Navigating ISO 15189: A Comprehensive Guide to IHC Assay Validation for Reliable Diagnostics

Abstract

This article provides researchers, scientists, and drug development professionals with a detailed roadmap for implementing ISO 15189 standards in Immunohistochemistry (IHC) assay validation. It covers the foundational principles of the standard, step-by-step methodological applications for different test types, strategies for troubleshooting and process optimization, and a comparative analysis with other regulatory frameworks like CLIA and CLSI. The guidance is designed to help laboratories achieve technically sound, clinically relevant, and internationally recognized validation outcomes that ensure patient safety and data integrity in biomedical research and diagnostics.

Understanding ISO 15189: The Cornerstone of Quality in IHC Laboratories

What is ISO 15189? Defining Quality and Competence for Medical Laboratories

ISO 15189:2022 - Medical laboratories — Requirements for quality and competence is an international standard specifically designed for medical laboratories. It specifies the requirements for quality and competence, serving as the benchmark for developing a robust Quality Management System (QMS) and demonstrating technical competence [1] [2]. Unlike general quality standards like ISO 9001 or broad laboratory standards like ISO/IEC 17025, ISO 15189 is tailored to the unique environment of medical laboratories, addressing all steps of the total testing process (TTP), from sample collection to interpretation and reporting of results [3] [2].

The primary objective of this standard is to "promote the welfare of patients and satisfaction of laboratory users through confidence in the quality and competence of medical laboratories" [4]. For researchers and drug development professionals, ISO 15189 provides a critical framework that ensures laboratory data reliability, essential for making informed decisions in clinical trials and diagnostic development.

Key Requirements and Structure of the Standard

ISO 15189 is structured around two fundamental sets of requirements that medical laboratories must implement and maintain.

Management Requirements

The management requirements focus on establishing an effective Quality Management System that oversees all laboratory operations. Key elements include:

• Management System Documentation: Comprehensive documentation of policies, processes, and procedures [3].
• Control of Records: Maintaining records to demonstrate conformity to requirements [3].
• Addressing Risks and Opportunities: Implementing proactive risk management thinking throughout the laboratory system [4].
• Corrective Actions: Establishing systematic processes for addressing nonconformities [3].
• Management Review: Conducting regular reviews of the management system to ensure its continuing suitability, adequacy, and effectiveness [3].

Technical Requirements for Competence

The technical requirements focus on the scientific and technical competence of laboratory personnel and processes. Critical components include:

• Personnel Competence: Ensuring all laboratory staff are qualified, competent, and receive ongoing training [3].
• Laboratory Accommodation and Environmental Conditions: Maintaining appropriate facilities and conditions for reliable testing [3].
• Equipment Management: Ensuring all equipment is properly calibrated, maintained, and functioning correctly [3].
• Pre-examination Processes: Standardizing procedures for test requests, patient preparation, and sample collection, handling, and transport [3].
• Examination Processes: Implementing validated methods and procedures for performing tests [3].
• Ensuring Quality of Examination Results: Establishing internal quality control systems and participating in interlaboratory comparisons [5] [3].
• Post-examination Processes: Standardizing result reporting, interpretation, and release procedures [3].
• Reporting of Results: Ensuring results are accurate, clear, and contain all essential information [3].

Table: Key Technical Competence Requirements in ISO 15189

Requirement Area	Specific Components	Impact on Laboratory Quality
Personnel	Qualifications, training, competence assessment	Reduces operator-dependent variation
Equipment	Calibration, maintenance, performance records	Ensures measurement traceability and reliability
Pre-examination	Sample collection, transport, handling	Minimizes pre-analytical errors
Examination	Method validation, quality control, measurement uncertainty	Ensures analytical reliability and identifies error sources
Post-examination	Result interpretation, reporting format, turnaround time	Enhances clinical usefulness of laboratory data

ISO 15189:2012 vs. ISO 15189:2022 - Key Updates

The 2022 edition of ISO 15189 supersedes the 2012 version and introduces several important updates that laboratories must address to maintain accreditation.

Table: Comparison of ISO 15189:2012 and ISO 15189:2022

Aspect	ISO 15189:2012	ISO 15189:2022
Status	Withdrawn [6]	Current International Standard [1]
Point-of-Care Testing	Addressed in separate standard (ISO 22870) [4]	Requirements incorporated into main standard [4]
Risk Management	Implicit in requirements	Explicit emphasis on risk-based thinking and patient safety [4]
Internal Quality Control	Laboratory "shall design" IQC systems [5]	Laboratory "shall have" IQC procedure; more detailed requirements [5]
Measurement Uncertainty	Determine "where relevant and possible" [5]	MU must be evaluated, compared to specifications, and reviewed regularly [5]
Alignment with other Standards	-	Referenced to align with ISO/IEC 17025:2017 [4]

Accreditation Process and Implementation

The Accreditation Pathway

The journey to ISO 15189 accreditation follows a systematic process that typically takes 3-6 months to complete [2]. The following diagram illustrates the key stages a laboratory undergoes to achieve and maintain accreditation:

Practical Implementation Steps

Successful implementation requires a structured approach. Laboratories at St. Martin de Porres Hospital in Taiwan followed these key steps when transitioning to the 2022 standard [4]:

• Kickoff Meeting: Form a project team and divide tasks according to personnel expertise, including thoroughly reading and understanding the changes in the revised ISO.
• Gap Analysis: Compare new requirements against the current status of the laboratory's quality system.
• Decision to Change: Management team discusses the gap analysis and decides what changes need to be made.
• In-house Transition Plan: Develop a detailed plan with checklists, schedules, and responsible personnel.
• Implement Changes: Execute the transition plan, including any necessary personnel training.
• Monitor Changes: Continuously monitor implemented changes to ensure the laboratory achieves the new requirements.

Application to Immunohistochemistry (IHC) Assay Validation

IHC Validation Framework Under ISO 15189

For researchers and drug development professionals, ISO 15189 provides the critical foundation for validating immunohistochemistry assays, ensuring they are reliable and reproducible for biomarker detection in clinical settings [7]. The standard mandates evidence-based validation procedures that address the accuracy, specificity, and reproducibility of each test, along with management of measurement uncertainty [8].

The College of American Pathologists (CAP) has established specific validation protocols that align with ISO 15189 requirements. The 2024 CAP guideline update affirms and expands on previous recommendations to ensure accuracy and reduce variation in IHC laboratory practices [9]. The validation workflow for IHC assays involves multiple critical steps, as shown in the following diagram:

Essential Research Reagent Solutions for IHC Validation

Table: Essential Research Reagents for IHC Assay Validation

Reagent/Material	Function in Validation	Validation-Specific Considerations
Primary Antibodies	Target antigen detection	Specificity, sensitivity, and optimal dilution must be established during validation [9]
Control Cell Lines	Known protein content calibrators	Serve as reference materials for determining assay performance characteristics [9]
Tissue Microarrays	Multiplex tissue analysis platform	Enable high-throughput validation across multiple tissue types in a single experiment
Reference Standards	Benchmark for comparison	Comparator for new assay performance against validated methods or clinical trial data [9]
IQC Material	Monitoring ongoing validity	Third-party controls detect reagent or calibrator lot-to-lot variation [5]

Regulatory Considerations for IHC Assays

ISO 15189 compliance intersects with various regulatory frameworks depending on the intended use of the IHC assay and the geographic region of implementation:

• CLIA Regulations: Apply broadly to laboratory testing but don't specify how to satisfy performance requirements [7].
• FDA Requirements: Premarket approval submissions require studies exceeding CLIA requirements [7].
• EU IVDR: Classifies companion diagnostics as Class C devices, requiring technical dossier submission [7].

For global assay commercialization, manufacturers must plan parallel validation strategies that meet both CLIA and IVDR requirements, designing comprehensive validation packages from the outset to avoid duplicating efforts [7].

Benefits and Global Impact

ISO 15189 accreditation provides significant benefits to medical laboratories, healthcare systems, and researchers:

• Enhanced Patient Safety: Systematic risk management focuses on reducing potential harm to patients [4] [2].
• Improved Data Reliability: Robust quality systems decrease the probability of invalid results [2].
• International Recognition: ILAC endorsement facilitates global data sharing and collaboration [2].
• Operational Efficiency: Standardized processes save money by implementing more efficient practices and avoiding costly mistakes [2].
• Competitive Advantage: Accreditation increases opportunities for preferred supplier status and participation in clinical trials [2].

For the research and drug development community, ISO 15189 accreditation provides assurance that laboratory data supporting diagnostic and therapeutic development meets internationally recognized standards for quality and competence. This is particularly crucial for immunohistochemistry assays, where standardization and validation directly impact their reliability in guiding treatment decisions and providing prognostic information [8].

ISO 15189 represents the international benchmark for quality and competence in medical laboratories. Its specific design for medical laboratory environments, comprehensive coverage of the total testing process, and focus on both management and technical requirements make it uniquely valuable for ensuring reliable laboratory results. The 2022 revision strengthens these aspects with enhanced risk management requirements and incorporation of point-of-care testing.

For researchers and drug development professionals working with immunohistochemistry and other complex assays, understanding and applying ISO 15189 principles is essential for ensuring data integrity, regulatory compliance, and ultimately, patient safety. As laboratory medicine continues to evolve with advancing technologies, ISO 15189 provides a stable framework for maintaining quality and competence amidst these changes.

Immunohistochemistry (IHC) is a cornerstone technique in diagnostic pathology and biomarker development, playing a vital role in identifying biomarkers within tissue samples for both research and clinical decision-making [7]. The reliability of IHC results depends on rigorous analytical validation to ensure tests are accurate, reproducible, and fit for their intended purpose. The core principles of impartiality, confidentiality, and patient-centered focus form the ethical and operational foundation for this validation process, directly impacting the quality and safety of patient care.

The international standard for medical laboratory quality and competence, ISO 15189, provides a critical framework for implementing these principles. The recent update to ISO 15189:2022 significantly elevates the focus on patient-centered risk management, requiring laboratories to plan and implement actions to address risks and opportunities for improvement across all testing processes [4] [10]. This update, alongside specific guidelines from organizations like the College of American Pathologists (CAP), demonstrates an evolving recognition that technical excellence must be underpinned by unwavering ethical commitment to patient welfare.

Core Principles in Practice

Impartiality in Validation and Testing

Impartiality in IHC validation requires that laboratory processes and decisions are objective, unbiased, and based solely on valid scientific evidence. This principle is operationalized through standardized procedures and adherence to recognized guidelines that minimize inter-laboratory variation.

• Standardized Validation Protocols: Evidence shows that the publication of evidence-based guidelines has significantly improved consistency in IHC assay validation. Following the release of CAP guidelines, laboratories with written validation procedures for predictive markers increased from 45.9% to 73.8%, demonstrating a move toward more objective, standardized approaches [11].
• Objective Evidence-Based Decision Making: The CAP guidelines emphasize that validation must be based on objective evidence, recommending various comparators for validation study design, listed here from most to least stringent [9]:
  • Comparison to IHC results from cell lines with known protein amounts ("calibrators")
  • Comparison with results from a non-immunohistochemical method
  • Comparison with results from testing the same tissues in another laboratory using a validated assay
  • Comparison with prior testing of the same tissues in the same laboratory

Confidentiality in Laboratory Operations

Confidentiality protects patient data and proprietary laboratory information throughout the testing process. While the search results focus more on technical validation, this principle is embedded in quality management systems complying with standards like ISO 15189, which includes requirements for data protection and information security management systems.

• Data Protection in Validation Studies: Validation studies using patient-derived tissues and clinical data require stringent confidentiality safeguards. This includes de-identifying specimens and protecting associated clinical information during the validation process [12] [13].
• Secure Information Management: Laboratories must implement systems to ensure that patient data and test results are accessible only to authorized personnel, maintaining confidentiality from sample receipt through result reporting and data storage.

Patient-Centered Focus and Risk Management

A patient-centered focus prioritizes patient safety and outcomes in all laboratory decisions, with risk management as the primary tool for implementation. The updated ISO 15189:2022 standard makes this explicit by requiring laboratories to identify and mitigate risks that could impact patients [4] [14].

• Risk-Based Validation Approach: The level of validation required for an IHC assay should correspond to its clinical impact. Assays for predictive biomarkers (e.g., HER2, PD-L1) that directly influence treatment decisions require more extensive validation than those for non-predictive markers [9] [15].
• Patient Safety as Validation Goal: The fundamental purpose of rigorous validation is to prevent patient harm from inaccurate results. This is reflected in the ISO objective to "promote the welfare of patients and satisfaction of laboratory users through confidence in the quality and competence of medical laboratories" [4].

Table 1: Evolution of IHC Validation Practices Reflecting Core Principles

Aspect	Pre-Guideline Practice (2010)	Post-Guideline Practice (2015)	Principle Demonstrated
Written Validation for Predictive Markers	45.9% (299/651 labs) [11]	73.8% (795/1077 labs) [11]	Impartiality
Validated Most Recent Predictive Marker	74.9% (326/435) [11]	99.0% (101/102) [11]	Patient-Centered Focus
Primary Challenge in Implementation	N/A	Finding cases for rare antigens; Resource limitations [11]	Impartiality

Experimental Validation Protocols and Data Presentation

Validation Workflow for IHC Assays

The following diagram illustrates the comprehensive workflow for validating IHC assays, integrating risk management and the core principles at each stage:

Quantitative Validation Data and Performance Metrics

Robust validation requires predefined performance metrics and acceptance criteria. The CAP guidelines and other international recommendations provide specific targets for various performance characteristics.

Table 2: IHC Assay Validation Performance Requirements and Acceptance Criteria

Performance Characteristic	Validation Requirement	Acceptance Criteria	Applicable Test Types
Accuracy/Concordance	Comparison to reference method [9] [15]	≥90% concordance for all IHC assays [9]	All types
Number of Cases	Sufficient positive and negative cases [9] [12]	Minimum 10 positive and 10 negative cases for alternative fixatives [9]	All types
Repeatability	Same result when tested repeatedly in same run [15]	≥95% agreement	All types
Reproducibility	Same result over different runs [15]	≥90% agreement	All types
Analytical Sensitivity	Smallest amount accurately detected [15]	Established during LOD study	LDTs, RUO with reference
Analytical Specificity	Ability to detect specific substance [15]	No cross-reactivity with non-target antigens	LDTs, RUO with reference

Case Study: Validation of Cytology Cell Block IHC

Applying IHC assays to cytology cell block material presents unique challenges due to different fixation methods. One academic institution developed a practical validation protocol applying CAP guidelines [12]:

• Sample Selection: Ten consecutive lung resection specimens were collected within 45 minutes of removal. Each lesion was scraped twice and suspended in both alcohol-based fixative and 10% formalin, then processed as cell blocks using the plasma-thrombin method.
• Comparison Standard: A section of each lesion was processed as formalin-fixed paraffin-embedded (FFPE) tissue to serve as the gold standard.
• Staining and Evaluation: IHC tests on cytology cell block material were performed identically to validated surgical material. Results were scored by percentage of cells staining (0%, 1%-25%, 26%-50%, 51%-75%, >75%) with concordance defined as staining within the same quartile.
• Antibody Panel: The most frequently utilized diagnostic IHC studies were selected for validation, including TTF-1, p40, p63, AE1/3, CK7, CD56, synaptophysin, chromogranin, Ki-67, and CK8/18.

This systematic approach ensured that IHC tests performed on cytology specimens met the same reliability standards as those on traditional surgical pathology material, demonstrating impartial application of validation standards across different specimen types.

The Scientist's Toolkit: Essential Reagents and Materials

Table 3: Key Research Reagent Solutions for IHC Assay Validation

Reagent/Material	Function in Validation	Specific Application Notes
Primary Antibodies	Detect specific target antigens	Critical to validate specificity; different requirements for CE-IVD vs. LDTs [15]
Control Cell Lines	Provide known antigen content as calibrators	Used as most stringent comparator for validation [9]
Multitissue Blocks	Assess antibody specificity across tissues	Normal tissue panels required for establishing specificity [13]
Antigen Retrieval Solutions	Unmask epitopes altered by fixation	Variation from IFU requires revalidation [15]
Detection Systems	Visualize antibody-antigen binding	Must be validated with specific primary antibody [16]
Reference Standards	Serve as gold standard for comparison	Can include other IHC methods, non-IHC methods, or validated assays [9]

The core principles of impartiality, confidentiality, and patient-centered focus are not abstract concepts but essential components of technically sound IHC assay validation. The evolving regulatory landscape, including the updated ISO 15189:2022 standard and recent CAP guideline updates, reflects a growing emphasis on integrating these ethical principles with rigorous scientific practice.

Successful implementation requires a risk-based approach that prioritizes patient safety throughout the validation process and ongoing quality monitoring. By embedding these principles into standard operating procedures, laboratories can ensure the reliability of IHC testing, maintain stakeholder trust, and ultimately contribute to improved patient outcomes. As standardization continues to advance globally, these core principles will remain fundamental to laboratory competence and quality in IHC testing.

Immunohistochemistry (IHC) is a cornerstone technique in research and diagnostic laboratories, playing a vital role in identifying biomarkers within tissue samples [7]. For researchers and drug development professionals, validating IHC assays to ensure reliability and reproducibility is paramount, particularly in the context of precision medicine and companion diagnostic development [7] [17]. The International Standard ISO 15189, specifically designed for medical laboratories, provides a critical framework for quality and competence across all phases of testing [3] [18]. The updated 2022 version of this standard, which laboratories must transition to by December 2025, introduces significant enhancements that directly impact IHC assay validation [14] [4]. This guide examines the key requirements of ISO 15189:2022—structural governance, resource management, and process control—providing a detailed comparison with previous practices and experimental approaches for implementation.

Structural and Governance Requirements

Defined Leadership and Organizational Responsibility

Clause 5 of ISO 15189:2022 establishes rigorous structural and governance requirements, mandating that laboratories have a defined legal status and clear organizational responsibilities [18]. The standard requires designated leadership, specifically a Laboratory Director, who holds ultimate accountability for all operations [18]. This framework ensures clear lines of authority and accountability, which is essential for maintaining the integrity of IHC validation data, especially in regulated drug development environments.

Impartiality and Confidentiality

Under Clause 4, laboratories must establish and document procedures to ensure impartiality by avoiding conflicts of interest and safeguarding patient information [18]. This is achieved through enforceable agreements, secure data access controls, and training all personnel—including contractors—on confidentiality obligations [18]. For researchers, this translates to protocols that protect subject data and ensure unbiased analysis, which is critical for clinical trial validity.

Risk Management Integration

A pivotal update in the 2022 version is the enhanced focus on risk management, making patient safety central to the laboratory's ethos [4]. Laboratories must now implement robust risk management processes aligned with ISO 22367 and ISO 35001, requiring them to identify, assess, and mitigate potential risks across all testing phases [14] [18]. This proactive approach is particularly crucial for IHC assays used in predictive biomarker testing (e.g., HER2, PD-L1), where inaccurate results directly impact patient treatment decisions [17].

Resource Management Requirements

Personnel Competence and Training

Clause 6 of ISO 15189 mandates that all laboratory personnel are competent and adequately trained for their assigned tasks, with regular competence assessments [18]. This is especially critical for IHC, where the complex, multi-step nature of the assay means that "millions of different protocols can be generated for each biomarker with great risk of poor reproducibility compromising patient safety" [17]. Ensuring staff can consistently execute and validate these protocols is fundamental to reliable data generation.

Equipment and Calibration Management

The standard requires that all equipment, including automated stainers and microscopes, must be selected for suitability, calibrated, maintained, and monitored for metrological traceability [18]. Proper calibration ensures staining intensity and interpretation consistency, which are vital for accurate, reproducible IHC results in longitudinal studies.

Facilities and Environmental Conditions

Laboratories must maintain controlled environments that safeguard personnel safety and ensure the reliability of clinical results [18]. For IHC, this includes proper ventilation for chemical handling, stable storage conditions for temperature-sensitive antibodies, and appropriate space to prevent cross-contamination—all factors that directly impact assay performance.

Process Control Requirements

Pre-Examination to Post-Examination Process Control

Clause 7 of ISO 15189 focuses on implementing robust processes across the entire testing cycle: pre-examination, examination, and post-examination phases [18]. For IHC, this includes standardized procedures for sample collection, fixation, processing, staining, and result interpretation. The "total test approach" to IHC standardization emphasizes that the end result is influenced by multiple parameters across all phases, making integrated process control essential [17].

Examination Procedures: Verification and Validation

A core requirement is that all laboratory examination methods must be verified or validated for their intended use [18]. The approach depends on the test type and origin, with distinct evidence requirements as shown in Table 1.

Table 1: IHC Test Verification and Validation Requirements Based on Test Type

Test Type and Origin	Validation Level	Accuracy	Repeatability/ Reproducibility	Analytical Sensitivity	Analytical Specificity	Robustness
CE-IVD (used per IFU)	Verification	Required	Required	Not Required	Not Required	Required*
Modified CE-IVD (with reference)	Limited Validation	Required	Required	Not Required	Not Required	Required*
Modified CE-IVD (no reference)	Validation	Required	Required	Required	Required	Required*
Non-CE-IVD/RUO (with reference)	Simplified Validation	Required	Required	Required	Required	Required*
In-house developed LDT	Extensive Validation	Required	Required	Required	Required	Required*

Note: *Risk analysis determines parameters to verify. Based on Belgian recommendations [15].

The College of American Pathologists (CAP) provides specific guidance on validation study design, recommending comparison against appropriate comparators such as other validated methods, expected antigen localization, or published positive rates [9]. For predictive markers with distinct scoring systems (e.g., PD-L1, HER2), laboratories must separately validate each assay-scoring system combination [9].

Quality Assurance: IQC and EQA

The standard mandates implementation of both internal quality control (IQC) and external quality assessment (EQA) schemes to monitor performance and result accuracy [18]. The 2025 IFCC recommendations support using Westgard Rules and Sigma-metrics for planning IQC strategies, including determining control frequency based on the clinical significance of the analyte and the robustness of the method [5]. EQA participation, such as in the Nordic Immunohistochemical Quality Control (NordiQC) program, provides essential external validation, with Fit-For-Purpose principles ensuring assessments align with the test's intended use [17].

Table 2: Experimental Design Requirements for IHC Assay Validation

Validation Parameter	Experimental Approach	Acceptance Criteria	Special Considerations
Accuracy	Compare with gold standard (reference staining, EQA, non-IHC test) [15]	90% concordance for predictive markers [9]	Use well-characterized cell lines or known positive/negative tissues
Repeatability	Test same sample repeatedly in the same run (intrarun) [15]	Consistent staining intensity and distribution	Conduct over multiple days with different operators
Reproducibility	Test same sample over different runs (interrun) [15]	Consistent scoring results between runs	Include different instrument and reagent lots if possible
Analytical Sensitivity (LOD)	Test tissues with low antigen expression levels [15]	Detection of appropriate low-expressing iCAPCs	Use immunohistochemical critical assay performance controls (iCAPCs) [17]
Analytical Specificity	Test on tissues with known cross-reactive antigens [15]	No nonspecific staining in negative tissues	Include normal tissues known to express similar epitopes
Robustness	Deliberate variations in key parameters (antigen retrieval time, primary Ab incubation) [15]	Consistent results with minor protocol variations	Focus on pre-analytical variables like fixation time

Experimental Protocols for IHC Validation

Core Validation Protocol for IHC Assays

A comprehensive IHC validation experiment should include the following steps derived from regulatory guidelines and quality standards [15] [9]:

• Test Categorization: Classify the IHC test according to its purpose (diagnostic, prognostic, predictive) and origin (CE-IVD, modified, LDT) to determine appropriate validation level [15].
• Sample Selection: Obtain a minimum of 10 positive and 10 negative well-characterized cases for initial validation [9]. For predictive markers, include samples spanning the entire scoring range (0, 1+, 2+, 3+ for HER2).
• Comparison Method: Select an appropriate comparator based on availability, with the following hierarchy: validated non-IHC method (e.g., FISH)>validated IHC in reference laboratory>expected staining pattern based on literature [9].
• Staining and Interpretation: Perform IHC staining across multiple runs using different operators, equipment, and reagent lots when possible. Ensure readers are blinded to expected results.
• Data Analysis: Calculate concordance rates for categorical tests; for semiquantitative tests (e.g., PD-L1), use statistical methods like intraclass correlation coefficient to assess agreement.
• Documentation: Maintain comprehensive records of all validation data, including raw results, calculations, and final conclusions regarding assay performance.

Protocol for Cytology Specimens

For IHC performed on cytology specimens with different fixatives (e.g., alcohol-based) than those used for validation, a separate validation with a minimum of 10 positive and 10 negative cases is required to account for potential alterations in antigen sensitivity [9].

The Scientist's Toolkit: Essential Research Reagent Solutions

Table 3: Key Research Reagent Solutions for IHC Validation

Reagent/Resource	Function in IHC Validation	Quality Considerations
Primary Antibodies	Target antigen detection	Specificity, sensitivity, lot-to-lot consistency; CE-IVD preferred for clinical use [15]
Immunohistochemical Critical Assay Performance Controls (iCAPCs)	Evaluate analytical sensitivity and specificity; pseudo-reference materials [17]	Tissues with known low antigen expression; well-characterized normal and neoplastic tissues
Reference Standard Materials	Provide designated "true value" for method comparison [17]	Commercially available CDx assays for type 2 biomarkers; well-validated in-house methods for type 1
Cell Line Calibrators	Compare new assay results against known protein amounts [9]	Cell lines containing known amounts of target protein; formalin-fixed and paraffin-embedded
Detection Systems	Amplify and visualize antibody-antigen interaction	Sensitivity, low background, compatibility with automation
Automated Staining Platforms	Standardize staining process across runs	Reproducibility, integration with laboratory information systems, maintenance requirements

Workflow Diagram: IHC Assay Validation Process

The following diagram illustrates the key stages and decision points in the IHC assay validation process, integrating structural, resource, and process control requirements.

The ISO 15189:2022 standard provides a comprehensive framework for IHC assay validation that integrates structural governance, resource management, and process control into a cohesive quality management system. The updated standard's enhanced focus on risk management and patient safety aligns with the increasing importance of IHC in precision medicine and companion diagnostic development [4] [17]. By implementing these requirements through systematic experimental validation and ongoing quality control, research laboratories and drug development professionals can ensure the reliability, reproducibility, and clinical relevance of their IHC assays, ultimately supporting the development of more effective targeted therapies.

Immunohistochemistry (IHC) serves as a cornerstone technique in both research and diagnostic laboratories, playing a vital role in identifying biomarkers within tissue samples for patient care and treatment decisions [7]. The reliability of IHC results directly impacts clinical outcomes, particularly in areas such as breast cancer subtyping and therapy selection. Despite decades of quality control efforts, studies continue to reveal variability in biomarker assessment, underscoring the necessity for robust standardization frameworks [19].

The International Standard ISO 15189 provides specific requirements for quality and competence in medical laboratories, establishing a critical framework for ensuring IHC assay reliability [7]. With the recent publication of the ISO 15189:2022 version, which must be fully implemented by December 2025, laboratories face updated requirements that significantly impact IHC validation and quality control practices [14]. This standard's enhanced focus on risk management and structured quality control approaches addresses the very pre-analytical, analytical, and post-analytical variables that contribute to IHC variability [14] [20].

Within the context of biomarker detection, ISO 15189 serves not merely as an accreditation standard but as a foundational framework for ensuring that IHC results are accurate, reproducible, and clinically meaningful. This guide examines the practical application of ISO 15189 in IHC through the lens of current research and validation studies, providing researchers and drug development professionals with evidence-based insights for implementing these critical standards.

ISO 15189 Framework: Requirements for IHC Validation

Core Principles and Updated Requirements

ISO 15189:2022 brings a comprehensive overhaul to quality and competence requirements for medical laboratories, with several key changes directly impacting IHC practices [14]. The standard emphasizes a risk-management approach, requiring laboratories to implement robust processes to identify, assess, and mitigate potential risks affecting service quality [14]. This is particularly relevant for IHC, where multiple procedural variables can impact results.

The updated standard also provides more detailed requirements regarding laboratory structure and governance, including clearer definitions of roles and responsibilities and the need for documented policies and objectives [14]. For IHC laboratories, this translates to the need for comprehensive documentation of validation protocols, standard operating procedures, and quality control measures.

A significant update in ISO 15189:2022 is the integration of Point of Care Testing (POCT) requirements previously outlined in ISO 22870:2016, streamlining accreditation across different testing environments [14]. While primarily affecting POCT, this integration reflects the standard's evolving approach to managing testing quality across diverse operational contexts.

Analytical Validation Fundamentals

For IHC tests, the extent of analytical verification or validation is determined by three key factors: the purpose of the test related to its intended use, the IVDR classification, and the origin of the test [15]. ISO 15189 requires laboratories to establish performance specifications for each test method, with the validations being "as extensive as are necessary to meet the needs in the given application or field of application" [21].

The Belgian Practice Guideline, based on ISO 15189:2012, provides a refined framework for IHC test validation that demonstrates practical implementation of the standard's requirements [15]. This guideline categorizes tests based on their origin and establishes tailored validation requirements for each category, recognizing that a one-size-fits-all approach is impractical for diverse laboratory contexts.

Table: IHC Test Classification and Validation Requirements Based on ISO 15189 Principles

Test Origin	Definition	Validation Level	Key Performance Characteristics
CE-IVD	Used strictly according to manufacturer's IFU	Verification	Accuracy, Repeatability, Reproducibility
Modified CE-IVD with reference	Modifications not in IFU but with reference protocol available	Limited Validation	Accuracy, Repeatability, Reproducibility
Modified CE-IVD without reference	Modifications without reference documentation	Validation	All performance characteristics including analytical sensitivity and specificity
Non-CE-IVD with reference	RUO products with reference documentation	Simplified Validation	Accuracy, Repeatability, Reproducibility, Analytical Sensitivity, Analytical Specificity
Non-CE-IVD without reference (LDT)	In-house developed tests without reference	Extensive Validation	All performance characteristics including clinical performance

Key Performance Characteristics for IHC Validation

Under ISO 15189, IHC assays must demonstrate specific performance characteristics appropriate to their intended use. The Belgian recommendations, which align with ISO 15189 requirements, define key parameters that must be evaluated [15]:

• Accuracy: The degree of concordance with a gold standard (e.g., reference staining, EQA result, non-IHC test)
• Analytical Sensitivity: The smallest amount of substance in a tissue sample that can accurately be detected
• Analytical Specificity: The ability to detect one particular substance rather than others
• Repeatability: The ability to produce the same result when repeatedly tested in the same run
• Reproducibility: The ability to produce the same result when tested over different runs
• Robustness: The ability to remain unaffected by small variations in procedural parameters

The implementation of these requirements is not merely about compliance but about establishing a systematic approach to quality that ensures IHC results are reliable across different operators, equipment, and timepoints, ultimately supporting reproducible biomarker detection in research and clinical decision-making.

Experimental Data: ISO 15189-Compliant Validation in Action

HER2 IHC Scoring Concordance Studies

Recent research on HER2 scoring in breast cancer provides compelling evidence for the importance of standardized validation approaches aligned with ISO 15189 principles. A 2025 study examining HER2-low and HER2-ultralow scoring in breast cancer demonstrated that pathologists can achieve substantial agreement (κ = 0.69 across HER2 scores; κ = 0.79 for HER2-low cutoff) when supported by appropriate training and standardized methodologies [22].

This large-scale study involving 500 breast cancer samples highlighted that specific training in HER2 scoring per ASCO/CAP 2018 guidelines significantly improved reproducibility, particularly for the relatively new HER2-low and HER2-ultralow categories [22]. The study utilized the VENTANA HER2 (4B5) assay following manufacturer instructions, with all procedures conducted in a CAP/CLIA, ISO 15189 certified facility [22]. This experimental design exemplifies how ISO 15189-compliant environments support the generation of reliable, reproducible data.

The research compared real-world HER2 scoring performed prior to HER2-low being defined as clinically actionable with centralized scoring by trained pathologists, finding substantial agreement (κ = 0.72) between real-world pathologists and central consensus for the HER2-low cutoff [22]. This demonstrates that implementation of standardized approaches can yield consistent results even across different laboratory settings.

Method Comparison Studies: IHC vs. RT-qPCR

Further evidence supporting robust validation approaches comes from a 2021 study comparing routine IHC with RT-qPCR for breast cancer biomarkers [19]. This investigation revealed important insights about method-specific reproducibility challenges, particularly for Ki67 assessment, where 29.2% of cases showed discrepant categorization between IHC and RT-qPCR methods [19].

Table: Biomarker Concordance Between IHC and RT-qPCR in Breast Cancer

Biomarker	Correlation Between Methods	Key Discrepancy Findings	Clinical Implications
ER	High correlation	Minimal discrepancies	Both methods reliable for endocrine therapy decisions
PR	High correlation	Minimal discrepancies	Both methods reliable for endocrine therapy decisions
HER2	Generally high correlation	10/96 discrepant cases involved equivocal categorization	RT-qPCR may resolve equivocal IHC/FISH cases
Ki67	Moderate correlation	28 cases (29.2%) categorized differently; most changed from low to high	RT-qPCR offers more precise assessment of proliferation

The researchers noted that methods with wider dynamic range and higher reproducibility, such as RT-qPCR, may offer more precise assessment of endocrine responsiveness, improve Ki67 standardization, and help resolve HER2 cases that remain equivocal by IHC/FISH [19]. These findings underscore the importance of the method validation requirements in ISO 15189, particularly when implementing laboratory-developed tests or modifying existing protocols.

Error Rate Analysis in Predictive Biomarker Testing

A comprehensive study of error rates in predictive biomarker testing for lung and colorectal cancer provided valuable insights into the practical challenges in IHC standardization [23]. Analysis of External Quality Assessment (EQA) schemes between 2014-2018 revealed that errors occurred mostly in the post-analytical phase (48.1%) for NSCLC testing, with interpretation being a significant challenge [23].

The study found that accredited laboratories (including those accredited to ISO 15189) were less likely to make errors in EQA schemes and were more likely to complete root cause analysis surveys when errors did occur [23]. This suggests that implementation of quality management systems according to ISO 15189 not only reduces errors but also fosters a culture of continuous quality improvement.

For IHC specifically, the technical assessment of staining quality was reflected in a high percentage of analytical issues as contributing factors for problems [23]. This highlights the importance of the robust validation of analytical performance required by ISO 15189, particularly for laboratory-developed tests and modified protocols.

Diagram: IHC Testing Process with Error Risk Distribution. The post-analytical phase, particularly interpretation and scoring, accounts for nearly half of all errors in biomarker testing according to EQA scheme data [23].

Implementation Guide: ISO 15189-Compliant IHC Validation

Step-by-Step Validation Protocol

Implementing ISO 15189-compliant IHC validation requires a systematic approach. Based on international guidelines and practical recommendations, the following protocol ensures comprehensive method validation:

1. Define Intended Use and Performance Specifications

• Establish clear clinical or research application for the IHC test
• Define acceptable performance criteria based on intended use
• Classify test according to IVDR categories and validation requirements [15]

2. Perform Risk Assessment

• Identify potential failure modes in pre-analytical, analytical, and post-analytical phases
• Prioritize variables based on impact on patient safety or research outcomes
• Document risk mitigation strategies [14]

3. Select Appropriate Validation Samples

• Include adequate representation of positive, negative, and borderline cases
• Ensure sample size provides sufficient statistical power (typically 20-50 cases for LDTs)
• Incorporate samples with known reactivity from well-characterized sources [15]

4. Execute Validation Experiments

• Test accuracy against reference standard or method
• Assess repeatability (intra-run precision) and reproducibility (inter-run, inter-operator, inter-day)
• Determine analytical sensitivity and specificity
• Evaluate robustness by deliberately varying critical parameters [15] [21]

5. Establish Ongoing Quality Control Procedures

• Define QC frequency based on risk assessment and Sigma metrics
• Implement multi-rule QC procedures (e.g., Westgard rules) where appropriate
• Document all QC results and corrective actions [5]

The Scientist's Toolkit: Essential Research Reagent Solutions

Table: Key Reagent Solutions for Validated IHC Assays

Reagent Category	Specific Examples	Function in IHC Validation	Quality Considerations
Primary Antibodies	VENTANA anti-HER2/neu (4B5); CE-IVD and LDT versions	Target antigen detection	Specificity, sensitivity, lot-to-lot consistency
Detection Systems	Polymer-based detection; chromogenic substrates	Signal amplification and visualization	Sensitivity, background noise, linear range
Control Materials	Cell line controls; tissue microarrays; commercial control slides	Process verification and validation	Stability, commutability, matrix effects
Antigen Retrieval Solutions	Citrate buffer; EDTA-based solutions; enzyme retrieval	Epitope exposure and standardization	pH stability, retrieval efficiency, batch consistency
Blocking Reagents	Normal serum; protein blocks; endogenous enzyme blockers	Reduction of non-specific binding	Effectiveness, compatibility with detection system

Internal Quality Control and Uncertainty Measurement

The updated ISO 15189:2022 emphasizes the importance of structured internal quality control (IQC) procedures. The 2025 IFCC recommendations for IQC practices, aligned with ISO 15189:2022, support the continued use of Westgard Rules and Sigma-metrics for planning IQC strategies [5].

Laboratories must determine both the frequency of IQC and the size of the series (number of patient samples between IQC events) based on multiple factors [5]:

• Clinical significance and criticality of the analyte
• Time frame for result release and subsequent use
• Feasibility of re-analyzing samples
• Analytical robustness measured by Sigma-metrics

For measurement uncertainty (MU), ISO 15189:2022 requires that "the MU of measured quantity values shall be evaluated and maintained for its intended use, where relevant" [5]. The standard now mandates that MU be compared against performance specifications, regularly reviewed, and made available to laboratory users on request [5]. The IFCC recommends a "top-down" approach using IQC and EQA data rather than the complex "bottom-up" approach that estimates uncertainty of each variable in the measurement process [5].

Diagram: ISO 15189-Compliant IHC Validation Workflow. The process emphasizes risk-based approach to determine appropriate validation level based on test type and intended use [15].

The implementation of ISO 15189 standards in IHC laboratories represents a critical evolution from artisanal techniques to rigorously validated analytical procedures. Evidence from recent studies demonstrates that standardized approaches significantly improve reproducibility, particularly for challenging interpretations such as HER2-low scoring in breast cancer [22]. The framework provided by ISO 15189 addresses the entire testing process, from pre-analytical specimen handling to post-analytical interpretation and reporting, creating a systematic defense against the variability that has historically plagued IHC.

For researchers and drug development professionals, adherence to these standards provides not only regulatory compliance but, more importantly, generates data of consistently high quality that can be reliably compared across studies and institutions. As biomarker detection becomes increasingly central to personalized medicine approaches, the role of ISO 15189 in ensuring accurate and reproducible IHC results will only grow in significance. The December 2025 deadline for implementing ISO 15189:2022 [14] offers a timely opportunity for laboratories to strengthen their validation protocols and quality management systems, ultimately advancing the reliability of biomarker detection in both research and clinical applications.

In the field of medical laboratory sciences, particularly in specialized areas like immunohistochemistry (IHC) assay validation, researchers and drug development professionals navigate a complex landscape of standards and guidelines. Two foundational frameworks govern this space: ISO 15189, which sets requirements for quality and competence in medical laboratories, and guidelines from the Clinical and Laboratory Standards Institute (CLSI), which provide detailed, practical implementation guidance. Rather than competing, these frameworks function in a synergistic relationship that strengthens laboratory quality systems [24] [25]. ISO 15189 establishes the "what" - the overarching requirements for quality management systems - while CLSI provides the "how" - detailed protocols and procedures for meeting these requirements [25]. This complementary relationship is especially valuable in IHC assay validation, where technical complexity demands both rigorous standards and practical implementation methodologies. For researchers and drug development professionals, understanding this synergy is crucial for developing robust, defensible validation protocols that meet international standards while remaining practically implementable in real-world laboratory settings.

Understanding the Core Standards: ISO 15189 and CLSI

ISO 15189: The Quality Management Standard

ISO 15189 is an international standard specifically designed for medical laboratories, with requirements focused on quality and competence [24] [26]. Unlike broader quality standards, ISO 15189 emphasizes both technical competence and the clinical relevance of laboratory results, ensuring they reliably support patient diagnosis, treatment, and monitoring [24]. The standard is structured into two principal components: management requirements and technical requirements [26].

The management requirements focus on the quality management system (QMS) structure and operational governance, including organization and management responsibility, document control, service agreements, resolution of complaints, and continual improvement [26]. The technical requirements address competency-related elements, including personnel qualifications, accommodation and environmental conditions, laboratory equipment, pre-examination, examination, and post-examination processes, and ensuring quality of examination results [26]. A key strength of ISO 15189 is its process-oriented approach, which encourages laboratories to systematically identify and document their processes and interactions [26]. This approach facilitates continual improvement through regular internal audits, management reviews, and root cause analysis of nonconformities [26].

CLSI Guidelines: Practical Implementation Tools

The Clinical and Laboratory Standards Institute (CLSI) develops consensus-based guidelines through collaboration among laboratorians, clinicians, regulators, and industry experts [24]. Unlike ISO standards, which are formal and certifiable, CLSI documents serve as practical implementation tools that bridge the gap between international requirements and daily laboratory practice [24]. CLSI guidelines are characterized by their standardization of methods, usability and accessibility, and alignment with global standards [24].

CLSI documents provide detailed, methodology-specific guidance for various laboratory procedures. For example, the ILA21 guideline offers comprehensive recommendations for the clinical evaluation of immunoassays, including planning, design, execution, and analysis of evaluation studies [27]. Similarly, POCT05 provides specific metrics for evaluating continuous glucose monitoring systems [28]. These guidelines translate high-level quality principles into operational procedures, making them indispensable resources for laboratories implementing complex testing protocols [24].

Table 1: Core Characteristics of ISO 15189 and CLSI Guidelines

Characteristic	ISO 15189	CLSI Guidelines
Nature	Formal certification standard	Practical implementation guidance
Development	Through International Organization for Standardization	Consensus-based through expert collaboration
Focus	Overall quality management system and technical competence	Specific procedures and methodological details
Implementation	System-level requirements	Step-by-step protocols for daily operations
Flexibility	Allows laboratory-specific implementation	Provides standardized approaches for consistency

The Complementary Relationship in Practice

How CLSI Guidelines Support ISO 15189 Requirements

The relationship between CLSI guidelines and ISO 15189 is fundamentally complementary rather than conflicting [25]. CLSI guidelines serve as a practical translation tool, converting the high-level requirements of ISO 15189 into specific, actionable laboratory procedures [25]. This translation function is particularly valuable for meeting the technical requirements of ISO 15189, where laboratories must establish validated methods, quality control procedures, and competency assessment protocols [24].

For instance, ISO 15189 requires laboratories to ensure the quality of examination procedures but does not prescribe detailed methodologies for specific assay types [26]. CLSI documents fill this gap by providing technical specifications for various testing methodologies. When implementing IHC assays, CLSI guidelines offer detailed protocols for analytical validation, including study design requirements, statistical methods, and acceptance criteria [7]. This detailed guidance helps laboratories generate the robust evidence needed to demonstrate compliance with ISO 15189's requirements for method validation and verification [7].

Similarly, ISO 15189's requirements for continual improvement are supported by CLSI's ongoing revision and updating of guidelines based on technological advancements and emerging evidence [24]. For example, CLSI regularly updates documents to address new technologies and global applications, as seen in the ILA21-A2 guideline for immunoassay evaluation [27]. This dynamic updating process provides laboratories with current best practices to maintain and enhance their quality management systems over time.

Integration in IHC Assay Validation

The integration of ISO 15189 and CLSI guidelines is particularly evident in the validation of immunohistochemistry assays, where regulatory compliance and technical robustness are both essential. The validation process for IHC assays must address multiple dimensions, including analytical performance, clinical validity, and quality control [7].

ISO 15189 provides the overarching framework for validation within a quality management system, emphasizing process management, document control, and risk management [26]. Meanwhile, CLSI guidelines provide the specific methodological guidance for designing and executing validation studies appropriate for IHC assays [7]. This includes recommendations on sample selection, specimen library collections, reference panels, commutability issues, and sample size considerations [27].

For drug development professionals, this integration is crucial when navigating different regulatory environments. As noted by Precision for Medicine, "CLSI guidelines provide recommendations on study designs, requirements, statistical methods, and acceptance criteria for evaluating all aspects of an assay for commercialization" [7]. By implementing CLSI guidelines within an ISO 15189 quality framework, laboratories can efficiently meet both CLIA requirements in the United States and IVDR requirements in the European Union [7].

Diagram 1: The complementary relationship between ISO 15189 and CLSI guidelines in achieving robust assay validation. ISO 15189 provides the quality management framework, while CLSI offers practical implementation tools.

Experimental Protocols and Methodologies

Validation Study Design Based on CLSI and ISO 15189

The integration of CLSI guidelines with ISO 15189 requirements produces a comprehensive methodological framework for validation studies. This integration is particularly important for immunoassay validation, where CLSI document ILA21-A2 provides specific guidance that supports ISO 15189's requirements for method validation [27]. The experimental approach encompasses four critical elements, each with specific protocols:

1. Development Plan Formulation A robust development plan establishes the foundation for effective evaluation. CLSI ILA21-A2 recommends creating a comprehensive plan that includes intended use statement, performance criteria, and acceptance criteria [27]. This aligns with ISO 15189's requirements for defining examination procedures [26]. The plan should explicitly address unique characteristics of the specific assay format and define statistical approaches for data analysis.

2. Planning and Design Considerations CLSI guidelines emphasize meticulous planning of evaluation studies, including specimen selection, reference methods, and clinical sites [27]. For IHC assays, this includes selecting appropriate tissue specimens that represent the intended use population and ensuring adequate sample size for statistical power [27] [7]. These design considerations directly support ISO 15189's technical requirements for pre-examination processes [26].

3. Evaluation Execution and Monitoring The execution phase requires careful monitoring and data management. CLSI recommends establishing procedures for database management, monitoring protocol compliance, and documenting deviations [27]. This aligns with ISO 15189's requirements for control of records and documentation [26]. Implementation should include regular data review to identify trends or issues requiring corrective action.

4. Analytical Performance Assessment Before clinical evaluation, CLSI emphasizes establishing robust analytical performance data, including precision, accuracy, and measuring range [27]. This foundational work supports ISO 15189's requirement for assuring quality of examination procedures [26]. For IHC assays, this includes establishing staining consistency, antigen retrieval reproducibility, and interpretation concordance [7].

Table 2: Key Research Reagent Solutions for IHC Assay Validation

Reagent Category	Specific Examples	Function in Validation	CLSI/ISO Guidance Alignment
Primary Antibodies	Monoclonal, polyclonal, validated clones	Target detection specificity	CLSI: Specificity testing; ISO: Reagent validation
Detection Systems	Polymer-based, ABC, labeled streptavidin	Signal amplification and visualization	CLSI: Sensitivity optimization; ISO: Procedure standardization
Antigen Retrieval	Citrate, EDTA, enzymatic retrieval	Epitope exposure and standardization	CLSI: Pre-examination variables; ISO: Process control
Controls	Positive, negative, tissue controls	Assay performance monitoring	CLSI: Quality control; ISO: Quality assurance
Validation Panels	Characterized tissue specimens	Analytical performance assessment	CLSI: Specimen selection; ISO: Examination processes

Data Analysis and Interpretation Framework

The integration of CLSI and ISO frameworks extends to data analysis and interpretation, where statistical rigor and clinical relevance must be balanced. CLSI guidelines provide specific recommendations for statistical approaches appropriate for different validation parameters [27]. These analytical protocols support ISO 15189's requirement for ensuring quality of examination results through appropriate data analysis methods [26].

For IHC assays, the data analysis framework should include concordance assessment between readers, comparison to reference standards, and reproducibility evaluation across multiple runs [7]. CLSI guidelines recommend specific statistical measures for each of these parameters, including Cohen's kappa for interobserver agreement, sensitivity and specificity for accuracy, and coefficients of variation for reproducibility [27]. These quantitative assessments provide objective evidence of assay performance that satisfies ISO 15189's requirement for monitoring the validity of examination results [26].

The interpretation of validation data should also consider clinical utility and intended use, reflecting ISO 15189's patient-centered approach [24]. For companion diagnostics, this includes assessing the predictive value for treatment response and establishing clinically relevant scoring thresholds [7]. This integrated approach ensures that validation demonstrates both technical robustness and clinical applicability.

Comparative Analysis with Other Regulatory Frameworks

CLIA vs. ISO 15189 with CLSI Support

In the United States, the Clinical Laboratory Improvement Amendments (CLIA) establish mandatory requirements for laboratory testing, while ISO 15189 adoption is voluntary [29]. However, CLSI guidelines serve as a bridge between these frameworks, providing implementation guidance recognized by U.S. regulators [7].

CLIA focuses primarily on regulatory compliance with specific requirements for proficiency testing, patient test management, and quality control [29]. In contrast, ISO 15189 emphasizes continuous improvement and risk management within a comprehensive quality management system [29]. CLSI guidelines help laboratories meet both sets of requirements by providing detailed procedures that satisfy CLIA's regulatory standards while supporting ISO 15189's quality management principles [7].

For IHC assays, CLIA establishes general requirements for test validation but does not provide specific guidance on how to satisfy these requirements [7]. CLSI documents fill this gap by offering detailed recommendations on validation study design, performance criteria, and acceptance standards [27] [7]. This makes CLSI guidelines particularly valuable for laboratories operating under CLIA regulations while pursuing ISO 15189 accreditation.

Global Regulatory Alignment

The synergy between ISO 15189 and CLSI guidelines facilitates global regulatory alignment, enabling laboratories to efficiently meet requirements across different jurisdictions. This is particularly important for pharmaceutical companies developing companion diagnostics for global markets [7].

In the European Union, the In Vitro Diagnostic Regulation (IVDR) requires rigorous validation of diagnostic assays, with companion diagnostics uniformly classified as Class C devices [7]. ISO 15189 provides the quality management framework for IVDR compliance, while CLSI guidelines offer the technical protocols for generating the necessary validation data [7]. Similarly, in the United States, the FDA recognizes CLSI guidelines for satisfying regulatory requirements for premarket submissions [27] [7].

This regulatory alignment allows laboratories to design comprehensive validation studies that simultaneously address multiple regulatory frameworks. As noted by Precision for Medicine, "Validation studies performed in US-based laboratories can be designed to meet both CLIA and CLSI standards while simultaneously supporting EU regulatory submissions under ISO 13485 and good clinical laboratory practice (GCLP) guidelines" [7]. This integrated approach streamlines the regulatory process and reduces duplication of efforts.

Diagram 2: Integrated validation workflow showing how CLSI guidelines and ISO 15189 framework complement each other throughout the assay development and commercialization process.

For researchers, scientists, and drug development professionals working with IHC assays, the strategic integration of CLSI guidelines within an ISO 15189 quality framework offers a comprehensive approach to assay validation. This synergy combines the structured quality management of ISO 15189 with the technical specificity of CLSI guidelines, creating a robust foundation for generating reliable, defensible data [24] [25].

The complementary nature of these standards is particularly valuable in the context of companion diagnostic development, where regulatory requirements are stringent and technical complexity is high [7]. By implementing CLSI's detailed protocols within ISO 15189's quality management system, laboratories can efficiently address both U.S. and EU regulatory expectations while maintaining operational efficiency [7].

This integrated approach ultimately supports the development of clinically relevant and technically robust IHC assays that reliably inform treatment decisions. As laboratory medicine continues to evolve with new technologies and increasing regulatory expectations, the synergistic relationship between ISO 15189 and CLSI guidelines will remain essential for advancing patient care through accurate and reliable diagnostic testing.

Implementing ISO 15189: A Step-by-Step Framework for IHC Assay Validation

For researchers and drug development professionals, validating an immunohistochemistry (IHC) assay is a critical step that bridges scientific discovery and clinical application. The scope and rigor of this validation are not arbitrary but are fundamentally dictated by three interdependent pillars: the assay's intended use, its regulatory classification under frameworks like the In Vitro Diagnostic Regulation (IVDR), and its origin as a commercial product or laboratory-developed test (LDT). Under the IVDR, which became fully applicable in May 2022, the regulatory landscape has shifted significantly, bringing most LDTs under its scope and mandating a risk-based classification system [30] [31]. This guide objectively compares how these factors determine validation requirements, providing a structured framework for designing compliant and scientifically sound validation protocols within a quality management system aligned with ISO 15189 [30].

Core Concepts and Regulatory Frameworks

The Foundational Role of "Intended Purpose"

The intended purpose is the manufacturer's definitive statement of what the device does and its clinical application. It is the primary determinant for the device's qualification, risk classification, and the extent of performance evaluation required [32].

For an IHC assay, the intended purpose must precisely define several key aspects [32]:

• Analyte and Function: The specific biomarker detected and the assay's function (e.g., screening, diagnosis, prognosis, or companion diagnostics).
• Clinical Context: The physiological/pathological state, patient population, and predispositions to be identified.
• Specifications: The type of specimen required, whether the test is qualitative or quantitative, and the intended user profile. Inconsistencies in articulating the intended purpose across technical documentation, marketing materials, and validation protocols are a common source of non-compliance, as they can lead to a mismatch between claims and the evidence provided [32].

The IVDR Risk-Based Classification System

The IVDR introduced a rule-based classification system with four risk classes (A to D), which directly dictates the conformity assessment route and the depth of scrutiny from a Notified Body [31].

Table 1: IVDR Risk Classification and Examples for IHC Assays

IVDR Class	Level of Risk	Example IHC Assays	Notified Body Involvement
Class A	Low individual and public risk	Laboratory equipment, specimen receptacles	Self-certification (lowest oversight)
Class B	Moderate risk	Self-tests (with exceptions), certain SARS-CoV-2 tests [33]	Mandatory for ~80-90% of IVDs
Class C	High individual risk and/or moderate public risk	Cancer staging, companion diagnostics, blood grouping [7] [31]
Class D	High individual and public risk	Detection of emerging SARS strains, HIV/hepatitis testing [33] [31]	Highest level of scrutiny

Test Origin: Commercial IVD vs. Laboratory-Developed Test (LDT)

The origin of a test creates distinct regulatory pathways. An LDT, often referred to as an "in-house" device, is developed and used within a single legal entity [30]. Under IVDR, LDTs benefit from an exemption under Article 5(5) only if they meet strict conditions, including [30]:

• Non-transferability: The device is not transferred to another legal entity.
• Clinical Justification: There is no equivalent CE-marked device available to meet the specific patient need.
• Quality Framework: The device is manufactured and used within an environment subject to a quality management system, such as one certified to ISO 15189 [30].

Comparative Analysis: How Scope Dictates Validation Rigor

The interplay between intended use, classification, and origin directly translates into vastly different validation requirements, as detailed in the following experimental protocols and data.

Analytical Validation Protocols by Intended Use

The College of American Pathologists (CAP) provides evidence-based guidelines for the analytical validation of IHC assays. The required experimental rigor scales significantly with the clinical consequence of the test result [9].

Table 2: CAP Recommended IHC Assay Validation Sample Sizes (2024 Update)

Intended Use / Assay Type	Minimum Positive Cases	Minimum Negative Cases	Target Concordance	Key Methodological Notes
General IHC Assays (e.g., for tumor classification)	10	10	≥90%	Use a tiered comparator list, from known protein calibrators to expected staining patterns [9]
Predictive Marker with Multiple Scoring Systems (e.g., HER2, PD-L1)	10 per scoring system	10 per scoring system	≥90%	Each distinct scoring system (by tumor site/type) requires separate validation [9]
Assays on Cytology Specimens with Alternative Fixatives	10	10	N/A	Mandatory separate validation if fixation differs from original FFPE validation [9]
Verification of FDA-Cleared/Approved Assays	As per manufacturer's instructions	As per manufacturer's instructions	As per manufacturer's specifications	Follow the manufacturer's verified protocol [9]

The following workflow outlines the decision process for determining the appropriate validation protocol based on the assay's characteristics:

Validation Requirements by IVDR Class and Test Origin

The regulatory pathway and corresponding validation burden are a direct function of the IVDR classification. The transition from the old In Vitro Diagnostic Directive (IVDD) to the IVDR represents a paradigm shift, increasing the proportion of devices requiring Notified Body involvement from about 15% to 80-90% [31]. The following table compares the core validation data packages required for different regulatory scenarios.

Table 3: Comparative Validation Requirements by Regulatory Pathway

Validation Component	CLIA Lab Validation (LDT)	EU IVDR Class C (e.g., Companion Dx)	US FDA PMA (Class III)
Scientific Validity	Required	Required (Pillar 1 of Performance Evaluation)	Required
Analytical Performance	CLIA-based + guidelines [9]	Comprehensive (Pillar 2) per IVDR Annex XIII	Extensive, often exceeding CLIA; pre-submission meeting advised [7]
Clinical Performance	Often linked to lab's patient population	Required (Pillar 3) in target population and intended user	Required for intended use population
Multi-site Reproducibility	Not typically required for single site	Required for IVD kits	Required
Quality System	ISO 15189 or equivalent [30]	ISO 13485 QMS	21 CFR Part 820 (transitioning to ISO 13485) [7]
Post-Market Surveillance	Per lab QMS	Mandatory PMPF and PMS plans [31]	Mandatory post-approval studies

The relationship between the core components of performance evaluation under the IVDR and their role in the device lifecycle is illustrated below:

The Scientist's Toolkit: Essential Research Reagent Solutions

A robust IHC validation relies on critical reagents and materials, each serving a specific function in ensuring analytical precision and accuracy.

Table 4: Essential Materials for IHC Assay Validation

Research Reagent / Material	Function in Validation	Key Considerations
FFPE Tissue Sections	The primary biological matrix for validating staining protocols and specificity.	Must include known positive and negative controls; should represent the intended patient population and tissue types [9].
Cell Line Microarrays	Serve as calibrators or controls with known antigen expression levels for quantitative assessment.	Useful for standardizing staining intensity and monitoring assay drift [9].
Reference Standards	Well-characterized reagents (antibodies, tissues) used to establish expected results for comparator methods.	Critical for method comparison studies; can be commercial, WHO International Standards, or internally characterized materials.
Control Materials (Positive/Negative)	Run concurrently with patient samples to verify the assay is performing within established parameters.	Should challenge all aspects of assay performance (sensitivity, specificity); third-party controls are recommended [5].
External Quality Assessment (EQA) Materials	Specimens provided by an independent EQA scheme (e.g., NordiQC) to assess inter-laboratory performance.	Provides an external benchmark and supports compliance with IVDR and ISO 15189 requirements for continued competence [30] [5].

Defining the validation scope for an IHC assay is a systematic process guided by the trinity of intended use, IVDR classification, and test origin. As the regulatory landscape evolves, with the IVDR's full implementation and ongoing updates to standards like the CAP guidelines, a proactive and strategic approach to validation is paramount. For global drug development, this means designing validation studies from the outset that can meet the most stringent requirements of multiple jurisdictions, thereby streamlining the path from research to clinical application and ultimately ensuring the delivery of safe, effective, and reliable diagnostic tools to patients.

In the field of immunohistochemistry (IHC), the performance characteristics of assays—accuracy, precision, sensitivity, and specificity—form the cornerstone of reliable diagnostic testing. These parameters determine the technical quality of IHC results and directly impact patient diagnosis, treatment selection, and clinical outcomes. Within medical laboratories, the ISO 15189:2022 standard specifies requirements for quality and competence, providing a critical framework for ensuring these performance characteristics are rigorously validated and maintained [34] [18]. This standard emphasizes verification and validation of examination procedures, measurement accuracy, and traceable results to minimize diagnostic errors [18].

The evolving landscape of cancer diagnostics, particularly with the emergence of new therapeutic targets like HER2-low breast cancer, has highlighted limitations in conventional IHC assays and underscored the urgent need for enhanced validation protocols [35] [36]. This guide objectively compares performance characteristics across different IHC validation approaches, examining traditional methods against innovative solutions such as deep learning and refined scoring systems, all within the context of ISO 15189 standards for medical laboratory quality management.

Fundamental Concepts and Regulatory Context

Defining Key Performance Characteristics

• Accuracy: Refers to the closeness of agreement between a test result and the accepted reference value. In IHC, this represents how well immunohistochemical staining reflects true antigen expression patterns.
• Precision: Indicates the closeness of agreement between independent test results obtained under stipulated conditions. Precision includes repeatability (same operating conditions) and reproducibility (different conditions, laboratories, or operators).
• Analytical Sensitivity: The lowest concentration of an analyte that an assay can reliably detect. For IHC, this translates to the minimum antigen quantity detectable above background staining.
• Analytical Specificity: The ability of an assay to detect only the intended target antigen without cross-reacting with similar epitopes or non-target structures.

The ISO 15189:2022 Framework

The ISO 15189:2022 standard provides comprehensive requirements for quality management in medical laboratories, with significant implications for IHC validation [34] [18]. Key updates in the 2022 version include enhanced focus on risk management, integration of point-of-care testing requirements previously outlined in ISO 22870, and clarified structural and governance roles [14] [18]. Laboratories must transition to this updated standard by December 2025 [14].

ISO 15189:2022 mandates that laboratories establish procedures for validation and verification of examination methods, including determination of accuracy, precision, sensitivity, and specificity relevant to the intended use [18]. The standard's Clause 7 specifically addresses process requirements across the pre-examination, examination, and post-examination phases, requiring verification and validation of testing methods with defined procedures for sample handling and result reporting [18].

Table: ISO 15189:2022 Clauses Relevant to IHC Performance Characteristics

Clause	Title	Key Requirements for IHC Performance
Clause 4	General Requirements	Impartiality, confidentiality, and patient-focused obligations
Clause 6	Resource Requirements	Personnel competence, equipment calibration, and suitable facilities
Clause 7	Process Requirements	Verification/validation of methods, sample handling procedures, quality assurance
Clause 8	Management System Requirements	Quality management system, risk management, corrective actions, continuous improvement

Standardization and Validation Guidelines

CAP Guidelines for IHC Assay Validation

The College of American Pathologists (CAP) published updated guidelines in 2024 establishing evidence-based protocols for IHC analytical validation [37] [9]. These guidelines provide specific requirements for various assay types:

• Laboratory-developed non-predictive assays: Minimum of 10 positive and 10 negative cases
• Laboratory-developed predictive assays: Minimum of 20 positive and 20 negative cases
• Unmodified FDA-approved assays: Verification following manufacturer instructions or minimum 20 positive and 20 negative cases
• Assays with distinct scoring systems: Separate validation for each assay-scoring system combination

The CAP guidelines require laboratories to achieve at least 90% overall concordance between new and comparator assays during validation [37] [9]. For cytology specimens not fixed identically to tissues used for initial validation, separate validations with minimum 10 positive and 10 negative cases are mandated [9].

Comparative Regulatory Frameworks

Different regulatory jurisdictions employ varying approaches to IHC assay validation:

• United States: Clinical Laboratory Improvement Amendments (CLIA) regulations establish federal standards, but do not define specific validation methodologies [7]. The FDA provides additional oversight for in vitro diagnostics, with pre-market approval (PMA) submissions requiring studies exceeding CLIA requirements [7].
• European Union: The In Vitro Diagnostic Regulation (IVDR) classifies companion diagnostics as Class C devices, requiring technical dossiers with analytical and clinical data, plus audit of quality management systems by notified bodies [7].
• International Standards: ISO 15189:2022 accreditation is mandatory for all medical laboratories in some countries (e.g., France, Hungary), while others implement partial mandates for specific test types [18].

Experimental Approaches for Validation

Methodologies for Establishing Performance Characteristics

The CAP guidelines outline multiple approaches for IHC assay validation, ordered here from most to least stringent [9]:

• Comparison to cell line calibrators: Using cell lines with known protein quantities
• Non-immunohistochemical method comparison: Such as flow cytometry or fluorescent in-situ hybridization
• Inter-laboratory comparison: Testing same tissues in another laboratory using a validated assay
• Intra-laboratory comparison: Comparison with prior testing of same tissues with a validated assay
• Clinical trial laboratory comparison: Comparing with results from laboratories that performed testing for clinical trials
• Antigen localization assessment: Comparing with expected architectural and subcellular localization
• Published clinical trial data comparison: Comparing against percent positive rates in published trials
• Proficiency testing challenges: Comparison with previously graded tissues from formal proficiency testing programs

Workflow for IHC Assay Validation

The following diagram illustrates the comprehensive validation workflow for immunohistochemical assays, integrating multiple comparative methodologies and quality control checkpoints:

Comparative Performance Data

Traditional IHC Assay Performance

Conventional IHC assays demonstrate variable performance characteristics across different biomarkers and applications:

Table: Performance Characteristics of Conventional HCC IHC Markers [38]

Marker	Target/Antigen	Sensitivity (%)	Specificity (%)	Staining Pattern
HepPar1	CPS1 (mitochondrial)	70–87	71–97	Granular cytoplasmic
Arg-1	Urea cycle enzyme	76.6–96.0	97.5	Cytoplasmic
GPC3	Cell-surface proteoglycan	63–92	94–100	Cytoplasmic/membranous
Albumin ISH	Albumin mRNA	99	100	Cytoplasmic mRNA dots
pCEA	Biliary glycoprotein	60–90	95–100	Canalicular

The CASI-01 study, a consortium of 54 IHC laboratories in Europe and the United States, evaluated HER2 assay performance and found substantial variation in predicate assay sensitivity across laboratories, with detection thresholds ranging from 30,000 to 60,000 HER2 molecules per cell [35]. These assays showed 85.7% sensitivity and 100% specificity for identifying HER2-positive tumors but performed poorly in classifying HER2-low tumors due to limited dynamic range [35].

Emerging Technologies and Enhanced Approaches

Deep Learning-Based IHC Prediction

A 2025 study developed deep learning models to predict IHC staining directly from H&E whole slide images for gastrointestinal cancer subtyping [39]. The study automatically extracted 415,463 tiles from H&E slides for model construction based on annotation transfer from IHC slides, creating five IHC biomarker prediction models (P40, Pan-CK, Desmin, P53, Ki-67) [39].

Table: Performance of Deep Learning-Based IHC Prediction Models [39]

Biomarker	AUC	Accuracy (%)	Clinical Concordance with Conventional IHC (%)
P40	0.96	90.81	96.67-100
Pan-CK	0.90	83.04	96.67-100
Desmin	0.92	85.00	96.67-100
P53	0.94	88.57	70.00
Ki-67	0.92	86.67	Quantitative variability: 17.35% ±16.2%

The multi-reader multi-case (MRMC) validation study demonstrated substantial concordance between AI-generated and conventional IHC across most markers, supporting its potential as an assistive tool in routine diagnostics [39]. The Ki-67 proliferation index showed an intraclass correlation coefficient of 0.415 (P = 0.015) between AI and conventional IHC [39].

Specialized Scoring Systems for HER2-Low Detection

Research has demonstrated that conventional HER2 IHC assays, originally designed to detect HER2 overexpression, lack the dynamic range for accurate HER2-low classification [35]. A specialized HER2-low focused immunohistochemistry scoring system was independently validated to enhance pathologist precision and consistency [36].

Key components of this optimized approach include:

• Explicit identification of scoring pitfalls and heterogeneity challenges
• Requirement to evaluate all tumor tissue at ×40 magnification before scoring as 0
• Use of the 4B5 Rabbit Monoclonal Primary Antibody with appropriate controls
• Implementation of a 5-month washout period for learning retention assessment

This specialized approach achieved concordance levels of 75% to 86.67% with κ values in the moderate-to-excellent range, significantly improving upon historical performance with conventional scoring methods [36].

The Scientist's Toolkit: Essential Research Reagents

Table: Key Research Reagents for IHC Validation Studies

Reagent/Cell Line	Function in Validation	Application Example
Cell Line Calibrators	Provide known protein quantities for quantitative reference	Establishing analytical sensitivity thresholds [9]
HER2 4B5 Antibody	Rabbit monoclonal primary antibody for HER2 detection	HER2-low scoring system validation [36]
FFPE Tissue Sections	Standardized tissue preparation for validation studies	CAP-recommended validation sets [37]
Automated Image Analysis Software	Objective quantification of staining intensity	CASI-01 study showing 6-fold improvement in HER2-low detection [35]
HER2 Control Slides	Calibration and quality assurance for HER2 testing	Including positive, negative, and low-expression controls [36]
H&E Whole Slide Images	Basis for deep learning model training	AI-IHC prediction model development [39]

The comparison of IHC performance characteristics across different methodologies reveals both challenges and opportunities in assay validation. Traditional IHC assays demonstrate solid performance for their intended targets but show limitations in emerging applications like HER2-low detection, where specialized scoring systems and enhanced analytical sensitivity become crucial.

Deep learning-based approaches show promising results with AUCs ranging from 0.90 to 0.96 across multiple biomarkers, potentially offering alternatives to conventional IHC while maintaining substantial concordance [39]. However, the variable performance for specific markers like P53 (70% concordance) highlights the need for continued refinement and validation against established methods.

The ISO 15189:2022 standard provides an essential framework for ensuring rigorous validation of these performance characteristics, with requirements for verification procedures, personnel competence, and quality management systems [18]. As IHC technologies evolve toward more sensitive detection and quantitative applications, adherence to these standards and evidence-based guidelines like the 2024 CAP recommendations will be critical for maintaining diagnostic accuracy and patient safety in an era of increasingly targeted therapies.

In the field of diagnostic immunohistochemistry (IHC), adhering to standardized processes for test implementation is not merely a regulatory formality but a cornerstone of patient safety and reliable research. For scientists and drug development professionals, navigating the distinct requirements for in vitro diagnostic (IVD) tests and laboratory-developed tests (LDTs) is crucial. The international standard ISO 15189 provides the framework for quality and competence in medical laboratories, mandating specific approaches to ensure that tests are accurate, reliable, and clinically meaningful [40]. This guide objectively compares the performance of commercial CE-IVD tests and LDTs, detailing the experimental protocols that underpin their verification and validation.

Defining Verification and Validation in the Laboratory

A clear understanding of the terms "verification" and "validation" is the first critical step, as they apply to different scenarios and entail different levels of effort.

• Verification is the process of confirming that a commercial IVD test performs as claimed by the manufacturer when used within your specific laboratory environment. It ensures that your lab can reproduce the manufacturer's stated performance claims for parameters like accuracy and precision using your own equipment and personnel [40]. For a CE-marked PCR assay, this means verifying that it achieves the expected sensitivity and specificity in your facility before it is used for patient diagnosis [40].

• Validation is a more extensive process that establishes the performance characteristics of an LDT or a significantly modified IVD test. It demonstrates and documents that the new or altered test is fit for its intended purpose and delivers clinically accurate results [40]. Developing an in-house next-generation sequencing (NGS) test for oncology, for instance, requires a full validation to establish its sensitivity, specificity, and reproducibility [40].

The table below summarizes the key distinctions.

Table 1: Core Differences Between Verification and Validation

Feature	Verification	Validation
Definition	Confirming manufacturer's performance claims in your lab [40]	Establishing performance for a new or modified test [40]
Applicability	Commercial IVD tests (e.g., CE-IVD, FDA-cleared) [40]	Laboratory-developed tests (LDTs) or modified IVDs [40]
Regulatory Basis	Required under ISO 15189 for commercial tests [40]	Mandatory for LDTs under ISO 15189 and IVDR [40]
Process Complexity	Less extensive [40]	More extensive [40]

Decision Workflow for Test Implementation

Regulatory Frameworks and Performance Standards

The regulatory landscape for IHC assays is complex, with requirements varying based on the test's intended use and geographic market. For IVDs used in clinical trials, a significant risk (SR) assessment is often required. This evaluation determines if the test's use for patient stratification or treatment decisions necessitates an Investigational Device Exemption (IDE) submission to the FDA [7]. A key difference between the US and EU systems is the classification of companion diagnostics (CDx); in the EU, they are uniformly Class C devices under the In Vitro Diagnostic Regulation (IVDR) [7].

The College of American Pathologists (CAP) provides continuously updated guidelines for the analytic validation of IHC assays. A key update in 2024 harmonized the validation requirements for all predictive markers (like HER2 and PD-L1), setting a uniform 90% concordance requirement for the new assay compared to a validated method [9]. Furthermore, the guideline now stipulates that laboratories must separately validate each unique combination of an assay and its scoring system, acknowledging that performance can depend on tumor site and clinical indication [9].

Comparative Experimental Data: IVD vs. LDT

Objective performance data is essential for selecting the appropriate testing pathway. A 2022 academic study provides a compelling comparison, modeling the cost-effectiveness of PD-L1 testing in non-small cell lung cancer (NSCLC) using IVDs versus LDTs [41].

The study found that IVDs demonstrated significantly higher accuracy in diagnosing NSCLC, with a 93% success rate compared to 73% for LDTs. This 20-percentage-point difference translates to a substantially greater chance of misdiagnosis with LDTs. Consequently, approximately one in four patients could receive incorrect treatment based on LDT results. From a health economic perspective, the use of IVDs led to a 19% increase in successful diagnosis and treatment, with only a minimal (0.4%) increase in direct diagnostic costs, thereby reducing overall healthcare costs associated with disease progression and adverse event management [41].

Table 2: Performance and Outcomes of IVD vs. LDT in NSCLC PD-L1 Testing

Parameter	IVD	LDT
Diagnostic Accuracy	93% [41]	73% [41]
Risk of Misdiagnosis	Lower	20% greater [41]
Impact on Treatment	Higher alignment	1 in 4 patients potentially incorrect [41]
Cost vs. Benefit	+0.4% cost, +19% successful outcomes [41]	Less cost-effective

Detailed Protocols for Validation and Verification

Protocol for Verifying a CE-IVD IHC Assay

When a lab introduces a new commercial IVD, verification focuses on confirming the manufacturer's claims.

• Step 1: Define Performance Parameters. The key parameters to verify typically include accuracy (or concordance) and precision. According to CAP guidelines, for an unmodified FDA-approved/cleared assay, verification involves testing a minimum of 10 positive and 10 negative cases [9]. The overall concordance with the expected results must meet or exceed the 90% threshold established by CAP [9].
• Step 2: Execute the Verification Study. The selected cases are run using the new IVD assay according to the manufacturer's instructions. The results are then compared against a validated method or a known gold standard.
• Step 3: Analyze and Document. Calculate the positive, negative, and overall percent agreement. If the results meet the pre-defined acceptance criteria (e.g., ≥90% overall concordance), the assay is verified for clinical use [9].

Protocol for Validating an LDT IHC Assay

Validation of an LDT is a more comprehensive process, as the performance characteristics are not pre-established.

• Step 1: Establish the Intended Use. Clearly define the test's purpose, including the target analyte, tumor type, and scoring system. This is critical for determining the scope of validation [9].
• Step 2: Select a Comparator. CAP guidelines list several acceptable comparators, ordered from most to least stringent. These include comparison to IHC results from protein calibrators, a non-IHC method (like FISH), or testing in another validated laboratory [9].
• Step 3: Determine Sample Size and Assess Performance. The updated CAP guideline recommends a minimum of 30 positive and 30 negative cases for a new LDT. However, for predictive markers with distinct scoring systems, each scoring system must be validated separately. Similarly, a separate validation with 10 positive and 10 negative cases is required for specimens fixed in alternative fixatives (e.g., cytology specimens) if they differ from the tissues used in the initial validation [9].
• Step 4: Assess Analytical Sensitivity and Specificity. The protocol must be optimized to balance sensitivity and specificity. This is particularly challenging for IHC assays that detect a loss of protein expression, where "intermediate" staining patterns can complicate interpretation [42]. The choice of tissue for analytical validation is critical and must be carefully considered [42].

Case Study & Emerging Challenges

The evolution of cancer therapeutics continuously presents new diagnostic challenges. The emergence of therapies targeting HER2-low breast cancer has exposed limitations in current standard HER2 IHC assays. The CASI-01 study, a consortium of 54 IHC laboratories, found that while current FDA-cleared assays are reliable for identifying HER2-positive tumors (85.7% sensitivity, 100% specificity), they perform poorly in classifying HER2-low tumors due to a limited dynamic range [35].

This case highlights a critical consideration for researchers: standard IVDs, while robust for their approved indications, may lack the analytical sensitivity required for new clinical paradigms. The CASI-01 study demonstrated that higher-sensitivity assays combined with image analysis delivered a six-fold gain in accurate HER2-low detection [35]. This underscores the potential role for optimized LDTs or next-generation IVDs in advancing precision medicine.

The Scientist's Toolkit: Key Research Reagent Solutions

The following table details essential materials and their functions in IHC assay development and validation.

Table 3: Essential Reagents and Materials for IHC Validation

Item	Function in Validation
Validated Comparator	Serves as a reference standard for accuracy and concordance testing [9].
Cell Line Calibrators	Tissues or cell lines with known protein levels provide a stringent comparator for assay optimization [9].
Proficiency Testing Challenges	Previously graded tissues from formal programs help benchmark laboratory performance [9].
Internal Control Tissues	Tissues with known antigen expression ensure the assay is functioning correctly across runs [42].
Tissues with Alternative Fixatives	Essential for validating assay performance on cytology or non-FFPE specimens [9].

The choice between verifying a CE-IVD and validating an LDT is a fundamental decision that impacts data reliability, regulatory compliance, and ultimately patient care. Verification offers a streamlined path for implementing robust, commercially available tests, while validation provides the flexibility to develop novel assays for cutting-edge research. As the field moves towards increasingly precise biomarker detection, the principles outlined in standards like ISO 15189 and CAP guidelines provide an indispensable roadmap. By tailoring their approach with a clear understanding of these processes, scientists can ensure their IHC assays are both scientifically sound and regulatorily compliant, thereby reliably supporting drug development and clinical research.

For researchers and scientists in drug development, the validation of immunohistochemical (IHC) assays represents a critical juncture between basic research and clinical application. Within the framework of ISO 15189 standards for medical laboratory quality and competence, validation ensures that laboratory-developed tests (LDTs) produce reliable, reproducible results that can withstand regulatory scrutiny [43] [15]. The process confirms, through provision of objective evidence, that specific requirements for a particular intended use have been fulfilled, encompassing level of certainty, test performance characteristics, and fit-for-purpose application [43]. With the implementation of the In Vitro Diagnostic Regulation (IVDR) in the European Union and evolving FDA expectations in the United States, the demands for rigorous validation have intensified, particularly for predictive biomarkers used in patient selection for targeted therapies [15] [7]. This comparative guide examines the experimental approaches and data requirements for building robust validation sets that meet these escalating standards across different regulatory environments.

Core Components of IHC Assay Validation

Defining Validation and Verification Parameters

Within the IHC validation landscape, precise terminology governs the required processes. Validation constitutes demonstration through objective evidence that requirements for a specific intended use are met, while verification confirms that previously validated specifications can be fulfilled in a new laboratory environment [15]. For ISO 15189-compliant laboratories, this distinction dictates the extent of testing required when implementing new assays. The key performance characteristics assessed during these processes include:

• Accuracy: The degree of concordance with a gold standard method [15]
• Analytical Sensitivity: The smallest amount of antigen that can be accurately detected [15]
• Analytical Specificity: The ability to detect one particular substance rather than others [15]
• Repeatability: Consistency of results when testing is repeated within the same run [15]
• Reproducibility: Consistency of results across different runs, operators, and days [15]
• Robustness: Performance stability despite deliberate variations in procedural parameters [15]
• Readout Precision: Intra-observer and inter-observer reproducibility in interpretation [15]

Sample Size Requirements Across Assay Types

The composition of the validation set varies significantly based on the assay type and its intended use. The College of American Pathologists (CAP) provides evidence-based recommendations that have been widely adopted as standards.

Table 1: Sample Size Requirements for IHC Assay Validation

Assay Type	Positive Cases	Negative Cases	Special Considerations	Primary Citation
Laboratory-developed predictive markers	20	20	Should include high and low expressors; span expected clinical range	[9] [37]
Non-predictive laboratory-developed assays	10	10	Rare antigens may require documented rationale for fewer cases	[37]
FDA-cleared/approved assays (verification)	20	20	Follow manufacturer instructions; use this minimum if not specified	[37]
Assays with distinct scoring systems	20	20	Each assay-scoring system combination must be validated separately	[9] [37]
Cytologic specimens (different fixation)	10	10	Required for each new analyte and fixation method combination	[9] [37]

For predictive biomarkers, which determine patient eligibility for targeted therapies, the 2024 CAP guideline update harmonized requirements across all markers, establishing the 20 positive/20 negative case minimum regardless of the specific biomarker [9]. The validation set must include both high and low expressors to adequately span the expected range of clinical results, which is particularly important for biomarkers with continuous scoring systems like PD-L1 or HER2 [37].

Comparative Analysis of International Validation Standards

Methodological Approaches for Validation Studies

The design of validation studies requires careful selection of appropriate comparators. CAP guidelines prioritize these approaches from most to least stringent:

Table 2: Validation Study Design Comparators

Comparator Approach	Description	Relative Stringency	Application Context
Protein calibrators	Comparison to cell lines with known protein quantities	Most stringent	Ideal for quantitative assays
Non-IHC methods	Comparison to FISH, flow cytometry, or other non-IHC techniques	High	Useful for genetic alterations
Inter-laboratory comparison	Testing same tissues in another laboratory with validated assay	Medium	Common for LDT transfer
Intra-laboratory comparison	Comparison to prior testing of same tissues in same lab	Medium	Assay modification
Clinical trial laboratory	Comparison to laboratory that performed clinical trial testing	Medium	Companion diagnostic alignment
Expected antigen localization	Comparison to expected architectural and subcellular patterns	Low	Initial assay development
Published clinical trials	Comparison against percent positive rates in literature	Low	Rare biomarkers
Proficiency testing challenges	Comparison to formally graded tissue challenges	Variable	Ongoing validation

The choice of comparator methodology significantly influences the validation data's regulatory acceptability. For companion diagnostic development or substantial modifications, the more stringent approaches (calibrators, non-IHC methods) are preferred [9] [37].

Acceptance Criteria and Performance Metrics

Across validation frameworks, specific numerical acceptance criteria establish whether an assay performs adequately. The CAP guidelines stipulate at least 90% overall concordance between the new assay and the comparator method for initial analytic validation or verification [37]. This harmonized concordance requirement applies uniformly across all IHC assays, replacing previous variable requirements for different biomarkers [9].

For predictive biomarkers, the Belgian recommendations further classify validation requirements based on test origin and modification status, requiring more extensive validation for laboratory-developed tests (LDTs) without reference methods [15]. The emergence of new therapeutic categories, such as HER2-low breast cancer, has highlighted limitations in traditional validation approaches, demonstrating that while current HER2 assays show 85.7% sensitivity and 100% specificity for HER2-positive identification, they perform poorly for HER2-low classification due to limited dynamic range [35].

Experimental Protocols for IHC Assay Validation

Specimen Selection and Processing Protocols

The validation set must reflect the actual clinical specimens that will be tested routinely. Specimen selection should encompass:

• Tissue types: Various tumor types and normal tissues where applicable
• Fixation methods: Consistent with routine clinical processing (primarily FFPE)
• Expression range: For quantitative assays, cases spanning the entire scoring spectrum
• Control tissues: Known positive and negative controls for each batch

CAP guidelines specify that validation tissues should be processed using the same fixative and processing methods as clinical cases whenever possible [37]. When validating assays for cytologic specimens with different fixation methods (alcohol-based fixatives), separate validation with minimum 10 positive and 10 negative cases is required [9].

For biomarker-specific challenges, such as HER2-low detection, the CASI-01 study implemented a protocol using higher-sensitivity assays combined with image analysis, demonstrating a six-fold improvement in accurate HER2-low detection (P = .0017) compared to standard methods [35]. This highlights how traditional validation approaches may require augmentation for novel therapeutic applications.

Statistical Evaluation Methods

The statistical assessment of validation data must demonstrate both accuracy and reliability. Key evaluation methods include:

• Concordance analysis: Calculation of overall agreement, positive agreement, and negative agreement compared to reference method
• Cohen's kappa statistic: Measures inter-rater agreement beyond chance, with values >0.6 indicating substantial agreement
• Receiver Operating Characteristic (ROC) analysis: For assays with continuous scoring systems, determines optimal cutoff points
• Intraclass correlation coefficient (ICC): Assesses agreement for continuous measures, with ICC >0.9 indicating excellent reliability

In the development of AI-based IHC prediction models, recent studies have employed area under the curve (AUC) metrics ranging from 0.90 to 0.96 for virtual staining models, with accuracies between 83.04% and 90.81% across five different IHC biomarkers [44]. For dual-modality AI approaches combining H&E and IHC images, AUCs exceeding 0.97 for MSI/MMRd prediction in colorectal cancer and 0.96 for PD-L1 prediction in breast cancer demonstrate the potential for advanced computational methods to meet rigorous validation standards [45].

IHC Assay Validation Workflow - The sequential process for developing and validating IHC assays according to international standards.

The Scientist's Toolkit: Essential Research Reagents and Materials

Table 3: Essential Research Reagents for IHC Validation Studies

Reagent/Category	Function in Validation	Specific Application Notes
CE-IVD Antibodies	Provide standardized reagents with documented performance	First choice under IVDR; used according to manufacturer's IFU
Reference Standards	Serve as calibrators for quantitative assessment	Cell lines with known antigen expression levels
Control Tissues	Monitor assay performance across batches	Known positive and negative tissues for each marker
Detection Systems	Signal amplification and visualization	HRP-based systems with DAB chromogen most common
Antigen Retrieval Solutions	Epitope recovery for formalin-fixed tissues	Citrate buffer (pH 6.0) or EDTA (pH 9.0) most used
Automation Platforms	Standardize staining conditions across runs	Platforms from Ventana, Leica, Dako commonly used
Image Analysis Software	Objective quantification of staining	Particularly valuable for continuous scoring systems
Whole Slide Scanners	Digital pathology integration	Enable computational pathology approaches

The selection of appropriate reagents directly impacts validation outcomes. Under IVDR regulations, CE-IVD tests should be the first choice, with LDTs reserved for situations where no suitable commercial kit exists [15]. For modified CE-IVD tests, the Belgian recommendations classify based on availability of reference methods, with "modified CE-IVD with reference" requiring limited validation similar to verification, while "modified CE-IVD without reference" necessitates full validation [15].

The landscape of IHC assay validation continues to evolve with advancing therapeutic paradigms and computational approaches. The fundamental requirements for robust validation sets - appropriate sample sizes, stringent acceptance criteria, and comprehensive statistical evaluation - remain foundational to assay credibility. For researchers and drug development professionals, understanding the comparative requirements across regulatory frameworks enables efficient development of assays that meet both current and emerging standards. The integration of artificial intelligence and computational pathology into validation workflows promises enhanced objectivity and reproducibility, particularly for complex biomarkers with continuous scoring systems. As targeted therapies expand to include increasingly subtle molecular phenotypes, the rigor applied to validation set development will directly impact patient access to precision medicine.

Within a Quality Management System (QMS) such as ISO 15189, the analytical validation of Immunohistochemistry (IHC) assays is a critical, multi-stage process that ensures test results are reliable, reproducible, and fit for their intended diagnostic purpose. This guide objectively compares validation pathways for different types of IHC tests, supported by experimental data and structured within the framework of international standards.

A robust QMS, like that defined in ISO 15189, provides the scaffolding for all laboratory activities, ensuring quality and competence. This standard emphasizes a systems-oriented approach that is as relevant to anatomic pathology as it is to clinical pathology, focusing on continual improvement, risk management, and the integration of all processes from pre-examination to result reporting [26]. For IHC assays, which are pivotal in diagnosis, prognosis, and predicting response to therapy, analytical validation is a cornerstone of this system. It confirms through objective evidence that an assay consistently performs according to its predefined specifications [11] [15].

The fundamental principle is that the intended use of an assay dictates the rigor of its validation [7]. Assays with higher clinical impact, such as predictive markers (e.g., HER2, PD-L1), require more extensive validation than those used for non-predictive purposes. Recent guidelines, including the 2024 update from the College of American Pathologists (CAP), have worked to harmonize these requirements, for instance, by setting a uniform 90% concordance threshold for all predictive IHC assays [9].

Comparative Analysis of IHC Test Validation Pathways

The validation pathway an IHC test must follow is determined by its regulatory origin and whether it is used as intended by the manufacturer or modified. The following table synthesizes recommendations from international guidelines and peer-reviewed literature to compare these pathways [15].

Table 1: Validation Pathways for Different Types of IHC Tests

Test Type & Definition	Validation Level	Key Performance Characteristics to Assess
CE-IVD / FDA-Cleared: Used strictly per manufacturer's Instructions for Use (IFU).	Verification	Accuracy, Repeatability, Reproducibility, Robustness [15].
Modified CE-IVD / LDT with Reference: Protocol changed but supported by published data or EQA.	Limited Validation	Accuracy, Repeatability, Reproducibility, Robustness, plus Analytical Sensitivity and Specificity [15].
LDT / RUO without Reference: In-house developed or "Research Use Only" test without external reference.	Full Validation	All characteristics from "Limited Validation," plus demonstration of clinical performance [15].

Experimental Protocol for a Full Validation

For a laboratory developing a novel IHC assay as an LDT, a full validation is required. A key experiment is the accuracy and precision study, which often employs a method-comparison approach.

• Objective: To demonstrate the agreement (accuracy) between the new IHC assay and a validated comparator, and to establish the test's repeatability and reproducibility (precision).
• Methodology:
  • Sample Selection: A minimum of 10 positive and 10 negative cases is often recommended, though this number can vary based on the biomarker's prevalence and the intended use of the assay [9] [15]. Cases should be selected to represent a range of expression levels.
  • Comparator: The new assay's results are compared against a "gold standard" or validated reference method. The CAP guideline lists potential comparators in order of stringency, including:
    • Comparison to IHC results from protein-calibrated cell lines.
    • Comparison to a non-IHC method (e.g., flow cytometry, FISH).
    • Comparison to results from a validated assay in another laboratory [9].
  • Testing Protocol: Each sample is tested with the new IHC assay across multiple runs, on different days, and by different technologists to incorporate variables that affect real-world performance.
  • Blinded Evaluation: Stained slides are evaluated by pathologists who are blinded to the expected results and to each other's scores to assess inter-observer reproducibility (readout precision).
• Data Analysis:
  • Accuracy: Calculate the percent concordance between the new assay and the comparator. For predictive markers, a concordance of ≥90% is a common acceptance criterion [9].
  • Precision: Calculate the percentage agreement for intra-run, inter-run, and inter-observer comparisons.

This rigorous protocol was applied in the CASI-01 study, which evaluated HER2 testing. The study found that while standard FDA-cleared assays were reliable for identifying HER2-positive tumors (85.7% sensitivity, 100% specificity), they performed poorly in classifying the "HER2-low" subtype due to limited dynamic range [35]. This finding underscores the need for robust, fit-for-purpose validation, especially as therapeutic paradigms evolve.

Visualizing the IHC Validation Workflow within a QMS

The journey from protocol establishment to final report is a multi-step, iterative process embedded within the QMS. The following diagram illustrates this workflow and its key decision points.

Diagram 1: IHC validation workflow in QMS.

The Scientist's Toolkit: Key Reagents and Materials for IHC Validation

A successful IHC validation relies on a suite of critical reagents and materials. The selection of these components is a key variable that must be controlled and documented during the validation process.

Table 2: Essential Research Reagent Solutions for IHC Assay Validation

Reagent / Material	Critical Function in Validation
Primary Antibodies	The core reagent that specifically binds the target antigen. Source, clone, and dilution must be optimized and locked down during validation [42] [15].
Control Cell Lines & Tissues	Calibrators with known antigen expression levels used to standardize staining and establish assay sensitivity. Crucial for validating assays that detect loss of protein expression [9] [42].
Antigen Retrieval Solutions	Unmask target epitopes altered by tissue fixation. The pH and type of retrieval solution are key protocol variables affecting analytical sensitivity and specificity [42] [15].
Detection Kits	Amplify the primary antibody signal. The kit's sensitivity must be matched to the abundance of the target protein to achieve optimal dynamic range [35].
Reference Materials	Well-characterized tissue samples used as a benchmark for comparing accuracy across different laboratories and platforms [35].

Navigating the path from protocol to report under a QMS framework requires a disciplined, documented approach tailored to the IHC test's inherent risk and novelty. As the field advances towards more precise quantification, especially for biomarkers like HER2-low, the integration of higher-sensitivity methods and image analysis will become increasingly important [35]. A well-documented validation process, compliant with standards like ISO 15189 and evidence-based guidelines, is not merely a regulatory hurdle. It is the fundamental practice that ensures the analytical reliability of IHC results, thereby directly supporting accurate diagnosis and effective patient treatment.

Beyond Basics: Troubleshooting Common IHC Validation Challenges Under ISO 15189

In the field of immunohistochemistry (IHC), the reliability of assay results is fundamentally rooted in the rigorous management of pre-analytical variables. The 2022 update to the ISO 15189 standard, which specifies requirements for quality and competence in medical laboratories, places a significant new emphasis on risk management, requiring laboratories to control all processes that could impact patient results [4]. For researchers and drug development professionals, this formalizes the critical need to standardize the steps before staining even begins: specimen collection, fixation, processing, and antigen retrieval. These pre-analytical factors collectively represent the most significant source of variability in IHC outcomes, directly influencing the accuracy of biomarker discovery and validation [46] [47].

Variations in pre-analytical handling can degrade biomolecules, leading to inaccurate data and potentially compromising research conclusions and drug development decisions. This guide provides a detailed, evidence-based comparison of methodologies for managing pre-analytical variables, framed within the context of ISO 15189:2022's patient-centered risk management approach [4]. It is designed to equip scientists with the protocols and data necessary to build robust, reproducible, and standardized IHC workflows.

Fixation Variables and Optimization Strategies

Fixation is the first critical defense in preserving tissue morphology and antigen integrity. The primary goal is to halt degradation instantly while preserving the native state of antigens for antibody binding.

Fixative Types and Their Biochemical Impact

The choice of fixative dictates the extent of macromolecular cross-linking or precipitation, which directly impacts antigenicity.

• Formalin (Formaldehyde): The most common fixative, 10% Neutral Buffered Formalin (NBF), works by creating methylene bridges between proteins, providing excellent morphological preservation [48]. A key drawback is the potential for epitope masking due to these cross-links, which must be reversed later through antigen retrieval [49] [50].
• Precipitative Fixatives: Alcohols (e.g., methanol, ethanol) and acetone precipitate proteins rather than cross-linking them. They often better preserve antigenicity for some targets but can result in poorer cellular morphology and greater tissue shrinkage [48]. They are frequently used for cytology specimens and frozen sections [46].

The following diagram illustrates the decision-making workflow for tissue fixation and the consequences of improper handling.

The Critical Role of Ischemia and Fixation Time

Two of the most impactful variables are cold ischemic time and fixation duration. Cold ischemic time is defined as the time between tissue devascularization and immersion in fixative. Prolonged ischemia triggers enzymatic degradation, leading to a measurable loss of antigenicity. Studies on HER2 have shown that extended cold ischemia can cause a one- or two-step reduction in IHC score (e.g., from 2+ to 1+ or 0) [47]. The 2023 ASCO-CAP guideline recommends keeping this time "as short as possible," ideally under one hour [47].

Similarly, fixation time must be tightly controlled. Under-fixation (<6 hours in NBF) fails to preserve tissue architecture and antigens, while over-fixation (>72 hours) creates excessive cross-links that can permanently mask epitopes, making them irretrievable [47]. The following table summarizes the effects of these key variables, using HER2 as a model biomarker.

Table 1: Impact of Pre-Analytical Variables on HER2 IHC Results

Pre-Analytical Variable	Definition	Optimal Condition (per guideline)	Consequence of Deviation	Histomorphologic Clue
Cold Ischemic Time [47]	Time from devascularization to fixation.	As short as possible; ≤ 1 hour.	Protein degradation; false negative IHC (e.g., 2+ → 1+/0) [47].	Necrosis, cytoplasmic vacuoles, pyknotic nuclei in tissue center [47].
Fixation Time [47]	Total time in 10% NBF.	6 to 72 hours.	Under-fixation (<6h): Poor morphology, antigen loss.Over-fixation (>72h): Epitope masking.	Zonal staining variation; poor overall staining [47].
Fixative Type [46]	Chemical solution used.	10% Neutral Buffered Formalin.	Non-standard fixatives (e.g., alcohol) may alter antigen conformation, requiring re-validation [46].	Altered staining intensity/pattern compared to FFPE.

Tissue Processing and Specimen-Specific Considerations

After fixation, tissue processing and the type of specimen submitted for analysis introduce another layer of pre-analytical complexity.

Standard Processing and Cytology Challenges

Routine processing of formalin-fixed, paraffin-embedded (FFPE) tissue for sectioning is a well-established standard. However, the rising use of small biopsies and cytology specimens (e.g., fine-needle aspirations, effusions) for ancillary testing presents unique challenges. These specimens may be fixed in alcohol-based solutions or prepared as smears, which have different protein preservation properties than FFPE tissue [46].

The College of American Pathologists (CAP) updated its guidelines in 2024 to explicitly address this. It states that IHC assays performed on cytology specimens not fixed identically to the tissues used for initial validation require a separate validation study, recommending a minimum of 10 positive and 10 negative cases to verify performance [9]. This underscores the necessity of tailoring the pre-analytical and analytical workflow to the specific specimen type.

Antigen Retrieval: Reversing Fixation-Induced Epitope Masking

Antigen retrieval is a revolutionary technique that restores the antigenicity of formalin-fixed tissues by breaking the methylene cross-links formed during fixation [49] [50]. Selecting and optimizing the correct retrieval method is often the single most important factor in achieving successful IHC staining.

Methods of Antigen Retrieval

The two primary retrieval methods are Heat-Induced Epitope Retrieval (HIER) and Proteolytic-Induced Epitope Retrieval (PIER).

• Heat-Induced Epitope Retrieval (HIER): This is the most widely used method. It involves heating tissue sections in a specific buffer (e.g., citrate, Tris-EDTA) to a high temperature (95-100°C) to hydrolyze and break the cross-links [49] [50]. The equipment used can vary, with pressure cookers, steamers, and scientific microwaves being common.
• Proteolysis-Induced Epitope Retrieval (PIER): This older method uses proteolytic enzymes (e.g., trypsin, pepsin) to digest the proteins surrounding the epitope. While gentler for some fragile tissues or specific antigens, it carries a higher risk of destroying the antigen itself or damaging tissue morphology if not meticulously optimized [50].

Table 2: Comparison of Antigen Retrieval Methods

Characteristic	Heat-Induced Epitope Retrieval (HIER)	Proteolysis-Induced Epitope Retrieval (PIER)
Mechanism	Heat energy hydrolyzes formalin cross-links [49].	Enzymatic digestion of proteins obscuring the epitope [50].
Primary Advantage	Broader range of antigens, particularly nuclear; less tissue damage [50].	Preferred for some difficult-to-recover epitopes; less damaging to delicate tissues [50].
Primary Disadvantage	Overheating can damage tissues/antigens; uneven heating in microwaves [49].	High risk of destroying antigen and morphology; requires precise calibration [50].
Key Buffers	Citrate (pH 6.0), Tris-EDTA (pH 9.0), EDTA (pH 8.0/9.0) [49].	Trypsin, Pepsin, Proteinase K (concentration and time critical) [50].

Optimizing Heat-Induced Epitope Retrieval (HIER)

For HIER, the pH of the retrieval buffer is a critical determinant of success. The effect of pH on staining is antigen-dependent and can be categorized [50]:

• Stable Type: pH has minimal effect (e.g., PCNA, CD20).
• V Type: Good staining at both high and low pH, poor around pH 4-5 (e.g., ER, Ki-67).
• Increasing Type: Staining improves with increasing pH (e.g., HMB45).
• Decreasing Type: Staining weakens with increasing pH (rare, e.g., MOC31).

While citrate buffer at pH 6.0 is traditionally used, evidence suggests that EDTA-based buffers at higher pH (8.0-9.0) are often more effective, especially for nuclear antigens [50]. Optimization requires empirical testing using a matrix of time, temperature, and buffer pH.

Table 3: Example Matrix for Optimizing HIER Conditions

Heating Time	Citrate Buffer (pH 6.0)	Tris-EDTA Buffer (pH 9.0)	EDTA Buffer (pH 8.0)
8 minutes	Slide #1	Slide #2	Slide #3
12 minutes	Slide #4	Slide #5	Slide #6
15 minutes	Slide #7	Slide #8	Slide #9

Detailed Experimental Protocols

Protocol A: Heat-Induced Epitope Retrieval (HIER) Using a Pressure Cooker

This method is favored for its uniform heating and rapid temperature achievement [49].

• Materials: Domestic stainless steel pressure cooker, hot plate, slide rack, antigen retrieval buffer (e.g., 10mM Sodium Citrate pH 6.0, 1mM EDTA pH 8.0, or 10mM Tris-EDTA pH 9.0) [49].
• Procedure: a. Add antigen retrieval buffer to the pressure cooker and place it on a hot plate at full power. Rest the lid on top without securing it. b. While the buffer is heating, deparaffinize and rehydrate the tissue sections. c. Once the buffer is boiling, carefully transfer the slides from water to the buffer-filled pressure cooker. d. Secure the lid according to the manufacturer's instructions. e. Once full pressure is reached, time for 3 minutes. f. After 3 minutes, turn off the hotplate, move the cooker to a sink, and run cold water over it to release pressure and cool. g. Once depressurized, open the lid and run cold water over the slides for 10 minutes to cool. h. Continue with the IHC staining protocol [49].

Protocol B: Proteolysis-Induced Epitope Retrieval (PIER)

This method requires careful optimization of enzyme concentration and incubation time [50].

• Materials: 37°C incubator, humidified chamber, enzymatic retrieval solution (e.g., 0.1% Trypsin).
• Procedure: a. Prepare the enzyme solution and pre-warm it to 37°C. b. Pipette the enzyme solution onto the tissue section, ensuring complete coverage. c. Place the slides in a humidified container and incubate at 37°C for 10-30 minutes. The optimal time must be determined empirically. d. Transfer the slides to a container of tap water to stop the enzymatic reaction. e. Rinse under running water for 3 minutes. f. Proceed with the IHC staining protocol [50].

The Scientist's Toolkit: Essential Reagents and Materials

A successful IHC workflow relies on consistent, high-quality reagents. The following table lists key solutions and materials for managing pre-analytical variables.

Table 4: Research Reagent Solutions for Pre-Analytical Optimization

Item	Function/Description	Key Considerations
10% Neutral Buffered Formalin (NBF) [47]	Standard cross-linking fixative for tissue preservation.	Maintains a neutral pH to prevent artifact formation; fixation time must be controlled [47].
HIER Buffer (Citrate, pH 6.0) [49]	Low-pH retrieval buffer for heat-mediated unmasking.	A traditional, widely used buffer; effective for many antigens.
HIER Buffer (EDTA, pH 8.0/9.0) [49] [50]	High-pH retrieval buffer for heat-mediated unmasking.	Often more effective for nuclear antigens and a broader range of targets [50].
Proteolytic Enzymes (e.g., Trypsin) [50]	Enzyme solution for proteolytic-induced epitope retrieval.	Concentration, time, and temperature are critical and must be tightly optimized to avoid tissue damage [50].
Liquid-Based Cytology (LBC) Fixatives [46]	Alcohol-based fixatives for cytology specimens.	Requires separate IHC validation as performance may differ from FFPE [46].

The journey to reliable IHC data begins the moment a tissue sample is collected. As detailed in this guide, variables like cold ischemia time, fixation duration, and antigen retrieval methodology have a profound and measurable impact on staining results. The updated ISO 15189:2022 standard reinforces this by mandating a proactive, risk-management approach to all laboratory processes, with the ultimate goal of ensuring reliable results and patient safety [4].

For researchers and drug developers, this means that robust, documented control of pre-analytical variables is not just a best practice—it is a fundamental component of assay validation and quality. By adopting the standardized protocols and optimization strategies outlined here, scientists can significantly reduce variability, enhance the reproducibility of their IHC assays, and generate data that meets the rigorous standards required for both groundbreaking research and robust drug development.

Within the framework of ISO 15189 standards for medical laboratory quality, robust revalidation strategies are fundamental to ensuring the ongoing reliability of immunohistochemistry (IHC) assays. Revalidation is a mandatory process for quality assurance, essential for maintaining the validated status of equipment, manufacturing processes, and computer systems against a backdrop of evolving reagents, protocols, and regulatory demands [51]. For researchers and drug development professionals, establishing a disciplined approach to managing change is not merely a regulatory checkbox but a critical component of scientific integrity. The complexity of IHC, as a multimodality analytical method, makes it particularly vulnerable to pre-analytical, analytical, and post-analytical variations [17]. A "Fit-For-Purpose" philosophy, aligned with the intended use of an IHC test—whether for diagnostic (type 1), prognostic (type 2a), or pharmaco-predictive (type 2b) purposes—must underpin all revalidation activities [17] [15]. This guide outlines a systematic framework for revalidation, providing strategies to navigate changes in reagents and protocols while upholding the stringent requirements of ISO 15189 research.

The Regulatory and Standards Framework for Revalidation

Adherence to international standards and regulations forms the backbone of any effective revalidation strategy. Laboratories must operate within a complex landscape defined by several key documents and oversight bodies.

• ISO 15189:2012: This international standard specifies requirements for quality and competence in medical laboratories. It provides the overarching framework for a laboratory's Quality Management System (QMS), under which all validation and revalidation activities must be documented and performed [15].
• CLSI Guidelines: The Clinical Laboratory Standards Institute (CLSI) develops globally recognized standards for laboratory testing. Their guidelines are critical for designing statistically sound validation studies, though their application to IHC requires careful interpretation, sometimes necessitating pre-submission meetings with regulators for alignment [7].
• IVDR (In Vitro Diagnostic Regulation): Effective in the European Union since May 2022, the IVDR classifies most IHC tests as Class C devices. It demands rigorous performance evaluation and places specific obligations on laboratories using "in-house developed tests" under Article 5(5) [15].
• 21 CFR Part 820: This US regulation defines Quality System Requirements (QSR) for medical devices. For companion diagnostics (CDx) commercialized in the US, compliance with this regulation and successful passage of a Bioresearch Monitoring (BIMO) audit are required prior to FDA approval [7].

A pivotal concept in this framework is the classification of tests by origin and risk, which directly determines the rigor of the required revalidation. The Belgian recommendations provide a clear model for this, categorizing tests and linking them to specific verification or validation activities [15].

Table 1: Test Classification and Corresponding Revalidation Activities

Test Type	Description	Recommended Revalidation Activity
CE-IVD	Used strictly according to manufacturer's IFU	Verification
Modified CE-IVD (with reference)	Altered protocol, but a reference (e.g., publication, EQA) exists	Limited Validation
Modified CE-IVD (without reference)	Altered protocol with no available reference	Validation
Non-CE-IVD / LDT (with reference)	RUO antibody used with a published protocol	Simplified Validation
Non-CE-IVD / LDT (in-house)	Developed entirely in-house with no predicate	Full Validation

Triggers for Revalidation: A Risk-Based Approach

A proactive, risk-based approach to revalidation is essential for maintaining assay reproducibility. The triggers for revalidation can be broadly categorized into scheduled and event-driven activities.

Scheduled Revalidation is a proactive measure to ensure ongoing compliance and effectiveness. This is typically performed at regular intervals, such as annually or biennially, as defined by internal quality procedures and regulatory expectations [51] [52]. This periodic check serves as a systematic review to confirm that the assay's performance characteristics have not drifted over time.

Event-Driven Revalidation is necessitated by specific changes in the testing environment. Key triggers include [51]:

• Changes in Reagents: This includes lot-to-lot changes of critical reagents (e.g., primary antibodies), switching to a new antibody clone, or introducing new detection systems or antigen retrieval solutions. Even changes in buffer composition or concentration can impact performance.
• Changes in Protocol: Any alteration to the staining protocol, such as incubation times, temperatures, retrieval methods (e.g., moving from citrate to EDTA retrieval), or washing steps, requires revalidation.
• Changes in Equipment: The introduction of new automated stainers, microscopes, or changes to critical equipment like tissue processors or incubators can affect results and necessitate revalidation.
• Changes in Manufacturing Processes: In a pharmaceutical context, changes in the manufacturing process of a drug product that is monitored by an IHC assay may require revalidation of that assay.
• Performance Issues: Deviations in External Quality Assessment (EQA) performance, internal quality control trends, or unexpected staining results are critical triggers for an immediate revalidation investigation.

The following decision pathway visualizes the structured process for determining the scope and necessity of revalidation based on the change event and test classification.

Quantitative Comparison of IHC Assay Performance

Understanding the performance landscape of different IHC assays is crucial for making informed decisions during revalidation. Data from External Quality Assessment (EQA) programs like Nordic Immunohistochemical Quality Control (NordiQC) and analytical studies provide valuable benchmarks.

A comprehensive survey of over 600 laboratories across more than 60 countries conducted by NordiQC between 2017 and 2021 evaluated 65 different biomarkers and 31,967 results. The data reveals a wide spectrum of performance, heavily influenced by the biomarker's maturity and clinical application [17].

Table 2: IHC Assay Performance Based on NordiQC EQA (2017-2021)

Biomarker Category	Example Biomarkers	Average Pass Rate	Key Challenges & Observations
Predictive (Type 2b)	ER, PR, HER2	89% - 92%	High standardization due to long-standing clinical use and CDx status.
Diagnostic (Type 1)	PAX8, SOX10, Cytokeratins	~75% (Range: 37% - 95%)	Wide performance range attributed to antibody specificity/purpose and validation rigor.
New Diagnostic	7 newly introduced markers	Below 75% average	Inferior pass rates highlight challenges in optimizing/validating novel assays.
PD-L1 (Type 2b)	PD-L1 TPS/CPS (NSCLC)	65% - 79%	Performance varies significantly by specific clinical context and scoring algorithm.

Furthermore, a quantitative comparison of PD-L1 IHC assays using calibrators traceable to the National Institute of Standards and Technology (NIST) Standard Reference Material (SRM) 1934 revealed critical analytical differences. The study demonstrated that different FDA-cleared PD-L1 assays represent distinct levels of analytical sensitivity (lower Limit of Detection - LOD) and possess disparate dynamic ranges. This explains why a tissue sample can be positive by one assay and negative by another, and why some harmonization attempts have failed [53]. When revalidating or changing an assay, understanding these fundamental analytic parameters is vital for ensuring consistent patient stratification.

A Step-by-Step Protocol for Revalidation

A rigorous, documented protocol is essential for a successful revalidation. The following steps provide a template that can be adapted to a laboratory's specific needs and the scope of the change.

Step 1: Develop a Revalidation Plan

Before any laboratory work begins, a detailed plan must be drafted and approved. This plan should define the scope, objectives, and methodology of the revalidation. It must include a rationale for the revalidation, a detailed protocol outlining specific procedures, sampling methods, analytical techniques, and a clear timeline [52]. This plan is the foundation for all subsequent activities.

Step 2: Conduct a Risk Assessment

A formal risk assessment is the cornerstone of a "Fit-For-Purpose" strategy. This process involves identifying potential risks associated with the change, such as alterations to the assay's analytical sensitivity or specificity. The likelihood and impact of each risk are evaluated to appropriately focus the revalidation efforts and resources [15] [52]. Tools like FMEA (Failure Mode and Effects Analysis) can be applied here.

Step 3: Execute Revalidation Studies

The wet-lab work should be conducted according to the approved plan. Key performance characteristics must be evaluated, which typically include [15] [11]:

• Accuracy: The degree of concordance with a gold standard, which can be an EQA sample, a reference staining performed by a validated method, or a non-IHC test.
• Precision: This encompasses both repeatability (intra-run precision) and reproducibility (inter-run, inter-instrument, inter-operator precision).
• Analytical Sensitivity (LoD): Determining the smallest amount of the analyte that can be reliably detected. Using immunohistochemical critical assay performance controls (iCAPCs) is recommended for this purpose [17].
• Analytical Specificity: Confirming the assay detects the intended target and does not exhibit cross-reactivity.
• Robustness: Assessing the assay's ability to remain unaffected by small, deliberate variations in procedural parameters (e.g., incubation time ± 5%, antigen retrieval time ± 10%).

Step 4: Analyze Data and Document Results

All data from the revalidation studies must be compiled and analyzed against pre-defined acceptance criteria. Statistical tools should be employed to confirm that the cleaning procedures meet these criteria [52]. A comprehensive revalidation report must be generated, including the objectives, methodology, raw data, analysis, and a final conclusion on the assay's validated status. This documentation is crucial for demonstrating compliance during audits [51] [52].

Step 5: Review, Approve, and Implement

The final step is the formal review and approval of the revalidation report by the Laboratory Director and Quality Assurance personnel. Once approved, the updated assay protocol and any changes to Standard Operating Procedures (SOPs) can be officially implemented for clinical use [52].

The Scientist's Toolkit: Essential Materials for Revalidation

Table 3: Key Research Reagent Solutions and Materials for IHC Revalidation

Item	Function in Revalidation
iCAPCs (Immunohistochemical Critical Assay Performance Controls)	Tissue controls with defined levels of antigen expression used to evaluate analytical sensitivity (LLOD) and ensure test reproducibility [17].
NIST-Traceable Calibrators	Reference materials with units of measure traceable to a national standards body (e.g., NIST SRM 1934) to quantify assay parameters like LOD and dynamic range [53].
Tissue Microarrays (TMAs)	Slides containing multiple tissue cores from different cases, used as efficient tools for testing assay performance across a wide range of tissues and antigen expression levels during validation [17].
CE-IVD Marked Assays	Commercially available, fully validated assays that serve as predicate devices and reference standards, especially for type 2 biomarkers like PD-L1 and HER2 [17] [15].
External Quality Assessment (EQA) Samples	Commercially provided tissue samples used to objectively compare a laboratory's staining results with a designated "true value" and peer performance [17].

In the context of ISO 15189 research and modern drug development, managing change through robust revalidation is not an optional exercise but a fundamental pillar of quality and reproducibility. A successful strategy integrates a deep understanding of the regulatory landscape, a disciplined risk-based approach to identifying revalidation triggers, and the execution of rigorous, documented experimental protocols. The quantitative data from EQA programs and analytical studies underscore the very real consequences of inadequate validation—inter-assay discordance, diagnostic misclassification, and ultimately, compromised patient safety in the clinical realm. By adopting the structured framework outlined in this guide—from test classification and risk assessment to the systematic evaluation of performance characteristics—researchers and scientists can ensure their IHC assays remain "Fit-For-Purpose," delivering reliable data that stands up to scientific and regulatory scrutiny.

Immunohistochemistry (IHC) validation has evolved from a general quality practice to a highly specialized and regulated process, particularly for applications in pharmaceutical development and clinical trials. The International Standard ISO 15189:2012 provides the foundational framework for quality management in anatomical pathology laboratories, emphasizing the need for standardized verification and validation procedures to ensure reliable patient results [15]. For researchers and drug development professionals, adhering to these standards is not merely about regulatory compliance but is crucial for generating reproducible, trustworthy data that can support drug approvals and clinical trial outcomes.

Recent updates to key guidelines, including the 2024 College of American Pathologists (CAP) update, reflect the growing complexity of IHC applications, introducing specific requirements for predictive biomarkers, cytology specimens, and assays with multiple scoring systems [9]. This guide objectively compares validation methodologies across these specific contexts, providing structured data and experimental protocols aligned with both ISO 15189 standards and the latest regulatory expectations for the pre-market analytical verification and validation of IHC tests [15].

Validation Fundamentals: Key Definitions and a Risk-Based Approach

A clear understanding of terminology is essential for proper validation design. The following definitions are central to the process:

• Verification: Confirmation through objective evidence that the laboratory can fulfill specified requirements established by a manufacturer for a specific intended purpose. It applies to CE-IVD tests used according to instructions for use (IFU) [15].
• Validation: Demonstration through objective evidence that performance characteristics meet predefined criteria for a specific intended use. This is required for laboratory-developed tests (LTs) and modified CE-IVD tests [15].
• Analytical Sensitivity: The smallest amount of substance in a tissue sample that can be accurately detected [15].
• Analytical Specificity: The ability of an IHC assay to detect one particular substance rather than others [15].
• Robustness: The ability of a test to remain unaffected by small, deliberate variations in procedural parameters [15].

A risk-based approach, mandated by the EU In Vitro Diagnostic Regulation (IVDR), governs the extent of validation required. The classification depends on the test's origin and the availability of reference data, leading to different levels of validation rigor [15].

The diagram below illustrates this decision-making process for categorizing tests and determining the appropriate validation pathway.

Comparative Analysis of Validation Requirements by Context

The intended use of an IHC assay dictates its validation strategy. The table below summarizes the core performance characteristics that must be evaluated for different test types and contexts, synthesizing recommendations from international guidelines [15] [9].

Table 1: Performance Characteristics for Different Test Types and Contexts

Test Type / Context	Accuracy	Repeatability & Reproducibility	Analytical Sensitivity	Analytical Specificity	Robustness	Readout Precision
CE-IVD (per IFU)	Required [15]	Required [15]	Not Required [15]	Not Required [15]	Recommended [15]	Context-Dependent [15]
Predictive Marker (e.g., HER2, PD-L1)	Required (90% concordance) [9]	Required [9]	Required [15]	Required [15]	Required [15]	Required [9]
Cytology Specimens	Required (Separate Validation) [9]	Required [9]	Required [15]	Required [15]	Recommended [15]	Context-Dependent [15]
Laboratory-Developed Test (LDT)	Required [15]	Required [15]	Required [15]	Required [15]	Required [15]	Required [15]

Quantitative Validation Targets by Scenario

The number of cases required for a robust validation study varies significantly based on the test's application and regulatory risk. The following table provides specific, quantitative targets based on current guidelines.

Table 2: Sample Size Requirements for Validation Studies

Validation Scenario	Minimum Positive Cases	Minimum Negative Cases	Key Performance Target	Additional Requirements
Predictive Marker (e.g., ER, PR, HER2)	20-40 [9]	20-40 [9]	≥90% Overall Concordance [9]	Validate each scoring system separately [9]
Cytology Specimens (Alternative Fixatives)	10 [9]	10 [9]	Comparable performance to FFPE validation set [9]	Separate validation from FFPE tissues is mandatory [9]
Laboratory-Developed Test (LDT)	20-40 [15] [9]	20-40 [15] [9]	Meet pre-defined criteria for all performance characteristics [15]	Must demonstrate clinical performance [15]

Experimental Protocols for Targeted Contexts

Protocol 1: Validating Predictive Markers with Multiple Scoring Systems

Objective: To independently validate the analytical performance of a predictive IHC marker (e.g., PD-L1) for which different scoring algorithms are used for different tumor indications.

Methodology:

• Assay Design: Classify the test per the risk-based flowchart (Section 2). A modified CE-IVD PD-L1 assay without reference would require a full Validation [15].
• Case Selection: For each tumor type and its associated scoring system (e.g., PD-L1 in Lung NSCLC vs. Gastric Carcinoma), procure a minimum of 20 positive and 20 negative cases [9]. Cases should be representative of the real-world spectrum of expression.
• Comparator Selection: Use a validated non-IHC method (e.g., Flow Cytometry) or testing in another accredited laboratory as a comparator, ordered from most to least stringent [9].
• Staining and Analysis: Run the validation set over different days, using different instruments and personnel to incorporate reproducibility testing [9].
• Data Analysis: Calculate the concordance between the new assay and the comparator. The overall concordance must be ≥90% for all predictive markers, per the updated CAP guidelines [9]. This must be achieved for each unique assay-scoring system combination.

Protocol 2: Validating IHC on Cytology Specimens

Objective: To validate an IHC assay for use on cytology specimens (e.g., cell blocks) that are fixed differently from the standard FFPE tissues used in the initial validation.

Methodology:

• Scope Definition: This is a conditional requirement when specimens are fixed in alternative fixatives (e.g., alcohol-based) common in cytology, which can alter assay sensitivity [9].
• Sample Cohort: Perform a separate validation study using a minimum of 10 positive and 10 negative cytology samples [9]. These should be well-characterized cell blocks.
• Reference Standard: Compare the IHC results from the cytology samples to a gold standard, which could be the same sample type tested with a different validated method, or the expected antigen localization pattern [9].
• Performance Assessment: Evaluate accuracy, sensitivity, and specificity. The staining intensity and quality should be comparable to the established FFPE assay, with no increase in background or non-specific staining.

The Scientist's Toolkit: Essential Reagents and Platforms

A successful validation study relies on high-quality, standardized reagents and platforms. The following table details key solutions for ensuring reproducibility and compliance.

Table 3: Essential Research Reagent Solutions for IHC Validation

Tool Category	Specific Examples	Function in Validation
Automated IHC Stainers	Leica BOND RX, Ventana Discovery Ultra, Roche Benchmark Ultra [54]	Standardize analytical variables; provide consistent tissue conditioning, antibody incubation, and detection; essential for GLP-compliant work [54] [55].
Validated Antibodies	Carrier-free recombinant antibodies (e.g., for mIHC), FDA-approved/cleared IVD kits [56]	Ensure specificity and sensitivity. Carrier-free antibodies are critical for reproducible metal conjugation in multiplex IHC (mIHC) and mass spectrometry [56].
Third-Party Quality Controls	Standardized cell line FFPE blocks (e.g., for CLDN18.2, HER2) with defined expression levels (++, +, Negative) [57]	Monitor daily staining performance, validate new antibody lots, and facilitate inter-laboratory comparison. Provides an objective "answer key" for assay performance [57].
Multiplex IHC Solutions	Validated antibody panels (e.g., PD-L1/PD1 multiplex panel), Opal/TSA detection systems [56]	Enable simultaneous detection of multiple biomarkers on a single slide, crucial for complex analyses like tumor microenvironment characterization [56].

Visualizing the Validation Workflow for Complex Assays

The path to robust validation of complex assays, such as multiplex IHC for predictive biomarkers, involves a multi-stage process that ensures analytical reliability and clinical utility. This is especially critical in clinical trials where pathology data is used for patient stratification.

The diagram below outlines the key phases from initial analytical validation through to quality assurance for trial deployment.

Validation of IHC assays for predictive markers, cytology specimens, and multiple scoring systems requires a meticulous, context-driven approach grounded in international standards like ISO 15189:2012. The key trends shaping the future of IHC validation include the widespread adoption of multiplex IHC (mIHC) for complex biomarker panels [56], the integration of digital pathology and image analysis for more objective scoring [55], and the increasing availability of standardized third-party quality control materials to ensure inter-laboratory reproducibility [57].

For researchers and drug developers, adhering to the updated CAP guidelines and a rigorous risk-based model is not just a regulatory hurdle. It is a scientific imperative that underpins the integrity of clinical trial data, ensures patient safety, and ultimately accelerates the development of new therapies by providing a reliable and standardized evidence base.

The validation of immunohistochemistry (IHC) assays operates within an increasingly complex framework of regulatory standards and quality requirements. The recent publication of ISO 15189:2022, which must be fully implemented by December 2025, signifies a substantial shift in laboratory accreditation standards, with a pronounced emphasis on risk management and patient-centered care [14] [4]. This updated standard integrates requirements for point-of-care testing (POCT) and aligns more closely with ISO/IEC 17025:2017, but its most significant change is the heightened focus on establishing robust risk management processes throughout all laboratory operations [4] [18].

For researchers, scientists, and drug development professionals, this evolution presents both challenges and opportunities. A risk-based approach to IHC assay validation no longer represents a best practice but an expectation embedded within international standards. This methodology allows laboratories to direct resources toward the most critical aspects of their validation processes, particularly for predictive marker assays that directly influence patient treatment decisions [11] [7]. Evidence from comparative studies demonstrates that the dissemination and adoption of evidence-based guidelines have already produced measurable improvements in laboratory validation practices, with compliance for predictive marker assays rising from 74.9% to 99% between 2010 and 2015 surveys [11].

This guide objectively compares traditional versus risk-based validation approaches, providing experimental data and methodologies to help laboratories navigate this transition efficiently. By strategically applying risk management principles, organizations can streamline validation workflows, focus resources on critical parameters, and maintain compliance within the evolving regulatory ecosystem governed by ISO 15189:2022, CLSI guidelines, and regional regulatory frameworks [7] [4].

Comparative Analysis: Traditional vs. Risk-Based Validation Approaches

Fundamental Philosophical Differences

The transition from traditional to risk-based validation represents a paradigm shift in quality assurance for IHC assays. Traditional approaches often employed standardized, one-size-fits-all validation protocols with fixed parameters such as predetermined sample sizes and uniform acceptance criteria across all assay types. This method frequently allocated equal resources and scrutiny to both high-risk predictive assays and lower-risk assays, potentially leading to inefficiencies [11] [58].

In contrast, the risk-based validation framework advocates for a resource allocation strategy proportional to the assay's clinical impact and complexity [59] [7]. This approach, aligned with Quality by Design (QbD) principles, builds quality into the validation process from its inception rather than merely verifying it afterward [59]. It requires thorough identification of Critical Quality Attributes (CQAs) and Critical Process Parameters (CPPs) through systematic risk assessment tools, directing validation efforts toward factors with the greatest potential impact on clinical outcomes [59] [58].

Documented Impact on Laboratory Performance

Comparative data from large-scale laboratory surveys reveals the tangible benefits of adopting risk-based validation methodologies. The evidence demonstrates significant improvement in validation practices following the implementation of evidence-based guidelines that incorporated risk-based principles.

Table 1: Impact of Risk-Based Guideline Implementation on IHC Validation Practices

Validation Aspect	2010 Baseline (Pre-Guideline)	2015 Follow-up (Post-Guideline)	Statistical Significance
Written validation procedures for predictive markers	45.9% (299/651 labs)	73.8% (795/1077 labs)	P < 0.001 [11]
Written validation procedures for non-predictive markers	68.3% (496/726 labs)	80.4% (866/1077 labs)	P < 0.001 [11]
Validated most recent predictive marker assay	74.9% (326/435 labs)	99.0% (101/102 labs)	P < 0.001 [11]
Awareness and adoption of evidence-based guidelines	Not Applicable	79.9% (550/688 aware labs) adopted some/all recommendations	Not Provided [11]

The most striking improvement is observed in the validation of predictive marker assays, where compliance increased to nearly universal adoption (99%) following guideline implementation [11]. This demonstrates how risk-based approaches specifically target areas with the greatest potential impact on patient care. Laboratories reported that the implementation of these guidelines helped address previous inconsistencies in validation practices and created a more standardized, evidence-based approach to IHC assay validation [11].

Resource Allocation and Efficiency Outcomes

The risk-based approach demonstrates particular advantages in optimizing resource utilization during validation processes. Traditional methods often struggled with rare antigens, where finding sufficient validation cases presented significant challenges [11]. The risk-based framework provides methodologies for managing these constraints through alternative validation strategies rather than rigid case number requirements.

Implementation of the process lifecycle validation model, which incorporates risk management at each stage, has shown effectiveness in reducing non-value-added activities while maintaining rigorous quality standards [59] [58]. This approach applies risk assessment to determine the appropriate depth of validation required based on factors such as assay complexity, clinical context, and technological familiarity [59]. The integration of Six Sigma methodologies, particularly DMAIC (Define, Measure, Analyze, Improve, Control), further enhances this approach by providing statistical rigor and structured problem-solving techniques [58].

Experimental Protocols and Methodologies

Risk Assessment Protocol for IHC Assay Validation

A robust risk assessment protocol forms the foundation for streamlining validation activities. The following methodology provides a systematic approach for identifying and prioritizing factors that influence IHC assay performance:

• Define Intended Use and Critical Quality Attributes (CQAs): Precisely document the assay's clinical purpose, including whether it will be used for diagnosis, prognosis, predictive therapy selection, or research. Define CQAs such as staining intensity, specificity, sensitivity, and reproducibility that directly impact the assay's ability to fulfill its intended use [59] [7]. This definition directly influences the regulatory pathway and validation requirements [7].

• Identify Potential Failure Modes: Conduct a systematic analysis of potential failures across the entire testing process using Failure Mode and Effects Analysis (FMEA). Evaluate each step from sample preparation through result interpretation, assessing potential causes of failure and their effects on CQAs [59] [58].

• Assess Risk Priority: Evaluate each identified failure mode based on three criteria: severity (impact on patient outcome), occurrence (probability of failure), and detection (likelihood of detecting the failure before result reporting). Calculate Risk Priority Numbers (RPN) to objectively prioritize risks [59].

• Define Control Strategies: Develop specific control measures for high-priority risks, which may include additional validation experiments, enhanced quality control measures, personnel training requirements, or specific equipment qualifications [59] [58].

• Document and Review: Maintain comprehensive documentation of the risk assessment process and outcomes. Establish a schedule for periodic review, particularly when introducing new equipment, reagents, or when process changes occur [59] [18].

Diagram 1: Risk assessment workflow for IHC validation

Risk-Based Validation Experimental Design

Building upon the risk assessment, the following experimental protocol enables targeted validation activities focused on high-risk areas:

• Determine Sample Size Using Risk-Based Approach: For assay validation, establish minimum case numbers based on assay risk classification rather than fixed requirements. Higher-risk assays (e.g., predictive markers) require larger sample sizes with appropriate positive and negative controls. For rare antigens, employ statistical methods to determine minimum clinically relevant sample sizes, potentially using alternative validation approaches such as cell line models or computational prediction when patient samples are truly limited [11] [7].

• Define Risk-Based Acceptance Criteria: Establish acceptance criteria weighted according to risk priority. High-risk failure modes should have stricter acceptance criteria with appropriate safety margins. For example, assays where false positives could lead to inappropriate therapy administration require higher specificity thresholds [59] [58].

• Conduct Robustness Testing for High-Risk Parameters: Design experiments to challenge critical process parameters identified as high-risk in the assessment. This includes evaluating reagent stability, operator-to-operator variability, equipment performance boundaries, and environmental condition effects [59] [58].

• Implement Statistical Controls: Apply appropriate statistical methods based on risk classification, including confidence intervals, power analysis for sample size determination, and statistical process control for monitoring assay performance over time [58] [5].

• Design Risk-Based Verification Studies: For established assays being reintroduced or modified, design verification studies focused specifically on the aspects most likely to be affected by the change, rather than repeating full validation [59] [58].

Visualization of Risk-Based Validation Workflows

Process Lifecycle Validation Integration

The process lifecycle validation model, as outlined in FDA guidance and international standards, provides a structured framework for implementing risk management throughout a product's lifespan. This approach organizes validation activities into three interconnected stages with risk assessment at each phase.

Diagram 2: Risk-based process lifecycle validation

This continuous lifecycle approach ensures that risk management is not a one-time activity but an ongoing process that adapts to new information and changing conditions [59] [58]. The feedback loop from Stage 3 to Stage 1 enables continuous improvement based on real-world performance data, allowing laboratories to refine their processes and control strategies over time [59].

Integrated Risk Management System

A comprehensive risk management system for IHC validation integrates multiple components across the testing continuum. The following workflow illustrates how risk assessment connects different quality management elements within a medical laboratory.

Diagram 3: Integrated risk management system for IHC validation

This integrated system demonstrates how risk management connects various quality management components, with the CAPA (Corrective and Preventive Action) system serving as the feedback mechanism that closes the loop between quality monitoring and risk assessment [4] [18] [5]. This alignment with ISO 15189:2022 requirements ensures that risk management is not an isolated activity but permeates all laboratory operations [4] [18].

The Scientist's Toolkit: Essential Research Reagent Solutions

Implementing risk-based IHC validation requires specific reagents and materials designed to address identified risks and ensure assay robustness. The following table details essential solutions with their specific functions in risk-based validation.

Table 2: Essential Research Reagent Solutions for Risk-Based IHC Validation

Reagent Solution	Primary Function in Risk-Based Validation	Risk Mitigation Category
Third-Party Control Materials	Detects lot-to-lot reagent/calibrator variation; provides unbiased performance assessment independent of manufacturer controls [5].	Analytical Risk Control
Multiplex Validation Panels	Enables simultaneous validation of multiple biomarkers using scarce clinical specimens; optimizes resource utilization for rare antigens [11].	Resource Optimization
Reference Standard Materials	Establishes metrological traceability; enables bias estimation and correction in measurement uncertainty calculations [5].	Metrological Traceability
Stability-Tested Reagents	Reduces variation from reagent degradation; supports robustness testing under edge-of-failure conditions [59].	Process Robustness
Cell Line Models	Provides renewable reference material for rare antigens; enables validation when patient samples are limited [11].	Rare Antigen Validation
Calibration Verification Panels	Monitors measurement system stability; detects drift before it impacts clinical results [58] [5].	Continuous Monitoring

These specialized reagents directly address common challenges identified through risk assessment, particularly the difficulties associated with rare antigens and reagent variability cited as significant implementation challenges in laboratory surveys [11]. By strategically deploying these tools, laboratories can target their validation efforts more effectively while maintaining rigorous quality standards.

Regulatory Framework Navigation

ISO 15189:2022 Risk Management Requirements

The updated ISO 15189:2022 standard significantly enhances requirements for risk management throughout medical laboratory operations. Key elements most relevant to IHC assay validation include:

• Risk-Based Decision Making: Laboratories must now plan and implement actions to address risks and opportunities, with the overarching objective of promoting patient welfare and ensuring confidence in laboratory quality and competence [4] [18]. This represents a shift from merely detecting nonconformities to proactively preventing them.

• Process Approach: Requirements are designed to ensure that risk to patients becomes central to the laboratory's quality management design and processes [4]. This includes applying risk assessment to all phases of testing - pre-examination, examination, and post-examination processes [18].

• Resource Allocation: The standard implicitly requires that resources be allocated according to risk priority, with greater attention to processes and assays with higher potential impact on patient outcomes [4] [18].

• Point-of-Care Testing Integration: The incorporation of POCT requirements (previously in ISO 22870) introduces additional risk considerations related to testing environments outside the traditional laboratory setting [14] [18].

Implementation guidance suggests a structured approach including gap analysis, development of transition plans with clear responsibilities and schedules, staff training, and monitoring of implemented changes [4]. Laboratories successful in implementation report focusing on the healthcare quality goals of being safe, effective, patient-centered, timely, efficient, and equitable throughout their risk management activities [4].

Comparative Regulatory Pathways

Risk classification directly influences the regulatory pathway for IHC assays, particularly those intended as in vitro diagnostics (IVDs) or companion diagnostics (CDx). The risk-based approach varies significantly between regulatory jurisdictions.

Table 3: Risk-Based Regulatory Pathways for IHC Assays

Regulatory Aspect	United States Framework	European Union Framework
Risk Classification	Class II or Class III based on intended use and risk determination [7]	Uniformly Class C under IVDR for companion diagnostics [7]
Risk Determination	Study Risk Determination (SRD) assesses if assay use represents significant risk; FDA is ultimate arbiter [7]	Based on medical purpose and assay use; classification rules in Annex VIII of IVDR [7] [60]
Pre-Submission Process	SRD Q-submission to FDA or assessment by Institutional Review Board (IRB) [7]	Annex XIV submission to national competent authority in each country where samples are collected [7]
Quality Management	21 CFR Part 820 (transitioning to integration with ISO 13485 by 2026) [7]	ISO 13485 quality management system required [7]
Clinical Evidence	Modular PMA process preferred for CDx; requires BIMO audit [7]	Technical dossier with clinical data; consultation with competent authority/EMA [7]

The adoption of a risk-based approach for IHC assay validation must account for these regulatory differences, particularly for laboratories operating in multiple jurisdictions or developing assays for global markets. A successful global strategy involves designing validation studies that simultaneously meet CLIA requirements while supporting EU regulatory submissions under ISO 13485 and good clinical laboratory practice (GCLP) guidelines [7].

The integration of risk management principles into IHC assay validation represents both a regulatory imperative and an opportunity for substantial efficiency gains. The implementation of ISO 15189:2022 with its enhanced focus on risk management, coupled with evidence from laboratory surveys showing significantly improved validation practices following guideline adoption, demonstrates the tangible benefits of this approach [11] [4].

Successful implementation requires a systematic methodology beginning with comprehensive risk assessment, followed by targeted validation activities focused on high-priority risks, and culminating in ongoing monitoring and continuous improvement [59] [58]. The experimental protocols and workflows presented provide a framework for laboratories to develop their own risk-based validation approaches tailored to their specific assay portfolio and clinical contexts.

As regulatory frameworks continue to evolve globally, with the EU IVDR and updated FDA requirements, the ability to effectively apply risk management principles will become increasingly critical for maintaining compliance while optimizing resource utilization [7] [60]. Laboratories that successfully implement these approaches position themselves not only to meet current accreditation standards but also to adapt more efficiently to future regulatory changes and technological advancements in IHC and digital pathology.

Within the framework of ISO 15189, which specifies requirements for quality and competence in medical laboratories, ongoing verification is not a single activity but a continuous process integral to the quality management system (QMS) [18]. For immunohistochemical (IHC) assays, which play a critical role in diagnostic, prognostic, and predictive pathology, this process rests on two main pillars: Internal Quality Control (IQC) and External Quality Assessment (EQA), also known as Proficiency Testing (PT) [18] [24]. ISO 15189 mandates that laboratories must implement both IQC and EQA schemes to monitor performance and result accuracy continually [18].

The goal of ongoing verification is to ensure that every IHC test result remains reliable, reproducible, and clinically meaningful over time. IQC provides daily, internal monitoring of assay performance, while EQA offers an external, unbiased assessment of a laboratory's competence against peer groups or reference standards [61] [62]. Together, they form a robust system for detecting drift, identifying errors, and implementing corrective actions, thereby directly supporting patient safety and the credibility of laboratory results [18] [24]. This guide objectively compares the components, methodologies, and outcomes of these two essential practices within the context of IHC assay validation and ISO 15189 compliance.

Comparative Analysis: IQC vs. EQA

While Internal Quality Control and External Quality Assessment are complementary, they serve distinct purposes, operate on different frequencies, and provide unique insights into laboratory performance. The following table summarizes their core characteristics for easy comparison.

Table 1: A Comparative Overview of Internal Quality Control (IQC) and External Quality Assessment (EQA) in IHC

Feature	Internal Quality Control (IQC)	External Quality Assessment (EQA)
Primary Objective	Daily monitoring of assay precision and reproducibility [24]	Inter-laboratory comparison to assess accuracy and competence [61] [62]
Scope of Evaluation	Intra-laboratory (within-laboratory) performance	Inter-laboratory (between-laboratory) performance
Focus	Analytical process control (staining, equipment) [24]	Overall testing process (analytical, pre-/post-examination) [61]
Frequency	With each batch of tests or according to a defined schedule [18]	Periodic (e.g., quarterly, biannually) [61]
Key Performance Metrics	Repeatability, expected staining patterns, control results [15]	Trueness, concordance with target value, peer group performance [61]
Corrective Action	Immediate (e.g., repeat assay, troubleshoot reagents)	Investigative and systemic (e.g., process review, retraining)
ISO 15189 Reference	Clause 7: Process Requirements (Examination processes) [18]	Clause 7: Process Requirements (Quality assurance) [18]

Performance Data from Implementation Studies

The implementation of standardized guidelines for IHC assay validation, which include ongoing verification protocols, has demonstrated a measurable positive impact on laboratory performance. A major survey conducted by the College of American Pathologists (CAP) compared practices in 2010, before a key guideline was published, with those in 2015, after its dissemination [11].

Table 2: Impact of Guideline Implementation on IHC Validation Practices (2010 vs. 2015 CAP Survey Data) [11]

Validation Practice	2010 Survey (%)	2015 Survey (%)	P Value
Had written validation procedure for predictive markers	45.9	73.8	< .001
Had validated their most recently introduced predictive marker assay	74.9	99.0	< .001
Participated in EQA/Proficiency Testing for non-predictive markers	85.8	94.7	< .001

The data shows significant improvement across all measured metrics, underscoring how formalizing procedures for validation and ongoing verification leads to higher compliance and, presumably, better quality outcomes. The near-universal validation of predictive markers (99%) in 2015 highlights a major step forward in quality assurance for critical assays [11].

Experimental Protocols for Ongoing Verification

Protocol for Internal Quality Control in IHC

A robust IQC protocol for IHC assays involves multiple control levels and continuous monitoring.

1. Sample and Control Selection:

• Tissue Controls: Include known positive and negative tissue controls in every staining batch. For quantitative assays, tissues with low-positive expression levels are critical for monitoring analytical sensitivity [15] [63].
• Multi-tissue Blocks: Control tissues can be assembled into multi-tissue blocks to conserve material and simultaneously assess multiple antigens.

2. Staining and Interpretation:

• Run Controls: Process control tissues identically to patient samples throughout the pre-examination, examination, and post-examination phases.
• Assessment: Evaluate control tissues for expected staining intensity, localization, and absence of background. The results must meet pre-defined acceptance criteria before patient results are released.

3. Data Recording and Monitoring:

• Logging: Document all IQC results for each batch.
• Trend Analysis: Use statistical process control (e.g., Levey-Jennings charts) to monitor for trends, shifts, or increased variation in staining intensity over time, which can indicate reagent degradation or equipment calibration issues.

Protocol for External Quality Assessment (Proficiency Testing)

EQA is a formal process for inter-laboratory comparison, mandated by ISO 15189 for accreditation [61] [18].

1. Enrollment and Sample Reception:

• Program Selection: Enroll in a recognized EQA scheme relevant to the IHC assays performed (e.g., NordiQC, UK NEQAS) [62].
• Sample Handling: Upon receipt, EQA samples must be treated as patient samples and processed according to the laboratory's standard operating procedures. ISO/IEC 17043 requires EQA providers to ensure sample integrity during transportation, which may involve monitoring temperature indicators [61].

2. Testing and Reporting:

• Blinded Testing: Analyze the EQA samples without prior knowledge of the expected result.
• Result Submission: Report results (including staining intensity, distribution, and interpretation) to the EQA provider within the specified deadline.

3. Performance Assessment and Corrective Action:

• Evaluation: The EQA provider compares the laboratory's results to the target value (assigned by a reference method or as a consensus of participant peers) and issues a performance report [61].
• Remedial Measures: If performance is unsatisfactory, the laboratory must conduct a root cause analysis, implement corrective actions (e.g., staff retraining, protocol modification, reagent re-validation), and document all steps taken [61] [62]. This process of continuous improvement is a core requirement of ISO 15189 [18].

Logical Workflow for Ongoing Verification

The following diagram illustrates the logical relationship and workflow between Internal Quality Control and External Quality Assessment within a continuous quality improvement cycle, as guided by ISO 15189 principles.

The Scientist's Toolkit: Key Reagents and Materials

Successful ongoing verification relies on a set of essential reagents and materials. The following table details key solutions required for implementing IQC and EQA in an IHC laboratory.

Table 3: Essential Research Reagent Solutions for IHC Ongoing Verification

Reagent/Material	Function in Ongoing Verification
Characterized Tissue Control Slides	Serve as the primary material for both IQC and EQA. They provide a biological substrate with a known antigen expression profile to verify staining accuracy and reproducibility in every run [15].
Multi-tissue Control Blocks	Maximize efficiency by incorporating multiple control tissues into a single block, allowing simultaneous verification of several assays and conserving valuable tissue resources.
Reference Standard Materials	For quantitative IHC, materials with units traceable to a reference standard (e.g., NIST SRM 1934) enable calibration and objective assessment of analytical sensitivity (LOD) and dynamic range, moving beyond subjective comparison [63].
Stable Control Sera/Reagents	Used to monitor the performance of individual reagents (e.g., primary antibodies, detection systems) over time, helping to identify the specific source of variation or failure in an assay.
EQA/PT Program Samples	Commercially provided or exchanged samples that are central to external assessment. Their commutability with clinical patient samples is a critical factor for a meaningful evaluation [61].

Global Compliance: Comparing ISO 15189 with CLIA, CLSI, and IVDR for IHC

For researchers, scientists, and drug development professionals, particularly those working on immunohistochemistry (IHC) assay validation, navigating the landscape of laboratory standards is crucial. The choice of a quality framework is not merely procedural; it fundamentally shapes assay development, validation protocols, and the global acceptability of data. Two systems dominate this landscape: the International Organization for Standardization's ISO 15189 and the United States' Clinical Laboratory Improvement Amendments (CLIA). While CLIA provides the federal regulatory baseline for clinical testing in the U.S., ISO 15189 represents an international accreditation standard that emphasizes a laboratory's overall quality management system and technical competence [64] [65]. Understanding the distinction between a mandatory regulation (CLIA) and a voluntary accreditation (ISO 15189) is the first step in selecting the appropriate pathway for ensuring the reliability and reproducibility of laboratory data, especially for complex assays like IHC.

This guide objectively compares these two frameworks, focusing on their application within a research and development context, to inform strategic decision-making for assay validation and commercialization.

At a Glance: Core Comparison of CLIA and ISO 15189

The following table summarizes the fundamental characteristics of CLIA and ISO 15189, highlighting their primary distinctions in focus, scope, and application.

Table 1: Core Characteristics of CLIA and ISO 15189

Feature	CLIA (Clinical Laboratory Improvement Amendments)	ISO 15189 (Medical Laboratories)
Nature	U.S. Federal regulation (42 CFR 493) [64].	International accreditation standard [65].
Primary Focus	Analytical test performance and error reduction; a baseline for operation [7].	Overall quality management system and technical competence of the entire laboratory [65] [4].
Geographic Scope	Primarily the United States.	International; recognized globally.
Enforcement	Mandatory for U.S. labs testing human specimens for health assessment.	Voluntary, though often required for market access or as a mark of excellence.
Key Emphasis	Compliance with specific regulatory requirements for testing.	Risk management, patient-centered outcomes, and continuous improvement [14] [4].
IHC Assay Validation	Applies broadly but does not define specific study designs; often supplemented with other guidance [7].	Requires laboratories to demonstrate method validity (Section 5.5.2) [66].

---

Detailed Analysis: Key Differences and Their Implications

Scope, Philosophy, and Application in IHC Assay Validation

The divergence between CLIA and ISO 15189 extends beyond their legal status to their core philosophy.

• CLIA establishes a minimum federal standard for all clinical laboratory testing in the U.S. Its requirements are designed to ensure the accuracy, reliability, and timeliness of patient test results, focusing heavily on analytical performance [7]. For IHC assay validation, CLIA provides the umbrella regulation but does not prescribe detailed methodologies. Consequently, laboratories often turn to additional guidance, such as those from the Clinical Laboratory Standards Institute (CLSI) or evidence-based guidelines from the College of American Pathologists (CAP), to design their validation studies [7] [11].

• ISO 15189 embodies a comprehensive quality management system. It integrates the principles of ISO 17025 (for testing laboratories) and adds specific requirements for the medical laboratory environment [4]. A key update in the ISO 15189:2022 version is the enhanced focus on risk management, making it a central ethos of the laboratory's operations [14] [4]. This means laboratories must proactively identify and mitigate risks to patients throughout the entire testing process, from sample receipt to reporting. The standard also incorporates requirements for point-of-care testing (POCT), previously covered by ISO 22870 [14] [4].

Validation Protocols and Performance Assessment

For researchers validating IHC assays, the approach to demonstrating analytical validity differs under each framework.

Under CLIA, the laboratory has flexibility but must ensure the assay is "fit-for-purpose." A survey of IHC validation practices showed that dissemination of a CAP evidence-based guideline significantly improved laboratory compliance, with 99% of respondents validating their most recently introduced predictive marker assay in 2015, compared to 74.9% in 2010 [11]. This indicates that while CLIA sets the requirement, professional guidelines provide the de facto methodology.

Under ISO 15189, the laboratory is required to validate or verify each examination procedure to ensure it is fit for its intended use [66]. This involves designing a validation protocol that evaluates specific performance characteristics. An example protocol for an in-house method, as illustrated in a study on lead testing, can be adapted for IHC assays and typically includes the parameters in the table below [66].

Table 2: Example Validation Protocol Parameters for an In-House Method

Parameter	Purpose	Example from Analytical Chemistry [66]
Selectivity/Specificity	Ensure the method specifically detects the target analyte.	Verified by analyzing blank samples to confirm no interference.
Calibration Model	Establish the relationship between analyte concentration and instrument response.	Five calibration standards run in duplicate; linearity confirmed via statistical tests (Fisher's test).
Precision	Measure the random variation (repeatability and reproducibility).	Intra-assay (30 measurements in one run) and inter-assay (20 days) imprecision calculated (CV <10%).
Accuracy	Determine the closeness of results to the true value.	Analyte recovery assessed (within 80-120%).
Measurement Uncertainty	Estimate the doubt associated with a measurement result.	Combined standard uncertainty from precision and calibration bias, multiplied by a coverage factor.
Stability	Assess analyte stability under routine storage conditions.	Samples analyzed over 14 days; results within ±10% of nominal value were acceptable.
Contamination	Evaluate potential carryover or interference.	Protocol involving high and low controls; contamination <1% was acceptable.

Regulatory Pathways and Commercialization

The regulatory strategy for commercializing an assay, such as a companion diagnostic (CDx), is deeply intertwined with these laboratory standards.

• In the U.S., CLIA certification is necessary for the clinical laboratory performing the test. However, if the IHC assay is intended for use as an In Vitro Diagnostic (IVD) or CDx, it typically requires separate approval or clearance from the Food and Drug Administration (FDA) [7]. The FDA pathway (e.g., Premarket Approval or PMA) involves studies that exceed CLIA requirements and must comply with quality system regulations like 21 CFR Part 820 [7]. The FDA favors a modular PMA process for CDx commercialization, with a review timeline of approximately 12-24 months [7].

• In the European Union, the In Vitro Diagnostic Regulation (IVDR) applies. Under IVDR, CDxes are uniformly classified as Class C devices [7]. The regulatory authority is a notified body, and the process for CE marking involves a technical dossier, clinical data, and an audit of the quality management system, often based on ISO 13485 [7]. If an assay has a medical purpose in a clinical trial within the EU, it requires submissions to national competent authorities, adding complexity [7].

For global commercialization, manufacturers must plan parallel validation strategies, designing studies that meet both CLIA and international standards like ISO 15189 from the outset to avoid duplicated efforts [7].

The Scientist's Toolkit: Essential Reagents and Materials

The following table lists key reagents and materials commonly used in IHC assay development and validation, detailing their critical functions in the process.

Table 3: Key Research Reagent Solutions for IHC Assay Validation

Item	Function in IHC Assay Validation
Primary Antibodies	The core reagent that specifically binds to the target antigen (biomarker). Validation confirms its specificity, sensitivity, and optimal dilution.
Antigen Retrieval Solutions	To reverse formaldehyde-induced cross-linking and expose epitopes, which is critical for consistent and robust staining.
Detection Kits (e.g., HRP-based)	To visualize the antibody-antigen binding. Validation ensures the system has high sensitivity and low background noise.
Control Cell Lines/Tissues	Essential for accuracy and precision studies. Positive controls express the target antigen, while negative controls do not, establishing assay specificity.
Reference Materials	Well-characterized samples with known antigen expression levels used to standardize results across runs and laboratories, supporting accuracy claims.
Matrix Modifiers	In analytical chemistry, used to minimize interference from the sample matrix [66]. Analogous to blocking sera in IHC to reduce non-specific background staining.

Visualizing the Regulatory Pathway and Standard Integration

The following diagram illustrates the relationship between key standards and a typical regulatory pathway for an IHC assay, from development to commercialization.

Figure 1: Regulatory Pathways for IHC Assays. This workflow shows how CLIA and ISO 15189 fit into laboratory operations and support broader regulatory submissions for in vitro diagnostics (IVDs) in different markets. The path diverges based on the target market, with the FDA overseeing the U.S. and Notified Bodies under the IVDR overseeing the EU.

In the field of diagnostic immunohistochemistry (IHC), the path from establishing high-level quality standards to implementing consistent, reliable laboratory practices presents a significant challenge. Two key frameworks govern this landscape: the International Organization for Standardization's ISO 15189, which specifies requirements for quality and competence in medical laboratories, and guidelines from the Clinical and Laboratory Standards Institute (CLSI), which provide actionable implementation pathways [6] [26]. While ISO 15189 outlines what laboratories must achieve, CLSI documents, such as ILA28 - Quality Assurance for Design Control and Implementation of Immunohistochemistry Assays, provide the critical how-to guidance for meeting these requirements [67]. This guide objectively compares these complementary frameworks within the context of IHC assay validation, examining their respective roles, implementation tools, and practical impact on laboratory operations for researchers and drug development professionals.

The necessity for such practical implementation tools is underscored by studies revealing significant interlaboratory variation in IHC validation practices. Evidence shows that dissemination of structured, evidence-based guidelines has a markedly positive impact on laboratory performance, with nearly 80% of laboratories adopting recommended practices following guideline publication, a substantial increase from pre-guideline compliance rates [11]. This demonstrates the critical importance of effective implementation tools in translating theoretical standards into reliable diagnostic outcomes.

Comparative Analysis of Standards Frameworks

Defining the Roles: ISO 15189 versus CLSI Guidelines

ISO 15189 represents a comprehensive quality management system specifying requirements for quality and competence in medical laboratories. As a standards framework, it establishes what laboratories must achieve but remains largely non-prescriptive about how to meet these requirements [26]. Its structure encompasses both management requirements (quality system, document control, management review) and technical requirements (personnel competence, testing processes, result reporting) [26]. The standard encourages laboratories to create systems that are "failure resistant" and focus on continual improvement, but provides limited practical guidance on specific implementation methodologies for specialized techniques like IHC [26].

In contrast, CLSI guidelines fill this implementation gap by providing detailed, evidence-based procedural guidance. The CLSI ILA28 document specifically addresses the "total test system" for IHC assays, covering design control, pre-examination, examination, and post-examination processes [67]. Rather than competing with ISO 15189, CLSI guidelines function synergistically with it, offering the technical specificity needed to comply with the broader quality framework. The FDA has formally recognized CLSI guidelines as approved consensus standards for satisfying regulatory requirements, further validating their role in the implementation ecosystem [67].

Comparative Framework Analysis

Table 1: Comparison of ISO 15189 and CLSI Implementation Characteristics

Characteristic	ISO 15189	CLSI Guidelines
Primary Focus	Quality Management System (QMS) requirements	Technical implementation and analytical validation
Implementation Specificity	Non-prescriptive framework	Highly specific procedural guidance
Technical Detail Level	Generic laboratory requirements	IHC-specific protocols and methodologies
Documentation Approach	Process-oriented documentation	Step-by-step validation protocols
Validation Guidance	General requirements for test validation	Specific case numbers, acceptance criteria
Regulatory Recognition	International accreditation standard	FDA-recognized for regulatory compliance

CLSI Implementation Toolkit for IHC Validation

CLSI's value as an implementation tool lies in its comprehensive suite of practical resources designed to translate standards into laboratory practice. These include:

• Education and Training Programs: CLSI provides structured eLearning programs, webinars, and hands-on laboratory training with continuing education credits to build foundational skill sets [68].
• Customized Laboratory Assessments: Partnership programs offer customized implementation support through in-person, online, and hybrid team training specifically tailored to laboratory needs [68].
• Implementation Guides and Tools: Step-by-step instructions, interactive worksheets, forms, and database tools facilitate practical application of standards [68].
• Method Navigator: This specialized resource enables users to easily reference and access resources for establishing and implementing laboratory-developed tests (LDTs) or other regulatory-approved commercial test methods [69].

For IHC specifically, the CLSI ILA28 document provides comprehensive guidance on the "total product life cycle" of IHC reagents, kits, and systems, emphasizing that accurate and reliable results require attention to the entire test system [67]. This aligns directly with the ISO 15189 process approach while providing the technical specificity needed for implementation.

Experimental Validation Protocols and Methodologies

CLSI guidelines provide detailed methodological frameworks for IHC assay validation that operationalize the quality requirements of ISO 15189. The following experimental protocols represent standard approaches derived from CLSI-informed practices:

Protocol 1: Assay Validation for Predictive Markers

• Purpose: Validate IHC assays used for predicting response to therapeutics (e.g., HER2, PD-L1, hormone receptors)
• Sample Requirements: Minimum of 20 positive and 20 negative cases using tissues fixed and processed identically to clinical specimens [70]
• Comparator Standards: Use validated non-IHC methods (FISH, flow cytometry), testing at reference laboratories, or clinical trial data as gold standards [9]
• Acceptance Criteria: ≥90% overall concordance with comparator method; investigation required for any discordances [9]
• Statistical Analysis: Calculate positive percent agreement, negative percent agreement, and overall concordance with 95% confidence intervals

Protocol 2: Verification of FDA-Cleared/Approved Assays

• Purpose: Verify performance of commercially manufactured IHC tests used according to manufacturer instructions
• Sample Requirements: Follow manufacturer specifications; if not provided, use validation requirements for modified assays [70]
• Performance Characteristics: Confirm accuracy, repeatability, reproducibility, and robustness under local conditions [15]
• Acceptance Criteria: Meet or exceed manufacturer's stated performance specifications
• Documentation: Record lot numbers, expiration dates, and any deviations from intended use

Table 2: Validation Requirements Based on Test Type and Purpose

Test Type	Intended Use	Validation Cases	Performance Characteristics	Concordance Threshold
FDA-Cleared/Approved (Unmodified)	Diagnostic/Prognostic	Manufacturer specifications	Accuracy, Repeatability	Manufacturer claims
Predictive Marker IHC	Treatment selection	20 positive, 20 negative [70]	Accuracy, Analytical Sensitivity, Specificity	≥90% [9]
Non-Predictive IHC	Diagnostic aid	10 positive, 10 negative [70]	Accuracy, Specificity	≥90%
Cytology Specimens	Diagnostic	10 positive, 10 negative [9]	Accuracy, Robustness	≥90%
Rare Antigens	Diagnostic	Collaborate with other labs or use cell lines [70]	Accuracy, Specificity	Documented justification

Implementation Workflow and Signaling Pathways

The following diagram illustrates the relationship between standards frameworks and the practical implementation pathway for IHC validation:

Figure 1: Pathway from Standards to Practice. This diagram illustrates how CLSI implementation tools bridge the critical gap between high-level quality standards and standardized laboratory practice.

The validation workflow for IHC assays follows a structured process that incorporates both ISO 15189 quality principles and CLSI technical guidance:

Figure 2: IHC Assay Validation Workflow. This process flow diagram outlines the key stages in implementing a validated IHC assay according to established standards and guidelines.

The Scientist's Toolkit: Essential Research Reagent Solutions

Successful implementation of IHC validation standards requires specific research reagents and materials. The following table details essential components of the validation toolkit:

Table 3: Essential Research Reagents and Materials for IHC Validation

Reagent/Material	Function in Validation	Implementation Consideration
Commercial Primary Antibodies	Antigen detection with defined specificity	Select clones with published performance data (NordiQC, CAP) [70]
Control Tissue Materials	Validation of assay performance	Include known positive/negative tissues with similar pre-analytical factors [70]
Reference Standard Materials	Accuracy determination	Use validated cell lines, EQA samples, or previously characterized tissues [9]
Detection Systems	Signal amplification and visualization	Match to primary antibody host species and platform requirements
Antigen Retrieval Solutions	Epitope recovery	Optimize for specific antibody-antigen combinations [15]
Tissue Microarrays	Efficient validation testing	Enable multiple validations on single slide; conserve rare tissues [70]

Impact Assessment: Quantitative Data on Implementation Effectiveness

The effectiveness of implementation tools can be measured through comparative studies of laboratory practices before and after guideline adoption. A comprehensive survey conducted by the College of American Pathologists (CAP) demonstrated significant improvements in validation practices following the publication of evidence-based guidelines informed by CLSI principles [11].

Table 4: Impact of Guidelines on Laboratory Validation Practices

Validation Practice Metric	Pre-Guideline (2010)	Post-Guideline (2015)	Change
Laboratories with written validation procedures	68.3%	80.4%	+12.1%
Predictive marker assays validated before use	74.9%	99.0%	+24.1%
Laboratories using minimum case requirements	Significantly fewer	Majority	Substantial increase
Guideline awareness and adoption	Not available	79.9%	Benchmark established

The most significant improvement was observed in predictive marker validation, where the percentage of laboratories properly validating these clinically critical assays before use increased from 74.9% to 99% [11]. This demonstrates the substantial impact that implementation-focused guidelines have on standardizing critical laboratory practices.

For researchers and drug development professionals working with IHC assays, CLSI guidelines serve as an essential implementation tool that translates the high-level requirements of ISO 15189 into actionable laboratory practices. The quantitative data demonstrates that structured implementation guidance significantly improves standardization and reliability of IHC testing [11]. The most effective quality management approach integrates both frameworks: using ISO 15189 as the overarching quality system and CLSI guidelines as the technical implementation roadmap.

As IHC technologies evolve and regulatory landscapes become more complex—particularly with the implementation of the European Union's In Vitro Diagnostic Regulation (IVDR) and evolving FDA requirements—the role of CLSI as an implementation tool grows increasingly important [7] [15]. By providing evidence-based, practical guidance for validation and quality assurance, CLSI resources enable laboratories to navigate these complexities while maintaining compliance with international quality standards and ensuring reliable patient results.

The European Union's In Vitro Diagnostic Regulation (IVDR) (EU) 2017/746 has fundamentally transformed the regulatory landscape for immunohistochemistry (IHC) assays. This comprehensive analysis examines how the IVDR's stringent requirements impact validation workflows for commercial manufacturers and laboratory-developed tests (LDTs). We compare regulatory pathways under the new framework, provide detailed experimental protocols for compliance, and visualize critical decision workflows. Within the context of ISO 15189 standards for laboratory quality, we demonstrate that the IVDR mandates more rigorous analytical and clinical evidence, particularly for Class C companion diagnostics and LDTs, creating both challenges and opportunities for researchers and drug development professionals seeking to maintain seamless product supply while ensuring patient safety.

The In Vitro Diagnostic Regulation (IVDR) (EU) 2017/746 represents a paradigm shift from the previous Directive, establishing a more transparent and sustainable regulatory framework with significantly elevated standards for clinical safety and performance [71]. Effective from 26 May 2022, the IVDR introduces a risk-based classification system with heightened requirements for technical documentation, clinical evidence, and post-market surveillance [72] [73]. For IHC assays, which are crucial for predictive biomarker detection in precision oncology, these changes necessitate substantial modifications to validation approaches and quality management systems.

A cornerstone of the IVDR is its expanded scope to include in-house devices (IHDs), commonly known as laboratory-developed tests (LDTs), under Article 5.5 [71]. This inclusion directly impacts pathology laboratories and research institutions developing internal IHC assays, requiring them to justify the use of LDTs over commercially available alternatives and comply with General Safety and Performance Requirements (GSPRs) outlined in Annex I [73]. The regulation establishes a phased implementation timeline for these requirements, extending through May 2028, providing laboratories with transitional periods to align their validation processes with the new standards [73] [71].

Comparative Analysis of IVDR Validation Pathways

The IVDR establishes distinct validation and conformity assessment routes based on device classification and origin (commercial versus in-house). Understanding these pathways is essential for selecting the appropriate regulatory strategy.

Table 1: IVDR Classification and Requirements for IHC Assays

Device Type	IVDR Risk Class	Conformity Assessment	Key Validation Requirements	Transition Deadline
Companion Diagnostic IHC	Class C	Notified Body	Full technical documentation, clinical performance data, QMS audit	26 May 2026 [74] [71]
Other IHC Assays (e.g., prognostic)	Class B or C	Notified Body	Analytical performance data, clinical evidence, QMS	26 May 2027 (Class B) [74]
IHC Instruments	Class A (typically)	Self-certification (if non-sterile)	Manufacturer declaration of conformity	26 May 2022 (passed) [73]
In-House IHC Assays (LDTs)	N/A (Article 5.5)	Health Institution	Justification, QMS (ISO 15189), performance validation	26 May 2024-2028 [73]

Table 2: Comparison of Commercial IVD vs. LDT Validation Under IVDR

Validation Aspect	Commercial CE-IVD IHC Assay	In-House IHC Assay (LDT)
Clinical Evidence	Required for all classes; level depends on risk	Required, with justification when no equivalent CE-IVD exists
Quality System	ISO 13485:2016 required	ISO 15189:2012 required by 26 May 2024 [73]
Performance Verification	Full analytical and clinical validation per IVDR Annex XIII	Validation following professional guidelines (e.g., CAP) [9]
Documentation	Comprehensive technical documentation assessed by Notified Body	Internal documentation subject to competent authority review
Post-Market Surveillance	Systematic procedure required	Review of experience from clinical use required

For commercial manufacturers, the IVDR mandates a robust quality management system (QMS) aligned with IVDR requirements by 26 May 2025 for all legacy devices [74]. Class D IVDs and those covered by an IVDD CE Certificate must apply to a Notified Body for an IVDR certificate by the same date to benefit from extended transition periods [74]. The first IVDR-certified IHC portfolios are now emerging, exemplified by Leica Biosystems' achievement of IVDR certification for 63 BOND Ready-to-Use antibody products and the Bond Oracle HER2 IHC System, a companion diagnostic [75].

Experimental Protocols for IVDR-Compliant IHC Validation

Analytical Validation for IHC Assays

The College of American Pathologists (CAP) "Principles of Analytic Validation of Immunohistochemical Assays: Guideline Update" provides evidence-based protocols essential for IVDR compliance [9]. The 2024 update harmonizes validation requirements across predictive markers, establishing a 90% concordance threshold for all IHC assays, including ER, PR, and HER2 [9]. The recommended protocol includes:

• Sample Selection and Sizing: For new IHC assays, validate with a minimum of 60 cases (30 positive and 30 negative) established by a reference method. For assays with distinct scoring systems (e.g., PD-L1 with different tumor indications), separately validate each assay-scoring system combination [9].

• Alternative Fixative Validation: For IHC performed on cytology specimens with different fixatives (e.g., CytoRich, PreservCyt), perform separate validations with 10 positive and 10 negative cases to address variable sensitivity compared to FFPE tissues [9].

• Comparator Selection: Employ a hierarchical approach to validation comparators, ranging from cell lines with known protein content to previously validated assays or expected architectural localization of antigen [9].

Indirect Clinical Validation for Predictive Biomarkers

For LDTs targeting predictive biomarkers, indirect clinical validation (ICV) is mandatory when companion diagnostic assays are unavailable or not used [43]. The International Quality Network for Pathology (IQN Path) recommends categorizing biomarkers into ICV Groups with distinct validation approaches:

• ICV Group 1 (e.g., ALK, NTRK fusions): Focus on detecting specific biological events with minimal tumor heterogeneity. Validation requires demonstrating high accuracy in identifying the specific alteration using established reference methods.

• ICV Group 2 (e.g., PD-L1, TMB): Characterized by tumor heterogeneity and clinically validated cut-offs. Validation must demonstrate diagnostic equivalence to the gold standard assay used in clinical trials by stratifying patients identically into "positive" and "negative" categories [43].

• ICV Group 3 (e.g., screening assays): Technical screening assays require comparison to definitive biomarker assays to establish diagnostic accuracy for excluding negative patients [43].

The following workflow diagram illustrates the decision process for implementing indirect clinical validation:

The Scientist's Toolkit: Essential Research Reagent Solutions

Successfully navigating IVDR compliance requires strategic selection and validation of research reagents and platforms. The following table details essential components for establishing IVDR-compliant IHC workflows:

Table 3: Research Reagent Solutions for IVDR-Compliant IHC Validation

Tool/Reagent	Function in Validation	IVDR Compliance Considerations
IVDR-Certified Antibodies	Primary detection reagents for IHC	Select from manufacturers with IVDR transition progress (e.g., Leica BOND Ready-to-Use antibodies) [75]
Automated IHC Stainers	Standardized assay platform	Ensure platform validation and IVDR status (e.g., BOND-PRIME immunostainer) [75]
Reference Standard Materials	Establish assay accuracy	Use well-characterized cell lines or tissue samples with known antigen expression [9]
Control Tissues (Positive/Negative)	Monitor assay performance	Include in each run; validate with appropriate fixatives [9]
Image Analysis Software	Quantitative readout standardization	Validate algorithm performance, especially for cut-off dependent biomarkers [43]

Manufacturers are progressively achieving IVDR certification for their IHC portfolios, with instruments typically classified as Class A and reagents and assays falling into Class B, C, or D depending on their intended use and associated risk [73] [75]. When developing LDTs, laboratories must document why available IVDR-compliant devices do not meet their specific needs, a requirement that becomes mandatory by 26 May 2028 [73].

The IVDR establishes a significantly more demanding regulatory environment for IHC assay validation, with profound implications for researchers, manufacturers, and healthcare institutions. Successful navigation requires understanding the risk-based classification system, implementing rigorous analytical validation protocols aligned with updated professional guidelines, and addressing the specific requirements for indirect clinical validation of LDTs. The phased implementation timeline provides a critical window for laboratories to align their quality management systems with ISO 15189 requirements and establish robust validation protocols. As the IVDR continues to be implemented, maintaining awareness of evolving guidance from professional societies and notified bodies will be essential for ensuring compliance while advancing precision medicine through reliable IHC-based biomarker testing.

In the field of diagnostic and research pathology, immunohistochemistry (IHC) serves as a critical technique for biomarker detection, directly influencing patient diagnosis, treatment selection, and drug development. For scientists and researchers operating in international markets, the absence of a unified regulatory framework presents a significant challenge. Divergent requirements across regions can lead to delayed project timelines, increased costs, and barriers to the global adoption of validated assays. A harmonized validation strategy is, therefore, not merely a regulatory formality but a scientific necessity to ensure that data is reliable, reproducible, and universally accepted. This guide objectively compares the regulatory and validation requirements of major international markets, providing a foundational strategy for navigating this complex landscape. Adopting a harmonized approach from the outset streamlines the path to commercialization and ensures that high-quality, accurate IHC assays can be deployed consistently worldwide [7].

Comparative Analysis of International Regulatory Landscapes

The regulatory frameworks for IHC assays, particularly when used as In Vitro Diagnostics (IVDs) or companion diagnostics (CDx), vary considerably between the United States (US) and the European Union (EU). These differences influence the validation design, data package requirements, and the overall path to market.

The table below provides a high-level comparison of these two major systems:

Table 1: Comparison of US and EU Regulatory Frameworks for IHC Assays

Aspect	United States (US)	European Union (EU)
Primary Regulatory Authority	Food and Drug Administration (FDA) [7]	Notified Bodies [7]
Governing Regulation	Clinical Laboratory Improvement Amendments (CLIA) for lab tests; FDA pre-market review for IVDs [7]	In Vitro Diagnostic Regulation (IVDR) [7]
Companion Diagnostic (CDx) Classification	Class II or Class III devices [7]	Uniformly Class C devices [7]
Key Validation Standards	CLIA, CLSI guidelines, FDA feedback [7]	ISO 15189, ISO 13485, IVDR [7] [18]
Clinical Trial Use (Country-specific)	Investigational Device Exemption (IDE) may be required for significant risk studies [7]	Annex XIV submission and ethics committee approval required in each country [7]

A critical distinction lies in the validation philosophy. In the US, the Clinical Laboratory Improvement Amendments (CLIA) provides a baseline for laboratory testing but does not specify detailed methodologies for satisfying performance requirements [7]. For FDA approval, particularly through the Pre-Market Approval (PMA) pathway, studies must exceed basic CLIA standards and are often guided by Clinical Laboratory Standards Institute (CLSI) guidelines [7]. The European system, under the In Vitro Diagnostic Regulation (IVDR), is risk-based and integrates quality management system standards like ISO 13485 and the competence standard for medical laboratories, ISO 15189 [7] [18]. The core objective of ISO 15189 is to ensure that laboratories deliver accurate, timely, and reliable results, fostering confidence in diagnostic services [18].

For global clinical trials, the regulatory pathways further diverge. In the US, an assay used for patient stratification may require a risk determination and potentially an Investigational Device Exemption (IDE) [7]. In the EU, if the assay has a medical purpose, it requires an Annex XIV submission to each national competent authority where samples are collected, adding a layer of complexity due to local requirements [7].

Analytical Validation Standards and Experimental Protocols

The analytical validation of an IHC assay is the cornerstone of its reliability. Evidence-based guidelines, such as those from the College of American Pathologists (CAP), provide a framework for establishing analytical validity. A 2015 survey demonstrated that the dissemination of the 2014 CAP guideline had a significantly positive impact, with nearly 80% of respondent laboratories adopting some or all of its recommendations [11]. This underscores the scientific community's drive towards standardized, evidence-based practices.

Key Validation Performance Metrics

The validation process must establish key performance characteristics for the assay. The updated 2024 CAP guideline harmonizes requirements, moving away from marker-specific criteria to a more unified approach [9].

Table 2: Key Analytical Validation Metrics from CAP Guidelines

Performance Characteristic	CAP Guideline Recommendation	Experimental Consideration
Accuracy/Concordance	Minimum concordance of 90% for predictive biomarkers (previously variable for HER2, ER/PR) [9].	Compare new assay results to a validated comparator (e.g., another validated IHC assay, FISH, or flow cytometry) [9].
Sample Size for Validation	Minimum of 10 positive and 10 negative cases for most assays; required for alternative fixatives (e.g., cytology specimens) [9].	Cases should be selected to represent the full spectrum of antigen expression and the intended tissue types.
Assay-Scoring System Validation	Each unique assay-scoring system combination (e.g., PD-L1 in different tumor types) must be validated separately [9].	Validation must be performed for each clinical indication and tumor site, as scoring criteria and thresholds may differ.

Detailed Experimental Protocol for a Global Validation Study

To meet the requirements of both US and EU regulators, a comprehensive validation study should be designed. The following workflow outlines a robust methodology for a global analytical validation.

Diagram 1: IHC Assay Global Validation Workflow

Step 1: Define Intended Use and Target Markets

• Objective: Clearly articulate the assay's clinical or research purpose, the specific biomarkers, and the tissue types. Simultaneously, identify all intended geographic markets (US, EU, etc.) [7].
• Protocol: Document the intended use statement. This will dictate the risk classification and the level of validation rigor required by respective regulatory bodies [7].

Step 2: Assay Development and Optimization

• Objective: Establish a robust and reproducible staining protocol.
• Protocol: Optimize key reagents (primary antibody, detection system) and pre-analytical conditions (epitope retrieval, fixation time) using control tissues with known antigen expression levels [76].

Step 3: Select Validation Comparator

• Objective: Choose an appropriate reference method to establish accuracy.
• Protocol: Based on CAP guidelines, select from a hierarchy of comparators, ordered from most to least stringent:
  • Cell lines with known protein content ("calibrators") [9].
  • A non-IHC method (e.g., flow cytometry, FISH) [9].
  • A validated IHC assay in another laboratory [9].
  • Expected architectural and subcellular localization of the antigen [9].

Step 4: Procure Tissue Cohort

• Objective: Acquire a well-characterized set of tissue samples that reflect real-world use.
• Protocol: For a new assay, procure a minimum of 60 cases is a common practice, ensuring representation of positive, negative, and borderline expression levels, as well as relevant tissue types and fixatives [9]. For assays using alternative fixatives (e.g., in cytology), a minimum of 10 positive and 10 negative cases is required [9].

Step 5: Establish Scoring Methodology

• Objective: Define a precise, objective scoring system to minimize inter-observer variation.
• Protocol: For predictive markers like PD-L1, pre-define the scoring algorithm (e.g., Tumor Proportion Score, Combined Positive Score). If multiple scoring systems are used for different indications, each must be validated separately [9].

Step 6: Execute Staining Runs

• Objective: Generate staining results under controlled conditions that mimic routine laboratory operation.
• Protocol: Run the validation cohort over several days, using different instruments and personnel if possible, to incorporate sources of pre-analytical and analytical variation into the validation [9]. Include appropriate positive and negative controls in each run.

Step 7: Perform Statistical Analysis

• Objective: Quantify assay performance.
• Protocol: Calculate concordance (aim for ≥90% for predictive markers), precision (repeatability and reproducibility), sensitivity, and specificity based on the comparison to the chosen comparator [11] [9].

Step 8: Compile Evidence Dossier

• Objective: Create a comprehensive report for regulatory submissions.
• Protocol: The dossier should include the intended use statement, standard operating procedures, raw data, statistical analysis, and proof of quality management system adherence (e.g., ISO 13485, ISO 15189) [7] [18].

Developing an Integrated Global Validation Strategy

A proactive, parallel strategy is essential for efficient global market access. Rather than validating sequentially for different regions, manufacturers should design a single, comprehensive validation package that satisfies the key requirements of all target markets from the outset. This involves building a validation plan that integrates CLIA standards, CLSI guidelines, and ISO standards such as ISO 15189 for laboratory competence and ISO 13485 for quality management systems [7] [18].

Engaging with regulatory agencies early is a critical component of this strategy. The FDA encourages pre-submission meetings to align on analytical validation study designs before they are conducted [7]. Similarly, understanding the requirements of the EU's Notified Bodies early in the process can prevent costly missteps. This integrated approach avoids duplication of effort, reduces overall development time, and creates a robust, defensible validation package that demonstrates the assay's reliability to any regulator or client worldwide.

The Scientist's Toolkit: Essential Reagents and Controls

The quality of an IHC assay is intrinsically linked to the quality of its components. The table below details essential tools and reagents, highlighting the critical role of controls in a harmonized validation strategy.

Table 3: Research Reagent Solutions for IHC Validation

Tool/Reagent	Function in Validation	Considerations for Global Strategy
Validated Primary Antibodies	Binds specifically to the target antigen of interest.	Select antibodies with extensive characterization data. Vendor-provided validation for IHC is crucial for reproducibility [76].
Control Cell Lines	Serve as consistent, renewable sources of positive and negative controls with defined antigen expression levels.	Essential for standardizing staining across runs and sites. AI-based monitoring of these controls can objectively track staining performance over time [77].
Multiplex IHC Platforms	Allow simultaneous detection of multiple biomarkers on a single tissue section.	Vital for complex biomarker studies. Requires validation of each antibody pair to ensure no cross-reactivity and that staining patterns are accurate [76].
Tissue Microarrays (TMAs)	Contain dozens of tissue cores on one slide, enabling high-throughput screening of antibody performance across multiple tissues.	Accelerate the validation process by allowing simultaneous staining of many different tissue types under identical conditions.
Image Analysis & AI Software	Provides quantitative, objective assessment of staining intensity and distribution.	Reduces scorer subjectivity and is key to automating quality control. Tools like "Qualitopix" can trace subtle staining variations that may be missed visually [77].
ISO 15189 Framework	Defines requirements for quality and competence in medical laboratories.	Provides the over-arching operational structure for impartiality, personnel competence, equipment management, and process control, ensuring reliable results [18].

Navigating the international regulatory landscape for IHC assay validation is a complex but manageable endeavor. Success hinges on moving beyond a region-specific, reactive approach and adopting a forward-looking, globally harmonized strategy. This requires a deep understanding of the distinct yet overlapping requirements of major markets like the US and EU, a commitment to rigorous analytical validation based on evolving evidence-based guidelines like those from CAP, and the integration of robust quality systems such as ISO 15189. By leveraging a parallel validation approach, engaging with regulatory bodies early, and utilizing high-quality, standardized reagents and controls, researchers and drug developers can create a streamlined path to global commercialization. This strategy not only ensures regulatory compliance but, more importantly, guarantees the delivery of accurate, reliable, and reproducible IHC data—a fundamental requirement for advancing cancer diagnostics and improving patient lives worldwide.

ISO 15189 is the international standard specifying requirements for quality and competence in medical laboratories. The latest iteration, ISO 15189:2022, was published in December 2022 and requires laboratories with existing accreditation to transition to the new standard within three years, implementing changes by December 2025 [14] [4]. This standard promotes patient welfare and satisfaction through confidence in laboratory quality and competence, outlining requirements for laboratories to plan and implement actions to address risks and opportunities for improvement [4]. For immunohistochemistry (IHC) laboratories, compliance with ISO 15189 provides a critical framework ensuring the analytical validity of assays that are essential for diagnostic, prognostic, and predictive biomarker evaluation in cancer and other diseases.

A significant shift in the updated standard is its enhanced focus on risk management and patient-centered care [14] [4]. Laboratory-supported point-of-care testing (POCT) requirements, previously outlined in ISO 22870, have now been fully incorporated into ISO 15189 [14]. The standard has also been structurally aligned with ISO/IEC 17025:2017 for testing and calibration laboratories [4]. These changes collectively ensure that risk to patients is central to the laboratory's quality management design and processes, requiring robust risk management processes to identify, assess, and mitigate potential risks impacting service quality [14].

The ISO 15189:2022 Framework: Key Requirements for IHC Laboratories

Structural and Governance Requirements

The updated standard introduces more detailed requirements regarding laboratory structure and governance. Laboratories must be legal entities or defined parts of legal entities legally responsible for their activities [78]. The standard requires clearly defined roles and responsibilities, documented policies and objectives, and specified lines of authority and communication [14] [78]. A crucial requirement is that the laboratory must be directed by qualified personnel with specified competence, delegated authority, responsibility, and resources to fulfill standard requirements [78]. The laboratory director maintains ultimate responsibility for overall operations, even when duties are delegated to qualified personnel [78].

Resource and Process Requirements

ISO 15189:2022 emphasizes competent personnel, properly maintained equipment, validated systems, and appropriate support services [78]. Laboratories must ensure all personnel are competent and work impartially and ethically according to the management system [78]. For IHC laboratories, this includes specific competency requirements for pathologists and technicians performing and interpreting stains.

Process requirements encompass the entire testing cycle: pre-examination, examination, and post-examination phases [18]. This includes procedures for sample handling, examination method verification and validation, and result reporting. The standard mandates both internal quality control and external quality assessment schemes to monitor performance and result accuracy [18].

Table: Key ISO 15189:2022 Clause Requirements for IHC Laboratories

Clause	Title	Key Requirements for IHC Laboratories
Clause 4	General Requirements	Impartiality, confidentiality, patient rights, informed consent, incident disclosure [78] [18]
Clause 5	Structural & Governance	Legal entity status, defined director responsibilities, organizational structure [78]
Clause 6	Resource Requirements	Competent personnel, validated equipment, appropriate facilities and environmental conditions [78]
Clause 7	Process Requirements	Sample handling procedures, validated examination methods, result reporting, complaint handling [18]
Clause 8	Management System	Quality management system, risk management, corrective actions, internal audits, continual improvement [18]

The Audit Process: From Preparation to Surveillance

Pre-Audit Preparation and Gap Analysis

Successful accreditation begins with thorough preparation. Laboratories should start by conducting a comprehensive gap analysis to identify areas needing improvement relative to the updated standard's requirements [14] [4]. This involves comparing current laboratory practices against each requirement of ISO 15189:2022.

Essential preparatory steps include:

• Forming a project team with tasks divided according to personnel expertise [4]
• Thoroughly reviewing and understanding changes in the revised standard [4]
• Developing a detailed transition plan with timelines, responsibilities, and specific actions [14] [4]
• Investing in staff training on updated requirements [14]
• Engaging with accreditation bodies early in the process [14]

Documentation review is critical at this stage, requiring organization of essential documents including quality manuals, documented procedures, staff competency records, equipment calibration and maintenance logs, test method validation records, sample handling procedures, quality control data, and incident reports [79].

Internal Audit and Assessment Execution

Internal audits using the ISO 15189:2022 checklist provide laboratories an opportunity to evaluate conformance before the formal accreditation assessment [78] [79]. These assessments should systematically evaluate each section of the standard:

• General & Structural Requirements: Assessing legal identity, impartiality policies, confidentiality safeguards, and ethical practices [79]
• Resource Requirements: Verifying personnel competence, training programs, appropriate infrastructure, and validated IT systems [78] [79]
• Pre-Analytical Processes: Reviewing patient preparation, specimen collection, labeling, transport, storage, and rejection criteria [79]
• Analytical Processes: Validating examination procedures, quality control measures, reagent management, and equipment calibration [79]
• Post-Analytical Processes: Ensuring result review, validation, reporting timeliness, and critical result management [79]
• Risk Management & Continual Improvement: Confirming risk assessments, corrective actions, and management review processes [79]

Digital audit tools can streamline this process by enabling real-time documentation of findings, attachment of evidence, assignment of corrective actions, and generation of compliance reports [79].

External Audit and Corrective Action Management

The formal accreditation assessment involves evaluation by an independent accreditation body. Laboratories must choose a recognized accrediting organization, such as A2LA, which offers combined programs assessing both ISO 15189:2022 and CLIA requirements during a single assessment [80].

During the external audit, assessors evaluate implementation of and compliance with all standard requirements. They examine documentation, interview personnel, observe processes, and review records and quality indicators. Following the assessment, any identified nonconformities must be addressed through a structured corrective action process:

• Creating specific tasks for each finding with assigned roles and deadlines [79]
• Implementing corrective and preventive actions (CAPA) to address root causes [18]
• Documenting resolution evidence such as revised SOPs or retraining records [79]
• Undergoing follow-up assessments to verify effectiveness of corrections [79]

Successful accreditation leads to a certificate of accreditation, typically valid for a defined period with ongoing surveillance assessments to ensure continued compliance [80].

IHC Assay Validation: Core Requirements and Controls

Essential Experimental Controls for IHC Validation

Valid interpretation of IHC assays requires appropriate positive and negative controls that must be documented and included in laboratory procedures and publications [81]. The absence of proper controls can lead to incorrect interpretations, irreproducible results, and clinical misdiagnoses [81].

Positive controls are specimens containing the target molecule in its known location. The most rigorous approach uses positive anatomical controls where antigen presence in the specimen is known a priori [81]. This can be a known site of expression in the specimen (internal positive control) or a separate specimen known to contain the target molecule (external positive control) [81]. For example, an antibody specific for insulin should include pancreas sections with islets of Langerhans and visualize only insulin-producing islet beta cells [81].

Negative controls must demonstrate that the reaction visualized results specifically from interaction between the target epitope and antibody paratope. A common but inadequate practice is omitting the primary antibody - this only controls for nonspecific secondary antibody binding, not primary antibody specificity [81]. The proper negative control involves substitution with serum or isotype-specific immunoglobulins at the same protein concentration as the primary antibody [81].

Table: Essential IHC Controls for ISO 15189 Compliance

Control Type	Purpose	Proper Implementation	Common Pitfalls
Positive Control	Verify antibody detects target antigen	Use tissue with known antigen expression in correct location	Using cell lines with abnormal expression levels; poorly characterized tissues
Negative Control (Specificity)	Demonstrate staining specificity	Substitute primary antibody with isotype-matched immunoglobulins at same concentration	Only omitting primary antibody (tests secondary only)
Antibody Validation	Confirm antibody specificity	Western blot of same tissue/cells; knockout/knockdown controls	Relying solely on manufacturer claims without verification
Process Controls	Monitor staining procedure	Include control tissues in each staining run	Inconsistent control quality; heterogeneous control tissues

Method Validation and Verification Protocols

ISO 15189 requires that examination procedures are verified or validated for their intended use [18]. For IHC assays, this includes establishing protocols for antibody validation, staining procedures, and interpretation criteria.

Key methodological requirements include:

• Antibody specificity validation using techniques like western blot with proteins from the same tissue or cells used for IHC [81]
• Staining optimization with appropriate titrations to determine optimal antibody concentrations [81]
• Interpretation criteria established with defined thresholds for positivity [45]
• Regular monitoring of staining quality using control tissues with known expression levels [77]

Recent advances incorporate artificial intelligence and image analysis to objectively monitor IHC staining quality. One study implemented an AI algorithm (Qualitopix) to measure stain quality control of standardized cell lines for HER2 and PD-L1 IHC over 24 months, successfully identifying unexpected variations and prompting maintenance that reduced variation [77].

Emerging Technologies: AI-Enhanced IHC and Quality Control

Deep Learning for IHC Biomarker Prediction

Advanced computational approaches are emerging as powerful tools for enhancing IHC quality and interpretation. Deep learning-based IHC biomarker prediction models represent a transformative innovation with significant implications for laboratory accreditation.

Recent research has developed automated pipelines for constructing deep learning models that generate AI-driven virtual IHC staining results directly from H&E-stained whole slide images (WSIs) [44]. One study created five IHC biomarker models (P40, Pan-CK, Desmin, P53, Ki-67) for gastrointestinal cancers, achieving AUCs of 0.90-0.96 and accuracies between 83.04-90.81% [44]. In a multi-reader multi-case study comparing AI-IHC to conventional IHC, pathologists showed high consistency rates for Desmin, Pan-CK and P40 (96.67-100%), with moderate consistency for P53 (70.00%) [44].

Dual-Modality AI Frameworks for Enhanced Prediction

Integration of multiple data types through unified AI frameworks demonstrates promising improvements in biomarker prediction accuracy. One study developed DuoHistoNet, a dual-modality transformer-based framework integrating both H&E and IHC stained images for predicting MSI/MMRd and PD-L1 status [45]. This approach achieved exceptional performance with AUROC exceeding 0.97 for MSI/MMRd prediction in colorectal cancer and 0.96 for PD-L1 prediction in breast cancer [45].

Notably, the AI-predicted biomarker status demonstrated significant prognostic value, with patients having biomarker-positive model predictions showing prolonged time-on-treatment and overall survival when treated with pembrolizumab [45]. For breast cancer patients, the model's predictions were superior to PD-L1 IHC in stratifying patients with improved outcomes on pembrolizumab, suggesting a reevaluation of existing PD-L1 status thresholds may be warranted [45].

Table: Performance Comparison of AI-Based IHC Assessment Methods

Study	Technology	Biomarkers	Performance Metrics	Clinical Validation
Deep Learning IHC Prediction [44]	H&E to IHC translation using ResNet-50	P40, Pan-CK, Desmin, P53, Ki-67	AUC: 0.90-0.96Accuracy: 83.04-90.81%	MRMC study with pathologists (n=150 WSIs)
AI Quality Monitoring [77]	Qualitopix algorithm on control cell lines	HER2, PD-L1	Detected staining variations over 24 months	Identified inter-stainer and intra-run variations
Dual-Modality Framework [45]	Transformer integrating H&E and IHC	MSI/MMRd, PD-L1	AUROC: >0.97 (CRC)AUROC: >0.96 (BRCA)	Survival analysis showing improved patient stratification

The Scientist's Toolkit: Essential Research Reagent Solutions

Implementing and maintaining ISO 15189 compliance requires specific reagents and materials that ensure assay validity and reproducibility. The following tools are essential for IHC laboratories:

• Standardized Control Cell Lines: Commercially available cell lines with characterized antigen expression levels for consistent staining controls, particularly valuable for proteins like HER2 and PD-L1 where staining intensity must be carefully calibrated [77].

• Validated Antibody Panels: Antibodies specifically validated for IHC applications with documented specificity evidence, including western blot data and optimal working concentrations [81].

• Automated Staining Systems: Standardized autostainers from manufacturers like Agilent, Roche, and Leica that ensure consistent reagent application and incubation times across runs [77] [45].

• Whole Slide Imaging Systems: High-resolution scanners from manufacturers such as Philips, Leica, and KFBIO capable of digitizing slides at 20x-40x magnification for both archival and AI analysis [44] [45].

• Image Analysis Software: Both commercial and custom computational tools for quantitative assessment of staining intensity and distribution, including open-source platforms like QuPath [77] [45].

• Reference Standard Materials: Commercially available tissue microarrays with well-characterized biomarker status for assay validation and proficiency testing [45].

• Quality Monitoring Algorithms: AI-based quality control systems like Qualitopix that continuously monitor staining performance across multiple instruments and batches [77].

The path to ISO 15189 accreditation requires meticulous attention to both traditional quality management principles and emerging technological innovations. The updated 2022 standard emphasizes risk management, patient-centered care, and robust validation processes that collectively enhance laboratory competence and patient safety. For IHC laboratories, this means implementing comprehensive control strategies, validated protocols, and continual monitoring systems.

Emerging AI technologies offer promising avenues for enhancing IHC quality, interpretation consistency, and operational efficiency. From virtual IHC prediction to automated quality monitoring, these tools can significantly support accreditation efforts when properly validated and implemented. However, these technological advances must be grounded in fundamental quality principles including proper controls, method validation, and personnel competence.

As the December 2025 transition deadline approaches, laboratories should prioritize gap analysis, staff training, and process refinement. Successful accreditation demonstrates commitment to quality and positions laboratories to reliably deliver the accurate, reproducible IHC results essential for modern precision medicine.

Conclusion

Successful IHC assay validation under ISO 15189 is not merely a regulatory hurdle but a fundamental component of quality that directly impacts patient care and research integrity. By integrating the standard's management system requirements with rigorous technical validation protocols, laboratories can establish a robust, reproducible, and clinically relevant IHC practice. The future of IHC validation will see an increased emphasis on risk-based approaches, global harmonization of standards like ISO 15189 and IVDR, and the development of more sophisticated tools for ongoing quality monitoring. For researchers and drug developers, mastering this framework is essential for generating reliable data, accelerating diagnostic commercialization, and ultimately, advancing precision medicine.

References

• 1. Medical laboratories - ISO 15189:2022 [https://www.iso.org/standard/76677.html]
• 2. What is ISO 15189 and why is it important? [https://www.ideagen.com/thought-leadership/blog/what-is-iso-15189-and-why-is-it-important]
• 3. ISO 15189 Accreditation: Navigation Between Quality ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC6287216/]
• 4. Insights on updated accreditation guidelines ISO 15189:2022 [https://diagnostics.roche.com/global/en/article-listing/lab-leaders/article/iso-15189-updated-accreditation-guidelines.html]
• 5. 2025 IFCC Recommendations for IQC [https://www.westgard.com/essays/quality-requirements-and-standards/ifcc-iqc-2025.html]
• 6. Medical laboratories - ISO 15189:2012 [https://www.iso.org/standard/56115.html]
• 7. Optimizing Immunohistochemistry Validation and ... [https://www.precisionformedicine.com/blog/optimizing-ihc-validation-regulatory-strategies]
• 8. Immunohistochemistry should undergo robust validation ... [https://pubmed.ncbi.nlm.nih.gov/26280782/]
• 9. Principles of Analytic Validation of Immunohistochemical ... [https://www.cap.org/protocols-and-guidelines/cap-guidelines/current-cap-guidelines/principles-of-analytic-validation-of-immunohistochemical-assays]
• 10. Novelties in the ISO 15189:2023 standard - PMC [https://pmc.ncbi.nlm.nih.gov/articles/PMC10724875/]
• 11. Analytic Validation of Immunohistochemical Assays [https://pmc.ncbi.nlm.nih.gov/articles/PMC6938252/]
• 12. Validation of immunohistochemical tests performed on ... [https://cytojournal.com/validation-of-immunohistochemical-tests-performed-on-cytology-cell-block-material-practical-application-of-the-college-of-american-pathologists-guidelines/]
• 13. Guidance for Submission of Immunohistochemistry ... [https://www.fda.gov/medical-devices/guidance-documents-medical-devices-and-radiation-emitting-products/guidance-submission-immunohistochemistry-applications-food-and-drug-administration-final-guidance]
• 14. ISO 15189 Quality Requirements by December 2025 [https://theproficientlab.com/navigating-the-transition-iso-15189-quality-requirements-by-december-2025/]
• 15. Belgian Recommendations for Analytical Verification and ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC10695338/]
• 16. A critical evaluation of strategies used for validation ... [https://www.sciencedirect.com/science/article/pii/S1574789114000568]
• 17. Lessons Learned, Challenges Taken, and Actions Made for ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC10396077/]
• 18. ISO 15189: Definition, Requirements, Implementation, and ... [https://simplerqms.com/iso-15189/]
• 19. Comparison between routine methods and RT-qPCR [https://pubmed.ncbi.nlm.nih.gov/34555047/]
• 20. Exploring technical aspects of biomarker assays - NCBI - NIH [https://www.ncbi.nlm.nih.gov/books/NBK513094/]
• 21. A Practical Guide to Immunoassay Method Validation - PMC [https://pmc.ncbi.nlm.nih.gov/articles/PMC4541289/]
• 22. Accuracy of human epidermal growth factor receptor 2 (HER2 ... [https://diagnosticpathology.biomedcentral.com/articles/10.1186/s13000-025-01624-3]
• 23. Causes behind error rates for predictive biomarker testing [https://link.springer.com/article/10.1007/s00428-020-02966-7]
• 24. Key principles of ISO , 15189 /IEC 17025, and ISO for Lab... | QIA CLSI [https://www.qi-a.com/learning-center/key-principles-of-iso-15189-iso-iec-17025-and-clsi-for-lab-qms-standards/]
• 25. Module BASICS OF THE QUALITY SYSTEM CONCEPT - SlideServe [https://www.slideserve.com/lois/module-basics-of-the-quality-system-concept-powerpoint-ppt-presentation]
• 26. International Organization for Standardization (ISO) 15189 [https://pmc.ncbi.nlm.nih.gov/articles/PMC5500734/]
• 27. CLSI ILA21 - Clinical Evaluation of Immunoassays [https://clsi.org/shop/standards/ila21/]
• 28. CLSI POCT05 Glucose Monitoring Metrics | News [https://clsi.org/about/news/clsi-publishes-new-edition-of-poct05-performance-metrics-for-continuous-interstitial-glucose-monitoring/]
• 29. USA CLIA Standards vs. International ISO 15189 Standards [https://www.linkedin.com/pulse/comparing-quality-management-usa-clia-standards-vs-international-yf26e]
• 30. Understanding In Vitro Diagnostic Regulation (IVDR) [https://sakura.eu/tdx-knowledge-hub/ivdr]
• 31. EU IVDR Regulation: What It Is, Compliance Guide & ... [https://www.obelis.net/news/eu-ivdr-regulation-what-it-is-compliance-guide-requirements/]
• 32. Intended Purpose and Intended Use [https://blog.johner-institute.com/regulatory-affairs/intended-purpose/]
• 33. The Medical Device Coordination Group In Vitro Diagnostic ... [https://www.medtecheurope.org/2025/04/03/the-medical-device-coordination-group-in-vitro-diagnostic-medical-devices-classification-guidance-revises-sars-cov-2/]
• 34. Comparative analysis of ISO 15189:2022 with earlier ... [https://pubmed.ncbi.nlm.nih.gov/40985154/]
• 35. New Findings Show Standard HER2 Assays Miss Key ... [https://www.pharmacytimes.com/view/new-findings-show-standard-her2-assays-miss-key-her2-low-cases]
• 36. Independent Validation of a HER2-Low Focused ... [https://www.sciencedirect.com/science/article/abs/pii/S0893395224002734]
• 37. Principles of Analytic Validation of Immunohistochemical ... [https://www.guidelinecentral.com/guideline/24377]
• 38. An Update of Immunohistochemistry in Hepatocellular ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC12428263/]
• 39. Development and clinical validation of deep learning-based ... [https://bmcgastroenterol.biomedcentral.com/articles/10.1186/s12876-025-04045-0]
• 40. Verification vs. Validation: When do I need which in the lab? [https://platomics.com/verification-vs-validation-when-do-i-need-which-in-the-lab/]
• 41. IVD vs LDT - Which should you be using? | LabLeaders [https://diagnostics.roche.com/global/en/article-listing/lab-leaders/article/ivd-vs-ldt.html]
• 42. Technical Challenges Faced in Validation of ... [https://pubmed.ncbi.nlm.nih.gov/41246894/]
• 43. Indirect clinical validation for predictive biomarkers in ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC12488805/]
• 44. Development and clinical validation of deep learning ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC12211442/]
• 45. Synergistic H&E and IHC image analysis by AI predicts ... [https://www.nature.com/articles/s43856-025-01045-9]
• 46. Optimizing cytology and small biopsy specimen processing ... [https://www.sciencedirect.com/science/article/pii/S221329452500078X]
• 47. Impact of pre-analytic variables on HER2 IHC results [https://www.her2know.com/pathology-perspectives/spotlight-series/impact-of-pre-analytic-variables-on-her2-ihc-results.html]
• 48. Immunohistochemistry (IHC): The Complete Guide [https://www.antibodies.com/applications/immunohistochemistry]
• 49. IHC antigen retrieval protocol [https://www.abcam.com/en-us/technical-resources/protocols/ihc-antigen-retrieval]
• 50. Antigen Retrieval in Immunohistochemistry [https://www.bosterbio.com/blog/post/antigen-retrieval-in-immunohistochemistry?srsltid=AfmBOoqQ4uyEC16wpeiknFmnJsazzwoyo5axVyCgXNPyeMpi8QiJTA0z]
• 51. Revalidation of Equipment and Process in Pharmaceuticals [https://www.pharmaguideline.com/2017/12/revalidation-of-equipment-process.html]
• 52. The Vital Role of Periodic Cleaning Revalidation [https://pharmallies.com/ensuring-safety-and-compliance-the-vital-role-of-periodic-cleaning-revalidation/]
• 53. Quantitative comparison of PD-L1 IHC assays against NIST ... [https://pubmed.ncbi.nlm.nih.gov/34389791/]
• 54. 免疫组织化学（IHC）测定 [https://www.labcorp.com/content/labcorp/zh/ch/biopharma/nonclinical/disciplines/pathology/specialty/immunohistochemistry.html]
• 55. Quality assurance guidance for scoring and reporting for ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC6463860/]
• 56. 從臨床試驗到IVD 應用，解鎖Multiplex IHC 的價值與挑戰 [https://www.blossombio.com/eNews/20230720/index.html]
• 57. IHC第三方质控品——病理质控基石，精准诊断起点 [https://www.cobioer.com/solution/index1114.html]
• 58. Process Validation in Six Sigma: A Complete Guide [https://www.6sigma.us/process-improvement/process-validation/]
• 59. Process Lifecycle Validation: Applying Risk Management [https://www.biopharminternational.com/view/process-lifecycle-validation-applying-risk-management]
• 60. Regulating the future of laboratory medicine: European re... [https://www.degruyterbrill.com/document/doi/10.1515/cclm-2025-0482/html?lang=en&srsltid=AfmBOopTCne5yf_NdtFUMQjVnnvCjSiaoJd9kvcgF0BDaZLJ7Jcf5DVO]
• 61. Navigating Quality Assessment Hurdles in Clinical Laboratory ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC10929212/]
• 62. Importance of External Quality Assessement in Immuno- ... [https://www.sciencedirect.com/science/article/pii/S0923753419515678]
• 63. Quantitative comparison of PD-L1 IHC assays against NIST ... [https://www.sciencedirect.com/science/article/pii/S089339522200309X]
• 64. Review and comparison of quality standards, guidelines ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC5644525/]
• 65. ISO 13485 Certification vs ISO 15189 Accreditation for ... [https://a2la.org/iso-13485-certification-vs-iso-15189-accreditation-for-ldts/]
• 66. Working Towards Accreditation by the International ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC4151005/]
• 67. ILA28 | Quality Assurance for Design Control and ... [https://clsi.org/shop/standards/ila28/]
• 68. Standards Implementation [https://clsi.org/standards-implementation/]
• 69. CLSI: Clinical & Laboratory Standards Institute [https://clsi.org/]
• 70. Frequently Asked Questions from Immunohistochemistry ... [https://www.cap.org/member-resources/councils-committees/immunohistochemistry-topic-center/frequently-asked-questions-from-immunohistochemistry-laboratories]
• 71. Structure and content of the EU-IVDR [https://pmc.ncbi.nlm.nih.gov/articles/PMC9896441/]
• 72. New Regulations - Public Health - European Commission [https://health.ec.europa.eu/medical-devices-sector/new-regulations_en]
• 73. In Vitro Diagnostic Regulation (IVDR)—Frequently Asked ... [https://www.thermofisher.com/us/en/home/clinical/ivdr/faqs.html]
• 74. IVDR 2025 Deadline: Key Requirements for Legacy IVDs ... [https://www.obelis.net/news/ivdr-2025-legacy-deadline/]
• 75. IVDR Certification Update for IHC Portfolio [https://www.leicabiosystems.com/en-be/news/update-on-leica-biosystems-progress-toward-in-vitro-diagnostics-regulation-ivdr/]
• 76. Validating Antibodies for Research Applications in IHC [https://www.leicabiosystems.com/us/life-science-solutions/webinars/validating-antibodies-for-research-applications-in-ihc/]
• 77. Monitoring Immunohistochemical Staining Variations Using ... [https://www.sciencedirect.com/science/article/pii/S0023683725000157]
• 78. ISO 15189:2022 Checklist [https://goaudits.com/checklist/iso-15189-2022-checklist/920/35/]
• 79. ISO 15189:2022 Checklist [https://eauditor.app/2025/07/04/iso-151892022-checklist/]
• 80. Clinical Testing Lab Accreditation (ISO 15189 & CLIA) [https://a2la.org/accreditation/clinical-testing/]
• 81. Controls for Immunohistochemistry: The Histochemical ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC4212362/]