Erythrocytes

Electrical impedance of cells is a sensitive indicator of changes in cellular structure and biophysical characteristics. Integration of electrical impedance sensing in microfluidics can be a useful tool for characterization of blood cells for their disease state, such as sickle cell disease and malaria. The first part of this dissertation presents application of a microfluidics-based electrical impedance sensor for the study of sickle cell disease. Dynamic cell sickling-unsickling process of blood cells in response to cyclic hypoxia was measured. Strong correlation was found between the electrical impedance data and patients’ hematological parameters such as levels of sickle hemoglobin and fetal hemoglobin. In addition, application of electrical impedance spectroscopy in narrow microfluidic channel was used for label-free flow cytometry and non-invasive assay of single sickle cells under controlled oxygen level. We demonstrate the capability of this new technique in differentiating normal red blood cells from sickle cells, as well as sickled cells from unsickled cells, using normoxic and hypoxic conditions. The second part of this dissertation reports an application of electrical impedance sensing for the study of placental malaria. Testing conditions were optimized so that electrical impedance can be used for real time monitoring of different cellular and molecular level variations in this in vitro model of placental malaria. Impedance characteristics of cell proliferation, syncytial fusion and long-term response of BeWo cells to adhesion of infected erythrocytes were obtained and related to the immunostaining results and inflammatory cytokines measurements. Comparing to the conventional optical microscope-based methods, electrical impedance sensing technique can provide a label-free, real-time monitoring tool to study erythrocytes and cytoadhesion, and can further be extended to other disease models and cell types.

Polyethylene glycol (PEG)-induced aggregation, hemolysis and
fusion of chicken red blood ceils (CRBC's) was studied in relation to
the following parameters: osmotic pressure, polymer concentration,
ionic strength, electrolyte species, surface charge removal and
glutaraldehyde fixation. Indirect evidence indicated that osmotic
pressure does not play a primary role in aggregation and fusion of red
blood cells. Maximum aggregation and fusion was obtained in solutions
of 20 and 40% PEG, respectively. In contrast, hemolysis increased
almost linearily with PEG concentration. The negative electrostatic
charge due to the presence of sialic acid on the membrane surface was
found to be the primary factor affecting aggregation, hemolysis and
fusion. Removal of the charge by neuraminidase or screening with cations
enhanced aggregation and fusion while inhibiting hemolysis. The
inhibition of aggregation by glutaraldehyde fixation, and hemolysis
and fusion results not attributed to surface charges, are discussed
in terms of intramembrane interactions with PEG.

Transmission electron micrographs provided evidence that fusion of
erythrocytes occurred after treatment with proteolytic enzymes and
polyethylene glycol (PEG). Fusion was not completed until after the
elution of PEG. Scanning electron micrographs indicated that the
outer membranes of adjacent cells were fused. The effects of the
proteolytic enzymes employed in the cell fusion process were examined.
Treatment of erythrocytes with protease alone was found superior to a
combination of trypsin and protease or to trypsin alone.

The effectiveness of polyethylene glycol as a fusogenic
agent tor avian erythrocytes was evaluated. Parameters
affecting the efficiency of fusion included number of pre-fusion
washes, age of cells, length of exposure to enzyme
solutions at varied pH values, composition of wash solutions
and molecular weights of polyethylene glycol. Fusion was
measured as the percentage of visible polykaryons. Maximal
fusion obtained using polyethylene glycol was approximately
60-70%. Other methods of cell fusion (lysolecithin, Sendai
virus, high calcium concentration at high pH) were compared
to polyethylene glycol. All other means yielded lesser
amounts of fused products. A photographic comparison of the
various methods is presented, and possible mechanisms of
fusion are discussed.