The AquaLite® LH Bioluminescent Immunoassay (BIA) Kit (or the AquaLite® LH assay) is intended to be used for the quantitative determination of human LH in sera and plasma. The AquaLite® LH assay is for in vitro diagnostic use.

Device Description

The AquaLite® LH Bioluminescent Immunoassay Kit uses a polyclonal anti-LH aitibody that is pre-coated onto polystyrene tubes (solid phase). Samples (serum or plasma and appropriate calibrators or controls, are pipetted (50 uL) into the pre-coated tubes. Anti-LH Conjugate (150uL) is then added to the tubes. The conjugate uses the photoprotein, AquaLite® (recombinant aequorin; Patent Nos. 5, 422, 266 and 5, 486, 455 which is covalently linked to an anti-LH monoclonal antibody. LH in the sample simultaneously combines with polyclonal 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 60-minute incubation period at room temperature (18°C to 25°C) 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. An injected calcium trigger solution causes AquaLite® to oxide its self-contained luciferin molecule. This reaction produces a flash of light at 469 nm, which is measured by the luminometer. The intensity of the light is directly proportional to the concentration of the LH in the sample. To calculate results, the light intensity (in relative light units. RLU) of the LH calibrators is plotted against LH concentration (in International Units per liter, IU/L) to yield a calibration curve. This curve is used to relate the light intensity generated from the samples and controls to LH concentration in IU/L. Note that the numerical value for LH in mIU/mL is the same as for IU/L (International System). For example, 15.6 mIU/mL equals 15.6 IU/L.

Note: Samples that generate signals greater than the signal from the highest calibrator are off-scale. These samples must be diluted with Calibrator A 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 acceptance criteria and study detailed in the provided text for the SeaLite Sciences, Inc. AquaLite® LH immunoassay, structured as requested:

Acceptance Criteria and Device Performance (AquaLite® LH)

Note: This document does not explicitly state pre-defined "acceptance criteria" but rather reports the performance characteristics observed during testing. The reported performance is the criteria met for demonstrating safety and effectiveness compared to other commercially available assays for LH.

1. Table of Acceptance Criteria and Reported Device Performance

Performance Characteristic	Acceptance Criteria (Implicitly met by reported performance)	Reported Device Performance
Sensitivity	Detection limit to be clinically relevant	0.01 IU/L
Specificity (Cross-reactivity)	Minimal cross-reactivity with related hormones	TSH: 4.9%
		FSH: 0.68%
		hCG: 1.2%
High Dose Hook Effect	No hook effect within expected clinical range	No high dose hook effect prior to 5,000 IU/L LH
Intra-Assay Precision (% CV)	Acceptable variability for quantitative measurement	7.55% (at 1.069 IU/L)
		5.9% (at 9.821 IU/L)
		6.8% (at 40.129 IU/L)
Inter-Assay Precision (% CV)	Acceptable variability across different runs	8.2% (at 1.585 IU/L)
		8.17% (at 11.303 IU/L)
		8.9% (at 40.09 IU/L)
Method Comparison (vs. commercially available kit)	Strong correlation and agreement with existing assays	Slope: 0.89
		Y-intercept: 1.53
		Correlation coefficient: 0.938
Linearity	Measured values should approximate expected values upon dilution	Percent Recovered ranging from 95.4% to 130% across various dilutions (example values for SLS#4, SLS#18, SLS#26)
Spike and Recovery	High percentage of spiked LH should be recovered	Percent Recovered ranging from 91.6% to 109.3%
Recovery in Serum and Plasma	Consistent results across different sample types	No significant differences among serum and SST serum, or among serum and heparin, EDTA, and citrate plasmas. Oxalate plasmas not recommended.
Effect of Common Interferents	Minimal impact on LH measurement	Not significantly affected by hemoglobin (500 mg/dL), bilirubin (20 mg/dL), human serum albumin (12 mg/dL), or triglycerides (3000 mg/dL) at levels tested.

2. Sample Size and Data Provenance (Test Set)

Sensitivity: 20 replicates of the zero-level calibrator.
Specificity (Cross-reactivity): Aliquots of WHO/NIBSC preparations (specific quantities not detailed beyond "diluted to the following levels").
Intra-assay precision: N = 20 per solution (three different concentrations).
Inter-assay precision: N = 20 (10 assays, duplicate measurements per assay) per solution (three different concentrations).
Method Comparison: N = 62 patient samples.
Linearity and Nonparallelism: Three human serum samples (SLS#4, SLS#18, SLS#26) subjected to serial dilutions.
Spike and Recovery: Eight normal male human serum samples.
Recovery in Serum and Plasma: Blood samples from 2 normal subjects, processed into various types of serum and plasma.
Effect of Common Interferents: Pooled normal male human serum.

Data Provenance:

Country of Origin: Not explicitly stated for all samples. However, human sialoglycoprotein hormones for specificity were supplied by the World Health Organization's National Institute for Biological Standards and Control (London, England).
Retrospective or Prospective: Unclear for patient samples. The study involved controlled laboratory experiments using commercial controls, patient samples, and spiked samples.

3. Number of Experts and Qualifications for Ground Truth (Test Set)

Number of Experts: Not applicable. The ground truth for this in vitro diagnostic (IVD) device is typically established by:
- Calibrators: Precisely defined concentrations of the analyte.
- Reference Methods: Established methods (e.g., the "commercially available kit" for method comparison) that are accepted as accurate.
- Known Spiked Concentrations: Artificially created samples with a defined amount of analyte.
Qualifications of Experts: Not applicable in the context of establishing ground truth for an IVD test.

4. Adjudication Method (Test Set)

Not applicable for an in vitro diagnostic (IVD) device where ground truth is based on quantitative measurements against known standards or established reference methods, not expert interpretation of images or clinical data requiring adjudication.

5. Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study

Not done. This is an in vitro diagnostic (IVD) device for quantitative measurement of an analyte. MRMC studies are typically for imaging devices or devices requiring human interpretation.

6. Standalone Performance (Algorithm Only)

Yes, implied. The entire study describes the performance of the AquaLite® LH assay itself, which is a standalone in vitro test kit. There is no human-in-the-loop performance component beyond operating the luminometer and interpreting the calibration curve.

7. Type of Ground Truth Used

Reference/Assigned Values:
- Calibrators: For sensitivity, intra-assay, and inter-assay precision.
- World Health Organization's National Institute for Biological Standards and Control (WHO/NIBSC) preparations: For specificity/cross-reactivity.
- Commercial Reference Kit: For method comparison (the "commercially available kit").
- Known Spiked Concentrations: For linearity and spike & recovery.
- Expected values based on dilution: For linearity.

8. Sample Size for the Training Set

The document describes performance studies for a finished in vitro diagnostic kit. It does not mention a "training set" in the typical machine learning sense. The device is a chemical immunoassay, not an AI/ML algorithm that requires training on a dataset in that manner. Any "training" would refer to the internal development and optimization of the assay by SeaLite Sciences, Inc., the details of which are not provided here.

9. How the Ground Truth for the Training Set was Established

Not applicable. As stated above, this is not an AI/ML device with a "training set" in the conventional sense. The development of the assay would have involved various optimization steps using known concentrations of LH and other related substances, but these would fall under assay development rather than "training set ground truth establishment."

Summary

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K9604925

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JAN 2 2 1997

EXHIBIT D

SUMMARY OF SAFETY AND EFFECTIVENESS

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GENERAL INFORMATION I.

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

Common or usual name - Bioluminescent immunoassay (BIA)

Classification name - FDA has classified LH test systems intended for the measurement of LH in the diagnosis of gonadal dysfunction.

INDICATIONS FOR USE II.

III. DEVICE DESCRIPTION

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ALTERNATIVE PRACTICES AND PROCEDURES IV.

There are several assay technologies commonly employed to measure the presence of human LH in serum or plasma. They include: radioimmunoassay (RIA) and enzymelinked immunosorbent immunoassay (ELISA).

MARKETING HISTORY V.

The modified SeaLite Sciences, Inc. AquaLite® LH that is the subject of this submission is not currently marketed.

POTENTIAL ADVERSE EFFECTS OF THE DEVICE ON HEALTH VI.

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 virro 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.

VII. SUMMARY OF STUDIES

Studies on SeaLite Sciences, Inc. AquaLite® LH were conducted at SeaLite Sciences. The results are summarized below:

Performance Characteristics

1. Sensitivity

The sensitivity or detection limit of the AquaLite® LH assay is 0.01 IU/L. Sensitivity is determined by adding the mean signal of twenty (20) replicates of the zero level calibrator plus two (2) standard deviations above this mean. The LH concentration (IU/L) corresponding to this calculated signal is defined as the analytical sensitivity of the assay.

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2. Specificity

The AquaLite® LH assay measures intact LH. The following human sialoglycoprotein 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 zero calibrator and assayed. Percent crossreactivity (%) is reported below:

Substance	WHO/NIBSCLot Number	Tested at (IU/L)	% Cross-reactivity
TSH	2nd IRP 80-558	1,000	4.9
FSH	I.S. 83-575	1,000	0.68
hCG	IRP 75-551	2,500	1.2

High Dose Hook Effect No high dose hook effect occurs prior to 5,000 IU/L LH. 3.
1. Precision
Intra-assay precision. Tri-level commercial controls containing LH at the following (a) concentrations were assayed to determine intra-assay precision. (Total N = 20 per solution.)

Mean LH Level (IU/L)	SD	% CV (calibration values)
1.069	0.0807	7.55%
9.821	0.578	5.9%
40.129	2.728	6.8%

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Inter-assay precision. Tri-level commercial controls containing LH at the following (b) concentrations were assayed in duplicate repetitively. Ten assays were performed using ten sets of calibration values. Inter-assay precision observed for the solutions (Total N = 2 x 10 = 20) is shown below.

Mean LH Level (IU/L)	SD	% CV
1.585	0.131	8.2%
11.303	0.924	8.17%
40.09	3.60	8.9%

ડ. Method Comparison

)

The AquaLite® LH assay was used to test patient samples (N=62) that were previously assayed by a commercially available kit. The samples ranged from 0.5 to 62.0 IU/L. A slope of 0.89 with a y-intercept of 1.53 was obtained. The correlation coefficient was 0.938.

6. Linearity and Nonparallelism

Three human serum samples containing the levels of endogenous LH shown below were diluted in parallel using Calibrator A with 0.9% sodium chloride added.

SampleID	DilutionFactor	Found(IU/L)	Expected(IU/L)	Percent(%)
SLS#4	Undiluted	80.83	--	--
	1:2	39.55	40.42	97.9
	1:4	19.575	20.21	96.9
	1:8	13.4	10.1	130
SLS#18	Undiluted	63.82	--	--
	1:2	30.436	31.91	95.4
	1:4	15.84	15.95	99.4
	1:8	9.09	8.0	113
SLS#26	Undiluted	75.46	--	--
	1:2	44.4	37.73	115
	1:4	19.65	18.86	104
	1:8	10.4	9.43	111

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7. Spike and Recovery

Normal male human serum samples were spiked with LH in the amounts noted below. The spiked samples were assayed using the AquaLite® LH assay. All values are in IU/L.

EndogenousLH	AddedLH	MeasuredLH	ExpectedLH	PercentRecovered
6.83	50	60.35	56.83	106.1
11.82	50	56.59	61.82	91.6
3.14	50	57.69	53.14	108.6
0.922	50	48.88	51.0	96
2.17	50	53.29	52.17	101.5
0.16	50	52.7	50.2	104.6
2.06	50	56.89	52.06	109.3
2.53	50	56.02	52.53	106.6

8. Recovery in Serum and Plasma

Blood Samples from 2 normal subjects were prepared as sera (standard technique and SST tubes) as well as heparin, EDTA, oxalate, and citrate plasmas. LH was quantified using the AquaLite® LH assay. Recovered LH was compared with LH recovered in serum (standard technique). The data demonstrate that there are no significant differences among serum and SST serum nor among serum and heparin, EDTA, and citrate plasmas when using the AquaLite® LH assay. Oxalate plasmas is not recommended.

9. Effect of Common Interferents

Pooled normal male human serum was spiked with preparations of hemoglobin, bilirubin, human serum albumin and triglycerides to the levels shown below. Equal amounts of LH were spiked into normal male serum as well as the normal male serum aliquots containing potential interferents. LH was quantified using the AquaLite® LH assay. Recovered LH was compared with the LH recovered in normal male serum. The data (in IU/L) demonstrate that the AquaLite® LH assay is not

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significantly affected by hemoglobin, bilirubin, human serum albumin or triglycerides at the levels tested.

LH	Neat	Hemoglobin(at 500 mg/dL)		Bilirubin(at 20 mg/dL)		Albumin(at 12 mg/dL)		Triglycerides(at 3000 mg/dL)
Spike	Serum	IU/L	%	IU/L	%	IU/L	%	IU/L	%
0	4.8	-	-	-	-	-	-	-	-
65	70.0	68.7	98.2	72.4	103	71.0	101	69.1	98.7

VIII. CONCLUSIONS DRAWN FROM STUDIES

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

Regulation Number and Section

§ 862.1485 Luteinizing hormone test system.

(a)
Identification. A luteinizing hormone test system is a device intended to measure luteinizing hormone in serum and urine. Luteinizing hormone measurements are used in the diagnosis and treatment of gonadal dysfunction.(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.