Supernates from stored red blood cells inhibit platelet aggregation

BACKGROUND: Previously, we reported that red blood cells (RBCs) stored in AS‐5 accumulated proinflammatory substances during storage. We observed in those studies that supernates from nonleukoreduced (NLR) RBCs reduced mean anti‐CD41a‐fluorescein isothiocyanate (FITC) fluorescence on platelets (PLTs), indicative of decreased expression of glycoprotein (GP)IIb/IIIa on the PLT membrane. The objective of this study was to determine if supernates from stored RBCs impaired PLT aggregation as a consequence of reduction in GPIIb/IIIa expression. STUDY DESIGN AND METHODS: Leukoreduced (LR) and NLR RBC units were prepared in AS‐5 and stored at 1 to 6°C for 6 weeks. Supernates from RBC samples collected every 2 weeks were mixed with freshly collected type‐matched blood and incubated for 30 minutes at 37°C. PLTs in each incubated blood sample were evaluated for GPIIb/IIIa expression by flow cytometry and for aggregation response to collagen by whole blood aggregometry. RESULTS: Supernates from stored NLR RBCs reduced CD41a‐FITC fluorescence on PLTs by 15% to 31%. A reduction in fluorescence was induced by supernates of RBCs stored for 14 days and increased as storage time increased. Supernates from Day 42 NLR RBCs reduced the mean amplitude of PLT aggregation by 31% compared to Day 0 supernates and lengthened the time before onset of aggregation by 21%. In addition, amplitude correlated directly and lag time correlated inversely with CD41a‐FITC fluorescence in all samples. Supernates from prestorage LR RBCs did not affect PLT CD41a‐FITC fluorescence or aggregation response. CONCLUSIONS: Substances that decrease expression of GPIIb/IIIa and inhibit PLT aggregation accumulate in NLR RBCs. Accumulation of this material is prevented by leukoreduction.     

Function of platelets in apheresis platelet concentrates and in patient blood after transfusion as assessed by Impact‐R

BACKGROUND: Platelet (PLT) transfusion is a mainstream therapy for preventing or treating bleeding episodes in patients with thrombocytopenia. The efficacy is usually estimated from the corrected count increment of PLTs after transfusion, which does not assess PLT function. We therefore evaluated PLT function in blood samples of patients with thrombocytopenia before and after transfusion. STUDY DESIGN AND METHODS: PLT function was assessed in 24 chemotherapy‐treated patients and in the PLT concentrates (PCs) by the Impact‐R (DiaMed). This device evaluates PLT adhesion and aggregation recorded as surface coverage (%) and size of aggregates (AS µm²). P‐selectin expression was determined by flow cytometry. RESULTS: The PCs were stored for a median of 70 hours before transfusion. An analysis stratified by the median storage of PCs (<70 hr or >70 hr) showed no differences in the SC, the AS, and P‐selectin expression between these concentrates' groups. Transfusion resulted in an increase of adhering PLTs in the patients after transfusion. There were no differences in the AS and in P‐selectin expression before and after transfusion, but the AS increased after transfusion upon ex vivo exposure to adenosine 5′‐diphosphate. P‐selectin expression was significantly lower in the patient group receiving PCs stored for more than 70 hours. CONCLUSION: The current trial shows the feasibility of using the Impact‐R to assess the function of transfused PLTs in the patient's blood stream.     

Effect on the quality of blood components after simulated blood transfusions using volumetric infusion pumps

BACKGROUND: It is unknown whether the use of volumetric infusion pumps for the transfusion of red blood cells (RBCs) or platelet (PLT) concentrates (PCs) affects the quality of the blood components. We therefore investigated the in vitro quality of these components after use of infusion pumps. STUDY DESIGN AND METHODS: Ten different volumetric infusion pumps were used to simulate transfusion with RBCs and PCs. To prevent donor‐dependent differences multiple units were pooled and divided into equal portions. The storage time of RBCs was 30 to 35 days (n = 10 experiments), and for PCs, either 2 (n = 5) or 7 days (n = 5). For RBCs an infusion rate of 100 or 300 mL/hr was used, and for PCs, 600 mL/hr. Transfusions without an infusion pump served as a reference. RESULTS: None of the infusion pumps induced an increase of free hemoglobin, annexin A5 binding, or formation of echinocytes in RBCs compared to reference units. In 2‐ and 7‐day‐old PCs no effect was shown on PLT concentration, annexin A5 binding, mean PLT volume, and morphology score compared to the reference. The CD62P expression of 2‐day‐old PCs was significantly lower after transfusion compared to the reference, that is, 11.7 ± 2.1% versus 8.1 ± 1.3% (p < 0.01). CONCLUSION: There was no adverse effect on the in vitro quality of RBCs or PCs after simulated transfusion using volumetric infusion pumps. A decrease in PLT activation was observed, which can probably be explained by capturing of activated or damaged PLTs in the 200‐µm filter present in the infusion system.     

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.     

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.     

Active cooling of whole blood to room temperature improves blood component quality

BACKGROUND: Many countries use cooling plates to actively cool collected whole blood (WB) to room temperature. Until now, no paired comparison had been performed, and it was our aim to compare the effect of active versus no active cooling on the in vitro quality of WB and subsequently prepared blood components. STUDY DESIGN AND METHODS: Two units of WB were pooled and divided shortly after donation. One unit was placed under a butane‐1,4‐diol plate to obtain active cooling; the other was placed in an insulated box with other warm units to mimic worst‐case holding conditions. WB was held overnight and processed into a white blood cell (WBC)‐reduced red blood cells (RBCs), buffy coat (BC), and plasma. The BCs were further processed into platelet (PLT) concentrates. RBCs were stored for 42 days, and PLT concentrates for 8 days (n = 12 paired experiments). RESULTS: After overnight storage, ATP content of the RBCs was 4.9 ± 0.3 µmol/g Hb for actively cooled WB versus 4.5 ± 0.4 µmol/g Hb for not actively cooled WB (p < 0.001). On Day 42 of storage, RBCs prepared from this WB contained 3.1 ± 0.3 µmol ATP/g Hb with active cooling versus 2.6 ± 0.3 µmol/g Hb without (p < 0.001). Hemolysis on Day 42 was 0.35 ± 0.08% with active cooling and 0.67 ± 0.21% without (p < 0.001). No effect was observed on the in vitro quality of plasma, BC, or PLT concentrates. CONCLUSIONS: Active cooling of WB results in improved ATP levels and less hemolysis in WBC‐reduced RBCs, although the clinical implications are unclear. It has no effect on the in vitro quality of plasma or PLT concentrates.     

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.     

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

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

Transfusion reactions: a comparative observational study of blood components produced before and after implementation of semiautomated production from whole blood

BACKGROUND: A semiautomated method of component production from whole blood was implemented at Canadian Blood Services. To assess safety of the new components, the frequency of adverse transfusion events (ATEs) to platelet components (PCs) and red blood cell (RBCs) produced before and after implementation of the new method was surveyed and compared. STUDY DESIGN AND METHODS: This retrospective, observational, noninferiority study was conducted in 12 sentinel hospitals across Canada. The control group received RBCs in additive solution‐3 (AS‐3) and platelet‐rich plasma (PRP)‐produced platelets (PLTs) for 3 to 11 months before implementation of semiautomated production, and the study group received RBCs in saline‐adenine‐glucose‐mannitol (SAGM) and buffy coat (BC)‐produced PLTs for 3 to 11 months after implementation. ATE definitions at each hospital and standard practice for reporting did not change between control and study periods. Data for analysis were obtained from databases and original report forms. RESULTS: The pooled risk ratio of a reaction to SAGM versus AS‐3 RBCs was 0.77 (95% confidence interval [CI], 0.66‐0.90), suggesting that SAGM products had significantly lower reaction rates than AS‐3 products (p < 0.01). Reported allergic reactions to RBCs decreased from 0.07% (AS‐3) to 0.04% (SAGM). For PLTs, the difference in reaction rates between BC and PRP was not significant (p = 0.37), and the pooled risk ratio of BC versus PRP was 1.14 (95% CI, 0.86‐1.50). CONCLUSION: The change in manufacturing method was associated with lower reaction rates to SAGM RBCs than to AS‐3 RBCs. Pooled BC PLTs were noninferior to random‐donor PRP PLTs with respect to ATEs.     

Platelet capacity of various platelet pooling systems for buffy coat-derived platelet concentrates

BACKGROUND: Platelet (PLT) storage lesions might depend on the total PLT count in the storage container and also on the PLT pooling system, especially the storage container, that is used for preparation of PLT concentrates (PCs). In this study, the PLT capacity of four commercially available PLT pooling systems was studied. MATERIALS AND METHODS: Four PCs were prepared in pooling systems of Baxter, Fresenius, Terumo, or Pall. The PCs were pooled and divided with various total PLT counts over the four storage containers (<225 × 10⁹, 225 × 10⁹‐324 × 10⁹, 325 × 10⁹‐424 × 10⁹, and >424 × 10⁹ PLTs). Volumes were kept equal by adding plasma to PCs with less than 425 × 10⁹ PLTs until a same volume as for PCs with more than 424 × 10⁹ PLTs was reached. PCs were stored at room temperature and tested for various in vitro variables on Days 1, 3, 5, 7, and 9. Paired experiments were repeated for each system five times. RESULTS: In vitro variables remained good for 9 days, that is, swirling score of 2 or more, pH value of 6.8 or more, glucose level of 10 mmol per L or more, lactate level of less than 25 mmol per L, and CD62p expression of less than 50 percent, for PCs in Baxter systems with more than 225 × 10⁹ PLTs, for PCs in Fresenius and Terumo systems with 225 × 10⁹ to 424 × 10⁹ PLTs, and for PCs in Pall systems with fewer than 425 × 10⁹ PLTs. CONCLUSION: PLT capacity depended on the PLT pooling systems used. All systems provide acceptable storage conditions. The Baxter system was the only system with capacity for more than 424 × 10⁹ PLTs per PC.     

Platelet counting in platelet concentrates with various automated hematology analyzers

BACKGROUND: Hematology analyzers use impedance, optical, and/or immunologic techniques for counting platelets (PLTs). PLT counting in whole blood has been validated thoroughly; however, this is not the case for PLT counting in PLT concentrates (PCs), in which red cells (RBCs) are absent. Therefore, this study is focused on PLT counting in PCs to study use of ethylenediaminetetraacetate (EDTA), carryover, and accuracy of the analyzers. STUDY DESIGN AND METHODS: In total six hematology analyzers (AcT 8, Beckman Coulter; ADVIA 2,120, Bayer; Cell-Dyn 4,000, Abbott; Onyx, Beckman Coulter; K4,500, Sysmex; and XT 2,000i, Sysmex) were tested for PLT counting. PC samples with various PLT concentrations were made (0-1,700 x 10(9)/L) and measured 10 times. Carryover was determined five times. RESULTS: PC samples (1,000 x 10(9) PLTs/L) in EDTA tubes showed significantly higher PLT counts than samples in "dry" tubes for all analyzers except for the Cell-Dyn 4,000 with the impedance technique. Carryover was not more than 0.3 percent for all analyzers. The K4,500 showed the most accurate results, whereas the Cell-Dyn 4,000 with the impedance technique had low accuracy due to an overestimation of more than 20 percent. CONCLUSION: Most tested analyzers seemed to be suitable for counting PLTs in PCs. All hematology analyzers should be validated for counting PLTs in absence of RBCs as is the case in PCs, in addition to validation of PLT counting in whole blood.     

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.     

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.     

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.     

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.     

In vitro properties of platelets stored in three different additive solutions

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

Level of platelet-derived cytokines in leukoreduced red blood cells is influenced by the processing method and type of leukoreduction filter

BACKGROUND: In contrast to the well-documented effect of white blood cells on the quality of red blood cells (RBCs), the effect of platelets (PLTs) has received little consideration. In this study, the PLT content and level of PLT-derived cytokines in RBCs prepared using different types of leukoreduction methods were investigated.
STUDY DESIGN AND METHODS: Buffy coat–poor RBCs and five types of leukofiltered (LF) RBCs, including RBCs prepared with a whole blood (WB) PLT-saving filter, were prepared and stored according to standard blood bank conditions. PLT content was measured on Day 1, and levels of PLT-derived cytokines were measured by enzyme-linked immunosorbent assay at nominated timepoints during 42 days of storage.
RESULTS: The PLT content of leukoreduced RBCs varied widely depending on the processing method and/or leukoreduction filter used, with some types of RBCs containing very low PLT counts while other units contained PLT counts comparable to those of unprocessed WB. The PLT content of RBCs directly influenced the concentration and accumulation of PLT-derived cytokines. Several PLT-derived factors exhibited significant accumulation throughout 42 days of storage. RBCs with high PLT content exhibited concentrations of RANTES (CCL5) and soluble CD40 ligand equivalent to those previously reported to show significant biologic and clinical effects.
CONCLUSION: The PLT content and levels of PLT-derived cytokines in leukoreduced RBCs are influenced by the processing method and types of leukoreduction filters used. It may be inappropriate to consider LF-RBCs prepared with different types of leukoreduction filters as equivalent products based on their differing levels of PLT factors.     

Effects of storage duration and volume on the quality of leukoreduced apheresis‐derived platelets: implications for pediatric transfusion medicine

BACKGROUND: Platelet (PLT) storage adversely affects PLT structure and function in vitro and is associated with decreased PLT recovery and function in vivo. In pediatric transfusion medicine, it is not uncommon for small residual volumes to remain in parent units after aliquot preparation of leukoreduced apheresis‐derived PLTs (LR‐ADP). However, limited data exist regarding the impact of storage on residual small‐volume LR‐ADP. STUDY DESIGN AND METHODS: Standard metabolic testing was performed on residual volumes of LR‐ADP after aliquot removal and PLT aggregometry using a dual agonist of ADP and collagen was performed on stored, small‐volume aliquots (10‐80 mL) created from an in vitro model of PLT storage. RESULTS: Seventy‐seven LR‐ADP underwent metabolic (n = 67) or metabolic and aggregation (n = 10) studies. All products maintained a pH value of more than 6.89 throughout storage. Lactate and pCO₂ increased proportionally with longer storage time. Regardless of acceptable metabolism during storage, aggregation in 10‐ to 20‐mL aliquots was impaired by Day 4 and aliquots less than 40 mL demonstrated the most dramatic decrease in aggregation from baseline. CONCLUSIONS: Despite maintenance of acceptable metabolic conditions, residual volumes of LR‐ADP develop impaired aggregation in vitro that may adversely affect PLT survival and function in vivo. At volumes below 40 mL, LR‐ADP revealed reduced aggregation. As a result, it is recommended to monitor and record volumes of LR‐ADP used for pediatric transfusion. Moreover, once LR‐ADP attain a volume of 50 mL or less on Day 4 or Day 5 of storage, consider discarding these products until their in vivo efficacy can be studied.     

In vitro platelet function of platelet concentrates prepared using three different apheresis devices determined by impedance and optical aggregometry

BACKGROUND: Testing the functional capacity of platelets (PLTs) from platelet concentrates (PC) is a main issue in transfusion medicine. Therefore, the aim was to study agonist-inducible PLT aggregation of PLTs obtained by three apheresis devices. A semiautomated impedance aggregometry-based whole-blood method (multiplate electrode PLT aggregometry [MEA]) was modified for the use of PLTs from PC and data were compared with light transmission aggregometry (LTA).
STUDY DESIGN AND METHODS: PLT function was determined in 135 PCs without reconstitution with red blood cells (RBCs), obtained by three devices: Amicus, Trima Accel collection system, and MCS+. PLT function was assessed by the Multiplate TRAP test, and the area under the curve (AUC) was quantified. TRAP-6–inducible maximal PLT aggregation (MA%) by LTA was used for analyses.
RESULTS: The AUC was significantly lower in the Amicus PLTs compared to the Trima and MCS+ PLTs (Amicus versus Trima, p = 0.007; Amicus versus MCS+, p < 0.001). The Amicus PLTs were significantly less responsive to TRAP-6–inducible PLT aggregation than Trima (p = 0.002) or MCS+ PLTs (p < 0.001) by LTA, and Trima PLTs responded significantly less than MCS+ PLTs (p = 0.001). There was only a weak correlation between MEA and LTA (r = 0.29, p = 0.019).
CONCLUSION: PLTs obtained by the Amicus system show significantly less aggregation response to thrombin receptor stimulation compared to those obtained with other cell separators, examined by MEA and LTA. Testing PLT function in PCs by the MEA is a simple and rapid method without the need of adding RBCs. However, LTA and MEA appear to measure different aspects of PLT function.     

Correlation between platelet thrombus formation on collagen-coated beads and platelet aggregation induced by ADP

BackgroundThe thrombus-forming ability is a critical in vitro parameter to assess platelets (PLTs), but flow-based methods using collagen-coated materials generally require multistep, proficiency, and advanced analysis.Study design and methodsCommercially available collagen-coated bead columns were examined to assess thrombus-forming ability of PLTs. The retention rate as an index of thrombus formation was calculated using the PLT count before and after column passage. Thrombi were imaged by anti-CD41 using a fluorescent microscope. PLT aggregation was measured by light-transmitting aggregometry.ResultsThe retention rate was low when apheresis-collected PLT concentrates (PCs) were suspended in plasma either with or without Ca²⁺. Reconstitution of PCs with red blood cells (RBCs) increased the retention rate with good reproducibility on repeated-measurements, and therefore, PLT samples were reconstructed with RBCs in subsequent experiments. The retention rate of PCs varied widely in a product-dependent manner, and was correlated with the aggregation rate induced by ADP, but not that by collagen. Using platelet-rich-plasma, antagonists of P2Y₁ or P2Y₁₂ receptors for ADP reduced both the retention and aggregation of PLTs. Acetylsalicylic acid reduced retention, although it had no effect on ADP-induced aggregation. Prostaglandin E₁ significantly inhibited both retention and aggregation. These anti-PLT reagents resulted in reduced or no thrombus formation on the beads.ConclusionThe collagen-coated bead column was useful to readily examine the thrombus-forming ability of PLTs. Variance of the PLT retention rate was correlated with responsiveness to ADP. Results from anti-PLT reagents revealed that thrombus formation on collagen-coated beads was similar to in vivo thrombus development.