Basic principles for evaluation of less deformable erythrocyte subpopulations with the Microfiltrometer.

The Microfiltrometer consists of a filtration system for diluted erythrocyte suspensions, through a filter containing 30 cylindrical micropores, 5 microm in diameter, under the influence of a driving pressure. A feeding sinusoidal alternating current of 40 kHz, 300 microA is delivered to the filter. The change in impedance is collected for each temporary flow of erythrocytes through a given micropore. Two main parameters are measured for individually explored erythrocytes: the entry time tau in the micropore and the maximal variation of impedance deltaZ occurring for the transitory flow. The slope deltaZ/tau defines the velocity of pore blockage. A "Microfiltrometer Deformability Index" (MDI) is established by using this slope. When MDI > or = 1, the erythrocyte is considered to be deformable and, conversely, when MDI < 1, the erythrocyte is considered to be undeformable. Using this procedure, less than 2% undeformable erythrocytes in healthy blood samples are identified, with a specificity of 99% and a sensitivity of 97.5%.

The Microfiltrometer (MicroFM): a new filtration device for the assessment of less deformable erythrocyte subpopulations.

The hardware of a new filtration device, the Microfiltrometer (MicroFM), is described. The different components of the device; impedance meter, power supply, measuring cell and its 5-micron Oligopore filter are described and it is shown how they are interrelated and interfaced to a computer for data acquisition. The properties of the filter and the general functioning principle of the device are also elucidated. For each run, the MicroFM generates elementary signals from individual passages of many hundreds of red blood cells (RBCs) through micropores of a given 5-micron Oligopore filter. Analysis of each elementary signal provides two complementary parameters, the transit time tau of the explored RBC and the change in electrical impedance deltaZ caused by the temporary flow of the considered RBC through a particular micropore of the filter. These two parameters can be utilized for reliable assessment of erythrocyte deformability on a cellular level.

A standardized parameter for the distribution of individual red blood cell deformability with the cell transit analyzer.

The purpose of this study was to identify a parameter allowing the standardization of the Cell Transit Analyzer (CTA) in order to study the individual deformability of each explored Red Blood Cell (RBC). Using theoretical arguments based on the principle of the CTA, we calculated the thickness "x" of the crown of fluid surrounding each RBC during its entry phase into the micropore. A mathematical equation (x = 62.5/magnitude of dU) was established between the difference of potential (dU, mV) that occurs during this phase and the corresponding thickness ("x", micron) of the crown. By exploring fresh control RBCs of healthy subjects and assuming that the rigid RBCs proportion in a fresh blood sample of healthy subject is less than 3.5%, we performed a thresholding of "x" to distinguish rigid RBC from deformable ones. That thresholding was necessary to stamp the variability of counts linked to polycarbonate filters (PF) used to carry out measures. According to the PF, the value of the threshold "Tx" provided by the thresholding ranged between 0.222 and 0.246 micron. Using the values of "Tx", we counted approximately 10-25% rigid RBCs in the pathological samples of four patients with sickle cells SS disease and of one diabetic patient with splenomegaly. We observed in addition that the percentages of rigid RBCs counted after thresholding are identical from a filter to another one with an absolute error less than 2% in fresh RBCs (normal or pathological) samples. We concluded that the method of standardization by thresholding presented here could be used in clinic routine to count the rigid RBCs percentage contained in a given sample.