Long-term storage in liquid nitrogen does not affect cell viability in cardiac valve allografts.

Liquid nitrogen is the most common medium used by tissue banks for the storage of cryopreserved heart valves. This study evaluates the effect of the length of storage on human cryopreserved heart valves. Human tissues (14 aortic and 13 pulmonary) were frozen in a controlled-rate freezer (1 degrees C/min) and stored in the liquid phase of a nitrogen tank for 9.1+/-1.6 years. The preservative solution was medium M199 containing 5% human serum albumin and 10% Me(2)SO. After thawing in a water bath at 42 degrees C, the cryoprotectant was removed. Then, fragments from vascular wall and leaflet were dissected. Explant cultures and histological studies were performed in order to assess cell viability and structural integrity. CD90 and CD31 expression was analysed in cultured cells using flow cytometry. Light microscopy, immunofluorescence staining and laser scanning confocal microscopy were used to evaluate cell viability and extracellular matrix components. Electron microscopy was used for ultrastructural study. Cell cultures could be obtained from all the specimens assayed. Cells grew from explants showing a fibroblastic phenotype. CD90 expression was common in cultured cells but a low percentage of cells expressed CD31. Histological results showed a good preservation estructure in both leaflets and vascular walls. Morphological features of cellular irreversible damage were very rare. No differences which could be due to length of allograft storage period were observed. We concluded that allografts stored in liquid nitrogen up to 13 years did not significantly undergo loss of cell viability other than that due to disinfection, freezing and thawing protocols.

Automated separation of cord blood units in top and bottom bags using the Compomat G4.

Cord blood (CB) has become a real alternative source of haematopoietic stem cells for bone marrow reconstitution in a variety of malignant disorders. As a response to this increasing activity, CB banks have been developed to guarantee the quality of processed CB units. Volume reduction of CB units maximizes storage space and also has other advantages. The aim of this study was to develop a program for the volume reduction of CB in the Compomat G4 device. We also compared two different top and bottom systems for CB fractionation (Compomat G4 and Optipress II). We empirically designed three different programs for volume reduction of CB with Compomat G4: two for final BC volume of 41 ml (CB1 and CB2) and the other one for buffy coat (BC) volume of 25 ml (CB3). Significantly worse recoveries were achieved for CB processed with program CB3. A RBC depletion of >or=50%, >or=60% and >or=70% were achieved for 67%, 39% and 9% of all units respectively. When comparing Compomat G4 and Optipress II, total nucleated cell recovery was similar for both methods, while lymphocytes recovery was significantly better for Optipress II.

Optimización de las condiciones de cultivo de las células mesenquimales de médula ósea humana para su uso en traumatología / Optimization of culture conditions of the human bone marrow mesenchymal stem cells for traumatology use

Las células mesenquimales (MSC) de la médula ósea(MO) humana constituyen un candidato celular muy prometedorpara regenerar el cartílago dañado. Las MSC generadasin vitro contienen dos poblaciones celulares condistinta capacidad de expansión y de diferenciación. En esteestudio, utilizando muestras de MO procedente de biopsiasde hueso, se definen las condiciones de cultivo quepermiten optimizar el rendimiento de los cultivos enriquecidosen células más pluripotentes. También se ha determinadoel contenido en células progenitoras de las MSC de laMO. Tras exposición a medios de diferenciación específicos,las MSC obtenidas en cultivo demostraron su capacidadosteogénica y condrogénica. Finalmente hemos demostradoque el plasma rico en plaquetas puede sustituiral factor de crecimiento transformante (TGF)-β1 utilizadopara inducir la condrogénesis no sólo en las MSC agregadasen micromasas, sino también en las crecidas en monocapay embebidas en un gel de fibrina

Mesenchymal stem cells (MSC) from human bone marrow(BM) represent a promising candidate cell type fordamaged cartilage repair. In vitro generated MSC containtwo cell populations with a distinct capability of expansionand differentiation. We have used BM samples derivedfrom human bone biopsies and we have defined cultureconditions for optimizing the yields of cultures enrichedfor pluripotent cells. Moreover, it has been determined thecontent of progenitor MSC in human BM. MSC could differentiateinto osteocytic and chondrocytic lineage afterculturing with defined culture mediums. Finally we demonstratedthat platelet-rich plasma can replace transforminggrowth factor (TGF)-β1 used to induce chondrogenicdifferentiation in MSC pelleted into micromasses but alsoin MSC cultured in monolayer and immersed in a fibrin gel

Utility of bag segment and cryovial samples for quality control and confirmatory HLA typing in umbilical cord blood banking.

Many cord blood (CB) banks have been established worldwide as a response to the increasing number of CB transplantations. In this study, we describe a quality control program in which the utility of an integral bag segment and cryovial containing aliquots of cryopreserved product as haematopoietic content control and HLA typing confirmation for CB units has been evaluated. For this purpose, every month one stored CB unit and its satellite cryovials were thawed and washed. Nucleated cell counts, viability and clonogenic assays were performed from the bag and cryovial before washing. After washing, total nucleated cell, CD34+ counts, viability, and clonogenic assays were performed from the bag. In order to assure the ability of bag segments to confirm hematopoietic potential of CB units, clonogenic assays and viability were performed from attached segments of 10 CB units and the results were compared with those from bags and cryovials. When comparing all variables between thawed bag and cryovial samples, they showed similar results. Mean colony-forming unit (CFU) content of segment samples was 118.8 +/- 93.72 x 10(4) that resulted similar to bags and cryovials haematopoietic content. In conclusion, the quality control system described in this paper demonstrates that CB units are processed preserving the quantity and quality of the progenitor cells. The contiguous segment haematopoietic content is representative of the final product.

Optimizing donor selection in a cord blood bank.

OBJECTIVES: The main limitation factor for the wide use of umbilical cord blood (UCB) as a source of hematopoietic progenitor for transplantation is cell dose. One of the specific areas identified by some studies for improvement of UCB collection is donor selection. METHODS: Over a 3-mth period, 391 consecutive maternal-neonatal pairs were evaluated during the pre-partum period in the maternity ward at La Fe University Hospital (Valencia) by the Cord Blood Bank staff. Reasons for discarding umbilical cord blood donors and collected UCB units at the Cord Blood Bank in Valencia have been analysed. Obstetric factors influencing TNC content of 1300 collected UCB units have been determined, in order to establish obstetric criteria for cord blood donors selection in our geographic area. RESULTS: Only 32.5% of potential cord blood donors were refused. Among 1300 UCB collected, 506 (38.9%) were discarded before cryopreservation, mainly due to low cell counts. Multivariate analyses showed that the main significant factors influencing nucleated cell count were the weight of the placenta, sex of newborn and mode of collection. CONCLUSIONS: Our study shows that maternal medical histories must be completely reviewed by medical staff before collection of the UCB. Obstetrical factors influence cell content of UCB and could be added to standard cord blood donor criteria in order to improve the bank efficiency.

Comparison between two strategies for umbilical cord blood collection.

The use of cord blood (CB) for transplantation has increased greatly in recent years. The collection strategy is the first step in collecting good-quality CB units. There are two main techniques for collecting CB from the umbilical vein: in the delivery room while the placenta is still in the uterus by midwives and obstetricians or in an adjacent room after placental delivery by CB bank trained personnel. In this study, the benefits and disadvantages between the two different CB collection strategies were evaluated, in order to improve CB bank methodology. Valencia CB bank maintains the two different collection strategies. CB was obtained from 569 vaginal and 70 caesarean deliveries and obstetrical and clinical charts were reviewed. Before processing CB units, volume was calculated and samples were drawn for cell counts. After processing and before cryopreservation samples were drawn for cell counts, CD34+cell analysis, viability, clonogenic assays and microbiology were drawn directly from the bags. We compared the efficiency of the two collection techniques. Obstetric data and umbilical CB were obtained from 569 vaginal (264 collected in utero and 305 collected ex utero) and 70 caesarean deliveries. The proportion of excluded CB units before processing was 33% for vaginal ex utero, 25% for vaginal in utero and 46% for caesarean deliveries. Differences were statistically significant. For vaginal deliveries a larger volume and a higher number of nucleated cells, percentage of CD34+ cells and colony-forming units (CFUs) were harvested in the in utero collection group. There was no statistical difference between CB collected after placental expulsion from vaginal and caesarean deliveries. Comparison between all vaginal and caesarean deliveries did not show any difference. We conclude that the mode of collection influences the haematopoietic content of CB donations. Collection before placental delivery is the best approach to CB collection and allows optimisation of CB bank methodology. Caesarean deliveries seem to contain similar progenitor content to vaginal deliveries.

Practices and attitudes towards quality assurance, inspection and accreditation in blood collection establishments in the European community.

In the framework of a European Community (EC)-supported project, a survey of practices and attitudes towards quality assurance, inspection and accreditation in Blood Collection Establishments (BCEs) in the EC member states was carried out. Analysis of 352 responses to a structured questionnaire revealed a preference for national standards over international, and an introspective and reactive view to quality management. Four broad categories of operational performance in relation to safety were formed: initial, repeatable, managed and optimising, with the majority of responses (209) being characterized as initial. Although a direct relationship between the size of the BCE and the range and level of quality management practices is apparent in the data, further analysis shows that small BCEs have much higher ratio of personnel per blood unit collected/processed than large BCEs and thus seem to have an inherent potential for improvement. Overall, a clear preference for inspection and accreditation by professional peers at the national level was indicated.

A new method for phenotyping red blood cells using microplates.

BACKGROUND AND OBJECTIVES: The supply of phenotyped red blood cells (RBC) for patients with several RBC antibodies presents a difficult task to hospital blood banks and regional blood centers. The aim of this study was to establish a low-cost typing system to allow extensive phenotyping of regular blood donors for clinically significant RBC antigens. MATERIALS AND METHODS: We developed a new buffer that greatly intensifies the antigen-antibody reaction and thus reduces the quantity of serum needed for phenotyping. The procedure was carried out on microplates. RESULTS: A total of 20,435 regular blood donors have been typed to date. For 752 units required for transfusion, 3,584 phenotyping tests were performed, validating the results by tube or gel typing methods; agreement was achieved in all cases. CONCLUSION: This technique seems adequate for phenotyping a large number of RBC units at very low cost, thus facilitating the availability of phenotyped blood.

A monolayer coagglutination microplate technique for typing red blood cells.

BACKGROUND AND OBJECTIVES: Serologic agglutination tests have the disadvantage of lack of an objective endpoint that can be easily read and quantitated. We have developed a new method for ABO and Rh typing based on producing a red blood cell (RBC) monolayer on microplates and the mixed hemagglutination reaction. MATERIALS AND METHODS: We have employed a new red cell fixation buffer to prepare the RBC monolayer. As the technique for grouping, we used a mixed agglutination reaction between the RBC monolayer and the RBCs to be typed. RESULTS: Compared with an automated hemagglutination system in 34,519 samples, the method is shown to be more sensitive, free of false positive reactions, and cost-effective. CONCLUSIONS: The microplate coagglutination method is accurate and lower in cost than conventional methods.

A new microplate red blood cell monolayer technique for screening and identifying red blood cell antibodies.

A new method has been developed to immobilize red blood cells in wells of microplates using a cell fixation buffer. This method has been employed for detecting and identifying red blood cell antibodies with greater sensitivity than haemagglutination antiglobulin tests, without loss of specificity. This method decreases the amount of test erythrocytes and anti-human globulin reagents employed per test, consequently lowering the cost.