ARUP Laboratories, Salt Lake City, UT.
Department of Pathology, University of Utah, Salt Lake City.
Am J Clin Pathol. 2020 Jan 2;153(2):251-257. doi: 10.1093/ajcp/aqz166.
Many commonly used FLT3 mutational assay protocols require a tedious blast enrichment step. We investigated whether elimination of this step would still give equivalent results and compared the accuracy of variant allele fraction (VAF) between polymerase chain reaction/capillary electrophoresis (PCR/CE) vs next-generation sequencing (NGS) methods.
Total leukocyte vs blast-enriched whole-blood aliquots were tested for FLT3 internal tandem duplication (ITD) and tyrosine kinase domain mutations by PCR/CE. VAF of the ITD mutations was also compared with NGS VAF.
Blast-enriched vs total leukocyte specimens showed 100% concordance in the 25 positive specimens. VAF was consistently lower by NGS, with poorer fidelity to PCR/CE VAF as the ITD size increased.
Our study supports elimination of the blast enrichment step without compromising results or sensitivity. In addition, since NGS shows a loose correlation with PCR/CE quantitative results, NGS VAF should not be reported for FLT3 ITDs.
许多常用的 FLT3 突变检测分析方案需要繁琐的 Blast 富集步骤。我们研究了省略该步骤是否仍能得到等效的结果，并比较了聚合酶链反应/毛细管电泳（PCR/CE）与下一代测序（NGS）方法之间变异等位基因分数（VAF）的准确性。
通过 PCR/CE 对白细胞和富含白细胞的全血等分试样进行 FLT3 内部串联重复（ITD）和酪氨酸激酶结构域突变检测。还比较了 ITD 突变的 NGS VAF。
在 25 份阳性标本中，富集的白细胞与白细胞标本之间具有 100%的一致性。NGS 始终显示较低的 VAF，随着 ITD 大小的增加，与 PCR/CE VAF 的一致性更差。
我们的研究支持省略 Blast 富集步骤，而不会影响结果或敏感性。此外，由于 NGS 与 PCR/CE 定量结果相关性较弱，不应报告 NGS VAF 用于 FLT3 ITD。
