Preserved bronchoalveolar lavage (BAL) cells on cytospin slides are laborious to collect and deserve to undergo quantifiable, high throughput analysis that also considers the four primary cell types of BAL: macrophages, neutrophils, eosinophils and lymphocytes. Therefore, for the first time, we used the NanoStrings GeoMx spatial analysis to quantify a multiplex of proteins per cell type on cytospin slides.

To date, BAL cytospins had not been used in this tissue-section optimised equipment, therefore there was some concerns including: pre-preservation of the cells, the height of the spun cells relative to the collection probe, cell permeabilization, cell density for the minimum area collected and, post-preservation of the stained slide.

Testing showed that methanol preserved slides gently permeabilised with Tween-20 (without formalin fixation nor antigen retrieval) were viable for the GeoMx equipment. We found three morphology markers that specifically stained the macrophages (CD68(KP1)-AF647, santa cruz), neutrophils (NE(950334)-AF532, novus bio) and eosinophils (Siglec-8-AF594, clone#837535, R&D). Lymphocytes were collected as ?the rest? of the SYTO-13 stained nuclei. For protein panel analyses, pooling of several maximum sized collection areas per cell type provided sufficient collection area for all 40 protein targets tested.

By determining the morphology stains and adjusted staining protocols for cytospins of BAL cells, NanoString GeoMx can potentially assess up to 80 surface and internal proteins for each primary cell type. Potential analysis of BAL cytospin slides from healthy, mild asthma and severe asthma are currently under investigation using this technique.