Does This Look Straight to You? Linearity Studies Explained

In the realm of clinical laboratory testing, ensuring the accuracy and reliability of test results is paramount. The Centers for Medicare & Medicaid Services (CMS) plays a crucial role in regulating laboratory testing through the Clinical Laboratory Improvement Amendments (CLIA). One key aspect of this regulation is the requirement for laboratories to conduct linearity studies, which are essential for validating the performance characteristics of quantitative assays. This summary explores the principles, methodologies, and significance of linearity studies as outlined by CMS and general good lab practice.

Linearities: So much confusion about a straight line.

What is Linearity?

Linearity refers to the ability of an analytical method to produce results that are directly proportional to the concentration of an analyte within a given range. In other words, a linear relationship between the measured values and the actual concentrations indicates that the assay can accurately quantify the analyte across its specified reportable range (also commonly known as the analytical range). Linearity studies (also referred to as Calibration Verification, or "Cal Ver") are performed to confirm this proportional relationship, ensuring that the assay provides reliable and reproducible results.

Importance of Linearity Studies

Linearity studies are critical for several reasons:

1. Validation of Assay Performance: They validate the accuracy and precision of the assay over a specified range of concentrations.

2. Regulatory Compliance: Compliance with CLIA and CMS guidelines requires documented evidence of assay linearity withing the reportable range.

3. Quality Assurance: They help in maintaining high standards of laboratory practice by ensuring consistent and accurate test results.

4. Troubleshooting and Calibration: Identifying and correcting non-linearity issues ensures that the assay remains reliable.

What to Use for Linearities?

There are specially made linearity materials that can be used for studies. These often have their own statistical analysis software available too. The only drawback is the expense of these materials.

To do a home cooked version, serial dilutions of pre-analyzed standards can be used. But you will need to do your own analysis.

Are you hitting your target? Linearity studies can help.

CMS Guidelines on Linearity Studies: what are the actual requirements?

CMS, through CLIA, provides specific guidelines for performing linearity studies. These guidelines ensure that laboratories follow a standardized approach, maintaining the integrity and reliability of test results.

Frequency of Linearity Studies

According to CMS guidelines, the frequency of linearity studies depends on several factors, including the type of assay, changes in the testing system, and the laboratory's quality control policies. Key points regarding the frequency of linearity studies include:

1. Initial Validation: Linearity studies must be performed during the initial validation of a new assay or instrument. This is sometimes referred to as the verification of the reportable range.

2. Periodic Verification: CMS recommends periodic verification of linearity, typically on a six-month basis, to ensure ongoing accuracy and reliability.

3. Changes in Testing System: Linearity studies should be conducted whenever there are significant changes in the testing system, such as new reagents, major instrument maintenance, or software updates.

4. Quality Control Failures: If quality control results indicate a potential issue with linearity, additional studies should be performed to investigate and rectify the problem.

When do I NOT have to do Linearity Studies (aka CalVers)?

Factory calibrated analyzers do not need linearities after the initial study. This pertains to many point-of-care (POC) systems. Of course, they may be done if deemed helpful.

Analytes that have a three-or-more-point calibrator that is run at least every six months. For this to satisfy the requirements, the high and low calibrators need to have values near the highs and lows of your reportable range.

Qualitative assays do not need linearity studies.

If you are not sure if you need to run linearities, ask the manufacturer.

Steps for Doing Linearity Studies

Defining the Range

Before conducting a linearity study, it is essential to define the range over which the assay is expected to be linear. This range should encompass the lower and upper limits of the analyte concentrations that the assay will measure in clinical practice.

Preparation of Standards

Linearity studies typically involve the use of standards or reference materials with known concentrations of the analyte. These standards should cover the entire defined range and include multiple points to adequately assess linearity. These can be specially ordered.

Replicate Measurements

For each concentration point, multiple replicate measurements are performed to ensure accuracy and reproducibility. The number of replicates can vary, but CMS recommends a minimum of three replicates per concentration.

Data Analysis

The results from the linearity study are plotted on a graph, with the measured values on the y-axis and the known concentrations on the x-axis. A linear regression analysis is performed to assess the linearity of the assay. Key parameters to evaluate include:

• Slope: Indicates the rate of change of the measured values with respect to the concentration.

• Intercept: Represents the point where the regression line intersects the y-axis.

• Correlation Coefficient (R²): A measure of how well the data fit the linear model, with values close to 1 indicating strong linearity

And finally, the laboratory director must sign off on the studies so that they may be accepted.

Troubleshooting

If linearity studies fail, attempt to re-calibrate and re-run the samples. If you can't get the assay to pass linearities for your defined range, the reportable range may be adjusted to account for only the points that passed linearity testing. Consult with your Laboratory Medical Director if you would like to amend the ranges. Just make sure you have a range that includes important clinical decision points.

Conclusion

Linearity studies are a fundamental component of clinical laboratory quality assurance, ensuring the accuracy and reliability of quantitative test results. CMS guidelines provide a framework for conducting these studies, specifying the frequency and the analytes that require them. By adhering to these guidelines, laboratories can maintain high standards of analytical performance, contributing to better patient care and compliance with regulatory requirements.

References

• Centers for Medicare & Medicaid Services. (n.d.). Clinical Laboratory Improvement Amendments (CLIA).

• Clinical and Laboratory Standards Institute (CLSI). (2014). EP06-A: Evaluation of the Linearity of Quantitative Measurement Procedures.

• U.S. Food and Drug Administration (FDA). (2001). Guidance for Industry: Bioanalytical Method Validation.