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IASLC ATLAS OF ALK TESTING IN LUNG CANCER
DNA quality. In addition to commercial products, published protocols for cell block construction are available, and three commonly used ones are the so-called cell button method, the sodium alginate method, and the plasma-thrombin Box 1. Protocols for Preparation of Cytology Cell Blocks method (Box 1) (Orell 2011, Kalhor Cell Button Method (Orell 2011, Kalhor 2013) 2013, Noda 2010, Jing 2013).
ALK FISH Analysis in Pulmonary Cytology
FISH is a robust technology that is applicable to almost all types and formats of cytologic specimens. The protocols and criteria for ALK FISH Sodium Alginate Method (Noda 2010) a. Suspend centrifuge-corrected fluid material and fix in analysis are identical to those for his 10% buffered formalin for 2-3 hours. tology. However, a significant subset b. Collect the pellet with fixed cells by centrifuge and of cell blocks contain too few or no decant off supernatant formalin and wash with cancer cells for molecular analysis distilled water. c. Centrifuge and resuspend the pellet with 0.5mL of (Knoepp 2013), and differentiating 1% sodium alginate. tumor cells from adjacent reactive d. Add sodium calcium (1M) to the solution and allow to cells is more challenging than in con gelatinate. ventional cytology, especially during e. Harvest the gelatinated material with forceps and process in the same manner as a small biopsy specimen. FISH analysis. Therefore, ALK analysis of cytologic specimens is another Plasma-Thrombin Method (protocol used at the valid option that is preferred by some University Hospitals of Basel and Zurich, Switzerland) laboratories (Figure 2) (Betz 2013, a. Centrifuge the cytologic material for 10 minutes at Savic 2013). An important advantage 2,500 rpm. b. Remove the supernatant. of conventional cytology is the ability c. Pipette two drops of the sediment into a small to select the optimal cytologic slide tapered tube. among all previously stained slides d. Add 200 ?l plasma, and vortex the specimen briefly. for FISH analysis, with no need for e. Add 50 ?l thrombin, and vortex the specimen briefly. f . Incubate the specimen for 5 minutes. additional unstained slides. In addi g. Put the clot into an embedding cassette (between two tion to a lack of nuclear truncation filter pads), and close the cassette. and related artifacts, the DNA quality h. Fix the material in 10% buffered formalin. i . Take out the fixed material and process it in the same in air-dried or alcohol-fixed cytologic manner as a small biopsy specimen. specimens is better than that after formaldehyde fixation, which leads to crosslinking and chemical modification of nucleotides. This fact provides an explanation for a success rate of up to 100% for ALK FISH analysis in conventional cytology and a failure rate of up to 19% for histologic specimens (McLeer-Florin 2011, Savic 2013). FISH is applicable to almost all types of cytologic specimens, including conventional smears, cytospins, or liquid-based preparations (e.g., ThinPrep, Hologic; or SurePath, BD Diagnostics) regardless of fixation type (air-dried and alcohol-based fixatives). The use of adhesive-coated or positively charged slides in lung cytology is recommended, as these slides improve the adherence of the cells and prevent them from floating off during technical FISH procedures. FISH works equally well Method 3 Method 2
a. Gently expel a drop of aspirated or cytospinned material on to a glass slide without spreading or smearing. b. After a few seconds for adhering, carefully immerse the slide in ethanol for fixation. c. Gently detach the fixed drop (like a button) with a scalpel blade and process in the same manner as a small biopsy specimen.
Method 1