干血斑样本用于HIV抗体检测方法学优化与应用

Optimization and application of HIV antibody detection method based on dry blood spot samples

目的探讨用于干血斑样本中艾滋病病毒（HIV）抗体检测的最佳洗脱条件。方法选取并制备中值（24）与低值（26）的干血斑样本,利用HIV抗体诊断试剂盒（酶联免疫吸附试验法）,对不同洗脱温度、不同洗脱液组分、不同洗脱液体积、不同洗脱时间分别进行组合后,进行干血斑（DBS）样本洗脱液中抗体OD值测定分析,从而选出最适宜、最方便用于HIV抗体检测的DBS洗脱条件。结果室温与4℃条件下洗脱样本差异无统计学意义（P=0.979,P=0.704）;不同洗脱液体积对样本洗脱的影响差异无统计学意义（P=0.183）;洗脱液组分对低值（26）样本的洗脱效果有统计学意义（P=0.033）,其中以含0.5‰Tween20的PBS对样本洗脱的影响更为显著（P=0.02,P=0.004）;洗脱时间对中值（24）与低值（26）样本均有显著影响（P=0.00,P=0.00）,其中4小时和6小时为显著差异分界点。670份临床已知样本的检测应用中,试剂盒现成Cutoff值计算判定阳性样本时,存在2.63%的假阳性,无假阴性。结论用于DBS中HIV抗体最佳洗脱条件为：用300μL含有0.5‰Tween20的PBS在室温下（24±2℃）洗脱4～6小时。试剂盒Cutoff值不适用于DBS样本结果判定,需进一步研究设定用于该方法的Cutoff值。

Objective To optimize the elutriation conditions of HIV antibody detection method based on DBS.Methods The low and median DBS samples（24 and 26）were selected and prepared by using HIV negative blood series to dilute anti-HIV positive blood.HIV antibody diagnostic kit（enzyme-linked immunoassay）was used to test the OD value of the DBS eluent,obtained under different combination conditions including elution temperature,elution composition,elution volume and elution time.Then the most suitable and convenient elution combination conditions were chosen.Results There was no statistically significant difference in the dry blood spot samples eluted at room temperature and 4℃（P=0.979,P=0.704）and different elution volume（P=0.183）.There was a significant effect when the low value DBS samples were eluted by different elution composition（P=0.033）,and the PBS with 0.5‰ Tween20 more significant（P=0.02,P=0.004）.There was significant effect between the different elution time（P=0.00,P=0.00）,and 6 hours and 4 hours were the significant difference demarcation points.There was 2.63%false positive and no false negative were found during the application of 670 clinically dry blood spot samples.Conclusion The best elution conditions for HIV antibody detection based on DBS are use of 300 ul contain0.5‰ Tween20 PBS at room temperature（24±2℃）elution for 6 hours.And we need further to setup the cutoff value for this method and carefully evaluate the methodology.