Predicate Devices

Reference Devices

Not Found

AI/ML (Artificial Intelligence / Machine Learning)

No
The device is a blood collection tube with an additive for DNA stabilization. The description focuses on the physical characteristics of the tube, the additive, and the performance of the collected blood sample for downstream molecular diagnostic testing. There is no mention of any computational or analytical components that would utilize AI or ML.

Therapeutic

No.

The device is intended to collect, anticoagulate, stabilize, transport, and store whole blood samples for the preparation of human DNA for use with molecular diagnostic test methods, not to provide therapy.

Diagnostic

No

This device is designed to collect, anticoagulate, stabilize, transport, and store a blood sample for DNA preparation, which is then used with molecular diagnostic test methods. It is a sample collection and preservation device, not a device that itself performs a diagnostic assay or makes a diagnosis.

SaMD (Software as a Medical Device)

No

The device is a physical blood collection tube with chemical additives and a vacuum, clearly a hardware medical device, not software.

IVD (In Vitro Diagnostic)

Based on the provided information, the PAXgene Blood DNA Tube is an IVD (In Vitro Diagnostic) device.

Here's why:

Intended Use: The intended use explicitly states that the device is for collecting, stabilizing, transporting, and storing blood samples for "molecular diagnostic test methods." This directly aligns with the definition of an IVD, which are devices used to examine specimens from the human body to provide information for diagnostic purposes.
Performance Characteristics: The document details performance studies conducted to demonstrate the suitability of the collected DNA for use with specific FDA-cleared molecular diagnostic tests. This is a key requirement for IVD devices.
Clinical Testing: The document describes clinical testing performed with FDA-cleared molecular diagnostic tests using blood collected in the device. This type of testing is characteristic of IVD device validation.
Predicate Device: A predicate device (BD Vacutainer® PPT™ Plasma Preparation Tube) is listed, which is common in the regulatory submission process for IVD devices.

While the device itself is a sample collection and preparation device, its intended use is specifically to provide a sample suitable for in vitro diagnostic testing. Therefore, it falls under the regulatory scope of IVD devices.

PCCP (Predetermined Change Control Plan) Authorized

N/A

Overview

Intended Use / Indications for Use

The PAXgene Blood DNA Tube is intended to collect, anticoagulate, stabilize, transport, and store a venous whole blood sample for preparation of human DNA for use with molecular diagnostic test methods that require DNA.

The performance characteristics of this device have not been established for molecular diagnostic assays in general. Users must validate use of product for their specific molecular diagnostic assay.

Product codes

PJE

Device Description

The PAXgene® Blood DNA Tube is a sterile, single use, plastic, evacuated blood collection tube with a BD Hemogard™ closure assembly and a measured quantity of K2EDTA additive. The additive quantity dispensed into each tube is designed to match the nominal blood draw volume of 2.5 mL. The tube is made of polyethylene terephthalate (PET) plastic which functions to maintain vacuum within the tube, allowing for accurate and consistent blood draw for the duration of the shelf life of the tube. A predetermined vacuum is drawn inside the tube that is sealed with a BD Hemogard™ closure which consists of a rubber stopper plus BD Hemogard™ shield.

The tube is intended to be placed inside a tube holder or an adaptor that contains a needle designed to pierce the tube closure and allow blood to flow into the tube. Once the vein has been penetrated (using either a standard blood collection needle or a blood collection set), the tube is pushed into the holder, and the blood enters the tube. Once a tube has drawn the appropriate amount of blood, it is disengaged from the holder and inverted the recommended number of times (8–10) to mix the additive with the blood.

The DNA in whole blood collected in the PAXgene® Blood DNA Tube has been shown to be suitable for molecular diagnostic testing for 14 days at room temperature (18–25°C), 28 days refrigerated (2–8°C), 3 days at 35°C, up to 52 weeks frozen (–20°C), or when subjected to up to three freeze-thaw cycles. The PAXgene® Blood DNA Tube is robust with respect to mishandling including reduced inversions and partial blood draw. The product shelf life is one year from the date of manufacture including limited storage temperature excursions which simulate shipping conditions from –20°C to 45°C.

The PAXgene® Blood DNA Tube is available as a 13 x 75 mm tube with a 2.5 mL nominal blood draw. The referenced first dimension represents the diameter of the tube and the second dimension represents the length of the tube.

Mentions image processing

Not Found

Mentions AI, DNN, or ML

Not Found

Input Imaging Modality

Not Found

Anatomical Site

Venous whole blood sample

Indicated Patient Age Range

≥ 18 years of age

Intended User / Care Setting

Not Found

Description of the training set, sample size, data source, and annotation protocol

Not Found

Description of the test set, sample size, data source, and annotation protocol

Not Found

Summary of Performance Studies (study type, sample size, AUC, MRMC, standalone performance, key results)

Performance Characteristics of PAXgene Blood DNA Tube by Sample Preparation Method:
- DNA Yield, Concentration and Purity Summary: Data from 581 specimens from approximately 200 consented subjects was used with an automated magnetic bead based DNA extraction kit. Data from 540 specimens from 152 consented subjects was used with an automated silica membrane based DNA extraction kit. Key results showed mean DNA yield, concentration, and purity, with 95% of samples meeting specified thresholds.
Performance Characteristics of PAXgene Blood DNA Tube by Molecular Diagnostic Test Methods:
- Clinical testing was performed on blood collected in both the subject and predicate devices with five (5) FDA cleared DNA based molecular diagnostic tests across four (4) external clinical test sites and one (1) internal test site.
- Test Results by Site (Table 4): Overall high percentage of correct calls (ranging from 97.3% to 100%) for CF Assay, HLA Assay 1, Thrombophilia, HLA Assay 2, and HLA Assay 3 across multiple sites.
- Test Results by Study (Table 5): Consistent 100% correct calls for reproducibility and lot-to-lot variation studies. Tube performance studies showed 99.1% to 100% correct calls.
Reproducibility:
- Site-to-site reproducibility study: Three tubes from one lot collected from each of 20 donors and sent to 3 different sites. DNA extracted using silica membrane based kit and tested with CF Assay and HLA Assay 1. All samples showed 100% concordant results.
- Lot-to-lot device reproducibility study: One tube from each of 3 lots collected from each of 20 donors and sent to one site. DNA extracted using silica membrane based kit and tested with CF Assay and HLA Assay 1. All samples showed 100% concordant results.
Product Stability - Shelf Life Study:
- Evaluated unused devices stored at room temperature for up to 13 months, with and without temperature cycling.
- Products tested for DNA yield, concentration, purity, and HLA Assay 2 concordance with a control EDTA tube.
- The data supports a product shelf life of up to 12 months at room temperature, with 100% assay concordance for all tested groups (Table 6).
DNA Stability - Whole blood storage in tube:
- Stability of blood stored in the tube was tested for DNA concentration and purity, and HLA Assay 2 concordance with a control EDTA tube.
- Data supports storage of blood at various conditions: 18-25°C for up to 14 days, 2-8°C for up to 28 days, -20°C for up to 12 months, and 1, 2, or 3 freeze-thaw cycles. Also 35°C for up to 3 days. All conditions showed 100% assay concordance (Table 7) where applicable.
Interference:
- Potentially interfering substances (Hemoglobin, Bilirubin, Triglycerides, Albumin) were added separately to the PAXgene Blood DNA Tube.
- No effect on FDA cleared assay performance (HLA Assay 3) was observed. All samples were concordant with control tubes.
Sample Handling – Mixing and Underfilling of Tubes:
- Robustness of DNA from samples subjected to various handling conditions (underfilling to 2.5 ml, 1.25 ml, 0.70 ml; 0, 1, 5, 8 tube inversions) was tested.
- All conditions resulted in 100% assay concordance with control EDTA tube (Table 11).

Key Metrics (Sensitivity, Specificity, PPV, NPV, etc.)

% Correct calls: Ranged from 97.3% to 100% across different assays and sites.
95% CI lower bound for % Correct calls: Ranged from 86.2% to 99.5%.
DNA yield (magnetic bead): Mean 6.05 ± 1.61 µg DNA / 200 µl blood. 95% of samples > 3.64 µg.
DNA concentration (magnetic bead): Mean 30.2 ± 8.0 ng DNA / µl eluate. 95% of samples > 18.2 ng/µl.
DNA purity (magnetic bead): Mean 1.85 ± 0.04 (A260/A280). 95% of samples 1.75-1.93.
DNA yield (silica membrane): Mean 4.89 ± 2.48 µg DNA / 200 µl blood. 95% of samples ≥ 1.86 µg.
DNA concentration (silica membrane): Mean 48.85 ± 24.75 ng DNA / µl eluate. 95% of samples ≥ 18.56 ng/µl.
DNA purity (silica membrane): Mean 1.86 ± 0.08 (A260/A280). 95% of samples 1.67-2.06.
Overall probe concordance (HLA Assay 1): 99.95% with 95% CI lower bound of 99.9%.
DNA concentration with interfering substances (magnetic bead): ≥ 43.6 ng/ul.
DNA purity with interfering substances (magnetic bead): 1.7-1.9.
DNA concentration with interfering substances (silica membrane): ≥ 24.0 ng/ul.
DNA purity with interfering substances (silica membrane): 1.7-1.9.
DNA concentration with underfilling/mixing: ≥ 16.7 ng/ul (underfilling), ≥ 16.8 ng/ul (mixing).
DNA purity with underfilling/mixing: 1.8-1.9.
Assay concordance for interference and handling studies: 100%.

Predicate Device(s):

K972075: BD Vacutainer® PPT™ Plasma Preparation Tube

Reference Device(s):

Not Found

Predetermined Change Control Plan (PCCP) - All Relevant Information

Not Found

Regulation Number and Section

§ 862.1675 Blood specimen collection device.

(a)
Identification. A blood specimen collection device is a device intended for medical purposes to collect and to handle blood specimens and to separate serum from nonserum (cellular) components prior to further testing. This generic type device may include blood collection tubes, vials, systems, serum separators, blood collection trays, or vacuum sample tubes.(b)
Classification. Class II.

Summary

0

Image /page/0/Picture/1 description: The image shows the logo for the U.S. Department of Health and Human Services. The logo consists of a circular seal with the text "DEPARTMENT OF HEALTH & HUMAN SERVICES - USA" arranged around the perimeter. Inside the circle is a stylized design featuring three overlapping human profiles, creating a sense of depth and connection.

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

PREANALYTIX GMBH C/O PASQUALE AMATO STAFF SPECIALIST REGULATORY AFFAIRS 1 BECTON DRIVE FRANKLIN LAKES, NJ 07417

September 9, 2015

Re: K142821

Trade/Device Name: PAXgene® Blood DNA Tube Regulation Number: 21 CFR 862.1675 Regulation Name: Blood specimen collection device Regulatory Class: II Product Code: PJE Dated: August 27, 2015 Received: August 28, 2015

Dear Pasquale Amato:

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.

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.

1

If you desire specific advice for your device on our labeling regulations (21 CFR Parts 801 and 809), please contact the Division of Industry and Consumer Education at its toll-free number (800) 638 2041 or (301) 796-7100 or at its Internet address

http://www.fda.gov/MedicalDevices/Resourcesfor You/Industry/default.htm. 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

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 (800) 638-2041 or (301) 796-7100 or at its Internet address

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

Sincerely yours,

Katherine Serrano -S

For : Courtney H. Lias, Ph.D. Director Division of Chemistry and Toxicology Devices Office of In Vitro Diagnostics and Radiological Health Center for Devices and Radiological Health

Enclosure

2

Indications for Use

510(k) Number (if known) K142821

Device Name PAXgene® Blood DNA Tube

Indications for Use (Describe)

The PAXgene Blood DNA Tube is intended to collect, anticoagulate, stabilize, transport, and store a venous whole blood sample for preparation of human DNA for use with molecular diagnostic test methods that require DNA.

The performance characteristics of this device have not been established for molecular diagnostic assays in general. Users must validate use of product for their specific molecular diagnostic assay.

Type of Use (Select one or both, as applicable)

X Prescription Use (Part 21 CFR 801 Subpart D)
Over-The-Counter Use (21 CFR 801 Subpart C)

CONTINUE ON A SEPARATE PAGE IF NEEDED.

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DO NOT SEND YOUR COMPLETED FORM TO THE PRA STAFF EMAIL ADDRESS BELOW.

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3

510(K) SUMMARY 21 CFR 807.92(c) PAXgene® Blood DNA Tube

| Submitter

Information	Submitter Name:	PreAnalytiX GmbH
	Submitter Address:	Feldbachstrasse
Hombrechtikon, CH 8634
	Contact Person:	Pasquale Amato
Staff Specialist Regulatory Affairs
1 Becton Drive, Franklin Lakes, NJ 07417
Phone: 201-847-4513
Email: Pasquale_Amato@bd.com
	Alternate Contact:	Alex Wesolowski
Vice President, BDX Corporate/Shared Services
Phone: 201-847-5051
Email: Alex_F_Wesolowski@bd.com
	Date of Preparation:	September 8, 2015
Device
Information	Trade Name:	PAXgene® Blood DNA Tube
	Common Name:	Blood Collection Device
	Classification Name:	Blood Specimen Collection Device (21 CFR 862.1675)
	Classification:	Class II
	Product Code:	PJE
Predicate Device	Trade Name:	BD Vacutainer® PPT™ Plasma Preparation Tube
Device Description	The PAXgene® Blood DNA Tube is a sterile, single use, plastic, evacuated
blood collection tube with a BD Hemogard™ closure assembly and a measured
quantity of K2EDTA additive. The additive quantity dispensed into each tube is
designed to match the nominal blood draw volume of 2.5 mL. The tube is made
of polyethylene terephthalate (PET) plastic which functions to maintain vacuum
within the tube, allowing for accurate and consistent blood draw for the duration
of the shelf life of the tube. A predetermined vacuum is drawn inside the tube
that is sealed with a BD Hemogard™ closure which consists of a rubber stopper
plus BD Hemogard™ shield

4

| | The tube is intended to be placed inside a tube holder or an adaptor that contains
a needle designed to pierce the tube closure and allow blood to flow into the
tube. Once the vein has been penetrated (using either a standard blood collection
needle or a blood collection set), the tube is pushed into the holder, and the
blood enters the tube. Once a tube has drawn the appropriate amount of blood, it
is disengaged from the holder and inverted the recommended number of times
(8–10) to mix the additive with the blood. |
|----------------------------------------|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| | The DNA in whole blood collected in the PAXgene® Blood DNA Tube has
been shown to be suitable for molecular diagnostic testing for 14 days at room
temperature (18–25°C), 28 days refrigerated (2–8°C), 3 days at 35°C, up to 52
weeks frozen (–20°C), or when subjected to up to three freeze-thaw cycles. The
PAXgene® Blood DNA Tube is robust with respect to mishandling including
reduced inversions and partial blood draw. The product shelf life is one year
from the date of manufacture including limited storage temperature excursions
which simulate shipping conditions from –20°C to 45°C. |
| | The PAXgene® Blood DNA Tube is available as a 13 x 75 mm tube with a 2.5
mL nominal blood draw. The referenced first dimension represents the diameter
of the tube and the second dimension represents the length of the tube. |
| Intended
Use/Indications
for Use | The PAXgene® Blood DNA Tube is intended to collect, anticoagulate, stabilize,
transport, and store a venous whole blood sample for preparation of human DNA
for use with molecular diagnostic test methods that require DNA.
The performance characteristics of this device have not been established for
molecular diagnostic assays in general. Users must validate use of product for
their specific molecular diagnostic assay. |
| Technological
Characteristics | The technological characteristics of the subject device, the PAXgene® Blood
DNA Tube, are equivalent to that of the predicate device, the BD Vacutainer®
PPT™ Plasma Preparation Tube with respect to device design and operating
principle. The PAXgene® Blood DNA Tube utilizes identical component
materials to the BD Vacutainer® PPT™ Plasma Preparation Tube. Aside from
intended use/indications for use, the only changes are with the amount of
K2EDTA present in the tube and lack of gel barrier material. These differences
do not raise any new questions of safety or effectiveness. |

5

	Subject/Evaluation Device	Predicate Device
Key Parameters	PAXgene® Blood DNA Tube	[510(k):K972075] BD Vacutainer® PPTTM Plasma Preparation Tube
Intended Use/Indications
for Use	The PAXgene® Blood DNA Tube
is intended to collect,
anticoagulate, stabilize, transport,
and store a venous whole blood
sample for preparation of human
DNA for use with molecular
diagnostic test methods that require
DNA.

The performance characteristics of
this device have not been
established for molecular
diagnostic assays in general. Users
must validate use of product for
their specific molecular diagnostic
assay. | The Vacutainer® Brand PPTTM
Plasma Preparation Tube with EDTA
anticoagulant and a gel barrier
material are evacuated blood
collection tubes which provide a
means of collecting, processing and
transporting blood in a closed plastic
tube. When the Tube is used together
with Vacutainer® Brand Needles and
Holders, it is a closed system for thecollection of venous blood with the
same indications identified here.

Summary Comparison between the PAXgene® Blood DNA Tube and Predicate Device:

6

Summary Results of Performance Testing:

1. Performance Characteristics of PAXgene Blood DNA Tube by Sample Preparation Method

DNA Yield, Concentration and Purity Summary

DNA yield, concentration and purity (A26) A280/A280) were determined by UV absorbance for samples purified using both magnetic bead and silica membrane sample preparation technologies. Data from 581 specimens from approximately 200 consented subjects was used to support the performance characteristics of the PAXgene Blood DNA Tube with a commercially available, automated magnetic bead based DNA extraction kit. Data from 540 specimens from 152 consented subjects was used to support the performance characteristics of the PAXgene Blood DNA Tube with a commercially available, automated silica membrane based DNA extraction kit.

The following histograms and table summarize the DNA yield, DNA concentration and A260 A280 ratio results obtained using the automated magnetic bead based DNA extraction kit (elution volume: 200 µl).

Image /page/6/Figure/6 description: The image is a histogram displaying DNA concentration on the x-axis and the number of samples on the y-axis. The x-axis, labeled "DNA Concentration (ng DNA / µl eluate)," ranges from 0 to 60. The y-axis, labeled "Number of Samples," ranges from 0 to 80. The histogram shows a distribution of DNA concentrations, with the highest number of samples around 25-30 ng DNA / µl eluate.

DNA Eluate Concentrations using an Automated, Magnetic Bead Bead-Based DNA Purification System

Figure 1

Blood was drawn from a donor pool of approximately 200 consented subjects ≥ 18 years of age into PAXgene Blood DNA Tubes. Tubes were processed within 24 hours at room temperature. Total DNA was purified from 581 specimens using a commercially available, automated magnetic bead based DNA extraction kit.

7

Image /page/7/Figure/0 description: The image is a histogram displaying the distribution of DNA purity measurements. The x-axis represents DNA purity (A260/A280) ranging from 1.65 to 2.00, while the y-axis represents the number of samples. The histogram shows a distribution that is skewed to the left, with a peak around 1.85, indicating that most samples have a DNA purity value close to 1.85.

DNA Purity using an Automated, Magnetic Bead Bead-Based DNA Purification System

Figure 2

Blood was drawn from a donor pool of approximately 200 consented subjects ≥ 18 years of age into PAXgene Blood DNA Tubes. Tubes were processed within 24 hours at room temperature. Total DNA was purified from 581 specimens using a commercially available, automated magnetic bead based DNA extraction kit.

| | Yield
( $\mu$ g DNA / 200 $\mu$ l blood) | Concentration
(ng DNA / $\mu$ l eluate) | Purity
(A260/A280) |
|---------------------|---------------------------------------------|--------------------------------------------|-----------------------|
| n | 581 | 581 | 581 |
| Mean ± SD | 6.05 ± 1.61 | 30.2 ± 8.0 | 1.85 ± 0.04 |
| Median | 5.77 | 28.9 | 1.86 |
| Interquartile range | 4.88–7.22 | 24.4-36.1 | 1.83–1.88 |
| Range | 2.43-10.79 | 12.2-54.0 | 1.69–1.94 |
| 95% of samples | > 3.64 | > 18.2 | 1.75-1.93 |

Table 1: Performance testing summary (magnetic bead based DNA purification)

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The following histograms and table summarize the DNA yield, DNA concentration and A260 A280 ratio results obtained using the automated silica membrane based DNA extraction kit (elution volume: 100 µl).

Image /page/8/Figure/1 description: This image is a histogram showing the distribution of DNA concentrations in a number of samples. The x-axis represents the DNA concentration in nanograms per microliter eluate, ranging from 0 to 210. The y-axis represents the number of samples, ranging from 0 to 120. The histogram shows that the majority of samples have a DNA concentration between 30 and 60 ng/μl eluate.

DNA Eluate Concentrations using an Automated, Silica Membrane-Based DNA Purification System

Figure 3

Blood was drawn from 152 consented subjects ≥ 18 years of age into PAXgene Blood DNA Tubes were stored at room temperature for ≤ 14 days. Total DNA was purified from 540 specimens using a commercially available, automated silica membrane based DNA extraction kit.

9

DNA Purity using an Automated, Silica Membrane-Based DNA Purification System

Image /page/9/Figure/1 description: The image is a histogram showing the distribution of DNA purity (A260/A280) across a number of samples. The x-axis represents DNA purity, ranging from 1.6 to 2.2, while the y-axis represents the number of samples. The distribution is centered around a DNA purity of 1.9, with the highest number of samples at this value. The number of samples decreases as the DNA purity moves away from 1.9.

Figure 4

Blood was drawn from 152 consented subjects ≥ 18 years of age into PAXgene Blood DNA tubes. Tubes were stored at room temperature for ≤ 14 days. Total DNA was purified from 540 specimens using a commercially available, automated silica membrane based DNA extraction kit.

| | Yield
(µg DNA / 200 µl blood) | Concentration
(ng DNA / µl eluate) | Purity
(A260/A280) |
|---------------------|----------------------------------|---------------------------------------|-----------------------|
| n | 540 | 540 | 540 |
| Mean ± SD | 4.89 ± 2.48 | 48.85 ± 24.75 | 1.86 ± 0.08 |
| Median | 4.49 | 44.90 | 1.88 |
| Interquartile range | 3.27-5.71 | 32.73-57.10 | 1.81-1.92 |
| Range | 0.75-21.1 | 7.46-211.10 | 1.65-2.19 |
| 95% of samples | ≥ 1.86 | ≥ 18.56 | 1.67-2.06 |

Table 2: Performance testing summary (silica membrane based DNA purification)

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2. Performance Characteristics of PAXgene Blood DNA Tube by Molecular Diagnostic Test Methods

Evaluations of the PAXgene Blood DNA Tube have been performed for selected FDA cleared assays on certain instrument platforms. See Table 3 for sample preparation, instrument, and assay information.

Assay	Cystic Fibrosis Assay	HLA Assay 1	Thrombophilia Assay	HLA Assay 2	HLA Assay 3
Assay instrument	Multiplex fluorescent
microsphere
based flow
cytometry	Multiplex fluorescent
microsphere
based flow
cytometry	Electrochemical detection based
DNA microarray	N/A, gel-based readout	N/A, gel-based readout
DNA isolation kit and
instrument technology	Silica membrane	Silica membrane	Silica membrane	Magnetic bead	Magnetic bead
and silica
membrane

Table 3: Assay and DNA Sample Preparation Information:

The performance of the PAXgene Blood DNA Tube was assessed relative to an EDTA tube control using FDA cleared molecular diagnostic assays. Assays were evaluated at either 1 or 3 sites. See Table 4 and Table 5 for testing results:

| Site | Assay | Samples
tested | Correct
calls | Incorrect
calls | No-
calls | % Correct
calls | 95% CI
lower bound |
|--------|----------------------------|-------------------|------------------|--------------------|--------------|--------------------|-----------------------|
| Site A | CF Assay | 40 | 40 | 0 | 0 | 100% | 91.2% |
| | HLA Assay 1 | 40 | 40 | 0 | 0 | 100% | 91.2% |
| Site B | CF Assay, After Retesting† | 37 | 36 | 0 | 1 | 97.3% | 86.2% |
| | HLA Assay 1* | 40 | 40 | 0 | 0 | 100% | 91.2% |
| Site C | CF Assay | 40 | 40 | 0 | 0 | 100% | 91.2% |
| | HLA Assay 1, After Retest‡ | 40 | 40 | 0 | 0 | 100% | 91.2% |
| Site D | Thrombophilia | 80 | 80 | 0 | 0 | 100% | 95.2% |
| Site E | HLA Assay 2, After Retest§ | 698 | 698 | 0 | 0 | 100% | 99.5% |
| | HLA Assay 3 | 100 | 100 | 0 | 0 | 100% | 96.3% |

Table 4: Test Results by Site:

In addition to the two-field concordance presented in the table, probe hit patterns were analyzed and a total of 7 probes out of 14,400 (200 comparisons > 72 probes) were found to be discordant. The overall probe concordance was 99.95% with a 95% CI lower bound of 99.9%.

T CF Assay, after retest, includes 1 sample from Site B showing a result of "No Call" that was not retested. Three previous runs at Site B included up to 38 samples showing a result of "No Call" due to a degraded enzyme in the CF Assay Kit. Run 4 used a new enzyme to perform the test at Site B. The results exclude 3 subjects from Site B where the assay was not repeated for 3 evaluation tubes.

HLA Assay 1, after retest, includes 4 samples from Site C that were re-extracted and retested due to a labeling error.

S HLA Assay 2, after retest, includes 2 repeat testing samples due to labeling errors and removes 12 samples (3 concordant with previous results, 9 discordant with previous results due to labeling errors) that could not be retested.

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Objective	Sites	Assay	Samples tested	Correct calls	Incorrect calls	No-calls	% Correct calls	95% CI lower bound
Site-to-site
reproducibility	A, B, C	CF Assay	20	20	0	0	100%	N/A
Site-to-site
reproducibility	A, B, C	HLA Assay 1*	20	20	0	0	100%	N/A
Lot-to-lot
variation	A	CF Assay	20	20	0	0	100%	N/A
Lot-to-lot
variation	A	HLA Assay 1	20	20	0	0	100%	N/A
Tube
performance	A, B, C	CF Assay, After Retest†	117	116	0	1	99.1%	95.3%
Tube
performance	A, B, C	HLA Assay 1, After
Retest‡*	120	120	0	0	100%	96.9%
	D	Thrombophilia Assay	80	80	0	0	100%	95.4%
	E	HLA Assay 2, After Retest§	698	698	0	0	100%	99.5%
Interference	E	HLA Assay 3	100	100	0	0	100%	96.3%

Table 5: Test Results by Study:

In addition to the two-field concordance presented in the table, probe hit patterns were analyzed and a total of 7 probes out of 14.400 (200 comparisons > 72 probes) were found to be discordant. The overall probe concordance was 99.95% with a 95% CI lower bound of 99.9%.
CF Assay, after retest, includes 1 sample from Site B showing a result of "No Call" that was not retested. Three previous runs at Site B included up to 38 samples showing a result of "No Call" due to a degraded enzyme in the CF Assay Kit. Run 4 used a new enzyme to perform the test at Site B. The results exclude 3 subjects from Site B where the assay was not repeated for 3 evaluation tubes.
HLA Assay 1, after retest, includes 4 samples from Site C that were re-extracted and retested due to a labeling error.

S HLA Assay 2, after retest, includes 2 repeat testing samples due to labeling errors and removes 12 samples (3 concordant with previous results, 9 discordant with previous results due to labeling errors) that could not be retested.

3. Reproducibility

Two reproducibility studies were performed.

The site-to-site reproducibility study was conducted at three sites. Three tubes from one lot were collected from each of 20 donors and sent to 3 different sites for DNA extraction and testing. DNA was extracted using a commercially available, automated silica membrane based DNA extraction kit, followed by determination of DNA concentration and purity for all samples were tested using the CF Assay and HLA Assay 1 for concordance. All samples gave 100% concordant results.
The lot-to-lot device reproducibility study was conducted at three sites. One tube from each of 3 lots was collected from each of 20 donors and sent to one site for DNA extraction and testing. DNA was extracted using a commercially available, automated silica membrane based DNA extraction kit, followed by determination of DNA concentration and purity for all samples were tested using the CF Assay and HLA Assay 1 for concordance. All samples gave 100% concordant results.

4. Product stability - Shelf Life Study

Product shelf life was evaluated by storing unused devices at room temperature for up to 13 months, with and without temperature cycling for 10 days at 45°C and 5 days at -20°C to simulate temperature extremes in transport. DNA was purified using a commercially available, automated magnetic bead based DNA extraction kit and samples were tested for DNA yield, concentration and purity, as well as HLA Assay 2 concordance with a control EDTA tube.

The data supports a product shelf life of up to 12 months at room temperature.

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The following handling conditions were tested:

| Storage
condition | Time
points | Groups | Blood storage
condition | Subjects | Assay
concordance |
|--------------------------------------------------------------------------------------|------------------|---------------------------------------------------------------------------------------|----------------------------|---------------------------------|----------------------|
| 25°C | 12, 13
months | 6: 3 lots per time point | 14 days at 18–25°C | 24 (12 per time
point/group) | 100% (72/72) |
| 25°C | 7, 13
months | 2: 1 lot per time point | 14 days at 18–25°C | 24 (12 per time
point/group) | 100% (24/24) |
| 25°C with
temperature
cycling for 10
days at 45°C
and 5 days at
-20°C | 7, 13
months | 3: 1 at 7 months and 2
at 13 months
(temperature cycling at
6 and 12 months) | 14 days at 18–25°C | 24 (12 per time
point/group) | 100% (36/36) |

Table 6: Tube storage conditions for samples tested with HLA Assay 1

DNA concentration and purity were assessed for the PAXgene Blood DNA Tube using a commercially available, automated magnetic bead based DNA extraction kit (elution volume: 200 µl). DNA concentration was ≥ 15.2 ng/ul and DNA purity was between 1.7-1.9 for all samples.

5. DNA Stability - Whole blood storage in tube

Stability of blood stored in the tube was tested for DNA concentration and purity, as well as HLA Assay 2 concordance with a control EDTA tube. DNA was purified using a commercially available, automated magnetic bead based DNA extraction kit (elution volume: 200 µl). The data supports storage of blood in the tube for the following conditions:

Storage time	Storage	Sample	Concentration Purity		Assay	95% CI
	temperature	size	(ng DNA /	(A260/A280)	concordance	lower
			ul eluate)			bound
0, 3, 7, 14 days	18—25°C	12	> 17.5	1.8–1.9	100% (48/48)	92.6%
0. 14 days	18—25°C	60	> 16.7	1.7-1.9	100% (120/120)	196.9%
0, 7, 14, 21, 28 days	2-8°C	12	> 13.4	1.8–1.9	100% (36/36)	90.4%
0, 28 days	2-8°C	60	> 16.3	1.7—1.9	100% (120/120)	196.9%
0, 1, 6, 12 months	-20°C	12	> 15.3	1.8–1.9	100% (48/48)	92.6%
1, 2, 3 freeze-thaw cycles	-20°C / 18-25°C 12		> 16.1	1.7-1.9	100% (24/24)	86.2%
1,2,3 freeze-thaw cycles	-20°C / 18-25°C 60		> 13.1	1.7—1.9	N/A	N/A
6 hours, 1, 2, 3 days	35°C	12	> 14.1	1.7–1.9	100% (60/60)	94.0%

Table 7: Whole blood storage conditions and assay results

DNA concentration and purity were assessed for the PAXgene Blood DNA Tube using a commercially available, automated magnetic bead based DNA extraction kit (elution volume: 200 ul). DNA concentration was ≥ 13.1 ng/ul and DNA purity was between 1.7-1.9 for all samples.

6. Interference

Potentially interfering substances were added separately to the PAXgene Blood DNA Tube. The addition of these substances did not have an effect on the FDA cleared assay performance (HLA Assay 3). All samples were concordant with a PAXgene Blood DNA Tube from the same subject without the added substances and control EDTA tube. The following substances were evaluated, using both a commercially available, automated magnetic bead based DNA extraction kit and a commercially available, automated silica membrane based DNA extraction kit:

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Table 8. Interfering substances - Concentrations tested

Interfering substance	Hemoglobin	Bilirubin	l riglycerides	Albumin
Concentration	200 g/L*	200 mg/L	18.2 g/L	27.4 g/L
------------------------------------------------------------------------------------------------------------------------------------------------------------------------------

Total concentration includes endogenous and added hemoglobin

T Concentration of interferent added to sample

DNA concentration and purity were assessed for the PAXgene Blood DNA Tube with potentially interfering substances in comparison to the PAXgene Blood DNA Tube with no potential interferents, using both a commercially available, automated magnetic bead based DNA extraction kit (elution volume: 200 µl) and a commercially available, automated silica membrane based DNA extraction kit (elution volume: 100 µl):

Table 9. Interfering substances - Study summary (magnetic bead based DNA purification)

Interfering substance	None	Hemoglobin	Bilirubin	Triglycerides	Albumin
Sample size	10	10	10	10	10
Concentration (ng DNA / µl eluate)	$\ge 69.0$	$\ge 59.2$	$\ge 58.8$	$\ge 44.6$	$\ge 43.6$
Purity (A260/A280)	1.8-1.9	1.8-1.9	1.8-1.9	1.8-1.9	1.7-1.9
Assay concordance	100% (10/10)	100% (10/10)	100% (10/10)	100% (10/10)	100% (10/10)

Table 10. Interfering substances - Study summary (silica membrane based DNA purification)

Interfering substance	None	Hemoglobin	Bilirubin	Triglycerides	Albumin
Sample size	10	10	10	10	10
Concentration (ng DNA / µl eluate)	$\geq$ 28.2	$\geq$ 27.0	$\geq$ 29.2	$\geq$ 34.4	$\geq$ 24.0
Purity (A260/A280)	1.7-1.9	1.7-1.9	1.8-1.9	1.7-1.8	1.7-1.9
Assay concordance	100% (10/10)	100% (10/10)	100% (10/10)	100% (10/10)	100% (10/10)

DNA concentration was ≥ 43.6 ng/ul for magnetic bead based DNA extraction kit samples and ≥ 24.0 ng/ul for silica membrane based DNA extraction kit samples. DNA purity was between 1.7-1.9 for all samples.

7. Sample Handling – Mixing and Underfilling of Tubes

Robustness of DNA from samples subjected to a range of handling conditions was tested for DNA yield, concentration and purity, as well as HLA Assay 2 concordance with a control EDTA tube. DNA was purified using a commercially available, automated magnetic bead based DNA extraction kit (elution volume: 200 ul). The following handling condition were tested:

| Handling | Test conditions | Subjects | Concentration
(ng DNA / µl eluate) | Purity
(A260/A280) | Assay
concordance |
|--------------|----------------------------|----------|---------------------------------------|-----------------------|----------------------|
| Underfilling | 2.5 ml, 1.25 ml, 0.70 ml | 10 | ≥ 16.7 | 1.8—1.9 | 100% (30/30) |
| Mixing | 0, 1, 5, 8 tube inversions | 10 | ≥ 16.8 | 1.8—1.9 | 100% (20/20) |

Table 11: Whole blood handling conditions and assay results

Substantial

Equivalence

DNA concentration and purity were assessed for the PAXgene Blood DNA Tube using a commercially available, automated magnetic bead based DNA extraction kit (elution volume: 200 ul). DNA concentration was ≥ 16.7 ng/ul and DNA purity was between 1.8-1.9 for all samples.

Based on a comparison of the device design, operational use, and the intended use and performance for venous whole blood specimen collection, anticoagulation, stabilization, transportation and storage for the preparation of human DNA, the PAXgene® Blood DNA Tube is as safe, as effective and performs as well as the commercially available predicate device, the BD Vacutainer® PPTTM Plasma Preparation Tube. For the specific intended use, the PAXgene® Blood DNA Tube is substantially equivalent to the predicate device.