Kinetics of sickle cell biorheology and implications for painful vasoocclusive crisis

Abstract

We developed a microfluidics-based model to quantify cell-level processes modulating the pathophysiology of sickle cell disease (SCD). This in vitro model enabled quantitative investigations of the kinetics of cell sickling, unsickling, and cell rheology. We created short-term and long-term hypoxic conditions to simulate normal and retarded transit scenarios in microvasculature. Using blood samples from 25 SCD patients with sickle hemoglobin (HbS) levels varying from 64 to 90.1%, we investigated how cell biophysical alterations during blood flow correlated with hematological parameters, HbS level, and hydroxyurea (HU) therapy. From these measurements, we identified two severe cases of SCD that were also independently validated as severe from a genotype-based disease severity classification. These results point to the potential of this method as a diagnostic indicator of disease severity. In addition, we investigated the role of cell density in the kinetics of cell sickling. We observed an effect of HU therapy mainly in relatively dense cell populations, and that the sickled fraction increased with cell density. These results lend support to the possibility that the microfluidic platform developed here offers a unique and quantitative approach to assess the kinetic, rheological, and hematological factors involved in vasoocclusive events associated with SCD and to develop alternative diagnostic tools for disease severity to supplement other methods. Such insights may also lead to a better understanding of the pathogenic basis and mechanism of drug response in SCD.

Keywords: Aes-103; capillary obstruction ratio; cell deformability; sickle cell anemia; vasoocclusion.

Conflict of interest statement

Conflict of interest statement: E.D., M.D.-S., M.D., and S.S. have filed a patent based on the work presented in this paper.

Figures

Fig. 1.

Microfluidic platform for investigation of biophysical alterations in sickle RBCs under transient hypoxia conditions. (A) Schematic of microfluidic device with O2 control for studying kinetics of cell sickling and unsickling. (B) Identification of cell sickling events from a microscopic image (arrows indicate sickled RBCs). (C) Schematic of microfluidic device with capillary-inspired structures for single-cell rheology study. Note: Schematics are not drawn to scale, and the dimensions are in microns.

Fig. 2.

Profiles of cell sickling and unsickling under transient hypoxia conditions with 2% for the lowest O2 concentration. (A and B) Profile of short-term transient DeOxy (O2 concentration less than 5% for ∼25 s) (A) and profile of long-term DeOxy (O2 concentration less than 5% for ∼220 s) (B). (C) Profiles of the sickled fraction of multiple SCD samples during long-term transient DeOxy (each curve represents an individual patient sample).

Fig. 3.

Kinetics of cell sickling. Delay time of cell sickling (A) for 5% sickled fraction and (B) for 10% sickled fraction. Distributions of maximum sickled fractions under (C) short-term transient DeOxy state (O2 concentration less than 5% for ∼25 s) and (D) long-term transient DeOxy state (O2 concentration less than 5% for ∼220 s). Arrows indicate severe cases, defined as those where sickling delay time was less than 25 s. Red circles represent on-HU and blue circles represent off-HU. Error bars indicate standard deviations.

Fig. 4.

Individual sickle RBC rheology under transient hypoxia. (A) Time sequence of RBCs traveling through capillary-inspired structures. Arrows indicate sickled cells that are unable to pass through the microgates, thereby obstructing RBC flow. (B) Representative velocity profile of RBC flow, with each data point representing the average speed of an individual RBC traveling through five of the periodic microgates under a pressure difference of 15 mL water in a 60-mL Terumo plastic syringe tube (equivalent to 22.6 mm H2O). The shaded area indicates an O2 concentration lower than 5%. (C) Cell capillary obstruction ratio as a function of %HbS. The arrow indicates a severe case with the highest capillary obstruction ratio. Error bars indicate standard deviations.

Fig. 5.

Role of cell density. (A) Delay time of cell sickling and (B) sickled fraction under the short-term DeOxy state. Error bars indicate standard deviations.