Quantification of the contribution of individual coagulation factors to haemostasis using a microchip flow chamber system and reconstituted blood from deficient plasma.

BACKGROUND AND OBJECTIVES: Quantifying the contribution of individual coagulation factors to haemostasis may aid our understanding of the haemostatic function in patients with rare coagulation deficiencies (RCDs) and the exploration of suitable treatments. MATERIALS AND METHODS: Reconstituted blood prepared from specific coagulation factor-deficient plasma (factor [F]II; prothrombin, FV, FVII, FVIII, FIX, FX, FXI or FXII) and red blood cell/platelet products were used to simulate the whole blood of patients with RCD. We prepared in vitro treatment models for patients with prothrombin deficiency using coagulation factor agents and fresh frozen plasma. Haemostatic function was measured using a microchip flow chamber system at 600 s-1. RESULTS: The haemostatic function was low, especially in blood samples reconstituted with prothrombin- and FX-deficient plasma. In a plasma transfusion model of prothrombin deficiency, haemostatic function recovered after 10% replacement with normal plasma and reached a plateau at ≧60% replacement. A treatment model of prothrombin deficiency with prothrombin complex concentrates revealed dose-dependent therapeutic effects in the range of 0-50 IU/kg. CONCLUSION: Microchip flow chamber system-based quantification of haemostatic function using reconstituted blood could predict haemostasis and therapeutic effects of treatments in patients with prothrombin deficiency.

A novel haemodilution chip mounted on the total thrombus-formation analysis system, a flow chamber system, enables stable analysis of platelet products under low platelet concentration/high shear conditions.

BACKGROUND AND OBJECTIVES: The total thrombus-formation analysis system (T-TAS) can quantitatively analyse the contribution of platelets to haemostasis using reconstituted blood samples. However, it is unsuitable in cases with low platelet counts. We introduced a haemodilution (HD) chip with a shallow chamber depth, adapted to low platelet counts and high shear conditions (1500 s-1). MATERIALS AND METHODS: Blood samples were prepared by mixing red blood cell products, standard human plasma and platelet products; the final platelet count was 50 × 103/µL. Aggregation tests were performed by using the aggregation inducers collagen, adenosine diphosphate (ADP) and ristocetin. Samples with 2-, 4- and 9-day-old platelet products (N = 10) were evaluated. RESULTS: The HD chip enabled the stable analysis of the haemostatic function of all samples at a platelet count of 50 × 103/µL. Haemostatic function was correlated with ADP aggregation (time to 10 kPa [T10]: r = -0.53; area under the curve for 30 min: r = 0.40) and storage period (T10: r = 0.44). CONCLUSION: The HD chip-mounted T-TAS can stably analyse haemostatic function under low platelet counts and high shear conditions; this approach is expected to serve as a bridge to in vivo haemostatic tests with experimental animals.

X-irradiated umbilical cord blood cells retain their regenerative effect in experimental stroke.

Although regenerative therapy with stem cells is believed to be affected by their proliferation and differentiation potential, there is insufficient evidence regarding the molecular and cellular mechanisms underlying this regenerative effect. We recently found that gap junction-mediated cell-cell transfer of small metabolites occurred very rapidly after stem cell treatment in a mouse model of experimental stroke. This study aimed to investigate whether the tissue repair ability of umbilical cord blood cells is affected by X-irradiation at 15 Gy or more, which suppresses their proliferative ability. In this study, X-irradiated mononuclear (XR) cells were prepared from umbilical cord blood. Even though hematopoietic stem/progenitor cell activity was diminished in the XR cells, the regenerative activity was surprisingly conserved and promoted recovery from experimental stroke in mice. Thus, our study provides evidence regarding the possible therapeutic mechanism by which damaged cerebrovascular endothelial cells or perivascular astrocytes may be rescued by low-molecular-weight metabolites supplied by injected XR cells in 10 min as energy sources, resulting in improved blood flow and neurogenesis in the infarction area. Thus, XR cells may exert their tissue repair capabilities by triggering neo-neuro-angiogenesis, rather than via cell-autonomous effects.

A novel quantitative method to evaluate the contribution of platelet products to white thrombus formation in reconstituted blood under flow conditions.

BACKGROUND AND OBJECTIVES: Currently, the quality of platelet (PLT) products is evaluated using a series of in vitro tests, which only analyse PLTs as an inspection material. However, it would be ideal to assess the physiological functions of PLTs under conditions similar to the sequential blood haemostatic process. In this study, we attempted to establish an in vitro system where the thrombogenicity of PLT products was evaluated in the presence of red blood cells (RBCs) and plasma using a microchamber under constant shear stress (600/s). MATERIALS AND METHODS: Blood samples were reconstituted by mixing PLT products, standard human plasma (SHP) and standard RBCs. Each component was serially diluted keeping the other two components fixed. The samples were applied onto a flow chamber system (Total Thrombus-formation Analysis System [T-TAS]), and white thrombus formation (WTF) was assessed under large arterial shear conditions. RESULTS: We observed a good correlation between the PLT numbers in the test samples and WTF. The WTF of samples containing ≦10% SHP was significantly lower than those containing ≧40% SHP, and no difference was observed in WTF among samples containing 40%-100% SHP. WTF significantly declined in the absence of RBCs, whereas no change in WTF was observed in the presence of RBCs, over haematocrit range of 12.5%-50%. CONCLUSION: The WTF assessed on the T-TAS using reconstituted blood may serve as a new physiological blood thrombus test to quantitatively determine the quality of PLT products.

Gap Junction-Mediated Transport of Metabolites Between Stem Cells and Vascular Endothelial Cells.

We have previously demonstrated that small molecular transfer, such as glucose, between hematopoietic stem cells (HSCs) or mesenchymal stem cells (MSCs) and vascular endothelial cells via gap junctions constitutes an important mechanism of stem cell therapy. Cell metabolites are high-potential small-molecule candidates that can be transferred to small molecules between stem cells and vascular endothelial cells. Here, we investigated the differences in metabolite levels between stem cells (HSCs and MSCs), vascular endothelial cells, and the levels of circulating non-hematopoietic white blood cells (WBCs). The results showed remarkable differences in metabolite concentrations between cells. Significantly higher concentrations of adenosine triphosphate (ATP), guanosine triphosphate (GTP), total adenylate or guanylate levels, glycolytic intermediates, and amino acids were found in HSCs compared with vascular endothelial cells. In contrast, there was no significant difference in the metabolism of MSCs and vascular endothelial cells. From the results of this study, it became clear that HSCs and MSCs differ in their metabolites. That is, metabolites that transfer between stem cells and vascular endothelial cells differ between HSCs and MSCs. HSCs may donate various metabolites, several glycolytic and tricarboxylic acid cycle metabolites, and amino acids to damaged vascular endothelial cells as energy sources and activate the energy metabolism of vascular endothelial cells. In contrast, MSCs and vascular endothelial cells regulate each other under normal conditions. As the existing MSCs cannot ameliorate the dysregulation during insult, exogenous MSCs administered by cell therapy may help restore normal metabolic function in the vascular endothelial cells by taking up excess energy sources from the lumens of blood vessels. Results of this study suggested that the appropriate timing of cell therapy is different between HSCs and MSCs.

Automated preparation of washed platelet concentrates through spinning-membrane filtration.

BACKGROUND: Washed platelet concentrates (WPC), prepared with an automated system cell processor (ACP), have recently been approved to be manufactured and marketed in Japan. From the perspective of risk management, it is preferable to secure alternative technologies for ACP. Here, we conducted a study to evaluate the quality of WPC prepared using an automated membrane filtration-based system, Lovo. STUDY DESIGN AND METHODS: Replaced PCs prepared from apheresis PCs were equally divided into control and test units, and subsequently washed using ACP and Lovo respectively. Work and operational efficiencies were evaluated by in vitro analyses, including total handling time, platelet recovery, and plasma protein removal rate. Product quality, including a set of biochemical and physiological indicators of platelets and supernatants, were assessed before and 3 days after washing. RESULTS: In vitro platelet recovery rates and plasma protein removal rates were >85% and >95%, respectively, in both groups. The pH values on day 0 were significantly high (6.97 vs. 6.86) due to low pCO2 in the test group, while no significant differences in glucose consumption and lactate production were observed between the two groups. The levels of hypotonic shock responses, aggregation response, platelet shape, CD62P expression, and sCD62P concentration were similar in both groups during the 3-day storage period. CONCLUSION: Platelet washing with Lovo provides platelet quality equivalent to, or better than, conventional washing with ACP. Thus, the new automated system, Lovo, can be considered as an alternative to ACP for WPC preparation.

Production and nonclinical evaluation of an autologous iPSC-derived platelet product for the iPLAT1 clinical trial.

Donor-derived platelets are used to treat or prevent hemorrhage in patients with thrombocytopenia. However, ∼5% or more of these patients are complicated with alloimmune platelet transfusion refractoriness (allo-PTR) due to alloantibodies against HLA-I or human platelet antigens (HPA). In these cases, platelets from compatible donors are necessary, but it is difficult to find such donors for patients with rare HLA-I or HPA. To produce platelet products for patients with aplastic anemia with allo-PTR due to rare HPA-1 mismatch in Japan, we developed an ex vivo good manufacturing process (GMP)-based production system for an induced pluripotent stem cell-derived platelet product (iPSC-PLTs). Immortalized megakaryocyte progenitor cell lines (imMKCLs) were established from patient iPSCs, and a competent imMKCL clone was selected for the master cell bank (MCB) and confirmed for safety, including negativity of pathogens. From this MCB, iPSC-PLTs were produced using turbulent flow bioreactors and new drugs. In extensive nonclinical studies, iPSC-PLTs were confirmed for quality, safety, and efficacy, including hemostasis in a rabbit model. This report presents a complete system for the GMP-based production of iPSC-PLTs and the required nonclinical studies and thus supports the iPLAT1 study, the first-in-human clinical trial of iPSC-PLTs in a patient with allo-PTR and no compatible donor using the autologous product. It also serves as a comprehensive reference for the development of widely applicable allogeneic iPSC-PLTs and other cell products that use iPSC-derived progenitor cells as MCB.

Comparison of the programmed freezer method and deep freezer method in the manufacturing of frozen red blood cell products.

BACKGROUND AND OBJECTIVES: Frozen-thawed red blood cells (FTRCs) are useful blood components to patients with rare blood phenotypes. However, frozen red blood cells (FRCs) sometimes cause significant haemolysis after thawing due to the freeze/thaw process. In this study, we aimed to focus on the former process and reduce process-related haemolysis. MATERIALS AND METHODS: Five-day-old red blood cells (RBCs) (5D) or 9-week-old RBCs (9 W) were glycerolized, pooled and split into two aliquots. RBCs were frozen using either the programmed freezer (PF) method or the deep freezer (DF) method. After 4-8 weeks, the FRCs were thawed and washed. In vitro characteristics were compared between the PF and DF methods. Nine week were used as a starting material for FTRCs with the assumption that they can mimic disqualified FTRCs with respect to Hb recovery. RESULTS: The PF method resulted in a significantly higher Hb recovery rate than the DF method (5D: 85.9 ± 2.1 vs. 81.1% ± 3.5%, p < 0.001) (9 W: 56.8 ± 4.0 vs. 52.4% ± 3.5%, p < 0.001). Both 5D and 9W-derived FTRCs immediately after preparation prepared by the PF method were more resistible to haemolysis than those prepared by the DF method. On the other hand, there were no significant differences between PF and DF methods in Adenosine 5'-triphosphate (ATP) and 2,3-diphosphoglycerate (2,3-DPG). CONCLUSION: The PF method was more suitable for RBC freezing than the DF method in terms of Hb recovery in FTRCs. Although it was only 4%-5%, the improvement in the Hb recovery rate will contribute to a more stable supply.