(210 days)
Not Found
P200010
No
The device is a blood collection tube and does not perform any analysis or processing that would typically involve AI/ML. The performance studies focus on the tube's ability to preserve cfDNA for downstream analysis by a separate assay.
No.
This device is a blood collection tube intended for the collection and transport of whole blood samples for diagnostic assays, not for therapeutic purposes.
No
The device is a blood collection tube intended for collecting and transporting blood samples for use with other diagnostic assays. It does not perform the diagnostic analysis itself.
No
The device description explicitly states it is comprised of physical components: a glass tube, rubber stopper, anticoagulant, and cell preservatives. It is a blood collection tube, which is a hardware device.
Yes, this device is an IVD (In Vitro Diagnostic).
Here's why:
- Intended Use: The intended use explicitly states that the device is for the "collection, and transport of venous whole blood samples for use in conjunction with cell-free DNA next-generation sequencing liquid biopsy assays". These assays are performed in vitro (outside the body) on biological samples to provide information for diagnostic purposes.
- Device Description: The device is a blood collection tube designed to collect a biological specimen (whole blood) for subsequent laboratory analysis.
- Performance Studies: The performance studies described focus on evaluating the device's ability to preserve the sample (cfDNA) for use in a specific diagnostic assay (Guardant360 CDx assay). This directly relates to the device's function as a component in an in vitro diagnostic process.
- Reference Device: The device is intended for use with the Guardant360 CDx assay (P200010), which is itself a diagnostic assay.
The device is a sample collection and preservation device specifically designed to support in vitro diagnostic testing. Therefore, it falls under the definition of an IVD.
N/A
Intended Use / Indications for Use
Cell-Free DNA BCT is a direct-draw venous whole blood collection device intended for the collection, and transport of venous whole blood samples for use in conjunction with cell-free DNA next-generation sequencing liquid biopsy assays that have been cleared or approved for use with samples collected in the Cell-Free DNA BCT device.
Product codes (comma separated list FDA assigned to the subject device)
QMA
Device Description
Cell-Free DNA BCT is a sterile, single use, direct-draw blood collection tube comprised of 3 components (i.e. glass tube with rubber stopper, anticoagulant, and cell preservatives). The blood collection tube manufactured with USP Type III glass containing cerium oxide (to prevent color change associated with gamma irradiation). Each tube includes 200 uL ± 10% of liquid reagent composition includes an anticoagulant K3EDTAand a preservative.
The device 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 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 (10 mL), it is disengaged from the holder and inverted 10 times to mix the blood. The specimen is then transported to the lab for plasma isolation and extraction of cfDNA.
Mentions image processing
Not Found
Mentions AI, DNN, or ML
Not Found
Input Imaging Modality
Not Found
Anatomical Site
Not Found
Indicated Patient Age Range
Not Found
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)
Repeatability
Within-lot (between tube) variability was evaluated using native specimens collected from 33 patients with advanced stage solid tumors run on the Guardant360 CDx assay (P200010). Venous whole blood was drawn into 4 Cell-Free DNA BCTs from a single lot per subject. A single lot of Guardant360 CDx sample preparation kit was used by the blood collection vendor to isolate plasma within 7 days after blood collection. Plasma was then shipped to Guardant Health (GH) on dry ice and stored at -80°C until further processing. Sequencing was performed using 6 NextSeq 550 Sequencers (Illumina). Of the 132 samples processed, 131 samples passed all GH Quality Control metrics and were included in the final analysis. The sample that was found to have evidence of contamination during sample processing. Variability between replicates for each patient was evaluated based on variant call agreement for somatic variants. A concordant positive call reflects detection of an identical sequencing alteration between replicates, and a discordant call reflects the presence of an alteration in one replicate and the absence of that same alteration in another replicate. Average positive agreement (APA) and average negative agreement (ANA) were calculated.
Reproducibility
Lot to lot variability was evaluated using native specimens collected from 30 patients with advanced stage solid tumors run on the Guardant Health Guardant360 CDx assay (P200010). Venous whole blood was drawn into Cell-Free DNA BCTs representing 3 different lots. All Cell-Free DNA BCTs were processed into plasma within 7 days after whole blood collection, and the plasma was frozen at -80°C ± 10°C until analysis using the Guardant360 CDx Test. Variability between Cell-Free DNA BCT lot was evaluated based on variant call agreement for somatic variants. APA and ANA were calculated as described previously.
Shelf-life
To validate device performance across the claimed shelf life when the device is stored prior to blood collection under the recommended storage conditions (2-30°C), venous whole blood was collected from 30 healthy subjects. Three lots of Cell-Free DNA BCTs were tested under the following conditions: without storage (T0), 8.5 months after manufacture (T8.5), 12.5 months after manufacture (T12.5), 18.5 months after manufacture (T18.5), and 24.5 months after manufacture (T24.5). Prior to blood collection, Cell-Free DNA BCTs were stored at 2, 22, or 30°C. For T0, venous whole blood was collected into 2 Cell-Free DNA BCTs for each of the 3 lots. After collection, T0 Cell-Free DNA BCTs were held at 22°C for a maximum of 4 hours. For the remaining timepoints (8.5, 12.5, 18.5, 24.5 months), 7 Cell-Free DNA BCTs were collected at each aged timepoint from each subject representing the following conditions: 1 conventional KiEDTA Vacutainerstored at 22°C for a maximum of 4 hours (which was considered the baseline condition), 2 Cell-Free DNA BCTs stored at 2°C, 2 Cell-Free DNA BCTs stored at 22°C, and 2 Cell-Free DNA BCTs stored at 30°C. Each set of Cell-Free DNA BCTs collected from the same patient, sharing the same pre-collection storage temperature, were divided into two conditions: immediate processing (D0) and storage for 8 days (D8). Plasma was then isolated from the whole blood samples and cfDNA was extracted using the same kit as used in the Guardant360 CDx assay (P200010). cfDNA concentration was assessed using droplet digital PCR (ddPCR) and fluorometry, and size and overall cfDNA sample integrity was assessed by electrophoress. At T0, ctDNA concentration, size, and integrity was compared between Cell-Free DNA BCTs that were not stored and tubes that were stored at 22°C for 8 days after blood collection. The results support that cfDNA concentration, size, and integrity were maintained for 8 days when stored at 22°C after collection. At each timepoint (8.5, 12.5, 18.5, 24.5 months), samples drawn into Cell-Free DNA BCTs were compared to blood samples drawn into K2EDTA tubes that were processed into plasma on the same day as blood collection. The results support that devices stored at 2-30°C for 18.5 months prior to blood collection are able to maintain cfDNA concentration and integrity when blood specimens are stored for up to 8 days after blood collection.
Additional Studies
Additional studies were conducted to assess robustifingation, draw volume, stopper closure assembly stability, stopper pullout force, stopper resealing, and anticoagulant effectiveness. Study protocols, acceptance criteria, and results for these studies were provided and found to be acceptable.
Preservative
To validate that the preservative formulation does not interfere with the Guardant Health Guardant 360 CDX assay (P200010), three venous whole blood specimens were collected into candidate devices from each of 12 subjects with advanced stage solid tumors. Cell-Free DNA BCTs were manufactured to reflect the normal preservative formulation ("reference"), 2x Preservative A, or 2x Preservative B. After collection, whole blood samples were processed into plasma and then shipped to Guardant Health for cfDNA extraction and sequencing. cfDNA analysis was conducted using the Guardant 360CDx assay. Performance was evaluated based on sample-level molecule recovery. exon-level molecule recovery, and variant call concordance relative to the reference Cell-Free DNA BCT. Each variant called in the reference sample was evaluated in the experimental condition samples by positive percent agreement (PPA). The total number of concordant and discordant calls for all reference positive calls in a given experimental condition was counted across patients and used to calculate PPA. For each reference - treatment sample pair, each eligible site that is negative in the reference sample will be assessed for presence of a somatic call in the treatment sample via Negative Percent Agreement. The total number of concordant and discordant calls for all reference negative calls in a given experimental condition will be counted across patients and used to calculate NPA. The results confirmed that increasing either component of the preservative does not interfere with the ability of the Cell-Free DNA BCT to preserve cfDNA suitable for assay performance.
Incomplete Mixing
The instructions for use indicate that the tube should be inverted 10 times after collection. To evaluate the impact of variations in mixing after blood collection, specimens were collects with advanced stage solid tumors. Venous whole blood was collected into three Cell-Free DNA BCTs per subject. Cell-Free DNA BCTs collected from each patient were inverted 10 times. 5 times. After collection, whole blood samples were processed into plasma and then shipped to Guardant Health for ctDNA extraction and sequencing, cfDNA analysis was conducted using the Guardant 360CDx assav. Performance was assessed based on (D) (4) relative to 10 inversions. As described above under the Preservative interference study. PPA and (b) (4) NPA were used to assess variant call concordance. The results indequate or overmixing may result in diminished performance.
Short Draw
To evaluate potential interference caused by underfilling Cell-Free DNA BCTs, specimens were collected from 15 subjects with advanced stage solid tumors. Venous whole blood was collected into three Cell-Free DNA BCTs per subject. Cell-Free DNA BCTs were manufactured to reflect the normal reagent (e.g. preservative and anticoagulant) formulation ("reference"), 2x volume of Reagent, or (0) (4) Reagent. These conditions were intended to reflect 10 mL whole blood collected, 5 mL whole blood collected, and 10) (4) whole blood collection, whole blood samples were processed into plasma and then shipped to (b) (4) for cfDNA extraction and sequencing. cfDNA analysis was conducted using the Guardant 360CDx assay. The results indicated that underfilling of the Cell-Free DNA BCTs with less than 5 mL of blood may lead to poor product performance. A precaution has been included in the labeling warning users to fill the tube completely.
Tube Stopper
Extractable and leachable studies were performed to identify substances within the tube stopper that may interact with patient specimens and interfere with the ability of the tube to preserve cfDNA. The results support that extractables from the tube stopper are not anticipated to interfere with device performance.
cfDNA Stability
To validate that specimens collected into the device maintain their integrity throughout the claimed shelf life, the sponsor collected specimens from patients with advanced stage solid tumors into aged Cell-Free DNA BCTs that were between 1 month and 17 months post-manufacture. Cell-Free DNA BCTs were stored at room temperature and blood collection. For each patient, venous whole blood was collected into four Cell-Free DNA BCTs: two Cell-Free DNA BCTs from the youngest lot (approximately 1 month post-manufacture) which served as the reference conditions (R1 and R2) and one Cell-Free DNA BCT from each of the older lots (8 months post-manufacture) which served as test. All Cell-Free DNA BCTs were processed to plasma within 7 days after whole blood collection, and the plasma was frozen at -80°C ± 10°C. cfDNA analysis was conducted using the Guardant 360CDx assay. The sponsor assessed the stability of the Cell-Free DNA BCTs in terms of variant call concordance (PPA and NPA) between the reference condition (1 month) and the test conditions (8 and 17 months). The results support that cfDNA isolated from the device is of sufficient quantity, quality, and integrity for the intended downstream application throughout the of the device.
Post-Collection Storage of Venous Whole Blood prior to plasma separation
To characterize preservation of cfDNA in the candidate device compared to K2EDTA tubes prior to plasma separation when venous whole blood samples are stored for up to 7 days post-blood collection, venous whole blood was collected from 30 healthy subjects. The candidate device and K2EDTA tubes were tested under the following conditions: plasma isolation immediately after collection (Day 0, held a (b) (4) for " (hours), stored at (b)(4) for 7 days, or stored at (b) (0) for 7 days. A total of eight tubes are collected from each donor (4 K2EDTA tubes and 4 Cell-Free DNA BCTs) and each tube was held at one of the conditions described previously. Plasma was then isolated from the whole blood samples and cfDNA was extracted using the same kit as used in the Guardant Health Guardan1360 CDx assay (P200010). cfDNA concentration was assessed using (b) (4) and size and overall cfDNA sample integrity was assessed by (b) (4) . For each condition evaluated, the Day 0 (b) (4) tube was used as the baseline. The results demonstrated that the candidate device is able to preserve cfDNA specimen concentration and integrity better than K2EDTA tubes when Cell-Free DNA BCTs are stored post-blood collection for up to 8 days at (0) (4).
To validate that storage of Cell-Free DNA BCTs at 2-30°C prior to whole blood collection or storage of whole blood specimens in the Cell-Free DNA BCT for up to 7 days after blood collection does not impact Guardant Health Guardant360 CDx assay (P200010) performance. the sponsor collected whole blood specimens with advanced stage solid tumors. Cell-Free DNA BCTs used in this study represented: as soon as feasible after manufacture (T0), 3.2 months (T3.2), 7.2 months (T12.2), 18.2 months (T18.2), and 24.2 months (T24.2) after manufacturing. T0 Cell-Free DNA BCTs were not stored prior to blood collection. T3.2, T7.2, and T12.2 Cell-Free DNA BCTs were stored at the extremes of the recommended empty storage temperature range (b) (4) prior to blood collection. At each timepoint, venous whole blood was collects and then plasma was either isolated one day after blood collection (D1) or after 7 days of storage (D7). Plasma was stored at (B) (4) until c(DNA extraction and analysis using the Guardant Health Guardant360 CDx assay (P200010). Variability between conditions (Cell-Free DNA BCT storage are (4) 0r (4) (4) prior to collection ; plasma isolation one day after blood collection of plasma isolation after 7 days of storage) was evaluated based on variant call agreements. APA and ANA was calculated as described previously. The APA values between D1 and D7 within each combination of time point. storage temperature, and Cell-Free DNA BCT lot are shown below. The results demonstrate that storage of whole blood specimens in the Cell-Free DNA BCT for up to 7 days after blood collection does not impact Guardant Health Guardant360 CDx assay (P200010) performance. The results demonstrate that storage of Cell-Free DNA BCTs at 2-30°C prior to whole blood collection does not impact Guardant Health Guardant360 CDx assay (P200010) performance.
Shipping Stability
The sponsor conducted a series of tests to demonstrate that the device is robust to extreme temperature (b) (4) and physical stress conditions (e.g. vibration, drops, compression) that may be encountered during before or after sample collection. Study protocols, acceptance criteria, and results for these studies were found to be accepable.
Cell-Free DNA BCT vs. K2EDTA BCT Concordance
The purpose of this study was to establish concordance between test results obtained with cfDNA isolated from the Cell-Free DNA BCT and test results obtained with cfDNA isolated from K2EDTA BCTs since K2EDTA BCTs were used to collect samples in the clinical studies supporting the safety and effectiveness of the Guardant360 CDx (P200010). Blood from non-small cell lung cancer Stage III or IV patients, prescreened externally for CDx positive and negative markers EGFR L858R, EGFR 1790M, EGFR exon 19 deletions), were collected by utilizing two K2EDTA BCTs and two Cell-Free DNA BCTs. The second K2EDTA BCT was not processed for this study. A total of 59 patients were enrolled, some with and others without CDx variants, and whole blood samples were tested from three tubes, two Cell-Free DNA BCTs and one K2EDTA tube. The performance of K2EDTA BCTs relative to Cell-Free DNA BCTs was evaluated through a call agreement analysis which tests the difference of the (b) (4). Of the one-hundred and seventy seven (01(4) aliquots ( " samples across 3 Cell-Free DNA BCT designations), 176 (4) (4) passed in-process and post-sequencial of (A) (A) (A) (A) (A) (A) (A) (A) (A) (A) (A) (A) (A) (A) (A) (A) (A) (A) (A) (A) (A) (A) (A) (A) (A) (A) (A) (A) (A) (A) (A) (A) ( b) (4) patients replicates. In total, " patients met study criteria for including " distinct CDx variants observed in at least one tube. Call concordance across tubes, aggregating across all CDx variants, is shown. K2EDTA tubes and Cell-Free DNA BCTs demonstrated expected levels of positive agreement, PPA(0)(4) for CDx variants. Discordant detection was observed below LoD, with agreement above LoD being (b) (4) KxEDTA and Cell-Free DNA BCTs demonstrated expected levels of negative agreement, NPA(b) (4) for CDx variants. The delta PPA and delta NPA values, shown below, were within acceptable limits. The data confirmed concordance between test results obtained with cfDNA isolated from Cell-Free DNA BCT and test results obtained with cfDNA isolated from K2EDTA BCTs.
Key Metrics (Sensitivity, Specificity, PPV, NPV, etc.)
Average positive agreement (APA), Average negative agreement (ANA), Positive percent agreement (PPA), Negative percent agreement (NPA)
Predicate Device(s): If the device was cleared using the 510(k) pathway, identify the Predicate Device(s) K/DEN number used to claim substantial equivalence and list them here in a comma separated list exactly as they appear in the text. List the primary predicate first in the list.
Not Found
Reference Device(s): Identify the Reference Device(s) K/DEN number and list them here in a comma separated list exactly as they appear in the text.
Not Found
Predetermined Change Control Plan (PCCP) - All Relevant Information for the subject device only (e.g. presence / absence, what scope was granted / cleared under the PCCP, any restrictions, etc).
Not Found
§ 862.1676 Blood collection device for cell-free nucleic acids.
(a)
Identification. A blood collection device for cell-free nucleic acids is a device intended for medical purposes to collect, store, transport, and handle blood specimens and to stabilize and isolate cell-free nucleic acid components prior to further testing.(b)
Classification. Class II (special controls). The special controls for this device are:(1) Design verification and validation documentation must include appropriate design inputs and design outputs that are essential for the proper functioning of the device for its intended use, including all of its indications for use, and must include the following:
(i) Documentation demonstrating that appropriate, as determined by FDA, measures are in place (
e.g., validated device design features and specifications) to ensure that users of blood collection device for cell-free nucleic acids devices are not exposed to undue risk of bloodborne pathogen exposure and operator injury during use of the device, including blood collection, transportation, and centrifugation processes.(ii) Documentation demonstrating that appropriate, as determined by FDA, measures are in place (
e.g., validated device design features and specifications) to ensure that the device reproducibly and reliably collects, transports, stabilizes, and isolates cell-free nucleic acids of sufficient yield and quality suitable for downstream applications as appropriate for its intended use. At a minimum, these measures must include:(A) Data demonstrating that blood samples collected in the device have reproducible cell-free nucleic acid yields that are suitable, as determined by FDA, for downstream testing as appropriate for the intended use, including estimates of within-lot, within-device, and lot-to-lot variability;
(B) Data demonstrating that cell-free nucleic acid yields isolated from blood specimens collected into the device do not add clinically significant bias to test results obtained using the downstream application(s) described in the intended use. For devices indicated for use with multiple downstream applications, data demonstrating acceptable performance for each type of claimed use or, alternatively, an appropriate, as determined by FDA, clinical justification for why such data are not needed;
(C) Data demonstrating that the device appropriately stabilizes cell-free nucleic acids after sample collection, during storage, and during transport over the claimed shelf life of the device;
(D) Data demonstrating that samples collected in the device have minimal levels of contamination with other types of nucleic acids present in cells or cellular components, and that these levels of contamination do not interfere with downstream testing;
(E) Data from analytical or clinical studies that demonstrate that, when used as intended, the device consistently draws a blood sample volume that is within the indicated fill range;
(F) Data from analytical or clinical studies that demonstrate that, when used as intended, cell-free nucleic acid yield, stability, and quality are not significantly impacted by interference due to other parts of the device (such as reduced or excess active ingredient) or specimen collection and processing procedures (such as hemolysis, centrifugation, or mixing of blood with anticoagulant or additives); and
(G) Data from analytical studies that demonstrate that the device is suitable for its intended use across all storage and sample handling conditions described in the device labeling, including device shelf life and shipping conditions (
e.g., temperature, humidity, duration).(iii) A protocol, reviewed and determined acceptable by FDA, that specifies the verification and validation activities that will be performed for anticipated device modifications to reevaluate performance claims or performance specifications. This protocol must include a process for assessing whether a modification to technology, engineering, performance, materials, specifications, or indications for use, or any combination thereof, could significantly affect the safety or effectiveness of the device. The protocol must include assessment metrics, acceptance criteria, and analytical methods for the performance testing of changes.
0
EVALUATION OF AUTOMATIC CLASS III DESIGNATION FOR Cell-Free DNA BCT
DECISION SUMMARY
A. DEN Number:
B. Purpose for Submission:
New device
C. Measurand:
Not applicable
D. Type of Test:
Not applicable
E. Applicant:
Streck, Inc.
F. Proprietary and Established Names:
Cell-Free DNA BCT
G. Regulatory Information:
| Regulation | Name | Product Code | Panel |
|---|---|---|---|
| 21 CFR 862.1676 | Blood collection device | ||
| for cell-free nucleic | |||
| acids | QMA | Chemistry (75) |
H. Indications for Use:
1. Indication(s) for use:
Cell-Free DNA BCT is a direct-draw venous whole blood collection device intended for the collection, and transport of venous whole blood samples for use in conjunction with cell-free DNA next-generation sequencing liquid biopsy assays that have been cleared or approved for use with samples collected in the Cell-Free DNA BCT device.
-
- Special conditions for use statement(s):
- Performance characteristics for this device have only been established on the Guardant360 CDx assay (P200010), which was . performed at one Guardant Health laboratory.
- Prescription use only. .
- · Do not store outside of established conditions.
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- Do not transfer samples drawn into tubes containing other anti-coagulants and/or preservatives into Cell-Free DNA BCT. ●
- Do not use past expiration date printed on label. .
- Do not use for clinical chemistry assays other than liquid biopsy next-generation sequencing. ●
- Do not use for collection of materials to be injected into patients. ●
- Cell-Free DNA BCT is not intended for the stabilization of RNA nor is it intended for viral or microbial nucleic acids. .
-
- Special instrument requirements:
Not applicable.
I. Device Description:
Cell-Free DNA BCT is a sterile, single use, direct-draw blood collection tube comprised of 3 components (i.e. glass tube with rubber stopper, anticoagulant, and cell preservatives). The blood collection tube manufactured with USP Type III glass containing cerium oxide (to prevent color change associated with gamma irradiation). Each tube includes 200 uL ± 10% of liquid reagent composition includes an anticoagulant K3EDTAand a preservative.
The device 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 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 (10 mL), it is disengaged from the holder and inverted 10 times to mix the blood. The specimen is then transported to the lab for plasma isolation and extraction of cfDNA.
J. Standard/Guidance Document Referenced (if applicable):
CLSI EP25-A (Replaces EP25-P) Evaluation of Stability of In Vitro Diagnostic Reagents: Approved Guideline.
ISO 11137-1: Sterilization of health care products-Radiation: Part 1: Requirements for development, validation and routine control of a sterilization process for medical devices
K. Test Principle:
Not applicable.
L. Performance Characteristics (if/when applicable):
-
- Analytical performance:
- a. Precision/Reproducibility:
Repeatability
Within-lot (between tube) variability was evaluated using native specimens collected from 33 patients with advanced stage solid tumors run on the Guardant360 CDx assay (P200010). Venous whole blood was drawn into 4 Cell-Free DNA BCTs from a single lot per subject. A single lot of Guardant360 CDx sample preparation kit was used by the blood collection vendor to isolate plasma within 7 days after blood collection. Plasma was then shipped to Guardant Health (GH) on dry ice and stored at -80°C until further processing. Sequencing was performed using 6 NextSeq 550 Sequencers (Illumina). Of the 132 samples processed, 131 samples passed all GH Quality Control metrics and were included in the final analysis. The sample that was found to have evidence of contamination during sample processing. Variability between replicates for each patient was evaluated based on variant call agreement for somatic variants. A concordant positive call reflects detection of an identical sequencing alteration between replicates, and a discordant call reflects the presence of an alteration in one replicate and the
2
absence of that same alteration in another replicate. Average positive agreement (APA) and average negative agreement (ANA) were calculated as follows:
$$APA = \frac{# \text{ concordant positives}}{# \text{ concordant positives } + \frac{# \text{ discordant calls}}{2}}$$
$$ANA = \frac{# \text{ concordant negatives}}{# \text{ concordant negatives } + \frac{# \text{ discordant calls}}{2}}$$
Average positive agreement (APA) was calculated overall and for those variants detected at mutation allele frequency (MAF) ≥ 1x the limit of detection (LoD), whereas average negative agreement (ANA) was reported panelwide across all reportable positions and variants. This statistic includes all called variant sites, not just the eligible variants sites based on LoD in the source samples, so includes positions with expected stochastic detection due to low mutant molecule count. The results are summarized below:
3
| Concordant
positive calls
(overall ≥ 1x
LoD) | Discordant
calls
(overall ≥ 1x
LoD) | Overall
APA | APA
(≥ 1x LoD) | Panel-Wide
ANA |
|---------------------------------------------------------|------------------------------------------------|----------------|-------------------|-------------------|
| 40 32 | 11 5 | (b) (4) | | |
Reproducibility
Lot to lot variability was evaluated using native specimens collected from 30 patients with advanced stage solid tumors run on the Guardant Health Guardant360 CDx assay (P200010). Venous whole blood was drawn into Cell-Free DNA BCTs representing 3 different lots. All Cell-Free DNA BCTs were processed into plasma within 7 days after whole blood collection, and the plasma was frozen at -80°C ± 10°C until analysis using the Guardant360 CDx Test. Variability between Cell-Free DNA BCT lot was evaluated based on variant call agreement for somatic variants. APA and ANA were calculated as described previously. The results are summarized below.
| Lot
Comparison | Overall
APA
(b) (4) | APA
(≥ 1x LoD) | Panel-Wide
ANA |
|-------------------|---------------------------|-------------------|-------------------|
| 1 vs 2 | | | |
| 1 vs 3 | | | |
| 2 vs 3 | | | |
-
b. Linearity/assay reportable range:
Not applicable. -
c. Traceability, Stability, Expected values:
Shelf-life
To validate device performance across the claimed shelf life when the device is stored prior to blood collection under the recommended storage conditions (2-30°C), venous whole blood was collected from 30 healthy subjects. Three lots of Cell-Free DNA BCTs were tested under the following conditions: without storage (TO), 8.5 months after manufacture (T8.5), 12.5 months after manufacture (T12.5), 18.5 months after manufacture (T18.5), and 24.5 months after manufacture (T24.5). Prior to blood collection, Cell-Free DNA BCTs were stored at 2, 22, or 30°C. For T0, venous whole blood was collected into 2 Cell-Free DNA BCTs for each of the 3 lots. After collection, T0 Cell-Free DNA BCTs were held at 22°C for a maximum of 4 hours. For the remaining timepoints (8.5, 12.5, 18.5, 24.5 months), 7 Cell-Free DNA BCTs were collected at each aged timepoint from each subject representing the following conditions: 1 conventional KiEDTA Vacutainerstored at 22°C for a maximum of 4 hours (which was considered the baseline condition), 2 Cell-Free DNA BCTs stored at 2°C, 2 Cell-Free DNA BCTs stored at 22°C, and 2 Cell-Free DNA BCTs stored at 30°C. Each set of Cell-Free DNA BCTs collected from the same patient, sharing the same pre-collection storage temperature, were divided into two conditions: immediate processing (D0) and storage for 8 days (D8). Plasma was then isolated from the whole blood samples and cfDNA was extracted using the same kit as used in the Guardant360 CDx assay (P200010). cfDNA concentration was assessed using droplet digital PCR (ddPCR) and fluorometry, and size and overall cfDNA sample integrity was assessed by electrophoress. At TO, ctDNA concentration, size, and integrity was compared between Cell-Free DNA BCTs that were not stored and tubes that were stored at 22°C for 8 days after blood collection. The results support that cfDNA concentration, size, and integrity were maintained for 8 days when stored at 22°C after collection. At each timepoint (8.5, 12.5, 18.5, 24.5 months), samples drawn into Cell-Free DNA BCTs were compared to blood samples drawn into K2EDTA tubes that were processed into plasma on the same day as blood collection. The results support that devices stored at 2-30°C for 18.5 months prior to blood collection are able to maintain cfDNA concentration and integrity when blood specimens are stored for up to 8 days after blood collection.
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Additional Studies
Additional studies were conducted to assess robustifingation, draw volume, stopper closure assembly stability, stopper pullout force, stopper resealing, and anticoagulant effectiveness. Study protocols, acceptance criteria, and results for these studies were provided and found to be acceptable.
-
d. Detection limit:
Not applicable. -
e. Analytical specificity:
Preservative
To validate that the preservative formulation does not interfere with the Guardant Health Guardant 360 CDX assay (P200010), three venous whole blood specimens were collected into candidate devices from each of 12 subjects with advanced stage solid tumors. Cell-Free DNA BCTs were manufactured to reflect the normal preservative formulation ("reference"), 2x Preservative A, or 2x Preservative B. After collection, whole blood samples were processed into plasma and then shipped to Guardant Health for cfDNA extraction and sequencing. cfDNA analysis was conducted using the Guardant 360CDx assay. Performance was evaluated based on sample-level molecule recovery. exon-level molecule recovery, and variant call concordance relative to the reference Cell-Free DNA BCT. Each variant called in the reference sample was evaluated in the experimental condition samples by positive percent agreement (PPA). The total number of concordant and discordant calls for all reference positive calls in a given experimental condition was counted across patients and used to calculate PPA. For each reference - treatment sample pair, each eligible site that is negative in the reference sample will be assessed for presence of a somatic call in the treatment sample via Negative Percent Agreement. The total number of concordant and discordant calls for all reference negative calls in a given experimental condition will be counted across patients and used to calculate NPA using the following equation. expressed as a percentage. PPA and NPA were calculated as follows:
$$PPA \text{ (Experimental + | Reference +)} = \frac{# \text{concordant positive calls}}{# \text{reference positive calls}}$$
$$NPA \text{ (Experimental - | Reference -)} = \frac{# \text{concordant negative calls}}{# \text{reference negative calls}}$$
PPA was calculated overall and for those variants detected at mutation allele frequency (MAF) > 1x the limit of detection (LoD), whereas average negative agreement (ANA) was reported panel-wide across all reportable positions and variants. The results confirmed that increasing either component of the preservative does not interfere with the ability of the Cell-Free DNA BCT to preserve cfDNA suitable for assay performance.
| Endpoint | Metric | 2x Preservative A | 2x Preservative B |
|---|---|---|---|
| Sample-level | |||
| Molecule | |||
| Recovery | |||
| Difference | |||
| (fold change) | Median of NSC fold | ||
| (condition/reference) | (b) (4) | ||
| Relative exon- | |||
| level coverage | Lower bound of | ||
| 95% CI | |||
| Variant call | |||
| concordance | PPA | ||
| PPA (>1x LoD) | |||
| NPA |
5
Incomplete Mixing
The instructions for use indicate that the tube should be inverted 10 times after collection. To evaluate the impact of variations in mixing after blood collection, specimens were collects with advanced stage solid tumors. Venous whole blood was collected into three Cell-Free DNA BCTs per subject. Cell-Free DNA BCTs collected from each patient were inverted 10 times. 5 times. After collection, whole blood samples were processed into plasma and then shipped to Guardant Health for ctDNA extraction and sequencing, cfDNA analysis was conducted using the Guardant 360CDx assav. Performance was assessed based on (D) (4)
relative to 10 inversions. As described above under the Preservative interference study. PPA and (b) (4) NPA were used to assess variant call concordance. The results indequate or overmixing may result in diminished performance.
| Endpoint | Metric | 5 Inversions | 15 Inversions |
|---|---|---|---|
| Sample-level | |||
| Molecule | |||
| Recovery | |||
| Difference | |||
| (fold change) | Median of NSC fold | ||
| (condition/reference) | (b) (4) | ||
| Relative exon- | |||
| level coverage | Lower bound of | ||
| 95% CI | |||
| Variant call | |||
| concordance | PPA | ||
| PPA (>1x LoD) | |||
| NPA |
Short Draw
To evaluate potential interference caused by underfilling Cell-Free DNA BCTs, specimens were collected from 15 subjects with advanced stage solid tumors. Venous whole blood was collected into three Cell-Free DNA BCTs per subject. Cell-Free DNA BCTs were manufactured to reflect the normal reagent (e.g. preservative and anticoagulant) formulation ("reference"), 2x volume of Reagent, or (0) (4) Reagent. These conditions were intended to reflect 10 mL whole blood collected, 5 mL whole blood collected, and 10) (4) whole blood collection, whole blood samples were processed into plasma and then shipped to (b) (4) for cfDNA extraction and sequencing. cfDNA analysis was conducted using the Guardant 360CDx assay. Performance of the Guardant360 CDx assay was assessed based on (4) (4) (b) (4) . As described above under the
Preservative interference study, PPA and NPA were used to assess variant call concordance. The results indicated that underfilling of the Cell-Free DNA BCTs with less than 5 mL of blood may lead to poor product performance. A precaution has been included in the labeling warning users to fill the tube completely.
| Endpoint | Metric | 2x Reagent
(5mL whole blood) | (b) (4) Reagent
(b) (4) whole blood) |
|---------------------------------------------------------------------|---------------------------------------------|---------------------------------|-----------------------------------------|
| Sample-level
Molecule
Recovery
Difference
(fold change) | Median of NSC fold
(condition/reference) | (b) (4) | |
| Relative exon-
level coverage | Lower bound of
95% CI | | |
| Variant call
concordance | PPA | | |
| | PPA (≥1x LoD) | | |
| | NPA | | |
6
|--|
7
Tube Stopper
Extractable and leachable studies were performed to identify substances within the tube stopper that may interact with patient specimens and interfere with the ability of the tube to preserve cfDNA. The results support that extractables from the tube stopper are not anticipated to interfere with device performance.
f. Assay Cut-off:
Not applicable.
g. Specimen Stability
cfDNA Stability
To validate that specimens collected into the device maintain their integrity throughout the claimed shelf life, the sponsor collected specimens from patients with advanced stage solid tumors into aged Cell-Free DNA BCTs that were between 1 month and 17 months post-manufacture. Cell-Free DNA BCTs were stored at room temperature and blood collection. For each patient, venous whole blood was collected into four Cell-Free DNA BCTs: two Cell-Free DNA BCTs from the youngest lot (approximately 1 month post-manufacture) which served as the reference conditions (R1 and R2) and one Cell-Free DNA BCT from each of the older lots (8 months post-manufacture) which served as test. All Cell-Free DNA BCTs were processed to plasma within 7 days after whole blood collection, and the plasma was frozen at -80°C ± 10°C. cfDNA analysis was conducted using the Guardant 360CDx assay. The sponsor assessed the stability of the Cell-Free DNA BCTs in terms of variant call concordance (PPA and NPA) between the reference condition (1 month) and the test conditions (8 and 17 months). The results support that cfDNA isolated from the device is of sufficient quantity, quality, and integrity for the intended downstream application throughout the of the device.
Positive Percent Agreement (PPA) Variant Call Concordance
| Condition | $\Delta$ PPA1
(PPAr1t-
PPAr1r2) | 95%
Confidence
Interval of
$\Delta$ PPA1 | $\Delta$ PPA2
(PPAr2t-
PPAr2r1) | 95%
Confidence
Interval of
$\Delta$ PPA2 |
| ----------- | --------------------------------------- | --------------------------------------------------- | --------------------------------------- | --------------------------------------------------- |
|---|
8
| Condition | ΔNPA1
(NPAr1t-
NPAr1r2) | 95%
Confidence
Interval of
ΔNPA1 | ΔNPA2
(NPAr2t-
NPAr2r1) | 95%
Confidence
Interval of
ΔNPA2 |
|-----------|-------------------------------|-------------------------------------------|-------------------------------|-------------------------------------------|
| (b) (4) | | | | |
Negative Percent Agreement (NPA) Variant Call Concordance
Post-Collection Storage of Venous Whole Blood prior to plasma separation
To characterize preservation of cfDNA in the candidate device compared to K2EDTA tubes prior to plasma separation when venous whole blood samples are stored for up to 7 days post-blood collection, venous whole blood was collected from 30 healthy subjects. The candidate device and K2EDTA tubes were tested under the following conditions: plasma isolation immediately after collection (Day 0, held a (b) (4) for " (hours), stored at (b)(4) for 7 days, or stored at (b) (0) for 7 days. A total of eight tubes are collected from each donor (4 K2EDTA tubes and 4 Cell-Free DNA BCTs) and each tube was held at one of the conditions described previously. Plasma was then isolated from the whole blood samples and cfDNA was extracted using the same kit as used in the Guardant Health Guardan1360 CDx assay (P200010). cfDNA concentration was assessed using (b) (4) and size and overall cfDNA sample integrity was assessed by (b) (4) . For each condition evaluated, the Day 0 (b) (4) tube was used as the baseline. The results demonstrated that the candidate device is able to preserve cfDNA specimen concentration and integrity better than K2EDTA tubes when Cell-Free DNA BCTs are stored post-blood collection for up to 8 days at (0) (4)
To validate that storage of Cell-Free DNA BCTs at 2-30°C prior to whole blood collection or storage of whole blood specimens in the Cell-Free DNA BCT for up to 7 days after blood collection does not impact Guardant Health Guardant360 CDx assay (P200010) performance. the sponsor collected whole blood specimens with advanced stage solid tumors. Cell-Free DNA BCTs used in this study represented: as soon as feasible after manufacture (T0), 3.2 months (T3.2), 7.2 months (T12.2), 18.2 months (T18.2), and 24.2 months (T24.2) after manufacturing. T0 Cell-Free DNA BCTs were not stored prior to blood collection. T3.2, T7.2, and T12.2 Cell-Free DNA BCTs were stored at the extremes of the recommended empty storage temperature range (b) (4) prior to blood collection. At each timepoint, venous whole blood was collects and then plasma was either isolated one day after blood collection (D1) or after 7 days of storage (D7). Plasma was stored at (B) (4) until c(DNA extraction and analysis using the Guardant Health Guardant360 CDx assay (P200010). Variability between conditions (Cell-Free DNA BCT storage are (4) 0r (4) (4) prior to collection ; plasma isolation one day after blood collection of plasma isolation after 7 days of storage) was evaluated based on variant call agreements. APA and ANA was calculated as described previously. The APA values between D1 and D7 within each combination of time point. storage temperature, and Cell-Free DNA BCT lot are shown below. The results demonstrate that storage of whole blood specimens in the Cell-Free DNA BCT for up to 7 days after blood collection does not impact Guardant Health Guardant360 CDx assay (P200010) performance.
| APA and ANA Variant Call Concordance between D1 and D7 within each time point, storage temperature, and lot. | |||||
|---|---|---|---|---|---|
| Condition | APA | APA
(above LoD) | ANA | ANA
(above LoD) |
|-----------|-----|--------------------|-----|--------------------|
| (b) (4) | | | | |
9
(b) (4)
The APA and NPA values between 2°C and 30°C within each combination of time point, storage temperature, and Cell-Free DNA BCT lot are shown below. The results demonstrate that storage of Cell-Free DNA BCTs at 2-30°C prior to whole blood collection does not impact Guardant Health Guardant360 CDx assay (P200010) performance.
| Condition | APA | APA
(above LoD) | ANA | ANA
(above LoD) |
|-----------|-----|--------------------|-----|--------------------|
| (b) (4) | | | | |
APA and NPA Variant Call Concordance between 2°C and 30°C within each time point and lot.
h. Shipping Stability
The sponsor conducted a series of tests to demonstrate that the device is robust to extreme temperature (b) (4) and physical stress conditions (e.g. vibration, drops, compression) that may be encountered during before or after sample collection. Study protocols, acceptance criteria, and results for these studies were found to be accepable.
2. Comparison studies:
- Method comparison with predicate device: a.
Cell-Free DNA BCT vs. K2EDTA BCT Concordance
The purpose of this study was to establish concordance between test results obtained with cfDNA isolated from the Cell-Free DNA BCT and test results obtained with cfDNA isolated from K2EDTA BCTs since K2EDTA BCTs were used to collect samples in the clinical studies supporting the safety and effectiveness of the Guardant360 CDx (P200010).
Blood from non-small cell lung cancer Stage III or IV patients, prescreened externally for CDx positive and negative markers EGFR L858R, EGFR 1790M, EGFR exon 19 deletions), were collected by utilizing two K2EDTA BCTs and two Cell-Free DNA BCTs. The second K2EDTA BCT was not processed for this study. A total of 59 patients were enrolled, some with and others without CDx variants, and whole blood samples were tested from three tubes, two Cell-Free DNA BCTs and one K2EDTA tube.
The performance of K2EDTA BCTs relative to Cell-Free DNA BCTs was evaluated through a call agreement analysis which tests the difference of the (b) (4)
Of the one-hundred and seventy seven (01(4) aliquots ( " samples across 3 Cell-Free DNA BCT designations), 176 (4) (4) passed in-process and post-sequencial of (A) (A) (A) (A) (A) (A) (A) (A) (A) (A) (A) (A) (A) (A) (A) (A) (A) (A) (A) (A) (A) (A) (A) (A) (A) (A) (A) (A) (A) (A) (A) (A) (A) ( b) (4) patients replicates.
In total, " patients met study criteria for including " distinct CDx variants observed in at least one tube. Call concordance across tubes, aggregating across all CDx variants, is shown below.
10
CDx Call concordance
| S1+ | S1- | |||
|---|---|---|---|---|
| S2+ | S2- | S2+ | S2- | |
| K1+ | (b) (4) | |||
| K1- |
The PPA and NPA values across the entire set of CDx variants (aggregated), and for each CDx variants is summarized below. K2EDTA tubes and Cell-Free DNA BCTs demonstrated expected levels of positive agreement, PPA(0)(4) for CDx variants. Discordant detection was observed below LoD, with agreement above LoD being (b) (4) KxEDTA and Cell-Free DNA BCTs demonstrated expected levels of negative agreement, NPA(b) (4) for CDx variants. The delta PPA and delta NPA values, shown below, were within acceptable limits. The data confirmed concordance between test results obtained with cfDNA isolated from Cell-Free DNA BCT and test results obtained with cfDNA isolated from K2EDTA BCTs.
PPA and NPA Values for all CDx variants
| | | All EGFR
CDx Variants | | EGFR
T790M | | EGFR
L858R | | EGFR
Exon 19 Deletion | |
|-------------------------------------------------------|---------|--------------------------|-----|---------------|-----|---------------|-----|--------------------------|--|
| Tube Pairing | PPA | NPA | PPA | NPA | PPA | NPA | PPA | NPA | |
| K2EDTA
Cell-Free
DNA BCT #1 | (b) (4) | | | | | | | | |
| K2EDTA
Cell-Free
DNA BCT #2 | | | | | | | | | |
| Cell-Free DNA
BCT #2 Cell-
Free DNA
BCT #1 | | | | | | | | | |
| Cell-Free DNA
BCT #1 Cell-
Free DNA
BCT #2 | | | | | | | | | |
11
Delta PPA and NPA values for all CDx variants
| Condition | Value |
|---|---|
| ΔPPA1 | |
| (K2EDTA Cell-Free DNA BCT #1 - | |
| Cell-Free DNA BCT #2 Cell-Free DNA BCT #1) | (b) (4) |
| ΔPPA2 | |
| (K2EDTA Cell-Free DNA BCT #2 - | |
| Cell-Free DNA BCT #1 Cell-Free DNA BCT #2) | |
| ΔNPA1 | |
| (K2EDTA Cell-Free DNA BCT #1 - | |
| Cell-Free DNA BCT #2 Cell-Free DNA BCT #1) | |
| ΔNPA2 | |
| (K2EDTA Cell-Free DNA BCT #2 - | |
| Cell-Free DNA BCT #1 Cell-Free DNA BCT #2) |
-
b. Matrix Comparison:
Not applicable. -
- Clinical studies:
- a. Clinical Sensitivity:
Not applicable.
-
b. Clinical specificity:
Not applicable. -
c. Other clinical supportive data (when a. and b are not applicable):
Refer to P200010. -
- Clinical cut-off:
Not applicable.
- Clinical cut-off:
-
- Expected values/Reference range:
Not applicable.
- Expected values/Reference range:
M. Proposed Labeling:
The labeling supports the decision to grant the De Novo request for this device.
N. Identified Risks to Health and Mitigation Measures:
| Identified Risk | Mitigation Measures |
|---|---|
| Blood pathogen exposure/Injury | Certain design verification and validation |
12
| Identified Risk | Mitigation Measures |
|---|---|
| Failure to collect and transport sample | Certain design verification and validation |
| Insufficient sample quantity and quality | Certain design verification and validation |
O. Benefit/Risk Analysis:
Summary of the Assessment of Benefit For the Proposed Indications for Use
The Cell-Free DNA BCT may be used to collect and stabilize whole blood for further processing and testing of cell-free DNA (cfDNA). The tube may preserve cfDNA and minimize cellular contamination for up to 7 days following collection and storage at 18-25 degrees Celsius. Standard blood collection in an EDTA containing tube is typically stored for no longer than 2 hours prior to centrifugation. After the sample undergoes centrifugation, the plasma is stored in cold temperatures, then it is shipped to a laboratory which can perform the assay. The device can be realized in situations where blood cannot be processed to plasma immediately, and when limiting the anount of genomic DNA contamination is critical to assay performance.
Summary of the Assessment of Risk
For the Proposed Indications for Use
According to the National Cancer Institute's Biorepositories and Biospecimen Research Branch of DNA specific Guidelines. "cfDNA concentrations and cellular DNA contamination are greatly influenced by the type of blood collection tube used as well as the processing steps employed, which include the durations of a pre-centrifygation delay and parameters relating to centrifugation or filtration." Additionally, they state, "the mutant allele frequency in cfDNA samples is also sensitive to blood collection tube type, storage durations, and a combination of storage conditions and extraction methods." The risks associated with the candidate blood collection tube (BCT) device can include preanalytical issues, including delay in patient results due to insufficient cfDNA vield, insufficient anticoagulation, insufficient draw volume, and other analytical isks. Injury to laboratory personnel can occur due to broken BCTs during transport/centrifugation or leaking/breakage/splashing during handling of BCTs. Additionally. false results can occur due to insufficient vield and quality deficiencies, such as cellular DNA contamination, insufficient inversions/mixing, cfDNA instability, interference, lot-to-lot variability, etc.
The extent of the clinical risks from false results using a Cell-Free DNA BCT are dependent on the target tumor cell mutation and the purpose for testing (e.g. companion diagnostic testing for targeted cancer treatment, recurrence after surgery, understanding molecular profile of tumor(s) before and after treatment). For example, if used for the purposes of blood collection to identify a tumor mutation of targeted drug therapy, a falsely positive result can lead to potential unnecessary exposure to toxicity from the drug. A falsely negative result due to poor analytical performance of the Cell-Free DNA BCT can contribute to a missed opportunity to treat a patient with targeted cancer treatment. These clinical risks cannot be controlled by the candidate BCT device since the clinical risks of false results are dependent on the target tested.
Summary of the Assessment of Benefit-Risk For the Proposed Indications for Use
Despite the benefits of the device described above, the analytical performance of the Cell-Free DNA BCT involves risks that must be mitigated to ensure safe and effective performance of the device. General controls are insufficient to mitigate the risks of the device. However, the probable clinical benefits would outweigh the probable risks for the considering the mitigation of the risks provided for in the special controls. Device design verification , including verification and validation of design features and specifications as special controls, helps ensure that users of the device are not exposed to undue risks of blood-borne pathogen exposure or injury during use of the device, including blood collection, and
13
centrifugation processes. Additionally, further design verification and validation, including the clinical and analytical studies specified in the special controls, helps ensure that the device reproducibly collects, transports, stabilizes, and isolates cell-free nucleic acids, resulting in cell-free DNA samples that are of sufficient quantity and quality to be suith a next-generation sequencing liquid biopsy test system. Once the combination of the required general controls and the special controls established for this device are taken into consideration, the probable benefits would outweigh the probable risks.
Patient Perspectives
This submission did not include specific information on patient perspectives for this device.
Summary of the Benefit/Risk Conclusion
For the Proposed Indications for Use
Given the combination of the required general controls established for this device, the probable benefits would outweigh the probable risks.
P. Conclusion:
The De Novo request is granted and the device is classified under to the special controls identified in the letter granting the De Novo request: Product Code: Device Type: Blood collection device for cell-free nucleic acids Class: II (special controls) Regulation: 21 CFR 862.1676