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.

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.

AI/ML Overview

The provided document describes the PAXgene® Blood DNA Tube, a blood collection device, and its performance characteristics as part of a 510(k) premarket notification. The study aims to demonstrate substantial equivalence to a legally marketed predicate device.

Here's an analysis of the acceptance criteria and study findings based on the provided text, while noting the limitations of the document regarding certain specific study parameters that are often found in AI/ML performance studies:

1. Table of Acceptance Criteria and Reported Device Performance

The document does not explicitly state formal "acceptance criteria" in a typical table format with pre-defined thresholds. Instead, it presents performance data for DNA yield, concentration, purity, and concordance with FDA-cleared molecular diagnostic assays to demonstrate the device's suitability for its intended use and substantial equivalence to the predicate device.

However, based on the summary results and the nature of this submission, which is for a blood collection tube rather than an AI/ML diagnostic algorithm, the acceptance criteria are implicitly met if the device consistently yields high-quality DNA that produces 100% correct calls in various molecular diagnostic assays, and if its stability and robustness are demonstrated.

Here's a synthesized table based on the various performance summaries:

Performance Metric	Implicit Acceptance Criteria (based on reported results)	Reported Device Performance (PAXgene® Blood DNA Tube)
DNA Yield (µg DNA / 200 µl blood)	Consistent yield for downstream applications	Magnetic Bead: Mean 6.05 ± 1.61 µg; Median 5.77 µg; 95% samples > 3.64 µg. Silica Membrane: Mean 4.89 ± 2.48 µg; Median 4.49 µg; 95% samples ≥ 1.86 µg.
DNA Concentration (ng DNA / µl eluate)	Sufficient concentration for downstream applications	Magnetic Bead: Mean 30.2 ± 8.0 ng/µl; Median 28.9 ng/µl; 95% samples > 18.2 ng/µl. Silica Membrane: Mean 48.85 ± 24.75 ng/µl; Median 44.90 ng/µl; 95% samples ≥ 18.56 ng/µl.
DNA Purity (A260/A280)	Optimal range (typically 1.7-2.0)	Magnetic Bead: Mean 1.85 ± 0.04; Median 1.86; 95% samples 1.75-1.93. Silica Membrane: Mean 1.86 ± 0.08; Median 1.88; 95% samples 1.67-2.06. Interference/Handling: 1.7-1.9 or 1.8-1.9.
Assay Concordance (with FDA-cleared MDx assays)	100% correct calls / High concordance	Primary Testing: 100% correct calls in all assessed assays (CF Assay, HLA Assay 1, Thrombophilia, HLA Assay 2, HLA Assay 3) across multiple sites, with 95% CI lower bounds generally >90%, and for large sample sets >99%. One exceptional case of 97.3% for CF Assay at Site B with one "No Call" due to degraded enzyme, which was not attributed to the device itself. Device Shelf-Life: 100% concordance. DNA Stability (Blood in Tube): 100% concordance across various storage conditions. Interference: 100% concordance with interfering substances. Sample Handling (Underfilling/Mixing): 100% concordance.
Product Shelf Life	At least 12 months at room temperature	Supported for up to 12 months at room temperature, including temperature cycling (45°C and -20°C).
Blood Stability in Tube	Up to 14 days (RT), 28 days (refrigerated), 52 weeks (frozen), 3 freeze-thaw cycles	As stated: 14 days at 18-25°C; 28 days at 2-8°C; 1, 6, 12 months at -20°C; 1, 2, 3 freeze-thaw cycles; up to 3 days at 35°C.
Robustness (Underfilling/Mixing)	Consistent performance under mishandling	Demonstrated consistent DNA concentration, purity, and 100% assay concordance for underfilling (2.5ml, 1.25ml, 0.70ml) and mixing (0, 1, 5, 8 inversions).
Interference	No impact on assay performance	No effect on FDA-cleared assay performance (HLA Assay 3) with tested interfering substances (Hemoglobin, Bilirubin, Triglycerides, Albumin).

2. Sample sizes used for the test set and the data provenance

The "test set" here refers to the samples used for performance evaluation, as this is not an AI/ML device in the traditional sense that uses training/test splits.

DNA Yield, Concentration, and Purity Summary:
- Magnetic Bead based DNA extraction kit: 581 specimens from approximately 200 consented subjects.
- Silica Membrane based DNA extraction kit: 540 specimens from 152 consented subjects.
Molecular Diagnostic Test Methods (Assay Concordance):
- Individual assays varied: CF Assay (40 samples per site, total 117 after retest); HLA Assay 1 (40 samples per site, total 120 after retest); Thrombophilia Assay (80 samples); HLA Assay 2 (698 samples after retest); HLA Assay 3 (100 samples).
- Reproducibility studies: 20 donors for site-to-site and lot-to-lot.
Device Shelf-Life Study: Tested with 24 subjects (12 per time point/group) for 12, 13 months, and with temperature cycling.
DNA Stability (Whole Blood Storage in Tube): Sample sizes of 12 or 60 for various storage conditions and time points.
Interference Study: 10 samples per interfering substance for both magnetic bead and silica membrane purification methods.
Sample Handling (Underfilling and Mixing): 10 subjects for each handling condition.

Data Provenance: The document states that blood was drawn from "consented subjects" and mentions testing at "four (4) external clinical test sites and one (1) internal test site". This strongly indicates prospective data collection from human subjects. The document does not specify the country of origin of the data, but given the FDA submission, it likely includes data from the US or regions compliant with FDA standards.

3. Number of experts used to establish the ground truth for the test set and the qualifications of those experts

This is not applicable in the context of this device. The ground truth for this device (a blood collection tube) is objectively measured through DNA quantification (yield, concentration, purity) and the concordance of results with established, FDA-cleared molecular diagnostic assays. The "ground truth" for the molecular diagnostic assays themselves would be derived from clinical diagnosis or confirmed genetic status, not expert consensus on image interpretation. No human experts are used for establishing ground truth for device performance in this manner.

4. Adjudication method (e.g. 2+1, 3+1, none) for the test set

Not applicable. This is not a study requiring reader adjudication of ambiguous cases, as it measures objective biochemical and molecular assay outcomes.

5. If a multi reader multi case (MRMC) comparative effectiveness study was done, If so, what was the effect size of how much human readers improve with AI vs without AI assistance

Not applicable. This is not an AI/ML medical device, and no human readers are involved in interpreting results that would be enhanced by AI assistance. The performance is assessed on the ability of the tube to collect and preserve DNA for downstream molecular diagnostic tests.

6. If a standalone (i.e. algorithm only without human-in-the-loop performance) was done

Yes, in a sense, the primary studies are "standalone" performance evaluations of the device itself (the blood collection tube) to ensure it performs its function of preserving DNA for analysis. The "algorithm" here is the device's mechanism (anticoagulation, stabilization) and the subsequent molecular diagnostic assays. The results presented (DNA metrics and assay concordance) are direct outputs of using the PAXgene Blood DNA Tube in conjunction with standard laboratory procedures and FDA-cleared assays, without human interpretation as part of the 'device's' output.

7. The type of ground truth used (expert consensus, pathology, outcomes data, etc)

The ground truth used for evaluating the PAXgene Blood DNA Tube's performance is multi-faceted:

Quantitative DNA metrics: Measured values (yield, concentration, purity) obtained through standard laboratory equipment (e.g., UV absorbance).
Concordance with FDA-cleared Molecular Diagnostic Assays: The "ground truth" for the assay results themselves is that the assay should correctly identify the genetic markers it is cleared to detect. The study establishes that DNA collected in the PAXgene tube yields results (e.g., genotypes) that are 100% concordant with those obtained from control (predicate or EDTA) tubes, demonstrating that the sample preparation did not alter the diagnostic outcome.

8. The sample size for the training set

Not applicable. This is a blood collection device, not an AI/ML algorithm that requires a training set.

9. How the ground truth for the training set was established

Not applicable. No training set is used for this device.

Summary

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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.

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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

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Indications for Use

510(k) Number (if known) K142821

Device Name PAXgene® Blood DNA Tube

Indications for Use (Describe)

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)

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

SubmitterInformation	Submitter Name:	PreAnalytiX GmbH
	Submitter Address:	FeldbachstrasseHombrechtikon, CH 8634
	Contact Person:	Pasquale AmatoStaff Specialist Regulatory Affairs1 Becton Drive, Franklin Lakes, NJ 07417Phone: 201-847-4513Email: Pasquale_Amato@bd.com
	Alternate Contact:	Alex WesolowskiVice President, BDX Corporate/Shared ServicesPhone: 201-847-5051Email: Alex_F_Wesolowski@bd.com
	Date of Preparation:	September 8, 2015
DeviceInformation	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, evacuatedblood collection tube with a BD Hemogard™ closure assembly and a measuredquantity of K2EDTA additive. The additive quantity dispensed into each tube isdesigned to match the nominal blood draw volume of 2.5 mL. The tube is madeof polyethylene terephthalate (PET) plastic which functions to maintain vacuumwithin the tube, allowing for accurate and consistent blood draw for the durationof the shelf life of the tube. A predetermined vacuum is drawn inside the tubethat is sealed with a BD Hemogard™ closure which consists of a rubber stopperplus BD Hemogard™ shield

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	The tube is intended to be placed inside a tube holder or an adaptor that containsa needle designed to pierce the tube closure and allow blood to flow into thetube. Once the vein has been penetrated (using either a standard blood collectionneedle or a blood collection set), the tube is pushed into the holder, and theblood enters the tube. Once a tube has drawn the appropriate amount of blood, itis 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 hasbeen shown to be suitable for molecular diagnostic testing for 14 days at roomtemperature (18–25°C), 28 days refrigerated (2–8°C), 3 days at 35°C, up to 52weeks frozen (–20°C), or when subjected to up to three freeze-thaw cycles. ThePAXgene® Blood DNA Tube is robust with respect to mishandling includingreduced inversions and partial blood draw. The product shelf life is one yearfrom the date of manufacture including limited storage temperature excursionswhich 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 2.5mL nominal blood draw. The referenced first dimension represents the diameterof the tube and the second dimension represents the length of the tube.
IntendedUse/Indicationsfor Use	The PAXgene® Blood DNA Tube is intended to collect, anticoagulate, stabilize,transport, and store a venous whole blood sample for preparation of human DNAfor use with molecular diagnostic test methods that require DNA.The performance characteristics of this device have not been established formolecular diagnostic assays in general. Users must validate use of product fortheir specific molecular diagnostic assay.
TechnologicalCharacteristics	The technological characteristics of the subject device, the PAXgene® BloodDNA Tube, are equivalent to that of the predicate device, the BD Vacutainer®PPT™ Plasma Preparation Tube with respect to device design and operatingprinciple. The PAXgene® Blood DNA Tube utilizes identical componentmaterials to the BD Vacutainer® PPT™ Plasma Preparation Tube. Aside fromintended use/indications for use, the only changes are with the amount ofK2EDTA present in the tube and lack of gel barrier material. These differencesdo not raise any new questions of safety or effectiveness.

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	Subject/Evaluation Device	Predicate Device
Key Parameters	PAXgene® Blood DNA Tube	[510(k):K972075] BD Vacutainer® PPTTM Plasma Preparation Tube
Intended Use/Indicationsfor Use	The PAXgene® Blood DNA Tubeis intended to collect,anticoagulate, stabilize, transport,and store a venous whole bloodsample for preparation of humanDNA for use with moleculardiagnostic test methods that requireDNA.The performance characteristics ofthis device have not beenestablished for moleculardiagnostic assays in general. Usersmust validate use of product fortheir specific molecular diagnosticassay.	The Vacutainer® Brand PPTTMPlasma Preparation Tube with EDTAanticoagulant and a gel barriermaterial are evacuated bloodcollection tubes which provide ameans of collecting, processing andtransporting blood in a closed plastictube. When the Tube is used togetherwith Vacutainer® Brand Needles andHolders, it is a closed system for thecollection of venous blood with thesame indications identified here.Blood collected in a tube containingEDTA anticoagulant and gel barriermaterial is used primarily to provideundiluted plasma for use in moleculardiagnostic test methods including butnot limited to PCR (PolymeraseChain Reaction) and bDNA(branched DNA). The specimen mayalso be used for other testing thatrequires an undiluted plasma sampleas determined by the laboratory.
Design/Function	Evacuated blood collection tube	Same
Dimensions	13 mm x 75 mm	13 mm x 100 mm / 16 mm x 100 mm
Nominal Draw Volume	2.5 mL	5.0 mL / 8.5 mL
Closure	BD HemogardTM closure consistingof a rubber stopper plus BDHemogardTM shield	Same
Anticoagulant	K₂EDTA	Same
Tube Material	Polyethylene terephthalate (PET)	Same
Tube Stopper Lubricant	Silicone	Same
Tube Sterility	Sterile	Same
Sterilization Method	Gamma irradiation	Same
Sterility Assurance Level	10-6	Same
Shelf Life	12 months	Same
Injection Molding(Tube/HemogardTMClosure)	Injection molded	Same
Rubber Molding (Stopper)	Compression molded rubber	Same
Interior Coating	Spray coated/Dried	Same
Evacuation	Vacuum chamber	Same
Shelf Pack Level	Shrink-wrapped expandedpolystyrene (EPS) tray	Same
Shipper/Case Level	Corrugated cardboard	Same
Performance Characteristics	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. The test results demonstrated that the subject device performance was substantially equivalent to the legally marketed predicate device for the collection, anticoagulation, stabilization, transportation and storage of venous whole blood for the preparation of human DNA for use with molecular diagnostic test methods that required DNA.

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

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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.

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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

	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.

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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 fluorescentmicrospherebased flowcytometry	Multiplex fluorescentmicrospherebased flowcytometry	Electrochemical detection basedDNA microarray	N/A, gel-based readout	N/A, gel-based readout
DNA isolation kit andinstrument technology	Silica membrane	Silica membrane	Silica membrane	Magnetic bead	Magnetic beadand silicamembrane

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	Samplestested	Correctcalls	No-calls	% Correctcalls	95% CIlower bound
Site A	CF Assay	40	40	0	100%	91.2%
	HLA Assay 1	40	40	0	100%	91.2%
Site B	CF Assay, After Retesting†	37	36	1	97.3%	86.2%
	HLA Assay 1*	40	40	0	100%	91.2%
Site C	CF Assay	40	40	0	100%	91.2%
	HLA Assay 1, After Retest‡	40	40	0	100%	91.2%
Site D	Thrombophilia	80	80	0	100%	95.2%
Site E	HLA Assay 2, After Retest§	698	698	0	100%	99.5%
	HLA Assay 3	100	100	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	No-calls	% Correct calls	95% CI lower bound
Site-to-sitereproducibility	A, B, C	CF Assay	20	20	0	100%	N/A
Site-to-sitereproducibility	A, B, C	HLA Assay 1*	20	20	0	100%	N/A
Lot-to-lotvariation	A	CF Assay	20	20	0	100%	N/A
Lot-to-lotvariation	A	HLA Assay 1	20	20	0	100%	N/A
Tubeperformance	A, B, C	CF Assay, After Retest†	117	116	1	99.1%	95.3%
Tubeperformance	A, B, C	HLA Assay 1, AfterRetest‡*	120	120	0	100%	96.9%
	D	Thrombophilia Assay	80	80	0	100%	95.4%
	E	HLA Assay 2, After Retest§	698	698	0	100%	99.5%
Interference	E	HLA Assay 3	100	100	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:

Storagecondition	Timepoints	Groups	Blood storagecondition	Subjects	Assayconcordance
25°C	12, 13months	6: 3 lots per time point	14 days at 18–25°C	24 (12 per timepoint/group)	100% (72/72)
25°C	7, 13months	2: 1 lot per time point	14 days at 18–25°C	24 (12 per timepoint/group)	100% (24/24)
25°C withtemperaturecycling for 10days at 45°Cand 5 days at-20°C	7, 13months	3: 1 at 7 months and 2at 13 months(temperature cycling at6 and 12 months)	14 days at 18–25°C	24 (12 per timepoint/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
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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)	Assayconcordance
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.

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.