Storing apheresis platelets without agitation with simulated shipping conditions during two separate periods: immediately after collection and subsequently between Day 2 and Day 3

BACKGROUND: Apheresis platelet (PLT) units are not routinely agitated during transit. Our study compared the in vitro properties of apheresis PLT units that were stored with continuous agitation (CA) and without continuous agitation (WCA) during two separate periods, immediately after collection and between Day 2 and Day 3 of storage. STUDY DESIGN AND METHODS: Two identical apheresis PLTs units were prepared from collections with Amicus (n = 11, Fenwal, Inc.) and Trima (n = 10, CaridianBCT) cell separators. One apheresis PLT unit was continuously agitated, starting routinely within 30 minutes of collection, and an identical apheresis PLT unit was held without agitation initially for 7 to 8 hours and subsequently for 24 hours between Day 2 and Day 3 of storage. The apheresis PLT units were maintained WCA at 20 to 24°C in a shipping box. In vitro PLT properties were evaluated on Day 1 (day after collection), after 5 and 7 days of storage. RESULTS: With both Amicus and Trima apheresis PLT units, the mean PLT content and concentration of CA and WCA were comparable and essentially constant throughout storage. Mean pH levels (±1 SD) after 5 days for Amicus apheresis PLT units were 6.97 ± 0.20 (WCA) and 7.13 ± 0.16 (p < 0.001, CA) and for Trima apheresis PLT units 6.97 ± 0.21 (WCA) and 7.22 ± 0.17 (p < 0.001, CA). In vitro variables, including percentage of disc PLTs, extent of shape change, and hypotonic stress levels, after 5 days of storage, showed mean differences between WCA and CA that were less than 15%. CONCLUSION: The in vitro results show that apheresis PLT units can be stored without agitation for 7 to 8 hours immediately after collection and also subsequently during storage for 24 hours with minimal influence on in vitro PLT properties compared to continuously agitated PLTs.     

A rest period does not affect in vitro storage properties in apheresis platelets collected from the buffy coat layer

BACKGROUND: A previous study demonstrated that several in vitro storage properties of apheresis platelets (PLTs) that are isolated by sedimentation against the collection container and subsequently resuspended can benefit from a rest period before continuous agitation. This study examines whether the in vitro storage properties of apheresis PLTs isolated by collection from the buffy coat layer benefit from a rest period before agitation. STUDY DESIGN AND METHODS: Freshly collected apheresis PLTs (Trima, GambroBCT) were divided into five 60‐mL aliquots. One aliquot was immediately placed on a flat‐bed agitator; the other aliquots were held on a laboratory bench for 1, 2, 4, and 6 hours before continuous agitation. Samples were obtained on Days 1, 5, and 7 for standard in vitro PLT assays. The experiment was repeated 12 times. RESULTS: For each sampling day, no significant differences were observed in aliquots held with or without a rest period for any of the following PLT properties: PLT content, mean PLT volume, pH, pCO₂, bicarbonate, glucose, lactate, hypotonic shock response, extent of shape change, aggregation, morphology, CD62P, CD63, and CD42b. Although regression analysis identified several in vitro properties whose mean levels appeared to improve with increasing length of the rest period, maximum differences in mean levels were small (<6%). CONCLUSION: The in vitro storage properties of Trima apheresis PLTs isolated from the buffy coat layer do not benefit from a rest period.     

A rest period before agitation may improve some in vitro apheresis platelet parameters during storage

BACKGROUND: Whole blood‐derived platelets (PLTs) prepared by the PLT‐rich plasma method are subjected to a recommended 1‐hour rest period after the second centrifugation to avoid excessive PLT activation. Different apheresis PLT preparation methods demonstrate different levels of PLT activation and ability to form macroscopic aggregates immediately after collection. PLT collections are lost on Day 1 of storage if aggregates are not dispersed. It is possible that a rest period may help to disperse PLT aggregates. It is not established whether apheresis PLTs require a rest period before agitation and what the length of this period should be. STUDY DESIGN AND METHODS: Apheresis PLTs (Amicus, Fenwal, Inc.) were divided into five identical aliquots. One aliquot was placed on the flatbed agitator immediately after division. The other aliquots were subjected to agitation after 1, 2, 4, and 6 hours of rest. Samples were taken on Days 1, 5, and 7 for standard PLT assays. RESULTS: No differences during 7‐day storage were observed in PLT content, mean PLT volume, pH levels, bicarbonate, glucose, lactate, oxygen and carbon dioxide levels, hypotonic shock response, aggregation, and activation markers in PLT aliquots subjected to different rest periods or without a rest period. In contrast, values of extent of shape change, percentage of discoid PLTs, and expression of GP1b‐α were greater in aliquots subjected to different periods of rest compared to those of PLTs without a rest period. CONCLUSION: A rest period from 1 to 6 hours may improve some but not all in vitro PLT storage parameters.     

Maintenance of storage properties of pediatric aliquots of apheresis platelets in fluoroethylene propylene containers

BACKGROUND: Platelet (PLT) aliquots for pediatric use have been shown to retain in vitro properties when stored in gas‐impermeable syringes for up to 6 hours. As an alternative, PLT aliquots can be stored for longer periods in containers used for storage of whole blood–derived PLTs. These containers are not available separate from whole blood collection sets and PLT volumes less than 35 mL either have not been evaluated or may be unsuitable for PLT storage. Gas‐permeable fluoroethylene propylene (FEP) containers have been used in the storage of cell therapy preparations and are available in multiple sizes as single containers but have not been evaluated for PLT storage. STUDY DESIGN AND METHODS: A single apheresis unit was divided on Day 3 into small aliquots with volume ranging from 20 to 60 mL, transferred using a sterile connection device, and stored for an additional 2 days either in CLX (control) or in FEP containers. PLT storage properties of PLTs stored in FEP containers were compared to those stored in CLX containers. Standard PLT in vitro assays were performed (n = 6). RESULTS: PLT storage properties were either similar to those of CLX containers or differed by less than 20% excepting carbon dioxide levels, which varied less than 60%. CONCLUSION: Pediatric PLT aliquots of 20, 30, and 60 mL transferred on Day 3 into FEP cell culture containers adequately maintain PLT properties for an additional 2 days of storage.     

Acquired cytochrome C oxidase impairment in apheresis platelets during storage: a possible mechanism for depletion of metabolic adenosine triphosphate

BACKGROUND: Intracellular adenosine triphosphate (ATP) levels decline significantly during storage of platelet (PLT) products, in part due to PLT degranulation. However, metabolic ATP stores also become depleted during storage through an unclear mechanism. Since both anaerobic glycolysis and oxidative phosphorylation are important for PLT ATP production, it is possible that the reduction in metabolic ATP reflects impaired oxidative phosphorylation. To assess this, we evaluated the kinetic activity and protein expression of cytochrome C oxidase (CcOX) in stored apheresis PLTs. STUDY DESIGN AND METHODS: Apheresis PLTs were collected and stored with agitation at 22 ± 2°C for 7 days. In vitro measurements of PLT metabolic state, function, and activation were performed on Days 0, 2, 4, and 7 of storage. Total PLT ATP content, steady‐state CcOX kinetic activity, and protein immunoblotting for CcOX Subunits I and IV were also performed using isolated PLT mitochondria from simultaneously collected samples. RESULTS: Intra‐PLT ATP and steady‐state PLT CcOX activity declined significantly and in a progressive manner throughout storage while steady‐state levels of CcOX I and IV protein remained unchanged. Time‐dependent decline in CcOX activity correlated with progressive ATP depletion over time. CONCLUSION: During storage of apheresis PLTs for 7 days, the parallel decline in CcOX function and intra‐PLT ATP suggests development of an acquired impairment in PLT oxidative phosphorylation associated with perturbed ATP homeostasis in stored PLTs.     

Volume-reduced platelet concentrates: optimization of production and storage conditions

BACKGROUND: Plasma can be removed from platelet (PLT) concentrates (PCs) when volume reduction for PLT transfusion is indicated. Volume‐reduced PCs are currently produced from pooled buffy coat (BC) PCs or apheresis PCs by pretransfusion volume reduction, followed by transfer to a syringe for immediate transfusion. We evaluated the maximal storage time of the volume‐reduced PCs in gas‐permeable containers. STUDY DESIGN AND METHODS: Volume‐reduced PCs were produced from BC‐derived and apheresis PCs by hard‐spin centrifugation. Supernatant was removed and the PLTs were resuspended in 20 mL of retained original PC and had PLT concentrations ranging from 10.8 × 10⁹ to 13.8 × 10⁹ PLTs/mL. Volume‐reduced PCs were stored either in syringes or in containers made from diethylhexyl phthalate (DEHP)‐polyvinylchloride (PVC) or butyryl trihexyl citrate (BTHC)‐PVC plastic. Units were sampled at t = 0, 1, 3, and 6 hours for in vitro measurements. RESULTS: When prepared from 2‐day‐old PCs (n = 4), volume‐reduced PCs from BCs in a syringe had a pH_37°C of 5.76 ± 0.04 at t = 6 hours after volume reduction. In the DEHP‐PVC container, pH was 5.85 ± 0.15 (not significant), and in the BTHC‐PVC, 6.34 ± 0.16 (p < 0.001), at t = 6 hours. When made from 7‐day‐old PCs, pH was lower for all storage conditions: 5.68 ± 0.06 in the syringe, 5.70 ± 0.09 in the DEHP‐PVC container (not significant), and 6.07 ± 0.24 in the BTHC‐PVC container (p < 0.01) at t = 6 hours. Volume‐reduced 2‐day‐old apheresis PCs had a pH of 6.47 ± 0.20 at t = 6 hours. CONCLUSIONS: Adult‐dose PCs derived from BC or apheresis can be volume‐reduced to approximately 20 mL in a closed gas‐permeable system. Volume‐reduced PCs in BTHC‐PVC containers retain a mean pH of more than 6.0 up to 6 hours after production. Syringes allow only 3 hours of storage.     

Effect of storage on levels of nitric oxide metabolites in platelet preparations

BACKGROUND: Nitric oxide (NO), a potent signaling molecule, is known to inhibit platelet (PLT) function in vivo. We investigated how the levels of NO and its metabolites change during routine PLT storage. We also tested whether the material of PLT storage containers affects nitrite content since many plastic materials are known to contain and release nitrite. STUDY DESIGN AND METHODS: For nitrite and nitrate measurement, leukoreduced apheresis PLTs and concurrent plasma (CP) were collected from healthy donors using a cell separator. Sixty‐milliliter aliquots of PLT or CP were stored in CLX or PL120 Teflon containers at 20 to 24°C with agitation and daily samples were processed to yield PLT pellet and supernatant. In a separate experiment, PLTs were stored in PL120 Teflon to measure NO generation using electron paramagnetic resonance (EPR). RESULTS: Nitrite level increased markedly in both PLT supernatant and CP stored in CLX containers at a rate of 58 and 31 nmol/L/day, respectively. However, there was a decrease in nitrite level in PLTs stored in PL120 Teflon containers. Nitrite was found to leach from CLX containers and this appears to compensate for nitrite consumption in these preparations. Nitrate level did not significantly change during storage. CONCLUSION: PLTs stored at 20 to 24°C maintain measurable levels of nitrite and nitrate. The nitrite decline in nonleachable Teflon containers in contrast to increases in CLX containers that leach nitrite suggests that it is consumed by PLTs, residual white blood cells, or red blood cells. These results suggest NO‐related metabolic changes occur in PLT units during storage.     

Comparison of the in vitro properties of apheresis platelets during 7-day storage after interrupting agitation for one or three periods

BACKGROUND: Many platelet (PLT) components undergo multiple periods of shipment before transfusion. We have previously conducted studies investigating maintenance of apheresis PLT in vitro quality measures during a single 24‐ or 30‐hour interruption of agitation, but data are not available for multiple periods without agitation. STUDY DESIGN AND METHODS: Apheresis PLTs were collected with both the Amicus (Fenwal, Inc.) and the Trima (Gambro BCT) cell separators to provide two identical PLT products, each with approximately 4 × 10¹¹ to 5 × 10¹¹ PLTs. One product was subjected to a single contiguous 24‐ or 30‐hour period of interrupted agitation between Days 2 and 3 of storage by placement in a standard shipping box at room temperature. The matched product was not agitated on each of 3 days (Days 0, 1, and 3) for specified intervals totaling an identical period of time. RESULTS: Interrupting agitation for three periods resulted in greater maintenance of pH during storage than that observed using one contiguous period. These differences were significant for units held without agitation for 24 hours (Day 5, 0.08 pH units, p < 0.0001; Day 7, 0.10 pH units, p = 0.0059) and were also significant for units held without agitation for 30 hours (Day 5, 0.15, p < 0.0001; Day 7, 0.20, p < 0.0001). The two different interruption of agitation scenarios did not result in significant differences in the extent of shape change and hypotonic shock response variables after 5 or 7 days of storage. CONCLUSION: Apheresis PLTs subjected to three periods without agitation maintained overall pH levels slightly greater than those of matched units subjected to one contiguous period without agitation. Other measures showed comparability of PLT in vitro variables with the two scenarios for interruption of agitation.     

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.     

Extended storage of platelet‐rich plasma–prepared platelet concentrates in plasma or Plasmalyte

BACKGROUND: Using bacterial detection or pathogen reduction, extended platelet (PLT) storage may be licensed if PLT viability is maintained. The Food and Drug Administration (FDA)'s poststorage PLT acceptance guidelines are that autologous stored PLT recoveries and survivals should be 66 and 58% or greater, respectively, of each donor's fresh PLT data. STUDY DESIGN AND METHODS: Nonleukoreduced PLT concentrates were prepared from whole blood donations. Autologous PLT concentrates from 62 subjects were stored in 100% plasma (n = 44) or 20% plasma/80% Plasmalyte (n = 18), an acetate‐based, non–glucose‐containing crystalloid solution previously used for PLT storage. Fresh PLTs were obtained on the day the donor's stored PLTs were to be transfused. The fresh and stored PLTs were alternately radiolabeled with either ⁵¹chromium or ¹¹¹indium, and in vitro measurements were performed on the stored PLTs. RESULTS: The FDA's PLT recovery criteria were met for 7 days of plasma storage, but PLT survivals maintained viability for only 6 days. Plasmalyte‐stored PLTs did not meet either acceptance criteria after 6 days of storage. After 7 days of storage, PLT recoveries averaged 43 ± 4 and 30 ± 4% and survivals 4.1 ± 0.4 and 2.0 ± 0.2 days for plasma‐ and Plasmalyte‐stored PLTs, respectively (p = 0.03 for recoveries and p < 0.001 for survivals). Poststorage PLT recoveries correlated with the commonly used in vitro PLT quality measurements of hypotonic shock response and annexin V binding, while survivals correlated with extent of shape change, morphology score, and pH. CONCLUSION: There is a progressive decrease in recoveries and survivals of plasma‐stored PLTs over time. PLT viability is better maintained in plasma than Plasmalyte.     

Comparison of the in vitro storage properties of Amicus apheresis platelets collected using single- and double-needle procedures from the same donors

BACKGROUND: Amicus apheresis platelets (PLTs) can be collected using either a single‐ (SN) or a double‐needle (DN) procedure. To investigate whether the method of PLT collection using the same instrument influences PLT quality, the in vitro storage properties of Amicus PLTs were evaluated in the same donors collected by SN and DN procedures. STUDY DESIGN AND METHODS: Single apheresis PLT collections with concurrent plasma were performed on donors using the Amicus with a target yield of 4 × 10¹¹. A PLT unit was collected from a donor assigned to either a SN or a DN procedure; a subsequent donation from the same individual was collected by the other procedure (n = 10). Units were stored at 20 to 24°C with continuous agitation, assayed for 19 PLT storage variables, and analyzed by paired t test, with differences between values obtained with SN and DN collections considered significant with p values of less than 0.001. RESULTS: PLT units collected by SN procedure had contents and concentrations similar to those collected by DN procedures (4.1 × 10¹¹ ± 0.3 × 10¹¹ vs. 4.0 × 10¹¹ ± 0.3 × 10¹¹ and 1396 × 10⁹ ± 131 × 10⁹ vs. 1367 × 10⁹ ± 110 × 10⁹ PLTs/L). On Day 7, SN and DN PLTs had comparable pH values (7.07 ± 0.09 vs. 6.99 ± 0.17), morphology (52.4 ± 18.7% vs. 56.0 ± 13.3% discoid), aggregation (87.1 ± 11.5% vs. 91.3 ± 5.4%), and activation (45.8 ± 11.9% vs. 48.2 ± 8.7% CD62P), as well as all other variables (p > 0.05; Day 7 CO₂, p = 0.0304). CONCLUSION: The in vitro storage properties of apheresis PLTs collected from the same donors using a SN and DN procedure with the Amicus instrument were maintained through 7 days of storage and yielded comparable results.     

Quantitation of coated platelet potential during collection, storage, and transfusion of apheresis platelets

BACKGROUND: Coated platelets (PLTs), a subpopulation of PLTs observed upon dual agonist stimulation with collagen and thrombin, are known to retain several procoagulant α‐granule proteins on their surface. By formation of a highly active membrane‐bound prothrombinase complex, these PLTs represent an important step in the coagulation cascade as a consequence of their ability to generate thrombin at the site of vascular injury. Various clinical observations suggest that higher levels of coated PLTs are associated with thrombosis while a deficiency of coated PLTs results in a bleeding diathesis. Current quality control guidelines for in vitro PLT storage measure PLT viability but no routine evaluation of the hemostatic function of stored PLTs and particularly no estimation of coated PLT potential is performed. Our primary objective was to evaluate if the process of apheresis and storage of PLT units alters the levels of coated PLTs. In addition, we sought to determine how transfusion of stored PLTs into patients with thrombocytopenia affects the patient's coated PLT levels. STUDY DESIGN AND METHODS: Coated PLT levels were analyzed in 13 voluntary PLT donors before donation, in the fresh apheresis product (Trima, CaridianBCT) and in the stored apheresis product just before transfusion. In addition, 10 patients with thrombocytopenia were analyzed for coated PLTs before and after transfusion of a stored PLT product. RESULTS: Coated PLT levels were significantly decreased after the process of apheresis (17% relative decline; p < 0.01) and with prolonged storage (1 to 5 days; 53% relative decline; p < 0.001). Transfusion of stored PLT units did not result in significant increment of coated PLT levels in patients with thrombocytopenia as expected considering the low level of coated PLTs in stored PLT units. Furthermore, there was no suggestion of regeneration of coated PLT potential upon reinfusion. CONCLUSIONS: Isolation and storage of apheresis PLTs by standard blood bank procedures results in a significant decline in coated PLT potential. Reinfusion of stored apheresis PLTs into patients with thrombocytopenia resulted in a predictable change in coated PLT potential with no suggestion of regeneration of lost coated PLT potential.     

Evaluation of the automated collection and extended storage of apheresis platelets in additive solution

BACKGROUND: Collecting apheresis platelets (PLTs) into additive solution has many potential benefits. The new Trima software (Version 6.0, CaridianBCT) allows automated addition of PLT additive solution (PAS) after collection, compared to Trima Version 5.1, which only collects PLTs into plasma. The aim of this study was to compare PLT quality during extended storage, after collection with the different Trima systems. STUDY DESIGN AND METHODS: Apheresis PLTs were collected using both Trima Accel apheresis systems. The test PLT units (n = 12) were collected using the new Trima Version 6.0 into PLT AS (PAS‐IIIM), while the control units (n = 8) were collected into autologous plasma using Trima Version 5.1. All units were stored for 9 days, and in vitro cell quality variables were evaluated during this time. RESULTS: PLTs collected in PAS‐IIIM maintained a stable pH between 7.2 and 7.4, whereas plasma‐stored apheresis units exhibited significantly increased acidity during storage, due to lactate accumulation and bicarbonate exhaustion. Plasma‐stored PLTs also demonstrated a more rapid consumption of glucose. However, there was little difference in PLT activation or cytokine secretion between PAS‐IIIM and control PLTs. CONCLUSION: These data indicate that apheresis PLT concentrates collected in PAS‐IIIM, using Trima Version 6.0 software, maintained acceptable PLT metabolic and cellular characteristics until Day 9 of storage.     

Maintenance of platelet in vitro properties during 7-day storage in M-sol with a 30-hour interruption of agitation

BACKGROUND: Extensive periods without agitation can occasionally occur during platelet (PLT) shipment and can affect PLT quality during 5‐ to 7‐day storage. The use of buffer‐containing PLT additive solutions (ASs) may better preserve PLT quality during storage by maintaining PLT pH and other in vitro variables. A newly described bicarbonate‐containing AS, M‐sol, was compared to plasma for preservation of whole blood–derived PLT concentrates in which a 30‐hour interruption of agitation was included. STUDY DESIGN AND METHODS: ABO‐identical PLT‐rich plasma intermediate products were pooled in sets of four, split, and centrifuged with subsequent plasma expression (n = 12). Two units were resuspended with M‐sol AS to produce a 70 percent solution/30 percent plasma PLT concentrate; 2 units were resuspended in 100 percent plasma. One M‐sol resuspended unit and 1 plasma unit were held on a laboratory bench in a standard shipping box for 30 hours between Day 2 and Day 3, while the other M‐sol and plasma unit were continuously agitated. Standard in vitro testing for PLT quality variables on each set of 4 units was performed during storage (n = 12). RESULTS: Interrupting agitation of PLTs suspended in M‐sol resulted in less of a pH decrement during storage than that of PLTs suspended in 100 percent plasma. On Days 5 and 7, the pH differences between M‐sol and plasma units were 0.56 and 0.75 pH units, respectively (p < 0.0003). In addition, PLTs suspended in M‐sol and subjected to an interruption of agitation had lesser Day 7 CD62+ cells, glucose utilization, and lactate production and greater hypotonic stress response, morphology, swirling, and aggregation response than those suspended in plasma (p ≤ 0.005). CONCLUSION: The in vitro properties of PLTs suspended in 70 percent M‐sol/30 percent plasma and subjected to a 30‐hour interruption of agitation are better maintained during 7‐day storage than those of matched units suspended in plasma.     

The influence of simulated shipping conditions (24- or 30-hr interruption of agitation) on the in vitro properties of apheresis platelets during 7-day storage

BACKGROUND: Platelet (PLT) components undergo interruption of agitation during shipment. Studies have demonstrated maintenance of PLT quality of whole blood–derived PLT concentrates during a 24-hour interruption of agitation, but data are not available for apheresis PLTs in 100 percent plasma.
STUDY DESIGN AND METHODS: Apheresis PLTs were collected with one of two commercially available separators (Amicus, Fenwal, Inc.; or Trima Accel, Gambro BCT) to provide two identical PLT products, each with approximately 3 × 10¹¹ to 4.5 × 10¹¹ PLTs. The control product was continuously agitated. The test product was subjected to a continuous 24- or 30-hour period of interrupted agitation between Day 2 and Day 3 of storage by placement in a standard shipping box at room temperature.
RESULTS: Interrupting agitation for 24 or 30 hours influenced in vitro PLT properties to various degrees. After 5 days of storage, pH levels were judged to be well maintained after 24 hours without agitation for PLTs collected with both separators (pH < 6.2, Trima [0/12] and Amicus [0/12]). The changes in other variables associated with the retention of postinfusion viability were also considered limited and acceptable in units subjected to a 24-hour interruption of agitation. After 7 days of storage including a 24-hour interruption of agitation, Trima PLTs better maintained PLT properties compared to Amicus PLTs. With a 30-hour period, both Trima and Amicus PLTs were deemed satisfactory for pH at 5 days, but not at 7 days (pH < 6.2: Day 5, Trima [0/23] and Amicus [1/22]; Day 7, Trima [5/23] and Amicus [4/17]).
CONCLUSION: Based on the retention of pH levels of at least 6.2, apheresis PLT quality was maintained for 5 days with a 24-hour and a 30-hour interruption of agitation.     

Platelet-white blood cell (WBC) interaction, WBC apoptosis, and procoagulant activity in stored red blood cells

BACKGROUND: Nonleukoreduced units of red blood cells (RBCs) contain activated platelets (PLTs) that interact with white blood cells (WBCs) and may promote inflammation and thrombosis in the recipient. The aim of this study was to characterize PLT‐WBC interactions (PLT‐WBC aggregates [PLAs]), WBC apoptosis, WBC death, and the development of procoagulant activity in RBCs during storage. STUDY DESIGN AND METHODS: RBCs were prepared from volunteer donor blood and stored. Samples were analyzed with flow cytometry between Days 1 and 15 to measure PLT‐monocyte aggregate (PMA) and PLT‐neutrophil aggregate (PNA) formation, WBC apoptosis (annexin V binding), and cell death (binding of 7‐aminoactinomycin D). Procoagulant activity in the supernatant of four RBC preparations was assessed between Days 1 and 39 using a clotting assay with and without the addition of an inhibitory anti‐tissue factor (TF) antibody, αTF‐5. RESULTS: PLA formation was extensive and maximal on Day 3 of storage (PNA, 23 ± 13%; PMA, 93 ± 4%; n = 6). Apoptosis was progressive throughout storage, with 95 ± 4% of neutrophils and 73 ± 19% of monocytes binding annexin V on Day 15. Cell death became measurable after apoptosis. Procoagulant activity was observed in all RBCs but with varying temporal patterns. It was partially TF dependent and removed with high‐speed centrifugation, suggestive of an association with microparticles. CONCLUSION: The activation of PLTs during the storage of RBCs induces PLA formation that precedes WBC apoptosis and death. Procoagulant activity, likely associated with microparticles derived from apoptotic WBCs, may contribute to adverse effects of stored, nonleukoreduced RBCs.     

Platelet concentrates prepared after a 20- to 24-hour hold of the whole blood at 22°C

BACKGROUND: The Food and Drug Administration (FDA) requires that red blood cells must be refrigerated within 8 hours of whole blood collection. Longer storage of whole blood at 22°C before component preparation would have many advantages. STUDY DESIGN AND METHODS: Two methods of holding whole blood for 20 to 24 hours at room temperature were evaluated, refrigerated plates or a 23°C incubator. After extended whole blood storage, platelet (PLT) concentrates were prepared from PLT‐rich plasma on Day 1 postdonation, and the PLTs were stored for 6 more days. On Day 7 of PLT storage, blood was drawn from each subject to prepare fresh PLTs. The stored and fresh PLTs were radiolabeled and transfused into their donor. RESULTS: Eleven subjects' whole blood was stored using refrigerated butanediol plates (Compocool, Fresenius), and 10 using an incubator. Poststorage PLT recoveries averaged 47 ± 13% versus 53 ± 11% and survivals averaged 4.6 ± 1.7 days versus 4.7 ± 0.9 days for Compocool versus incubator storage, respectively (p = NS). With all results, poststorage PLT recoveries averaged 75 ± 10% of fresh and survivals 57 ± 13% of fresh; PLT recoveries met FDA guidelines for poststorage PLT viability but not survivals. CONCLUSION: Seven‐day poststorage PLT viability is comparable when whole blood is stored for 22 ± 2 hours at 22°C using either refrigerated plates or an incubator to maintain temperature before preparing PLT concentrates.     

Thrombin receptor levels in platelet concentrates during storage and their impact on platelet functionality

BACKGROUND: Quality control of platelet (PLT) concentrates is challenging, due to PLT lesions, which are difficult to detect with routine methods. The search for reliable PLT lesion biomarkers is focused on the role of PLTs in primary hemostasis. PLT transfusions also have a significant impact on secondary hemostasis. In this phase, responsiveness of PLTs to small amounts of thrombin is crucial. PAR1 and PAR4 are protease‐activated receptors and are responsible for thrombin reactivity of human PLTs. This study should elucidate if levels of those two receptors are changing in PLT concentrates during storage and if those changes have an impact on PLT aggregation and support of thrombin generation. STUDY DESIGN AND METHODS: PLT concentrates from buffy coat preparations were stored in SSP+ solution for 9 days at 22 ± 2°C on a horizontal flatbed agitator, and samples were taken daily for analysis. PAR1 and PAR4 levels were evaluated using Western blot analysis. PLT aggregation was measured using Born aggregometry and specific PAR1 or PAR4 agonists. Thrombin generation was measured using calibrated automated thrombography. RESULTS: Levels of both receptors (PAR1 and PAR4) started to decrease after 5 days of storage. PAR1‐mediated PLT aggregation remained constant, whereas PAR4‐mediated PLT aggregation decreased with storage time. Rate of thrombin generation was accelerated after 5 days of storage. CONCLUSION: Decreasing levels of PARs in PLT concentrates after 5 days of storage influenced PAR4‐mediated, but not PAR1‐mediated, aggregation. Thrombin generation with senescent PLTs was increased, which may be attributed to other mechanisms promoting increased phosphatidylserine exposure.     

A randomized controlled trial comparing autologous radiolabeled in vivo platelet (PLT) recoveries and survivals of 7-day-stored PLT-rich plasma and buffy coat PLTs from the same subjects

BACKGROUND: A recent review concluded that there was inadequate evidence to show a difference between buffy coat (BC) and platelet (PLT)‐rich plasma (PRP) PLT concentrates prepared from whole blood. We hypothesized that 7‐day‐stored BC‐PLTs would have superior autologous recoveries and survivals compared to PRP‐PLTs and that both would meet the Food and Drug Administration (FDA) criteria for poststorage viability. STUDY DESIGN AND METHODS: This was a randomized, crossover study design in healthy subjects who provided informed consent. Each participant donated a unit of whole blood on two occasions. In random order, either BC‐PLTs or PC‐PLTs were prepared after a 20 ± 2°C overnight hold of the whole blood. PLTs were stored under standard conditions. On Day 7, fresh PLTs were prepared from 43 mL of autologous whole blood. The fresh PLTs paired with either BC‐PLTs or PRP‐PLTs were alternately labeled with ¹¹¹In or ⁵¹Cr and simultaneously reinfused to determine recoveries and survivals. In vitro assays were performed on Days 1 and 7. RESULTS: Fourteen subjects completed the study at two sites. No differences in poststorage PLT viabilities were observed between BC‐PLTs and PRP‐PLTs; recovery differences averaged 3.7 ± 2.4% (±SE, p = 0.15) and survival differences averaged 0.48 ± 0.56 days (p = 0.41). Neither type of PLTs met the current FDA criteria for either poststorage PLT recoveries or survivals. CONCLUSION: We were unable to demonstrate that single‐unit BC‐PLTs stored for 7 days have superior poststorage viability compared to PRP‐PLTs. Failure to meet the minimum FDA criteria for poststorage PLT viability raises questions regarding the acceptance thresholds of these metrics.     

Evaluation of in vitro storage properties of prestorage pooled whole blood–derived platelets suspended in 100 percent plasma and treated with amotosalen and long-wavelength ultraviolet light

BACKGROUND: Amotosalen, a psoralen, has been utilized for photochemical treatment (PCT) of apheresis platelets (PLTs) and pooled buffy coat PLTs suspended in additive solution. In the United States, the source of many PLT transfusions is from whole blood–derived PLTs prepared by the PLT‐rich plasma (PRP) method. This study investigated the in vitro PLT properties of amotosalen‐PCT of leukoreduced pools of PLTs prepared by the PRP method and suspended in 100 percent plasma. STUDY DESIGN AND METHODS: On Day 1 of storage, 12 leukoreduced (n = 6) or 10 leukoreplete (n = 6) ABO‐identical PLT concentrates were pooled, separated into two pools of 6 or 5 units, respectively, and leukoreduced (leukoreplete pools only). Each pool of 5 or 6 units was then photochemically treated (designated “test”: amotosalen plus 3.0 J/cm² long‐wavelength ultraviolet light followed by amotosalen/photoproduct removal) while the remaining identical pool (designated “control”) was untreated. PLT in vitro assays were performed on test and control pools during 7‐day storage. RESULTS: PCT resulted in slightly reduced pH in test pools compared to that of matched control pools after 5 days of storage (5‐unit pools: test, 6.96 ± 0.12 vs. control, 7.15 ± 0.09, p = 0.0033; 6‐unit pools: test, 6.90 ± 0.10 vs. control, 7.07 ± 0.09, p < 0.0001). Test pools adequately maintained many other in vitro properties including PLT morphology, hypotonic shock response, and extent of shape change parameters during 5‐day storage, which, like pH, also differed from those of controls. The pH of test and control pools declined on Day 7, with 1 of 6 test pools (either 5 or 6 units) having a pH value of less than 6.20, while all control pools had pH values of more than 6.66. CONCLUSION: PCT of leukoreduced PLT pools of whole blood–derived PLTs in 100 percent plasma maintained adequate PLT in vitro variables through 5 days of storage.