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SUMMARY: New platelet storage systems, such as changes in the plastic of the storage bags, require validation. In this study, pooled buffy coat platelets stored in Fresenius/NPBI polyolefin bags were compared with those stored in Fresenius/NPBI butyryl-trihexyl citrate (BTHC) plasticized polyvinyl chloride (PVC). The CompoSelect thrombocyte polishing filter system (1000 mL polyolefin bag) and the CompoStop F730 system (1300 mL BTHC-PVC bag) were used to prepare paired, plasma-suspended, buffy coat platelet concentrates. Samples were taken up to day 7 for in vitro analysis. In a separate experiment, 12 units were prepared using the CompoStop F730 system and samples taken after leucofiltration for FXIIa assay. By day 7, platelet concentrates stored in BTHC-PVC demonstrated significantly higher pH levels (7.32 +/- 0.05 vs. 7.26 +/- 0.05) and a greater degree of cell lysis as shown by increased lactate dehydrogenase levels (497 +/- 107 vs. 392 +/- 81 U L(-1)). The supernatants contained higher concentrations of soluble P-selectin and the chemokine 'regulated on activation, normal T-cell expressed and presumably secreted', which are released from the alpha-granules during activation. The ATP concentrations were significantly lower in BTHC-PVC. Platelet counts, mean platelet volume and hypotonic shock response were similar for both bags. FXIIa antigen concentrations were 0.6 +/- 0.2 ng mL(-1) indicating that activation of the contact factor pathway had not occurred. Although the CompoStop F730 leucoreduction filter did not activate the contact system, platelets stored in 100% plasma in BTHC-PVC bags demonstrated different in vitro characteristics from those stored in polyolefin. Further work is required to demonstrate whether these differences will affect in vivo recovery and survival.

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BACKGROUND: This study was designed to determine which in vitro assays would be most useful for studying the effects of cold storage on platelet concentrates and to establish an in vivo model for platelet recovery and survival. STUDY DESIGN AND METHODS: Paired, plasma-suspended, leucoreduced, buffy-coat-derived platelet concentrates were stored either at 22 or 4 degrees C. Prior to storage and after 18 h, 5 days and 7 days, samples were taken and various assays were performed. On day 6, in vivo studies were carried out using a model system. Galactosylation of the platelets, prior to cold storage, was also tested. RESULTS: Hypotonic shock response, collagen-induced aggregation, RANTES and P-selectin binding site measurements demonstrated differences between platelets stored at 22 and 4 degrees C. The glycocalicin assay was able to demonstrate microvesicle formation at 4 degrees C. The in vivo model showed that there was at least a 50% decrease in recovery and survival when the platelets were stored in the cold. Galactosylation did not improve these results. CONCLUSIONS: Several assays, both in vitro and in vivo, were able to detect differences in platelet-storage characteristics and in vivo recovery and survival in a model system. Galactosylation did not correct these cold-induced changes.

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BACKGROUND AND OBJECTIVES: The DiaMed Impact R tests platelet function under close to physiological flow conditions using cone and plate technology. An image analyser quantifies the adhered platelets and results are expressed as percentage of well surface covered by aggregates (SC %) as an index of adhesion and average size of the aggregates (AS microm(2)) as an index of aggregation. The machine is designed to use whole blood and the aim of this study was to determine if it could be used to assess platelet function in platelet concentrates (PC). MATERIAL AND METHODS: Platelet concentrates were mixed with various ratios of platelet-poor plasma (PPP) or ABO-compatible routine leucoreduced red cells in saline-adenine-glucose-mannitol. The effects of platelet counts, haematocrit, shear rate and time of activation upon SC and AS were evaluated to identify optimized assay conditions. Routine PCs were then tested on Days 2, 5 and 7. Samples were also stored at 4 or 37 degrees C for 1 to 2 h before assay to see if function was altered. RESULTS: Platelet concentrate in PPP resulted in no detectable platelet adhesion. However, addition of red cells to PC resulted in measurable platelet adhesion and aggregation. Optimal conditions were identified as shear rate of 1800 per second, 4-min activation, platelet count between 250 and 400 x 10(6) per ml, haematocrit between 30 and 40%. When stored PCs were tested under these conditions we observed median values of 8.2% for SC and 32.7 microm(2) for AS at 2 days, which reduced to 6.9% and 25.0 microm(2), respectively, after 5-day storage and 6.8% and 21.0 microm(2) after 7 days. CONCLUSION: We were able to reconstitute PCs by adding red cells and identified conditions to allow platelet adhesion and aggregation functions of PCs to be measurable in the DiaMed Impact R. Platelet functions of adhesion and aggregation were shown to decrease during storage but improved after a 1-h treatment at 37 degrees C.

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Variant Creutzfeldt-Jakob disease (vCJD) poses a serious risk of secondary transmission and the need to detect infectivity in asymptomatic individuals is therefore of major importance. Following infection, it is assumed that minute amounts of disease-associated prion protein (PrP(Sc)) replicate by conversion of the host cellular prion protein (PrP(C)). Therefore, methods of rapidly reproducing this conversion process in vitro would be valuable tools in the development of such tests. We show that one such technique, protein misfolding cyclic amplification (PMCA), can amplify vCJD PrP(Sc) from human brain tissue, and that the degree of amplification is dependent upon the substrate PRNP codon 129 polymorphism. Both human platelets and transgenic mouse brain are shown to be suitable alternative substrate sources, and amplified PrP(Sc) can be detected using a conformation-dependent immunoassay (CDI), allowing the detection of putative proteinase K sensitive forms of PrP(Sc).

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BACKGROUND AND OBJECTIVES: The aim of this study was to investigate the effects of extended storage of pooled random platelets in SSP+ additive solution (MacoPharma). MATERIALS AND METHODS: Eight buffy coat-derived, pooled, leucoreduced platelet concentrates were prepared in 75% SSP+, 25% plasma using Fresenius/NPBI Composelect thrombocyte polishing filter (TPF) systems. Platelet concentrates were stored for 19 days in polyolefin storage bags and samples for in vitro analysis were taken at various time-points during storage. RESULTS: Platelet yields were lower than seen routinely when platelets are prepared in 100% plasma. The in vitro quality of the platelets stored in SSP+ was maintained until day 9. Glucose was depleted by day 12 and this was accompanied by a rapid fall in pCO2, a rise in pO2 and a cessation of lactate production. ATP and bicarbonate concentrations fell, the platelets began to swell and the ability to swirl decreased. Soluble P-selectin, glycocalicin, and regulated on activation, normal, T-cell expressed, and secreted (RANTES) concentrations increased, as did P-selectin expression. Loss of platelets and an increase in lacate hydrogenase concentration indicated that lysis had occurred. However, the pH remained between 6.4 and 7.4. CONCLUSIONS: The results suggest that SSP+ could be used for platelet storage for up to 9 days. However, the preparation of platelets in the additive requires some optimization. In vivo studies are required to confirm these in vitro results.

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The impact of vCJD upon blood transfusion practice hinges on its lymphoreticular involvement. B lymphocytes play a key supporting role for the capture and replication of infectivity by follicular dendritic cells of the lymphoid tissue in animal models of transmissible spongiform encephalopathies (TSE) and tonsils, spleen and appendix in man can harbour vCJD infectivity, a situation not seen with the other human TSEs. Leucodepletion of blood donations in the UK was implemented to reduce possible vCJD transmission and preliminary data suggests that white cell associated infectivity will be effectively removed although plasma infectivity will not. Blood screening assays are under development but none yet are ready for application. The conformation dependant immunoassay, based on differences in secondary and tertiary structure between normal and TSE-associated abnormal prion protein, has a sensitivity now approaching the best bioassay. Even so further development is needed to detect the fg/ml levels likely in the event that vCJD blood does contain abnormal prion, which is as yet unproven. Surrogate assays, such as for erythroid associated factor, may provide additional means of identifying donors harbouring vCJD. Validation of clearance of TSEs from pooled plasma products consistently demonstrates effective removal of the agents in downscaled systems and studies comparing vCJD, BSE and scrapie agents yield similar results. Many approaches to therapy are under investigation, in cell culture and animal models, targeted to normal or abnormal prion metabolism, including chemical and immunological interventions. Efficacy of quinacrine/chlorpromazine and pentosan polysulphate in a clinical setting, and agents yet to be used, will be more accurately known following recent agreement of clinical drug evaluation protocols.

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BACKGROUND AND OBJECTIVES: The risk of haemophiliacs contracting variant Creutzfeldt-Jakob disease (vCJD) via treatment with factor VIII concentrates is not known. Therefore, in order to determine the extent to which the vCJD agent might be removed during the preparation of factor VIII concentrate, the partitioning of a bovine spongiform encephalopathy (BSE)-derived agent was measured over the main purification step used to prepare the Scottish National Blood Transfusion Service high-purity factor VIII concentrate (Liberate). MATERIALS AND METHODS: Murine-passaged BSE (strain 301V), in the form of a microsomal fraction prepared from infected brain, was used to 'spike' a solution of factor VIII of intermediate purity. The 'spiked' starting material was subjected to solvent-detergent treatment and then to anion-exchange chromatography with Toyopearl DEAE-650M. All fractions were tested for 301V infectivity using a murine bioassay, including the procedures used to clean the ion-exchange media after use. RESULTS: BSE 301V infectivity was reduced by 2.9 log(10) in the fibrinogen fraction and by 2.7 log(10) in the factor VIII fraction. Over 99% of the added 301V infectivity remained bound to the ion-exchange column after elution of factor VIII. A large quantity of infectivity was subsequently removed by washing the ion-exchange media with 2 m NaCl. No further BSE 301V infectivity was detected in column eluates after treatment with 0.1 m NaOH or a second wash with 2 m NaCl. CONCLUSIONS: Results using a BSE-derived agent suggest that vCJD infectivity would be substantially removed by the ion-exchange process used in the preparation of fibrinogen and factor VIII concentrate. Although 301V infectivity remained bound to the ion-exchange matrix following elution of factor VIII, this appeared to be eliminated by the procedure used for cleaning the ion-exchange media after each use.

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BACKGROUND AND OBJECTIVES: There is still uncertainty over how the agent of variant Creutzfeld-Jakob disease (vCJD) would partition during the manufacture of plasma derivatives. In this study, a BSE-derived agent was used as a vCJD model to determine the extent to which infectivity could be removed by selected steps used in the manufacture of intravenous immunoglobulin (IVIG). MATERIALS AND METHODS: Murine-passaged BSE (strain 301V), in the form of a microsomal fraction prepared from infected brain, was used to "spike" the starting material in three experiments. The partitioning of BSE infectivity was measured over Fraction I+III precipitation, borosilicate microfibre depth filtration and Seitz depth filtration, with these steps being examined individually and in series. RESULTS: Most 301V infectivity partitioned into Fraction I+III (log reduction 2.1). Infectivity remaining in Supernatant I+III was reduced by AP20 glass-fibre depth filtration (log reduction 0.6) and subsequently removed to below the limit of detection by Seitz KS80 depth filtration, giving an overall log reduction of > or = 2.9 for the three steps in series. By contrast, glass-fibre depth filtration gave a log reduction of 2.4 when challenged directly with "spiked" feedstock. Seitz KS80 depth filtration gave a log reduction of > or = 3.1 when challenged directly with 'spiked' feedstock and also removed residual infectivity to below the limit of detection when applied as the final step in series. CONCLUSIONS: Results using a BSE-derived agent suggest that vCJD infectivity should be substantially removed from immunoglobulin G (IgG) solutions by Fraction I+III precipitation and Seitz KS80 depth filtration. The three different process steps examined acted in a complementary manner to one another when operated in series. However, the data demonstrated that it would be inappropriate to add together the reduction factors that had been derived for each step in isolation.

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BACKGROUND AND OBJECTIVES: The concern that variant Creutzfeldt-Jakob disease could be transmitted via blood transfusion has prompted studies of blood infectivity in animal models. As normal prion protein acts as a substrate for conversion to the abnormal form associated with infectivity, we have quantified its distribution in mice and hamsters, the most commonly used animal models. MATERIALS AND METHODS: A time-resolved fluoroimmunoassay was used to measure normal prion protein in hamster and mouse tissues, including blood. RESULTS: Levels of prion protein in hamster blood were remarkably low compared with human blood. In contrast, levels in mouse blood were quite similar to human blood; however, there were differences in the distribution of normal prion between cellular and cell-free fractions. CONCLUSION: Differences between levels of normal prion in blood of animal models and humans should be considered as a possible contributor to infectivity study outcomes in these models.

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OBJECTIVE: Esophagogastrectomy is an established surgical treatment for esophageal malignancy. The postoperative period may be complicated by the development of acute lung injury syndromes and thus, may provide a useful model in which to study the early pathogenic mechanisms of inflammatory lung injury. DESIGN: Open, prospective study. SETTING: High dependency and intensive therapy units. PATIENTS: Eight healthy male volunteers and 20 patients in the early postoperative period INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: The lung protein accumulation index (PAI) of radiolabeled transferrin was determined by using a portable, double-isotope system. The following circulating inflammatory markers-thought to reflect neutrophil-endothelial activation and injury including circulating neutrophil elastase-soluble L-, E-, and P-selectins and thrombomodulin and von Willebrand factor antigen were assayed from venous blood samples The PAI for healthy volunteers was median -0.5 (range, -1.73 to 0.27) x 10(-3)/min and for patients undergoing esophagogastrectomy -0.005 (range, -1.53 to 2.28) x 10(-3)/min. There was no statistical difference between the two groups. In the postesophagogastrectomy group, a significant elevation in circulating levels of neutrophil elastase, soluble P- and E-selectin, thrombomodulin, and von Willebrand factor antigen were observed relative to the control group but only circulating plasma elastase demonstrated a significant correlation with the PAI (r2 = .23, p =.03). CONCLUSIONS: The data suggest patients undergoing esophagogastrectomy develop a inflammatory response but this is not a surrogate of permeability and other factors are likely to determine persistent injury to the alveolar-capillary barrier function in this patient group.

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Universal leucodepletion is being introduced in the U.K. to reduce a theoretical risk of Creutzfeldt-Jakob disease (CJD) transmission. If CJD infectivity is associated with leucocytes, any cell fragmentation associated with filtration could reduce the potential benefit. Four types each of whole blood, red cell and platelet leucodepletion filters were assessed after holding of blood units for at least 4 h at 22 degrees C. In all cases the mean residual leucocyte content was <1 000 000 per unit, with only two individual filtered whole blood units having a leucocyte content exceeding this. Evidence of leucocyte fragmentation during filtration was sought but not found by assay of soluble elastase, beta-thromboglobulin and normal prion protein, as well as by isotopic labelling of leucocyte external membrane. These preliminary studies indicate that it was possible to prepare leucodepleted blood components by filtration at room temperature, and that this appeared not to be associated with overt cell fragmentation. Definitive demonstration that fragmentation does not occur requires the development of improved general (non-specific) assays for cell membrane fragments.

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It is well known that 10-20% of severe haemophiliacs are likely to develop inhibitors to factor VIII, usually soon after the commencement of therapy. Two recent inhibitor outbreaks have occurred in patients treated for a number of years on switching to a product subjected to additional virus inactivation. Hence the incidence of inhibitor formation may be affected by the type of product used for treatment and the potential for processing to result in 'neoantigens'. Examination of the parts of factor VIII interacting with inhibiting antibodies, and the effect of various therapies on these, can teach us something about the mechanisms involved in antibody formation. However, the development of pre-clinical assays to assess products and processes for neoantigen formation should allow the prevention of inhibitor outbreaks. This review summarizes current in vitro and in vivo approaches to this problem, concluding that most available assays are inadequate for this purpose, with competitive immunoassay and phospholipid binding providing the most hopeful route forward.

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Pulmonary microvascular injury, one of the earliest events in adult respiratory distress syndrome (ARDS), is caused by the release of injurious products from stimulated neutrophils and other inflammatory cells in the lung microvessels. An increased level of the endothelial cell-surface anticoagulant protein thrombomodulin (TM) in plasma from patients with ARDS as shown in this study may be one consequence of this, and our objective in this investigation was to define the mechanisms by which TM is modulated by neutrophils, using an endothelial tissue culture system. Human neutrophils in contact with endothelium caused a fourfold reduction in cell-surface TM activity, but only after prior neutrophil priming and activation. Neutrophil release products elastase and cathepsin G caused rapid dose-related reductions in endothelial cell-surface TM activity, with complete abolition at 5 microg/ml in the absence of endothelial detachment. H2O2 also reduced TM activity. TM antigen accumulated in culture supernatant after treatment of endothelium with cathepsin G, indicating proteolytic release of cell-surface TM. We conclude that primed activated neutrophils are potent modulators of endothelial TM in vitro.

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Recent studies using assays for surrogate markers of thrombogenicity in man have demonstrated that activation of the coagulation system occurs following infusion of clinical doses of prothrombin complex concentrates (PCC) but not after the same doses of high-purity factor IX concentrates (HP-FIX) in patients with haemophilia B. Here we have investigated the mechanism of such thrombogenesis by applying assays that detect early-through to late-events in coagulation system activation in a pharmacokinetic cross-over study of 50 IU/kg PCC and a new HP-FIX product in haemophilia B patients. Satisfactory recoveries and half-lives were observed for both concentrates. HP-FIX caused no increases in thrombin-antithrombin III complex (TAT), prothrombin activation peptide fragment F1+2 (F1+2), factor X activation peptide (FXAP) or factor VIIa (FVIIa). In contrast the same dose of factor IX in the form of PCC was followed by significant increases over pre-infusion levels of TAT, F1+2 and FXAP, but not FVIIa. Elevations of FIXAP occurred after both HP-FIX and PCC but did not reach normal levels and were attributed to normalisation of the FIX concentration in those patients whose levels of FIXAP were initially low. We conclude that the thrombogenic trigger associated with PCC infusion occurs at the level of factor X activation. In the absence of any increase in FVIIa, we would attribute this to the likely presence of FIXa in the PCC.

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OBJECTIVE: To assess the relationship between neutrophil activation and indices of disease severity in patients with chronic liver disease. METHODS: Plasma neutrophil elastase was measured by radioimmunoassay as a marker of neutrophil activation, and disease severity assessed by standard clinical, biochemical, haematological and histological techniques. PATIENTS: Eighty-eight patients with chronic liver disease were studied, Thirty-nine had alcohol-induced liver disease (ALD), 18 autoimmune chronic hepatitis, 13 cryptogenic cirrhosis, seven primary biliary cirrhosis, six primary sclerosing cholangitis, three haemochromatosis and two secondary biliary cirrhosis. Seventy-three patients were cirrhotic and 15 were non-cirrhotic, confirmed by biopsy. RESULTS: Levels of neutrophil elastase were raised in Childs C cirrhotic patients with ALD compared with Childs A or B patients with ALD (P < 0.01), Childs A or B patients with non-ALD (P < 0.01), and Childs C patients with non-ALD (P = 0.02). In patients with ALD, neutrophil elastase correlated with prothrombin time (r = 0.679, P = 0.001), bilirubin (r = 0.587, P < 0.001), Child-Pugh score (r = 0.546, P < 0.001) and inversely with serum albumin (r = -0.511, P < 0.001). In patients with non-ALD, there were no correlations with the measurements of with transaminase levels. CONCLUSION: Neutrophil activation, as measured by plasma neutrophil elastase, is a marker of disease severity in patients with alcohol-induced chronic liver damage, but not in those with other causes of liver disease.

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The aim of this study was to investigate whether abnormalities in the fibrinolytic system and in the naturally occurring anticoagulant proteins could contribute to the thrombotic risk in essential thrombocythemia. Euglobulin lysis time, fibrin plate lysis area, tissue plasminogen activator antigen, and activity and plasminogen activator inhibitor antigen were measured before and after venous occlusion in a group of 16 patients with essential thrombocythemia and in 16 healthy age and sex matched controls. In addition, resting levels of antithrombin III, D-dimer, prothrombin fragment 1 + 2, and protein C and S were assessed. The results were related to the presence or absence of a thrombotic history. The results demonstrated that the patients had a significantly elevated fibrin plate lysis area and significantly decreased plasminogen activator antigen, both at baseline and after venous occlusion. They also had significantly decreased levels of plasma protein C and total protein S. There was a modest, non-significant elevation in the plasma concentration of D-Dimer and F 1 + 2. Those patients with a history of thrombosis had significantly lower protein C levels compared with individuals without a thrombotic history. We conclude that patients with essential thrombocythemia have evidence of activated fibrinolysis in the resting state and after stimulation. This, and the decreased levels of protein C and total protein S, may be secondary to chronic clinically occult thrombosis occurring in myeloproliferative disorders.

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We used monoclonal antibody ELISAs, antigen molecular size distribution, competition ELISA and neonatal mouse immune tolerance methods to detect potential neoantigen formation and increased immunogenicity following severe dry-heat treatment of high-purity factor VIII (Liberate) and factor IX concentrates. To provide positive controls, concentrates were heated in solution (70 degrees C for 2 h) to produce denaturation on purpose. The competition ELISA applied to factor IX proved particularly useful for quantifying differences between the positive control and the dry-heated/unheated concentrates. None of the test systems employed by us indicated any detectable neoantigen formation or any alteration in immunogenicity following terminal severe dry-heat treatment of the high-purity concentrates, and this finding is supported by clinical experience so far.

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