Cell block and liquid based cytology

• 1. Points to be discussed Cell block- its advantages/disadvantages and preparation methods and applications. Liquid based cytology-its uses/disadvantages. Role of liquid based preparation in cervical cytology Bethesda system for reporting cervical cytology

2. What is a cell block???? 3. Salvaging of grossly visible flecks of tissue or sediment Fixation by formalin or any other fixative Paraffin embedding Cutting Staining 4. Cell blocks offer the opportunity to examine the histological structure and allows the use of ancillary tests. Cytology Histopathology Bridge Cell blocks 5. Advantages of cell block Slides are more readily interpretable by histopathologists Availability of a block facilitates more sections. Imp residual material is salvaged which is generally not available in cytology smears .Loose cells, cell aggregates and microscopic tissue fragments are easily recoverable. Concentrated in a small area of the slide so examination less time consuming Special stains mucicarmine, congo stain, melanin etc 6. Stains for immunocytochemistry For microorganisms esp fungi and bacteria Pattern and architectural recognition of tumor possible CB is simple ,reproducible and readily available in routine laboratory No necessity of biopsy Storage of cell blocks is easy than unstained slides 7. Disadvantages Compared routine smears takes longer time Sparse cellularity Distortion artifacts 8. Specimen FNA material Sputum Effusion fluids Urine sediment Various washings and lavages 9. Material needed 10. Methods of cell block preparation Direct processing of tissue fragments present in fluids Fixed sediment method Bacterial agar method Simplified cell block technique using alcohol, acetone and paraffin Compact cell block technique Plasma thrombin method Cell blocks from millipore 11. Histogel method Gelatin embedding Celloidin bag Cell block preparation from scraping of cytology smears Automated cell block preparation Albumin method 12. . 13. Residual clot /tissue in the hub of the needle is carefully removed & rinsed in 50% ethanol Centrifuged at 4000rpm for 6min in a 10ml disposable to form pellets Supernatant is discarded & fixative is added to the deposit After 1 hr deposit is taken and put into filter paper & processed as regular paraffin specimen and then stained routinely by H&E 14. Cytologic studies of body fluids are done to gain knowledge about the etiology of effusion. Most common reason for evalaution is to determine whether or not it contains malignant cells. Cytologic evaluation is the best method to detect malignancy in body cavity fluids Diagnostic accuracy for malignancy ranges from50-80 % with a false negative rate of about 25%, to increase diagnostic accuracy, cell block preparation is done Cell blocks in body fluids 15. Few advantages: Spontaneous formed clot in the body fluid may enmesh virtually all cells Induced clot prepared from the sediment obtained by centrifugation of the fluid specimen would also contain many cells .Useful in haemorrhagic fluids Identification of primary site of malignancy can be enhanced with the use of cell block technique Histologic patterns of cancer(acinar ,papillary ,duct like)can be readily identified on cell block. 16. Psammoma bodies ,granulation tissue ,cholestrol clefts ,microorganisms, fragments of collageous stroma, hyperpalstic mesothelium,etc can be easily identified easily. Fluid samples or tissue fragments can be preserved in a refrigerator for 72 hrs before processing in cell blocks. IHC on cell blocks are comparable to that of surgical specimen Microarray technique, Molecular tests like FISH ,In situ PCR. Electron microscopy. 17. Role of cell block in non neoplastic effusion Mesothelial cells are often misinterpretated as metastatic adenocarcinoma Differentiation between polymorphous lymphocytes of non neoplastic effusion & monomorphous lymphoid cells of lymphoproliferative diseases Rheumatic effusion(elongated & giant cell multinucleated histiocytes ,granular background , cholestrol crystals) L E cells, granulomas in TB, number of parasitic, fungal and viral diseases can be picked up 18. Cell blocks in neoplastic effusion Adenocarcinoma Signet ring cell carcinoma Small cell anaplastic carcinoma Mesotheliomas 19. Cell block is simple, reliable technique suitable for all types of cytology specimens & does contribute to better final cytological diagnosis and so should be considered wherever possible. 20. Liquid Based Cytology 21. Liquid based cytology It means cytology (study of cells )through a liquid medium. Cells are collected from cervix(any other site) are placed directly into liquid preservative, rather than transferred to slide. Sample is processed and resultant thin smear easy to screen. 22. Samples to Process Exfoliative samples Urines Sputum Bronchial washings Bronchial brushings Body fluids Aspiration samples FNA Head and neck FNA Lymph nodes FNA Lung FNA Liver FNA Breast 23. Advantages l Simplifies the collection process for the smear taken Reduced inadequate rate Cells better preserved and not obscured by blood, mucus, or inflammatory cells Infectious organisms retained and better preserved Quicker to screen and report Multiple slides can be produced allowing further testing Residual material in the vial can be made available for further tests eg; HPV tests Facilitates computer assisted screening which can be available in future(automated cytology) 24. More costly than conventional pap smears Preparation is more labor intensive than conventional Loss of background material Some differences in architecture and morphology Requires training for both the screeners and the smear takers Disadvantages 25. Conventional Pap Smear Overcoming the Inherent Limitations of the Conventional Pap Smear Majority of cells not captured Non-representative transfer of cells Clumping and overlapping of cells Obscuring material Virtually all cells of sample are collected Randomized, representative transfer of cells Even distribution of cells Minimizes obscuring material Liquid-based Cytology* 26. Conventional smear 27. What we see under the microscope conventional smear 28. The prepared slide with the new ThinPrep 29. What we see under the microscope. Notice the clean back ground and how well the cells are dispersed rendering easier to interpretation. 30. Liquid based cytology techniques Thin Prep: Filtration and collection of vacuum packed cells on a membrane & transferring to glass slide Sure Path: Centrifugation & sedimentation through a density gradient 31. Thin prep Path sure 32. Thin prep 33. 1. Dispersion 2. Cell Collection 3. Cell Transfer Principle 34. Path sure 35. Morphology Clean background Cells are shrunken ,more circular Cytoplasmic and nuclear features are more easily identified Cells with Low grade dyskaryosis ,especially koilocytosis Lower nuclear hyperchromasia Glandular neoplasms show characteristic picture in thin film cytology 36. Adequacy Conventional pap: 8,000-12,000 squamous cells Liquid based cytology: 5000 squamous cells 37. Screening methods Area to be sceened is smaller & circular. Abnormal cells may be located anywhere and it should cover 100% of deposit & allow for overlap Screening is more intensive, 10 x and 40 x Screener must take proper breakes not exceeding 5 hrs/day. 38. Bethesda System 2001 Nomenclature of cervical cytology on pap test. 39. Bethesda System 2001 Specimen Type: Indicate conventional Pap smear vs. liquid-based vs. other Specimen Adequacy Satisfactory for evaluation (describe presence or absence of endocervical/transformation zone component and any other quality indicators--e.g., partially obscuring blood, inflammation, etc.) Unsatisfactory for evaluation (specify reason) Specimen rejected/not processed (specify reason) Specimen processed and examined, but unsatisfactory for evaluation of epithelial abnormality because of (specify reason) 40. General Categorization Negative for intraepithelial lesion or malignancy Epithelial cell abnormality: See interpretation/result (specify squamous or glandular as appropriate) Other: See interpretation/result (e.g., endometrial cells in a woman 40 years of age) Automated Review: If case examined by automated device, specify device and result Ancillary Testing: Provide a brief description of the test methods and report the result so that it is easily understood by the clinician Bethesda System 2001 (continued) 41. Interpretation/Result Negative for Intraepithelial Lesion or Malignancy (when there is no cellular evidence of neoplasia, state this in the General Categorization above and/or in the Interpretation/Result section of the report, whether or not there are organisms or other non-neoplastic findings) Organisms Trichomonas vaginalis Fungal organisms morphologically consistent with Candida spp Shift in flora suggestive of bacterial vaginosis Bacteria morphologically consistent with Actinomyces spp. Cellular changes consistent with Herpes simplex virus Other Non-Neoplastic Findings (optional to report; list not inclusive): Reactive cellular changes associated with Inflammation (includes typical repair) Radiation Intrauterine contraceptive device (IUD) Glandular cells status post hysterectomy Atrophy Bethesda System 2001 (continued) 42. Bethesda System 2001 (continued) Other (list not inclusive) Endometrial cells (in a woman 40 years of age) (specify if negative for squamous epithelial lesion) Epithelial Cell Abnormalities Squamous Cell Atypical squamous cells Of undetermined significance (ASC-US) Cannot exclude HSIL (ASC-H) Low-grade squamous intraepithelial lesion (LSIL) Encompassing: HPV/mild dysplasia/CIN1 High-grade squamous intraepithelial lesion (HSIL) Encompassing: moderate and severe dysplasia, CIS/CIN2 and CIN3 With features suspicious for invasion (if invasion suspected) Squamous cell carcinoma 43. Bethesda System 2001 (continued) Glandular Cell Atypical Endocervical cells (NOS* or specify in comments) Endometrial cells (NOS or specify in comments) Glandular cells (NOS or specify in comments) Atypical Endocervical cells, favor neoplastic Glandular cells, favor neoplastic Endocervical adenocarcinoma in situ Adenocarcinoma Endocervical Endometrial Extra uterine NOS Other Malignant Neoplasms: (specify) * NOS = Not otherwise specified 44. The Bethesda System 2001 LSIL = HPV / mild dysplasia / CIN1 HSIL = moderate and severe dysplasia / CIS / CIN2 and CIN3 ASCUS = ASC-US (undetermined significance) or ASC-H (cannot exclude HSIL) 45. Bethesda 2001 Changes SBLB eliminated Unsatisfactory: specimen rejected/not processed; or specimen processed/examined, but unsatisfactory because of (specify reason) WNL and BCC are now Negative for Intraepithelial Lesions or Malignancy; includes BCC (e.g., organisms and reactive changes) as descriptor only The multiple subcategories of ASCUS have been reduced to ASC- US or ASC-H, with no other modifiers The subcategories of AGUS (now AGC) have been expanded to allow for a more descriptive diagnosis of glandular abnormalities; AIS is now a distinct subcategory 46. Unsatisfactory 47. Satisfactory 48. Actinomyces 49. ASC-US 50. Low grade-sqamous intraepithelial lesion(LSIL) 51. High grade squamous intrepithelial lesion(HSIL) 52. Squamous cell carcinoma (keratinising) 53. Squamous cell carcinoma (non keratinising) 54. Atypical endocervical cells Atypical endometrial cells 55. Adenocarcinoma (endocervical) 56. Adenocarcinoma (endometrial) 57. Adenocarcinoma (extrauterine) 58. REPORTING FOR CERVICAL CYTOLOGY Specimen adequacy Presence or absence of TZ zone Other quality indicators Interpretation/Result Negative for intraepthelial lesion or malignancy: organisms (T.vaginalis, Candida ,Actinomyces, Lactobacilli & bacterial vaginosis), other non neoplastic findings (inflammation ,radiation changes, IUD), atrophy, glandular cells Endometrial cells > 40 yrs of age Epithelial abnormalities Squamous cell Atypical squamous cells: ASC-US and ASC-H LSIL(low grade squamous intraepithelial lesion) HSIL(high grade squamous intraepithelial lesion) SCC(squamous cell carcinoma) Glandular cells Atypical : Endocervical, Endometrial, Glandular Endocervical adenocarcinoma in situ Adenocarcinoma: Endocervical, Endometrial ,extrauterine, NOS 59. References Bales CE .Cytologic techniques. In Koss LG, editor.Diagnostic cytopathology and its histopathological bases,4th ed.Philadelphia: J B Lippincort & company,1992,vol2p1472-74. Tapar M, Mishra RK et al. Critical analysis of cell block versus smear examination in effusions .J Cytol 2009;26:60-64. Marluce Bibbo, Comprehensive cytopathology;2nd ed .Saunders.1997.p. Massima Pignatelli, James C. E Underwood. Recent advances in Histopathology.22.p127-144. Diane Solomon, Ritu Nayar. The Bethesda System for reporting Cervical cytology.2nd ed.Springer.2004.