Glutaraldehyde – A Subtle Tool in the Investigation of Healthy and Pathologic Red Blood Cells (1)
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.
Especially for rare red blood cell (RBC)-related diseases, cell shape is an important diagnostic parameter and fixation is an approach to circumvent the subtle sample transportation challenge.
PBS solutions (39.4 ml) with various concentrations of glutaraldehyde (various suppliers) were prepared in 50 ml tubes. Packed RBCs (600 μl) were pipetted slowly into the solution. The large tube and the high solution/cell volume ratio (65:1) was chosen to make sure the sample had sufficient volume to fix as individual cells and would not form aggregates. The tubes were placed on a tube roller for 1 h to allow for glutaraldehyde to fix. After the fixation procedure, glutaraldehyde was removed by washing the cells 3 times with PBS and resuspended in the same solution.
Percentages above 0.01% showed almost no hemoglobin in the supernatant. At lower concentrations, increasing in glutaraldehyde percentage increases the hemolysis rate compared to unfixed control cells until glutaraldehyde reaches a concentration where RBCs are fixed. Very low concentrations of glutaraldehyde (here: 0.0001%) resemble the mechanical behavior of living cells, while concentrations above 0.01% glutaraldehyde are indistinguishable in their elongation index plot. Closer shapes were shown in the latter range compared living RBCs with 0.001 and 0.0025% glutaraldehyde treated RBCs. It is sufficient to dilute the blood sample to hematocrits of 5% in a solution of lower viscosity, e.g., PBS.
In a glutaraldehyde solution monomers and polymers coexist. Both monomers and polymers have different “fixation properties.” In particular, the higher efficiency in crosslinking due to monomers or polymers is discussed controversially. The ratio of absorbance between 280 nm (monomer) and 235 nm (polymer) can be taken as the monomer-polymer ratio. The storage temperature of glutaraldehyde is among the most important parameters for a stable preservation of the stock solution. Glutaraldehyde is most stable at -20°C. The higher the temperature, the faster the increase in polymer formation.
Long staining with high (1%) concentrations of glutaraldehyde prevents binding of the antibodies.
(i) Store glutaraldehyde at -20°C in aliquots to avoid unnecessary thawing of the whole bottle for each use. Use these aliquots to prepare fresh solutions before treating the cells. This allows a maximal consistency within a series of measurements in a particular laboratory over time.
(ii) The glutaraldehyde concentration to be used depends on the purpose and needs to be determined for the particular application. In general, concentrations between 0.05 and 0.1%.
(iii) Measure the osmolality of the glutaraldehyde containing solution and correct it by diluting the suspension buffer when fixing with 0.5% or higher concentrations of glutaraldehyde.
(iv) Check the monomer/polymer ratio of the glutaraldehyde using a spectrometer and provide this ratio in the publications as a way to help interlaboratory comparability.
(v) Consider the appropriate staining dye and its concentration in relation to the induced autofluorescence. Staining based on the recognition of protein structures (antibodies) should be pre-tested on glutaraldehyde treated positive controls to show the feasibility.