ATHENA MULTI-LYTE TREPONEMA PALLIDUM IGG PLUS TEST SYSTEM

{0} 1 # 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION SUMMARY ASSAY ONLY TEMPLATE A. 510(k) Number: K093837 B. Purpose for Submission: To obtain a Substantial Equivalence determination for the AtheNA Multi-Lyte Treponema pallidum IgG Plus Test System C. Measurand: Antibodies to Treponema pallidum (T. pallidum) D. Type of Test: A multiplex flow qualitative immunoassay E. Applicant: Zeus Scientific, Inc. F. Proprietary and Established Names: AtheNA Multi-Lyte Treponema pallidum IgG Plus Test System; Treponema pallidum treponemal test reagents G. Regulatory Information: 1. Regulation section: 21 CFR § 866.3830, Treponema pallidum treponemal test reagents 2. Classification: Class II 3. Product code: LIP - Enzyme linked immunoabsorption assay, Treponema pallidum {1} 4. Panel: Microbiology (83) H. Intended Use: 1. Intended use: The Zeus Scientific, Inc. AtheNA Multi-Lyte® Treponema pallidum IgG Plus Test System is a multiplex flow immunoassay intended for the qualitative detection of specific human IgG class antibodies to Treponema pallidum in human serum. The presence of antibodies to Treponema pallidum specific antigen, in conjunction with non treponemal laboratory tests and clinical findings, may aid in the diagnosis of syphilis infection. This test is for in vitro diagnostic use only. This test is not intended for screening blood or plasma donors. 2. Indications for use: The Zeus Scientific, Inc. AtheNA Multi-Lyte® Treponema pallidum IgG Plus Test System is a multiplex flow immunoassay intended for the qualitative detection of specific human IgG class antibodies to Treponema pallidum in human serum. The presence of antibodies to Treponema pallidum specific antigen, in conjunction with non treponemal laboratory tests and clinical findings, may aid in the diagnosis of syphilis infection. This test is for in vitro diagnostic use only. This test is not intended for screening blood or plasma donors. 3. Special condition for use statement: For prescription use only. 4. Special instrument requirements: Luminex 200 IS xMAP instrument and AtheNA Multi-Lyte software version 3.0.9. 2 {2} I. Device Description: The AtheNA Multi-Lyte® Treponema pallidum IgG Plus Test System is an immunoassay system that employs traditional sandwich immunoassay techniques to measure antibody in human serum samples. The platform for this system is the Luminex 200 IS xMAP platform. This is an open platform consisting of solid phase microparticles on which the immunoassays are built and a modified flow cytometer used to interpret the reactions on the microparticles. The system contains the AtheNA Multi-Lyte Software version 3.0.9. In this data analysis software, raw data ("output.csv" file) from a test performed on the Luminex is converted from the MFI per specimen/per analyte to a result based on the specific assay calibration data (from their Intra-Well Calibration Technology) and reported to the end user. J. Substantial Equivalence Information: 1. Predicate device name: - Trep-Chek Treponemal Antibody EIA 2. Predicate 510(k) number: - K001552 1. Comparison with predicate: | Similarities | | | | --- | --- | --- | | Item | Device | Predicate | | Intended Use | AtheNA Multi-Lyte® Treponema pallidum IgG Plus Test System is a multiplex flow immunoassay intended for the qualitative detection of specific human IgG class antibodies to treponema pallidum in human serum. The AtheNA Multi-Lyte® Treponema pallidum | The Phoenix Bio-Tech Corp. Syphilis Trep-Chek Test Kit is a confirmatory immunoassay for the qualitative detection of Treponema pallidum IgG antibodies in human serum or plasma. This product is not cleared (approved) by the U.S. Food and Drug Administration | {3} | Similarities | | | | --- | --- | --- | | Item | Device | Predicate | | | IgG Plus Test System is not intended for use in screening blood or plasma donors. | (FDA) for use in screening blood or plasma donors. | | Assay type | Enzyme labeled, immunoassay | Enzyme labeled, immunoassay | | Analyte Measured | Human IgG | Human IgG | | Differences | | | | --- | --- | --- | | Item | Device | Predicate | | Sample Dilution | 1:21 in SAVe Diluent | 1:20 in phosphate buffer based diluent | | Detection Method | Fluorescent | Colorimetric | | Scale | Intra-Well Calibration determines a unit value for each sample from the regression curve | Calculate the index value of unknown samples by comparing their OD to the cut off OD | | Sample Dilution | 1:21 in SAVe Diluent | 1:20 in phosphate buffer based diluent | | Specimen Tested | Human Serum | Human Serum or plasma | | Calibration | Includes Intra-Well Calibration that provides a separate calibration curve for every sample | Includes Calibrator (human serum) | | Cut-Offs | Negative is < 100, Positive is > 120 and Equivocal is 100-120 AU/mL | Negative is <= 0.90, Positive is >= 1.10 and Equivocal is 0.90 - 1.09 | # K. Standard/Guidance Document Referenced: - CLSI EP07-A2: Interference Testing in Clinical Chemistry - CLSI EP5: Evaluation of Precision Performance of Clinical Chemistry Devices-Second Edition - Guidance for the Content of Premarket Submissions for Software Contained in Medical Devices, May 11, 2005 {4} # L. Test Principle: The test procedure involves two incubation steps where patient sera are diluted and the diluted test sera are incubated in a vessel containing a multiplexed mixture of the bead suspension. The multiplexed bead suspension contains a mixture of distinguishable sets of polystyrene microspheres; each set conjugated with Treponema pallidum antigen. The bead mix also contains one bead set designed to detect non-specific antibodies in the patient sample (if present) and four separate bead sets used for assay calibration. If present in patient sera, the individual antibodies will bind to the corresponding immobilized antigen bead set. The microspheres are rinsed to remove non-reactive serum proteins. Phycoerythrin-conjugated goat anti-human IgG is added to the vessel and the plate is incubated. The conjugate will react with IgG antibody immobilized on the beads in step 1. The bead suspension is then analyzed by the AtheNA Multi-Lyte® instrument using software version 3.0.9. The bead set(s) are sorted (identified) and the amount of reporter molecule (PE conjugate) is determined for each bead set. Using the Intra-Well Calibration Technology®, internal calibration bead sets are used to convert raw fluorescence into outcome (units). # M. Performance Characteristics: # 1. Analytical performance: # a. Precision/Reproducibility: Precision was evaluated internally at the manufacturer's site. The study was conducted as follows: Nine samples (negative, high negative, near cut-off, low positive and high positive) were identified and/or prepared (by Zeus Scientific, Inc.) for use in the study based upon their activity on the AtheNA Multi-Lyte assay. To assess precision, on each day of testing, each sample was diluted twice and tested. This was repeated by a second technologist in a separate run and resulted in four results per day. The test results for negative samples, less than $20\mathrm{AU / mL}$ is consistent with a high $\% \mathrm{CV}$ which is acceptable. This was repeated for twenty days and the resulting data used to assess precision. Please see section M.1.f. for the assays interpretive criteria. AtheNA Multi-Lyte Treponema pallidum Precision Study | Panel Member | Sample N | Mean AU/mL | Within-Run | | Within-Day | | Between-Run | | Total | | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | | | | SD | %CV | SD | %CV | SD | %CV | SD | %CV | | High Positive 1 | 80 | 352.68 | 19.2 | 5.4% | 9.1 | 6.4% | 8.8 | 2.5% | 24.0 | 6.8% | | High Positive 2 | 80 | 462.59 | 19.2 | 4.1% | 11.1 | 4.3% | 10.8 | 2.3% | 26.8 | 5.8% | | High Positive 3 | 80 | 481.24 | 24.8 | 5.2% | 8.4 | 5.5% | 12.4 | 2.6% | 27.9 | 5.8% | | Moderate Positive 1 | 80 | 210.11 | 16.2 | 7.4% | 6.5 | 6.0% | 12.3 | 5.5% | 22.3 | 10.6% | {5} Reproducibility was evaluated internally and at two external clinical sites. The study was conducted as follows: Nine samples (negative, high negative, near cut-off, low positive and high positive) were identified and/or prepared (by Zeus Scientific, Inc.) for use in the study based upon their activity on the AtheNA Multi-Lyte assay. To assess reproducibility, on each day of testing, each sample was diluted twice and each dilution was run in triplicate. This process was repeated by a second technologist resulting in twelve results per day. This was repeated for five days at each site and the resulting data used to assess reproducibility. The test results for negative samples, less than 20 AU/mL is consistent with a high $\% \mathrm{CV}$ which is acceptable. Summary Of Multi-Site Reproducibility - AtheNA T. pallidum IgG Plus | Panel Member | Sample N | Mean AU/mL | Within-Run | | Within-Day | | Between-Run | | Between-Site | | Total | | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | | | | SD | %CV | SD | %CV | SD | %CV | SD | %CV | SD | %CV | | High Positive 1 | 180 | 326 | 19.2 | 5.8% | 21.8 | 6.6% | 12.7 | 3.8% | 24.2 | 7.4% | 26.6 | 8.2% | | High Positive 2 | 180 | 436 | 21.9 | 5.0% | 26.6 | 6.1% | 19.2 | 4.4% | 29.3 | 6.7% | 32.6 | 7.5% | | High Positive 3 | 180 | 459 | 23.8 | 5.2% | 30.0 | 6.5% | 21.2 | 4.6% | 30.9 | 6.7% | 33.1 | 7.2% | | Mod Positive 1 | 180 | 196 | 10.6 | 5.4% | 12.2 | 6.2% | 7.0 | 3.6% | 13.5 | 6.9% | 14.6 | 7.5% | | Mod Positive 2 | 180 | 304 | 16.3 | 5.3% | 18.7 | 6.1% | 11.2 | 3.7% | 19.3 | 6.4% | 21.2 | 7.0% | | Mod Positive 3 | 180 | 234 | 14.0 | 5.9% | 18.4 | 7.8% | 13.9 | 5.9% | 19.1 | 8.2% | 21.1 | 9.0% | | Low Positive 1 | 180 | 170 | 10.1 | 5.9% | 12.1 | 7.1% | 8.2 | 4.8% | 13.0 | 7.7% | 15.5 | 9.1% | | Low Positive 2 | 180 | 137 | 7.8 | 5.7% | 9.8 | 7.1% | 6.9 | 4.9% | 10.9 | 8.0% | 11.7 | 8.5% | | Low Positive 3 | 180 | 156 | 8.7 | 5.5% | 11.2 | 7.1% | 8.0 | 5.1% | 12.3 | 7.9% | 14.5 | 9.3% | | Near Cut-off 1 | 180 | 106 | 6.6 | 6.1% | 8.2 | 7.7% | 6.1 | 5.7% | 8.6 | 8.1% | 9.8 | 9.2% | | Near Cut-off 2 | 180 | 103 | 5.9 | 5.7% | 7.2 | 6.9% | 4.9 | 4.7% | 7.5 | 7.3% | 8.9 | 8.6% | | Near Cut-off 3 | 180 | 108 | 6.3 | 5.8% | 7.0 | 6.4% | 3.3 | 3.1% | 8.1 | 7.5% | 9.4 | 8.8% | | High Negative 1 | 180 | 73 | 4.6 | 6.3% | 5.3 | 7.2% | 2.8 | 3.8% | 5.5 | 7.6% | 8.5 | 8.8% | | High Negative 2 | 180 | 81 | 5.6 | 6.9% | 6.5 | 8.1% | 3.9 | 4.8% | 6.9 | 8.5% | 7.4 | 9.2% | | High Negative 3 | 180 | 88 | 5.2 | 5.8% | 6.3 | 7.2% | 4.1 | 4.6% | 6.8 | 7.7% | 7.6 | 8.7% | | Negative 1 | 180 | 11 | 2.1 | 19.3% | 2.2 | 20.1% | 0.8 | 7.0% | 2.2 | 20.1% | 2.5 | 22.8% | | Negative 2 | 180 | 9 | 1.8 | 20.2% | 1.9 | 20.9% | 0.5 | 6.0% | 2.0 | 21.9% | 2.2 | 24.9% | | Negative 3 | 180 | 17 | 2.3 | 13.7% | 2.4 | 14.0% | 0.9 | 5.0% | 2.4 | 13.8% | 2.5 | 14.7% | | Non-Reactive Control | 90 | 12 | 2.3 | 18.7% | 2.4 | 19.9% | 1.2 | 9.7% | 2.5 | 20.2% | 2.8 | 23.3% | | Reactive Control | 90 | 886 | 32.2 | 3.6% | 35.4 | 4.0% | 18.3 | 2.1% | 36.9 | 4.2% | 38.9 | 4.4% | Results from the precision and reproducibility studies are acceptable. {6} b. Linearity/assay reportable range: Three strong positive samples were evaluated using a FDA cleared test system to determine the samples' reactivity. A negative sample was used as the diluent to prepare serial dilutions of the positive samples. Each dilution was tested in duplicate, the mean calculated and the result plotted. The total %CV between the three samples was determined to be 0.3%. The linearity is acceptable if the coefficient of variation for each sample is greater than 0.95. c. Traceability, Stability, Expected values (controls, calibrators, or methods): Not applicable. d. Detection limit: The Master Cutoff of the assay was determined by calibrating against a panel of confirmed syphilis positive and confirmed syphilis negative samples. A minimum of 25 samples were tested using the predicate ELISA test system and confirmed as negative and then tested with the investigational device. Using the mean and standard deviations of this negative population theoretical cut-off was calculated. Known positive samples, selected for their reactivity with the predicate devices, were tested with the investigational device and checked to ascertain that the results fell above the theoretical cut-off. e. Analytical specificity: Interference Testing The effect of potential interfering substances on sample results generated using the AtheNA Multi-Lyte test system was evaluated with the following possible interfering substances based on the guidelines established in CLSI EP7-A2: albumin, bilirubin, cholesterol, hemoglobin, triglycerides and intralipids. The samples were exposed to the possible interfering substances and tested. An increase or reduction of signal equal to or less than 20% is considered acceptable. The negative sample may have a signal change greater than 20% if there is no change in the qualitative result of the sample. All samples showed less than a 20% recovery of signal with the following exceptions: {7} - The positive sample showed a recovery of signal of 76% with the low spike of triglycerides, 60% with the high spike of hemoglobin and 74% with the high spike of intralipids. - The borderline sample showed a recovery of signal of 74% with the low spike of albumin and 77% with the high spike of albumin. - The high spikes of triglycerides and hemoglobin resulted in a recovery of signal of 76% for both substances. The negative sample showed a change of signal with several of the potential interfering substance at both the high and low spikes. The negative samples stayed below the cut-off of 100 AU/ml. All signal changes greater than 20% was specified in the Limitations section of the package insert. ## Cross-Reactivity Study To test for possible cross-reactivity, multiple patient samples containing different concentrations of potentially cross-reactive antibodies were analyzed with the AtheNA Multi-Lyte® Treponema pallidum IgG Plus Test System that were sero-positive to EBV, ANA, RF IgM, Rubella, HIV, HSV 1, HSV 2, pregnancy, Hepatitis B, VZV IgG, VZV IgM, CMV, Toxoplasma, Lyme G/M and Hepatitis C. No cross-reactivity was exhibited. 8 {8} | Organism | Total # Samples | # Reactive/ Total # Samples | | --- | --- | --- | | Cytomegalovirus IgG | 10 | 0/10 | | Epstein-Barr Virus | 10 | 0/10 | | Anti-Nuclear Antibodies | 10 | 0/10 | | Herpes Simplex Virus 1 | 10 | 0/10 | | Herpes Simplex Virus 2 | 10 | 0/10 | | Rubella IgG | 10 | 0/10 | | Toxoplasma IgG | 10 | 0/10 | | Rheumatoid Factor | 10 | 0/10 | | Borrelia burgdorferi IgG/ IgM (US strain) | 10 | 0/10 | | Anti-Hepatitis B Surface Antigen | 10 | 0/10 | | Hepatitis C Virus | 10 | 0/10 | | Hepatitis B Surface Antigen | 10 | 0/10 | | HIV | 10 | 0/10 | | HCG | 10 | 0/10 | | VZV | 10 | 0/10 | | VZV IgM | 10 | 0/10 | f. Assay cut-off: The cut-off for each assay was established using a negative population for each marker. The AtheNA Multi-Lyte results were determined for this population, and the cut-off was set at approximately the mean plus three times the standard deviation. $\geq 100 \mathrm{AU} / \mathrm{mL}$ Negative 100 to 120 AU/mL Equivocal &lt;120 AU/mL Positive 2. Comparison studies: a. Method Comparison with Predicate: Not applicable. b. Matrix comparison: Not applicable. {9} 10 3. Clinical Studies: a. Clinical Sensitivity: A total of 1000 serum samples were collected and tested in the study. The samples were tested at a hospital laboratory located in the Mid-Atlantic, the Northeast and at Zeus Scientific, Inc. The hospital laboratories tested 400 samples from patients with a syphilis test ordered and 400 vendor purchased samples from pregnant women with syphilis test ordered. Zeus Scientific, Inc. tested 100 samples from patients with a syphilis tests ordered and 100 samples from pregnant women. | | AtheNA Multi-Lyte Treponema pallidum IgG Plus Comparative Testing Result Summary Presented with 95% CI Banked sera from patients with syphilis test ordered | | | | | | | | --- | --- | --- | --- | --- | --- | --- | --- | | | | Predicate | | | | | | | | | Positive | Equivocal | Negative | Site Total | PPA NPA | 95% CI | | AtheNA Multi-Lyte Treponema pallidum IgG Plus | | | | | | | | | | Positive Equivocal | 6 | | 13 4 | 19 4 | 100.0% | 60.7 - 100% | | | Negative Invalid | | | 477 | 477 0 | 96.6% | 94.6 - 98% | | | Site Total | 6 | 0 | 494 | 500 | | | {10} 11 | | AtheNA Multi-Lyte Treponema pallidum IgG Plus Comparative Testing Result Summary Presented with 95% CI Banked purchased sera from pregnant women with syphilis test ordered | | | | | | | | --- | --- | --- | --- | --- | --- | --- | --- | | | | Predicate | | | | | | | | | Positive | Equivocal | Negative | Site Total | PPA NPA | 95% CI | | AtheNA Multi-Lyte Treponema pallidum IgG Plus | | | | | | | | | | Positive Equivocal | 1 | | 3 | 4 | 100.0% | 50 - 100% | | | Negative Invalid | | | 494 | 0 | 99.4% | 98.3 - 99.9% | | Site Total | 1 | 0 | 497 | 498* | | | | *Note that 500 specimens were originally obtained; however, two serum specimens did not meet the inclusion criteria for the study. ## Prospectively Collected Population of Unselected Hospitalized Patients: Additional clinical performance was assessed in a population of 1000 hospitalized patients. These samples were pulled from a hospital laboratory routine workload of patient testing and were tested at the three sites. | | AtheNA Multi-Lyte Treponema pallidum IgG Plus Comparative Testing Result Summary Presented with 95% CI Unselected hospitalized patients | | | | | | | | --- | --- | --- | --- | --- | --- | --- | --- | | | | Predicate | | | | | | | | | Positive | Equivocal | Negative | Site Total | PPA NPA | 95% CI | | AtheNA Multi-Lyte Treponema pallidum IgG Plus | | | | | | | | | | Positive Equivocal | 18 1 | 1 | 32 8 | 50 10 | 94.7% | 74 - 99.9% | | | Negative Invalid | | | 932 4 | 932 4 | 95.9% | 94 - 96.7% | | | Site Total | 19 | 1 | 976 | 996* | | | *4 samples QNS for testing {11} Retrospective HIV-1 Positive Samples: A total of 223 banked known positive HIV-1 samples were acquired from a commercial vendor. | | AtheNA Multi-Lyte Treponema pallidum IgG Plus Comparative Testing Result Summary Presented with 95% CI Banked purchased known HIV-1 positive serum samples | | | | | | | | --- | --- | --- | --- | --- | --- | --- | --- | | | | Predicate | | | | | | | | | Positive | Equivocal | Negative | Site Total | PPA NPA | 95% CI | | AtheNA Multi-Lyte Treponema pallidum IgG Plus | | | | | | | | | | Positive Equivocal | 46 | 3 2 | 5 2 | 54 4 | 97.9% | 88.7 - 100% | | | Negative Invalid | | 1 | 164 | 165 0 | 94.3% | 91.8 - 98.3% | | | Site Total | 46 | 6 | 171 | 223 | | | Retrospective TPPA /RPR Positive: A total of 280 samples requested to be RPR/TPPA positive were purchased from a commercial vendor. | | AtheNA Multi-Lyte Treponema pallidum IgG Plus Comparative Testing Result Summary Presented with 95% CI Banked purchased sera requested to be RPR/TPPA reactive | | | | | | | | --- | --- | --- | --- | --- | --- | --- | --- | | | | Predicate | | | | | | | | | Positive | Equivocal | Negative | Site Total | PPA NPA | 95% CI | | AtheNA Multi-Lyte Treponema pallidum IgG Plus | | | | | | | | | | Positive Equivocal | 275 1 | | | 275 1 | 96.3% | 81 - 99.9% | | | Negative Invalid | | | 1 | 1 0 | 96.0% | 92.8 - 98.1% | | | Site Total | 276 | 0 | 1 | 277* | | | *Three of the original 300 specimens were QNS for testing. Retrospective TPPA Negative and TPPA Positive Samples Collected from Pregnant Women: A total of 250 samples requested to be collected from pregnant women and requested to be syphilis antibody negative were purchased from a commercial vendor. Only 27 samples requested to be collected from pregnant women and requested to be RPR/TPPA positive {12} were available. These samples were purchased from a vendor. | | AtheNA Multi-Lyte Treponema pallidum IgG Plus Comparative Testing Result Summary Presented with 95% CI Banked purchased sera from pregnant women requested to be TPPA positive (27) RPR/TPPA non-reactive (250) | | | | | | | | --- | --- | --- | --- | --- | --- | --- | --- | | | | Predicate | | | | | | | | | Positive | Equivocal | Negative | Site Total | PPA NPA | 95% CI | | AtheNA Multi-Lyte Treponema pallidum IgG Plus | | | | | | | | | | Positive Equivocal | 26 | | 9 1 | 35 1 | 96.3% | 81 – 99.9% | | | Negative Invalid | 1 | | 240 | 241 0 | 96.0% | 92.8 – 98.1% | | | Site Total | 27 | 0 | 1 | 277 | | | CDC Syphilis Panel: A total of 164 categorized samples with various reactivity to syphilis were evaluated. Performance with CDC Characterized Serum Panel. | Clinical Diagnosis | AtheNA Multi-Lyte Treponema pallidum Results | | | | % Agreement with Clinical Diagnosis Presented with 95% CI | | --- | --- | --- | --- | --- | --- | | | Positive | Equivocal | Negative | Total | | | Primary Treated | 10 | 0 | 1 | 11 | 90.9% 58.7-99.8% | | Secondary Untreated | 40 | 0 | 3 | 43 | 93.0% 80.8-98.5% | | Secondary Treated | 39 | 0 | 0 | 39 | 100.0% 92.6-100% | | Latent Untreated | 6 | 0 | 5 | 11 | 54.5% 23.4-83.3% | | Latent Treated | 45 | 1 | 6 | 52 | 86.5% 74.2-94.4% | {13} 14 | Congenital | 2 | 0 | 1 | 3 | 66.7% | 9.4-99.2% | | --- | --- | --- | --- | --- | --- | --- | | Total | 141 | 1 | 17 | 159 | 88.7% | 93.2% | *Only 159 of the original 164 specimens were available for testing by AtheNA Multi-Lyte. Performance of AtheNA Multi-Lyte versus Panel of Sera obtained from the CDC: A total of 359 uncharacterized serum samples obtained from the CDC were evaluated. Performance of AtheNA Multi-Lyte Treponema pallidum vs. Panel of Samples from the CDC | | AtheNA Multi-Lyte Treponema pallidum IgG Plus Comparative Testing Result Summary Presented with 95% CI CDC Syphilis Panel Results | | | | | | | | --- | --- | --- | --- | --- | --- | --- | --- | | | | TPPA | | | | | | | | | Positive | Equivocal | Negative | Site Total | PPA NPA | 95% CI | | AtheNA Multi-Lyte Treponema pallidum IgG Plus | | | | | | | | | | Positive Equivocal | 246 | 1 | 3 | 250 | 96.5% | 93.4 - 98.2% | | | Negative Invalid | 9 | | 94 | 103 | 93.1% | 86.2 - 97.2% | | | Site Total | 255 | 1 | 100 | 356 | | | *Three of the original 359 specimens to be included in this study were indeterminate by the CDC TPPA method and were therefore excluded from the study. b. Clinical Specificity See section M.3.a c. Other clinical supportive data (when a. and b. are not applicable): Not applicable. 4. Clinical cut-off: Not applicable {14} 15 5. Expected Values/Reference Range: To determine expected values in the populations tested, internal and external investigators assessed the device’s performance with 500 masked samples prospectively collected from patients with a syphilis test ordered and 498 samples from pregnant women with a syphilis test ordered. The samples were requested to be random, unselected sera submitted for syphilis antibody testing. Additional studies were conducted in a population of 1000 unselected hospitalized patients. Site 1 was Zeus Scientific. Site 2 was a hospital laboratory located in the northeast and site 3 was a hospital laboratory located in the mid-Atlantic region. 19/500 samples tested positive from the target population of patients with a syphilis test ordered ranging in age from &lt;1 to &gt;70 years old. The observed prevalence in this population was 3.8%. From this positive group of 19 individuals, 68.4% were females ranging in age from 30 to &gt;70 (13/19, or 68.4%) and 31.6% were males ranging in age from 30 to &gt;70 (6/19 or 31.6%). In the target group of pregnant women ranging in age from 17 to 49, 4/498 samples tested positive. The positive samples were from women in the 30 to 49 age group. The observed prevalence in this group of 498 pregnant women is 0.8%. In the population of unselected hospitalized patients ranging in age from &lt;1 to &gt;70 years old, 50/1000 samples tested positive. The observed prevalence in this population was 5.0%. From the group of fifty positive specimens, 22/50 positive samples were from females ranging in age of 20 to &gt;70 (44%) and 28/50 positive samples were from males ranging in age from &lt;1 to &gt;70 (56%). No additional studies or testing was done on any of the discrepant specimens to determine the definitive serological status of these patient sera. N. Proposed Labeling: The labeling is sufficient and it satisfies the requirements of 21 CFR Part 809.10. O. Conclusion: The submitted information in this premarket notification is complete and supports a substantial equivalence decision.