ISOLAB'S T4 TEST KIT by ISOLAB, INC.

The Isolab's T4 Test Kit is a sequential, competitive enzyme immunossay with fluorometric detection in which T4 is eluted from dried blood disks and simultaneously forms a complex with the biotin-labeled monoclonal antibody which binds to the streptavidin coated solid phase. After sufficient incubation with biotinylated antibody, a conjugate of T4 with horseradish peroxidase is added to the wells and incubated with the reaction mixture. This conjugate competes with the analyte for the free on the bound monoclonal antibody. Excess of unbound reagent is washed away after a sequential incubation with biotinylated antibody and conjugate, and the enzymatic reaction with a fluorogenic substrate 3-phydroxyphenylpropionic acid (HPPA) [9] takes place. The reaction is stopped by addition of glycine buffer and the fluorescence in each well is measured with Fluoroscan II Neonate at 405 nm (excitation being 320 nm).

AI/ML Overview

Here's an analysis of the provided text regarding the Isolab T4 Test Kit's acceptance criteria and studies:

Device: Isolab T4 Test Kit for determining total thyroxine (T4) in blood specimens dried on filter paper.

Intended Use: Screening for congenital hypothyroidism (CH) in neonates. The results are considered accurate for screening purposes, but due to hematocrit value differences, they should not be assumed as accurate as serum-based assays.

Acceptance Criteria and Reported Device Performance

Acceptance Criteria Category	Specific Criteria/Metric	Target/Expected Value (Implicit)	Reported Device Performance
Linearity	Range of linearity	Not explicitly stated, but generally as wide as physiologically relevant.	0 to 25 ug/dL of T4
Recovery	% Recovery	Preferably close to 100%	88% at low T4 levels (6.25 µg/dL spiked), 97% at normal T4 levels (12.5 µg/dL spiked)
Sensitivity (Detection Limit)	Lower limit of detection	As low as clinically relevant, ideally below the CH cutoff.	1.0 ug/dL
Reproducibility (Precision)	Total SD / Total CV	Lower values indicate better precision.	T4-depleted neonate (6.25 µg/dL spiked): - Mean: 5.5 µg/dL - Total SD: 0.57 - Total CV: 10.4% - Within-run SD: 0.43 - Within-run CV: 7.8% Normal neonate (12.5 µg/dL spiked): - Mean: 12.1 µg/dL - Total SD: 1.89 - Total CV: 15.6% - Within-run SD: 1.52 - Within-run CV: 12.6%
Method Comparison (Cutoff Values)	Agreement with established methods for cutoff.	Cutoff value generally accepted for screening. For CH, typically based on lower 10%.	Isolab: 8.5 µg/dL (State 1), 8.1 µg/dL (State 2) Neometrics: 11.7 µg/dL (State 2) Wallac Oy: 10.2 µg/dL (State 1)
Interference	Minimal interference from common substances or therapeutic agents.	Interference should not significantly impact results.	D-T4: ~100% increase (expected). DL-T, TA3: Slight interference. 5-propyl-2-thiouracil (PTU): Slight interference at tested concentration (likely higher than neonatal therapeutic levels).

Study Details

Sample Sizes Used for Test Set and Data Provenance:
- Linearity: Unspecified number of whole blood samples, but each spiked concentration (6.25, 12.5, 25 ug/dL, plus unspiked) was run five times. This suggests a small sample size per concentration. Data provenance not specified (country, retrospective/prospective).
- Recovery: Not a separate sample set; derived from the same data as Reproducibility.
- Sensitivity (Detection Limit): Sample size for determining detection limit is not specified.
- Reproducibility: Two sample pools (T4-depleted neonate and normal neonate) were used. Each pool was run in duplicate, twice a day, for 20 days. This means 80 measurements per pool (2 assays/day * 20 days * 2 duplicates). Data provenance not specified.
- Comparison of Methods: Not explicitly stated how many samples were tested for the comparison. It mentions "the lower 10% of the values for each of the assays are shown," implying a larger dataset from which these cutoffs were derived. The comparative assays (Neometrics and Wallac Oy) were "performed at two state laboratory facilities," suggesting the data comes from these facilities (country not specified). It is likely retrospective data as the comparison is against existing methods.
- Interference Testing: A control sample was assayed seven times. Each spiked substance sample was also assayed seven times. Data provenance not specified.
Number of Experts Used to Establish Ground Truth for the Test Set and Qualifications of Experts:
- No information is provided about the use of experts to establish ground truth for the test set in the traditional sense (e.g., for diagnostic accuracy).
- For the "Comparison of Methods," the cut-off values for the comparative assays were "performed at two state laboratory facilities." These facilities would presumably employ qualified laboratory personnel, but their specific qualifications are not detailed.
- The "ground truth" for linearity, recovery, and reproducibility studies is based on the known spiked concentrations and repeated measurements, not expert consensus on diagnostic outcomes.
Adjudication Method for the Test Set:
- Not applicable/Not described. The studies focus on analytical performance (linearity, precision, recovery, interference) and comparison of analytical cutoffs, not diagnostic adjudication.
Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study:
- No. This type of study (comparing human readers with and without AI assistance affecting diagnostic performance) is not applicable to an immunoassay like the T4 Test Kit. The device is a standalone diagnostic test, not an AI-assisted interpretation tool.
Standalone (Algorithm Only) Performance:
- Yes, all the described studies (linearity, recovery, sensitivity, reproducibility, interference, and method comparison) represent the standalone performance of the Isolab T4 Test Kit as an analytical device. There is no human-in-the-loop component for these evaluations.
Type of Ground Truth Used:
- Known Spiked Concentrations: For Linearity, Recovery, and Reproducibility, the ground truth is established by adding known amounts of T4 to samples.
- Analytical Measurement: For Sensitivity (detection limit), the ground truth is the reliably detectable lowest concentration.
- Reference Method/Established Cutoffs: For the Comparison of Methods, the "ground truth" for comparison involves the established performance and cutoff values from Neometrics Accuscreen T4 and Wallac Oy Delphia Neonatal T4 assays, as interpreted by state laboratory facilities.
- Known Interferent Presence: For Interference testing, the ground truth is the presence of known interfering substances.
Sample Size for the Training Set:
- Not applicable/Not explicitly mentioned. The Isolab T4 Test Kit is a chemical immunoassay, not a machine learning or AI algorithm that requires a "training set" in the computational sense. The development of the assay itself would have involved extensive R&D and optimization, but this isn't typically referred to as a "training set" for the final product in regulatory submissions.
How the Ground Truth for the Training Set Was Established:
- Not applicable, as there's no "training set" for this type of device in the context of AI/ML. The "ground truth" in the development of such assays is based on established biochemical principles, purified standards, and clinical validation against reference methods to ensure the assay accurately measures T4.

Summary

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K951014

ISOLAB INC.

510(K)

T4 TEST KIT EXHIBIT C

Summary of Safety and Effectiveness

OCT 18 1996

Isolab's T4 Test Kit is intended for use in determining the total thyroxine (T4) in blood specimens dried on filter paper.

Isolab has two kit sizes available for the T4 Test Kit. Test Kit NT-1000 contains enough reagents and Microstrips to run 800 patient samples. Additonal wells are available for Calibrators and controls. Test Kit NT-4000 contains enough reagents and coated Microstrips to run 4000 patient samples. Additonal wells are available for Calibrators and Controls.

Code	Code	Description
NT-1000	NT-4000	Item
10 plates	50 plates	Streptavidin coated Microstrips®
2 x 90 ml	10 x 90 ml	Biotinylated Antibody (Bottle 1)
2 X 30 ml	10 X 30 ml	T4-HRP Conjugate (Bottle 2)
210 ml	5 X 210 ml	HPPA Substrate(Bottle 3)
45 ml	5 X 45 ml	H2O2 Solution(Bottle 4)
200 ml	2 X 500 ml	Stopping Solution(Bottle 5)
220 ml	2 X 500 ml	Wash Solution(Bottle 6)
1 card	5 cards	T4 Calibrators
10	50	Microstrip Covers(Plastic sheets to cover theMicrostrips® during incubation.)

The T4 values given for the Controls, Calibrators and thus patient results are given in ug/dL. This unit of measure reflects the estimated amount that would be found in serum. These values should be considered accurate for the purposes of screening. Due to differences in hematocrit values, these values should not be assumed as accurate as those from serum-based assays. The discrimination between normal and presumptive positives for CH is based on a predetermined fixed cut-off value generally regarded to be based on the lower 10%. The cut-off value that represents the lower 10% for this assay is 8.5 µe/d! Anv result at this value or below should be confirmed by another diagnestic test or procedure to confirm that the patient is positive for congenital hypothyrcidism.

Linearity

Results of the linearity study show that the assay is linear from concentration of 0 to 25 ug/dL of T4. Concentrations of T4 in the amounts of 6.25, 12.5, and 25 ug/dL were added to a whole blood sample and spotted onto filter Schleicher & Schuell No. 903 paper. Each spiked sample along with the unspiked sample was run five times.

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Recovery

The recovery was determined from the same data as shown under Reproducibility. The recovery can be calculated by the following formula:

mean
              X 100
                   = % recovery
amount added

వ్.వ X 100 = 88% 6.25 12.1 X 100 = 97% 12.5 The recovery is shown at the low T4 levels to be around 88% and at normal levels around 97%.

Sensitivity

The detection limit for the T4 assay 1.0 ug/dL.

Reproducibility

Two sample pools were run in duplicate twice a day for 20 days. The summary of the results are shown:

	amountadded	mean	totalSD	totalCV	within-run SD	within-run CV
T4depletedneonate	6.25µg/dL	5.5µg/dL	0.57	10.4%	0.43	7.8%
normalneonate	12.5µg/dL	12.1µg/dL	1.89	15.6%	1.52	12.6%

Comparison of Methods

Isolab's T4 Test Kit was compared to Neometric's Accuscreen T4 and Wallac Oy's Delphia Neonatal T4. The values for the Neometrics and Wallac Oy assays were performed at two state laboratory facilities. the lower 10% of the values for each of the assays are shown.

Test	State 1 Cutoff(µg/dL)	State 2 Cutoff(µg/dL)
Isolab	8.5	8.1
Neometrics	-	11.7
Wallac Oy	10.2	-

The distribution of values as well as the mean values, standard deviations and median values for the Isolab methods and the two comparative rest kit methods are shown on the next page.

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Image /page/2/Figure/0 description: The image contains two bar charts that compare the distribution of T4 values. The chart on the left compares Isolab and Wallack, while the chart on the right compares T4 Neometrics and Isolab distribution. Both charts show the frequency of T4 values in different ranges, with the x-axis representing T4 levels in ug/dL and the y-axis representing frequency. The charts also include tables with statistical data such as mean, standard deviation, and median for each group.

Interference testing was designed and executed using NCCLS document EP-7 as guidance [14]. The following substances were added to a control sample and tested as possible interferents to the T4 Test Kit. The control sample was assayed seven times and each of the whole spiked with the following substances were also assayed seven times. The two thyroxines (D-T4) added about 100% to the calculated values as expected. Two other thyroxine anologs (DL-T and TA3) also produced a slight interference. The drug, 5-propyl-2-thiouracil (PTU) also caused a slight interference at the concentration tested. This level would probably be higher than in a neonate on this drug.

Regulation Number and Section

§ 862.1700 Total thyroxine test system.

(a)
Identification. A total thyroxine test system is a device intended to measure total (free and protein bound) thyroxine (thyroid hormone) in serum and plasma. Measurements obtained by this device are used in the diagnosis and treatment of thyroid diseases.(b)
Classification. Class II (special controls). The device is exempt from the premarket notification procedures in subpart E of part 807 of this chapter subject to the limitations in § 862.9.