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The Invader® MTHFR 677 test is an in vitro diagnostic test intended for the detection and genotyping of a single point mutation (C to T at position 677) of the human 5.10-methylenetetrahydrofolate reductase (MTHFR) gene in isolated genomic DNA obtained from whole blood Potassium EDTA samples from patients with suspected thrombophilia.

The Invader MTHFR 677 test consists of the following components:
MTHFR 677 Oligo Mix
Universal Buffer
Universal Enzyme Mix
No DNA Control
MTHFR 677 Wild Type Control
MTHFR 677 Heterozygous Control
MTHFR 677 Mutant Control
Invader Call Reporter™ Software

The Invader® MTHFR 677 test is an in vitro diagnostic test for detecting and genotyping the C to T mutation at position 677 of the MTHFR gene.

1. Table of Acceptance Criteria and Reported Device Performance:

The document doesn't explicitly state "acceptance criteria" but demonstrates performance through several studies of 100% agreement with the ground truth for most tests. The primary performance metric is agreement with bi-directional sequencing.

2. Sample Sizes and Data Provenance:

• Inter-laboratory Reproducibility (Study #1):
  • Test set sample size: 9 whole blood samples (3 wild type, 3 heterozygous, 3 homozygous mutant). Tested in duplicate over 5 non-consecutive days by 6 operators (2 from each of 3 sites), resulting in 90 samples tested per operator, totaling 540 data points.
  • Data provenance: Not explicitly stated, but includes 2 external sites and 1 internal site, suggesting geographically diverse testing within a controlled study. Prospective based on the study design.
• Lot-to-Lot Reproducibility (Study #9):
  • Test set sample size: 9 genomic DNA samples (3 wild type, 3 heterozygous, 3 mutant). Tested in quadruplicate using 3 different kit lots, totaling 108 data points.
  • Data provenance: Not explicitly stated, but part of analytical performance studies, likely within a controlled laboratory setting.
• Real-Time Stability Study (Study #5):
  • Test set sample size: Samples representing all 3 genotypes (including 3 controls and 4 gDNA samples) tested in quadruplicate at each time point.
  • Data provenance: Not explicitly stated, likely internal laboratory testing.
• Reagent Freeze-Thaw Stability Study (Study #6):
  • Test set sample size: Genomic DNA isolated from cell lines, representing all possible genotypes.
  • Data provenance: Not explicitly stated, likely internal laboratory testing.
• Analytical Sensitivity (Study #3):
  • Test set sample size: 3 genomic DNA samples (WT, HET, MUT). Each sample diluted to 8 different concentrations and tested in replicates of 40, totaling 960 data points.
  • Data provenance: Whole blood collected in potassium EDTA, likely laboratory-controlled study.
• Analytical Specificity (Interfering Substances) (Study #4):
  • Test set sample size: 9 whole blood samples of different genotypes (3 WT, 3 HET, 3 MUT).
  • Data provenance: Not explicitly stated, likely laboratory-controlled study.
• Pre-Analytical Equivalency Study/Genomic DNA Extraction Reproducibility (Study #7):
  • Test set sample size: 30 human whole blood samples and 10 leukocyte-depleted whole blood samples spiked with cell lines (total 40 samples). These were analyzed with 4 different extraction methods, leading to 160 extracted DNA samples tested.
  • Data provenance: Human whole blood and spiked cell lines, likely from a controlled laboratory setting.
• Instrument Equivalency (Study #8):
  • Test set sample size: 29 human whole blood samples and 10 leukocyte-depleted whole blood samples spiked with cell lines (total 39 samples). Tested across 3 thermal cyclers and 3 fluorometers (39*3 = 117 tests per fluorometer in Table 9, but recorded as 78 of 78 = 100% for each cell, implying 78 distinct samples or tests per fluorometer/thermal cycler combination).
  • Data provenance: Human whole blood and spiked cell lines, likely from a controlled laboratory setting.
• Method Comparison (Bi-directional Sequencing) (Study #2):
  • Test set sample size: 361 human whole blood samples.
  • Data provenance: Human whole blood samples. Not specified if retrospective or prospective or country of origin, but generally such studies involve controlled collection.

3. Number of Experts and Qualifications for Ground Truth:

The document does not explicitly mention the number or qualifications of experts used to establish the ground truth. However, the ground truth for all performance studies is established by bi-directional DNA sequencing, which is a widely accepted gold standard for determining genetic variants. This method is inherently objective and does not rely on expert interpretation in the same way as, for example, image-based diagnostics.

4. Adjudication Method:

Given that the ground truth is established by bi-directional DNA sequencing, which is a definitive molecular method, there is no mention of an adjudication method in the context of human interpretation (e.g., 2+1, 3+1). The testing process involves comparing the device's output to the objective sequencing results. In cases of discrepancies or invalid results (e.g., in Study #2), these are noted and factored into the agreement calculations.

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

No, a multi-reader multi-case (MRMC) comparative effectiveness study was not done. The device is an in vitro diagnostic test for genetic analysis, not an imaging device or one requiring human interpretation of complex visual data. Therefore, the concept of "human readers improve with AI vs without AI assistance" is not applicable here.

6. Standalone Performance:

Yes, a standalone performance study (algorithm only without human-in-the-loop performance) was done. All the analytical performance studies (precision, stability, sensitivity, specificity, equivalency, and method comparison against sequencing) represent the standalone performance of the Invader® MTHFR 677 test, as it directly outputs genotype calls from fluorescence data without human interpretation influencing the result. The Invader Call Reporter™ software performs the data analysis and determines results autonomously following the import of fluorescence data.

7. Type of Ground Truth Used:

The primary type of ground truth used across all studies is bi-directional DNA sequencing. This is explicitly stated for the method comparison study and implied as the reference method for verifying all genotype calls throughout the analytical performance sections (e.g., "percent agreement between Invader® MTHFR 677 test and sequencing").

8. Sample Size for the Training Set:

The document does not specify the sample size used for a "training set." This device is based on a well-established molecular biology technique (Invader Plus® chemistry, PCR, FRET) and software for data interpretation, rather than a machine learning or AI model that typically requires a distinct training phase with large datasets. The controls included with the kit (Wild Type, Heterozygous, Mutant, No DNA Control) serve to ensure proper functioning and calibration for each run, effectively acting as internal references for the test.

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

Since there is no explicit mention of a "training set" in the context of machine learning, the question of how its ground truth was established is not directly applicable. The performance is validated against the gold standard of bi-directional DNA sequencing, which inherently provides the "ground truth" for the MTHFR 677 genotype. The development and optimization of the assay itself would have involved molecular biology techniques to ensure accurate detection of the specific SNP.

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The Invader® MTHFR 1298 test is an in vitro diagnostic test intended for the detection and genotyping of a single point mutation (A to C at position 1298) of the human 5,10-methylenetetrahydrofolate reductase (MTHFR) gene in isolated genomic DNA obtained from whole blood potassium EDTA samples from patients with suspected thrombophilia.

The Invader MTHFR 1298 test consists of the following components:
MTHFR 1298 Oligo Mix
Universal Buffer
Universal Enzyme Mix
No DNA Control
MTHFR 1298 Wild Type Control
MTHFR 1298 Heterozygous Control
MTHFR 1298 Mutant Control
Invader Call Reporter™ Software
Invader® MTHFR 1298 Software

Here's an analysis of the Invader® MTHFR 1298 device based on the provided text, addressing your specific questions.

1. Table of Acceptance Criteria and Reported Device Performance

(Note: The document does not explicitly state "acceptance criteria" in a separate section with pass/fail thresholds for each study. Instead, it presents study results and concludes that the information "supports a substantial equivalence decision." The table below infers the implicit acceptance criterion as 100% agreement/concordance where achieved, and notes the precise percentage reported for other metrics.)

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

• Inter-laboratory Reproducibility (Study #1):
  • Test Set Sample Size: 9 (panel of genomic DNA samples representing 3 genotypes: wild type, heterozygous, homozygous mutant).
  • Total Tests Performed: 540 (9 samples x 2 operators/site x 3 sites x 2 duplicates/day x 5 days of testing).
  • Data Provenance: Not specified, but involved 2 external sites and 1 internal site. The samples were "whole blood samples specific for each of the three possible genotypes." Implied retrospective as specific genotypes were selected.
• Lot-to-Lot Reproducibility (Study #9):
  • Test Set Sample Size: 9 (genomic DNA samples: 3 wild type, 3 heterozygous, 3 mutants).
  • Total Tests Performed: 108 (9 samples x 4 replicates x 3 lots).
  • Data Provenance: Not specified, but samples were "genomic DNA samples." Implied retrospective.
• Real-time Stability (Study #5):
  • Test Set Sample Size: 3 genomic DNA samples (WT, HET, MUT) + 3 controls.
  • Total Tests Performed: Performed in quadruplicate at each time point (initial, 4 months, 7 months) for 3 lots. Total: (3 gDNA + 3 controls) x 4 replicates x 3 lots x 3 time points = 216 tests.
  • Data Provenance: Not specified.
• Freeze-Thaw Stability (Study #6):
  • Test Set Sample Size: 3 genomic DNA samples (WT, HET, MUT) + 3 controls.
  • Total Tests Performed: The table indicates varying numbers of tests per sample per freeze/thaw cycle. Total for gDNA: 36 (WT), 48 (HET), 36 (MUT). Total for controls: 45 each. Total overall is 255.
  • Data Provenance: Genomic DNA isolated from cell lines.
• Analytical Sensitivity (Detection Limit) (Study #3):
  • Test Set Sample Size: 3 genomic DNA samples (1 MUT, 1 HET, 1 WT).
  • Total Tests Performed: 960 (3 samples x 8 concentrations x 40 replicates).
  • Data Provenance: "Genomic DNA samples with different genotypes (i.e. WT, HET, MUT) were extracted from whole blood collected in potassium EDTA." Implied retrospective.
• Analytical Specificity (Interfering Substances) (Study #4):
  • Test Set Sample Size: 9 whole blood samples (3 WT, 3 HET, 3 MUT).
  • Total Tests Performed: 18 tests per substance (9 samples x 2 replicates). 7 substances + 1 control = 8 conditions. Total: 18 x 8 = 144 tests.
  • Data Provenance: "human whole blood samples." Implied retrospective.
• Pre-Analytical Equivalency (Genomic DNA Extraction Reproducibility) (Study #7):
  • Test Set Sample Size: 30 human whole blood samples + 10 leukocyte depleted whole blood spiked with cell lines = 40 unique samples.
  • Total Tests Performed: 160 (40 samples x 4 extraction methods, analyzed in singlicate).
  • Data Provenance: "human whole blood samples and leukocyte depleted whole blood spiked with cell lines." Implied retrospective.
• Instrument Equivalency (Study #8):
  • Test Set Sample Size: 29 human whole blood samples + 10 leukocyte depleted whole blood samples = 39 unique samples.
  • Total Tests Performed: 78 tests were reported per combination of fluorometer/thermal cycler. Overall: 39 samples x 2 extraction methods x 3 thermal cyclers x 3 fluorometers (but results are combined for each fluorometer/cycler pairing). The table shows 78 total tests for each of the 9 combinations.
  • Data Provenance: "human whole blood samples and leukocyte depleted whole blood samples spiked with cell lines." Implied retrospective.
• Method Comparison (Bi-directional Sequencing) (Study #2):
  • Test Set Sample Size: 348 human whole blood samples.
  • Data Provenance: "Human whole blood samples (n = 348)." Implied retrospective.

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

The document does not explicitly state the number or qualifications of experts used to establish ground truth.

For ground truth established by bi-directional sequencing, it's a laboratory method that typically involves trained molecular biologists or laboratory technicians. The results are considered highly accurate.

4. Adjudication Method for the Test Set

The document does not describe an adjudication method for the test sets. The ground truth was primarily established by bi-directional sequencing, which is considered a definitive method, minimizing the need for adjudication in the traditional sense (e.g., of discrepant expert reads).

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

This section is not applicable. The Invader® MTHFR 1298 is an in vitro diagnostic molecular test for genotyping a specific gene mutation. It is not an imaging or interpretive AI device that involves human "readers" or assesses "AI assistance." The device outputs a direct genotype call (WT, HET, MUT).

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

Yes, the device's performance studies were conducted in a standalone manner. The Invader® MTHFR 1298 test is an automated system where the Invader Call Reporter™ Software processes raw fluorescence data to determine genotypes. Human input is in sample preparation and loading, and reviewing the generated report, but the genotyping call itself is algorithm-driven without real-time human interpretation embedded in the performance measurement. The various studies (reproducibility, sensitivity, specificity, method comparison) directly evaluate this standalone algorithmic performance against a known ground truth (sequencing).

7. The Type of Ground Truth Used

The primary ground truth used for all performance studies was bi-directional DNA sequencing. This is considered the gold standard for confirming specific genetic variations. For some studies, expected genotypes based on controls or known cell lines were also used, which would have initially been characterized by sequencing.

8. The Sample Size for the Training Set

The document primarily describes validation studies and does not explicitly mention a "training set" in the context of device development. For IVD devices, a training set generally refers to the data used to initially develop and optimize the assay parameters and interpretation algorithms. Such details are typically proprietary and not extensively disclosed in 510(k) summaries, which focus on performance validation. The studies described are validation and verification studies using independent "test sets."

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

As no specific "training set" details are provided, the method for establishing its ground truth is also not described. However, it can be inferred that any samples used for initial assay development and algorithm optimization would also have had their MTHFR 1298 genotype established using a highly accurate method, most likely bi-directional DNA sequencing, similar to the validation studies.

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