K Number

Device Name

INPLEX CF MOLECULAR TEST

Manufacturer

THIRD WAVE TECHNOLOGIES, INC.

Date Cleared

2008-03-13

(448 days)

Product Code

NUA

Regulation Number

866.5900

Intended Use

InPlex™ CF Molecular Test is an in vitro diagnostic device used to simultaneously detect and identify a panel of mutations and variants in the cystic fibrosis transmembrane conductance regulator (CFTR) gene in genomic DNA samples isolated from human peripheral whole blood specimens. The panel includes mutations and variants recommended by the 2004 American College of Medical Genetics (ACMG). The InPlex™ CF Molecular Test is a qualitative genotyping test that provides information intended to be used for cystic fibrosis carrier screening as recommended by ACMG and the 2005 American College of Obstetricians and Gynecologists (ACOG) for adults of reproductive age, as an aid in newborn screening for cystic fibrosis, and in confirmatory diagnostic testing for cystic fibrosis in newborns and children. The test is not indicated for use in fetal diagnostic or pre-implantation testing. This test is also not indicated for stand-alone diagnostic purposes and results should be used in conjunction with other available laboratory and clinical information.

Device Description

InPlex™ CF Molecular Test amplifies specific regions of the CF7R gene in genomic DNA extracted from human whole peripheral blood. Each amplified DNA sample is subsequently mixed with Cleavase® enzyme and buffer then added to a loading port on an InPiex™ microfluidic card. An InPlex™ card contains eight sample-loading ports, each connected to 48 independent reaction chambers. Twenty-eight of these reaction chambers contain dried assay mixes specific for reporting the 23 ACMG/ACOG recommended CF7R mutations and variants. The remaining chambers consist of a "No Invader" Control", an Independent Quality Control. and several unused chambers. After an InPlex™ card is loaded; the channels are mechanically sealed using a micro-fluidic card sealer, isolating each individual reaction chamber from all other chambers. The card is then incubated to allow individual Invader® reactions to occur. Following incubation, the card is read in a multi-well fluorometer and the raw signal data are imported into the InPlex™ CF Molecular Test Call Reporting Software for final result analysis.

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Predicate Devices

K043011

Reference Devices

Not Found

AI/ML (Artificial Intelligence / Machine Learning)

No
The description focuses on traditional molecular biology techniques and data analysis software, with no mention of AI or ML.

Therapeutic

No
The device is described as an "in vitro diagnostic device" used for testing purposes and is not indicated for treatment or therapy.

Diagnostic

Yes

The intended use explicitly states this device is an "in vitro diagnostic device" and is used for "confirmatory diagnostic testing for cystic fibrosis in newborns and children."

SaMD (Software as a Medical Device)

The device description explicitly details hardware components like microfluidic cards, a micro-fluidic card sealer, and a multi-well fluorometer, in addition to the software.

IVD (In Vitro Diagnostic)

Yes, this device is an IVD (In Vitro Diagnostic).

Here's why:

Intended Use: The "Intended Use / Indications for Use" section explicitly states that the InPlex™ CF Molecular Test is an "in vitro diagnostic device".
Nature of the Test: The device performs a test on a biological sample (genomic DNA isolated from human peripheral whole blood specimens) to detect and identify specific genetic mutations. This is a hallmark of in vitro diagnostics, which are used to examine specimens from the human body to provide information for diagnosis, treatment, or prevention of disease.
Purpose: The test is intended for cystic fibrosis carrier screening, newborn screening, and confirmatory diagnostic testing. These are all diagnostic or screening purposes.

PCCP (Predetermined Change Control Plan) Authorized

N/A

Overview

Intended Use / Indications for Use

The test is not indicated for use in fetal diagnostic or pre-implantation testing. This test is also not indicated for stand-alone diagnostic purposes and results should be used in conjunction with other available laboratory and clinical information.

Product codes

NUA

Device Description

After an InPlex™ card is loaded; the channels are mechanically sealed using a micro-fluidic card sealer, isolating each individual reaction chamber from all other chambers. The card is then incubated to allow individual Invader® reactions to occur. Following incubation, the card is read in a multi-well fluorometer and the raw signal data are imported into the InPlex™ CF Molecular Test Call Reporting Software for final result analysis.

Mentions image processing

Not Found

Mentions AI, DNN, or ML

Not Found

Input Imaging Modality

Not Found

Anatomical Site

Not Found

Indicated Patient Age Range

adults of reproductive age, newborns and children.

Intended User / Care Setting

Not Found

Description of the training set, sample size, data source, and annotation protocol

Not Found

Description of the test set, sample size, data source, and annotation protocol

DNA Extraction Equivalency:

Sample Size: 7 CF positive genomic DNA samples isolated from whole peripheral blood.
Data Source: Internal processing involved the purification of the same 7 samples using four commonly used, commercially available DNA extraction kits. Samples were also genotyped by DNA sequencing to confirm results.
Annotation Protocol: Genotype calls as generated by the InPlex™ CF Molecular Test were compared to DNA sequencing based genotyping for all extraction methods.

Fluorometer Equivalency:

Sample Size: A panel of eight genomic DNA (gDNA) samples, either positive for a subset of the CFTR mutations or normal.
Data Source: The eight samples were tested in triplicate.
Annotation Protocol: InPlex™ CF Molecular Test genotype results were compared to DNA sequencing genotype results.

Incubator Equivalency:

Sample Size: A panel of eight gDNA samples.
Data Source: Tested in triplicate using each of three thermal incubators.
Annotation Protocol: Not explicitly stated, but results were compared to known genotypes.

Interfering Substances:

Sample Size: Eight, CF positive, genomic DNA samples isolated from whole peripheral blood.
Data Source: Samples were tested in the absence (untreated) and presence (treated) of potential interfering substances (bilirubin, triglycerides, potassium EDTA, Qiagen® Buffer AW2, hemoglobin).
Annotation Protocol: Genotype calls for the DNA samples containing an interfering substance were compared to the untreated samples for calculation of percent agreement.

Limit of Detection:

Sample Size: A panel of eight gDNA samples.
Data Source: Samples were prepared and tested at eight concentrations ranging from 1ng/uL to 150ng/uL (total input DNA range of 5-750ng / reaction).
Annotation Protocol: Genotype call results for all eight characterized samples were assessed for percent agreement at each concentration. The lower limit of detection was defined as the lowest DNA concentration in which a 99% or greater concordance with DNA sequencing was observed.

Lot-to-Lot Equivalency:

Sample Size: A panel of 23 CFTR gDNA samples.
Data Source: Tested in singlicate with each of three lots of InPlex™ CF Molecular Test kits.
Annotation Protocol: Genotype calls were compared to pre-characterized gDNA genotypes.

Accuracy and Repeat Rate:

Sample Size: 123 unique samples containing 144 positive CFTR calls and 2808 normal (negative) CFTR calls. Genomic DNA samples isolated from peripheral whole blood and cell lines.
Data Source: InPlex™ CF Molecular Test genotyping results were compared to bi-directional DNA sequence analysis.
Annotation Protocol: Genotype calls were compared between the DNA sequencing results and the InPlex™ CF Molecular Test results for the calculation of overall agreement. Positive and negative agreement for each mutation was calculated. The repeat rate was determined by the number of samples that generated an invalid genotype call for one or more mutations with the InPlex™ CF Molecular Test on the first attempt.

Freeze-Thaw Tolerance:

Sample Size: A panel of 23 CFTR gDNA samples.
Data Source: Tested after 2, 4, 6, 8, 10 and 12 freeze-thaw cycles.
Annotation Protocol: Compared to DNA sequencing for all freeze-thaw cycles tested.

Real-Time Stability:

Sample Size: A panel of seven CFTR gDNA samples and a panel of eight control samples comprised of pooled amplicons that cover the mutations in the InPlex™ CF Molecular Test.
Data Source: Samples were tested in duplicate with each of three master lots for each storage condition (recommended, substandard, ship-stressed) at each time point.
Annotation Protocol: Not explicitly stated, but implies comparison for percent agreement.

Reproducibility Study:

Sample Size: Performance phase used 23 samples containing mutations representing the ACMG recommended panel. Proficiency phase used eight (8) pre-characterized, gDNA samples.
Data Source: Samples from pre-characterized commercially available reference materials. Tested at three investigative sites by two technicians each, with tests being run on each of five (5) nonconsecutive days.
Annotation Protocol: Data generated during the proficiency phase was to demonstrate technician proficiency only and not used for clinical performance. Percent agreement between operators and sites was assessed.

Summary of Performance Studies (study type, sample size, AUC, MRMC, standalone performance, key results)

DNA Extraction Equivalency:

Study type: Analytical study – Equivalency
Sample size: 7 CF positive genomic DNA samples
Key results: All calls were concordant with the DNA sequencing based genotyping, providing an overall agreement of 100% (99.5%, 1-sided lower 95% Confidence Limit).

Fluorometer Equivalency:

Study type: Analytical study – Equivalency
Sample size: 8 genomic DNA (gDNA) samples, tested in triplicate (1,656 data points)
Key results: All InPlex™ CF Molecular Test genotype results for the characterized gDNA samples described were concordant with DNA sequencing genotype results and in 100% (99.8% 1-sided lower 95% Confidence Limit) agreement regardless of the fluorometer used.

Incubator Equivalency:

Study type: Analytical study – Equivalency
Sample size: 8 gDNA samples, tested in triplicate (1,656 data points)
Key results: The percent agreement to known genotypes was 99.88% (99.74%, 1-sided 95% Confidence Limit).

Interfering Substances:

Study type: Analytical study – Evaluation of interfering substances
Sample size: 8 CF positive genomic DNA samples
Key results: The InPlex™ CF Molecular Test achieved a 100% agreement (99.7%, 1-sided lower 95% Confidence Limit) between the genotypes of the samples containing potential interfering substances and bidirectional sequencing.

Limit of Detection:

Study type: Analytical study – Limit of Detection
Sample size: 8 gDNA samples, tested at 8 concentrations (1,472 data points)
Key results: A 5ng/μL DNA concentration (input DNA of 25ng) provided a percent agreement of 100% (98.4%, 1-sided lower 95% Confidence Limit) with DNA sequencing qualifying it as the lower limit of detection. At the highest DNA concentration tested, 150ng/uL (input DNA of 750ng), a 99.5% percent agreement was obtained (98.6%, 1 sided lower 95% Confidence Limit). The remaining DNA concentrations tested, 10, 20, 50, and 100ng/ul (input DNA of 50, 100, 250 and 500ng) all obtained a 100 percent agreement (98.4%, 1-sided lower 95% Confidence Limit) with expected results.

Lot-to-Lot Equivalency:

Study type: Analytical study – Equivalency
Sample size: 23 CFTR gDNA samples, tested in singlicate with each of 3 lots (529 genotype calls per lot)
Key results: All genotype calls were in 100% (99.4% 1-sided lower 95% Confidence Limit) agreement to pre-characterized gDNA genotypes for each lot tested.

Accuracy and Repeat Rate:

Study type: Analytical study – Accuracy and Repeat Rate
Sample size: 123 unique samples (144 positive CFTR calls and 2808 normal (negative) CFTR calls)
Key results: The InPlex™ CF Molecular Test achieved 99.96% (2951/2952) overall agreement (99.9%, 1-sided lower 95% Confidence Limit), 100% (144/144) positive agreement (97.9%, 1-sided lower 95% Confidence Limit), and 99.96% (2807/2808) negative agreement (99.9%, 1-sided lower 95% Confidence Limit). There was one "Invalid" call out of 123 tests, resulting in a repeat rate of 0.8%.

Freeze-Thaw Tolerance:

Study type: Analytical study – Freeze-Thaw Tolerance
Sample size: 23 CFTR gDNA samples (3,703 data points across test points)
Key results: The InPlex™ CF Molecular Test achieved an overall percent agreement of 100% (99.92%, 1sided lower 95% Confidence Limit) as compared to DNA sequencing for all freeze-thaw cycles tested.

Real-Time Stability:

Study type: Analytical study – Real-Time Stability
Sample size: 15 samples (7 CFTR gDNA samples + 8 control samples), tested in duplicate with 3 master lots.
Key results: All lots met the passing criterion for the Real-Time Stability performance evaluation study with an observed percent agreement of 100% (99.1%, 1-sided lower 95% Confidence Limit).

Reproducibility Study:

Study type: Analytical study – Reproducibility
Sample size: 23 samples (performance phase); 8 gDNA samples (proficiency phase)
Key results: Percent agreement ranged from 99.962% to 100% at each of the three sites and 99.987% to 99.994% across all sites. The overall accuracy rate was 31,738/31,740 = 99.994% (99.986, 1-sided lower 95% Confidence Limit).

Key Metrics (Sensitivity, Specificity, PPV, NPV, etc.)

DNA Extraction Equivalency: Overall agreement of 100% (99.5%, 1-sided lower 95% Confidence Limit).
Fluorometer Equivalency: 100% (99.8% 1-sided lower 95% Confidence Limit) agreement.
Incubator Equivalency: 99.88% (99.74%, 1-sided 95% Confidence Limit) agreement.
Interfering Substances: 100% agreement (99.7%, 1-sided lower 95% Confidence Limit).
Limit of Detection:
- 5ng/μL DNA concentration: 100% percent agreement (98.4%, 1-sided lower 95% Confidence Limit).
- 150ng/uL DNA concentration: 99.5% percent agreement (98.6%, 1 sided lower 95% Confidence Limit).
- 10, 20, 50, and 100ng/ul DNA concentrations: 100 percent agreement (98.4%, 1-sided lower 95% Confidence Limit).
Lot-to-Lot Equivalency: 100% (99.4% 1-sided lower 95% Confidence Limit) agreement.
Accuracy and Repeat Rate:
- Overall agreement: 99.96% (2951/2952) (99.9%, 1-sided lower 95% Confidence Limit).
- Positive agreement: 100% (144/144) (97.9%, 1-sided lower 95% Confidence Limit).
- Negative agreement: 99.96% (2807/2808) (99.9%, 1-sided lower 95% Confidence Limit).
- Repeat rate: 0.8%.
Freeze-Thaw Tolerance: Overall percent agreement of 100% (99.92%, 1sided lower 95% Confidence Limit).
Real-Time Stability: Observed percent agreement of 100% (99.1%, 1-sided lower 95% Confidence Limit).
Reproducibility Study:
- Percent agreement across sites: 99.987% to 99.994%.
- Overall accuracy rate: 99.994% (99.986, 1-sided lower 95% Confidence Limit).

Predicate Device(s)

K043011

Reference Device(s)

Not Found

Predetermined Change Control Plan (PCCP) - All Relevant Information

Not Found

Regulation Number and Section

§ 866.5900 Cystic fibrosis transmembrane conductance regulator (CFTR) gene mutation detection system.

(a)
Identification. The CFTR gene mutation detection system is a device used to simultaneously detect and identify a panel of mutations and variants in the CFTR gene. It is intended as an aid in confirmatory diagnostic testing of individuals with suspected cystic fibrosis (CF), carrier identification, and newborn screening. This device is not intended for stand-alone diagnostic purposes, prenatal diagnostic, pre-implantation, or population screening.(b)
Classification. Class II (special controls). The special control is FDA's guidance document entitled “Class II Special Controls Guidance Document: CFTR Gene Mutation Detection System.” See § 866.1(e) for the availability of this guidance document.

Summary

K063787

510(k) SUMMARY

TRADE NAME:

MAR 1 3 2008

InPlex™ CF Molecular Test

COMMON NAME:

CFTR Gene Mutation Detection System

CLASSIFICATION NAME:

Class II, 21 CFR 866.5900: Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Gene Mutation Detection System

PREDICATE DEVICE:

Tag-It™ Cystic Fibrosis Kit, K043011

DEVICE DESCRIPTION:

INDICATIONS FOR USE / INTENDED USE:

InPlex™ CF Molecular Test 510(k) Submission

genotyping test that provides information intended to be used for cystic fibrosis carrier screening as recommended by ACMG and the 2005 American College of Obstetricians and Gynecologists (ACOG) for adults of reproductive age, as an aid in newborn screening for cystic fibrosis, and in confirmatory diagnostic testing for cystic fibrosis in newborns and children.

Characteristics	Tag-It™ Cystic Fibrosis Kit	InPlex™ CF Molecular Test
Intended Use	The Tag-It™ Cystic Fibrosis Kit is a
device used to simultaneously detect and
identify a panel of mutations and variants
in the cystic fibrosis transmembrane
conductance regulator (CFTR) gene in
human blood specimens. The panel
includes mutations and variants currently
recommended by the American College
of Medical Genetics and American
College of Obstetricians and
Gynecologists (ACMG/ACOG), plus
some of the worlds most common and
North American-prevalent mutations.
The Tag-It™ Cystic Fibrosis Kit is a
qualitative genotyping test which
provides information intended to be used
for carrier testing in adults of
reproductive age, as an aid in newborn
screening, and in confirmatory diagnostic
testing in newborns and children.

The kit is not indicated for use in fetal
diagnostic or pre-implantation testing.
This kit is also not indicated for stand-alone diagnostic purposes. | Same as the predicate device
for the ACMG/ACOG panel,
but with no further mutations
beyond the ACMG/ACOG
panel. |
| Gene mutation and
variant screening | The Tag-It™ Cystic Fibrosis Kit
simultaneously screens for the 23 cystic
fibrosis transmembrane conductance
regulator (CFTR) gene mutations, plus
16 other mutations and 4 variants
(polymorphisms) recommended by the
American College of Medical Genetics
(ACMG) and American College of
Obstetricians and Gynecologists (ACOG) | The InPlex™ CF Molecular
Test simultaneously screens
for the 23 cystic fibrosis
transmembrane conductance
regulator (CFTR) gene
mutations and IVS8 variants
(polymorphisms)
recommended by the
American College of Medical
Genetics (ACMG) and
American College of
Obstetricians and
Gynecologists (ACOG) |

SUMMARY OF TECHNOLOGICAL CHARACTERISTICS:

Characteristics	Tag-It™ Cystic Fibrosis Kit	InPlex™ CF Molecular Test
Technology:
Genomic DNA
sample preparation	Genomic DNA Sample Preparation from
whole blood	Same as the predicate device
Technology: Assay
reactions	(1)Multiplex PCR
(2) Multiplex Allele Specific Primer
Extension (ASPE)
(3) PCR samples are treated with
enzymes (Alkaline Phosphatase
(SAP) to inactivate any remaining
nucleotides (especially dCTP), and
with Exonuclease I (EXO) to degrade
any primers left over from the PCR
reaction)
(4) Multiplex allele-specific primer
extension (ASPE) for genotyping
(5)Hybridization using 5 $ μ $ L ASPE with
45 $ μ $ L bead	(1)Multiplex PCR
(2)Amplified samples are
mixed with enzyme
(Cleavase®) and buffer
and added to micro-fluidic
cards
(3)Invader® reactions occur
whereby two
oligonucleotides (a
discriminatory Primary
Probe and an Invader®
oligonucleotide) hybridize
in tandem to the target
DNA to form an
overlapping structure that
is subsequently cleaved by
an enzyme (Cleavase®).
Data Analysis	Assay result data is analyzed by a
software program which provides a final
genotype	Same as the predicate device

PERFORMANCE DATA:

ANALYTICAL STUDIES

DNA Extraction Equivalency

Equivalency between four different DNA purification methodologies and their subsequent performance using the InPlex™ CF Molecular Test was evaluated using 7 CF positive genomic DNA samples isolated from whole peripheral blood. Internal processing involved the purification of the same 7 samples using four commonly used, commercially available DNA extraction kits. These samples were also genotyped by DNA sequencing to confirm results.

Genotype calls as generated by the InPlex™ CF Molecular Test were compared to DNA sequencing based genotyping for all extraction methods.

The seven CF positive genomic DNA samples extracted from human peripheral whole blood using four different DNA extraction kits generated a total of 644 calls with 28 positive and 616 normal (negative) calls. Each extraction method provided 161 data points. All calls were concordant with the DNA sequencing based genotyping, providing an overall agreement of 100% (99.5%, 1-sided lower 95% Confidence Limit).

Fluorometer Equivalency

Equivalency between three multi-well fluorometers meeting indicated specifications in generating signal data from the InPlex™ CF Molecular Test micro-fluidic card was determined using a panel of eight genomic DNA (gDNA) samples. The genomic DNA samples were either positive for a subset of the CFTR mutations listed as part of the ACMG recommended panel or normal. The eight samples were tested in triplicate providing a minimum of 1,656 data points (8 samples x 23 assays x 3 replicates x 3 plate reader data).

All InPlex™ CF Molecular Test genotype results for the characterized gDNA samples described were concordant with DNA sequencing genotype results and in 100% (99.8% 1-sided lower 95% Confidence Limit) agreement regardless of the fluorometer used.

Incubator Equivalency

Three thermal incubators were evaluated for equivalency in generating concordant genotype data using the InPlex™ CF Molecular Test. A panel of eight gDNA samples was tested in triplicate using each incubator. A total of 1,656 data points (8 samples x 23 assays x 3 replicates x 3 incubators - 552 data points per incubator) were generated in this study. The percent agreement to known genotypes was 99.88% (99.74%, 1-sided 95% Confidence Limit).

Interfering Substances

The performance of the InPlex™ CF Molecular Test was evaluated using eight, CF positive, genomic DNA samples isolated from whole peripheral blood in the absence (untreated) and presence (treated) of potential interfering substances included compounds that are endogenous to the blood sample matrix, compounds associated with blood collection and those that may result from sample preparation solutions. Depending on the nature of the substance, compounds were added either directly to the blood sample or to purified genomic DNA. Compounds added to the blood sample included bilirubin, triglycerides, and potassium EDTA (blood collection anti-coagulant). Compounds added to the purified genomic DNA sample included Qiagen® Buffer AW2 and hemoglobin. Following DNA extraction, eight whole peripheral blood samples were each tested as treated and untreated samples. The number of samples provided 1,104 data points (8 blood samples x 23 assays x 6 treatments).

The InPlex™ CF Molecular Test detected a total of 23 CFTR mutations. The genotype calls for the DNA samples containing an interfering substance were compared to the untreated samples for calculation of percent agreement. The results of this study showed that the InPlex™ CF Molecular Test achieved a 100% agreement (99.7%, 1-sided lower 95% Confidence Limit)

between the genotypes of the samples containing potential interfering substances and bidirectional sequencing.

Limit of Detection

An input genomic DNA concentration range was evaluated with the InPlex™ CF Molecular Test. A panel of eight gDNA samples were prepared and tested at eight concentrations ranging from 1ng/uL to 150ng/uL (total input DNA range of 5-750ng / reaction). The number of samples analyzed provided 1,472 data points (8 samples x 8 concentrations x 23 assays). One hundred and eighty four genotype calls were generated per DNA concentration tested (8 samples x 1 concentration x 23 assays).

Genotype call results for all eight characterized samples were assessed for percent agreement at each concentration. The lower limit of detection was defined as the lowest DNA concentration in which a 99% or greater concordance with DNA sequencing was observed.

Based on these results, a 5ng/μL DNA concentration (input DNA of 25ng) provided a percent agreement of 100% (98.4%, 1-sided lower 95% Confidence Limit) with DNA sequencing qualifying it as the lower limit of detection. At the highest DNA concentration tested, 150ng/uL (input DNA of 750ng), a 99.5% percent agreement was obtained (98.6%, 1 sided lower 95% Confidence Limit). The remaining DNA concentrations tested, 10, 20, 50, and 100ng/ul (input DNA of 50, 100, 250 and 500ng) all obtained a 100 percent agreement (98.4%, 1-sided lower 95% Confidence Limit) with expected results (see Table 3).

Table 3. Percent Agreement for Each DNA Concentration
DNA
Concentration
(ng/µL)	# of
Concordant
Calls	# of Low
Signal
Calls	# of
Equivocal
Calls	Total #
of Calls	Percent
Agreement	1 sided lower
95% CI
1	148	27	9	184	80.40%	75.6%
2	178	4	2	184	96.80%	94.6%
5	184	0	0	184	100.00%	98.4%
10	184	0	0	184	100.00%	98.4%
20	184	0	0	184	100.00%	98.4%
50	184	0	0	184	100.00%	98.4%
100	184	0	0	184	100.00%	98.4%
150	183	0	1	184	99.50%	98.6%

Lot-to-Lot Equivalency

Equivalency between three lots of InPlex™ CF Molecular Test kits was evaluated. A panel of 23 CFTR gDNA samples was tested in singlicate with each lot. Five hundred twenty-nine (529) genotype calls were generated for each lot tested (23 samples x 23 assays). All genotype calls were in 100% (99.4% 1-sided lower 95% Confidence Limit) agreement to pre-characterized gDNA genotypes for each lot tested.

Accuracy and Repeat Rate

Accuracy and repeat rate of the InPlex™ CF Molecular Test was determined by comparing InPlex™ CF Molecular Test genotyping results from of a panel of unique genomic DNA samples to bi-directional DNA sequence analysis. The sample panel tested consisted of genomic DNA samples isolated from peripheral whole blood and cell lines. A total of 23 CFTR mutations (as well as the IVS8-5T/7T/9T variants) were tested in this study. Genotype calls were compared between the DNA sequencing results and the InPlex™ CF Molecular Test results for the calculation of overall agreement. In addition, positive and negative agreement for each mutation was calculated. The repeat rate was determined by the number of samples that generated an invalid genotype call for one or more mutations with the InPlex™ CF Molecular Test on the first attempt.

This study was performed on a total of 123 unique samples containing 144 positive CFTR calls and 2808 normal (negative) CFTR calls.

The results of this study showed that the InPlex™ CF Molecular Test achieved 99.96% (2951/2952) overall agreement (99.9%, 1-sided lower 95% Confidence Limit), 100% (144/144) positive agreement (97.9%, 1-sided lower 95% Confidence Limit), and 99.96% (2807/2808) negative agreement (99.9%, 1-sided lower 95% Confidence Limit). There was one "Invalid" call out of 123 tests, resulting in a repeat rate of 0.8%. This overall agreement of 99.96% is derived without repeat testing. No repeat testing was done to support this study.

Of the 123 unique samples in the initial study, four (4) samples (negative for R117H) were confirmed by sequencing as 77/9T, but miscalled as 7T/7T by the InPlex™ CF Molecular Test. The four miscalled samples were tested in a reproducibility study once a day for 5 days in conjunction with two 7T/7T and two 7T/9T samples, which were called correctly in the initial study. Upon retesting all four (4) miscalled samples gave the correct 7T/9T result on each of the five consecutive days. The root cause of the miscalls was determined to potentially be due to the InPlex cards failing to be rotated during incubation resulting in temperature inconsistencies

InPlex™ CF Molecular Test 510(k) Submission .

(e.g., hot spots) near the heat source causing the 9T/7T ratio to fall below the cut-off for the 9T/7T call. The 7T/7T miscall was reproduced in one of the four samples experimentally.

Freeze-Thaw Tolerance

A study was performed to establish the tolerance of the InPlex™ CF Molecular Test to various freeze-thaw cycles. The InPlex™ CF Molecular Test kit were subjected to 2, 4, 6, 8, 10 and 12 freeze-thaw cycles (test points) followed by functional testing of the product with control samples. Each freeze-thaw cycle consisted of freezing at -20°C±3°C for at least 24 hours and thawing at room temperature for 30 minutes. For the multiple freeze-thaw cycles the cards were returned to -20°C±3 °C storage after 30 minutes at room temperature.

This study used a panel of 23 CFTR gDNA samples to evaluate the 23 CFTR mutations at each freeze thaw cycle. The samples generated 3,703 data points (23 samples x 23 assays x 7 test points). Each Freeze-Thaw cycle generated 529 data points (23 samples x 23 assays).

The InPlex™ CF Molecular Test achieved an overall percent agreement of 100% (99.92%, 1sided lower 95% Confidence Limit) as compared to DNA sequencing for all freeze-thaw cycles tested. See Table 4 below.

Table 4. Percent Agreement for Each Freeze-Thaw Period
Freeze-Thaw
Cycle	Concordant Calls	Total # of Calls	Percent
Agreement	1-sided lower
95% CI
0	529	529	100%	99.4%
2	529	529	100%	99.4%
4	529	529	100%	99.4%
6	529	529	100%	99.4%
8	529	529	100%	99.4%
10	529	529	100%	99.4%
12	529	529	100%	99.4%
Total	3,703	3,703	100%	99.92%

Eight or fewer freeze-thaw cycles for the InPlex™ Molecular Test are recommended to ensure the InPlex™ CF Molecular Test maintains the ability to generate accurate genotype calls.

Real-Time Stability

A study was performed with the objective to establish the stability of the InPlex™ CF Molecular Test when stored under pre-defined storage conditions.

Three lots of the InPlex™ micro-fluidic cards and three lots of InPlex™ reagents were used to create three master lots of the InPlex™ CF Molecular Test used in this study. A panel of seven CFTR gDNA samples and a panel of eight control samples comprised of pooled amplicons that cover the mutations in the InPlex™ CF Molecular Test were used to assess the performance. The samples were tested in duplicate with each lot for each storage condition at each time point.

Three lots of the InPlex™ Reagents are being maintained at the recommended storage temperature of (-20°C +/-3°C). The InPlex™ micro-fluidic cards are stored at the recommended temperature (-20°C +/-3°C), substandard temperature (4°C -- 8°C), and ship-stressed conditions (37°C±1°C for 48 hours, then room temperature (25°C+/-3°C).

A total of 345 calls were evaluated (15 samples x 23 assays) for each lot tested. All lots met the passing criterion for the Real-Time Stability performance evaluation study with an observed percent agreement of 100% (99.1%, 1-sided lower 95% Confidence Limit) (see Table 5).

Table 5: Percent Agreement for Each Lot at Each Storage Condition
Time
point	Storage Condition	Master Lot	# of
Concordant
Calls	Total # of Calls	Percent
Agreement	1-sided
lower 95%
CI
T=0	NA	C04AC	345	345	100%	99.1%
T=0	NA	C05AA	345	345	100%	99.1%
T=0	NA	C06AA	345	345	100%	99.1%
T=1	-20°C±3°C	C04AC	345	345	100%	99.1%
		C05AA	345	345	100%	99.1%
		C06AA	345	345	100%	99.1%
T=1	4-8°C	C04AC	345	345	100%	99.1%
		C05AA	345	345	100%	99.1%
		C06AA	345	345	100%	99.1%
T=1	37°C±1°C for 48
hours,
then hold at room
temp	C04AC	345	345	100%	99.1%
		C05AA	345	345	100%	99.1%
		C06AA	345	345	100%	99.1%

This study showed that multiple lots of the InPlex™ CF Molecular Test produce equivalent results after storage for one month under the conditions tested. This is an ongoing study for which the results from future time points will be used to extend shelf-life dating.

Reproducibility Study

Samples from pre-characterized commercially available reference materials were used to assess the reproducibility of the InPlex™ CF Molecular Test. The study was conducted in two main phases, a proficiency phase, and a performance phase. The purpose of the proficiency phase was to ensure that each site had the required expertise in the fundamental methodologies so that meaningful assay reproducibility measures could be calculated from the data generated during the performance phase. The proficiency phase was carried out in two steps, one for the training on the product methodology, and one for the confirmation of proficiency. Proficiency testing was performed using eight (8) pre-characterized, gDNA samples. Technicians at each of three investigative sites ran the same DNA samples in duplicate. Data generated during this phase of the study was to demonstrate technician proficiency only and not used to demonstrate the clinical performance of the test.

The reproducibility testing for the performance phase was conducted using 23 samples containing mutations representing the ACMG recommended panel. Each site ran the same samples tested in duplicate by each technician, with tests being run on each of five (5) nonconsecutive days. There were two technicians at each test site.

Percent agreement ranged from 99.962% to 100% at each of the three sites and 99.987% to 99.994% across all sites. Out of 31,740 calls generated (23 [samples] x 23 [assays] x 2 [replicates] x 2 [operators] x 5 [days] x 3 [sites]), the within operator agreement for two calls at one site vielded an equivocal result for both replicates of a single mutation. The overall accuracy rate was 31,738/31,740 = 99.994% (99.986, 1-sided lower 95% Confidence Limit).

DEPARTMENT OF HEALTH & HUMAN SERVICES

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Food and Drug Administration 2098 Gaither Road Rockville MD 20850

MAR 1 3 2008

Third Wave Technologies, Inc. c/o Andrew A. Lukowiak, Ph.D. Associate Vice President of Product Development 502 S. Rosa Road. Madison, WI 53719-1256

Re: K063787

Trade/Device Name: InPlex™ CF Molecular Test Regulation Number: 21 CFR 866.5900 Regulation Name: Cystic fibrosis transmembrane conductance regulator (CFTR) gene mutation detection system Regulatory Class: Class II Product Code: NUA Dated: March 7, 2008 Received: March 10, 2008

Dear Dr. Lukowiak:

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 the Code of Federal Regulations, Title 21, Parts 800 to 898. 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 Part 801); good manufacturing practice requirements as set forth in the quality systems (QS) regulation (21 CFR Part 820). This letter will allow you to begin marketing your device as described in your Section 510(k) premarket notification. The

Page 2 –

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 (240) 276-0450. Also, please note the regulation entitled, "Misbranding by reference to premarket notification" (21 CFR Part 807.97). For questions regarding postmarket surveillance, please contact CDRH's Office of Surveillance and Biometric's (OSB's) Division of Postmarket Surveillance at 240-276-3474. For questions regarding the reporting of device adverse events (Medical Device Reporting (MDR)), please contact the Division of Surveillance Systems at 240-276-3464. 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 (240) 276-3150 or at its Internet address http://www.fda.gov/cdrh/industry/support/index.html.

Sincerely vours.

Rola Becker

Robert L. Becker, Jr., M.D., Ph.D. Director Division of Immunology and Hematology Devices Office of In Vitro Diagnostic Device Evaluation and Safety Center for Devices and Radiological Health

Enclosure

INDICATIONS FOR USE STATEMENT

510(k) Number (if known): K063787

Device Name: InPlex™ CF Molecular Test

Indications For Use:

InPlex™ CF Molecular Test is an in vitro diagnostic device used to simultaneously detect and identify a panel of mutations and variants in the cystic fibrosis transmembrane regulator (CFTR) gene in genomic DNA samples isolated from human peripheral whole blood specimens. The panel includes mutations and variants recommended by the 2004 American College of Medical Genetics (ACMG), plus other more common and North American-prevalent mutations. The InPlex™ CF Molecular Test is a qualitative genotyping test that provides information intended to be used for cystic fibrosis carrier screening as recommended by ACMG and the 2005 American College of Obstetricians and Gynecologists (ACOG) for adults of reproductive age, as an aid in newborn screening for cystic fibrosis, and in confirmatory diagnostic testing for cystic fibrosis in newborns and children.

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

Concurrence of CDRH, Office of Device Evaluation (ODE)

Prescription Use

Over-The-Counter Use

(Per 21 CFR 801.109)

ia m c

(Optional Format 1-2-96)

Division Sign-01

Office of In Vitro Diagn Device Evaluation and

510(k) K063787