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The BCR-ABL1 (p210) %IS Kit (Digital PCR Method) is an in vitro nucleic acid amplification test for the quantitation of BCR-ABL1 and ABL1 transcripts in total RNA from whole blood of diagnosed t (9;22) positive Chronic Myeloid Leukemia (CML) adult patients expressing BCR-ABL1 fusion transcripts type e13a2 and/or e14a2. The BCR-ABL1 (p210) %IS Kit (Digital PCR Method) is a reverse transcription-quantitative PCR performed on the Sniper Digital PCR All-in-One System and is intended to measure BCR-ABL1 to ABL1, expressed as a log molecular reduction (MR value) from a baseline of 100% on the International Scale, in t (9:22) positive CML patients during monitoring of treatment with Tyrosine Kinase Inhibitors (TKIs).

The BCR-ABL1 (p210) %IS Kit (Digital PCR Method) is intended for use only on the Sniper Digital PCR All-in-One System.

The test does not differentiate between e13a2 or e14a2 fusion transcripts and does not monitor other rare fusion transcripts resulting from t (9:22). This test is not intended for the diagnosis of CML.

The BCR-ABL1 (p210) %IS Kit (Digital PCR Method) is designed for detection of the BCR-ABL1 fusion gene (p210) and ABL1 gene, with specific primers and specific fluorescence probes. The test process includes three parts. The first part is to extract ribonucleic acid (RNA) from peripheral blood of CML patients. The second part is to detect BCR-ABL1 fusion gene (p210) and ABL1 internal reference gene in RNA samples by RT-dPCR (Reverse Transcription-Droplet PCR) reaction solution using the Sniper Digital PCR All-in-One System (DQ24-Dx). The third part is to analyze the results.

The Sniper Digital PCR All-in-One System consists of one instrument, which can be used together with it's supporting consumables and BCR-ABL1 (p210) %IS Kit (Digital PCR Method) to complete the detection of samples.

The Sniper Digital PCR All-in-One System divides the sample into about 20000 droplets and carries out PCR amplification, read the number of positive and negative droplets through fluorescent signals, and then calculate the concentration of nucleic acid quantitatively according to the volume of the droplets and the principle of Poisson Distribution.

DQ24-Dx-Sight Software (v1.0.2) is used to control the system and analyze test results. This software is embedded in the Sniper Digital PCR All-in-One System.

Here's a breakdown of the acceptance criteria and study detailed in the provided document:

Acceptance Criteria and Device Performance

Study Details

1. A table of acceptance criteria and the reported device performance: See table above.

2. Sample size used for the test set and the data provenance:

   • Precision/Reproducibility (Multi-site): 5 positive pools (3 variants, 5 different MR concentrations). 36 replicates per sample (2 replicates/run, 2 runs/day, 3 days, 3 sites). Total 540 observations.
   • Precision between batches: 2 positive pools (2 variants, 4 different MR concentrations). 108 replicates per sample (3 replicates/run, 2 runs/day, 3 days, 1 site with 2 instruments, 3 reagent lots). Total 864 observations.
   • RNA Extraction Method: 3 positive pools (3 variants, 5 different MR concentrations) each derived from multiple positive peripheral blood samples (6 e13a2, 7 e14a2) or K562 cells. Total of 180 results (samples extracted 2 times by 2 operators/day for 3 days).
   • Linearity/Assay reportable range: 2 positive pools (2 variants, 10 different MR concentrations). 4 replicates per sample.
   • Limit of Blank: 144 negative samples.
   • Limit of Detection/Limit of Quantitation: 2 positive pools (2 variants, 3 different MR concentrations). 20 replicates per day for 3 days with 2 reagent lots. Total 120 replicates.
   • Analytical Specificity (Interference): 1 sample pool (MR ~3.0). 2 replicate extractions, each tested in 3 replicates (6 tests per sample type). Potential interfering substances evaluated.
   • Primer Specificity: 4 positive samples (p190, p230, p210 e13a2, p210 e14a2) at 4 different concentrations. 4 replicates per sample.
   • Carryover Contamination: 1 high positive pool and 1 negative pool. 64 total test samples (32 high positive wells and 32 negative wells).
   • RNA Input: 2 positive pools (2 variants, 4 different MR concentrations). 3-5 replicates per run across 6 different RNA input amounts.
   • Real-Time Stability: 1 positive pool (1 variant, 3 different MR concentrations). 3 lots tested across 7 time points (T0, T3, T6, T9, T11, T12, T13).
   • Freeze-thaw Stability: Same samples as real-time stability. 1 lot tested with 3, 5, and 6 freeze-thaw cycles.
   • Specimen Stability: 3 fresh peripheral blood samples (1 variant, 3 different MR values). RNA extracted on Day 0, 1, 2. Each RNA sample tested 6-8 replicates.
   • Method Comparison with Predicate Device: 112 clinical samples (retrospective) collected from 2 hospitals. All samples were from patients diagnosed with t(9;22) positive CML with MR values distributed between 0.32 and 4.47.
   • Data Provenance: The document explicitly mentions that for the Method Comparison Study, clinical samples were collected from 2 hospitals and were retrospective. Other analytical studies used various types of prepared RNA samples or pools. The specific country of origin is not explicitly stated for all samples, but the submitting company is Suzhou Sniper Medical Technologies Co., Ltd. from China, suggesting the data collection likely occurred there.

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

   • Ground Truth Type: For the analytical studies, the ground truth was established by known concentrations/MR values of prepared RNA samples or specific variants (e.g., in linearity, LOD/LOQ, primer specificity). For the method comparison study, the ground truth was the result from the predicate device, the QXDx BCR-ABL %IS Kit.
   • Experts: The document does not explicitly state the use of external "experts" (e.g., radiologists, pathologists) to establish ground truth for this in vitro diagnostic (IVD) kit. The ground truth for analytical performance was based on the physical properties of the prepared samples (e.g., known dilutions, concentrations). For the clinical comparison, the predicate device served as the reference.

4. Adjudication method for the test set:

   • Not applicable. As this is an IVD kit for quantitative measurement and not an image-based AI device requiring human interpretation, there is no mention of an adjudication method like 2+1 or 3+1 for establishing ground truth. Raw data from instrument readings are compared against predefined analytical or clinical reference methods/values.

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

   • Not applicable. This is an in vitro diagnostic device, not an imaging AI device involving human interpretation/readers. Therefore, an MRMC study with human readers and AI assistance is not relevant or described.

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

   • The study primarily details the standalone performance of the device (BCR-ABL1 (p210) %IS Kit on the Sniper Digital PCR All-in-One System) itself, without direct human-in-the-loop interaction for result interpretation beyond running the assay and reviewing the automatically generated results. The device "is intended to measure BCR-ABL1 to ABL1, expressed as a log molecular reduction (MR value) from a baseline of 100% on the International Scale." The "results are interpreted automatically by the embedded Software DQ24-Dx-Sight from measured droplet counts, fluorescent signals, and embedded calculation algorithms." This indicates a standalone algorithmic performance.

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

   • For the analytical performance section, the ground truth was based on known concentrations/values of prepared RNA samples and comparisons to the 1st WHO International Genetic Reference Panel for BCR-ABL translocation quantitation.
   • For the method comparison study, the ground truth was established by the results from the legally marketed predicate device (QXDx BCR-ABL %IS Kit).

8. The sample size for the training set:

   • The document describes the analytical and method comparison studies for validation of the device. It does not mention a separate "training set" in the context of machine learning, as this is a digital PCR assay kit and not an AI/ML-based diagnostic system that typically requires distinct training, validation, and test sets.

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

   • Not applicable, as no "training set" is described for an AI/ML context.

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The Xpert BCR-ABL Ultra test is an in vitro diagnostic test for the quantitation of BCR-ABL1 and ABL1 mRNA transcripts in peripheral blood specimens of diagnosed t(9;22) positive Chronic Myeloid Leukemia (CML) patients expressing BCR-ABLI fusion transcripts type e13a2 and/or e14a2. The test utilizes automated, quantitative, real-time reverse transcription polymerase chain reaction (RT-qPCR). The Xpert BCR-ABL Ultra test is intended to measure BCR-ABL1 to ABL1 percent ratios on the International Scale (IS), and also expressed as a log molecular reduction (MR value) from a baseline of 100% (IS), in t(9;22) positive CML patients during of treatment with Tyrosine Kinase Inhibitors (TKIs).

The test does not differentiate between e13a2/b2a2 or e14a2/b3a2 fusion transcripts and does not monitor other rare fusion transcripts resulting from t(9;22). This test is not intended for the diagnosis of CML.

The Xpert BCR-ABL Ultra test is intended for use only on the Cepheid GeneXpert® Dx System and the GeneXpert Infinity System.

The Xpert BCR-ABL Ultra test is an automated in vitro diagnostic test for quantifying the amount of BCR-ABLI (BCR-ABL, hereafter) mRNA transcript as a ratio of BCR-ABL/ABL per the International Scale (IS).

The test is performed on the Cepheid GeneXpert® Dx System and GeneXpert Infinity System (referred to as the GeneXpert systems). The GeneXpert systems require the use of single-use, disposable cartridges that hold the PCR reagents and host the PCR process. Because the cartridges are self-contained and specimens never come into contact with working parts of the instrument modules, cross-contamination between samples is minimized. The GeneXpert systems have 1 to 80 randomly accessible modules, depending upon the instrument, that are each capable of performing separate sample preparation and real-time PCR and RT-PCR tests. Each module contains a syringe drive for dispensing fluids (i.e., the syringe drive activates the plunger that works in concert with the rotary valve in the cartridge to move fluids between chambers), and a proprietary I-CORE® thermocycler for performing real-time PCR and RT-PCR and detection.

The Xpert BCR-ABL Ultra test includes reagents to detect BCR-ABL fusion genes resulting from two major breakpoints, translocation e13a2/b2a2 and e14a2/b3a2 and the ABL transcript as an endogenous control in peripheral blood specimens. The amount of BCR-ABL transcript in the patient sample is reported as a percent ratio of BCR-ABL/ABL on the International Scale (IS), and also expressed as a log molecular reduction (MR value) from a baseline of 100% (IS), using the GeneXpert software.

There are two controls included in each Xpert BCR-ABL Ultra test, which are the ABL Endogenous Control and the Probe Check Control (PCC). The ABL Endogenous Control normalizes the BCR-ABL target and ensures that sufficient sample is used in the test. The PCC verifies reagent rehydration. PCR tube filling, and that all reaction components, including probes and dyes, are present and functional in the cartridge.

Here's an analysis of the acceptance criteria and the study that proves the device meets them, based on the provided text:

Acceptance Criteria and Device Performance for Xpert® BCR-ABL Ultra

Note: The provided document is a 510(k) Summary, which typically focuses on demonstrating substantial equivalence to a predicate device rather than explicitly stating pre-defined "acceptance criteria" in a quantitative, pass/fail manner. For this analysis, I will infer relevant performance metrics from the non-clinical and clinical studies presented as evidence of substantial equivalence. The "Acceptance Criteria" column will represent the demonstrated performance that FDA found acceptable for classification, and the "Reported Device Performance" will be the specific results from the studies.

1. Table of Acceptance Criteria and Reported Device Performance

2. Sample Size Used for the Test Set and Data Provenance

The primary clinical performance study used:

• Total specimens initially enrolled: 266
• Specimens available for analysis: 208 (after exclusions)
• Specimens within quantitative reportable range for both assays: 147
  • Frozen specimens: 117
  • Fresh, prospectively collected specimens: 30

Data Provenance:

• Country of Origin: U.S. (evaluated at four institutions in the U.S. for the clinical study).
• Retrospective/Prospective: The study included both:
  • Prospective: Fresh, prospectively collected EDTA whole blood specimens from CML patients.
  • Retrospective/Archived: Leftover specimens stored as frozen lysates (from the same patient population).

3. Number of Experts Used to Establish the Ground Truth for the Test Set and Qualifications

The document states that the Xpert BCR-ABL Ultra test performance was compared to an "FDA-cleared molecular assay" (the predicate device). This implies the ground truth for the clinical study was established by the predicate device's results.

• Number of Experts: Not applicable in the sense of human experts reviewing diagnostic images or pathology slides. The ground truth was based on the quantitative results from the predicate molecular assay.
• Qualifications of Experts: Not applicable, as the "ground truth" was an analytical measurement from another device, not human expert consensus.

4. Adjudication Method for the Test Set

Not applicable. The study compares the new device's quantitative results directly against those of an existing FDA-cleared molecular assay. There is no mention of a human adjudication process for discrepancies between the two assays; rather, statistical methods (Deming regression, Bland-Altman) are used to assess agreement and bias.

5. If a Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study Was Done

No. An MRMC study is typically performed for diagnostic imaging devices where human readers interpret cases, and AI assistance might improve their performance. This device is an in vitro diagnostic (IVD) based on RT-qPCR, where the result is generated automatically by the instrument, not interpreted by human readers. Therefore, an MRMC study comparing human readers with and without AI assistance is not relevant or applicable for this type of device.

6. If a Standalone (i.e., algorithm only without human-in-the-loop performance) Was Done

Yes, the studies described are standalone performance evaluations of the Xpert BCR-ABL Ultra system. The system performs automated sample preparation, RT-qPCR, and result interpretation without human intervention in the result generation process. The "Non-Clinical Studies" (Linearity, LoD, LoQ, Specificity, Interfering Substances, Carry-Over, Traceability) and the "Clinical Performance" study (comparison to predicate device) all evaluate the device's performance as an automated, standalone system.

7. The Type of Ground Truth Used

The ground truth used for the clinical performance evaluation was the quantitative results obtained from an FDA-cleared molecular assay (the predicate device). The predicate device measures BCR-ABL1 and ABL1 mRNA transcripts using similar RT-qPCR principles.

8. The Sample Size for the Training Set

The document does not explicitly state the sample size for a "training set" for the Xpert BCR-ABL Ultra assay. This is common for IVD assays based on established PCR technology, where the assay design is based on molecular biology principles and analytical validation, rather than machine learning models requiring large datasets for "training." The method is "Reverse transcription, quantitative, polymerase chain reaction (RT-qPCR) based nucleic acid amplification," which is a deterministic analytical method, not an AI/ML algorithm that is "trained" on data in the traditional sense.

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

As noted in point 8, the concept of a "training set" and "ground truth for training" in the context of machine learning (AI/ML) does not directly apply to this device. The development of an IVD assay like Xpert BCR-ABL Ultra involves:

• Assay Design: Primers and probes are designed based on known genetic sequences (BCR-ABL1 and ABL1 mRNA transcripts).
• Analytical Validation: Extensive non-clinical studies (linearity, LoD, LoQ, specificity, interference, etc.) are performed using characterized samples (e.g., CML patient specimens, spiked controls, cell lines, WHO reference panels) to establish the assay's performance characteristics. This is a process of characterizing the assay, not "training" it.

The "ground truth" for these analytical validation steps would be:

• Known concentrations in serially diluted samples.
• Characterized cell lines with specific breakpoints.
• Reference materials (e.g., WHO International Genetic Reference Panel) with assigned values.
• Negative controls (healthy donor blood, non-CML leukemic specimens).

These ground truths are established through laboratory preparation, internationally recognized standards, and biological characterization, rather than human expert consensus on a dataset.

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The QXDx™ BCR-ABL %IS Kit is an in vitro nucleic acid amplification test for the quantitation of BCR-ABL1 and ABL1 transcripts in total RNA from whole blood of diagnosed (9;22) positive Chronic Myeloid Leukemia (CML) patients expressing BCR-ABL1 fusion transcripts type e13a2 and/or e14a2. The QXDx BCR-ABL %IS Kit is a reverse transcription-quantitative PCR performed on the Bio-Rad QXDx™ AutoDG™ ddPCR System and is intended to measure BCR-ABL1 to ABL1, expressed as a log molecular reduction (MR value) from a baseline of 100% on the International Scale, in (9;22) positive CML patients during monitoring of treatment with Tyrosine Kinase Inhibitors (TKIs).

The QXDx BCR-ABL %IS Kit is intended for use only on the Bio-Rad QXDx AutoDG ddPCR System.

The test does not differentiate between e13a2 or e14a2 fusion transcripts and does not monitor other rare fusion transcripts resulting from t(9;22). This test is not intended for the diagnosis of CML.

The QXDx AutoDG ddPCR System consists of two instruments, the QXDx Automated Droplet Generator and the QXDx Droplet Reader, and their associated consumables. The QXDx Automated Droplet Generator partitions samples into approximately 20,000 nanoliter-sized droplets and, after PCR on a thermal cycler, droplets from each sample are analyzed individually on the QXDx Droplet Reader. PCR-positive and PCR-negative droplets are counted to provide direct quantification of nucleic acid in digital form. Results are analyzed on QXDx Software running on a Windows based computer.

The QXDx AutoDG ddPCR System contains:

• QXDx Automated Droplet Generator ●
• QXDx Droplet Reader ●
• Laptop Computer with QXDx Software .
• Accessory components: ●
  • o ddPCR Dx AutoDG Consumable Pack
    • Automated Droplet Generation Oil for Probes ■
    • DG32 Cartridges w/ Gaskets ■
    • ddPCR Pipet Tips
    • . ddPCR 96 Well Plates
    • . ddPCR Pierceable Foil Seals
  • o ddPCR Dx AutoDG Droplet Reader Oil Pack

Components of the kit QXDx BCR-ABL %IS KIT:

• QXDXTM BCR-ABL primers & probes
• QXDXTM Nuclease Free Water
• QXDXTM iScript Advanced Reverse Transcriptase
• QXDXTM 5x iScript Select Reaction Mix
• QXDXTM RT Primers
• QXDXTM 2X ddPCRTM Supermix
• QXDXTM BCR-ABL ~0.1%IS
• QXDXTM BCR-ABL ~10%IS
• QXDXTM BCR-ABL Neg-CTRL
• QXDXTM BCR-ABL H-CTRL
• QXDXTM BCR-ABL L-CTRL

The provided document details the analytical and clinical performance of the QXDx BCR-ABL %IS Kit for use on the QXDx AutoDG ddPCR System. This device is an in vitro nucleic acid amplification test for the quantitation of BCR-ABL and ABL1 transcripts in total RNA from whole blood of diagnosed Chronic Myeloid Leukemia (CML) patients.

The document does not describe the acceptance criteria and study for an AI/ML powered medical device, but rather for an in vitro diagnostic (IVD) kit. Therefore, many of the typical acceptance criteria and study elements associated with AI/ML devices (e.g., human-in-the-loop performance, expert consensus for ground truth on images, MRMC studies) are not applicable.

Below is a breakdown of the acceptance criteria and study details provided for this IVD kit, adapted to the requested format where possible, and noting where specific requested information (relevant to AI/ML devices) is not available for this type of device.

Acceptance Criteria and Reported Device Performance

The core acceptance criteria are related to the analytical performance of the kit, primarily precision, reproducibility, cross-reactivity, interference, linearity, and detection capability. The performance is consistently reported in terms of Molecular Reduction (MR) value or %International Scale (%IS), which are standardized measures for BCR-ABL levels.

Table 1: Acceptance Criteria of the QXDx BCR-ABL %IS Kit and Reported Performance

Study Details for the QXDx BCR-ABL %IS Kit

As this is an in vitro diagnostic (IVD) kit and not an AI/ML powered device, several of the requested sections are not applicable.

1. Sample sizes used for the test set and data provenance:

   • Precision and Multisite Reproducibility: 36 replicates per sample (2 reps x 2 runs x 3 days x 3 sites x 1 reagent lot). Two panels of eight test samples each (six contrived, two controls).
   • Lot to Lot Reproducibility: 108 data points per sample (3 replicates x 2 operators x 3 days x 3 lots x 2 instruments). Sixteen (16) BCR-ABL1 negative and one hundred (100) BCR-ABL1 positive RNA samples (pooled).
   • Cross Reactivity: Each dilution tested with 4 or 8 replicates (N). Two prepared samples (p190, p230 variants).
   • Interference: Ten (10) tests per sample type (5 replicate extractions x 2 tests per extraction). Various interferents tested.
   • Assay Linearity: Not explicitly stated, but typically involves multiple dilutions. Two positive BCR-ABL RNA patient pools (E13a2, E14a2) and one negative RNA pool.
   • Detection Capability (LoD): 160 replicates per sample (20 replicates x 4 days x 2 lots x 1 instrument) for two contrived samples (e13a2, e14a2 variants).
   • Kit, Calibrator and Control Stability: 3 kit lots at various time points (T0, T1, T2.5, T5, T11, T12, T19, T25 months). Freeze-thaw: 4 kits from one lot tested through several cycles.
   • Specimen Stability (Whole Blood Stability): Positive blood sample and 3 negative blood samples. Dilutions tested with 4 or 11 replicate extractions.
   • WHO Standard Quantification: 7 kit lots. Four (4) levels of WHO primary standards tested in four (4) replicates per lot.
   • Carryover Contamination: 288 initial replicate tests; 286 valid replicates (from 2 plates, tested on 3 instruments).
   • Clinical Performance (Method Comparison): 139 samples. Samples acquired from at least two geographically distinct regions. Samples were de-identified leftover RNA samples, previously collected. The study was conducted at a single testing lab.
   • Data Provenance (Retrospective/Prospective, Country): For the clinical method comparison study, samples were "de-identified leftover RNA samples that have been previously collected from a minimum of two (2) sites." This indicates a retrospective data collection approach. The country of origin is not explicitly stated, but the submission is to the US FDA, so it's likely from the US or a region with compliant medical standards.

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

   • Not Applicable. For this IVD device, the "ground truth" is established by the known concentrations/compositions of the samples (e.g., contrived samples with specific BCR-ABL:ABL ratios, WHO primary reference standards with assigned values, or comparison to a legally marketed predicate device). There are no human experts "reading" or annotating images or clinical data to establish ground truth in the way described for AI/ML devices.

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

   • Not Applicable. As there are no human expert readings of the test set requiring adjudication. The ground truth refers to the biochemical composition or an independent, established reference assay.

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

   • Not Applicable. This is an in vitro diagnostic device, not an AI-powered system designed to assist human readers (e.g., radiologists interpreting images). The "method comparison" study compares the device's analytical performance against a predicate device, not human performance.

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

   • This entire document describes the standalone performance of the IVD kit and its associated system (QXDx AutoDG ddPCR System). The output is a quantitative MR value or %IS, which is then used by clinicians for patient management. There is no "human-in-the-loop performance" for the device's operation itself, beyond standard laboratory procedures and interpretation of the quantitative results.

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

   • The ground truth extensively used in the analytical studies is based on:
     • Contrived samples: Samples prepared by mixing known concentrations of BCR-ABL positive and negative RNA, allowing for precise control of target ratios.
     • Reference materials: Specific WHO International Standards (WHO-IS primaries) with assigned values used for calibration and verification.
     • Predicate device comparison: The Asuragen Quantidex qPCR BCR-ABL IS Kit (IVD) served as the comparator in the clinical method comparison study, establishing a "ground truth" for comparative performance.
     • Known sample characteristics: E.g., for cross-reactivity and interference studies, samples with known absence of target or presence of interfering substances are used.

7. The sample size for the training set:

   • Not applicable in the usual AI/ML sense. This is an IVD kit involving biochemical reactions and a detection system, not a machine learning algorithm that undergoes 'training' on a dataset in the same way. The development and optimization of the kit's components and parameters would be based on internal R&D, but there isn't a "training set" in the context of typical AI/ML submissions.

8. How the ground truth for the training set was established:

   • Not Applicable. As above, there's no "training set" in the AI/ML sense. The ground truth for the analytical studies (which could be considered analogous to validation/testing stages for IVDs) was established by carefully prepared samples with known concentrations/compositions or recognized international standards.

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The MolecularMD MRDx BCR-ABL Test is an in vitro diagnostic test for the quantitative detection of BCR-ABL 1 transcripts (e13a2/b2a2 and/or e14a2/63a2) and the ABL1 endogenous control mRNA in peripheral blood specimens from patients previously diagnosed with t(9:22) positive chronic myeloid leukemia (CML). The ratio of BCR-ABL1 is calculated and reported on the WHO International Scale. The test utilizes quantitative, real-time reverse transcription polymerase chain reaction performed on the Applied Biosystems 7500 Fast Dx instrument.

The MolecularMD MRDx BCR-ABL Test is intended to measure BCR-ABL mRNA transcript levels in patients diagnosed with t(9;22) positive CML during monitoring of treatment with Tyrosine Kinase Inhibitors (TKIs).

The device is also intended to be used in the serial monitoring for BCR-ABL mRNA transcript levels as an aid in identifying CML patients in the chronic phase being treated with nilotinib who may be candidates for treatment discontinuation and for monitoring of treatment-free remission.

The test does not differentiate between e13a2 or e14a2 fusion transcripts and does not monitor other rare fusion franscripts resulting from t(9:22). The test is not intended for the diagnosis of CML.

The MolecularMD MRDx® BCR-ABL Test is a quantitative, real-time polymerase chain reaction test that provides quantitation of BCR-ABL1 (hereafter BCR-ABL) transcript e13a2/b2a2 or e14a2/b3a2 and ABL1 (hereafter ABL) transcript levels in RNA extracted from peripheral blood samples collected from CML patients. Peripheral blood is collected in either EDTA or PAXgene Blood RNA Tubes. Each collection tube type requires a specified RNA extraction method and RNA input amount. Total RNA is extracted from peripheral blood and serves as the template for RT-qPCR. The test is performed using a onestep RT-qPCR protocol wherein the reverse transcription and quantitative, real-time PCR reactions are performed in the same well. BCR-ABL and ABL amplicons are generated and detected in real-time using TaqMan® MGB probes. The MolecularMD MRDx BCR-ABL Test is performed on the Applied Biosystems 7500 Fast Dx Real-Time PCR Instrument. The instrument integrates a thermal cycler, a fluorometer and application specific software. The ABI 7500 Fast Dx instrument software v1.4.1 calculates the BCR-ABL and ABL copy numbers using a standard curve generated with calibrators on each individual plate. The data are exported as a CSV file for further analysis in the MRDx® BCR-ABL Test Software. The BCR-ABL/ABL ratio is calculated and converted to the International Scale by the MRDx BCR-ABL Test Software. The MRDx BCR-ABL Test Software is used to analyze all test results. This software, provided with the MRDx BCR-ABL Test, is used to calculate the BCR-ABL/ABL % IS and MR value for patient sample using the conversion factor for the MRDx BCR-ABL Test after validating each MRDx BCR-ABL Test result against the run acceptance criteria and sample acceptance criteria.

The MolecularMD MRDx BCR-ABL Test is an in vitro diagnostic test for the quantitative detection of BCR-ABL1 transcripts and ABL1 endogenous control mRNA in peripheral blood specimens from patients previously diagnosed with t(9;22) positive chronic myeloid leukemia (CML). It calculates the ratio of BCR-ABL1 to ABL1 and reports it on the WHO International Scale. The device is intended to measure BCR-ABL mRNA transcript levels in CML patients during monitoring of treatment with Tyrosine Kinase Inhibitors (TKIs). It is also intended for serial monitoring of BCR-ABL mRNA transcript levels to aid in identifying CML patients in the chronic phase being treated with nilotinib who may be candidates for treatment discontinuation and for monitoring of treatment-free remission.

Here's an overview of the acceptance criteria and study data:

1. Table of Acceptance Criteria and Reported Device Performance

The document does not explicitly present a consolidated table of acceptance criteria for all performance characteristics. However, individual acceptance criteria are mentioned within the description of each study. Below is a summary of acceptance criteria and the device's reported performance, extracted from the document:

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

• Traceability to WHO IS: Tested over 20 days for each of 3 MRDx BCR-ABL Test kit lots. A different WHO primary panel (four levels A-D) was reconstituted daily, with each level tested multiple times (implicitly, across the 20 days). MolecularMD WHO BCR-ABL Reference Panel Secondary Standards were also tested.
• Accuracy (Correlation to ddPCR):
  • Test Set Size: 217 samples total. 119 evaluable EDTA samples and 98 evaluable PAXgene samples.
  • Data Provenance: Samples were from CML patients. The document does not specify the country of origin but implies clinical samples. The study involved comparison to a reference method, suggesting an analytical, retrospective-like analysis of patient samples.
• Limit of Blank (LoB): 30 BCR-ABL negative samples, tested in duplicate using 3 assay kit lots, totaling 180 replicates.
• Limit of Detection (LoD): 6 CML patient specimens (3 e13a2, 3 e14a2) used to create serial dilutions. Sufficient RNA for each panel (5 levels) was made to allow for testing of 60 total replicates (20 replicates x 3 lots) per level. 1725 valid EDTA samples and 1595 valid PAXgene samples were tested.
• Analytical Specificity (Interfering Substances): CML samples at 2 levels (MR3.0 and MR4.5) extracted from both EDTA and PAXgene tubes, spiked with test substances. No specific number of replicates or distinct samples given for this part.
• Analytical Specificity (Primer Specificity): RNA from six CML patient samples (three e13a2, three e14a2) were used. Additionally, ABL2 IVT RNA and RNA from an e19a2 positive cell line were tested.
• Analytical Specificity (Specimen Cross Contamination):
  • PAXgene: 30 replicates.
  • EDTA: 40 replicates.
• Precision (Repeatability): Contrived patient samples (e13a2 and e14a2 at MR3.0, MR4.0, MR4.5). Each MR sample level for each transcript was tested in 17 replicates on individual MRDx BCR-ABL Test plates.
• Precision (Reproducibility): Two series of samples (e13a2 and e14a2 CML patient samples diluted into non-diseased blood) at 5 levels each.
  • EDTA: 1800 total tests (60 replicates x 3 sites x 2 transcripts x 5 panel members). N for individual levels ranged from 149-152.
  • PAXgene: 900 total tests (90 measurements per panel member x 2 transcripts x 5 panel members, implying 90 measurements per level). N for individual levels ranged from 77-92.
• Linear Range (Assay Range): Two dilution series (one e13a2, one e14a2) for each tube type. Each series had 10 BCR-ABL target levels, with 5 replicates at each level.
• Kit Real-Time and Freeze-Thaw Stability: 6 kits from each of 3 lots for each time point were tested.
• Reagent Preparation and Reaction Intermediate Stability (In Use): 2 kits were run for each condition (standard/extreme) on each of 2 days, with 2 sets of calibration curves, RNA controls, and samples (total n=8 per condition).
• Specimen Stability:
  • PAXgene: Samples at three levels (MR3.0, MR4.0, MR4.5) tested in quadruplicate at 0, 24, 48, 60 hours (for 2-8°C storage) and at 0, 14, 28, 30 days (for -30 to -15°C storage).
  • EDTA: 17 CML patient specimens, tested in quadruplicate at 0, 24, 48, 60 hours.
• Shipping Stability: Multiple types of containers and shipping conditions, varying load. Functional testing using samples across the assay range. Specific N not provided for samples.
• PAXgene-EDTA Tube Equivalency: 100 CML patient paired blood samples.

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

Not applicable for analytical performance studies. The "ground truth" for these studies is established through defined reference methods (e.g., WHO International Genetic Reference Panel, ddPCR, precisely prepared dilutions/spikes, or established consensus sequences/protocols), not expert consensus in the human interpretation sense.

For the clinical studies (ENESTfreedom and ENESTop), the efficacy outcome (treatment-free remission, loss of MMR) is based on the quantitative results of the MRDx BCR-ABL Test itself per pre-defined thresholds, rather than expert interpretation of individual clinical cases as a ground truth.

4. Adjudication method for the test set

Not applicable. For the analytical studies, the ground truth is quantitative and objectively determined (e.g., standard concentrations, reference assay results, sequencing). For the clinical studies, outcomes are determined by the device's quantitative measurements against pre-defined clinical thresholds, not by human adjudication of conflicting interpretations.

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

Not applicable. This device is an in vitro diagnostic test (RT-qPCR assay) and not an AI-assisted diagnostic imaging or interpretation tool that involves human "readers." Therefore, no MRMC study, or assessment of human reader improvement with AI, was performed or is relevant to this device.

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

Yes, the analytical performance studies explicitly demonstrate the standalone performance of the MolecularMD MRDx BCR-ABL Test. This device is an automated quantitative assay (RT-qPCR) followed by software analysis. The outputs are quantitative values (BCR-ABL/ABL % IS and MR values), and the performance characteristics (accuracy, precision, LoD, LoQ, linearity, etc.) are reported for the assay system itself, without human-in-the-loop performance influencing the primary measurements or calculations. While interpretation of results for clinical decision-making is done by clinicians, the device's measurement function is standalone.

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

• Traceability to WHO IS: The ground truth was the 1st WHO International Genetic Reference Panel for quantitation of BCR-ABL translocation by RQ-PCR, which represents a harmonized international standard.
• Accuracy (Correlation to ddPCR): The ground truth was derived from a validated BCR-ABL reverse transcription droplet digital PCR (ddPCR) assay, which served as the reference method for comparison.
• Limit of Blank (LoB): "BCR-ABL negative samples" served as the negative ground truth.
• Limit of Detection (LoD), Linear Range: Ground truth was established using serial dilutions of CML patient RNA and non-diseased subject blood RNA, allowing for precise control of target concentrations.
• Analytical Specificity: Ground truth included precisely spiked interfering substances (endogenous and exogenous), known ABL2 IVT RNA, and e19a2 positive cell line RNA, and alternation of known high-positive and negative samples for cross-contamination.
• Precision (Repeatability and Reproducibility): Ground truth was created using contrived patient samples (CML patient RNA and non-diseased subject RNA) with targeted MR levels.
• PAXgene-EDTA Tube Equivalency: Ground truth was established by comparing results from paired CML patient blood samples processed via two different collection and extraction methods.
• Clinical Studies (ENESTfreedom and ENESTop): The clinical validity was demonstrated against clinical outcomes data from two prospective clinical trials (ENESTfreedom and ENESTop). The ground truth for identifying candidates for treatment discontinuation and monitoring remission relied on sustained molecular response (MR4.5/MR4.0) as defined by the measured BCR-ABL levels.

8. The sample size for the training set

The document does not explicitly specify a "training set" for the development of the device's algorithm in the way that would typically be described for a machine learning model. This is an RT-qPCR assay, not a machine learning-based AI device.

However, if "training set" is broadly interpreted as data used during the development and optimization phases:

• The quantitative methodology is based on established RT-qPCR principles and standard curve generation using calibrators.
• The "conversion factor" to the International Scale is derived from harmonization studies with the WHO International Genetic Reference Panel.
• The "MRDx BCR-ABL Test Software" uses established equations and validated run/sample acceptance criteria, which would have been developed and refined using internal data.
• The specific number of samples or data points used for these internal development and optimization processes is not provided.

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

As noted above, there isn't a "training set" in the machine-learning sense for this device. The ground truth for analytical performance studies, which serve to validate the device's performance, is established through:

• International Reference Standards: The 1st WHO International Genetic Reference Panel for BCR-ABL quantitation.
• Reference Methods: A validated droplet digital PCR (ddPCR) assay.
• Controlled Experiments: Creation of precisely defined mock samples or panels with known concentrations or characteristics (e.g., serial dilutions of CML patient RNA, non-diseased RNA, spiked samples, targeted MR levels in contrived samples).
• Clinical Trial Data: Long-term clinical follow-up in the ENESTfreedom and ENESTop studies, where the MRDx BCR-ABL Test itself determined the molecular response status that defined the clinical ground truth for treatment discontinuation and remission monitoring.

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The QuantideX qPCR BCR-ABL IS Kit is an in vitro nucleic acid amplification test for the quantitation of BCR-ABL1 and ABL1 transcripts in total RNA from whole blood of diagnosed t(9,22) positive Chronic Myeloid Leukemia (CML) patients expressing BCR-ABL1 fusion transcripts type e13a2 and/or e14a2. The QuantideX qPCR BCR-ABL IS Kit is a reverse transcription-quantitative PCR performed on the Applied Biosystems 7500 Fast Dx Real-Time PCR Instrument and is intended to measure BCR-ABL1 to ABL1, expressed as a log molecular reduction (MR value) from a baseline of 100% on the International Scale, in t(9;22) positive CML patients during monitoring of treatment with Tyrosine Kinase Inhibitors (TKIs).

The test does not differentiate between e13a2 or e14a2 fusion transcripts and does not monitor other rare fusion transcripts resulting from t(9:22). This test is not intended for the diagnosis of CML.

The QuantideX qPCR BCR-ABL IS Kit reagents are adapted for use on the ABI 7500 Fast Dx Real-Time PCR Instrument. The assay includes reagents sufficient for 60 reactions. A description of the reagents provided is described below in Table 1.

Here's a summary of the acceptance criteria and study that proves the device meets them, based on the provided text:

1. Table of Acceptance Criteria and Reported Device Performance

The document describes both analytical and clinical performance acceptance criteria.

Analytical Performance - Precision/Reproducibility

Analytical Performance - Linearity

Analytical Performance - Limit of Quantitation (LoQ)

Analytical Performance - Interfering Substances

Analytical Performance - Primer Specificity

Analytical Performance - Specimen Carryover Contamination

Analytical Performance - RNA Input

Analytical Performance - Traceability

Analytical Performance - Specimen Stability (Whole Blood)

Clinical Performance

2. Sample Size Used for the Test Set and Data Provenance

The primary test set for clinical performance involved a retrospective study with:

• Sample Size: 137 evaluable samples from 96 subjects (out of 139 samples from 98 patients initially collected).
• Data Provenance: Retrospective, collected at 2 clinical sites in the US (OHSU and Hospital of the University of Pennsylvania).

For analytical performance studies, various sample compositions and quantities were used:

• Precision (within-lab & site-to-site): 25 samples formulated from 5 human RNA specimens positive for BCR-ABL1 diluted into RNAs from CML-negative blood. The site-to-site reproducibility study involved 1200 measurements from this 25-sample pool.
• Linearity/Reportable Range: 2 separate RNA specimens (one e13a2, one e14a2) diluted into RNAs from CML-negative whole blood (ranging from MR 0.1 to MR 4.8).
• Limit of Blank (LoB): 30 non-leukemic human RNA specimens.
• Limit of Detection (LoD): 4 separate human RNA specimens positive for BCR-ABL1, serially diluted to 28 levels.
• Limit of Quantitation (LoQ): 6 specimens derived from 6 human RNA positive for BCR-ABL (diluted to target MR 4.7).
• Interfering Substances: Residual CML-positive blood diluted to approximately MR 4.0 into RNA from CML-negative whole blood (tested in 9 replicates).
• Primer Specificity: 11 leukemic specimens (CML, AML, ALL) and 2 non-leukemic RNA specimens.
• Specimen Carryover Contamination: Serially diluted total RNA from residual clinical CML-positive blood into RNA from CML-negative whole blood in a checkerboard pattern (25 high positive, 25 negative).
• RNA Isolation: CML-positive white blood cells serially diluted across 4 levels (MR 1-4) into CML-negative human anti-coagulated whole blood.
• RNA Input: 30 samples derived from 2 primary RNA samples diluted into non-leukemic human RNA (total 216 evaluable observations).
• Traceability: WHO Reference Panel (4 panel members tested in duplicate).
• Reagent Stability: 5 samples diluted to approximately MR 1, 2, 3, 4, and < LoD (15 replicates per time point).
• Specimen Stability (Whole Blood): 13 CML positive specimens and 3 CML negative specimens.

3. Number of Experts Used to Establish the Ground Truth for the Test Set and Their Qualifications

The document does not explicitly state the number of experts or their specific qualifications (e.g., radiologist with X years of experience) used to establish the ground truth for the test set.

For the clinical study, the "ground truth" for event-free survival (EFS) was based on clinical outcomes defined by specific criteria:

• Death by any cause
• Development of accelerated-phase or blast crisis CML
• Loss of complete hematologic response
• Loss of complete cytogenetic response
• Appearance of mutation
• Change in TKI treatment not due to toxicity and not due to the results of another BCR-ABL assay

These outcomes would typically be determined by treating physicians and medical records, often relying on established clinical guidelines and diagnostic criteria. While not explicitly stated as "experts establishing ground truth," the medical documentation and clinical assessments would serve this role.

4. Adjudication Method for the Test Set

The document does not describe an explicit adjudication method (e.g., 2+1, 3+1, none) for a test set, particularly in the context of expert review. The ground truth for the clinical study was based on observed clinical outcomes and treatment changes, which are derived from patient records and physician decisions rather than expert consensus on image or assay interpretation.

5. If a Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study Was Done

No, an MRMC comparative effectiveness study was not done. This device is a quantitative in vitro diagnostic assay, not an imaging device or a device requiring human interpretation in the loop in the same way an MRMC study evaluates. The clinical study assessed the device's ability to predict clinical outcomes (EFS) based on its quantitative output (MR value), rather than comparing human reader performance with and without AI assistance.

6. If a Standalone (Algorithm Only Without Human-in-the-loop Performance) Was Done

Yes, the primary clinical performance and all analytical performance studies represent a standalone assessment of the device. The QuantideX qPCR BCR-ABL IS Kit is an in vitro diagnostic test that provides quantitative results (MR values) directly. There is no human-in-the-loop interpretation of the assay's raw output described; the device itself calculates the BCR-ABL1 to ABL1 ratio and MR value. The clinical study then correlated these device-generated MR values with patient outcomes.

7. The Type of Ground Truth Used

• Analytical Ground Truth: For many analytical performance studies (precision, linearity, LoD, LoQ, interference), the ground truth was established by known concentrations or dilutions of BCR-ABL1 and ABL1 transcripts, or by using characterized RNA specimens (e.g., CML-positive or CML-negative clinical samples, or cell lines with specific translocations) to create spiked samples or panels. Traceability was established against the First (1st) WHO International Genetic Reference Panel.
• Clinical Ground Truth: For the clinical performance study, the ground truth was patient outcomes data, specifically "event-free survival" at 32-40 months after TKI treatment. "Events" were defined by specific clinical criteria related to disease progression, death, or changes in therapy.

8. The Sample Size for the Training Set

The document does not specify a separate training set size. As an in vitro diagnostic kit, the "training" analogous to machine learning would be the extensive analytical validation and optimization performed by the manufacturer during product development, which informs the assay's design, protocols, and reagent formulations. The clinical and analytical studies described are primarily for validation and verification of the final device, not for training a specific algorithm.

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

Since a distinct "training set" in the machine learning sense is not explicitly mentioned or applicable for this type of IVD kit, the concept of establishing ground truth for a training set does not directly apply. Instead, the analytical characteristics (like limit of detection, linearity, precision) are established using controlled experiments with known concentrations and reference materials, and clinical utility is demonstrated by correlating the device's measurements with observed patient clinical outcomes. These outcomes in the clinical study are determined by medical definitions and patient records, as described in point 3.

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