The AquaLite® hGH Bioluminescent Immunoassay (BIA) Kit (or the AquaLite® hGH Assay) is an in vitro diagnostic product intended for use in clinical laboratories for the quantitative determination of human growth hormone in serum. Human Growth Hormone measurements are used in the diagnosis and treatment of disorders involving the anterior lobe of the pituitary gland. The AquaLite® Human Growth Hormone Assay is for in vitro diagnostic use.

Device Description

The AquaLite® hGH Bioluminescent Immunoassay Kit uses a mouse monoclonal anti-hGH antibody that is pre-coated onto polystyrene tubes (solid phase). Serum samples or appropriate calibrators or controls, are pipetted (150 uL) into the precoated tubes. A sheep anti-hGH antibody covalently linked to AquaLite® (100 uL) is then added to the tubes. hGH in the sample combines with the antibody on the solid phase and conjugate antibody to form an immune complex or "sandwich" bound to the solid phase. Complex formation is complete after a 120-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 intensity of the light emitted from antibody bound to the tubes is directly proportional to the concentration of the hGH in the sample. To calculate results, the light intensity (in relative light units, RLU) of the hGH calibrators is plotted against hGH concentration (in ng per mL) to yield a calibration curve. This curve is used to relate the light intensity generated from the samples and controls to hGH concentration in ng/mL.

Note: Samples that generate signals greater than the signal from the highest calibrator are off-scale. These samples must be diluted and re-assayed. Remember to multiply the results from diluted samples by the dilution factor used.

AI/ML Overview

Here's an analysis of the provided text regarding the AquaLite® Human Growth Hormone Assay, broken down by your requested categories:

1. A table of acceptance criteria and the reported device performance

The document does not explicitly state "acceptance criteria" as a set of pre-defined thresholds. Instead, it presents performance characteristics determined through studies. The implication is that these reported performance characteristics were deemed acceptable by the FDA for substantial equivalence to the predicate device.

Performance Characteristic	Reported Device Performance (AquaLite® hGH Assay)
Sensitivity	0.005 ng/mL
Specificity (Cross-reactivity)
Prolactin	0.09% (at 500 ng/mL), 0.17% (at 1,000 ng/mL)
hPL	< 0.01% (at 10,000 ng/mL), < 0.01% (at 100,000 ng/mL)
LH	< 0.01% (at 250 mIU/L), < 0.01% (at 500 mIU/L)
TSH	< 0.01% (at 250 µIU/L), < 0.01% (at 500 µIU/L)
hCG	< 0.01% (at 10,000 mIU/L), < 0.01% (at 50,000 mIU/L)
FSH	< 0.01% (at 250 mIU/L), < 0.01% (at 500 mIU/L)
High Dose Hook Effect	No high dose hook effect occurs prior to 500 ng/mL
Precision (Intra-assay)
3.33 ng/mL hGH level	4.80 % CV
6.20 ng/mL hGH level	4.97 % CV
12.30 ng/mL hGH level	6.95 % CV
Precision (Inter-assay)
2.89 ng/mL hGH level	6.4 % CV
5.19 ng/mL hGH level	9.8 % CV
9.42 ng/mL hGH level	11.8 % CV
Method Comparison (vs. commercially available chemiluminescence immunoassay)
Slope	1.2831
Y-intercept	0.146
Correlation coefficient (R)	0.9665
Linearity and Non-parallelism (Recovery)
Sample A (dilutions)	92-101%
Sample B (dilutions)	98-103%
Sample C (dilutions)	100-103%
Spike and Recovery
Sample 1 (mixtures)	92-103%
Sample 2 (mixtures)	99-110%

2. Sample size used for the test set and the data provenance (e.g. country of origin of the data, retrospective or prospective)

Sensitivity: 20 replicates of the zero-level calibrator.
Specificity: Aliquots of hormones from the World Health Organization's National Institute for Biological Standards and Control (London, England), diluted in human serum.
Precision (Intra-assay): Total N = 20 replicates per concentration level (three levels).
Precision (Inter-assay): Total n = 20 (2 duplicates x 10 assays over 3 weeks).
Method Comparison: N = 60 patient serum samples.
Linearity and Nonparallelism: Three human serum samples, each diluted at three levels (1:2, 1:4, 1:8), assayed in duplicate.
Spike and Recovery: Two hGH serum samples, each mixed in three ratios (2:1, 1:1, 1:2), assayed in duplicate.

The origin of the data is stated as "Studies on the AquaLite® hGH Assay were conducted at SeaLite Sciences." The location of SeaLite Sciences, Inc. is Norcross, GA, USA. The information does not explicitly state if the studies were retrospective or prospective, but the nature of the "Performance Characteristics" section suggests prospective testing of the device.

3. Number of experts used to establish the ground truth for the test set and the qualifications of those experts (e.g. radiologist with 10 years of experience)

This device is an in vitro diagnostic assay, so the "ground truth" is typically established by reference methods or validated standards rather than expert consensus on images.

For Specificity, the "ground truth" for the interfering substances was provided by the World Health Organization's National Institute for Biological Standards and Control (London, England) and their specified lot numbers and concentrations.
For Method Comparison, the "ground truth" for the 60 patient samples was established by a "commercially available chemiluminescence immunoassay" (the predicate device or another validated method).

No "experts" in the sense of human visual assessment for establishing ground truth are mentioned or applicable for this type of device.

4. Adjudication method (e.g. 2+1, 3+1, none) for the test set

Adjudication methods like "2+1" or "3+1" are relevant for subjective interpretations, often in imaging studies. For an in vitro diagnostic assay that produces quantitative results, such adjudication is not applicable. The results are quantitative measurements, and consistency/accuracy is assessed through statistical methods (e.g., %CV for precision, correlation for method comparison, % recovery for linearity/spike and recovery).

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

No such study was conducted or is applicable here. The device is an automated in vitro diagnostic assay, not an AI-assisted diagnostic tool for human readers.

6. If a standalone (i.e. algorithm only without human-in-the-loop performance) was done

Yes, the studies presented here are entirely standalone performance evaluations of the AquaLite® hGH Assay as an algorithm/device-only system. It is an automated laboratory test, not designed for human-in-the-loop interaction in the diagnostic process.

7. The type of ground truth used (expert consensus, pathology, outcomes data, etc.)

The ground truth used for various performance characteristics includes:

Sensitivity: Defined as the concentration associated with the mean + 2 standard deviations of a zero calibrator signal, which is a common practice for analytical sensitivity.
Specificity (Cross-reactivity): Known concentrations of specific hormones provided by a reputable scientific organization (WHO/NIBSC).
Precision: Commercial controls with known hGH levels.
Method Comparison: Results from a "commercially available chemiluminescence immunoassay" on patient samples, serving as a reference.
Linearity and Nonparallelism: Expected concentrations derived from serial dilutions of human serum samples with endogenous hGH.
Spike and Recovery: Expected concentrations derived from mixing hGH serum samples in known ratios.

In general, the ground truth relies on established laboratory standards, controls, and comparisons to predicate methods.

8. The sample size for the training set

The document does not describe a "training set" in the context of machine learning. This is a traditional in vitro diagnostic device, not an AI/ML-based algorithm that requires a separate training set. All data presented are for evaluating the performance of the developed assay.

9. How the ground truth for the training set was established

As there is no "training set" in the AI/ML sense, this question is not applicable. The development of the assay itself would have involved empirical testing and optimization against known standards, but this is a different concept from an AI training set.

Summary

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SEP 25 .997

millillel

EXHIBIT D

510(k) Summary For AquaLite® Human Growth Hormone Assay

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510(k) SUMMARY

I. GENERAL INFORMATION

Trade or proprietary name - SeaLite Sciences, Inc. AquaLite® hGH Assay

Common or usual name - Bioluminescent immunoassay (BIA)

Classification name - FDA has classified human growth hormone test systems intended for the measurement of human growth hormone in the diagnosis and treatment of disorders involving the anterior lobe of the pituitary glands. Class I devices (21 C.F.R. § 862.1370)

Submitter's Name and Address:	Cathryn N. CambriaDirector, Regulatory Affairs andQuality AssuranceSeaLite Sciences, Inc.3000 Northwoods ParkwaySuite 200Norcross, GA 30071(800) 874-4471, ext. 227
Submission Date:	July 23, 1997
Legally Marketed DeviceTo Which Claim SubstantialEquivalence:	Nichols InstituteHuman Growth Hormone Assay

II. INDICATIONS FOR USE

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DEVICE DESCRIPTION III.

SUMMARY OF STUDIES AND TECHNOLOGICAL CHARACTERISTICS IV.

Studies on the AquaLite® hGH Assay were conducted at SeaLite Sciences. The results are summarized below:

Performance Characteristics

1. Sensitivity
  The sensitivity or detection limit of the AquaLite® hGH Assay is 0.005 ng/mL. Sensitivity is determined by adding the mean signal of 20 replicates of the zero level calibrator plus two (2) standard deviations above this mean. The hGH concentration (ng/mL) associated with this calculated signal is defined as the sensitivity of the assay.

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Specificity 2.

The AquaLite® hGH Assay measures human growth hormone. The following human hormones were supplied by the World Health Organization's National Institute for Biological Standards and Control (London, England). Aliquots of these preparations were diluted to the following levels in human serum and assayed. Percent cross-reactivity (%) is reported below:

SUBSTANCE	WHO/NIBSCLOT NUMBER	TESTED AT	CROSS-REACTIVITY
Prolactin	3rd I.S. 84/500	500 ng/mL1,000 ng/mL	0.090.17
hPL	1st IRP 73/545	10,000 ng/mL100,000 ng/mL	< 0.01< 0.01
LH	2nd I.S. 80/552	250 mIU/L500 mIU/L	< 0.01< 0.01
TSH	2nd IRP 80/558	250 µIU/L500 µIU/L	<0.01< 0.01
hCG	3rd I.S. 75/537	10,000 mIU/L50,000 mIU/L	< 0.01< 0.01
FSH	1st I.S. 83/575	250 mIU/L500 mIU/L	< 0.01< 0.01

3. High Dose Hook Effect

No high dose hook effect occurs prior to 500 ng/mL.

4. Precision

Intra-assay precision. Three serum commercial controls containing hGH (a) at the following concentrations were assayed to determine intra-assay precision. (Total N = 20 per concentration level.)

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hGH Level (ng/mL)	% CV
3.33	4.80
6.20	4.97
12.30	6.95

Commercial controls containing hGH at the Inter-assay precision. (b) following concentrations were assayed in duplicate over a period of 3 weeks. 20 assays were performed using 3 sets of calibration values. The inter-assay precision observed for the solutions (Total n = 2 x 10 = 20) are shown below.

hGH Level (ng/mL)	% CV
2.89	6.4%
5.19	9.8%
9.42	11.8%

5. Method Comparison

The AquaLite® hGH Assay was used to assay patient samples (N=60) that were previously assayed by a commercially available chemiluminescence immunoassay. The serum samples ranged from 0.5 to 20.3 ng/ml of human growth hormone. Correlation by linear regression analysis gave a slope of 1.2831 with a y-intercept of 0.146. The correlation coefficient was 0.9665.

6. Linearity and Nonparallelism

Three human serum samples containing the levels of endogenous hGH shown below were diluted as indicated using AquaLite® hGH Calibrator A and assayed in duplicate using AquaLite® hGH. All concentrations are in ng/ml.

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SAMPLEID	DILUTIONFACTOR	OBSERVED(ng/mL)	hGHEXPECTED(ng/mL)	RECOVERY(%)
A	Undiluted1:21:41:8	10.034.622.541.27	---5.022.511.25	---92101100
B	Undiluted1:21:41:8	6.423.331.580.80	---3.211.610.81	---10398100
C	Undiluted1:21:41:8	5.542.761.440.71	---2.771.390.69	---100103103

7. Spike and Recovery

hGH serum samples were mixed in 2 to 1, 1 to 1, and 1 to 2 ratios and assayed in duplicate using the AquaLite® hGH Assay. All values are in ng/mL.

SAMPLEID	DILUTION	hGHOBSERVED	hGHEXPECTED	%RECOVERED
1	Undiluted A2A:1B1A:1B1A:2BUndiluted B	11.377.236.234.560.93	---7.896.154.41---	---92%101%103%---
2	Undiluted C2C:1D1C:1D1C:2DUndiluted D	12.248.377.285.110.94	---8.476.594.71---	---99%110%108%---

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POTENTIAL ADVERSE EFFECTS OF THE DEVICE ON HEALTH V.

Use Universal Precautions. No known test method can offer complete Caution: assurance that products derived from human serum are pathogen-free; therefore, handle all materials of human origin as though they were potentially infectious.

Sodium azide is used as a preservative. This preservative may react with metallic plumbing to from explosive metal azides. Flush with large volumes of water when disposing of materials containing sodium azide.

As an in vitro diagnostic test, there are not direct adverse effects on the health of a patient from the use of this product. However, failure of the device to perform as indicated, the contamination of reagents, the use of reagents past the labeled expiration dates, the use of improper specimens, or human error during the performance of the test may lead to erroneous results and possible improper patient management.

CONCLUSIONS DRAWN FROM STUDIES VI.

The data from the studies conducted demonstrate that the performance of SeaLite Sciences, Inc. AquaLite® hGH Assay is similar and substantially equivalent to that of other commercially available assays for hGH.

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Image /page/7/Picture/2 description: The image is a black and white logo for the U.S. Department of Health and Human Services. The logo features a stylized caduceus symbol, which is a staff with two snakes coiled around it. The symbol is surrounded by the text "DEPARTMENT OF HEALTH & HUMAN SERVICES USA" in a circular arrangement. The text is in all caps and is evenly spaced around the symbol.

SEP 2 5 1997

Food and Drug Administration 2098 Gaither Road Rockville MD 20850

Cathryn Cambria . Director, Regulatory Affairs and Quality Assurance SeaLite Sciences, Inc. 3000 Northwoods Parkway, Suite 200 Norcross, Georgia 30071

Re: K972761 AquaLite® Human Growth Hormone Requlatory Class: I Product Code: CFL Dated: July 23, 1997 Received: July 24, 1997

Dear Ms. Cambria:

We have reviewed your Section 510(k) notification of intent to market the device referenced above and we have determined the device is substantially equivalent (for the indications for use stated in the enclosure) to devices marketed in interstate commerce prior to May 28, 1976, the enactment date of the Medical Device Amendments, or to devices that have been reclassified in accordance with the provisions of the Federal Food, Druq, and Cosmetic Act (Act). You may, therefore, market the device, subject to the general controls provisions The general controls provisions of the Act of the Act. include requirements for annual reqistration, listing of devices, good manufacturing practice, labeling, and prohibitions aqainst misbranding and adulteration.

If your device is classified (see above) into either class II (Special Controls) or class III (Premarket Approval), it may be subject to such additional controls. Existing major regulations affecting your device can be found in the Code of Federal Requlations, Title 21, Parts 800 to 895. A substantially equivalent determination assumes compliance with the Current Good Manufacturing Practice requirements, as set forth in the Quality System Requlation (QS) for Medical Devices: General requlation (21 CFR Part 820) and that, through periodic QS inspections, the Food and Drug Administration (FDA) will verify such assumptions. Failure to comply with the GMP regulation may result in regulatory In addition, FDA may publish further announcements action. concerning your device in the Federal Register. Please note:

this response to your premarket notification submission does not affect any obligation you might have under sections 531 through 542 of the Act for devices under the Electronic Product Radiation Control provisions, or other Federal laws or regulations.

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Page 2

Under the Clinical Laboratory Improvement Amendments of 1988 (CLIA-88), this device may require a CLIA complexity categorization. To determine if it does, you should contact the Centers for Disease Control and Prevention (CDC) at (770) 488-7655.

This letter will allow you to beqin marketing your device as described in your 510(k) premarket notification. The FDA finding of substantial equivalence of your device to a legally marketed predicate device results in a classification for your device and thus, permits your device to proceed to the market.

If you desire specific advice for your device on our labeling regulation (21 CFR Part 801 and additionally 809.10 for in vitro diagnostic devices), please contact the Office of Compliance at (301) 594-4588. Additionally, for questions on the promotion and advertising of your device, please contact the Office of Compliance at (301) 594-4639. Also, please note the regulation entitled, "Misbranding by reference to
premarket notification" (21 CFR 807.97). Other general information on your responsibilities under the Act may be obtained from the Division of Small Manufacturers Assistance at its toll-free number (800) 638-2041 or (301) 443-6597 or at its internet address "http://www.fda.gov/cdrh/dsmamain.html".

Sincerely yours,
Steven Litman

Steven I. Gutman, M.D., M.B.A. Director Division of Clinical Laboratory Devices Office of Device Evaluation Center for Devices and Radiological Health

Enclosure

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Page 1 of of 1 -

510(k) Number (if known): ____________________________________________________________________________________________________________________________________________________

Device Name:

AquaLite® hGH Bioluminescent Immunoassay (BIA) Kit (or the AquaLite® hGH Assay)

Indications for Use:

The AquaLite® hGH Bioluminescent Immunoassay (BIA) Kit (or AquaLite® hGH Assay) is an in vitro diagnostic product intended for use in clinical laboratories for the quantitative measurement of growth hormone in human serum. Human growth hormone measurements are used in the diagnosis and treatment of disorders involving the anterior lobe of the pituitary gland.

Image /page/9/Picture/6 description: The image shows a black circle with a small, black, crescent shape to the left of it. The circle is solid black and appears to be the main focus of the image. The crescent shape is smaller and positioned slightly off-center to the left of the circle. The background is white.

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(PLEASE DO NOT WRITE BELOW THIS LINE CONTINUE ON ANOTHER PAGE IF NEEDED)

Concurrence of CDRH, Office of Device Evaluation (ODE)

Prescription Use (Per 21 CFR 801.109)

Over-The-Counter Use

(Optional Format

1-2-96)

(Division Sign-Off)
Division of Clinical Laboratory Devices
510(k) Number. R972761

455551.1

Regulation Number and Section

§ 862.1370 Human growth hormone test system.

(a)
Identification. A human growth hormone test system is a device intended to measure the levels of human growth hormone in plasma. Human growth hormone measurements are used in the diagnosis and treatment of disorders involving the anterior lobe of the pituitary gland.(b)
Classification. Class I (general controls). The device is exempt from the premarket notification procedures in subpart E of part 807 of this chapter subject to § 862.9.