Calibration of a flow cytometer against a microphotometer for morphologic cell identification

Area of Science:

• Biomedical Engineering
• Cell Biology
• Analytical Chemistry

Background:

• Flow cytometry is a powerful tool for cell analysis.
• Correlating cell morphology with fluorescence intensity is crucial for accurate subpopulation identification.
• Current methods may require specialized equipment like cell sorters.

Purpose of the Study:

• To describe a method for calibrating flow cytometers using microphotometers.
• To establish a correlation between cell morphology and fluorescence intensity.
• To provide an alternative to cell sorting for cell subpopulation identification.

Main Methods:

• Calibration of a flow cytometer against a microphotometer.
• Utilizing three human lymphoblastoid cell lines.
• Optimizing photomultiplier amplification for linearity between fluorescence intensity and DNA content.
• Comparing fluorescence intensity profiles from both instruments.

Main Results:

• Optimal linearity was established between fluorescence intensity and DNA content for both instruments.
• Satisfactory linear agreement was observed between flow cytometer and microphotometer fluorescence intensity profiles.
• Minimal day-to-day variation was achieved.
• The procedure proved effective for morphologic identification of cell subpopulations.

Conclusions:

• The described calibration procedure allows for the correlation of cell morphology with fluorescence intensity.
• This method offers a viable alternative to cell sorting for identifying cell subpopulations in flow cytometric analysis.
• The approach enhances the diagnostic capabilities of flow cytometry.