ImmuLisa Enhanced Centromere Antibody ELISA

{0} 1 # 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION SUMMARY A. 510(k) Number: K151559 B. Purpose for Submission: New device C. Measurand: IgG autoantibodies specific for Centromere protein D. Type of Test: Immunoassay, qualitative and semi-quantitative E. Applicant: IMMCO Diagnostics Inc. F. Proprietary and Established Names: ImmuLisa™ Enhanced Centromere Antibody ELISA 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 4. Panel: Immunology (82) H. Intended Use: 1. Intended use(s): An enzyme linked immunoassay (ELISA) for the qualitative or semiquantitative detection of anti-centromere antibodies in human serum or plasma to aid in the diagnosis of limited cutaneous systemic sclerosis / CREST in conjunction with other laboratory tests and clinical findings. {1} 2. Indication(s) for use: Same as intended use 3. Special conditions for use statement(s): For prescription use only 4. Special instrument requirements: An ELISA microplate reader capable of reading absorbance values at 450 nm. If dual wavelength microplate reader is available, the reference filter should be set at 600–650 nm. An automatic microplate washer capable of accurately dispensing 200 µL of fluid is also required. I. Device Description: Each kit consists of 12- 1 x 8 antigen coated microwell strips, negative control, positive control, five assay calibrators, anti-human IgG-horse radish peroxidase conjugate, TMB substrate, stop solution, wash buffer and diluent. J. Substantial Equivalence Information: 1. Predicate device name(s): QUANTA Lite™ Centromere ELISA 2. Predicate 510(k) number(s): K003959 3. Comparison with predicate: | Similarities | | | | --- | --- | --- | | Item | ImmuLisa™ Centromere | Predicate Device | | Intended Use | An enzyme linked Immunoassay (ELISA) for the qualitative or semi-quantitative detection of anti-centromere antibodies in human serum or plasma to aid in the diagnosis of limited cutaneous systemic sclerosis / CREST in conjunction with other laboratory tests and clinical findings. | QUANTA Lite Centromere (CENP-A & CENP-B) is an enzyme-linked immunosorbent assay (ELISA) for the semi-quantitative detection of Centromere antibodies, specifically reactive to the CENP-A and CENP-B antigens in human sera. Detection of these antibodies is an aid in diagnosis of certain connective tissue diseases such as the CREST variant of scleroderma. | | Assay Type | ELISA | Same | | Type of Test | Semi-quantitative and qualitative | Semi-quantitative | {2} | Similarities | | | | --- | --- | --- | | Item | ImmuLisa™ Centromere | Predicate Device | | Capture Antigen | Recombinant purified CENP-A and CENP-B Centromere antigens | Same | | Conjugate | HRP conjugated anti-human IgG | Same | | Substrate | TMB | Same | | Traceability | International Reference Preparation is not available. Results are traceable to in-house standards. | Same | | Sample Type | Serum | Same | | Screening Dilution | 1:101 | Same | | Negative Cut-off | < 20 EU/mL | < 20 units | | Conjugate | HRP conjugated anti-human IgG | Same | | Instrumentation | Spectrophotometer 450 nm | Same | | Signal | Optical density | Same | | Differences | | | | --- | --- | --- | | Item | ImmuLisa™ Centromere | Predicate Device | | Positive Cutoff | >25 EU/mL | >20 units | | Indeterminate Region | 20–25 EU/mL | Not applicable | | Calibrators | Set of 5; values in EU/mL: 160,80 40, 20,1 and Cal D (20 EU/mL) for the single point calibration method | Single point calibrator | | Linear Range | 3.9 EU/mL–160 EU/mL | Not specified | | Limit of Detection | 3.9 EU/mL | Not specified | # K. Standard/Guidance Document Referenced: 1. Evaluation of Precision Performance of Quantitative Measurement Methods (CLSI EP05-A2) {3} 2. Evaluation of the Linearity of Quantitative Measurement Procedures: A Statistical Approach; Approved Guideline (CLSI EP6-A) 3. Interference Testing in Clinical Chemistry; Approved Guideline - Second Edition (CLSI EP07-A2) 4. Method Comparison and Bias Estimation Using Patient Samples (CLSI EP9-A2) 5. User Protocol for Evaluation of Qualitative Test Performance (CLSI EP12-A2) 6. Protocols for Determination of Limits of Detection and Limits of Quantitation; Approved Guideline (CLSI EP17-A) L. Test Principle: The test is performed as a solid phase immunoassay. Controls, calibrators and patient sera are incubated in the antigen coated wells to allow specific antibodies present in the serum to bind to the centromere antigen. Unbound antibodies and other serum proteins are removed by washing the microwells. Bound antibodies are detected by adding an enzyme labeled anti-human IgG conjugate to the microwells. Unbound conjugate is removed by washing. Enzyme substrate (TMB) is then added to the wells and the presence of antibodies is detected by a color change produced by the conversion of TMB substrate to a colored reaction product. The reaction is stopped and the intensity of the color change, which is proportional to the concentration of antibody, is read by a spectrophotometer at 450 nm. Semi-quantitative results are determined from a series of five calibrators (160 EU/mL, 80 EU/mL, 40 EU/mL, 20 EU/mL, and 1 EU/mL). Values less than 20 EU/mL are considered negative results while values greater than 25 EU/mL are considered positive; results between 20 EU/mL and 25 EU/mL are considered 'indeterminate'. Qualitative results are determined using a ratio of the absorbance of the sample to the absorbance of the cut-off calibrator (20 EU/mL). The ratio is multiplied by the concentration of the cut-off calibrator to give a numerical value. Values greater than 20 EU/mL are reported as positive. M. Performance Characteristics: 1. Analytical performance: a. Precision/Reproducibility: Semi-Quantitative Precision: Sera from seven patients from the Intended Use population were selected to cover the analytical measuring range of the assay and included samples in the negative range, ~20% below cut-off, around the cut-off, ~20% above cut-off and in the moderate positive range of the assays. Samples were run in duplicate, twice per day for 20 days (n = 80 replicates per sample). Assays were run by two operators on two different sets of equipment, each using a multichannel pipettor, microplate washer and microplate reader. The manufacturer's pre-determined acceptance criteria met for all measures. 4 {4} Semi-Quantitative Precision: | | | Repeatability | | Between Days | | Inter-operator | | Total | | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | | EU/mL Mean | SD | %CV | SD | %CV | SD | %CV | SD | %CV | | 1 | 8.5 | 0.3 | 4.1 | 0.3 | 3.3 | 0.2 | 2.8 | 0.4 | 5.2 | | 2 | 16.4 | 0.5 | 3.0 | 0.8 | 5.2 | 0.6 | 3.6 | 1.0 | 6.0 | | 3 | 20.4 | 0.9 | 4.4 | 0.5 | 2.3 | 0.5 | 2.5 | 1.0 | 5.0 | | 4 | 23.8 | 0.5 | 1.9 | 0.8 | 3.5 | 0.5 | 2.2 | 0.9 | 4.0 | | 5 | 49.0 | 1.5 | 3.0 | 2.2 | 4.5 | 1.5 | 3.1 | 2.6 | 5.4 | | 6 | 105.1 | 1.7 | 1.6 | 2.7 | 2.6 | 2.4 | 2.3 | 3.2 | 3.0 | | 7 | 150.6 | 4.7 | 3.1 | 2.7 | 1.8 | 0.0 | 0.0 | 5.4 | 3.6 | # Operator-to-Operator Reproducibility: The sponsor provided operator-to-operator reproducibility for the manual method. This study incorporated three operators running five replicates per assay over three days. The data are summarized in the following table: | Sample | Mean EU/mL | Operator 1 %CV | Operator 2 %CV | Operator 3 %CV | Total %CV | | --- | --- | --- | --- | --- | --- | | Low Negative | 7.9 | 10.1 | 12.4 | 10.5 | 10.8 | | Cut-off - 20% | 16.3 | 7.5 | 6.6 | 5.1 | 6.5 | | Cut-off | 20.6 | 6.1 | 6.3 | 4.5 | 5.8 | | Cut-off + 20% | 23.5 | 5.4 | 5.0 | 5.4 | 5.3 | | Moderate Positive | 43.0 | 5.9 | 5.6 | 4.2 | 5.2 | | High Positive | 95.8 | 3.2 | 3.2 | 3.7 | 3.8 | | High Positive | 154.0 | 3.9 | 3.8 | 2.8 | 3.5 | The manufacturer's pre-determined acceptance criteria were met for all measures. # Qualitative Reproducibility: Studies were performed under the guidance of CLSI EP12-A2 "User Protocol for Evaluation of Qualitative Test Performance." Eighty replicates each of sera in the negative range: $20\%$ below the cut-off, at the cut-off, $20\%$ above the cut-off and in the moderate positive range of the assays were tested to evaluate the reproducibility of the qualitative method of analysis. The results were calculated using single-point (qualitative) analysis as indicated in the product insert. The manufacturer's predetermined acceptance criteria were met. Results are summarized below. | Sample | Mean EU/mL | % Negative | % Positive | | --- | --- | --- | --- | | Low Negative | 7.9 | 100 | 0 | | Cut-off -20% | 16.2 | 100 | 0 | | Cut-off | 20.4 | 37 | 63 | {5} | Sample | Mean EU/mL | % Negative | % Positive | | --- | --- | --- | --- | | Cut-off +20% | 23.4 | 1 | 99 | | Moderate Positive | 44.5 | 0 | 100 | | High Positive | 79.3 | 0 | 100 | | High Positive | 149.4 | 0 | 100 | Lot-to-Lot Reproducibility: Inter-lot reproducibility was tested by using samples spanning the assay range. Three lots of material were used in the study. Seven samples were tested in duplicate on three different lots of the kit. Assays were run by two operators on two different sets of equipment, each using a multichannel pipettor, microplate washer and microplate reader. There was no recalibration of this equipment over the course of this study. The manufacturer's pre-determined acceptance criteria were met for all measures. Lot-to-Lot Reproducibility: | Sample | EU/mL | Lot 1 %CV | Lot 2 %CV | Lot 3 %CV | Between Lots %CV | | --- | --- | --- | --- | --- | --- | | 1 | 8.0 | 9.2 | 8.7 | 8.2 | 9.0 | | 2 | 16.2 | 7.1 | 6.6 | 7.0 | 6.9 | | 3 | 20.2 | 6.3 | 5.2 | 7.2 | 6.7 | | 4 | 23.0 | 6.2 | 5.1 | 5.8 | 6.2 | | 5 | 43.3 | 5.0 | 5.7 | 6.4 | 6.1 | | 6 | 96.5 | 3.6 | 3.1 | 3.1 | 3.7 | | 7 | 154.0 | 4.2 | 4.6 | 3.8 | 4.4 | Control Material Precision Studies: Positive and negative control materials were tested in duplicate, twice per day for 20 days for a total of 80 replicates per sample. Assays were run by two operators on two different sets of equipment. | | | Repeatability | | Between Runs | | Total | | | --- | --- | --- | --- | --- | --- | --- | --- | | | Mean | SD | %CV | SD | %CV | SD | %CV | | Negative Control | 5.3 | 0.1 | 2.2 | 0.4 | 7.6 | 0.4 | 7.3 | | Positive Control | 82.6 | 2.4 | 2.9 | 5.1 | 6.2 | 5.1 | 6.2 | b. Linearity/assay reportable range: Positive samples with values throughout the measuring range were selected and assayed in duplicate, at equidistant dilutions to determine linear range of the assays. {6} The linear range of the assay was determined to be 3.9–160 EU/mL. | Sample | Dilution Range (EU/mL) | Slope (95% CI) | Y-Intercept (95% CI) | R² | % Recovery | | --- | --- | --- | --- | --- | --- | | 1 | 3.2–46.0 | 1.05 (0.97 –1.13) | -0.67 (-2.88 –1.53) | 0.994 | 92 –116 | | 2 | 6.3–123.1 | 1.02 (0.95 –1.10) | -0.18 (-5.64 –5.29) | 0.995 | 93 –104 | | 3 | 4.9–188.1 | 1.03 (0.95 –1.11) | 1.42 (-7.15 –9.98) | 0.984 | 87 –102 | High dose hook effect: High concentrations specimens were serially diluted (from approximated 407 EU/mL) to assess the presence of a decrease in assay signal associated with antigen excess (hook effect). A hook effect at concentrations up to 407 EU/mL was not seen. c. Traceability, Stability, Expected values (controls, calibrators, or methods): i) Traceability: There are currently no recognized international standards for the measurement of anti-centromere antibodies. Calibrator and Control values are directly traceable to in-house standards. ii) Value Assignment: Calibrators and Positive Controls are dilutions of pooled Centromere antibody-positive sera. The sponsor formulates new calibrator and control lots from an array of antibody positive sera obtained from commercial plasma centers stored frozen at -70°C. The calibrators and controls are taken from different pooled sera. All source sera has been tested and found negative for infectious disease as stated in the product insert. Manufactured calibrator sets are stored in aliquots frozen at -70°C. As new lots of calibrators are developed, comparison studies are performed to calibrate values against original calibrators. Each lot of calibrator is also tested in comparison with normal human sera, clinical samples and internal standards. iii) Stability: Shelf life stability: Accelerated and open kit studies for each device were performed on three lots of components/reagents. Accelerated studies were conducted with materials incubated at 37°C. In these conditions, one day is considered equivalent to one month stored at 2°–8°C. Materials are removed from the incubator for testing at three-day intervals for a minimum of 18 days. Real time stability of three kit lots was tested using five specimens with reactivities across the analytical measuring range; current results support a six month shelf life stability claim. {7} 8 Open Kit Stability: For open kit stability studies, materials are opened and stored as required for bench-top usage, then assayed at 15, 45 and 90 day intervals. Open vial stability studies demonstrate opened reagents are stable at 45 days, but the sponsor chose a more restrictive one month open kit stability claim. d. Detection limit: The Limit of Blank (LoB) and the Limit of Detection (LoD) were determined using 60 replicates of the blank and 10 replicates each of 6 low-level (normal human) samples by following CLSI EP17-A. Sixty samples of diluent were run as blank samples and six different normal human sera were each assayed 10 times. LoB was determined to be 3.5 and LoD was determined to be 3.9 EU/ml. e. Analytical specificity: Interference: The studies were performed according to CLSI EP07-A2, Interference Testing in Clinical Chemistry, Approved Guideline- Second Edition. Interference was studied by mixing sera with known Centromere antibody levels with serum samples containing potentially interfering substances and studying deviation from expected results. Samples were selected from the negative range, near the assay cutoff and in the low, moderate and high positive range less than the value of the top calibrator. No significant interference (defined as &gt; 15% deviation from expected) was demonstrated in the Centromere assay for the following substances at the levels indicated: Hemoglobin (2 g/L), Bilirubin (342 μmol/L), Rheumatoid Factor (100 EU/mL), Triglycerides (37 mmol/L) and Cholesterol (13 mmol/L). Comparison to Reference Sera: Twelve ANA human reference sera from the Centers for Disease Control and Prevention were tested with the ImmuLisa Centromere Antibody ELISA. As expected, the CDC sample known to contain anti-Centromere antibodies tested strongly positive. The eleven other samples were negative. The other samples represent other ANA-type antigens such as SS-A, SS-B, Jo-1, etc. Ten ANA human reference sera from the Association of Medical Laboratory Immunologists were tested with the ImmuLisa Centromere Antibody ELISA. As expected, the AMLI sample known to contain anti-Centromere antibodies tested strongly positive. The nine other samples were negative. The other samples represent other ANA-type antigens such as SS-A, SS-B, Jo-1, etc. f. Assay Cut-off: Positive assay cut-offs were assigned a unit values of 25 EU/mL for the semi-quantitative method and 20 EU/mL for the qualitative method, based on the standardized method used by other IMMCO products. Indeterminate/borderline results (defined as 20–25 EU/mL) for the semi-quantitative method, are {8} recommended to be retested and evaluated along with other laboratory methods for detection of antibodies to ENA. ## 2. Comparison studies: a. Method comparison with predicate device: A method comparison study was performed with the QUANTA Lite™ Centromere ELISA kit, using 119 samples from patients with limited system sclerosis (LcSSc)/ CREST Syndrome (SSc)] and 288 disease controls. Sample values were within the analytical measuring range for the device. Performance relative to the predicate was determined by calculating agreement where borderline values were considered positive or negative. Qualitative results were the same as those obtained when semi-quantitative indeterminate values are considered positive. | Borderline values considered positive | Quanta Lite Centromere Antibody ELISA | | | | | --- | --- | --- | --- | --- | | | | Positive | Negative | Total | | IMMCO Centromere Ab ELISA | Positive | 55 | 16 | 71 | | | Negative | 4 | 332 | 336 | | | Total | 59 | 348 | 407 | Positive Percent Agreement: 93.2% (95% CI 88.7–97.8%) Negative Percent Agreement: 95.4% (95% CI 92.5–97.3%) Total Agreement: 95.1% (95% CI 92.4–96.9%) | Borderline values considered negative | Quanta Lite Centromere Antibody ELISA | | | | | --- | --- | --- | --- | --- | | | | Positive | Negative | Total | | IMMCO Centromere Ab ELISA | Positive | 51 | 13 | 64 | | | Negative | 8 | 335 | 343 | | | Total | 59 | 348 | 407 | Positive Percent Agreement: 86.4% (95% CI 74.5–93.6%) Negative Percent Agreement: 96.3% (95% CI 93.5–97.9%) Total Agreement: 94.8% (95% CI 92.2–96.6%) The manufacturer’s pre-determined acceptance criteria were met for all measures. {9} b. Matrix comparison: Not applicable. # 3. Clinical studies: # a. Clinical Sensitivity and Specificity: The clinical sensitivity and specificity of the IMMCO Centromere assay was evaluated in 989 serum samples representing various autoimmune and infectious conditions; the performance was assessed against the clinical diagnosis. Borderline results were considered positive and negative for these analyses. When results were calculated by the qualitative method, sensitivity and specificity are identical to the semi-quantitative results when borderline results are considered positive. | Borderline values considered positive | Diagnosis -lcSSC | | | | | --- | --- | --- | --- | --- | | | | lcSSC/ CREST | Controls | Total | | IMMCO Centromere Ab ELISA | Positive | 66 | 39 | 105 | | | Negative | 58 | 826 | 884 | | | Total | 124 | 865 | 989 | Sensitivity $= 53.2\%$ (95% C.I. $= 44.1 - 62.2\%$ ) Specificity $= 95.5\%$ (95% C.I. $= 93.8 - 96.7\%$ | Borderline values considered negative | Diagnosis -lcSSC | | | | | --- | --- | --- | --- | --- | | | | lcSSC/ CREST | Controls | Total | | IMMCO Centromere Ab ELISA | Positive | 61 | 35 | 96 | | | Negative | 63 | 830 | 893 | | | Total | 124 | 865 | 989 | Sensitivity $= 49.2\%$ (95% C.I. $= 40.2 - 58.3\%$ ) Specificity $= 96.0\%$ (95% C.I. $= 94.4 - 97.2\%$ ) {10} The distribution of the Centromere positivity rate in the cohort is in the Table below: | Cohort | n | #Pos1 | %Pos1 | | --- | --- | --- | --- | | Limited cutaneous systemic sclerosis | 124 | 66 | 53.2% | | Diffuse cutaneous scleroderma | 39 | 1 | 2.6% | | Anti-phospholipid syndrome | 36 | 4 | 11.1% | | Anti-phospholipid syndrome with SLE | 20 | 0 | 0.0% | | Celiac Disease | 35 | 2 | 11.4% | | Churg-Strauss syndrome | 27 | 0 | 0.0% | | Crohn's disease | 25 | 0 | 0.0% | | Granulomatosis with polyangiitis | 27 | 0 | 0.0% | | Graves' disease | 20 | 0 | 0.0% | | Hashimoto's thyroiditis | 20 | 0 | 0.0% | | Osteoarthritis | 20 | 0 | 0.0% | | Polymyositis/Dermatomyositis | 30 | 1 | 3.3% | | Rheumatoid Arthritis | 24 | 1 | 4.2% | | Sjögren's Syndrome | 37 | 2 | 5.4% | | Systemic Lupus Erythematosus | 92 | 1 | 1.1% | | Thrombocytopenia | 15 | 2 | 13.3% | | Ulcerative Colitis | 25 | 0 | 0.0% | | Autoimmune hepatitis | 30 | 4 | 13.3% | | Mixed Connective Tissue Disease | 30 | 2 | 6.7% | | Primary Biliary Cirrhosis | 30 | 6 | 20.0% | | IDDM | 30 | 1 | 3.3% | | CMV | 20 | 0 | 0.0% | | Hepatitis C | 20 | 0 | 0.0% | | HSV-1 | 20 | 3 | 15.0% | | HSV-2 | 20 | 0 | 0.0% | | Lyme disease | 20 | 0 | 0.0% | | Mononucleosis | 20 | 1 | 5.0% | | Rubella | 20 | 0 | 0.0% | | Syphilis | 17 | 0 | 0.0% | | Toxoplasmosis | 20 | 0 | 0.0% | | Advanced chronic renal failure | 30 | 3 | 10.0% | | Cancers2 | 46 | 3 | 6.5% | 1. Indeterminate results were considered positive 2. Cancers included breast, colorectal, lung, ovarian and pancreatic. The manufacturer's pre-determined acceptance criteria were met for all measures. Clinical cut-off: Not applicable {11} 4. Expected values/Reference range: Test results in a normal population are expected to be negative. A study of 117 normal, apparently disease-free samples tested with the anti-Centromere assay yielded no borderline results (0.0%) and no positive results (0.0%). N. Proposed Labeling: The labeling is sufficient and it satisfies 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. 12