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.     

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.     

A randomized controlled trial evaluating recovery and survival of 6% dimethyl sulfoxide–frozen autologous platelets in healthy volunteers

BACKGROUND: Availability of platelets (PLTs) is severely limited by shelf life in some settings. Our objective was to determine and compare to Food and Drug Administration (FDA) criteria the PLT recovery and survival of autologous PLTs cryopreserved at ?65°C or less in 6% dimethyl sulfoxide (DMSO) reconstituted with a no‐wash method (cryopreserved PLTs [CPPs]) compared to autologous fresh PLTs. STUDY DESIGN AND METHODS: This was a randomized, Phase I study analyzing PLT viability and in vitro function in consenting healthy subjects. Apheresis PLTs (APs) were collected in plasma. APs were suspended in 6% DMSO, concentrated, and placed at not more than ?65°C for 7 to 13 days. Frozen CPPs were thawed at 37°C and resuspended into 25 mL of 0.9% NaCl. Control PLTs (fresh autologous) and CPPs were labeled with ¹¹¹In or ⁵¹Cr, and recovery and survival after reinfusion were determined using standard methods. A panel of in vitro assays was completed on APs and CPPs. RESULTS: After frozen storage, CPPs retained 82% of AP yield and showed increased PLT associated P‐selectin and reduced responses to agonists. CPP 24‐hour recovery (41.6 ± 9.7%) was lower than for fresh PLTs (68.4 ± 8.2%; p < 0.0001) and did not meet the current FDA criterion. CPPs had diminished survival compared to fresh PLTs (7.0 ± 2.1 days vs. 8.4 ± 1.2 days, respectively; p = 0.018), but did meet and exceed the FDA criterion for survival. CONCLUSION: While 24‐hour recovery does not meet FDA criteria for liquid‐stored PLTs, the CPP survival of circulating PLTs was surprisingly high and exceeded the FDA criteria. These data support proceeding with additional studies to evaluate the clinical effectiveness of CPPs.     

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.     

Viability and function of 8-day-stored apheresis platelets

BACKGROUND: Methods of bacterial detection and pathogen inactivation of platelets (PLTs) may allow extended storage of PLTs as long as PLT quality is maintained. STUDY DESIGN AND METHODS: Twenty normal volunteers had their PLTs collected with an apheresis machine (Haemonetics Corp.). A variety of in vitro PLT function and metabolic assays were performed both on Day 0 and after 8 days of storage. On Day 8, a small blood sample was drawn from each donor to obtain fresh PLTs. The fresh and stored autologous PLTs were labeled with either (51)Cr or (111)In, and the radiolabeled PLTs were transfused. Posttransfusion serial blood samples were drawn to determine the relative posttransfusion recoveries and survivals of the fresh versus the stored PLTs. RESULTS: Although the in vitro assays showed some differences between the two trial sites, the results were generally within the ranges expected for fresh and stored PLTs. Overall, PLT recoveries averaged 66 +/- 16 percent versus 53 +/- 20 percent and survivals averaged 8.5 +/- 1.6 days versus 5.6 +/- 1.6 days, respectively, for fresh compared to 8-day-stored PLTs. There were no significant differences in the in vivo PLT data between the trial sites or based on the radiolabel used for the measurements. CONCLUSION: After 8 days of storage, the in vivo posttransfusion recovery and survival of autologous Haemonetics apheresis PLTs meet the proposed standards for poststorage PLT quality.     

Implementation of buffy coat platelet component production: comparison to platelet-rich plasma platelet production

BACKGROUND: Buffy coat (BC) production of platelets (PLTs) has been successfully used in Europe for more than two decades. Currently, Canadian Blood Services is implementing the BC method. This article summarizes results of the validation testing performed to qualify the process of PLT production from whole blood and compares the quality of PLTs produced in routine production by either the PLT‐rich plasma method (PRP‐PCs) or the BC method (BC‐PCs). STUDY DESIGN AND METHODS: Validation data included variables used for routine quality control (QC; pH, PLT count, volume, sterility, residual white blood cell count) as well as nonroutine testing of PLTs for PLT activation, metabolic changes during storage, and PLT responsiveness to hypotonic shock and the extent of shape change induced by adenosine 5′‐diphosphate. BC‐PCs were tested on Days 1 and 6. QC of production runs included the same routine tests performed on Day 6. RESULTS: PLTs produced by the BC method during validation and pilot implementation met all Canadian Standards Association standards with respect to yield, volume, pH, and leukoreduction. Additional validation testing indicated a moderate level of PLT storage lesion development. In comparison to PRP‐PCs, in vitro variables of BC‐PCs, either pH in this study, or other markers compared to the literature were better, suggesting that BC‐PCs have less evidence of production‐related damage and improved PLT quality during storage. CONCLUSIONS: PLT concentrates produced from whole blood by the BC method after an overnight hold have laboratory variables suggestive of a higher quality than those concentrates produced by the PRP method.     

Frequency and specificity of the Trima Accel "verify WBCs" advisory: considerations for product management

BACKGROUND: The Trima Accel displays a “verify WBCs” message if the plateletpheresis product (PLT) may not be leukoreduced (LR). Most blood banks require sensitive white blood cell (WBC) testing of these PLTs by flow or Nageotte. We evaluated how often these PLTs were non‐LR by European or US Food and Drug Administration (FDA) criteria and whether sensitive WBC testing is necessary. STUDY DESIGN AND METHODS: Phase 1 reviewed the frequency of this message with various procedure types and the flow WBC results for PLTs with or without the message. Phase 2 assessed how many FDA LR failures were detectable by a hematology analyzer. In Phase 3, PLTs were managed by hematology analyzer results. RESULTS: In Phase 1, 3.8% of PLT‐only and 11.1% of PLT‐plasma collections had the “verify WBCs” message. Only 1% of “verify” PLTs contained more than 1 × 10⁶ WBCs and only 0.5% were FDA LR failures. In Phase 2, 10 of 670 “verify” PLTs and one nonflagged PLT were FDA LR failures. Six of 11 LR failures had hematology analyzer WBC concentrations of 0.4 × 10⁹/L or higher. In Phase 3, “verify” PLTs were allowed in inventory if hematology analyzer WBC concentration was below 0.4 × 10⁹/L; inventory quality control showed no FDA LR failures by flow. Trima Version 6.0 software lowered the “verify” message frequency in PLT‐plasma procedures but not in PLT‐only procedures. CONCLUSION: Four percent of Trima PLT collections have the “verify WBCs” message but almost all of these are LR by European and FDA criteria. Fifty percent of FDA LR failures were detectable by a hematology analyzer. Sensitive WBC testing of all “verify WBCs” PLTs may not be necessary to satisfy LR quality assurance requirements.     

In vitro and in vivo evaluation of a whole blood platelet‐sparing leukoreduction filtration system

BACKGROUND: In‐line leukoreduction (LR) filters decrease adverse clinical sequelae caused by residual white blood cells (WBCs). Such filtration, however, can remove platelets (PLTs) needed for production of PLT concentrates (PCs). This study measured in vitro and in vivo efficacy of a new whole blood PLT‐sparing LR filter (WBPSF) system that performs whole blood (WB) LR using a single closed‐system filtration step. The WBPSF provides three final LR products: AS‐5 red blood cells (RBCs), citrate‐phosphate‐dextrose (CPD) PLTs, and CPD plasma. STUDY DESIGN AND METHODS: Volunteers (n = 59) donated WB processed using the WBPSF system. WB filtration time was recorded, and LR WB was processed into AS‐5 LR RBCs, CPD LR PLTs, and LR plasma. Final components were assayed for in vitro indices, and in vivo characteristics for LR AS‐5 RBCs and CPD PLTs were assayed after radiolabeling. RESULTS: WB filtration time averaged 37 minutes. Transfusion products obtained after WBPSF met all in vitro and in vivo Food and Drug Administration (FDA) requirements. Radiolabeling of LR AS‐5 RBCs after WBPSF showed a 24‐hour RBC recovery of 81.3 ± 5.3% after 42 days of storage. In vivo dual ¹¹¹In/⁵¹Cr radiolabeling of PCs manufactured using WBPSF showed a Day 5 recovery ratio of 80 ± 19% versus fresh autologous PLTs and a survival ratio of 81 ± 17% that of fresh autologous PLTs. CONCLUSION: All WBPSF‐derived transfusion products met or exceeded in vitro and in vivo FDA guidelines. This filtration system is suitable for routine blood center or hospital use in the production of LR AS‐5 RBCs, CPD PLTs, and CPD plasma.     

In vitro and in vivo quality of leukoreduced apheresis platelets stored in a new platelet additive solution

BACKGROUND: Platelets (PLTs) stored in additive solutions (PASs) may reduce the risk of several plasma‐associated adverse transfusion reactions such as allergic reactions and potentially transfusion‐associated lung injury. The objective of this study was to determine the in vitro characteristics and the in vivo radiolabeled recovery and survival of apheresis PLTs (APs) stored in a new PAS and compare the latter to Food and Drug Administration (FDA) criteria. STUDY DESIGN AND METHODS: Hyperconcentrated APs were collected from healthy subjects in a paired crossover study comparing PAS (35% plasma) and 100% plasma‐stored APs (Part 1) up to 7 days and, in Part 2, to determine the in vivo recovery and survival of PAS stored AP at 5 days compared to fresh PLT controls. In vitro and in vivo assays were performed following standard methods. RESULTS: Sixty‐six and 25 evaluable subjects successfully completed Parts 1 and 2, respectively. pH for PAS AP was maintained above 6.6 for 5 days of storage. P‐selectin values were consistent with published values for commonly transfused PLT products. The PAS in vivo PLT recovery (54.3 ± 8.1%) was 86.7% of the fresh control, and survival (6.4 ± 1.3 days) was 78.0% of the fresh control, both meeting the FDA performance criteria. CONCLUSION: APs stored in PAS with 35% plasma carryover maintained pH over 5 days of storage and met current FDA criteria for radiolabeled recovery and survival. The use of PAS for storage of single‐donor PLTs in clinical practice represents an acceptable transfusion product that reduces the volume of plasma associated with PLT transfusion.     

Freezing of buffy coat-derived, leukoreduced platelet concentrates in 6 percent dimethyl sulfoxide

BACKGROUND: There has recently been renewed interest in freezing platelets (PLTs) in dimethyl sulfoxide (DMSO) for the treatment of major traumatic injuries, especially in military situations. This study examined PLTs that were frozen in small volumes of 6 percent DMSO at ?80°C. STUDY DESIGN AND METHODS: Buffy coat–derived pooled leukoreduced PLT concentrates were frozen in 6 percent DMSO and stored at ?80°C. Assays included hypotonic shock response (HSR); aggregation; glycoprotein (GP)Ibα and P‐selectin binding sites; annexin V binding to phosphatidylserine, glycocalicin, and lactate dehydrogenase (LDH). Cone and plate technology (DiaMed Impact‐R, DiaMed) was used to test PLT function under near physiologic conditions. RESULTS: The freeze‐thaw loss of PLTs was 23 percent. HSR was 17 ± 7 percent. Cytometry demonstrated two populations of PLTs: one with normal levels of GPIbα binding sites (27 × 10³ ± 3 × 10³/PLT) and one with reduced levels (5.5 × 10³ ± 1.2 × 10³/PLT). There were 1.4 × 10³ ± 0.2 × 10³ P‐selectin binding sites per PLT. Annexin V binding to phosphatidylserine was 50 ± 9 percent and LDH was 496 ± 207 IU per 10¹² PLTs. Surface coverage and aggregate size, as measured by the DiaMed Impact‐R, were similar to those observed with PLTs stored for 2 days at 22°C. CONCLUSION: Some degree of activation was demonstrated by the proportion of PLTs with reduced levels of GPIbα binding sites, increased P‐selectin expression, and increased Annexin V binding. LDH concentrations indicated a degree of lysis. The DiaMed Impact‐R results showed that the PLTs were still capable of adhering to surfaces and forming aggregates under shear force.     

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.     

Temperature cycling improves in vivo recovery of cold‐stored human platelets in a mouse model of transfusion

BACKGROUND: Platelet (PLT) storage at room temperature (RT) is limited to 5 days to prevent growth of bacteria, if present, to high levels. Storage in cold temperatures would reduce bacterial proliferation, but cold‐exposed PLTs are rapidly cleared from circulation by the hepatic Ashwell‐Morell (AM) receptor, which recognizes PLT surface carbohydrates terminated by β‐galactose. We cycled storage temperature between 4 and 37°C to preserve PLT function and reduce bacterial growth. STUDY DESIGN AND METHODS: Temperature‐cycled (TC) human PLTs were stored at 4°C for 12 hours and then incubated at 37°C for 30 minutes before returning back to cold storage. PLTs stored at RT or at 4°C (COLD) or TC for 2, 5, and 7 days were infused into SCID mice and the in vivo recovery was determined at 5, 20, and 60 minutes after transfusion. RESULTS: PLTs stored for 2 days in COLD had significantly lower in vivo recoveries than RT PLTs. TC PLTs had improved recoveries over COLD and comparable to RT PLTs. After 5‐ and 7‐day storage, TC PLTs had better recoveries than RT and COLD PLTs. PLT surface β‐galactose was increased significantly for both COLD and TC PLTs compared to RT. Blocking of the AM receptor by asialofetuin increased COLD but not TC PLT recovery. CONCLUSION: TC cold storage may be an effective method to store PLTs without loss of in vivo recovery. The increased β‐galactose exposure in TC PLTs suggests that mechanisms in addition to AM receptors may mediate clearance of cold‐stored PLTs.     

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.     

Effects of pretransfusion warming of platelets to 35°C on posttransfusion platelet viability

BACKGROUND: Three of four prior studies suggested that warming platelets (PLTs) to 37°C before transfusion into patients with thrombocytopenia gave improved corrected PLT count increments.
STUDY DESIGN AND METHODS: Eighteen normal subjects had apheresis PLTs collected that were stored at 22°C for 5 days in two storage bags. One bag of PLTs was warmed to 35°C before infusion, and one remained at 22°C. Three different methods of warming the donor's autologous PLTs before reinfusion were evaluated: warming PLTs to 35°C for 10 or 60 minutes followed by radiolabeling or radiolabeling the PLTs followed by warming to 35°C for 60 minutes. In the first two methods, the warmed PLTs would have returned to 22°C before infusion, and in the third, the PLTs would still be warm when injected. The paired test and control PLTs were radiolabeled with either ¹¹¹In or ⁵¹Cr to determine posttransfusion PLT recoveries and survivals. PLT morphology score, pH, hypotonic shock response, extent of shape change, and annexin V binding were determined just before transfusion.
RESULTS: There were no differences in posttransfusion autologous radiolabeled PLT recoveries and survivals or in the in vitro measurements for the PLTs maintained at 22°C versus those warmed to 35°C using any of the three methods of PLT warming before infusion.
CONCLUSION: Based on these 5-day-stored autologous radiolabeled PLT recovery and survival measurements, there is no evidence that warming PLTs to 35°C before infusion improves postinfusion PLT viability.     

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.     

Effects of immediate or delayed addition of platelet additive solution on the in vitro quality of apheresis platelets

BACKGROUND: There is little knowledge how different hold times of hyperconcentrated platelet (PLT) suspensions (HPSs) before the addition of platelet additive solution (PAS) might affect PLT quality. We compared the in vitro quality of single‐donor PLT concentrates (SDPs) with immediate or delayed PAS addition and studied the quality of collected concurrent plasma (CP). STUDY DESIGN AND METHODS: We collected 6 × 10¹¹ PLTs in 175 of mL plasma and CP from 31 donors. The HPSs were split into two parts, with 162 mL of modified PAS III (PAS‐IIIM) added immediately (0hr‐SDP) or 2 hours later (2hr‐SDP). Final SDPs had a targeted concentration of 1.2 × 10¹² PLTs/L and a PAS proportion of 65%. On Days 1, 5, and 7 we determined glucose and lactate concentration, pH, P‐selectin expression, hypotonic shock response (HSR), and extent of shape change (ESC). Clotting Factor V (FV) and VIII (FVIII) activities and D‐dimer concentration were determined in CP and donor. RESULTS: Glucose utilization, lactate production, and pH were similar for both kinds of products. Low P‐selectin expression indicated no relevant PLT activation during storage. HSR and ESC were similarly well preserved. Recoveries of FV and FVIII were 100.0 ± 14.0 and 98.6 ± 14.9%, respectively. Concentrations of D‐dimers in the donor and CP were 173.7 ± 90.1 and 177.6 ± 91.2 ng/dL, respectively. CONCLUSIONS: Adding PAS immediately or 2 hours after collection does not result in different in vitro quality of PLTs stored up to 7 days. The good recovery of clotting factors with no signs of activation indicates a good quality of CP.     

In vitro properties of apheresis platelet during extended storage in plasma treated with anandamide

BackgroundIn China apheresis platelets (PLTs) are stored in plasma for only 5 days, resulting in PLT inventory pressures. Anandamide (ANA) was reported to be a potential agent to inhibit PLT apoptosis. The aim of this study was to evaluate the characteristics of extended storage PLTs in plasma treated with ANA in vitro.MethodsApheresis PLTs (n = 20) were prepared in plasma treated with ANA, and stored at 22 °C for up to 11 days. On day 1, 3, 5, 7, 9 and 11, PLTs were tested for PLT count, mean PLT volume (MPV), PLT distribution width (PDW), pH, pCO₂, pO₂, hypotonic shock response (HSR), phosphatidylserine (PS) exposure and soluble P-selectin content.ResultsPLTs stored in plasma with/without ANA didn't show significant differences during the first 5 days of storage. From the 7^th day on, PLTs stored in plasma with ANA displayed significantly lower PS expression, soluble P-selectin content and higher HSR scores than those stored in plasma without ANA (P <0.05), respectively.ConclusionThe extended storage of PLTs in plasma treated with 0.5 µmol/l ANA showed better characteristics of the PLTs, compared with the control group, which was suggested to potentially alleviate the PLT storage lesion.     

Mitochondrial dysfunction of platelets stored in first‐ and second‐generation containers is,in part,associated with elevated carbon dioxide levels

BACKGROUND: The gas permeability of platelet (PLT) storage bags influences the retention of in vitro PLT parameters during storage. The aim of this study was to evaluate mitochondrial function of PLTs stored in first‐ and second‐generation bags with different gas permeabilities. STUDY DESIGN AND METHODS: Identical whole blood–derived PLT concentrates were stored in second‐generation CLX (Pall Corp.) and first‐generation PL146 (Baxter Healthcare Corp.) bags (n = 12). 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 on Days 1, 4, 5, 6, and 7. Results were analyzed by paired t test and by multiple regression analysis. RESULTS: With PLTs stored in PL146 bags that underwent large pH declines, there was greater superoxide production, greater peroxide accumulation, and greater mitochondrial membrane depolarization. Superoxide anion generation was correlated with higher levels of carbon dioxide (p = 0.0001) and lower oxygen levels (p = 0.0064; multiple regression R² = 0.9204). Changes in MMP were correlated with higher levels of carbon dioxide (p = 0.0288) and PLT activation (p = 0.0178; multiple regression R² = 0.9511). CONCLUSION: Prolonged periods of elevated carbon dioxide levels, potentially coupled with other factors, is associated with PLT mitochondria dysfunction and poor pH control during 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.     

Storage of interim platelet units for 18 to 24 hours before pooling: in vitro study

BACKGROUND: The Atreus 3C system (CaridianBCT) automatically produces three components from whole blood (WB), a red blood cell (RBC) unit, a plasma unit, and an interim platelet (PLT) unit (IPU) that can be pooled with other IPUs to form a PLT dose for transfusion. The Atreus 3C system also includes a PLT yield indicator (PYI), which is an advanced algorithm that provides an index that is shown to correlate well with the amount of PLTs that finally end up in the IPU bag. The aim of our in vitro study was to compare the effects of holding WB overnight versus processing WB fresh (2‐8 hr), both with 18‐ to 24‐hour storage of the IPUs before pooling into a transfusable PLT dose. STUDY DESIGN AND METHODS: WB was processed either fresh (within 8 hr after collection, Atreus F) or after overnight storage (14‐24 hr, Atreus S) without agitation at 22 ± 2°C. After a subsequent resting time of 18 to 24 hours on a flat‐bed shaker, five IPUs were selected for pooling with 200 mL of PAS II for in vitro quality assessments during a 7‐day storage period (n = 10 in each arm). IPUs were selected for pooling using the PYI of the Atreus 3C system. RESULTS: During storage, the glucose concentration was lower (p < 0.05) and the lactate concentration was higher (p < 0.05) in Atreus S pools, but no differences in the glucose consumption rate were noted. Adenosine triphosphate levels and hypotonic shock response reactivity were higher in Atreus S (p < 0.05). No significant differences in PLT counts, contents, mean PLT volume, lactate dehydrogenase, pO₂, pCO₂, extent of shape change, and CD62P between groups were detected. pH was maintained higher than 6.8 (Day 7). With exception of 2 units in the Atreus S arm, swirling remained at greater than 2 in all units at all times. CONCLUSION: Our results suggest that PLTs prepared from fresh or overnight‐stored WB and pooled after 18 to 24 hours meet necessary in vitro criteria without any relevant differences between both groups. Using the PYI, comparable yields can be obtained between WB processed within 2 to 8 hours and WB stored overnight.