(18 days)
The AquaLite® Free T Bioluminescent Immunoassay (BIA) Kit (or the AquaLite® Free T . Assay) is an in vitro diagnostic product intended for use in clinical laboratories for the quantitative determination of human free T4 in serum.
The AquaLite® FT4 Bioluminescent Immunoassay (BIA) Kit (or the AquaLite® F14 Assay) is an in vitro diagnostic product intended for use in the quantitative measurement of FT4 in human serum in clinical laboratories. Free thyroxine measurements are used to diagnosis and treat diseases of the thyroid.
The AquaLite® Free T. Assay is a single-site, competitive inhibition bioluminescent immunoassay kit. A T carrier complex is immobilized on polystyrene tubes (solid phase). Serum samples, appropriate calibrators or controls, are pipetted (50 uL) into the pre-coated tubes. A mouse monoclonal anti-T antibody covalently linked to AquaLite® (100 uL) is then added to the tubes. Free T. in the sample competes with immobilized T for the available T binding sites of the anti-T antibody conjugate. Complex formation is complete after a 90-minute incubation period at room temperature on a standard orbital shaker. The tubes are then washed to remove unbound conjugate.
The washed tubes are placed in a luminometer that is capable of reading a triggered, flash-type reaction in 12 x 75 mm tubes. Injection of the calcium trigger buffer causes AquaLite® to oxide its self-contained luciferin molecule, producing a flash of light, which is measured by the luminometer. The presence of Ta in the sample or calibrator reduces the binding of the conjugate to the immobilized T . pre-coated on the tubes. The amount of signal inhibition is indirectly proportional to the Free T concentration. To calculate results, the luminometer uses a cubic spline curve fit applied to a log-log transformation of the light intensity (in relative light units, RLU) of the Free T calibrators versus Free T, concentration (in ng/dL).
The document describes a 510(k) submission for the SeaLite Sciences, Inc. AquaLite® Free T Assay, a bioluminescent immunoassay kit for the quantitative determination of human free T4 in serum. The submission aims to demonstrate substantial equivalence to the Nichols Institute Free T4 Assay.
1. Table of Acceptance Criteria and Reported Device Performance
The acceptance criteria are not explicitly stated as pass/fail thresholds in the provided document for all tests, but rather implied by the reported performance and comparison to a legally marketed predicate device. The performance characteristics were generally deemed acceptable by the FDA through the 510(k) clearance process.
| Acceptance Criteria (Implied) | Reported Device Performance |
|---|---|
| 1. Sensitivity/Detection Limit | 0.007 ng/dL (calculated at 95% confidence level of twenty replicates of A Calibrator (0 ng/dL)). |
| 2. Specificity and Cross Reactivity (undetectable interference) | Triiodo-L-thyronine (20 µg/dL): undetectable cross-reactivity. |
| Triiodothyroacetic acid (20 µg/dL): undetectable cross-reactivity. | |
| Monoiodotyrosine (20 µg/dL): undetectable cross-reactivity. | |
| Diiodo-L-tyrosine (20 µg/dL): undetectable cross-reactivity. | |
| Methimazole (0.4 mg/mL): undetectable cross-reactivity. | |
| 6-n-Propyl-2-thiouracil (0.4 mg/mL): undetectable cross-reactivity. | |
| 3. Drift (no significant "end of run" effect) | Two patient samples showed consistent Free T4 levels across 20-30, 50-60, and 90-100 tubes, indicating no significant drift over 100 tubes. (Example: Sample 1: 2.8, 2.9, 2.8 ng/dL; Sample 2: 4.5, 4.5, 4.5 ng/dL). |
| 4. Precision and Reproducibility | Intra-Assay Precision (n=20 per level): |
- 0.75 ng/dL: 10.1% CV
- 2.6 ng/dL: 4.1% CV
- 4.4 ng/dL: 2.9% CV
Inter-Assay Precision (n=2x20=40 over 2 weeks): - 0.6 ng/dL: 13.0% CV
- 2.6 ng/dL: 5.0% CV
- 4.2 ng/dL: 3.4% CV |
| 5. Method Comparison (good correlation with predicate) | Linear regression analysis with a commercially available chemiluminescence immunometric assay for Free T4 yielded: - Slope: 1.1
- y-intercept: -0.2
- Correlation coefficient (r): 0.92
(for Free T4 ranging from 0.27 to 3.67 ng/dL in n=127 serum samples). |
| 6. Kinetics (optimal incubation time) | 90-minute incubation shown to be optimal based on parallel assays of three human serum samples at 60, 90, and 120 minutes. |
| 7. Recovery in Serum and Plasma (no significant differences) | No significant differences found for Free T4 recovery between standard serum, SST tubes, heparin plasma, and EDTA plasma in blood samples from 7 normal subjects. |
| 8. Effect of Hemolysis (not significantly affected) | Not significantly affected by mild, moderate, or severe hemolysis (tested with three patient samples spiked with hemoglobin preparations). |
| 9. Effect of TBG (no interference at normal levels) | No interference at normal TBG levels (15-34 mg/L). Slight inhibition of %B/Bo observed at 200 and 300 mg/L TBG (5.9 and 8.8 times normal physiologic level), potentially leading to an apparent increase in Free T concentration at very high supra-physiological levels. |
| 10. Effect of Albumin (not significantly affected) | Not significantly affected by albumin at concentrations of 15, 25, and 75 mg/mL (tested with three patient samples). |
| 11. Effect of Nonesterified Fatty Acids (not significantly affected) | Not significantly affected by oleic acid at 2.5, 5, and 10 mmol/L (tested with three patient samples). |
| 12. Effect of Bilirubin (not significantly affected) | Not significantly affected by bilirubin at 10 and 20 mg/dL (tested with three patient samples). |
| 13. Effect of Salicylate (not significantly affected) | Not significantly affected by salicylate at 1, 5, 10, and 25 mg/dL (tested with three patient samples). |
| 14. Effect of Phenytoin (potential effect at high levels) | "Not significantly affected by phenytoin at the levels tested" is stated, but the data shows increasing Free T4 values with increasing phenytoin concentrations, particularly at 10 µg/mL and 25 µg/mL (e.g., Sample 1: 1.1 (Neat) to 2.22 (25µg/mL); Sample 2: 1.5 (Neat) to 2.2 ng/mL (25µg/mL)). The accompanying text "Assay is needing to patients falling Should be interpreted with courtion" (sic: caution) suggests a recognition of potential interference at higher levels. This implies that while low levels might not significantly affect, higher levels might, requiring clinical judgment. |
| 15. Effect of Phenylbutazone (not significantly affected) | Not significantly affected by phenylbutazone at 1, 5, and 10 µg/mL (tested with three patient samples). |
2. Sample Sizes Used for the Test Set and Data Provenance
The document doesn't explicitly refer to "test sets" in the modern AI/machine learning sense, but rather "studies" for performance characterization.
- Sensitivity: 20 replicates of a 0 ng/dL calibrator.
- Drift: Two patient samples, each run in 5 duplicates, across 100 tubes (implied data from 10 groups of 10 tubes).
- Intra-assay precision: 20 replicates per concentration level for three serum controls (total 60 measurements).
- Inter-assay precision: Duplicates in 20 assays for three serum controls (total 40 measurements * 3 levels = 120 control measurements, plus each assay included a standard curve).
- Method Comparison: 127 serum samples.
- Kinetics: Three human serum samples.
- Recovery in Serum and Plasma: Blood samples from 7 normal subjects.
- Effect of Hemolysis: Three patient samples.
- Effect of TBG: TBG added to calibrator A (0 ng/dL Free T4) at various concentrations.
- Effect of Albumin: Three patient samples.
- Effect of Nonesterified Fatty Acids: Three patient samples.
- Effect of Bilirubin: Three patient samples.
- Effect of Salicylate: Three patient samples.
- Effect of Phenytoin: Two patient samples.
- Effect of Phenylbutazone: Three patient samples.
Data Provenance: The studies were conducted at SeaLite Sciences, Inc. The samples used (serum controls, patient samples, normal subjects' blood) are implied to be retrospective clinical samples or laboratory-prepared samples. The country of origin is not explicitly stated, but since SeaLite Sciences, Inc. is based in Norcross, GA, and the submission is to the US FDA, it is highly likely the data originated from the USA.
3. Number of Experts Used to Establish the Ground Truth for the Test Set and Qualifications of Those Experts
This information is not provided in the document. For an in vitro diagnostic immunoassay, "ground truth" often refers to reference methods, certified standards, or clinical diagnoses. The document compares the device's performance to established analytical methods and other commercially available assays, implying that these existing methods or values serve as the reference. There's no mention of expert consensus for establishing ground truth for the performance studies themselves, as is common for subjective image interpretation.
4. Adjudication Method for the Test Set
This is not applicable and therefore not described. Adjudication methods (like 2+1, 3+1) are typically used in studies involving subjective interpretation (e.g., by radiologists) where discrepancies need to be resolved. For an objective quantitative immunoassay, the result is a numerical value, and "adjudication" in this sense is not performed.
5. If a Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study was Done
No, an MRMC comparative effectiveness study was not done. Such studies are relevant for devices where human interpretation is involved and improved by AI assistance (e.g., AI for radiology). This device is an in-vitro diagnostic immunoassay kit, where the result is quantitative and read by a luminometer, not interpreted by a human reader in the same way.
6. If a Standalone (i.e., algorithm only without human-in-the-loop performance) was Done
Yes, this entire submission describes the standalone performance of the AquaLite® Free T Assay. As an in-vitro diagnostic assay, it is inherently a "standalone" device providing a quantitative measurement. Human involvement is in sample preparation, running the assay, and interpreting the numerical result in a clinical context, but the assay's performance characteristics (sensitivity, precision, accuracy, etc.) are evaluated intrinsically.
7. The Type of Ground Truth Used
The ground truth or reference used for assessing performance varied by study:
- Sensitivity: Defined using replicates of a 0 ng/dL calibrator.
- Specificity/Cross-reactivity: Measured against known concentrations of potentially interfering compounds.
- Drift, Precision, Kinetics, Interference Studies: Measured against the device's own internal calibration/control system, with consistency and expected behavior serving as the "ground truth."
- Method Comparison: A commercially available chemiluminescence immunometric assay kit for Free T4 was used as the comparator, which serves as a widely accepted reference method in the field for demonstrating substantial equivalence.
- Recovery in Serum and Plasma: Compared to standard serum samples.
Essentially, the ground truth relies on established laboratory practices, verified calibrators, and comparison to a legally marketed predicate device (the Nichols Institute Free T4 Assay) which itself would have been validated against accepted reference methods.
8. The Sample Size for the Training Set
This information is not provided and is not typically described in a 510(k) submission for an immunoassay. Immunoassay development relies on chemical and biological optimization, rather than a "training set" in the machine learning sense. The "standard curve" generated for each assay run is analogous to a form of internal calibration or "on-the-fly training," but this differs from a large, predefined training dataset for an algorithm.
9. How the Ground Truth for the Training Set was Established
As there is no "training set" in the machine learning context for this device, this question is not applicable. The underlying principles of the immunoassay (antibody-antigen binding, bioluminescence) are based on established scientific knowledge. The "ground truth" for the calibrators used in each assay's standard curve would have been established through a robust process of analytical validation, likely involving established reference materials and methods, but the details are not in this document.
§ 862.1695 Free thyroxine test system.
(a)
Identification. A free thyroxine test system is a device intended to measure free (not protein bound) thyroxine (thyroid hormone) in serum or plasma. Levels of free thyroxine in plasma are thought to reflect the amount of thyroxine hormone available to the cells and may therefore determine the clinical metabolic status of thyroxine. Measurements obtained by this device are used in the diagnosis and treatment of thyroid diseases.(b)
Classification. Class II.