XTAG CYSTIC FIBROSIS 39 KIT V2, (CFTR 39 KIT V2), MODEL I027C0231, I027D0266, I027E0267
Device Facts
| Record ID | K083846 |
|---|---|
| Device Name | XTAG CYSTIC FIBROSIS 39 KIT V2, (CFTR 39 KIT V2), MODEL I027C0231, I027D0266, I027E0267 |
| Applicant | Luminex Molecular Diagnostics, Inc. |
| Product Code | NUA · Immunology |
| Decision Date | Sep 1, 2009 |
| Decision | SESE |
| Submission Type | Traditional |
| Regulation | 21 CFR 866.5900 |
| Device Class | Class 2 |
| Attributes | Pediatric |
Indications for Use
The xTAG™ Cystic Fibrosis 39 Kit v2 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 world's most common and North American prevalent mutations. The xTAG™ Cystic Fibrosis 39 Kit v2 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.
Device Story
The xTAG Cystic Fibrosis 39 kit v2 is an in vitro diagnostic test for detecting 39 CFTR gene mutations and 4 variants in human whole blood. The process involves multiplex PCR amplification of CFTR gene segments, followed by multiplex allele-specific primer extension (ASPE) using primers with unique 5' tags. These tagged primers hybridize to complementary sequences on spectrally distinguishable polystyrene beads. A biotinylated reporter molecule (streptavidin-phycoerythrin) binds to the extended primers. The Luminex 100 or 200 instrument uses two lasers to identify the bead (genotype) and the presence of the reporter (allele detection). The xTAG Data Analysis Software (TDAS) processes the MFI signals to provide a qualitative genotype. The device is used in clinical laboratories by trained personnel. Results assist clinicians in carrier screening and diagnostic confirmation of cystic fibrosis. The device provides a standardized, high-throughput method for identifying common CFTR mutations, facilitating timely clinical decision-making and patient management.
Clinical Evidence
Bench testing only. Accuracy assessed using 319 samples (clinical specimens, cell lines, plasmids) confirmed by bi-directional sequencing; 100% agreement with predicate. Precision/reproducibility evaluated across 3 sites, 3 lots, and multiple operators; >99.54% precision. LoD established at 1.56 ng/µL. Interference testing showed no significant inhibition by hemoglobin, bilirubin, or triglycerides.
Technological Characteristics
Multiplex PCR and Allele Specific Primer Extension (ASPE) assay. Uses polystyrene beads internally dyed with red/infrared fluorochromes. Analyzed on Luminex 100/200 flow cytometry-based platform. Software-based genotype calling. Requires PCR thermal cycler and Luminex instrument. Reagents include Platinum TFI DNA Polymerase, Shrimp Alkaline Phosphatase, and Exonuclease I.
Indications for Use
Indicated for carrier testing in adults of reproductive age, as an aid in newborn screening, and in confirmatory diagnostic testing in newborns and children. Not for fetal diagnostic, pre-implantation, or stand-alone diagnostic use.
Regulatory Classification
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.
Special Controls
*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.
Predicate Devices
- xTAG™ Cystic Fibrosis Kit (k043011, k060627)
Related Devices
- K083845 — XTAG CF60 KIT V2 · Luminex Molecular Diagnostics, Inc. · Dec 11, 2009
- DEN050004 — TAG-IT CYSTIC FIBROSIS KIT · Tm Bioscience Corporation · May 9, 2005
- K163347 — xTAG Cystic Fibrosis 39 Kit v2 · Luminex Molecular Diagnostics, Inc. · Dec 15, 2016
- K060627 — TAG-IT CYSTIC FIBROSIS KIT · Tm Bioscience Corporation · Jun 7, 2006
Submission Summary (Full Text)
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# 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION SUMMARY
A. 510(k) Number:
k083846
B. Purpose for Submission:
New Device
C. Measurand:
CFTR (Cystic Fibrosis transmembrane conductance regulator) gene from human blood specimens
D. Type of Test:
Multiplex PCR followed by multiplex allele specific primer extension for genotyping, hybridized to multiplexed fluorescing microparticles, detected by flow cytometry
E. Applicant:
Luminex Molecular Diagnostics Inc.
F. Proprietary and Established Names:
xTAG™ Cystic Fibrosis 39 Kit v2
G. Regulatory Information:
1. Regulation section:
21 CFR 866.5900, CFTR (cystic fibrosis transmembrane conductance regulator) gene mutation detection system
2. Classification:
Class II
3. Product code:
NUA, system, test, CFTR (cystic fibrosis transmembrane conductance regulator) gene mutation detection
4. Panel:
Immunology (82)
H. Intended Use:
1. Intended use(s):
The xTAG™ Cystic Fibrosis 39 Kit v2 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 world's most common and North American prevalent mutations. The xTAG™ Cystic Fibrosis 39 Kit v2 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.
2. Indication(s) for use:
Same as Intended use.
3. Special conditions for use statement(s):
For Prescription use only
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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
4. Special instrument requirements:
Luminex 100 or 200 IS instruments with IS v2.3 (see k073506 for details) and xPONENT v3.1
I. Device Description:
The xTAG™ Cystic Fibrosis 39 Kit v2 is comprised of the following components: 1 vial (240 μL) xTAG CFTR PCR Primer Mix v2; 1 vial (192 μL) xTAG CFTR ASPE Primer Mix A v2; 2 vials (5 units/μL, 115 μL each) Platinum Tfi Exo(-) DNA Polymerase; 4 vials (1.3 mL each) 5X Platinum Tfi Reaction buffer; 2 vials (1 mL each) Tfi 50 mM MgCl₂; 2 vials (10 units/μL, 48 μL each) xTAG Exonuclease I; 2 vials (1 unit/μL, 120 μL each) xTAG Shrimp Alkaline Phosphatase (SAP); 1 vial (1 mg/mL, 108 μL) xTAG Streptavidin, R-Phycoerythrin Conjugate; 1 vial (2.16 mL) xTAG CFTR Bead Mix A v2; and 1 bottle (12 mL) xTAG 10X Buffer.
J. Substantial Equivalence Information:
1. Predicate device name(s):
xTAG™ Cystic Fibrosis Kit
2. Predicate 510(k) number(s):
k043011, k060627
3. Comparison with predicate:
| Similarities | | |
| --- | --- | --- |
| Item | Device | Predicate |
| Intended Use | The xTAG™ Cystic Fibrosis 39 Kit v2 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 world's most common and North American prevalent mutations. | Same |
| Indications for Use | The xTAG™ Cystic Fibrosis 39 Kit v2 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. | Same |
| Contra-Indications | This kit is not intended for use in fetal diagnostic or pre-implantation testing. This kit is also not indicated for stand-alone diagnostic purposes. | Same |
| Type of Test | Multiplex PCR followed by multiplex allele specific primer extension for genotyping, hybridized to multiplex fluorescent microparticles, detected by flow cytometry | Same |
| Product Description | Tests for 39 mutations and 4 variants in the CFTR gene (23 of which are recommended by ACMG/ACOG). | Same |
| Specimen type | Peripheral human whole blood | Same |
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| Differences | | |
| --- | --- | --- |
| Item | Device | Predicate |
| Software | TDAS CFTR contains 1 template to detect a panel of 39 mutations and 4 variants. Software has a masking function where user can choose to display results for either the ACMG/ACOG 23 mutations or for the full panel of mutations. | TDAS CF-1 contains 1 template to detect a panel of 39 mutations and 4 variants only. |
| Instrument system | Luminex 100 or 200 IS v.2.3 | Luminex 100 IS v2.3 |
| Instrument software | IS v2.3 and xPONENT v3.1 | IS v2.3 only |
## K. Standard/Guidance Document Referenced (if applicable):
- American College of Medical Genetics (ACMG) / American College of Obstetricians and Gynecologists Technical Standards and Guidelines for CFTR Mutation Testing and Standards and Guidelines for Clinical Genetic Laboratories
- Cystic Fibrosis Foundation / Center for Disease Control Recommendations on Newborn Screening for CF
- FDA Class II Special Controls Guidance: Quality Control Material for Cystic Fibrosis Nucleic Acid Assays (Jan 2007)
- FDA Class II Special Controls Guidance: CFTR Gene Mutation Detection Systems (Oct 2005)
- CDRH Draft Guidance on Multiplex Tests for Heritable DNA Markers, Mutations and Expression Patterns (Feb 2003)
- CDRH Draft Guidance on Statistical Guidance on Reporting Results from Studies Evaluating Diagnostic Tests (Mar 2003)
- CDRH Guidance for the Content of Pre-Market Submission for Software Contained in Medical Devices (May 1998)
- CDRH Guidance on General Principles of Software Validation (Jan 2002)
- CDRH Guidance on Format for Traditional and Abbreviated 510ks (Aug 2005)
- MM01-A2: Molecular Diagnostic Methods for Genetic Diseases
- MM13-PE: Collection, Transport, Preparation, and Storage of Specimens for Molecular Methods
- MM17-A: Verification and Validation of Multiplex Nucleic Acid Assays
- EP05-A2: Evaluation of Precision Performance of Clinical Chemistry Devices
- EP07-A2E: Interference Testing in Clinical Chemistry
- EP12-A: User Protocol for Evaluation of Qualitative Test Performance
- EP17-A: Protocols for Determining Limits of Detection and Limits of Quantitation
## L. Test Principle:
The xTAG™ CFTR 39 Kit v2 incorporates multiplex Polymerase Chain Reaction (PCR) and multiplex Allele Specific Primer Extension (ASPE) with Luminex’s proprietary Universal Tag sorting system on the Luminex® 100 or 200 xMAP™ platform.
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The amplimer sizes range from 179 bp to 465 bp. Purified genomic DNA is extracted from each sample and a multiplex PCR reaction is carried out under optimized conditions. Each sample undergoes a multiplex allele specific primer extension (ASPE) reaction, where an aliquot of the PCR product is run through the ASPE A reaction. This step allows for detection of each allele (wild-type or mutant) of a given locus using an allele-specific probe (ASP) which contains a unique DNA sequence (tag) at its 5' end. Each bi-allelic locus has 2 ASPs and each tri-allelic loci has 3 ASPs included in the ASPE Mix. For each ASP, the 3' end of the primer is a perfect match for its allele, but will have a 3' mismatch on any other allele. Both these ASPs however are tagged with a common tag at their 5' end. The DNA polymerase will only extend the primer when there is a perfect match on the 3' end, so that the primer is only extended if its target allele is present in the sample. Biotin-dCTP is incorporated into the extending chain if extension occurs.
For the hybridization reaction, the ASPE reaction product is added directly to microwells containing aliquots of the Bead Mix. Each coupled bead is spectrally distinguishable from the other coupled beads in a given bead mix. A fluorescent reporter molecule (streptavidin-phycoerythrin) is bound to the biotin on the extended primers. Each tagged primer hybridizes only to its unique anti-tag complement; therefore, each colored bead represents a specific allele, through the bead/anti-tag/tagged primer association. The beads are then analyzed by the Luminex instrument, which contains two lasers: one identifies the color-coded bead, and the other identifies the presence or absence of extended allele specific primer through the phycoerythrin reporter. Thus, the genotype of that locus is identified by the presence of phycoerythrin signal attached to one or both ASPs.
For each sample analyzed by the CFTR 39 kit v2, an output file containing MFI signals from the Luminex Instrument is generated. The proprietary software component of this product analyzes this output data file, to provide a final qualitative genotype for the sample. The software includes a feature which enables users to select between 2 options for the final output:
Option 1: Full Panel (39 mutations + 4 variants).
Option 2:ACMG/ACOG panel (23 mutations).
When Option 2 is chosen, the non-ACMG/ACOG recommended panel of mutations/variants will not be available to the user.
Mutations and 4 variants (variants italized) included in the CFTR 39 Kit v2
| ΔF508* | 1717-1G>A* | W1282X* | 2307insA |
| --- | --- | --- | --- |
| ΔI507* | R560T* | 1078delT | Y1092X |
| G542X* | R553X* | 394delTT | M1101K |
| G85E* | G551D* | Y122X | S1255X |
| R117H* | 1898+1G>A* | R347H | 3876delA |
| 621+1G>T* | 2184delA* | V520F | 3905insT |
| 711+1G>T* | 2789+5G>A* | A559T | 5/7/9T |
| N1303K* | 3120+1G>A* | S549N | F508C |
| R334W* | R1162X* | S549R | I507V |
| R347P* | 3659delC* | 1898+5G>T | I506V |
| A455E* | 3849+10kbC>T* | 2183AA>G | |
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*denotes ACMG/ACOG panel
# M. Performance Characteristics (if/when applicable):
# 1. Analytical performance:
# a. Precision/Reproducibility:
A multi-centre, multi-operator, multi-lot, blinded study design was used to evaluate total variability of the xTAG CFTR v2 system. The experimental design generally follows CLSI recommendations (EP5-A and MM17-A) and requirements laid forth by the FDA in I080024.
The study included two arms. Arm A evaluated the reproducibility of the sample extraction step using 18 unique clinical (whole blood) samples representing wild-type and 3 mutant genotypes. DNA was extracted using 3 different methods and tested using the same assay lot by 2 operators, across 9 non-consecutive days. Each operator performed 3 runs/extraction methods, and each assay point was run in duplicate. An extraction was performed for each assay run by each operator. The number of replicates per sample was: (3 extraction methods) x (2 operators/extraction method) x (3 runs/operator) x (2 replicates/run) = 36 replicates. The results are summarized in the following table.
Summary of Results from Reproducibility Study Arm A
| Extraction Method | Number of Samples | Total Number of Assays | CFTR v2 Results |
| --- | --- | --- | --- |
| a | 18 | 18 x 12 = 216 | All assays passed (correct calls) |
| b | 18 | 18 x 12 = 216 | All assays passed (correct calls) |
| c | 18 | 18 x 12 = 216 | All assays passed (correct calls) |
Arm B evaluated the reproducibility of the analytical (post-extraction) steps of the assays at 3 external sites using in order of preference and availability, purified genomic DNAs extracted from clinical (whole blood) samples, purified genomic DNA extracted from lymphoid cell lines, and/or plasmids. Each set contained samples representing all mutations and variants probed by the CFTR 39 Kit v2. There were 2 operators per site, each performing 1 run/day across 3 non-consecutive days (3 runs per operator or 6 runs per site). Within a given run, each assay point was run in duplicate. A total of three assay lots were tested (1 lot/site). This arm of the study also provided information on the ability to resolve different sources of variability in the post-extraction (analytical) step of the assay. For example, it was possible to analyze data from a particular site to estimate the operator-to-operator variability.
The results indicated a precision of $>99.54\%$ across the 3 sites. There was one "no-call" after 1 re-run at Site 3, and 3 mis-calls (from a single plasmid sample) at Site 1. Reproducibility of a compound heterozygote dF508/F508C was also demonstrated in
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this study. 36 of 30 replicates of one sample generated a dF508 call and 6 generated a dF508 Mu D call (Mu D calls are defined as detection of only a mutant allele).
Between Site/ Operator Reproducibility Data for xTAG CFTR 39 Kit v2
| Sample ID | Genotype | Operator - to - Operator | | | | | | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | | Site 1 | | | | Site 2 | | | | Site 3 | | | |
| | | | +Op 1N ¥ | Op 1% corr | Op2N | Op2% corr | Op1N | Op1% corr | Op2N | Op2% corr | Op1N | Op1% corr | Op2N | Op2% corr |
| 3893 | 711+1G>T | dF508 | 438 | 100 | 438 | 100 | 438 | 100 | 438 | 100 | 438 | 100 | 438 | 100 |
| 4919 | 1717-1G>A | - | 438 | 100 | 438 | 100 | 438 | 100 | 438 | 100 | 438 | 100 | 438 | 100 |
| 9354 | G542X | R117H | 438 | 100 | 438 | 100 | 438 | 100 | 438 | 100 | 438 | 100 | 438 | 100 |
| 10561 | A455E | - | 438 | 100 | 438 | 100 | 438 | 100 | 438 | 100 | 438 | 100 | 438 | 100 |
| 10839 | 3659delC | - | 438 | 100 | 438 | 100 | 438 | 100 | 438 | 100 | 438 | 100 | 438 | 100 |
| 10988 | R1162X | dF508 | 438 | 100 | 438 | 100 | 438 | 100 | 438 | 100 | 438 | 100 | 438 | 100 |
| 11688 | 3849+10kbC>T | - | 438 | 100 | 438 | 100 | 438 | 100 | 438 | 100 | 438 | 100 | 438 | 100 |
| 15249 | W1282X | - | 438 | 100 | 438 | 100 | 438 | 100 | 438 | 100 | 438 | 100 | 438 | 100 |
| 15560 | 1078delT | dF508 | 438 | 100 | 438 | 100 | 438 | 100 | 438 | 100 | 438 | 100 | 438 | 100 |
| 17594 | A559T | - | 438 | 100 | 438 | 100 | 438 | 100 | 438 | 100 | 438 | 100 | 438 | 100 |
| 30116 | S549N | - | 438 | 100 | 438 | 100 | 438 | 100 | 438 | 100 | 438 | 100 | 438 | 100 |
| 41406 | G551D | R347P | 438 | 100 | 438 | 100 | 438 | 100 | 438 | 100 | 438 | 100 | 438 | 100 |
| 48308 | 3905insT | - | 438 | 100 | 438 | 100 | 438 | 100 | 438 | 100 | 438 | 100 | 438 | 100 |
| 53948 | R560T | dF508 | 438 | 100 | 438 | 100 | 438 | 100 | 438 | 100 | 438 | 100 | 438 | 100 |
| 63436 | 394delTT | - | 438 | 100 | 438 | 100 | 438 | 100 | 438 | 100 | 438 | 100 | 438 | 100 |
| 69711 | R553X | - | 438 | 100 | 438 | 100 | 438 | 100 | 438 | 100 | 438 | 100 | 438 | 100 |
| 70268 | 2184delA | - | 438 | 100 | 438 | 100 | 438 | 100 | 438 | 100 | 438 | 100 | 438 | 100 |
| 71713 | 1898+1G>A | dF508 | 438 | 100 | 438 | 100 | 438 | 100 | 438 | 100 | 438 | 100 | 438 | 100 |
| 77590 | Y1092X-C>A | dF508 | 438 | 100 | 438 | 100 | 438 | 100 | 438 | 100 | 438 | 100 | 438 | 100 |
| 89633 | 2183AA>G | - | 438 | 100 | 438 | 100 | 438 | 100 | 438 | 100 | 438 | 100 | 438 | 100 |
| 98748 | V520F | 3120+1G>A | 438 | 100 | 438 | 100 | 438 | 100 | 438 | 100 | 438 | 100 | 438 | 100 |
| LMD002-08 111 | R334W | - | 438 | 100 | 438 | 100 | 438 | 100 | 438 | 100 | 438 | 100 | 438 | 100 |
| LMD002-08 33 | 2789+5G>A | - | 438 | 100 | 438 | 100 | 438 | 100 | 438 | 100 | 438 | 100 | 438 | 100 |
| LMD002-08 58 | 612+1 G>A | - | 438 | 100 | 438 | 100 | 438 | 100 | 438 | 100 | 438 | 100 | 438 | 100 |
| LMD002-08 64 | dI507 | - | 438 | 100 | 438 | 100 | 438 | 100 | 438 | 100 | 438 | 100 | 438 | 100 |
| LMD002-08 73 | dF508 (+ F508C variant) | - | 439* | 100 | 442** | 100 | 438*** | 100 | 438*** | 100 | 438*** | 100 | 439* | 100.00 |
| LMD002-08 86 | G85E | - | 438 | 100 | 438 | 100 | 438 | 100 | 438 | 100 | 438 | 100 | 438 | 100 |
| LMD002-08 90 | N1303K | - | 438 | 100 | 438 | 100 | 438 | 100 | 438 | 100 | 438 | 100 | 438 | 100 |
| NA07857 | M1101K | M1101K | 438 | 100 | 438 | 100 | 438 | 100 | 438 | 100 | 438 | 100 | 438 | 100 |
| NA18802 | Y122X | R1158X | 438 | 100 | 438 | 100 | 438 | 100 | 438 | 100 | 438 | 100 | 438 | 100 |
| NA20737 | R347H | - | 438 | 100 | 438 | 100 | 438 | 100 | 438 | 100 | 438 | 100 | 438 | 100 |
| NA20741 | 3876delA | - | 438 | 100 | 438 | 100 | 438 | 100 | 438 | 100 | 438 | 100 | 438 | 100 |
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| NA20915 | S549R | - | 438 | 100 | 438 | 100 | 438 | 100 | 438 | 100 | 438 | 100 | 438 | 100 |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| NA21551 | dF508 | - | 438 | 100 | 438 | 100 | 438 | 100 | 438 | 100 | 438 | 100 | 437 | 99.77 |
| Plasmids 10B+10Cठ| dF508(+I506V variant) | V520F | 18 | 100 | 18 | 100 | 18 | 100 | 18 | 100 | 18 | 100 | 18 | 100 |
| Plasmid 12B | 1898+5G>T | - | 6 | 100 | 6 | 100 | 6 | 100 | 6 | 100 | 6 | 100 | 6 | 100 |
| Plasmid 13E | 2307insA | 2055del9>A | 12 | 91.67 | 12 | 83.33 | 12 | 100 | 12 | 100 | 12 | 100 | 12 | 100 |
| Plasmid 19E | 3791delC | - | 12 | 100 | 12 | 100 | 12 | 100 | 12 | 100 | 12 | 100 | 12 | 100 |
| Plasmid 17bB | Y1092X-C>G | - | 6 | 100 | 6 | 100 | 6 | 100 | 6 | 100 | 6 | 100 | 6 | 100 |
| Plasmid 19A | S1255X (ex.19) | - | 6 | 100 | 6 | 100 | 6 | 100 | 6 | 100 | 6 | 100 | 6 | 100 |
| Plasmid 20A | S1255X (ex.20) | W1282X | 12 | 100 | 12 | 100 | 12 | 100 | 12 | 100 | 12 | 100 | 12 | 100 |
Site 1: Hartford Hospital, Connecticut, USA; Site 2: Luminex Molecular Diagnostics, Toronto, CA; Site 3: Hospital for Sick Children, Toronto, CA
† Op = operator (1 or 2)
¥ N = number of calls
‡ % corr = percent correct
* Total number of calls 438 + 1 = 439, because TDAS made one dF508 Mu D call (and the F508C variant call is unmasked)
** Total number of calls 438 + 4 = 442, because TDAS made 4 dF508 Mu D calls (and the F508C variant call is unmasked)
*** Total number of calls = 438, because TDAS made all dF508 HET calls (the F508C variant call is masked)
b. Linearity/assay reportable range:
Not Applicable
c. Traceability, Stability, Expected values (controls, calibrators, or methods):
Previous stability studies support a shelf-life of 1 year when kit reagents are stored at -25°C to -15°C. Repeated freeze-thaw cycles (up to 4) do not compromise the integrity of the xTAG™ CFTR 39 Kit v2.
Assay controls: The package insert recommends that at least two negative controls and a rotating set of positive controls (including a dF508 mutation) are included with each run.
d. Detection limit:
The package insert recommends that purified DNA have at least a UV 260/280 ratio of >1.5 depending on the extraction method used. The assay has been optimized for use with 50 ng of total DNA, but reliable results were previously demonstrated with DNA amounts between 10 ng to 1.5 µg.
The detection limits were determined by serially diluting 10 gDNA samples, obtained from commercially available cell lines at 10 levels ranging from 300 - 0.39 ng/µL. The LoD is defined as the lowest amount of genomic DNA in a sample for which the assay can detect genotypes with a positive agreement rate of ≥ 95%. Eight of the samples were heterozygous, one (1) was homozygous, and one (1) contained a non-panel mutation which would be called as wild type by the assay. Three of the heterozygous samples were compound heterozygotes, but the second allele was a non-panel mutation and therefore called as wild-type by the assay. At each
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concentration, 22 replicates from each of the samples were run. Based on initial results, a $\mathrm{LoD} = 1.56\mathrm{ng / \mu L}$ was proposed. An additional study was performed using $2\mathrm{ng / \mu L}$ for each of the 22 sample replicates. Two samples exhibited 21/22 correct calls, corresponding to a $95.4\%$ positive agreement. No failures were observed at the $300\mathrm{ng / \mu L}$ concentration.
# e. Analytical specificity:
To examine the effects of potential interferents that might be found in whole blood samples, a study was conducted using 8 whole blood samples (4 wild-type for CFTR mutations, 1 N1303K Het, 1 V520F Het, and 2 $\Delta$ F508 Het). Each sample was split into 6 parts, and incubated in the absence or presence of one of 3 potential interferents (hemoglobin at final concentration of $1,500~\mu \mathrm{g / mL}$ , bilirubin at a final concentration of $200~\mu \mathrm{g / mL}$ , and a mixture of triglycerides at a final concentration of $30~\mathrm{mg / mL}$ ). Samples were extracted and assayed with the kit according to the instructions. Dideoxy- sequencing of the CFTR exons in each sample was conducted to confirm the genotypes of each sample. Results indicated that none of the substances at the concentrations tested produced a significant inhibitory effect on kit performance.
f. Assay cut-off:
Not Applicable
# 2. Comparison studies:
# a. Method comparison with predicate device:
Accuracy of the xTAG CFTR 39 Kit v2 was assessed through evaluation of samples representing all alleles (mutations and polymorphisms) probed by the assay. Three hundred nineteen (319) samples were used in this method comparison study. These samples were left-over, anonymized, clinical specimens, eight were genomic DNAs from EBV-transformed lymphoid cell lines, and 2 were custom-designed plasmids engineered to contain 1-2 CFTR mutations. The genotype of all samples was confirmed by bi-directional sequencing. The xTAG Cystic Fibrosis Mutation Detection Kit was used as the comparator method.
For all exons, the results indicated $100\%$ agreement between the xTAG Cystic Fibrosis 39 Kit v2 and the xTAG Cystic Fibrosis Mutation Detection Kit. There were 0 no-calls and 0 mis-calls in the accuracy study.
Table: Overall accuracy for the xTAG Cystic Fibrosis 39 Kit v2
| | | Number of Samples tested per mutation | | | Before Allowable Rerun | | | After Allowable Rerun | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Exon or Intron | Mutations | Independent Clinical Samples | Cell lines | Plasmids | Repeats due to mis-calls | Repeats due to no-calls | % Accuracy prior to repeats | Repeats due to mis-calls | Repeats due to no-calls | Final % Accuracy (after repeats) | LB of 95% CI* | UB of 95% CI* |
| Ex 3 | G85E# | 2 | 0 | 0 | 0 | 0 | 100 | 0 | 0 | 100 | 15.81 | 100 |
| | 394delTT | 2 | 0 | 0 | 0 | 0 | 100 | 0 | 0 | 100 | 15.81 | 100 |
| Ex 4 | R117H | 36 | 0 | 0 | 0 | 0 | 100 | 0 | 0 | 100 | 90.51 | 100 |
| | Y122X | 1 | 1 | 0 | 0 | 0 | 100 | 0 | 0 | 100 | 15.81 | 100 |
| | 621+1G>T# | 6 | 0 | 0 | 0 | 0 | 100 | 0 | 0 | 100 | 54.07 | 100 |
| Ex 5 | 711+1G>T# | 3 | 0 | 0 | 0 | 0 | 100 | 0 | 0 | 100 | 29.24 | 100 |
| Ex 7 | 1078delT | 3 | 0 | 0 | 0 | 0 | 100 | 0 | 0 | 100 | 29.24 | 100 |
| | R334W# | 3 | 0 | 0 | 0 | 0 | 100 | 0 | 0 | 100 | 29.24 | 100 |
| | R347Pmut# | 6 | 0 | 0 | 0 | 0 | 100 | 0 | 0 | 100 | 54.07 | 100 |
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| | | Number of Samples tested per mutation | | | Before Allowable Rerun | | | After Allowable Rerun | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | R347Hmut | 3 | 1 | 0 | 0 | 0 | 100 | 0 | 0 | 100 | 39.76 | 100 |
| Ex 9 | A455E# | 3 | 0 | 0 | 0 | 0 | 100 | 0 | 0 | 100 | 29.24 | 100 |
| Ex 10 | Dl507mt# | 9 | 0 | 0 | 0 | 0 | 100 | 0 | 0 | 100 | 66.37 | 100 |
| | dF508mut# | 162 | 1 | 0 | 0 | 0 | 100 | 0 | 0 | 100 | 97.87 | 100 |
| | V520F | 2 | 0 | 0 | 0 | 0 | 100 | 0 | 0 | 100 | 15.81 | 100 |
| Ex 11 | 1717-1G>A# | 5 | 0 | 0 | 0 | 0 | 100 | 0 | 0 | 100 | 47.82 | 100 |
| | G542X# | 13 | 0 | 0 | 0 | 0 | 100 | 0 | 0 | 100 | 75.29 | 100 |
| | S549N | 1 | 1 | 0 | 0 | 0 | 100 | 0 | 0 | 100 | 15.81 | 100 |
| | S549R | 2 | 1 | 0 | 0 | 0 | 100 | 0 | 0 | 100 | 47.82 | 100 |
| | G551D# | 12 | 0 | 0 | 0 | 0 | 100 | 0 | 0 | 100 | 73.54 | 100 |
| | R553X | 7 | 0 | 0 | 0 | 0 | 100 | 0 | 0 | 100 | 59.04 | 100 |
| | A5559T | 2 | 0 | 0 | 0 | 0 | 100 | 0 | 0 | 100 | 29.24 | 100 |
| | R560T# | 4 | 0 | 0 | 0 | 0 | 100 | 0 | 0 | 100 | 36.76 | 100 |
| Ex12 | 1898+1G>A# | 2 | 0 | 0 | 0 | 0 | 100 | 0 | 0 | 100 | 15.81 | 100 |
| | 1898+5G>T | 0 | 0 | 2 | 0 | 0 | 100 | 0 | 0 | 100 | 15.81 | 100 |
| Ex13 | 2183AA>G | 2 | 0 | 0 | 0 | 0 | 100 | 0 | 0 | 100 | 15.81 | 100 |
| | 2184delA# | 1 | 0 | 0 | 0 | 0 | 100 | 0 | 0 | 100 | 2.50 | 100 |
| | 2307insA | 3 | 0 | 0 | 0 | 0 | 100 | 0 | 0 | 100 | 29.24 | 100 |
| Ex 14b | 2789+5G>A# | 5 | 0 | 0 | 0 | 0 | 100 | 0 | 0 | 100 | 47.82 | 100 |
| Ex 16 | 3120+1G>A | 7 | 0 | 0 | 0 | 0 | 100 | 0 | 0 | 100 | 59.04 | 100 |
| Ex17b | Y1092X-C>G | 0 | 0 | 2 | 0 | 0 | 100 | 0 | 0 | 100 | 15.81 | 100 |
| | Y1092X-C>A | 2 | 0 | 0 | 0 | 0 | 100 | 0 | 0 | 100 | 15.81 | 100 |
| | M1101K | 0 | 2 | 0 | 0 | 0 | 100 | 0 | 0 | 100 | 15.81 | 100 |
| Ex 19 | R1162X# | 5 | 0 | 0 | 0 | 0 | 100 | 0 | 0 | 100 | 47.82 | 100 |
| | 3659delC# | 4 | 0 | 0 | 0 | 0 | 100 | 0 | 0 | 100 | 39.76 | 100 |
| | S1255X(19) | 4 | 0 | 0 | 0 | 0 | 100 | 0 | 0 | 100 | 39.76 | 100 |
| Intron 19 | 3849+10kb# | 13 | 0 | 0 | 0 | 0 | 100 | 0 | 0 | 100 | 75.29 | 100 |
| Ex 20 | S1255X(20) | 4 | 0 | 0 | 0 | 0 | 100 | 0 | 0 | 100 | 39.76 | 100 |
| | 3876delA | 1 | 1 | 0 | 0 | 0 | 100 | 0 | 0 | 100 | 29.24 | 100 |
| | 3905insT | 2 | 0 | 0 | 0 | 0 | 100 | 0 | 0 | 100 | 15.81 | 100 |
| | W1282X# | 8 | 0 | 0 | 0 | 0 | 100 | 0 | 0 | 100 | 63.06 | 100 |
| Ex 21 | N1303K# | 6 | 0 | 0 | 0 | 0 | 100 | 0 | 0 | 100 | 54.07 | 100 |
| Ex 10 | I506V-var tg | 3 | 0 | 0 | 0 | 0 | 100 | 0 | 0 | 100 | 29.24 | 100 |
| Ex 10 | I506V-variant | 5 | 0 | 0 | 0 | 0 | 100 | 0 | 0 | 100 | 47.82 | 100 |
| | I507V-variant | 0 | 1 | 0 | 0 | 0 | 100 | 0 | 0 | 100 | 2.50 | 100 |
| | F508C-variant | 5 | 0 | 0 | 0 | 0 | 100 | 0 | 0 | 100 | 47.82 | 100 |
# member of ACMG-23 panel
*Clopper-Pearson CI calculator provided by John C. Pezzullo (Kissimmee, Florida, USA) and is available at http://statpages.org/confint.
# b. Matrix comparison:
Not applicable. This test is only for use with human whole blood collected using
EDTA as the anticoagulant.
# 3. Clinical studies:
# a. Clinical Sensitivity:
{9}
The clinical sensitivity can be estimated based on the published studies of mutation frequencies in various ethnicities and based on the results of analytical studies described in this submission.
b. Clinical specificity:
The clinical specificity can be estimated based on the published literature and the results of analytical studies described in this submission.
c. Other clinical supportive data (when a. and b. are not applicable):
Not applicable.
4. Clinical cut-off:
Not applicable
5. Expected values/Reference range:
Cystic fibrosis (CF) is the most common autosomal recessive disorder in the Caucasian population, with an incidence of approximately 1 in 3,200 live births. The incidence of CF in other ethnic groups varies, as seen in the following table.
| Ethnic Group | Incidence of Cystic Fibrosis |
| --- | --- |
| North American Caucasian | 1 in 3200 |
| Ashkenazi Jewish | 1 in 3300 |
| Hispanic | 1 in 9500 |
| African American | 1 in 15 300 |
| Asian American | 1 in 32 100 |
| Native American (Pueblo) | 1 in 3970 |
| Native American (Zuni) | 1 in 1347 |
N. Instrument Name:
Luminex System (100 IS or 200 including ISv2.3 or xPONENT v3.1 software, calibrators, and controls)
O. System Descriptions:
See k073506 for a description of the Luminex System and base software environment
Results:
For each sample analyzed by the CFTR 39 kit v2, an output file containing MFI signals from the Luminex Instrument is generated. The proprietary software component of this product analyzes this output data file, to provide a final qualitative genotype for the sample. The software includes a feature which enables users to select between 2 options for the final output:
Option 1: Full Panel (39 mutations/deletions + 4 variants).
Option 2: ACMG/ACOG panel (23 mutations and deletions).
When Option 2 is chosen, the non-ACMG/ACOG recommended panel of mutations/variants will not be available to the user.
FDA has reviewed applicant's Hazard Analysis and software development processes for this line of product types:
{10}
Yes ☐ x ☐ or No ☐
1. Specimen Identification:
Users must fill in Batch Information by providing a unique batch Name, Description and Creator. Users have to enter appropriate patient information, i.e. number of samples, and sample IDs.
2. Specimen Sampling and Handling:
Genomic DNA preparation, multiplex (16-plex) PCR, amplicon treatment, multiplex ASPE, bead hybridization and incubation with reporter are performed semi-manually. Subsequently, beads are analyzed by the Luminex® 100 xMAP™ system, and data generated analyzed by TDAS CFTR Analysis Software CF-I (TDAS CF-I) to provide final genotype for the sample. User must pre-select sample view, either “ACMG panel” or “Full Panel” for each unique patient sample to be analyzed. The user will not be able to unmask any hidden data for samples for which the “ACMG panel” is selected, and visa-versa.
3. Calibration:
The Luminex system should be prepared and calibrated according to procedures described in the Luminex 100 IS 2.3 user Manual or Luminex 200 System user Manual.
The xTAG Cystic Fibrosis v2 T-A IS template must be installed on the computer that controls the Luminex system.
4. Quality Control:
Negative Controls:
At least two DNase, RNase free water controls are required with each run. The wells which are negative controls must be designated when analyzing data with the TDAS CFTR software.
Positive Controls:
It is recommended to routinely include in each run a set of rotating positive controls for CFTR mutations probed by the kit. Since the CF dF508 mutation is the most common (accounting for 30-88% of all CF mutations depending on ethnic group*) it is recommended that a control sample wit this mutation be included in every run. Luminex Molecular Diagnostics recommends the use of genomic DNA controls similar to the specimen type whenever feasible although spiked controls (using synthetic DNA) may be used when specimen samples are not available.
(*Gibson, Moskowitz et al, 2001)
P. Other Supportive Instrument Performance Characteristics Data Not Covered In The "Performance Characteristics" Section above:
None
Q. Proposed Labeling:
The labeling is sufficient and it satisfies the requirements of 21 CFR Part 809.10, labeling for in vitro diagnostic products.
R. Conclusion:
The submitted information in this premarket notification is complete and supports a substantial equivalence decision.
