BIOPLEX 200 ANA SCREEN ON THE BIOPLEX 2200 MULTI-ANALYTE DETECTION SYSTEM, MODEL BIOPLEX 2200
Device Facts
| Record ID | K041658 |
|---|---|
| Device Name | BIOPLEX 200 ANA SCREEN ON THE BIOPLEX 2200 MULTI-ANALYTE DETECTION SYSTEM, MODEL BIOPLEX 2200 |
| Applicant | Bio-Rad Laboratories, Inc. |
| Product Code | LKJ · Immunology |
| Decision Date | Dec 20, 2004 |
| Decision | SESE |
| Submission Type | Traditional |
| Regulation | 21 CFR 866.5100 |
| Device Class | Class 2 |
Indications for Use
The Bio-Rad ANA Screen is intended for the qualitative screening of specific antinuclear antibodies (ANA), the quantitative detection of antibody to dsDNA, and the semi-quantitative detection of ten (10) separate antibody assays (Chromatin, Ribosomal Protein, SS-A, SS-B, Sm, Sm/RNP, RNP, Scl-70, Jo-1, and Centromere B,) in human serum and/or EDTA or heparinized plasma. The test system is used as an aid in the diagnosis of systemic rheumatic diseases. The ANA Screen is intended for use with the Bio-Rad BioPlex 2200 System.
Device Story
The BioPlex 2200 ANA Screen is a multiplex flow immunoassay system used in clinical laboratories to detect autoantibodies in human serum or plasma. It utilizes 13 populations of fluorescently dyed beads, each coated with a specific autoantigen (dsDNA, Chromatin, Ribosomal Protein, SS-A 60/52, SS-B, Sm, SmRNP, RNP A/68, Scl-70, Jo-1, Centromere B). Patient samples are incubated with these beads; captured antibodies are detected using a phycoerythrin-conjugated anti-human IgG. The system uses flow cytometry to identify bead regions via fluorescence and quantify antibody binding. Internal Standard Beads (ISB), Serum Verification Beads (SVB), and Blank Beads (BB) monitor system performance and sample addition. Results are reported as IU/mL for dsDNA or Antibody Index (AI) for other analytes. By simultaneously identifying multiple autoantibodies in a single reaction vessel, the device aids clinicians in diagnosing systemic rheumatic diseases, potentially streamlining diagnostic workflows compared to traditional single-analyte EIA methods.
Clinical Evidence
Clinical performance evaluated via prospective (N=908) and retrospective (N=440) studies comparing the BioPlex 2200 ANA Screen to commercially available microplate EIA methods. Combined prospective and retrospective analysis showed overall agreement ranging from 86% (Chromatin) to 99.5% (Jo-1) across the 11 analytes. Reproducibility studies (intra-assay and inter-assay) conducted at three sites demonstrated CVs generally ranging from 2% to 12%.
Technological Characteristics
Multiplex flow immunoassay using 13 populations of dyed, antigen-coated beads. Detection via fluorescence (phycoerythrin conjugate). Automated system with on-board refrigerated reagent storage, sheath fluid, and barcode sample identification. Calibration: 6-point 4PL curve (dsDNA) or 4-point point-to-point curve (others). Connectivity: supports LIS integration. Standards: NCCLS EP5-A, EP7-A.
Indications for Use
Indicated for qualitative screening of ANA, quantitative detection of anti-dsDNA, and semi-quantitative detection of 10 specific autoantibodies (Chromatin, Ribosomal Protein, SS-A, SS-B, Sm, Sm/RNP, RNP, Scl-70, Jo-1, Centromere B) in human serum/plasma. Used as an aid in diagnosing systemic rheumatic diseases (SLE, MCTD, UCTD, Sjögren's, Scleroderma, Dermatomyositis, Polymyositis, RA, CREST, Raynaud's) in conjunction with clinical findings.
Regulatory Classification
Identification
An antinuclear antibody immunological test system is a device that consists of the reagents used to measure by immunochemical techniques the autoimmune antibodies in serum, other body fluids, and tissues that react with cellular nuclear constituents (molecules present in the nucleus of a cell, such as ribonucleic acid, deoxyribonucleic acid, or nuclear proteins). The measurements aid in the diagnosis of systemic lupus erythematosus (a multisystem autoimmune disease in which antibodies attack the victim's own tissues), hepatitis (a liver disease), rheumatoid arthritis, Sjögren's syndrome (arthritis with inflammation of the eye, eyelid, and salivary glands), and systemic sclerosis (chronic hardening and shrinking of many body tissues).
Predicate Devices
- Zeus Athena Multi-Lyte ANA Test System (K011244)
- Diagnostic Products Corporation (DPC) Immulite 2000 Automated Immunoassay Analyzer (K970227)
- Bio-Rad (Helix) Autoimmune EIA ANA Screening Test (954723)
- Inova Diagnostics, Inc. QUANTA Lite SSA (922830)
- Bio-Rad (Helix) Autoimmune EIA SS-B / La Test (932419)
- Inova Diagnostics, Inc. QUANTA Lite Sm (922831)
- Inova Diagnostics, Inc. QUANTA Lite RNP (Sm/RNP) (922833)
- Pharmacia Varelisa RNP Antibodies (993589)
- Inova Diagnostics, Inc. QUANTA Lite Ribosome P (981237)
- Inova Diagnostics, Inc. QUANTA Lite Chromatin ELISA (982603)
- Pharmacia Varelisa ds-DNA ANTIBODY EIA Kit (950031)
- Bio-Rad (Helix) Autoimmune EIA Anti-Centromere Test (000489)
- Bio-Rad (Helix) Autoimmune EIA Anti-Scl-70 Test (951798)
- Bio-Rad (Helix) Autoimmune EIA Anti-Jo-1 Test (951850)
Related Devices
- K113610 — ANA-SCREEN WITH MDSS · Bio-Rad Laboratories · Jul 9, 2012
- K021103 — MODIFICATION TO ZEUS SCIENTIFIC'S ATHENA MULTI-LYTE ANA TEST SYSTEM · Zeus Scientific, Inc. · Apr 24, 2002
Submission Summary (Full Text)
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# 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION SUMMARY
A. 510(k) Number:
k041658
B. Purpose for Submission:
New device
C. Measurands:
Anti-SS-A, anti-SS-B, anti-Sm, anti-Sm/RNP, anti-RNP, anti-ribosomal protein, anti-chromatin, anti-dsDNA, anti-centromere, anti-Scl-70, and anti-Jo1 antibodies.
D. Type of Test:
Multiplex flow, bead-based immunoassay
E. Applicant:
Bio-Rad Laboratories
F. Proprietary and Established Names:
BioPlex 2200 ANA Screen on the BioPlex 2200 Multi-Analyte Detection System
G. Regulatory Information:
1. Regulation sections:
21 CFR 866.5100 Antinuclear Antibody Immunological Test System
2. Classification:
Class II
3. Product Code:
LKJ, Antinuclear Antibody, Antigen, Control
LRM, Anti-DNA Antibody (Enzyme-Labeled), Antigen, Control
MQA, Anti-Ribosomal P Antibodies
LKO, Anti-RNP Antibody, Antigen, Control
LJM, Antinuclear Antibody (Enzyme-Labeled), Antigen, Controls
LLL, Extractable Antinuclear Antibody, Antigen and Controls
4. Panel:
Immunology 82
H. Intended Use:
1. Intended use(s):
The Bio-Rad ANA Screen is intended for the qualitative screening of specific antinuclear antibodies (ANA), the quantitative detection of antibody to dsDNA, and the semi-quantitative detection of ten (10) separate antibody
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assays (Chromatin, Ribosomal Protein, SS-A, SS-B, Sm, Sm/RNP, RNP, Scl-70, Jo-1, and Centromere B,) in human serum and/or EDTA or heparinized plasma. The test system is used as an aid in the diagnosis of systemic rheumatic diseases. The ANA Screen is intended for use with the Bio-Rad BioPlex 2200 System.
2. **Indication(s) for use:**
The Bio-Rad ANA Screen is intended for the qualitative screening of specific antinuclear antibodies (ANA), the quantitative detection of antibody to dsDNA, and the semi-quantitative detection of ten (10) separate antibody assays (Chromatin, Ribosomal Protein, SS-A, SS-B, Sm, Sm/RNP, RNP, Scl-70, Jo-1, and Centromere B,) in human serum and/or EDTA or heparinized plasma. The ANA Screen is intended for use with the Bio-Rad BioPlex 2200 System.
**Uses:**
The test system is used to screen serum or plasma (EDTA and heparin) samples and detect the presence of antinuclear antibodies as an aid in the diagnosis of systemic rheumatic diseases (systemic lupus erythematosus (SLE), Sjogren’s syndrome, mixed connective tissue disease (MCTD), undifferentiated connective tissue disease, scleroderma, dermatomyositis, polymyositis, rheumatoid arthritis, CREST syndrome, and Raynaud’s phenomenon) in conjunction with clinical findings and other laboratory tests.
3. **Special condition for use statement(s):**
For prescription use only
4. **Special instrument Requirements:**
BioPlex 2200 Multi-Analyte Detection System
I. **Device Description:**
The device components include the following: Bead Set containing dyed beads coated with dsDNA, Chromatin, Ribosomal Protein, SS-A 60, SS-A 52, SS-B, Sm, SmRNP, RNP A, RNP 68, Scl-70, Jo-1, Centromere B, Internal Standard (ISB), Serum Verification (SVB), and a Reagent Blank Bead (RBB), with glycerol and protein stabilizers; Conjugate containing murine monoclonal anti-human IgG/phycoerythrin (PE) conjugate and sheep anti-human FXIII/phycoerythrin conjugate in a phosphate buffer; Sample Diluent; Calibrator Set of 6 vials containing antibodies to dsDNA, and a set of 4 vials for the other 12 analytes; Positive and Negative Controls; Sheath Fluid; Wash Solution; and the BioPlex 2200 system.
J. **Substantial Equivalence Information:**
1. **Predicate device name(s) and K numbers:**
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## Instrument
| BioPlex2200 System | Comparative FDA Cleared PREDICATE DEVICE | 510(k) Number |
| --- | --- | --- |
| BioPlex 2200 ANA Screen on the BioPlex 2200 System | Zeus Athena Multi-Lyte ANA Test System | K011244 |
| | Diagnostic Products Corporation (DPC) Immulite 2000 Automated Immunoassay Analyzer | K970227 |
## Assay
| BioPlex2200 ANA Screen | Comparative FDA Cleared Device | 510(k) Number |
| --- | --- | --- |
| ANA Screen (based on results of all analytes listed below) | Bio-Rad (Helix) Autoimmune EIA ANA Screening Test | 954723 |
## Analytes
| BioPlex2200 ANA Screen Analyte | Comparative FDA Cleared PREDICATE DEVICE | 510(k) Number |
| --- | --- | --- |
| SSA (SSA 60 and SSA 52) | Inova Diagnostics, Inc. QUANTA Lite™ SSA | 922830 |
| SSB | Bio-Rad (Helix) Autoimmune EIA SS-B / La Test | 932419 |
| Sm | Inova Diagnostics, Inc. QUANTA Lite™ Sm | 922831 |
| SmRNP | Inova Diagnostics, Inc. QUANTA Lite™ RNP (Sm/RNP) | 922833 |
| RNP (RNP 68 and RNP A) | Pharmacia Varelisa® RNP Antibodies | 993589 |
| Ribosomal Protein | Inova Diagnostics, Inc. QUANTA Lite™ Ribosome P | 981237 |
| Chromatin | Inova Diagnostics, Inc. QUANTA Lite™ Chromatin ELISA | 982603 |
| dsDNA (quantitative /semi-quantitative) | Pharmacia Varelisa® ds-DNA ANTIBODY EIA Kit | 950031 |
| Centromere | Bio-Rad (Helix) Autoimmune EIA Anti-Centromere Test | 000489 |
| Scl-70 | Bio-Rad (Helix) Autoimmune EIA Anti-Scl-70 Test | 951798 |
| Jo-1 | Bio-Rad (Helix) Autoimmune EIA Anti-Jo-1 Test | 951850 |
2. Predicate K number(s):
See table above
3. Comparison with predicate:
| Similarities | | |
| --- | --- | --- |
| Item | Device | Predicate |
| Instruments | BioPlex 2200 System | |
| Detection | Based on Luminex Corporation’s multiplex, bead-based technology | Zeus Athena Multi-Lyte: Based on Luminex Corporation’s multiplex, bead-based technology |
| Sample handling/ | Automated sample | Immulite 2000 Automated |
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| Similarities | | |
| --- | --- | --- |
| Item | Device | Predicate |
| processing | handling and processing | Immunoassay Analyzer Automated sample handling and processing |
| Reagent storage | On-board, refrigerated reagent storage | Immulite 2000 Automated Immunoassay Analyzer On-board, refrigerated reagent storage |
| Components/materials | | Autoimmune EIA's: |
| Reagents | Wash buffer, sample diluent | Wash buffer, sample diluent |
| Calibrators | Quantitative and semi-quantitative analytes | Quantitative and semi-quantitative analytes |
| Controls | Single negative control | Single negative control |
| Function and Use | | Autoimmune EIA's: |
| Intended use | Quantitative (dsDNA only) and semi-quantitative autoimmune antibody detection | Quantitative (dsDNA only) and semi-quantitative autoimmune antibody detection |
| Matrix | Serum and plasma (EDTA and heparin) for all analytes | Serum and plasma (Jo-1, dsDNA, RNP, SS-B) |
| Differences | | |
| Item | Device | Predicate |
| Instruments | BioPlex 2200 System | |
| Detection | Based on Luminex Corporation's multiplex, bead-based technology | Immulite 2000 Automated Immunoassay Analyzer: luminometer (photomultiplier tube) to detect chemiluminescence |
| Sample handling/processing | Automated sample handling and processing | Zeus AtheNA Multi-Lyte: No automated sample handling or processing capabilities |
| Reagent storage | On-board, refrigerated reagent storage | Zeus AtheNA Multi-Lyte: Off-line reagent storage |
| Components/materials | | Autoimmune EIA's: |
| Solid phase | Dyed antigen coated beads | 96 well antigen coated microwells |
| Conjugate | Anti-human IgG/phycoerythrin | Anti-human IgG/horseradish peroxidase with TMB substrate |
| Calibrators | Qualitative and semi-quantitative analytes | Cut-off /low positive controls (qualitative analytes) |
| Controls | One multi-analyte positive control containing all 11 analytes | One positive control per analyte |
| Sheath fluid | Used to suspend the bead reagent and introduce it | Not utilized in EIA's |
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| Similarities | | |
| --- | --- | --- |
| Item | Device | Predicate |
| | into the detector | |
| Function and use | | Autoimmune EIA’s |
| Matrix | Serum and plasma (EDTA and heparin) for all analytes | Serum (ANA Screen, SS-A, Sm, Sm/RNP, Ribosome P, Chromatin, Centromere, Scl-70) |
K. Standard/Guidance Document Referenced (if applicable):
NCCLS EP5-A and NCCLS EP7-A.
L. Test Principle:
The ANA Screen detects the presence of circulating autoantibodies in serum or plasma using a group of autoantigens. Beads are individually coated with individual antigens, so that the presence of each antinuclear and autoimmune antibody can be individually determined. Fluorescence detection facilitates the differentiation of normal and abnormal antibody concentrations.
The ANA Screen uses multiplex flow immunoassay, a methodology that resembles traditional EIA, but permits simultaneous detection and identification of several antibodies in a single tube. Thirteen (13) different populations of dyed beads are coated with antigens associated with systemic autoimmune disease (dsDNA, Chromatin, Ribosomal Protein, SS-A 60, SS-A 52, SS-B, Sm, Sm/RNP, RNP A, RNP 68, Scl-70, Jo-1 and Centromere B)*. The BioPlex 2200 System combines an aliquot of patient sample, sample diluent, and bead reagent into a reaction vessel and the mixture is incubated at 37°C. After a wash cycle, murine monoclonal anti-human IgG antibody, conjugated to phycoerythrin (PE), is added to the dyed beads and this mixture is incubated at 37°C. The excess conjugate is removed in another wash cycle, and the beads are re-suspended in wash buffer.
The bead mixture is suspended in sheath fluid, passes through the detector and the identity of the dyed beads is determined by the fluorescence of the dyes. Based on its fluorescent signature, each bead is classified to its own unique region. The detector measures at least 200 beads for each analyte, per specimen. The BioPlex 2200 ANA Screen utilizes one of these regions for each of the 13 analytes it detects. Three additional regions are assigned to beads used for quality control purposes. While the identity of the dyed beads is determined by the unique fluorescence intensity of the dyes, the amount of antibody captured by the antigen is determined by the fluorescence of the attached PE. Raw data is calculated in relative fluorescence intensity (RFI) and fluorescence ratio (FR).
Three additional dyed beads, Internal Standard Bead (ISB), Serum Verification Bead (SVB) and a Reagent Blank Bead (RBB) are present in each reaction mixture to verify detector response, the addition of serum or plasma to the reaction vessel and the absence of significant non-specific binding in serum or plasma. The instrument is calibrated using sets of distinct calibrators. For dsDNA, six (6) vials, representing six (6) different levels of antibody concentrations, are used for quantitative calibration, and results for patient samples are expressed in IU/mL. Results of ≤4 IU/mL are
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negative, 5 - 9 IU/mL are indeterminate, and results of 10 IU/mL or higher are considered positive for dsDNA antibody. For the other twelve (12) beads, four (4) vials representing four (4) different antibody concentrations are used for semi-quantitative calibration. The result for each of these antibodies is expressed as an antibody index (AI). An AI of 1.0 indicates an antibody cut-off concentration that corresponds to approximately the 99th percentile of values obtained from a non-diseased population; results of 1.0 or higher are reported as positive. Results of <1.0 are reported as negative.
* In cases where either SS-A 60 and/or SS-A 52 are positive, results are reported as positive for SS-A; and when either RNP A and/or RNP 68 are positive, results are reported as positive for RNP.
## M. Performance Characteristics (if/when applicable):
### 1. Analytical performance:
#### a. Precision/Reproducibility
Reproducibility testing for the BioPlex 2200 ANA Screen was conducted at three clinical sites located in the U.S. A reproducibility panel consisting of 33 panel members was prepared at Bio-Rad Laboratories. Each of 10 positive panel members was prepared by combining one or more antibody positive patient samples for one or more of the 13 analytes contained in the BioPlex 2200 ANA Screen (dsDNA, Chromatin, SS-A 52, SS-A 60, SS-B, Sm, RNP 68, RNP A, Sm/RNP, Centromere, Ribosomal Protein, Scl-70, Jo-1 antibodies). Five of the 10 members had levels near the cut-off. One panel member was negative for all 13 analytes. Each panel was made in serum (N=11), EDTA (N=11) and lithium heparin (N=11); a total of 33 panel members. In addition, three controls (positive, dilute positive, and negative) were included and tested as panel members (a total of 36 panel members).
Each testing facility evaluated reproducibility using a different kit lot of the Bio-Rad ANA Screen. The 36 panel members (including the Autoimmune Control Set) were tested in duplicate on 2 runs per day (one in the morning and one in the afternoon), for 10 days using one lot of BioPlex 2200 ANA Screen Reagent Pack and one lot of BioPlex 2200 ANA Screen Calibrator set at each of the 3 sites (2 times x 2 runs x 10 days = 40 replicates per panel member per site). The combined total of replicates for all three sites is 120 replicates per panel member. The data were analyzed for intra-assay and inter-assay reproducibility according to NCCLS EP5-A Vol. 19 No. 2, p 24 Eq. C2 and p.25 Eq's. C3 and C4. Intra-run %CV ranged from 1.2 to 11.1% and inter-run %CV ranged from 2.8 to 12.8%
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| Site | A | B | C |
| --- | --- | --- | --- |
| Instrument | 1 | 2 | 3 |
| Reagent Pack Lot | X | Y | Z |
| Serum | | | |
| Intra-run %CV | 1.7 – 7.9% | 2.1 – 8.6% | 2.1 – 11.1% |
| Inter-run %CV | 2.8 - 12.8% | 5.8 - 11.9% | 3.6 - 12.0% |
| EDTA Plasma | | | |
| Intra-run %CV | 1.7 - 9.4% | 2.7 - 7.3% | 1.2 - 7.6% |
| Inter-run %CV | 3.7 - 10.9% | 5.5 - 11.6% | 3.8 - 9.6% |
| Lithium Heparin Plasma | | | |
| Intra-run %CV | 2.0 - 7.5% | 2.3 - 9.0% | 2.1 - 7.2% |
| Inter-run %CV | 3.2 - 8.9% | 6.2 - 12.2% | 3.9 - 8.3% |
# b. Linearity/assay reportable range:
All analytes except dsDNA antibodies are reported as positive or negative compared to an internally established cut-off value with a measuring range of $0.2 - 8.0$ AI (antibody index). Linearity for the anti-dsDNA assay was demonstrated.
In order to find samples within the measuring range of the assay (1 IU/mL to 300 IU/mL), 4 very high dsDNA positive patient samples were initially diluted with negative serum. The samples were then diluted to the following dilution ratios: neat, 1:1.25, 1:1.67, 1:2.5, 1:5, and 1:10. Each dilution was tested a minimum of 4 times. The replicates were separated by time and the dilutions were randomized on the sample racks. Percent recoveries ranged from 83 to $105\%$ .
| Sample n=6 | % Recovery range |
| --- | --- |
| 1 | 83 – 100% |
| 2 | 93 – 100% |
| 3 | 90 – 100% |
| 4 | 97 – 105% |
In addition, serial dilutions of the WHO W0/80 dsDNA Standard were made to obtain dsDNA antibody concentrations throughout the assay range. The dilutions were assayed and the actual results compared to the expected results. Linear regression analysis of the comparison yielded $y = 0.9854x + 2.773$ , $r^2 = 0.9994$ .
# c. Traceability (controls, calibrators, or method):
Traceability for dsDNA values in the calibrator set were established using the WHO First International Standard for Anti-double stranded DNA (dsDNA), Human Code: W0/80 (W1065). The WHO W0/80 standard is a lyophilized powder and is reconstituted to a concentration of $200\mathrm{IU / mL}$ . Serial dilutions of this material were
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made. The average percent recovery for all dilutions of the standard was 111%.
d. Detection limit:
Not given/not applicable
e. Analytical specificity:
A cross-reactivity study was performed to determine if samples from various disease states and other potentially interfering factors interfere with test results when tested with the ANA Screen. A panel of ten (10) specimens with potentially interfering disease states was tested with the ANA Screen for each of the eleven (11) autoantibodies. Samples that elicited a positive response by the BioPlex 2200 ANA Screen were also tested by a corresponding commercially available EIA test method. Some cross-reactivity did occur with the new and the predicate devices. This information is included in the package insert for the user's information.
| Cross Reactants | N | Method | Centromere B | Chromatin | dsDNA | Jo1 | Ribosomal P | RNP | SCL-70 | Smith | Sm-RNP | SSA | SSB |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | | # of Positive Samples | | | | | | | | | | |
| Cardiolipin | 10 | BioPlex | 0 | 3 | 1 | 1 | 0 | 0 | 0 | 1 | 2 | 4 | 0 |
| | | EIA | NT | 3 | 1 | 1 | NT | NT | NT | 0 | 2 | 4 | NT |
| Mitochondrial M2 | 10 | BioPlex | 6 | 3 | 1 | 0 | 0 | 2 | 0 | 0 | 1 | 4 | 1 |
| | | EIA | 4 | 2 | 0 | NT | NT | 1 | NT | NT | 1 | 4 | 1 |
| Single-stranded DNA (ssDNA) | 10 | BioPlex | 1 | 4 | 4 | 0 | 2 | 1 | 2 | 1 | 1 | 4 | 2 |
| | | EIA | 1 | 4 | 4* | NT | 1 | EQ | 1 | 1 | 1 | 4 | 2 |
| Histone | 10 | BioPlex | 0 | 7 | 6 | 0 | 4 | 5 | 1 | 5 | 6 | 3 | 1 |
| | | EIA | NT | 7 | 6 | NT | 2 | 5 | 1 | 5 | 6 | 3 | 1 |
| Myeloperoxidase (MPO) | 10 | BioPlex | 1 | 1 | 2 | 0 | 2 | 0 | 0 | 0 | 0 | 1 | 0 |
| | | EIA | 0 | 0 | 0 | NT | 0 | NT | NT | NT | NT | 0 | NT |
| Proteinase 3 (PR3) | 10 | BioPlex | 1 | 1 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| | | EIA | 1 | 0 | NT | NT | NT | NT | NT | NT | NT | NT | NT |
| Gliadin | 10 | BioPlex | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 1 | 0 |
| | | EIA | NT | NT | NT | NT | NT | NT | NT | NT | NT | 1 | NT |
| Smooth Muscle | 10 | BioPlex | 0 | 2 | 2 | 0 | 2 | 1 | 0 | 1 | 2 | 1 | 0 |
| | | EIA | NT | 2 | 2 | NT | 2 | 1 | NT | 1 | 2 | 1 | NT |
| Thyroid Peroxidase (TPO) | 10 | BioPlex | 2 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| | | EIA | 2 | NT | NT | NT | NT | NT | NT | NT | NT | NT | NT |
| Thyroglobulin (Tg) | 10 | BioPlex | 2 | 2 | 1 | 0 | 0 | 2 | 0 | 1 | 1 | 1 | 0 |
| | | EIA | 2 | 0 | 0 | NT | NT | 1 | NT | 0 | 1 | 0 | NT |
| Rheumatoid Factor (RF) | 10 | BioPlex | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| | | EIA | NT | NT | NT | NT | NT | NT | NT | NT | NT | NT | NT |
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NT= Not Tested
Testing for interfering substances was conducted according to NCCLS EP7-A (Vol. 22, no. 27). No significant interference was observed in any of the substances tested. The following substances were tested (N=20) at up to and including the maximum levels in duplicate on one lot.
| Substance | Concentration |
| --- | --- |
| Hemoglobin | ≤ 500 mg/dl |
| Bilirubin (unconjugated) | ≤ 20 mg/dl |
| Triglycerides | ≤ 3000 mg/dl |
| Protein (total) | ≤ 12 g/dl |
| Cholesterol | ≤ 500 mg/dl |
| Red blood cells | ≤ 0.4% concentrate |
| Gamma-globulin | ≤ 2.5 g/dl |
| Ascorbic acid | ≤ 3.0 mg/dl |
f. Assay cut-off:
A total of 719 samples were used for the study. The negative population consisted of 285 samples from normal donors and confirmed to be negative by the predicate screening device. The remaining 434 samples were positive for one or more of the analytes by predicate EIA methods.
| Antigen | Total number of EIA positive samples |
| --- | --- |
| dsDNA | 100 |
| Chromatin | 50 |
| RNP A + RNP 68 | 115 |
| SSB | 110 |
| SSA 52 + SSA 60 | 49 |
| Scl-70 | 95 |
| Sm | 52 |
| Centromere | 93 |
| Sm/RNP | 51 |
| Ribosome Protein | 83 |
| Jo-1 | 99 |
The 719 samples were assayed in singlicate. Calibrators, controls and WHO dsDNA standards were assayed in quadruplicate, duplicate and duplicate, respectively, in each run. There were a total of 11 runs on 3 different instruments over a period of 16 days.
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Cut-offs were established as follows: for dsDNA, results of $\leq 4$ IU/mL are negative, 5-9 IU/mL are indeterminate, and results of $\geq 10$ IU/mL are reported as positive; and for all other assays, results of $< 1.0$ AI (antibody index) are negative, and results of 1.0 or greater are reported as positive. Using these cut-offs, Receiver Operator Curve (ROC) analysis of the data showed sensitivities ranging from $67\%$ to $100\%$ . Specificities ranged from $94\%$ to $100\%$ .
| Antigen | Sensitivity | Specificity |
| --- | --- | --- |
| dsDNA | 67% | 99% |
| Chromatin | 94% | 100% |
| RNP A + RNP 68 | 96% | 94% |
| SSB | 100% | 98% |
| SSA 52 + SSA 60 | 99% | 98% |
| Scl-70 | 98% | 98% |
| Sm | 96% | 100% |
| Centromere | 98% | 98% |
| Sm/RNP | 96% | 99% |
| Ribosome Protein | 82% | 98% |
| Jo-1 | 95% | 100% |
# 2. Comparison studies:
a. Method comparison with predicate devices:
Comparison of ANA Screen and ANA Microplate EIA Screen: The ability of BioPlex 2200 System ANA Screen qualitative screening format to detect positive results in serum samples collected prospectively from patients was compared to results of testing with the Bio-Rad (Helix) Autoimmune EIA ANA Screening test. A total of 908 prospective serum samples were tested at 3 U.S. clinical testing sites. The samples were collected from patients seen at a rheumatology clinic and suspected of having systemic autoimmune disease. Results are summarized in the table below.
| | Positive ANA Screen | Negative ANA Screen | Total |
| --- | --- | --- | --- |
| Positive EIA | 351 | 149 | 500 |
| Negative EIA | 39 | 369 | 408 |
| Total | 390 | 518 | 908 |
Positive Agreement: $351 / 500 = 70.2\%$ (95% CI: 66.1 - 74.4%)
Negative Agreement: $369 / 408 = 90.4\%$ (95% CI: 87.5 - 93.4%)
Over-all Agreement: $720 / 908 = 79.3\%$ (95% CI: 76.6 - 82%)
Results presented below are for patients with a diagnosis of targeted connective tissue disease (SLE, scleroderma, Raynaud's phenomenon, CREST, Sjogren's syndrome, mixed connective tissue
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disease, undifferentiated connective tissue disease and/or dermatomyositis/polymyositis) as well as for those patients diagnosed with connective tissue disease other than the targeted diseases and for those patients in whom connective tissue disease was not a diagnosis. Results for targeted connective tissue disease are presented for both those diagnosed by a qualified M.D. rheumatologist (Physician Diagnosed Targeted CTD) and those diagnosed using adherence to American College of Rheumatology or literature disease classification criteria (Criteria Diagnosed Targeted CTD). A diagnosis of connective tissue disease other than the targeted diseases was made on the basis of physician diagnosis.
Comparison of BioPlex 2200 ANA Screen (Qualitative Screening Format) to Bio-Rad (Helix) Autoimmune EIA ANA Screening Test in Patients with Physician Diagnosed Targeted CTD (n=413):
| | BioPlex 2200 ANA Screen | | |
| --- | --- | --- | --- |
| Bio-Rad (Helix) EIA | Positive | Negative | Total |
| Positive | 256 | 76 | 332 |
| Negative | 9 | 72 | 81 |
| Total | 265 | 148 | 413 |
Positive Agreement: 77.1% (256/332)
Negative Agreement: 88.9% (72/81)
Overall Agreement 79.4% (328/413)
Comparison of BioPlex 2200 ANA Screen (Qualitative Screening Format) to Bio-Rad (Helix) Autoimmune EIA ANA Screening Test in Patients with Criteria Diagnosed Targeted CTD (n=408):
| | BioPlex 2200 ANA Screen | | |
| --- | --- | --- | --- |
| Bio-Rad (Helix) EIA | Positive | Negative | Total |
| Positive | 268 | 67 | 335 |
| Negative | 9 | 64 | 73 |
| Total | 277 | 131 | 408 |
Positive Agreement: 80.0% (268/335)
Negative Agreement: 87.7% (64/73)
Overall Agreement 81.4% (332/408)
Comparison of Selected Antibodies to EIA Methods (n=1047):
Testing to evaluate the performance of the BioPlex 2200 ANA Screen compared to the individual predicate EIAs was conducted at three sites located in the U.S. using 907 samples collected prospectively from consecutive patients being seen in a rheumatology clinic and suspected of, or with a history consistent
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with an autoimmune/connective tissue disease. Additional selected retrospective (frozen) samples, known to be positive for one or more of the 11 analytes detected by the Bio-Rad BioPlex 2200 ANA Screen were selected for inclusion in the study. Forty positive samples for each of the 11 analytes were included (440 total positive samples) in the study. One-hundred selected retrospective samples, known to be ANA EIA negative, were also included in the study. This resulted in n=1047 (907+40+100) for each analyte.
## Combined Prospective and Retrospective Comparison Testing
| Antibody/antibody group | ds-DNA | Chrom | Rib-P | SSA | SSB | Sm | Sm/RNP | RNP | Scl-70 | Jo-1 | Cent. |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| N = | 955 | 1046 | 1046 | 1047 | 1048 | 1047 | 1047 | 1017 | 1047 | 1038 | 1048 |
| Bio-Rad and EIA positive | 102 | 109 | 32 | 193 | 96 | 60 | 128 | 116 | 22 | 36 | 67 |
| Bio-Rad positive and EIA negative | 25 | 72 | 22 | 17 | 20 | 25 | 13 | 27 | 18 | 0 | 10 |
| Bio-Rad and EIA negative | 800 | 788 | 986 | 821 | 920 | 947 | 881 | 854 | 995 | 997 | 970 |
| Bio-Rad negative and EIA positive | 28 | 77 | 6 | 16 | 12 | 15 | 25 | 20 | 12 | 5 | 1 |
| %Positive agreement | 78.5 | 58.6 | 84.2 | 92.3 | 88.9 | 80 | 83.7 | 85.3 | 64.7 | 87.8 | 98.5 |
| %Negative agreement | 96.7 | 91.6 | 97.8 | 97.8 | 97.9 | 97.4 | 98.5 | 96.9 | 98.2 | 100 | 99 |
| % Overall agreement | 94.5 | 85.7 | 97.8 | 96.8 | 96.9 | 96.2 | 96.4 | 95.4 | 97.1 | 99.5 | 98.9 |
| 95% CI | 93-96 | 84-88 | 96-98 | 96-98 | 96-98 | 95-97 | 95-98 | 94-97 | 96-98 | 99-99.9 | 98-99.6 |
| Ind./equiv.-results omitted | 92* | | | | | | | 31** | | | |
| Low SVB*** -results omitted | 1 | 2 | 2 | 1 | | 1 | 1 | | 1 | 10 | |
*92 BioPlex dsDNA or predicate dsDNA results omitted from the calculations
**31 predicate RNP results omitted from the calculations
*** 19 low Serum Verification Bead results omitted from the calculations
## Additional anti-dsDNA assay comparisons:
A correlation was made of the BioPlex ANA dsDNA assay versus the Pharmacia Varelisa dsDNA EIA by testing the prospective population of 907 samples. For test results under 25 IU/mL, and
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where indeterminate and equivocal results were included (n=850), the linear regression analysis showed $y = 0.05x + 1.08$ , $r = 0.536$ . For all results and where indeterminate and equivocal results were excluded (n=812), the linear regression analysis showed $y = 0.23x - 0.76$ and $r = 0.544$ . In another study, the BioPlex ANA Calibrators were run on the Pharmacia Varelisa dsDNA assay and recoveries ranged from $36\%$ to $92\%$ . The BioPlex calibrators were also run on a second commercially available dsDNA antibody assay and recoveries ranged from $27\%$ to $121\%$ .
Bio-Rad states that the differences between the dsDNA antibody assays can be caused by several factors including the type of capture antigen, whether the assays detect both ssDNA and dsDNA antibodies simultaneously and how well the assays capture high versus low avidity dsDNA antibodies. This description and information is included in the package insert.
## b. Matrix comparison:
A subset of 214 subjects from the prospective population was collected and tested as matched serum, EDTA (N=214), and sodium heparinized plasma (N=214) samples. Compared to the serum values recoveries for the EDTA specimens ranged from 95.3% to 100.2% and for sodium heparin, recoveries ranged from 90.5% to 103.6%. The performance of the assay using lithium heparin plasma was demonstrated in the reproducibility studies.
| Analyte | EDTA % Recovery | Sodium Heparin % Recovery |
| --- | --- | --- |
| dsDNA | 98.0% | 96.1% |
| Chromatin | 100.2% | 90.5% |
| Ribosomal-P | 98.3% | 101.2% |
| SSA | 98.3% | 100.2% |
| SSA-52 | 97.1% | 102.0% |
| SSA-60 | 99.0% | 100.5% |
| SSB | 98.3% | 102.6% |
| Sm | 100.2% | 101.4% |
| Sm/RNP | 99.8% | 103.6% |
| RNP | 99.1% | 100.2% |
| RNP-A | 99.1% | 100.4% |
| RNP-68 | 98.5% | 100.3% |
| Scl-70 | 95.3% | 95.9% |
| Jo-1 | 99.2% | 100.0% |
| Centromere B | 97.4% | 98.0% |
## 3. Clinical studies:
a. Clinical sensitivity and specificity:
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The clinical sensitivity and specificity was calculated by running 908 prospectively collected disease positive samples and comparing the result to the clinical status. These calculations do not take into consideration the prevalence (incidence) antibodies found in the disease states.
**BioPlex 2200 versus Patient Clinical Status**
| Disease status: | BioPlex Positive | BioPlex Negative | Total |
| --- | --- | --- | --- |
| Positive | 277 | 131 | 408 |
| Negative | 113 | 387 | 500 |
| Total | 390 | 518 | 908 |
Sensitivity: 67.9% (277/408) [95% CI 63.2 – 72.6%]
Specificity: 77.4% (387/500) [95% CI 73.6 – 81.2%]
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:**
Expected values for the ANA Screen test are presented in the following table for a U.S. population of normal blood donors (N=222). For dsDNA, results of ≤4 IU/mL are negative, 5 – 9 IU/mL are indeterminate, and ≥10 IU/mL or higher are reported as positive. For the other assays, results of <1.0 are negative and results of ≥1.0 AI are reported as positive.
| Result | Positive | | Negative | | Indeterminate | |
| --- | --- | --- | --- | --- | --- | --- |
| N = 222 | # | (%) | # | (%) | # | (%) |
| ANA Screen* | 15 | 6.8 | 207 | 93.2 | N/A | N/A |
| dsDNA | 3 | 1.4 | 211 | 95.0 | 8 | 3.6 |
| Chromatin | 2 | 0.9 | 220 | 99.1 | N/A | N/A |
| Ribosomal protein | 0 | 0 | 222 | 100.0 | | |
| SS-A | 2 | 0.9 | 220 | 99.1 | | |
| SS-B | 0 | 0 | 222 | 100 | | |
| Sm | 1 | 0.5 | 221 | 99.5 | | |
| SmRNP | 2 | 0.9 | 220 | 99.1 | | |
| RNP | 6 | 2.7 | 216 | 97.3 | | |
| Scl-70 | 1 | 0.5 | 221 | 99.5 | | |
| Jo-1 | 0 | 0.0 | 222 | 100.0 | | |
| Centromere B | 3 | 1.4 | 219 | 98.6 | | |
*results calculated based on testing of all analytes.
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Expected values for the ANA Screen test are presented in the following table for patients from the BioPlex 2200 ANA Screen prospective study conducted at three clinical sites (N=908).
| Result | Positive | | Negative | | Indeterminate | |
| --- | --- | --- | --- | --- | --- | --- |
| N = 908 | # | (%) | # | (%) | # | (%) |
| ANA Screen | 390 | 43.0 | 518 | 57.0% | N/A | N/A |
| dsDNA | 119 | 13.1 | 741 | 81.6% | 49 | 5.4% |
| Chromatin | 168 | 18.5 | 740 | 81.5% | N/A | N/A |
| Ribosomal Protein | 37 | 4.1% | 871 | 95.9% | | |
| SS-A | 173 | 19.0 | 735 | 81.0% | | |
| SS-B | 76 | 8.4% | 832 | 91.6% | | |
| Sm | 60 | 6.6% | 848 | 93.4% | | |
| SmRNP | 103 | 11.3 | 805 | 88.7% | | |
| RNP | 112 | 12.3 | 796 | 87.7% | | |
| Scl-70 | 23 | 2.5% | 885 | 97.5% | | |
| Jo-1 | 6 | 0.7% | 902 | 99.3% | | |
| Centromere B | 38 | 4.2% | 870 | 95.8% | | |
# N. Instrument Name:
BioPlex™ 2200 Multi-Analyte Detection System
# O. System Descriptions:
1. Modes of Operation:
Calibrators, controls and patient samples are loaded by the operator into the sample racks and then loaded onto the instrument. The operator schedules calibration (if needed), quality control (QC) and patient samples by interaction with instrument software. Samples are processed automatically by the instrument. Results are displayed for the QC and patient samples in tabular format with warnings and/or error messages appearing if problems occur. Results are verified by the operator and can be printed out or sent to a laboratory information system if present.
2. Software:
FDA has reviewed applicant's Hazard Analysis and software development processes for this line of product types: Yes
3. Sample Identification:
Samples are identified by a bar code reader from a bar code label on each sample tube.
4. Specimen Sampling and Handling:
Specimen sampling and processing is automatically performed by the instrument.
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5. **Assay Types:**
The BioPlex 2200 uses a multiplex bead-based flow immunoassay.
6. **Reaction Types:**
Antigen/antibody binding is detected and measured.
7. **Calibration:**
Calibration is performed by loading calibrator sets and assaying at a minimum in duplicate every 14 days or with each new reagent pack lot. For dsDNA antibodies, a 6-point 4PL fitted curve is used to calculate quantitative results. For the other analytes, a point-to-point curve fit using four calibrators is used to calculate semi-quantitative results.
8. **Quality Control:**
The control set should be run at the beginning of each day that the BioPlex 2200 assays are used. The ANA Screen Control Set should be run at least once per day, and with each new reagent pack lot.
## P. Other Supportive Instrument Performance Characteristics Data Not Covered In The "L. Performance Characteristics" Section Of The SE Determination Decision Summary:
## Q. Conclusion:
The submitted material in this premarket notification is complete and supports a substantial equivalence decision. The labeling is sufficient and it satisfies the requirements of 21 CFR Part 809.10. Each human donor unit used to manufacture the BioPlex reagents was tested by FDA accepted methods and found non-reactive for Hepatitis B surface antigen (HBsAg), antibody to Hepatitis C (HCV) and antibody to HIV-1/HIV-2.
