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

K202573 · DiaSorin, Inc. · LSR · Feb 18, 2021 · Microbiology

Device Facts

• Record ID: K202573
• Device Name: LIAISON Lyme IgM, LIAISON Lyme IgM 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 IgM assay uses chemiluminescent immunoassay (CLIA) technology for the qualitative detection of IgM antibodies to Borrelia burgdorferi in human serum and plasma samples (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 antibodies and exposure to Borrelia burgdorferi. In addition, the LIAISON® Lyme IgM 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 IgM assay should be confirmed through additional testing with a Standard two-tier test (STT) methodology using an IgM 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 IgM assay should not be used to exclude Lyme disease. The test must be performed on the LIAISON® XL Analyzer. The DiaSorin LIAISON® Lyme IgM Control Set is intended for use as assayed quality control samples to monitor the performance of the LIAISON® Lyme IgM assay. The performance characteristics of LIAISON® Lyme IgM controls have not been established for any other assays or instrument platforms different from the LIAISON® XL.

Device Story

LIAISON Lyme IgM is an automated indirect chemiluminescent immunoassay (CLIA) for qualitative detection of IgM antibodies to Borrelia burgdorferi in human serum/plasma. Device uses magnetic particles coated with recombinant Borrelia antigens (OspC, VlsE) and isoluminol-antibody conjugate. Performed on LIAISON XL Analyzer; instrument handles incubation, washing, and signal measurement (RLU). Healthcare providers use results as part of two-tiered testing algorithms (STTT or MTTT) to support clinical diagnosis of Lyme disease. Output is index value (positive, negative, equivocal). Benefits include automated, standardized testing compared to manual methods, aiding in diagnosis of early Lyme disease.

Clinical Evidence

Clinical performance evaluated using 2,621 prospective samples and 280 characterized CDC reference samples. In prospective testing, MTTT algorithm showed 93.0% PPA (95% CI: 86.3-96.6%) and 57.6% NPA (95% CI: 48.8-65.9%) compared to STTT. Precision/reproducibility studies showed total CVs ranging from 4.5% to 15.2%. Cross-reactivity and interference studies confirmed no significant impact from common endogenous substances or cross-reactive disease states.

Technological Characteristics

Indirect CLIA; magnetic particles coated with recombinant OspC and VlsE antigens; isoluminol-antibody conjugate; automated on LIAISON XL Analyzer; chemiluminescent signal detection; serum/plasma samples; 0.01-8.0 Index measuring range; calibration via RFID-tagged Reagent Integral; software-controlled processing.

Indications for Use

Indicated for symptomatic patients or those suspected of having Lyme disease to assess presence of IgM antibodies to Borrelia burgdorferi. Used as first-tier test in Standard Two-Tiered Test (STTT) or second-tier test in Modified Two-Tiered Test (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 IgM Test System (K900196/K191240)

Submission Summary (Full Text)

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Image /page/0/Picture/0 description: The image shows the logo of the U.S. Food and Drug Administration (FDA). On the left is the Department of Health & Human Services USA seal. To the right of the seal is the FDA logo in blue, with the words "U.S. FOOD & DRUG" stacked on top of the word "ADMINISTRATION".

February 18, 2021

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

Re: K202573

Trade/Device Name: LIAISON Lyme IgM, LIAISON Lyme IgM 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)

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

#### Indications for Use (Describe)

The LIAISON® Lyme IgM assay uses chemiluminescent immunoassay (CLIA) technology for the qualitative detection of IgM antibodies to Borrelia burgdorferi in human serum and plasma samples (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 antibodies and exposure to Borrelia burgdorferi. In addition, the LIALSON® Lyme IgM 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 IgM assay should be confirmed through additional testing with a Standard two-tier test (STT) methodology using an IgM 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 IgM assay should not be used to exclude Lyme disease.

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

The DiaSorin LIAISON® Lyme IgM Control Set is intended for use as assayed quality control samples to monitor the performance of the LIAISON® Lyme IgM assay. The performance characteristics of LIAISON® Lyme IgM 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) |
|---------------------------------------------------------------------------------------------------------|
| <span style="font-family: Arial, sans-serif;"> Prescription Use (Part 21 CFR 801 Subpart D) </span> |
| <span style="font-family: Arial, sans-serif;"> Over-The-Counter Use (21 CFR 801 Subpart C) </span> |

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### 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: | May 05, 2020 |
| NAME OF DEVICE:<br>Trade Name: | LIAISON® Lyme IgM<br>LIAISON® Lyme IgM Control Set |
| Common Names/Descriptions: | Borrelia burgdorferi IgM assay and<br>Borrelia burgdorferi IgM controls |
| Classification Names: | Treponema pallidum; treponemal testreagents<br>Class II, 21 CFR: 866.3830; Microbiology |
| Product Code: | LSR and QCH |
| Predicate Device: | ZEUS ELISA Borrelia burgdorferi IgM Test System<br>(K900196)/modified (K191240) |

### INTENDED USE:

The LIAISON® Lyme IgM assay uses chemiluminescent immunoassay (CLIA) technology for the qualitative detection of IgM antibodies to Borrelia burgdorferi in human serum and plasma samples (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 antibodies and exposure to Borrelia burgdorferi. In addition, the LIAISON® Lyme IgM 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 IgM assay should be confirmed through additional testing with a Standard two-tier test (STTT) methodology using an IgM 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 IgM assay should not be used to exclude Lyme disease. The test must be performed on the LIAISON® XL Analyzer.

The DiaSorin LIAISON® Lyme IgM Control Set is intended for use as assayed quality control samples to monitor the performance of the LIAISON® Lyme IgM assay. The performance characteristics of LIAISON® Lyme IgM 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 IgM assay is an indirect chemiluminescence immunoassay (CLIA) for the qualitative detection of IgM 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 IgM mouse monoclonal antibody linked to an isoluminol derivative (isoluminol-antibody conjugate). 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 IgM 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 IgM antibodies present in calibrators, samples or controls.

| Table 1: Table of Similarities | | |
|--------------------------------|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Characteristic | Candidate Device<br>LIAISON® Lyme IgM | Predicate Device<br>ZEUS ELISA<br>Borrelia burgdorferi IgM Test System –<br>K900196/K191240 |
| Intended Use | Qualitative detection of IgM antibodies to Borrelia<br>burgdorferi in human serum and plasma samples.<br>This assay is intended for use on samples from<br>patients with signs and symptoms consistent with or<br>patients suspected of having Lyme disease to<br>assess the presence of antibodies and exposure to<br>Borrelia burgdorferi.<br>In addition, the LIAISON® Lyme IgM 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>If used as a first stage test, positive or equivocal<br>results with the LIAISON® Lyme IgM 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>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>Negative results by the LIAISON® Lyme IgM assay<br>should not be used to exclude Lyme disease. | Qualitative detection of IgM 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>Positive and equivocal test results with the<br>ZEUS ELISA Borrelia burgdorferi IgM Test<br>System for the presence of Borrelia<br>burgdorferi antibodies must be confirmed<br>through additional testing by one of the<br>following approaches:<br>(1) Standard two-tier test methodology (STTT)<br>using IgM Western blot testing following current<br>guidelines; or -<br>(2) Modified two-tier test methodology using<br>the ZUES ELISA Borrelia VIsE1/pepC10<br>IgG/IgM Test System.<br>Positive test results by either the STTT or<br>MTTT methodology are supportive evidence for<br>the presence of antibodies and exposure to<br>Borrelia burgdorferi, the cause of Lyme<br>disease.<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 IgM | Predicate Device<br>ZEUS ELISA<br><i>Borrelia burgdorferi</i> IgM Test System –<br>K900196/K191240 |
|----------------------------------------|------------------------------------------------------------------------------------------------------|----------------------------------------------------------------------------------------------------------------------|
| Measurand | IgM 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 IgM antibodies. | Uses <i>B. burgdorferi</i> antigen on coated<br>solid phase (wells) to bind with<br>IgM antibodies in patient sample |
| Conjugate<br>antibody<br>specificities | Anti-human IgM | Anti-human IgM |
| Assay Output | Index | Same |

| Table 2: Table of Differences | | |
|-------------------------------|----------------------------------------------------------------------------------------------------------------|-----------------------------------------------------------------------------------------------------|
| Feature | Candidate Device<br>LIAISON® Lyme IgM | Predicate Device<br>ZEUS ELISA<br><i>Borrelia burgdorferi</i> IgM Test System –<br>K900196/K191240 |
| Test Format | CLIA (indirect chemiluminescent assay) | ELISA |
| Sample Type | Human serum, serum separator tubes, K2-EDTA, lithium heparin plasma | Human serum |
| Reporter Molecule | Isoluminol derivative conjugated to anti-human IgM | TMB (as a substrate for Horseradish peroxidase conjugated to anti-human IgM). |
| Antigen | Recombinant antigens:<br>OspC ( <i>B. afzelii</i> strain pKo).and<br>VlsE ( <i>B. burgdorferi</i> strain B31) | Whole cell antigen from<br><i>B. burgdorferi</i> (B31 strain) |
| Assay Procedure | Automated (on the LIAISON® XL Analyzer) | Manual |
| Calibration | Two-point verification (in triplicate) of stored 10 point master curve | Single Cut-off Calibrator assayed in triplicate |
| Output Signal | Flash chemiluminescent response is integrated over a 3 second reading period to generate a relative light unit | Microtiter well O.D. (450 nm) is measured after the enzyme reaction is halted by 1M H2SO4/0.7M HCl. |
| Measurement 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 IgM assay on the LIAISON® XL was performed in three (3) laboratories (2 external and internally at DiaSorin).

| | Predicate Assay (ELISA IgM) | | | |
|------------------|-----------------------------|-----------|----------|-------|
| LIAISON Lyme IgM | Positive | Equivocal | Negative | Total |
| Positive | 164 | 8 | 51 | 223 |
| Equivocal | 13 | 5 | 28 | 46 |
| Negative | 87 | 59 | 2206 | 2352 |
| Total | 264 | 72 | 2285 | 2621 |

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

### Agreement Results

| Positive % Agreement* | 56.5% (190/336) | 95% CI: 51.2% - 61.7% |
|-------------------------------------------|-------------------|-----------------------|
| Negative % Agreement | 96.5% (2206/2285) | 95% CI: 95.7% - 97.2% |
| *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 guidelines 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 IgM + | WB IgM - |
|-------------------------------------|-----------------|----------|----------|
| Predicate assay | 336 | 119 | 217 |
| LIAISON® Lyme IgM | 269 | 125 | 144 |
| Predicate assay + LIAISON® Lyme IgM | 190 | 109 | 81 |

### Agreement Results:

| 2nd Tier PPA | 91.6% (109/119) | 95% Cl: 85.2% - 95.4% |
|--------------|-------------------|-----------------------|
| 2nd Tier NPA | 99.4% (2486/2502) | 95% Cl: 99.0% - 99.6% |

## 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 (IgM) Western Blot.

Using the MTTT algorithm, the positive/equivocal specimens (n=225) were tested on the LIAISON® Lyme IgM assay. The second-tier LIAISON® Lyme IgM 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 (IgM) | | |
|----------------|-------|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-------|-------|
| | | + | | Total |
| XL -MTTT (IgM) | t | ત્વે ઉત્પત્તર તે જેવી સવલતો પ્રાપ્ય થયેલી છે. આ ગામમાં પ્રાથમિક શાળા, પંચાયતઘર, આંગણવાડી તેમ જ દૂધની ડેરી જેવી સવલતો પ્રાપ્ય થયેલી છે. આ ગામમાં પ્રાથમિક શાળા, પંચાયતઘર, આંગણ | ર્દિર | 146 |
| | | 7 | 72 | 79 |
| | Total | 100 | 125 | 225 |

### Table 5: MTTT LIAISON Lyme IgM compared to STTT WB IgM

Agreement Results

| PPA: | 93.0% (93/100) | 95% CI: 86.3% - 96.6% |
|------|----------------|-----------------------|
| NPA: | 57.6% (72/225) | 95% CI: 48.8% - 65.9% |

### 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 IqM assay with a characterized serum panel. This does not imply an endorsement of the assay by the CDC.

#### Table 6: Testing of CDC Lyme Reference Sera

| CDC Reference Classification | | | | | | | | | |
|------------------------------|-----|------------------|-----|-----|---------------------------------|---------------|-----|-----|-----------------------------------|
| Sample Category | N | LIAISON Lyme IgM | | | PPA 95% Wilson CI | Predicate IgM | | | PPA 95% Wilson CI |
| | | Pos | Neg | Eqv | | Pos | Neg | Eqv | |
| Acute | 39 | 27 | 11 | 1 | 71.8% (28/39)<br>56.2% - 83.5% | 28 | 10 | 1 | 74.4% (29/39)<br>(58.9% - 85.4%) |
| Convalescent | 31 | 26 | 4 | 1 | 87.1% (27/31)<br>71.1% - 94.9% | 25 | 6 | 0 | 80.6% (25/31)<br>(63.7% - 90.8%) |
| Late | 20 | 9 | 11 | 0 | 45.0% (9/20)<br>25.8% - 65.8% | 17 | 2 | 1 | 90.0% (18/20)<br>(69.9% - 97.2%) |
| Look-alike Diseases | 90 | 6 | 80 | 4 | 88.9% (80/90)<br>80.7% - 93.9% | 8 | 79 | 3 | 87.8% (79/90)<br>(79.4% - 93.0%) |
| Healthy controls | 100 | 2 | 97 | 1 | 97.0% (97/100)<br>91.5% - 99.0% | 2 | 97 | 1 | 97.0% (97/100)<br>(91.5% - 99.0%) |

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### 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 IgM Western Blot (STTT) or the LIAISON® Lyme IgM assay (MTT). The results of MTTT-IgM compared to WB-STTT (IgM).

| | Stage I<br>(n=60) | | Stage II<br>(n=10) | | Stage III<br>(n=20) | | Healthy Controls<br>(n=99) | | Disease Controls<br>(n=90) | |
|-----------------------|-------------------|----------------|--------------------|----------------|---------------------|----------------|----------------------------|----------------|----------------------------|----------------|
| | STTT<br>WB IgM | MTTT<br>XL IgM | STTT<br>WB IgM | MTTT<br>XL IgM | STTT<br>WB IgM | MTTT<br>XL IgM | STTT<br>WB IgM | MTTT<br>XL IgM | STTT<br>WB IgM | MTTT<br>XL IgM |
| Positive | 30 | 44 | 9 | 9 | 7 | 9 | 0 | 0 | 0 | 0 |
| Negative | 30 | 16 | 1 | 1 | 13 | 11 | 99 | 99 | 90 | 90 |
| Sensitivity or<br>PPA | 50.0<br>% | 73.3<br>% | 90.0<br>% | 90.0<br>% | 35.0<br>% | 45.0<br>% | N/A | N/A | N/A | N/A |
| Specificity or<br>NPA | N/A | N/A | N/A | N/A | N/A | N/A | 100% | 100% | 100% | 100% |

### PRECISION STUDY

A 12 day precision/repeatability was conducted at DiaSorin Inc. on 2 lots of the LIAISON® Lyme IgM assay. Six (6) serum samples and two (2) lots of LIAISON® Lyme IgM 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 1 | 48 | 2550* | 84.7 | 3.3% | 95.6 | 3.8% | 53.1 | 2.1% | 138.4 | 5.4% |
| Positive Control Lot 1 | 48 | 2.39 | 0.06 | 2.3% | 0.07 | 2.8% | 0.14 | 6.0% | 0.17 | 7.0% |
| Negative Control Lot 2 | 48 | 1500* | 50.9 | 3.4% | 50.7 | 3.4% | 39.4 | 2.6% | 81.9 | 5.5% |
| Positive Control Lot 2 | 48 | 2.33 | 0.08 | 3.5% | 0.00 | 0.0% | 0.13 | 5.5% | 0.15 | 6.3% |
| LM-QC3 | 48 | 1.54 | 0.06 | 3.8% | 0.00 | 0.0% | 0.1 | 6.8% | 0.12 | 7.8% |
| LM-QC11 | 48 | 1.51 | 0.03 | 2.3% | 0.06 | 3.7% | 0.07 | 4.9% | 0.1 | 6.5% |
| LM-QC12 | 48 | 4.65 | 0.12 | 2.7% | 0.08 | 1.8% | 0.31 | 6.6% | 0.34 | 7.3% |
| LM-QC13 | 48 | 0.29 | 0.01 | 2.9% | 0.01 | 4.5% | 0.02 | 7.1% | 0.03 | 8.9% |
| LM-QC16 | 48 | 0.8 | 0.04 | 4.5% | 0.01 | 1.8% | 0.06 | 7.4% | 0.07 | 8.8% |
| LM-QC17 | 48 | 0.78 | 0.02 | 3.0% | 0.01 | 1.3% | 0.05 | 6.9% | 0.06 | 7.6% |

*RLU - Index was below measuring range of assay

## 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 IgM assay.

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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. CLSI document EP15-A3 was consulted in the preparation of the testing protocol.

| Sample ID | | | Within Run | | Between Day | | Between Run | | Between Site | | | TOTAL |
|------------------|----|-------|------------|------|-------------|------|-------------|------|--------------|-------|-------|-------|
| | n | mean | SD | %CV | SD | %CV | SD | %CV | SD | %CV | SD | %CV |
| Negative Control | 90 | 1277* | 56.99 | 4.5% | 29.39 | 2.3% | 0.000 | 0.0% | 331.56 | 26.0% | 337.7 | 26.4% |
| Positive Control | 90 | 2.74 | 0.104 | 3.8% | 0.082 | 3.0% | 0.077 | 2.8% | 0.104 | 3.8% | 0.185 | 6.8% |
| LM-QC3 | 90 | 1.62 | 0.050 | 3.1% | 0.047 | 2.9% | 0.036 | 2.2% | 0.103 | 6.4% | 0.129 | 8.0% |
| LM-QC11 | 90 | 1.50 | 0.048 | 3.2% | 0.056 | 3.8% | 0.020 | 1.4% | 0.061 | 4.1% | 0.098 | 6.5% |
| LM-QC12 | 90 | 4.40 | 0.105 | 2.4% | 0.047 | 1.1% | 0.078 | 1.8% | 0.139 | 3.2% | 0.197 | 4.5% |
| LM-QC13 | 90 | 0.312 | 0.012 | 3.8% | 0.015 | 4.8% | 0.000 | 0.0% | 0.043 | 13.9% | 0.047 | 15.2% |
| LM-QC16 | 90 | 0.791 | 0.028 | 3.6% | 0.029 | 3.7% | 0.011 | 1.4% | 0.045 | 5.6% | 0.061 | 7.7% |
| LM-QC17 | 90 | 0.774 | 0.024 | 3.1% | 0.03 | 3.9% | 0.013 | 1.7% | 0.056 | 7.2% | 0.069 | 9.0% |

*RLU - Index was below measuring range of assay

### CROSS-REACTIVITY STUDY

The cross-reactivity study was designed to evaluate 191 specimens from nineteen (19) 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 Infected or Disease State | Samples<br>Tested (n) | Pos or Eqv |
|------------------------------------|-----------------------|------------|
| <b>Tick Borne Diseases</b> | | |
| Anaplasmosis IgM | 1 | 1 |
| Babesiosis IgM | 10 | 4^ |
| <b>Autoimmune Disorders</b> | | |
| Anti-Nuclear Antibodies (ANA) | 10 | 0 |
| Multiple Sclerosis | 10 | 0 |
| <b>Viral Diseases</b> | | |
| Cytomegalovirus (CMV) IgM | 10 | 1^ |
| Epstein-Barr Virus (EBV) | 10 | 0 |
| VCA and/or heterophile Ab IgM | 10 | 0 |
| Epstein-Barr Virus (EBV) VCA IgM | 10 | 2\$ |
| Herpes Simplex Virus (HSV) IgM | 10 | 1^ |
| Human Immunodeficiency Virus (HIV) | 10 | 0 |
| Influenza Virus | 10 | 0 |
| Parvovirus IgM | 10 | 1 |
| Varicella Zoster Virus (VZV) IgM | 10 | 1^ |
| <b>Bacterial Diseases</b> | | |
| <i>H. pylori</i> | 10 | 0 |
| Syphilis | 10 | 2 |
| <b>Rheumatic Diseases</b> | | |
| Fibromyalgia | 10 | 0 |
| Rheumatoid Arthritis | 10 | 0 |
| Rheumatoid Factor | 10 | 1^ |
| Systemic Lupus Erythematosus (SLE) | 10 | 1 |
| <b>Additional Markers</b> | | |
| Chronic Fatigue Syndrome | 10 | 0 |
| Human Anti-mouse Antibodies (HAMA) | 10 | 1 |
| <b>Total</b> | <b>191</b> | <b>16</b> |

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### INTERFERING SUBSTANCES

"Controlled studies of potentially interfering substances from endogenous interferents spiked into serum specimens containing B. burgdorferi IgM 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 | 12 g/dL |
| Cholesterol | 400 mg/dL |
| Biotin | 3600 ng/mL |

### MATRIX EQUIVALENCE STUDY:

Thirty-two (32) matched patient sets of 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 IgM assay. A summary of the results are shown in the following table.

| Comparison to Serum | | Bias | 95% Cl |
|------------------------|--------------|-------|----------------|
| Serum SST | Constant | -0.01 | -0.03 to 0.00 |
| | Proportional | 1.03 | 1.00 to 1.06 |
| EDTA Plasma | Constant | -0.02 | -0.05 to 0.03 |
| | Proportional | 1.03 | 0.97 to 1.08 |
| Lithium Heparin Plasma | Constant | -0.02 | -0.05 to -0.00 |
| | Proportional | 1.03 | 1.00 to 1.07 |

### CONCLUSION:

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