Microbiological assessment of arterial allografts processed in a tissue bank.

The transmission of microbial infection through tissue allografts is one of the main risks that must be controlled in tissue banks. Therefore, microbiological monitoring controls and validated protocols for the decontamination of tissues during processing have been implemented. This study is based on the evaluation of data from microbiological cultures of arteries (mainly long peripheral arteries) processed in the tissue bank of Valencia (Spain). Donors' profile, pre- and post-disinfection tissue samples were assessed. The presence of residual antibiotics in disinfected tissues was determined and the antimicrobial potential of these tissues was tested. Our overall contamination rate was 23.69%, with a disinfection rate (after antibiotic incubation) of 87.5%. Most (76.09%) of the microbial contaminants were identified as Gram positive. Arterial allografts collected from body sites affected by prior organ removal showed higher risk of contamination. Only vancomycin was detected as tissue release. The antimicrobial effect on Candida albicans was lower than that for bacterial species. Risk assessment for microbial contamination suggested the donor's skin and the environment during tissue collection as the main sources for allograft contamination. Antibiotic-disinfected arterial allografts showed antimicrobial potential.

Risk assessment of arterial allograft contamination from tissue donors colonized by Candida auris.

BACKGROUND: Microbiological contamination is one of the main risks that must be controlled in tissue banking practices. For this reason, strict donor selection criteria are applied, disinfection protocols are used, and microbiological monitoring is performed at various stages. AIM: To detect Candida auris in arterial allografts and assess its origin. METHODS: Data on two multi-tissue donations with positive microbiological cultures for C. auris were analysed. Risk factors for microbiological contamination were assessed at procurement, processing and post storage. FINDINGS: C. auris was only isolated in cultures from arteries, and was not detected in cultures from cornea, musculoskeletal tissue or skin (even in the axillary-rectal sample taken from one donor). CONCLUSION: The donor's own skin was identified as the most likely source to explain the contamination of arteries by C. auris. Due to the pathogenicity of this fungus and difficulties associated with its correct identification, the implementation of measures for its detection in tissue donations is recommended.

Sequential hepatogenic transdifferentiation of adipose tissue-derived stem cells: relevance of different extracellular signaling molecules, transcription factors involved, and expression of new key marker genes.

Adipose tissue contains a mesenchymal stem cell (MSC) population known as adipose-derived stem cells (ASCs) capable of differentiating into different cell types. Our aim was to induce hepatic transdifferentiation of ASCs by sequential exposure to several combinations of cytokines, growth factors, and hormones. The most efficient hepatogenic protocol includes fibroblastic growth factors (FGF) 2 and 4 and epidermal growth factor (EGF) (step 1), hepatocyte growth factor (HGF), FGF2, FGF4, and nicotinamide (Nic) (step 2), and oncostatin M (OSM), dexamethasone (Dex), and insulin-tranferrin-selenium (step 3). This protocol activated transcription factors [GATA6, Hex, CCAAT/enhancer binding protein alpha and beta (CEBPalpha and beta), peroxisome proliferator-activated receptor-gamma, coactivator 1 alpha (PGC1alpha), and hepatocyte nuclear factor 4 alpha (HNF4alpha)], which promoted a characteristic hepatic phenotype, as assessed by new informative markers for the step-by-step hepatic transdifferentiation of hMSC [early markers: albumin (ALB), alpha-2-macroglobuline (alpha2M), complement protein C3 (C3), and selenoprotein P1 (SEPP1); late markers: cytochrome P450 3A4 (CYP3A4), apolipoprotein E (APOE), acyl-CoA synthetase long-chain family member 1 (ACSL1), and angiotensin II receptor, type 1 (AGTR1)]. The loss of adipose adult stem cell phenotype was detected by losing expression of Thy1 and inhibitor of DNA binding 3 (Id3). The reexpression of phosphoenolpyruvate corboxykinase (PEPCK), apolipoprotein C3 (APOCIII), aldolase B (ALDOB), and cytochrome P450 1A2 (CYP1A2) was achieved by transduction with a recombinant adenovirus for HNF4alpha and finally hepatic functionality was also assessed by analyzing specific biochemical markers. We conclude that ASCs could represent an alternative tool in clinical therapy for liver dysfunction and regenerative medicine.

Microbiological analysis of cryopreserved human heart valves after storage: a survey of 3 banks in Spain.

Several reports have shown liquid nitrogen containers as not being sterile. Microorganism transmission has been observed in different cells and tissues stored under this condition, but there is no data on contamination of stored human valves. We performed a survey on heart valve banking in Spain. Regarding the questionnaire, we have a complete microbiological analysis of 304 thawed tissues prior to implant. In six cases positive culture results were observed. Patient follow-up did not reveal any adverse effects. Although some other possibilities should be stated, contamination of heart valves during storage in liquid nitrogen should be considered as a risk element in tissue banking. Strategies to asses and prevent microbial transmission from liquid nitrogen to heart valve banking ought to be further developed.

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.

Donor screening for hepatitis B virus infection in a cell and tissue bank.

BACKGROUND AND OBJECTIVES: Hepatitis B virus (HBV) has been transmitted by tissue transplantation. In order to reduce the risk of HBV transmission, testing for antibody to HBV core antigen (anti-HBc) is used in addition to testing for hepatitis B surface antigen (HBsAg) in many blood centers and tissue banks. DESIGN AND METHODS: We retrospectively analyzed the results of HBV assays in tissue donors. All tissue donors were tested for HBsAg and anti-HBc. All anti-HBc positive sera were tested for the antibody to HBsAg (anti-HBs). From July 2006, an HBV nucleic acid testing (NAT) assay was also performed. RESULTS: A total of 6855 tissue donors from January 1999 till July 2007 were tested for HBV assays: 4756 women and 2099 men. Positive HBsAg was found in 23 (0.36%) living donors, while no multiorgan or cord blood (CB) donor was found to be positive for HBsAg. Positive anti-HBc was found in 80 multiorgan donors (12.94%), 599 living donors (17.84%), and 103 CB donors (3.57%) (P<0.005), while isolated anti-HBc was found in 12 multiorgan (1.94%), in 126 living tissue donors (3.75%), and in 8 CB donors (0.28%). A total of 1310 donors were analyzed for single-sample DNA HBV NAT assay. DISCUSSION: We consider that anti-HBc and NAT assays must both still be performed in addition to HBsAg assay for HBV screening in tissue donors. All these tests will be useful in order to define an algorithm for safe and efficient management of the tissue bank.

Human mesenchymal stem cells from adipose tissue: Differentiation into hepatic lineage.

Adipose tissue represents an accessible source of mesenchymal stem cells (ADSCs), with similar characteristics to bone marrow-derived stem cells. The aim of this work was to investigate the transdifferentiation of ADSCs into hepatic lineage cells in vitro. ADSCs were obtained from human adipose tissue from lipectomy. Cells were grown in medium containing 15% AB human serum. Cultures were serum deprived for two days and exposed to a two-step protocol with two different media using growth factors and cytokines. Hepatic differentiation was assessed by RT-PCR of liver-marker genes. ADSCs exhibited a fibroblastic morphology that changed to a cuboidal shape when cells differentiated. Expression of liver genes increased when using one of the two studied media consisting of DMEM supplemented with HGF, bFGF and nicotinamide for 14 days. The results indicate that, under certain specific inducing conditions, ADSCs can be induced to differentiate into hepatic lineage in vitro. Adipose tissue may be an ideal source of high amounts of autologous stem cells.

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.

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.

Comparison between two cord blood collection strategies.

BACKGROUND AND OBJECTIVES: Collection strategy is the first step for collecting good quality cord blood (CB) units. There are two principal different techniques to collect 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 two different CB collection strategies were evaluated in order to improve CB bank methodology. DESIGN AND METHODS: Valencia CB bank maintains the two different collection strategies aforementioned. Before processing CB units, volume was calculated and samples were drawn for cell counts. After processing and before cryopreservation, samples for cell counts, CD34 analysis, viability, clonogenic assays and microbiology were drawn directly from the bags. We compared the efficiency of the two collection techniques. RESULTS: Obstetric date and umbilical CB was obtained from 848 vaginal (484 collected in uterus and 364 collected ex uterus). The proportion of excluded CB units before processing was 33% for ex uterus and 25% for in uterus. The difference was statistically significant. A larger volume and a higher number of total nucleated cells, CD34+ cells and CFUs were harvested in the in uterus collection group. INTERPRETATION AND CONCLUSIONS: Based on our findings, we conclude that the mode of collection influences the hematopoietic content of CB donations. Collection before placental delivery is the best approach to CB collection and allows optimizing CB bank methodology.

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.

Quality analysis of blood components obtained by automated buffy-coat layer removal with a top & bottom system (Optipress (R)II).

BACKGROUND AND OBJECTIVE: There are Council of Europe recommendations for the quality of blood components. We analyzed the quality of blood components processed by a top & bottom system (Optipress((R)) II), the routine method used in our blood bank, to test whether the components reached the recommended quality. DESIGN AND METHODS: Blood was collected in triple CPD-SAGM bags (Optipac((R)) Baxter). Whole blood (WB) was centrifuged at 4,158 g for 14 min before separation by an automated top & bottom system (Optipress((R) )II). Platelet concentrate (PC) was prepared by pooling four isogroup buffy-coat (BC) units before low-speed centrifugation, and transferring the supernatant (4 BC-PC) to a 5-day storage bag (PL732, Baxter). An alternative approach involved PC preparation from a single BC unit by adding approximately 70 mL of plasma before centrifugation, followed by transfer of the platelet concentrate (1BC-PC) to a 300 mL Teruflex((R)) transfer bag. Both 4 BC-PC and 1 BC-PC were stored in a flat agitator at 22 degrees C for up to 5 days after collection. Cell counts were determined, along with hemoglobin and hematocrit in a Sysmex K-800 cell counter. The pH was determined on day 5 at 22 degrees C. Weights were measured and volumes were calculated based on specific gravity. Statistical analyses were carried out using the Kolmogorov-Smirnov test as a normality distribution test, the t-test for parametric values and Wilcoxon's test as a non-parametric test. Statistical significance between samples was considered to have been reached when p<0.05. RESULTS: The best parameters for configuring the system were: strength 25; BC volume 33-55; level of BC 5.5. Red blood cell (n = 1,434) volume was 279+/-20 mL, with 54.92+/-7.16 g of hemoglobin. More than 96% of units had fewer than 1.2x10(9) white blood cells. Fresh plasma volume (n = 803) averaged 279+/-19 mL, with a white blood cell contamination of fewer than 0.1x10(9)/L in all samples examined (n = 23). Platelet recovery in BC was 92+/-9% of platelets present in WB; the percentage of removed leukocytes was 74+/-10%, and between 13 and 15% of RBCs were lost in the BC (95% confidence interval). The BC volume (n = 1,037) fitted the target volume of 60 mL, except for some devices, when Optipress II((R)) lost the configuration for this parameter. Of 4 BC-PCs 80.3% yielded more than 0.6x10(11) platelets per unit, whereas this criterion was only met by 59.7% of 1 BC-PCs, and a greater proportion of 1 BC-PCs (58.8%) showed pH values within the range of 6.5-7.4 after 5 days of storage in comparison with 4 BC-PCs (44.25%). INTERPRETATION AND CONCLUSIONS: Optipress II((R)) provides standardized, leukocyte-poor blood components. Council of Europe requirements were met in a large percentage of red-cell concentrates, with less than 92 and 74% of the original platelets and leukocytes, respectively, and a small hemoglobin loss per unit. The system gave an optimal yield in terms of plasma volume. The top & bottom technique allowed us to reduce the number of blood units per platelet concentrate from 6 to 4 units, with similar platelet yields compared with traditional procedures. Nevertheless, the storage conditions must be improved to satisfy all Council of Europe requirements for platelet concentrates.

[Analysis of the quality of hemoderivatives obtained using a buffy-coat extraction system with a top-and-bottom technique (Optipress II)]. / Análisis de calidad de hemoderivados obtenidos mediante un sistema de extracción de la capa leucoplaquetar con tecnología Top & Bottom (Optipress II).

PURPOSE: The aim of the present study is to know the results of the quality analysis of blood components processed with a Top & Bottom system (Optipress II) as a routine method in our blood bank, and compare it with the CE recommendations for quality of blood components. MATERIAL AND METHODS: Blood was collected in triple CPD-SAGM bags (Optipac, Baxter) and whole blood (WB) were centrifuged at 4,158 g, 14 min. Blood separation was performed by an automated Top & Bottom system (Optipress II), in which parameters were individually configured in preliminary trials. The buffy-coat (BC) layer was maintained within the configured levels during the separation process and remained into the original bag, whereas red cells (RBC) were collected into the bottom satellite bag (with 100 mL of SAGM) and fresh plasma (FP) was sent to the top satellite bag. Platelet concentrate (PC) was prepared by two different ways: 4 isogroup buffy-coats units were pooled by means of a sterile connector device (TSCD-201, Terumo) before a low centrifugation (1,040 g, 9 min) and the supernatant (4BC-PC) was transferred into a PL732 bag (Fenwal, Baxter); the other PC was prepared from one unit of BC by additioning approximately 70 mL of FP before centrifugation (321 g, 6 min) and following transference of the platelet concentrate (1BC-CP) into a 300 mL (Teruflex, Terumo) transfer bag. Both, 4BC-PC and 1BC-PC, were stored in a flat agitator at 22 degrees C to up five days after collection. We determined cell counts, haemoglobin, and hematocrit in a Sysmex K-800 cell counter in WB and blood components. Nageotte chamber was used when low white blood cells (WBC) counts were obtained. We also determined pH values on day five at 22 degrees C in a Crison 2000. Weights were measured and volumes were calculated using specificity gravity. Statistical analysis were carried out by Kolmogorov-Smirnov test as a normality distribution test, t-test for parametrical values and Wilcoxon-test as a no parametrical test (p < 0.05 was considered as Wilcoxon a significant value between different samples). RESULTS: The best parameters to configure the system were: strength: 25; BC volume: 33-35; level of BC: 5.5. RBCs (n: 1434) volume was 279 +/- 20 mL with 54.92 +/- 7.16 g of haemoglobin. More than 96% units had less than 1.2 x 10(9) WBC. FP volume (n: 803) averaged 279 +/- 19 mL with a WBC contamination less than 0.1 x 10(9)/L in all examined samples (n: 23). Platelet recovery in BC 92 +/- 9 percent of platelets present in WB, the percentage of removed leukocytes was 74 +/- 10 and between 13 and 15% of RBCs were lost in the BC (CI 95%). The BC volume (n: 1037) fitted the target volume of 60 mL (59-61 mL, CI 95%) except in some devices, where Optipress II lost the configuration for this parameter. 4BC-CPs (n: 325) showed a platelet yield per unit greater than 1BC-CPs (226). In addition, 80.3% of 4BC-CPs yielded more than 0.6 x 10(11) platelets per unit, whereas this criteria was only met in 59.7% of 1BC-CPs (p < 0.001). The ratio volume oper 10(9) platelets in 1 BC-CPs was significantly higher (1.57 mL) than 4BC-CPs (1.31 mL), and a greater level of 1BC-CPs (58.8%) showed pH values within 6.5-7.4 after 5 days of storage in comparison with 4BC-CPs (44.25%) (p < 0.001). CONCLUSIONS: Optipress II provides standardized and poor leukocytes blood components. CE requirements were met in a great percentage of red-cell concentrates with less than 92 and 74 percent of original platelets and leukocytes, respectively and a low loss of haemoglobin per unit. Plasma volume obtained with this system represents an optimal yield. Top and Bottom technique allowed us to reduce the number of blood units per platelet concentrate, from six to four units with similar platelet yield compared to traditional procedures. Nevertheless, we must improve the storage conditions, in orter to satisfy all the CE requirements for platelet concentrates.

In vitro proliferation, differentiation and immuno-magnetic bead purification of human myoblasts.

OBJECTIVES: In vitro culture of myoblasts and subsequent grafting into injured myocardium represents a new therapeutic approach for the treatment of myocardial infarct. A major limitation to developing enough myoblasts to engrafting purpose is the isolation and purification. In the present work we purified myoblast from primary culture using an immunomagnetic bead technique. METHODS: Primary culture was obtained by trypsin-EDTA digestion of human muscle biopsies. Cells were cultured in DMEM growth medium containing 10% FBS, 2 mM L-glutamine and antibiotics. Immunotechniques using both monoclonal anti-myosin heavy chain (skeletal fast) and 5.1.H11 antibody combining with flow cytometry did identification of myoblasts. Positive selection was on myoblasts bound to 5.1.H11 incubating with human antimouse IgG coated magnetic beads (Dynabead) and subsequent isolation by magnet, releasing cells from beads with DNAse. RESULTS: More than 59% of primary cell culture are positive to 5.1.H11 and decreasing with passage. The coating of culture dish surface increased specific growth rate of myoblast clones twice. Positive selection allows to increasing concentration of myoblasts from 8.4% in mixed culture to more than 90% without affecting neither viability nor platting efficiency. CONCLUSION: Purification procedure reported here is easy, efficient and requires small amount of sample, which will facilitate the purpose of autologous implant.

HLA-DQA, -DQB and -DRB allele contribution to narcolepsy susceptibility.

The association of narcolepsy with HLA class I antigens and HLA class II alleles was studies in a series of Spanish narcoleptic patients. The haplotype DRB1*1501-DRB5*0101-DQA1*0102-DQB1*0602 was found to be significantly associated with the disease, while the haplotype DRB1*0701-DRB4*01-DQA1*0201-DQB1*02 might confer a slight protective effect against narcolepsy. Gene dose-effect was not seen in any of the involved alleles, and linkage disequilibrium between the positively associated alleles was found to be stronger in patients than in controls. Statistical analysis applied to identify the HLA allele truly responsible for the association did not clearly discriminate between the contribution of DRB1*1501 and that of DQB1*0602, but it proved that the association with DQA1*0102 is secondary to that with DRB1*1501/DQB1*0602. Analysis of the diagnostic value of typing for the narcolepsy-associated alleles demonstrated a very high negative predictive value and revealed that this test can be convenient for exclusion of narcolepsy in cases when the diagnosis is not evident after clinical evaluation and the marker haplotype is absent. Finally, a family study indicated that narcolepsy is a multifactorial disorder that involves HLA genes under an incomplete penetrance model, with possible influences from environmental factors or other genes different to HLA genes.