TRANSFUSION MEDICINE

REVIEWS Vol 13, No 3 July 1999

I I

The Efficacy of Synthetic Media in the Storage of Human Platelets for Transfusion

Scott Murphy

] ~ O R THREE DECADES, specialists in transfu- . ] ~ sion medicine have become accustomed to preparing, storing, and transfusing red blood cells suspended in synthetic storage solutions. The idea of a simple nutrient solution containing adenine and glucose for this purpose was apparently first described in 1965.1 It is now widely accepted practice. During storage at 4~ red cells maintain their viability longer in these solutions than they do in plasma.

Since the first report of Rock et al 2 in 1985, many investigators have directed their efforts toward developing :an analogous solution for platelets. Potential advantages include superior platelet qual- ity after storage; availability of more plasma for fractionation; and the reduction in frequency and severity of transfusion complications related to components of the suspending plasma. Although

�9 progress has been slower than was originally hoped, some succ;essful solutions have been devel- oped and are in use in some transfusion services in Europe.

The purpose of this review is to describe the events of the last 15 years that have led to our current circumstances. Inevitably, it will reflect the experience (and the biases) of the author. Where strong disagreements have been expressed, these are noted and addressed. Attention is confined to the storage ofplatelets with appropriate agitation at 20~ to 24~ 3 within plastic containers with gas permeability adequate to maintain the oxidative metabolism of the platelets being stored. 4 The optimal method for assessing the quality of stored platelets is the measurement of their capacity to circulate in vivo after reinfusion. Because of the

complexity and expense of such in vivo studies, some of the reports described have used in vitro correlates for in vivo viability. 5

Most of the studies reviewed have used platelets prepared by the platelet-rich plasma (PRP) method from units of whole blood. In this method, whole blood is centrifuged at low speed to prepare PRP, which is then centrifuged at high speed to prepare a platelet button, which is resuspended in approxi- mately 50 mL of autologous plasma, yielding a platelet concentrate (PC). A PC made by this method is referred to as PRP-PC. Typically, when PRP-PC have been used with synthetic media, the button is resuspended in medium, leaving a 10% to 20% plasma carryover. In later studies, the buffy coat (BC) method was used, in which the whole blood is centrifuged at high speed to prepare a BC from which a BC-PC is prepared. Finally, two studies used platelet concentrates prepared by apher- esis (AP-PC).

INITIAL STUDIES USING SYNTHETIC MEDIA FOR THE STORAGE OF PLATELETS

These studies used PRP-PC and studied the storage of platelets for 72 hours within a PL-146 container (poorly permeable to oxygen), using either plasma or a modified Tyrode's solution buffered with either histidine or phosphate. 2 Re-

From the American Red Cross Blood Services, Penn-Jersey Region, Philadelphia, PA.

Address reprint requests to Scott Murphy, MD, Chief Medical Officer, American Red Cross Blood Services, Penn-Jersey Region, 700 Spring Garden St, Philadelphia, PA 19123.

Copyright �9 1999 by W.B. Saunders Company 0887- 7963/99/1303-0001 $3.00/0

Transfusion Medicine Reviews, Vol 13, No 3 (July), 1999: pp 153-163 153

1 5 4 S C O T T M U R P H Y

sults of platelet aggregation studies were similar after storage in plasma or synthetic solutions, although there frequently was a frequent fall in pH in both groups because of the poor oxygen perme- ability of the container. Nonetheless, the results were encouraging.

In a subsequent report from the same laboratory, 6 platelets were resuspended in a medium prepared by mixing 0.5 mL 46.7% trisodium citrate and 0.5 mL 50% dextrose with 59 mL Plasma-Lyte A (Travenol Canada Inc, Mississauga, Ontario; 90 mmol/L NaC1, 23 mmol/L sodium gluconate, 27 mmol/L sodium acetate, 5 mmol/L KC1, 3 mmol/L MgC12, 294 mOsm/L, pH 7.4). After 5 days of storage, in vitro results from 14 studies and in vivo results using chromium-51 labeling from five stud- ies were excellent. In this study and those that were to follow, the authors noted a decline in the rate of production of lactate, during storage in their me- dium relative to that seen in plasma. This was ascribed simply to storage in a medium and not to any particular component of the medium used.

The authors recognized that glucose inclusion in the medium introduced a potential manufacturing disadvantage because glucose caramelizes during steam sterilization at neutral or alkaline pH; it does not do so at acid pH. In their next study, they performed in vitro studies suggesting that glucose could be omitted from the storage medium. 7 They reasoned that the small amount of glucose present as a result of plasma carryover (approximately 10% to 20% relative to total volume) was enough to support platelets for 5 days. In the final study in this series, 8 the authors performed further in vitro and in vivo studies to show that citrate could be omitted as

well as the glucose. Essentially, they were saying that Plasma-Lyte A (Table 1) alone with 11% to 17% plasma carryover was sufficient to allow successful platelet storage for 5 days. Clearly, the authors' work required confirmation, and there were many remaining questions: (1) Which of the components of Plasma-Lyte A were crucial? (2) What were the "optimal concentration ranges for these components? (3) What was the minimal amount of plasma carryover required for successful storage? (4) What was the explanation for the decrease in the rate of lactate production during storage in Plasma-Lyte A?

EVOLVING UNDERSTANDING OF PLATELET METABOLISM DURING STORAGE

During the 1980s, new knowledge was reported concerning the patterns of intermediary metabo- lism of platelets during storage at 20~ to 24~ 9 There are two major metabolic pathways: glycoly- sis, which does not require oxygen, and oxygen consumption through the tricarboxylic acid (TCA) cycle (Fig 1). In glycolysis, one molecule of glucose is converted into two molecules of lactic acid so that the lactate concentration rises by approximately 2.5 mmol/L day. It is crucial to recognize that little, if any, of the pyruvate interme- diate, derived from glucose, enters the TCA cycle. For platelets in this setting, glucose is not a significant substrate for oxidative metabolism. Rather, the major substrate appears to be plasma free fatty acids. 10

The rates of production of lactic acid and con- sumption of oxygen are essentially the same, approximately 1.0 to 1.5 mmol/day/10 ]2 platelets:

Table 1. Platelet Storage Media

Plasma Ly te A PAS P S M I - p H Setoso l PAS-2 PAS-3 Rock et al 8 H o l m e et a112 M u r p h y et al TM S h i m i z u et a 1 2 7 Gull ikssoi144 Corash 48

N a C I 9 0 . 0 1 1 0 . 0 9 8 . 0 9 0 . 0 1 1 5 . 5 7 7 . 0

K CI 5 . 0 5.1 5 . 0 5 .0 - - - -

CaC I2 - - 1 .7 . . . .

M g C I 2 3 . 0 - - - - 3 . 0 - - - -

M g S O 4 - - 0 . 8 . . . .

N a 3 c i t r a t e - - 1 5 . 2 2 3 . 0 1 7 . 0 1 0 . 0 1 2 . 3

C i t r i c a c i d - - 2 .7 . . . .

N a H C O 3 - - 3 5 . 0 . . . .

N a p h o s p h a t e - - 2 . 7 2 5 . 0 2 5 . 0 - - 2 8 . 0

N a a c e t a t e 2 7 . 0 - - - - 2 3 . 0 3 0 . 0 4 2 . 0

N a g l u c o n a t e 2 3 . 0 . . . . .

G l u c o s e - - 3 5 . 5 - - 2 3 . 5 - - - -

M a l t o s e - - - - - - 2 8 . 8 - - - -

N O T E . C o m p o n e n t s g i v e n i n m m o l / L .

PLATELET STORAGE IN SYNTHETIC MEDIA 155

Fig 1. Metabolic pathways used by platelets during storage in plasma or synthetic media. For energy production, platelets use glucose and free fatty acids from plasma and acetate if the latter is added to plasma or a synthetic medium. Almost all glucose used is converted through pyrbvate to lactate and a hydrogen ion. In plasma, the hydrogen ion is buffered by bicar- bonate, which is thereby con- verted to carbon dioxide, which can leave the medium through the walls of the container. Very little, if any, pyruvate is decarbox- ylated to acetyl coenzyme A (CoA) and COz; therefore, this pathway is indicated by inter- rupted lines. Rather, acetyl CoA for entry into the tricarboxylic acid (TCA) cycle is derived from free fatty acids in plasma or ac- etate if it has been, ~ added. The cycle oxidizes carbon atoms from acetyl CoA to CO2. Redrawn with minor modifications from Mur- phy ~1 with permission.

GLUCOSE ~, PYRUVATE z i

s i t I I

"4

F A T T ~ c A ; I A D T E - ~ T Y L ? " '"" ' -- . .~ C O 2 ~ "

o

/ TCA )

+ t, LACTATE + H

HCO3

Because the production of one lactate molecule fuels the regeneration of one adenosine triphos- phate (ATP) molecule and the consumption of one oxygen molecule regenerates six ATP molecules, the cell must derive approximately 85% of its ATP

�9 regeneration from oxygen Consumption through the TCA cycle and 15% through glycolysis. Because glucose metabolism contributes relatively little to ATP regeneration, these results lent support to the idea of omitting glucose from the platelet storage medium.

The end product of oxidative metabolism is carbon dioxide, a volatile acid that can leave the platelet suspension through the walls of the plastic container. Lactate and its accompanying hydrogen ion cannot do this. During storage in plasma, the hydrogen ion must be buffered by the only signifi- cant plasma buffer, bicarbonate. As each hydrogen ion is buffered, bicarbonate is converted into water and carbon dioxide. The latter leaves through the wall of the container. There is only enough bicarbon- ate in plasma to accommodate a rise in lactate

concentration to approximately 20 mmol/L. Be- yond that concentration, the pH will fall rapidly to levels in the mid to low 6s, where platelet viability will be lost. u

Again, such results were crucial in the design of subsequent platelet storage media. There is not enough bicarbonate in the plasma carried over into a platelet product to buffer all of the lactic acid produced should all of the glucose carried over be metabolized. This problem is magnified if glucose is included in the medium without an equivalent amount of buffer. Thus, successful storage solu- tions need to reduce the rate of glycolysis to a minimum and provide a mechanism to buffer the hydrogen ion that is produced by glycolysis. In thinking about the experiments of Rock et al 2,6,7,8 during that period, the source of the buffering capacity in Plasma-Lyte A was a mystery.

STUDIES OF HOLME ET AL

These authors developed a medium (platelet additive solution [PAS]) (Table 1) that contained

156 SCOTT MURPHY

the major, nonprotein ingredients in citrated plasma, including a high concentratio n of glucose, 35.5 mmol/L, and sodium bicarbonate, 35 mmol/L.t 2 Contrary to the conclusions of Rock et al, glucose was found to be essential to allow the maintenance of ATP levels33 In vitro results after 10 days of storage in PAS were similar to those for platelets stored for only 7 days in plasma. 14 In vivo results confirmed the in vitro results. 12

Results were improved still further by adding to the PAS a combination of inhibitors of platelet activation; 300 nmol/L prostaglandin E 1 (PGE1) and 1.9 mmol/L theophylline. In vitro findings indicating the possibility of extending storage t O 15 to 20 days,iS and subsequent in vivo studies 16 were interpreted as supporting storage for 14 days. However, mean in vivo recovery using isotopic labeling was only 23% after 14 days of storage. This is substantially poorer than results for storage for 5 to 7 days in plasma, which are generally 40% O r higher.

Furthermore, the authors acknowledged that PAS presented manufacturing problems. The carameliza- tion of glucose during sterilization at neutral pH has been mentioned. Furthermore, if the bicarbon- ate in a solution is packaged in a gas-permeable container at neutral or acid pH, it will be in continuous equilibrium with carbon dioxide, which can leave the solution through the walls of the container. Therefore, in a final iteration, 17 PAS was manufactured as two separate solutions. The first acid solution (pH = 5.5) contained citric acid, citrate, dextrose, adenine, sodium chloride, potas- sium chloride, calcium chloride, and magnesium chloride in water. The second alkaline solution (pH = 8.5) contained sodium bicarbonate and so- dium phosphate in water. The two solutions were sterilized and stored separately and added together at the time of platelet collection with a CS-3000 Plus Blood Cell Separator (Baxter Healthcare Corp, Deerfield, IL). The in vitro and in vivo characteris- tics of platelets stored in this solution for 5 days were excellent, equivalent to those of platelets stored in plasma.

With all of the work carried out by Holme et al,t2q7 there is little doubt that platelets stored in PAS have very high quality after 5 to 7 days of storage and perhaps longer, particularly if inhibi- tors of platelet activation are included in the medium. Nonetheless, there appears to have been very little work in the past 5 years with PAS or any

other solutions containing glucose and bicarbonate. Apparently, the complexity and expense of using two separate solutions have led to the conclusion that this approach would not be cost effective.

OMISSION OF GLUCOSE AND BICARBONATE

Murphy et a118 explored the idea that glucose might be omitted from a storage medium because it contributes only 15% to ATP regeneration. They did so recognizing that Holme et al had concluded that the presence of glucose was crucial. Because the production of lactic acid would be limited to that produced from platelet glycogen and the glucose in the plasma carryover, Murphy et al hoped that bicarbonate could be omitted as well. They began with an extraordinarily simple solution (called PSM-2) containing only sodium citrate, sodium chloride, potassium chloride, magnesium sulfate, and calcium chloride. In these studies, mean plasma carryover was 14.4%.

Unfortunately, as predicted by the metabolic considerations reviewed above, they observed a mean pH on day 5 of storage of 6.35 with values as low as 6.0. This follows from the basic fact that, in any volume of plasma, there is not enough bicarbon- ate t o buffer the lactic acid produced by the complete metabolism of the glucose present. These results again raised the question: what accounts for much greater stability of pH when platelets are stored in Plasma-Lyte A because neither acetate nor gluconate have buffering capacity a t physio- logic pH?

Murphy et al also studied PSM-lpH (Table 1), which was similar to PSM-2 but also contained 25 mmol/L Sodium phosphate to provide a buffering effect. Indeed, the phosphate was able to buffer the lactic acid produced to a great extent So that final pH was in the range 6.7 to 6.95. However, using isotopic labeling, three laboratories found a 10% to 35% reduction in in vivo recovery after 5 days of storage in PSM- lpH. 18

The cause of the inferior results in these studies is not known with certainty. The most straightfor- ward explanation is that Holme et al are right, and continuous availability of glucose-is required for optimal storage. Alternatively, the pH range at the end of storage, 6.7 to 6.95, is considerably lower than that characteristic of storage in plasma. Al- though these pHs are not dramatically low, they are sustained over several days in these circumstances and therefore might be deleterious. Interestingly,

PLATELET STORAGE IN SYNTHETIC MEDIA 157

free fatty acid levels were 90 to 155 and 150 to 175 ~mol/L on days 1 and 5; respectively. The authors' evidence suggests that, under these circumstances, p!atelets oxidize free fatty acids, Which are re- placed by free fatty acids made available by the hydrolysis of plasma triglycerides: 1~ Thus, there is not a problem with availability of free fat ty acids for oxidative metabolism even when the plasma carryover is quite low.

Nonetheless, these results were not Considered to be satisfactory, and there seemed to be a need foi new ideas.

UNDERSTANDING THE ROLE OF ACETATE IN PLATELET METABOLISM

A publication in 1990 introduced such an idea. GUppy et aP 9 studied the incubation of washed platelet suspensions in the presence of 14C-labeled substrateS such aS glucose and acetate, They con- firmed previous work 9 indicating that glucose is oxidized to a limited extent and that oXidative metabolism fUelS 70% to 100% of ATP turnover, They reported the presence of an unidentified "endogenous fuel" that could be oxidized if no exogenous substrate were added. However, most importantly, added acetate could completely re- place this "endogenous fuel." If present, its rate of oxidation was sufficient to account for all of the oxygen being consumed. They proposed that a platelet storage medium should contain an oxidiz- able fuel such as acetate. Finally, they performed preliminary storage studies and came to the conclu- sion that platelets were better preserved during storage, in the absence of glucose.

Of course, these results directed attention to the results of Rock et al and the acetate that was present in Plasma-Lyte A. Perhaps it is a crucially active, not an inert, component. Murphy has reviewed acetate's role in the metabolism of many ceils in many species and reported studies of his own concerning its metabolism by human platelets under the conditions of storage of PRP-PC in plasma. 2~ This review and report was published several years after the studies of storage in media containing acetate, which will be discussed. Its contents are presented at this point, out of chrono- logical order, to facilitate understanding of the subsequent material in this article.

Acetate is present in normal human plasma but at very low concentrations. It contributes very little to normal metabolism? ~ However, in other species as

diverse as the termite 22 and the cowY acetate is the major oxidative substrate. These creatures are ca- pable Of ingesting wood and grass, respectively. These are degraded by symbiotic microorganisms in the hindgut or tureen to acetate, which then enters their circulation to be metabolized. In hu- mans, acetate has been used as the anion against which patients with renal failure are dialyzed. Under these circumstances, acetate enters the circu- lation of the patient to achieve concentrations of approximately 5 mmol/L and provides 55~ to 73% of the body's oxidized fuel. z4 Benefit during homo- dialysis is derived from the strong alkalinizing effect of the oxidation of this fuel~ Its oxidation requires that a hydrogen ion be brought with the anion into the TCA cycle~ that is. acetate is metabolized as acetic acid (Fig 2). Thus. the alkalinizing effect is achieved as acetate anions and an equal number of hydrogen ions are metabolized to C02 by the tissues.

Murphy's work 2~ confirmed several of the strik- ing findings from Guppy et al. ~9 When acetate was added to platelets during storage in plasma, the acetate was vigorously metabolized, approximately 0.5 mmol/day/1012 platelets. Ninety percent of the metabolized acetate was found as CO2. Because two oxygen molecules are required to oxidize one acetate anion, acetate oxidation accounted for all of the oxygen being consumed, approximately 1.0 mmol/day/1012 platelets. Just as Guppy et al had found that the oxidation of acetate suppressed the oxidation of their "endogenous fuel," Murphy found that it suppressed the oxidation of free fatty acids. Fatty acids must be transferred into the mitochondria for oxidation by a complex series of reactions, using carnitine as a carrier, and then be broken down to acetyl CoA by the equally complex process of beta-0xidationY Acetate does not re- quire such a complex pathway. It is likely that acetyl CoA is formed relatively easily from it, and that acetyl CoA is a major inhibitor of the beta- oxidation of fatty acids. 26 Thus, suppression of fatty acid oxidation would be expected.

Furthermore, as predicted from the experience from hemodialysis patients and the concepts pre- sented in Figure 2, the oxidation of acetate had an alkalinizing effect during platelet storage. It spared the consumption of bicarbonate and allowed pro- longed stabilization of pH. The extent to which the rate of bicarbonate consumption was reduced was similar to the rate at which acetate was being

158 SCOTT MURPHY

GLUCOSE ~ LACTATE

H +

HCO3 ACETATE

(ACETIC ACID)

CO2 ACETYL COA

02

Fig 2. Beneficial effect of ac- etate metabolism on acid-base homeostasis during platelet stor- age. As shown in Figure 1, glu- cose is converted to lactate and hydrogen ion in glycolysis. This hydrogen ion may be buffered by bicarbonate, but acetate me- tabolism provides another buffer- ing mechanism. For acetate to be converted to acetyl CoA, it must carry a hydrogen ion with it. The equation for this conver- sion is: CH3COO + H + + CoA- SH = CH3CO-S-CoA + HzO. The oxidation of acetyl CoA in the TCA cycle results in the genera- tion of COz. Thus, buffering of hydrogen ion, by either bicarbon- ate or acetate metabolism, re- sults in the formation of the vola- tile acid, carbon dioxide, which can leave the platelet suspen- sion through the walls of the container.

metabolized. This would be predicted from the idea that the metabolism of each acetate anion uses a hydrogen ion.

In retrospect, acetate was probably included in Plasma-Lyte A to provide the functions described above when used for fluid replacement in patients. It provides carbon for metabolism and an alkaliniz- ing effect to offset the acidosis commonly seen in the sick and traumatized. Today, at the author's hospital, it is often included in solutions for paren- teral nutrition to achieve these twin purposes. To this day, the author of this article has been unable to determine why gluconate was included in Plasma- LyteA.

SETOSOL, A SOLUTION CONTAINING ACETATE, GLUCOSE, AND PHOSPHATE

Shimizu et al first reported on Setosol in 1992. 27 As shown in Table 1, Setosol combined many of the features of Plasma-Lyte A, PAS, and PSMI-pH. The gluconate in Plasma-Lyte A was replaced with phosphate as in PSMI-pH to provide buffering

capacity. Acetate was retained from Plasma-Lyte A to stimulate oxidative metabolism. Glucose and citrate were introduced as in PAS. After the conclu- sions of Holme et al, glucose was considered necessary for energy metabolism. The solution was autoclaved in a nitrogen rather than an oxygen atmosphere to prevent the caramelization of glu- cose already described as occurring at neutral pH. Finally, maltose was introduced to reduce osmotic fragility of the platelets. Storage was carried out with approximately 10% plasma carryover using PRP-PC.

During platelet storagefor 5 days in Setosol, the pH was uniformly very well maintained, mean = 7.03 on day 5; and the in vitro parameters rettectin~ platelet quality were excellent. In a-direct compari- son with platelets stored in Plasma-Lyte A, platelets stored in Setosol were superior in the hypotonic shock response test and in the prevention of platelet swelling, which was interpreted as a sign of platelet injury. The platelet concentrations were somewhat higher, mean = 1.7 • 109/mE, in these studies than

PLATELET STORAGE IN SYNTHETIC MEDIA 159

in the earlier studies of Rock et al. 68 Shimizu et a128 pointed out that glucose from plasma carryover was depleted by day 3 of storage in Plasma-Lyte A at these higher platelet concentrations. In a subse- quent study, platelets were washed to achieve less than 1% plasma carryover, and platelet quality appeared to be successfully maintained during storage for 3 days in Setosol. 28

Shimizu et al performed two subsequent stud- ies 29,3~ to define the mechanisms by which the superior results had been achieved with Setosol. Close to the results in Murphy's studies of storage of PRP-PC in plasma, acetate consumption rate was 0.6 mmol/day/101~ platelets, accounting for 85% of the oxygen consumed. In Setosol, the rate of lactate production was 60% of that in plasma, and, because of the mechanism defined in Figure 2, the fall in pH at any lactate-level achieved was markedly reduced relative to that seen with plate- lets stored in the absence of acetate. In fact, the mean ratio of acetate consumption/lactate produc- tion was 0.88, suggesting that, essentially by chance, approximately one molecule of acetate is con- sumed for each molecule of lactate that is pro- duced. This balat~ce explains the stability of pH according to the concept in Figure 2. The results clearly established acetate as a crucial component of a platelet storage medium.

The suppression of lactate production by the oxidation of acetate, which is obviously advanta- geous for platelet storage, has several potential explanations. The acetyl CoA formed from acetate condenses with oxaloacetate to form citrate, which then proceeds through the TCA cycle, where oxida- tive phosphorylation results in the synthesis of ATE Both citrate and ATP inhibit phosphofructokinase, the rate-limiting step in glycolysis, thus reducing the rate of lactate production. 31,32

A totally new finding in the studies of Shimizu et al was that at least 10 mmol/L phosphate was required to assure pH stability during storage in Setosol. In 8 Of 20 studies z9,3~ in which phosphate was omitted from the Setosol, there was acceler- ated lactate production beginning early in storage and marked fall in pH during storage. This accelera- tion of lactate production was never observed in approximately 50 studies in which at least 10 mmol/L phosphate was present. The mechanism of this beneficial effect of phosphate is not known. It may provide a buffering effect early in storage or prevent depletion of adenine nucleotides through

its inhibition of AMP deaminase? 3 The results contrast with those of Rock et al, who apparently never identified this problem using acetate alone in Plasma-Lyte A, which contains no phosphate.

Finally, in the last series of studies, 3~ it was shown that maltosewas not required for success.

STORAGE OF PLATELETS DERIVED FROM POOLED BUFFY COATS IN SYNTHETIC MEDIA

CONTAINING ACETATE

To this point in the discussion, with the excep- tion of Holme et al , 17 the platelets being stored have been from PRP-PC. In 1985, Pietersz et a134 described a method for obtaining platelets for transfusion from buffy coats. In 1989, Bertolini et aP 5 were the first to describe the use of pooled buffy coats and a synthetic storage medium essen- tially equivalent to Plasma-Lyte A. They were followed quickly in 1990 by Eriksson and Hog- man, 36 with a similar concept but a different medium. 36

The approach of Bertolini et al involved centrifu- gation of the whole blood unit at high speed to concentrate the platelets in the buffy coat, Seven bully coats were pooled with 350 mL Plasma-Lyte A and centrifuged slowly so that the platelet-rich supernatant (about 350 mL With a 30% plasma carryover and a platelet concentration about 1,0 • 109/mL) could be expressed into a 1,000-mL stor- age container with high oxygen permeability. In a subsequent iteration, 37 this supernatant was passed through a leukoreduction filter to obtain a leukore- duced product before storage,

Bertolini et al used in vitro and in vivo methods to demonstrate the very high quality of these preparations after prolonged storage. The hypo- tonic shock response and morphology score after 10 days of storage were equivalent to PRP-PC stored for only 5 days. In thrombocytopenic pa- tients, platelets prepared by this new method pro- duced in vivo increments, after 9 to 12 days of storage, similar to those produced by PRP-PC after 1 to 3 days of storage. Prolonged bleeding times were reduced after transfusion as well.

Two subsequent studies 38,39 were directed toward understanding how this method produced its out- standing results. Much that was difficult to under- stand at the time of publication is now clear with the new knowledge Concerning acetate metabo- lism. In vitro measurements reflecting platelet quality on day 15 of storage were the same as those

160 SCOTT MURPHY

obtained on day 7 for PRP-PC. It was shown that acetate conferred benefit and that gluconate did not, and that acetate was oxidized under theSe condi- tions Of storage. 39

The rate of Oxygen consumption was increased in .platelets prepared from buffy Coats relative to PRP-PC, and the rates of glucose consumption and lactate production were dramatically reduced even When compared with the rates seen during the storage of PRP-PC in Setosol. Thus, the mean lactate concentrations On day l and day t5 of storage Were 4.0 and 13.7 mmol/L, a rise Of 0.7 mmol/L/day. The rate of rise at similar platelet Concentrations in platelets prepared by the PRP method would have been approximately 1.7 mmol/ L/day, 2.5 times greater. 9

The glucose concentration at the start of storage was approXimately 6.0 mmol/L because of the 30% plasma Carryover. Glucose consumption rate, half of the lactate production rate, was 0.35 mmol/Ll day. Thus, there was enough glucose at the start to allow glucose to be present for a 17-day storage interval. The starting bicarbonate concentration was 8.3 mmol/L, provided by the plasma carryover. It did not change over the 15-day storage interval in spite of the lactate being produced. This is now easily understood (Fig 2),-because the rate of acetate oxidation (not measured in Bertolini et a138'39) was undoubtedly equal to the rate of lactate production.

Bertolini et al also studied PRP-PC stored under the same circumstances as platelets prepared from pooled buffy coats, that is, pooled concentrates in 30% plasma and 70% Plasma-Lyte A. Briefly stated, measurements reflecting platelet quality and metabolism were intermediate relative to the excel- lent results obtained with platelets from pooled buffy coats, and the poorer results obtained with PRP-PC prepared and stored in conventional fash- ion. Thus, as developmental work goes forward, it is very likely that we will find a storage medium with a given level of plasma carryover will be effective with platelets prepared by one method but not by another. As a final caveat, the platelet concentrations in the studies of Bertolini et al were low, approximately 0 .8 to 1.0 • 109/mL. It re- mained to be seen whether similar excellent results would be obtained at higher concentrations.

In the 3 years that followed the publications of Bertolini et at, several other groups have reported

that platelets prepared from buffy coats could be stored in synthetic media containing acetate, Three publications 4~ showed that platelets harveSted from single buffy coats coUtd be stored successfully in a medium essentially identical to Plasma-Lyte A with a plasma carryover as low as 10%. 41 In essence, these reports confirmed the results of Rock et al: using a different method of harvesting plates lets from donations Of whole blood.

Two Other groups 43-45 reported success with storage of pooled buffy coat plateletS in-,acetate - containing media using in vitro measurements refleCting platelet quality, and a third 4~ reported good results infusing such platelets into thr0mbocy- topenic patients. Gullikson et a143,44 expressed concern about the citrate concentration during storage. They reported that concentrations above 15 mmol/L were associated with excessive lactate production. Conversely, concentrations less than 8 mmol/L might be associated with fibrin formation. These concerns have lead m the introduction of PAS-2 (Table 1). which contains 310 mrnol/L citrate and 30 mmol/L acetate. PAS-2 is now in clinical use in Europe for the preparation and storage of platelets obtained from pooled buffy . coats.

STORAGE OF APHERESIS PLATELETS IN SYNTHETIC MEDIUM

The work of Holme et aP 7 using PAS, which contains glucose and bicarbonate but no acetate, has already shown that it is possible to store AP-PC in a synthetic medium with good in vivo viability. There has been very little work with AP-PC stored in acetate-containing media, in fact, the results of Klinger et a147 were poor. They suspended platelets obtained with a Baxter CS-3000 instrument in 10% plasma and 90% PAS-2. Glucose from the small amount of plasma carryover had been consumed by day 3 of storage, and the morphology of the platelets had deteriorated markedly by the end of storage relative to control platelets stored in plasma.

Conversely, Corash et al48 have reported in vivo viability in normal volunteers of mindium-Jabeled platelets prepared with the CS-3000 and stored for 5 days with 35% plasma and 65% new acetate- containing additive solution called PAS-3 (Table 1). Its major difference from PAS-2 is the inclusion of 28 mmol/L phosphate. Mean pH on day 5 was 7.0. Mean posttransfusion recovery and survival

PLATELE.T STORAGE IN SYNTHETIC MEDIA 161

were 50.3% and 6 days, respectively. One suspects that these improved results relative to those of Klinger et at derive from the higher plasma car- ryover and not the addition of phosphate to the storage medium, but this remains to, be determined.

DISSENTING POINTS OF VIEW

The research of Guppy et al has ted ttiem to a different point of v ieN particularly as regards glucose and ptasma caryover. 49 They have used PRP-PC from whole, blood obtained with a low- glucose anticoagulant and resuspended the plate- lets, virtually free of plasma and glucose, in a simple medium containing NaC1, KCt, Na phos- phate, Na citrate, and magnesium chloride. They report adequate storage by adding just one fuel, acetate. The literature does not contain a direct refutation of these contentions. However, taken as a whole, the data suggest that there may he a need for glucose, at Ieas~ in the plasma carryover, to obtain optimal results. Furthermore; media that are success, ful with 30% plasma carryover sometimes: fail when the: plasma carryover is reduced to approxi- mately I0%. Thus~ the data of: Guppy et al are of great interest but await confirmation.

CONCLUSIONS

The success of in vivo studies with the use. of synthetic media for storing platelets from pools of buffy coats 4~ and from apheresis48 convince the au~or of this article that such. media have great potential for wide use in ~ansfusion practice. Acetate appears to make a valuable contribution to such media,. The optimal concentratio~ of acetate remains to be de,ermined, but R should be at least 2 mmot/L for each day of projected storage. This was

the maximum rate of acetate consumption per day from the: data given in Murphy et al. 3~

The practice over the last few years has been to not ~nclude glucose, undoubtedly because of the: problems that it creates for manufacturing: The greatest success has been with a high rate of plasma carryover, approximately 30%, which provides glucose for the final: product, approximately 7.5 trffnol/L at the start of storage. The rate of glucose utilization rarely exceeds 1.0 mmol/L/day ~~ and is generally considerably less;, if acetate is present.. Therefore, this is enough glucose forat least 7 days. of storage, The need for glucose is debate& be- cause it contributes to, only approximately ~5% of ATP regeneration. Nonetheless, it may be required for the activity of the:hexose monophosphate shunt and for the provision of ATP for specific membrane functions such as Na+/K+ exchange, s~ This ques- tion deserves more study.

The issues with moving to: 10% to 20% plasma carryover are not well define& We f0und29,3~ that a significant minority of preparations showed acceler- ated lactate production in spite of the presence of acetate, unless at teas~ 10 mmol/L phosphate was present. Finally, the optimal concentration o f ci- trate is not defined.: Some 44: have recommended at least 8 mmot/L. It is expected that answers to these interesting questions will be provided over the next few years.

ACKNOWLEDGEMENTS

The author thanks the many friends and colleagues who have worked in this area over the lasr 15 years. In; particular, he wishes to recognize the inteIligence, creativity, and:hard work of Drs Stein Holme, Tetsuo Shimizu;. and Francesco BertolinL Without their input, this would have been a mucfi; shorter manuscript.

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