ACCUMETRICS,INC. VERIFYNOW PRUTEST, ACCUMETRICS, INC. VERIFYNOW PRUTEST

K141427 · Accumetrics, Inc. · JOZ · Jul 24, 2015 · Hematology

Device Facts

• Record ID: K141427
• Device Name: ACCUMETRICS,INC. VERIFYNOW PRUTEST, ACCUMETRICS, INC. VERIFYNOW PRUTEST
• Applicant: Accumetrics, Inc.
• Product Code: JOZ · Hematology
• Decision Date: Jul 24, 2015
• Decision: SESE
• Submission Type: Traditional
• Regulation: 21 CFR 864.5700
• Device Class: Class 2

Intended Use

The VerifyNow™ PRUTest is a whole blood test used in the laboratory or point of care setting to measure the level of platelet P2Y12 receptor blockade. For in vitro diagnostic use. For professional use only.

Device Story

VerifyNow PRUTest measures platelet P2Y12 receptor blockade in citrated whole blood; used in laboratory or point-of-care settings by professionals. Device contains three lyophilized reagent pellets: ADP (for P2Y12-mediated aggregation), TRAP (internal control), and No-Agonist Pellet. Instrument automatically draws blood from closed vacuum collection tube; uses fibrinogen-coated microparticles to bind activated platelet GP IIb/IIIa receptors. Turbidimetric optical detection measures aggregation; output reported in P2Y12 Reaction Units (PRU). Results assist clinicians in assessing platelet inhibition levels. System includes internal controls for sample filling, fluid transfer, and reaction monitoring; requires factory calibration via barcode scanning. Electronic Quality Control (EQC) ensures instrument optics, pneumatics, and calculation accuracy.

Clinical Evidence

Bench testing only. Precision and reproducibility studies (n=9 donors) showed between-instrument variability ≤10%. Method comparison against predicate (n=119 patients) yielded a slope of 1.01 (95% CI: 0.97–1.05) and correlation coefficient R=0.98. Stability studies support 9-month reagent shelf life. Interference testing confirmed no significant impact from common medications/substances. Reference range established using 152 healthy individuals (182-335 PRU).

Technological Characteristics

Turbidimetric optical detection system. Disposable test device contains lyophilized reagent pellets (ADP, TRAP, No-Agonist) with fibrinogen/BSA-coated beads. Instrument-based electronic quality control (EOC) and wet quality controls (WQC). Factory calibration. Results reported in PRU. Standalone instrument. No humidity sensor in test device.

Indications for Use

Indicated for professional use in laboratory or point-of-care settings to measure platelet P2Y12 receptor blockade in whole blood samples from patients, including those with acute coronary syndrome (ACS).

Regulatory Classification

Identification

An automated platelet aggregation system is a device used to determine changes in platelet shape and platelet aggregation following the addition of an aggregating reagent to a platelet-rich plasma.

Predicate Devices

• VerifyNow™ P2Y12 Assay (K051231)

Submission Summary (Full Text)

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Image /page/0/Picture/1 description: The image shows the logo for the U.S. Department of Health & Human Services. The logo consists of a circular seal with the words "DEPARTMENT OF HEALTH & HUMAN SERVICES - USA" around the perimeter. Inside the circle is an abstract image of a stylized eagle with three human profiles incorporated into its design.

Food and Drug Administration 10903 New Hampshire Avenue Document Control Center - WO66-G609 Silver Spring, MD 20993-0002

October 30, 2015

ACCUMETRICS, INC. FRANK LADUCA CHIEF SCIENTIFIC OFFICER 3985 SORRENTO VALLEY BLVD. SUITE B SAN DIEGO. CA92121

Re: K141427

Trade/Device Name: VerifyNow PRUTest Regulation Number: 21 CFR 684.5700 Regulation Name: Automated platelet aggregation system Regulatory Class: Class II Product Code: JOZ Dated: July 23, 2015 Received: July 24, 2015

Dear Dr. LaDuca:

This letter corrects our substantially equivalent letter of July 24, 2015.

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. 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.

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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 Parts 801 and 809); medical device reporting (reporting of medical device-related adverse events) (21 CFR 803); good manufacturing practice requirements as set forth in the quality systems (QS) regulation (21 CFR Part 820); and if applicable, the electronic product radiation control provisions (Sections 531-542 of the Act); 21 CFR 1000-1050.

If vou desire specific advice for your device on our labeling regulation (21 CFR Part Parts 801 and 809), please contact the Division of Industry and Consumer Education at its toll-free number [phone] or [phone] or at its Internet address

http://www.fda.gov/MedicalDevices/ResourcesforYou/Industry/default.htm. Also, please note the regulation entitled. "Misbranding by reference to premarket notification" (21CFR Part 807.97). For questions regarding the reporting of adverse events under the MDR regulation (21 CFR Part 803), please go to

http://www.fda.gov/MedicalDevices/Safety/ReportaProblem/default.htm for the CDRH's Office of Surveillance and Biometrics/Division of Postmarket Surveillance.

You may obtain other general information on your responsibilities under the Act from the Division of Industry and Consumer Education at its toll-free number [phone] or [phone] or at its Internet address

http://www.fda.gov/MedicalDevices/ResourcesforYou/Industry/default.htm.

Sincerely yours,

# Leonthena R. Carrington -S

Leonthena R. Carrington, MS. MBA, MT (ASCP) Director Office of In Vitro Diagnostics and Radiological Health Center for Devices and Radiological Health

Enclosure

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# Indications for Use

510(k) Number (if known) K141427

Device Name VerifyNow PRUTest

Indications for Use (Describe)

The VerifyNow PRUTest is a whole blood test used in the laboratory or point of care setting to measure the level of platelet P2Y12 receptor blockade. For in vitro diagnostic use. For professional use only.

| Type of Use (Select one or both, as applicable) | |
|--------------------------------------------------------------------------------------------------|-------------------------------------------------------------------------------------------------|
| <div> <span style="font-size:100%;">☑ Prescription Use (Part 21 CFR 801 Subpart D)</span> </div> | <div> <span style="font-size:100%;">☐ Over-The-Counter Use (21 CFR 801 Subpart C)</span> </div> |

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#### 510(k) Summary

This 510(k) summary is submitted in accordance with 21 CFR §807.92.

| Owner: | Accumetrics, Inc.<br>3985 Sorrento Valley Blvd.<br>San Diego, CA 92121 USA<br>Contact: Frank M. LaDuca, PhD; Chief Scientific Officer<br>Telephone: [phone]<br>Fax: [phone] |
|----------------------|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Prepared: | 22 July 2015 |
| Trade name: | Accumetrics, Inc. VerifyNow PRUTest ™ |
| Common name: | Platelet reactivity test |
| Classification name: | Automated platelet aggregation system<br>(21 CFR §864.5700; Product Code: JOZ) |
| Predicate device: | VerifyNow P2Y12 Assay, Accumetrics, Inc., k051231 |

#### Device description:

The VerifyNow System is a turbidimetric-based optical detection system that measures plateletinduced aggregation. The system consists of an instrument, a disposable test device and quality control materials. Quality control measures include an instrument based electronic quality control (EOC). two levels of wet quality controls (WOC), internal quality controls, and shipping controls. The instrument controls all assay sequencing, temperature, reagent-sample mixing and performs self-diagnostics. The degree of platelet function is determined and the result is displayed.

The VerifyNow PRUTest device contains three lyophilized reagent pellets in separate reaction chambers within the test device: 1) ADP pellet consisting of a preparation of Fibrinogen and BSA coated beads, adenosine-5-diphosphate (ADP), prostaglandin E1 (PGE1), dye, buffer, and a preservative; 2) TRAP pellet (Internal Control) consisting of a preparation of iso-TRAP (Thrombin Receptor Activating Peptide), Fibrinogen and BSA coated beads, buffer, dye, and a preservative; and 3) No-Agonist Pellet (NAP) consisting of a preparation of BSA coated beads, dye, buffer, and a preservative.

## Test principle:

The patient sample is citrate-anticoagulated whole blood, which is automatically dispensed from the blood collection tube into the test device by the instrument, with no blood handling required by the user. Fibrinogen-coated microparticles are used in the VerifyNow PRUTest device to bind activated platelet GP IIb/IIIa receptors. ADP is incorporated into the assay to activate platelets. and the reagent is formulated to specifically measure P2Y12-mediated platelet aggregation.

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When the activated platelets are exposed to the fibrinogen-coated microparticles, aggregation occurs in proportion to the number of activated platelet receptors. The VerifyNow PRUTest reports results in P2Y12 Reaction Units (PRU).

## Intended use:

The VerifyNow PRUTest is a whole blood test used in the laboratory or point of care setting to measure the level of platelet P2Y12 receptor blockade. For in vitro diagnostic use. For professional use only.

## Indications for use:

Same as the intended use.

## Summary of the technological characteristics of the VerifyNow PRUTest (subject device) compared to VerifyNow P2Y12 Assay (predicate device):

The technological characteristics of the Accumetrics Inc. VerifyNow PRUTest are compared with the VerifyNow P2Y12 Assay (k051231) in the table below. This comparison demonstrates the substantial equivalence of this device to the predicate device. The devices are similar in intended use, principle of operation and specimen type. Both devices have similar analytical performance. The modifications from the predicate device are:

#### Reagents:

- ADP pellet: The reagent pellet was reformulated to contain 80% Fibrinogen/20% BSA coated beads from the previous 100% Fibrinogen coated beads.
- TRAP pellet: This reagent was moved from channel 4 to channel 3. The formulation . was unchanged. This channel is used as an internal assay control.
- NAP pellet: The No-Agonist pellet was introduced to channel 4. The purpose of this ● channel is to prevent a software error and provide no further input into the algorithm for generating the PRU result.

## Controls (WQC):

- Level 1 (B-Carbon Sol Filler): This control was reformulated to contain BSA instead of ● Fish Skin Gelatin.
- . Level 2: The GPRP peptide conjugate length was modified from 2 x 10° MW AD to 500 MW AD.

## Test Device/Instrumentation:

- . The humidity sensor was removed from the test device.
- Port cover added to instrument. ●
- The instrument reports results for the ADP pellet measurement in PRU units.

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| Item | Accumetrics, Inc.<br>VerifyNow PRUTest<br>(Subject Device) | Accumetrics, Inc.<br>VerifyNow P2Y12 Assay (k051231)<br>(Predicate Device) |
|---------------------------|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| | Similarities | |
| Principle of<br>Operation | Fibrinogen bead / platelet aggregation<br>with optical detection. | Same. |
| Specimen Type | Citrate-anticoagulated whole blood. | Same. |
| Testing Site | Point of care or laboratory. | Same. |
| Assay Results | P2Y12 Reaction Units (PRU). | Same. |
| Controls | Pre-packaged, two-level Wet Quality<br>Controls (WQC); Internal control in<br>each assay device. | Same. |
| Test Procedure | 1. Add cartridge to instrument.<br>2. Attach blood collection tube.<br>No other operator intervention. | Same. |
| Calibration | Factory. | Same. |
| Time to result | 3 minutes. | Same. |
| Analytical Claims | Hematocrit range = 33-52% | Same. |
| | Modifications | |
| Intended Use | The Accumetrics, Inc. VerifyNow<br>PRUTest is a whole blood test used in<br>the laboratory or point of care setting<br>to measure the level of platelet P2Y12<br>receptor blockade. For <i>in vitro</i><br>diagnostic use. For professional use<br>only. | The VerifyNow P2Y12 Assay is a<br>whole blood assay used in the<br>laboratory or point of care setting to<br>measure the level of platelet P2Y 12<br>receptor blockade. |
| Reagents | ADP Pellet contains 80%<br>Fibrinogen/20% BSA coated beads | ADP Pellet contains 100% Fibrinogen<br>coated beads |
| | TRAP Pellet found in channel 3. | TRAP Pellet found in channel 4. |
| | NAP Pellet introduced into channel 4. | No NAP Pellet |
| | Level 1 (B-Carbon Sol Filler) contains<br>BSA | Level 1 (F-Carbon Filler) contains Fish<br>Skin Gelatin |
| | Level 2 GPRP Peptide Conjugate<br>length truncated to 500,000 kDa | Level 2 GPRP |
| Test Device | No humidity sensor | Contains humidity sensor |
| Analytical Claims | Stability = 9 months (ongoing real-<br>time study in process) | Stability = 18 months |

# Table A. Comparison of Predicate to Subject Device

## Reference range:

A reference range study (Healthy Donor PRU Reference Range) was conducted to measure platelet function in a healthy donor population using VerifyNow PRUTest. A total of 152 evaluable healthy donors were recruited and their blood samples tested with the VerifyNow PRUTest. The data were evaluated as recommended in EP28-A3c "Defining, Establishing and

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Verifying Reference Intervals in the Clinical Laboratory; Approved Guideline – Third Edition. " The reference range determined from this study, expressed as the central 95% Confidence Interval of the mean, is 182-335 PRU. The data are summarized in the table below.

Table B. Reference Range

| N | Mean | SD | 95% confidence interval (CI) | |
|-----|------|----|------------------------------|----------------------|
| | | | Lower limit (95% CI) | Upper limit (95% CI) |
| 152 | 266 | 42 | 182 (116–197) | 335 (324–354) |

## Expected PRUTest values:

## Treatment-naïve patients in the intended use population

Whole blood samples from 84 patients with acute coronary syndrome (ACS) that were not receiving a P2Y12 receptor inhibiting drug were tested with VerifyNow PRUTest. The data were evaluated as recommended in EP28-A3c "Defining, Establishing and Verifying Reference Intervals in the Clinical Laboratory: Approved Guideline – Third Edition." The range of values expressed as the central 95% Confidence Interval of the mean is 180-376 PRU. The data are summarized in the table below.

Table C. Expected PRU values in treatment-naïve patients

| N | Mean<br>(PRU) | SD<br>(PRU) | 95% confidence interval (CI) | |
|----|---------------|-------------|---------------------------------------|---------------------------------------|
| 84 | 274 | 48 | Lower limit (95% CI)<br>180 (160–200) | Upper limit (95% CI)<br>376 (358–395) |

#### ACS patients receiving treatment with clopidogrel

Whole blood samples from 71 patients with ACS receiving dual treatment with aspirin and clopidogrel were tested with VerifyNow PRUTest. The data were evaluated as recommended in EP28-A3c "Defining, Establishing and Verifying Reference Intervals in the Clinical Laboratory: Approved Guideline - Third Edition. " The range of values expressed as the central 95% Confidence Interval of the mean is 6-300 PRU. The data are shown in the table below.

Table D. Expected PRU values in patients receiving clopidogrel treatment

| N | Mean<br>(PRU) | SD<br>(PRU) | 95% confidence interval (CI) | |
|----|---------------|-------------|------------------------------|---------------|
| 71 | 156 | 73 | 6 (0-34) | 300 (269-329) |

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## Performance studies

## Method Comparison

A method comparison study (VerifyNow P2Y12 and PRUTest Method Comparison Study) was performed using the VerifyNow P2Y12 Assay System (predicate device) and the VerifyNow PRUTest System (subject device). A total of three investigational intended use sites were used to gather data for this investigation. The VerifyNow test analysis was performed at each site by intended users who were trained by the manufacturer. Test device and instrument configurations are described below.

## Results:

The PRU results from both the VerifyNow P2Y12 (predicate device) and VerifyNow PRUTest (subject device) demonstrate comparable performance, with a slope not statistically significantly different from 1.0 and an intercept not statistically significantly different from 0, and a correlation (r) greater than 0.9 as shown in the table below.

| | | Regression | Slope | Slope | Intercept | Correlation |
|-----------------|-----|---------------|-------------|---------|--------------|---------------|
| Comparison | N | Method | (95% CI) | p-value | (95% CI) | Coefficient R |
| P2Y12 Rep 1 vs. | 119 | Ordinary | 1.01 | | -0.77 | 0.98 |
| PRUTest Rep 1 | | Least Squares | (0.97—1.05) | 0.56 | (-8.00-6.50) | |
| P2Y12 Avg. vs. | 119 | Ordinary | 1.04 | | -4.57 | |
| PRUTest Avg. | | Deming | (1.00-1.07) | 0.07 | (-11.2-2.05) | 0.98 |

## Table E. Method Comparison

# CLSI Precision Studies

Complex precision for the VerifyNow PRUTest was assessed using Wet Quality Controls (WOC). Levels 1 and 2, and intermediate precision was assessed using whole blood samples from healthy donors and from ACS patients receiving clopidogrel with or without aspirin by testing three different lots of test devices and three instruments. Data were analyzed in accordance with Clinical Laboratory Standards Institute (CLSI) guidance EP05-A2, "Evaluation of Precision Performance of Quantitative Measurement Methods; Approved Guideline – Second Edition."

Testing over several days with samples from the same patient is expected to yield variable results between days because platelet reactivity in response to orally ingested medications known to vary based upon time between dosages, as well as the time between dosage and blood collection. A summary of the precision verification studies performed is shown in Table F.

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## Table F. Description of precision studies

| Type | Description |
|---------------------------------------------------------------------------------------------------------|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Complex precision using WQC Levels 1<br>and 2 | Measured intermediate precision with WQC<br>Level 2 samples over multiple days. |
| Complex precision between instruments<br>using WQC Level 2 | Measured Level 2 WQC between-instrument<br>precision over multiple days using a single lot of<br>PRUTest devices. |
| Intermediate precision using whole blood<br>samples from donors and ACS patients | Measured intermediate precision and instrument-<br>to-instrument precision with whole blood<br>samples over multiple days using subjects on<br>dual antiplatelet therapy, healthy subjects dosed<br>in vivo with P2Y12 clopidogrel / P2Y12<br>inhibitor, and normal healthy donors |
| Intermediate precision between<br>instruments using whole blood samples<br>from donors and ACS patients | Measured whole blood between-instrument<br>precision over multiple days using a single lot of<br>PRUTest devices. |

Complex precision using WQC Levels 1 and 2:

WQC Level 1 and Level 2 samples were tested over 20 nonconsecutive days. WQC Level 1 is a negative control (blank) from which no aggregation is expected. All WQC Level 1 results fell within the acceptable range of ≤ 30 PRU and data are not shown. WQC Level 2 results are detailed below. Analysis of variance (ANOVA) was used to estimate the components of variance in accordance with CLSI guidance EP05-A2.

# Table G. Reproducibility using WQC Level 2

# Between-lot

| N | Mean<br>PRU | Within-run | | Between-run | | Between-day | | Between-lot | | Total | |
|-----|-------------|------------|-----|-------------|-----|-------------|-----|-------------|-----|-------|-----|
| | | SD | %CV | SD | %CV | SD | %CV | SD | %CV | SD | %CV |
| 240 | 300.5 | 13.6 | 4.5 | 7.7 | 2.5 | 8.3 | 2.8 | 13.5 | 4.5 | 22.2 | 7.4 |

## Between instrument

| N | Mean<br>PRU | Within-run | | Between-run | | Between-day | | Between-lot | | Total | |
|-----|-------------|------------|-----|-------------|-----|-------------|-----|-------------|-----|-------|-----|
| | | SD | %CV | SD | %CV | SD | %CV | SD | %CV | SD | %CV |
| 240 | 302.7 | 22.6 | 7.5 | 0.0 | 0.0 | 8.5 | 2.8 | 0.0 | 0.0 | 24.2 | 8.0 |

Intermediate precision using whole blood samples

Whole blood samples from nine donors were drawn once or twice a day, run in duplicate, using three device lots on three instruments over a minimum of five non-consecutive days. Analysis of variance (ANOVA) was used to estimate the components of variance within individual donors in accordance with CLSI guidance EP05-A2. The results of these precision measurements are

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detailed in the following two tables. Table H evaluates precision across multiple PRUTest device lots using whole blood samples. Table I evaluates precision across multiple VerifyNow instruments using whole blood samples.

| ID | Rx1 | Days | Runs | Reps | Mean PRU | SD | Within-run<br>%CV | SD | Between-run<br>%CV | SD | Between-day<br>%CV | SD | Between-lot<br>%CV |
|----|-----|------|------|----------------------|----------|------|-------------------|------|--------------------|------|--------------------|------|--------------------|
| | | | | Acceptance criteria: | | | <10 | | <10 | | N/A2 | | <10 |
| 1 | 2 | 5 | 1 | 30 | 64 | 4.7 | 7.4 | --- | --- | 9.6 | 15.0 | 0.03 | 0.03 |
| 2 | 0 | 10 | 2 | 118 | 244 | 10.4 | 4.2 | 0.0 | 0.0 | 21.9 | 9.0 | 0.0 | 0.0 |
| 3 | 1 | 5 | 2 | 60 | 162 | 6.3 | 3.9 | 5.1 | 3.1 | 17.7 | 10.9 | 0.0 | 0.0 |
| 4 | 1 | 5 | 2 | 30 | 190 | 12.0 | 6.3 | --- | --- | 15.1 | 7.9 | 0.0 | 0.0 |
| 5 | 0 | 10 | 2 | 120 | 289 | 11.2 | 3.9 | 7.7 | 2.7 | 27.0 | 9.3 | 0.0 | 0.0 |
| 6 | 2 | 5 | 1 | 30 | 221 | 8.9 | 4.0 | --- | --- | 17.0 | 7.7 | 0.0 | 0.0 |
| 7 | 1 | 8 | 2 | 96 | 123 | 9.6 | 7.8 | 18.7 | 15.1 | 57.6 | 46.7 | 0.0 | 0.0 |
| 8 | 1 | 10 | 2 | 114 | 216 | 13.0 | 6.0 | 7.3 | 3.4 | 16.5 | 7.6 | 0.0 | 0.0 |
| 9 | 2 | 5 | 1 | 30 | 163 | 10.9 | 6.7 | --- | --- | 13.3 | 8.2 | 0.0 | 0.0 |

Table H: Precision of whole blood samples - multiple lot experiment

Table I: Precision of whole blood samples - multiple instrument experiment

| ID | Rx1 | Days | Runs | Reps | Mean PRU | SD | Within-run %CV | SD | Between-run %CV | SD | Between-day %CV | SD | Between-lot %CV |
|----|-----|------|------|------|----------------------|------|----------------|------|-----------------|------|-----------------|------|-----------------|
| | | | | | Acceptance criteria: | | | | <10 | | N/A2 | | <10 |
| 1 | 2 | 5 | 1 | 30 | 67 | 7.3 | 10.8 | --- | --- | 9.3 | 13.8 | 0.03 | 0.03 |
| 2 | 0 | 8 | 2 | 89 | 253 | 13.6 | 5.4 | 0.0 | 0.0 | 20.4 | 8.1 | 9.3 | 3.7 |
| 3 | 1 | 5 | 2 | 60 | 160 | 9.3 | 5.8 | 4.1 | 2.5 | 18.1 | 11.3 | 3.6 | 2.3 |
| 44 | 1 | 0 | 0 | 0 | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 5 | 0 | 9 | 2 | 101 | 290 | 10.7 | 3.7 | 5.4 | 1.9 | 21.4 | 7.4 | 11.4 | 3.9 |
| 6 | 2 | 5 | 1 | 30 | 229 | 7.9 | 3.5 | --- | --- | 14.4 | 6.3 | 8.1 | 3.5 |
| 7 | 1 | 5 | 2 | 60 | 115 | 8.8 | 7.6 | 20.4 | 17.7 | 65.9 | 57.1 | 0.0 | 0.0 |
| 8 | 1 | 5 | 2 | 54 | 231 | 14.7 | 6.4 | 12.1 | 5.3 | 9.2 | 4.0 | 7.9 | 3.4 |
| 9 | 2 | 5 | 1 | 30 | 160 | 11.2 | 7.0 | --- | --- | 11.6 | 7.3 | 0.0 | 0.0 |

1 0 = healthy, no treatment; 1 = dual therapy with clopidogrel plus aspirin; 2 = healthy, given clopidogrel Key: 75 mg/day: 2 not applicable as acceptance criteria for this parameter is undefined in the text; 3 between-lot SD of 0 and %CV of 0 can be observed when the contribution of one source of variability is very small relative to other sources of variability, or if all of the sample elements are the same; " Donor 4 had insufficient blood volume to allow for inclusion in the multiple instrument experiment

# Open Pouch Device Stability

Extended Open Pouch Stability at Elevated Humidity was conducted to evaluate the stability of the reagents in the PRUTest test device when exposed to environmental humidity. The results of the study are detailed below.

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| Time<br>unpouched | PRU whole blood | | PRU WQC Level 2 |
|-------------------|-----------------|---------------|-------------------------|
| | Mean (n=3) | Pct. recovery | Allowable range 230–362 |
| 0 | 274 | 100.0 | 286 |
| 10 | 277 | 101.1 | 254 |
| 14 | Attention 32* | N/A | 3 |
| 18 | Attention 32* | N/A | 7 |
| 22 | Attention 32* | N/A | 18 |
| 26 | Attention 32* | N/A | 21 |

Table J. Open Pouch Device Stability

* Attention 32 is generated because the initial DC value is too high, and the VerifyNow system recognizes that either the blood sample and reagent is too clear, or the reagent pellet is not performing as intended

Percent recovery of VerifyNow PRUTest values in whole blood met the acceptance criteria after exposure to 61-73% RH and 21-24°C temperatures for 10 hours. PRU result with WQC Level 2 remained within the allowable range for 10 hours under the same conditions. These findings support a claim of 10 hour open-pouch stability.

#### Sample Wait Time and Sample Stability

A sample stability and sample wait time study was conducted to determine the appropriate values. Sample wait time was determined to be 10 minutes, meaning that a blood sample must be stored for a minimum of 10 min after collection by venipuncture. Sample stability was determined to be four hours, meaning that the blood sample must be assayed prior to four hours from the time of blood draw.

#### Interfering substances

An "interference screen," was performed which followed the recommendations outlined in Clinical Laboratory Standards Institute (CLSI) guidance EP07-A2, "Interference Testing in Clinical Chemistry; Approved Guideline - Second Edition." Compounds were tested at a worstcase concentration - at least three-fold greater than therapeutics concentrations. Samples from 11 different donors were assayed in singlicate across eight VerifyNow PRUTest instruments with one device lot of VerifyNow PRUTest using a randomized scheme. Interference testing was performed with six donors using native blood samples and blood samples spiked with 2-MeSAMP, a P2Y12 receptor inhibitor.

| | | | 2-MeSAMP not added | | 2-MeSAMP added | | | | | |
|----------------------------------|---------|-------------|--------------------|----------|----------------|-------------|----------|--|--|--|
| | | Mean<br>PRU | Mean<br>PRU | Pct. | Mean<br>PRU | Mean<br>PRU | Pct. | | | |
| Interferent / Test concentration | | Control | Test | Recovery | Control | Test | Recovery | | | |
| A3P5PS2 | 100 µM | 274.3 | 265.0 | 96.6 | 96.8 | 97.0 | 100.3 | | | |
| Acetaminophen | 1.32 mM | 274.3 | 263.7 | 96.2 | 96.8 | 99 1 | 102.5 | | | |
| Betamethasone | 64 uM | 274.3 | 270.5 | 98.6 | 96.8 | 95.1 | 98.3 | | | |
| Caffeine | 306 uM | 274.3 | 264.8 | 96.5 | 96.8 | 99.5 | 102.8 | | | |

#### Table K. Interfering Substance

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| Captopril | 23 μΜ | 274.3 | 262.9 | 95.9 | 96.8 | 100.0 | 103.4 |
|---------------------|-----------|-------|-------|-------|-------|-------|-------|
| Catechin | 86 μΜ | 274.3 | 273.1 | 99.6 | 96.8 | 99.0 | 102.3 |
| Celecoxib | 8.5 µg/mL | 318.3 | 319.1 | 98.0 | 129.9 | 128.0 | 98.6 |
| Cilostazol | 60 μΜ | 328.8 | 326.6 | 99.4 | 139.6 | 141.8 | 101.5 |
| Cimetidine | 79 μΜ | 328.8 | 315.3 | 95.9 | 139.6 | 140.9 | 100.9 |
| DMSOb | 0.11% | 318.3 | 322.1 | 98.9 | 129.9 | 131.9 | 101.5 |
| Dipyridamole | 20 μΜ | 328.8 | 319.9 | 97.3 | 139.6 | 146.8 | 105.1 |
| Diltiazem | 15 μΜ | 328.8 | 321.1 | 97.7 | 139.6 | 139.9 | 100.2 |
| Ethanol | 87 mM | 328.8 | 321.1 | 97.3 | 139.6 | 141.5 | 101.3 |
| Fish oil | 32 mg/mL | 328.8 | 317.1 | 96.5 | 139.6 | 142.0 | 101.7 |
| Glucosamine HCl | 9.4 μΜ | 201.4 | 205.8 | 102.2 | 139.4 | 137.2 | 98.4 |
| Heparin, LMWc | 1833 U/L | 201.4 | 208.6 | 103.6 | 139.4 | 141.8 | 101.7 |
| Hydrochlorothiazide | 20 μΜ | 201.4 | 204.3 | 101.4 | 139.4 | 138.5 | 99.4 |
| Ibuprofen | 2.4 mM | 201.4 | 200.0 | 99.3 | 139.4 | 137.4 | 98.6 |
| Insulin | 3 ng/mL | 201.4 | 208.9 | 103.7 | 139.4 | 134.9 | 96.8 |
| Lidocaine | 51 μΜ | 201.4 | 206.8 | 102.7 | 139.4 | 137.5 | 99.6 |
| Nitroglycerin | 0.1 µg/mL | 249.5 | 241.0 | 96.6 | 129.8 | 132.1 | 101.8 |
| Norfluoxitine | 7.27 μΜ | 249.5 | 241.4 | 96.7 | 129.8 | 132.4 | 102.0 |
| Norverapamil | 4.5 μΜ | 249.5 | 243.3 | 97.5 | 129.8 | 133.8 | 103.1 |
| Omeprazole | 4.5 μΜ | 269.1 | 265.6 | 98.8 | 142.0 | 143.9 | 101.3 |
| Oxypurinol | 99 μΜ | 249.5 | 242.0 | 97.0 | 129.8 | 131.3 | 101.2 |
| Pravastatin | 56 μΜ | 249.5 | 249.1 | 99.9 | 129.8 | 129.9 | 100.1 |
| Propranolol | 7.7 μΜ | 249.5 | 242.8 | 97.3 | 129.8 | 135.6 | 104.5 |
| Salicylic acid | 4.3 mM | 249.5 | 241.1 | 96.6 | 129.8 | 128.9 | 99.3 |
| Streptokinase | 400 U/mL | 325.6 | 317.6 | 97.5 | 129.9 | 132.6 | 102.1 |
| Theophylline | 220 μΜ | 325.6 | 318.3 | 97.8 | 129.9 | 131.4 | 101.2 |
| L-Thyroxine | 32 nM | 201.4 | 198.5 | 98.6 | 139.4 | 138.4 | 99.3 |
| α-Tocopherol | 58 μΜ | 318.3 | 311.1 | 96.5 | 129.9 | 133.1 | 102.5 |
| Triglycerides | 37 mM | 318.3 | 315.9 | 97.0 | 129.9 | 129.3 | 99.5 |
| Warfarin | 32 μΜ | 318.3 | 319.1 | 98.0 | 129.9 | 129.4 | 99.6 |

ª Putative thiamine-phosphate pyrophosphorylase

b DMSO = dimethyl sulfoxide c LMW = low molecular weight

Each interferent spiked into whole blood samples at the above concentrations showed percent recovery which deviated less than 10% from baseline PRUTest values. Therefore, none of these substances significantly interfere with VerifyNow PRUTest results.

## Stability

# Wet Quality Controls (WQC)

Device stability of the VerifyNow PRUTest reagent device has been evaluated over nine months. Three lots of WQC level 1, three lots of WQC Level 2 and three lots of reagent devices are stored at -70°C and at 20±5 °C. Each of the three lots of WQC levels 1 and 2 were tested with the three lots of reagent devices at time zero, 3, 6, and 9 months under both storage conditions.

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# WQC Level 1

WQC Level 1 simulates a patient sample with highly inhibited platelets and does not produce a significant aggregation profile. The CVs for Level 1 WOC are not calculated because the nominal PRU value is zero. The acceptance criterion for stability of WOC Level 1 was a PRU result < 30. At the nine-month time point, the range of PRU values generated with WOC Level 1 did not exceed 30 PRU.

## WQC Level 2

The acceptance criterion for stability for WQC Level 2 was percent recovery ≥ 90 to ≤ 110% of the baseline PRU result. At the nine-month time point, percent recovery of WQC Level 2 passed acceptance criterion for stability when compared to time zero.

#### Isochronous reagent stability study with donor whole blood samples

Each of three reagent lots was tested at time zero, 3, 6, and 9 months using whole blood samples from normal donors. The acceptance criterion for stability for whole blood samples was percent recovery ≥ 90 to ≤ 110% of the baseline PRU result. At the nine-month time point, percent recovery of PRUTest results in whole blood passed acceptance criterion for stability when compared to time zero. This further supports the nine month stability claim for VerifyNow™ PRUTest reagent storage at 20±5 ℃.

#### Conclusion

The results of this study indicate that the PRUTest device is stable for up to nine months when stored at ambient temperature (20°C ± 5°C).

## Table L. WQC Level 1 Results: 3, 6, and 9 Month Stability

| 20 ± 5°C Condition | | | | | | | |
|--------------------|-----------|----------|-----------|-----|-----------|-----|-----------|
| Time Zero | 3 Months | 6 Months | 9 Months | | | | |
| N | PRU Range | N | PRU Range | N | PRU Range | N | PRU Range |
| 324 | 1 - 5 | 108 | 1 - 3 | 108 | 0 - 4 | 108 | 0 - 3 |

At all test intervals, all Level 1 WQC results met the acceptance criteria of ≤30.

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| | | 3 Months | | | | | 6 Months | | | | | | | 9 Months | | | | | | | | |
|-------|------------------|----------|------|------|----|------|----------|------------------------------------------------|----|------|------|----|------|----------|------------------------------------------------|----|------|------|----|------|------|----------------------------------------------|
| | | N | Mean | SD | N | Mean | SD | %<br>Difference<br>from -<br>70°C<br>Condition | N | Mean | SD | N | Mean | SD | %<br>Difference<br>from -<br>70°C<br>Condition | N | Mean | SD | N | Mean | SD | %<br>Difference<br>from<br>70°C<br>Condition |
| WQC | PRUTest<br>Lot 1 | 6 | 296 | 11.6 | 6 | 289 | 6.0 | -2.1 | 6 | 297 | 12.1 | 6 | 291 | 8.2 | -2.0 | 6 | 286 | 4.9 | 6 | 287 | 13.5 | 0.3 |
| Lot 1 | PRUTest<br>Lot 2 | 6 | 281 | 9.0 | 6 | 276 | 14.2 | -2.0 | 6 | 280 | 17.9 | 6 | 288 | 10.4 | 3.2 | 6 | 285 | 13.0 | 6 | 273 | 7.2 | -4.2 |
| | PRUTest<br>Lot 3 | 6 | 271 | 3.8 | 6 | 265 | 11.8 | -2.0 | 6 | 265 | 11.9 | 6 | 281 | 10.3 | 6.2 | 6 | 276 | 18.0 | 6 | 270 | 13.2 | -2.1 |
| WQC | PRUTest<br>Lot 1 | 6 | 285 | 7.7 | 6 | 293 | 7.0 | 2.8 | 6 | 287 | 22.8 | 6 | 291 | 10.3 | 1.1 | 6 | 287 | 9.2 | 6 | 277 | 9.3 | -3.5 |
| Lot 2 | PRUTest<br>Lot 2 | 6 | 278 | 6.5 | 6 | 270 | 7.8 | -3.0 | 6 | 292 | 11.6 | 6 | 280 | 8.4 | -4.0 | 6 | 282 | 12.0 | 6 | 268 | 11.1 | -4.9 |
| | PRUTest<br>Lot 3 | 6 | 272 | 7.9 | 6 | 262 | 7.2 | -3.6 | 6 | 280 | 11.8 | 6 | 266 | 10.0 | -4.9 | 6 | 274 | 8.6 | 6 | 263 | 11.8 | -4.0 |
| WQC | PRUTest<br>Lot 1 | 6 | 276 | 11.2 | 6 | 288 | 5.5 | 4.3 | 6 | 291 | 22.6 | 6 | 285 | 16.1 | -2.2 | 6 | 295 | 11.5 | 6 | 285 | 4.6 | -3.4 |
| Lot 3 | PRUTest<br>Lot 2 | 6 | 271 | 9.8 | 6 | 266 | 6.9 | -1.7 | 6 | 292 | 12.6 | 6 | 286 | 20.6 | -2.0 | 6 | 278 | 12.0 | 6 | 284 | 13.4 | 2.0 |
| | PRUTest<br>Lot 3 | 6 | 269 | 11.9 | 6 | 263 | 9.8 | -2.2 | 6 | 267 | 8.7 | 6 | 270 | 23.3 | 1.4 | 6 | 264 | 4.8 | 6 | 272 | 5.6 | 3.0 |
| | Aggregated | 54 | 278 | 11.8 | 54 | 275 | 14.4 | -1.1 | 54 | 283 | 18.0 | 54 | 282 | 15.5 | -0.5 | 54 | 281 | 13.4 | 54 | 275 | 12.5 | -1.9 |

| Table M. WQC Level 2 Results (Comparison to Control [-70°C]): 3, 6, and 9 months | | |
|----------------------------------------------------------------------------------|--|--|
|----------------------------------------------------------------------------------|--|--|

The table above shows Level 2 WQC Results from the three, six, and nine month time point results using control PRUTest devices stored at ≤ -70 ℃ compared to those stored at 20 ± 5° C. All lots fell within ± 10% of the control condition (≤ -70 °C)

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| | | | 9 Months | | | | | |
|---------|---------------|---|-----------------|------|--------------------|----------|------|-----------------------------------|
| | | | -70 C Condition | | 20 ± 5°C Condition | | | |
| | | N | PRU Mean | SD | N | PRU Mean | SD | % Difference From -70 C Condition |
| Donor 1 | PRUTest Lot 1 | 3 | 254 | 12.1 | 3 | 235 | 23.4 | -7.6 |
| Donor 1 | PRUTest Lot 2 | 3 | 262 | 14.6 | 3 | 239 | 22.0 | -8.9 |
| Donor 1 | PRUTest Lot 3 | 3 | 256 | 4.2 | 3 | 274 | 65.0 | 7.2 |
| Donor 1 | Aggregated | 9 | 257 | 10.4 | 9 | 249 | 40.8 | -3.2 |
| Donor 2 | PRUTest Lot 1 | 3 | 184 | 5.8 | 3 | 195 | 6.9 | 6.2 |
| Donor 2 | PRUTest Lot 2 | 3 | 196 | 9.9 | 3 | 193 | 4.2 | -1.5 |
| Donor 2 | PRUTest Lot 3 | 3 | 184 | 10.0 | 3 | 213 | 45.7 | 15.7 |
| Donor 2 | Aggregated | 9 | 188 | 9.6 | 9 | 200 | 25.2 | 6.6 |

Table N. Stability at nine-month time point in whole blood compared to -70°C Condition

Whole blood PRU results are shown above Table . Donors were tested at the nine month time point with PRUTest devices stored at 20 ± 5°C and compared to devices stored at ≤ -70°C. For all three PRUTest device lots and both donors the aggregated lot data at (20 ± 5°C) fell within ± 10% of the control condition (≤ -70 ℃).

Table O. Stability at nine-month time point in whole blood inhibited with 2-MeSAMP compared to newly manufactured devices

| | | Recently<br>Manufactured Lot | | | 20 ± 5°C Condition | | | | |
|---------------|-------------------------------------------|------------------------------|-------------|------|--------------------|----|-------------|------|---------------------------------------------------|
| | | N | PRU<br>Mean | SD | | N | PRU<br>Mean | SD | % Difference From<br>Recently<br>Manufactured Lot |
| Donor<br>399 | Recently<br>Manufactured<br>Lot (Control) | 5 | 140 | 13.8 | | 5 | 140 | 11.9 | -0.4 |
| | | | | | | 5 | 125 | 13.0 | -10.7 |
| | | | | | | 5 | 136 | 9.0 | -2.7 |
| | | | | | Aggregated | 15 | 134 | 12.4 | -4.6 |
| Donor<br>1077 | Recently<br>Manufactured<br>Lot (Control) | 5 | 130 | 20.3 | | 5 | 140 | 10.3 | 7.4 |
| | | | | | | 5 | 130 | 27.3 | 0.3 |
| | | | | | | 5 | 144 | 16.8 | 2.4 |
| | | | | | Aggregated | 15 | 138 | 18.9 | 6.1 |

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Inhibited whole blood PRU results are shown above Table. Donors were tested at the nine month time point with PRUTest devices stored at 20 ± 5℃ and compared to newly manufactured devices. For all three PRUTest device lots and both donors the aggregated lot data at (20 ± 5°C) fell within ± 10% of the control condition.

# Conclusions drawn from the nonclinical and clinical performance studies:

The results of the bench and clinical performance testing demonstrate that the VerifyNow PRUTest (subject device) is substantially equivalent in performance to the predicate VerifyNow P2Y12 Assay (predicate device).