Novel RBC Adhesion and Deformability Assays Reveal Deleterious Effect of Diabetes on RBC Health

Diabetes is one of the most important health issues in the world. The number of people living with diabetes has quadrupled since 1980¹. Cardiovascular disease (CVD), mainly coronary and artery disease, remains the most common cause for morbidity and mortality in people living with diabetes^2,3. A better understanding of the effect of diabetes on the deformability and adhesiveness of red blood cells (RBCs) is essential in assessing disease status and evaluating the risk of CVD, as well as in investigating the effectiveness of potential therapies ^4-6. In this work we investigate the deformability of RBCs and their adhesion to erythrocytes using two proprietary microfluidic assays, OcclusionChip⁷ and Adhesion Biochip^8, respectively. We previously developed these assays for investigating RBC properties.

Occlusion chip assay, consisting of micropillar arrays ranging from 20 µm spacing to 4 µm spacing along the flow direction, creating a gradient of microcapillaries, were fabricated using soft lithography and perfused with isolated RBCs at a constant inlet pressure. Venous samples from diabetic or non-diabetic individuals were collected in EDTA tubes and tested within 48 hours of collection. RBCs were isolated from whole blood and resuspended in PBS at 20% hematocrit, then flown through the chip using a pressure pump for 20 min. The microchannel was then washed and imaged using an inverted microscope. The cells trapped within the micropillar arrays were manually counted to calculate the occlusion index, which we previously established as a biomarker for cell deformability⁹.

To quantify RBC adhesion to endothelial cells, human umbilical vein endothelial cells (HUVECs) were cultured within a microfluidic channel under flow for 48 h. HUVECs were then activated by heme at a concentration of 40 µM for 1 h at 37 °C. Isolated RBCs resuspended in basal cell culture medium at 20% hematocrit supplemented by 10 mM of HEPES were injected in the microchannel for 15 min. Non-adherent RBCs were rinsed with a basal cell culture medium containing HEPES. The remaining adherent erythrocytes were imaged using an inverted microscope and manually quantified.

Our results show that RBCs isolated from individuals with diabetes (A1c > 6.5%) tend to form more occlusion events (OI) than RBCs collected from healthy individuals (A1c < 5.7%), reflecting a loss of deformability (Fig. 1). For comparison we evaluated the elongation index (EI) using ektacytometry (data not shown), and no statistically significant difference was observed. Moreover, RBCs with diabetes tend to be more adhesive to the endothelium than those from healthy individuals, as evident from the count of adherent RBCs (Fig. 2).

In conclusion, our assays revealed that diabetes presents a significant adverse effect on the deformability and adhesion of RBCs. These assays provide a much-needed tool for the quantification of these effects for the assessment of CVD risk and the evaluation of treatment efficacy. Future work will focus on conducting a larger-scale study to confirm our preliminary results. We will test samples from patients in all clinically relevant ranges of disease status, i.e. healthy, pre-diabetes, and diabetes.

References

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