Impact of various handling and storage conditions on quantitative detection of hepatitis C virus RNA
Impact of various handling and storage conditions on quantitative detection of hepatitis C virus RNA
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DOI：
10.1016/s0168-8278(96)80116-4
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发表时间：
1996-09-01
影响因子：
25.7
通讯作者：
Cartouzou, G
中科院分区：
文献类型：
--
作者：
Halfon, P;Khiri, H;Cartouzou, G
背景/目标：HCV RNA病毒载量和HCV基因型是决定慢性丙型肝炎患者对干扰素应答的重要预测因素。为了研究加工和储存条件是否会影响HCV RNA的稳定性以及是否会改变血清中HCV RNA的浓度，采用分支DNA检测法对HCV RNA进行定量。我们研究了从7名经组织学证实的慢性丙型肝炎患者中获得的血清样本，这些患者经历了以下身体条件：（1）立即定量，（2）在室温下储存5天，（3）在4 ℃下储存5天，（4）在-20 ℃下储存5天，（5）在-80 ℃下储存5天，（6）5次冻融循环，（7）血液在室温下不旋转4小时，然后离心并在-80 ℃下储存5天，（8）在4 ℃下储存6个月，（9）在-20 ℃下储存6个月，（10）在-80 ℃下储存6个月，结果：在室温下储存5天后观察到HCV RNA滴度损失100%，然后在4 ℃下储存6个月，在-20 ℃保存5天的血清中观察到HCV RNA水平下降15.6%，5次冻融循环导致HCV RNA水平下降16%，当在室温下延迟4小时后进行离心时，观察到HCV RNA滴度显著损失29.5%，长期稳定性在-80 ℃下观察到HCV RNA滴度轻微下降约10%，但在-20 ℃下观察到HCV RNA显著降低23%。对5份患者样本进行了重复8次的bDNA检测，平均变异系数为9.1%。这些数据证实了储存和处理在测量临床样本中HCV RNA量方面的重要性。
Background/Aims: Both HCV RNA viral load and HCV genotype have been described as important predicting factors determining the response to interferon in chronic hepatitis C, To investigate whether processing and storage conditions might influence the stability and could alter the concentration of the HCV RNA in serum, quantification of HCV RNA was performed by branched DNA assay,Methods: We studied serum samples obtained from seven patients with histologically proven chronic hepatitis C, These were subjected to the following physical conditions: (1) immediate quantification, (2) storage at room temperature for 5 days, (3) storage at 4 degrees C for 5 days, (4) storage at -20 degrees C for 5 days, (5) storage at -80 degrees C for 5 days, (6) five freeze-thaw cycles, (7) blood unspun for 4 h at room temperature then centrifuged and stored at -80 degrees C for 5 days, (8) storage at 4 degrees C for 6 months, (9) storage at -20 degrees C for 6 months, (10) storage at -80 degrees C for 6 months,Results: A loss of 100% HCV RNA titers was observed after storage at RT for 5 days and then storage at 4 degrees C for 6 months, A surprising decrease of HCV RNA titer (15.6%) was observed in sera stored for 5 days at -20 degrees C, Five freeze-thaw cycles resulted in a 16% decrease of the HCV RNA level, When centrifugation was performed after a 4 h delay at room temperature, a significant loss of HCV RNA titers of 29.5% was observed, Long-term stability (6 months) was observed at -80 degrees C with a slight loss of about of 10% HCV RNA titers, but a significant decrease in HCV RNA of 23% was observed at -20 degrees C, The reproducibility of the bDNA assay on five patient samples was performed eight times in duplicate and showed an average coefficient of variation of 9.1%,Conclusions: These data confirm the importance of storage and handling in measuring the amount of HCV RNA in clinical samples.