Development of an efficient, ready to use, blood platelet-release device based on two new flow regime parameters: The periodic hydrodynamic loading and the shear stress accumulation.

In vitro production of blood platelets for transfusion purposes is gaining interest. While platelet production is now possible on a laboratory scale, the challenge is to move towards industrial production. Attaining this goal calls for the development of platelet release devices capable of producing large quantities of platelets. To this end, we have developed a continuous-flow platelet release device composed of five spherical chambers each containing two calibrated cones placed in a staggered configuration. Following perfusion of proplatelet-bearing cultured megakaryocytes, the device achieves a high yield of about 100 bona-fide platelets/megakaryocyte, at a flow rate of ∼80 mL/min. Performances and operating conditions comply with the requirements of large-scale platelet production. Moreover, this device enabled an in-depth analysis of the flow regimes through Computational Fluid Dynamics (CFD). This revealed two new universal parameters to be taken into account for an optimal platelet release: i.e. a periodic hydrodynamic load and a sufficient accumulation of shear stress. An efficient 16 Pa.s shear stress accumulation is obtained in our system at a flow rate of 80 mL/min.

Leukodepletion Filters-Derived CD34+ Cells As a Cell Source to Study Megakaryocyte Differentiation and Platelet Formation.

The in vitro expansion and differentiation of human hematopoietic progenitors into megakaryocytes capable of elongating proplatelets and releasing platelets allows an in-depth study of the mechanisms underlying platelet biogenesis. Available culture protocols are mostly based on hematopoietic progenitors derived from bone marrow or cord blood raising a number of ethical, technical, and economic concerns. If there are already available protocols for obtaining CD34 cells from peripheral blood, this manuscript proposes a straightforward and optimized protocol for obtaining CD34+ cells from leukodepletion filters readily available in blood centers. These cells are isolated from leukodepletion filters used in the preparation of blood transfusion products, corresponding to eight blood donations. These filters are meant to be discarded. A detailed procedure to collect hematopoietic progenitors identified as CD34+ cells from these filters is described. The method to obtain mature megakaryocytes extending proplatelets while discussing their phenotypic evolution is also detailed. Finally, the protocol present a calibrated pipetting method, to efficiently release platelets that are morphologically and functionally similar to native ones. This protocol can serve as a basis for evaluating pharmacological compounds acting at various steps of the process to dissect the underlying mechanisms and approach the in vivo platelet yields.

Human platelets labeled at two discrete biotin densities are functional in vitro and are detected in vivo in the murine circulation: A promising approach to monitor platelet survival in vivo in clinical research.

BACKGROUND: The production of platelet concentrates (PCs) is evolving, and their survival capacity needs in vivo evaluation. This requires that the transfused platelets (PLTs) be distinguished from those of the recipient. Labeling at various biotin (Bio) densities allows one to concurrently trace multiple PLT populations, as reported for red blood cells. STUDY DESIGN AND METHODS: A method is described to label human PLTs at two densities of Bio for future clinical trials. Injectable-grade PLTs were prepared in a sterile environment, using injectable-grade buffers and good manufacturing practices (GMP)-grade Sulfo-NHS-Biotin. Sulfo-NHS-Biotin concentrations were chosen to maintain PLT integrity and avoid potential alloimmunization while enabling the detection of circulating BioPLTs. The impact of biotinylation on human PLT recirculation was evaluated in vivo in a severe immunodeficient mouse model using ex vivo flow cytometry. RESULTS: BioPLTs labeled with 1.2 or 10 µg/ml Sulfo-NHS-Biotin displayed normal ultrastructure and retained aggregation and secretion capacity and normal expression of the main surface glycoproteins. The procedure avoided detrimental PLT activation or apoptosis signals. Transfused human BioPLT populations could be distinguished from one another and from unlabeled circulating mouse PLTs, and their survival was comparable to that of unlabeled human PLTs in the mouse model. CONCLUSIONS: Provided low Sulfo-NHS-Biotin concentrations (<10 µg/ml) are used, injectable-grade BioPLTs comply with safety regulations, conserve PLT integrity, and permit accurate in vivo detection. This alternative to radioisotopes, which allows one to follow different PLT populations in the same recipient, should be valuable when assessing new PC preparations and monitoring PLT survival in clinical research.

Removal of citrate from PAS-III additive solution improves functional and biochemical characteristics of buffy-coat platelet concentrates stored for 7 days, with or without INTERCEPT pathogen reduction.

BACKGROUND: Deterioration in quality of platelet concentrates (PCs) during storage results from the appearance of storage lesions affecting the hemostatic functions and posttransfusion survival of platelets. These lesions depend on the preparation and pathogen inactivation methods used, duration of storage, and platelet additive solutions (PASs) present in storage bags. METHODS: We investigated the effects of citrate contained in third-generation PAS (PAS-III) on storage lesions in buffy-coat PCs with or without photochemical (amotosalen-ultraviolet A) treatment over 7 days. RESULTS: Platelet counts were conserved in all groups during storage, as was platelet swirling without appearance of macroscopic aggregates. Glycoprotein (GP) IIbIIIa and GPVI expression remained stable, whereas GPIbα declined similarly in all groups during storage. Removal of citrate from PAS-III, resulting in global reduction of citrate from 11 to 5 mM, led to a significant decrease in glucose consumption, which largely countered a modest deleterious effect of photochemical treatment. Citrate reduction also resulted in decreased lactate generation and better maintenance of pH during storage, while photochemical treatment had no impact on these parameters. Moreover, citrate-free storage significantly reduced exposure of P-selectin and the apoptosis signal phosphatidylserine, thereby abolishing the activating effect of photochemical treatment on both parameters. Citrate reduction benefited platelet aggregation to various agonists up to Day 7, whereas PCT had no impact on these responses. CONCLUSION: Removal of citrate from PAS-III has a beneficial impact on platelet metabolism, spontaneous activation, and apoptosis, and improves platelet aggregation, irrespective of photochemical treatment, which should allow transfusion of platelets with better and longer-lasting functional properties.