HemosIL Liquid Anti-Xa

K223187 · Instrumentation Laboratory CO · QLU · Jun 23, 2023 · Hematology

Device Facts

• Record ID: K223187
• Device Name: HemosIL Liquid Anti-Xa
• Applicant: Instrumentation Laboratory CO
• Product Code: QLU · Hematology
• Decision Date: Jun 23, 2023
• Decision: SESE
• Submission Type: Traditional
• Regulation: 21 CFR 864.7295
• Device Class: Class 2

Indications for Use

HemosIL Liquid Anti-Xa is an automated chromogenic assay for in vitro diagnostic use by laboratory professionals in clinical laboratories. The assay provides quantitative results on 3.2% citrated human plasma for the following analytes based on the calibrators used: · When used with HemosIL Heparin Calibrators: Quantitative determination of unfractionated heparin (UFH) and low molecular weight heparin (LMWH) activity on the ACL TOP Family and ACL TOP Family 50 Series. · When used with HemosIL Apixaban Calibrators: Quantitative determination of apixaban on the ACL TOP Family 50 Series through measurement of factor Xa activity, which is inversely proportional to the apixaban level. With HemosIL Apixaban Calibrators, the assay is intended to measure apixaban concentrations in patients on apixaban therapy in the following situations where measurement of apixaban levels could be useful to have as additional information: - Patients at risk for major bleeding - Patients experiencing a bleeding episode · When used with HemosIL Rivaroxaban Calibrators: Quantitative determination of rivaroxaban on the ACL TOP Family and ACL TOP Family 50 Series through measurement of factor Xa activity, which is inversely proportional to the rivaroxaban level. With HemosL Rivaroxaban Calibrators, the assay is intended to measure rivaroxaban concentrations in patients on rivaroxaban therapy in the following situations where measurement of rivaroxaban levels could be useful to have as additional information: - Patients at risk for major bleeding - Patients experiencing a bleeding episode The assay is not a stand-alone test and the results should be used in conjunction with other clinical and laboratory findings. For use in adult population. For prescription use only.

Device Story

HemosIL Liquid Anti-Xa is an automated chromogenic assay for quantitative measurement of rivaroxaban, apixaban, or heparin in 3.2% citrated human plasma. The device is used in clinical laboratories on ACL TOP Family and ACL TOP Family 50 Series instruments. Operation involves adding excess bovine Factor Xa to a patient sample; rivaroxaban in the sample partially neutralizes the Factor Xa. Residual Factor Xa is then quantified by adding a synthetic chromogenic substrate (S-2732), which releases paranitroaniline (pNA). The rate of pNA release is monitored kinetically at 405 nm; the measured absorbance is inversely proportional to the drug concentration. Results are reported in ng/mL and used by clinicians alongside other clinical and laboratory findings to assess patients at risk of or experiencing bleeding. The assay is not intended for routine therapeutic monitoring or dosage adjustment.

Clinical Evidence

Method comparison study (CLSI EP09c) performed at 3 sites on 337 patient samples (rivaroxaban-treated). Compared to LC-MS/MS, the assay showed high correlation (r=0.995, slope 0.971, mean bias -2.8%). Precision studies (CLSI EP05-A3) demonstrated total CVs ranging from 1.0% to 9.8% across various concentrations. LoD is 8 ng/mL; LoQ is 20 ng/mL. Interference testing confirmed no significant impact from common substances (hemoglobin, bilirubin, triglycerides, warfarin, etc.) up to specified limits.

Technological Characteristics

One-stage chromogenic assay. Reagents: purified bovine Factor Xa, Tris-Buffer, EDTA, dextran sulfate, NaCl, bovine serum albumin, and chromogenic substrate S-2732. Instrumentation: ACL TOP Family and ACL TOP Family 50 Series. Measurement: kinetic absorbance at 405 nm. Reportable range: 20-1000 ng/mL. Stability: 1 month open-vial (2-8°C), 4 days on-board (15-25°C).

Indications for Use

Indicated for adult patients on rivaroxaban or apixaban therapy, or heparin therapy (UFH/LMWH), who are at risk for major bleeding or experiencing a bleeding episode. For use by laboratory professionals in clinical laboratories. Not for monitoring or dosage adjustment.

Regulatory Classification

Identification

A heparin and direct oral factor Xa inhibitor drug test system is intended for the detection of heparin and direct oral factor Xa inhibitors in human specimens collected from patients taking heparin or direct oral factor Xa inhibitors. This device is intended to aid in the management of therapy in conjunction with other clinical and laboratory findings.

Special Controls

*Classification.* Class II (special controls). The special controls for this device are:(1) Design verification and validation must include the following: (i) Detailed documentation of analytical device performance studies and results demonstrating acceptable analytical performance with a sufficient number of specimens tested in order to obtain unbiased estimates of analytical performance. This documentation shall include the following as appropriate to the technology, specimen types tested, and intended use of the device: (A) Studies and results for that demonstrate device precision including repeatability and reproducibility, using quality controls and clinical samples, when appropriate. Precision studies must assess specimens for each indicated drug at concentrations throughout the measuring range of the device including near clinically relevant levels, as appropriate. The study must evaluate different sources of variability including, as appropriate, between-run, between-operator, between-lot, between-instrument, between-day, and between-site; (B) Studies and results that demonstrate that the device is free from clinically significant interference, from endogenous and exogenous interferents associated with the target population(s), and interferents that are specific for, or related to, the technology or methodology of the device; (C) Data to demonstrate appropriate specimen stability for the intended sample matrices under the intended conditions for specimen collection, handling, and storage described in the device labeling; (D) Studies and results that demonstrate the linear range, limit of blank (LoB), limit of detection (LoD), and limit of quantitation (LoQ), as applicable to the technology of the device; and (E) For any devices intended for use for near patient testing, studies and results that demonstrate the robustness of the device in the hands of the intended user, including the entire testing procedure, pre-analytical specimen processing steps, and results interpretation. (ii) Detailed documentation of clinical performance testing in which the performance is analyzed relative to a comparator that FDA has determined is appropriate. Specimens must be representative of the intended use population(s) and must cover the full range of the device output and any clinically relevant decision points as appropriate. (2) The labeling required under § 809.10(b) of this chapter must include: (i) Identification of any known interferents, including all endogenous, exogenous, technology-specific, and patient population-specific interferents, specific to the test outputs. The information must include the concentration(s) or level(s) of the interferent at which clinically significant interference was found to occur, and the concentration range or levels at which interference was not found to occur; (ii) A prominent statement that the device is not intended for use in monitoring patients taking heparin or direct oral factor Xa inhibitors; and (iii) Limiting statements indicating, as applicable: (A) That the device should only be used in conjunction with information available from clinical evaluations and other diagnostic procedures; and (B) That the device is not specific to the direct oral factor Xa inhibitor that has been evaluated and may detect the presence of other direct factor Xa inhibitors that have not been evaluated.

Predicate Devices

• HemosIL Liquid Anti-Xa (DEN190032)
• HemosIL Liquid Anti-Xa (K213464)

Submission Summary (Full Text)

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FDA U.S. FOOD & DRUG ADMINISTRATION

# 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION SUMMARY

ASSAY ONLY

## I Background Information:

A 510(k) Number

K223187

B Applicant

Instrumentation Laboratory Co.

C Proprietary and Established Names

- HemosIL Liquid Anti-Xa
- HemosIL Rivaroxaban Calibrators
- HemosIL Rivaroxaban Controls
- HemosIL Apixaban Calibrators
- HemosIL Apixaban Controls
- HemosIL Heparin Calibrators
- HemosIL UF Heparin Controls
- HemosIL LMW Heparin Controls

D Regulatory Information

| Product Code(s) | Classification | Regulation Section | Panel |
| --- | --- | --- | --- |
| QLU | Class II | 21 CFR 864.7295 - Heparin And Direct Oral Factor Xa Inhibitor Drug Test System | HE - Hematology |

## II Submission/Device Overview:

A Purpose for Submission:

Clearance of HemosIL Liquid Anti-Xa for Rivaroxaban measurement with HemosIL Rivaroxaban Calibrators.

B Measurand:

Heparin, Apixaban, Rivaroxaban

Food and Drug Administration

10903 New Hampshire Avenue

Silver Spring, MD 20993-0002

www.fda.gov

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C Type of Test:

Anti-Factor Xa chromogenic assay

III Intended Use/Indications for Use:

A Intended Use(s):

See Indications for Use below.

B Indication(s) for Use:

HemosIL Liquid Anti-Xa is an automated chromogenic assay for in vitro diagnostic use by laboratory professionals in clinical laboratories. The assay provides quantitative results on 3.2% citrated human plasma for the following analytes based on the calibrators used:

- When used with HemosIL Heparin Calibrators:

Quantitative determination of unfractionated heparin (UFH) and low molecular weight heparin (LMWH) activity on the ACL TOP Family and ACL TOP Family 50 Series.

- When used with HemosIL Apixaban Calibrators:

Quantitative determination of apixaban on the ACL TOP Family and ACL TOP Family 50 Series through measurement of Factor Xa activity, which is inversely proportional to the apixaban level. With HemosIL Apixaban Calibrators, the assay is intended to measure apixaban concentrations in patients on apixaban therapy in the following situations where measurement of apixaban levels could be useful to have as additional information:

- Patients at risk for major bleeding
- Patients experiencing a bleeding episode

- When used with HemosIL Rivaroxaban Calibrators:

Quantitative determination of rivaroxaban on the ACL TOP Family and ACL TOP Family 50 Series through measurement of Factor Xa activity, which is inversely proportional to the rivaroxaban level. With HemosIL Rivaroxaban Calibrators, the assay is intended to measure rivaroxaban concentrations in patients on rivaroxaban therapy in the following situations where measurement of rivaroxaban levels could be useful to have as additional information:

- Patients at risk for major bleeding
- Patients experiencing a bleeding episode

The assay is not a stand-alone test and the results should be used in conjunction with other clinical and laboratory findings.

For use in adult population. For prescription use only.

C Special Conditions for Use Statement(s):

Rx - For Prescription Use Only
For use in adult population only
For in vitro diagnostic use only

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D Special Instrument Requirements:

ACL TOP Family (K160276): ACL TOP 300 CTS; ACL TOP 500 CTS; ACL TOP 700; ACL TOP 700 CTS; ACL TOP 700 LAS

ACL TOP Family 50 Series (K150877): ACL TOP 350 CTS; ACL TOP 550 CTS; ACL TOP 750; ACL TOP 750 CTS; ACL TOP 750 LAS

IV Device/System Characteristics:

A Device Description:

HemosIL Liquid Anti-Xa is a one stage chromogenic assay based on a synthetic chromogenic substrate and on Factor Xa inactivation. The assay provides quantitative heparin, apixaban, and rivaroxaban results on 3.2% citrated human plasma when used with HemosIL Heparin Calibrators, HemosIL Apixaban Calibrators and/or Rivaroxaban Calibrators.

The HemosIL Liquid Anti-Xa assay consists:

4mL Kit Vial Size
- Factor Xa reagent 5 x 2.5 mL vial of a liquid preparation containing purified bovine Factor Xa, Tris-Buffer, EDTA, dextran sulfate, sodium chloride and bovine serum albumin
- Chromogenic substrate 5 x 3 mL vial of liquid chromogenic substrate S-2732 and bulking agent

10mL Kit Vial Size
- Factor Xa reagent 5 x 5 mL vial of a liquid preparation containing purified bovine Factor Xa, Tris-Buffer, EDTA, dextran sulfate, sodium chloride and bovine serum albumin
- Chromogenic substrate 5 x 6 mL vial of liquid chromogenic substrate S-2732 and bulking agent

The assay requires the following components which are not included in the assay kit:
- HemosIL Rivaroxaban Calibrators- two levels (0 and 500 ng/mL) of lyophilized calibrators prepared from human citrated plasma containing rivaroxaban, buffers, and stabilizers.
- HemosIL Rivaroxaban Controls- two levels (80 and 300 ng/mL) of lyophilized controls prepared from human citrated plasma containing rivaroxaban, buffers, and stabilizers.
- HemosIL Apixaban Calibrators and Controls refer to DEN190032.
- HemosIL Heparin Calibrator and Controls (LMWH and UFH) refer to K090209.
- Cleaning solution
- Cleaning agent
- Factor diluent

B Principle of Operation:

The HemosIL Liquid Anti-Xa is an anti-factor Xa one-stage chromogenic assay. When an excess amount of factor Xa is added to a sample containing rivaroxaban, factor Xa is partially

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neutralized by rivaroxaban. Residual factor Xa is quantified when the synthetic chromogenic substrate is added to the patient sample. The released paranitroaniline (pNA) is released and monitored kinetically at 405 nm. The measured pNA is inversely proportional to rivaroxaban levels. Thus, maximum absorption occurs with low levels of drug, and minimum absorption occurs with high levels of drug.

V Substantial Equivalence Information:

A Predicate Device Name(s): HemosIL Liquid Anti-Xa

B Predicate 510(k) Number(s): DEN190032, K213464

C Comparison with Predicate(s):

| Device & Predicate Device(s): | K223187 | K213464 |
| --- | --- | --- |
| General Device Characteristic Similarities | | |
| Device Trade Name | HemosIL Liquid Anti-Xa | HemosIL Liquid Anti-Xa |
| Intended Use/Indications For Use | HemosIL Liquid Anti-Xa is an automated chromogenic assay for in vitro diagnostic use by laboratory professionals in clinical laboratories. The assay provides quantitative results on 3.2% citrated human plasma for the following analytes based on the calibrators used:
• When used with HemosIL Heparin Calibrators:
Quantitative determination of unfractionated heparin (UFH) and low molecular weight heparin (LMWH) activity on the ACL TOP Family and ACL TOP Family 50 Series.
• When used with HemosIL Apixaban Calibrators:
Quantitative determination of apixaban on the ACL TOP Family and ACL TOP Family 50 Series through measurement of Factor Xa activity, which is inversely proportional to the apixaban level. With HemosIL Apixaban Calibrators, the assay is intended to measure apixaban concentrations in patients on apixaban therapy in the following situations where measurement of apixaban levels could be useful to have as additional information:
- Patients at risk for major bleeding | HemosIL Liquid Anti-Xa is an automated chromogenic assay for in vitro diagnostic use by laboratory professionals in clinical laboratories. The assay provides quantitative results on 3.2% citrated human plasma for the following analytes based on the calibrators used:
• When used with HemosIL Heparin Calibrators:
Quantitative determination of unfractionated heparin (UFH) and low molecular weight heparin (LMWH) activity on the ACL TOP Family, ACL TOP Family 50 Series.
• When used with HemosIL Apixaban Calibrators:
Quantitative determination of apixaban on the ACL TOP Family and ACL TOP Family 50 Series through measurement of Factor Xa activity, which is inversely proportional to the apixaban level. With HemosIL Apixaban Calibrators, the assay is intended to measure apixaban concentrations in patients on apixaban therapy in the following situations where measurement of apixaban levels could be useful to have as additional information: |

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| | - Patients experiencing a bleeding episode

• When used with HemosIL Rivaroxaban Calibrators: Quantitative determination of rivaroxaban on the ACL TOP Family and ACL TOP Family 50 Series through measurement of Factor Xa activity, which is inversely proportional to the rivaroxaban level. With HemosIL Rivaroxaban Calibrators, the assay is intended to measure rivaroxaban concentrations in patients on rivaroxaban therapy in the following situations where measurement of rivaroxaban levels could be useful to have as additional information:
- Patients at risk for major bleeding
- Patients experiencing a bleeding episode

The assay is not a stand-alone test and the results should be used in conjunction with other clinical and laboratory findings. For use in adult population. For prescription use only.

*Note: Same as predicate except for addition of Rivaroxaban. | - Patients at risk for major bleeding
- Patients experiencing a bleeding episode

The assay is not a stand-alone test and the results should be used in conjunction with other clinical and laboratory findings. For use in adult population. For prescription use only. |
| --- | --- | --- |
| Technical Method/Test Methodology | One stage chromogenic assay based on a synthetic chromogenic substrate and on factor Xa inactivation. The test detects residual factor Xa using a chromogenic substrate. The signal or optical density is compared to a drug-specific calibration curve and results are reported as nanograms per milliliter (ng/mL). | Same |
| Measurement | Quantitative | Same |
| Sample Matrix | 3.2% citrated plasma | Same |
| Composition | The HemosIL Anti-Xa includes the following components:
• Factor Xa reagent: Liquid preparation containing purified bovine Factor Xa (approximately 5.5 nkat/mL), Tris-Buffer, EDTA, dextran sulfate, sodium chloride and bovine serum albumin. | Same |

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| | • Chromogenic substrate: liquid chromogenic substrate S-2732 (approximately 1.2 mg/mL) and bulking agent. | |
| --- | --- | --- |
| Instrumentation | ACL TOP Family (K160276)
ACL TOP 50 Series (K150877) | Same |
| Quality Control | Automated QC | Same |
| Open Reagent Stability | 1 month | Same |
| On-board Stability | 4 Days | Same |
| General Device Characteristic Differences | | |
| Measurand | Heparin
Apixaban
Rivaroxaban | Heparin
Apixaban |
| Assay Volume | 4 mL Vial Size
Factor Xa reagent 5 x 2.5 mL
Chromogenic substrate 5 x 3 mL
10 mL Kit Vial Size
Factor Xa reagent 5 x 5 mL
Chromogenic substrate 5 x 6 mL | 4 mL Vial Size
Factor Xa reagent 5 x 2.5 mL
Chromogenic substrate 5 x 3 mL |
| Limit of Detection | 8 ng/mL for Rivaroxaban | N/A |
| Linearity | 20 ng/mL to 1000 ng/mL
for Rivaroxaban | N/A |
| Calibrators
(Sold Separately) | HemosIL Heparin Calibrators and
HemosIL Apixaban Calibrators,
HemosIL Rivaroxaban Calibrators;
Target Levels: 0 and 500 ng/mL | HemosIL Heparin Calibrators;
Target levels: 0, 0.8 and 2.0 IU/mL
HemosIL Apixaban Calibrators;
Target Levels: 0 and 500 ng/mL |
| Controls
(Sold Separately) | HemosIL Heparin Controls and
HemosIL Apixaban Controls,
HemosIL Rivaroxaban Controls;
Target Levels: 80 and 300 ng/mL | HemosIL Heparin (LMW and UF)
Controls; Target Levels: low and high
HemosIL Apixaban Controls;
Target Levels: 75 and 300 ng/mL |

VI Standards/Guidance Documents Referenced:

EP05-A3. Evaluation of Precision of Quantitative Measurement Procedures; Approved Guideline–Third Edition, October 2014 (R2019).

EP06-A2. Evaluation of Linearity of Quantitative Measurement Procedure-Second Edition, April 2003.

EP07-Ed. 3. Interference Testing in Clinical Chemistry-Third Edition, November 2005 (R2022).

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EP17-A2. Evaluation of Detection Capability for Clinical Laboratory Measurement Procedures; Approved Guideline–Second Edition, October 2004 (R2017).

EP34-Ed. 1. Establishing and Verifying an Extended Measuring Interval Through Specimen Dilution and Spiking-First Edition, August 2018.

EP37-Ed. 1. Supplement Tables for Interference Testing in Clinical Chemistry-First Edition, April 2018.

## VII Performance Characteristics (if/when applicable):

### A Analytical Performance:

Analytical performance studies for apixaban, low-molecular weight heparin (LMWH) and unfractionated heparin (UFH) were previously reported in DEN190032 (apixaban) and K090209 (LMWH, UFH), respectively.

#### 1. Precision/Reproducibility:

Four precision studies were performed following the recommendations of the CLSI EP05-A3 guideline.

Study 1: This study was conducted over 20 days, two runs per day and two replicates per run for a total of 480 determinations (i.e., 80 determinations per reagent lot). The study design included three representative ACL TOP Family instrument models, two reagent lots (each lot was tested on all instrument models), as well as three spiked plasma samples and one patient pool. Precision estimates were calculated for each of the following variance component: within-run, between-run, between-day, between-lot and within laboratory. The results are provided in the summary table below.

| Sample | N | Rivaroxaban Mean ng/mL | Repeatability (Within-Run) | | Between-Run | | Between-Day | | Between-Lot | | Within-Laboratory | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | | SD | %CV | SD | %CV | SD | %CV | SD | %CV | SD | %CV |
| Spiked 1 | 480 | 50.2 | 1.57 | 3.1 | 1.12 | 2.2 | 0.59 | 1.2 | 1.15 | 2.3 | 2.32 | 4.6 |
| Spiked 2 | 480 | 787.2 | 11.25 | 1.4 | 12.00 | 1.5 | 6.18 | 0.8 | 6.70 | 0.9 | 23.71 | 3.0 |
| Spiked 3 | 480 | 928.2 | 11.91 | 1.3 | 17.18 | 1.9 | 17.55 | 1.9 | 0.00 | 0.0 | 33.77 | 3.6 |
| Plasma Pool | 480 | 127.2 | 1.77 | 1.4 | 0.98 | 0.8 | 1.00 | 0.8 | 2.04 | 1.6 | 3.04 | 2.4 |

Study 2: This study was conducted over 20 days, two runs per day and two replicates per run for a total of 720 determinations (i.e., 80 determinations per reagent lot). The study design included three representative ACL TOP Family 50 Series instrument models, three reagent lots (each lot was tested on all instrument models), as well as three spiked plasma samples and one patient pool. Precision estimates were calculated for each of the following variance component: within-run, between-run, between-day, between-lot and within laboratory. The results are provided in the summary table below.

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| Sample | N | Rivaroxaban Mean ng/mL | Repeatability (Within-Run) | | Between-Run | | Between-Day | | Between-Lot | | Within-Laboratory | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | | SD | %CV | SD | %CV | SD | %CV | SD | %CV | SD | %CV |
| Spiked 1 | 720 | 50.7 | 2.10 | 4.1 | 0.71 | 1.4 | 0.93 | 1.8 | 0.75 | 1.5 | 2.78 | 5.5 |
| Spiked 2 | 720 | 777.2 | 19.90 | 2.6 | 4.48 | 0.6 | 6.28 | 0.8 | 11.03 | 1.4 | 38.80 | 5.0 |
| Spiked 3 | 720 | 923.4 | 17.40 | 1.9 | 4.44 | 0.5 | 10.32 | 1.1 | 4.52 | 0.5 | 40.36 | 4.4 |
| Plasma Pool | 720 | 127.9 | 2.44 | 1.9 | 1.39 | 1.1 | 1.19 | 0.9 | 1.92 | 1.5 | 5.19 | 4.1 |

Study 3: This study was conducted over 20 days, two runs per day and two replicates per run for a total of 480 determinations (i.e., 80 determinations per reagent lot). The study design included two representative ACL TOP Family and ACL TOP Family 50 Series instrument models, three reagent lots (each lot was tested on all instrument models), native MDL trough and peak rivaroxaban plasma samples. Precision estimates were calculated for each of the following variance component: within-run, between-run, between-day, between-lot and within laboratory. The results are provided in the summary table below.

| Sample | N | Rivaroxaban Mean ng/mL | Repeatability (Within-Run) | | Between-Run | | Between-Day | | Between-Lot | | Within-Laboratory | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | | SD | %CV | SD | %CV | SD | %CV | SD | %CV | SD | %CV |
| Native 5 (Trough) | 480 | 32.4 | 1.41 | 4.4 | 0.39 | 1.2 | 0.66 | 2.0 | 0.00 | 0.0 | 1.70 | 5.2 |
| Native 6 (Peak) | 480 | 428.0 | 6.32 | 1.5 | 0.00 | 0.0 | 1.64 | 0.4 | 5.54 | 1.3 | 11.06 | 2.6 |

Study 4: This study was conducted at three sites by three operators (1 operator per site), over 5 days, two runs per day and three replicates per run for a total of 270 determinations (i.e., 30 determinations per reagent level). The study design included three different ACL TOP Family instruments, three reagent lots (i.e., one per instrument and the same reagent lots were used across all sites), as well as two quality controls, three spiked plasma samples and two patient pools. Precision estimates were calculated for each of the following variance component: within-run, between-run, between-day, between-site, between-lot, and reproducibility. The results are provided in the summary table below.

| Pooled 3 Site Data | | | | | | | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Level | Mean (ng/mL) | N | Repeatability (Within-Run) | | Between-Run | | Between-Day | | Between-Site | | Between-Lot | | Reproducibility (Total) |
| | | | SD | %CV | SD | %CV | SD | %CV | SD | %CV | SD | %CV | SD |
| Low Control | 72.6 | 270 | 2.28 | 3.1% | 1.03 | 1.4% | 0.00 | 0.0% | 1.44 | 2.0% | 0.80 | 1.1% | 3.00 |
| High Control | 281 | 270 | 4.58 | 1.6% | 4.10 | 1.5% | 0.00 | 0.0% | 2.15 | 0.8% | 0.17 | 0.1% | 6.51 |
| Spiked 1 | 45.0 | 270 | 2.55 | 5.7% | 0.88 | 2.0% | 0.56 | 1.3% | 3.38 | 7.5% | 0.66 | 1.5% | 4.41 |
| Spiked 2 | 750 | 270 | 19.68 | 2.6% | 1.44 | 0.2% | 6.30 | 0.8% | 22.12 | 3.0% | 5.26 | 0.7% | 30.76 |
| Spiked 3 | 939 | 270 | 25.49 | 2.7% | 3.43 | 0.4% | 7.41 | 0.8% | 22.98 | 2.4% | 8.83 | 0.9% | 36.36 |
| Plasma Pool 1 | 51.8 | 270 | 2.46 | 4.7% | 1.30 | 2.5% | 0.84 | 1.6% | 1.55 | 3.0% | 0.83 | 1.6% | 3.39 |
| Plasma Pool 2 | 118 | 270 | 3.71 | 3.1% | 4.29 | 3.6% | 2.76 | 2.3% | 5.71 | 4.8% | 1.64 | 1.4% | 8.67 |

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2. Linearity:

Linearity studies were performed following the CLSI EP06-A2 guideline using three different reagent lots tested on a representative ACL TOP Family and an ACL TOP Family 50 series model. Normal Pooled Plasma spiked with known concentrations of rivaroxaban was tested in four replicates. Two separate linear regressions were performed. For the first, samples with thirteen different concentrations were evaluated to establish the analytical measuring interval; and for the second, samples with nine different concentrations were evaluated to establish the extended measuring interval.

Based on the results of the Linearity studies and Detection Limit studies, the claimed assay reportable range is 20–1000 ng/mL.

3. Analytical Specificity/Interference:

Interference studies were conducted based on the CLSI EP07-A2 guideline and CLSI EP37 guideline. The study design included one representative instrument model and one regent lot. Potentially interfering endogenous and exogenous substances were spiked into the test samples (rivaroxaban at 50 ng/mL, 180 ng/mL or 400 ng/mL native donor samples collected at peak and trough), and each sample level were run in five replicates on an ACL TOP family instrument.

Rivaroxaban results on ACL TOP Family and ACL TOP Family 50 Series are not affected by the interferents listed up to the concentrations detailed in the table below.

| Interfering Substances | Concentration without Interference |
| --- | --- |
| Hemoglobin | 600 mg/dL |
| Bilirubin (unconjugated) | 40 mg/dL |
| Bilirubin (conjugated) | 40 mg/dL |
| Triglycerides | 921 mg/dL |
| Lupus anticoagulant | dRVVT Screen/Confirm Ratio 2.47
(dRVVT: Diluted Russell Viper Venom Time) |
| Acetylsalicylic acid | 3.00 mg/dL |
| Atorvastatin | 0.075 mg/dL |
| Isosorbide dinitrate | 0.600 mg/dL |
| Ticagrelor | 0.188 mg/dL |
| Warfarin | 7.50 mg/dL |

Rivaroxaban results may be falsely elevated in samples tested post-andexanet alfa administration.

The assay is not intended for the monitoring and dosage adjustment of rivaroxaban.

Administer a reversal agent based on current clinical guidance, and take into consideration other clinical and laboratory findings including the Factor Xa inhibitor dose and time since last dose. For additional information, refer to current clinical guidance and reversal agent prescribing information.

4. Assay Reportable Range:

HemosIL Liquid Anti-Xa Assay Reportable Range: 20–1000 ng/mL rivaroxaban.

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5. Traceability, Stability, Expected Values (Controls, Calibrators, or Methods):

**Traceability**

Calibrator value assignments are traceable to Rivaroxaban supplied by the manufacturer and quantitated in plasmas assayed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Two levels of lyophilized calibrators prepared from human citrated plasma by means of a dedicated process at two different concentrations containing rivaroxaban, buffers, and stabilizers. A house standard lot of HemosIL Rivaroxaban Calibrators is used to value assign each new lot of Rivaroxaban Calibrator 2.

Expected values:

Calibrator 1: 0 ng/mL

Calibrator 2: 500 ng/mL

HemosIL Liquid Anti-Xa assay uses Rivaroxaban Controls when calibrated with HemosIL Rivaroxaban Calibrators. Two levels of lyophilized HemosIL Rivaroxaban controls are prepared from human citrated plasma by means of a dedicated process at two different concentrations containing rivaroxaban, buffers, and stabilizers.

Expected values:

Low Control: 80 ng/mL

High Control: 300 ng/mL

**Sample Stability**

Sample stability studies were performed to support the recommended storage and handling instructions detailed in device labeling. Citrated plasma samples were tested after storage in the following temperature ranges 15–25°C and 2–8°C. The study was performed using one reagent lot with three contrived samples and two native (trough and peak) samples. The contrived samples were tested in quadruplicate and native samples were tested in 8 replicates for baseline then in quadruplicate for various time intervals. The study demonstrated specimens are stable up to 24 hours at 15–25°C and up to 7 days at 2–8°C.

**Reagent Stability**

The results support the following claim: Open Vial 2–8°C for 1 month, On-Board Instrument 15–25°C for 4 days, Shelf-life 2–8°C for 30 Months.

6. Detection Limit:

The limit of blank (LoB), limit of detection (LoD), and limit of quantitation (LoQ) for the test system was determined following CLSI EP17-A2 guideline. Each study design included three reagent lots, an ACL TOP Family and ACL TOP Family 50 Series instruments.

The LoB was determined using four citrated plasma pools containing no rivaroxaban. Three replicate measurements were tested for each of the four plasma pools on five different days on two different instrument models for 60 determinations per reagent lot per instrument model. The LoB was determined to be 2.4 ng/mL.

The LoD was determined using four citrated plasma pools containing low levels of rivaroxaban around 10 ng/mL. Three replicate measurements were tested for each of the four

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plasma pools on five different days on two different instrument models for 60 determinations per reagent lot per instrument model. The LoD was determined to be 8 ng/mL.

The LoQ was determined using four citrated plasma pools spiked with four target concentration 15, 20, 30, 50 ng/mL of rivaroxaban. Eight replicate measurements were tested for each of the four plasma pools on five different days on two different instrument models for 40 determinations per reagent lot per instrument model. The LoQ was determined to be 20 ng/mL.

7. Assay Cut-Off:

Not Applicable.

B Comparison Studies:

1. Method Comparison with Predicate Device:

The method comparison study was conducted using the HemosIL Liquid Anti-Xa assay on three ACL TOP Family instrument models by testing 337 clinical samples collected from patients receiving rivaroxaban at three different US clinical sites. Results from the HemosIL Liquid Anti-Xa were compared to results from a validated rivaroxaban liquid chromatography tandem mass spectrometry (LC-MS/MS) method. Weighted Deming regression analysis was performed for the dataset collected and the result is shown in the following table.

| Sample Number | Rivaroxaban Range (ng/mL) | r | Slope (95% CI) | Intercept (95% CI) |
| --- | --- | --- | --- | --- |
| 337 | 20.3–940.4 | 0.995 | 0.971 (0.953, 0.990) | -0.697 (-3.087, 1.692) |

2. Matrix Comparison:

Not Applicable.

C Clinical Studies:

1. Clinical Sensitivity:

Not Applicable.

2. Clinical Specificity:

Not Applicable.

3. Other Clinical Supportive Data (When 1. and 2. Are Not Applicable):

Not Applicable.

D Clinical Cut-Off:

Not Applicable.

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E Expected Values/Reference Range:

The HemosIL Liquid Anti-Xa assay is not intended to monitor patients on rivaroxaban therapy. Rivaroxaban does not require therapeutic monitoring, thus, there is no standard therapeutic range as the on-therapy range may be different for each individual patient.

VIII Proposed Labeling:

The labeling supports the finding of substantial equivalence for this device.

IX Conclusion:

The submitted information in this premarket notification is complete and supports a substantial equivalence decision.