New methods for the investigation of blood-biomaterial interaction.

Quantitative microscopy with integrated image processing is a useful tool for investigation of the interaction of blood components with biomaterials. We have developed new automated measuring devices suitable for simultaneously characterizing biological cells (size, shape, localization, migration, electrophoresis), synthetic particles (electrophoretic fingerprinting), and dialysis membranes (morphology, electric charge). These techniques are useful for the investigation of cell adherence on biomaterials, localization of cells in membrane filters (Chemotaxis), characterization of the protein adsorption on model systems, detection of cytokines (produced after lymphocyte-biomaterial contact), and estimation of morphological properties and charge distribution in dialysis membranes.

Behaviour of white blood cells and the complement system.

Seven different types of dialysers were investigated in five dialysis centres in four countries with respect to behaviour of white blood cells and the complement system. The results of this controlled crossover study demonstrated significant differences in the dialysers. Those containing cuprammonium cellulose (G10-3N and G120 M) showed the greatest changes in white blood cell count, including monocytes and neutrophils, as well as the greatest complement activation. With regard to lymphocytes the subpopulation of low-mobility cells, which were predominantly the B-cells, showed the greatest mobility with dialysers containing cuprammonium cellulose. The PAN copolymer- and PMMA-containing dialysers Filtral and T 150 clearly caused the least changes in white blood cells and complement factors. Dialysers containing cellulose acetate and polysulphone membranes (Duo-Flux Artificial Kidney, CD 4000, and F 60) produced only a moderate decrease of WBC, monocyte, neutrophil, and lymphocyte counts, and this result corresponded to a relatively small change in complement factors.

Detection of charges and their distribution on dialysis membranes with cationic/anionic dyes using confocal laser scanning microscopy.

Methods for the detection of positive or negative charges on the surface of biomaterials/membranes and inside a membrane are important for the characterisation of such materials. We tested different dyes and optimized staining procedures. Under standardized conditions negatively charged membranes were stained with cationic triarylmethane compounds such as crystal violet and positively charged membranes with the anionic anthraquinone dye anthralan blue B. There was no staining of uncharged cellulose membranes. The applicability of these methods was demonstrated on membranes coated to varying degrees with charged compounds such as heparin, these changes in charge being detectible quantitatively by photometry. The distribution of charges inside a membrane was detected by optical sectioning across the stained (FITC labelled poly-L-lysine) membrane using confocal laser scanning microscopy (LSM). LSM offers a completely new application possibility in biomaterial and biocompatibility research.

Biomedical and clinical applications of automated single cell electrophoresis.

The automated single cell electrophoresis microscope Parmoquant has been used for the discrimination of lymphocytes and the study of the interaction of substances with cells and synthetic particles. Electrophoretic histograms allowed the determination of changes in the proportion of lymphocyte populations after kidney transplantation, during dialysis treatment, open heart surgery and during pregnancy. Discrimination of leukemic cells on the basis of electrophoresis was used as an additional parameter in diagnosis. In a mouse tumor model, histogram determination enabled the in vivo effect of the tumor necrosis factor on immune cells to be evaluated. Cell electrophoresis was shown to be suitable to detect the influence of antibodies, lectins and bacteria on the cell surface. Protein adsorption was studied on synthetic particles using cell electrophoresis. This method was applied to investigate the phenomena of blood interaction with biomaterials used in artificial organs and to determine differences in the protein composition of serum or other body fluids connected with diseases.

Chemical modification of cellulosic membranes and their blood compatibility.

Blood compatibility is determined by interactions at the blood-material interface that depend on the material surface chemical structure. Through selective modifications of the chemically reactive hydroxyl groups of cellulose, the aim was to improve the biocompatibility of cellulose membranes. The number of potentially reactive hydroxyl groups on the cellulose membrane surface were reduced through isocyanate cross-links or through the introduction of hydrophilic, hydrophobic, or ionic functionalities by graft copolymerization. To assess blood compatibility, levels of C3a desArg were determined in plasma after membrane contact. Using the electrophoretic mobility test, the release of cytokines were measured after in-vitro incubation of mononuclear cells with membranes. Adsorption of 131J-human fibrinogen was additionally investigated. With respect to the biocompatibility parameters selected, the modified cellulose membranes show improved in-vitro blood compatibility in comparison to unmodified cellulose membranes.

[Correlation of complement activation, cytokine liberation and beta 2 microglobulin in the use of various dialysis and hemofiltration membranes]. / Zusammenhang zwischen Komplementaktivierung, Zytokinfreisetzung und beta 2-Mikroglobulin bei der Anwendung verschiedener Dialyse- bzw. Hämofiltrations-Membranen.

The release of biological active factors from blood cells, e.g. interleukin 1, is of importance with regard to the bioavailability testing of membranes for extracorporeal blood purification. The data from an in vitro system of cytokine release may show a connection between complement activation and cytokine (interleukin) release of membranes. In contrast the results of the generation rate of beta 2-microglobulin did show no definite reference to complement activation or cytokine release. Further time-dependent investigations of the generation rate beta 2-microglobulin are necessary.

Cell electrophoretic investigations of peripheral blood lymphocytes during hemodialysis with different membranes.

Determining the relative percentage of LMC in PBL histograms of RDT patients during hemodialysis we observed a temporary decrease of the LMC proportion during the first 15 minutes of hemodialysis using Cuprophan in contrast to AN 69S and MC Cellulose. These results correlate with the increase of C3a plasma concentration and the decrease of total count of granulocytes. In all investigated RDT patients higher proportions of LMC could be found in relation to healthy donors already before dialysis treatment. Our study therefore seems to indicate that the application of cell electrophoresis is a useful method for the characterization of lymphocytes during extracorporeal circulation as an additional parameter of blood compatibility.

[Cell electrophoresis studies of peripheral blood lymphocytes in patients following kidney transplantation]. / Zellelektrophoretische Untersuchungen an peripheren Blutlymphozyten von Patienten nach Nierentransplantation.

In 111 patients (control group, dialysis patients, kidney recipients) the behaviour of T lymphocytes was observed using an automatic measuring apparatus ( Parmoquant cytopherometer ) and the spontaneous rosette test. The Parmoquant method gives no information on the process of rejection, but it helps to identify risk situations due to bacteria of viruses. In cases of rejection after the second post-operative week, the results of the two methods are reversed.

Fetal pulmonary maturity as determined by particle electrophoresis.

Particle electrophoresis has been used to investigate particles obtained from amniotic fluid. These particles are defined mainly as unilamellar and multilamellar liposomes by electron microscopy. A total of 172 samples of amniotic fluids from 132 pregnancies were included in this study. The electrophoretic results were compared to the total phospholipids and to neonatal outcomes in 42 cases. At least two kinds of particles from amniotic fluids with different electrophoretic mobilities (EPM) are evident. With a ratio of F greater than 1.0, the presence of electrophoretically faster particles was found to be associated with apparent fetal lung maturity. On the other hand when the ratio was F less than 1.0, this seems to indicate an immature fetal lung. Seventy-seven per cent of F values correspond to the total phospholipid content P regarding the prediction of fetal pulmonary maturity resp. immaturity. The correct prediction of fetal lung maturity was demonstrated in 42 patients delivered within 48 hours of amniotic fluid sampling. In 28 cases with a mature F value (greater than 1.0) the correct prediction of respiratory distress syndrome (RDS) was 100 per cent. Eight out of 14 patients with immature F values (less than 1.0) developed RDS. The F values were found to increase as pregnancy progressed. The electrophoretically differentiated amniotic fluid particles behave differently at low pH. Selection and electrophoretic measurement of particles were made in a subjective manner. This preliminary investigation suggests that the results correspond to those obtained by objective cell electrophoretic measurements.

The in vitro effect of horse anti-human lymphocyte globulin on the electrophoretic mobility of human lymphocytes and platelets.

In accordance with earlier results the electrophoretic mobility of lymphocytes is decreased by horse anti-human lymphocyte globulin (AHLG), which correlates with the results of the rosette inhibition test. Under the influence of AHLG, the distribution of the electrophoretic mobilities of lymphocytes shows a characteristic change. In some of the AHLG samples, the antilymphocyte action is accompanied by an antithrombocyte effect. The resulting decrease in the electrophoretic mobility of platelets correlates with the thrombocytopenic effect in vivo. The variation of pH or ionic strength leads to changes of the AHLG effects. The in vivo influence of AHLG on the distribution of the electrophoretic mobility of peripheral blood lymphocytes and platelets is discussed.