EL-ANCA: ANTI-MPO, ANTI-PR3, ANTI-MPO AND ANTI-PR3

K061376 · Theratest Laboratories, Inc. · MOB · Jul 20, 2006 · Immunology

Device Facts

• Record ID: K061376
• Device Name: EL-ANCA: ANTI-MPO, ANTI-PR3, ANTI-MPO AND ANTI-PR3
• Applicant: Theratest Laboratories, Inc.
• Product Code: MOB · Immunology
• Decision Date: Jul 20, 2006
• Decision: SESE
• Submission Type: Traditional
• Regulation: 21 CFR 866.5660
• Device Class: Class 2

Intended Use

TheraTest EL-ANCA™: anti-MPO and EL-ANCA™: anti-PR3 are intended for use in clinical laboratories as an in vitro diagnostic tests for the detection and measurement of autoantibodies in human serum directed against human neutrophil myeloperoxidase and proteinase3, respectively. Measurement of anti-MPO aids in the diagnosis of microscopic polyangiitis. Measurement of anti-PR3 aids in the diagnosis of Wegener’s granulomatosis.

Device Story

TheraTest EL-ANCA™ consists of in vitro diagnostic tests for detecting autoantibodies in human serum. Input: human serum samples. Principle: enzyme-linked immunosorbent assay (ELISA) to detect anti-MPO and anti-PR3 antibodies. Output: quantitative or qualitative measurement of autoantibodies. Used in clinical laboratories by trained laboratory personnel. Results assist clinicians in diagnosing microscopic polyangiitis (anti-MPO) and Wegener's granulomatosis (anti-PR3).

Clinical Evidence

No clinical data provided in the document; substantial equivalence is based on technological characteristics and intended use.

Technological Characteristics

Solid-phase enzyme immunoassay (ELISA). Polystyrene microwells coated with autoantigens. Detection via HRP-labeled goat anti-human IgG (Fc gamma specific) and TMB substrate. Requires microplate reader (450 nm) and automatic washer. Semi-quantitative output in U/mL. Reagents provided ready-to-use.

Indications for Use

Indicated for clinical laboratory use to detect and measure autoantibodies against human neutrophil myeloperoxidase (anti-MPO) and proteinase 3 (anti-PR3) in human serum. Anti-MPO measurement aids diagnosis of microscopic polyangiitis; anti-PR3 measurement aids diagnosis of Wegener's granulomatosis.

Regulatory Classification

Identification

A multiple autoantibodies immunological test system is a device that consists of the reagents used to measure by immunochemical techniques the autoantibodies (antibodies produced against the body's own tissues) in serum and other body fluids. Measurement of multiple autoantibodies aids in the diagnosis of autoimmune disorders (disease produced when the body's own tissues are injured by autoantibodies).

Submission Summary (Full Text)

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# 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION SUMMARY

A. 510(k) Number:
k061376

B. Purpose for Submission:
This is a new device.

C. Measurand:
Antineutrophil Cytoplasmic Antibodies (ANCA)
Anti-MPO (myeloperoxidase)
Anti-PR3 (proteinase3)

D. Type of Test:
ELISA (Semi-quantitative)

E. Applicant:
TheraTest Laboratories Inc.

F. Proprietary and Established Names:
TheraTest EL-ANCA™: anti-MPO
TheraTest EL-ANCA™: anti-PR3
TheraTest EL-ANCA™: anti-MPO and anti-PR3

G. Regulatory Information:
1. Regulation section:
21 CFR § 866.5660 Multiple Antibodies Immunological Test System
2. Classification:
Class II
3. Product code:
MOB, Anti-neutrophil cytoplasmic antibodies (ANCA)
4. Panel:
(82) Immunology

H. Intended Use:
1. Intended use(s):
TheraTest EL-ANCA™: anti-MPO and EL-ANCA™: anti-PR3 are intended for use in clinical laboratories as an *in vitro* diagnostic tests for the detection and measurement of autoantibodies in human serum directed against human neutrophil myeloperoxidase and proteinase3, respectively.
Measurement of anti-MPO aids in the diagnosis of microscopic polyangiitis.
Measurement of anti-PR3 aids in the diagnosis of Wegener’s granulomatosis.
2. Indication(s) for use:
Same as above
3. Special conditions for use statement(s):
The device is for prescription use only.
4. Special instrument requirements:
- Microplate reader capable of reading absorbance values at 450 nm. If dual wavelength is available, the reference filter should be set at 620-690 nm.
- Automatic microplate washer

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I. Device Description:

The device consists of the following: a foil package containing 12 (1 X 8) microwell strips with holder, calibrator, a negative control, a positive control, ANCA Specimen diluent, Wash Buffer concentrate, HRP-labeled goat anti-human IgG conjugate (fc γ specific) and conjugate diluent. All reagents supplied in the kit are ready for use. The kits contain all necessary reagents to perform one or both of the following tests:

1. TheraTest EL-ANCA™: anti-MPO
2. TheraTest EL-ANCA™: anti-PR3
3. TheraTest EL-ANCA™: anti-MPO and anti-PR3

J. Substantial Equivalence Information:

1. Predicate device name(s):
Wielisa™ MPO ANCA
Wielisa™ PR3 ANCA

2. Predicate 510(k) number(s):
k974166
k974167

3. Comparison with predicate:

| Similarities | | |
| --- | --- | --- |
| Item | Device | Predicate |
| | TheraTest EL-ANCA™: anti-MPO and anti-PR3 | Wielisa™ MPO ANCA Wielisa™ PR3 ANCA |
| Intended Use | Detection of anti-MPO and anti-PR3 autoantibodies | Same |
| Sample type | Serum | Same |
| Type of test | Semi-quantitative | Same |
| Assay type | Elisa | Same |
| Detection Method | Colorimetric | Same |
| Solid phase capture | Microwells | Same |
| Differences | | |
| --- | --- | --- |
| Item | Device | Predicate |
| Calibrator | One level | 5 levels |
| Conjugate | HRP-goat anti-human IgG | Alkaline phosphatase-goat anti-human IgG |
| Substrate | TMB | pNPP |
| Absorbance | 450 nm/620 or 690 nm | 405 nm |

K. Standard/Guidance Document Referenced (if applicable):

None referenced.

L. Test Principle:

The TheraTest EL-ANCA™: anti-MPO and anti-PR3 ELISA test system are solid phase enzyme immunoassay tests. The wells of a polystyrene plate have been coated

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with autoantigens. The wells are incubated with specimens, controls and calibrators. During the incubation, the antibody present in the test sample binds to the solid phase. The wells are washed and pre-diluted horseradish peroxidase labeled goat anti-human (Fc gamma specific) is incubated in the wells. Unbound antibody is removed by aspiration and washing. A specific substrate is added and the autoantibody binding is detected by a color change, which is analyzed using a spectrophotometric enzyme immunoassay reader.

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

### 1. Analytical performance:

#### a. Precision/Reproducibility:

Intra-assay reproducibility was determined by assaying 3 samples 20 times on one plate. Results are shown on the table below.

| Test | Mean conc. | Mean %CV |
| --- | --- | --- |
| Anti-MPO | 54 | 8.5% |
| Anti-MPO | 101 | 4.4% |
| Anti-MPO | 201 | 2.9% |
| Anti-PR3 | 25 | 8.3% |
| Anti-PR3 | 52 | 4.9% |
| Anti-PR3 | 75 | 3.5% |

Inter-assay reproducibility was determined by assaying 3 samples in 20 different runs. Results are shown on the table below.

| Test | Mean conc. | Mean %CV |
| --- | --- | --- |
| Anti-MPO | 50 | 11.1% |
| Anti-MPO | 107 | 9.7% |
| Anti-MPO | 218 | 11.2% |
| Anti-PR3 | 31 | 6.8% |
| Anti-PR3 | 59 | 6.0% |
| Anti-PR3 | 78 | 6.0% |

#### b. Linearity/assay reportable range:

Linearity is not claimed for this assay.

#### c. Traceability, Stability, Expected values (controls, calibrators, or methods):

An international reference material for anti-MPO and anti-PR3 antibodies is not available. The assays are calibrated in relative arbitrary units (U/mL).

Accelerated and real time stability studies were performed for the kit and its components. Results were acceptable.

#### d. Detection limit:

Not applicable

#### e. Analytical specificity:

##### Cross-reactivity

To assess cross-reactivity of other autoantibodies, 200 serum samples representing 125 blood bank donor samples and 75 disease controls were tested. The disease control group consisted of 25 patients with rheumatoid

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arthritis, 25 with scleroderma, and 25 with SLE.

Cross-reactivity was observed in 4 SLE patients for anti-MPO and 1 SLE patient for anti-PR3. One scleroderma patient tested positive for anti-MPO.

## Interfering substance

The ranges for potentially interfering substances have not been determined. A statement that specimens that are lipemic, hemolyzed or icteric should not be used was added to the instruction manual.

Possible interference of rheumatoid factor in the test system was tested. Three different RF samples were added (10% by volume) to anti-MPO and anti-PR3 positive samples. These mixtures were tested using the EL-ANCA assays. Data showed rheumatoid factor does not appear to affect the reactivity in the EL-ANCA™ test system.

## f. Assay cut-off:

The cut-off value was determined by percentile ranking of 100 blood bank donors. The population was represented by 51 females, 49 males, with a median age of 29 years and a range from 16y to 70y. The race distribution was 14% Hispanic, 8% black, 4% Asian and 74% other. Since both anti-MPO and anti-PR3 are rarely found in normal healthy individuals, 99-100 percentiles were used to establish the upper limit of normal.

| Test | Percentile | Normal | Equivocal | Abnormal |
| --- | --- | --- | --- | --- |
| Anti-MPO | 99-100 | ≤ 20 U/mL | 21-25 U/mL | >25 U/mL |
| Anti-PR3 | 99-100 | ≤ 10 U/mL | 11-20 U/mL | >20 U/mL |

## 2. Comparison studies:

### a. Method comparison with predicate device:

The TheraTest EL-ANCA™: anti-MPO and anti-PR3 was compared to the predicate devices Wielisa MPO-ANCA and Wielisa PR3-ANCA for a total of 280 specimens: 125 blood bank donors, 37 expected positive for anti-MPO antibodies, 43 expected positive for anti-PR3 antibodies, 25 with SLE, 25 with scleroderma, and 25 with rheumatoid arthritis (RA). Equivocal samples were considered negative in the agreement calculations. There were four samples that were discrepant for anti-MPO in the SLE group. No discrepancy was observed for anti-PR3. Results are summarized in the following two tables.

| | Wielisa MPO-ANCA | | | | |
| --- | --- | --- | --- | --- | --- |
| | | Positive | Equivocal | Negative | Total |
| TheraTest EL-ANCA™: anti-MPO | Positive | 40 | 0 | 4 | 44 |
| | Equivocal | 0 | 1 | 0 | 1 |
| | Negative | 0 | 0 | 135 | 135 |
| | Total | 40 | 1 | 139 | 180 |

Positive Agreement = 100.0% (40/40)

Negative Agreement = 98.3% (236/240)

Total Agreement = 98.6% (276/280)

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| | Wielisa PR3-ANCA | | | | |
| --- | --- | --- | --- | --- | --- |
| | | Positive | Equivocal | Negative | Total |
| TheraTest EL-ANCA™: anti-PR3 | Positive | 46 | 0 | 0 | 46 |
| | Equivocal | 0 | 1 | 5 | 6 |
| | Negative | 0 | 0 | 128 | 128 |
| | Total | 46 | 1 | 133 | 180 |

Positive Agreement = 100% (46/46)
Negative Agreement = 100% (234/234)
Total Agreement = 100% (280/280)

b. Matrix comparison:
Serum is the only recommended matrix.

3. Clinical studies:

a. Clinical Sensitivity and Clinical specificity:
Clinical sensitivity for the TheraTest EL-ANCA™: anti-MPO and anti-PR3 was determined by testing sera (N=37) from patients expected positive for anti-MPO and (N=43) from patients expected positive for anti-PR3. Anti-MPO and anti-PR3 are rarely found in the same patient. Two PR3 patient samples were found anti-MPO positive and 4 MPO patient samples were found equivocal for anti-PR3. If the equivocal samples were considered negative clinical sensitivity of the TheraTest EL-ANCA™: anti-MPO test and anti-PR3 was 100%.

Clinical specificity was determined by evaluating samples from 125 healthy blood donors, 25 RA, 25 SLE and 25 scleroderma patients. None of the samples from the healthy blood donors were tested positive for the anti-MPO and anti-PR3. One patient with scleroderma and 4 patients in the SLE group were found positive for anti-MPO. One patient in the SLE group was found positive for anti-PR3. If the equivocal samples were considered negative, the clinical specificity of the TheraTest EL-ANCA™: anti-MPO test and anti-PR3 in the disease groups was 94.1% (111/118) and 99.1% (111/112) respectively (see table below).

| Group tested | N | MPO Pos. | MPO Equiv | PR3 Pos. | PR3 Equiv. |
| --- | --- | --- | --- | --- | --- |
| Expected MPO Pos | 37 | 37 | 0 | 0 | 4 |
| Expected PR3 Pos | 43 | 2 | 0 | 43 | 0 |
| Seropositive RA | 25 | 0 | 0 | 0 | 0 |
| SLE | 25 | 4 | 1 | 1 | 2 |
| Scleroderma | 25 | 1 | 0 | 0 | 0 |

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:

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The expected value in the normal population is 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.

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