Background Platelets play an important role in acute bleeding.Whether 4℃cold stored platelets (CSPs) can be an effective substitute for platelets at room temperature (RTPs) is rarely reported, we carried out the clinical application observation trial of CSPs in China for the first time. Method A prospective, double-blind, randomized clinical trial was conducted on surgical patients with related bleedingto assess the hemostatic potential of CSPs compared with RTPs. The primary outcomes measured were drainage volume, platelet counts, and Thrombelastography-maximum amplitude. Secondary outcomes included hospital stays, length of stay in the intensive care unit, medical expenses. Results A total of 62 patients were completed the final clinical observation. There were 31 cases ineach of the CSPs group and RTPs group. With in 1–12 hours, 13–24 hours, 25–48 hours, and 49–72 hours after platelet transfusion, drainage volume: 8.5ml/h vs 20.83ml/h ,0.52ml/h vs 5.0ml/h, 3.5ml/h vs 5.0ml/h, 0.63ml/h vs 4.1ml/h. platelet counts:58×109/L vs 79×109/L, 54×109/Lvs77×109/L,63×109/L vs 75×109/L, 66×109/Lvs79×109/L. TEG-MA: 50.1mm vs 52.0mm ,50.1mm vs 54.8mm, 53.0mm vs 56.6, 56.0mm vs 53.2mm.There was no overall difference between the two groups by Generalized estimation equation at different times (Pdrainage=0.933,PPLT counts=0.473,PTEG−MA=0.246). The secondary outcomes (hospital stay, ICU stay time, medical cost, discharge outcome) were no difference between the CSPs group and RTPs group (P > 0.05). There were no significant differences in adverse platelet transfusion events between the groups (P > 0.05). Conclusion CSPs and RTP have equivalent efficacy and safety in the treatment of surgery-related bleeding. This trial provides reliable evidence to support the clinical application of CSPs. Trial registration Chinese Clinical Trial Registry: ChiCTR2000036353;Principal investigator's name: Jiangcun Yang. Date of registration: 22/08/2020.

BACKGROUND:Assessing cytotoxicity is fundamental to studying natural killer (NK) cell function. Various radioactive and non-radioactive cytotoxicity assays measuring target cell death have been developed. Among these methods, the most commonly used 51 Chromium-release assay (CRA) and flow cytometry-based cytotoxicity assays (FCCs) are the major representatives. Nonetheless, several drawbacks, including dye leakage and the potential effects of prior labeling on cells, curb the broad applicability of the FCCs. METHODS:Here, we report a rapid FCC for quantifying target cell death after co-incubation with NK cells. In this assay, after 4 hours of NK cell-target cell co-incubation, fluorochrome-conjugated CD2 antibody was used to identify NK cells, and SYTOX Green and Annexin V-FITC were further used to detect target cell death in CD2-negative population. In parallel, both CRA and FCC assay using CFSE/ 7-AAD were performed to validate the reproducibility and replicability. RESULTS:We observed that CD2 is exclusively positive on NK cells other than the most common hematological target tumor cells, such as K562, HL60, MOLM13, Raji, NCI-H929, rpmi8226, MM.1S, and KMS11. Assessment of target cell death using the CD2-based FCC shows a significantly higher percent specific lysis of the target cells compared to the standard CRA and the FCC assay using CFSE and 7-AAD. CONCLUSIONS:We demonstrated that this CD2-based FCC is a fast, simple, and reliable method for evaluating NK cell cytotoxicity.

This study compared the corrective effects of storage of platelets at 4 degrees C and at 22 degrees C in an in vitro model of massive blood loss and thrombocytopenia to provide an experimental basis for the storage of platelets for clinical applications. In vitro model of massive blood loss and thrombocytopenia were constructed by the in vitro hemodilution method and cell washing method. Using storage of platelets at 4 degrees C (1, 3, 5, 7, 10, 14 days) and at 22 degrees C (1, 3, 5 days) to correct the coagulation condition of the different models, by thromboelastography and by routine blood indices. Platelets stored at 4 degrees C (1, 3, 5,7, 10, 14 days) and at 22 degrees C (1, 3, 5 days) to correct the in vitro model of massive blood loss. Platelet count results improved from 17 to 27 x 10(9)/L to greater than 120 x 10(9)/L for 4 degrees C storage, and 20 to 27 x 10(9)/L to greater than 120 x 10(9)/L for 22 degrees C storage. Thromboelastography maximum amplitude (TEG-MA) results improved from 8.8 to 15.4mm to greater than 43mm for 4 degrees C storage, and 12.2 to 14.4mm to greater than 44.8mm for 22 degrees C storage. Thromboelastography reaction time values decreased from 9.9-24.9 minutes to 3.8-5.5 minutes for 4 degrees C storage, and 9.9-22.7 minutes to 4.3-4.5 minutes for 22 degrees C storage. Platelets stored at 4 degrees C (1, 3, 5,7, 10, 14 days) and at 22 degrees C (1, 3, 5 days) to correct the in vitro model of thrombocytopenia. Platelet count results improved from 12 to 34 x 10(9)/L to greater than 99 x 10(9)/L for 4 degrees C storage, and 12 to 34 x 10(9)/L to greater than 120 x 10(9)/L for 22 degrees C storage. TEG-MA results improved from 21.4 to 32.1mm to greater than 49.1mm for 4 degrees C storage, and 21.4 to 31.6mm to greater than 50.5mm for 22 degrees C storage. Platelets stored at 4 degrees C and 22 degrees C have the same correcting effect for 1, 3, and 5 days. Platelets stored at 4 degrees C for 7 to 14 days have similarly hemostatic effect on the in vitro model of massive blood loss and thrombocytopenia.

Objective To compare the metabolic changes in platelets stored at 4℃ and those with agitation at 22℃ in order to provide an experimental basis for the preservation technology of platelets.Methods Blood samples were collected for platelets stored at 4℃ for 1,3,5,7,10,14,and 21 days as well as for those stored with agitation at 22℃ for 1,3,and 5 days.The levels of gas indicators,biochemical indicators,and hypotonic shock response (HSR) were measured for platelets stored under different conditions.Results There were no significant changes in the pH,Na+,Ca,and Cl-levels of platelets stored at 4℃ for 7 days (P＞0.05).There were significant changes in the pH,Na+,Ca+,Cl-,pCO2,pO2,lactate (Lac),HCO3-,ctCO2,and Gap(K+) levels of platelets stored with agitation at 22℃ for 5 days (P＜0.005).On the 5th day,platelets stored at 22℃ showed significantly lower pH values than platelets stored at 4℃,while the lactate dehydrogenase (LDH) and Lac levels were significantly higher than for platelets stored at 4℃ (P＜0.005).The LDH,pH,K+,pCO2,pO2,Lac,HCO3-,ctCO2,and Gap(K+) levels between the two groups showed significant differences (P＜0.05).Platelets stored at 4℃ for 5 days showed no significant changes in the recovery from HSR (P＞0.05),while those stored at 22℃ showed a reduced rate of HSR recovery of 53.77％ between the 5th and 1st day.Conclusion The comparison of platelet metabolism between platelets stored at 4℃ and those agitated at 22℃ reveals that platelets stored under cold conditions show low metabolism,low lactate production,low reduction in pH,and low glucose consumption.Platelets stored at 4℃ for 10-14 days still show some HSR recovery.In terms of metabolic data,platelets stored at 4℃ are recommended to be stored for 10-14 days.

Objective To evaluate the corrective effects of platelets stored at 4℃ with the help of a in vitro hemorrhage model Platelets agitated at 22℃ were used as comparison.we hope this study would provide valuable experimental bata for the preservation of platelets in clinical applications.Methods Sample platelets with a total volume of 5×200 mL were divided into 2× 100 mnL each 200 mL for storage at 4℃ and 22℃,respectively.An in vitro model of hemorrhage was established with the in vitro hemodilution method where blood was diluted with normal saline (NS) with a ratio of 1∶9.The in vitro corrective effects of the platelets stored at 4℃ and 22℃ were evalutaed by Thromboelastography (TEG) test and routine blood marker level tests,where these two groups of platelets were prepared in a mix of suspended erythrocytes and fresh frozen plasma with a ratio of 1 ∶ 1 ∶ 1.Results Platelets stored at 4℃ and those agitated at 22℃ for 1,3,and 5 days were determined to be corrected in the in vitro hemorrhagie model,where platelet counts (× 109/L) were corrected from 20-27 to 127-161 and 128-160,respectively (P＞0.05).The TEG-MA (mm) were corrected from 12.7-14.4 to 45-51 and 47-50 while TEG-R values were corrected from 27.7-9.9 to 4.4-4.3 and 4.5-4.7,respectively (P＞0.05).For platelets stored at 4℃ for 7-14 days,platelet counts (× 109/L) were corrected from 18-27 to 162-161 and TEG-MA(mm)were corrected from 8.8-14.5 to 46-43 with TEG-R(min) corrected from 24-13 to 5.5-5.2(P＜0..05).Conclusion Correction has been detected using the hemorrhage model.The platelet counts,TEG-R,and TEG-MA values have been corrected,thus supporting the claim that platelets stored at 4℃ for 10-14 days presents fine coagulation quality.

Objective To study platelet count and morphological changes of platelets stored under different conditions (at 4℃,and agitated at 22℃),in order to provide experimental data for the preservation technology of platelets.Methods Blood samples were collected for manual preparation of platelets,which were stored at 4℃ for 1,3,5,7,10,14,and 21 days (experimental group) and at 22℃ for 1,3,5,7,and 10 days.The platelet count,Wright staining,and scanning electron microscopy of the platelets from these two groups were compared.Results Changes in the platelet count for platelets stored at 4℃ for 21 days and those agitated at 22℃ for 5 days were not significant (P＞0.05).Differences in the mean platelet volume (MPV) and platelet distribution width (PDW) values of platelets stored with agitation at 22℃ within 5 days showed an increasingly statistically significant trend (P＜0.05).Additionally,there were no significant changes in the MPV and PDW values for platelets stored at 4℃for 14 days (P＞0.05).Wright staining revealed that the size and morphology of platelets preserved at 4℃ for 21 days were not significantly changed.The platelet cytoplasm appeared uniformly dense and darker in color;shape was irregular with most cells appearing to be nearly round,size varied,and minimal platelet aggregation occurred.For platelets stored with agitation at 22℃,the platelet cytoplasm appeared sparse and lighter in color and platelet number was lower.Scanning electron microscopy of platelets stored at 4℃ for 10-14 days revealed significant platelet activation,aggregation,uneven surface,and significant long pseudopodia formation.For platelets stored with agitation at 22℃for 7 and 10 days,the platelet number and aggregation were lower.Some of these platelets also appeared to have an empty halo,nearly round shape,no obvious formation of pseudopodia,and no obvious platelet activation.Conclusion In comparison,the platelet count,cell morphology,platelet membrane,and cytoplasmic structure of cells stored at 4℃for 10-14 days were better than those stored with agitation at 22℃for 5 days.

Objective To comparison and analysis the changes in functional of platelets stored.at 4℃ and those agitated at 22℃ were compared to provide an experimental basis for the cryopreservation technology of platelets.Methods Blood samples were collected for platelets stored at 4℃ for 1,3,5,7,10,14,and 21 days,as well as for those stored with agitation at 22℃ for 1,3,and 5 days.Thromboelastography (TEG),light transmittance aggregometry,and flow cytometry were used to test platelets that had been stored under the different conditions.Results The thromboelastograph of platelets stored at 4℃ showed that their MA values were still within the normal range after 21 days of storage.The five TEG indicators of platelets stored with agitation at 22℃ for 5 days showed low coagulation (P＜0.05).The maximum platelet aggregation rate of platelets stored at 4℃ for 1 day was higher than 50％ and was higher than that of platelets stored with agitation at 22℃ (P＜ 0.05).After 5 days of storage,the COLL-and ACA-induced maximum aggregation rate of platelets stored at 4℃ was maintained at above 80％,while the maximum aggregation rate by the four inducers for platelets agitated at 22℃ was less than 5％.For platelets stored at 4℃ for 10-14 days,although the four aggregation rates were reduced,they were significantly higher those of platelets stored with agitation at 22℃ for 5 days (P＜0.05).Analysis of activated procaspase-activating compound 1 (activated Ⅱb/Ⅲa) and CD62P (P-selectin) on the platelet surface revealed that a large number of platelets stored at 4℃ after 10-14 days remained in the mid and late activation states.Conclusion Platelets stored at 4℃ showed better aggregation,coagulation,and higher activity than platelets stored with agitation at 22℃.Platelets can be stored in vitro for 10-14 days.

Objective To compare the corrective effects on platelets in storage at 4℃ and agitation at 22℃ on the in vitro thrombocytopenic mode in order to provide an experimental basis for the preservation of platelets for clinical application.Methods Thromboelastography and platelet count were used to evaluate the in vitro corrective effects on thrombocytopenic platelets stored at 4℃ and those agitated at 22℃ for various durations.Results Platelets stored at 4℃ and those agitated at 22℃ for 1,3,and 5 days were corrected using thrombocytopenic platelets prepared using the same blood samples.The platelet counts were corrected from 10-30× 109/L to greater than 100× 109/L.Platelets that had been stored at 4℃ for 7-14 days achieved similar correction values.Platelets that had been stored at 4℃ for 10-14 days and those agitated at 22℃ for 5 days were corrected using thrombocytopenic platelets.Their corrected thromboelastography (TEG)-MA values were within the normal range,and thus the corrective effects were achieved.Conclusion Platelets stored at 4℃ for 10-14 days and those agitated at 22℃ for 5 days were corrected using the in vitro thrombocytopenia mode.Based on the platelet counts,the TEG-MA values showed that corrective effects were achieved,thus supporting the claim that platelets stored at 4℃ for 10-14 days could achieve good coagulation effects.

目的 研究观察悬浮红细胞内血小板在冷藏条件下功能变化,为冷藏保存血小板研究提供依据.方法 随机抽取不同保存时间(3-33 d)的悬浮红细胞50 U,检测观察其TEG指标及血小板CD62P活化功能,同时观察11U悬浮红细胞在保存3、7、14、21和28 d时血液常规指标、凝血指标、TEG指标的变化.结果 悬浮红细胞内含有大量有活性的血小板(PAC1+ 62P+,PAC1-62P+),甚至保存28d时,悬浮红细胞TEG-MA指标仍显示正常范围,血小板功能良好.结论 悬浮红细胞内血小板在冷藏保存下具有较高的活性,为研究冷藏血小板提供了信息.

In order to provide Chinese clinicians with guidelines for the management of massive blood loss, we investigated the correlation between the frequency of blood tests and the mortality rate in patients undergoing massive blood transfusion (MBT). The aim of this study is to provide Chinese clinicians with guidelines for the management of massive blood loss. We retrospectively reviewed the medical records of patients who underwent massive blood transfusion (MBT) from 20 tertiary hospitals in 5 regions of China. The frequency of blood tests performed within 24 or 72 hours was compared between patients infused with < 10 and ≥ 10 U of red blood cells (RBC). The correlation between the frequency of blood tests and the mortality rate was determined. A high frequency of blood tests was associated with a low mortality rate in MBT cases. The frequency of all blood tests performed within 24 hours was negatively correlated with the mortality rate in patients infused with ≥ 10 U of RBC, while the frequency of blood coagulation tests performed within 72 hours was negatively correlated with the mortality rate in both patients infused with ≥ 10 and < 10 U of RBC. In conclusions: Measuring the blood indices frequently within the first 24 hours of MBT links to lower mortality rate. Coagulation indices in MBT patients should be closely monitored in the long term to help improve survival.

Layer by layer (LbL) self-assemble technique attracts much attention in the field of drug delivery because of its simplicity in procedure and fine tuning of the microstructure. Considering the acting force between the assemble material and the drug molecule, the authors chose a beta-cyclodextrin derivative as assemble material. Therefore, a polycarboxymethyl-beta-cyclodextrin (polyCM-beta-CD)/polyetherimide (PEI) multilayer for drug delivery was designed and constructed in this work. Orfloxacin was loaded into the multilayer during the self-assemble procedure by incorporating drugs in the PEI assemble solution as well as in polyCM-beta-CD assemble solution, which was confirmed by the largest drug absorbance of UV spectrum. Twenty assembled layers were chosen in view of the relative stable properties of the hydrogel surface. Since the pH value was an important factor to the properties of the polyelectrolyte multilayer, the effects of pH value on the drug release behaviour were further investigated in this work. It was found that the drug release behaviour was influenced by the drug loading method as well as by the pH value of the released medium.

PURPOSE:This study aimed to determine the optimal composition and timing for the administration of blood supplements during in vivo blood transfusion with red blood cells suspension (pRBC), fresh frozen plasma (FFP), and apheresis platelet (PLT) administered for the correction of anemia and coagulation dysfunction caused by in vitro hemodilution.MATERIALS AND METHODS:We collected blood samples from 24 healthy volunteers and prepared various dilutions of whole blood with normal saline: 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, and 1:9. The diluted blood samples were then supplemented with blood components at various proportions and then analyzed to determine the values of the routine blood indices, coagulation indices, and thromboelastogram measures.RESULTS:At hemodilutions of 40%, 50%, and 60%, the hemoglobin, coagulation indices, and platelet number and function reached critical levels, necessitating supplementation with pRBC, FFP, and PLT, respectively. When hemodilution was 90%, the supplementation required was approximately 1:1.3:0.9 of pRBC/FFP/PLT.CONCLUSION:The use of pRBC, FFP, and PLT in appropriate proportions can correct the blood coagulation dysfunction and anemia caused by in vitro hemodilution, and these proportions can be used as guidelines for in vivo massive transfusion.

OBJECTIVE:To provide a basis for the cold-storage of human platelets as a way to assess changes in platelet function.METHODS:Red blood cell suspensions (11 U and 50 U) were randomly selected at different storage times (3-28 days) and evidence of platelet activation (CD62P) and thromboelastography (TEG) reaction times were investigated.RESULTS:After 21 days of storage at 4°C, a large number of activated platelets (PAC1+62P+, PAC1-62P+) within the red blood cell suspension (RBCs) retained their function and had TEG-maximum amplitude (TEG-MA) indices in the normal range.CONCLUSION:We report that platelets in RBC suspensions retain high activity when stored at 4°C for 21 days. The results provide important information for studies that involve storing platelets under cold conditions.

Clinical observations suggest that red blood cells (RBCs) participate directly in hemostasis. We designed an in-vitro system aimed at evaluating the hemostatic function of RBCs. Blood samples were collected from 20 healthy volunteers and packed RBCs (PRBCs) were supplied by the Shaanxi Province Blood Center. We investigated the effect of RBCs and hemoglobin concentration on the hemostatic function in vitro by thromboelastography. The activation of platelets was evaluated by detecting their active markers through flow cytometry. PRBCs ameliorated the coagulation disorders induced by dilution of the blood in vitro. However, addition of hemoglobin did not increase the blood coagulation, as the level of hemoglobin was negatively correlated to the clot index. Furthermore, washing PRBCs to remove contaminating residual clotting factors and platelets excluded that the coagulation effect of the PRBCs transfusion was because of the RBCs itself. Platelet activity in PRBCs exposed to storage greater than 3 weeks was not significantly reduced consistent with it being a possible contributor. Therefore, we postulate that the suspected coagulation effects ascribed to the PRBCs at transfusion may simply be because of residual clotting factors and active platelets incompletely removed in the preparation of PRBCs rather than because of the red cell membrane or its contents.

It was previously reported that several kinds of intercellular adhesion molecules are closely related to chronic HBV infection. The complex of CD2 and CD58 plays an important role in enhancing the adhesion of T lymphocytes to target cells, and promoting hyperplasia and activation of T lymphocytes. In this study, we detected the level of CD2 expressed on the surface of PBMC, the expression level of CD2 mRNA in PBMC and the percentage of CD2 positive cells in PBMC of patients with chronic HBV infection and compared them with the expression level of normal controls. We also determined the level of serum HBV DNA from patients with chronic HBV infection and from normal controls. The clinical characteristics of hepatic function were tested as well. The results showed that the expression of CD2 significantly increased with the severity of chronic HBV infection, which suggested that CD2 might contribute to the hepatocyte damage in chronic HBV infection.

Hemorrhagic fever with renal syndrome (HFRS) is an acute viral disease characterized by endothelial dysfunction. The cellular immune response, especially the virus-specific CD8+ T lymphocytes, is known to attack vascular endothelial cells (VEC) and to contribute to the diffuse damage and penetrability increasing of VEC. Lymphocyte function associated antigen 3 (LFA-3) is expressed on T lymphocytes and VEC, which is contributed to the activation of T lymphocytes. The expression of LFA-3 on the activated T lymphocytes and VEC is highly increased, which can exfoliate into plasma to increase the level of soluble LFA-3 (sLFA-3) in plasma. So the change of sLFA-3 levels is correlated with the activation of T lymphocytes. In this study we detected the levels of sLFA-3 in plasma of patients with HFRS. We examined the levels of sLFA-3 in plasma samples collected from 53 HFRS patients by double antibody sandwich ELISA. We found variable, but persistently elevated levels of sLFA-3 throughout the various phases and types of the HFRS disease, which suggest that sLFA-3 levels have correlation with disease stages. Moreover, elevated sLFA-3 levels are closely correlated to the severity of HFRS and the degree of kidney damage.