Analysis of flow acceleration during erythrocyte filtration: Dependence of hematocrit and cell rigidity

At flow onset the blood filtration rate accelerates to a steady state, this may affect the interpretation of red blood cell (RBC) filterability. We studied the acceleration of flow while the pressure is built up across the filter to analyse effects of various hematocrits and RBC rigidity by glutaraldehyde (GA) hardening. This was analysed by a new filtration system with high time resolution and unlimited filtration volume. The system uses a digital balance that samples the accumulated weight (e.g., filtration rate through 5 μm Nuclepore membranes) with on-line computer communication. The filtration is computer controlled via a pneumatic valve. White blood cells (WBC) were removed prior to filtration by a WBC-eliminating filter to avoid clogging artifacts. When flow is initiated a steady state is reached at 0.3–0.4 s. This timing was also tested and confirmed by a video monitoring technique of filtration flow into a horizontal pipette. The digital balance has a mathematical function to reduce the effects of vibration noise; when this function was activated the apparent acceleration was retarded to 1.2 s. With any of these techniques the steady state timing did not vary with the hematocrit, however, the volume of filtered suspension during acceleration varied with both the hematocrit and the GA hardening (p < 0.001). Extrapolation to yield the initial filtration rate from the relative flow curve (RBC suspension divided by buffer flow) varied depending on if the acceleration phase was included or not. In the most unfavourable situation, with GA-hardened RBC, this difference was 340% (p < 0.01). The slope to calculate clogging rate was affected in a similar way. Moreover, with the most GA-hardened RBC a delay in flow onset was observed with this technique. The acceleration phenomenon may cause artifacts in systems employing volumederived filtration kinetics because of fixed volumes of filtrated medium.     

Crosslinking of membrane proteins in red blood cells from vitamin E-deficient lead-poisoned rats

Red blood cells from vitamin E-deficient rats lose their filterability after incubation in vitro and concurrent lead poisoning of the rats accelerates this decline. This decreased red cell filterability is associated with an increased crosslinking of red cell membrane proteins. Previous reports by others suggested an association between red cell glutathione levels and filterability, but we found no such association in red cells treated with N-ethylmaleimide. Increased aggregation of red cell membrane proteins may play a role in the discocyte/ spherocyte shape change that accounts for the impaired filterability of red blood cells from vitamin E-deficient lead-poisoned rats.     

Kinetics of decrease in erythrocyte filterability under the effect of ephazol]

The effect of palladium-containing complex Ephazol on the filtration rate of erythrocyte suspensions through nuclear filters was studied by the constant-pressure filtration method. It was shown that the filterability of red blood cells incubated with ephazol decreased. If the time necessary for a fixed volume of red blood cell suspension to pass through a filter was plotted against the time of incubation with Ephazol or against its initial concentration, the curves typical of autoaccelerated processes were obtained. From analysis of kinetic models, it was concluded that the effects observed are due to the nonlinear dependence of the filtration rate w on the rate at which an erythrocyte passes through a pore and the influence of Ephazol on the distribution of erythrocytes with respect to w. Several models describing changes in the distribution of erythrocytes with respect to w in the presence of Ephazol and possible mechanisms relating the filtration kinetics to the incubation parameters are discussed.     

A new simple method for determination of erythrocyte filtrability.

A new simple filtration technique designed for measuring red cell filtrability in the routine laboratory use was developed. The suspension of the whole blood in saline (1:20,000 dilution) was processed on the Sartorius filter membranes, pore size 8 micron. The percentage of passed erythrocytes indicating red cell filtrability was determined. The suitability and perspective applicability of this method for studying various hematological disorders is proposed.     

Regulation of red blood cell filterability by Ca2+ influx and cAMP-mediated signaling pathways

To investigate the mechanism of theregulation of human red blood cell deformability, we examined thedeformability under mechanical stress. Washed human red blood cellswere rapidly injected through a fine needle, and their filterabilitywas measured using a nickel mesh filter. The decrease in filterabilityshowed a V-shaped curve depending on the extracellularCa²⁺ concentration; the maximumdecrease was achieved at ~50 µM. The decreased filterability wasaccompanied by no change in cell morphology and cell volume, indicatingthat the decrease in filterability can be ascribed to alterations ofthe membrane properties. Ca²⁺entry blockers (nifedipine and felodipine) inhibited the impairment offilterability under mechanical stress. ProstaglandinsE₁ and E₂, epinephrine, andpentoxifylline, which are thought to modulate the intracellularadenosine 3',5'-cyclic monophosphate (cAMP) level of redblood cells, improved or worsened the impaired filterability accordingto their expected actions on the cAMP level of the cells. These resultsstrongly suggest that the membrane properties regulating red blood celldeformability are affected by the signal transduction system, includingCa²⁺-dependent and cAMP-mediatedsignaling pathways.

     

Filterability of blood cells: methods and clinical applications

It could be claimed that expansion in clinical aspects of haemorheology has largely been fueled by the development of a simple test of blood flow properties, i.e., analysis of filterability. With time, the level of sophistication in equipment, theory and sample preparation has increased. Theories for the development of flow in the filter have been described and these enable cellular parameters, such as transit times, to be calculated from experimental data. These theories can be quite general, and applied to filtration of red or white cells. Ideally, experimental design requires an understanding of cell behaviour at the filter and of the effects of factors such as the sample concentration and volume, and the presence of different types of cells or subpopulations. Otherwise, results are susceptible to misinterpretation, particularly if impurities or mixed populations of cells are present. It is thus very important to know accurately the constituents of the test suspension. In clinical applications the trend has been to move away from whole blood filtration, toward use of relatively pure suspension of separated red cells and white cells. In the area of red cell filtration this has led to reappraisal of some previously reported abnormalities. The relatively new study of white cell filtration should benefit from previous experience, but there is the added problem of the reactivity of the cells under test, and their mixed nature. In any case, critical evaluation of the meaning and clinical relevance of results is necessary.     

Role of leukocyte adhesion in filterability of blood cell suspensions]

We investigated blood cells suspension filterability of 16 donors. The filtration was performed trough 5 microns-pore nuclear filters at constant perfusion pressure 10(5) din/cm2. We estimated also the adherence of leukocytes and platelets on nylon. The adherence of platelets and mononuclear leukocytes reduced the level of the suspension filterability only by 21% (p > 0.05). The presence of nonadhesive polynuclear leukocytes in the suspensions did not change practically their filterability. The addition in the suspensions of adhesive polynuclear leukocytes reduced suspension filterability dramatically.     

Use of polyester leukocyte elimination filters in blood filterability research

We tested a new routine to eliminate leukocytes for blood rheology measurements using commercial leukocyte absorbing filters (here PALL RC400). These filters were punched out and fitted in smaller chambers through which blood was filtered under controlled suction pressure (< 30 mm Hg). This technique resulted in a very effective leukocyte elimination to 0.0022% but also a platelet reduction to 0.2%. The process causes a small but significant hemolysis with free hemoglobin, of the order of 0.06% of the filtered erythrocytes. A small fraction of the erythrocytes were retained in the filter, versus plasma, to reduce the hematocrit on the order of 1.4%. The leukocyte filtration did not cause any detectable functional trauma to the erythrocytes, measured as micro-pore filterability of normal and glutaraldehyde (GA) hardened erythrocytes. However, when 10% of the erythrocytes were hardened with GA, which caused an increase in pore clogging slope (p < 0.05), the additional passage through the leukocyte elimination filter removed this measured change in clogging. This observation suggests that the leukocyte elimination filter may selectively remove, not only leukocytes and platelets, but also hardened erythrocytes. Reticulocyte counting did not reveal any selective removal of young erythrocytes. In general, we find the presented method reproducible, efficient and easy for eliminating leukocytes for blood rheology research although the risk of removing undeformable erythrocytes must be considered.     

Blood filterability in peripheral obstructive arterial disease

The aim of this paper is to test whether an alteration of blood flow in microcirculation and in particular of red cell deformability is present in chronic arterial occlusive disease. To this end we determined by the method proposed by Reid and Dormandy (J. Clin. Pharmacol. 1976, 29, 855) whole-blood filterability in 18 patients with peripheral vascular disease, in 15 clinically healthy subjects and in 99 subjects without clinical evidence of ischemic pathology displaying one or more vascular risk factors. Blood filterability turned out to be significantly lower in cases of arterial disease that in the controls (p less than 0,001), and we found a constant reduction of blood filterability with the increase of number of risk factors. Comparison of the results obtained in arteriopatics and in controls displaying risk factors shows that though risk factors have an important impact on blood filterability, their presence alone is not enough to explain the reduction of blood filterability which, risk factors being equal, in vascular disease is always significantly lower than in controls. We also found a significant correlation between levels of fibrinogen and reduction of blood filterability.     

Mammalian cell retention in a spinfilter perfusion bioreactor

A spinning cylindrical filter is often used to retain mammalian cells in a continuous perfusion bioreactor. This device, known as a spinfilter, has typically been with pore size smaller than the cell particles (single cells or aggregates) in order to achieve cell separation. For single cells in suspension, such an operation cannot be sustained over a long period of time because of clogging of the filter surface. Recently, screens with openings larger than the average cell size have been used to reduce the incidence of clogging. In this article, we have investigated how the screen size affects cell retention. We also showed why it is necessary to optimize the rotational speed of the spinfilter in order to achieve cell retention and reduce screen clogging. Effects of bulk mixing and perfusion rate on screen fouling cell retention, and cell washout were also investigated. (c) 1992 John Wiley & Sons, Inc.     

Erythrocyte filtrability measurement by the initial flow rate method

A new filtration technique, based on the initial filtration rate of a diluted RBC suspension through 5 mu Nucleopore filter is described. As only a few hundreds RBCs traverse each pore and as the measurement are made in a few seconds, the method is by large insensitive to filter plugging and to sedimentation effects. The results are given as a filtration index IF which is, as a first order approximation, independent of the filter conductance and of the suspending medium viscosity. The filtration times are measured electronically. The filters are re-used many times. The influence on the results reproducibility of RBC washing, of the anticoagulant, of the blood sample and the suspension storage times are considered. With our technical procedure, the relative incertitude on the measurement of I.F. is about +/- 10%. The filtration index is shown to be an intrinsic RBC filterability property.     

Blood filterability in cerebral vascular disease

The aim of this paper is to test whether an alteration of blood flow in microcirculation and in particular of red cell deformability is present in cerebrovascular insufficiency. To this end we determined by the method proposed by Reid and Dormandy (J. Clin. Pharmacol. 1976, 29, 855) whole-blood filterability in 31 patients with cerebrovascular disease, in 15 clinically healthy subjects and in 99 subjects without clinical evidence of ischemic pathology displaying one or more vascular risk factors. Blood filterability turned out to be significantly lower in cases of cerebrovascular disease than in the controls (p less than 0,001), while data about presence of risk factors in cerebrovascular insufficiency and in controls are almost overlapping. Reduction of blood filterability in cerebrovascular disease may therefore be attributed directly to the risk factors associated to the disease, which can significantly reduce red cell deformability. Further we didn't find any significant correlation between blood filterability and levels of fibrinogen, platelets, triglycerides and cholesterol in blood.     

Changes in membrane properties of rat red blood cells induced by cadmium accumulating in the membrane fraction

When rat red blood cells were incubated in a cadmium (Cd)-free medium following 1-h pretreatment with 0.5 mM CdCl2, incorporated Cd was retained in the cell during 14-h incubation and progressively accumulated in the membrane fraction, especially in the cytoskeleton fraction. In parallel to this accumulation, red cell filterability decreased and echinocytic cells increased, although intracellular ATP was maintained at the control level. The echinocytic shape was maintained in ghosts and cytoskeletons prepared from the Cd-loaded cells. In addition, the association of bands 2.1, 3, 4.2, and 4.5 with cytoskeletons increased and dissociation of cytoskeletal networks at low ionic strength decreased as the incubation time increased. Pretreatment of red blood cells with Cd also induced a release of small vesicles. These vesicles contained hemoglobin but were depleted of spectrin and actin, showing a phospholipid composition similar to that of red cell ghosts. These results suggest that the organization of cell membranes, especially cytoskeletal networks, is altered by Cd accumulation in the cytoskeleton fraction, which results in acceleration of age-related changes of red blood cells such as shape change and decreased filterability.     

Blood filterability in ischemic heart disease

The aim of this paper is to test whether an alteration of blood flow in microcirculation and in particular of red cell deformability is present in myocardial ischaemia. To this end we determined by the method proposed by Reid and Dormandy (J. Clin. Pharmacol. 1976, 29, 855) whole-blood filterability in 23 patients with myocardial ischaemia, in 15 clinically healthy subjects and in 99 subjects without clinical evidence of ischemic pathology displaying one or more vascular risk factors. Blood filterability turned out to be significantly lower in cases of coronary diseases than in controls (p less than 0,001), while it was lower but not significantly so, than in subjects displaying risk factors. We conclude therefore that the presence of risk factors is enough important to be considered one of the fundamental elements (though non the only one) in causing the reduction of blood-filterability encountered in ischemic cardiopathology.     

Wall shear stress in backward-facing step flow of a red blood cell suspension.

An experimental investigation of the wall shear stress distribution downstream of a backward-facing step is carried out. The wall shear stress distribution was determined by measuring the deformation of a gel layer, attached to the wall downstream of the step. Speckle pattern interferometry was applied to measure the deformation of the gel layer. The measured deformation, combined with the properties of the gel layer, served as an input for a finite element solid mechanics computation to determine the stress distribution in the gel layer. The wall shear stress, required to generate the measured deformation of the gel layer, was determined from these computations. A Newtonian buffer solution and a non-Newtonian red blood cell suspension were used as measuring fluids. The deformation of the gel layer was determined for a Newtonian buffer solution to evaluate the method and to obtain the properties of the gel layer. Subsequently, the wall shear stress distribution for the non-Newtonian red blood cell suspension was determined for three different flow rates. The inelastic non-Newtonian Carreau-Yasuda model served as constitutive model for the red blood cell suspension. Using this model, the velocity and wall shear stress distribution were computed by means of a finite element fluid mechanics computation. From the comparison between the numerical and the experimental results, it can be concluded that wall shear stresses, induced by the red blood cell suspension, can be modeled accurately by employing a Carreau-Yasuda model.     

Scaleup of spinfilter perfusion bioreactor for mammalian cell retention

A spinning cylindrical filter, known as a spinfilter, permits the mammalian cell bioreactor operation at high perfusion rates leading to very high cell densities (10(7) mL(-1)). Filter screens with openings (25 mum) slightly larger than the average cell size have been used to retain single cells in suspension over a long period of operation without clogging. We have previously shown why it is necessary to optimize the rotational speed of the spinfilter in order to achieve efficient cell retention and avoid potential screen clogging. Effects of bulk mixing and perfusion rate on screen fouling and cell retention were also investigated. Based on this analysis, in this article, we suggest strategies for scaleup of spinfilters. Experimental data from 12- and 175-L (working volume) bioreactors is shown in support of the scaleup analysis. (c) 1994 John Wiley & Sons, Inc.     

Differences between cation-osmotic hemolysis and filterability in exaprolol- and glutaraldehyde-treated human red blood cells

The changes in human red blood cell microrheology in different glutaraldehyde (3.0 and 5.0 x 10(-6) mol x l(-1)) and exaprolol (2.5 and 5.0 x 10(-4) mol x l(-1)) concentrations were studied. The method of millipore filtration was compared with the method of cation-osmotic hemolysis. Both drugs prolonged the filtration time. Cation-osmotic hemolysis in glutaraldehyde-treated cells was significantly lower in comparison with the control group. On the other hand, there was a significant increase in cation-osmotic hemolysis in exaprolol-treated cells. Besides cation-osmotic hemolysis and filterability of erythrocytes, we evaluated the medium cell volume (MCV) and the medium cell hemoglobin concentration (MCHC). No changes in MCV and MCHC in glutaraldehyde-treated cells were observed. However, the MCV was significantly lower and the MCHC was significantly higher in exaprolol-treated cells. In conclusion, we suggest that the method of cation-osmotic hemolysis is more sensitive than the filtration method for determination of red blood cell microrheology.     

Deformability of stored erythrocytes as a criteria for their in vivo survival rate

The loss of deformability observed in erythrocytes stored as whole blood for 36 days (ACD-AG) or as buffy-coat free erythrocyte concentrate (EK) was characterized by measuring their filterability. During the first 3 weeks the index of filterability for ACD-AG erythrocytes increased only slightly and rose to about 140% of its initial value on the 36th day. In contrast, a heavy loss of deformability (increase of the filterability index to more than 600%) was detected for erythrocytes from EK, which, from a rheological point of view, is apt to raise doubts of using this stored blood. An incubation of 1 hour at 37 degrees C in fresh plasma did not result in improving the deformability. A cell volume loss of more than 20% connected with an increase of the inner viscosity to more than 400% was found to be the cause of this decrease of deformability. These rheological differences are also reflected in the 24 hours in vivo survival rate (SR), if the "early loss" of damaged erythrocytes immediately after transfusion is taken into account. Whereas the SR values of 80% for whole blood erythrocytes do not change significantly, the SR values for EK values can be found to reach 54% approximately.     

Membrane filtration of food suspensions.

Factors affecting the membrane filtration of food suspensions were studied for 58 foods and 13 membrane filters. Lot number within a brand, pore size (0.45 or 0.8 micrometer), and time elapsed before filtration had little effect on filterability. Brand of membrane filter, flow direction, pressure differential, age (microbiological quality) of the food, duration of the blending process, temperature, and concentration of food in the suspension had significant and often predictable effects. Preparation of suspensions by Stomacher (relative to rotary blender) addition of surfactant (particularly at elevated temperature) and prior incubation with proteases sometimes had dramatic effects of filterability. In contrast to popular opinion, foods can be membrane filtered in quantities pertinent to the maximums used in conventional plating procedures. Removal of growth inhibitors and food debris is possible by using membrane filters. Lowering of the limits of detection of microorganisms by concentration on membrane filters can be considered feasible for many foods. The data are particularly relevant to the use of hydrophobic grid-membrane filters (which are capable of enumerating up to 9 X 10(4) organisms per filter) in instrumented methods of food microbiological analysis.     

EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. I. Collection of Virus Samples

EPA Method 1615 was developed with a goal of providing a standard method for measuring enteroviruses and noroviruses in environmental and drinking waters. The standardized sampling component of the method concentrates viruses that may be present in water by passage of a minimum specified volume of water through an electropositive cartridge filter. The minimum specified volumes for surface and finished/ground water are 300 L and 1,500 L, respectively. A major method limitation is the tendency for the filters to clog before meeting the sample volume requirement. Studies using two different, but equivalent, cartridge filter options showed that filter clogging was a problem with 10% of the samples with one of the filter types compared to 6% with the other filter type. Clogging tends to increase with turbidity, but cannot be predicted based on turbidity measurements only. From a cost standpoint one of the filter options is preferable over the other, but the water quality and experience with the water system to be sampled should be taken into consideration in making filter selections.