Original Paper Vox Sang 1992;62:87-93
K. 7: ShirnizuJ Shibata’ S. Kora
’
a
Aichi Center, Japanese Red Cross Blood Transfusion Service. Seto: Terumo Corporation, R& D Center, Kanagawa. Japan
First Autoclave-Sterilized Platelet= Additive Solution Containing Glucose with a PhysiologicalpH for the Preparation of Plasma-Poor Platelet Concentrates
................................................................................................. Abstract
The glucose-free platelet-additive solution (termed AR solution), developed by Adams and Rock [Transfusion 1988;28:217-2201, was modified by adding glucose as an energy substrate for platelets and maltose to prevent platelet lysis and by replacing sodium gluconate with sodium phosphate for better p H maintenance. The new platelet-additive solution (termed Set0 solution) contained 90 mM NaCl, 5 mM KCI, 3 mM MgC12, 17 mM tri-sodium citrate, 4.9 mMNaH2POJ,20.1 mM Na2HP04,23 mM sodium acetate, 28.8 mM maltose, and 23.5 mM glucose with a pH of 7.4. The solution was sterilized by autoclaving in plastic bags in nitrogen to prevent glucose caramelization at high pH. Plasma-poor platelet concentrates prepared by adding Set0 solution to the pelleted platelet buttons were stored in a LE-2 polyolefin bag at 22°C with constant agitation for 5 days. The platelets suspended in Set0 solution maintained oxygen consumption at a rate of 1.1 nmol/min/lOYplatelets after 5-day storage, with glucose consumption and lactate production rates of 0.5 f 0.2 and 1.2 f 0.2 nmol/min/lOYplatelets, respectively. This resulted in a final mean pH of 7.0. Those suspended in AR solution ceased glycolysis within 3 days because residual plasma glucose had been consumed. This was associated with decreases in percent hypotonic shock response and aggregation induced by adenosine diphosphate and collagen. Lactate dehydrogenase discharge in AR solution was 5 and 8 times higher at day 3 and day 5 , respectively, than that of Set0 solution. Morphologically, there were no ballooned platelets after storage in Set0 solution. Platelets stored in Set0 solution were nearly equal to those stored in plasma. Conclusions: Steam-sterilized Seto-additive solution caused better storage of platelets for 5 days than AR solution, suggesting that glucose is important for optimal storage. Phosphate acts as an effective buffer and maltose stabilizes platelet volumes.
Kece1vcd: June 211. IYW Revised rn:iiiu\cript received: May 17. I V J I Acccpted: May 18. I Y Y I
Tersuo Shimizu. PhD Aichi Center, Japanese Red Cross Blood Transfusion Seivice S3Y-3 Minami-Yamaguchi Seto. Aichi 4XY (Japan)
1YY2 S. Karger AG. Basel IKN2-9007/Y?/~Ki22-0087 $>.7S/I) h)
Table 1. Chemical compositions of platelet-additive solutions
Introduction
Studies by Adams et al. [l]and Holme et al. [2] showed that blood platelets could be stored in synthetic media for 5-7days with acceptable in vivo recovery and survival. In addition, Bertolini et al. [3] reported that the infusion of platelets stored in the synthetic medium, originally developed by Adams and Rock [4] (termed A R solution in this article), reduced the bleeding time in leukemic patients. Plasma-poor platelet concentrates (PCs) may have numerous advantages over those preserved in plasma. The ability to control the environment during storage should lead to a more consistent product. The removal of plasma from PCs should diminish the risk of transfusion of infectious agents, toxins, allergens and drugs, while providing additional plasma for fractionation. Platelet-additive solutions so far reported have a pH level of 7.4. Standard autoclave sterilization may, therefore, produce significant amounts of caramelized glucose in additive solutions. The additive solutions reported in references [l] and [2] were sterilized by filtration through a sterile filter. The A R solution lacked glucose. The present study was designed to develop a plateletadditive solution (termed Set0 solution) containing glucose with a physiological pH. Seto solution was autoclaved in plastic bags using technology developed to prevent formation of caramelized glucose. We compared in vitro changes in platelets stored in the new Set0 solution with those of the A R solution.
Materials and Methods Compositions of Additive Solutions The compositions of Seto solution and AR solution are shown in table 1. In Seto solution, sufficient amounts of glucose were included for the duration of storage, and sodium gluconate was replaced by sodium phosphate for stronger buffer activity. The concentration of trisodium citrate was slightly increased in Seto solution. In addition, maltose was added to the medium with the exception that it would prevent platelet lysissince maltose is effective in reducing the osmotic fragility of red cells [ 5 ] .Fourhundred milliliters of these experimental solutions were autoclave sterilized in 450-ml standard PVC bags at 121"C for 20 min in an apparatus for preventing glucose caramelization, in which, just before introducing steam into the autoclave apparatus, atmospheric oxygen was replaced by inert nitrogen gas. Colorization of Seto solution was checked by the absorbance at 410 nm with a spectrophotometer. Increase in absorbance was 0.01 after steam sterilization of Seto solution while, in the usual sterile condition, a 0.24 absorbance appeared.
NaCl KCI MgClz Na gluconate NaH?P04 Na2HP04 Na acetate Na, citrate Maltose Glucose PH
'
Seto additive solution
Glucose-free A R solution'
90 5 3
s9 5 3 22.8
-
4.9 20.1 23
17 28.8 23.5 7.4
-
26.8 13.5 -
-
7.4
Concentrations are given as millimoles. -=Not included. See ref. [4].
Platelet Preparation Within 3 h after apheresis platelet collection into a 1.5- LE-2 polyolefin (PO) bag [6] (Nissho Corporation, Osaka, Japan) with the CS-3000 system (Baxter Healthcare Corporation), the PCs were divided into at least 3 portions, each of which was transferred to a 300-1111 di-n-decyl phtalate (DnDP)-plasticized PVC bag (Terumo Corporation, Tokyo, Japan) [7]. The DnDP bags were then centrifuged at 2,740 g for 6 min to obtain pelleted platelet buttons. The centrifuged bag was laid on its side on the flat top surface of the flat table. The supernatant plasma was then transferred by gravity to a satellite PVC bag positioned 30 cm below the centrifuged bag. The bottom of the centrifuged bag was then raised so as to express as much residual plasma as possible. About 15 min later, 35 ml of Seto solution, A R solution or plasma were introduced at 2144°C into the centrifuged bag containing the platelet button, using asterile connection device (SCD) (SCD312 DuPontlHaemonetics). All of this was accomplished within4 h after platelet collection. After 1 h standing at room temperature (21-24°C). the PCs were agitated overnight on a flatbed rotator at 55 strokes/min to resuspend the pelleted buttons. Theresuspended PCswerefurtherstoredforupto5 daqsat22"C with continuous agitation on a to-and-fro flatbed agitator at 60 strokes/ min. About 450 ml of platelet-rich plasma (PRP) was collected in a 0.6LE-2 PO bag [8] usingaplasma collection system (PCS: Haemonetics Corporation). While, PRP derived from 400 ml CPD-anticoagulated whole blood was transferred to a 300-1111 LE-2 PO bag. Within 3 h after collection, PRPwascentrifuged at 2,740 gfor 6 min: all plasma in the bag was emptied into a satellite PVC bag as soon as possible, leaving 5-7 ml per bag. Within 15 min, 70 ml and 45 ml of Seto solution, respectively, were added to the pelleted platelet buttons derived from apheresis PRP and whole blood using the SCD. The resuspension and storage procedures were then the same as those described above. Platelet Function Tests PCs were diluted to2.5 X 10H/mlwith autologous plasma frozen on day 0. For PCs in plasma, one part of Set0 solution was included to
88
Shimizu/Shibata/Kora
Plasma-Poor PCs
standardize the test conditions. Hypotonic shock response (YOHSR) was performed using an NKK aggregometer (Hematracer, SSR Engineering. Tokyo, Japan) as described elsewhere [2], with some modifications. The diluted PC sample was kept in a closed plastic tube at 37°C for 30 min before testing. Testing was done by addition of a half-volume of either prewarmed distilled water or phosphate-buffered saline (pH 7.4). The YO HSR was determined after 10 min by expressing the decrease in light transmission occurring during recovery as a percentage of the total increase in light transmissioncorrected for dilution. Platelet aggregation responses were also determined using the aggregometer. After 40-50 min prewarming of diluted PCs sample at 37"C, aggregating agents such as collagen (final concentration 10 pglml). adenosine diphosphate (ADP; 10 p M ) , and the calcium inophore A23187 (10 p M ) were added. Aggregation was defined as the maximum change in transmittance after stimulation and expressed as percent of PPP. For the determination of the formation of platelet thromboxane Bz, supernatant samples of aggregates were frozen at -40°C for I week.
beads (calibrite, Becton Dickinson). The thousand platelets were analyzed in each sample to quantify the amount of platelet-bound antibody. Antibody binding was expressed as the mode fluorescence intensity. Statistical Analysis Values cited here are the arithmetic mean f SD (unless otherwise stated). A repeated measure ANOVA followed by a Duncan's multiple range test were used to determine significant diferences between the three groups of Seto solution, AR solution, and plasma. Significance level of (p
K. 7: ShirnizuJ Shibata’ S. Kora
’
a
Aichi Center, Japanese Red Cross Blood Transfusion Service. Seto: Terumo Corporation, R& D Center, Kanagawa. Japan
First Autoclave-Sterilized Platelet= Additive Solution Containing Glucose with a PhysiologicalpH for the Preparation of Plasma-Poor Platelet Concentrates
................................................................................................. Abstract
The glucose-free platelet-additive solution (termed AR solution), developed by Adams and Rock [Transfusion 1988;28:217-2201, was modified by adding glucose as an energy substrate for platelets and maltose to prevent platelet lysis and by replacing sodium gluconate with sodium phosphate for better p H maintenance. The new platelet-additive solution (termed Set0 solution) contained 90 mM NaCl, 5 mM KCI, 3 mM MgC12, 17 mM tri-sodium citrate, 4.9 mMNaH2POJ,20.1 mM Na2HP04,23 mM sodium acetate, 28.8 mM maltose, and 23.5 mM glucose with a pH of 7.4. The solution was sterilized by autoclaving in plastic bags in nitrogen to prevent glucose caramelization at high pH. Plasma-poor platelet concentrates prepared by adding Set0 solution to the pelleted platelet buttons were stored in a LE-2 polyolefin bag at 22°C with constant agitation for 5 days. The platelets suspended in Set0 solution maintained oxygen consumption at a rate of 1.1 nmol/min/lOYplatelets after 5-day storage, with glucose consumption and lactate production rates of 0.5 f 0.2 and 1.2 f 0.2 nmol/min/lOYplatelets, respectively. This resulted in a final mean pH of 7.0. Those suspended in AR solution ceased glycolysis within 3 days because residual plasma glucose had been consumed. This was associated with decreases in percent hypotonic shock response and aggregation induced by adenosine diphosphate and collagen. Lactate dehydrogenase discharge in AR solution was 5 and 8 times higher at day 3 and day 5 , respectively, than that of Set0 solution. Morphologically, there were no ballooned platelets after storage in Set0 solution. Platelets stored in Set0 solution were nearly equal to those stored in plasma. Conclusions: Steam-sterilized Seto-additive solution caused better storage of platelets for 5 days than AR solution, suggesting that glucose is important for optimal storage. Phosphate acts as an effective buffer and maltose stabilizes platelet volumes.
Kece1vcd: June 211. IYW Revised rn:iiiu\cript received: May 17. I V J I Acccpted: May 18. I Y Y I
Tersuo Shimizu. PhD Aichi Center, Japanese Red Cross Blood Transfusion Seivice S3Y-3 Minami-Yamaguchi Seto. Aichi 4XY (Japan)
1YY2 S. Karger AG. Basel IKN2-9007/Y?/~Ki22-0087 $>.7S/I) h)
Table 1. Chemical compositions of platelet-additive solutions
Introduction
Studies by Adams et al. [l]and Holme et al. [2] showed that blood platelets could be stored in synthetic media for 5-7days with acceptable in vivo recovery and survival. In addition, Bertolini et al. [3] reported that the infusion of platelets stored in the synthetic medium, originally developed by Adams and Rock [4] (termed A R solution in this article), reduced the bleeding time in leukemic patients. Plasma-poor platelet concentrates (PCs) may have numerous advantages over those preserved in plasma. The ability to control the environment during storage should lead to a more consistent product. The removal of plasma from PCs should diminish the risk of transfusion of infectious agents, toxins, allergens and drugs, while providing additional plasma for fractionation. Platelet-additive solutions so far reported have a pH level of 7.4. Standard autoclave sterilization may, therefore, produce significant amounts of caramelized glucose in additive solutions. The additive solutions reported in references [l] and [2] were sterilized by filtration through a sterile filter. The A R solution lacked glucose. The present study was designed to develop a plateletadditive solution (termed Set0 solution) containing glucose with a physiological pH. Seto solution was autoclaved in plastic bags using technology developed to prevent formation of caramelized glucose. We compared in vitro changes in platelets stored in the new Set0 solution with those of the A R solution.
Materials and Methods Compositions of Additive Solutions The compositions of Seto solution and AR solution are shown in table 1. In Seto solution, sufficient amounts of glucose were included for the duration of storage, and sodium gluconate was replaced by sodium phosphate for stronger buffer activity. The concentration of trisodium citrate was slightly increased in Seto solution. In addition, maltose was added to the medium with the exception that it would prevent platelet lysissince maltose is effective in reducing the osmotic fragility of red cells [ 5 ] .Fourhundred milliliters of these experimental solutions were autoclave sterilized in 450-ml standard PVC bags at 121"C for 20 min in an apparatus for preventing glucose caramelization, in which, just before introducing steam into the autoclave apparatus, atmospheric oxygen was replaced by inert nitrogen gas. Colorization of Seto solution was checked by the absorbance at 410 nm with a spectrophotometer. Increase in absorbance was 0.01 after steam sterilization of Seto solution while, in the usual sterile condition, a 0.24 absorbance appeared.
NaCl KCI MgClz Na gluconate NaH?P04 Na2HP04 Na acetate Na, citrate Maltose Glucose PH
'
Seto additive solution
Glucose-free A R solution'
90 5 3
s9 5 3 22.8
-
4.9 20.1 23
17 28.8 23.5 7.4
-
26.8 13.5 -
-
7.4
Concentrations are given as millimoles. -=Not included. See ref. [4].
Platelet Preparation Within 3 h after apheresis platelet collection into a 1.5- LE-2 polyolefin (PO) bag [6] (Nissho Corporation, Osaka, Japan) with the CS-3000 system (Baxter Healthcare Corporation), the PCs were divided into at least 3 portions, each of which was transferred to a 300-1111 di-n-decyl phtalate (DnDP)-plasticized PVC bag (Terumo Corporation, Tokyo, Japan) [7]. The DnDP bags were then centrifuged at 2,740 g for 6 min to obtain pelleted platelet buttons. The centrifuged bag was laid on its side on the flat top surface of the flat table. The supernatant plasma was then transferred by gravity to a satellite PVC bag positioned 30 cm below the centrifuged bag. The bottom of the centrifuged bag was then raised so as to express as much residual plasma as possible. About 15 min later, 35 ml of Seto solution, A R solution or plasma were introduced at 2144°C into the centrifuged bag containing the platelet button, using asterile connection device (SCD) (SCD312 DuPontlHaemonetics). All of this was accomplished within4 h after platelet collection. After 1 h standing at room temperature (21-24°C). the PCs were agitated overnight on a flatbed rotator at 55 strokes/min to resuspend the pelleted buttons. Theresuspended PCswerefurtherstoredforupto5 daqsat22"C with continuous agitation on a to-and-fro flatbed agitator at 60 strokes/ min. About 450 ml of platelet-rich plasma (PRP) was collected in a 0.6LE-2 PO bag [8] usingaplasma collection system (PCS: Haemonetics Corporation). While, PRP derived from 400 ml CPD-anticoagulated whole blood was transferred to a 300-1111 LE-2 PO bag. Within 3 h after collection, PRPwascentrifuged at 2,740 gfor 6 min: all plasma in the bag was emptied into a satellite PVC bag as soon as possible, leaving 5-7 ml per bag. Within 15 min, 70 ml and 45 ml of Seto solution, respectively, were added to the pelleted platelet buttons derived from apheresis PRP and whole blood using the SCD. The resuspension and storage procedures were then the same as those described above. Platelet Function Tests PCs were diluted to2.5 X 10H/mlwith autologous plasma frozen on day 0. For PCs in plasma, one part of Set0 solution was included to
88
Shimizu/Shibata/Kora
Plasma-Poor PCs
standardize the test conditions. Hypotonic shock response (YOHSR) was performed using an NKK aggregometer (Hematracer, SSR Engineering. Tokyo, Japan) as described elsewhere [2], with some modifications. The diluted PC sample was kept in a closed plastic tube at 37°C for 30 min before testing. Testing was done by addition of a half-volume of either prewarmed distilled water or phosphate-buffered saline (pH 7.4). The YO HSR was determined after 10 min by expressing the decrease in light transmission occurring during recovery as a percentage of the total increase in light transmissioncorrected for dilution. Platelet aggregation responses were also determined using the aggregometer. After 40-50 min prewarming of diluted PCs sample at 37"C, aggregating agents such as collagen (final concentration 10 pglml). adenosine diphosphate (ADP; 10 p M ) , and the calcium inophore A23187 (10 p M ) were added. Aggregation was defined as the maximum change in transmittance after stimulation and expressed as percent of PPP. For the determination of the formation of platelet thromboxane Bz, supernatant samples of aggregates were frozen at -40°C for I week.
beads (calibrite, Becton Dickinson). The thousand platelets were analyzed in each sample to quantify the amount of platelet-bound antibody. Antibody binding was expressed as the mode fluorescence intensity. Statistical Analysis Values cited here are the arithmetic mean f SD (unless otherwise stated). A repeated measure ANOVA followed by a Duncan's multiple range test were used to determine significant diferences between the three groups of Seto solution, AR solution, and plasma. Significance level of (p
