The ASI Automated RPR (rapid plasma reagin) Test for Syphilis, for use on the ASI Evolution Automated Syphilis Analyzer, is a qualitative nontreponemal flocculation test for the detection of reagin antibodies in human serum and plasma as a screening test for serological evidence of syphilis. All reactive RPR test samples should be further tested with a treponemal test. The ASI Automated RPR Test for Syphilis for use on the ASI Evolution produces only a reactive or non-reactive result and does not report RPR titers for reactive samples. The ASI Automated RPR Test for Syphilis is for professional use only. The test is intended to be used for in vitro diagnostic testing. The ASI Evolution is intended to be used as a fully automated analyzer to objectively interpret the results of the ASI automated RPR test for syphilis. The ASI Evolution is designed to provide standardized test interpretation and to provide for storage, retrieval, and transmittal of the test results. It is intended to be acquired, possessed and used only by health care professionals. The ASI Evolution analyzer, in conjunction with the ASI Automated RPR Test is intended to be used for in vitro diagnostic testing. The ASI Automated RPR Test for Syphilis for use on the ASI Evolution produces only a reactive or non-reactive result and does not report RPR titers for reactive samples.

Device Description

The ASI Evolution is an integrated digital particle analyzer designed to objectively interpret certain slide agglutination tests manufactured by Arlington Scientific, Inc. (ASI). The ASI Evolution fully automates the sample and reagent handling steps of the test procedure. Qualitative tests are performed by laboratory professionals who use the ASI Evolution to provide standardized test interpretation using criteria that define reactive and nonreactive aqqlutination reactions. The ASI Evolution employs a camera that uses light reflectance to create a highly sensitive and high-resolution image of the agglutination immunoassay. This image is then analyzed by the proprietary software algorithm to interpret the agglutination pattern. The ASI Evolution further provides tools that enable the creation, storage, retrieval and transmittal of the test results. The ASI Automated RPR Test for Syphilis reagents include the following: CARBON ANTIGEN - 0.003% cardiolipin, 0.020-0.022% lecithin, 0.09% cholesterol, charcoal (activated) as visual enhancer, phosphate buffer, 0.1% sodium azide as preservative and stabilizers. CONTROLS (REACTIVE, WEAK REACTIVE, NONREACTIVE) - Human serum or defibrinated plasma (liquid), with 0.1% sodium azide as preservative. Reagents have two-year expiration dating from date of manufacture. The specific expiration date is located on the label on the vial.

AI/ML Overview

This document describes the regulatory approval for the ASI Automated RPR Test for Syphilis ASI Evolution. The product is a qualitative nontreponemal flocculation test for the detection of reagin antibodies in human serum and plasma for syphilis screening. It is designed to be used with the ASI Evolution Automated Syphilis Analyzer, which objectively interprets test results as reactive or non-reactive.

Here is a summary of the acceptance criteria and study information:

1. Table of Acceptance Criteria and Reported Device Performance

The acceptance criteria for the ASI Automated RPR Test for Syphilis ASI Evolution are based on demonstrating substantial equivalence to its predicate device, the ASI RPR Card Test for Syphilis on the ASiManager-AT. The key performance metrics are positive and negative agreement rates.

Performance Metric	Acceptance Criteria (Implicitly based on predicate equivalence)	Reported Device Performance
Prospective Serum Samples (N=1068)
Positive Agreement (Reactive)	High agreement with predicate device (ASiManager-AT)	99.13% (95% CI: 95.25% - 99.98%)
Negative Agreement (Nonreactive)	High agreement with predicate device (ASiManager-AT)	99.9% (95% CI: 99.42% - 100%)
Retrospective Serum Samples (N=10)
Positive Agreement (Reactive)	High agreement with predicate device (ASiManager-AT)	100% (95% CI: 59.04% - 100%)
Negative Agreement (Nonreactive)	High agreement with predicate device (ASiManager-AT)	100% (95% CI: 29.24% - 100%)
Retrospective Plasma Samples (N=1003)
Positive Agreement (Reactive)	High agreement with predicate device (ASiManager-AT)	100% (95% CI: 69.15% - 100%)
Negative Agreement (Nonreactive)	High agreement with predicate device (ASiManager-AT)	100% (95% CI: 99.63% - 100%)
Precision	100% repeatability for a variety of reactive and nonreactive samples	100% for all 10 tested samples
Reproducibility (3 sites, 7 samples)	100% agreement with expected results across sites and operators	100% for all 7 tested samples across all 3 sites
Reproducibility (3 sites, 448 samples)	100% agreement with expected results across sites	RPR Nonreactive: 100% (144/144) RPR Reactive: 100% (1200/1200)
Cross-Reactivity/Interfering Substances	No interference or cross-reactivity with specified conditions	No interference observed in all tested categories
Carry-Over	No contamination of nonreactive samples from adjacent reactive samples	All results as expected (no carry-over)
On-board Stability	100% agreement for samples and reagent for 8 hours on instrument	100% agreement for all samples and operators for 8 hours
Frozen vs. Refrigerated Testing	100% agreement between frozen and refrigerated samples	100% agreement for all samples

2. Sample Sizes Used for the Test Set and Data Provenance

Prospective Samples: 1,068 individual samples. Collected at two different Departments of Public Health Labs. Data provenance is prospective.
Retrospective Serum Samples (Agreement Study): 10 individual samples. Collected from various reference labs and serum and plasma vendors from across the United States. Data provenance is retrospective.
Retrospective Plasma Samples (Agreement Study): 1,003 individual samples. Collected from various reference labs and serum and plasma vendors from across the United States. Data provenance is retrospective.
ASI Evolution Characterized Specimen Testing (Clinical Diagnosis): Total of 1,068 + 1,013 = 2,081 samples (Total samples from prospective and retrospective studies)
- Prospective Random Samples:
  - Site a: 567 Serum samples (country not specified, likely US based on other descriptions)
  - Site b: 501 Serum samples (country not specified, likely US based on other descriptions)
- Retrospective Samples:
  - Site c: 17 Known infected samples (serum & plasma, from across the United States)
  - Site c: 996 Known uninfected samples (serum & plasma, from across the United States)
  - Total Retrospective samples: 1013
Performance with Samples from Pregnant Women: 250 non-reactive and 30 reactive samples from pregnant women. Serum samples. Country not specified, likely US.
Precision Testing: 10 samples (3 nonreactive, 7 reactive at various titers), each tested 192 times (total 1920 tests).
Reproducibility Testing (7 samples): 7 samples (2 nonreactive, 5 reactive at various titers), tested at 3 sites, each sample tested 180 times across various conditions (total 1260 tests).
Reproducibility Testing (448 samples): 448 retrospective serum samples (48 nonreactive, 400 reactive), tested at 3 sites (total 1344 tests). Samples collected from various reference labs and serum and plasma vendors from across the United States.
Cross Reactivity/Interfering Substances: 368 samples across various categories (e.g., ANA (+), HIV (+), pregnant women, etc.).
Carry-Over: 48 aliquots per run (24 reactive, 24 nonreactive). Run over 5 days.
On-board Stability Testing: 4 samples (1 nonreactive, 3 reactive at various titers), each run in triplicate morning and afternoon for 5 days by 2 operators (total 120 tests).
Frozen vs. Refrigerated Testing: Total of 55 samples (20 non-reactive, 35 reactive at various titers) tested as both frozen and refrigerated.

3. Number of Experts Used to Establish the Ground Truth for the Test Set and Qualifications

The document does not explicitly state the "number of experts" or their "qualifications" for establishing the ground truth for the test set in the comparative studies.

For the comparative studies against the ASiManager-AT, the predicate device's results (interpreted by "trained laboratory professionals") served as the reference. These professionals are implicitly qualified by their training to interpret RPR tests.
For the "ASI Evolution Characterized Specimen Testing," the "Expected results are based on known clinical diagnosis." This implies that clinical experts (e.g., physicians, specialists) established the diagnosis, which then served as a proxy for ground truth.

4. Adjudication Method for the Test Set

The document does not explicitly describe an adjudication method like 2+1 or 3+1. For the comparative studies, the results of the ASiManager-AT, interpreted by trained laboratory professionals, were considered the comparator. In cases of discrepancies or challenges, no specific adjudication process is detailed for these studies. For the Characterized Specimen Testing, the "known clinical diagnosis" itself serves as the ground truth, implying no further adjudication on the test results against the diagnosis.

5. If a Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study was done

No, a traditional MRMC comparative effectiveness study, where multiple human readers interpret cases with and without AI assistance to measure improvement in human performance, was not performed. The study focused on the standalone performance of the ASI Evolution device compared to the predicate device (ASiManager-AT) and known clinical diagnoses. When human operators were involved (e.g., for ASiManager-AT interpretation), they were trained professionals, but the study did not measure their improvement with AI assistance.

6. If a Standalone (i.e., algorithm only without human-in-the-loop performance) was done

Yes, the studies primarily assessed the standalone performance of the ASI Evolution. The device is described as a "fully automated analyzer to objectively interpret the results" of the RPR test. The comparison against the ASiManager-AT (which involves human interpretation of results) and known clinical diagnoses evaluates the ASI Evolution's direct output.

7. The Type of Ground Truth Used

The ground truth used varied:

Comparative Studies: The results obtained from the predicate device, the ASI RPR Card Test for Syphilis on the ASiManager-AT, interpreted by trained laboratory professionals, served as the comparator/reference.
Characterized Specimen Testing: "Known clinical diagnosis" was used as the ground truth. This implies diagnoses established by medical professionals through various clinical and laboratory indicators.
Precision, Reproducibility, Cross-Reactivity, Carry-Over, On-board Stability, Frozen vs. Refrigerated Testing: Expected results for controls and characterized samples (e.g., "RPR nonreactive," "RPR reactive 1:1 titered samples") were used as the ground truth. These expected results are typically established during the characterization and manufacturing of the control materials themselves.

8. The Sample Size for the Training Set

The document does not explicitly state the sample size used for the training set of the ASI Evolution's algorithm. It mentions that the "same proprietary interpretive algorithm used in the predicate device (ASiManager-AT) is used in the ASI Evolution." This suggests that the algorithm was developed and potentially trained using data prior to this submission, possibly with data collected for the ASiManager-AT validation. However, details of that training are not provided here.

9. How the Ground Truth for the Training Set Was Established

As the document does not specify a training set for the ASI Evolution's algorithm, it also does not detail how the ground truth for such a set was established. Given the claim that the algorithm is the "same proprietary interpretive algorithm" as in the predicate device, it is likely that the ground truth for any initial algorithm development would have been established through expert human interpretation of RPR flocculation patterns over a large set of samples, potentially correlated with confirmatory treponemal tests or clinical diagnoses.

Summary

{0}------------------------------------------------

Image /page/0/Picture/0 description: The image contains the logo of the U.S. Food and Drug Administration (FDA). On the left is the Department of Health & Human Services logo. To the right of that is the FDA logo, with the letters "FDA" in a blue square, followed by the words "U.S. FOOD & DRUG" in blue, and then the word "ADMINISTRATION" in a smaller font below.

June 14, 2018

Arlington Scientific, Inc. (ASI) David Binks COO/VP Regulatory 1840 North Technology Dr. Springville, Utah 84663

Re: K173376

Trade/Device Name: ASI Automated RPR Test for Syphilis ASI Evolution Regulation Number: 21 CFR 866.3820 Regulation Name: Treponema pallidum nontreponemal test reagents Regulatory Class: Class II Product Code: GMO Dated: June 7, 2018 Received: June 8, 2018

Dear David Binks:

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 Part 801 and Part 809); medical device 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

{1}------------------------------------------------

Part 820); and if applicable, the electronic product radiation control provisions (Sections 531-542 of the Act); 21 CFR 1000-1050.

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 comprehensive regulatory information about medical devices and radiation-emitting products, including information about labeling regulations, please see Device Advice (https://www.fda.gov/MedicalDevices/DeviceRegulationandGuidance/) and CDRH Learn (http://www.fda.gov/Training/CDRHLearn). Additionally, you may contact the Division of Industry and Consumer Education (DICE) to ask a question about a specific regulatory topic. See the DICE website (http://www.fda.gov/DICE) for more information or contact DICE by email (DICE@fda.hhs.gov) or phone (1-800-638-2041 or 301-796-7100).

Sincerely.

Steven R. Gitterman -S for

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

Enclosure

{2}------------------------------------------------

Indications for Use

510(k) Number (if known)

K173376

Device Name

ASI Automated RPR Test for Syphilis ASI Evolution

Indications for Use (Describe)

The ASI Automated RPR Test for Syphilis is for professional use only. The test is intended to be used for in vitro diagnostic testing.

The ASI Evolution is intended to be used as a fully automated analyzer to objectively interpret the results of the ASI automated RPR test for syphilis. The ASI Evolution is designed to provide standardized test interpretation and to provide for storage, retrieval, and transmittal of the test results. It is intended to be acquired, possessed and used only by health care professionals. The ASI Evolution analyzer, in conjunction with the ASI Automated RPR Test is intended to be used for in vitro diagnostic testing. The ASI Automated RPR Test for Syphilis for use on the ASI Evolution produces only a reactive or non-reactive result and does not report RPR titers for reactive samples.

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.

This section applies only to requirements of the Paperwork Reduction Act of 1995.

DO NOT SEND YOUR COMPLETED FORM TO THE PRA STAFF EMAIL ADDRESS BELOW.

The burden time for this collection of information is estimated to average 79 hours per response, including the time to review instructions, search existing data sources, gather and maintain the data needed and complete and review the collection of information. Send comments regarding this burden estimate or any other aspect of this information collection, including suggestions for reducing this burden, to:

Department of Health and Human Services Food and Drug Administration Office of Chief Information Officer Paperwork Reduction Act (PRA) Staff PRAStaff@fda.hhs.gov

"An agency may not conduct or sponsor, and a person is not required to respond to, a collection of information unless it displays a currently valid OMB number."

{3}------------------------------------------------

Arlington Scientific, Inc.

5.0 510(k) Summary

5.1 Preparation Date: 04/23/2018

Submitted By

David Binks MT (ASCP), MBA COO/Vice President Regulatory Arlington Scientific, Inc. 1840 North Technology Dr. Springville, UT 84663 Phone 801-489-8911 / Fax 801-489-5552

5.2 Trade Name - ASI Automated RPR Test for Syphilis ASI Evolution
Regulation section: (21 CFR 866.3820) Treponema pallidum test reagents

Classification: Class II

Product Code: GMQ

Panel: Microbiology

5.3 Predicate Device(s) – ASI RPR Card Test for Syphilis on the ASiManager-AT (K851504)

Device Similarities and Differences
Item	ASI Evolution	ASIManager-AT
Intended Use	The ASI Automated RPR (rapid plasmareagin) Test for Syphilis, for use on the ASIEvolution Automated Syphilis Analyzer, isa qualitative nontreponemal flocculationtest for the detection of reagin antibodies inhuman serum and plasma as a screeningtest for serological evidence of syphilis. Allreactive RPR test samples should befurther tested with a treponemal test. TheASI Automated RPR Test for Syphilis foruse on the ASI Evolution produces only areactive or non-reactive result and doesnot report RPR titers for reactive samples.The ASI Automated RPR Test for Syphilisis for professional use only. The test isintended to be used for in vitro diagnostictesting.The ASI Evolution is intended to be usedas a fully automated analyzer to objectively	The ASI RPR (rapid plasmareagin)Card Test for Syphilis is aqualitative and semiquantitativenontreponemal flocculation test forthe detection of reagin antibodiesin human serum and plasma as ascreening test for serologicalevidence of syphilis. This test isalso intended for use in screeningblood donors and cadaveric (non-heart beating) donor specimens fortissue donation when the test isread and interpreted with theASIManager-AT. The ASI RPRCard Test for Syphilis is forprofessional use only.
	interpret the results of the ASI automatedRPR test for syphilis. The ASI Evolution isdesigned to provide standardized testinterpretation and to provide for storage,retrieval, and transmittal of the test results.It is intended to be acquired, possessedand used only by health careprofessionals. The ASI Evolution analyzer,in conjunction with the ASI AutomatedRPR Test is intended to be used for in vitrodiagnostic testing. The ASI AutomatedRPR Test for Syphilis for use on the ASIEvolution produces only a reactive or non-reactive result and does not report RPRtiters for reactive samples.	The ASiManager-AT is intended tobe used as an integrated digitalparticle analyzer to objectivelyinterpret the results of the ASIRPR Card Test for Syphilis. TheASiManager-AT is designed toprovide standardized testinterpretation and to provide forstorage, retrieval, and transmittalof the test results. It is intended tobe acquired, possessed and usedonly by health care professionals.The ASiManager-AT is intended tobe used for in vitro diagnostics,blood donor and cadaveric (non-heart beating) donor screening.
TechnologyInstruments	The ASI Evolution is an integrated digitalparticle analyzer designed to objectivelyinterpret certain agglutination testsmanufactured by Arlington Scientific, Inc.(ASI). The ASI Evolution fully automatesthe sample and reagent handling steps ofthe test procedure. Laboratoryprofessionals use the ASI Evolution toprovide standardized test interpretationusing criteria that define reactive andnonreactive agglutination reactions. Thesame proprietary interpretive algorithmused in the predicate device (ASiManager-AT) is used in the ASI Evolution.	The ASiManager-AT is anintegrated digital particle analyzerdesigned to objectively interpretcertain slide agglutination testsmanufactured by ArlingtonScientific, Inc. (ASI). Qualitativeand semiquantitative tests areperformed by laboratoryprofessionals who use theASiManager-AT to providestandardized test interpretationusing criteria that define reactiveand nonreactive agglutinationreactions.
	The ASI Evolution employs a camera tocreate a highly sensitive and high-resolution image of the agglutinationimmunoassay. This image is thenanalyzed by the proprietary softwarealgorithm to interpret the agglutinationpattern.The ASI Evolution further provides toolsthat enable the creation, storage, retrievaland transmittal of the test results.	The ASiManager-AT employs acamera to create a highly sensitiveand high-resolution image of theagglutination immunoassay. Thisimage is then analyzed by theproprietary software algorithm tointerpret the agglutination pattern.The ASiManager-AT furtherprovides tools that enable thecreation, storage, retrieval andtransmittal of the test results.
TechnologyReagents	Flocculation Test	Same
Antigen	ASI RPR Carbon Antigen	Same
Reported Results	Reactive, Nonreactive	Same
Interpretation	Automated	Same
Sample Processing	Automated	Manual
Reagent Volumeused per Sample	110 $μ$ l	105 $μ$ l
Sample Type	Serum or Plasma	Same
Controls	Reactive, Weak Reactive, Nonreactive	Same
Test card	48 well plastic test plate	30, 15, or 10 well plastic coatedtest card
Target Population	Used for in vitro diagnostic testing and	Used for in vitro diagnosticstesting, blood donor and cadaveric

Device Similarities and Differences

{4}------------------------------------------------

Arlington Scientific, Inc.

{5}------------------------------------------------

Arlington Scientific, Inc.

		(non-heart beating) donor screening.
--	--	--------------------------------------

5.4 Device Description – The ASI Evolution is an integrated digital particle analyzer designed to objectively interpret certain slide agglutination tests manufactured by Arlington Scientific, Inc. (ASI). The ASI Evolution fully automates the sample and reagent handling steps of the test procedure. Qualitative tests are performed by laboratory professionals who use the ASI Evolution to provide standardized test interpretation using criteria that define reactive and nonreactive aqqlutination reactions.
The ASI Evolution employs a camera that uses light reflectance to create a highly sensitive and high-resolution image of the agglutination immunoassay. This image is then analyzed by the proprietary software algorithm to interpret the agglutination pattern.

The ASI Evolution further provides tools that enable the creation, storage, retrieval and transmittal of the test results.

The ASI Automated RPR Test for Syphilis reagents include the following:

CARBON ANTIGEN - 0.003% cardiolipin, 0.020-0.022% lecithin, 0.09% cholesterol, charcoal (activated) as visual enhancer, phosphate buffer, 0.1% sodium azide as preservative and stabilizers.

CONTROLS (REACTIVE, WEAK REACTIVE, NONREACTIVE) - Human serum or defibrinated plasma (liquid), with 0.1% sodium azide as preservative.

Reagents have two-year expiration dating from date of manufacture. The specific expiration date is located on the label on the vial.

Intended Use – The ASI Evolution is intended to be used as a fully automated analyzer to objectively interpret the results of the ASI Automated RPR Test for Syphilis. The ASI Evolution is designed to provide standardized test interpretation and to provide for storage, retrieval, and transmittal of the test results. It is intended to be acquired, possessed and used only by health care professionals. The ASI Evolution analyzer, in conjunction with the ASI Automated RPR Test for Syphilis is intended to be used for in vitro diagnostic testing. The ASI Automated RPR Test for Syphilis for use on the ASI Evolution produces only a reactive or non-reactive result and does not report RPR titers for reactive samples.

{6}------------------------------------------------

Arlington Scientific, Inc.

Test for Syphilis for use on the ASI Evolution produces only a reactive or nonreactive result and does not report RPR titers for reactive samples. The ASI Automated RPR Test for Syphilis is for professional use only. The test is intended to be used for in vitro diagnostic testing.

5.5 Clinical Data - A comparison of the digital interpretation of the results of testing samples with the ASI Automated RPR Test for Syphilis using the ASI Evolution and the ASiManager-AT by trained laboratory professionals was conducted to show substantial equivalence.
The ASI Evolution was evaluated for equivalence, in its pattern of reactivity, aqainst the ASiManager-AT. A total of 1,068 individual prospective samples, with identifiers removed. were collected at two different Departments of Public Health Labs and tested by the ASI Evolution in comparison with the ASiManager-AT at each of the facilities. Reactive, Weak Reactive and Nonreactive controls were run on each day of testing.

Prospective Serum Sample Testing - 1,068 Samples ASI RPR Card Test for Syphilis on the ASiManager-AT Result ASI Automated Reactive Nonreactive RPR Test for Syphilis on the 114 1 ASI Evolution Reactive Result Nonreactive 1 952

The following data are the results from two testing sites:

Using the data from the prospective performance results above, the positive agreement of the ASI Evolution can be calculated as:

114/(114 + 1) = 99.13% 95% Cl = 95.25% - 99.98%

Using the data from the prospective performance results above, the negative agreement of the ASI Evolution can be calculated as:

952/(952 + 1) = 99.9% 95% Cl = 99.42% - 100%

A total of 1,013 individual retrospective samples, with identifiers removed, were collected from various reference labs and serum and plasma vendors from across the United States and tested by the ASI Evolution in comparison with the ASiManager-AT

{7}------------------------------------------------

Arlington Scientific, Inc.

in-house. Reactive, Weak Reactive and Nonreactive controls were run on each day of testing.

{8}------------------------------------------------

Arlington Scientific, Inc.

Retrospective Serum Sample Testing – 10 Samples
ASI RPR Card Test for Syphilis on the ASiManager-AT Result
	Reactive	Nonreactive
ASI AutomatedRPR Test forSyphilis on theASI EvolutionResult	7	0
Nonreactive	0	3

Retrospective Serum Sample Testing – 10 Samples

Serum positive agreement is calculated as:

7/(7 + 0) = 100% 95% Cl = 59.04% - 100%

Serum negative agreement is calculated as:

3/(3 + 0) = 100% 95% Cl = 29.24% - 100%

Retrospective Plasma Sample Testing – 1,003 Samples
ASI AutomatedRPR Test forSyphilis on theASI EvolutionResult	ASI RPR Card Test for Syphilis on the ASiManager-ATResult
	Reactive	Nonreactive
Reactive	10	0
Nonreactive	0	993

Sodium Citrate positive agreement is calculated as:

10/(10 + 0) = 100% 95% Cl = 69.15% - 100%

Sodium Citrate negative agreement is calculated as:

993/(993 + 0) = 100% 95% Cl = 99.63% - 100%

{9}------------------------------------------------

Arlington Scientific, Inc.

	Prospective random samples			Retrospective samples
Site	Plasma	Serum	Total	Serum & Plasma		Total	Grand Total
				Known infected	Known Uninfected
a	0	567	567	0	0	0	567
b	0	501	501	0	0	0	501
c	0	0	0	17	996	1,013	1,013
total	0	1,068	1,068	17	996	1,013	2,081

Distribution of Samples

ASI Evolution Characterized Specimen Testing

Testing was conducted at: .
- o Arlington Scientific, Inc. Site C
Carbon antigen lot used Lot CA5K01RBA ●
Each sample was tested by an operator with experience in performing the ASI ● Automated RPR Test for Syphilis and operating the ASI Evolution.
ASI Evolution unit 5800-0102 (#1) used for testing .
Expected results are based on known clinical diagnosis ●
All samples were serum .

The results of the testing are contained in tables below:

ClinicalDiagnosis	No. Reactive	% Agreement	95% CI
Primary Treated	25	100%	86.28 – 100%
Primary Untreated	18	100%	81.47 – 100%
Secondary Treated	25	100%	86.28 – 100%
Secondary Untreated	25	100%	86.28 – 100%
Latent Treated	25	100%	86.28 – 100%
Latent Untreated	25	100%	86.28 – 100%

Performance with Samples from Pregnant Women:

Samples were collected from 250 pregnant women and tested on the ASI Evolution for reactivity. These samples were also tested using the ASiManager-AT. The age of these women ranged in age from 15 to 41 years old. (with a median age of 28 years old). All samples were nonreactive for syphilis when tested on the ASI Evolution. Samples were collected from 30 pregnant women that also had been diagnosed as having syphilis and were tested on the ASI Evolution for reactivity. The women ranged in age from 18 to 40 years old (with a median age of 28 years old). All samples were reactive for syphilis when tested on the ASI Evolution. All the samples were serum.

{10}------------------------------------------------

Arlington Scientific, Inc.

Pregnant Women Testing

ASI AutomatedRPR Test forSyphilis on theASI EvolutionResult	ASI RPR Card Test for Syphilis on the ASiManager-ATResult
	Reactive	Nonreactive
Reactive	30	0
Nonreactive	0	250

Conclusion:

The positive and negative percent agreement for the ASI Evolution and the ASiManager-AT demonstrate that they have a very similar performance.

ASI Evolution Performance Characteristics

Precision

The interpretation of 10 samples using the ASI Automated RPR Test for Syphilis and the ASI Evolution were evaluated for reactivity. The testing requirements were as follows:

1. All qualitative testing was conducted according to the procedure listed in the package insert.
1. Each qualitative sample was tested 192 times. The number of replicates were chosen since the instrument can hold 4 - 48 well plates for a total of 192 wells. The 192 samples will test each well position.

A total of 10 samples were evaluated to determine repeatability of reactivity. Of the 10 samples:

3 were nonreactive

4- RPR reactive 1:1 titered samples
1- RPR reactive 1:2 titered sample
1- RPR reactive 1:8 titered sample
1- RPR reactive 1:256 titered sample

Each of the 10 samples was repeated 192 times to evaluate the reactivity using the ASI Evolution. An aliquot of the same sample was dispensed into 192 tubes. All 192 tubes were placed into the ASI Evolution and the run was performed. In this manner all 192 wells were tested with the same sample to show well to well and plate to plate repeatability using four test plates.

{11}------------------------------------------------

Arlington Scientific, Inc.

Precision Testing
Sample	Result		% Agreement
	Sample ID	Titer
1	R7C21R	1:8	R	192/192	100%
2	N7D04	NR	NR	192/192	100%
3	11114B	1:1	R	192/192	100%
4	11114C	1:1	R	192/192	100%
5	11114F	1:1	R	192/192	100%
6	02287	NR	NR	192/192	100%
7	08296	1:256	R	192/192	100%
8	11114D	1:1	R	192/192	100%
9	W7E26R	1:2	R	192/192	100%
10	N7H03	NR	NR	192/192	100%

Precision Testing

The data above show that the ASI Evolution has a high degree of repeatability.

Reproducibility

Reproducibility testing was conducted at three sites. The testing consisted of:

. Testing seven (7) samples
- o 2 RPR nonreactive samples
- o 1 RPR reactive 1:2 titered samples
- o 1 RPR reactive 1:4 titered sample
- o 2 RPR reactive 1:8 titered sample
- o 1 RPR reactive 1:16 titered sample
Each sample was run in duplicate within the panel. .
Each sample was tested each day for five non-consecutive days by an operator . with experience in performing the ASI Automated RPR Test for Syphilis and operating the ASI Evolution.
Each sample was tested a second time on each of the days referenced above . separated by approximately 2 hours.
. Testing at each site was performed on the same ASI Evolution.

{12}------------------------------------------------

Arlington Scientific, Inc.

RPR		Site 1		Site 2		Site 3		Total
Sample	Sample#	N	ExpectedResult	95%ConfidenceInterval	%ExpectedResult	95%ConfidenceInterval	%ExpectedResult	95%ConfidenceInterval	%ExpectedResult	95%ConfidenceInterval
RPRnonreactive	02287	180	100%(60/60)	93.98 - 100	100%(60/60)	93.98 - 100	100%(60/60)	93.98 - 100	100%(180/180)	97.91 - 100
RPRnonreactive	N6K14	180	100%(60/60)	93.98 - 100	100%(60/60)	93.98 - 100	100%(60/60)	93.98 - 100	100%(180/180)	97.91 - 100
RPR reactive1:2	05225B	180	100%(60/60)	93.98 - 100	100%(60/60)	93.98 - 100	100%(60/60)	93.98 - 100	100%(180/180	97.91 - 100
RPR reactive1:4	07035	180	100%(60/60)	93.98 - 100	100%(60/60)	93.98 - 100	100%(60/60)	93.98 - 100	100%(180/180)	97.91 - 100
RPR reactive1:8	05225A	180	100%(60/60)	93.98 - 100	100%(60/60)	93.98 - 100	100%(60/60)	93.98 - 100	100%(180/180)	97.91 - 100
RPR reactive1:8	R7C21R	180	100%(60/60)	93.98 - 100	100%(60/60)	93.98 - 100	100%(60/60)	93.98 - 100	100%(180/180)	97.91 - 100
RPR reactive1:16	07117	180	100%(60/60)	93.98 - 100	100%(60/60)	93.98 - 100	100%(60/60)	93.98 - 100	100%(180/180	97.91 - 100

The data shows a very high degree of reproducibility.

Another reproducibility study was conducted at the three sites. All qualitative testing was conducted according to the procedure listed in the package insert. The testing consisted of:

. Testing 448 samples
- o 48 RPR nonreactive samples
- o 400 RPR reactive samples
The same samples were tested at all three sites described above for the clinical ● and the previous reproducibility studies.
. All samples were retrospective serum.
Samples, with identifiers removed, were collected from various reference labs ● and serum and plasma vendors from across the United States.
Testing at each site was performed on the same ASI Evolution. .
Results were as follows: ●

RPR		Site 1		Site 2		Site 3		Total
Sample	N	ExpectedResult	95%ConfidenceInterval	%ExpectedResult	95%ConfidenceInterval	%ExpectedResult	95%ConfidenceInterval	% ExpectedResult	95%ConfidenceInterval
RPRnonreactive	48	100%(48/48)	92.60 - 100	100%(48/48)	92.60 - 100	100%(48/48)	92.60 - 100	100%(144/144)	97.47 - 100
RPR reactive	400	100%(400/400)	99.08 - 100	100%(400/400)	99.08 - 100	100%(400/400)	99.08 - 100	100%(1200/1200)	99.69 - 100

{13}------------------------------------------------

Arlington Scientific, Inc.

Cross Reactivity/Interfering Substances

A study was conducted to evaluate potential interference or cross reactivity from different disease conditions. Results are listed below:

Specimen Category	Number of Samples	Expected Result	# of samples with expected result/# of samples tests
ANA (+) Syphilis (-)	3	NR	3/3
ASO (+) Syphilis (-)	2	NR	2/2
CRP (+) Syphilis (-)	2	NR	2/2
Infectious Mono (+) Syphilis (-)	3	NR	3/3
RF (+) Syphilis (-)	12	NR	12/12
Rubella (+) Syphilis (-)	12	NR	12/12
Lyme's (+) Syphilis (-)	12	NR	12/12
HIV (+) Syphilis (-)	50	NR	50/50
HIV (+) Syphilis (+)	24	R	24/24
Pregnancy (+) Syphilis (-)	250	NR	250/250
Pregnancy (+) Syphilis (+)	30	R	30/30
Bilirubin 20 mg/dl	2	NR	2/2
Hemoglobin 10 mg/ml	2	NR	2/2
Triglycerides 1000mg/dl	2	NR	2/2

The positive infectious mono samples were heterophile positive. EBV testing was not conducted.

The study showed no interference.

Carry-Over

A study was conducted to evaluate if contamination of a nonreactive sample due to carryover from an adjacent reactive sample can occur.

Testing was conducted at: ●
- o Arlington Scientific, Inc.
Testing was conducted using two different samples: ●
- RPR reactive 1:64 tittered sample (high reactive) 06237 o
- o RPR nonreactive sample Lot 06127
The same samples were used for all testing.
The same lot of carbon antigen was used - Lot CA7D24R
Each test run was completed each day for five days by an operator with experience in performing the ASI Automated RPR Test for Syphilis and operating the ASI Evolution.
The test consisted of alternating 24 aliquots of the samples listed above in the sample rack and completing a run of 48 tests.

{14}------------------------------------------------

Arlington Scientific, Inc.

Testing was performed on the same ASI Evolution. ●

	Expected Results
ASI AutomatedRPR Test forSyphilis on theASI EvolutionResult
	Reactive	Nonreactive
Reactive	24	0
Nonreactive	0	24

Carry-Over Testing

This data demonstrates that all testing results were as expected and there was no evidence of contamination or carry-over.

ASI Evolution On-board Stability Testing

Testing was conducted at: ●
- o Arlington Scientific, Inc. Site C
Testing was conducted using four different samples: .
- RPR nonreactive sample Lot 12313 o
- RPR reactive 1:2 tittered sample (low reactive) LR0646530 O
- RPR reactive 1:16 tittered sample (moderate reactive) LB0482464 o
- RPR reactive 1:64 tittered sample (high reactive) OL0500976 o
The same samples were used for all testing. ●
The same lot of carbon antigen was used Lot CA5K01RBA ●
. Each sample was tested each morning for five days by an operator with experience in performing the ASI Automated RPR Test for Syphilis and operating the ASI Evolution.
. Each sample was tested each afternoon for five days by a different operator with experience in performing the ASI Automated RPR Test for Syphilis and operating the Evolution.
The carbon antigen reagent was left on the ASI Evolution between runs for a total elapsed time of 8 hours.
The carbon antigen was stored at 2-8° C between the afternoon test run and the next morning test run (overnight)
Each of the four samples was run in triplicate on the morning and afternoon . runs.
ASI Evolution unit 5800-0102 (#1) used for testing
All samples were serum. .

{15}------------------------------------------------

The results of the testing are contained in table below:

Sample	Expected	Results					%Agreement
		ASI Evolution
Operator 1		Day 1	Day 2	Day 3	Day 4	Day 5
12313	NR	3/3	3/3	3/3	3/3	3/3	100%
LR0646530	R	3/3	3/3	3/3	3/3	3/3	100%
LB0482464	R	3/3	3/3	3/3	3/3	3/3	100%
OL0500976	R	3/3	3/3	3/3	3/3	3/3	100%
Operator 2
12313	NR	3/3	3/3	3/3	3/3	3/3	100%
LR0646530	R	3/3	3/3	3/3	3/3	3/3	100%
LB0482464	R	3/3	3/3	3/3	3/3	3/3	100%
OL0500976	R	3/3	3/3	3/3	3/3	3/3	100%

On-board Stability Testing

Using this data the following evaluations can be made:

All testing results were as expected.

The carbon antigen reagent is stable on the instrument for up to 8 hours.

ASI Evolution Frozen vs Refrigerated Testing

Testing was conducted at: .
- o Arlington Scientific, Inc. Site C
Carbon antigen Lot used Lot CA5K01RBA .
Each sample was tested by an operator with experience in performing the ASI ● Automated RPR Test for Syphilis and operating the ASI Evolution.
ASI Evolution unit 5800-0102 (#1) used for testing. .
Each sample was split into two aliquots. One was stored at 2-8° C and the other ● one was frozen. The frozen specimens were allowed to remain frozen for 1 month and then were thawed.
All testing was done on the ASI Evolution. ●
. All specimens were allowed to come to room temperature before testing.

{16}------------------------------------------------

Sample Titer	N	Fresh Samples% Expected Result(# expectedresult/N)	Frozen Samples% Expected Result(# expectedresult/N)
Not Reactive	20	100% (20/20)	100% (20/20)
1:1	20	100% (20/20)	100% (20/20)
1:2	5	100% (5/5)	100% (5/5)
1:4	5	100% (5/5)	100% (5/5)
1:8	3	100% (3/3)	100% (3/3)
1:16	2	100% (2/2)	100% (2/2)

Frozen vs. Refrigerated Testing

All samples gave the expected results.

Regulation Number and Section

§ 866.3820

Treponema pallidum nontreponemal test reagents.(a)
Identification. Treponema pallidum nontreponemal test reagents are devices that consist of antigens derived from nontreponemal sources (sources not directly associated with treponemal organisms) and control sera (standardized sera with which test results are compared) used in serological tests to identify reagin, an antibody-like agent, which is produced from the reaction of treponema microorganisms with body tissues. The identification aids in the diagnosis of syphilis caused by microorganisms belonging to the genusTreponema and provides epidemiological information on syphilis.(b)
Classification. Class II (performance standards).