Washing Of Cells Research Articles

Lymphocyte Cultures and FISH Analysis after Long-term Storage of Chernobyl Cleanup Worker Samples

The aim was to establish optimal culture conditions for lymphocytes after long-term liquid nitrogen storage and to determine the frequency of translocations after fluorescence in situ hybridization (FISH) painting of samples from Estonian Chernobyl cleanup workers with average recorded doses of about 130 mSv. Lymphocyte samples from more than 2000 cleanup workers and controls have been stored at STUK in liquid nitrogen during 1995-1997. In the present study, 150 cell samples from exposed cleanup workers and population controls were thawed during 2004-2005 and cultured with routine methods. The most critical factors for successful cultures were: a) adequate freezing procedure of the samples, b) careful washing of cells to remove DMSO, c) cells kept in foetal serum after washing before cultures were set up, d) minimum cell density of 0.4 to 0.6 x 106 per ml in culture medium. More than 90% of the cultures were successful, i.e. the mitotic index and metaphase quality were adequate for FISH analysis. Chromosome aberrations from FISH painted slides were scored from 37 Estonian unexposed controls and 101 cleanup workers. One thousand metaphases were analysed per person. In preliminary analyses, the FISH translocation frequencies in the control group and the cleanup worker group were relatively similar. However, slightly higher translocation frequencies were observed among workers with recorded doses of more than 200 mSv. Results from regression analyses considering translocation yields with respect to age, exposure to ionizing radiation and smoking will be presented.

Detection of human cytomegalovirus-specific T lymphocytes in human blood: comparison of two methods

Human cytomegalovirus (HCMV) infection remains a major cause of morbidity and mortality in immunocompromised patients undergoing allogeneic stem cell transplantation (SCT). In the case of HCMV reactivation, the well-defined detection of virus-specific effector cells in patients might positively impact antiviral treatment. We examined blood samples from healthy volunteers serologically typed for HCMV IgG. Based on multicolor flow cytometry analysis, we addressed HCMV-specific CD8(+) effector T lymphocytes using HCMV-specific tetramers for the respective major histocompatibility complex (MHC) class I type. As a second approach, we employed the cytokine secretion assay (CSA), which allows the indirect detection of target-specific CD4(+) and CD8(+) T cells via their interferon (IFN)-gamma secretion upon HCMV pp65 in vitro stimulation. We hypothesized the detection of HCMV-specific lymphocytes in >50% of healthy Caucasians that were IgG-seropositive for HCMV. In terms of specificity, both assays showed comparably good results (specificity 100%, confidence interval >95%). Regarding sensitivity, both assays met the zero hypothesis. However, with 45/52 (86.5%) the tetramer technology was superior to the CSA, which detected 34/52 (65.4%) based on CD8(+) T cells and 41/52 (78.8%) based on both CD4(+) and CD8(+) T cells. A good correlation was observed between both assays, although the tetramers addressed only CD8(+) HCMV-specific T cells, whereas IFN-gamma secretion was detected on all T-cell types. Disadvantages of the CSA are the time-consuming stimulation, the extensive cell washing steps and the fact that the target cells are detected indirectly. The analysis with tetramers is rapid and reliable but their general use is hampered because of the restriction to a few HLA types.

Microbial colonization of anin vitromodel of a tissue engineered human skin equivalent – a novel approach

This was a preliminary investigation to define the conditions of colonization of a human skin equivalent (SE) model with cutaneous microorganisms. SEs of 24 mm diameter were constructed with a dermal matrix of fibrin containing fibroblasts and a stratified epidermis. Microbial colonization of the SEs was carried out in a dry environment, comparable to 'in vivo' skin, using a blotting technique to remove inoculation fluid. The microbial communities were sampled by scrub washing and viable cells enumerated on selective growth medium. Staphylococcus epidermidis, Propionibacterium acnes and Malassezia furfur (human skin commensals) and Staphylococcus aureus (transient pathogen) were colonized at inoculum densities of 10(2)-10(6) CFU SE(-1) on the surface of replicate SEs. Growth of all species was supported for upto 72-120 h, with recovery densities of between 10(4)-10(9) CFU SE(-1). A novel, real-time growth monitoring method was also developed, using S. aureus containing a lux cassette. Light output increased from 20 to 95 h, and colonization increased from 10(2) to 10(8) CFU SE(-1), as confirmed by conventional recovery. Thus, the SE model has potential to investigate interactions between resident and transient microbial communities with themselves and their habitat, and for testing treatments to control pathogen colonization of human skin.

93. Enhanced functional recovery of cryopreserved peripheral blood stem cells using a novel cell washing device

93. Enhanced functional recovery of cryopreserved peripheral blood stem cells using a novel cell washing device

Reduction of Prion Infectivity in Packed Red Cells by Separation of Whole Blood and Washing of the Red Cell Fraction.

Although prion diseases are rare in humans, the established link between a new variant form of CJD (vCJD) and the consumption of cattle meat contaminated by BSE have raised concerns about a possible outbreak of a large epidemic in the human population. Over the past few years, BSE has become a significant health concern in several countries, and it now seems apparent that vCJD can also be iatrogenically transmitted from human to human by blood transfusion. Exacerbating this state of affairs is the lack of a reliable test to identify individuals incubating the disease during the long and silent period from the onset of infection to the appearance of clinical symptoms. The purpose of this research study was to evaluate the effectiveness of separation of whole blood and washing of the red cell fraction for the removal of infectious scrapie prion protein (PrPSc) from red blood cell (RBC) suspensions. Samples of human, whole blood were spiked with 5 × 106 LD50 263K PrPSc. Analysis of the treated sample supernatants by Western blot revealed that approximately >88% of the PrPSc was removed with the initial plasma expression and the equivalent of 6% was detected in a saline wash (300 mL; 0.9% saline). The final sample of RBCs revealed no detectable levels of PrPSc by Western blots. Further analysis of the treated RBCs using the PMCA assay indicated detectable amounts of PrPSc only after 2 consecutive amplification rounds. Semi-quantitative analysis of PMCA amplification enabled us to estimate that the treated RBCs contained less than 1 × 104 LD50 PrPSc. This corresponded to removal levels exceeding ≥99% of spiked material in whole blood. These in vitro estimations were confirmed by in vivo infectivity studies in a hamster model of disease transmission.Results from in vivo studies displayed significant differences in the incubation periods of the spiked blood inoculated hamsters (100.1 ± 1.7) versus washed RBCs (135.8 ± 6.7). Moreover, a substantial difference in the attack rate (6/15: 40% in washed RBC, versus 13/13: 100% in spiked blood) further indicated a substantial removal of infectious prions. Comparison of this data with results of animals inoculated with different dilutions of infectious material, indicated a >99.94% reduction of infectivity. Washed, packed human red cells produced by this procedure were able to be stored in standard additive solutions (AS-3) for 42 days while still meeting all in vitro blood bank standards for acceptable red cell quality.Conclusion This data suggests that separation of plasma coupled with a simple, low volume wash of red cells may represent an efficient method to remove prions from red blood cell fractions, thus reducing possible infectivity of these products.

Live Cell Imaging of Outward and Inward Vesiculation Induced by the Complement C5b-9 Complex

Cells resist death induced by the complement membrane attack complex (MAC, C5b-9) by removal of the MAC from their surface by an outward and/or inward vesiculation. To gain an insight into the route of MAC removal, human C9 was tagged with Alexa Fluor 488 and traced within live cells. Tagged C9-AF488 was active in lysis of erythrocytes and K562 cells. Upon treatment of K562 cells with antibody and human serum containing C9-AF488, C9-AF488 containing MAC bound to the cells. Within 5-10 min, the cells started shedding C5b-9-loaded vesicles (0.05-1 mum) by outward vesiculation. Concomitantly, C9-AF488 entered the cells and accumulated in a perinuclear, late recycling compartment, co-localized with endocytosed transferrin-Texas Red. Similar results were obtained with fixed cells in which the MAC was labeled with antibodies directed to a C5b-9 neoepitope. Inhibition of protein kinase C reduced endocytosis of C5b-9. Kinetic analysis demonstrated that peripheral, trypsin-sensitive C5b-9 was cleared from cells at a slower rate relative to fully inserted, trypsin-resistant C5b-9. MAC formation is controlled by CD59, a ubiquitously expressed membrane complement regulator. Analysis at a cell population level showed that the amount of C5b-9-AF488 bound to K562 cells after complement activation was highly heterogeneous and inversely correlated with the CD59 level of expression. Efficient C9-AF488 vesiculation was observed in cells expressing low CD59 levels, suggesting that the protective impact of MAC elimination by vesiculation increases as the level of expression of CD59 decreases.

GAPex-5 Mediates Ubiquitination, Trafficking, and Degradation of Epidermal Growth Factor Receptor

Upon ligand stimulation, epidermal growth factor receptor (EGFR) is rapidly ubiquitinated, internalized, and sorted to lysosomes for degradation. Rab5 has been shown to play an important role in the early stages of EGFR trafficking. GAPex-5 is a newly described Rab5 exchange factor. Herein, we investigate the role of GAPex-5 on EGFR trafficking and degradation. Down-regulation of GAPex-5 by RNA interference decreases epidermal growth factor-stimulated EGFR degradation. Moreover, ubiquitination of EGFR is impaired by depletion of GAPex-5. This inhibitory effect is due to a decrease in the interaction between the adapter protein c-Cbl and EGFR, but not the phosphorylation state of EGFR. Consistently, when examined by immunofluorescence microscopy in cells depleted of GAPex-5, ligand-bound EGFR appeared trapped in early endosomes and the trafficking of internalized receptor from early to late endosomes was impaired. In agreement with the depletion studies, EGFR degradation is enhanced by overexpressing GAPex-5 wild type, but not GAPex-5DeltaGAP, a mutant lacking the Ras GTPase-activating protein (GAP) domain. This is consistent with the finding that c-Cbl binds specifically to the Ras GAP domain. Finally, overexpression of dominant negative Rab5a or depletion of all three isoforms of Rab5 does not inhibit ubiquitination of EGFR, which suggests that GAPex-5-mediated EGFR ubiquitination is independent of Rab5 activation. Collectively, the results suggest a novel mechanism by which EGF-stimulated receptor ubiquitination and trafficking are mediated via GAPex-5.

Transamidation of Wheat Flour Inhibits the Response to Gliadin of Intestinal T Cells in Celiac Disease

Celiac disease is characterized by activation of HLA-DQ2/DQ8-restricted intestinal gluten-specific CD4(+) T cells. In particular, gluten becomes a better T-cell antigen following deamidation catalyzed by tissue transglutaminase. To date, the only available therapy is represented by adherence to a gluten-free diet. Here, we examined a new enzyme strategy to preventively abolish gluten activity. Enzyme modifications of the immunodominant alpha-gliadin peptide p56-68 were analyzed by mass spectrometry, and peptide binding to HLA-DQ2 was simulated by modeling studies. Wheat flour was treated with microbial transglutaminase and lysine methyl ester; gliadin was subsequently extracted, digested, and deamidated. Gliadin-specific intestinal T-cell lines (iTCLs) were generated from biopsy specimens from 12 adult patients with celiac disease and challenged in vitro with different antigen preparations. Tissue transglutaminase-mediated transamidation with lysine or lysine methyl ester of p56-68 or gliadin in alkaline conditions inhibited the interferon gamma expression in iTCLs; also, binding to DQ2 was reduced but not abolished, as suggested by in silico analysis. Lysine methyl ester was particularly effective in abrogating the activity of gliadin. Notably, a block in the response was observed when iTCLs were challenged with gliadin extracted from flour pretreated with microbial transglutaminase and lysine methyl ester. Transamidation of wheat flour with a food-grade enzyme and an appropriate amine donor can be used to block the T cell-mediated gliadin activity. Considering the crucial role of adaptive immunity in celiac disease, our findings highlight the potential of the proposed treatment to prevent cereal toxicity.

Characterizing doxorubicin‐induced apoptosis in HepG2 cells using an integrated microfluidic device

Apoptosis has now established its importance in numerous areas of biology and is recently receiving great attention as an important topic related to the development of diseases. In this work, an integrated microfluidic device was developed to characterize doxorubicin-induced apoptosis in human hepatocellular carcinoma (HepG2) cells. A continuous concentration gradient of stimulator (doxorubicin) was generated in the upstream network and used to perfuse downstream cultured HepG2 cells. The appropriate fluorescent dyes were introduced into cells from the inlets connected to the cell culture chambers, allowing one to distinguish apoptotic cells from nonapoptotic or necrotic cells. The resultant fluorescence of cellular population was monitored and quantified with single-cell resolution to infer the apoptosis process being studied. The feasibility of studying apoptosis was demonstrated by measuring several apoptotic events, including morphological alterations, plasma membrane phosphatidylserine externalization, and mitochondrial membrane potential collapse. This microfluidic device, integrating the cell culture, stimulation, staining, and washing steps into a single device, can simultaneously generate a number of experimental conditions and investigate multiple parameters relating stimulation to apoptosis. It offers a unique platform to characterize various cellular responses in a high-throughput fashion, which is otherwise impossible with conventional methods.

Glycated haemoglobin (HbG) as a stable indicator of blood glucose status in ostrich

Glycated haemoglobin (HbG) concentration is a retrospective measure of mean blood glucose level and is not affected by recent stresses, food ingestion or exercise. HbG has been determined in various wild and domestic animals such as kestrels, mankhor, mouflon, aoudad, deer, goat, sheep, dog, camel and horse. But there is no information regarding HbG in ostrich and its relation to blood glucose. The purposes of this study were to determine the normal values of HbG in ostriches and to investigate its relation to fasting plasma glucose. Blood samples were collected from jugular veins of 30 clinically healthy ostriches after 12 h of fasting. After separation and washing of red blood cells, haemolysate was prepared and subjected to weak cation exchange chromatography for determination of HbG. Glucose was measured in plasma samples. Fasting plasma glucose and HbG were 11.23 ± 0.80 mmol/l and 1.20 ± 0.2% of total haemoglobin, respectively. It was shown that HbG percent and fasting plasma glucose were correlated (P < 0.05; r = 0.93). To determine if low HbG percent in ostrich is related to low permeability of erythrocytes to blood glucose, haemolysates incubated with glucose (11.21 mmol/l final glucose concentrations) were subjected to chromatography under the same conditions. It was shown that HbG was significantly increased in glycosylated haemolysates. It was concluded that low HbG percent in ostrich is related to low glucose permeability of erythrocytes.

Washing of irradiated red blood cells prevents hyperkalaemia during cardiopulmonary bypass in neonates and infants undergoing surgery for complex congenital heart disease

High concentrations of potassium and lactate in irradiated red cells transfused during cardiopulmonary bypass may have detrimental effects on infants and neonates undergoing cardiac surgery. The effects of receiving washed and unwashed irradiated red cells from the cardiopulmonary circuit on serum potassium and lactate concentrations were compared. The study population included neonates and infants undergoing heart surgery for complex congenital heart disease. A control group (n=11) received unwashed irradiated red cells and the study group (n=11) received irradiated red cells washed in a cell saver (Dideco Electa) using 900ml of 0.9% saline prior to pump priming. Potassium and lactate concentrations were compared before, during and after bypass. Washing irradiated red cells reduced donor blood [potassium] from>20 to 0.8+/-0.1mmol/l, and [lactate] from 13.7+/-0.5 to 5.0+/-0.3mmol/l (p<0.001). The resulting prime had significantly lower [potassium] and [lactate] than the unwashed group (potassium 2.6+/-0.1 vs 8.1+/-0.4mmol/l, p<0.001; lactate 2.6+/-0.2 vs 4.6+/-0.3mmol/l, p<0.001). Peak [potassium] in the unwashed group occurred 3 minutes after going on bypass (4.9+/-0.3mmol/l) and during rewarming (4.9+/-0.4mmol/l). These were significantly higher than the washed group (3.1+/-0.1, p<0.001 and 3.0+/-0.1mmol/l, p<0.001). The [potassium] was greater than 6.0mmol/l for 4 out of these 11 unwashed patients compared with none of the washed group. Immediately post-bypass the washed group had significantly lower serum [potassium] (3.2+/-0.1 vs 4.2+/-0.2mmol/l, p=0.002). There was no significant difference in [lactate] between groups during and after cardiopulmonary bypass. The washing of irradiated red cells reduces potassium and lactate loads and prevents hyperkalaemia during cardiopulmonary bypass. The washing of irradiated red cells should be considered in neonates and infants undergoing cardiac surgery for complex congenital heart disease.

Is Membrane Transport of FFA Mediated by Lipid, Protein, or Both?

POINT/COUNTERPOINTIs Membrane Transport of FFA Mediated by Lipid, Protein, or Both?Published Online:01 Feb 2007https://doi.org/10.1152/physiologyonline.2007.22.1.29MoreSectionsPDF (68 KB)Download PDF ToolsExport citationAdd to favoritesGet permissionsTrack citations ShareShare onFacebookTwitterLinkedInWeChat Points of Agreement The movement of long-chain fatty acids across the plasma membrane is likely facilitated by membrane-associated proteins. The exact molecular mechanisms of this protein-facilitated movement across the plasma membrane are not fully understood. A number of membrane-associated proteins involved in facilitating the uptake of fatty acids into metabolically important tissues have been identified (i.e., FAT/CD36, FABPpm, FATP1–6). However, at this point, it is not clear as to 1) why there are so many different proteins, 2) whether they directly interact with each other, 3) whether some or all of these proteins enhance uptake through means (e.g., esterification or oxidation) other than transport, or 4) whether they associate with other proteins to channel fatty acids to different metabolic fates.In heart and muscle, physiological stimuli (insulin, contraction) induce the translocation of FABPpm and/or FAT/CD36 from an endosomal pool to the plasma membrane, resulting in an increased rate of fatty acid uptake. Evidence for endocrine (insulin) regulation of adipocyte fatty acid uptake remains contradictory. It cannot be discounted that other proteins or groups of proteins participate in the transmembrane transport of fatty acids, particularly in adipose tissue where the roles of known fatty acid transport proteins remain elusive. Given the very different metabolic roles of muscle tissues and adipose tissue, it would not be surprising if, in these tissues, the fatty acid transport system and its regulation differ considerably. Points of Disagreement The role of FAT/CD36 and FABPpm in mediating transport of long-chain fatty acids across membranes. Bonen et al. find that FAT/CD36 and FABPpm mediate fatty acid transport into giant sarcolemmal vesicles, whereas Kampf and Kleinfeld conclude that neither FAT/CD36 nor FABPpm mediate membrane transport of long-chain fatty acids in adipocyte cell lines.The role of lipid phase (diffusion) and protein-mediated mechanisms in the membrane transport of long-chain fatty acids. Bonen et al. find evidence for a small contribution by a lipid phase (diffusion) mechanism and a quantitatively larger protein-mediated mechanism in giant sarcolemmal vesicles, whereas Kampf and Kleinfeld conclude that fatty acids transport in adipocytes occurs solely through a protein-mediated mechanism.Whether inhibitors of fatty acid transport have been identified. Kampf and Kleinfeld have not observed transport inhibition in adipocytes, whereas Bonen et al find that specific reagents inhibit fatty acid transport across the plasma membrane of giant sarcolemmal vesicles.Whether cells can be washed without removing the bulk of the unesterified fatty acids. Kampf and Kleinfeld have found that washing cells removes virtually all unesterified fatty acid from adipocytes, whereas Bonen et al. find that the bulk of the transported fatty acids observed after washing remains unesterified. Fatty Acid Nomenclature: A Source of Confusion.In papers dealing with fatty acids and their derivatives, as in the present Point/ Counterpoint, there is a considerable lack of uniformity in the terms used to designate these molecules (Glatz and van der Vusse. Trends Biochem Sci 13: 167–168, 1988). For example, the term “free fatty acid” is often used to refer to nonesterified fatty acids, which is itself an ambiguous term because a fatty acid is by definition not esterified. A more accurate term for the covalently modified fatty acid found in, for example, triacylglycerols is “fatty ester,” as recommended by the IUPACIUB. An additional problem is that the fatty acid may be solubilized in the aqueous phase or bound to a macromolecule such as a protein or a membrane. Glatz and van der Vusse have proposed the designation “fatty acid” (FA) for the aqueous phase form (also known as unbound FA) and protein (macromolecule)-bound FA for the nonaqueous form. Bonen et al. have used this nomenclature. To emphasize the direct measurement of the aqueous form of the FA, Kampf and Kleinfeld have designated the aqueous FA as FFAu and have used the term FFA to designate the sum of the noncovalently bound and unbound FA. Another complication is that the aqueous FA at neutral pH is largely ionized (and therefore not an acid), whereas the macromolecular bound FA may be either ionized or protonated. Both sides of this Point/Counterpoint discussion agree that the nomenclature proposed by Glatz and van der Vusse represents a consistent and more accurate terminology than is currently in general use.This article has no references to display. Download PDF Previous Back to Top Next FiguresReferencesRelatedInformation More from this issue > Volume 22Issue 1February 2007Pages 29-29 Copyright & Permissions© 2007 Int. Union Physiol. Sci./Am. Physiol. Soc.https://doi.org/10.1152/physiologyonline.2007.22.1.29History Published online 1 February 2007 Published in print 1 February 2007 Metrics

Acid wash in determining cellular uptake of Fab/cell‐permeating peptide conjugates

Successful intracellular delivery of various bioactive molecules has been reported using cell-permeating peptides (CPPs) as delivery vectors. To determine the effects of CPPs on the cellular uptake of immunoglobulin Fab fragment, conjugates of a radio-iodinated Fab fragment with CPPs (CPP-(125)I-Fab) derived from HIV-1 TAT, HIV-1 REV, and Antennapedia (ANP) were prepared. These vectors are rich in basic amino acids, and their strong adsorption on cell surfaces often results in overestimation of internalized peptides. Cell wash with an acidic buffer (0.2M glycine-0.15M NaCl, pH 3.0) was thus employed in this study to remove cell-surface adsorbed CPP-(125)I-Fab conjugates. This procedure enabled clearer understanding of the methods of internalization of CPP-(125)I-Fab conjugates. The kinetics of internalization of REV-(125)I-Fab conjugate was rapid, and a considerable fraction of REV-(125)I-Fab was taken up by HeLa cells as early as 5 min after administration. It was also shown that cellular uptake of these conjugates was significantly inhibited in the presence of endocytosis/ macropinocytosis inhibitors, in the order REV-(125)I-Fab > or = TAT-(125)I-Fab > or = ANP-(125)I-Fab; this order was the same as for effectiveness of intracellular delivery. Simultaneous cell washing with phosphate-buffered saline (PBS) and this acidic buffer effectively separated the internalized conjugates from the cell-surface-adsorbed ones, and considerable differences were observed in these amounts dependent on the employed CPPs.

Washing of stored red blood cells by an autotransfusion device before transfusion

The use of an autotransfusion device to wash blood of the incision site is increasing. After washing, this blood is retransfused without side effects caused by activated plasma factors and cell release products. This procedure could be extended to washing of donor blood, which may be particularly useful for red blood cells (RBCs) stored for more than 4 weeks that contain high concentrations of free haemoglobin, potassium, lactate and other metabolites. It is not known whether stored RBCs can withstand the cell washing procedure with the use of an autotransfusion device, while keeping their primary functions intact. The objective of this study was to determine the quality of RBCs, after cell washing in comparison to untreated RBCs. RBCs were studied in terms of integrity (free haemoglobin), stored energy (2,3-diphosphoglycerate, adenosine triphosphate), metabolites (lactate, potassium) and physical characteristics (osmotic resistance, aggregability, deformability). After washing, free lactate and potassium were significantly reduced as compared to the levels before washing. The osmotic resistance of RBCs slightly improved, whereas aggregation capacity reduced after washing. Fifteen per cent of haemoglobin was lost during washing. The deformability and free haemoglobin levels remained unchanged. Washing stored blood before transfusion may be of benefit, because the waste products are effectively removed from the stored RBC.

Atomic Force Microscopy Analysis of Bacterial Surface Morphology Before and After Cell Washing

Atomic Force Microscopy Analysis of Bacterial Surface Morphology Before and After Cell Washing

Utilization of Vero Cells for Primary and Chronic BK Virus Infection

BK virus (BKV) nephropathy has a poor prognosis for renal allograft survival with 30% to 60% risk of allograft loss over 1 year. In the past decade, BKV nephropathy has occurred in 1% to 10% of renal transplant patients, with higher rates observed in patients with increased immunosuppression exposure and renal allograft injury. Vero cells (Green monkey kidney cell origin) were optimized for BKV primary and chronic infection inclusive of culture requirements for 60-day growth and monolayer confluence. Quantification of BKV replication in the culture supernatant (SN) and cells was by real-time polymerase-chain reaction (PCR) using the Roche Lightcycler 2.0. Primary BKV infection of Vero cells is achieved by 2 hour incubation with 6.5 × 10 5 BKV copies with subsequent washing of cells leading to steady-state cellular infection of 10 2 to 10 3 BKV copies. Primary infection is demonstrated within 7 to 10 days by a >10-fold increase of BKV copies in SN. Thereafter, a BKV viral load reduction in SN to a chronic/latent level (<10 2 BKV copies in SN) is observed by 14 days. Vero cells with chronic low-level BKV infection (10 2–10 3 BKV copies in cells) exhibited reactivation (>10 5 BKV copies in SN) in >72% of late culture wells after 40 days. Vero cells can accommodate primary and chronic BKV infection followed by viral reactivation in late culture. The performance characteristics of 3 different pathogenic BKV strains obtained from patients with BKV nephropathy had infectivity profiles that correlated well the relative clinical profile in this Vero cell culture system.

Comparison of Cord Blood Transplant Outcome between U.S. Versus Non-U.S. Transplant Centers for Cord Blood from a Single Source.

Background: Cord blood transplantations (CBT) are performed in transplant centers (TC) worldwide; however, conditions for TC located around the world may be quite different, in particular, few studies have focused on CBT outcome in Asian TC. Factors such as patient populations, experiences, disease indications, available resources, and protocols may affect outcome as some have suggested.Methods: In an effort to compare CBT performed by U.S. (n=129) and non-U.S. TC (n=63), a retrospective audited analysis was performed for all 192 patients with available engraftment and/or survival data using cord blood units (CBU) from a single source to minimize potential differences in quality.Results: Kaplan-Meier estimates of engraftment for ANC500, platelet 20K and 50K were 91±3%, 80±5% and 74±5% for U.S. respectively, and 81±5%, 85±6%, and 81±7% for non-U.S. TC respectively. The relapse rate for the U.S. and non-U.S. TC were 26±5% and 17±5% respectively. TRM were 31±4% for U.S. TC and 22±5% for non-U.S. TC. The 3-year OS and DFS were 51±5% and 41±7% for U.S. and 68±6% and 61±7% for non-U.S. centers respectively. Factors including recipient age, CD34+ cell dose, #HLA matches, malignancy and wash status of the CBU were taken into consideration in a multivariate analysis which will be presented. In this study, non-U.S. TC were more likely to forego post-thaw wash of CB and perform CBT for benign indications, which appeared to affect outcome. Upon closer inspection of the individual TC, it appeared that there may be more heterogeneity in the TC results overseas, and that centers in the U.S. produced more consistent outcome. While it is possible that, due to many complex reasons, outcome at some non-U.S. TC may be somewhat uneven, many including several in Asia, produced outstanding results that rival the best TC anywhere.Conclusion: It appears that many factors, including available resources, post-thaw wash, and CBT experience affect outcome, and outstanding outcome can be achieved, regardless of TC location.

An Alternative Technique To Wash out DMSO from Thawed PBSC for Autotransplant.

Introduction Dimethilsulfoxyde (DMSO) is a cryoprotectant routinely used for stem cell cryopreservation. At reinfusion, DMSO is toxic for the recipient and the risk of adverse effects (chills, flushing, nausea, vomiting, abdominal pain, chest tightness, blood pressure changes) are proportional to the DMSO amount. DMSO may also induce lifethreatening complications (cardiac and pulmonary arrest, acute renal failure) in low body weight patients or in subjects with a pre-existing cardiac disease. DMSO related complications are emphasized in “bad mobilizer” patients (CD34+/mL <20) in which multiple leukapheresis collections (large volume to reinfuse with high DMSO content) are needed to reach the stem cell dose necessary for transplant. The techniques (manual or automated) adopted to remove DMSO imply a various degree of stem cell loss, the risk of cell clotting and bacterial contamination.Herein we present our experience in washing out DMSO from thawed PBSC with an alternative technique.Material and Methods 48 PBSC bags, with a mean volume of 110 ml (range: 107–112) containing 10% DMSO were thawed for autotransplant in 8 patients affected with NHL-B (3), HL (3) and AML (2). 2 patients suffered from cardiac arrhythmia and 6 patients weighted less than 40 kgs (range 31–39.5). Each bag was thawed in a water bath at 37°C and immediately after was made a sterile connection with a bag containing a solution of the same volume (saline and 20% ACD-A) previously refrigerated at 4°C to minimize the DMSO cell damage. Afterward, the diluted PBSC bag was centrifuged at 1200g× 5mins at 4°C and the excess supernatant was removed using a plasma extractor. Finally, the cells were resuspended in an equal volume of refrigerated saline solution and human serum albumin (2%) and promptly reinfused to the patient. Pre-freezing, post thawing and post washing samples were collected to determine TNC, MNC, CD34+ cell content, viability and microbial contamination.Results Pre-freezing, post thawing and post washing TNC, MNC, CD34+ cell content, viability and pre-freezing versus post washing cell recovery are shown in table 1. Data are expressed as mean and range (min-max). No microbial contamination was detected in all samples analyzed. No infusion related toxicity was recorded. No clotting was observed. No delay in WBC and PLT engrafment was documented.Conclusions Our DMSO wash out technique is feasible, simple and safe and permits a satisfying recovery of MNC and CD34+ cells, preserving cell viability. PBSC manipulation at 4°C employing refrigerated solutions during every step mantains the quality of the thawed graft, minimizing DMSO stem cell damage with no delay in engraftment time. Finally, our method may be considered expecially when DMSO infusion could be lifetreathening in patients at high risk for a pre-existing cardiac, pulmonary or renal disease or in low body weight patients.Table 1Pre-freezingPost-thawingPost-washingRecovery (%)TNC (109)46.2 (41.2–53.1)40.4 (37.6–46.3)31.7 (26.2–35.8)67.3 (64–71.2)MNC (109)24.5 (20.3–28.6)22.4 (17.1–26.8)20.3 (14.7–26.4)83.3 (81–85.2)CD34+ (106)35.7 (29–38.6)31.3 (25–35.4)27.2 (21.1–30.5)77.1 (73.1–80)Viability (%)98.9 (97.5–99.9)87 (86.2–89.4)79 (76.7–81.9)

Pathogen-reduction systems for blood components: The current position and future trends

The current multi-layered interventional approaches to blood safety have dramatically reduced the risk of viral contamination of blood components. Nowadays most of the residual transfusion transmitted infections (TTI) occur as the result of the interval between the time the donor is infected and the moment at which tests are capable of detecting the agent, the so called “window period” which has been considerably reduced by the increased sensitivity of nucleic acid testing (NAT). However, the residual risk of bacterial contamination and the unexpected appearance of some other emerging pathogens, almost every five years, are still of major concern to the public, politicians, regulatory agencies and place immense pressures on the organisations responsible for the provision of safe blood and its components. In view of these bleak scenarios, the use of human blood as a raw biological source is inherently unsafe, and screening/testing alone cannot exclude all the potential human pathogens; hence we need to put in place some sort of safer alternatives and/or additional preventative safety measures. Recently, several substitutes (alternatives) to virtual blood components have been developed and tried. Moreover, various mechanical methods such as cell washing and leukofiltration have been implemented as additional preventative safety measures but with limited success in abrogating the risk of transfusion transmitted cell-associated agents. The most promising approaches, so far, are methods that target pathogen nucleic acids (Methylene blue; Psolaren and Riboflavin UV light treatment). These procedures have undergone considerable in vitro studies to ensure their extremely high safety margins in terms of toxicity to the cells or to the recipients. In essence, while the technology of targeting nucleic acid to stop viral proliferation is common to the above three strategies, in practice these procedures differ in terms of operational, physicochemical and biological characteristics; including the potential impacts of their metabolites and photo-adducts; their effects on the spectrum of pathogens affected and the log reductions in culture infective studies. Accordingly, any strategy that involves addition of an extraneous agent or physicochemical manipulation of blood must balance the benefits of pathogen reduction against the loss or alteration to the cells and plasma functional integrity, short and long term toxicity to the cells and to the recipients, as well as the risk to the personnel involved and the community at large. Moreover, it must be noted that each method will have a different profile of adverse reactions and may differ in terms of the risk to particularly vulnerable groups of patients, requiring in depth clinical trials, while taking into consideration the cost benefit of the final process. Newer diagnostic procedures must be in place to establish the storage stability of products that have undergone pathogen inactivation, in particular tests reflecting the release of platelet-derived cytokines, cellular apoptosis or microvesiculation and their role in immunosupressiveness. This overview aims to provide an update on the continual improvements in blood component safety, in particular using methods that target pathogen nucleic acid. Emphasis is placed on methylene blue light treatment (MBLT) and Intercept or Mirasol PRT systems for platelets and plasma. The status of pathogen reduction of whole blood and red cells is also highlighted, though the progress in this area has been virtually stopped after the finding of antibody development in the clinical trial.

Simulation of removing permeable cryoprotective agents from cryopreserved blood with hollow fiber modules

A new method for removing permeable cryoprotective agents (CPAs) from cryopreserved blood with hollow fiber modules is presented and simulated theoretically, and CPA concentration variation and cell volume response during the removal process are investigated in detail. Then, the comparison between the results from this paper and the ones from traditional methods is carried out, which shows the advantage of this new method in decreasing maximal cell volume and shortening washing time. Lastly, the effects of hydraulic and solute permeabilities of hollow fibers and flow rates of washing solution and cell suspension on maximal cell volume and washing time is studied. The result reveals that by means of adjusting the above controllable parameters properly, it is convenient for this method to decrease osmotic damage of cells and increase clearance efficiency of CPAs in practice.