Part 1 of webinar: Multiplexed Serodiagnostic Test with Advanced Machine Learning Classifier for Single-Tier Diagnosis of Lyme Disease

Lyme disease is a high-incidence infectious disease with a global prevalence of 14.5% of the world's population. Two-tier serology is currently the primary diagnostic test for Lyme disease; however, this approach has limitations and does not aid in diagnosis at the earliest, most treatable stage. Here, we present an innovative multiplexed serodiagnostic test aiming to improve the diagnostic accuracy of Lyme disease testing. The assay measures both IgM and IgG antibodies against multiple Borrelia proteins and peptides utilizing Luminex xMAP® Technology, and includes a unique calibrator for quantification. Luminex’s output is interpreted with an advanced machine learning classifier to provide an unambiguous test result. The test is performed using Luminex analyzers already in place in many clinical laboratories. This test could potentially improve early diagnosis of Lyme disease, enabling earlier treatment to reduce the risk of Lyme-associated chronic illness attributed to undiagnosed cases. This single test achieves high sensitivity without losing specificity in people with both early- and late-stage Lyme disease.

Learning Objectives: 

1. Discuss the challenges of diagnosing Lyme disease and the limitations of the current two-tier serological tests.

2. Explain how the combination of IgM and IgG antibody detection with a machine-learning classifier significantly improves test accuracy.

3. Compare test performance of the multiplexed immunoassay with two tier serology, and consider how improved early detection of Lyme disease would improve patient short- and long-term outcomes.
 

Part 2 of webinar: A Novel Blood Test for Axial Spondyloarthritis: Anti-14-3-3 eta Multiplex Test Validation

Background:

New Day Diagnostics, LLC validated the Anti-14-3-3η Multiplex Laboratory Developed Test (LDT) for precision, analytical sensitivity, specificity, accuracy, reportable range, and robustness. The assay qualitatively detects anti-14-3-3η autoantibodies against five peptide targets in human serum using xMAP® multi-analyte immunoassay technology. Elevated anti-14-3-3η levels are associated with axial spondyloarthritis (axSpA), a type of arthritis that causes inflammation in the spine and large joints that results in pain and stiffness.

Methods:

Precision followed a 20-day, 2-run-per-day, 2-replicate-per-run design (80 data points per sample). Analytical sensitivity and hook-effect studies used CLSI EP17-A2 guidelines. Clinical performance was assessed with 269 retrospective serum samples (158 axSpA, 111 healthy) and assessed by cross-validation. Accuracy, linearity, robustness, interference, and stability were evaluated under protocol-specific acceptance criteria.

Results:

Repeatability ranged 4.5–11.4 %CV and within-laboratory precision 8.3–14 %CV. Limit of Blank (LoB) & Limit of Detection (LoD) were determined for all targets. No significant hook effect or clinically relevant cross-reactivity was observed except for minor signal reduction for target 7 ≥ 160 mg/dL. Cross-validation yielded 60.2 % positive percent agreement (PPA) and 75.7 % negative percent agreement (NPA), exceeding acceptance thresholds (≥ 49.5 % PPA, ≥ 66.3 % NPA). Accuracy reached 96.0 % agreement. All targets exhibited linearity. Robustness and stability studies passed; serum remained stable ≥ 5 weeks at –80 °C, 48 h at 2–8 °C, and 48 h at room temperature.

Conclusions:

The Anti-14-3-3η Multiplex LDT met or exceeded all analytical and clinical validation criteria. With 60.2 % sensitivity, 75.7 % specificity, and 96 % accuracy, the assay demonstrates reliable performance suitable for clinical application in evaluating patients for axial spondyloarthritis.

Part 1 – first 30-minutes of Bio-Techne’s Webinar

Revolutionizing Bladder Cancer Care How Multiplex Assays Are Changing the Game

Hematuria may occur in up to 10% of the general population and results in costly evaluation to ensure it is of no consequence, i.e., no bladder cancer (BCa) is present since hematuria is typically the initial sign of BCa. Furthermore, 75% of patients with newly diagnosed BCa have non-muscle-invasive disease (NMIBC), which has a very high recurrence rate (>70%). Because of this, it is recommended that the patients have adjuvant intravesical bacillus Calmette-Guerin (BCG) instillation to reduce this risk. Despite BCG treatment, up to 50% of patients fail to respond and 20% progress to muscle invasive bladder cancer mandating more radical treatment (i.e., cystectomy - removal of the urinary bladder). Moreover, the delay in radical treatment can negatively impact survival rates, hence, a test that could predict treatment response to intravesical BCG, ensuring the right patient, gets the right treatment at the right time is urgently required. Our group has developed and tested a multiplex immunoassay towards a BCa signature with extremely encouraging results in subjects evaluated for hematuria and in subjects destined to be treated with BCG. This research will open the door for improving on the non-invasive methods for detecting BCa and predicting treatment response as such it will have a marked impact on patient care.

Learning Objectives:

1. Explore how multiplex assays are advancing non-invasive approaches for bladder cancer detection.

2. See how a novel assay may help predict patient response to intravesical BCG therapy. 

3. Discover a multiplex immunoassay delivering impressive results in both hematuria evaluations and BCG-treated patient groups.

Part 2 – second 30-minutes of Bio-Techne’s Webinar

Multiplex measurement of tumor biomarkers associated with colorectal adenocarcinoma using novel sample types

Discovery of new tumor biomarkers and their roles in cancer progression is an intense research area. Testing strategies that utilize a combination of proteins associated with cancers may improve diagnosis and monitoring chemotherapy in patients. Multiplex immunoassays are a valuable tool for investigating potential biomarkers in serum associated with cancer. We report here using a 28-plex Luminex performance immunoassay to measure tumor biomarkers in serum, plasma (EDTA, heparin, and Streck), and tissue lysates. Sera from late-stage colorectal cancer (CRC) patients had several potential tumor biomarkers at higher levels than sera from healthy donors. Tissue lysates prepared from late-stage CRC and healthy adjacent fresh-frozen tissue were used to measure CEA, CA19-9, IL-8, and MIF. The levels of MIF and IL-8 in these tissue lysates measured using Luminex was validated using the Jess™ Simple Western System from Bio-Techne. Stability testing of tumor biomarkers in EDTA, heparin, Streck® Cell-Free BCT®, and Streck Protein Plus plasma samples from healthy donors after 0, 3 and 5 days at room temperature revealed stabilization of most analyte concentrations using Streck tubes, with the exceptions of ENO-2 and CYFRA21-1. Multiplex immunoassays are a valuable tool for the discovery and development of soluble tumor biomarkers associated with cancer.

Learning Objectives:

1. Discover how multiplex immunoassays are a valuable tool for the discovery and development of soluble tumor biomarkers associated with cancer. 

2. See result of measuring biomarkers associated with colon cancer using serum, plasma, and tissue lysates using the R&D Systems Human Tumor Biomarker Performance Assay. 

3: Find out how the R&D Systems Human Tumor Biomarker Performance Assay enables the simultaneous quantitation of multiple tumor biomarkers in more sample types than any other Luminex assay.