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• The A/C Enzymatic Homocysteine Assay is intended for the quantitative determination of total homocysteine (tHCY) in human plasma or serum.
• The device can assist in the diagnosis and treatment of patients suspected of having hyperhomocysteinemia.
• The A/C Enzymatic Homocysteine Assay Kit is only for in vitro diagnostic use.

The A/C Enzymatic Homocysteine Assay is calibrated with A/C Enzymatic Homocysteine Assay Calibrators. The A/C Enzymatic Homocysteine Assay is assayed for the verification of the accuracy and precision on the Hitachi 912 Automatic Analyzer.

The A/C Enzymatic Homocysteine Assay measures tHCY. The principle of the assay is that recombinant homocysteinase (rHCYase) produces hydrogen sulfide (H2S) from tHCY, which is quantified by use of N,N-dibutyl phenylene diamine (DBPDA), the combination of which forms a chromophore.

The A/C Enzymatic Homocysteine Assay on the Hitachi 912 Automatic Analyzer used four reagents, a number compatible with implementation on the Hitachi 912 Automatic Analyzer. We used 30 uL of EDTA plasma in a dithiothreitol (DTT) reduction reaction (1 mmol/L DTT, 0.2% Triton X-100 in 40 mmol/L sodium phosphate buffer [pH 8.3]) for 1.5 minutes to release bound homocysteine. The rHCYase reaction (0.05 mg/ml in 40 mmol/L sodium phosphate buffer [pH 8.3] with 20 umol/L pyridoxal 5-phosphate [PLP]) is then run for 3.5 minutes. The DBPDA chromophore (12.5 mmol/L DBPDA in 1.5 N H2SO4) is then added and 5 minutes later, an oxidant, potassium ferricyanide (5 mmol/L K3Fe(CN), in 10 mmol/L sodium phosphate buffer [pH 7.6]), is added. Five minutes after addition of oxidant, the end-points are read at absorbances of 700 and 660 nm. As the assay is based on an increase in absorbance over baseline, no blank without enzyme was used. The detection limit of the assay is 1.5 umol/L defined by quantification of a serial dilution of a plasma sample of tHCY diluted to 0.77 umol/L. The limit of quantification is defined as the lowest concentration measured having a CV <20%. The linear range extends to at least 80 umol tHCY/L as determined by measuring varying amounts of homocysteine in phosphate buffered saline.

The within-run imprecisions (CV) run over 8 repeats were 4.8% at 8.9 umol/L tHCY; 3.0% for 14.9 umol/L tHCY; and 4.5% for 25 umol/L tHCY. Between-assay imprecisions (CV) over 10 days were 7.8% for 8.8 umol/L tHCY: 5.9% for 15 umol/L tHCY; and 4.9% for 25 umol/L tHCY. These imprecisions are with in ranges reported for currently-used assays including the Bio-Rad HCY assay on HPLC, which is used as a comparison method in the present study.

The results showed that L-cysteine (L-CYS) in the physiological concentrations (0-200 µmol/L) had less than 10% interference and L-methionine (L-MET) in the physiological concentrations (0-200 umol/L) had no interference in the A/C Enzymatic Homocysteine Assay on the Hitachi 912 Automatic Analyzer.

The A/C Enzymatic Homocysteine Assay on the Hitachi 912 Automatic Analyzer is a four-step reaction. The total assay takes 15 minutes, and the through-put is 360 tests per hour.

Here's an analysis of the provided text regarding the A/C Enzymatic Homocysteine Assay, focusing on acceptance criteria and the supporting study:

1. Table of Acceptance Criteria and Reported Device Performance

The document doesn't explicitly state formal "acceptance criteria" in a clear, quantified manner for regulatory approval. Instead, it describes performance characteristics and compares them to a predicate device. The performance is deemed acceptable because it is within ranges reported for currently-used assays including the predicate.

Here's a table summarizing the performance characteristics presented, with an interpretation of implied acceptance based on the comparison to existing assays:

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

• Sample Size for Test Set: 121 plasma samples.
• Data Provenance: Not explicitly stated. The document refers to "plasma samples," but does not specify their country of origin or whether they were collected retrospectively or prospectively.

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

This device is an in vitro diagnostic (IVD) assay for quantitative determination of a biomarker (homocysteine), not an imaging or clinical decision support AI device that requires human expert review for "ground truth." Therefore, the concept of "experts establishing ground truth" as it applies to subjective assessments is not relevant here.

• The "ground truth" for the test set is established by the predicate device, the Bio-Rad Homocysteine by HPLC (k 993107). The performance of the new device is compared directly to the measurements obtained from this established method.

4. Adjudication Method for the Test Set

Not applicable. As this is a quantitative chemical assay, there's no "adjudication" in the sense of resolving conflicting interpretations by multiple human readers. The comparison is statistical between two quantitative measurement methods.

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

No. An MRMC study is relevant for medical imaging or subjective interpretation tasks where multiple human readers assess cases. This document describes the performance of a chemical assay, so an MRMC study is not applicable.

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

Yes, in the sense that the A/C Enzymatic Homocysteine Assay operates as a standalone analytical system on the Hitachi 912 Automatic Analyzer. Its performance characteristics (detection limit, linearity, imprecision, interference) and its comparison to the predicate are presented as the algorithm's direct output. There is no human interpretation component being measured against a human-augmented performance.

7. The Type of Ground Truth Used

The primary "ground truth" used for comparison and establishing equivalence in this study is the measurements obtained from a legally marketed predicate device (Bio-Rad Homocysteine by HPLC). This is a common method for IVD assays seeking 510(k) clearance by demonstrating "substantial equivalence."

8. The Sample Size for the Training Set

Not applicable. This device is a chemical assay, not a machine learning or AI algorithm in the contemporary sense that typically requires a "training set." The assay's parameters would have been developed and optimized through laboratory work and internal studies, but not in the framework of a distinct "training set" and "test set" common in AI/ML validation.

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

Not applicable, as there isn't a "training set" in the context of an AI/ML algorithm. The assay's chemical reagents and reaction conditions were presumably optimized through standard chemical and enzymatic assay development processes based on biochemical principles and laboratory experimentation.

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