QUANTA Flash M2 (MIT3), QUANTA Flash M2 (MIT3) Calibrators, QUANTA Flash M2 (MIT3) Controls

{0} 1 # 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION MEMORANDUM A. 510(k) Number: K163525 B. Purpose for Submission: New assay device on a previously cleared instrument C. Measurand: IgG antibodies specific for M2 protein (MIT3) D. Type of Test: Chemiluminescent immunoassay, semi-quantitative E. Applicant: Inova Diagnostics, Inc. F. Proprietary and Established Names: QUANTA Flash® M2 (MIT3) QUANTA Flash® M2 (MIT3) Calibrators QUANTA Flash® M2 (MIT3) Controls G. Regulatory Information: 1. Regulation section: 21 CFR §866.5090, Antimitochondrial antibody immunological test system 21 CFR §862.1150, Calibrator 21 CFR §862.1660, Quality control material (assayed and unassayed) 2. Classification: Class II (Assay and Calibrator) Class I (Controls) 3. Product code: DBM, Antimitochondrial Antibody, Indirect Immunofluorescent, Antigen, Control {1} JIT, Calibrator, Secondary JJX, Single (Specified) Analyte Controls (assayed and unassayed) 4. Panel: Immunology (82) (Assays) Clinical Chemistry (75) (Calibrator) Clinical Chemistry (75) (Controls) H. Intended Use: 1. Intended uses: QUANTA Flash M2 (MIT3) is a chemiluminescent immunoassay for the semi-quantitative determination of IgG anti-mitochondrial antibodies in human serum. The presence of anti-mitochondrial antibodies, in conjunction with clinical findings and other laboratory tests, is an aid in the diagnosis of primary biliary cholangitis. QUANTA Flash M2 (MIT3) Calibrators are intended for use with the QUANTA Flash M2 (MIT3) chemiluminescent immunoassay for the determination of IgG anti-mitochondrial antibodies in human serum. Each calibrator establishes a point of reference for the working curve that is used to calculate unit values. QUANTA Flash M2 (MIT3) Controls are intended for use with the QUANTA Flash M2 (MIT3) chemiluminescent immunoassay for quality control in the determination of IgG anti-mitochondrial antibodies in human serum. 2. Indications for use: Same as intended use 3. Special conditions for use statement: For prescription use only 4. Special instrument requirements: For use on the BIO-FLASH Instrument (K094060) I. Device Description: The QUANTA Flash M2 (MIT3) Reagents include: a. One QUANTA Flash M2 (MIT3) Reagent Cartridge contains the following reagents for 50 determinations: i. Recombinant M2 (MIT3) coated paramagnetic beads, lyophilized. 2 {2} ii. Tracer IgG - Isoluminol labeled anti-human IgG antibodies in buffer, containing protein stabilizers and preservative. iii. Assay Buffer - colored pink, containing protein stabilizers and preservatives. b. One Resuspension Buffer c. One Transfer Pippet The QUANTA Flash M2 (MIT3) Calibrators are sold separately and contain human antibodies to M2 (MIT3) in stabilizers and preservatives. The Calibrators include: a. QUANTA Flash M2 (MIT3) Calibrator 1: Two (2) barcode labeled tubes containing $0.3\mathrm{mL}$ prediluted, ready to use reagent b. QUANTA Flash M2 (MIT3) Calibrator 2: Two (2) barcode labeled tubes containing $0.3\mathrm{mL}$ prediluted, ready to use reagent c. QUANTA Flash M2 (MIT3) Calibrator 3: Two (2) barcode labeled tubes containing $0.3\mathrm{mL}$ prediluted, ready to use reagent The QUANTA Flash M2 (MIT3) Controls are sold separately and contain human antibodies to M2 (MIT3) in stabilizers and preservatives. The Controls include: a. QUANTA Flash M2 (MIT3) Negative Control: Two (2) barcode labeled tubes containing $0.5\mathrm{mL}$ , ready to use reagent b. QUANTA Flash M2 (MIT3) Positive Control: Two (2) barcode labeled tubes containing $0.5\mathrm{mL}$ , ready to use reagent # J. Substantial Equivalence Information: 1. Predicate device name: QUANTA Lite™ M2 EP (MIT3) ELISA 2. Predicate 510(k) number: K052262 3. Comparison with predicate: The QUANTA Flash M2 (MIT3) Reagents | Similarities | | | | --- | --- | --- | | Item | New Device QUANTA Flash M2 (MIT3) | Predicate Quanta Lite M2 EP (MIT3) ELISA | | Intended Use/ Indications for Use | QUANTA Flash M2 (MIT3) is a chemiluminescent immunoassay for the semi-quantitative determination of IgG anti- mitochondrial antibodies in human serum. The presence of anti- | The QUANTA LITE™ M2 EP (MIT3) ELISA is an enzyme- linked immunosorbent assay (ELISA) for the semi-quantitative detection of mitochondria antibodies in human | {3} | Similarities | | | | --- | --- | --- | | Item | New Device QUANTA Flash M2 (MIT3) | Predicate Quanta Lite M2 EP (MIT3) ELISA | | | mitochondrial antibodies, in conjunction with clinical findings and other laboratory tests, is an aid in the diagnosis of primary biliary cholangitis. | serum. The presence of mitochondria antibodies can be used in conjunction with clinical findings and other laboratory tests to aid in the diagnosis of primary biliary cirrhosis. | | Antigen | Recombinant antigen (MIT3) containing immunodominant portions of PDC-E2, BCOADC-E2, and OGDC-E2 | Same | | Assay Format | Semi-quantitative | Same | | Assay Methodology | Solid phase immunoassay | Same | | Sample Matrix | Serum | Same | | Reagent Shelf Life | One year at 2-8°C | Same | | Differences | | | | --- | --- | --- | | Item | New Device QUANTA Flash M2 (MIT3) | Predicate Quanta Lite M2 EP (MIT3) ELISA | | Assay Type | Chemiluminescent immunoassay | Manual ELISA | | Solid Phase | Antigen-coated paramagnetic beads | 96-well polystyrene plate | | Sample Dilution | 1:276 (automated instrument dilution) | 1:101 (manual dilution only) | | Reaction Temperature | 37°C (controlled) | Room temperature (20-26°C) | | Incubation times | Diluted patient samples: 9.5 min. Conjugate: 9.5 min. | High positive, low positive and negative controls, diluted patient samples: 30 min. Conjugate: 30 min. Substrate: 30 min (in dark). | | Detection Antibody (Conjugate) | Isoluminol conjugated mouse monoclonal anti-human IgG antibody | Goat anti-human IgG horseradish peroxidase | | Substrate/Chromogen | Trigger 1 (Fe(III) coproporphyrin in sodium hydroxide solution) and Trigger 2 (urea-hydrogen peroxide in sodium chloride solution) | Tetramethylbenzidene (TMB) | | Stop Solution | N/A | Sulfuric Acid (0.344 M) | | Signal | Chemiluminescence | Optical density | | Instrumentation | BIO-FLASH instrument | Microwell plate reader (450 nm) (or 620 for dual wavelength readings) | {4} 5 | Differences | | | | --- | --- | --- | | Item | New Device QUANTA Flash M2 (MIT3) | Predicate Quanta Lite M2 EP (MIT3) ELISA | | Calibration | Lot specific seven point, digital Master Curve plus three calibrators | Single-point calibration (Low positive), included with kit | | Calibration Curve | Lot specific calibration curve stored internally | Single point calibrator | | Reported Unit | CU (chemiluminescent units) | U/mL | | Limit of detection | 0.61 CU | Not specified | | Measuring Range | 1.2 CU–3,000.0 CU | Not specified | | Reportable Range | 1.2 CU–60,000.0 CU | Not specified | | Results Interpretation | Negative: < 20 CU Positive: ≥ 20 CU | Negative: < 20 U/mL Equivocal: 20.1–24.9 U/mL Positive: > 25 U/mL | ## QUANTA Flash M2 (MIT3) Calibrators | Similarities and Differences | | | | --- | --- | --- | | Item | New Device QUANTA Flash M2 (MIT3) Calibrators | Predicate | | Intended Use | For use with the QUANTA Flash M2 (MIT3) Reagents Each calibrator establishes a point of reference for the working curve that is used to calculate unit values. | No separate intended use; calibrator is part of the kit. | | Level | Three calibrators with values assigned at 12 CU, 400 CU and 2550 CU, sold separately | Single-point calibrator (Low positive) included with kit | | Method | QUANTA Flash M2 (MIT3) chemiluminescent immunoassay | QUANTA Lite M2 EP (MIT3) ELISA | | Analyte | Anti-mitochondrial antibodies | Same | | Matrix | Human serum, stabilizer and preservative | Human serum, buffer, protein stabilizer and preservative | | Unit | CU, arbitrary | units, arbitrary | | Physical state | Liquid, prediluted, ready to use | Same | | Shelf Life | One Year at 2–8°C | Same | {5} QUANTA Flash M2 (MIT3) Controls | Similarities and Differences | | | | --- | --- | --- | | Item | New Device QUANTA Flash M2 (MIT3) Controls | Predicate | | Intended Use | For use with the QUANTA Flash M2 (MIT3) reagents for quality control in the determination of IgG anti-mitochondrial autoantibodies in human serum. | No separate intended use; controls are part of the kit. | | Levels | Two (negative and positive) | Two (ELISA negative and high positive) | | Method | QUANTA Flash M2 (MIT3) chemiluminescent immunoassay | QUANTA Lite M2 EP (MIT3) ELISA | | Analyte | Anti-mitochondrial antibodies | Same | | Matrix | Human serum, stabilizer and preservative | Human serum, buffer, protein stabilizer and preservative | | Unit | CU (chemiluminescent units, arbitrary) | units, arbitrary | | Physical state | Liquid, ready to use | Same | | Shelf Life | One Year at 2–8°C | Same | K. Standard/Guidance Document Referenced: CLSI EP05-A3, Evaluation of Precision of Quantitative Measurement Procedures; Approved Guideline - Third Edition CLSI EP6-A, Evaluation of the Linearity of Quantitative Measurement Procedures: A Statistical Approach; Approved Guideline CLSI EP07-A2, Interference Testing in Clinical Chemistry; Approved Guideline - Second Edition CLSI EP09-A3, Measurement Procedure Comparison and Bias Estimation Using Patient Samples; Approved Guideline - Third Edition CLSI EP17-A2, Protocols for Determination of Limits of Detection and Limits of Quantitation; Approved Guideline CLSI C28-A3c, Defining, Establishing, and Verifying Reference Intervals in the Clinical Laboratory; Approved Guideline - Third Edition L. Test Principle: The QUANTA Flash M2 (MIT3) assay is a microparticle chemiluminescent immunoassay designed for use on the BIO-FLASH instrument. The instrument platform is a fully {6} automated closed system with continuous load and random access capabilities that automatically processes the samples, runs the assay and reports the results. It includes liquid handling hardware, luminometer and computer with software-user interface. The QUANTA Flash M2 (MIT3) assay utilizes a reagent cartridge format, which is compatible with the BIO-FLASH instrument. A patient's serum is diluted with buffer and mixed with M2 (MIT3) antigen-coated beads (final serum dilution: 1:276). Any antibodies specific for the mitochondrial antigens present on the beads will bind to the beads. Timed incubation and wash steps are followed by the addition of isoluminol conjugated monoclonal anti-human IgG (known as Tracer IgG). Following additional incubation and wash steps, the isoluminol conjugate is oxidized when Trigger 1 (Fe(III)coproporphyrin in sodium hydroxide solution) and Trigger 2 (ureahydrogen peroxide in sodium chloride solution) are added to the cuvette, and the flash of light produced from this reaction is measured as Relative Light Units (RLU) by the BIOFLASH optical system. The measured RLU is proportional to the amount of bound isoluminol conjugate, which in turn is proportional to the amount of anti-mitochondrial antibodies bound to the antigen on the beads. # M. Performance Characteristics: 1. Analytical performance: For all studies presented here, the results met the sponsor's predefined acceptance criteria. a. Precision/Reproducibility: The precision/reproducibility studies were performed in accordance with CLSI EP05-A3. # Precision: The precision of the QUANTA Flash M2 (MIT3) assay was evaluated on five patient samples, along with negative and positive controls which contained anti-mitochondrial antibodies ranging from 8.5–2187.3 CU. Samples were run in duplicate, twice a day, for 20 days on one lot of reagent. Within-run, between-run, between-day and total precision were calculated and summarized in the table below. | Sample | N | Mean (CU) | Within-Run | | Between-Run | | Between-Day | | Total | | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | | | | SD | CV (%) | SD | CV (%) | SD | CV (%) | SD | CV (%) | | 1 | 80 | 8.5 | 0.4 | 4.2 | 0.2 | 2.4 | 0.0 | 0.3 | 0.4 | 4.8 | | 2 | 80 | 18.3 | 0.5 | 2.6 | 0.2 | 1.1 | 0.2 | 0.8 | 0.5 | 2.9 | | 3 | 80 | 21.2 | 0.7 | 3.1 | 0.5 | 2.3 | 0.5 | 2.3 | 1.0 | 4.5 | | 4 | 80 | 45.4 | 1.2 | 2.7 | 1.0 | 2.2 | 0.6 | 1.3 | 1.7 | 3.7 | | 5 | 80 | 277.3 | 7.4 | 2.7 | 5.3 | 1.9 | 0.0 | 0.0 | 9.1 | 3.3 | | 6 | 80 | 1007.5 | 39.3 | 3.9 | 25.3 | 2.5 | 15.0 | 1.5 | 49.1 | 4.9 | | 7 | 80 | 2183.7 | 119.6 | 5.5 | 78.7 | 3.6 | 3.9 | 3.9 | 166.6 | 7.6 | {7} 8 # Site-to-Site Reproducibility: Nine samples were tested at three different sites. Samples were run in replicates of five, once a day, for five days, to generate 25 data points per sample, per site (one instrument/site). | Sample | N | Mean (CU) | Between-Site | | | --- | --- | --- | --- | --- | | | | | SD | CV (%) | | 1 | 75 | 9.3 | 0.8 | 9.1 | | 2 | 75 | 11.1 | 0.4 | 3.4 | | 3 | 75 | 22.9 | 1.4 | 6.2 | | 4 | 75 | 30.5 | 1.1 | 3.5 | | 5 | 75 | 53.7 | 2.8 | 5.2 | | 6 | 75 | 404.3 | 31.4 | 7.8 | | 7 | 75 | 617.8 | 50.4 | 8.2 | | 8 | 75 | 939.4 | 59.1 | 6.3 | | 9 | 75 | 2182.7 | 44.9 | 2.1 | # Lot-to-Lot Reproducibility Six samples were tested with three different lots of reagents in five replicates for five days, to generate 25 data points per lot (75 data points total for each sample). | Sample | N | Mean (CU) | Between Lots | | | --- | --- | --- | --- | --- | | | | | SD | CV (%) | | 1 | 75 | 8.3 | 0.5 | 6.5 | | 2 | 75 | 18.9 | 1.2 | 6.4 | | 3 | 75 | 19.7 | 0.9 | 4.7 | | 4 | 75 | 42.4 | 1.7 | 4.0 | | 5 | 75 | 869.4 | 37.9 | 4.4 | | 6 | 75 | 2186.7 | 191.0 | 8.7 | ## b. Linearity/assay reportable range: The linearity of the AMR (1.2–3,000 CU) was evaluated by a study according to CLSI EP6-A. Six anti-mitochondrial antibody positive serum samples were diluted with negative serum in 10% increments (from 0% to 90% negative serum) to obtain values from 1.2–3,373.5 CU. The dilutions were assayed in duplicates. Percent recovery of obtained mean results was calculated compared to the expected mean results (based on the dilution factor). The obtained values were plotted against expected values. The linear regression analysis with samples falling within AMR resulted in the following equation. {8} | Sample | Test Range (CU) | Slope (95% CI) | Intercept (95% CI) | R² | Average Recovery % | | --- | --- | --- | --- | --- | --- | | 1 | 3,373.5–481.9 | 1.08 (0.92–1.24) | -146.8 (-486–192.5) | 0.98 | 98.5 | | 2 | 1,496–149.7 | 1.01 (0.96–1.06) | 26.0 (-21.7–73.7) | 1.00 | 105.2 | | 3 | 281.5–28.1 | 1.03 (1.00–1.06) | -1.6 (-7.2–4.0) | 1.00 | 105.2 | | 4 | 52.5–5.3 | 1.00 (0.98–1.03) | 0.4 (-0.5–1.2) | 1.00 | 102.0 | | 5 | 22.7–2.3 | 0.98 (0.94–1.02) | 1.0 (0.4–1.6) | 1.00 | 109.2 | | 6 | 11.8–1.2 | 0.96 (0.92–1.00) | 0.1 (-0.2–0.4) | 1.00 | 100.3 | | Combined | 3,373–1.2 | 1.02 (1.00–1.04) | -2.2 (-18.7–14.4) | 1.00 | 102.9 | The combined data supported the claimed analytical measuring range. **High dose hook effect:** Hook effect was assessed with serial dilution of a high concentration sample. RLU values increased with increasing analyte concentrations, thereby confirming that high positive specimens above the AMR do not show hook effect up to 256,113 CU (theoretical value calculated using the highest value in the Working Curve and its dilution factor) in the QUANTA Flash M2 (MIT3) assay. c. Traceability, Stability, Expected values (controls, calibrators, or methods): i) Traceability, Stability, Expected values (controls, calibrators, or methods): **Traceability** No international standard material for anti-M2 (MIT3) antibodies is available for the standardization of anti-M2 (MIT3) antibody assays. Calibrators and controls values are directly traceable to the in-house standards. **Value Assignment** **Calibrators** – The calibrators are human serum with stabilizer and preservative. There are seven levels of standards that are used to create a Master Curve that is specific for each lot of reagents, with assigned values from 6.7–3474.6 CU, plus zero CU. The parameters for the Master Curve are provided with the reagent lot. Three levels of commercial calibrators are marketed separately, traceable to the Master Lot and used to calibrate the Master Curve on the BIO-FLASH® Instrument. Calibrators are tested on at least two instruments, on at least two lots {9} of reagent cartridge, in replicates of ten to determine final value assignment. The target values and ranges for the Calibrators can be found in the table below. Controls – Controls contain human anti-mitochondrial antibodies in stabilizer. Controls are tested on at least two instruments, on at least two lots of reagent cartridge, in replicates of ten to determine final value assignment. Control values are directly traceable to the in-house Standards that are used to create the Master Curves for the QUANTA Flash M2 (MIT3) assay. The target values and ranges for the Controls can be found in the table below. | | Target Value (CU) | Target Range (CU) | | --- | --- | --- | | QUANTA Flash M2 (MIT3) Calibrators | | | | Calibrator 1 | 12 | 10–14 | | Calibrator 2 | 400 | 360–440 | | Calibrator 3 | 2,550 | 2,450–2,650 | | QUANTA Flash M2 (MIT3) Controls | | | | Negative Control | 10 | 8–12 | | Positive Control | 50 | 40–60 | ii) Kit Stability: Shelf-life stability To establish the initial claim for shelf life, accelerated stability studies were performed for up to 4 weeks at $37^{\circ}\mathrm{C} \pm 3^{\circ}\mathrm{C}$, where one week is equal to six months at $5 \pm 3^{\circ}\mathrm{C}$. Accelerated stability testing was performed on each of the following sealed components of the QUANTA Flash M2 (MIT3) to establish initial stability claim: the beads, the two Calibrators, the Negative and Positive Controls and two high positive samples (&gt;2000 CU). Each week a new sealed component was placed in the incubator, and all components were tested at the end of the experiment together with the one that was stored at $5 \pm 3^{\circ}\mathrm{C}$. The recovery of the measured values was calculated for each time point by comparing results of sealed components stored at $37 \pm 3^{\circ}\mathrm{C}$ (test) for 1, 2, 3, and (optional) 4 weeks to those stored at $5 \pm 3^{\circ}\mathrm{C}$ (control), where one week is equal to six months at $5 \pm 3^{\circ}\mathrm{C}$. A real-time stability study is underway and currently available data supports a 12-month stability claim when stored at $2 - 8^{\circ}\mathrm{C}$. On-board/in-use stability Calibrators: Calibrators were placed uncapped, onboard the instrument, and calibration was performed altogether five times over nine hours. Controls and a panel of characterized patient specimens were run on each calibration curve. Testing verified that calibrators can be used for up to four calibrations over an eight hour period. 10 {10} Controls: Two vials of each control were assayed once a day for a total of 20 runs. The first run was used to establish baseline value, by running each vial in duplicate, and then additional 19 runs were performed, by running each vial in singlicate. During runs, the controls were left uncapped, onboard the instrument for 15 minutes per run. When not in use, the controls were capped, and stored at $5 \pm 3^{\circ}\mathrm{C}$. Results obtained support the claim that controls can be used for up to 15 times, at ten minutes per use. Reagent Cartridge: Two lots of cartridges were tested with four serum specimens. The specimens were tested periodically up to 70 days. Percent recoveries were calculated compared to the average values at Day 0, and linear regression analysis was performed by plotting % recovery against the number of days. Based on this study, the in-use (on-board) stability of M2 (MIT3) reagent cartridge was set up to 60 days. iii) Sample Storage: Four samples, encompassing negative, around the cut-off, and positive samples were tested in duplicates for up to 21 days while stored at $2 - 8^{\circ}\mathrm{C}$, up to 48 hours while stored at room temperature and after repeated freeze/thaw cycles up to 3 cycles. Results were compared to those obtained on control samples (day zero, stored at $2 - 8^{\circ}\mathrm{C}$). Based on these results, the sponsor recommends that samples could be stored up to 48 hours at room temperature, up to 14 days at $2 - 8^{\circ}\mathrm{C}$, and can be subjected to up to 3 freeze/thaw cycles (when samples are stored at or below $-20^{\circ}\mathrm{C}$). d. Detection limit: The Limit of Blank (LoB), Limit of Detection (LoD) and Limit of Quantitation (LoQ) were determined based on the studies designed according to CLSI EP17-A2. The LoB was determined by testing four blank samples (System Rinse). Samples were run in replicates of five on two reagent lots, once per day, for 3 days, with 60 data points generated on each lot. The LoB for each lot was calculated separately at the 95th percentile. The claimed LoB is 0.43 CU. The LoD was determined by testing four low level samples with two reagent lots, consistent with CLSI EP17-A2. The LoD was established with less than $5\%$ false positive and false negative results based on 240 determinations; 60 measurements on blank samples from LoB study and 60 measurements of low level samples with two reagent lots. The claimed LoD of the QUANTA Flash M2 (MIT3) assay is 0.61 CU and is below the lower limit of the analytical measuring range (AMR) of the assay. The LoQ was determined based on the data generated from the LoD study with a total error (TE) goal of $25\%$. The claimed LoQ for the assay is 1.2 CU and is the lower 11 {11} limit of the AMR. # e. Analytical specificity: # i) Endogenous Interference: The interference study was performed according to CLSI EP07-A2. Five specimens, one high positive (1422.5 CU), one moderately positive (392.4 CU), two near the cutoff (13.9 and 22.1 CU), and one negative (9.9 CU) sample were tested. Interfering substances (hemoglobin, conjugated bilirubin, triglycerides, cholesterol, human IgG, glutamate oxacatate transaminase and high density lipoprotein) were spiked into every specimen at three different concentrations in $10\%$ of total specimen volume, and the resulting samples were assessed in triplicates with the QUANTA Flash M2 (MIT3) assay. Six additional samples were tested for rheumatoid factor (RF) interference by combining them with different concentrations of a high positive RF IgM serum sample $(1,534~\mathrm{IU / mL})$ . Recovery of the unit values was calculated compared to control samples spiked with the same volume of diluents $(10\%$ of total sample volume). No interference was detected in the samples up to the concentrations listed in the table below. | Potential Interfering Compound | Test Concentration | % Recovery | | --- | --- | --- | | Bilirubin (conjugated) | 1 mg/mL | 93% – 100% | | Hemoglobin | 2 mg/mL | 94% – 101% | | Triglycerides | 1000 mg/dL | 94% – 102% | | Cholesterol | 332.5 mg/dL | 97% – 104% | | Human IgG | 35 mg/mL | 91% – 115% | | Rheumatoid factor IgM | 153.4 IU/mL | 96% – 111% | | Glutamate Oxacatate Transaminase | 0.12 IU/mL | 98% – 115% | | High Density Lipoprotein | 3.5 mg/mL | 95% – 105% | In addition, the same protocol was used to evaluate the interference of six drugs commonly used to treat primary biliary cholangitis (PBC). The tested concentrations for the drugs were three times the therapeutic concentration. No interference was detected in the samples up the concentrations listed in the table below. | Potential Interfering Therapeutics | Test Concentration | % Recovery | | --- | --- | --- | | Ursodeoxycholic acid (UDCA) | 0.75 mg/mL | 97% – 104% | | Prednisone | 0.3 mg/L | 100% – 105% | | Methotrexate | 9.1 mg/mL | 96% – 103% | | Cholestyramine | 18 mg/mL | 97% – 100% | | Colestipol | 18 mg/mL | 98% – 104% | | Hydroxyzine | 1.0 mg/L | 102% – 108% | {12} ii) Cross-reactivity: Refer to clinical study section iii) Carry-over: Not applicable f. Assay cut-off: The reference population for establishing the reference interval for the M2 (MIT3) assay consisted of 180 subjects: | Sample Group | N | | --- | --- | | Apparently healthy blood donors | 100 | | Infectious disease | 30 | | Rheumatoid arthritis | 30 | | Celiac Disease | 20 | The cut-off was established in accordance to CLSI EP28-A3c. One high positive sample confirmed to be true positive with the predicate device was excluded from the calculations as an outlier. The $99^{\text{th}}$ percentile of the remaining obtained values was calculated as 3,835 RLU. Additionally, three diagnosed PBC patient specimens were assayed to aid in the determination of the cut-off. Based on these results, the cut-off was increased to 6,000 RLU to ensure optimal differentiation between negatives and positives, and a 20 CU value was assigned to this RLU value. No reference patients tested positive at this new cut-off level. 2. Comparison studies: a. Method comparison with predicate device: Samples for method comparison analysis included 409 samples from the clinical validation study plus an additional eight contrived samples to yield a total of 417 samples with 20 around the cutoff (15–25 CU). These samples were tested on both the QUANTA Flash M2 (MIT3) and on the predicate QUANTA Lite M2 EP (MIT3) ELISA. A total of 174 samples within the AMRs of both assays were included in the method comparison analysis to evaluate negative percent agreement (NPA), positive percent agreement (PPA), and total percent agreement (TPA). The data are presented in the following tables. {13} 14 | Equivocal from Predicate Considered as Negative | QUANTA Lite M2 EP (MIT3) ELISA | | | Percent Agreement (95% CI) | | | --- | --- | --- | --- | --- | --- | | | | Negative | Positive | | Total | | QUANTA Flash M2 (MIT3) | Negative | 73 | 11 | 84 | NPA: 85.9% (76.9–91.7%) | | | Positive | 12 | 78 | 90 | PPA: 87.6% (79.2–93.0%) | | | Total | 85 | 89 | 174 | TPA: 86.8% (80.9–91.0%) | | Equivocal from Predicate Considered as Positive | QUANTA Lite M2 EP (MIT3) ELISA | | | Percent Agreement (95% CI) | | | --- | --- | --- | --- | --- | --- | | | | Negative | Positive | | Total | | QUANTA Flash M2 (MIT3) | Negative | 71 | 13 | 84 | NPA: 91.0% (82.6–95.6%) | | | Positive | 7 | 83 | 90 | PPA: 86.5% (78.2–91.9%) | | | Total | 78 | 96 | 174 | TPA: 88.5% (82.9–92.4%) | b. Matrix comparison: Not applicable 3. Clinical studies: a. Clinical sensitivity and specificity: A total of 586 characterized samples were included in the clinical evaluation for the QUANTA Flash M2 (MIT3). This validation set of samples included 151 samples from patients diagnosed with primary biliary cholangitis (PBC) and 435 control samples from patients with various types of liver and gastroenterological diseases, other autoimmune syndromes, and various infectious diseases. Literature indicates a relatively high prevalence of PBC in Limited cutaneous systemic sclerosis (LSSc) patients, as compared to the general population. LSSc samples were therefore excluded from sensitivity and specificity calculations. Additionally, four samples with AIH/PBC overlap were excluded for all sensitivity and specificity calculations. Clinical sensitivity and specificity for PBC is summarized in the following table: | | Diagnosis of PBC | | | Analysis (95% CI) | | | --- | --- | --- | --- | --- | --- | | | | Positive | Negative | | Total | | QUANTA Flash M2 (MIT3) | Positive | 127 | 4 | 131 | Sensitivity: 84.1% (77.4–89.1%) Specificity: 99.0% (97.6–99.6%) | | | Negative | 24 | 413 | 437 | | | | Total | 151 | 417 | 568 | | b. Cross Reactivity The cross-reactivity with autoantibodies and infection-induced antibodies was further evaluated based on the results from the clinical validation study above plus additional {14} 14 samples from patients with limited cutaneous systemic sclerosis and four samples from patients with Autoimmune Hepatitis (AIH)/PBC overlap. The composition of the cohort $(N = 586)$ and the anti-mitochondrial positivity rate is shown in the Table below. | Patient Group | N | Number of Positive | % Positive | | --- | --- | --- | --- | | Target Disease Samples | | | | | PBC | 151 | 27 | 84.1% | | | | | | | Control Samples Used in Specificity | | | | | Autoimmune Hepatitis type 1 (AIH1) | 41 | 2 | 4.9% | | LKM1 antibody positive Autoimmune Hepatitis 2 (AIH2) | 28 | 0 | 0.0% | | Primary Sclerosing Cholangitis (PSC) | 21 | 0 | 0.0% | | Liver Cancer | 10 | 0 | 0.0% | | Celiac Disease | 19 | 0 | 0.0% | | Hepatitis B virus (HBV) | 10 | 0 | 0.0% | | Hepatitis C virus (HCV) | 25 | 0 | 0.0% | | Syphilis | 10 | 0 | 0.0% | | Ulcerative Colitis | 26 | 0 | 0.0% | | Crohn's Disease | 10 | 0 | 0.0% | | Alcoholic Liver Disease | 20 | 0 | 0.0% | | Idiopathic inflammatory myopathies (IIM) | 8 | 0 | 0.0% | | Systemic lupus erythematosus (SLE) | 8 | 0 | 0.0% | | Sjögren's syndrome (SS) | 4 | 0 | 0.0% | | Sicca syndrome | 20 | 0 | 0.0% | | Type 1 Diabetes | 30 | 0 | 0.0% | | Osteoporosis | 28 | 1 | 3.6% | | Chronic fatigue | 30 | 1 | 3.3% | | Skin conditions | 30 | 0 | 0.0% | | Drug-induced hepatotoxicity | 9 | 0 | 0.0% | | Hypothyroidism | 30 | 0 | 0.0% | | Total | 417 | 4 | 4.0% | | | | | | | Others | | | | | Limited cutaneous systemic sclerosis (LSSc)* | 14 | 5 | 35.7% | | AIH/PBC overlap** | 4 | 4 | 100% | | | | | | | Total | 586 | | | c. Other clinical supportive data (when a. and b. are not applicable): Not applicable {15} 4. Clinical cut-off: Not applicable 5. Expected values/Reference range: The expected value in the normal population is “negative”. Anti-mitochondrial antibody levels were analyzed in a cohort of 100 apparently healthy blood donors (50 females and 50 males, ages 17 to 57 years, with an average and median age of 34 years) using the QUANTA Flash M2 (MIT3). This patient population was different from the one that was used to establish the cutoff, and was only used to assess expected values. With the cut-off of 20 CU, two samples were positive on the QUANTA Flash M2 (MIT3). The mean concentration was 2.8 CU, and the values ranged from &lt;1.2 to 91.4 CU. N. Proposed Labeling: The labeling is sufficient and it satisfies the requirements of 21 CFR Part 809.10. O. Conclusion: 1. The submitted information in this premarket notification is complete and supports a substantial equivalence decision. 16