- Email: [email protected]
Influence of cryopreservation on antigenicity of aortal allografts in rats
Abstracts / Cryobiology 71 (2015) 537e573
S25. INFLUENCE OF CRYOPRESERVATION ON ANTIGENICITY OF AORTAL ALLOGRAFTS IN RATS 1, 6 ri J. Hrubý 1, 6, R. Spunda , P. Me cka 2, 6, M. Ml cek 3,6, K. Splith 4, 6, M. cek 1, 6, I. Schmelzle 4,5, 6, F. Krenzien 7, 6, J. Lindner 1, 6, M. Spa Matia 3, 7, 6. 1 2nd Department of Surgery, Department of Cardiovascular Surgery, 1st Faculty of Medicine, Charles University in Prague and General University Hospital in Prague, Prague, Czech Republic; 2 Tissue Bank of the University Hospital Hradec Kralove, Hradec Kralove, Czech Republic; 3 Institute of Physiology, 1st Faculty of Medicine, Charles University, Prague, Czech Republic; 4 Translational Centre for Regenerative Medicine, University of Leipzig, Leipzig, Germany; 5 Department of General, Visceral and €tsmedizin, Berlin, Germany; Transplantation Surgery, Charit e Universita 6 Transplant Surgery Research Laboratory and Division of Transplant Surgery, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts, USA; 7 Department of Visceral, Transplant, Thorax and Vascular Surgery, University of Leipzig, Leipzig, Germany
E-mail address: [email protected]
Cryopreserved and cold-stored arterial allografts are used in the treatment of vascular prosthesis infection. The influence of both types of conservation protocols on arterial antigenicity is still not clear. The aim of the study is to compare the acute antibody and cell-mediated rejection of both cryopreserved and cold-stored arterial allografts 30 days after their transplantation in rats. Material and Methods: Infrarenal aortal grafts obtained from Brown-Norway rats were stored either in liquid nitrogen or Custodiol solution and subsequently transplanted to Lewis rats. Histological and immunohistochemical evaluations were performed on day 30 post transplant. Flow cytometry analysis of recipient’s sera was performed to determinate the presence of donor-specific anti MHC class I and class II antibodies. Results: Both cryopreserved and cold-stored allografts induced strong donor specific anti-MHC class I and anti-MHC class II antibody production on day 30. However, immunoglobuline G mediated destruction of tunica media was observed only in cold-stored allografts. In addition, cold-stored arterial allografts showed more extensive intimate hyperplasia and more massive infiltration of tunica adventitia by MHC class II+, CD8+ and CD4+ cells of recipient origin compared to cryopreserved grafts. Conclusion: Cryopreserved aortal allografts showed markedly lower acute antibody and cell-mediated rejection compared to cold-stored allografts in our experimental study. Funding: N/A Conflict of Interests: N/A S26. BIOBANKING IN HAEMATO-ONCOLOGY jek, Petra Vrublova . Hemato-oncology Clinic, University Hospital Roman Ha Ostrava, Ostrava, Czech Republic E-mail address: [email protected]
Biobanking means storage of biological material for research use. We specialize in collecting samples from patients with haematooncological diagnoses, mainly monoclonal gammopathies (multiple myeloma, monoclonal gammopathy of undetermined significance, AL-amyloidosis and € m macroglobulinemia), but also other diagnosies such as Waldenstro lymphomas, leukemias and myeloproliferative disorders. Biological material (blood, bone marrow, tissue, etc.) is collected after an informed consent is signed. The samples are further processed according to standard operating procedures, thus ensuring a high quality of samples for downstream research analyses and also reproducibility of results. Blood is mainly used for storage of plasma, serum and DNA. From bone marrow (and also from blood) we are able to separate different types of cells by recognizing special surface molecules using either magnetic or fluorescence activated cell sorting, by which we obtain a highly enriched fraction of our targeted cells. All samples are then frozen and can be stored over a long period of time. Thus the samples are ready for research use. Funding: N/A Conflict of Interests: N/A
543
S27. BIOBANKING FOR HEPARIN INDUCED THROMBOCYTOPENIA J. Gumulec, P. Vrublov a. Department of Haematooncology, University Hospital Ostrava and the Faculty of Medicine, University of Ostrava, Ostrava, Czech Republic E-mail address: [email protected]
Heparin-induced thrombocytopenia (HIT) is a life-threatening disorder that follows exposure to unfractionated or less commonly low molecular weight heparin. Patients usually have a low platelet count or a relative decrease of 50% or more from baseline. Thrombotic complications develop in approximately 20-50% of patients and the risk of thrombosis remains high for days to weeks after discontinuation of heparin, even after the platelet count normalizes. HIT is caused by platelet activating antibodies against complexes of platelet factor 4 (PF4) and heparin. It is uncertain why complications occur in some patients but not in others. The goals of management of HIT are to reduce the thrombotic risk by reducing platelet activation and thrombin generation. All sources of heparin should be discontinued when the clinical suspicion of HIT is intermediate or high, and alternative anticoagulant therapy should be initiated. The clinical criteria of HIT include the presence of thrombocytopenia and/or thrombosis bearing a temporal relationship to an immunizing exposure to heparin. The laboratory criterion is the detection of platelet-activating antibodies in acute patient serum or plasma. The high diagnostic sensitivity of the PF4/heparin solid-phase enzyme-immunoassays (EIAs) is accompanied by a parallel problem in diagnostic specificity for HIT. This problem arises because EIAs also have high sensitivity for detecting clinically insignificant anti-PF4/heparin antibodies. Diagnostic accuracy for HIT is optimized by combining the anti-PF4/heparin EIA with a functional, platelet activation test. Platelet activation assays like serotonin-release assay and heparin-induced platelet activation tests are much more specific for HIT than are EIAs. The functional assays require strict quality controls. Most important is the inclusion of a weak-positive HIT serum control to ensure adequate donor platelet reactivity in maintaining high test sensitivity, the use of high heparin inhibition, and an appropriate negative control. The biggest problem is the selection of a suitable donor of platelets, because platelet responsiveness to HIT antibodies varies widely among normal donors (approximately one in seven donors being responsive). The main objectives of this project are: Archiving of plasma, serum, DNA, and platelet rich plasma of patients with HIT and creating of the database of patients with HIT. Diagnosis of HIT using a set of available diagnostic procedures and the introduction of new diagnostic methods. Validation of the proposed diagnostic algorithms using samples archived in bio-bank. Monitoring of the effectiveness, side effects and pitfalls of established and novel therapeutic approaches. Monitoring the dynamics of procoagulant activity HIT antibodies after discontinuation of heparin. Comparison of the different parameters of the donor platelets sensitive and insensitive to HIT antibodies including of the occurrence of selected polymorphisms of platelet receptors. Coordination of care for patients with HIT in Czech Republic. Funding: N/A Conflict of Interests: N/A S28. CORD BLOOD BANKING IN CZECH REPUBLIC Eva Matejkova, Dagmar Hruzova, Zdenek Koristek. National Cell and Tissue Center Inc., Brno, Czech Republic E-mail address: [email protected]
The history of cord blood (CB) transplantation in clinical practice started in 1988. In the Czech Republic the first CB unit was collected in August 1994 and transplanted in the same year. In the Czech Republic allogeneic and autologous donations have been performed. For the Donor Program, the public Cord Blood Bank was established in Prague. Currently it has about
S25. INFLUENCE OF CRYOPRESERVATION ON ANTIGENICITY OF AORTAL ALLOGRAFTS IN RATS 1, 6 ri J. Hrubý 1, 6, R. Spunda , P. Me cka 2, 6, M. Ml cek 3,6, K. Splith 4, 6, M. cek 1, 6, I. Schmelzle 4,5, 6, F. Krenzien 7, 6, J. Lindner 1, 6, M. Spa Matia 3, 7, 6. 1 2nd Department of Surgery, Department of Cardiovascular Surgery, 1st Faculty of Medicine, Charles University in Prague and General University Hospital in Prague, Prague, Czech Republic; 2 Tissue Bank of the University Hospital Hradec Kralove, Hradec Kralove, Czech Republic; 3 Institute of Physiology, 1st Faculty of Medicine, Charles University, Prague, Czech Republic; 4 Translational Centre for Regenerative Medicine, University of Leipzig, Leipzig, Germany; 5 Department of General, Visceral and €tsmedizin, Berlin, Germany; Transplantation Surgery, Charit e Universita 6 Transplant Surgery Research Laboratory and Division of Transplant Surgery, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts, USA; 7 Department of Visceral, Transplant, Thorax and Vascular Surgery, University of Leipzig, Leipzig, Germany
E-mail address: [email protected]
Cryopreserved and cold-stored arterial allografts are used in the treatment of vascular prosthesis infection. The influence of both types of conservation protocols on arterial antigenicity is still not clear. The aim of the study is to compare the acute antibody and cell-mediated rejection of both cryopreserved and cold-stored arterial allografts 30 days after their transplantation in rats. Material and Methods: Infrarenal aortal grafts obtained from Brown-Norway rats were stored either in liquid nitrogen or Custodiol solution and subsequently transplanted to Lewis rats. Histological and immunohistochemical evaluations were performed on day 30 post transplant. Flow cytometry analysis of recipient’s sera was performed to determinate the presence of donor-specific anti MHC class I and class II antibodies. Results: Both cryopreserved and cold-stored allografts induced strong donor specific anti-MHC class I and anti-MHC class II antibody production on day 30. However, immunoglobuline G mediated destruction of tunica media was observed only in cold-stored allografts. In addition, cold-stored arterial allografts showed more extensive intimate hyperplasia and more massive infiltration of tunica adventitia by MHC class II+, CD8+ and CD4+ cells of recipient origin compared to cryopreserved grafts. Conclusion: Cryopreserved aortal allografts showed markedly lower acute antibody and cell-mediated rejection compared to cold-stored allografts in our experimental study. Funding: N/A Conflict of Interests: N/A S26. BIOBANKING IN HAEMATO-ONCOLOGY jek, Petra Vrublova . Hemato-oncology Clinic, University Hospital Roman Ha Ostrava, Ostrava, Czech Republic E-mail address: [email protected]
Biobanking means storage of biological material for research use. We specialize in collecting samples from patients with haematooncological diagnoses, mainly monoclonal gammopathies (multiple myeloma, monoclonal gammopathy of undetermined significance, AL-amyloidosis and € m macroglobulinemia), but also other diagnosies such as Waldenstro lymphomas, leukemias and myeloproliferative disorders. Biological material (blood, bone marrow, tissue, etc.) is collected after an informed consent is signed. The samples are further processed according to standard operating procedures, thus ensuring a high quality of samples for downstream research analyses and also reproducibility of results. Blood is mainly used for storage of plasma, serum and DNA. From bone marrow (and also from blood) we are able to separate different types of cells by recognizing special surface molecules using either magnetic or fluorescence activated cell sorting, by which we obtain a highly enriched fraction of our targeted cells. All samples are then frozen and can be stored over a long period of time. Thus the samples are ready for research use. Funding: N/A Conflict of Interests: N/A
543
S27. BIOBANKING FOR HEPARIN INDUCED THROMBOCYTOPENIA J. Gumulec, P. Vrublov a. Department of Haematooncology, University Hospital Ostrava and the Faculty of Medicine, University of Ostrava, Ostrava, Czech Republic E-mail address: [email protected]
Heparin-induced thrombocytopenia (HIT) is a life-threatening disorder that follows exposure to unfractionated or less commonly low molecular weight heparin. Patients usually have a low platelet count or a relative decrease of 50% or more from baseline. Thrombotic complications develop in approximately 20-50% of patients and the risk of thrombosis remains high for days to weeks after discontinuation of heparin, even after the platelet count normalizes. HIT is caused by platelet activating antibodies against complexes of platelet factor 4 (PF4) and heparin. It is uncertain why complications occur in some patients but not in others. The goals of management of HIT are to reduce the thrombotic risk by reducing platelet activation and thrombin generation. All sources of heparin should be discontinued when the clinical suspicion of HIT is intermediate or high, and alternative anticoagulant therapy should be initiated. The clinical criteria of HIT include the presence of thrombocytopenia and/or thrombosis bearing a temporal relationship to an immunizing exposure to heparin. The laboratory criterion is the detection of platelet-activating antibodies in acute patient serum or plasma. The high diagnostic sensitivity of the PF4/heparin solid-phase enzyme-immunoassays (EIAs) is accompanied by a parallel problem in diagnostic specificity for HIT. This problem arises because EIAs also have high sensitivity for detecting clinically insignificant anti-PF4/heparin antibodies. Diagnostic accuracy for HIT is optimized by combining the anti-PF4/heparin EIA with a functional, platelet activation test. Platelet activation assays like serotonin-release assay and heparin-induced platelet activation tests are much more specific for HIT than are EIAs. The functional assays require strict quality controls. Most important is the inclusion of a weak-positive HIT serum control to ensure adequate donor platelet reactivity in maintaining high test sensitivity, the use of high heparin inhibition, and an appropriate negative control. The biggest problem is the selection of a suitable donor of platelets, because platelet responsiveness to HIT antibodies varies widely among normal donors (approximately one in seven donors being responsive). The main objectives of this project are: Archiving of plasma, serum, DNA, and platelet rich plasma of patients with HIT and creating of the database of patients with HIT. Diagnosis of HIT using a set of available diagnostic procedures and the introduction of new diagnostic methods. Validation of the proposed diagnostic algorithms using samples archived in bio-bank. Monitoring of the effectiveness, side effects and pitfalls of established and novel therapeutic approaches. Monitoring the dynamics of procoagulant activity HIT antibodies after discontinuation of heparin. Comparison of the different parameters of the donor platelets sensitive and insensitive to HIT antibodies including of the occurrence of selected polymorphisms of platelet receptors. Coordination of care for patients with HIT in Czech Republic. Funding: N/A Conflict of Interests: N/A S28. CORD BLOOD BANKING IN CZECH REPUBLIC Eva Matejkova, Dagmar Hruzova, Zdenek Koristek. National Cell and Tissue Center Inc., Brno, Czech Republic E-mail address: [email protected]
The history of cord blood (CB) transplantation in clinical practice started in 1988. In the Czech Republic the first CB unit was collected in August 1994 and transplanted in the same year. In the Czech Republic allogeneic and autologous donations have been performed. For the Donor Program, the public Cord Blood Bank was established in Prague. Currently it has about