| Objective:Study on the influence of hemolysis,fatty blood,storage temperature and storage time on HCV RNA,HIV RNA and HBV DNA detection in blood with Roche MPX V2.0 kit.To search the key factors for the correct processing and preservation of specimens for NAT detection.Methods: 1.Using ELISA to detect the 41552 blood samples which collected from blood donators from January 1,2016 to September 30,2016,and distinguish the blood samples of HBsAg,anti-HCV,anti-HIV positively from negatively;2.Roche MPX V2.0 kit was used to detect for HCV RNA,HIV RNA,HBV DNA for all samples by the mode of every 6 samples mixture for one pool detection,firstly,and for positive mixture samples,an one by one detection for each sample was followed.The single positive samples of HCV RNA,HIV RNA,HBV DNA were used to make the nucleic acid positive samples of 12-30 times reagent limit of detection(LOD)concentration,respectively;3.The prepared nucleic acid positive samples were stored at 4℃,25℃,37℃ and-30℃ for 4h,24 h,48h,72 h,1w,4w according to design.All specimens were detected by NAT for HCV RNA,HIV RNA and HBV DNA,and the Ct values were analyzed.4.The red blood cell suspension and the severe fatty plasma were prepared for double dilution hemolytic samples(1 、 1/2 、 1/4 、 1/8 、1/16),hemolytic standard samples(1、1/2、1/4、1/8),double dilution fat plasma samples(1、1/2、1/4、1/8),the standard sample of fatty plasma(1/2、1/6、1/12、1/24、1/48).The concentration of the standard sample of hemoglobin(Hb)by HiCN method and triglyceride(TG)by glycerol lipase oxidase was detected,and the Ct values for HCV RNA,HIV RNA and HBV DNA with Roche MPX V2.0 kit were tested for each sample.Results: 1.There were 205(0.49%)positive samples for HBsAg,82(0.20%)for anti–HCV and 35(0.08%)for anti-HIV in 41552 blood samples from blood donators by ELISA.2.The concentration of viral nucleic acid for HBV DNA,HCV RNA and HIV RNA in single positive sample was 100IU/ml,2.7×107 IU/ml and 9.6×104 IU/ml,respectively.3.In the experiment of preservation time and storage temperature,the Ct values of HCV RNA,HIV RNA,HBV DNA were no significant difference when samples were stored at-30℃ for 4h and 4w(P>0.00333).When the samples were kept under 4 ℃ storage condition,their Ct values of HCV RNA for 4h,24 h,48h and 72 h were statistically different from the values from the samples stored for 1w and 4w,respectively(P<0.00333),but there were no difference among themselves(P > 0.00333).Under the storage condition of 25 ℃,there were no significant difference for the Ct values of HCV RNA between 4h and 72h(P>0.00333),except from the compares between 4h and 1w(P<0.00333).Under the storage condition of 37℃,there were statistical significances for the Ct values of the HCV RNA for samples between the storage time of 4h and 24h(P<0.00333),but not for the Ct values of HIV RNA and HBV DNA(P>0.00333).The Ct values of HCV RNA were significant difference when compared the group stored under-30℃ and 37℃with preservation time for 24h(P<0.00833),and it was also significant difference when compared the group stored under 4℃ and 37℃(P<0.00833).With preservation time for 72 h,the Ct values of HBV DNA were statistically significant(P<0.00833)when compared the group stored under-30℃ and 37℃.4.When Hb concentration reached 97g/L,the results of HBV DNA and HIV RNA were negative,but HCV RNA was still able to be detected(Ct:46.3±7.9).When the concentration of Hb downregulated to 34g/L,17g/L,8g/L,5g/L and 3g/L,compared with the control group,there were no significant difference in Ct values of HIV RNA and HBV DNA(P>0.05).When the concentration of Hb was more than 8g/L,compared with the control group(Ct:36.4±0.2),there were significant difference in Ct values of HCV RNA(Ct:46.3±7.9、37.5±0.4、37.3±0.3、37.6±0.5).When the concentration of Hb was less than 5g/L,compared with the control group(Ct:36.4±0.2),there were no significant difference in Ct values of HCV RNA(Ct:36.4±0.6、36.2±0.5);5.HCV RNA,HIV RNA,HBV DNA NAT were not affected by the fat blood in the lipid factor experiment,when the TG concentration was less than 7.93mmol/L,and there were no significant difference in Ct values for them between the control group and the TG group(P>0.05);Conclusions: 1.When the concentration of TG is less than 7.93mmol/L,the results of Roche MPX V2.0 kit for the detection of HCV RNA,HIV RNA and HBV DNA are not significantly affected;2.When the concentration of Hb is more than 8g/L,the results of Roche MPX V2.0 kit for the detection of HCV RNA have constant influence;When the concentration of Hb is less than 34g/L,the results of Roche MPX V2.0 kit for the detection of HIV RNA and HBV DNA are not significantly affected;3.HCV RNA samples preserved at 4℃ for 72 h and HIV RNA or HBV DNA samples stored at room temperature(25 ℃)in 4W are best detected by Roche MPX V2.0 kit. |
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