Lab-on-a-disk extraction of PBMC and metered plasma from whole blood: An advanced event-triggered valving strategy

Abstract

In this paper, we present a centrifugal microfluidic concept employing event-triggered valving for automated extraction of metered plasma and peripheral blood mononuclear cells (PBMCs). This "lab-on-a-disk" system has been developed for retrieving different density layers from a liquid column by "overflowing" the layers sequentially using the pressure exerted by a density-gradient liquid. Defined volumes of plasma and PBMCs were efficiently forwarded into designated microfluidic chambers as a sample preparation step prior to further downstream processing. Furthermore, the extracted PBMCs were counted directly on-disk using an automated optical unit by object-based image analysis, thus eliminating the requirement for the post-processing of the extracted PBMCs. This study is a direct continuation of our previous work¹ where we demonstrated combined on-disk detection of C-reactive protein and quantification of PBMCs following on-disk extraction of plasma and PBMCs from a single blood sample using a centrifugo-pneumatic valving mechanism. However, the former valving technique featured limited PBMC extraction efficiency. Here, integrating the novel concept along with event-triggered valving mechanism, we eliminated the occurrence of a specific microfluidic effect, which led us to increase PBMC extraction efficiency to 88%. This extraction method has the potential to be utilized for efficiently separating multiple density layers from a liquid sample in relevant biomedical applications.

FIG. 1.

(a) Schematics of the assembly of the multi-layer disks with dissolvable valves and multi-layered microchannels. (b) Schematics of the top-down view of the basic event-triggered design. (c) Schematics of top-down view of the “overflow” design.

FIG. 2.

(a) Schematic of a single functional unit of the microfluidic disk with the basic event-triggered valving design. (b) Sequence of the entire microfluidic process. The lower channels are marked with blue when pressurized and marked with green when vented. Similarly, the DF valves are marked with blue when intact and marked with green when dissolved. The three DFs are numbered (e.g., DF1, DF2) according to the sequence of their dissolution. (c) Picture frames of the event-triggered mechanism to sequentially forward plasma and PBMCs to their distinct receiving chambers.

FIG. 3.

(a) Schematic of the overflow design as an operational unit of the microfluidic disk. The four DFs (blue-colored) are numerically named (e.g., DF1, DF2) indicating the sequence of their dissolution. The microchannels are embedded into two separate layers so that the microchannels at the lower layer (bottle green-colored) can pass beneath the chambers on the upper layer. (b) Representative schematics of the entire microfluidic process. The lower channels are marked with blue when pressurized and marked with green when vented. Similarly, the DF valves are marked with blue when intact and marked with green when dissolved. The four DFs are numbered (e.g., DF1, DF2) according to the sequence of their dissolution. (c) Frame sequence of plasma and PBMC separation using the overflow design. (i) Blood is fractionated into plasma, PBMC, and RBC by DGM-based centrifugation. (ii) Elevation of the spin rate drives DGM in OD1 to dissolve DF1. The dissolution of DF1 enables DGM in OD1 to enter the BP chamber through lower microchannel to increase the liquid column height and eventually leading to plasma overflow into the outlet. (iii) The overflowed plasma enters the PH chamber and dissolves DF2. (iv) The plasma enters the plasma chamber from the PH chamber and the dissolution of DF2 triggers the venting of associated pneumatic lower microchannel causing DF3 to be dissolved by the remaining plasma in overflow chamber. The dissolution of DF3 enables the remaining plasma to enter the CC chamber. (v) Further elevation of the spin rate leads DGM in OD2 to dissolve DF4 and enter the BP chamber through lower microchannel causing the overflow of PBMC to the overflow chamber and subsequently to the CC chamber. (vi) PBMCs along with some plasma and DGM are held in the CC chamber for quantification by optical imaging.

FIG. 4.

(a) A raw image of a part of the CC chamber scanned by the imaging unit. Platelets (blue-circled) and RBCs (red-circled) can be seen in the pool of PBMCs (green-circled). Only two PBMCs have been marked with green for presentation purpose. (b) A view of the segmented images after the segmentation process demonstrating some RBC contamination (red-marked). (c) A view of the segmented images containing only PBMCs after the deletion of the segmented RBCs. The orange-marked segmented images shows that there are few cases where two adjacent cells are counted as one segmented image, which reduces the count of PBMCs present in the CC chamber. (d) PBMC quantification data from one healthy subject comparing the PBMC count among the basic design, overflow design, and the clinical data. The error bars represent the standard deviation obtained from triplicate measurements.