← Product Code [LSR](/submissions/MI/subpart-d%E2%80%94serological-reagents/LSR) · K202573

# LIAISON Lyme IgM, LIAISON Lyme IgM Control Set, LIAISON Lyme Total Antibody Plus (K202573)

_DiaSorin, Inc. · LSR · Feb 18, 2021 · Microbiology · SESE_

**Canonical URL:** https://fda.innolitics.com/submissions/MI/subpart-d%E2%80%94serological-reagents/LSR/K202573

## Device Facts

- **Applicant:** DiaSorin, Inc.
- **Product Code:** [LSR](/submissions/MI/subpart-d%E2%80%94serological-reagents/LSR.md)
- **Decision Date:** Feb 18, 2021
- **Decision:** SESE
- **Submission Type:** Traditional
- **Regulation:** 21 CFR 866.3830
- **Device Class:** Class 2
- **Review Panel:** Microbiology

## Indications for 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.

## Regulatory 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](/device/K900196.md)/[K191240](/device/K191240.md))

## Submission Summary (Full Text)

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>
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FDA U.S. FOOD & DRUG ADMINISTRATION

# 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION SUMMARY
ASSAY AND INSTRUMENT

## I Background Information:

A 510(k) Number
K202573

B Applicant
DiaSorin Inc.

C Proprietary and Established Names
LIAISON Lyme IgM, LIAISON Lyme IgM Control Set, LIAISON Lyme Total Antibody Plus

D Regulatory Information
|  Product Code(s) | Classification | Regulation Section | Panel  |
| --- | --- | --- | --- |
|  LSR | Class II | 21 CFR 866.3830 - Treponema Pallidum
Treponemal Test Reagents | MI – Microbiology  |
|  QCH | Class II | 21 CFR 866.3920 – Assayed quality control material for clinical microbiology assays | MI – Microbiology  |

## II Submission/Device Overview:

### A Purpose for Submission:

To obtain a substantial equivalence determination and FDA clearance for a new device and to obtain a substantial equivalence determination and FDA clearance for a modified intended use of a previously cleared medical device. This regulatory filing follows the FDA guidance document titled “Bundling Multiple Devices or Multiple Indications in a Single Submission”¹. For these devices, bundling is appropriate since the device review presented scientific and regulatory issues that were most efficiently addressed during a single review. In determining whether a bundled submission was appropriate FDA considered that: (i) the supporting data are similar; (ii)

¹ https://www.fda.gov/media/73500/download

Food and Drug Administration
10903 New Hampshire Avenue
Silver Spring, MD 20993-0002
www.fda.gov

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primarily one review division/group will be involved; and (iii) the devices or indications for use are similar.

## B Measurand:
Anti-Borrelia burgdorferi antibodies

## C Type of Test:
Chemiluminescent immunoassay (CLIA)

## III Intended Use/Indications for Use:

### A Intended Use(s):
See Indications for Use below.

### B Indication(s) for Use: LIAISON Lyme IgM

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 (K2-EDTA, Li-heparin) specimens. 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 IgM antibodies and exposure to Borrelia burgdorferi. In addition, the LIAISON Lyme IgM assay may be used as a confirmatory test in the Modified Two-tiered test (MTTT) methodology 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-Tiered 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.

### LIAISON Lyme IgM Control Set

The 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.

### LIAISON Lyme Total Antibody Plus

The LIAISON Lyme Total Antibody Plus assay uses chemiluminescent immunoassay (CLIA) technology for the qualitative determination of IgG and IgM antibodies of Borrelia burgdorferi in

K202573 - Page 2 of 23

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human serum and plasma (K2-EDTA, Li-heparin) samples. This assay is intended for use on samples from patients with signs and symptoms that are consistent with Lyme disease.

Positive or equivocal results with the LIAISON® Lyme Total Antibody Plus should be confirmed with additional testing by one of the following methodologies:

a). Standard two-tier test (STTT) methodology using an IgG and/or IgM Western blot test for Borrelia burgdorferi.

or -

b). Modified two-tier test (MTTT) methodology using one or more of the following LIAISON® assays: LIAISON Lyme IgM and/or LIAISON Lyme IgG

Positive supplemental results either by the STTT or MTTT are supportive evidence of the presence of antibodies and of 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.

## C Special Conditions for Use Statement(s):

Rx - For Prescription Use Only

## D Special Instrument Requirements:

LIAISON XL Analyzer

## IV Device/System Characteristics:

## A Device 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.

## B Principle of Operation:

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Chemiluminescence immunoassay (CLIA)

# C Instrument Description Information:

1. Instrument Name:
LIAISON XL Analyzer

2. Specimen Identification:
Specimens are identified by barcode and each test parameter is identified via information encoded in the Reagent Integral Radio Frequency Identification transponder (RFID Tag).

Control identification is detected by the bar code label or may be manually programmed into the instrument. Follow the analyzer operator's manual to start the run. Return controls to the refrigerator immediately after each use.

3. Specimen Sampling and Handling:
Either human serum, SST serum, K2-EDTA plasma and Lithium Heparin plasma may be used in this assay. Blood should be collected aseptically by venipuncture. Serum samples should be allowed to clot. Centrifuge samples and separate serum or plasma from the clot as soon as possible. Samples having particulate matter, turbidity, lipemia, or erythrocyte debris may require clarification by filtration or centrifugation before testing. Grossly hemolyzed or lipemic samples as well as samples containing particulate matter or exhibiting obvious microbial contamination should not be tested. Check for and remove air bubbles before assaying. Samples are stable at room temperature for up to three days. If the assay is performed within 8 days of sample collection, the samples should be kept at 2-8°C; otherwise they should be stored frozen (-20°C or below). If samples are stored frozen, mix thawed samples well before testing. Samples may be frozen-thawed 5 times. Self-defrosting freezers are not recommended for sample storage.

4. Calibration:
The DiaSorin LIAISON Lyme IgM assay generates a continuous response (relative light units, RLU) which is used in sample grading to provide a qualitative (positive, negative, or equivocal) reportable result. The sample grading is based on the use of a calibration curve referenced to an 'In-house' anti-Lyme IgG reference standard curve (Master Curve) and is controlled by the use of two calibrators (Calibrator 1 and Calibrator 2) provided in the Reagent Integral. Two point kit calibrators are used to establish specific working curves based on assay master curves stored on the analyzer.

Calibration is required every 8 weeks, or whenever one of the following conditions occurs:

- If Quality Control results are out of the acceptable range
- With each new lot of reagents (Reagent Integral or Starter Reagents)

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- After each servicing of the LIAISON XL Analyzer

The assay measuring range is 0.01 to 8.0 Index value. Samples that read lower than the measuring curve are reported as < 0.01 Index, and samples that read above the measuring curve are reported as > 8.0 Index.

## 5. Quality Control:

Quality control is required to be performed once per day of use, or according to the guidelines or requirements of local regulations or accredited organizations. It is recommended that the user refer to CLSI C24-A3 and 42 CFR 493.1256 (c) for guidance on appropriate quality control practices.

LIAISON Lyme IgM controls are intended to monitor for substantial reagent failure. Single replicates of LIAISON controls should be run to monitor the assay performance. If control values lie within the expected ranges provided on the certificate of analysis, the test is valid. If control values lie outside the expected ranges, the test is invalid and patient results cannot be reported. Assay calibration should be performed if a control failure is observed and controls and patient specimens must be repeated.

The performance of other controls should be evaluated for compatibility with this assay before they are used. Appropriate value ranges should be established for all quality control materials used.

The range of concentrations of each control is reported on the certificate of analysis and indicates the limits established by DiaSorin for control values that can be obtained in reliable assay runs.

## V Substantial Equivalence Information:

### A Predicate Device Name(s):

ZEUS ELISA Borrelia burgdorferi IgM Test System; LIAISON Lyme Total Antibody Plus

### B Predicate 510(k) Number(s):

K900196, K191240; K193051

### C Comparison with Predicate(s):

|  Device & Predicate Device(s): | K202573 | K900196 / K191240 | K193051  |
| --- | --- | --- | --- |
|  Device Trade Name | Device 1: LIAISON Lyme IgM
Device 2: LIAISON Lyme Total Antibody Plus | ZEUS ELISA Borrelia burgdorferi IgM Test System | LIAISON Lyme Total Antibody Plus  |
|  General Device |  |  |   |

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|  Characteristic Similarities |  |  |   |
| --- | --- | --- | --- |
|  Intended Use/Indications For Use | These are two indirect chemiluminescence assays for the qualitative detection of B. burgdorferi antibodies in human serum. For a complete description of the Intended Use please see Item III B. above | The ZEUS ELISA Borrelia burgdorferi IgM Test System is an enzyme-linked immunosorbent assay (ELISA) for the qualitative detection of IgM class antibodies to Borrelia burgdorferi in human serum. The assay is intended for testing serum samples from symptomatic patients or those suspected of Lyme disease.

Positive and equivocal test results with the ZEUS ELISA Borrelia burgdorferi IgM Test System
for the presence of Borrelia burgdorferi antibodies must be confirmed through additional testing by one of the following methods:
(1) Standard two-tier test methodology (STTT) using IgM Western blot testing following current interpretation guidelines;
or
(2) Modified two-tier test methodology (MTTT) using the ZEUS ELISA Borrelia VlsE1/pepC10 IgG/IgM Test System.

Positive test results by either the STTT or MTTT methodology are supportive evidence for the | LIAISON Lyme Total Antibody Plus: The LIAISON Lyme Total Antibody Plus assay uses chemiluminescent immunoassay (CLIA) technology for the qualitative determination of IgG and IgM antibodies of Borrelia burgdorferi in human serum and plasma (EDTA, Li-heparin) samples. This assay is intended for use on samples from patients with signs and symptoms that are consistent with Lyme disease. Positive or equivocal results should be supplemented by testing with a standardized Western blot procedure. Positive supplemental results provide evidence of exposure to B. burgdorferi and can be used to support a clinical diagnosis of Lyme disease. Negative results by LIAISON Lyme Total Antibody Plus assay should not be used to exclude Lyme disease. The test has to be performed on the LIAISON XL Analyzer.  |

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|   |  | presence of antibodies and exposure to Borrelia burgdorferi, the cause of Lyme disease. A diagnosis of Lyme disease should be made based on the presence of Borrelia burgdorferi antibodies, history, symptoms, and other laboratory data. |   |
| --- | --- | --- | --- |
|  Assay Type | Qualitative | Same | Same  |
|  General Device Characteristic Differences |  |  |   |
|  Assay Technology | CLIA (Indirect chemiluminescent assay) | ELISA | CLIA (Indirect chemiluminescent assay)  |
|  Sample Type | Serum, Serum Separator Tubes, K2-EDTA, lithium heparin plasma | Serum | Serum, Serum Separator Tubes, K2-EDTA, lithium heparin plasma  |
|  Instrumentation | LIAISON XL Analyzer | None | LIAISON XL Analyzer  |
|  Signal detection | Chemiluminescence | Colorimetric | Chemiluminescence  |
|  Antigen | Recombinant antigens: OspC (B. afzelii strain pKo) and VIsE (B. burgdorferi strain B31 and from B. garinii strain pbi) | Whole cell antigen from B. burgdorferi (B31 strain) | Recombinant antigens: VlsE from B. Burgdorferi strain B31 and B. garinii strain Pbi
OspC antigen from B. afzelii  |
|  Assay Procedure | Automated | Manual | Automated  |

VI Standards/Guidance Documents Referenced:

- CLSI EP05-A3, Evaluation of Precision of Quantitative Measurement Methods; Approved Guideline – Third Edition
- CLSI EP15-A3, User Verification of Precision and Estimation of Bias; Approved Guideline
- CLSI EP07-A3, Interference Testing in Clinical Chemistry; Approved Guideline, Second Edition

VII Performance Characteristics (if/when applicable):

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# A Analytical Performance:

Note: This clearance is to support use a new device, the LIAISON Lyme IgM assay, as both a first-tier test as part of a standard two-tiered test algorithm (STTT) and a second-tier test when used as part of a modified two-tiered test (MTTT) algorithm with the already cleared LIAISON Lyme Total Antibody Plus assay. The LIAISON Lyme Total Antibody Plus intended use has been modified to include this new indication.

# 1. Precision/Reproducibility:

12 day precision/repeatability study was conducted at DiaSorin Inc. on two lots of the LIAISON Lyme IgM assay. Six serum samples corresponding to one negative specimen, two high negative specimens, two low positive specimens, and one moderate positive specimen were evaluated with two lots of LIAISON Lyme IgG controls. Testing was performed by multiple technicians over 12 days with two runs per day and two replicates per run for a total of 48 replicates per lot spanning two calibration cycles.

Table 1. Precision Study Results - Combined

|  Sample ID | N | Mean | Between Lot |   | Total (Across Lots)  |   |
| --- | --- | --- | --- | --- | --- | --- |
|   |   |   |  SD | % CV | SD | %CV  |
|  Sample 1 | 96 | 1.49 | 0.06 | 4.2% | 0.09 | 5.8%  |
|  Sample 2 | 96 | 1.55 | 0.05 | 3.6% | 0.08 | 5.0%  |
|  Sample 3 | 96 | 4.53 | 0.17 | 3.7% | 0.25 | 5.6%  |
|  Sample 4 | 96 | 0.28 | 0.02 | 8.8% | 0.02 | 7.4%  |
|  Sample 5 | 96 | 0.80 | 0.01 | 0.8% | 0.05 | 6.5%  |
|  Sample 6 | 96 | 0.78 | 0.01 | 1.5% | 0.05 | 5.9%  |
|  Negative Control Lot 1 | 96 | 2595* | 63.61 | 2.5% | 120.86 | 4.7%  |
|  Positive Control Lot 1 | 96 | 2.39 | 0.01 | 0.3% | 0.12 | 5.2%  |
|  Negative Control Lot 2 | 96 | 1499* | 1.74 | 0.1% | 71.23 | 4.8%  |
|  Positive Control Lot 2 | 96 | 2.41 | 0.01 | 4.3% | 0.11 | 4.7%  |

*RLU. Index value was below measuring range of the assay

Table 2. Precision Study Results Single - Kit Lot 1

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|  Sample ID | N | Mean | Within Run |   | Between Run |   | Between Day |   | Total  |   |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|   |   |   |  SD | % CV | SD | % CV | SD | % CV | SD | %CV  |
|  Sample 1 | 48 | 1.45 | 0.02 | 1.1% | 0.02 | 1.3% | 0.02 | 1.1% | 0.03 | 2.0%  |
|  Sample 2 | 48 | 1.59 | 0.02 | 1.5% | 0.03 | 1.9% | 0.02 | 1.5% | 0.05 | 2.9%  |
|  Sample 3 | 48 | 4.41 | 0.08 | 1.8% | 0.05 | 1.2% | 0.07 | 1.7% | 0.12 | 2.7%  |
|  Sample 4 | 48 | 0.26 | 0.00 | 1.8% | 0.01 | 4.4% | 0.00 | 1.2% | 0.01 | 4.9%  |
|  Sample 5 | 48 | 0.81 | 0.01 | 1.8% | 0.01 | 1.4% | 0.01 | 1.5% | 0.02 | 2.8%  |
|  Sample 6 | 48 | 0.79 | 0.02 | 1.9% | 0.02 | 2.1% | 0.02 | 2.1% | 0.03 | 3.5%  |
|  Negative Control Lot 1 | 48 | 2640* | 63.61 | 2.2% | 62.69 | 2.4% | 51.53 | 2.0% | 100.32 | 3.8%  |
|  Positive Control Lot 1 | 48 | 2.39 | 0.01 | 1.4% | 0.04 | 1.5% | 0.02 | 0.9% | 0.05 | 2.2%  |
|  Negative Control Lot 2 | 48 | 1498* | 1.74 | 2.3% | 41.92 | 2.8% | 22.81 | 1.5% | 58.61 | 3.9%  |
|  Positive Control Lot 2 | 48 | 2.48 | 0.10 | 1.9% | 0.03 | 1.1% | 0.04 | 1.5% | 0.06 | 2.6%  |

*RLU. Index value was below measuring range of the assay

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.

Table 3. Multi-Site Reproducibility Study Results

|  Sample ID | N | Mean | Within Run |   | Between Run |   | Between Day |   | Between Site |   | Total  |   |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|   |   |   |  SD | % CV | SD | % CV | SD | % CV | SD | % CV | SD | %CV  |
|  Sample 1 | 90 | 1.62 | 0.050 | 8.0% | 0.036 | 2.2% | 0.047 | 2.9% | 1.62 | 6.4% | 0.129 | 8.0%  |
|  Sample 2 | 90 | 1.50 | 0.048 | 6.5% | 0.020 | 1.4% | 0.056 | 3.8% | 1.50 | 4.1% | 0.098 | 6.5%  |
|  Sample 3 | 90 | 4.40 | 0.105 | 4.5% | 0.078 | 1.8% | 0.047 | 1.1% | 4.40 | 3.2% | 0.197 | 4.5%  |
|  Sample 4 | 90 | 0.312 | 0.012 | 15.2% | 0.000 | 0.0% | 0.015 | 4.8% | 0.312 | 13.9% | 0.047 | 15.2%  |
|  Sample 5 | 90 | 0.791 | 0.028 | 7.7% | 0.011 | 1.4% | 0.029 | 3.7% | 0.791 | 5.6% | 0.061 | 7.7%  |
|  Sample 6 | 90 | 0.774 | 0.024 | 9.0% | 0.013 | 1.7% | 0.03 | 3.9% | 0.774 | 7.2% | 0.069 | 9.0%  |
|  Negative Control | 90 | 1277* | 56.99 | 26.4% | 0.000 | 0.0% | 29.39 | 2.3% | 331.56 | 26.0% | 337.7 | 26.4%  |
|  Positive Control | 90 | 2.74 | 0.104 | 6.8% | 0.077 | 2.8% | 0.082 | 3.0% | 0.104 | 3.8% | 0.185 | 6.8%  |

*RLU. Index value was below measuring range of the assay

2. Linearity:

Not Applicable.

3. Analytical Specificity/Interference:

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Cross-reactivity study: The cross-reactivity study evaluated 191 specimens from 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 late manifestations of Lyme disease and cause false positive results.

Table 4. Cross-reactivity Study Results

|  Organism/Disease State | Samples Tested (N) | LIAISON Lyme IgG Positive or Equivocal Result  |
| --- | --- | --- |
|  Tick Borne Diseases  |   |   |
|  Anaplasmosis IgM | 1 | 1  |
|  Babesiosis IgG | 10 | 4a  |
|  Autoimmune Disorders  |   |   |
|  Anti-Nuclear Antibodies (ANA) | 10 | 0  |
|  Multiple Sclerosis | 10 | 0  |
|  Viral Diseases  |   |   |
|  Cytomegalovirus (CMV) IgM | 10 | 1a  |
|  Epstein-Barr Virus (EBV IgM) | 10 | 0  |
|  Epstein-Barr Virus (EBV) VCA IgM | 10 | 2b  |
|  Herpes Simplex Virus (HSV) IgM | 10 | 1a  |
|  Human Immunodeficiency Virus (HIV) | 10 | 0  |
|  Influenza Virus | 10 | 0  |
|  Parvovirus IgM | 10 | 1  |
|  Varicella Zoster Virus (VZV) IgM | 10 | 1a  |
|  Bacterial Diseases  |   |   |
|  H. pylori | 10 | 0  |
|  Syphilis | 10 | 2  |
|  Rheumatic Diseases  |   |   |
|  Fibromyalgia | 10 | 0  |
|  Rheumatoid Arthritis | 10 | 0  |
|  Rheumatoid Factor | 10 | 1a  |
|  Systemic Lupus Erythematosus (SLE) | 10 | 1  |
|  Additional Markers  |   |   |
|  Chronic Fatigue Syndrome | 10 | 0  |
|  Human Anti-mouse Antibodies (HAMA) | 10 | 1  |

a Positive by the predicate device

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b One out of two samples was positive by the predicate device

Interfering Substances: Controlled studies of potentially interfering substances from endogenous interferents spiked into equivocal B. burgdorferi serum specimens showed that assay performance was not affected at the concentration for each substance listed below.

Table 5. Interference Testing Results

|  Substance | Concentration  |
| --- | --- |
|  Hemoglobin | 1000 mg/dL  |
|  Triglycerides | 1500 mg/dL  |
|  Cholesterol | 400 mg/dL  |
|  Bilirubin (conjugated) | 40 mg/dL  |
|  Bilirubin (unconjugated) | 40 mg/dL  |
|  Total protein | 12 g/dL  |
|  Biotin | 3600 ng/mL  |

Class Specificity Study: Dithiothreitol (DTT) was used to specifically inactivate IgM antibodies without affecting IgG antibodies. Five Borrelia burgdorferi positive specimens containing various levels of IgG antibodies and known to be IgM positive by Western blot were used for this testing. Samples were divided into two aliquots: one aliquot was spiked with DTT to a final concentration of 10 mM and the second aliquot was spiked with the same volume of vehicle control. Results are summarized in the table below.

Table 6. Class Specificity Study Results

|  Sample ID | Vehicle Control IgM Assay | DTT Treated IgM Assay | Vehicle Control IgG Assay | DTT Treated IgG Assay | % Difference IgG  |
| --- | --- | --- | --- | --- | --- |
|  1 | 1.78 | <0 | 41.1 | 40.0 | 2.7%  |
|  2 | 6.13 | 0.0264 | 28.6 | 28.5 | 0.3%  |
|  3 | 7.70 | 0.134 | 39.6 | 41.0 | 3.5%  |
|  4 | 5.31 | <0 | 4.35 | 4.7 | -9.7%  |
|  5 | 1.19 | <0 | 5.68 | 5.54 | 2.5%  |

4. Assay Reportable Range:

Not Applicable.

5. Traceability, Stability, Expected Values (Controls, Calibrators, or Methods):

Specimen Stability

Freeze-thaw Study

A study was performed using six samples for each of the following sample types: serum (no preservative), Serum-Separating Tube (SST) serum, EDTA plasma and lithium heparin

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plasma. Each sample type includes neat or contrived serum samples spiked to the following levels: one negative, two high negative, two low positive, and one moderate positive.

Samples were aliquoted, frozen and thawed for up to six freeze/thaw cycles before being tested in duplicate in the same assay.

Table 7. Freeze-thaw (FT) Sample Stability Results (Mean Index Value)

|  Serum  |   |   |   |   |   |   |
| --- | --- | --- | --- | --- | --- | --- |
|  Sample | 0 FT | 1 FT | 3 FT | 4 FT | 5 FT | 6 FT  |
|  Negative | 0.0844 | 0.0934 | 0.0944 | 0.0899 | 0.0871 | 0.087  |
|  High Negative 1 | 0.633 | 0.664 | 0.679 | 0.689 | 0.67 | 0.692  |
|  High Negative 2 | 0.624 | 0.628 | 0.584 | 0.644 | 0.644 | 0.62  |
|  Low Positive 1 | 1.33 | 1.42 | 1.46 | 1.37 | 1.42 | 1.42  |
|  Low Positive 2 | 1.31 | 1.35 | 1.42 | 1.41 | 1.38 | 1.41  |
|  Moderate Positive | 4.03 | 4.14 | 4.25 | 4.07 | 4.09 | 3.98  |
|  SST Serum  |   |   |   |   |   |   |
|  Sample | 0 FT | 1 FT | 2 FT | 4 FT | 5 FT | 6 FT  |
|  Negative | 0.0804 | 0.0998 | 0.0874 | 0.0852 | 0.0894 | 0.0711  |
|  High Negative 1 | 0.634 | 0.670 | 0.653 | 0.685 | 0.664 | 0.683  |
|  High Negative 2 | 0.637 | 0.657 | 0.619 | 0.618 | 0.678 | 0.635  |
|  Low Positive 1 | 1.35 | 1.37 | 1.41 | 1.36 | 1.38 | 1.41  |
|  Low Positive 2 | 1.32 | 1.41 | 1.45 | 1.37 | 1.42 | 1.47  |
|  Moderate Positive | 3.8 | 4.04 | 3.81 | 4.09 | 3.86 | 4.24  |
|  K2-EDTA Plasma  |   |   |   |   |   |   |
|  Sample | 0 FT | 1 FT | 2 FT | 4 FT | 5 FT | 6 FT  |
|  Negative | 0.0616 | 0.0458 | 0.0562 | 0.0637 | 0.0695 | 0.0641  |
|  High Negative 1 | 0.687 | 0.677 | 0.658 | 0.673 | 0.679 | 0.675  |
|  High Negative 2 | 0.732 | 0.78 | 0.783 | 0.704 | 0.711 | 0.735  |
|  Low Positive 1 | 1.56 | 1.61 | 1.70 | 1.60 | 1.63 | 1.60  |
|  Low Positive 2 | 1.50 | 1.54 | 1.52 | 1.52 | 1.48 | 1.61  |
|  Moderate Positive | 3.84 | 3.92 | 3.89 | 3.98 | 3.75 | 4.19  |
|  Lithium Heparin Plasma  |   |   |   |   |   |   |

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Regression of the mean percent difference versus freeze thaw counts does not cross $\pm 10\%$ for any of the sample types tested and thus demonstrate the stability of all claimed specimen types over the sponsor's stability claim of five freeze-thaw cycles.

## Room Temperature Stability

Another stability study was performed to demonstrate analyte stability when stored at room temperature. Six samples containing the same analyte levels as described above were aliquoted and stored at $22^{\circ}\mathrm{C}$ for the indicated times prior to testing. Samples were tested in duplicate at the following time points: 0, 4, 8, 9, 24, 25, 48, 49, 72, and 73 hours. Data is summarized in the table below.

Table 8. Room Temperature Stability Results (Mean Index Values)

|  Serum  |   |   |   |   |   |   |   |   |   |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|  Sample | T0 | 4 h | 8 h | 9 h | 24 h | 25 h | 48 h | 49 h | 72 h  |
|  Negative | 0.083 | 0.077 | 0.088 | 0.080 | 0.084 | 0.082 | 0.082 | 0.091 | 0.103  |
|  High Negative 1 | 0.721 | 0.647 | 0.651 | 0.650 | 0.690 | 0.676 | 0.721 | 0.672 | 0.814  |
|  High Negative 2 | 0.624 | 0.574 | 0.609 | 0.570 | 0.585 | 0.608 | 0.604 | 0.618 | 0.653  |
|  Low Positive 1 | 1.35 | 1.26 | 1.29 | 1.32 | 1.25 | 1.31 | 1.35 | 1.37 | 1.43  |
|  Low Positive 2 | 1.35 | 1.31 | 1.32 | 1.31 | 1.27 | 1.35 | 1.35 | 1.39 | 1.38  |
|  Moderate Positive | 4.61 | 4.10 | 4.04 | 4.14 | 4.08 | 4.07 | 4.34 | 4.41 | 4.60  |
|  SST Serum  |   |   |   |   |   |   |   |   |   |
|  Sample | T0 | 4 h | 8 h | 9 h | 24 h | 25 h | 48 h | 49 h | 72 h  |
|  Negative | 0.087 | 0.075
3 | 0.073
4 | 0.084
8 | 0.075
1 | 0.077
1 | 0.084
5 | 0.088
5 | 0.096
2  |
|  High Negative 1 | 0.730 | 0.640 | 0.667 | 0.662 | 0.689 | 0.671 | 0.683 | 0.717 | 0.741  |
|  High Negative 2 | 0.774 | 0.653 | 0.576 | 0.577 | 0.596 | 0.631 | 0.621 | 0.607 | 0.630  |

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|  Low Positive 1 | 1.34 | 1.27 | 1.31 | 1.33 | 1.31 | 1.37 | 1.37 | 1.40 | 1.46 | 1.48  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|  Low Positive 2 | 1.43 | 1.26 | 1.32 | 1.31 | 1.33 | 1.29 | 1.35 | 1.35 | 1.46 | 1.43  |
|  Moderate Positive | 3.93 | 4.06 | 4.06 | 4.06 | 4.17 | 4.14 | 4.28 | 4.35 | 4.61 | 4.69  |
|  K2 EDTA Plasma  |   |   |   |   |   |   |   |   |   |   |
|  Sample | T0 | 4 h | 8 h | 9 h | 24 h | 25 h | 48 h | 49 h | 72 h | 73 h  |
|  Negative | 0.065 | 0.041 | 0.047 | 0.057 | 0.052 | 0.057 | 0.066 | 0.069 | 0.079 | 0.077  |
|  High Negative 1 | 0.721 | 0.628 | 0.638 | 0.654 | 0.662 | 0.682 | 0.700 | 0.668 | 0.740 | 0.798  |
|  High Negative 2 | 0.735 | 0.646 | 0.671 | 0.600 | 0.609 | 0.603 | 0.623 | 0.665 | 0.646 | 0.667  |
|  Low Positive 1 | 1.65 | 1.46 | 1.45 | 1.49 | 1.48 | 1.52 | 1.64 | 1.71 | 1.70 | 1.67  |
|  Low Positive 2 | 1.55 | 1.39 | 1.47 | 1.44 | 1.56 | 1.50 | 1.54 | 1.50 | 1.67 | 1.74  |
|  Moderate Positive | 3.93 | 4.03 | 3.83 | 3.96 | 4.28 | 4.31 | 4.39 | 4.41 | 4.83 | 4.91  |
|  Sample | T0 | 4 h | 8 h | 9 h | 24 h | 25 h | 48 h | 49 h | 72 h | 73 h  |
|  Negative | 0.070 | 0.056 | 0.069 | 0.062 | 0.069 | 0.070 | 0.077 | 0.077 | 0.087 | 0.086  |
|  High Negative 1 | 0.851 | 0.752 | 0.738 | 0.736 | 0.751 | 0.743 | 0.774 | 0.804 | 0.842 | 0.856  |
|  High Negative 2 | 0.722 | 0.603 | 0.621 | 0.584 | 0.607 | 0.602 | 0.616 | 0.623 | 0.637 | 0.662  |
|  Low Positive 1 | 1.54 | 1.46 | 1.40 | 1.39 | 1.44 | 1.49 | 1.54 | 1.56 | 1.64 | 1.69  |
|  Low Positive 2 | 1.73 | 1.36 | 1.44 | 1.50 | 1.47 | 1.39 | 1.62 | 1.60 | 1.72 | 1.58  |
|  Moderate Positive | 4.07 | 4.09 | 4.43 | 4.63 | 4.60 | 4.35 | 4.64 | 4.48 | 4.73 | 4.99  |

No significant trend over a period of 73 hours was observed and the mean percent difference versus time does not cross  $\pm 10\%$  over the course of the study. These data are sufficient to support the sponsor's recommended storage claim of three days at room temperature.

An additional specimen stability study was performed to assess stability of samples when stored at  $4^{\circ}\mathrm{C}$ . Six samples containing the same analyte levels as described above were aliquoted and stored at  $22^{\circ}\mathrm{C}$  for the indicated times prior to testing. Samples were tested in duplicate at the following time points: 0, 1, 3, 4, 7, 8, 14 and 15 days. Data is summarized in the table below.

Table 9. Storage at  ${4}^{ \circ  }\mathrm{C}$  Stability Results (Mean Index Values)

|  Serum  |   |   |   |   |   |   |   |   |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
|  Sample | 0 d | 1 d | 3 d | 4 d | 7 d | 8 d | 14 d | 15 d  |
|  Negative | 0.134 | 0.127 | 0.117 | 0.129 | 0.122 | 0.119 | 0.133 | 0.126  |

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|  High
Negative 1 | 0.782 | 0.745 | 0.716 | 0.751 | 0.714 | 0.717 | 0.745 | 0.734  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
|  High
Negative 2 | 0.740 | 0.633 | 0.637 | 0.659 | 0.640 | 0.639 | 0.674 | 0.689  |
|  Low
Positive 1 | 1.40 | 1.31 | 1.32 | 1.41 | 1.29 | 1.34 | 1.38 | 1.41  |
|  Low
Positive 2 | 1.40 | 1.31 | 1.32 | 1.41 | 1.29 | 1.34 | 1.38 | 1.41  |
|  Moderate
Positive | 4.28 | 3.82 | 3.79 | 4.03 | 4.04 | 3.88 | 4.03 | 4.23  |
|  SST Serum  |   |   |   |   |   |   |   |   |
|  Sample | 0 d | 1 d | 3 d | 4 d | 7 d | 8 d | 14 d | 15 d  |
|  Negative | 0.130 | 0.124 | 0.116 | 0.122 | 0.120 | 0.123 | 0.119 | 0.121  |
|  High
Negative 1 | 0.832 | 0.733 | 0.701 | 0.734 | 0.676 | 0.710 | 0.711 | 0.734  |
|  High
Negative 2 | 0.758 | 0.740 | 0.620 | 0.653 | 0.627 | 0.632 | 0.652 | 0.657  |
|  Low
Positive 1 | 1.39 | 1.28 | 1.34 | 1.38 | 1.35 | 1.37 | 1.38 | 1.39  |
|  Low
Positive 2 | 1.44 | 1.30 | 1.32 | 1.38 | 1.35 | 1.39 | 1.39 | 1.37  |
|  Moderate
Positive | 4.09 | 3.70 | 3.81 | 3.85 | 3.84 | 3.94 | 4.07 | 4.01  |
|  K_{2} EDTA Plasma  |   |   |   |   |   |   |   |   |
|  Sample | 0 d | 1 d | 3 d | 4 d | 7 d | 8 d | 14 d | 15 d  |
|  Negative | 0.107 | 0.101 | 0.098 | 0.109 | 0.091 | 0.098 | 0.109 | 0.116  |
|  High
Negative 1 | 0.777 | 0.685 | 0.694 | 0.698 | 0.670 | 0.676 | 0.705 | 0.725  |
|  High
Negative 2 | 0.749 | 0.725 | 0.653 | 0.690 | 0.677 | 0.664 | 0.682 | 0.704  |
|  Low
Positive 1 | 1.65 | 1.57 | 1.60 | 1.61 | 1.57 | 1.53 | 1.69 | 1.56  |
|  Low
Positive 2 | 1.58 | 1.42 | 1.48 | 1.47 | 1.58 | 1.51 | 1.54 | 1.54  |
|  Moderate
Positive | 4.08 | 4.00 | 3.83 | 3.90 | 3.82 | 3.79 | 4.12 | 4.22  |
|  Lithium Heparin Plasma  |   |   |   |   |   |   |   |   |
|  Sample | 0 d | 1 d | 3 d | 4 d | 7 d | 8 d | 14 d | 15 d  |
|  Negative | 0.121 | 0.117 | 0.107 | 0.109 | 0.107 | 0.106 | 0.114 | 0.118  |
|  High
Negative 1 | 0.830 | 0.760 | 0.791 | 0.809 | 0.773 | 0.762 | 0.875 | 0.841  |
|  High
Negative 2 | 0.743 | 0.655 | 0.710 | 0.677 | 0.632 | 0.657 | 0.659 | 0.659  |
|  Low
Positive 1 | 1.60 | 1.43 | 1.47 | 1.56 | 1.52 | 1.48 | 1.52 | 1.58  |
|  Low
Positive 2 | 1.55 | 1.35 | 1.48 | 1.55 | 1.46 | 1.52 | 1.56 | 1.58  |

{15}

The regression of mean percent difference versus time does crossed  $10\%$  following the 14 day time point for SST serum specimens. These data support stability of all claimed specimen types over a period of 8 days when stored at  $4^{\circ}\mathrm{C}$ .

Finally, sample stability was also assessed when stored at  $-20^{\circ}\mathrm{C}$ . Six samples containing the same analyte levels as described above were aliquoted and stored at  $22^{\circ}\mathrm{C}$  for the indicated times prior to testing. Samples were tested in duplicate at designated intervals of 1, 2, 3, 6, 7, 9, 10, 12 and 13 months. Results for up to three months have been obtained, additional testing is currently ongoing. Available data is summarized in the table below.

Table 10. Frozen Sample Stability Results (Mean Index Values)

|  Serum  |   |   |   |   |
| --- | --- | --- | --- | --- |
|  Sample | 0 Mo. | 1 Mo. | 2 Mo. | 3 Mo.  |
|  Negative | 0.101 | 0.097 | 0.096 | 0.092  |
|  High Negative 1 | 0.710 | 0.690 | 0.762 | 0.708  |
|  High Negative 2 | 0.663 | 0.718 | 0.604 | 0.581  |
|  Low Positive 1 | 1.36 | 1.50 | 1.32 | 1.36  |
|  Low Positive 2 | 1.35 | 1.28 | 1.33 | 1.38  |
|  Moderate Positive | 4.31 | 4.16 | 4.20 | 4.39  |
|  SST Serum  |   |   |   |   |
|  Sample | 0 Mo. | 1 Mo. | 2 Mo. | 3 Mo.  |
|  Negative | 0.099 | 0.103 | 0.093 | 0.091  |
|  High Negative 1 | 0.730 | 0.831 | 0.764 | 0.747  |
|  High Negative 2 | 0.723 | 0.62 | 0.622 | 0.746  |
|  Low Positive 1 | 1.36 | 1.33 | 1.34 | 1.41  |
|  Low Positive 2 | 1.40 | 1.61 | 1.43 | 1.43  |
|  Moderate Positive | 3.94 | 3.92 | 4.02 | 4.22  |
|  K2EDTA Plasma  |   |   |   |   |
|  Sample | 0 Mo. | 1 Mo. | 2 Mo. | 3 Mo.  |
|  Negative | 0.078 | 0.076 | 0.082 | 0.055  |
|   |  | 6 | 1 | 8  |

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|  High Negative 1 | 0.730 | 0.747 | 0.849 | 0.700  |
| --- | --- | --- | --- | --- |
|  High Negative 2 | 0.739 | 0.746 | 0.649 | 0.815  |
|  Low Positive 1 | 1.62 | 1.47 | 1.51 | 1.53  |
|  Low Positive 2 | 1.54 | 1.43 | 1.55 | 1.45  |
|  Moderate Positive | 3.95 | 4.17 | 3.91 | 4.74  |
|  Lithium Heparin Plasma  |   |   |   |   |
|  Sample | 0 Mo. | 1 Mo. | 2 Mo. | 3 Mo.  |
|  Negative | 0.089 | 0.101 | 0.091 | 0.081  |
|  High Negative 1 | 0.800 | 0.769 | 0.756 | 0.837  |
|  High Negative 2 | 0.717 | 0.799 | 0.619 | 0.713  |
|  Low Positive 1 | 1.54 | 1.61 | 1.57 | 1.48  |
|  Low Positive 2 | 1.56 | 1.38 | 1.48 | 1.43  |
|  Moderate Positive | 4.10 | 3.97 | 4.08 | 4.24  |

6. Detection Limit:

Not Applicable.

7. Assay Cut-Off:

The cut-off for the LIAISON Lyme IgM Assay was determined by evaluating U.S. sourced characterized samples from Lyme disease patients and routine clinical samples sent to the laboratory for Lyme disease serology testing. Based on available clinical and laboratory data and by comparison with other cleared serology assays, the samples were classified as expected positive or negative for B. burgdorferi antibodies and evaluated with the LIAISON Lyme IgM assay. A cut-off index of 1.0 was determined to provide the best balance of sensitivity and specificity. An equivocal zone of 0.90-1.10 was applied to the assay to account for normal measurement imprecision.

B Comparison Studies:

1. Method Comparison with Predicate Device:

Prospective Cohort Testing: A total of 2,643 human serum specimens were obtained from four approved commercial vendors. The specimens were collected from non-selected subjects sent to the lab for Lyme disease testing in 14 states which represented five distinct geographical regions. Twenty-two (22) samples did not have enough volume to complete all

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testing and were excluded from the analysis. A total of 2,621 samples were evaluated of which 44.1% were male, 55.7% were female, and 0.2% did not have a gender reported.

Samples were tested with the LIAISON Lyme IgM assay on the LIAISON XL instrument at three laboratories (two external and one internal at DiaSorin). Results were evaluated for first-tier testing and compared to the predicate device.

Table 11. First-Tier Percent Agreement with the Predicate Device

|   | Predicate Assay (ZEUS ELISA B. Burgdorferi IgM Test System)  |   |   |   |
| --- | --- | --- | --- | --- |
|  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  |

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 results combined

Western blot testing was performed on samples positive or equivocal by the test device and the predicate. The following results were obtained:

Table 12. Second-Tier Testing Results

|  Test System | Tier 1+ or Eqv. | Western Blot IgM + | Western Blot IgM -  |
| --- | --- | --- | --- |
|  Predicate Assay | 336 | 119 | 217  |
|  LIAISON Lyme IgM | 269 | 125 | 144  |
|  Predicate and LIAISON Lyme IgM | 190 | 109 | 81  |

Despite a larger number of initially positive specimens on the predicate device, the LIAISON Lyme IgM test yields a larger number of samples positive by second-tier western blot testing. Furthermore, of the 146 specimens positive by the predicate device and negative by the LIAISON Lyme IgM test (Table 11), 124 (84.9%) were negative by second-tier western blot.

Performance agreement of the Lyme IgM assay when utilized as a first-tier test in a standard two-tiered testing algorithm (STTT) is summarized below and compared to results for the predicate assay.

Table 13. Second-Tier Agreement – Standard Two-tiered Testing Algorithm

|   | Predicate Assay IgM - STTT  |   |   |
| --- | --- | --- | --- |
|  LIAISON Lyme IgM - STTT | Positive | Negative | Total  |
|  Positive | 109 | 16 | 125  |
|  Negative | 10 | 2486 | 2496  |
|  Total | 119 | 2502 | 2621  |

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Second-Tier Positive % Agreement: 91.6% (109/119) 95% CI: 85.2-95.4%

Second-Tier Negative % Agreement: 99.4% (2486/2502) 95% CI: 99.0-99.6%

Cumulatively, these data support equivalence of the LIAISON Lyme IgM test to the predicate device as a first-tier test for Lyme disease.

Performance of the LIAISON Lyme IgM was also evaluated when utilized as a second-tier test as part of a Modified Two-tiered Testing (MTTT) algorithm. Specifically, the 2621 prospective specimens were tested initially with the cleared assay, LIAISON Lyme Total Antibody Plus. Positive or equivocal results on the LIAISON Lyme Total Antibody Plus were subsequently evaluated by the LIAISON Lyme IgM assay with positive or equivocal results considered MTTT positive. Agreement of the MTTT algorithm was also assessed against use of the cleared chemiluminescent immunoassay (CLIA) LIAISON Lyme Total Antibody Plus followed by a cleared western blot test as part of an STTT protocol. These schemes are summarized below.

![img-0.jpeg](img-0.jpeg)
Figure 1. STTT IgM Western Blot Algorithm (WB-STTT [IgM])
Figure 2. MTTT IgM CLIA Algorithm (CLIA-MTTT [IgM])

![img-1.jpeg](img-1.jpeg)

Performance of Lyme IgM assay as a second tier test was considered on only those specimens positive by the first-tier test. A total of 225 specimens exhibited positive or equivocal results when tested on Lyme Total Antibody Plus assay as a first-tier test.

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Table 14. Performance of the Lyme IgM MTTT Algorithm compared to STTT using WB IgM

|   | WB-STTT (IgM)  |   |   |   |
| --- | --- | --- | --- | --- |
|   |   |  Positive | Negative | Total  |
|  CLIA-MTTT (IgM) | Positive | 93 | 53 | 146  |
|   |  Negative | 7 | 72 | 79  |
|  Total |   | 100 | 125 | 225  |

Positive % Agreement: 93.0% (93/100) 95% CI: 91.2-98.9%

Negative % Agreement: 57.6% (72/125) 95% CI: 48.8-65.9%

Retrospective Cohort Testing: A retrospective cohort was also evaluated that consisted of 280 panel members obtained from CDC. The retrospective panel contained 90 specimens from patients known to have Lyme disease, 90 specimens from patients with diseases other than Lyme disease, and 50 specimens each from healthy controls in both endemic and non-endemic regions.

The CDC Lyme panel was tested internally at DiaSorin on the LIAISON Lyme IgM assay and the predicated device (ZEUS ELISA B. burgdorferi IgM assay). No repeat testing of equivocal samples was performed on the ZEUS IgM assay due to limited sample volume. No western blots were performed on any of the positive or equivocal results. Summary of agreement is included in the table below.

Table 15. First-Tier Performance Comparison to Predicate Device - CDC Reference Panel

|  Sample Category | LIAISON Lyme IgM |   |   |   |   | ZEUS B. Burgdorferi IgM  |   |   |   |   |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|   |  Pos. | Eqv. | Neg. | PPA/NPA | 95% CI | Pos. | Eqv. | Neg. | PPA/NPA | 95% CI  |
|  Acute | 27 | 1 | 11a | 71.8% | 56.2-83.5% | 28 | 1 | 10 | 74.4% | 58.9-85.4%  |
|  Convalescent | 26 | 1 | 4b | 87.1% | 71.1-94.9% | 25 | 0 | 6 | 80.6% | 63.7-90.8%  |
|  Late | 9 | 0 | 11c | 45.0% | 25.8-65.8% | 17 | 1 | 2 | 90.0% | 69.9-97.2%  |
|  Healthy Controls (N = 100) | 2d | 1d | 97 | 97% | 91.6-99.0% | 2 | 1 | 97 | 97.0% | 91.5-99.0%  |
|  Disease Controls (N = 90) | 6e | 4e | 80 | 88.9% | 80.7-93.9% | 8 | 3 | 79 | 87.8% | 79.4-93.0%  |

a Of the 11 negative discordant specimens, 10 were Early Lyme-EM and 1 Cardiac Lyme, 10 were negative by CDC 2-tier IgM WB, 9 were Zeus negative
b Of 4 negative discordant S2, 4 were negative by CDC 2-tier IgM WB, 4 were negative by Zeus IgM
c Of the 11 discordant, all were Lyme arthritis, and 10 were CDC 2-tier IgM negative
d All 3 discordant healthy controls were non-endemic controls
e The 10 discordant look-alike diseases were the following specimens: 6 Syphilis, 2 Mononucleosis, 1 Rheumatoid arthritis, and 1 Multiple Sclerosis

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These results establish that LIAISON Lyme IgG assay performance is equivalent to the ZEUS B. Burgdorferi IgG test for the pre-determined CDC Reference Panel cohort classifications.

The STTT and MTTT testing algorithms utilized for this study are the same as identified above in Figures 1 and 2. One sample from the endemic negative control group did not have sufficient volume for testing; therefore, a total of 279 retrospective samples were evaluated.

Table 16. Performance Comparisons between EIA-MTTT (IgG) and WB-STTT (IgG) methods - CDC Panel Results

|   | Stage I (N = 60) |   | Stage II (N = 10) |   | Stage III (N = 20) |   | Healthy Controls (N=99) |   | Disease Controls (N = 90)  |   |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|   |  WB-STTT (IgM) | CLIA-MTTT (IgM) | WB-STTT (IgM) | CLIA-MTTT (IgM) | WB-STTT (IgM) | CLIA-MTTT (IgM) | WB-STTT (IgM) | CLIA-MTTT (IgM) | WB-STTT (IgM) | CLIA-MTTT (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 PPA | 50% | 73.3% | 90.0% | 90.0% | 35.0% | 45.0% | N/A | N/A | N/A | N/A  |
|  Specificity or PPA | N/A | N/A | N/A | N/A | N/A | N/A | 100% | 100% | 100% | 100%  |

Based on the above results, use of the LIAISON Lyme IgM assay as a second-tier test, successfully detects a greater proportion of early Lyme disease cases. Use of the LIAISON Lyme IgM assay is therefore considered acceptable for use with the LIAISON Lyme Total Antibody Plus assay following the Modified Two-Tiered Testing Methodology.

Analytical Specificity: The LIAISON Lyme IgM assay was used to test 300 samples from apparently healthy individuals in the U.S. This population was  $57.3\%$  female,  $42.7\%$  male with a mean age of 50 years. Fifty percent of the samples were collected in a Lyme disease endemic region and  $50\%$  were collected from a Lyme disease non-endemic region.

Table 17. Analytical Specificity Study Results

|  Population | N | % Positivity  |
| --- | --- | --- |
|  Endemic | 150 | 5.3% (8/150)  |
|  Non-endemic | 150 | 4.7% (7/150)  |
|  All specimens | 300 | 5.0% (15/300)  |

2. Matrix Comparison:

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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 &amp; 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.

Table 18. Matrix Equivalency Study Results

|  Comparison to Serum | Bias | 95% CI  |
| --- | --- | --- |
|  SST Serum Constant | -0.01 | -0.03 – 0.00  |
|  SST Serum Proportional | 1.03 | 1.00 – 1.06  |
|  K2-EDTA Constant | -0.02 | -0.05 – 0.03  |
|  K2-EDTA Proportional | 1.03 | 0.97 – 1.08  |
|  Lithium Heparin Constant | -0.02 | -0.05 – 0.00  |
|  Lithium Heparin Proportional | 1.03 | 1.00 – 1.07  |

C Clinical Studies:

1. Clinical Sensitivity:
N/A

2. Clinical Specificity:
N/A

3. Other Clinical Supportive Data (When 1. and 2. Are Not Applicable):
N/A

D Clinical Cut-Off:
N/A

E Expected Values/Reference Range:

Internal and external investigators assessed the device’s performance with 2,621 masked specimens prospectively collected from patients between the ages of 2-103 that were submitted for Borrelia antibody testing. Specimens were acquired from five distinct geographical regions within the U.S. The specimens were randomly distributed among three testing sites, one of which was the manufacturer’s research facility for testing. The device’s performance was also assessed at the manufacturer’s research facility with specimens from an asymptomatic population obtained from both endemic and non-endemic regions. Available subject demographics, quantity of specimens tested and number of specimens which tested positive or equivocal for each population are summarized in Table 18.

Table 19. Positivity Rates Among Study Populations

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|  Populations | Number Tested | Gendera |   | Age Range | Positive/Tested  |
| --- | --- | --- | --- | --- | --- |
|   |   |  Male | Female  |   |   |
|  Prospective | 2,621 | 1,156 | 1,459 | 2-103 | 269/2,621  |
|  Endemic | 150 | 49 | 101 | 18-87 | 8/150  |
|  Non- Endemic | 150 | 79 | 71 | 19-71 | 7/150  |

a Six specimens did not include gender information

## VIII Proposed Labeling:

The labeling supports the finding of substantial equivalence for this device.

## IX Conclusion:

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

K202573 - Page 23 of 23

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**Source:** [https://fda.innolitics.com/submissions/MI/subpart-d%E2%80%94serological-reagents/LSR/K202573](https://fda.innolitics.com/submissions/MI/subpart-d%E2%80%94serological-reagents/LSR/K202573)

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