11 Sep Label-Free Microfluidic Blood Processing for Platelet and Leukocyte Analysis
Platelets and leukocytes are important participants in thrombosis, inflammation, and immune responses, while interactions between these cells can provide useful information about disease and treatment response. Measuring platelet activation and platelet-leukocyte aggregates, however, depends strongly on how blood is processed before analysis. Conventional centrifugation requires relatively large sample volumes and several handling steps, and the mechanical stresses involved can unintentionally activate platelets or disturb native cell-cell interactions. These challenges are particularly important when only small blood samples are available or when repeated sampling is needed. A microfluidic approach could provide a gentler way to prepare blood samples while reducing the required volume and processing time.
The researchers developed a two-stage, label-free microfluidic device based on Dean Flow Fractionation, or DFF. Rather than relying on antibodies, magnetic particles, or centrifugation to separate cells, the microfluidic chip uses size-dependent inertial and Dean forces inside curved channels. The first stage separates leukocytes, red blood cells, and platelets, while the second stage reconcentrates the leukocyte fraction by removing excess sheath fluid. This arrangement allows platelet and leukocyte-enriched samples to be obtained from approximately 100 µL of whole blood in about five minutes.
“Microfluidic blood fractionation using DFF. (A) Centrifugation-free workflow illustrating microfluidic blood processing followed by flow cytometric analysis of platelet activation and neutrophil–platelet aggregates (NPAs). (B) Schematic of the integrated two-stage DFF chip incorporating a downstream leukocyte concentrator. In stage 1, larger leukocytes are inertially focused toward the inner wall of the spiral channel due to the balance between size-dependent inertial forces (FL) and Dean drag forces (FD), while smaller platelets remain entrained in Dean-induced secondary flows and migrate toward the outer wall for collection at outlet 5 (O5). In stage 2, the leukocytes-enriched stream enters a serpentine channel, where inertial focusing further aligns leukocytes toward the channel centreline for collection at outlet 2 (O2). (C) Composite fluorescence images showing distinct equilibrium positions of 2 μm beads (mimic platelets) and 10 μm beads (mimic leukocytes) near the outer and inner channel walls, respectively, in stage 1, (red boxes) and enhanced alignment of 10 μm beads at the channel centreline in the serpentine leukocyte concentrator (stage 2, green boxes). Scale bar: 100 μm”. Reproduced from Kay Khine Maw, Kerem Delikoyun, Si Ko Myo, et. al; Label-free microfluidic small-volume blood processing for analysis of platelet activation and neutrophil–platelet aggregates. Lab Chip 2026; with permission from The Royal Society of Chemistry.
The microfluidic channel design was first evaluated using PDMS devices microfabricated by standard photolithography and soft lithography. After validating the separation behaviour, the researchers transferred the design to PMMA to improve mechanical robustness and support scalable manufacturing. For the PMMA microfluidic chip, channels and collection reservoirs were produced by CNC machining and the two PMMA layers were thermally bonded. Enlarged outlet reservoirs also allowed the sorted fractions to be collected directly by pipette, reducing dead volume during small-volume processing.
During operation, whole blood was diluted tenfold and introduced into the outer inlet of the spiral channel at 150 µL/min, while sheath buffer was supplied through the inner inlet at approximately 900 to 1000 µL/min. Larger leukocytes experienced stronger inertial lift forces and focused toward the inner wall, while much smaller platelets followed the Dean-induced secondary flow toward the outer wall. Red blood cells were predominantly directed to a central waste outlet. The leukocyte-rich stream then entered a serpentine channel, where the cells were focused toward the centre while roughly 70-80% of the surrounding sheath fluid was removed through side outlets, producing a more concentrated leukocyte sample.
The PMMA microfluidic platform removed approximately 96% of red blood cells while recovering about 70-80% of leukocytes and 45% of platelets. Importantly, the sorted platelets showed little P-selectin expression in untreated samples, indicating that the separation process itself caused minimal platelet activation. When compared with conventional platelet-rich plasma preparation and commercial red blood cell depletion, cells processed by DFF also showed lower expression of platelet and neutrophil activation markers. The microfluidic system maintained biologically relevant neutrophil-platelet interactions as well.
The researchers also tested the microfluidic workflow beyond controlled laboratory samples. Blood from 20 fever patients was processed using the PMMA microfluidic device, and the measurements reproduced expected trends such as reduced platelet counts and increased leukocyte counts relative to healthy controls. The separated samples were compatible with both conventional haematology analysis and digital holographic microscopy. In a diabetic mouse model, the ability to work with approximately 100–200 µL of blood also allowed platelet activation and neutrophil–platelet interactions to be followed at multiple time points in the same animals rather than relying on terminal blood collection.
This study shows how established inertial microfluidics can be integrated into a practical workflow for functional blood analysis. By combining spiral Dean Flow Fractionation with on-chip leukocyte reconcentration, the microfluidic platform separates platelets and leukocytes from small blood samples without centrifugation while limiting handling-induced cellular activation. The transition from PDMS prototypes to a CNC-machined PMMA device also addresses practical considerations related to robustness and manufacturing. Although further optimization is needed to improve recovery of smaller leukocyte populations and the current workflow still relies on off-chip analytical instruments, the microfluidic system provides a useful approach for studying platelet activation, platelet-leukocyte interactions, and other blood-based functional biomarkers when sample volume is limited.
Figures are reproduced from Kay Khine Maw, Kerem Delikoyun, Si Ko Myo, Koh Kai Bing, Johannes Krell, Lingyan Gong, Hui Min Tay, Feng Chen, Wei Wang, Yew Guan Goh, John Tshon Yit Soong, Win Sen Kuan, Matthew Edward Cove, Siu Ling Wong, Oliver Hayden, Han Wei Hou; Label-free microfluidic small-volume blood processing for analysis of platelet activation and neutrophil–platelet aggregates. Lab Chip 2026; https://doi.org/10.1039/d6lc00413j with permission from The Royal Society of Chemistry.
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