July 2019
Volume 60, Issue 9
Open Access
ARVO Annual Meeting Abstract  |   July 2019
Comparing Liquid Biopsy Fixatives for Preservation of DNA and RNA integrity: Identifying the optimum fixative for Primary Vitreoretinal Lymphoma diagnostics
Author Affiliations & Notes
  • Mona Meng Wang
    Translational Ophthalmic Pathology, Singapore Eye Research Institute, Singapore
  • Wei Jian Tan
    A. Menarini Biomarkers Singapore Pte Ltd, Singapore
  • Paola Ricciardi-Castagnoli
    A. Menarini Biomarkers Singapore Pte Ltd, Singapore
  • Tong Seng Lim
    A. Menarini Biomarkers Singapore Pte Ltd, Singapore
  • Anita Sook Yee Chan
    Translational Ophthalmic Pathology, Singapore Eye Research Institute, Singapore
    Singapore National Eye Centre, Singapore
  • Footnotes
    Commercial Relationships   Mona Wang, A. Menarini Biomarkers Singapore Pte Ltd (F), A. Menarini Biomarkers Singapore Pte Ltd (P), Singapore Eye Research Institute (E), Singapore Eye Research Institute (P); Wei Jian Tan, A. Menarini Biomarkers Singapore Pte Ltd (E), A. Menarini Biomarkers Singapore Pte Ltd (P); Paola Ricciardi-Castagnoli, A. Menarini Biomarkers Singapore Pte Ltd (C), A. Menarini Biomarkers Singapore Pte Ltd (P); Tong Seng Lim, A. Menarini Biomarkers Singapore Pte Ltd (E), A. Menarini Biomarkers Singapore Pte Ltd (P); Anita Chan, A. Menarini Biomarkers Singapore Pte Ltd (F), A. Menarini Biomarkers Singapore Pte Ltd (P), Singapore Eye Research Institute (E), Singapore Eye Research Institute (P)
  • Footnotes
    Support  This study was supported by the funding of research collaboration agreement between Menarini Biomarkers Singapore and Singapore Eye Research Institution.
Investigative Ophthalmology & Visual Science July 2019, Vol.60, 3545. doi:
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      Mona Meng Wang, Wei Jian Tan, Paola Ricciardi-Castagnoli, Tong Seng Lim, Anita Sook Yee Chan; Comparing Liquid Biopsy Fixatives for Preservation of DNA and RNA integrity: Identifying the optimum fixative for Primary Vitreoretinal Lymphoma diagnostics. Invest. Ophthalmol. Vis. Sci. 2019;60(9):3545.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : Primary vitreoretinal lymphoma diagnostics are greatly limited by cellular preservation of morphology, DNA and RNA integrity. Molecular analysis and staining properties of liquid biopsies are affected by the type of fixative and fixation protocols. This study aims to determine the optimal fixative and fixation method to obtain efficiently high-quality RNA and DNA for further downstream molecular analyses and cellular markers staining for flow cytometry (FACS).

Methods : Six different common fixative solutions (RNAlater™, HOLOGIC-PreservCyt Solution, PAXgene Tissue Container, Shandon™ Cytospin™ Collection Fluid, Allprotect Tissue Reagent and HOPE® I solution) were included in the comparison. Fresh unfixed Germinal center B-cell-like DLBCL cell line Pfeiffer (ATCC CRL2632) as controls. The equivalent amount of Pfeiffer cells were fixed immediately after harvesting and stored at −4°C for seven days. Each fixation method was evaluated by DNA and RNA yield, integrity analysis by Agilent 2100 Bioanalyzer (Agilent Technologies) and gel electrophoresis and antibody staining by subsequent flow cytometry (FACS) analysis.

Results : PreservCyt fixed cells were found to have similar results as the fresh cell line (Table 1). The fixation protocol using methanol-based PreservCyt Solution showed the best performance for the preservation of high-quality RNA, DNA and staining properties studied cell markers concurrently. Furthermore, this is a commonly used cytological fixative that allows cytology to be performed for PVRL diagnosis.

Conclusions : PreservCyt solution provides both morphological preservation as well as RNA and DNA integrity for downstream analysis, even after 7 days of fixation. This makes it the ideal diagnostic and research preservative for collecting PVRL vitreous samples.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.

 

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