(159 days)
Binding assay for the in vitro quantitative determination of total 25-hydroxyvitamin D in human serum and plasma. This assay is to be used as an aid in the assessment of vitamin D sufficiency in adults.
The electrochemiluminescence binding assay is intended for use on cobas e immunoassay analyzers.
Elecsys Vitamin D total III is a binding assay for the in vitro quantitative determination of total 25-hydroxyvitamin D in human serum and plasma. The assay is to be used as an aid in the assessment of vitamin D sufficiency in adults. The assay is intended for use on the cobas e immunassay analyzers. The cobas e family of analyzers employ the electrochemiluminescence "ECLIA" technology.
Elecsys Vitamin D total III utilizes a competition test principle and has a total test duration of 27 minutes:
- 1st incubation: By incubating the sample (15 µL ) with pretreatment reagent 1 and 2, bound 25-hydroxyvitamin D is released from the vitamin D binding protein (VDBP).
- 2nd incubation: By incubating the pretreated sample with the ruthenium labeled VDBP, a complex between the 25-hydroxyvitamin D and the ruthenylated VDBP is formed. A specific unlabeled antibody binds to 24,25-dihydroxyvitamin D present in the sample and inhibits cross-reactivity to this vitamin D metabolite.
- 3rd incubation: After addition of streptavidin-coated microparticles and 25-hydroxyvitamin D labeled with biotin, unbound ruthenylated labeled VDBP become occupied. A complex consisting of the ruthenylated VDBP and the biotinylated 25-hydroxyvitamin D is formed and becomes bound to the solid phase via interaction of biotin and streptavidin.
- The reaction mixture is aspirated into the measuring cell where the microparticles are magnetically captured onto the surface of the electrode. Unbound substances are then removed with ProCell/ProCell M. Application of a voltage to the electrode then induces chemiluminescent emission which is measured by a photomultiplier.
Results are determined via a calibration curve which is instrument-specifically generated by a 2-point calibration and a master curve provided via the reagent barcode or e-barcode.
The reagent working solutions include the reagent rackpack (M, R1, R2) and the pretreatment reagents (PT1, PT2);
PT1 Pretreatment reagent 1 (white cap), 1 bottle, 4 mL: Dithiothreitol 1 g/L, pH 5.5
PT2 Pretreatment reagent 2 (gray cap), 1 bottle, 4 mL: Sodium hydroxide 57.5 g/L
M Streptavidin-coated microparticles (transparent cap), 1 bottle, 6.5 mL: Streptavidin-coated microparticles 0.72 mg/ml; preservative
R1 Vitamin D binding protein-Ru/(bpy) (gray cap), 1 bottle, 9 mL: Ruthenium labeled vitamin D binding protein 150 µg/L; bis-tris propane buffer 200 mmol/L; albumin (human) 25 g/L; pH 7.5; preservative
R2 25-hydroxyvitamin Dbiotin (black cap), 1 bottle, 8 5 mL: Biotinylated 25-hydroxyvitamin D 20 µg/L; bis-tris propane buffer 200 mmol/L; pH 8.6; preservative
This document describes the Elecsys Vitamin D total III assay, a binding assay for the in vitro quantitative determination of total 25-hydroxyvitamin D in human serum and plasma. It is intended to aid in the assessment of vitamin D sufficiency in adults and is designed for use on cobas e immunoassay analyzers.
1. Acceptance Criteria and Reported Device Performance
The provided text details several performance characteristics and the testing conducted to meet acceptance criteria. While explicit "acceptance criteria" values are not listed in a separate table for all tests, the document states that "All predefined acceptance criteria was met" for various studies. The reported performance for each study is summarized below:
| Study/Performance Characteristic | Acceptance Criteria (Implied by Met/Not Met) | Reported Device Performance |
|---|---|---|
| Precision | Predefined acceptance criteria for repeatability and intermediate precision | All predefined acceptance criteria met. |
| Lot-to-Lot Reproducibility | Predefined acceptance criteria | All predefined acceptance criteria met using three reagent lots. |
| Limit of Blank (LoB) | Not explicitly stated; based on CLSI EP17-A2 guidelines. | LoB claim: 2.0 ng/mL |
| Limit of Detection (LoD) | Not explicitly stated; based on CLSI EP17-A2 guidelines. | LoD claim: 3.0 ng/mL |
| Limit of Quantitation (LoQ) | Not explicitly stated; based on CLSI EP17-A2 guidelines. | LoQ claim: 6.0 ng/mL |
| Linearity | Confirmation within a specified range | Confirmed in the range of 2.04 - 129 ng/mL. Measuring range claim: 6.00 - 120 ng/mL. |
| High-Dose Hook Effect (HDHE) | No hook effect up to a certain concentration | No hook effect seen up to 10,000 ng/mL for both samples tested. |
| HAMA Interference | No HAMA interference observed | No HAMA interference observed. |
| Endogenous Interference | Predefined acceptance criteria | All predefined acceptance criteria met for 10 endogenous substances. |
| Cross-Reactivity | Evaluation of percent cross-reactivity with vitamin D metabolites. | - 25-hydroxyvitamin D3 (50 ng/mL): 100% |
- 25-hydroxyvitamin D2 (50 ng/mL): 103.3%
- 24,25-dihydroxyvitamin D3 (100 ng/mL): 8.1%
- 3-epi-25-hydroxyvitamin D3 (50 ng/mL): 121.6%
- 3-epi-25-hydroxyvitamin D2 (50 ng/mL): 102.7%
- 1,25-dihydroxyvitamin D3 (100 ng/mL): not determined
- 1,25-dihydroxyvitamin D2 (100 ng/mL): 0.9%
- Vitamin D3 (1000 ng/mL): 0.9%
- Vitamin D2 (1000 ng/mL): 0.7% |
| Exogenous Interference | Predefined acceptance criteria | All predefined acceptance criteria met for 20 pharmaceutical compounds; no interference observed. |
| Method Comparison (LC-MS/MS) | Not explicitly stated; assessed by Deming and Passing Bablok regression. | Deming: y = 0.981x + 0.795, r = 0.982
Passing Bablok: y = 0.979x + 0.675, T = 0.908 |
| Method Comparison (Predicate Device) | Not explicitly stated; assessed by Passing Bablok regression. | Passing Bablok: y = 0.896x + 2.63, T = 0.913 |
| Anticoagulant Effects | Predefined acceptance criteria for various sample types. | All predefined acceptance criteria met. Serum, SST, Li-Heparin, K2-EDTA, K3-EDTA plasma primary tubes are acceptable. |
| Plasma Separation Tubes (PSTs) Effects | Predefined acceptance criteria for PSTs from various manufacturers. | All predefined acceptance criteria met. PSTs are an acceptable sample type. |
| Reagent Stability (After First Opening) | Up to a specified duration | Up to 8 weeks (56 days) when stored at 2-8°C. |
| On-board Reagent Stability | Up to a specified duration | Up to 28 days (4 weeks) with a recommended new calibration every 7 days. |
| Lot Calibration Frequency | Up to a specified duration | Recommended every 12 weeks (3 months). |
| On-board Calibration Frequency | Up to a specified duration | Up to 7 days without a new calibration. |
| Reference Range Study | Determination of a 95% reference range | Calculated 95% reference range: 10.2 – 49.4 ng/mL (25.4 – 123 nmol/L). |
2. Sample Sizes Used for the Test Set and Data Provenance
- Precision Measurements: Sample size not specified, but conducted with multiple measurements over 21 days according to CLSI guideline EP05-A3. Data provenance is implied to be internal testing by Roche Diagnostics.
- Lot-to-Lot Reproducibility: Sample size not specified, but performed using three reagent lots. Data provenance is implied to be internal testing by Roche Diagnostics.
- Limit of Blank (LoB), Limit of Detection (LoD), Limit of Quantitation (LoQ): Sample size not specified; determined according to CLSI EP17-A2. Data provenance is implied to be internal testing by Roche Diagnostics.
- Linearity: Sample size not specified, but evaluated on one cobas e 601 analyzer. Data provenance is implied to be internal testing by Roche Diagnostics.
- High-Dose Hook Effect (HDHE): Two-fold determination with two samples on one cobas e 601 analyzer. Data provenance is implied to be internal testing by Roche Diagnostics.
- HAMA Interference: Sample size not specified, assessed on one cobas e 601 analyzer. Data provenance is implied to be internal testing by Roche Diagnostics.
- Endogenous Interference: 10 endogenous substances evaluated. Sample size not specified for each substance. Data provenance is implied to be internal testing by Roche Diagnostics.
- Cross-Reactivity: Cross-reactivity with various vitamin D metabolites and related compounds. Sample size not specified for each compound. Data provenance is implied to be internal testing by Roche Diagnostics.
- Exogenous Interference: 17 commonly and 3 specially used pharmaceutical compounds evaluated. Sample size not specified for each compound. Data provenance is implied to be internal testing by Roche Diagnostics.
- Method Comparison (LC-MS/MS): 157 single donor serum samples. Data provenance: CDC Verification Samples provided by the Vitamin D Standardization and Certification Program, with assigned values by the candidate Reference Method Procedure: ID-LCMS/MS at the CDC Vitamin D Reference Laboratory. This indicates a prospective and externally validated dataset.
- Method Comparison (Predicate Device): 151 human serum samples. Data provenance is implied to be internal testing by Roche Diagnostics, comparing with their own predicate device (Elecsys Vitamin D total II).
- Anticoagulant Effects: Single-donor samples (number not specified) drawn into serum, SST, Li-Heparin, K2-EDTA plasma primary tubes. Data provenance is implied to be internal testing by Roche Diagnostics.
- Plasma Separation Tubes (PSTs) Effects: Single-donor samples (number not specified) drawn into PSTs from 3 separate manufacturers. Data provenance is implied to be internal testing by Roche Diagnostics.
- Reagent Stability: Tested on one cobas e 601 analyzer. Data provenance is implied to be internal testing by Roche Diagnostics.
- On-board Reagent Stability: Tested on one cobas e 601 analyzer. Data provenance is implied to be internal testing by Roche Diagnostics.
- Lot Calibration Frequency: Tested on one cobas e 601 analyzer. Data provenance is implied to be internal testing by Roche Diagnostics.
- On-board Calibration Frequency: Tested on one cobas e 601 analyzer. Data provenance is implied to be internal testing by Roche Diagnostics.
- Reference Range Study: A total of 827 subjects enrolled; 463 eligible subjects included in the final determination after exclusions. Data provenance: Serum samples collected from adult subjects during summer and winter months from three geographically diverse locations in the U.S. (Northern regions). One clinical laboratory was contracted to measure samples. This indicates prospective, multi-site clinical data.
Note: For internal testing studies (precision, linearity, interference, stability), the data provenance is implied to be prospective testing conducted by Roche Diagnostics in a controlled laboratory setting.
3. Number of Experts Used to Establish the Ground Truth for the Test Set and the Qualifications of Those Experts
The document does not describe the use of "experts" to establish ground truth in the traditional sense of medical image analysis or diagnostic interpretation by physicians.
For the Method Comparison (LC-MS/MS), the "ground truth" (or reference values) for the 157 samples was established by the CDC Verification Samples with assigned values by the candidate Reference Method Procedure: ID-LCMS/MS at the CDC Vitamin D Reference Laboratory. This implies a highly standardized and validated reference method for quantifying vitamin D, rather than expert clinical judgment.
For the Reference Range Study, "characterization testing" was used to determine whether samples met stated inclusion/exclusion criteria. This involved a variety of characterization assays on cobas e 601 or cobas c 501 analyzers, not human expert adjudication.
4. Adjudication Method
Not applicable, as the studies described primarily involve analytical performance testing using quantitative measurements against reference methods or predefined criteria, rather than qualitative assessments requiring human adjudication.
5. Multi Reader Multi Case (MRMC) Comparative Effectiveness Study
Not applicable. The Elecsys Vitamin D total III is an in vitro diagnostic assay, not an AI-powered image analysis or diagnostic tool that assists human readers. Therefore, an MRMC study and the concept of "human readers improve with AI vs without AI assistance" are not relevant to this device's evaluation.
6. Standalone Performance Study
Yes, a standalone study of the algorithm (in this case, the assay system's performance) was done. All the non-clinical and clinical tests described, such as precision, accuracy (method comparison), linearity, limits of detection, interference, and stability, demonstrate the standalone performance of the Elecsys Vitamin D total III assay system. The device performs the quantitative analysis without human intervention in the measurement process itself, once the sample is loaded and the assay initiated.
7. Type of Ground Truth Used
The primary ground truth used for performance evaluation, particularly for accuracy, was:
- Reference Method/Standard: For the method comparison study, the CDC Verification Samples with values assigned by the ID-LCMS/MS (Isotope Dilution Liquid Chromatography-Mass Spectrometry/Mass Spectrometry) at the CDC Vitamin D Reference Laboratory served as the reference method or "ground truth" for 25-hydroxyvitamin D concentration. This is a highly accurate and precise analytical method.
- Predicate Device Comparison: The predicate device, Elecsys Vitamin D total II, also served as a reference for comparison, indicating substantial equivalence.
- Analytical Standards/Controls: For other analytical performance tests (e.g., precision, linearity, limits), ground truth is established through the use of certified reference materials, calibrators, and specified spiked samples with known concentrations.
8. Sample Size for the Training Set
The document does not explicitly mention a "training set" in the context of machine learning or AI algorithm development. The Elecsys Vitamin D total III is a binding assay with a defined chemical and immunological principle, not a machine learning algorithm that is "trained" on a dataset in the conventional sense. Therefore, the concept of a training set size for an AI model is not directly applicable here.
However, the assay's development and optimization would have involved extensive testing and "training" by researchers and developers in a broader, non-AI sense to establish reagent formulations, reaction conditions, and calibration parameters before formal validation studies. This is not detailed in the provided regulatory document.
9. How the Ground Truth for the Training Set Was Established
As explained in point 8, the concept of a "training set" for AI is not directly applicable to this device. Therefore, the method for establishing ground truth for such a set is also not discussed. The assay's fundamental performance characteristics (e.g., binding kinetics, signal generation) are based on established biochemical principles and extensive internal research and development, which would have leveraged known concentrations of 25-hydroxyvitamin D and its metabolites in control samples and reference materials.
§ 862.1825 Vitamin D test system.
(a)
Identification. A vitamin D test system is a device intended for use in clinical laboratories for the quantitative determination of 25-hydroxyvitamin D (25-OH-D) and other hydroxylated metabolites of vitamin D in serum or plasma to be used in the assessment of vitamin D sufficiency.(b)
Classification. Class II (special controls). Vitamin D test systems must comply with the following special controls:(1) Labeling in conformance with 21 CFR 809.10 and
(2) Compliance with existing standards of the National Committee on Clinical Laboratory Standards.