P38 mitogen activated protein kinase inhibitor improves platelet in vitro parameters and in vivo survival in a SCID mouse model of transfusion for platelets stored at cold or temperature cycled conditions for 14 days.

Platelets for transfusion are stored at room temperature (20-24°C) up to 7 days but decline in biochemical and morphological parameters during storage and can support bacterial proliferation. This decline is reduced with p38MAPK inhibitor, VX-702. Storage of platelets in the cold (4-6°C) can reduce bacterial proliferation but platelets get activated and have reduced circulation when transfused. Thermocycling (cold storage with brief periodic warm ups) reduces some of the effects of cold storage. We evaluated in vitro properties and in vivo circulation in SCID mouse model of human platelet transfusion of platelets stored in cold or thermocycled for 14 days with and without VX-702. Apheresis platelet units (N = 15) were each aliquoted into five storage bags and stored under different conditions: room temperature; cold temperature; thermocycled temperature; cold temperature with VX-702; thermocycled temperature with VX-702. Platelet in vitro parameters were evaluated at 1, 7 and 14 days. On day 14, platelets were infused into SCID mice to assess their retention in circulation by flow cytometry. VX-702 reduced negative platelet parameters associated with cold and thermocycled storage such as an increase in expression of activation markers CD62, CD63 and of phosphatidylserine (marker of apoptosis measured by Annexin binding) and lowered the rise in lactate (marker of increase in anaerobic metabolism). However, VX-702 did not inhibit agonist-induced platelet aggregation indicating that it does not interfere with platelet hemostatic function. In vivo, VX-702 improved initial recovery and area under the curve in circulation of human platelets infused into a mouse model that has been previously validated against a human platelet infusion clinical trial. In conclusion, inhibition of p38MAPK during 14-days platelet storage in cold or thermocycling conditions improved in vitro platelet parameters and platelet circulation in the mouse model indicating that VX-702 may improve cell physiology and clinical performance of human platelets stored in cold conditions.

Validation of a SCID mouse model for transfusion by concurrent comparison of circulation kinetics of human platelets, stored under various temperature conditions, between human volunteers and mice.

BACKGROUND: Initial evaluation of new platelet (PLT) products for transfusion includes a clinical study to determine in vivo recovery and survival of autologous radiolabeled PLTs in healthy volunteers. These studies are expensive and do not always produce the desired results. A validated animal model of human PLTs in vivo survival and recovery used pre-clinically could reduce the risk of failing to advance product development. STUDY DESIGN AND METHODS: An immunodeficient (SCID) mouse model to evaluate recovery of human PLTs was compared to a radiolabeling study in human volunteers. Autologous apheresis PLTs stored for 7 days at room temperature (RT), thermo-cycled (TC), and cold temperature (CT) were radiolabeled and infused into healthy humans (n = 16). The same PLTs, non-radiolabeled, were also infused into mice (n = 160) on the same day. Blood samples from humans and mice were collected to generate clearance curves of PLTs in circulation. Flow cytometry was used to detect human PLTs in mouse blood. RESULTS: Human and mouse PLTs were cleared with one phase exponential clearance. Relative differences for initial recovery and AUC, expressed as ratio of test and control PLTs, were similar in humans and mice. The initial recovery ratio of TC/RT was 0.73 ± 0.07 in humans and 0.67 ± 0.14 in mice. The ratio for CT/TC was 0.53 ± 0.06 in humans and 0.75 ± 0.18 in mice. CONCLUSION: The SCID mouse model can provide information on relative differences of initial in vivo recovery and AUC between control and alternatively stored/processed human PLTs that is predictive of performance in healthy human volunteers.

Temperature cycling during platelet cold storage improves in vivo recovery and survival in healthy volunteers.

BACKGROUND: Room temperature (RT) storage of platelets (PLTs) can support bacterial proliferation in contaminated units, which can lead to transfusion-transmitted septic reactions. Cold temperature storage of PLTs could reduce bacterial proliferation but cold exposure produces activation-like changes in PLTs and leads to their rapid clearance from circulation. Cold-induced changes are reversible by warming and periodic rewarming during cold storage (temperature cycling [TC]) has been proposed to alleviate cold-induced reduction in PLT circulation. STUDY DESIGN AND METHODS: A clinical trial in healthy human volunteers was designed to compare in vivo recovery, survival, and area under the curve (AUC) of radiolabeled autologous apheresis PLTs stored for 7 days at RT or under TC or cold conditions. Paired comparisons of RT versus TC and TC versus cold PLTs were conducted. RESULTS: Room temperature PLTs had in vivo recovery of 55.7 ± 13.9%, survival of 161.3 ± 28.8 hours, and AUC of 5031.2 ± 1643.3. TC PLTs had recovery of 42.6 ± 16.4%, survival of 48.1 ± 14.4% hours, and AUC of 1331.3 ± 910.2 (n = 12, p < 0.05). In a separate paired comparison, cold PLTs had recovery of 23.1 ± 8.8%, survival of 33.7 ± 14.7 hours, and AUC of 540.2 ± 229.6 while TC PLTs had recovery of 36.5 ± 12.9%, survival of 49.0 ± 17.3 hours, and AUC of 1164.3 ± 622.2 (n = 4, AUC had p < 0.05). CONCLUSION: TC storage for 7 days produced PLTs with better in vivo circulation kinetics than cold storage but is not equivalent to RT storage.

Increase of plasma concentration to 10% improves a number of in vitro storage parameters of apheresis platelets suspended in a bicarbonate-containing additive solution and stored with a 24-hour interruption of agitation.

BACKGROUND: A previous study demonstrated several pH failures during 7-day storage of platelets suspended in 5% plasma/95% PAS-5 following a 24-hour interruption of agitation. The aim of this study was to investigate whether pH control improves in platelets stored in PAS-5 with 10% plasma following interruption of agitation. MATERIALS AND METHODS: Four aliquots were prepared from a single unit of apheresis platelets: two each with 5% and 10% plasma. After resting for 1 hour, the aliquots were placed on an agitator. On day 2, agitation of one aliquot with 5% plasma and another with 10% plasma was interrupted for 24 hours before the aliquots were returned to agitator. The two control aliquots remained on the agitator. An array of platelet parameters was measured on days 2, 5 and 7. RESULTS: On day 7, aliquots containing 10% plasma and subjected to interruption of agitation had a significantly higher mean pH compared to those of similarly treated aliquots containing 5% plasma (6.80±0.54 vs 6.41±0.57, p≤0.05). Platelets containing 10% plasma/95% PAS-5 subjected to interruption of agitation had a greater hypotonic stress response, greater shape change, higher mitochondrial membrane potential, decreased glucose utilisation and lower CD62P levels compared to those of similarly treated platelets suspended in 5% plasma. DISCUSSION: Increasing plasma concentration to 10% improves pH control and some in vitro platelet properties during 7 days of storage of platelets suspended in PAS-5 after a 24-hour interruption of agitation compared to those of similarly treated platelets suspended in 5% plasma/95% PAS-5.

Value of calcium and phosphate in a bicarbonate-containing platelet additive solution with low plasma levels in maintaining key in vitro platelet storage parameters.

BACKGROUND: Use of recently developed platelet (PLT) additive solutions (PAS) with 5% plasma levels may reduce the frequency and/or severity of transfusion reactions attributed to plasma. PLTs suspended in bicarbonate-containing PAS-5 with 5% plasma levels can maintain key PLT parameters during 7-day storage. This study evaluates the role of calcium and phosphate, as constituents of PAS-5, in maintaining PLT parameters. STUDY DESIGN AND METHODS: An Amicus apheresis PLT unit (n = 13) was equally divided into four 60-mL aliquots in CF-250 polyolefin bags. Four different formulations of PAS-5 were prepared: PAS-5, PAS-5 without phosphate (-PO4 ), PAS-5 without calcium (-Ca), and PAS-5 without Ca and phosphate (-Ca/-PO4 ). PLTs were centrifuged, and the supernatant was expressed and replaced with the respective PAS, yielding PLTs suspended in 95% PAS and 5% plasma. PLTs were stored at 20 to 24ºC with agitation for 7 days. PLT in vitro parameters were evaluated on Days 1, 5, and 7. RESULTS: In PLT PAS-5 aliquots, pH levels were maintained better compared with those in -Ca and -Ca/-PO4 aliquots. Glycolysis was greater in -Ca and -Ca/-PO4 PLT aliquots compared with PAS-5 aliquots. Hypotonic stress response and morphology were less and p-selectin (CD62P) binding was greater in -Ca/-PO4 PLT aliquots. The accumulation of reactive oxygen species was greater in -Ca/-PO4 PLTs. Phosphorylation of p38 mitogen-activated protein kinase (MAPK) was greater in -Ca and -Ca/-PO4 PLT aliquots during storage. CONCLUSION: The removal of calcium and phosphate from PAS-5 leads to the activation of p38 MAPK and deterioration of key PLT storage parameters.

Automated cold temperature cycling improves in vitro platelet properties and in vivo recovery in a mouse model compared to continuous cold storage.

BACKGROUND: Platelets (PLTs) stored at cold temperatures (CTs) for prolonged time have dramatically reduced bacterial growth but poor survival when infused. A previous study demonstrated that human PLTs stored with manual cycling between 4 °C (12 hr) and 37 °C (30 min) and infused into severe combined immunodeficient (SCID) mice had survivals similar to or greater than those stored at room temperature (RT). In this study, the in vitro and in vivo properties of PLTs stored in an automated incubator programmed to cycle between 5 °C (11 hr) and 37 °C (1 hr) were evaluated. STUDY DESIGN AND METHODS: A Trima apheresis unit (n = 12) was aliquoted (60 mL) in CLX bags. One sample was stored with continuous agitation (RT), a second sample was stored at 4-6 °C without agitation (CT), and a third sample was placed in an automated temperature cycler with 5 minutes of agitation during the warm-up period (thermocycling [TC]). PLTs were assayed for several relevant quality variables. On Day 7, PLTs were infused into SCID mice and in vivo recovery was assessed at predetermined time points after transfusion. RESULTS: The glucose consumption rate, morphology score, hypotonic shock recovery level, and aggregation levels were increased and mitochondrial reactive oxygen species accumulations were decreased in TC-PLTs compared to those of CT-PLTs. The pH and Annexin V binding were comparable to those of RT-PLTs. All TC-PLTs had greater recovery than CT-PLTs and were comparable to RT-PLTs. CONCLUSION: PLTs stored under automated TC conditions have improved in vivo recovery and improved results for a number of in vitro measures compared to CT-PLTs.

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.

In vitro variables of apheresis platelets are stably maintained for 7 days with 5% residual plasma in a glucose and bicarbonate salt solution, PAS-5.

BACKGROUND: Platelet additive solutions (PASs) facilitate improved recovery of plasma and may reduce the severity and/or frequency of plasma-associated transfusion reactions. Current apheresis platelet (PLT) PAS products contain approximately 30 to 40% residual plasma. In an effort to further decrease the residual plasma, two in vitro studies were conducted with PLTs suspended in 5% plasma and a reformulated PAS-3, named PAS-5, that contains additional salts, glucose, and bicarbonate. STUDY DESIGN AND METHODS: In Study 1, PLTs suspended in 5% plasma/95% PAS-5 were prepared directly on a separator (Amicus, Fenwal, Inc.) without additional centrifugation or washing. In Study 2, a double unit of hyperconcentrated Amicus PLTs in plasma was collected, divided, and centrifuged to prepare a control unit in 100% plasma and a paired test unit in 5% plasma/95% PAS-5. The in vitro properties of PLTs were assessed in both studies during 7-day storage at 20 to 24°C with continuous agitation. RESULTS: In Study 1, PLT concentration, pH, mean PLT volume (MPV), HCO(3)(-), pCO(2), pO(2), lactate dehydrogenase, and hypotonic shock response (HSR) did not significantly change during storage. By Day 7, glucose levels and morphology scores modestly decreased (17.6 and 14.4%, respectively) and lactate levels modestly increased (to 7.2 mmol/L). In Study 2, MPV, pH, glucose, pO(2), HSR, and morphology were comparable in control and test PLTs during 7-day storage. Glucose consumption and lactate production were significantly less in test versus control PLTs (p≤0.0015). Extent of shape change and %CD62P-positive test PLTs were less than those of controls (p<0.001). CONCLUSION: Apheresis PLTs suspended in 5% plasma/95% PAS-5 maintained in vitro properties during 7-day storage.

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

Comparison of the in vitro properties of apheresis platelets during 7-day storage after interrupting agitation for one or three periods.

BACKGROUND: Many platelet (PLT) components undergo multiple periods of shipment before transfusion. We have previously conducted studies investigating maintenance of apheresis PLT in vitro quality measures during a single 24- or 30-hour interruption of agitation, but data are not available for multiple periods without agitation. STUDY DESIGN AND METHODS: Apheresis PLTs were collected with both the Amicus (Fenwal, Inc.) and the Trima (Gambro BCT) cell separators to provide two identical PLT products, each with approximately 4 x 10(11) to 5 x 10(11) PLTs. One product was subjected to a single contiguous 24- or 30-hour period of interrupted agitation between Days 2 and 3 of storage by placement in a standard shipping box at room temperature. The matched product was not agitated on each of 3 days (Days 0, 1, and 3) for specified intervals totaling an identical period of time. RESULTS: Interrupting agitation for three periods resulted in greater maintenance of pH during storage than that observed using one contiguous period. These differences were significant for units held without agitation for 24 hours (Day 5, 0.08 pH units, p < 0.0001; Day 7, 0.10 pH units, p = 0.0059) and were also significant for units held without agitation for 30 hours (Day 5, 0.15, p < 0.0001; Day 7, 0.20, p < 0.0001). The two different interruption of agitation scenarios did not result in significant differences in the extent of shape change and hypotonic shock response variables after 5 or 7 days of storage. CONCLUSION: Apheresis PLTs subjected to three periods without agitation maintained overall pH levels slightly greater than those of matched units subjected to one contiguous period without agitation. Other measures showed comparability of PLT in vitro variables with the two scenarios for interruption of agitation.

Platelet products prepared by different methods of sedimentation undergo platelet activation differently during storage.

BACKGROUND: The activation marker CD40 ligand (CD40L) has recently been demonstrated to be released from the cytoplasm of platelets (PLTs) during storage. CD40L may be associated with some adverse transfusion reactions including febrile responses and transfusion-related acute lung injury. CD62P has been traditionally measured to assess PLT activation. This study compares the surface levels of CD40L and CD62P and accumulation of the soluble forms of these activation markers in the plasma of stored PLTs, prepared by PLT-rich plasma (PRP) or buffy coat (BC) methods and with two apheresis instruments. STUDY DESIGN AND METHODS: Individual PLT concentrates (PCs) were prepared in 100 percent plasma from a pool of two ABO-identical whole-blood units. Apheresis PLTs (APs) were prepared in 100 percent plasma using one of two commercially available cell separators (Amicus, Baxter Healthcare; and Trima, Gambro BCT). Surface expression of CD40L and CD62P was measured by flow cytometry, and secretion of soluble CD40L (sCD40L) and soluble CD62 (sCD62) was measured by enzyme-linked immunosorbent assay during 7 days of PLT storage. RESULTS: Secretion of sCD40L was greater in Amicus APs than in Trima APs during the first 3 days of storage. It was also greater in the PRP-PC preparations than in BC-PC preparations through the first day of storage. Surface expression of CD40L was low in all PLT preparations. Secretion of sCD62P was greater in Amicus APs than in Trima APs during the entire storage period and greater in PRP-PC than in BC during the first 5 days of storage. The percentage of CD62P-positive PLTs was greater in Amicus units than Trima units and greater in PRP-PC than BC-PC preparations during the first 5 and 3 days of storage, respectively. CONCLUSION: The kinetics of the secretion of CD40L are influenced by the method used to prepare PLTs for storage. The patterns for CD40L membrane association and secretion are different than those observed for CD62P during storage.

The influence of simulated shipping conditions (24- or 30-hr interruption of agitation) on the in vitro properties of apheresis platelets during 7-day storage.

BACKGROUND: Platelet (PLT) components undergo interruption of agitation during shipment. Studies have demonstrated maintenance of PLT quality of whole blood-derived PLT concentrates during a 24-hour interruption of agitation, but data are not available for apheresis PLTs in 100 percent plasma. STUDY DESIGN AND METHODS: Apheresis PLTs were collected with one of two commercially available separators (Amicus, Fenwal, Inc.; or Trima Accel, Gambro BCT) to provide two identical PLT products, each with approximately 3 x 10(11) to 4.5 x 10(11) PLTs. The control product was continuously agitated. The test product was subjected to a continuous 24- or 30-hour period of interrupted agitation between Day 2 and Day 3 of storage by placement in a standard shipping box at room temperature. RESULTS: Interrupting agitation for 24 or 30 hours influenced in vitro PLT properties to various degrees. After 5 days of storage, pH levels were judged to be well maintained after 24 hours without agitation for PLTs collected with both separators (pH < 6.2, Trima [0/12] and Amicus [0/12]). The changes in other variables associated with the retention of postinfusion viability were also considered limited and acceptable in units subjected to a 24-hour interruption of agitation. After 7 days of storage including a 24-hour interruption of agitation, Trima PLTs better maintained PLT properties compared to Amicus PLTs. With a 30-hour period, both Trima and Amicus PLTs were deemed satisfactory for pH at 5 days, but not at 7 days (pH < 6.2: Day 5, Trima [0/23] and Amicus [1/22]; Day 7, Trima [5/23] and Amicus [4/17]). CONCLUSION: Based on the retention of pH levels of at least 6.2, apheresis PLT quality was maintained for 5 days with a 24-hour and a 30-hour interruption of agitation.