Hintergrund: Es erscheint aus vielen Gründen wünschenswert, Thrombozytenkonzentrate über mehr als 5 Tage prätrans-fusionell lagern zu können. Daher wurden in den letzten Jah-ren verschiedene Methoden zur Kryokonservierung dieser Zellen entwickelt, wobei sich Dimethylsulfoxid (DMSO) als Kryoprotektivum am geeignetsten erwies. Ziel der vorliegenden Untersuchung war es, Veränderungen der Ultrastruktur von Thrombozyten während des Kryovorgangs aufzuzeigen und die Ergebnisse mit funktionellen Parametern zu vergleichen. Material und Methoden: Einzelspender-Thrombozyten-konzentrate wurden mit DMSO in flüssigem Stickstoff kryokonserviert. Zu verschiedenen Zeitpunkten wurden Proben für die elektronenmikroskopische Analyse entnommen. Am Anfang und Ende des Vorgangs wurden die Fibrinogenbin-dungsfähigkeit der Zellen sowie aggregatorische Eigenschaf-ten gemessen. Ergebnisse: Während sich nach der Isolierung sowie nach Zugabe von DMSO eine nahezu normale Ultrastruktur der Zellen nachweisen läβt, zeigen sich nach dem Auftauvorgang sowie nach Resuspension in autologem Plasma deutliche Zeichen einer beginnenden Zellnekrose. Entsprechend sind sowohl die Aggregationsfähigkeit als auch die Fibrinogenbindungsfähigkeit am Ende der Kryokonservierung reduziert. Schluβfolgerung: Obwohl kryokonservierte Thrombozyten durchaus hämostatische Effekte in vivo aufweisen, scheinen diese durch methodische Weiterentwicklungen noch verbesserbar zu sein.

BACKGROUND During the past decade much work has been carried out towards establishing the optimum method for cryopreservation of platelets. Among the various cryoprotectants dimethyl sulfoxide (DMSO) has been shown to be the most effective. This report describes ultrastructural and functional changes of platelets during the deep-freezing process with DMSO. MATERIALS AND METHODS Single-donor platelet concentrates were cryopreserved in liquid nitrogen by use of DMSO. Before, during and after the freezing process samples were taken for analysis of ultrastructure and platelet function. RESULTS While after isolation and addition of DMSO a normal ultrastructure of platelets could be observed, clear signs of beginning cell necrosis were detected after thawing and resuspension in autologous plasma. Fibrinogen-binding capacity and platelet aggregation were significantly diminished. CONCLUSIONS Although cryopreserved platelets are characterized by hemostatic effects in vivo, it seems conceivable that these effects could be improved by further development of platelet-freezing techniques.

Background: During the past decade much work has been carried out towards establishing the optimum method for cryopreservation of platelets. Among the various cryoprotectants dimethyl sulfoxide (DMSO) has been shown to be the most effective. This report describes ultrastructural and functional changes of platelets during the deep-freezing process with DMSO. Materials and Methods: Single-donor platelet concentrates were cryopreserved in liquid nitrogen by use of DMSO. Before, during and after the freezing process samples were taken for analysis of ultrastructure and platelet function. Results: While after isolation and addition of DMSO a normal ultrastructure of platelets could be observed, clear signs of beginning cell necrosis were detected after thawing and resuspension in autologous plasma. Fibrinogen-binding capacity and platelet aggregation were significantly diminished. Conclusions: Although cryopreserved platelets are characterized by hemostatic effects in vivo, it seems conceivable that these effects could be improved by further development of platelet-freezing techniques.

We investigated the ultrastructural effects of recombinant tumor necrosis factor alpha (TNF) on primary leukemia cells of 8 patients (4 cases of acute myelogenous leukemia and 4 cases of chronic myelogenous leukemia) as well as on bone marrow cells of 1 normal control. The cells were kept in liquid culture for up to 92 h in the presence of up to 10,000 U/ml of recombinant human TNF without adding colony-stimulating factors. Under these conditions the concentration of viable leukemic cells decreased by 14 to 53%, compared to control cultures. In acute myelogenous leukemia, all cases to some degree developed an enlargement of mitochondria; in 2 cases prominent cytoplasmic processes, and in 2 cases cytoplasmic vacuoles were observed. In chronic myelogenous leukemia, an enlargement and deformation of all cell types was observed to varying degrees. In the normal bone marrow sample only minor cytoplasmic changes occurred. In all cultures apoptotic changes were rarely observed and--if present--were observed also in cultures without TNF. When the DNA of leukemic cells treated with TNF was separated on agarose, no fragments characteristic of apoptosis were visible. Our results demonstrate that TNF does not induce direct cytotoxicity or apoptosis in acute or chronic myeloid leukemias and are compatible with the notion that some leukemic cells may be activated or stimulated by TNF. The mitochondrion appears to be one of the primary targets of TNF. Electron microscopy is useful for monitoring the changes induced by TNF.

We report here that tumor necrosis factor alpha (TNF) induces peculiar cytoplasmic vesicles in the human erythromyeloid leukemia cell line K 562, sensitized to the cytotoxic action of TNF by a treatment with the inhibitor of transcription actinomycin D. These vesicles are well delineated ultrastructurally. The formation of these vesicles is characteristic for the combination of actinomycin D with TNF and precedes the changes of apoptosis and cellular disintegration. These vesicles correspond to an intermediate step in the cytotoxicity caused by TNF and may indicate that reactive metabolites are involved in the mechanism of action of TNF.

A newly developed adsorption filter has been evaluated for the removal of white cells (WBCs) from platelet concentrates. It consists of surface-modified polyester fibers and can be used at the patient's bedside. WBC mean removal rate was 98.8 +/- 0.9 percent, and platelet recovery was 99.0 +/- 0.7 percent. The aggregation curves (ADP, collagen) and hypotonic shock response of platelets were not influenced by the filtration, and the plasma electrolyte and lactate dehydrogenase concentrations were unaffected. Transmission and scanning electron micrographs showed no difference in platelet ultrastructure before and after filtration. Determination of posttransfusion platelet recovery did not reveal any significant difference from that of nonfiltered platelets. The tested filter seems to be a suitable device for WBC depletion of single-donor platelet concentrates at the bedside.

A newly developed filter-system (PL 100) for the removal of leukocytes from platelet concentrates was tested. It removes contaminating leukocytes by 99.7 +/- 0.6%. The platelet loss was calculated as 25.7 +/- 9.5%. The filtration process does not influence the filtered platelets; plasma-electrolytes, LDH-concentrations, platelet aggregation curves and serotonin-release remain constant. In addition, platelet ultrastructure is not influenced by the filtration. In spite of the high platelet loss, the tested filter-system seems to be a suitable alternative to the conventional methods for the removal of contaminating leukocytes from platelet concentrates.

A newly developed filter-system (PL 100) for the removal of leukocytes from platelet concentrates was tested. It removes contaminating leukocytes by 99.7 +/- 0.6%. The platelet loss was calculated as 25.7 +/- 9.5%. The filtration process does not influence the filtered platelets; plasma-electrolytes, LDH-concentrations, platelet aggregation curves and serotonin-release remain constant. In addition, platelet ultrastructure is not influenced by the filtration. In spite of the high platelet loss, the tested filter-system seems to be a suitable alternative to the conventional methods for the removal of contaminating leukocytes from platelet concentrates.