Feasibility evaluation of two novel systems for the automated preparation and extended storage of DMSO cryopreserved platelets.

BACKGROUND: Manufacturing methods for dimethyl sulfoxide (DMSO)-cryopreserved platelets (CPPs) are manual and labor intensive. Thawing and prepare-for-transfusion steps are in an open system that requires transfusion within 4 h. A fill-and-finish system (CUE) can automate the manufacturing process. A newly configured bag system allows freezing, thawing, and use of resuspension solutions while maintaining the functionally closed system, and extending the post-thaw shelf life beyond 4 h. Our objective is to evaluate the feasibility of the CUE system and the functionally closed bag system. STUDY DESIGN AND METHODS: DMSO was volumetrically added to double-dose apheresis platelets, concentrated, and delivered to a 50- or 500-mL ethylene-vinyl acetate (EVA) bag by the CUE (n = 12). The functionally closed bag system contained 25 mL platelet additive solution 3 (PAS-3) in a 50-mL EVA bag. Control CPP (n = 2) were manually prepared. PAS-3 and CPP were thawed together. CPP were stored up to 98 h (20-24°C) and tested using a standard assay panel. RESULTS: CUE prepared CPP met the design targets: volume, platelet content, and DMSO concentration. CUE CPP P-selectin was high. CD42b, phosphatidylserine (PS) expression, and live cell percentage were favorable compared to controls and favorably maintained over storage. The thrombin generation potency was slightly reduced compared to controls. The 50 mL EVA bag maintained pH for up to 30 h, and the 500 mL EVA bag beyond 76 h. DISCUSSION: The CUE system presents a technically feasible method to prepare CPP. A functionally closed bag system with resuspension solution was successful and can extend the post-thaw storage time of CPP.

No inflammatory effects after acute inhalation of barium sulfate particles in human volunteers.

BACKGROUND: Most threshold limit values are based on animal experiments. Often, the question remains whether these data reflect the situation in humans. As part of a series of investigations in our exposure lab, this study investigates whether the results on the inflammatory effects of particles that have been demonstrated in animal models can be confirmed in acute inhalation studies in humans. Such studies have not been conducted so far for barium sulfate particles (BaSO4), a substance with very low solubility and without known substance-specific toxicity. Previous inhalation studies with zinc oxide (ZnO), which has a substance-specific toxicity, have shown local and systemic inflammatory respones. The design of these human ZnO inhalation studies was adopted for BaSO4 to compare the effects of particles with known inflammatory activity and supposedly inert particles. For further comparison, in vitro investigations on inflammatory processes were carried out. METHODS: Sixteen healthy volunteers were exposed to filtered air and BaSO4 particles (4.0 mg/m3) for two hours including one hour of ergometric cycling at moderate workload. Effect parameters were clinical signs, body temperature, and inflammatory markers in blood and induced sputum. In addition, particle-induced in vitro-chemotaxis of BaSO4 was investigated with regard to mode of action and differences between in vivo and in vitro effects. RESULTS: No local or systemic clinical signs were observed after acute BaSO4 inhalation and, in contrast to our previous human exposure studies with ZnO, no elevated values of biomarkers of inflammation were measured after the challenge. The in vitro chemotaxis induced by BaSO4 particles was minimal and 15-fold lower compared to ZnO. CONCLUSION: The results of this study indicate that BaSO4 as a representative of granular biopersistent particles without specific toxicity does not induce inflammatory effects in humans after acute inhalation. Moreover, the in vitro data fit in with these in vivo results. Despite the careful and complex investigations, limitations must be admitted because the number of local effect parameters were limited and chronic toxicity could not be studied.

Separation of in-vitro-derived megakaryocytes and platelets using spinning-membrane filtration.

In-vitro-derived platelets (PLTs) could potentially overcome problems associated with donated PLTs, including contamination and alloimmunization. Although several groups have produced functional PLTs from stem cells in vitro, the challenge of developing this technology to yield transfusable PLT units has yet to be addressed. The asynchronous nature of in vitro PLT generation makes a single harvest point infeasible for collecting PLTs as soon as they are formed. The current standard of performing manual centrifugations to separate PLTs from nucleated cells at multiple points during culture is labor-intensive, imprecise, and difficult to standardize in accordance with current Good Manufacturing Practices (cGMP). In an effort to develop a more effective method, we adapted a commercially-available, spinning-membrane filtration device to separate in-vitro-derived PLTs from nucleated cells and recover immature megakaryocytes (MKs), the precursor cells to PLTs, for continued culture. Processing a mixture of in-vitro-derived MKs and PLTs on the adapted device yielded a pure PLT population and did not induce PLT pre-activation. MKs recovered from the separation process were unaffected with respect to viability and ploidy, and were able to generate PLTs after reseeding in culture. Being able to efficiently harvest in-vitro-derived PLTs brings this technology one step closer to clinical relevance.