Fine Needle Aspiration Biopsy (FNA) Techniques - Dr. Britt Marie Ljung
PURPOSE Toluidine blue can be used as a temporary stain for Fine Needle Aspiration direct smears serving during ROSE (Rapid OnSite Evaluation) for adequacy check and preliminary diagnosis in a subset of cases at the time of sampling. It provides excellent nuclear detail and also stains the cytoplasm. Toluidine blue is also the preferred stain for epoxy embedded material typically used for electron microscopy. REFERENCES •Dawes, C.J., Biological Techniques in Electron Microscopy, Barnes & Noble, 1971. Section 10.6, page148 SAFETY Wear gloves, lab coat and apron when preparing this stain. The stain powder must be handled carefully to prevent dusting of surrounding lab equipment. MATERIALS AND EQUIPMENT •2 x 1 L brown-glass bottles •Stir plate and stir bar •Filter paper Whatman No. 1 •120mm Funnel REAGENTS •5 gm Toluidine Blue O (EM Sciences #22050) •2.5 gm Sodium bicarbonate (Fisher #BP 328-) or Sodium borate •500 ml distilled water PROCEDURE 1. Measure 500 ml of distilled water into a 1L brown bottle with stir bar and place on stirring plate. 2. Weigh Toluidine Blue O and transfer into the water while it is stirring. 3. Add Sodium bicarbonate. 4. Stir 3-4 hours. Let settle at room temperature overnight. 5. Filter stain into a clean brown bottle using Whatman No 1 filter paper. 6. Label and assign a one-year expiration date. Store at Room Temperature. 7. Filter again just before use. If using frequently after filtering, fill a small bottle with a narrow neck suitable for dispensing a few drops or a container with a fitted dropper. Keep container in biopsy procedure room or carry to radiology suite etc. Label small container with expiration date and refilter as needed based on appearance of stain when used. 8. Immerse smear in alcohol based fixative for 10 -20 seconds. 9. Remove smear from fixative. 10. Place smear on flat absorbent material for example paper towel. 11. With dropper or narrow spout place one to two drops of stain solution on smeared material. 12. Cover with coverslip large enough to cover smeared area. 13. Let stain penetrate for about 15 seconds, a bit longer if there are large/fibrous appearing clusters in the smear. 14. Turn slide over to gently blot out surplus stain. 15. Dry off bottom of slide to avoid adherens to microscope stage. 16. Examine slide. 17. Return slide to alcohol fixative container. The cover slip will fall away from the slide and the stain with dissolve almost completely in the alcohol. Use care when removing stained slide, use tweezer. 18. Stain with Papanicolaou stain using routine protocol, or other stains typically used for alcohol fixed smears. 19. There may be slight alteration of coloration in the final permanent stain due to the exposure to Toluidine stain. Results Nuclei Dark blue Cytoplasm Light blue Glycogen deposition Violet Myelin Blue/black CLEANING AND MAINTENANCE Remove toluidine blue stains with alcohol-wetted gauze or paper towel.