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Assessing immunophenotyping performance: proficiency-validation for adopting improved flow cytometry methods
Lance E Hultin1, Frederick A Menendez, Patricia M Hultin
1Department of Medicine, Cellular Immunology and Cytometry, University of California, Los Angeles, California, USA.
Insights
Implementing new flow cytometry methods for T-cell analysis can be streamlined. Evaluating performance using proficiency testing panels offers an alternative to repeated "switch" studies, accelerating adoption of superior techniques.
Area of Science:
- Immunology
- Laboratory Science
- Clinical Diagnostics
Background:
- Advancing immune cell phenotyping necessitates regular updates to laboratory methods and technology.
- National Institute of Allergy and Infectious Diseases (NIAID)-sponsored research protocols require flow cytometry laboratories to conduct "switch" studies to validate new T-cell subset analysis methods.
Purpose of the Study:
- To evaluate the impact of adopting a new 3-color, lyse no-wash flow cytometry method in two Multicenter AIDS Cohort Study (MACS) laboratories.
- To assess alternatives to repeating failed "switch" studies for method validation.
Main Methods:
- Comparison of a 2-color, lyse-wash method with a 3-color, lyse no-wash method across four MACS flow cytometry laboratories.
- Evaluation of performance using the Immunology Quality Assurance (IQA) proficiency-testing program for laboratories that adopted the new method after failing initial switch studies.
Main Results:
- Laboratory performance at the two sites significantly improved after adopting the new method, as evidenced by IQA standard test results.
- Reduced variation was observed across the four MACS sites and among replicate samples with the new method.
Conclusions:
- Alternative validation strategies to repeating failed "switch" studies should be considered for adopting new laboratory methods.
- Testing the new method against a proficiency testing reference panel or demonstrating superior performance on reference samples with both old and new methods are viable alternatives.
- These alternatives can expedite the transition to superior methodologies, bypassing the need for multiple, serial "switch" studies.
Background:
The continuous improvement and evolution of immune cell phenotyping requires periodic upgrading of laboratory methods and technology. Flow cytometry laboratories that are participating in research protocols sponsored by the NIAID are required to perform "switch" studies to validate performance before methods for T-cell subset analysis can be changed.
Methods:
Switch studies were conducted among the four flow cytometry laboratories of the Multicenter AIDS Cohort Study (MACS), comparing a 2-color, lyse-wash method and a newer, 3-color, lyse no-wash method. Two of the laboratories twice failed to satisfy the criteria for acceptable differences from the previous method. Rather than repeating more switch studies, these laboratories were allowed to adopt the 3-color, lyse no-wash method. To evaluate the impact of the switch to the new method at these two sites, their results with the new method were evaluated within the context of all laboratories participating in the NIH-NIAID-Division of AIDS Immunology Quality Assurance (IQA) proficiency-testing program.
Results:
Laboratory performance at these two sites substantially improved relative to the IQA standard test results. Variation across the four MACS sites and across replicate samples was also reduced.
Conclusions:
Although switch studies are the conventional method for assessing comparability of laboratory methods, two alternatives to the requirement of repeating failed switch studies should be considered: (1) test the new method and assess performance on the proficiency testing reference panel, and (2) prior to adoption of the new methods, use both the old and the new method on the reference panel samples and demonstrate that performance with the new method is better according to standard statistical procedures. These alternatives may help some laboratories' transition to a new and superior methodology more quickly than if they are required to attempt multiple, serial switch studies.
