Characteristics of prion-filtered red cells suspended in pathogen-inactivated plasma (MB treated or solvent-detergent treated) for neonatal exchange transfusion.

BACKGROUND AND OBJECTIVES: Neonates undergoing exchange transfusion require <5-day-old red cells suspended in plasma. This study assesses the effect of replacing the saline, adenine, glucose and mannitol (SAGM) of prion reduced (P-Capt) red cells with either methylene blue-treated plasma (MBTFFP) or OctaplasLG to reduce the risk of variant Creutzfelt-Jakob disease transmission. MATERIALS AND METHODS: Twenty leucoreduced red cell units in SAGM were prion reduced on day 1. The SAGM was replaced by MBTFFP (n=10) or OctaplasLG (n=10). The units were irradiated and stored at 4°C for 24 h. A further 20 units were stored for 5 days before being processed as above. Haemolysis (%), potassium, ATP, 2,3-DPG and plasma proteins were measured. RESULTS: Haemolysis remained low (≤0·16%). Following irradiation and storage, red cells in both types of plasma showed similar changes in potassium and ATP concentrations. The 2,3-DPG concentrations were well maintained although lower in red cells in OctaplasLG compared with those in MBTFFP (4·79 vs. 6·83 µmoles/g Hb on day 6). MBTFFP contained lower concentrations of fibrinogen, FV and FVIII. In OctaplasLG, alpha-2-antiplasmin was approximately 0·4 U/ml lower than in MBTFFP. After 24 h at 4°C, free protein S in OctaplasLG fell from 0·82 to 0·57 IU/ml. Other plasma proteins, in both types of plasma, were stable. CONCLUSIONS: Red cells in both types of plasma demonstrated similar storage characteristics. The plasma proteins, except protein S in OctaplasLG, were stable over 24 h at 4°C in both types of plasma, and low FVIII concentrations were noted in the MBTFFP (group O) units used.

Cold storage of pooled, buffy-coat-derived, leucoreduced platelets in plasma.

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.

Platelet storage in Fresenius/NPBI polyolefin and BTHC-PVC bags: a direct comparison.

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.

Neutrophil elastase: a determinant of endothelial damage and reperfusion injury after liver transplantation?

Reperfusion injury has been implicated in the development of primary graft dysfunction (PGD) after liver transplantation. Neutrophil migration and activation may be involved in the pathogenesis of this injury. We studied neutrophil activation and its role in the etiology of PGD by measuring neutrophil elastase by radioimmunoassay, in serial blood samples of 19 patients before, during, and for 24 hr after transplantation. In a subgroup of patients, we also measured soluble thrombomodulin at the same time points as a marker of endothelial damage. The pretransplant elastase level was significantly raised (40.13+/-4.84 ng/ml, mean+/-SEM) compared with levels of healthy controls (18.7+/-5.6 ng/ml, P<0.05). A marked increase in elastase activity followed reperfusion, with a peak at 2 hr (370+/-50.5 ng/ml, P<0.01). Thereafter, there was a decline, but elastase remained elevated at 24 hr (186+/-60.94 ng/ml). The mean increase in neutrophil elastase after reperfusion correlated significantly with markers of graft function (P<0.05) and with the mean rise in soluble thrombomodulin (P=0.042), which increased from a pretransplant level of 81.2+/-11.32 to 186+/-50.4 ng/ml, 6 hr after reperfusion (P<0.05). The results of this study indicate that marked neutrophil activation and endothelial cell damage occurs after graft reperfusion during orthotopic liver transplantation, and the degree of activation correlates with markers of graft function, which may suggest a role in the etiology of PGD.

A novel rat model of thrombogenicity: its use in evaluation of prothrombin complex concentrates and high purity factor IX concentrates.

A non-stasis rodent model of thrombogenicity has been used for dose-ranging studies with a conventional prothrombin complex concentrate (PCC) and to evaluate high purity factor IX concentrates from different manufacturers. Fibrin monomer (soluble fibrin) and fibrinopeptide A (FPA) were monitored before and after infusion of test solution. FPA was found to be the more sensitive and reproducible indicator of thrombogenicity and exhibited a dose-related elevation after infusion of the PCC at doses of between 100-300 IU/kg. In contrast the amounts of FPA generated after 300 IU/kg of the high purity factor IX products were similar to control infusions of albumin.

Processing of whole femoral head allografts: a method for improving clinical efficacy and safety.

Femoral heads removed during primary hip replacement surgery are widely utilised as a source of allograft bone. Despite evidence that processing these grafts to remove blood and marrow elements improves both the clinical performance and safety of these allografts, many are transplanted without any processing being applied at all. The goal of this study was to investigate the efficiency of an allograft processing protocol which incorporates pasteurisation, (3 h, 56-60 degrees C) centrifugation, (1850g, 2 x 15 min, 40 degrees C) sonication, and repeated washing in warm (56-60 degrees C, 19 h) distilled, sterile water to remove blood and marrow elements from the graft. The protocol also involves applying heat treatment to the grafts which has been demonstrated to inactivate many pathogenic viruses. Following the processing procedure, the grafts are lyophilised and sterilised with ethylene oxide gas. The amount and rate of removal of 4 different components of blood and marrow from 6 whole femoral head allografts were measured. These were lipid, soluble protein, elastase and chloride ions. Lipid removal was assessed gravimetrically by solvent extraction of dried samples, soluble protein by the Bradford assay, elastase by radioimmunoassay and choride ion content by a modified commercially available colorimetric assay. Removing lipid from grafts has been shown to increase the rate of incorporation when the graft is used clinically. Elastase was studied as a marker of leukocyte removal, as evidence suggests the majority of potentially infective transmissible spongiform encephalopathy (TSE) activity resides in a sub-population of leukocytes. Soluble protein was studied as a marker of plasma removal, as a smaller amount of TSE infectivity resides here. Chloride removal was measured as this is a necessary pre-requisite to terminal sterilisation with ethylene oxide. The results showed that the protocol removed 74.5% (range: 68.0-90.8) of the lipid content, 96.4% (range: 94.8-98.4) of the soluble protein content, 97.7% (range: 97.1-100) of the elastase content and 98.8% (range: 98.0-99.2) of the chloride ion content. We have shown that processing designed to improve the clinical efficiency and safety of bone allografts can be accomplished without compromising the structural and biological properties of the graft.

Prion protein in patients with renal failure.

We previously found elevated levels of prion protein (PrP(C)) in the blood plasma of 16 patients with renal failure. We studied a further 20 patients with renal failure, and all had a significantly higher PrP(C) concentration than healthy normal subjects (P < 0.0001). Renal dialysis did not remove plasma PrP(C) in these patients. Because dialysis patients receive heparin during dialysis, which could potentially bind to PrP(C), the concentration of PrP(C) was measured in patients receiving heparin for cardiopulmonary bypass and was found to be similar to normal controls. We also studied several other groups with chronic illnesses and found that patients with thrombotic thrombocytopenic purpura and sickle cell anaemia had normal plasma PrP(C) levels, but that those with beta-thalassaemia had slightly elevated levels of plasma PrP(C). This suggests that the observations in renal failure were not just part of a generalized response to chronic illness or acute phase reaction. The mechanism of elevated plasma PrP(C) levels in renal disease is unknown, but this shows that plasma PrP(C) is not a specific marker of neurological disease or Creutzfeldt-Jakob disease.

Extended storage of platelets in SSP platelet additive solution.

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.

Separation of whole blood into plasma and red cells by using a hollow-fibre filtration system.

BACKGROUND AND OBJECTIVES: The aim of this study was to assess the separation of whole blood into red cells and plasma by using the Sangofer device, which is a gravity-fed, hollow-fibre system. The components would then be compared with those produced by the use of more elaborate technical equipment. MATERIALS AND METHODS: Ten whole-blood units were leucoreduced by using a WBF2 filter and immediately separated into red cells and plasma by using the Sangofer blood-separation device. Red cells were stored in additive solution and tested on days 1 and 42. The plasma was assayed for levels of various coagulation factors and for markers of both coagulation and complement activation. RESULTS: The red-cell parameters were similar to those obtained when routine centrifugation methods were used. The filter did not cause haemolysis. Levels of plasma factor VIII and factor XI were lower than those seen in routinely produced leucoreduced plasma units but there was no evidence of activation of the coagulation and complement systems. CONCLUSIONS: The Sangofer device is simple and straightforward to use and eliminates the need for both centrifugation and automated separation steps during the processing of whole blood into red cells and plasma components. Minor changes are required to make the procedure easier to incorporate into routine use.

Species differences in the blood content of the normal cellular isoform of prion protein, PrP(c), measured by time-resolved fluoroimmunoassay.

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.

Serum quality: an analysis of its components.

Foetal and new born bovine sera, horse serum, human serum and human plasma, and protein solutions prepared from the by-products of human plasma fractionation have been analysed. Foetal bovine sera were found to have lower total protein (g/l) and % of gamma-globulin than the other sera studied while the potassium (mmol/1) was higher. Protease inhibitors could be detected in all specimens tested.

In vivo models of thrombogenic potential: usefulness and limitations.

The thrombogenicity of prothrombin complex concentrates (PCCs) has been known as a risk factor since their first clinical use about 30 years ago. The development of in vivo models to define the thrombogenic components in PCCs was instrumental in providing a logical basis for selecting in vitro assays to screen for the distribution of such components during the manufacture of PCCs, and to minimize their appearance in the final product. Even so, these thrombogenic components are not completely removed, as shown in our canine nonstasis model of thrombogenicity: PCCs were still found to elicit a thrombogenic response, shown by increased fibrinopeptide A, fibrin(ogen) degradation products, activated partial thromboplastin time, and decreased fibrinogen and platelet counts when clinically relevant doses were used. The new generation of high-purity factor IX (HP-FIX) concentrates differs from PCCs because these products contain only negligible amounts of clotting factors other than factor IX, lower amounts of activated clotting factors, and, in products we have assayed, no coagulant-active phospholipids. When we infused a number of HP-FIX products in the canine nonstasis model, no thrombogenic response was observed at doses considerably greater than PCC doses that did elicit a response. Likewise, HP-FIX products were much less thrombogenic than PCCs when tested in small-animal stasis and nonstasis thrombogenicity models. Small-animal models are also useful for evaluating the role of factor IXa as a potential thrombogenic contaminant of concentrates and ensuring minimal amounts in the final product. The limitations associated with extrapolating in vivo model data will be shown to be minimal if ongoing clinical studies continue to demonstrate the low thrombogenic potential of HP-FIX concentrates in humans.

Development, optimization and use of an enzyme linked immunosorbent assay (ELISA) to measure factor VIII antigen utilizing monoclonal antibodies.

An enzyme linked immunosorbent assay (ELISA) has been developed to measure VIII:Ag in plasma and concentrates. The assay utilizes two commercially available monoclonal antibodies to VIII:Ag and provides an alternative to the established immunoradiometric assay (IRMA). It has the advantage of not requiring the use of radioactive material and human antibodies. The assay sensitivity is 0.006 u/ml and the interassay coefficient of variation is 6.3%. Forty-eight samples with VIII:Ag levels ranging from 0.006 to 1.5 u/ml were assayed by both ELISA and IRMA. The coefficient of correlation between the two assays was 0.89. In addition to measuring human VIII:Ag, it is also possible to detect antigen in several animal plasma and sera.

In vitro properties of red cells prepared from half-strength citrate CPD/RAS-2 (Erythro-sol) donations in PL-146 plastic.

Whole blood donations were collected into 0.5CPD anticoagulant in PL-146 plastic. This was shown to improve the stability of plasma FVIII levels when compared with CPD. RAS-2 was used as additive and this improved the in vitro properties of the red cells, such that post processing 2,3-DPG levels were maintained for 21 days and ATP levels were maintained for 28 days. Whether or not such improvements in red cell properties yield a benefit in clinical use remains to be established.

The release of prion protein from platelets during storage of apheresis platelets.

BACKGROUND: Recent studies using a time-resolved fluoroimmunoassay method (dissociation-enhanced lanthanide fluoroimmunoassay) showed that platelets and plasma are the main reservoir of the normal isoform of cell-associated prion protein (PrPc) in human blood. The aims of the present study were to monitor PrPc levels in various fractions of apheresis platelets during storage by using the DELFIA method and to assess the association of this release with alpha-granule protein ss-thrombo-globulin and cytoplasmic LDH. STUDY DESIGN AND METHODS: Units of apheresis platelets (n = 6) were obtained from volunteer donors by the use of a cell separator and stored up to 10 days. Samples (7-9 mL) were aseptically collected from each unit on storage Days 1, 2, 3, 4, 5, 8, and 10. Platelet-poor plasma and apheresis platelets were prepared and the former split into two fractions, one centrifuged at 40,000 x g for 2 hours at 4 degrees C to remove microparticles. The spun microparticles, apheresis platelets and platelet samples, platelet-poor plasma, and high-spun plasma fractions were stored in a frozen state until they were tested. RESULTS: The results showed that the mean overall levels of PrPc throughout storage remained within 15 percent of Day 1 levels. In contrast, the mean cellular levels in platelets significantly decreased to 46 percent of Day 1 levels by Day 10 of storage (p<0.01), while the corresponding levels in plasma significantly rose as much as 329 percent (p<0.01). Moreover, although microparticle-bound PrPc was released during storage, it was increasingly superseded by soluble protein. PrPc and ss-thrombo-globulin release exhibited very similar patterns (p<0.01). In contrast, LDH showed a significant increase in high-spun plasma only toward the end of the storage period (p<0.01). CONCLUSION: These results indicate that PrPc is released from platelets during the storage of apheresis platelets and that this release is probably due mainly to platelet activation and alpha-granule release in the first few days of storage. Moreover, the released PrPc is increasingly composed of soluble proteins, as the storage period exceeds 5 days.

A potentially improved approach to methylene blue virus inactivation of plasma: the Maco Pharma Maco-Tronic system.

Plasma was subjected to methylene blue (MB) photochemical virus inactivation using the Maco Pharma Maco-Tronic system which allows three units to be illuminated together, thus reducing processing time. The plasma bag system used incorporates an integral membrane plasma filter and a dry MB pill which dissolves in the plasma to give a 1-microM concentration. There is computer-controlled processing and datalogging. In an assessment of 10 pools of Group A plasma, the losses of coagulation factors, following MB/light treatment, were 23% fibrinogen, 10% FV, 26% FVIII, 11% FIX and 13% FXI. Group O, Group B and Group AB plasmas were not tested. Von Willebrand factor (vWf) multimers showed no substantial change when treated with MB, and no losses were seen for antithrombin III (ATIII), protein C and vWf:Ag. Measurements of C3a, C5a, prothrombin fragment 1+2 and FXIIa indicated that there was no activation as a result of filtration.

Application of a time-resolved fluoroimmunoassay for the analysis of normal prion protein in human blood and its components.

BACKGROUND AND OBJECTIVES: To quantify the cellular isoform of prion protein (PrP(c)) in human blood using a new time-resolved dissociation-enhanced fluoroimmunoassay (DELFIA). MATERIALS AND METHODS: The DELFIA was optimised for human blood samples and applied to isolated cell and plasma fractions from blood donations. The physicochemical properties of PrP(c) were analysed. RESULTS: 26. 5% of blood PrP(c) was associated with the platelet fraction, 0.8% with polymorphonuclear leucocytes, 2.4% with mononuclear leucocytes, 1.8% with red cells and 68.5% with plasma (mean values from 4 processed donations). CONCLUSION: The majority of blood PrP(c) is found in the platelet and plasma compartments.