AXIS-SHIELD 3-REAGENT HOMOCYSTEINE ASSAY FOR SYNCHRON

{0} 1 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION SUMMARY ASSAY ONLY TEMPLATE A. 510(k) Number: k112790 B. Purpose for Submission: New device C. Measurand: Homocysteine D. Type of Test: Quantitative, enzymatic assay E. Applicant: Axis-Shield Diagnostics Ltd. F. Proprietary and Established Names: Axis-Shield 3-Reagent Homocysteine Assay for Synchron G. Regulatory Information: 1. Regulation section: 21 CFR § 862.1377, Urinary Homocysteine (Nonquantitative) Test System 2. Classification: Class II 3. Product code: LPS 4. Panel: Chemistry (75) {1} H. Intended Use: 1. Intended use(s): See indication(s) for use below 2. Indication(s) for use: The 3-Reagent Homocysteine Assay for Beckman Coulter SYNCHRON® and UniCel® systems is intended for in vitro quantitative determination of total homocysteine in human serum and plasma. The device can assist in the diagnosis and treatment of patients suspected of having hyperhomocysteinemia and homocystinuria. 3. Special conditions for use statement(s): For prescription use only. The following black box warning is included in the labeling: WARNING: Specimens from patients who are on drug therapy involving S-adenosyl-methionine may show falsely elevated levels of homocysteine. Patients who are taking methotrexate, carbamazepine, phenytoin, nitrous oxide, anticonvulsants, or 6-azauridine triacetate may have elevated levels of homocysteine due to their effect on the pathway. Refer to the LIMITATIONS FOR USE section in this assay package insert. 4. Special instrument requirements: For use on the Beckman Coulter Synchron LX 20 Pro and Unicel DxC 600 I. Device Description: Each reagent kit is made up of one cartridge containing the three enzymatic homocysteine reagents and two calibrators: - Reagent 1: 250 mM Tris Buffer with L-Serine, Magnesium Chloride 6-Hydrate, β-NADH II, Brij 35, Lipase, L-Glutamic Acid, Alpha Cyclodextrin, Alcohol Dehydrogenase, Absolute Ethanol, and Sodium Azide - Reagent 2: 25mM Tris (2-carboxyethyl) Phosphine Hydrochloride (TCEP) with α-ketoglutaric acid {2} - Reagent 3: 100mM Potassium Phosphate Enzyme Buffer (containing sodium azide) with CBS and CBL cycling enzymes. - Calibrator 0 and Calibrator 28: 100 μM L-Cystine with Homocysteine (in form of L-Homocysteine, the Homocysteine dimmer) spiked to 0 μM (Cal 0) and '28' μM (Cal '28') in an aqueous matrix. The calibrators (included in the kit) are prepared gravimetrically and are traceable to NIST SRM 1955, confirmed by a designated measurement procedure (HPLC). # J. Substantial Equivalence Information: 1. Predicate device name(s): Axis-Shield Liquid Stable (LS) 2-part Homocysteine Reagent 2. Predicate 510(k) number(s): k083222 3. Comparison with predicate: | Similarities | | | | --- | --- | --- | | Item | Axis-Shield 3-Reagent Homocysteine Assay for Synchron | Axis-Shield Liquid Stable (LS) 2-part Homocysteine Reagent | | Intended Use | In vitro quantitative determination of total homocysteine in human serum and plasma. The device can assist in the diagnosis and treatment of patients suspected of having hyperhomocysteinemia and homocystinuria. | Same | | Technology | Enzymatic Assay | Same | | Active Assay Composition | Lactate dehydrogenase Cystathionine beta-Synthase Cystathionine beta-Lyase TCEP (tris(2- | Same | {3} | Similarities | | | | --- | --- | --- | | Item | Axis-Shield 3-Reagent Homocysteine Assay for Synchron | Axis-Shield Liquid Stable (LS) 2-part Homocysteine Reagent | | | carboxyethyl)phosphine) | | | Detection Method | The rate of NADH conversion to NAD+ (D A340 nm) is directly proportional to the concentration of homocysteine | Same | | Storage Conditions | Reagents and calibrators must be stored at 2 – 8 C. | Same | | On-board reagent stability | 30 days | Same | | Calibrator traceability | Traceable to NIST SRM 1955 Standard Reference Material | Same | | Units of measurement | μmol/L | Same | | Calibration | Quantitative assay using 2 gravimetrically prepared L-homocysteine calibrators, assigned 0 and 28 umol/L | Same | | Limit of Detection | 0.89 umol/L | 0.33 umol/L | | Sample carryover | Less than LoD | Same | | Specimen type | EDTA plasma, lithium heparin plasma, serum and serum separator tubes | Same | | Differences | | | | --- | --- | --- | | Item | Device | Predicate | | Assay format | 3 reagent enzymatic assay | 2 reagent enzymatic assay | | Reagent #1 composition | Pre-treatment buffer | Reductant reagent: lactate dehydrogenase (38 KU/L) TCEP (830 mg/L) | | Reagent #2 composition | Reductant reagent: TCEP (3.0 g/L) | Enzyme reagent: Cystathionine beta-synthase (0.748 KU/L) Cystathionine beta-Lyase (16.4 KU/L) | | Reagent #3 composition | Enzyme reagent: Cystathionine beta-synthase (0.748 KU/L) Cystathionine beta-Lyase | Not applicable | {4} | Differences | | | | --- | --- | --- | | Item | Device | Predicate | | | (16.4 KU/L) | | | Calibration frequency | 14 days | 30 days | | Instrument | BC Synchron LX 20 Pro BC Unicel DxC 600 | Olympus AU400 | | Assay range | 1-50 μmol/L | 1-46 μmol/L | ## K. Standard/Guidance Document referenced (if applicable): 1. Evaluation of Precision Performance of Quantitative Measurement Methods; Approved Guideline – Second Edition, EP5-A2 2. Evaluation of the Linearity of Quantitative Measurement Procedures; Approved Guideline, EP6-A 3. Interference Testing in Clinical Chemistry; Approved Guideline – Second Edition, EP7-A2 4. Method Comparison and Bias Estimation Using Patient Samples; Approved Guideline – Second Edition, EP09-A2 5. Protocol for Determination of Limits of Detection and Limits of Quantitation; Approved Guideline, EP17-A 6. Format for Traditional and Abbreviated 510(k)s – Guidance for Industry and FDA Staff ## L. Test Principle: Bound or dimerized homocysteine (HCT) (oxidized form) is reduced to free HCT, which then reacts with serine catalyzed by cystathionine beta-synthase (CBS) to form cystathionine. Cystathionine is then broken down by cystathionine beta-lyase (CBL) to form HCT, pyruvate, and ammonia. Pyruvate is then converted by lactate dehydrogenase (LDH) to lactate with nicotinamide adenine dinucleotide (NADH) as a coenzyme. The rate of NADH conversion to NAD+ is directly proportional to the concentration of HCT ($\Delta$ A340 nm). ## M. Performance Characteristics (if/when applicable): ### 1. Analytical performance: #### a. Precision/Reproducibility: Precision studies on the Synchron LX 20 Pro and the UniCel DxC600 were performed following CLSI EP5-A2. For each instrument, three HCT controls and three human plasma samples were assayed using two lots of reagents, in replicates of two, in two runs per day for 20 days on one instrument (n = 80). Results are summarized below: {5} Synchron LX 20 Pro | Sample | Reagent Lot | Mean (μmol/L) | Within-Run | | Between-Run | | Total | | | --- | --- | --- | --- | --- | --- | --- | --- | --- | | | | | SD | %CV | SD | %CV | SD | %CV | | Low Control | 1 | 5.73 | 0.30 | 5.2 | 0.00 | 0.0 | 0.31 | 5.3 | | | 2 | 5.61 | 0.23 | 4.2 | 0.10 | 1.9 | 0.30 | 5.4 | | Medium Control | 1 | 11.01 | 0.28 | 2.5 | 0.09 | 0.9 | 0.32 | 2.9 | | | 2 | 10.97 | 0.29 | 2.6 | 0.08 | 0.7 | 0.33 | 3.0 | | High Control | 1 | 23.18 | 0.33 | 1.4 | 0.17 | 0.7 | 0.46 | 2.0 | | | 2 | 23.08 | 0.32 | 1.4 | 0.30 | 1.3 | 0.47 | 2.0 | | Sample P1 | 1 | 6.99 | 0.34 | 4.9 | 0.10 | 1.4 | 0.37 | 5.4 | | | 2 | 6.80 | 0.31 | 4.6 | 0.10 | 1.5 | 0.42 | 6.2 | | Sample P2 | 1 | 32.10 | 0.42 | 1.3 | 0.30 | 0.9 | 0.64 | 2.0 | | | 2 | 32.03 | 0.40 | 1.2 | 0.42 | 1.3 | 0.75 | 2.3 | | Sample P3 | 1 | 43.93 | 0.58 | 1.3 | 0.22 | 0.5 | 0.79 | 1.8 | | | 2 | 44.05 | 0.52 | 1.2 | 0.45 | 1.0 | 0.88 | 2.0 | Unicel DxC 600 | Sample | Reagent Lot | Mean (μmol/L) | Within-Run | | Between-Run | | Total | | | --- | --- | --- | --- | --- | --- | --- | --- | --- | | | | | SD | %CV | SD | %CV | SD | %CV | | Low Control | 1 | 6.15 | 0.36 | 5.9 | 0.00 | 0.0 | 0.49 | 8.0 | | | 2 | 6.37 | 0.30 | 4.7 | 0.00 | 0.0 | 0.40 | 6.3 | | Medium Control | 1 | 11.65 | 0.49 | 4.2 | 0.36 | 3.1 | 0.63 | 5.4 | | | 2 | 11.90 | 0.33 | 2.8 | 0.21 | 1.8 | 0.62 | 5.2 | | High Control | 1 | 24.13 | 0.64 | 2.6 | 0.43 | 1.8 | 1.09 | 4.5 | | | 2 | 24.37 | 0.63 | 2.6 | 0.59 | 2.4 | 1.13 | 4.6 | | Sample P1 | 1 | 7.43 | 0.47 | 6.3 | 0.13 | 1.7 | 0.53 | 7.2 | | | 2 | 7.63 | 0.27 | 3.5 | 0.11 | 1.4 | 0.48 | 6.3 | | Sample P2 | 1 | 33.20 | 0.85 | 2.6 | 0.62 | 1.9 | 1.42 | 4.3 | | | 2 | 33.58 | 0.79 | 2.4 | 0.65 | 1.9 | 1.83 | 5.4 | | Sample P3 | 1 | 45.38 | 1.08 | 2.4 | 1.27 | 2.8 | 1.92 | 4.2 | | | 2 | 45.61 | 0.96 | 2.1 | 0.62 | 1.4 | 2.44 | 5.4 | b. Linearity/assay reportable range: A linearity study was performed to validate the claimed measuring range of 1-50 $\mu \mathrm{mol} / \mathrm{L}$ . A high level sample was serially diluted with zero level calibrator, to yield a concentration range from $0.35 - 68.82\mu \mathrm{mol} / \mathrm{L}$ . A total of 11 samples for each instrument, (Synchron LX 20 Pro and Unicel DxC600) were analyzed for linearity with the following regression results: | Parameter | Synchron LX 20 Pro | Unicel DxC 600 | | --- | --- | --- | | Slope of Regression Line | 1.0108 | 1.0076 | | Y-intercept | -0.2605 | -0.0741 | | Correlation Coefficient | 0.9997 | 0.9990 | {6} 7 # Validation for out-of-range specimens Three EDTA samples with approximate HCT levels of 90, 150, and 500 $\mu$ mol/L were used for manual dilution recovery of out-of-range samples. All three levels were first diluted using a 1:9 dilution factor and measured on the reference instrument (Olympus AU400) to establish a baseline. The neat samples were then measured on the Synchron LX 20 Pro and Unicel DxC 600 in replicates of two and the mean was calculated to demonstrate the need for dilution at very high HCT levels. The neat samples were then diluted with a factor of 1:2 (for the 90 and $150~\mu \mathrm{mol} / \mathrm{L}$ levels) and a factor of 1:9 (for the $500~\mu \mathrm{mol} / \mathrm{L}$ level). The $\%$ recoveries for the samples when corrected by the dilution factors were within $10\%$ of the reference instrument values. The sponsor included the following in the package insert with regard to high levels of HCT: The linear range of the 3-Reagent Homocysteine Assay for Synchron LX 20 Pro and UniCel DxC 600 when run as directed is $1 - 50~\mu \mathrm{mol} / \mathrm{L}$ . Specimens $>50~\mu \mathrm{mol} / \mathrm{L}$ should be diluted 1 part specimen to 2 parts Cal 0 $\mu \mathrm{mol} / \mathrm{L}$ or 1 part specimen to 9 parts Cal 0 $\mu \mathrm{mol} / \mathrm{L}$ as appropriate. c. Traceability, Stability, Expected values (controls, calibrators, or methods): The calibrators were cleared under k083222 and are included in the kit. d. Detection limit: The determination of LoB and LoD followed guidance from CLSI document EP17-A. A zero calibrator was used to determine LoB. For the LoD estimation, five EDTA plasma samples were prepared with a concentration between LoB and 4x LoB. Samples were diluted in the zero calibrator to obtain the required concentrations and confirmed by testing on the Olympus AU400. ## Limit of Blank For the Synchron LX 20 Pro and Unicel DxC 600, 20 replicates of the zero calibrator were run using each reagent lot on each of 3 days ( $n = 60$ for each reagent lot), i.e. 1 run per day for each lot of reagents and calibrators. The results are summarized in the table below. ## Limit of Detection For the Synchron LX 20 Pro and Unicel DxC 600, five replicates of each of the five low level homocysteine samples were run with each reagent lot on each of three separate days ( $n = 75$ for each reagent lot) i.e. 1 run per day for each lot of reagents and calibrators. The results are summarized in the table below. {7} | Instrument | Reagent Lot | LoB (μmol/L) | LoD (μmol/L) | | --- | --- | --- | --- | | Synchron LX 20 Pro | 1 | 0.47 | 0.82 | | | 2 | 0.46 | 0.89 | | Unicel DxC 600 | 1 | 0.2 | 0.51 | | | 2 | 0.2 | 0.54 | The linearity studies and detection limit studies support the sponsor's claimed measuring range of 1.0 to $50~\mu \mathrm{mol / L}$ . # e. Analytical specificity: The studies for interfering substances were carried out according to the CLSI document EP7-A2. Control samples were in the medically important range of $12 - 18\mu \mathrm{mol} / \mathrm{L}$ . Each control sample was tested in replicates of five to determine a homocysteine baseline. Each control sample was then spiked with the relevant interfering compound up to the concentrations indicated in the table below. All samples were tested in replicates of five. No significant interference was defined as a difference of less than $\pm 10\%$ of the control value. The summary data is below: | Substance | Maximum Concentrations Tested | Highest concentration with no significant interference : | | --- | --- | --- | | Bilirubin | 20 mg/dL | 20 mg/dL | | Hemoglobin | 554 mg/dL | 500 mg/dL | | Triglyceride | 1500 mg/dL | 1000 mg/dL | | Glutathione | 3000 μmol/L | 1000 μmol/L | | Methionine | 800 μmol/L | 800 μmol/L | | Cysteine | 400 μmol/L | 200 μmol/L | | Pyruvate | 1704 μmol/L | 1250 μmol/L | | Total Protein | 126 mg/mL | 120 mg/mL | # Other limitations: - Cystathionine is measured with homocysteine, but in the general population the cystathionine level (0.065 to $0.3\mu \mathrm{mol} / \mathrm{L}$ ) has a negligible effect. In very rare cases, end stage renal disease and patients with severe metabolic disturbances, cystathionine levels may rise dramatically and in severe cases cause greater than $20\%$ interference. - Carbamazepine, methotrexate, phenytoin, nitrous oxide, or 6 azauridine triacetate may affect the homocysteine concentration - Specimens from patients on drug therapy involving S-adenosyl- {8} methionine may show falsely elevated levels of homocysteine. Patients who are taking methotrexate, carbamazepine, phenytoin, nitrous oxide, anti-convulsants, or 6 azauridine triacetate may have elevated levels of homocysteine due to their effect on the pathway. - Specimens containing particulate matter (fibrin, red blood cells, or other matter) and visibly lipemic specimens should not be used with the assay. Results from these specimens may be inaccurate. f. Assay cut-off: Not applicable 2. Comparison studies: a. Method comparison with predicate device: Fifty EDTA plasma samples were tested in the method comparison study. Of the 50 samples, 10 were spiked with L-homocysteine to obtain samples across the assay range. The samples were assayed in singlicate. The correlation of the candidate device used on the Synchron LX 20 Pro and Unicel DxC 600 to the predicate device on the Olympus AU400 was determined using Passing-Bablok method. The correlation coefficient was determined using Pearson Correlation. The summary of results is below: | Parameter | Synchron LX 20 Pro | Unicel DxC 600 | | --- | --- | --- | | Measuring Range | 5.95 – 46.25 μmol/L | 6.72 – 46.09 μmol/L | | Slope of Regression Line | 1.01 (95% CI: 0.99 – 1.04) | 0.99 (95% CI: 0.97 – 1.02) | | Y-intercept | 0.07 (95% CI: -0.30 – 0.44) | 0.74 (95% CI: 0.30 – 1.02) | | Correlation Coefficient | 0.997 (95% CI: 0.99 – 1.00) | 0.994 (95% CI:0.99 – 1.00) | b. Matrix comparison: Each of EDTA plasma, heparin plasma, serum, and serum separator tube types were used to collect samples from 23 in-house volunteers. Samples were divided into three concentration ranges: 1-10 μmol/L, 10 - 20 μmol/L, and 20 - 50 μmol/L. Samples in the range 20 - 50 μmol/L were spiked. Each sample from all four tube types was tested in duplicate on the Unicel DxC 600 with one lot of the reagent and calibrators. The first set of individual concentrations of the replicates of each sample was used for analysis, along with the percent recovery. The first set of the individual replicate values for each sample was calculated to be ±10% of the EDTA Sample control tube type. Each of the four collection tube types is considered suitable for use. {9} Below is the summary of statistics for the study performed on the Unicel DxC 600: | | EDTA vs Serum | 95% CI | EDTA vs. SST | 95% CI | EDTA vs. LiHep | 95% CI | | --- | --- | --- | --- | --- | --- | --- | | Slope | 1.03 | 0.99–1.07 | 1.01 | 0.98–1.06 | 1.02 | 0.99-1.04 | | y-intercept | -0.18 | -0.63–0.35 | -0.18 | -0.56-0.09 | -0.23 | -0.46-0.13 | | R value | 0.99 | N/A | 0.99 | N/A | 0.99 | N/A | 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): 4. Clinical cut-off: Not applicable 5. Expected values/Reference range: Expected values are based on literature references cited in the package insert. Expected values in adult males and females range between 5 and $15\mu \mathrm{mol} / \mathrm{L}$ , men having higher values than women, and post-menopausal women having higher values than pre-menopausal women. Homocysteine values will normally increase with age, giving a reference range among an elderly population (>60 years) of 5 - 20 $\mu \mathrm{mol} / \mathrm{L}$ . 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.