Interview Questions for In-Depth Knowledge of IVD (in vitro Diagnostics).

The key difference between IVDs (In Vitro Diagnostics) and LDTs (Laboratory Developed Tests) lies in their regulatory pathways and manufacturing processes. IVDs are commercially manufactured diagnostic tests that undergo rigorous premarket review and approval by regulatory bodies like the FDA (in the US) or the EMA (in Europe). They adhere to strict quality control standards throughout their manufacturing and distribution. Think of them as standardized, ready-to-use kits available to any qualified laboratory.

LDTs, on the other hand, are diagnostic tests designed, manufactured, and used within a single laboratory. They are not subject to the same level of premarket scrutiny as IVDs. While they often provide unique solutions for specific clinical needs, their quality control and validation are typically less standardized and subject to internal laboratory quality control processes. An analogy would be comparing a mass-produced car (IVD) to a custom-built car (LDT) – both serve the same basic function (transportation/diagnosis), but their development, quality assurance, and overall accessibility differ significantly.

I have extensive experience working under ISO 13485, the internationally recognized quality management system standard for medical devices. This includes involvement in every stage of the product lifecycle, from design and development to manufacturing and post-market surveillance. My responsibilities have encompassed implementing the standard’s requirements, conducting internal audits, and managing corrective and preventive actions (CAPAs). This experience directly translates to ensuring that products meet the highest quality standards, regulatory requirements and customer expectations.

Beyond ISO 13485, I’m familiar with other relevant IVD regulations such as the EU In Vitro Diagnostic Regulation (IVDR) and FDA’s 21 CFR Part 820, covering Good Manufacturing Practices (GMP). I’ve also been involved in navigating other regulatory requirements specific to individual countries and adapting product documentation to meet these diverse stipulations. In one project, we had to completely overhaul our quality systems to conform to the newly implemented IVDR, involving significant documentation updates and process adjustments. This provided invaluable experience in navigating the complexities of international regulatory compliance.

I am very familiar with the FDA’s regulations for IVD medical devices, including 21 CFR Part 820 (GMP) and the specific requirements for different classes of IVDs based on their risk classification. I understand the different pathways for submitting premarket notifications (510(k)) or premarket approvals (PMA), the importance of design controls, and the need for robust quality systems. I’m also aware of the post-market surveillance requirements and the importance of reporting adverse events.

My experience involves creating and reviewing submissions, understanding labeling requirements and handling interactions with FDA inspectors. For example, I was involved in a 510(k) submission for a new molecular diagnostic assay, where we had to meticulously document our equivalence to a predicate device and demonstrate the safety and efficacy of our product. This involved extensive testing, data analysis, and close collaboration with regulatory consultants to meet all requirements. I have a deep understanding of the complexities involved and the rigorous testing needed to ensure market authorization.

Key performance characteristics of an IVD assay are crucial for its reliability and clinical utility. They can be broadly categorized into:

• Analytical Performance: This includes sensitivity (ability to detect low concentrations of the analyte), specificity (ability to correctly identify the target analyte without cross-reactivity), accuracy (closeness of the measured value to the true value), precision (reproducibility of measurements), and linearity (consistent response across the assay’s measurement range).
• Clinical Performance: This focuses on the assay’s performance in a real-world clinical setting. Key parameters include diagnostic sensitivity (the proportion of truly diseased individuals correctly identified by the test), diagnostic specificity (the proportion of truly non-diseased individuals correctly identified), positive predictive value (probability of a truly positive result given a positive test result), negative predictive value (probability of a truly negative result given a negative test result), and clinical utility (overall benefit of using the test in a specific clinical context).
• Operational Characteristics: Aspects like ease of use, turnaround time, cost-effectiveness, required equipment, and storage conditions also significantly impact the overall performance and practical usability of the assay.

Understanding these characteristics is vital for selecting, validating, and using an appropriate IVD assay, ensuring its reliable contribution to patient diagnosis and management.

IVD assay validation is a critical process to demonstrate that the assay performs reliably and accurately as intended. It’s a systematic approach to verify the analytical and clinical performance characteristics mentioned earlier. The process typically involves the following stages:

1. Assay Design and Development: Defining the assay’s purpose, selecting appropriate methods, and optimizing assay parameters.
2. Analytical Validation: This focuses on the analytical performance characteristics using appropriate samples and statistical methods. It includes tests for linearity, accuracy, precision, limit of detection (LOD), limit of quantitation (LOQ), and specificity.
3. Clinical Validation: This verifies the assay’s performance in a clinical setting, often involving comparison to a reference standard (gold standard test) using samples from a relevant patient population. This stage helps establish diagnostic sensitivity and specificity.
4. Documentation and Reporting: Meticulous documentation of all procedures, data, and results is crucial. A comprehensive validation report summarizes the findings and conclusions.

Successful validation provides strong evidence that the assay meets its intended purpose and can be reliably used for its clinical application. Failure to properly validate an assay can lead to inaccurate results, misdiagnosis, and potentially harm patients.

Ensuring the quality and accuracy of IVD test results is paramount. This involves a multi-faceted approach focusing on the entire testing process:

• Pre-analytical Phase: Proper sample collection, handling, storage, and transportation are crucial to prevent degradation or contamination. Standardized protocols and well-trained personnel are essential.
• Analytical Phase: Using validated assays, employing appropriate quality controls, performing regular instrument maintenance and calibration, and following standardized operating procedures are critical elements.
• Post-analytical Phase: Accurate data entry, proper interpretation of results, and timely reporting are necessary. Quality management systems (QMS) including robust internal quality control, external quality assessment (EQA) participation, and regular audits help to maintain consistency and detect any deviations from expected performance.
• Personnel Training and Competency: Ensuring that laboratory personnel receive adequate training on all aspects of testing, from sample handling to result interpretation, is fundamental. Regular competency assessments can ensure ongoing proficiency.

By meticulously managing each phase, laboratories can ensure that IVD test results are reliable, accurate, and contribute meaningfully to patient care.

My experience encompasses various IVD technologies, including:

• ELISA (Enzyme-Linked Immunosorbent Assay): I’ve worked extensively with ELISA, particularly in developing and validating assays for detecting various biomarkers in serum or plasma samples. This includes experience with both direct and indirect ELISA methods, optimization of assay parameters, and troubleshooting common issues.
• PCR (Polymerase Chain Reaction): My experience includes designing and optimizing PCR-based assays for the detection of nucleic acids (DNA or RNA) from various sample types. This encompasses real-time PCR (qPCR) for quantitative analysis and conventional PCR for qualitative detection. I understand the principles of primer and probe design, optimization of PCR conditions, and interpretation of PCR results. I’ve also worked with multiplexing PCR applications for simultaneously detecting several targets.
• Flow Cytometry: I’m experienced in using flow cytometry for the analysis of cells and their characteristics. This includes experience in experimental design, data acquisition, and analysis using specialized software. My work involves analyzing cell populations based on their size, granularity, and expression of specific surface markers. I’ve applied flow cytometry techniques in immunology research and clinical diagnostics.

Each technology presents unique challenges and opportunities. Understanding their strengths and limitations is critical for selecting the most appropriate technology for a specific diagnostic application. For example, PCR’s high sensitivity makes it ideal for detecting low levels of pathogens, while flow cytometry is powerful for characterizing immune cell populations in immunological disorders.

The Limit of Detection (LOD) and Limit of Quantification (LOQ) are crucial parameters in In Vitro Diagnostics (IVDs) that define the assay’s sensitivity and precision. The LOD represents the lowest concentration of an analyte that can be reliably distinguished from background noise, essentially determining if a signal is present. The LOQ, on the other hand, is the lowest concentration of an analyte that can be measured with acceptable accuracy and precision. Think of it like this: LOD tells you if something is there, while LOQ tells you how much is there.

For example, imagine testing for a specific hormone in a blood sample. The LOD might be 0.5 ng/mL, meaning the assay can reliably detect concentrations above 0.5 ng/mL. However, the LOQ might be 2 ng/mL because below that concentration, the measurements become too variable to be accurate. We typically use statistical methods, often involving analyzing blank samples and low-concentration samples, to determine the LOD and LOQ. These calculations often involve establishing thresholds based on the standard deviation of the background signal.

Troubleshooting IVD assay performance issues requires a systematic approach. My strategy typically involves a series of steps, starting with a review of the assay protocol, reagents, and equipment. Let’s say we observe unexpectedly high variability in results. First, I’d check the quality of the reagents – are they within their expiration dates, have they been stored correctly, and are there any signs of degradation? Then, I’d examine the instrument calibration and maintenance logs to rule out any instrument-related issues. If the reagents and instrument are fine, I would assess the sample preparation techniques to ensure consistent handling and processing.

If the problem persists, I would move on to investigating potential contamination of the reagents or samples. Finally, if all these checks yield no results, a deeper investigation including statistical analysis of the data and perhaps even redesigning certain aspects of the assay procedure might be required. Throughout the process, maintaining meticulous documentation is crucial for tracing the root cause and implementing corrective actions.

I have extensive experience in designing and conducting IVD clinical trials, from concept development to final report writing. This includes defining study objectives, selecting appropriate study designs (e.g., prospective, retrospective, randomized controlled trials), and identifying and recruiting suitable participants. I’m familiar with regulations like Good Clinical Practice (GCP) and relevant guidelines for IVD studies. I’ve worked on trials assessing the diagnostic accuracy of various IVDs, including those for infectious diseases and cardiovascular markers, and I’m proficient in managing data collection, analysis, and interpretation in accordance with the study protocol. A specific example includes a clinical trial I managed for a new rapid diagnostic test for influenza, where we compared its performance to the gold standard method using a rigorous statistical analysis.

I’m very familiar with the statistical methods used in IVD studies. My expertise encompasses descriptive statistics, inferential statistics, and regression analysis. For assessing diagnostic accuracy, I routinely use methods like sensitivity, specificity, positive and negative predictive values, likelihood ratios, and receiver operating characteristic (ROC) curve analysis. Understanding and applying these techniques allows for proper interpretation of results, and drawing meaningful conclusions about the performance of the IVD. For example, during a study evaluating a new cardiac marker assay, I employed ROC curve analysis to determine the optimal cut-off value for maximizing the test’s diagnostic accuracy. I’m also proficient in using statistical software packages like SAS and R for data analysis and visualization.

The development of an IVD product typically involves several distinct stages. It starts with the concept and design phase, where the target analyte, intended use, and assay principle are defined. This is followed by prototyping and pre-clinical development, where the assay is developed and optimized in the laboratory. Analytical validation is next, where parameters like sensitivity, specificity, precision, and accuracy are rigorously determined. Then comes clinical validation, involving clinical trials to assess the performance of the IVD in real-world settings. After successful clinical validation, regulatory submissions are prepared and submitted to the relevant regulatory bodies (e.g., FDA, EMA). Finally, manufacturing and commercialization involves scaling up the manufacturing process, quality control, and marketing the product.

Ensuring the stability and shelf life of an IVD product is critical for its quality and reliability. This involves a comprehensive approach involving various studies. Firstly, accelerated stability studies are conducted under stressed conditions (e.g., high temperature, humidity) to predict the product’s long-term stability. Secondly, real-time stability studies are performed under normal storage conditions to monitor changes in the product’s performance over time. These studies assess factors like reagent degradation, changes in assay performance, and the integrity of the packaging. The data collected from these studies helps to determine the expiration date and appropriate storage conditions to maintain the product’s quality and performance within acceptable limits. Careful consideration is given to all aspects of the product, including the packaging to ensure optimal protection from environmental factors.

My experience in managing IVD manufacturing processes includes overseeing all aspects of production, from raw material procurement to finished product release. This involves ensuring compliance with Good Manufacturing Practices (GMP), establishing quality control procedures, and implementing robust process validation protocols. I’m familiar with various manufacturing techniques used in IVD production, such as liquid handling automation, lyophilization, and packaging. Furthermore, I possess expertise in managing and optimizing manufacturing processes to improve efficiency, reduce costs, and maintain product quality. For example, in a previous role, I implemented a new automated liquid handling system, which increased the throughput of the manufacturing process by 30% while simultaneously reducing error rates.

Ensuring compliance with regulatory requirements in IVD manufacturing is paramount. It’s not just about ticking boxes; it’s about building a culture of quality and safety. This involves a multi-faceted approach encompassing design control, manufacturing processes, quality control, and post-market surveillance.

• Design Control: We meticulously document every aspect of the product design, from initial concept to final validation, ensuring traceability and compliance with regulations like the FDA’s 21 CFR Part 820 (in the US) or the EU’s IVDR. This involves rigorous risk assessments, verification and validation testing, and meticulous record-keeping.
• Manufacturing Processes: We adhere to strict Good Manufacturing Practices (GMP) guidelines, implementing robust quality control checks at each stage of production. This includes calibration of equipment, proper handling of materials, environmental monitoring, and operator training. We utilize statistical process control (SPC) to monitor our processes and identify potential deviations early on.
• Quality Control: Thorough quality control testing is performed on raw materials, in-process materials, and finished products to ensure they meet predefined specifications. This often includes performance verification, stability testing, and sterility testing depending on the device type. We employ a robust quality management system (QMS) and utilize software to track and manage all aspects of quality control.
• Post-Market Surveillance: Even after a product is launched, we actively monitor its performance through post-market surveillance activities, promptly addressing any reported issues or identified trends. This involves collecting and analyzing data on product performance, adverse events, and complaints.

For example, in one project, we implemented a new automated system for filling and sealing cartridges. Before launch, we performed extensive validation testing, including simulations of worst-case scenarios, to verify its reliability and reproducibility under different environmental conditions. This ensured that the system consistently delivered the desired product quality while complying with GMP.

Risk management is woven into the fabric of every stage of IVD development and manufacturing. It’s not a separate activity but an integral part of our process. We use a structured approach, commonly employing a Failure Modes and Effects Analysis (FMEA) and risk prioritization techniques.

• Hazard Identification: We systematically identify potential hazards throughout the product lifecycle, from design to use. This includes potential risks to patients, operators, and the environment.
• Risk Assessment: We evaluate the likelihood and severity of each identified hazard. Factors such as the probability of occurrence and the potential impact are considered.
• Risk Control: We develop and implement controls to mitigate identified risks. This might involve design modifications, improved manufacturing processes, enhanced labeling, or changes to the user instructions.
• Risk Monitoring and Review: We continuously monitor and review implemented controls to ensure their ongoing effectiveness. This often involves periodic audits and risk reassessments.

For instance, in the development of a rapid diagnostic test, we conducted an FMEA to identify potential failure modes, such as reagent degradation or improper sample handling. We implemented controls such as using stabilized reagents, providing detailed instructions, and incorporating visual indicators to minimize these risks.

Handling complaints about IVD products requires a structured and systematic approach to ensure patient safety and product improvement. Our process centers around prompt investigation, detailed documentation, and proactive corrective actions.

• Complaint Receipt and Recording: All complaints are promptly acknowledged and logged in our complaint management system, ensuring complete traceability.
• Investigation: A thorough investigation is conducted to determine the root cause of the complaint. This often involves reviewing the product’s history, production records, and potentially conducting further testing.
• Corrective and Preventive Actions (CAPA): Based on the investigation’s findings, we implement appropriate corrective actions to address the immediate problem and preventive actions to prevent similar issues from recurring. This might involve design changes, improved training, or revised manufacturing processes.
• Reporting: We maintain detailed records of all complaints and the actions taken. We also report significant complaints to regulatory authorities as required.
• Customer Communication: We keep the customer informed of the investigation’s progress and the actions taken to resolve the complaint. This fosters trust and strengthens customer relations.

In one case, we received complaints about inconsistent results from a particular batch of a diagnostic test. Our investigation revealed a problem with a specific reagent used in that batch. We immediately initiated a recall of the affected batch, replaced it with a properly manufactured batch, and investigated the root cause of the reagent issue, implementing improvements to our manufacturing process to prevent recurrence.

Post-market surveillance (PMS) is critical for ensuring the continued safety and effectiveness of IVD devices after they’ve been launched into the market. It’s a continuous process involving data collection, analysis, and action.

• Data Collection: We collect data from various sources, including post-market reports, adverse event reports, customer feedback, and field performance data. This data provides valuable insights into the product’s real-world performance.
• Data Analysis: We analyze the collected data to identify trends, patterns, and potential problems. Statistical methods and data visualization techniques are used to identify potential issues early on.
• Action and Reporting: Based on the data analysis, we take appropriate actions, which could range from minor process adjustments to major product recalls. We also report any significant findings to regulatory agencies, as required.

For example, through post-market surveillance of a blood glucose monitoring system, we detected a slight increase in inaccurate readings under specific environmental conditions. This led us to revise the user instructions with additional recommendations and initiate a software update to improve the device’s accuracy under those conditions.

Staying current in the rapidly evolving IVD field requires a proactive and multifaceted approach.

• Scientific Literature: We regularly review peer-reviewed scientific journals and publications to stay abreast of the latest research and advancements.
• Industry Conferences and Trade Shows: Attending industry conferences and trade shows allows us to network with peers, learn about new technologies, and gain insights into market trends.
• Professional Organizations: Membership in professional organizations like the AACC (American Association for Clinical Chemistry) or similar organizations provides access to educational resources and networking opportunities.
• Online Resources: We utilize online resources such as databases, webinars, and online courses to expand our knowledge and expertise.
• Collaboration: We collaborate with researchers and other industry experts to share knowledge and stay informed about cutting-edge developments.

For example, I recently attended a conference on next-generation sequencing technologies and learned about promising new diagnostic applications, potentially impacting our future product development plans.

The global IVD market is a dynamic and rapidly growing sector, driven by factors like increasing prevalence of chronic diseases, technological advancements, and growing healthcare expenditure globally. The market is highly segmented, with various product categories like clinical chemistry, immunoassay, molecular diagnostics, microbiology, and point-of-care testing.

Key trends include the rising adoption of automation and digitalization in IVD labs, the growth of personalized medicine and companion diagnostics, and the increasing demand for cost-effective and rapid diagnostic tests. Different regions show varying growth rates, with emerging markets in Asia and Africa showing particularly strong potential for expansion. Regulatory landscapes vary globally, which impacts product development and market access strategies.

Understanding these market dynamics is essential for developing successful IVD products and strategies. We regularly monitor market trends and regulatory changes to adapt our strategies and optimize product offerings for specific regional markets.

Developing effective marketing strategies for IVD products requires a deep understanding of the target audience, the competitive landscape, and the regulatory environment. We employ a multi-pronged approach tailored to the specific product and market.

• Target Audience Definition: We clearly define our target audience – whether it’s clinicians, laboratory technicians, or healthcare administrators. This helps us tailor our messaging and choose the appropriate channels.
• Value Proposition: We clearly articulate the unique value proposition of our IVD product, emphasizing its clinical benefits, ease of use, cost-effectiveness, and any unique features. This is central to effective communication.
• Marketing Channels: We utilize a range of channels to reach our target audience, including scientific publications, industry conferences, online marketing, direct sales, and partnerships with key opinion leaders.
• Regulatory Compliance: All marketing materials must be compliant with relevant regulations and avoid any misleading or unsubstantiated claims.
• Data-Driven Optimization: We continuously track marketing campaign performance to measure their effectiveness and make data-driven improvements.

For a new molecular diagnostic test, we focused our marketing efforts on demonstrating its improved accuracy and speed compared to existing methods. We collaborated with key opinion leaders to present our data at scientific conferences and published articles showcasing the test’s clinical utility. This strategy led to strong adoption of the new test in leading hospitals.

Interpreting IVD assay results involves a multi-step process that goes beyond simply reading a numerical value. It requires understanding the assay’s methodology, the clinical context of the patient, and potential sources of error. First, we compare the obtained result to the assay’s established reference intervals (or cut-offs for qualitative assays). This interval defines the range of values expected in a healthy population. Values outside this range may indicate a disease or condition.

Next, we assess the assay’s performance characteristics, such as sensitivity and specificity. Sensitivity refers to the assay’s ability to correctly identify individuals with the condition, while specificity measures its ability to correctly identify individuals without the condition. Low sensitivity might lead to false negatives, while low specificity results in false positives.

Furthermore, we must consider pre-analytical factors such as proper sample collection, handling, and storage, all of which significantly impact the accuracy of results. Finally, we interpret the findings within the clinical context by considering other relevant patient information (medical history, other test results, symptoms) to arrive at a comprehensive diagnosis. For example, an elevated troponin level (a cardiac marker) may indicate a heart attack, but this conclusion needs corroboration with other clinical data and the patient’s history.

The analysis may also involve evaluating the assay’s control values. Internal controls assess the assay’s performance during each run, while external controls are used for monitoring long-term performance and compliance with quality standards. Deviations from expected control values highlight potential problems requiring investigation.

Clinical samples used in IVD testing are diverse, reflecting the wide range of analytes and diseases diagnosed. Common examples include:

• Blood samples (serum, plasma, whole blood): These are used for a vast array of tests, ranging from basic hematology and blood chemistry to more specialized assays for detecting infections, hormones, and tumor markers.
• Urine samples: Used for diagnosing urinary tract infections, kidney disorders, and metabolic diseases. Analysis often involves dipstick tests for initial screening and more sophisticated techniques for detailed assessments.
• Saliva samples: Increasingly used for genetic testing, drug monitoring, and infectious disease diagnostics, leveraging their non-invasive nature.
• Tissue samples (biopsies): Crucial for histopathological examination (microscopic examination of tissues) and molecular diagnostic tests like PCR or immunohistochemistry for cancer diagnostics.
• Cerebrospinal fluid (CSF): Collected through lumbar puncture and used for diagnosing neurological conditions such as meningitis and encephalitis.
• Other body fluids (e.g., synovial fluid, pleural fluid): Used in specific situations to analyze the composition of fluids in body cavities and aid in the diagnosis of joint diseases (synovial fluid) or lung-related issues (pleural fluid).

The choice of sample type depends heavily on the nature of the analyte being measured and the diagnostic question being asked.

Data management systems (DMS) are vital for efficient and compliant operation of IVD testing laboratories. A robust DMS integrates sample tracking, instrument interfacing, result reporting, quality control data management, and often, electronic medical record (EMR) integration. This is accomplished using LIMS (Laboratory Information Management Systems). They ensure data integrity, accuracy, and traceability throughout the testing process.

My experience includes working with various LIMS, from small, laboratory-specific systems to enterprise-level solutions. I am familiar with data import/export functions, audit trails, user roles, and security protocols vital for data confidentiality and compliance with regulations such as HIPAA and GDPR. I understand the importance of proper data validation and verification procedures to prevent errors and maintain data quality. A well-implemented DMS helps streamline workflows, improve turnaround times, and minimize manual data entry, thereby reducing the risk of human error. It also plays a critical role in generating reports for regulatory compliance and quality audits.

Traceability in IVD testing is the ability to track and document the entire life cycle of a sample or a reagent, from its origin to its final disposition. This includes tracking sample collection, handling, processing, testing, and storage. It’s crucial for ensuring the accuracy and reliability of test results and for investigating any potential discrepancies or errors.

A comprehensive traceability system typically incorporates unique identifiers (e.g., barcodes or RFID tags) assigned to each sample and reagent. This allows for accurate tracking through various stages of the testing process. Maintaining detailed records of all procedures performed, personnel involved, instrument calibrations, and quality control checks is equally important. In case of a problem with a result, traceability helps quickly pinpoint the source of error, allowing for corrective actions and preventing similar occurrences in the future. In essence, traceability enhances the quality and reliability of the entire IVD process and helps to ensure compliance with regulatory standards.

Developing and implementing quality control (QC) procedures is paramount in IVD testing to ensure the accuracy, reliability, and validity of results. My experience encompasses various aspects of QC, from designing and validating QC protocols to performing routine QC checks and troubleshooting issues. This involves selecting appropriate QC materials (e.g., controls with known values), establishing acceptance criteria based on assay performance characteristics, and documenting all QC activities. I am familiar with the use of control charts (e.g., Levey-Jennings charts) to monitor assay performance over time and detect trends indicating a potential problem.

Furthermore, I have experience in performing proficiency testing (PT), which involves participating in external quality assessment programs to compare the laboratory’s performance against other labs. PT helps identify areas for improvement and ensures that the lab is meeting external benchmarks for accuracy and precision. The QC processes are usually detailed in a standard operating procedure (SOP). This SOP also details the corrective actions to take if the QC results indicate a problem with the assay. This might involve recalibrating instruments, repeating the assay, or even investigating potential problems with reagents or sample handling.

During a routine quality control check for a newly implemented ELISA assay for detecting a specific antibody, we observed consistently high absorbance values for the negative control wells. This indicated a possible problem with the assay’s background. Initial troubleshooting steps, including checking reagents, washing procedures, and instrument calibration, did not resolve the issue.

To systematically approach the problem, we employed a step-wise troubleshooting approach: We first repeated the assay with a new batch of reagents to rule out reagent degradation. Next, we investigated potential contamination issues by thoroughly cleaning the equipment and changing the working solutions. Still, no improvement. Then, we carefully reviewed the assay protocol, and we noticed a possible error in the washing step. By carefully examining each step, we found that the washing buffer was being dispensed inconsistently, leaving residual antibody that was causing the elevated background absorbance. After correcting the washing procedure and implementing more stringent quality control checks on the washing process, the issue was resolved, demonstrating the importance of systematic troubleshooting, attention to detail, and the value of meticulous documentation in resolving complex IVD assay issues.