Objective To evaluate the quality of blood after marine storage and transportation,to establish a quantitative energy metabolism platform supported by extracellular flux(XF)technique to determine the glycolysis level of red blood cells(RBC), and to introduce the "functional dose", as a valuable supplement, into the quality evaluation of RBCs after marine storage and transportation.Methods The extracellular acidification rate(ECAR)of erythrocyte glycolysis was detected by XF.The levels of glycolysis, ATP contents and free hemoglobin of RBCs were detected before and after marine navigation.RBC suspensions stored at 4℃in a blood bank on land served as control.Results The results showed that the glycolysis level of RBCs was detected by XF.Indicators of blood quality and energy metabolism of RBCs were detected in the twice marine storage and transportation.Blood samples transported around the Gulf of Bohai had no significant change,indicating that the samples were well preserved.The energy indicators of blood samples navigated in the Pacific Ocean decreased significantly,and it was recommended that the samples be discarded.Conclusion XF technique has been applied to the measurement of erythrocyte glycolysis,enabling high-throughput and multi-indicator dynamic detection of viable cells.The energy metabolism of erythrocyte should be recommended as an important indicator for evaluating blood after marine storage and transportation.

OBJECTIVE: To analyze the expression of CCR7 and Tim-3 in childhood patients with acute lymphoblastic leukemia (ALL) and their predictive value for prognosis. METHODS: Eighty-six newly diagnosed ALL childhood patients from January 2007 to January 2017 treated in our hospital were selected. The expression level of CCR7 and Tim-3 in bone marrow isolated cells of ALL patients were detected by flow cytometry, all the patients were divided into the recurrence group and non-recurrence group according to the follow-up results, the differences in the expressions of CCR7, Tim-3 between the two groups were compared. The correlation between the expression of CCR7 , Tim-3 and the clinicopathologic features of ALL patients were analyzed, the predictive value of CCR7 and Tim-3 for the prognosis of newly ALL patients were evaluated by ROC curve, and the relationship between serum CCR7, Tim-3 and prognosis were analyzed. RESULTS: The expression levels of CCR7 and Tim-3 in recurrence group were significantly higher than those in non-recurrence group(P<0.05). The critical value of CCR7 for diagnosis of recurrence was 45.97%, the sensitivity was 66.7%, the specificity was the 84.5% and the area under ROC curve (AUC) was 0.798 (95CI 0.777-0.939). The critical value of Tim-3 for diagnosis of recurrence was 53.54%, the sensitivity was 73.3%, the specificity was the 80.3% and the AUC was 0.806 (95CI 0.792-0.947). The AUC of the combined detection of CCR7 and Tim-3 was 0.895 (95CI 0.914-0.996), sensitivity 86.6%, specificity 78.9% (P<0.05); There was no significant correlation between CCR7, Tim-3 expression and age, sex, hemoglobin concentration, number of white blood cells, bone marrow blasts, platelets, central nervous system invasion, fusion gene (P>0.05). The exogenous infiltration rate of patients with high expression of CCR7 and Tim-3 was significantly higher than those in low expression group (P<005). The high expression rate 76.9% of Tim-3 in patients with T-ALL was significantly higher than that of B-ALL patients with Tim-3 high expression rate 45.2% (P<0.05). The median OS of patients with CCR7 level ≥45.97% and <45.97% were 9.3 months and 13.6 months respectively(P=0.004), and the Tim-3≥53.54% and Tim-3<53.54% were 9.1 months and 13.6 months respectively(P=0.001). The results of Cox's multi-factor regression analysis showed that CCR7 level(HR=1.024, 95 CI 1.001-1.049) and Tim-3 level (HR=1.879, 95 CI 1.183- 2.985) were the independent risk factors that affect the OS in ALL patients(P<0.05). CONCLUSION The expression of CCR7 and Tim-3 in bone marrow isolated cells of ALL patients shows good predictive value for prognosis, and the combination of CCR7 and Tim-3 can improve the sensitivity of the detection, the higher expression of CCR7 and Tim-3 can be used as potential indexes in prognosis evaluate.
Bone Marrow ; Child ; Hepatitis A Virus Cellular Receptor 2 ; Humans ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; Prognosis ; Receptors, CCR7

Objective: To produce latent membrane protein 2A (LMP2A) chimeric antigen receptor (CAR)-T cells and detect the lethal effect of LMP2A CAR-T cells on nasopharyngeal carcinoma (NPC) cells. Methods: The study was conducted from September 2016 to December 2017.Genetic engineering technology was used to construct anti-LMP2A CAR lentiviral expression vector and sequencing was identified. The expression of anti-LMP2A CAR in the 293T cells was confirmed by western blot. CCK8 assay was used to evaluate the cytotoxicity of LMP2A CAR-T cells to NPC cells. ELISA assay was performed to test IL-2 and IFN-γ releasing of activated LMP2A CAR-T cells. The inhibition effect of LMP2A CAR-T cells on NPC xenograft tumor was observed in vivo. Statistical analysis was performed by statistical software SPSS 21.0. Results: The results of PCR and sequencing showed that anti-LMP2A CAR lentiviral expression vector was constructed successfully. The result of western blot indicated the expression of anti-LMP2A CAR in the 293T cells effectively. The results of CCK-8 assay showed that the killing activities of LMP2A CAR-T cells to LV-LMP2A-CNE1 cells were (72.11±9.75)%, (54.65 ±5.42)% and (36.68±3.80)% at 20∶1, 10∶1 and 5∶1 ratio of effective cells to target cells, and had a statistical difference compared to CD19 CAR-T cells and T cells (P<0.05). There was no significant difference in the killing activities of LMP2A CAR-T cells to CNE1 cells compared with CD19 CAR-T cells and T cells. The results of ELISA showed that the content of IL-2 and IFN-γ in the co-culture supernatant of LMP2A CAR-T cells and LV-LMP2A-CNE1 cells was significantly higher than that of LMP2A CAR-T cells and CNE1 cells which had statistical difference (P<0.05); In vivo experiment, the volume of LMP2A CAR-T cell group was (80.3±10.0) mm³ which was significantly lower than that of the control groups, and the difference was statistically significant (P<0.05). Conclusion LMP2A CAR-T cells are successfully prepared and have an obvious targeting cytotoxicity on LMP2A-positive NPC cells.