CLSI EP25 vs ISO 15189 method validation

By Dr. Zubair Khalid, DVM, MS, PhD · 2026-08-21

Quick Answer

• CLSI EP25 supplies the technical protocol for precision evaluation, while ISO 15189 establishes the quality management framework that requires method validation and verification.
• Laboratory managers should apply CLSI EP25 experimental designs to generate precision data that satisfy ISO 15189 examination process requirements and accreditation expectations.
• Neither document alone covers all validation needs, and laboratories must integrate both with biological variation acceptance criteria and documented procedures for bias, reference intervals, and operator competence.

Understanding the Regulatory Landscape for Laboratory Method Validation

Clinical laboratory managers face a complex environment when implementing new diagnostic assays or modifying existing methods. Two dominant frameworks shape this work, the Clinical and Laboratory Standards Institute (CLSI) documents and the International Organization for Standardization (ISO) 15189 standard. Each serves a distinct purpose, yet laboratories pursuing accreditation must understand how they interact.

CLSI documents function as detailed technical protocols. They describe experimental designs, statistical calculations, and interpretation criteria for specific validation activities. EP25, formally titled Evaluation of Precision Performance of Quantitative Measurement Methods, provides the statistical framework for assessing random error in measurement procedures. This protocol guides how many runs, days, and replicates a laboratory needs to estimate imprecision reliably.

ISO 15189 operates at a different level. It establishes requirements for quality and competence in medical laboratories. The standard addresses management system elements, pre-examination processes, examination processes, and post-examination processes. Method validation and verification appear within the examination process requirements, but the standard does not prescribe the detailed statistical procedures. Instead, it requires laboratories to have documented policies for confirming that methods perform acceptably for their intended use.

The relationship between these frameworks matters for practical implementation. A laboratory can use CLSI EP25 to generate the precision data required by ISO 15189, but the standard also demands more. Bias estimation, measurement uncertainty, reference interval verification, and ongoing quality control all fall within the ISO 15189 scope. The National Library of Medicine provides access to foundational texts on laboratory methods that help practitioners understand the scientific basis for these requirements.

Accreditation bodies assess compliance with ISO 15189, not with CLSI documents directly. However, CLSI protocols serve as the accepted technical means for meeting many ISO 15189 requirements. This creates a practical workflow where laboratory managers select the appropriate CLSI document for each validation activity and then document how the results satisfy ISO 15189 clauses.

Scope and Purpose Differences

CLSI EP25 Precision Evaluation

CLSI EP25 addresses one specific analytical performance characteristic, precision. Precision describes the closeness of agreement between independent measurement results obtained under stipulated conditions. The protocol distinguishes between repeatability, which is precision under the same operating conditions over a short time interval, and reproducibility, which is precision under changed conditions such as different operators, instruments, or days.

The EP25 document provides detailed experimental designs. A typical precision evaluation involves running multiple levels of control materials or patient samples across multiple days. The protocol specifies minimum numbers of runs and replicates to achieve statistically meaningful estimates of within-run, between-run, and between-day components of variation. These components combine into an estimate of total imprecision that can be compared against allowable performance criteria.

Laboratories use EP25 data to calculate coefficients of variation for each level of analyte tested. These coefficients serve multiple purposes. They support method comparison studies, help establish quality control limits, and provide inputs for measurement uncertainty calculations. The National Institute of Standards and Technology emphasizes that measurement science depends on understanding and quantifying all sources of variability, and EP25 provides the structured approach for doing so in clinical chemistry.

ISO 15189 Validation and Verification Requirements

ISO 15189 takes a broader view. The standard requires laboratories to validate methods that they introduce or modify. Validation means confirming through examination and provision of objective evidence that the method meets specified requirements for the intended use. Verification means confirming through objective evidence that the method performs as claimed by the manufacturer.

The distinction between validation and verification is critical. A laboratory using a commercial assay with regulatory clearance typically performs verification. This involves confirming that the assay performs according to the manufacturer's claims in the laboratory's own setting. A laboratory developing an in-house method or modifying an established method must perform full validation, which includes establishing performance characteristics such as accuracy, precision, analytical sensitivity, analytical specificity, and measuring interval.

ISO 15189 does not specify how many samples to test or which statistical tests to use. This flexibility allows laboratories to choose appropriate protocols based on the method and its intended use. In practice, most laboratories turn to CLSI documents for the technical details. The MIQE guidelines provide a parallel example from molecular biology, where detailed reporting requirements ensure that qPCR experiments are transparent and interpretable.

Flexible Scope Accreditation

The concept of flexible scope accreditation deserves attention because it changes how laboratories approach method validation. Under a fixed scope, a laboratory must notify the accreditation body before introducing any new method. Under a flexible scope, the laboratory can introduce methods within defined categories without prior approval, provided the laboratory has a documented validation procedure and meets specified criteria.

A study published in Biochemia Medica demonstrated this approach in practice. The laboratory validated two biochemistry analyzers using CLSI protocols, evaluated imprecision and bias for 30 chemistry assays, and used biological variation as the acceptance criteria. The study concluded that a laboratory with a well-structured and documented validation procedure can successfully operate under a flexible scope of accreditation.

This finding has practical implications. Laboratories pursuing flexible scope accreditation must invest in robust validation procedures before seeking this status. The validation work itself becomes the foundation for the laboratory's ability to introduce new methods efficiently. The study also noted that performing validation activities before using methods on patient samples can reveal technical issues that must be corrected to minimize impact on patient results.

Quality Control Requirements Compared

Statistical Quality Control Under CLSI EP25

CLSI EP25 provides the statistical foundation for quality control by quantifying the imprecision that quality control rules are designed to detect. The protocol's estimates of within-run and between-run variation directly inform the selection of quality control materials, the number of control levels, and the control rules applied to each analytical run.

The relationship between precision estimates and quality control design follows established statistical principles. Laboratories use the standard deviation estimates from EP25 studies to calculate control limits. These limits typically span the mean plus or minus two or three standard deviations. The choice of control rules depends on the clinical significance of errors and the analytical performance of the method.

EP25 also supports the evaluation of quality control data over time. Laboratories can compare ongoing quality control performance against the precision estimates established during initial validation. Significant changes in observed imprecision may indicate instrument deterioration, reagent lot changes, or operator issues that require investigation.

ISO 15189 Quality Control Framework

ISO 15189 requires laboratories to establish a comprehensive quality control program that goes beyond statistical calculations. The standard mandates that laboratories use control materials that are as similar as possible to patient samples, that control materials are tested at appropriate intervals, and that quality control data are reviewed and documented.

The standard also requires laboratories to participate in external quality assessment or proficiency testing programs. These programs provide an independent assessment of laboratory performance by comparing results across multiple laboratories using the same or similar methods. The World Health Organization Laboratory Biosafety Manual provides context on the broader quality and safety framework within which laboratory testing operates.

ISO 15189 emphasizes the importance of traceability in quality control. Laboratories must ensure that calibrators and control materials are traceable to reference materials or reference methods where available. This requirement connects quality control to the broader measurement traceability chain that supports reliable patient results.

Integrating EP25 Data into ISO 15189 Quality Control

The practical integration of EP25 data into an ISO 15189 quality control program follows a logical sequence. First, the laboratory conducts an EP25 precision study to establish baseline imprecision estimates. Second, these estimates inform the selection of quality control rules and limits. Third, the laboratory implements ongoing quality control using these rules. Fourth, the laboratory reviews quality control data periodically to confirm that method performance remains within acceptable limits.

This integration requires documentation. The laboratory must record the precision study design, the results obtained, and the rationale for the quality control rules selected. This documentation demonstrates compliance with ISO 15189 requirements and provides evidence for accreditation assessments.

Validation Expectations and Experimental Design

Precision Studies Following CLSI EP25

Designing a precision study under CLSI EP25 requires attention to several experimental parameters. The laboratory must select appropriate test materials, determine the number of runs and replicates, and specify the conditions under which measurements are performed.

Test material selection depends on the analyte and the method. Commercial quality control materials are commonly used because they are stable and available in multiple concentration levels. Patient samples may be used when commercial materials are not available or when matrix effects are a concern. The laboratory should use at least two concentration levels spanning the clinically relevant range.

The number of runs and replicates affects the statistical power of the study. More runs and replicates provide more reliable estimates of imprecision components but require more time and resources. CLSI EP25 provides guidance on minimum experimental designs that balance statistical needs with practical constraints.

The Addgene protocols illustrate a parallel principle in molecular biology, where experimental design decisions directly affect the reliability and interpretability of results. While these protocols address molecular biology workflows instead of clinical chemistry, the underlying principle of careful experimental planning applies across laboratory disciplines.

Bias and Accuracy Assessment

Precision alone does not establish method acceptability. Laboratories must also assess bias, which is the systematic difference between the method's results and the true value or a reference method's results. ISO 15189 requires laboratories to evaluate accuracy as part of method validation or verification.

Bias assessment typically involves comparing the new method against a reference method or analyzing samples with known values. The Biochemia Medica study evaluated bias for 30 chemistry assays and calculated total error for all tests. The study used biological variation as the acceptance criteria, which represents a clinically meaningful approach to defining allowable performance.

The combination of precision and bias estimates yields total error, which describes the overall uncertainty of a measurement result. Total error calculations combine the random error from imprecision with the systematic error from bias. Laboratories compare total error against allowable total error criteria to determine whether a method is acceptable for clinical use.

Analytical Sensitivity and Specificity

ISO 15189 requires laboratories to evaluate analytical sensitivity and analytical specificity as part of method validation. Analytical sensitivity refers to the method's ability to detect small quantities of the analyte, often expressed as the limit of detection or limit of quantification. Analytical specificity refers to the method's ability to measure the intended analyte without interference from other substances.

These performance characteristics are particularly important for methods used in infectious disease diagnosis. A study of malaria microscopic diagnosis conducted at a hospital in Senegal demonstrated the practical application of validation principles. The study assessed microscopists' ability to detect parasites, determine parasite density, and identify parasite species. The study used a minimum threshold of 50% concordance for comparison and found variable performance across different tasks.

This study illustrates that validation extends beyond instrument performance to include operator competence. ISO 15189 requires laboratories to ensure that personnel are qualified for their assigned tasks. Method validation must therefore include assessment of the people performing the testing, beyond the analytical system.

Reference Interval Verification

Reference intervals are critical for interpreting patient results. A reference interval defines the range of values expected in a healthy population. Laboratories must verify that the reference intervals they use are appropriate for their patient population and method.

A nationwide survey in Nepal examined knowledge, attitudes, and practices regarding reference interval utilization. The survey found that most laboratories relied on manufacturer-provided reference intervals without local validation. Accredited laboratories demonstrated better knowledge of reference intervals and higher confidence in using their current intervals.

This finding has direct implications for method validation. When a laboratory introduces a new method, the manufacturer's reference intervals may not apply. The laboratory must verify reference intervals using its own patient population or transfer reference intervals using established protocols. This verification is part of the overall validation process required by ISO 15189.

Practical Implementation Steps

Step 1, Define the Validation Scope

Before beginning any validation activity, the laboratory must clearly define what is being validated and why. This definition should address the method's intended use, the analytes being measured, the specimen types accepted, and the clinical decisions the results will support.

The validation scope also determines whether the laboratory performs full validation or verification. A laboratory introducing a modified version of a commercial method must validate the modifications. A laboratory implementing an unmodified commercial method with regulatory clearance typically verifies performance. The National Library of Medicine provides background on the scientific principles that distinguish these activities.

Step 2, Select Appropriate CLSI Protocols

Once the validation scope is defined, the laboratory selects the appropriate CLSI protocols. EP25 addresses precision. Other CLSI documents address accuracy, linearity, interference, and other performance characteristics. The laboratory should select protocols that match the performance characteristics relevant to the method and its intended use.

The selection process should be documented. The laboratory should record which protocols were selected, why they were selected, and how they will be implemented. This documentation supports the ISO 15189 requirement for a documented validation procedure.

Step 3, Conduct the Precision Study

The precision study follows the experimental design specified in CLSI EP25. The laboratory prepares test materials, assigns operators, schedules runs, and collects data according to the protocol. The study should be conducted under conditions that reflect routine laboratory operations to ensure that the precision estimates are representative.

During the study, the laboratory should monitor for unexpected events that could affect results. Instrument malfunctions, reagent problems, or operator errors should be documented and investigated. The National Institute of Standards and Technology emphasizes that measurement quality depends on controlling and documenting all factors that could influence results.

Step 4, Analyze the Data

Data analysis follows the statistical procedures specified in CLSI EP25. The laboratory calculates within-run, between-run, and between-day standard deviations. These components combine into an estimate of total imprecision. The laboratory also calculates coefficients of variation for each concentration level tested.

The results are compared against acceptance criteria. These criteria may come from biological variation, regulatory requirements, or manufacturer claims. The Biochemia Medica study used biological variation as acceptance criteria, which represents a clinically meaningful standard for method acceptability.

Step 5, Document the Results

Documentation is essential for ISO 15189 compliance. The laboratory must record the validation protocol, the results obtained, the acceptance criteria used, and the conclusions reached. This documentation must be retained and made available for accreditation assessments.

The documentation should also address any performance characteristics that did not meet acceptance criteria. The laboratory must describe the investigation conducted, the corrective actions taken, and the final disposition of the method. This level of documentation demonstrates the laboratory's commitment to quality and provides evidence of competence.

Records and Measurements

Essential Records for Method Validation

Laboratories must maintain comprehensive records of all validation and verification activities. These records serve multiple purposes. They demonstrate compliance with ISO 15189 requirements, provide evidence for accreditation assessments, and support ongoing quality monitoring.

Essential records include the validation protocol, raw data from precision studies, statistical calculations, acceptance criteria, and final conclusions. The laboratory should also record any deviations from the protocol and the actions taken to address them. This level of detail supports the transparency required by ISO 15189.

The MIQE guidelines provide a useful model for documentation standards. While developed for qPCR experiments, the guidelines emphasize the importance of reporting sufficient information to allow others to evaluate the quality of the work. Clinical laboratory validation records should follow a similar principle.

Measurements to Track Over Time

Method validation is not a one-time activity. Laboratories must monitor method performance continuously to detect changes that could affect patient results. This monitoring includes ongoing quality control, periodic calibration verification, and regular review of quality control data.

Key measurements to track include control values, calibration parameters, and quality control rule violations. Trends in these measurements may indicate developing problems that require investigation. The laboratory should have procedures for reviewing these data at defined intervals and for taking corrective action when needed.

External Quality Assessment Records

Participation in external quality assessment or proficiency testing programs provides an independent check on laboratory performance. The laboratory must document its participation, its results, and any corrective actions taken in response to unsatisfactory performance.

External quality assessment results complement internal quality control data. While internal quality control monitors day-to-day consistency, external quality assessment evaluates accuracy against other laboratories. Both are required by ISO 15189 and both contribute to the overall assessment of method performance.

Common Failure Patterns

Insufficient Number of Runs or Replicates

A common failure in precision studies is using too few runs or replicates to obtain reliable estimates. This failure produces imprecision estimates with wide confidence intervals that may not reflect true method performance. Laboratories may be tempted to shorten the study to save time, but this compromises the validity of the results.

CLSI EP25 provides guidance on minimum experimental designs. Laboratories should follow this guidance unless they have documented justification for a different design. The National Institute of Standards and Technology emphasizes that measurement science requires adequate data to support conclusions, and precision studies are no exception.

Inappropriate Acceptance Criteria

Another common failure is using acceptance criteria that are not appropriate for the method or its intended use. Some laboratories use manufacturer claims without considering whether those claims are clinically meaningful. Others use criteria that are too lenient or too strict for the clinical application.

Biological variation provides a clinically meaningful basis for acceptance criteria. The Biochemia Medica study demonstrated this approach by using biological variation to evaluate 30 chemistry assays. Laboratories should consider biological variation when defining acceptance criteria for their methods.

Failure to Verify Reference Intervals

Many laboratories adopt manufacturer-provided reference intervals without verification. This practice can lead to incorrect interpretation of patient results if the reference intervals do not apply to the laboratory's patient population. The survey from Nepal found that 87.5% of laboratories relied on manufacturer-provided reference intervals, highlighting the prevalence of this practice.

Reference interval verification should be part of the overall validation process. The laboratory must confirm that the reference intervals it uses are appropriate for its method and patient population. This verification may involve testing samples from healthy individuals or transferring reference intervals using established protocols.

Inadequate Operator Training

Method validation must include assessment of operator competence. A method that performs well in the hands of an experienced operator may perform poorly when used by someone with inadequate training. The malaria microscopy study demonstrated that operator performance varied across different tasks, with some tasks showing poor concordance among microscopists.

ISO 15189 requires laboratories to ensure that personnel are qualified for their assigned tasks. This requirement extends to method validation. The laboratory must document that operators have the necessary training and competence to perform the method correctly.

Biosafety and Regulatory Context

Biosafety Considerations in Method Validation

Method validation often involves handling biological materials, including patient samples, control materials, and potentially infectious agents. Laboratories must follow appropriate biosafety practices to protect personnel and the environment.

The CDC and NIH Biosafety in Microbiological and Biomedical Laboratories provides guidance on biosafety principles, risk assessment, and containment. This guidance applies to all laboratory activities, including method validation. Laboratories must conduct risk assessments for their validation procedures and implement appropriate containment measures.

The World Health Organization Laboratory Biosafety Manual provides additional international guidance on biosafety practices. Laboratories should consult this guidance when developing their biosafety procedures.

Recombinant and Synthetic Nucleic Acid Oversight

Laboratories working with recombinant or synthetic nucleic acid molecules must comply with additional oversight requirements. The NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules establish the framework for this oversight.

These guidelines apply to research activities, including method development and validation involving recombinant or synthetic nucleic acids. Laboratories must ensure that their activities comply with applicable requirements, including institutional biosafety committee review where required.

Regulatory Requirements for Diagnostic Methods

Diagnostic methods used in clinical laboratories may be subject to regulatory requirements in addition to ISO 15189. In the United States, the Food and Drug Administration regulates in vitro diagnostic devices. Laboratories that develop their own tests may be subject to different requirements than laboratories using commercially available assays.

Laboratory managers must understand the regulatory requirements that apply to their methods. This understanding informs the validation approach and the documentation required. The National Library of Medicine provides access to information on regulatory and scientific aspects of laboratory testing.

Professional Escalation Criteria

When to Escalate Validation Issues

Laboratory managers must know when to escalate validation issues to higher levels of the organization. Escalation is appropriate when validation results indicate a method is not suitable for clinical use, when corrective actions do not resolve identified problems, or when there is disagreement about the interpretation of validation data.

The decision to escalate should be based on documented evidence. The laboratory should have clear criteria for when a method is considered unacceptable and when issues require escalation. These criteria should be established before validation begins and applied consistently.

Documenting Escalation Decisions

Escalation decisions must be documented. The documentation should describe the issue, the evidence supporting the decision, the individuals involved, and the outcome. This documentation provides a record of the laboratory's decision-making process and supports accountability.

The Biochemia Medica study noted that validation activities may reveal technical issues that must be corrected to minimize impact on patient results. When such issues are identified, the laboratory must decide whether to correct the issue, modify the method, or discontinue use. These decisions should be documented and escalated as appropriate.

Involving Laboratory Leadership

Laboratory leadership should be involved in validation decisions that have significant implications for patient care or laboratory operations. This involvement ensures that decisions are made with appropriate oversight and that resources are allocated appropriately.

Leadership involvement is particularly important when validation results indicate that a method cannot meet acceptance criteria. In these situations, the laboratory must decide whether to invest additional resources in method optimization, seek an alternative method, or accept the method with documented limitations. These decisions have financial and clinical implications that warrant leadership attention.

At a Glance

Validation Workflow Decision Table

Common Failure Patterns in Implementation

Treating CLSI and ISO as Interchangeable

A significant failure pattern is treating CLSI EP25 and ISO 15189 as interchangeable frameworks. These documents serve different purposes and cannot substitute for one another. CLSI EP25 provides the technical protocol for precision evaluation, while ISO 15189 establishes the quality management framework within which all validation activities occur.

Laboratories that rely solely on CLSI EP25 may miss the broader requirements of ISO 15189, such as bias assessment, reference interval verification, and ongoing quality monitoring. Conversely, laboratories that focus only on ISO 15189 requirements may lack the detailed statistical guidance needed to conduct rigorous precision studies.

Incomplete Validation Documentation

Another common failure is incomplete documentation of validation activities. Some laboratories conduct the experimental work but fail to document the protocol, results, and conclusions adequately. This failure creates problems during accreditation assessments and makes it difficult to defend method performance.

Complete documentation should include the validation protocol, raw data, statistical calculations, acceptance criteria, and final conclusions. The documentation should also address any deviations from the protocol and the actions taken. The MIQE guidelines provide a model for the level of detail expected in scientific documentation.

Ignoring Biological Variation

Laboratories that use arbitrary acceptance criteria may approve methods that do not meet clinically meaningful performance standards. Biological variation provides a scientifically sound basis for defining allowable performance. The Biochemia Medica study demonstrated the application of biological variation criteria in method validation.

Laboratories should consider biological variation when establishing acceptance criteria for precision, bias, and total error. This approach ensures that method performance is evaluated against clinically meaningful standards instead of arbitrary thresholds.

Neglecting Operator Competence

Method validation that focuses exclusively on instrument performance may miss operator-related issues. The malaria microscopy study demonstrated that operator performance can vary significantly, even when using the same method and materials.

ISO 15189 requires laboratories to ensure that personnel are competent for their assigned tasks. Method validation should include assessment of operator competence, and ongoing competency assessment should be part of the laboratory's quality program.

Safety and Welfare Context

Protecting Laboratory Personnel

Method validation activities expose laboratory personnel to potential hazards, including biological agents, chemicals, and sharps. Laboratories must implement appropriate safety measures to protect personnel during validation activities.

The CDC and NIH Biosafety in Microbiological and Biomedical Laboratories provides guidance on risk assessment and containment. Laboratories should conduct risk assessments for validation procedures and implement appropriate controls, including personal protective equipment, engineering controls, and administrative controls.

Ensuring Sample Integrity

Method validation often involves testing patient samples or control materials. Laboratories must ensure that samples are handled appropriately to maintain their integrity. This includes proper collection, storage, and processing procedures.

Sample integrity is essential for obtaining valid validation data. If samples are degraded or contaminated, the validation results will not reflect true method performance. Laboratories must follow established procedures for sample handling throughout the validation process.

Maintaining Ethical Standards

Method validation must be conducted according to ethical standards. This includes obtaining appropriate approvals for the use of human samples, protecting patient confidentiality, and ensuring that validation activities do not compromise patient care.

Laboratories should have policies in place to address ethical considerations in method validation. These policies should be consistent with applicable regulations and professional standards.

Building a Practical Decision Framework for Method Validation

Laboratory managers often struggle to translate the requirements of CLSI EP25 and ISO 15189 into daily operational decisions. The documents describe what to do but not always how to choose between competing priorities when resources are limited. A practical decision framework helps laboratory managers determine which validation activities to perform, how deeply to investigate each performance characteristic, and when to accept or reject a method.

Tiered Validation Approach Based on Risk

A risk-based tiering system provides a structured way to allocate validation effort. This approach classifies methods into three tiers based on the clinical impact of errors and the maturity of the method.

Tier 1 methods are those with high clinical impact where errors could lead to serious patient harm. Examples include methods used for critical care decisions, therapeutic drug monitoring, and infectious disease diagnosis. These methods require full validation including precision studies, bias assessment, analytical sensitivity and specificity evaluation, and reference interval verification. The laboratory should conduct multiple precision studies across different concentration levels and operators to ensure robust performance estimates.

Tier 2 methods have moderate clinical impact where errors may affect patient management but are less likely to cause immediate harm. These methods require verification of manufacturer claims instead of full validation. The laboratory should conduct a precision study following CLSI EP25, verify bias using a limited number of samples, and confirm that reference intervals are appropriate for the local population.

Tier 3 methods have low clinical impact where errors rarely affect clinical decisions. These methods require minimal validation, typically a brief precision check and confirmation that the method produces results consistent with the manufacturer's specifications. The laboratory should document the rationale for assigning a method to this tier and review this classification periodically.

The Biochemia Medica study demonstrated that a structured validation procedure enables laboratories to manage multiple methods efficiently. The study validated 30 chemistry assays using a consistent approach, which allowed the laboratory to identify the three parameters that did not meet acceptance criteria and take corrective action before patient testing began.

Decision Points for Method Acceptance

The laboratory must establish clear decision points for determining whether a method is acceptable for clinical use. These decision points should be defined before validation begins and applied consistently throughout the process.

The first decision point occurs after the precision study. The laboratory compares the observed imprecision against the acceptance criteria. If the method meets the precision criteria, the laboratory proceeds to bias assessment. If the method fails the precision criteria, the laboratory must investigate the cause, which may include instrument problems, reagent issues, or operator error. The laboratory may repeat the study after corrective action or reject the method.

The second decision point occurs after bias assessment. The laboratory combines the precision and bias estimates to calculate total error. If the total error meets the acceptance criteria, the method proceeds to reference interval verification. If the total error exceeds the acceptance criteria, the laboratory must decide whether to optimize the method, accept the method with documented limitations, or reject the method.

The third decision point occurs after reference interval verification. The laboratory confirms that the reference intervals are appropriate for the local patient population. If the reference intervals are not appropriate, the laboratory must either establish new reference intervals or transfer reference intervals using established protocols. The survey from Nepal found that 87.5% of laboratories relied on manufacturer-provided reference intervals without local validation, which highlights the importance of this decision point.

Resource Allocation Matrix

Laboratories face competing demands for time, personnel, and materials. A resource allocation matrix helps laboratory managers plan validation activities and ensure that resources are directed to the highest priority methods.

The matrix should consider the number of methods requiring validation, the complexity of each method, the availability of personnel, and the time required for each validation activity. The laboratory should also consider the cost of control materials, calibrators, and other consumables needed for validation studies.

For laboratories with limited resources, the matrix can help identify opportunities to batch validation activities. For example, multiple methods using the same instrument platform may share precision study runs. The laboratory can also use the same control materials for multiple methods, reducing the cost and time required for validation.

The National Institute of Standards and Technology emphasizes that measurement quality depends on understanding and controlling all sources of variability. A resource allocation matrix helps laboratories achieve this understanding by ensuring that validation activities are conducted systematically and completely.

Escalation Criteria for Validation Failures

The laboratory must define clear criteria for escalating validation failures to laboratory leadership. Escalation is appropriate when a method fails to meet acceptance criteria, when corrective actions do not resolve identified problems, or when there is disagreement about the interpretation of validation data.

The escalation process should include a documented review of the validation data, a discussion of the options available, and a decision about the method's disposition. The laboratory should document the escalation, the individuals involved, and the outcome. This documentation provides a record of the laboratory's decision-making process and supports accountability.

The malaria microscopy study demonstrated that validation failures may relate to operator performance instead of instrument performance. The study found that microscopists obtained variable concordance across different tasks, with some tasks showing poor agreement. When validation failures relate to operator performance, the laboratory must address training and competency issues in addition to any instrument or method issues.

Integrating Validation Data into Ongoing Quality Monitoring

Validation data should not sit in a file after the validation is complete. The laboratory should use validation data to establish baseline performance for ongoing quality monitoring. This integration ensures that the laboratory can detect changes in method performance over time.

The precision estimates from the validation study inform the selection of quality control rules and limits. The laboratory should compare ongoing quality control data against the validation baseline to detect shifts or trends that may indicate developing problems. The laboratory should also review validation data periodically to confirm that the acceptance criteria remain appropriate for the method and its clinical use.

The MIQE guidelines emphasize the importance of transparency and documentation in scientific work. Clinical laboratory validation records should follow a similar principle, providing sufficient detail to allow others to evaluate the quality of the work and to support ongoing quality monitoring.

Practical Implementation Checklist

Laboratory managers can use the following checklist to implement a practical decision framework for method validation.

First, classify each method into a validation tier based on clinical impact and method maturity. Document the classification and the rationale for the assignment.

Second, define acceptance criteria for each performance characteristic before beginning validation. Use biological variation as the primary basis for acceptance criteria where available.

Third, select the appropriate CLSI protocols for each validation activity. EP25 addresses precision, while other protocols address bias, linearity, and interference.

Fourth, conduct the validation studies according to the selected protocols. Document all data, including raw data, statistical calculations, and any deviations from the protocol.

Fifth, evaluate the results against the acceptance criteria at each decision point. Document the evaluation and the decision reached.

Sixth, escalate any validation failures according to the laboratory's escalation criteria. Document the escalation and the outcome.

Seventh, integrate the validation data into ongoing quality monitoring. Use the precision estimates to establish quality control rules and limits.

Eighth, review the validation data periodically to confirm that the acceptance criteria remain appropriate and that the method continues to perform acceptably.

This checklist provides a practical structure for implementing the decision framework. The National Library of Medicine provides access to foundational texts on laboratory methods that can help laboratory managers understand the scientific basis for these activities and refine their implementation approach.

Frequently Asked Questions

What is the main difference between CLSI EP25 and ISO 15189?

CLSI EP25 is a technical protocol that specifies experimental designs and statistical calculations for evaluating measurement precision. ISO 15189 is a quality management standard that establishes requirements for all aspects of medical laboratory operations, including method validation and verification. Laboratories use CLSI EP25 to generate precision data that help satisfy ISO 15189 requirements.

Does ISO 15189 require laboratories to follow CLSI EP25?

ISO 15189 does not mandate specific CLSI protocols. The standard requires laboratories to validate or verify methods using appropriate procedures, but it does not prescribe which protocols to use. In practice, many laboratories use CLSI EP25 because it provides detailed, accepted guidance for precision evaluation.

How many runs and replicates does CLSI EP25 require?

CLSI EP25 provides guidance on minimum experimental designs for precision studies. The specific number of runs and replicates depends on the study design and the desired statistical confidence. Laboratories should follow the guidance in the current version of EP25 and document their experimental design.

What acceptance criteria should laboratories use for precision?

Acceptance criteria should be based on the clinical intended use of the method. Biological variation provides a scientifically sound basis for defining allowable imprecision. Laboratories may also use manufacturer claims or regulatory requirements as acceptance criteria, but these should be evaluated for clinical meaningfulness.

Can a laboratory use manufacturer precision data instead of conducting its own study?

For verification of a commercial method, a laboratory may compare its own precision data with manufacturer claims. However, the laboratory must still conduct its own precision study to confirm that the method performs acceptably in its setting. Manufacturer data cannot substitute for local verification.

What is the difference between validation and verification under ISO 15189?

Validation is the confirmation that a method meets specified requirements for its intended use, typically required for in-house methods or modified commercial methods. Verification is the confirmation that a method performs as claimed by the manufacturer, typically required for unmodified commercial methods. The distinction affects the scope of the evaluation required.

How does flexible scope accreditation affect method validation?

Flexible scope accreditation allows a laboratory to introduce new methods within defined categories without prior approval from the accreditation body. This approach requires the laboratory to have a well-documented validation procedure and to demonstrate competence in conducting validations. The Biochemia Medica study demonstrated that a laboratory with a structured validation procedure can successfully operate under a flexible scope.

What records must a laboratory maintain for method validation?

Laboratories must maintain records of the validation protocol, raw data, statistical calculations, acceptance criteria, and conclusions. Records should also document any deviations from the protocol and corrective actions taken. These records demonstrate compliance with ISO 15189 and support accreditation assessments.

Related Diagnostic Guides

• Method Validation in Clinical Laboratories: A Practical Protocol
• Laboratory Quality Management System: A Guide to ISO 15189 Accreditation
• ISO 17025 vs ISO 15189: Choosing the Right Quality Standard for Your Laboratory
• Laboratory Inspection Checklists: Ensuring Compliance with CAP and ISO Standards
• Laboratory Quality Management Systems: A Practical Implementation Framework for Diagnostic Laboratories

References and Further Reading

• Molecular Biology and Laboratory Methods. National Library of Medicine.
• Biosafety in Microbiological and Biomedical Laboratories. CDC and NIH.
• NIH Guidelines for Recombinant or Synthetic Nucleic Acid Molecules. National Institutes of Health.
• Addgene Protocols. Addgene.
• MIQE Guidelines. Clinical Chemistry.
• NIST Laboratory Programs. National Institute of Standards and Technology.
• Laboratory Biosafety Manual. World Health Organization.
• NCBI Literature Resources. National Center for Biotechnology Information.
• The importance of having a flexible scope ISO 15189 accreditation and quality specifications based on biological variation - the case of validation of the biochemistry analyzer Dimension Vista. Biochemia Medica, 2013.
• Quality assessment of malaria microscopic diagnosis at the Aristide Le Dantec University Hospital of Dakar, Senegal, in 2020. BMC Research Notes, 2024.
• Nationwide Survey on Knowledge, Attitudes, and Practices regarding Reference Interval Utilization in Clinical Laboratories in Nepal. EJIFCC, 2026.

This article is educational and does not replace validated laboratory procedures, institutional biosafety review, manufacturer instructions, or professional interpretation.