An inhibition of p38 mitogen activated protein kinase delays the platelet storage lesion.

BACKGROUND AND OBJECTIVES: Platelets during storage undergo diverse alterations collectively known as the platelet storage lesion, including metabolic, morphological, functional and structural changes. Some changes correlate with activation of p38 mitogen activated protein kinase (p38 MAPK). Another MAPK, extracellular signal-related kinase (ERK), is involved in PLT activation. The aim of this study was to compare the properties of platelets stored in plasma in the presence or absence of p38 and ERK MAPK inhibitors. MATERIALS AND METHODS: A single Trima apheresis platelet unit (n = 12) was aliquoted into five CLX storage bags. Two aliquots were continuously agitated with or without MAPK inhibitors. Two aliquots were subjected to 48 hours of interruption of agitation with or without MAPK inhibitors. One aliquot contained the same amount of solvent vehicle used to deliver the inhibitor. Platelets were stored at 20-24°C for 7 days and sampled on Days 1, 4, and 7 for 18 in vitro parameters. RESULTS: Inhibition of p38 MAPK by VX-702 leads to better maintenance of all platelet in vitro storage parameters including platelet mitochondrial function. Accelerated by interruption of agitation, the platelet storage lesion of units stored with VX-702 was diminished to that of platelets stored with continuous agitation. Inhibition of ERK MAPK did not ameliorate decrements in any in vitro platelet properties. CONCLUSION: Signaling through p38 MAPK, but not ERK, is associated with platelet deterioration during storage.

Maintenance of storage properties of pediatric aliquots of apheresis platelets in fluoroethylene propylene containers.

BACKGROUND: Platelet (PLT) aliquots for pediatric use have been shown to retain in vitro properties when stored in gas-impermeable syringes for up to 6 hours. As an alternative, PLT aliquots can be stored for longer periods in containers used for storage of whole blood-derived PLTs. These containers are not available separate from whole blood collection sets and PLT volumes less than 35 mL either have not been evaluated or may be unsuitable for PLT storage. Gas-permeable fluoroethylene propylene (FEP) containers have been used in the storage of cell therapy preparations and are available in multiple sizes as single containers but have not been evaluated for PLT storage. STUDY DESIGN AND METHODS: A single apheresis unit was divided on Day 3 into small aliquots with volume ranging from 20 to 60 mL, transferred using a sterile connection device, and stored for an additional 2 days either in CLX (control) or in FEP containers. PLT storage properties of PLTs stored in FEP containers were compared to those stored in CLX containers. Standard PLT in vitro assays were performed (n =6). RESULTS: PLT storage properties were either similar to those of CLX containers or differed by less than 20% excepting carbon dioxide levels, which varied less than 60%. CONCLUSION: Pediatric PLT aliquots of 20, 30, and 60mL transferred on Day 3 into FEP cell culture containers adequately maintain PLT properties for an additional 2days of storage.

A rest period before agitation may improve some in vitro apheresis platelet parameters during storage.

BACKGROUND: Whole blood-derived platelets (PLTs) prepared by the PLT-rich plasma method are subjected to a recommended 1-hour rest period after the second centrifugation to avoid excessive PLT activation. Different apheresis PLT preparation methods demonstrate different levels of PLT activation and ability to form macroscopic aggregates immediately after collection. PLT collections are lost on Day 1 of storage if aggregates are not dispersed. It is possible that a rest period may help to disperse PLT aggregates. It is not established whether apheresis PLTs require a rest period before agitation and what the length of this period should be. STUDY DESIGN AND METHODS: Apheresis PLTs (Amicus, Fenwal, Inc.) were divided into five identical aliquots. One aliquot was placed on the flatbed agitator immediately after division. The other aliquots were subjected to agitation after 1, 2, 4, and 6 hours of rest. Samples were taken on Days 1, 5, and 7 for standard PLT assays. RESULTS: No differences during 7-day storage were observed in PLT content, mean PLT volume, pH levels, bicarbonate, glucose, lactate, oxygen and carbon dioxide levels, hypotonic shock response, aggregation, and activation markers in PLT aliquots subjected to different rest periods or without a rest period. In contrast, values of extent of shape change, percentage of discoid PLTs, and expression of GP1b-α were greater in aliquots subjected to different periods of rest compared to those of PLTs without a rest period. CONCLUSION: A rest period from 1 to 6 hours may improve some but not all in vitro PLT storage parameters.

Comparative in vitro evaluation of apheresis platelets stored with 100% plasma or 65% platelet additive solution III/35% plasma and including periods without agitation under simulated shipping conditions.

BACKGROUND: A comparative study evaluated the retention of apheresis platelet (A-PLT) in vitro properties prepared with PLT additive solution (PAS)-III or 100% plasma and stored with continuous agitation (CA) and without continuous agitation (WCA). STUDY DESIGN AND METHODS: PLTs collected with the Amicus cell separator (Fenwal, Inc.) were utilized to prepare two matched components, each with approximately 4 × 10(11) PLTs. In the primary study, one component contained 65% PAS-III/35% plasma and the other 100% plasma. Four storage scenarios were used, one with CA and three with periods without agitation under simulated shipping conditions. In vitro assays were used early and after 5 days of storage. RESULTS: pH levels after 5 days with CA were less with PAS-III components than 100% plasma components, with levels always above 6.6 in any component. With CA, a number of other variables were reduced even early during storage with PAS-III including morphology, extent of shape change, hypotonic stress response, adhesion, and aggregation. Storage WCA resulted in only a limited increase in the magnitude of the assay differences between PAS-III and 100% plasma components. Periods WCA did not reduce the pH below 6.6. The thromboelastograph variable associated with the strengthening of clots by PLTs was essentially comparable with PAS-III and plasma components throughout storage with CA or WCA. CONCLUSION: The data indicate that a 100% plasma medium provides for better retention of specific in vitro PLT properties, with CA and WCA, although the clinical significance of these in vitro decrements due to PAS-III is unknown.

Mitochondrial dysfunction of platelets stored in first- and second-generation containers is, in part, associated with elevated carbon dioxide levels.

BACKGROUND: The gas permeability of platelet (PLT) storage bags influences the retention of in vitro PLT parameters during storage. The aim of this study was to evaluate mitochondrial function of PLTs stored in first- and second-generation bags with different gas permeabilities. STUDY DESIGN AND METHODS: Identical whole blood-derived PLT concentrates were stored in second-generation CLX (Pall Corp.) and first-generation PL146 (Baxter Healthcare Corp.) bags (n = 12). PLTs were assayed for standard in vitro PLT assays as well as for mitochondrial membrane potential (MMP), accumulation of reactive oxygen species, Annexin V binding, mitochondrial mass, and activity of mitochondrial reduction power on Days 1, 4, 5, 6, and 7. Results were analyzed by paired t test and by multiple regression analysis. RESULTS: With PLTs stored in PL146 bags that underwent large pH declines, there was greater superoxide production, greater peroxide accumulation, and greater mitochondrial membrane depolarization. Superoxide anion generation was correlated with higher levels of carbon dioxide (p = 0.0001) and lower oxygen levels (p = 0.0064; multiple regression R(2) = 0.9204). Changes in MMP were correlated with higher levels of carbon dioxide (p = 0.0288) and PLT activation (p = 0.0178; multiple regression R(2) = 0.9511). CONCLUSION: Prolonged periods of elevated carbon dioxide levels, potentially coupled with other factors, is associated with PLT mitochondria dysfunction and poor pH control during storage.

Influence of apheresis container size on the maintenance of platelet in vitro storage properties after a 30-h interruption of agitation.

We have previously conducted studies investigating maintenance of apheresis platelet in vitro quality measures during storage under simulated shipping conditions in which agitation was interrupted. This study examines the effect of increasing bag surface area on the preservation of in vitro platelet properties during storage with continuous agitation and with a 30 h interruption of agitation. Apheresis platelets were collected in 100% plasma with the Amicus separator to provide two identical platelet products, each with approximately 4-5 x 10(11) platelets. After collection, the volume was divided equally between 1.0 and 1.3 L PL2410 containers. In an initial study, both products were continuously agitated. In a second study, both products were subjected to a single 30-h period of interrupted agitation between Days 2 and 3 of storage by placement in a standard shipping box at room temperature. In each study, units were assayed during storage for standard in vitro platelet quality measures. Platelets stored in the 1.3 L container maintained slightly greater mean pH during 7 day storage with either continuous agitation (n=6) or with a 30-h interruption of agitation (n=12) than those of similarly treated identical platelets stored in the 1.0 L container. Most noteworthy, in experiments with products stored in the 1.0 L container in which there was a large decrease in pH to levels <6.7 or <6.3 on days 5 or 7, respectively, the pH in the matched product stored in the 1.3 L container was substantially greater (0.17+/-06 and 0.37+/-0.09 pH units greater, n=4, respectively). Other measures showed either small differences or comparability of platelet in vitro parameters with storage in the two containers after an interruption of agitation.

Periods without agitation diminish platelet mitochondrial function during storage.

BACKGROUND: Prolonged periods without agitation produce platelet (PLT) storage lesions that result in reduced in vitro assay parameters and an increase of apoptotic markers during storage. The aim of this study was to evaluate the influence of periods without agitation on PLT mitochondrial function, blood gases, and activation. STUDY DESIGN AND METHODS: Apheresis PLT units (n = 12) were collected using a cell separator and each was equally divided among five storage bags (50 mL of PLT suspension in 300-mL nominal volume containers). Four bags were held without agitation for 24, 48, 72, and 96 hours in a standard shipping box at room temperature and the fifth bag was continuously agitated. PLTs were assayed for standard in vitro PLT assays as well as for mitochondrial membrane potential (MMP), accumulation of reactive oxygen species, Annexin V binding, mitochondrial mass, and activity of mitochondrial reduction power (MRP) immediately after removal of units from the shipping container on Days 1, 2, 3, 4, and 7. RESULTS: Increasing periods without agitation resulted in increased superoxide anion generation and PLT activation as well as reduced PLT MMP and MRP. Increasing periods without agitation resulted in increasing Annexin V binding. PLTs that had undergone periods without agitation showed increased oxygen and carbon dioxide levels immediately after storage without agitation. The superoxide anion generation was highly correlated with the loss of MMP, increasing Annexin V binding, and pH decline. CONCLUSIONS: PLTs, if stored without agitation, produce a lesion that leads PLTs to apoptosis. The severity of the lesion depends on the length of the period without agitation. Prolonged periods without agitation induce formation of superoxides and depolarization of MMP along with a presentation of apoptotic markers.

Calcium is a key constituent for maintaining the in vitro properties of platelets suspended in the bicarbonate-containing additive solution M-sol with low plasma levels.

BACKGROUND: Commercially available additive solutions (ASs) require 30% to 35% plasma for optimal storage of platelets (PLTs). PLTs suspended in M-sol, a bicarbonate-based experimental platelet additive solution (PAS), maintain in vitro PLT properties during storage with low levels of plasma (< or =5%). STUDY DESIGN AND METHODS: Four different formulations of M-sol were prepared at the optimal pH (6.1): M-sol, M-sol without calcium, M-sol without citric acid, and M-sol without calcium and citric acid. Apheresis PLT units (100% plasma) were equally divided into five 50-mL aliquots in PL732 containers, centrifuged, and resuspended to prepare units suspended in the four different PASs (95%) with 5% plasma and 1 unit in 100% plasma. Units (n = 10) were stored under standard conditions and assayed for in vitro properties on Days 1, 5, and 7. The data were analyzed by analysis of variance for repeated measures (n = 10, p < 0.001). RESULTS: On Day 5 of storage, PLTs suspended in the M-sol formulation containing calcium but lacking citric acid had similar pH, extent of shape change (ESC) values, and percentage of CD62-positive PLTs and greater hypotonic shock response (HSR) and percentage of discoid PLTs compared to those of PLTs suspended in 100% plasma. In contrast, PLTs suspended in the M-sol formulation lacking calcium had lesser ESC values, greater percentage of CD62-positive PLTs, and similar HSR values and percentage of discoid PLTs compared to those of PLTs suspended in 100% plasma on Day 5 (p < 0.001). CONCLUSIONS: Calcium plays an important role in maintaining CD62-negative PLTs and relatively high ESC in 5% plasma. The removal of citric acid from M-sol may improve PLT storage properties with low plasma levels.

Evaluation of in vitro storage properties of prestorage pooled whole blood-derived platelets suspended in 100 percent plasma and treated with amotosalen and long-wavelength ultraviolet light.

BACKGROUND: Amotosalen, a psoralen, has been utilized for photochemical treatment (PCT) of apheresis platelets (PLTs) and pooled buffy coat PLTs suspended in additive solution. In the United States, the source of many PLT transfusions is from whole blood-derived PLTs prepared by the PLT-rich plasma (PRP) method. This study investigated the in vitro PLT properties of amotosalen-PCT of leukoreduced pools of PLTs prepared by the PRP method and suspended in 100 percent plasma. STUDY DESIGN AND METHODS: On Day 1 of storage, 12 leukoreduced (n = 6) or 10 leukoreplete (n = 6) ABO-identical PLT concentrates were pooled, separated into two pools of 6 or 5 units, respectively, and leukoreduced (leukoreplete pools only). Each pool of 5 or 6 units was then photochemically treated (designated "test": amotosalen plus 3.0 J/cm(2) long-wavelength ultraviolet light followed by amotosalen/photoproduct removal) while the remaining identical pool (designated "control") was untreated. PLT in vitro assays were performed on test and control pools during 7-day storage. RESULTS: PCT resulted in slightly reduced pH in test pools compared to that of matched control pools after 5 days of storage (5-unit pools: test, 6.96 +/- 0.12 vs. control, 7.15 +/- 0.09, p = 0.0033; 6-unit pools: test, 6.90 +/- 0.10 vs. control, 7.07 +/- 0.09, p < 0.0001). Test pools adequately maintained many other in vitro properties including PLT morphology, hypotonic shock response, and extent of shape change parameters during 5-day storage, which, like pH, also differed from those of controls. The pH of test and control pools declined on Day 7, with 1 of 6 test pools (either 5 or 6 units) having a pH value of less than 6.20, while all control pools had pH values of more than 6.66. CONCLUSION: PCT of leukoreduced PLT pools of whole blood-derived PLTs in 100 percent plasma maintained adequate PLT in vitro variables through 5 days of storage.

Maintenance of platelet in vitro properties during 7-day storage in M-sol with a 30-hour interruption of agitation.

BACKGROUND: Extensive periods without agitation can occasionally occur during platelet (PLT) shipment and can affect PLT quality during 5- to 7-day storage. The use of buffer-containing PLT additive solutions (ASs) may better preserve PLT quality during storage by maintaining PLT pH and other in vitro variables. A newly described bicarbonate-containing AS, M-sol, was compared to plasma for preservation of whole blood-derived PLT concentrates in which a 30-hour interruption of agitation was included. STUDY DESIGN AND METHODS: ABO-identical PLT-rich plasma intermediate products were pooled in sets of four, split, and centrifuged with subsequent plasma expression (n = 12). Two units were resuspended with M-sol AS to produce a 70 percent solution/30 percent plasma PLT concentrate; 2 units were resuspended in 100 percent plasma. One M-sol resuspended unit and 1 plasma unit were held on a laboratory bench in a standard shipping box for 30 hours between Day 2 and Day 3, while the other M-sol and plasma unit were continuously agitated. Standard in vitro testing for PLT quality variables on each set of 4 units was performed during storage (n = 12). RESULTS: Interrupting agitation of PLTs suspended in M-sol resulted in less of a pH decrement during storage than that of PLTs suspended in 100 percent plasma. On Days 5 and 7, the pH differences between M-sol and plasma units were 0.56 and 0.75 pH units, respectively (p < 0.0003). In addition, PLTs suspended in M-sol and subjected to an interruption of agitation had lesser Day 7 CD62+ cells, glucose utilization, and lactate production and greater hypotonic stress response, morphology, swirling, and aggregation response than those suspended in plasma (p

Toward closed-system culture of blood origin endothelial cells.

BACKGROUND: Blood outgrowth endothelial cells (BOECs) are thought to arise from very rare progenitors that are present in the mononuclear fraction of marrow or peripheral blood. Recently, BOECs have been expanded from progenitors present in buffy coat into confluent monolayers on fibronectin- or collagen-coated polystyrene surfaces. A method for sterile closed-system culture of these cells has not been described, however. Here, efforts are described toward developing closed-system culture of BOECs derived from progenitors present in a mononuclear apheresis unit by use of a cord blood filter, a sterile connection device, and a fibronectin-coated polycarbonate cassette. STUDY DESIGN AND METHODS: Strongly adherent cells from a mononuclear apheresis unit were eluted from a cord blood filter and resuspended in EGM-2 with 10 percent serum. Approximately 2 x 10(8) eluted cells were introduced into human fibronectin-coated polycarbonate cassettes. Medium was introduced and removed from cassettes with a sterile connection device and changed every 2 days. After expansion, cells were either cryopreserved or characterized by fluorescence-activated cell sorting analysis and ability to take up Dil-Ac-LDL. RESULTS: After 2 to 3 weeks of culture, 3 to 28 colonies with cobblestone morphology were observed in cassettes and passed to new cassettes within 3 to 4 weeks. By approximately 5 weeks of culture, 2 x 10(6) cells were typically obtained. BOECs uniformly took up Dil-Ac-LDL and were CD31+, CD105+, CD146+, CD45-, and CD14-. A population of BOECs was HLA-ABC+ or CD34+. CONCLUSION: BOEC progenitors can be isolated from mononuclear apheresis units with cord blood filters, expanded with fibronectin-coated polycarbonate cassettes, and cryopreserved.

Monocyte enrichment of mononuclear apheresis preparations with a multistep back-flush procedure on a cord blood filter.

INTRODUCTION: Monocytes or mononuclear cells have been investigated for the treatment of chronic wounds and spinal cord injuries, as well as serve as a source for dendritic or endothelial cell culture. Because these cells may have clinical benefit yet no rapid and inexpensive closed system for monocyte purification is commercially available, a method was investigated to enrich monocytes from mononuclear apheresis units using a cord blood filter. METHODS: A 4-step method for monocyte enrichment was developed which involved 1) filtering a mononuclear apheresis unit through a cord blood filter, 2) chasing with medium to remove non-adherent residual cells and plasma, 3) back-flushing under low shear conditions to remove loosely adherent lymphocytes, and 4) back-flushing under high shear conditions to collect a fraction enriched in monocytes. Apheresis units and enriched monocyte preparations were characterized by cell count and differential, filter-isolated preparations were cryopreserved, and thawed preparations were assayed for viability, and phagocytosis. Enriched monocyte preparations were also assayed for inflammatory cytokines secretion and secretion of prostaglandin E2 during short-term culture. RESULTS: Monocytes were viable, capable of phagocytosis, and enriched using the multistep filter elution technique to represent 42 +/- 13-percent of white cells in the final preparation. Fifty-three-percent of monocytes were recovered in the final preparation, while total cell counts of red cells, platelets, neutrophils and lymphocytes were reduced to 3.0, 3.0, 4.5 and 16-percent, respectively, from levels present in mononuclear apheresis units. Filter enriched monocyte preparations secreted IL-8, IL-6, MCP-1, and MIP-1alpha, during short term culture. CONCLUSION: The use of a multi-step back flush procedure with a cord blood filter resulted in rapid enrichment of viable and functional monocytes from mononuclear apheresis units with significant reduction of contaminating platelets and red cells.

Sterile isolation and cryopreservation of human adherent monocytes/macrophages from mononuclear cell apheresis preparations.

There is evidence from animal and human study that suggests clinical use of monocytes/macrophages may be of benefit in wound healing, tissue regeneration, and cancer therapy. To facilitate further study, a method was developed for sterile isolation and cryopreservation of adherent monocyte/macrophages from mononuclear cell apheresis units collected from unstimulated normal human donors. Preparations contained approximately 1 x 108 total cells and were comprised of approximately 60% monocytes, 38% lymphocytes, and 2% granulocytes. Cells could be cryopreserved for up to 8 months and subsequently thawed and stored at 1-6 degrees C for up to 4 hours with retention of viability and adequate phagocytic function. These cells can be used in clinical trials to determine their possible therapeutic benefit, e.g., whether administration of exogenously supplied cells improves the healing of chronic wounds or promotes the regeneration of transected nerves.