510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION SUMMARY A. 510(k) Number: K160447 B. Purpose for Submission: New Device C. Measurand: Fecal calprotectin D. Type of Test: Quantitative, ELISA E. Applicant: Eurospital S.p.A. F. Proprietary and Established Names: Calprest®NG G. Regulatory Information: 1. Regulation section: 21 CFR§866.5180 – Fecal calprotectin immunological test system 2. Classification: Class II 3. Product code: NXO, Calprotectin, Fecal 4. Panel: Immunology (82) 1 H. Intended Use: 1. Intended use(s): Calprest®NG is a quantitative ELISA for detecting concentration of fecal calprotectin, which can be used as an in vitro diagnostic to 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) in conjunction with other clinical and laboratory findings. 2. Indication(s) for use: Same as Intended Use 3. Special conditions for use statement(s): Prescription use only 4. Special instrument requirements: Microtiter plate reader (450 nm filter) I. Device Description: The Calprest®NG kit contains the following materials: • Microtiter plate coated with rabbit anti-calprotectin antibodies (12 strips, 8 wells/strip) • Horse-radish peroxidase (HRP)-labeled mouse anti-calprotectin antibody (1 x 15 mL) • Substrate (1 x 15 mL) • Stop solution (1 x 15 mL, 0.5M H2SO4) • 20x Washing solution (1 x 50 mL) • 10x Diluent solution (1 x 20 mL) • 2.5x Extraction solution (2 x 50 mL) • Calibrators (6 x 1.5 mL, 6 vials containing 1.5 mL/vial calprotectin solution at concentration of 0, 2.5, 12.5, 25, 50, 150 ng/mL) • Control 1 (low) (1 x 1.5 mL) • Control 2 (high) (1 x 1.5 mL) J. Substantial Equivalence Information: 1. Predicate device name: Calprest® 2. Predicate 510(k) number: 2 K130945 3. Comparison with predicate: Similarities Item Device Predicate Calprest®NG Calprest® Intended to aid in the diagnosis of Same Use/Indication for Use Inflammatory Bowel Diseases (IBD), specifically Crohn’s disease and ulcerative colitis, and to differentiate IBD from Irritable Bowel Syndrome (IBS) in conjunction with other clinical and laboratory findings Analyte Calprotectin Same Assay format Quantitative Same Method Colorimetric ELISA Same Sample type Fecal Same Specimen Requirement 1.0–5.0 g stool Same Stability Washing buffer: 20–25°C for 30 days Same (Working solution) Extraction buffer: 2–8°C for 3 months Dilution buffer: 2–8°C for 30 days Stability Conjugate: 2–8°C for 30 days Same (Open vial reagents) Substrate: 2–8°C for 90 days Calibrators: 2–8°C for 30 days Controls: 2–8°C for 30 days Sample storage Stored at 2–8°C for up to 4 days Same before testing. If not tested immediately, freeze stored samples at -20°C. Differences Item Device Predicate Calprest®NG Calprest® OD reading 450 nm 405 nm Detection Antibody HRP-labeled mouse anti- Alkaline-phosphatase- calprotectin labeled rabbit anti- calprotectin Substrate TMB pNPP Stop solution H2SO4 (0.5M) None Sample dilution 1:20,000 1:2500 Control 2 controls 2 controls Control 1: 10–20 ng/mL Control 1: 20–40 ng/mL Control 2: 30–70 ng/mL Control 2: 40–85 ng/mL Calibrators 6 levels: 6 levels: 3 Differences Item Device Predicate Calprest®NG Calprest® 0, 2.5, 12.5, 25, 50, 150 6.25, 12.5, 25, 50, 100, ng/mL 200 ng/mL Conversion factor 20 2.5 Reportable range 27.1–3000 mg/kg 15.6–500 mg/kg Results interpretation Normal: 27.1– <50 mg/kg Normal: <15.6–50 mg/kg Borderline: 50–120 mg/kg Borderline: 50–120 mg/kg Abnormal: >120 mg/kg Abnormal: >120 mg/kg ELISA Procedure : Sample incubation 60 min 45 min Conjugate incubation 30 min 45 min Substrate incubation 15 min 30 min K. Standard/Guidance Document Referenced (if applicable): • CLSI EP5-A3, Evaluation of Precision of Quantitative Measurement Procedures; Approved Guideline—Third Edition • CLSI EP6-A, Evaluation of the Linearity of Quantitative Measurement Procedures: A Statistical Approach; Approved Guideline • CLSI EP7-A2, Interference Testing in Clinical Chemistry; Approved Guideline—Second Edition • CLSI EP17-A2, Evaluation of Detection Capability for Clinical Laboratory Measurement Procedures; Approved Guideline—Second Edition • Guidance for Industry and FDA Staff–Class II Special Controls Guidance Document: Fecal Calprotectin Immunological Test Systems L. Test Principle: Calprest®NG is a colorimetric enzyme-linked immunosorbent assay (ELISA). The test is performed on stool samples collected without preservatives. An extract is prepared by combining the stool sample with extraction buffer and mixing for 30 minutes followed by centrifugation. The assay uses a polyclonal rabbit antibody against calprotectin as the capture antibody. Calprotectin present in the diluted stool extract sample is bound by the antibody adsorbed onto the surface of the microtiter plate. HRP-conjugated antibodies bind to the captured calprotectin. The enzyme catalyzes the conversion of the substrate (TMB) to a colored product and the optical density (OD) at 450 nm of the sample is read on an ELISA plate reader. The intensity of the color is proportional to the amount of conjugate bound, and thus to the amount of captured calprotectin. The concentration of calprotectin in the extracted sample is interpreted from a standard curve generated from the six calibrators and converted to mg of calprotectin per kg of stool using a conversion factor supplied in the package insert. M. Performance Characteristics: 4 1. Analytical performance: All results presented below were within the sponsor’s pre- determined acceptance criteria for each study. a. Precision/Reproducibility: Precision: The precision of the Calprest®NG assay was evaluated by extracting seven stool samples containing various concentration of calprotectin. Each extracted sample was tested in duplicate, two runs per day for 10 days by three different operators using one lot of reagent (total of 120 replicates per sample). The results are summarized in the table below Between- Between- Between- Total Within-Run Mean Run Day Operator (mg/kg) CV CV CV CV CV SD SD SD SD SD (%) (%) (%) (%) (%) 1 42.60 1.28 3.0 4.30 10.1 0.00 0.0 0.55 1.3 4.52 10.6 2 170.88 3.40 2.0 6.77 4.0 5.51 3.2 0.80 0.5 9.40 5.5 3 248.74 6.44 2.6 11.98 4.8 12.21 4.9 0.00 0.0 18.28 7.3 4 741.60 17.38 2.3 43.12 5.8 55.37 7.5 0.00 0.0 72.30 9.7 5 1006.17 41.69 4.1 39.67 3.9 55.80 5.5 28.24 2.8 84.98 8.4 6 1193.10 52.91 4.4 73.33 6.1 118.78 10.0 60.32 5.1 161.01 13.5 7 2267.29 223.08 9.8 83.22 3.7 155.52 6.9 58.33 2.6 290.31 12.8 Reproducibility: The lot-to-lot reproducibility was done by extracting four stool samples with calprotectin concentrations of 64.7, 259.2, 778.2, and 2005.2 mg/kg. Each extracted sample was tested in replicates of five using three different lots of the reagents. Mean and %CV for each sample were calculated and %CV values for between-lot reproducibility were 3.1%–7.3% for all samples. The site-to-site reproducibility was evaluated by testing a total of eight stool samples at three sites. The samples were run in replicates of five per day for five days at each site, and a total of 75 data points were generated for each sample. Data were analyzed for within-run, between-run, within-site, between site and total reproducibility. The results are summarized in the table below: Within-Run Between- Within-Site Between- Total Mean Day Site (mg/kg) SD CV SD CV SD CV SD CV SD CV (%) (%) (%) (%) (%) 1 36.54 3.69 10.1 3.54 9.7 5.11 14.0 0.00 0.0 5.11 14.0 2 62.08 4.19 6.8 3.78 6.1 5.64 9.1 6.60 10.6 8.68 14.0 3 356.84 12.00 3.4 27.57 7.7 30.07 8.4 42.34 11.9 51.93 14.6 4 433.80 13.43 3.1 62.56 14.4 63.99 14.8 8.47 2.0 64.55 14.9 5 682.76 35.88 5.3 34.21 5.0 49.57 7.3 61.45 9.0 78.95 11.6 6 1180.64 71.08 6.0 139.04 11.8 156.16 13.2 0.00 0.0 156.16 13.2 7 1629.44 96.29 5.9 191.96 11.8 214.76 13.2 73.04 4.5 226.84 13.9 8 2152.20 173.58 8.1 254.59 11.8 308.13 14.3 50.20 2.3 312.19 14.5 5 The extraction reproducibility was evaluated by using four stool samples with calprotectin concentrations of 29.6, 52.7, 227.0, and 1973.3 mg/kg. Each stool sample was extracted 10 times and each stool extract was tested in duplicate. The %CVs for these four stool samples are 14.6%, 11.1%, 6.8%, and 5.9%, respectively. b. Linearity/assay reportable range: Linearity: The linearity of Calprest®NG was evaluated according to the CLSI guideline EP6-A. Four sample dilution series each with nine dilution levels were prepared by serially diluting the high concentration stool extract sample pools with the low concentration stool extract sample pools.