Fig 1.
The passive, centrifugation-free system for separating WB into components.
(a) Photograph of the WB separation system, including the custom compression/expressor apparatus and components of the disposable kit. A one cent U.S. coin is shown for size reference. (b) A schematic illustration of the system’s operation: (i) WB is separated into PRP and RBCs via natural sedimentation in a shallow blood bag held within a custom compression/expressor apparatus, (ii) PRP is expressed from the sedimentation bag, (iii) PRP passing through the microfluidic platelet concentrator (MPC) is separated into PC and PPP, and (iv) sedimented RBCs are mixed with additive solution and expressed from the sedimentation bag through a standard leukoreduction filter (LRF).
Fig 2.
Separation of WB into RBCs and PRP via RBC sedimentation (Stage 1).
(a) Schematic illustration of the process of natural RBC sedimentation at 1×g gravity: (i) RBCs form rouleaux that (ii) gradually assemble into a large network, which then (iii) collapses as PRP ‘channels’ upward through the network of RBC aggregates falling towards to the bottom of the vessel. (b) For a given erythrocyte sedimentation rate (ESR = Δh/Δt), a WB sample in a shallow vessel achieves a greater separation of PRP and RBCs, in a given amount of time. (c) The custom compression/expressor apparatus, utilizing the constant force regime of an air spring to provide a consistent flow rate through the platelet concentration module. (d) Separation of RBCs from PRP via passive sedimentation over time: i) 250 mL of freshly-drawn WB in a 1 L sedimentation bag, placed on a slightly (10°) angled base plate and gently compressed by an air spring (~2 PSI); ii) within ~60 min the ‘shallow pool’ of WB within the sedimentation bag has largely separated into pRBCs and PRP; iii) after 150 min, the pressure in the bag is increased to ~10 PSI, and the exit valve is opened, expressing the PRP layer through the Stage 2 microfluidic platelet concentrator (MPC) downstream (see Fig 3).
Fig 3.
The microfluidic platelet concentrator (MPC) of passive WB separation system (Stage 2).
(a) Components of the MPC: (i) top layer, containing five inlet channel branches for distributing PRP throughout the device, and two outlet channels for collecting the streams of separated PC and PPP; (ii) two device layers each consisting of ten individual CIF microfluidic devices in parallel, which perform the separation; (iii) a flat bottom layer for sealing the device. Inset shows a single CIF microdevice schematically; arrows indicate the direction of flow. (b) Photograph of an assembled MPC device. A one cent U.S. coin is shown for size reference. (c) Photograph of PRP being expressed from the sedimentation bag, through the MPC module, separated into PC and PPP.
Fig 4.
Schematic illustration of the design of the dual arm, split unit study comparing the quality of blood components produced using the new passive separation system versus conventional centrifugation.
Table 1.
Fig 5.
Activation of platelets due to processing of a typical WB unit into components using the two separation methods.
Each panel shows i) the level of platelet activation (primary y-axis: all platelets, green; secondary y-axis: platelet aggregates, blue), and ii) the associated formation of platelet aggregates, for (a) the WB unit prior to processing, and the two PC units produced (b) via conventional centrifugation, and (c) using the passive separation system. Platelets were labelled with CD42b-PerCP, and activated platelets were identified using CD62p (P-selectin)-FITC. The threshold of positive P-selectin expression was defined using an isotype control (IgG-FITC). A manual gate was applied to SSC vs. CD42b scatter plots to demarcate the presence of any (typically highly activated) platelet aggregates, which would be observed in the form of a characteristic ‘comet tail’ near the upper-right edge of a platelet distribution.