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Objective To establish a new method for quantitating leukocyte fragments (LFs) in apheresis platelet concentrates (AP-PCs) by using real-time quantitative polymerase chain reaction (RQ-PCR) and flow cytometry(FCM) and discuss the factors influencing LFs concentrations such as storage time, filtration and PLT concentration. Methods 67 qualified donors were selected. Each of them donated one therapeutic dose of AP-PCs. AP-PCs samples were collected as soon as possible and divided into si xfractions. One was analyzed by hematology analyzer. For the Others, DNA was extracted under differen tconditions (filtrated or unfiltrated, before or after centrifugation) at 4 hours, 24 hours, 48 hours, 72 hours, 96 hours after blood draw, respectively. Then the amounts of albumin gene of the AP-PCs and the cell-free DNA in supematant were quantitatively determined using RQ-PCR and the results were calculated into leukocytes equivalent(WBCs/μl). Intact leucocytes were counted by FCM. The concentrations of LFs were calculated by subtracting cell-frce DNA and intact leucocytes from the total DNA amount. Then the differences of LFs concentrations among groups with different storage time were compared and the differences of LFs concentrations between unfihrated and filtrated groups were also compared. After grouping all the AP-PCs according to their PLT concentrations, LFs contents of AP-PCs before filtration among groups were compared. Meanwhile, bivariate correlation analysis between PLT concentrations and LFs contents was carried out. ResultsLFs contents of all the AP-PCs samples were quantitated successfully.The concentrations of LFs in AP-PCs before filtration in 4 hours,24 hours,48 hours,72 hours , 96 houres after blood draw were(31.4±17. 6), (47.5±25.3), (100.7±53.5), (89.5 ±47.2) and (16.1±7.8) WBCs/μl ; After filtration the results were (16. 9±8. 7), (24. 3 ± 12. 2), (83. 1±42. 6), (78.2 ±40. 2) and (13.6 ± 6. 6) WBCs/μl respectively. There were statistically significant differences among groups of different storage time (Fwithin subjects = 472. 756,P < 0.01). The concentrations of LFs kept on increasing within 48 hours after collections, and then decreased gradually. The peaks appeared between 48 hours and 72 hours after collections. The differences of LFs contents between unfiltered and filtered AP-PCs in 4 hours, 24 hours, 48 hours, 72 hours, 96 hours after collections were 14. 5, 23. 2, 17. 6, 11.3 and 2. 5 WBCs/μl, respectively.There was statistically significant difference between unfiltered and filtered samples (Fbetween subjects=9. 216,P < 0. 05). The differences were considerable within 48 hours, and then declined gradually. The results of bivariate correlation analysis showed that there were no statistically significant correlation between PLT concentrations and LFs contents (at 4, 24, 48, 72, 96 hours after collections the correlation coefficients rs were -0.002, 0.015, 0.027, 0.042 and 0. 037,respectively,P2-tailed>0.05). ConclusionsRQ-PCR and FCM can be used to quantitate LFs in AP-PCs. The concentration of LFs in AP-PCs is influenced by storage time and filtration, but it is not affected by PLT concentration.
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Objective To explore the feasibility of fluorescence quantitative PCR(FQ-PCR)in prenatal diagnosis of the fetal RhD genotyping using free DNA from RhD-negative pregnant women.Methods The fetal RhD gene was amplified from 78 RhD-negative pregnant women with single fetus maternal plasma (gestation from 11 to 40 weeks).Rhe existence of fetal DNA was confirmed by amplification ofnine different polymorphic short tandem repeat loci(STR)and sex-determining region Y chromosome(SRY)gene.Exon5,7,10 and intron 4 were amplified by real-time polymerase chain reaction with TaqMan probe.The results of fetal RhD genotyping were evaluated retrospectively by the serologic analysis of infant cord blood.Results Among the 78 specimens,the SRY positive signals were detected from samples of 41 and were all identified male fetal through 8ex observation after newborn infants delivered from the women enrolled.The mean concentration of SRY gene reached(214.7±120.9)eopies/ml.RhD genotyping results of 70 cases were in complete concordance with the resets through serological detection of fetal cord blood after delivery.In addition,5 cases were false-positive.3 cages were considered inconclusive.The coincidence rate was 90%(70/78).From 5 false-positive cases,4 cases were identified as RhDel phenotype by detecting RHDl227A allele gene.The final accuracy rate of FQ-PCR was 95%(74/78)in the fetal RhD genotyping.Conclusion FQ-PCR analysis for noninvasive prenatal of fetal RhD genotyping could be useful in prevention and diagnosis of hemolytic disease of newborn.
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Objective To separate the CD1c+ MDC and CD304 PDC subsets and analyze their immunobiologic properties.Methods MDC or PDC were separated by immunomagnetic beads and then induced by TNF-α or CpG2006 OND.The phenotype of different DC subsets and allogeneic T-cell-stimulating capacity were detected,respectively.Results The purlty of separated DC was about ninety-five percent.MDC expressed HLA-DR,CD11c,CD4 and CD33,PDC expressed HLA-DR,CD4 and CD45RA,and the percentage of CD40,CD80 were up-regulation after induced to mature.The results of mixed lymphocyte reaction(MLR):MDC had strong potential to stimulate allogeneic T lymphocytes proliferation,while PDC had the weak stimslate potential.The supematant IFN-r in all groups was increased.which the groups of MDC and mMDC were more obviously.The supematant IL-10 was increased obviously in groups of PDC and mPDC.The expression of IFN-r in CD4+ cells were increased obviously after stimulated by MDC or mMDC.PDC and mPDC had the effect of inducing of CIM+ CD25high regulatory T lymphocyte in MLR.The expressing of Foxp3 mRNA had no evident difference in all groups.Conclusion Two different DC subsets in the donors peripheral blood which mobilized with G-CSF were in the state of immature and could be induced to mature in the suitable condition.although expressed high constitutive levels of HLA,DR.No matter how MDC was mature or not,they had strong potential to stimulate allogeneic T lymphocytes proliferation.MDC enhanced the production of IFN-r among T lymphocytes,and the increase of IFN-r was probably the result of TH1 polarization.Unlike MDC,PDC was less efficient present antigens and had poor T-cell-stimulating capacity,probably because of their lesser ability to phagocytose,process and present antigens to the MHC molecules.Although PDC could promote the production of IFN-r,it seemed that did not produced by TH 1.PDC could not promote TH2 cells to product IL-4,and the polarization of TH2 probably reflect the production of IL-10.PDC could induce the generation of CD4+ CD25high regulatory T cells.
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0.05).The volumes of PBSC collected each time in the 3 groups were about 60 ml,and there were(3.67—10.25)?1010 erythrocytes per pack in the ABO major incompatible group,and 22—38 ml of plasma in ABO minor incompatible component.All the PBSC were infused to recipients without the removal of erythrocytes or plasma,and no haemolytic reaction was observed in any recipients and all their hematopoietic functions were re-established.Conclusion Enough PBSC can be harvested by using CS-3000 Plus blood cell separator from ABO incompatible allogeneic donors.By modification of separator parameters,the number of erythrocytes in the collects can be reduced.The collected PBSC can be safely infused into the blood group incompatible recipients without removal of erythrocytes or plasma.