ProMark Sample Preparation

TISSUE REQUIREMENTS

ProMark, a proteomic prognostic biopsy test run on unstained tissue sections, is compatible with standard formalin fixed, paraffin-embedded (FFPE) biopsy tissue. Tissue should be fixed in 10% neutral buffered formalin and processed with a standard ethanol/xylene/paraffin sequence. The use of eosin or other tissue penetrating stains during tissue processing is not compatible with ProMark. Microwaved tissue is not compatible with ProMark. If biopsy tissue color contrast is desired when embedded in the paraffin for cutting, a simple and quick ink staining method conducted before FFPE processing is recommended, as described below.

Patient inclusion criteria

Patients with biopsy Gleason scores of 3+3 or 3+4 are eligible. Five (5) consecutive sections from one tissue block with minimally 1mm tumor area are required. The block should have biopsy cores with the highest Gleason pattern. If the Gleason pattern is the same across cores, use the block with the largest tumor area.

Tissue processing requirements

• FORMALIN FIXATION: 10% NBF (neutral buffered formalin). Do not use a microwave.
• TISSUE PROCESSING: Use standard FFPE processing protocols. Do not use a microwave.
• DYES AND INKS: Do not use tissue-penetrating dyes (eosin, floxine, methylene blue, etc) during fixation or tissue processing. See alternative ink staining protocol below.
• CUTTING SECTIONS: Do not pre-treat FFPE blocks with any chemicals before cutting. Water soaking is fine. Use care in cutting to avoid local folding, wrinkles, or knife marks on tissue sections. Do not use additional reagents in the water bath (H2O only). Use only 5 um sections, one section per slide, placed in the center of each slide.
• SPECIAL SLIDES: TRUBOND 380 slides are required for tissue mounting and provided by Metamark (as part of the ProMark kit). Further details can be provided upon request.

Protocol for biopsy staining with ink (optional pre-processing staining step)

1. Prepare an ink solution (see list below) 1:50 in distilled water or 1:5000 in 70% EtOH.

2. Dip the fixed biopsy core for 3-5 seconds into the ink solution.

3. Briefly touch the stained biopsy core to a paper towel to remove excess ink.

4. Dip the core into 70% ethanol for a few seconds.

5. Place the core into a tissue processing cassette and process as usual for FFPE.

RECOMMENDED INKS (American Master Tech: #STD2227BL, #STD2227BK, #STD2227GR; Cancer Diagnostics Inc: Green - #0728-3; Davidson Marking System: Black – #3408-3, Green – #3408-1)

If cores are already placed into cassettes

1. Place cassettes with cores into a basket and dip into an ink solution for ~1 minute.

2. Place the basket on paper towels to remove excess ink.

3. Dip the basket several times

Also look for sample preparation instructions, included in your ProMark kit.

Questions? We’re happy to help.

• Contact your local Metamark representative
• Call our Client Services team at 1 (877) 743-3338

For information on lab verification, please contact your local Metamark representative or call our Lab Verification Team at: 1 (617) 721-8698.