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P Rebulla

Publications and source records attributed to P Rebulla.

At least 19 recordsLinked to original sources
PubMed2006

Animal model for liver cell banking from non-heart beating donors after prolonged ischaemia time.

BACKGROUND AND AIM: Although there is a growing interest on the use of non-heart beating donors to enlarge the liver donor pool, livers with prolonged warm ischaemia time are not currently considered for organ transplantation. We hypothesised that these organs may represent a source of hepatocytes for cell transplantation and/or use in bioartificial liver devices. Thus, we investigated if prolonged ischaemia could influence the recovery and viability of functional hepatocytes dissociated from rat livers. METHODS: Hepatocytes were isolated from the liver within 15 min after death (t=15 min) and after 4, 8 and 12h of ischaemia. Cells were either maintained in culture or cryopreserved. In all products, we evaluated cell recovery and viability, hepatocyte markers and cellular functions, including albumin and urea production. RESULTS: The number of cells per gram of tissue was similar at 15 min, 4 and 8h, while it was significantly decreased at 12h. About 0.2 x 10(6) viable cells expressing hepatocyte markers and producing albumin and urea were isolated up to 8h of ischaemia per gram of tissue. CONCLUSIONS: Recovery of viable and functional hepatocytes seems possible after prolonged ischaemia time. These data warrant the evaluation of hepatocyte isolation from human livers of non-heart beating donors.

Animals↗
PubMed2006

Multi-laboratory evaluation of procedures for reducing the volume of cord blood: influence on cell recoveries.

BACKGROUND: Various procedures can be used to isolate stem and progenitor cells from cord blood. This study evaluated the hydroxyethyl starch sedimentation (HES) with two centrifugation steps, and the top and bottom (T&B) isolation of buffy coat following a single centrifugation, and two filter systems for processing cord blood, one developed by Asahi Kasei Medical (filter A) and the second by Terumo (filter B). METHODS: Each of seven laboratories was randomly assigned the evaluation of either the HES or T&B method and one of the filter methods (n=8 cord blood units, per laboratory, for each method). The leukocyte-containing fraction with the stem/progenitor cells was recovered from the filters by reverse flushing. Utilizing the routine traditional processing and testing procedures of each laboratory, in vitro parameters were determined, with samples obtained after collection, after processing and after freezing/thawing. The results were expressed as the percentage recovery of viable cells in processed vs. collected samples (performance 1; PF1) and in thawed vs. processed samples (performance 2; PF2). The composite results obtained by the seven laboratories were summarized. RESULTS: The median PF1 percentage recovery of total nucleated cells (TNC) was comparable with both traditional methods (HES 79%, T&B 86%) and statistically reduced with both filtration procedures (filter A 58%, filter B 61%). Mononuclear cell (MNC) PF1 recovery was highest statistically with the T&B method (91%) and reduced on using filter A (77%) and filter B (70%) and the HES method (72%). CD34+ cell recovery was judged to be essentially comparable with the four methods, although the range of unit recoveries differed. The percentage recovery of TNC and MNC in PF1 was influenced by the volume of the collected cord blood, especially with use of the filtration procedures. This correlated with TNC content. A greater percentage of red cells and platelets was removed during processing with both filter methods. The time to process cord blood preparations with filter A was significantly shorter than the other methods. Processing with the HES method took the longest time. The recoveries for TNC, MNC and CD34+ cells in PF2 did not appear to be influenced by the specific processing procedure. DISCUSSION: These data indicate that filters that capture stem and progenitor cells may be an appropriate methodology for processing cord blood collected for banking.

Antigens, CD34↗
PubMed2006

Transfusion recipient identification.

Recent reports from different haemovigilance systems indicate that errors in the whole-blood transfusion chain - from initial recipient identification to final blood administration - occur with a frequency of approximately 1 in 1000 events. Although mistakes occur also within the blood transfusion service, about two-thirds of errors are associated with incorrect blood recipient identification at the patient's bedside. To prevent the potentially fatal consequences of such mistakes, specific tools have been developed, including patient identification bracelets with barcodes and/or radio frequency identification devices, mechanical or electronic locks preventing access to bags assigned to other patients, and palm computers suitable for transferring blood request and administration data from the patient's bedside to the blood transfusion service information system in real time. The effectiveness of these systems in preventing mistransfusion has been demonstrated in a number of studies.

Blood Group Incompatibility↗
PubMed2006

Assessment of selective homing and contribution to vessel formation of cryopreserved peripherally injected bone marrow mononuclear cells following experimental myocardial damage.

In view of a potential clinical use we aimed this study to assess the selective homing to the injured myocardium and the definitive fate of peripherally injected labeled and previously cryopreserved Bone Marrow Mononuclear cells (BMMNCs). The myocardial damage (cryoinjury) was produced in 59 rats (45 treated, 14 controls). From 51 donor rats 4.4 x 10(9) BMMNCs were isolated and cryopreserved (slow-cooling protocols); the number of CD34+ and the viability of pooled cells was assessed by flow-cytometry analysis before and after cryopreservation and simulated delivery through a 23G needle. Seven days after injury, BMMNCs were thawed, labeled with PKH26 dye and peripherally injected (20 x 10(6) cells in 500 microl) in recipient rats. Two weeks after experimental injury, the heart, lungs, liver, kidneys, spleen and thymus were harvested to track transplanted cells. Except a small amount in the spleen, PKH26+ cells were found only in the infarcted myocardium of the treated animals. Typical vascular structures CD34+ were found in the infarcted areas of all animals; treated rats showed a significantly higher number of these structures if compared with untreated. Morphological ultra-structural examination of infarcted areas confirmed in treated rats the presence of early-stage PKH26+ vascular structures derived from injected BMMNCs. The estimated mean CD34+ cells loss due to the cryopreservation procedure and to the system of delivery was 0.24% and 0.1%, respectively, confirming the feasibility of the procedure. This study supports the possible therapeutic use of cryopreserved peripherally injecetd BMMNCs as a source of CD34+ independent vascular structures following myocardial damage.

Animals↗
PubMed2005

High-altitude trekking in the Himalayas increases the activity of circulating endothelial cells.

Circulating endothelial progenitor cells (EPCs) are believed to contribute to vascular homeostasis; unfortunately, the response of EPCs in physiological conditions remains largely unknown. Herein we report our observations of a 44-year-old healthy subject after a trek in the Himalayas that support high-altitude hypoxia and exercise oxygen demands are strong stimuli for clonogenic endothelial cell activation and activity, as shown by the increase in the number of mature EPCs and in the endothelial colony-forming unit capacity. Both of these effects were completely reverted at sea level, 45 days after the subject's trek.

Adult↗
PubMed2004

Prophylactic platelet transfusion for haemorrhage after chemotherapy and stem cell transplantation.

BACKGROUND: Platelet transfusions are used in modern clinical practice to prevent and treat bleeding in thrombocytopenic patients with bone marrow failure. Although considerable advances have been made in platelet transfusion therapy in the last 30 years, some areas continue to provoke debate, especially the use of prophylactic platelet transfusions for the prevention of thrombocytopenic bleeding. OBJECTIVES: To determine the optimal use of platelet transfusion for the prevention of haemorrhage (prophylactic platelet transfusion) in patients with haematological malignancies undergoing chemotherapy or stem cell transplantation. SEARCH STRATEGY: Randomised controlled trials (RCTs) were searched for in the Cochrane Central Register of Controlled Trials (CENTRAL). Searching was also undertaken on the OVID versions of MEDLINE and EMBASE using an RCT search filter strategy. SELECTION CRITERIA: Randomised controlled trials involving transfusions of platelet concentrates, prepared either from individual units of whole blood or by apheresis, and given prophylactically to prevent bleeding in patients with haematological malignancies and receiving treatment with chemotherapy and/or stem cell transplantation. DATA COLLECTION AND ANALYSIS: All electronically derived citations and abstracts of papers identified by the review search strategy were initially screened for relevancy by one reviewer. Studies clearly irrelevant were excluded at this stage. The full text of all potentially relevant trials was then formally assessed for eligibility by two reviewers independently. Two reviewers completed data extraction independently. Missing data were requested from the original investigators, as appropriate. Disagreements were resolved by discussion with the other reviewers. MAIN RESULTS: Eight completed published trials, with a total of 390 participants in the intervention groups and 362 participants in the control groups, were included in the review for further analysis. The eight studies were classified as: * three trials relevant to prophylactic platelet transfusions versus therapeutic platelet transfusions; * three trials relevant to prophylactic platelet transfusion with one trigger level versus prophylactic platelet transfusion with another trigger level; * two trials relevant to prophylactic platelet transfusion with one dose schedule versus prophylactic platelet transfusion with another dose schedule. The few reports of controlled trials addressing prophylactic versus therapeutic transfusions contained small numbers of patients and were all undertaken over 25 years ago. None of these three studies explicitly clarified whether the lack of a reported difference was a reflection of insufficient power in the trials. The findings of the meta-analyses for this group of three small studies must be interpreted with caution. In contrast, more contemporary trials addressed the question of what platelet count thresholds should apply for prophylactic transfusion; three identified studies broadly compared platelet transfusion thresholds of 10 versus 20 x 109/litre for different clinical groups of patients. There were no statistically significant differences between the groups with regards to mortality, remission rates, number of participants with severe bleeding events or red cell transfusion requirements. However, it was unclear whether the studies had sufficient power to demonstrate in combination non-inferiority in terms of safety of the lower threshold, 10 x 109/litre. Insufficient randomised trials have been undertaken to make clinically relevant conclusions about the effect of different platelet doses. REVIEWERS' CONCLUSIONS: There are no reasons to change current practice but uncertainty about the practice of prophylactic transfusion therapy should be recognised, particularly in the light of concerns about the scenario that blood products, including platelets, could become an increasingly scarce resource in the future and for which adequate alternatives do not exist. Consideration should be given to developing adequately powered trials comparing strategies of prophylaxis versus therapeutic platelet transfusion.

Hemorrhage↗
PubMed2004

Quality-related variables at hepatological websites.

BACKGROUND: The amount of hepatology-related information available on the Internet has substantially increased, but little is known about the characteristics and quality of the websites. AIM: The aim of this study was to describe analytically and evaluate critically the information concerning three diseases of hepatological interest: chronic hepatitis, hemochromatosis and Caroli's disease. METHODS: In accordance with a validated method, the three search terms were entered into four English language search engines and the first five links of each were considered (a total of 60 sites). The characteristics of the websites were described and their quality was evaluated by three independent reviewers who assigned scores of 1-5 for accuracy, reliability and depth. The relationships between the site characteristics and quality scores were analysed by means of multiple logistic regression. RESULTS: The overall rating score was sufficient (>3) in 51% (95% confidence interval: 38-65%) of cases. The majority of the sites (73%) were aimed at patients rather than at physicians. Commercial sponsorship was significantly more frequent among the chronic hepatitis sites (45%) than among the hemochromatosis (15%) or Caroli's disease sites (0%) (P = 0.002); 61% of the commercial sites did not include a financial disclosure. The only variable that independently related to poor quality was the presence of commercial sponsorship (odds ratio 18.1; 95% confidence interval: 1.7-192.5). CONCLUSIONS: Hepatological websites are characterised by poor quality and are mainly aimed at patients. Quality is negatively affected by commercial interests, which are often undeclared. Guidelines for the certification and surveillance of websites relating to liver diseases are highly advisable.

Authorship↗
PubMed2004

Clinical grade cell manipulation.

In the past decade, a new form of therapy based on biological rather than pharmacological intervention has been developed. The term 'cell therapy', as applied to this new therapeutic tool, means the administration of living, non-germline somatic cells to humans for diagnostic or therapeutic purposes. Cell therapy products (CTPs) are generated by ex vivo processes, which comprise cell harvesting from patients or healthy donors, in vitro manipulation and administration of the manipulated cells to patients. The aim of ex vivo processes is to obtain cell subsets with defined functional properties that are capable of replacing or repairing damaged tissues or organs. Some examples of cell therapy are transplantation of expanded haematopoietic stem cells (HSCs), adoptive immunotherapy and dendritic cell vaccination to augment or restore the immune response for the treatment of malignant or infectious diseases. The types of cells most frequently used for cell therapy include haematopoietic pluripotent progenitor and stem cells from the bone marrow and peripheral blood, T-cell clones and dendritic cells. Although CTPs should be produced according to good manufacturing practice, they differ from traditional pharmaceutical products with regard to quality control and safety aspects. These differences prompted the development of a number of documents issued by regulatory bodies, which specifically address CTPs. This review discusses several issues related to the design, construction and validation of a hospital-based facility for the production of CTPs, the implementation of cell-manipulation processes and quality control of the final products.

Animals↗
PubMed2004

Methods for the analysis of bleeding outcomes in randomized trials of PLT transfusion triggers.

BACKGROUND: A number of methodologic challenges arise in the analysis of bleeding data from clinical trials of PLT transfusion triggers. It is important to understand the assumptions and role of the various methods of analysis to interpret published trials and to design future studies appropriately. STUDY DESIGN AND METHODS: The methods of analysis used for testing the effectiveness and safety of transfusion strategies are reviewed from several recent PLT transfusion trigger trials. The underlying assumptions of these methods are discussed, as well as the clinical interpretations of the resulting summary statistics. Four methods of analysis were applied to data from a large PLT transfusion trigger study to illustrate the differences in the interpretations that can arise from various approaches. RESULTS: PLT transfusion trigger trials of patients with leukemia have based their primary analyses on 1) simple dichotomous classifications of whether or not at least 1 day of clinically important bleeding was experienced; 2) the time to the first day of clinically important bleeding; and 3) the proportion of days at risk with clinically important bleeding. Recurrent event methods provide a robust alternative approach to the analysis of this kind of data and should be considered if interest is in capturing the overall burden of bleeding over time. These four methods differ in the extent to which they utilize information on the number of days with bleeding and the temporal variation in bleeding patterns. Inferences drawn regarding the relative safety and efficacy of different transfusion triggers can vary depending on the method of analysis. CONCLUSION: To rigorously design and analyze future PLT transfusion studies based on bleeding outcomes, it is important to have a clear understanding of the interpretation of the different ways of analyzing bleeding outcomes. The analysis strategy should be selected based on the clinical question being addressed.

Hemorrhage↗
PubMed2001

Platelet transfusion for patients with cancer: clinical practice guidelines of the American Society of Clinical Oncology.

OBJECTIVE: To determine the most effective, evidence-based approach to the use of platelet transfusions in patients with cancer. OUTCOMES: Outcomes of interest included prevention of morbidity and mortality from hemorrhage, effects on survival, quality of life, toxicity reduction, and cost-effectiveness. EVIDENCE: A complete MedLine search was performed of the past 20 years of the medical literature. Keywords included platelet transfusion, alloimmunization, hemorrhage, threshold and thrombocytopenia. The search was broadened by articles from the bibliographies of selected articles. VALUES: Levels of evidence and guideline grades were rated by a standard process. More weight was given to studies that tested a hypothesis directly related to one of the primary outcomes in a randomized design. BENEFITS/HARMS/COST: The possible consequences of different approaches to the use of platelet transfusion were considered in evaluating a preference for one or another technique producing similar outcomes. Cost alone was not a determining factor. RECOMMENDATIONS: Appendix A summarizes the recommendations concerning the choice of particular platelet preparations, the use of prophylactic platelet transfusions, indications for transfusion in selected clinical situations, and the diagnosis, prevention, and management of refractoriness to platelet transfusion. VALIDATION: Five outside reviewers, the ASCO Health Services Research Committee, and the ASCO Board reviewed this document. SPONSOR: American Society of Clinical Oncology

Cost-Benefit Analysis↗
PubMed2001

Platelet transfusion trigger in difficult patients.

There is general consensus that a prophylactic pre-transfusion trigger at 10.000 platelets/microL in stable oncohematological patients is as safe as the traditional trigger of 20.000/microL, and that perioperative triggers at 50.000 and 100.000/microL are adequate in most surgical and neurosurgical conditions respectively. Guidelines on the trigger and other issues related to platelet transfusion can be found in nine documents published during 1987-2001 by the National Institutes of Health (NIH), the British Committee on Standardization in Hematology, the Royal College of Physicians of Edinburgh, the College of American Pathologists, the American Society of Anesthesiology and the American Society of Clinical Oncology (ASCO). Although consensus may be less evident on specific triggers for 'difficult' patients, the following triggers, listed by progressively increasing levels, have been proposed in the literature and have found general agreement: a stable oncohematological recipient: 10.000; lumbar puncture in a stable pediatric leukemic patient: 10.000; thrombocytopenia secondary to gpIIb/IIIa receptor inhibitors [corrected]:10.000; bone marrow aspiration and biopsy: 20.000; gastrointestinal endoscopy in cancer: 20.000-40.000; disseminated intravascular coagulation: 20.000-50.000; fiber-optic bronchoscopy in a bone marrow transplant recipient: 20.000-50.000; neonatal alloimmune thrombocytopenia: 30.000; major surgery in leukemia: 50.000; thrombocytopenia secondary to massive transfusion: 50.000; invasive procedures in cirrhosis: 50.000; cardiopulmonary bypass: 50.000-60.000; liver biopsy: 50.000-100.000; a nonbleeding premature infant: 60.000; neurosurgery: 100.000. The proposed values must be considered within the context of careful clinical evaluation of each individual patient, and attention should be given to the power of discrimination of platelet counters at low counts and to the prompt availability of good quality platelet products in the case of emergency.

Adolescent↗
PubMed2001

Evaluation of the effect of cryopreservation on ex vivo expansion of hematopoietic progenitors from cord blood.

In previous studies, we identified a cytokine cocktail including thrombopoietin, Flt-3 ligand, interleukin (IL)-6 and IL-11 in serum-free medium, suitable to induce significant and sustained ex vivo expansion of primitive hematopoietic stem cells (HSCs) from cord blood (CB) for up to 10 weeks. The aim of the present study was to evaluate the effects of cryopreservation on ex vivo expansion of HSCs and their committed progenitors. CD34+ cells were purified from CB units, each of which was processed in part as such and in part as cryopreserved and thawed, then expanded for 5 weeks in serum-free medium with the cytokine cocktail described above. We determined the number of nucleated cells (NC), CD34+, CD34+/38(-)/33(-), CD34+/61+, CD61+ cells and the clonogenic potential. After 2 weeks the median fold expansion of NC, CD34+ and CD34+/38(-)/33(-) cells was around two log both with fresh and cryopreserved CB and the expansion continued similarly until week 5. Our data suggest that this serum free protocol induces similar ex vivo expansion of HSCs and their committed progenitors from both fresh and cryopreserved CB. Our findings can be useful in view of clinical applications, since CB used for transplantation is stored in the cryopreserved state.

Blood Preservation↗
PubMed2001

Long-term expansion and maintenance of cord blood haematopoietic stem cells using thrombopoietin, Flt3-ligand, interleukin (IL)-6 and IL-11 in a serum-free and stroma-free culture system.

Although cord blood (CB) compares favourably with other haematopoietic stem cell (HSCs) sources, its use in large patients is limited by the low number of cells available. Ex vivo expansion of CB HSCs has been used to overcome this limitation. In this study, we investigated the effect of different cytokine cocktails, including interleukin (IL)-6, IL-11, Flt3-ligand (FL) and thrombopoietin (TPO) combined with serum or serum-free medium on the ex vivo expansion of CD34+ cells from CB. Initial experiments showed that expansion could be slightly improved using serum, but we chose to use serum-free medium in the subsequent investigations to apply good medical practice (GMP) conditions suitable for clinical use. The highest expansion of CD34+ cells was obtained with a cocktail containing FL + TPO + IL-6 + IL-11. The median (range) fold expansions of CD34+ cells at 5 and 10 weeks with serum-free medium were 235.6 (131.3-340) and 5205.6 (4736.6-5674.7) respectively. The absence of IL-11 was associated with a similar fold expansion after 5 weeks (median 215.6, range 149.8-281.5), but after 10 weeks expansion was slightly lower (median 1314.7, range 645-1984.4). Our data support the possibility of maintaining long-term expansion of CB HSCs in a simple stroma- and serum-free system.

Antigens, CD34↗
PubMed2001

The 'cherry buffy-coat syndrome', a cause of decreased platelet yield in platelet concentrates obtained from buffy-coats.

BACKGROUND AND OBJECTIVES: A large number of European blood centres, including our own, use the buffy-coat method for platelet production. In this article we describe a previously unnoticed phenomenon shown by a proportion of buffy-coats, which display an unusually bright cherry colour and low platelet counts. MATERIALS AND METHODS: We performed bacterial cultures, platelet counts, pO2, pCO2 and pH, and evaluated platelet activation by flow cytometry in cherry versus normal-colour (control) buffy-coats. In addition, we compared donor characteristics in the two groups and platelet counts in the packed red blood cells (RBC) obtained from the original donations. Finally, we monitored the frequency of cherry buffy-coats in the bags of three manufacturers, and determined the concordance rate of two trained technicians in detecting cherry buffy-coats. RESULTS: Bacterial cultures were negative. Cherry buffy-coats contained significantly fewer platelets, more O2, less CO2 and had a significantly higher pH than normal buffy coats. Platelet activation was slightly higher in cherry buffy-coats. RBC from donations yielding cherry buffy-coats contained a significantly higher number of platelets than controls. Donor characteristics were not significantly different. Cherry buffy-coats were significantly more frequent with bags from one manufacturer (24%) than from others (9% and 11.6%). The concordance study showed excellent agreement. CONCLUSIONS: Our hypothesis is that the cherry colour is caused by O2 accumulation in buffy-coats with low platelet counts. The latter may be caused by platelet activation and aggregation during blood processing. Further work is needed to determine the cause of this phenomenon, its frequency in different laboratories and means to prevent it.

Blood Platelets↗