The effect of storage time of human red cells on intestinal microcirculatory oxygenation in a rat isovolemic exchange model.

OBJECTIVE: To determine whether the storage time of human leukodepleted red blood cell concentrates compromises intestinal microvascular oxygen concentration oxygen (muPo(2)) during isovolemic exchange transfusion at low hematocrit. DESIGN: Prospective, randomized, controlled study. SETTING: University research institute laboratory. SUBJECTS: Male Wistar rats. INTERVENTIONS: Intestinal muPo(2) was determined by Pd-porphyrin phosphorescence life-time measurements. MEASUREMENTS AND MAIN RESULTS: Rats were brought near to a state of oxygen supply dependency by hemodilution with a pasteurized plasma protein solution to a hematocrit of 14.3 +/- 1.1% (n = 24). Subsequently, an isovolemic exchange transfusion with human leukodepleted red blood cells, stored for 2-6 days (fresh, n = 8), 2-3 wks (intermediate, n = 8), or 5-6 wks (old, n = 8), was performed to determine whether intestinal muPo(2) would be preserved. Immunologic reactions were avoided by washing the red blood cell concentrates three times before use. Isovolemic exchange with fresh and intermediate red blood cells maintained muPo(2) whereas old cells decreased muPo(2) with 26%. Subsequent transfusion with red blood cells (hematocrit approximately 60%) until reaching a hematocrit of 32.4 +/- 2.1 % (n = 24) increased intestinal muPo(2) in all three groups to the same extent between 28% and 32%. No changes in red blood cell deformability, as determined by a Laser-assisted Optical Rotational Cell Analyzer, could be demonstrated during 5 wks of storage. CONCLUSION: This study shows that at low hematocrit, the oxygen-delivering capacity of human red blood cells stored 5-6 wks is reduced compared with fresh cells and red blood cells stored for an intermediate period. Although red blood cells stored for 2-3 wks are completely devoid of 2,3-diphosphoglycerate, their oxygen-delivering capacity to the intestines was the same as fresh red blood cells. Our study showed that red blood cell deformability was preserved during storage, suggesting that other mechanisms may account for the observed decrease in oxygen delivery by red blood cells stored 2-3 wks.

Improved platelet compatiblity of water vapour glow discharge treated non-woven poly(ethylene terephthalate) leukocyte-reduction filters for different types of platelet concentrates.

Non-woven poly[ethylene terephthalate] (NW-PET) filter fabric, usually used for leucocyte removal of red cells, was modified by water vapour glow discharge (WVGD) treatment to improve platelet compatibility. Modified filter material was evaluated with different kinds of platelet concentrates (PCs). In addition, modified filter materials were gamma-sterilized and tested after different time intervals at different storage conditions. Modification of the filter material resulted in an improved platelet recovery after filtration of PC from 57 to about 80%. No significant difference in platelet recovery was observed when filtering either freshly prepared (79 +/- 3.5%, mean +/- SD), overnight-stored single BC-PC (78 +/- 3.3%), overnight-stored single PRP-PC (75 +/- 8.8%) or overnight-stored pooled BC-PC (79 +/- 8.9%). However, freshly prepared pooled BC-PC gave a significantly higher platelet recovery (84 +/- 3.5%). Leukocyte depletion did not differ significantly between the different types of PC. gamma-Sterilization and subsequent storage of the modified filter material for 5, 14 and 26 weeks at 20 degrees C or 37 degrees C had no significant influence on the filtration results of overnight-stored pooled BC-PC. The results of the present study show that WVGD-treated NW-PET is platelet compatible and can be used for leucocyte removal from preferably BC-PC. It can be gamma-sterilized and stored for at least 6 months prior to filtration without affecting the platelet recovery and leucocyte removal.

Increase in glycocalicin levels in platelet concentrates stored in plasma or synthetic medium for 8 days: comparison with other platelet activation markers.

BACKGROUND AND OBJECTIVES: Glycocalicin (GC) is a proteolytic fragment of GpIb and can conveniently be measured in supernatants of platelet concentrates (PCs) by means of a sandwich ELISA. Because of the convenience of the assay and easy sample storage, we tested its suitability as a sensitive platelet activation parameter during PC storage. MATERIAL AND METHODS: Filtered PCs in plasma or additive solution were made from 5 pooled buffy coats and were subsequently stored during 8 days at 22+/-2 degrees C. Correlation coefficients (r) were calculated after comparison of GC levels with platelet parameters. RESULTS: A significant increase in GC concentration was found on all subsequent sampling days. PC stored in plasma showed GC levels that correlated well with the soluble P-selectin levels (r = 0.7506), P-selectin (CD62P) expression on platelet membranes (r = 0. 8843), morphology scores according to Kunicki (r = -0.7102), lactate concentrations (r = 0.9216), glucose concentrations (r = -0.8913) and beta-thromboglobulin (beta-TG) concentrations (r = 0.8913). In PCs stored in additive solution, the correlation coefficients with these markers were 0.9209 with soluble P-selectin, 0.7161 with CD62P expression, -0.7474 with morphology score, -0.8908 with glucose concentrations, 0.8923 with lactate concentrations and 0.8908 with beta-TG concentrations. CONCLUSIONS: The GC concentration correlates well with sensitive platelet (activation) parameters, rendering it a sensitive and convenient parameter for platelet activation.

Comparison between a new PVC platelet storage container (UPX80) and a polyolefin container.

During storage of platelet concentrates (PCs), the quality of the platelets deteriorates gradually, partially dependent on gas exchange. UPX80 (JMS, Japan) 1-L platelet storage PVC containers with increased gas transport capacity were compared with 1- and 1. 5-L polyolefin (PO) containers (NPBI, the Netherlands) with filtered PCs stored either in GAC (gluconate-acetate-citrate, < 10% plasma) or in plasma, for 8 days. In total 32 PCs were made (260-330 x 109 platelets per concentrate), equally divided over different bags and storage media. During storage, gas exchange, metabolic, physical and activation parameters were measured. No consistent differences for all parameters were observed between UPX80 and PO containers (1-L or 1.5-L). Blood gas parameters indicated better gas exchange for UPX80 containers compared with PO containers. Good morphology was observed in UPX80 and metabolic functions were not significantly different compared with PO containers. During prolonged storage (after day 6), some significant differences in CD62P and CD63 expression were found, indicating a higher degree of platelet activation in UPX80 containers, especially in GAC. UPX80 PC containers are suitable for storage of PCs. Although in UPX80 better gas exchange is demonstrated, as compared with PO containers, this does not improve the platelet quality during storage for 6 days, indicating that gas exchange above the level of PO containers has no effect on the switch to aerobic metabolism in platelets.

Soluble P-selectin as parameter for platelet activation during storage.

Platelet concentrates stored at room temperature deteriorate. The so-called storage lesion is characterised by morphological changes and a loss of functionality. To find an assay for early platelet activation in platelet concentrates the morphological score, beta-TG release and P-selectin expression were determined, and compared with the amount of soluble P-selectin. An ELISA was used to quantify soluble P-selectin in the storage medium. We found a significant correlation between the amount of soluble P-selectin and the percentage of P-selectin positive platelets (flow-cytometric analysis) (r = 0.7449; p < 0.0001) or the amount of beta-TG release (r = 0.6837; p < 0.0001). The morphological score also correlated significantly (negative) with the amount of soluble P-selectin (r = -0.7669; p = 0.0002). From day 0 till day 8, the amount of soluble P-selectin increased constantly from 219 +/- 49.2 ng/ml to 556 +/- 102.3 ng/ml. The detection of soluble P-selectin can be used to quantify activation of platelets during storage. The immuno-assay for soluble P-selectin is more sensitive than flow-cytometric analysis of the percentage of P-selectin-positive cells and allows earlier detection of platelet activation.

Pooled platelet concentrates prepared by the platelet-rich-plasma method and filtered with three different filters and stored for 8 days.

Pooled platelet concentrates (PC) prepared by the platelet-rich plasma (PRP) method were filtered with three different filters and stored for 8 days at room temperature. The effect of filtration on leukocyte contamination, platelet concentration, and the in vitro function, morphology, metabolism and activation of platelets were studied. Eight pools of 20 PRP-PC were used, each pool was split into 4 equal volumes; 3 were filtered over a PL50HF, a PL-10A and a Bio P10 filter, the 4 served as a control. After filtration, leukocyte counts exceeded 3 x 10(5) in none of the pooled PC. Platelet loss induced by filtration was about 17%. During storage, no differences in pH, PCO2, and lactate and glucose concentration were found between the filtered and the unfiltered units, nor were any differences observed between filtered and unfiltered pooled PC in aggregation upon stimulation with collagen and/or ADP, adhesion capacity to collagen in flowing blood, nucleotide content of the platelets and nucleobase concentration in the plasma, expression of activation-dependent antigens, or platelet morphology as observed by light microscopy and by the swirling effect. Selective removal of beta-thromboglobulin (22%) by the PL50HF filter was observed. Pooled PC prepared by the PRP-method can be filtered and stored for 8 days without detrimental effect on platelet function, metabolism or activation.

The platelet adhesion capacity to subendothelial matrix and collagen in a flow model during storage of platelet concentrates for 7 days.

The influence of storage of platelet concentrates (PC) on the adhesion capacity of platelets was studied. Twenty-four PC, 12 prepared by the buffy coat (BC) method and 12 by the platelet-rich plasma (PRP) method, were stored for 7 days at room temperature. On days 1, 3 and 7 of storage, the platelet adhesion capacity to subendothelial matrix (SEM) and collagen was studied in a rectangular perfusion system under flow conditions in conjunction with the platelet aggregation capacity after stimulation and the adenine nucleotide content. The platelet adhesion capacity to collagen was constant until day 3 of storage and decreased to about 80% of the starting value on day 7 of storage. The adhesion capacity to SEM, however, had already decreased on day 3 to about 75% of the value of day 1 and was even more decreased on day 7 to about 45% of the starting value. On day 1, platelets prepared by the BC method displayed a higher adhesion capacity to collagen and a higher aggregation capacity after stimulation by collagen alone or in combination with ADP, compared to platelets prepared by the PRP method. No other significant differences in adhesion or aggregation capacity were observed between the PC prepared by the two different methods. Both platelet adhesion and aggregation response decreased during storage, as did the total adenine nucleotide content. This study shows that platelet function, as measured by the aggregation and adhesion capacity, of platelets prepared by the PRP method is more severely impaired during the first 3 days of storage as compared to the function of platelets prepared by the BC method.

In vitro evaluation of platelet concentrates, prepared from pooled buffy coats, stored for 8 days after filtration.

BACKGROUND: Posttransfusion complications can be prevented by pretransfusion removal of donor white cells from platelet concentrate. The filtration used for this removal seems to have little effect on platelet function and activation, but more information is needed on its effect on function during subsequent long-term storage of concentrate. STUDY DESIGN AND METHODS: The effect of prestorage filtration of buffy coat-prepared platelet concentrates (PCs) on platelet function, metabolism, and activation was investigated. A pool of three PCs, each made of four buffy coats, was split into three equal volumes; two were filtered over two different filters and the third served as a control. Variables monitored immediately after filtration and during the subsequent 8-day storage period at 22 degrees C included aggregation upon stimulation with collagen and/or ADP, platelet adhesion capacity to collagen and fibrinogen in flowing blood, nucleotide content of and nucleobase release by the platelets, expression of activation-dependent antigens, and beta-thromboglobulin release by the platelets. RESULTS: No differences were observed between the PCs filtered over two different filters and the nonfiltered control PCs immediately after filtration and during storage, except for a selective removal (20%) of beta-thromboglobulin by one filter. CONCLUSION: PCs prepared from a pool of four buffy coats can be filtered and subsequently stored for 8 days (starting +/- 24 hours after whole blood collection) without detriment to platelet function, metabolism, or activation.

Platelets stored for 6 days in a polyolefin container in a synthetic medium with minimal plasma carry-over.

Platelet concentrates (PC) were stored for 6 days in either polyolefin (PO) or polyvinylchloride/di-(2-ethylhexyl)phtalate (PVC/DEHP) bags in 100% plasma or in a synthetic medium with 35 or 10% plasma. For all conditions studied the usual in vitro parameters were well maintained, with a pH above 6.8. In both bag types platelets can be satisfactorily stored for 6 days in a synthetic medium with minimal amounts of residual plasma. For this medium, the PO bag offers a slight advantage with respect to the preservation of platelet ATP content (> 80 versus > 70% in the PVC bags) and aggregation and adhesion capacity. The adhesion capacity increased in the PO bags, while it decreased in the PVC bags.

Stored platelets release nucleotides as inhibitors of platelet function.

It is well known that the function of platelets decreases progressively during storage of platelet concentrates at room temperature. To investigate this phenomenon in more detail, we have resuspended platelets that had been stored for 24 h or 72 h in fresh plasma, and we have measured the aggregation response and the ATP secretion. Conversely, the effect of plasma in which platelet concentrates (PC) had been stored for 24 h or 72 h, was tested on fresh platelets. Both the aggregation response to collagen and ADP and the collagen-induced ATP secretion of stored platelets partially recovered after incubation with fresh plasma (p < 0.05). The same parameters measured with fresh platelets incubated in stored PC-plasma were found to be significantly reduced in comparison with the response of fresh platelets in fresh plasma (p < 0.05). Finally, platelets were stored in a plasma-free medium, suitable for platelet storage and the supernatant was tested. This supernatant inhibited the function of fresh platelets in a storage time-dependent fashion. Boiling of these supernatants did not change the inhibiting capacities, whereas filtration over active charcoal did. Analysis of this supernatant revealed AMP and diadenosine tetraphosphate, which both inhibit platelet function. These data show that stored platelets release nucleotides that inhibit platelet function in a reversible manner. This phenomenon may contribute to the decrease of platelet function during storage and the recovery of platelet function after transfusion.

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.

Platelet activation during preparation of platelet concentrates: a comparison of the platelet-rich plasma and the buffy coat methods.

The activation of platelets during the preparation of platelet concentrates (PCs) by two methods was compared. To eliminate interdonor differences, 2 units of whole blood were pooled and subsequently divided into two batches. From one batch, the platelets were harvested as pelleted platelets from platelet-rich plasma (PRP) and from the other as nonpelleted platelets from the buffy coat (BC). The activation of platelets in these PCs was studied immediately after preparation and during storage for up to 9 days at 22 degrees C with gentle agitation. The binding of monoclonal antibodies (MoAbs) against the GP IIb/IIIa complex and against activation-dependent antigens (GMP 140 from the alpha granules and a 53-kDa glycoprotein from the lysosomal granules) was measured. Beta-thromboglobulin (beta-TG) release was also determined. Disc-to-sphere transformation was quantitated by measuring on an aggregometer the difference in light transmission during stirring at different rates and also by light microscopy. Immediately after preparation, platelets derived from PRP had a more spheric morphology (p less than 0.01), had a higher beta-TG release (p less than 0.01), bound more MoAbs against GP IIb/IIIa (p less than 0.01), and expressed more GMP 140 and 53-kDa glycoprotein (p less than 0.01) than did BC-derived platelets. However, these differences had disappeared after 2 days of storage. It was concluded that, immediately after preparation, PRP-derived platelets are more activated than BC-derived platelets. This is most likely a result of the pelleting that follows the second high-speed centrifugation of the PRP.

Depletion of dense granule nucleotides during storage of human platelets.

The energy metabolism of human platelets was studied during storage of platelet concentrates. The platelets were prepared from buffy coats in PVC/DEHP bags and stored for 7 days at room temperature at a concentration of 1.0 x 10(9)/ml with horizontal agitation. The total amount of ATP and ADP decreased with 40% during this storage. This decrease correlated with the disc-to-sphere transformation associated with the loss of platelet viability. During storage, the ability to incorporate 3H-adenosine into metabolic ATP and ADP (45 min at 37 degrees C) decreased with 50%. Via measurement of the specific activity of actin-bound ADP and the amount of incorporated radioactivity into total ATP and ADP, we calculated the content of the metabolic and storage pools of ATP and ADP. The results indicate that the decrease in adenine nucleotide levels during storage was mainly caused by a depletion of ATP and ADP from the storage pool, whereas the metabolic pool remained nearly intact. After 7 days, the ATP:ADP ratio of the storage pool had decreased from 1.0 to 0.3, indicating hydrolysis of ATP. Diadenosine-triphosphate and diadenosine-tetraphosphate (present in the storage pool) decreased with only 30%, and the serotonin content remained nearly constant. Therefore, it is unlikely that the storage pool was completely secreted. Probably, the storage pool of nucleotides serves as an internal supply for maintaining the contents of the metabolic pool of ATP and ADP during storage of platelets.