Purchase this article with an account.
Wei Jian Tan, Mona Meng Wang, Paola Castagnoli, Anita SY Chan, TongSeng Lim; Single cell vitreous analysis increases sensitivity of MYD88L265P detection and allows detailed genotyping. Invest. Ophthalmol. Vis. Sci. 2019;60(9):2776.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Myeloid differentiation primary response 88 (MYD88) mutational screening is increasingly being used to aid diagnosis of vitreoretinal lymphoma (VRL). However, current studies are performed on bulk vitreous fluid, and thus the detection of MYD88L265P is dependent on the proportion of wild type (WT) versus mutant MYD88. Here, we interrogate the use of single cell analysis to increase the sensitivity of detecting MYD88L265P mutation in vitreous biopsies.
Study was performed in compliance with the Declaration of Helsinki with informed consent and SingHealth institutional review board approval. Single CD19+CD20+ B cells were isolated from residual biopsy proven VRL and control (inflammatory) vitreous biopsies leftover from the clinical diagnostic laboratory. Single cells from VRL patients (n=4) and inflammatory control cases (n=3) were subjected to whole genome amplification to increase the amount of genomic DNA for single cell-MYD88 polymerase chain reaction (PCR). Profiling of MYD88 mutation was performed using Sanger sequencing.
MYD88 mutational profiling was successfully performed using single cells isolated from the vitreous (Figure 1). As compared to bulk cell sequencing with detection sensitivity of 30%, single cell-MYD88 sequencing enabled the characterization of individual cell as WT, heterozygous or homozygous mutation profile of individual cell at 100% purity. Additionally, we were able to observe predominant homozygous MYD88L265P mutation in single cells isolated from cytology-proven VRL patients (n=2).
Single cell-MYD88 mutational profiling allows genetic characterization of B cells for VRL diagnosis, which cannot be achieved by standard cytology or bulk cell analysis. This approach enabled the precise genotyping of MYD88 mutation at single cell level which is otherwise impossible to achieve with bulk sample analysis. This single cell based molecular screening is particularly suitable for small volume and rare target cell detection with the potential to help refine VRL diagnosis.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
This PDF is available to Subscribers Only