayed quality control materials intended for in vitro diagnostic use in the quantitative verification of calibration and reportable range of the LIAISON® Calprotectin assay when performed on the LIAISON® XL Analyzer. The LIAISON® Q.S.E.T. Buffer (Quantitative Stool Extraction and Test) is intended for use in the preparation of human stool specimens for testing in designated DiaSorin stool assays. The DiaSorin LIAISON® Q.S.E.T. (Quantitative Stool Extraction and Test) is intended for use in the preparation of human stool specimens for testing in the LIAISON® Calprotectin assay.

Device Story

System consists of LIAISON Calprotectin assay reagents and optional Q.S.E.T. extraction device; used on LIAISON XL Analyzer. Input: human stool samples; processed via manual weight normalization or Q.S.E.T. device. Principle: quantitative sandwich chemiluminescent immunoassay (CLIA); paramagnetic microparticles coated with monoclonal mouse anti-human calprotectin capture antibodies; isoluminol-labeled detection antibodies. Analyzer automates incubation, washing, and light measurement. Output: quantitative calprotectin concentration (μg/g). Used in clinical laboratories by trained personnel. Results interpreted as normal (<50 μg/g), borderline (50–120 μg/g), or elevated (>120 μg/g). Output aids clinicians in diagnosing IBD and differentiating from IBS in conjunction with clinical evaluation.

Clinical Evidence

Prospective clinical study (n=240) evaluated performance in patients undergoing endoscopy for IBD/IBS symptoms. IBD diagnosis confirmed via endoscopic scores; IBS via Rome III criteria. Sensitivity for IBD was 98.0% (borderline as elevated) or 88.2% (borderline as normal). Specificity for IBD vs non-IBD was 66.8% (borderline as elevated) or 90.6% (borderline as normal). Reference range study (n=127) established normal values in healthy individuals.

Technological Characteristics

Solid-phase chemiluminescent immunoassay (CLIA). Magnetic particles coated with mouse monoclonal antibodies. Isoluminol-conjugated monoclonal antibody detection. Analytical measuring range: 5-800 µg/g. Automated on LIAISON XL Analyzer. Reagents stored at 2-8°C. Q.S.E.T. device provides standardized stool sampling (10.5 mg ± 0.1 mg).

Indications for Use

Indicated for quantitative measurement of fecal calprotectin in human stool as an aid in the diagnosis of inflammatory bowel diseases (IBD), specifically Crohn's disease and ulcerative colitis, and to differentiate IBD from irritable bowel syndrome (IBS). For use in patients with signs and symptoms suggestive of IBD or IBS.

Regulatory Classification

Identification

A fecal calprotectin immunological test system is an in vitro diagnostic device that consists of reagents used to quantitatively measure, by immunochemical techniques, fecal calprotectin in human stool specimens. The device is intended for in 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.

Special Controls

The device is classified as Class II under regulation 21 CFR 866.5180 with special controls. The special control guidance document, "Class II Special Controls Guidance Document: Fecal Calprotectin Immunological Test Systems" will be available shortly.

*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).

Predicate Devices

• PhiCal™ Test Fecal Calprotectin Immunoassay (DEN060001)

Submission Summary (Full Text)

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# 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION MEMORANDUM

A. 510(k) Number:
K182698

B. Purpose for Submission:
New device

C. Measurand:
Calprotectin

D. Type of Test:
Quantitative, automated immunoassay

E. Applicant:
DiaSorin, Inc.

F. Proprietary and Established Names:
LIAISON Calprotectin
LIAISON Q.S.E.T. Device

G. Regulatory Information:

1. Regulation section:
21CFR §866.5180 – Fecal Calprotectin Immunological Test

2. Classification:
Class II

3. Product code:
NXO – Calprotectin, Fecal

4. Panel:
Immunology (82)

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H. Intended Use:

1. Intended use:

The DiaSorin LIAISON Calprotectin assay is an in vitro diagnostic chemiluminescent immunoassay (CLIA) intended for the quantitative measurement, in human stool, of fecal calprotectin, a neutrophilic protein that is a marker of mucosal inflammation. The LIAISON Calprotectin assay can be used 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 (IBS). Test results are to be used in conjunction with information obtained from the patients' clinical evaluation and other diagnostic procedures. The test has to be performed on the LIAISON XL Analyzer.

The DiaSorin LIAISON Q.S.E.T. Device (Quantitative Stool Extraction and Test) is intended for use in the preparation of human stool specimens for testing in the LIAISON Calprotectin assay.

2. Indications for use:

Same as Intended Use

3. Special conditions for use statement:

For prescription use only

4. Special instrument requirements:

LIAISON XL Analyzer (K181464)

I. Device Description:

The device is an IVD reagent system consisting of:

- paramagnetic capture particles coated with monoclonal mouse antibodies against human calprotectin
- conjugated monoclonal mouse anti-human calprotectin detection antibodies, labeled with an isoluminol derivative
- Q.S.E.T. sample extraction buffer
- sample diluent
- wash buffer
- calibrators and controls

- controls: two levels of calprotectin, low (50 µg/g) and high (250 µg/g)
- calibrators: two levels of recombinant calprotectin, low (28.5 µg/g) and high (451 µg/g)
- calibration verifiers: four levels of recombinant calprotectin, 30–505 µg/g

- (optional) Q.S.E.T. device: a probe and container, for sampling and extraction of fecal samples

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J. Substantial Equivalence Information:

1. Predicate device name:
Genova Diagnostics PhiCal Test

2. Predicate 510(k) number:
DEN060001/K050007

3. Comparison with predicate:
| Similarities | | |
| --- | --- | --- |
| Item | Device | Predicate |
| Intended Use | The DiaSorin LIAISON Calprotectin assay is an in vitro diagnostic chemiluminescent immunoassay (CLIA) intended for the quantitative measurement, in human stool, of fecal calprotectin, a neutrophilic protein that is a marker of mucosal inflammation. The LIAISON Calprotectin assay can be used 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 (IBS). Test results are to be used in conjunction with information obtained from the patients' clinical evaluation and other diagnostic procedures. The test has to be performed on the LIAISON XL Analyzer. The DiaSorin LIAISON Q.S.E.T. Device (Quantitative Stool Extraction and Test) is intended for use in the preparation of human stool specimens for testing in the LIAISON Calprotectin assay. | The PhiCal test is a quantitative ELISA for measuring, in human stool, concentrations of fecal calprotectin, a neutrophilic protein that is a marker of mucosal inflammation. The PhiCal test 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. |
| Assay Methodology | Solid-phase (heterogeneous) immunoassay | Same |
| Assay Output | Quantitative | Same |
| Measurand | Human calprotectin | Same |
| Antigen | Recombinant human calprotectin | Same |
| Interpretation | Normal: <50 μg/g | Same |

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| Similarities | | |
| --- | --- | --- |
| Item | Device | Predicate |
| | Borderline: 50 – 120 μg/g
Elevated: >120 μg/g | |
| Differences | | |
| --- | --- | --- |
| Item | Device | Predicate |
| Assay processing | Automated | Manual |
| Instrumentation | LIAISON XL Analyzer | Generic ELISA Reader |
| Pre-analytical sample processing | Manual weight normalization
-or-
Q.S.E.T device | Manual weight normalization |
| Solid-phase | Paramagnetic microparticles | 96-well polystyrene plate |
| Capture antibodies | Mouse monoclonal anti-human calprotectin | Rabbit polyclonal anti-human calprotectin |
| Detection antibodies | Mouse monoclonal anti-human calprotectin | Rabbit polyclonal anti-human calprotectin |
| Detection chemistry | Isoluminol chemiluminescence | Chromogenic alkaline phosphatase |
| Primary measurement units | Relative Light Units (RLU) | Optical Density (OD) |
| Analytical Measuring Range | 5.0 – 800.0 μg/g | 27.1 – 3000 μg/g |
| Extended Analytical Measuring Range | Automated 1:10 dilution: 800.0 – 8000 μg/g | n/a |

## K. Standard/Guidance Document Referenced (if applicable):

| Org | Std ID | Ver | Date | Title |
| --- | --- | --- | --- | --- |
| CLSI | EP05 | A3 | Sep 2014 | Evaluation of Precision Performance of Quantitative Measurement Methods |
| CLSI | EP06 | A | Apr 2003 | Evaluation of the Linearity of Quantitative Measurement Procedures |
| CLSI | EP07 | A2 | Dec 2002 | Interference Testing in Clinical Chemistry |
| CLSI | EP12 | A2 | Jan 2014 | User Protocol for Evaluation of Qualitative Test Performance |
| CLSI | EP15 | A3 | Sep 2014 | User Verification of Precision and Estimation of Bias |
| CLSI | EP17 | A2 | Jun 2012 | Evaluation of Detection Capability for Clinical Laboratory Measurement Procedure |
| CLSI | EP28 | A3c | Oct 2010 | Defining, Establishing and Verifying Reference Intervals in the Clinical Laboratory |

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L. Test Principle:

The assay is a quantitative sandwich immunoassay, in which capture antibodies, immobilized to the paramagnetic solid phase bind calprotectin from processed fecal samples. Washing eliminates non-specific interactions, allowing for specific detection by conjugate antibodies against calprotectin. Subsequent measurement of light from the bound, conjugated isoluminol is in proportion to the quantity of bound calprotectin. The LIAISON XL Analyzer automates the assay process steps including: incubations, washing, calibration, measurement, and analysis.

Pre-analytical processing of fecal samples is accomplished by either manual weight normalization, followed by homogenization in Q.S.E.T. buffer; or using the optional Q.S.E.T. device for unitized collection and homogenization.

Following measurement, the assay reports calprotectin concentrations in units of microgram-per-gram of starting fecal sample material (μg/g). Interpretation of fecal calprotectin concentration ([Cal]) is as follows:

| [Cal] (μg/g) | Interpretation |
| --- | --- |
| < 50 μg/g | Negative |
| 50 – 120 μg/g | Borderline |
| >120 μg/g | Positive |

M. Performance Characteristics (if/when applicable):

1. Analytical performance:

All analytical studies met the manufacturer's pre-specified acceptance criteria.

a. Precision/Reproducibility:

Within-laboratory Precision:

Six stool samples spanning the analytical measuring range (AMR) were selected for testing and stored frozen. Aliquots were extracted by manual weighing method on each day of testing. Testing was performed by 3 operators × 2 runs/day × 12 days × 2 replicates × 1 lot, for a total of 144 datapoints per sample. The results are summarized in the table below:

| Within-Laboratory Precision | | | | | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Sample ID | Mean [Cal] (μg/g) | Within Run | | Between Run | | Between Day | | Between Operator | | Total | |
| | | SD | %CV | SD | %CV | SD | %CV | SD | %CV | SD | %CV |
| 1 | 21.6 | 0.7 | 3.1% | 0.4 | 1.7% | 2.9 | 13.5% | 0.6 | 2.9% | 3.1 | 14.3% |
| 2 | 24.9 | 0.7 | 3.0% | 0.7 | 2.6% | 2.9 | 11.7% | 1.0 | 4.2% | 3.3 | 13.0% |
| 3 | 39.9 | 0.9 | 2.2% | 0.8 | 1.9% | 3.4 | 8.6% | 0.0 | 0.0% | 3.6 | 9.1% |
| 4 | 155 | 4.0 | 2.6% | 3.5 | 2.2% | 11.3 | 7.2% | 8.3 | 5.3% | 14.9 | 9.6% |
| 5 | 253 | 7.0 | 2.8% | 8.2 | 3.2% | 20.2 | 8.0% | 2.9 | 1.1% | 23.0 | 9.1% |

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| Within-Laboratory Precision | | | | | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Sample ID | Mean [Cal] (μg/g) | Within Run | | Between Run | | Between Day | | Between Operator | | Total | |
| | | SD | %CV | SD | %CV | SD | %CV | SD | %CV | SD | %CV |
| 6 | 639 | 15.9 | 2.5% | 25.3 | 4.0% | 39.2 | 6.1% | 21.0 | 3.3% | 53.6 | 8.4% |

## Multi-site Reproducibility:

Six stool samples, spanning the AMR, were selected for testing and stored frozen. Aliquots were extracted by manual weight method on each day of testing. Testing was performed by 3 sites × 2 operators/site × 1 run/day × 5 days × 6 replicates × 1 lot, for a total of 180 datapoints per sample. The results are summarized in the table below:

| Multi-site Reproducibility | | | | | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Sample ID | Mean [Cal] (μg/g) | Within Run | | Between Day | | Within Site | | Site-to-Site | | Total | |
| | | SD | %CV | SD | %CV | SD | %CV | SD | %CV | SD | %CV |
| 1 | 23.4 | 0.6 | 2.6% | 2.6 | 11.2% | 2.7 | 11.5% | 0.0 | 0.0% | 2.5 | 10.5% |
| 2 | 25.9 | 0.7 | 2.6% | 2.2 | 8.3% | 2.3 | 8.7% | 0.0 | 0.0% | 2.2 | 8.4% |
| 3 | 42.1 | 0.8 | 1.8% | 5.3 | 12.5% | 5.3 | 12.6% | 1.9 | 4.4% | 5.6 | 13.4% |
| 4 | 173 | 4.7 | 2.7% | 20.4 | 11.9% | 20.9 | 12.1% | 20.6 | 11.9% | 29.3 | 17.0% |
| 5 | 281 | 5.6 | 2.0% | 26.9 | 9.6% | 27.4 | 9.7% | 40.1 | 14.3% | 48.6 | 17.3% |
| 6 | 695 | 18.9 | 2.7% | 66.1 | 9.5% | 68.3 | 9.8% | 39.6 | 5.7% | 78.9 | 11.3% |

## Lot-to-Lot Reproducibility:

Six stool samples, spanning the AMR, were selected for testing and stored frozen. Aliquots were extracted by manual weight method on each day of testing. Testing was performed by 1 site × 1 instrument × 3 lots × 5 days × 6 replicates, for a total of 90 datapoints per sample. The results are summarized in the table below:

| Lot-to-Lot Reproducibility | | | | | |
| --- | --- | --- | --- | --- | --- |
| Sample ID | Mean [Cal] (μg/g) | Between Lot | | Total | |
| | | SD | %CV | SD | %CV |
| 1 | 27.6 | 2.5 | 9.0% | 4.5 | 16.4% |
| 2 | 31.7 | 2.5 | 7.8% | 4.1 | 12.9% |
| 3 | 53.0 | 3.0 | 5.7% | 13.0 | 24.5% |
| 4 | 179 | 11.3 | 6.3% | 18.3 | 10.2% |
| 5 | 288 | 16.7 | 5.8% | 33.8 | 11.7% |
| 6 | 743 | 62.2 | 8.4% | 88.3 | 11.9% |

## Q.S.E.T Extraction Device Validation:

Seven stool specimens, representing a range of qualitative consistency (Bristol stool form scale 2–6), were used to evaluate the reproducibility of sample weight collected by the Q.S.E.T. extraction device. Specimens were sampled with the Q.S.E.T.

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extraction device by three operators × five replicates per sample per operator. The Q.S.E.T. device was weighed before and after sampling; the sample weight (final weight – tare weight) was calculated for precision parameters, summarized in the table below:

| Precision of Q.S.E.T. Device sample weight | | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Sample ID | BSFS | Mean Weight (g) | Repeatability | | Between-Operator | | Total | |
| | | | SD | %CV | SD | %CV | SD | %CV |
| 1 | 2 | 10.7 | 0.39 | 3.6% | 0.28 | 2.6% | 0.52 | 4.9% |
| 2 | 3 | 10.4 | 0.39 | 3.7% | 0.18 | 1.7% | 0.48 | 4.7% |
| 3 | 4 | 10.3 | 0.25 | 2.5% | 0.12 | 1.1% | 0.32 | 3.1% |
| 4 | 4 | 10.3 | 0.56 | 5.4% | 0.25 | 2.4% | 0.70 | 6.8% |
| 5 | 4 | 10.7 | 0.42 | 3.9% | 0.37 | 3.4% | 0.59 | 5.5% |
| 6 | 5 | 10.6 | 0.03 | 2.6% | 0.21 | 2.0% | 0.37 | 3.5% |
| 7 | 6 | 10.6 | 0.15 | 1.4% | 0.42 | 4.0% | 0.40 | 3.8% |

Repeatability of sample collection weight was calculated across all seven samples, replicates, and operators. The sample weight collected by the Q.S.E.T. Extraction Device is described in the table below:

| Sample collection performance of Q.S.E.T. Device | |
| --- | --- |
| Mean Sample Weight | 10.5 mg |
| Median Sample Weight | 10.5 mg |
| Range | 9.0–12.0 mg |
| 95% CI | 10.4–10.6 mg |
| Std Dev | 0.51 mg |
| %CV | 4.9% |

## Q.S.E.T. Extraction Device Reproducibility:

Five stool samples spanning the AMR were each extracted a total of 10 times using the Q.S.E.T device over three days and tested in triplicate using one reagent lot. The results are summarized in the table below:

| Q.S.E.T. Extraction Device Reproducibility | | | | | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Sample ID | Mean [Cal] (μg/g) | Repeatability | | Between-Day | | Within-Operator | | Between-Operator | | Total | |
| | | SD | %CV | SD | %CV | SD | %CV | SD | %CV | SD | %CV |
| 1 | 21.9 | 0.64 | 2.9% | 3.18 | 14.5% | 3.23 | 14.8% | 0.95 | 4.3% | 3.05 | 13.9% |
| 2 | 26.2 | 0.81 | 3.1% | 1.95 | 7.4% | 2.09 | 7.9% | 1.84 | 7.0% | 2.64 | 10.1% |
| 3 | 38.6 | 1.43 | 3.7% | 4.73 | 12.2% | 4.90 | 12.7% | 1.34 | 3.5% | 4.62 | 12.0% |
| 4 | 166 | 6.14 | 3.7% | 9.11 | 5.5% | 10.7 | 6.5% | 8.14 | 4.9% | 12.8 | 7.7% |
| 5 | 297 | 11.3 | 3.8% | 28.6 | 9.6% | 30.4 | 10.2% | 6.59 | 2.2% | 28.4 | 9.6% |
| 6 | 505 | 18.6 | 3.7% | 59.4 | 11.8% | 61.8 | 12.2% | 34.4 | 6.8% | 65.5 | 13.0% |

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| Q.S.E.T. Extraction Device Reproducibility | | | | | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Sample ID | Mean [Cal] (μg/g) | Repeatability | | Between-Day | | Within-Operator | | Between-Operator | | Total | |
| | | SD | %CV | SD | %CV | SD | %CV | SD | %CV | SD | %CV |
| 7 | 2381 | 62.6 | 2.6% | 410.4 | 17.2% | 414.4 | 17.4% | 254.7 | 10.7% | 450.5 | 18.9% |
| 8 | 4140 | 126.8 | 3.1% | 309.2 | 7.5% | 330.1 | 8.0% | 228.9 | 5.5% | 377.1 | 9.1% |

b. Linearity/assay reportable range:

Linearity:

A pool of three stool extracts with endogenous calprotectin levels above the AMR was diluted into 13 intervals over the range of the AMR. Diluted samples were tested in triplicate, in random sequence, using one reagent lot. The results are summarized in the table below:

| Linearity | |
| --- | --- |
| [Cal] Range (μg/g) | 5.0 – 800 μg/g |
| Slope (95% CI) | 0.96 (0.93–0.99) |
| Y-Intercept (95% CI) | 4.14 (−13.82–22.1) |
| R² | 1.00 |

Extended Analytical Measuring Range (AMR):

The AMR for the assay was set as the linear range of 5.0 – 800 μg/g. An extended AMR for the LIAISON Calprotectin assay was evaluated using auto-dilution features incorporated into the LIAISON XL Analyzer. To evaluate the extended AMR (800-8000 μg/g), automated dilutions were compared to manual dilutions for three stool samples with calprotectin concentrations above 800 μg/g, extracted using the Q.S.E.T. device, and tested in triplicate. The results are summarized in the table below:

| Extended Analytical Measuring Range Dilution | | | | |
| --- | --- | --- | --- | --- |
| Sample ID | BSFS | 1:10 Dilution [Cal] (μg/g ± SD) | | % Difference |
| | | Manual | Automated | |
| 1 | 7 | 3646 ± 132 | 3827 ± 146 | 4.93% |
| 2 | 5 | 3807 ± 11.5 | 4127 ± 167 | 8.14% |
| 3 | 7 | 7557 ± 405 | 7990 ± 567 | 5.69% |

Hook Effect:

Antigen excess was evaluated by testing three stool extracts spiked with exogenous recombinant calprotectin to a final concentration of 100,000 μg/g. The spiked samples were serially diluted 1:10 to a final concentration of 10 μg/g. Dilutions, the undiluted

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spiked samples, and the unspiked native samples were tested in triplicate, using one reagent lot. No high-dose hook effect was observed for calprotectin concentrations up to 100,000 µg/g.

## Assay Recovery:

To evaluate recovery, five high concentration stool extracts and five low concentration stool extracts were tested neat. Recovery samples were prepared by mixing defined ratios of high and low samples (i.e. 2:1, 1:1, 1:2), and tested in five replicates using one reagent lot. Recovery was calculated as the observed calprotectin concentration, as a percent ratio of the expected calprotectin concentration. Mean recovery across all five sample pairs, replicates, and dilution ratios was 103% (± 3%).

c. Traceability, Stability, Expected values (controls, calibrators, or methods):

## Traceability:

Recognized international standards for calprotectin measurement are not available. The LIAISON calprotectin calibrators are referenced to an in-house standard preparation of recombinant calprotectin. Reagent integral lots include two calibrators to establish working curves based on 10-point master curves stored on the analyzer.

## Stability:

Independent evaluations of the stability of the LIAISON Calprotectin kit, the Q.S.E.T extraction buffer, calibrators, controls, calibration verifiers, and Q.S.E.T. device are summarized below.

| Kit Stability: LIAISON Calprotectin Test Components | | |
| --- | --- | --- |
| Component | Storage Conditions | Shelf Life |
| Kit | 2-8°C | 18 months |
| Q.S.E.T. Extraction Buffer | 2-8°C | 18 months |
| Calibration Curve | onboard instrument | 28 days |
| Open Kit | onboard instrument | 56 days |
| Open Q.S.E.T. Extraction Buffer | 2-8°C | 56 days |
| Reconstituted Calibrators | 2-8°C | 28 days |
| Reconstituted Calibrators | RT | 6 hours |
| Kit Control Materials | 2-8°C | 18 months |
| Reconstituted Control Materials | 2-8°C | 28 days |
| Reconstituted Control Materials | RT | 6 hours |
| Reconstituted Control Materials | -20°C | 8 weeks |
| Reconstituted Control Materials | freeze/thaw cycles | 4 cycles |
| Calibration Verifiers | 2-8°C | 18 months |
| Reconstituted Calibration Verifiers | 2-8°C | 28 days |
| Reconstituted Calibration Verifiers | RT | 6 hours |

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| Kit Stability: LIAISON Calprotectin Test Components | | |
| --- | --- | --- |
| Component | Storage Conditions | Shelf Life |
| Reconstituted Calibration Verifiers | −20°C | 8 weeks |
| Reconstituted Calibration Verifiers | freeze/thaw cycles | 4 cycles |

In addition, critical storage parameters for stool samples were evaluated for stability, using six samples. In addition to storing native, unextracted sample, extracts derived from the manual extraction method or the Q.S.ET. device were stored in different conditions, and evaluated. Data from these sample stability studies are summarized below:

| Sample Stability: Human Stool Samples tested by LIAISON Calprotectin | | |
| --- | --- | --- |
| Sample Extraction for Storage | Condition | Stability |
| none, native | Refrigerated: 2–8°C | 72 hours |
| none, native | Frozen: −20°C | 16 weeks |
| none, native | freeze/thaw cycles | 3 cycles |
| Manual | Refrigerated: 2–8°C | 7 days |
| Q.S.E.T. Device, without centrifugation | Refrigerated: 2–8°C | 6 hours |
| Q.S.E.T. Device, with centrifugation | Refrigerated: 2–8°C | 8 days |
| Manual | Room Temperature | 8 hours |
| Q.S.E.T. Device, without centrifugation | Room Temperature | 4 hours |

d. Detection limit:

Limit of Blank (LoB): Five blank extraction buffer samples were tested in duplicate in six runs × two operators/instruments × two lots. The LoB was calculated by the parametric method as the 95th percentile of a corrected, normal distribution on the mean and standard deviation. The greater value from two lots, calculated independently, was selected.

Limit of Detection (LoD): Four low stool samples were tested in duplicate in six runs over three days × two operators/instruments × two lots. The LoD was calculated by the parametric method, as the 95th percentile of a corrected, normal distribution on the mean and standard deviation, added to the LoB. The greater value from two lots, calculated independently, was selected.

Limit of Quantitation (LoQ): Nine low stool samples were tested in duplicate in six runs over three days × two operators/instruments × two lots. The LoQ was calculated by precision profile, calculated from the inverse power regression function intercepting a precision target of 20%. The greater value from two lots, calculated independently, was selected; a third lot was used for verification.

Values for detection capabilities are summarized in the table below:

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| Detection Limits for LIAISON Calprotectin | |
| --- | --- |
| LoB | 0.107 μg/g |
| LoD | 0.395 μg/g |
| LoQ | 0.400 μg/g |

e. Analytical specificity:

One stool sample extract, containing a calprotectin concentration of ~50 μg/g was tested in triplicate in the presence of each interferent or vehicle buffer, using one assay reagent lot. A total of four endogenous, six microbial, and 23 exogenous interferents were evaluated, each at a single concentration. Interference was calculated as a percent ratio of the observed calprotectin concentration to the expected calprotectin concentration.

Interference studies are summarized in the table below:

| Interference testing for LIAISON Calprotectin | | |
| --- | --- | --- |
| Interferent | Concentration | Interference |
| Endogenous interferents | | |
| Hemoglobin | 6.7 mg/mL | 1.6% |
| Mucin | 3.33 mg/mL | -4.0% |
| Palmitic acid | 1.3 mg/mL | -4.0% |
| S100A12 | 21 μg/mL | 0.3% |
| Stearic acid | 2.65 mg/mL | -4.5% |
| Microbial interferents | | |
| Escherichia coli | 1.2 × 10⁸ cfu | 3.0% |
| Klebsiella pneumonia | 1.2 × 10⁸ cfu | 4.0% |
| Salmonella enterica | 1.2 × 10⁸ cfu | -1.8% |
| Shigella boydii | 1.2 × 10⁸ cfu | -1.4% |
| Yersinia enterocolitica | 1.2 × 10⁸ cfu | -1.9% |
| Citrobacter freundii | 1.2 × 10⁸ cfu | 1.5% |
| Exogenous interferents | | |
| Barium sulfate | 5.0 mg/mL | -1.0% |
| Imodium AD | 6.67 mg/mL | 2.5% |
| Kaopectate | 0.87 mg/mL | 2.4% |
| Mylanta | 4.2 mg/mL | -1.9% |
| Pepto-Bismol | 0.87 mg/mL | 0.4% |
| Tums (CaCO₃) | 0.5 mg/mL | -0.8% |
| Polyethylene glycol 3350 | 79.05 mg/mL | -2.1% |
| Simethicone | 0.625 mg/mL | -0.4% |
| Lansoprazole (Prevacid) | 0.2 mg/mL | 2.2% |
| Omeprazole (Prilosec) | 0.5 mg/mL | -2.6% |
| Cimetidine (Tagamet) | 0.5 mg/mL | 1.9% |
| Ciprofloxacin | 1.25 mg/mL | -1.8% |

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| Interference testing for LIAISON Calprotectin | | |
| --- | --- | --- |
| Interferent | Concentration | Interference |
| Metronidazole | 12.5 mg/mL | −0.8% |
| Sulfamethoxazole | 1.6 mg/mL | 0.1% |
| Vancomycin HCl | 2.5 mg/mL | −1.3% |
| Azathioprine | 0.2 mg/mL | 1.2% |
| Mesalamine (5-ASA) | 5.0 mg/mL | 7.3% |
| Prednisone | 0.3 mg/mL | 0.3% |
| Provitamin A | 5.0 mg/mL | −0.6% |
| Vitamin C | 0.1 mg/mL | −0.2% |
| Vitamin D₃ | 0.1 μg/mL | −0.3% |
| Vitamin E | 0.3 mg/mL | −0.4% |

f. Assay cut-off:

| Assay cut-off | |
| --- | --- |
| Normal: | <50 μg/g |
| Borderline: | 50–120 μg/g |
| Elevated: | >120 μg/g |

2. Comparison studies:

a. Method comparison with predicate device:

Clinical stool samples (n=182) were collected prospectively and tested in in-singlicate using the LIAISON calprotectin assay and the predicate device, tested in duplicate according to the predicate instructions for use. Samples above (n=16) or below (n=2) the AMR for either respective assay were excluded. Results of the Passing-Bablok regression of the comparison are summarized in the table below:

| Method Comparison: Passing-Bablok regression | |
| --- | --- |
| [Cal] Range (μg/g) | 27.1 – 800 μg/g |
| Slope (95% CI) | 0.97 (0.89 – 1.00) |
| Y-Intercept (95% CI) | 1.50 (−1.55 – 5.40) |
| R² | 0.96 |

In addition, qualitative interpretative agreement between the two assays was compared.

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| | Method Comparison: Qualitative agreement | | | | |
| --- | --- | --- | --- | --- | --- |
| | | Predicate | | | Totals |
| | | Abnormal | Borderline | Normal | |
| LIAISON Calprotectin | Elevated | 90 | 4 | 0 | 94 |
| | Borderline | 2 | 25 | 4 | 34 |
| | Normal | 0 | 4 | 32 | 36 |
| | Totals | 92 | 36 | 36 | 164 |
| Borderline results considered elevated/abnormal (95% CI) | | | |
| --- | --- | --- | --- |
| PPA: | 124/128 | 96.9% | (92.2–99.1%) |
| NPA | 32/36 | 88.9% | (73.9–96.9%) |
| Borderline results considered normal (95% CI) | | | |
| PPA: | 90/92 | 97.8% | (92.4–99.7%) |
| NPA: | 68/72 | 94.4% | (86.3–98.5%) |

The sponsor evaluated the accuracy of the Q.S.E.T. extraction device, comparing calprotectin values for the LIAISON calprotectin assay obtained by the manual weight extraction method, to LIAISON calprotectin values for the same 128 samples, selected to represent the AMR, obtained using the Q.S.E.T. device. Extracts were tested in singlicate using one reagent lot. Results were evaluated by linear regression by the method of Passing and Bablok, summarized in the table below:

| Q.S.E.T. Device Accuracy: Passing-Bablok regression | |
| --- | --- |
| [Cal] Range (μg/g) | 5.31 – 744 μg/g |
| Slope (95% CI) | 0.96 (0.92 – 1.02) |
| Y-Intercept (95% CI) | -1.12 (-2.81 – 0.60) |
| R² | 0.97 |
| Bias (at 50 μg/g, w/95%CI) | -2.91 (-5.15 – -0.53) |
| Bias (at 120 μg/g, w/95%CI) | -5.43 (-9.58 – -0.45) |

The comparison of outputs between calprotectin the Q.S.E.T. device was also evaluated for qualitative outcomes, presented in the table below:

| | Method Comparison: Qualitative agreement of Q.S.E.T. values with manual extraction | | | | |
| --- | --- | --- | --- | --- | --- |
| | | Manual Extraction | | | Totals |
| | | Elevated | Borderline | Normal | |
| Q.S.E.T. Extraction | Elevated | 36 | 0 | 0 | 36 |
| | Borderline | 6 | 23 | 3 | 32 |
| | Normal | 0 | 7 | 53 | 60 |
| | Totals | 42 | 30 | 56 | 128 |

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| Borderline results considered elevated (95% CI) | | | |
| --- | --- | --- | --- |
| PPA: | 65/72 | 90.3% | (81.3–95.2%) |
| NPA | 53/56 | 94.6% | (85.4–98.2%) |
| Intermediate results considered normal (95% CI) | | | |
| PPA: | 36/42 | 85.7% | (72.2–93.3%) |
| NPA: | 86/86 | 100% | (95.7–100%) |

b. Matrix comparison:

Stool is the only matrix for this assay.

3. Clinical studies:

To study the clinical performance of the LIAISON calprotectin assay, prospective specimens were collected at 14 sites within the U.S. The population consisted of subjects undergoing endoscopy for evaluation of signs and symptoms of Inflammatory Bowel Disease (IBD) or Irritable Bowel Syndrome (IBS). A total of 240 evaluable subjects meeting inclusion and exclusion criteria remained (see table below) after an initial 411 subjects were recruited. These subjects included 160 female, 80 male, and 19 pediatric (&lt;22 years of age) subjects.

| Inclusion Criteria | Exclusion Criteria | Sample Ineligibility |
| --- | --- | --- |
| ≥4 years of age
Signs and symptoms of IBD or IBS
Diagnosis of IBD, IBS, or other GI disorder by colonoscopy
Informed consent
Able to follow sample collection procedure | Surgical resection or diversion procedure
Currently taking NSAIDs within seven days of colonoscopy and sample collection
Immunomodulator or biologic therapy within six months of colonoscopy and sample collection
Currently pregnant or lactating
Unable or unwilling to provide informed consent
Unable or unwilling to perform required study procedures | Insufficient quantity (<5g)
Collected with urine and/or toilet water
Not stored frozen
Not received frozen by clinical site |

Of the 240 evaluable subjects, additional metadata collected included demographic information (age, sex, race/ethnicity), current medications, signs and symptoms, clinical lab test results, and colonoscopy and biopsy results.

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For 102 IBD patients, disease status was based on the Simple Endoscopic Score for Crohn's disease and the Mayo Endoscopic Score for Ulcerative Colitis and Indeterminate Colitis. For 67 IBS patients, diagnosis was based on negative colonoscopy within the previous year and the Rome III criteria. Of the 71 patients labeled with other GI conditions, seven diagnoses other than IBD or IBS were represented, also based in part on negative colonoscopic findings. In order of representation, these Other GI conditions included (with total number): diverticular disease (35), chronic diarrhea (16), recurrent abdominal pain (11), Clostridium difficile infection (3), other GI infection (3), celiac disease (2), and small bowel obstruction (1). Qualitative interpretation for the LIAISON Calprotectin test for these disease categories is summarized in the table below:

| | Clinical Study: Qualitative comparison | | | | |
| --- | --- | --- | --- | --- | --- |
| | | Diagnostic Category | | | Totals |
| | | IBD | IBS | Other GI | |
| LIAISON Calprotectin | Elevated | 90 | 8 | 5 | 103 |
| | Borderline | 10 | 15 | 15 | 40 |
| | Normal | 2 | 44 | 51 | 97 |
| | Totals | 102 | 67 | 71 | 240 |
| Clinical Study: Qualitative evaluation of IBD versus non-IBD | | | |
| --- | --- | --- | --- |
| Borderline results considered elevated (95% CI) | | | |
| Sensitivity: | 100/102 | 98.0% | (93.1–99.8%) |
| Specificity: | 95/138 | 66.8% | (60.4–76.7%) |
| Borderline results considered normal (95% CI) | | | |
| Sensitivity: | 90/102 | 88.2% | (80.4–93.8%) |
| Specificity: | 125/138 | 90.6% | (84.4–94.9%) |

In addition to the above determination, where the specificity calculation is based on including both IBS and Other GI as a "Non-IBD" category (n=138), sensitivity and specificity values were also calculated for differential comparison of IBD and IBS:

| Clinical Study: Qualitative evaluation of IBD versus IBS | | | |
| --- | --- | --- | --- |
| Borderline results considered elevated (95% CI) | | | |
| Sensitivity: | 100/102 | 98.0% | (93.1–99.8%) |
| Specificity: | 44/67 | 65.7% | (53.1–76.9%) |
| Borderline results considered normal (95% CI) | | | |
| Sensitivity: | 90/102 | 88.2% | (80.4–93.8%) |
| Specificity: | 59/67 | 88.1% | (77.8–94.7%) |

4. Clinical cut-off:

See assay cut-off

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# 5. Expected values/Reference range:

Stool samples from apparently healthy individuals were obtained from multiple sources. The study population fitting the inclusion and exclusion criteria comprised 112 adults and 15 children (3–21 years of age).

| Inclusion Criteria | Exclusion Criteria | Sample Eligibility |
| --- | --- | --- |
| · Informed consent | · History of prolonged abdominal complaints
· History of IBD, IBS, or other chronic intestinal disorders
· Currently taking NSAIDs | · Received frozen within 16 weeks of collection date |

The values obtained for the study are summarized in the table below:

| Reference Range | |
| --- | --- |
| Mean: | 23.5 μg/g |
| Median: | 15.0 μg/g |
| Range: | 5.0 to 103 μg/g |
| 90% Central Interval (95% CI) | 5.0 μg/g (5.0–5.4 μg/g)
to
79.8 μg/g (55.6–96.8 μg/g) |

Qualitative interpretation for the LIAISON Calprotectin test for the reference population is summarized in the table below:

| | Reference Range: Qualitative evaluation | | | |
| --- | --- | --- | --- | --- |
| | | Reference Population | | Totals |
| | | Adult (≥22yo) | Pediatric (<22yo) | |
| LIAISON Calprotectin | Elevated | 0 (0%) | 0 (0%) | 0 (0%) |
| | Intermediate | 12 (10.7%) | 3 (20%) | 15 (11.8%) |
| | Normal | 100 (89.3%) | 12 (80%) | 112 (88.2%) |
| | Totals | 112 | 15 | 127 |

# N. Proposed Labeling:

The labeling is sufficient and it satisfies the requirements of 21 CFR Parts 801 and 809, as applicable, and the special controls for this device type under 21 CFR §866.5180.

# O. Conclusion:

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The submitted information in this premarket notification is complete and supports a substantial equivalence decision.

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