[Evaluation of Storage Performance of Preserving Bags for Manually Separated Platelets].

OBJECTIVE: To evaluate the storage performance of the domestically made platelet storage bags (experimental group) and the United States Trima set platelet storage bags (control group). METHODS: The manually separated platelets were divided in two equal parts, which was added to control blood bags and experimental blood bags respectively, all samples were stored at a 22 °C ± 2 °C. The platelet count, mean volume, aggregation activity (ADP, THR), pH, glucose, lactate concentration, lactate dehydrogenase concentration, hypotonic shock reaction, CD62P and phosphatidic acid serine content were detected at day 0, 3, 5 and 7 of storage. RESULTS: There was no significant difference of platelet quality at day 5 after storage between the experimental group and the control group (T-test, P > 0.05). CONCLUSION: Two kinds of platelet storage bags have the similar storage performance.

Manganese superoxide dismutase gene therapy protects against irradiation- induced intestinal injury.

Radiation-induced intestinal injury is a common complication in radiotherapy for solid organ malignancies in abdomen or pelvis. However, currently there are no approved medical countermeasures for radiation-induced intestinal injury. Therefore, it is urgent to develop new treatments for radiation-induced intestinal injury. In the present study, we demonstrated that bone marrow derived mesenchymal stem cells (MSCs) and overexpression of human manganese superoxide dismutase (MnSOD) could ameliorate radiation-induced intestinal syndrome. NOD/SCID mice received abdominal irradiation at a selected dose of 5 Gy, and then infused intravenously with MnSOD-MSCs. Mice body weight, survival and diarrhea were monitored for 30-days. Colonization and differentiation of MnSOD-MSCs in the irradiated intestine were analyzed by histological and immunohistochemical methods. Consequently, our data demonstrated that intravenous administration of MnSOD-MSCs improved survival, decreased diarrhea occurrence and protected the small intestinal structural integrity of irradiated mice. Moreover, intravenously transplanted MnSOD-MSCs could colonize the irradiated intestine and repair injured sites. These findings suggested that MnSOD-MSCs may be an attractive and potential option for radiation-induced intestinal injury.

[Effects of loaded buffer with epigallocatechin gallate on physiological functions of platelets].

This study was aimed to explore the change of aggregation and activation of platelets loaded with epigallocatechin gallate (EGCG). The platelets were treated by loading buffer with different concentrations of EGCG (0, 2.5, 5, 7.5, 10, 12.5, 15, 20 and 30 mmol/L) and were divided into 2.5, 5, 10 mmol/L groups and control group. The physiological and biochemical functions of platelets were observed, including recovery rate, aggregation and activation of platelets. The platelet counts were determined by Counter Cell-DYN 1200. The aggregation activities were tested through turbidimetry, the platelet apoptosis was detected by flow cytometry. The results showed that the concentrations of EGCG loading in platelets of 2.5, 5 and 10 mmol/L groups were 0.4006 ± 0.12, 1.0527 ± 0.1503, 1.6902 ± 0.1112 mmol/L respectively. Along with the increasing of EGCG concentrations in loading-buffer, the EGCG absorbed by platelets increased too. When the concentration of EGCG in loading-buffer exceeded 15 mmol/L, the EGCG absorbed by platelets did not increase. The recovery rate in 2.5 mmol/L loading buffer group was 82.45 ± 0.360% which was lower than that in control group (90.33 ± 1.115%) (p < 0.05). As compared with control group, the recovery rate in 5 mmol/L loading buffer group (57.51 ± 2.468)% and 10 mmol/L loading buffer group (47.45 ± 2.030)% were even significantly lower (p < 0.01). When ADP was used as the inducer, the maximal aggregation rate (MAR) in control group was (63.6 ± 4.037)%, which was higher than that in other EGCG-loading groups (p < 0.01). And the aggregation activity of platelets negatively correlated with the concentration of EGCG in loading-buffer. When THR was used as the inducer, the MAR in control group was (89.3 ± 6.533)% and higher than that those in other groups (p < 0.05), especially in groups with loading-buffer higher than 10 mmol/L EGCG (70.1 ± 5.400%) (p < 0.01). In the experiment of cellular apoptosis, the early apoptosis easy appeared in platelets loaded with EGCG. It is concluded that the EGCG loading in platelets markedly influences the physiological and biochemical functions of platelets, the apoptosis easy occurs in platelets loaded with EGCG. The EGCG accelerates the course of platelet apoptosis.

[Healing effect of lyophilized platelets on rat chronic wound model].

Platelets carry over 20 growth factors, which all have been shown to improve wound healing, particularly recalcitrant wound healing. The aim of this study was to investigate the healing effect of lyophilized platelets on the chronic wounds through establishing diabetic rat chronic wound model. Healthy male SD rats were intraperitoneally injected with streptozotocin (STZ) solution at the dose of 65 mg/kg. The blood glucose and weights were observed every week. The re-epithelialization rates of normal control group (NDR), diabetic group (DR) and diabetic treatment group (TLP) was analysed. Two full thickness skin wounds were incised in the back of the rats. The re-epithelialization rates were observed at 1, 3, 5, 7 and 12 days. The results showed that after induced by streptozotocin for 72 hours, the blood glucose of the DR group was higher than 16.7 mmol/L. 1 week after induced by STZ, the weight of the DR group was significant lighter than that of the NDR group (p < 0.05). The re-epithelialization rate of DR group were lower than that of NDR. After 12 day treatment, the re-epithelialization rates of NDR and TLP groups were 88.1% and 81.8%, which were significantly higher than that of DR group (62.8%). It is concluded that diabetic rat model established by the intraperitoneal injection of streptozotocin can be used as a better diabetic chronic wound model. And the lyophilized platelets have healing effect on diabetic chronic wounds.

Addition of oligosaccharide decreases the freezing lesions on human red blood cell membrane in the presence of dextran and glucose.

Although incubation with glucose before freezing can increase the recovery of human red blood cells frozen with polymer, this method can also result in membrane lesions. This study will evaluate whether addition of oligosaccharide (trehalose, sucrose, maltose, or raffinose) can improve the quality of red blood cell membrane after freezing in the presence of glucose and dextran. Following incubation with glucose or the combinations of glucose and oligosaccharides for 3h in a 37°C water bath, red blood cells were frozen in liquid nitrogen for 24h using 40% dextran (W/V) as the extracellular protective solution. The postthaw quality was assessed by percent hemolysis, osmotic fragility, mean corpuscle volume (MCV), distribution of phosphatidylserine, the postthaw 4°C stability, and the integrity of membrane. The results indicated the loading efficiency of glucose or oligosaccharide was dependent on their concentrations. Moreover, addition of trehalose or sucrose could efficiently decrease osmotic fragility of red blood cells caused by incubation with glucose before freezing. The percentage of damaged cell following incubation with glucose was 38.04±21.68% and significantly more than that of the unfrozen cells (0.95±0.28%, P<0.01). However, with the increase of the concentrations of trehalose, the percentages of damaged cells were decreased steadily. When the concentration of trehalose was 400mM, the percentage of damaged cells was 1.97±0.73% and similar to that of the unfrozen cells (P>0.05). Moreover, similar to trehalose, raffinose can also efficiently prevent the osmotic injury caused by incubation with glucose. The microscopy results also indicated addition of trehalose could efficiently decrease the formation of ghosts caused by incubation with glucose. In addition, the gradient hemolysis study showed addition of oligosaccharide could significantly decrease the osmotic fragility of red blood cells caused by incubation with glucose. After freezing and thawing, when both glucose and trehalose, sucrose, or maltose were on the both sides of membrane, with increase of the concentrations of sugar, the percent hemolysis of frozen red blood cells was firstly decreased and then increased. When the total concentration of sugars was 400mM, the percent hemolysis was significantly less than that of cells frozen in the presence of dextran and in the absence of glucose and various oligosaccharides (P<0.01). However, when both glucose and trehalose were only on the outer side of membrane, with increase of the concentrations of sugars, the percent hemolysis was increased steadily. Furthermore, addition of oligosaccharides can efficiently decrease the osmotic fragility and exposure of phosphatidylserine of red blood cells frozen with glucose and dextran. In addition, trehalose or raffinose can also efficiently mitigate the malignant effect of glucose on the postthaw 4°C stability of red blood cells frozen in the presence of dextran. Finally, addition of trehalose can efficiently protect the integrity of red blood cell membrane following freezing with dextran and glucose. In conclusion, addition of oligosaccharide can efficiently reduce lesions of freezing on red blood cell membrane in the presence of glucose and dextran.

In vivo circulation of mouse red blood cells frozen in the presence of dextran and glucose.

Loading with monosaccharide can improve the quality of human red blood cells (hRBCs) frozen with polymer. But in vivo life span of hRBCs frozen with polymer and sugar is not determined. In this study, following incubation with glucose, mouse red blood cells (mRBCs) were frozen in liquid nitrogen for 24h using dextran as the extracellular protectant. After thawing, hemolysis, exposure of PS, and osmotic fragility of frozen mRBCs were determined in vitro. After transfusion of fluorescein isothiocyanate (FITC)-labeled mRBCs, the 24h recovery and half life span of frozen mRBCs were determined. The data indicated the postthaw hemolysis of mRBCs frozen with dextran and glucose were significantly less than that of cells frozen with dextran (17.23%+/-5.21% vs 25.96%+/-10.07%, P=0.034). But freezing can also result in exposure of phosphatidylserine and increase of osmotic fragility of mRBCs. After transfusion, the 24h recovery of mRBCs frozen in the absence or presence of glucose was similar to that of the control cells (P=0.748 and 0.971). However, the half life span of mRBCs frozen in the absence or presence of glucose was significantly less than that of the control cells (P=0.000). In addition, incubation with glucose can not increase the life span of frozen red blood cells (7.16+/-0.93 d vs 7.15+/-0.34 d, P=0.982). In conclusion, incubation with monosaccharide could significantly increase the recovery of mRBCs frozen with polymer. Although freezing can significantly shorten the half life span of frozen cells, it can not influence the 24h recovery of frozen mRBCs. In addition, incubation with monosaccharide before freezing can not increase the life span of frozen mRBCs. So according to the above data, to increase the life span of hRBCs frozen with polymer and monosaccharide, the osmotic fragility of the frozen RBCs must be decreased in the future.

[Research advance on application of platelet-rich plasma in wound repair -- review].

Platelets (Plts) have been shown to play a critical role in tissue repair mechanisms such as chemotaxis, cell proliferation, angiogenesis and extracellular matrix deposition. These effects are largely due to the contents of platelet granules, among which there are a number of important growth factors contributing to the wound repairing process. Platelet-rich plasma (PRP) has been traditionally used as a source of platelet growth factors (PGFs). After connecting activator, platelet gel (PG) is formed and numerous PGFs such as PDGF, TGF, VEGF, IGF, and EGF are released. It is important in different stages of the wound-healing cascade and greatly influence mitogenic and cellular differentiation activities. The aim of this review is to present knowledge about properties of PRP and possibilities of using PRP in the treatment of wound, as well as the success of the clinical studies performed so far, finally the future of PRP therapies are also described.

[Inhibitory effect of trehalose on phosphatidylserine exposure, osmotic fragility and membrane lipid peroxidation damage of erythrocytes induced by high concentration of glucose].

Though high concentration of glucose can benefit the survival of lyophilized human red blood cells, the high concentration of glucose can result in serious damage of red blood cells. This study was aimed to investigate the inhibitory effect of trehalose on damage of red blood cells induced by high concentration of glucose. After incubation with the high concentration of glucose buffer containing different concentrations of trehalose for three hours at 37 degrees C, the phosphatidylserine exposure and the osmotic fragility of cells were analyzed by flow cytometry and the lipid peroxidation of membrane was evaluated by TBA method. The results showed that the high concentration of glucose could lead to phosphatidylserine exposure, osmotic fragility increase, and lipid peroxidation damage which were dependent on the glucose concentrations and incubation temperature. However, trehalose could effectively prevent the phosphatidylserine exposure, osmotic fragility increase, and lipid peroxidation damage induced by high concentration glucose. With increase of the trehalose concentrations. As the trehalose concentration increases, the phosphatidylserine exposure, maloni-aldehyde concentration and cell debris rate decreased gradually. In conclusion, the high concentration of glucose can lead to phosphatidylserine exposure, osmotic fragility increase, and lipid peroxidation damage of red blood cells. However, trehalose can inhibit the damaging effects of high concentration of glucose on red blood cells, which may be useful for the application of sugars to lyophilization of red blood cells.

[Ultrastructural and functional assessment on platelets loaded with small molecule carbohydrates].

The aim of this study was to investigate the effect of loading some small molecule carbohydrates into human platelets on ultrastucture and function. The ultrastructure of platelets were observed by transmission electron microscope (TEM); the platelet counts and mean platelet volume (MPV) were measured by hemocytometer, the maximal platelet aggregation rate was measured optically in an aggregometer; the surface marker of platelet membranes CD62p and phosphatidyl serine were analyzed by flow cytometry. The results showed that no significant changes of the ultrastructure of platelets loaded with small molecule carbohydrates were seen. The aggregation responsiveness of platelets loaded with small molecule carbohydrates reached to 60% of the fresh control platelets. The values of platelet counts and MPV showed no significant differences. The expression level of CD62p and the binding rate with Annexin V before and after loading small molecule carbohydrates into platelets were no different. It is concluded that the platelets after loading with small molecule carbohydrates remained fine ultrastructure and function.

Inhibition of high glucose-induced erythrocyte phosphatidylserine exposure by leupeptin and disaccharides.

High glucose can lead to serious phosphatidylserine exposure of erythrocytes which may influence the protective effect of glucose on lyophilization of erythrocytes. In this study, caspase activation has not occurred during phosphatidylserine exposure of erythrocytes. However, leupeptin can efficiently inhibit phosphatidylserine exposure of erythrocytes induced by high glucose. With increase of the leupeptin concentrations, the percentages of cells with exposed phosphatidylserine were decreased steadily. In addition, trehalose and sucrose can significantly inhibit phosphatidylserine exposure and cell shrinkage of erythrocytes induced by high glucose through increasing tolerance to osmotic shock. When the disaccharide concentrations were more than 100 mM, the percentages of cells with exposed phosphatidylserine were similar to those of control cells. Moreover, addition of disaccharides in the glucose buffer can result in high osmotic pressure which may facilitate uptake of glucose and disaccharides into erythrocytes and higher cellular glucose and disaccharide concentrations can provide more protection for lyophilized erythrocytes. Although disaccharides can increase the osmotolerance and decrease the phosphatidylserine exposure of erythrocytes exposed to high glucose, whether disaccharides can prevent phosphatidylserine exposure of lyophilized erythrocytes still needs further researches.

[Research advance in platelet function assays].

Platelets play a key role in the pathogenesis of atherothrombosis, and also perform the physiological hemostasis. The platelet function assays have values in the investigating patients with suspected platelet disorders and in studying the effects of anti-platelet drugs. There are increasing assays for investigating platelet function, including assessment of platelet adhesion, activation and aggregation, etc. However, all of these assays have certain limited sensitivity. It is necessary to develop a simple, sensitive assay that measures the activated platelet. This article reviewed advances of researches on platelet function assays, including assay for general function of platelet, assay of platelet adhesion function, platelet activation assay, platelet aggregation assay, platelet coagulation assay and application of flow cytometry in assessment of platelet functions, etc. and looked forward to research prospect in this field.

[Comparison of modification of surface xenoantigens on bovine and porcine erythrocytes].

This study was aimed to explore impact of removal of cell membrane G alalpha1-3Gal beta1-4Glc NAc epitopes (called alpha-Gal) and chemical modification of other xenoantigen on bovine red blood cell (bRBC) and porcine red blood cell (pRBC) antigenicity and to compare their modified erythrocytes, in order to provide basis for development of human blood substitute with rich source, high safety and efficacy. bRBC and pRBC were subjected to both enzymatic removal of membrane alpha-Gal with recombinant coffee bean alpha-galactosidase (rC alpha-GalE) and covalent attachment of benzotriazole carbonate-linked methoxypolyethylene glycol (mPEG-BTC, MW = 20 kD). The effects of treatment were measured by hemagglutination, flow cytometric assay of IgG binding and clinical cross-match testing to human sera. The results showed that although alpha-galactosidase treatment reduced hemagglutination titers to levels similar to negative control, the combination of the treatments was most effective. Clinically used cross-match tests between bRBC, pRBC and human sera demonstrated increased compatibility. Bovine RBC were more robust than pRBC, and had less xenoantigens, and had longer half life than pRBC in vivo. These characteristics suggested that bRBCs were more suitable to investigation as an alternatives to hRBC in clinical transfusion than pRBC. These data suggested that strategies to remove or mask xenoantigens on bRBC reduce antigenicity sufficiently to allow in vitro cross-match compatibility to human sera, and therefore bRBC following modification may be considered as human blood substitute.

[A pilot study on establishment of human/pig hematopoietic chimera model in fetal and neonatal pigs].

This study was aimed to explore the feasibility of transplanting human cord blood stem cells (HSC) into pre-immune fetal and neonatal pigs, and to investigate the self-renewal of HSC in the recipient pigs. The fetus and neonate were manipulated in sterile separated room and human donor cells were injected into fetus via fetus muscle or umbilical vein (dissectted womb) or into neonate via umbilical vein before cutting it. Human CD45(+) cells s were detected by labeling with human anti-CD45 antibody and analyzed by fluorescence activated cell sorting (FACS). The results showed that tested pigs developed as well as control and a definite proportion of human cells existed in peripheral blood of chimeric pig on day 60 after transplantation. In conclusion, the fetus and neonate pigs can tolerate a definite proportion of human antigens, and to establish the human/pig model of hematopoietic chimerism is possible.

[Preliminary study on conversion of RhD positive red blood cells to RhD negative by modification with methoxy polyethylene glycol].

Rh is a very important blood group like ABO blood system in transfusion medicine. It causes severe transfusion reaction and hemolytic disease of the newborn (HDN) if RhD blood group does not match between the donor and the recipient. The population of RhD negative is only about 0.2% - 0.5% in Chinese. Conversion of RhD positive RBCs to RhD negative is very important in clinical transfusion. This study was to try to modify RhD antigen located on the surface of A, B, O and AB red blood cells in order to convert RhD positive to RhD negative by the modification of four kinds of methoxypolyethylene glycol (mPEG) derivatives and to observe the effect of mPEG modification on cell morphology, structure and function. The result demonstrated that modification efficiency of mPEG-BTC (mPEG-benzotriazole carbonate) was better than other three kinds of mPEG derivatives. It could camouflage RhD antigen efficiently when the concentration reached to 1 mmol/L. The result also showed that there were no harmful effects of mPEG modification on cell morphology, osmotic fragility, hemolysis, AchE, cholesterol, ATP, 2,3-DPG and deformability. It is suggested that success in converting RhD positive RBCs to RhD negative was preliminarily achieved.

[Recent advance on blood group antigen modification of porcine erythrocytes].

Advances in the field of xenotransplantation raise the intriguing possibility of using porcine red blood cells (pRBCs) as an alternative source for blood transfusion. Serologically, pRBCs share a number of characteristics with human red blood cells (RBCs), so pRBCs are considered the most likely donor for xenotransfusion. However, xenoantigens on porcine erythrocytes play major roles in antibody-mediated RBC destruction. Although the alphaGal epitope (Galalpha1, 3Galbeta1, 4GalNAc-R) is the major xenoantigen on porcine erythrocytes and is responsible for the binding of the majority of human natural antibodies, other non-alphaGal xenoantigens have been identified. The importance of these non-alphaGal xenoantigens in binding human natural antibodies and subsequently triggering immunological responses cannot be underestimated.