(89 days)
The i-STAT TBI test is a panel of in vitro diagnostic immunoassays for the quantitative measurements of glial fibrillary acidic protein (GFAP) and ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) in whole blood and a semi-quantitative interpretation of test results derived from these measurements, using the i-STAT Alinity instrument. 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), which may include one of the following four clinical criteria: 1) any period of loss of consciousness, 2) any loss of memory for events immediately before and after the accident, 3) any alteration in mental state at the time of accident, and/or 4) focal neurological deficits, within 24 hours of injury, to assist in determining the need for a CT (computed tomography) scan of the head. A 'Not Elevated' test interpretation is associated with the absence of acute traumatic intracranial lesions visualized on a head CT scan.
The test is to be used with venous whole blood collected with EDTA anticoagulant in point of care or clinical laboratory settings by a healthcare professional.
The i-STAT TBI cartridge is a multiplex immunoassay that contains assays for both ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) and glial fibrillary acidic protein (GFAP). The assays test for the presence of these biomarkers in a whole blood sample and vield a semi-quantitative test interpretation based on measurements of both UCH-L1 and GFAP in approximately 15 minutes. The i-STAT TBI cartridge is designed to be run only on the i-STAT Alinity instrument.
The i-STAT Alinity instrument is a handheld, in vitro diagnostic device. The instrument is the main user interface of the i-STAT Alinity System and functions as the electro-mechanical interface to the test cartridge. The instrument executes the test cycle, acquires and processes the electrical sensor signals converting the signals into quantitative results. These functions are controlled by a microprocessor.
The i-STAT Alinity System is comprised of the i-STAT Alinity instrument, the i-STAT test cartridges and accessories (i-STAT Alinity Base Station, Electronic Simulator and Printer).
Assaved quality control materials are also available for use with the i-STAT TBI cartridge and include i-STAT TBI Control Level 1, i-STAT TBI Control Level 2, and the i-STAT TBI Calibration Verification Levels 1-3.
The i-STAT TBI Controls are available to monitor the performance of glial fibrillary acidic protein (GFAP) and ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) assays on the i-STAT Alinity instrument.
The i-STAT TBI Calibration Verification Materials are available to verify the calibration of glial fibrillary acidic protein (GFAP) and ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) assays throughout the reportable range on the i-STAT Alinity instrument.
The provided text describes the analytical and clinical performance of the i-STAT TBI cartridge with the i-STAT Alinity System, which measures GFAP and UCH-L1 to aid in the evaluation of patients with suspected mild traumatic brain injury (TBI). The information is presented to support a 510(k) premarket notification for substantial equivalence to a predicate device.
Here's an analysis of the acceptance criteria and the study proving the device meets them, based on the provided document:
1. A Table of Acceptance Criteria (Implied) and Reported Device Performance
The document does not explicitly present a "table of acceptance criteria" with predefined thresholds. Instead, it describes performance characteristics that are presumably deemed acceptable for demonstrating substantial equivalence. The core clinical performance criterion for this device, a TBI assessment test, is its ability to correctly identify patients not needing a head CT scan, which translates to high sensitivity and negative predictive value (NPV) for the absence of acute intracranial lesions.
Here's a summary of the reported core performance:
| Performance Metric | Reported Device Performance (i-STAT TBI cartridge with i-STAT Alinity System) |
|---|---|
| Clinical Sensitivity (for acute traumatic intracranial lesions) | 96.5% (273/283) [95% CI: 93.6%, 98.1%] |
| Clinical Specificity (for absence of acute traumatic intracranial lesions) | 40.3% (277/687) [95% CI: 36.7%, 44.0%] |
| Negative Predictive Value (NPV) | 96.5% (277/287) [95% CI: 93.7%, 98.1%] |
| Adjusted NPV at 6% prevalence | 99.4% [95% CI: 99.0%, 99.7%] |
| Positive Predictive Value (PPV) | 40.0% (273/683) [95% CI: 38.4%, 41.5%] |
| False Negative Rate | 3.5% (10/283) |
Key Implied Acceptance Criteria based on Regulatory Context:
- High Clinical Sensitivity: The device must reliably identify patients with acute intracranial lesions, minimizing false negatives to ensure patient safety and avoid missing critical injuries. A 96.5% sensitivity is presented as acceptable.
- High Negative Predictive Value (NPV): Crucially, the device's main utility is to aid in determining the need for a CT scan. A high NPV means that a "Not Elevated" result reliably indicates the absence of acute traumatic intracranial lesions. The 96.5% NPV (and higher adjusted NPV) supports this.
- Acceptable False Negative Rate: The reported 3.5% false negative rate, with the additional detail that "None of these ten (10) subjects with false negative results required surgical intervention related to their head injury as no neurosurgical lesions were identified by CT scan in these subjects," addresses a critical safety aspect.
- Analytical Performance: The document provides extensive data on analytical precision (semi-quantitative and qualitative, 20-day and multi-site), linearity, hook effect, traceability, reference interval, detection limit, analytical specificity (interference, cross-reactivity, cross-talk), and hematocrit sensitivity. These are all standard analytical performance characteristics that would need to meet predefined criteria (often internal to the manufacturer or based on regulatory guidance) to ensure the assay's reliability and robustness. While specific numerical acceptance criteria for each are not stated (e.g., "CV must be <X%"), the presentation of the data implies these were within acceptable ranges.
2. Sample Size Used for the Test Set and Data Provenance
- Sample Size for Clinical Performance (Test Set): 970 subjects were included in the clinical performance analysis.
- Data Provenance: The data was collected from a prospective, multi-center, observational study conducted at 20 external point-of-care clinical sites across the United States. The study was designed to evaluate the clinical performance in classifying the intended use population.
3. Number of Experts Used to Establish the Ground Truth for the Test Set and Qualifications of Those Experts
- Number of Experts: At least two neuroradiologists interpreted the CT images for ground truth establishment.
- Qualifications of Experts: The experts were "neuroradiologists." No specific details on their years of experience or board certifications are provided in this summary, but being neuroradiologists implies specialized training in interpreting neurological imaging.
4. Adjudication Method for the Test Set
- Adjudication Method: The clinical outcome (ground truth) was based on the "consensus interpretation between two neurologists" (which appears to be a slight inconsistency as the previous sentence referred to neuroradiologists, but the intent is clear: expert consensus). This implies a method where if the initial two readers disagreed, they would discuss and reach a consensus. If they couldn't reach consensus, a third reader might be involved, though the document only explicitly mentions "consensus interpretation between two neurologists." "Procedures for scoring images were established before conducting image review," suggesting a structured approach to adjudication.
5. If a Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study was Done
- No, a Multi-Reader Multi-Case (MRMC) comparative effectiveness study comparing human readers with and without AI assistance was not reported. This device is an in-vitro diagnostic (IVD) blood test, not an imaging AI algorithm designed to assist human readers in interpreting images. Its function is to provide a biomarker-based "Elevated" or "Not Elevated" interpretation to aid in clinical decision-making regarding CT scans, rather than augment human image interpretation.
6. If a Standalone (Algorithm Only Without Human-in-the-Loop Performance) Was Done
- Yes, the clinical performance study evaluated the device in a standalone (algorithm only) manner. The i-STAT TBI cartridge provides a "semi-quantitative interpretation of test results" (Elevated/Not Elevated) based on measurements of GFAP and UCH-L1. This interpretation is then compared directly to the CT scan ground truth. While it's used in conjunction with other clinical information by healthcare professionals, the reported sensitivity, specificity, and NPV are measures of the device's inherent performance.
7. The Type of Ground Truth Used
- The ground truth used was based on the presence or absence of acute traumatic intracranial lesions visualized on a head CT scan, as determined by consensus interpretation of at least two neuroradiologists/neurologists. "Acute intracranial lesion was defined as any trauma induced or related finding visualized upon head CT scan." This is a form of imaging-based ground truth, established by expert consensus.
8. The Sample Size for the Training Set
- The training set for determining the assay cut-offs for GFAP and UCH-L1 involved 420 subjects (274 males and 146 females) with suspected mild traumatic brain injury (GCS 13-15).
9. How the Ground Truth for the Training Set Was Established
- The ground truth for the training set used to establish the cut-off values was based on CT scan determinations. "Subjects who had blood drawn within 12 hours of injury and a head CT scan determination, were included in the analysis." The process involved "Using a 10-fold cross validation and bootstrapping method" to select the cut-off values of 65 pg/mL (GFAP) and 360 pg/mL (UCH-L1), using a selection criteria of "adjusted NPV (to 10%) ≥98.5% and sensitivity ≥97%". This indicates the CT scan results served as the ground truth for optimizing these cut-off values during model development.
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Image /page/0/Picture/0 description: The image contains the logo of the U.S. Food and Drug Administration (FDA). On the left is the Department of Health & Human Services logo. To the right of that is the FDA logo, which is a blue square with the letters "FDA" in white. To the right of the blue square is the text "U.S. FOOD & DRUG ADMINISTRATION" in blue.
March 27, 2024
Abbott Point of Care Brian Ma Principal Specialist. Regulatory Affairs 400 College Road East Princeton, New Jersey 08540
Re: K234143
Trade/Device Name: i-STAT TBI Cartridge with the i-STAT Alinity System Regulation Number: 21 CFR 866.5830 Regulation Name: Brain Trauma Assessment Test Regulatory Class: Class II Product Code: QAT Dated: December 28, 2023 Received: December 29, 2023
Dear Brian Ma:
We have reviewed your section 510(k) premarket notification of intent to market the device referenced above and have determined the device is substantially equivalent (for the indications for use stated in the enclosure) to legally marketed predicate devices marketed in interstate commerce prior to May 28, 1976, the enactment date of the Medical Device Amendments, or to devices that have been reclassified in accordance with the provisions of the Federal Food, Drug, and Cosmetic Act (the Act) that do not require approval of a premarket approval application (PMA). You may, therefore, market the device, subject to the general controls provisions of the Act. Although this letter refers to your product as a device, please be aware that some cleared products may instead be combination products. The 510(k) Premarket Notification Database available at https://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfpmn/pmn.cfm identifies combination product submissions. The general controls provisions of the Act include requirements for annual registration, listing of devices, good manufacturing practice, labeling, and prohibitions against misbranding and adulteration. Please note: CDRH does not evaluate information related to contract liability warranties. We remind you, however, that device labeling must be truthful and not misleading.
If your device is classified (see above) into either class II (Special Controls) or class III (PMA), it may be subject to additional controls. Existing major regulations affecting your device can be found in the Code of Federal Regulations, Title 21, Parts 800 to 898. In addition, FDA may publish further announcements concerning your device in the Federal Register.
Additional information about changes that may require a new premarket notification are provided in the FDA guidance documents entitled "Deciding When to Submit a 510(k) for a Change to an Existing Device" (https://www.fda.gov/media/99812/download) and "Deciding When to Submit a 510(k) for a Software Change to an Existing Device" (https://www.fda.gov/media/99785/download).
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Your device is also subject to, among other requirements, the Quality System (QS) regulation (21 CFR Part 820), which includes, but is not limited to, 21 CFR 820.30, Design controls; 21 CFR 820.90, Nonconforming product; and 21 CFR 820.100, Corrective and preventive action. Please note that regardless of whether a change requires premarket review. the OS regulation requires device manufacturers to review and approve changes to device design and production (21 CFR 820.30 and 21 CFR 820.70) and document changes and approvals in the device master record (21 CFR 820.181).
Please be advised that FDA's issuance of a substantial equivalence determination does not mean that FDA has made a determination that your device complies with other requirements of the Act or any Federal statutes and regulations administered by other Federal agencies. You must comply with all the Act's requirements, including, but not limited to: registration and listing (21 CFR Part 807); labeling (21 CFR Part 801 and Part 809); medical device reporting of medical device-related adverse events) (21 CFR Part 803) for devices or postmarketing safety reporting (21 CFR Part 4, Subpart B) for combination products (see https://www.fda.gov/combination-products/guidance-regulatory-information/postmarketing-safetyreporting-combination-products); good manufacturing practice requirements as set forth in the quality systems (OS) regulation (21 CFR Part 820) for devices or current good manufacturing practices (21 CFR Part 4, Subpart A) for combination products; and, if applicable, the electronic product radiation control provisions (Sections 531-542 of the Act); 21 CFR Parts 1000-1050.
Also, please note the regulation entitled, "Misbranding by reference to premarket notification" (21 CFR 807.97). For questions regarding the reporting of adverse events under the MDR regulation (21 CFR Part 803), please go to https://www.fda.gov/medical-device-safety/medical-device-reportingmdr-how-report-medical-device-problems.
For comprehensive regulatory information about mediation-emitting products, including information about labeling regulations, please see Device Advice (https://www.fda.gov/medicaldevices/device-advice-comprehensive-regulatory-assistance) and CDRH Learn (https://www.fda.gov/training-and-continuing-education/cdrh-learn). Additionally, you may contact the Division of Industry and Consumer Education (DICE) to ask a question about a specific regulatory topic. See the DICE website (https://www.fda.gov/medical-device-advice-comprehensive-regulatoryassistance/contact-us-division-industry-and-consumer-education-dice) for more information or contact DICE by email (DICE@fda.hhs.gov) or phone (1-800-638-2041 or 301-796-7100).
Sincerelv.
Ying Mao -S
Ying (Katelin) Mao, Ph.D. Branch Chief Division of Immunology and Hematology Devices OHT7: Office of In Vitro Diagnostics Office of Product Evaluation and Quality Center for Devices and Radiological Health
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Indications for Use
Submission Number (if known)
K234143 Device Name
i-STAT TBI cartridge with the i-STAT Alinity System
Indications for Use (Describe)
The i-STAT TBI test is a panel of in vitro diagnostic immunoassays for the quantitative measurements of glial fibrillary acidic protein (GFAP) and ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) in whole blood and a semi-quantitative interpretation of test results derived from these measurements, using the i-STAT Alinity instrument. 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), which may include one of the following four clinical criteria: 1) any period of loss of consciousness, 2) any loss of memory for events immediately before and after the accident, 3) any alteration in mental state at the time of accident, and/or 4) focal neurological deficits, within 24 hours of injury, to assist in determining the need for a CT (computed tomography) scan of the head. A 'Not Elevated' test interpretation is associated with the absence of acute traumatic intracranial lesions visualized on a head CT scan.
The test is to be used with venous whole blood collected with EDTA anticoagulant in point of care or clinical laboratory settings by a healthcare professional.
Type of Use (Select one or both, as applicable)
Prescription Use (Part 21 CFR 801 Subpart D)
Over-The-Counter Use (21 CFR 801 Subpart C)
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Image /page/3/Picture/0 description: The image shows the Abbott logo. The logo consists of a stylized letter "a" in blue, followed by the word "Abbott" in black, bold font. The letter "a" is made up of three horizontal lines connected by a vertical line on the right side, forming a square shape with an open left side.
510(k) Summary
The information in this 510(k) summary is being submitted in accordance with the requirements of 21 CFR 807.92.
1. Submitter Information
| Owner | Abbott Point of Care Inc.400 College Road EastPrinceton, NJ 08540 |
|---|---|
| Contact | Primary: Brian Ma, PhDPrincipal Specialist Regulatory AffairsPhone: 613-688-5949Secondary: Mojgan SoleimaniAssociate Director, Regulatory AffairsPhone: 613-295-0932 |
| Date Prepared | March 25, 2024 |
2. Device Information
Proprietary Name: i-STAT TBI cartridge with the i-STAT Alinity System
Glial fibrillary acidic protein (GFAP) Common Name: Ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1)
K234143 510(k) Number
| ProductCode | Device Classification Name | RegulationNumber | Class | Panel |
|---|---|---|---|---|
| QAT | Brain trauma assessment test | 866.5830 | II (specialcontrols) | Immunology |
Predicate Device 3.
i-STAT TBI Plasma cartridge with the i-STAT Alinity System Proprietary Name 510(k) Number K201778
| ProductCode | Device Classification Name | RegulationNumber | Class | Panel |
|---|---|---|---|---|
| QAT | Brain trauma assessment test | 866.5830 | II (specialcontrols) | Immunology |
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4. Device Description
The i-STAT TBI cartridge is a multiplex immunoassay that contains assays for both ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) and glial fibrillary acidic protein (GFAP). The assays test for the presence of these biomarkers in a whole blood sample and vield a semi-quantitative test interpretation based on measurements of both UCH-L1 and GFAP in approximately 15 minutes. The i-STAT TBI cartridge is designed to be run only on the i-STAT Alinity instrument.
The i-STAT Alinity instrument is a handheld, in vitro diagnostic device. The instrument is the main user interface of the i-STAT Alinity System and functions as the electro-mechanical interface to the test cartridge. The instrument executes the test cycle, acquires and processes the electrical sensor signals converting the signals into quantitative results. These functions are controlled by a microprocessor.
The i-STAT Alinity System is comprised of the i-STAT Alinity instrument, the i-STAT test cartridges and accessories (i-STAT Alinity Base Station, Electronic Simulator and Printer).
Assaved quality control materials are also available for use with the i-STAT TBI cartridge and include i-STAT TBI Control Level 1, i-STAT TBI Control Level 2, and the i-STAT TBI Calibration Verification Levels 1-3.
The i-STAT TBI Controls are available to monitor the performance of glial fibrillary acidic protein (GFAP) and ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) assays on the i-STAT Alinity instrument.
The i-STAT TBI Calibration Verification Materials are available to verify the calibration of glial fibrillary acidic protein (GFAP) and ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) assays throughout the reportable range on the i-STAT Alinity instrument.
5. Intended Use Statement
The i-STAT TBI test is a panel of in vitro diagnostic immunoassays for the quantitative measurements of glial fibrillary acidic protein (GFAP) and ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) in whole blood and a semi-quantitative interpretation of test results derived from these measurements, using the i-STAT Alinity instrument. 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), which may include one of the following four clinical criteria: 1) any period of loss of consciousness, 2) any loss of memory for events immediately before and after the accident. 3) any alteration in mental state at the time of accident. and/or 4) focal neurological deficits, within 24 hours of injury, to assist in determining the need for a CT (computed tomography) scan of the head. A "Not Elevated" TBI test interpretation is associated with the absence of acute traumatic intracranial lesions visualized on a head CT scan.
The test is to be used with venous whole blood collected with EDTA anticoagulant in point of care or clinical laboratory settings by a healthcare professional.
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| Similarities and Differences: System (Test and Instrument) | ||
|---|---|---|
| Feature orCharacteristic | Candidate Device:i-STAT TBI cartridge with the i-STATAlinity System | Predicate Device:i-STAT TBI Plasma cartridge with thei-STAT Alinity System(K201778) |
| Intended Use | The i-STAT TBI test is a panel of in vitrodiagnostic immunoassays for thequantitative measurements of glialfibrillary acidic protein (GFAP) andubiquitin carboxyl-terminal hydrolaseL1 (UCH-L1) in whole blood and asemi-quantitative interpretation oftest results derived from thesemeasurements, using the i-STAT Alinityinstrument. The interpretation of testresults is used, in conjunction withother clinical information, to aid in theevaluation of patients, 18 years of ageor older, presenting with suspectedmild traumatic brain injury (GlasgowComa Scale score 13-15), which mayinclude one of the following fourclinical criteria: 1) any period of loss ofconsciousness, 2) any loss of memoryfor events immediately before andafter the accident, 3) any alteration inmental state at the time of accident,and/or 4) focal neurological deficits,within 24 hours of injury, to assist indetermining the need for a CT(computed tomography) scan of thehead. A 'Not Elevated' testinterpretation is associated with theabsence of acute traumatic intracraniallesions visualized on a head CT scan.The test is to be used with venouswhole blood collected with EDTAanticoagulant in point of care orclinical laboratory settings by ahealthcare professional. | The i-STAT TBI Plasma test is apanel of in vitro diagnosticimmunoassays for the quantitativemeasurements of glial fibrillary acidicprotein (GFAP) and ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) inplasma and a semi- quantitativeinterpretation of test results derivedfrom these measurements, using thei-STAT Alinity Instrument. Theinterpretation of test results is used,in conjunction with other clinicalinformation, to aid in the evaluationof patients, 18 years of age or older,presenting with suspected mildtraumatic brain injury (Glasgow ComaScale score 13-15) within 12 hours ofinjury, to assist in determining theneed for a CT (computed tomography)scan of the head. A 'Not Elevated' testinterpretation is associated with theabsence of acute traumaticintracranial lesions visualized on ahead CT scan.The test is to be used with plasmaprepared from EDTA anticoagulatedspecimens in clinical laboratorysettings by a healthcare professional.The i-STAT TBI Plasma test is notintended to be used in point of caresettings. |
| Similarities and Differences: System (Test and Instrument) | ||
| Feature orCharacteristic | Candidate Device:i-STAT TBI cartridge with the i-STATAlinity System | Predicate Device:i-STAT TBI Plasma cartridge with thei-STAT Alinity System(K201778) |
| Intended UseSetting | Clinical Laboratory and Point of Care | Clinical Laboratory |
| Measurands | GFAP and UCH-L1 | Same |
| AssayTechnology | Enzyme-linked immunosorbent assay | Same |
| Assay Format | Single use multiplex cartridge (bothassays (GFAP and UCH-L1) in onecartridge) | Same |
| DetectionTechnology | Electrochemical | Same |
| Sample Type | Whole Blood | Plasma |
| Sample Volume | 20 μL | Same |
| Automation | Test and wash cycles are fullyautomated after sample loading step | Same |
| AnalyticalMeasuringInterval | GFAP: 47 - 10,000 pg/mLUCH-L1: 87 - 3,200 pg/mL | GFAP: 30 - 10,000 pg/mLUCH-L1: 200 - 3,200 pg/mL |
| Time to Result | 15 minutes | Same |
| ReportableResult | Quantitative results for GFAP andUCH-L1 and semi-quantitativeinterpretation | Same |
| InstrumentPlatform | i-STAT Alinity | Same |
| Calibration | No calibration needed by the enduser, calibration is pre-set duringmanufacture of the cartridge | Same |
| Controls | GFAP and UCH-L1 combined:i-STAT TBI Controls (Levels 1 and 2)i-STAT TBI Calibration VerificationMaterials (Levels 1, 2, 3) | Same |
| Traceability | GFAP and UCH-L1 values assigned toi-STAT controls and calibrationverification materials are traceable toAbbott's working calibrators preparedusing recombinant GFAP and UCH-L1(expressed and purified from E. coli). | Same |
| Assay Cut-off | GFAP: 65 pg/mlUCH-L1: 360 pg/ml | GFAP: 30 pg/mlUCH-L1: 360 pg/ml |
6. Summary Comparison of Technological Characteristics
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Performance Characteristics 7.
A. Analytical Performance
a. Precision/Reproducibility:
Semi-quantitative 20-day precision: The precision of the GFAP and UCH-L1 assays in the i-STAT TBI cartridge with the i-STAT Alinity System was evaluated using plasma samples spiked with native or recombinant GFAP and UCH-L1 antigens at various levels across the reportable range of the GFAP and UCH-L1 assays, and two (2) controls (i- STAT TBI Control L1 and Control L2). The study was executed over 20 non-consecutive days, two (2) runs per day that were separated by minimum two (2) hours, by at least two (2) operators using three (3) lots of i-STAT TBI cartridges. Due to the inability to store or freeze whole blood samples to maintain sample stability over multiple days, plasma samples were used for this study. The study followed the standard single-site 20x2x2 experimental design based on guidance provided in CLSI EP05-A3; Evaluation of Precision of Quantitative Measurement Procedures; Approved Guideline -Third Edition. The components of variability were estimated for GFAP and UCH-L1 and the precision results for the plasma panel are shown in Table 2, and for the i-STAT TBI Controls in Table 3.
| Table 1: Estimate of GFAP Assay Precision | ||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Repeatability | Between-run | Between-day | Between-lot | Within-Laboratory | ||||||||
| Plasma | N | Mean | SD | CV | SD | CV | SD | CV | SD | CV | SD | CV |
| Sample | (pg/mL) | (pg/mL) | (%) | (pg/mL) | (%) | (pg/mL) | (%) | (pg/mL) | (%) | (pg/mL) | (%) | |
| 1B | 240 | 78.8 | 3.04 | 3.86 | 0.85 | 1.07 | 0.57 | 0.72 | 2.17 | 2.76 | 3.90 | 4.95 |
| 2B | 240 | 98.6 | 6.03 | 6.12 | 1.40 | 1.42 | 0.72 | 0.73 | 2.57 | 2.61 | 6.78 | 6.87 |
| 3A | 240 | 880.6 | 21.29 | 2.42 | 15.78 | 1.79 | 1.66 | 0.19 | 9.76 | 1.11 | 28.79 | 3.27 |
| 4A | 240 | 4415.3 | 144.73 | 3.28 | 67.27 | 1.52 | 17.25 | 0.39 | 135.59 | 3.07 | 212.16 | 4.81 |
| 5A | 240 | 8346.7 | 285.03 | 3.41 | 151.07 | 1.81 | 56.69 | 0.68 | 347.63 | 4.16 | 479.49 | 5.74 |
్ Pooled plasma from normal donors spiked with <5% v/v recombinant GFAP antigen
8 Pooled plasma from normal donors spiked with <5% v/v GFAP from pooled TBI patient plasma
| Table 2: Estimate of UCH-L1 Assay Precision | ||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Repeatability | Between-run | Between-day | Between-lot | Within-Laboratory | ||||||||
| PlasmaSample | N | Mean(pg/mL) | SD(pg/mL) | CV(%) | SD(pg/mL) | CV(%) | SD(pg/mL) | CV(%) | SD(pg/mL) | CV(%) | SD(pg/mL) | CV(%) |
| 1A | 240 | 159.9 | 11.91 | 7.45 | 3.44 | 2.15 | 0.76 | 0.48 | 4.92 | 3.08 | 13.54 | 8.47 |
| 2A | 240 | 255.7 | 18.11 | 7.08 | 4.97 | 1.94 | 3.17 | 1.24 | 6.21 | 2.43 | 20.33 | 7.95 |
| 3B | 240 | 488.8 | 26.47 | 5.42 | 15.02 | 3.07 | 5.93 | 1.21 | 11.56 | 2.37 | 33.53 | 6.86 |
| 4A | 240 | 826.2 | 49.40 | 5.98 | 23.07 | 2.79 | 12.72 | 1.54 | 24.38 | 2.95 | 61.92 | 7.49 |
| 5A | 240 | 1763.7 | 100.60 | 5.70 | 26.56 | 1.51 | 29.57 | 1.68 | 84.62 | 4.80 | 138.92 | 7.88 |
| 6A | 240 | 2190.3 | 126.88 | 5.79 | 46.79 | 2.14 | 23.66 | 1.08 | 105.13 | 4.80 | 176.27 | 8.05 |
A Pooled plasma from normal donors spiked with <5% v/v recombinant UCH-L1 antigen
8 Pooled plasma from normal donors spiked with <5% v/v UCH-L1 from pooled TBI patient plasma
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| Table 3: Estimate of GFAP and UCH-L1 Assay Precision with i-STAT TBI Controls | ||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| ControlLevel | N | Mean(pg/mL) | Repeatability | Between-run | Between-day | Between-lot | Within-Laboratory | |||||
| SD(pg/mL) | CV(%) | SD(pg/mL) | CV(%) | SD(pg/mL) | CV(%) | SD(pg/mL) | CV(%) | SD(pg/mL) | ||||
| GFAP Assay | ||||||||||||
| L1 | 240 | 161.2 | 6.80 | 4.22 | 1.77 | 1.10 | 1.78 | 1.11 | 2.54 | 1.57 | 7.76 | 4.81 |
| L2 | 240 | 4645.0 | 166.40 | 3.58 | 45.04 | 0.97 | 45.91 | 0.99 | 148.68 | 3.20 | 234.60 | 5.05 |
| UCH-L1 Assay | ||||||||||||
| L1 | 240 | 466.2 | 28.55 | 6.12 | 7.27 | 1.56 | 6.43 | 1.38 | 16.60 | 3.56 | 34.75 | 7.45 |
| L2 | 240 | 1597.6 | 93.98 | 5.88 | 39.23 | 2.46 | 19.79 | 1.24 | 65.97 | 4.13 | 124.87 | 7.82 |
Qualitative 20-day precision: The qualitative agreement of cartridge results relative to the expected sample result (mean) was evaluated for the 80 measurements per sample per cartridge lot for each assay above. The mean, total number of replicates, total number of elevated results, and % correct call for each sample level is presented in Table 4 for GFAP and Table 5 for UCH-L1.
| Table 4: GFAP Assay Results for Qualitative Precision Analysis | |||||
|---|---|---|---|---|---|
| PlasmaSample | Mean(pg/mL) | Total Numberof Replicates | Qualitative Agreement | %Correct Call | |
| Total Number ofResults at orAbove the Cut-off | Total Number ofResults Belowthe Cut-off | ||||
| 1b | 78.8 | 240 | 239 | 1 | 99.6%* |
| 2c | 98.6 | 240 | 240 | 0 | 100.0% |
| 3c | 880.6 | 240 | 240 | 0 | 100.0% |
| 4c | 4415.3 | 240 | 240 | 0 | 100.0% |
| 5c | 8346.7 | 240 | 240 | 0 | 100.0% |
| i-STAT TBI Control | |||||
| L1c | 161.2 | 240 | 240 | 0 | 100.0% |
| L2c | 4645.0 | 240 | 240 | 0 | 100.0% |
ª Below cut-off; b Near cut-off; § Above cut-off
*Determination of correct call based on test material mean. Replicates for sample with mean near cut-off can have replicates below cut-off or at/above cut-off.
| Table 5: UCH-L1 Assay Results for Qualitative Precision Analysis | ||||||
|---|---|---|---|---|---|---|
| Qualitative Agreement | ||||||
| PlasmaSample | Mean(pg/mL) | Total Numberof Replicates | Total Number ofResults at orAbove the Cut-off | Total Number ofResults Belowthe Cut-off | %Correct Call | |
| 1a | 159.9 | 240 | 0 | 240 | 100.0% | |
| 2a | 255.7 | 240 | 0 | 240 | 100.0% |
{9}------------------------------------------------
| Table 5: UCH-L1 Assay Results for Qualitative Precision Analysis | |||||
|---|---|---|---|---|---|
| Qualitative Agreement | |||||
| Plasma Sample | Mean (pg/mL) | Total Number of Replicates | Total Number of Results at or Above the Cut-off | Total Number of Results Below the Cut-off | %Correct Call |
| 3b | 488.8 | 240 | 240 | 0 | 100.0% |
| 4c | 826.2 | 240 | 240 | 0 | 100.0% |
| 5c | 1763.7 | 240 | 240 | 0 | 100.0% |
| 6c | 2190.3 | 240 | 240 | 0 | 100.0% |
| i-STAT TBI Control | |||||
| L1b | 466.2 | 240 | 240 | 0 | 100.0% |
| L2c | 1597.6 | 240 | 240 | 0 | 100.0% |
ª Below cut-off; b Near cut-off; ^ Above cut-off
Semi-quantitative multi-site precision performance of the GFAP and UCH-L1 assays in the i-STAT TBI cartridge on the i-STAT Alinity System was evaluated using an 8member panel of plasma-based test materials spiked with native or recombinant sourced antigens, which included six (6) levels of GFAP and six (6) levels of UCH-L1. The test materials were tested in point-of-care settings at three (3) clinical sites. At each site, each of the test materials was tested once per day for five (5) days by two (2) different operators, with each operator using three (3) i-STAT TBI cartridges on three (3) i-STAT Alinity instruments. Due to the inability to store or freeze whole blood samples to maintain sample stability over multiple days, plasma samples were used for this study. The study followed a 3x5x2x3 design based on CLSI EP05-A3: Evaluation of Precision of Quantitative Measurement Procedures; Approved Guideline – Third Edition. The estimates of GFAP and UCH-L1 precision are shown in Table 6 and Table 7.
| Table 6: i-STAT TBI Multi-site Precision – GFAP (pg/mL) Assay - All Sites | ||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| TestMaterial | Mean(Min, Max) | Repeatability | Between-Day | Between-Operator | Within-Site | Between-Site | Reproducibility | |||||||
| N | SD(95% CI) | %CV | SD(95% CI) | %CV | SD(95% CI) | %CV | SD(95% CI) | %CV | SD(95% CI) | %CV | SD(95% CI) | %CV | ||
| 2+ | 90 | 66.4(50, 77) | 3.85(3.57, 4.17) | 5.79 | 0.28(0.20, 0.46) | 0.42 | 0.56(0.43, 0.81) | 0.85 | 3.90(3.63, 4.21) | 5.87 | 0.00(0.00, 0.00) | 0.00 | 3.90(3.63, 4.21) | 5.87 |
| 4 * | 90 | 86.0(60, 109) | 5.94(5.52, 6.44) | 6.91 | 0.00(0.00, 0.00) | 0.00 | 0.17(0.13, 0.25) | 0.20 | 5.95(5.54, 6.42) | 6.92 | 0.61(0.32, 3.82) | 0.71 | 5.98(5.57, 6.46) | 6.95 |
| 5 * | 90 | 980.9(912, 1147) | 35.38(32.86, 38.33) | 3.61 | 18.04(12.93, 29.78) | 1.8 | 8.11(6.24, 11.59) | 0.83 | 40.53(36.55, 45.50) | 4.13 | 13.86(7.21, 87.09) | 1.41 | 42.84(36.65, 51.55) | 4.37 |
| 6* | 90 | 2785.5(2617, 2941) | 59.55(55.29, 64.51) | 2.14 | 22.55(16.17, 37.23) | 0.81 | 26.04(20.03, 37.21) | 0.93 | 68.79(62.80, 76.07) | 2.47 | 0.00(0.00, 0.00) | 0.00 | 68.79(62.76, 76.12) | 2.47 |
| 7* | 90 | 5357.3(4873, 5927) | 135.38(125.71, 146.68) | 2.53 | 120.86(86.67, 199.51) | 2.26 | 54.17(41.68, 77.41) | 1.01 | 189.4(160.74, 230.58) | 3.54 | 46.07(23.99, 289.52) | 0.86 | 194.92(162.82, 242.89) | 3.64 |
| 8* | 90 | 7652.6(7192, 8239) | 166.85(154.93, 180.77) | 2.18 | 84.16(60.35, 138.92) | 1.10 | 0.00(0.00, 0.00) | 0.00 | 186.87(168.34, 210.02) | 2.44 | 114.05(59.38, 716.75) | 1.49 | 218.92(167.49, 316.14) | 2.86 |
*pooled K₂EDTA plasma spiked with <5% v/v recombinant GFAP antigen
†pooled K₂EDTA plasma spiked with <5% v/v GFAP from TBI patient plasma
{10}------------------------------------------------
| Table 7: i-STAT TBI Multi-site Precision - UCH-L1 (pg/mL) Assay - All Sites | ||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| TestMaterial | N | Mean(Min, Max) | Repeatability | Between-Day | Between-Operator | Within-Site | Between-Site | Reproducibility | ||||||
| SD(95% CI) | %CV | SD(95% CI) | %CV | SD(95% CI) | %CV | SD(95% CI) | %CV | SD(95% CI) | %CV | SD(95% CI) | ||||
| 1* | 90 | 206.5(163, 251) | 13.25(12.31, 14.36) | 6.42 | 4.60(3.30, 7.59) | 2.23 | 10.14(7.80, 14.49) | 4.91 | 17.31(15.35, 19.85) | 8.38 | 0.00(0.00, 0.00) | 0.00 | 17.31(15.35, 19.85) | 8.38 |
| 3† | 93 | 384.1(284, 460) | 21.02(19.96, 22.20) | 5.47 | 0.00(0.00, 0.00) | 0.00 | 22.75(17.51, 32.52) | 5.92 | 30.98(26.50, 37.28) | 8.06 | 0.00(0.00, 0.00) | 0.00 | 30.98(26.64, 37.01) | 8.06 |
| 4* | 90 | 681.8(575, 800) | 35.91(33.34, 38.90) | 5.27 | 16.13(11.57, 26.63) | 2.37 | 35.67(27.44, 50.98) | 5.23 | 53.12(46.02, 62.85) | 7.79 | 0.00(0.00, 0.00) | 0.00 | 53.12(45.98, 62.92) | 7.79 |
| 5* | 90 | 1225.9(1028, 1460) | 82.64(76.74, 89.54) | 6.74 | 0.00(0.00, 0.00) | 0.00 | 9.25(7.12, 13.22) | 0.75 | 83.16(77.46, 89.77) | 6.78 | 31.55(16.43, 198.30) | 2.57 | 88.94(77.63, 104.14) | 7.26 |
| 6* | 90 | 2051.2(1646, 2403) | 98.89(91.83, 107.14) | 4.82 | 24.98(17.91, 41.24) | 1.22 | 99.04(76.20, 141.54) | 4.83 | 142.17(122.50, 169.43) | 6.93 | 0.00(0.00, 0.00) | 0.00 | 142.17(122.83, 168.80) | 6.93 |
| 8* | 90 | 2851.8(2488, 3331) | 137.56(127.73, 149.03) | 4.82 | 39.27(28.16, 64.83) | 1.38 | 110.46(84.99, 157.86) | 3.87 | 180.74(159.43, 208.68) | 6.34 | 0.00(0.00, 0.00) | 0.00 | 180.74(159.60, 208.38) | 6.34 |
*pooled K2EDTA plasma spiked with <5% v/v recombinant UCH-L1 antigen †pooled K₂EDTA plasma spiked with <5% v/v UCH-L1 from TBI patient plasma
Qualitative multi-site precision: The qualitative agreement of cartridge results relative to the expected sample result was evaluated for all measurements per test material for each assay above. The mean value of the test material was used as the expected result to classify the sample as below cut-off, near cut-off (overall mean ± 25% of cut-off), or above cut-off for each assay. The mean, total number of replicates, total number of elevated results, and % correct call for each test material is presented in Table 8 for GFAP and Table 9 for UCH-L1.
| Table 8: Qualitative Precision Analysis - GFAP Assay - All Sites | |||||
|---|---|---|---|---|---|
| Test Material | Mean (pg/mL) | N | Qualitative Agreement | % Correct call | |
| Total # of results at or above the assay cut-off | Total # of results below the assay cut-off | ||||
| 2B | 66.4 | 90 | 74 | 16 | 82.22* |
| 4C | 86.0 | 90 | 89 | 1 | 98.89 |
| 5C | 980.9 | 90 | 90 | 0 | 100.00 |
| 6C | 2785.5 | 90 | 90 | 0 | 100.00 |
| 7C | 5357.3 | 90 | 90 | 0 | 100.00 |
| 8C | 7652.6 | 90 | 90 | 0 | 100.00 |
A Below cut-off; 8 Near cut-off (overall mean ± 25%); ^ Above cut-off
*Determination of correct call based on test material mean. Replicates for sample with mean near cut-off can have replicates below cut-off or at/above cut-off.
{11}------------------------------------------------
| Table 9: Qualitative Precision Analysis - UCH-L1 Assay – All Sites | |||||
|---|---|---|---|---|---|
| Test Material | Mean (pg/mL) | N | Qualitative Agreement | ||
| Total # of results at or above the cut-off | Total # of results below the assay cut-off | % Correct call | |||
| 1A | 206.5 | 90 | 0 | 90 | 100.00 |
| 3B | 384.1 | 93 | 83 | 10 | 89.25* |
| 4C | 681.8 | 90 | 90 | 0 | 100.00 |
| 5C | 1225.9 | 90 | 90 | 0 | 100.00 |
| 6C | 2051.2 | 90 | 90 | 0 | 100.00 |
| 8C | 2851.8 | 90 | 90 | 0 | 100.00 |
A Below cut-off; 8 Near cut-off (overall mean ± 25%); 6 Above cut-off
- Determination of correct call based on test material mean. Replicates for sample with mean near cut-off can have replicates below cut-off or at/above cut-off
Semi-quantitative whole blood precision: The precision performance of the GFAP and UCH-L1 assays in the i-STAT TBI cartridge on the i-STAT Alinity System was evaluated in point of care settings at three (3) clinical sites following a modified design based on CLSI EP05-A3: Evaluation of Precision of Quantitative Measurement Procedures; Approved Guideline – Third Edition. At each site, test samples across the reportable ranges of each assay were prepared by spiking prospectively collected venous whole blood specimens with recombinant GFAP and/or UCH-L1 or human plasma sample from traumatic brain injury (TBI) patients with native GFAP and UCH-L1. Eight (8) GFAP and eight (8) UCH-L1 samples were prepared at Site 1; eight (8) GFAP and 13 UCH-L1 samples were prepared at Site 2; seven (7) GFAP and eight (8) UCH-L1 samples were prepared at Site 3 At each site, each whole blood sample was tested in three (3) runs, by two (2) different operators, each operator using four (4) i-STAT TBI cartridges on four (4) i-STAT Alinity instruments (1 replicate/instrument/run) for a total of 24 replicates/specimen. For samples with target ranges near the GFAP and UCH-L1 assay cut-offs, a minimum of 2 samples were prepared and tested using the i-STAT TBI cartridges at each clinical site. The estimates of GFAP and UCH-L1 precision are shown in Table 10 and Table 11.
{12}------------------------------------------------
| Table 10: Whole Blood Precision at Point-of-Care Sites - GFAP Assay | ||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Site | WholeBloodSample | N | Mean | Repeatability | Between-Instrument | Between-Operator | Within-Site | |||||
| SD(pg/mL) | CV(%) | SD(pg/mL) | CV(%) | SD(pg/mL) | CV(%) | SD(pg/mL) | CV(%) | |||||
| 01 | 1+ | 24 | 63.3 | 9.84 | 15.53 | 0.00 | 0.00 | 2.98 | 4.70 | 10.28 | 16.23 | |
| 2+ | 23‡ | 64.3 | 11.72 | 18.23 | 6.76 | 10.51 | 4.25 | 6.61 | 14.18 | 22.06 | ||
| 3* | 24 | 103.5 | 10.85 | 10.48 | 0.00 | 0.00 | 2.22 | 2.14 | 11.07 | 10.70 | ||
| 4* | 23‡ | 128.5 | 14.51 | 11.29 | 0.00 | 0.00 | 0.00 | 0.00 | 14.51 | 11.29 | ||
| 5* | 24 | 986.3 | 88.48 | 8.97 | 0.00 | 0.00 | 0.00 | 0.00 | 88.48 | 8.97 | ||
| 6* | 24 | 3431.6 | 338.46 | 9.86 | 0.00 | 0.00 | 104.36 | 3.04 | 354.19 | 10.32 | ||
| 7* | 24 | 6371.3 | 637.41 | 10.00 | 0.00 | 0.00 | 162.96 | 2.56 | 657.91 | 10.33 | ||
| 8* | 24 | 7836.9 | 730.91 | 9.33 | 0.00 | 0.00 | 102.96 | 1.31 | 738.13 | 9.42 | ||
| 02 | 1+ | 24 | 57.7 | 7.24 | 12.56 | 4.60 | 7.97 | 5.28 | 9.16 | 10.07 | 17.47 | |
| 2+ | 24 | 60.9 | 11.08 | 18.18 | 0.00 | 0.00 | 2.15 | 3.53 | 11.28 | 18.52 | ||
| 3* | 24 | 83.7 | 6.98 | 8.34 | 0.00 | 0.00 | 0.00 | 0.00 | 6.98 | 8.34 | ||
| 4* | 24 | 148.1 | 12.08 | 8.16 | 0.00 | 0.00 | 0.00 | 0.00 | 12.08 | 8.16 | ||
| 5* | 24 | 900.6 | 28.89 | 3.21 | 10.84 | 1.20 | 0.00 | 0.00 | 30.85 | 3.43 | ||
| 6* | 24 | 3731.1 | 161.63 | 4.33 | 0.00 | 0.00 | 121.29 | 3.25 | 202.08 | 5.42 | ||
| 7* | 24 | 5762.3 | 289.18 | 5.02 | 0.00 | 0.00 | 0.00 | 0.00 | 289.18 | 5.02 | ||
| 8* | 24 | 8310.3 | 499.50 | 6.01 | 0.00 | 0.00 | 0.00 | 0.00 | 499.50 | 6.01 | ||
| 03 | 1+ | 23‡ | 58.9 | 4.47 | 7.59 | 2.60 | 4.41 | 0.00 | 0.00 | 5.17 | 8.78 | |
| 2+ | 22§ | 67.2 | 16.54 | 24.62 | 0.00 | 0.00 | 0.00 | 0.00 | 16.54 | 24.62 | ||
| 3* | 24 | 145.4 | 10.54 | 7.25 | 0.00 | 0.00 | 3.28 | 2.26 | 11.03 | 7.59 | ||
| 4* | 24 | 962.1 | 56.81 | 5.90 | 24.53 | 2.55 | 0.00 | 0.00 | 61.88 | 6.43 | ||
| 5* | 24 | 2954.5 | 167.36 | 5.66 | 0.00 | 0.00 | 3.12 | 0.11 | 167.39 | 5.67 | ||
| 6* | 24 | 6226.4 | 246.48 | 3.96 | 18.23 | 0.29 | 20.69 | 0.33 | 248.02 | 3.98 | ||
| 7* | 23¶ | 8366.9 | 502.57 | 6.01 | 0.00 | 0.00 | 168.21 | 2.01 | 529.97 | 6.33 |
*prospectively collected KչEDTA venous whole blood spiked with <5% v/v recombinant GFAP antigen
†prospectively collected K₂EDTA venous whole blood spiked with <5% v/v GFAP from TBI patient plasma
one (1) result not obtained due to a quality check failure (QCF) or star-out error
§ two (2) results not obtained due to a quality check failure (QCF) or star-out error
¶ one (1) result not obtained due to operator error
| Table 11: Whole Blood Precision at Point-of-Care Sites - UCH-L1 Assay | ||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Whole | Repeatability | Between-Instrument | Between-Operator | Within-Site | ||||||||
| Site | Blood | N | Mean | SD | CV | SD | CV | SD | CV | SD | CV | |
| Sample | (pg/mL) | (%) | (pg/mL) | (%) | (pg/mL) | (%) | (pg/mL) | (%) | ||||
| 1* | 23‡ | 215.7 | 16.58 | 7.69 | 0.00 | 0.00 | 0.00 | 0.00 | 16.58 | 7.69 | ||
| 2* | 24 | 243.5 | 19.35 | 7.95 | 0.00 | 0.00 | 11.14 | 4.57 | 22.33 | 9.17 | ||
| 3+ | 24 | 333.7 | 28.69 | 8.60 | 12.79 | 3.83 | 17.15 | 5.14 | 35.79 | 10.7 | ||
| 01 | 4+ | 22 | 438.9 | 55.79 | 12.71 | 0.00 | 0.00 | 0.00 | 0.00 | 55.79 | 12.7 | |
| 5* | 24 | 486.7 | 25.57 | 5.25 | 6.37 | 1.31 | 9.33 | 1.92 | 27.96 | 5.74 | ||
| 6* | 24 | 1451.4 | 106.30 | 7.32 | 0.00 | 0.00 | 70.00 | 4.82 | 127.28 | 8.77 | ||
| 7* | 24 | 1746.3 | 96.10 | 5.50 | 0.00 | 0.00 | 0.00 | 0.00 | 96.10 | 5.50 | ||
| 8* | 22‡ | 3020.3 | 146.12 | 4.84 | 20.19 | 0.67 | 57.99 | 1.92 | 158.50 | 5.25 | ||
| 1* | 24 | 183.0 | 15.28 | 8.35 | 2.89 | 1.58 | 0.00 | 0.00 | 15.55 | 8.50 | ||
| 2* | 24 | 220.2 | 19.75 | 8.97 | 0.00 | 0.00 | 0.00 | 0.00 | 19.75 | 8.97 | ||
| 02 | 3* | 24 | 232.3 | 15.71 | 6.76 | 0.00 | 0.00 | 0.00 | 0.00 | 15.71 | 6.76 | |
| 4+ | 24 | 360.8 | 26.99 | 7.48 | 18.27 | 5.06 | 0.00 | 0.00 | 32.59 | 9.03 | ||
| 5+ | 24 | 413.0 | 38.18 | 9.25 | 10.68 | 2.59 | 5.52 | 1.34 | 40.03 | 9.69 |
{13}------------------------------------------------
| Whole | Repeatability | Between-Instrument | Between-Operator | Within-Site | |||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| Site | BloodSample | N | Mean | SD(pg/mL) | CV(%) | SD(pg/mL) | CV(%) | SD(pg/mL) | CV(%) | SD(pg/mL) | CV(%) |
| 6* | 24 | 535.1 | 61.35 | 11.47 | 0.00 | 0.00 | 10.79 | 2.02 | 62.29 | 11.64 | |
| 7* | 23‡ | 630.5 | 49.93 | 7.92 | 0.00 | 0.00 | 5.97 | 0.95 | 50.29 | 7.98 | |
| 8* | 24 | 675.0 | 50.54 | 7.49 | 20.74 | 3.07 | 0.00 | 0.00 | 54.63 | 8.09 | |
| 9* | 23‡ | 935.1 | 62.83 | 6.72 | 20.06 | 2.15 | 0.00 | 0.00 | 65.95 | 7.05 | |
| 10* | 21§ | 1114.1 | 59.38 | 5.33 | 0.00 | 0.00 | 0.00 | 0.00 | 59.38 | 5.33 | |
| 11* | 23‡ | 2286.3 | 121.21 | 5.30 | 0.00 | 0.00 | 0.00 | 0.00 | 121.21 | 5.30 | |
| 12 | 24 | 2319.1 | 139.38 | 6.01 | 42.48 | 1.83 | 66.90 | 6.91 | 160.34 | 6.91 | |
| 13 | 21# | 2945.8 | 141.67 | 4.81 | 0.00 | 0.00 | 0.00 | 0.00 | 141.67 | 4.81 | |
| 03 | 1* | 24 | 182.5 | 14.24 | 7.80 | 0.00 | 0.00 | 0.48 | 0.26 | 14.25 | 7.81 |
| 2* | 24 | 204.2 | 16.51 | 8.08 | 0.00 | 0.00 | 11.65 | 5.71 | 20.21 | 9.90 | |
| 3+ | 24 | 357.1 | 35.46 | 9.93 | 0.00 | 0.00 | 5.71 | 1.60 | 35.92 | 10.06 | |
| 4+ | 24 | 392.8 | 39.52 | 10.06 | 12.15 | 3.09 | 0.00 | 0.00 | 41.35 | 10.53 | |
| 5* | 24 | 522.6 | 42.40 | 8.11 | 0.00 | 0.00 | 9.55 | 1.83 | 43.46 | 8.32 | |
| 6* | 24 | 1213.4 | 54.20 | 4.47 | 34.69 | 2.86 | 0.00 | 0.00 | 64.35 | 5.30 | |
| 7* | 24 | 1947.1 | 118.94 | 6.11 | 0.00 | 0.00 | 0.00 | 0.00 | 118.94 | 6.11 | |
| 8* | 21ঠ| 2829.4 | 150.88 | 5.33 | 59.80 | 2.11 | 81.45 | 2.88 | 181.59 | 6.42 |
*prospectively collected K₂EDTA venous whole blood spiked with <5% v/v recombinant UCH-L1 antigen +prospectively collected K2EDTA venous whole blood spiked with <5% v/v UCH-L1 from TBI patient plasma ‡ one (1) result not obtained due to a quality check failure (QCF) or star-out error
§ three (3) results not obtained due to a quality check failure (QCF) or star-out error
¶ one (1) result not obtained due to operator error
|| one (1) result not measurable due to being above the measurement range
three (3) results not measurable due to being above the measurement range
Qualitative Whole Blood Precision: The qualitative agreement of cartridge results relative to the expected sample result was evaluated for all measurements per test material for each assay above. The mean (or median) value of the test material was used as the expected result to classify the sample as below cut-off, near cut-off (overall mean ± 25% of cut-off), or above cutoff for each assay. The mean (or median), total number of replicates, total number of elevated results, and % correct call for each test material is presented in Table 12 for GFAP and Table 13 for UCH-L1.
| Table 12: Qualitative Precision Analysis – GFAP Assay | ||||||
|---|---|---|---|---|---|---|
| Site | Sample | Mean/Median(pg/mL) | N | Qualitative Agreement | ||
| # of GFAP Results at orAbove the Cut-off | # of GFAP ResultsBelow the Cut-off | % CorrectCall | ||||
| 01 | 1B | 61.0+ | 24 | 8 | 16 | 66.67%* |
| 2B | 66.0+ | 23 | 16 | 7 | 69.57%* | |
| 3C | 103.5 | 24 | 24 | 0 | 100.00% | |
| 4C | 128.5 | 23 | 23 | 0 | 100.00% | |
| 5C | 986.3 | 24 | 24 | 0 | 100.00% | |
| 6C | 3431.6 | 24 | 24 | 0 | 100.00% |
{14}------------------------------------------------
| Table 12: Qualitative Precision Analysis – GFAP Assay | ||||||
|---|---|---|---|---|---|---|
| Site | Sample | Mean/Median(pg/mL) | N | # of GFAP Results at orAbove the Cut-off | # of GFAP ResultsBelow the Cut-off | % CorrectCall |
| 02 | 7C | 6371.3 | 24 | 24 | 0 | 100.00% |
| 8C | 7836.9 | 24 | 24 | 0 | 100.00% | |
| 1B | 58.5+ | 24 | 5 | 19 | 79.17%* | |
| 2B | 62.5+ | 24 | 9 | 15 | 62.50%* | |
| 3C | 83.7 | 24 | 24 | 0 | 100.00% | |
| 4C | 148.1 | 24 | 24 | 0 | 100.00% | |
| 5C | 900.6 | 24 | 24 | 0 | 100.00% | |
| 6C | 3731.1 | 24 | 24 | 0 | 100.00% | |
| 7C | 5762.3 | 24 | 24 | 0 | 100.00% | |
| 8C | 8310.3 | 24 | 24 | 0 | 100.00% | |
| 03 | 1B | 60.0+ | 23 | 1 | 22 | 95.65%* |
| 2B | 67.0+ | 22 | 14 | 8 | 63.64%* | |
| 3C | 145.4 | 24 | 24 | 0 | 100.00% | |
| 4C | 962.1 | 24 | 24 | 0 | 100.00% | |
| 03 | 5C | 2954.5 | 24 | 24 | 0 | 100.00% |
| 03 | 6C | 6226.4 | 24 | 24 | 0 | 100.00% |
| 03 | 7C | 8366.9 | 23 | 23 | 0 | 100.00% |
†Median value shown.
^ Below cut-off; 8 Near cut-off (overall mean ± 25%); ^ Above cut-off
*Determination of correct call based on test material mean. Replicates for sample with mean near cut-off can have replicates below cut-off or at/above cut-off.
| Table 13: Qualitative Precision Analysis – UCH-L1 Assay | ||||||
|---|---|---|---|---|---|---|
| Site | Sample | Mean(pg/mL) | N | Qualitative Agreement | ||
| # of UCH-L1 Results at orAbove the Cut-off | # of UCH-L1 ResultsBelow the Cut-off | % CorrectCall | ||||
| 01 | 1A | 215.65 | 23 | 0 | 23 | 100.00% |
| 2A | 243.54 | 24 | 0 | 24 | 100.00% | |
| 3B | 438.91 | 22 | 22 | 0 | 100.00% | |
| 4B | 333.71 | 24 | 5 | 19 | 79.17%* | |
| 5C | 486.71 | 24 | 24 | 0 | 100.00% | |
| 6C | 1451.38 | 24 | 24 | 0 | 100.00% | |
| 7C | 1746.33 | 24 | 24 | 0 | 100.00% | |
| 8C | 3020.27 | 22 | 22 | 0 | 100.00% | |
| 02 | 1A | 182.96 | 24 | 0 | 24 | 100.00% |
| 2A | 220.17 | 24 | 0 | 24 | 100.00% | |
| 3A | 232.33 | 24 | 0 | 24 | 100.00% | |
| 4B | 360.75 | 24 | 13 | 11 | 54.17%* | |
| 5B | 413.00 | 24 | 22 | 2 | 91.67%* | |
| 6C | 675.04 | 24 | 24 | 0 | 100.00% | |
| 7C | 630.52 | 23 | 23 | 0 | 100.00% |
{15}------------------------------------------------
| Table 13: Qualitative Precision Analysis – UCH-L1 Assay | |||||||
|---|---|---|---|---|---|---|---|
| Site | Sample | Mean(pg/mL) | N | Qualitative Agreement | |||
| # of UCH-L1 Results at orAbove the Cut-off | # of UCH-L1 ResultsBelow the Cut-off | % CorrectCall | |||||
| 8C | 535.13 | 24 | 24 | 0 | 100.00% | ||
| 9C | 1114.14 | 21 | 21 | 0 | 100.00% | ||
| 10C | 935.13 | 23 | 23 | 0 | 100.00% | ||
| 11C | 2286.26 | 23 | 23 | 0 | 100.00% | ||
| 12C | 2319.13 | 24 | 24 | 0 | 100.00% | ||
| 13C | 2945.81 | 21 | 21 | 0 | 100.00% | ||
| 1A | 204.21 | 24 | 0 | 24 | 100.00% | ||
| 2A | 182.46 | 24 | 0 | 24 | 100.00% | ||
| 3B | 357.08 | 24 | 11 | 13 | 54.17%* | ||
| 4B | 392.75 | 24 | 21 | 3 | 87.50%* | ||
| 03 | 5C | 522.63 | 24 | 24 | 0 | 100.00% | |
| 6C | 1213.38 | 24 | 24 | 24 | 0 | 100.00% | |
| 7C | 1947.08 | 24 | 24 | 0 | 100.00% | ||
| 8C | 2829.38 | 21 | 21 | 0 | 100.00% |
A Below cut-off; 8 Near cut-off (overall mean ± 25%); ^ Above cut-off
*Determination of correct call based on test material mean. Replicates for sample with mean near cut-off can have replicates below cut-off or at/above cut-off.
b. Linearity/assay reportable range:
i. Linearity
The linearity of the GFAP and UCH-L1 assays in the i-STAT TBI cartridge on the i-STAT Alinity System was established by testing whole blood samples of varying GFAP and UCH-L1 levels over the reportable range of each assay. The study was designed based on CLSI EP06-Ed2: Evaluation of the Linearity of Quantitative Measurement Procedures, 2nd Edition. The study was conducted using whole blood samples of varying GFAP and UCH-L1 levels prepared by admixture with either native antigens from a TBI patient sample or prepared by admixture with recombinant antigens. The regression summary of the results obtained for each assay in the i-STAT TBI cartridge (y-axis) versus the expected values (x-axis) is provided in Table 14 below.
| Table 14: Linearity Across Reportable Range | |||||||||
|---|---|---|---|---|---|---|---|---|---|
| Assay | Test SampleRange (pg/mL) | ReportableRange (pg/mL) | Slope | Intercept(pg/mL) | R2 | ||||
| GFAP | 17.6 – 11303.9 | 47 – 10000 | 1.01 | -3.49 | 0.9990 | ||||
| UCH-L1 | 86.4 - 3281.5 | 87 - 3200 | 0.98 | 2.66 | 0.9977 |
ii. Hook Effect
The GFAP and UCH-L1 assays in the i-STAT TBI cartridge on the i-STAT Alinity System were evaluated for high dose hook effect. The testing was conducted using whole blood samples spiked to a high antigen level for each assay. Each sample was tested to verify that the measured signal is greater than that of a nominal GFAP target of 10,000 pg/mL and a nominal UCH-L1 target of 4000 pg/mL. Hook effect was not observed for the GFAP and UCH-L1 assays using whole blood samples with antigen concentrations exceeding 100,000 pg/mL.
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c. Traceability, Calibration and Reference Interval
i. Traceability and Calibration
There are no internationally recognized standard reference materials available for either glial fibrillary acidic protein (GFAP) or ubiquitin carboxy-terminal hydrolase L1 (UCH-L1). Therefore, the traceability of both the GFAP and UCH-L1 assays in the i-STAT TBI cartridge has been established against reference materials created using recombinant GFAP and UCH-L1 antigens (expressed and purified from E. coli) as the metrologically highest-level material in the metrological calibration hierarchy.
ii. Reference Interval
A reference interval study was conducted with a general population. Whole blood specimens from 150 apparently healthy subjects, aged ≥ 18 years of age reporting no history of neurological disease were tested with the i-STAT TBI cartridge with the i-STAT Alinity System to determine GFAP and UCH-L1 levels. Based on the test results, a 95% reference interval of an apparently healthy population of each biomarker was determined to be as shown in Table 15 below.
| Table 15: Reference Interval | |||||||||
|---|---|---|---|---|---|---|---|---|---|
| Assay | N | Mean(pg/mL) | SD(pg/mL) | Median(pg/mL) | Reference Interval (2.5th to 97.5thpercentile) (pg/mL) | ||||
| GFAP | 150 | 18.7 | 14.51 | 18.0 | <47 - 53 | ||||
| UCH-L1 | 150 | 89.3 | 50.99 | 81.5 | <87 - 251 |
Based on the test results with the i-STAT TBI cartridge with the i-STAT Alinity System, 0.7% (1/150) of the individuals from an apparently healthy population had a test interpretation of "elevated" for biomarkers.
d. Detection Limit
Limit of Quantitation (LoQ) i.
The LoQ was determined for the GFAP and UCH-L1 assays in the i-STAT TBI cartridge in a study based on CLSI EP17-A2: Evaluation of Detection Capability for Clinical Laboratory Measurement Procedures; Approved Guideline–Second Edition. The testing was conducted on six (6) days using three (3) lots of i-STAT TBI cartridges with fresh whole blood from 12 apparently healthy donors altered to achieve 11 low-level samples of GFAP and 12 low level samples of UCH-L1. The estimated LoQ for the i-STAT TBI cartridge tested on the i-STAT Alinity instrument from this study was 47 pg/mL for the GFAP assay and 32 pg/mL for the UCH-L1 assay. Based on the concentration range in the linearity study, the established LoQ of the UCH-L1 assay is 87 pg/mL
e. Analytical Specificity
i. Interference
The interference performance of the GFAP and UCH-L1 assays in the i-STAT TBI cartridge on the i-STAT Alinity Sustem was evaluated using whole blood samples based on CLSI EP07 ED3:
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Interference Testing in Clinical Chemistry, Third Edition. The effect of each substance was evaluated by comparing the performance of a control sample, spiked with blank solvent solution, with the test results from a sample spiked with the potentially interfering substance at the toxic/pathological concentration based on CLSI EP37 ED1: Supplemental Tables for Interference Testing in Clinical Chemistry, First Edition, as applicable. A substance was identified as an interferent if the difference between the control and test samples was outside of a pre-determined acceptable range for each assay. Table 16 below contains the list of potentially interfering substances tested for the GFAP and UCH-L1 assays and the interference results.
| Table 16: Interfering Substances Testing | |||||
|---|---|---|---|---|---|
| Substance | Test Concentration | Assay | Interference (Yes/No) | Comment | |
| µmol/L | mg/dL | ||||
| Acetaminophen a | 1324 | 20 | GFAPUCH-L1 | NoNo | |
| Acetylsalicylic acid a | 3620 | 65.22 | GFAPUCH-L1 | NoNo | |
| Albumin | 150 g/L | 15 g/dL | GFAPUCH-L1 | NoYes | Decreased results at >12.1 g/dL. The highest concentration in the reference interval reported by CLSI EP37 is 5.2 g/dL. |
| Amphetamine | 2.44 | 0.033 | GFAPUCH-L1 | YesNo | Decreased results at >1.83 µmol/L. The highest drug concentration under therapeutic treatment reported by CLSI EP37 is 0.815 µmol/L. |
| Ascorbic acid | 298 | 5.90 | GFAPUCH-L1 | NoNo | |
| Benzoylecgonine a | 8.64 | 2.5 µg/mL | GFAPUCH-L1 | NoNo | |
| Bilirubin | 684 | 40 | GFAPUCH-L1 | NoNo | |
| Bilirubin (conjugated) | 475 | 40 | GFAPUCH-L1 | NoNo | |
| Caffeine | 556 | 10.8 | GFAPUCH-L1 | NoNo | |
| Chloramphenicol | 241 | 7.79 | GFAPUCH-L1 | NoNo | |
| Clopidogrel a | 21.4 | 0.90 | GFAPUCH-L1 | NoNo | |
| Cocaine a | 3.46 µg/mL | 0.346 | GFAPUCH-L1 | NoYes | Decreased results at >2.595 µg/mL. The mean maximum |
| Substance | Test Concentration | Assay | Interference(Yes/No) | Comment | |
| Diazepam | 105 | 2.99 | GFAPUCH-L1 | NoNo | plasma concentration(Cmax) per literature is 0.115 µg/mLc. |
| Diclofenac | 81 | 2.58 | GFAPUCH-L1 | NoNo | |
| Dopamine | 4.06 | 0.077 | GFAPUCH-L1 | NoNo | |
| EDDPa | 0.33 | 125 ng/mL | GFAPUCH-L1 | NoNo | |
| Erythromycin | 188 | 13.80 | GFAPUCH-L1 | NoNo | |
| Ethanol | 130 mmol/L | 599 | GFAPUCH-L1 | NoNo | |
| Hemoglobin | 10 g/L | 1000 | GFAPUCH-L1 | NoNo | |
| Human anti-mouseantibodies (HAMA) a | >80x b | N/A | GFAPUCH-L1 | NoNo | |
| Ibuprofen a | 2425 | 50.0 | GFAPUCH-L1 | NoNo | |
| Intralipid (Intralipid 20%) | N/A | 7075 | GFAPUCH-L1 | NoNo | |
| Methadone | 10.3 | 0.319 | GFAPUCH-L1 | YesNo | Decreased results at >7.725 µmol/L. The highest drugconcentration undertherapeutic treatmentreported by CLSI EP37 is 3.43 µmol/L. |
| d-Methamphetamine a | 1.865 | 278.3 ng/mL | GFAPUCH-L1 | YesNo | Decreased results at >208.8 ng/mL. The mean maximumplasma concentration(Cmax) per literature is 92.8 ng/mL.d |
| Methaqualone a | 32.36 | 8.1 µg/mL | GFAPUCH-L1 | NoNo | |
| Metoprolol a | 18.7 | 1.28 | GFAPUCH-L1 | NoYes | Decreased results at >14.025 µmol/L. The highest drugconcentration undertherapeutic treatment |
| Table 16: Interfering Substances Testing | |||||
| Test Concentration | Interference | ||||
| Substance | μmol/L | mg/dL | Assay | (Yes/No) | Comment |
| Morphine | 27.3 | 0.78 | GFAP | No | reported by CLSI EP37 is1.875 μmol/L. |
| UCH-L1 | No | ||||
| Nicardipinehydrochloride | 0.97 | 0.05 | GFAP | No | |
| UCH-L1 | No | ||||
| Nicotine | 5.97 | 0.0097 | GFAP | No | |
| UCH-L1 | No | ||||
| Oxazepam | 15.1 | 0.432 | GFAP | No | |
| UCH-L1 | No | ||||
| Phencyclidine a | 0.0357 | 8.7 ng/mL | GFAP | No | |
| UCH-L1 | No | ||||
| Phenytoin | 238 | 6.0 | GFAP | No | |
| UCH-L1 | No | ||||
| Propoxyphene a | 9.46 | 0.32 | GFAP | No | |
| UCH-L1 | Yes | Decreased results at >7.095µmol/L. The highest drugconcentration undertherapeutic treatmentreported by CLSI EP37 is 3.15μmol/L. | |||
| Rheumatoid Factor (RF) a | 1000 IU/mL | N/A | GFAP | No | |
| UCH-L1 | Yes | Decreased results at >875IU/mL | |||
| Secobarbital | 66.8 | 1.59 | GFAP | No | |
| UCH-L1 | No | ||||
| Triglycerides a | 33.88 mmol/L | 3000 | GFAP | No | |
| UCH-L1 | No | ||||
| Warfarin | 243 | 7.5 | GFAP | No | |
| UCH-L1 | No |
{18}------------------------------------------------
{19}------------------------------------------------
†2-Ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine
ª The test concentration used for this substance is not from CLSI guideline EP37 1* edition
b The 'x' factor listed indicates the number of times more activity than a known negative sample for its ability to crosslink antibodies in a mouse system assay.
° Scheidweiler, K. B., Spargo, E. A., Kelly, T. L., Cone, E. J., Barnes, A. J., and Huestis, M. A. (2010) Pharmacokinetics of cocaine and metabolites in human oral fluid and correlations after controlled administration. Ther Drug Monit. 32, 628-637
d Karch, S. (2008) Dissociative Anesthetics. In: Karch's Pathology of Drug Abuse, 4th ed. Boca Raton, FL: CRC Press
ii. Cross-reactivity
The i-STAT TBI cartridge on the i-STAT Alinity System were evaluated in the presence of potentially cross-reactive endogenous substances in whole blood specimens based on CLSI guidance EP07-ED3: Interference Testing in Clinical Chemistry, 3rd edition. The effect of each
{20}------------------------------------------------
substance was evaluated by comparing the performance of a test sample spiked with a potentially cross-reactive substance and a control sample spiked with an equal volume of blank plasma diluent as per CLSI EP07 ED3. Table 17 below lists the proteins with significant homology to GFAP and UCH-L1 that were tested at highest known physiological levels. None (0) of the nine (9) substances tested were found to cross-react with the GFAP or UCH-L1 assays in the i-STAT TBI cartridge tested on the i-STAT Alinity System.
| Table 17 : Cross-Reactivity | |||
|---|---|---|---|
| Assay | Substance | Substance TestConcentration(pg/mL) | Outcome (Cross-Reactivity/No Cross-Reactivity) |
| GFAP | Keratin type II | 10,000 | No Cross-Reactivity |
| Internexin | 77,000 | No Cross-Reactivity | |
| Neurofilament Medium | 8,600 | No Cross-Reactivity | |
| Neurofilament Heavy | 77,000 | No Cross-Reactivity | |
| Neurofilament Light | 68 | No Cross-Reactivity | |
| Peripherin Protein | 5,000 | No Cross-Reactivity | |
| Desmin | 127,000 | No Cross-Reactivity | |
| Vimentin | 354,000 | No Cross-Reactivity | |
| UCH-L1 | Ubiquitin Carboxyl-Terminal Hydrolase L3(UCH-L3) | 354,000 | No Cross-Reactivity |
iii. Cross-talk
The GFAP and UCH-L1 assays in the i-STAT TBI cartridge were evaluated for potential crosstalk to determine if high levels of the antigen (GFAP or UCH-L1) of one assay have potential to the impact the result of the other assay. Whole Blood samples spiked to low and high GFAP and UCH-L1 levels were evaluated in the presence of a single high level of the other antigen being evaluated for potential cross-talk. As presented in Table 18 no cross-talk effect was observed as the results demonstrated that the GFAP result is not affected when UCH-L1 is present in a sample, and that the UCH-L1 result is not affected when GFAP is present in a sample.
| Table 18 : Cross-talk | ||||
|---|---|---|---|---|
| Assay | Level | Substance | Substance TestConcentration(pg/mL) | Cross-talk |
| GFAP | Low-positive | UCH-L1 | 100,000 | No |
| GFAP | Moderate-positive | UCH-L1 | 100,000 | No |
| UCH-L1 | Low-positive | GFAP | 100,000 | No |
| UCH-L1 | Moderate-positive | GFAP | 100,000 | No |
{21}------------------------------------------------
f. Hematocrit Sensitivity
The effect of hematocrit on the GFAP and UCH-L1 assays in the i-STAT TBI cartridge was assessed across a hematocrit range of 15-60% packed cell volume (PCV). The study was conducted using two (2) lots of i-STAT TBI cartridges and i-STAT Alinity instruments. Whole blood samples from six (6) donors were altered to target three (3) GFAP and UCH-L1 levels (low, moderate and high) across the reportable range for each respective assay. Each sample was evaluated at three (3) hematocrit (HCT) levels, with the nominal hematocrit level as control condition and low and high hematocrit levels as test conditions. The hematocrit sensitivity at each GFAP and UCH-L1 level was assessed by comparing the results at the low and high hematocrit levels (test conditions) to the nominal hematocrit level (control condition). Imprecision (CV) and bias exceeding 10% were observed for low level GFAP samples with hematocrit levels above 56% PCV.
g. Assay Cut-Off
The assay cut-offs were determined by analyzing a training set with GFAP and UCH-L1 results from a total of 420 (274 males and 146 females) with suspected mild traumatic brain injury (TBI; Glasgow Coma Scale score of 13-15). Subjects who had blood drawn within 12 hours of injury and a head CT scan determination, were included in the analysis. Using a 10-fold cross validation and bootstrapping method, the cut-off values of 65 pg/mL (GFAP assay) and 360 pg/mL (UCH-L1 assay) were selected for the i-STAT TBI Cartridge using the selection criteria with an adjusted NPV (to 10%) ≥98.5% and sensitivity ≥97%.
B. Clinical Sensitivity and Specificity
A prospective, multi-center, observational study was conducted to evaluate the clinical performance of the i-STAT TBI cartridge in classifying intended use population subjects with suspected mild TBI for the likely absence of acute intracranial lesions visualized by a head CT scan. Venous whole blood specimens were used for i-STAT TBI cartridge testing.
Venous whole blood specimens were collected in KJEDTA within 24 hours of the head injury from prospectively enrolled subjects, 18 years of age or older, who had experienced a head injury and presented to the health care facility or the emergency department (ED) with suspected mild TBI, with a GCS score of 13-15; and who had a head CT scan ordered as part of their standard of clinical care. Each specimen was tested for GFAP and UCH-L1 using two (2) i-STAT TBI cartridges and two (2) i-STAT Alinity instruments. Testing was performed at 20 external point of care clinical sites across the United States.
CT scans were performed in accordance with the clinical site's standard of care. Images were transmitted to a central data capture system. Images were interpreted by at least two neuroradiologists who were masked to other clinical and laboratory data: procedures for scoring images were established before conducting image review. The clinical outcome was based on the consensus interpretation between two neurologists. Outcomes were positive or negative as defined by the presence or absence of acute traumatic intracranial lesions, respectively. Acute intracranial lesion was defined as any trauma induced or related finding visualized upon head CT scan.
Specimens from 970 subjects were included in the analysis.
{22}------------------------------------------------
The demographic characteristics of the subjects represented in the clinical performance analysis are summarized in Table 19 below.
| Table 19 : Demographic Characteristics | |||
|---|---|---|---|
| Characteristic | Head CT Scan ResultPositive | Negative | Total |
| N | 283 | 687 | 970 |
| Age (Years) | |||
| Mean | 51.1 | 45.0 | 46.8 |
| Median | 52.0 | 42.0 | 46.0 |
| Standard Deviation | 19.68 | 18.92 | 19.33 |
| Minimum | 18 | 18 | 18 |
| Maximum | 96 | 97 | 97 |
| Gender, N (%) | |||
| Male | 187 (66.1%) | 434 (63.2%) | 621 (64.0%) |
| Female | 94 (33.2%) | 252 (36.7%) | 346 (35.7%) |
| Unspecified/ Not Reported | 2 (0.7%) | 1 (0.1%) | 3 (0.3%) |
| Race, N (%) | |||
| White | 224 (79.2%) | 441 (64.2%) | 665 (68.6%) |
| Black or African American | 20 (7.1%) | 152 (22.1%) | 172 (17.7%) |
| Asian | 11 (3.9%) | 38 (5.5%) | 49 (5.1%) |
| Native Hawaiian/Pacific Islander | 4 (1.4%) | 6 (0.9%) | 10 (1.0%) |
| American Indian or Alaska Native | 4 (1.4%) | 8 (1.2%) | 12 (1.2%) |
| Asian, White | 2 (0.7%) | 3 (0.4%) | 5 (0.5%) |
| Asian, Black or African American | 0 (0.0%) | 1 (0.1%) | 1 (0.1%) |
| Black or African American, American Indian orAlaska Native | 0 (0.0%) | 2 (0.3%) | 2 (0.2%) |
| White, Black or African American | 0 (0.0%) | 5 (0.7%) | 5 (0.5%) |
| Not Reported | 10 (3.5%) | 19 (2.8%) | 29 (3.0%) |
| Unknown | 8 (2.8%) | 12 (1.7%) | 20 (2.1%) |
| Ethnicity, N (%) | |||
| Hispanic or Latino | 67 (23.7%) | 120 (17.5%) | 187 (19.3%) |
| Not Hispanic or Latino | 209 (73.9%) | 552 (80.3%) | 761 (78.5%) |
| Unknown | 6 (2.1%) | 6 (0.9%) | 12 (1.2%) |
| Not Reported | 1 (0.4%) | 9 (1.3%) | 10 (1.0%) |
{23}------------------------------------------------
The head injury characteristics of the 970 subjects 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 in Table 20 below.
| Table 20 : Head Injury Characteristics | |||
|---|---|---|---|
| Head CT Scan Result | |||
| Assessment | Positive | Negative | Total |
| N | 283 | 687 | 970 |
| Time from head injury to Initial Assessment (hours)* | |||
| Mean | 2.0 | 1.3 | 1.5 |
| Median | 1.0 | 0.8 | 0.9 |
| Standard Deviation | 2.01 | 1.45 | 1.67 |
| Range | (1.0, 10.2) | (0.8, 10.0) | (0.8, 10.2) |
| Time from head injury to CT scan (hours)* | |||
| Mean | 2.6 | 2.5 | 2.6 |
| Median | 1.7 | 2.0 | 1.9 |
| Standard Deviation | 2.37 | 1.80 | 1.98 |
| Range | (0.2, 11.4) | (0.3, 10.7) | (0.2, 11.4) |
| Time from head injury to blood draw (hours)* | |||
| Mean | 14.5 | 8.8 | 10.4 |
| Median | 13.5 | 5.8 | 8.1 |
| Standard Deviation | 6.65 | 6.43 | 6.99 |
| Range | (2.0, 24.0) | (1.5, 24.0) | (1.5, 24.0) |
| Glasgow Coma Score - N (%) | |||
| 13 | 28 (9.9%) | 11 (1.6%) | 39 (4.0%) |
| 14 | 79 (27.9%) | 90 (13.1%) | 169 (17.4%) |
| 15 | 176 (62.2%) | 586 (85.3%) | 762 (78.6%) |
| Neurological assessment - N (%) of subjects experiencing: | |||
| Loss of Consciousness (LOC) | 225 (79.5%) | 450 (65.5%) | 675 (69.6%) |
| Confusion/Alteration of Consciousness (AOC) | 195 (68.9%) | 504 (73.4%) | 699 (72.1%) |
| Vomiting | 24 (8.5%) | 21 (3.1%) | 45 (4.6%) |
| Post Traumatic Amnesia (PTA) | 196 (69.3%) | 409 (59.5%) | 605 (62.4%) |
| Post Traumatic Seizures | 3 (1.1%) | 0 (0.0%) | 3 (0.3%) |
| Subjects with Drug Intoxication at the Time ofPresentation to Facility | 48 (17.0%) | 66 (9.6%) | 114 (11.8%) |
| Subjects with Alcohol Intoxication at the Time ofPresentation to Facility | 49 (17.3%) | 61 (8.9%) | 110 (11.3%) |
| Mechanism of Injury† - N (%) of subjects affected: | |||
| Table 20 : Head Injury Characteristics | |||
| Assessment | Head CT Scan Result | Total | |
| Positive | Negative | ||
| Acceleration/Deceleration | 68 (24.0%) | 221 (32.2%) | 289 (29.8%) |
| Direct Impact (blow to head) | 44 (15.5%) | 85 (12.4%) | 129 (13.3%) |
| Direct Impact (head against object) | 157 (55.5%) | 437 (63.6%) | 594 (61.2%) |
| Crush | 0 (0.0%) | 3 (0.4%) | 3 (0.3%) |
| Blast | 0 (0.0%) | 1 (0.1%) | 1 (0.1%) |
| Ground level fall | 82 (29.0%) | 170 (24.7%) | 252 (26.0%) |
| Fall from Height > 1 meter (3 feet) | 39 (13.8%) | 79 (11.5%) | 118 (12.2%) |
| Other | 15 (2.2%) | 7 (2.5%) | 22 (2.3%) |
| Physical Evidence‡– N (%) of subjects with: | |||
| Visible Trauma Above Clavicle | 214 (75.6%) | 422 (61.4%) | 636 (65.6%) |
| Signs of Basal Skull Fracture | 37 (13.1%) | 7 (1.0%) | 44 (4.5%) |
{24}------------------------------------------------
*Based on time subject arrived at the study hospital for neurological assessment.
†A subject could have experienced head injury due to multiple mechanisms of injury. No subjects experienced head injury due to gunshot or fragment (including shell/shrapnel).
‡Prior to head CT scan.
Of the 970 evaluable subjects, 283 subjects had positive CT scan results. Of these 283 subjects with positive CT scan results, 273 had an 'Elevated' i-STAT TBI test interpretation (sensitivity = 96.5% (273/283)). Ten (10) subjects with CT scan positive results had an i-STAT TBI test interpretation that was 'Not Elevated'. The rate of false negative (FN) results was 3.5% (10/283). None of these ten (10) subjects with false negative results required surgical intervention related to their head injury as no neurosurgical lesions were identified by CT scan in these subjects.
Of the 687 subjects with negative CT scan results, 277 had an i-STAT TBI test interpretation that was 'Not Elevated' (277/687, specificity = 40.3 %). The rate of false positive (FP) results was 59.6% (410/687).
In the clinical study, the prevalence of adjudicated CT scan positive subjects was 29.2% (283/970). Overall, there were 287 subjects with i-STAT TBI test interpretations of Not Elevated'. Of these, 277 subjects had negative CT scan results. The negative predictive value (NPV) of the assay was 96.5% (277/287). Table 21 below provides the clinical performance estimates of i-STAT TBI cartridge with i-STAT Alinity instrument.
{25}------------------------------------------------
| Table 21 : Clinical Performance | |||
|---|---|---|---|
| i-STAT TBI TestInterpretation | All Evaluable Results (0-24h) | Total | |
| Adjudicated Head CT ScanPositive | Adjudicated Head CT ScanNegative | ||
| Elevated | 273 | 410 | 683 |
| Not Elevated | 10 | 277 | 287 |
| Total | 283 | 687 | 970 |
| Clinical Performance Parameters | N=970 | 95% CI | |
| Clinical Sensitivity | 96.5% (273/283) | (93.6%, 98.1%)+ | |
| Clinical Specificity | 40.3% (277/687) | (36.7%, 44.0%)+ | |
| Negative Predictive Value (NPV) * | 96.5% (277/287) | (93.7%, 98.1%)‡ | |
| Positive Predictive Value (PPV) | 40% (273/683) | (38.4%, 41.5%)‡ | |
| Likelihood Ratio Negative (LRN) | 0.09 | (0.05, 0.16)§ | |
| Likelihood Ratio Positive (LRP) | 1.62 | (1.52, 1.73)§ |
- Adjusted NPV at 6% prevalence is 99.4% (95% Cl: 99.0%, 99.7%).
†95% confidence intervals are calculated using the Wilson score method for a binomial portion (see CLSI EP12-Ed3) : 195% confidence intervals for predictive values are calculated based on the confidence intervals of the
corresponding likelihood ratios
§95% confidence intervals are calculated using asymptotic method for a ratio of two binomial proportion
8. Conclusion
The results of these studies demonstrate that performance of the GFAP and UCH-L1 assays in the i-STAT TBI cartridge with the i-STAT Alinity System are substantially equivalent to the predicate device.
§ 866.5830 Brain trauma assessment test.
(a)
Identification. A brain trauma assessment test is a device that consists of reagents used to detect and measure brain injury biomarkers in human specimens. The measurements aid in the evaluation of patients with suspected mild traumatic brain injury in conjunction with other clinical information to assist in determining the need for head imaging per current standard of care.(b)
Classification. Class II (special controls). The special controls for this device are:(1) The 21 CFR 809.10(b) compliant labeling must include detailed descriptions of and results from performance testing conducted to evaluate precision, accuracy, linearity, analytical sensitivity, interference, and cross-reactivity. This information must include the following:
(i) Performance testing of device precision must, at minimum, use one unmodified clinical specimen from the intended use population with concentration of the brain injury biomarker(s) near the medical decision point. Contrived specimens that have been generated from pooling of multiple samples or spiking of purified analyte to cover the measuring range may be used, but the contrived samples must be prepared to mimic clinical specimens as closely as possible. This testing must evaluate repeatability and reproducibility using a protocol from an FDA-recognized standard.
(ii) Device performance data must be demonstrated through a clinical study and must include the following:
(A) Data demonstrating clinical validity including the clinical sensitivity and specificity, and positive and negative predictive value of the test in the intended use population of patients with suspected mild traumatic brain injury (
i.e., Glasgow Coma Score (GCS) of 13-15), or equivalent standard of care for determination of severity of traumatic brain injury (TBI).(B) Study must be performed using the operators and in settings that are representative of the types of operators and settings for which the device is intended to be used.
(C) All eligible subjects must meet the well-defined study inclusion and exclusion criteria that define the intended use population. The prevalence of diseased or injured subjects in the study population must reflect the prevalence of the device's intended use population, or alternatively, statistical measures must be used to account for any bias due to enrichment of subpopulations of the intended use population.
(D) All eligible subjects must have undergone a head computerized tomography (CT) scan or other appropriate clinical diagnostic standard used to determine the presence of an intracranial lesion as part of standard of care and must also be evaluated by the subject device. All clinical diagnostic standards used in the clinical study must follow standard clinical practice in the United States.
(E) Relevant demographic variables and baseline characteristics including medical history and neurological history. In addition, head injury characteristics, neurological assessments, and physical evidence of trauma must be provided for each subject. This information includes but is not limited to the following: Time since head injury, time from head injury to CT scan, time from head injury to blood draw, GCS score or equivalent, experience of loss of consciousness, presence of confusion, episodes of vomiting, post-traumatic amnesia characteristics, presence of post-traumatic seizures, drug or alcohol intoxication, mechanism of injury, acute intracranial lesion type, neurosurgical lesion, and cranial fracture.
(F) Each CT scan or other imaging result must be independently evaluated in a blinded manner by at least two board-certified radiologists to determine whether it is positive or negative as defined by the presence or absence of acute intracranial lesions. This independent review must be conducted without access to test results of the device. Prior to conducting the review, the criteria and procedures to be followed for scoring the images must be established, including the mechanism for determining consensus.
(G) All the clinical samples must be tested with the subject device blinded to the TBI status and the neurological-lesion-status of the subject.
(H) Details on how missing values in data are handled must be provided.
(I) For banked clinical samples, details on storage conditions and storage period must be provided. In addition, a specimen stability study must be conducted for the duration of storage to demonstrate integrity of archived clinical samples. The samples evaluated in the assay test development must not be used to establish the clinical validity of the assays.
(iii) Performance testing of device analytical specificity must include the most commonly reported concomitant medications present in specimens from the intended use population. Additionally, potential cross-reacting endogenous analytes must be evaluated at the highest concentration reported in specimens from the intended use population.
(iv) Expected/reference values generated by testing a statistically appropriate number of samples from apparently healthy normal individuals.
(2) The 21 CFR 809.10(a) and (b) compliant labeling must include the following limitations:
(i) A limiting statement that this device is not intended to be used a stand-alone device but as an adjunct to other clinical information to aid in the evaluation of patients who are being considered for standard of care neuroimaging.
(ii) A limiting statement that reads “A negative result is generally associated with the absence of acute intracranial lesions. An appropriate neuroimaging method is required for diagnosis of acute intracranial lesions.”
(iii) As applicable, a limiting statement that reads “This device is for use by laboratory professionals in a clinical laboratory setting.”