Traumatic brain injury (TBI) test

K223602 · Abbott Laboratories · QAT · Mar 2, 2023 · Immunology

Device Facts

• Record ID: K223602
• Device Name: Traumatic brain injury (TBI) test
• Applicant: Abbott Laboratories
• Product Code: QAT · Immunology
• Decision Date: Mar 2, 2023
• Decision: SESE
• Submission Type: Traditional
• Regulation: 21 CFR 866.5830
• Device Class: Class 2

Intended 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.

Device Story

TBI test is a panel of two automated chemiluminescent microparticle immunoassays (CMIA) for quantitative measurement of GFAP and UCH-L1 in human plasma/serum; performed on Alinity i system. Input: patient plasma/serum sample. Operation: two-step immunoassay; sample incubated with paramagnetic microparticles coated with anti-GFAP or anti-UCH-L1; washed; acridinium-labeled conjugate added; chemiluminescent reaction measured as relative light units (RLU). Output: quantitative GFAP/UCH-L1 concentrations and semi-quantitative TBI interpretation (positive/negative) based on cut-offs (35.0 pg/mL GFAP; 400.0 pg/mL UCH-L1). Used in clinical laboratories by healthcare professionals. Output aids clinical decision-making regarding necessity of head CT scan; negative result associated with absence of acute intracranial lesions on CT. Benefits patient by potentially reducing unnecessary CT scans.

Clinical Evidence

Pivotal study (N=1899) using archived plasma specimens from patients with suspected mild TBI (GCS 13-15) within 12 hours of injury. Primary endpoint: sensitivity/NPV for predicting absence of intracranial lesions on head CT. Results: Sensitivity 96.7% (95% CI: 91.7-98.7%), Specificity 40.1%, NPV 99.4% (95% CI: 98.6-99.8%). Supplemental fresh specimen study (N=97) showed 100% sensitivity and 100% NPV. Bench testing confirmed precision, linearity, and interference profiles.

Technological Characteristics

Automated two-step chemiluminescent microparticle immunoassay (CMIA). Reagents: monoclonal anti-GFAP/anti-UCH-L1 coated paramagnetic microparticles, acridinium-labeled conjugates. Platform: Alinity i system. Sample types: K2 EDTA plasma, serum. Analytical measuring interval: 6.1–42,000 pg/mL (GFAP); 26.3–25,000 pg/mL (UCH-L1). Connectivity: Alinity i system software.

Indications for Use

Indicated for patients 18+ years old presenting with suspected mild traumatic brain injury (GCS 13-15) within 12 hours of injury to assist in determining the need for a head CT scan.

Regulatory Classification

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.

Special Controls

*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.”

Predicate Devices

• Banyan Brain Trauma Indicator (BTI) (DEN170045)

Submission Summary (Full Text)

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March 2, 2023

Abbott Laboratories Lisa Kelly Regulatory Affairs Associate Director 100 Abbott Park Road Abbott Park, Illinois 60064

Re: K223602

Trade/Device Name: TBI Regulation Number: 21 CFR 866.5830 Regulation Name: Brain trauma assessment test Regulatory Class: Class II Product Code: QAT Dated: November 30, 2022 Received: December 2, 2022

Dear Lisa Kelly:

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 (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 located 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.

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

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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 803) for devices or postmarketing safety reporting (21 CFR 4, Subpart B) for combination products (see https://www.fda.gov/combination-products/guidance-regulatory-information/postmarketing-safety-reportingcombination-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 4, Subpart A) for combination products; and, if applicable, the electronic product radiation control provisions (Sections 531-542 of the Act); 21 CFR 1000-1050.

Also, please note the regulation entitled, "Misbranding by reference to premarket notification" (21 CFR Part 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 ([e-mail]) or phone [phone] or 301-796-7100).

Sincerely,

# Ying Mao -S

Ying 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

Enclosure

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# Indications for Use

510(k) Number (if known) K223602

Device Name TBI

#### Indications for Use (Describe)

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 cominction, 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.

| Type of Use (Select one or both, as applicable) | |
|--------------------------------------------------------------------------------|-------------------------------------------------------------------------------|
| <div> <span>☑</span> Prescription Use (Part 21 CFR 801 Subpart D) </div> | <div> <span>☐</span> Over-The-Counter Use (21 CFR 801 Subpart C) </div> |

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## 510(k) Summary (Summary of Safety and Effectiveness)

This summary of the 510(k) safety and effectiveness information is being submitted in accordance with the requirements of SMDA 1990 and 21 CFR § 807.92.

#### I. Applicant Name

Abbott Diagnostics Department 09AA, Building CP01 100 Abbott Park Road Abbott Park, IL 60064

Primary contact person for all communications:

Lisa Kelly, Associate Director of Regulatory Affairs Abbott Core Diagnostics [e-mail] Phone [phone] Fax [phone]

Secondary contact person for all communications:

Noah Lermer PhD, Director of Regulatory Affairs Abbott Core Diagnostics [e-mail] Phone [phone] Fax [phone]

Date Summary Prepared: November 30, 2022

# II. Device Name

TBI

#### Reagents

Trade Name: Glial fibrillary acidic protein (GFAP) Ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) Device Classification: Class II (Special controls) Classification Name: Brain trauma assessment test Governing Regulation: 21 CFR § 866.5830 Product Code: QAT

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# III. Predicate Device

Banyan Brain Trauma Indicator (BTI) (DEN170045)

# IV. Description of Device

# A. Reagents

The kit configurations of the GFAP and UCH-L1 reagent kits are described below.

| | List Number | |
|------------------------------|--------------|----------------|
| | GFAP 04W1722 | UCH-L1 04W1922 |
| Tests per cartridge | 100 | 100 |
| Number of cartridges per kit | 2 | 2 |
| Tests per kit | 200 | 200 |
| Microparticles | 7.1 mL | 7.1 mL |
| Conjugate | 6.4 mL | 12.5 mL |
| Assay Specific Diluent | 6.4 mL | 10.5 mL |

# GFAP Reagent Kit

| Microparticles: | Anti-GFAP (rabbit, monoclonal) coated microparticles in<br>TRIS buffer with protein (bovine) stabilizer. Minimum<br>concentration: 0.05% solids. Preservative: ProClin 300. |
|----------------------------|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Conjugate: | Anti-GFAP (mouse, monoclonal) acridinium-labeled<br>conjugate in MES buffer with protein (bovine) stabilizer.<br>Minimum concentration: 0.2 mg/L. Preservative:<br>ProClin 300. |
| Assay Specific<br>Diluent: | TRIS buffer with protein (bovine) stabilizer. Preservative:<br>ProClin 300. |

# UCH-L1 Reagent Kit

| Microparticles: | Anti-UCH-L1 (mouse, monoclonal) coated microparticles<br>in TRIS buffer with protein (bovine) stabilizer. Minimum<br>concentration: 0.05% solids. Preservative: sodium azide. |
|----------------------------|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Conjugate: | Anti-UCH-L1 (mouse, monoclonal) acridinium-labeled<br>conjugate in MES buffer with protein (bovine) stabilizer.<br>Minimum concentration: 0.2 mg/L. Preservative:<br>ProClin 300. |
| Assay Specific<br>Diluent: | TRIS buffer with protein (bovine) stabilizer. Preservative:<br>sodium azide. |

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#### B. Biological Principles of the Procedure

The TBI test is a panel of in vitro diagnostic quantitative measurements of GFAP and UCH-L1 and provides a semi-quantitative interpretation of GFAP and UCH-L1 in human plasma and serum.

### GFAP

This assay is an automated, two-step immunoassay for the quantitative measurement of GFAP in human plasma and serum using chemiluminescent microparticle immunoassay (CMIA) technology.

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.

# UCH-L1

This assay is an automated, two-step immunoassay for the quantitative measurement of UCH-L1 in human plasma and serum using CMIA technology.

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. There is a direct relationship between the amount of UCH-L1 in the sample and the RLU detected by the system optics.

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# C. Interpretation of Results

The assay cutoffs were established to be 35.0 pg/mL (35.0 ng/L) for GFAP and 400.0 pg/mL (400.0 ng/L) for UCH-L1.

The GFAP and UCH-L1 results are reported separately and the software provides a TBI interpretation relative to the respective cutoff values as shown in the following table.

| Specification for Constituent Assay Results | TBI Result | TBI Interpretation |
|---------------------------------------------|------------|--------------------|
| GFAP and UCH-L1 below (<) cutoff | 0 | Negative |
| GFAP and/or UCH-L1 above (≥) cutoff | 1 | Positive |

The following table provides a detailed summary of the TBI interpretation based on potential results.

| GFAP Assay Result<br>(Relative to Cutoff of<br>35.0 pg/mL [35.0 ng/L])* | UCH-L1 Assay Result<br>(Relative to Cutoff of<br>400.0 pg/mL<br>[400.0 ng/L])* | 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 cutoff. Below means less than the cutoff.

** 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 cutoff 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 cutoff and no result is obtained for the other assay.

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In the case of a flagged ">" or "<" result for either assay, the TBI interpretation should be evaluated manually. A result flagged ">" should be considered above the cutoff and a result flagged "<" should be considered below the cutoff.

#### V. Intended Use of the Device

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.

#### VI. Comparison of Technological Characteristics

The TBI test is a panel of in vitro diagnostic quantitative measurements of GFAP and UCH-L1 and provides a semi-quantitative interpretation of GFAP and UCH-L1 in human plasma and serum.

The GFAP assay (subject device) is an automated immunoassay for the quantitative measurement of GFAP in plasma and serum using chemiluminescent microparticle immunoassay (CMIA) technology on the Alinity i system.

The UCH-L1 assay (subject device) is an automated immunoassay for the quantitative measurement of UCH-L1 in plasma and serum using chemiluminescent microparticle immunoassay (CMIA) technology on the Alinity i system.

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The similarities and differences between the subject device and the predicate device are presented in the following table.

| Similarities and Differences Between Subject & Predicate Device | | |
|-----------------------------------------------------------------|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| | Subject Device:<br>TBI | Predicate Device:<br>Banyan BTI (DEN170045) |
| | General Device Characteristic Similarities | |
| Intended Use and<br>Indications for<br>Use | The TBI test is a panel of <i>in vitro</i><br>diagnostic chemiluminescent<br>microparticle immunoassays<br>(CMIA) used for the quantitative<br>measurements of glial fibrillary<br>acidic protein (GFAP) and ubiquitin<br>carboxyl-terminal hydrolase L1<br>(UCH-L1) in human plasma and<br>serum and provides a semi-<br>quantitative interpretation of test<br>results derived from these<br>measurements using the Alinity i<br>system.<br>The interpretation of test results is<br>used, in conjunction with other<br>clinical information, to aid in the<br>evaluation of patients, 18 years of<br>age or older, presenting with<br>suspected mild traumatic brain<br>injury (Glasgow Coma Scale score<br>13-15) within 12 hours of injury, to<br>assist in determining the need for a<br>CT (computed tomography) scan of<br>the head. A negative test result is<br>associated with the absence of acute<br>intracranial lesions visualized on a<br>head CT scan.<br>The TBI test is intended for use in<br>clinical laboratory settings by<br>healthcare professionals. | The Banyan BTI is an <i>in vitro</i><br>diagnostic chemiluminescent enzyme-<br>linked immunosorbent assay (ELISA).<br>The assay provides a semi-quantitative<br>measurement of the concentrations of<br>ubiquitin C-terminal hydrolase-L1<br>(UCH-L1) and glial fibrillary acidic<br>protein (GFAP) in human serum and<br>is used with the Synergy 2<br>Multi-mode Reader.<br>The assay results obtained from serum<br>collected within 12 hours of suspected<br>head injury are used, along with other<br>available clinical information, to aid in<br>the evaluation of patients 18 years of<br>age and older with suspected traumatic<br>brain injury (Glasgow Coma Scale<br>score 13-15). A negative assay result<br>is associated with the absence of acute<br>intracranial lesions visualized on a<br>head CT (computed tomography)<br>scan. |
| Intended Use<br>Setting | Clinical Laboratory | Same |
| Measurands | GFAP and UCH-L1 | Same |
| Assay<br>Technology | Chemiluminescent microparticle<br>immunoassays (CMIA) | Enzyme-linked immunosorbent assay |
| Similarities and Differences Between Subject & Predicate Device | | |
| | Subject Device:<br>TBI | Predicate Device:<br>Banyan BTI (DEN170045) |
| Reportable Result | Quantitative results for GFAP and<br>UCH--L1 and semi-quantitative<br>interpretation for TBI | Same |
| Assay Format | Two separate test kits – one for<br>GFAP and one for UCH-L1 | Two test kits on separate 96-well<br>microtiter plates – one for GFAP and<br>one for UCH-L1 |
| Detection<br>Technology | Chemiluminescence | Same |
| General Device Characteristic Differences | | |
| Platform | Alinity i | Synergy 2 Multi-mode Reader<br>(BioTek Instruments, Inc.) |
| Specimen Type | Serum and Plasma | Serum |
| Sample Volume | GFAP kit: 200 µL<br>UCH-L1 kit: 150 µL | GFAP kit: 150 µL<br>UCH-L1 kit: 100 µL |
| Time to Result | Approximately 18 minutes | Approximately 4 hours |
| Reportable<br>Interval | Analytical Measuring Interval:<br>GFAP: 6.1 - 42,000.0 pg/mL<br>UCH-L1: 26.3 – 25,000.0 pg/mL<br><br>Reportable Interval:<br>GFAP: 3.2 - 42,000.0 pg/mL<br>UCH-L1: 18.3 - 25,000.0 pg/mL | GFAP: 10 - 320 pg/mL<br>UCH-L1: 80 - 2560 pg/mL |
| GFAP Cutoff | 35.0 pg/mL | 22 pg/mL |
| UCH-L1 Cutoff | 400.0 pg/mL | 327 pg/mL |

# Comparison of Subject Device (TBI for Alinity i) to Predicate Device (Banyan BTI)

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# VII. Summary of Nonclinical Performance

# A. Reportable Interval

Based on representative data, the ranges over which results can be reported are provided below according to the definitions from CLSI EP34, 1st ed.

| | GFAP<br>(pg/mL, ng/L) | UCH-L1<br>(pg/mL, ng/L) |
|--------------------------------------|-----------------------|-------------------------|
| Analytical Measuring Interval (AMI)a | 6.1 - 42,000.0 | 26.3 - 25,000.0 |
| Reportable Intervalb | 3.2 - 42,000.0 | 18.3 - 25,000.0 |

a AMI: The AMI extends from the limit of quantitation (LoQ) to the upper limit of quantitation (ULoQ). This is determined by the range of values in pg/mL (ng/L) that demonstrated acceptable performance for linearity, imprecision, and bias.

b The reportable interval extends from the limit of detection (LoD) to the upper limit of the AMI.

NOTE: The default Low Linearity value of the assay file corresponds to the lower limit of the reportable interval.

# B. Within-Laboratory Precision

# GFAP

A study was performed based on guidance from CLSI EP05-A3. T Testing was conducted using 2 lots of the GFAP reagents, 2 lots of the GFAP Calibrators, 1 lot of the GFAP Controls, and 2 instruments. Three controls and eight human plasma panels (one native panel and seven panels supplemented with GFAP analyte) were tested in a minimum of 2 replicates at 2 separate times per day on 20 days on 4 reagent lot/calibrator lot/instrument combinations, where a unique reagent lot and a unique calibrator lot are paired with both instruments. The performance is shown in the following table.

^{^} Clinical and Laboratory Standards Institute (CLSI). Establishing and Verifying an Extended Measuring Interval Through Specimen Dilution and Spiking. 1st ed. CLSI Guideline EP34. Wayne, PA: CLSI; 2018.

^* Clinical and Laboratory Standards Institute (CLSI). Evaluation of Quantitative Measurement Procedures: Approved Guideline-Third Edition. CLSI Document EP05-A3. Wayne, PA: CLSI; 2014.

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| | | Mean | Within-Run | | Between-Run | | Between-Day | | Between-Lot | | Between-<br>Instrument | | Overall<br>Within-<br>Laboratoryª | |
|------------------|-----|------------------|------------|-----|-------------|-----|-------------|-----|-------------|-----|------------------------|-----|-----------------------------------|-----|
| Sample | N | (pg/mL,<br>ng/L) | SD | %CV | SD | %CV | SD | %CV | SD | %CV | SD | %CV | SD | %CV |
| 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 | 4.9 |
| 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 | 3.7 |
| High Control | 479 | 31608.5 | 758.80 | 2.4 | 846.98 | 2.7 | 288.19 | 0.9 | 968.97 | 3.1 | 423.35 | 1.3 | 1579.35 | 5.0 |
| Panel 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 | 5.0 |
| Panel 2 (Native) | 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 | 3.8 |
| Panel 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 | 4.1 |
| Panel 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 | 3.8 |
| Panel 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 | 4.2 |
| Panel 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 | 4.3 |
| Panel 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 | 4.8 |
| Panel 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 | 5.9 |

ª Overall within-laboratory variability contains within-run, between-lot, and between-instrument variance components.

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# Qualitative Precision: Results Relative to Cutoff

The qualitative analysis of precision results relative to the cutoff (35.0 pg/mL) was performed using the precision data generated for all instruments and reagent lots.

The GFAP mean (pg/mL), number of results greater than or equal to the cutoff, and % correct call for each GFAP panel and GFAP control levels for all instruments and reagent lots are presented in the table below.

| Sample | N | Mean<br>(pg/mL) | # of Results<br>≥ 35.0 (pg/mL) / N | % of Correct<br>Calla |
|-------------------|-----|-----------------|------------------------------------|-----------------------|
| Low Controlb | 479 | 25.4 | 0 / 479 | 100.0 |
| Medium Controld | 480 | 504.8 | 480 / 480 | 100.0 |
| High Controld | 479 | 31,608.5 | 479 / 479 | 100.0 |
| Panel 1b | 479 | 20.4 | 0 / 479 | 100.0 |
| Panel 2 (Native)c | 474 | 37.7 | 462 / 474 | 100.0 |
| Panel 3 c | 479 | 40.2 | 479 / 479 | 100.0 |
| Panel 4d | 480 | 95.6 | 480 / 480 | 100.0 |
| Panel 5d | 480 | 3097.2 | 480 / 480 | 100.0 |
| Panel 6d | 478 | 7586.3 | 478 / 478 | 100.0 |
| Panel 7d | 480 | 15,462.7 | 480 / 480 | 100.0 |
| Panel 8d | 478 | 36,874.7 | 478 / 478 | 100.0 |

a Replicates for positive samples should always be > cutoff, replicates for negative samples should always be < cutoff, and replicates for samples near medical decision points can have replicates < cutoff or ≥ cutoff.

- b Negative samples
- C Samples near medical decision point

d Positive samples

# UCH-L1

A study was performed based on guidance from CLSI EP05-A3. Testing was conducted using 2 lots of the UCH-L1 reagents, 2 lots of the UCH-L1 Calibrators, 1 lot of the UCH-L1 Controls, and 2 instruments. Three controls and eight human plasma panels (one native panel and seven panels supplemented with UCH-L1 analyte) were tested in a minimum of 2 replicates at 2 separate times per day on 20 days on 4 reagent

^* Clinical and Laboratory Standards Institute (CLSI). Evaluation of Quantitative Measurement Procedures: Approved Guideline-Third Edition. CLSI Document EP05-A3. Wayne, PA: CLSI; 2014.

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lot/calibrator lot/instrument combinations, where a unique reagent lot and a unique calibrator lot are paired with both instruments. The performance is shown in the following table.

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| | | Mean | Within-Run | | Between-Run | | Between-Day | | Between-Lot | | Between-Instrument | | Overall<br>Within-<br>Laboratorya | |
|------------------|-----|------------------|------------|-----|-------------|-----|-------------|-----|-------------|-----|--------------------|-----|-----------------------------------|-----|
| Sample | N | (pg/mL,<br>ng/L) | SD | %CV | SD | %CV | SD | %CV | SD | %CV | SD | %CV | SD | %CV |
| 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 |
| Panel 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 |
| Panel 2 (Native) | 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 |
| Panel 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 |
| Panel 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 |
| Panel 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 |
| Panel 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 |
| Panel 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 |
| Panel 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 |

ª Overall within-laboratory variability contains within-run, between-lot, and between-instrument variance components.

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# Qualitative Precision: Results Relative to Cutoff

The qualitative analysis of precision results relative to the cutoff (400.0 pg/mL) was performed using the precision data generated for all instruments and reagent lots.

The UCH-L1 mean (pg/mL), number of results greater than or equal to the cutoff, and % correct call for each UCH-L1 panel and UCH-L1 control levels for all instruments and reagent lots are presented in the table below.

| Sample | N | Mean<br>(pg/mL) | # of Results<br>≥ 400.0 (pg/mL) / N | % of Correct<br>Calla |
|-------------------|-----|-----------------|-------------------------------------|-----------------------|
| Low Controlb | 479 | 247.5 | 0 / 479 | 100.0 |
| Medium Controld | 479 | 2019.6 | 479 / 479 | 100.0 |
| High Controld | 477 | 15,179.4 | 477 / 477 | 100.0 |
| Panel 1b | 480 | 177.6 | 0 / 480 | 100.0 |
| Panel 2 (Native)c | 471 | 391.8 | 164 / 471 | 100.0 |
| Panel 3c | 479 | 419.8 | 391 / 479 | 100.0 |
| Panel 4d | 477 | 823.8 | 477 / 477 | 100.0 |
| Panel 5d | 476 | 1553.8 | 476 / 476 | 100.0 |
| Panel 6d | 479 | 4793.5 | 479 / 479 | 100.0 |
| Panel 7d | 480 | 7974.6 | 480 / 480 | 100.0 |
| Panel 8d | 472 | 19,165.5 | 472 / 472 | 100.0 |

a Replicates for positive samples should always be ≥ cutoff, replicates for negative samples should always be < cutoff, and replicates for samples near medical decision points can have replicates < cutoff or ≥ cutoff.

- b Negative samples
- c Samples near medical decision point
- d Positive samples

# C. Lower Limits of Measurement

The claimed limit of blank (LoB), limit of detection (LoD), and limit of quantitation (LoQ) values are summarized in the table below. The LoD is in aligmment with the low end of the reportable interval and the LoQ is in alignment with the low end of the AMI for GFAP and UCH-L1.

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| | GFAP<br>pg/mL, ng/L | UCH-L1<br>pg/mL, ng/L |
|-----|---------------------|-----------------------|
| LoB | 1.6 | 6.1 |
| LoD | 3.2 | 18.3 |
| LoQ | 6.1 | 26.3 |

A study was performed based on guidance from CLSI EP17-A2.* Testing was conducted using 3 lots of the GFAP and UCH-L1 reagents on each of 2 instruments over a minimum of 3 days.

- . The LoB represents the 95th percentile from n > 60 replicates of zero-analyte samples. The observed GFAP LoB was 1.6 pg/mL (1.6 ng/L). The observed UCH-L1 LoB was 6.1 pg/mL (6.1 ng/L).
- . The LoD represents the lowest concentration at which the analyte can be detected with 95% probability based on n ≥ 60 replicates of low-analyte level samples. The observed GFAP LoD was 2.2 pg/mL (2.2 ng/L). The observed UCH-L1 LoD was 16.1 pg/mL (16.1 ng/L).
- . The LoQ is defined as the lowest concentration at which a maximum allowable precision of 20.0 %CV was met and was determined from n ≥ 60 replicates of low-analyte level samples. The observed GFAP LoQ was 2.4 pg/mL (2.4 ng/L). The observed UCH-L1 LoQ was 16.1 pg/mL (16.1 ng/L).

# D. Linearity

# GFAP

A study was performed based on guidance from CLSI EP06, 2nd ed.1

This assay is linear across the analytical measuring interval of 6.1 to 42,000.0 pg/mL

(6.1 to 42,000.0 ng/L).

# UCH-LI

A study was performed based on guidance from CLSI EP06, 2nd ed.+

This assay is linear across the analytical measuring interval of 26.3 to 25.000.0 pg/mL

(26.3 to 25,000.0 ng/L).

Clinical and Laboratory Standards Institute (CLSI). Evaluation of Detection Capability for Clinical Laboratory Measurement Procedures; Approved Guideline-Second Edition. CLSI Document EP17-A2. Wayne, PA: CLSI; 2012.

^് Clinical and Laboratory Standards Institute (CLSI). Evaluation of the Linearity of Quantitative Measurement Procedures. 2nd ed. CLSI Guideline EP06. Wayne, PA: CLSI: 2020.

⁺ Clinical and Laboratory Standards Institute (CLSI). Evaluation of the Linearity of Quantitative Measurement Procedures. 2nd ed. CLSI Guideline EP06. Wayne, PA: CLSI; 2020.

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# E. Analytical Specificity

# Interference

# Potentially Interfering Endogenous Substances

A study was performed based on guidance from CLSI EP07, 3rd ed.* Each substance was tested at 2 levels of the GFAP analyte (approximately 25 pg/mL and 10,000 pg/mL) and at 2 levels of the UCH-L1 analyte (approximately 280 pg/mL and 5000 pg/mL).

No significant interference (interference within ± 10.0%) was observed at the following concentrations.

| No Significant Interference (Interference within ± 10.0%) | | |
|-----------------------------------------------------------|-------------------|--------------|
| | Interferent Level | |
| Potentially Interfering Substance | 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 |
| Total Protein | 10 g/dL | 9 g/dL |
| Glucose | 1000 mg/dL | 1000 mg/dL |
| Heterophilic Antibodies (HAMA) | 80x activity | 80x activity |
| Rheumatoid Factor (RF) | 500 IU/mL | 500 IU/mL |

^* Clinical and Laboratory Standards Institute (CLSI). Interference Testing in Clinical Chemistry. 3rd ed. CLSI Guideline EP07. Wayne, PA: CLSI; 2018.

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# Interference beyond ± 10.0% (based on 95% Confidence Interval [CI]) was

observed at the concentrations shown below for the following substances.

| Interference beyond ± 10.0% (based on 95% CI) | | | | |
|-----------------------------------------------|--------------------------------------|----------------------|------------------|----------------------------|
| Assay | Potentially Interfering<br>Substance | Interferent<br>Level | Analyte<br>Level | % Interference<br>(95% CI) |
| GFAP | Intralipid | 2000 mg/dL | 10,000 pg/mL | -10.3%<br>(-11.3%, -9.3%) |
| GFAP | Total Protein | 15 g/dL | 10,000 pg/mL | -15.6%<br>(-16.7%, -14.6%) |
| UCH-L1 | Unconjugated Bilirubin | 40 mg/dL | 280 pg/mL | 9.2%<br>(7.7%, 10.7%) |
| UCH-L1 | Total Protein | 15 g/dL | 280 pg/mL | -19.0%<br>(-21.5%, -16.5%) |
| UCH-L1 | Total Protein | 15 g/dL | 5000 pg/mL | -16.9%<br>(-18.5%, -15.4%) |

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# Potentially Interfering Drugs

A study was performed based on guidance from CLSI EP07, 3rd ed. * Each substance was tested at 2 levels of the GFAP analyte (approximately 25 pg/mL and 10,000 pg/mL) and at 2 levels of the UCH-L1 analyte (approximately 280 pg/mL and 5000 pg/mL).

# No significant interference (interference within ± 10.0%) was observed at the

following concentrations.

| No Significant Interference (Interference within ± 10.0%) | | |
|-----------------------------------------------------------|-------------------|--------------|
| Potentially 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 |…