Millions of blood components including red blood cells, platelets, and granulocytes are transfused each year in the United States. The transfusion of these blood products may be associated with adverse clinical outcomes in some patients due to residual proteins and other contaminants that accumulate in blood units during processing and storage. Blood products are, therefore, often washed in normal saline or other media to remove the contaminants and improve the quality of blood cells before transfusion. While there are numerous methods for washing and volume reducing blood components, a vast majority utilize centrifugation-based processing, such as manual centrifugation, open and closed cell processing systems, and cell salvage/autotransfusion devices. Although these technologies are widely employed with a relatively low risk to the average patient, there is evidence that centrifugation-based processing may be inadequate when transfusing to immunocompromised patients, neonatal and infant patients, or patients susceptible to transfusion-related allergic reactions. Cell separation and volume reduction techniques that employ centrifugation have been shown to damage blood cells, contributing to these adverse outcomes. The limitations and disadvantages of centrifugation-based processing have spurred the development of novel centrifugation-free methods for washing and volume reducing blood components, thereby causing significantly less damage to the cells. Some of these emerging technologies are already transforming niche applications, poised to enter mainstream blood cell processing in the not too distant future.

Background: Hypothermic storage transforms red blood cells (RBC) from smooth biconcave discocytes into increasingly spherical spiculated echinocytes and, ultimately, fragile spherocytes (S). Individual cells undergo this transformation at different rates, producing a heterogeneous mixture of RBCs at all stages of echinocytosis in each unit of stored blood. Here we investigated how washing (known to positively affect RBC properties) changes morphology of individual RBCs at the single-cell level. Study Design and Methods: We tracked the change in shape of individual RBCs (n = 2870; drawn from six 4-to 6-week-old RBC units) that were confined in an array of microfluidic wells during washing in saline (n = 1095), 1% human serum albumin (1% HSA) solution (n = 999), and the autologous storage supernatant (control, n = 776). Results: Shape recovery proceeded through the disappearance of spicules followed by the progressive smoothening of the RBC contour, with the majority of changes occurring within the initial 10 minutes of being exposed to the washing solution. Approximately 57% of all echinocytes recovered by at least one morphologic class when washed in 1% HSA (36% for normal saline), with 3% of cells in late-stage echinocytosis restoring their discoid shape completely. Approximately one-third of all spherocytic cells were lysed in either washing solution. Cells washed in their autologous storage supernatant continued to deteriorate during washing. Conclusion: Our findings suggest that the replacement of storage supernatant with a washing solution during washing induces actual shape recovery for RBCs in all stages of echinocytosis, except for S that undergo lysis instead.

Sickle cell disease (SCD) is the most common inherited blood disorder, affecting approximately 100,000 patients in the U.S. and millions more worldwide. Patients with SCD experience a wide range of clinical complications, including frequent pain crises, stroke, and early mortality, all originating from a single-point mutation in the β-globin subunit. The RBC changes resulting from the sickle mutation lead to a host of rheological abnormalities that diminish microvascular blood flow, and produce severe anemia due to RBC hemolysis, and ischemia from vaso-occlusion initiated by sticky, rigid sickle RBCs. While the pathophysiology and mechanisms of SCD have been investigated for many years, therapies to treat the disease are limited. In addition to RBC transfusion, there are only two US Food and Drug Administration (FDA)-approved drugs to ameliorate SCD complications: hydroxyurea (HU) and L-glutamine (Endari™). The only curative therapy currently available is allogeneic hematopoietic stem cell transplantation (HSCT), which is generally reserved for individuals with a matched related donor, comprising only 10–15% of the total SCD population. Potentially curative advanced gene therapy approaches for SCD are under investigation in ongoing clinical trials. The ultimate goal of any curative treatment should be to repair the hemorheological abnormalities caused by SCD, and thus normalize blood flow and prevent clinical complications. Our mini-review highlights a set of key hemorheological biomarkers (and the current and emerging technologies used to measure them) that may be used to guide the development of novel curative and palliative therapies for SCD, and functionally assess outcomes. Impact statement Severe impairment of blood rheology is the hallmark of SCD pathophysiology, and one of the key factors predisposing SCD patients to pain crises, organ damage, and early mortality. As novel therapies emerge to treat or cure SCD, it is crucial that these treatments are functionally evaluated for their effect on blood rheology. This review describes a comprehensive panel of rheological biomarkers, their clinical uses, and the technologies used to obtain them. The described technologies can produce highly sensitive measurements of the ability of current treatments to improve blood rheology of SCD patients. The goal of curative therapies should be to achieve blood rheology biomarkers measurements in the range of sickle cell trait individuals (HbAS). The use of the panel of rheological biomarkers proposed in this review could significantly accelerate the development, optimization, and clinical translation of novel therapies for SCD.

Leukapheresis, the extracorporeal separation of white blood cells (WBCs) from red blood cells (RBCs) and platelets (PLTs), is a life-saving procedure used for treating patients with cancer and other conditions, and as the initial step in the manufacturing of cellular and gene-based therapies. Well-tolerated by adults, leukapheresis poses a significant risk to neonates and low-weight infants because the extracorporeal volume (ECV) of standard centrifugation-based machines represents a particularly large fraction of these patients’ total blood volume. Here we describe a novel high-throughput microfluidic device (with a void volume of 0.4 mL) based on controlled incremental filtration (CIF) technology that could replace centrifugation for performing leukapheresis. The CIF device was tested extensively using whole blood from healthy volunteers at multiple hematocrits (5–30%) and flow rates (10–30 mL/min). In the flow-through regime, the CIF device separated WBCs with > 85% efficiency and 10–15% loss of RBCs and PLTs while processing whole blood diluted with saline to 10% hematocrit at a flow rate of 10 mL/min. In the recirculation regime, the CIF device demonstrated a similar level of separation performance, virtually depleting WBCs in the recirculating blood (~ 98% reduction) by the end of a 3.5-hour simulated leukapheresis procedure. Importantly, the device operated without clogging or decline in separation performance, with minimal activation of WBCs and PLTs and no measurable damage to RBCs. Compared to the typical parameters of centrifugation-based leukapheresis, the CIF device had a void volume at least 100-fold smaller, removed WBCs about twice as fast, and lost ~ 2–3-fold fewer PLTs, while operating at a flow rate compatible with the current practice. The hematocrit and flow rate at which the CIF device operated were significantly higher than previously published for other microfluidic cell separation methods. Finally, this study is the first to demonstrate a highly efficient separation of cells from recirculating blood using a microfluidic device. Overall, these findings suggest the feasibility of using high-throughput microfluidic cell separation technology to ultimately enable centrifugation-free, low-ECV leukapheresis. Such a capability would be particularly useful in young children, a vulnerable group of patients who are currently underserved.

Washed red blood cells (RBCs) are indicated for immunoglobulin A (IgA) deficient recipients. Centrifugation-based cell processors commonly used by hospital blood banks cannot consistently reduce IgA below the recommended levels, hence double washing is frequently required. Here, we describe a prototype of a simple, portable, disposable system capable of washing stored RBCs without centrifugation, while reducing IgA below 0.05 mg/dL in a single run. Samples from RBC units (n = 8, leukoreduced, 4-6 weeks storage duration) were diluted with normal saline to a hematocrit of 10%, and then washed using either the prototype washing system, or via conventional centrifugation. The efficiency of the two washing methods was quantified and compared by measuring several key in vitro quality metrics. The prototype of the washing system was able to process stored RBCs at a rate of 300 mL/hour, producing a suspension of washed RBCs with 43 ± 3% hematocrit and 86 ± 7% cell recovery. Overall, the two washing methods performed similarly for most measured parameters, lowering the concentration of free hemoglobin by >4-fold and total free protein by >10-fold. Importantly, the new washing system reduced the IgA level to 0.02 ± 0.01 mg/mL, a concentration 5-fold lower than that produced by conventional centrifugation. This proof-of-concept study showed that centrifugation may be unnecessary for washing stored RBCs. A simple, disposable, centrifugation-free washing system could be particularly useful in smaller medical facilities and resource limited settings that may lack access to centrifugation-based cell processors.

Biomarker development is a key clinical research need in sickle cell disease (SCD). Hemorheological parameters are excellent candidates as abnormal red blood cell (RBC) rheology plays a critical role in SCD pathophysiology. Here we describe a microfluidic device capable of evaluating RBC deformability and adhesiveness concurrently, by measuring their effect on perfusion of an artificial microvascular network (AMVN) that combines microchannels small enough to require RBC deformation, and laminin (LN) coating on channel walls to model intravascular adhesion. Each AMVN device consists of three identical capillary networks, which can be coated with LN (adhesive) or left uncoated (non-adhesive) independently. The perfusion rate for sickle RBCs in the LN-coated networks (0.18 ± 0.02 nL/s) was significantly slower than in non-adhesive networks (0.20 ± 0.02 nL/s), and both were significantly slower than the perfusion rate for normal RBCs in the LN-coated networks (0.22 ± 0.01 nL/s). Importantly, there was no overlap between the ranges of perfusion rates obtained for sickle and normal RBC samples in the LN-coated networks. Interestingly, treatment with poloxamer 188 decreased the perfusion rate for sickle RBCs in LN-coated networks in a dose-dependent manner, contrary to previous studies with conventional assays, but in agreement with the latest clinical trial which showed no clinical benefit. Overall, these findings suggest the potential utility of the adhesive AMVN device for evaluating the effect of novel curative and palliative therapies on the hemorheological status of SCD patients during clinical trials and in post-market clinical practice.

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Changes in red blood cell (RBC) morphology distribution have emerged as a quantitative biomarker for the degradation of RBC functional properties during hypothermic storage. Previously published automated methods for classifying the morphology of stored RBCs often had insufficient accuracy and relied on proprietary code and datasets, making them difficult to use in many research and clinical applications. Here we describe the development and validation of a highly accurate open-source RBC morphology classification pipeline based on ensemble deep learning (DL). The DL-enabled pipeline utilized adaptive thresholding or semantic segmentation for RBC identification, a deep ensemble of four convolutional neural networks (CNNs) to classify RBC morphology, and Kalman filtering with Hungarian assignment for tracking changes in the morphology of individual RBCs over time. The ensembled CNNs were trained and evaluated on thousands of individual RBCs from two open-access datasets previously collected to quantify the morphological heterogeneity and washing-induced shape recovery of stored RBCs. Confusion matrices and reliability diagrams demonstrated under-confidence of the constituent models and an accuracy of about 98% for the deep ensemble. Such a high accuracy allowed the CNN ensemble to uncover new insights over our previously published studies. Re-analysis of the datasets yielded much more accurate distributions of the effective diameters of stored RBCs at each stage of morphological degradation (discocyte: 7.821 ± 0.429 µm, echinocyte 1: 7.800 ± 0.581 µm, echinocyte 2: 7.304 ± 0.567 µm, echinocyte 3: 6.433 ± 0.490 µm, sphero-echinocyte: 5.963 ± 0.348 µm, spherocyte: 5.904 ± 0.292 µm, stomatocyte: 7.080 ± 0.522 µm). The effective diameter distributions were significantly different across all morphologies, with considerable effect sizes for non-neighboring classes. A combination of morphology classification with cell tracking enabled the discovery of a relatively rare and previously overlooked shape recovery of some sphero-echinocytes to early-stage echinocytes after washing with 1% human serum albumin solution. Finally, the datasets and code have been made freely available online to enable replication, further improvement, and adaptation of our work for other applications.