Integrated automated particle tracking microfluidic enables high‐throughput cell deformability cytometry for red cell disorders

Abstract

Investigating individual red blood cells (RBCs) is critical to understanding hematologic diseases, as pathology often originates at the single-cell level. Many RBC disorders manifest in altered biophysical properties, such as deformability of RBCs. Due to limitations in current biophysical assays, there exists a need for high-throughput analysis of RBC deformability with single-cell resolution. To that end, we present a method that pairs a simple in vitro artificial microvasculature network system with an innovative MATLAB-based automated particle tracking program, allowing for high-throughput, single-cell deformability index (sDI) measurements of entire RBC populations. We apply our technology to quantify the sDI of RBCs from healthy volunteers, Sickle cell disease (SCD) patients, a transfusion-dependent beta thalassemia major patient, and in stored packed RBCs (pRBCs) that undergo storage lesion over 4 weeks. Moreover, our system can also measure cell size for each RBC, thereby enabling 2D analysis of cell deformability vs cell size with single cell resolution akin to flow cytometry. Our results demonstrate the clear existence of distinct biophysical RBC subpopulations with high interpatient variability in SCD as indicated by large magnitude skewness and kurtosis values of distribution, the "shifting" of sDI vs RBC size curves over transfusion cycles in beta thalassemia, and the appearance of low sDI RBC subpopulations within 4 days of pRBC storage. Overall, our system offers an inexpensive, convenient, and high-throughput method to gauge single RBC deformability and size for any RBC population and has the potential to aid in disease monitoring and transfusion guidelines for various RBC disorders.

Deformability of Stored Red Blood Cells

“…In the initial period of studying the effect of cold-storage on the deformability of PRBCs was carried out using the filtration method (Kucera et al, 1985;Wegner and Kucera, 1989) and ektacytometry (Cluitmans et al, 2012;Sosa et al, 2014;Marin et al, 2021;van Cromvoirt et al, 2021). In the last decade, measurements with a flow chamber (Relevy et al, 2008;Barshtein et al, 2014Barshtein et al, , 2020bOrbach et al, 2017) or microfluidic systems (Guruprasad et al, 2019;Islamzada et al, 2020;Man et al, 2020) have become more frequently used.…”

“…Research directly targeting unit-to-unit variations in PRBCs deformability has only recently been initiated, with several authors have demonstrated the significance of this phenomenon (Frank et al, 2013;Matthews et al, 2015;Guruprasad et al, 2019;Jeon et al, 2019;Barshtein et al, 2020b;Islamzada et al, 2020;van Cromvoirt et al, 2021). It was found that two factors primarily cause variability among units: the difference in the initial properties of donated RBCs and the differing ability of the cells to retain their deformability during storage (Barshtein et al, 2020b;Islamzada et al, 2020).…”

“…As mentioned above, research in this area began in the mid-1980s. At present, with the advent of new approaches (microfluidics) which enable us to determine the distribution of deformability in the cell population (Relevy et al, 2008;Guruprasad et al, 2019;Barshtein et al, 2020b;Islamzada et al, 2020;Man et al, 2020), the scientific interest in this field has increased. This led to an increase in the number of research groups working in this area.…”

Vascularized Microfluidics and Their Untapped Potential for Discovery in Diseases of the Microvasculature

“…As such, blood cannot be treated as a simple continuum, and it is important to consider the stiffness and size of single blood cells. In the smallest capillaries, RBCs must deform to pass, so changes in microvasculature function and transport occur due to increases in RBC stiffness or adhesion, which is common in many cases, including sickle cell disease ( 16 , 17 ), malaria ( 18 , 19 ), or even various intravenous fluids ( 16 ). Adding to this complexity, the shear ( 20 ) and oxygen levels ( 21 ) in the local microenvironment also affect RBC adhesiveness.…”

Sorting of capsules according to their stiffness: from principle to application

“…3 RBC disorders such as sickle cell disease are also associated with increasing cell stiffness -an approximately threefold increase in membrane shear modulus 4 -and a broadening of the cell size distribution. 5 RBCs are examples of natural capsules consisting of a fluid core encapsulated by a semi-permeable membrane. 6 In fact, the transport and flow-induced deformation of RBCs and other biological cells are commonly modelled with idealised capsules and soft beads.…”