FACTOR II (PROTHROMBIN) G20210A KIT

K033612 · Roche Diagnostics Corp. · NPR · Dec 18, 2003 · Hematology

Device Facts

• Record ID: K033612
• Device Name: FACTOR II (PROTHROMBIN) G20210A KIT
• Applicant: Roche Diagnostics Corp.
• Product Code: NPR · Hematology
• Decision Date: Dec 18, 2003
• Decision: SESE
• Submission Type: Traditional
• Regulation: 21 CFR 864.7280
• Device Class: Class 2

Indications for Use

The Factor II (Prothrombin) G20210A Kit is an in vitro diagnostic test for the detection and genotyping of a single point mutations (G to A at position 20210) of the human Factor II gene, from DNA isolated from human whole peripheral blood. The Factor II (Prothrombin) G20210A Kit is indicated as an aid to diagnosis in the evaluation of patients with suspected thrombophilia

Device Story

In vitro diagnostic test for detection/genotyping of Factor II (Prothrombin) G20210A mutation; utilizes DNA isolated from human whole peripheral blood. Operates on LightCycler Instrument using PCR for DNA amplification and fluorigenic target-specific hybridization for genotyping. Used in clinical laboratory settings. Healthcare providers use output to aid diagnosis of patients with suspected thrombophilia. Benefits include identification of genetic risk factor for thrombosis.

Clinical Evidence

Clinical validation performed by comparing kit results against DNA sequencing method. Study included 572 samples; observed 99.5% agreement between methods.

Technological Characteristics

In vitro diagnostic kit; utilizes PCR amplification and fluorigenic target-specific hybridization. Designed for use on LightCycler Instrument with SW 3.5. Analytical sensitivity < 50 allele copies per reaction. Reagents stable at -15 to -25°C.

Indications for Use

Indicated for the detection and genotyping of the Factor II (Prothrombin) G20210A mutation as an aid to diagnosis in the evaluation of patients with suspected thrombophilia.

Regulatory Classification

Identification

Factor V Leiden mutation detection system is a device that allows the detection and genotyping of a single point mutation of the human Factor V gene, referred to as Factor V Leiden mutation, from DNA isolated from human whole peripheral blood. The system consists of different reagents and instruments which includes polymerase chain reaction (PCR) primers, hybridization matrices, thermal cyclers, imagers, and software packages. The detection system is an aid to diagnosis in the evaluation of patients with suspected thrombophilia.

Special Controls

*Classification.* Class II (special controls). The special control is FDA's guidance entitled “Class II Special Controls Guidance Document: Factor V Leiden DNA Mutation Detection Systems.” (See § 864.1(d) for the availability of this guidance document.)

Submission Summary (Full Text)

{0}

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

A. 510(k) Number:
K033612

B. Analyte:
Factor II (Prothrombin)

C. Type of Test:
Qualitative

D. Applicant:
Roche Diagnostics Corporation

E. Proprietary and Established Names:
Factor II (Prothrombin) G20210A Kit

F. Regulatory Information:
1. Regulation section:
864.7280
2. Classification:
Class II
3. Product Code:
NPR
4. Panel:
81 Hematology

G. Intended Use:
1. Intended use(s):
The Factor II (Prothrombin) G20210A Kit is an in vitro diagnostic test for the detection and genotyping of a single point mutations (G to A at position 20210) of the human Factor II gene, from DNA isolated from human whole peripheral blood.
2. Indication(s) for use:
The Factor II (Prothrombin) G20210A Kit is indicated as an aid to diagnosis in the evaluation of patients with suspected thrombophilia
3. Special condition for use statement(s):
None.
4. Special instrument Requirements:
The test is intended to be used on the LightCycler instrument. The sample preparation must be performed according to a workflow procedure described in the package insert.

{1}

Page 2 of 5

# H. Device Description

The Factor II (Prothrombin) G20210A Kit is an in vitro diagnostic test for the detection and genotyping of the Factor II (Prothrombin) G20210A mutation, from DNA isolated from human whole peripheral blood. The test incorporates fluorogenic target-specific hybridization to PCR-amplified Factor II gene DNA to detect the presence of the mutation. Melting curve analysis of the probe-target complex reveals the presence of wild type and mutant alleles by their different melting temperatures.

# I. Substantial Equivalence Information:

1. Predicate device name(s): Factor V Leiden Kit
2. Predicate K number(s): K033607
3. Comparison with predicate:

| Similarities | | |
| --- | --- | --- |
| Item | Device | Predicate |
| Specimen type | Blood | Same |
| Sample type | Purified DNA | Same |
| Instrumentation | LightCycler Version 1.2 | Same |
| Thermal cycling program | (95, 55, 72°C) X 45 | Same |
| Precision | Tm1: 0.23-0.28% CV
Tm2: 0.18-0.30% CV
ΔTm: 1.09-1.35% CV | Tm1: 0.19-0.33% CV
Tm2: 0.22-0.44% CV
ΔTm: 1.23-1.53% CV |
| Sensitivity | 50 allele copies/reaction | Same |
| Interferences | Heparin | Same |
| Comparator | Sequence | Same |
| Performance | 98.0% sens., 99.6% spec. overall (nonclinical and clinical samples) | 100% sens., 99.8% spec. overall (nonclinical and clinical samples) |
| Differences | | |
| Item | Device | Predicate |
| Oligonucleotide probes and primers | Specific for Factor II (prothrombin) G20210A | Specific for Factor V Leiden |
| Melting curve | 45-75°C, 0.1°C/sec ramp | 40-70°C, 0.1°C/sec ramp |
| Melting temp. | 49°C, 55°C | 57, 65°C |
| ΔTm | 10 ±1.5°C | 8±1.5°C |

# J. Standard/Guidance Document Referenced (if applicable):

Class II Special Controls Guidance Document: Factor V Leiden DNA mutation detection system

# K. Test Principle:

Fluorogenic detection of PCR-amplified Factor II locus by melting curve analysis.

{2}

Page 3 of 5

# L. Performance Characteristics (if/when applicable):

## 1. Analytical performance:

a. Precision/Reproducibility:

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

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:

Crossing point &lt;31

c. Traceability (controls, calibrators, or method):

Not applicable

d. Detection limit:

50 copies of locus

e. Analytical specificity:

5/5 heterozygous samples correctly called for human and plasmid control DNA

f. Assay cut-off:

Not applicable

## 2. Comparison studies:

a. Method comparison with predicate device:

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:

{3}

Page 4 of 5

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&lt;0.0001) than those in EDTA or citrate. Heparin has been excluded as an allowable matrix for testing.

3. Clinical studies:
a. Clinical sensitivity:
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.
b. Clinical specificity:
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:
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, reviewed and cleared in K033607

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:

{4}

Page 5 of 5

PCR, fluorogenic detection of melting curve

7. Calibration:
N/A

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.
MagnaPure DNA purification instrument was reviewed and cleared in K033607, and may be used to purify sample and set up reactions.

P. Conclusion:
This device is found to be substantially equivalent to K033607 Factor V Leiden Kit.