(341 days)
No
The device description details a gene expression assay using RT-qPCR to quantify specific RNA transcripts and combine them into a score. There is no mention of AI or ML in the description of the scoring algorithm or interpretation. The "Mentions AI, DNN, or ML" section explicitly states "Not Found".
No
This device is an in vitro diagnostic (IVD) test used to aid in the differentiation of infection-positive from infection-negative systemic inflammation in patients suspected of sepsis. It helps in diagnosis rather than directly treating or preventing disease.
Yes
The device is explicitly stated to be an "in-vitro diagnostic test" and its intended use is "as an aid to differentiate infection-positive (sepsis) from infectionnegative systemic inflammation in patients suspected of sepsis."
No
The device is an in vitro diagnostic test that involves physical components (cartridge, reagents) and runs on a specific hardware system (Biocartis Idylla System) to perform gene expression analysis. While it generates a score, the core functionality is tied to the physical assay and hardware, not solely software.
Yes, this device is an IVD (In Vitro Diagnostic).
Here's why:
- Intended Use: The "Intended Use / Indications for Use" section explicitly states: "SeptiCyte ® RAPID is intended for in-vitro diagnostic use on the Biocartis IdyllaTM System."
- Device Description: The "Device Description" section also begins by stating: "SeptiCyte RAPID is an in vitro diagnostic test..."
- Function: The device performs a gene expression assay on a biological sample (whole blood) to aid in the differentiation of infection-positive from infection-negative systemic inflammation. This is a classic function of an in vitro diagnostic device.
- Specimen: It uses a biological specimen (whole blood) collected from a patient.
- Analysis: It analyzes the components of the biological specimen (RNA transcripts) to provide information about the patient's health status.
All of these points align with the definition of an In Vitro Diagnostic device.
N/A
Intended Use / Indications for Use
The SeptiCyte ® RAPID test is a gene expression assay using reverse transcription polymerase chain reaction to quantify the relative expression levels of host response genes isolated from whole blood collected in the PAXgene ® Blood RNA Tube. The SeptiCyte ® RAPID test is used in conjunction with clinical assessments and other laboratory findings as an aid to differentiate infection-positive (sepsis) from infectionnegative systemic inflammation in patients suspected of sepsis on their first day of ICU admission. The SeptiCyte ® RAPID test generates a score (SeptiScore®) that falls within one of four discrete Interpretation Bands based on the increasing likelihood of infectionpositive systemic inflammation. SeptiCyte ® RAPID is intended for in-vitro diagnostic use on the Biocartis IdyllaTM System.
Product codes
PRE
Device Description
SeptiCyte RAPID is an in vitro diagnostic test for simultaneous amplification and detection of two RNA transcripts (PLA2G7 and PLAC8) using total RNA extracted from human blood. The test has been designed, manufactured, and validated for use on the Biocartis Idylla real-time PCR system. The SeptiCyte RAPID test is performed with an Idylla Cartridge, a single-use, disposable, multi-chambered fluidic cartridge that runs on the Biocartis Idylla System. In an automated fashion, all reaction steps take place within the cartridge, including sample extraction/purification, RT-qPCR for the detection and relative quantification of the two human mRNA targets PLAC8. PLA2G7. Test results (measured Cg values and calculated SeptiScore) are available in about 65 minutes.
The specimen used for the SeptiCyte RAPID is a sample of whole blood collected in a PAXgene blood RNA tube (FDA 510k number K042613). The cartridge contains all of the necessary reagents to perform RNA isolation from the sample.
SeptiCyte RAPID uses quantitative, real-time determination of the amount of each transcript in the sample based on the detection of fluorescence by the Biocartis Idylla qPCR instrument function. The cartridge includes the reagents for reverse transcription and PCR. Transcripts PLAC8 and PLA2G7 are amplified and quantified. These values are combined to produce the SeptiScore, which is interpreted and categorized into four discrete bands, which are associated with a sequentially higher likelihood of sepsis.
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
Adults (≥20 years of age), specifically patients suspected of sepsis on their first day of ICU admission.
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
The validation and 510(k) clearance of SeptiCyte RAPID was based on samples and clinical data collected from the following observational trials, which recruited critically ill subjects suspected of sepsis:
- Netherlands-based Molecular diagnosis And Risk Stratification of sepsis (MARS) clinical trial (NCT01905033) - retrospective study
- USA-based Validation of septic gene Expression Using SeptiCvte (VENUS) clinical trial (NCT02127502) - retrospective study
- USA-based NEar PatienT MolecUlar TestiNg in Sepsis (NEPTUNE) clinical trial - prospective study
The overall clinical accuracy of the SeptiCyte RAPID test was established by combining data from the retrospective (N= 356, frozen specimens) and prospective (N = 30, fresh specimens) clinical studies for a combined dataset of size N = 386. The prospective study was terminated early due to enrollment restrictions during the COVID-19 pandemic.
In both retrospective and prospective studies, the clinical status of each subject (SIRS, sepsis or indeterminate) was determined by clinical case review or retrospective physician diagnosis (RPD) conducted by an external three-member expert panel not involved in the care of the patients. Three different methods of RPD were used in analysis, as briefly described below:
Consensus: Two or three RPD panelists agree that patient is either sepsis or SIRS. Indeterminates are excluded.
Unanimous: All three RPD panelists, as well as the consensus discharge evaluation of the site investigators, agree that patient is either sepsis or SIRS. (A unanimous call of indeterminate is also theoretically possible).
Forced: Applies to patients that are initially called "indeterminate" by the RPD panelists. The RPD panelists are then forced to make a consensus or unanimous call of sepsis or SIRS.
Summary of Performance Studies (study type, sample size, AUC, MRMC, standalone performance, key results)
Analytical Performance Data:
- Precision/Reproducibility:
- Repeatability: Evaluated using the Low sample pool (SeptiScore mean 4.56, CV 4.0%) over three days by two operators running three replicates per day on a single instrument.
- Intermediate Precision: Evaluated with one cartridge lot over 12 days using three instruments and two operators. Each operator's runs conducted over 4 non-sequential days. Low pool (SeptiScore mean 4.48, CV 5.0%), Med pool (SeptiScore mean 6.33, CV 4.3%), High pool (SeptiScore mean 10.33, CV 2.0%). Low CVs observed for individual PLA2G7 and PLAC8 Cq's.
- Lot-to-Lot reproducibility: Performed with Low, Medium, and High SeptiScore pools using three cartridge lots, over 3 non-consecutive days, using two instruments and one operator. Low pool (SeptiScore mean 4.36, SD 0.14, CV 3.3%), Medium pool (SeptiScore mean 6.25, SD 0.19, CV 3.0%), High pool (SeptiScore mean 10.20, SD 0.19, CV 1.8%). Low CVs observed for individual PLA2G7 and PLAC8 Cq's.
- Linearity/assay reportable range: Tested by spiking varying amounts of white blood cells (WBC) into platelet-reduced plasma (25 cells/uL to 50,000 cells/μL). An inverse log-linear response (Pearson's r2 > 0.99) was observed for PLA2G7 and PLAC8. SeptiScore compensated for Hook Effect at high input concentrations.
- Detection limit (LoD) and Quantitation limit (LoQ): Determined by spiking serial dilutions of WBCs into platelet-reduced plasma. LoD and LoQ were both 25 WBC/uL blood (19/20 replicates generated a SeptiScore for LoD, and with a standard deviation of 1.0 Score units or less for LoQ).
- Analytical specificity:
- Interfering Substances: No interference found for substances listed in Table 7-3 (e.g., Rheumatoid Factor, Heparin, Bilirubin, Hemoglobin, etc.) at tested concentrations.
- Lack of Reactivity Towards Blanks or Genomic DNA: SeptiCyte RAPID test did not exhibit reactivity toward blank samples (20 aliquots of nuclease-free PBS in PAXgene stabilizer, plus 10 empty cartridges) or human genomic DNA (10 replicates of 500 ng and 1000 ng gDNA per reaction).
- Carryover: Alternating blank, low, and high SeptiScore samples (21 cartridges total). All blank samples failed to generate a valid SeptiScore, low SeptiScore samples generated Band 1, and high SeptiScore samples generated Band 4, confirming no carryover contamination.
- PAXgene Blood RNA Tube Stability: Matched healthy donor PAXgene blood samples (15 donors) did not differ significantly with 2-hour incubation or fresh vs. frozen/thawed samples, confirming stability.
Clinical Studies:
- Study Types: Retrospective (MARS, VENUS) and Prospective (NEPTUNE, terminated early due to COVID-19 pandemic enrollment restrictions).
- Sample Size: Combined dataset of N = 386 (356 frozen retrospective specimens, 30 fresh prospective specimens).
- Primary Endpoint Acceptance Criteria:
- Monotonic increase in probability of sepsis across the four SeptiScore interpretation bands.
- Non-overlapping 80% confidence intervals for the probability of sepsis between non-adjacent SeptiScore interpretation bands (i.e., between bands 1 and 3; and between bands 2 and 4).
- Key Results (Prevalence of Sepsis across SeptiScore Bands):
- Forced RPD Method (N=386):
- Band 1 (0-4.9): 0.12 (80% CI: 0.08 - 0.18)
- Band 2 (5.0-6.1): 0.24 (80% CI: 0.18 - 0.3)
- Band 3 (6.2-7.3): 0.48 (80% CI: 0.4 - 0.56)
- Band 4 (7.4-15): 0.80 (80% CI: 0.74 - 0.84)
- Consensus RPD Method (N=348):
- Band 1 (0-4.9): 0.10 (80% CI: 0.06 - 0.15)
- Band 2 (5.0-6.1): 0.22 (80% CI: 0.16 - 0.29)
- Band 3 (6.2-7.3): 0.45 (80% CI: 0.37 - 0.54)
- Band 4 (7.4-15): 0.81 (80% CI: 0.75 - 0.85)
- Unanimous RPD Method (N=254):
- Band 1 (0-4.9): 0.11 (80% CI: 0.06 - 0.18)
- Band 2 (5.0-6.1): 0.20 (80% CI: 0.14 - 0.29)
- Band 3 (6.2-7.3): 0.46 (80% CI: 0.36 - 0.56)
- Band 4 (7.4-15): 0.82 (80% CI: 0.76 - 0.87)
- Forced RPD Method (N=386):
Key Metrics (Sensitivity, Specificity, PPV, NPV, etc.)
Sepsis Likelihood Ratio based on SeptiScore Interpretation Band:
- Forced Diagnosis:
- Band 4 (7.4 - 15.0): 5.23
- Band 3 (6.2 - 7.3): 1.24
- Band 2 (5.0 - 6.1): 0.41
- Band 1 (0 - 4.9): 0.19
- Consensus Diagnosis:
- Band 4 (7.4 - 15.0): 5.86
- Band 3 (6.2 – 7.3): 1.17
- Band 2 (5.0 – 6.1): 0.40
- Band 1 (0 – 4.9): 0.16
- Unanimous Diagnosis:
- Band 4 (7.4 – 15.0): 6.03
- Band 3 (6.2 – 7.3): 1.10
- Band 2 (5.0 – 6.1): 0.33
- Band 1 (0 – 4.9): 0.16
Predicate Device(s)
Reference Device(s)
Not Found
Predetermined Change Control Plan (PCCP) - All Relevant Information
Not Found
§ 866.3215 Device to detect and measure non-microbial analyte(s) in human clinical specimens to aid in assessment of patients with suspected sepsis.
(a)
Identification. A device to detect and measure non-microbial analyte(s) in human clinical specimens to aid in assessment of patients with suspected sepsis is identified as an in vitro device intended for the detection and qualitative and/or quantitative measurement of one or more non-microbial analytes in human clinical specimens to aid in the assessment of patients with suspected sepsis when used in conjunction with clinical signs and symptoms and other clinical and laboratory findings.(b)
Classification. Class II (special controls). The special controls for this device are:(1) Premarket notification submissions must include the device's detailed Indications for Use statement describing what the device detects and measures, the results provided to the user, whether the measure is qualitative and/or quantitative, the clinical indications for which the test is to be used, and the specific population(s) for which the device use is intended.
(2) Premarket notification submissions must include detailed documentation of the device description, including (as applicable), all device components, software, ancillary reagents required but not provided, explanation of the device principle and methodology, and for molecular devices include detailed documentation of the primer/probe sequence, design, and rationale for sequence selection.
(3) Premarket notification submissions must include detailed documentation of applicable analytical studies, such as, analytical sensitivity (Limit of Detection, Limit of Blank, and Limit of Quantitation), precision, reproducibility, analytical measuring range, interference, cross-reactivity, and specimen stability.
(4) Premarket notification submissions must include detailed documentation of a prospective clinical study or, if appropriate, results from an equivalent sample set. This detailed documentation must include the following information:
(i) Results must demonstrate adequate device performance relative to a well-accepted comparator.
(ii) Clinical sample results must demonstrate consistency of device output throughout the device measuring range likely to be encountered in the Intended Use population.
(iii) Clinical study documentation must include the original study protocol (including predefined statistical analysis plan), study report documenting support for the Indications for Use(s), and results of all statistical analyses.
(5) Premarket notification submissions must include evaluation of the level of the non-microbial analyte in asymptomatic patients with demographic characteristics (
e.g., age, racial, ethnic, and gender distribution) similar to the Intended Use population.(6) As part of the risk management activities performed under 21 CFR 820.30 design controls, you must document an appropriate end user device training program that will be offered as part of your efforts to mitigate the risk of failure to correctly operate the instrument.
(7) A detailed explanation of the interpretation of results and acceptance criteria must be included in the device's 21 CFR 809.10(b)(9) compliant labeling, and a detailed explanation of the interpretation of the limitations of the samples (
e.g., collected on day of diagnosis) must be included in the device's 21 CFR 809.10(b)(10) compliant labeling.
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November 29, 2021
Image /page/0/Picture/1 description: The image shows the logo of the U.S. Food and Drug Administration (FDA). The logo consists of two parts: the Department of Health and Human Services seal on the left and the FDA acronym along with the full name of the agency on the right. The FDA acronym is in a blue square, and the full name "U.S. Food & Drug Administration" is in blue text.
Immunexpress, Inc Lakesha Hunt-Dickens Director, Quality and Regulatory Affairs 425 Pontius Avenue North, Suite 470 Seattle, Washington 98109
Re: K203748
Trade/Device Name: SeptiCyte RAPID Regulation Number: 21 CFR 866.3215 Regulation Name: Rt-Qpcr Assay For Mrna Transcript Immune Biomarkers Regulatory Class: Class II Product Code: PRE Dated: December 21, 2020 Received: December 23, 2020
Dear Lakesha Hunt-Dickens:
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. Although this letter refers to your product as a device, please be aware that some cleared products may instead be combination products. The 510(k) Premarket Notification Database located at https://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfpmn/pmn.cfm identifies combination product submissions. 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
1
requirements, including, but not limited to: registration and listing (21 CFR Part 807); labeling (21 CFR Part 801 and Part 809); medical device reporting of medical device-related adverse events) (21 CFR 803) for devices or postmarketing safety reporting (21 CFR 4, Subpart B) for combination products (see https://www.fda.gov/combination-products/guidance-regulatory-information/postmarketing-safety-reportingcombination-products); good manufacturing practice requirements as set forth in the quality systems (OS) regulation (21 CFR Part 820) for devices or current good manufacturing practices (21 CFR 4, Subpart A) for combination products; and, if applicable, the electronic product radiation control provisions (Sections 531-542 of the Act); 21 CFR 1000-1050.
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 https://www.fda.gov/medical-device-safety/medical-device-reportingmdr-how-report-medical-device-problems.
For comprehensive regulatory information about mediation-emitting products, including information about labeling regulations, please see Device Advice (https://www.fda.gov/medicaldevices/device-advice-comprehensive-regulatory-assistance) and CDRH Learn (https://www.fda.gov/training-and-continuing-education/cdrh-learn). Additionally, you may contact the Division of Industry and Consumer Education (DICE) to ask a question about a specific regulatory topic. See the DICE website (https://www.fda.gov/medical-device-advice-comprehensive-regulatoryassistance/contact-us-division-industry-and-consumer-education-dice) for more information or contact DICE by email (DICE@fda.hhs.gov) or phone (1-800-638-2041 or 301-796-7100).
Sincerely,
Maria Ines Garcia, Ph.D. Assistant Director Division of Microbiology Devices OHT7: Office of In Vitro Diagnostics and Radiological Health Office of Product Evaluation and Quality Center for Devices and Radiological Health
Enclosure
2
Indications for Use
510(k) Number (if known) K203748
Device Name SeptiCyte® RAPID
Indications for Use (Describe)
The SeptiCyte ® RAPID test is a gene expression assay using reverse transcription polymerase chain reaction to quantify the relative expression levels of host response genes isolated from whole blood collected in the PAXgene ® Blood RNA Tube. The SeptiCyte ® RAPID test is used in conjunction with clinical assessments and other laboratory findings as an aid to differentiate infection-positive (sepsis) from infectionnegative systemic inflammation in patients suspected of sepsis on their first day of ICU admission. The SeptiCyte ® RAPID test generates a score (SeptiScore®) that falls within one of four discrete Interpretation Bands based on the increasing likelihood of infectionpositive systemic inflammation. SeptiCyte ® RAPID is intended for in-vitro diagnostic use on the Biocartis IdyllaTM System.
| 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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7. 510(k) Summary
A. 510(k) Number:
-
B. Purpose for Submission:
To obtain a substantial equivalence determination for SeptiCyte® RAPID -
C. Measurand:
Two mRNA transcript immune biomarkers: PLA2G7, PLAC8
D. Type of Test:
Reverse transcription + quantitative PCR (RT-qPCR)
E. Applicant:
Immunexpress, Inc.
- F. Proprietary and Established Names:
SeptiCyte® RAPID
G. Regulatory Information:
-
- Regulation section:
21 CFR 866.3215; Device to detect and measure non-microbial analyte(s) in human clinical specimens to aid in assessment of patients with suspected sepsis
- Regulation section:
-
- Classification:
Class II (Special Controls)
- Classification:
-
- Product code: PRE
-
- Panel:
83: Microbiology
H. Intended Use:
-
- Intended use(s):
The SeptiCyte® RAPID test is a gene expression assay using reverse transcription polymerase chain reaction to quantify the relative expression levels of host response genes isolated from whole blood collected in the PAXgene® Blood RNA Tube. The SeptiCyte® RAPID test is used in conjunction with clinical assessments and other laboratory findings as an aid to differentiate infection-positive (sepsis) from infectionnegative systemic inflammation in patients suspected of sepsis on their first day of ICU admission. The SeptiCyte® RAPID test generates a score (SeptiScore®) that falls within one of four discrete Interpretation Bands based on the increasing likelihood of infectionpositive systemic inflammation. SeptiCyte® RAPID is intended for in-vitro diagnostic use on the Biocartis Idylla™ System.
- Intended use(s):
4
-
- Indication(s) for use:
Same as Intended Use.
- Indication(s) for use:
-
- Special conditions for use statement(s):
Whole blood is collected in a PAXgene® blood RNA tube (K042613)
- Special conditions for use statement(s):
-
- Special instrument requirements:
SeptiCyte RAPID is designed to run on the Biocartis Idylla system.
- Special instrument requirements:
Device Description: -
SeptiCyte RAPID is an in vitro diagnostic test for simultaneous amplification and detection of two RNA transcripts (PLA2G7 and PLAC8) using total RNA extracted from human blood. The test has been designed, manufactured, and validated for use on the Biocartis Idylla real-time PCR system. The SeptiCyte RAPID test is performed with an Idylla Cartridge, a single-use, disposable, multi-chambered fluidic cartridge that runs on the Biocartis Idylla System. In an automated fashion, all reaction steps take place within the cartridge, including sample extraction/purification, RT-qPCR for the detection and relative quantification of the two human mRNA targets PLAC8. PLA2G7. Test results (measured Cg values and calculated SeptiScore) are available in about 65 minutes.
The specimen used for the SeptiCyte RAPID is a sample of whole blood collected in a PAXgene blood RNA tube (FDA 510k number K042613). The cartridge contains all of the necessary reagents to perform RNA isolation from the sample.
SeptiCyte RAPID uses quantitative, real-time determination of the amount of each transcript in the sample based on the detection of fluorescence by the Biocartis Idylla qPCR instrument function. The cartridge includes the reagents for reverse transcription and PCR. Transcripts PLAC8 and PLA2G7 are amplified and quantified. These values are combined to produce the SeptiScore, which is interpreted and categorized into four discrete bands, which are associated with a sequentially higher likelihood of sepsis.
J. Substantial Equivalence Information:
-
- Predicate device name(s): SeptiCyte LAB
-
- Predicate 510(k) number(s): K163260
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3. Comparison with predicate:
Similarities:
| Item | Proposed Device:
SeptiCyte RAPID | Predicate device:
SeptiCyte LAB |
|-------------------------|---------------------------------------------------------|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Assay Method | Same | Reverse transcription + quantitative PCR (RT-qPCR) |
| Assay Principle | Same | Quantitative gene expression assay, based on real-time generation of fluorescence from hydrolysis of dye-quencher hydrolysis probes during cycles of PCR amplification of nucleic acid templates |
| Detection Method | Same | Multi-channel fluorescence detection |
| Intended Use Population | Same | Patients suspected of sepsis on their first day of Intensive Care Unit (ICU) admission |
| Specimen Type | Same | Whole blood collected in PAXgene Blood RNA tube |
| Analyte(s) | Two mRNA transcript immune biomarkers: PLA2G7 and PLAC8 | Four mRNA transcript immune biomarkers: LAMP1, CEACAM4, PLA2G7, PLAC8 |
Differences:
| Item | Proposed Device:
SeptiCyte RAPID | Predicate device:
SeptiCyte LAB |
|-------------------------------------------|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Specimen Processing | Automated extraction of material
using the Idylla System | 1. Manual extraction of material in
PAXgene Blood RNA Tube, using
the IVD version of the QIAGEN
PAXgene Blood RNA Kit
2. Semi-automated extraction
PAXgene Blood RNA Tube, using
the QIAGEN QIAcube System |
| PCR Chemistry | One-step RT-qPCR; Three
Singleplex reactions; Dried format | Two-Step RT-qPCR; one Singleplex,
one Triplex reaction; Wet format |
| Fluorescent 5'-Probes
and 3'-Quenchers | PLA2G7: 5'-FAM / 3'-BHQ1
PLAC8: 5'-ATTO532 / 3'-BHQ1
MS2: 5'-ATTO647N / 3'-BHQ2 | PLA2G7: 5'-FAM / 3'-BHQ1
PLAC8: 5'-Q670 / 3'-BHQ3
CEACAM4: 5'-JOE / 3'-BHQ1
LAMP1: 5'-FAM / 3'-BHQ1 |
| Controls | MS2 bacteriophage particles, serving
as sample processing control (SPC),
i.e., a within-cartridge positive control
for both the sample extraction step
and the coupled RT-qPCR step | High Positive Control, Low Positive
Control and Negative Control for each
in vitro transcript (LAMP1, CEACAM4,
PLA2G7, PLAC8 (IVTs) formulated in
neutral buffer, with IVT concentrations
designed to produce high, medium or
low SeptiScore values |
| Instrument Platform | Biocartis Idylla System | Applied Biosystems 7500 Fast Dx
Real-Time PCR Instrument |
6
| Standard Number | Standard Name |
|---|---|
| EP05-A3 | Evaluation of Precision of Quantitative Measurement Procedures |
| EP07-A2 | Interference Testing in Clinical Chemistry |
| EP17-A2 | Evaluation of Detection Capability for Clinical Laboratory |
| Measurement Procedures | |
| EP25-A | Evaluation of Stability of In Vitro Diagnostic Reagents |
| Guidance | |
| FDA Guidance for the Content of Premarket Submissions for Software Contained in | |
| Medical Devices (issued 11 May 2005) |
K. Standard/Guidance Document Referenced (if applicable):
L. Test Principle:
The SeptiCyte RAPID test employs a coupled reverse transcription - quantitative polymerase chain reaction (RT-qPCR) system to measure the expression levels (Cq values) of the host response genes PLA2G7 and PLAC8 isolated from whole blood. Amplicons generated by the RT-qPCR process are detected and quantitated by fluorescent dyes, upon exonucleolytic release of the dyes from oligonucleotide probes that specifically bind to the amplicons. The test is configured to run on the Biocartis Idylla System. The assay-specific software uses the measured PLA2G7 and PLAC8 Cq values to generate a SeptiScore, which falls into four discrete bands associated with a sequentially higher likelihood of sepsis.
M. Performance Characteristics:
1. Analytical Performance:
a. Precision/Reproducibility
Repeatability and intermediate precision were measured according to CLSI EP5-A3, using replicate PAXgene blood samples prepared from low, medium and high SeptiScore pools of PAXgene blood. Standard deviations were calculated for different subsets of data across operators, days, and instruments. Two sub-studies were performed:
Sub-Study #1 - The primary purpose of this sub-study was to evaluate repeatability (within-run precision). The study was performed using the Low sample pool, and was performed over three days, by two operators running three replicates per day, with all runs on a single instrument. Sub-study #1 was performed with a single lot of cartridges.
7
Sub-Study #2 - The purpose of this sub-study was to evaluate intermediate precision. Sub-study #2 was performed with one cartridge lot, over a total span of 12 days, using three instruments and two operators. Each operator's runs were conducted over 4 non-sequential days within the 12-day study window. Each combination of day, operator and instrument was tested with two replicates. At each level of sub-study #2, aggregate means and standard deviations were calculated. These were then further summarized by taking the average of the aggregates as presented in the summary table.
Table 7-1 presents a summary of results for the repeatability and intermediate precision studies for SeptiScore. Low CVs were also observed for the individual PLA2G7 and PLAC8 Cq's.
| Sub-study-1 | Sub-study-2 | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| Repeatability | Across | |||||||||
| Days | Across | |||||||||
| Operators | Across | |||||||||
| Instruments | Total | |||||||||
| Variation | ||||||||||
| Pool | MEAN | CV | ||||||||
| (%) | MEAN | CV | ||||||||
| (%) | MEAN | CV | ||||||||
| (%) | MEAN | CV | ||||||||
| (%) | MEAN | CV | ||||||||
| (%) | ||||||||||
| Low | 4.56 | 4.0 | 4.48 | 4.5 | 4.48 | 4.8 | 4.48 | 4.9 | 4.48 | 5.0 |
| Med | NA | NA | 6.33 | 4.0 | 6.33 | 3.9 | 6.33 | 4.3 | 6.33 | 4.3 |
| High | NA | NA | 10.33 | 1.8 | 10.33 | 1.9 | 10.33 | 1.9 | 10.33 | 2.0 |
Table 7-1: Summary of Repeatability and Intermediate Precision Results
-NA's: Only the Low pool was used for Repeatability (Sub-study-1), Medium and High pools were not run for Repeatability
b. Lot-to-Lot reproducibility
Lot-to-lot reproducibility was performed with the three sample types (Low, Medium, and High pools, as described in the previous study) using three cartridge lots, over 3 non-consecutive days, using two instruments and one operator. Each combination of lot, day, and instrument was tested with two replicates.
The samples used in the study were prepared from banked samples in PAXgene Blood RNA Tubes. The banked samples were pooled to prepare a High Pool, Medium Pool, and Low Pool, with high, medium, and low SeptiScore results, respectively. Table 7-2 summarizes the results for the lot-to-lot reproducibility study for SeptiScore. Low CVs were also observed for the individual PLA2G7 and PLAC8 Cq's.
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| SeptiScore | |||
|---|---|---|---|
| Pool | Mean | SD | CV (%) |
| Low | 4.36 | 0.14 | 3.3 |
| Medium | 6.25 | 0.19 | 3.0 |
| High | 10.20 | 0.19 | 1.8 |
Table 7-2: Lot-to-lot reproducibility of SeptiCyte RAPID
ﻥ Linearity/assay reportable range
A specimen panel to test the linearity of quantitation of the two mRNA targets (PLA2G7, PLAC8) was constructed by spiking varying amounts of white blood cells (WBC) into a matrix consisting of platelet-reduced plasma. A broad range of WBC input amounts (25 cells/uL to 50,000 cells/μL) was tested. An inverse log-linear response (Pearson's r2 > 0.99) was observed for the individual SeptiCyte RAPID assay targets (PLA2G7 and PLAC8) when calculated across the entire input WBC concentration range. At the level of the individual analytes (PLA2G7, PLAC8), there appears to be a small Hook Effect (non-linearity) at the highest input concentrations (25,000 cells/uL and above). However, the SeptiScore, which is calculated based on relative expression of the individual analytes, compensates for the Hook Effect at the high input concentrations.
d. Traceability, Stability, Expected Values (controls, calibrators, or methods)
Controls:
A sample processing control is present in each cartridge. This consists of a predefined quantity of dried bacteriophage MS2 particles, which become mixed with the sample after sample addition but before further processing. The sample processing control is taken through the entire sample processing path, ultimately generating a PCR curve and Cq value, thereby serving as a positive control for both the extraction process and for the correct general functioning of the RT-qPCR enzymology.
Real-Time Stability:
Three manufactured lots of SeptiCyte RAPID cartridges were found to be stable for a minimum of 12 months when stored between 2°C and 30°C in their packaging as delivered (i.e., in their sealed pouches) .
In-Use Stability:
The results of the in-use stability study confirm that the SeptiCyte RAPID cartridge exhibits stability under two tested conditions:
- When unwrapped for up to 3 days before loading the sample and running on the . ldylla instrument, and
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- When held for up to 9 hours after receiving a sample, before running on the Idylla . instrument.
These tests were conducted at the upper end of the range of expected laboratory conditions (30 ± 2 °C and 75 ± 5 % relative humidity).
Shipping Stability:
The SeptiCyte RAPID test is packaged in secondary packaging that is consistent with other tests manufactured for the Idylla platform. The shipping stability study consisted of thermal stress and shock/vibration components. Cartridges subjected to thermal stress or shock vibration had similar SeptiScore values when compared to their respective reference condition (cartridges tested during QC release). All Cq and SeptiScore values were valid and within ±10% of their respective reference values obtained at the time of QC release.
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e. Detection limit
To determine the LoD and LoQ, platelet-reduced plasma was spiked with serial dilutions of white blood cells (WBCs) and 20 replicates of each level of WBC tested with the SeptiCyte RAPID Test. The lowest level at which 19/20 replicates generated a SeptiScore is reported as the LoD. The LoQ is the lowest level of WBC for which 19/20 replicates generate a SeptiScore with a standard deviation of 1.0 Score units or less. The LoD and LoQ were both determined to be 25 WBC/uL blood. Additionally, a platelet-reduced plasma sample with no spiked WBC (matrix only) was included and tested with 10 replicates to demonstrate that a SeptiScore would not be generated for any of the matrix replicates. -
f. Analytical specificity
Interfering Substances
Based on the CLSI Guidance EP07-A2, "Interference Testing in Clinical Chemistry" SeptiCyte RAPID was evaluated in the presence of interfering substances. Each substance was added to a blood sample in a PAXgene Blood RNA tube. No interference was found for any of the substances in Table 7-3 at the concentration listed.
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| Potential Interferent | Concentration |
|---|---|
| Rheumatoid Factor | 45 IU/mL |
| Heparin | 3000 U/L |
| Imipenem | 1.2 mg/mL |
| Bilirubin | 20 mg/dL |
| Triglycerides | 500 mg/dL |
| Vancomycin | 70 µmol/L |
| Cefotaxime | 670 µmol/L |
| Dopamine | 6.0 µmol/L |
| C-reactive protein (CRP) | 4 mg/dL |
| Noradrenaline | 700 pg/mL |
| Dobutamine | 12 µg/mL |
| Hemoglobin | 20 g/dL |
| Albumin | 5 g/dL |
| Furosemide | 180 µmol/L |
| soluble CD14 (sCD14) | 5 µg/mL |
| Interleukin 6 (IL-6) | 15 pg/mL |
| Lipopolysaccharide binding protein (LBP) | 45 µg/mL |
Table 7-3: Interferents and Testing Levels
Lack of Reactivity Towards Blanks or Genomic DNA
This analytical specificity study was designed to confirm that the SeptiCyte RAPID test does not generate a valid signal against blank samples or in the presence of genomic DNA (gDNA).
Blank samples were prepared by adding a volume of nuclease-free phosphate buffered saline solution (PBS) to PAXgene stabilizer at the same volume ratio of specimen to RNA stabilizer expected during the standard sample collection process for PAXgene Blood RNA tubes. Twenty aliquots (0.9 mL) of this blank preparation were then tested with individual SeptiCyte RAPID cartridges. In addition to the blank samples, 10 cartridges were run empty, with no sample added to the lysis chamber
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prior to running on the Biocartis Idylla system. For absence of reactivity towards gDNA. testing was conducted on 10 replicates of each of two commercially procured total human gDNA samples. Each lot was tested at 500 ng and 1000 ng gDNA input per reaction.
The results of the study conclude that (1) the SeptiCyte RAPID test does not exhibit reactivity toward blank samples and (2) the SeptiCyte RAPID test does not exhibit reactivity toward human genomic DNA, at input quantities of either 500 ng or 1000 ng per reaction.
g. Carryover
To evaluate the potential for carryover events, a series of 21 cartridges were tested with alternating blank samples, low SeptiScore positive samples, and high SeptiScore positive samples. The results of the study showed that all blank samples failed to generate a valid SeptiScore. All of the low SeptiScore samples generated a result in Band 1, and all of the high SeptiScore samples generated a result in Band 4. The results from the carryover study confirm that no carryover contamination events occur during the processing of individual SeptiCyte RAPID test cartridges.
h. PAXgene Blood RNA Tube Stability
Two matched PAXgene Blood RNA samples were collected from each of 15 healthy donors. The first sample was processed in real time, by removing two 0.9 mL aliquots for SeptiCyte RAPID analysis at the following times after phlebotomy: immediately (i.e., less than 5 min) and after 120 minutes. During this period of time, both tubes were held at room temperature (18 - 25°C) to approximate the sample handling conditions in a near-patient setting (e.g., STAT laboratory). The second PAXgene Blood RNA sample was frozen and stored at