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Flow rate calibration I: a novel approach for performing absolute cell counts
Ian Storie1, Alex Sawle, Karen Goodfellow
1UK NEQAS for Leucocyte Immunophenotyping, Royal Hallamshire Hospital, Sheffield, United Kingdom.
Cytometry. Part B, Clinical Cytometry
|September 2, 2003
Summary
Flow rate calibration (FRC) enables accurate absolute leucocyte counting. This method, using a simple calibration procedure and lysing solution calibration factor, provides results comparable to established methods.
Area of Science:
- Immunology
- Hematology
- Analytical Chemistry
Background:
- Flow rate (FR) is reported as constant in benchtop flow cytometers.
- Constant FR implies consistent volume acquisition, enabling absolute leucocyte counting via flow rate calibration (FRC).
Purpose of the Study:
- To evaluate the feasibility of FRC for absolute leucocyte enumeration.
- To establish a lysing solution calibration factor (CF) for FRC.
Main Methods:
- FR stability was assessed using TruCount beads on a FACSCalibur.
- A CF was determined for two lysing solutions.
- Absolute CD4(+) T-lymphocyte counts were compared between FRC and the predicate TruCount/MultiTEST method.
Main Results:
- FRC demonstrated high linearity across a wide range of CD4(+) T-lymphocyte counts.
- FRC results showed no significant difference from the predicate method when using the appropriate lysing solution CF.
Conclusions:
- FRC is a viable and easily implementable method for absolute leucocyte enumeration.
- Incorporating a lysing solution CF is crucial for accurate FRC results.