(112 days)
The ALPCO Calprotectin Chemiluminescence ELISA is an in vitro diagnostic chemiluminescent assay intended for the quantitative measurement of fecal calprotectin, a neutrophilic protein that is a marker of intestinal mucosal inflammation, in human stool. The ALPCO Calprotectin Chemiluminescence ELISA is intended for in vitro diagnostic use as an aid in the diagnosis of inflammatory bowel disease (IBD), specifically Crohn's disease (CD) and ulcerative colitis (UC), and as an aid in the differentiation of IBD from irritable bowel syndrome (IBS) in conjunction with other clinical and laboratory findings.
The ALPCO Easy Stool Extraction Device is intended for use in the preparation of human stool specimens for testing in the ALPCO Calprotectin Chemiluminescence ELISA.
The ALPCO Calprotectin Chemiluminescence ELISA is performed on stool samples, collected without preservatives. After an extraction procedure of the stool sample, using either the manual weighing or Easy Extraction Device procedure, the test allows the selective measurement of calprotectin-antigen by sandwich ELISA. A monoclonal capture antibody (mAb) highly specific to the calprotectin heterodimeric and polymeric complexes, respectively, is coated onto the microtiter plate. Calibrators, controls and specimen extracts are incubated. After a washing step, a biotinylated secondary monoclonal detection antibody detects the calprotectin molecules bound to the antibody coated onto the plate. After incubation and a further washing step, a Streptavidin-Horseradish Peroxidase Enzyme conjugate binds to the available biotin on the immobilized secondary antibody. A chemiluminescent substrate is added and read when the substrate glows as a result of its oxidation with the enzyme. The signal is then read on a chemiluminescent plate reader.
Here's an analysis of the acceptance criteria and study detailed in the provided text for the ALPCO Calprotectin Chemiluminescence ELISA and ALPCO Easy Stool Extraction Device:
1. Table of Acceptance Criteria and Reported Device Performance
The document doesn't explicitly state "acceptance criteria" for all performance aspects in a unified table. However, based on the narrative, acceptance implies successful demonstration of performance within established guidelines (e.g., CLSI standards) and acceptable ranges. Here's a table summarizing the performance evaluation and implied acceptance:
| Performance Characteristic | Acceptance Criteria (Implied) | Reported Device Performance |
|---|---|---|
| Precision/Reproducibility | ||
| Within-run Precision (%CV) | Low (typ. <15-20%) | 2.6% - 5.6% (various samples) |
| Between-run Precision (%CV) | Low (typ. <15-20%) | 3.2% - 8.1% (various samples) |
| Within-day Precision (%CV) | Low (typ. <15-20%) | 4.5% - 7.9% (various samples) |
| Between-day Precision (%CV) | Low (typ. <15-20%) | 3.6% - 8.9% (various samples) |
| Total Precision (%CV) | Low (typ. <15-20%) | 5.7% - 15.5% (various samples) |
| Lot-to-Lot Reproducibility (%CV) | Low (typ. <15-20%) | 8.8% - 13.9% (various samples) |
| Site-to-Site Reproducibility (%CV) | Low (typ. <15-20%) | 9.9% - 15.0% (various samples) |
| Operator-to-Operator Reproducibility (%CV) | Low (typ. <15-20%) | 8.5% - 15.5% (various samples) |
| Extraction Method Reproducibility (%CV) | Low (typ. <15-20%) | 5.0% - 12.7% (Easy Extraction) 4.3% - 9.9% (Manual Weighing) |
| Analytical Sensitivity | ||
| Limit of Blank (LoB) | Established Value | 3.6 µg/g |
| Limit of Detection (LoD) | Established Value | 7.7 µg/g |
| Limit of Quantitation (LoQ) | Established Value | 7.9 µg/g |
| Linearity | Linear or acceptable curve fit | Linear (R² close to 1) |
| Matrix Linearity (R²) | Near 1 | 1.00 |
| Aqueous Linearity (R²) | Near 1 | 0.99 |
| Average Recovery (%) | Within acceptable range (e.g., 90-110%) | 99.2% (Matrix) & 100.6% (Aqueous) |
| Accuracy/Recovery (Spiking) | Recovery within acceptable range | 89.7% - 110.8% |
| Interfering Substances | No significant interference | Recovery 91.0 – 108.1%, no interference observed |
| Product Stability | Established stability duration | Reagents: 18 months (unopened), 7 days (opened), 14 days (transport) Raw Stool: 14 days (2-8°C, -20°C), 3 cycles (freeze/thaw) Stool Extract: 3 days (2-8°C), 14 days (-80°C), 3 cycles (freeze/thaw) |
| Stool Sample Extraction Method Comparison | Strong agreement between methods | Slope 1.014, Y-Intercept -1.296, Correlation r 0.997 (Analytical) PPA 96.2-100%, NPA 100%, TPA 98.4-100% (Qualitative) |
| Comparison with Predicate Device (Qualitative) | High agreement (PPA, NPA, TPA) | PPA 46.8-51.4%, NPA 93.1-93.6%, TPA 75.3-85.8% |
| Comparison with Predicate Device (Analytical) | Similar performance (slope, intercept, correlation) | Slope 0.6919, Y-Intercept -18.35, Correlation-r 0.784 |
| Clinical Performance (IBD vs Non-IBD) | High sensitivity, specificity, PPV, NPV | Equivocal = positive: Sensitivity 92.1%, Specificity 92.5%, PPV 72.9%, NPV 98.2% Equivocal = negative: Sensitivity 65.8%, Specificity 96.3%, PPV 79.4%, NPV 92.8% |
| Clinical Performance (IBD vs IBS) | High sensitivity, specificity, PPV, NPV | Equivocal = positive: Sensitivity 92.1%, Specificity 95.1%, PPV 92.1%, NPV 95.1% Equivocal = negative: Sensitivity 65.8%, Specificity 98.4%, PPV 96.2%, NPV 82.2% |
| Expected Values (Normal Population) | Low values for asymptomatic individuals | 130/131 samples normal/negative (< 50 µg/g) |
2. Sample Size Used for the Test Set and Data Provenance
The "test set" implies data used for the clinical performance evaluation.
- Clinical Performance (IBD vs Non-IBD): 424 prospectively collected stool specimens. The document does not specify the country of origin for these samples. The study is prospective as it used prospectively collected specimens.
- Clinical Performance (IBD vs IBS): A subset of the above, N = 198 (76 IBD + 122 IBS patients).
- Comparison with Predicate Device: 400 samples, likely a subset of or related to the clinical performance samples, tested on both the new device and the predicate.
- Expected Values: 131 samples from apparently healthy individuals, a separate study cohort.
3. Number of Experts Used to Establish Ground Truth and Qualifications
The ground truth for the clinical performance study (IBD diagnosis, differentiation from IBS) was established by clinical investigator/gastroenterologist(s). The document states:
- "IBD diagnosis was based on endoscopy results and/or histology of biopsies taken during the endoscopy."
- "IBS diagnosis was based on the Rome IV criteria and confirmed by negative endoscopy including the colon and terminal ileum."
The number of experts is not specified (e.g., "a single gastroenterologist" or "a panel of gastroenterologists"). Their specific experience (e.g., "10 years of experience") is also not provided.
4. Adjudication Method for the Test Set
The document does not describe an adjudication method for establishing the clinical ground truth (IBD/IBS diagnosis). It suggests direct diagnosis by a clinical investigator/gastroenterologist based on established clinical criteria (endoscopy, histology, Rome IV). There is no mention of independent expert review, consensus methods (e.g., 2+1), or tie-breaking procedures.
5. If a Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study Was Done
No, a Multi-Reader Multi-Case (MRMC) comparative effectiveness study was not conducted. This device is an in vitro diagnostic (IVD) assay that aims for quantitative measurement of a biomarker, not image interpretation or a human-in-the-loop diagnostic aid. Therefore, there's no mention of human readers or "AI assistance" in the context of improving human reader performance.
6. If a Standalone (i.e., algorithm only without human-in-the-loop performance) Was Done
Yes, this study is inherently a standalone performance evaluation of the ALPCO Calprotectin Chemiluminescence ELISA. The assay system itself processes the stool sample extracts and generates quantitative calprotectin values. Its performance (sensitivity, specificity, etc.) in aiding diagnosis is then compared against established clinical ground truth, without human-in-the-loop interaction with the assay's output during the diagnostic phase.
7. The Type of Ground Truth Used
The ground truth used for the clinical performance evaluations was a combination of:
- Clinical Diagnosis: Established by clinical investigator(s)/gastroenterologist(s).
- Endoscopy Results: Direct visualization of the GI tract.
- Histology of Biopsies: Microscopic examination of tissue samples for IBD confirmation.
- Rome IV Criteria: Standardized diagnostic criteria for functional gastrointestinal disorders like IBS.
8. The Sample Size for the Training Set
The document does not provide information on a training set for the device. As an ELISA assay, it is a biochemical test procedure rather than a machine learning algorithm that typically requires a distinct training phase with labeled data. The development of such assays involves optimization, calibration, and verification steps, but these are distinct from "training sets" in the AI/ML context.
9. How the Ground Truth for the Training Set Was Established
Since there is no mention of a training set, the establishment of its ground truth is not applicable. The device's "training" or development would involve standard analytical chemistry and immunoassay development practices to optimize reagents, protocols, and ensure accurate detection and quantification of calprotectin.
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Image /page/0/Picture/0 description: The image contains the logos of the Department of Health and Human Services and the Food and Drug Administration (FDA). The Department of Health and Human Services logo is on the left, and the FDA logo is on the right. The FDA logo includes the letters "FDA" in a blue square, followed by the words "U.S. FOOD & DRUG ADMINISTRATION" in blue text.
October 25, 2019
ALPCO Jeffrey Freedman Regulatory Affairs Specialist 26 Keewaydin Drive, Unit G Salem, New Hampshire 03079
Re: K191807
Trade/Device Name: ALPCO Calprotectin Chemiluminescence ELISA, ALPCO Easy Stool Extraction Device Regulation Number: 21 CFR 866.5180 Regulation Name: Fecal Calprotectin Immunological Test System Regulatory Class: Class II Product Code: NXO Dated: July 3, 2019 Received: July 5, 2019
Dear Jeffrey Freedman:
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
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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,
Douglas Jeffery, Ph.D. Chief 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
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Indications for Use
510(k) Number (if known) K191807
Device Name
ALPCO Calprotectin Chemiluminescence ELISA ALPCO Easy Stool Extraction Device
Indications for Use (Describe)
The ALPCO Calprotectin Chemiluminescence ELISA is an in vitro diagnostic chemiluminescent assay intended for the quantitative measurement of fecal calprotectin, a neutrophilic protein that is a marker of intestinal mucosal inflammation, in human stool. The ALPCO Calprotectin Chemiluminescence ELISA is intended for in vitro diagnostic use as an aid in the diagnosis of inflammatory bowel disease (IBD), specifically Crohn's disease (CD) and ulcerative colitis (UC), and as an aid in the differentiation of IBD from irritable bowel syndrome (IBS) in conjunction with other clinical and laboratory findings.
The ALPCO Easy Stool Extraction Device is intended for use in the preparation of human stool specimens for testing in the ALPCO Calprotectin Chemiluminescence ELISA.
Type of Use (Select one or both, as applicable)
| Prescription Use (Part 21 CFR 801 Subpart D) |
|---|
| Over-The-Counter Use (21 CFR 801 Subpart C) |
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510(k) SUMMARY
Date of Summary:
October 22, 2019
Product Name:
ALPCO Calprotectin Chemiluminescence ELISA; ALPCO Easy Stool Extraction Device
Sponsor:
ALPCO 26 Keewaydin Drive, Unit G Salem, NH 03079
Correspondent:
Jeffrey Freedman, Regulatory Affairs Specialist 26 Keewaydin Drive, Unit G Salem, NH 03079 Phone: (603)893-8914 Fax: (603)898-6854
Common Name:
Fecal calprotectin immunological test system
Regulation Number: 21 CFR 866.5180
Classification: NXO, Class II
Predicate Device:
Calprest®NG (QUANTA Lite® Calprotectin Extended Range ELISA), 510(k) number: K160447
Intended Use
The ALPCO Calprotectin Chemiluminescence ELISA is an in vitro diagnostic chemiluminescent assay intended for the quantitative measurement of fecal calprotectin, a neutrophilic protein that is a marker of intestinal mucosal inflammation, in human stool. The ALPCO Calprotectin Chemiluminescence ELISA is intended for in vitro diagnostic use as an aid in the diagnosis of inflammatory bowel disease (IBD), specifically Crohn's disease (CD) and ulcerative colitis (UC), and as an aid in the differentiation of IBD from irritable bowel syndrome (IBS) in conjunction with other clinical and laboratory findings.
The ALPCO Easy Stool Extraction Device is intended for use in the preparation of human stool specimens for testing in the ALPCO Calprotectin Chemiluminescence ELISA.
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Methodology
The ALPCO Calprotectin Chemiluminescence ELISA is performed on stool samples, collected without preservatives. After an extraction procedure of the stool sample, using either the manual weighing or Easy Extraction Device procedure, the test allows the selective measurement of calprotectin-antigen by sandwich ELISA. A monoclonal capture antibody (mAb) highly specific to the calprotectin heterodimeric and polymeric complexes, respectively, is coated onto the microtiter plate. Calibrators, controls and specimen extracts are incubated. After a washing step, a biotinylated secondary monoclonal detection antibody detects the calprotectin molecules bound to the antibody coated onto the plate. After incubation and a further washing step, a Streptavidin-Horseradish Peroxidase Enzyme conjugate binds to the available biotin on the immobilized secondary antibody. A chemiluminescent substrate is added and read when the substrate glows as a result of its oxidation with the enzyme. The signal is then read on a chemiluminescent plate reader.
Substantial Equivalency & Comparison with Predicate
The ALPCO Calprotectin Chemiluminescence ELISA has the same intended use and assay principle as the predicate device. A comparison of the similarities and differences between the devices are provided in the following tables:
| Characteristic | ALPCOALPCO Calprotectin ChemiluminescenceELISA(New Device) | Eurospital S.p.A.Calprest®NGK160447(Predicate Device) |
|---|---|---|
| Similarities | ||
| Intended Use | The ALPCO Calprotectin ChemiluminescenceELISA is an in vitro diagnosticchemiluminescent assay intended for thequantitative measurement of fecalcalprotectin, a neutrophilic protein that is amarker of intestinal mucosal inflammation, inhuman stool. The ALPCO CalprotectinChemiluminescence ELISA is intended for invitro diagnostic use as an aid in the diagnosisof inflammatory bowel disease (IBD),specifically Crohn's disease (CD) andulcerative colitis (UC), and as an aid in thedifferentiation of IBD from irritable bowelsyndrome (IBS) in conjunction with otherclinical and laboratory findings. | Calprest® NG is a quantitative ELISAfor detecting concentration of fecalcalprotectin, which can be used as anin vitro diagnostic to aid in thediagnosis of Inflammatory BowelDiseases (IBD), specifically Crohn'sdisease and ulcerative colitis, and todifferentiate IBD from Irritable BowelSyndrome (IBS) in conjunction withother clinical and laboratory findings. |
| Analyte | Fecal Calprotectin | Same |
| Assay Format | Quantitative | Same |
| Platform | 96 well microtiter plate | Same |
| Characteristic | ALPCOALPCO Calprotectin ChemiluminescenceELISA(New Device) | Eurospital S.p.A.Calprest®NGK160447(Predicate Device) |
| Detection antibody | Monoclonal anti-calprotectin antibody | Same |
| Assay process | Manual | Same |
| Differences | ||
| Capture Antibody | Monoclonal anti-calprotectin antibody | Polyclonal anti-calprotectin antibody |
| AnalyticalMeasuring Range | $7.9 - 6000 \mu g/g$ | $27.1-3000.0 mg/kg$ |
| Units | $\mu g/g$ | $mg/kg$ |
| Method | Chemiluminescent | Colorimetric |
| PrimaryMeasurement Units | Relative Light Units (RLU) | Optical Density (OD) |
| Calibrators | 8 levels:0, 5, 20, 40, 156, 625, 2500, 10000 µg/g | 6 levels:0, 2.5, 12.5, 25, 50, 150 ng/mL |
| Controls | 3 levels | 2 levels |
| Calibrator/ControlAnalyte | Native human calprotectin | Recombinant calprotectin antigen(rAg) |
| Calibrator/ControlPhysio-chemicalCharacteristics | Lyophilized | Ready to use |
| Sample Dilution | 1:25000 | 1:20000 |
| Stop Solution | None | H2SO4 |
| Substrate | Luminol | TMB |
| Incubation Time | 30-30-30-5 minutes at room temp | 60-30-15 minutes at room temp |
| Pre-analyticalsample processing | Manual weighing extraction method or EasyExtraction Device method | Manual weighing extraction method |
| ResultsInterpretation | Normal: $< 50 \mu g/g$Borderline: $50 – 100 \mu g/g$Abnormal: $> 100 \mu g/g$ | Normal: $< 50 mg/kg$Borderline: $50 – 120 mg/kg$Abnormal: $> 120 mg/kg$ |
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Standard Documents Referenced:
- CLSI EP05-A3 Evaluation of Precision of Quantitative Measurement Procedures; Approved Guideline – Third Edition
- CLSI EP06-A Evaluation of The Linearity of Quantitative Measurement Procedures: A Statistical Approach; Approved Guideline
- CLSI EP07-ED3 Interference Testing in Clinical Chemistry, Third Edition .
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- CLSI EP17-A2 Evaluation of Detection Capability for Clinical Laboratory Measurement Procedures; Approved Guideline - Second Edition
- CLSI EP28-A3c Defining, Establishing, and Verifying Reference Intervals in the Clinical Laboratory - Third Edition
Performance Data
Precision/Reproducibility Studies
Precision
The precision of the ALPCO Calprotectin Chemiluminescence ELISA was evaluated internally at ALPCO in accordance with CLSI EP05-A3, Evaluation of Quantitative Measurement Procedures. The study was performed using one (1) kit lot of ALPCO Calprotectin Chemiluminescence ELISA using one (1) ELISA reader by one (1) operator. Eight (8) stool samples containing various fecal calprotectin concentrations covering a significant portion of the reportable range of the ALPCO Calprotectin Chemiluminescence ELISA were extracted using the Easy Extraction method and the resultant extracts were analyzed in duplicate, twice per day, for 20 days to generate a total of 80 replicates per sample. Data were analyzed using Analyse-it Method Validation edition and acceptance criteria were met. The results are as follows:
| Within-run | Between-run | Within-Day | Between-day | Total | ||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Mean(µg/g) | SD(µg/g) | %CV | SD(µg/g) | %CV | SD(µg/g) | %CV | SD(µg/g) | %CV | SD(µg/g) | %CV | ||
| 1 | 80 | 288.7 | 9.5 | 3.3% | 12.3 | 4.2% | 15.5 | 5.4% | 8.6 | 3.0% | 17.7 | 6.1% |
| 2 | 80 | 5323.5 | 220.0 | 4.1% | 359.8 | 6.8% | 421.7 | 7.9% | 410.8 | 7.7% | 588.8 | 11.1% |
| 3 | 80 | 680.3 | 19.7 | 2.9% | 26.2 | 3.8% | 32.8 | 4.8% | 29.2 | 4.3% | 43.9 | 6.4% |
| 4 | 80 | 908.3 | 25.7 | 2.8% | 29.2 | 3.2% | 38.9 | 4.3% | 56.6 | 6.2% | 68.7 | 7.6% |
| 5 | 80 | 116.6 | 3.0 | 2.6% | 4.3 | 3.6% | 5.2 | 4.5% | 4.2 | 3.6% | 6.7 | 5.7% |
| 6 | 80 | 32.9 | 1.3 | 3.9% | 1.2 | 3.7% | 1.8 | 5.3% | 2.9 | 8.9% | 3.4 | 10.3% |
| 7 | 80 | 82.2 | 3.7 | 4.5% | 3.3 | 4.0% | 4.9 | 6.0% | 3.8 | 4.6% | 6.2 | 7.6% |
| 8 | 80 | 39.1 | 1.8 | 4.7% | 1.6 | 4.0% | 2.4 | 6.1% | 2.8 | 7.2% | 3.7 | 9.5% |
Lot-to-Lot Reproducibility
The lot-to-lot reproducibility of the ALPCO Calprotectin Chemiluminescence ELISA was evaluated internally at ALPCO in accordance with CLSI EPO5-A3, Evaluation of Quantitative Measurement Procedures. The study was performed using three (3) kit lots of ALPCO Calprotectin Chemiluminescence ELISA using one (1) ELISA reader by one (1) operator. Seven (7) stool samples containing various fecal calprotectin concentrations covering a fraction of the
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reportable range of the ALPCO Calprotectin Chemiluminescence ELISA were extracted using the Easy Extraction method and analyzed in replicates of 5, once per day, for 5 days using each of the kit lots to generate a total of 75 replicates per sample. Data were analyzed using Analyse-it Method Validation edition and acceptance criteria were met. The results are as follows:
| Sample | N | Within-run | Between-day | Within-lot | Between-lot | Total | ||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Mean(µg/g) | SD(µg/g) | %CV | SD(µg/g) | %CV | SD(µg/g) | %CV | SD(µg/g) | %CV | SD(µg/g) | %CV | ||
| 1 | 75 | 288.6 | 8.5 | 2.9% | 16.5 | 5.7% | 18.5 | 6.4% | 17.2 | 5.9% | 25.3 | 8.8% |
| 2 | 75 | 4480.2 | 321.3 | 7.2% | 445.1 | 9.9% | 548.9 | 12.3% | 298.0 | 6.7% | 624.6 | 13.9% |
| 3 | 75 | 684.2 | 23.3 | 3.4% | 45.1 | 6.6% | 50.8 | 7.4% | 49.6 | 7.2% | 71.0 | 10.4% |
| 4 | 75 | 919.2 | 38.1 | 4.1% | 72.3 | 7.9% | 81.7 | 8.9% | 60.8 | 6.6% | 101.9 | 11.1% |
| 5 | 75 | 110.8 | 7.7 | 6.9% | 7.3 | 6.6% | 10.6 | 9.5% | 7.2 | 6.5% | 12.8 | 11.5% |
| 6 | 75 | 35.4 | 1.8 | 5.2% | 2.1 | 6.0% | 2.8 | 8.0% | 1.8 | 5.2% | 3.4 | 9.5% |
| 7 | 75 | 76.7 | 3.4 | 4.4% | 4.5 | 5.9% | 5.6 | 7.3% | 1.7 | 2.2% | 5.9 | 7.7% |
Site-to-Site Reproducibility
The site-to-site reproducibility of the ALPCO Calprotectin Chemiluminescence ELISA was evaluated internally at ALPCO and at two other analytical test sites in accordance with CLSI EP05-A3, Evaluation of Precision of Quantitative Measurement Procedures. The study was performed using one (1) kit lot of ALPCO Calprotectin Chemiluminescence ELISA by one (1) operator per test site. Seven (7) stool samples containing various fecal calprotectin concentrations of the reportable range of the ALPCO Calprotectin Chemiluminescence ELISA were extracted using the Easy Extraction method at ALPCO, frozen, and then shipped to the two additional sites. Each extract was analyzed in replicates of 5, once per day, for 5 days at each site to generate 25 replicates per site and a total of 75 replicates per sample. Data were analyzed using Analyse-it Method Validation edition and acceptance criteria were met. The results are as follows:
| Within-run | Between-day | Within-site | Between-site | Total | ||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Sample | N | Mean(μg/g) | SD (μg/g) | %CV | SD(μg/g) | %CV | SD(μg/g) | %CV | SD(μg/g) | %CV | SD(μg/g) | %CV |
| 1 | 75 | 46.3 | 2.6 | 5.6% | 3.7 | 8.1% | 4.5 | 9.8% | 0.7 | 1.6% | 4.6 | 9.9% |
| 2 | 75 | 195.2 | 8.9 | 4.6% | 21.6 | 11.0% | 23.3 | 12.0% | 0.0 | 0.0% | 23.3 | 12.0% |
| 3 | 75 | 20.0 | 2.4 | 12.1% | 1.7 | 8.6% | 3.0 | 14.9% | 0.3 | 1.4% | 3.0 | 15.0% |
| 4 | 75 | 446.5 | 21.5 | 4.8% | 27.6 | 6.2% | 35.0 | 7.8% | 0.0 | 0.0% | 35.0 | 7.8% |
| 5 | 75 | 138.2 | 10.1 | 7.3% | 15.8 | 11.4% | 18.7 | 13.5% | 0.0 | 0.0% | 18.7 | 13.5% |
| 6 | 75 | 21.1 | 2.2 | 10.4% | 1.7 | 7.9% | 2.8 | 13.1% | 0.0 | 0.0% | 2.8 | 13.1% |
| 7 | 75 | 2353.7 | 125.3 | 5.3% | 172.9 | 7.3% | 213.5 | 9.1% | 137.1 | 5.8% | 253.7 | 10.8% |
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Operator-to-Operator Reproducibility
The operator-to-operator reproducibility of the ALPCO Calprotectin Chemiluminescence ELISA was evaluated internally at ALPCO in accordance with CLSI EP05-A3, Evaluation of Precision of Quantitative Measurement Procedures. The study was performed using one (1) kit lot of ALPCO Calprotectin Chemiluminescence ELISA by three (3) operators. Seven (7) stool samples containing various fecal calprotectin concentrations covering a significant portion of the reportable range of the ALPCO Calprotectin Chemiluminescence ELISA were extracted using the Easy Extraction method and analyzed by each operator independently in replicates of 5, once per day, for 5 days to generate 25 replicates per sample, per operator and a total of 75 replicates per sample. Data were analyzed using Analyse-it Method Validation edition and acceptance criteria were met. The results are as follows:
| Sample | N | Mean(µg/g) | Within-run | Between-run | Within-operator | Between-operator | Total | |||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| SD (µg/g) | %CV | SD (µg/g) | %CV | SD (µg/g) | %CV | SD (µg/g) | %CV | SD (µg/g) | %CV | |||
| 1 | 75 | 4468.6 | 299.8 | 6.7% | 470.4 | 10.5% | 557.9 | 12.5% | 225.5 | 5.0% | 601.7 | 13.5% |
| 2 | 75 | 555.8 | 22.2 | 4.0% | 26.2 | 4.7% | 34.4 | 6.2% | 70.9 | 12.8% | 78.8 | 14.2% |
| 3 | 75 | 765.0 | 29.5 | 3.9% | 81.3 | 10.6% | 86.5 | 11.3% | 42.5 | 5.6% | 96.4 | 12.6% |
| 4 | 75 | 98.8 | 3.9 | 3.9% | 4.8 | 4.8% | 6.1 | 6.2% | 5.8 | 5.8% | 8.4 | 8.5% |
| 5 | 75 | 26.7 | 0.9 | 3.5% | 2.1 | 8.0% | 2.3 | 8.7% | 2.3 | 8.6% | 3.3 | 12.3% |
| 6 | 75 | 68.1 | 2.1 | 3.1% | 5.8 | 8.5% | 6.1 | 9.0% | 8.4 | 12.3% | 10.4 | 15.3% |
| 7 | 75 | 32.5 | 1.6 | 5.0% | 3.2 | 9.7% | 3.6 | 10.9% | 3.6 | 11.0% | 5.0 | 15.5% |
Extraction Method Reproducibility
The extraction reproducibility of the ALPCO Calprotectin Chemiluminescence ELISA was evaluated internally at ALPCO. The study was performed using one (1) kit lot of ALPCO Calprotectin Chemiluminescence ELISA by one (1) operator. Sets of seven (7) stool samples containing various fecal calprotectin concentrations, including stool samples that are near the clinical cut-offs of the ALPCO Calprotectin Chemiluminescence ELISA were extracted 10 times using both the Easy Extraction method and manual weighing method. Each stool sample extract was analyzed in duplicate to generate 20 replicates per sample, per extraction method. Data were analyzed using Analyse-it Method Validation edition and acceptance criteria were met. The results are as follows:
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| Within-Extraction | BetweenExtraction | TotalImprecision | ||||||
|---|---|---|---|---|---|---|---|---|
| Sample | N | Mean(µg/g) | SD(µg/g) | %CV | SD(µg/g) | %CV | SD(µg/g) | %CV |
| 1 | 20 | 129.0 | 1.8 | 1.4% | 6.2 | 4.8% | 6.5 | 5.0% |
| 2 | 20 | 45.9 | 4.6 | 9.9% | 2.8 | 6.1% | 5.3 | 11.6% |
| 3 | 20 | 4298.5 | 317.2 | 7.4% | 426.9 | 9.9% | 531.8 | 12.4% |
| 4 | 20 | 123.7 | 6.9 | 5.6% | 3.9 | 3.1% | 7.9 | 6.4% |
| 5 | 20 | 1748.5 | 186.5 | 10.7% | 121.4 | 6.9% | 222.6 | 12.7% |
| 6 | 20 | 68.3 | 3.5 | 5.1% | 3.2 | 4.7% | 4.8 | 7.0% |
| 7 | 20 | 33.4 | 1.7 | 5.1% | 0.0 | 0.0% | 1.7 | 5.1% |
Easy Extraction method reproducibility using the Easy Extraction Device:
Extraction reproducibility using the manual weighing method:
| Sample | N | Mean(µg/g) | Within-Extraction | BetweenExtraction | TotalImprecision | ||||
|---|---|---|---|---|---|---|---|---|---|
| SD(µg/g) | %CV | SD(µg/g) | %CV | SD(µg/g) | %CV | ||||
| 1 | 20 | 4063.3 | 389.2 | 9.6% | 0.0 | 0.0% | 389.2 | 9.6% | |
| 2 | 20 | 1336.1 | 42.4 | 3.2% | 113.5 | 8.5% | 121.2 | 9.1% | |
| 3 | 20 | 122.0 | 5.1 | 4.1% | 8.9 | 7.3% | 10.2 | 8.4% | |
| 4 | 20 | 25.7 | 1.1 | 4.5% | 1.6 | 6.4% | 2.0 | 7.8% | |
| 5 | 20 | 103.7 | 2.5 | 2.4% | 3.7 | 3.6% | 4.5 | 4.3% | |
| 6 | 20 | 69.6 | 3.2 | 4.6% | 0.3 | 0.5% | 3.2 | 4.6% | |
| 7 | 20 | 298.6 | 13.5 | 4.5% | 26.2 | 8.8% | 29.5 | 9.9% |
Analytical Sensitivity Studies
Limit of Blank (LoB)
The limit of blank of the ALPCO Calprotectin Chemiluminescence ELISA was determined in accordance with CLSI EP17-A2. Four blank stool samples were extracted and tested in replicates of six on two reagent lots across three days. The LoB was determined to be 3.6 µg/g by the nonparametric method.
Limit of Detection (LoD)
The limit of detection of the ALPCO Calprotectin Chemiluminescence ELISA was determined in accordance with CLSI EP17-A2. Four stool samples containing a low level of calprotectin were extracted and tested in replicates of five on two reagent lots across three days. The LoD was determined to be 7.7 µg/g by the parametric method.
Limit of Quantitation (LoQ)
The limit of quantitation of the ALPCO Calprotectin Chemiluminescence ELISA was determined in accordance with CLSI EP17-A2. Six stool samples containing a low level of calprotectin were extracted and tested in replicates of five on two reagent lots across three days. The mean and
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within laboratory precision were calculated for each reagent lot. For each reagent lot, the observed precision (%CV) (Y-axis) was plotted vs. the sample calprotectin concentration (X-axis) to give a precision profile, and fit by a constant variance function using Analyse-it Method Validation edition. The LoQ estimate for each reagent lot was determined as the measurand concentration at the intersection of the precision profile curve with the accuracy goal of 20 %CV. The LoQ was determined to be 7.9 µg/g.
Linearity
The matrix linearity and aqueous linearity of the analytical measuring range of the ALPCO Calprotectin Chemiluminescence ELISA were evaluated internally at ALPCO in accordance with CLSI EPOG-A, Evaluation of the Linearity of Quantitative Measurement Procedures: A Statistical Approach. To evaluate matrix linearity, a stool sample extract containing a high concentration of fecal calprotectin was serially diluted 1:2 with a stool sample extract containing a low concentration of fecal calprotectin to obtain dilution levels with values that cover the entire AMR. Stool sample extracts were obtained using the Easy Extraction method. Each stool sample extract combination was assayed in duplicate in a single analysis using one reagent lot of the ALPCO Calprotectin Chemiluminescence ELISA. To evaluate aqueous linearity, a sample made by diluting native antigen in standard diluent buffer containing stabilizers and preservatives (the same buffer used to make the controls and calibrators) was serially diluted 1:2 with standard diluent buffer to obtain dilution levels with values that cover the entire AMR. Each aqueous sample dilution was assayed in duplicate in a single analysis using one reagent lot of the ALPCO Calprotectin Chemiluminescence ELISA. Results were analyzed using Analyse-it Method Validation edition to determine the best fitting polynomial. For linearity to be claimed, the best fitting polynomial had to be linear (first order) or the difference between the best fitting nonlinear polynomial (second or third order) and the linear polynomial could not exceed ±15%. The plots were further analyzed by linear regression. The slope and y-intercept, and the 95% confidence intervals thereof, and Rof the regression analysis were calculated. In addition, the recovery of each replicate value included in the regression analysis for the linearity dilutions was calculated and the average recovery was calculated. Acceptance criteria were met, the ALPCO Calprotectin Chemiluminescence ELISA is acceptably linear over the analytical measuring range. The results are as follows:
| Sample Type | Test Range (μg/g) | Slope(95% CI) | Y-Intercept(95% CI) | R² | Average Recovery(%) |
|---|---|---|---|---|---|
| Matrix | 2.5 — 5780.1 | 1.003(0.994 to 1.013) | 1.77(-15.87 to 19.42) | 1.00 | 99.2 |
| Aqueous | 3.0 – 6221.7 | 0.99(0.98 to 1.00) | 0.03(-22.42 to 22.47) | 0.99 | 100.6 |
Accuracy/Recovery
The accuracy/recovery of the ALPCO Calprotectin Chemiluminescence ELISA was evaluated using seven (7) extracted stool samples containing various concentrations of calprotectin across the analytical measuring range of the assay, samples were extracted using the Easy Extraction
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method. Native calprotectin diluted in standard diluent was used as the spiking material. The stool sample extracts were mixed with the spiking material in a proportion of 9:1 (9 parts sample:1 part spiking material) to calculate recovery: Samples with calprotectin concentrations lower than 200 µg/g were spiked with 42.8 µg/g. Samples with calprotectin concentrations higher than 200 µg/g were spiked with 129.3 µg/g. Each baseline sample extract, control spike and spiked sample were tested in duplicate in the same assay run. Recovery was calculated compared to the baseline result and acceptance criteria were met. The results are as follows:
| Sample | MeanBaselineResult (µg/g) | Spike Value(µg/g) | TheoreticalPost-SpikeResult (µg/g) | ObservedPost-SpikeResult (µg/g) | Recovery(%) |
|---|---|---|---|---|---|
| 1 | 248.5 | 129.3 | 353.0 | 391.0 | 110.8 |
| 2 | 5392.3 | 129.3 | 4982.4 | 4469.3 | 89.7 |
| 3 | 615.5 | 129.3 | 683.2 | 754.1 | 110.4 |
| 4 | 94.8 | 42.8 | 128.1 | 123.5 | 96.4 |
| 5 | 28.1 | 42.8 | 68.1 | 72.1 | 105.9 |
| 6 | 77.7 | 42.8 | 112.7 | 123.6 | 109.7 |
| 7 | 36.2 | 42.8 | 75.3 | 79.0 | 104.9 |
Interfering Substances
The interference testing of the ALPCO Calprotectin Chemiluminescence ELISA was evaluated internally at ALPCO using CLSI EP07-ED3, Interference Testing in Clinical Chemistry as a guideline. The study was performed using seven stool samples containing various concentrations of fecal calprotectin. Interfering substances were spiked into each stool sample at 10% of the total specimen volume. The solvent used to create the interferent stock was spiked into each stool sample at 10% of the total specimen volume to generate a control sample. The stool sample containing the interferents and the control stool sample were extracted using the Easy Extraction method. Recovery of the test samples ranged from 91.0 – 108.1%, no interference was observed at the concentrations tested. The list of interferents and the concentrations tested are as follows:
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| Oral Pharmaceuticals | ||
|---|---|---|
| Trade Name | Active Component | Spiking Concentration |
| Ferro-Gradumet | Iron (II) sulfate | 0.02 mg/50 mg stool |
| Prednisone | Prednisone | 0.065 mg/50 mg stool |
| Imurek | Azathioprine | 0.035 mg/50 mg stool |
| Pentasa/Asacol | Mesalamine; 5-ASA | 1.0 mg/50 mg stool |
| Prevacid | Lansoprazol | 0.035 mg/50 mg stool |
| Vancocin | Vancomycin | 0.40 mg/50 mg stool |
| Sulfamethoxazole | Sulfamethoxazole | 0.32 mg/50 mg stool |
| Trimethoprim | Trimethoprim lactate | 0.065 mg/50 mg stool |
| Cipro | Ciprofloxacin | 0.04 mg/50 mg stool |
| Nutritional Supplements | ||
| Trade Name | Active Component | Spiking Concentration |
| Vitamin E | DL-α Tocopherol Acetate | 0.06 mg/50 mg stool |
| Multiple Vitamin | A, B1, B2, B3, B5, B6, B8, B9, B12,C, D, E, and minerals | 0.215 mg/50 mg stool |
| Biotin | B7 | 1750 ng/50 mg stool |
| Other | ||
| Human anti-mouse antibody (HAMA) | 0.5 µg/50 mg stool | |
| Hemoglobin | 0.25 mg/50 mg stool | |
| Microorganisms | ||
| Escherichia coli | 1.5 x $10^7$ cfu/mL | |
| Salmonella enterica subsp. enterica | 1.5 x $10^7$ cfu/mL | |
| Klebsiella pneumoniae subsp. pneumonia | 1.5 x $10^7$ cfu/mL | |
| Citrobacter freundii | 1.5 x $10^7$ cfu/mL | |
| Shigella flexneri | 1.5 x $10^7$ cfu/mL | |
| Yersinia enterocolitica subsp. enterocolitica | 1.5 x $10^7$ cfu/mL |
Product Stability
Real-Time Reagent Stability Summary - The ALPCO Calprotectin Chemiluminescence ELISA reagents are stable for 18 months at 2-8°C.
Open Vial Stability Summary – The ALPCO Calprotectin Chemiluminescence ELISA reagents are stable for 7 days at 2-8°C once opened.
Transport Stability Summary - The ALPCO Calprotectin Chemiluminescence ELISA reagents are stable at 28°C for 14 days.
Raw Stool Sample Stability
| Storage Conditions | Stability Duration |
|---|---|
| 2-8°C | 14 days |
| -20°C | 14 days |
| Freeze/Thaw Cycles | 3 cycles |
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Stool Sample Extract Stability
| Storage Conditions | Stability Duration |
|---|---|
| 2-8°C | 3 days |
| -80°C | 14 days |
| Freeze/Thaw Cycles | 3 cycles |
Stool Sample Extraction Method Comparison
The extraction methods of the ALPCO Calprotectin Chemiluminescence ELISA were evaluated internally at ALPCO to determine if the extraction methods provide similar results. The study was performed using one (1) kit lot of ALPCO Calprotectin Chemiluminescence ELISA by one (1) operator. Sixty-eight (68) stool samples varying in consistency and containing various fecal calprotectin concentrations over a significant fraction of reportable range and near the clinical cut-offs of the ALPCO Calprotectin Chemiluminescence ELISA were extracted in parallel using both the Easy Extraction method and manual weighing method. Each resultant stool sample extract was analyzed in singlicate. 61 of the samples were within the AMR of the ALPCO Calprotectin Chemiluminescence ELISA and included in the analysis. A scatter plot was created by plotting the values obtained from the Easy Extraction method extracts (Y-axis) against the manual weighing method extracts (X-axis) and analyzed by Passing-Bablok regression analysis using Analyse-it Method Validation Edition. The Y-Intercept, slope, and bootstrap 95% confidence intervals thereof, and correlation r were calculated. The results are as follows:
| Stool Sample Extraction Analytical Method Comparison | |
|---|---|
| N | 61 |
| Slope (95% CI) | 1.014 (1.004 to 1.031) |
| Y-Intercept (95% CI) | -1.296 (-1.986 to -0.3179) |
| Correlation r | 0.997 |
Qualitative agreement analysis between the Easy Extraction method and manual weighing method was also conducted to calculate the positive percent agreement, negative percent agreement, and total percent and Wilson 95% Cl considering equivocal results as both positive and negative. The results are as follows:
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| Manual weighing method | |||||
|---|---|---|---|---|---|
| EasyExtractionmethod | Positive | Equivocal | Negative | Totals | |
| Positive | 25 | 0 | 0 | 25 | |
| Equivocal | 1 | 13 | 0 | 14 | |
| Negative | 0 | 0 | 22 | 22 | |
| Totals | 26 | 13 | 22 | 61 | |
| Equivocal results considered positive (95% CI) | |||||
| PPA | 39/39 | 100% | (91.0 - 100%) | ||
| NPA | 22/22 | 100% | (85.1 - 100%) | ||
| TPA | 61/61 | 100% | (94.1 - 100%) | ||
| Equivocal results considered negative (95% CI) | |||||
| PPA | 25/26 | 96.2% | (81.1 - 99.3%) | ||
| NPA | 35/35 | 100% | (90.1 - 100%) | ||
| TPA | 60/61 | 98.4% | (91.3 - 99.7%) |
Stool Sample Extraction Qualitative Method Comparison
Comparison with Predicate Device
method comparison study was performed comparing the ALPCO Calprotectin A Chemiluminescence ELISA and the predicate device. 400 samples that were used to determine the clinical performance characteristics of the ALPCO Calprotectin Chemiluminescence ELISA were tested on the predicate device according to the manufacturer-supplied labeling.
A qualitative agreement analysis between the ALPCO Calprotectin Chemiluminescence ELISA and the predicate device was conducted including all samples that were tested on both devices (N=400) using the cut-offs of both products to calculate the positive percent agreement (PPA), negative percent agreement (NPA), and total percent agreement (TPA), and 95% Cls (Wilson score method) thereof considering equivocal results as both positive and negative. The results are as follows:
| ALPCO Calprotectin Chemiluminescence ELISA | ||||
|---|---|---|---|---|
| Predicate | Normal(< 50 μg/g) | Equivocal(50 – 100 μg/g) | Abnormal(> 100 μg/g) | Total |
| Normal (< 50 mg/kg) | 229 | 5 | 12 | 246 |
| Equivocal (50 - 120 mg/kg) | 60 | 11 | 9 | 80 |
| Abnormal (> 120 mg/kg) | 22 | 14 | 38 | 74 |
| Total | 311 | 30 | 59 | 400 |
| Equivocal results considered positive (95% CI) | ||||
| PPA | 72/154 | 46.8% | (39.0 – 54.6%) | |
| NPA | 229/246 | 93.1% | (89.2 – 95.6%) | |
| TPA | 301/400 | 75.3% | (70.8 – 79.2%) | |
| Equivocal results considered negative (95% CI) | ||||
| PPA | 38/74 | 51.4% | (40.2 – 62.4%) | |
| NPA | 305/326 | 93.6% | (90.4 – 95.7%) | |
| TPA | 343/400 | 85.8% | (82.0 – 88.8%) |
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An analytical method comparison was conducted including the samples that were within the analytical measuring range of both assays (N=169); results had to measure within 27.1 – 3000 mg/kg on the predicate device and 7.9 - 6000 µg/g on the ALPCO Calprotectin Chemiluminescence ELISA. A scatter plot was created by plotting the values obtained with the ALPCO Calprotectin Chemiluminescence ELISA (Y-axis) against the predicate device (X-axis) and analyzed by Passing-Bablok regression analysis using Analyse-it Method Validation Edition. The slope, y-intercept, and correlation-r were determined. The 95% Cl of the slope and intercept was determined using the bootstrap technique. The results are as follows:
| Analytical Method Comparison with Predicate Device | |
|---|---|
| N | 169 |
| Slope (95% CI) | 0.6919 (0.5760 – 0.8777) |
| Y-Intercept (95% CI) | -18.35 (-29.54 - -11.13) |
| Correlation-r | 0.784 |
Clinical Performance
The estimates of clinical sensitivity, clinical specificity, positive predictive value (PPV), and negative predictive value (NPV) of the ALPCO Calprotectin Chemiluminescence ELISA were determined by comparing analytical test results of the prospectively collected stool specimens against the clinical diagnosis made by the clinical investigator/gastroenterologist (reference standard):
- IBD diagnosis was based on endoscopy results and/or histology of biopsies taken during the endoscopy.
- IBS diagnosis was based on the Rome IV criteria and confirmed by negative endoscopy including the colon and terminal ileum.
- Subjects were diagnosed with "Other Gl conditions" when they did not meet the diagnostic criteria for IBD or IBS (Rome IV).
| Clinical Diagnosis | Number of Subjects |
|---|---|
| IBD | 76 |
| Ulcerative Colitis (UC) | 34 |
| Crohn's Disease (CD) | 30 |
| Indeterminant/Undefined | 12 |
| IBS | 122 |
| Other Gl conditions | 226 |
| Total | 424 |
| Number of Results in ALPCO Calprotectin ChemiluminescenceELISA Range | ||||
|---|---|---|---|---|
| Clinical Diagnosis | <50 μg/g | 50 – 100 μg/g | >100 μg/g | Total |
| IBD | 6 | 20 | 50 | 76 |
| IBS | 116 | 4 | 2 | 122 |
| GI Other | 206 | 9 | 11 | 226 |
| Total | 328 | 33 | 63 | 424 |
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Estimates of sensitivity, specificity, PPV, and NPV, along with 95% confidence intervals (Wilson score method for sensitivity/specificity and Mercaldo-Wald logit method for predictive value) were calculated for the ALPCO Calprotectin Chemiluminescence ELISA as an aid in the diagnosis of IBD (n = 424). The estimates of sensitivity, specificity, PPV, and NPV were calculated considering equivocal results as both positive and negative:
| ALPCO Test Result | |||
|---|---|---|---|
| Equivocal results = positive | > 50 | <= 50 | Total |
| Clinical Diagnosis | > 50 | <= 50 | Total |
| IBD | 70 | 6 | 76 |
| Non-IBD | 26 | 322 | 348 |
| Total | 96 | 328 | 424 |
| Fraction | % | 95% CI | |
| Sensitivity | 70/76 | 92.1 | 83.8 - 96.3 |
| Specificity | 322/348 | 92.5 | 89.3 - 94.9 |
| PPV | 70/96 | 72.9 | 64.9 - 79.7 |
| NPV | 322/328 | 98.2 | 96.1 - 99.1 |
| Equivocal results = negative | ALPCO Test Result | ||
|---|---|---|---|
| Clinical Diagnosis | > 100 | <= 100 | Total |
| IBD | 50 | 26 | 76 |
| Non-IBD | 13 | 335 | 348 |
| Total | 63 | 361 | 424 |
| Fraction | % | 95% CI | |
| Sensitivity | 50/76 | 65.8 | 54.6 – 75.5 |
| Specificity | 335/348 | 96.3 | 93.7 – 97.8 |
| PPV | 50/63 | 79.4 | 68.8 – 87.0 |
| NPV | 335/361 | 92.8 | 90.4 – 94.6 |
Estimates of sensitivity, specificity, PPV, and NPV, along with 95% confidence intervals (Wilson score method for sensitivity/specificity and Mercaldo-Wald logit method for predictive value) were calculated for the ALPCO Calprotectin Chemiluminescence ELISA as an aid in the differentiation of IBD vs IBS (n = 198). The estimates of sensitivity, specificity, PPV, and NPV were calculated considering equivocal results as both positive and negative:
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| Equivocal results = positive | ALPCO Test Result | ||
|---|---|---|---|
| Clinical Diagnosis | > 50 | <= 50 | Total |
| IBD | 70 | 6 | 76 |
| IBS | 6 | 116 | 122 |
| Total | 76 | 122 | 198 |
| Fraction | % | 95% CI | |
| Sensitivity | 70/76 | 92.1 | 83.8 – 96.3 |
| Specificity | 116/122 | 95.1 | 89.7 – 97.7 |
| PPV | 70/76 | 92.1 | 84.2 – 96.2 |
| NPV | 116/122 | 95.1 | 90.0 – 97.7 |
| Equivocal results = negative | ALPCO Test Result | ||
|---|---|---|---|
| Clinical Diagnosis | > 100 | <= 100 | Total |
| IBD | 50 | 26 | 76 |
| IBS | 2 | 120 | 122 |
| Total | 52 | 146 | 198 |
| Fraction | % | 95% CI | |
| Sensitivity | 50/76 | 65.8 | 54.6 – 75.5 |
| Specificity | 120/122 | 98.4 | 94.2 – 99.5 |
| PPV | 50/52 | 96.2 | 86.2 – 99.0 |
| NPV | 120/146 | 82.2 | 77.1 – 86.3 |
Expected Values
To verify the low clinical cut-off value (50 µg/g), normal stool samples were obtained from asymptomatic individuals with no abdominal complaints and no history of IBS, IBD or other chronic intestinal disorders; this study cohort was separate from those used to establish estimates of the clinical performance of the test device. The expected result for the "normal"/asymptomatic population is < 50 µg/g (normal). Calprotectin levels were analyzed using the ALPCO Calprotectin Chemiluminescence ELISA on 131 samples obtained from apparently healthy individuals with the following demographics:
- Gender: 65 females / 66 males
- Age: 22 to 82 years old, mean age 42.4 years
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With a cut-off of 50 µg/g, 130/131 of the samples were normal/negative with the ALPCO Calprotectin Chemiluminescence ELISA. Values ranged from <7.9 µg/g to 75.1 µg/g, with 86/131 samples measuring below the lower end of the AMR. The 90% confidence intervals for the lower and upper 95% reference limits of the 131 healthy individuals were determined by the nonparametric quantile method, (N+1)p in accordance with CLSI EP28-A3c, Defining, Establishing, and Verifying Reference Intervals in the Clinical Laboratory, 3rd Edition using Analyse-it Method Validation Edition. The results are as follows:
Lower Limit (90% CI): 0.7 μg/g (0.5 – 0.9 µg/g)
Upper Limit (90% CI): 37.7 μg/g (30.9 – 75.1 μg/g)
§ 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).