Learn Fine Needle Aspiration (FNA) techniques from a leader in the field of Cytology.
By studying this video you will get guidance on materials to use and how to best position the patient and the operator. Included are illustrations of how to handle the US probe, including pitfalls, position for palpation guided sampling, effective application of local anesthesia, placement of the needle for palpation guided sampling and in relation to the US-probe for image guided FNA. The principles of effective placement and manipulation of the needle during sampling are discussed and illustrated using animations. Triaging of samples based on Rapid OnSite Evaluation are discussed with examples of cell blocks used for IHC and flow cytometry for lymphoma etc. Detailed illustrations of sampling preparation with focus on quality smears, optimal use of the harvest including for cell block, flow cytometry and molecular markers.
Advantages of FNA 0:38
Equipment 2:52
Equipment Checklist 4:45
Rapid On Site Evaluation (ROSE) 5:16
Palpation Guided FNA 7:44
Sampling Highly Vascular Targets 9:52
FNA Sampling Procedure 10:51
Suction vs No Suction Sampling 16:17
Ultrasound Guided FNA 18:03
Ultrasound Guided FNA - Parallel Needle Placement 20:06
Ultrasound Guided FNA - Perpendicular Needle Placement 21:05
Pitfalls of Ultrasound Guided FNA 22:48
Thyroid Ultrasound Guided FNA 24:13
Distribution and Triage 26:01
Troubleshooting 27:14
Preparing the Sample 28:14
Dividing and Smearing Technique 29:38
Flip Technique 32:40
Closing 35:28
To view the video on how to prepare/process a cell block, click this link: • UCSF Collodion Bag Cel...
TOLUIDINE STAIN PROTOCOL
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.
16 июл 2024
