Detection of fluorescently labeled microparticles in blood

BACKGROUND: A microsphere-based detoxification system is an adsorption system, whereby microadsorbent particles having diameters of 1-20 microm circulate in an extracorporeal filtrate circle. A thin-wall hollow-fiber membrane filter separates the microparticle-plasma suspension from the bloodstream. For patient safety, it is necessary to have a means to detect membrane ruptures that could lead to a release of microparticles into the patient's bloodstream. METHODS: An optical detection system was developed to monitor the venous bloodstream for the presence of microparticles from the filtrate circuit. For detection purposes, cellulose microspheres, both ferromagnetic and fluorescence labeled, were included with the microsphere adsorbant particles. In the case of a membrane rupture, the labeled particles would also be released into the bloodstream. By illuminating a small volume of blood with an excitation wavelength (590 nm) of the fluorescence marker, the particles can be detected by their emission light at 620 nm. The detector sensitivity is increased by collecting the ferromagnetic and fluorescently labeled microparticles using a magnetic trap. The efficiency of magnetic trap arrangements was tested by adjusting the magnet placements. RESULTS: In vitro experiments were performed by pumping whole blood and labeled microparticles through the fluorescence detector. The efficiency of a magnetic trap arrangement was determined. With an optimal trap setup, 5-10 microl of labeled microparticles can be clearly detected in streaming whole blood. CONCLUSION: An easy to handle microparticle detector was developed, ready for use in particle based blood detoxification systems. The microparticle detection system fulfills the medical and technical requirements to bring the MDS into clinical tests.     

Comparison of specific adsorbents for tumor necrosis factor-alpha and therapeutic anti-tumor necrosis factor-alpha antibodies: an in vitro sepsis model

BACKGROUND: Tumor necrosis factor alpha (TNF-alpha), as a key mediator, represents a major point of attack in sepsis. Since it has been shown that systemic anti-TNF-alpha antibodies do not improve the situation of septic patients, the use of specific adsorption technology in the treatment of sepsis could have beneficial effects. METHODS: Magnetic beads coated with polyclonal or with monoclonal anti-TNF-alpha antibodies were investigated in vitro in order to analyze their ability to prevent TNF-alpha induced adhesion of peripheral blood mononuclear cells (PBMCs) at human umbilical vein endothelial cells (HUVECs). Additionally, therapeutical monoclonal anti-TNF-alpha antibodies were proofed for inhibitory effects of TNF-alpha mediated activation of HUVECs. RESULTS: We have shown, in vitro, that beads coated with polyclonal or monoclonal anti-TNF-alpha antibodies were able to significantly reduce monocyte adhesion. It was possible to decrease monocyte adhesion from nearly 9% to 3% within 2 hours and from 18% to 2% within 6 hours of TNF-alpha treatment by the simultaneous use of beads coated with polyclonal anti-TNF-alpha antibodies. Beads coated with monoclonal anti-TNF-alpha antibodies could even prevent monocyte adhesion within the first 2 hours, and reduced monocyte adhesion to 2% during 6 hours of incubation with TNF-alpha. On the other hand, application of therapeutic anti-TNF-alpha antibodies showed no significant difference compared to the measured monocyte adhesion values of activated endothelial cells. CONCLUSION: Adsorption techniques using specific adsorbents, possibly used in MDS (Microspheres-Based Detoxification System), are efficient in specific reduction of TNF-alpha and pathophysiological consequences, since monocyte adhesion at activated HUVECs was shown to be reduced.     

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.     

Serum complement and protein metabolism in chronic dialysis patients.

1. Patients receiving regular hemodialysis treatment who are permitted to select their own diets are in danger of a protein deficiency manifested by a significant reduction in serum transferrin and in complement C3c. 2. Correlation analysis within the dialysis group revealed no secure connexion between the complement C3c and total hemolytic activity respectively, and transferrin levels. 3. Analysis of diets showed that protein intake and the serum transferrin level correlate. No such correlation was found for the complement C3c or total hemolytic activity. 4. Low transferrin levels in the serum appear to result in more severe anemia among dialysis patients. 5. Knowledge of the serum transferrin level permits much more exact assessment of the protein metabolism in regular hemodialysis patients than knowledge of the total hemolytic activity or the level of the complement C3c.     

Application of a mathematical model to the penetration of amino acids through a dialysis membrane in chronic hemodialysis]

With the help of a one-pool-model from the clearance values measured for 4 different dialysator models the elimination rates for amino acids are calculated with special regard to the essential amino acids. In these cases the body-weight of the patient also belongs to the calculation of the elimination of the amino acids. The loss of the amino acids under the dialysis procedes corresponding to an e-function and is particularly high during the first period of dialysis.     

Stimulation of particle-induced chemiluminescence in human granulocytes by various Escherichia coli strains

The patterns of neutrophil chemiluminescence stimulated by groups of E. coli strains opsonized with pooled normal human serum were compared. All strains of E. coli were obtained from patients with chronic pyelonephritis. 'O' serovars which lacked capsular (K) antigen produced significantly higher chemiluminescence than strains possessing a variety of K antigens. Chemiluminescence produced by a range 'O' serovars bearing the K1 antigen did not differ significantly from those containing other K antigens. The lowest chemiluminescence values were obtained with a group of O2:K1 strains. Rough strains containing K1 antigen were also poor stimulators of chemiluminescence.     

Epstein-Barr virus infections in childhood.

IgM and IgG antibodies against Epstein-Barr virus (EBV) capsid antigen and antibodies against EBV nuclear antigen and heterophil antibodies were investigated in 115 paired sera of children with acute infections and in 100 sera of healthy controls of the same age and sex. EBV-specific IgM antibodies could be recognized in 13.7% of the patients and in 7% of the controls. Antibodies against EBV nuclear antigen were not detected in the IgM-positive sera.     

Influence of membranes on generation of beta 2 M and release of leukocyte lysosomal enzymes

Normal leukocyte functional capacity was investigated by evaluation of phagocytosis of opsonised yeast cells in a radiometric test system. After incubation with dialysis membranes (different cellulosic membranes, polysulfon membrane (PS), polymethylmetacrylate membrane (PMMN), the phagocytosis index, expressed as percent decrease with respect to initial values without membrane, decreased by 10%-25%. The most pronounced effect was observed with PS, cuprophane, modified cellulose and PMMA. The results are not related to differences in the viability of PMN during the test procedure; dead PMN amounted to about 4-6.5%. A significant increase in beta-NAG and beta-Gluc activities was released in the supernatants of the phagocytosis suspensions. This increase activity can be explained by the phagocytosis of PMN but it was not influenced by membrane contact. There was no influence of membrane contact or phagocytosis activity of PMN on the beta 2 M concentration in the supernatant demonstrating that no in vitro generation during incubation with either membrane exists.     

Excretion of antibody-coated bacteria in chronic pyelonephritis

Of 168 urine sediments, which were obtained from 55 patients with chronic pyelonephritis in the course of 3 years when a significant bacteriuria with E. coli was present, we demonstrated antibody-coated bacteria in 81 cases (48.21%). In the active stage of the disease with 54.10% were found significantly more than in the inactive with 32.61%. In obstructive chronic pyelonephritis the positive rate was 54.79% in contrast to 43.16% in non-obstructive chronic pyelonephritis. While in the active stage of the obstructive chronic pyelonephritis with 57.41% more antibody-coated bacteria were excreted only ACB+-, 34.29% only ACB-- and 40% ACB+- and ACB--germs in the course of the disease. The ACB-positive quote was in rough forms with 62.50% statistically significantly increased in contrast to 45.54% in O-typable and 42.86% in non-O-typable strains. In the ACB+-group the immunofluorescence titres to the homologous strain and the C3-activator in the serum as well as the urine lysozyme were significantly higher than in the ACB--group.