Laura M. Currie

Recovery of in vitro functional activity of platelet concentrates stored at 4 ° C and treated with second-messenger effectors

BACKGROUND: The potential for bacterial contamination limits the storage of platelets at 22 degrees C to 5 days. Refrigerated storage at 4 degrees C would abrogate this problem but would also result in a rapid loss of in vitro viability and functional activity and in vivo viability. The inhibition of platelets during storage by a combination of specific, reversible, second‐messenger effectors has been investigated to allow prolonged storage at 4 degrees C with significant retention of in vitro viability and functional activity. STUDY DESIGN AND METHODS: The combination of effectors was added directly to platelet concentrates, and this step was followed by storage at 4 degrees C. Control units were incubated at 4 degrees C without the effectors and at 22 degrees C according to standard blood‐banking techniques. At 1, 5, and 9 days, the units were tested for recovery of cell number, recovery of in vitro functional activity and viability, and expression of platelet surface markers. RESULTS: Treated platelets stored at 4 degrees C for 9 days, while spherical in shape, displayed no loss of cell number and had a recovery of viability and functional activity, as compared with control platelets stored at 22 degrees C for 5 days, as follows: ADP and collagen aggregation responses of 250 and 100 percent, respectively; a 70‐percent recovery of hypotonic shock response; and a 60‐percent recovery of extent of shape change. The treated platelets also expressed an equivalent amount of the surface marker glycoprotein lb and a lower amount of the activation marker alpha‐granule membrane protein‐140 on the membrane surface. CONCLUSION: Second‐messenger effectors added to platelets significantly maintained in vitro functional activity with storage at 4 degrees C. In vitro analysis demonstrates the potential for extended 4 degrees C storage of platelets with numerical and functional recovery comparable to that achieved with current methods. Refrigerated storage of platelet concentrates has the potential to reduce the risk of bacterial contamination.

Inhibition of cytokine accumulation and bacterial growth during storage of platelet concentrates at 4 degrees C with retention of in vitro functional activity

BACKGROUND: The potential for bacterial contamination limits the storage of platelet concentrates (PCs) at 22° C to 5 days. In addition, storage of platelets under conventional protocols for longer times (> 3 days), in the absence of white cell filtration, has been correlated with incidents of cytokine‐associated febrile reaction in recipients. It has been demonstrated that the addition of a reagent mixture of second‐messenger effectors allows platelets stored at 4°C to maintain significant in vitro functional activity. Thus, the effects of 4°C storage on the growth of bacteria and the accumulation of cytokines by the white cell fraction of PCs were analyzed to demonstrate the benefits of this refrigerated storage system.

Cryopreservation of single-donor platelets with a reduced dimethyl sulfoxide concentration by the addition of second-messenger effectors: enhanced retention of in vitro functional activity

BACKGROUND: The potential for bacterial contamination limits the storage of platelets at 22 degrees C to 5 days. This creates an inventory problem, which could be overcome by the use of cryopreservation to allow long‐term storage of platelets. It has been demonstrated that the addition to platelets of a mixture of second‐ messenger effectors (platelet storage solution), allows these cells to retain significant in vitro functional activity following cold storage. Analysis is needed of the ability of this second messenger effector mixture both to protect platelets during cryopreservation and to reduce the need for a cryoprotectant. STUDY DESIGN AND METHODS: Fresh single‐ donor platelet units (n = 8) were divided into three samples and treated with 6‐percent dimethyl sulfoxide (DMSO), 2‐percent DMSO or the platelet storage solution and 2‐percent DMSO. The samples were placed directly into a −80 degrees C freezer and stored for 1 week, after which they were thawed and analyzed for in vitro functional activity. RESULTS: Platelets cryopreserved with the platelet storage solution and 2‐percent DMSO displayed statistically higher retention of functional activity and viability‐including cell number, percent of discoid cells, extent of shape change, and hypotonic shock response‐than did platelets stored by the method using 6‐percent DMSO. In addition, the treated platelets displayed statistically lower expression of p‐ selectin. The treated platelets showed no loss of cell number, > 88‐ percent retention of discoid morphology, and > 75‐percent retention of ristocetin‐induced aggregation as compared to values for these measures in fresh platelets. CONCLUSION: The use of this platelet storage solution in the cryopreservation of platelets yields a significant improvement in their postthaw in vitro recovery and allows for a reduction of the DMSO concentration from 6 to 2 percent, with superior maintenance of in vitro viability and function.

Quality assessment of platelet concentrates supplemented with second-messenger effectors

BACKGROUND: While reducing the potential for bacterial contamination, the storage of platelet concentrates (PCs) at refrigerated temperatures is not routine, because of the induction of the so‐called platelet storage lesion. As the modulation of second‐messenger levels might help to overcome this drawback, a quality assessment of PCs treated with a mixture of second‐messengers effectors known as ThromboSol was performed.

Enhanced retention of in vitro functional activity of platelets from recombinant human thrombopoietin-treated patients following long-term cryopreservation with a platelet-preserving solution (ThromboSol) and 2% DMSO

Chemotherapy‐induced thrombocytopenia represents a significant clinical problem in the management of patients with malignancy. Recombinant human thrombopoietin (rhTPO) is a potent stimulator of platelet production in vivo. The ability to cryopreserve rhTPO‐derived platelets would enable the use of autologous platelets during the period of thrombocytopenia. ThromboSolTM is a platelet‐stabilizing formulation consisting of second messenger effectors that inhibit specific activation pathways endogenous to platelets. To investigate the effect of ThromboSol cryopreservation, platelets from rhTPO‐treated patients (n = 23) and normal donors were treated with ThromboSol and 2% DMSO and cryopreserved for up to 6 months. The platelets were thawed at different intervals and tested for retention of platelet functional activity in vitro. Following a short‐term storage (1 week), the cryopreserved platelets from patients treated with rhTPO exhibited significantly higher retention of functional activities including discoid morphology (70% v 57%), extent of shape change (19% v 13%) stirring shape change (15% v 11%) and hypotonic shock response (56% v 25%), as compared to the cryopreserved platelets from controls. Furthermore, there was no further significant loss of functional activity following cryopreservation for up to 6 months. These findings suggest that cryopreservation of platelets from rhTPO‐treated donors may provide a useful novel strategy for autologous or allogeneic donation for subsequent transfusions to manage treatment‐related thrombocytopenia.

Prevention of monocyte adhesion and inflammatory cytokine production during blood platelet storage: An in vitro model with implications for transfusion practice

A novel platelet additive solution [ThromboSoltrade mark (TS)] was designed to allow extended refrigerated platelet storage. It has been shown to preserve platelet function and prevent cytokine accumulation in platelet concentrates stored for up to 9 days. It consists of amiloride, adenosine, sodium nitroprusside, dipyridamole, quinacrine, and ticlopidine. We hypothesized that the cytokine inhibition may be due to prevention of monocyte (MC) adhesion and activation on the surfaces of platelet storage bag plastic polymers. In an in vitro model, we incubated purified peripheral blood MCs on discs of polyolefin and polyvinylchloride from platelet storage bags, and on polystyrene, in the presence of TS for up to 7 days. We found that after incubation with TS, adherent MC numbers were decreased by >80-95% compared with controls on all surfaces examined. Levels of cytokines [interleukin (IL)-1beta, IL-1RA, IL-6, IL-8, and tumor necrosis factor-alpha] were low in wells with TS but rose progressively in the controls during incubation. Amiloride alone had similar effects on adhesion and cytokine release as the complete TS preparation. Removing amiloride from TS abrogated these effects. These findings suggest an important role for TS and amiloride in monocyte function, and have implications for the development of agents designed for prolonged platelet storage.