Helen Awatefe - Academia.edu (original) (raw)

Papers by Helen Awatefe

PLoS ONE, 2013

Background and Objectives: Platelets during storage undergo diverse alterations collectively know... more 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. 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 20-24uC 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. Signaling through p38 MAPK, but not ERK, is associated with platelet deterioration during storage.

Transfusion, 2010

BACKGROUND: The gas permeability of platelet (PLT) storage bags influences the retention of in vi... more 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 R2 = 0.9204). Changes in MMP were correlated with higher levels of carbon dioxide (p = 0.0288) and PLT activation (p = 0.0178; multiple regression R2 = 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.

Transfusion, 2004

BACKGROUND: Photodynamic dyes have been used to investigate pathogen reduction methods in RBC sus... more 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.

Photochemistry and Photobiology, 2006

The efficacy of two chalcogenoxanthylium photosensitizers to reduce pathogens in red cell suspens... more 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.

Transfusion, 2005

BACKGROUND: Progress in developing photochemical methods for pathogen reduction of red blood cell... more 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 µmol per L DP and less than or equal to 100 µmol per L TP, illuminated with 1.1 J/cm2 of red light, and titered. RBC suspensions containing 200 µmol per L DP and 160 µmol 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.

Photochemistry and Photobiology, 2006

The efficacy of two chalcogenoxanthylium photosensitizers to reduce pathogens in red cell suspens... more 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 loglo) and species-dependent bactericidal actidties that ranged from 0.6 to 8.7 loglo. Addition of DP to red cell suspensions containing TMR-S or DJD-42 reduced Day 42 photoinduced 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.

Transfusion, 2006

BACKGROUND: Development of a robust pathogen reduction system for red cells (RBCs) utilizing pho... more 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 log10 of 5 tested viruses and from 2.3 to greater than 7.0 log10 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.

Transfusion, 2010

BACKGROUND: Prolonged periods without agitation produce platelet (PLT) storage lesions that resul... more 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.

Transfusion, 2010

BACKGROUND: The gas permeability of platelet (PLT) storage bags influences the retention of in vi... more 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 R2 = 0.9204). Changes in MMP were correlated with higher levels of carbon dioxide (p = 0.0288) and PLT activation (p = 0.0178; multiple regression R2 = 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.

Transfusion, 2009

BACKGROUND: Commercially available additive solutions (ASs) require 30% to 35% plasma for optimal... more 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 (≤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.

Transfusion, 2004

BACKGROUND: Photodynamic dyes have been used to investigate pathogen reduction methods in RBC sus... more 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.

Transfusion and Apheresis Science, 2010

We have previously conducted studies investigating maintenance of apheresis platelet in vitro qua... more 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 Ò separator to provide two identical platelet products, each with approximately 4-5 Â 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.

Photochemistry and Photobiology, 2006

The efficacy of two chalcogenoxanthylium photosensitizers to reduce pathogens in red cell suspens... more 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.

Transfusion, 2008

BACKGROUND: Extensive periods without agitation can occasionally occur during platelet (PLT) ship... more 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 ≤ 0.005).CONCLUSION: The in vitro properties of PLTs suspended in 70 percent M-sol/30 percent plasma and subjected to a 30-hour interruption of agitation are better maintained during 7-day storage than those of matched units suspended in plasma.

Transfusion, 2005

BACKGROUND: Progress in developing photochemical methods for pathogen reduction of red blood cell... more 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 µmol per L DP and less than or equal to 100 µmol per L TP, illuminated with 1.1 J/cm2 of red light, and titered. RBC suspensions containing 200 µmol per L DP and 160 µmol 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.

Photochemistry and Photobiology, 2006

The efficacy of two chalcogenoxanthylium photosensitizers to reduce pathogens in red cell suspens... more 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 loglo) and species-dependent bactericidal actidties that ranged from 0.6 to 8.7 loglo. Addition of DP to red cell suspensions containing TMR-S or DJD-42 reduced Day 42 photoinduced 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.

Transfusion, 2009

BACKGROUND: Amotosalen, a psoralen, has been utilized for photochemical treatment (PCT) of aphere... more 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/cm2 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.

Transfusion, 2006

BACKGROUND: Development of a robust pathogen reduction system for red cells (RBCs) utilizing pho... more 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 log10 of 5 tested viruses and from 2.3 to greater than 7.0 log10 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.

Transfusion, 2010

BACKGROUND: Prolonged periods without agitation produce platelet (PLT) storage lesions that resul... more 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.

Transfusion, 2009

BACKGROUND: Commercially available additive solutions (ASs) require 30% to 35% plasma for optimal... more 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 (≤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.

PLoS ONE, 2013

Background and Objectives: Platelets during storage undergo diverse alterations collectively know... more 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. 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 20-24uC 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. Signaling through p38 MAPK, but not ERK, is associated with platelet deterioration during storage.

Transfusion, 2010

BACKGROUND: The gas permeability of platelet (PLT) storage bags influences the retention of in vi... more 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 R2 = 0.9204). Changes in MMP were correlated with higher levels of carbon dioxide (p = 0.0288) and PLT activation (p = 0.0178; multiple regression R2 = 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.

Transfusion, 2004

BACKGROUND: Photodynamic dyes have been used to investigate pathogen reduction methods in RBC sus... more 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.

Photochemistry and Photobiology, 2006

The efficacy of two chalcogenoxanthylium photosensitizers to reduce pathogens in red cell suspens... more 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.

Transfusion, 2005

BACKGROUND: Progress in developing photochemical methods for pathogen reduction of red blood cell... more 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 µmol per L DP and less than or equal to 100 µmol per L TP, illuminated with 1.1 J/cm2 of red light, and titered. RBC suspensions containing 200 µmol per L DP and 160 µmol 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.

Photochemistry and Photobiology, 2006

The efficacy of two chalcogenoxanthylium photosensitizers to reduce pathogens in red cell suspens... more 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 loglo) and species-dependent bactericidal actidties that ranged from 0.6 to 8.7 loglo. Addition of DP to red cell suspensions containing TMR-S or DJD-42 reduced Day 42 photoinduced 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.

Transfusion, 2006

BACKGROUND: Development of a robust pathogen reduction system for red cells (RBCs) utilizing pho... more 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 log10 of 5 tested viruses and from 2.3 to greater than 7.0 log10 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.

Transfusion, 2010

BACKGROUND: Prolonged periods without agitation produce platelet (PLT) storage lesions that resul... more 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.

Transfusion, 2010

BACKGROUND: The gas permeability of platelet (PLT) storage bags influences the retention of in vi... more 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 R2 = 0.9204). Changes in MMP were correlated with higher levels of carbon dioxide (p = 0.0288) and PLT activation (p = 0.0178; multiple regression R2 = 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.

Transfusion, 2009

BACKGROUND: Commercially available additive solutions (ASs) require 30% to 35% plasma for optimal... more 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 (≤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.

Transfusion, 2004

BACKGROUND: Photodynamic dyes have been used to investigate pathogen reduction methods in RBC sus... more 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.

Transfusion and Apheresis Science, 2010

We have previously conducted studies investigating maintenance of apheresis platelet in vitro qua... more 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 Ò separator to provide two identical platelet products, each with approximately 4-5 Â 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.

Photochemistry and Photobiology, 2006

The efficacy of two chalcogenoxanthylium photosensitizers to reduce pathogens in red cell suspens... more 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.

Transfusion, 2008

BACKGROUND: Extensive periods without agitation can occasionally occur during platelet (PLT) ship... more 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 ≤ 0.005).CONCLUSION: The in vitro properties of PLTs suspended in 70 percent M-sol/30 percent plasma and subjected to a 30-hour interruption of agitation are better maintained during 7-day storage than those of matched units suspended in plasma.

Transfusion, 2005

BACKGROUND: Progress in developing photochemical methods for pathogen reduction of red blood cell... more 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 µmol per L DP and less than or equal to 100 µmol per L TP, illuminated with 1.1 J/cm2 of red light, and titered. RBC suspensions containing 200 µmol per L DP and 160 µmol 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.

Photochemistry and Photobiology, 2006

The efficacy of two chalcogenoxanthylium photosensitizers to reduce pathogens in red cell suspens... more 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 loglo) and species-dependent bactericidal actidties that ranged from 0.6 to 8.7 loglo. Addition of DP to red cell suspensions containing TMR-S or DJD-42 reduced Day 42 photoinduced 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.

Transfusion, 2009

BACKGROUND: Amotosalen, a psoralen, has been utilized for photochemical treatment (PCT) of aphere... more 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/cm2 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.

Transfusion, 2006

BACKGROUND: Development of a robust pathogen reduction system for red cells (RBCs) utilizing pho... more 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 log10 of 5 tested viruses and from 2.3 to greater than 7.0 log10 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.

Transfusion, 2010

BACKGROUND: Prolonged periods without agitation produce platelet (PLT) storage lesions that resul... more 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.

Transfusion, 2009

BACKGROUND: Commercially available additive solutions (ASs) require 30% to 35% plasma for optimal... more 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 (≤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.