[Analytical Methods] Differential leukocyte counting via fluorescent detection and image processing
Centrifugal microfluidics has received much attention in the last decade for the automation of blood testing at the point-of-care, specifically for the detection of chemistries, proteins, and nucleic acids. However, the detection of common blood cells on-disc, particularly leukocytes, remains a challenge. In this paper, we present two methods for enumerating leukocytes on a centrifugal platform using a custom-built fluorescent microscope, nuclear staining, and image processing. In the first method, cell analysis is performed in glass capillary tubes; in the second, acrylic chips are used. A bulk-cell analysis approach is implemented in both cases where the pixel areas of fractionated lymphocyte/monocyte and granulocyte layers are correlated with cell counts. Generating standard curves using porcine blood sample controls, we observed strong linear fits to measured cell counts using both methods. Analyzing the pixel intensities of the fluorescing white cell region, we are able to differentiate lymphocytes from monocytes via pixel clustering, demonstrating the capacity to perform a 3-part differential. Finally, a discussion of pros and cons of the bulk-cell analysis approach concludes the paper.
Max L. Balter,*a Alvin I. Chen,a C. Amara Colinco,a Alexander Gorshkov,a Brian Bixon,a Vincent Martin,a Alexander Fromholtz,a Timothy J. Maguireb and Martin L. Yarmushac Author affiliations * Corresponding authors a Rutgers University, Department of Biomedical Engineering, Piscataway, USA E-mail: firstname.lastname@example.org Fax: +1-732-445-3753 Tel: +1-617-721-2064 b VascuLogic LLC, Piscataway, USA c Massachusetts General Hospital, Boston, USA