Differential Leukocyte Counting via Fluorescent Detection and Image Processing on a Centrifugal Microfluidic Platform

Area of Science:

• Biomedical Engineering
• Microfluidics
• Point-of-Care Diagnostics

Background:

• Centrifugal microfluidics enables automated point-of-care blood testing for various analytes.
• Detecting common blood cells, especially leukocytes, on-disc remains a significant challenge in microfluidic platforms.

Purpose of the Study:

• To develop and validate analytical methods for enumerating leukocytes on a centrifugal microfluidic platform.
• To assess the feasibility of performing a three-part differential white blood cell count using this platform.

Main Methods:

• Utilized centrifugal microfluidics with two configurations: glass capillary tubes and acrylic chips.
• Employed acridine orange nuclear staining, a custom-built fluorescent microscope, and image processing for cell analysis.
• Implemented a bulk-cell analysis approach correlating fractionated cell layer pixel areas with cell counts.

Main Results:

• Achieved strong linear correlations between measured cell counts and pixel areas using both glass capillary and acrylic chip methods.
• Demonstrated the ability to differentiate lymphocytes from monocytes via pixel intensity clustering, enabling a three-part differential.
• Validated methods using porcine blood samples and generated standard curves.

Conclusions:

• The developed methods offer a viable approach for on-disc leukocyte enumeration and differentiation using centrifugal microfluidics.
• This technology has the potential to advance point-of-care blood testing capabilities for white blood cell analysis.
• The bulk-cell analysis approach presents specific advantages and disadvantages that warrant further discussion for practical implementation.