(231 days)
EUROIMMUN's 25-OH Vitamin D ELISA is intended for the quantitative determination of 25-OH Vitamin D and other hydroxylated vitamin D metabolites in human serum and plasma (EDTA, Li-heparin). Results are to be used in conjunction with other clinical and laboratory data to assist the clinician in the assessment of vitamin D sufficiency in adult populations.
EUROIMMUN 25-OH Vitamin D ELISA consists of a microwell ELISA plate coated with (sheep) monoclonal anti-25-OH vitamin D antibodies, 6 calibrators, 2 controls, Biotin, sample buffer, conjugate. wash buffer concentrate. enzyme TMB chromogen/substrate solution and stop solution.
This ELISA test kit is designed for the in vitro determination of 25-OH vitamin D in human serum or plasma samples. In the first analysis step, the calibrators and patient samples are diluted with biotin-labelled 25-OH vitamin D and added to microplate wells coated with (sheep) monoclonal anti-25-OH vitamin D antibodies. During the incubation an unknown amount of 25-OH vitamin D in the patient sample and a known amount of biotinlabelled 25-OH vitamin D compete for the antibody binding sites in the microplate wells plate. Unbound 25-OH vitamin D is removed by washing. For the detection of bound biotin-labelled 25-OH vitamin D, a second incubation is performed using peroxidase-labelled streptavidin. In a third incubation using the peroxidase substrate tetramethylbenzidine (TMB) the bound peroxidase promotes a colour reaction. The colour intensity is inversely proportional to the 25-OH vitamin D concentration in the sample. Results for the samples can be calculated directly using a standard curve.
Antibodies: sheep monoclonal antibodies which identify specifically 25-OH vitamin D3 and 25-OH vitamin D2.
Here's a summary of the acceptance criteria and the study that proves the device meets them, based on the provided text:
1. Table of Acceptance Criteria and Reported Device Performance
| Performance Characteristic | Acceptance Criteria | Reported Device Performance |
|---|---|---|
| Reproducibility | CV's 10 ng/ml; CV's 10% deviation) seen with 1000 mg/dl of hemoglobin._ | |
| Method Comparison (vs. Predicate) | High correlation to predicate device | n: 240 samples. Concentration Range (Predicate): 4.1 - 110.4 ng/ml. Concentration Range (Candidate): 4.1 - 119.1 ng/ml. Regression Equation (y = Candidate, x = Predicate): y = 0.78 + 1.08 x. 95% C.I. of Intercept: -0.06 - 1.63. 95% C.I. of Slope: 1.06 - 1.11. Correlation Coefficient R: 0.9858. 95% C.I. of R: 0.9817 - 0.9890. |
| Matrix Comparison (Plasma vs. Serum) | Regression equation near ideal correlation (intercept 0; slope 1.0); Coefficients of determination > 0.99; %recovery compared to serum in range of 87 to 127 % | EDTA plasma (n=38): Y = 0.29 + 0.99x (95% CI Intercept: -0.37 - 1.18, 95% CI Slope: 0.93 - 1.02); R² = 0.996; Mean %recovery = 100% (Range: 87 - 116%). Li-heparin plasma (n=38): Y = 0.55 + 0.97x (95% CI Intercept: -0.65 - 1.33, 95% CI Slope: 0.93 - 1.04); R² = 0.993; Mean %recovery = 101% (Range: 87 - 127%). |
| Stability | Predicted shelf-life of at least 12 months at 2-8°C for control materials and calibrators; Open-vial stability of 3 months at 2-8°C for kit. | Predicted shelf-life of at least 12 months at 2-8°C for control materials and calibrators (based on accelerated testing at 37°C, real-time ongoing). Open-vial stability of 3 months when stored at 2-8°C. Biotin stable for at least 2 weeks when diluted to working strength. |
2. Sample Sizes Used for the Test Set and Data Provenance
- Reproducibility:
- Intra- and Inter-Assay: Minimum of 8 natural serum samples (2 spiked) for CV determination; 40 determinations for intra-assay CVs; 40 determinations performed in 10 different runs on 5 different days (with 4 replicates per run) for inter-assay CVs.
- Lot-to-Lot: Minimum of 8 natural serum samples (2 spiked); 32 determinations performed in 8 different runs on 4 different lots (with 2 runs per lot and 4 replicates per run).
- Data Provenance: "natural serum samples collected freshly in-house from obvious healthy blood donors".
- LoD: 200 determinations from 5 samples in the low range (2-10 ng/ml), measured in 5 independent runs with 8 replicates per run.
- Linearity/Assay Reportable Range: Sets of 11 sample preparations (natural negative sample + high positive samples), tested in double determinations.
- Cross-Reactivity: 25-OH Vitamin D free sample aliquoted and spiked with 7 potential cross-reacting Vitamin D metabolites.
- Interference: Sera at different 25-OH Vitamin D concentrations spiked with 6 potential interfering substances.
- Method Comparison w/Predicate Device:
- Sample Size: 240 samples in total.
- Data Provenance:
- 141 prospective samples for 25-OH-Vitamin D testing from a clinical laboratory.
- 28 samples from 25-OH-Vitamin D quality assessment programs (harvested from blood donated by venesection undergoing therapeutic for patients with hemochromatosis or polycythemia).
- 5 samples from a 25-OH-Vitamin D quality control panel (untreated routine patient material).
- 30 samples from normal blood donors.
- 36 samples (15%) were spiked with 25-OH Vitamin D3 stock solution to ensure concentration distribution.
- All patients gave informed consent. Samples are natural and not treated.
- Matrix Comparison: 38 sample pairs of serum and corresponding plasma (EDTA, Li-heparin) from donors. 3 sample pairs were spiked.
- Expected Values/Reference Range: 206 samples from healthy subjects (70 men, 136 women; average age 64 years; age range: 22–99 years) from a US commercial source (normal US blood donors from the US Midwest region, drawn early Oct 2010).
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 qualifications for establishing ground truth for the test set in terms of clinical interpretation. However, the performance studies (e.g., reproducibility, linearity) rely on in-house personnel and laboratory procedures, with calibrator and control value assignments confirmed by "2 different technicians" and "a different person for final release." Method comparison was against an "FDA-cleared reference assay," implying the predicate device serves as a reference for comparison.
4. Adjudication Method for the Test Set
No specific adjudication method (e.g., 2+1, 3+1) is mentioned or implied for the evaluation of the test set results for clinical interpretation. The performance studies describe analytical methods and statistical comparisons.
5. If a Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study was done
No, a Multi-Reader Multi-Case (MRMC) comparative effectiveness study was not done. This device is an in-vitro diagnostic (IVD) assay, not an imaging device typically evaluated with MRMC studies. The "Method Comparison" section compares the device's analytical results against a predicate device, not human readers with and without AI assistance.
6. If a Standalone (i.e., algorithm only without human-in-the-loop performance) was done
Yes, the studies described are for the standalone performance of the EUROIMMUN 25-OH Vitamin D ELISA assay, which is an algorithm-only (test kit) device. Its performance characteristics like linearity, reproducibility, limits of detection, and interference are evaluated intrinsically, and its results are then compared to a predicate device. There is no human-in-the-loop component for the device's operation, only human involvement in performing the laboratory tests (technicians) and interpreting the output for clinical use.
7. The Type of Ground Truth Used
The ground truth used varies based on the type of study:
- Reproducibility, LoB, LoD, Functional Sensitivity, Linearity, Cross-Reactivity, Interference: The ground truth is effectively the known concentrations of the analytes in the prepared samples (e.g., spiked samples, calibrators, controls, or precisely characterized samples). These are established through gravimetric methods, UV spectrophotometric analysis, and comparison with NIST and DEQAS standards, as well as predicate device measurements and HPLC.
- Method Comparison w/Predicate Device: The ground truth for comparison is the results obtained from the FDA-cleared predicate device, which is considered the reference method for this type of comparison.
- Matrix Comparison: The ground truth is the serum sample concentrations when comparing to plasma.
- Expected Values/Reference Range: The ground truth for establishing population reference intervals is the 25-OH Vitamin D levels measured in healthy subjects using the new device itself.
8. The Sample Size for the Training Set
The document does not describe a "training set" in the context of machine learning, as this is an in-vitro diagnostic ELISA kit. The term "training set" is not applicable here. The assay is based on chemical reactions and optical density measurements, not a learned algorithm in the typical sense.
9. How the Ground Truth for the Training Set was Established
As noted in #8, there isn't a "training set" in the machine learning context. The calibration and standardization of the assay (which could be considered analogous to "establishing ground truth" for the assay's internal function) involves:
- Gravimetric calibration using UV-Vis verified stock standards.
- Comparison with NIST standards and DEQAS quality assessment data.
- In-house quality control sera.
- Initial values for quality control sera assigned using the predicate device in conjunction with liquid chromatography (HPLC).
- Final calibrator values verified and assigned by adjusting initial values to meet specified ranges when tested against predicate assays.
§ 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.