GOLD STANDARD DIAGNOSTICS ANTI-NUCLEAR ANTIBODY (ANA) SCREEN ELISA TEST KIT

{0} Page 1 of 12 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION SUMMARY ASSAY ONLY A. 510(k) Number: k131330 B. Purpose for Submission: New device C. Measurand: Anti-Nuclear Antibodies (ANA) D. Type of Test: Manual, qualitative enzyme linked immunosorbent assay (ELISA) E. Applicant: Gold Standard Diagnostics Corporation F. Proprietary and Established Names: Proprietary Name: Gold Standard Diagnostics Anti-nuclear Antibody (ANA) Screen ELISA Test Kit Established Name: Antinuclear Antibody (Enzyme-Labeled), Antigen, Controls G. Regulatory Information: 1. Regulation section: 21 CFR §866.5100 Antinuclear antibody immunological test system 2. Classification: Class II 3. Product code: LJM, antinuclear antibody (enzyme-labeled), antigen, controls {1} 4. Panel: 82 (Immunology) H. Intended Use: 1. Intended use(s): The Gold Standard Diagnostics Antinuclear Antibody (ANA) Screen ELISA Test Kit is a qualitative assay for the detection of ANAs in human serum. The assay collectively detects in one well ANAs against double stranded DNA (dsDNA), SSA (Ro60 and Ro52), SSB (La), Sm, Sm/RNP, Scl-70, Jo-1, Ribosomal P, and Centromeric antibodies along with sera positive for immunofluorescent HEp-2 ANAs. The assay is used as an aid in the diagnosis of Systemic Lupus Erythematosus, Mixed Connective Tissue Disease, Sjögren's Syndrome, Progressive Systemic Sclerosis, and Polymyositis/Dermatomyositis, and should be used in conjunction with other laboratory tests and clinical findings. 2. Indication(s) for use: Same as Intended Use 3. Special conditions for use statement(s): Prescription use only 4. Special instrument requirements: Microwell plate reader capable of measuring OD at 450 nm and at 620 nm for dual wavelength readings. I. Device Description: The device, as described in the labeling, is a kit composed of the following reagents: 1. One microtiterplate consisting of 96 wells with coated antigen (lyophilized) from which single wells can be broken. 2. Sample Buffer (orange cap) 100 mL ready to use solution, with preservative and Tween 20. 3. Wash Buffer (blue cap), 100 mL 10x concentrate solution, with preservative and Tween 20. Add to 900 mL deionized water. 4. Negative Control, 2.0 mL, human serum with protein-stabilizer and preservative, ready to use. 5. Positive Control, 2.0 mL, human serum containing antibodies positive for ANA with Page 2 of 12 {2} protein-stabilizer and preservative, ready to use. 6. Cutoff Control, 2.0 mL, human serum containing antibodies positive for ANA with protein-stabilizer and preservative, ready to use. 7. Conjugate (red cap), 14 mL ready to use solution, peroxidase-labeled goat antihuman IgG conjugate with protein-stabilizer and preservative. 8. Substrate (3, 3', 5, 5'-TetraMethylBenzidine), 14 mL ready to use solution. 9. Stop Solution, 14 mL ready to use solution, contains acid. ## J. Substantial Equivalence Information: 1. Predicate device name(s) and 510(k) number(s): Aeskulisa ANA Hep2, k040953 2. Comparison with predicate: | Similarities | | | | --- | --- | --- | | Item | Device | Predicate | | Intended Use | A qualitative assay for the detection of ANAs in human serum | Same | | Measured analytes | Antibodies to double stranded DNA (dsDNA), SS-B (La), Sm, RNP/Sm, Scl-70, Jo-1 and centromeric antigens, and antibodies detected against Hep2 cells by immunofluorescence test | Same | | Assay technology | Colorimetric enzyme immunoassay | Same | | Immunoglobulin detected | Human IgG autoantibodies | Same | | Captured antigens | Lysed Hep-2 extract Purified native: Ro60, Sm Recombinant: SSB, RNP, Scl-70, Jo-1, CENP-B | Same | | Detection antibody | Horseradish peroxidase (HRP) labeled goat anti-human IgG | Same | | Substrate | 3, 3', 5, 5'-tetramethylbenzidine (TMB) | Same | | Controls | Positive, negative and cut-off controls | Same | | Sample matrix | Serum | Same | {3} | Differences | | | | --- | --- | --- | | Item | Device | Predicate | | Measured analytes and captured antigens | Antibodies to SSA [Ro60 (purified native) and Ro52 (recombinant)] Ribosomal P (recombinant) | Antibodies to SS-A (Ro) only Ribosomal P not detected | | Interpretation | Convert to units: Negative <0.83 units; Equivocal units 0.83-1.2 units Positive >1.2 units | Convert to units: Negative <1.0 units; no equivocal zone Positive >1.0 units | K. Standard/Guidance Document Referenced (if applicable): 1. CLSI EP07-A2 Interference Testing in clinical chemistry 2. FDA Guidance for Industry and Staff. Recommendations for anti-nuclear antibody (ANA) test systems premarket submissions L. Test Principle: The Antinuclear Antibody (ANA) Screen ELISA Test kit is an enzyme linked immunosorbent assay where natural and recombinant Extractable Nuclear Antigens (ENAs) are collectively bound to microwells in polystyrene microtiter plates. Human serum containing ANA (IgG) antibodies is added to the wells and any ANAs bind to their cognate antigen(s). After incubation, the wells of the plate are washed to remove unbound serum components and non-ANA IgGs. A detection antibody conjugated with HRP is added to detect human IgG antibodies bound to antigens on the microtiter well. After an incubation period, the wells of the plate are washed to remove unbound enzyme-labeled anti-human IgG. Upon its addition to the washed wells the substrate TMB is converted by bound enzyme-conjugate antibody producing a blue end product. The Stop Solution is an acid that stops the reaction and turns the substrate yellow. The amount of color produced is proportional to the amount of antibody is present in the patient serum. The antigens used in the Screen ELISA Test are a lysed HEp-2 cell extract with added purified antigens. The assay collectively detects in one well antibodies against double stranded DNA (dsDNA), SSA (Ro60 and Ro52), SSB (La), Sm, Sm/RNP, Scl-70, Jo-1, Ribosomal P, and Centromeric antibodies along with antibodies with reactivity against HEp-2 cells. Optical density values read by the spectrophotometer are converted into 3 categories based upon a ratio of optical density of the samples (or controls) to the optical density of a cutoff control reagent present in the assay kit. Three result categories are determined by optical density ratio values as follows: {4} | Optical density ratio | Interpretation | | --- | --- | | < 0.83 units | negative | | 0.83 – 1.2 units | equivocal | | > 1.2 units | positive | # M. Performance Characteristics (if/when applicable): # 1. Analytical performance: # a. Precision/Reproducibility: Assessment of the repeatability of the assay was performed on seven samples; three positive samples, one equivocal sample, and three negative samples. One positive sample was diluted with normal human serum to give a unit value $20\%$ above the positive ratio cutoff (1.36 U). The other two positive samples gave assay units approximately 2.56 and 5.60. One negative sample was diluted to $20\%$ below the negative cutoff ratio (approximately 0.70 units). The other two negative samples gave assay units approximately 0.3 and 0.15. Each sample was tested twice a day for ten days for a total of 20 replicates. All the observed results matched the expected results. The results, presented as ratio Units, are summarized in the following table: # Repeatability: | Sample | Mean (U) | Range (U) | Expected qualitative result | % Observed matching expected result | | --- | --- | --- | --- | --- | | 1 | 5.60 | 5.18–5.82 | Positive | 100% | | 2 | 2.56 | 2.13–2.96 | Positive | 100% | | 3 | 1.36 | 1.24–1.67 | Positive | 100% | | 4 | 1.06 | 0.93–1.30 | Equivocal | 90% | | 5 | 0.73 | 0.62–0.90 | Negative | 90% | | 6 | 0.34 | 0.27–0.38 | Negative | 100% | | 7 | 0.16 | 0.12–0.20 | Negative | 100% | The reproducibility of the assay (between-lab imprecision) was done by testing three samples in duplicate for five days, twice a day, at three sites with two technicians per site for a total of 60 replicates. The mean results, reported as ratio Units, are summarized in the table below: {5} Reproducibility: | Sample | Mean (U) | Site | Range (U) | Expected qualitative result | % Observed result | | --- | --- | --- | --- | --- | --- | | 1 | 0.20 | 1 | 0.18-0.24 | Negative | 100% | | 1 | | 2 | 0.18-0.26 | Negative | 100% | | 1 | | 3 | 0.20-0.27 | Negative | 100% | | 2 | 1.45 | 1 | 1.05-1.57 | Equivocal | 95% | | 2 | | 2 | 1.06-1.45 | Equivocal | 95% | | 2 | | 3 | 1.4-1.55 | Equivocal | 100% | | 3 | 3.08 | 1 | 2.94-3.14 | Positive | 100% | | 3 | | 2 | 2.51-3.18 | Positive | 100% | | 3 | | 3 | 3.04-3.37 | Positive | 100% | Lot to Lot: Three samples were tested five times each on three different lots. The mean results, reported as ratio Units, are summarized in the table below: | Sample | Mean (U) | Lot Number | Range (U) | Expected qualitative result | % Observed result matching expected result | | --- | --- | --- | --- | --- | --- | | 1 | 1.474 | 1 | 1.42-1.51 | Positive | 100% | | 1 | | 2 | 1.48-1.60 | Positive | 100% | | 1 | | 3 | 1.39-1.47 | Positive | 100% | | 2 | 0.960 | 1 | 0.92-0.96 | Equivocal | 100% | | 2 | | 2 | 0.92-1.01 | Equivocal | 100% | | 2 | | 3 | 0.96-1.00 | Equivocal | 100% | | 3 | 0.519 | 1 | 0.51-0.58 | Negative | 100% | | 3 | | 2 | 0.50-0.52 | Negative | 100% | | 3 | | 3 | 0.50-0.51 | Negative | 100% | b. Linearity/assay reportable range: Linearity: Not applicable. Assay reportable range: Not applicable. Hook effect: To evaluate the hook effect on the assay, five sera with high antibody concentrations were diluted 1:100 to 1:12,000 and the units were calculated after completion of the testing. No hook effect was seen at the maximum measurement of 12 U. c. Traceability, Stability, Expected values (controls, calibrators, or methods): {6} Page 7 of 12 ## Traceability: There is no recognized standard or reference material, so the assay is calibrated in arbitrary units. Each new lot of the cutoff control is traceable to an internal master control. ## Positive and Negative Control: Positive control was prepared from a pool of eight pathological sera obtained from various plasma brokers determined to be positive for ANAs by HEp-2 immunofluorescence. Negative control was prepared by diluting the positive control pool 100-fold with sample diluent and protein stabilizer. The package insert states that the negative control must have an OD of &lt;0.15; the positive control must have an OD of 0.6 to 1.6. ## Cut-off Control: Cutoff controls are derived from a pool of eight pathological sera obtained from various plasma brokers determined to be positive by HEp-2 immunofluorescence. The cutoff control is prepared by diluting the serum pool with analyte-free serum to the appropriate concentration. The OD value of the cutoff control is compared with the master cutoff control. The unit ratio (OD cutoff control / OD master cutoff control) during the in-house QC procedure has to be 0.87 – 1.15. Each new lot of the cutoff controls, which are prepared by dilution of the pool stock, is compared with the master control and must meet the pre-specified acceptance criteria. Further, a set of 12 sera representing the antigen specificities is measured for each new lot of the controls and have to fall within their pre-specified ranges (which was obtained from several tests with different lots). ## Calibrators: Not applicable Stability: The sponsor provided data demonstrating real-time stability of an opened kit for 6 months when stored at 2-8°C. ## d. Detection limit: Not applicable ## e. Analytical specificity: The effect of endogenous substances in serum specimens that could cause interference with the ANA ELISA Screen Test was evaluated. Five samples ranging from 1.4 to 2.5 U were spiked with the concentrations of hemoglobin, bilirubin, rheumatoid factor and triglycerides indicated below. The recovery in relation to the un-spiked sample without interferent was calculated. In addition, to assess the interference in the assay from heterophilic antibodies, two samples were spiked with three concentrations of HAMA type 1 antibody. The recommended concentrations from the guideline “Interference Testing in Clinical Chemistry” from the Clinical and Laboratory Standards Institute were used (CLSI EP7-A2). The tested substances as listed below did not affect the performance of the ELISA Screen Test. {7} | Substance | Concentration | Mean Percent Inhibition | | --- | --- | --- | | Hemoglobin | 2 g/L | -2.1% | | Bilirubin | 20 mg/dL | -3.0% | | Rheumatoid Factor | 100 IU/mL | 8.8% | | Triglycerides | 3000 mg/dL | -1.4% | | Heterophile | 65 μg/mL | 5.4% | | Heterophile | 32.5 μg/mL | 2.2% | | Heterophile | 16.25 μg/mL | -0.6% | Ten CDC and AMLI samples were tested in duplicate. The following table summarizes the specific reactivity of each sample and the result of each replicate. | Sample | Specificity | Result 1 | Result 2 | | --- | --- | --- | --- | | CDC #1 | DNA | Positive | Positive | | CDC #2 | SS-A/SS-B | Positive | Positive | | CDC #3 | RNP, SS-A, SS-B | Positive | Positive | | CDC #4 | RNP | Positive | Positive | | CDC #5 | Sm | Positive | Positive | | CDC #7 | SS-A/Ro | Positive | Positive | | CDC #8 | CENP-B | Positive | Positive | | CDC #9 | Scl-70 | Positive | Positive | | CDC #10 | Jo-1 | Positive | Positive | | CDC #12 | Ribosomal-P | Positive | Positive | | AMLI #1 | Negative | Negative | Negative | | AMLI #2 | SS-A/SS-B | Positive | Positive | | AMLI #3 | SmRNP | Positive | Positive | | AMLI #4 | SS-A/Ro | Positive | Positive | | AMLI #5 | SS-A/SS-B | Positive | Positive | | AMLI #6 | Scl-70 | Positive | Positive | | AMLI #7 | Jo-1 | Positive | Positive | | AMLI #8 | CENP-B | Positive | Positive | | AMLI #9 | dsDNA | Positive | Positive | | AMLI #10 | Negative | Negative | Negative | f Assay cut-off The cutoff was determined by testing 99 normal blood donors and three known negative samples. The mean value plus three standard deviations of the negative samples was established as the cutoff. To encourage a repeat testing of samples close to the cutoff, an equivocal range of $\pm 20\%$ of the cutoff was established. 2. Comparison studies: a. Method comparison with predicate device: A comparison of the performance of the test device and the predicate was performed at three different sites using a total of 848 samples. The sera used for this study were prospectively obtained from the reference labs that were from samples that were {8} submitted for ANA serology testing. The results are summarized in the following table with the percent positive (PPA) and percent negative (NPA) agreements: | | Predicate device | | | | --- | --- | --- | --- | | Gold Standard Diagnostic ANA ELISA | Positive | Negative | Total | | Positive | 251 | 22 | 273 | | Equivocal | 7 | 23 | 30 | | Negative | 14 | 531 | 545 | | Total | 272 | 576 | 848 | Equivocals considered as Positive: $\mathrm{PPA} = 94.9\%$ (95% C.I. 91.5% - 97.2%), $\mathrm{NPA} = 92.2\%$ (95% C.I. 89.7% - 94.2%), $\mathrm{Overall} = 93.0\%$ (95% C.I. 91.1% - 95.7%), Equivocals considered as Negative: $\mathrm{PPA} = 92.3\%$ (95% C.I. 88.4% - 95.2%) $\mathrm{NPA} = 96.2\%$ (95% C.I. 94.3% - 97.6%) $\mathrm{Overall} = 94.9\%$ (95% C.I. 93.2% - 96.3%) To demonstrate the proposed test has comparable performance to the individual analyte assays, five samples known to be positive for each analyte (dsDNA, SS-A/Ro 60, SS-A/Ro 52, SS-B, Sm, Sm/RNP, Scl-70, Jo-1, Ribosomal P, Centromere, and HEp-2 IFA, total of 55 samples) were obtained and were tested on the proposed ANA Screening test, on an FDA-cleared test that measures each analyte individually, and by ANA HEp-2 IFA test. The percentage of the five samples from each analyte that were positive in the different tests is shown in the following table: | Sample reactivity | GSD test | Individual analyte assay- % positive | | | | | | | | | | | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | | | DNA | SSA (Ro60) | SSA (Ro52) | SSB (Ro52) | Sm | SM/RNP | SCL-70 | Jo-1 | Ribosomal P | Centromere | Hep-2 | | dsDNA | 100% | 100% | 60% | 20% | 20% | 40% | 40% | 0% | 0% | 20% | 0% | 100% | | SSA (Ro60) | 100% | 60% | 100% | 40% | 20% | 0% | 0% | 0% | 0% | 20% | 0% | 100% | | SSA (Ro52) | 80% (1 equivocal) | 20% | 80% | 100% | 20% | 0% | 0% | 0% | 0% | 0% | 0% | 80% | | SSB | 100% | 20% (1 equivocal) | 100% | 60% | 100% | 0% | 0% | 0% | 0% | 0% | 0% | 100% | | Sm | 100% | 60% | 0% | 0% | 0% | 100% | 100% | 0% | 0% | 0% | 0% | 100% | | Sm/RNP | 100% | 80% | 20% | 0% | 0% | 60% | 100% | 0% | 0% | 40% | 0% | 100% | | SCL-70 | 100% | 20% | 0% | 0% | 0% | 0% | 0% | 100% | 0% | 0% | 0% | 100% | | Jo-1 | 100% | 0% | 20% | 40% | 0% (1 equivocal) | 0% | 0% | 0% | 100% | 0% | 0% | 20% | | Ribosomal | 100% | 80% | 40% | 0% | 0% | 0% | 40% | 0% | 0% | 100% | 0% | 80% | | Centromere | 100% | 0% | 0% | 0% | 0% | 0% | 0% | 0% | 0% | 0% | 100% | 100% | | Hep-2 | 100% | 40% | 40% | 0% | 20% | 20% | 40% | 20% | 0% | 40% | 0% | 100% | {9} b. Matrix comparison: Not applicable. # 3. Clinical studies: # a. Clinical Sensitivity and Specificity: A comparison of the assay results to clinical diagnosis was performed using 753 retrospective samples with associated clinical diagnosis and demographic information obtained from serum brokers. No NHS or proficiency samples were used in the Clinical Sensitivity and Specificity study. | Clinical Diagnosis | Number Tested | Positive (%) | Equivocal (%) | Negative (%) | | --- | --- | --- | --- | --- | | Systemic Lupus Erythematosus (SLE) | 322 | 269 (82.9) | 11 (3.4) | 42 (13) | | Systemic Sclerosis (SSc) | 40 | 29 (72.5) | 1 (2.5) | 10 (25) | | Polymyositis (PM) | 12 | 4 (33.3) | 2 (16.7) | 6 (50) | | Dermatomyositis (DM) | 15 | 4 (26.7) | 2 (13.3) | 9 (60) | | PM or DM Overlap | 5 | 1 (20) | 1 (20) | 3 (60) | | Myositis | 10 | 4 (40) | 1 (10) | 5 (50) | | Mixed Connective Tissue Disease (MCTD) | 28 | 28 (100) | 0 | 0 | | Undifferentiated CTD (UTCD) | 3 | 3 (100) | 0 | 0 | | Sjögren's Syndrome (SjS) | 75 | 63 (84) | 4 (5.3) | 8 (10.7) | | Total (CTD) | 510 | 405 (79.4) | 22 (4.3) | 83 (16.3) | | Clinical Diagnosis | Number Tested | Positive (%) | Equivocal (%) | Negative (%) | | --- | --- | --- | --- | --- | | Rheumatoid Arthritis (RA) | 100 | 2 (2) | 0 | 98 (98) | | Osteoarthritis | 20 | 5 (25) | 0 | 15 (75) | | Primary Biliary Cirrhosis (PBC) | 8 | 1 (12.5) | 0 | 7 (87.5) | | Autoimmune Hepatitis (AIH) | 2 | 0 | 0 | 2 (100) | | Hashimoto's Thyroiditis | 17 | 0 | 0 | 17 (100) | | Grave's Disease | 17 | 0 | 0 | 17 (100) | | Ulcerative Colitis | 5 | 0 | 0 | 5 (100) | | Celiac Disease | 5 | 0 | 0 | 5 (100) | | Primary Anti-phospholipid Syndrome (PAPS) | 22 | 0 | 0 | 22 (100) | | Granulomatosis with polyangitis (Wegener's) | 5 | 0 | 0 | 5 (100) | | Total (non-CTD) | 201 | 8 (4) | 0 | 193 (96) | {10} Page 11 of 12 | Clinical Diagnosis | Number Tested | Positive (%) | Equivocal (%) | Negative (%) | | --- | --- | --- | --- | --- | | Herpes Simplex Virus (HSV) | 7 | 0 | 0 | 7 (100) | | Epstein Barr Virus (EBV) | 7 | 0 | 0 | 7 (100) | | Syphilis | 7 | 0 | 0 | 7 (100) | | Varicella Zoster Virus (VZV) | 7 | 0 | 0 | 7 (100) | | Mumps | 7 | 0 | 0 | 7 (100) | | Rheumatoid Factor | 7 | 0 | 0 | 7 (100) | | Total (other) | 42 | 0 | 0 | 42 (100) | Equivocals considered as Positive: Sensitivity = 83.5% (95% C.I. = 79.1% - 86.0%) Specificity = 96.7% (95% C.I. = 93.6% - 98.6%) Overall = 87.8% (95% C.I. = 85.2% - 90.0%) Equivocals considered as Negative: Sensitivity = 79.2% (95% C.I. = 75.4% - 82.7%) Specificity = 96.7% (95% C.I. = 93.6% - 98.6%) Overall = 84.9% (95% C.I. = 82.1% - 87.4%) b. Other clinical supportive data (when a. is not applicable): Not applicable 4. Clinical cut-off: Not applicable. 1) Expected values/Reference range: The expected value is negative; however, it is known that a certain percentage of NHS will be positive. In the sponsor's cutoff study, of the 99 samples from normal healthy donors, two were positive, for a prevalence of ANAs in the healthy population detected by this assay of 7%. The results are presented below. | Total | | | --- | --- | | N | 99 | | Positives | 2 | | Equivocals | 7 | | Negatives | 92 | | Prevalence | 7.0 % | | Ratio Mean (SD) (U) | 0.59 (0.2) | | Ratio Range (U) | 0.1 – 1.58 | {11} | By Gender | Number Tested | Mean Units | SD | 95^{th} Percentile | | --- | --- | --- | --- | --- | | Male | 51 | 0.62 | 0.24 | 1.09 | | Female | 48 | 0.55 | 0.13 | 0.82 | N. Proposed Labeling: The labeling meets the requirements of 21 CFR Part 809.10. O. Conclusion: The submitted information in this premarket notification is complete and supports a substantial equivalence decision. Page 12 of 12