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.

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.

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.

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.

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.

Use of the DiaMed Impact R to test platelet function in stored platelet concentrates.

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.

In vitro amplification and detection of variant Creutzfeldt-Jakob disease PrPSc.

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).

Calcium-sensitive mitochondrial membrane potential in human platelets and intrinsic signals of cell death.

The mechanisms involved in storage-induced damage in platelets are not well understood, but membrane signalling via Ca2+ ion flux may affect mitochondrial H+ gradients and metabolism and the intrinsic pathways of cell death, platelet survival and function. In this study, the effects of blood bank storage conditions, including reduced plasma concentration and interrupted agitation, were evaluated in platelets from 136 healthy donors. Mitochondrial membrane potential (DeltaPsim), an indicator of intrinsic cell death, and its sensitivity to Ca2+ ionophore A23187, were monitored using JC-1 by flow cytometry and fluorescence microscopy. Platelet survival was examined using lactate dehydrogenase release, annexin V binding and caspase-3/7 activity. Decreased plasma concentration and interrupted agitation affected DeltaPsim and caspase-3/7. Over 7 days in 30% plasma DeltaPsim showed a significant reduction (86.3 +/- 1.1% platelets with polarised mitochondria day 1; 79.9 +/- 2.1% day 5; 75.1 +/- 3.8% day 7, P = 0.01 day 1 vs. day 7). Whilst DeltaPsim in agitated platelets in 100% plasma was unchanged up to day 7, interruption of agitation was associated with a 44% reduction in the proportion of platelets with polarised mitochondria after 5 days (56 +/- 11%). The Ca2+ sensitivity of DeltaPsim changed earlier: 5 microM A23187 caused a 20-30% change in the fraction of platelets with polarised mitochondria by day 5. Ca2+ sensitivity also increased during interrupted agitation and reduced plasma concentration. DeltaPsim also correlated with indicators of platelet death, caspase-3 activity and annexin V binding (correlation coefficients of 0.8). In conclusion, changes in Ca2+-sensitive DeltaPsim are involved in the initiation of storage-induced cell death signals that influence platelet count and function in vivo.

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.

Cryoprecipitate prepared from plasma treated with methylene blue plus light: increasing the fibrinogen concentration.

When cryoprecipitate is prepared from plasma which has been treated with methylene blue plus light (MB) for the purpose of virus inactivation, clottable fibrinogen content is 40% lower compared with units prepared from untreated plasma. Initial studies showed that when frozen MB plasma units were removed to +2 to +6 degrees C for 4 h and then returned to -40 degrees C prior to cryoprecipitation, fibrinogen recoveries increased from 24 to 42%. Although fibrinogen yield improved when plasma units were stored at +2 to +6 degrees C for varying lengths of time, FVIII levels decreased with increasing time. Conditioning for 8 h was studied in more detail. Groups of two plasma units were mixed together, divided into two equal units, frozen/thawed and treated with MB. One of each pair was stored continually at -40 degrees C, whereas the other was removed to +2 to +6 degrees C for 8 h. Samples were assayed for fibrinogen, FVIII, VWF:Ristocetin cofactor activity (RCo), VWF:Ag and VWF:Collagen binding (CB). The cryoprecipitate fibrinogen content increased to a mean of 207 mg unit(-1). VWF:Ag, VWF:RCo and VWF:CB recoveries also increased. FVIII recovery decreased from 50 to 45% (mean 124 iu unit(-1)). Conditioning has been validated for routine production of cryoprecipitate from imported plasma.

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.

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.

Coagulation factor content of cryoprecipitate prepared from methylene blue plus light virus-inactivated plasma.

Levels of factor VIII (FVIII) and fibrinogen were assessed in control cryoprecipitate and cryoprecipitate prepared in two centres from plasma subjected to methylene blue (MB) photochemical virus inactivation. The level of coagulation FVIII activity was reduced in plasma by approximately 30% after MB photoinactivation, with only 44% (centre A) and 31% (centre B) of units meeting the current UK specification of 0.7 iu/ml. A revised specification of 0.5 iu/ml is suggested. Losses of less than 11% were seen for von Willebrand factor (VWF)-related activities. Cryoprecipitate prepared from group O or group A MB-treated plasma contained 27-40% less FVIII than control units. This reflected the lower levels in MB-treated plasma. The concentrating power of the cryoprecipitation process was not reduced for FVIII or fibrinogen in MB-treated units. MB cryoprecipitate from centre A still met the UK guideline specification for FVIII and fibrinogen content, whereas at centre B only 62.5% of the group O cryoprecipitates contained > 70 iu FVIII/unit. This may reflect the lower product volume and lower FVIII content of group O plasma used at centre B and suggests that maintenance of total coagulation factor recovery in MB-treated cryoprecipitate will require the higher product volume.

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.