IG_PLEX CELIAC DGP PANEL

{0} 1 # 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION MEMORANDUM A. 510(k) Number: k140691 B. Purpose for Submission: New assay and instrument C. Measurand: Autoantibodies to deamidated gliadin peptide (DGP) IgG and IgA classes Autoantibodies to tissue transglutaminase (tTG) IgG and IgA classes D. Type of Test: Multiplexed Immunoassay E. Applicant: SQI Diagnostic Systems, Inc. F. Proprietary and Established Names: SQI Ig_plex Celiac DGP Panel G. Regulatory Information: 1. Regulation section: 21 CFR §866.5750: Radioallergosorbent (RAST) immunological test system 21 CFR §866.5660: Multiple autoantibodies immunological test system 21 CFR §862.2570: Instrumentation for Clinical Multiplex Test Systems 2. Classification: Class II 3. Product code: MST: Antibodies, gliadin MVM: Autoantibodies, Endomysial (Tissue transglutaminase) NSU: Instrumentation for Clinical Multiplex Test Systems {1} 4. Panel: Immunology (82) H. Intended Use: 1. Intended use(s): The Ig_plex Celiac DGP Panel is an in vitro diagnostic test for the semi-quantitative detection of the IgA and IgG immunoglobulin classes of antibodies to deamidated gliadin peptide (DGP) and tissue transglutaminase (tTG) in human serum. The test is intended for use in clinical laboratories as an aid in the diagnosis of celiac disease in conjunction with other laboratory and clinical findings, and requires the sqid-X system. 2. Indication(s) for use: Same as intended use. 3. Special conditions for use statement(s): For prescription use only 4. Special instrument requirements: For use on the sqid-X system I. Device Description: 1. Microarray plate: 1 package, storage 2-8°C The Ig_plex Celiac DGP Panel microarray plate consists of an array of capture replicate spots, covalently bound to the surface of coated glass within each well of a 96-well SBS compliant assay plate. All wells have the identical eight by eight grid containing: - Eighteen (18) capture spots each of DGP and tTG - Six (6) capture spots each of human IgG control and human IgA/IgG mix control - Four (4) spots of Dy649-BSA - Twelve (12) blanks The control IgG and IgG/IgA captures are used to confirm that the reporter mix is performing within specification and for other internal quality checks. The fluorescently tagged BSA spots are used as “Landing Lights” for the spot finding algorithm’s grid registration. The spots are approximately 200μm in diameter. The outer plate holder is labeled with a barcode indicating the control number which the system reads to identify the assay type and lot number. 2. Reporter mix: mouse or goat anti-human antibodies, fluorescently labeled with Cy5 or Cy3 1 amber bottle, store ≤ -15°C {2} 3. Standards: Eight (8) 2.0 mL tubes, store ≤ -15°C 4. Positive Control #1: Human One (1) 2.0 mL tube, store ≤ -15°C 5. Positive Control #2: Human One (1) 2.0 mL tube, store ≤ -15°C 6. Negative Control: One (1) 2.0 mL tube, store ≤ -15°C 7. Sample Diluent: Three (3) Translucent bottles, store ≤ -15°C 8. Wash Buffer Concentrates: Four (4) Translucent bottles, store 2-8°C 9. Ig_plex software: The Ig_plex configuration software contains several modules: washer, scanner, spot finding algorithm, data analysis algorithm, report generation algorithm, and the user interface. 10. The sqid-X system consists of the following components: | Component | Description | Manufacturer | | --- | --- | --- | | FLAIR™ Scanner | High resolution plate imaging instrument | Sensovation AG | | ELx405UCWVS™ Plate Washer | Microarray plate washing instrument | BioTek Instruments Inc. | | Drying Station | Microarray plate final drying station | SQI Diagnostics Systems Inc. | | ME 4C NT Vario | Vacuum pump for drying microarray | VacuuBrand Inc. | | Microplate Shaker | Plate shaker used during incubation steps | VWR | | Computer System | Computer running Microsoft Windows™ XP with peripherals | Hewlett Packard, Microsoft etc. | | sqid-X software | Ig_plex Celiac DGP panel configuration | SQI Diagnostics Systems Inc. | The system combines manual liquid handling (samples and reagents) including sample pipetting into the plate, with automated steps for washing, scanning, data analyses and reporting. Results for each patient sample are obtained simultaneously and independently for each of the four Ig_plex Celiac DGP Panel markers: DGP IgA, DGP IgG, tTG IgA and tTG IgG using the data from one well containing one sample of the patient's serum. The system uses an integrated hardware and software solution to create a procedure that is semi-automated. The system is configured with assay specific software to perform the automated steps of the assay protocol. J. Substantial Equivalence Information: 1. Predicate device names and numbers: Assays: INOVA Diagnostics Inc. Quanta Lite™ Gliadin IgA II ELISA (k052143); Quanta Lite™ Gliadin IgG II ELISA (k052142); Quanta Lite™ h-tTG IgA ELISA (k011566) and Quanta Lite™ h-tTG IgG ELISA (k011570) Instrument: SQI Diagnostics SQiDworks Diagnostics Platform (k102490). {3} 2. Comparison with predicate: | Similarities- All devices | | | | | | | --- | --- | --- | --- | --- | --- | | Item | Device | Predicates | | | | | | SQI Ig_plex Celiac DGP Panel | Quanta Lite™ Gliadin IgA II (k052143) | Quanta Lite™ Gliadin IgG II (k052142) | Quanta Lite™ h-tTG IgA (k011566) | Quanta Lite™ h-tTG IgA (k011570) | | Intended Use | Detection of autoantibodies to aid in the diagnosis of celiac disease. | Same | Same | Same | Same | | Assay format | Semi-quantitative, manual washing and preparation | Same | Same | Same | Same | | Sample | Human serum | Same | Same | Same | Same | | Assay substrate | 96-well plate | Same | Same | Same | Same | | Calibration | On each plate | Same | Same | Same | Same | | Differences- All devices | | | | | | | --- | --- | --- | --- | --- | --- | | Item | Device | Predicates | | | | | | SQI Ig_plex Celiac DGP Panel | Quanta Lite™ Gliadin IgA II (k052143) | Quanta Lite™ Gliadin IgG II (k052142) | Quanta Lite™ h-tTG IgA (k011566) | Quanta Lite™ h-tTG IgA (k011566) | | Sample Dilution | 1:151 | 1:101 | 1:101 | 1:101 | 1:101 | | Automation | Semi-automated | Manual | Manual | Manual | Manual | | Technology | Microarray-based Fluorescence detection | ELISA | ELISA | ELISA | ELISA | | Mutiplexed assay | Yes | No | No | No | No | | Shelf life Stability | 6 months | Not stated in months | Not stated in months | One year | One year | | Controls/Standards | Eight standards, three controls: Negative, Positive #1 and Positive #2 | Three controls: Negative High positive, low positive | Three controls: Negative High positive, low positive | Three controls: Negative High positive, low positive | Three controls: Negative High positive, low positive | {4} | Device-specific similarities and differences | | | | --- | --- | --- | | Item | Ig_plex Celiac DGP Panel DGP IgG | Quanta Lite™ Gliadin IgA II (k052143) | | Analyte | Deamidated gliadin peptide (DGP) IgA antibodies | Gliadin IgA antibodies | | Capture antigen | Synthetic DGP | Similar | | Measuring range | 8.0–110.0 U/mL | Not specified | | Cut-off | 15.0 U/mL | 20.0 U/mL | | Device-specific similarities and differences | | | | --- | --- | --- | | Item | Ig_plex Celiac DGP Panel tTG IgA | Quanta Lite™ h-tTG IgA (k011566) | | Analyte | Tissue transglutaminase (tTG) IgA antibodies | Human tissue Transglutaminase (h-tTG) IgA antibodies | | Capture antigen | Recombinant human tTG | Native human red blood cell tTG | | Measuring range | 16.0–140.0 U/mL | Not specified | | Cut-off | 20.0 U/mL | 20.0 U/mL | | Device-specific similarities and differences | | | | --- | --- | --- | | Item | Ig_plex Celiac DGP Panel tTG IgG | Quanta Lite™ h-tTG IgG (k011570) | | Analyte | Tissue transglutaminase (tTG) IgG antibody. | Human tissue Transglutaminase (h-tTG) IgG antibodies | | Capture antigen | Recombinant human tTG | Native human red blood cell tTG | | Measuring range | 24.0–100.0 U/mL | Not specified | | Cut-off | 36.0 U/mL | 20.0 U/mL | {5} | Item | Sqid-X platform | SQiDworks Diagnostics Platform | | --- | --- | --- | | Sample, Reagent and Plate Handling | Manual | Automated | | Detector | Multichannel fluorescence LED camera scanner | Multichannel fluorescence CCD camera scanner | # K. Standard/Guidance Document Referenced (if applicable): 1. CLSI EP05-A2: Evaluation of Precision Performance of Quantitative Measurement Methods; Approved Guideline 2. CLSI EP06-A: Evaluation of the Linearity of Quantitative Analytical Methods 3. CLSI EP07-A2: Interference Testing in Clinical Chemistry, Approved Guideline 4. CLSI EP09-A2: Method Comparison Bias Estimate using Patient Samples 5. CLSI C28-A3: Defining, Establishing, and Verifying Reference Intervals in the Clinical Laboratory; Approved Guideline- Third Edition 6. CLSI EP17-A: Protocols for Determination of Limits of Detection and Limits of Quantitation, Approved Guideline # L. Test Principle: The system is a multiplex immunoassay analyzer that semi-automates the protocol of a specific Ig_plex assay from plate washing to reporting of all assay markers for each individual patient sample. The system combines manual liquid handling (samples and reagents) with automated steps for washing, scanning, data analyses and reporting. The system uses graphical user interface software to lead the operator through manual sample, reagent, and plate handling operations. The software controls the instruments, analysis and reporting using the same core software as the predicate instrument. The sqid-X software also provides access to maintenance procedures. The system controls the washer to ensure that the same wash programs are used every time and in the correct sequence. Once the assay's biochemical reactions are complete and the plate is placed in the scanner, the instrument automatically performs a multi- wavelength fluorescent scan of each well in the microarray plate, analyzes the data, and generates a report containing results for all assay markers. The sqid-X system also includes numerous internal quality checks. Results for each patient sample are obtained simultaneously for each of the four Ig_plex Celiac DGP Panel markers: DGP IgA, DGP IgG, tTG IgA and tTG IgG using the data from one well containing one sample of the patient's serum. The system does not interpret the results with respect to diagnosis; it simply reports each result independently. The biochemical reactions and analysis for the measurements are found in 4.5 Principle of Operation following the description of the device components. {6} M. Performance Characteristics (if/when applicable): 1. Analytical performance: a. Precision/Reproducibility: The sample sets used in these studies together covered the measuring range for each assay, although not every sample contained all four analytes. The specific number of samples for each element is described below. The manufacturer's acceptance criterion is that all reproducibility results should have % CVs of ≤10%. i. Within-Run (intra-assay) Reproducibility Testing Negative and positive samples covering the measuring range for each device, including at least 10% of total samples within ±25% of the cut-off for each assay, were run in replicates of twelve (12) on one kit. A total of four (4) kits were run on different days. | DGP IgA | | DGP IgG | | tTG IgA | | tTG IgG | | | --- | --- | --- | --- | --- | --- | --- | --- | | Mean (U/mL) | %CV | Mean (U/mL) | %CV | Mean (U/mL) | %CV | Mean (U/mL) | %CV | | 11.33 | 3.5% | 11.97 | 3.4% | 17.43 | 2.9% | 32.58 | 4.5% | | 17.99 | 2.6% | 15.23 | 3.4% | 27.26 | 3.4% | 35.78 | 3.4% | | 20.08 | 4.3% | 27.31 | 2.7% | 38.35 | 4.8% | 40.82 | 4.9% | | 32.30 | 3.1% | 38.42 | 2.6% | 41.80 | 2.8% | 44.53 | 5.2% | | 43.23 | 2.2% | 54.69 | 3.5% | 56.68 | 3.9% | 58.51 | 3.5% | | 56.08 | 4.6% | 69.51 | 2.7% | 64.03 | 3.3% | 61.70 | 5.2% | | 64.03 | 6.3% | 86.69 | 3.8% | 86.56 | 1.9% | 71.01 | 2.7% | | | | 117.26 | 2.3% | 125.66 | 3.1% | 78.36 | 3.2% | | | | | | | | 98.78 | 4.8% | ii. Day-to-Day Reproducibility Testing Negative and positive samples covering the measuring range for each device, including at least 10% of total samples within ±25% of the cut-off for each assay, were run in replicates of two (2) per run, two (2) runs per day, for 20 non-consecutive days, on one system by two operators (31 runs by operator 1 and 9 runs by operator 2) using one kit lot for a maximum of 80 results per sample per analyte (2×2×20=80). | DGP IgA- Day to day reproducibility | | | | | | | | | | --- | --- | --- | --- | --- | --- | --- | --- | --- | | Mean (U/mL) | Within Run | | Between Run | | Between Day | | Total Precision | | | | SD | %CV | SD | %CV | SD | %CV | SD | %CV | {7} | DGP IgA- Day to day reproducibility | | | | | | | | | | --- | --- | --- | --- | --- | --- | --- | --- | --- | | Mean (U/mL) | Within Run | | Between Run | | Between Day | | Total Precision | | | | SD | %CV | SD | %CV | SD | %CV | SD | %CV | | 9.13 | 0.43 | 4.70% | 0.31 | 3.30% | 0.00 | 0.00% | 0.52 | 5.70% | | 15.83 | 0.64 | 4.00% | 0.27 | 1.70% | 0.31 | 2.00% | 0.76 | 4.80% | | 20.28 | 0.70 | 3.50% | 0.98 | 4.80% | 0.00 | 0.00% | 1.21 | 6.00% | | 28.47 | 1.68 | 5.90% | 1.23 | 4.30% | 0.66 | 2.30% | 2.18 | 7.70% | | 36.2 | 2.02 | 5.60% | 1.14 | 3.20% | 0.59 | 1.60% | 2.39 | 6.60% | | 47.84 | 1.83 | 3.80% | 2.59 | 5.40% | 1.16 | 2.40% | 3.38 | 7.10% | | 54.63 | 2.97 | 5.40% | 1.82 | 3.30% | 1.56 | 2.90% | 3.81 | 7.00% | | 78.94 | 4.75 | 6.00% | 3.47 | 4.40% | 1.43 | 1.80% | 6.06 | 7.70% | | 104.24 | 3.80 | 3.60% | 5.39 | 5.20% | 0.00 | 0.00% | 6.60 | 6.30% | | DGP IgG- Day to day reproducibility | | | | | | | | | | --- | --- | --- | --- | --- | --- | --- | --- | --- | | Mean (U/mL) | Within Run | | Between Run | | Between Day | | Total Precision | | | | SD | %CV | SD | %CV | SD | %CV | SD | %CV | | 15.38 | 0.71 | 4.60% | 0.47 | 3.00% | 0.70 | 4.50% | 1.10 | 7.10% | | 22.09 | 1.01 | 4.60% | 1.01 | 4.60% | 0.75 | 3.40% | 1.61 | 7.30% | | 31.89 | 1.26 | 4.00% | 0.61 | 1.90% | 0.91 | 2.90% | 1.67 | 5.20% | | 41.51 | 2.02 | 4.90% | 0.61 | 1.50% | 0.69 | 1.70% | 2.22 | 5.30% | | 50.08 | 2.61 | 5.20% | 1.42 | 2.80% | 1.17 | 2.30% | 3.19 | 6.40% | | 71.35 | 2.74 | 3.80% | 2.64 | 3.70% | 0.00 | 0.00% | 3.80 | 5.30% | | 85.57 | 3.22 | 3.80% | 2.34 | 2.70% | 0.00 | 0.00% | 3.98 | 4.60% | | 119.12 | 6.06 | 5.10% | 2.27 | 1.90% | 0.00 | 0.00% | 6.47 | 5.40% | | tTG IgA- Day to day reproducibility | | | | | | | | | | --- | --- | --- | --- | --- | --- | --- | --- | --- | | Mean (U/mL) | Within Run | | Between Run | | Between Day | | Total Precision | | | | SD | %CV | SD | %CV | SD | %CV | SD | %CV | | 17.29 | 0.64 | 3.70% | 0.67 | 3.90% | 0 | 0.00% | 0.93 | 5.40% | | 24.46 | 1.00 | 4.10% | 0.62 | 2.60% | 0.53 | 2.20% | 1.29 | 5.30% | | 32.35 | 1.62 | 5.00% | 0.56 | 1.70% | 0 | 0.00% | 1.71 | 5.30% | | 35.19 | 1.38 | 3.90% | 0.74 | 2.10% | 1.71 | 4.90% | 2.32 | 6.60% | | 43.29 | 1.95 | 4.50% | 1.79 | 4.10% | 0 | 0.00% | 2.64 | 6.10% | | 60.05 | 3.26 | 5.40% | 1.42 | 2.40% | 1.21 | 2.00% | 3.75 | 6.30% | | 86.24 | 3.88 | 4.50% | 1.95 | 2.30% | 2.86 | 3.30% | 5.20 | 6.00% | | 120.73 | 7.52 | 6.20% | 1.90 | 1.60% | 2.92 | 2.40% | 8.28 | 6.90% | | 136.63 | 6.42 | 4.70% | 3.29 | 2.40% | 1.84 | 1.30% | 7.44 | 5.40% | {8} 9 | tTG IgG- Day to day reproducibility | | | | | | | | | | --- | --- | --- | --- | --- | --- | --- | --- | --- | | Mean (U/mL) | Within Run | | Between Run | | Between Day | | Total Precision | | | | SD | %CV | SD | %CV | SD | %CV | SD | %CV | | 27.56 | 1.04 | 3.80% | 1.19 | 4.30% | 0.69 | 2.50% | 1.72 | 6.20% | | 28.22 | 2.01 | 7.10% | 1.18 | 4.20% | 1.09 | 3.90% | 2.58 | 9.10% | | 33.72 | 2.54 | 7.50% | 0.00 | 0.00% | 1.02 | 3.00% | 2.73 | 8.10% | | 40.65 | 2.30 | 5.70% | 2.19 | 5.40% | 1.39 | 3.40% | 3.47 | 8.50% | | 48.39 | 3.40 | 7.00% | 1.49 | 3.10% | 0.00 | 0.00% | 3.71 | 7.70% | | 50.61 | 3.19 | 6.30% | 0.00 | 0.00% | 1.39 | 2.70% | 3.48 | 6.90% | | 78.54 | 4.84 | 6.20% | 2.00 | 2.50% | 2.99 | 3.80% | 6.03 | 7.70% | | 87.78 | 3.72 | 4.20% | 2.44 | 2.80% | 3.28 | 3.70% | 5.53 | 6.30% | | 99.21 | 6.20 | 6.20% | 3.51 | 3.50% | 3.22 | 3.20% | 7.82 | 7.90% | iii. Lot-to-Lot Reproducibility Testing Negative and positive samples covering the measuring range for each device, including at least 10% of total samples within ±25% of the cut-off for each assay, were run in replicates of five (5) per kit, two (2) kits per lot, from three (3) kit lots were tested on one instrument by one operator for a maximum of 30 results (5x2x3) per sample per analyte. Ten (10) results per sample per analyte, per lot were evaluated. | Lot-to-lot reproducibility | | | | | | | | | --- | --- | --- | --- | --- | --- | --- | --- | | DGP IgA | | DGP IgG | | tTG IgA | | tTG IgG | | | Mean (U/mL) | %C.V. | Mean (U/mL) | %C.V. | Mean (U/mL) | %C.V. | Mean (U/mL) | %C.V. | | 9.35 | 9.4% | 11.74 | 5.1% | | | | | | 11.90 | 7.2% | 15.42 | 5.8% | 19.54 | 5.8% | 29.58 | 6.6% | | 15.04 | 6.0% | 17.98 | 4.7% | 32.60 | 6.7% | 34.40 | 8.1% | | 16.65 | 6.9% | 33.08 | 6.3% | 38.05 | 6.1% | 43.83 | 6.8% | | 18.66 | 4.7% | 46.42 | 4.6% | 44.31 | 6.8% | 50.13 | 9.5% | | 20.07 | 5.0% | 74.15 | 6.7% | 60.49 | 6.1% | 62.01 | 7.6% | | 59.91 | 5.7% | 85.92 | 6.0% | 95.28 | 4.5% | 65.06 | 7.5% | | 65.01 | 7.1% | 116.14 | 4.5% | 135.20 | 6.8% | 103.74 | 8.4% | iv. Instrument-to-Instrument Reproducibility Testing Two studies were performed. In the first study, the same samples from the lot-to-lot study were tested in replicates of five (5) per kit, two (2) kits per lot, {9} from three (3) kit lots on three (3) instruments at three (3) different sites (one internal, one external in Canada and one external in USA) by three (3) operators for a maximum 30 results (5x2x3) per instrument per analyte and 90 results (5x2x3x3) per sample per analyte. | Instrument-to-instrument Study #1 | | | | | | | | | --- | --- | --- | --- | --- | --- | --- | --- | | DGP IgA | | DGP IgG | | tTG IgA | | tTG IgG | | | Mean (U/mL) | % C.V. | Mean (U/mL) | % C.V. | Mean (U/mL) | % C.V. | Mean (U/mL) | % C.V. | | 8.58 | 9.6% | 16.61 | 9.3% | 19.22 | 5.5% | 29.52 | 9.9% | | 16.64 | 7.8% | 32.52 | 8.0% | 32.12 | 6.4% | 47.76 | 9.8% | | 18.73 | 7.5% | 88.87 | 6.9% | 46.76 | 8.6% | 51.00 | 10.1% | | 21.01 | 7.9% | 123.18 | 6.0% | 65.78 | 6.4% | 63.68 | 9.9% | | 61.71 | 9.0% | | | 177.91 | 5.7% | 105.62 | 8.6% | In study two, a set of fourteen (14) celiac samples with results near the cut-off for one or more analytes were tested at a single site in duplicates on each of three instruments for a maximum of six (6) results per sample. Three kits from the same lot were used. A minimum of four (4) samples were reported per analyte. Two of the three instruments used in the study were used in the original instrument-to-instrument study (SQX1 and SQX 2) and the third instrument is a new one (SQX 3). All three runs were performed by one operator at SQI. | Instrument-to-instrument Study #2 | | | | | | | | | --- | --- | --- | --- | --- | --- | --- | --- | | DGP IgA | | DGP IgG | | tTG IgA | | tTG IgG | | | Mean (U/mL) | % C.V. | Mean (U/mL) | % C.V. | Mean (U/mL) | % C.V. | Mean (U/mL) | % C.V. | | 15.37 | 6.27 | 13.63 | 7.18 | 24.64 | 6.30 | 35.49 | 6.46 | | 15.81 | 6.06 | 11.41 | 3.63 | 23.35 | 4.18 | 29.34 | 8.91 | | 9.77 | 6.43 | 15.75 | 5.73 | 23.59 | 4.40 | 33.11 | 7.07 | | 18.85 | 3.93 | 17.90 | 5.56 | 17.69 | 2.91 | 37.53 | 5.61 | | | | 16.69 | 4.57 | | | 30.59 | 6.19 | | | | 17.82 | 2.86 | | | 50.12 | 2.84 | b. Linearity/assay reportable range: i. Linearity Serial dilutions of high positive clinical samples in normal serum were assayed across each analyte range. A minimum of seven levels with three replicates at each level {10} were evaluated. Because the instrument reports values below the cut-off as “&lt;” instead of with a number, the concentration of analyte in normal serum was arbitrarily assigned a value below the cut-off so expected concentrations could be calculated. Using the analyte concentration values for different dilutions, a graph of observed (measured) values vs. expected concentrations was plotted. The claimed upper limit is rounded off to the nearest tenth of the result. | Linearity | | | | | | | | --- | --- | --- | --- | --- | --- | --- | | Analyte | Sample | Test Range (U/mL) | R² | Intercept | Slope | Linear Range (U/mL) | | DGP IgA | S1 | 6.80–115.01 | 0.994 | -3.427 | 1.030 | 8.0 – 110.0 | | | S2 | 9.27–73.86 | 0.992 | -1.846 | 0.977 | | | | S3 | 6.34–44.87 | 0.992 | -0.614 | 0.995 | | | DGP IgG | S1 | 11.18–129.70 | 0.997 | -1.506 | 1.036 | 9.0 – 120.0 | | | S2 | 5.20–86.34 | 0.992 | -2.302 | 1.033 | | | | S3 | 5.84–60.09 | 0.996 | -0.466 | 0.991 | | | tTG IgA | S1 | 10.42–142.72 | 0.996 | 1.879 | 1.015 | 16.0-140.0 | | | S2 | 11.37–94.50 | 0.996 | 0.082 | 1.013 | | | | S3 | 11.89–94.7211 | 0.991 | 0.697 | 1.045 | | | tTG IgG | S1 | 13.07–103.60 | 0.991 | 2.996 | 1.017 | 24.0 – 100.0 | | | S2 | 12.19–87.89 | 0.995 | 1.331 | 1.016 | | | | S3 | 11.71–82.17 | 0.984 | 4.226 | 1.013 | | ii. Analytical Measuring Range: The measuring range was defined as lower and upper limit of reporting range: DGP IgA: 8.0–110.0 U/mL DGP IgG: 9.0–120.0 U/mL tTG IgA: 16.0–140.0 U/mL tTG IgG: 24.0–100.0 U/mL c. Traceability, Stability, Expected values (controls, calibrators, or methods): i. Controls and Calibrators a. Assay Standards (Calibrators) There are no international standard materials available for celiac anti-DGP IgA and IgG, and anti-tTG IgA and IgG. The calibrators were assigned with arbitrary units. The raw material for the standards is celiac positive patient {11} serum sample with high titers for anti-DGP IgA and IgG and anti-tTG IgA and IgG. During development, the raw material samples were diluted to a desired anchorage point, e.g., 2000x, 4000x and 16000x for a U/mL value assignment. These anchorage levels were selected to ensure coverage of the hypothetical cut-off points. A standard curve was constructed using the chosen dilution levels for each. The external standards are eight serial dilutions of a sample derived from human sera containing an appropriate representation of each of the analytes to be reported. Each assay has a specific standard range that is present in the standards; these are encoded in the bar code for each standard. In-house standards are prepared, and are assigned with arbitrary units. For new standards, the repetitiveness of the standard curves and bias of fit versus measured values were considered. Eight standard levels were then selected. Curve fitting algorithms were configured for each analyte. Finally, the standards were validated using celiac positive and normal samples. In this study, cut-off values and sensitivity/specificity of the assay were evaluated and to a large extent were found within industry range. The positive and negative control samples are derived from human samples. The positive controls were prepared by diluting celiac positive patient serum sample to give results above cut-off for applicable analytes and negative control was prepared by diluting normal human serum sample to give results below cut-off for all four analytes. Expected results for each applicable analyte of each control are provided in the Certificate of Analysis. b. Antigen The DGP peptides are a mix of custom sequences, which are conjugated and then lyophilized. Upon receipt, the Certificate of Analysis is confirmed and an incoming acceptance test is performed using an HPLC method to verify the purity and consistency of the peptides. The tTG is recombinant tissue transglutaminase in a buffer solution. The Certificate of Analysis is verified upon receipt of the material and incoming acceptance testing confirms protein concentration, content, and characteristics using Bradford Protein Assay, Western Blot and SDS-Page methods, respectively. c. Reporter_mix The reporter mix is comprised of fluorescently labeled secondary antibodies, Mouse anti-human IgG- Cy5; goat anti-human IgA: Cy3. The same dyes are used for anti-DGP and anti-tTG, but different capture spots in the microarray are analyzed to provide the results for each of the four analytes. 12 {12} ii. Hook Effect: High positive samples were selected and tested at a starting dilution of 1:51. Therefore, since the assay's dilution as per package insert is 1:151, at least three fold higher values can be achieved. All of the samples were serially diluted to evaluate any hook effect in the assays. It was observed that the results were above the assay's reportable range, therefore, during this development study, a manual analysis was performed to see how high above the reportable range the actual values were. The instrument saturated at $&gt;200\mathrm{U / mL}$ for DGP IgG and IgA, so no hook effect was detected. No hook effect was detected at $1354\mathrm{U / mL}$ for tTG IgA and $835\mathrm{U / mL}$ for tTG IgG. iii. Stability: a. Sample stability - The sponsor provided data supporting a claim of 1 week stability at $2 - 8^{\circ}\mathrm{C}$ . b. Shelf life stability - the sponsor provided data supporting a claim of 6 months stability when stored according to the package insert. c. Open-vial stability - The directions for use state that the kit is single-use only; therefore, open vial stability is not applicable. d. Detection limit: i. Limit of Blank (LoB): Sample diluent from the kit was tested in replicates of seventy three (73) on one kit to determine the software-derived values for each analyte. Values are reported in fluorescence intensity units. All measurements are below the bottom of the AMR. | LoB | DPG IgA | DPG IgG | tTG IgA | tTG IgG | | --- | --- | --- | --- | --- | | Mean | 159.6 | 192.0 | 125.2 | 77.8 | | STDEV | 64.5 | 37.0 | 108.4 | 33.0 | | LoB | 265.6 | 252.7 | 303.4 | 132.1 | ii. Limit of Detection (LoD): Thirteen (13) normal human serum samples were tested in replicates of five (5), on two runs (total $n = 130$ ) using two different kit lots and two different instruments to determine the software-derived LoD values for each analyte. For analytes, where some replicates reported numerical values, the probability {13} of values being the same as the intensity values was calculated and reported as a percentage. | | DPG IgA | DPG IgG | tTG IgA | tTG IgG | | --- | --- | --- | --- | --- | | LoB | 265.6 | 252.7 | 303.4 | 132.1 | | STDEV | 116.7 | 241.6 | 125.3 | 196.1 | | LoD | 457.5 | 650.1 | 509.4 | 454.7 | iii. Limit of Quantitation (LoQ): Eleven (11) diluted celiac positive samples (to report three samples per analyte) were tested over five (5) runs, nine (9) replicates per run. Two kit lots were used for a total of ten (10) runs. Bias was calculated based on the expected value and mean, SD and %CV were calculated for each sample. The claimed LoQs meet the manufacturer's required acceptance criteria. | Analyte | Result for LoQ (U/mL) | LoQ Claim (U/mL) | Bias (%) | CV (%) | | --- | --- | --- | --- | --- | | DGP IgA | 8.25 | 8.0 | 0.4 | 8.9 | | DGP IgG | 9.02 | 9.0 | 2.2 | 6.2 | | tTG IgA | 16.23 | 16.0 | 4.5 | 7.9 | | tTG IgG | 24.30 | 24.0 | 1.8 | 6.0 | e. Analytical specificity: i. Cross-reactivity This information is contained in the Clinical Sensitivity and Specificity section. ii. Interference This study was performed according to the recommendations in CLSI EP7-A2, Interference Testing in Clinical Chemistry, Approved Guideline. Eight celiac positive samples (from the SQI Diagnostics' Sample Bank) and one normal (negative sample) were included in the study in order to report two levels per analyte. The individual specimens were spiked with three concentrations of the interferents, starting at the maximum concentration and titering down to low levels. The samples were assayed in replicates of $n = 5$ for control and for unspiked, and the mean result reported. Interference was calculated as the analyte recovery in the presence of interferent, relative to the measurement of the analyte {14} in a control, unspiked sample. The level of non-interference claimed was that level where there was $\leq 15\%$ difference between "control results" and "spiked results." Interferent testing concentrations: | Interferent | Interferent Test Level (20x stock) | | | | --- | --- | --- | --- | | | Test Level 1 | Test Level 2 | Test Level 3 | | Bilirubin (conjugated) | 3.0 mg/mL | 1.5 mg/mL | 0.8 mg/mL | | Hemoglobin | 100.0 mg/mL | 50.0 mg/mL | 25.0 mg/mL | | Triglycerides | 100.0 mg/mL | 50.0 mg/mL | 25.0 mg/mL | | IgG | 10.0 mg/mL | 5.0 mg/mL | 2.5 mg/mL | There was no interference detected by any of the interfering substances at the concentrations tested. # f. Assay cut-off: Specimens from 110 celiac diagnosed patients, 126 normal donors and 56 other autoimmune diseases (total =292) were collected from commercial sources and collaborative laboratories representative of North American and European populations. Specimens were tested according to standard procedure, and all specimens were assayed in duplicate on separate kits using two lots of assay kits on two different squid-X instruments for the determination of mean values. These samples were not used in any other validation assays. The samples and demographic information are presented below: | Condition | N | Gender | | | Age (years) | | | Demographic | | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | | | M | F | Unk | Min | Max | Ave. | North America | Europe | | Celiac, Biopsy Confirmed | 10 | 4 | 6 | 0 | 21 | 46 | 29 | 10 | 0 | | Celiac, EMA Positive (adult) | 50 | 12 | 38 | 0 | 22 | 90 | 45 | 12 | 38 | | Celiac, EMA positive (pediatric) | 50 | 16 | 30 | 4 | 0 | 21 | 12 | 10 | 40 | | Normal Female Serum | 54 | 0 | 54 | 0 | 20 | 63 | 40 | 54 | 0 | | Normal Male Serum | 72 | 72 | 0 | 0 | 18 | 64 | 41 | 72 | 0 | | Crohn's Disease | 5 | 2 | 3 | 0 | 26 | 66 | 46 | 5 | 0 | {15} 16 | Condition | N | Gender | | | Age (years) | | | Demographic | | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | | | M | F | Unk | Min | Max | Ave. | North America | Europe | | Ulcerative Colitis | 5 | 3 | 2 | 0 | 17 | 68 | 38 | 5 | 0 | | Systemic Lupus Erythematous | 14 | 2 | 12 | 0 | 24 | 63 | 38 | 10 | 4 | | Rheumatoid Arthritis (RA) | 22 | 15 | 7 | 0 | 45 | 72 | 58 | 22 | 0 | | Vasculitis | 10 | 0 | 10 | 0 | 26 | 63 | 44 | 0 | 10 | The cut-offs were selected by balancing sensitivity and specificity using ROC curves using Analyze-it v.2.30. The chosen cut-offs for each assay are: | Analyte | Cut-off (U/mL) | Sensitivity (%) | 95% CI | Specificity (%) | 95% CI | | --- | --- | --- | --- | --- | --- | | DGP IgA | 15.0 | 85.5 | 0.775 – 0.915 | 99.5 | 0.970 to 1.00 | | DGP IgG | 13.0 | 94.5 | 0.885 – 0.980 | 97.8 | 0.945 to .994 | | tTG IgA | 20.0 | 97.3 | 0.922 – 0.994 | 97.8 | 0.945 to .994 | | tTG IgG | 36.0 | 58.2 | 0.484 – 0.675 | 97.8 | 0.944 to .994 | ## 2. Comparison studies: a. Method comparison with predicate device: 229 positive celiac patient samples and 150 non-celiac disease samples including presumptively normal samples were assayed with the Ig_plex Celiac DGP Panel and with the available predicates' ELISA method for each of the analytes. The celiac patient samples were classified using the following characteristics: 147 samples from celiac diagnosed, biopsy confirmed patients, 27 Endomysial Antibody (EMA) positive pediatric patient samples, 45 EMA positive adult patient samples, nine celiac diagnosed without additional information, and one IgA deficient celiac sample. The non-celiac samples included 132 presumptively normal samples and 18 other autoimmune disease samples. The sponsor analyzed the discrepant samples around the cut-off; the majority that were positive in the Ig_plex assays were Celiac-biopsy positive. i. DGP IgA | | Quanta Lite DGP IgA | | Total | PPA and NPA | | | | | --- | --- | --- | --- | --- | --- | --- | --- | | | | Positive | | Negative | | % Agreement | 95% CI | | Ig_plex DGP IgA | Positive | 126 | 9 | 135 | Positive | 93.3 | 87.8–96.5 | | | Negative | 9 | 173 | 182 | Negative | 95.1 | 90.9–97.4 | | | Total | 135 | 182 | 317 | Overall | 94.3 | 91.2–96.4 | {16} ii. DGP IgG | | Quanta Lite DGP IgG | | Total | PPA and NPA | | | | | --- | --- | --- | --- | --- | --- | --- | --- | | | | Positive | | Negative | | % Agreement | 95% CI | | Ig_plex DGP IgG | Positive | 129 | 19 | 148 | Positive | 98.5 | 94.6–99.6 | | | Negative | 2 | 176 | 178 | Negative | 90.3 | 85.3–93.7 | | | Total | 131 | 195 | 326 | Overall | 93.6 | 90.4–95.7 | iii. tTG IgA | | Quanta Lite tTG IgA | | Total | PPA and NPA | | | | | --- | --- | --- | --- | --- | --- | --- | --- | | | | Positive | | Negative | | % Agreement | 95% CI | | Ig_plex tTG IgA | Positive | 133 | 23 | 156 | Positive | 100.0 | 97.2–100.0 | | | Negative | 0 | 167 | 167 | Negative | 87.9 | 82.5–91.8 | | | Total | 133 | 190 | 323 | Overall | 92.9 | 89.5–95.2 | iv. tTG IgG | | Quanta Lite tTG IgG | | Total | PPA and NPA | | | | | --- | --- | --- | --- | --- | --- | --- | --- | | | | Positive | | Negative | | % Agreement | 95% CI | | Ig_plex tTG IgG | Positive | 48 | 35 | 83 | Positive | 94.1 | 84.1–98.0 | | | Negative | 3 | 192 | 195 | Negative | 84.6 | 79.3–88.7 | | | Total | 51 | 227 | 277 | Overall | 86.3 | 81.8–89.9 | b. Matrix comparison Not applicable 3. Clinical studies: a. Clinical Sensitivity and specificity: The study included 378 samples, collected from the following groupings: 128 celiac diagnosed by Marsh III criteria, biopsy confirmed, and 250 samples from other non-celiac autoimmune diseases. Samples were assayed according to the Ig_plex Celiac DGP Panel Instructions for Use. Clinical sensitivity was calculated from celiac disease patients. Clinical specificity was calculated for each disease subgroup and the overall specificity for the total of non-celiac disease patients and normal subject samples were also calculated. The sample distribution included both pediatric and adult subjects: {17} | Disease Code | Disease Category | Age (years) | | --- | --- | --- | | Celiac Biopsy | Celiac Disease Biopsy Confirmed | 4-82 | | SLE | SLE | 21-85 | | SS | Sjögren’s Syndrome | 30-76 | | Wheat Allergy | Wheat Allergy | 2 (3 unknown) | | IBD (Crohn's Disease) | IBD (Crohn's Disease) | 23-92 | | IBD (Ulcerative Colitis) | IBD (Ulcerative Colitis) | 19-68 | | Lactose Intolerant | Lactose Intolerant | 32-60 | | Osteoarthritis | Osteoarthritis | 46-83 | | Celiac 1° Relative | Celiac 1o Relative | 20-59 | | IgA Def, non-Celiac | IgA Deficient, non-Celiac | 60-101 | | RA | Rheumatoid Arthritis (RA) | 45-72 | | HT | Hashimoto’s Thyroiditis | 30-57 | | GT | Grave’s Thyroiditis | 27-67 | | Vasculitis | Vasculitis | 26-62 | | EBV Positive | EBV Positive | 34-54 | | Syphilis Positive | Syphilis Positive | 18-60 | | Diabetes Mellitus (Types 1 and 2) | Diabetes Mellitus | 39-70 | | Suspected Celiac, biopsy negative | Suspected Celiac, biopsy negative | 20-73 | | OAD | Other Autoimmune Diseases | 20-83 | | Normal Female | Normal Female | 18-66 | | Normal Male | Normal Male | 19-90 | The manufacturer's pre-specified acceptance criteria are: Specificity for each analyte should be $&gt;95\%$ ; Sensitivity should be $&gt;72\%$ for DGP IgA, $&gt;85\%$ for DGP IgG, $&gt;90\%$ for tTG IgA and $&gt;45\%$ for tTG IgG. i. DGP IgA | DGP IgA | Celiac Disease | | Total | | | --- | --- | --- | --- | --- | | | | Positive | | Negative | | Ig_plex | Positive | 102 | 2 | 104 | | | Negative | 26 | 248 | 274 | | | Total | 128 | 250 | 378 | Sensitivity: $79.7\%$ (95% CI 76.1% - 83.2%) Specificity: $99.2\%$ (95% CI 98.6% - 99.8%) {18} 19 | DGP IgA Sample Group | Sample Disease Classification | Number of Samples | Ig_plex | Sens | | --- | --- | --- | --- | --- | | TP | FN | | Celiac Disease (Sensitivity) | Celiac Disease Biopsy confirmed | 128 | 102 | 26 | 79.7% | | | TN | FP | Spec | | Non-Celiac Diseases (Specificity) | SLE | 30 | 0 | 30 | 100% | | Sjögren’s syndrome | 14 | 0 | 14 | 100% | | Wheat allergy | 4 | 0 | 4 | 100% | | IBD (Crohn’s Disease) | 24 | 0 | 24 | 100% | | IBD (Ulcerative Colitis) | 25 | 0 | 25 | 100% | | Lactose Intolerant | 8 | 1 | 7 | 87.5 | | Osteoarthritis | 10 | 0 | 10 | 100.0% | | Celiac 1° relative | 5 | 0 | 5 | 100% | | IgA Deficient, non-Celiac | 3 | 1 | 2 | 66.7% | | Rheumatoid Arthritis | 26 | 0 | 26 | 100% | | Hashimoto’s thyroiditis | 20 | 0 | 20 | 100% | | Vasculitis | 16 | 0 | 16 | 100% | | EBV positive | 6 | 0 | 6 | 100% | | Syphilis positive | 7 | 0 | 7 | 100% | | Type 2 Diabetes | 2 | 0 | 2 | 100% | | Type I Diabetes | 10 | 0 | 10 | 100% | | Grave’s Thyroiditis | 12 | 0 | 12 | 100% | | Suspected celiac, negative biopsy | 10 | 0 | 10 | 100% | | Other autoimmune disease | 16 | 0 | 16 | 100% | | | Total non-celiac diseases | 250 | 2 | 248 | 99.2% | ii. DGP IgG | DGP IgG | Celiac Disease | | Total | | | --- | --- | --- | --- | --- | | | | Positive | | Negative | | Ig_plex | Positive | 114 | 1 | 115 | | | Negative | 14 | 249 | 263 | | | Total | 128 | 250 | 378 | Sensitivity: 89.1% (95% CI 86.3% - 91.8%) Specificity: 99.6% (95% CI 99.2% - 100.0%) {19} 20 | DGP IgG Sample Group | Sample Disease Classification | Number of Samples | Ig_plex | Sens | | --- | --- | --- | --- | --- | | TP | FN | | Celiac Disease (Sensitivity) | Celiac Disease Biopsy confirmed | 128 | 114 | 14 | 89.1% | | | TN | FP | Spec | | Non-Celiac Diseases (Specificity) | SLE | 30 | 0 | 30 | 100% | | Sjögren’s syndrome | 14 | 0 | 14 | 100% | | Wheat allergy | 4 | 0 | 4 | 100% | | IBD (Crohn’s Disease) | 24 | 0 | 24 | 100% | | IBD (Ulcerative Colitis) | 25 | 0 | 25 | 100% | | Lactose Intolerant | 8 | 0 | 8 | 100% | | Osteoarthritis | 10 | 0 | 10 | 100% | | Celiac 1° relative | 5 | 0 | 5 | 100% | | IgA Deficient, non-Celiac | 3 | 0 | 3 | 100% | | Rheumatoid Arthritis | 26 | 1 | 25 | 96.2% | | Hashimoto’s thyroiditis | 20 | 0 | 20 | 100% | | Vasculitis | 16 | 0 | 16 | 100% | | EBV positive | 6 | 0 | 6 | 100% | | Syphilis positive | 7 | 0 | 7 | 100% | | Type 2 Diabetes | 2 | 0 | 2 | 100% | | Type 1 Diabetes | 10 | 0 | 10 | 100% | | Other autoimmune disease | 16 | 0 | 16 | 100% | | Grave’s Thyroiditis | 12 | 0 | 12 | 100% | | Suspected celiac, negative biopsy | 10 | 0 | 10 | 100% | | | Total non-celiac diseases | 250 | 1 | 249 | 99.6% | iii. tTG IgA | tTG IgA | Celiac Disease | | Total | | | --- | --- | --- | --- | --- | | | | Positive | | Negative | | Ig_plex | Positive | 126 | 0 | 126 | | | Negative | 2 | 250 | 252 | | | Total | 128 | 250 | 378 | Sensitivity: 98.4% (95% CI 97.3% - 99.5%) Specificity: 100.0% (95% CI 100.0% - 100.0%) {20} 21 | tTG IgA Sample Group | Sample Disease Classification | Number of Samples | Ig_plex | Sens | | --- | --- | --- | --- | --- | | TP | FN | | Celiac Disease (Sensitivity) | Celiac Disease Biopsy confirmed | 128 | 126 | 2 | 98.4% | | | TN | FP | Spec | | Non-Celiac Diseases (Specificity) | SLE | 30 | 0 | 30 | 100% | | Sjögren’s syndrome | 14 | 0 | 14 | 100% | | Wheat allergy | 4 | 0 | 4 | 100% | | IBD (Crohn’s Disease) | 24 | 0 | 24 | 100% | | IBD (Ulcerative Colitis) | 25 | 0 | 25 | 100% | | Lactose Intolerant | 8 | 0 | 8 | 100% | | Celiac 1° relative | 5 | 0 | 5 | 100% | | IgA Deficient, non-Celiac | 3 | 0 | 3 | 100% | | Rheumatoid Arthritis | 26 | 0 | 26 | 100% | | Hashimoto’s thyroiditis | 20 | 0 | 20 | 100% | | Vasculitis | 16 | 0 | 16 | 100% | | EBV positive | 6 | 0 | 6 | 100% | | Syphilis positive | 7 | 0 | 7 | 100% | | Type 2 Diabetes | 2 | 0 | 2 | 100% | | Type I Diabetes | 10 | 0 | 10 | 100% | | Other autoimmune disease | 16 | 0 | 16 | 100% | | Grave’s Thyroiditis | 12 | 0 | 12 | 100% | | Suspected celiac, negative biopsy | 10 | 0 | 10 | 100% | | | Total non-celiac diseases | 250 | 0 | 250 | 100% | iv. tTG IgG | tTG IgG | Celiac Disease | | Total | | | --- | --- | --- | --- | --- | | | | Positive | | Negative | | Ig_plex | Positive | 60 | 3 | 63 | | | Negative | 68 | 247 | 315 | | | Total | 128 | 250 | 378 | Sensitivity: 46.9% (95% CI 42.5% - 51.3%) Specificity: 98.8% (95% CI 98.1% - 99.5%) {21} | tTG IgG Sample Group | Sample Disease Classification | Number of Samples | Ig_plex | Sens | | --- | --- | --- | --- | --- | | TP | FN | | Celiac Disease (Sensitivity) | Celiac Disease Biopsy confirmed | 128 | 60 | 68 | 46.9% | | | TN | FP | Spec | | Non-Celiac Diseases (Specificity) | SLE | 30 | 1 | 29 | 96.7% | | Sjögren’s syndrome | 14 | 0 | 14 | 100% | | Wheat allergy | 4 | 0 | 4 | 100% | | IBD (Crohn’s Disease) | 24 | 0 | 24 | 100% | | IBD (Ulcerative Colitis) | 25 | 0 | 25 | 100% | | Lactose Intolerant | 8 | 1 | 7 | 87.5% | | Osteoarthritis | 10 | 0 | 10 | 100% | | Celiac 1° relative | 5 | 0 | 5 | 100% | | IgA Deficient, non-Celiac | 3 | 0 | 3 | 100% | | Rheumatoid Arthritis | 26 | 1 | 25 | 96.2% | | Hashimoto’s thyroiditis | 20 | 1 | 19 | 95.0% | | Vasculitis | 16 | 0 | 16 | 100% | | EBV positive | 6 | 0 | 6 | 100% | | Syphilis positive | 7 | 0 | 7 | 100% | | Type 2 diabetes | 2 | 0 | 2 | 100% | | Type 1 diabetes | 10 | 0 | 10 | 100% | | Grave’s Thyroiditis | 12 | 0 | 12 | 100% | | Suspected celiac, negative biopsy | 10 | 0 | 10 | 100% | | Other autoimmune disease | 16 | 0 | 16 | 100% | | | Total non-celiac diseases | 250 | 3 | 247 | 98.8% | c. Other clinical supportive data (when a. and b. are not applicable): Not applicable 4. Clinical cut-off: See assay cut-off 5. Expected values/Reference range: Specimens from 328 presumptively normal donors (including 167 males ranging in age from 19 to 90 years, and 161 females ranging in age from 18 to 66 years) were collected from commercial sources. Specimens were tested according to standard procedure, and all specimens were assayed in single replicates. The normal response is negative. {22} | Analyte | DGP IgA | DGP IgG | tTG IgA | tTG IgG | | --- | --- | --- | --- | --- | | N (% Positive) | 4 (1.22%) | 10 (3.05%) | 2 (0.61%) | 2 (0.61%) | | Range (U/mL) | <8.00 – 46.30 | <9.00 – 55.04 | <16.00 – 100.96 | <24.00 – 54.33 | | Negative Result (U/mL) | <15.0 | <13.0 | <20.0 | <36.0 | | Positive Result (U/mL) | ≥15.0 | ≥13.0 | ≥20.0 | ≥36.0 | N. Instrument Name: sqid-X system O. System Descriptions: 1. Modes of Operation: Semi-automated: All sample handling and preparation is performed by the user, and adds reagents to each well. The instrument incubates, washes, scans and analyzes the results. The sqid-X software provides processing instructions and analysis which are configured for the Ig_plex Celiac DGP Panel. 2. Software: FDA has reviewed applicant’s Hazard Analysis and software development processes for this line of product types: Yes ☐ x ☐ or No ☐ 3. Specimen Identification: Each 96-well plate has a barcode. The user types the sample information into the computer file. 4. Specimen Sampling and Handling: The system uses graphical user interface software to lead the operator through manual sample, reagent, and plate handling operations. The system controls the washer to ensure that the same wash programs are used every time and in the correct sequence. Once the assay’s biochemical reactions are complete and the plate is placed in the scanner, the instrument automatically performs a multi-wavelength fluorescent scan of each well in the microarray, analyzes the data, and generates a report containing results for all assay markers. Results for each patient sample are obtained simultaneously for each of the four 23 {23} Ig_plex Celiac DGP Panel markers: DGP IgA, DGP IgG, tTG IgA and tTG IgG using the data from one well containing one sample of the patient's serum. The system does not interpret the results with respect to diagnosis; it reports each result independently. The sqid-X system also includes numerous internal quality checks. 5. Calibration: The assay has eight calibrators/standards that are aliquoted into specific wells of the 96-well plate. These wells do not have the microarray of antigens/controls that are in the sample wells. 6. Quality Control: In addition to the results calculation, a set of internal quality control rules are invoked to evaluate the data that is produced. These rules were identified by safety and hazard analysis to mitigate risks such as general failures in the reaction. They include quality control rules for checking the fitness of the standardization curves, as well as thresholds of controls measuring reporter activity. When data is found that does not fit the required control levels or rules, further processing of the data is halted and the value "No Result" is reported. The internal quality control rules, or invalidation rules, are at each level of data processing. Single analyte results in a well may be invalidated due to high CV of the replicate capture spots; results for an entire well may be invalidated due to failing to meet a control threshold, and all results for an analyte on a plate may be invalidated due to an improper standard curve. P. Other Supportive Instrument Performance Characteristics Data Not Covered In The "Performance Characteristics" Section above: Not applicable. Q. Proposed Labeling: The labeling is sufficient and it satisfies the requirements of 21 CFR Part 809.10. R. Conclusion: The submitted information in this premarket notification is complete and supports a substantial equivalence decision. 24