Red Blood Cell Adhesion Index in a Patient with Sickle Cell Disease Prior and Post Hematopoietic Stem Cell Transplant (HSCT)

Introduction: Sickle cell disease (SCD) is one of the most common inherited blood disorders and affects over 100,000 individuals in US only. SCD is caused by a single mutation in the β-globin gene that leads to the production of sickle hemoglobin (HbS). Red blood cells (RBCs) from patients with SCD are sticky, rigid, and prone to hemolysis, resulting in a wide range of acute and chronic complications, such as vaso-occlusive crises, acute chest syndrome, cerebrovascular disease, and multi-organ damage. Hematopoietic stem cell transplant (HSCT) is a curative therapy for SCD, that results in stabilization of organ function and gradual amelioration of cerebrovascular and pulmonary complications, as well vaso-occlusive crises. We report a clinically applicable microfluidic device (SCD Biochip) that enables quantitative evaluation of RBC adhesion to endothelium-associated protein-immobilized microchannels before and after HSCT.

Methods: Venous blood samples were collected in EDTA tubes from a 40-year-old male patient with SCD at University Hospitals, Cleveland Medical Center. The sample was tested for abnormal RBCs adhesion with the SCD Biochip at multiple time points from 2016 to 2021. The transplant took place at University Hospitals and we collected the post-transplant blood at two, three and five months. The therapy regimen for the patient before the transplant consisted of transfusions and Hydroxyurea. Fabrication of the microfluidic channels consisted of a glass surface functionalized with Intercellular Adhesion Molecule 1 (ICAM-1) and Laminin (LN), a poly(methyl methacrylate) plastic top (encompassing inlets and outlets), and a sandwiched 50-μm-thick double-sided adhesive tape that defined the height and shape of the microchannels. The samples were injected into the ICAM-1 and LN-immobilized microchannels. An inverted microscope and microscopy camera were used to obtain high-resolution images of whole channel for processing (Adobe Photoshop, San Jose, CA) and quantification of adhered RBCs per unit area (32 mm ²). Adhesion index for ICAM-1 and LN was calculated as the square root of the number of adherent cells counted on the selected areas.

Results: RBC adhesion to immobilized ICAM-1 and LN was analyzed under physiologic flow using a standardized microfluidic platform. Three data points were collected both before and after the HCST. The ICAM-1 RBCs adhesion index before and after HSCT is shown in Figure 1a. The LN RBCs adhesion index before and after HSCT is shown in Figure 1b, the green rectangle represents the normal range value for the HbAA controls as established in our previous studies (1,2). The patient had high adhesion indexes in both ICAM-1 and LN before HSCT, a decrease in adhesion indexes to the normal range was observed after HSCT.

Conclusion: It has been observed that the adhesion index for ICAM-1 and LN were abnormal in the patient with SCD but they decreased to normal levels post-transplant. These results suggest that the SCD Biochip microfluidic devices could be an effective platform to estimate the RBC adhesion level and to validate the efficacy of HSCT in SCD population.

References:

1. Kucukal et al. Blood Adv. 2020 4(15):3688-3698.

2. Kim et al., Microcirculation, 2017, 24, e12374.

Figure 1

View largeDownload slide

Figure 1

View largeDownload slide

Close modal