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Determination of optimal replicate number for validation of imprecision using fluorescence cell based assays:
Bruce H Davis1, Christine E McLaren, Anthony J Carcio
1Trillium Diagnostics, LLC, 4 Union Street, Bangor, Maine, 04401, USA.
Cytometry. Part B, Clinical Cytometry
|July 23, 2013
Summary
Practical guidelines for flow cytometer precision testing are needed. This study found 3-4 replicates are sufficient for most cell-based assays, reducing validation time and cost.
Area of Science:
- Clinical laboratory science
- Flow cytometry
- Assay validation
Background:
- Assay validation requires determining imprecision across the reportable range.
- Specific guidelines for cell-based flow cytometry precision are lacking.
Purpose of the Study:
- To establish practical guidelines for determining precision in cell-based flow cytometry assays.
- To identify optimal replicate numbers for assay validation.
Main Methods:
- Flow cytometric assays (CD64, fetal RBC, hENT1, CD34+ HSC) were run on multiple platforms.
- Replicates (10-20) were analyzed for mean, 95%CI, SD, and CV.
- A novel 'variance factor' (VF) was derived to determine optimal replicates.
Main Results:
- Fewer than 5 replicates were adequate for most assays to meet <5-10% CV repeatability.
- The variance factor (VF) provided a data-driven method for selecting replicate numbers.
- Optimal replicate numbers varied by assay and instrument platform.
Conclusions:
- The novel variance factor (VF) aids in selecting optimal replicates for flow cytometry precision validation.
- 3-4 replicates are generally sufficient for flow cytometric assays, fewer than CLSI guidelines for soluble analytes.
- This simplifies and optimizes the assay validation process.
