Commercially available blood storage containers.

Plastic blood bags improve the safety and effectiveness of blood component separation and storage. Progress towards optimal storage systems is driven by medical, scientific, business and environmental concerns and is limited by available materials, consumer acceptance and manufacturing and regulatory concerns. Blood bag manufacturers were invited to submit lists of the bags they manufacture. The lists were combined and sorted by planned use. The lists were analysed by experts to assess the degree to which the products attend to scientific problems. Specific issues addressed included the use of di-ethylhexyl phthalate (DEHP) as plasticizer for polyvinyl chloride (PVC) blood bags, the size, material and thickness of platelet bags, and the fracture resistance of plasma bags. Alternatives to DEHP for red blood cell (RBC) storage exist, but are mostly in a developmental stage. Plastic bags (DEHP-free, PVC-free) for platelet storage with better gas diffusion capabilities are widely available. Alternatives for plasma storage with better fracture resistance at low temperatures exist. Most RBC products are stored in DEHP-plasticized PVC as no fully satisfactory alternative exists that ensures adequate storage with low haemolysis. A variety of alternative platelet storage systems are available, but their significance - other than improved oxygen transport - is poorly understood. The necessity to remove DEHP from blood bags still needs to be determined.

Component pathogen inactivation: a critical review.

BACKGROUND AND OBJECTIVES: Pathogen inactivation (PI)-treated plasma and platelets are increasingly becoming the products of choice, where licensed. This review summarizes the clinical evidence available for licensed component PI technologies and red cell PI under development. MATERIALS AND METHODS: Available literature on licensed technologies was reviewed. RESULTS: For the plasma and platelets technologies available, evidence for the inactivation of most pathogens is good, except for certain nonenveloped viruses. Clinical trials and haemovigilance programmes suggest the observed loss of potency is of little clinical significance, with some technology-specific exceptions. Concerns over adverse toxicological effects or neoantigen formation have not been confirmed for currently licensed products. CONCLUSION: While platelet PI has been adopted to reduce bacterial contamination, the ability of PI methods to replace testing for emerging bloodborne infections, or as a substitute for selective pathogen testing, gamma-irradiation or even leucodepletion, make adoption of PI for components increasingly attractive.

Platelet concentrates from fresh or overnight-stored blood, an international study.

BACKGROUND: Whole blood and also buffy coats (BCs) can be held for a few hours or overnight before processing into blood components or platelet concentrates (PCs). Individual studies have reported a range of outcomes regarding in vitro variables for PCs prepared from fresh and stored whole blood. In this multicenter study, effects of storage of whole blood or BCs on the in vitro quality of PCs were studied. STUDY DESIGN AND METHODS: The leukoreduced BC PCs were prepared from fresh BCs (2-8 hr after collection; fresh/fresh), from BCs at 20 to 24 hours after collection (fresh/stored), or from BCs prepared from whole blood stored for 20 to 24 hours (stored/fresh). PCs were stored on a flat-bed shaker at 20 to 24°C for 7 days. PCs were tested on Days 0 (only fresh/fresh), 1, 5, and 7 for in vitro quality. There were six participating centers that tested all three conditions with n = 6 per condition. RESULTS: In comparison to fresh/stored and stored/fresh PCs, fresh/fresh PCs exhibited a lower platelet (PLT) count (Day 1-220 × 10(9) ± 70 × 10(9) vs. 324 × 10(9) ± 50 × 10(9) and 368 × 10(9) ± 56 × 10(9) PLTs/PC), lactate, pCO(2), and hypotonic shock response (HSR; Days 5 and 7; Day 7-50 ± 13% vs. 57 ± 12 and 63 ± 11%) and a higher pH, glucose, pO(2), and CD62P expression (than stored/fresh PCs only; Day 7-33 ± 10% vs. 28 ± 12 and 24 ± 11%; p < 0.05). No differences were observed for volume, swirling effect, white blood cell count, annexin V binding, or aggregation between these conditions. CONCLUSION: Based on PLT count, HSR, and PLT activation, PCs are best prepared after 20 to 24 hours hold of the whole blood or BCs.

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.

Evaluation of noninvasive methods for the estimation of haemoglobin content in red blood cell concentrates.

OBJECTIVES: Red blood cell concentrates (RCCs) are the major blood component transfused to patients. There is a great variability in patient response, depending on both the patient's blood volume and haemoglobin content in the RCC. Standardisation of transfusion practice is needed to improve the prediction of patient outcome. AIM: We hypothesise that labelling of RCCs with haemoglobin content will add possibilities for the standardisation of transfusion practice. METHODS: Data from multiple international transfusion services regarding haemoglobin content and weight or volume of RCC were collected and analysed. RESULTS: We demonstrate a strong and highly significant correlation between haemoglobin content with both weight and volume of the RCCs. A linear regression model was used to assess these relationships, and it demonstrates how haemoglobin content can be estimated for different cell production processes. CONCLUSIONS: We recommend the use of weight or volume of the RCCs as the basis of estimating haemoglobin in the RCC and postulate that this can be used in future studies to explore the effects of a haemoglobin dose-based transfusion system. As the weight - and sometimes the volume - of the blood bag is easily accessible, in contrast to direct haemoglobin measurements from each individual unit, this method is feasible and simple.

Stored red-blood-cells inhibit platelet function under physiologic flow.

BACKGROUND AND OBJECTIVES: The DiaMed Impact R tests platelet function under close to physiological flow conditions. The machine is designed to use whole blood but by adding back compatible red cells, it can be used to study stored platelet concentrates. To date, red cells ≤14 days old have been used. In this study, the effect on the assay of using red cells stored for up to 60 days was examined. MATERIAL AND METHODS: This study looked at buffy coat-derived platelet concentrates on day 2 of storage along with various stored red-blood-cells (RBC). To determine whether the age of the RBC is a factor in supporting adhesion and aggregation, platelets were assayed with either RBC stored between 2 and 60 days or with separated 'young' and 'old' red cell populations obtained using a centrifugation method and confirmed by percoll gradient analysis. RESULTS: A statistically significant difference was observed between red-blood-cells stored for ≤20 days compared with those which have been stored for 21-60 days in respect of their ability to support platelet adhesion (SC) and aggregation (AS) (P<0·01). Separating red cells by centrifugation into top (young population) and bottom (old population) showed that the effect of storage was much greater than was any difference between young and old at the individual time-points e.g. 'young' red cells from stored units were poorer at supporting platelet adhesion and aggregation than 'young' red cells from fresh units. CONCLUSION: Results suggest that the red cells should be stored for less than 21 days when using this assay. This assay may also allow assessment of red cell functionality.

A pilot study of the possibility and the feasibility of haemoglobin dosing with red blood cells transfusion.

BACKGROUND AND OBJECTIVES: Red blood cell concentrates (RBCs) are the major blood component transfused. Although the haemoglobin content is variable, the transfusion dose is prescribed as units of red cell concentrates. Thus, by chance, large volume patients may receive a low haemoglobin dose and low volume patients may be transfused with haemoglobin-rich RBCs. The aim of this study was to evaluate whether the haemoglobin increment (grams per litre) in the patient can be predicted from the haemoglobin dose (in grams) transfused, with and without correction for estimated blood volume. If this is true, it may be possible to achieve the predicted transfusion outcome by selecting RBCs for each patient. MATERIALS AND METHODS: Haemodynamically stable patients scheduled for day treatment with transfusion of RBCs were recorded. A total of 52 transfusions episodes, 27 for women and 25 for men, were recorded. Blood volumes were estimated, haemoglobin content in the RBCs was measured before transfusion, and pre- and post-transfusion haemoglobin concentrations were obtained. RESULTS: The haemoglobin content of the RBCs prepared for transfusion showed a wide range, varying from 38.7 g/unit to 69.0 g/unit. There were statistically significant correlations between haemoglobin concentration in the RBCs and haemoglobin increment in patients. CONCLUSION: Post-transfusion increment in circulating haemoglobin can be predicted from the haemoglobin content of transfused cells, but knowledge of the patient's blood volume improves the accuracy of prediction. It may be feasible to select the high haemoglobin content RBC for patients with largest blood volume and vice versa.

A report on transmissible spongiform encephalopathies and transfusion safety.

The incidence of BSE in Europe is in continued decline. At present, iatrogenic transmission from person to person is considered a serious threat to public health. This report of the International Society of Blood Transfusion Working Party on Transmissible Spongiform Encephalopathy will focus on the state of the art in relation to blood components and plasma safety. Latest information on the pathogenicity of the infectious agent, the frequency and dynamics of infection in blood and transfusion transmissibility will be documented. Preventive measures including donor deferral policies, technologies for prion removal from labile blood components and for prion detection in plasma, the absence of a sensitive and rapid reference assay able to confirm the positive results from any putative blood screening assay will be updated. At last, as many uncertainties remain and a number of assumptions await confirmation, the areas to continue to explore are listed.

Acceptability of blood and blood substitutes.

Alternatives to donor blood have been developed in part to meet increasing demand. However, new biotechnologies are often associated with increased perceptions of risk and low acceptance. This paper reviews developments of alternatives and presents data, from a field-based experiment in the UK and Holland, on the risks and acceptance of donor blood and alternatives (chemical, genetically modified and bovine). UK groups perceived all substitutes as riskier than the Dutch. There is a negative association between perceived risk and acceptability. Solutions to increasing acceptance are discussed in terms of implicit attitudes, product naming and emotional responses.

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.

The gamma-carboxy glutamic acid content of human and bovine prothrombin following warfarin treatment.

A form of prothrombin induced by Warfarin therapy, has been isolated which is adsorbed onto insoluble barium salts, but has a reduced biological activity. This protein contains, on average, seven out of a possible ten gamma-carboxy glutamic acid residues. A second form of prothrombin is also described, which is not adsorbed into barium slats, and has less than 1% the activity of the normal protein, contains only four gamma-carboxy glutamic acid residues. The significance of these results is discussed.

A variant of prothrombin induced in cattle by prolonged administration of warfarin.

Cows on long term Warfarin therapy produce a form of prothrombin which, although it binds to barium citrate, has a low biological activity. Activation experiments on this form of prothrombin show that it is only slowly converted to thrombin in the presence of Ca2+, although the thrombin produced has normal activity. Further experiments show that the Fragment 1 region of the molecule has a reduced calcium binding capacity. The results indicate the existance of a partially carboxylated form of prothrombin.

Impacts and concerns for vCJD in blood transfusion: current status.

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.

Neoantigens and antibodies to factor VIII.

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.

Reductive amination for solid-phase coupling of protein. A practical alternative to cyanogen bromide.

For coupling proteins to Sephacryl gels periodate oxidation of these gels was investigated as an alternative method to cyanogen bromide activation. Optimum conditions were studied with respect to periodate concentration, time of oxidation, pH and type of coupling buffer, concentration of protein, temperature and time of protein uptake, and protein leakage after coupling. The effects of sodium cyanoborohydride and ascorbic acid as reducing agents, and of manganese ions as a potential catalyst were investigated. Using the derived optimum conditions, stable solid-phased antibodies were produced in high yield and used to adsorb factor VIII from plasma. These gels were stable for many weeks, as was the intermediate oxidised gel. Reductive amination for coupling proteins to oxidised Sephacryl gels results in increased binding and lower leakage than is obtained with cyanogen bromide activated agarose.