Time from apheresis platelet donation to cold storage: Evaluation of platelet quality and bacterial growth.

BACKGROUND: Cold storage reduces posttransfusion survival of platelets; however, it can improve platelet activation, lower risk of bacterial contamination, and extend shelf-life compared to room temperature (RT) storage. To facilitate large-scale availability, manufacturing process optimization is needed, including understanding the impact of variables on platelet potency and safety. Short time requirements from collection to storage is challenging for large blood centers to complete resuspension and qualify platelets for production. This study evaluated the impact of time from platelet component collection to cold storage on in vitro properties and bacterial growth. STUDY DESIGN AND METHODS: Double-apheresis platelet components were collected from healthy donors, suspended in 65% PAS-III/35% plasma, and split into 2 equal units. One unit was placed into cold storage within 2 h and the other unit after 8 h. Eight matched pairs were evaluated for 12 in vitro parameters. Twenty-four matched pairs were evaluated with 8 bacterial strains tested in triplicate. Samples were tested throughout 21 days of storage. RESULTS: In vitro properties were not different between 2 and 8 h units, and trends throughout storage were similar between arms. Time to cold storage did not significantly impact bacterial growth, with <1 log10 difference at all timepoints between units. DISCUSSION: Our studies showed that extending time to cold storage from 2 to 8 h from collection did not significantly increase the bacterial growth, and the platelet component quality and function is maintained. The ability to extend the time required from collection to storage will improve blood center logistics to feasibly produce CSPs.

Preliminary characterization of the properties of cold-stored apheresis platelets suspended in PAS-III with and without an 8-hour room temperature hold.

BACKGROUND: Use of extended cold storage of platelets promises to increase PLT availability and the bacterial safety of bleeding patients. No information is currently available on the preservation of apheresis PLT in vitro quality parameters when PLTs are held at room temperature early in the storage period prior to transfer to cold storage. STUDY DESIGN AND METHODS: Double units of platelets suspended in 35% plasma/65% PAS-III were collected from normal consenting research donors and rested at room temperature for 1-2 hours. One of the units was then stored at 1-6°C while the other unit was placed on an agitator at 20-24°C. Eight hours after collection, the unit stored at room temperature was transferred to 1-6°C storage without agitation. Units were sampled for an array of PLT in vitro parameters on Days 1, 7, 14, and 21. RESULTS: As expected, PLTs held for 8 hours at 20-24°C prior to 1-6°C storage had greater lactate levels and reduced glucose levels and pH compared to PLTs subjected to a 1-2-hour room temperature hold prior to cold storage (P < .05). Unexpectedly, platelets held for 8 hours at room temperature had less aggregation response to collagen, ADP, and TRAP compared to PLTs held 1-2 hours at room temperature prior to cold storage (P < .05, n = 8). CONCLUSION: Decline of aggregation response should be considered when evaluating longer than necessary room temperature holds prior to cold storage of platelets.

Sodium citrate contributes to the platelet storage lesion.

BACKGROUND: Sodium citrate has become the preferred anticoagulant used for apheresis collection and has been included in commercial platelet additive solutions (PASs) since PAS-II. It was suggested that citrate be included in PASs to prevent spontaneous aggregation. Reports in cell lines and cord blood have demonstrated that concentrations of citrate present in PAS formulations (10 mM) cause apoptosis. We evaluated whether the removal of citrate from PAS-III could improve platelet storage. STUDY DESIGN AND METHODS: Study 1 evaluated the effects of a citrate dose response on the storage of platelets in 65% PAS containing sodium chloride, sodium acetate, and phosphate. Study 2 compared the cell quality and function of platelets stored in 65% citrate-free PAS-III or PAS-III containing 10 mM of citrate. Measurements included cell count, blood gases, flow cytometry analysis of surface activation markers, and aggregation. RESULTS: Study 1 identified that inclusion of citrate in PAS resulted in a dose-dependent increase in glucose utilization, lactate formation, P-selectin expression, phosphatidylserine (PS) exposure, and reactive oxygen species (ROS) formation. Study 2 showed similar results in which platelets stored in citrate-free PAS-III benefited through better maintenance of glucose utilization with less lactate production, P-selectin expression, PS exposure, and ROS formation compared to citrate-containing PAS-III. Platelets stored in citrate-free PAS-III had aggregation responses that were at least 10% greater than those platelets stored in PAS-III. CONCLUSION: Storage of apheresis platelets in citrate-free PAS-III improved multiple storage parameters including glucose utilization, lactate production, P-selection expression, PS exposure, and ROS formation and resulted in a modest increase in aggregation.

Value of calcium and phosphate in a bicarbonate-containing platelet additive solution with low plasma levels in maintaining key in vitro platelet storage parameters.

BACKGROUND: Use of recently developed platelet (PLT) additive solutions (PAS) with 5% plasma levels may reduce the frequency and/or severity of transfusion reactions attributed to plasma. PLTs suspended in bicarbonate-containing PAS-5 with 5% plasma levels can maintain key PLT parameters during 7-day storage. This study evaluates the role of calcium and phosphate, as constituents of PAS-5, in maintaining PLT parameters. STUDY DESIGN AND METHODS: An Amicus apheresis PLT unit (n = 13) was equally divided into four 60-mL aliquots in CF-250 polyolefin bags. Four different formulations of PAS-5 were prepared: PAS-5, PAS-5 without phosphate (-PO4 ), PAS-5 without calcium (-Ca), and PAS-5 without Ca and phosphate (-Ca/-PO4 ). PLTs were centrifuged, and the supernatant was expressed and replaced with the respective PAS, yielding PLTs suspended in 95% PAS and 5% plasma. PLTs were stored at 20 to 24ºC with agitation for 7 days. PLT in vitro parameters were evaluated on Days 1, 5, and 7. RESULTS: In PLT PAS-5 aliquots, pH levels were maintained better compared with those in -Ca and -Ca/-PO4 aliquots. Glycolysis was greater in -Ca and -Ca/-PO4 PLT aliquots compared with PAS-5 aliquots. Hypotonic stress response and morphology were less and p-selectin (CD62P) binding was greater in -Ca/-PO4 PLT aliquots. The accumulation of reactive oxygen species was greater in -Ca/-PO4 PLTs. Phosphorylation of p38 mitogen-activated protein kinase (MAPK) was greater in -Ca and -Ca/-PO4 PLT aliquots during storage. CONCLUSION: The removal of calcium and phosphate from PAS-5 leads to the activation of p38 MAPK and deterioration of key PLT storage parameters.

Automated cold temperature cycling improves in vitro platelet properties and in vivo recovery in a mouse model compared to continuous cold storage.

BACKGROUND: Platelets (PLTs) stored at cold temperatures (CTs) for prolonged time have dramatically reduced bacterial growth but poor survival when infused. A previous study demonstrated that human PLTs stored with manual cycling between 4 °C (12 hr) and 37 °C (30 min) and infused into severe combined immunodeficient (SCID) mice had survivals similar to or greater than those stored at room temperature (RT). In this study, the in vitro and in vivo properties of PLTs stored in an automated incubator programmed to cycle between 5 °C (11 hr) and 37 °C (1 hr) were evaluated. STUDY DESIGN AND METHODS: A Trima apheresis unit (n = 12) was aliquoted (60 mL) in CLX bags. One sample was stored with continuous agitation (RT), a second sample was stored at 4-6 °C without agitation (CT), and a third sample was placed in an automated temperature cycler with 5 minutes of agitation during the warm-up period (thermocycling [TC]). PLTs were assayed for several relevant quality variables. On Day 7, PLTs were infused into SCID mice and in vivo recovery was assessed at predetermined time points after transfusion. RESULTS: The glucose consumption rate, morphology score, hypotonic shock recovery level, and aggregation levels were increased and mitochondrial reactive oxygen species accumulations were decreased in TC-PLTs compared to those of CT-PLTs. The pH and Annexin V binding were comparable to those of RT-PLTs. All TC-PLTs had greater recovery than CT-PLTs and were comparable to RT-PLTs. CONCLUSION: PLTs stored under automated TC conditions have improved in vivo recovery and improved results for a number of in vitro measures compared to CT-PLTs.

Influence of apheresis collection device and container on the storage properties of platelets in 90% PAS-5/10% plasma.

BACKGROUND: The storage properties of apheresis platelets suspended in the experimental additive solution PAS-5 and 10% plasma may be affected by the collection instrument or storage container. METHODS AND EXPERIMENTAL DESIGN: The same consenting 12 donors provided A or T platelets with concurrent plasma on four occasions in 100% plasma. Following collection and resting, the platelets were centrifuged, and plasma was expressed and resuspended in PAS-5 to yield units with 10% plasma. Platelets were either maintained in the original storage container or transferred to another of the manufacturer's storage containers. On days 1, 5 and 7, units were assayed for an array of in vitro tests. RESULTS: Average unit volume, yield and percent plasma was 291±11 mL, 3.7±0.4×1011, and 10.3±0.7%, respectively, and were comparable between collections with either of the apheresis instruments and stored with either of the manufacturer's containers. Day 1 platelet activation (CD62P+) was 40±22% and was similar in either of the collection instruments or containers. Except for pH (days 1, 5), CO2 (days 1, 5, 7), and extent of shape change (day 5), every other in vitro parameter was similar between apheresis platforms or the manufacturer's container. pH values of all units on all days of storage were ≥6.8, except one unit that was collected on T and stored in an A container, which had pH values of 6.8 and 5.7 on days 5 and 7, respectively. DISCUSSION: Storage of platelets suspended in PAS-5 with 10% plasma is feasible in the original manufacturer's container for seven days. Based on CO2 levels, T containers have greater gas exchange than A containers.

Increase of plasma concentration to 10% improves a number of in vitro storage parameters of apheresis platelets suspended in a bicarbonate-containing additive solution and stored with a 24-hour interruption of agitation.

BACKGROUND: A previous study demonstrated several pH failures during 7-day storage of platelets suspended in 5% plasma/95% PAS-5 following a 24-hour interruption of agitation. The aim of this study was to investigate whether pH control improves in platelets stored in PAS-5 with 10% plasma following interruption of agitation. MATERIALS AND METHODS: Four aliquots were prepared from a single unit of apheresis platelets: two each with 5% and 10% plasma. After resting for 1 hour, the aliquots were placed on an agitator. On day 2, agitation of one aliquot with 5% plasma and another with 10% plasma was interrupted for 24 hours before the aliquots were returned to agitator. The two control aliquots remained on the agitator. An array of platelet parameters was measured on days 2, 5 and 7. RESULTS: On day 7, aliquots containing 10% plasma and subjected to interruption of agitation had a significantly higher mean pH compared to those of similarly treated aliquots containing 5% plasma (6.80±0.54 vs 6.41±0.57, p≤0.05). Platelets containing 10% plasma/95% PAS-5 subjected to interruption of agitation had a greater hypotonic stress response, greater shape change, higher mitochondrial membrane potential, decreased glucose utilisation and lower CD62P levels compared to those of similarly treated platelets suspended in 5% plasma. DISCUSSION: Increasing plasma concentration to 10% improves pH control and some in vitro platelet properties during 7 days of storage of platelets suspended in PAS-5 after a 24-hour interruption of agitation compared to those of similarly treated platelets suspended in 5% plasma/95% PAS-5.

An inhibition of p38 mitogen activated protein kinase delays the platelet storage lesion.

BACKGROUND AND OBJECTIVES: Platelets during storage undergo diverse alterations collectively known as the platelet storage lesion, including metabolic, morphological, functional and structural changes. Some changes correlate with activation of p38 mitogen activated protein kinase (p38 MAPK). Another MAPK, extracellular signal-related kinase (ERK), is involved in PLT activation. The aim of this study was to compare the properties of platelets stored in plasma in the presence or absence of p38 and ERK MAPK inhibitors. MATERIALS AND METHODS: A single Trima apheresis platelet unit (n = 12) was aliquoted into five CLX storage bags. Two aliquots were continuously agitated with or without MAPK inhibitors. Two aliquots were subjected to 48 hours of interruption of agitation with or without MAPK inhibitors. One aliquot contained the same amount of solvent vehicle used to deliver the inhibitor. Platelets were stored at 20-24°C for 7 days and sampled on Days 1, 4, and 7 for 18 in vitro parameters. RESULTS: Inhibition of p38 MAPK by VX-702 leads to better maintenance of all platelet in vitro storage parameters including platelet mitochondrial function. Accelerated by interruption of agitation, the platelet storage lesion of units stored with VX-702 was diminished to that of platelets stored with continuous agitation. Inhibition of ERK MAPK did not ameliorate decrements in any in vitro platelet properties. CONCLUSION: Signaling through p38 MAPK, but not ERK, is associated with platelet deterioration during storage.