Alegria Flash ENA Screen

{0} FDA U.S. FOOD &amp; DRUG ADMINISTRATION # 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION SUMMARY ASSAY ONLY ## I Background Information: A 510(k) Number K250408 B Applicant ZEUS Scientific C Proprietary and Established Names Alegria Flash ENA Screen D Regulatory Information | Product Code(s) | Classification | Regulation Section | Panel | | --- | --- | --- | --- | | LLL | Class II | 21 CFR 866.5100 - Antinuclear Antibody Immunological Test System | IM - Immunology | ## II Submission/Device Overview: A Purpose for Submission: New assay B Measurand: Human IgG autoantibodies to Jo-1, SSA-52, SSA-60, SS-B, Scl-70, Sm, and SmRNP C Type of Test: Automated qualitative 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 ENA Screen kit is a chemiluminescent immunoassay (CLIA) for the qualitative screening of IgG autoantibodies to Jo-1, SSA-52, SSA-60, SS-B, Scl-70, Sm, or SmRNP in human serum. The presence of these autoantibodies is intended for use as an aid in the diagnosis of the connective tissue diseases (CTD) Sjögren's syndrome (SS), systemic lupus erythematosus (SLE), systemic sclerosis (SSc), mixed connective tissue disease (MCTD), Limited Cutaneous Systemic Sclerosis (CREST Syndrome), polymyositis, and dermatomyositis along with other laboratory and clinical findings. The test must be performed on the Alegria Flash instrument. C Special Conditions for Use Statement(s): Rx - For Prescription Use Only D Special Instrument Requirements: For use with the Alegria Flash instrument (Zeus Solinas α instrument, K230863). IV Device/System Characteristics: A Device Description: Each Alegria Flash ENA Screen kit contains the following materials: - One (1) Assay Cartridge containing bead suspension, sample diluent, and conjugate reagents. - Bead suspension: magnetic particles that are coated with a cocktail of ENA antigens in 5 mL of storage buffer. The suspension contains Tween-20, bovine serum albumin, phosphate-buffered-saline, and &lt;0.1% sodium azide. Ready to use. - Sample diluent: 10 mL of phosphate-buffered-saline solution containing detergent, proteins, &lt;0.1% sodium azide, and ProClin 300. Ready to use. - Conjugate: 10 mL of anti-human IgG that is conjugated with Acridinium Ester in a buffered solution containing, ProClin 300. Ready to use. - One (1) Calibrator 1 (blue-capped vial): 0.5 mL of human serum that contains anti-ENA autoantibodies at levels above the cutoff value and &lt;0.1% sodium azide. Ready to use. Assay and lot specific. K250408 - Page 2 of 11 {2} - One (1) Calibrator 2 (white-capped vial): 0.5 mL of human serum containing anti-ENA autoantibodies at levels near the cutoff value and &lt;0.1% sodium azide. Ready to use. Assay and lot specific. A separate Alegria Flash ENA Control kit is sold separately. The Alegria Flash Control kit contains: - One (1) positive control (“CTRL +”): a 1.0 mL vial of human serum containing anti-ENA antibodies at levels above the cutoff value and &lt;0.1% sodium azide. Ready to use and assay specific. - One (1) negative control (“CTRL –”): a 1.0 mL vial of human serum containing anti-ENA antibodies at levels below the cutoff value and &lt;0.1% sodium azide. Ready to use and assay specific. ## B Principle of Operation: The Alegria Flash ENA Screen kit is designed to detect human anti-Jo-1, SSA-52, SSA-60, SS-B, Scl-70, Sm, or SmRNP IgG autoantibodies, in human serum. The Alegria Flash ENA Screen Kit test procedure involves three main steps. Sample diluent, test sera, and antigen-coated magnetic particles are added to a reaction cuvette. During the initial incubation, autoantibodies specific to Jo-1, SSA-52, SSA-60, SS-B, Scl-70, Sm, or SmRNP present in the serum will bind to the immobilized antigens. The beads are then washed to remove unbound antibodies and other serum components. An acridinium ester-conjugated anti-human IgG solution is then added to the reaction cuvette. During the second incubation, the conjugate reacts with IgG autoantibodies that are immobilized on the magnetic particles in step 1. The beads are then washed to remove unbound conjugate. 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 the signal is reported as relative light units (RLU). RLU values are converted to CLIA Units using the instrument’s Alegria Flash software. CLIA Unit values that are above or equal to the cut-off value of 100 CLIA Units are indicative of the presence of selected anti-ENA antibodies within the original serum sample. ## V Substantial Equivalence Information: ### A Predicate Device Name(s): Zeus ENA Screen ELISA Test System ### B Predicate 510(k) Number(s): K941014 K250408 - Page 3 of 11 {3} C Comparison with Predicate(s): | Device & Predicate Device(s): | K250408 (Candidate Device) | K941014 (Predicate Device) | | --- | --- | --- | | Device Trade Name | Alegria Flash ENA Screen | ENA Screen ELISA Test System | | General Device Characteristic Similarities | | | | Intended Use/Indications For Use | The Alegria Flash ENA Screen kit is a chemiluminescent immunoassay (CLIA) for the qualitative screening of IgG autoantibodies to Jo-1, SSA-52, SSA-60, SS-B, Scl-70, Sm, or SmRNP in human serum. The presence of these autoantibodies is intended for use as an aid in the diagnosis of the connective tissue diseases (CTD) Sjögren's syndrome (SS), systemic lupus erythematosus (SLE), systemic sclerosis (SSc), mixed connective tissue disease (MCTD), Limited Cutaneous Systemic Sclerosis (CREST Syndrome), polymyositis, and dermatomyositis along with other laboratory and clinical findings. The test must be performed on the Alegria Flash instrument. | The ZEUS ELISA ENA Screen Test System is a qualitative screening assay designed to detect antibodies to extractable nuclear antigens (anti-ENA) in human sera. When performed according to the enclosed instructions, this test is capable of detecting all anti-ENAs commonly tested for, such as those against Jo-1, Sm, Sm/RNP, SSA, SSB, and Scl-70. This device is for In Vitro diagnostic use | | Sample matrix | Serum | Same | | Detection | Qualitative | Same | | General Device Characteristic Differences | | | | Antigens | Jo-1, SSA-52, SSA-60, SS-B, Scl-70, Sm, SmRNP | SSA 60, SS-B, Jo-1, Scl70, SmRNP, Sm | | Technology | Chemiluminescent Immunoassay (CLIA) | Enzyme Linked Immunosorbent Assay (ELISA) | | Solid Phase | Magnetic microscopic beads | 96 well polystyrene ELISA | | Cut-off | 100 CLIA Units | 0.90 and 1.10 Units | | Interpretation | Negative: <100 CLIA Units Positive: ≥100 CLIA Units | Negative: ≤0.90 Units Equivocal: 0.91–1.09 Units Positive: ≥1.10 Units | VI Standards/Guidance Documents Referenced: The following Clinical and Laboratory Standards Institute (CLSI) guidelines were used: - CLSI EP07-Ed3 – Interference Testing in Clinical Chemistry - CLSI EP12-Ed3 – Evaluation of Qualitative, Binary Output Examination Performance K250408 - Page 4 of 11 {4} - CLSI EP17-A2 – Evaluation of Detection Capability for Clinical Laboratory Measurement Procedures - CLSI EP25-Ed2 – Evaluation of Stability of In Vitro Medical Laboratory Test Reagents - CLSI EP28-A3c – Defining, Establishing and Verifying Reference Intervals in the Clinical Laboratory ## VII Performance Characteristics (if/when applicable): ### A Analytical Performance: #### 1. Precision/Reproducibility: The precision of the Alegria Flash ENA Screen was evaluated based on the CLSI EP12-Ed3. **Within-laboratory precision:** To evaluate the within-laboratory precision of the Alegria Flash ENA Screen, 11 samples were prepared by mixing positive native serum samples with negative serum samples. The prepared samples, containing various concentrations 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 percent of positive (% Pos), with 95% confidence interval (CI), for each sample was calculated. The results are summarized in the following table. | Sample | Expected Result | (CLIA Units) | | Positive | | | | --- | --- | --- | --- | --- | --- | --- | | | | Mean | Range | n(+)/N | % Pos. | (95% CI) | | 1 | Positive | 457.4 | 398.6–594.5 | 80/80 | 100 | (95.4–100) | | 2 | Positive | 303.3 | 254.6–386.6 | 80/80 | 100 | (95.4–100) | | 3 | Positive | 262.3 | 213.9–342.6 | 80/80 | 100 | (95.4–100) | | 4 | Positive | 259.5 | 227.7–337.6 | 80/80 | 100 | (95.4–100) | | 5 | Positive | 167.4 | 124.7–204.8 | 80/80 | 100 | (95.4–100) | | 6 | Positive | 113.7 | 102.3–131.9 | 80/80 | 100 | (95.4–100) | | 7 | Negative | 97.2 | 76.0–120.3 | 28/80 | 35.0 | (25.4–45.9) | | 8 | Negative | 78.4 | 64.7–91.3 | 0/80 | 0.0 | (0.0–4.6) | | 9 | Negative | 47.2 | 35.9–65.0 | 0/80 | 0.0 | (0.0–4.6) | | 10 | Negative | 17.2 | 9.8–29.4 | 0/80 | 0.0 | (0.0–4.6) | | 11 | Negative | 11.5 | 7.4–21.5 | 0/80 | 0.0 | (0.0–4.6) | **Lot-to-lot imprecision:** To evaluate the between-lot imprecision of the Alegria Flash ENA Screen, eight samples were prepared by diluting positive native serum samples with negative serum samples. The samples, containing various concentrations of autoantibodies, were assayed were assayed in quintuplicate, once a day, for 5 days, using three reagent lots, using one instrument, for a total of 75 replicates per sample. The results are summarized in the following table. K250408 - Page 5 of 11 {5} | Sample | Expected Result | Total (across lots) | | | | Lot 1 | | Lot 2 | | Lot 3 | | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | | | Mean (u) | Range (u) | n(+)/N | % Pos | n(+)/N | % Pos | n(+)/N | % Pos | n(+)/N | % Pos | | 1 | Positive | 663.0 | 422.2 – 864.7 | 75/75 | 100 | 25/25 | 100 | 25/25 | 100 | 25/25 | 100 | | 2 | Positive | 243.9 | 188.8 – 379.7 | 75/75 | 100 | 25/25 | 100 | 25/25 | 100 | 25/25 | 100 | | 3 | Positive | 144.5 | 124.9 – 174.8 | 75/75 | 100 | 25/25 | 100 | 25/25 | 100 | 25/25 | 100 | | 4 | Positive | 115.5 | 96.6 – 129.7 | 74/75 | 98.7 | 25/25 | 100 | 25/25 | 100 | 24/25 | 96.0 | | 5 | Positive | 103.8 | 87.1 – 121.1 | 48/75 | 64.0 | 24/25 | 96.0 | 16/25 | 64.0 | 8/25 | 32.0 | | 6 | Negative | 89.2 | 75.9 – 125.6 | 3/75 | 4.0 | 0/25 | 0.0 | 3/25 | 12.0 | 0/25 | 0.0 | | 7 | Negative | 79.2 | 59.8 – 126.2 | 2/75 | 2.7 | 0/25 | 0.0 | 2/25 | 8.0 | 0/25 | 0.0 | | 8 | Negative | 16.7 | 5.1 – 60.3 | 0/75 | 0.0 | 0/25 | 0.0 | 0/25 | 0.0 | 0/25 | 0.0 | Site-to-site imprecision: The reproducibility of the Alegria Flash ENA Screen was evaluated at three sites using 11 samples that were prepared by mixing positive native serum samples with negative serum samples. The samples, containing various concentrations of autoantibodies, were assayed in quintuplicate, once a day, for five days to generate 25 data points per sample per site for a total of 75 replicates per sample. One reagent lot was used in the study. The instrument and operator variables were nested within the multiple site component. The resulting data are summarized in the following table: | Sample | Expected Result | Total (across sites) | | | | Site 1 | | Site 2 | | Site 3 | | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | | | Mean (u) | Range (u) | n(+)/N | % Pos | n(+)/N | % Pos | n(+)/N | % Pos | n(+)/N | % Pos | | 1 | Positive | 512.7 | 417.8 – 644.2 | 75/75 | 100 | 25/25 | 100 | 25/25 | 100 | 25/25 | 100 | | 2 | Positive | 352.9 | 284.9 – 426.3 | 75/75 | 100 | 25/25 | 100 | 25/25 | 100 | 25/25 | 100 | | 3 | Positive | 288.3 | 228.1 – 374.3 | 75/75 | 100 | 25/25 | 100 | 25/25 | 100 | 25/25 | 100 | | 4 | Positive | 287.0 | 233.3 – 420.6 | 75/75 | 100 | 25/25 | 100 | 25/25 | 100 | 25/25 | 100 | | 5 | Positive | 197.0 | 144.2 – 242.4 | 75/75 | 100 | 25/25 | 100 | 25/25 | 100 | 25/25 | 100 | | 6 | Positive | 115.6 | 84.6 – 154.3 | 66/75 | 88.0 | 24/25 | 96.0 | 18/25 | 72.0 | 24/25 | 96.0 | | 7 | Negative | 97.0 | 80.3 – 123.5 | 22/75 | 29.3 | 0/25 | 0 | 1/25 | 4.0 | 21/25 | 84.0 | | 8 | Negative | 85.1 | 70.3 – 99.5 | 0/75 | 0 | 0/25 | 0 | 0/25 | 0 | 0/25 | 0 | | 9 | Negative | 62.5 | 45.3 – 81.8 | 0/75 | 0 | 0/25 | 0 | 0/25 | 0 | 0/25 | 0 | | 10 | Negative | 30.4 | 17.0 – 49.4 | 0/75 | 0 | 0/25 | 0 | 0/25 | 0 | 0/25 | 0 | | 11 | Negative | 24.8 | 14.6 – 41.3 | 0/75 | 0 | 0/25 | 0 | 0/25 | 0 | 0/25 | 0 | 2. Linearity: Not applicable 3. Analytical Specificity/Interference: Endogenous and Exogenous Interference: Two serum samples (a negative and a positive serum samples), were spiked (&lt;10% volume/volume) with the test endogenous or exogenous test substance at the indicated concentrations or with an appropriate blank vehicle solvent. The samples were tested in triplicate using an Alegria Flash instrument. The percent recovery of the spiked samples was K250408 - Page 6 of 11 {6} determined relative to the blank vehicle solvent spike in sample. None of the interferents changed the expected result at the indicated testing concentrations. | 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 | | 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 ENA Screen to illustrate the analytical specificity of the assay. The results are outlined below. | Sample ID | CDC Description | Alegria Flash ENA Screen Result | | --- | --- | --- | | ANA 01 | ANA homogeneous positive/ anti-native DNA | Negative | | ANA 02 | ANA speckled positive/ anti-SSB/La | Positive | K250408 - Page 7 of 11 {7} | Sample ID | CDC Description | Alegria Flash ENA Screen Result | | --- | --- | --- | | ANA 03 | ANA speckled positive/ anti-U1 RNP, SSB/La, SSA/Ro | Positive | | ANA 04 | Anti-U1 RNP positive | Positive | | ANA 06 | ANA nucleolar (U3RNP) positive | Negative | | ANA 07 | Anti-SSA Ro positive | Positive | | ANA 08 | ANA centromere positive | Negative | | ANA 09 | ANA anti-Scl70 positive | Positive | | ANA 10 | Anti-Jo-1 positive | Positive | | ANA 11 | Anti-PM/Scl positive | Negative | | ANA 12 | Anti-Ribosomal P positive | Negative | | ANA 15 | Anti-MPO positive | Negative | | ANA 16 | Anti-PR3 positive | Negative | 4. Assay Reportable Range: Not applicable 5. Traceability, Stability, Expected Values (Controls, Calibrators, or Methods): Traceability: Currently there are no recognized international standards for the measurement of Jo-1, SSA-52, SSA-60, SS-B, Scl-70, Sm, and SmRNP. The calibrators and controls are directly traceable to in house standards. Reagent stability: Shelf-life: The reagent stability studies were designed and conducted following CLSI EP25-Ed2. To assess the shelf-life stability of the Alegria Flash ENA Screen kits, six serum samples and two controls were assessed in singlicate using Alegria Flash ENA Screen kits that were stored for 1 day, or for up to 6 months at 2-8°C. The results support that the Alegria Flash ENA Screen kits are stable when stored unopened at 2-8°C for up to five months. On-board stability: The onboard storage stability of the Alegria Flash ENA Screen kit was determined by testing three serum samples and two controls using Alegria Flash ENA Screen reagents stored opened within the Alegria Flash's refrigerated reagent bay or within the refrigerator at 2-8°C. The opened Alegria Flash ENA Screen kits were assessed after up to 5 weeks of storage. The results of the study support that Alegria Flash ENA Screen reagents are stable when stored onboard or at 2-8°C for four weeks. Sample stability: The sample stability studies were designed and conducted following CLSI EP25-Ed2. To assess the stability of patient samples, three serum samples were stored up to 20 days at 2-8°C or at room temperature. The samples were then assessed using three replicates and the K250408 - Page 8 of 11 {8} 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 19 days at 2-8°C or at room temperature. To determine the stability of patient samples subjected to multiple freeze/thaw cycles, three serum samples were subjected up to 5 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 using three replicates 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 4 times. 6. Detection Limit: Not applicable 7. Assay Cut-Off: The cut-off of the Alegria Flash ENA Screen is 100 Units. To validate this cut-off, 30 ANA positive clinically characterized serum samples and 90 apparently healthy serum donor samples that were not used in any other studies were tested using the Alegria Flash ENA Screen. All known ANA positive samples were found to be positive for the Alegria Flash ENA Screen. B Comparison Studies: 1. Method Comparison with Predicate Device: The 943 samples used in the Alegria Flash ENA Screen clinical validation study (Section C.1 below) were compared to the predicate in a method comparison analysis. All samples were tested in singlicate. Positive percent agreement (PPA) and negative percent agreement (NPA), with their associated 95% confidence intervals (CIs), were calculated. The results are summarized in the following tables. | | Predicate | | | Total | | | --- | --- | --- | --- | --- | --- | | | | Positive | Indeterminate | | Negative | | Alegria Flash ENA Screen | Positive | 352 | 14 | 55 | 421 | | | Negative | 12 | 19 | 491 | 522 | | | Total | 364 | 33 | 546 | 943 | | Indeterminate as positive | | | | --- | --- | --- | | PPA | 92.2% | 95% CI: 89.1 – 94.2% | | NPA | 89.9% | 95% CI: 87.1 – 92.2% | | Indeterminate as negative | | | | PPA | 96.7% | 95% CI: 94.3 – 98.1% | | NPA | 88.1% | 95% CI: 85.2 – 90.5% | K250408 - Page 9 of 11 {9} 2. Matrix Comparison: Not applicable C Clinical Studies: 1. Clinical Sensitivity and Specificity: A cohort of characterized samples were used to validate the clinical performance of the Alegria Flash ENA Screen. The clinical validation study included 589 from ENA-associated connective tissue diseases (CTDs) and 354 samples from diseases that might be considered in the differential diagnosis of CTDs. The results stratified by the disease conditions are summarized in the table below: | Diagnosis | n | Alegria Flash ENA Screen | | | | | --- | --- | --- | --- | --- | --- | | | | No. of Positives | % of Positives | 95% CI | | | Sensitivity Cohort | Sjögren's syndrome | 135 | 114 | 84.4 | 77.4 - 89.6 | | | Systemic Sclerosis | 55 | 33 | 60.0 | 46.8 - 71.9 | | | Systemic Sclerosis (CREST Syndrome) | 55 | 25 | 45.4 | 33.0 - 58.5 | | | Polymyositis | 51 | 25 | 49.0 | 35.9 - 62.3 | | | Dermatomyositis | 53 | 26 | 49.1 | 36.1 - 62.1 | | | Mixed Connective Tissue Disease | 60 | 41 | 68.3 | 55.7 - 78.7 | | | Systemic Lupus Erythematosus | 180 | 129 | 71.7 | 64.7 - 77.7 | | Specificity Cohort | Autoimmune Hepatitis | 30 | 4 | 13.3 | 5.3 - 29.7 | | | Antiphospholipid Syndrome | 20 | 3 | 15.0 | 5.2 - 36.0 | | | Cancer | 20 | 0 | 0 | 0 - 16.1 | | | Celiac Disease | 24 | 0 | 0 | 0 - 13.8 | | | Drug Induced Lupus | 5 | 2 | 40.0 | 11.7 - 76.9 | | | Fibromyalgia | 20 | 2 | 10.0 | 2.8 - 30.1 | | | Ulcerative Colitis | 29 | 0 | 0 | 0 - 11.0 | | | Crohn's Disease | 31 | 0 | 0 | 0 - 11.7 | | | Hepatitis B Virus infection | 18 | 3 | 16.7 | 5.8 - 39.2 | | | Hepatitis C Virus infection | 10 | 1 | 10.0 | 1.8 - 40.4 | | | HIV infection | 12 | 1 | 8.3 | 1.5 - 35.4 | | | Herpes Simplex Virus infection | 10 | 0 | 0 | 0 - 27.8 | | | Primary Biliary Cholangitis | 30 | 3 | 10.0 | 3.5 - 25.6 | | | Rheumatoid Arthritis | 20 | 3 | 15.0 | 5.2 - 36.0 | | | Vasculitis* | 16 | 2 | 12.5 | 3.5 - 36.0 | | | Atrophic Gastritis | 20 | 1 | 5.0 | 0.9 - 23.6 | | | Graves' Disease | 20 | 1 | 5.0 | 0.9 - 23.6 | | | Hashimoto's Thyroiditis | 19 | 3 | 15.8 | 5.5 - 37.6 | | Total | | 943 | | | | * The vasculitis sample panel consisted of five ANCA-associated vasculitis samples, five large/medium vessel vasculitis samples, and six undefined vasculitis sample. K250408 - Page 10 of 11 {10} Clinical sensitivity and specificity for the Alegria Flash ENA Screen are summarized in the table below: | | Diagnosis | | | | | --- | --- | --- | --- | --- | | | | ENA-Associated CTDs | Non ENA-Associated CTD | Totals | | Alegria Flash ENA Screen | Positive ≥100.0 | 393 | 29 | 422 | | | Negative <100.0 | 196 | 325 | 521 | | | Total | 589 | 354 | 943 | | Sensitivity | 66.7% (393/589) | 95% CI: 62.8; 70.4% | | --- | --- | --- | | Specificity | 91.8% (325/354) | 95% CI: 88.5; 94.2% | # D Clinical Cut-Off: Refer to the assay cut-off section (VII.A.7) above. # E Expected Values/Reference Range: The frequency of ENA autoantibody positivity was investigated in a cohort of 200 serum samples from apparently healthy donors. The samples were equally distributed across age and sex. The samples were tested using the Alegria Flash ENA Screen on the Alegria Flash instrument. Eight samples $(4.0\%)$ were positive. # 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. K250408 - Page 11 of 11