Standard Blood Bank Conditions Research Articles

Platelet activation and apoptosis are different phenomena: evidence from the sequential dynamics and the magnitude of responses during platelet storage

Platelet activation and apoptosis are different phenomena: evidence from the sequential dynamics and the magnitude of responses during platelet storage

The level of matrix metalloproteinase‐2 in donor plasma strongly correlates with post‐transfusion recovery of stored platelet concentrates

A simple test accurately predicting post-transfusion recovery of stored platelet concentrates (PCs) is needed. Matrix metalloproteinase-2 (MMP-2) was determined in fresh-frozen plasma of normal donors using enzyme-linked immunosorbent assay (ELISA)-based assay. Post-transfusion recovery of human PCs was determined in rabbits with an inhibited reticuloendothelial system by flow cytometry. We demonstrated a strong positive (r = 0.91) and highly significant (P = 0.0015) correlation between the concentration of MMP-2 in plasma of normal donors and post-transfusion recovery of PCs stored for 5 days at 22 degrees C under standard blood banking conditions. A higher level of donor plasma MMP-2 is associated with improved recovery of transfused platelets. These observations suggest that pre-transfusion MMP-2 assay of donor's plasma may be a predictor of post-transfusion recovery of PCs. However, the usefulness of this assay should be elucidated in clinical transfusion settings.

Interruption of agitation of platelet concentrates: a multicenter in vitro study by the BEST Collaborative on the effects of shipping platelets

Transported platelets (PLTs) are not under continuous agitation. The aim of this study was to determine whether PLTs shipped between 24 and 48 hours would be able to maintain a pH(22 degrees C) value of 6.5 at the end of 7 days of storage. Six laboratories prepared leukoreduced PLTs. PLT pools were divided into low and high PLT concentration with paired shipped (20-43 hr) and unshipped controls. Units were under continuous agitation at 22 +/- 2 degrees C when not being transported. In vitro measures including pH, pO(2), and pCO(2) were determined over 7 days. Ninety-two PLT components from 24 pools were eligible for analysis. One unshipped control and three shipped products failed to maintain a pH(22 degrees C) value of 6.5 through 7 days. In vitro characteristics were maintained slightly better over 7 days of storage in the unshipped control arms. PLT concentration, shipping time, and their interaction were significant determinants of the final pH at the end of storage (p < 0.05). Lactate generation rate increased by 35 +/- 2 (mean +/- SE) micromol per 10(12) PLTs per hour over baseline during shipping (p < 0.0001). After restoration of standard blood banking conditions with agitation, this rate dropped 24 +/- 2 micromol per 10(12) PLTs per hour (p < 0.0001). PLTs in plasma shipped for at least 20 to 24 hours maintain a pH(22 degrees C) value of 6.5 for 7 days. A longer shipping time may result in a pH(22 degrees C) value of 6.5. During shipping, glycolysis was up regulated in these PLTs resulting in increased lactic acid production. After restoration of agitation, shipped products down regulated glycolysis. These effects should be accounted for in the development of PLT storage and transportation systems.

Thrombin Is Able To Trigger Apoptosis of Human Platelets.

Although primarily known as a coagulation factor and as an inducer of platelet activation and aggregation, thrombin can modulate apoptosis in nucleated cells. Over the last decade, it has been recognized that apoptosis occurs not only in nucleated cells but also in anucleated cytoplasts and platelets. The current study investigated whether thrombin can impact apoptosis in anucleated human platelets. Using flow cytometry, we studied platelet apoptosis at the single cell level, analyzing markers of mitochondrial and cytoplasmic apoptosis (Leytin et al, Biochem Biophys Res Commun 320:303, 2004; Leytin et al, Br J Haematol 133:78, 2006). Western blotting was also employed, in addition to flow cytometry, for determining the expression of proapoptotic Bax and Bak proteins. We found that, in comparison to untreated platelets, human alpha-thrombin (1 U/mL) significantly induced four key manifestations of platelet apoptosis: (i) mitochondrial inner transmembrane potential depolarization (P<0.01), (ii) expression of pro-apoptotic Bax (P=0.002) and Bak (P=0.04) proteins, (iii) caspase-3 activation (P=0.0009), and (iv) phosphatidylserine (PS) exposure (P<0.0001). We also compared the magnitude of thrombin effects with those of A23187 and in vitro platelet storage under standard blood banking conditions. We demonstrated that the maximal level of both caspase-3 activation and PS exposure is achieved in A23187-stimulated platelets, indicating that A23187 is a useful positive control for quantifying these apoptosis events. Thrombin triggered caspase-3 activation to a level equal to that in A23187-induced platelets and significantly higher than in platelets stimulated with control buffer (P<0.001) and stored for 0, 6 (P<0.001) and 13 days at 22°C (P<0.05). PS exposure was also markedly enhanced in thrombin-stimulated platelets resulting in increase of annexin V-positive cells from 1.2 ± 0.1% to 21.2 ± 2.5% (P=0.0002); platelet storage increased annexin V-positive cells from 1.4 ± 0.4% (Day 0) to 6.0 ± 0.6% (Day 6, P=0.006) and 47.6 ± 5.6% (Day 13 platelets, P=0.0013) and much higher PS exposure was observed with 10 μM A23187 (97.8 ± 0.4%, P<0.0001). Thus, PS exposure induced by 1 U/mL thrombin is significantly higher than in platelets stored for 6 days (P<0.001), but lower than in 13 day-old platelets (P<0.001) and A23187-stimulated platelets (P<0.0001). This study demonstrates that, aside from its ‘classical' function as a coagulation factor and an inducer of platelet activation, thrombin can trigger platelet apoptosis. Thrombin appears to trigger platelet apoptosis by impacting on several intracellular apoptotic targets, including shifting the balance between Bcl-2 regulatory proteins in a pro-apoptotic direction, depolarizing the inner mitochondrial membrane, activating the executioner caspase-3, and stimulating aberrant exposure of phosphatidylserine on the platelet surface. Thrombin-induced platelet apoptosis may contribute to the pathophysiology of thrombocytopenia in diseases associated with enhanced thrombin generation, such as sepsis and disseminated intravascular coagulation.

Platelet Apoptosis Events Can Be Triggered by Shear Stresses and Chemical Agonists.

Apoptosis, or programmed cell death, is appreciated as the main physiologic mechanism that regulates cell life-span and serves for controlled deletion of unwanted cells. Since its discovery in 1972, apoptosis was long attributed exclusively to nucleate cells. It took more than 20 years to recognize apoptosis in enucleated cells cytoplasts and anucleate platelets. During the following years, apoptosis has been demonstrated in platelets treated with natural and artificial agonists, in platelet concentrates aged during storage under standard blood banking conditions, and in animal models of suppressed thrombopoiesis and thrombocytopenia. Other studies documented that mechanical forces (shear stresses) stimulate platelet activation and signaling in the absence of exogenous chemical stimuli. We analysed whether shear stresses can trigger platelet apoptosis, a question that has not yet been studied. Using a cone-and-plate viscometer (CAP-2000, Brookfield Engineering Labs, Inc., Middleboro, MA), we exposed human platelet-rich plasma to different shear stresses, ranging from physiologic arterial and arterioles levels (10-44 dynes/cm2) to pathologic high levels (117–388 dynes/cm2) occurring in stenosed coronary, peripheral or cerebral arteries. We found that pathologic shear stresses induce not only platelet activation (P-selectin upregulation and GPIbα downregulation) but also trigger apoptosis events, including mitochondrial transmembrane potential depolarization, caspase 3 activation, phosphatidylserine exposure, and platelet shrinkage and fragmentation into microparticles, whereas physiologic shear stresses are not effective. Platelets subjected to pathologic shear stresses are characterized by impaired platelet function as shown by the absence of ADP-induced platelet aggregation. Apoptosis changes were also induced by the treatment of platelets with calcium ionophore A23187 (10 μM) and thrombin (1 U/mL). Thus, in the present work, we have demonstrated that platelet apoptosis can be induced by chemical stimuli and by mechanical rheological forces (pathologic high shear stresses). Most of shear-induced activation and apoptosis events occur inside of the platelet, including translocation of CD62 from α-granules to the platelet surface, depolarization of mitochondrial inner membrane potential, activation of cytosolic enzyme caspase 3, and translocation of phosphatidylserine from the inner to the outer plasma membrane leaflet. These data suggest that the effects of shear stress on platelet activation and apoptosis are mediated by mechanoreceptor(s) that transmit activation and apoptosis signals to the cell interior. The platelet paradigm of apoptosis induced by chemical agonists and shear stresses suggests that apoptotic cytoplasmic machinery may function without nuclear participation.

Chemical Agonists and High Shear Stress Induce Apoptosis in Human Platelets.

Apoptosis, or programmed cell death, is appreciated as the main physiologic mechanism that regulates cell life-span and serves for controlled deletion of unwanted cells. Since its discovery in 1972, apoptosis was long attributed exclusively to nucleate cells. It took more than 20 years to recognize apoptosis in enucleated cells cytoplasts and anucleate platelets. During the following years, apoptosis has been demonstrated in platelets treated with natural and artificial agonists, in platelet concentrates aged during storage under standard blood banking conditions, and in animal models of suppressed thrombopoiesis and thrombocytopenia. Other studies documented that mechanical forces (shear stresses) stimulate platelet activation and signaling in the absence of exogenous chemical stimuli. We analysed whether shear stresses can trigger platelet apoptosis, a question that has not yet been studied. Using a cone-and-plate viscometer (CAP-2000, Brookfield Engineering Labs, Inc., Middleboro, MA), we exposed human platelet-rich plasma to different shear stresses, ranging from physiologic arterial and arterioles levels (10–44 dynes/cm2) to pathologic high levels (117–388 dynes/cm2) occurring in stenosed coronary, peripheral or cerebral arteries. We found that pathologic shear stresses induce not only platelet activation (P-selectin upregulation and GPIb-alpha downregulation) but also trigger apoptosis events, including mitochondrial transmembrane potential depolarization, caspase 3 activation, phosphatidylserine exposure, and platelet shrinkage and fragmentation into microparticles, whereas physiologic shear stresses are not effective. Platelets subjected to pathologic shear stresses are characterized by impaired platelet function as shown by the absence of ADP-induced platelet aggregation. Apoptosis changes were also induced by the treatment of platelets with calcium ionophore A23187 (10 μM) and thrombin (1 U/mL). Thus, in the present work, we have demonstrated that platelet apoptosis can be induced by chemical stimuli and by mechanical rheological forces (pathologic high shear stresses). Most of shear-induced activation and apoptosis events occur inside of the platelet, including translocation of CD62 from alpha-granules to the platelet surface, depolarization of mitochondrial inner membrane potential, activation of cytosolic enzyme caspase 3, and translocation of phosphatidylserine from the inner to the outer plasma membrane leaflet. These data suggest that the effects of shear stress on platelet activation and apoptosis are mediated by mechanoreceptor(s) that transmit activation and apoptosis signals to the cell interior. The platelet paradigm of apoptosis induced by chemical agonists and shear stresses suggests that apoptotic cytoplasmic machinery may function without nuclear participation.

Transfusion of Red Blood Cells under Shock Conditions in the Rat Microvasculature.

Background RBC transfusion is the mainstay of therapy for resuscitation of patients with hemorrhagic shock. Ex-vivo storage of RBCs for transfusions is associated with a storage lesion which decreases erythrocyte deformability and increases their adhesiveness to vascular endothelium. This may impair microcirculatory flow with deleterious effects on oxygen delivery post-transfusion. The objective of this study was to determine whether transfused stored rat blood impairs the microcirculation following resuscitation from hemorrhagic shockMethods Donor Sprague Dawley rat RBCs were collected and stored in CPDA-1 under standard blood bank conditions. RBCs were labelled with FITC (fluorescein isothiocyanate) 24 hours before the experiment. RBCs stored for less than 24 hours (fresh) RBCs (n=3) were compared to RBCs stored for 7 days (n=3). To induce shock, forty percent of blood volume was removed from male Sprague Dawley rats. Animals were then resuscitated by injecting 1.5mL of labelled packed RBCs and sufficient saline to restore blood volume via arterial line. The number of adherent labelled RBCs in the extensor digitorum longus muscle (EDL) was detected using intravital video microscopy (20X magnification). RBC velocity, capillary diameters, capillary Hct (hematocrit), arterial Hct, and lactate data was collected at three time points (baseline, shock and resuscitation).Results Lactate levels increased significantly during shock in both the fresh and stored groups. Arterial Hct decreased in shock and was restored to baseline levels with resuscitation. The stored group had an increased number of stopped cells in the microcirculation compared to their fresh counterparts (p=0.004). There was no statistical significant difference in capillary diameters between groups or time points. Both groups showed an increase in RBC velocity from baseline to resuscitation (stored group doubled, fresh group tripled). Capillary Hct did not change in the fresh group but doubled in the old group from baseline to resuscitation.Conclusion In our shock model, transfusion of stored RBCs resulted in an increased number of stopped cells and lower RBC velocity in capillaries as compared to fresh RBCs. Stored RBCs also had an increased capillary Hct from baseline to resuscitation and a diminished hyperemic response as compared to fresh RBCs. We conclude that stored RBCs following transfusion behave differently than native or fresh transfused cells in the microcirculation. Further studies are needed to determine the impact of these microcirculatory changes on oxygen availability in transfusion.

Platelet apoptosis in stored platelet concentrates and other models

Apoptosis or programmed cell death was discovered in nucleate cells 30 years ago and has been well documented. In contrast, apoptosis in anucleate platelets has only a five-year research history and as yet but few publications related to it. In this review, we will present the data on platelet apoptosis in several models. These include in vitro models where platelet apoptosis was induced by calcium ionophores, natural platelet agonists, storage in capped tubes at 37 °C and storage at room temperature under standard blood banking conditions, and in vivo models where apoptosis was provoked by suppression of thrombopoiesis, malaria infection and injection of tumor necrosis factor or anti-platelet antibodies. Understanding of platelet apoptosis and its role in the platelet storage lesion is an exciting challenge; future research is likely to provide us with further insight into this field.

Cell age-related monovalent cations content and density changes in stored human erythrocytes

Conversion of erythrocyte membrane protein 4.1b to 4.1a occurs through a non-enzymatic deamidation reaction in most mammalian erythrocytes, with an in vivo half-life of approximately 41 days, making the 4.1a/4.1b ratio a useful index of red cell age [Inaba and Maede, Biochim. Biophys. Acta 944 (1988) 256–264]. Normal human erythrocytes distribute into subpopulations of increasing cell density and cell age when centrifuged in polyarabinogalactan density gradients. We have observed that, when erythrocytes were stored at 4°C under standard blood bank conditions, the deamidation was virtually undetectable, as cells maintained the 4.1a/4.1b ratio they displayed at the onset of storage. By measuring the 4.1a/4.1b values in subpopulations of cells of different density at various time points during storage, a modification of the normal ‘cell age/cell density’ relationship was observed, as erythrocytes were affected by changes in cell volume in an age-dependent manner. This may stem from a different impact of storage on the imbalance of monovalent cations, Na + and K +, in young and old erythrocytes, related to their different complement of cation transporters.

Accumulation of the neutrophil-derived protein YKL-40 during storage of various blood components.

Post transfusion infectious complications associated with allogeneic blood components may depend on storage time and may be related to extracellular accumulation of bioactive substances during storage. YKL-40 is a glycoprotein located in the specific granules of the neutrophils. While exocytosed it may play a role in inflammation and remodelling of the extracellular matrix. We studied the potential accumulation of YKL-40 in blood components during storage. Using a RIA method extracellular accumulation of YKL-40 was determined in supernatants from whole blood, plasma-reduced whole blood, buffy-coat-depleted SAGM (saline-adenine-glucose-mannitol) blood, whole blood leukocyte depleted by prestorage filtration, and whole blood leukocyte depleted by bedside filtration. The blood was donated by volunteer, healthy blood donors, and stored under standard blood bank conditions for 35 days. Extracellular accumulation of YKL-40 increased significantly in a time-dependent manner during storage for 35 days of non-filtered whole blood, plasma-reduced whole blood, and SAGM blood, respectively. Prestorage leukocyte depletion of whole blood prevented extracellular YKL-40 accumulation, while YKL-40 accumulation was not reduced by bedside leukocyte depletion. YKL-40 appears to accumulate extracellularly in a time-dependent manner in standard erythrocyte components. Prestorage leukocyte depletion by filtration of whole blood may be an effective procedure to prevent extracellular YKL-40 accumulation during storage of erythrocyte components.

The mechanism of apoptosis in human platelets during storage.

Although it is usually involved only in nucleated cells (NCs), artificially enucleated cells also lose viability by a programmed process of cell death called apoptosis. Because platelets undergo loss of viability during storage, an attempt was made to determine whether platelets contained the apoptotic mechanisms and whether it was activated during platelet storage. Platelet viability was measured by reduction of a tetrazolium dye (MTS) and annexin V binding. Members of the death receptor, caspase, and Bcl-2 families were detected by RNase protection assay and Western blotting. Caspase 3 activation was measured by enzyme and Western blot assays and by cleavage of gelsolin. After 5 days of storage under standard blood banking conditions, platelets display biochemical signs of apoptosis by losing MTS activity and increasing the amount of phosphatidylserine on their surface. The mRNA and the proenzyme for several members of the caspase, death receptor, and Bcl-2 families are expressed at high levels in platelets. An increase in caspase 3 activity and the amount of the biologically active p17 subunit of active caspase 3 were observed to coincide with the appearance of apoptotic markers during storage. These effects were not due to platelet activation. The caspase 3 substrate, gelsolin, began to undergo proteolysis after 3 to 4 days of storage, and the addition of the caspase inhibitor z-VAD-fmt substantially inhibited this process. Platelets contain many of the components of the apoptotic mechanism and show activation of caspase 3 and consequent cleavage of gelsolin during storage, independent of platelet activation. Evaluation of the mechanism of apoptosis in platelets may provide a basis for developing novel strategies to enhance platelet viability during storage.

The effect of storage on the expression of platelet membrane phosphatidylserine and the subsequent impacton the coagulant function of stored platelets.

Platelet concentrates (PCs) derived from whole blood and stored under standard blood bank conditions undergo changes that are referred to as the platelet storage lesion. This study assesses the effect of PC preparation and storage on the distribution of phosphatidylserine (PS) in the platelet membrane and the effect that this distribution may have on the thrombogenic potential of stored PCs. Fresh platelets and PCs donated by healthy donors were obtained. PCs derived from platelet-rich plasma were studied on Day 1, Day 3, and Day 6 of storage under blood bank conditions. Platelet aggregation after exposure to the platelet agonists ADP and epinephrine singly declined progressively, but, when ADP and epinephrine in combination and collagen and thrombin in combination were used as agonists, the decline in platelet aggregation was less marked. PS expression as measured by Annexin V binding (mean and SD) was 2.02 +/- 0.93 percent in fresh platelet samples and increased to 5.39 +/- 4.2 percent on Day 1, 22. 1 +/- 7.1 percent on Day 3, and 39.5 +/- 12.1 percent on Day 6. Platelet prothrombinase activity (mean +/- SD) as measured by thrombin generation increased from 1.49 +/- 0.7 micro per mL in fresh platelet samples to 3.68 +/- 1.1 micro per mL in Day 1 platelets (p<0.001), 5.15 +/- 2.5 micro per mL in Day 3 platelets (p<0.001), and 4.65 +/- 2.48 micro per mL in Day 6 platelets (p<0. 001). These results show that PS expression increases after preparation of PCs from platelet-rich plasma and rises progressively during platelet storage under blood bank conditions. Furthermore, the greater PS expression is associated with increased platelet- dependent thrombin-generating capacity.

In vitro analysis of platelet concentrates stored in the presence of modulators of 3′,5′ adenosine monophosphate, and organic anions

The storage of conventional platelet concentrates (PCs) under standard blood bank conditions is limited to five days, in part because longer storage periods lead to increasing damage in platelet integrity and functionality. The growing demand of PCs for clinical use, raises the interest to develop agents that would potentially permit a more extended period of storage. We have evaluated and compared the in vitro quality of PCs treated with: (1) Modulators of levels of cAMP (PGE 1, foskolin, theophylline and isobutyl-methyl-xanthine [IBMX]); and (2) organic anions that function as alternative substrates of platelets (pyruvate and acetate). Platelet rich plasma (PRP) from pools ( n=6) of PCs was distributed into storage bags, and the agents to be tested were added, using saline as a control substance. PCs were stored at 22°C with continuous agitation for up to 10 days. At 0, 5 and 10 days of storage, samples were analyzed for platelet counts, mean platelet volume (MPV), metabolic markers, and expression of glycoproteins (GPs). The addition of modulators of levels of cAMP, at the concentration used in the study, did not lead to substantial improvement in the parameters being evaluated, with respect to those in control units. The supplementation with organic anions, while not affecting the surface levels of GPs, favored the maintenance of metabolic values, such as pH, PCO 2, and bicarbonate concentrations, as well as the preservation of MPV ( p values <0.05 respect to control units both at 5 and 10 days of storage). Our results indicate that while the use of modulators of levels of cAMP do not provide substantial benefit in the prevention of platelet storage lesions, organic anions have some advantageous effect in the storage promoted metabolic changes of PCs. These data might be considered when designing strategies to improve PC storage.

Platelet Membrane Early Activation Markers during Prolonged Storage

The relationship between platelet aging and early markers of membrane activation have not been defined clearly. Activation markers expressed during prolonged storage are similar if not identical to those that appear after exposure to thrombin. Using flow cytometry, we investigated platelet membrane expression of CD62P, CD63, and annexin V binding (i.e., loss of membrane asymmetry) in platelets stored for up to 11 days under standard blood banking conditions. We compared five apheresis platelets to two random donor platelet concentrates, and to one pooled platelet preparation from six single platelet concentrates before and after exposure to thrombin. CD62P, CD63 expression, and annexin V binding increased during storage albeit with different kinetics. The differential increments observed between resting and thrombin (1 unit/ml) activated platelets showed an inverse correlation to storage time for CD62P, CD63, and annexin V binding, which was identical to published survival curves. A difference between apheresis platelets and platelet concentrates was observed only on day 1. Our data indicate that the in vitro platelet reserve activity to thrombin activation mirrors that of radiolabeled platelet survival in vivo and that platelet cross-sectional residual life span can explain their diminished capacity to respond to thrombin as a function of viability.

Time-dependent, spontaneous release of white cell- and platelet-derived bioactive substances from stored human blood.

The mechanisms of the detrimental effects of perioperative allogeneic blood transfusion are still unclear. Previous studies have suggested a higher incidence of adverse effects after the use of blood stored for prolonged time. Therefore, a possible time-dependent release of various white cell- and platelet-derived bioactive substances in stored human red cell suspensions was studied. Whole blood (6 units), plasma-reduced whole blood (6 units), and saline-adenine-glucose-mannitol blood (6 units) from 18 unpaid, normal blood donors were stored under standard blood bank conditions at 4 degrees C for 35 days. After refrigeration, samples were collected from all blood bags on Days 0, 2, 5, 9, 14, 21, 28, and 35 of storage. Extracellular concentrations of eosinophil cationic protein, eosinophil protein X, plasminogen activator inhibitor 1, myeloperoxidase, and interleukin 6 were analyzed by enzyme-linked immunosorbent assay and radioimmunoassay. The total intracellular and donor plasma levels of these substances also were analyzed at the time of blood donation. Eosinophil cationic protein, eosinophil protein X, and myeloperoxidase increased 10- to 25-fold (p < 0.05) in a time-dependent manner in whole blood, plasma-reduced whole blood, and saline-adenine-glucose-mannitol blood during storage for 35 days. Plasminogen activator inhibitor 1 increased threefold to sixfold (p < 0.05) in whole blood and plasma-reduced whole blood, but not in saline-adenine-glucose-mannitol blood. Interleukin 6 was not detected in either plasma or samples obtained from the blood bags. Stored whole blood, plasma-reduced whole blood, and saline-adenine-glucose-mannitol blood may release white cell- and platelet-derived bioactive substances in a time-dependent manner, which may be related to the detrimental effects of perioperative blood transfusions. Therefore, prestorage white cell reduction should be considered for further improvement of red cell suspensions.

Tirilazad mesylate protects stored erythrocytes against osmotic fragility

The hypoosmotic lysis curve of freshly collected human erythrocytes is consistent with a single Gaussian error function with a mean of 46.5 ± 0.25 mM NaCl and a standard deviation of 5.0 ± 0.4 mM NaCl. After extended storage of RBCs under standard blood bank conditions the lysis curve conforms to the sum of two error functions instead of a possible shift in the mean and a broadening of a single error function. Thus, two distinct sub-populations with different fragilities are present instead of a single, broadly distributed population. One population is identical to the freshly collected erythrocytes, whereas the other population consists of osmotically fragile cells. The rate of generation of the new, osmotically fragile, population of cells was used to probe the hypothesis that lipid peroxidation is responsible for the induction of membrane fragility. If it is so, then the antioxidant, tirilazad mesylate (U-74,006f), should protect against this degradation of stored erythrocytes. We found that tirilazad mesylate, at 17 μM (1.5 mol% with respect to membrane lecithin), retards significantly the formation of the osmotically fragile RBCs. Concomitantly, the concentration of free hemoglobin which accumulates during storage is markedly reduced by the drug. Since the presence of the drug also decreases the amount of F 2-isoprostanes formed during the storage period, an antioxidant mechanism must be operative. These results demonstrate that tirilazad mesylate significantly decreases the number of fragile erythrocytes formed during storage in the blood bank.

In vitro changes in platelet function and metabolism following increasing doses of ultraviolet-B irradiation.

Ultraviolet-B (UV-B) irradiation of platelet concentrates (PCs) may prevent the development of posttransfusion HLA alloimmunization. This study evaluated the effect of increasing doses of UV-B radiation on stored PCs. Pooled PCs were irradiated at UV-B doses of 600, 2400 or 10,000 mJ per cm2 and stored up to 96 hours under standard blood bank conditions. Compared to nonirradiated room-temperature and 37 degrees C controls, the irradiated units showed no significant changes in platelet count, white cell count, discharge of lactate dehydrogenase, release of beta-thromboglobulin, metabolism of ATP, ADP, ammonia, glutamine, glutamate, hypoxanthine, pCO2, or pO2 at any time of storage following any of the three UV-B doses. However, after a dose of 10,000 mJ per cm2, there were significant decreases in in vitro assays of platelet function-specifically, osmotic recovery and morphology score. Some metabolic systems were also affected by the 10,000 mJ per cm2 radiation dose, as shown by a decline in pH and bicarbonate and an increase in glucose consumption and lactate production (p < 0.05). The changes in these latter assays appeared only after 96 hours of postirradiation storage. Such changes were not seen in either the room-temperature or 37 degrees C control groups. Thus, heat generated during irradiation, per se, did not appear responsible for the observed in vitro changes in platelet function and metabolism. On the basis of the assays analyzed, it is concluded that UV-B irradiation of PCs at doses up to 10,000 mJ per cm2 does not induce significant metabolic or functional derangements following short-term storage (24-48 hours).(ABSTRACT TRUNCATED AT 250 WORDS)

Progressive platelet activation with storage: evidence for shortened survival of activated platelets after transfusion

Platelets are known to become activated during storage, but it is unclear whether such activation affects recovery or survival after platelet concentrate (PC) transfusion. With the use of flow cytometry to determine the percentage of platelets expressing the alpha-granule membrane protein 140 (GMP-140), a known adhesive ligand appearing on the platelet surface after activation, several studies were conducted. These investigations evaluated 1) the occurrence of significant platelet activation over time in PCs (n = 46) stored under standard blood bank conditions; 2) the correlation between platelet activation and platelet recovery in normal subjects after PC storage (n = 12), as assessed by the recovery of Indium-labeled platelets; and 3) the recovery of activated and unactivated platelets in thrombocytopenic cancer patients transfused with standard PCs (n = 11). It was determined 1) that an increasing duration of storage of PC was associated with increasing platelet activation as measured by the percentage of platelets expressing GMP-140, progressing from a mean of 4 +/- 2 percent (SD) on the day of collection to a mean of 25 +/- 8 percent by 5 days of storage: 2) that, in normal subjects, posttransfusion recovery of autologous platelets stored for 2 to 4 days and then labeled with In111 was inversely correlated with the percentage of activated platelets in the transfused PC (r = -0.55, p = 0.05); and 3) that, when thrombocytopenic patients were transfused with standard PCs, the recovery of the activated platelets in the transfused PCs averaged only 38 +/- 15 percent of the number predicted by the absolute platelet increment.(ABSTRACT TRUNCATED AT 250 WORDS)

Cryopreserved platelets have decreased adhesive capacity

The technique of freezing blood platelets has proven very useful in transfusion support of some patients who have become alloimmunized by prior transfusions. Although transfused frozen platelets have an acceptable life span in vivo, functional defects have been found when these cells were tested in vitro. The adhesive properties of frozen platelets were investigated by use of a modified Baumgartner chamber to perform paired perfusion studies of fresh versus frozen platelets or fresh versus 5-day-stored platelets from the same whole blood unit. Platelets were either frozen in liquid nitrogen with dimethyl sulfoxide as the cryopreservative or stored under standard blood bank conditions for 5 days. The freeze-thaw recovery of platelets was 73 +/- 8 percent. Frozen platelets exhibited a significant decrease in platelet adhesion as compared to fresh platelets from the same unit; adhesion of frozen platelets was only 53 percent of that of fresh platelets (p = 0.04). A slight, but insignificant decrease was noted with platelets stored for 5 days (86%, p = 0.197). These findings indicate that frozen-thawed platelets have a significant defect in adhesive capacity as compared to fresh platelets, and that platelets stored under blood bank conditions for 5 days maintain adhesive capacity well.

Possibility to Improve Preservation of Whole Blood by Leukocyte‐Depletion before Storage

In order to evaluate the influence of leukocytes in whole blood on the standard blood bank conditions for storage, we compared several parameters of red cells, leukocytes and platelets in leukocyte-rich and leukocyte-poor whole blood over a storage period of 5 weeks. The leukocyte-depleted units were prepared by filtration with a leukocyte trapping filter (Sepacell R500A). Our data indicate improved storage conditions, made evident by significantly lower cell damage: smaller increases of elastase, beta-thromboglobulin and lactate dehydrogenase in leukocyte-depleted blood. This improvement was achieved in a simple two-bag system using only filtration, without addition of rejuvenating solutions. In addition, this procedure allows greater flexibility in the use of filtrated blood for transfusions.