Recent advances in microfluidic cell separation to enable centrifugation-free, low extracorporeal volume leukapheresis in pediatric patients.

Leukapheresis is a common extracorporeal procedure for leukodepletion and cellular collection. During the procedure, a patient's blood is passed through an apheresis machine to separate white blood cells (WBCs) from red blood cells (RBCs) and platelets (PLTs), which are then returned to the patient. Although it is well-tolerated by adults and older children, leukapheresis poses a significant risk to neonates and low-weight infants because the extracorporeal volume (ECV) of a typical leukapheresis circuit represents a particularly large fraction of their total blood volume. The reliance of existing apheresis technology on centrifugation for separating blood cells limits the degree to which the circuit ECV could be miniaturized. The rapidly advancing field of microfluidic cell separation holds excellent promise for devices with competitive separation performance and void volumes that are orders of magnitude smaller than their centrifugation-based counterparts. This review discusses recent advancements in the field, focusing on passive separation methods that could potentially be adapted to perform leukapheresis. We first outline the performance requirements that any separation method must meet to replace centrifugation-based methods successfully. We then provide an overview of the passive separation methods that can remove WBCs from whole blood, focusing on the technological advancements made in the last decade. We describe and compare standard performance metrics, including blood dilution requirements, WBC separation efficiency, RBC and PLT loss, and processing throughput, and discuss the potential of each separation method for future use as a high-throughput microfluidic leukapheresis platform. Finally, we outline the primary common challenges that must still be overcome for these novel microfluidic technologies to enable centrifugation-free, low-ECV leukapheresis in the pediatric setting.

A high-throughput microfluidic device based on controlled incremental filtration to enable centrifugation-free, low extracorporeal volume leukapheresis.

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.

Concurrent Assessment of Deformability and Adhesiveness of Sickle Red Blood Cells by Measuring Perfusion of an Adhesive Artificial Microvascular Network.

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.

Traditional and emerging technologies for washing and volume reducing blood products.

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.

Substituting Sodium Hydrosulfite with Sodium Metabisulfite Improves Long-Term Stability of a Distributable Paper-Based Test Kit for Point-of-Care Screening for Sickle Cell Anemia.

Sickle cell anemia (SCA) is a genetic blood disorder that is particularly lethal in early childhood. Universal newborn screening programs and subsequent early treatment are known to drastically reduce under-five SCA mortality. However, in resource-limited settings, cost and infrastructure constraints limit the effectiveness of laboratory-based SCA screening programs. To address this limitation our laboratory previously developed a low-cost, equipment-free, point-of-care, paper-based SCA test. Here, we improved the stability and performance of the test by replacing sodium hydrosulfite (HS), a key reducing agent in the hemoglobin solubility buffer which is not stable in aqueous solutions, with sodium metabisulfite (MS). The MS formulation of the test was compared to the HS formulation in a laboratory setting by inexperienced users (n = 3), to determine visual limit of detection (LOD), readout time, diagnostic accuracy, intra- and inter-observer agreement, and shelf life. The MS test was found to have a 10% sickle hemoglobin LOD, 21-min readout time, 97.3% sensitivity and 99.5% specificity for SCA, almost perfect intra- and inter-observer agreement, at least 24 weeks of shelf stability at room temperature, and could be packaged into a self-contained, distributable test kits comprised of off-the-shelf disposable components and food-grade reagents with a total cost of only $0.21 (USD).

A rapid paper-based test for quantifying sickle hemoglobin in blood samples from patients with sickle cell disease.

Quantification of sickle hemoglobin (HbS) in patients with sickle cell disease (SCD) undergoing hydroxyurea or chronic transfusion therapy is essential to monitoring the effectiveness of these therapies. The clinical monitoring of %HbS using conventional laboratory methods is limited by high per-test costs and long turnaround times usually associated with these methods. Here we demonstrate a simple, rapid, inexpensive paper-based assay capable of quantifying %HbS in blood samples from patients with SCD. A 20 µL droplet of whole blood and hemoglobin solubility buffer was deposited on chromatography paper. The relative color intensities of regions of the resulting blood stain, determined by automated image analysis, are used to estimate %HbS. We compared the paper-based assay with hemoglobin electrophoresis (comparison method) using blood samples from 88 subjects. The test shows high correlation (R(2) = 0.86) and strong agreement (standard deviation of difference = 7%HbS) with conventional Hb electrophoresis measurement of %HbS, and closely approximates clinically predicted change in %HbS with transfusion therapy (mean difference 2.6%HbS, n = 5). The paper-based assay can be completed in less than 35 min and has a per-test cost less than $0.25. The assay is accurate across a wide range of HbS levels (10-97%) and hemoglobin concentrations (5.6-12.9 g/dL) and is unaffected by high levels of HbF (up to 80.6%). This study demonstrates the feasibility of the paper-based %HbS assay. The paper-based test could improve clinical care for SCD, particularly in resource-limited settings, by enabling more rapid and less expensive %HbS monitoring.