Method Validation in Clinical Laboratories
A comprehensive guide to validation and verification of quantitative, qualitative, and semi-quantitative examination methods according to ISO 15189:2022 and international recommendations.
What is Method Validation?
Method validation is the process of confirming, through the provision of objective evidence, that a method is fit for its intended purpose. In clinical laboratories, it ensures that an examination procedure produces results that are accurate, reliable, and clinically useful before it is used for patient testing.
ISO 15189:2022 requires laboratories to validate methods that they develop or modify, and to verify methods that are already validated by the manufacturer (commercial methods) before introducing them into routine use.
Validation vs Verification
| Aspect | Validation | Verification |
|---|---|---|
| Definition | Confirming that a method is suitable for its intended use (usually for laboratory-developed or modified methods) | Confirming that a manufacturer-validated method performs as claimed under the laboratory’s own conditions |
| When required | New methods, modified methods, laboratory-developed tests (LDTs) | Commercial kits and methods already validated by the manufacturer |
| Extent | More extensive performance studies | Usually limited to key performance characteristics |
Types of Examination Methods
Method validation/verification requirements differ according to the nature of the test:
1. Quantitative Methods
These methods produce numerical results (e.g., glucose, creatinine, haemoglobin, TSH, viral load). Performance characteristics typically evaluated include:
- Trueness / Bias
- Precision (repeatability and intermediate precision)
- Linearity / Measuring interval
- Limit of Detection (LoD) and Limit of Quantitation (LoQ)
- Analytical specificity / Interference
- Reference interval verification (when applicable)
- Measurement uncertainty (where relevant)
2. Qualitative Methods
These methods give binary or categorical results such as Positive/Negative, Reactive/Non-reactive, Detected/Not detected (e.g., many infectious disease serology tests, some molecular assays, urine pregnancy tests). Key performance characteristics include:
- Diagnostic sensitivity
- Diagnostic specificity
- Positive and Negative Predictive Values (where relevant)
- Limit of Detection (analytical sensitivity)
- Cross-reactivity / Analytical specificity
- Robustness and reproducibility
3. Semi-Quantitative Methods
These methods produce results in categories or titres (e.g., urine dipstick, ANA titres, some agglutination tests). Evaluation usually combines elements of both quantitative and qualitative approaches, focusing on:
- Agreement with a reference method or predicate method
- Precision around clinical decision points
- Limit of detection / cut-off verification
Key Performance Characteristics Explained
Trueness (Bias)
The closeness of agreement between the average value obtained from a large series of test results and an accepted reference value. Assessed by comparison with a reference method, certified reference materials, or recovery studies.
Precision
The closeness of agreement between independent test results obtained under stipulated conditions.
- Repeatability – same operator, same instrument, short time interval
- Intermediate precision – different operators, different days, different calibrations (within-laboratory)
- Reproducibility – different laboratories (usually assessed in multi-centre studies)
Linearity / Measuring Interval
The ability of a method to give results that are directly proportional to the concentration of the analyte. Laboratories must verify that the method is linear across the clinically relevant measuring range.
Limit of Detection (LoD) and Limit of Quantitation (LoQ)
- LoD – lowest concentration that can be reliably distinguished from blank (detected but not necessarily quantified accurately)
- LoQ – lowest concentration that can be quantitatively determined with acceptable precision and accuracy
Analytical Specificity / Interference
The ability of the method to measure only the intended analyte without interference from other substances (haemolysis, icterus, lipaemia, drugs, related compounds, heterophile antibodies, etc.).
Diagnostic Sensitivity and Specificity (Qualitative Tests)
- Diagnostic sensitivity – ability to correctly identify true positives
- Diagnostic specificity – ability to correctly identify true negatives
Practical Approach to Method Validation / Verification
- Define the intended use of the method and the clinical requirements.
- Select performance characteristics to be evaluated based on the type of method (quantitative / qualitative / semi-quantitative).
- Prepare a validation/verification protocol including acceptance criteria.
- Perform the experiments using appropriate samples (patient samples, control materials, reference materials, spiked samples).
- Analyse the data using statistical methods (regression, difference plots, precision profiles, contingency tables, etc.).
- Compare results against acceptance criteria.
- Document the study comprehensively (protocol, raw data, analysis, conclusions, approval).
- Train staff and implement the method only after successful validation/verification and authorization.
Setting Acceptance Criteria
Acceptance criteria should be based on:
- Clinical requirements and allowable total error
- Manufacturer’s claims (for verification studies)
- Professional recommendations (CLSI, IFCC, EQAS data)
- Biological variation (for some analytes)
- Regulatory or accreditation body expectations
Documentation Requirements
A complete validation/verification file should typically contain:
- Purpose and scope of the study
- Description of the method and equipment
- Protocol with acceptance criteria
- Sample details and experimental design
- Raw data and statistical analysis
- Summary of results against acceptance criteria
- Conclusions and statement of fitness for purpose
- Approval by authorized personnel
- Any limitations or special considerations
ISO 15189:2022 Requirements Related to Method Validation
Key clauses include:
- The laboratory shall validate methods that it develops or modifies, or that are used outside their intended scope.
- The laboratory shall verify that it can properly perform manufacturer-validated methods before introducing them into routine use.
- Performance specifications shall be related to the intended use of the examination results.
- Validation/verification shall be as extensive as necessary to meet the needs of the given application or field of application.
- Records of validation/verification shall be retained.
Summary
Method validation and verification are essential activities that ensure examination procedures are fit for their intended clinical purpose. The extent of the study depends on whether the method is laboratory-developed, modified, or manufacturer-validated, and on whether the method is quantitative, qualitative, or semi-quantitative.
A well-planned and properly documented validation/verification study provides confidence that patient results will be reliable and clinically meaningful, and supports compliance with ISO 15189:2022 and other accreditation requirements.
- ISO 15189:2022 – Medical Laboratories Requirements
- What is Quality Control in Clinical Laboratories?
- Westgard Rules in Clinical Laboratory Quality Control
- Measurement Uncertainty in Laboratory Medicine
Educational Disclaimer: This article is intended for educational and professional reference purposes. Laboratories should design validation and verification studies according to ISO 15189:2022, CLSI guidelines, manufacturer instructions, and their own quality management system requirements.
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