Size and density measurements of single sickle red blood cells using microfluidic magnetic levitation

Abstract

Single cells have unique biophysical signatures that can rapidly change during various disease states. For instance, cellular density is an inherent property differing between cell types. Characterizing changes in fundamental density properties down to the single-cell level can reveal sub-populations in pathological states. Here, we have developed a microfluidic, magnetic levitation-based assay (MagDense) that detects minute density differences of individual red blood cells (RBCs) down to 0.0001 g mL^-1 resolution. This assay fractionates RBCs based on their density profiles in a non-ionic paramagnetic medium flowing in a capillary microchannel placed between magnets with same poles facing each other. Based on precisely measured levitation height and density of individual RBCs at their specific equilibrium state, we demonstrated that MagDense can accurately analyze the density of sickle hemoglobin (HbS)-containing RBCs and normal hemoglobin (HbA)-containing RBCs. In addition, the precise density and cell size measurements at the single cell level showed three different sub-populations of RBCs in blood samples from individuals with homozygous sickle cell disease receiving blood transfusions; where less dense, HbA-containing RBCs levitated higher, while the denser, HbS-containing RBCs levitated lower. We compared the mean RBC densities of sickle cell disease subjects with healthy controls and found distinctly separated bands of RBC density for each group denoting the likely range of cell densities seen in the blood samples. The high resolution of our method enabled measurement of deviation from the mean RBC density. Moreover, we introduced a new term as a measure of density dispersion, "RBC levitational density width, RLDW". Mean RBC density in sickle cell disease associated with hemoglobin from complete blood count (p = 0.032, linear regression) and RLDW associated with absolute reticulocyte count (ARC) and RBC distribution width (RDW) from complete blood count (p = 0.002 for ARC and p = 003 for RDW, linear regression). Our magnetic levitation-based assay enables rapid, accurate, density-based imaging, profiling and label-free monitoring of single RBCs. Our approach can be broadly applicable to investigate blood cell disorders and the effects of emerging pharmacological and curative therapies in patient outcomes.

Figure 1.. Microfluidic magnetic levitation-based red blood cell (RBC) density measurement assay at the single RBC level.

(A) MagDense is composed of a microchannel placed between two permanent magnets. Tilted mirrors provide real-time imaging and efficient assessment of blood samples. (B) Diluted mixture of sickle and healthy RBCs are loaded into the microfluidic device and allowed to equilibrate under the effects of gravitational and magnetic levitation forces inside a paramagnetic medium. (C) Representative illustration of levitation height (μm, y-axis) versus cell number (x-axis) depicts distributions of denser, hemoglobin S containing RBCs (HbSS RBCs) compared to a normal control (HbAA RBCs). (D) Representative microscopic images of levitating RBCs before and after equilibrium. (E) Based on precisely measured levitation height and density of cells in levitation state, the density of HbS containing RBCs and HbA containing normal RBCs can be accurately analyzed. Less dense HbA RBCs levitate higher, while the denser HbS RBCs levitate lower in the channel. (F) Representative levitation image of density-based separation of reference beads within the MagDense device. Scale bar is 100 μm (G) The relationship between the bead density and levitation heights in 30 mM paramagnetic medium. Linear fitting curve of each data point provides a standard function for measuring densities of particles. The slope of the curve represents the amount of density change per micrometer levitation height, which is converted to density change per pixel of microscope image. 3.08×10⁻⁴ g/ml/μm corresponds to 0.98×10⁻⁴ g/ml/pixel under 20x objective.

Figure 2.. Size and density analysis of red blood cells (RBCs) at a single cell level provides RBC classification by cluster analysis with respect to RBC density and size.

(A) The histogram plots for density distributions of an HbAA control subject with no known hemoglobinopathy (light blue) and from two subjects with homozygous sickle cell disease; a subject who receives hydroxyurea (red) and a subject who recently received hematopoietic stem cell transplant (HSCT, purple). HbAA RBCs have a narrower distribution of both density and size, compared to HbSS RBCs. (B) Scatter plots of cell density versus cell size (major axis length) for HbAA and HbSS RBCs with confidence ellipses (95% coverage). Confidence ellipse of HU subject with high HbS and HbF shows greater heterogeneity, probably due to presence of both dehydration resistant cells and cells with higher density.

Figure 3.. Three groups of red blood cells (RBCs) revealed by clustering of scatter plots from a transfused homozygous sickle cell disease (HbSS SCD) subject.

HbSS SCD subjects cannot produce HbAA containing RBCs and blood transfusions are needed to replace sickle RBCs with HbAA RBCs. Some of sickle RBCs may be dehydration resistant, while some of them become irreversibly sickled after cycles of hemoglobin polymerization. Scatter plots of individual subjects can be post processed to explore different RBC clusters of distinct size and density. Clusters were identified with agglomerative hierarchical clustering method. Representative images of RBCs with different shapes were depicted as inset figures next to their corresponding dots.

Figure 4.. RBC density, measured by MagDense, is significantly greater (p<0.001, ANOVA) for subjects with sickle cell disease (SCD) (HbSS, N=16) compared with non-sickle control (HbAA, N=12).

(A) Bands represent anticipated range for the RBC density for each group and are defined as interquartile ranges. Blue dashed rectangle represents the HbAA band, which is the anticipated results for the assay following fully curative therapy, with complete reversal of the abnormal red cell phenotype in SCD. Scale bars are 80 μm. (B) Receiver operating curve (ROC) demonstrate sensitivity and 1-specificity for differentiation of HbSS from HbAA subjects based on mean RBC density. Enumerated bullets on the curve denotes four different thresholds of mean RBC density. The first threshold represents the lower boundary of the HbSS band. The second threshold has the minimum distance to unity among four. (C) ROC analysis outputs are tabulated for each threshold shown in the previous panel.

Figure 5.. Dispersion of red blood cell (RBC) density from the mean can be assessed by MagDense with high resolution.

The weighted standard deviation of the averaged RBC density curves was found to be greater in HbSS, compared to HbAA. Red lines represent subjects with sickle cell disease (SCD) and blue lines represent non-sickle, control subjects. Thin lines depict the RBC density distribution for individuals and thick lines show the averaged RBC distribution of each group of subjects.

Figure 6.. Red blood cell (RBC) density parameters, mean RBC density and RBC levitational density width (RLDW) are positively related with clinical laboratory test results in sickle cell disease (SCD).

(A) Hemoglobin is one of the most abundant protein in RBCs. The hemoglobin concentration from the clinical laboratory results is associated with mean RBC density (PCC=0.54, p=0.032, Pearson correlation and linear regression). (B) Absolute reticulocyte count (ARC) is considered as a disease severity marker in SCD. There was no significant association between ARC and mean RBC density, however, (C) there was a significant relation of increased ARC with increased RLDW (PCC=0.69, p=0.003, Pearson correlation and linear regression), which implies greater intra-patient RBC density heterogeneity as measured by MagDense platform. (D) The RDW as measured by central clinical laboratory and RLDW was significantly and positively related (PCC=0.70, p=0.002, Pearson correlation and linear regression). Blue boxes represent likely regions for HbAA subjects in each plot.

Figure 7.. MagDense magnetic levitation-based red blood cell (RBC) density measurement assay at the single RBC level can be used to predict patient response to the clinical interventions.

(A-B) Levitational height change after blood transfusion treatment can be observed visually from microscope images of RBCs within the MagDense channel. Shown are representative microscope images of red blood cells within MagDense capillary (i) before and (ii) after the SCD subjects receive blood transfusion. (C-D) Mean RBC density of samples from individuals with sickle cell disease (SCD) approaches to HbAA control range following a transfusion treatment. (E) Levitational height (i) before and (ii) after hematopoietic stem cell transplantation (HSCT). HSCT is a curative therapy (F) Mean RBC density decreased from 1.124 g/ml to 1.107 g/ml following HSCT, along with an increase in RLDW. Scale bars are 300 μm.