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The ipsogen JAK2 RGQ PCR Kit is a qualitative in vitro diagnostic test for the JAK2 V617F/G1849T allele in genomic DNA extracted from EDTA whole blood. The ipsogen JAK2 RGQ PCR Kit is a real time PCR test performed on the QIAGEN Rotor-Gene Q MDx instrument. The test is intended for use as an adjunct to evaluation of suspected Myeloproliferative Neoplasm in conjunction with other clinicopathological factors.

This test does not detect less common JAK2 mutations associated with Myeloproliferative Neoplasm including mutations in exon 12 and is not intended for stand-alone diagnosis of Myeloproliferative Neoplasm.

The ipsogen® JAK2 RGQ PCR Kit is a qualitative in vitro diagnostic test for the detection of the JAK2 V617F/G1849T allele in genomic DNA extracted from whole blood. The ipsogen JAK2 RGQ PCR Kit uses polymerase chain reaction (PCR), and ARMS (amplification refractory mutation system) technology on the Rotor-Gene O MDx instrument.

Samples are extracted and prepared using the QIAsymphony SP instrument (QSSP) with the QIAsymphony® DSP DNA Mini Kit, followed by assay setup amplification and detection are carried out using the ipsogen JAK2 RGQ PCR Kit with the Rotor-Gene Q MDx and Rotor-Gene AssayManager software version 2.1.x**. Rotor-Gene AssayManager Gamma MDx plug-in installed, version 1.0.x** and an associated JAK2 Assay profile (from file AP ipsogen JAK2 blood US Vx x x**.iap).

The extraction step on the OIAsymphony instrument (OSSP) ensures that enough gDNA (of adequate quality) is purified during each run (no repeated extraction for the same sample is expected unless the instrument states that the step failed).

The ipsogen JAK2 RGQ PCR Kit is designed to be used with the Rotor-Gene Q MDx (RGO) instrument which is a real-time PCR analyzer designed for rapid thermal cvcling and real-time detection of PCR assays. The Rotor-Gene AssayManager (RGAM) controls and monitors PCR reactions and allows the determination of the mutation status based upon PCR results.

The presence of the JAK2 mutation is indicated by the fluorescent signal generated through the use of fluorescently labeled oligonucleotide probes. The probes do not generate a signal unless they are specifically bound to the amplified product. The amplification cycle at which fluorescent signal is detected by the RGO MDx is inversely proportional to the DNA target concentration present in the original specimen.

The ipsogen JAK2 RGQ PCR Kit contains reagents for two separate PCR reactions: one mutation-specific reaction and one wild-type specific reaction mix uses a mutant-specific or wild-type specific primer (Reverse primer), together with a common Forward primer and a labeled probe, to amplify and detect the G1849T mutation or the wild-type sequence in the JAK2 gene.

In addition, each Reaction Mix contains reagents (unlabeled primers, probe and oligonucleotide template) for an internal control reaction. The internal control is designed to be a reaction that does not compete significantly with the mutation-specific or wildtype specific, reaction mixes. and serve to demonstrate that the entire assay process has proceeded correctly for each specimen.

Note: ** x≥ 0, x corresponds to the latest version available on OIAGEN Website

The provided text describes the performance characteristics of the ipsogen® JAK2 RGQ PCR Kit, a qualitative in vitro diagnostic test for the detection of the JAK2 V617F/G1849T allele. This is a molecular diagnostic test, not an AI/imaging device, therefore, many of the requested criteria such as expert adjudication, MRMC studies, and AI-specific ground truth methodologies are not applicable.

Here's an attempt to extract and present the information as per your request, adapting to the nature of the device:

Acceptance Criteria and Device Performance Study for ipsogen® JAK2 RGQ PCR Kit

Note: This device is a molecular diagnostic PCR kit, not an AI-based imaging device. Therefore, criteria related to image analysis, expert readers, MRMC studies, and AI-specific ground truth establishment (like pathology or outcomes data for imaging) are not directly applicable. The "acceptance criteria" here relate to the analytical accuracy of the test in detecting a specific genetic mutation compared to a reference method.

1. Table of Acceptance Criteria and Reported Device Performance

The document does not explicitly state pre-defined quantitative acceptance criteria in a table format. However, the "Performance Characteristics - Non-Clinical Studies" section describes the study's objective and results, implying that the observed accuracy demonstrates the device's acceptable performance. The primary metric presented is concordance with a reference method (Sanger Bi-directional Sequencing, BDS, and subsequently Next Generation Sequencing, NGS).

Based on the study results, we can infer the performance:

2. Sample Size and Data Provenance

• Test Set Sample Size: 473 specimens.
  • 276 with suspected Polycythemia Vera (PV)
  • 98 with Myelofibrosis (ET)
  • 99 with Primary Myelofibrosis (PMF)
• Data Provenance: Not explicitly stated, but clinical samples from subjects suspected of having myeloproliferative neoplasms. It is likely retrospective as the samples appear to be collected and then tested. No country of origin is specified.
• Training Set Sample Size: Not applicable for this type of analytical validation study. The kit is a diagnostic reagent system, not a machine learning model that undergoes training.

3. Number of Experts and Qualifications for Ground Truth

• Number of Experts: Not applicable. Ground truth was established using standard molecular diagnostic methods (Sanger Bi-directional Sequencing and Next Generation Sequencing), not by human expert consensus as would be typical for image interpretation.
• Qualifications of Experts: N/A as per above. These are laboratory-based reference methods.

4. Adjudication Method for the Test Set

• Adjudication Method: Not applicable. The ground truth was established by laboratory-based objective methods (BDS and NGS). Discordances were investigated by using a more sensitive reference method (NGS).

5. Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study

• MRMC Study: No. This is a molecular diagnostic kit, not an AI/imaging device requiring human reader interpretation. Therefore, an MRMC study and
  assessment of human reader improvement with AI assistance are not relevant.

6. Standalone Performance (Algorithm Only)

• Standalone Performance: Yes, in a way. The "algorithm" here is the chemical and enzymatic process of the PCR kit and the associated instrument's detection capabilities. The study assesses the performance of the kit itself (the "device") in detecting the mutation, independent of human interpretation beyond typical lab procedures and result reporting. The kit's results are compared directly to the reference methods.

7. Type of Ground Truth Used

• Type of Ground Truth:
  • Primary Ground Truth: Sanger Bi-directional Sequencing (BDS).
  • Confirmatory/Higher Sensitivity Ground Truth for discordant samples: Validated Next Generation Sequencing (NGS).
  • The document notes that BDS is "not as sensitive as the JAK2 assay" (which reportedly detects down to 1%), and NGS was used to confirm the 15 discordant samples. This suggests that the ground truth evolved or was refined, with NGS serving as a more robust ground truth for low-level mutations.

8. Sample Size for the Training Set

• Training Set Sample Size: Not applicable. As stated before, this is a diagnostic kit, not an AI model requiring a training set. The "design" of the kit is based on established molecular biology principles, not data-driven learning.

9. How the Ground Truth for the Training Set Was Established

• Ground Truth for Training Set Establishment: Not applicable, as there is no training set for this type of device. The kit's design and validation rely on its analytical performance against known samples or samples characterized by established, robust reference methods.

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The ipsogen JAK2 RGQ PCR Kit is a qualitative in vitro diagnostic test for the detection of the JAK2 V617F/G1849T allele in genomic DNA extracted from EDTA whole blood. The ipsogen JAK2 RGQ PCR Kit is a real time PCR test performed on the QIAGEN Rotor-Gene Q MDx instrument. The test is intended for use as an adjunct to evaluation of suspected Polycythemia Vera, in conjunction with other clinicopathological factors.

This test does not detect less common mutations associated with Polycythemia Vera including mutations in exon 12 and is not intended for stand-alone diagnosis of Polycythemia Vera.

The ipsogen® JAK2 RGQ PCR Kit (hereafter also referred to as the JAK2 Kit or JAK2 assay) employs allele-specific, quantitative, polymerase chain reaction (PCR) using an amplification refractory mutation system (ARMS). DNA is extracted from K2-EDTA anticoagulated whole blood using the QIAsymphony instrument (QSSP) and QIAsymphony® DSP DNA Mini Kit. Purified DNA must be diluted to 10 ng/ul using the TE buffer provided in the JAK2 Kit. Each PCR reaction of the Rotor-Gene Q MDx is optimized for 50 ng of purified gDNA diluted in a final volume of 5 ul. A total of 100 ng per tested sample (50 ng for each reaction) is needed. The Kit contains sufficient reagents to test 24 reactions.

The provided text describes the performance evaluation of the ipsogen® JAK2 RGQ PCR Kit. Here's a breakdown of the acceptance criteria and the study that proves the device meets them, organized as requested:

Acceptance Criteria and Reported Device Performance

Table 1: Acceptance Criteria and Reported Device Performance

1. A260/280 OD within 1.7-2.0 and gDNA concentrations $\ge$ 10 ng/µL with a 90% pass rate.
2. Variability between sites must be less than that of within-laboratory study. | Study 1 met pre-specified acceptance criteria. Variability was acceptable. For the 1.02% JAK2 MUT mean, 25/92 tests were positive. For 0% JAK2 MUT mean, 0/92 tests were positive. (Tables 4, 5) |
   | | Site-to-Site Reproducibility (Study 2 - Clinical Samples): All calls (positive/negative) must be correctly identified with 100% concordance (95% CI, 90.3%, 100%). Observed variability to be lower than in the precision study for comparable mutation levels. | For all 6 clinical specimens, 100% of calls were correctly positive or negative (95% CI, 90.3%, 100%). Observed variability was lower than in the precision study. (Table 6) |
   | | Precision of Controls: Acceptable repeatability and reproducibility including variations between operators, kits, instruments, and days for mutant and wild-type controls, and internal controls (HEX channel Ct values 25 to 37.79). | Mutant Control: Mean 99.98% JAK2 MT, SD 0.02, %CV 0.02. Wild Type Control: Mean 0.00% JAK2 MT, SD 0.00, %CV 0.00. HEX Mutant Ct: Mean 33.09, Total SD 0.70, %CV 2.11. HEX Wild Type Ct: Mean 32.90, Total SD 0.83, %CV 2.53. Controls were acceptable. (Tables 7, 8) |
   | | DNA Extraction Method Reproducibility: No evidence of cross-contamination greater than the clinical cut-off (1%). | One sample in the control extraction run produced 0.012% MUT and one in the test run 0.001% MUT; both below the 1% cut-off. The study established the lack of cross-contamination. |
   | Analytical Performance: Linearity/Reportable Range and DNA Input | Linearity: Assay must be linear for tested DNA input levels across the measuring interval (0-70% mutation). At 1% cutoff, degree of linearity must not be statistically different from 0. | Regression analysis demonstrated linearity at DNA inputs of 5, 10, and 20 ng/uL. The assay is not linear at 2 or 30 ng/uL. Significant effect when DNA input was 2 ng/uL and %MUT was LoB >90% of the time. | LoB: All results below LoD, supporting that LoB is below LoD and not detectable. LoD: Verified to be 0.042% MUT. The 10th percentile was calculated and determined to be greater than the LoB >90% of the time. |
   | Analytical Performance: Analytical Specificity | Interfering Substances: No interference with assay performance (except Proteinase K, which was further examined). Key acceptance for Proteinase K: control and control+TE not different, internal control Ct > 38.51, A260/280 1.7-2.0, gDNA $\ge$ 10 ng/µL, %MUT comparison p-value 1%. | All testing met acceptance criteria for reagent stability, transport conditions, and freeze-thaw cycles. Supports a shelf life of months (specific duration redacted) and freeze-thaw cycles for the JAK2 Assay kit with the study ongoing. (Tables 13, 14, 20) |
   | | Specimen Stability (Whole Blood): Stability for up to 4 days (96 hrs) after collection in K2 EDTA tubes at 2-8°C and room temperature. | Study designed to support 96 hours stability, consistent with literature. (Specific results not fully detailed, but implies acceptance criteria were met). |
   | | Specimen Stability (Genomic DNA): Stability of extracted DNA at -15 to -30°C and after 4 freeze/thaw cycles for 15 months (studies ongoing to extend to 24 months). | Testing parameters within specifications to 18 months, with studies ongoing to 25 months. The extracted gDNA can be stored up to 15 months with studies ongoing to extend this claim. |
   | Comparison Studies: Accuracy Method Comparison | Agreement with Bi-directional Sanger Sequencing: High positive percent agreement (PPA) and negative percent agreement (NPA). | PPA: 100% (71/71 subjects; 95% CI: 94.4%, 100%). NPA: 99.5% (204/205 subjects; 95% CI: 97.3%, 100%). One discordant case with Sanger negative but JAK2 Kit positive (5.6% MUT, below Sanger LoD). (Table 15) |
   | Clinical Performance | Sensitivity: High sensitivity (expected to detect PV in vast majority of subjects with disease).
   Specificity: High specificity (expected to rule out PV in vast majority of subjects without PV). | Sensitivity: 94.6% (53/56 subjects; CI: 85.1%, 98.8%). Specificity: 98.1% (157/160 subjects; 95% CI: 94.6%, 99.6%). (Tables 18, 19) |

Study Details

2. Sample Size and Data Provenance

• Test Set Sample Size:
  • Analytical Performance (Precision):
    • Within-laboratory: 11 samples (0% to 70% mutation), 108 measurements per sample.
    • Site-to-Site Reproducibility Study 1 (cell lines): 8 samples (50% to 0% MUT), 96 data points per mutation level.
    • Site-to-Site Reproducibility Study 2 (clinical samples): 6 clinical samples (4 positive, 2 negative), 36 measurements per sample.
  • Analytical Performance (Linearity): 11 different mutation percentages (0-70%) at 5 DNA input levels (30, 20, 10, 5, 2 ng/uL), 4 data points per %MUT per DNA input.
  • Analytical Performance (LoD): First study: 3 positive + 3 negative clinical samples, 6-point dilution series, 20 replicates each in 3 kit lots (180 total measurements). Second study: 2 samples, 30 replicates each (60 data points).
  • Analytical Performance (Interfering Substances): Samples at LoD, with various interfering substances. Proteinase K study used pooled healthy and PV whole blood, spiked, 4 replicates per sample.
  • Accuracy Method Comparison: 276 clinical specimens.
  • Clinical Performance: 216 evaluable subjects (from 286 consented subjects).
• Data Provenance:
  • Country of Origin: Clinical samples for the primary clinical study were collected from 9 study sites in the United States (7 enrolled subjects), 12 study sites in France (all 12 enrolled subjects), and 9 study sites in Italy (5 enrolled subjects).
  • Retrospective/Prospective: The clinical performance study was a multicenter, international, prospective, interventional study. Analytical studies primarily used prepared or pooled samples, with some clinical samples, making them mostly prospective for the specific test scenario.

3. Number of Experts and Qualifications for Ground Truth

• Number of Experts: Not explicitly stated as a number of individual experts.
• Qualifications: Ground truth for the clinical performance study was established by independent assessment of patient status at the clinical site based on the 2008 WHO diagnostic criteria, combined with bidirectional Sanger sequencing and, in some cases, bone marrow biopsy with histologic and cytogenetic analysis. This implies a consensus among clinical professionals (hematologists, pathologists) using established diagnostic guidelines. For the accuracy method comparison, bidirectional Sanger sequencing served as the comparator method.

4. Adjudication Method for the Test Set

• The text describes a process for establishing "clinical truth" in the clinical performance study. The reference for JAK2 status determination was independent assessment of patient status at the clinical site based on the 2008 WHO diagnostic criteria. If initial blood tests by bidirectional assessment (BDS) were negative, bone marrow from patients was tested by bidirectional sequencing. This suggests an adjudicated process based on a diagnostic algorithm and multiple testing modalities to arrive at a final clinical diagnosis for ground truth. There's no explicit mention of an external, blinded adjudication panel in a "X+Y" format, but rather a structured clinical diagnostic pathway.

5. Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study

• No, an MRMC comparative effectiveness study was not performed as described. This is an automated IVD kit, not an imaging device requiring human reader interpretation. The performance of the device (ipsogen JAK2 RGQ PCR Kit) was compared against a reference standard (bidirectional Sanger sequencing for analytical accuracy and WHO diagnostic criteria combined with BDS for clinical performance), not against human readers' performance with and without AI assistance.

6. Standalone Performance

• Yes, standalone performance was done. The entire performance evaluation described focuses on the analytical and clinical performance of the ipsogen JAK2 RGQ PCR Kit as a standalone diagnostic test (without human-in-the-loop assistance for interpretation beyond standard laboratory procedures). The device outputs a qualitative result ("Mutation detected," "No Mutation Detected," or "Invalid") and a quantitative %MUT value, which are then used by clinicians as an adjunct to diagnosis. The "Rotor-Gene AssayManager (RGAM) software associated to the JAK2 Plug-in" automates data analysis, meaning no manual analysis required.

7. Type of Ground Truth Used

• Analytical Studies:
  • Known concentrations/dilutions: For precision, linearity, LoD, and stability studies, samples were prepared to contain known percentages of mutated DNA or were derived from characterized cell lines (MUTZ-9, HEL, K-562).
  • Orthogonal methods: For traceability/calibration of standards, an independent orthogonal method was used to confirm values.
  • Bi-directional Sanger sequencing: Used as the comparator method to establish accuracy.
• Clinical Performance Study:
  • The primary ground truth for the clinical study was the 2008 World Health Organization (WHO) diagnostic criteria for myeloproliferative neoplasms, with specific emphasis on PV. This standard incorporates various clinicopathological factors, including hemoglobin levels, hematocrit, EPO levels, and JAK2 mutation status (determined by bidirectional Sanger sequencing). Bone marrow biopsy and cytogenetic analysis were performed when required by the algorithm to confirm diagnosis.

8. Sample Size for Training Set

• The document describes a commercial in vitro diagnostic kit, not an AI/ML model that undergoes "training" in the traditional sense with a distinct training dataset. The development and validation of such a kit typically involve optimization studies and design verification using various samples (cell lines, pooled DNA, clinical samples) which inform the final design and performance characteristics, but these are not referred to as a "training set" in the context of machine learning. Therefore, a specific "training set sample size" as one would expect for an AI model is not applicable/not provided in this document.

9. How Ground Truth for Training Set was Established

• As above, given this is an IVD kit and not an AI/ML model, the concept of a "training set" with ground truth in the AI context does not directly apply. The kit's design and analytical parameters were likely developed and optimized using:
  • Quantified DNA samples: Known concentrations of mutant and wild-type DNA, often from cell lines (like MUTZ-8, HEL, K-562) with characterized mutational status.
  • Clinical samples: Utilized during development and analytical validation to ensure the test performs as expected with real-world variability.
  • Established molecular biology techniques: Such as quantitative PCR, and knowledge of the JAK2 V617F mutation's characteristics.
    The "ground truth" during this development (rather than "training") would be based on these highly characterized materials and established genomic methods.

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