(49 days)
The PAXgene™ Blood RNA System consists of a blood collection tube (PAXgene™ Blood RNA Tube) and nucleic acid purification kit (PAXgene™ Blood RNA Kit). It is intended for the collection, storage, and transport of blood and stabilization of intracellular RNA in a closed tube and subsequent isolation and purification of host RNA from whole blood for RT-PCR used in molecular diagnostic testing.
Performance characteristics for the PAXgene™ Blood RNA System have only been established with "cfos and IL1B." The user is responsible for establishing appropriate PAXgene™ Blood RNA System performance characteristics for other target transcripts.
The PAXgene™ Blood RNA System consists of:
- the PAXgene™ Blood RNA tubes and .
- the PAXgene™ Blood RNA kit. .
The PAXgene™ Blood RNA tube is of a sterile, plastic, evacuated blood collection tube containing stabilization solution (tetradecyl trimethyl-ammonium oxalate and tartaric acid. These components serve to lyse cells, protect RNA molecules from degradation by ribonucleases (RNases) and prevent induction of gene expression.
The kit consists of 5 aqueous buffer solutions for resuspending, binding, washing, and eluting RNA, RNase-free water, proteinase K, an RNase-Free DNase set, spin columns, microcentrifuge tubes, processing tubes, and secondary blood collection tube closures.
The provided text details the performance characteristics and studies for the PAXgene™ Blood RNA System, intended for the collection, storage, and purification of intracellular RNA from whole blood for RT-PCR.
1. Table of Acceptance Criteria and Reported Device Performance
| Parameter | Acceptance Criteria | Reported Device Performance |
|---|---|---|
| RNA Yield | ≥ 3 µg/tube (A260 nm) | Achieved (specific values not always given, but "all samples... fulfilled the acceptance criteria" and "in the expected ranges") |
| RNA Purity | A260/A280 ratio between 1.8 and 2.2 | Achieved ("all samples... fulfilled the acceptance criteria" and "in the expected ranges") |
| Integrity (CFOS/18S rRNA) | CFOS within 2.34 CT | Achieved ("within the acceptance range") |
| Integrity (IL1B/18S rRNA) | IL1B within 1.94 CT | Achieved ("within the acceptance range") |
| Genomic DNA (gDNA) Contamination | Percentage of gDNA in total nucleic acid preparation | All samples "fulfilled the acceptance criteria" |
| RT-PCR Inhibition | Kit components do not introduce inhibition for an RT-PCR assay | "All samples demonstrated that the kit components do not introduce any inhibition" |
| Reproducibility (% CV) | Within-run, inter-user, inter-lot | Varies: |
- Repeatability (quadruplicate per donor pool, per lot/user): Min/max % CV 3.4-28.8, overall 11.9%
- Reproducibility (per user, between lots): Min/max % CV 8.7-20.1, overall 14.9%
- Reproducibility (between lots and users): Min/max % CV 8.7-23.1, overall 16.4% |
| PAXgene Tube Stability (25°C) | Draw volume, liquid additive volume, closure performance, pH, conductivity, density, RNA yield, purity, CFOS/18S rRNA & IL1B/18S rRNA stability | All physical/chemical attributes and functional performance were within expected ranges for 6 months (sponsor claims 19 months based on accelerated studies) |
| PAXgene Kit Component Stability | Recovery of input RNA, variability of recovery, degree of inhibition, pH, conductivity, density, Proteinase K & DNase I activity, bioburden | All parameters in expected ranges for 6 months real-time storage; no bioburden; acceptable performance after extreme temperature simulation |
| RNA In Situ Stability (various temperatures/times/freeze-thaw) | RNA yield, purity, integrity (CFOS/18S rRNA, IL1B/18S rRNA) within acceptance range | All samples at all time points, temperatures, and freeze/thaw cycles were within the acceptance range (with one unspecified exception) |
| Linearity/Reportable Range (CFOS) | 0.2 to 7.8 ng total input nucleic acid; up to 25% DNA without affecting performance | Met |
| Linearity/Reportable Range (IL1B) | 0.1 to 7.8 ng total input nucleic acid; up to 5% DNA without affecting performance | Met |
2. Sample Sizes and Data Provenance
- Test Set (Precision/Reproducibility of RT-PCR Assays):
- K2EDTA samples: 5 donors, 5 tubes/donor (25 tubes total), 10 ml blood/tube. RNA isolated via OIAzol and RNeasy, pooled, concentrated.
- PAXgene samples: 48 donors, 8 tubes/donor (384 PAXgene tubes total), 2.5 ml blood/tube. RNA purified, DNase treated, concentrated, pooled.
- Reproducibility Design: 3 different component lots, 3 different experimenters, 3 different laboratories (equipment), on 3 different days.
- Each "run" (9 runs total) involved: 2 RNA samples (K=calibrator, T=test), each with 10 replicates. This generated 180 ΔCT values and 90 ΔΔCT values.
- Test Set (Precision/Reproducibility of PAXgene Blood RNA System - Experiment 1):
- 14 donors (WBC: 4.8-11.0 x 10^6 cells/ml blood)
- 12 tubes/donor (168 PAXgene tubes total)
- Test Set (Precision/Reproducibility of PAXgene Blood RNA System - Experiment 2):
- 30 donors (WBC: 4.8-11.0 x 10^6 cells/ml blood)
- 12 tubes/donor (360 PAXgene tubes total)
- Blood from 3 donors pooled and aliquoted into empty tubes to create 10 donor pools with 36 tubes each.
- Test Set (RNA Purity, DNA Contamination, RNA Yield):
- 10 donors
- 2 PAXgene tubes/donor (20 tubes total) + 1 EDTA tube/donor (10 EDTA tubes) for WBC count.
- Test Set (RT-PCR Inhibition):
- 22 blank eluates from columns.
- HeLa cell RNA used as template.
- Test Set (PAXgene Tube Stability):
- Functional tests: 60 tubes/time point (TTP) for accelerated studies (120 tubes total), 60 tubes/TTP for real-time study (810 tubes scheduled, 6 months data presented).
- Functional performance: 10 donors per time point for RNA yield, purity, CFOS/IL1B relative levels (3 time points for blood-filled tubes storage at 18-25°C x 2 preparations = 60 measurements).
- Test Set (PAXgene Kit Component Stability):
- Two reagent sets per time point for functional stability (4 blank eluates, 8 RNA eluates).
- Test Set (RNA In Situ Stability):
- Multiple experiments with 10 donors each. Number of PAXgene tubes per donor varied (6, 10, 12, 14 tubes/donor), resulting in totals of 60, 100, 120, and 140 tubes for different conditions.
- Data Provenance: The studies appear to be prospective, performed by the applicant (PreAnalytiX GmbH) to demonstrate the device's performance. The applicant is based in Switzerland (GmbH). The data is generated in a lab setting, not clinical real-world data from multiple countries.
3. Number of Experts and Qualifications for Ground Truth
The document does not mention the use of experts to establish a ground truth for the "test set" in the context of diagnostic interpretation (e.g., radiologists evaluating images). This device is a sample collection and purification system, not an interpretative diagnostic device. The ground truth for its performance is instead based on quantitative measurements of RNA yield, purity, integrity, and the absence of inhibitors. These are objective measures determined by laboratory instrumentation (e.g., spectrophotometers, Q-RT-PCR machines, BioAnalyzer).
However, "experimenters" and "technicians" are mentioned:
- Precision/Reproducibility of O-RT-PCR Duplex Assays: "3 different experimenters"
- Precision/Reproducibility of PAXgene Blood RNA System (both Experiments 1 & 2): "3 technicians"
Their specific qualifications (e.g., years of experience) are not provided, but they are implied to be trained laboratory personnel.
4. Adjudication Method
Not applicable. The "ground truth" for this device is based on objective, quantifiable laboratory measurements (e.g., RNA yield, purity ratios, Ct values), not on subjective expert interpretation requiring adjudication.
5. Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study
No. An MRMC comparative effectiveness study is not applicable for this type of device (RNA collection and purification system). Such studies are typically used for diagnostic imaging or interpreted assays where human readers are involved in making diagnostic decisions, and the AI aims to assist or replace them. This device is a sample preparation tool, and its "performance" is measured analytically, not by human reader accuracy.
6. Standalone (Algorithm Only) Performance
Yes, the studies described are essentially "standalone" performance evaluations of the device and its components. The PAXgene Blood RNA System's analytical performance (e.g., RNA yield, purity, integrity) is assessed directly through laboratory assays, without human intervention in the "decision-making" loop. Its function is to prepare RNA for downstream molecular diagnostic testing (RT-PCR), meaning its performance is evaluated on its ability to produce high-quality RNA suitable for such assays. The Q-RT-PCR assays run on the RNA extracted by the system are designed to evaluate the RNA quality produced by the device, not to perform a diagnosis.
7. Type of Ground Truth Used
The ground truth used is primarily based on analytical measurements derived from standard laboratory techniques:
- Spectrophotometric measurements: Absorbance at 260 nm (for RNA yield), A260/A280 ratio (for RNA purity).
- Quantitative Reverse Transcription-Polymerase Chain Reaction (Q-RT-PCR) assays: Using CFOS and IL1B transcripts normalized to 18S rRNA (for RNA integrity and relative transcription levels). This involves comparing threshold cycle (Ct) values.
- Electropherograms (BioAnalyzer): For RNA purity and integrity.
- Beta-actin PCR: To determine genomic DNA contamination.
- Biological assays: Proteinase K and DNase I activity tests.
- Physical and chemical property tests: pH, conductivity, density, draw volume, liquid additive volume, closure performance.
- Microbiological assays: Bioburden analysis.
8. Sample Size for the Training Set
The document does not explicitly describe a separate "training set" in the context of machine learning or AI algorithms, as this is not an AI-based device. Instead, the studies detail the performance validation of the system. The various studies use different cohorts of human donors for the collection of blood samples to assess different aspects of the device's performance (e.g., 5 donors for K2EDTA comparator, 48 donors for PAXgene in initial RT-PCR assay validation, 14 and 30 donors for system precision/reproducibility, 10 donors for purity/DNA contamination, 10 donors for each in situ stability condition).
9. How the Ground Truth for the Training Set Was Established
As mentioned above, there is no "training set" in the AI sense for this device. The establishment of "ground truth" for the various performance metrics (RNA yield, purity, integrity, etc.) is through well-established and standardized laboratory analytical methods as described in point 7. These methods themselves are considered the "gold standard" for measuring these specific biochemical and molecular parameters.
§ 866.4070 RNA Preanalytical Systems.
(a)
Identification. RNA Preanalytical Systems are devices intended to collect, store, and transport patient specimens, and stabilize intracellular RNA from the specimens, for subsequent isolation and purification of the intracellular RNA for RT-PCR used in in vitro molecular diagnostic testing.(b)
Classification. Class II (special controls). The special control is FDA's guidance document entitled “Class II Special Controls Guidance Document: RNA Preanalytical Systems (RNA Collection, Stabilization and Purification System for RT-PCR Used in Molecular Diagnostic Testing).” See § 866.1(e) for the availability of this guidance document.