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The ARK Methotrexate II Assay is a homogeneous enzyme immunoassay intended for the quantitative determination of methotrexate in human serum or plasma on automated clinical chemistry analyzers. The measurements obtained are used in monitoring levels of methotrexate to help ensure appropriate therapy. Specimens from patients who have received glucarpidase (carboxypeptidase G2) as a high dose methotrexate rescue therapy should not be tested with the ARK Methotrexate II Assay.

The ARK Methotrexate II Assay is a homogeneous immunoassay based on competition between drug in the specimen and methotrexate labeled with the recombinant enzyme glucose-6phosphate dehydrogenase (rG6PDH) for binding to the antibody reagent. As the latter binds antibody, enzyme activity decreases. In the presence of drug from the specimen, enzyme activity increases and is directly related to the drug concentration. Active enzyme converts the coenzyme nicotinamide adenine dinucleotide (NAD) to NADH that is measured spectrophotometrically as a rate of change in absorbance. Endogenous serum G6PDH does not interfere with the results because the coenyzme NAD functions only with the bacterial enzyme (rG6PDH) used in the assay. The ARK Methotrexate II Assay consists of reagents R1 anti-methotrexate monoclonal antibody with substrate and R2 methotrexate labeled with recombinant G6PDH enzyme. The test system includes the ARK Methotrexate II Calibrator, ARK Methotrexate II Control, and ARK Methotrexate II Dilution Buffer.

The ARK Methotrexate II Assay is a homogeneous enzyme immunoassay for the quantitative determination of methotrexate in human serum or plasma.

Here's an analysis of the acceptance criteria and the study proving the device meets them:

1. Table of Acceptance Criteria and Reported Device Performance:

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

• Clinical Accuracy (vs. LC-MS/MS): 90 patient samples.
• Method Comparison (vs. Predicate Device): 123 patient samples.
• LoQ, Recovery, Linearity, Precision, Interference, Analytical Specificity: These studies involved spiked human serum samples, pooled human serum, and controls, rather than a specific number of unique patient samples for the test set. For LoQ, 40 replicates were tested. For Recovery, 6 replicates per sample were tested. For Linearity, 6 replicates per sample were tested. For Precision, 160 replicates were tested for each of the 6 control levels and 6 human serum levels. For Interference and Analytical Specificity, 6 replicates per sample were tested.
• Data Provenance: "Leftover specimens were obtained from persons receiving high-dose methotrexate therapy" for the method comparison studies. The document does not specify the country of origin of the data or whether the studies were retrospective or prospective, though "leftover specimens" suggests a retrospective collection.

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

• The ground truth for the clinical accuracy study was established by an LC-MS/MS reference method. This is an analytical laboratory technique, not based on expert consensus. Therefore, no "experts" in the traditional sense (e.g., radiologists) were used for ground truth establishment. The performance of LC-MS/MS as a reference method typically implies its results are considered the gold standard.

4. Adjudication Method for the Test Set:

• Not applicable. The ground truth for the clinical accuracy and method comparison studies was established using objective analytical methods (LC-MS/MS and the predicate device), not through expert review requiring an adjudication method.

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 (IVD) assay for quantitative determination of a substance in human samples. It does not involve human readers interpreting medical images or data.

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

• Yes, the performance data presented (Limit of Quantitation, Measurement Range, Recovery, Linearity, Clinical Accuracy, Method Comparison, Precision, Interference, Analytical Specificity) reflects the standalone performance of the ARK Methotrexate II Assay (an automated immunoassay) without human intervention in the result generation. The device itself is designed to provide quantitative measurements.

7. The Type of Ground Truth Used:

• Clinical Accuracy: Liquid Chromatography with Tandem Mass Spectrometry (LC-MS/MS) was used as the reference method (gold standard) for ground truth.
• Method Comparison: The legally marketed predicate device, ARK™ Methotrexate Assay (K111904), was used to compare results.
• Other performance metrics (LoQ, Recovery, Linearity, Precision, Interference, Analytical Specificity): Ground truth was established by preparing samples with known concentrations of methotrexate or interfering substances, typically by spiking human serum with certified reference materials.

8. The Sample Size for the Training Set:

• Not applicable. This device is an immunoassay (laboratory test kit), not a machine learning or AI algorithm that requires a "training set" in the conventional sense of computational models. The development and optimization of the assay would involve various experiments and reagent formulations, but not a distinct "training set" like for AI models.

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

• Not applicable, as there is no "training set" in the context of an AI/ML algorithm for this type of medical device. The "ground truth" for the development of the assay components would involve standard analytical chemistry practices, such as using reference materials and established analytical methods to verify the concentration and purity of substances used in the reagents.

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