Journal: Virchows Archiv

Article Title: The spectrum of nodular lymphocyte predominant Hodgkin lymphoma: a report of the lymphoma workshop of the 20th meeting of the European Association for Haematopathology

doi: 10.1007/s00428-023-03554-1

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Article Snippet: RNA was isolated from formalin-fixed paraffin-embedded unstained slides as submitted to the EAHP-SH Workshop according to standard methods using the RNeasy FFPE kit (Qiagen, Hilden, Germany) following the manufacturer’s instructions. Digital-multiplexed gene expression (DMGE) profiling was performed with Nanostring Pancancer Immune Profiling Panel (NanoString Technologies, Seattle, WA, USA) platform, a 770-plex gene expression panel to measure the human immune response.20 In brief, 200 ng purified RNA was used as input material. The custom codeset was hybridized to the total RNA overnight, before being purified and fixated using the Nanostring Prep Station. Gene expression data were obtained using the Immunepanel nCounter Digital Analyzer, which automatically performs quality control, normalization, and data analysis. Data quality was assessed by the ratio of preset fields of view (FOV) to observed FOV, which should not be below 0.8. The FOV were set to a high resolution of 555. In addition, only samples with a binding density between 0.1 and 2.25 were included. Raw data QC and normalization were done using nSolver software (NanoString Technologies, version 4.0). For differential gene expression analyses using two group analyses Qlucore software (Qlucore, Lund, Sweden; version 3.6) was applied. Next, deconvolution was performed using Cibersort to assign gene expression profiles to immune cell populations ( http://cibersort.stanford.edu/).21.

Techniques: Isolation, Formalin-fixed Paraffin-Embedded, Gene Expression, Purification, Control, Binding Assay, Software