LIAISON Lyme IgG, LIAISON Lyme IgG Control Set, LIAISON Lyme Total Antibody Plus

K202574 · DiaSorin, Inc. · LSR · Feb 18, 2021 · Microbiology

Device Facts

• Record ID: K202574
• Device Name: LIAISON Lyme IgG, LIAISON Lyme IgG Control Set, LIAISON Lyme Total Antibody Plus
• Applicant: DiaSorin, Inc.
• Product Code: LSR · Microbiology
• Decision Date: Feb 18, 2021
• Decision: SESE
• Submission Type: Traditional
• Regulation: 21 CFR 866.3830
• Device Class: Class 2

Intended Use

The LIAISON® Lyme IgG assay uses chemiluminescent immunoassay (CLIA) technology for the qualitative detection of IgG antibodies to Borrelia burgdorferi in human serum and plasma specimens (K2-EDTA, Li-heparin). This assay is intended for use on samples from patients with signs and symptoms consistent with or patients suspected of having Lyme disease to assess the presence of IgG antibodies and exposure to Borrelia burgdorferi. In addition, the LIAISON® Lyme IgG assay may be used as a confirmatory test in the modified two-tier test (MTTT) in combination with the DiaSorin LIAISON® Lyme Total Antibody Plus assay. If used as a first stage test, positive or equivocal results with the LIAISON® Lyme IgG assay should be confirmed through additional testing with a Standard two-tier test (STT) methodology using an IgG Borrelia burgdorferi Western blot test following current guidelines. Positive supplemental results are supportive evidence of the presence of antibodies and exposure to Borrelia burgdorferi and may be used along with patient history, symptoms and other laboratory data to support a clinical diagnosis of Lyme disease. Negative results by the LIAISON® Lyme IgG assay should not be used to exclude Lyme disease. The test must be performed on the LIAISON® XL Analyzer. The DiaSorin LIAISON® Lyme IgG Control Set is intended for use as assayed quality control samples to monitor the performance of the LIAISON® Lyme IgG assay. The performance characteristics of LIAISON® Lyme IgG controls have not been established for any other assays or instrument platforms different from the LIAISON® XL.

Device Story

Indirect chemiluminescent immunoassay (CLIA) for qualitative detection of IgG antibodies to Borrelia burgdorferi; utilizes magnetic particles coated with recombinant Borrelia antigens (VIsE and C6 peptide). Patient serum/plasma samples incubated with solid phase; anti-Borrelia antibodies bind; unbound material washed; anti-human IgG mouse monoclonal antibody conjugated to isoluminol derivative added; secondary incubation; wash cycle; starter reagents added to induce flash chemiluminescence. Photomultiplier measures light signal (RLU) proportional to antibody concentration. Performed on LIAISON XL Analyzer in clinical laboratory settings by trained technicians. Output is index value; assists clinicians in diagnosing Lyme disease when combined with patient history and symptoms. Benefits include automated processing and potential for use in modified two-tier testing algorithms.

Clinical Evidence

Prospective cohort study (N=2,621) and retrospective CDC reference panel (N=280). Evaluated performance as first-tier (STTT) and second-tier (MTTT) test. STTT agreement: 89.0% PPA, 99.5% NPA. MTTT agreement: 96.9% PPA, 30.2% NPA. Analytical specificity evaluated in 300 healthy individuals (4.0% positivity).

Technological Characteristics

Indirect CLIA; magnetic particle solid phase coated with recombinant VIsE and C6 peptide antigens; isoluminol-antibody conjugate; flash chemiluminescence detection via photomultiplier. Automated on LIAISON XL Analyzer. Sample types: serum, SST, K2-EDTA, lithium heparin plasma. Calibration: two-point verification of stored 10-point master curve.

Indications for Use

Indicated for patients with signs, symptoms, or suspicion of Lyme disease to detect IgG antibodies to Borrelia burgdorferi. Used as a first-tier test in Standard Two-Tiered Testing (STTT) or as a second-tier test in Modified Two-Tiered Testing (MTTT) with LIAISON Lyme Total Antibody Plus. Not for excluding Lyme disease.

Regulatory Classification

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), the Treponema pallidum immobilization test (T.P.I.), and other treponemal tests used to identify antibodies to Treponema pallidum directly from infecting treponemal organisms in serum. The identification aids in the diagnosis of syphilis caused by bacteria belonging to the genus Treponema and provides epidemiological information on syphilis.

Predicate Devices

• ZEUS ELISA Borrelia burgdorferi IgG Test System (K895292)
• LIAISON Lyme Total Antibody Plus (K191398, K193051)

Submission Summary (Full Text)

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Image /page/0/Picture/0 description: The image contains the logo of the U.S. Food and Drug Administration (FDA). On the left, there is a symbol representing the Department of Health & Human Services. To the right of the symbol, there is the FDA logo in blue, followed by the words "U.S. FOOD & DRUG" in a larger font and "ADMINISTRATION" in a smaller font below it, also in blue.

February 18, 2021

DiaSorin Inc. Carol Depouw Principal Regulatory Affairs Specialist 1951 Northwestern Avenue Stillwater, Minnesota 55082

Re: K202574

Trade/Device Name: LIAISON Lyme IgG, LIAISON Lyme IgG Control Set, LIAISON Lyme Total Antibody Plus Regulation Number: 21 CFR 866.3830 Regulation Name: Treponema Pallidum Treponemal Test Reagents Regulatory Class: Class II Product Code: LSR Dated: September 2, 2020 Received: September 4, 2020

Dear Carol Depouw:

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. Although this letter refers to your product as a device, please be aware that some cleared products may instead be combination products. The 510(k) Premarket Notification Database located at https://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfpmn/pmn.cfm identifies combination product submissions. 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

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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) for devices or postmarketing safety reporting (21 CFR 4, Subpart B) for combination products (see https://www.fda.gov/combination-products/guidance-regulatory-information/postmarketing-safety-reportingcombination-products); good manufacturing practice requirements as set forth in the quality systems (OS) regulation (21 CFR Part 820) for devices or current good manufacturing practices (21 CFR 4, Subpart A) for combination products; 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 https://www.fda.gov/medical-device-safety/medical-device-reportingmdr-how-report-medical-device-problems.

For comprehensive regulatory information about mediation-emitting products, including information about labeling regulations, please see Device Advice (https://www.fda.gov/medicaldevices/device-advice-comprehensive-regulatory-assistance) and CDRH Learn (https://www.fda.gov/training-and-continuing-education/cdrh-learn). 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 (https://www.fda.gov/medical-device-advice-comprehensive-regulatoryassistance/contact-us-division-industry-and-consumer-education-dice) for more information or contact DICE by email ([e-mail]) or phone [phone] or 301-796-7100).

Sincerely,

Maria Ines Garcia, Ph.D. Branch Chief Division of Microbiology Devices OHT7: Office of In Vitro Diagnostics and Radiological Health Office of Product Evaluation and Quality Center for Devices and Radiological Health

Enclosure

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# Indications for Use

510(k) Number (if known) K202574

Device Name LIAISON® Lyme IgG assay: LIAISON® Lyme IgG Control Set

#### Indications for Use (Describe)

The LIAISON® Lyme IgG assay uses chemiluminescent immunoassay (CLIA) technology for the qualitative detection of IgG antibodies to Borrelia burgdorferi in human serum and plasma specimens (K2-EDTA, Li-heparin). This assay is intended for use on samples from patients with signs and symptoms consistent with or patients suspected of having Lyme disease to assess the presence of IgG antibodies and exposure to Borrelia burgdorferi. In addition, the LIAISON® Lyme Ig G assay may be used as a confirmatory test in the modified two-tier test (MTTT) in combination with the DiaSorin LIAISON® Lyme Total Antibody Plus assay.

If used as a first stage test, positive or equivocal results with the LIAISON® Lyme IgG assay should be confirmed through additional testing with a Standard two-tier test (STT) methodology using an IgG Borrelia burgdorferi Western blot test following current guidelines.

Positive supplemental results are supportive evidence of the presence of antibodies and exposure to Borrelia burgdorferi and may be used along with patient history, symptoms and other laboratory data to support a clinical diagnosis of Lyme disease.

Negative results by the LIAISON® Lyme IgG assay should not be used to exclude Lyme disease.

The test must be performed on the LIAISON® XL Analyzer.

The DiaSorin LIAISON® Lyme IgG Control Set is intended for use as assayed quality control samples to monitor the performance of the LIAISON® Lyme IgG assay. The performance characteristics of LIAISON® Lyme IgG controls have not been established for any other assays or instrument platforms different from the LIAISON® XL.

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

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## 5.0 510(k) SUMMARY

| SUBMITTED BY: | Carol A. DePouw<br>DiaSorin Inc.<br>1951 Northwestern Avenue<br>Stillwater, MN 55082-0285<br>Phone [phone]<br>Fax [phone]<br>Email [e-mail] |
|--------------------------------|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| DATE PREPARED: | September 01, 2020 |
| NAME OF DEVICE:<br>Trade Name: | LIAISON® Lyme IgG<br>LIAISON® Lyme IgG Control Set |
| Common Names/Descriptions: | Borrelia burgdorferi IgG assay and<br>Borrelia burgdorferi IgG controls |
| Classification Names: | Treponema pallidum; treponemal test reagents<br>Class II, 21 CFR: 866.3830; Microbiology |
| Product Code: | LSR and QCH |
| Predicate Device: | ZEUS ELISA B. burgdorferi IgG Test System<br>(K895292)/modified (K191398) |

#### INTENDED USE:

The LIAISON® Lyme IgG assay uses chemiluminescent immunoassay (CLIA) technology for the qualitative detection of IqG antibodies to Borrelia burqdorferi in human serum and plasma specimens (K2-EDTA, Li-heparin). This assay is intended for use on samples from patients with signs and symptoms consistent with or patients suspected of having Lyme disease to assess the presence of IgG antibodies and exposure to Borrelia burgdorferi. In addition, the LIAISON® Lyme IgG assay may be used as a confirmatory test in the modified two-tier test (MTT) in combination with the DiaSorin LIAISON® Lyme Total Antibody Plus assay.

If used as a first stage test, positive or equivocal results with the LIAISON® Lyme IgG assay should be confirmed through additional testing with a Standard two-tier test (STTT) methodology using an IgG Borrelia burgdorferi Western blot assay following current quidelines.

Positive supplemental results are supportive evidence of the presence of antibodies and exposure to Borrelia burgdorferi and may be used along with patient history, symptoms and other laboratory data to support a clinical diagnosis of Lyme disease.

Negative results by the LIAISON® Lyme IgG assay should not be used to exclude Lyme disease.

The test must be performed on the LIAISON® XL Analyzer.

The LIAISON® Lyme IgG Control Set is intended for use as assayed quality control samples to monitor the performance of the LIAISON® Lyme IgG assay. The performance characteristics of LIAISON® Lyme IgG controls have not been established for any other assays or instrument platforms different from the LIAISON® XL.

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## KIT DESCRIPTION:

The LIAISON® Lyme IgG assay is an indirect chemilyminescence immunoassay (CLIA) for the qualitative detection of IgG antibodies to Borrelia burgdorferi in human serum and plasma samples. All assay steps (with the exception of magnetic particle resuspension) and incubations are performed by the Analyzer. The principal components of the test are magnetic particles (solid phase) coated with recombinant Borrelia antigens and a conjugate reagent containing an anti-human IgG mouse monoclonal antibody linked to an isoluminol derivative (isoluminol-antibody coniugate). During the first incubation, anti-Borrelia burgdorferi antibodies present in calibrators, samples or controls bind to the solid phase. Unbound material is then removed with a wash cycle. During the second incubation, the antibody conjugate reacts with anti-Borrelia burgdorferi IgG antibodies that have bound to the solid phase. Excess antibody conjugate is then removed with a wash cycle. Subsequently, the starter reagents are added and a flash chemiluminescence reaction is induced. The light signal, and hence the amount of isoluminol-antibody conjugate, is measured by a photomultiplier as relative light units (RLU) and is indicative of the presence of Borrelia burgdorferi IgG antibodies present in calibrators, samples or controls.

| Table 1: Table of Similarities | |
|--------------------------------|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Characteristic | Candidate Device<br>LIAISON® Lyme IgG |
| Intended Use | Qualitative detection of IgG antibodies to Borrelia<br>burgdorferi in human serum and plasma<br>specimens. This assay is intended for use on<br>samples from patients with signs and symptoms<br>consistent with or patients suspected of having<br>Lyme disease to assess the presence of IgG<br>antibodies and exposure to Borrelia burgdorferi.<br><br>In addition, the LIAISON® Lyme IgG assay may be<br>used as a confirmatory test in the modified two-tier<br>test (MTTT) in combination with the DiaSorin<br>LIAISON® Lyme Total Antibody Plus assay.<br><br>If used as a first stage test, positive or equivocal<br>results with the LIAISON® Lyme IgG assay should<br>be confirmed through additional testing with a<br>Standard two-tier test (STTT) methodology using<br>an IgM Borrelia burgdorferi Western blot test<br>following current guidelines.<br><br>Positive supplemental results are supportive<br>evidence of the presence of antibodies and<br>exposure to Borrelia burgdorferi and may be used<br>along with patient history, symptoms and other<br>laboratory data to support a clinical diagnosis of<br>Lyme disease.<br><br>Negative results by the LIAISON® Lyme IgG assay<br>should not be used to exclude Lyme disease. |
| | Predicate Device<br>ZEUS ELISA<br>Borrelia burgdorferi IgG Test System –<br>K895292/K191398 |
| | Qualitative detection of IgG class antibody to<br>Borrelia burgdorferi in human serum.<br>The assay is intended for testing serum<br>samples from symptomatic patients or those<br>suspected of Lyme Disease.<br><br>Positive and equivocal test results with the<br>ZEUS ELISA Borrelia burgdorferi IgG Test<br>System for the presence of Borrelia burgdorferi<br>antibodies must be confirmed through additional<br>testing by one of the following approaches:<br>(1) Standard two-tier test methodology (STTT)<br>using IgG Western blot testing following current<br>guidelines; or -<br>(2) Modified two-tier test methodology using the<br>ZUES ELISA Borrelia VIsE1/pepC10 IgG/IgM<br>Test System.<br><br>Positive test results by either the STTT or MTTT<br>methodology are supportive evidence for the<br>presence of antibodies and exposure to Borrelia<br>burgdorferi, the cause of Lyme disease.<br><br>A diagnosis of Lyme disease should be made<br>based on the presence of Borrelia burgdorferi<br>antibodies history, symptoms and other<br>laboratory data. |
| Results | Qualitative |
| | Same |

# COMPARISON TO PREDICATE DEVICE

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| Characteristic | Candidate Device<br>LIAISON® Lyme IgG | Predicate Device<br>ZEUS ELISA<br><i>Borrelia burgdorferi</i> IgM Test System –<br>K895292/K191398 |
|----------------------------------------|------------------------------------------------------------------------------------------------------|----------------------------------------------------------------------------------------------------------------------|
| Measurand | IgG antibodies to <i>Borrelia burgdorferi</i> | Same |
| Intended<br>Population | Patients with signs and symptoms<br>consistent with <i>Borrelia</i> infection<br>(Lyme disease) | Same |
| Assay<br>Principle | Uses <i>Borrelia</i> antigens coated on a solid phase<br>to capture specific patient IgG antibodies. | Uses <i>B. burgdorferi</i> antigen on coated<br>solid phase (wells) to bind with<br>IgG antibodies in patient sample |
| Conjugate<br>antibody<br>specificities | Anti-human IgG | Same |
| Assay Output | Index | Same |

| Table 2: Table of Differences | | |
|-------------------------------|----------------------------------------------------------------------------------------------------------------------|-----------------------------------------------------------------------------------------------------------|
| Feature | Candidate Device<br>LIAISON® Lyme IgG | Predicate Device<br>ZEUS ELISA<br>Borrelia burgdorferi IgM Test System –<br>K895292/K191398 |
| Test Format | CLIA (indirect chemiluminescent<br>assay) | ELISA |
| Sample Type | Human serum, serum separator<br>tubes, K2-EDTA, lithium heparin plasma | Human serum |
| Reporter Molecule | Isoluminol derivative conjugated to<br>anti-human IgG | TMB (as a substrate for Horseradish<br>peroxidase conjugated to anti-human IgG). |
| Antigen | Recombinant antigens:<br>VIsE ( <i>B. burgdorferi</i> strain B31)<br>Peptide for C6 region of VIsE | Whole cell antigen from<br><i>B. burgdorferi</i> (B31 strain) |
| Assay Procedure | Automated<br>(on the LIAISON® XL Analyzer) | Manual |
| Calibration | Two-point verification (in triplicate) of<br>stored 10 point master curve | Single Cut-off Calibrator assayed in triplicate |
| Output Signal | Flash chemiluminescent response is<br>integrated over a 3 second reading<br>period to generate a relative light unit | Microtiter well O.D. (450 nm) is measured<br>after the enzyme reaction is halted by<br>1M H2SO4/0.7M HCl. |
| Measurement<br>System | Photomultiplier<br>(flash chemiluminescence reader) | Spectrophotometer<br>(EIA Microtiter plate reader) |

#### PERFORMANCE DATA: METHOD COMPARISON:

Two thousand six hundred twenty one (2621) human serum specimens were collected in 14 states which represented five (5) distinct U.S. geographical regions. Of the 2621 samples: 44.1% were male, 55.7% were female, 0.2% gender unknown, the ages ranged from 2 years to 103 years of age.

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Testing with the LIAISON® Lyme IgG assay on the LIAISON® XL was performed in three (3) laboratories (2 external and internally at DiaSorin).

| | Predicate Assay (ELISA IgG) | | | |
|------------------|-----------------------------|-----------|----------|-------|
| LIAISON Lyme IgG | Positive | Equivocal | Negative | Total |
| Positive | 156 | 4 | 86 | 246 |
| Equivocal | 6 | 0 | 24 | 30 |
| Negative | 119 | 16 | 2210 | 2345 |
| Total | 281 | 20 | 2320 | 2621 |

| | | | | Table 3: First Tier Percent Agreement with Predicate Device |
|--|--|--|--|-------------------------------------------------------------|
|--|--|--|--|-------------------------------------------------------------|

## Agreement Results

| Positive % Agreement* | 55.1% (166/301) | 95% CI: 49.5% - 60.7% |
|-------------------------------------------|-------------------|-----------------------|
| Negative % Agreement | 96.5% (2210/2320) | 95% CI: 94.3% - 96.1% |
| *Includes Positive and Equivocal combined | | |

# Standard Two-Tier Testing Methodology:

Western blot testing was performed on the samples that were positive or equivocal by the test device and the predicate following the current quidelines for Standard Two-Tier testing methodology. The following results were obtained:

| Table 4: Standard Two-Tier Western Blot | | |
|-----------------------------------------|--|--|
| | | |

| Test System | Tier 1 + or Eqv | WB IgG + | WB IgG - |
|-------------------------------------|-----------------|----------|----------|
| Predicate assay | 301 | 109 | 192 |
| LIAISON® Lyme IgG | 276 | 109 | 167 |
| Predicate assay + LIAISON® Lyme IgG | 166 | 97 | 69 |

## Agreement Results:

| 2nd Tier PPA | 89.0% (97/109) | 95%CI: 81.7% - 93.6% |
|--------------|-------------------|----------------------|
| 2nd Tier NPA | 99.5% (2500/2512) | 95%CI: 99.2% - 99.7% |

## Modified Two Tier Testing Methodology – Prospective Population

All 2621 prospective (all comer) specimens were tested with the first-tier assay, LIAISON® Lyme Total Antibody Plus. There were 202 positive and 23 equivocal results. In the STTT protocol, the specimens that are positive or equivocal (n=225) are then tested with a B. burgdorferi (IgG) Western Blot.

Using the MTTT algorithm, the positive/equivocal specimens (n=225) were tested on the LIAISON® Lyme IgG assay. The second-tier LIAISON® Lyme IgG equivocal and positive results were considered positive. The equivocal and positive results were added together, and the results compared with the STTT positive results. The results obtained are shown in Table 5.

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| | WB-STTT (IgG) | | | |
|----------------|---------------|----|-------|-----|
| | + | - | Total | |
| XL -MTTT (IgG) | + | 93 | 90 | 183 |
| | - | 3 | 39 | 42 |
| | Total | 96 | 129 | 225 |

## Table 5: MTTT | IAISON | vme lgG compared to STTT WB lgG

Agreement Results

| PPA: | 96.9% (93/96) | 95% CI: 91.2% - 98.9% |
|------|----------------|-----------------------|
| NPA: | 30.2% (39/129) | 95% CI: 23.0% - 38.6% |

# Characterized Lyme Panel:

Two hundred eighty samples of various reactivity were acquired from the CDC and evaluated internally at the manufacture's site. The results of the testing are presented here as a means of conveying further information on the performance of the LIAISON® Lyme IgG assay with a characterized serum panel. This does not imply an endorsement of the assay by the CDC.

| CDC Reference Classification | | | | | | | | |
|------------------------------|-----|------------------|-----|-----|---------------|-----|-----|---------------------------------|
| Sample Category | N | LIAISON Lyme IgG | | | Predicate IgG | | | |
| | | Pos | Neg | Eqv | Pos | Neg | Eqv | PPA<br>95% Wilson CI |
| Acute | 39 | 28 | 10 | 1 | 23 | 15 | 1 | 74.4% (29/39)<br>58.9% - 85.4% |
| Convalescent | 31 | 29 | 2 | 0 | 22 | 6 | 3 | 93.5% (29/31)<br>79.3% - 98.2% |
| Late | 20 | 20 | 0 | 0 | 20 | 0 | 0 | 100% (20/20)<br>83.9% - 100% |
| Look-alike Diseases | 90 | 9 | 81 | 0 | 9 | 77 | 4 | 90% (81/90)<br>82.1% - 94.6% |
| Healthy controls | 100 | 3 | 97 | 0 | 1 | 98 | 1 | 97.0% (97/100)<br>91.5% - 99.0% |

## Table 6: Testing of CDC Lyme Reference Sera

## Modified Two Tier Testing Methodology-Retrospective population

The 280 retrospective samples from the CDC were first tested with the LIAISON® Lyme Total Antibody Plus. One (1) sample, belonging to endemic negative control group, did not have sufficient volume for testing; therefore 279 retrospective samples were evaluated. The LIAISON® Lyme Total Antibody Plus assay vielded 82 positive and equivocal samples. The 82 positive and equivocal samples were then tested by the IgG Western Blot (STTT) or the LIAISON® Lyme IgG assay (MTTT).

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| | Stage I (n=60) | | Stage II (n=10) | | Stage III (n=20) | | Healthy Controls (n=99) | | Disease Controls (n=90) | |
|--------------------|-----------------|-----------------|-----------------|-----------------|------------------|-----------------|-------------------------|-----------------|-------------------------|-----------------|
| | STTT-<br>WB IgG | MTTT-<br>XL IgG | STTT-<br>WB IgG | MTTT-<br>XL IgG | STTT-<br>WB IgG | MTTT-<br>XL IgG | STTT-<br>WB IgG | MTTT-<br>XL IgG | STTT-<br>WB IgG | MTTT-<br>XL IgG |
| Positive | 18 | 48 | 5 | 9 | 20 | 20 | 0 | 0 | 0 | 2 |
| Negative | 42 | 12 | 5 | 1 | 0 | 0 | 99 | 99 | 90 | 88 |
| Sensitivity or PPA | 30% | 80% | 50% | 90% | 100% | 100% | N/A | N/A | N/A | N/A |
| Specificity or NPA | N/A | N/A | N/A | N/A | N/A | N/A | 100% | 100% | 100% | 97.8% |

The results of MTTT-IqG compared to WB-STTT (IgG) are in the following table.

# PRECISION STUDY

A 12 day precision/repeatability was conducted at DiaSorin Inc. on two (2) lots of the LIAISON® Lyme IgG assay. Six (6) serum samples and two (2) lots of LIAISON® Lyme IgG Controls were tested for 12 days, 2 runs/day, and 2 reps per run by multiple technicians for a total of 48 replicates per lot spanning 2 calibration cycles. One (1) lot is presented below. The CLSI Document EP5-A3 was consulted in the preparation of the testing protocol.

| Sample ID | N | Mean | Within Run | | Between Run | | Between Day | | TOTAL<br>(Within-lot) | |
|-------------------------|----|------|------------|------|-------------|------|-------------|------|-----------------------|------|
| | | | SD | %CV | SD | %CV | SD | %CV | SD | %CV |
| Negative Control Lot V1 | 48 | 0.46 | 0.02 | 4.2% | 0.02 | 4.7% | 0.00 | 0.0% | 0.03 | 5.9% |
| Positive Control Lot V1 | 48 | 2.22 | 0.10 | 4.4% | 0.11 | 4.8% | 0.03 | 1.5% | 0.15 | 6.7% |
| Negative Control LotV2 | 48 | 0.09 | 0.00 | 4.1% | 0.00 | 3.5% | 0.00 | 4.7% | 0.01 | 7.2% |
| Positive Control Lot V2 | 48 | 2.39 | 0.07 | 2.9% | 0.21 | 8.6% | 0.00 | 0.0% | 0.17 | 7.2% |
| LG-QC1 | 48 | 0.77 | 0.03 | 3.9% | 0.04 | 5.4% | 0.00 | 0.0% | 0.05 | 6.1% |
| LG-QC3 | 48 | 1.37 | 0.04 | 2.7% | 0.05 | 3.8% | 0.04 | 2.7% | 0.07 | 5.4% |
| LG-QC4 | 48 | 4.89 | 0.11 | 2.3% | 0.11 | 2.3% | 0.11 | 2.2% | 0.19 | 3.9% |
| LG-QC5 | 48 | 0.82 | 0.05 | 6.4% | 0.04 | 5.2% | 0.04 | 5.4% | 0.08 | 9.8% |
| LG-QC6 | 48 | 1.47 | 0.05 | 3.3% | 0.06 | 4.2% | 0.00 | 0.0% | 0.08 | 5.2% |
| LG-QC7 | 48 | 0.48 | 0.02 | 3.3% | 0.01 | 1.7% | 0.01 | 2.3% | 0.02 | 4.4% |

# REPRODUCIBILITY STUDY

A five (5) day precision/reproducibility study was performed internally at DiaSorin Inc. and at two (2) external U.S. laboratories with one (1) lot of the LIAISON® Lyme IgG assay.

The study was performed for 5 days, 2 runs/day, and 3 replicates/run. Each day, two operators, at each testing site performed the testing for a total of 30 replicates at each site. The combined results from the 3 sites are provided. CLSI document EP15-A3 was consulted in the preparation of the testing protocol.

| Sample<br>ID | n | mean | Within Run | | Between Day | | Between Run | | Between Site | | TOTAL | |
|------------------|----|-------|------------|------|-------------|------|-------------|------|--------------|------|-------|------|
| | | | SD | %CV | SD | %CV | SD | %CV | SD | %CV | SD | %CV |
| Negative Control | 90 | 0.128 | 0.006 | 4.3% | 0.004 | 3.3% | 0.00 | 0.0% | 0.006 | 4.8% | 0.009 | 7.3% |
| Positive Control | 90 | 2.25 | 0.064 | 2.9% | 0.048 | 2.1% | 0.037 | 1.7% | 0.113 | 5.0% | 0.144 | 6.4% |
| LG-QC1 | 90 | 0.820 | 0.018 | 2.2% | 0.032 | 4.0% | 0.016 | 2.0% | 0.043 | 5.3% | 0.060 | 7.3% |
| LG-QC3 | 90 | 1.40 | 0.039 | 2.8% | 0.035 | 2.5% | 0.041 | 2.9% | 0.039 | 2.8% | 0.077 | 5.5% |
| LG-QC4 | 90 | 5.05 | 0.113 | 2.2% | 0.142 | 2.8% | 0.068 | 1.3% | 0.203 | 4.0% | 0.280 | 5.5% |
| LG-QC5 | 90 | 0.735 | 0.021 | 2.8% | 0.030 | 4.1% | 0.017 | 2.2% | 0.034 | 4.6% | 0.053 | 7.2% |
| LG-QC6 | 90 | 1.63 | 0.048 | 3.0% | 0.029 | 1.8% | 0.035 | 2.1% | 0.065 | 4.0% | 0.092 | 5.7% |
| LG-QC7 | 90 | 0.542 | 0.013 | 2.5% | 0.020 | 3.7% | 0.006 | 1.1% | 0.033 | 6.2% | 0.041 | 7.7% |

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

# CROSS-REACTIVITY STUDY

The cross-reactivity study was designed to evaluate 222 specimens from twenty-two (22) disease states either known to contain potentially cross reactive antibodies to B. burgdorferi or from patients with diagnoses that can exhibit signs and symptoms similar to Lyme disease and cause false positive results.

| Organism/Disease State | Samples<br>Tested<br>(n) | LIAISON® Lyme IgG<br>Pos or Eqv |
|------------------------------------|--------------------------|---------------------------------|
| Tick Borne Diseases* | | |
| Babesiosis IgG | 10 | 5 |
| Autoimmune Disorders | | |
| Anti-Nuclear Antibodies (ANA) | 10 | 0 |
| Multiple Sclerosis | 10 | 0 |
| Viral Diseases | | |
| Cytomegalovirus (CMV) IgG | 10 | 0 |
| Epstein-Barr Virus (EBV) IgG | 10 | 0 |
| Epstein-Barr Virus (EBV) EBNA IgG | 10 | 1 |
| Epstein-Barr Virus (VCA) IgG | 10 | 0 |
| Herpes Simplex Virus (HSV) IgG | 10 | 0 |
| Human Immunodeficiency Virus (HIV) | 10 | 0 |
| Influenza Virus | 10 | 0 |
| Parvovirus IgG | 10 | 0 |
| Varicella Zoster Virus (VZV) IgG | 10 | 0 |
| Bacterial Diseases | | |
| H. pylori | 10 | 1 |
| Syphilis | 10 | 0 |
| Leptospirosis | 2 | 1 |
| Rheumatic Diseases | | |
| Fibromyalgia | 10 | 0 |
| Rheumatoid Arthritis | 10 | 0 |
| Rheumatoid Factor | 10 | 0 |
| Systemic Lupus Erythematosus (SLE) | 10 | 0 |
| Sjogrens Syndrome | 10 | 1 |
| Additional Markers | | |
| Chronic Fatigue Syndrome | 10 | 1 |
| Human Anti-mouse Antibodies (HAMA) | 10 | 0 |
| E. coli | 10 | 0 |
| Total | 222 | 10 |

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

## INTERFERING SUBSTANCES

"Controlled studies of potentially interfering substances from endogenous interferents spiked into serum specimens containing B. burgdorferi IgG antibodies at levels near the cut-off showed that assay performance was not affected at the concentration for each substance listed below. The testing was based on CLSI-EP7-A3.

| Substances | Tested<br>Concentrations |
|---------------|--------------------------|
| Hemoglobin | 1000 mg/dL |
| Triglycerides | 1500 mg/dL |
| Bilirubin | 40 mg/dL |
| Total protein | 15 g/dL |
| Cholesterol | 400 mg/dL |
| Biotin | 3600 ng/mL |

#### MATRIX EQUIVALENCE STUDY:

Thirty-two (32) matched patient sets of serum, SST serum, K2-EDTA plasma and lithium heparin plasma samples were tested to determine if these sample types provide equivalent results. Sample regression analysis was done by Passing & Bablok regression. All sample types met acceptance criteria for use in the LIAISON® Lyme IgG assay. A summary of the results are shown in the following table.

| Comparison to Serum | | Bias | 95% Cl |
|------------------------|--------------|-------|---------------|
| Serum SST | Constant | -0.01 | -0.04 to 0.00 |
| | Proportional | 1.04 | 1.01 to 1.10 |
| EDTA Plasma | Constant | 0.00 | -0.01 to 0.03 |
| | Proportional | 0.96 | 0.93 to 1.01 |
| Lithium Heparin Plasma | Constant | 0.01 | -0.04 to 0.04 |
| | Proportional | 0.97 | 0.91 to 1.03 |

## CONCLUSION:

The material submitted in this premarket notification supports a substantial equivalence decision. The labelling satisfies the requirements of 21CFR 809.10.