IMMUNOSCAN CCPLUS
Device Facts
| Record ID | K091657 |
|---|---|
| Device Name | IMMUNOSCAN CCPLUS |
| Applicant | Euro-Diagnostica AB |
| Product Code | NHX · Immunology |
| Decision Date | Nov 19, 2009 |
| Decision | SESE |
| Submission Type | Traditional |
| Regulation | 21 CFR 866.5775 |
| Device Class | Class 2 |
| Attributes | Real-World Evidence |
Real-World Evidence
| Submission | Device | Sponsor | RWD Sources | RWE Use Summary | Key Tags |
|---|---|---|---|---|---|
| K091657 · Nov 19, 2009 | IMMUNOSCAN CCPLUS | Euro-Diagnostica AB | Retrospective clinical serum samples; Patient medical records/clinical characterization | Retrospective clinical serum samples were used to evaluate the performance of the modified device, including method comparison against the predicate and assessment of clinical sensitivity and specificity across various disease states. | Retrospective study; Clinical sensitivity; Clinical specificity; Method comparison |
Clinical Evidence
| Study Design | Population | Comparator | Key Endpoints |
|---|---|---|---|
| Retrospective method comparison | RA patients and apparently healthy blood donors; Sample Size: 659 | Immunoscan RA anti-CCP (predicate) | Percent agreement (Positive, Negative, Overall) |
| Retrospective clinical performance evaluation | Patients with clinically defined RA, non-RA diseased patients, and asymptomatic individuals; Sample Size: 1180 | Not applicable for this study | Clinical sensitivity and specificity |
Indications for Use
The Immunoscan CCPlus® test kit is an enzyme-linked immunosorbent assay (ELISA) for qualitative and semi-quantitative determination of IgG antibodies to Cyclic Citrullinated Peptides (CCP) in human sera. The assay is used to detect antibodies in a single serum specimen. The results of the assay are to be used as an aid to the diagnosis of Rheumatoid Arthritis (RA), in conjunction with other laboratory and clinical findings. The analysis should be performed by trained laboratory professionals. "For in vitro diagnostic use".
Device Story
Immunoscan CCPlus® is an ELISA-based in vitro diagnostic test for detecting IgG antibodies to Cyclic Citrullinated Peptides (CCP) in human serum. The device uses microtitre plates coated with synthetic CCP. Patient serum is incubated in the wells, allowing specific antibodies to bind to the antigen. After washing, an alkaline phosphatase-labeled anti-human IgG conjugate is added, followed by a substrate solution (TMB). The resulting color intensity, measured as optical density (OD) at 450 nm, correlates to the concentration of bound antibodies. Results are calculated against a calibrator curve and expressed in arbitrary units. The test is performed by trained laboratory professionals to assist clinicians in diagnosing Rheumatoid Arthritis alongside other clinical and laboratory findings.
Clinical Evidence
Performance evaluated using 628 retrospective frozen sera (368 RA patients, 260 healthy donors) and 1180 clinically characterized samples. Overall percent agreement with predicate was 98.9% (95% CI: 97.7-99.6%). Clinical sensitivity for RA was 77.4% (95% CI: 73.3-81.5%). Clinical specificity ranged from 94.1% to 100% across various disease controls and healthy donors. Precision studies (intra-assay and inter-assay) showed %C.V. values ranging from 1.0% to 17.0%. Interference studies confirmed no significant impact from bilirubin, hemoglobin, lipids, or rheumatoid factor.
Technological Characteristics
ELISA-based immunoassay. Materials: Polystyrene microtitre plate with MaxiSorp surface. Energy source: Photometric measurement at 450 nm. Connectivity: Standalone. Sterilization: Not applicable (in vitro diagnostic kit). Software: None (manual/automated plate reader processing).
Indications for Use
Indicated for qualitative and semi-quantitative detection of IgG antibodies to Cyclic Citrullinated Peptides (CCP) in human serum specimens to aid in the diagnosis of Rheumatoid Arthritis (RA). For use by trained laboratory professionals in clinical settings.
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
- Immunoscan RA anti-CCP Test kit (K052133)
Submission Summary (Full Text)
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510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION
DECISION SUMMARY
ASSAY ONLY TEMPLATE
A. 510(k) Number:
k091657
B. Purpose for Submission:
Device modification to k052133
The modifications are:
a. Double the highest calibrator level from 1600 U/mL to 3200 U/mL
b. Replace strips of twelve wells with a 96-well microtiter plate
C. Measurand:
Anti-cyclic citrullinated peptide (CCP) IgG autoantibodies
D. Type of Test:
Qualitative and Semi-quantitative ELISA
E. Applicant:
Euro-Dagnostica AB
F. Proprietary and Established Names:
Immunoscan CCP Plus®
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 (CCP)
4. Panel:
Immunology (82)
H. Intended Use:
1. Intended use(s):
The Immunoscan CCP Plus® test kit is an enzyme-linked immunosorbent assay (ELISA) for qualitative and semi-quantitative determination of IgG antibodies to cyclic citrullinated peptides (CCP) in human sera. The assay is used to detect antibodies in a single serum specimen. The results of the assay are to be used as an aid in the diagnosis of Rheumatoid Arthritis (RA), in conjunction with other laboratory and clinical findings. The analysis should be performed by trained laboratory professionals. “For in vitro diagnostic use”.
2. Indication(s) for use:
Same as intended use.
3. Special conditions for use statement(s):
For prescription use only.
4. Special instrument requirements:
Microplate reader capable of measuring OD at 450 nm
Microplate washer (300μL volume).
I. Device Description:
Each device contains the following ready to use reagents: 1 sealed (96 wells) CCP
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peptide-coated microtiter plate; 5 vials containing calibrators (25, 50, 200, 800, 3200 U/ml); reference, positive and negative controls (human serum in diluent); conjugate solution (peroxidase conjugated to anti human IgG antibodies); substrate solution TMB(3, 3', 5, 5'-tetramethylbenzidin); dilution buffer; stop solution (0.5 M sulfuric acid); wash buffer 20 x concentrated.
# J. Substantial Equivalence Information:
1. Predicate device name(s):
Immunoscan RA anti-CCP Test Kit
2. Predicate 510(k) number(s):
k052133
3. Comparison with predicate:
Similarities
| Item | Modified Device Immunoscan CCPlus® | Predicate Device Immunoscan RA anti-CCP |
| --- | --- | --- |
| Intended use | The Immunoscan CCPlus® test kit is an enzyme-linked immunosorbent assay (ELISA) for qualitative and semi-quantitative determination of IgG antibodies to CCP in human sera. The assay is used to detect antibodies in a single serum specimen. The results of the assay are to be used as an aid to the diagnosis of RA, in conjunction with other laboratory and clinical findings. The analysis should be performed by trained laboratory professionals. “For in vitro diagnostic use”. | Same |
| Type of test | Qualitative and semi-quantitative | Same |
| Analyte measured | Anti-CCP | Same |
| Coated antigen | Synthetic CCP | Same |
| Conjugate | Horse radish peroxidase (HRP) labelled Anti-human IgG | Same |
| Substrate | TMB (3, 3', 5, 5'-tetramethylbenzidin) | Same |
| Wavelength | 450 nm | Same |
| Incubation time | 60 min + 30 min + 30 min | Same |
| Cut-off | ≥25 U/mL | Same |
| Sample | Serum | Same |
| Sample preparation | Dilute 1:50 | Same |
| Microtiter plate | Material: PolystyreneSurface: MaxiSorp | Same |
| Controls | Reference control, Negative control Positive control | Same |
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Differences
| Item | Modified Device Immunoscan CCPlus® | Predicate Device Immunoscan RA anti-CCP |
| --- | --- | --- |
| Microtiter plate | 96 individual wells plate | 8 strips x 12 wells plate |
| Calibrators | 25 U/mL, 50 U/mL, 200 U/mL, 800 U/mL, 3200 U/mL
Note: The same material is used to produce the calibrator | 25 U/mL, 50 U/mL, 200 U/mL, 800 U/mL, 1600 U/mL |
K. Standard/Guidance Document Referenced (if applicable):
None provided
L. Test Principle:
The Immunoscan CCPlus® test kit is an enzyme-linked immunosorbent assay (ELISA) for detection and semi-quantitation of IgG antibodies to CCP in human sera. The test utilizes microtiter plate wells coated with citrullinated synthetic peptides (antigen). Diluted patient serum is applied to the wells and incubated. If specific antibodies are present, they will bind to the antigen in the wells. Unbound material is washed away and any bound antibody is detected by adding horse radish peroxidase (HRP) labelled anti-human IgG, followed by a second washing step then incubation with substrate. The presence of reacting antibodies will result in the development of colour, which is proportional to the quantity of bound antibody, and this is determined photometrically.
M. Performance Characteristics (if/when applicable):
1. Analytical performance:
a. Precision/Reproducibility:
Intra-assay precision was determined by testing six different samples across the measuring range (two high eight times each). The results are summarized in the table below:
| | High | | | Medium | Low | |
| --- | --- | --- | --- | --- | --- | --- |
| Mean (U/mL). | 2672 | 2685 | 1150 | 239 | 56 | 60 |
| S.D. (U/mL) | 138 | 205 | 55.3 | 2.3 | 2.1 | 1.2 |
| % C.V. | 5.2 | 7.6 | 4.8 | 1.0 | 3.8 | 1.9 |
Two additional samples were run (8 replicates each) – a weak positive sample and one just below the cut off of 25 U/ml. The results are summarized in the table below:
| | < 25 U/ml | Weak positive |
| --- | --- | --- |
| Mean (U/mL) | 19 | 28 |
| S.D. (U/mL) | 0.2 | 0.5 |
| % C.V. | 1.1 | 3.6 |
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Inter-assay precision/reproducibility was determined by testing six samples eight times each. Results were obtained for three different runs:
| | High | | | Medium | Low | |
| --- | --- | --- | --- | --- | --- | --- |
| Mean (U/mL) | 2696 | 2600 | 1168 | 242 | 59 | 62 |
| S.D. (U/mL) | 328 | 299 | 101.7 | 5.0 | 3.1 | 3.0 |
| % C.V. | 12.2 | 11.5 | 8.7 | 2.1 | 5.2 | 4.9 |
Two additional samples were run in 3 plates and 8 replicates each— a weak positive sample and one just below the cut off of $25\mathrm{U / ml}$ . The results are summarized below:
| | < 25 U/ml | Weak positive |
| --- | --- | --- |
| Mean (U/mL) | 19 | 28 |
| S.D. (U/mL) | 0.5 | 0.5 |
| % C.V. | 2.6 | 1.8 |
Lot to lot variation was determined by testing six different samples eight replicates each. Results were obtained for three different lots. The results are summarized below:
| | High | | | Medium | Low | |
| --- | --- | --- | --- | --- | --- | --- |
| Mean (U/mL) | 2896 | 2870 | 1530 | 259 | 60 | 62 |
| S.D. (U/mL) | 405 | 335 | 260.4 | 21.8 | 4.2 | 6.6 |
| % C.V. | 14.0 | 11.7 | 17.0 | 8.4 | 6.9 | 10.8 |
Reproducibility: The reproducibility of the qualitative protocol around the assay cut-off was determined by testing two samples eight times each on three different days $(n = 24)$ . All results were negative for the sample with 19 Units/ml concentration and all results were positive for the sample with 28 Units/ml.
# b. Linearity/assay reportable range:
Dilution recovery of three high concentration samples was determined by testing five serial dilutions of each sample. The results are summarized in the table below:
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| Sample | Dilution of sample | Observed Mean conc (Units/mL) | Expected Mean conc (Units/mL) | Dilution % recovery |
| --- | --- | --- | --- | --- |
| 1 | 1/50 – 1/800 | 114 – 1506 | 94 – 1506 | 100 – 121 |
| 2 | 1/50 – 1/800 | 63 – 921 | 58 – 921 | 100 – 112 |
| 3 | 1/50 – 1/800 | 194 – 2962 | 185 – 2962 | 94 - 105 |
Four additional samples were tested with concentrations 164, 180, 321 and 395 U/mL. The results are shown in the table below:
| Sample | Dilution of sample | Observed Mean conc (Units/ml) | Expected Mean conc (Units/ml) | Dilution % recovery |
| --- | --- | --- | --- | --- |
| MK-RA 039 | 1/50 – 1/1600 | 6.0 – 164 | 6.0 – 164 | 98 – 105 |
| MK-RA 086 | 1/50 – 1/1600 | 6.8 – 180 | 6.0 – 180 | 92 – 113 |
| MK-RA 120 | 1/50 – 1/3200 | 6.2 – 321 | 6.0 – 321 | 99 - 110 |
| MK-RA 169 | 1/50 – 1/3200 | 7.8 – 395 | 6.5 – 395 | 98 – 120 |
The measuring assay range is 25-2962 U/mL.
c. Traceability, Stability, Expected values (controls, calibrators, or methods): There is no widely accepted reference standard for anti-CCP. The calibrators and controls (positive and negative) are prepared in-house and arbitrary units are assigned during the development process.
d. Detection limit:
The limit of blank (LoB) was determined by running the zero standard 14 times on three different lots. The LoB of 1.6 U/mL was calculated by finding the mean plus two standard deviations.
e. Analytical specificity:
Interference Study/Endogenous compounds: Three low positive samples were spiked with bilirubin at 0.2 mg/mL, hemoglobin at 400 mg/dL, lipid at 15 mg/mL and rheumatoid factor at 200 IU/mL. The data indicates that the assayed concentrations do not interfere with the anti-CCP results. Please refer to the predicate (k052133) for more details on the studies conducted for the device.
f. Assay cut-off:
The cut-off of 25 U/mL was established in the predicate assay. The proposed modification did not alter the cut-off.
Semi-Quantitative protocol:
Samples with results < 25 U/mL are defined as negative.
Samples ≥25 U/mL are defined as positive.
Qualitative protocol:
Euro-Diagnostica recommends the following cut-off:
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Absorbance ratio Result Interpretation
<0.95 Negative
$\geq 0.95$ to $\leq 1.0$ Borderline - recommend repeat testing
>1.0 Positive
Users should calculate a cut-off between positive and negative samples that is specific to their patient populations.
# 2. Comparison studies:
a. Method comparison with predicate device:
A study was done to evaluate the percent agreement between the Immunoscan CCPlus® kit compared to Immunoscan RA anti-CCP. A total of 659 frozen retrospective sera were assayed (399 samples were from RA patients and 260 samples were from apparently healthy blood donors). The following table summarizes the results:
| | | Immunoscan RA anti-CCP | | |
| --- | --- | --- | --- | --- |
| Immunoscan CCPlus® Kit | | Positive | Negative | Total |
| | Positive | 275 | 5 | 280 |
| | Negative | 2 | 346 | 348 |
| | Total | 277 | 351 | 628 |
Positive Percent Agreement: $275 / 277 = 99.3\%$ (95% CI = 97.4 - 99.9)
Negative Percent Agreement: $346 / 351 = 98.6\%$ (95% CI = 96.7 - 99.5)
Overall Percent Agreement: $621 / 628 = 98.9\%$ (95% CI = 97.7 - 99.6)
b. Matrix comparison:
Not applicable
# 3. Clinical studies:
a. Clinical Sensitivity:
A total of 1180 frozen retrospective sera with clinical characterization were assayed. The following table summarizes the results:
| | n | negative | positive | Sensitivity (95% CI) |
| --- | --- | --- | --- | --- |
| Patients with clinically defined RA | 399 | 90 | 309 | 77.4% (73.1%-81.5%) |
b. Clinical specificity:
For the clinical specificity study, non-RA diseased patients and asymptomatic individuals (healthy blood donors) were used. The results are summarized below:
Blood donors 257/260 = 98.8% 95% CI = 96.7 - 99.8%
WG 18/20 = 90.0% 95% CI = 68.3 - 98.8%
MP 20/20 = 100% 95% CI = 83.2 - 100%
SLE 64/66 = 97.0% 95% CI = 89.5 - 99.6%
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| Sjogren’s | 13/13 = 100 % | 95% CI = 75.3 - 100% |
| --- | --- | --- |
| IBD | 95/98 = 96.9% | 95% CI = 91.3 - 99.4% |
| Osteoarthritis | 21/21 = 100% | 95% CI = 83.9 - 100% |
| Thyroiditis | 20/20 = 100% | 95% CI = 83.2 - 100% |
| Infectious Disease | 85/86 = 98.8% | 95% CI = 93.7 - 100% |
| Scleroderma | 16/17 = 94.1% | 95% CI = 71.3 - 99.8% |
| Multiple Sclerosis | 20/20 = 100% | 95% CI = 83.2 - 100% |
| IDDM | 20/20 = 100% | 95% CI = 83.2 - 100% |
| PM/DM | 20/20 = 100% | 95% CI = 83.2 - 100% |
| MCTD | 19/20 = 95.0% | 95% CI = 75.1 - 99.9% |
| Suspected autoimmune disease
(samples undefined) | 78/80 = 97.5 % | 95% CI = 91.3 - 99.7 % |
| Overall specificity | =766/781=98.1% | 95% CI = 96.9 – 98.9% |
c. Other clinical supportive data (when a. and b. are not applicable):
Not applicable
4. Clinical cut-off:
Same as assay cut-off.
5. Expected values/Reference range:
Expected values in the normal population should be negative.
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.
