LIGHTCYCLER INSTRUMENT VERSION 1.2

K033734 · Roche Diagnostics Corp. · JJF · Dec 17, 2003 · Clinical Chemistry

Device Facts

• Record ID: K033734
• Device Name: LIGHTCYCLER INSTRUMENT VERSION 1.2
• Applicant: Roche Diagnostics Corp.
• Product Code: JJF · Clinical Chemistry
• Decision Date: Dec 17, 2003
• Decision: SESE
• Submission Type: Traditional
• Regulation: 21 CFR 862.2170
• Device Class: Class 1

Indications for Use

The LightCycler Instrument is a fully automated amplification and detection system for nucleic acids using fluorescence detection. The LightCycler is intended to be used by laboratory professionals trained in laboratory techniques and on the use of the analyzer.

Device Story

LightCycler Version 1.2 is a benchtop analyzer for automated PCR amplification and detection of nucleic acids. Input: purified nucleic acids in glass capillaries. Operation: programmable thermal cycling using heated air to control DNA polymerase activity; integrated microvolume fluorimeter measures fluorescence. Output: real-time PCR amplification data, DNA concentration estimates, and melting curves generated by monitoring dissociation of fluorogenic probes. Used in clinical laboratories by trained professionals. Healthcare providers use melting curve analysis and amplification data to identify DNA mutations (e.g., Factor V Leiden) by distinguishing alleles based on melting temperature (Tm) shifts. Benefits include rapid, automated detection of genetic mutations compared to manual or traditional sequencing methods.

Clinical Evidence

Performance validated via reference to K033607 (Factor V Leiden Kit). Bench testing included precision/reproducibility (total CV 0.36%), linearity, and analytical specificity. Method comparison against DNA sequencing on 441 retrospective samples showed 99.3% agreement. Clinical study of 112 patient samples demonstrated 100% sensitivity and 100% specificity for Factor V Leiden mutation detection. Matrix interference testing excluded heparin due to melting temperature shifts.

Technological Characteristics

Benchtop analyzer; hot-air thermal cycler; microvolume fluorimeter; 32-sample capacity; glass capillary sample containers; 3 optical detection channels (530, 640, 710 nm); temperature range 40-98 °C; accuracy ± 0.4 °C. Connectivity via PC with instrument-specific software. Fluorescence resonance energy transfer (FRET) detection chemistry.

Indications for Use

Indicated for laboratory professionals to perform automated nucleic acid amplification and detection using fluorescence-based PCR and melting curve analysis.

Regulatory Classification

Identification

A micro chemistry analyzer for clinical use is a device intended to duplicate manual analytical procedures by performing automatically various steps such as pipetting, preparing filtrates, heating, and measuring color intensity. The distinguishing characteristic of the device is that it requires only micro volume samples obtainable from pediatric patients. This device is intended for use in conjunction with certain materials to measure a variety of analytes.

Predicate Devices

• COBAS TaqMan Analyzer (K012966)

Submission Summary (Full Text)

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510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION
DECISION SUMMARY
DEVICE AND INSTRUMENT TEMPLATE

A. 510(k) Number:
K033734

B. Analyte:
Analyte used to validate instrumentation is Factor V Leiden DNA mutation

C. Type of Test:
Qualitative

D. Applicant:
Roche Diagnostics Corporation

E. Proprietary and Established Names:
LightCycler Instrument Version 1.2

F. Regulatory Information:
1. Regulation section:
862.2170
2. Classification:
Analyzer, Chemistry, Micro, for clinical use, Class I
3. Product Code:
JJF
4. Panel:
75 Clinical Chemistry

G. Intended Use:
1. Intended use(s):
The LightCycler Instrument is a fully automated amplification and detection system for nucleic acids using fluorescence detection. The LightCycler is intended to be used by laboratory professionals trained in laboratory techniques and on the use of the Analyzer
2. Indication(s) for use:
Not specific
3. Special condition for use statement(s):
Professional use only
4. Special instrument Requirements:
None

H. Device Description
The LightCycler Version 1.2 is a flexible, benchtop analyzer that automates the amplification and detection steps of the Polymerase Chain Reaction (PCR) through

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programmable thermal cycling steps. The instrument uses heated air to control the activity of DNA polymerase that makes copies of sample DNA fragments. The LightCycler also calculates approximate DNA concentration of the amplified sample when compared to a standard, and generates melting curves of amplified DNA by measuring the dissociation of a fluorogenic probe sequence from the amplified DNA fragments.

I. Substantial Equivalence Information:

1. Predicate device name(s): COBAS TaqMan Analyzer
2. Predicate K number(s): K012966
3. Comparison with predicate:

| Similarities | | |
| --- | --- | --- |
| Item | Device | Predicate |
| Intended use | The LightCycler Instrument is a fully automated amplification and detection system for nucleic acids using fluorescence detection. The LightCycler is intended to be used by laboratory professionals trained in laboratory techniques and on the use of the Analyzer. | The COBAS TaqMan Analyzer is a fully automated amplification and detection system for nucleic acids using 5’ nuclease technology. The COBAS TaqMan Analyzer is intended to be used by laboratory professionals trained in laboratory techniques and on the use of the Analyzer |
| Primary operational components | Integrated thermocycler and microvolume fluorimeter for walkaway PCR amplification and detection | Same |
| Detection procedure | Optical detection of stimulated fluorescence | Same |
| Specimen type | Purified nucleic acids | Same |
| Specimen preparation | Performed off-line | Same |
| Temperature range | 40-98°C | Same |
| User Interface | PC with instrument-specific software (LightCycler version 3.5 of higher) | PC with instrument-specific software (Amplilink Software version 3.0 or higher) |
| Differences | | |
| Item | Device | Predicate |
| Heating method thermal cycling | Hot air cycling with glass capillaries | Peltier device with sample block |

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| Number of thermal cyclers | One | Four |
| --- | --- | --- |
| Sample positions | 32 | 96 |
| Sample size | 10-20 uL in glass capillaries | 100 uL in 200 uL K-tubes |
| Number of optical detection channels | Three with fixed wavelengths (530 nm, 640 nm, 710 nm) | Four with wavelength ranges 510-710 nm |
| Detection chemistry | Paired hybridization probes using fluorescence resonance energy transfer (FRET) | 5' nuclease hydrolysis probes using FRET (TaqMan technology) |
| Detection timing | Detection occurs at defined intervals during PCR cycle and can be viewed in real-time | Detection occurs only at end of each PCR cycle and be viewed at completion of run |
| Absolute temperature accuracy | ± 0.4°C | ± 1.5°C |

J. Standard/Guidance Document Referenced (if applicable):
EC Declaration of Conformity, TUV Certificate, IEC CB Test Certificate, and USL and CSL statement (standards); FDA "Guidance for the Content of Premarket Submissions for Software Contained in Medical Devices".

K. Test Principle:
Fluorogenic detection of PCR-amplified DNA fragments by melting curve analysis.

L. Performance Characteristics (if/when applicable):
1. Analytical performance:
a. Precision/Reproducibility:
(Incorporated by reference to K033607, Factor V Leiden Kit)
Determined at three sites, with three instruments and three operators.
Six replicates of each (control template and human DNA) were run at each site in ten separate runs with no more than 2 runs per day.
Imprecision was calculated according to NCCLS EP-5A.

Within-run:
Tm1: 0.14-0.26% CV
Tm2: 0.14-0.24% CV
ΔTm: 0.58-0.90% CV

Total:
Tm1: 0.19-0.33% CV
Tm2: 0.22-0.44% CV
ΔTm: 1.23-1.53% CV

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Overall median: 0.36% CV

Lot-to-lot: Eight heterozygous human samples were run in a single PCR using three lots of reagent. All lots performed within specifications.

b. Linearity/assay reportable range:
(Incorporated by reference to K033607 Factor V Leiden Kit)
Crossing point <31

c. Traceability (controls, calibrators, or method):
N/A

d. Detection limit:
(Incorporated by reference to K033607 Factor V Leiden Kit) 50 copies of locus

e. Analytical specificity:
(Incorporated by reference to K033607 Factor V Leiden Kit) 5/5 heterozygous samples correctly called for human and plasmid control DNA

f. Assay cut-off:
N/A

2. Comparison studies:

a. Method comparison with predicate device:
(Incorporated by reference to K033607 Factor V Leiden Kit)
Comparison is with DNA sequencing on Amersham MegaBASE 500 sequencer equipped with SW 3.0 and Cimarron Base Caller 3.12.
Sequence method was validated and preferred sequencing direction determined by quality score of 50 samples. All remaining samples were sequenced in the preferred direction.
Agreement between sequence and Factor II kit was 99.3% on 441 qualified retrospective repository samples. 19 repository samples could not be sequenced and were excluded from the study.

b. Matrix comparison:
(Incorporated by reference to K033607 Factor V Leiden Kit) Blood samples from 50 volunteers were collected into EDTA, citrate and heparin anticoagulants. Student’s t-test showed that samples collected in heparin had lower melting temperatures (P<0.0001) than those in EDTA or citrate. Heparin has been excluded as an allowable matrix for testing.

3. Clinical studies:

a. Clinical sensitivity:
(Incorporated by reference to K033607 Factor V Leiden Kit) One hundred twelve fresh samples from patients referred to hospital for thrombophilia testing were collected under IRB approval. Citrate (14) and EDTA (98) were used as anticoagulants. There was 100% (5/5) agreement between the sequence and the Factor II Kit results, with all 5 heterozygotes being correctly called by the Factor II kit.

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b. Clinical specificity:
(Incorporated by reference to K033607 Factor V Leiden Kit) In the same 112 samples cited above, there was 100% specificity (107/107), with no wild-type samples being called as Factor II mutations by the Factor II Kit

c. Other clinical supportive data (when a and b are not applicable):

4. Clinical cut-off:
Not applicable

5. Expected values/Reference range:
(Incorporated by reference to K033607 Factor V Leiden Kit) ΔTm’s must be 10 ± 1.5°C for wild type and mutant alleles. Melting point temperatures of 49°C and 59°C (± 2.5°C for wild type and mutant, respectively.

M. Instrument Name:
LightCycler V 1.2

N. System Descriptions:

1. Modes of Operation:
Software driven, PCR, melting curve analysis

2. Software:
FDA has reviewed applicant’s Hazard Analysis and software development processes for this line of product types:
Yes ☐ X ☐ or No ☐

3. Sample Identification:
By position in carousel

4. Specimen Sampling and Handling:
Sample applied manually or by MagnaPure instrumentation to capillaries inserted into carousel. No further handling.

5. Assay Types:
Melting curve, any DNA change that can be distinguished from wild type by ΔTm

6. Reaction Types:
PCR, fluorogenic detection of melting curve

7. Calibration:
Not applicable

8. Quality Control:
Positive control material, user inspection of melting curves.

O. Other Supportive Instrument Performance Characteristics Data Not Covered In The “L. Performance Characteristics” Section Of The SE Determination Decision Summary.

All nonclinical and clinical data is incorporated by reference to K033607, Factor V Leiden Kit.

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P. Conclusion:
This device is substantially equivalent to the COBAS TaqMan analyzer by similarity of device configuration and intended use, and demonstration of performance against a reference method (DNA sequencing) using reagents for the detection of Factor V Leiden mutations.