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Cytology Protocols

  • Histological and cytological specimens should be reviewed jointly to select the most appropriate specimens for biomarker analysis
  • ALK analysis (IHC and FISH) is applicable to both cytospins/smears and cell blocks
  • ALK analysis of cytospins and liquid-based specimens requires different technical protocols to those used with cell blocks and histology

Dietel M et al. Thorax. 2016;71:177-84.

 

Detailed protocols

ALK IMMUNOCYBIOCHEMISTRY ON CYTOLOGY ON BOND-MAX IMMUNOSTAINER
(LEICA 5A4 ANTIBODY)

  • ICC was performed directly on cytology smears fixed in Delaunay solution and stained according to Papanicolaou. After uncovering the glass slides, the ICC reaction was performed using the automated immunostainer BOND-MAX according to the manufacturer’s recommendations with minor modifications (Leica Microsystems, Wetzlar, Germany).
  • Briefly, the slides were pre-treated in Epitope Retrieval Solution 2 for 5 minutes at 100°C and then incubated with the mouse monoclonal antibody for ALK (dilution 1:50) for 30 minutes, at room temperature (Novocastra™, Clone 5A4, Leica Biosystems, Newcastle Upon Tyne, United Kingdom). Antibody binding was detected using the Bond Polymer Refine Detection Kit, which through small multifunctional linkers enhances cell penetration and detection sensitivity, with 3,3’-Diaminobenzidine (DAB) as chromogen (Leica Microsystems, Wetzlar, Germany).
  • FFPE cell blocks were sectioned at 4 μm thickness and processed in a similar way to cytology smears, except for an additional deparaffinisation step, a longer prep-treatment (30 minutes) and a higher antibody concentration (dilution 1:10) with a shorter incubation (15 min).
  • Cytospins and cell blocks sections of an ALK-rearranged and FISH-validated cell line (H3122) were used as positive controls.

Savic S et al. J Thorac Oncol. 2013;8:1004-11.

 

BASEL (SWITZERLAND) PROTOCOL FOR FISH ON CYTOLOGICAL SPECIMENS
(PAPANICOLAOU-STAINED SMEARS OR CYTOSPINS); VYSIS ALK BREAK-APART ASSAY

Day 1:

  • Place the uncovered slide(s) for 2 minutes in 2xSSC at 73°C in a microwave
  • Place slide(s) for 30 minutes in 0.5 mg/Im Pepsin in 0.01N HCL at 37°C in a microwave
  • Place slide(s) for 5 minutes in formalin 1%/100X MgCI2 (25 ml 10% formalin, 74 ml PBS and 1 ml 2M MgCI2) at room temperature (RT)
  • Place slide(s) for 5 minutes in PBS at RT
  • Place slide(s) for 1 minute in 70% ethanol at RT
  • Place slide(s) for 1 minute in 80% ethanol at RT
  • Place slide(s) for 1 minute in 100% ethanol at RT
  • Let slide(s) dry for 10 minutes at RT
  • Warm up heating plate to 45°C
  • Place slide(s) for 2 minutes on the heating plate
  • Apply 10 μl ALK probe “ready to use” to the target area of the slide
  • Cover area with coverslip (20x20 mm)
  • Seal slide(s) with rubber cement or parafilm
  • Place slide(s) on the surface of the HYBrite™ (or ThermoBrite™) denaturation/ hybridisation system. Fill in empty slots with blank glass slides
  • Close the lid of the HYBrite™ and start the following programme:
    - Co-denaturation: 8 minutes at 74°C
    - Hybridisation: overnight at 37°C

Day 2:

  • Warm up heating plate to 37°C
  • Heat up water bath to 73°C together with a coplin jar containing 2xSSC/0.3% NP40 wash solution
  • Remove the rubber cement or parfilm and the coverslip from the slide(s)
  • Place slide(s) in the 2x SSC/0.3% NP40 at 73°C. When all slides are in the coplin jar incubate for 2 minutes
  • Remove the slide(s) from the wash solution and place them in a coplin jar containing 2x SSC/0.1% NP40 and incubate the slides for 1 minute at RT
  • Remove the slide(s) from the wash solution and place them vertically in the dark on a paper towel for drying
  • Apply 10 μl DAPI I counterstain onto the target area and place a 24x32 mm coverslip over the DAPI solution, avoiding air bubbles

Direct smears

 

Thin prep

Cell blocks