Differential leukocyte counting via fluorescent detection and image processing on a centrifugal microfluidic platform

Abstract

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 analytical methods for enumerating leukocytes on a centrifugal platform using a custom-built fluorescent microscope, acridine orange nuclear staining, and image processing techniques. 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.

Automated end-to-end blood testing at the point-of-care: Integration of robotic phlebotomy with downstream sample processing

“…Specifically, with a lower number of beads (or cells) in the sample, a thinner buffy coat is formed after centrifugation, and thus it is more difficult to segment and quantify the buffy coat area. In previous work, we demonstrated feasibility of our blood analysis approach using porcine blood samples with clinically relevant cell concentrations 27 .…”

“…After centrifugation, the reservoir holds the packed red blood cells, and a band of white cells sits atop this layer in the detection channel. To image the white cell layer, a miniaturized fluorescent microscope was developed 27 . After the white cell layer is detected by the system and brought into focus, an image processing step is performed and the thickness of the white cell region is quantified.…”

“…Following the robotically guided venipuncture, the blood sample is then extracted and transferred to the analysis unit, which provides blood measurements on sample volumes of 25 μ l using microcentrifugation and optical detection. Currently, the device is aimed at performing key components of the complete blood count — specifically, a 3-part white blood cell differential and hemoglobin measurement 27 .…”

Microfluidic Adaptation of Density-Gradient Centrifugation for Isolation of Particles and Cells

“…Al-Faqheri et al present an excellent review summarizing centrifugal force-based microfluidic efforts for cell separations [ 24 ]. Other works of importance to the method discussed in this paper include a manuscript by Balter et al who used centrifugal microfluidics to label and count leukocyte populations [ 25 ], a manuscript by Yu et al where they use centrifugal forces to drive fluid flow and accomplish leukocyte capture on immuno-modified surfaces [ 26 ], Ramachandraiah et al [ 27 ] developed a lab-on-a-DVD for labeling and counting of CD4 + cells, a centrifugally driven immunoassay where antibody-coated beads are transported via centrifugal forces and an ELISA-like readout is used to facilitate accurate dosing of VEGF [ 28 ], Schaff et al [ 29 ] developed an immunoassay using centrifugal microfluidics for evaluation of biomarkers in blood, and another manuscript by Zhang et al [ 30 ] where a centrifugal microfluidic platform was used to separate plasma from the blood cells and used to separate plasma and determine hematocrit. There are also few examples of density-gradient centrifugation using miniaturized platforms.…”