Overview of FNA procedure- Strengths, weaknesses, and training S. K. Navale Medical College, Pune, India. Dec 23, 2011

1. Vinod B. Shidham , MD, FRCPath, FIAC Professor Co-editor-in-chief & Executive editor, CytoJournal (www.cytojournal.com) Vice-chair - AP Director of Cytopathology, Residency training program, Cytotechnology School, Cytopathology fellowship, & GI fellowship Dept of Pathology, Wayne State University Medical School Karmanos Cancer Institute & Detroit Medical Center Detroit, MI 48201, USA [email_address] Overview of FNA procedure - Strengths, weaknesses, and training S. K. Navale Medical College, Pune, India Dec 23, 2010

4. Acknowledgement Shidham & Atkinson Cytopathologic Diagnosis of Serous Fluids Elsevier (W. B. Saunders Company) Some of the sketches and tables used are from the following reference.

5. Introduction FNAB has two important components- Performing the procedure & Interpretation of the aspirated specimen

6. Introduction With following two important considerations Onsite adequacy evaluation- with appropriate triage for appropriate ancillary tests Preferably the performer and interpreter is same to allow benefits of continuity of the process from clinical insight into the lesion to cytomorphologic scrutiny . If the performer and interpreter can not be the same all efforts and resources should be available to achieve continuity amongst the entities involved. Any compromise will lead to suboptimum results in long run.

7. Onsite adequacy evaluation- with appropriate triage For appropriate ancillary tests Preferably the performer and interpreter is same To allow benefits of continuity of the process From clinical insight into the lesion To cytomorphologic scrutiny If the performer and interpreter can not be the same (due to logistics or local conditions)- Resources should be available to achieve the continuity Any compromise risks suboptimum results in long run Introduction (contd) With following two important considerations

8. Performing the procedure is relatively simple if appropriate sequences are followed with strict order with proper application and release of vacuum as needed Although simple, being a skill it demands training and practice Introduction (contd)

9. Cytopathology (FNA Biopsy)

10. Cytopathology (FNA Biopsy)

11. Surgical pathology (Core Biopsy)

12. Surgical pathology (Core Biopsy)

13. Surgical pathology (Core Biopsy) Tissue section 4 to 6 micron section

14. Cytopathology (FNA Biopsy)

15. Cytopathology (FNA Biopsy)

17. Aspiration of loose cells

18. Aspiration of loose cells

19. Aspiration of loose cells

20. Aspiration of loose cells

21. Aspiration of loose cells

22. Aspiration of loose cells

23. Aspiration of loose cells

24. Aspiration of loose cells

25. Aspiration of loose cells

26. Aspiration of loose cells

27. Aspiration of loose cells

28. Aspiration of loose cells

29. Aspiration of loose cells

30. Aspiration of loose cells

32. Smear Cytopathology (FNA Biopsy)

34. FNA Biopsy Critical steps 2. Apply vacuum 1. Locate the lesion, insert the needle tip 3. Maintain vacuum & sample different areas of the lesion by inserting back & forth 4. Release the vacuum completely by releasing the syringe piston 5. Remove the needle

36. Pancreatic mucinous cyst (Pap stained LBC ) DD - Mucinous cystic neoplasm (MCN) versus Intraductal papillary mucinous neoplasm (IPMN)

37. From: Shidham VB and Atkinson BF. Cytopathologic Diagnosis of Serous Fluids , Elsevier (Saunders) 2007 (ISBN-13: 9781416001454). Preparation of direct smears.

38. Tissue section Wet-fixed smear Air-dried smear Effect of processing on cell sizes and details

41. Processing of Smear

42. Processing of Smear

43. Processing of Smear

44. Air-dried Wet-fixed

45. Air-dried Wet-fixed

46. Pancreatic ductal adenocarcinoma- Mod to poorly differentiated (Diff-Quik stain- Direct Smear)

47. Pancreatic ductal adenocarcinoma- Mod to poorly differentiated (Diff-Quik stain- Direct Smear)

48. Pancreatic ductal adenocarcinoma- Mod to poorly differentiated (Pap stain- Direct Smear-RH)

49. Pancreatic ductal adenocarcinoma- Mod to poorly differentiated (Pap stain- Direct Smear-RH)

51. Contamination & Sampling artifact

53. Gut lumen Biopsy needle Lesions to be sampled

57. FNA of cystic lesions. FNA of solid lesions. Potential of sampling artifact

59. Practicing FNAB on phantom lesion

60. How? Without risk to the patients Suitable approach to train & practice needed Phantom lesion described is easy to make Training FNAB procedure

61. Video Preparation of Phantom and practicing FNAB procedure Shidham V.B., Varsegi G.M., D’Amore K., Shidham A. (2009). Preparation and Using Phantom Lesions to Practice Fine Needle Aspiration Biopsies. JoVE. 31. doi: 10.3791/1404 Video article is available FREE on web as open access at- http://www.jove.com/index/Details.stp?ID=1404

62. Preparation of Phantom and practicing FNAB procedure 9.26 minutes demo video article FREE on web at- http://www.jove.com/index/Details.stp?ID=1404

63. Certificate of training & practice provided after the training prior to beginning FNAs on patients

65. Which is also relatively simple if appropriately trained as surgical pathologist with cytopathology training the physician component is the backbone from performing the procedure to final correlation with various clinical components. Ultimate aim is final interpretation of the aspirated specimen

91. Immunoprofile evaluation- Immunohstochemistry on cell block sections, Flowcytometry On cytospins and other cytology preparations (if required) Molecular studies for- Diagnosis, prognosis, and surveillance Retrieval of cells for other indications- Cell harvesting (tissue banking etc), Cell transplantation (islet cells etc) In addition to morphologic studies:

92. FNAB passes for Cell block

93. Shidham V.B., Hunt B., Jardeh S.S., Barboi A.C., Devata S., Hari P. (2010). Performing and Processing FNA of Anterior Fat Pad for Amyloid. JoVE. 44 . doi: 10.3791/1747 Video article is available FREE on web as open access at- http://www.jove.com/index/Details.stp?ID=1747

94. Performing FNAB predominantly for cell block FREE on web at- http://www.jove.com/index/Details.stp?ID=1747 9.41 minutes demo video article

95. Processing of FNA aspirate to be submitted to laboratory for Cell block Let the remaining aspirate clot in the syringe for 5 to 7 minutes (slightly longer than the clotting time). 1

96. Processing of FNA aspirate to be submitted to laboratory for Cell block Let the remaining aspirate clot in the syringe for 5 to 7 minutes (slightly longer than the clotting time). 1 Aspirate 10% formalin from the container in which the specimen is to be submitted for cell block processing. This dislodges the clot from syringe wall. 2

97. Processing of FNA aspirate to be submitted to laboratory for Cell block Let the remaining aspirate clot in the syringe for 5 to 7 minutes (slightly longer than the clotting time). 1 Gently and firmly remove the plunger of the syringe . 3 Aspirate 10% formalin from the container in which the specimen is to be submitted for cell block processing. This dislodges the clot from syringe wall. 2

98. Processing of FNA aspirate to be submitted to laboratory for Cell block Let the remaining aspirate clot in the syringe for 5 to 7 minutes (slightly longer than the clotting time). 1 Transfer the aspirated formalin with dislodged cot in to the specimen container with 10% formalin fixative 4 Gently and firmly remove the plunger of the syringe . 3 Aspirate 10% formalin from the container in which the specimen is to be submitted for cell block processing. This dislodges the clot from syringe wall. 2

99. HE stained cell block section Cell-block sections Important for immunocharacterization

100. GIST (Cell-block- HE & Immuno) HE CK S-100 SMMS vimentin CD117

101. Proper processing - Including direct smear preparation For PAP and Romanowski stains Proper triage - Based on clinical and on-site cytomorphologic evaluation Cell block (submit directly in 10% formalin ) Flow cytometry (& cytogenetics)- RPMI Microbiology cultures Electron microscopy ( 2.5% glutaraldehyde ) Molecular studies Onsite adequacy evaluation Onsite adequacy evaluation by a trained cytotech, pathologist, or cytopathologist is crucial component of FNA for:

103. Aspirate triage & processing Direct smears Air-dried Romanowski stain Pap stain (postfixed in 95% ethanol after saline rehydration)

104. Aspirate triage & processing Direct smears Air-dried Romanowski stain Pap stain (postfixed in 95% ethanol after saline rehydration) Wet-fixed in 95% ethanol (for PAP stain ) &/OR

105. Aspirate triage & processing Needle rinse Direct smears Air-dried Romanowski stain Pap stain (postfixed in 95% ethanol after saline rehydration) Pap stain (SurePPath) Pap stain (ThinPrep) OR other LBC fluid (weak fixative) Wet-fixed in 95% ethanol (for PAP stain ) &/OR

106. Aspirate triage & processing Needle rinse Direct smears Air-dried Romanowski stain Pap stain (postfixed in 95% ethanol after saline rehydration) Pap stain (SurePPath) Pap stain (ThinPrep) OR other LBC fluid (weak fixative) Wet-fixed in 95% ethanol (for PAP stain ) &/OR OR Needle rinses in isotonic fluid For Cytspins or smears by other methods from the centrifuged cell buttons

107. Aspirate triage & processing Needle rinse Cell- block Direct smears Air-dried Romanowski stain Pap stain (postfixed in 95% ethanol after saline rehydration) Pap stain (SurePPath) Pap stain (ThinPrep) OR other 10% Formalin PEFF cell-block HE & immuno- stained cell block sections LBC fluid (weak fixative) Wet-fixed in 95% ethanol (for PAP stain ) &/OR OR Needle rinses in isotonic fluid For Cytspins or smears by other methods from the centrifuged cell buttons

108. (contd) Aspirate triage & processing Flow cytometry Cytogenetics (RPMI) EM (2.5% glutaraldehyde)

109. (contd) For other tests such as Microbiology cultures Molecular tests Aspirate triage & processing Flow cytometry Cytogenetics (RPMI) EM (2.5% glutaraldehyde)

111. Summary Strength FNAB is well established, minimally invasive, low cost, simple procedure .

112. Weaknesses (Relative limitations) Although simple, being a skill it demands training and practice structured training and practice session is highly recommended for continued efficiency of this very promising and critical modality in patient care in this era with increasing incidence of cancer. Summary (contd)

113. Weaknesses (Relative limitations) Although simple, being a skill it demands training and practice structured training and practice session is highly recommended for continued efficiency of this very promising and critical modality in patient care in this era with increasing incidence of cancer. Precise execution of vacuum application & release is critical. On-site adequacy evaluation and proper triage is highly recommended - To avoid inadequate sampling To minimize chances of sampling artifact To prevent inappropriate processing with suboptimal results Summary (contd)

114. Weaknesses (Relative limitations) Although simple, being a skill it demands training and practice structured training and practice session is highly recommended for continued efficiency of this very promising and critical modality in patient care in this era with increasing incidence of cancer. Precise execution of vacuum application & release is critical. On-site adequacy evaluation and proper triage is highly recommended - To avoid inadequate sampling To minimize chances of sampling artifact To prevent inappropriate processing with suboptimal results Lack of continuity from performing to interpretation. Contamination of non-representative material such as- mucin and mucosal lining in EUS FNAs Sampling artifact due to difficult lesions (accessibility, technical limitations, sclerotic, necrotic etc). Summary (contd)

117. Peer-reviewed, open access,

118. Peer-reviewed, open access, teaching material with many pictures.

119. Peer-reviewed, open access, teaching material with many pictures. Hard copy and online availability.

120. Peer-reviewed, open access, teaching material with many pictures. Hard copy and online availability. Opportunity for frequent updates

121. Q/A

122. Detroit [email_address] Thank you