Vox Sanguinis (2014) 107, 205–212 © 2014 International Society of Blood Transfusion DOI: 10.1111/vox.12172
REVIEW
Platelet storage media H. Gulliksson Department of Clinical Immunology and Transfusion Medicine, Karolinska University Hospital, and Karolinska Institutet, Stockholm, Sweden
Received: 6 February 2014, revised 8 May 2014, accepted 27 May 2014, published online 27 June 2014
Present platelet storage media often designated platelet additive solutions (PAS) basically contain acetate, citrate and phosphate and recently also potassium and magnesium. However, there seems to be an increasing interest in developing PASs that can be used also after further reduction of residual plasma content below 15–20% plasma. Inclusion of glucose but also calcium and bicarbonate in such solutions have been suggested to improve platelet (PLT) storage, especially when plasma content is reduced to very low levels. Results from a limited number of studies using novel PAS alternatives have been presented during the last years, such as InterSol-G, PAS-5, M-sol, PAS-G and SAS. Most of them are experimental solutions. The combined results presented in those studies suggest that presence of glucose may be necessary during PLT storage, primarily to maintain ATP at acceptable levels. At plasma inclusion below 15–20%, the content of glucose will generally be too low to support PLT metabolism for more than a few days making glucose addition in PAS necessary. Significant effects associated with presence of calcium was observed in PLTs stored in PAS with 5% inclusion but not with 20–35% plasma inclusion, suggesting that the content of plasma could be of importance. Bicarbonate only seems to be of importance for pH regulation, primarily when plasma inclusion is reduced to about 5%. Reduction in rate of glycolysis was observed in some PAS alternatives containing potassium and magnesium but not in others. Differences in pH or in concentrations of the various compounds included in PAS may be possible explanations. Additionally, novel PAS containing glucose, calcium and bicarbonate does not seem to be associated with improved in vitro results as compared to SSP+ or CompoSol when PLTs are stored with 35% plasma inclusion. The results would then also suggest that excess of glucose in novel PAS environment may not be associated with additional positive effects on PLT metabolism. This review is based on the few publications on novel PAS available, and additional studies would be needed in the future. Key words: bicarbonate, calcium, glucose, platelet additive solutions, platelet additive solutions-G, platelet storage media.
Introduction Correspondence: Hans Gulliksson, Department of Clinical Immunology and Transfusion Medicine, Karolinska University Hospital, Huddinge C2 66, SE-141 86 Stockholm, Sweden E-mail: [email protected] Review article by invitation by Dr. Leo van de Watering. This is a review of available literature about effects on platelets associated with novel platelet storage media supplemented with glucose, calcium and bicarbonate.
First, it should be emphasized that characteristics and quality of platelet (PLT) preparations are influenced by several factors, viz. (1) preparation method, such as whole-blood-derived or apheresis platelets, (2) storage environment, plasma or platelet storage medium often designated platelet additive solution (PAS) and (3) the platelet storage container that may be produced of different plastic materials and with different size. In this context, the storage environment including PAS
205
206 H. Gulliksson
storage environment are useful tools to optimize platelet storage conditions. The composition of some PAS alternatives of the present generation is presented in Table 2. They primarily include acetate, citrate and phosphate and the most recent PASs also potassium and magnesium corresponding to categories PAS-B to PAS-F. The scientific background of present PAS alternatives primarily represented by PAS-II and PAS-III was published in the 1990s and to some extent also in the late 1980s. The most recent PASs of the present generation primarily represented by PAS-IIIM (SSP+) is based on publications from the first decade of the 21st century [10]. In general, various PAS alternatives have been used for apheresis as well as BC-derived platelets. The storage medium normally is composed of a mixture of plasma (generally 20–50%) and PAS (50–80%). One significant difference is that in apheresis platelets, ACD is often preferred to CPD anticoagulant, because resuspension of platelets after preparation is facilitated. Glucose is not included in the present PAS-A to PAS-F generation (cf. Table 1). Glucose is generally supplied by a plasma fraction included as a complement to PAS. Consequently, initial glucose levels of 5–10 mM are normal. On the other hand, results of early studies by Holme et al. [11] and Gulliksson et al. [12] on the effects of PAS indicated that the presence of glucose during the entire platelet storage period is crucial for platelet metabolism. Effects observed after depletion of glucose involved rapid decrease in adenine nucleotide levels, cessation in lactate production and finally disintegration of platelets. A slight correlation with in vivo viability in terms of recovery below 50% occurs at ATP levels below 40 lmol/1011 PLTs [13]. Such ATP levels are often observed after depletion of glucose [12]. Glucose is metabolized to lactic acid by the glycolytic pathway resulting in decreasing pH levels. Glucose may also be further metabolized in the respiratory chain to carbon dioxide and water. Depletion of glucose is generally preceded by change in platelet metabolism in terms of increased rate of glycolysis and fall in pH levels. Those results suggested that depletion of glucose, not the pH
consequently is only one of those factors. This implies that results of platelet in vitro or in vivo studies using a certain storage environment may not be similar if the other factors involved are different. A fourth factor is the specific blood or platelet donor who may have platelets with specific characteristics as a consequence of genomic variation among donors. A number of PAS alternatives have been developed [1–8]. During the last decades, such additive solutions have been made available for transfusion purposes in many countries. PAS is generally used as a substitute for plasma to (1) reduce the amount of plasma transfused with platelets and to recover plasma for other purposes, primarily fractionation into plasma products; (2) avoid transfusion of large volumes of plasma to reduce incidence of adverse reactions and circulatory overload; (3) make possible photochemical treatment for the inactivation of bacteria and other pathogens in platelets using certain technique and (4) improve storage conditions [6]. A standard terminology for PAS has been developed to avoid confusion, for instance to avoid that two solutions with the same formulation will be given different official designations [9]. In order to simplify the terminology of PAS and the ISBT 128 product codes of their associated platelet products, a generic naming system for PAS has been adopted. At the moment, PAS is categorized from A to G (PAS-A to PAS-G) based on the components included in the specific PAS, but additional groups will most likely follow in the future. The composition associated with the naming system is outlined in Table 1.
The present generation of PAS The use of PAS has offered the possibility of including components with specific effects on platelets in the storage solution that are not present in plasma or in the anticoagulant. A number of effects have been observed that can be assigned to certain components [1–8]. Reducing platelet activation and improving platelet metabolism and function by inclusion of key components in the platelet
Table 1 Terminology of platelet additive solutions PAS
Citrate
Phosphate
PAS-A PAS-B PAS-C PAS-D PAS-E PAS-F PAS-G
X X X X X
X
X
X
X X
Acetate
Magnesium
Potassium
Gluconate
Glucose
X X X X X X X
X X X X
X X X X
X X X
© 2014 International Society of Blood Transfusion Vox Sanguinis (2014) 107, 205–212
Platelet storage media 207
Table 2 Composition of PAS alternatives of the present generation including commercial designations. As the compositions of solutions may be slightly different from each other, the exact composition is not indicated. The concentration of citrate is generally about 10 mM and the concentration of acetate about 30 mM Components
PlasmaLyte A
PAS-II/PAS-B (T-Sol, SSP)
PAS-III/PAS-C (InterSol)
Composol PS/PAS-D
PAS-III M/PAS-E (SSP+)
NaCl KCl MgCl2 Na3-citrate NaH2PO4/Na2HPO4 Na-acetate Na-gluconate
X X X – – X X
X – – X – X –
X – – X X X –
X X X X – X X
X X X X X X –
level alone, may be detrimental to platelets during storage. In contrast to plasma, the fall in pH during storage of platelets in the present generation of PAS will stop at a significantly higher level than about 60, often at pH levels of 65–68 as a result of the limited amount of glucose generally available in plasma-PAS storage media [12]. To improve this situation, efforts to increase buffering capacity for instance by addition of phosphate in PAS-C have been made (cf. Table 1). A number of effects have been observed that can be assigned to specific components either in PAS or even in the anticoagulant. However, the effects on PLT function and metabolism associated with different components in PAS are only partly known. There is also a complexity of effects and interdependence associated with such components. Citrate is derived from both the anticoagulant and PAS and is primarily included to avoid activation of coagulation. On the other hand, citrate and magnesium may also modify potassium efflux through the platelet membrane [14]. Citrate also induces platelets into a state of heightened responsiveness to some activating agents of PLTs such as ADP [15]. In addition, effects on the rate of glucose consumption and lactate production related to the concentration of citrate in PAS have been observed [16]. PLTs stored in a PAS medium with a citrate concentration of 8 mM produced only half the quantity of lactate as platelets in a similar medium with a citrate concentration of 14–26 mM [16]. Because no negative effects on in particular adenine nucleotide levels were observed, the results suggested that PAS preferably should include citrate at low concentrations to avoid excessive lactate production and acid pH. On the other hand, increased production of lactate associated with higher concentrations of citrate can be neutralized by addition of acetate [16]. However, a citrate concentration of at least 8 mM seems to be a minimum level, as clotting problems due to activation of coagulation can be expected at lower concentrations [17]. © 2014 International Society of Blood Transfusion Vox Sanguinis (2014) 107, 205–212
The metabolism of acetate present in PAS significantly stabilizes the pH level. In parallel with glucose, acetate is used as a substrate for platelet oxygen-dependent metabolism. Acetate is entered into the tricarboxylic acid cycle to form carbon dioxide and is further oxidized in the respiratory chain to water [18, 19]. The oxidation of organic ions like acetate in cells is preceded by its conversion to acetic acid. The hydrogen ion necessary for the conversion can be derived primarily from lactic acid. In this way, production of hydrogen ions by glycolysis may be balanced by removal of hydrogen ions by oxidation of acetate [20]. Thus, by formation of bicarbonate from the carbon dioxide produced by acetate, very stable pH levels are maintained during platelet storage. Furthermore, acetate has been evidenced to reduce production of lactate and increase oxygen consumption by platelets when present in synthetic media [20, 21]. Phosphate may have two different roles during storage of platelets: (1) as a buffer to prevent fall in pH and (2) as a stimulant of platelet glycolysis to increase production of lactic acid [21]. These two effects theoretically may compete and thereby at least partly neutralize each other. There are no indications of net utilization or production of phosphate during storage of platelets [21]. The most recent PAS alternatives with designation PAS-D and PAS-E (Composol PS and PAS-IIIM/SSP+) and the early Plasma Lyte A (PAS-F) contain magnesium and potassium. Except for the presence of citrate, Composol PS basically has the same composition as Plasma Lyte A. Magnesium, in combination with calcium, potassium and citrate, is associated with complexity of effects and interdependence. Effects on platelet membrane function and platelet activation as well as rate of glycolysis similar to those of the concentration of citrate in PAS have been described, and the various effects may even be combined [22–25]. Presence of extracellular magnesium ions significantly inhibits exposure of P-selectin, decreases binding of fibrinogen to ADP-activated platelets and significantly decreases agonist-induced platelet aggregation [22, 23].
208 H. Gulliksson
A new generation of PAS The present generation of PAS includes quite simple salt solutions lacking primarily glucose. Inclusion of glucose but also bicarbonate in such solutions was considered unpractical, as they could not be easily steam sterilized. Thus, from a production point of view, glucose-free solutions are preferred, as the presence of glucose would cause manufacturing problems due to the instability and caramelization of glucose during steam sterilization. Standard steam sterilization generally requires pH ranges lower than 55 to avoid caramelization of sugars. Such low pH levels are not compatible with successful platelet preservation. For this reason, designing of different PAS alternatives very much focused on non-glucose alternatives. In addition, bicarbonate is an unstable component in this context as it can escape as carbon dioxide. However, there seems to be an increasing interest in developing PASs that can be used in combination with further reduction of residual plasma levels below 15–20% plasma inclusion. In studies performed by Holme in the 1990s, unsatisfactory in vitro results were observed with platelets in a medium with
REVIEW
Platelet storage media H. Gulliksson Department of Clinical Immunology and Transfusion Medicine, Karolinska University Hospital, and Karolinska Institutet, Stockholm, Sweden
Received: 6 February 2014, revised 8 May 2014, accepted 27 May 2014, published online 27 June 2014
Present platelet storage media often designated platelet additive solutions (PAS) basically contain acetate, citrate and phosphate and recently also potassium and magnesium. However, there seems to be an increasing interest in developing PASs that can be used also after further reduction of residual plasma content below 15–20% plasma. Inclusion of glucose but also calcium and bicarbonate in such solutions have been suggested to improve platelet (PLT) storage, especially when plasma content is reduced to very low levels. Results from a limited number of studies using novel PAS alternatives have been presented during the last years, such as InterSol-G, PAS-5, M-sol, PAS-G and SAS. Most of them are experimental solutions. The combined results presented in those studies suggest that presence of glucose may be necessary during PLT storage, primarily to maintain ATP at acceptable levels. At plasma inclusion below 15–20%, the content of glucose will generally be too low to support PLT metabolism for more than a few days making glucose addition in PAS necessary. Significant effects associated with presence of calcium was observed in PLTs stored in PAS with 5% inclusion but not with 20–35% plasma inclusion, suggesting that the content of plasma could be of importance. Bicarbonate only seems to be of importance for pH regulation, primarily when plasma inclusion is reduced to about 5%. Reduction in rate of glycolysis was observed in some PAS alternatives containing potassium and magnesium but not in others. Differences in pH or in concentrations of the various compounds included in PAS may be possible explanations. Additionally, novel PAS containing glucose, calcium and bicarbonate does not seem to be associated with improved in vitro results as compared to SSP+ or CompoSol when PLTs are stored with 35% plasma inclusion. The results would then also suggest that excess of glucose in novel PAS environment may not be associated with additional positive effects on PLT metabolism. This review is based on the few publications on novel PAS available, and additional studies would be needed in the future. Key words: bicarbonate, calcium, glucose, platelet additive solutions, platelet additive solutions-G, platelet storage media.
Introduction Correspondence: Hans Gulliksson, Department of Clinical Immunology and Transfusion Medicine, Karolinska University Hospital, Huddinge C2 66, SE-141 86 Stockholm, Sweden E-mail: [email protected] Review article by invitation by Dr. Leo van de Watering. This is a review of available literature about effects on platelets associated with novel platelet storage media supplemented with glucose, calcium and bicarbonate.
First, it should be emphasized that characteristics and quality of platelet (PLT) preparations are influenced by several factors, viz. (1) preparation method, such as whole-blood-derived or apheresis platelets, (2) storage environment, plasma or platelet storage medium often designated platelet additive solution (PAS) and (3) the platelet storage container that may be produced of different plastic materials and with different size. In this context, the storage environment including PAS
205
206 H. Gulliksson
storage environment are useful tools to optimize platelet storage conditions. The composition of some PAS alternatives of the present generation is presented in Table 2. They primarily include acetate, citrate and phosphate and the most recent PASs also potassium and magnesium corresponding to categories PAS-B to PAS-F. The scientific background of present PAS alternatives primarily represented by PAS-II and PAS-III was published in the 1990s and to some extent also in the late 1980s. The most recent PASs of the present generation primarily represented by PAS-IIIM (SSP+) is based on publications from the first decade of the 21st century [10]. In general, various PAS alternatives have been used for apheresis as well as BC-derived platelets. The storage medium normally is composed of a mixture of plasma (generally 20–50%) and PAS (50–80%). One significant difference is that in apheresis platelets, ACD is often preferred to CPD anticoagulant, because resuspension of platelets after preparation is facilitated. Glucose is not included in the present PAS-A to PAS-F generation (cf. Table 1). Glucose is generally supplied by a plasma fraction included as a complement to PAS. Consequently, initial glucose levels of 5–10 mM are normal. On the other hand, results of early studies by Holme et al. [11] and Gulliksson et al. [12] on the effects of PAS indicated that the presence of glucose during the entire platelet storage period is crucial for platelet metabolism. Effects observed after depletion of glucose involved rapid decrease in adenine nucleotide levels, cessation in lactate production and finally disintegration of platelets. A slight correlation with in vivo viability in terms of recovery below 50% occurs at ATP levels below 40 lmol/1011 PLTs [13]. Such ATP levels are often observed after depletion of glucose [12]. Glucose is metabolized to lactic acid by the glycolytic pathway resulting in decreasing pH levels. Glucose may also be further metabolized in the respiratory chain to carbon dioxide and water. Depletion of glucose is generally preceded by change in platelet metabolism in terms of increased rate of glycolysis and fall in pH levels. Those results suggested that depletion of glucose, not the pH
consequently is only one of those factors. This implies that results of platelet in vitro or in vivo studies using a certain storage environment may not be similar if the other factors involved are different. A fourth factor is the specific blood or platelet donor who may have platelets with specific characteristics as a consequence of genomic variation among donors. A number of PAS alternatives have been developed [1–8]. During the last decades, such additive solutions have been made available for transfusion purposes in many countries. PAS is generally used as a substitute for plasma to (1) reduce the amount of plasma transfused with platelets and to recover plasma for other purposes, primarily fractionation into plasma products; (2) avoid transfusion of large volumes of plasma to reduce incidence of adverse reactions and circulatory overload; (3) make possible photochemical treatment for the inactivation of bacteria and other pathogens in platelets using certain technique and (4) improve storage conditions [6]. A standard terminology for PAS has been developed to avoid confusion, for instance to avoid that two solutions with the same formulation will be given different official designations [9]. In order to simplify the terminology of PAS and the ISBT 128 product codes of their associated platelet products, a generic naming system for PAS has been adopted. At the moment, PAS is categorized from A to G (PAS-A to PAS-G) based on the components included in the specific PAS, but additional groups will most likely follow in the future. The composition associated with the naming system is outlined in Table 1.
The present generation of PAS The use of PAS has offered the possibility of including components with specific effects on platelets in the storage solution that are not present in plasma or in the anticoagulant. A number of effects have been observed that can be assigned to certain components [1–8]. Reducing platelet activation and improving platelet metabolism and function by inclusion of key components in the platelet
Table 1 Terminology of platelet additive solutions PAS
Citrate
Phosphate
PAS-A PAS-B PAS-C PAS-D PAS-E PAS-F PAS-G
X X X X X
X
X
X
X X
Acetate
Magnesium
Potassium
Gluconate
Glucose
X X X X X X X
X X X X
X X X X
X X X
© 2014 International Society of Blood Transfusion Vox Sanguinis (2014) 107, 205–212
Platelet storage media 207
Table 2 Composition of PAS alternatives of the present generation including commercial designations. As the compositions of solutions may be slightly different from each other, the exact composition is not indicated. The concentration of citrate is generally about 10 mM and the concentration of acetate about 30 mM Components
PlasmaLyte A
PAS-II/PAS-B (T-Sol, SSP)
PAS-III/PAS-C (InterSol)
Composol PS/PAS-D
PAS-III M/PAS-E (SSP+)
NaCl KCl MgCl2 Na3-citrate NaH2PO4/Na2HPO4 Na-acetate Na-gluconate
X X X – – X X
X – – X – X –
X – – X X X –
X X X X – X X
X X X X X X –
level alone, may be detrimental to platelets during storage. In contrast to plasma, the fall in pH during storage of platelets in the present generation of PAS will stop at a significantly higher level than about 60, often at pH levels of 65–68 as a result of the limited amount of glucose generally available in plasma-PAS storage media [12]. To improve this situation, efforts to increase buffering capacity for instance by addition of phosphate in PAS-C have been made (cf. Table 1). A number of effects have been observed that can be assigned to specific components either in PAS or even in the anticoagulant. However, the effects on PLT function and metabolism associated with different components in PAS are only partly known. There is also a complexity of effects and interdependence associated with such components. Citrate is derived from both the anticoagulant and PAS and is primarily included to avoid activation of coagulation. On the other hand, citrate and magnesium may also modify potassium efflux through the platelet membrane [14]. Citrate also induces platelets into a state of heightened responsiveness to some activating agents of PLTs such as ADP [15]. In addition, effects on the rate of glucose consumption and lactate production related to the concentration of citrate in PAS have been observed [16]. PLTs stored in a PAS medium with a citrate concentration of 8 mM produced only half the quantity of lactate as platelets in a similar medium with a citrate concentration of 14–26 mM [16]. Because no negative effects on in particular adenine nucleotide levels were observed, the results suggested that PAS preferably should include citrate at low concentrations to avoid excessive lactate production and acid pH. On the other hand, increased production of lactate associated with higher concentrations of citrate can be neutralized by addition of acetate [16]. However, a citrate concentration of at least 8 mM seems to be a minimum level, as clotting problems due to activation of coagulation can be expected at lower concentrations [17]. © 2014 International Society of Blood Transfusion Vox Sanguinis (2014) 107, 205–212
The metabolism of acetate present in PAS significantly stabilizes the pH level. In parallel with glucose, acetate is used as a substrate for platelet oxygen-dependent metabolism. Acetate is entered into the tricarboxylic acid cycle to form carbon dioxide and is further oxidized in the respiratory chain to water [18, 19]. The oxidation of organic ions like acetate in cells is preceded by its conversion to acetic acid. The hydrogen ion necessary for the conversion can be derived primarily from lactic acid. In this way, production of hydrogen ions by glycolysis may be balanced by removal of hydrogen ions by oxidation of acetate [20]. Thus, by formation of bicarbonate from the carbon dioxide produced by acetate, very stable pH levels are maintained during platelet storage. Furthermore, acetate has been evidenced to reduce production of lactate and increase oxygen consumption by platelets when present in synthetic media [20, 21]. Phosphate may have two different roles during storage of platelets: (1) as a buffer to prevent fall in pH and (2) as a stimulant of platelet glycolysis to increase production of lactic acid [21]. These two effects theoretically may compete and thereby at least partly neutralize each other. There are no indications of net utilization or production of phosphate during storage of platelets [21]. The most recent PAS alternatives with designation PAS-D and PAS-E (Composol PS and PAS-IIIM/SSP+) and the early Plasma Lyte A (PAS-F) contain magnesium and potassium. Except for the presence of citrate, Composol PS basically has the same composition as Plasma Lyte A. Magnesium, in combination with calcium, potassium and citrate, is associated with complexity of effects and interdependence. Effects on platelet membrane function and platelet activation as well as rate of glycolysis similar to those of the concentration of citrate in PAS have been described, and the various effects may even be combined [22–25]. Presence of extracellular magnesium ions significantly inhibits exposure of P-selectin, decreases binding of fibrinogen to ADP-activated platelets and significantly decreases agonist-induced platelet aggregation [22, 23].
208 H. Gulliksson
A new generation of PAS The present generation of PAS includes quite simple salt solutions lacking primarily glucose. Inclusion of glucose but also bicarbonate in such solutions was considered unpractical, as they could not be easily steam sterilized. Thus, from a production point of view, glucose-free solutions are preferred, as the presence of glucose would cause manufacturing problems due to the instability and caramelization of glucose during steam sterilization. Standard steam sterilization generally requires pH ranges lower than 55 to avoid caramelization of sugars. Such low pH levels are not compatible with successful platelet preservation. For this reason, designing of different PAS alternatives very much focused on non-glucose alternatives. In addition, bicarbonate is an unstable component in this context as it can escape as carbon dioxide. However, there seems to be an increasing interest in developing PASs that can be used in combination with further reduction of residual plasma levels below 15–20% plasma inclusion. In studies performed by Holme in the 1990s, unsatisfactory in vitro results were observed with platelets in a medium with
