BIOPLEX 2200 ANTI-CCP KIT ON THE BIOPLEX 2200 MULTI-ANALYTE DETECTION SYSTEM

• K Number: K093954
• Device Name: BIOPLEX 2200 ANTI-CCP KIT ON THE BIOPLEX 2200 MULTI-ANALYTE DETECTION SYSTEM
• Manufacturer: Bio-Rad Laboratories
• Date Cleared: 2010-08-24 ( 244 days)
• Product Code: NHX , JIX , JJY
• Regulation Number: 866.5775
• Type: Traditional
• Panel: IM
• Reference & Predicate Devices: K023285

Intended Use

The BioPlex™ 2200 Anti-CCP Kit is a multiplex flow immunoassay intended for the semi-quantitative detection of IgG antibodies to Cyclic Citrullinated Peptide (CCP) in human serum and EDTA or heparinized plasma. Detection of CCP antibodies may be used as an aid in the diagnosis of rheumatoid arthritis and should be used in conjunction with other clinical information.

The BioPlex 2200 Anti-CCP kit is intended for use with the Bio-Rad BioPlex 2200 system.

The BioPlex™ 2200 Anti-CCP Calibrator Set is intended for the calibration of the BioPlex 2200 Anti-CCP Reagent Pack.

The BioPlex™ 2200 Anti-CCP Control Set is intended for use as an assayed quality control to monitor the overall performance of the BioPlex 2200 instrument and BioPlex 2200 Anti-CCP Reagent Pack in the clinical laboratory. The performance of the BioPlex 2200 Anti-CCP Control Set has not been established with any other Anti-CCP assay.

Device Description

The BioPlex™ 2200 Anti-CCP IgG kit uses multiplex flow immunoassay, a methodology that greatly resembles traditional EIA, but permits simultaneous detection and identification of many antibodies in a single tube. "CCP IgG" is an acronym for the detection of IgG antibodies to Cyclic Citrullinated Peptide.

One (1) population of fluorescent beads is coated with antigens associated with cyclic citrullinated peptide (CCP). Three populations of fluorescent beads function as assay controls (see below). The BioPlex™ 2200 System combines an aliquot of patient sample, sample diluent, and bead reagent into a reaction vessel and incubates the mixture at 37°C. After a wash cycle to remove unbound antibody, anti-human IgG conjugated to phycoerythrin is added and the mixture is incubated at 37°C. Excess conjugate is removed in another wash cycle and the washed beads are re-suspended in wash buffer. The bead mixture then passes through the detector. The identity of the assay and control beads is determined by the fluorescence embedded in the surface of the bead and the amount of immobilized antibody is determined by the fluorescence of the anti-IgG reporter conjugate. Raw data are calculated in relative fluorescence intensity (RFI).

Three additional dyed beads, Internal Standard Bead (ISB), Serum Verification Bead (SVB) and Reagent Blank Bead (RBB) are present in each reaction mixture to verify detector response, the addition of serum or plasma to the reaction and the absence of significant non-specific binding. Refer to the BioPlex™ 2200 System Operation Manual for more information.

The instrument is calibrated using a set of six distinct calibrator vials, supplied separately by Bio-Rad Laboratories. The six vials representing six different analyte concentrations are used for calibration. The cut-off value and assignment of the calibrators are determined by performing concordance and Receiver Operator Characteristic (ROC) analysis using Axis-Shield DIAStat Anti-CCP predicate results as the reference. The result for anti-CCP is expressed as arbitrary units (U/mL). Results of 3 U/mL as positive.

The BioPlex 2200 Anti-CCP Control Set is intended for use as an assayed quality control to monitor the overall performance of the BioPlex 2200 Instrument and BioPlex 2200 Anti-CCP Reagent Pack in the clinical laboratory. The Control Set includes a negative and a positive control. The BioPlex Anti-CCP Positive Control is manufactured to give positive results, with values above the cut-off for the analyte. The BioPlex Anti-CCP Negative Control is manufactured to give negative results, with values below the cut-off for the analyte. The recommended frequency for performing quality control is once every 24-hour testing period. Performing quality control is also necessary after each new assay calibration and certain service procedures.

AI/ML Overview

Here's a summary of the acceptance criteria and study details for the BioPlex™ 2200 Anti-CCP Kit, based on the provided 510(k) summary report:

Acceptance Criteria and Device Performance

Acceptance Criteria Category	Acceptance Criteria (Specifics from text)	Reported Device Performance
Precision/Reproducibility	Based on CLSI EP5-A2 and EP15-A2 guidelines.	CLSI EP5-A2 Reproducibility Study: Total precision (%CV) for serum samples ranged from 6.5% to 8.2%. For EDTA plasma samples, it ranged from 3.8% to 9.2%. For Heparin plasma samples, it ranged from 3.7% to 8.2%. CLSI EP15-A2 Reproducibility Study: Within-run precision (%CV) for positive samples near the cut-off (3 U/mL) in all sample matrices ranged from 3.5% to 5.3%. Total precision (%CV) for positive samples near the cut-off (3 U/mL) ranged from 4.0% to 5.3%. Specific %CV values for various samples and matrices are provided in the tables above, all falling within acceptable ranges.
Linearity/Reportable Range	Linear regression analysis of anti-CCP IgG recovery U/mL vs. sample dilution to determine if dilution curves exhibit statistically significant non-linear regression based on CLSI guideline EP06-A. For onboard dilution features, recovery must be within ±20% of manual dilution, and precision (U/mL CV) must be ≤ 10%.	The assay demonstrated linearity from 0 to 300 U/mL, with R-squared values for samples A, B, and C near 1.0 (0.9985, 0.9990, 0.9978 respectively). For onboard dilutions (1:4, 1:10, 1:100), recovery ranged from 87% to 104%, and onboard CVs ranged from 2.8% to 4.7%, meeting the specified criteria.
Limit of Detection (LoD)	Determined according to CLSI EP17-A.	The calculated LoD for the anti-CCP IgG assay is 0.2 U/mL.
Interfering Substances	No interference observed with specified endogenous and exogenous substances at maximum tested levels (based on CLSI EP7-A2).	No interference was observed with any of the substances tested (Hemoglobin, Bilirubin, Cholesterol, Red Blood Cells, Gamma Globulin, Triglycerides, Protein, Rheumatoid Factor, Ascorbic Acid, Lithium Heparin, Sodium Heparin, EDTA) at their maximum tested concentrations.
Cross-Reactivity	Evaluation with samples from various disease states and other potentially interfering factors.	Possible cross-reactivity observed with ANA samples (11%, including Centromere B at 23% and SS-A at 12%) and Myeloma IgG samples (30%). TPO IgG, VCA IgG, CMV IgG, and Lyme IgG showed 0% cross-reactivity. T. gondii IgG showed 10% cross-reactivity.
Assay Cut-off	Based on concordance and ROC analysis using predicate results as the standard, aiming for specified positive and negative agreement.	A cutoff of 3.0 U/mL was obtained, achieving a positive agreement of 92.9% and a negative agreement of 98.2% with the predicate Axis-Shield DIASTAT anti-CCP assay among 1394 patient samples.
Method Comparison	Performance evaluated against predicate device, Axis-Shield DIASTAT Anti-CCP immunoassay.	Positive Agreement: 97.5% (95% CI: 95.4 - 98.7%) (358/367). Negative Agreement: 91.4% (95% CI: 88.5 - 93.7%) (416/455).
Matrix Comparison	Plasma U/mL values compared to matched serum U/mL values (CLSI EP9-A2). The scatter plots and regression analysis (slope, intercept, correlation coefficient) should demonstrate strong correlation between matrices.	EDTA vs. Serum: Slope = 0.9636 (95% CI: 0.8753, 1.0519), Intercept = 2.5368 (95% CI: -2.5799, 7.6536), Correlation (r) = 0.9824 (95% CI: 0.9670, 0.9906). Heparin vs. Serum: Slope = 0.9642 (95% CI: 0.8995, 1.0289), Intercept = 2.5264 (95% CI: -1.4891, 6.5419), Correlation (r) = 0.9852 (95% CI: 0.9723, 0.9921). These results indicate a strong correlation, demonstrating acceptable matrix equivalency.
Clinical Sensitivity and Specificity	Evaluate on a cohort of healthy blood donors, diagnosed RA patients, and other rheumatic disease patients.	Clinical Sensitivity (for diagnosed RA): 83.1% (412/496) (95% CI: 79.5 – 86.1%). Clinical Specificity (for healthy blood donors and other rheumatic diseases): 97.8% (490/501) (95% CI: 96.1 – 98.8%).

Study Details

1. Sample Sizes and Data Provenance (Test Set):

   • Precision/Reproducibility:
     • CLSI EP5-A2: 3 panels (serum, EDTA plasma, heparinized plasma), each with 10 samples plus 2 controls. Each panel member was assayed 80 times (2 times in 2 separate daily runs over 20 days).
     • CLSI EP15-A2: 3 panels (serum, EDTA plasma, heparinized plasma), each with 10 samples plus 2 controls. Each panel member was tested in quadruplicate over 5 days (20 replicates).
   • Linearity: 3 high anti-CCP IgG positive patient samples (ranging 241-270 U/mL) and their dilutions, assayed in replicates of four. For onboard dilution, 3 high positive samples for each dilution (1:4, 1:10, 1:100), assayed in replicates of ten.
   • Limit of Detection: Low positive, high negative, and blank samples in replicates of fifty (50).
   • Interfering Substances: Specific substances at various concentrations (e.g., Hemoglobin ≤ 500 mg/dL). Number of samples not explicitly stated for this specific test, but design follows CLSI EP7-A2.
   • Cross-Reactivity: 163 ANA samples and 72 other samples containing potential cross-reactants.
   • Assay Cut-off Determination: 1394 patient samples (177 normal, 504 RF-tested/positive, 82 older, 287 RA, 344 non-RA).
   • Method Comparison: 997 specimens (300 healthy blood donors, 496 diagnosed RA patients, 201 other rheumatic disease patients). 822 samples within the detection range were evaluated.
   • Matrix Comparison: 41 matched sets of serum and plasma (EDTA and heparin) from the same donor, spiked with high positive anti-CCP IgG serum, evaluated in replicates of two.
   • Clinical Sensitivity and Specificity: Same 997 specimens as Method Comparison.
   • Expected Values/Reference Range: 300 apparently healthy donors.
   • Prevalence: 300 apparently healthy blood donors and 300 clinical samples submitted for RF/anti-CCP testing.

   Data Provenance:

   • The 997 specimens for Method Comparison and Clinical Sensitivity/Specificity were purchased from commercial suppliers and were frozen serum.
   • Adjudication for RA diagnosis in these patients was by physician's diagnosis or ICD-9 code 714.0.
   • The CLSI EP15-A2 Reproducibility Study was performed at "one clinical trial site."
   • The report does not specify the country of origin for the data; it implies a single clinical site for some studies. The phrase "purchased from commercial suppliers" for patient specimens suggests retrospective data.

2. Number of Experts and Qualifications (Ground Truth for Test Set):

   • The report does not explicitly state the number of experts or their specific qualifications for establishing the ground truth beyond:
     • For RA patients: "Patients previously diagnosed with RA were selected by either physicians' diagnosis or an ICD-9 code 714.0." This implies a physician's diagnosis as the primary ground truth for RA status.
     • For the assay cut-off, the BioPlex 2200 results were compared against the Axis-Shield DIASTAT anti-CCP predicate assay as the reference for confirming positive or negative status.
   • This is not a traditional "expert consensus" in the sense of multiple radiologists reviewing images, but rather relies on a predicate device and existing clinical diagnoses.

3. Adjudication Method (Test Set):

   • For RA diagnosis (ground truth): "Physicians' diagnosis or an ICD-9 code 714.0" serves as the primary adjudication for RA status for the clinical samples.
   • For assay cut-off determination: "All samples were confirmed positive or negative by the Axis-Shield DIASTAT anti-CCP predicate assay." This indicates the predicate device acted as the gold standard for concordance.
   • No "2+1" or "3+1" expert adjudication method is mentioned.

4. Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study:

   • No, a multi-reader multi-case (MRMC) comparative effectiveness study was not explicitly described. This report focuses on the standalone performance of the in vitro diagnostic device (IVD) compared to a predicate IVD, rather than comparing human readers with and without AI assistance. The device is an automated assay, not an AI-based image analysis tool for human readers.

5. Standalone Performance:

   • Yes, the entire study focuses on the standalone performance of the BioPlex™ 2200 Anti-CCP Kit relative to its intended use and a predicate device. The performance characteristics (precision, linearity, LoD, specificity, method comparison, clinical sensitivity/specificity) are all measures of the algorithm/device itself, without human interpretation as part of the primary diagnostic step.

6. Type of Ground Truth Used:

   • Predicate Device Results: For assay cut-off determination and method comparison, the Axis-Shield DIASTAT Anti-CCP predicate immunoassay was used as the reference/ground truth.
   • Clinical Diagnosis: For clinical sensitivity and specificity, the ground truth for "diagnosed RA patients" relied on physicians' diagnosis or ICD-9 code 714.0.
   • Apparent Health Status: For healthy donor groups, "apparently healthy blood donors" formed the negative ground truth.

7. Sample Size for Training Set:

   • The report does not explicitly mention a "training set" in the context of machine learning. This is a traditional IVD device, not an AI/ML-based diagnostic.
   • The calibrators are fundamental to the assay's function and can be considered analogous to a training component. The BioPlex 2200 Anti-CCP Calibrator Set consists of six distinct calibrator vials. The cut-off value and assignment of the calibrators are determined by performing concordance and Receiver Operator Characteristic (ROC) analysis using Axis-Shield DIAStat Anti-CCP predicate results as the reference (as stated in section 2).

8. How Ground Truth for Training Set Was Established:

   • As this is not an AI/ML device with a distinct "training set" in that sense, the concept of ground truth for a training set does not directly apply.
   • For the calibrators, which define the assay's measurement scale and interpretation: "The BioPlex 2200 Anti-CCP Calibrators are assigned relative units from predicate Axis-Shield DIASTAT Anti-CCP Calibrators." BioPlex 2200 Anti-CCP Calibrators are prepared by blending defibrinated and delipidated human plasma units with known anti-CCP IgG activity in a processed human serum matrix. This means the ground truth for calibrator assignment is derived from measurements established by the predicate device and characterized human disease state plasma.

Regulation Number and Section

§ 866.5775 Rheumatoid factor immunological test system.

(a)
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.(b)
Classification. Class II (performance standards).