1. Enrichment of Hodgkin and Reed-Sternberg (HRS) cells using size-based microfiltration

Large, multinucleated Reed-Sternberg and mononuclear Hodgkin (HRS) cells (50-100 µm and 20-30 µm, respectively) are pathognomonic features in Hodgkin Lymphoma (HL). Current methods available to isolate these cells are challenging, time-consuming, and low yield, making them unsuitable for clinical assays. To address these challenges, we sought to develop a method for HRS cell enrichment using size-based microfiltration. Originally created to enrich rare cells in the blood, these microfilters have 40,000, 8 µm pores which will remove small lymphocytes while capturing larger HRS cells of interest.

Using mixtures of L428s, an HL cell line, spiked into dissociated lymph node (LN), we demonstrated the ability to capture on average 93% of the input L428s. In our model, CD30, a cell surface marker expressed on HRS cells, was employed to identify the L428s from the background via immunofluorescent staining on-chip. Concurrently, we optimized a method for DNA extraction directly from the microfilter and reliably yielded over 3 µg of DNA. This DNA was then used for single nucleotide polymorphism genotyping which further verified our ability to enrich L428s from the LN. Finally, these experiments were replicated with a primary human HL sample with 3.75% HRS cells pre-enrichment and 58.25% HRS cells post-filtration. This level of enrichment and DNA yield will be sufficient to accomplish downstream clinical assays including high-throughput sequencing, liquid biopsy, and fluorescence in situ hybridization. Clinically, this workflow may allow us to ask new questions about the initiation of Hodgkins and the driving factors leading to metastasis and relapse.