Lights, camera, occlusion! watch sickle cells in real time

In this issue of Blood Advances, Morakis et al¹ have captured the direct images of vaso-occlusion forming in the capillary blood vessels of people with sickle cell disease, revealing how red blood cells initiate blockages through adhesion to vessel walls.

Sickle cell disease arises from a mutation in hemoglobin that causes red blood cells to become rigid and sticky when oxygen levels drop. Stiff and adhesive sickle red blood cells interacting with adhesive white blood cells lead to vaso-occlusion, which is blocking of blood flow of small vessels.^2,3 Vaso-occlusions can lead to intense pain, organ damage, and reduced life expectancy.⁴ Understanding exactly how and why the sickle red blood cell and vessel wall interactions and blockages happen has been difficult due to the small size and inaccessibility of capillary blood vessels in the human body. Previous studies relied on laboratory models or animal studies to image and analyze these interactions. Human studies using other imaging methods could observe overall blood flow but lacked the resolution to see individual cells or the precise sequence of events.

In this work, Morakis et al expertly used oblique back-illumination microscopy, a label-free technique that provides high-contrast images of cells deep in tissue using angled light illumination.⁵ By imaging the underside of the tongue (sublingual), where capillaries are close to the surface, they recorded videos of blood cells in motion in patients before and after red blood cell transfusions and in healthy controls.

In the oblique back-illumination microscopy videos from patients with sickle cell disease, the team observed red blood cells sticking to the endothelium, the inner wall of the capillary vessels. These adhered cells, many displaying the characteristic elongated and stiff shaped sickled red cells, formed aggregations leading to stopped flow as additional cells piled up behind them. Occlusions formed and sometimes resolved within seconds to 1 minute. The authors quantified key aspects of blood behavior: the percentage of vessels with fast, slow, or no flow, and the number of adhered red blood cells per vessel.

As expected, patients with sickle cell disease showed poorer flow, fewer fast vessels and more completely stopped ones, and more adhered cells than healthy individuals. Transfusions, which introduce normal red blood cells to blood stream and lower the fraction of abnormal hemoglobin, improved these measures significantly, with better flow and less adhesion.

This study offers a new perspective on sickle cell disease vaso-occlusion formation. While red blood cell involvement is uncontested, the precise order of events leading up to vaso-occlusion remains unclear. In vitro and ex vivo experiments observed that adhesion of deformable red blood cells, followed by trapping of dense sickle red blood cells, drives occlusion.^3,4 Observing the cascade of events leading to capillary occlusion in humans opens new avenues for clinical studies that could improve treatment monitoring with new therapies in sickle cell disease.

Microfluidic in vitro models have greatly advanced our understanding of these processes under tightly controlled conditions.^6,7 Our group has engineered microfluidic platforms that mimic human capillaries to measure red blood cell adhesion, dynamic deformability, and transit times under physiologic flow and hypoxia, including assays that predict clinical responses to treatments.8, 9, 10, 11, 12 Caruso et al has developed endothelialized “microvasculature-on-a-chip” systems that recapitulate multicellular interactions and vaso-occlusive events.^13,14 Wood and colleagues have created high-throughput microfluidic assays that quantify single-cell mechanical properties and oxygen-dependent changes in sickle erythrocytes.^15,16 These in vitro microphysiological models have enabled detailed biophysical studies and therapeutic testing not previously possible in humans.

Notably, Bennewitz et al, Jimenez et al, and Perkins et al have made landmark contributions using intravital microscopy in transgenic sickle cell mouse models.17, 18, 19 Their innovative quantitative intravital lung imaging has revealed that neutrophil-platelet aggregates and thromboinflammatory pathways drive pulmonary vaso-occlusion, often independent of simple red blood cell trapping.^20,21 These studies highlight organ-specific mechanisms and the critical roles of P-selectin and neutrophil extracellular traps in acute lung injury during crisis.

In contrast, murine sickle cell disease intravital microscopy observed white blood cell–endothelial adhesion as the trigger in larger vessels.²² Such conflicting evidence may indicate that occlusions arise through context-dependent mechanisms: occlusions in smaller vessels may be red blood cell-initiated, while those in larger vessels or those triggered by inflammation may be white blood cell–initiated. The authors observed red blood cell-initiated occlusions in capillaries in vivo, including some transient occlusions that form or dissipate in seconds to 1 minute. The association of these occlusions with pain or ischemic damage remains unknown, especially since no subjects were experiencing pain crises. These findings may align with this context-dependent framework and do not exclude the possibility of white blood cell-initiated occlusions in other vascular or inflammatory settings.

Oblique back-illumination microscopy-derived hemorheological indices show potential as new biomarkers of sickle cell disease severity. Hematologic indices, including protein biomarkers, have limited ability to clearly and reliably indicate sickle cell disease severity.²³ While relationships with hemoglobin S percentage, absolute reticulocyte count, and hemoglobin were relatively weak, these hemorheological indices clearly reflected transfusion-induced improvements and differences between controls and sickle cell disease.

Phase contrast of oblique back-illumination microscopy and the design of quantification metrics improve sensitivity in flow evaluation compared to previous studies.²⁴ The superior resolution and phase contrast of oblique back-illumination microscopy allowed visualization of cell shapes, empty vessels, and dynamic events.

There are several limitations of this study. The study population was small and all females. Paired pre- and posttransfusion hemoglobin S measurements were not available for all subjects. Though ventral tongue imaging maximized oblique back-illumination microscopy contrast in superficial capillaries, motion prevented continuous imaging in some areas.

Overall, Morakis and colleagues have opened a new chapter in sickle cell research. By making visible the invisible processes inside living human capillaries, this approach holds great potential to accelerate development of better therapies and deepen our knowledge of disease pathobiology. Congratulations to the authors for this important work.

Disclaimer: This article's contents are solely the author’s responsibility and do not necessarily represent the official views of the National Institutes of Health.

Conflict-of-interest disclosure: U.A.G. and Case Western Reserve University have financial interests in Hemex Health Inc, BioChip Labs Inc, and Xatek Inc. Financial interests include licensed intellectual property, stock ownership, research funding, employment, and consulting. Hemex Health Inc offers point-of-care diagnostics for hemoglobin disorders and anemia. BioChip Labs Inc offers commercial clinical microfluidic biomarker assays for inherited or acquired blood disorders. Xatek Inc offers point-of-care global assays to evaluate the hemostatic process. The competing interests of Case Western Reserve University employees are overseen and managed by the conflict of interests committee according to a conflict-of-interest management plan.