Viability of Human Arterial Grafts Monitored by Comet Assay.

An analytical method for studying DNA degradation by electrophoresis after cell lysis and visualization of DNA fragments with fluorescent dye, comet assay, was used to evaluate the viability of the endothelial layer of human arterial grafts with the aim of identifying the procedure that will least damage the tissue before cryopreservation. Four groups of samples were studied: cryopreserved arterial grafts that were thawed in two different ways, slowly lasting 2 hours or rapidly for approx. 7 minutes. Arterial grafts that were collected as part of multiorgan procurement with minimal warm ischemia time. Cadaveric grafts were taken as part of the autopsy, so they have a more extended period of warm ischemia. The HeadDNA (%) parameter and others commonly used parameters like TailDNA (%). TailMoment, TailLength, OliveMoment, TailMoment to characterize the comet were used to assess viability in this study. The ratio of non-decayed to decayed nuclei was determined from the values found. This ratio for cadaveric grafts was 0.63, for slowly thawed cryopreserved grafts 2.9, for rapidly thawed cryopreserved grafts 1.9, and for multi-organ procurement grafts 0.68. The results of the study confirmed the assumption that the allografts obtained from cadaveric donors are the least suitable. On the other hand, grafts obtained from multiorgan donors are better in terms of viability monitored by comet assay. Keywords: Arterial grafts, Cryopreservation, Cadaveric, Multiorgan procurement, Viability, Comet assay.

The role of cryopreservation techniques in manufacturing, transport, and storage of Car-T therapy products.

Several clinical trials have proved the efficacy and safety of T-cells chimeric antigen receptor (CAR-T cells) in treatment of malignant lymphoma and the first products were registered in the European Union in 2018. The shelf-life of CAR-T cell products in the liquid state is short, so cryopreservation offers a significant benefit for logistics in manufacturing and patient management. Direct shipment of the cryopreserved CAR-T cell therapy products to the clinical department is feasible, nevertheless, intermediate storage in the hospital cryostorage facility gives significant advantage in planning of their administration to patients. Moreover, some manufacturers prefer transport of the starting material cryopreserved at the collection site. The cryopreservation protocol used for starting material by the authors is based on combining dimethyl sulphoxide (DMSO) with hydroxyethyl starch (HES) and slow controlled cooling in cryobags housed in metal cassettes. This achieves the mononuclear cell post-thaw viability of 98.8 ± 0.5 % and recovery of 72.8, ± 10.2 %. Transport of the starting material to the manufactures and return transport of the CAR-T therapy product is performed by authorized courier companies. Intermediate cryostorage of the final CAR-T cell therapy product is performed in a separate dry-storage liquid nitrogen container. On the day of infusion, the cryopreserved products are transported to the clinical department in a dry shipper. On the wards the product is removed from the cassette, inserted into a sterile plastic bag, thawed in a 37 degree C water bath followed by immediate intravenous administration. The authors discuss the adherence of the used technology to good manufacturing practice (GMP) principles and genetic safety assurance rules. Doi: 10.54680/fr23310110112.

Cardiovascular changes associated with infusion of hematopoietic cell grafts in oncohematological patients -- impact of cryopreservation with dimethylsulfoxide.

AIM: Dimethylsulfoxide (DMSO) is the most frequently used agent for hematopoietic cell (HC) graft cryopreservation. This study aimed to monitor blood pressure and heart rate (HR) during HC graft infusion and assess the impact of cryopreservation with DMSO. METHODS: 153 HC graft infusions in 153 consecutive hematological patients (mean age 49.1 -/+ 12.6 years; 80 males) were evaluated. Cryopreservation with DMSO was used in 133 grafts (DMSO group). Twenty grafts were infused directly without cryopreservation (control group). Systolic blood pressure (SBP), diastolic blood pressure (DBP) and HR were measured immediately before and after HC graft infusion. RESULTS: SBP and DBP increased significantly after graft infusions cryopreserved with DMSO ( p<0.0001 for SBP; p<0.01 for DBP). Increases (> 10mmHg) in SBP were seen in 42 (31.6%) patients; in DBP in 31 (23.3%) patients. Changes in HR were non-significant in DMSO group. Increases in BP and HR correlated with increasing DMSO dose (p<0.01; p<0.05, respectively). Changes in SBP, DBP and HR were non-significant in control group. CONCLUSION: HC graft infusions cryopreserved with DMSO could cause statistically significant increases in SBP and DBP, without changes in HR. These changes were mostly transient and asymptomatic, not requiring therapeutic intervention. However, they might cause complications, especially in patients with preexisting cardiovascular disease, who should be monitored closely during HC transplantation.

Intensive cyclic chemotherapy and transplantation of autologous peripheral blood progenitor cells (PBPC) or whole blood in high-risk breast cancer - follow up at 10 years.

AIM: The main aim of our paper is to contribute to objectification of currently widely discussed results of overall survival (OS), disease free survival (DFS) and time from relapse to tumor progression (TTP) in women with breast cancer. METHODS: Forty consecutive patients fulfilling the eligibility criteria were admitted to the study. Fifty-six women were included in the control group. All patients received 6 cycles of adjuvant intensive cyclic combined chemotherapy with epirubicin 150 mg/m(2) and cyclophosphamide 1250 mg/m(2) (EC) applied each 14 days. To overcome haematological toxicity transplantations of autologous peripheral blood progenitor cells (PBPCs) or whole blood enriched of PBPC were used. RESULTS: We found statistically significant difference in OS regardless of the stage of the disease to the benefit of women treated by intensive cyclic EC chemotherapy when compared with the control group. In evaluation of DFS no statistically significant difference was found in survival between the control group and the group with all stages of the disease. TTP in women without relation to the stage was statistically significantly longer than in the control group. CONCLUSION: In our study intensive cyclic EC chemotherapy did not show better curative effect when compared with conventional dosage chemotherapy.

Current trends in safety assurance for tissue grafts used in burn treatment.

The author presents a summary of current safety standards for allogeneic and xenogeneic biological skin grafts. The fundamental document relevant to allogeneic transplants, establishing the minimal level of safety guaranteed in European Union states, is the European Parliament and Council Directive (2004/23/EC) from March 31st 2004. This Directive determines that grafts will be prepared by a licensed or accredited tissue bank, and that this arrangement must be put in place by the member states within 2 years. In the Czech Republic licensing of tissue banks took place immediately after issuance of the Directive. Licensing was also a condition for product reimbursement by insurance companies. To gain a licence, tissue banks had to fulfil many safety criteria associated with screening of living or deceased donors for health suitability, providing traceability of the donor-recipient route, prevention of secondary and cross-contamination during processing and storage of the harvested tissues, proof of product microbiology check up, and cold chain control. The Tissue Bank of the Faculty Hospital in Hradec Králové is one of the two tissue banks that gained the broader type of 'multifunctional' licence and was granted registration number MTB 006. Obtaining the licence was facilitated by completion of a new workplace project conceived as a combination of cryogenic and clean-room technology. Currently, this tissue bank prepares cryopreserved dermoepidermal and dermal grafts as well as amnion and chorioamnion grafts. All tissue banks will have to renew their licences again according to the conditions established by a new law about human tissues and cells which is currently in preparation. Neither the Directive of the European Parliament nor the Transplantation Law of the Czech Republic regulates the issue of xenografts. Since availability of allogeneic biological covers is limited, it is significant that the WHO perspective on the use of xenogeneic biological covers, as established in 2005, is positive. This attitude should also be taken in the Czech Republic. The directives of the European Union pertinent to medical devices of biological origins can be applied only to devitalized tissues--and moreover, the domestic pig is not on the risk animal list. The author presumes that to guarantee safe use of viable xenografts it is necessary to follow the general principles of quality control as applied in the workplace for many years, as well as general medicinal product safety principles, including strict veterinary control of breed of animals whose tissues are used for the preparation of xenografts.

Prevention of infection transmission during stem cell transplantation.

Group of 152 patients (investigated before autologous transplantation) and 35 healthy donors for allogeneic transplantation was examined for the risk of infection transmission that can be associated with the infusion of cryopreserved peripheral blood progenitor cells to the patient and/or cross-contamination of stored grafts. No laboratory signs of active infection were found in 22 donors (63 %) and in 91 patients (60%). The most common was active infection by herpes viruses--50 cases in patients, 21 cases in donors; hepatitis B was found in only two cases. The rate of clinically unsuspected (but dangerous) infections in donors and patients thus remains relatively high in spite of the fact that the system of donor search and the whole transplantation procedure have improved in the last years. The system of safety assurance is extremely important and the whole palette of preventive tests according to EBMT (European Blood and Marrow Transplantation Group) and ISHAGE (International Society for Hemotherapy and Graft Engineering) is fully justified.

Potential risk of infection transmission during storage and transplantation of hematological progenitor cells. Safety assurance.

In a group of 71 patients and 22 donors the danger of infection transmission by infusion of cryopreserved peripheral blood progenitor cells to the patient and/or cross contamination of stored grafts was evaluated. No laboratory signs of active infection were found in 15 donors (13 related, 2 unrelated; 68%) and in 55 patients (77%). Active infection by herpesviruses was the most common (in 13 patients and 7 donors), hepatitis B being found in only one case. The cytomegalovirus IgG test was the most common marker of previous infection; it was found in 14 donors and 55 patients. The rate of clinically unsuspected infections in donors and patients including cases requiring immediate treatment among the patients group is relatively high and fully justifies the practice of prophylactic serological testing in the whole range of tests according to the European Blood and Marrow Transplantation Group and International Society for Hematotherapy and Graft Engineering in both autologous and allogeneic transplantations of hematopoietic stem cells.

New safety assurance for biological skin covers.

The described system of safety assurance of cryopreserved allogeneic and xenogeneic dermoepidermal transplants comprises serological examination of deceased tissue donors, long-term storage of sera of all donors, microbiological control of prepared allogeneic and xenogeneic dermoepidermal grafts, labelling of released tissue grafts and monitoring of temperatures inside the mechanical freezer (-80 degrees C). From a total number of 76 donors from whom tissues were collected for transplantation during 1999-2001, tissues were discarded in two instances. One because of a positive HBsAg test, the others second one because of presence of anti-HTLV antibodies. In xenogeneic dermoepidermal grafts, out of a total number of 1,203 grafts prepared during the same period, 84 (6.9%) were discarded because of the presence of pathogeneic or potentially pathogeneic microbes. The system of labelling released grafts makes unequivocal identification of the pathway from recipient to donor possible, while at the same time respecting the anonymous character of the donor's data. In xenogeneic grafts it ensures the identification of the appropriate batch. Storage of cryopreserved biological skin covers at a temperature of -80 degrees C in low temperature cabinets with emergency back-up cooling with liquid nitrogen and a supplementary source of electric power, proved very useful. The system responds to the gradual implementation of the principles of Quality Management System ISO 9000 and Good Manufacturing Practice into the activities of tissue banks. Further tightening of the demands for the safety of allo- and xeno-transplantation is foreseen in conjunction with the occurrence of transmissible spongiform encephalopathy and porcine retroviruses.

The importance of stored supplies of biological skin covers in territorial management of mass burn casualties.

This study provides supportive evidence of a possible role played by planning supplies of biological covers needed in fire disaster, based on the experience of the authors. The major steps to be taken are these: 1. Providing a technically and technologically adequate base for collection and long-term storage of cells and tissues ready for use in case of catastrophe. 2. Developing a method for estimating the amount of reserved tissue grafts. 3. Solution of logistical problems associated with providing supplies for operating theatres treating disaster casualties. 4. Organisation of national and international network of graft exchange capable of supporting local skin banks in times of need. In contrast to the situation in the 1970s and 1980s, nowadays the Czech Republic can deliver any required amount of biological covers without having to face technological difficulties. The idea of collaborating in the management of a fire disaster emerged from experience gained by the authors during the Bashkir disaster in 1989, relating particularly to an inadequate reserve stock of skin grafts and the impossibility of increasing their production. Intensified demands for the safety of grafts and recent experience from the US emphasise the need for an immediate conceptual solution concerning production of reserves of biological covers that should be ready for transportation to wherever needed. Another urgent necessity is the establishment of conditions enabling effective international collaboration at a disaster event.

[Preservation of venous grafts]. / Príspevek ke konzervaci zilních stepu.

The objective of the work was to find an optimal preservation medium for short-term preservation of venous grafts which could be subsequently used to line metal stents. The external jugular vein of dogs (n = 15) was removed surgically, divided into portions and immersed into preservation media. For hypothermic preservation (+4 degrees C) solutions of Optisol (Chiron, USA), University of Wisconsin (Baxter, USA), Eurocollins (Fresenius, GFR) and saline (Bieffe Medital, Italy) were used. For normothermic preservation (+37 degrees C) in an atmosphere with 5% CO2 Dulbecc's medium for tissue cultures (Sigma, USA) was used. During hypothermic preservation the specimens were kept for 24 hours, 3 and 7 days, during normothermic preservation in Dulbecc s medium also for 24 hours, 3 and 7 days. The specimens were evaluated by light microscopy and raster electron microscopy. The results revealed that minimal changes on the endothelia of venous grafts occurred during normothermic preservation in Dulbecc's medium where after 7 days the endothelium did not become detached and the vitality of cells did not change. During hypothermic preservation the solution of Wisconsin University proved most suitable. By addition of 5% human albumin to this solution it proved possible moreover to reduce pyknosis of the endothelial cell nuclei. The specimens kept in saline displayed deformities of the nuclei, oedema and loss of endothelia incl. incipient oedema of the cellular wall already after 24 hours of hypothermic preservation. The authors consider the use of this solution unsuitable for preservation.

Application of whole blood and peripheral blood progenitor cells (PBPC) and new strategies for rescue after intensive cyclic chemotherapy in high-risk breast cancer.

The efficacy of autologous peripheral stem cells given as mobilized whole blood or leukapheresis product for hematopoietic rescue after intensive chemotherapy was studied in 34 consecutive female patients with high-risk breast cancer. All patients received six cycles of chemotherapy regimen EC (epirubicin 150 mg/m2 and cyclophosphamide 1250 mg/m2) at 14-day intervals. In the first cycle, chemotherapy was given on day 1, and 24 h later mobilization of PBPC was started with G-CSF at a dose of 5 microg/kg/day for 13 days. In all other cycles, G-CSF was given at the same dose from day 7. On days 11, 12, and 13, leukaphereses were performed, and whole blood was collected on day 14 (the peak incidence of colony-forming units-granulocyte-macrophage [CFU-GM] burst-forming units-erythrocyte [BFU-E], and colony-forming unit-granulocyte-erythrocyte-macrophage-megakaryocyte [CFU-GEMM]). The second cycle of chemotherapy was started on day 15, and 24 h later, whole blood (collected in the first cycle) was reinfused, and the same was done in the third cycle. In the fourth to sixth chemotherapy cycles, leukapheresis product was used for hematopoietic rescue. The median increment of absolute values in both whole blood and leukapheresis product was as follows: CD34+ cells over baseline was approximately 17.4-fold, CFU-GM was 85.3-fold, BFU-E was 95.9-fold, and CFU-GEMM was 44.2-fold. In the cycles with whole blood support, the mean values of applied progenitors per cycle were CD34+ cells 1.52 x 10(6)/kg, CFU-GM, 1.18 x 10(5)/kg, BFU-E 2.54 x 10(5)/kg, CFU-GEMM 0.31 x 10(5)/kg. In the courses with PBPC support, the mean values of progenitors were CD34+ 2.04 x 10(6)/kg, CFU-GM 1.59 x 10(5)/kg, BFU-E 2.87 x 10(5)/kg, and CFU-GEMM 0.34 x 10(5)/kg. Leukopenia in patients supported with whole blood versus leukapheresed PBPC was as follows: grade 4, 13/6 (38.2%/17.6%), grade 3, 19/23 (55.9%/70.6%), and grade 2, 1/4 (2.9%/11.8%), respectively. Thrombocytopenia was grade 4, 11/6 (32.4%/17.6%), grade 3, 10/7 (29.4%/20.6%), grade 2, 7/13 (20.6%/38.2%), and grade 1, 6/6 (17.6%/17.6%), respectively. The median follow-up analysis was at 24.6 (7-36) months. High-risk patients previously treated with surgery and adjuvant chemotherapy (n = 5) were not evaluated for response. In 21 patients with locally advanced or inflammatory breast carcinoma the response rate (RR) was 94%, CR was 90%, and PR was 15%. No response to therapy was observed in 1 patient. In 8 patients with metastatic disease, RR was 75%, there was no CR, and PR was 75%. Two patients died during therapy. Relapse-free survival (RFS) in the adjuvant group was 23.7 (range 12-36) months and in the group with locally advanced disease was 18.2 (range 7-27) months. In the group with metastatic disease, time to tumor progression (TTP) was 12.1 (range 1-16) months. Mean duration of hospital stay for whole blood reinfusion in the second and third chemotherapy cycles was 6.7 (range 5-8) days and for PBPC in the fourth to sixth cycles was 6.2 (range 4-8) days, which at p < 0.001 was not statistically significant.

Autologous vein stent-graft: feasibility study.

PURPOSE: To evaluate expandable stents healed into vein wall as autologous vein stent-grafts for endoluminal grafting. MATERIALS AND METHODS: Balloon expandable stents were placed into external jugular veins of eight dogs. Stent and vein patency was followed by ultrasonography. Five weeks after stent placement, jugular veins with endothelialized stent were harvested. The autologous vein stent-grafts were divided into two groups. In group A, autologous vein stent-grafts (n = 3) were placed immediately into Baker solution for microscopic examination. In group B, autologous vein stent-grafts (n = 3) underwent mechanical manipulation; they were compressed, mounted on angioplasty balloon, pushed through a 9-F sheath and dilated. The autologous vein stent-graft endothelialization and changes after mechanical manipulation were evaluated by light and electron microscopy. RESULTS: Stent placement was successful in seven dogs. One stent migrated into the pulmonary artery. One well placed stent was damaged by compression dressing and thrombosed. At 5 weeks, gross and microscopic examinations revealed the autologous vein stent-grafts were fully covered by a 0.115- +/- 0.036-mm-thick neointimal layer. Small wall thrombus was observed in one autologous vein stent-graft. Repeated manipulations did not result in any intimal damage or stent loosening in the autologous vein stent-grafts. CONCLUSION: Expandable stents healed into a vein have potential to be used as autologous vein stent-grafts for endoluminal grafting without risk of disruption during percutaneous transcatheter introduction.

[Evaluation of the results of surgery of intracranial supratentorial tumors in 363 children over the past 50 years (1948-1997)]. / Zhodnocení výsledku u 363 detí operovaných pro nitrolebecní supratentoriální nádory za 50 let (1948-1997).

At the Department of Neurosurgery, Hradec Králové, in the course of 50 years (1948-1997) 363 children, 199 boys and 164 girls (1.2:1) aged under 18 years were operated on for intracranial supratentorial tumours. The average age in children at the time of first operation was 9.3 years; most frequently they participated those of 8 and 11 to 14 years of age. Children aged 1, 2 and 3 years constituted 4.7%, 4.4% and 5% of operations. The tumours were located in: cerebral hemispheres 123 (33.8%), lateral ventricles 17 (4.7%), IIIrd ventricle 5 (1.4%), hypothalamus 26 (7.2%), thalamus 19 (5.2%), basal ganglia 24 (6.6%), sellar region 86 (23.7%), chiasmatic region 38 (10.5%) and pineal region 19 (5.2%). 223 of tumours (61.4%) were located in the midline and 140 of them (38.6%) laterally (in hemispheres and lateral ventricles of the brain). 268 of tumours were histologically verified (73.8%) and 95 of cases were evaluated according to the neurosurgeon's point of view and/or to the clinical and CT controls (26.2%), because of the biopsy (especially in the pre-CT era) was highly riskfull. Histological typing of tumours was retrospectively reevaluated according to the present WHO classification. Summarized 53 types of tumours were differentiated. The most frequent lesions were various variants of astrocytic gliomas (135 = 37.2%). Further on the craniopharyngiomas dominated (73 = 20.1%). The tumours were operated on through craniotomies 299 times, by primarily drainage operations 52 times, functions 6 times, stereotactically 8 times and or by combination of these operations 82 times. Reoperation was needed for postoperative complications in 1.7% (6 times) and for delayed recurrence in 11.3% (41 times). The postoperative mortality (up to 1 month after initial surgery) was in 156 children operated on in pre-CT era (between 1948 and 1977), as compared with 207 children operated on in the era of CT (between 1978 and 1997) in astrocytomas 3.8:0%, pilocytic astrocytomas 6.5:2.8%, craniopharyngiomas 15.4:0% and in all tumours 12.2:2.9%. 16 children with orbital tumours (the average age 5.8 years) operated on with orbitofrontal approach were also evaluated. 14 of them survive for 5-37 years (on the average 16.6 years). The chronological development of diagnostic and operative processes of supratentorial tumours in children's care is discussed. The prognostic elements of present histobiological classification of tumours are positively evaluated.

Brief history of the tissue bank, charles university hospital, hradec králové, czech republic.

A brief history of the Tissue Bank (TB) of the University Hospital Hradec Králové, Czech Republic, established by Dr. R. Klen in 1952 is presented. In Dr. Klen's original concept the TB was defined as a department specialised in the harvesting, processing, preservation, storage and distribution of various kinds of tissue for clinical and experimental practice. The first kinds of tissue collected in cadaveric donors were corneas, bone and skin. Xenogeneic cartilage and bone grafts were prepared at the same time. Later, preparation of soft connective tissues and chorion-amnion was introduced. During the first 15 years of activity a total of 11,443 grafts preserved by hypothermy at +4 degrees C or freezing in absence of cryoprotectants (-20 degrees C) were prepared. In the 60's freeze-drying of tissue grafts was introduced and the bank of cryopreserved cell lines was established. In the 80's cryopreservation of haematopoietic progenitor cells for clinical transplantations was started and the spectrum of tissue grafts was enlarged (xenogeneic pericardium and allogeneic specially treated dura mater for neurosurgical operations, pigskin for burn treatment, demineralised bone for parodontology and implantology). In the 90's human keratinocyte culture for treatment of burns and chronic skin defects was started. The human milk bank and organ bank co-operating with the Regional Transplantation Centre are component parts of the TB as well. The TB is an institutional member of the European Association of Tissue Banks and annually delivers approximately 1000 grafts that are used in University and county hospitals as well as in surgeons' private practices. Health insurance companies reimburse all grafts on a non-profit and tax-free basis.

The increase of the rate of hemopoietic recovery and clinical benefit of the erythropoietin (EPO) and granulocyte colony-stimulating factor (G-CSF) with peripheral blood progenitor cells (PBPC) after intensive cyclic chemotherapy in high-risk breast cancer patients.

The role of erythropoietin (EPO) plus granulocyte-colony stimulating factor (G-CSF) combination in hemopoietic recovery was studied in patients with high-risk breast carcinoma and compared to a control group of previously treated identical patients who were not given EPO plus G-CSF. Eleven consecutive patients admitted to this study had Stage III or IV breast cancer. They received 6 cycles of intensive chemotherapy (epirubicin 150 mg/m2 and cyclophosphamide 1300 mg/m2). The 1st cycle served for mobilization of peripheral blood progenitor cells (PBPC). At its end leukaphereses collections of PBPC were performed to be used as hematologic support (PBPCT) in the 5 remaining cycles. The administration of EPO plus G-CSF was started when leukocyte (WBC) count in peripheral blood dropped below 1 x 10(9)/l and hemoglobin (Hb) level fell below 100 g/l. The treatment was stopped when leukocyte count rose to 5 x 10(9)/l and Hb to 130 g/l. EPO plus G-CSF combination after PBPCT produced significant effects in terms of hemopoietic recovery, clinical benefit and supportive care requirements when compared with 12 historic control patients: Periods of leukopenia were shorter which resulted in reduced risk of infectious complications. The grades of leukopenia in the study and control groups were as follows: grade 4 (36 vs. 18%), grade 3 (57 vs. 30%), grade 2 (7 vs. 13%) respectively. Significantly shorter was the time of PLT recovery < 50 x 10(9)/l (p < 0.001). The grades of thrombocytopenia were: grade 4 (29 vs. 11%), grade 3 (21 vs. 12%), grade 2 (25 vs. 36%) respectively. The number of necessary transfusions was significantly reduced as well as the length of hospital stay (p < 0.001). In conclusion, our results obtained in this study confirm that combination of EPO plus G-CSF not only increases the rate of hemopoietic recovery, reduces the number of necessary red blood cell and platelet transfusions but, at the same time, simplifies the clinical management and is more tolerable for the patients.

[Treatment of ptosis in children and adults]. / Moznosti resení ptózy v detství i v dospelosti.

Pathological ptosis of the eyelids can be manifest already at birth but may develop also in advanced age. In children the congenital form predominates with or without affection of the elevators of the eyes (Congenital fibrous syndrome or Misdirection syndrome). A special form is blepharophimosis, juvenile blepharochalasis and Marcus-Gunn's syndrome. In adult age the etiology of acquired ptosis of the upper eyelid is more varied. There are mechanical causes incl. dermatochalasis or it develops on a myogenic and neurogenic background or as the aponeurotic form. The group comprises 46 patients 3-70 years old where in the course of the last three years at the Ophthalmological Clinic of the Faculty Hospital Prague Vinohrady a total of 54 operations of ptoses were made. Resection of the tarsal plate according to Fasanella-Servat was used in 29 instances. In four eyes a plastic operation of the aponeurosis of the levator was performed. Frontotarsal suspension in the authors modification of Fox method using deep freeze-dried allogenei fasciae was used in the paretic form of ptosis in 21 eyes. The authors give also an account of the technological preparation of a fascial graft kept at a temperature of -80 degrees C.

Posterior cranial fossa surgery in 454 children. Comparison of results obtained in pre-CT and CT era and after various types of management of dura mater.

At the Department of Neurosurgery, Hradec Kralové, 454 children (aged under 18 years) were operated on for posterior cranial fossa lesions in a period of 49 years (1948-1996). The majority (402) had tumours: cerebellar astrocytomas 149 (37.1%), medulloblastomas 139 (34.6%), brain stem gliomas 46 (11.4%), ependymomas 28 (7.0%), and others 40 (9.9%). Postoperative mortality was compared for the pre-CT era (1948-1977) and the CT era (1978-1996): astrocytomas (8.6%:4.7%), medulloblastomas (14.9%:2.9%), brain stem gliomas (21.7%:19.0%), ependymomas (18.2%:6.3%), and others (40.0%:7.4%). The initially high mortality was due to insufficient intracranial decompression, brain oedema and disturbances of cerebrospinal fluid circulation. Obstructive hydrocephalus was treated in 53 children with tumours and 25 with aqueduct stenoses, by Torkildsen's drainage in 5.5%, and/or by catheterisation of aqueduct in 12.3%. The main postoperative complications of medial posterior fossa surgery in 429 children operated on were: pseudomeningocele (12.3%), active hydrocephalus (6.2%) and CSF leakage (4.6%). Only 8.2% had shunts placed for these complications. We presume that this low percentage of shunts used results from a frequent use of duraplasties and drains installed at the primary operation. The dura mater was initially (1948-1954) left open (50 cases), and later (1955-1958) also sutured (37 cases), and from 1958, onward, and especially from 1961, reconstructed by a medial approach by means of various grafts (377 cases). In all, duraplasty was performed in 81.6% of cases. The grafts used for dura mater reconstruction were prepared from autogeneic (1.6%), allogeneic (72.3%), xenogeneic (24.8%), or synthetic (1.3%) material. They were successful in 99.2% of cases (all materials). Our own suture technique for posterior fossa duraplasty is presented.

Mobilization of peripheral blood progenitor cells (PBPC) through a combination of chemotherapy and G-CSF in breast cancer patients and a possibility of unprocessed whole blood collection.

To support multicyclic, dose-intensive chemotherapy in breast cancer, we assessed the effects of reinfusing hematopoietic progenitors either as a leukapheresis product or as mobilized unprocessed whole blood. In this clinical study, 16 consecutive female breast cancer patients were given six cycles of chemotherapy regimen EC (epirubicin (150 mg/m2) and cyclophosphamide (1250 mg/m2) on day 1). In the first cycle, 24 h after chemotherapy, mobilization of the peripheral blood progenitor cells (PBPC) was started with growth factor G-CSF (Neupogen; Amgen-Roche) at a dose of 5 microg/kg/day for 13 days. In all other cycles G-CSF had been given at the same dose from day 7. On days 11, 12 and 13 the leukaphereses were performed and their products cryopreserved. On day 14 whole blood was collected. The median peak incidence of CFU-GM (granulocyte-macrophage colony-forming unit) in peripheral blood was approximately 50 times the baseline level. The leukapheresed PBPC were divided into portions and reinfused after the fourth, fifth and sixth chemotherapy courses. The support with mobilized whole blood was given after the second and third cycles. The effects of the support of whole blood vs leukapheresed PBPC on hematopoietic recovery were compared. The best yields of leukaphereses were achieved on day 13 after initiation of the chemotherapy. The mean number of CD34+ cells was 4.93 x 10(6)/kg (s.d. 2.7; range 0.36-10.54 x 10(6)/kg) the amount of CFU-GM was 2.18 x 10(5)/kg (s.d. 1.3; range 0.07-4.2 x 10(5)/kg). The yields of CFU-GM in 450 ml whole blood collected on day 14 reached 0.51 x 10(5)/kg (s.d. 0.28; range 0.05-1.5 x 10(5)/kg) and of CD34+ cells were 1.3 x 10(6)/kg (s.d. 0.8, range 0.18-2.58 x 10(6)/kg). PBPC yields in 450 ml of unprocessed whole blood were in some cases not sufficient for good hematopoietic recovery after the EC cycles. Grade 4 leukopenias and thrombocytopenias were two times higher in cycles with whole blood support than in cycles with cryopreserved PBPC support. An increase of PBPC harvest can be simply achieved by collecting larger amounts of unprocessed blood, as used by some authors.

Detailed evaluation of 2959 allogeneic and xenogeneic dense connective tissue grafts (fascia lata, pericardium, and dura mater) used in the course of 20 years for duraplasty in neurosurgery.

Surgical experience with 2959 allogeneic and xenogeneic dense connective tissue grafts (1767 of fascia lata, 909 of pericardium, and 283 of dura mater), used in 2665 neurosurgical operations performed in the course of 20 years (1976 to 1995) is reported. Duraplasty using either allogeneic or xenogeneic grafts has had a similar, and favourable clinical outcome. Nevertheless, the pliable deep frozen fascia lata grafts, which could be used in any location, have been reserved for sella turcica plugging, anterior cranial base plasty, aneurysmal wrapping, and surgery of lipomyelomeningocele. Pericardium and dura mater grafts were in the majority of cases used over the brain convexity and posterior cranial fossa. Ovine pericardium proved to be superior to bovine and allogeneic pericardia because of its workability, flexibility, reduced thickness, and better transparency. Postsurgical complications occurred in 7.3%, and they were: 1) cerebrospinal fluid fistulas in 2.8%; 2) meningites in 2.3% (aseptic 1.4%, bacterial 0.8%, and tumoural 0.1% meningites); 3) pseudomeningoceles in 2.2%; 4) wound infections in 0.6%; 5) malresorptive hydrocephalus in 0.5%; and 6) adhesions to nerve tissue in 0.5%. The majority of complications healed without surgery. Forty-eight grafts (1.6%) failed to fulfil the requirements of the surgeon, and 46 of them were re-operated upon. Though another thirty-nine grafts healed successfully, 39 shunts (1.5%) had to be performed for malresorptive hydrocephalus (0.9%), and/or for a big pseudomeningocele (0.6%). So, the pure complication rate in 2665 duraplasties was 3.1%. The complex evaluation of the allogeneic and xenogeneic grafts (fascia, pericardium, and dura mater), used for duraplasty in neurosurgery during the last 20 years proved them, as remarkably good, with a success rates of 96.9%.