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.

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

Use of a red cell band 3-ligand/antioxidant to improve red cell storage properties following virucidal phototeatment with chalcogenoxanthylium photosensitizers.

The efficacy of two chalcogenoxanthylium photosensitizers to reduce pathogens in red cell suspensions while maintaining red cell storage properties was investigated in the presence and absence of the red cell band-3 ligand and antioxidant, dipyridamole (DP). In the presence of DP, both sensitizers, TMR-S and DJD-42, displayed potent virucidal photoactivity (>6 log10) and species-dependent bactericidal activities that ranged from 0.6 to 8.7 log10. Addition of DP to red cell suspensions containing TMR-S or DJD-42 reduced Day 42 photo-induced hemolysis by approximately eight-fold and four-fold, respectively. Red cell binding studies revealed only a small degree of competition between DP and TMR-S, and no competition between DP with DJD-42 for binding to red cell membranes, suggesting that protection of red cells by DP in this system may primarily stem from its antioxidant properties.

Thiazole orange, a DNA-binding photosensitizer with flexible structure, can inactivate pathogens in red blood cell suspensions while maintaining red cell storage properties.

BACKGROUND: Development of a robust pathogen reduction system for red cells (RBCs) utilizing photosensitive dyes has been constrained by hemolysis, usually mediated by reactive oxygen species emanating from dye free in solution as well as dye bound to the RBC membrane. The RBC binding properties of thiazole orange (TO), a flexible nucleic acid intercalating cyanine dye that predominantly acts as a photosensitizer only when bound, were assessed along with its virucidal, bactericidal, and light-induced hemolytic activities. STUDY DESIGN AND METHODS: Leukodepleted 20% hematocrit RBCs suspended in Erythrosol (RAS-2) were oxygenated, inoculated with test organisms, incubated with TO, and illuminated. Control and treated samples were analyzed by appropriate assay. Identically prepared, but uncontaminated samples were phototreated, concentrated to 45% hematocrit, and assayed for potassium leakage, hemolysis, and ATP during storage. RESULTS: Approximately 21 percent TO bound to RBCs. Phototreatment inactivated from 5.4 to 7.1 log(10) of 5 tested viruses and from 2.3 to greater than 7.0 log(10) of 8 tested bacteria. Phototreated RBCs exhibited only slightly increased hemolysis, moderately elevated potassium efflux, and similar levels of ATP compared to controls. CONCLUSION: TO can photoinactivate several model viruses and pathogens in RBCs under conditions that produce limited hemolysis without the addition of quenchers or competitive inhibitors.

Use of a flexible thiopyrylium photosensitizer and competitive inhibitor for pathogen reduction of viruses and bacteria with retention of red cell storage properties.

BACKGROUND: Progress in developing photochemical methods for pathogen reduction of red blood cells (RBCs) has been hampered by hemolysis. A flexible, nucleic acid-intercalating thiopyrylium (TP) dye that is only photochemically active in the bound state and a competitive inhibitor of RBC membrane binding, dipyridamole (DP), was used to reduce photoinduced hemolysis stemming from free- and membrane-bound dye. STUDY DESIGN AND METHODS: Oxygenated leukodepleted 20% hct RBC suspensions were deliberately inoculated with virus or bacteria, incubated with 200 micromol per L DP and less than or equal to 100 micromol per L TP, illuminated with 1.1 J/cm(2) of red light, and titered. RBC suspensions containing 200 micromol per L DP and 160 micromol per L TP were identically phototreated, concentrated to 45% hct, and assayed for RBC storage properties. RESULTS: In RBC suspensions containing DP, TP photoinactivated vesicular stomatitis virus, pseudorabies virus, duck hepatitis B virus, bovine virus diarrhea virus, extracellular human immunodeficiency virus (HIV) to the limit of detection and 6.2 log intracellular HIV. More than 5 log inactivation of 6 bacterial species was demonstrated. DP prevented approximately 30% of TP binding to RBCs. Phototreated RBCs that were subsequently stored for 42 days exhibited acceptable levels of hemolysis, morphology scores, extracellular pH, ATP, glucose utilization rates, and lactate production. Treated samples exhibited substantially increased potassium efflux compared to controls. CONCLUSION: Use of TP photosensitizer and DP enables significant levels of pathogen reduction while retaining most, but not all RBC properties during 42 day storage.

Evaluation of the sensitivity of red blood cell markers to detect photodynamic membrane damage.

BACKGROUND: Photodynamic dyes have been used to investigate pathogen reduction methods in RBC suspensions, but treatment with sensitizers and light can produce unwanted membrane damage during routine blood bank storage. This study compares the relative sensitivity of three assays for detecting RBC membrane damage. STUDY DESIGN AND METHODS: RBCs were treated with dimethylmethylene blue and red light-emitting diode light under four conditions differing in photodynamic stringency and subsequently stored under refrigerated conditions for up to 42 days. Hemolysis, potassium release, and the apoptosis marker annexin V binding were assayed immediately after phototreatment and following storage. RESULTS: In terms of increasing sensitivity for detecting photodynamic RBC membrane damage was enhanced ion leakage >> hemolysis > annexin V binding. CONCLUSION: Although very stringent photodynamic treatment conditions generate annexin V-positive RBCs, the assay will not detect more subtle membrane damage that is readily detected by other well-established methods. In addition, many RBCs destined for photo-induced hemolysis later in storage are annexin V-negative throughout the storage period, suggesting that the two measures are not directly related.