Clinical experience with transfusion of leukocyte-poor platelet concentrates prepared by filtration with prostacyclin

Repeated transfusions with platelets from randomly selected donors lead to HLA alloimmunization in about 50% of patients due to lymphocyte contamination of platelet concentrates. Attempts to remove the leukocytes from the platelet concentrates by additional centrifugation steps led to substantial loss of platelets. We report a new procedure for removal of almost all leukocytes with excellent platelet recoveries. Single donor concentrates are treated with 50 ng/mL prostacyclin to inactivate the platelets transiently. The concentrates are then passed through a cellulose-acetate filter to remove the leukocytes. In 30 concentrates this treatment reduced the contamination by leukocytes to less than 0.1 million per concentrate with a platelet recovery of 89% +/- 1% (mean +/- SEM). Thirty filtered platelet concentrates transfused to ten thrombocytopenic patients within one hour after filtration were well tolerated and led to corrected count increments of (22.0 +/- 1.1) X 10(6)/mL blood after one hour and normal survival thereafter. In four of five patients these concentrates reduced the bleeding time. We conclude that transient inactivation of platelets by prostacyclin enables optimal removal of leukocytes and may help to reduce alloimmunization during frequent transfusions with platelet concentrates.     

Comparison of various dimethylsulphoxide-containing solutions for cryopreservation of leucoreduced platelet concentrates

BACKGROUND AND OBJECTIVES: Leucoreduced platelet concentrates (LR-PCs) can be stored at 20-24 degrees C for 5-7 days. When LR-PCs are cryopreserved they can be stored for several years. For cryopreservation to become applicable in blood-bank practice, an off-the-shelf cryoprotectant is needed that can be added to the LR-PC in a sterile manner. For this, we varied the composition of the cryopreservation medium and studied various parameters of cryopreserved LR-PCs for up to 24 h after thawing at room temperature. MATERIALS AND METHODS: LR-PCs in plasma or Composol were concentrated and divided into 2 units. To each unit, an equal part of 10% dimethylsulfoxide (DMSO) in plasma, Composol with or without 5% albumin, or GPO (pasteurized plasma-protein solution) was added. Freezing occurred at 1 degrees C/min and LR-PCs were placed in the vapour phase of nitrogen. LR-PCs were thawed at 37 degrees C and stored at room temperature. LR-PCs were tested for morphology, platelet recovery, swirling effect, and activation antigens at various time-points thereafter. RESULTS: LR-PCs in 100%, 65% and 50% plasma supplemented with Composol showed good morphology scores (>250), limited CD62P expression (<35%), low CD63 expression (<20%) and a swirling effect of about 2, at 24 h after thawing. At the same time-point, platelet recovery was >80% under all conditions and CD42b expression varied between 70 and 85%. Results of LR-PCs in 15% plasma and Composol, with or without plasma substitutes, were not acceptable at 24 h after thawing, i.e. the morphology score was <200 and the CD62P expression was >40%. CONCLUSIONS: A minimum of 50% plasma in the cryopreserved LR-PC is necessary to maintain an acceptable in vitro quality of platelets up to 24 h after thawing. Composol is a good candidate for using to prepare an off-the-shelf cryoprotectant.     

Multicentre standardisation of a clinical grade procedure for the preparation of allogeneic platelet concentrates from umbilical cord blood

BackgroundIn addition to a largely prevalent use for bleeding prophylaxis, platelet concentrates from adult blood have also been used for many years to prepare platelet gels for the repair of topical skin ulcers. Platelet gel can be obtained by activation of fresh, cryopreserved, autologous or allogeneic platelet concentrates with calcium gluconate, thrombin and/or batroxobin. The high content of tissue regenerative factors in cord blood platelets and the widespread availability of allogeneic cord blood units generously donated for haematopoietic transplant but unsuitable for this use solely because of low haematopoietic stem cell content prompted us to develop a national programme to standardise the production of allogeneic cryopreserved cord blood platelet concentrates (CBPC) suitable for later preparation of clinical-grade cord blood platelet gel.ResultsDuring 14 months, 13 banks produced 1,080 CBPC with mean (± standard deviation) volume of 11.4±4.4 mL and platelet concentration of 1,003±229×10⁹/L. Total platelet count per CBPC was 11.3±4.9×10⁹. Platelet recovery from cord blood was 47.7±17.8%. About one-third of cord blood units donated for haematopoietic transplant could meet the requirements for preparation of CBPC. The cost of preparation was € 160.92/CBPC. About 2 hours were needed for one technician to prepare four CBPC.DiscussionThis study yielded valuable scientific and operational information regarding the development of clinical trials using allogeneic CBPC.     

Therapeutic effectiveness of frozen platelet concentrates for transfusion

Six patients received platelet concentrate transfusions from their HLA- identical siblings. Platelet concentrates were administered either fresh, or after being frozen in 10% dimethylsulfoxide, at a slow controlled rate (1 degree C/min) or rapidly (approximately 8 degrees C/min) in the vapor-phase of a liquid nitrogen refrigerator. The median freeze-thaw loss was 13.5%. The mean 1-hr and 20-hr corrected increments in platelet count were calculated for fresh platelet concentrates transfused before and after transfusion with controlled- rate frozen and vapor-phase frozen platelet concentrates. There was no significant difference among the first and second transfusion of fresh platelet concentrates, nor was the difference observed between fresh and controlled-rate frozen platelet concentrates significant. The difference between fresh and vapor-phase frozen platelet concentrates, and between controlled-rate frozen and vapor-phase frozen platelet concentrates were highly significant (p < 0.01). In vitro tests of aggregation using ristocetin and platelet ultrastructural studies paralleled the transfusion experience. Our results indicate that HLA- identical platelet concentrates can be successfully frozen and thawed for transfusion if a slow, controlled rate of freezing is employed. The use of HLA-identical frozen platelet concentrates may be important in emergency situations for the refractory patient and potentially for the establishment of a platelet concentrate bank.     

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.     

A shear-induced in vitro platelet function test can assess clinically relevant anti-thrombotic effects

Morphological features of haemostatic plugs formed in vitro under high shear forces were investigated. Electron microscopy confirmed the relevance of such haemostatic plug to a platelet-rich arterial thrombus, which is formed in vivo . In rat blood samples, the effects of anticoagulants and various antiplatelet agents on platelet reactivity (rate of haemostatic plug formation) and subsequent coagulation of the flowing blood were investigated. Haemostasis did not occur in citrated blood, and heparin greatly inhibited the shear-induced platelet reaction. Aspirin (1 mM), a thromboxane A(2) receptor antagonist (5 microM), a stable prostacyclin (0.55 nM), a stable prostaglandin E(1) (141 nM) and a phosphodiesterase inhibitor (100 microM) were tested. All these agents exerted significant inhibitory effect on shear-induced platelet reaction, including the inhibition of the very first phase of platelet plug formation, due to aggregation of shear-activated platelets. Except for the phosphodiesterase inhibitor, which prolonged clotting time, none of the above agents affected dynamic coagulation. These results suggest that the employed in vitro shear-induced thrombosis/haemostasis test can reveal in vivo the antithrombotic effect of various agents independently of their mechanism of action.     

Female sex hormones and platelet/endothelial cell interactions

The effects of estradiol and progesterone added to the growth medium of human umbilical vein endothelial cells for 72 h on the formation and release of prostacyclin were investigated. The influence on collagen-induced platelet aggregation and on the platelet formation of thromboxane A2 following aggregation, of the growth medium collected before and after thrombin stimulation of the endothelial cells, was studied simultaneously. Under basal conditions, endothelial cells grown with progesterone released significantly less prostacyclin into the growth medium than did controls (p less than 0.05). Following thrombin stimulation, endothelial cells grown with estradiol (p less than 0.05) or a combination of estradiol and progesterone (p less than 0.01) contained significantly less prostacyclin than controls. No significant effects on the platelet aggregation or platelet thromboxane formation could be found. This study indicates a lowering effect of both female sex hormones on the endothelial cell prostacyclin formation and release. This may be of significance for the increased risk of vascular disease in pregnant women and oral contraceptive users, but can hardly explain the consequences of the hormonal loss occurring at the menopause.     

Efficacy of leukocyte-depleted platelet concentrates for prevention of HLA-alloimmunization in patients with frequent platelet transfusions: a prospective multi-institutional study using a polyester platelet filter]

A prospective multi-institutional study was conducted to assess the efficacy of leukocyte-depleted platelet concentrates, prepared by a method using a newly developed polyester filter, in the prevention of HLA-alloimmunization in patients with hematological disorders. Patients who were expected frequent platelet transfusions, were assigned into two groups, receiving either standard platelet concentrates (control group) or leukocyte-depleted platelet concentrates prepared through a polyester platelet filter, Sepacell-PL (filtered group). All patients received leukocyte-depleted red cell products. Of III patients enrolled, 72 were evaluable, 23 in the control and 49 in the filtered group. Both groups were comparable according to age, sex ratio, underlying disorders, previous exposure to alloantigens by transfusion and/or pregnancy. There was no statistically significant difference in the number and duration of transfusion in the two groups. There were significant differences in HLA-alloimmunization rate (9 cases out of 23, 39% in the control group versus 4 cases out of 49, 8% in the filtered group; p less than 0.01) and refractoriness to platelet transfusion from random donors (6 cases out of 23, 26% in the control group versus 2 cases out of 49, 4% in the filtered group; p less than 0.05). These results indicated that leukocyte-depleted platelet concentrates prepared through the polyester platelet filter are beneficial to reduce HLA-alloimmunization in patients with frequent platelet transfusions.     

Platelet regulatory prostanoids and platelet release products in sickle cell disease.

Changes in platelet function have been observed for sickle cell disease (SCD). Levels of the arachidonic acid metabolites, thromboxane A2 (released by stimulated platelets) and prostacyclin (released from vascular endothelium), which stimulate and inhibit platelets, respectively, have been implicated in overall regulation of platelet function. Circulating basal levels of thromboxane and prostacyclin were determined in 1) a group of SCD volunteers (n = 21; at half-yearly steady state intervals and also at 24 hr, 72 hr, and 7 days after start of pain crisis) and 2) an age-, sex-, and race-matched control group (n = 18; single determinations). Circulating levels of beta-thromboglobulin (beta-TG), as well as thrombin (clotting)-stimulated platelet release of thromboxane, were also determined. Statistically significant decreases were found for prostacyclin, basal thromboxane, and thrombin-induced (maximal) thromboxane (alone or per platelet), for steady state SCD vs. normal controls. In addition, significant increases in maximal thromboxane were identified in crises (24, 72 hr) compared with steady state. Crisis beta-TG (24 hr) was significantly elevated compared with controls or steady state SCD. The ratio of basal thromboxane to prostacyclin was increased in crisis, but not significantly. Crisis frequency may correlate in part with changes in platelet function: steady state maximal thromboxane and released thromboxane per platelet were significantly lower in SCD volunteers who had crises during the study vs. those who did not (equivalent study time). The data support altered platelet function in SCD, possibly refractoriness (desensitization), manifest as decreased thromboxane release, to thrombin and/or other stimuli: alternate explanations are discussed.     

The evolution of platelet procoagulant activity of remnant platelets in stored platelet concentrates prepared by the platelet-rich plasma method and the buffy coat method

 Platelets stored as concentrates are gradually activated (storage lesion), a process associated with changes in the expression of platelet procoagulant activity (PPCA). The aim of the present study was to evaluate the evolution of PPCA and the mean platelet volume (MPV) of stored platelets prepared according to the platelet-rich method (PRM) and the buffy coat method (BCM). Using the platelet factor 3 availability clotting test (PF3AT) on appropriately diluted concentrate samples, we found a decrease in PPCA expression of remnant platelets as a function of storage time (0.025<p<0.01 between day 1 and 7) in PRM-derived but not in BCM-derived platelet concentrates. Using the PF3AT reduction test we found a more important clotting time reduction in samples obtained from BCM than in samples obtained from PRM platelet concentrates, suggesting a higher PPCA expression of BCM platelets, not significant after 1 day but highly significant after 3 days (p<0.0005) and after 7 days (p<0.0005) of storage, as compared with PRM platelets. For both PRM and BCM concentrates there were no significant MPV changes as a function of storage time, but at any storage day the MPV of BCM concentrates was significantly higher (p<0.0005) than the MPV of PRM concentrates. We conclude that the decrease of PPCA expression in PRM-derived concentrates as a function of storage time is in agreement with the gradual decrease of the platelet activation status in PRM concentrates during storage. There are probably several factors or variables causing platelets of BCM concentrates to express higher PPCA than those of PRM concentrates. Higher PPCA expression in BCM concentrates may be explained by an intrinsic platelet property, such as a difference in MPV between the two kinds of concentrates, or it may be related to an extrinsic factor such as different storage media, e.g., undiluted autologous plasma in PRM concentrates versus Plasmalyte A-diluted autologous plasma in BCM concentrates. Whether the difference in PPCA expression of remnant platelets in PRM and BCM concentrates is just an in vitro laboratory finding or may have consequences for the therapeutic efficiency of the concentrates is an interesting, still unresolved question. Received: April 3, 1998 / Accepted: October 1, 1998     

A shear-induced in vitro platelet function test can assess clinically relevant anti-thrombotic effects

Morphological features of haemostatic plugs formed in vitro under high shear forces were investigated. Electron microscopy confirmed the relevance of such haemostatic plug to a platelet-rich arterial thrombus, which is formed in vivo. In rat blood samples, the effects of anticoagulants and various antiplatelet agents on platelet reactivity (rate of haemostatic plug formation) and subsequent coagulation of the flowing blood were investigated. Haemostasis did not occur in citrated blood, and heparin greatly inhibited the shear-induced platelet reaction. Aspirin (1 mM), a thromboxane A₂ receptor antagonist (5 μM), a stable prostacyclin (0.55 nM), a stable prostaglandin E₁ (141 nM) and a phosphodiesterase inhibitor (100 μM) were tested. All these agents exerted significant inhibitory effect on shear-induced platelet reaction, including the inhibition of the very first phase of platelet plug formation, due to aggregation of shear-activated platelets. Except for the phosphodiesterase inhibitor, which prolonged clotting time, none of the above agents affected dynamic coagulation. These results suggest that the employed in vitro shear-induced thrombosis/haemostasis test can reveal in vivo the antithrombotic effect of various agents independently of their mechanism of action.     

The effect of plasma removal from apheresis platelet concentrates on platelet function

The effect of plasma removal on platelet function has scarcely been investigated. Plasma removal from apheresis platelet concentrates was achieved by centrifugation at 5000 g for 6 min or 2000 g for 10 min. After resting for 1 h, platelet concentrates were resuspended in 0·9% NaCl. Platelet function was tested before centrifugation and after resuspension by multiple electrode impedance aggregometry (MEA) and light transmission aggregometry (LTA). Plasma removal resulted in 10-14% lower response to TRAP-6 by MEA using both washing procedures, whereas TRAP-6-inducible aggregation by LTA increased slightly (2-5%). Neither plasma removal method affected collagen-induced aggregation. Thus, platelet function did not deteriorate significantly by either method.     

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.     

Sustained elevation of intracellular cyclic 3'-5' adenosine monophosphate is necessary for preservation of platelet integrity during long-term storage at 22 degrees C

Preservation of platelet integrity and responsiveness was examined in platelet concentrates prepared in the presence of various formulations and combinations of platelet-activation inhibitors affecting intracellular levels of cyclic 3'-5' adenosine monophosphate (cAMP). Platelet concentrates were prepared and stored in an artificial medium for two weeks at 22 degrees C. Markers of metabolic activity (pH, lactate, pO2, pCO2 in the medium), aggregation response, hypotonic shock response, and glycoprotein Ib (GPIb) expression were assessed along with direct measurements of cAMP in platelet pellets and thromboxane B2 (TxB2) in the supernate. The platelet concentrates prepared with only adenylate-cyclase stimulators (prostaglandin E-1 or forskolin) showed less maintenance of the integrity and responsiveness markers and greater loss of GPIb than concentrates prepared with phosphodiesterase inhibitors (theophylline or caffeine) or combinations with the above. These results were correlated with the ability of these compounds to sustain elevation of cAMP above basal level during the entire extended-storage period. The strong correlation (rs = -0.67) between elevation of cAMP levels and suppression of TxB2 production suggests that the phosphodiesterase inhibitors provided better protection than stimulators of adenylate cyclase alone through a reduction in platelet activation and its deleterious effects on preservation of platelets during storage.     

Transfusion of platelet concentrates cryopreserved with ThromboSol plus low-dose dimethylsulphoxide in patients with severe thrombocytopenia: a pilot study

We have recently reported the possibility of supporting the phase of severe thrombocytopenia after high-dose chemotherapy (HDC) and stem cell transplantation using 5% dimethylsulphoxide (DMSO)-cryopreserved autologous platelet concentrates (PCs). The aim of the present study was to evaluate the therapeutic potential of ThromboSol (a recently developed platelet storage solution) plus PCs cryopreserved in 2% DMSO in patients undergoing myeloablative chemotherapy and autologous transplantation. PCs were collected from 14 women with breast cancer by a single plateletapheresis and cryopreserved in ThromboSol/2% DMSO by either direct insertion in a -80 degrees C freezer or in liquid nitrogen after computer-controlled rate (CR) freezing. When required, PCs were thawed, centrifuged to remove the cryoprotectants and transfused. In vitro studies on thawed platelets showed loss of epitopes of surface glycoproteins and a marked reduction of functional activity compared with fresh platelets. Transfusion of CR-frozen PCs was associated with a mean 1 h corrected count increment (CCI) of 9.2 +/- 5.4 x 109/l and only one allogeneic PC was required in this group. In contrast, six out of seven patients required additional allogeneic transfusions in the -80 degrees C group (CCI = 2.7 +/- 1.4 x 109/l). ThromboSol-treated PCs have the ability to overcome thrombocytopenia if processed by a CR freezing protocol, but appear ineffective when frozen by direct placing at -80 degrees C.     

A pharmacological approach to the inhibition of platelet adhesion and platelet aggregation. Wright-Schulte Lecture

A first level of pharmacological interference with platelet function is located at the site of the agonist-receptor interaction (receptors for collagen, adenosine diphosphate, serotonin, fibrinogen). A second level of interaction is to prevent the mobilization of intracellular calcium ions which can be obtained by inhibition of phosphodiesterase, activation of cAMP (e.g. with prostacyclin analogues), inhibition of thromboxane A2 formation or blocking of its receptors. Compounds preventing the platelet contractile process or platelet secretion could theoretically also prevent some platelet functions.     

Measurement of platelet aggregation in diabetics using the new electronic platelet aggregometer

Platelet function has been studied in diabetic subjects using a new electronic platelet aggregometer which enables platelet aggregation to be studied in whole blood. This may be a more physiological approach to the assessment of platelet behaviour as centrifugation is avoided and platelets are studied in the presence of other blood elements which may be important modulators of platelet function in vivo. Twenty insulin-dependent diabetic subjects were studied along with 20 age and sex-matched controls. Platelet aggregation to collagen (1 microgram/ml) and arachidonic acid (1 mM) was significantly increased in the diabetic group. In addition the sensitivity of diabetic platelets to the antiaggregatory effects of prostacyclin was significantly reduced. A significant inverse correlation was found between platelet sensitivity to prostacyclin and glycosylated haemoglobin concentration in the diabetic group. It is unlikely that the platelet abnormalities in this diabetic group are due to underlying vascular disease as none of the patients had evidence of diabetic complications. These findings may have important implications for the development of vascular disease in diabetics.     

Biochemical evidence of platelet activation in patients with persistent unstable angina

Thromboxane released from activated platelets and prostacyclin of the vessel wall may act as potent antagonistic modulators of platelet aggregability and coronary vascular tone. Therefore, urinary excretion of their major metabolites, 2,3-dinor-thromboxane B2 and 2,3-dinor-6-ketoprostaglandin F1 alpha, was studied in 16 patients presenting with prolonged angina at rest. The 10 patients whose condition did not improve under vigorous antianginal treatment within 48 hours exhibited higher thromboxane metabolite excretion than did the 6 patients who responded to therapy (2,208 +/- 1,542 versus 609 +/- 312 ng/g creatinine; p less than 0.001). Elevated values were also found in four of eight patients with sustained postinfarction angina. Enhanced thromboxane metabolite excretion was frequently associated with angiographic evidence of thrombus formation. When nine patients were restudied in a stable phase after 11 +/- 5 months, thromboxane metabolite excretion was consistently normal or high normal. Excretion of prostacyclin metabolites was not depressed in any patient but correlated weakly with thromboxane (r = 0.41). Thus, enhanced thromboxane production as an index of platelet activation may identify patients with active thrombus formation who could benefit most from platelet inhibitory treatment.     

The effects of selective serotonin reuptake inhibitors on platelet function in whole blood and platelet concentrates

Several studies report that patients who are treated with selective serotonin reuptake inhibitors (SSRIs) for depression may have increased risk of bleeding, particularly from the gastrointestinal tract. This may be related to low intraplatelet serotonin concentrations. Several blood banks do not store platelets from donors using SSRIs for transfusion, although the possible effects of SSRIs on platelet storage are not documented. We conducted a case-control pilot study of apheresis platelet concentrates prepared from donors using SSRIs (n?=?8) and from donors without medication (n?=?10). The platelet concentrates were stored for 5 days. Light transmission aggregometry (LTA), thrombelastography (TEG), and flow cytometric analyses were preformed for in vitro measurements of platelet function. Platelet function and platelet serotonin content were investigated in whole blood and in platelet concentrates stored for up to 5 days. LTA, TEG, and flow cytometric analysis of glycoprotein expression did not reveal any significant differences between the two groups. All 18 platelet concentrates performed well according to the standards set for platelet quality in relation to transfusion. Blood donors using SSRIs had significantly lower platelet serotonin compared to blood donors without medication. The results from our pilot study indicate that platelets from donors using SSRIs may be suitable for transfusion after storage for 5 days, but further laboratory and clinical studies are necessary to confirm this.