Predicate Devices

Reference Devices

Not Found

AI/ML (Artificial Intelligence / Machine Learning)

No
The device description and performance studies focus on standard molecular biology techniques (PCR, OLA, electrophoresis) and software analysis based on size and fluorescent labels, with no mention of AI or ML.

Therapeutic

No.
This device is an in vitro diagnostic device used for genotyping mutations and variants in the CFTR gene, providing information for carrier screening, newborn screening, and confirmatory diagnostic testing. It does not provide any therapeutic intervention.

Diagnostic

Yes

The "Intended Use / Indications for Use" section explicitly states that the device is a "qualitative in vitro diagnostic device" and provides information "intended to be used for carrier screening... as an aid in newborn screening, and in confirmatory diagnostic testing in newborns and children."

SaMD (Software as a Medical Device)

No

The device description explicitly states that the device includes "reagents" and relies on a "Celera CEGA-16™ Instrument System" for electrophoresis and detection. While software is used for analysis, it is part of a larger system that includes hardware and chemical components.

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 it is a "qualitative in vitro diagnostic device".
Specimen Type: It is used to genotype mutations and variants in genomic DNA isolated from "human whole blood specimens". This is a biological specimen taken from the human body.
Purpose: The purpose is to provide information for "carrier screening", "newborn screening", and "confirmatory diagnostic testing". These are all diagnostic or screening purposes.
Method: The device performs laboratory tests (PCR, OLA, electrophoresis) on the biological specimen to obtain results.

All of these characteristics align with the definition of an In Vitro Diagnostic device.

PCCP (Predetermined Change Control Plan) Authorized

N/A

Overview

Intended Use / Indications for Use

The Cystic Fibrosis Genotyping Assay is a qualitative in vitro diagnostic device used to genotype a panel of mutations and variants in the cystic fibrosis transmembrane conductance regulator (CFTR) gene in genomic deoxyribonucleic acid (DNA) isolated from human whole blood specimens. The panel includes mutations and variants recommended by the American College of Medical Genetics (ACMG, 2004) and the American College of Obstetricians and Gynecologists (ACOG, 2005) plus additional multiethnic mutations. '23 The Cystic Fibrosis Genotyping Assay 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 of newborns and children. This test is not indicated for use in fetal diagnostic or pre-implantation testing. This test is also not indicated for stand-alone diagnostic purposes.

Product codes

NUA

Device Description

The Cystic Fibrosis Genotyping Assay is designed to genotype the normal and mutant alleles at 30 loci of the CFTR gene using purified human genomic DNA. Genotype coverage includes the panel of 23 mutations recommended by the American College of Medical Genetics (ACMG) 2004 guidelines for use in CF population carrier screening. Coverage also includes 9 additional mutations as part of an expanded core panel to support genetic diversity of multiethnic populations that may be underserved by the ACMG panel alone (e.g. Hispanic, African American). In addition to core panel coverage, the assay is designed to detect polythymidine variants (5/7/9T) within intron 8 of the CFTR gene and polymorphisms (1506V, I507V, and F508C) within Exon 10 of the CFTR gene, in accordance with ACMG guidelines.

Purified genomic human DNA is prepared by standard purification methods. A multiplex polymerase chain reaction (PCR) is then performed to amplify the genomic DNA sample with 16 pairs of PCR primers and DNA polymerase. Next, the oligonucleotide ligation assay (OLA) is performed on the CFTR amplicons. Allele-specific OLA probes hybridize to the respective normal, mutant, and variant alleles and become ligated with fluorescent-labeled common probes by the ligase enzyme. The OLA probes are varied in length due to the addition of inert mobility modifiers. The ligated, fluorescent-labeled DNA fragments are separated on the Celera CEGA-16™ Instrument System by electrophoresis. Detection is based on size and fluorescent label. The ligation products are then identified and genotyped by analysis with the CEGA-16 software and assay-specific configuration disk.

The CF GT Assay also contains Reflex OLA reagents for the detection of the polythymidine 5/7/9T variants in intron 8 of the CFTR gene and for the detection of the I506V, I507V, and F508C polymorphisms in Exon 10 of the CFTR gene. The CFTR R117H mutation, along with the 5T variant of the 5/7/9T polymorphism in intron 8 on the same chromosome (cis), can cause classical CF if another CF mutation is present on the other chromosome. As a result, reflex testing for the 5/7/9T variant with the CF 5/7/9T Reflex OLA assay is recommended when the R117H mutation is detected. The CF Exon 10 Reflex OLA assay is used to verify a homozygous deletion of the F508 or 1507 codon and to exclude a potential false-positive result due to interference by certain non-CF causing variants at codons 506, 507, and 508. The CF Exon 10 Reflex OLA assay will distinguish between a true homozygous F508del or I507del from a sample containing one F508del or I507del allele plus the benign variants of I506V, I507V or F508C, respectively. The same software contained on the configuration disk is used to report reflex testing genotyping information.

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

The study was conducted using a set of 201 samples where each core mutation was present within a minimum of 4 samples. The sample set was comprised of 163 unique samples representing purified genomic DNA (51), frozen whole blood (98), fresh whole blood (6) and frozen cell line pellets (8). Most of the samples (65%) contained a single, heterozygous core mutation.
Bi-directional dideoxy DNA sequencing was performed on all clinical trial samples by an independent supplier operating under applicable good laboratory practices (GLPs) and current good manufacturing practices (cGMP) according to 21 CFR Parts 58, 211 and 820, respectively. Polymerase Chain Reaction (PCR) amplification was performed for fifteen (15) regions of interest (ROI) within each sample. Amplicons were sequenced to provide a minimum of double strand sequence data (4-fold coverage, 2 reads per strand) or, for regions containing a heterozygote insertion/deletion. 4-fold coverage from a single strand.

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

Accuracy Study:

Study Type: Clinical accuracy study comparing Cystic Fibrosis Genotyping Assay results to bi-directional dideoxy DNA sequencing (gold standard).
Sample Size: 201 samples (163 unique samples + 38 duplicates generated from unique samples). Each mutation was present in a minimum of 4 samples (except 394delTT with 3). Samples were tested twice (2 replicates per sample) at two test sites. Total replicates tested = (Total Samples with Mutation) x (2 replicates/sample) x (2 test sites).
Key Results: Analysis of results from the core OLA assay indicated 100% agreement with the reference results obtained through sequencing for all 32 tested mutations. Analysis of results from the two reflex OLA assays indicated nearly 100% agreement with sequencing. One sample replicate produced a 5T/7T/9T genotype call per the 5/7/9T OLA Reflex Assay where sequencing indicated only 5T and 7T were present. Overall percent agreement between the Cystic Fibrosis Genotyping Assay and sequencing was 99.996%.
System Failure Rate: 0.004% failure rate (1 incorrect genotype call out of 24,954 calls), associated with the 5/7/9T Reflex OLA Assay.
Retest Rate: 2.5% (20 out of 804 sample replicates) required retest due to poor reaction, PCR failure, or questionable GeneMapper quality flags. 6.6% (53 samples) had injection failures on the genetic analyzer and required reinjection.

Reproducibility Study:

Study Type: Single-blinded study conducted at three external test sites to determine clinical reproducibility.
Sample Size: 144 reproducibility study samples, consisting of 3 replicates each of 48 unique genomic DNA samples (25 from frozen whole blood, 23 from frozen cell line pellets). Each site conducted 3 independent assays x 144 samples/assay = 432 results per site. Total results = 1,296 (48 samples x 27 replicates).
Key Results: The Cystic Fibrosis Genotyping Assay showed 100% reproducibility, with zero (0) missed calls. Statistical analysis of all 1,296 results indicated 100% agreement between the Core OLA Assay and sequencing, with a one-sided 95% lower confidence limit equal to 99.8%. Reflex testing also showed 100% agreement.

Precision Study:

Study Type: Analysis of precision based on site-to-site, operator-to-operator, and lot-to-lot comparisons.
Sample Size: A total of 1,296 test results for each mutation (48 samples x 27 replicates). For reflex assays, 81 test results for each sample (27 sample replicates x 3 tests each).
Key Results: The Core OLA assay detected all 23 mutations and normal (wild type) alleles with a precision of 100%. Exon 10 reflex testing and 5/7/9T reflex testing also showed 100% precision.

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

Accuracy:

Percent Agreement (Sequencing vs. Test Results - Core OLA Assay): 100% for all individual target loci.
Overall Percent Agreement (Sequencing vs. Test Results - Core + Reflex OLA Assays): 99.996% (24,953 concordant calls out of 24,954 total calls).
One-sided 95% lower confidence limit for overall agreement: 99.98%

Reproducibility:

Reproducibility: 100% (zero missed calls).
One-sided 95% lower confidence limit for agreement: 99.8% (overall) and 99.1% (for each individual sample).

Precision:

Core OLA Assay: 100% correct calls across sites, operators, and lots for all 23 mutations.
Reflex Assays (Exon 10 and 5/7/9T): 100% correct calls across sites, operators, and lots.

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

0

510(k) SUMMARY

Cystic Fibrosis Genotyping Assay

SEP -7 2007

This summary of 510(k) safety and effectiveness information is being submitted in accordance with the requirements of SMDA 1990 and CFR 807.92.

1. Submitter name, address, contact person and date prepared:
Submitter:	Celera, An Applera Corporation Business
	1401 Harbor Bay Parkway
	Alameda, CA 94502
	T: (510) 749-4200

Company Contact:	Victoria Mackinnon
	VP, RA / CA
	T: (510) 749-4389
	F: (510) 749-1803

Date Summary Prepared: September 4, 2007

Device Name:

Device Generic Name:	Cystic Fibrosis Genotyping Assay
Device Trade Name:	Cystic Fibrosis Genotyping Assay
Product Code:	NUA
Classification:	Class II (21 CFR 866.5900)
510(k) Number:	#K062028

1

3. Equivalence to Legally Marketed Device

The Celera Cystic Fibrosis Genotyping (CF GT) Assay is equivalent to the Tm Bioscience Corporation Tag-It™ Cystic Fibrosis Kit 510(k) #K043011 and to bidirectional DNA sequence analysis (per FDA Special Controls Guidance, CFTR Gene Mutation Detection Systems, issued October 26, 2005).

4. Device Description

The Cystic Fibrosis Genotyping Assay is designed to genotype the normal and mutant alleles at 30 loci of the CFTR gene using purified human genomic DNA. Genotype coverage includes the panel of 23 mutations recommended by the American College of Medical Genetics (ACMG) 2004 guidelines for use in CF population carrier screening. Coverage also includes 9 additional mutations as part of an expanded core panel to support genetic diversity of multiethnic populations that may be underserved by the ACMG panel alone (e.g. Hispanic, African American). In addition to core panel coverage, the assay is designed to detect polythymidine variants (5/7/9T) within intron 8 of the CFTR gene and polymorphisms (1506V, I507V, and F508C) within Exon 10 of the CFTR gene, in accordance with ACMG guidelines.

Purified genomic human DNA is prepared by standard purification methods. A multiplex polymerase chain reaction (PCR) is then performed to amplify the genomic DNA sample with 16 pairs of PCR primers and DNA polymerase. Next, the oligonucleotide ligation assay (OLA) is performed on the CFTR amplicons. Allele-specific OLA probes hybridize to the respective normal, mutant, and variant alleles and become ligated with fluorescent-labeled common probes by the ligase enzyme. The OLA probes are varied in length due to the addition of inert mobility modifiers. The ligated, fluorescent-labeled DNA fragments are separated on the Celera CEGA-16™ Instrument System by electrophoresis. Detection is based on size and fluorescent label. The ligation products are then identified and genotyped by analysis with the CEGA-16 software and assay-specific configuration disk.

The CF GT Assay also contains Reflex OLA reagents for the detection of the polythymidine 5/7/9T variants in intron 8 of the CFTR gene and for the detection of the I506V, I507V, and F508C polymorphisms in Exon 10 of the CFTR gene. The CFTR R117H mutation, along with

2

CELERA

the 5T variant of the 5/7/9T polymorphism in intron 8 on the same chromosome (cis), can cause classical CF if another CF mutation is present on the other chromosome. As a result, reflex testing for the 5/7/9T variant with the CF 5/7/9T Reflex OLA assay is recommended when the R117H mutation is detected. The CF Exon 10 Reflex OLA assay is used to verify a homozygous deletion of the F508 or 1507 codon and to exclude a potential false-positive result due to interference by certain non-CF causing variants at codons 506, 507, and 508. The CF Exon 10 Reflex OLA assay will distinguish between a true homozygous F508del or I507del from a sample containing one F508del or I507del allele plus the benign variants of I506V, I507V or F508C, respectively. The same software contained on the configuration disk is used to report reflex testing genotyping information.

5. Intended Use

The Cystic Fibrosis Genotyping Assay is a qualitative in vitro diagnostic device used to genotype a panel of mutations and variants in the cystic fibrosis transmembrane conductance regulator (CFTR) gene in genomic deoxyribonucleic acid (DNA) isolated from human whole blood specimens. The panel includes mutations and variants recommended by the American College of Medical Genetics (ACMG, 2004) and the American College of Obstetricians and Gynecologists (ACOG, 2005) plus additional multiethnic mutations. '23 The Cystic Fibrosis Genotyping Assay 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 of newborns and children. This test is not indicated for use in fetal diagnostic or pre-implantation testing. This test is also not indicated for stand-alone diagnostic purposes.

6. Substantial Equivalence / Performance Characteristics

The following table compares the Celera CF GT Kit with the TM Tag-IT CF Kit (#K043011) on basic system characteristics. The clinical trial for the Celera CF GT Kit was performed using sequencing as a gold standard. The performance characteristics the Celera CF GT Kit are well-documented and comparable to the TM Bioscience's product.

3

Parameter	Celera CF GT Assay	TM Bioscience
Intended Use	The Cystic Fibrosis Genotyping Assay
is a qualitative in vitro diagnostic
device used to genotype a panel of
mutations and variants in the cystic
fibrosis transmembrane conductance
regulator (CFTR) gene in genomic
deoxyribonucleic acid (DNA) isolated
from human whole blood specimens.
The panel includes mutations and
variants recommended by the
American College of Medical
Genetics (ACMG, 2004) and the
American College of Obstetricians
and Gynecologists (ACOG, 2005) plus
additional multiethnic mutations. The
Cystic Fibrosis Genotyping Assay
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 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.
Parameter	Celera CF GT Assay	TM Bioscience
Contra-indications	This device is not intended for use in fetal diagnostic or pre-implantation testing. This test is also not indicated for stand-alone diagnostic purposes.	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.
Product Description	The Cystic Fibrosis Genotyping Assay is designed to genotype the normal and mutant alleles at 30 loci of the CFTR gene using purified human genomic DNA. Genotype coverage includes the panel of 23 mutations recommended by the 2004 American College of Medical Genetics (ACMG) guidelines for use in CF population carrier screening. Coverage also includes 9 additional mutations as part of an expanded core panel to support genetic diversity of multiethnic populations. The assay is also designed to detect polythymidine variants (5/7/9T) within Intron 8 of the CFTR gene and polymorphisms (I506V, I507V, and F508C) within Exon 10 of the CFTR gene, in accordance with ACMG guidelines.	The Tag-It™ Cystic Fibrosis Kit tests for 39 mutations and 4 polymorphisms in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. These mutations include those currently recommended for testing by the ACMG/ACOG plus 16 mutations shown to be associated with CF phenotypes in Caucasian Americans, Hispanic Americans and African Americans.
Parameter	Celera CF GT Assay	TM Bioscience
Type of Test	Multiplex PCR followed by DNA
sequencing and OLA reflex testing.	Multiplex PCR followed by
multiplex allele specific primer
extension for genotyping,
hybridized to multiplexed
fluorescing microparticles,
detected by flow cytometry.
Specimen Type	Peripheral Human whole blood
(EDTA)	Peripheral Human whole blood
(EDTA)
Instrument
Systems	CEGA-16TM Instrument System	Luminex 100 IS
(Integrated System)
Software	Analysis Software:
CEGA-16TM Instrument Software with
supplied settings and parameters	Tag-IT Data Analysis Software
TDAS CF-I
No Template
(Negative)
Control	CF Sample Diluent
(included in Celera CF Genotyping
Assay Kit, Set-Up Module)	ddH2O Control
(included in Tag-IT CF Kit)
Positive Control	CFTR Wild Type Control
(included in Celera CF Genotyping
Assay Kit, Set Up Module)	Recommendation is to use
genomic DNA controls similar
to sample type with $\triangle$ F508
mutation
Reproducibility	100%	>99.99%
Precision	100%	>99.99%
Accuracy	>99.99% Agreement
(vs. bi-directional sequencing)	100% Agreement
	delF508	$\triangle$ F508
	dell507	$\triangle$ I507
Parameter	Celera CF GT Assay	TM Bioscience
Mutations	G542X	G542X
Detected	G551D	G551D
	W1282X	W1282X
	N1303K	N1303K
	R553X	R553X
	621+1G→T	621+1G→T
	R117H	R117H
	1717-1G→A	1717-1G→A
	A455E	A455E
	R560T	R560T
	R1162X	R1162X
	G85E	G85E
	R334W	R334W
	R347P	R347P
	711+1G→T	711+1G→T
	1898+1G→A	1898+1G→A
	2184delA	2184delA
	1078delT	1078delT
	3849+10kbC→T	3849+10kbC→T
	2789+5G→A	2789+5G→A
	3659delC	3659delC
	3120+1G→A	I148T
	394delTT	3120+1G→A
	S549N	394delTT
	S549R	Y122X
	V520F	R347H
Parameter	Celera CF GT Assay	TM Bioscience
	3876delA	V520F
	2183AA→G	A559T
	R347H	S549N
	3905insT	S549R
	5/7/9/T	2307insA
	1506V	Y1092X
	1507V	M1101K
	F508C	S1255X
		3876delA
		3905insT
		5/7/9T
		F508C
		1507V
		1898+5G→T
		2183AA→G
		1506V

4

:

.

5

6

7

Stability

The expiration date for the Celera CF GT Assay will be based on real-time stability testing.

Analytical Specificity / Interfering Substances

Commonly occurring biological substances that are present in patient blood were tested for their potential to interfere with the performance of the CF Genotyping Assay in detecting mutations. Potential interfering substances (hemoglobin, bilirubin, triglycerides, and protein) were added to whole blood prior to sample preparation process for purified genomic DNA.

8

CELERA

Results indicate that these four substances at the following concentrations did not interfere with the CF GT Assay:

Hemoglobin	600 to 820 mg/dL
Bilirubin	20 mg/dL
Triglycerides	500 mg/dL
Protein	8 g/dL

Clinical Testing

Overview

Clinical studies were performed to determine the performance characteristics of the Cystic Fibrosis Genotyping Assay. All studies were performed using materials, equipment and procedures described within the Cystic Fibrosis Genotyping Assay Operator's Manual. Every assay run included the CFTR Wild Type Control (allelic ladder/positive assay control) and No Template Control (CF Sample Diluent as a negative control), which are provided within the assay kit. Multiple reagent lots, testing sites, and operators were involved in performing the studies. Samples were blinded and randomized prior to use by the testing sites.

Bi-directional dideoxy DNA sequencing was performed on all clinical trial samples by an independent supplier operating under applicable good laboratory practices (GLPs) and current good manufacturing practices (cGMP) according to 21 CFR Parts 58, 211 and 820, respectively. Polymerase Chain Reaction (PCR) amplification was performed for fifteen (15) regions of interest (ROI) within each sample. Amplicons were sequenced to provide a minimum of double strand sequence data (4-fold coverage, 2 reads per strand) or, for regions containing a heterozygote insertion/deletion. 4-fold coverage from a single strand.

9

Accuracy

The objective of this study was to assess the clinical accuracy of the Cystic Fibrosis Genotyping Assay for the targeted normal and mutant alleles. The samples tested included genomic DNA obtained from frozen whole blood and frozen pellets from commercially available cell lines. Results from bi-directional dideoxy DNA sequencing by an independent testing service were used as the gold standard for purposes of assessing accuracy of the Cystic Fibrosis Genotyping Assay.

Sample Set

The study was conducted using a set of 201 samples where each core mutation was present within a minimum of 4 samples. The sample set was comprised of 163 unique samples representing purified genomic DNA (51), frozen whole blood (98), fresh whole blood (6) and frozen cell line pellets (8). Most of the samples (65%) contained a single, heterozygous core mutation. Full segmentation of the sample set is summarized in the following table:

Sample Segments	Number of Unique Samples
One Core CF Mutation	109
➤ Homozygotes	3 (all delF508)
➤ Heterozygotes	106
Two Core CF Mutations	23
Exon 10 Polymorphisms
without Core CF Mutation*	6
Normal for all targeted loci	25
	Total = 163

*Included to assess the assay's ability to detect the polymorphism when present.

To achieve the target minimum of 5 samples per mutation (one exception: 394delTT), additional independent aliquots were generated from 38 of the unique samples. Thus, the complete sample set consisted of 201 samples (i.e., 163 unique + 38 duplicates). Ultimately, all samples were blinded and randomized prior to shipment to the study sites. An overview of the mutation, polymorphism and variant distribution within each of the complete sample set is provided in the following Mutation, Polymorphism and Variant Distribution Table:

10

CYSTIC FIBROSIS GENOTYPING ASSAY 510(K) SUMMARY [#K062028]

	Mutations & P/V	Number of Unique Samples with Mutation(s)	Subset with a Second Mutation	Subset with Core P/V	Number of Unique Samples with P/V only	Additional Independent Aliquots from the Unique Sample Set	Total Samples with Mutation (Unique + Additional Independent Aliquots)	Total Samples with P/V	Total Replicates Tested
1	1078delT	4	—	N/A	N/A	1	5	N/A	20
2	1717-1G>A	5	2 of 5 (delF508)	N/A	N/A	-	5	N/A	20
3	1898+1G>A	3	—	N/A	N/A	2	5	N/A	20
4	2183AA>G	1	1 of 1 (delF508)	N/A	N/A	4	5	N/A	20
5	2184delA	4	1 of 4 (delF508)	N/A	N/A	1	5	N/A	20
6	2789+5G>A	5	1 of 5 (delF508)	N/A	N/A	—	5	N/A	20
7	3120+1G>A	4	—	N/A	N/A	1	5	N/A	20
8	3659delC	4	1 of 4 (G542X))	N/A	N/A	—	5	N/A	20
9	3849+10kbC>T	5	—	N/A	N/A	—	5	N/A	20
10	3876delA	5	—	N/A	N/A	1	6	N/A	24
11	3905insT	3	—	N/A	N/A	2	5	N/A	20
12	394delTT	3	—	N/A	N/A	1	4	N/A	16
13	621+1G>T	5	1 of 5 (711+1G>T)
1 of 5 (delF508)
1 of 5 (G85E)	N/A	N/A	4	9	N/A	36
14	711+1G>T	4	1 of 4 (621+1G>T)	N/A	N/A	1	5	N/A	20
15	A455E	5	1 of 5 (delF508)	N/A	N/A	—	5	N/A	20
16	delF508	26	2 of 26 (1717-1G>A)
1 of 26 (2183AA>G)
1 of 26 (2184delA)
1 of 26 (2789+5G>A)
1 of 26 (621+1G>T)
1 of 26 (R117H+5T)
1 of 26 (A455E)
3 of 26 (G542X)
2 of 26 (G551D)
1 of 26 (N1303K)
2 of 26 (R553X)
1 of 26 (R560T)
1 of 26 (S549N)
1 of 26 (W1282X)	2 (1506V)
1 (1507V)	N/A	7	33	N/A	132
17	delF507	7	—	0	N/A	1	8	N/A	32
18	G542X	5	1 of 5 (3659delC)
3 of 5 (delF508)	N/A	N/A	—	5	N/A	20
19	G551D	5	2 of 5 (delF508)	N/A	N/A	—	5	N/A	20
20	G85E	4	1 of 4 (621+1G>T)	N/A	N/A	1	5	N/A	20
21	N1303K	5	1 of 5 (delF508)	N/A	N/A	—	5	N/A	20
22	R1162X	3	—	N/A	N/A	2	5	N/A	20
23	R117H	5	1 of 5 (delF508)	2 (5T/7T)
3 (7T/7T)	N/A	—	5	N/A	20
24	R334W	3	—	N/A	N/A	2	5	N/A	20
25	R347H	4	1 of 4 (R553X)	N/A	N/A	1	5	N/A	20
26	R347P	5	—	N/A	N/A	—	5	N/A	20
27	R553X	4	2 of 4 (delF508)
1 of 4 (R347H)	N/A	N/A	1	5	N/A	20
28	R560T	4	1 of 4 (delF508)	N/A	N/A	1	5	N/A	20
29	S549N	3	1 of 3 (delF508)	N/A	N/A	2	5	N/A	20
30	S549R	2	—	N/A	N/A	3	5	N/A	20
31	V520F	4	—	N/A	N/A	1	5	N/A	20
32	W1282X	6	1 of 6 (delF508)	N/A	N/A	—	6	N/A	24
33a	5T	N/A	N/A	2 (R117H)	0	—	N/A	2	8
33b	7T	N/A	N/A	5 (R117H)	0	—	N/A	5	20
33c	9T	N/A	N/A	0	0	—	N/A	0	0
34	1506V	N/A	N/A	2 (delF508)	1	1	N/A	4	16
35	1507V	N/A	N/A	1 (delF508)	2	3	N/A	6	24
36	F508C	N/A	N/A	0	3	2	N/A	5	20

11

a. P = Exon 10 polymorphisms (F508C, I506V, and I507V); V = Intron 8 variants (5T, 7T, and 9T)
b. Represents samples that contained an Exon 10 polymorphism without a corresponding core mutation (of either homozygous delF508 or delI507).
Represents independent aliquots of individual, unique samples that were used to achieve the target minimum of 4 samples per mutation.
sumples per mulation:
d. Total Replicates Tested = [Total Samples with Mutation] x [2 replicates/sample] x [2 test sites]

e. “——” = zero

·

e. "—" = zero
f. N/A = not applicable

12

Accuracy Results

Analysis of results from the core OLA assay indicated 100% agreement with the reference results obtained through sequencing. The following table summarizes the Cystic Fibrosis Genotyping Assay results from the core OLA assay relative to the sequencing results. For each sample. analysis by both methods confirmed that normal alleles were present and detected for all loci excluding the loci that contained the expected mutations.

| Target Locus
by Mutations | Number of Sample with a Mutation Identified at
the Target Locus | Number Normal/Wild Type Allele Identified the Target Locus | Percent between
Sequencing and
Test Results (%) |
|------------------------------|--- | 1078delT | 20 | 1717-1G>A | 20 | 1898+1G>A | 20 | 2183AA>G | 20 | 2184delA | 20 | 2789+5G>A | 20 | 3120+1G>A | 20 | 3659delC | 20 | 3849+10kbC>T | 20 | 3876delA | 24 | 3905insT | 20 | 394delTT | 16 | 621+1G>T | 36 | 711+1G>T | 20 | A455E | 20 | delF508 | 132 | dell507 | 32 | G542X | 20 | G551D | 20 | G85E | 20 | N1303K | 20 | R1162X | 20 | R117H | 20 | R334W | 20 | R347H | 20 | R347P | 20 | R553X | 20 | R560T | 20 | S549N | 20 | S549R | 20 | V520F | 20 | W1282X | 24 Replicates
of Sample Replicates with a
at
Agreement
-------------------------------------------------------------------------------|-------------------------------------------------------------------------------------------------|--------------------------------------------------------------------|
| 804 | 100 |
| 804 | 100 |
| 804 | 100 |
| 804 | 100 |
| 804 | 100 |
| 804 | 100 |
| 804 | 100 |
| 804 | 100 |
| 804 | 100 |
| 804 | 100 |
| 804 | 100 |
| 804 | 100 |
| 804 | 100 |
| 804 | 100 |
| 804 | 100 |
| 792* | 100 |
| 804 | 100 |
| 804 | 100 |
| 804 | 100 |
| 804 | 100 |
| 804 | 100 |
| 804 | 100 |
| 804 | 100 |
| 804 | 100 |
| 804 | 100 |
| 804 | 100 |
| 804 | 100 |
| 804 | 100 |
| 804 | 100 |
| 804 | 100 |
| 804 | 100 |
| 804 | 100 |

Three samples that contained the delF508 mutation were homozygous mutants. Since these three samples did not contain any normal/wild type alleles at the F508 locus, the number of sample replicates with a wild type allele is lower |804 -- (3 samples/set x 2 sets/site x 2 sites) = 792].

13

Analysis of results from the two reflex OLA assays indicated nearly 100% agreement with sequencing, with the exception of one sample replicate that produced a 5T/77/9T genotype call per the 5/7/9T OLA Reflex Assay. Sequencing indicated that only the 5T and 7T alleles were present. The results from the two reflex OLA assays are summarized in the following table:

| P/Va | Number of
Replicates with
P/V Detected | Test Results (Reflex OLA Assays) | Number of Replicates that Did
Not Meet the Requirements
for Reflex Testingb | Number of Replicates
with Normal/Wild
Type Calls | Percent Agreement
between Sequencing and
Test Results (%) |
|-------|----------------------------------------------|----------------------------------|-----------------------------------------------------------------------------------|--------------------------------------------------------|-----------------------------------------------------------------|
| 5T | 8 | | N/Ac | 0 | 100 |
| 7T | 0 | | N/A | 16 | 100 |
| 9T | 0 | | N/A | 5d | 80 (4/5) |
| F508C | 0 | | 20 | 0c | 100 |
| I506V | 12 | | 4 | 16e | 100 |
| I507V | 4 | | 20 | 1e | 100 |

P = Exon 10 polymorphisms (F508C, 1506V, and 1507V); V = Intron 8 variants (5T, 7T and 9T) പ.

These are counted as correct results since in all cases the Core OLA Assay correctly made a wild type (normal)eall in the b. presceee of heterozygous F508C, 1506V or 1507V polymorphism (as indicated by sequencing).Reflex testing was not performed for these samples per the clinical trial protocol, which included a reflex testing algorithm based on ACMG recommendations

N/A = not applicable C.

The 5/7/9T OLA Reflex Assay generated a 51/77/91' call for one replicate of sample 6043, which did not match the ದೆ. reference sequencing call (5T/7T).

The Exon 10 OLA Reflex Assay is not designed to detect the normal allele F508. In the presence of deletion mutations, c. the 1506 and 1507 normal alleles are not seen by the Exon 10 OLA Reflex Assay in most cases; thus, Exon 10 results must always be analyzed with results from the Core OLA Assay.

All 201 samples, classified below into one of four genotypic categories, were tested twice for the presence of 32 mutant alleles, 30 normal alleles, 3 Exon 10 polymorphisms, and 3 polythymidine variants by each of the two independent sites:

25: normal DNA with none of the CFTR mutations within the test panel
140: contained one mutant allele within the core panel
35: contained either two mutant alleles within the core panel or one mutant allele + one polymorphism/variant
1: contained two mutant alleles within the core panel + one variant

Percent agreement between the Cystic Fibrosis Genotyping Assay and sequencing is summarized in the following table for all 24,954 genotype calls within the sample set. The results for the Core OLA Assay were compared to sequencing with respect to the mutations and normal alleles that the core assay is designed to identify in accordance with ACMG guidelines. 12 When reflex testing was indicated by the Core OLA Assay, the results for the polymorphisms or variants identified by the reflex testing were compared with sequencing. The

14

Cystic Fibrosis Genotyping Assay uses a testing algorithm based on the ACMG guidelines; therefore, testing for polymorphisms within Exon 10 and variants within the polythymidine tract of Intron 8 is only warranted when specific results are observed in the Core OLA Assay.

Sequencing Result	Test Result (Core + Reflex OLA Assays)			Concordant Call	Total (%)
	Normal /
Wild Type Call	Heterozygous
Call	Homozygous
Call
Normal / Wild Type	$\frac{24,145}{24,146}^a$	0	0	$24,145^a$	99.996
Heterozygous	0	796	0	796	100
Homozygous	0	0	12	12	100
Total	$\frac{24,145}{24,146}^a$	796	12	24,953	$99.996^b$

The 5/7/9T Reflex OLA Assay crroncously identified the 9T variant in one replicate of sample 6043.Sequencing દ્ધા. confirmed that this sample contained a heterozygous 5T variant and the 7T normal allele, which were also detected by the 5/7/9T Reflex OLA Assay (along with 9T).

b. The one-sided 95% lower confidence limit = 99.98%.

System Failure Rate

A total of 24.954 genotype calls was generated by the Cystic Fibrosis Genotyping Assay during the accuracy study, and all but one call matched the sequencing result (failure rate = 0.004%). The single incorrect genotype call was associated with the 5/7/9T Reflex OLA Assay result from a single replicate of sample 6043. The reflex assay erroneously indicated the presence of a 9T variant. One 5T and one 7T variant were also detected; thus, the genotype call was 5T/7T/9T. Sequencing confirmed that the sample contained a heterozygous 5T variant and the 7T normal allele. All other replicates of this sample produced the correct 5T/7T result.

Retest Rate

Of the 804 sample replicates tested, a total of 20 (2.5%) required retest due to a poor reaction or PCR failure, or GeneMapper quality flags that indicated that the sample should be repeated when data results were considered questionable or invalid. For samples that did not initially report a genotype result, the correct result was reported upon repeat testing.

Additionally, injection failures on the genetic analyzer were observed for 53 samples (6.6%). The injections were repeated using the same sample-specific electrophoretic mixes that were prepared for the original injection. Therefore, the genotype call was classified as 'delayed'

15

since repeating the assay was not required. In some cases, the user chose to limit the reinjection to the individual affected samples. In other cases, all samples within the capillary run or entire plate were reinjected, based on convenience. Reinjections were usually performed immediately after the previous run and were always performed within 48 hours of the addition of Hi-Di™ Formamide to the OLA sample. Upon reinjection, valid results were obtained for each sample. In cases where partial or entire plates were reinjected, the initial, correct genotype calls did not change upon reinjection.

An overview of the entire accuracy study results by mutation is provided in the following Accuracy Study by Mutation Table:

| Genotype by DNA
Sequencing | Number of Unique
Samples and/or
Replicates | Number CF GT Calls Before Repeat Testing (Based on Initial Results) | | | | Number of CF GT Calls After Repeat Testing (30 calls per sample x number of replicates) | | | | | | | |
|----------------------------------|--------------------------------------------------------|---------------------------------------------------------------------|------------------------------------------------------------------------|-------------------------|---------------------------------------|-----------------------------------------------------------------------------------------|----------|---------------------------------------------------------------------------------------------------|-------------------------|---------------------------|-------------------------------------|-------------------------------------------|---------------------------------------------------|
| | Site A | Site B | Correct Calls
Site A | Correct Calls
Site B | Delayed Calls (Re-injections) | Percent Agreement with Sequencing for Initial Results | Repeatsd | Correct Calls
Site A | Correct Calls
Site B | Missed (In-correct) Calls | Total Number of Correct Calls | Percent Agreement with Sequencing (%) | |
| 3659delC | 2 | 2 | 60 | 60 | 0 | 100 | 0 | 60 | 60 | 0 | 120 | 100 | |
| 621+1G>T/
delF508 | 2 | 2 | 60 | 60 | 0 | 100 | 0 | 60 | 60 | 0 | 120 | 100 | |
| 1078delT | 10 | 10 | 270 | 210 | 90 | 95.0 | 30 | 300 | 300 | 0 | 600 | 100 | |
| 1717-1G>A | 6 | 6 | 180 | 180 | 0 | 100 | 0 | 180 | 180 | 0 | 360 | 100 | |
| 1717-1G>A/
delF508 | 4 | 4 | 90 | 90 | 60 | 100 | 0 | 120 | 120 | 0 | 240 | 100 | |
| 1898+1G>A | 10 | 10 | 270 | 300 | 30 | 100 | 0 | 300 | 300 | 0 | 600 | 100 | |
| 2183AA>G/
delF508 | 10 | 10 | 270 | 270 | 30 | 95.0 | 30 | 300 | 300 | 0 | 600 | 100 | |
| 2184delA | 6 | 6 | 180 | 150 | 0 | 91.7 | 30 | 180 | 180 | 0 | 360 | 100 | |
| 2184 del A/
delF508 | 4 | 4 | 120 | 120 | 0 | 100 | 0 | 120 | 120 | 0 | 240 | 100 | |
| 2789+5G>A | 8 | 8 | 240 | 240 | 0 | 100 | 0 | 240 | 240 | 0 | 480 | 100 | |
| 2789+5G>A/
delF508 | 2 | 2 | 60 | 30 | 30 | 100 | 0 | 60 | 60 | 0 | 120 | 100 | |
| 3120+1G>A | 10 | 10 | 270 | 240 | 60 | 95.0 | 30 | 300 | 300 | 0 | 600 | 100 | |
| 3659delC | 6 | 6 | 180 | 180 | 0 | 100 | 0 | 180 | 180 | 0 | 360 | 100 | |
| 3849+10kbC>T | 10 | 10 | 300 | 270 | 0 | 95.0 | 30 | 300 | 300 | 0 | 600 | 100 | |
| 3876delA | 12 | 12 | 330 | 360 | 30 | 100 | 0 | 360 | 360 | 0 | 720 | 100 | |
| 3905insT | 10 | 10 | 240 | 300 | 60 | 100 | 0 | 300 | 300 | 0 | 600 | 100 | |
| 394delTT | 8 | 8 | 240 | 240 | 0 | 100 | 0 | 240 | 240 | 0 | 480 | 100 | |
| 621+1G>T | 8 | 8 | 210 | 240 | 30 | 100 | 0 | 240 | 240 | 0 | 480 | 100 | |
| 621+1G>T/
711+1G>T | 4 | 4 | 120 | 120 | 0 | 100 | 0 | 120 | 120 | 0 | 240 | 100 | |
| 711+1G>T | 6 | 6 | 150 | 150 | 30 | 91.7 | 30 | 180 | 180 | 0 | 360 | 100 | |
| A455E | 8 | 8 | 180 | 210 | 60 | 93.8 | 30 | 240 | 240 | 0 | 480 | 100 | |
| A455E/
delF508 | 2 | 2 | 60 | 60 | 0 | 100 | 0 | 60 | 60 | 0 | 120 | 100 | |
| delF508 | 2 | 2 | 30 | 60 | 30 | 100 | 0 | 60 | 60 | 0 | 120 | 100 | |
| delF508/
delF508e | 6 | 6 | 186e | 96e | 90 | 100 | 0 | 186e | 186e | 0 | 372e | 100 | |
| delI507 | 16 | 16 | 480 | 480 | 0 | 100 | 0 | 480 | 480 | 0 | 960 | 100 | |
| F508C | 10 | 10 | 270 | 270 | 30 | 95.0 | 30 | 300 | 300 | 0 | 600 | 100 | |
| Genotype by
DNA
Sequencing | Number of
Unique
Samples
and/or
Replicates | | Number CF GT Calls Before Repeat Testing (Based on
Initial Results) | | | Percent
Agreement
with
Sequencing
for Initial
Resultsc | Repeatsd | Number of CF GT Calls After
Repeat Testing (30 calls per
sample x number of
replicates)e | | | Missed
(In-
correct)
Calls | Total
Number
of
Correct
Calls | Percent
Agreement
with
Sequencing
(%) |
| | Site A | Site B | Site A | Site B | Delayed
Callsb (Re-
injections) | | | Site A | Site B | | | | |
| G542X | 2 | 2 | 60 | 30 | 30 | 100 | 0 | 60 | 60 | 0 | 120 | 100 | |
| G542X/
3659delC | 2 | 2 | 60 | 30 | 30 | 100 | 0 | 60 | 60 | 0 | 120 | 100 | |
| G542X/
delF508 | 6 | 6 | 180 | 150 | 30 | 100 | 0 | 180 | 180 | 0 | 360 | 100 | |
| G551D | 6 | 6 | 180 | 150 | 30 | 100 | 0 | 180 | 180 | 0 | 360 | 100 | |
| G551D/
delF508 | 4 | 4 | 120 | 90 | 30 | 100 | 0 | 120 | 120 | 0 | 240 | 100 | |
| G85E | 6 | 6 | 120 | 150 | 60 | 91.7 | 30 | 180 | 180 | 0 | 360 | 100 | |
| G85E/
621+1G>T | 4 | 4 | 90 | 120 | 30 | 100 | 0 | 120 | 120 | 0 | 240 | 100 | |
| 1506V | 2 | 2 | 30 | 60 | 30 | 100 | 0 | 60 | 60 | 0 | 120 | 100 | |
| 1506V/
delF508 | 6 | 6 | 156c | 155c | 30 | 91.7 | 31e | 186c | 186c | 0 | 372c | 100 | |
| 1507V | 10 | 10 | 300 | 300 | 0 | 100 | 0 | 300 | 300 | 0 | 600 | 100 | |
| 1507V/
delF508 | 2 | 2 | 32c | 62c | 30 | 100 | 0 | 62c | 62c | 0 | 124c | 100 | |
| N1303K | 8 | 8 | 240 | 240 | 0 | 100 | 0 | 240 | 240 | 0 | 480 | 100 | |
| N1303K/
delF508 | 2 | 2 | 60 | 30 | 30 | 100 | 0 | 60 | 60 | 0 | 120 | 100 | |
| R1162X | 10 | 10 | 300 | 270 | 0 | 95.0 | 30 | 300 | 300 | 0 | 600 | 100 | |
| R117H/7Tf | 6 | 6 | 126c | 125c | 90 | 91.7 | 31e | 186c | 186c | 0 | 372c | 100 | |
| R117H/5T/
7Tf | 2 | 2 | 64c | 63c,f | 0 | 99.2 | 0 | 64c | 63c | 1 | 127c | 99.2 | |
| R117H/5T/9T
/ delF508c | 2 | 2 | 64c | 64c | 0 | 100 | 0 | 64c | 64c | 0 | 128c | 100 | |
| R334W | 10 | 10 | 300 | 300 | 0 | 100 | 0 | 300 | 300 | 0 | 600 | 100 | |
| R347H | 8 | 8 | 180 | 210 | 60 | 93.8 | 30 | 240 | 240 | 0 | 480 | 100 | |
| R347H/R553X | 2 | 2 | 60 | 30 | 30 | 100 | 0 | 60 | 60 | 0 | 120 | 100 | |
| R347P | 10 | 10 | 240 | 300 | 60 | 100 | 0 | 300 | 300 | 0 | 600 | 100 | |
| R553X | 4 | 4 | 120 | 120 | 0 | 100 | 0 | 120 | 120 | 0 | 240 | 100 | |
| R553X/
delF508 | 4 | 4 | 90 | 60 | 90 | 100 | 0 | 120 | 120 | 0 | 240 | 100 | |
| R560T | 6 | 6 | 150 | 180 | 30 | 100 | 0 | 180 | 180 | 0 | 360 | 100 | |
| R560T/
delF508 | 4 | 4 | 90 | 120 | 30 | 100 | 0 | 120 | 120 | 0 | 240 | 100 | |
| S549N | 8 | 8 | 240 | 240 | 0 | 100 | 0 | 240 | 240 | 0 | 480 | 100 | |
| S549N/
delF508 | 2 | 2 | 60 | 30 | 0 | 75.0 | 30 | 60 | 60 | 0 | 120 | 100 | |
| S549R | 10 | 10 | 270 | 270 | 30 | 95.0 | 30 | 300 | 300 | 0 | 600 | 100 | |
| V520F | 10 | 10 | 300 | 300 | 0 | 100 | 0 | 300 | 300 | 0 | 600 | 100 | |
| W1282X | 10 | 10 | 300 | 270 | 30 | 100 | 0 | 300 | 300 | 0 | 600 | 100 | |
| W1282X/
delF508 | 2 | 2 | 60 | 30 | 30 | 100 | 0 | 60 | 60 | 0 | 120 | 100 | |
| Wild Type | 50 | 50 | 1,440 | 1,290 | 90 | 94.0 | 180 | 1,500 | 1,500 | 0 | 3,000 | 100 | |
| Overall Testing | 402 | 402 | 11,128 | 10,825 | 1,590 | 97.9 | 632 | 12,088 | 12,087 | 1 | 24,175 | 99.996 | |

16

CYSTIC FIBROSIS GENOTYPING ASSAY 510(K) SUMMARY [#K062028]

a. Two lots were used in this study.

.

Reinjection of same OLA reaction used for the first injection. Represents cases where users chose to reinject b. individual samples as well as entire or partial plates (usually based on convenience). In cases where partial or entire plates were reinjected, the initial, correct genotype calls did not change upon reinjection.

c. Results reflect percent agreement with sequencing prior to repeat testing.

17

d. Repeat from DNA/PCR. Repeat testing was performed due to a poor reaction. PCR failure, or GeneMapper quality flags indicating questionable or invalid data.
Reflex testing included. Exon 10: Additional calls per site (1 call per sample x number of replicates x 2 lots). 5/7/9T: ల. Additional calls per site (1 or 2 variant calls per sample x number of replicates x 2 lots).
Initial reflex testing made an erroneous call for the 9T variant for 1 of 4 replicates of sample C6043; correct calls f were made for the 5/7T variants in this sample. Operator did not perform follow-up testing for this sample, therefore, it remains a missed call.

Reproducibility

To determine clinical reproducibility for the Cystic Fibrosis Genotyping Assay, a singleblinded study was conducted at three external test sites. Each test site had one set of instruments, two operators, and three lots of reagents. Each site also processed the same set of samples. The samples tested included genomic DNA obtained from frozen whole blood and frozen pellets from commercially available cell lines. The expected genotype call for every unique sample was confirmed by DNA sequencing.

Sample Set

The set of 144 reproducibility study samples consisted of three (3) replicates each of forty-eight (48) unique genomic DNA samples derived from frozen whole blood (n = 25), and frozen cell line pellets (n = 23). The frozen whole blood samples contained homozygous normal alleles for each of the targeted loci within the Core OLA Assay, whereas the frozen cell line specimens contained either a single heterozygous mutation, a single homozygous mutation, or compound heterozygous mutations for CF. Each of three test sites received sufficient aliquots of each test panel member to conduct three independent assays x 144 samples/assay = 432 results per site). Each assay was performed with a different reagent lot (n = 3) on a different day. Reflex testing was performed as indicated per results of the Core OLA Assay and in accordance with ACMG guidelines. These details are summarized in the following table:

| Unique Samples | Replicates per Panel | Independent Core OLA
Assays Performed per
Test Site | Test Sites | Total Results |
|----------------------------------------------------------|----------------------|-----------------------------------------------------------|------------|--------------------------------------------------|
| 25
DNA from Frozen
Whole Blood
(normal alleles) | 3 | 3 | 3 | 1.296
=432 results per
site x 3 test sites |
| 23
DNA from Frozen
Cell Lines
(mutant alleles) | | | | |

18

Reproducibility Results

Results of the study indicate that the Cystic Fibrosis Genotyping Assay has a reproducibility of 100%, with zero (0) missed calls, as shown in the Reproducibility Table below.

Statistical analysis of all 1,296 results within the sample set (48 samples x 27 replicates) indicates 100% agreement between the Core OLA Assay and sequencing, with a one-sided 95% lower confidence limit equal to 99.8%.

Based on results of the Core OLA Assay, two samples required reflex testing. Sample #S009346 had a homozygous delF508 call by the Core OLA Assay. Both sequencing and Exon 10 reflex testing for all replicates (9) confirmed the homozygous delF508 call. The Core OLA Assay result for sample #S009369 contained an R117H mutation, therefore, reflex testing for variants in the polythymidine tract on Intron 8 (i.e., 5/7/9T) was conducted. Specifically, the 5T and 9T variant alleles were identified in this sample. Both sequencing and reflex testing results were in 100% agreement for all calls (18).

| Sample
Identification | Disease Causing Mutation(s)
Confirmed by Sequencing Call | Number
of Sites | Number of Sample
Replicates Per
Each Site
= 3 sample
replicates
x 3 lots | Number of Assay
Calls Per Each
Site
= 30 calls per
sample x 3
replicates x 3 lots | Missed
Calls | Percent
Agreement with
Sequencing
(%) | |
|--------------------------|-------------------------------------------------------------|------------------------------|-----------------------------------------------------------------------------------------|--------------------------------------------------------------------------------------------------|-----------------|------------------------------------------------|-----|
| 1 | S009346 | delF508; delF508 | 3 | 9 | 279 | 0 | 100 |
| 2 | S009347 | 3120+1G>A; 621+1G>T | 3 | 9 | 270 | 0 | 100 |
| 3 | S009348 | delF508; R553X | 3 | 9 | 270 | 0 | 100 |
| 4 | S009349 | G551D | 3 | 9 | 270 | 0 | 100 |
| 5 | S009350 | 3659delC; delF508 | 3 | 9 | 270 | 0 | 100 |
| 6 | S009351 | delI507 | 3 | 9 | 270 | 0 | 100 |
| 7 | S009352 | 621+1G>T; 711+1G>T | 3 | 9 | 270 | 0 | 100 |
| 8 | S009353 | 621+1G>T; delF508 | 3 | 9 | 270 | 0 | 100 |
| 9 | S009354 | 621+1G>T; G85E | 3 | 9 | 270 | 0 | 100 |
| 10 | S009355 | A455E; delF508 | 3 | 9 | 270 | 0 | 100 |
| 11 | S009356 | delF508; R560T | 3 | 9 | 270 | 0 | 100 |
| 12 | S009357 | N1303K | 3 | 9 | 270 | 0 | 100 |
| 13 | S009358 | G542X; G542X | 3 | 9 | 270 | 0 | 100 |
| 14 | S009359 | W1282X | 3 | 9 | 270 | 0 | 100 |
| 15 | S009360 | 2789+5G>A; 2789+5G>A | 3 | 9 | 270 | 0 | 100 |
| 16 | S009361 | 3849+10kb C>T; 3849+10kb C>T | 3 | 9 | 270 | 0 | 100 |
| 17 | S009362 | 1717-1G>A | 3 | 9 | 270 | 0 | 100 |
| 18 | S009363 | R1162X | 3 | 9 | 270 | 0 | 100 |
| 19 | S009364 | G551D; R347P | 3 | 9 | 270 | 0 | 100 |
| 20 | S009365 | R334W | 3 | 9 | 270 | 0 | 100 |
| 21 | S009369 | R117H; 5T/9T; delF508 | 3 | 9 | 288 | 0 | 100 |
| 22 | S009370 | 2184delA; delF508 | 3 | 9 | 270 | 0 | 100 |

Reproducibility Table

19

CYSTIC FIBROSIS GENOTYPING ASSAY 510(K) SUMMARY [#K062028]

| Sample
Identification | Disease Causing Mutation(s)
Confirmed by Sequencing Call | Number
of Sites | Number of Sample
Replicates Per
Each Site
= 3 sample
replicates
x 3 lots | Number of Assay
Calls Per Each
Site
= 30 calls per
sample x 3
replicates x 3 lots | Missed
Calls | Percent
Agreement with
Sequencing
(%) | |
|--------------------------|-------------------------------------------------------------|--------------------|-----------------------------------------------------------------------------------------|--------------------------------------------------------------------------------------------------|-----------------|------------------------------------------------|-----|
| 23 | S009371 | 1898+1G>A; delF508 | 3 | 9 | 270 | 0 | 100 |
| 24 | T001 | WT (wild type) | 3 | 9 | 270 | 0 | 100 |
| 25 | T002 | WT | 3 | 9 | 270 | 0 | 100 |
| 26 | T004 | WT | 3 | 9 | 270 | 0 | 100 |
| 27 | T005 | WT | 3 | 9 | 270 | 0 | 100 |
| 28 | T006 | WT | 3 | 9 | 270 | 0 | 100 |
| 29 | T007 | WT | 3 | 9 | 270 | 0 | 100 |
| 30 | T008 | WT | 3 | 9 | 270 | 0 | 100 |
| 31 | T009 | WT | 3 | 9 | 270 | 0 | 100 |
| 32 | T010 | WT | 3 | 9 | 270 | 0 | 100 |
| 33 | T011 | WT | 3 | 9 | 270 | 0 | 100 |
| 34 | T012 | WT | 3 | 9 | 270 | 0 | 100 |
| 35 | T013 | WT | 3 | 9 | 270 | 0 | 100 |
| 36 | T014 | WT | 3 | 9 | 270 | 0 | 100 |
| 37 | T015 | WT | 3 | 9 | 270 | 0 | 100 |
| 38 | T016 | WT | 3 | 9 | 270 | 0 | 100 |
| 39 | T0017 | WT | 3 | 9 | 270 | 0 | 100 |
| 40 | T018 | WT | 3 | 9 | 270 | 0 | 100 |
| 41 | T019 | WT | 3 | 9 | 270 | 0 | 100 |
| 42 | T020 | WT | 3 | 9 | 270 | 0 | 100 |
| 43 | T021 | WT | 3 | 9 | 270 | 0 | 100 |
| 44 | T022 | WT | 3 | 9 | 270 | 0 | 100 |
| 45 | T023 | WT | 3 | 9 | 270 | 0 | 100 |
| 46 | T024 | WT | 3 | 9 | 270 | 0 | 100 |
| 47 | T025 | WT | 3 | 9 | 270 | 0 | 100 |
| 48 | T026 | WT | 3 | 9 | 270 | 0 | 100 |

a. Missed Calls are defined as discrepant results relative to the sequencing result.

b. The one-sided 95% lower confidence limit = 99.1% for each individual sample.

c. An additional 9 calls per site (1 call per sample x 3 replicates x 3 lots) were generated as a result of Exon 10 reflex testing (sample ID S009346).

d. An additional 18 calls per site (2 calls per sample x 3 replicates x 3 lots) were generated as a result of 5/7/9T reflex testing (sample ID S009369).

20

Precision

The Core OLA assay detected all 23 mutations tested under the study protocol, as well as normal (wild type) alleles, with a precision of 100%. Precision of the assay was determined by comparing the results between sites (a total of 3 sites), between operators (a total of six operators, 2 per site) and between reagent lots (a total of 3 lots) as described in the following table:

| Mutation | Percent Correct Calls Made for Each Mutation per the Test Method
(Cystic Fibrosis Genotyping Assay) (%) | | |
|--------------|------------------------------------------------------------------------------------------------------------|-------------------------------------------------------------------------------------------------------------|-----------------------------------------------|
| | Site to Site
3 Sites, n = 432 results per site | Operator to Operator
6 Operators
(n = 288 results for 3 operators
n = 144 results for 3 operators) | Lot to Lot
3 Lots, n = 432 results per lot |
| 1717-1G>A | 100 | 100 | 100 |
| 1898+1G>A | 100 | 100 | 100 |
| 2184delA | 100 | 100 | 100 |
| 2789+5G>A | 100 | 100 | 100 |
| 3120+1G>A | 100 | 100 | 100 |
| 3659delC | 100 | 100 | 100 |
| 3849+10kbC>T | 100 | 100 | 100 |
| 621+1G>T | 100 | 100 | 100 |
| 711+1G>T | 100 | 100 | 100 |
| A455E | 100 | 100 | 100 |
| del1507 | 100 | 100 | 100 |
| delF508 | 100 | 100 | 100 |
| G542X | 100 | 100 | 100 |
| G551D | 100 | 100 | 100 |
| G85E | 100 | 100 | 100 |
| N1303K | 100 | 100 | 100 |
| R1162X | 100 | 100 | 100 |
| R117H | 100 | 100 | 100 |
| R334W | 100 | 100 | 100 |
| R347P | 100 | 100 | 100 |
| R553X | 100 | 100 | 100 |
| R560T | 100 | 100 | 100 |
| W1282X | 100 | 100 | 100 |

Each of the three reagent lots was tested on a different day at each site. Comparisons by site, operator and reagent lot were calculated for each of the 23 mutations tested. There were a total of 1,296 test results for each mutation (48 samples x 27 replicates).

Exon 10 reflex testing detected all polymorphisms as "present" or "not present," and 5/7/9T reflex testing detected all variants as "present" or "not present," with a precision of 100%. These results are limited to two samples and 81 test results for each sample (27 sample

21

replicates x 3 tests each). The following table summarizes the precision results for the two reflex assays.

| Reflex Assay | Percent Correct Calls Made by Exon 10 or 5/7/9T Reflex Testing (%)
Site to Site | Operator to Operator | Lot to Lot |
|--------------|------------------------------------------------------------------------------------|------------------------------------------------------------------------------------|--------------------------------|
| | 3 Sites, n = 27 results per site | 6 Operators
(n = 9 results for 3 operators,
n = 18 results, for 3 operators) | 3 Lots, n = 27 results per lot |
| 5/7/9T | 100 | 100 | 100 |
| Exon 10 | 100 | 100 | 100 |

An overview of the entire reproducibility study results by mutation is provided in the following Reproducibility Study by the Mutation Table:

| Genotype by
DNA
Sequencing | Number of
Samples
Replicates | Site
A | Site
B | Site
C | Number CF GT Calls Before Repeat Testing
(Based on Initial Results) | | | Percent
Agreement
with
Sequencing
for Initial
Resultsb | Repeatsc | Number of CF GT Calls
After Repeat Testing
(30 calls per sample
x 3 replicates x 3 lots) | | | | Total
Number
of
Correct
Calls | Percent
Agreement
with
Sequencing
(%) | |
|----------------------------------|------------------------------------|-----------|-----------|-----------|------------------------------------------------------------------------|-----------|-----------|-----------------------------------------------------------------------|----------|---------------------------------------------------------------------------------------------------|----------------------------|-----------|-----------|-------------------------------------------|---------------------------------------------------|--------------------------------|
| | | Site
A | Site
B | Site
C | Correct Calls
Site
A | Site
B | Site
C | | | Delayed
Callsa (Re-
injection) | Correct Calls
Site
A | Site
B | Site
C | | | Missed
(Incorrect)
Calls |
| 1717-1G>A | 9 | 9 | 9 | 270 | 270 | 120 | 60 | 88.9 | 90 | 270 | 270 | 270 | 0 | 810 | 100 | |
| 1898+1G>A/
del F508 | 9 | 9 | 9 | 240 | 270 | 180 | 30 | 88.9 | 90 | 270 | 270 | 270 | 0 | 810 | 100 | |
| 2184delA/
delF508 | 9 | 9 | 9 | 240 | 270 | 120 | 90 | 88.9 | 90 | 270 | 270 | 270 | 0 | 810 | 100 | |
| 2789+5G>A/
2789+5G>A | 9 | 9 | 9 | 240 | 270 | 180 | 30 | 88.9 | 90 | 270 | 270 | 270 | 0 | 810 | 100 | |
| 3120+1G>A/
621+1G>T | 9 | 9 | 9 | 240 | 270 | 120 | 90 | 88.9 | 90 | 270 | 270 | 270 | 0 | 810 | 100 | |
| 3659delC/
delF508 | 9 | 9 | 9 | 210 | 270 | 120 | 120 | 88.9 | 90 | 270 | 270 | 270 | 0 | 810 | 100 | |
| 3849+10kbC>T/
3849+10kbC>T | 9 | 9 | 9 | 240 | 270 | 180 | 30 | 88.9 | 90 | 270 | 270 | 270 | 0 | 810 | 100 | |
| 621+1G>T/
711+1G>T | 9 | 9 | 9 | 240 | 270 | 120 | 90 | 88.9 | 90 | 270 | 270 | 270 | 0 | 810 | 100 | |
| 621+1G>T/
delF508 | 9 | 9 | 9 | 240 | 270 | 120 | 90 | 88.9 | 90 | 270 | 270 | 270 | 0 | 810 | 100 | |
| 621+1G>T/
G85E | 9 | 9 | 9 | 240 | 270 | 180 | 30 | 88.9 | 90 | 270 | 270 | 270 | 0 | 810 | 100 | |
| A455E/delF508 | 9 | 9 | 9 | 240 | 270 | 180 | 30 | 88.9 | 90 | 270 | 270 | 270 | 0 | 810 | 100 | |
| delF508/
delF508 | 9 | 9 | 9 | 249d | 276d | 159d | 60d | 88.9 | 93d | 279d | 279d | 279d | 0 | 837d | 100 | |
| del1507 | 9 | 9 | 9 | 240 | 240 | 120 | 90 | 85.2 | 120 | 270 | 270 | 270 | 0 | 810 | 100 | |
| G542X/G542X | 9 | 9 | 9 | 240 | 270 | 180 | 30 | 88.9 | 90 | 270 | 270 | 270 | 0 | 810 | 100 | |
| G551D | 9 | 9 | 9 | 240 | 270 | 150 | 60 | 88.9 | 90 | 270 | 270 | 270 | 0 | 810 | 100 | |
| G551D/R347P | 9 | 9 | 9 | 270 | 270 | 180 | 60 | 96.3 | 30 | 270 | 270 | 270 | 0 | 810 | 100 | |
| N1303K | 9 | 9 | 9 | 240 | 270 | 180 | 30 | 88.9 | 90 | 270 | 270 | 270 | 0 | 810 | 100 | |
| R1162X | 9 | 9 | 9 | 210 | 270 | 180 | 60 | 88.9 | 90 | 270 | 270 | 270 | 0 | 810 | 100 | |
| R11711/5T/9T/
delF508 | 9 | 9 | 9 | 258d | 282d | 138d | 90d | 88.9 | 96d | 288d | 288d | 288d | 0 | 864d | 100 | |
| R334W | 9 | 9 | 9 | 270 | 270 | 180 | 60 | 96.3 | 30 | 270 | 270 | 270 | 0 | 810 | 100 | |
| R553X/delF508 | 9 | 9 | 9 | 240 | 270 | 150 | 60 | 88.9 | 90 | 270 | 270 | 270 | 0 | 810 | 100 | |
| R560T/delF508 | 9 | 9 | 9 | 240 | 270 | 180 | 30 | 88.9 | 90 | 270 | 270 | 270 | 0 | 810 | 100 | |
| W1282X | 9 | 9 | 9 | 240 | 270 | 120 | 90 | 88.9 | 90 | 270 | 270 | 270 | 0 | 810 | 100 | |
| Wild Type | 225 | 225 | 225 | 5,430 | 6,660 | 3,420 | 2,820 | 90.5 | 1,920 | 6,750 | 6,750 | 6,750 | 0 | 20,250 | 100 | |
| Overall Testing | 432 | 432 | 432 | 11,007 | 12,858 | 6,957 | 4,230 | 89.4 | 3,909 | | | | 0 | 38,961 | 100 | |
| | | | | | 35,052 | | | | | | | | | | | |

22

a. Reinjection of same OLA reaction. Represents cases where users chose to reinject individual samples as well as entire or partial plates (usually based on convenience). In cases where reinjected, initial, correct genotype calls did not change with repeat testing.
b. Results reflect percent agreement with sequencing prior to repeat testing.
c. Repeat from DNA/PCR. Repeat testing was performed due to a poor reaction or PCR failure, or GeneMapper quality flags indicating questionable or invalid data.
d. Reflex testing included. Exon 10: Nine additional calls per site (1 call per sample x 3 lots). 5/7/9/T: Eighteen additional calls per site (2 calls per sample x 3 replicates x 3 lots).

7. Conclusions

The Cystic Fibrosis Genotyping Assay accurately identifies the normal and mutant alleles at 30 loci of the CFTR gene from purified human genomic DNA.

8. References Cited:

1. Gordy, W.W. et al. 2001. Laboratory standards and guidelines for population-based cystic fibrosis carrier screening. Genetics in Medicine 3 (2): 149-154.
1. Watson, M. et al. 2004. Cystic fibrosis population carrier screening: 2004 revision of American College of Medical Genetics mutation panel. Genetics in Medicine 6 (5): 387-391.
Update on carrier screening for cystic fibrosis. ACOG Committee Opinion No. 325. 3. American College of Obstetricians and Gynecologists. Obstet Gynecol 2005; 106: 1465-8.

23

DEPARTMENT OF HEALTH & HUMAN SERVICES

Image /page/23/Picture/1 description: The image shows the logo for the U.S. Department of Health and Human Services. The logo consists of a stylized eagle or bird-like symbol with three curved lines representing its wings or body. The logo is surrounded by a circular arrangement of text that reads "DEPARTMENT OF HEALTH & HUMAN SERVICES USA".

Food and Drug Administration 2098 Gaither Road Rockville MD 20850

Celera Diagnostics c/o Ms Victoria Mackinnon Vice President, RA/CA 1401 Harbor Bay Pkwy Alameda, CA 94502

SEP - 7 2007

Re: K062028

Trade/Device Name: Cystic Fibrosis Genotyping Assay Regulation Number: 21 CFR 866.5900 Regulation Name: CFTR (cystic fibrosis transmembrane conductance regulator) gene mutation detection system Regulatory Class: Class II Product Code: NUA Dated: August 6, 2007 Received: August 7, 2007

Dear Ms. Mackinnon:

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

24

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 yours,

Robert H. Booker

Robert L. Becker, Jr., M.D 1. 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

25

Indications for Use

510(k) Number:

Device Name: Celera Cystic Fibrosis Genotyping Assay

Indications for Use:

The Cystic Fibrosis Genotyping Assay is a qualitative in vitro diagnostic device used to genotype a panel of mutations and variants in the cystic fibrosis transmembrane conductance requlator (CFTR) gene in genomic DNA isolated from human whole blood specimens. The panel includes mutations and variants recommended by the American College of Medical Genetics (ACMG, 2004) and the American College of Obstetricians and Gynecologists (ACOG, 2005) plus additional multiethnic mutations. The Cystic Fibrosis Genotyping Assay provides information intended to be used for carrier screening in adults of reproductive age, as an aid in newborn screening, and in confirmatory diagnostic testing in newborns and children.

This test is not indicated for use in fetal diagnostic or pre-implantation testing. This test is also not indicated for stand-alone diagnostic purposes.

Prescription Use × (Part 21 CFR 801 Subpart D) AND/OR

Over-The-Counter Use (21 CFR 801 Subpart C)

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

Concurrence of CDRH, Office of In Vitro Diagnostic Devices (OIVD)

m chan
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Office of In Vitro Diagnostic Device Evaluation and Sufety

51000 K062028