A high-throughput microfluidic approach for 1000-fold leukocyte reduction of platelet-rich plasma

Abstract

Leukocyte reduction of donated blood products substantially reduces the risk of a number of transfusion-related complications. Current 'leukoreduction' filters operate by trapping leukocytes within specialized filtration material, while allowing desired blood components to pass through. However, the continuous release of inflammatory cytokines from the retained leukocytes, as well as the potential for platelet activation and clogging, are significant drawbacks of conventional 'dead end' filtration. To address these limitations, here we demonstrate our newly-developed 'controlled incremental filtration' (CIF) approach to perform high-throughput microfluidic removal of leukocytes from platelet-rich plasma (PRP) in a continuous flow regime. Leukocytes are separated from platelets within the PRP by progressively syphoning clarified PRP away from the concentrated leukocyte flowstream. Filtrate PRP collected from an optimally-designed CIF device typically showed a ~1000-fold (i.e. 99.9%) reduction in leukocyte concentration, while recovering >80% of the original platelets, at volumetric throughputs of ~1 mL/min. These results suggest that the CIF approach will enable users in many fields to now apply the advantages of microfluidic devices to particle separation, even for applications requiring macroscale flowrates.

Figure 1. Design of a reduced shear (RS) CIF device (c.d. = 7 μm).

(a) Overall layout of the device. (b) Device inlet: (i) the initial width of the centre channel is w_c(i = 0) = 300 μm; the channel is flanked by a series of serpentine side segments; (ii) the width of the side segments, G_S = 22.8 μm, is set slightly larger than the width of the subsequent inter-post gaps, G = 16 μm. (c) Side channel architecture transition: (i) progressively-shortening serpentine segments ultimately become rectilinear side channels, after which the width of the centre channel w_c(i), initially constant, progressively narrows; (ii) the width of the side channels at the first set of pill-shaped posts, w_s(i = 1), is set to equal the desired width of the inter-post gap, i.e. the smallest feature of the device. (d) Central and side channel progression: (i) the width of the central channel, w_c(i) gradually decreases until it reaches 150 μm, and remains constant thereafter; (ii) the width of the side channels continues to gradually increase, w_s(i + 1) > w_s(i), throughout the remainder of the device. (e) Loop transition: (i) the first leg of device transitions into the first loop; (ii) to maintain appropriate fluidic streamlines, the width of the inner side channel decreases from w_s(i) = 69.5 μm to w_s(loop 1_inner) = 57.6 μm; correspondingly, the width of the outer side channel increases to w_s(loop 1_outer) = 77.4 μm (not shown). Arrows indicate direction of fluid flow. Scale bars: (a), 5 mm; (b–e)(i), 250 μm; (b–e)(ii), 50 μm.

Figure 2. Estimated shear rates within original (OR) and reduced shear (RS) CIF devices (c.d. = 7 μm), under a driving pressure of 25 PSI.

(a) Schematics of the (i) OR-7 and (ii) RS-7 devices. (b) Maximum shear in a given device [OR-7, diamonds; RS-7 circles] segment typically occurs on the post surface that faces the centre channel. (c–e) Results of simulations in COMSOL Multiphysics illustrating the shear rate in the two devices at their inlet area, first loop, and channel outlets, respectively. Each image shows colour-scaled surface shear values of the cross-sectioned device segments at their centre plane and below (i.e. z ≤ 74.5 μm), with a wireframe rendering of the balance of the design. Scale Bars: (a), 5 mm; (c–e), 200 μm.

Figure 3. Images of an RS-CIF device performing leukocyte reduction of PRP.

(a) Schematic illustration of the RS-7 device. (b-i) Fluorescent images of DNA-stained leukocytes within a PRP sample as they are progressively concentrated in the centre channel, while flowing through the device: (b) device inlet; (c) side channel architecture transition; (d) end of the second (right) and fourth (left) loop of the device; (e) the third (right) and fifth (left) legs of the device; (f) the seventh leg of the device; (g) device outlet; (h) central channel (retentate) collection port for highly-concentrated leukocytes; (i) side channel (filtrate) collection port for leukocyte reduced PRP. Filtrate-to-retentate volumetric flow ratio is approximately 15:1. Scale Bars: (a), 5 mm; (b–i), 250 μm.

Figure 4. Performance of OR-7, and RS-7/8/9 CIF devices at driving pressures of 6.25, 12.5 and 25 PSI.

(a) Volumetric throughput. (b) Leukocyte reduction (in log depletion and percent depletion) of collected filtrate relative to input PRP sample. (c) Platelet recovery in filtrate. All values shown as mean ± s.d. (n = 5). Asterisks represent a significant difference (p < 0.01) between different devices at a given driving pressure or a given device at different driving pressures.

Figure 5. Platelet metrics for leukocyte reduced PRP (filtrate; open symbols) and leukocyte concentrated PRP (retentate; filled symbols) output from CIF devices.

Plots of: (a) absolute MPV values, (b) MPV normalized to the corresponding inlet sample, (c) platelet P-selectin expression, and (d) normalized P-selectin expression; shown as mean ± s.d. (n = 5) for the four CIF devices under different driving pressures. Outlet sample data for the OR-7, RS-7, RS-8, and RS-9 devices are represented by diamonds, circles, triangles, and squares, respectively. Retentate measurements in all plots are significantly higher than corresponding filtrate data (p < 0.05). Grey boxes represent mean ± s.d. of: (a,c) the inlet sample measurements, or (b,d) the cumulative retentate and filtrate values, weighted by their respective platelet recovery. Dotted lines (b,d) represent the normalized inlet values, by definition equal to unity. Average filtrate results show slightly lower MPV (in all 9 data points; B) and P-selectin expression (in 6 of 9; (d)) than corresponding inlet values of the three devices found best suited to perform leukocyte reduction (i.e. OR-7, RS-7, and RS-8). Cumulative weighted MPV data show no significant difference with inlet MPV, while cumulative weighted P-selectin data are on average ~10% higher than the corresponding inlet values; indicating a small degree of additional platelet activation in the retentate channel but no increased platelet aggregation in either channel. Asterisks represent significant difference (p < 0.01) in paired data between the devices or driving pressures indicated.