ABSTRACT
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.
ABSTRACT
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.
ABSTRACT
Objective To study the in-vitro antibacterial activity of Niaoluqing Decoction on Ureaplasma Urealyticum (Uu) . Methods Sixty-three clinical strains of Uu were identified to determine their serology and antibiotic susceptibilities by the metabolic inhibition test(MIT). These strains were subcultured for 3 generations and then cultured with Niaoluqing Decoction with a series of concentrations from 250 mg/mL to 0.48 mg/mL. The minimal inhibitory concentration (MIC) was determined when the color of culture medium did not change from yellow to red in 72 h.Results For 63 clinical strains of Uu, the range of MIC of Niaoluqing Decoction was from 0.48 mg/mL to 15.63 mg/mL,MIC50≤1.95 mg/mL,MIC90≤3.91 mg/mL. Clinical strains of Uu had a higher sensitive to Niaoluqing Decoction than to tetracycline(?2=18.38,P 0.05).Clinical strains of Uu including all of its serotypes had a higher antibiotic susceptibility to Niaoluqing Decoction (MIC≤3.91 mg/mL) except for serotype 11, 1, 2 in this experiment contains (MIC≥7.81 mg/mL). Conclusion Niaoluqing Decoction showed a significant in-vitro antibacterial activity on clinical Uu strains with different serotypes.