Storage of platelets in additive solutions: effects of magnesium and/or potassium

BACKGROUND: Previous studies indicate that platelet concentrates (PCs) in a platelet additive solution (PAS) containing citrate, acetate, and sodium chloride (PAS-2) show a significantly higher increase of CD62+ platelets than PCs in other brands of PAS containing Mg(2+) and K(+). To investigate whether this difference can be explained by the presence of Mg(2+) and/or K(+) in the storage medium, we performed paired studies comparing storage of PCs in PAS-2 to PAS-2 with either Mg(2+) or K(+) or both in combination. STUDY DESIGN AND METHODS: PCs from pooled buffy coats were prepared in either PAS-2 or PAS-2 with Mg(2+) or K(+) or both in combination (PAS-2 modified). Different volumes of MgCl(2) solution (1 mol/L) and/or KCl solution (1 mol/L) were added to PAS-2 to obtain various concentrations. After preparation and during storage (at Days 3 and 7), pH, pCO(2), pO(2), HCO(3)(-), and CD62 (%) were measured. RESULTS: During 7 days of storage, pH was very stable (6.9-7.2) in all PCs. At Day 7, platelet CD62 expression was 49 percent (PAS-2), 41 percent (PAS-2 with 1.5 mmol/L Mg(2+)), and 38 percent (PAS-2 with 4.5 mmol/L Mg(2+)). With added K(+), at Day 7, expression of CD62 was 55 percent (PAS-2), 39 percent (PAS-2 with 4.5 mmol/L K(+)), and 35 percent (PAS-2 with 9.0 mmol/L K(+)). In PAS-2 modified (PAS-2 with 1.5 mmol/L Mg(2+) and 4.5 mmol/L K(+)) and CPD plasma, the corresponding CD62 values were 23 and 35 percent, respectively. CONCLUSION: The combination of Mg(2+) and K(+) gave significantly (p < 0.05) lower platelet CD62 expression in the storage medium than in PAS-2. The effects of these differences on platelet metabolism and in vivo properties remain to be investigated.     

Storage of platelets in additive solutions: a new method for storage using sodium chloride solution

The in vitro effect of 6-day storage of platelets prepared from 6 pooled buffy coat (BC) units and stored in a platelet storage medium containing approximately 40 percent CPD-plasma and 60 percent platelet additive solution (PAS) was evaluated. PAS is composed of sodium and potassium chloride, citrate, phosphate, and mannitol. The total count of platelets per pooled unit included in the in vitro studies (n = 25) was 376 +/- 59 x 10(9) (mean +/- SD). The present study included three steps. 1. Evaluation of platelet storage in one (n = 7) and two (n = 6) 1000-mL polyolefin containers using PAS. During storage in one container, significantly lower values were found for pH, pO2, glucose, ATP, and the ratio of ATP to AMP+ADP+ATP. The values for mean platelet volume, pCO2, lactate, and extracellular adenylate kinase activity were significantly higher. These results indicate that storage in only one polyolefin container is not appropriate for maintaining satisfactory platelet quality. During storage in two polyolefin containers, a remarkably decreased lactate production (0.07 +/- 0.02 mmol/day/10(11) platelets) was noted. 2. PAS was substituted for saline during 6-day storage in two 1000-mL polyolefin containers (n = 12). The composition of the platelet preparations was the same in all other respects. Similar in vitro results were noted with PAS and saline, which indicated that PAS has no specific effect on the storage of platelets different from that of saline.(ABSTRACT TRUNCATED AT 250 WORDS)     

Storage of platelets in additive solutions: the effect of citrate and acetate in in vitro studies

The in vitro effects of storage of platelets prepared from 6 pooled buffy coat units and stored in a platelet storage medium consisting of CPD and plasma and different platelet additive solutions were evaluated. The total count of platelets per pooled unit included in the present investigation (n = 20) was 335 +/− 35 × 10(9) (mean +/− SD). Measurements of pH, pO2, pCO2, glucose, lactate, ATP, total adenine nucleotide content, and extracellular adenylate kinase activity were performed in a three-part study. The observations were 1) During storage in saline and citrate (10 mmol/L of citrate), the consumption of glucose and the production of lactate were significantly increased over the values with storage in saline, which were used as a reference. The values for pH at Day 6 were significantly lower. 2) The effects of different concentrations (10, 20, and 30 mmol/L) of acetate in saline were studied. With the exception of significantly higher pH values in saline and acetate, no significant differences were seen in the effects with saline and those with saline and acetate. 3) The combined effect of citrate and acetate was evaluated. The consumption of glucose and the production of lactate, the values for pO2, and extracellular adenylate kinase activity were significantly lower with saline and citrate and acetate than with saline and citrate. Significantly higher values for pH were found at Day 5.(ABSTRACT TRUNCATED AT 250 WORDS)     

Prolonged storage of red cells at 4 degrees C

One of the events associated with red cell storage at 4 degrees C is the development of an increasing proportion of echinocytes. Vesicles also may bud off the spicules, presumably leading to a decreased surface-to-volume ratio and decreased deformability. Pursuing the hypothesis that increasing the surface tension of the cells by increasing their volume might reduce the tendency toward echinocytosis and extend refrigerated storage time, packed red cells were resuspended in a solution hypotonic (210 mOsm) with respect to solutes that do not penetrate the cell. Since a reduced ionic concentration results in increased membrane permeability for cations, normal ionic concentration was maintained by the addition of NH4C1, which readily penetrates red cells and therefore contributes no osmotic support. Adenine, glucose, mannitol, citrate, and phosphate also were included. Unexpectedly, the predominant effect of red cell storage in this solution was a remarkable elevation of adenosine triphosphate (ATP). At 4, 8, and 10 weeks, (ATP) levels averaged 165, 135, and 110 percent of initial values, respectively. At 16 weeks, ATP still averaged 50 percent of initial values. Twenty-four-hour in vivo survival of red cells measured at 12 to 18 weeks ranged between 70 and 80 percent, and hemolysis ranged from 0.3 to 7.1 percent. Both the hypotonicity and the ammonium salt appear to be necessary for the high ATP.     

Generation of annexin V-positive platelets and shedding of microparticles with stimulus-dependent procoagulant activity during storage of platelets at 4 degrees C

BACKGROUND: The ability of propyl gallate to activate platelet factor 3 has been determined through the activated partial thromboplastin time, but its effect on phosphatidylserine has not been established. STUDY DESIGN AND METHODS: A novel platelet activator, propyl gallate, was introduced to a study of platelets stored at 4 degrees C. The effects of storage on platelet coagulation activity, on phosphatidylserine, and on the shedding of activated and activable membrane particles (microparticles) were examined by activated plasma clotting time, and the effect on annexin V binding was examined by gated flow cytometry. The ratios of annexin V binding and microparticle shedding in stored platelet samples were compared with those in fresh platelets stimulated with propyl gallate. RESULTS: Microparticle shedding by stored platelets compensated for the diminished procoagulant potential of intact platelets (shown as the total propyl gallate-dependent platelet factor 3 activity), which did not change during prolonged (20-day) storage, but levels of phosphatidylserine confined to microparticles increased dramatically as platelet counts fell. Both annexin V binding and microparticle shedding increased spontaneously with storage and artificially with propyl gallate stimulation. However, at the same level of annexin V binding, stored platelets shed more microparticles than did fresh platelets stimulated with propyl gallate. CONCLUSION: Propyl gallate induces platelet procoagulant activity and annexin V binding. Stored platelets differ from fresh platelets in a lower reactivity to propyl gallate activation and a higher rate of microparticle shedding.     

Washing platelets with new additive solutions: aspects on the in vitro quality after 48 hours of storage

BACKGROUND: Rare clinical conditions cause the need for washed platelet (PLT) concentrates (PCs). Saline-washed PCs can only be stored shortly, however, owing to lack of substrates for PLT metabolism. New PLT additive solutions (PASs) contain such substrates and might be used alternatively. The in vitro quality of apheresis PCs washed with Composol-PS or modified PAS-III (PAS-IIIM) stored up to 48 hours after wash was compared. STUDY DESIGN and METHODS: Twelve blood donors underwent two apheresis procedures (A and B) collecting 6.0 x 10(11) PLTs in 500 mL of plasma with a least 2 weeks in between. The PCs collected by Apheresis A were stored for 3 days and then split in two equal units before washing with Composol-PS or PAS-IIIM. The PCs collected by Apheresis B were split after collection. One unit was released for transfusion and 1 unit was stored unwashed up to Day 6 and used as reference unit. In vitro testing was performed before and after washing as well as 24 and 48 hours after wash. RESULTS: After 48 hours of postwash storage, the units washed with either PAS showed acceptable results for hypotonic shock response (HSR), P-selectin expression, and pH, whereas PLT aggregability was significantly impaired. Throughout the storage, unwashed units showed better in vitro quality. HSR and P-selectin expression were similar before and immediately after the washing procedure. CONCLUSION: Based on these in vitro results, 48-hour postwash storage of washed PCs with the two PASs seems to be feasible. In vivo recovery studies, however, must confirm this finding in the future.     

Elimination and multiplication of bacteria during preparation and storage of buffy coat-derived platelet concentrates

BACKGROUND: The prevalence of bacterial contamination of random-donor platelet concentrates (PCs) is considerably lower than that of blood donations. Which key steps of the preparation procedure contribute to the elimination of bacteria was investigated. STUDY DESIGN AND METHODS: Ten bacteria species were used. Blood donations were spiked with bacteria and stored at 22 degrees C for 8 hours. The buffy coats were kept for 6 hours. PCs were prepared from pools of 4 buffy coats. At each preparation step and during PC storage, bacteria contents were measured. In additional experiments, the titers of spiked blood and buffy coats were determined after storage at 20, 22, or 24 degrees C for 8 and up to 24 hours, respectively. RESULTS: Enterobacter cloacae, Escherichia coli, Pseudomonas aeruginosa, Serratia marcescens, and Yersinia enterocolitica were completely inactivated during storage in blood or buffy coats. Titer reduction was between 3.32 and 4.62 log. Bacillus cereus, Propionibacterium acnes, Staphylococcus aureus, and Staphylococcus epidermidis did not multiply. Compared with their values in spiked blood the titers in the PCs were reduced by 1.7 to 2.8 log. Klebsiella pneumoniae was the only species that grew in blood. With the exception of P. acnes, those species that were not removed by the preparation process multiplied in the PCs. Remarkable donor-to-donor variations of the bactericidal activities of buffy coats were detected when the storage time was prolonged to 24 hours. CONCLUSIONS: Bacteria are significantly eliminated by the preparation procedure for random donor PCs. Also, blood and buffy coats are bactericidal for most species. When buffy-coat storage is prolonged, it cannot, however, be predicted whether specific strains vanish or multiply.     

In vitro cell quality of buffy coat platelets in additive solution treated with pathogen reduction technology

BACKGROUND: Pathogen reduction technologies (PRTs) may induce storage lesion in platelet (PLT) concentrates. To investigate this, buffy coat PLTs (BCPs) in PLT additive solution (AS; SSP+) with or without Mirasol PRT (CaridianBCT Biotechnologies) were assessed by quality control tests and four‐color flow cytometry. STUDY DESIGN AND METHODS: In vitro comparison of PRT and control pooled‐and‐split BCPs after 2, 3, 6, 7, and 8 days of storage was made. PLT concentration, count per unit, swirl, metabolism, activation (CD62P, PAC1, CD42b/GPIb, CD63, CD40L/CD154, CD40, annexin V), and microparticle, sCD40L, and sCD62P release were evaluated. RESULTS: PRT induced a minor initial PLT loss (Day 2 [mean ± SD], 302 × 10⁹ ± 44 × 10⁹ PLTs/unit vs. 325 × 10⁹ ± 46 × 10⁹ PLTs/unit; p < 0.001) but the decline was comparable to control BCP. Swirling was comparable and declined with similar rates in PRT‐treated and control BCPs during storage. PRT enhanced PLT metabolism and activation, evidenced by lower pH₂₂; increased glucose consumption and lactate production rates (p < 0.01); early increases in CD62P‐, PAC1‐, CD63‐, CD40L‐, CD40‐, and annexin V–positive PLTs; reduced GPIb expression; and enhanced release of PLT‐derived MPs and sCD40L (all p < 0.05). CD62P and PAC1 expression changed with different kinetics during storage and varying GPIb expression was displayed within the CD62P/PAC1‐positive PLT subsets. CONCLUSION: PRT treatment of BCP in AS induced a minor initial PLT loss and enhanced metabolism and PLT activation. The clinical relevance for PLT function in vivo of these findings will be investigated in a clinical trial.     

In vitro properties of platelets stored in three different additive solutions

BACKGROUND: New platelet (PLT) additive solutions (PASs) contain compounds that might improve the storage conditions for PLTs. This study compares the in vitro function, including hemostatic properties (clot formation and elasticity), of PLTs in T‐Sol, Composol, or SSP+ during storage for 5 days. STUDY DESIGN AND METHODS: Fifteen buffy coats were pooled and divided into three parts. PLT concentrates (PCs) with 30% plasma and 70% PAS (T‐Sol, Composol, or SSP+) were prepared (n = 10). Swirling, PLT count, blood gases, metabolic variables, PLT activation markers, and coagulation by free oscillation rheometry (FOR) were analyzed on Days 1 and 5. RESULTS: Swirling was well preserved and pH acceptable (6.4‐7.4) during storage for all PASs. Storage of PLTs in T‐Sol led to a decrease in PLT count whereas the number of PLTs was unchanged in Composol or SSP+ PCs. PLTs in T‐Sol showed higher glucose metabolism than PLTs in Composol or in SSP+. At the end of storage PLTs in T‐Sol had higher spontaneous activation and lower ability to respond to an agonist than PLTs in Composol or SSP+. PLTs in all the PASs had a similar ability to promote clot formation and clot elasticity. CONCLUSION: Storage of PLTs in Composol or in SSP+ improved the quality of PCs in terms of better maintained PLT count, lower glucose metabolism, lower spontaneous activation, and improved response to a PLT agonist compared to PLTs in T‐Sol. PLTs stored in the various PASs had similar hemostatic properties. These findings make Composol and SSP+ interesting alternatives as PASs.     

Storage of platelets in balanced salt solutions: a simple platelet storage medium

When a commercially available intravenous solution, plasmalyte-A, was added to platelet pellets as a platelet storage medium (PSM) after more than 85 percent of the plasma had been expressed, the platelets functioned equally well, after 5 days of storage, in platelet aggregation, release reaction, and serotonin uptake as did platelets stored in plasma. The level of fibrinopeptide A was significantly different in the PSM. Similarly, lower levels of kallikrein and complement components in the PSM-stored platelets indicated reduced activation of plasma enzyme systems. Morphology scores showed better shape maintenance in PSM, with values of 255 versus values of 185 in plasma (time zero, 280); electron microscopy also showed somewhat better structural maintenance in PSM. Glucose consumption, lactate and ammonia production, and fatty acid oxidation were also reduced in PSM. No significant differences in pH, white cell count, pO2, pCO2, or lactic dehydrogenase were observed after 5 days of storage in plasma or PSM; however, platelet counts were reduced after 5 days in plasma but remained constant in PSM. The beta-thromboglobulin release was significantly lower in the PSM-stored platelets after 5 days. The volume of platelet membrane glycoprotein lb (which is responsible for platelet adhesion) decreased 15 percent in plasma following storage, whereas a decrease of only 5 percent was noted in PSM. There was less leaching of plasticizers in PSM than in plasma. Survival and recovery using 51Cr-labeled autologous platelets showed that, after 5 days of storage, the recovery of PSM-stored platelets was 63 percent and the survival was 7.7 +/- 0.8 days (multiple hit model).(ABSTRACT TRUNCATED AT 250 WORDS)     

Liquid storage at 4 degrees C of previously frozen red cells

Fresh human blood was collected in citrate-phosphate-dextrose, frozen by a high-glycerol technique, and stored at -80 degrees C. The red cells were thawed, deglycerolized, and resuspended in a final wash solution, ADSOL (Fenwal Laboratories), or an additive solution (AS) containing glucose, adenine, mannitol, and phosphate. The cells were then stored at 4 to 6 degrees C for 21 days and assayed weekly for adenosine triphosphate and 2,3 diphosphoglycerate, pH, glucose use, and lysis. AS and, to a lesser extent, ADSOL produced metabolic profiles similar to or better than profiles of cells not frozen and stored in commercially available additive solutions. AS offers a potential post-thaw preservative solution for red cells that would greatly increase the flexibility and reduce the expense of using frozen blood. A sterile post-thaw storage capability will make the stockpiling of frozen red cells a practical concept for both military and civilian blood banks.     

Increased thrombin sensitivity of human platelets after storage at 4 C

In vitro function of fresh platelets and platelets stored for 24 hours at 4 C and 22 C was studied using the release of ¹⁴C-5-hydroxytryptamine (5-HT), adenine nucleotides and calcium following stimulation with thrombin. Storage of platelets did not affect ¹⁴C-5-HT uptake capability, adenine nucleotide content or adenine nucleotide release. Platelets stored in 4 C varied from fresh platelets and 22 C-stored platelets with respect to 5-HT and calcium release. For measurement of 5-HT release, different thrombin-to-platelet concentrations were used. At thrombin-to-platelet concentrations of 0.013 to 0.02 U/10⁸ platelets, 4 C-stored platelets released more 5-HT than did fresh platelets. Differences between 4 C-stored platelets and 22 C stored platelets were observed over a wider range of thrombin-to-plate-lets concentrations. The 4 C-stored platelets released significantly greater amounts of 5-HT at thrombin-to-platelet concentrations of 0.008-0.02 L/ml. Calcium release, measured with the calcium electrode, was studied at high (1.0 L/ml) and low (0.06 U/ml) thrombin concentrations and at a platelet concentration of 8 × 10⁸ platelets/ml. At the high thrombin concentration, the amount of calcium released was the same for all platelets studied. At the low thrombin concentration, platelets stored at 4 C released a greater amount of calcium than did either fresh platelets or platelets stored at 22 C. The results obtained with different thrombin-to-platelet concentrations suggest that there is only a narrow range of thrombin concentrations per platelet at which the subtle differences between platelet populations may be detected. The range may reflect the threshold concentration of thrombin that is required to initiate a release reaction. The apparently lower thrombin threshold required by 4 C platelets to undergo a release reaction may be a factor contributing to their reduced circulation and superior hemostatic effectiveness.     

The new generation of platelet additive solution for storage at 22 degrees C: development and current experience

The storage of platelets (PLTs) in PLT additive solutions (PASs) might have several advantages. It can reduce allergic and febrile transfusion reactions, facilitate AB0-incompatible PLT transfusions, enable pathogen inactivation, and make more plasma available for other purposes (eg, for fractionation). For this reason, there has been considerable focus on the development of new PASs that assure maintenance of good PLT quality throughout storage. Several compounds in PASs such as citrate, acetate, phosphate, potassium, and magnesium have all turned out to be important, and the same applies to the necessary amount of glucose as determined by the plasma carryover. The latest generation of PASs, the modified PAS-III and Composol-PS, contains most or all of those compounds. Recently published data on the in vitro quality of either buffy coat- or apheresis-derived PLT concentrates stored in 70% or even 80% of PAS might encourage transfusion specialists to consider using these PASs in routine blood banking. However, because in vitro tests do not adequately predict clinical effectiveness of PLTs after transfusion, in vivo studies are still needed to assess the quality of PAS-stored PLTs.     

Serotonin uptake at 22 degrees C of stored platelets

In order to ascertain the possibility that platelet serotonin uptake may occur during storage of platelet concentrates (PC) at 22 degrees C with agitation, the high-performance liquid chromatographic procedure was applied to determine serotonin uptake by platelets. Studies at 22 degrees C showed that platelets stored for 4 days exhibited a significant serotonin uptake with a Vmax value of 2.4 X 10(-19) mole/platelet/min and a Km value of 0.62 X 10(-6) M. Incubation of PC with 5 X 10(-6) M serotonin for 1 day at 22 degrees C increased their serotonin contents from 2.2 to 4.2 X 10(-7) mole/10(11) platelets. Thrombin stimulation caused about 80% release of intracellular serotonin from fresh as well as stored platelets, which contained standard serotonin in the same amount as the original amount. These results suggest that a significant serotonin uptake of platelets might occur during in vitro storage at 22 degrees C and stored platelets have retained abilities to sequester extracellular serotonin into dense granules.     

Platelet storage lesion of WBC-reduced, pooled, buffy coat-derived platelet concentrates prepared in three in-process filter/storage bag combinations

BACKGROUND: With the implementation of universal WBC reduction in the United Kingdom, in-process WBC-reduction filters for pooled buffy coat (BC)-derived platelet concentrates (PCs) are used in routine production. The effects of three filter/storage bag combinations on platelet activation and microvesiculation and on the activation of coagulation were investigated. STUDY DESIGN AND METHODS: Using pooled BCs from the same donors, three filter/storage bag combinations (Autostop BC/CLX, Pall Biomedical; Sepacell PLX5/PL2410, Asahi Medical; and Imugard III-PL 4P/Teruflex, Terumo) were compared with unfiltered controls for their effects on microvesiculation and other storage-induced changes in platelets. Process efficiency was measured by platelet yield and residual WBC count. The storage changes were assessed: pH, activation of platelets measured by CD62P on the platelet surface and in supernatant plasma, quantitation of platelet-derived and RBC-derived microvesicles, cellular injury measured by annexin V in the supernatant plasma, and activation of the coagulation system measured by kallikrein-like and thrombin-like activities, prothrombin fragment 1+2, and thrombin-antithrombin complex. RESULTS: All three filters were comparable in terms of platelet recovery and WBC removal, and none induced immediate platelet activation or microvesiculation. With storage, platelet activation or microvesiculation increased in platelets prepared by all three filters and in unfiltered controls, but these effects were significantly less in the Imugard PCs than in controls. These findings were consistent with those for annexin V in the supernatant plasma, which were lower in Imugard PCs than in other products. Sepacell and Imugard filters reduced RBC-derived microvesicles to 50 percent of control levels, but the Autostop filter had no effect. On storage, levels of RBC-derived microvesicles in filtered products remained static, but levels in the unfiltered control doubled. Kallikrein- and thrombin-like activities were generated only by the Autostop filter without any further increment on storage. CONCLUSION: WBC-reduced pooled BC-PCs prepared by various filter/bag combinations were equivalent on Day 1 but differed during storage in terms of platelet activation or microvesiculation.     

血小板保养液与血浆混合保存血小板的初步研究

目的探讨血小板保养液与不同比例血浆混合保存单采血小板的效果。方法选用枸橼酸钠、醋酸钠、磷酸钠和氯化钠等组成血小板保养液,(22±2)℃振荡条件下保存单采血小板7d;按保养液与血浆混合比例,实验组分为实验Ⅰ组(50%保养液+50%血浆)和实验Ⅱ组(80%保养液+20%血浆),分别于1、3、5、7d取样检测血小板数(Plt)、pH值、葡萄糖消耗量、乳酸产生量和血小板膜CD62p的表达情况,并与100%血浆保存的血小板(对照组)比较。结果血小板保存到5d时,实验组与对照组比较Plt差异无统计学意义(P>0.05),其pH值较对照组均有明显下降(P<0.05),但pH仍>6.0;实验组血小板膜CD62p阳性表达率分别为32%和36%,比对照组的28%略高(P<0.05)。血小板保存到第7天时,Plt、pH和血小板膜CD62p阳性表达率,实验各组较对照组为差(P<0.05);1—7d葡萄糖平均消耗量实验Ⅱ组比对照组和实验Ⅰ组为高(P<0.05),而乳酸平均产生量则实验各组较对照组明显为高(P<0.05)。结论采用血小板保养液与20%或50%比例的血浆混合,短期(5d)保存单采血小板的pH值和血小板数量与用100%血浆保存单采血小板的效果基本相同,但保存在保养液与血浆混合液中的血小板更易激活。     

Altered processing of thawed red cells to improve the in vitro quality during postthaw storage at 4 degrees C

BACKGROUND: The use of a functionally closed system (ACP215, Haemonetics) for the glycerolization and deglycerolization of red blood cell (RBC) units allows for prolonged postthaw storage. In this study, the postthaw quality of previously frozen, deglycerolized RBCs resuspended in saline-adenine-glucose-mannitol (SAGM) or additive solution AS-3 was investigated. STUDY DESIGN AND METHODS: Leukoreduced RBC units were frozen with 40 percent glycerol and stored at -80 degrees C for at least 14 days. The thawed units were deglycerolized with the ACP215, resuspended in SAGM or AS-3, and stored at 2 to 6 degrees C for up to 21 days. RESULTS: The mean +/- standard deviation in vitro freeze-thaw-wash recovery was 81 +/- 5 percent. During storage, hemolysis of deglycerolized cells remained below 0.8 percent for 2 days in SAGM and for 14 days in AS-3. This difference was explained by the protective effect of citrate, which is present in AS-3. Cells stored in AS-3 showed a lower glycolytic activity and a faster decline in adenosine 5'-triphosphate (ATP) than cells in SAGM. Increasing the internal pH of cells before storage in AS-3 by use of phosphate-buffered saline (PBS) in the deglycerolization procedure resulted in elevated lactate production and better maintenance of intracellular ATP content. After 3 weeks of storage, the ATP content of PBS-washed cells amounted to 2.5 +/- 0.5 micromol per g of hemoglobin (Hb), whereas for saline/glucose-washed cells this value was decreased to 1.0 +/- 0.3 micromol per g of Hb. CONCLUSIONS: Leukoreduced, deglycerolized RBCs can be stored for 48 hours in SAGM. Improved ATP levels during refrigerated storage can be observed with thawed cells, resuspended in AS-3, when PBS is used as a washing solution.