Evaluation of a flow cytometric method for simultaneous leukocyte phenotyping and quantification by fluorescent...

Area of Science:

• Hematology
• Immunology
• Biotechnology

Background:

• Accurate cell quantification is crucial for clinical diagnostics and research.
• Traditional cell counting methods can be labor-intensive and prone to cell loss.
• Flow cytometry is a powerful tool for cell analysis but often requires separate quantification steps.

Purpose of the Study:

• To introduce and evaluate a novel flow cytometric method for precise single-cell quantification.
• To assess the accuracy and reproducibility of this method using fluorochrome-containing microspheres.
• To demonstrate the method's utility in conjunction with immunophenotyping without additional sample preparation.

Main Methods:

• Development of fluorochrome-containing microspheres (Flow Count fluorospheres) for cell quantification.
• Utilizing flow cytometry to detect and distinguish microspheres from cellular events based on light-scattering and fluorescence.
• Evaluating the method's performance by determining CD45+ leukocyte counts in 48 blood samples and comparing with conventional techniques.
• Assessing cell and microsphere sedimentation rates during analysis.

Main Results:

• The flow cytometric method accurately quantifies cells at a single-cell level.
• Results for leukocyte counts were reliable and comparable to conventional methods.
• The method demonstrated a limit of detection for white blood cell concentration down to 100 cells/microL.
• No additional cell loss or preparation steps were required when combined with immunophenotyping.

Conclusions:

• The developed flow cytometric method offers a precise, accurate, and reproducible means of cell quantification.
• This technique simplifies cell analysis by enabling simultaneous quantification and immunophenotyping.
• The method has broad applicability in clinical decision-making, including monitoring HIV patients and optimizing stem cell transplantation.