TITLE: TMA-TNP  Section Map and Slide Processing - Phase 3
AUTHORS: Heidi S Feiler

[DESCRIPTION]
Human Tumor Atlas Tissue MicroArrary TNP  (TMA-TNP)

The Tissue MicroArray (TMA) TNP will extend the  SARDANA-TNP characterization and analytics methodologies for evaluation and validation on a large array of breast tumor samples that provide a broad spectrum of disease state and subtype. A commercially available anonymized breast tumor TMA was purchased and sections distributed to participating HTAN Centers. Some deidentified basic clinical data will also be provided. The participating HTAN Centers will characterize the FFPE specimens (e.g. by imaging) and generate a spatially resolved cell type/state census using each Center’s method of choice. Data will then be recorded in a common repository to enable joint analysis.

This TNP has two specific Aims focused on (i) Data Collection and (ii) Data Coordination and Analysis. Individual Centers can participate in one or both aims.

The custom TMA design for this project was generated by Dr. Koei Chin (OHSU) and sent to Pantomics for custom TMA FFPE block generation and sectioning.  The TMA design and clinical sample descriptions can be found here:  
dx.doi.org/10.17504/protocols.io.bn2fmgbn .

This protocol describes the procedure by which the OHSU OMS Atlas HTAN Center sectioned and distributed TMA sections for Phase 3 analysis.

[STEPS]
SECTION: Preparation
1. Verify the identity of the FFPE block to be cut against written request for sectioning.  The FFPE block (TMA1) will be utilized for TMA-TNP Phase 3.
SECTION: Preparation
2. Each slide was labeled with a  unique OHSU Slide ID corresponding to the FFPE section map (below).

                   Slide ID            Section #  Description      Institution  Thickness (μm)    OHSU_TMA1_000  0  QC H&E Pantomics    OHSU  4    OHSU_TMA1_001  1  cmIF (G3B Panel) Cyclic   Immunoflourescence  OHSU  4    OHSU_TMA1_002  2  cmIF (G3A Panel ) Cyclic   Immunoflourescence  OHSU  4    OHSU_TMA1_003  3  H&E      OHSU  4    OHSU_TMA1_004  4  t-CycIF (Tumor Panel)   Cyclic Immunoflourescence  HMS  4    OHSU_TMA1_005  5  t-CycIF (Immune Panel)   Cyclic Immunoflourescence  HMS  4    OHSU_TMA1_006  6   mIHC Multiplex IHC (Discovery Panel)  OHSU  4    OHSU_TMA1_007  7  cmIF (G3A Panel) Cyclic   Immunoflourescence  OHSU  4    OHSU_TMA1_008  8  cmIF (G3B Panel) Cyclic   Immunoflourescence  OHSU  4    OHSU_TMA1_009  9  H&E      OHSU  4    OHSU_TMA1_010  10  t-CycIF (Tumor Panel)   Cyclic Immunoflourescence  HMS  4    OHSU_TMA1_011  11  t-CycIF (Immune Panel)   Cyclic Immunoflourescence  HMS  4    OHSU_TMA1_012  12  mIHC Multiplex IHC   (Discovery Panel)  OHSU  4    OHSU_TMA1_013  13  cmIF (reserve) Cyclic   Immunoflourescence  OHSU  4    OHSU_TMA1_014  14  cmIF (reserve) Cyclic   Immunoflourescence  OHSU  4  
 

SECTION: Sectioning
3.  Align block on microtome to minimize tissue loss.
SECTION: Sectioning
4. Face into block at 5μm until full section of tissue is achieved.
SECTION: Sectioning
5. Cut adequate ribbon at 5μm to cover all serial sections.
SECTION: Sectioning
6.  Mount tissue sections onto Tanner slides to maintain serial order and orientation of sections.  Sections for Nanostring must be placed in a shifted orientation to capture the cores of interest (2 sections/slides to be combined for full representation of TMA1).  
SECTION: Slide Processing and Shipping
7. All Slides are immediately baked in a 45 °C oven  (~45-48C) for 2 hours prior to distribution.

SECTION: Slide Processing and Shipping
8. In the lab, the slides are baked in an oven at  55 °C  720 min (except for #30-33 for Nanostring) , and then at  65 °C  for 30 min

Note: Slides should be baked at 65 °C for at least 30 minutes.

The  slides are stored in  a 4 °C  cold room until deparaffinization or shipping.
SECTION: Slide Processing and Shipping
9. Slides were shipped  on 4 °C  ice packs by express shipping per the FFPE spatial map noted above.