Traumatic brain injury (TBI) test

{0} FDA U.S. FOOD &amp; DRUG ADMINISTRATION # 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION SUMMARY ASSAY ONLY ## I Background Information: A 510(k) Number K223602 B Applicant Abbott Laboratories C Proprietary and Established Names TBI D Regulatory Information | Product Code(s) | Classification | Regulation Section | Panel | | --- | --- | --- | --- | | QAT | Class II | 21 CFR 866.5830 - Brain Trauma Assessment Test | IM - Immunology | ## II Submission/Device Overview: A Purpose for Submission: New device B Measurand: Glial fibrillary acidic protein (GFAP) and ubiquitin caboxyl-terminal hydrolase-L1 (UCH-L1) C Type of Test: Automated chemiluminescent microparticle immunoassay, semi-quantitative Food and Drug Administration 10903 New Hampshire Avenue Silver Spring, MD 20993-0002 www.fda.gov {1} K223602 - Page 2 of 28 ## III Intended Use/Indications for Use: ### A Intended Use(s): See Indications for Use below. ### B Indication(s) for Use: The TBI test is a panel of *in vitro* diagnostic chemiluminescent microparticle immunoassays (CMIA) used for the quantitative measurements of glial fibrillary acidic protein (GFAP) and ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) in human plasma and serum and provides a semi-quantitative interpretation of test results derived from these measurements using the Alinity i system. The interpretation of test results is used, in conjunction with other clinical information, to aid in the evaluation of patients, 18 years of age or older, presenting with suspected mild traumatic brain injury (Glasgow Coma Scale score 13-15) within 12 hours of injury, to assist in determining the need for a CT (computed tomography) scan of the head. A negative test result is associated with the absence of acute intracranial lesions visualized on a head CT scan. The TBI test is intended for use in clinical laboratory settings by healthcare professionals. ### C Special Conditions for Use Statement(s): For prescription use only ### D Special Instrument Requirements: Alinity i system ## IV Device/System Characteristics: ### A Device Description: The TBI test consists of two reagent kits: the GFAP Reagent Kit for the GFAP assay components and the UCH-L1 Reagent Kit for the UCH-L1 assay components. The GFAP Reagent Kit and UCH-L1 Reagent Kit are provided separately. The configurations of the two reagent kits are described below. | | GFAP Reagent Kit | UCH-L1 Reagent Kit | | --- | --- | --- | | Tests per cartridge | 100 | 100 | | Number of cartridges per kit | 2 | 2 | | Tests per kit | 200 | 200 | | Microparticles per cartridge | 7.1 mL | 7.1 mL | | Conjugate per cartridge | 6.4 mL | 12.5 mL | | Assay-specific diluent per cartridge | 6.4 mL | 10.5 mL | {2} 1. GFAP Reagent Kit: - Microparticles: Anti-GFAP (rabbit, monoclonal) coated microparticles in TRIS buffer with stabilizer and preservative. - Conjugate: Anti-GFAP (mouse, monoclonal) acridinium-labeled conjugate in MES buffer with stabilizer and preservative. - Assay-specific diluent: TRIS buffer with stabilizer and preservative. 2. UCH-L1 Reagent Kit: - Microparticles: Anti-UCH-L1 (mouse, monoclonal) coated microparticles in TRIS buffer with stabilizer and preservative. - Conjugate: Anti-UCH-L1 (mouse, monoclonal) acridinium-labeled conjugate in MES buffer with stabilizer and preservative. - Assay-specific diluent: TRIS buffer with stabilizer and preservative. 3. Materials required but not provided: - TBI assay file package - GFAP Calibrators: 6 levels (0, 50, 400, 2000, 20000, and 50000 pg/mL) - GFAP Controls: 3 levels (25, 500, and 30000 pg/mL) - UCH-L1 Calibrators: 6 levels (0, 200, 500, 1000, 5000, and 25000 pg/mL) - UCH-L1 Controls: 3 levels (250, 2000, and 15000 pg/mL) - Alinity Pre-Trigger Solution - Alinity Trigger Solution - Alinity i-series Concentrated Wash Buffer B Principle of Operation: The TBI test is a panel of in vitro diagnostic quantitative measurements of GFAP and UCH-L1 in human plasma and serum using an automated, two-step chemiluminescent microparticle immunoassay (CMIA) on the Alinity i system, and provides a semi-quantitative interpretation based on the results of GFAP and UCH-L1. For GFAP, sample, anti-GFAP coated paramagnetic microparticles, and assay specific diluent are combined and incubated. The GFAP present in the sample binds to the anti-GFAP coated microparticles. The mixture is washed. Anti-GFAP acridinium-labeled conjugate is added to create a reaction mixture and incubated. Following a wash cycle, Pre-Trigger and Trigger Solutions are added. The resulting chemiluminescent reaction is measured as a relative light unit (RLU). There is a direct relationship between the amount of GFAP in the sample and the RLU detected by the system optics. For UCH-L1, sample, anti-UCH-L1 coated paramagnetic microparticles, and assay specific diluent are combined and incubated. The UCH-L1 present in the sample binds to the anti-UCH-L1 coated microparticles. The mixture is washed. Anti-UCH-L1 acridinium-labeled conjugate is added to create a reaction mixture and incubated. Following a wash cycle, Pre-Trigger and Trigger Solutions are added. The resulting chemiluminescent reaction is measured as an RLU. K223602 - Page 3 of 28 {3} There is a direct relationship between the amount of UCH-L1 in the sample and the RLU detected by the system optics. The assay cut-offs were established to be $35.0~\mathrm{pg / mL}$ $(35.0~\mathrm{ng / L})$ for GFAP and $400.0~\mathrm{pg / mL}$ $(400.0~\mathrm{ng / L})$ for UCH-L1. The GFAP and UCH-L1 results are reported separately, and the Alinity i system software provides a TBI interpretation relative to the respective cut-off values as shown in the following table. | Specification for Constituent Assay Results | TBI Result | TBI Interpretation | | --- | --- | --- | | GFAP and UCH-L1 below (<) cut-off | 0 | Negative | | GFAP and/or UCH-L1 above (≥) cut-off | 1 | Positive | The following table provides a detailed summary of the TBI interpretation based on potential results. | GFAP Assay Result (relative to 35.0 pg/mL cut-off)* | UCH-L1 Assay Result (relative to 400.0 pg/mL cut-off)* | TBI Interpretation | | --- | --- | --- | | Below | Below | Negative** | | Below | Above | Positive** | | Above | Below | Positive** | | Above | Above | Positive** | | No result | Below | Not reportable*** | | No result | Above | Positive*** | | Below | No result | Not reportable*** | | Above | No result | Positive*** | | No result | No result | Not reportable*** | * Above means greater than or equal to the cut-off. Below means less than the cut-off. ** The GFAP and UCH-L1 results can be found on the Result Details screen under Constituent Information on the User Interface. *** An automated TBI interpretation will not be reported for specimens without a result for GFAP and/or UCH-L1. The GFAP and/or UCH-L1 assay(s) may be retested if needed to obtain a result and a manual TBI interpretation may be required. The TBI interpretation for a specimen is considered positive if the result for either constituent assay (GFAP or UCH-L1) is greater than or equal to the cut-off and no result is obtained for the other assay. The TBI interpretation for a specimen is not reportable if the result for either constituent assay is less than the cut-off and no result is obtained for the other assay. In the case of a flagged "&gt;" or "&lt;" result for either assay, the TBI interpretation should be evaluated manually. A result flagged "&gt;" should be considered above the cut-off and a result flagged "&lt;" should be considered below the cut-off. K223602 - Page 4 of 28 {4} V Substantial Equivalence Information: A Predicate Device Name(s): Banyan BTI B Predicate 510(k) Number(s): DEN170045 C Comparison with Predicate(s): | Device & Predicate Device(s): | Device K223602 | Predicate DEN170045 | | --- | --- | --- | | Device Trade Name | TBI test | Banyan BTI | | General Device Characteristic Similarities | | | | Intended Use/Indications for Use | The TBI test is a panel of in vitro diagnostic chemiluminescent microparticle immunoassays (CMIA) used for the quantitative measurements of glial fibrillary acidic protein (GFAP) and ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) in human plasma and serum and provides a semi-quantitative interpretation of test results derived from these measurements using the Alinity i system. The interpretation of test results is used, in conjunction with other clinical information, to aid in the evaluation of patients, 18 years of age or older, presenting with suspected mild traumatic brain injury (Glasgow Coma Scale score 13-15) within 12 hours of injury, to assist in determining the need for a CT (computed tomography) scan of the head. A negative test result is associated with the absence of acute intracranial lesions visualized on a head CT scan. The TBI test is intended for use in clinical laboratory settings by healthcare professionals. | The Banyan BTI is an in vitro diagnostic chemiluminescent enzyme-linked immunosorbent assay (ELISA). The assay provides a semi-quantitative measurement of the concentrations of ubiquitin C-terminal hydrolase-L1 (UCH-L1) and glial fibrillary acidic protein (GFAP) in human serum and is used with the Synergy 2 Multi-mode Reader. The assay results obtained from serum collected within 12 hours of suspected head injury are used, along with other available clinical information, to aid in the evaluation of patients 18 years of age and older with suspected traumatic brain injury (Glasgow Coma Scale score 13-15). A negative assay result is associated with the absence of acute intracranial lesions visualized on a head CT (computed tomography) scan. | K223602 - Page 5 of 28 {5} | Device & Predicate Device(s): | Device K223602 | Predicate DEN170045 | | --- | --- | --- | | Intended Use Setting | Clinical laboratory | Same | | Measurands | GFAP and UCH-L1 | Same | | Assay Technology | Immunoassay (Chemiluminescent microparticle immunoassay) | Immunoassay (Enzyme-linked immunosorbent assay) | | Calibrators | GFAP: 6 levels UCH-L1: 6 levels | Same | | Reportable Result | Quantitative results for GFAP and UCH-L1 and semi-quantitative interpretation for TBI | Same | | Assay Format | Two separate test kits | Same | | Detection Technology | Chemiluminescence | Same | | General Device Characteristic Differences | | | | Platform | Alinity i system | Synergy 2 Multi-mode Reader (BioTek Instruments, Inc.) | | Specimen Type | Serum and plasma | Serum | | Controls | GFAP: 3 levels (25, 500, and 30000 pg/mL) UCH-L1: 3 levels (250, 2000, and 15000 pg/mL) | GFAP: 2 levels (Control 1-Low, Control 2-High) UCH-L1: 2 levels (Control 1-Low, Control 2-High) | | Sample Volume | GFAP: 200 μL UCH-L1: 150 μL | GFAP: 150 μL UCH-L1: 100 μL | | Time to Result | Approximately 18 minutes | Approximately 4 hours | | Analytical Measurement Interval | GFAP: 6.1 – 42,000.0 pg/mL UCH-L1: 26.3 – 25,000.0 pg/mL | GFAP: 10 – 320 pg/mL UCH-L1: 80 – 2560 pg/mL | | Cut-off | GFAP: 35.0 pg/mL UCH-L1: 400.0 pg/mL | GFAP: 22 pg/mL UCH-L1: 327 pg/mL | | Assay Procedure | Automated immunoassay | Manual ELISA | | Result Interpretation | Software | Performed by user | VI Standards/Guidance Documents Referenced: - CLSI EP05-A3: Evaluation of Precision of Quantitative Measurement Procedures; Approved Guideline – Third Edition - CLSI EP06, 2nd ed.: Evaluation of Linearity of Quantitative Measurement Procedures, 2nd Edition; Approved Guideline – Second Edition - CLSI EP07 Ed3: Interference Testing in Clinical Chemistry – Third Edition - CLSI EP17-A2: Evaluation of Detection Capability for Clinical Laboratory Measurement Procedures; Approved Guideline – Second Edition K223602 - Page 6 of 28 {6} - CLSI EP25-A: Evaluation of Stability of In Vitro Diagnostic Reagents; Approved Guideline - CLSI EP28-A3c: Defining, Establishing, and Verifying Reference Intervals in the Clinical Laboratory; Approved Guideline – Third Edition - CLSI EP35 Ed1: Assessment of Equivalence or Suitability of Specimen Types for Medical Laboratory Measurement Procedures; Approved Guideline – First Edition - CLSI EP37 Ed1: Supplemental Tables for Interference Testing in Clinical Chemistry – First Edition ## VII Performance Characteristics (if/when applicable): ### A Analytical Performance: All results met the manufacturer's pre-determined acceptance criteria. #### 1. Precision/Reproducibility: ##### a. Within-laboratory precision A study was conducted per CLSI guideline EP05-A3 to evaluate the within-laboratory precision of the GFAP and UCH-L1 assays. K2 EDTA human plasma samples in the test panels were prepared to achieve target concentrations of GFAP and UCH-L1 that cover the respective measuring ranges of the individual assays and tested at one site using two reagent lots of each assay and two Alinity i instruments. The panel consisted of a native plasma sample near the assay cut-off (panel member 2) and pooled plasma samples (panel members 1 and 3–8) spiked with recombinant analytes (&lt; 0.02% of sample volume). The panel members were tested along with three controls in three replicates in two runs per day for 20 days (3 x 2 x 20 =120 replicates per reagent lot/instrument combination) on the four reagent lot/instrument combinations to generate a total of 480 replicates per test panel (i.e., N=120 for lot 1/instrument 1 combination, N=120 for lot 2/instrument 1 combination, N=120 for lot 2/instrument 1 combination, and N=120 for lot 2/instrument 2 combination). The performance across the four reagent lots/instrument combinations were analyzed. In addition, the qualitative analysis of precision results relative to the cut-offs (35.0 pg/mL for GFAP and 400 pg/mL for UCH-L1) was performed using the same 20-day within-laboratory precision study data. The % correct call was calculated for each plasma sample and was based on the number of replicates providing the expected GFAP / UCH-L1 results (Not Elevated or Elevated) based on the mean analyte concentration for the sample (either below cut-off or at/above cut-off, respectively). Results are summarized in the tables below for each assay. K223602 - Page 7 of 28 {7} i. GFAP: | GFAP Assay - Within-Laboratory Precision (Quantitative Analysis) | | | | | | | | | | | | | | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | Panel Member | N | Mean (pg/mL) | Within-Run | | Between-Run | | Between-Day | | Between-Lot | | Between-Instrument | | Within-Laboratory | | | | | SD | %CV | SD | %CV | SD | %CV | SD | %CV | SD | %CV | SD | | 1 | 479 | 20.4 | 0.69 | 3.4 | 0.64 | 3.1 | 0.36 | 1.8 | 0.02 | 0.1 | 0.19 | 0.9 | 1.03 | | 2 | 474 | 37.7 | 1.11 | 2.9 | 0.80 | 2.1 | 0.37 | 1.0 | 0.15 | 0.4 | 0.20 | 0.5 | 1.43 | | 3 | 479 | 40.2 | 1.20 | 3.0 | 1.03 | 2.6 | 0.42 | 1.0 | 0.19 | 0.5 | 0.00 | 0.0 | 1.65 | | 4 | 480 | 95.6 | 2.41 | 2.5 | 2.34 | 2.4 | 0.00 | 0.0 | 0.36 | 0.4 | 1.29 | 1.3 | 3.62 | | 5 | 480 | 3097.2 | 72.89 | 2.4 | 63.34 | 2.0 | 55.85 | 1.8 | 55.24 | 1.8 | 36.55 | 1.2 | 129.74 | | 6 | 478 | 7586.3 | 168.16 | 2.2 | 187.23 | 2.5 | 84.73 | 1.1 | 177.85 | 2.3 | 48.77 | 0.6 | 323.30 | | 7 | 480 | 15462.7 | 346.62 | 2.2 | 389.78 | 2.5 | 289.53 | 1.9 | 441.47 | 2.9 | 92.99 | 0.6 | 747.96 | | 8 | 478 | 36874.7 | 969.22 | 2.6 | 1233.45 | 3.3 | 0.00 | 0.0 | 1435.60 | 3.9 | 509.41 | 1.4 | 2186.60 | | Low Control | 479 | 25.4 | 0.76 | 3.0 | 0.70 | 2.7 | 0.12 | 0.5 | 0.43 | 1.7 | 0.53 | 2.1 | 1.24 | | Medium Control | 480 | 504.8 | 12.89 | 2.6 | 11.67 | 2.3 | 0.00 | 0.0 | 1.40 | 0.3 | 6.38 | 1.3 | 18.58 | | GFAP Assay - Within-Laboratory Precision (Qualitative Analysis) | | | | | | --- | --- | --- | --- | --- | | Panel Member a | Replicates (N) | Mean (pg/mL) | Elevated Results (at/above cut-off) / Total Replicates (n/N) | % of Correct Call b | | 1 | 479 | 20.4 | 0 / 479 | 100.0 | | 2 | 474 | 37.7 | 462 / 474 | 100.0 | | 3 | 479 | 40.2 | 479 / 479 | 100.0 | | 4 | 480 | 95.6 | 480 / 480 | 100.0 | | 5 | 480 | 3097.2 | 480 / 480 | 100.0 | | 6 | 478 | 7586.3 | 478 / 478 | 100.0 | | 7 | 480 | 15,462.7 | 480 / 480 | 100.0 | | 8 | 478 | 36,874.7 | 478 / 478 | 100.0 | | Low Control | 479 | 25.4 | 0 / 479 | 100.0 | | Medium Control | 480 | 504.8 | 480 / 480 | 100.0 | | High Control | 479 | 31,608.5 | 479 / 479 | 100.0 | a Panel 1: negative; Panel 2 (native) and 3: Samples near medical decision point; Panel 4-8: positive; Low Control: negative; Medium and High Controls: positive. b Replicates for positive samples should always be $\geq$ cutoff, replicates for negative samples should always be $&lt;$ cutoff, and replicates for samples near medical decision points can have replicates $&lt;$ cutoff or $\geq$ cutoff. K223602 - Page 8 of 28 {8} ii. UCH-L1 | UCH-L1 Assay - Within-Laboratory Precision (Quantitative Analysis) | | | | | | | | | | | | | | | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | Panel Member | N | Mean (pg/mL) | Within-Run | | Between-Run | | Between-Day | | Between-Lot | | Between-Instrument | | Within-Laboratory | | | | | | SD | %CV | SD | %CV | SD | %CV | SD | %CV | SD | %CV | SD | %CV | | 1 | 480 | 177.6 | 7.20 | 4.1 | 5.15 | 2.9 | 1.68 | 0.9 | 0.00 | 0.0 | 5.59 | 3.1 | 10.60 | 6.0 | | 2 | 471 | 391.8 | 15.53 | 4.0 | 4.50 | 1.1 | 7.18 | 1.8 | 1.27 | 0.3 | 14.89 | 3.8 | 23.15 | 5.9 | | 3 | 479 | 419.8 | 14.47 | 3.4 | 8.81 | 2.1 | 6.95 | 1.7 | 0.00 | 0.0 | 14.86 | 3.5 | 23.58 | 5.6 | | 4 | 477 | 823.8 | 27.42 | 3.3 | 21.32 | 2.6 | 8.46 | 1.0 | 0.00 | 0.0 | 25.33 | 3.1 | 43.81 | 5.3 | | 5 | 476 | 1553.8 | 53.15 | 3.4 | 28.09 | 1.8 | 25.67 | 1.7 | 9.83 | 0.6 | 21.27 | 1.4 | 69.44 | 4.5 | | 6 | 479 | 4793.5 | 164.54 | 3.4 | 138.24 | 2.9 | 0.00 | 0.0 | 0.00 | 0.0 | 115.93 | 2.4 | 244.18 | 5.1 | | 7 | 480 | 7974.6 | 269.27 | 3.4 | 161.73 | 2.0 | 72.92 | 0.9 | 21.25 | 0.3 | 145.84 | 1.8 | 354.54 | 4.4 | | 8 | 472 | 19165.5 | 619.10 | 3.2 | 428.85 | 2.2 | 162.96 | 0.9 | 0.00 | 0.0 | 290.87 | 1.5 | 823.62 | 4.3 | | Low Control | 479 | 247.5 | 9.16 | 3.7 | 4.99 | 2.0 | 3.13 | 1.3 | 1.62 | 0.7 | 1.07 | 0.4 | 11.06 | 4.5 | | Medium Control | 479 | 2019.6 | 47.72 | 2.4 | 28.81 | 1.4 | 19.81 | 1.0 | 17.23 | 0.9 | 0.00 | 0.0 | 61.62 | 3.1 | | High Control | 477 | 15179.4 | 367.43 | 2.4 | 219.00 | 1.4 | 120.20 | 0.8 | 0.00 | 0.0 | 59.99 | 0.4 | 448.35 | 3.0 | | UCH-L1 Assay - Within-Laboratory Precision (Qualitative Analysis) | | | | | | --- | --- | --- | --- | --- | | Panel Member a | Replicates (N) | Mean (pg/mL) | Elevated Results (at/above cut-off) / Total Replicates (n/N) | % of Correct Call b | | 1 | 480 | 177.6 | 0 / 480 | 100.0 | | 2 | 471 | 391.8 | 164 / 471 | 100.0 | | 3 | 479 | 419.8 | 391 / 479 | 100.0 | | 4 | 477 | 823.8 | 477 / 477 | 100.0 | | 5 | 476 | 1553.8 | 476 / 476 | 100.0 | | 6 | 479 | 4793.5 | 479 / 479 | 100.0 | | 7 | 480 | 7974.6 | 480 / 480 | 100.0 | | 8 | 472 | 19,165.5 | 472 / 472 | 100.0 | | Low Control | 479 | 247.5 | 0 / 479 | 100.0 | | Medium Control | 479 | 2019.6 | 479 / 479 | 100.0 | | High Control | 477 | 15,179.4 | 477 / 477 | 100.0 | a Panel 1: negative; Panel 2 (native) and 3: Samples near medical decision point; Panel 4-8: positive; Low Control: negative; Medium and High Controls: positive. b Replicates for positive samples should always be $\geq$ cutoff, replicates for negative samples should always be $&lt;$ cutoff, and replicates for samples near medical decision points can have replicates $&lt;$ cutoff or $\geq$ cutoff. K223602 - Page 9 of 28 {9} b. Multi-site reproducibility A study was conducted per CLSI guideline EP05-A3 to evaluate the multi-site reproducibility precision of the GFAP and UCH-L1 assays. Two separate test panels, each consisting of seven human K2 EDTA plasma samples with levels of GFAP or UCH-L1 that cover the measuring range of the respective assays, were tested along with three controls. The panel included one native sample near the assay cut-off (panel member 2) and pooled plasma samples (panel members 1 and 3–7) spiked with recombinant analytes. Each sample was tested in four replicates per run, two runs per day, for five days at three sites in the U.S. with one instrument at each site, using three reagent lots to generate a total of 360 replicates (i.e., $4 \times 2 \times 5 \times 3 \times 3$). The quantitative and qualitative analysis were conducted as described above. Results are summarized in the tables below for each assay. i. GFAP | GFAP Assay - Multi-Site Reproducibility (Quantitative Analysis) | | | | | | | | | | | | | | | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | Panel Member | N | Mean (pg/mL) | Within-Run | | Between-Run | | Between-Day | | Between-Lot | | Between-Site | | Reproducibility | | | | | | SD | %CV | SD | %CV | SD | %CV | SD | %CV | SD | %CV | SD | %CV | | 1 | 360 | 20.4 | 0.61 | 3.0 | 0.26 | 1.3 | 0.53 | 2.6 | 0.19 | 0.9 | 0.34 | 1.7 | 0.94 | 4.6 | | 2 | 360 | 37.4 | 0.74 | 2.0 | 0.49 | 1.3 | 0.91 | 2.4 | 0.29 | 0.8 | 0.83 | 2.2 | 1.54 | 4.1 | | 3 | 360 | 94.9 | 1.66 | 1.8 | 0.89 | 0.9 | 1.87 | 2.0 | 0.00 | 0.0 | 1.26 | 1.3 | 2.94 | 3.1 | | 4 | 360 | 3072.8 | 50.95 | 1.7 | 33.14 | 1.1 | 43.49 | 1.4 | 17.37 | 0.6 | 11.66 | 0.4 | 77.60 | 2.5 | | 5 | 360 | 7449.5 | 135.20 | 1.8 | 84.45 | 1.1 | 102.16 | 1.4 | 0.00 | 0.0 | 0.00 | 0.0 | 189.34 | 2.5 | | 6 | 360 | 15269.2 | 252.80 | 1.7 | 152.99 | 1.0 | 207.63 | 1.4 | 68.84 | 0.5 | 227.57 | 1.5 | 432.38 | 2.8 | | 7 | 360 | 36101.1 | 852.46 | 2.4 | 368.77 | 1.0 | 568.78 | 1.6 | 781.48 | 2.2 | 1037.82 | 2.9 | 1695.28 | 4.7 | | Low Control | 360 | 24.9 | 0.61 | 2.4 | 0.18 | 0.7 | 0.57 | 2.3 | 0.26 | 1.1 | 0.37 | 1.5 | 0.97 | 3.9 | | Medium Control | 360 | 494.6 | 8.33 | 1.7 | 4.46 | 0.9 | 8.06 | 1.6 | 3.46 | 0.7 | 0.94 | 0.2 | 12.93 | 2.6 | | High Control | 360 | 30520.7 | 630.70 | 2.1 | 404.86 | 1.3 | 230.74 | 0.8 | 252.37 | 0.8 | 428.65 | 1.4 | 928.64 | 3.0 | K223602 - Page 10 of 28 {10} | GFAP Assay - Multi-Site Reproducibility (Qualitative Analysis) | | | | | | --- | --- | --- | --- | --- | | Panel Member a | Replicates (N) | Mean (pg/mL) | Elevated Results (at/above Cut-off) / Total Replicates (n/N) | % of Correct Call b | | 1 | 360 | 20.4 | 0 / 360 | 100.0 | | 2 | 360 | 37.4 | 337 / 360 | 100.0 | | 3 | 360 | 94.9 | 360 / 360 | 100.0 | | 4 | 360 | 3072.8 | 360 / 360 | 100.0 | | 5 | 360 | 7449.5 | 360 / 360 | 100.0 | | 6 | 360 | 15269.2 | 360 / 360 | 100.0 | | 7 | 360 | 36101.1 | 360 / 360 | 100.0 | | Low Control | 360 | 24.9 | 0 / 360 | 100.0 | | Medium Control | 360 | 494.6 | 360 / 360 | 100.0 | | High Control | 360 | 30520.7 | 360 / 360 | 100.0 | a Panel 1: negative; Panel 2: Samples near medical decision point; Panel 3-7: positive; Low Control: negative; Medium and High Controls: positive. b Replicates for positive samples should always be $\geq$ cutoff, replicates for negative samples should always be $&lt;$ cutoff, and replicates for samples near medical decision points can have replicates $&lt;$ cutoff or $\geq$ cutoff. ii. UCH-L1 | UCH-L1 Assay - Multi-Site Reproducibility (Quantitative Analysis) | | | | | | | | | | | | | | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | Panel Member | N | Mean (pg/mL) | Within-Run | | Between-Run | | Between-Day | | Between-Lot | | Between-Site | | Reproducibility | | | | | SD | %CV | SD | %CV | SD | %CV | SD | %CV | SD | %CV | SD | | 1 | 360 | 187.3 | 5.29 | 2.8 | 2.03 | 1.1 | 4.45 | 2.4 | 1.68 | 0.9 | 9.93 | 5.3 | 12.39 | | 2 | 360 | 402.4 | 10.91 | 2.7 | 4.55 | 1.1 | 3.58 | 0.9 | 5.52 | 1.4 | 13.24 | 3.3 | 18.93 | | 3 | 359 | 838.9 | 20.67 | 2.5 | 6.18 | 0.7 | 7.22 | 0.9 | 13.02 | 1.6 | 32.20 | 3.8 | 41.52 | | 4 | 360 | 1568.0 | 40.83 | 2.6 | 21.71 | 1.4 | 3.97 | 0.3 | 15.74 | 1.0 | 49.33 | 3.1 | 69.54 | | 5 | 360 | 4792.8 | 125.95 | 2.6 | 48.58 | 1.0 | 61.63 | 1.3 | 23.27 | 0.5 | 112.59 | 2.3 | 187.72 | | 6 | 360 | 8011.9 | 188.84 | 2.4 | 62.49 | 0.8 | 77.68 | 1.0 | 30.53 | 0.4 | 192.57 | 2.4 | 289.16 | | 7 | 360 | 19606.6 | 513.60 | 2.6 | 197.00 | 1.0 | 252.28 | 1.3 | 201.66 | 1.0 | 571.99 | 2.9 | 856.78 | | Low Control | 360 | 249.9 | 5.83 | 2.3 | 1.63 | 0.7 | 4.85 | 1.9 | 1.62 | 0.6 | 4.29 | 1.7 | 9.01 | | Medium Control | 360 | 1983.7 | 36.07 | 1.8 | 14.64 | 0.7 | 14.46 | 0.7 | 2.40 | 0.1 | 31.50 | 1.6 | 52.18 | | High Control | 360 | 14945.1 | 243.83 | 1.6 | 124.28 | 0.8 | 187.94 | 1.3 | 75.42 | 0.5 | 183.94 | 1.2 | 386.96 | K223602 - Page 11 of 28 {11} | UCH-L1 Assay - Multi-Site Reproducibility (Qualitative Analysis) | | | | | | --- | --- | --- | --- | --- | | Panel Member a | Replicates (N) | Mean (pg/mL) | Elevated Results (at/above cut-off) / Total Replicates (n/N) | % of Correct Call b | | 1 | 360 | 187.3 | 0 / 360 | 100.0 | | 2 | 360 | 402.4 | 230 / 360 | 100.0 | | 3 | 359 * | 838.9 | 359 / 359 | 100.0 | | 4 | 360 | 1568.0 | 360 / 360 | 100.0 | | 5 | 360 | 4792.8 | 360 / 360 | 100.0 | | 6 | 360 | 8011.9 | 360 / 360 | 100.0 | | 7 | 360 | 19606.6 | 360 / 360 | 100.0 | | Low Control | 360 | 249.9 | 0 / 360 | 100.0 | | Medium Control | 360 | 1983.7 | 360 / 360 | 100.0 | | High Control | 360 | 14945.1 | 360 / 360 | 100.0 | a Panel 1: negative; Panel 2: Samples near medical decision point; Panel 3-7: positive; Low Control: negative; Medium and High Controls: positive. b Replicates for positive samples should always be $\geq$ cutoff, replicates for negative samples should always be $&lt;$ cutoff, and replicates for samples near medical decision points can have replicates $&lt;$ cutoff or $\geq$ cutoff. * One (1) no result with aspiration error message code 2. Linearity: The linearity of the GFAP and UCH-L1 assays on the Alinity i system was evaluated in accordance with the CLSI guideline EP6 $2^{\mathrm{nd}}$ Edition. Two studies for each assay were conducted in parallel and the data from the studies were analyzed for evaluating linearity for each assay. a. GFAP The linear range of the GFAP assay was established using a native human plasma sample set and three recombinant GFAP sample sets. One unique native sample set was prepared using a native high K2 EDTA plasma combined with analyte-depleted plasma to create a series of 10 dilutions with GFAP concentrations spanning from 3.8 to $23868.7~\mathrm{pg / mL}$ . Each sample dilution was tested in five replicates using one reagent lot. Three recombinant sample sets (A, B, and C) were prepared using a high recombinant GFAP stock diluted with three unique sets of analyte-depleted plasma to create a series of dilutions with GFAP concentrations spanning from 5.2 to $53720.5~\mathrm{pg / mL}$ for set A, from 4.8 to $49245.4~\mathrm{pg / mL}$ for set B, and from 5.1 to $42556.9~\mathrm{pg / mL}$ for set C. Each recombinant sample set consisted of at least nine dilutions, each tested in ten replicates. Deviations from linearity were calculated for the pooled results of the native human plasma sample set and each recombinant sample set individually (i.e., native set and recombinant set A, native set and recombinant set B, and native set and recombinant set C). The representative deviation from linearity analysis using the native set and recombinant set A is shown in the table below. K223602 - Page 12 of 28 {12} | Analyte Source | Sample No. | N | Mean Observed (pg/mL) | Expected (pg/mL) | Predicted Value (pg/mL) | Deviation from Linearity (pg/mL) | %Deviation from Linearity | | --- | --- | --- | --- | --- | --- | --- | --- | | Native | 1 | 5 | 23868.7 | 23868.7 | 22731.8 | 1136.9 | 5.0 | | Native | 2 | 5 | 19233.7 | 19095.0 | 18185.5 | 1048.2 | 5.8 | | Native | 3 | 5 | 14498.4 | 14321.2 | 13639.1 | 859.3 | 6.3 | | Native | 4 | 5 | 9582.6 | 9547.5 | 9092.7 | 489.9 | 5.4 | | Native | 5 | 5 | 4739.0 | 4773.7 | 4546.4 | 192.7 | 4.2 | | Native | 6 | 5 | 2136.1 | 2100.4 | 2000.4 | 135.7 | 6.8 | | Native | 7 | 5 | 230.7 | 238.7 | 227.3 | 3.4 | 1.5 | | Native | 8 | 5 | 57.9 | 59.7 | 56.8 | 1.1 | 1.8 | | Native | 9 | 5 | 12.8 | 14.3 | 13.6 | -0.8 | -6.2 | | Native | 10 | 5 | 3.8 | 4.8 | 4.5 | -0.8 | * | | Recombinant | 1 | 10 | 53720.5 | 53720.5 | 51161.7 | 2558.8 | 5.0 | | Recombinant | 2 | 10 | 42269.4 | 43647.9 | 41568.9 | 700.5 | 1.7 | | Recombinant | 3 | 10 | 31945.8 | 33575.3 | 31976.1 | -30.2 | -0.1 | | Recombinant | 4 | 10 | 19557.5 | 20145.2 | 19185.6 | 371.8 | 1.9 | | Recombinant | 5 | 10 | 9603.3 | 10072.6 | 9592.8 | 10.4 | 0.1 | | Recombinant | 6 | 10 | 3093.7 | 3357.5 | 3197.6 | -103.9 | -3.3 | | Recombinant | 7 | 10 | 312.1 | 335.8 | 319.8 | -7.6 | -2.4 | | Recombinant | 8 | 10 | 73.9 | 83.9 | 79.9 | -6.1 | -7.6 | | Recombinant | 9 | 10 | 18.6 | 21.0 | 20.0 | -1.4 | -6.8 | | Recombinant | 10 | 10 | 5.2 | 5.2 | 5.0 | 0.2 | * | * Not applicable for % bias as acceptance criteria is: ± 1.0 pg/mL for samples less than 10.0 pg/mL and ± 10.0% for samples greater than or equal to 10.0 pg/mL The study demonstrated linearity across a series of dilutions spanning an interval of 3.8 to 42,556.9 pg/mL. The claimed linearity of the GFAP assay is 6.1 to 42,000.0 pg/mL based on the pooled analysis of native and non-native samples. b. UCH-L1 The linear range of the UCH-L1 assay was established using a native human plasma sample set. One unique sample set was prepared using a native high K2 EDTA plasma combined with analyte-depleted plasma to create a series of 10 dilutions of UCH-L1 concentrations that span across the analytical measuring interval of the assay. Each sample dilution was tested in five replicates using one reagent lot. For each sample, the mean value of the measured values, predicted value and the deviation from linearity were calculated. K223602 - Page 13 of 28 {13} | Analyte Source | Sample No. | N | Mean Observed (pg/mL) | Expected (pg/mL) | Predicted Value (pg/mL) | Deviation from Linearity (pg/mL) | %Deviation from Linearity | | --- | --- | --- | --- | --- | --- | --- | --- | | Native | 1 | 5 | 23235.1 | 28235.1 | 27517.9 | 717.2 | 2.6 | | Native | 2 | 5 | 22054.2 | 22588.1 | 22014.3 | 39.9 | 0.2 | | Native | 3 | 5 | 16514.3 | 16941.1 | 16510.7 | 3.6 | 0.0 | | Native | 4 | 5 | 10774.9 | 11294.0 | 11007.2 | -232.2 | -2.1 | | Native | 5 | 5 | 5492.3 | 5647.0 | 5503.6 | -11.2 | -0.2 | | Native | 6 | 5 | 2367.5 | 2484.7 | 2421.6 | -54.1 | -2.2 | | Native | 7 | 5 | 271.7 | 282.4 | 275.2 | -3.5 | -1.3 | | Native | 8 | 5 | 66.6 | 70.6 | 68.8 | -2.2 | -3.2 | | Native | 9 | 5 | 17.6 | 16.9 | 16.5 | 1.0 | 6.4 | | Native | 10 | 5 | 10.1 | 8.5 | 8.3 | 1.9 | * | * Not applicable for % bias as acceptance criteria: ± 16.0 pg/mL for samples less than 160.0 pg/mL and ± 10.0% for samples greater than or equal to 160.0 pg/mL The study demonstrated linearity across a series of dilutions produced with a native specimen and spanning an interval of 10.1 to 28,235.1 pg/mL. The claimed linearity of the UCH-L1 assay is 26.3 to 25,000 pg/mL. 3. Analytical Specificity/Interference: a. Interference Assay interference was assessed in accordance with the CLSI guideline EP07-3rd Edition by testing pooled human plasma spiked with either recombinant GFAP at final concentrations of 25 and 10,000 pg/mL or recombinant UCH-L1 at final concentrations of 280 pg/mL and 5000 pg/mL. Test samples were created by spiking with the potentially interfering substances. Control samples were spiked only with the appropriate solvent used to create the interfering substances panel. Test and control samples were analyzed in replicates of five, in one assay run, with one reagent lot of each assay on the Alinity i system. The %Interference of each analyte was calculated by determining the percent difference between the test and control samples: %Interference = [(Meantest - Meancontrol)/Meancontrol] x 100%. No significant interference was observed (≤10% difference from the control sample) for the GFAP and UCH-L1 assays up to the concentrations of the endogenous and exogenous substances tested as shown in the tables below: K223602 - Page 14 of 28 {14} | Potentially Endogenous Interfering Substance | Interferent Level | | | --- | --- | --- | | | GFAP | UCH-L1 | | Conjugated Bilirubin | 40 mg/dL | 40 mg/dL | | Unconjugated Bilirubin | 40 mg/dL | 20 mg/dL | | Hemoglobin | 1000 mg/dL | 1000 mg/dL | | Intralipid | 1500 mg/dL | 2000 mg/dL | | Human Albumin | 10 g/dL | 9 g/dL | | Glucose | 1000 mg/dL | 1000 mg/dL | | Human anti-mouse antibody (HAMA) | 80x activity | 80x activity | | Rheumatoid Factor (RF) | 500 IU/mL | 500 IU/mL | | Potentially Exogenous Interfering Substance | Interferent Level | | | --- | --- | --- | | | GFAP | UCH-L1 | | Acetaminophen | 20 mg/dL | 20 mg/dL | | Acetylcysteine | 15 mg/dL | 9 mg/dL | | Acetylsalicylic Acid | 65 mg/dL | 65 mg/dL | | Amphetamine | 33 μg/dL | 33 μg/dL | | Ampicillin-Na | 7.5 mg/dL | 7.5 mg/dL | | Ascorbic Acid | 5.25 mg/dL | 5.25 mg/dL | | Benzoylecgonine | 200 μg/dL | 200 μg/dL | | Biotin | 4250 ng/mL | 4250 ng/mL | | Brivaracetam | 1.05 mg/dL | 1.05 mg/dL | | Calcium dobesilate | 6 mg/dL | 2 mg/dL | | Cannabinoids | 50 ng/mL | 50 ng/mL | | Carbamazepine | 4.5 mg/dL | 4.5 mg/dL | | Cefoxitin | 660 mg/dL | 660 mg/dL | | Celecoxib | 879 μg/dL | 879 μg/dL | | Clopidogrel (Plavix) | 9 μg/mL | 9 μg/mL | | Codeine | 141 μg/dL | 141 μg/dL | | Cyclobenzaprine | 10.2 μg/dL | 10.2 μg/dL | | Cyclosporine | 0.18 mg/dL | 0.18 mg/dL | | Diazepam | 3 mg/dL | 3 mg/dL | | Doxycycline | 1.8 mg/dL | 1.8 mg/dL | | EDDP* | 318 μg/dL | 318 μg/dL | | Ethanol | 3000 mg/dL | 1000 mg/dL | | Fentanyl | 0.03 mg/dL | 0.03 mg/dL | | Heparin | 330 U/dL | 330 U/dL | | Ibuprofen | 50 mg/dL | 50 mg/dL | | Imipramine | 0.0315 mg/dL | 0.0315 mg/dL | | Levodopa | 0.75 mg/dL | 0.75 mg/dL | | Methadone | 318 μg/dL | 318 μg/dL | | d-Methamphetamine | 400 μg/dL | 400 μg/dL | K223602 - Page 15 of 28 {15} | Potentially Exogenous Interfering Substance | Interferent Level | | | --- | --- | --- | | | GFAP | UCH-L1 | | Methaqualone | 200 μg/dL | 200 μg/dL | | Methyldopa | 2.25 mg/dL | 2.25 mg/dL | | Methylenedioxy methamphetamine | 500 ng/mL | 500 ng/mL | | Metoprolol | 0.5 mg/dL | 0.5 mg/dL | | Metronidazole | 12.3 mg/dL | 12.3 mg/dL | | Morphine | 780 μg/dL | 780 μg/dL | | Naproxen | 36 mg/dL | 36 mg/dL | | Nicardipine | 46.5 μg/dL | 46.5 μg/dL | | Ondansetron | 34.2 μg/dL | 34.2 μg/dL | | Oxazepam | 425 μg/dL | 432 μg/dL | | Phencyclidine | 20 μg/dL | 20 μg/dL | | Propoxyphene | 321 μg/dL | 321 μg/dL | | Rifampicin | 4.8 mg/dL | 4.8 mg/dL | | Secobarbital | 1.59 mg/dL | 1.59 mg/dL | | Theophylline | 6 mg/dL | 6 mg/dL | | Warfarin (Coumadin) | 7.5 mg/dL | 7.5 mg/dL | *2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine ## b. Cross-reactivity To assess test performance in the presence of putative cross-reactants, a panel comprised of proteins that have significant homology to either GFAP or UCH-L1 was evaluated. Samples containing the cross-reactants at the concentrations listed in the table below were prepared in GFAP-depleted plasma or UCH-L1 depleted plasma and tested with the GFAP and UCH-L1 assays on the Alinity i system in 30 replicates. The % cross-reactivity was calculated, and the results showed none of the potentially cross-reactants tested were found to interfere with the GFAP and UCH-L1 assays. | Cross-reactant | Cross-reactant Concentration | % Cross-reactivity (95% CI) | | --- | --- | --- | | GFAP | | | | Desmin | 130,000 pg/mL | 0.0% (0.0%, 0.0%) | | Internexin | 80,000 pg/mL | 0.0% (0.0%, 0.0%) | | Keratin type II | 12,000 pg/mL | 0.0% (0.0%, 0.0%) | | Neurofilament light | 70 pg/mL | -0.4% (-0.5%, -0.2%) | | Neurofilament medium | 9000 pg/mL | 0.0% (0.0%, 0.0%) | | Neurofilament heavy | 80,000 pg/mL | 0.0% (0.0%, 0.0%) | | Peripherin | 6000 pg/mL | 0.0% (0.0%, 0.0%) | | Vimentin | 360,000 pg/mL | 0.0% (0.0%, 0.0%) | | UCH-L1 | | | | Ubiquitin carboxyl-terminal hydrolase L3 (UCH-L3) | 360,000 pg/mL | 0.0% (0.0%, 0.0%) | K223602 - Page 16 of 28 {16} c. Carryover / cross-contamination A study was conducted to evaluate if cross-contamination and/or carryover occurs between samples during the assay procedure by comparing the result of a protected low sample (analyte-depleted pooled plasma sample before a high sample) to that of an unprotected low sample (analyte-depleted pooled plasma sample after a high sample). The high sample was a pooled plasma sample spiked with recombinant GFAP analyte stock targeted to greater than or equal to 500,000 pg/mL and UCH-L1 analyte stock targeted to greater than or equal to 100,000 pg/mL. There were four testing steps (wash buffer – protected low sample – high sample – unprotected) in each testing iteration and the cycle of the four testing steps was repeated for a total of 27 iterations across five runs on the Alinity i system. No sample carryover was observed and the GFAP and UCH-L1 assays are not susceptible to sample carryover. 4. Assay Reportable Range: The analytical measuring internal (AMI) is 6.1–42,000 pg/mL for GFAP and 26.3–25,000 pg/mL for UCH-L1. 5. Traceability, Stability, Expected Values (Controls, Calibrators, or Methods): **Traceability** The GFAP and UCH-L1 Calibrators are manufactured gravimetrically and are referenced to an internal reference standard at each concentration level. The internal reference standards are stored frozen at ≤ -70°C. The GFAP and UCH-L1 Controls are traceable to an internal reference standard at each concentration level. **Stability** a. Sample storage stability A study was performed to evaluate serum and plasma specimens when subjected to various storage conditions (-28 to -20°C, 2 to 8°C, and 15 to 25°C) and tested with the GFAP and UCH-L1 assays. Native specimens spanning the respective AMI of each assay were collected from patients with suspected TBI in either K2 EDTA or serum separator tubes and tested in two or three replicates to determine sample storage stability. For the on-the-cell storage conditions, testing was performed directly from the blood collection tubes. For the off-the-cell storage conditions, multiple collection tubes were obtained from each subject and their respective serum or plasma was pooled prior to baseline testing. Sample storage stability was evaluated individually for the K2 EDTA or the serum separator tube type. The results support the use of the GFAP and UCH-L1 assays when testing serum and plasma specimens that have been stored at the following conditions: - room temperature (15 to 25°C): up to 8 hours on and off-the-cells - 2 to 8°C: up to 8 hours on-the-cells/clot/gel and up to 7 days off-the-cells/clot/gel K223602 - Page 17 of 28 {17} - -28 to -20°C: up to 1 month off-the-cells/clot/gel with up to 1 freeze/thaw cycle The TBI Package Insert ‘Limitations of the Procedure’ states ‘Avoid more than 1 freeze/thaw cycle’. b. On-board sample stability A study was performed to evaluate the stability of the samples when stored on the Alinity i system and tested with the GFAP and UCH-L1 assays using two levels of samples (GFAP at 25 and 10,000 pg/mL, UCH-L1 at 280 and 5000 pg/mL). Minimum sample volume was 200 µL for the GFAP assay and 150 µL for the UCH-L1 assay. Samples may be stored on the Alinity i system for up to 2 hours. c. Assay kit stability An assay kit stability study was performed using a real-time stability study design in accordance with the CLSI guideline EP25-A. Three lots of each GFAP and UCH-L1 assay kit were stored at 2 to 8°C for the evaluation of real-time stability. An additional lot of each GFAP and UCH-L1 assay kit was opened and stored in the Alinity i system for the evaluation of on-board stability. Plasma samples at three analyte levels for each GFAP (~11, ~43, and ~26,000 pg/mL) and UCH-L1 (~90, ~400, and ~15,000 pg/mL) were tested in a minimum of 10 replicates at each testing time point from the baseline time point (0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, and 13 months for real-time stability; at 0, 2, 4, 6, 8, 10, 12, and 13 months for on-board stability). The data from the stability study support expiration dating up to 12 months at 2 to 8°C and 30 days on-board. 6. Detection Limit: The Limit of Blank (LoB), Limit of Detection (LoD), and Limit of Quantitation (LoQ) studies were conducted in accordance with the CLSI guideline EP17-A2. The studies evaluated three reagents lots of the GFAP and UCH-L1 assays on each of two Alinity i systems. A description of each study and the results obtained are summarized below: The LoB was determined as the 95th percentile of measurements from 120 replicates of four analyte-depleted pooled human plasma samples that were each tested in five replicates for three days in two instruments per lot. The highest observed LoB of the three lots was 1.6 pg/mL for GFAP and 6.1 pg/mL for UCH-L1. The claimed LoB is 1.6 pg/mL for GFAP and 6.1 pg/mL for UCH-L1. For determination of LoD, five plasma samples with low GFAP concentrations ranged 1.6 to 8.8 pg/mL and six plasma samples with low UCH-L1 concentrations ranged 10.1 to 50.7 pg/mL were measured in 20 replicates in a run per day over three days in two instruments for a total of 120 replicates per lot (60 replicates per lot / instrument) for each sample. The highest observed GFAP LoD was 2.2 pg/mL of three lots. The highest observed UCH-L1 LoD was 16.1 pg/mL of three lots. The claimed LoD is 3.2 pg/mL for GFAP and 18.3 pg/mL for UCH-L1. K223602 - Page 18 of 28 {18} The LoQ was defined as the lowest concentration at which a maximum allowable precision of 20.0 %CV and determined from 120 replicates per lot (60 replicates per lot / instrument) for each low-analyte level sample. The highest observed GFAP LoQ was 2.4 pg/mL. The highest observed UCH-L1 LoQ was 16.1 pg/mL of three lots. The claimed LoQ is 6.1 pg/mL for GFAP and 26.3 pg/mL for UCH-L1. 7. Assay Cut-Off: The GFAP and UCH-L1 assay cut-offs were determined by analyzing a training dataset from a completely independent study population that is distinct from subjects evaluated in the pivotal study to validate the assay cut-offs. Frozen EDTA plasma samples from two study cohorts; ATO-04b (ClinicalTrials.gov Identifier: NCT01295346) and ATO-06x (ClinicalTrials.gov Identifier: NCT02439736) were utilized to establish the assay cut-offs. A total of 354 subjects with Glasgow Coma Scale (GCS) scores between 13–15 who had blood specimens collected within 12 hours from the time of suspected head injury, a head CT scan determination, and were 18 years or older at the time of injury were included in the training dataset. Of the 354 subjects, 37.3% (132/354) had a positive CT result. Using a 10-fold cross validation and bootstrapping method, the optimal cut-off values were selected as 35 pg/mL for the GFAP assay and 400 pg/mL for the UCH-L1 assay. B Comparison Studies: 1. Method Comparison with Predicate Device: Not applicable 2. Matrix Comparison: Sample matrix equivalence was evaluated in accordance with the CLSI guideline EP35 1st ed for samples collected with six different blood collection tube types: K2 EDTA, serum, serum separator tube, K3 EDTA, lithium heparin, and lithium heparin separator tube. Samples ranged across the AMI (45–47 contrived and 54–55 native samples per tube type for the GFAP assay; 46 contrived and 53–54 native samples per tube type for the UCH-L1 assay) and tested using one instrument and one reagent lot in duplicates for each sample. The test results of the samples from each tube type (except K2 EDTA) were compared to the test results of the samples from the primary tube type (K2 EDTA) ranged 12.8 to 41,121.0 pg/mL. A Passing-Bablok regression analysis was performed using the primary tube (K2 EDTA) first replicate concentration compared to other tube type first replicate concentration as summarized in the table below. K223602 - Page 19 of 28 {19} GFAP | Candidate Tube | N | Candidate Tube (pg/mL) | | Intercept | | Slope | | Correlation Coefficient (r) | | --- | --- | --- | --- | --- | --- | --- | --- | --- | | | | Min | Max | Estimate | 95% CI | Estimate | 95% CI | | | K3 EDTA | 102 | 11.5 | 38808.4 | 0.5 | (0.1, 1.1) | 0.96 | (0.95, 0.97) | 1.000 | | Serum | 100 | 13.2 | 44850.6 | 0.4 | (-0.3, 1.1) | 1.03 | (1.02, 1.04) | 0.999 | | Serum Separator Tube | 99 | 12.4 | 41490.4 | 0.7 | (-0.1, 1.4) | 1.02 | (1.02, 1.03) | 1.000 | | Lithium Heparin | 102 | 12.0 | 42579.4 | 0.5 | (-0.1, 1.1) | 1.01 | (1.00, 1.01) | 1.000 | | Li-Hep (Plasma Separator) | 99 | 13.2 | 40402.5 | 0.7 | (0.4, 1.5) | 0.99 | (0.99, 1.00) | 1.000 | UCH-L1 | Candidate Tube | N | Candidate Tube (pg/mL) | | Intercept | | Slope | | Correlation Coefficient (r) | | --- | --- | --- | --- | --- | --- | --- | --- | --- | | | | Min | Max | Estimate | 95% CI | Estimate | 95% CI | | | K3 EDTA | 100 | 46.5 | 18226.9 | 4.8 | (1.7, 8.5) | 0.98 | (0.97, 0.99) | 0.999 | | Serum | 100 | 46.3 | 19428.8 | 5.7 | (0.1, 10.8) | 1.06 | (1.04, 1.07) | 0.999 | | Serum Separator Tube | 99 | 57.2 | 18508.7 | 2.8 | (-3.8, 7.1) | 1.06 | (1.05, 1.07) | 0.999 | | Lithium Heparin | 100 | 44.9 | 18632.1 | 2.2 | (-3.2, 5.5) | 1.03 | (1.01, 1.04) | 0.999 | | Li-Hep (Plasma Separator) | 99 | 51.0 | 19133.1 | 2.0 | (-2.3, 4.8) | 1.02 | (1.01, 1.03) | 0.999 | ## C Clinical Studies: 1. Clinical performance with frozen plasma specimens from the ALERT-TBI study: A pivotal study using archived (frozen) plasma specimens was conducted to evaluate the clinical performance of the TBI test on the Alinity i system. The clinical specimens tested in this study were previously collected by Banyan Biomarkers from subjects participating in their study "A Prospective Clinical Evaluation of Biomarkers of Traumatic Brain Injury" (ALERT-TBI, protocol ATO-06). The ALERT-TBI study enrolled subjects at 18 years of age or older who presented to a health care facility (HCF) or emergency department (ED) with head injuries and had a blood drawn within 12 hours of head injury and a standard of care computed tomography (CT) scan of the head. Subjects were enrolled at 22 clinical sites in three countries: United States (U.S.), Germany, and Hungary. Under the ALERT-TBI study protocol, CT scans were performed in accordance with the clinical site's standard of care and a subject's CT scan was classified as positive if intracranial lesions were present. The standard of care head CT scans was previously classified as positive or negative for intracranial lesions as part of the ALERT-TBI study. K223602 - Page 20 of 28 {20} Additional details regarding the evaluation of CT scans and definition of intracranial lesion can be found in DEN170045. Of the 1994 subjects enrolled in the ALERT-TBI study with GCS scores of 13 to 15, 72 subjects (3.6%, 72/1994) were not included in the clinical performance study due to lack of consent for future testing, withdrawn consent, and no specimen available. Specimens from 23 subjects (1.2%, 23/1994) were not included in the analysis due to unreadable, inconclusive, or no CT scan results; unknown time of blood draw or blood draw more than 12 hours after injury; and/or no recorded time of injury. A total of 1899 frozen and de-identified K2 EDTA plasma samples collected from these subjects were included in the clinical performance evaluation. The plasma specimens were divided into aliquots and frozen in cryovials before being provided to three testing sites in the U.S. Each specimen was associated with a standard of care computed tomography (CT) scan of the head from the same study subject. The demographic characteristics of the subjects enrolled subjects evaluated with the TBI test are presented in the table below. | ALERT-TBI study | | | | | --- | --- | --- | --- | | Demographic Characteristics | Head CT Scan Results | | Total (N) | | | Positive | Negative | | | n (n/N %) | 120 (6.3%) | 1779 (93.7%) | 1899 | | Age (Years)a | | | | | Mean (SD) | 58.8 (18.29) | 48.5 (21.01) | 49.1 (20.99) | | Median | 58.5 | 48.0 | 49.0 | | Range (minimum, maximum) | (20, 95) | (18, 98) | (18, 98) | | Gender, n (%) | | | | | Male | 70 (58.3%) | 1003 (56.4%) | 1073 (56.5%) | | Female | 50 (41.7%) | 776 (43.6%) | 826 (43.5%) | | Ethnicity, n (%) | | | | | Hispanic or Latino | 1 (0.8%) | 89 (5.0%) | 90 (4.7%) | | Not Hispanic or Latino | 118 (98.3%) | 1689 (94.9%) | 1807 (95.2%) | | Not Reported | 1 (0.8%) | 1 (0.1%) | 2 (0.1%) | | Race, n (%) | | | | | White | 97 (80.8%) | 1237 (69.5%) | 1334 (70.2%) | | Black or African American | 16 (13.3%) | 477 (26.8%) | 493 (26.0%) | | Asian | 4 (3.3%) | 24 (1.3%) | 28 (1.5%) | | Native Hawaiian or other Pacific Islander | 0 (0.0%) | 2 (0.1%) | 2 (0.1%) | | American Indian or Alaska Native | 0 (0.0%) | 4 (0.2%) | 4 (0.2%) | | White/American Indian or Alaska Native b | 1 (0.8%) | 4 (0.2%) | 5 (0.3%) | | White/Black or African American b | 0 (0.0%) | 3 (0.2%) | 3 (0.2%) | | White/Black or African American/American Indian or Alaska Native b | 0 (0.0%) | 1 (0.1%) | 1 (0.1%) | | Asian/Native Hawaiian or other Pacific Islander b | 1 (0.8%) | 0 (0.0%) | 1 (0.1%) | | Unknown | 1 (0.8%) | 27 (1.5%) | 28 (1.5%) | a Age was calculated relative to the date of informed consent. b Subjects indicated more than 1 race. K223602 - Page 21 of 28 {21} The head injury characteristics of the subjects represented by the 1899 specimens included in the performance analysis were tabulated. Information regarding time from head injury to exam, head injury to CT scan, and head injury to blood draw, as well as GCS, neurological assessment and physical evidence of trauma, categorized by head CT scan results, are shown below. | ALERT-TBI study | | | | | --- | --- | --- | --- | | Head Injury Characteristics | Head CT Scan Result | | Total (N) | | | Positive | Negative | | | n | 120 | 1779 | 1899 | | Time from head injury to examination (hours)a | | | | | Mean (SD) | 1.9 (1.73) | 1.6 (1.71) | 1.6 (1.71) | | Median | 1.2 | 1.0 | 1.1 | | Range (minimum, maximum) | (0.3, 7.8) | (0.1, 10.7) | (0.1, 10.7) | | Time from head injury to CT scan (hours)a | | | | | Mean (SD) | 2.8 (1.95) | 2.7 (1.93) | 2.7 (1.93) | | Median | 2.1 | 2.2 | 2.1 | | Range (minimum, maximum) | (0.5, 8.9) | (0.2, 13.3) | (0.2, 13.3) | | Time from head injury to blood draw (hours)a | | | | | Mean (SD) | 3.8 (1.91) | 3.5 (1.88) | 3.5 (1.89) | | Median | 3.3 | 3.1 | 3.2 | | Range (minimum, maximum) | (0.3, 9.3) | (0.3, 11.9) | (0.3, 11.9) | | GCS score | | | | | 13 | 7 (5.8%) | 15 (0.8%) | 22 (1.2%) | | 14 | 19(15.8%) | 71 (4.0%) | 90 (4.7%) | | 15 | 94 (78.3%) | 1693 (95.2%) | 1787 (94.1%) | | Neurological assessment - Number (%) of subjects experiencing | | | | | Loss of Consciousness (LOC) | 82 (68.3%) | 720 (40.5%) | 802 (42.2%) | | Confusion | 44 (36.7%) | 312 (17.5%) | 356 (18.7%) | | Alteration of Consciousness (AOC) | 92 (76.7%) | 976 (54.9%) | 1068 (56.2%) | | Vomiting | 14 (11.7%) | 128 (7.2%) | 142 (7.5%) | | Vomiting Two or More Episodes | 10 (8.3%) | 60 (3.4%) | 70 (3.7%) | | Post Traumatic Amnesia (PTA) | 81 (67.5%) | 544 (30.6%) | 625 (32.9%) | | Post Traumatic Seizures | 2 (1.7%) | 11 (0.6%) | 13 (0.7%) | | Subjects with Drug or Alcohol Intoxication at Time of Presentation to Facility | 33 (27.5%) | 369 (20.7%) | 402 (21.2%) | | Dangerous Mechanism of Injuryb | 27 (22.5%) | 369 (20.7%) | 396 (20.9%) | | Physical Evidencec | | | | | Visible Trauma Above the Clavicle | 101 (84.2%) | 1102 (61.9%) | 1203 (63.3%) | | Suspected Open or Depressed Skull Fracture | 14 (11.7%) | 46 (2.6%) | 60 (3.2%) | | Signs of Basal Skull Fracture | 10 (8.3%) | 26 (1.5%) | 36 (1.9%) | | Presence of Neurosurgical Lesions | 5 (4.2%) | 0 (0.0%) | 5 (0.3%) | a Time since head injury calculated relative to time that the subject was first examined by medical personnel at facility. b Dangerous mechanism of injury was pedestrian struck by a motor vehicle, an occupant ejected from a motor vehicle, or a fall from an elevation of 3 or more feet or 5 stairs. Prior to head CT. K223602 - Page 22 of 28 {22} The most common head CT findings in the 120 subjects with CT-positive scans were scalp injury (96.7%), subarachnoid hemorrhage (59.2%), the presence of incidental findings (57.5%), and acute subdural hematoma (47.5%). Other frequently reported findings included cranial fractures (26.7%), parenchymal hematoma (20.0%), facial fractures (16.7%), skull based fractures (15.0%), and indeterminate extra-axial lesions (15.0%). All other findings occurred in less than 10% of CT-positive subjects. To estimate the clinical performance characteristics, the TBI test result was compared to the adjudicated head CT scan result for each subject. The performance estimates are summarized in the 2x2 table below. Of the 1899 subjects, 120 had positive CT scan results. Of these 120 subjects with positive CT scan results, 116 had a positive TBI test result (clinical sensitivity= 96.7%). The remaining four CT scan positive subjects had a negative TBI test result. The rate of false negative (FN) results was 3.3% (4/120). Five subjects in the study were identified with lesion requiring surgical intervention; none of these five subjects had a FN result, suggesting that the TBI test correctly classified all these five CT-positive subjects with a positive TBI test result. Of the 1779 subjects with negative CT scan results, 713 had a negative TBI interpretation (clinical specificity=40.1%). The rate of false positive (FP) results was 59.9% (1066/1779). Overall, there were 717 subjects with a negative TBI test result. Of these, 713 specimens were associated with negative CT scan results. The negative predictive value (NPV) of the assay was 99.4% (713/717). The potential benefit of the assay would be a reduction in unnecessary CT scans by approximately 40.1% (713 of 1779 subjects had true negative assay results). The positive predictive value (PPV) of the assay was 9.8% (116/1182). The Likelihood Ratio Negative (LRN) of the assay was 0.08. The Likelihood Ratio positive (LRP) of the assay was 1.61. | | Head CT scan result | | Total | | | --- | --- | --- | --- | --- | | | | Positive | | Negative | | TBI test result | Positive | 116 | 1066 | 1182 | | | Negative | 4 | 713 | 717 | | | Total | 120 | 1779 | 1899 | Clinical sensitivity = 96.7% (116/120); 95% CI: 91.7 - 98.7% Clinical specificity = 40.1% (713/1779); 95% CI: 37.8 - 42.4% Negative predictive value (NPV) a = 99.4% (713/717); 95% CI: 98.6 - 99.8% Positive predictive value (PPV) b = 9.8% (116/1182); 95% CI: 8.2 - 11.6% Likelihood ratio negative (LRN) = 0.08; 95% CI: 0.03 - 0.22 Likelihood ratio positive (LRP) = 1.61; 95% CI: 1.53 - 1.70 CT scan positive prevalence rate in study = 6.3% (120/1899) a Adjusted NPV for 6% CT scan positive prevalence* = 99.5%; 95% CI: 98.6 - 99.8% b Adjusted PPV for 6% CT scan positive prevalence* = 9.3%; 95% CI: 8.9 - 9.8% Analyses of assay performance by gender and time from injury relative to blood draw are shown in the table below. There was little variation in NPV and PPV between males and females and with increasing time from injury. These data indicate that gender differences and K223602 - Page 23 of 28 {23} differences between head injury characteristics did not translate into statistically significant differences in assay performance. | ALERT-TBI study | | | | | | | | | | | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | Category | Head CT scan results | | | | Sensitivity (%) (N) (95% CI) | Specificity (%) (N) (95% CI) | Adj. PPV* (%) (95% CI) | Adj. NPV* (%) (95% CI) | LRP (95% CI) | LRN (95% CI) | | | Positive | | Negative | | | | | | | | | | TBI | | TBI | | | | | | | | | | Pos | Neg | Pos | Neg | | | | | | | | All subjects N = 1899 | 116 | 4 | 1066 | 713 | 96.7 (116 /120) (91.7, 98.7) | 40.1 (713 /1779) (37.8, 42.4) | 9.3 (8.9, 9.8) | 99.5 (98.6,99.8) | 1.61 (1.53,1.70) | 0.08 (0.03, 0.22) | | Gender | | | | | | | | | | | | Male n = 1073 (56.5%) | 68 | 2 | 601 | 402 | 97.1 (68 / 70) (90.2, 99.2) | 40.1 (402 /1003) (37.1, 43.1) | 9.4 (8.8, 9.9) | 99.5 (98.2,99.9) | 1.62 (1.52,1.73) | 0.07 (0.02,0.28) | | Female n = 826 (43.5%) | 48 | 2 | 465 | 311 | 96.0 (48 / 50) (86.5, 98.9) | 40.1 (311 /776) (36.7, 43.6) | 9.3 (8.6, 10.0) | 99.4 (97.6,99.8) | 1.60 (1.48,1.74) | 0.10 (0.03,0.39) | | Time from injury to blood draw | | | | | | | | | | | | 0 - 4 hours n = 1443 (76.0%) | 84 | 2 | 823 | 534 | 97.7 (84 / 86) (91.9, 99.4) | 39.4 (534 /1357) (36.8, 42.0) | 9.3 (8.9,9.8) | 99.6 (98.5,99.9) | 1.61 (1.53,1.70) | 0.06 (0.01,0.23) | | > 4 - 8 hours n = 378 (19.9%) | 27 | 1 | 198 | 152 | 96.4 (27 / 28) (82.3, 99.4) | 43.4 (152 /350) (38.3, 48.7) | 9.8 (8.8,10.9) | 99.5 (96.5,99.9) | 1.70 (1.52,1.91) | 0.08 (0.01,0.57) | | 0 - 8 hours n = 1821 (95.9%) | 111 | 3 | 1021 | 686 | 97.4 (111 /114) (92.5, 99.1) | 40.2 (686 /1707) (37.9, 42.5) | 9.4 (9.0,9.8) | 99.6 (98.7,99.9) | 1.63 (1.55,1.71) | 0.07 (0.02,0.20) | | > 8 - 12 hours n = 78 (4.1%) | 5 | 1 | 45 | 27 | 83.3 (5 / 6) (43.6, 97.0) | 37.5 (27 / 72) (27.2, 49.0) | 7.8 (5.4,11.3) | 97.2 (85.2,99.5) | 1.33 (0.89,1.99) | 0.44 (0.07,2.73) | Since specimens from the ALERT-TBI study were archived frozen samples, a specimen stability study was conducted to demonstrate the integrity of clinical samples, as per special control b(1)(ii)(i) of 21 CFR 866.5830. The study demonstrated stability of plasma samples covering a range of GFAP and UCH-L1 analyte levels stored frozen at $-70^{\circ}\mathrm{C}$ . # 2. Clinical performance with fresh plasma samples: To supplement the results of the pivotal study (N=1899) using archived (frozen) plasma specimens from the ALERT-TBI described above, a prospective clinical validation study (Protocol No CS-2018-0009: Clinical Evaluation of the i-STAT TBI Test study) was conducted using freshly collected plasma specimens from consenting men and women 18 K223602 - Page 24 of 28 {24} years of age or older who presented to a health care facility (HCF) or emergency department (ED) with suspected mild TBI, with initial GCS scores of 13–15, and who had a CT scan of the head performed. A total of 97 subjects were enrolled across five clinical sites in the United States. Similar to the pivotal study, CT scans were performed in accordance with the clinical site’s standard of care. CT adjudication was performed by an independent central panel consisting of board-certified radiologists blinded to the TBI test results and each other’s evaluation to determine the presence or absence of an acute intracranial lesion. The intracranial lesions were defined as any trauma-induced or related finding visualized upon head CT scan, and may have included acute epidural hematomas, acute subdural hematomas, indeterminate extra-axial lesions, cortical contusions, parenchymal hematomas, non-hemorrhagic contusions, ventricle compression, ventricular trapping, brain herniation, intraventricular hemorrhage, hydrocephalus, subarachnoid hemorrhage, petechial hemorrhage, global or focal brain edema and post traumatic ischemia. A head CT scan result of a subject was classified as positive if intracranial lesions were present. Whole blood was collected into K2 EDTA blood collection tubes from each subject using venipuncture and centrifuged to obtain plasma. Specimens were collected within 12 hours of head injury. The demographic characteristics of the subjects are presented in the table below: | Supplemental fresh plasma specimen study | | | | | --- | --- | --- | --- | | Demographic Characteristics | Head CT Scan Result | | Total (N) | | | Positive | Negative | | | n (n/N %) | 14 (14.4%) | 83 (85.6%) | 97 | | Age (Years) | | | | | Mean (SD) | 41.5 (20.33) | 48.4 (20.24) | 47.4 (20.29) | | Median | 33.5 | 49.0 | 43.0 | | Range (minimum, maximum) | (19.0, 73.0) | (18.0, 85.0) | (18.0, 85.0) | | Gender, n (%) | | | | | Male | 10 (71.4%) | 51 (61.4%) | 61 (62.9%) | | Female | 4 (28.6%) | 32 (38.6%) | 36 (37.1%) | | Ethnicity, n (%) | | | | | Hispanic or Latino | 4 (28.6%) | 22 (26.5%) | 26 (26.8%) | | Not Hispanic or Latino | 10 (71.4%) | 60 (72.3%) | 70 (72.2%) | | Race, n (%) | | | | | White | 13 (92.9%) | 56 (67.5%) | 69 (71.1%) | | Black or African American | 0 (0.0%) | 12 (14.5%) | 12 (12.4%) | | Asian | 1 (7.1%) | 7 (8.4%) | 8 (8.2%) | | Native Hawaiian or Other Pacific Islander | 0 (0.0%) | 1 (1.2%) | 1 (1.0%) | The head injury characteristics of the subjects in the supplemental fresh plasma specimen study including information regarding time from head injury to CT scan and head injury to blood draw, as well as GCS, neurological assessment, physical evidence of trauma, and mechanism of injury, categorized by head CT scan results, are shown below. K223602 - Page 25 of 28 {25} K223602 - Page 26 of 28 | Supplemental fresh plasma specimen study | | | | | --- | --- | --- | --- | | Head Injury Characteristics | Head CT Scan Result | | Total (N) | | | Positive | Negative | | | n | 14 | 83 | 97 | | Time from head injury to CT scan (hours) | | | | | Mean (SD) | 2.2 (1.47) | 3.1 (1.76) | 3.0 (1.74) | | Median | 1.8 | 2.7 | 2.6 | | Range (minimum, maximum) | (0.8, 6.5) | (0.8, 9.9) | (0.8, 9.9) | | Time from head injury to blood draw (hours) | | | | | Mean (SD) | 8.4 (2.90) | 5.9 (2.64) | 6.3 (2.81) | | Median | 8.6 | 5.2 | 5.4 | | Range (minimum, maximum) | (4.1, 11.8) | (1.4, 12.0) | (1.4, 12.0) | | GCS score – Number (%) | | | | | 13 | 1 (7.1%) | 0 (0.0%) | 1 (1.0%) | | 14 | 3 (21.4%) | 4 (4.8%) | 7 (7.2%) | | 15 | 10 (71.4%) | 79 (95.2%) | 89 (91.8%) | | Neurological Assessment – Number (%) of subjects experiencing: | | | | | Loss of Consciousness (LOC) | 10 (71.4%) | 31 (37.3%) | 41 (42.3%) | | Alteration of Consciousness (AOC) | 11 (78.6%) | 51 (61.4%) | 62 (63.9%) | | Vomiting | 3 (21.4%) | 3 (3.6%) | 6 (6.2%) | | Post Traumatic Amnesia (PTA) | 10 (71.4%) | 35 (42.2%) | 45 (46.4%) | | Subjects with Drug in System at the Time of Presentation to Facility | 5 (35.7%) | 8 (9.6%) | 13 (13.4%) | | Subjects with Alcohol in System at the Time of Presentation to Facility | 7 (50.0%) | 6 (7.2%) | 13 (13.4%) | | Physical Evidence – n (%) | | | | | Subdural Hematoma | 10 (71.4%) | 0 (0.0%) | 10 (10.3%) | | Subarachnoid Hemorrhage | 10 (71.4%) | 1 (1.2%) | 11 (11.3%) | | Acute Skull Fracture | 8 (57.1%) | 1 (1.2%) | 9 (9.3%) | | Contusion | 6 (42.9%) | 0 (0.0%) | 6 (6.2%) | | Intracerebral Hemorrhage | 1 (7.1%) | 0 (0.0%) | 1 (1.0%) | | Epidural Hematoma | 1 (7.1%) | 0 (0.0%) | 1 (1.0%) | | Traumatic Axonal Injury | 1 (7.1%) | 0 (0.0%) | 1 (1.0%) | | Midline Shift Supratentorial | 2 (14.3%) | 0 (0.0%) | 2 (2.1%) | | Cisternal Compression | 2 (14.3%) | 0 (0.0%) | 2 (2.1%) | | Edema | 1 (7.1%) | 0 (0.0%) | 1 (1.0%) | | Brain Atrophy or Encephalomalacia | 0 (0.0%) | 2 (2.4%) | 2 (2.1%) | | Brain Swelling | 2 (14.3%) | 0 (0.0%) | 2 (2.1%) | | Visible Trauma Above the Clavicle | 13 (92.9%) | 55 (66.3%) | 68 (70.1%) | | Signs of Basal Skull Fracture | 2 (14.3%) | 0 (0.0%) | 2 (2.1%) | | Mechanism of Injury – n (%) | | | | | Acceleration/Deceleration | 2 (14.3) | 26 (31.3) | 28 (28.9) | | Direct impact: Blow to Head | 4 (28.6) | 13 (15.7) | 17 (17.5) | {26} | Supplemental fresh plasma specimen study | | | | | --- | --- | --- | --- | | Head Injury Characteristics | Head CT Scan Result | | Total (N) | | | Positive | Negative | | | Direct impact: Head Against Object | 11 (78.6) | 54 (65.1) | 65 (67.0) | | Fall from height > 1 meter (3 ft) | 2 (14.3) | 4 (4.8) | 6 (6.2) | | Ground level fall | 4 (28.6) | 26 (31.3) | 30 (30.9) | To estimate clinical performance characteristics, the TBI test results from the supplemental plasma fresh specimen study were compared to the consensus head CT scan results. The results are summarized in the table below. | | Head CT scan result | | Total | | | --- | --- | --- | --- | --- | | | | Positive | | Negative | | TBI test Result | Positive | 14 | 60 | 74 | | | Negative | 0 | 23 | 23 | | | Total | 14 | 83 | 97 | Clinical sensitivity $(\%) = 100.0$ (14/14); $95\%$ CI: 78.5 -100.0% Clinical specificity $(\%) = 27.7$ (23/83); $95\%$ CI: 19.2 - 38.2% Negative predictive value (NPV) $(\%)^{\mathrm{a}} = 100.0$ (23/23); $95\%$ CI: 85.7-100.0% Positive predictive value (PPV) $(\%)^{\mathrm{a}} = 18.9$ (14/74); $95\%$ CI: 11.6 - 29.3% Likelihood Ratio Negative (LRN) = 0.12; 95% CI: 0.01 - 1.91 Likelihood Ratio Positive (LRP) = 1.38; 95% CI: 1.21 - 1.58 a NPV and PPV are estimated at $14.43\%$ prevalence of CT scan positive rate for suspected mild TBI subjects in the supplemental fresh plasma specimen study cohort. If NPV and PPV are adjusted to $6\%$ prevalence rate (comparable to the pivotal study cohort), $\mathrm{NPV} = 99.2\%$ (95% CI: 89.1-99.9%), and $\mathrm{PPV} = 8.1$ (95% CI: 7.2-9.1%) The results summarized in the table above show that the TBI test, when evaluated with fresh plasma samples, is characterized by high sensitivity and NPV supportive of its clinical utility as an aid in the evaluation of the need for a CT scan in subjects presenting with a GCS score of 13 to 15 and a negative TBI test. ## D Clinical Cut-Off: Refer to Assay Cut-off ## E Expected Values/Reference Range: A study was performed in accordance with the CLSI guideline C28-A3c to establish the expected values from 160 apparently healthy individuals (≥ 18 years old) in the U.S. population ranging in age from 18 to 91 years (mean 49.2 years) who did not have acute injury to the head (or history of neurological disease or disorder, neurosurgery, motor vehicle accident or injury requiring K223602 - Page 27 of 28 {27} medical attention for head/neck/spine within the last one year, or active cancer / diagnosis within the last 5 years. Frozen K2 EDTA plasma specimens stored for up to 5 months at -70°C or colder were tested with both the GFAP and UCH-L1 assays on the Alinity i system. The 2.5th and 97.5th percentiles (95% reference interval) and their respective 95% confidence intervals for GFAP and UCH-L1 were determined non-parametrically and presented in the table below: | Analyte | n | Mean (pg/mL) | SD | Median (pg/mL) | Reference Interval (2.5th to 97.5th percentile) (pg/mL) | | --- | --- | --- | --- | --- | --- | | GFAP | 160 | 23.5 | 13.79 | 20.5 | 6.6, 70.9 | | UCH-L1 | 160 | 108.1 | 45.28 | 98.0 | 44.7, 226.8 | There were 21 healthy donors who tested positive for GFAP only and no donors were positive for both GFAP and UCH-L1. The results summarized in the table below show that 86.9% have a negative TBI test result and 16% have a positive TBI test result. | GFAP Result (Relative to Cutoff of 35.0 pg/mL)^{a} | UCH-L1 Result (Relative to Cutoff of 400.0 pg/mL)^{a} | TBI test Interpretation Result | N (Percentage) | | --- | --- | --- | --- | | Above | Above | Positive | 0 (0/160 = 0.0%) | | Below | Above | Positive | 0 (0/160 = 0.0%) | | Above | Below | Positive | 21 (21/160 = 13.1%)^{b} | | Below | Below | Negative | 139 (139/160 = 86.9%) | a Above means greater than or equal to the cut-off. Below means less than the cut-off. b Although 13.1% of an apparently healthy population was found to have a positive TBI interpretation, it is important to note GFAP and UCH-L1 cut-offs were optimized in a population of patients with head injury. It is the responsibility of each laboratory to establish its own reference ranges for the population of patients it serves, as expected values may be affected by different factors including age. ## VIII Proposed Labeling: The labeling supports the finding of substantial equivalence for this device. ## IX Conclusion: The submitted information in this premarket notification is complete and supports a substantial equivalence decision. K223602 - Page 28 of 28