Fluorescent Ghosts

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Johanna Clauser

Chief Scientist Research & Validation

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+49 241 80 89356

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Background

Artificial cardiovascular support systems, such as blood pumps or heart valves, are used in many different ways in the therapy of cardiovascular diseases. Despite constant progress in development, their use is often associated with serious complications, such as the increased destruction of red blood cells, hemolysis.
During hemolysis, the red blood pigment, hemoglobin, leaks into the blood plasma and can thus no longer contribute to the vital transport of oxygen. Minimizing hemolysis is therefore a key objective in the development of blood-bearing medical devices.
To investigate the flow in blood-carrying medical devices, methods of optical flow analysis, such as Particle Image Velocimetry (PIV) with a blood substitute fluid are used.
In standard-compliant in-vitro experiments, blood is used as the test medium. These experiments do not allow conclusions to be drawn about the exact site of hemolysis and only the blood damage to the entire system can be quantified. The PIV measurement, on the other hand, allows for a spatially resolved flow investigation and can reveal possible points of origin for hemolysis. Instead of blood, however, a PIV fluid is used, which in many respects does not match the characteristics of blood.
These two aspects can be combined with the help of so-called ghost cells. Ghost cells are erythrocytes from which the hemoglobin has been partially removed. The shape and size of ghost cells is comparable to the original erythrocytes, but the ghost cells are much more transparent.

Method

For the production of Ghostcells, porcine erythrocytes are brought from the slaughterhouse to controlled lysis in a strongly hypotonic buffer solution by osmotic pressure. The membrane becomes permeable for ions and larger molecules such as hemoglobin, which diffuse into the surrounding fluid. The structure of the membrane remains intact during the process. After lysis, the membrane is sealed and the original membrane permeability is restored. Ghost cells have the same shape and size as normal erythrocytes, but are more transparent to visible light due to the lower hemoglobin content. Figure 1 shows a microscopic image of the ghost cells under phase contrast.
In addition to optical measurements on blood-analogous Ghostcell suspensions, it is possible to load the Ghostcells with a calcium dicitrato complex during controlled lysis. This allows for the first time the visualization and spatial measurement of hemolysis.
A sensitive fluorescent calcium indicator is dissolved in the carrier fluid outside the cells. First, indicator and calcium are separated by the intact membrane of the ghost cells. If, for example, a mechanically induced hemolysis of the cell occurs, indicator and calcium come into contact and fluoresce under excitation of an external light source. The fluorescence signal is recorded by a camera.
The innovation of the Fluorescent Hemolysis Detection (FHD) method is of particular interest with large volume production, which requires larger amounts of test fluid to test medical devices. Based on this motivation, a Large Volume Batch Production System (LVBPS) has already been realized in the institute, with which the efficiency for the production of ghost cells could be significantly increased. With the LVBPS, sufficient test fluid could be produced to simulate a typical in-vitro hemolysis test with two comparison circuits of up to 450 ml blood volume each. Furthermore, it could be shown that loaded ghost cells are comparable to erythrocytes in essential properties such as rheology and impermeability.

Project objective: In vitro validation

In the Ghostcell follow-up project, the FHD is to be combined, optimized and validated with PIV in vitro to finally enable spatially resolved hemolysis detection in blood-bearing medical devices.
Therefore, the influence of PIV particles on the flow properties of a Ghostcell suspension will be investigated. Furthermore, the refractive index of the fluid used for PIV measurements has to be adjusted to that of the blood-carrying components by means of additives in order to guarantee the best possible optical resolution of the measurements.

The mechanical load capacity of Ghostcells due to shear forces

To compare the mechanical stress capacity of intact red blood cells and Ghostcells, a scale will be developed that relates the plasma hemoglobin (fPHb) released by hemolysis directly to the Ca2+ released from Ghostcells as a function of shear stress.
To validate the measurement accuracy of FHD, a study will be performed to investigate the spatial and temporal resolution limit of several hemolysis hotspots. Finally, after successful completion