LIAISON QuantiFERON - TB Gold Plus, LIAISON Control QuantiFERON - TB Gold Plus and LIAISON QuantiFERON Software

P180047 · DiaSorin, Inc. · NCD · Nov 26, 2019 · Microbiology

Device Facts

• Record ID: P180047
• Device Name: LIAISON QuantiFERON - TB Gold Plus, LIAISON Control QuantiFERON - TB Gold Plus and LIAISON QuantiFERON Software
• Applicant: DiaSorin, Inc.
• Product Code: NCD · Microbiology
• Decision Date: Nov 26, 2019
• Decision: APPR
• Device Class: Class 3

Indications for Use

The LIAISON QuantiFERON-TB Gold Plus assay is an in vitro diagnostic test for the detection of interferon-γ (IFN-γ) in human lithium heparin plasma by chemiluminescence immunoassay (CLIA) using the LIAISON XL Analyzer. QIAGEN QuantiFERON-TB Gold Plus Blood Collection Tubes, containing a peptide cocktail simulating ESAT-6, and CFP-10 proteins, are used in conjunction with the LIAISON QuantiFERON-TB Gold Plus assay to stimulate cells in heparinized whole blood. Detection of IFN-γ is used to identify in vitro responses to these peptide antigens that are associated with Mycobacterium tuberculosis infection. The assay is a qualitative indirect test for M. tuberculosis infection (including disease) and is intended for use in conjunction with risk assessment, radiography, and other medical and diagnostic evaluations to assist the clinician in making individual patient management decisions. The LIAISON QuantiFERON-TB Gold Plus assay must be performed using the LIAISON XL Analyzer. The LIAISON Control QuantiFERON-TB Gold Plus is intended for use as assayed quality control samples to monitor the performance of the LIAISON QuantiFERON-TB Gold Plus assay. The performance characteristics of LIAISON Control QuantiFERON-TB Gold Plus have not been established for any other assays or instrument platforms other than the LIAISON XL Analyzer.

Device Story

The LIAISON QuantiFERON-TB Gold Plus is an IGRA chemiluminescence immunoassay (CLIA) performed on the LIAISON XL Analyzer. It requires whole blood collected in four specific QIAGEN QuantiFERON-TB Gold Plus tubes (TB1, TB2, Mitogen, Nil) for antigen stimulation. The analyzer processes plasma samples via a sandwich immunoassay using magnetic particles coated with anti-IFN-γ monoclonal antibodies and isoluminol-conjugated antibodies. The resulting light signal (RLU) is measured by a photomultiplier and converted to IFN-γ concentration (IU/mL). The LIAISON QuantiFERON Software (LQS) optionally automates result interpretation (positive, negative, indeterminate) based on an algorithm comparing TB1/TB2 responses against Nil and Mitogen controls. Used in clinical laboratories by technicians to aid clinicians in diagnosing M. tuberculosis infection. Results are interpreted alongside patient risk factors and radiography to guide clinical management, such as initiating antimicrobial therapy or monitoring, potentially reducing disease transmission and morbidity.

Clinical Evidence

Clinical agreement study across three cohorts (Active TB, Low Risk, Mixed Risk) totaling 1,042 subjects. Active TB cohort (n=160) showed 84.4% sensitivity. Low Risk cohort (n=312) showed 96.9% specificity. Mixed Risk cohort (n=570) showed 99.1% overall agreement with the comparator. Reproducibility study (5-day, multi-site) demonstrated acceptable precision across sites and lots. No clinical safety hazards reported.

Technological Characteristics

Chemiluminescence immunoassay (CLIA) using magnetic particles coated with mouse anti-human IFN-γ monoclonal antibody. Energy source: LIAISON XL Analyzer (electrical). Connectivity: Networked/standalone analyzer. Software: LIAISON XL Software and optional LIAISON QuantiFERON Software. Reagents include lyophilized calibrators and controls (recombinant human IFN-γ).

Indications for Use

Indicated for the detection of IFN-γ in human lithium heparin plasma to identify in vitro responses associated with Mycobacterium tuberculosis infection. Intended for use in conjunction with risk assessment, radiography, and other medical evaluations to assist in patient management decisions. No specific age or gender contraindications.

Reference Devices

• QIAGEN QuantiFERON-TB Gold Plus

Submission Summary (Full Text)

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SUMMARY OF SAFETY AND EFFECTIVENESS DATA (SSED)

I. GENERAL INFORMATION

Device Generic Name: Test, Immunity, Cell mediated, Mycobacterium tuberculosis

Device Trade Name: LIAISON QuantiFERON-TB Gold Plus, LIAISON Control QuantiFERON-TB Gold Plus, LIAISON QuantiFERON Software

Device Procode: NCD

Applicant's Name and Address: DiaSorin Inc.
1951 Northwestern Avenue
Stillwater, MN 55082-0285

Date(s) of Panel Recommendation: None

Premarket Approval Application (PMA) Number: P180047

Date of FDA Notice of Approval: November 26, 2019

II. INDICATIONS FOR USE

The LIAISON QuantiFERON-TB Gold Plus assay is an in vitro diagnostic test for the detection of interferon-γ (IFN-γ) in human lithium heparin plasma by chemiluminescence immunoassay (CLIA) using the LIAISON XL Analyzer. QIAGEN QuantiFERON-TB Gold Plus Blood Collection Tubes, containing a peptide cocktail simulating ESAT-6, and CFP-10 proteins, are used in conjunction with the LIAISON QuantiFERON-TB Gold Plus assay to stimulate cells in heparinized whole blood. Detection of IFN-γ is used to identify in vitro responses to these peptide antigens that are associated with Mycobacterium tuberculosis infection.

The assay is a qualitative indirect test for M. tuberculosis infection (including disease) and is intended for use in conjunction with risk assessment, radiography, and other medical and diagnostic evaluations to assist the clinician in making individual patient management decisions. The LIAISON QuantiFERON-TB Gold Plus assay must be performed using the LIAISON XL Analyzer.

The LIAISON Control QuantiFERON-TB Gold Plus is intended for use as assayed quality control samples to monitor the performance of the LIAISON QuantiFERON-TB Gold Plus assay. The performance characteristics of LIAISON Control QuantiFERON-TB Gold Plus have not been established for any other assays or instrument platforms other than the LIAISON XL Analyzer.

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The LIAISON QuantiFERON Software (LQS) is optional software intended to analyze the data generated by the LIAISON QuantiFERON-TB Gold Plus assay on the LIAISON XL Analyzer. LQS reports assay results as positive, negative, or indeterminate by an algorithm that combines the individual results associated with the four QIAGEN QuantiFERON-TB Gold Plus Blood Collection Tubes into a final result.

## III. CONTRAINDICATIONS

There are no known contraindications.

## IV. WARNINGS AND PRECAUTIONS

The warnings and precautions specific to the LIAISON QuantiFERON-TB Gold Plus assay, the LIAISON Control QuantiFERON-TB Gold Plus, and the LIAISON QuantiFERON Software (LQS), can be found in the respective package insert labeling.

## V. DEVICE DESCRIPTION

The LIAISON QuantiFERON-TB Gold Plus [an interferon-γ release assay IGRA] is a qualitative chemiluminescence immunoassay for assay performed on the LIAISON XL Analyzer designed to be used in conjunction with QuantiFERON-TB Gold Plus (QFT-Plus) Blood Collection Tubes which are manufactured by QIAGEN Sciences LLC¹. The four QFT-Plus Blood Collection Tubes are used for the collection and stimulation of whole blood samples and are an essential component of the LIAISON QuantiFERON-TB Gold Plus assay.

1) QFT-Plus TB1 and TB2 Blood Collection Tubes contain a peptide cocktail simulating ESAT-6 and CFP-10 proteins of Mycobacterium tuberculosis that elicit IFN-γ production.

2) QFT-Plus Mitogen and Nil Blood Collection Tubes serve as assay positive and negative controls, respectively. The QFT-Plus Mitogen tube contains phytohemagglutinin that stimulates T-cells and the production of IFN-γ. The QFT-Plus Nil tube contains no antigens and is used to adjust for pre-existing IFN-γ (i.e., The IFN-γ level of the Nil tube sample is subtracted from the IFN-γ levels of the TB1, TB2, and Mitogen tube samples).

Each whole blood sample collected using the four QFT-Plus Blood Collection Tubes is handled and processed in accordance with QFT-Plus Specimen Collection and Handling procedures in the QFT-Plus Package Insert. The resulting plasma samples are transferred to the LIAISON XL² Analyzer for detection of the analyte IFN-γ.

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¹ QIAGEN Sciences LLC is the contract manufacturer of the QFT-Plus Blood Collection Tubes. The QFT-Plus Blood Collection Tubes are a critical component of the LIAISON QuantiFERON-TB Gold Plus assay.
² The LIAISON XL Analyzer instrument and software were reviewed and cleared as part of 510(k) K181464.

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The LIAISON XL Analyzer, controlled by LIAISON XL Software, is fully automated and performs sample processing (sample pre-dilutions, sample and reagent dispensing, incubations, wash processes) as well as measurement and evaluation.

The LIAISON QuantiFERON-TB Gold Plus detects the analyte IFN-γ by direct sandwich chemiluminescence immunoassay (CLIA). During the first incubation, anti-IFN-γ monoclonal (mouse) antibodies with solid phase magnetic particles, and anti-IFN-γ monoclonal (mouse) antibodies conjugated with isoluminol, will bind to any IFN-γ present in the test sample (or controls, or calibrators) and form a sandwich. Any unbound material in the sample is removed with a wash cycle. During the second incubation, Assay Buffer W is added to reduce sample related non-specific binding, followed by a second wash cycle. Next the starter reagents of the LIAISON XL Analyzer are added and a flash chemiluminescent reaction is induced. The light signal which reflects the amount of isoluminol-antibody conjugate (indicative of the amount of IFN-γ in the test sample, control, or calibrator), is measured by a photomultiplier as relative light units (RLUs). The RLUs are evaluated and translated to amount of IFN-γ is generated, in International Units (IU)/mL.

The LIAISON QuantiFERON-TB Gold Plus assay results are interpreted via the use of an algorithm (Table 1) that combines the results from each of the four QFT-Plus Blood Collection Tubes. The final assay result is qualitative (i.e., positive, negative, indeterminate).

The LIAISON QuantiFERON Software (LQS) is optional software that may be used to assist the user with assay data analysis and results interpretation. The LQS calculates results as positive, negative, or indeterminate, based on an algorithm that combines the individual results associated with the four QIAGEN QuantiFERON-TB Gold Plus Blood Collection Tubes.

Table 1. LIAISON QuantiFERON-TB Gold Plus Assay Results Interpretation Algorithm

| Nil (IU/ml) | TB1 minus Nil (IU/ml) | TB2 minus Nil (IU/ml) | Mitogen minus Nil (IU/ml) | LIAISON QuantiFERON - TB Gold Plus result | Report/ Interpretation |
| --- | --- | --- | --- | --- | --- |
| ≤8.0 | ≥0.35 and ≥25% of Nil | Any | Any | Positive | M. tuberculosis infection likely |
| | Any | ≥0.35 and ≥25% of Nil | | | |
| | <0.35 OR ≥0.35 and <25% of Nil | <0.35 OR ≥0.35 and <25% of Nil | ≥0.5 | Negative | M. tuberculosis infection NOT likely |
| | <0.35 OR ≥0.35 and <25% of Nil | <0.35 OR ≥0.35 and <25% of Nil | <0.5 | Indeterminate | Likelihood of M.tuberculosis infection cannot be determined |
| >8.0 | Any | | | | |

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# Components of the LIAISON QuantiFERON-TB Gold Plus Assay

The LIAISON QuantiFERON-TB Gold Plus assay kit (200 tests) consists of three reagents (Magnetic particles, Diluent, Assay Buffer W) provided in individual compartments within a plastic container called a Reagent Integral, and four additional reagents (Calibrator A, Calibrator B, Buffer R, and Conjugate) are provided in individual glass vials. The contents of each reagent are further described below:

## Reagent Integral:

a. MAGNETIC PARTICLES – Magnetic particles coated with mouse anti-human IFN-γ monoclonal antibody, BSA, phosphate buffer, &lt; 0.1% sodium azide. One vial, total volume is 2.5 mL. Ready to use.
b. DILUENT- BSA, casein, phosphate buffer, EDTA, 0.2% ProClin 300, Polyclonal Mouse Nonspecific IgG, gentamycin sulfate 0.1 g/L. One vial, total volume is 18 mL. Ready to use.
c. ASSAY BUFFER W – BSA, casein, phosphate buffer, EDTA, 0.2% ProClin 300, and an inert blue dye. Two vials, volume of each vial is 23 mL. Ready to use.

## Individual Reagent Vials:

d. CALIBRATOR A – Recombinant human IFN-γ (produced in E.coli), HEPES buffer, BSA, bovine serum, 0.4% ProClin 300, 0.2 μg/L gentamycin sulfate, detergents. One vial, lyophilized. Total volume once reconstituted is 2.0 mL.
e. CALIBRATOR B – Recombinant human IFN-γ (produced in E.coli), HEPES buffer, BSA, bovine serum, 0.4% ProClin 300, 0.2 μg/L gentamycin sulfate, detergents. One vial, lyophilized. Total volume once reconstituted is 2.0 mL
f. BUFFER R – Streptavidin conjugated with isoluminol derivative, BSA, casein, phosphate buffer, 0.2% ProClin 300, gentamycin sulphate 0.1 g/L, Polyclonal Mouse Nonspecific IgG, detergents. Two vials, volume of each vial is 4.5 mL. Ready to use.
g. CONJUGATE – Biotinylated mouse anti-human IFN-γ monoclonal antibody, HEPES buffer, BSA, casein, Polyclonal Mouse Nonspecific IgG, 0.2% ProClin 300, gentamycin sulphate 0.1 g/L, detergents, anti-proteases. Two vials, lyophilized. Volume of each vial once reconstituted is 4 mL.

# Components of the LIAISON Control QuantiFERON-TB Gold kit:

LIAISON Control QuantiFERON-TB Gold Plus kit is an additional material required to perform LIAISON QuantiFERON-TB Gold Plus assay, and is used for monitoring assay performance. Each kit consists of two controls, each with a specific concentration that is predetermined to be within a defined range of analyte (IFN-γ) concentration. The analyte concentration is established by DiaSorin (the manufacturer of the controls), and is described in the Certificate of Analysis (CoA). Two vials of each control are provided lyophilized with each kit. Each control vial contains enough reagent for 20 tests. The contents of each of the controls are further described below:

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a. Control 1 – Recombinant human IFN-γ (produced by E.coli), HEPES buffer, BSA, bovine serum, 0.4% ProClin 300, 0.2 g/L gentamycin sulfate. Total volume of each vial once reconstituted is 2 mL.
b. Control 2 – Recombinant human IFN-γ (produced by E.coli), HEPES buffer, BSA, bovine serum, 0.4% ProClin 300, 0.2 g/L gentamycin sulfate. Total volume of each vial once reconstituted is 2 mL.

Additional Components Required by Not Provided in the LIAISON QuantiFERON-TB Gold Plus assay kit.

LIAISON XL Analyzer
LIAISON XL Cuvettes (REF X0016)
LIAISON XL Disposable Tips (REF X0015)
LIAISON XL Starter Kit (REF 319200)
LIAISON XL Wash/System Liquid (REF 319100)
LIAISON XL Waste Bags (REF X0025)

Required Materials from Other Supplier
QIAGEN QuantiFERON-TB Gold Plus Blood Collection Tubes:
- #622536 – 200 Count (50 each of Nil, TB1, TB2, and Mitogen tube)
- #622433 – 25 Dispenser Packs per carton (each pack includes 1 Nil, 1 TB1, 1 TB2, and 1 Mitogen tube)
- #623536 – High Altitude 200 Count (50 each of Nil, TB1, TB2, and Mitogen tube)

Quality Control Procedures

a. Assay Calibration - LIAISON QuantiFERON-TB Gold Plus Assay

Assay calibration must be tested and validated using two kit calibrators (Calibrator A, Calibrator B), to compensate for variability in assay reagent kit lots, LIAISON XL analyzers, and environmental conditions. The results from the calibrators are used to establish working curves when compared to assay master curves stored in the LIAISON XL analyzer. More specifically, the stored master curve is generally defined with 10 master curve base points. The two assay kit calibrators with defined analyte concentrations are measured. The measurement signals [in relative light units (RLU)] of both calibrators are compared to the master curve signals of the corresponding calibrator concentrations. The measurement signals of the calibrators allow the shift of all master curve points to a working curve, corresponding with the actual conditions during measurement. Calibration is required every 4 weeks or when quality control test results fall outside of acceptable limits. In addition, calibration is required when a new reagent lot is tested or after servicing of the LIAISON XL analyzer.

b. Quality Control (QC) - LIAISON Control QuantiFERON-TB Gold Plus

A set of assay controls must be tested to monitor the reagents and performance of the LIAISON QuantiFERON-TB Gold Plus assay. The analyte concentration of each control

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indicates the limits established by DiaSorin for control values that can be obtained in reliable assay test runs, and is indicated on the accompanying Certificate of Analysis (CoA). QC testing should be performed at least once per day of use, or according to guidelines or requirements of accredited organizations or local regulations. When QC test results fall outside the expected range of analyte concentration, calibration should be repeated and controls and samples retested. Patient sample results should not be reported until QC test results fall within the analyte expected range. Once reconstituted, assay controls are stable for four weeks, when stored at 2-8°C.

## VI. ALTERNATIVE PRACTICES AND PROCEDURES

There are two FDA approved IVD IFN-γ release assays currently on the market. Similar to the LIAISON QuantiFERON-TB Gold Plus assay, these devices are qualitative indirect tests intended for use in conjunction with risk assessment, radiography, and other medical and diagnostic evaluations to aid the diagnosis of Mycobacterium tuberculosis infection (including disease).

## VII. MARKETING HISTORY

The LIAISON QuantiFERON-TB Gold Plus assay and LIAISON Control QuantiFERON-TB Gold (CE-marked products) are currently marketed in multiple countries. These devices have not been withdrawn from the market in any country to date for any reasons related to safety and effectiveness. A list of countries where the CE-marked products are currently marketed is illustrated in Table 2 below.

Table 2. Countries Currently Marketing CE-marked LIAISON QuantiFERON-TB Gold Plus assay and LIAISON Control QuantiFERON-TB Gold

| • Austria | • Norway |
| --- | --- |
| • Belgium | • Portugal |
| • Denmark | • Spain |
| • Finland | • Sweden |
| • France | • Switzerland |
| • Germany | • United Kingdom |
| • Italy | |
| • Luxembourg | |
| • Netherlands | |

## VIII. POTENTIAL ADVERSE EFFECTS OF THE DEVICE ON HEALTH

When used according to the instructions in the package insert, the LIAISON QuantiFERON-TB Gold Plus poses no known major direct adverse effects to health.

Minor adverse reactions may occur during blood collection associated with venipuncture to include pain and/or redness at the site of venipuncture, slight risk of bleeding,

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hematoma, and skin infection. Other mild reactions associated with venipuncture may include: agitation, sweating, pallor, coldness, sense of weakness, nausea, or fainting.

Failure of the product to perform as intended, or incorrect performance of the assay, may lead to false positive or false negative results. The risk of false positive or false negative results negatively impacting the determination of infection with *M. tuberculosis*, and subsequent patient care, is mitigated by the requirement that all LIAISON QuantiFERON-TB Gold Plus assay results be interpreted in conjunction with patient risk factors, radiography, and other medical and diagnostic evaluations.

## IX. SUMMARY OF NONCLINICAL STUDIES

### A. 20-Day Precision Study

A precision study was conducted at DiaSorin S.p.A. in Italy. A coded test panel of 10 contrived samples was prepared by spiking analyte negative lithium heparinized plasma samples with native IFN-γ. Native IFN-γ was obtained from plasma harvested after incubation/stimulation of whole blood in QFT-Plus Mitogen blood collection tubes. The 10-member test panel was prepared with established IFN-γ concentrations that spanned the LIAISON QuantiFERON-TB Gold Plus assay result range (Table 3). The 10-member sample panel was tested along with the two LIAISON QuantiFERON-TB Gold controls (one kit lot). Testing was conducted in two runs per day, two replicates per run, using two LIAISON QuantiFERON-TB Gold Plus kit lots (#291001, and #291003) for a total of 160 test results per sample member. Study testing was conducted by a total of three operators and spanned two calibration cycles.

Table 3. 20-Day Precision Study Sample Test Panel

| PANEL | Sample ID | Classification IU/mL |
| --- | --- | --- |
| PRECISION PANEL | QFTB-01-P01 | 0.000 - 0.350 |
| | QFTB-01-P02 | 0.000 - 0.350 |
| | QFTB-01-P03 | 0.350 - 2.00 |
| | QFTB-01-P04 | 0.350 - 2.00 |
| | QFTB-01-P05 | 0.350 - 2.00 |
| | QFTB-01-P06 | 2.00 - 5.00 |
| | QFTB-01-P07 | 2.00 - 5.00 |
| | QFTB-01-P08 | 5.00 – 10.0 |
| | QFTB-01-P09 | 5.00 – 10.0 |
| | QFTB-01-P10 | 5.00 – 10.0 |
| Kit Control | #7124010 | ≤ 0.200 |
| | #7125010 | 0.826 – 2.18 |

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The results obtained are relatively consistent across both LIAISON QuantiFERON-TB Gold Plus kit lots and appear to demonstrate good precision. A summary of the study results is illustrated in Tables 4 and 5 below.

Table 4. 20-Day Precision Study Summary Results #1 - LIAISON QuantiFERON-TB Gold Plus Kit Lots 291001 and 291003

| Panel Member | n | Mean IFN-γ IU/mL | Within Lot - #291001 | | Within Lot - #291003 | |
| --- | --- | --- | --- | --- | --- | --- |
| | | | SD | %CV | SD | %CV |
| QFTB-01-P01 | 160 | 0.241 | 0.021 | 9.014 | 0.021 | 8.449 |
| QFTB-01-P02 | 160 | 0.294 | 0.028 | 9.679 | 0.024 | 8.156 |
| QFTB-01-P03 | 160 | 0.525 | 0.040 | 7.568 | 0.038 | 7.270 |
| QFTB-01-P04 | 160 | 0.797 | 0.065 | 8.162 | 0.064 | 8.059 |
| QFTB-01-P05 | 160 | 1.556 | 0.126 | 8.030 | 0.139 | 9.032 |
| QFTB-01-P06 | 160 | 2.988 | 0.269 | 8.845 | 0.263 | 8.956 |
| QFTB-01-P07 | 160 | 3.913 | 0.367 | 9.257 | 0.358 | 9.257 |
| QFTB-01-P08 | 160 | 5.494 | 0.498 | 8.920 | 0.483 | 8.935 |
| QFTB-01-P09 | 160 | 5.851 | 0.485 | 8.186 | 0.542 | 9.379 |
| QFTB-01-P10 | 160 | 6.678 | 0.603 | 8.948 | 0.640 | 9.686 |
| #7124010 (Control Level 1) | 160 | 0.074 | 0.018 | 29.995 | 0.019 | 21.134 |
| #7125010 (Control Level 2) | 160 | 1.520 | 0.122 | 8.335 | 0.148 | 9.442 |

Table 5. 20-Day Precision Study Summary Results #2 - LIAISON QuantiFERON-TB Gold Plus Kit Lots 29100 and 291003

| Panel Member | N | Mean | Repeatability | | Between-Run | | Between-Day | | Between-Lot | | Within-Lab (Total) | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | | SD | %CV | SD | %CV | SD | %CV | SD | %CV | SD | %CV |
| QFTB-01-P01 | 160 | 0.241 | 0.008 | 3.121 | 0.005 | 2.242 | 0.019 | 7.836 | 0.005 | 1.932 | 0.022 | 8.939 |
| QFTB-01-P02 | 160 | 0.294 | 0.011 | 3.590 | 0.007 | 2.263 | 0.023 | 7.854 | 0.003 | 1.169 | 0.026 | 9.003 |
| QFTB-01-P03 | 160 | 0.525 | 0.012 | 2.250 | 0.011 | 2.076 | 0.035 | 6.674 | 0.000 | 0.000 | 0.039 | 7.343 |
| QFTB-01-P04 | 160 | 0.797 | 0.020 | 2.514 | 0.013 | 1.637 | 0.059 | 7.455 | 0.000 | 0.000 | 0.064 | 8.036 |
| QFTB-01-P05 | 160 | 1.556 | 0.035 | 2.252 | 0.033 | 2.149 | 0.123 | 7.899 | 0.000 | 0.000 | 0.132 | 8.490 |
| QFTB-01-P06 | 160 | 2.988 | 0.090 | 3.006 | 0.034 | 1.125 | 0.248 | 8.301 | 0.039 | 1.318 | 0.269 | 8.997 |
| QFTB-01-P07 | 160 | 3.913 | 0.090 | 2.308 | 0.081 | 2.079 | 0.340 | 8.700 | 0.000 | 0.000 | 0.361 | 9.238 |
| QFTB-01-P08 | 160 | 5.494 | 0.121 | 2.201 | 0.093 | 1.687 | 0.466 | 8.487 | 0.053 | 0.967 | 0.493 | 8.980 |
| QFTB-01-P09 | 160 | 5.851 | 0.121 | 2.068 | 0.155 | 2.644 | 0.474 | 8.108 | 0.000 | 0.000 | 0.513 | 8.775 |
| QFTB-01-P10 | 160 | 6.678 | 0.173 | 2.585 | 0.243 | 3.633 | 0.542 | 8.124 | 0.000 | 0.000 | 0.619 | 9.267 |
| #7124010 | 160 | 0.074 | 0.009 | 11.943 | 0.009 | 11.554 | 0.013 | 18.207 | 0.022 | 29.419 | 0.028 | 38.381 |
| #7125010 | 160 | 1.520 | 0.036 | 2.345 | 0.040 | 2.613 | 0.125 | 8.231 | 0.067 | 4.398 | 0.152 | 9.971 |

# B. Analytical Specificity/Cross-Reactivity

B.1 Study One - Potential Interference/Cross-Reactivity of Endogenous Substances

Study One was performed to evaluate potential LIAISON QuantiFERON-TB Gold Plus assay interference/cross-reactivity due to endogenous substances.

Test samples were prepared using confirmed analyte-negative plasma samples spiked with native IFN- $\gamma$ . A total of three samples, each prepared at a high non-reactive, around the cutoff (0.35 IU/mL), and low-reactive (0.5-1.0 IU/mL) concentration were used in this study. Each sample was divided into two aliquots, the first aliquot spiked with a volume of

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the potential interfering substance to achieve a high concentration, and the second aliquot spiked with the same volume of water to serve as a control. The endogenous potential interfering substances and the concentrations tested are illustrated in Table 6 below. All samples were tested in the same run, in 26 replicates each, using one lot of the LIAISON QuantiFERON-TB Gold Plus assay kit reagents, and one lot of kit controls.

Table 6. Study One - Potential Interfering Endogenous Substances and Concentrations Tested

| Substance | Concentrations Tested |
| --- | --- |
| IL-2 | 10 ng/mL |
| IL-4 | 5 ng/mL |
| IL-5 | 100 ng/mL |
| IL-6 | 100 ng/mL |
| IL-10 | 100 ng/mL |
| IL-12 | 100 ng/mL |
| IFN- alpha | 50 ng/mL |
| IFN- beta | 50 ng/mL |
| TNF-alpha | 5 ng/mL |

Test sample results were deemed acceptable if the percent interference fell within $\pm 10\%$ of that of the control samples. A summary of the study results are illustrated in Table 7 below. Test sample results are depicted to the right of the control sample results.

Table 7. Study One – Interference/Cross-Reactivity of Endogenous Substances Study Summary Results

| Sample | High Non-Reactive | | | Sample around cut off value | | | Low Reactive | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Substance | Mean IU/mL Control | Mean IU/mL | % INT | Mean IU/mL Control | Mean IU/mL | % INT | Mean IU/mL Control | Mean IU/mL | % INT |
| IL-2 10 ng/mL | 0.260 | 0.250 | 3.92 | 0.642 | 0.637 | 0.80 | 0.969 | 0.956 | -1.35 |
| IL-4 5 ng/mL | 0.265 | 0.267 | 0.81 | 0.702 | 0.696 | -0.86 | 1.03 | 1.02 | -0.28 |
| IL-5 100 ng/ml | 0.262 | 0.267 | 1.79 | 0.676 | 0.706 | 4.47 | 1.01 | 1.01 | 0.31 |
| IL-6 100 ng/mL | 0.256 | 0.259 | 1.34 | 0.626 | 0.641 | 2.41 | 0.993 | 0.993 | -0.03 |
| IL-10 100 ng/mL | 0.257 | 0.267 | 4.01 | 0.602 | 0.612 | 1.55 | 0.951 | 1.01 | 6.10 |
| IL-12 100 ng/mL | 0.252 | 0.258 | 2.52 | 0.621 | 0.631 | 1.64 | 0.997 | 0.954 | -4.00 |
| IFN- alpha 50 ng/mL | 0.216 | 0.210 | -2.49 | 0.545 | 0.538 | -1.19 | 0.831 | 0.867 | 4.38 |
| IFN- beta 50 ng/mL | 0.215 | 0.223 | 3.96 | 0.547 | 0.520 | -5.00 | 0.859 | 0.891 | 3.79 |
| TNF-alpha 5 ng/mL | 0.256 | 0.251 | -1.80 | 0.589 | 0.598 | 1.66 | 0.974 | 0.960 | -1.46 |

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B.2 Study Two - Potential Interference/Cross-reactivity of Endogenous and Pharmacological Substances

A second interference study (Study Two) was performed to evaluate the potential interference of endogenous and pharmacological substances at concentrations listed in Table 8 below. Study Two employed the same study design and acceptance criteria as Study One.

Table 8. Study Two - Potential Interfering Endogenous and Pharmacological Substances and Concentrations Tested

| Substance | Kind of substance | Normal range | Reference for test concentration | Concentration to be tested | Vehicle |
| --- | --- | --- | --- | --- | --- |
| Triglycerides | Endogenous | 0.34 – 3.7 mmol/L | EP7-A2 | 3000 mg/dL | Plasma |
| Hemoglobin | Endogenous | 1 – 2 g/L | EP7-A2 | 1000 mg/dL | Plasma |
| Unconjugated bilirubin | Endogenous | 5 - 21 μmol/L | EP7-A2 | 20 mg/dL | H_{2}O |
| Conjugated bilirubin | Endogenous | 0.0 - 0.34 mmol/L | EP7-A2 | 20 mg/dL | H_{2}O |
| Cholesterol | Endogenous | 2.95 -5.2 mmol/L | EP7-A2 | 350 mg/dL | Plasma |
| Prednisolone | Pharmacological | Not applicable | EP7-A2- Appendix C | 0.3 mg/dL | H_{2}O |
| Cyclosporine | Pharmacological | Not applicable | Information provided by Qiagen | 5 μg/mL | H_{2}O |
| Abacavir sulfate | Pharmacological | Not applicable | Information provided by Qiagen | 15 μg/mL | H_{2}O |
| Biotin | Pharmacological | Not applicable | Ref. L-13-10-064-M | 3500 ng/mL | H_{2}O |

A summary of the study results are illustrated in Table 9 below. Test sample results are depicted to the right of the control sample results.

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Table 9. Study Two – Interference/Cross-Reactivity of Endogenous and Pharmacological Substances Study Summary Results

| Sample | High Non-Reactive | | | Sample around cut off value | | | Low Reactive | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Substance | Mean IU/mL Control | Mean IU/mL | % INT | Mean IU/mL Control | Mean IU/mL | % INT | Mean IU/mL Control | Mean IU/mL | % INT |
| Triglycerides 3000 mg/dL | 0.217 | 0.211 | -3.05 | 0.569 | 0.565 | -0.70 | 1.18 | 1.20 | 1.37 |
| Hemoglobin 1000 mg/dL | 0.231 | 0.233 | 0.63 | 0.600 | 0.616 | 2.80 | 0.963 | 0.949 | -1.50 |
| Unconjugated bilirubin 20 mg/dL | 0.225 | 0.230 | 2.55 | 0.586 | 0.592 | 1.04 | 0.916 | 0.911 | -0.49 |
| Conjugated bilirubin 20 mg/dL | 0.225 | 0.227 | 1.11 | 0.586 | 0.590 | 0.68 | 0.916 | 0.879 | -3.99 |
| Cholesterol 350 mg/dL | 0.217 | 0.218 | 0.18 | 0.569 | 0.581 | 2.08 | 1.18 | 1.22 | 3.13 |
| Prednisolone 0.3 mg/dL | 0.197 | 0.198 | 0.86 | 0.542 | 0.546 | 0.76 | 0.892 | 0.899 | 0.73 |
| Cyclosporine 5 μg /mL | 0.192 | 0.196 | 1.84 | 0.489 | 0.469 | -4.04 | 0.782 | 0.795 | 1.67 |
| Abacavir sulfate 15 μg/mL | 0.225 | 0.231 | 2.43 | 0.557 | 0.525 | -5.73 | 0.927 | 0.928 | 0.14 |
| Biotin 3500 ng/mL | 0.227 | 0.230 | 1.54 | 0.554 | 0.558 | 0.66 | 0.915 | 0.924 | 1.01 |

B.3. Study Three - Potential Interference/Cross-Reactivity of Total Protein, Rheumatoid Factor, and Human Anti-Murine Antibody (HAMA).

A third interference study (Study Three) was performed to evaluate the potential interference of total protein, rheumatoid factor, and HAMA. Study Three employed the same study design and acceptance criteria as Study One. Each potential interferent and associated concentrations tested are listed in Table 10 below.

Table 10. Study Three - Potential Interferents - Total Protein, Rheumatoid Factor, HAMA, and Concentrations Tested

| Substance | Normal range | Reference for test concentration | Concentration to be tested |
| --- | --- | --- | --- |
| Total protein (high) | 60 – 80 g/L | EP7-A2 | 120 g/L |
| Total protein (low) | 60 – 80 g/L | EP7-A2 | < 60 g/L |
| Rheumatoid Factor | No reference level | | The highest RF concentration possible |
| HAMA | No reference level | | The highest HAMA concentration possible |

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A summary of the study results are illustrated in Table 11 below. Test sample results are depicted to the right of the control sample results.

Table 11. Study Three – Interference/Cross-Reactivity of Total Protein, Rheumatoid Factor, and HAMA Study Summary Results

| Sample | High Non-Reactive | | | Sample around cut off value | | | Low Reactive | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Substance | Mean IU/mL Control | Mean IU/mL | % INT | Mean IU/mL Control | Mean IU/mL | % INT | Mean IU/mL Control | Mean IU/mL | % INT |
| Total protein (high) 120 g/L | 0.340 | 0.329 | -3.2 | 0.499 | 0.477 | -4.4 | 0.964 | 0.948 | -1.6 |
| Total protein (low) 38 g/L | 0.340 | 0.341 | 0.27 | 0.499 | 0.535 | 7.1 | 0.964 | 0.986 | 2.4 |
| Rheumatoid Factor 469 IU/mL | 0.320 | 0.306 | -4.31 | 0.458 | 0.484 | 5.71 | 0.919 | 0.925 | 0.57 |
| HAMA 600 ng/mL | 0.325 | 0.315 | -2.95 | 0.524 | 0.486 | -7.27 | 0.918 | 0.889 | -3.16 |

With regard to all three interference/cross-reactivity studies, the concentrations of all potential interfering substances appear to provide sufficient challenge to the device, and the study results suggest the performance of the LIAISON QuantiFERON-TB Gold Plus assay is not affected by elevated levels of the substances evaluated.

## C. Limit of Blank (LoB)

A study was conducted to determine the Limit of Blank (LoB) of the LIAISON QuantiFERON-TB Gold Plus assay. The aim of the study was to determine the highest value (IU/mL) expected of a natural plasma sample that is confirmed analyte (IFN-γ) negative. The study was conducted using a test sample panel that consisted of five lithium heparin plasma samples, confirmed to be analyte negative. Testing was performed in duplicate on two different LIAISON XL Analyzer instruments. Testing was conducted using two assay kit lots and one control kit lot; one instrument and one operator per assay kit lot, and six test runs conducted over three days. A total of 60 blank results were obtained for each assay kit lot. The LoB of each assay kit lot tested was calculated using the following formula:

$$
\mathrm{LoB} = (\text{Mean of All Blank Samples tested}) + 1.653(\text{Standard Deviation of All Blank Samples tested})^3
$$

The LoB acceptance criterion was defined as the highest LoB value among product lots/instruments/operators. A summary of the LoB study results is illustrated in Table 12 below.

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Table 12. Limit of Blank (LoB) Study Summary Results

| LIAISON QuantiFERON-TB Gold Plus assay Kit Lot | #291006 | #291007 |
| --- | --- | --- |
| LIAISON XL Analyzer Instrument No. | 2206000004 | 2206000003 |
| Blank Dose Mean (IU/mL) | 0.032 | 0.035 |
| Blank Dose SD (IU/mL) | 0.020 | 0.010 |
| Calculated LoB (IU/mL) | 0.065 | 0.052 |

The highest LoB value obtained was 0.065 IU/mL, and is therefore determined to be the established LoB for the LIAISON QuantiFERON-TB Gold Plus assay.

## D. High Dose Hook Effect

A study was conducted to assess the saturation effect that may occur when testing samples containing very high levels of analyte, resulting in a decrease in the actual analyte concentration detected. Test samples were prepared by spiking confirmed analyte negative lithium heparin plasma with elevated concentrations of IFN-γ (either native or recombinant). The study summary results were illustrated in Table 13 and Graphs A, B, and C, below.

Table 13. High Dose Hook Effect Study Summary Results

| | Sample 24930100 | | | Sample 2 Pool 1 | | | Sample 3 Pool 2 | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Dilution factor (1/x) | Mean RLU | Mean IU/mL | IU/mL (based on dilution factor applied) | Mean RLU | Mean IU/mL | IU/mL (based on dilution factor applied) | Mean RLU | Mean IU/mL | IU/mL (based on dilution factor applied) |
| NEAT | 10834455 | >10 | 92400 | 7115561 | >10.0 | 620 | 7819727 | >10.0 | 566 |
| 1:2 | 17916338 | >10 | 46200 | 4145415 | >10.0 | 310 | 4269435 | >10.0 | 283 |
| 1:10 | 20624777 | >10 | 9240 | 1151196 | >10.0 | 62.0 | 1144409 | >10.0 | 56.6 |
| 1:100 | 8667421 | >10 | 924 | 127539 | 6.20 | 6.20 | 116160 | 5.66 | 5.66 |
| 1:500 | 2754273 | >10 | 185 | 28685 | 1.43 | 1.43 | 26937 | 1.34 | 1.34 |
| 1:1000 | 1490507 | >10 | 92.4 | 34329 | 1.72 | 1.72 | 14375 | 0.726 | 0.726 |
| 1:2000 | 838296 | >10 | 46.2 | 8418 | 0.430 | 0.430 | 7214 | 0.364 | 0.364 |
| 1:20000 | 93046 | 4.62 | 4.62 | n.a. | n.a. | n.a. | n.a. | n.a. | n.a. |
| 1:40000 | 46157 | 2.30 | 2.30 | n.a. | n.a. | n.a. | n.a. | n.a. | n.a. |

Sample 24930100 = Test sample prepared by spiking confirmed analyte negative human plasma with commercially available recombinant human IFN-γ.
Sample 2 Pool 1 and Sample 3 Pool 2 = Test sample prepared by spiking confirmed analyte negative human plasma with pooled residual native human IFN-γ.

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Graph A. Sample 24930100 - Signal (RLU) versus IFN- $\gamma$ Concentration

Graph B. Sample 2 Pool 1- Signal (RLU) versus IFN- $\gamma$ Concentration

Graph C. Sample 3 Pool 2 - Signal (RLU) versus IFN- $\gamma$ Concentration

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The study demonstrates that no hook effect was observed until IFN-γ levels reached 9240 IU/mL. However samples with an analyte level value up to 92,400 IU/mL still resulted in a signal above the maximum range of &gt;10 IU/mL.

## E. Analyte Carry-Over Study

The LIAISON XL Analyzer has one dedicated sample dispense needle, one dedicated reagent needle, and utilizes disposable tips for both sample and reagent pipetting; single use cuvettes are also employed. An analyte carry-over study was conducted to evaluate whether any significant amount of IFN-γ analyte is carried over from one sample into subsequent samples. The study used two types of samples:

a) Analyte Negative Sample - a heparinized plasma sample confirmed negative for IFN-γ, divided into five separate aliquots, and
b) Analyte Positive Sample - a heparinized plasma sample spiked with IFN-γ at a level of &gt;10 IU/mL.

Testing was performed using one LIAISON XL Analyzer, one lot of LIAISON QuantiFERON-TB Gold Plus assay kit, and one lot of LIAISON Control QuantiFERON-TB Gold Plus.

Study testing was conducted in two stages.

Stage A: Five aliquots of the negative sample were tested in duplicate in two separate runs to establish a mean signal of the negative sample.

Stage B: Analyte positive samples were tested in a series alternating with aliquots of analyte negative samples.

Study design for Stage A and Stage B are illustrated in Tables 14 and 15 below.

Table 14. Stage A Carry-Over Study Design

| Stage A | | | |
| --- | --- | --- | --- |
| 1st RUN | | 2nd RUN | |
| NEG Aliquot 1 | 2 replicates | NEG Aliquot 1 | 2 replicates |
| NEG Aliquot 1 | 2 replicates | NEG Aliquot 1 | 2 replicates |
| NEG Aliquot 2 | 2 replicates | NEG Aliquot 2 | 2 replicates |
| NEG Aliquot 2 | 2 replicates | NEG Aliquot 2 | 2 replicates |
| NEG Aliquot 3 | 2 replicates | NEG Aliquot 3 | 2 replicates |
| NEG Aliquot 3 | 2 replicates | NEG Aliquot 3 | 2 replicates |
| NEG Aliquot 4 | 2 replicates | NEG Aliquot 4 | 2 replicates |
| NEG Aliquot 4 | 2 replicates | NEG Aliquot 4 | 2 replicates |
| NEG Aliquot 5 | 2 replicates | NEG Aliquot 5 | 2 replicates |
| NEG Aliquot 5 | 2 replicates | NEG Aliquot 5 | 2 replicates |

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Table 15. Stage B Carry-Over Study Design

| Stage B | | | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 1st RUN | | 2nd RUN | | 3rd RUN | | 4th RUN | | 5th RUN | |
| HIGH POS | 1 replicate | HIGH POS | 1 replicate | HIGH POS | 1 replicate | HIGH POS | 1 replicate | HIGH POS | 1 replicate |
| NEG Aliquot 1 | 1 replicate | NEG Aliquot 1 | 1 replicate | NEG Aliquot 1 | 1 replicate | NEG Aliquot 1 | 1 replicate | NEG Aliquot 1 | 1 replicate |
| HIGH POS | 1 replicate | HIGH POS | 1 replicate | HIGH POS | 1 replicate | HIGH POS | 1 replicate | HIGH POS | 1 replicate |
| NEG Aliquot 2 | 1 replicate | NEG Aliquot 2 | 1 replicate | NEG Aliquot 2 | 1 replicate | NEG Aliquot 2 | 1 replicate | NEG Aliquot 2 | 1 replicate |
| HIGH POS | 1 replicate | HIGH POS | 1 replicate | HIGH POS | 1 replicate | HIGH POS | 1 replicate | HIGH POS | 1 replicate |
| NEG Aliquot 3 | 1 replicate | NEG Aliquot 3 | 1 replicate | NEG Aliquot 3 | 1 replicate | NEG Aliquot 3 | 1 replicate | NEG Aliquot 3 | 1 replicate |
| HIGH POS | 1 replicate | HIGH POS | 1 replicate | HIGH POS | 1 replicate | HIGH POS | 1 replicate | HIGH POS | 1 replicate |
| NEG Aliquot 4 | 1 replicate | NEG Aliquot 4 | 1 replicate | NEG Aliquot 4 | 1 replicate | NEG Aliquot 4 | 1 replicate | NEG Aliquot 4 | 1 replicate |
| HIGH POS | 1 replicate | HIGH POS | 1 replicate | HIGH POS | 1 replicate | HIGH POS | 1 replicate | HIGH POS | 1 replicate |
| NEG Aliquot 5 | 1 replicate | NEG Aliquot 5 | 1 replicate | NEG Aliquot 5 | 1 replicate | NEG Aliquot 5 | 1 replicate | NEG Aliquot 5 | 1 replicate |

The carry-over study summary results for Stage A and Stage B are illustrated below in Table 16 and Table 17, respectively.

Table 16. Stage A Carry-Over Study Summary Results

| | Stage A | | | | |
| --- | --- | --- | --- | --- | --- |
| | | 1st RUN | | 2nd RUN | |
| SAMPLE | REPLICATE | IU/mL | Result | IU/mL | Result |
| NEG Aliquot 1 | Mean of Replicates 1 and 2 | 0.0239 | neg | 0.0307 | neg |
| NEG Aliquot 1 | Mean of Replicates 1 and 2 | 0.0365 | neg | 0.0361 | neg |
| NEG Aliquot 2 | Mean of Replicates 1 and 2 | 0.0241 | neg | 0.0217 | neg |
| NEG Aliquot 2 | Mean of Replicates 1 and 2 | 0.0232 | neg | 0.0320 | neg |
| NEG Aliquot 3 | Mean of Replicates 1 and 2 | 0.0276 | neg | 0.0333 | neg |
| NEG Aliquot 3 | Mean of Replicates 1 and 2 | 0.0251 | neg | 0.0358 | neg |
| NEG Aliquot 4 | Mean of Replicates 1 and 2 | 0.0205 | neg | 0.0306 | neg |
| NEG Aliquot 4 | Mean of Replicates 1 and 2 | 0.0364 | neg | 0.0302 | neg |
| NEG Aliquot 5 | Mean of Replicates 1 and 2 | 0.0325 | neg | 0.0250 | neg |
| NEG Aliquot 5 | Mean of Replicates 1 and 2 | 0.0248 | neg | 0.0270 | neg |
| N° of samples tested | | 40 | | | |
| N° of NEGATIVE results for all aliquots of negative sample | | 40 (100%) | | | |
| N° of POSITIVE results for all aliquots of negative sample | | 0 | | | |
| % POSITIVE RESULTS | | 0% | | | |

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Table 17. Stage B Carry-Over Study Summary Results

| SAMPLES | IU/mL | Result | IU/mL | Result | IU/mL | Result | IU/mL | Result | IU/mL | Result |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| HIGH POS Aliquot 1 | 10.0 | POS | 10.0 | POS | 10.0 | POS | 10.0 | POS | 10.0 | POS |
| NEG Aliquot 1 | 0.03 | neg | 0.02 | neg | 0.03 | neg | 0.03 | neg | 0.03 | neg |
| HIGH POS Aliquot 2 | 10.0 | POS | 10.0 | POS | 10.0 | POS | 10.0 | POS | 10.0 | POS |
| NEG Aliquot 2 | 0.00 | neg | 0.03 | neg | 0.03 | neg | 0.02 | neg | 0.03 | neg |
| HIGH POS Aliquot 3 | 10.0 | POS | 10.0 | POS | 10.0 | POS | 10.0 | POS | 10.0 | POS |
| NEG Aliquot 3 | 0.02 | neg | 0.03 | neg | 0.03 | neg | 0.03 | neg | 0.02 | neg |
| HIGH POS Aliquot 4 | 10.0 | POS | 10.0 | POS | 10.0 | POS | 10.0 | POS | 10.0 | POS |
| NEG Aliquot 4 | 0.03 | neg | 0.03 | neg | 0.03 | neg | 0.03 | neg | 0.02 | neg |
| HIGH POS Aliquot 5 | 10.0 | POS | 10.0 | POS | 10.0 | POS | 10.0 | POS | 10.0 | POS |
| NEG Aliquot 5 | 0.03 | neg | 0.03 | neg | 0.03 | neg | 0.03 | neg | 0.03 | neg |

The percent negative results for all negative samples tested for the carry-over study was $100\%$ , consistently demonstrating no significant amount of IFN- $\gamma$ is carried over from positive samples into alternating negative samples in the series.

# F. Sample Handling and Stability Studies

A series of studies were conducted to verify stability and consistency of IFN- $\gamma$ detection of harvested plasma samples stored in secondary containers using a contrived sample test panel. Sample storage stability after multiple freeze-thaw cycles, as well as stability after storage at $20^{\circ}\mathrm{C}$ , $-20^{\circ}\mathrm{C}$ , and $30^{\circ}\mathrm{C} \pm 1^{\circ}\mathrm{C}$ was evaluated. Confirmed analyte negative lithium heparin plasma was collected from seven donors at DiaSorin (Italy). Plasma was divided into 7 aliquots; 6 aliquots were spiked with native IFN- $\gamma$ , and one aliquot was not spiked to create test panel samples that spanned the assay measuring range as indicated below:

3 low-reactive samples (0.35 - 1 IU/mL)
3 reactive samples (1.0 - 10 IU/mL)
1 non-spiked sample (&lt;0.35 IU/mL)

# F.2 Freeze-Thaw Study

Each test panel sample was initially tested fresh to determine the $T = 0$ value. Test panel samples were then divided into individual aliquots and frozen at $-20^{\circ}\mathrm{C}$ . Aliquots were subjected to one, two, three, four, and five freeze-thaw cycles. Sample aliquots were thawed at room temperature; once completely thawed were refrozen. Testing of all sample aliquots was then performed on a single occasion using one lot of LIAISON QuantiFERON-TB Gold Plus assay kit, and one lot of LIAISON Control QuantiFERON-TB Gold Plus.

Study results were evaluated as follows:

- Calculation of the mean % difference between the amount of detected IFN- $\gamma$ (in IU/mL) of the fresh condition versus the amount of detected IFN- $\gamma$ at each of the subsequent freeze-thaw cycles, for each sample.
- Plotting of the mean % difference between the amount of detected IFN- $\gamma$ (in IU/mL) of the fresh condition versus the amount of detected IFN- $\gamma$ at each subsequent freeze-thaw cycle number for low-reactive and reactive samples.

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- Fitting a regression line to the data to determine the freeze-thaw cycle at which the regression line exceeds $\pm 10\%$ difference from $T=0$.
- Calculation of $\% \mathrm{CV}$ across freeze-thaw cycles.

The stability of low-reactive and reactive samples was determined to be the freeze-thaw cycle that was one cycle less than the last freeze-thaw cycle tested before the regression line exceeds $\pm 10\%$ difference from $T=0$. The stability of non-reactive samples was considered acceptable if the non-reactive status of the sample is maintained one freeze-thaw cycle beyond the claimed freeze-thaw cycle.

Based on the real-time study results, plasma sample freeze-thaw stability is determined to be four freeze-thaw cycles for both reactive and non-reactive samples. This finding is reflected in the LIAISON QuantiFERON-TB Gold Plus assay package insert.

## F.3 Sample Storage Study $2 - 8^{\circ}\mathrm{C}$

Each test panel sample was initially tested fresh to determine the $\mathrm{T} = 0$ value. Test panel samples were then divided into individual sample aliquots and stored at $2 - 8^{\circ}\mathrm{C}$ for 7, 14, 21, 22, 28, 29, 30, and 44 days. All sample aliquots that were stored at different exposure times were then each tested in duplicate using one lot of LIAISON QuantiFERON-TB Gold Plus assay kit, and one lot of LIAISON Control QuantiFERON-TB Gold Plus.

Study results were evaluated as follows:

- Calculation of the mean $\%$ difference between the amount of detected IFN- $\gamma$ (in IU/mL) of the fresh condition versus the amount of detected IFN- $\gamma$ at each subsequent storage time point, for each sample.
- Plotting of the mean $\%$ difference between the amount of detected IFN- $\gamma$ (in IU/mL) of the fresh condition versus the amount of detected IFN- $\gamma$ at each subsequent storage time point for low-reactive and reactive samples.
- Fitting a regression line to the data to determine the storage time point at which the regression line exceeds $\pm 10\%$ difference from $T=0$.
- Calculation of $\% \mathrm{CV}$ across the storage time points.

The stability of low-reactive and reactive samples was determined to be the storage time point that was one cycle less than the last storage time point tested before the regression line exceeds $\pm 10\%$ difference from $T=0$. The stability of non-reactive samples was considered acceptable if the non-reactive status of the sample is maintained one storage time point beyond the claimed storage time point.

Based on the real-time study results, plasma sample stability when stored at $2 - 8^{\circ}\mathrm{C}$ is determined to be 28 days for both reactive and non-reactive samples. This finding reflected in the LIAISON QuantiFERON-TB Gold Plus assay package insert.

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F.4 Sample Storage Study -20°C

Each test panel sample was initially tested fresh to determine the T=0 value. Test panel samples were then divided into individual sample aliquots and stored at -20°C for 1, 3, 4, 6, and 7 months. All sample aliquots that were stored at different exposure times were then each tested in duplicate using one lot of LIAISON QuantiFERON-TB Gold Plus assay kit, and one lot of LIAISON Control QuantiFERON-TB Gold Plus.

Study results were evaluated as follows:

- Calculation of the mean % difference between the amount of detected IFN-γ (in IU/mL) of the fresh condition versus the amount of detected IFN-γ at each subsequent storage time point, for each sample.
- Plotting of the mean % difference between the amount of detected IFN-γ (in IU/mL) of the fresh condition versus the amount of detected IFN-γ at each subsequent storage time point for low-reactive and reactive samples.
- Fitting a regression line to the data to determine the storage time point at which the regression line exceeds ±10% difference from T=0.
- Calculation of %CV across the storage time points.

The stability of low-reactive and reactive samples was determined to be the storage time point that was one cycle less than the last storage time point tested before the regression line exceeds ±10% difference from T=0. The stability of non-reactive samples was considered acceptable if the non-reactive status of the sample is maintained one storage time point beyond the claimed storage time point.

Based on the real-time study results, plasma sample stability when stored at -20°C is determined to be 6 months for both reactive and non-reactive samples. This finding is reflected in the LIAISON QuantiFERON-TB Gold Plus assay package insert.

F.5 Sample Storage Study 30°C ± 1°C

Each test panel sample was initially tested fresh to determine the T=0 value. Test panel samples were then divided into individual sample aliquots and stored at 30°C ± 1°C for 8, 9, 24, 25 hours. All sample aliquots that were stored at different exposure times were then each tested in duplicate using one lot of LIAISON QuantiFERON-TB Gold Plus assay kit, and one lot of LIAISON Control QuantiFERON-TB Gold Plus.

Study results were evaluated as follows:

- Calculation of the mean % difference between the amount of detected IFN-γ (in IU/mL) of the fresh condition versus the amount of detected IFN-γ at each subsequent storage time point, for each sample.
- Plotting of the mean % difference between the amount of detected IFN-γ (in IU/mL) of the fresh condition versus the amount of detected IFN-γ at each subsequent storage time point for low-reactive and reactive samples.
- Fitting a regression line to the data to determine the storage time point at which the regression line exceeds ±10% difference from T=0.

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- Calculation of %CV across the storage time points.

The stability of low-reactive and reactive samples was determined to be the storage time point that was one cycle less than the last storage time point tested before the regression line exceeds ±10% difference from T=0. The stability of non-reactive samples was considered acceptable if the non-reactive status of the sample is maintained one storage time point beyond the claimed storage time point.

Based on the real-time study results, plasma sample stability when stored at 30°C ± 1°C is determined to be 24 hours for both reactive and non-reactive samples.

## G. LIAISON QuantiFERON-TB Gold Plus Assay Calibration Stability Studies

A study was conducted to assess the stability of the LIAISON QuantiFERON-TB Gold Plus assay calibration by simulating normal conditions of use. A coded test panel of six samples was prepared by spiking analyte negative lithium heparinized plasma samples with native IFN-γ, to achieve concentrations that spanned the assay result range. Native IFN-γ was obtained from plasma harvested after incubation/stimulation of whole blood in QFT-Plus Mitogen blood collection tubes.

LIAISON QuantiFERON-TB Gold Plus assay reagent integrals were opened and stored on board the LIAISON XL Analyzer in the refrigerated reagent bay.

Testing of samples was performed in duplicate, using one lot of the LIAISON QuantiFERON-TB Gold Plus assay kit, and one lot of LIAISON Control QuantiFERON-TB Gold Plus.

The performance of the LIAISON QuantiFERON-TB Gold Plus assay kit, using the open reagent integrals was evaluated weekly for a period of nine weeks. Sample and control test results were generated using the initial (Time 0) assay calibration, and using a freshly reconstituted conjugate vial every 14 days. A second calibration stability study, using the same study design, but using a freshly reconstituted conjugate for each run was also conducted.

Study results were evaluated as follows:

- The IU/mL value result, for each sample, was evaluated and compared to the defined acceptance criteria. The acceptance criteria were the sole means of evaluation for non-reactive samples.
- Calculation of the mean % difference in the amount of detected IFN-γ (in IU/mL) of each sample at Time 0 versus the amount of detected IFN-γ at each subsequent time point.
- Plotting of the mean % difference between the amount of detected IFN-γ (in IU/mL) of each reactive sample at Time 0 versus the amount of detected IFN-γ at each subsequent time point.
- Fitting a regression line to the sample data to determine the time point at which the regression line exceeds ±10% difference from T=0.

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The calibration stability was determined to be the time point that was one point less than the final time point tested before the regression line exceeds $\pm 10\%$ difference from $T=0$ for each reactive sample.

Regarding the initial calibration interval study that used a freshly reconstituted conjugate vial every 14 days, for the majority of samples tested, the fitted regression line did not exceed $\pm 10\%$ deviation from $T=0$ over 9 weeks of testing. However, for one sample the fitted regression line intercepted the $-10\%$ deviation from $T=0$ between the Week 8 and Week 9 time points.

Regarding the second calibration interval study that used a freshly reconstituted conjugate for each run, for all samples tested, the fitted regression line did not exceed $\pm 10\%$ deviation from $T=0$ over the entire 9 weeks of testing.

Based on the findings of both calibration interval studies, the stability of the LIAISON QuantiFERON-TB Gold Plus assay was determined to be 7 weeks. However, in an effort to foster a degree of consistency across assay kit reagents for ease of customer use, the stability of the LIAISON QuantiFERON-TB Gold Plus assay calibration was established as 4 weeks.

## H. LIAISON QuantiFERON-TB Gold Plus Reagent and LIAISON Control QuantiFERON-TB Gold Plus Storage and Shelf Life Studies

## H.1. LIAISON QuantiFERON-TB Gold Plus Reagent Storage and Shelf Life Study

A (real-time) study was conducted to evaluate the long term stability of the LIAISON QuantiFERON-TB Gold Plus assay kit reagents and to establish shelf life. Testing was conducted using a coded test panel of samples and controls (level 1 and level 2). The samples were prepared by spiking analyte negative lithium heparinized plasma samples with native IFN-$\gamma$, to achieve concentrations that spanned the assay result range. Native IFN-$\gamma$ was obtained from plasma harvested after incubation/stimulation of whole blood in QFT-Plus Mitogen blood collection tubes.

Testing was performed in duplicate using three lots of LIAISON QuantiFERON-TB Gold Plus assay kits, and three lots of LIAISON Control QuantiFERON-TB Gold Plus kits.

The performance of the LIAISON QuantiFERON-TB Gold Plus assay kit was evaluated at time points of 1, 2, 4, 6, 9, 11, 12, 13, and 14 months after kit manufacture.

Data analysis of the study test panel sample and control results included the following:

For test panel samples:
- Data for each sample tested, on each kit lot, was plotted separately.
- The mean amount of detected IFN-$\gamma$ was plotted against the corresponding study time point for each sample.

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- A linear regression was fitted to the test sample data.
- The y-intercept of the regression line was calculated to estimate the baseline value for the IFN-γ.
- The 95% confidence intervals for each time point were calculated.
- ±10% deviation from the regression intercepts were indicated on the plots as horizontal lines.

Similarly for controls:
- The mean amount of detected IFN-γ (IU/mL) for each of the controls, at each testing point, was evaluated by comparing it to the established range, as defined in the product Certificate of Analysis (CoA).
- Data for each control tested, on each kit lot, was plotted separately.
- The mean amount of detected IFN-γ was plotted against the corresponding study time point for each control.
- A linear regression was fitted to the test sample data.
- The y-intercept of the regression line was calculated to estimate the baseline value for the IFN-γ.
- ±10% deviation from the regression intercepts were indicated on the plots as horizontal lines.

The LIAISON QuantiFERON-TB Gold Plus assay kit reagent shelf life claim was determined to be the time point that is one time point less than the final time point tested before the regression line exceeds ±10% difference from the baseline value.

The test results of all three lots of positive and negative controls fell within the established range as defined in the product CoA. Regarding all three lots of positive controls tested, the fitted regression line did not exceed ±10% deviation from the regression intercept at any time point during the study. Regarding all three lots of negative controls tested, due to the very low result values, the absolute differences from the regression intercept, at each time point was evaluated, and found to minimal and acceptable.

Based on the real-time stability study findings, the claimed shelf life of the LIAISON QuantiFERON-TB Gold Plus assay was established at 13 months after the date of manufacture.

## H.2. LIAISON Control QuantiFERON-TB Gold Plus Kit Storage and Shelf Life Study

A (real-time) study was conducted to evaluate the long term stability of the LIAISON Control QuantiFERON-TB Gold Plus kit and to establish shelf life. Testing was performed in duplicate using three lots of LIAISON QuantiFERON-TB Gold Plus assay kits, and three lots of LIAISON Control QuantiFERON-TB Gold Plus kits.

The performance of the LIAISON Control QuantiFERON-TB Gold Plus kit was evaluated at time points of 1, 2, 4, 6, 9, 11, 12, 13, and 14.5 months after kit manufacture.

Data analysis of the study control results included the following:

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- The mean amount of detected IFN-γ (IU/mL) for each of the controls, at each testing point, was evaluated by comparing it to the established range, as defined in the product Certificate of Analysis (CoA).
- Data for each control tested, on each kit lot, was plotted separately.
- The mean amount of detected IFN-γ (IU/mL) was plotted against the corresponding study time point for each control.
- A linear regression was fitted to the control data.
- The y-intercept of the regression line was calculated to estimate the baseline value for the IFN-γ.
- The 95% confidence intervals for each time point were calculated.
- ±10% deviation from the regression intercepts were indicated on the plots as horizontal lines.

The LIAISON Control QuantiFERON-TB Gold Plus kit shelf life claim was determined to be the time point that is one time point less than the final time point tested before the regression line exceeds ±10% difference from the baseline value, and also within the established range, as defined in the CoA.

The test results of all three lots of positive and negative controls fell within the established range as defined in the product CoA. Regarding all three lots of positive controls tested, the fitted regression line did not exceed ±10% deviation from the regression intercept at any time point during the study. Regarding all three lots of negative controls tested, due to the very low result values, the absolute differences from the regression intercept, at each time point was evaluated, and found to be minimal and acceptable.

Based on the real-time stability study findings, the claimed shelf life of the LIAISON Control QuantiFERON-TB Gold Plus kit was established at 13 months after the date of manufacture when stored at 2-8°C.

## I. Open Reagent Kit Stability Studies

Studies were conducted to evaluate the stability of the LIAISON QuantiFERON-TB Gold Plus kit reagents by simulating normal conditions of use. The simulation included refrigerated storage of the reagent integral (Magnetic Particles, Diluent, and Assay Buffer W) on board LIAISON XL Analyzer for the duration of the study, and reconstitution and refrigerated storage of lyophilized kit reagents (Calibrator A, Calibrator B, Buffer R, and Conjugate) to mimic a customer's routine. Conjugate reconstituted at T=0 was kept for at least six hours on board the analyzer, then stored at 2-8°C. Controls and calibrators reconstituted at T=0 were kept at least four hours on board the analyzer, then stored at 2-8°C.

Testing was conducted using a coded test panel of samples and controls (level 1 and level 2). The samples were prepared by spiking analyte negative lithium heparinized plasma samples with native IFN-γ, to achieve concentrations that spanned the assay result range. Native IFN-γ was obtained from plasma harvested after incubation/stimulation of whole blood in QFT-Plus Mitogen blood collection tubes. Samples were tested in duplicate

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using one lot of LIAISON QuantiFERON-TB Gold Plus assay kit and one lot of LIAISON Control QuantiFERON-TB Gold Plus.

Data analysis of the study results included the following:

- The IU/mL value result, for each reactive sample and control was evaluated and compared to the defined acceptance criteria. The acceptance criteria were the sole means of evaluation for non-reactive samples and controls.
- Calculation of the mean % difference in the amount of detected IFN-γ (in IU/mL) of each reactive sample and control at Time 0 versus the amount of detected IFN-γ at each subsequent time point.
- Plotting of the mean % difference between the amount of detected IFN-γ (in IU/mL) of each reactive sample and control at Time 0 versus the amount of detected IFN-γ at each subsequent time point.
- Fitting a regression line to the sample data to determine the time point at which the regression line exceeds ±10% difference from T=0.

Open reagent stability was determined to be the time point that was one time point less than the final time point tested before the regression line exceeded ±10% difference from T=0 for each reactive sample.

## I.1. Open Conjugate Stability

The performance of the conjugate was evaluated at the following time points after reconstitution: Day 0, 3, 7, 10, 14, 18, 21, and 32. For all reactive samples and controls tested, the fitted regression line did not exceed ±10% deviation from T=0 over 32 days of testing. The non-reactive samples and controls met the defined acceptance criteria from T=0 over 32 days of testing.

Based on the study findings, the open stability of the conjugate was determined to be 21 days. However, in an effort to foster a degree of consistency across assay kit reagents for ease of customer use, the stability of the open conjugate was established at 14 days when stored at 2-8°C.

## I.2. On board Reagent Integral Stability Study

The performance of the reagent integral was evaluated at the following at time points after opening: Week 0, 1, 2, 3, 4, 5, 6, 7, 8, and 9. However, due to the limitation of the calibration stability of 7 weeks, the analysis of study data was limited to 7 weeks. For all reactive samples and controls tested, the fitted regression line did not exceed ±10% deviation from T=0 over 7 weeks of testing. The non-reactive samples and controls met the defined acceptance criteria from T=0 over 7 weeks of testing.

Based on the study findings, the open stability of the reagent integral was determined to be 6 weeks. However, in an effort to foster a degree of consistency across assay kit reagents for ease of customer use, the stability of the open reagent integral was established at 4 weeks when stored at 2-8°C.

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I.3. Open Calibrators Stability Study

The performance of the calibrators was evaluated at the following time points after opening: Week 0, 1, 2, 3, 4, 5, 6, 7, 8, and 9. However, due to the limitation of the on-board reagent integral stability of 5 weeks, the analysis of study data was limited to 5 weeks. For the majority of reactive samples and controls tested, the fitted regression line did not exceed ±10% deviation from T=0 over 5 weeks of testing. The non-reactive samples and controls met the defined acceptance criteria from T=0 over 5 weeks of testing.

Based on the study findings, the open stability of the calibrators was determined to be 5 weeks. However, in an effort to foster a degree of consistency across assay kit reagents for ease of customer use, the stability of the open calibrators was established at 4 weeks when stored at 2-8°C.

J. Open Control Kit Stability Studies

Studies were conducted to evaluate the stability of the LIAISON Control QuantiFERON-TB Gold Plus kit by simulating normal conditions of use. Controls were opened and reconstituted at T=0, stored on the LIAISON XL Analyzer for at least four hours, then stored at 2-8°C. Controls were tested in duplicate, weekly for a total of nine weeks. However due to the limitation of the on-board reagent integral stability of 5 weeks, the analysis of study data was limited to 5 weeks.

Data analysis of the study results included the following:

- The IU/mL value result, for each control was evaluated and compared to the defined acceptance criteria. The acceptance criteria were the sole means of evaluation for the non-reactive control.
- Calculation of the mean % difference in the amount of detected IFN-γ (in IU/mL) of the reactive control at Time 0 versus the amount of detected IFN-γ at each subsequent time point.
- Plotting of the mean % difference between the amount of detected IFN-γ (in IU/mL) of the control at Time 0 versus the amount of detected IFN-γ at each subsequent time point.
- Fitting a regression line to the control data to determine the time point at which the regression line exceeds ±10% difference from T=0.

Open control stability was determined to be the time point that was one time point less than the final time point tested before the regression line exceeded ±10% difference from T=0 for each reactive control. The fitted regression line did not exceed ±10% deviation from T=0 over 5 weeks of testing for the reactive control. The non-reactive control met the defined acceptance criteria from T=0 over 5 weeks of testing.

Based on the study findings, the open stability of the controls was determined to be 5 weeks. However, in an effort to foster a degree of consistency across assay kit reagents for ease of customer use, the stability of the open controls was established at 4 weeks when stored at 2-8°C.

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# K. Reagent and Control Transport Stability Studies

Studies were conducted to verify that LIAISON QuantiFERON-TB Gold Plus assay reagents, and LIAISON Control QuantiFERON-TB Gold Plus maintain performance after transport and delivery to customers.

## K.1. LIAISON QuantiFERON-TB Gold Plus Assay Reagent Kit Transport Stability Study

The shipment of the LIAISON QuantiFERON-TB Gold Plus assay reagent kit includes two parts:

**Part One: Intercontinental shipment from DiaSorin S.p.A in Saluggia, Italy to DiaSorin Inc., in Stillwater, Minnesota.** The product is shipped under storage conditions from 0-30°C and may take 2 to 10 days depending on duration of customs clearance. During the shipment, temperature exposure is monitored with a temperature data logger. In the event that the product is found to have been exposed to temperatures outside the range of 0-30°C, the product is deemed non-conformant and an investigation is initiated.

**Part Two: Shipment within the United States from DiaSorin Inc., in Stillwater, Minnesota to the end user.** This leg of the shipment is at ambient temperature (up to 35.6°C) and occurs overnight (up to a total of 27 hours) or worst case, over a few days.

In this study, reagent transport was simulated according to the following conditions:

- All assay kit reagents⁴ stored for five days (at least 120 hours) at 30°C, representing potential of removal from controlled refrigerated storage conditions (2 – 8°C) and exposure to room temperature (18-24°C) conditions for up to 5 days;
- All assay kit reagents stored for ten days (at least 240 hours) at 30°C, representing potential removal from controlled refrigerated storage conditions (2 – 8°C) and exposure to room temperature (18-24°C) conditions for up to 10 days;
- All assay kit reagents stored for one day (at least 24 hours) at 37°C, representing potential removal from controlled refrigerated storage conditions (2 – 8°C) and exposure to extreme temperature conditions for a short period of time, up to 1 day;
- All assay kit reagents stored for one day (at least 24 hours) at -20°C, representing the possible freezing of product.

The sample test panel used during the study was comprised of three samples:

- High-Negative (0.20 – 0.29 IU/mL)
- Close to Medical Decision Point (0.30 – 0.35 IU/mL)
- Positive (4.0 – 6.0 IU/mL)

These samples were prepared by spiking confirmed analyte-negative human (heparinized) plasma samples with native IFN-γ. Native IFN-γ was obtained via incubation of whole

⁴All assay kit reagents includes: Reagent Integral, Conjugate, Calibrator A, Calibrator B, and Buffer R

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blood in QFT-Plus Mitogen blood collection tubes, and subsequently stimulated plasma with analyte IFN- $\gamma$ was harvested.

The study was performed using one lot of the LIAISON QuantiFERON-TB Gold Plus assay kit and one lot of the LIAISON Control QuantiFERON-TB Gold Plus. Testing was performed once during the life of the assay kit (i.e., at the one month time point). Each simulated stress condition was performed versus the control condition (i.e., product stored at $2 - 8^{\circ}\mathrm{C}$ ). For each of the four stress conditions, and the control condition, a test result was determined by taking the mean of 12 sample test results generated over four days, with one run per day, and three replicates per run. The study design summary is illustrated in Table 18 below.

Table 18. LIAISON QuantiFERON-TB Gold Plus Assay Reagent Kit Transport Stability Study Design Summary

| Sample | Day 1 – 1 Run | Day 2 – 1 Run | Day 3– 1 Run | Day 4– 1 Run | Data point for testing occasion |
| --- | --- | --- | --- | --- | --- |
| High Negative | Replicate 1 | Replicate 1 | Replicate 1 | Replicate 1 | Mean of 12 replicates |
| | Replicate 2 | Replicate 2 | Replicate 2 | Replicate 2 | |
| | Replicate 3 | Replicate 3 | Replicate 3 | Replicate 3 | |
| Close to the Medical Decision Point | Replicate 1 | Replicate 1 | Replicate 1 | Replicate 1 | Mean of 12 replicates |
| | Replicate 2 | Replicate 2 | Replicate 2 | Replicate 2 | |
| | Replicate 3 | Replicate 3 | Replicate 3 | Replicate 3 | |
| Positive | Replicate 1 | Replicate 1 | Replicate 1 | Replicate 1 | Mean of 12 replicates |
| | Replicate 2 | Replicate 2 | Replicate 2 | Replicate 2 | |
| | Replicate 3 | Replicate 3 | Replicate 3 | Replicate 3 | |

The results of each sample tested for each stressed condition were evaluated in terms of the percent deviation from the unstressed reference condition. Results were considered acceptable if the amount of detected IFN- $\gamma$ for each stress condition did not exceed $\pm 10\%$ of the amount of detected IFN- $\gamma$ for the unstressed condition. A summary of the LIAISON QuantiFERON-TB Gold Plus assay reagent kit transport stability study results is illustrated in Table 19 below.

Table 19. LIAISON QuantiFERON-TB Gold Plus Assay Reagent Kit Transport Stability Study Summary Results

| Sample | Stress condition: | REFERENCE (2-8 °C) | 5 DAYS @ 30 °C | 10 DAYS @ 30 °C | 1 DAY @ 37 °C | 1 DAY @ -20 °C |
| --- | --- | --- | --- | --- | --- | --- |
| High Negative | MEAN IU/mL | 0.235 | 0.237 | 0.236 | 0.225 | 0.242 |
| | Percent Deviation from Unstressed Condition | | 0.67% | 0.14% | -4.18% | 2.80% |
| Close to Medical Decision Point | MEAN IU/mL | 0.299 | 0.319 | 0.319 | 0.294 | 0.319 |
| | Percent Deviation from Unstressed Condition | | 6.92% | 6.75% | -1.54% | 6.89% |
| Positive | MEAN IU/mL | 4.02 | 4.08 | 4.37 | 4.28 | 4.10 |
| | Percent Deviation from Unstressed Condition | | 1.35% | 8.57% | 6.25% | 1.84% |

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The acceptance criterion was met for all stress conditions tested demonstrating that the LIAISON Control QuantiFERON-TB Gold Plus assay reagents maintain acceptable performance after simulations that mimic product transport conditions.

## K.2. LIAISON Control QuantiFERON-TB Gold Plus Kit Transport Stability Study

The shipment of the LIAISON Control QuantiFERON-TB Gold Plus kit includes two parts:

**Part One: Intercontinental shipment from DiaSorin S.p.A in Saluggia, Italy to DiaSorin Inc., in Stillwater, Minnesota.** The product is shipped under storage conditions from 0-30°C and may take from 2 to 10 days depending on duration of customs clearance. During the shipment, temperature exposure is monitored with a temperature data logger. In the event that the product is found to have been exposed to temperatures outside the range of 0-30°C, the product is deemed non-conformant and an investigation is initiated.

**Part Two: Shipment within the United States from DiaSorin Inc., in Stillwater, Minnesota to the end user.** This leg of the shipment is at ambient temperature (up to 35.6°C) and is expected to occur overnight (up to a total of 27 hours) or worst case, over a few days.

In this study, reagent transport was simulated according to the following conditions:

- Controls stored for five days (at least 120 hours) at 30°C, representing potential of removal from controlled refrigerated storage conditions (2 – 8°C) and exposure to room temperature (18-24°C) conditions for up to 5 days;
- Controls stored for ten days (at least 240 hours) at 30°C, representing potential removal from controlled refrigerated storage conditions (2 – 8°C) and exposure to room temperature (18-24°C) conditions for up to 10 days;
- Controls stored for one day (at least 24 hours) at 37°C, representing potential removal from controlled refrigerated storage conditions (2 – 8°C) and exposure to extreme temperature conditions for a short period of time, up to 1 day;
- Controls stored for one day (at least 24 hours) at -20°C, representing the possible freezing of product.

The study was performed using one lot of the LIAISON QuantiFERON-TB Gold Plus assay kit and one lot of the LIAISON Control QuantiFERON-TB Gold Plus. Testing was performed once during the life of the control kit (i.e., at the one month time point). Each simulated stress condition was performed versus the control condition (i.e., product stored at 2-8°C). For each of the four stress conditions, and the control condition, a test result was determined by taking the mean of 12 test results generated over four days, with one run per day, and three replicates per run. The study design summary is illustrated in Table 20 below.

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Table 20. LIAISON Control QuantiFERON-TB Gold Plus Kit Transport Stability Study Design Summary

| Control | Day 1 – 1 Run | Day 2 – 1 Run | Day 3– 1 Run | Day 4– 1 Run | Data point for testing occasion |
| --- | --- | --- | --- | --- | --- |
| Negative Control RS 912 | Replicate 1 | Replicate 1 | Replicate 1 | Replicate 1 | Mean of 12 replicates |
| | Replicate 2 | Replicate 2 | Replicate 2 | Replicate 2 | |
| | Replicate 3 | Replicate 3 | Replicate 3 | Replicate 3 | |
| Positive Control RS 914 | Replicate 1 | Replicate 1 | Replicate 1 | Replicate 1 | Mean of 12 replicates |
| | Replicate 2 | Replicate 2 | Replicate 2 | Replicate 2 | |
| | Replicate 3 | Replicate 3 | Replicate 3 | Replicate 3 | |

The results of the controls (Negative RS 912, Positive RS 914) tested for each stress condition were evaluated in terms of the percent deviation from the unstressed reference condition. Results were considered acceptable if the amount of detected IFN-γ of each stressed condition did not exceed ±10% of the amount of detected IFN-γ of the unstressed condition. A summary of the LIAISON Control QuantiFERON-TB Gold Plus kit transport stability study results is illustrated in Table 21 below.

Table 21. LIAISON Control QuantiFERON-TB Gold Plus Kit Transport Stability Study Summary Results

| Control | Stress condition | REFERENCE (2-8 °C) | 5 DAYS @ 30 °C | 10 DAYS @ 30 °C | 1 DAY @ 37 °C | 1 DAY @ -20 °C |
| --- | --- | --- | --- | --- | --- | --- |
| RS 912 Negative Control | MEAN IU/mL | 0.042 | 0.041 | 0.038 | 0.041 | 0.043 |
| | Percent Deviation from Unstressed Condition | | -1.87% | -7.87% | -1.40% | 3.65% |
| RS 914 Positive Control | MEAN IU/mL | 0.96 | 1.01 | 0.96 | 1.00 | 1.00 |
| | Percent Deviation from Unstressed Condition | | 4.78% | -0.31% | 4.01% | 4.62% |

The acceptance criterion for all stress conditions tested was met demonstrating that the LIAISON Control QuantiFERON-TB Gold Plus kit maintains acceptable performance after simulations that mimic product transport conditions..

## L. Software

A software review was conducted, and all documentation provided was found to be appropriate and acceptable. A summary of the elements of the review are as follows:

- Level of Concern: Moderate; the level of concern is acceptable
- Device Hazard Analysis: Relevant software hazards were identified, and detailed risk assessments and mitigations were provided.
- Verification and Validation: Verification and validation plans, logs, and result summaries were provided for all versions of instrument software.
- Revision Level History: This is the first version of the LIAISON QuantiFERON Software to be submitted for review.
- Unresolved Anomalies: Documentation of all unresolved anomalies was found to be acceptable. Residual issues do not impact assay safety or effectiveness.

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A cybersecurity review was also conducted and found to be compliant/acceptable.

## X. SUMMARY OF PRIMARY CLINICAL STUDY

### A. Clinical Study Design

A multi-site clinical agreement study was conducted comparing the LIAISON QuantiFERON-TB Gold Plus assay to the QIAGEN QuantiFERON-TB Gold Plus (QFT-Plus). Samples were collected and tested from study subjects who had signs and symptoms consistent with active TB disease (Active TB cohort), subjects who had no identified risk factors for tuberculosis infection (Low Risk cohort), and subjects with at least one known risk factor for tuberculosis exposure and at risk for latent TB infection (Mixed Risk cohort).

### A.1 Clinical Performance Study Analysis

There is no definitive reference method for confirming or excluding the diagnosis of latent tuberculosis infection (LTBI); accordingly an estimate of sensitivity and specificity of the LIAISON QuantiFERON-TB Gold Plus assay cannot be directly evaluated. Therefore, the specificity of the LIAISON QuantiFERON-TB Gold Plus assay was approximated by testing plasma samples from a cohort of study subjects with no known risk factors of tuberculosis infection (low risk). Similarly, sensitivity was approximated by testing plasma samples from a cohort of study subjects with culture confirmed active tuberculosis (TB) disease. Assay performance was further evaluated by testing plasma samples from a cohort of healthy study subjects with identified risk factors for LTBI (mixed risk). Comparative performance of the LIAISON QuantiFERON-TB Gold Plus relative to QIAGEN QuantiFERON-TB Gold Plus test was also evaluated.

### A.2 Clinical Inclusion and Exclusion Criteria

#### Inclusion Criteria

##### Active TB Cohort

- Subjects with clinical symptoms consistent with suspicion for active TB disease;
- Subjects receiving, or are likely to receive, therapy for active TB (therapy must not have been initiated more than 2 weeks before recruitment into the study);
- Sputum and/or other relevant bodily samples collected from these individuals for AFB smear and culture testing to determine the presence of *M. tuberculosis* organisms. Alternatively, FDA approved nucleic acid amplification (NAA) methods in combination with culture testing were used to confirm the presence of *M. tuberculosis* organisms.

##### Low Risk Cohort

- Individuals with no identified risk factors for tuberculosis infection.

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# Mixed Risk Cohort

- Subjects with at least one of the following known risk factors for latent TB infection (LTBI)
- Recent contact with a known source case.
- Inmate at a correctional facility.
- Resident of a homeless shelter or nursing home.
- Healthcare worker.
- Time spent in a country with a high prevalence of tuberculosis.
- Subjects deemed free from tuberculosis disease and exhibiting no clinical signs/symptoms consistent with active tuberculosis disease.
- In addition, this group also included subjects who are HIV positive, and subjects who have been BCG vaccinated.

# Exclusion Criteria

## Active TB Cohort

- Subjects who have taken therapy for active tuberculosis infection or latent TB infection for more than 14 days.
- Subjects for which culture confirmation of *M. tuberculosis* was not obtained.
- Subjects aged less than 18 years or greater than 80 years.

## Low Risk Cohort

- Subjects who have resided for more than one (1) month in an area with a current active TB rate of &gt;50/100,000
- Subjects who have lived, volunteered, or worked for longer than one (1) month in a nursing home, hospital, homeless shelter, or other situation known to be associated with an increased risk of TB exposure.
- Individuals having exposure to someone who was sick with TB.
- Subjects with a previous history of TB diagnosis or treatment for TB.
- Subjects having immunosuppressive conditions that could interfere with a person’s ability to initiate a cell mediated immune response in the assay such as the following:
- HIV infection
- Cancer requiring chemotherapy in the preceding 3 months
- Rheumatoid arthritis
- Renal failure
- Systemic immunosuppressive usage
- Subjects aged less than 18 years or greater than 80 years.

## Mixed Exposure Risk Cohort

- Subjects not having at least one of the known risk factors for tuberculosis exposure (see Inclusion Criteria, Mixed Risk).
- Subjects presenting with clinical signs and symptoms consistent with active…