Quantification methods for human and large animal leukocytes using DNA dyes by flow cytometry.

Area of Science:

• Immunology
• Biotechnology
• Cell Biology

Background:

• Blood cell damage monitoring is crucial for in vitro testing of blood-handling medical devices like left ventricular assist devices (LVADs).
• Hematology analyzers provide cell counts but lack cell viability information.
• Anthraquinone DNA dyes CyTRAK Orange and DRAQ7 offer suitable properties for multicolor assays and improved staining protocols compared to existing dyes.

Purpose of the Study:

• To evaluate the combined use of CyTRAK Orange and DRAQ7 for flow cytometry-based viability and cell counting in large animal blood (bovine, ovine) and human leukocytes.
• To establish a protocol for viability and cell counting applicable to different species.

Main Methods:

• A protocol for viability and cell counting of bovine, ovine, and human leukocytes using CyTRAK Orange and DRAQ7 was developed.
• Four different absolute counting bead standards were assessed using Navios and FACSAria flow cytometers.
• Results were compared against counts from hematology analyzers.

Main Results:

• CyTRAK Orange effectively detected CD45(+) leukocytes across all tested species.
• DRAQ7 single-stained and CyTRAK Orange/DRAQ7 double-stained dead cell counts correlated well in human and bovine blood.
• A discrepancy was observed in ovine blood, potentially due to the blood source.

Conclusions:

• The combined use of CyTRAK Orange and DRAQ7 with absolute counting bead standards is effective for viability and cell counting in human and bovine blood using flow cytometry.
• This method offers a viable alternative for assessing cell viability in large animal blood samples.