Imaging Erythrocyte Sedimentation in Whole Blood

Abstract

The erythrocyte sedimentation rate (ESR) is one of the oldest medical diagnostic tools. However, currently there is some debate on the structure formed by the cells during the sedimentation process. While the conventional view is that erythrocytes sediment as separate aggregates, others have suggested that they form a percolating gel, similar to other colloidal suspensions. However, visualization of aggregated erythrocytes, which would settle the question, has always been challenging. Direct methods usually study erythrocytes in 2D situations or low hematocrit (∼1%). Indirect methods, such as scattering or electric measurements, provide insight on the suspension evolution, but cannot directly discriminate between open or percolating structures. Here, we achieved a direct probing of the structures formed by erythrocytes in blood at stasis. We focused on blood samples at rest with controlled hematocrit of 45%, from healthy donors, and report observations from three different optical imaging techniques: direct light transmission through thin samples, two-photon microscopy and light-sheet microscopy. The three techniques, used in geometries with thickness from 150 μm to 3 mm, highlight that erythrocytes form a continuous network with characteristic cracks, i.e., a colloidal gel. The characteristic distance between the main cracks is of the order of ∼100 μm. A complete description of the structure then requires a field of view of the order of ∼1 mm, in order to obtain a statistically relevant number of structural elements. A quantitative analysis of the erythrocyte related processes and interactions during the sedimentation need a further refinement of the experimental set-ups.

Early stage of erythrocyte sedimentation rate test: Fracture of a high-volume-fraction gel

“…We performed experiments using light sheet microscopy (Z1, Zeiss, Jena, Germany), as described in a previous methodological publication ( 26 ). This technique allows enough resolution to extract the velocity field of the RBCs in the obtained image sequences using particle image velocimetry (PIV, through PIVLab ( 30 )), but only probes a small part of the gel close to its lateral edge.…”

“…To investigate the larger scale structure of RBC gels, we utilized microscopy with infrared light transmission through thin samples. We followed a similar procedure outlined in a previous paper which used blue light ( 26 ); however, we replaced the blue LED source with a halogen lamp (Nikon, LHS-H100P-1). The emitted light was filtered by an infrared long pass filter with a cutting wavelength of 950 (Neewer, IR950).…”

“…We previously performed this kind of imaging with blue light, to obtain a better contrast. However, the transmission through RBCs is then so low that only part of the structure homogeneity could be observed ( 26 ). The lighter parts in the observed structures are therefore depleted in RBCs, as observed for the streamers in recent numerical simulations of gel settling ( 27 ).…”

“…The origin of colloidal gel sedimentation delay is still debated; however, it is likely to be associated with gel aging and the development of cracks for fluid flow within the gel ( 15 , 16 , 18 , 21 , 23–25 ). Surprisingly, contrary to colloidal hard sphere suspensions, an increase in attractive interactions between RBCs results in gel destabilization, leading to faster structure rearrangement and apparition of cell-depleted cracks, which collapses faster ( 13 , 26 ). This feature likely contributed to the establishment of the ESR as a medical tool, as a reduced delay and an increased collapse velocity are additive for the typical medical readout, which considers the average velocity of the interface during the first hour ( 1 , 5 , 6 ).…”

Red Blood Cell Sedimentation Index Using Shear Stress of Blood Flow in Microfluidic Channel

“…Without an external actuator, RBCs are sedimented owing to the gravitational force in a tube. According to this measurement method, the ESR is obtained as a sedimentation velocity (mm/h) from a visual detection of the sedimentation front with an elapse of 1 h. During the sedimentation of the RBCs, they aggregate and form continuous networks [ 10 ]. The networks of the colloidal gels then collapse [ 11 ].…”