Method Validation

Overview of Method Validation

Definition: Method validation is the process of demonstrating that an analytical procedure is suitable for its intended purpose. It is an essential step in ensuring the accuracy, reliability, and reproducibility of laboratory results
Purpose:
- Ensure Accuracy and Reliability: To confirm that the assay is measuring what it is supposed to measure and is providing consistent results
- Meet Regulatory Requirements: To comply with regulatory requirements and accreditation standards
- Support Clinical Decision-Making: To provide reliable data that can be used to make informed clinical decisions
Key Validation Characteristics:
- Accuracy
- Reproducibility/Precision
- Sensitivity
- Specificity
- Linearity
- Reference Range
- Robustness

Definition: The closeness of agreement between the result obtained by the method and the true value of the analyte
Assessment:
- Use Reference Materials: Analyze certified reference materials (CRMs) or standard reference materials (SRMs) with known values for the analyte
- Compare to a Gold Standard Method: Compare the results of the new method to those obtained using a well-established gold standard method
- Spike Recovery: Add a known amount of the analyte to a sample and measure the recovery
Acceptance Criteria:
- The mean value obtained by the method should be within a specified percentage of the true value (e.g., ±10%)
- The recovery of the analyte should be within a specified range (e.g., 90-110%)
Mathematical formula:
- Percent recovery = (measured concentration/ true concentration) * 100
Use at least 3 concentration points to determine accuracy

Definition: The closeness of agreement between independent test results obtained under stipulated conditions
Types of Precision:
- Repeatability (Intra-Assay Precision): The precision of the method when repeated within a single run
- Intermediate Precision (Inter-Assay Precision): The precision of the method when repeated on different days, by different operators, or using different instruments
- Reproducibility (Inter-Laboratory Precision): The precision of the method when performed in different laboratories
Assessment:
- Analyze Multiple Replicates: Analyze multiple replicates of the same sample within a single run and across multiple runs
- Calculate Standard Deviation (SD) and Coefficient of Variation (CV): Use SD and CV to quantify the variability of the results
Acceptance Criteria:
- The CV for repeatability should be within a specified range (e.g., <5%)
- The CV for intermediate precision should be within a specified range (e.g., <10%)
Use the same material over multiple days
The experimenter should use the same instrument

Definition: The ability of the method to detect small changes in the concentration of the analyte
Assessment:
- Limit of Detection (LOD): The lowest concentration of the analyte that can be detected but not necessarily quantified
- Limit of Quantitation (LOQ): The lowest concentration of the analyte that can be quantified with acceptable accuracy and precision
- Serial Dilutions: Analyze a series of serial dilutions of the analyte to determine the LOD and LOQ
Acceptance Criteria:
- The LOD should be below a specified level
- The LOQ should be within a specified range
Use a series of low concentration samples
The results should be compared to a blank sample

Definition: The ability of the method to measure only the analyte of interest without interference from other substances
Assessment:
- Analyze Samples Containing Potential Interferents: Analyze samples that contain substances that may interfere with the assay
- Compare Results to a Reference Method: Compare the results of the new method to those obtained using a reference method that is known to be specific for the analyte of interest
Acceptance Criteria:
- The method should not be significantly affected by the presence of potential interferents
- The results obtained by the new method should correlate well with those obtained by the reference method
Analyze a sample of the test substance only

Definition: The ability of the method to produce results that are directly proportional to the concentration of the analyte
Assessment:
- Analyze a series of samples with known concentrations of the analyte that span the expected range
- Plot the measured values against the known concentrations
- Calculate the correlation coefficient (r) and the slope of the line
Acceptance Criteria:
- The correlation coefficient should be close to 1 (e.g., >0.99)
- The slope of the line should be close to 1
Linearity should be performed at least in triplicate with at least 5 concentration points

Definition: The range of values that are expected to be found in healthy individuals
Establishment:
- Analyze Samples from a Representative Population: Analyze samples from a representative population of healthy individuals
- Determine the Mean and Standard Deviation: Calculate the mean and standard deviation of the results
- Establish the Reference Range: The reference range is typically defined as the mean ± 2 standard deviations
Considerations:
- Sample Size: A sufficient number of samples should be analyzed to establish a reliable reference range (e.g., >120 individuals)
- Demographics: The reference range may vary depending on age, sex, ethnicity, and other demographic factors
A large sample size will provide the most accurate reference range

Definition: The ability of the method to withstand small, deliberate variations in the experimental conditions
Purpose:
- Assess Procedure Ruggedness: Evaluate if small changes to procedure impacts results
Parameters to Manipulate:
- Incubation Time: Vary the incubation time by a small amount (e.g., ±10%)
- Temperature: Vary the temperature by a small amount (e.g., ±2°C)
- Reagent Volume: Vary the reagent volume by a small amount (e.g., ±5%)
What to look for:
- The method should still provide acceptable results even when small variations in the experimental conditions are introduced

Detailed Protocol:
- Create a detailed protocol for the method that includes all of the steps, reagents, and equipment required
Validation Report:
- Prepare a validation report that summarizes the results of the validation experiments
- The validation report should include all of the data, calculations, and conclusions
SOPs:
- Prepare Standard Operating Procedures (SOPs) for performing the method and for analyzing the data
- The SOPs should be clear, concise, and easy to follow

Failure to Meet Acceptance Criteria:
- Possible Causes:
  - Errors in the method
  - Suboptimal reagents
  - Instrument malfunction
- Troubleshooting Steps:
  - Review the method for errors
  - Replace suspect reagents
  - Calibrate instrument
Inconsistent Results:
- Possible Causes:
  - Lack of precision
  - Inadequate controls
- Troubleshooting Steps:
  - Standardize all measurements
  - Review and evaluate control results

Method Validation: Demonstrating that an analytical procedure is suitable for its intended purpose
Accuracy: Closeness of agreement between the result and the true value
Precision: Closeness of agreement between independent test results
Sensitivity: Ability to detect small changes in the concentration of the analyte
Specificity: Ability to measure only the analyte of interest
Linearity: Ability to produce results that are directly proportional to the concentration of the analyte
Reference Range: Range of values expected in healthy individuals
Robustness: Ability to withstand small variations in the experimental conditions