Valgent4 方案：对使用 SurePath 介质采集的宫颈样本进行 11 种 HPV 检测与基因分型的稳健分析和临床验证。
The Valgent4 protocol: Robust analytical and clinical validation of 11 HPV assays with genotyping on cervical samples collected in SurePath medium.
机构信息
Molecular Pathology Laboratory, Department of Pathology, Copenhagen University Hospital, Kettegårds Alle 30, 2650 Hvidovre, Denmark.
Molecular Pathology Laboratory, Department of Pathology, Copenhagen University Hospital, Kettegårds Alle 30, 2650 Hvidovre, Denmark.
出版信息
J Clin Virol. 2018 Nov;108:64-71. doi: 10.1016/j.jcv.2018.09.012. Epub 2018 Sep 17.
BACKGROUND
The VALidation of HPV GENoyping Tests (VALGENT) is an international initiative designed to validate HPV assays with genotyping capability. The VALGENT4 protocol differs from previous VALGENT installments as the sample collection medium is SurePath, and exclusively includes samples from women ≥30 years of age which is concordant with the majority of HPV primary screening guidelines. Here we present the protocol for the fourth installment of the VALGENT framework.
OBJECTIVES
In VALGENT4 11 HPV assays will be evaluated using two comparator assays based on PCR with the GP5+/6+ primers.
STUDY DESIGN
Overall, the VALGENT4 panel consists of 1,297 routine samples comprised of 998 unselected, consecutive samples, of which 51 samples had abnormal cytology with 13 women diagnosed with ≥CIN2, and 299 consecutive samples enriched for ≥ASCUS cytology (100 ASCUS, 100 LSIL, 99 HSIL) with 106 ≥CIN2 upon follow up. Manipulated and DNA extracted panel samples were characterized with respect to human beta globin (HBB) and overall DNA content and composition to quality assess the panel prior to distribution to the collaborating sites.
RESULT
The relative cellularity (mean CT value of HBB from the Onclarity assay) on the 1,297 LBC samples (CT=24.8) was compared with 293 un-manipulated routine cytology screening samples (CT=23.8). Furthermore, the DNA extracted panel samples was characterized using the Exome iPLEX pro assay, which reports amplifiable copies on individual samples as well as copies of five different base pair lengths. Here the data showed a slightly lower number of amplifiable DNA copies (ratio: 0.7, p=<0.01)) in the VALGENT4 panel samples compared to routine extracted cervical DNA samples CONCLUSION: The present manuscript details the manipulation, processing and quality assessment of samples used in VALGENT-4. This methodological document may be of value for future international projects of HPV test validation.
背景
VALidation of HPV GENoyping Tests（VALGENT）是一项旨在验证具有基因分型能力的 HPV 检测的国际计划。VALGENT4 方案与之前的 VALGENT 版本不同，因为样本采集介质是 SurePath，并且仅包括年龄≥30 岁的女性样本，这与大多数 HPV 初筛指南一致。在这里，我们介绍 VALGENT 框架的第四部分的方案。
目的
在 VALGENT4 中，将使用基于 PCR 与 GP5+/6+引物的两种比较检测方法评估 11 种 HPV 检测方法。
研究设计
总体而言，VALGENT4 小组由 1297 例常规样本组成，其中 998 例为未经选择的连续样本，其中 51 例有异常细胞学表现，其中 13 例女性被诊断为≥CIN2，299 例连续样本为≥ASCUS 细胞学（100 例 ASCUS、100 例 LSIL、99 例 HSIL），随访中有 106 例≥CIN2。在分发给合作机构之前，对处理和提取 DNA 的小组样本进行了人类β球蛋白（HBB）和总体 DNA 含量和组成的特征分析，以对小组进行质量评估。
结果
1297 例 LBC 样本的相对细胞密度（Onclarity 检测的 HBB 平均 CT 值）（CT=24.8）与 293 例未经处理的常规细胞学筛查样本（CT=23.8）进行了比较。此外，使用 Exome iPLEX pro 检测法对提取的 DNA 小组样本进行了特征分析，该检测法报告了个体样本的可扩增拷贝数以及五个不同碱基长度的拷贝数。在此，数据显示 VALGENT4 小组样本的可扩增 DNA 拷贝数略低（比值：0.7，p<0.01），与常规提取的宫颈 DNA 样本相比。
结论
本手稿详细介绍了 VALGENT-4 中使用的样本的操作、处理和质量评估。本方法文件可能对未来的 HPV 检测验证国际项目具有价值。
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