The eQ-PCR™ LC Warfarin Genotyping kit is an in vitro diagnostic test for the detection and genotyping of two single nucleotide polymorphisms (SNP) in the cytochrome P450 enzyme gene CYP2C9 known as CYP2C92 (C430T) and CYP2C93 (A1075C), and a SNP in the vitamin K epoxide reductase complex 1 gene VKORC1, known as VKORC1 (-1639G>A) obtained from human peripheral blood samples. The eQ-PCR LC Warfarin Genotyping kit is a qualitative assay for use in clinical laboratories upon prescription by the attending physician.

The eO-PCR™ LC Warfarin Genotyping kit is indicated for use as an aid in identifying patients who may be at risk of warfarin sensitivity.

Device Description

The eQ-PCR 1M LC Warfarin Genotyping Kit assay requires extracted DNA obtained by using any commercially available DNA extraction kit. The extracted DNA sample, within a range of 50-200ng of total DNA, is mixed with a PCR Mix, and an eQ-PCR "M specific Probe Mix reagent containing specific primers and fluorescent labeled probes for the CYP2C9 and/or VKORC1 gene polymorphisms. Amplification and detection are then performed in the Roche Diagnostics LightCycler® Real-Time PCR System instrument model 1.2 using conditions defined in the specific eQ-PCRTM LC Warfarin Genotyping Kit Product Insert. After the PCR reaction is completed, the Roche Diagnostics LightCycler® Real-Time PCR System instrument automatically proceeds to the melting curve-based detection method. This real time PCR test is a closed test system and does not require post PCR operations. It reduces human errors and eliminates post-PCR handling contamination.

The instrument's standard melting curve analysis software algorithm of peak patterns and melting temperatures (Tm) determine the genotype (wild type, mutant, heterozygous) for each of the three specified polymorphisms.

AI/ML Overview

This document describes the TrimGen eQ-PCR™ LC Warfarin Genotyping Kit, an in vitro diagnostic test for genotyping SNPs associated with warfarin sensitivity. The study aims to demonstrate substantial equivalence to a predicate device through clinical performance and reproducibility testing.

1. Table of Acceptance Criteria and Reported Device Performance:

The document doesn't explicitly state pre-defined quantitative acceptance criteria in a dedicated section. However, the performance is reported as "overall 100% clinical sensitivity" for the assay compared to bi-directional sequencing after re-sequencing of discrepant samples. The table below presents the detailed clinical sensitivity for each SNP and genotype, which can be inferred as the performance measures.

SNP	Genotype	Reported Clinical Sensitivity (# / total)	Reported Clinical Sensitivity (%)	95% Confidence Interval
2C9*2	Wild Type	123/126	97.6%	94.40%
	Heterozygous	27/28	96.4%	87.68%
	Variant	5/5	100%	47.98%
2C9*3	Wild Type	138/140	98.6%	96.09%
	Heterozygous	12/13	92.3%	75.32%
	Variant	5/6	83.3%	54.28%
VKORC1	Wild Type	77/79	97.5%	93.16%
	Heterozygous	62/63	98.4%	94.32%
	Variant	17/17	100%	80.52%

For reproducibility, the criteria were implicitly met by "no discordance among observed results or between observed and expected results across sites, days or operators" and all results matching bi-directional sequencing.

2. Sample Size Used for the Test Set and Data Provenance:

Sample Size: 159 donors provided whole blood samples for the clinical performance study. For the reproducibility study, a panel of 31 samples (whole blood) was used at each of the three sites across five days.
Data Provenance: The whole blood samples were collected from adult volunteers under IRB approval and with Informed Consent. The country of origin is not explicitly stated, but the submission is to the US FDA, implying testing within the US or for the US market. The clinical performance study is a prospective collection for testing.

3. Number of Experts Used to Establish the Ground Truth for the Test Set and Qualifications of Those Experts:

The ground truth for the clinical performance study was established by bi-directional sequencing performed at a "reference laboratory." The number of experts and their specific qualifications (e.g., geneticists, molecular biologists) are not specified within the provided text.

4. Adjudication Method for the Test Set:

The clinical performance study mentions that "Only 0.4% (2/450) of the results was discordant and resolved in agreement with the eQ-PCR LC Warfarin Genotyping Kit result when bi-directional sequencing was repeated." This indicates an adjudication method where initial discrepancies were re-evaluated by re-sequencing using the ground truth method (bi-directional sequencing). This could be considered a form of "tie-breaker" or confirmatory testing for discordant results.

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, a Multi-Reader Multi-Case (MRMC) comparative effectiveness study was not conducted. This device is an in vitro diagnostic (IVD) kit for genotyping, not an AI-assisted diagnostic imaging or interpretation tool. Therefore, the concept of human readers improving with AI assistance is not applicable here.

6. If a Standalone (i.e., algorithm only without human-in-the-loop performance) was Done:

Yes, the clinical performance study is essentially a standalone performance evaluation of the device (the eQ-PCR™ LC Warfarin Genotyping Kit) against the established ground truth (bi-directional sequencing). The device's algorithm for determining genotype from melting curve analysis is used independently.

7. The Type of Ground Truth Used:

The ground truth used was bi-directional sequencing. This is a highly accurate method for determining DNA sequences and is considered a gold standard for genotyping in many contexts.

8. The Sample Size for the Training Set:

The document describes a post-market clearance study demonstrating assay performance. It does not mention a separate training set for the device's algorithm. The algorithm for interpreting melting curves is likely pre-defined based on known principles of real-time PCR and melting curve analysis, rather than a machine learning model requiring a specific training dataset in the context presented here. The "training" of such a system would typically involve establishing robust assay conditions and expected melting curve profiles for different genotypes.

9. How the Ground Truth for the Training Set Was Established:

As a dedicated training set is not explicitly mentioned for an algorithm that learns from data, the establishment of ground truth for a training set is not applicable as per the provided text. The device relies on a pre-established methodology (real-time PCR and melting curve analysis) for genotype determination, where the "ground truth" for defining peak patterns and melting temperatures for different genotypes would be based on well-characterized samples and molecular biology principles.

Summary

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510(k) SUMMARY 1.

Feb 6, 2009

FEB - 6 2009

01307

Contact

Dr. Howard Doong TrimGen Corporation 34 Loveton Circle, Suite 210 Sparks, MD 21152

NAME OF DEVICE

Trade Name:	eQ-PCR TM LC Warfarin Genotyping kit
Regulation Number:	21 CFR 862.3360
Classification Name:	Drug metabolizing enzyme genotyping system

PREDICATE DEVICE

Verigene® Warfarin Metabolism Nucleic Acid Test (K070804)

INTENDED USE

The eO-PCR™ LC Warfarin Genotyping kit is indicated for use as an aid in identifying patients who may be at risk of warfarin sensitivity.

PRODUCT DESCRIPTION

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SUBSTANTIAL EQUIVALENCE

Clinical Performance

A clinical trial was conducted to test the performance of the eQ-PCRTM LC Warfarin Genotyping Kit compared to bi-directional sequencing using extracted nucleic acid from whole blood from 159 donors. Two EDTA-anticoagulated whole blood samples were collected from each of 159 adult volunteers. The samples were collected under IRB approval and with Informed Consent. The whole blood samples were extracted at a reference laboratory. The extracted DNA from the two samples from the same matrix per donor was pooled for testing in the study. Bi-directional sequencing was performed at a reference laboratory on the 159 DNA samples extracted from the whole blood samples.

Aliquots of the extracted nucleic acid samples were prepared at TrimGen at a concentration of 10ng/uL. Each testing site received aliquots of 50 whole blood (WB) DNA. and 9 commercial control DNA samples. The donor samples tested at each site were unique to that site and did not duplicate the donors at the other two sites. Ten (10) WB samples were tested with the eQ-PCR 104 LC Warfarin Genotyping Kit and the Roche LightCycler each day for a total of 5 testing days.

SNP	Genotype	Clinical Sensitivity		95% ConfidenceInterval
		#	%
2C9*2	Wild Type	123/126	97.6%	94.40%
	Heterozygous	27/28	96.4%	87.68%
	Variant	5/5	100%	47.98%
2C9*3	Wild Type	138/140	98.6%	96.09%
	Heterozygous	12/13	92.3%	75.32%
	Variant	5/6	83.3%	54.28%
VKORC1	Wild Type	77/79	97.5%	93.16%
	Heterozygous	62/63	98.4%	94.32%
	Variant	17/17	100%	80.52%

The results for the whole blood testing are summarized below.

The initial results showed overall 99.6% clinical sensitivity of the eQ-PCR ™ LC Warfarin Genotyping Kit compared to bi-directional results for the whole blood samples. Only 0.4% (2/450) of the results was discordant and resolved in agreement with the eQ-PCRTM LC Warfarin Genotyping Kit result when bidirectional sequencing was repeated. After re-sequencing the discrepant samples, the overall result demonstrates 100% clinical sensitivity of the assay's substantial equivalence to bi-directional sequencing.

Reproducibility

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A study was conducted at two external sites and at TrimGen Corporation to determine the reproducibility of the eQ-PCR™ LC Warfarin Genotyping Kit across days, operators, and sites. A panel of 31 samples was extracted and tested at each of the three sites on each of five days. The panel consisted of whole blood samples. At all sites, separate operators performed the testing on alternate days.

There was no discordance among observed results or between observed and expected results across sites, days or operators. All whole blood and controls gave consistent eQ-PCR™ LC Warfarin Genotyping Kit results within days, across days, across operators, and across sites. In addition, all genotyping results matched the bi-directional sequencing results.

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DEPARTMENT OF HEALTH & HUMAN SERVICES

Image /page/3/Picture/1 description: The image shows the logo for the U.S. Department of Health & Human Services. The logo consists of a circular seal with the text "DEPARTMENT OF HEALTH & HUMAN SERVICES - USA" arranged around the perimeter. Inside the circle is an emblem featuring a stylized eagle with its wings spread, symbolizing the department's mission to protect the health of all Americans.

Public Health Service

Food and Drug Administration 2098 Gaither Road Rockville MD 20850

TrimGen Corporation c/o Dr. Howard Doong President 34 Loveton Circle #210 Sparks, MD 21152

FEB - 6 2009

Re: K073071

Trade/Device Name: eQ-PCR™ LC Warfarin Genotyping Kit Regulation Number: 21 CFR 862.3360 Regulation Name: Drug metabolizing enzyme genotyping system. Regulatory Class: Class II Product Code: ODW, NSU, ODV Dated: January 20, 2009 Received: January 23, 2009

Dear Dr. Doong:

We have reviewed your Section 510(k) premarket notification of intent to market the device referenced above and have determined the device is substantially equivalent (for the indications for use stated in the enclosure) to legally marketed predicate 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, Drug, and Cosmetic Act (Act) that do not require approval of a premarket approval application (PMA). You may, therefore, market the device, subject to the general controls provisions of the Act. The general controls provisions of the Act include requirements for annual registration, listing of devices, good manufacturing practice, labeling, and prohibitions against misbranding and adulteration.

If your device is classified (see above) into either class II (Special Controls) or class III (PMA), it may be subject to such additional controls. Existing major regulations affecting your device can be found in Title 21, Code of Federal Regulations (CFR), Parts 800 to 895. In addition, FDA may publish further announcements concerning your device in the Federal Register.

Please be advised that FDA's issuance of a substantial equivalence determination does not mean that FDA has made a determination that your device complies with other requirements of the Act or any Federal statutes and regulations administered by other Federal agencies. You must comply with all the Act's requirements, including, but not limited to: registration and listing (21 CFR Part 807); labeling (21 CFR Parts 801 and 809); and good manufacturing practice requirements as set forth in the quality systems (OS) regulation (21 CFR Part 820).

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Page - 2

This letter will allow you to begin marketing your device as described in your Section 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 information about the application of labeling requirements to your device, or questions on the promotion and advertising of your device, please contact the Office of In Vitro Diagnostic Device Evaluation and Safety at (301) 594-3084. Also, please note the regulation entitled, "Misbranding by reference to premarket notification" (21CFR Part 807.97). You may obtain other general information on your responsibilities under the Act from the Division of Small Manufacturers, International and Consumer Assistance at its toll-free number (800) 638-2041 or (301) 443-6597 or at its Internet address http://www.fda.gov/cdrh/dsma/dsmamain.html.

Sincerely yours.

Corg C. He

Courtney C. Harper, Ph.D. Acting Director Division of Chemistry and Toxicology Office of In Vitro Diagnostic Device Evaluation and Safety Center for Devices and Radiological Health

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Indication for Use

510(k) Number (if known): K073071

Device Name: eQ-PCR LC Warfarin Genotyping Kit

Indication For Use:

The eQ-PCR™ LC Warfarin Genotyping kit is an in vitro diagnostic test for the detection and genotyping of two single nucleotide polymorphisms (SNP) in the cytochrome P450 enzyme gene CYP2C9 known as CYP2C92 (C430T) and CYP2C93 (A1075C), and a SNP in the vitamin K epoxide reductase complex 1 gene VKORC1, known as VKORC1 (-1639G>A) obtained from EDTA anticoagulated whole blood samples. The eQ-PCR LC Warfarin Genotyping kit is used as an aid in the identification of patients at risk for increased warfarin sensitivity. It is a qualitative assay for use in clinical laboratories upon prescription by the attending physician.

Prescription Use × (21 CFR Part 801 Subpart D)

And/Or

Over the Counter Use (21 CFR Part 801 Subpart C)

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

Concurrence of CDRH, Office of In Vitro Diagnostic Device Evaluation and Safety (OIVD)

Division Sign-Off Office of In Vitro Diagnostic Device Evaluation and Safety

510(k) IC073071

Regulation Number and Section

§ 862.3360 Drug metabolizing enzyme genotyping system.

(a)
Identification. A drug metabolizing enzyme genotyping system is a device intended for use in testing deoxyribonucleic acid (DNA) extracted from clinical samples to identify the presence or absence of human genotypic markers encoding a drug metabolizing enzyme. This device is used as an aid in determining treatment choice and individualizing treatment dose for therapeutics that are metabolized primarily by the specific enzyme about which the system provides genotypic information.(b)
Classification. Class II (special controls). The special control is FDA's guidance document entitled “Class II Special Controls Guidance Document: Drug Metabolizing Enzyme Genotyping Test System.” See § 862.1(d) for the availability of this guidance document.