DIAZYME MYOGLOBIN; ASSAY, CALIBRATOR SET, CONTROL SET

{0} 1 # 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION SUMMARY ASSAY ONLY TEMPLATE A. 510(k) Number: k123785 B. Purpose for Submission: New device C. Measurand: Myoglobin D. Type of Test: Quantitative Immunoturbidimetric assay E. Applicant: Diazyme Laboratories F. Proprietary and Established Names: Diazyme Myoglobin Assay Diazyme Myoglobin Calibrator Set Diazyme Myoglobin Control Set G. Regulatory Information: | Product Code | Classification | Regulation Section | Panel | | --- | --- | --- | --- | | Myoglobin, antigen, antiserum, control (DDR) | Class II | 21 CFR 866.5680 Myoglobin immunological test system | Immunology (82) | | Product Code | Classification | Regulation Section | Panel | | Calibrator, secondary (JIT) | Class II | 21 CFR 862.1150 Calibrator | Clinical Chemistry (75) | | Product Code | Classification | Regulation Section | Panel | | single (specified) analyte controls (assayed and unassayed) (JJX) | Class I, reserved | 21 CFR 862.1660 Quality control material (assayed) | Clinical Chemistry (75) | {1} H. Intended Use: 1. Intended use(s): See indication(s) for use below. 2. Indication(s) for use: The Diazyme Myoglobin Assay is for the quantitative determination of myoglobin in human serum and plasma. Measurement of myoglobin is used as an aid in the diagnosis of acute myocardial infarction. For in vitro diagnostic use only. The Diazyme Myoglobin Calibrator Set is intended for use in the calibration of the Diazyme Myoglobin Assay. For in vitro diagnostic use only. The Diazyme Myoglobin Control Set is intended for use as quality controls for the Diazyme Myoglobin Assay. For in vitro diagnostic use only. 3. Special conditions for use statement(s): For prescription use only 4. Special instrument requirements: Performance characteristics were provided for the Hitachi 917 analyzer. I. Device Description: The device is supplied as a ready-to-use two reagent kit. Calibrators and controls are sold separately. Reagent 1 contains 100 mM Tris-buffer with 0.09% sodium azide. Reagent 2 contains a suspension of goat anti-human Myoglobin-coated latex particles with 0.09% sodium azide. Ready-to-use liquid calibrators at 5 levels (5 x 1 mL) are prepared from 50% human serum spiked with purified human Myoglobin to targeted concentrations with 0.09% sodium azide added as a preservative. A sixth level (saline, not provided) is to be used as a zero to create a 6-point calibration. Ready-to-use liquid controls at 2 levels (2 x 1 mL) are prepared from 50% human serum spiked with purified human Myoglobin to targeted concentrations with 0.1% sodium azide added as a preservative. {2} Calibrator and control materials are provided separately from the Reagent Kit. All human source materials were tested by FDA approved methods and found to be negative for Hepatitis B Surface Antigen (HBsAg), antibodies to HIV I/II and antibodies to HCV.. # J. Substantial Equivalence Information: 1. Predicate device names and 510(k) numbers: Roche Tina-Quant Myoglobin Gen. 2 Test System and C.f.a.s. (Calibrators for automated systems) Myoglobin (k061683); Myoglobin Control Set (k973358) 2. Comparison with predicate: Diazyme Myoglobin Assay: | Similarities | | | | --- | --- | --- | | Item | Subject Device | Predicate (k061683) | | Intended use | Same | For the quantitative determination of myoglobin in human serum and plasma. For in vitro diagnostic use only. | | Principle | Same | Quantitative Immunoturbidimetric assay | | Sample type | Same | Serum or plasma (Li-Heparin and EDTA) | | Analyte Measured | Same | Myoglobin | | Differences | | | | --- | --- | --- | | Reportable range | 13.2 to 615.9 ng/mL | 20 to 580 ng/mL | | Reference Range | Men: 23.3-82.1 ng/mL Women: 15.2-56.5 ng/mL | Men: 19-89 ng/mL Women: 12.7-56.3 ng/mL | | Instrumentation | Hitachi 917 Analyzer | Hitachi 902, Hitachi 912, Hitachi 917, MODULAR Analyzers | {3} 4 | Assay Reagent Composition | **Reagent 1:** 100 mM Tris; preservative **Reagent 2:** Suspension of anti-human myoglobin coated latex particles, ready to use | **Reagent 1:** 170 mM Glycine, pH 8.3; 100 mM NaCl; 50 mM EDTA; preservative **Reagent 2:** Suspension of anti-human myoglobin coated latex particles, ready to use | | --- | --- | --- | | Reagent storage and stability | Store at 2 to 8 °C Shelf Life: until expiration date. On board stability: 8 weeks. | Store at 2 to 8 °C Shelf Life: until expiration date. On board stability: 12 weeks. | Diazyme Myoglobin Calibrator Set: | Similarities | | | | --- | --- | --- | | Item | Subject Device | Predicate (k061683) | | Intended use | Same | For the calibration of Myoglobin assay | | Analytes | Same | Human Myoglobin | | Vial Volume | Same | 1 mL | | Format | Same | Liquid | | Number of levels | Same | Five, plus saline for zero standard | | Differences | | | | --- | --- | --- | | Matrix | Human Serum | Bovine Serum Albumin | | Storage and Stability | Store at 2 to 8 °C Shelf Life: until expiration date. Open vial: 3 Months. | Store at 2 to 8 °C Shelf Life: until expiration date. Open vial: 4 weeks. | {4} Diazyme Myoglobin Control Set: | Similarities | | | | --- | --- | --- | | Item | Subject Device | Predicate (k973358) | | Intended use | Same | For use as assayed quality control samples to monitor the accuracy of assay. | | Analytes | Same | Human Myoglobin | | Vial Volume | 2 x 1 mL | 2 x 3 mL | | Matrix | Same | Human Serum | | Number of levels | Same | Two | | Differences | | | | --- | --- | --- | | Format | liquid | Lyophilized | | Storage and Stability | Store at 2 to 8 °C Shelf Life: until expiration date. Open vial: 3 Months. | Store at 2 to 8 °C Shelf Life: until expiration date. Open vial: 4 weeks. | ## K. Standard/Guidance Document Referenced (if applicable): CLSI EP5-A2: Evaluation of Precision Performance of Quantitative Measurement Methods CLSI EP6-A: Evaluation of Linearity of Quantitative Measurement Procedures, A Statistical Approach CLSI EP7-A: Interference Testing in Clinical Chemistry CLSI EP9-A2: Method Comparison and Bias Estimation Using Patient Samples CLSI EP17-A: Protocols for Determination of Limits of Detection and Limits of Quantitation CLSI C28-A3: Defining, Establishing, and Verifying Reference Intervals in the Clinical Laboratory ## L. Test Principle: The Diazyme Myoglobin Assay is based on a latex enhanced immunoturbidimetric assay. When an antigen-antibody reaction occurs between myoglobin in a sample and {5} anti-myoglobin antibodies which have been sensitized to latex particles, agglutination occurs. This agglutination is detected as an absorbance change (570 nm), with the magnitude of the change being proportional to the quantity of myoglobin in the sample. The actual concentration is then determined by the interpolation from a calibration curve prepared from calibrators of known concentration. ## M. Performance Characteristics (if/when applicable): Performance characteristics were established on the Hitachi 917 analyzer ## 1. Analytical performance: ### a. Precision/Reproducibility: The precision of the Diazyme Myoglobin Assay was evaluated following CLSI guidance document EP5-A2. Three levels of Myoglobin serum controls and three levels of serum specimens were tested in duplicate for 2 runs per day over 20 working days. One lot of reagent was used for this study. Within-run and total precision are presented in the table below. | | N | Mean (ng/mL) | Within Run | | Total | | | --- | --- | --- | --- | --- | --- | --- | | | | | SD | % CV | SD | % CV | | Control 1 | 80 | 66 | 2.45 | 3.71 | 3.37 | 5.10 | | Control 2 | 80 | 170 | 6.69 | 3.87 | 7.37 | 4.30 | | Control 3 | 80 | 335 | 11.9 | 3.54 | 14.9 | 4.40 | | Serum 1 | 80 | 35 | 1.77 | 4.69 | 1.97 | 5.20 | | Serum 2 | 80 | 150 | 3.53 | 2.37 | 5.32 | 3.58 | | Serum 3 | 20 | 414 | 19.7 | 4.80 | 21.8 | 5.3 | ### b. Linearity/assay reportable range: The claimed measuring range of the Diazyme Myoglobin assay is 13.2 to 615.9 ng/mL. Eleven levels of samples were prepared by diluting a serum-based sample with a high level of spiked Myoglobin with saline according to CLSI EP6-A to produce linearity samples ranging from 10.11 to 623.8 ng/mL. The linearity samples were tested in triplicate using 1 lot of Diazyme Myoglobin reagent. Linear regression analysis provided the following fit equation for a graph of the observed (y-axis) vs expected (x-axis) myoglobin concentrations: slope = 0.9798, intercept = -5.1076, r² = 0.9983. Percent Error for each of the dilutions is shown below: | Level | Recovery Mean | Expected Recovery | Difference | Percent Difference | | --- | --- | --- | --- | --- | | 11 | 623.83 | 625 | -1.17 | 0% | | 10 | 538.93 | 563.6 | -24.67 | -4% | {6} 7 | 9 | 480.52 | 502.2 | -21.68 | -4% | | --- | --- | --- | --- | --- | | 8 | 418.64 | 440.8 | -22.16 | -5% | | 7 | 372.14 | 379.4 | -7.26 | -2% | | 6 | 311.23 | 318 | -6.77 | -2% | | 5 | 240.2 | 256.6 | -16.4 | -6% | | 4 | 180.54 | 195.2 | -14.66 | -8% | | 3 | 121.28 | 133.8 | -12.52 | -9% | | 2 | 73.34 | 72.4 | 0.94 | 1% | | 1 | 10.11 | 11 | -0.89 | -8% | To test for High Dose “Hook Effect”, a dilution series of 20 samples, ranging from 260 to 49260 ng/mL was prepared from spiked serum samples. The samples were tested in triplicate using 1 lot of Diazyme Myoglobin assay, and showed that no High Dose “Hook Effect” occurred up to 15000 ng/mL of Myoglobin. A sample dilution study was performed to demonstrate that manual 1:1 dilution of serum samples exceeding 615.9 ng/mL with saline results in accurate myoglobin value determination. Three samples were analyzed in triplicate, undiluted and diluted, on the Diazyme Myoglobin assay and percent recovery of the diluted samples was determined. The study indicates that samples up to 849.07 ng/mL may be accurately determined by 1:1 dilution with saline prior to analysis. | Sample | Undiluted Value | Diluted Value x Dilution factor | Percent Recovery | | --- | --- | --- | --- | | #1 | 617.73 | 630.21 | 102% | | #2 | 702.49 | 735.45 | 105% | | #3 | 784.06 | 849.07 | 108% | c. Traceability, Stability, Expected values (controls, calibrators, or methods): **Diazyme Myoglobin Calibration Stability:** Myoglobin assay calibration stability was assessed, and the sponsor claims that calibration is stable for 28 days. The protocol and acceptance criteria have been provided and were found to be adequate to support the sponsor’s claims. **Calibrator Traceability and value assignment:** The calibrators for the Diazyme Myoglobin Assay are traceable to a commercially available, FDA cleared assay, and were prepared by spiking Myoglobin stock solution into a diluted human serum to the target concentrations. Value assignment of the myoglobin calibrators was established by transferring the target values from the predicate method to the in-house {7} master calibrators. ## Control value assignment: Myoglobin controls are prepared by spiking Myoglobin stock solution to diluted pooled human serum to the target Myoglobin concentrations. Diazyme Myoglobin reagents and calibrators were used in replicate analysis to determine the mean value of the control materials. Target values for Myoglobin level I and II controls are 50 ng/mL and 330 ng/mL, respectively. Lot specific target ranges are provided in the value assignment sheet. ## Control Stability: Real time stability testing demonstrated that the Diazyme Myoglobin controls were stable for 6 months at 2-8°C. Real time stability studies are on-going. Open vial stability testing at 2-8°C showed that calibrators are stable for at least 3 months after opening. Open vial stability studies are ongoing. ## Calibrator Stability: Real time stability testing demonstrated that the Diazyme Myoglobin calibrators were stable for 6 months at 2-°C. Real time stability studies are on-going. Open vial stability testing at 2-8°C showed that calibrators are stable for at least 3 months after opening. Open vial stability studies are ongoing. Stability study protocols and acceptance criteria have been provided and found to be adequate to support the sponsor's claims. ## d. Detection limit: The LoB, LoD, and LoQ of the Diazyme Myoglobin Assay were determined according to CLSI EP17-A. LoB tests were conducted using blank samples (7.5% BSA) tested with 60 replicates with one reagent lot and one instrument. LoB, calculated as the mean of the 57th and 58th highest blank values was determined to be 4.4 ng/mL. For LoD studies, five low, diluted serum samples were tested with 4 replicates, in each of 3 runs, with one reagent lot and one instrument. The LOD was calculated as the LOB + (1.645 * SD of LOD samples). The LoD was determined to be 8.2 ng/mL. Limit of Quantitation (LoQ) determination was based on interassay precision of ≤20% for 8 replicate measurements of 5 low serum samples, per day for 5 days, using 1 lot of Diazyme Myoglobin reagent and 1 instrument. The LoQ was determined to be 13.23 ng/mL for the Diazyme Myoglobin assay. 8 {8} # e. Analytical specificity: The sponsor evaluated the effect of known endogenous interferents on the Diazyme Myoglobin assay following Clinical and Laboratory Standards Institute EP7-A guidelines. Two serum samples, with normal and high levels of Myoglobin (50 and 275 ng/mL), were spiked with various concentrations of endogenous substances at four levels, or therapeutic drugs at 1 level, and the results were compared to the samples without added interferent. The samples were tested using one lot of reagent and one instrument. The following substances showed no significant interference as defined by the sponsor as less than $10\%$ deviation when tested at levels equal to the concentrations listed below: | Interferent | Concentration | | --- | --- | | Triglyceride | 1000 mg/dL | | Ascorbic Acid | 176 mg/dL | | Bilirubin | 40 mg/dL | | Bilirubin Conjugated | 40 mg/dL | | Hemoglobin | 1000 mg/dL | | Rheumatoid Factor | 100 IU/mL | | Heparin | 1.5 IU/mL | | N-acetylcysteine | 11.04 mM | | Acetylsalicylic Acid | 2.78 mM | | Ampicillin | 152 μM | | Carbamazepine | 0.13 mM | | Na2-Cefoxitin | 1549 μM | | Ibuprofen | 2425 μM | | Cyclosporin | 0.125 μM | | Levodopa | 30.4 mM | | Methyldopa | 71 μM | | Metronidazole | 701 μM | | Rifampicin | 78.1 μM | | Theophylline | 222 μM | | Phenylbutazone | 650 μM | | Valproic Acid, Sodium Salt | 3.5 mM | Significant interference of the Diazyme Myoglobin assay was observed with Intralipid concentrations above $125\mathrm{mg / dL}$ . Specimens from patients undergoing Intralipid therapy may have interference with the Myoglobin Assay. f. Assay cut-off: Not applicable. 2. Comparison studies: {9} 10 a. Method comparison with predicate device: Method Comparison of the Diazyme Myoglobin Assay versus the predicate assay was performed according to CLSI guidance document EP9-A2. Fifty-seven unaltered lithium heparin plasma samples and 9 altered lithium heparin plasma samples, ranging from 16.9 to 478.0 ng/mL, were tested in singlicate on the predicate and subject devices, using 1 lot of Myoglobin reagent. Deming regression analysis of a plot of the calculated concentrations from the Diazyme Myoglobin Assay (Y-axis) versus the predicate device (X-axis) produced an equation of Y = 0.959X - 5.141, r² = 0.9855. The 95% CI for slope and intercept are 0.930 to 0.988 and -10.16 to -0.12, respectively. b. Matrix comparison: Matrix comparison studies were completed by analyzing 40 paired Serum, EDTA plasma and Lithium Heparin plasma samples in singlicate using 1 lot of the Diazyme Myoglobin Assay. The samples had serum myoglobin values ranging from 25.35 to 509.07 ng/mL, and 4 were spiked with myoglobin. Linear regression analysis was used to determine the relationship between plasma and serum samples. For serum vs. K3 EDTA, the linear regression equation was y = 0.9829x - 2.865, r² = 0.9949. For serum vs. Lithium heparin plasma, the linear regression equation was y = 0.9999x + 2.6558, r² = 0.9978. The sponsor concluded that Lithium heparin plasma and EDTA plasma are acceptable anti-coagulants to be used with the Diazyme Myoglobin assay. 3. Clinical studies: a. Clinical Sensitivity: Not applicable b. Clinical specificity: Not applicable c. Other clinical supportive data (when a. and b. are not applicable): Not applicable 4. Clinical cut-off: Not applicable 5. Expected values/Reference range: The reference interval was determined using serum specimens from apparently healthy adults (120 females and 121 males ranging in age from age 21 - 77) {10} according to CLSI C28-A3. The samples were tested in singlicate using 1 lot of the Diazyme Myoglobin Assay. The 2.5th and 97.5th percentile intervals are 15.2 ng/mL and 56.5 ng/mL for females, and 23.3 ng/mL and 82.1 ng/mL for males. ## 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. 11