Glutaraldehyde – A Subtle Tool in the Investigation of Healthy and Pathologic Red Blood Cells

Abstract

Glutaraldehyde is a well-known substance used in biomedical research to fix cells. Since hemolytic anemias are often associated with red blood cell shape changes deviating from the biconcave disk shape, conservation of these shapes for imaging in general and 3D-imaging in particular, like confocal microscopy, scanning electron microscopy or scanning probe microscopy is a common desire. Along with the fixation comes an increase in the stiffness of the cells. In the context of red blood cells this increased rigidity is often used to mimic malaria infected red blood cells because they are also stiffer than healthy red blood cells. However, the use of glutaraldehyde is associated with numerous pitfalls: (i) while the increase in rigidity by an application of increasing concentrations of glutaraldehyde is an analog process, the fixation is a rather digital event (all or none); (ii) addition of glutaraldehyde massively changes osmolality in a concentration dependent manner and hence cell shapes can be distorted; (iii) glutaraldehyde batches differ in their properties especially in the ratio of monomers and polymers; (iv) handling pitfalls, like inducing shear artifacts of red blood cell shapes or cell density changes that needs to be considered, e.g., when working with cells in flow; (v) staining glutaraldehyde treated red blood cells need different approaches compared to living cells, for instance, because glutaraldehyde itself induces a strong fluorescence. Within this paper we provide documentation about the subtle use of glutaraldehyde on healthy and pathologic red blood cells and how to deal with or circumvent pitfalls.

Study by optical techniques of the dependence of aggregation parameters of human red blood cells on their deformability

“…Firstly, results in the Fig. 1a show a decrease in the deformability index with the increase of GA J of Biomedical Photonics & Eng 6 223 Jun 2020 © J-BPE 020305-3 concentration that corresponds to the conclusions from the study [16]. The deformability of all GA treated samples is lower than that of the control sample.…”

Heterogeneity of Red Blood Cells: Causes and Consequences

“…3D volume reconstruction of, e.g., confocal recordings are more informative than 2-dimentional images. High resolution 3D-imaging was performed for fixed RBC (Abay et al, 2019). First attempts to get the 3D imaging working for RBCs in flow are undertaken but is not yet available as a high-throughput mode (Quint et al, 2018).…”

An optofluidic “tweeze-and-drag” cell stretcher in a microfluidic channel

“…Glutaraldehyde has been known to be able to cross-link cellular proteins and thus increase the cell stiffness. 52,53 The stiffness of RBCs before and after treatment of glutaraldehyde with a relatively low concentration (0-0.01%) can have moderate differences. [53][54][55] It is known that the stiffness of treated RBCs exhibits a gradual increase with glutaraldehyde concentration.…”

“…52,53 The stiffness of RBCs before and after treatment of glutaraldehyde with a relatively low concentration (0-0.01%) can have moderate differences. [53][54][55] It is known that the stiffness of treated RBCs exhibits a gradual increase with glutaraldehyde concentration. [53][54][55] Therefore, glutaraldehyde treatment with low concentrations is often used to increase the stiffness of RBCs to mimic the change in the cell mechanical properties under physiological conditions and to evaluate the effectiveness of cell mechanical characterization techniques upon the resulting stiffening.…”