Use and Waste of Reconstituted Whole Blood Exchange Transfusions: An 11-year National Observational Study.

OBJECTIVES: To identify indications for exchange transfusions, assess the use and waste of exchange transfusion products (ie, reconstituted whole blood exchange transfusions), and determine nationwide distribution and prevalence of these transfusions in the Netherlands. STUDY DESIGN: All 9 neonatal intensive care units and 15 non-neonatal intensive care unit hospitals participated in this retrospective, observational, cohort study. We retrieved data on the indications for and use of all exchange transfusion products ordered by participating centers over an 11-year period. RESULTS: A total of 574 patients for whom 1265 products were ordered were included for analyses. Severe ABO (32.6%) and non-ABO (25.2%) immune hemolysis and subsequent hyperbilirubinemia were the most frequent indications. Rare indications were severe leukocytosis in Bordetella pertussis (2.1%) and severe anemia (1.5%). Approximately one-half of all ordered products remained unused. In 278 of 574 neonates (48.4%), ≥1 products were not used, of which 229 (82.7%) were due to the resolving of severe hyperbilirubinemia with further intensification of phototherapy. The overall prevalence of neonates who received an exchange transfusion was 14.6:100 000 liveborn neonates. CONCLUSIONS: A considerable proportion of products remained unused, and annually a limited number of patients are treated with an exchange transfusion in the Netherlands, highlighting the rarity of the procedure in the Netherlands.

Strategies to reduce wastage of red blood cell units.

BACKGROUND AND OBJECTIVES: Wastage of red blood cell units (RBCs) due to their inappropriate storage at the clinical ward has become both a financial and ethical challenge in the daily hospital practice. This study was aimed at identifying the extent of RBC wastage and evaluating the effects of various interventions in reducing this wastage. MATERIALS AND METHODS: From January 2011 to March 2011, baseline wastage level was evaluated using temperature-sensitive labels. Following this initial analysis, various interventions were implemented, including modifying the transfusion practice, intensifying training of and communication with the medical staff and improving the transport conditions. The impact of these interventions on wastage was measured during two periods, and results were compared with baseline wastage level. RESULTS: Based on the extent of label colouring, 7·5% of the units dispensed by the transfusion laboratory were determined as non-reusable at baseline. After implementation of the various interventions, wastage decreased to 1% of the units dispensed, potentially leading to an annual saving for our hospital of approximately €208·000/$230·600 on the total number of RBCs dispensed. CONCLUSION: Relative straightforward interventions, such as raising awareness among medical staff and particularly improving transport conditions, had a clear impact on the level of RBC wastage, accommodating the financial issue not to waste public money as well as the ethical issue that RBC wastage should be as low as possible.

Effects of bile salts on cholestan-3beta,5alpha,6beta-triol-induced apoptosis in dog gallbladder epithelial cells.

Oxysterols are cytotoxic agents. The gallbladder epithelium is exposed to high concentrations of oxysterols, and so elucidating the mechanisms of cytotoxicity in this organ may enhance our understanding of the pathogenesis of biliary tract disorders. We investigated the cytotoxic effects of the oxysterol cholestan-3beta,5alpha,6beta-triol (TriolC) on dog gallbladder epithelial cells. Apoptosis was the major form of cytotoxicity, as determined by analysis of nuclear morphologic changes and by multiparameter flow cytometry. Hydrophobic bile salts are known to have cytotoxic effects, whereas hydrophilic bile salts have cytoprotective effects. We therefore examined whether the hydrophobic bile acid taurodeoxycholic acid (TDC) and the hydrophilic bile acid tauroursodeoxycholic acid (TUDC) had modifying effects on oxysterol-induced cytotoxicity. TriolC caused an increase in the number of apoptotic cells from 14+/-11% (control) to 48+/-12% of total cells (P<0.01). After combining TriolC with TDC, cell apoptosis increased to 63+/-16% (P<0.05), whereas after addition of TUDC, the number of apoptotic cells decreased to 31+/-12% (P<0.05) of total cells. In summary, oxysterols such as TriolC induce apoptosis. Hydrophobic bile salts enhance TriolC-induced apoptosis, whereas hydrophilic bile salts diminish TriolC-induced apoptosis. These results suggest that interactions between oxysterols and bile salts play a role in the pathophysiology of biliary tract disorders.

Fresh frozen plasma transfusion fails to influence the hemostatic balance in critically ill patients with a coagulopathy.

BACKGROUND: Coagulopathy has a high prevalence in critically ill patients. An increased International Normalized Ratio (INR) is a common trigger to transfuse fresh frozen plasma (FFP), even in the absence of bleeding. Therefore, FFP is frequently administered to these patients. However, the efficacy of FFP in correcting hemostatic disorders in non-bleeding recipients has been questioned. OBJECTIVES: To assess whether INR prolongation parallels changes in the results of other tests investigating hemostasis, and to evaluate the coagulant effects of a fixed dose of FFP in non-bleeding critically ill patients with a coagulopathy. METHODS: Markers of coagulation, individual factor levels and levels of natural anticoagulants were measured. Also, thrombin generation and thromboelastometry (ROTEM) assays were performed before and after FFP transfusion (12 mL kg(-1) ) to 38 non-bleeding critically ill patients with an increased INR (1.5-3.0). RESULTS: At baseline, levels of factor II, FV, FVII, protein C, protein S and antithrombin were reduced, and thrombin generation was impaired. ROTEM variables were within reference ranges, except for a prolonged INTEM clot formation time. FFP transfusion increased the levels of coagulation factors (FII, 34% [interquartile range (IQR) 26-46] before vs. 44% [IQR 38-52] after; FV, 48% [IQR 28-76] before vs. 58% [IQR 44-90] after; and FVII, 25% [IQR 16-38] before vs. 37% [IQR 28-55] after), and the levels of anticoagulant proteins. Thrombin generation was unaffected by FFP transfusion (endogenous thrombin potential, 72% [IQR 51-88] before vs. 71% [IQR 42-89] after), whereas ROTEM EXTEM clotting time and maximum clot firmness slightly improved in response to FFP. CONCLUSION: In non-bleeding critically ill patients with a coagulopathy, FFP transfusion failed to induce a more procoagulant state.

Cholestan-3beta,5alpha,6beta-triol, but not 7-ketocholesterol, suppresses taurocholate-induced mucin secretion by cultured dog gallbladder epithelial cells.

In order to investigate oxysterol-mediated effects on the biliary system, we studied the effects of cholestan-3beta,5alpha,6beta-triol (TriolC) and 7-ketocholesterol (7KC) on gallbladder epithelial cells. We compared their cell proliferation effects in cultured dog gallbladder epithelial cells (DGBE) to their effects in cultured human pulmonary artery endothelial cells (HPAE). Oxysterols inhibited cell proliferation in a dose-dependent fashion. Oxysterols inhibited cell growth to 50% of control at a higher dose for DGBE cells than for HPAE cells. TriolC was more cytotoxic than 7KC. We also investigated the effect of oxysterols on bile salt-induced mucin secretion by DGBE cells. TriolC suppressed mucin secretion by DGBE cells, whereas 7KC did not. These findings support the hypothesis that biliary oxysterols affect gallbladder mucosal function.

Bile salts stimulate mucin secretion by cultured dog gallbladder epithelial cells independent of their detergent effect.

1. Bile salts stimulate mucin secretion by the gallbladder epithelium. We have investigated whether this stimulatory effect is due to a detergent effect of bile salts. 2. The bile salts taurocholic acid (TC) and tauroursodeoxycholic acid (TUDC) and the detergents Triton X-100 (12.5-400 microM) and Tween-20 (0.1-3.2 mM) were applied to monolayers of cultured dog gallbladder epithelial cells. Mucin secretion was studied by measuring the secretion of [3H]N-acetyl-d-glucosamine-labelled glycoproteins. We also attempted to alter the fluidity of the apical membrane of the cells through extraction of cholesterol with beta-cyclodextrin (2.5-15 mM). The effect on TUDC-induced mucin secretion was studied. Cell viability was assessed by measuring lactate dehydrogenase (LDH) leakage or 51Cr release. 3. In contrast with the bile salts, the detergents were not able to cause an increase in mucin secretion without causing concomitant cell lysis. Concentrations of detergent that increased mucin release (>100 microM Triton X-100, >0.8 mM Tween-20), caused increased LDH release. Incubation with beta-cyclodextrin resulted in effective extraction of cholesterol without causing an increase in 51Cr release. However, no effect of the presumed altered membrane fluidity on TUDC (10 mM)-induced mucin secretion was observed. 4. The stimulatory effect of bile salts on mucin secretion by gallbladder epithelial cells is not affected by the fluidity of the apical membrane of the cells and also cannot be mimicked by other detergents. We conclude that the ability of bile salts to cause mucin secretion by the gallbladder epithelium is not determined by their detergent properties.

Lipopolysaccharide from Escherichia coli stimulates mucin secretion by cultured dog gallbladder epithelial cells.

Biliary infection is associated with mucin hypersecretion by the biliary epithelium. Mucins have been identified as potent pronucleators of cholesterol in bile. The aim of the present study was to determine whether lipopolysaccharides (LPS) from different bacteria are capable of stimulating mucin secretion by cultured dog gallbladder epithelial (DGBE) cells, and to investigate the mechanism by which LPS stimulate mucin secretion. Mucin secretion by confluent monolayers of DGBE cells was quantified by measuring the secretion of [3H]-N-acetyl-D-glucosamine-labeled glycoproteins. Cell viability was evaluated by measuring the leakage of the enzyme, lactate dehydrogenase (LDH), into the culture medium. LPS, derived from Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa (200 microg/mL), all caused an increase in mucin secretion by the DGBE cells, without causing concomitant cell lysis. LPS from E. coli was found to be the most potent stimulator of mucin secretion, and increased mucin secretion by the DGBE cells to 252% +/- 14% of control. LPS from E. coli had no effect on intracellular cyclic adenosine monophosphate (cAMP) levels in the DGBE cells. Addition of the nitric oxide (NO)-releasing compound, NOR-4 (0.125-1 mmol/L), to the cells did not result in increased mucin secretion, and the NO synthase inhibitor, Nomega-nitro-L-arginine methyl ester (L-NAME) (4 or 10 mmol/L), did not inhibit the LPS-stimulated mucin secretion. Exogenous tumor necrosis factor alpha (TNF-alpha) (1-10 ng/mL) did cause a minor increase in mucin secretion by the DGBE cells, but the effect of LPS from E. coli on mucin secretion could not be inhibited by preincubation with a TNF-alpha antibody (10 microg/mL). We conclude that LPS stimulates mucin secretion by the gallbladder epithelium. Whether this stimulation is mediated by TNF-alpha remains to be determined.

Mechanism of bile salt-induced mucin secretion by cultured dog gallbladder epithelial cells.

1. Hypersecretion of gallbladder mucin has been proposed to be a pathogenic factor in cholesterol gallstone formation. Using cultured gallbladder epithelial cells, we demonstrated that bile salts regulate mucin secretion by the gallbladder epithelium. In the present study we have investigated whether established second messenger pathways are involved in bile salt-induced mucin secretion. 2. The effect of activators and inhibitors on mucin secretion was studied by measuring the secretion of [3H]N-acetyl-D-glucosamine-labelled glycoproteins. Intracellular cAMP content of the cells was measured using a radioimmunoassay. 3. Incubation of the cells with 10 mM taurocholate did not increase the intracellular cAMP content (25.7 versus control 22.8 pmol of cAMP/mg of protein). No stimulation of mucin secretion was observed after incubation with 1-100 microM concentrations of the calcium ionophores ionomycin and A23187. The stimulatory effect of 10 mM tauroursodeoxycholate (TUDC) on mucin secretion could not be inhibited by the addition of EDTA. Activation of protein kinase C (PKC) by 1 microgram/ml phorbol 12-myristate 13-acetate (PMA) caused an increase in mucin secretion (342% versus control 100%), comparable with the effect of 40 mM TUDC. The effect of 10 ng/ml PMA could partially be inhibited by a concentration of 2 microM of the PKC inhibitor staurosporin. Staurosporin had no inhibitory effect on mucin secretion induced by TUDC. 4. In gallbladder epithelial cells bile salts do not stimulate mucin secretion via one of the classical signal transduction pathways. We hypothesize that bile salts act on mucin secretion via a direct interaction with the apical membrane.

Mucous granule exocytosis and CFTR expression in gallbladder epithelium.

A mechanistic model of mucous granule exocytosis by columnar epithelial cells must take into account the unique physical-chemical properties of mucin glycoproteins and the resultant mucus gel. In particular, any model must explain the intracellular packaging and the kinetics of release of these large, heavily charged species. We studied mucous granule exocytosis in gallbladder epithelium, a model system for mucus secretion by columnar epithelial cells. Mucous granules released mucus by merocrine exocytosis in mouse gallbladder epithelium when examined by transmission electron microscopy. Spherules of secreted mucus larger than intracellular granules were noted on scanning electron microscopy. Electron probe microanalysis demonstrated increased calcium concentrations within mucous granules. Immunofluorescence microscopic studies revealed intracellular colocalization of mucins and the cystic fibrosis transmembrane conductance regulator (CFTR). Confocal laser immunofluorescence microscopy confirmed colocalization. These observations suggest that calcium in mucous secretory granules provides cationic shielding to keep mucus tightly packed. The data also suggests CFTR chloride channels are present in granule membranes. These observations support a model in which influx of chloride ions into the granule disrupts cationic shielding, leading to rapid swelling, exocytosis and hydration of mucus. Such a model explains the physical-chemical mechanisms involved in mucous granule exocytosis.

Model bile and bile salts accelerate mucin secretion by cultured dog gallbladder epithelial cells.

BACKGROUND & AIMS: Hypersecretion of gallbladder mucin has been proposed as a pathogenic factor in gallstone formation. We investigated whether mucin secretion is modulated by biliary constituents using normal, well-differentiated dog gallbladder epithelial cells. METHODS: Model biles or bile salts were applied to monolayers of epithelial cells. Mucin secretion was studied by measuring the secretion of [3H]N-acetyl-D-glucosamine-labeled glycoproteins. RESULTS: Model biles with different cholesterol saturation indices increased mucin secretion by the cells to an average 251% after 5 hours of incubation (P < 0.01). Mucin secretion remained elevated during a 24-hour period, suggesting a sustained effect on mucin secretion. There was no relation between the cholesterol or phospholipid concentration and the extent of stimulation of mucin secretion. Taurocholate caused a dose-dependent increase in mucin secretion, suggesting that bile salt was the bile component responsible for the stimulatory effect. At a concentration of 0.5 mmol/L, only the more hydrophobic bile salts taurochenodeoxycholate and taurodeoxycholate, but not the hydrophylic bile salts taurocholate and tauroursodeoxycholate, stimulated mucin secretion (P < 0.01). CONCLUSIONS: Bile salts play an important role in the regulation of mucin secretion. A shift in the bile salt composition of bile towards the more hydrophobic bile salts may cause mucin hypersecretion, thereby initiating cholesterol gallstone formation.

Mucin secretion by the human colon cell line LS174T is regulated by bile salts.

We recently reported that bile salts play a role in the regulation of mucin secretion by cultured dog gallbladder epithelial cells. In this study we have examined whether bile salts also influence mucin secretion by the human epithelial colon cell line LS174T. Solutions of bile salts were applied to monolayers of LS174T cells. Mucin secretion was quantified by measuring the secretion of [3H]GlcNAc labeled glycoproteins. Both unconjugated bile salts as well as taurine conjugated bile salts stimulated mucin secretion by the colon cells in a dose-dependent fashion. Hydrophobic bile salts were more potent stimulators than hydrophilic bile salts. Free (unconjugated) bile salts were more stimulatory compared with their taurine conjugated counterparts. Stimulation of mucin secretion by LS174T cells was found to occur at much lower bile salt concentrations than in the experiments with the dog gallbladder epithelial cells. The protein kinase C activators PMA and PDB had no stimulatory effect on mucin secretion. We conclude that mucin secretion by the human colon epithelial cell line LS174T is regulated by bile salts. We suggest that regulation of mucin secretion by bile salts might be a common mechanism, by which different epithelia protect themselves against the detergent action of bile salts, to which they are exposed throughout the gastrointestinal tract.

Heterogeneity of human gallbladder mucin in bile.

1. Human gallbladder mucin has been implicated to play a role in gallstone disease. In spite of this fact relatively little is known about the structure of human gallbladder mucin. In this study we have investigated the possible heterogeneity of mucin. For this purpose polyclonal and monoclonal antibodies against gallbladder mucin were raised. All antibodies reacted primarily with carbohydrate antigenic determinants. With these antibodies the immunoreactivity of gallbladder mucin from 60 patients with cholesterol gallstones and 20 subjects without stones was screened. In addition, reactivity with several lectins was studied. 2. Considerable heterogeneity was found with both antibody and lectin typing, but there was no significant difference in heterogeneity between mucin from patients with gallstones and control subjects. Immunoblotting revealed that there was similarity between the reaction of the polyclonal antibody and the Helix pomatia agglutinin. All mucin preparations reacting with the polyclonal antibody also bound to Helix pomatia agglutinin. Nineteen of the 21 reacting mucins (90%) were from patients with blood group A (18 patients) or AB (one patient) and expression of A antigen could be demonstrated on the mucin of these patients. The resulting two reacting mucins were from patients with type O. However, expression of the blood group antigen could not account for the lack of reactivity of the mucin of other patients. The Helix pomatia agglutinin partially blocked the reactivity of the polyclonal antibody, whereas anti-A antibody did not show inhibition, indicating that more than only blood group A epitopes were recognized by this antibody. 3. We conclude that considerable patient to patient heterogeneity of human gallbladder mucin exists.(ABSTRACT TRUNCATED AT 250 WORDS)

Heterogeneity of human biliary mucin: functional implications.

1. Human gallbladder mucin has been implicated as playing a role in the pathogenesis of gallstones. In previous studies no differences have been found in the content or composition of mucins derived from control bile or cholesterol gallstone bile. Until now, no differences were also found between these two groups of mucins with regard to their ability to cause cholesterol nucleation. In the accompanying paper we have reported that there is a strong heterogeneity of gallbladder mucins derived from individual patients (M. J. A. van Wijland, J. H. Klinkspoor, L. Th. de Wit, R. P. J. Oude Elferink, G. N. J. Tytgat and A. K. Groen, Clin Sci 1994; 86: 67-74). In the present study we further investigated a possible patient to patient heterogeneity of mucin by means of immunological and functional characterization of mucins isolated from hepatic bile of six different patients with gallstones. 2. Considerable heterogeneity was found. Two of the mucins barely reacted with a polyclonal anti-mucin antibody, whereas the other four mucins reacted very strongly. Lectin-binding studies indicated that the glycans of these two mucins expressed less D-N-acetylgalactosamine residues than the other four mucins. This was confirmed by analyses of the glycan compositions. These studies furthermore indicated that the glycans were of the O-linked type, contained alpha-D-N-acetylglucosamine and were fucosylated, sialylated and sulphated to different extents. Except for a strong heterogeneity in the sugar composition of the mucins, heterogeneity was also found in the biological activity of the mucins.(ABSTRACT TRUNCATED AT 250 WORDS)

Apical gene transfer into quiescent human and canine polarized intestinal epithelial cells by lentivirus vectors.

Intestinal epithelial cells secrete a protective luminal mucus barrier inhibiting viral gene transfer. Quiescent, polarized monolayers of primary epithelial cells from dog gallbladder and human colon are efficiently transduced through the apical mucus side by lentivirus vectors, suggesting their application to intestinal gene therapy.