Complement activation products in liquid stored plasma and C3a kinetics after transfusion of autologous plasma.

BACKGROUND AND OBJECTIVES: Keeping a small stock of liquid plasma readily available for transfusion is common practise in Sweden. We report data on complement activation markers in plasma components during storage in the liquid state and the kinetics of C3a-(desArg) after transfusion of autologous plasma with high content of C3a-(desArg). MATERIAL AND METHODS: Plasma components were prepared by apheresis or from whole blood. C3 fragments (C3a-(desArg), C3d,g, iC3), and soluble terminal complement complex (sC5b-9) were investigated. C3a-(desArg) kinetics was investigated in regular apheresis donors. RESULTS: Apheresis plasma prepared by membrane centrifugation had significantly higher level of C3a-(desArg), C3d,g and sC5b-9 from day 0 and low iC3, than plasma prepared by other methods. By storage day 7, C3a-(desArg)-levels were above the reference value in 88% of all components. After re-infusion of autologous plasma with high C3a-(desArg) content, there were rapid a(1) and a(2)-distribution followed by a slower b-elimination phase. CONCLUSION: Plasma components prepared by different methods and stored in the liquid phase differ significantly in the amount and timing of complement activation. C3a-(desArg) present in plasma is rapidly eliminated after transfusion. Autologous plasma could be used to study complement kinetics in different clinical situations.

Unexpected effects of donor gender on the storage of liquid plasma.

BACKGROUND: Swedish regulations in effect since 2006 allow the storage of plasma for transfusion up to 14 days at 2-6 degrees C and for 3 years at < or = -30 degrees C. In this study, the quality of currently used plasma components was investigated. MATERIALS AND METHODS: Plasma components, prepared from whole blood or by apheresis, either leucocyte depleted or not leucocyte depleted, were stored at 2-6 degrees C as liquid plasma or as thawed fresh-frozen plasma; 31% were from female donors. Concentration, function and activation markers of the plasma coagulation systems were investigated during storage for up to 42 days. RESULTS: Cold-induced contact activation was the dominant storage lesion, occurring earlier and at higher frequency in plasma from females. Increased kallikrein-like activity led to changes in activated partial thromboplastin time, prothrombin time, protein C and C1 inhibitor (C1INH). C1INH function dropped to 53% on Day 14 in cold-activated plasma components. CONCLUSION: Contact activation may be triggered before Day 14, especially in plasma from females, and may progress as a result of the consumption of C1INH. The data suggest that lack of cold-induced contact activation may be an important quality criterion. To achieve this, plasma from male donors could be selected for transfusion and the storage time limited to 7 days.

Oxidative stress markers in pre-uremic patients.

AIM: The present study was designed to investigate a complex of oxidative stress (OS) markers in patients with chronic renal failure (CRF) and to study the relationship between different OS markers and degree of renal failure. The following indices of OS were measured in plasma: oxidized glutathione (GSSG), reduced glutathione (GSH), total glutathione (TGSH), glutathione redox ratio (GSSG/GSH) and resistance of lipoprotein fraction to oxidation (lag phase of LPF). Baseline diene conjugation level of lipoprotein fraction (BDC-LPF), total antioxidative activity (TAA), diene conjugates (DC), lipid hydroperoxides (LOOH) and thiobarbituric acid-reactive substances (TBARS) were measured in serum. All markers in plasma and serum were measured both in patients with CRF and in healthy controls. SUBJECTS AND METHODS: Blood samples were obtained from 38 patients with CRF and from 61 healthy controls. Routine biochemical analyses were performed by using commercially available kits. RESULTS: Levels of DC, BDC-LPF, LOOH, GSSG and GSSG/GSH ratio were significantly increased and lag phase of LPF was significantly shortened in patients with CRF compared with healthy controls. Serum creatinine and urea levels correlated significantly with GSSG level and GSSG/GSH in patients with CRF. A significant inverse correlation was found between glutathione redox ratio and lag phase of LPF and between GSSG level and BDC-LPF. CONCLUSIONS: The findings suggest that renal patients are in a state of oxidative stress compared with healthy controls. The most informative indices to evaluate the degree of OS in CRF were: GSSG level, GSSG/GSH status, lag phase of LPF and BDC-LPF.

The supply of blood products in 10 different systems or countries.

Countries vary greatly in their ability to produce their own blood products including albumin and IVIgG. Part of this variability depends on the supply of plasma within the country. As has been seen most recently in the UK, the quality of the plasma and its acceptability for plasma fractionation must also be considered. Therefore concerns regarding the quality of the plasma have been added to those regarding the quantity.Only a few countries are nationally self sufficient in plasma. This has a marked effect on blood product availability and therefore the ability to treat patients. Unlike most pharmaceuticals, the plasma fractionation industry must rely, for its raw products, on plasma obtained from blood donors. As such this puts it in a potentially compromised situation since neither the supply nor the quality of the raw material can be assured and both of those will vary with time. This paper reviews the processes through which blood products are made available in 10 different systems including: Canada, England, France, Italy, Norway Scotland, Sweden, Switzerland, South Africa and USA. A series of specific questions were posed and the responses received from the various coauthors and other respondents provide comparative data on blood product availability in different areas of the world.

Stability of blood coagulation factors and inhibitors in blood drawn into half-strength citrate anticoagulant.

Drawing of blood into a citrate-phosphate-dextrose (CPD) solution with a reduced citrate concentration has been shown to improve the maintenance of coagulation factor VIII (F VIII) in plasma and to give possibilities to improve erythrocyte preservation. We studied the quality of plasma obtained from whole blood drawn under continuous mixing into CPD in which the citrate concentration was reduced by 50% (0.5CPD). The blood was stored at room temperature for 8 h before component preparation. We confirmed improved stability of F VIII by 0.5CPD. We found no clinically significant changes in inhibitors to the coagulation and kallikrein systems, and no signs of activation of these systems, during the 8-hour holding time. In control blood drawn into CPD, F VIII and coagulation factor IX decreased by 0.09 IU/ml (8%) and 0.07 U/ml (7%), respectively, otherwise we found no significant differences between 0.5CPD plasma and CPD plasma.

Freezing technique and quality of fresh-frozen plasma.

Cell-poor plasma was prepared by apheresis from 10 donors. From each donor, an amount of 200 ml was frozen rapidly to -40 degrees C in standard blood bags, and a further 200 ml was frozen slowly to -20 degrees C. Before freezing and after thawing, plasma samples were collected and frozen to -70 degrees C pending analysis. Coagulation factor VIII activity was reduced to 90% by rapid freezing and to 80% by slow freezing. Factor V was not influenced by rapid freezing, but slow freezing reduced the levels to 92% of the pre-freezing levels. In some of the plasma bags a slight increase in fibrinopeptide A occurred. However, soluble fibrin, thrombin-antithrombin complexes and spontaneous proteolytic activity were not altered by freezing. The beta-thromboglobulin increased slightly with slow freezing. Moreover, in a separate experiment, evaluating the possible effects of refreezing plasma samples, an increase in beta-thromboglobulin was also recorded, while the levels of factors VIII and V and von Willebrand factor were not affected. The changes in some variables, which were recorded in the cell-poor plasma, frozen soon after the blood donation at a slow freezing rate, must be regarded as insignificant in most clinical situations.

Blood component processing technique and plasma quality.

To maintain a closed system during the preparation of blood components, including the removal of buffy coat, many centers use a quadruple blood bag additive solution system which in this study has been reduced to a cheaper triple bag system. The buffy coat and plasma were after centrifugation transferred to the first satellite bag and, after a second spin, the plasma separated from the buffy coat was transferred to the second satellite bag and stored for a fortnight at 4 degrees C. This resulted in a statistically significant increase in platelet factor 4 and elastase activity levels. No significant changes were found in the levels of C1-esterase inhibitor and kallikrein inhibiting activity, thrombin-antithrombin complexes, soluble fibrin, fibrinopeptide A and spontaneous proteolytic activity. The changes observed must be regarded as clinically insignificant. The platelet count is low enough to meet the requirements for platelet poor plasma. Using this blood component separation technique, one can reduce the CPD/additive solution 4-pack blood bag system to a less expensive 3-pack blood bag system.

Allo-immunization during pregnancy. Clinical results from 1983 to 1989 in a Scandinavian university hospital.

From 1983 to 1989, 147,068 pregnancies were analyzed for allo-immunization against erythrocyte antigens. Approximately half of the cases were due to immunization against factor D and the others were due to allo-immunization against other antigens (K, c, E, etc.). In 61 cases exchange transfusion of the newborn was needed and in 115 cases diagnostic amniocentesis was done during pregnancy. Intrauterine transfusions were performed in 10 cases. Fetal and neonatal mortality was 4% in these moderate to severe cases, all due to immunization against D. Immunization against D was due to failure to give immunoglobulin anti-D in about 2/3 of the cases. Systematic prophylactic treatment with anti-D during pregnancy would probably not be cost-effective in this population.

A study of the effect of ABO incompatible plasma in platelet concentrates transfused to bone marrow transplant recipients.

Bone marrow transplant recipients at Huddinge Hospital have routinely received single-donor platelet concentrates (PC) from blood group O donors. These PC contain approximately 350 ml plasma, which is incompatible with patients of group A, B and AB. In 27 patients transplanted with an ABO-identical bone marrow, serological investigations have been performed every week after transplantation (median 6 weeks, range 3-14). Nine of 11 recipients with blood group A developed a positive direct antiglobulin test (DAT) after PC transfusions, while none of 15 patients of blood group O developed a positive DAT. Anti-A could be eluted in DAT-positive cases. In no case was there any clinical sign of hemolysis. Nor did recipients of groups A, B or AB (n = 34) require more red blood cells or PC transfusions compared to recipients of group O (n = 47).

The effect of different agitation modes on platelet metabolism, thromboxane formation, and alpha-granular release during platelet storage.

Platelet concentrates (PCs), prepared by plateletpheresis, were stored in aliquots in polyvinylchloride blood bags for 5 days at 22 degrees C under rapid, slow, or no agitation. Nonagitated PCs were also stored in a 98-percent oxygen atmosphere. In nonagitated PCs, pO2, lactate production, and platelet factor 4 (PF 4) concentration increased, whereas the ATP level and pH dropped rapidly. These changes were somewhat minimized in nonagitated PCs stored in oxygen. There was no significant difference between the two agitated groups. The increase in PF 4 correlated inversely to the decrease in ATP: r = -0.91, p less than 0.001, n = 24. The formation of thromboxane B2 (TxB2) after stimulation with arachidonic acid or collagen was significantly higher in slowly agitated PCs on Day 5 than on Day 0 (p less than 0.01). Nonagitated PCs produced lower levels of TxB2 (collagen stimulation) on Day 5 (p less than 0.05). In unstimulated PCs, the levels of TxB2 and ATP were inversely correlated on Day 5 (r = -0.70, p less than 0.001, n = 20). In vivo survival was performed after 72 hours of storage; mean survival (+/- SD) was 6.5 (+/- 0.3) days for nonagitated oxygenated PCs and 6.8 (+/- 0.7) days for agitated PCs. In nonagitated PCs, anaerobic metabolism increased, although oxygen diffusion through the container wall was sufficient. Agitation seems to facilitate the diffusion of oxygen through the storage medium. Nonagitated PCs were stored safely for 24 hours; this period can be extended to at least 72 hours when aerobic metabolism is maintained.

The platelet storage capability of different plastic containers.

Platelet concentrates (PC), prepared by platelet apheresis, were stored in four different types of blood bags. One of the bags, manufactured with a thinner PVC film than previously, was tested in three different bag volumes. From 25 donors a total number of 99 PC were prepared. Platelet numbers varied from 20 to 140 X 10(9) platelets per bag. The cell count, pH, pO2, pCO2 and lactate were determined initially and on days 1, 3 and 5 of storage. In a separate test, the oxygen diffusion capacity of the bags was determined by oxidation of sodium sulfite in the presence of cobaltous chloride. The oxygen diffusion capacity found was 16 (PL 732, 300 ml), 13.5 (Teruflexa 800 ml), 11.5 (PL 1240, 400 ml), 10.6 (Teruflexa 600 ml), 9 (Teruflexa 400 ml) and 4 (PL 146, 300 ml) mumol O2/h, respectively. For each bag type, the minimum and maximum platelet number stored with maintained pH levels (6.9-7.4) was defined. The maximum platelet number stored with maintained aerobic metabolism, correlated to the oxygen diffusion capacity of the bag, r = 0.998, p less than 0.001, n = 6; thus the maximum platelet number successfully stored for 5 days in each container can be predicted by determination of the oxygen diffusion capacity. In PC with a low platelet yield, pH values above 7.4 were observed after 1 and 3 days. When the results are compared with platelet yield data from routine blood banking, the optimal bags for platelet storage can be chosen. These conclusions must be further investigated in studies in vivo.

[Blood component therapy with additives]. / Komponententherapie mit Additiva.

Since 1988, 96% of all blood donations in Sweden are given in a multiple blood bag system using a SAGMAN-System. This system has many advantages over conventional blood bags without additive solution. We routinely eliminate 50-80% of the leucocytes and more than 80% of the platelets by separation of buffy-coat from red cells using such a system. We have compared the preparation results of a SAGMAN-System with a similar system containing top and bottom outlets (BAT) using 2 different semiautomatic separation systems. With these new bag systems and separation devices we received similar results. However by optimizing the procedure it should be possible to obtain a higher reduction of leucocytes and platelets at comparable red cell yield using the BAT-System.

Screening of factor VIII:C levels in blood donors.

A new chromogenic peptide substrate method, modified for assay with semi-micro tubes, Cobas Bio centrifugal analyser and microplates, was used for screening of F VIII:C in blood donors. The precision of the assays is high and the costs are reasonable. The assay with microplates is especially suitable for selection of donors for a plasma programme and for quality control in blood banks.

Activation of blood coagulation, fibrinolytic and kallikrein systems during storage of plasma.

This study deals with the question of how blood coagulation, kallikrein and fibrinolytic systems are affected by storage of plasma at +6 degrees C. Blood was collected into citrate phosphate dextrose adenine (CPD) or acid citrate dextrose (ACD) and the plasma samples were stored at +6 degrees C for 35 days. Samples were taken at weekly intervals for assays of various parameters of the different systems. No significant changes were observed in the levels of the main thrombin inhibitor, antithrombin III. At the end of the storage period, however, fibrinopeptide A levels increased markedly, particularly in the ACD plasma, indicating thrombin activation. There was no change in the plasminogen level, but a decrease in the levels of antiplasmin and urokinase inhibitors and an increase in the level of the fibrinogen degradation fragment B beta 15-42 were observed, indicating activation of the fibrinolytic system. The level of antikallikrein activity decreased sharply in ACD plasma; CPD plasma was less affected. This decrease was parallel to the increase in spontaneous proteolytic activity and correlated with the increase in fibrinopeptide A. Prolonged storage of plasma of +6 degrees C thus resulted in the activation of coagulation, fibrinolytic and kallikrein systems and decrease in inhibitors. The activation was much more pronounced in ACD than in CPD plasma.

Haemotherapy with red-cell concentrates and a new red-cell storage medium.

The effects on plasma proteins and haemostasis of haemotherapy with buffy-coat-poor red-cell concentrate and red cells suspended and stored in a new medium containing sodium chloride, adenine, glucose, and mannitol (SAGM) were studied in elective surgery. In patients with normal preoperative serum albumin levels no transfusion of plasma was necessary before 50% of blood volume had been lost. When three different types of haemotherapy were investigated in patients undergoing orthopaedic surgery, it was found that when whole blood was replaced by red-cell concentrate and, later, by red-cell suspension in SAGM medium, the peroperative bleeding pattern did not change. The volume of transfused plasma was reduced by 47% in the red-cell-concentrate series and by 72% in the red-cell-suspension series. No albumin preparations were given and the use of red cells was decreased by 26%. Haemotherapy with red cells suspended in SAGM was useful in elective surgery and saved plasma for other purposes.