A Whole Blood-Based Assay for Assessing Red Blood Cell-Mediated Microcapillary Occlusion in Sickle Cell Disease

Introduction

Sickle cell disease (SCD) is an inherited blood disorder characterized by the production of sickle hemoglobin (HbS). Under hypoxia, HbS polymerizes, causing red blood cells (RBCs) to become sickle-shaped and less deformable. These sickled RBCs can occlude capillaries and contribute to organ damage. We previously developed a microfluidic device to assess RBC-mediated microvascular occlusion under hypoxia using isolated RBCs, which required laborious pre-processing steps to remove white blood cells (WBCs) (Oshabaheebwa et al., 2023). These additional steps increased the assay's complexity, hindering its adoption beyond research laboratories. Here, we present an alternative method to assess RBC-mediated microvascular occlusion under hypoxia using whole blood. We also quantify WBCs' contribution to microcapillary occlusion. This approach significantly reduced the required blood volume, total assay time, number of steps, and overall operation complexity.

Methods

Microfluidic devices were fabricated using standard photolithography and polydimethylsiloxane (PDMS) micro-molding protocols. The device comprised 6 micropillar arrays with interpillar distances decreasing from 12 μm at the inlet to 3 μm at the outlet, mimicking the capillary network (Oshabaheebwa et al., 2024). Venous blood samples were collected in EDTA tubes from participants with HbAA (n=4) and HbSS (n=12) under an IRB-approved protocol. Whole blood was suspended in a solution containing 6% (v/v) EC-Oxyrase (an oxygen-reducing enzyme), 0.13M sodium lactate, and 1X PBS buffer, then incubated for 30 minutes at 37°C to achieve deoxygenation. The volume of whole blood (range: 4-7 µL) was adjusted to achieve 0.2% hematocrit. Samples were perfused through the microfluidic device at a constant inlet pressure of 200 mbar and the temperature was maintained at 37°C. After 15 minutes of perfusion, images were obtained with an Olympus IX83 microscope, and occluding RBCs and WBCs were counted manually in Adobe Photoshop. The occlusion index (OI) was calculated to represent the percent occlusion of the microcapillary network. The OI obtained from this novel whole blood assay was compared to the previously described method of measuring hypoxic OI from isolated RBCs (Oshabaheebwa et al., 2023). To optimize the incubation time, the rate of deoxygenation by EC-Oxyrase was measured using an oxygen sensor (Ocean Insight, Rochester, NY). Samples containing EC-Oxyrase, or a blank PBS buffer, were added to a conical tube, an oxygen probe was immediately inserted, and the partial pressure of oxygen (PO₂) was monitored continuously for 15 minutes.

Results

Hypoxic OI for HbSS whole blood samples (HOI-WB) was significantly higher than for HbAA (mean ± SD: 14.7% ± 4.79% vs. 1.83% ± 0.79%, P = 0.0011). Occlusions from WBCs alone (OI-WBC) in HbAA samples ranged from 0.23% to 1.65%, with total OI (HOI-WB, WBCs + RBCs) between 1% to 2.6%. In HbSS samples, OI-WBC ranged from 1.31% to 2.62%, while HOI-WB ranged from 8.15% to 21.7%. HOI-WB was strongly correlated (PCC=0.9391, P=0.0054) with Hypoxia OI measured with the previously reported assay that used isolated RBCs. The PO₂ in samples containing EC-Oxyrase mixed with either PBS, HbAA, or HbSS diluted whole blood reduced to an average of 0.61 ± 0.2 mmHg within 5 minutes of incubation. Conversely, the PO₂ in a blank PBS buffer without EC-Oxyrase remained at an average value of 158 mmHg.

Conclusion

WBCs made minimal contribution to the microcapillary occlusion thus HOI-WB was mainly driven by RBCs. Moreover, HOI-WB was significantly higher in SCD indicating the assay's sensitivity to HbS-induced RBC impairment. Importantly, HOI-WB strongly correlated with previous OI measurements that used isolated RBCs. While HOI-WB was obtained after 30 minutes of incubation with EC-Oxyrase, samples' full deoxygenation was detected in <5 minutes. Future studies will assess whether similar HOI-WB is observed with 5-minute incubation. This assay utilizes a small blood volume, eliminates need for auxiliary equipment for blood processing, and minimizes the expertise required to run the assay. These improvements in efficiency and simplicity bring us closer to rapid, bedside monitoring of RBC-mediated microcapillary occlusion, and enabling adoption in resource-limited settings.

References

Oshabaheebwa, S, et al. (2023) Blood 142, 5022

Oshabaheebwa, S, et al. (2024) Biosensors and Bioelectronics 258: 116352

Suster:XaTek Inc: Current equity holder in private company, Current holder of stock options in a privately-held company, Patents & Royalties, Research Funding. Mohseni:XaTek Inc: Current equity holder in private company, Current holder of stock options in a privately-held company, Patents & Royalties, Research Funding. Gurkan:XaTek Inc: Patents & Royalties; Hemex Health Inc: Consultancy, Current Employment, Current equity holder in private company, Current holder of stock options in a privately-held company, Patents & Royalties, Research Funding; BioChip Labs Inc: Consultancy, Current Employment, Current equity holder in private company, Current holder of stock options in a privately-held company, Patents & Royalties, Research Funding; DxNow Inc: Patents & Royalties.