Comparison of three commercially available buffy coat pooling sets for the preparation of platelet concentrates.

BACKGROUND: A disposable set for platelet concentrate (PC) preparation by the buffy coat method allows pooling of buffy coats, centrifugation and cell separation with in-line leucocyte filtration. This study compares three commercially available pooling sets in combination with INTERCEPT pathogen inactivation (PI). MATERIALS AND METHODS: Sets for pooling of buffy coats were from Fresenius Kabi (FRE), Macopharma (MAC) and Terumo BCT (TER). Platelet yield, recovery and concentration were compared before and after PI (n = 20). Platelet quality was assessed by annexin V binding, P-selectin expression and PAC1 binding. RESULTS: The TER pooling set had the highest platelet yield (5·39 ± 0·44 × 1011 ) compared with MAC (4·53 ± 0·77) and FRE (4·56 ± 0·51) prior to PI. This was the result of a significantly higher platelet concentration in the TER storage bag (1·41 ± 0·12 × 106 /µL) compared with MAC (1·18 ± 0·19) and FRE (1·28 ± 0·15). However, the TER platelet content decreased by 15·6% after PI, yielding 4·55 ± 0·47 × 1011 platelets compared with smaller reductions at 9·5% for MAC (4·10 ± 0·69) and 4·4% for FRE (4·36 ± 0·52). None of the individual PC contained >106 leucocytes. The pH in TER PC was lower compared with MAC and FRE caused by a higher lactic acid production rate. Consequently, PAC1 binding after TRAP activation was lowest for TER PC on day 6. P-selectin and annexin V were not different between suppliers. CONCLUSION: This study demonstrates the added value of evaluating the entire component production process when introducing a new consumable. This study helped to inform a decision on what pooling set is ideally suited for routine implementation taking into account PI.

High platelet content can increase storage lesion rates following Intercept pathogen inactivation primarily in platelet concentrates prepared by apheresis.

BACKGROUND: Pathogen inactivation methods for platelet concentrates are increasingly being used in blood banks worldwide. In vitro studies have demonstrated its effects on storage lesion, but little routine quality control data on blood banking outcomes have been reported. MATERIALS AND METHODS: Swirling of distributed products was monitored before and after implementation of Intercept pathogen inactivation. Metabolic parameters pH, glucose and lactic acid were determined in a random cohort of expired pathogen-inactivated products. Storage lesion indicators in apheresis concentrates with premature low swirling were compared to concentrates with normal swirling. RESULTS: During validation for implementing Intercept pathogen inactivation, pH and glucose levels decreased faster in apheresis platelet concentrates with high platelet content than with low platelet content or than in pathogen-inactivated pooled buffy coat-derived products. In routine products, glucose exhaustion was more often found in apheresis compared to buffy coat-derived platelet concentrates despite 3-7% more plasma carryover in the former. Annual incidence of premature low swirling increased significantly by 50% following implementation of pathogen inactivation implementation for apheresis but not for pooled buffy coat platelet concentrates. In addition, apheresis concentrates with premature low swirling had a significantly higher median platelet count (5·0 × 1011 ) than unaffected products (3·5 × 1011 ). CONCLUSION: The risk of increased storage lesion rates following Intercept pathogen inactivation is higher for apheresis than for buffy coat-derived platelet concentrates, especially when platelet contents are higher than 5·0 × 1011 .

The contribution of von Willebrand factor-GPIbα interactions to persistent aggregate formation in apheresis platelet concentrates.

BACKGROUND AND OBJECTIVES: Apheresis platelet concentrates sometimes contain persistent aggregates (PA). Because apheresis involves extracorporeal circulation, we hypothesized that interactions between GPIbα and von Willebrand factor (VWF) underlie their origin. MATERIALS AND METHODS: Platelets in donations with PA were compared to aggregate-free (AF) controls. Flow cytometry was used to determine platelet bound VWF. Degranulation was measured using P-selectin expression in flow cytometry and cytokine release using immunosorbent assays. Platelet adhesion to VWF was assessed in hydrodynamic flow and real-time video microscopy. RESULTS: Platelets in PA concentrates had significantly more (P = 0·009, n ≥ 8) bound VWF compared to AF platelets, but differences in VWF concentration, VWF collagen binding, activated VWF or GPIbα expression were not found. Degranulation was higher (P = 0·030, n = 7) in PA than AF concentrates on day 1 of storage, but adhesion to immobilized VWF under hydrodynamic flow conditions was normal at that moment. On day 6, however, significantly less VWF adhesion (P = 0·009, n ≥ 6) was found for PA platelets compared to AF, indicating accelerated storage lesion in PA products. In a model that mimicks PA formation by chemically induced binding of VWF to platelets, we found that degranulation, phosphatidylserine expression and metabolism did not differ with paired controls at any time during subsequent storage. CONCLUSION: Accelerated storage lesion is found in concentrates with PA, but this cannot be explained solely by increased platelet bound VWF following apheresis. Therefore, additional stressors are probably responsible for the increases observed in platelet degranulation and storage lesion in products with PA.

Persistent aggregates in apheresis platelet concentrates.

BACKGROUND: Aggregates often appear during apheresis. Sometimes, these persist throughout storage, causing product wastage. This study assessed product quality of apheresis concentrates containing persistent aggregates (PA) and aimed to identify the factors that contribute to their formation. METHODS: Donation (n = 180) and platelet indices (n ≥ 10) from apheresis concentrates with PA were compared with aggregate-free products. RESULTS: The proportion of donors with at least one previous PA donation was twofold higher in the PA group (P < 0·0001) indicating a donor dependence. Significantly higher donor whole blood platelet counts (286 ± 50 vs. 266 ± 49 × 10(3) /µl, P < 0·0001) and higher apheresis yields (6·0 ± 1·6 vs. 5·4 ± 1·5 × 10(11) , P < 0·0001) were noted in the PA group. Haematocrit was also slightly higher, but age, gender and body mass were similar. The pH of PA products on day six postdonation was significantly lower (P < 0·001), in line with higher lactic acid concentrations. Flow cytometry showed no differences in GPIbα levels or phosphatidylserine exposure. However, there was slightly more integrin activation as well as increased degranulation measured by P-selectin expression. Cytokine concentrations were also significantly higher in PA concentrates. Aggregation was normal in response to SFLLRN peptide and collagen stimulation, but agglutination at low-dose ristocetin was significantly higher (P = 0.01) in PA products. Finally, PA were disintegrated by plasmin-mediated thrombolysis but not by integrin αIIb ß3 inhibition. CONCLUSION: Products with PA have acceptable quality parameters, but additional functional studies are warranted. Furthermore, PA are more likely to recur in certain donors who have higher platelet counts.

Riboflavin and amotosalen photochemical treatments of platelet concentrates reduce thrombus formation kinetics in vitro.

BACKGROUND: Photochemical treatment (PCT) of platelet concentrates using photosensitizers and ultraviolet light illumination reduces the proliferation potential of pathogens by damaging biomolecules. MATERIALS AND METHODS: The impact of riboflavin (RF-PRT)- and amotosalen (AS-PCT)-based pathogen inactivation on platelets was studied using microfluidic flow chambers on immobilized collagen using standard platelet concentrates prepared from buffy coats in additive solution. Flow cytometry, metabolic parameters and light transmission aggregometry with thrombin-related peptide, collagen and ristocetin were determined concurrently. RESULTS: Both PCTs significantly decreased the platelet surface coverage kinetics in flow chambers over the course of the 7-day study. Platelet aggregation was affected following RF-PRT in response to all agonists, while AS-PCT mainly impacted low-dose ristocetin agglutination. RF-PRT induces premature platelet activation because integrin αII b ß3 was spontaneously activated, and α-degranulation, phosphatidylserine/-ethanolamine exposure and anaerobic metabolism significantly increased following treatment, which was not the case for AS-PCT. On the other hand, AS-PCT significantly diminished thrombus growth onto von Willebrand factor under shear flow. This defect was caused by fewer integrin αII b ß3 interactions, not by defective GPIbα-VWF binding as shown by adhesion experiments in the presence of tirofiban. Moreover, integrin αII b ß3 activation was also affected following the activation of platelets via GPVI-FcγRIIa or PAR1. Finally, amotosalen illumination as such is sufficient to induce platelet damage, with no additional measurable effect of the chemical adsorption step. Gamma irradiation caused no significant difference compared to controls on any time-point or for any parameter. CONCLUSION: Both PCTs significantly reduce thrombus formation rate but by different biochemical mechanisms.

Oxygen removal during pathogen inactivation with riboflavin and UV light preserves protein function in plasma for transfusion.

BACKGROUND AND OBJECTIVE: Photochemical pathogen inactivation technologies (PCT) for individual transfusion products act by inhibition of replication through irreversibly damaging nucleic acids. Concern on the collateral impact of PCT on the blood component's integrity has caused reluctance to introduce this technology in routine practice. This work aims to uncover the mechanism of damage to plasma constituents by riboflavin pathogen reduction technology (RF-PRT). METHODS: Activity and antigen of plasma components were determined following RF-PRT in the presence or absence of dissolved molecular oxygen. RESULTS: Employing ADAMTS13 as a sentinel molecule in plasma, our data show that its activity and antigen are reduced by 23 ± 8% and 29 ± 9% (n = 24), respectively, which corroborates with a mean decrease of 25% observed for other coagulation factors. Western blotting of ADAMTS13 shows decreased molecular integrity, with no obvious indication of additional proteolysis nor is riboflavin able to directly inhibit the enzyme. However, physical removal of dissolved oxygen prior to RF-PRT protects ADAMTS13 as well as FVIII and fibrinogen from damage, indicating a direct role for reactive oxygen species. Redox dye measurements indicate that superoxide anions are specifically generated during RF-PRT. Protein carbonyl content as a marker of disseminated irreversible biomolecular damage was significantly increased (3·1 ± 0·8 vs. 1·6 ± 0·5 nmol/mg protein) following RF-PRT, but not in the absence of dissolved molecular oxygen (1·8 ± 0·4 nmol/mg). CONCLUSIONS: RF-PRT of single plasma units generates reactive oxygen species that adversely affect biomolecular integrity of relevant plasma constituents, a side-effect, which can be bypassed by applying hypoxic conditions during the pathogen inactivation process.

Salmonella Enteritidis universal stress protein (usp) gene expression is stimulated by egg white and supports oviduct colonization and egg contamination in laying hens.

Salmonella enterica subspecies enterica serovar Enteritidis has caused a worldwide egg-associated pandemic since the mid 1980s. The exact mechanisms causing this egg tropism are still largely unknown, and only a few Salmonella genes have been implicated in the interaction with the oviduct or eggs. A in vivo expression technology screening performed previously, identified the uspA and uspB genes as being highly expressed in the chicken oviduct and in eggs. Here, we demonstrate that uspA and uspB gene expression is indeed induced after contact with egg white. Intra-oviduct inoculation of Salmonella Enteritidis uspB and uspBA mutant strains showed that the mutants had a decreased ability to colonize the magnum and isthmus of the oviduct, the organs that produce the egg white and eggshell membranes, respectively, at 7 days post-inoculation. Intravenous challenge showed that a Salmonella Enteritidis uspBA mutant strain had a decreased ability to contaminate eggs. Analogous to the function of universal stress proteins A and B in other bacterial species, we hypothesize that the Salmonella uspA and uspB genes are involved in long term persistence of Salmonella Enteritidis in harmful environments, such as in the oviduct and eggs, by conferring resistance against compounds that damage the bacterial cell membrane and DNA.

Bearded dragons (Pogona vitticeps) asymptomatically infected with Devriesea agamarum are a source of persistent clinical infection in captive colonies of dab lizards (Uromastyx sp.).

Devriesea agamarum causes dermatitis and septicaemia in a variety of lizards, notably those belonging to the genus Uromastyx, whereas other species such as bearded dragons (Pogona vitticeps) seem to be asymptomatic carriers. Using amplified fragment length polymorphism (AFLP), the relatedness between 69 D. agamarum isolates was examined. The isolates derived from 44 diseased lizards, of which 31 belonged to the genus Uromastyx, and from 25 healthy lizards, of which 21 were bearded dragons. Eight AFLP genotypes were obtained, four of which comprised 93% of the isolates. These four genotypes were each present in 2, 2, 8 and 13 different captive colonies. Up to three genotypes were isolated from a single infected colony simultaneously. On two occasions, the same genotype was found in healthy bearded dragons and diseased Uromastyx lizards from the same colony, confirming the role of the former as an asymptomatic source of infection for the latter. Two genotypes, comprising 12 isolates, were exclusively associated with diseased Uromastyx lizards, suggesting strain dependent host adaptation. Finally, D. agamarum was shown to be able to persist for at least seven years in a lizard colony, persistently causing severe disease in several lizard species.

The effect of starch gelatinisation degree on intestinal morphology, intestinal pH and bacteriology in pigeons.

The effect of starch gelatinisation degree in extruded feed on intestinal morphology, intestinal pH and faecal bacteriology was investigated in pigeons. Extruded complete pigeon diets would offer the principle advantage of providing equilibrated nutrients and energy, but factors such as starch gelatinisation require investigation before these diets are offered as main dietary items to pigeons. Birds were fed two diets with equal ingredient composition and nutrient content, but with a different degree of starch gelatinisation resulting from altered extrusion processing (high gelatinisation degree (HG) with 73.6% gelatinisation vs. low gelatinisation degree (LG) with 53.1% gelatinisation). Feed intake and weight gain changes were measured weekly. Blood samples were collected at day 28 and analysed for non-esterified fatty acids, lactate dehydrogenase and glucose. The pH values for fresh excreta were measured; thereafter fresh excreta were collected and cultured for measurement of colony-forming units for bacterial classes. At the end, morphological measurements were examined and the pH values throughout the gastrointestinal tract were recorded. Liver, pancreas and abdominal fat were weighed. There was a tendency (p= 0.07) towards higher numbers of Escherichia coli in the excreta of the LG group compared with those in the HG group. No dietary treatment effects were noted on the number of Lactobacillus sp. in the excreta. In proximal parts of the intestine, LG revealed a significantly lower pH than HG. Villus height and crypt depth were not affected by dietary treatment, but the duodenum muscularis thickness, liver weight and pancreas weight were significantly lower in the LG than that in HG group. This trial demonstrated that the lower level of starch gelatinisation degree of extruded feed leads to acidification of the proximal gut and altered gut morphology in pigeons. Hence, extruded pigeon diets should preferably contain low-gelatinised starch instead of high-gelatinised starch. In addition, future research must focus on the effects of starch gelatinisation on the correlation between the intestinal pH, microflora content and intestinal morphology.

Surgical repair of Ebstein's anomaly.

Ebstein's anomaly is a complex malformation that has been treated by various surgical techniques, with variable results. We have developed a repair-by plication of the free wall of the atrialized portion of the right ventricle, posterior tricuspid annuloplasty, and right atrial reduction--that has been used since 1972 in 16 patients. The repair is based on the construction of a monocusp valve by the use of the anterior leaflet of the tricuspid valve, which is usually enlarged in this anomaly. There were 14 operative survivors and no instances of complete heart block. Two patients with refractory dysrhythmias due to the Wolff-Parkinson-White syndrome underwent successful intraoperative epicardial and endocardial mapping and surgical division of accessory pathways. This repair has resulted in improvement in the operative mortality and in gratifying early and late results in most patients.