The maintenance of platelet properties upon limited discontinuation of agitation during storage

Platelet concentrates are routinely stored with continuous agitation but may need to be maintained without agitation for substantial time periods. Studies were conducted in vitro to assess the retention of platelet properties after the discontinuation of agitation. Platelets were maintained without agitation in an insulated cardboard container. In one study, platelet concentrates were kept at 20 to 24 degrees C for the entire 24-hour period. In another study, they were kept at 37 degrees C for 6 hours with subsequent storage at ambient room temperature for the remainder of the 24-hour holding period. Under the simulated shipping conditions, discontinuation of agitation for 24 hours between Days 2 and 3 of a 7-day storage period minimally influenced the maintenance of a series of in vitro platelet properties. The maintenance of platelet concentrates at 20 to 24 degrees C, sitting undisturbed on a bench top for 9 hours, after storage for 6 days with continuous agitation, also had no damaging influence.     

New insulation technology provides next-generation containers for "iceless" and lightweight transport of RBCs at 1 to 10 degrees C in extreme temperatures for over 78 hours

BACKGROUND: There is a universal need, in both civilian and military settings, for a lightweight container capable of maintaining RBCs at 1 to 10 degrees C in remote areas, during extended transit times, and under austere environments. The use of ice in insulated containers or small commercial coolers for these purposes often results in loss of RBCs due to failure to maintain temperatures within the requisite range. A lightweight and thermally efficient container capable of carrying 4 to 6 units of RBCs at 1 to 10 degrees C for over 72 hours under extreme conditions would help resolve current problems in RBC transportation. STUDY DESIGN AND METHODS: Six different prototype containers incorporating phase-change materials (PCMs) in their designs were evaluated for their ability to maintain RBCs between 1 and 10 degrees C while exposed to external temperatures of -24 degrees C and 40 degrees C. In separate experiments, a container was opened and a RBC unit removed. RESULTS: One container weighing 10 pounds with four units of RBCs was capable of maintaining the temperature of the units between 1 and 10 degrees C for over 78 hours, 96 hours, and 120 hours at 40 degrees C, -24 degrees C, and 23 degrees C, respectively. Opening the container decreased these times by 2 to 3 hours. CONCLUSIONS: An energy-efficient and lightweight container that maintains RBCs at 1 to 10 degrees C under austere environments for over 78 hours is now available. This container, known as the Golden Hour container (GHC), will facilitate transport of RBCs. The GHC will have additional applications (transport and/or storage of vaccines, other biologics, organs, reagents, etc).     

Dependence of the number of platelets maintaining aerobic metabolism on the size of storage containers

In order to elucidate the role of the size (surface area) of the storage container in maintaining the oxidative metabolism of platelets during in vitro storage at 22 degrees C, human platelets were stored as platelet concentrates (PC) with plasma in conventional polyvinylchloride plastic containers of various sizes for 3 days at 22 degrees C. Oxygen permeability of the container was 1.5 nmole/min/atm/cm2. Levels of the partial oxygen pressure (PO2) of PC depended on both container size and total number of platelets. The larger the containers, the higher was the PO2. PO2 linearly decreased to 16 mmHg with increasing platelet number. In well-oxygenated PC, 93% of the total ATP production was through the oxidative phosphorylation. With further increases in platelet number in the container, PO2 maintained low levels. The increases in glucose consumption and concomitant lactate production occurred to compensate the oxygen debt. The pH fall in PC depended on the degree of glycolysis. The partial carbon dioxide pressure (PCO2) increased with increasing platelet number. However, the larger the container, the lower was the PCO2. When PC were stored in 6 different-sized containers, the number of platelets, at which PO2 was 16 mmHg, was correlated with the size of the container. The present data indicate that the amounts of oxygen entering a container, which depended on container size, may determine the number of platelets maintaining oxidative metabolism.     

Storage of apheresis platelets after gamma radiation

BACKGROUND: There are conflicting data on the effect of irradiation and subsequent storage on the quality of platelet components. STUDY DESIGN AND METHODS: The retention of platelet properties during storage of gamma-irradiated apheresis suspensions was studied in 22 apheresis components obtained on a cell separator with a specialized centrifugation chamber. Immediately after collection, each suspension was divided equally into two 1-L polyolefin containers. On Day 1 (n = 12) and Day 3 (n = 10) one of each pair of suspension containers was gamma radiated with 2500 cGy. All platelet suspensions were stored for 5 days at 20 to 24 degrees C. Samples were drawn on Day 5 from each of the 22 pairs of containers for evaluation of an array of in vitro properties. Samples were taken from 10 pairs of containers for platelet labeling with either 51Cr or 111In for subsequent transfusion and concurrent in vivo measurement of recovery and survival. Posttransfusion samples were drawn after 24 hours for ex vivo whole blood aggregation. RESULTS: Comparable in vitro and in vivo properties were measured in irradiated and control platelets, whether irradiation was performed on Day 1 or Day 3. The mean +/− 1 SD in vivo recovery and survival time for controls and platelets irradiated on Day 1 was 52 +/− 14 percent and 146 +/− 34 hours and 51 +/− 7 percent and 147 +/− 36 hours, respectively. For Day 3 irradiation, the values were 46 +/− 12 percent and 150 +/− 60 hours and 47 +/− 9 percent and 151 +/− 53 hours, respectively. A small, but measurable adverse effect of irradiation on ex vivo platelet aggregation was present. CONCLUSION: These data indicate that storage of apheresis platelets after gamma radiation is without clinically significant, demonstrably adverse effects on platelet quality.     

Studies on platelets exposed to or stored at temperatures below 20 degrees C or above 24 degrees C

BACKGROUND : Platelet concentrates (PCs) may be subjected to temperatures outside 20 to 22 degrees C during shipping or storage, which may have an adverse effect on platelet quality. STUDY DESIGN AND METHODS : These studies systematically evaluated the effect of short- term exposure (≤ 24 hours) of platelets to temperatures above 22° or below 20° C as part of standard 5-day PC storage at 22° C, as well as the effect of long-term storage (5 days) at 24 and 26° C. For the short-term exposure studies, up to 6 units of Day 1 standard PCs were mixed, split, and returned to the containers. Test units were then stored without agitation in an incubator at a specific temperature (4, 12, 16, or 18° C) for various times up to 24 hours, after which they were stored with agitation at 22° C. One unit acted as control and was stored at 20 to 22° C throughout the 5-day storage period. Loss of platelet discoid shape was determined photometrically by the extent of shape change assay, by an increase in apparent platelet size by morphologic evaluation, and by swirling. RESULTS : A gradual loss of platelet discoid shape occurred at temperatures below 20° C. For similar periods, a greater difference between test and control PCs was observed in units held at 4° C than in those held at 16° C. The data were fitted to an equation to relate platelet discoid shape (% of control) to exposure temperature and time. Assuming that a 20-percent decrease or more in the extent of shape change assay represents a significant loss in platelet viability, the equation predicts that such a loss occurs when the platelets are exposed to 16° C for ≥16 hours, to 12° C for ≥10 hours, or to 4°C for ≥6 hours, whereas exposure to 18° C for ≤24 hours has no significant effect. Storage for 5 days at temperatures ≤26° C was not associated with any significant reduction in platelet discoid shape or other measures of platelet quality. CONCLUSION : There was a gradual loss of platelet discoid shape at exposure temperatures < 20°C, which worsened as temperatures decreased and exposure times increased to 24 hours. This relationship can be described in an equation that could be used as a guideline for allowable exposure conditions.     

Liquid storage, shipment, and cryopreservation of cord blood

BACKGROUND: Cord blood banking requires methods for shipping and storage. This study examines the influence of shipping via overnight courier on postthaw viability of cord blood. STUDY DESIGNS AND METHODS: Anticoagulated cord blood was divided with one sample diluted 1:1 using STM-sav (a storage solution) and the other undiluted. Units were shipped from Minneapolis to Memphis and returned, RBC-depleted, cryopreserved, stored for 14 days, and thawed. MNC counts, percent viable cells, quantity of CD34+ cells, and frequency of CFU-GM were measured. Temperature during shipment was continuously monitored. RESULTS: Preliminary studies showed the packing and processing protocol influenced the temperatures experienced during shipping. Samples achieved temperatures below 10 degrees C within 4 to 8 hours with a few units dropping near or below 1 degrees C with cold ambient temperatures. The MNC recovery, CD34+45+ recovery, and frequency of CFU-GM for samples that were shipped were comparable to those observed using static liquid storage. The postthaw viable cell recovery was greatest for storage and shipping times of 24 hours and decreased when the storage and shipping times were longer. CONCLUSION: Ambient conditions and the packing and processing protocol influence the temperature history of the sample. Samples stored beyond 24 hours in liquid storage and shipping exhibit a decreased postthaw recovery.     

Periods without agitation diminish platelet mitochondrial function during storage

BACKGROUND: Prolonged periods without agitation produce platelet (PLT) storage lesions that result in reduced in vitro assay parameters and an increase of apoptotic markers during storage. The aim of this study was to evaluate the influence of periods without agitation on PLT mitochondrial function, blood gases, and activation. STUDY DESIGN AND METHODS: Apheresis PLT units (n = 12) were collected using a cell separator and each was equally divided among five storage bags (50 mL of PLT suspension in 300‐mL nominal volume containers). Four bags were held without agitation for 24, 48, 72, and 96 hours in a standard shipping box at room temperature and the fifth bag was continuously agitated. PLTs were assayed for standard in vitro PLT assays as well as for mitochondrial membrane potential (MMP), accumulation of reactive oxygen species, Annexin V binding, mitochondrial mass, and activity of mitochondrial reduction power (MRP) immediately after removal of units from the shipping container on Days 1, 2, 3, 4, and 7. RESULTS: Increasing periods without agitation resulted in increased superoxide anion generation and PLT activation as well as reduced PLT MMP and MRP. Increasing periods without agitation resulted in increasing Annexin V binding. PLTs that had undergone periods without agitation showed increased oxygen and carbon dioxide levels immediately after storage without agitation. The superoxide anion generation was highly correlated with the loss of MMP, increasing Annexin V binding, and pH decline. CONCLUSIONS: PLTs, if stored without agitation, produce a lesion that leads PLTs to apoptosis. The severity of the lesion depends on the length of the period without agitation. Prolonged periods without agitation induce formation of superoxides and depolarization of MMP along with a presentation of apoptotic markers.     

The effect of the interruption of agitation on platelet quality during storage for transfusion

BACKGROUND: A considerable amount of data and the CFR suggest that platelet concentrates (PCs) should be stored with continuous, gentle agitation before transfusion. However, there are only limited data concerning the mechanisms of platelet damage that may occur when agitation is interrupted, and there are no CFR guidelines concerning shipment between periods of storage. STUDY DESIGN AND METHODS: PCs were prepared by the platelet-rich plasma method and stored for 5 days at 20 to 24 degrees C; agitation was interrupted for 1 to 3 days either by simply stopping the agitator or by placing the PCs in a stationary shipping container. Measurements of platelet metabolism and quality were made during storage and on Day 5. RESULTS: With interruption on the agitator, the production of lactic acid was increased during the interruption in proportion to the number of platelets in the PC and the duration of the interruption. The pO(2) was increased during agitation interruption, which suggested a decline in oxygen utilization. With the use of the hypotonic shock response and the extent of shape change as reflections of platelet quality, there was no evidence of platelet damage unless the pH fell to or below 6.5. No PC reached this level after an interruption of agitation for only 1 day, irrespective of which day was chosen for interruption. PCs whose agitation was interrupted for 2 and 3 days were at risk of having a pH less than 6.5 if their contents were greater than 1.25 x 10(11) and 0.75 x 10(11) platelets, respectively. Interruption of agitation for 1 day in the shipping container produced results essentially identical to those produced by interruption on the agitator. CONCLUSION: Interruption of agitation of PCs for 1 day, either on the agitator or in the shipping container, produces no platelet damage measurable by these in vitro techniques. However, an interruption of agitation for 2 days can result in significant damage in some components. Further studies will be required to learn more about the mechanisms that lead to the metabolic changes described and to determine if the same generalizations apply to apheresis PCs and PCs prepared from pooled buffy coats.     

Ultrastructure of human platelets processed for transfusion under standard blood bank conditions

Platelets processed for transfusion are routinely subjected to centrifugation, mechanical agitation and storage at either room temperature or 4 C. To evaluate morphologic changes associated with standard blood bank conditions, platelet units were grouped into four categories: linear agitation at room temperature, agitation at a 90 degrees arc with a Hemolater at room temperature, agitation at a 360 degrees arc with a commercial platelet agitator, and refirgeration at 4 C without agitation. Cold (4 C) preservation resulted in a surprisingly good preservation of platelet ultrastructure, even at 72 hours of storage. By contrast, harsh linear agitation at room temperature brought about marked alterations in platelet ultrastructure beginning at 24 hours. The Hemolator agitator with 90 degrees arc was associated with good preservation of platelet organelles even at 72 hours, whereas marked degenerative changes were observed at this time in platelets processed with a commercial agitator with a 360 degrees arc. These results indicate that platelets stored at 4 C or processed at room temperature with an agitator with 90 degrees arc show the best preservation of ultrastructure.     

Reduction of the volume of stored platelet concentrates for use in neonatal patients

Premature infants and neonatal patients who require platelet transfusions may develop circulatory overload when administered a 50-ml unit of platelet concentrate. We evaluated the influence of centrifugation and resuspension steps used to reduce the volume of stored platelet concentrates on platelet properties by in vitro methods and by determining post-transfusion increments in neonatal patients. In vitro studies were conducted with platelet concentrates stored at 20 to 24 degrees C for 1 and 5 days in CLX (Cutter) and PL732 (Fenwal) containers and for 1 and 2 days in PL146 containers (Fenwal). With platelets stored in any of the three containers, platelet morphology, mean platelet volume, hypotonic stress response, synergistic aggregation, and platelet factor 3 activity were not affected by the processing steps. The centrifugation and resuspension steps did not cause an enhanced discharge of lactate dehydrogenase from platelets. Similar results were obtained when the platelet concentrates were stored on either a flatbed or an end-over-end tumbler agitator. The in vitro platelet recovery following volume reduction was at least 85 percent. In vivo studies were conducted with platelets stored in the PL732 and PL146 containers. Infusion of platelet concentrates after volume reduction produced a mean corrected increment of 18,947 +/− 14,824 when platelets were stored in the PL146 container and 16,178 +/− 15,699 when platelets were stored in the PL732 container. These results indicate that the volume of stored platelet concentrates can be reduced in a manner which maintains platelet properties.     

Comparative Studies of the Viability of Human Platelets Stored at 4 C in Plastic and Glass Containers

Studies were done to investigate whether the surface of the container (plastic or glass) would influence the viability of human platelets stored at 4C for short intervals.
Cr⁵¹-labeled platelets prepared as concentrates suspended in plasma were preserved at 4 C for 24, 48 or 72 hours and their capacity to recirculate and survive after infusion into the respective donors ("viability") was determined. In addition, the clot retraction property of the stored platelets was measured.
Platelet viability was sharply reduced after storage at 4 C in all experiments. When the storage period was limited to 24 hours, survival curves of platelets preserved in plastic bags were similar to those of platelets preserved in glass bottles. However, after 48 hours of storage, viability of platelets preserved in plastic containers had values significantly higher than those of platelets preserved in glass containers. After 72 hours of storage, platelet viability was reduced to minimal values but was still greater for platelets stored in plastic bags.
The study of clot retraction confirmed data previously obtained and showed that this platelet property was preserved better by storage in glass bottles rather than in plastic bags. Platelet viability was, however, lost very rapidly during storage at 4 C in either type of container so that the unfavorable effect of plastic on the preservation of clot retraction was thought not to be of practical importance in platelet transfusion therapy.     

Stabilized viral nucleic acids in plasma as an alternative shipping method for NAT

BACKGROUND: Preservation of the integrity of viral nucleic acids in blood specimens during shipping and handling is crucial for NAT and viral load monitoring. An economical and convenient method is described for nucleic acid stabilization by using an RNA stabilizing solution (RNAlater, Ambion) in plasma that is designed for the shipment of samples to tropical countries. STUDY DESIGN AND METHODS: HCV, HIV, and HBV FFP were compared with RNAlater-treated plasma and dried plasma spots (DPSs) after incubation at 37 degrees C, which was chosen as an upper limit of ambient shipping temperature, for up to 28 days. HCV-infected chimpanzee plasma was shipped at either room temperature after RNAlater treatment or as frozen plasma in liquid nitrogen from Liberia to New York City. They were then compared for HCV RNA levels. The nucleic acid stabilities were determined by quantitative PCR by using a molecular beacon assay on a sequence detection system (ABI 7700, PE-Biosystems) and by visualizing the PCR components on an acrylamide gel. RESULTS: Quantitative PCR data showed that a 60:40 or greater ratio of RNAlater:plasma volume successfully stabilized HCV RNA and HIV RNA in plasma for up to 28 days at 37 degrees C. HBV DNA in plasma was stable for up to 14 days at 37 degrees C without any stabilizing solution. DPSs on filter paper stabilized viral nucleic acids, but the recoveries were 3 to 10 times less than those with frozen plasma. The integrity of the 5' UTR region of HCV RNA in RNA later-treated chimpanzee plasma was intact when its PCR component was viewed on an acrylamide gel. CONCLUSION: The DPS method stabilized nucleic acids, at least with the extraction method used, was less sensitive than use of RNAlater, and required tedious manual handling. RNAlater provides a convenient way of stabilizing viral nucleic acid in plasma at ambient temperature during sample transportation.     

Design and Test Considerations to Provide an Improved Blood Shipping Container

An improved blood shipping container, providing safer storage of blood in transit over longer periods of time, has been developed. This plastic foam "blood shipper" incorporates an insulated refrigerant system of wet ice. Icing and storage data have been collected for two ambient conditions, a "normal day" (up to 23 C) and a "hot day" (up to 36 C). The storage requirements for blood in transit are based upon the criteria of the standard 1 to 10C and the preferred 1 to 6C range. When the blood was precooled to 1 to 6 C and iced as directed with 1,800 Gm of wet ice, the shipper was found to maintain eight units within 1 to 10 C for 40 hours at normal temperatures and for 30 hours at elevated temperatures. The eight units were maintained within 1 to 6 C for 30 hours at normal temperatures and for 18 hours at elevated temperatures. Without ice, the units were maintained at 1 to 10C for only 6 hours at normal temperatures and for only 2.5 hours at elevated temperatures. Recommendations are made concerning methods of temperature measurement.     

低温保存的血小板胞内冰晶形成温度测定

血小板胞内冰晶形成（ⅡF）的温度是指导血小板低温保存最重要的物理参数之一。本研究通过生物学和物理学的两种方法同时测定血小板ⅡF温度范围，在分步降温法中，每降5℃取出血小板样本，测定37℃直接复温（T37℃）和经液氮处理后再37℃复温（LN）两种方法处理的血小板样本磷脂酰丝氨酸（PS），血浆乳酸脱氢酶（LDH）和血小板聚集功能的变化，同时利用差式扫描量热计（DSC）记录加和不加5%DMSO的血小板在降温过程中的放热峰。结果表明，PS，LDH和血小板聚集功能在-35℃左右不再发生变化，DSC测定结果表明，代表血小板ⅡF的第二个小放热峰在-35℃左右。结论：血小板ⅡF温度在-30℃至-40℃之间（-35℃左右）。     

Maintenance of in vitro properties of leukoreduced whole blood–derived pooled platelets after a 24-hour interruption of agitation

BACKGROUND: Continuous agitation during platelet concentrate (PC) storage is frequently interrupted during shipping. Studies have evaluated the effects of interrupted agitation in apheresis and single whole blood–derived PCs, but not PC pools. This study evaluated in vitro properties of pooled whole blood–derived platelets (PLTs) after a 24-hour interruption of agitation.
STUDY DESIGN AND METHODS: Eleven ABO-identical leukoreduced whole blood–derived PCs (Leukotrap RC-PL, Pall), pooled in a transfer container, were equally divided into each of two CLX-HP containers (Acrodose PL, Pall). One pool (test) was held in a shipping container unagitated for 24 hours between Day 2 and Day 3, while the other (control) was continuously agitated.
RESULTS: Ten pairs underwent in vitro assays after 5 and 7 days' storage. Pools contained a mean (±SD) of 5.0 × 10¹¹ ± 0.4 × 10¹¹ PLTs. Interrupting agitation for 24 hours reduced test pool pH versus control after 5 days' storage (6.77 ± 0.15 vs. 6.98 ± 0.06, p = 0.0005). Test and control pH differences were greater after 7 days' storage (6.17 ± 0.29 vs. 6.65 ± 0.14, p < 0.0001); 5 of 10 test pool pHs were less than 6.2 (vs. 0 of 10 controls). Other test pool key in vitro variables were reduced compared with controls after 5 days' storage, with greater differences after 7 days.
CONCLUSION: After 5 days' storage, pooled leukoreduced whole blood–derived-PCs in CLX-HP containers adequately maintained pH and other key in vitro variables after a 24-hour interruption of agitation. After 7 days' storage, 5 of 10 pools did not maintain a pH value of 6.2 or greater while matched continuously agitated units did.     

Comparison of ceftazidime degradation in glass bottles and plastic bags under various conditions

OBJECTIVE: To study the effect of type of container on ceftazidime stability in intravenous solutions. METHODS: One hundred millilitre polypropylene (PP) and polyvinyl chloride (PVC) bags and 100-mL glass bottles were filled with 5% dextrose or 0.9% sodium chloride solutions containing ceftazidime (Fortumset) at 40 mg/mL. Three containers of each solution were stored at 20 and 35 degrees C. One millilitre samples were drawn from each container at 0 and 20 h and assayed. Pyridine concentrations, the main degradation product of ceftazidime, were determined by high-pressure liquid chromatography. RESULTS: Pyridine levels increased during storage and were higher in PVC and PP bags than in glass bottles in both diluents. Solutions stored in PP bags showed better stability than in PVC bags. CONCLUSION: This study shows that ceftazidime undergoes slower degradation in PP than PVC containers although the difference is small. Glass bottles seems to be the better container for storing ceftazidime solutions, whatever storage temperature and diluent used.     

A prospective study of symptomatic bacteremia following platelet transfusion and of its management

BACKGROUND: The danger of bacteremia due to contaminated platelets is not well known. There are also no established guidelines for the management of febrile reactions after platelet transfusion. STUDY DESIGN AND METHODS: To determine the risk of symptomatic bacteremia after platelet transfusion, 3584 platelet transfusions given to 161 patients after bone marrow transplantation were prospectively studied. Platelet bags were routinely refrigerated for 24 hours after transfusion. Septic work-up was initiated for a temperature rise of more than 2 degrees C above the pretransfusion value within 24 hours of platelet transfusion or a temperature rise of more than 1 degree C that was associated with chills and rigor. Diagnosis of bacteremia after platelet transfusion was made only when the pairs of isolates from the blood and the platelet bags were identical with respect to their biochemical profile, antibiotic sensitivity, serotyping, or ribotyping. RESULTS: Thirty-seven febrile reactions, as defined above, occurred. Bacteremia subsequent to platelet transfusion was diagnosed in 10 cases. There was a 27-percent chance (95% CI, 15–43%) that these febrile reactions represented bacteremia that resulted from platelet transfusion. For a subgroup of 19 patients with a temperature rise of more than 2 degrees C, the risk of bacteremia was 42 percent (95% CI, 23–64%). Septic shock occurred in 4 of the 10 bacteremic patients. A rapid diagnosis was possible because the involved bacteria were demonstrated by direct Gram stain of the samples taken from the platelet bags of all 10 patients. CONCLUSION: Significant febrile reactions after platelet transfusion are highly likely to be indicative of bacteremia. Routine retention of platelet bags for subsequent microbiologic study was useful in the investigation of these febrile reactions. Empiric antibiotic therapy is indicated.     

Technical problems in platelet-concentrate preparation and their clinical significance

To obtain platelet concentrates the following procedure is in routine use in the German Red Cross Blood Bank in Ulm: 1. Harvesting of donor blood in a double bag system; 2. two step centrifugation of a) whole blood, and b) platelet rich plasma at room temperature (22 degrees C) at 300 X g for 20 min, and 1200 X g for 30 min respectively; 3, resuspension of platelets in a minimum of 50 ml of plasma. Platelet yield averages 60%. The white cell content is less than 5% as compared to the original material. Although red cells are nearly absent the ABO blood group system and the D antigen are considered for compatibility. Platelet concentrates are stores up to 72 hours at 22 degrees C at constant slow rotation. Technical and biological variables affecting platelet yield and viability are discussed in some detail.     

Platelet membrane integrity during storage and activation

BACKGROUND: The platelet cell membrane appears to undergo a lipid-phase transition on cooling from 23 degrees C to 4 degrees C. Consequences of this phase transition are leakage of cellular material and irreversible cellular damage. Whether agents, of known benefit in protecting membranes and proteins from cooling and drying injury, could also protect platelets was investigated. Leakage of cytosolic components was assessed by measuring the release of fluorescein into the surrounding medium. STUDY DESIGN AND METHODS: Fresh platelets were suspended in 5 percent dimethyl sulfoxide (DMSO) or in 5 mM of one the following agents: glucose, trehalose, sucrose, glycerol, ethylene glycol, 1,2-propanediol, or L-proline. Platelets were loaded with 10 nMfluorescein diacetate (FD), chilled at 4 degrees C for 24 hours or frozen at -1 degree C per minute to -70 degrees C, warmed rapidly at 37 degrees C, and centrifuged, and the supernatant was measured for the presence of fluorescein. The effect of FD on platelets was assessed by agglutination with ristocetin, aggregation with thrombin and ADP, platelet-induced clot retraction, and expression of p-selectin. Platelet function and activation before and after freezing or cooling were measured by the same methods. RESULTS: By flow cytometry, 98 percent of the platelets incorporated FD. The trapped fluorescein resulted in neither platelet activation (p = 0.9) nor reduction of platelet function (p = 0.12-0.94) from that in control platelets. Freezing of platelets in DMSO caused far less release of fluorescein than did freezing with other agents (p<0.001) or chilling of platelets at 4 degrees C for 24 hours (p<0.0001). Supernatant levels of fluorescein correlated inversely with platelet function. Fluorescein was also shown to be released during aggregation with thrombin or ADP but not during agglutination with ristocetin. CONCLUSIONS: Release of fluorescein into the surrounding medium indicated a loss of platelet membrane integrity and function. Cellular loading with FD is a simple method of studying membrane integrity of platelets and other cells.     

FATE OF CHLORBUTOL DURING STORAGE IN POLYETHYLENE DROPPER CONTAINERS AND SIMULATED PATIENT USE

The disappearance of chlorbutol from aqueous solutions stored in polyethylene containers under various experimental and simulated patient use conditions is described. Chlorbutol is lost by sorption into the container wall, container insert and by permeation into the external environment. The extent of chlerbutol loss from solution and uptake by the container components is dependent on temperature, chlorbutol concentration and the closure used. Aqueous chlorbutol solutions stored in the polyethylene container evaluated have a predicted shelf-life (time for 10% loss) of about 4 months at 25d?C and 8 months at 20d?C. Negligible loss is found during simulated patient use over 1 month.