Leucocyte and platelet-derived bioactive substances in stored blood: effect of prestorage leucocyte filtration

Abstract: Adverse reactions to transfusion of allogeneic blood may depend on content of leucocytes and platelets and on storage-time of the erythrocyte suspensions. Therefore, we studied the efficacy of prestorage leucocyte reduction by filtration on total content and extracellular accumulation of histamine, eosinophil cationic protein (ECP), eosinophil protein X (EPX), myeloperoxidase (MPO), plasminogen activator inhibitor type-1 (PAI-1) and interleukin-6 (IL-6) in samples obtained from 5 units of SAGM blood, 7 units of plasma-reduced whole-blood and 6 units of whole-blood before and after filtration, respectively. In addition, we analysed supernatants from the same units after storage at +4°C for 0, 21 and 35 d, respectively. The filtration was performed at room temperature within 2–4 h after donation. The substances were analysed by ELISA and RIA methods and we also analysed the donor plasma levels of the same bioactive substances. The total content of histamine, ECP, EPX, and MPO were 10–70-fold higher in all unfiltered erythrocyte products compared to donor plasma concentrations, while PAI-1 content was 15–20-fold higher only in plasma-reduced whole-blood and whole-blood. Prestorage leucocyte filtration significantly reduced the total histamine, ECP, EPX, MPO and PAI-1 content to levels similar to donor plasma levels in plasma-reduced whole-blood and whole-blood, while PAI-1 was still low in filtered SAGM blood. In addition, the levels of extracellular bioactive substances at d 0 after donation and filtration were within the range of concentrations in donor plasma, and there was no time-dependent accumulation during storage for 35 d at +4°C. IL-6 was not detected in either plasma or samples obtained from the blood bags. These results suggest prestorage leucocyte filtration to deplete leucocyte contents to levels, which prevent the previously shown time-dependent accumulation of leucocyte derived bioactive substances in various erythrocyte suspensions. In addition, the PAI-1 results suggest leucocyte filters to reduce the obligatory platelet content in whole-blood products.     

Peroxidative events in stored platelet concentrates

Changes in metabolic and functional activity of platelets stored as platelet concentrates in plastic bags highly permeable to gases were investigated. The following parameters were measured daily: pH, pO2, pCO2, HCO3, glucose, lactic acid, lactic dehydrogenase, cellular ATP and platelet aggregation induced by different agents (collagen and ADP). As indexes of lipid peroxidative damage, the cellular levels of conjugated dienes, malonyldialdehyde and some antioxidant molecules such as coenzyme Q10 and vitamin E were determined. A marked increase in pO2, conjugated dienes, malonyldialdehyde, lactic acid and lactic dehydrogenase activity was observed during the preservation. Platelet ATP content was unmodified and a remarkable decrease in platelet aggregability was found. pCO2, cyclooxygenase activity, vitamin E, coenzyme Q10, bicarbonate and glucose showed a rapid fall. Our data seem to indicate a preservation of platelet metabolic activity and a correlation between increased lipid peroxidation and functional impairement.     

Extracellular accumulation of bioactive substances during preparation and storage of various platelet concentrates

BACKGROUND: Side effects of platelet transfusion may be associated with infusion of bioactive substances. We therefore studied extracellular accumulation of histamine, plasminogen activator inhibitor (PAI)-1, vascular endothelial growth factor (VEGF), and interleukin (IL)-6 during preparation and storage of various platelet concentrates. METHODS: Twenty buffy-coat-derived platelet pools (BCPC) were prepared and stored in platelet additive solutions (PAS). Twelve apheresis platelet (APC) units were prepared using the COBE Spectra LRS, and 14 were prepared using the Fenwal Amicus Separator. After preparation half of the content was drawn from each APC unit. The normal ranges of the substances were determined in plasma from all donors, and the extracellular concentrations of the substances were determined in supernatants collected on days 0, 1, 3, 5, and 7 of storage from all platelet preparations. RESULTS: The platelet counts were not significantly different in BCPC units and APC units. The BCPC units had a significantly higher white cell count than the APC units (P < 0.0001), but the count was significantly higher in the Amicus APC units than in the COBE APC units (P < 0.0001). The extracellular histamine concentration was significantly (P < 0.001) increased in BCPC units after preparation and without further increase during storage, while there was no accumulation of histamine in APC units. After preparation the PAI-1 concentration was significantly (P < 0.02) higher in BCPC units than in APC units, but during storage PAI-1 increased significantly (P < 0.05) more in APC units than in BCPC units. Similarly, VEGF concentration was significantly (P < 0.05) higher in BCPC units than in APC units after preparation. During storage, however, VEGF increased more in BCPC units compared with COBE Spectra APC units (P < 0.05), but compared with Amicus Separator APC units only for the first 3 days of storage. At days 5 and 7 of storage the VEGF concentration was significantly higher in the Amicus APC units than in the COBE APC units (P < 0.05). IL-6 was not detectable in any of the concentrates after preparation or during storage. CONCLUSION: Platelet concentrates prepared by the apheresis method may contain less white cell derived bioactive substances than platelet concentrates prepared by the buffy-coat method. However, a substantial storage time dependent platelet derived bioactive substance accumulation takes place in all platelet concentrates tested, presumably due to platelet disintegration.     

Lipid composition of freshly prepared and stored platelet concentrates

To evaluate the possibility that changes in lipid composition might be related to the functional lesion that develops when platelets are stored as concentrates for several days, we measured lipid constituents of platelets in freshly prepared concentrates and in concentrates stored for 72 hr at 4 degrees C or at 20 degrees C under standard blood banking conditions. At 20 degrees C, but not at 4 degrees C, platelets lost about 15% of total cholesterol and 7%--11% of total phospholipid. The distribution of individual phospholipids remained unchanged. This was also true of the fatty acid distribution in total phospholipids and in individual phospholipids except for a statistically significant reduction of linoleic acid (18:2) and an increase in oleic acid (18:1) in phosphatidyl inositol (PI). Platelets collected in citrate-phosphate- dextrose (CPD) anticoagulant did not differ significantly in lipid composition from those collected in acid-citrate-dextrose (ACD) anticoagulant during the period of observation. These findings do not provide a basis to suggest that functional abnormalities developing in stored platelets are related to changes in lipid composition.     

Pre-transfusion non-invasive quality assessment of stored platelet concentrates

The Platelet Monitor System has been designed to assess routinely prepared platelet concentrates (P.C.) during storage. The system is based upon observations of changes in light transmission induced by platelets in intact packs. It is practical, provides constant gentle agitation for P.C., and non-invasively give continuous semi-quantitative evidence of the probable numbers of discoid platelets within each pack. The information yielded correlates both with established in vitro parameters of platelet behaviour, and with in vivo survival of platelets in normal healthy volunteers. The device safely provides the Blood Banker with the means to achieve on-going assessment of methods and improvements--or failings--in P.C. production. To the clinician it offers information throughout and even beyond present arbitrary storage limits, about which P.C. are likely to be of least or greatest value to a thrombocytopenic patient.     

Evaluation of biochemical parameters in platelet concentrates stored in glucose solution

Background

Therapeutic storage of platelet concentrate is a challenging problem for Transfusion Medicine, so that many studies have been carried out with the aim of improving the duration of storage of platelet concentrates. Little attention, however, has been given to the most appropriate biochemical methods for evaluating the quality of the stored platelet concentrates.

Material e methods

Platelet concentrates (n=10) were saved under gentle stirring at 22ºC for a total period of 8 days. Glucose 0.5% (w/v) was added either at the beginning of storage (time 0) or on the fifth day of storage. One millilitre of each concentrate was withdrawn at time 0 and after 5, 6, 7 and 8 days of storage for microbiological culture, evaluation of pH, lactate dehydrogenase (LDH), mean platelet volume, platelet haematocrit and analysis of metabolites of energy pathways (high energy phosphate derivatives, nucleosides, oxypurines and antioxidants) by high performance liquid chromatography.

Results

The addition of glucose 0.5% on day 5 did not produce significant differences in metabolites of energy pathways with respect to control platelet concentrates, whereas when the glucose was added at the beginning of storage (time 0) there was a recovery of ATP, GTP and a decrease of energetic catabolism, demonstrating a beneficial effect on energy metabolism. The changes in LDH values did not parallel those of the metabolites: indeed, only on day 7 of storage did the platelet concentrates treated with glucose on day 5 have significantly lower levels of this enzyme than those found in the other concentrates. The improvements produced by addition of glucose at time 0 were confirmed by morphological analyses (mean platelet volume, platelet haematocrit), and the pH.

Conclusions

The metabolic profile of glucose-enriched plasma concentrates on the fifth day of storage, and the different time course of increased LDH concentration, could represent valid parameters to interpret platelet vitality in the successive days of storage. These preliminary data also indicate that glucose might be a good additive for a new storage formulation.     

Comparison of platelet concentrates stored in plasma and in three plasmafree media

Platelet concentrates (PC) were stored for 7 days at 22°C in either CPDA-1 plasma (control) or one of three plasma-free media, namely a Tyrodes solution (BTST) fortified with citrate and with additional phosphate for buffering effect; Plasma-Lyte A solution (PCD) fortified with dextrose and citrate; and Ringers/CPD-50 (10:1) solution (RCPD) buffered with phosphate, all with a pH of 7.4. Parameters monitored included platelet count, pH, hypotonic shock response, aggregation response to single and paired agonists, and release by platelets of endogenous LDH, ATP and 5HT. BTST was shown to be a satisfactory plasma substitute over the entire storage period. PCD gave similar results over 4 days, although the decrease in pH was more marked. In all test systems RCPD was shown to be a poor plasma substitute.     

Von willebrand factor availability in platelet concentrates stored for 5 days

Von Willebrand factor (vWF) availability was assessed in platelet concentrates (PCs). After 5 days of storage, 82 ± 9% of basal levels of ristocetin cofactor activity (vWF:RCo) remained in PCs. vWF antigen (vWF:Ag) increased up to 166 ± 38% (P < 0.05) In the same period. Autoradiograph pattern of vW:Ag showed an increase in low molecular weight multimers, and fast migrating multimeric forms were visualized by crossed immunoeiectrophoresis on day 5. Studies carried out in platelet free plasma stored as PCs showed similar changes in vWF:RCo but increments in vWF:Ag were not detected. These data indicate that PCs maintain vWF:RCo levels of clinical value even after 5 days of storage and suggest that vWF comes out from platelets to plasma during storage. © 1994 Wiley-Liss, Inc.     

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.     

Post-transfusion recovery of function of 5-day stored platelet concentrates

Summary Platelets show a rapid reduction in their responsiveness to aggregating agents during storage for transfusion, but little is known about reversal of this defect in vivo after transfusion. In this study, fresh and stored platelets from the same donor ( n =12) were labelled with ¹¹¹In or ⁵¹Cr, respectively, mixed, and simultaneously infused. Blood samples were taken for up to 5 d post-infusion, and the functional behaviour of the labelled platelets ex vivo was measured by retention on glass bead columns, and by whole blood aggregability to ADP, epinephrine and ristocetin. Aggregation was determined by filtering aggregated samples through a column of cotton wool to remove the aggregates, and quantitated as per cent decrease in radioactive counts. The study showed that, although infused radiolabelled 5 d stored platelets had a significantly lower aggregability towards ADP and epinephrine immediately (1 h) after infusion (32% and 29%, respectively, of fresh platelet values), a complete restoration to fresh platelet levels was found 24–72 h post-infusion, with no further change observed over the ensuing 5 d with either fresh or stored labelled platelets. A slightly (6–9%) lower adhesion to both uncoated and collagen-coated beads was found for the stored platelets throughout the 5 d period of study post-infusion.
In conclusion, these studies show that, with ex vivo testing, platelets stored for 5 d quickly recovered adhesion and aggregability capabilities similar to that of fresh platelets, suggesting that the functional lesion developed during storage is quickly and completely reversed after infusion.     

Bioactive substances in buffy-coat-derived platelet pools stored in platelet-additive solutions

To determine if reducing the intensity of the mobilizing chemotherapy protocol used would alter the number and/or quality of the progenitors mobilized in patients with chronic myelogenous leukaemia (CML), we undertook a pilot study. 36 consecutive CML patients previously treated only with hydroxyurea were given mobilization therapy within 12 months of diagnosis. 17 patients were treated by the ICE protocol and 19 patients received the mini-ICE protocol. The leukapheresis product collected from 22/36 patients (62%) was entirely Ph-negative. The cytogenetic results between ICE and mini-ICE-treated protocols were not significant, although the reduction in median days of hospitalization required for the mini-ICE versus the ICE protocol was highly significant ( P  < 0.0001). There was no significant difference in the yield of CD34⁺ cells and CFU-GM collected. No patient in the mini-ICE protocol experienced high-grade oral mucositis and GI toxicity whereas three such cases occurred with the ICE protocol. No patient died of the mobilization procedure in either group.     

In vitro evaluation of buffy-coat-derived platelet concentrates stored in a synthetic medium

Human blood platelets, prepared by the buffy-coat method, were prepared and stored in different synthetic media. In a synthetic medium based on gluconate, acetate and citrate (GAC), the pH was 6.8 on day 6. This medium was chosen for further evaluation. The total platelet count and the leukocyte contamination were significantly lower in the platelet concentrates (PCs) prepared in GAC compared to PCs prepared in plasma. Platelets stored in plasma or in GAC were equally functional when tested for aggregation and adenosine triphosphate (ATP) secretion. Only stimuli that act through the arachidonic-acid pathway induced a lower platelet response in GAC. Platelet morphology was quantified by measuring the difference in light transmission during stirring at different rates in an aggregometer; no significant differences for platelets stored in GAC as compared to plasma were observed. Activation of platelets was measured by binding of monoclonal antibodies (McAb) against the Gp IIb/IIIa complex and against activation-dependent antigens (GMP 140 from the alpha-granules and a 53-kD glycoprotein from the lysosomal granules). There was no difference in binding of these McAb between platelets prepared and stored in plasma or GAC. We conclude that platelets prepared by the buffy-coat method and stored in GAC have the same in vitro qualities as platelets stored in plasma, except for the lower aggregation response by the arachidonic-acid pathway. This is probably due to an acetate-induced decrease in intracellular pH.     

Clinical evaluation of platelet concentrates stored for one to five days

There are no large-scale data available describing the increments obtained with platelet concentrates stored for varying durations. Platelet concentrates prepared by standard techniques were stored at 22 degrees C with horizontal agitation in PL-732 bags and administered to clinically stable, nonalloimmunized recipients known to respond well to random donor platelet transfusions. The platelet concentrates were stored in a mean volume of 65.0 mL (range 54-80 mL) with an average yield of .72 X 10(11) platelets per unit of platelet concentrates (N = 100 consecutive units). There was no significant deterioration of pH during storage. Mean corrected count increments ranged between 16,600 (N = 146 transfusions) after 1 day of storage to 13,300 (N = 34 transfusions) after 5 days of storage. Although these differences were statistically significant (P less than .003, analysis of variance), the overall deterioration in increments was quite modest. Platelet concentrates can be stored under standard conditions for 1 to 5 days with acceptable clinical results.     

Improved in vivo and in vitro viability of platelet concentrates stored for seven days in a platelet additive solution

An additive solution has been developed for storage of platelet concentrates (PC) which sustains improved in vivo and in vitro viability after 7 d of storage in second generation oxygen permeable containers. This platelet additive solution is a protein-free physiologic salt solution fortified with citrate, bicarbonate and glucose. The in vivo quality of the PC was evaluated by autologous radiolabelling with Indium-111-oxine to measure recovery and survival by multiple hit analysis. The in vitro quality was evaluated by total ATP content, hypotonic shock response and extent of shape change with ADP. Ten paired studies were performed with PC from the same donor being stored for 7 d at 22 degrees C in both CPDA-1 plasma and the additive solution. Mean recoveries and survivals were found to be substantially higher with PC stored in the additive solution than with PC stored in CPDA-1 plasma (51.0 +/- 7.8% and 144.1 +/- 15.9 h versus 36.6 +/- 10.7% and 110.4 +/- 31.6 h). The differences were statistically significant (P less than 0.001). The results of the in vitro assays described above parallelled the in vivo results, with statistically significantly superior results (P less than 0.01) for all parameters of PC stored in the additive solution. This study is the first to show that PC quality may be improved and storage extended using an additive solution.     

Comparison of posttransfusion recoveries achieved with either fresh or stored platelet concentrates

Summary The effectiveness of platelet concentrate transfusion depends on such variables as blood bag material, donor — recipient compatibility, and time elapsed between donation and transfusion. To study the latter a corrected thrombocyte increment for recovery in the recipients was evaluated with 108 platelet transfusions in 31 patients. In 83 treatment programs, the mean recovery at the one-hour post-transfusion time point was 8.6×10⁹ platelets/l with fresh platelets and 5.9×10⁹ platelets/l with stored platelets. Significantly better recovery was achieved with freshly prepared platelet over the total of platelet concentrates stored for up to 96 hours; however, if the recoveries in different patient groups given stored platelets were considered separately in terms of storage times of up to 48 h or 48–96 h, the good recovery with fresh platelets was significantly better only when compared to the older (p=0.034) but not to the younger group of stored platelets. In patients with signs indicating enhanced platelet destruction (fever, splenomegaly, disseminated intravascular coagulation) the transfusion with fresh platelet concentrates gave a significantly better recovery compared to stored platelet concentrates (p=0.028), whereas in the absence of such signs the recovery produced by fresh concentrates was not significantly higher than with stored concentrates. These findings may be relevant for the logistics in blood banking.     

Apheresis platelet concentrates contain platelet‐derived and endothelial cell‐derived microparticles

Background and Objectives Microparticles (MP) are membrane vesicles with thrombogenic and immunomodulatory properties. We determined MP subgroups from resting platelets, activated platelets and endothelial cells in donors and apheresis platelet concentrates (PC). Material and Methods MP were double stained with annexin V and CD61 (platelet‐derived MP; PMP), P‐selectin or CD63 (MP from activated platelets) and CD144 plus E‐selectin (endothelial cell‐derived MP; EMP) and detected by flow cytometry in platelet donors (n = 36) and apheresis PC (n = 11; Trima?). Results PC contained MP, mainly from resting platelets [93% (90–95)], and minor fractions of PMP from activated platelets [P‐selectin⁺ or CD63⁺; 4·8% (3·2–7·7) and 2·6% (2·0–4·0)]. Compared to donors, levels of annexin V+ MP, PMP, P‐selectin⁺ and CD63⁺ MP were 1·7‐, 2·3‐, 8·6‐ and 3·1‐fold higher in PC (all P < 0·05). During storage (1–5 days), levels of annexin V+ MP and PMP did not increase, although small increases in the fraction of P‐selectin⁺ or CD63⁺ MP occurred (both P < 0·05). PC also contained EMP, which were 2·6‐ to 3·7‐fold enriched in PC compared to donors (P < 0·05). Conclusions Transfusion of apheresis PC also results in transfusion of HLA‐carrying PMP and EMP. This might counteract the aim of reducing transfused HLA load by leucodepletion. The increases in PMP exposing P‐selectin or CD63 reflect mild platelet activation during storage. We conclude that in leucodepleted platelet apheresis using fluidized particle bed technology, MP are harvested mainly from the donor by apheresis. Improvement in apheresis technology might reduce MP load.     

Reduction of bioactive substances in stored donor blood: prestorage versus bedside leucofiltration.

Leucocyte filtration has been suggested to improve transfusion products. We studied the effect of prestorage versus bedside leucofiltration on reduction of bioactive substances and leucocyte content in donor blood. Forty-five units of whole blood from healthy blood donors were studied. Of these units, 9 were stored under standard conditions for 35 d, 9 were leucofiltered after donation and then stored for 35 d, and 3x9 units were stored for 7, 21 and 35 d, respectively, before leucofiltration. Samples were collected from blood units immediately after donation, and before and after leucofiltration, and analysed by ELISA and RIA methods for extracellular content of myeloperoxidase (MPO), eosinophil cationic protein (ECP), histamine (HIS) and plasminogen activator inhibitor-1 (PAI-1). Leucocyte content was counted in all samples. In non-filtered blood extracellular MPO, ECP, HIS and PAI-1 were accumulated in a storage time-dependent manner, while prestorage leucofiltration prevented this accumulation. Leucofiltration after storage for 7, 21 or 35 d did not significantly reduce the accumulated bioactive substances, which were similar to levels in non-filtered blood stored for the same period of time. Prestorage and bedside leucofiltration on day 7 reduced the leucocyte content to less than 0.5x10(6)/L, whereas the median content in blood stored for 21 or 35 d was only reduced to 32.0 and 52.2x10(6)/L, respectively. Prestorage leucofiltration may thus be advantageous to bedside leucofiltration.     

Stabilization of standard platelet concentrates and minimization of the platelet storage lesion by a prostacyclin analogue

Summary Platelet concentrates were pretreated with a stable synthetic prostacyclin analogue (Iloprost) at two different concentrations before the second centrifugation step (pelleting step) of preparation. This resulted in loss of platelet sensitivity to aggregating agents. To mimic the situation after transfusion and to assess the reversibility of platelet inhibition, platelets were washed during and after storage and resuspended in fresh-frozen autologous plasma. The Iloprost-treated and washed platelets exhibited an increased sensitivity to the aggregating agents, compared with the control platelets (p< 0.01). Post-storage recovery of the synergistic aggregation was more than 80% of prestorage aggregation, -thromboglobulin (TG) release and thromboxane B2 (TXB2) formation were significantly inhibited in Iloprost-treated platelets (p< 0.01). After the second centrifugation step, TG release was 0.7%±0.3%, compared with 2.7%±0.9% for the controls. TXB2 was 99±91 pg/ml, compared with 495±356 pg/ml for the controls. Platelet morphology and ultrastructure were well preserved during 5-day storage. In addition, Iloprost exerted a cytoprotective effect, as evidenced by the significant reduction in lactate dehydrogenase leakage. Post-storage LDH was 378±159 and 415±239 U/l respectively by the two Iloprost concentrations, compared with 1180±937 U/l for the control platelets. The inhibitory and cytoprotective effects of Iloprost were sustained throughout storage, in contrast to the effect of PGE1 (Prostin) which was limited to the early phase of storage.