Alegria Flash MPO; Alegria Flash PR3; Alegria Flash GBM

{0} [LOGO] FDA U.S. FOOD & DRUG ADMINISTRATION ### 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION SUMMARY ASSAY ONLY ### I Background Information: A 510(k) Number K261466 B Applicant ZEUS Scientific C Proprietary and Established Names Alegria Flash MPO Alegria Flash PR3 Alegria Flash GBM D Regulatory Information | Product Code(s) | Classification | Regulation Section | Panel | | --- | --- | --- | --- | | MOB MVJ | Class II | 21 CFR 866.5660 - Multiple autoantibodies immunological test system | IM - Immunology | ### II Submission/Device Overview: A Purpose for Submission: New Device B Measurand: Human IgG autoantibodies against MPO, PR3, and GBM C Type of Test: Automated, semi-quantitative, chemiluminescence immunoassay Food and Drug Administration 10903 New Hampshire Avenue Silver Spring, MD 20993-0002 www.fda.gov {1} ### III Intended Use/Indications for Use: #### A Intended Use(s): See Indications for Use below. #### B Indication(s) for Use: The Alegria Flash MPO kit uses chemiluminescent immunoassay (CLIA) technology for the semi-quantitative measurement of IgG antibodies to MPO in human serum. It is intended for use as an aid in the diagnosis of microscopic polyangiitis (MPA) in conjunction with other laboratory and clinical findings. The test must be performed on the Alegria Flash instrument. The Alegria Flash PR3 kit uses chemiluminescent immunoassay (CLIA) technology for the semi-quantitative measurement of IgG antibodies to PR3 in human serum. It is intended for use as an aid in the diagnosis of granulomatosis with polyangiitis (GPA) in conjunction with other laboratory and clinical findings. The test must be performed on the Alegria Flash instrument. The Alegria Flash GBM kit uses chemiluminescent immunoassay (CLIA) technology for the semi-quantitative measurement of IgG antibodies to GBM in human serum. It is intended for use as an aid in the diagnosis of Goodpasture's Syndrome in conjunction with other laboratory and clinical findings. The test must be performed on the Alegria Flash instrument. #### C Special Conditions for Use Statement(s): For Prescription Use Only #### D Special Instrument Requirements: For use with the Alegria Flash instrument (K230863). ### IV Device/System Characteristics: #### A Device Description: Each of test system (the Alegria Flash MPO, the Alegria Flash PR3, and the Alegria Flash GBM) contains the following components in sufficient quantities to perform the number of tests indicated on the packaging label. Assay Cartridge (1): includes three kit components: - Bead Suspension (1 vial): Magnetic particles coated with purified native antigen (MPO, PR3 or GBM, respectively for each test system) in 5 mL of storage buffer with Tween-20, bovine serum albumin, phosphate-buffered-saline, and <0.1% sodium azide. Ready to use. - Sample Diluent (2 vials): 10 mL of phosphate-buffered-saline solution containing detergent, proteins, <0.1% sodium azide, and ProClin 300. Ready to use. K261466 - Page 2 of 20 {2} - Conjugate (2 vials): 10 mL of anti-human IgG conjugated to Acridinium Ester. A protein, buffered solution containing, ProClin 300. Ready to use. Calibrators include: - Calibrator 1 (1 vial): Blue-capped vial. 0.5mL human serum containing anti-MPO antibodies at levels above the cutoff value. <0.1% sodium azide. Ready to use. Assay and Lot Specific - Calibrator 2 (1 vial): White-capped vial. 0.5mL human serum containing anti-MPO antibodies at levels near the cutoff value. <0.1% sodium azide. Ready to use. Assay and Lot Specific # Materials Required But Not Provided - Controls for each assay respectively: - The Alegria Flash MPO/PR3/GBM Control Kit (Positive and Negative) - 5× Wash Buffer concentrate - Trigger Solutions - Others: Reaction cuvettes, Sample tips, Biohazardous waste bags, End of day maintenance kit, Deionized water, One-liter graduated cylinder # B Principle of Operation: The Alegria Flash MPO kit is designed to detect IgG class antibodies against MPO antigen in human sera. The Alegria Flash PR3 kit is designed to detect IgG class antibodies against PR3 antigen in human sera. The Alegria Flash GBM kit is designed to detect IgG class antibodies against GBM antigen in human sera. The test procedure for each assay involves three steps: 1. Sample diluent, test sera, and antigen-coated magnetic particles are added to a reaction cuvette. During the initial incubation, anti-MPO, anti-PR3, or anti-GBM antibodies present in the serum will bind to the immobilized antigen. The beads are then washed to remove unbound antibodies and other serum components. 2. An acridinium ester-conjugated anti-human IgG solution is then added to the reaction cuvette. During the second incubation, the conjugate reacts with IgG antibodies immobilized on the magnetic particles in step 1. The beads are then washed to remove unbound conjugate. 3. Two trigger solutions are added to the cuvette containing immobilized acridinium ester conjugate, causing a flash chemiluminescence reaction to occur. The light signal released by the chemiluminescence reaction is measured by a photomultiplier within the Alegria Flash analyzer and reported as relative light units (RLU). RLU values are converted to Relative Units (RU) within the software. RU values above a predetermined cutoff are indicative of the presence of anti-MPO antibodies within the original serum sample. # V Substantial Equivalence Information: # A Predicate Device Name(s): AtheNA Multi-Lyte MPO/PR3 IgG Test System, AtheNA Multi-Lyte ANCA K261466 - Page 3 of 20 {3} # **B Predicate 510(k) Number(s):** K040111 K071219 # **C Comparison with Predicate(s):** | Device & Predicate: | K251440 (Candidate) | K040111 (Predicate) | | --- | --- | --- | | Device Trade Name | Alegria Flash MPO | AtheNA Multi-Lyte MPO/PR3 IgG Test System | | **General Device Characteristic Similarities** | | | | Sample Matrix | Serum | Same | | Detection | Semi-quantitative | Same | | **General Device Characteristic Differences** | | | | Intended Use/Indications For Use | The Alegria Flash MPO kit uses chemiluminescent immunoassay (CLIA) technology for the semi-quantitative measurement of IgG antibodies to MPO in human serum. It is intended for use as an aid in the diagnosis of microscopic polyangiitis (MPA) in conjunction with other laboratory and clinical findings. The test must be performed on the Alegria Flash instrument. | The ZEUS AtheNA Multi-Lyte Autoimmune Vasculitis (AIV) Plus Test System is intended for the qualitative and semi-quantitative detection of IgG class antibody to two separate antigens; (Myeloperoxidase and Proteinase 3) in human serum. The test system is intended to be used as an aid in the diagnosis of various autoimmune vasculitic disorders characterized by elevated levels of selected autoantibodies. MPO and/or PR-3 may be associated with autoimmune disorders such as Wegener's Granulomatosis, ICGN, MPA and PRS. This test is for In Vitro diagnostic use only. | | Antigens | Myeloperoxidase (MPO) | (MPO) and Proteinase 3(PR-3) | | Technology | Chemiluminescent Immunoassay (CLIA) | Fluorescent multiplexed addressable bead immunoassay | | Solid Phase | Paramagnetic microbeads | Polystyrene microspheres | | Analytical Measuring Interval | 24.06 – 570.65 Relative Units (RU) | 1 – 1905 Units | | Cut-Off | 100 RU | 100 Units | | Result Interpretation | Negative: <100 RU Positive: ≥100 RU | Negative: <100 Units Equivocal: 100-120 Units Positive: >120 Units | K261466 - Page 4 of 20 {4} | Device & Predicate Device(s): | K251440 (Candidate) | K040111 (Predicate) | | --- | --- | --- | | Device Trade Name | Alegria Flash PR3 | AtheNA Multi-Lyte MPO/PR3 IgG Test System | | **General Device Characteristic Similarities** | | | | Sample Matrix | Serum | Same | | Detection | Semi-quantitative | Same | | **General Device Characteristic Differences** | | | | Intended Use/Indications For Use | The Alegria Flash PR3 kit uses chemiluminescent immunoassay (CLIA) technology for the semi-quantitative measurement of IgG antibodies to PR3 in human serum. It is intended for use as an aid in the diagnosis of granulomatosis with polyangiitis (GPA) in conjunction with other laboratory and clinical findings. The test must be performed on the Alegria Flash instrument | The ZEUS AtheNA Multi-Lyte Autoimmune Vasculitis (AIV) Plus Test System is intended for the qualitative and semi-quantitative detection of IgG class antibody to two separate antigens; (Myeloperoxidase and Proteinase 3) in human serum. The test system is intended to be used as an aid in the diagnosis of various autoimmune vasculitic disorders characterized by elevated levels of selected autoantibodies. MPO and/or PR3 may be associated with autoimmune disorders such as Wegener's Granulomatosis, ICGN, MPA and PRS. Anti-GBM antibodies aid in the diagnosis of Goodpasture's syndrome. This test is for In Vitro diagnostic use only. | | Antigens | Proteinase 3 (PR3) | (MPO) and Proteinase 3(PR3) | | Technology | Chemiluminescent Immunoassay (CLIA) | Fluorescent multiplexed addressable bead immunoassay | | Solid Phase | Paramagnetic microbeads | Polystyrene microspheres | | Analytical Measuring Interval (AMI) | 24.44 – 550.43 Relative Units (RU) | 1 – 2728 Units | | Cut Off | 100 RU | 100 Units | | Result Interpretation | Negative: <100 RU Positive: ≥100 RU | Negative: <100 Units Equivocal: 100-120 Units Positive: >120 Units | K261466 - Page 5 of 20 {5} | Device & Predicate Device(s): | K251440 (Candidate) | K071219 (Predicate) | | --- | --- | --- | | Device Trade Name | Alegria Flash GBM | AtheNA Multi-Lyte ANCA | | **General Device Characteristic Similarities** | | | | Sample Matrix | Serum | Same | | Detection | Semi-quantitative | Same | | **General Device Characteristic Differences** | | | | Intended Use/Indications For Use | The Alegria Flash GBM kit uses chemiluminescent immunoassay (CLIA) technology for the semi-quantitative measurement of IgG antibodies to GBM in human serum. It is intended for use as an aid in the diagnosis of Goodpasture's Syndrome in conjunction with other laboratory and clinical findings. The test must be performed on the Alegria Flash instrument | The ZEUS AtheNA Multi-Lyte Autoimmune Vasculitis (AIV) Plus Test System is intended for the qualitative and semi-quantitative detection of IgG class antibody to 3 separate antigens; Glomerular Basement Membrane, Myeloperoxidase and Proteinase 3) in human serum. The test system is intended to be used as an aid in the diagnosis of various autoimmune vasculitic disorders characterized by elevated levels of selected autoantibodies. MPO and/or PR-3 may be associated with autoimmune disorders such as Wegener's Granulomatosis, ICGN, MPA and PRS. Anti-Glomerular Basement Membrane (GBM) antibodies aid in the diagnosis of Goodpasture's syndrome. | | Antigens | Glomerular Basement Membrane (GBM) | MPO, PR3, Glomerular Basement Membrane (GBM) | | Technology | Chemiluminescent Immunoassay (CLIA) | Fluorescent multiplexed addressable bead immunoassay | | Solid Phase | Paramagnetic microbeads | Polystyrene microspheres | | Analytical Measuring Interval (AMI) | 27.76 – 504.08 Relative Units (RU) | 1 – 970 Units | | Cut-Off | 100 RU | 100 Units | | Interpretation | Negative: <100 RU Positive: ≥100 RU | Negative: <100 Units Equivocal: 100-120 Units Positive: >120 Units | ### VI Standards/Guidance Documents Referenced: The following Clinical and Laboratory Standards Institute (CLSI) guidelines were used: - CLSI EP05-A3 – Evaluation of Precision of Quantitative Measurement Procedures K261466 - Page 6 of 20 {6} - CLSI EP06, 2nd Ed. – Evaluation of Linearity of Quantitative Measurement Procedures - CLSI EP07, 3rd Ed. – Interference Testing in Clinical Chemistry - CLSI EP12, 3rd Ed. – Evaluation of Qualitative, Binary Output Examination Performance - CLSI EP17-A2 – Evaluation of Detection Capability for Clinical Laboratory Measurement Procedures - CLSI EP25, 2nd Ed. – Evaluation of Stability of In Vitro Medical Laboratory Test Reagents - CLSI EP28-A3c – Defining, Establishing and Verifying Reference Intervals in the Clinical Laboratory - CLSI GP44-A4 – Procedures for the Handling and Processing of Blood Specimens for Common Laboratory Tests ### VII Performance Characteristics (if/when applicable): #### A Analytical Performance: ##### 1. Precision/Reproducibility: The precision of the Alegria Flash MPO, Alegria Flash PR3, and Alegria Flash GBM was evaluated based on the CLSI Guideline CLSI EP05-A3. Within-laboratory precision: To evaluate the within-laboratory precision of the Alegria Flash MPO Assay, Alegria Flash PR3 Assay, and Alegria Flash GBM Assay, seven samples were prepared by diluting positive native serum samples with negative serum samples. The prepared samples, containing various levels of autoantibodies, were assayed in duplicate, twice a day, for 20 days, using one reagent lot, one instrument, and one operator, for a total of 80 measurements per sample. The standard deviation (SD) and %CV of the within-run, between-run, between-day, and total within-laboratory imprecision were calculated for each sample. The results are summarized in the following tables. Alegria Flash MPO: | Sample | N | Mean (RU) | Within-Run | | Between-Run | | Within-Day | | Between-Day | | Total | | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | | | | SD | %CV | SD | %CV | SD | %CV | SD | %CV | SD | %CV | | 1 | 80 | 48.3 | 2.5 | 5.2 | 4.1 | 8.4 | 4.8 | 9.9 | 2.7 | 5.6 | 5.5 | 11.4 | | 2 | 80 | 83.8 | 3.6 | 4.3 | 3.4 | 4.0 | 4.9 | 5.9 | 4.6 | 5.5 | 6.7 | 8.1 | | 3 | 80 | 109.6 | 4.4 | 4.0 | 4.9 | 4.5 | 6.6 | 6.0 | 4.2 | 3.9 | 7.8 | 7.2 | | 4 | 80 | 160.7 | 7.2 | 4.5 | 6.2 | 3.9 | 9.5 | 5.9 | 8.9 | 5.5 | 13.0 | 8.1 | | 5 | 80 | 266.9 | 8.5 | 3.2 | 10.1 | 3.8 | 13.2 | 4.9 | 8.7 | 3.3 | 15.8 | 5.9 | | 6 | 80 | 337.9 | 11.7 | 3.5 | 11.3 | 3.3 | 16.2 | 4.8 | 13.3 | 3.9 | 21.0 | 6.2 | | 7 | 80 | 435.3 | 17.5 | 4.0 | 7.3 | 1.7 | 19.0 | 4.4 | 15.8 | 3.6 | 24.7 | 5.7 | K261466 - Page 7 of 20 {7} Alegria Flash PR3: | Sample | N | Mean (RU) | Within-Run | | Between-Run | | Within-Day | | Between-Day | | Total | | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | | | | SD | %CV | SD | %CV | SD | %CV | SD | %CV | SD | %CV | | 1 | 80 | 35.9 | 1.8 | 4.9 | 1.0 | 2.8 | 2.0 | 5.7 | 0.7 | 2.0 | 2.2 | 6.0 | | 2 | 80 | 63.0 | 2.6 | 4.2 | 2.2 | 3.5 | 3.4 | 5.5 | 0.7 | 1.1 | 3.5 | 5.6 | | 3 | 80 | 104.2 | 4.1 | 4.0 | 2.8 | 2.6 | 5.0 | 4.8 | 3.4 | 3.3 | 6.0 | 5.8 | | 4 | 80 | 136.4 | 5.0 | 3.7 | 3.2 | 2.4 | 6.0 | 4.4 | 2.8 | 2.0 | 6.6 | 4.8 | | 5 | 80 | 265.8 | 10.1 | 3.8 | 7.2 | 2.7 | 12.4 | 4.7 | 6.0 | 2.3 | 13.8 | 5.2 | | 6 | 80 | 344.1 | 13.3 | 3.9 | 11.0 | 3.2 | 17.3 | 5.0 | 3.2 | 0.9 | 17.6 | 5.1 | | 7 | 80 | 430.9 | 12.3 | 2.9 | 16.5 | 3.8 | 20.6 | 4.8 | 7.8 | 1.8 | 22.0 | 5.1 | Alegria Flash GBM: | Sample | N | Mean (RU) | Within-Run | | Between-Run | | Within-Day | | Between-Day | | Total | | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | | | | SD | %CV | SD | %CV | SD | %CV | SD | %CV | SD | %CV | | 1 | 80 | 46.8 | 2.1 | 4.6 | 0.0 | 0.0 | 2.1 | 4.6 | 2.0 | 4.2 | 2.9 | 6.2 | | 2 | 80 | 78.1 | 4.2 | 5.4 | 1.3 | 1.7 | 4.4 | 5.6 | 1.9 | 2.4 | 4.8 | 6.1 | | 3 | 80 | 102.0 | 5.7 | 5.5 | 0.0 | 0.0 | 5.7 | 5.5 | 3.2 | 3.2 | 6.5 | 6.4 | | 4 | 80 | 116.9 | 5.6 | 4.8 | 0.0 | 0.0 | 5.6 | 4.8 | 3.2 | 2.7 | 6.5 | 5.5 | | 5 | 80 | 192.3 | 8.5 | 4.4 | 4.3 | 2.2 | 9.5 | 4.9 | 9.8 | 5.1 | 13.6 | 7.1 | | 6 | 80 | 373.7 | 12.3 | 3.3 | 2.9 | 0.8 | 12.6 | 3.4 | 18.6 | 5.0 | 22.5 | 6.0 | | 7 | 80 | 463.1 | 17.0 | 3.7 | 8.5 | 1.8 | 19.0 | 4.1 | 17.9 | 3.9 | 26.1 | 5.6 | ### Between-instrument precision: To evaluate the between-instrument precision of the Alegria Flash MPO Assay, Alegria Flash PR3 Assay, and Alegria Flash GBM Assay, 6–7 samples were prepared by diluting positive native serum samples with negative serum samples. The prepared samples, containing various levels of autoantibodies, were assayed in quintuplicate once per day, on five different days, using three different Alegria FLASH instruments (n = 25 total replicates per sample for each instrument) for a total of 75 replicates per sample. The standard deviation (SD) and %CV of the within-day, between-day, within-instrument, between-instrument, and total imprecision were calculated for each sample. The results are summarized in the following tables: Alegria Flash MPO: | Sample | N | Mean (RU) | Within-Day | | Between-Day | | Within-Instrument | | Between-Instrument | | Total | | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | | | | SD | %CV | SD | %CV | SD | %CV | SD | %CV | SD | %CV | | 1 | 75 | 48.1 | 4.0 | 8.4 | 3.0 | 6.3 | 5.0 | 10.5 | 2.3 | 4.9 | 5.6 | 11.6 | | 2 | 75 | 79.3 | 4.8 | 6.1 | 3.6 | 4.6 | 6.0 | 7.6 | 1.0 | 1.3 | 6.1 | 7.7 | | 3 | 75 | 107.6 | 6.0 | 5.6 | 6.5 | 6.0 | 8.9 | 8.2 | 2.7 | 2.5 | 9.3 | 8.6 | K261466 - Page 8 of 20 {8} | Sample | N | Mean (RU) | Within-Day | | Between-Day | | Within-Instrument | | Between-Instrument | | Total | | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | | | | SD | %CV | SD | %CV | SD | %CV | SD | %CV | SD | %CV | | 4 | 75 | 155.0 | 9.6 | 6.2 | 7.4 | 4.7 | 12.1 | 7.8 | 5.6 | 3.6 | 13.3 | 8.6 | | 5 | 75 | 308.8 | 13.8 | 4.5 | 10.2 | 3.3 | 17.1 | 5.6 | 22.4 | 7.2 | 28.2 | 9.1 | | 6 | 75 | 400.0 | 19.6 | 4.9 | 18.6 | 4.7 | 27.1 | 6.8 | 27.2 | 6.8 | 38.4 | 9.6 | #### Alegria Flash PR3: | Sample | N | Mean (RU) | Within-Day | | Between-Day | | Within-Instrument | | Between-Instrument | | Total | | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | | | | SD | %CV | SD | %CV | SD | %CV | SD | %CV | SD | %CV | | 1 | 75 | 41.7 | 2.4 | 5.8 | 1.8 | 4.3 | 3.0 | 7.2 | 0.0 | 0.0 | 3.0 | 7.2 | | 2 | 75 | 68.0 | 2.8 | 4.0 | 4.0 | 5.9 | 4.9 | 7.2 | 0.0 | 0.0 | 4.9 | 7.2 | | 3 | 75 | 105.5 | 4.8 | 4.6 | 5.8 | 5.5 | 7.5 | 7.1 | 0.0 | 0.0 | 7.5 | 7.1 | | 4 | 75 | 144.4 | 6.1 | 4.2 | 7.5 | 5.2 | 9.7 | 6.7 | 0.0 | 0.0 | 9.7 | 6.7 | | 5 | 75 | 209.2 | 11.6 | 5.5 | 9.0 | 4.3 | 14.7 | 7.0 | 0.0 | 0.0 | 14.7 | 7.0 | | 6 | 75 | 373.0 | 18.0 | 4.8 | 17.8 | 4.8 | 25.3 | 6.8 | 11.9 | 3.2 | 28.0 | 7.5 | #### Alegria Flash GBM: | Sample | N | Mean (RU) | Within-Day | | Between-Day | | Within-Instrument | | Between-Instrument | | Total | | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | | | | SD | %CV | SD | %CV | SD | %CV | SD | %CV | SD | %CV | | 1 | 75 | 48.0 | 2.4 | 5.0 | 2.5 | 5.3 | 3.5 | 7.3 | 3.7 | 7.7 | 5.1 | 10.6 | | 2 | 75 | 81.2 | 3.4 | 4.2 | 3.5 | 4.3 | 4.8 | 6.0 | 3.0 | 3.6 | 5.7 | 7.0 | | 3 | 75 | 106.3 | 4.8 | 4.5 | 2.9 | 2.7 | 5.6 | 5.3 | 3.0 | 2.8 | 6.3 | 6.0 | | 4 | 75 | 119.5 | 4.7 | 3.9 | 8.6 | 7.2 | 9.7 | 8.2 | 0.0 | 0.0 | 9.7 | 8.2 | | 5 | 75 | 200.7 | 7.4 | 3.7 | 8.4 | 4.2 | 11.2 | 5.6 | 0.0 | 0.0 | 11.2 | 5.6 | | 6 | 75 | 377.6 | 12.5 | 3.3 | 17.1 | 4.5 | 21.2 | 5.6 | 0.0 | 0.0 | 21.2 | 5.6 | | 7 | 75 | 467.8 | 17.7 | 3.8 | 16.6 | 3.6 | 24.3 | 5.2 | 8.2 | 1.7 | 25.7 | 5.5 | #### *Lot-to-lot precision:* To evaluate the lot-to-lot precision of the Alegria Flash MPO Assay, Alegria Flash PR3 Assay, and Alegria Flash GBM Assay, 6–7 samples were prepared by diluting positive native serum samples with negative serum samples. The prepared samples, containing various levels of autoantibodies, were assayed in quintuplicate using three lots, once per day, on five different days ($n = 25$ total replicates per sample for each kit lot) using a single instrument for a total of 75 measurements per sample. The standard deviation (SD) and %CV of the within-day, between-day, within-lot, between-lot, and total imprecision were calculated for each sample. The results are summarized in the following tables: K261466 - Page 9 of 20 {9} ### Alegria Flash MPO: | Sample | N | Mean (RU) | Within-Day | | Between-Day | | Within-Lot | | Between-Lot | | Total | | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | | | | SD | %CV | SD | %CV | SD | %CV | SD | %CV | SD | %CV | | 1 | 75 | 34.8 | 2.9 | 8.4 | 1.2 | 3.6 | 3.2 | 9.2 | 1.3 | 3.7 | 3.4 | 9.9 | | 2 | 75 | 74.2 | 3.9 | 5.2 | 2.0 | 2.6 | 4.4 | 5.9 | 0.0 | 0.0 | 4.4 | 5.9 | | 3 | 75 | 104.4 | 5.1 | 4.8 | 4.2 | 4.0 | 6.6 | 6.3 | 3.1 | 3.0 | 7.3 | 7.0 | | 4 | 75 | 154.7 | 6.1 | 4.0 | 8.8 | 5.7 | 10.7 | 6.9 | 2.8 | 1.8 | 11.1 | 7.2 | | 5 | 75 | 356.6 | 11.6 | 3.3 | 13.5 | 3.8 | 17.9 | 5.0 | 15.9 | 4.5 | 23.9 | 6.7 | | 6 | 75 | 453.3 | 16.4 | 3.6 | 19.0 | 4.2 | 25.1 | 5.5 | 21.2 | 4.7 | 32.8 | 7.2 | ### Alegria Flash PR3: | Sample | N | Mean (RU) | Within-Day | | Between-Day | | Within-Lot | | Between-Lot | | Total | | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | | | | SD | %CV | SD | %CV | SD | %CV | SD | %CV | SD | %CV | | 1 | 75 | 34.9 | 1.8 | 5.0 | 1.9 | 5.5 | 2.6 | 7.4 | 2.0 | 5.8 | 3.3 | 9.4 | | 2 | 75 | 59.7 | 4.0 | 6.7 | 2.2 | 3.6 | 4.6 | 7.6 | 2.9 | 4.8 | 5.4 | 9.0 | | 3 | 75 | 100.6 | 4.2 | 4.2 | 4.5 | 4.4 | 6.2 | 6.1 | 0.6 | 0.6 | 6.2 | 6.2 | | 4 | 75 | 134.0 | 6.0 | 4.5 | 4.9 | 3.7 | 7.8 | 5.8 | 4.5 | 3.3 | 9.0 | 6.7 | | 5 | 75 | 275.5 | 11.5 | 4.2 | 8.4 | 3.1 | 14.2 | 5.2 | 2.3 | 0.8 | 14.4 | 5.2 | | 6 | 75 | 352.7 | 16.6 | 4.7 | 12.4 | 3.5 | 20.7 | 5.9 | 7.0 | 2.0 | 21.9 | 6.2 | | 7 | 75 | 445.1 | 17.4 | 3.9 | 20.5 | 4.6 | 26.9 | 6.0 | 3.1 | 0.7 | 27.1 | 6.1 | ### Alegria Flash GBM: | Sample | N | Mean (RU) | Within-Day | | Between-Day | | Within-Lot | | Between-Lot | | Total | | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | | | | SD | %CV | SD | %CV | SD | %CV | SD | %CV | SD | %CV | | 1 | 75 | 45.8 | 2.0 | 4.4 | 2.3 | 5.1 | 3.1 | 6.8 | 2.3 | 5.1 | 3.9 | 8.4 | | 2 | 75 | 77.6 | 4.3 | 5.5 | 3.8 | 4.8 | 5.7 | 7.3 | 4.7 | 6.0 | 7.4 | 9.5 | | 3 | 75 | 108.7 | 3.8 | 3.5 | 4.2 | 3.9 | 5.6 | 5.2 | 4.4 | 4.0 | 7.1 | 6.6 | | 4 | 75 | 120.4 | 4.9 | 4.1 | 6.1 | 5.1 | 7.8 | 6.5 | 4.3 | 3.5 | 8.9 | 7.4 | | 5 | 75 | 198.6 | 7.7 | 3.9 | 6.8 | 3.4 | 10.3 | 5.2 | 2.6 | 1.3 | 10.6 | 5.4 | | 6 | 75 | 378.6 | 13.6 | 3.6 | 22.6 | 6.0 | 26.4 | 7.0 | 8.2 | 2.2 | 27.6 | 7.3 | | 7 | 75 | 466.6 | 20.2 | 4.3 | 15.8 | 3.4 | 25.7 | 5.5 | 4.0 | 0.9 | 26.0 | 5.6 | ## 2. Linearity: Linearity for the Alegria Flash MPO, Alegria Flash PR3, and Alegria Flash GBM assays was evaluated based on CLSI EP06, 2$^{nd}$ Ed. For each assay, three dilution series were prepared by mixing one High native serum sample with a Low native serum sample. Dilutions were made in 10% increments that overlapped other samples' dilution series. Each sample was tested with five replicates per dilution level on one Alegria Flash Instrument. The percent (%) deviation from the weighted linear regression was used to assess the fit of the regression for each sample and analyte. The results are summarized in the following tables. K261466 - Page 10 of 20 {10} Alegria Flash MPO: | Sample ID | Test Range (RU) | Slope (95% CI) | Intercept (95% CI) | R² | % Deviation from Linearity | | --- | --- | --- | --- | --- | --- | | Panel 1 | 22.94 – 117.97 | 1.03 (0.99 to 1.07) | 1.25 (-5.36 to 0.58) | 1.00 | -4.48% to 4.39% | | Panel 2 | 91.61 –307.98 | 1.00 (0.90 to 1.10) | 8.41 (-12.9 to 28.3) | 0.99 | -8.40% to 4.56% | | Panel 3 | 231.46 – 570.65 | 1.08 (0.93 to 1.22) | 22.1 (-96.2 to 26.3) | 0.99 | -3.84% to 6.37% | Alegria Flash PR3: | Sample ID | Test Range (RU) | Slope (95% CI) | Intercept (95% CI) | R² | % Deviation from Linearity | | --- | --- | --- | --- | --- | --- | | Panel 1 | 24.04 – 115.67 | 0.99 (0.95 to 1.03) | 1.53 (-3.38 to 2.51) | 1.00 | -4.04% to 7.34% | | Panel 2 | 95.02 – 335.28 | 1.04 (0.98 to 1.10) | 5.49 (-19.2 to 9.33) | 1.00 | -4.57% to 3.99% | | Panel 3 | 185.14 – 550.43 | 0.99 (0.91 to 1.06) | 10.7 (-13.8 to 42.0) | 0.99 | -4.43% to 4.74% | Alegria Flash GBM: | Sample ID | Test Range (RU) | Slope (95% CI) | Intercept (95% CI) | R² | % Deviation from Linearity | | --- | --- | --- | --- | --- | --- | | Panel 1 | 27.76 – 126.24 | 1.03 (0.97 to 1.10) | 2.38 (-2.37 to 8.36) | 1.00 | -9.51% to 4.35% | | Panel 2 | 88.54 – 281.88 | 0.98 (0.90 to 1.05) | 5.66 (-3.21 to 26.2) | 0.99 | -5.87% to 5.17% | | Panel 3 | 154.41 – 504.08 | 1.00 (0.88 to 1.11) | 14.6 (-23.7 to 54.1) | 0.99 | -6.90% to 5.83% | The study results showed the following linear range for each assay: 24.06 – 570.65 RU for the Alegria Flash MPO; 24.44 – 550.43 RU for the Alegria Flash PR3, and 27.76 – 504.08 for the Alegria Flash GBM. ### 3. Analytical Specificity/Interference: Endogenous and Exogenous Interference: An interference study was performed according to CLSI EP07, 3rd Ed. and CLSI EP37, 1st Ed. for the Alegria Flash MPO, Alegria Flash PR3, and Alegria Flash GBM assays. Six endogenous and seventeen exogenous interfering substances were spiked into a low positive serum sample and a high negative serum sample. Five replicates of each spiked and non-spiked sample were tested. No significant interference observed for each assay at the concentration level tested for the substances listed in the following table. | Endogenous Substances | | | --- | --- | | Substance | Maximum Testing Concentration | | Bilirubin (unconjugated) | 0.15 mg/mL | | Cholesterol | 2.2 mg/mL | | Triglycerides | 2.5 mg/mL | | Albumin | 52 mg/mL | | Hemoglobin | 2 mg/mL | | Rheumatoid Factor (RF) | 400 U/mL | K261466 - Page 11 of 20 {11} | Exogenous Substances | | | --- | --- | | Substance | Maximum Testing Concentration | | Ibuprofen | 0.219 mg/mL | | Hydroxychloroquine | 0.024 mg/mL* | | Prednisone | 9.9×10^{-5} mg/mL | | Azathioprine | 2.58×10^{-3} mg/mL | | Diltiazem | 9×10^{-4} mg/mL | | Rituximab | 2 mg/mL | | Methotrexate | 1.36 mg/mL | | Enalapril | 8.19×10^{-4} mg/mL | | Omeprazole | 8.4×10^{-3} mg/mL | | Losartan | 3.06×10^{-4} mg/mL | | Atenolol | 9×10^{-3} mg/mL | | Erythromycin | 0.138 mg/mL | | Amoxicillin | 5.4×10^{-2} mg/mL | | Ranitidine | 1.05×10^{-2} mg/mL | | Furosemide | 1.59×10^{-2} mg/mL | | Alendronate | 3.4×10^{-5} mg/mL | | Atorvastatin | 7.5×10^{-4} mg/mL | * The concentration tested is lower than the recommended testing concentration of three times the maximum concentration observed in human serum. # Reference Sera: Selected Antinuclear Antibodies (ANA) IUIS Reference Standards samples, previously known as the CDC (Center for Disease Controls and Prevention) ANA Reference Panel, were tested in singlicate using one reagent lot of the Alegria Flash MPO, Alegria Flash PR3, and Alegria Flash GBM to illustrate the analytical specificity of the assay. Since no anti-GBM sample are available within the CDC panel, four ANCA-related serum samples were tested from a recent College of American Pathologists (CAP) survey sample set. The results are outlined below. | Sample ID | CDC Description | Alegria Flash MPO Result (RU) | Alegria Flash PR3 Result (RU) | Alegria Flash GBM Result (RU) | | --- | --- | --- | --- | --- | | ANA 01 | ANA homogeneous positive/anti-native DNA | 0.00 (Negative) | 6.16 (Negative) | 3.84 (Negative) | | ANA 02 | ANA speckled positive/anti-SSB/La | 0.00 (Negative) | 4.63 (Negative) | 3.39 (Negative) | | ANA 03 | ANA speckled positive/anti-U1 RNP, SSB/La, SSA/Ro | 0.00 (Negative) | 0.00 (Negative) | 1.35 (Negative) | | ANA 04 | Anti-U1 RNP positive | 0.00 (Negative) | 17.03 (Negative) | 8.69 (Negative) | | ANA 06 | ANA nucleolar (U3RNP) positive | 0.00 (Negative) | 0.00 (Negative) | 0.30 (Negative) | | ANA 07 | Anti-SSA Ro positive | 0.00 (Negative) | 0.00 (Negative) | 2.53 (Negative) | K261466 - Page 12 of 20 {12} | Sample ID | CDC Description | Alegria Flash MPO Result (RU) | Alegria Flash PR3 Result (RU) | Alegria Flash GBM Result (RU) | | --- | --- | --- | --- | --- | | ANA 08 | ANA centromere positive | 0.00 (Negative) | 0.00 (Negative) | 0.28 (Negative) | | ANA 09 | ANA anti-Scl-70 positive | 0.00 (Negative) | 9.70 (Negative) | 4.89 (Negative) | | ANA 10 | Anti-Jo-1 positive | 0.00 (Negative) | 0.00 (Negative) | 0.40 (Negative) | | ANA 11 | Anti-PM/Scl positive | 0.00 (Negative) | 9.85 (Negative) | 10.34 (Negative) | | ANA 12 | Anti-Ribosomal P positive | 29.01 (Negative) | 41.36 (Negative) | 14.83 (Negative) | | ANA 15 | Anti-MPO positive | 766.00 (Positive) | 30.25 (Negative) | 9.35 (Negative) | | ANA 16 | Anti-PR3 positive | 0.00 (Negative) | 2594.76 (Positive) | 0.00 (Negative) | | Sample ID | CAP Sample Category (Expected Results) | Alegria Flash MPO Result (RU) | Alegria Flash PR3 Result (RU) | Alegria Flash GBM Result (RU) | | --- | --- | --- | --- | --- | | S2-13 | ANCA (anti-MPO Negative, anti-PR3 Negative) | 0.00 (Negative) | 10.66 (Negative) | 14.87 (Negative) | | S2-14 | ANCA (anti-MPO Positive, anti-PR3 Negative) | 1396.54 (Positive) | 78.59 (Negative) | 32.82 (Negative) | | S2-17 | Anti-Glomerular (anti-GBM Positive) | 17.55 (Negative) | 26.14 (Negative) | 201.07 (Positive) | | S2-18 | Anti-Glomerular (anti-GBM Negative) | 0.00 (Positive) | 12.68 (Negative) | 15.51 (Negative) | #### 4. Detection Limit: Limit of Blank (LoB), Limit of Detection (LoD), and Limit of Quantitation (LoQ) for the Alegria Flash MPO, the Alegria Flash, PR3, and the Alegria Flash GBM assays were determined by following CLSI EP17-A2. ##### Limit of Blank (LoB): To determine the LoB for each assay, four analyte free serum samples were run in 20 replicates, once a day for three days using two reagent lots, for a total of 60 data points per sample per lot (240 total replicates per lot). The LoB was determined for each analyte, on each reagent lot separately, at the 95th percentile using the non-parametric method for each assay. ##### Limit of Detection (LoD): To determine the LoD for each assay, four low level samples were assayed in 20 replicates, for three days using two reagent lots, to generate a total of 60 data points for each sample on each reagent lot. The LoD was determined separately for each sample on each reagent lot and the highest LoD result was determined for the final LoD value, using the parametric method for each assay. K261466 - Page 13 of 20 {13} # Limit of Quantitation (LoQ): To determine the LoQ for each assay, the four LoD samples and an additional four low-level samples greater than the LoD were run in 20 replicates, for three days using two reagent lots, to generate 60 data points for each sample on each reagent lot. The LoQ was determined separately for each sample on each reagent lot by calculating the total imprecision of each sample. The LoQ was defined as the lowest concentration level that meets the within-laboratory imprecision of ≤15% for each lot. The claimed LoB, LoD and LoQ are shown in the following table for each assay | | LoB (RU) | LoD (RU) | LoQ (RU)* | | --- | --- | --- | --- | | Alegria Flash MPO | 17.23 | 21.90 | 24.06 | | Alegria Flash PR3 | 19.89 | 24.43 | 24.44 | | Alegria Flash GBM | 10.07 | 12.20 | 27.76 | *The value of the claimed LoQ is considered the lower limit of AMI for each assay. # 5. Assay Reportable Range The assay reportable range is the same as the analytical measuring intervals (AMIs): - Alegria Flash MPO: 24.06 – 570.65 RU - Alegria Flash PR3: 24.44 – 550.43 RU - Alegria Flash GBM: 27.76 – 504.08 RU # 6. Traceability, Stability, Expected Values (Controls, Calibrators, or Methods): Traceability: There is currently no recognized international standard for the measurement of IgG anti-MPO, anti-PR3, or anti-GBM antibodies. Reagent Stability: The reagent stability studies for Alegria Flash MPO, Alegria Flash PR3, and Alegria Flash GBM assays were designed and conducted following CLSI EP25, 2nd Ed. Shelf-life stability: To assess the shelf-life stability, six serum samples spanning the AMI with sample near the assay cut-off were assessed in triplicate on three lots of Alegria Flash MPO, Alegria Flash PR3, and Alegria Flash GBM kits at various time points up to 12 months. The results support that the assay kits are stable when stored unopened at 2-8°C for up to 9 months. On-board stability: To assess the on-board storage stability, five to six serum samples spanning the AMI with sample around the assay cut-off were assessed in duplicate on one Alegria Flash MPO, Alegria Flash PR3, and Alegria Flash GBM reagent kits and two assay-specific Alegria Flash controls. The Alegria Flash reagents stored opened within the Alegria K261466 - Page 14 of 20 {14} Flash's refrigerated reagent bay and assayed at various time points up to 5 weeks. The results support that the assay kits are stable when on-board the instrument for 4 weeks. Open cartridge stability: To assess the open cartridge stability, five to six serum samples spanning the AMI with sample around the assay cut-off were assessed in duplicate on one Alegria Flash MPO, Alegria Flash PR3, and Alegria Flash GBM reagent kits and two assay-specific Alegria Flash controls. The punctured reagent cartridges were removed from the instrument and then stored. The opened kits were then tested on the instrument at various time points up to 9 weeks. The results support that the assay kits are stable when opened for 8 weeks. Transport stability: To assess the transport stability, serum sample panels were assessed in singlicate on one Alegria Flash MPO, Alegria Flash PR3, and Alegria Flash GBM reagent kits and two assay-specific Alegria Flash controls. The kits were shipped to two locations within the US and one outside of US (OUS), then the results were compared to unshipped kits. The results support that the assay kits are stable in-transit. # Sample Stability: Sample stored at 2-8°C or RT: To assess the stability of patient samples, two serum samples – a low positive and a high negative – were stored up to 15 days at 2-8°C or at room temperature. The samples were then assessed in triplicate and the percent recovery in comparison to the unmanipulated, baseline sample was determined for each storage condition. The results support that patient serum samples are stable when stored for up to 14 days at 2-8°C or at room temperature. Freeze/thaw: To determine the stability of patient samples subjected to multiple freeze/thaw cycles, two serum samples – a low positive and a high negative – were subjected to five rounds of freeze/thaw cycles, consisting of ≥12 hours of storage at -20°C and a subsequent thaw. The samples were assessed for each condition in triplicate and the percent recovery in comparison to the unmanipulated, baseline sample was determined for each freeze/thaw cycle. The results support that patient serum samples are stable when frozen at -20°C and thawed for a maximum of four times. # 7. Assay Cut-Off: The cut-off for the Alegria Flash MPO, Alegria Flash PR3, and Alegria Flash GBM is 100 RU. # B Comparison Studies: # 1. Method Comparison with Predicate Device: A cohort of clinically characterized samples used in clinical study in Section C below were tested on the Alegria Flash MPO, Alegria Flash PR3, and Alegria Flash GBM assays and their respective predicate devices. Positive percent agreement (PPA) and negative percent agreement (NPA), with 95% confidence intervals were calculated for each analyte comparison using samples with values within the analytical measuring interval of both K261466 - Page 15 of 20 {15} candidate and predicate devices. The following tables summarize the assays' method comparison study results: Alegria Flash MPO | | Predicate | | | | | --- | --- | --- | --- | --- | | | | Positive | Negative | Total | | Alegria Flash MPO | Positive | 104 | 7 | 111 | | | Negative | 16 | 210 | 226 | | | Total | 120 | 217 | 337 | PPA: 86.7% (104/120) (95% CI: 79.4 – 91.6%) NPA: 96.8% (210/217) (95% CI: 93.5 – 98.4%) Alegria Flash PR3 | | Predicate | | | | | | --- | --- | --- | --- | --- | --- | | | | Positive | Equivocal | Negative | Total | | Alegria Flash PR3 | Positive | 89 | 0 | 16 | 105 | | | Negative | 4 | 1 | 209 | 214 | | | Total | 93 | 1 | 225 | 323 | Equivocal as Positive PPA: 94.7% (89/94) (95% CI: 88.2 – 97.7%) NPA: 92.9% (209/225) (95% CI: 88.7 – 95.6%) Equivocal as Negative PPA: 95.7% (89/93) (95% CI: 89.5 – 98.3%) NPA: 92.9% (210/226) (95% CI: 88.8 – 95.6%) Alegria Flash GBM | | Predicate | | | | | --- | --- | --- | --- | --- | | | | Positive | Negative | Total | | Alegria Flash GBM | Positive | 51 | 0 | 51 | | | Negative | 2 | 56 | 58 | | | Total | 53 | 56 | 109 | PPA: 96.2% (51/53) (95% CI: 87.3 – 99.0%) NPA: 100.0% (56/56) (95% CI: 93.6 – 100.0%) #### 2. Matrix Comparison: N/A ### C Clinical Studies: #### 1. Clinical Sensitivity and Specificity: A cohort of 609 characterized samples were used to validate the clinical performance of the Alegria Flash MPO Assay, the Alegria Flash PR3 Assay, and the Alegria Flash GBM Assay. Among the 609 samples, 123 samples were from patients diagnosed with microscopic polyangiitis (MPA), 90 samples were from patients diagnosed with granulomatosis with K261466 - Page 16 of 20 {16} polyangiitis (GPA), 46 samples were from patients diagnosed with Goodpasture's Syndrome, and 350 samples were from patients with diseases that might be considered in the differential diagnosis for the AAVs, including non-vasculitis disorders, as well as vasculitides not associated with ANCAs. The results stratified by the disease conditions are summarized in the table below: Alegria Flash MPO: | Diagnosis | N | Alegria Flash MPO Assay | | | | | --- | --- | --- | --- | --- | --- | | | | No. of Positives | % of Positives | 95% CI | | | Sensitivity Cohort | Microscopic polyangiitis | 123 | 100 | 81.3 | 73.5 - 87.2 | | Specificity Cohort | Granulomatosis with polyangiitis | 90 | 10 | 11.1 | 6.2 - 19.3 | | | Goodpasture's syndrome | 46 | 15 | 32.6 | 20.9 - 47.0 | | | Eosinophilic granulomatosis with polyangiitis | 8 | 5 | 62.5 | 30.6 - 86.3 | | | Asthma/COPD | 30 | 0 | 0.0 | 0.0 - 11.4 | | | Chronic kidney disease | 30 | 0 | 0.0 | 0.0 - 11.4 | | | Crohn's disease | 20 | 0 | 0.0 | 0.0 - 16.1 | | | Graves disease | 20 | 0 | 0.0 | 0.0 - 16.1 | | | Hashimoto's thyroiditis | 22 | 0 | 0.0 | 0.0 - 14.9 | | | HBV | 10 | 1 | 10.0 | 1.8 - 40.4 | | | HCV | 10 | 0 | 0.0 | 0.0 - 27.8 | | | HIV | 10 | 0 | 0.0 | 0.0 - 27.8 | | | Multiple myeloma | 20 | 0 | 0.0 | 0.0 - 16.1 | | | Myositis | 30 | 1 | 3.3 | 0.6 - 16.7 | | | Giant cell arteritis | 20 | 1 | 5.0 | 0.9 - 23.6 | | | Rheumatoid arthritis | 20 | 0 | 0.0 | 0.0 - 16.1 | | | Sinusitis/Rhinitis | 30 | 0 | 0.0 | 0.0 - 11.4 | | | Systemic lupus erythematosus | 30 | 0 | 0.0 | 0.0 - 11.4 | | | Systemic sclerosis | 20 | 0 | 0.0 | 0.0 - 16.1 | | | Ulcerative colitis | 20 | 0 | 0.0 | 0.0 - 16.1 | | Total | | 609 | | | | Clinical sensitivity and specificity for the Alegria Flash MPO are summarized in the table below: | | Diagnosis | | | | | --- | --- | --- | --- | --- | | | | MPA | Control | Total | | Alegria Flash MPO | Positive | 100 | 453 | 553 | | | Negative | 23 | 33 | 56 | | | Total | 123 | 486 | 609 | Sensitivity: 81.3% (100/123) (95% CI%: 73.5 – 87.2%) Specificity: 93.2% (453/486) (95% CI: 90.6 – 95.1%) K261466 - Page 17 of 20 {17} Alegria Flash PR3 | Diagnosis | N | Alegria Flash PR3 Assay | | | | | --- | --- | --- | --- | --- | --- | | | | No. of Positives | % of Positives | 95% CI | | | Sensitivity Cohort | Granulomatosis with polyangiitis | 90 | 77 | 85.6 | 76.8 - 91.4 | | Specificity Cohort | Microscopic polyangiitis | 123 | 21 | 17.1 | 11.5 - 24.7 | | | Goodpasture's syndrome | 46 | 4 | 8.7 | 3.4 - 20.3 | | | Eosinophilic granulomatosis with polyangiitis | 8 | 2 | 25.0 | 7.2 - 59.1 | | | Asthma/COPD | 30 | 1 | 3.3 | 0.6 - 16.7 | | | Chronic kidney disease | 30 | 3 | 10.0 | 3.5 - 25.6 | | | Crohns disease | 20 | 0 | 0.0 | 0.0 - 16.1 | | | Graves disease | 20 | 0 | 0.0 | 0.0 16.1 | | | Hashimoto's thyroiditis | 22 | 1 | 4.6 | 0.8 - 21.8 | | | HBV | 10 | 0 | 0.0 | 0.0 - 27.7 | | | HCV | 10 | 1 | 10.0 | 1.8 - 40.4 | | | HIV | 10 | 0 | 0.0 | 0.0 - 27.6 | | | Multiple myeloma | 20 | 0 | 0.0 | 0.0 - 16.1 | | | Myositis | 30 | 1 | 3.3 | 0.6 - 16.7 | | | Giant cell arteritis | 20 | 0 | 0.0 | 0.0 - 16.1 | | | Rheumatoid arthritis | 20 | 0 | 0.0 | 0.0 - 16.1 | | | Sinusitis/Rhinitis | 30 | 4 | 13.3 | 5.3 - 29.7 | | | Systemic lupus erythematosus | 30 | 0 | 0.0 | 0.0 - 11.4 | | | Systemic sclerosis | 20 | 0 | 0.0 | 0.0 - 16.1 | | | Ulcerative colitis | 20 | 0 | 0.0 | 0.0 - 16.1 | | Total | | 609 | | | | Clinical sensitivity and specificity for the Alegria Flash PR3 are summarized in the table below: | | Diagnosis | | | | | --- | --- | --- | --- | --- | | | | GPA | Control | Total | | Alegria Flash PR3 | Positive | 77 | 38 | 115 | | | Negative | 13 | 481 | 494 | | | Total | 90 | 519 | 609 | Sensitivity: 85.6% (77/90) (95% CI: 76.8 - 91.4%) Specificity: 92.7% (481/519) (95% CI: 90.1 - 94.6%) K261466 - Page 18 of 20 {18} Alegria Flash GBM: | Diagnosis | N | Alegria Flash GBM Assay | | | | | --- | --- | --- | --- | --- | --- | | | | No. of Positives | % of Positives | 95% CI | | | Sensitivity Cohort | Goodpasture's syndrome | 46 | 41 | 89.1 | 77.0 - 95.3 | | Specificity Cohort | Granulomatosis with polyangiitis | 90 | 0 | 0.0 | 0.0 - 4.1 | | | Microscopic polyangiitis | 123 | 0 | 0.0 | 0.0 - 3.0 | | | Eosinophilic granulomatosis with polyangiitis | 8 | 0 | 0.0 | 0.0 - 32.4 | | | Asthma/COPD | 30 | 0 | 0.0 | 0.0 - 11.4 | | | Chronic kidney disease | 30 | 0 | 0.0 | 0.0 - 11.4 | | | Crohns disease | 20 | 0 | 0.0 | 0.0 - 16.1 | | | Graves disease | 20 | 0 | 0.0 | 0.0 - 16.1 | | | Hashimoto's thyroiditis | 22 | 0 | 0.0 | 0.0 - 14.9 | | | HBV | 10 | 0 | 0.0 | 0.0 - 27.8 | | | HCV | 10 | 0 | 0.0 | 0.0 - 27.8 | | | HIV | 10 | 0 | 0.0 | 0.0 - 27.8 | | | Multiple myeloma | 20 | 0 | 0.0 | 0.0 - 16.1 | | | Myositis | 30 | 0 | 0.0 | 0.0 - 11.4 | | | Giant cell arteritis | 20 | 0 | 0.0 | 0.0 - 16.1 | | | Rheumatoid arthritis | 20 | 0 | 0.0 | 0.0 - 16.1 | | | Sinusitis/Rhinitis | 30 | 0 | 0.0 | 0.0 - 11.4 | | | Systemic lupus erythematosus | 30 | 0 | 0.0 | 0.0 - 11.4 | | | Systemic sclerosis | 20 | 0 | 0.0 | 0.0 - 16.1 | | | Ulcerative colitis | 20 | 0 | 0.0 | 0.0 - 16.1 | | Total | | 609 | | | | Clinical sensitivity and specificity for the Alegria Flash GBM are summarized in the table below: | | Diagnosis | | | | | --- | --- | --- | --- | --- | | | | Goodpasture's | Control | Total | | Alegria Flash GBM | Positive | 41 | 0 | 41 | | | Negative | 5 | 563 | 568 | | | Total | 46 | 563 | 609 | Sensitivity: 89.1% (41/46) (95% CI: 77.0 – 95.6%) Specificity: 100.0% (563/563) (95% CI: 99.3 – 100.0%) ### D Expected Values/Reference Range: A negative Alegria Flash MPO, Alegria Flash PR3, and Alegria Flash GBM test result is expected for apparently healthy individuals based on the cut-off of each assay. K261466 - Page 19 of 20 {19} To determine the reference interval for each of these assays, a panel of 200 apparently healthy blood donors were tested following CLSI EP28-A3c. The results are as follows: | | N | # of Samples >LoQ | # of Negatives (<100 RU) | % Negative | | --- | --- | --- | --- | --- | | Alegria Flash MPO | 200 | 11 | 200 | 100 | | Alegria Flash PR3 | 200 | 31 | 200 | 100 | | Alegria Flash GBM | 200 | 0 | 200 | 100 | ### VIII Proposed Labeling: The labeling supports the finding of substantial equivalence for this device. ### IX Conclusion: The submitted information in this premarket notification is complete and supports a substantial equivalence decision. K261466 - Page 20 of 20