Ghost Cells

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Malte Schöps

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+49 241 80 88352

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Background

Cardiovascular support systems, as for example blood pumps or heart valves, are used in a variety of therapies for cardiovascular diseases. Despite technical development, their application often causes serious complications, such as severe destruction of red blood cells, called hemolysis.

When red blood cells are damaged, hemoglobin, the red blood pigment, leaks into blood plasma and the cell loses its ability to carry oxygen. Therefore, one of the main targets of developing cardiovascular support systems is the minimization of hemolysis.

Currently, the blood flow in medical blood conducting systems can be estimated by optical flow measuring analysis by means of Particle Image Velocimetry (PIV). For those measurements, transparent blood substitute solutions are necessary.

Blood is used as the test medium in standard in-vitro experiments. These tests do not allow for drawing conclusions about the exact location of the hemolysis and only the blood damage to the entire system can be quantified. In turn, PIV measurements can be used to investigate the flow with spatial resolution, but cannot be conducted with blood. Unfortunately, the current available blood substitute solutions do not share sufficient characteristics with blood.

With the help of ghostcells, these two aspects can be combined. Ghostcells are erythrocytes that have been partially deprived of the hemoglobin. The shape and size of ghostcells is comparable to the original erythrocytes, but ghostcells are optically much more transparent.

  Microscopic image of red blood cells and ghost cells Copyright: AME Figure 1: microscopic image of ghostcells under phase contrast

Methods

To produce ghostcells, porcine erythrocytes are suspended in a hypotonic buffer solution for controlled lysis. Due to the osmotic pressure, the membrane becomes permeable to larger molecules such as hemoglobin that diffuse through the red blood cells membrane. The structure of the membrane remains intact throughout the procedure. After lysis, a resealing of the membrane takes place, restoring the original impermeability. Ghostcells have the same shape and size as normal erythrocytes but are more transparent to visible light due to the lower intracellular hemoglobin concentration. This makes them more transparent and they can be used for PIV-measurements.

Besides the possibility to perform optical flow measurements based on ghostcells, there is also the option to load the cells during controlled lysis with a calcium-dicitrato-complex. This procedure allows hemolysis hotspots to be visualized and spatially resolved hemolysis detection becomes possible, as shown in Figure 2.

  Fluorescent Hemolysis Detection Copyright: AME Figure 2: Process of an erythrocyte via controlled lysis to the loaded ghostcell   Fluorescent Hemolysis Detection Copyright: CVEAME Figure 3: Fluorescent Hemolysis Detection (FHD), extracellular fluorescence calcium indicator reacts with intracellular calcium

When a calcium sensitive fluorescent indicator is suspended in the artificial plasma surrounding the ghostcells, the indicator binds leaking calcium from damaged cells and emits a fluorescent signal that can be recorded with a camera, called Fluorescent Hemolysis Detection (FHD) and shown in Figure 3.

Projects

The functional principle of novel spatially resolved hemolysis detection has already been demonstrated by measurements in a 9.5 mm diameter channel with a hematocrit of 44 %, but unfortunately the production of ghostcells is currently not efficient. The yielded volume in this time-consuming process is too small for a ghostcell suspension to be used as a standard blood analogue fluid for in-vitro tests.

One of the key goals is to produce ghostcells semi-automatically in reproducible quality and large volumes in order to enable spatially resolved detection of hemolysis using Particle Image Velocimetry in blood conducting devices.

 
Founded by

This project was funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) – 321130633.