Establishment and identification of a human megakaryocytic cell line with low Ley expression / 中国输血杂志

【Objective】 To establish a stable human megakaryocytic cell line with low expression of Ley antigen to further study the role of Ley on activation of platelets. 【Methods】 The expression level of the Ley antigen in a human megakaryocytic cell line, DAMI, was determined using Western Blot and flow cytometry. The expression level of genes that encode fucosyltransferase (FUTs), which was involved in the biosynthesis of Ley antigen, was also determined to identify the candidate genes to be knocked out. The candidate FUT gene was knocked out via a CRISPR/Cas 9 gene knockout system and cells with low Ley antigen expression were sorted by flow cytometry. The sorted cell line was cultured and characterized. 【Results】 The Ley was expressed intensively on DAMI cell. FUT1 and FUT4 mRNA was expressed relatively higher, both may be key enzymes for the biosynthesis of the Ley antigen. In the DAMI cell line with the knockout of FUT1 gene, the expression of the Ley adntigen was remarkedly reduced, while cell proliferation was not affected compared to the wildtype control cells. 【Conclusions】 Since various FUTs contributes to the biosynthesis of the Ley antigen, the knockout of the primary one of them cannot totally block its biosynthesis, but only reduce its expression. In this study, a stable FUT gene knockout human megakaryocyticcell line is established using CRISPR/Cas 9 technology, which provides basis for the study of the impact of the Ley antigen on platelet functions.

β-Lactam antibiotics promoting aging and clearance of RBCs would deteriorate the DIIHA / 中国输血杂志

【Objective】 To analyze the influence of β-lactam antibiotics on RBC aging and clearance by detecting various indicators of aging and clearance on RBCs, as well as the differences in phagocytosis for erythrocytes before and after drugs treated in vitro. 【Methods】 RBCs were treated by β-lactam antibiotics, including Penicillin, Cefepime, Cefoperazone and Ceftazidime, and the changing of phosphatidylserine (PS) and clearance related CD markers, including CD35, CD47, CD55 and CD59 on the surface of the RBCs, were detected by flow cytometry at 0h and 24h after drugs treatment. The proportion of acanthocytes by microscope also at 0h and 24h after drugs treatment was calculated. The phagocytosis of drug-treated RBC was detected by monocyte monolayer assay (MMA). Untreated RBCs were incubated in PBS by the same condition as a negative control.The influence of β-lactam antibiotics on RBC aging and clearance by all the results above was studied. 【Results】 Compare to the untreated RBCs, the drug treated RBCs showed a higher PS level on the cell surface. The results showed by percentage as following(0 h vs 24 h): Penicillin 9.42% vs 93.30%, Cefepime 3.88% vs 57.27%, Cefoperazone 4.71% vs 75.75% and Ceftazidime 3.05% vs 43.19%. The acanthocytes ratio was as following(0 h vs 24 h): Penicillin 7.33% vs 86%, Cefepime 2.67% vs 52.67%, Cefoperazone 3.33% vs 67.67% and Ceftazidime 3.33% vs 90.67%. On the opposite, the clearance related CD markers, showed an obviously lower level after drugs treated(0 h vs 24 h): CD35: Penicillin 7.36% vs 11.87%, Cefepime 0.14% vs 28.51%, Cefoperazone 11.85% vs 21.55% and Ceftazidime 7.63% vs 8.73%; CD47: Penicillin 1.22% vs 9.13%, Cefepime 1.80% vs 0.86%, Cefoperazone 0.08% vs 6.85% and Ceftazidime 1.54% vs 5.50%; CD55: Penicillin 14.46% vs 44.31%, Cefepime 17.27% vs 38.41%, Cefoperazone 19.28% vs 33.28% and Ceftazidime 14.62% vs 34.13%; CD59: Penicillin 4.71% vs 20.56%, Cefepime 4.03% vs 7.60%, Cefoperazone 5.91% vs 22.38% and Ceftazidime 5.93% vs 30.89%. Drug-treated RBCs attached more to monocytes than untreated RBCs. 【Conclusion】 The β-lactam antibiotics could induce the changing of PS and the clearance of related CD markers on surface of RBCs. They also could lead acanthocytes and make the RBCs more susceptible to phagocytosis by monocytes. The β-lactam antibiotics could promote the RBCs aging and clearance, which might deteriorate the DIIHA.

Quantitative detection of red blood cell antibody-mediated complement activation / 中国输血杂志

【Objective】 To construct an in-vitro model of erythrocyte antibody-mediated complement activation, and establish quantitative detection methods based on flow cytometry and spectrophotometry, so as to explore the correlation of anti-body titers and complement activation speed, and provide a methodological basis for studying the adverse transfusion reactions of anti-body mediated complement hemolysis. 【Methods】 Mouse monoclonal antibody that recognized human C3b and fluorescent secondary antibody were used to label C3b fragments on erythrocytes, and the deposition of C3b fragments after complement activation was detected by flow cytometry. The absorbance at 540 nm of the supernatant in the complement activation reaction system was measured by spectrophotometry as the amount of hemoglobin released was related to the absorbance. 【Results】 The complement activation system was constructed according to the ratio of 3% red blood cell suspension (mixed for 6 people) 1∶anti-Tja 1∶complement 2. The repeatability was good (P value>0.05) as different red blood cell mixtures had been used to repeat the detection reaction system. When using 32×, 64× and 128× dilutions of anti-Tja mediated complement activation, the deposition of C3b fragments has been detected by flow cytometry at 30 s, 1 min and 2 min, respectively, and MFI peaked at 5 min, 10 min and 30 min, respectively. No obvious hemolysis has been observed within 1.5 h. 【Conclusion】 In vitro model of anti-Tja-mediated complement activation demonstrates the speed of complement activation is related to the concentration of antibody. At a certain antibody concentration, the speed of complement activation has been slowed down, and no obvious hemolysis observed.

Preparation of human monoclonal anti-C cell line from peripheral blood B lymphocytes of D--donor / 中国输血杂志

【Objective】 To establish human hybridoma cell lines, secreting monoclonal antibody against antigens of Rh blood system, from a donor with rare D--phenotype. 【Methods】 Peripheral blood B lymphocytes of an O type female donor, lacking C/c/E/e antigens on her erythrocyte, were transformed with Epstein-Barr virus (EBVs). EBVs were harvested from the cultural supernatant of B95-8 cells. The transformed lymphoblastoid cell line (LCL) secreting antibodies to C antigens were picked up and then hybridized with the myeloma SHM-D33 using electric fusion technique. Hybridoma cells were selected by HATD-Ouabain(HOTD)(Hypoxantine, Aminopterin, Thymidine, 2-Deoxycytide, and Ouabain)culture medium, microplate antibody screening and limited dilution subcloning. The monoclonal antibody was assayed by serological test and was confirmed by flow cytometry (FCM). 【Results】 From the cultural supernatant of D--peripheral blood transformed B lymphocytes, 3A6-C6, which agglutinated with R1R1(DCe/DCe)O-type RBCs but not with R2R2(DcE/DcE)O-type RBCs, was screened and preliminarily identified as anti-C. We established a hybridoma cell line secreting anti-C immunoglobulin M from B cells of D--individual successfully after hybridization with SHM-D33 myeloma cells. 【Conclusion】 The study had laid the groundwork for future research and development of human monoclonal antibodies against Rh antigens of RBC in future for diagnosis and screening purpose.

-D- phenotype screening by polybrene method / 中国输血杂志

【Objective】 To develop a novel screening reagent for -D- phenotype preliminary screening based on the difference in RhD antigen expression level of -D- phenotype and normal RhD phenotype. 【Methods】 RhD antigen expression of -D-phenotype and Rh D-- gene carrier were detected by flow cytometry. By adjusting the concentration of polybrene in the screening system, the red blood cells with high RhD antigen expression level agglutinated, and the preliminary screening of the -D-phenotype and its gene carriers was realized. 【Results】 According to the quantitative results of immunofluorescence intensity (MFI) analysis by flow cytometry, the expression level of RhD antigen in -D- phenotype cells (284 360±16 698, n=3) was about 3 times normal RhD positive cells (98 642±35 908, n=9)(P<0.01), while RhD antigen expression level of RhD-- gene carrier (181 109±39 455, n=4) was about 2 times normal RhD positive cells(P<0.01). RhD antigen expression (144 538±227 445, n=7) of the positive cells screened by 15 μL 3% fresh red blood cell suspension and screening system 35 μL (1 μL IgG anti-D, 29 μL polybrene polybrene, and 5 μL low ionic strength solution) was about 1.5 times normal RhD positive cells. 【Conclusion】 The polybrene preliminary screening system, which can be used for high-throughput screening of -D- phenotype, is a reliable technical method for frequency study of this phenotype.