Deformability based cell sorting enabling quality control of donated red blood cells

Red blood cell (RBC) transfusions are a critical component of patient care, with around 1.2 million units utilized annually in Canada alone. Patients with acute blood loss require temporary RBC transfusions until their erythropoiesis restores RBC levels. Those with chronic disorders, such as hemoglobinopathies and bone marrow cancers, rely on transfused RBCs for entire RBC circulatory lifespan. For these chronic recipients, selecting long-circulating RBC units could minimize transfusion-associated morbidity like iron overload and lung damage, yet established methods to identify these units are lacking. This dissertation studied the potential to use RBC deformability as a surrogate marker for the transfused RBC circulatory clearance time. Deformability is vital for RBC function and has been considered a physical biomarker of circulation time in transfusion recipients. Our team has developed a unique microfluidic ratchet device to analyze RBC deformability. This device operates by directing individual RBCs to deform through a series of micrometer-scale constrictions much smaller than RBC diameter. A consistent force gradient is applied to each cell as it deforms through constrictions, enabling measurement of RBC deformability with high sensitivity and repeatability. Using the microfluidic ratchet device, we show that RBC deformability varies across healthy donors, but is consistent for each donor over multiple donations. Furthermore, RBC deformability is preserved during the standard 42-day cold storage in blood bags but degrades significantly beyond this timeframe. Additionally, deformability can be estimated by sampling from the blood bag tubing segment instead of puncturing the bag. Finally, we use a mouse transfusion model to investigate whether low-deformability RBCs are preferentially cleared from circulation. We show the rigid fraction of transfused RBCs are rapidly cleared, while transfused RBCs remaining in circulation had a deformability profile closely matching that of endogenous RBCs from the recipient mouse. Our findings provide strong evidence that RBC deformability, measured using the microfluidic ratchet device, may be used to predict RBC circulation time in transfusion recipients. If validated using future clinical studies, this approach could be used to identify donors who can provide long-circulating RBC units and reserve these units for chronic transfusion recipients to reduce transfusion frequency and associated morbidity.Medicine, Faculty ofPathology and Laboratory Medicine, Department ofGraduat

Degradation of red blood cell deformability during cold storage in blood bags

Abstract Red blood cells (RBCs) stored in blood bags develop a storage lesion that include structural, metabolic, and morphologic transformations resulting in a progressive loss of RBC deformability. The speed of RBC deformability loss is donor‐dependent, which if properly characterized, could be used as a biomarker to select high‐quality RBC units for sensitive recipients or to provide customized storage timelines depending on the donor. We used the microfluidic ratchet device to measure the deformability of red blood cells stored in blood bags every 14 days over a span of 56 days. We observed that storage in blood bags generally prevented RBC deformability loss over the current standard 42‐day storage window. However, between 42 and 56 days, the deformability loss profile varied dramatically between donors. In particular, we observed accelerated RBC deformability loss for a majority of male donors, but for none of the female donors. Together, our results suggest that RBC deformability loss could be used to screen for donors who can provide stable RBCs for sensitive transfusion recipients or to identify donors capable of providing RBCs that could be stored for longer than the current 42‐day expiration window

Cell-­phoresis o fRed Blood Cells Revealing Biophysical Signatures in Falciparum Malaria

We describe the cell-phoresis mechanism for massively parallel analysis of red blood cell (RBC) deformability by transporting single cells through microstructures to measure their spatial dispersion. Analogous to gel electrophoresis, which transport molecules through nanostructures to measure their length, the spatial dispersion of RBCs within microstructures indicate their deformability. Similar to gel electrophoresis, cell-phoresis require minimal instrumentation, provide a simple image-based readout, and could be performed simultaneously on multiple samples as part of a biophysical assay. We applied the cell-phoresis mechanism to study the biophysical signatures of falciparum malaria where we demonstrate label-­‐free and calibration-­‐free detection of ring-­‐stage infection, as well as in vitro assessment of antimalarial drug efficacy. We show that all clinical antimalarial drugs rigidify RBCs infected P. falciparum and that recently discovered PfATP4 inhibitors show a distinct biophysical signature. We anticipate cell-phoresis to be a functional assay for screening new antimalarials and adjunctive agents, as well as for validating their mechanisms of action