Fig 1.
Physical configuration of the microfluidic disk.
(A) Each sample disk has eight lanes with a 60 mm radius. (B) Images of a loaded (top) and post-centrifugation (bottom) sample lane. (C) Sample lane shown with alternating colors of food coloring to demonstrate the ability of the capillary valves to keep sections separated prior to centrifugation. (D) Schematic channel view with examples of partition constituents following blood separation. Partition A is where the majority of red blood cells settle. Partitions B, C, and D are relatively cell-free areas, while partition E is contains the vast majority of leukocytes. Images taken on Nikon TI inverted microscope (40X DIC) and auto white balanced using the NIS Elements AR 4.12.01 software.
Fig 2.
Total time of centrifugation for our target cell as predicted by Eq 3.
Using the Histopaque® 1077 gradient, with an angular velocity of 500 RCF, we can predict the time it takes for RBCs, lymphocytes, monocytes, basophils, eosinophils, and neutrophils to reach various sections in our sample lane. Section F is where the sample is introduced and section A is most distal.
Fig 3.
Location of blood components post-centrifugation.
(A) Location and quantity of leukocytes after 4-min separation in density media as counted on disk via microscopy (figure is representative of n = 3). Background image denotes the orientation of the lane and location of the capillary valves. (B) The number of red blood cells and leukocytes extracted from section E, section C, and section A and counted with a Neubauer chamber. Results from three assay disks are shown to demonstrate disk-to-disk reproducibility. (WBCs are significantly enriched in section E versus section A, two-way ANOVA ** = p<0.05; RBC’s are significantly enriched in section A versus section E, * = p<0.05.)
Fig 4.
Typical field of view (FOV) for cell quantitation (Nikon TI 40X DIC).
Images of the disk can be used for direct cell enumeration. A TIFF format picture was taken of each FOV using a Nikon TI inverted microscope (1,800 FOVs/sample lane. This photo shows one such FOV in section E, where the predominant visualized cell type is the leukocyte. Inset shows a monocyte that was removed from a disk post- centrifugation and cultured until a macrophage phenotype was seen. Images taken on Nikon TI inverted microscope (40X DIC) and auto white balanced using the NIS Elements AR 4.12.01 software.
Table 1.
The specific densities and effective radii of common blood components.
Fig 5.
Sample processing time for our miniaturized centrifugal platform versus traditional methods.
(A) Comparison of protocol times for our miniaturized centrifugal platform and their corresponding traditional methods, in minutes. In both methodologies, buffy coat separation and modified Wright-Giemsa stain, the miniaturized centrifugal protocols were significantly shorter. (B) Modified Wright-Giemsa stained blood cells. Clockwise from bottom left: enriched cell FOV, basophil, neutrophil, monocyte, lymphocyte, and Rouleaux formation. All images were taken from section E with the exception of the Rouleaux formation which was taken from section B. Images were captured using a Nikon TI 40X DIC and auto white balanced using the NIS Elements AR 4.12.01 software.