The BioPlex Syphilis Total & RPR kit is a multiplex flow immunoassay intended for the qualitative detection of total (IgG/IgM) antibodies to Treponema pallidum and the qualitative detection and/or titer determination of non-treponemal reagin antibodies in human serum or plasma. The Syphilis Total or RPR assays may be used to supplement a previously determined reactive treponemal or non-treponemal test. The test system should be used in conjunction with other laboratory tests and clinical findings to aid in the diagnosis of syphilis infection.

The BioPlex 2200 Syphilis Total & RPR kit is not intended for use in screening blood or plasma donors

The BioPlex 2200 Syphilis Total & RPR kit is intended for use with the Bio-Rad BioPlex 2200 System.

The BioPlex 2200 Syphilis Total & RPR Control Set is intended for use as an assayed quality control to monitor the performance of the BioPlex 2200 Instrument and BioPlex 2200 Syphilis Total & RPR assay in the clinical laboratory. The performance of the BioPlex 2200 Syphilis Total & RPR Control Set has not been established with any other Syphilis Total & RPR assays.

The BioPlex 2200 Syphilis Total & RPR Calibrator Set is intended for the BioPlex 2200 Syphilis Total & RPR Reagent Pack.

Device Description

BioPlex 2200 Syphilis Total & RPR kit includes the following components:

One (1) 10 mL vial, containing dyed beads coated with recombinant Syphilis ● rTP47/rTP17 fusion protein, a cardiolipin antigen, an Internal Standard Bead (ISB) and a Serum Verification Bead (SVB) in MOPS (3-[N-Morpholino] propanesulfonic acid) buffer containing bovine proteins with protein stabilizers. ProClin 300 (≤ 0.3%), sodium benzoate (≤ 0.1%) and sodium azide (< 0.1%) are added as preservatives.
One (1) 5 mL vial, containing phycoerythrin conjugated murine monoclonal antihuman IgG and murine monoclonal anti-human IgM, and phycoerythrin conjugated murine monoclonal anti-human FXIII antibody in phosphate buffer supplemented with murine and bovine protein stabilizers. ProClin 300 (≤ 0.3%), sodium benzoate (≤ 0.1%) and sodium azide (< 0.1%) are added as preservatives.
One (1) 10 mL vial, containing bovine and murine protein stabilizers in MOPS (3-IN-Morpholino] propanesulfonic acid) buffer. ProClin 300 (< 0.3%), sodium benzoate (< 0.1%) and sodium azide (< 0.1%) are added as preservatives

BioPlex 2200 Syphilis Total & RPR Calibrator Set: Four (4) 0.5 mL vials, containing T. pallidum and reagin antibodies in a human serum matrix made from defibrinated plasma, and one (1) 0.5 mL vial containing human serum matrix made from defibrinated plasma for a total of five (5) calibrator vials. All calibrators contain ProClin 300 (≤ 0.3%), sodium benzoate (< 0.1%) and sodium azide (< 0.1%) as preservatives

BioPlex 2200 Syphilis Total & RPR Control Set: Two sets of three (3) control vials. Each set contains two (2) 1.5 mL Positive Control vials with antibodies to T. pallidum and reagin in a human serum matrix made from defibrinated plasma and one (1) 1.5 mL vial of Negative Control in a human serum matrix made from defibrinated plasma. ProClin 300 (≤ 0.3%) sodium benzoate (< 0.1%) and sodium azide (< 0.1%) are added as preservatives for all controls.

Additional materials required but not supplied include BioPlex 2200 Sheath Fluid containing Phosphate Buffered Saline (PBS), ProClin 300 (0.03%) and sodium azide (<0.1%) as preservatives; and BioPlex 2200 Wash Solution containing Phosphate Buffered Saline (PBS) and Tween 20. ProClin 300 (0.03%) and sodium azide (<0.1%) as preservatives.

AI/ML Overview

Here's an analysis of the provided text, extracting the acceptance criteria and study details as requested:

BIO-RAD BioPlex 2200 Syphilis Total & RPR Kit Evaluation

1. Table of Acceptance Criteria and Reported Device Performance

The document describes internal precision and reproducibility acceptance criteria implicitly through the reported values for %CV within CLSI guidelines. For clinical performance, acceptance criteria are not explicitly stated as numerical thresholds (e.g., minimum sensitivity/specificity), but agreement percentages are reported. The provided tables are direct results from the studies and serve as the reported performance against assumed internal/regulatory acceptance ranges.

For Syphilis Total Assay (Treponemal Test):

Performance Metric	Acceptance Criteria (Implicit from CLSI Guidelines/Observed Performance)	Reported Device Performance
Precision
Within Run %CV	Low %CV for various AI levels	Sample 1 (0.3 AI): 10.4% Sample 6 (3.2 AI): 2.5% Positive Control: 4.7%
Total Precision %CV	Low %CV for various AI levels	Sample 1 (0.3 AI): 13.4% Sample 6 (3.2 AI): 4.1% Positive Control: 5.1%
Reproducibility
Total %CV	Low %CV across sites for various AI levels	Sample 1 (0.5 AI): 18.2% Sample 5 (6.8 AI): 4.4% Positive Control: 7.0%
Matrix Comparison	Slope: 0.8-1.2, Intercept: -0.2-0.2, Correlation: >0.95	Lithium Heparin: Slope: 0.8421, Intercept: 0.0316, r: 0.9899 Sodium Heparin: Slope: 0.8333, Intercept: 0.0500, r: 0.9924 K2EDTA: Slope: 1.1923, Intercept: -0.0923, r: 0.9943 K3EDTA: Slope: 1.1667, Intercept: -0.0625, r: 0.9915
Clinical Performance (Overall Prospective)	High Positive Percent Agreement (PPA) and Negative Percent Agreement (NPA)	PPA: 92.5% (147/159) [87.3% - 95.6% CI] NPA: 97.9% (824/842) [96.6% - 98.6% CI]
Clinical Performance (Overall Retrospective - Known Positive)	High PPA and NPA (especially PPA for known positives)	PPA: 99.6% (486/488) [98.5% - 99.9% CI] NPA: 100% (56/56) [93.6% - 100% CI]
Clinical Performance (Clinically Diagnosed Syphilis - Total)	High PPA (often referred to as sensitivity here)	PPA: 96.8% (151/156) [92.7% - 98.6% CI]
Cross-Reactivity (Negative Agreement)	High % Negative Agreement (e.g., >95%)	Overall: 99.7% (331/332) (One Anti-Cardiolipin IgG sample was reactive)

For RPR Assay (Non-Treponemal Test):

Performance Metric	Acceptance Criteria (Implicit from CLSI Guidelines/Observed Performance)	Reported Device Performance
Precision
Within Run %CV	Low %CV for various AI levels	Sample 1 (0.2 AI): 11.2% Sample 6 (3.4 AI): 2.6% Positive Control: 2.4%
Total Precision %CV	Low %CV for various AI levels	Sample 1 (0.2 AI): 20.7% Sample 6 (3.4 AI): 6.5% Positive Control: 7.1%
Reproducibility
Total %CV	Low %CV across sites for various AI levels	Sample 1 (0.8 AI): 9.1% Sample 5 (7.4 AI): 6.9% Positive Control: 4.7%
RPR Titer On-Board Dilution Reproducibility	High agreement within ± 1 titer (e.g., 100%)	Negative Control: 100% Positive Control: 100% Low Reactive (1:8): 100% Moderately Reactive (1:16): 100% High Reactive (>1:64): 100%
Matrix Comparison	Slope: 0.8-1.2, Intercept: -0.2-0.2, Correlation: >0.95	Lithium Heparin: Slope: 0.8684, Intercept: 0.0908, r: 0.9893 Sodium Heparin: Slope: 0.8353, Intercept: 0.1147, r: 0.9849 K2EDTA: Slope: 1.0930, Intercept: -0.0314, r: 0.9864 K3EDTA: Slope: 1.0776, Intercept: -0.0233, r: 0.9810
Clinical Performance (Overall Prospective)	High PPA and NPA compared to predicate RPR	PPA: 81.5% (75/92) [72.4% - 88.1% CI] NPA: 96.5% (877/909) [95.1% - 97.5% CI]
Clinical Performance (Overall Retrospective - Known Positive)	High PPA and NPA compared to predicate RPR	PPA: 98.1% (422/430) [96.4% - 99.1% CI] NPA: 80.7% (92/114) [72.5% - 86.9% CI]
Clinical Performance (Clinically Diagnosed Syphilis - Total)	High PPA (referred to as sensitivity here)	PPA: 84.0% (131/156) [77.4% - 88.9% CI]
Cross-Reactivity (Negative Agreement)	High % Negative Agreement (e.g., >95%)	Overall: 99.4% (330/332) (One Anti-Cardiolipin IgA and one Systemic Lupus Erythematosus sample were reactive)

2. Sample Size and Data Provenance for Test Set

Total Samples for Clinical Studies: 2008 samples.
Test Set Breakdown:
- Prospective Samples: 1001 samples.
  - Country of Origin: Approximately 91% US (23.6% Northeast, 18.7% Southwest, 28.4% Southeast, 9.2% Midwest, 10.8% Unknown), 9% outside US (3.5% Argentina, 3.2% France/Europe, 1.4% China, 1.2% Others).
- Retrospective Samples: 661 samples.
  - Country of Origin: Not explicitly broken down for retrospective samples, but generally stated to be from "multiple commercial suppliers" with the 91% US / 9% outside US split likely encompassing both.
- Clinically Diagnosed Individuals: 160 individuals (separate cohort from prospective/retrospective samples with specific diagnoses).
Data Provenance: Both prospective and retrospective samples were used. Prospectively collected samples were from apparently healthy subjects, syphilis/RPR test ordered patients, pregnant women, and HIV positive individuals. Retrospective samples were from known RPR/VDRL positive, clinically diagnosed syphilis, pregnant syphilis positive, and HIV/Syphilis dual positive individuals.

3. Number of Experts and Qualifications for Ground Truth

The document does not explicitly state the number or qualifications of "experts" used for establishing the ground truth directly. Instead, it relies on a "Final Comparator Algorithm Result" for the Syphilis Total assay, which is derived from multiple commercially available syphilis assays (predicate devices). For the RPR assay, the ground truth is primarily based on a commercially available RPR Card Test (Predicate).

No specific number of human experts is mentioned for adjudicating individual cases or establishing the final ground truth. The "ground truth" seems to be established algorithmically or through established predicate diagnostic tests.

4. Adjudication Method

For BioPlex 2200 Syphilis Total assay: The ground truth was established using a "2 out of 3" rule from three commercially available syphilis assays:
1. Treponemal chemiluminescent immunoassay (primary predicate)
2. RPR Card Test (non-treponemal assay)
3. Treponema Pallidum Particle Agglutination (TP-PA) (second treponemal assay)
  If at least two of these predicate assays agreed, that was considered the final comparator result. If there was no agreement (e.g., 1 positive, 1 negative, 1 inconclusive), the result was deemed "indeterminate" and excluded from analysis.
For BioPlex 2200 RPR assay: The ground truth was established by comparing directly to a commercially available RPR Card Test (Predicate). No explicit "adjudication" among multiple assays is described here.

5. Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study

No MRMC or human-in-the-loop study is described in this document. The device is an automated immunoassay system, and its performance is compared to other diagnostic assays (predicate devices), not to human readers. Therefore, there is no effect size reported for human readers improving with or without AI assistance.

6. Standalone Performance Study

Yes, a standalone performance study was done. The entire document describes the standalone performance of the BioPlex 2200 Syphilis Total & RPR kit. The device is an algorithm only (automated immunoassay) without a human-in-the-loop component in the diagnostic process itself. The clinical performance tables explicitly show the agreement of the BioPlex 2200 with the "Final Comparator Algorithm Result" (for Syphilis Total) or the "Predicate RPR Result" (for RPR).

7. Type of Ground Truth Used

For Syphilis Total assay: A composite comparator algorithm based on the results of three commercially available diagnostic assays (predicate Treponemal CIA, RPR Card Test, TP-PA). This could be categorized as a form of expert consensus using established diagnostic tests rather than pathology or direct outcomes data for every individual case.
For RPR assay: A single predicate diagnostic assay (RPR Card Test).

8. Sample Size for Training Set

The document does not explicitly state the sample size used for the training set. It mentions the "feasibility phase of BioPlex 2200 Syphilis Total & RPR assay development" where cutoff values were established using "native human samples from apparently healthy subjects, patients sent to the laboratory for syphilis testing and patients diagnosed with syphilis infection." However, specific numbers for this development/training phase are not provided.

9. How Ground Truth for Training Set Was Established

The cutoff values for the Syphilis Total and RPR assays were established "in the feasibility phase" using Native human samples from:
- Apparently healthy subjects
- Patients sent to the laboratory for syphilis testing
- Patients diagnosed with syphilis infection
The establishment involved Receiver Operating Characteristics (ROC) analysis using "predicate results as standard". This indicates that the ground truth for establishing the cutoffs was derived from existing, legally marketed assays (predicates). Calibrator values were then adjusted based on this analysis to align with the 1.0 AI cutoff. Further confirmation of cutoff values involved comparing BioPlex results from patient samples to commercially available assays, and ultimately, clinical studies validated the cutoff.

Summary

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Food and Drug Administration 10903 New Hampshire Avenue Document Control Center - WO66-G609

Silver Spring, MD 20993-0002

May 11, 2017

BIO-RAD Laboratories Juang Wang Regulatory Affairs Representative, Clinical Immunology Division 5500 E. 2nd Street Benicia, CA 94510

Re: K170413

Trade/Device Name: Bioplex 2200 Syphilis Total & RPR Kit Bioplex 2200 Syphilis Total & RPR Calibrator Set Bioplex 2200 Syphilis Total & RPR Control Set Regulation Number: 21 CFR 866.3830 Regulation Name: Treponema pallidum treponemal test reagents Regulatory Class: II Product Code: LIP, GMQ, JIT, JJX Dated: February 9, 2017 Received: February 10, 2017

Dear Mr. Wang:

We have reviewed your Section 510(k) premarket notification of intent to market the device referenced above and have determined the device is substantially equivalent (for the indications for use stated in the enclosure) to legally marketed predicate devices marketed in interstate commerce prior to May 28, 1976, the enactment date of the Medical Device Amendments, or to devices that have been reclassified in accordance with the provisions of the Federal Food. Drug. and Cosmetic Act (Act) that do not require approval of a premarket approval application (PMA), You may, therefore, market the device, subject to the general controls provisions of the Act. The general controls provisions of the Act include requirements for annual registration, listing of devices, good manufacturing practice, labeling, and prohibitions against misbranding and adulteration. Please note: CDRH does not evaluate information related to contract liability warranties. We remind you, however, that device labeling must be truthful and not misleading.

If your device is classified (see above) into either class II (Special Controls) or class III (PMA), it may be subject to additional controls. Existing major regulations affecting your device can be found in the Code of Federal Regulations, Title 21, Parts 800 to 898. In addition, FDA may publish further announcements concerning your device in the Federal Register.

Please be advised that FDA's issuance of a substantial equivalence determination does not mean that FDA has made a determination that your device complies with other requirements of the Act or any Federal statutes and regulations administered by other Federal agencies. You must comply with all the Act's requirements, including, but not limited to: registration and listing (21 CFR Part 807); labeling (21 CFR Parts 801 and 809); medical device reporting (reporting of medical device-related adverse events) (21 CFR 803); good manufacturing practice requirements as set forth in the quality systems (QS) regulation (21 CFR Part 820); and if applicable, the

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electronic product radiation control provisions (Sections 531-542 of the Act); 21 CFR 1000-1050.

If you desire specific advice for your device on our labeling regulations (21 CFR Parts 801 and 809), please contact the Division of Industry and Consumer Education at its toll-free number (800) 638 2041 or (301) 796-7100 or at its Internet address

http://www.fda.gov/MedicalDevices/Resourcesfor You/Industry/default.htm. Also, please note the regulation entitled, "Misbranding by reference to premarket notification" (21 CFR Part 807.97). For questions regarding the reporting of adverse events under the MDR regulation (21 CFR Part 803), please go to

http://www.fda.gov/MedicalDevices/Safety/ReportaProblem/default.htm for the CDRH's Office of Surveillance and Biometrics/Division of Postmarket Surveillance.

You may obtain other general information on your responsibilities under the Act from the Division of Industry and Consumer Education at its toll-free number (800) 638-2041 or (301) 796-7100 or at its Internet address

http://www.fda.gov/MedicalDevices/ResourcesforYou/Industry/default.htm.

Sincerely yours,

Steven R. Gitterman -S

for Uwe Scherf, M.Sc., Ph.D. Director Division of Microbiology Devices Office of In Vitro Diagnostics and Radiological Health Center for Devices and Radiological Health

Enclosure

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Indications for Use

510(k) Number (if known) K170413

Device Name BioPlex 2200 Syphilis Total & RPR BioPlex 2200 Syphilis Total & RPR Control Set BioPlex 2200 Syphilis Total & RPR Calibrator Set

Indications for Use (Describe)

The BioPlex 2200 Syphilis Total & RPR kit is not intended for use in screening blood or plasma donors

The BioPlex 2200 Syphilis Total & RPR kit is intended for use with the Bio-Rad BioPlex 2200 System.

The BioPlex 2200 Syphilis Total & RPR Calibrator Set is intended for the BioPlex 2200 Syphilis Total & RPR Reagent Pack.

Type of Use (Select one or both, as applicable)
Prescription Use (Part 21 CFR 801 Subpart D)	Over-The-Counter Use (21 CFR 801 Subpart C)

CONTINUE ON A SEPARATE PAGE IF NEEDED.

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BioPlex 2200 Syphilis Total & RPR 510(k) Summary

Bio-Rad Laboratories hereby submits this 510(k) in accordance with the requirements of SMDA 1990 and 21 CFR 807.92. This summary of 510(k) safety and effectiveness information provides detail as a basis for a determination of substantial equivalence for the BioPlex 2200 Syphilis Total & RPR kit.

Applicant/Sponsor I.

Bio-Rad Laboratories, INC Clinical Immunology Division 5500 E. Second Street Benicia, CA 94510

Contact Person: Juang Wang, PhD, RAC Tel: 510-741-4609 FAX: 510-741-3941 Juang_wang@bio-rad.com

Date Prepared: May 09, 2017

Device Name II.

Proprietary Name: BioPlex 2200 Syphilis Total & RPR BioPlex 2200 Syphilis Total & RPR Calibrator BioPlex 2200 Syphilis Total & RPR Control

Classification Name:

Enzyme Linked Immunoabsorption Assay, Treponema pallidum
Antigens, Nontreponemal, All
Calibrator, Secondary
Single (specified) Analyte Controls (Assayed and Unassayed)

Regulatory Information: III.

Product Code	Classification	Regulation Section	Panel
Enzyme linkedimmunoabsorption assay,Treponema pallidum(LIP)	Class II	21 CFR §866.3830 – Treponema pallidum treponemal testreagents	Microbiology(83)
Antigens, Nontreponemal,All (GMQ)	Class II	21 CFR §866.3820 – Treponema pallidum nontreponemal testreagents	Microbiology(83)

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Product Code	Classification	Regulation Section	Panel
Calibrator, Secondary(JIT)	Class II	21 CFR § 862.1150 – Calibrator	ClinicalChemistry
Single (specified) analytecontrols (assayed andunassayed) (JJX)	Class I	21 CFR § 862.1660-Qualitycontrol Material (Assayed andUnassayed)	ClinicalChemistry

IV. Predicate Devices

LIAISON Treponema Assay, K061247 BD Macro-Vue RPR Card Tests, Pre-amendment prior to May 28, 1976

V. Device Description:

BioPlex 2200 Syphilis Total & RPR kit includes the following components:

One (1) 10 mL vial, containing dyed beads coated with recombinant Syphilis ● rTP47/rTP17 fusion protein, a cardiolipin antigen, an Internal Standard Bead (ISB) and a Serum Verification Bead (SVB) in MOPS (3-[N-Morpholino] propanesulfonic acid) buffer containing bovine proteins with protein stabilizers. ProClin 300 (≤ 0.3%), sodium benzoate (≤ 0.1%) and sodium azide (< 0.1%) are added as preservatives.
One (1) 5 mL vial, containing phycoerythrin conjugated murine monoclonal antihuman IgG and murine monoclonal anti-human IgM, and phycoerythrin conjugated murine monoclonal anti-human FXIII antibody in phosphate buffer supplemented with murine and bovine protein stabilizers. ProClin 300 (≤ 0.3%), sodium benzoate (≤ 0.1%) and sodium azide (< 0.1%) are added as preservatives.
One (1) 10 mL vial, containing bovine and murine protein stabilizers in MOPS (3-IN-Morpholino] propanesulfonic acid) buffer. ProClin 300 (< 0.3%), sodium benzoate (< 0.1%) and sodium azide (< 0.1%) are added as preservatives

VI. Intended Use:

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The BioPlex 2200 Syphilis Total kit is not intended for use in screening blood or plasma donors.

The BioPlex 2200 Syphilis Total & RPR kit is intended for use with the Bio-Rad BioPlex 2200 System.

The BioPlex 2200 Syphilis Total & RPR Calibrator Set is intended for the calibration of the BioPlex 2200 Syphilis Total & RPR Reagent Pack.

VII. Test Principle and Procedure

The BioPlex 2200 Syphilis Total & RPR kit employs Treponema pallidum fusion protein (rTP47/rTP17) and cardiolipin antigen-coated fluoromagnetic beads with unique fluorescent signatures to identify the presence of IgG and IgM antibodies to Treponema pallidum and reagin in a two-step assay format.

Dyed beads are coated with recombinant T. pallidum rTP47/rTP17 fusion protein or cardiolipin antigen.

The BioPlex 2200 System combines an aliquot of patient sample, sample diluent, and bead reagent into a reaction vessel. The mixture is incubated at 37℃. After a wash cycle, a mixture of murine monoclonal anti-human IgG and murine monoclonal antihuman IgM antibody conjugated to phycoerythrin (PE), is added to the dyed beads, and this mixture is incubated at 37℃. The excess conjugate is removed in another wash cycle and the beads are re-suspended in wash buffer. The bead mixture then passes through the detector. The identity of the dyed beads is determined by the fluorescence signature of the dyes, and the amount of antibody captured by the antigen is determined by the fluorescence intensity of the attached PE. Raw data is calculated in relative fluorescence intensity (RFI).

Two additional dyed beads, an Internal Standard Bead (ISB) and a Serum Verification Bead (SVB) are present in each reaction mixture to verify detector response and the presence of serum or plasma in the reaction vessel. Refer to the BioPlex 2200 System Operation Manual for more information.

The system is calibrated using a set of five (5) distinct calibrator vials, supplied separately by Bio-Rad Laboratories. One vial containing negative sample, and four

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vials containing human Treponema pallidum or human reagin antibodies, are used for qualitative calibration of assays. The results are expressed in antibody index (AI). The Syphilis Total assay results are reported as nonreactive (≤ 0.8 AI), equivocal (0.9, 1.0 AI) or reactive (≥ 1.1 AI); while the RPR assay results are reported as nonreactive (< 1.0 AI) or reactive (≥ 1.0 AI).

Technological Characteristics and Substantial Equivalence VIII.

The following tables summarize the similarities and differences between the BioPlex 2200 Syphilis Total & RPR kit and the predicate devices used in comparative studies with the BioPlex 2200 Syphilis Total & RPR kits.

Characteristics	New DeviceBioPlex 2200 Syphilis Total& RPR Kit	Predicate DeviceLIAISON TreponemaAssay, K061247
Intended Use	Multiplex flow immunoassayintended for the qualitativedetection of Total (IgG/IgM)antibodies to Treponemapallidum in human serum orplasma	Chemiluminescentimmunoassay intendedfor the qualitativedetermination of totalantibodies directedagainst Treponemapallidum in humanserum
Indications for Use	Used in conjunction withother serological tests andclinical findings to aid in thediagnosis of syphilisinfection.	Same
Measured Analyte	Total antibodies (IgG/IgM)to T. pallidum	Same
Assay Type	Qualitative	Same
Solid Phase	Antigen-coated paramagneticmicrobeads	Antigen coatedmagnetic particles
Cut-off Index	1.0 Antibody Index (AI)	Index 1.0
Equivocal Zone	0.9 - 1.0	0.9 - 1.1
Standardization	The calibrator is referencedto an internal referencematerial	The calibratorconcentrations arereferenced to an in-house antibodypreparation
Controls	2 (Negative and Positive)	Same

Device Differences
Characteristics	New Device	Predicate Device
	BioPlex 2200 Syphilis Total &RPR Kit	LIAISON TreponemaAssay, K061247

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Characteristics	New DeviceBioPlex 2200 Syphilis Total &RPR Kit	Predicate DeviceLIAISON TreponemaAssay, K061247
Assay Technology	Automated multiplex flowimmunoassay	Sandwichchemiluminescenceimmunoassay (CLIA)
Antigen	Recombinant fusion TP antigen:rTP17/rTP47	DNA-Tp17Recombinant antigen
Conjugate	Phycoerythrin conjugatedmurine monoclonal anti-humanIgG and murine monoclonalanti-human IgM	Conjugated to anIsoluminol derivative
Signal Detection	Fluorescence	Chemiluminescent
Sample Matrix	Serum or Plasma	Serum
Calibrator(s)	4 calibrator levels (soldseparately)	Two positivecalibrators
Open Pack Stability	60 days	4 weeks
Reagent PackCalibrationFrequency	Every 30 days	Every 14 days
Instrumentation	Bio-Rad BioPlex 2200 System	DiaSorin LIAISONAnalyzer

Characteristics	New DeviceBioPlex 2200 Syphilis Total &RPR Kit	Predicate DeviceBD Macro-Vue RPRCARD TEST,Pre-amendment
Intended Use	Multiplex flow immunoassayintended for the qualitativedetection and/or titerdetermination of non- Treponemapallidum reagin antibodies inhuman serum or plasma	A non-treponemaltesting procedure forthe serologicaldetection of syphilisin human serum orplasma
Measured Analyte	Non-Treponema pallidum reaginantibodies	Same
Antigen	Cardiolipin/lecithin/cholesterol	Same
Sample matrix	Serum or plasma	Same

Characteristics	New DeviceBioPlex 2200 Syphilis Total &RPR Kit	Predicate DeviceBD Macro-Vue RPRCARD TEST,Pre-amendment
Assay Technology	Automated multiplex flowimmunoassay	Macroscopicflocculation
Solid phase	Antigen-coated paramagnetic	Antigen carbon particle

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Characteristics	New DeviceBioPlex 2200 Syphilis Total &RPR Kit	Predicate DeviceBD Macro-Vue RPRCARD TEST,Pre-amendment
	microbeads	suspension
Conjugate	Phycoerythrin conjugatedmurine monoclonal anti-humanIgG and murine monoclonalanti-human IgM	None
Calibrator(s)	2 levels – negative and positive	None
Control(s)	2 (Negative and Positive)	3 (Negative and 2Positive)
Standardization	The calibrator is referenced toan internal reference material	None
Cut- off Index	1.0 Antibody Index (AI)	None
Signal Detection	Fluorescence	Flocculation by nakedeye
Reagent PackCalibrationFrequency	Every 30 days	None
Instrumentation	Bio-Rad BioPlex 2200 System	Card Test (Manual)

IX. Performance Characteristics

1. Analytical Performance:
- a. Precision/Reproducibility:

Internal Precision (CLSI EP05-A3)

Precision testing of the BioPlex 2200 Syphilis Total & RPR kit on the BioPlex 2200 instrument was performed in accordance with CLSI EP05-A3 guideline. A human serum panel consisting of 6 frozen samples spanning the measuring range was assayed in duplicate per run on two runs daily over 20 days (N=80) on one reagent lot. Two levels of the BioPlex Syphilis Total & RPR controls were also included. The data were analyzed for within-run, between-run, between-day. The total precision and the mean (AI), standard deviation (AI) and percent coefficient of variation (%CV) are summarized below:

SerumPanel	N	Mean AI	Within Run		Between Run		Between Day		Total Precision
			SD	%CV	SD	%CV	SD	%CV	SD	%CV
Sample 1	80	0.3	0.03	10.4%	0.00	0.0%	0.03	8.4%	0.04	13.4%
Sample 2	80	0.5	0.04	6.5%	0.02	3.5%	0.03	5.6%	0.05	9.3%
Sample 3	80	0.9	0.03	3.3%	0.02	2.2%	0.02	2.7%	0.04	4.8%
Sample 4	80	1.5	0.06	3.7%	0.02	1.0%	0.12	7.8%	0.13	8.7%
Sample 5	80	1.8	0.08	4.3%	0.00	0.0%	0.19	10.3%	0.20	11.2%
Sample 6	80	3.2	0.08	2.5%	0.04	1.4%	0.09	2.9%	0.13	4.1%
PositiveControl	80	2.6	0.13	4.7%	0.01	0.4%	0.05	1.8%	0.13	5.1%

BioPlex 2200 Syphilis Total -Precision (CLSI EP5-A3)

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SerumPanel	N	Mean AI	Within Run		Between Run		Between Day		Total Precision
			SD	%CV	SD	%CV	SD	%CV	SD	%CV
Sample 1	80	0.2	0.03	11.2%	0.02	6.5%	0.04	16.2%	0.05	20.7%
Sample 2	80	0.7	0.04	6.6%	0.06	9.0%	0.05	7.3%	0.09	13.4%
Sample 3	80	0.9	0.04	4.2%	0.06	6.5%	0.05	5.1%	0.09	9.3%
Sample 4	80	1.0	0.05	4.7%	0.08	7.5%	0.08	7.8%	0.12	11.8%
Sample 5	80	1.9	0.10	5.2%	0.10	5.3%	0.08	4.2%	0.16	8.6%
Sample 6	80	3.4	0.09	2.6%	0.16	4.8%	0.12	3.5%	0.22	6.5%
PositiveControl	80	2.0	0.05	2.4%	0.11	5.4%	0.08	4.0%	0.14	7.1%

BioPlex 2200 RPR-Precision (CLSI EP05-A3)

Reproducibility (CLSI EP15-A3)

The reproducibility was also evaluated in accordance with CLSI EP15-A3 guideline "User Verification of Precision and Estimation of Bias, Third Edition".

Reproducibility testing was performed at each of three (3) US testing facilities using a single lot of the BioPlex 2200 Syphilis Total & RPR reagent lot. A serum panel consisting of 5 samples spanning the measuring range were assayed in 4 replicates per run, two runs per day over 5 days (4 reps x 2 runs x 5 days x 3 sites = 120 total data points). The QC Controls were also included. The data were analyzed for within-run, between run, between day, between site/instrument and total precision and the mean AI, standard deviation and percent coefficient of variation (%CV) are summarized below:

Syphilis Total			Within Run		Between Run		Between Day		Between Site/Instrument		Total
Sample	N	Grand Mean (AI)	SD	%CV	SD	%CV	SD	%CV	SD	%CV	SD	%CV
Sample 1	120	0.5	0.02	5.1%	0.02	4.1%	0.00	0.9%	0.08	16.9%	0.08	18.2%
Sample 2	120	1.0	0.04	3.9%	0.02	1.6%	0.02	1.9%	0.08	7.5%	0.09	8.8%
Sample 3	120	1.5	0.05	3.5%	0.00	0.0%	0.02	1.5%	0.08	5.5%	0.10	6.7%
Sample 4	120	2.2	0.08	3.4%	0.03	1.5%	0.03	1.1%	0.01	0.5%	0.09	3.9%
Sample 5	120	6.8	0.22	3.2%	0.11	1.7%	0.17	2.6%	0.00	0.0%	0.30	4.4%
Positive Control	119	2.7	0.15	5.7%	0.07	2.7%	0.00	0.0%	0.08	3.1%	0.19	7.0%

BioPlex 2200 Syphilis Total - Reproducibility (CLSI EP15-A3)

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RPR			Within Run		Between Run		Between Day		Between Site/Instrument		Total
Sample	N	GrandMean(AI)	SD	%CV	SD	%CV	SD	%CV	SD	%CV	SD	%CV
Sample 1	120	0.8	0.05	5.9%	0.01	1.7%	0.04	5.4%	0.03	4.0%	0.07	9.1%
Sample 2	120	1.0	0.05	4.7%	0.01	0.9%	0.04	3.4%	0.04	3.5%	0.07	6.8%
Sample 3	120	2.0	0.08	4.0%	0.09	4.5%	0.09	4.7%	0.21	10.7%	0.26	13.2%
Sample 4	120	3.0	0.08	2.8%	0.10	3.2%	0.07	2.3%	0.07	2.4%	0.16	5.4%
Sample 5	120	7.4	0.26	3.6%	0.27	3.6%	0.24	3.3%	0.23	3.2%	0.51	6.9%
Positive Control	120	2.7	0.10	3.5%	0.04	1.5%	0.05	1.9%	0.05	1.9%	0.13	4.7%

BioPlex 2200 RPR - Reproducibility (CLSI EP15-A3)

RPR Titer On-board Dilution Reproducibility

The BioPlex 2200 System has a feature for the determination of an end point RPR titer result. All reactive RPR samples can be diluted on board at 1:4, 1:8, 1:16, 1:32, and 1:64. Four reactive RPR samples, negative and positive controls selected to evaluate the titer precision were tested in two runs per day in duplicate per run for 5 days for a total of 20 data points. The results are summarized below.

	End Point Titer Results								% Agreement
Sample Reactivity	Non-Reactive	Neat	<1:4	1:4	1:8	1:16	1:32	>1:64	within $\pm$ 1 titer(95% CI)
Negative Control	20	0	0	0	0	0	0	0	100%(83.9 - 100%)
Positive Control	0	20	20	0	0	0	0	0	100%(83.9 - 100%)
Low Reactive (1:8)	0	0	0	1	19	0	0	0	100%(83.9 - 100%)
Moderately Reactive(1:16)	0	0	0	0	0	20	0	0	100%(83.9 - 100%)
High Reactive (>1:64)	0	0	0	0	0	0	0	20	100%(83.9 - 100%)
High Reactive (>1:64)	0	0	0	0	0	0	0	20	100%(83.9 - 100%)

b. Traceability:

Value Assignment:

Calibrator assignment is established for the matched lot of BioPlex 2200 Syphilis Total & RPR kit using the internal reference standards. For each calibrator level, three vials are tested in replicates of five on three BioPlex 2200 analyzers for a total of 45 data points. The mean values obtained for each kit calibrator level are verified and must fall within specified acceptable range as shown below.

Calibrator Set	Assay	Range (AI)
Vial 1	Syphilis Totaland RPR	0.0 - 0.2
Vial 2	Syphilis Total	1.4 - 2.2
Vial 3	Syphilis Total	2.8 - 4.2
Vial 4	Syphilis Total	4.8 - 7.2

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Calibrator Set	Assay	Range (AI)
Vial 5	RPR	1.4 – 2.6

Calibrator

There is no international or certified reference material available. The BioPlex Syphilis Total & RPR Calibrators are traceable to internal standard. The internal reference standard is manufactured by spiking high positive plasma into non-reactive immunodepleted human plasma. The internal reference calibrators are frozen at ≤ -80℃.

The BioPlex Syphilis Total assay is calibrated using a set of four levels of distinct serum based calibrators whereas the BioPlex RPR assay is calibrated with a set of two levels of calibrators.

Controls

The BioPlex Syphilis & RPR Control Set contains one negative control, one positive control for Syphilis Total and one positive control for RPR. Positive controls are made from the human disease state serum containing anti-T. pallidum and reagin antibodies. The control set is provided in a human serum matrix stabilized with ≤ 0.3% ProClin 300, ≤0.1% sodium benzoate and <0.1% sodium azide.

For each control level, three vials are tested in multiple replicates using multiple reagent lots on three BioPlex 2200 analyzers for a minimum of 45 replicates per reagent lot. The minimum number of replicates for each control level is 90 when two reagent lots are used and 135 when three reagent lots are used. For each control level. the mean values were derived from replicate analyses and should fall within the corresponding ranges as shown below.

Control Set	Range (AI)
Negative Control	0.0 – 0.5
Syphilis Total Positive Control	1.8 - 3.8
RPR Positive Control	1.8 - 3.8

The manufacturing target ranges of the Control Sets are listed below.

Stability: C.

Stability studies have been performed to support the following claims:

Calibrator and Control:

BioPlex 2200 Syphilis Total & RPR Calibrator set is stable for 60 days after opening the vial the first time; Control Open Vial Stability (2 to 8°C), 60 days from first opening; Onboard Calibration Curve Stability, 30

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days; Calibrators and Controls Real Time Stability (2 to 8°C), 24 month claim or until the date of expiration; Calibrators and Controls Accelerated Stability at 25℃, 2 years predicted. Calibrators and controls freeze-thaw (-20℃ and -80℃), 5 freeze-thaw cycles.

Kit Stability:

BioPlex 2200 Syphilis Total & RPR Kit: Real Time (unopened) Kit Stability, 24 month claim or until the date of expiration when stored unopened on the instrument or at 2 to 8°C. The open kit claim is 60 days.

Sample Stability:

Sample stability studies were also performed: Sample stability fresh (2 to 8°C), 7 days; Sample stability frozen (-20 or -80°C), 24 months claimed; Sample Freeze-thaw (-20 or -80°C), up to 5-freeze thaw cycles at -20°C and 5-freeze thaw cycles at -80℃ acceptable.

d. Analytical specificity:

Interfering Substances

An interfering substances study was conducted to evaluate the potential interference of specific endogenous and exogenous substances with the BioPlex 2200 Syphilis Total & RPR kit according to CLSI EP7-A2 guideline.

No interference was observed with any of the substances tested. The substances and the maximum levels tested are shown in the table below:

Substance	Concentration
Hemoglobin	≤500 mg/dL
Bilirubin (unconjugated)	≤20 mg/dL
Bilirubin (conjugated)	≤30 mg/dL
Triglycerides	≤3300 mg/dL
Total Protein	≤12 g/dL
Cholesterol	≤450 mg/dL
Ascorbic Acid	≤3 mg/dL
Heparin Sodium	<8000 units/dL
EDTA	<800 mg/dL

Cross-Reactivity:

A study was conducted to determine if samples containing various cross reactants interfere with test results when tested with the BioPlex 2200 Syphilis Total & RPR kit. A panel of at least ten (10) specimens positive for each potential cross reactant was evaluated for possible cross reactivity with the BioPlex 2200 Syphilis Total & RPR kit for Syphilis Total and RPR assays.

Most of the samples evaluated were high positive (2x cut-off) for each cross reactant. The potential cross reactant samples were tested with

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commercially available predicate kits in order to confirm the negative status for the analytes intended to be measured. The potentially cross reactive samples were assayed in a single point. Cross reactivity, expressed as percent negative agreement is calculated by the ratio of the number of negative results to the total number of samples assayed for each cross reactant sample set. The results of each potential cross reactant are listed below:

Cross Reactant	Number	BioPlex Syphilis Total	BioPlex RPR
	Tested	% Negative Agreement	% Negative Agreement
Anti-HBs	12	100.0%	100.0%
Anti-Cardiolipin IgG	10	90.0% (9/10)	100.0%
Anti-Cardiolipin IgM	14	100.0%	100.0%
Anti-Cardiolipin IgA	10	100.0%	90.0% (9/10)
Anti-nuclear antibody (ANA)	10	100.0%	100.0%
B.burgdorferi IgG(US Strain)	10	100.0%	100.0%
B.burgdorferi IgG(EU Strain)	10	100.0%	100.0%
B.burgdorferi IgM(EU Strain)	12	100.0%	100.0%
Cytomegalovirus(anti-CMV IgG positive)	11	100.0%	100.0%
Cytomegalovirus(anti-CMV IgM positive)	12	100.0%	100.0%
E. coli	12	100.0%	100.0%
Epstein-Barr Virus(EBV IgG positive)	13	100.0%	100.0%
Epstein-Barr Virus(EBV IgM positive)	12	100.0%	100.0%
HBsAg	13	100.0%	100.0%
HCV	13	100.0%	100.0%
HIV	10	100.0%	100.0%
HSV(anti-HSV-2 IgG positive)	16	100.0%	100.0%
Hyper gamma-globulinemiaIgG	10	100.0%	100.0%
Hyper gamma-globulinemiaIgM	13	100.0%	100.0%
Leptospirosis	12	100.0%	100.0%
Pregnancy	17	100.0%	100.0%
Rheumatoid Factor (RF)	13	100.0%	100.0%
Rubella IgG	11	100.0%	100.0%
Rubella IgM	12	100.0%	100.0%
Systemic LupusErythematosus (SLE)	12	100.0%	91.7% (11/12)
Toxoplasma IgG positive	10	100.0%	100.0%
Toxoplasma IgM positive	10	100.0%	100.0%
Varicella Zoster Virus (anti-VZV IgG positive)	12	100.0%	100.0%
Total	332	99.7% (331/332)	99.4 %(330/332)

Assay cut-off: e.

The cutoff values were established in the feasibility phase of BioPlex 2200 Syphilis Total & RPR assay development using native human samples from apparently healthy subjects, patients sent to the laboratory

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for syphilis testing and patients diagnosed with syphilis infection. Based on the Receiver Operating Characteristics (ROC) analysis using predicate results as standard, calibrator values were adjusted such that the cut-off value was equal to 1.0 AI. Further confirmation of the cutoff values were compared the BioPlex Syphilis Total and RPR results from patient samples to those from the commercially available assays. Subsequently, clinical studies conducted at three sites in the US provided the final validation of the cut-off value.

For the Syphilis Total assay an equivocal range of ±10% was then set around the cut-off value. Thus, results above or equal to 1.1 AI are considered reactive and results below 0.9 AI are considered nonreactive. The 10% range was established based on the precision of the assay.

For RPR assay, no equivocal range was set around the cut-off value. Thus results <1.0AI are considered non-reactive and results ≥1.0 AI are considered reactive.

2. Comparison studies:

a. Method comparison with predicate device:
The results of the BioPlex 2200 Syphilis Total and RPR assays were compared to a composite comparator and RPR card test for Syphilis Total and RPR assays, respectively. The composite comparator is based on an algorithm of results obtained from three commercially available syphilis assays: (a) Treponemal chemiluminescent immunoassay (predicate). (b) a nontreponemal assay (RPR Card Test), and (c) a second treponemal assay (Treponema Pallidum Particle Agglutination (TP-PA). Additional details of how the final comparator result was determined are provided below in the "Clinical Studies" section.

b. Matrix comparison:

The effect of anticoagulants in the samples on the performance of the BioPlex 2200 Syphilis Total and RPR assays was determined by comparing values obtained from the native serum samples spiked with anti-T. pallidum and reagin antibodies to the values of the matched donor's plasma containing the following anti-coagulants:

Dipotassium EDTA ●
Tripotassium EDTA
Lithium heparin ●
Sodium heparin

A total of 113 serum/plasma pairs, screened to be non-reactive using the BioPlex 2200 Syphilis Total & RPR kit, were spiked with an equal amount of a highly reactive syphilis and RPR sample to obtain concentrations that span the assay measuring range.

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Each sample was tested in duplicate on one BioPlex 2200 instrument and the mean of the results for each matrix type was compared to the mean of the respective serum control sample by using Passing-Bablok analysis method to generate regression parameters (slope, intercept and correlation) along with 95% Confidence Interval. Sample results that exceeded the measuring range were excluded from the data analysis. Acceptance criteria for the regression analysis were: slope 0.8 to 1.2, intercept -0.2 to 0.2 and correlation >0.95.

Matrix	BioPlexAssay	Slope(95% CI)	Intercept(95% CI)	Correlation(r)	N
Lithiumheparin	SyphilisTotal	0.8421(0.8148 to 0.8694)	0.0316(-0.0312 to 0.0944)	0.9899	89
	RPR	0.8684(0.8333 to 0.9035)	0.0908(0.0625 to 0.1191)	0.9893	113
Sodiumheparin	SyphilisTotal	0.8333(0.8148 to 0.8519)	0.0500(0.0050 to 0.0950)	0.9924	89
	RPR	0.8353(0.8095 to 0.8611)	0.1147(0.0833 to 0.1461)	0.9849	113
K2EDTA	SyphilisTotal	1.1923(1.1538 to 1.230)	-0.0923(-0.1538 to -0.0308)	0.9943	83
	RPR	1.0930(1.0709 to 1.1151)	-0.0314(-0.0663 to 0.0035)	0.9864	112
K3EDTA	SyphilisTotal	1.1667(1.1290 to 1.2043)	-0.0625(-0.1073 to -0.0177)	0.9915	82
	RPR	1.0776(1.0500 to 1.1052)	-0.0233(-0.0484 to 0.0018)	0.9810	113

The resulting data support the package insert claim that serum, lithium heparin, sodium heparin, K2 EDTA, and K3EDTA specimens are acceptable for use with the BioPlex Syphilis Total and RPR assays.

Clinical studies: 3.

The clinical Performance of the BioPlex 2200 Syphilis Total & RPR kit was evaluated in three testing sites from November 2016 using a total of 2008 samples prospectively and retrospectively collected from multiple commercial suppliers (approximately 91% in the US and 9% from outside of the US). There were 6 samples that generated no values due to repeat instrument error flags. The tested specimens consisted of 1347 prospective samples (301 apparently healthy subjects, 201 syphilis and 200 RPR test ordered for a total of 401 test ordered samples, 295 pregnant women and 45 pregnant women with STD, 305 HIV positive individuals) and 661 retrospective samples (412 RPR/VDRL positive, 160 clinically diagnosed syphilis, 32 pregnant syphilis positive, 57 HIV/Syphilis dual positive).

The 2008 specimens enrolled in the multiple site study were collected in the following areas:

. US : 90.7%
- 23.6% Northeast (Maryland, Massachusetts, New York, Pennsylvania)

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-18.7% Southwest (California, Hawaii, New Mexico)
28.4% Southeast (Florida, Georgia) -
9.2% Midwest -
-10.8% Unknown
Outside US: 9.3% ●
- -3.5% Argentina
- -3.2% France/Europe
- -1.4% China
- 1.2% Others -

Performance of the BioPlex Syphilis Total assay was evaluated by calculating positive percent agreement and negative percent agreement of the assay with the final comparator results based on an algorithm of results from three commercially available assays: treponemal chemiluminescence immunoassay (Predicate), RPR Card Test, and a second treponemal assay (TP-PA). The final comparator results were determined using a "2 out of 3" rule (TP-CLIA, RPR Card and TP-PA) as shown below. Results with indeterminate final comparator results would be excluded from the data analysis.

Performance of the BioPlex RPR assay was also evaluated by calculating positive percent agreement and negative percent agreement against a commercially available RPR Card Test (Predicate).

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TreponemalIgG/IgM(Predicate)	Non-treponemal(Predicate)	2nd Treponemal(TP-PA)(Predicate)	Final ComparatorAlgorithm Result
Negative	Non-reactive	Reactive	Negative
		Non-reactive	Negative
		Inconclusive	Negative
Negative	Reactive	Reactive	Positive
		Non-reactive	Negative
		Inconclusive	Negative
Positive	Reactive	Reactive	Positive
		Non-reactive	Positive
		Inconclusive	Positive
Positive	Non-reactive	Reactive	Positive
		Non-reactive	Negative
		Inconclusive	Positive
Equivocal	Non-reactive	Reactive	Positive
		Non-reactive	Negative
		Inconclusive	Indeterminate
Equivocal	Reactive	Reactive	Positive
		Non-reactive	Negative
		Inconclusive	Indeterminate

a. Clinical Performance in Prospectively Collected Specimens in the Intended Use Population

The 1001 prospectively collected specimens in the intended use population evaluated with the BioPlex 2200 Syphilis Total & RPR kit consisted of 401 specimens sent for routine syphilis or RPR testing (194 females, 207 males,7 - 96 years old), 295 pregnant women (15 -42 years old), and 305 HIV positive individuals (97 females, 208 males, 17 -75 years old).

The comparison between the BioPlex Syphilis Total assay result and the final comparator result for the prospectively-collected specimens in the intended use population is shown below.

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Prospective Samples	Final Comparator Algorithm Result
	R (+)	NR(-)	Total	% PosAgreement(95% CI)	% NegAgreement(95% CI)
SyphilisTest Ordered	Pos (+)	26	3	29	89.7% (26/29)73.6%-96.4%	98.3% (169/172)95.0%-99.4%
	EQ	1	0	1
	Neg (-)	2	169	171
	Total	29	172	201
RPR Test Ordered	Pos (+)	31	0	31	96.9% (31/32)84.3%-99.4%	100% (168/168)97.8%-100%
	EQ	0	0	0
	Neg (-)	1	168	169
	Total	32	168	200
HIV Positive	Pos (+)	90	11	101	91.8% (90/98)84.7% - 95.8%	93.7%(194/207)89.6% - 96.3%
	EQ	2	2	4
	Neg (-)	6	194	200
	Total	98	207	305
Pregnant Women	Pos (+)	0	2	2	N/A	99.3% (293/295)97.6% - 99.8%
	EQ	0	0	0
	Neg (-)	0	293	293
	Total	0	295	295
Overall	Pos (+)	147	16	163	92.5% (147/159)87.3% - 95.6%	97.9% (824/842)96.6% - 98.6%
	EQ	3	2	5
	Neg (-)	9	824	833
	Total	159	842	1001
	BioPlex 2200 Syphilis Total Assay (Treponemal Test)

R- Reactive; EQ-Equivocal; NR-Non-reactive; Pos – Positive; Neg- Negative

The comparison between the BioPlex RPR assay result and the predicate RPR result is shown below.

	Predicate RPR Result
Prospective Samples	R (+)	NR (-)	Total	% PosAgreement(95% CI)	% NegAgreement(95% CI)
BioPlex 2200 RPR Assay(Non-Treponemal Test)	Syphilis TestOrdered	R (+)	19	5	24	79.2% (19/24)59.5%-90.8%	97.2% (172/177)93.6%-98.8%
		NR (-)	5	172	177
		Total	24	177	201
RPR TestOrdered	R (+)	27	2	29	77.1% (27/35)61.0%-87.9%	98.8% (163/165)95.7%-99.7%
	NR (-)	8	163	171
	Total	35	165	200
HIV Positive	R (+)	29	21	50	87.9% (29/33)72.7%-95.2%	92.3% (251/272)88.5%-94.9%
	NR (-)	4	251	255
	Total	33	272	305

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	R (+)
PregnantWomen	NR (-)	0	4	4	N/A	98.6% (291/295)96.6%-99.5%
	Total	0	291	291
		0	295	295
Overall	R(+)	75	32	107
	NR (-)	17	877	894	81.5% (75/92)72.4% -88.1%	96.5%(877/909)95.1% - 97.5%
	Total	92	909	1001

b. Clinical Performance in Retrospective known Syphilis Positive Samples

Clinical performance in the retrospective known syphilis positive samples was evaluated by testing a total of 544 samples including 412 known RRP/VDRL positive (106 females, 305 males, 1 unknown gender, <1 - 89 years old), 32 pregnant positive women (18- 36 years old), 45 pregnant women with a history of STD (20 - 39 years old), and 57 known HIV/Syphilis dual positive (24 females, 33 males, 20 - 67 years old). Two (2) samples from RPR/VDRL positive cohort obtained no values due to repeated instrument errors were excluded from analysis. The comparison between the BioPlex Syphilis Total results and the final comparator results is shown below.

Retrospective Samples	Final Comparator Algorithm Result
	R (+)	NR(-)	Total	% PosAgreement(95% CI)	% NegAgreement(95% CI)
RPR/VDRL PositivePregnant PositiveWomen	Pos (+)	404	0	404	100% (404/404)99.1%-100%	100% (6/6)61.0%-100%
	EQ	0	0	0
	Neg (-)	0	6	6
	Total	404	6	410
Pregnant Womenwith STD	Pos (+)	31	0	31	100% (31/31)89.0%-100%	100% (1/1)20.7%-100%
	EQ	0	0	0
	Neg (-)	0	1	1
	Total	31	1	32
HIV/Syphilis DualPositive	Pos (+)	1	0	1	100% (1/1)20.7% - 100%	100% (44/44)92.0% - 100%
	EQ	0	0	0
	Neg (-)	0	44	44
	Total	1	44	45
All knownpositiveRPR/VDRL	Pos (+)	50	0	50	96.2%(50/52)87.0% - 98.9%	100% (5/5)56.6% - 100%
	EQ	1	0	1
	Neg (-)	1	5	6
	Total	52	5	57

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Pos (+)	486	0	486
EQ	1	0	1
Neg (-)	1	56	57
Total	488	56	544

The comparison between the BioPlex RPR results and the predicate RPR results is shown below.

			Predicate RPR Result
	Retrospective Samples		R (+)	NR (-)	Total	% Pos Agreement (95% CI)	% Neg Agreement (95% CI)
	RPR/VDRLPositive	R (+)	390	11	401	98.5%(390/396)96.7%-99.3%	21.4% (3/14)7.6%-47.6%
		NR (-)	6	3	9
		Total	396	14	410
	PregnantPositiveWomen	R (+)	25	1	26	100% (25/25)86.7%-100%	85.7% (6/7)48.7%-97.4%
		NR (-)	0	6	6
		Total	25	7	32
	PregnantWomenwith STD	R (+)	0	1	1	N/A	97.8% (44/45)88.4% - 99.6%
BioPlex 2200 RPR Assay(Non-Treponemal Test)		NR (-)	0	44	44
		Total	0	45	45
	HIV/SyphilisDualPositive	R (+)	7	9	16	77.8% (7/9)45.3% - 93.7%	81.3% (39/48)68.1%- 89.8%
		NR (-)	2	39	41
		Total	9	48	57
	All knownpositive	R (+)	422	22	444	98.1%(422/430)96.4% -99.1%	80.7% (92/114)72.5% - 86.9%
		NR (-)	8	92	100
		Total	430	114	544

c. Clinical Performance in Clinically Diagnosed Individuals

Samples were collected from 160 individuals diagnosed with primary, secondary or latent syphilis with treatment status including 43 females (18 – 78 years old) and 117 males (19 – 64 years old). Four samples with no results were excluded due to repeated instrument error flags observed. The comparison results between BioPlex Syphilis Total and RPR assays and predicate results are shown below.

Sensitivity of the BioPlex Syphilis Total and RPR assays and predicate assays in individuals clinically diagnosed with syphilis along with the treatment status is shown below.

	TreatmentStatus	N	Reactivity in Medically Diagnosed Syphilis Patients (95% CI)
Syphilis Phase			BioPlexSyphilis Total	BioPlexRPR	FinalComparator	Predicate RPR
Primary	Untreated	26	96.2%	92.3%	100%	88.5%

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Syphilis Phase	TreatmentStatus	N	Reactivity in Medically Diagnosed Syphilis Patients (95% CI)
			BioPlexSyphilis Total	BioPlexRPR	FinalComparator	Predicate RPR
			(25/26)81.1% - 99.3%	(24/26)75.9% - 97.9%	(26/26)87.1% - 100%	(23/26)71.05 - 96.0%
	Treated	29	86.2%(25/29)69.4% - 94.5%	65.5%(19/29)47.3% - 80.1%	86.2%(25/29)69.4% - 94.5%	75.9%(22/29)57.9% - 87.8%
Secondary	Untreated	25	100%(25/25)86.7% - 100%	100%(25/25)86.7% - 100%	100%(25/25)86.7% - 100%	100%(25/25)86.7% - 100%
	Treated	26	100%(26/26)87.1% - 100%	88.5%(23/26)71.05 – 96.0%	100%(26/26)87.1% -100%	80.8%(21/26)62.1% - 91.6%
	Untreated	23	100%(23/23)85.7% - 100%	95.7%(22/23)79.0% - 99.2%	100%(23/23)85.7% - 100%	95.7%(22/23)79.0% - 99.2%
Latent	Treated	27	100%(27/27)85.1% -100%	66.7%(18/27)47.8% - 81.4%	100%(27/27)85.1% -100%	66.7%(18/27)47.8% - 81.4%
All Phases	Untreated	74	98.6%(73/74)92.7% - 99.8%	95.9%(71/74)88.7% - 98.6%	100%(74/74)95.1% - 100%	95.0%(70/74)86.9% - 97.9%
	Treated	82	95.1%(78/82)88.1% - 98.1%	73.2%(60/82)62.7% - 81.6%	95.1%(78/82)88.1% - 98.1%	74.4%(61/82)64.0% - 82.6%
Total		156	96.8% (151/156)92.7% - 98.6%	84.0% (131/156)77.4% - 88.9%	97.4% (152/156)93.6% - 99.3%	84.0% (131/156)77.4% - 88.9%

d. Clinical Performance in HIV Positive Individuals

Clinical performance in the HIV positive samples was evaluated by testing a total of 362 samples from 305 HIV positive individuals (97 females, 208 males, 17 -75 years old).and 57 known HIV/Syphilis dual positive individuals (24 females, 33 males, 20 – 67 years old). The comparison between the BioPlex Syphilis Total results and the final comparator results is shown below.

	HIV Positive			Final Comparator Algorithm Result
			R (+)	NR(-)	Total	% PosAgreement(95% CI)	% NegAgreement(95% CI)
BioPlex 2200 Syphilis Total Assay(Treponemal Test)	HIVPositive	Pos(+)	90	11	101	91.8% (90/98)84.7% - 95.8%	93.7%(194/207)89.6% - 96.3%
		EQ	2	2	4
		Neg(-)	6	194	200
		Total	98	207	305
	HIV /SyphilisDual Positive	Pos(+)	50	0	50	96.2%(50/52)87.0% - 98.9%	100% (5/5)56.6% - 100%
		EQ	1	0	1
		Neg(-)	1	5	6

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	Total	52	5	57
Overall	Pos(+)	140	11	151	93.3%(140/150)88.2% – 96.3%	93.9%(199/212)89.8% - 96.4%
	EQ	3	2	5
	Neg(-)	7	199	206
	Total	150	212	362

			Predicate RPR Result
HIV Positive			R (+)	NR (-)	Total	% PosAgreement(95% CI)	% NegAgreement(95% CI)
BioPlex 2200 RPR Assay(Non-Treponemal Test)	HIV Positive	R (+)	29	21	50	87.9% (29/33)72.7%-95.2%	92.3%(251/272)88.5%-94.9%
		NR (-)	4	251	255
		Total	33	272	305
	HIV /SyphilisDual Positive	R (+)	7	9	16	77.8% (7/9)45.3%- 93.7%	81.3% (39/48)68.1%-89.8%
		NR (-)	2	39	41
		Total	9	48	57
	Overall	R(+)	36	30	66	85.7% (36/42)72.2% -93.3%	90.6%(290/320)86.9% - 93.4%
		NR (-)	6	290	296
		Total	42	320	362

e. Clinical Performance in Pregnant Women

A total of 372 specimens were from 295 pregnant women (15 – 42 years old) including 295 samples sent for Syphilis testing ,32 preselected pregnant women (18 – 36 years old) with positive syphilis and 45 pregnant women with a history of STD (20 - 39 years old). The comparison results for pregnant women on the BioPlex Syphilis Total and RPR assay are shown below.

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		Final Comparator Algorithm Result
Pregnant Women		R (+)	NR(-)	Total	% PosAgreement(95% CI)	% NegAgreement(95% CI)
TestOrdered	Pos(+)	0	2	2	N/A	99.3%(293/295)97.6%-99.8%
	EQ	0	0	0
	Neg(-)	0	293	293
	Total	0	295	295
BioPlex 2200 Syphilis Total Assay(Treponemal Test)	SyphilisPositive	Pos(+)	31	0	31	100% (31/31)89.0%-100.0%	100% (1/1)20.7%-100%
		EQ	0	0	0
		Neg(-)	0	1	1
		Total	31	1	32
	SDT	Pos(+)	1	0	1	100% (1/1)20.7% - 100%	100% (44/44)92.0% - 100%
		EQ	0	0	0
		Neg(-)	0	44	44
		Total	1	44	45
	AllCombined	Pos(+)	32	2	34	100% (32/32)89.3%-100.0%	99.4%(338/340)97.9%-99.8%
		EQ	0	0	0
		Neg(-)	0	338	338
		Total	32	340	372

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Pregnant Women	Predicate RPR Result
	R (+)	NR(-)	Total	% PosAgreement(95% CI)	% NegAgreement(95% CI)
TestOrdered	R (+)	0	4	4	N/A	98.6% (291/295)98.6%-99.5%
	NR (-)	0	291	291
	Total	0	295	295
SyphilisPositive	R(+)	25	1	26	100% (25/25)86.7%-100%	85.7% (6/7)48.7%-97.4%
	NR (-)	0	6	6
	Total	25	7	32
STD	R (+)	0	1	1	N/A	97.8% (44/45)88.4%-99.6%
	NR (-)	0	44	44
	Total	0	45	45
AllCombined	R(+)	25	6	31	100% (25/25)86.7%-100%	98.3% (341/347)96.3%-99.2%
	NR (-)	0	341	341
	Total	25	347	372

f. Clinical Performance in Apparently Healthy Subjects

A total of 301 samples prospectively collected from apparently healthy subjects undergoing a routine check-up including 178 females (13 – 102 years old) and 123 males (8 – 93 years old) were evaluated with the BioPlex Syphilis Total & RPR. The comparison results of the BioPlex Syphilis Total and RPR assays are shown below.

	Apparently HealthySubjects	R (+)	NR(-)	Total	% PosAgreement(95% CI)	% NegAgreement(95% CI)
BioPlex 2200 SyphilisTotal Assay(Treponemal Test)	Pos (+)	3	1	4		99.0% (294/297)97.1% - 99.7%
	EQ	0	2	2	75,0% (3/4)30.1% - 95.5%
	Neg (-)	1	294	295
	Total	4	297	301

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Apparently HealthySubjects	Predicate RPR result			% PosAgreement(95% CI)	% NegAgreement(95% CI)
	R (+)	NR (-)	Total
BioPlex 2200RPR Assay(Non-TreponemalTest)	R(+)	0	6	6
	NR (-)	4	291	295	0% (0/4)0.0%-49.0%	98.0%(291/297)95.7% - 99.1%
Total	4	297	301

g. Summary of the Serological Test Profile

The serological test profile for all prospective samples tested in the clinical study is summarized below for both BioPlex Syphilis Total and RPR assays.

BioPlex Syphilis Total serological test profile
TrepAssay(predicate)	RPR(Predicate)	TPPA(Predicate)	FinalComparatorResult	BioPlexSyphilisTotal	Number ofSubjects
N	NR	Inconclusive	N	NR	7
N	NR	NR	N	NR	793
N	NR	NR	N	R	4
N	NR	R	N	NR	5
N	NR	R	N	R	4
N	R	NR	N	NR	9
EQ	NR	NR	N	NR	1
EQ	NR	R	P	NR	2
EQ	NR	R	P	R	1
P	NR	Inconclusive	P	R	2
P	NR	NR	N	EQ	2
P	NR	NR	N	NR	9
P	NR	NR	N	R	8
P	NR	R	P	EQ	3
P	NR	R	P	NR	5
P	NR	R	P	R	63
P	R	Inconclusive	P	NR	1
P	R	NR	P	R	2
P	R	NR	P	NR	1
P	R	R	P	NR	1
P	R	R	P	R	78

ﺎﻟﻤﺘﻔﺼﻠﻪ -1-1:- T-4-1 c: 1

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TrepAssay(predicate)	RPR(Predicate)	TPPA(Predicate)	FinalComparatorResult	BioPlexSyphilisTotal	Number ofSubjects
Total					1001

P- Positive; N - Negative; R- Reactive; NR - Non-reactive; EQ - Equivocal

BioPlex RPR serological test profile

Trep Assay(predicate)	RPR(Predicate)	TPPA(Predicate)	FinalComparatorResult	BioPlexRPR	Number ofSubjects
N	NR	Inconclusive	N	NR	7
N	NR	NR	N	NR	773
N	NR	NR	N	R	24
N	NR	R	N	NR	9
N	R	NR	N	NR	3
N	R	NR	N	R	6
EQ	NR	NR	N	NR	1
EQ	NR	R	P	NR	3
P	NR	Inconclusive	P	NR	2
P	NR	NR	N	NR	17
P	NR	NR	N	R	2
P	NR	R	P	NR	65
P	NR	R	P	R	6
P	R	Inconclusive	P	NR	1
P	R	NR	P	NR	1
P	R	NR	P	R	2
P	R	R	P	NR	24
P	R	R	P	R	55
Total					1001

P- Positive; N - Negative; R- Reactive; NR - Non-reactive; EQ - Equivocal

4. Expected values/Reference range:

There are a total of 1001 samples prospectively collected specimens for the intended use population that were tested with the BioPlex Syphilis Total and RPR kit. The three cohorts consisted of 401 specimens sent for routine syphilis or RPR testing (194 females, 207 males, 7 - 96 years old), 295 pregnant women (15 -42 years old), and 305 HIV positive individuals (97 females, 208 males, 17 -75 years old). The prevalence of each cohort is presented below.

Age (years)	Gender	N	BioPlex Syphilis Total N (%)			BioPlex RPR N (%)
			R(+)	EQ	NR(-)	R(+)	NR(-)
<21	Female	30	0 (0.0%)	0 (0.0%)	30 (100.0%)	1 (3.3%)	29 (96.7%)
	Male	22	2 (9.1%)	0 (0.0%)	20 (90.9%)	1 (4.5%)	21 (95.5%)
21-30	Female	56	1 (1.8%)	0 (0.0%)	55 (98.2%)	3 (5.4%)	53 (94.6%)

Prevalence of Subjects Sent for Syphilis Testing

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Age(years)	Gender	N	BioPlex Syphilis Total N (%)			BioPlex RPR N (%)
			R(+)	EQ	NR(-)	R(+)	NR(-)
31-40	Male	59	7 (11.9%)	1 (1.7%)	51 (86.4%)	5 (8.5%)	54 (91.5%)
	Female	42	3 (7.1%)	0 (0.0%)	39 (92.9%)	6 (14.3%)	36 (85.7%)
41-50	Male	50	10 (20.0%)	0 (0.0%)	40 (80.0%)	9 (18.0%)	41 (82.0%)
	Female	33	4 (12.2%)	0 (0.0%)	29 (87.9%)	3 (9.1%)	30 (90.9%)
51-60	Male	24	9 (37.5%)	0 (0.0%)	15 (62.5%)	6 (25.0%)	18 (75.0%)
	Female	13	5 (38.5%)	0 (0.0%)	8 (61.5%)	4 (30.8%)	9 (69.2%)
>61	Male	27	10 (37.0%)	0 (0.0%)	17 (63.0%)	7 (25.9%)	20 (74.1%)
	Female	20	2 (10%)	0 (0.0%)	18 (90.0%)	3 (15.0%)	17 (85.0%)
Overall7 – 96	Male	25	7 (28.0%)	0 (0.0%)	18 (72.0%)	5 (20.0%)	20 (80.0%)
	Female	194	15 (7.7%)	0 (0.0%)	179 (92.3%)	20 (10.3%)	174 (89.7%)
Combined		207	45 (21.7%)	1 (0.5%)	161 (77.8%)	33 (15.9%)	174 (84.1%)
		401	60 (15.0%)	1 (0.2%)	340 (84.8%)	53 (13.2%)	348 (86.8%)

Prevalence of Pregnant Women for whom syphilis testing is ordered

Age(years)	N	BioPlex Syphilis TotalN (%)			BioPlex RPRN (%)
		R(+)	EQ	NR(-)	R(+)	NR(-)
<21	45	0 (0.0%)	0 (0.0%)	45 (100%)	1 (2.2%)	44 (97.8%)
21-30	175	1 (0.6%)	0 (0.0%)	174 (99.4%)	1 (0.6%)	174 (99.4%)
31-40	73	1 (1.4%)	0 (0.0%)	72 (98.6%)	2 (2.7%)	71 (97.3%)
>41	2	0 (0.0%)	0 (0.0%)	2 (100%)	0 (0.0%)	2 (100%)
Overall15 - 42	295	2 (0.7%)	0 (0.0%)	293 (99.3%)	4 (1.4%)	291 (98.6%)

Prevalence of HIV Positive individuals

Age(years)	Gender	N	BioPlex Syphilis TotalN (%)			BioPlex RPRN (%)
			R(+)	EQ	NR(-)	R(+)	NR(-)
<21	Female	2	0 (0.0%)	0 (0.0%)	2 (100%)	0 (0.0%)	2 (100%)
	Male	5	1 (20%)	0 (0.0%)	4 (80.0%)	2 (40.0%)	3 (60.0%)
21-30	Female	6	0 (0.0%)	0 (0.0%)	6 (100%)	0 (0.0%)	6 (100%)
	Male	25	4 (16.0%)	0 (0.0%)	21 (84.0%)	4 (16.0%)	21 (84.0%)
31-40	Female	20	5 (25%)	0 (0.0%)	15 (75.0%)	4 (20.0%)	16 (80.0%)
	Male	26	8 (30.8%)	1 (3.8%)	17 (65.4%)	6 (23.1%)	20 (76.9%)
41-50	Female	42	12 (28.6%)	0 (0.0%)	30 (71.4%)	6 (14.3%)	36 (85.7%)
	Male	71	26 (36.6%)	1 (1.4%)	44 (62.0%)	12 (16.9%)	59 (83.1%)
51-60	Female	21	8 (38.1%)	0 (0.0%)	13 (61.9%)	3 (14.3%)	18 (85.7%)
	Male	61	30 (49.2%)	1 (1.6%)	30 (49.2%)	10 (16.4%)	51 (83.6%)
61 -70	Female	6	2 (33.3%)	0 (0.0%)	4 (66.7%)	0 (0.0%)	6 (100%)
	Male	16	5 (31.3%)	1 (6.3%)	10 (62.5%)	2 (12.5%)	14 (87.5%)

{28}------------------------------------------------

Age(years)	Gender	N	BioPlex Syphilis TotalN (%)			BioPlex RPRN (%)
			R(+)	EQ	NR(-)	R(+)	NR(-)
>71	Female	0	0 (0.0%)	0 (0.0%)	0 (0.0%)	0 (0.0%)	0 (0.0%)
	Male	2	0 (0.0%)	0 (0.0%)	2 (100%)	0 (0.0%)	2 (100%)
Unknown	Male	2	0 (0.0%)	0 (0.0%)	2 (100%)	0 (0.0%)	2 (100%)
All17-75	Female	97	27 (27.8%)	0 (0.0%)	70 (72.2%)	13 (13.4%)	84 (86.6%)
	Male	208	74 (35.6%)	4 (1.8%)	130 (62.5%)	37 (17.8%)	171 (82.2%)
Combined		305	101 (33.1%)	4 (1.3%)	200 (65.6%)	50 (16.4%)	255 (83.6%)

Instrument Name: X.

The BioPlex 2200 System, software version 4.3

XI. Conclusion:

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

Regulation Number and Section

§ 866.3830

Treponema pallidum treponemal test reagents.(a)
Identification. Treponema pallidum treponemal test reagents are devices that consist of the antigens, antisera and all control reagents (standardized reagents with which test results are compared) which are derived from treponemal sources and that are used in the fluorescent treponemal antibody absorption test (FTA-ABS), theTreponema pallidum immobilization test (T.P.I.), and other treponemal tests used to identify antibodies toTreponema pallidum directly from infecting treponemal organisms in serum. The identification aids in the diagnosis of syphilis caused by bacteria belonging to the genusTreponema and provides epidemiological information on syphilis.(b)
Classification. Class II (performance standards).