Expected Output

Output data contains the necessary data for image visualization and advanced analysis. The two classes of data files most important to the CODEX® Multiplex Analysis Viewer (MAV) are the FCS expression data and TIFF tile image data.

FCS Files

Example compensated FCS data from Human FFPE

Flow Cytometry Standard (FCS) is a file standard specified by the International Society for the Advancement of Cytometry Data Standards Task Force, last updated to FCS 3.1 in 2011. Publication is available here. CODEX® outputs FCS files in addition to CSV files for compatibility with many flow cytometry analysis platforms.

CODEX® FCS files contain integrated expression intensity of all markers for all cells. For each region, there are two FCS files, compensated and uncompensated. Compensated FCS file contains expressions compensated for spatial fluorescence spillover, while uncompensated FCS file contains the raw integrated expressions. In addition to marker expression, FCS files contain cell identifiers and metadata, such as cell size and location.

FCS files can be exported from CODEX® MAV.

Tile Images

Hu Tonsil FFPE / reg001_X01_Y01_t005_z005_c001.tif (DAPI)

Tile images are processed from the raw image data and organized into a folder structure compatible with CODEX® MAV. Within the 'tiles' folder, each folder contains all images corresponding to the region and tile location, 'reg001_X01_Y01'. Individual tile images are named 'reg001_X01_Y01_t001_z001_c001.tif', denoting region number, tile x-index, tile y-index, cycle number, z-slice number and channel number, respectively. Tile images are pseudo-colored white, blue, green and red, based on their respective channel number.

Stitched Images

Hu Tonsil FFPE / reg001_cyc005_cha001_DAPI5.tif

In addition to FCS and tile images, the processor creates stitched images for visualization outside of CODEX® MAV. Within the 'stitched' folder, each 'reg001' folder contains stitched image for each marker generated from best focus z-slices. Additionally, individual stitched images combined into a full stitched image.

reg001_cyc001_ch001_DAPI-1.tif
   |      |     |   |
   |      |     |   └ Biomarker name
   |      |     └ Channel number
   |      └ Cycle number
   └ Region number

All numbers are 1-index.

Hu Tonsil FFPE / seven markers

CD20 (Red) / CD21 (Green) / CD4 (Blue) / CD8 (White) / Pancytokeratin (Cyan) / SMA (Magenta) / CD15 (Yellow)