PROCOAGULANT AND PROFIBRINOLYTIC ACTIVITIES OF CRYOPRESERVED HUMAN MONOCYTES

The purpose of this study was to compare the ability of flesh and cryo preserved mononuclear cells to generate thrombin, induce fibrin format ion and finally resolve the fibrin formed, when exposed to plasma. Per ipheral blood mononuclear cells (PBM) from 4 donors were collected by gradient centrifugation on Lymfoprep, and cryopreserved in fetal calf serum and 10% dimethyl sulfoxide. Viability was tested by exclusion of trypan blue, as well as green/red fluorescence of fluorescein-diaceta te and ethidium bromide (FDA/EB). Fresh and frozen-thawed cells were s eeded, stimulated with lipopolysaccharide (LPS), and exposed to a stan dard heparinized overlay plasma. Plasma was harvested at intervals (0- 7 days). Thrombin generation and fibrin formation were measured by qua ntification of prothrombin fragment (F1+2) and fibrinopeptide A (FPA) and the fibrinolytic capacity of the cells as the amount of fibrin(oge n) degradation products (FbDP and FgDP). Recovery of cells after thawi ng was about 80%, and the viability of fresh and cryopreserved PBM was >95%. Compared to fresh, frozen cells fully retained their capability of Tissue Factor synthesis, leading to prothrombinase activity (F1+2) and fibrin formation (FPA). In contrast, the fibrinolytic capacity of frozen-thawed cells were significantly reduced. As expected there wer e significant variations between the donors in all the parameters meas ured. We conclude that cryopreservation of human blood mononuclear cel ls is possible with maintainance of the potential of the cells to medi ate coagulation in plasma upon LPS stimulation, whereas the fibrin res olving capacity apparently is reduced by the preservation procedure.