(256 days)
The DIAsource 25 OH Vitamin D Total ELISA is intended for the quantitative measurement of 25hydroxy vitamin D2 and D3 (25 OH-D2 and 25 OH-D3) in human serum. The results are to be used in conjunction with other clinical and laboratory findings to assess the Vitamin D status of a patient.
The DIAsource 25 OH Vitamin D Total ELISA Test is an enzyme linked immunosorbent assay to detect total 250H Vitamin D (D2 and D3) present in human serum. During the first incubation at room temperature, 250H Vitamin D is dissociated from binding serum proteins to fix on binding sites of a specific monoclonal antibody. After washing, a fixed amount of 250H Vitamin D-labeled with biotin in presence of horseradish peroxidase (HRP) compete with unlabeled 250H Vitamin D2 and 250H vitamin D3 present on the binding sites of the specific monoclonal antibody. After another incubation at room temperature, the microtiterplate is washed to stop the competition reaction. A chromogenic solution (TMB) is added and then stopped with a Stop Solution after the last incubation period. The amount of substrate turnover is determined colorimetrically by measuring the absorbance which is inversely proportional to the total 250H Vitamin D concentration.
The DIAsource 25OH Vitamin D Total ELISA Test is an in vitro diagnostic device for the quantitative measurement of 25-hydroxy vitamin D2 and D3 in human serum. The device's performance was evaluated through various non-clinical tests and a clinical comparison study to demonstrate substantial equivalence to a legally marketed predicate device (ImmunoDiagnostic Systems 25-Hydroxy Vitamin D EIA, K021163).
1. Table of Acceptance Criteria and Reported Device Performance:
| Test Category | Acceptance Criteria | Reported Device Performance |
|---|---|---|
| Stability (Accelerated) | 1. OD of Calibrator 0 and Calibrator 6: +/- 50% of values obtained with component/kit kept at 4°C. |
- (OD for Calibrator 1 / OD for calibrator 0) X 100: +/- 15% of the value obtained with the component kept at 4°C.
- Values of Positive and Negative controls: within the announced range with the component/kit kept at 4°C. | Accelerated stability studies indicated that the kit and components met the predefined criteria, supporting a 24-month stability for the kit and 48 months for Incubation Buffer and Conjugate Buffer. An expiration of 18 months at 4°C is given to the kits. |
| Open Vial Stability | Reconstituted Calibrators and Controls: Acceptable compared to a new kit after 7 days at 4°C, or after one, two, and three months at -20°C. | Results indicated that reconstituted Calibrators and Controls were acceptable after 7 days at 4°C and after one, two, and three months at -20°C. Therefore, Calibrators and Controls can be used up to 7 days when stored at 4°C or up to three months when stored at -20°C. |
| Precision | CV% values typically below 15-20% for inter-assay and intra-assay precision, with values decreasing at higher concentrations (general laboratory practice). | Intra-Assay (singlicate, 3 different lots, 20 days):
Sample A (5.6 ng/ml): CV (%) 7.8
Sample B (27.4 ng/ml): CV (%) 5.5
Sample C (43.0 ng/ml): CV (%) 2.7
Sample D (81.2 ng/ml): CV (%) 2.5
Inter-Assay (singlicate, 3 different lots, 20 days):
Sample A (17.7 ng/ml): CV (%) 7.4
Sample B (26.3 ng/ml): CV (%) 4.7
Sample C (42.0 ng/ml): CV (%) 4.5
Sample D (85.4 ng/ml): CV (%) 9.4
Reproducibility (60 serum samples, 3 concentrations, duplicate, 5 days, twice a day, 3 sites, 2 technicians/site):
Sample 1 (25.5 ng/ml): Total CV 10.2%
Sample 2 (52.9 ng/ml): Total CV 9.8%
Sample 3 (124.8 ng/ml): Total CV 5.0% |
| Limit of Detection (LoD) | LoD should be sufficiently low to detect clinically relevant low concentrations of 25OH Vitamin D. | LoB: 1.69 ng/ml
LoD: 2.81 ng/ml
LoQ: 4.32 ng/ml |
| Recovery | Typically within 80-120% for spiked samples. | Added 25OH-Vit D3:
25 ng/ml: 95%
50 ng/ml: 92%
Added 25OH-Vit D2:
25 ng/ml: 105%
50 ng/ml: 95% |
| Linearity | R² value close to 1.0, and recovery within an acceptable range (e.g., 90-110%) across the measurable range. | Sample 1:
Slope: 1.015, Y-Intercept: -0.298, R²: 0.99
Recoveries: 91.7% - 103%
Sample 2:
Slope: 1.005, Y-Intercept: 0.435, R²: 0.99
Recoveries: 97.4% - 105%
Linear range: 7.7 ng/ml to 122.9 ng/ml. |
| Time Delay | Assay results remain accurate, indicating minimal impact of time delay on dispensing. | Results showed minimal change in concentration for samples tested at 0, 10, and 20 minutes delay, indicating accuracy is maintained. |
| Analytical Specificity
(Cross-Reactivity) | % Cross Reaction should be low for structurally similar but distinct compounds. | 1,25(OH)2-Vitamin D3: 20.3%
1,25(OH)2-Vitamin D2: 1.9%
Vitamin D3: 2.9%
Vitamin D2: 1.3%
24,25(OH)2-Vitamin D3: >100%
25,26(OH)2-Vitamin D3: >100%
3-epi-25 hydroxy Vitamin D3: 0.07%
25 OH Vitamin D3 (reference): 100%
25 OH Vitamin D2: 83% |
| Analytical Specificity
(Interference) | Interference should be less than 10%. | Hemoglobin: -0.5% (at 250 mg/dL and 500 mg/dL)
Bilirubin Conjugated: -3.5% (at 50 mg/dL and 100 mg/dL)
Bilirubin Unconjugated: 2.5% (at 50 mg/dL and 100 mg/dL)
Triglyceride: -4.3% (at 7.5, 125, 250 mg/dL)
Vitamin C: 4.5% (at 7.5, 125, 500 mg/dL)
Biotin: 4.6% (at 1, 250, 500 mg/dL)
Zemplar: -4.3% (at 10, 100, 1000 ng/mL)
All tested substances resulted in interference less than 10%. |
| Method Comparison | Correlation coefficient (R-squared) should be high (e.g., >0.90) between the new device and the predicate device. Slope should be close to 1.0, and y-intercept close to 0. | Correlation coefficient (R) = 0.917 (95% CI: 87.6% - 93.6%)
Slope = 0.954
Y-intercept = 3.05
Range of samples: 8.0 ng/ml to 123.0 ng/ml. |
| Reference Range | Establish a representative reference range for the intended population. | Based on 150 healthy individuals from different US regions, collected in winter, not taking supplements, and without relevant medical conditions:
Highest Conc. (ng/mL): 88.6
Lowest Conc. (ng/ml): 4.9
Median Conc. (ng/ml): 17.2
Used Central 95% (2.5% - 97.5%) of the results. |
2. Sample size used for the test set and the data provenance:
- Precision: 4 serum samples for intra-assay and inter-assay (n varies, but typically 10-42 replicates); 60 serum samples (each at 3 concentrations) for reproducibility.
- Limit of Detection: Not explicitly stated as a separate test set, but calculations involved measuring blanks several times and testing 5 low-value samples 10 times.
- Recovery: Not explicitly stated as a dedicated test set, but involved adding different levels of 25OH Vitamin D to samples.
- Linearity: 2 samples (with concentrations known to be distributed throughout the measurable range).
- Time Delay: Not explicitly stated if a specific test set of samples was used; likely involved testing a few representative samples.
- Analytical Specificity (Cross-Reactivity): Sera with spiked and unspiked cross-reactants. Number of samples not specified.
- Analytical Specificity (Interference): Serum samples with different 25OH Vitamin D concentrations spiked with various interfering substances. Number of samples not specified for each interferent, but likely a small set per substance.
- Method Comparison: 356 samples.
- Reference Range: 150 apparently healthy individuals.
Data Provenance:
- Method Comparison: Implies human serum samples (not specified if retrospective or prospective or country of origin, but compared against a commercially available kit).
- Reference Range: Human serum samples obtained from a certified commercial source (Dx Biosamples, San Diego, CA.), collected from an FDA Licensed Donor Center with informed consent. Samples were from Northern US (Pennsylvania), Central US (Tennessee), and Southern US (Florida). Collected in the Winter season (January, February, March). This is prospective in nature as specific criteria for collection were followed.
3. Number of experts used to establish the ground truth for the test set and the qualifications of those experts:
For an ELISA test system like this, expert consensus or qualified experts are generally not used to establish the "ground truth" for quantitative measurements in the same way they would be for image interpretation. Instead, the ground truth for calibrators and controls is established through reference methods and rigorous validation.
- Calibrators Value Assignment: "The master calibrator stock is an ethanolic solution prepared in house by weighing 25OH Vitamin D3. The value assignment of the stock is made by UV absorbance at 254 nm using a molar extinction coefficient of 18000 L mol" cm". The stock solution of 25OH Vitamin D is then used to make calibrators by diluting into horse serum. Calibrator values have been determined using native serum samples that have been assayed by LC/MS-MS. The LC/MS-MS method has been validated against a reference method." The LC/MS-MS method acts as the "ground truth" reference here, but no specific number or qualifications of "experts" are mentioned for this process.
- Controls Value Assignment: "The mean values of 30 replicates obtained from 10 independent runs were assigned as the control target values." This is a statistical assignment, not expert consensus.
4. Adjudication method for the test set:
Not applicable in the context of a quantitative ELISA test. Adjudication, like 2+1 or 3+1, is typically used for qualitative or interpretation-based assessments (e.g., in medical image analysis) where inter-reader variability needs to be resolved to establish ground truth. For this device, values are measured quantitatively and compared against established methods or statistical criteria.
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 is a standalone diagnostic test (ELISA kit) for measuring a biomarker, not a device designed to assist human readers (e.g., radiologists) in interpretation. Therefore, no MRMC study or assessment of human reader improvement with/without AI assistance was performed.
6. If a standalone (i.e. algorithm only without human-in-the-loop performance) was done:
Yes, the entire submission describes the standalone performance of the DIAsource 25OH Vitamin D Total ELISA Test. This device is an analytical instrument and reagent system that provides quantitative results directly, without human interpretive input or human-in-the-loop performance in the clinical decision-making process for its output. The system itself generates the result.
7. The type of ground truth used (expert consensus, pathology, outcomes data, etc.):
The ground truth for the analytical performance of the device (calibration, linearity, accuracy/recovery, etc.) is primarily established through:
- Reference Methods: Specifically, LC/MS-MS for calibrator value assignment, which was itself "validated against a reference method."
- Statistical Derivation: For control value assignment.
- Spiked Samples: For recovery and linearity.
- Pharmacological Standards: For cross-reactivity.
- Comparison to a Predicate Device: For clinical correlation, where the predicate device's established performance serves as a comparative "truth."
8. The sample size for the training set:
For an ELISA assay like this, there isn't a traditional "training set" in the context of machine learning. The assay's performance characteristics (calibration curve, reagent formulations, incubation times, etc.) are developed and optimized through iterative experimental work much like an engineering process rather than a discrete "training phase" on a dataset. The stability, precision, linearity, and other studies are part of the validation and verification of the developed assay.
- However, if we consider any data used to develop the assay's parameters:
- Calibrators and Controls: Developed using in-house weighed standards and dilutions in horse serum, with values traced to UV absorbance and validated against LC/MS-MS results from native human serum. This development involves experimental data, but not a "training set" in the machine learning sense. Statistical data (e.g., 30 replicates from 10 independent runs for controls) are used to assign values, but these are part of the validation of the developed system, not a training set to build an algorithm.
9. How the ground truth for the training set was established:
As noted above, there isn't a distinct "training set" for an ELISA in the machine learning paradigm. The "ground truth" for the developmental stages of the assay (e.g., optimizing reagent concentrations, incubation times, measuring ranges) would be established through:
- Chemical/Biological Principles: Adherence to known antigen-antibody reaction kinetics and colorimetric detection.
- Gravimetric/Spectrophotometric Standards: For preparing primary calibrator stocks (e.g., weighing 25OH Vitamin D3 and measuring UV absorbance).
- Higher Order Reference Methods: The use of LC/MS-MS as a reference method to validate the calibrator values.
- Empirical Optimization: Iterative laboratory experiments to determine optimal conditions for sensitivity, specificity, and performance across the measuring range.
§ 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.