ELIA CCP, MODEL 14-5515-01; ELIA CCP CONTROL, MODEL 83-1009-01

K061165 · Sweden Diagnostics (Us), Inc. · NHX · Aug 3, 2006 · Immunology

Device Facts

• Record ID: K061165
• Device Name: ELIA CCP, MODEL 14-5515-01; ELIA CCP CONTROL, MODEL 83-1009-01
• Applicant: Sweden Diagnostics (Us), Inc.
• Product Code: NHX · Immunology
• Decision Date: Aug 3, 2006
• Decision: SESE
• Submission Type: Traditional
• Regulation: 21 CFR 866.5775
• Device Class: Class 2

Indications for Use

EliA CCP is intended for the in vitro semi-quantitative measurement of IgG antibodies directed to CCP in human serum and plasma. The presence of anti-CCP antibodies can be used in conjunction with clinical findings and other laboratory tests as an aid in the clinical diagnosis of rheumatoid arthritis (RA). EliA CCP uses the EliA IgG method on the instruments ImmunoCAP 100 and ImmunoCAP 250. EliA CCP Control is intended for laboratory use in monitoring the performance of in vitro measurement of anti-cyclic citrullinated peptide (CCP) antibodies with ImmunoCAP using the EliA IgG method. The device is for prescription use only.

Device Story

EliA CCP is an in vitro diagnostic test for semi-quantitative measurement of IgG anti-CCP antibodies. Device uses microwells coated with synthetic citrullinated peptides; patient serum/plasma samples are added. If anti-CCP antibodies are present, they bind to the antigen. After washing, enzyme-labeled anti-IgG (β-Galactosidase) is added to form antibody-conjugate complexes. Following further washing, a development solution (4-methylumbelliferyl-β-D-galactoside) is added; reaction is stopped with sodium carbonate. Fluorescence is measured by ImmunoCAP 100 or 250 instruments. Higher fluorescence indicates higher specific IgG concentration. Results are compared to calibrators to determine anti-CCP levels. Used in clinical laboratories by trained personnel to assist physicians in RA diagnosis. Output provides quantitative values used alongside clinical findings and other laboratory tests to support diagnostic decisions.

Clinical Evidence

Clinical study evaluated 534 subjects: 82 with confirmed RA and 452 with other conditions (CTD, infectious diseases, osteoarthritis, etc.). Clinical sensitivity was 87.8% (72/82) and clinical specificity was 96.7% (437/452). Method comparison with predicate (n=80) showed 98.1% positive agreement and 89.3% negative agreement. Analytical studies confirmed precision (CVs 2.7-7.0%), linearity, and lack of interference from bilirubin, hemoglobin, chyle, or RF.

Technological Characteristics

Fluoroenzyme-immunoassay; solid phase: microwells coated with synthetic citrullinated peptides. Detection: β-Galactosidase anti-IgG conjugate; substrate: 4-methylumbelliferyl-β-D-galactoside. Instrumentation: ImmunoCAP 100 or 250. Measuring range: 0.4 to ≥340 EliA U/mL. Calibrators traceable to WHO IRP 67/86. Software: automated instrument-based processing.

Indications for Use

Indicated for in vitro semi-quantitative measurement of IgG anti-CCP antibodies in human serum/plasma to aid in the clinical diagnosis of rheumatoid arthritis (RA). For prescription laboratory use.

Regulatory Classification

Identification

A rheumatoid factor immunological test system is a device that consists of the reagents used to measure by immunochemical techniques the rheumatoid factor (antibodies to immunoglobulins) in serum, other body fluids, and tissues. Measurement of rheumatoid factor may aid in the diagnosis of rheumatoid arthritis.

Predicate Devices

• DIASTAT Anti-CCP (k023285)

Submission Summary (Full Text)

{0}

1

# 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION SUMMARY

A. 510(k) Number:
k061165

B. Purpose for Submission:
New device

C. Measurand:
Anti-cyclic citrullinated peptides (CCP)

D. Type of Test:
Semi-quantitative fluoroenzymeimmunoassay

E. Applicant:
Phadia US, Inc. (formerly Sweden Diagnostics (US), Inc.)

F. Proprietary and Established Names:
EliA CCP, EliA CCP Control

G. Regulatory Information:
1. Regulation section:
21 CFR §866.5775 Rheumatoid Factor Immunological Test System
2. Classification:
Class II
3. Product code:
NHX, Antibodies, Anti-cyclic citrullinated peptide
4. Panel:
Immunology 82

H. Intended Use:
1. Intended use(s):
EliA CCP is intended for the in vitro semi-quantitative measurement of IgG antibodies directed to (CCP) in human serum and plasma. The presence of anti-CCP antibodies can be used in conjunction with clinical findings and other laboratory tests as an aid in the clinical diagnosis of rheumatoid arthritis (RA). EliA CCP uses the EliA IgG method on the instrument ImmunoCAP 100 [or ImmunoCAP 250].
2. Indication(s) for use:
Same as Intended Use.
3. Special conditions for use statement(s):
For prescription use
4. Special instrument requirements:
ImmunoCAP 100 or ImmunoCAP 250

I. Device Description:
The device components consist of: CCP Wells (coated with citrullinated synthetic peptides); CCP Controls: Negative, and Positive containing IgG antibodies to CCP; Sample Diluent; IgG Conjugate (β-Galactosidase anti-IgG mouse monoclonal antibodies; IgG Calibrators (0, 4, 10, 20, 100, 600 μg/L); IgG Curve Control; IgG Calibrator Well (coated with mouse monoclonal antibodies); Dummy Well; General Reagents (Development Solution of 1% 4-methylumbelliferyl-β-D-galactoside, Stop

{1}

Solution of $4\%$ sodium carbonate, and ImmunoCAP Washing Solution).

# J. Substantial Equivalence Information:

1. Predicate device name(s): Axis Shield Diagnostics, Ltd. DIASTAT Anti-CCP
2. Predicate 510(k) number(s): k023285
3. Comparison with predicate:

| Similarities | | |
| --- | --- | --- |
| Item | Device | Predicate |
| | EliA CCP | DIASTAT Anti-CCP |
| Intended Use | Semi-quantitative measurement of IgG antibodies directed to CCP in human serum and plasma as an aid in the clinical diagnosis of RA | Semi-quantitative/ qualitative ELISA for the detection of the IgG class of autoantibodies specific to cyclic citrullinated peptide (CCP) in human serum and plasma as an aid in the diagnosis of RA |
| Antigen | Synthetic citrullinated peptide | Same |
| Controls | Positive and negative | Same |
| Solid phase | Microwells | Same |
| Differences | | |
| --- | --- | --- |
| Item | Device | Predicate |
| | EliA CCP | DIASTAT Anti-CCP |
| Method | Fluoroenzyme-immunoassay | Enzyme-linked immunosorbent assay |
| Instrumentation | ImmunoCAP 100 or ImmunoCAP 250 | ELISA plate reader with 550 nm filter |
| Conjugated antibody | β-Galactosidase anti-IgG mouse monoclonal antibodies | Alkaline phosphatase-labeled murine monoclonal antibody to human IgG |
| Reference range | <7 U/mL = negative 7-10 U/mL = equivocal >10 U/mL = positive | Qualitative: absorbance ratio of <0.95 = negative; ≥0.95- ≤1.0 = borderline; > 1.0 = positive Semi-quantitative: ≤5 U/mL = negative >5 U/mL = positive |
| Substrate | 4-methylumbelliferyl-β-D-galactoside | Phenolphthalein monophosphate |
| Calibration | Total IgG calibrators (0, 4, 10, 20, 100, 600 μg/L) | Anti-CCP standards (0, 2, 8, 30, 100 U/mL) |

{2}

3

| Differences | | |
| --- | --- | --- |
| Item | Device | Predicate |
| Signal | Fluorescence | Optical density |
| Sample diluent | PBS containing BSA, detergent and sodium azide – ready to use | Phosphate buffer, protein stabilizer with sodium azide – 5X, dilute before use |
| Stop solution | Sodium carbonate | Sodium hydroxide |

K. Standard/Guidance Document Referenced (if applicable):
“Guidance for the Content of Premarket Submissions for Software Contained in Medical Devices”

L. Test Principle:
The EliA CCP Wells are coated with citrullinated synthetic peptides. If present in the patient’s specimen, antibodies to CCP bind to their specific antigen. After washing away non-bound antibodies, enzyme-labeled antibodies against human IgG antibodies (EliA IgG Conjugate) are added to form an antibody-conjugate complex. After incubation, non-bound conjugate is washed away and the bound complex is incubated with a Development Solution. After stopping the reaction, the fluorescence in the reaction mixture is measured. The higher the response value, the more specific IgG is present in the specimen. To evaluate test results, the response for patient samples is compared directly to the response for calibrators.

M. Performance Characteristics (if/when applicable):
1. Analytical performance:
a. Precision/Reproducibility:
Three positive samples were measured in duplicate on one instrument in 6 runs on 3 different CCP well batches together with 3 different conjugate batches. The studies yielded the following:

| Sample | Mean (U/mL) | Inter-run (CV %) | Intra-run (CV %) |
| --- | --- | --- | --- |
| 1 | 18.3 | 7.0 | 2.7 |
| 2 | 90.7 | 5.1 | 2.6 |
| 3 | 419 | 6.6 | 5.4 |

b. Linearity/assay reportable range:
Linearity by dilution was tested by using 5 high positive anti-CCP samples. The samples were tested in duplicates for dilutions at 1:2, 1:4, 1:8, and 1:16. The results of the dilutions were compared with their expected values and the ratios of observed-to-expected values were calculated. The following ratios were obtained.

| | Mean concentration (U/mL) | O/E (ratio) |
| --- | --- | --- |
| Sample 1 | 283.9 | 1.00 |
| 1:2 | 166.7 | 1.17 |
| 1:4 | 79.9 | 1.13 |
| 1:8 | 40.7 | 1.15 |
| 1:16 | 21.1 | 1.19 |

{3}

| | Mean concentration (U/mL) | O/E (ratio) |
| --- | --- | --- |
| Sample 2 | 287.8 | 1.00 |
| 1:2 | 155.2 | 1.08 |
| 1:4 | 73.3 | 1.02 |
| 1:8 | 40.6 | 1.13 |
| 1:16 | 20.4 | 1.14 |
| Sample 3 | 238.1 | 1.00 |
| 1:2 | 129.2 | 1.09 |
| 1:4 | 69.3 | 1.16 |
| 1:8 | 36.4 | 1.22 |
| 1:16 | 17.4 | 1.17 |
| Sample 4 | 74.6 | 1.00 |
| 1:2 | 39.5 | 1.06 |
| 1:4 | 20.0 | 1.07 |
| 1:8 | 10.7 | 1.14 |
| 1:16 | 5.7 | 1.22 |
| Sample 5 | 130.2 | 1.00 |
| 1:2 | 68.8 | 1.06 |
| 1:4 | 36.4 | 1.12 |
| 1:8 | 19.0 | 1.17 |
| 1:16 | 9.6 | 1.18 |

Two of the calculated ratios were above the specifications for this study. The package insert includes a statement that not all sera can be diluted linearly within the measuring range of the assay due to differing binding characteristics of the antibodies in the patient samples. The assay measuring range for the EliA CCP is from 0.4 to $\geq 340$ EliA U/mL/

c. Traceability, Stability, Expected values (controls, calibrators, or methods): The IgG calibrators are traceable (via an unbroken chain of calibrations) to the International Reference Preparation (IRP) 67/86 of Human Serum Immunoglobulins A, G and M from WHO. New batches of calibrators are compared to a secondary standard (standardized with the IRP) or the IRP directly and adjusted accordingly to meet the correct concentration (6 levels).

d. Detection limit:

The lower limit of the measuring range was determined by measuring dilutions (1:2, 1:4, and 1:8) of the $4\mu \mathrm{g} / \mathrm{L}$ calibrator in the Calibrator Wells. The results in Response Units (RU) were compared with the result of the sample diluent on CCP Wells. The discrimination ability of the assay should be $&gt;2$ . All samples were measured in triplicate.

| Results on Calibrator Wells | | |
| --- | --- | --- |
| Sample ID | Mean Response Units (RU) | SD |
| Sample Diluent | 31 | - |
| Calibrator 4.0 | 316 | - |
| Calibrator 4.0 at 1:2 | 127 | - |

{4}

5

| Results on Calibrator Wells | | |
| --- | --- | --- |
| Calibrator 4.0 at 1:4 | 89 | - |
| Calibrator 4.0 at 1:8 | 61 | 1.47 |
| Results on CCP Wells | | |
| Sample ID | Mean RU | SD |
| Sample Diluent | 20 | 10.6 |

The discrimination ability was 3.8 and the 1:8 dilution of the 4 µg/L calibrator could be discriminated from background. The lower limit of the measuring range was set at 0.5 µg/L, corresponding to 0.4 EliA U/mL.

e. Hook effect:

Hook effect was analyzed by using dilutions from a highly positive anti-CCP serum sample with an estimated anti-CCP IgG concentration around 4400 U/mL. The sample was measured in triplicate.

| Results on Calibrator Wells | | | | | |
| --- | --- | --- | --- | --- | --- |
| Sample ID | Mean RU | CV% | Mean conc. µg/L | CV% | |
| Cal-600 | 17533 | 0.2 | 600.9 | 0.6 | |
| Results on CCP Wells | | | | | |
| Sample | Mean RU | CV% | Mean conc. µg/L | CV% | Estimated conc. U/mL |
| 1:25 | 22236 | 2.5 | Over | | 4438 |
| 1:100 | 20882 | 2.1 | Over | | 1109 |
| 1:200 | 17766 | 7.5 | Over | | 555 |
| 1:400 | 13858 | 1.1 | 251.8 | 2.0 | 277 |

Dilutions showed that no hook effect occurred for this sample. The discrimination ability was 8.4 for the sample compared to the response received for Calibrator 600.

f. Analytical specificity:

The potential interferant and the corresponding blanks were added to two anti-CCP positive sera from patients with RA. The spiked samples were tested in triplicate.

| Additives | Concentration in raw sample | Final concentration in diluted sample (1:100) | Normal Values |
| --- | --- | --- | --- |
| Bilirubin F | 18.8 mg/dL | 0.188 | <1.0 |
| Bilirubin C | 20 mg/dL | 0.2 | <1.0 |
| Chyle | 236,000 Units/dL | 2360 | No data |
| Hemoglobin | 453 mg/dl | 4.5 | <2.0 |
| Rheumatoid Factor IgM | 550 IU/ml | 5.5 | <40.0 |

The ratio of the result of the sample spiked with the interfering substance and

{5}

the sample spiked with a buffer blank was determined:

| | | Positive Sample | | | Low Positive Sample | | |
| --- | --- | --- | --- | --- | --- | --- | --- |
| Additive | Blank/spiked sample | Conc. (U/mL) | CV % | Ratio | Conc. (U/mL) | CV % | Ratio |
| Bilirubin C | Blank | 67.1 | 8.4 | 0.98 | 13.4 | 6.2 | 1.01 |
| | Sample | 65.6 | 0.4 | | 13.5 | 8.3 | |
| Bilirubin F | Blank | 61.0 | 2.9 | 1.03 | 12.4 | 4.2 | 0.95 |
| | Sample | 62.9 | 2.6 | | 11.8 | 13.5 | |
| Hemoglobin | Blank | 63.6 | 1.6 | 0.92 | 12.6 | 0.7 | 0.93 |
| | Sample | 58.5 | 12.0 | | 11.7 | 6.7 | |
| Chyle | Blank | 64.3 | 4.3 | 1.01 | 13.0 | 8.5 | 1.02 |
| | Sample | 64.7 | 3.7 | | 13.2 | 10.0 | |
| RF | Blank | 62.5 | 4.8 | 0.96 | 13.5 | 9.5 | 0.90 |
| | sample | 59.8 | 4.9 | | 12.2 | 2.1 | |

The interfering substances listed did not appear to adversely affect the results of the new device.

g. Assay cut-off:

The purpose of the normal sera studies was to evaluate expected values in the normal population and to confirm the defined cut-off. Samples from 400 apparently healthy Caucasian adult blood donors were measured. The individuals were equally distributed by sex and age.

| | EliA U/mL |
| --- | --- |
| Mean | 2.6 |
| Median | 2.3 |
| Mean + 2 SD | 7.6 |
| Mean + 3 SD | 10.1 |
| 95th percentile | 4.3 |
| 99th percentile | 6.2 |

The results appeared to be equally distributed and not dependent on age or gender. The 99th percentile lies below the lower limit of the equivocal range of 7-10 U/mL.

2. Comparison studies:

a. Method comparison with predicate device:

Samples from 55 anti-CCP positive RA patients and 25 non-RA sample were tested.

| | N = 80 | Diastat anti-CCP | | |
| --- | --- | --- | --- | --- |
| | | Positive | Negative | Total |
| EliA CCP | Positive | 51 | 3 | 54 |
| | Negative | 1 | 25 | 26 |
| | Total | 52 | 28 | 80 |

{6}

Positive percent agreement 51/52 x 100 = 98.1%
Negative percent agreement 25/28 x 100 = 89.3%
Overall percent agreement 76/80 x 100 = 95%

b. Matrix comparison:

Fifty sets of samples from different donors were tested in double determinations. Each set contained serum, EDTA, heparin and citrate plasma samples taken at the same time from a single donor. All samples were tested and found to be negative for anti-CCP antibodies in all 4 sample types.

Antibodies were spiked into 25 of the sample sets to create 25 positive sample sets. Linear regression comparing the quotas between serum and each type of plasma for the positive samples was performed and showed:

| Plasma | r value | Slope |
| --- | --- | --- |
| Heparin | 0.9966 | 0.9861 |
| EDTA | 0.9975 | 0.9749 |
| Citrate | 0.9987 | 0.9912 |

The data show acceptable results for plasma versus serum in positive and negative samples.

c. Instrument platform comparison

For this comparison study, a total of 36 samples distributed over the measuring range were assayed: 4 negative samples, 1 equivocal sample and 31 positive samples. All samples were run on three ImmunoCAP 100 instruments and three ImmunoCAP 250 instruments in two runs and in single replicates. Results of the conformance study are summarized in the figure below:


Correlation Result: y=0.99x+1.0275 where x is ImmunoCAP 100 and y is ImmunoCAP 250

{7}

8

3. Clinical studies:

a. Clinical sensitivity and specificity:

The clinical studies were performed with 534 sera from subjects with clinically confirmed RA (n=82) and 452 other diseases (listed below).

| Other disease groups | No. of patients | No. of positives |
| --- | --- | --- |
| CTD | | |
| UCTD | 11 | 3 |
| SLE | 30 | 0 |
| Scleroderma | 39 | 3 |
| Sjögren’s syndrome | 20 | 3 |
| MCTD | 10 | 0 |
| Myositis | 10 | 0 |
| Total | 120 | 9 |
| | | |
| Infectious diseases | | |
| CMV | 25 | 0 |
| EBV | 30 | 1 |
| HBV | 10 | 0 |
| HCV | 10 | 0 |
| HIV | 15 | 0 |
| Other infectious diseases | 95 | 2 |
| Total | 185 | 3 |
| | | |
| Other | | |
| Osteoarthritis | 35 | 0 |
| Vasculitis | 21 | 0 |
| Autoimmune thyroid diseases | 20 | 0 |
| Crohn’s disease | 20 | 0 |
| Ulcerative colitis | 20 | 0 |
| Other misc. | 31 | 3 |
| Total | 147 | 3 |

Study results:

| | Rheumatoid Arthritis | | | |
| --- | --- | --- | --- | --- |
| | | Positive | Negative | Total |
| EliA CCP result | Positive | 72 | 15 | 87 |
| | Negative | 10 | 437 | 447 |
| | Total | 82 | 452 | 534 |

Clinical sensitivity: 87.8% (72/82)
Clinical specificity: 96.7% (437/452)
Overall agreement: 95.3% (509/534)

{8}

b. Other clinical supportive data (when a. is not applicable):
Not applicable

4. Clinical cut-off:
See assay cut-off.

5. Expected values/Reference range:
The expected value in the normal population is negative. The proportion of sera found positive for anti-CCP antibodies using the EliA CCP assay in the adult normal population tested was less than 1%.

N. Proposed Labeling:
The labeling is sufficient and it satisfies the requirements of 21 CFR Part 809.10.

O. Conclusion:
The submitted information in this premarket notification is complete and supports a substantial equivalence decision.

9