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Auxiliary partial liver transplantation: Intraoperative changes in fibrinolysis
133
ANTI-HEPATOCELLULAR-MEMBRANE-ANTIBODIES, CRITICAL ANALYSIS OF FREQUENCY OF OCCURENCE AND SPECIFICITY T. Poralla, A. Roth, U. Treichel, G. Gerken, M. Manns, K.H. Meyer zum BEischenfelde First Department of Internal Medicine, J. Gutenberg U n i v e r s i t y , Mainz, Fed. Rep. Germany
Recently several investigators have described anti-hepatocellular-membrane-antibodies (AHMA) in various liver diseases, predominantly autoimmune chronic active hepatitis (ai-CAH). Moreover these antibodies were suggested to be of pathogenic relevance in immune-mediated destruction of hepatocytes in inflammatory liver diseases. We, therefore, analysed the influence of different methods for the preparation of hepatocellular membranes on the results of AHMA-determinations. Following preliminary investigations four widely used techniques for the preparation of hepatocellular membranes were choosen for detailed analysis (Touster,O. et al. J. Cell Biol. 47:604-18, 1970, Toda,G. et al. Biochim. Biophys. Acta 413:52-64, 1975, H u b b a r d , A . L . et al. J. Cell Biol. 96:217-29, 1983, and Meier, P.J. et al. J. Cell Biol. 98:991-1000, 1984). The various preparations were compared by using SDS-Polyacrylamide-gelelectrophoresis, determination of marker-enzymes, electron-microscopical analysis and according to t h e i r reactivity to a number of monoclonal antibodies recognizing different subcellular structures of hepatocytes. Thereafter the different membrane preparations were used to determine the occurence of AHMA by using ELISA-tests. The different membrane preparations exhibited profound variations in terms of antigen yield, contamination with intracellular structures and enriched protein components. The reactivity of sera from 100 patients with various inflammatory liver diseases against the membrane preparations showed great variations, even though ai-CAH s e r a demonstrated the highest frequency of AHMA. In summary our results clearly demonstrate, that the frequency and specificity of AHMA largely depends on the membrane preparation used. Contamination with intracellular s t r u c t u res was common. Thus, AHMA-tests should be interpreted with caution.
134
AUXILIARY PARTIAL LIVER TRANSPLANTATION: INTPJ~OPERATIVE CHANGES IN FIBRINOLYSIS R.] Porte, EAR Knot, MPM de Maat, PJA Willemse, SW Schalm, J Stibbe, THN Groenland, OT Terpstra. Dept of Int Med I I , Hematology, Anesthesiology and Surgery, Univ Hosp Dijkzigt, Rotterdam, The Netherlands.
Liver transplantation is associated with severe hemostatic disturbances and a bleeding d~athesis. !qcreased f i b r i n o l y t i c a c t i v i t y has been mentioned as a possible causative factor in the occurance of uncontrollable bleeding. Lack of hepatic clearance during the anhepatic phase or release of plasminogen activators after graft-recirculation have been proposed as the underlying mechanisms in orthotopic l i v e r transplantation (OLT). During heterotopic transplantation of an auxiliary l i v e r graft (APLT), no anhepatic phase occurs. We examined f i b r i n o l y t i c a c t i v i t y in 8 cases of APLT by using thromboelastography (TEG) and specific measurement of plasma levels of tissue plasminogen activator a c t i v i t y (tPA-act) and antigen (tPA-Ag) and i t s inhibitor (PAl). In 6 cases no signs of intraoperative f i b r i n o l y s i s were detected by TEG, while increased f i b r i n o l y t i c was found in the period before graftrecirculation in one patient and 60 min after recirculation in another case. The 6 patients who did not develop significant f i b r i n o l y t i c a c t i v i t y had high plasma,levels of PAI( 25 IU/ ml; ref value: 0-25 IU/ml) during the operation and no or low levels of tPA ( 80 mIU/ml; ref value: 0-200 mIU/ml). In the two patients that demonstrated increased f i b r i n o l y t i c a c t i v i t y , very high levels (max. 8840 mIU/ml and 3760 mIU/ml) of tPA-act were measured. Increased tPAact lasted for 3 to 3.5 h and was caused by a sharp, 2-3 fold increase of tPA-Ag to 60ng/ml (ref value: 5-10 ng/ml) and a concurrent decrease of PAl. Since we observed increased f i b r i n o l y t i c a c t i v i t y in APLT, lack of hepatic clearance may not be the only cause of increased f i b r i n o l y s i s in OLT. Our findings suggest that f i b r i n o l y s i s in l i v e r transplantation is due to an increased release of tissue plasminogen activator (tPA).
$69
ANTI-HEPATOCELLULAR-MEMBRANE-ANTIBODIES, CRITICAL ANALYSIS OF FREQUENCY OF OCCURENCE AND SPECIFICITY T. Poralla, A. Roth, U. Treichel, G. Gerken, M. Manns, K.H. Meyer zum BEischenfelde First Department of Internal Medicine, J. Gutenberg U n i v e r s i t y , Mainz, Fed. Rep. Germany
Recently several investigators have described anti-hepatocellular-membrane-antibodies (AHMA) in various liver diseases, predominantly autoimmune chronic active hepatitis (ai-CAH). Moreover these antibodies were suggested to be of pathogenic relevance in immune-mediated destruction of hepatocytes in inflammatory liver diseases. We, therefore, analysed the influence of different methods for the preparation of hepatocellular membranes on the results of AHMA-determinations. Following preliminary investigations four widely used techniques for the preparation of hepatocellular membranes were choosen for detailed analysis (Touster,O. et al. J. Cell Biol. 47:604-18, 1970, Toda,G. et al. Biochim. Biophys. Acta 413:52-64, 1975, H u b b a r d , A . L . et al. J. Cell Biol. 96:217-29, 1983, and Meier, P.J. et al. J. Cell Biol. 98:991-1000, 1984). The various preparations were compared by using SDS-Polyacrylamide-gelelectrophoresis, determination of marker-enzymes, electron-microscopical analysis and according to t h e i r reactivity to a number of monoclonal antibodies recognizing different subcellular structures of hepatocytes. Thereafter the different membrane preparations were used to determine the occurence of AHMA by using ELISA-tests. The different membrane preparations exhibited profound variations in terms of antigen yield, contamination with intracellular structures and enriched protein components. The reactivity of sera from 100 patients with various inflammatory liver diseases against the membrane preparations showed great variations, even though ai-CAH s e r a demonstrated the highest frequency of AHMA. In summary our results clearly demonstrate, that the frequency and specificity of AHMA largely depends on the membrane preparation used. Contamination with intracellular s t r u c t u res was common. Thus, AHMA-tests should be interpreted with caution.
134
AUXILIARY PARTIAL LIVER TRANSPLANTATION: INTPJ~OPERATIVE CHANGES IN FIBRINOLYSIS R.] Porte, EAR Knot, MPM de Maat, PJA Willemse, SW Schalm, J Stibbe, THN Groenland, OT Terpstra. Dept of Int Med I I , Hematology, Anesthesiology and Surgery, Univ Hosp Dijkzigt, Rotterdam, The Netherlands.
Liver transplantation is associated with severe hemostatic disturbances and a bleeding d~athesis. !qcreased f i b r i n o l y t i c a c t i v i t y has been mentioned as a possible causative factor in the occurance of uncontrollable bleeding. Lack of hepatic clearance during the anhepatic phase or release of plasminogen activators after graft-recirculation have been proposed as the underlying mechanisms in orthotopic l i v e r transplantation (OLT). During heterotopic transplantation of an auxiliary l i v e r graft (APLT), no anhepatic phase occurs. We examined f i b r i n o l y t i c a c t i v i t y in 8 cases of APLT by using thromboelastography (TEG) and specific measurement of plasma levels of tissue plasminogen activator a c t i v i t y (tPA-act) and antigen (tPA-Ag) and i t s inhibitor (PAl). In 6 cases no signs of intraoperative f i b r i n o l y s i s were detected by TEG, while increased f i b r i n o l y t i c was found in the period before graftrecirculation in one patient and 60 min after recirculation in another case. The 6 patients who did not develop significant f i b r i n o l y t i c a c t i v i t y had high plasma,levels of PAI( 25 IU/ ml; ref value: 0-25 IU/ml) during the operation and no or low levels of tPA ( 80 mIU/ml; ref value: 0-200 mIU/ml). In the two patients that demonstrated increased f i b r i n o l y t i c a c t i v i t y , very high levels (max. 8840 mIU/ml and 3760 mIU/ml) of tPA-act were measured. Increased tPAact lasted for 3 to 3.5 h and was caused by a sharp, 2-3 fold increase of tPA-Ag to 60ng/ml (ref value: 5-10 ng/ml) and a concurrent decrease of PAl. Since we observed increased f i b r i n o l y t i c a c t i v i t y in APLT, lack of hepatic clearance may not be the only cause of increased f i b r i n o l y s i s in OLT. Our findings suggest that f i b r i n o l y s i s in l i v e r transplantation is due to an increased release of tissue plasminogen activator (tPA).
$69