Cytotechniques

different methods of preparation of cytological samples for diagnosis .

1. CYTOTECHNIQUES presenter : dr. tousif moderator : dr. harish s permi

2. Introduction The best morphologic presentation obtained from any cytologic specimen requires an understanding of the factors that went into collecting and preparing the specimen. to reduce the specimen to a cellular presentation, which can be interpreted and diagnosed. Principle of cytotechnique

3. History of cytodiagnosis The first era - 19 century - exfoliated cancer cells had been described in all types of specimen 1861 Pharyngeal secretion Post mortem Keratinizing squamous cell carcinoma

4. History of cytodiagnosis The second era - era of development and expansion -recognized the importance of of wet fixation of cytological specimens - screening of cervical cancer Dr. George N Papanicolaou

5. History of cytodiagnosis The third era - era of consolidation - technique of FNAC - Diagnostic cytology and its histopathologic basis the fourth era - the Bethesda system of reporting cervical/vaginal cytology diagnoses Dr. Leopold G Koss

6. CYTOLOGY SPECIMENS 1 . peritoneal, pericardial and pleural fluids 2. CSF 3. Nipple discharge 4 . Bronchial brushings / washings 5 . Sputum 6. Gastric washings 7. Urine sediment 8. Prostatic secretions 9. Cervicovaginal ( paps ) smear

7. Types of cytology samples Exfoliative cytology Aspiration cytology Fine Needle Aspiration Cytology (FNAC) Body fluids

8. Exfoliative cytology It is the study of cells that have been shed or removed from the epithelial surface of various organs. wash smear scraping brushing

9. Bronchial wash

10. Brushing

11. Scraping

12. Fine needle aspiration cytology ( fnac )

13. Body fluids Pleural fluid Pericardial fluid

14. Body fluids CSF ascitic fluid

15. Evaluation of specimen : Collection Preparation pH Protein content Enzymatic activity Bacteria +/- Fresh material Prefixation of material ?????? Prefixatives

16. Fresh material ↑↑ mucus ↑↑ protein Low mucus or protein Sputum Bronchial aspirates Mucocoele fluid Pleural Peritoneal pericardial Urine CSF 12 to 24 hrs if refrigerated 24 to 48 hrs Without refrigeration 1 to 2 hour delay even if refrigerated

17. Prefixation of material Ethyl alcohol 50 % Saccomanno ’ s fixative Shandon mucolexx Cytolyt

18. Centrifugation If too much fluid is obtained,. If little amount of fluid is aspirated (few drops), or if the fluid is thick, the centrifuge doesn’t required. Centrifuged for 5 mins Sediment

19. Cytocentrifugation It is a special machine that performs a centrifuge and collection of sediment on the center of the slides. This procedure is useful for hypocellular specimen .

20. Principle Hydraulic force Centrifugal force to concentrate cells within a defined area a filter card between the chamber sample and the glass slide resulting in cell to slide adhesion

21. Cytocentrifugation Shandon cytocentrifuge I Shandon cytocentrifuge II Shandon cytocentrifuge III Wescor cytopro Hettick cytocentrifuge Leif’s centrifugal cytology buckets

22. Methods of Smear Preparation: streaking spreading pull apart touch or impression smear

23. STREAKING - Used for preparing mucoid secretions , vaginal secretions, sputum and gastric content -use a spatula, dissecting needle or applicator stick and streak in a zigzag fashion

24. SPREADING used for thick mucoid secretions smears of fresh sputum and bronchial aspirates

25. PULL APART - for serous fluids, concentrated sputum, and enzymatic lavage form the GIT, smears of urinary sediment, vaginal pool and breast secretions.

26.              TOUCH IMPRESSION Impression cytology being collected From a patient , using a sterile glass slide with polished edges.

27. Squash smear preparation fairly accurate, simple and reliable tool for rapid intra-operative diagnosis of central nervous system lesions. Based on two essential factors: Availability of very small tissue fragments & good preservation of fine cellular details. Not effected by edema, hemorrhage, necrosis & calcification.

28. Cell block It is a procedure to convert cell sediment in to paraffin block further pathological procedures can be performed like immunohistochemistry (IHC).

29. 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

30. Methods of cell block preparation Cell blocks from millipore Histogel method Gelatin embedding Celloidin bag Cell block preparation from scraping of cytology smears Automated cell block preparation Albumin method

31. procedures Centrifugation 2400 rpm Pour off supernatant Add 5 ml of methanol +formalin (9:1) methanol+formalin for 30 to 60 min Spin at 2400 rpm remove hardened cell button submit in a cassette

32. Fixation of Cytology Specimens

33. Fixation of Cytology Specimens fixation means : - prevention of degeneration of cells and tissue - preservation of cells as close as possible to the living state specific periods of time changes the physical and chemical state of the cells

34. An appropriate fixative for cytodiagnostic purposes should perform the following functions Penetrate cells rapidly Minimize cell shrinkage Maintain morphologic integrity Deactivate autolytic enzymes Replace cellular water Facilitate diffusion of dyes across cell boundaries Help cells adhere to a glass surface Provide consistent results over time

35. FIXATION METHODS Wet Fixation Wet Fixation with Air Drying Spray Fixation Liquid-based Fixation for Papanicolaou Tests Lysing Fixation for Bloody Samples Air drying

36. Wet fixation 95% Ethyl Alcohol (Ethanol) ideal fixative recommended dehydrating agent desired amount of cell contraction yield optimal chromatin detail characteristics 100 % ethanol similar effect on cells more expensive Ether alcohol mixture ether and 95% ethyl alcohol 1 : 1 excellent fixative, but ether 100% Methanol produces less shrinkage than ethanol more expensive 80% Propanol and Isopropanol cause slightly more cell shrinkage Denatured alcohol 90 parts of 95% ethanol + 5 parts of 100% methanol + 5 parts of 100% isopropanol .

37. Time of Fixation Minimum 15 minutes fixation Can be Prolonged several days or even few weeks If smears are to be preserved over a long period of time in alcohol, it is better to store them in capped containers in the refrigerator.

38. Coating fixatives Aerosols or liquid base Dual action Carbowax (Polyethylene Glycol) fixative. Diaphine fixative Spray coating fixative (Hairspray) have practical value in situations where smears have to be mailed to a distant cytology laboratory for evaluation not recommended for bloody smears alcohol base - fixes the cells wax like substance - forms a thin protective coating

39. The distance from which the slides are sprayed with an aerosol fixative affects the cytology details - Prior to staining, the slides have to be kept overnight in 95% alcohol for removal of the coating fixative.

40. if the carbowax is not removed completely, Nuclei will then appear foggy and lack chromatinic detail and the cytoplasm may exhibit a pale blue color carbowax not removed Lack of chromatin details and hazy appearance of cell HSIL, Pap Hp

41. Carnoy’s fixative special purpose fixative for haemorrhagic samples absolute ethanol, chloroform and glacial acetic acid 6 : 3 : 1 acetic acid in the fixative haemolyses the red blood cells. Modified Carnoy’s an excellent nuclear fixative as well as a preservative of glycogen 95% ethanol Chloroform Glacial acetic acid 7 2.5 0.5 6 3 1 6 1

42. Other solutions used to lyse red blood cells: Clarke’s solution: absolute ethanol, glacial acetic acid (3 : 1) One drop of conc HCl per 500 mL of 95% ethanol Ten percent glacial acetic acid (this is followed by placing the slide in 95% ethanol) Commercially available fixatives such as CytoRich Red

43. Rehydration of Air Dried Smears Unfixed, air-dried gynaecological smears received from peripheral areas can be used for Papanicolaou staining by rehydration method The simplest rehydration technique is to place air dried cytological specimens 50 % aqueous solution of glycerine 2 rinses in 95% ethyl alcohol Pap staining

44. Liquid based cytology

45. Liquid-based cytology cytology (the 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

46. Liquid-based cytology techniques

47. collection of samples Ayre spatula Saline moistened cotton tip applicator

48. Thin prep Path sure

49. Thinprep Processor Cell dispersion Cell Collection Cell Transfer

50. (1)Cell dispersion Swirling the sampling device in the preservation solution Strong enough to separate debris and disperse mucus

51. (2) Cell Collection A gentle vacuum is created within the ThinPrep Pap Test Filter, which collects cells on the exterior surface of the membrane.

52. (3) cell transfer the ThinPrep Pap Test Filter is inverted and gently pressed against the ThinPrep Microscope Slide. Natural attraction and slight positive air pressure cause the cells to adhere .

53. path sure 1 4 3 2

54. Conventional smear Thin prep slide

55. Conventional smear Thin prep slide

56. Convetional papanicolaou ThinPrep SurePath Fixation Ethanol methanol Ethanol Collection Smear on slide Sample rinsed in vial Collection device left in vial Cell sample Random distribution Uniform distribution over 20 mm of slide Uniform distribution over 13 mm of slide

57. Collection device EC brush, spatula, Cervex - Brush EC brush, spatula, Cervex -Brush rinsed in vial Cervex -Brush most effective, tip deposited into vial Preservation artifacts Air drying, blood, inflammation, irregular distribution of cells All preservation artifacts greatly reduced All preservation artifacts greatly reduced

58. Automated processing Not applicable Vacuum pressure through TransCyte filter Gravity sedimentation process Imaging technology Not applicable Available Available Ancillary testing Not applicable HPV, Chlamydia, gonorrhea HPV, Chlamydia, gonorrhea

59. Staining Methods in Cytology Papanicolaou Staining Method May- Grunwald - Giemsa (MGG) Staining method

60. Papanicolaou Staining Method named after Dr. George N. Papanicolaou polychrome staining reaction display the many variations of cellular morphology showing degree of cellular maturity and metabolic activity.

61. principles Hydration and dehydration: – Hydration prepares the cell sample for uptake of the nuclear dye; – dehydration prepares the cell sample for uptake of the counterstains . Dehydration and clearing solutions result in cellular transparency and prepare the cell sample for the final steps

62. stains Nuclear staining : Hematoxylin Two cytoplasmic counter staining: (1) Orange G - (OG)-6, OG-5 and OG-8 is an acidic dye, stains keratin a bright, intense orange. (2) Eosin Azure (EA) - EA-36, EA-50 and EA-65 including three stains –Eosin Y –Light Green –Bismarck brown Y

63. The Papanicolaou Stain Steps of staining procedure (1) Fixation - 95% ethyl alcohol or in other substitutes - minimum of 15 minutes (2) Nuclear staining Harris haematoxylin - regressive staining method Gill I, - progressive staining method. Gill II, - progressive staining method. Gill III - progressive staining method

64. (3) Cytoplasmic staining (4) Dehydration Rinse the smears in absolute alcohol for two or three changes for the removal of water. Alternative to 100% ethanol are 100% isopropanol and 100% denatured alcohol. (5) Clearing alcohol is being replaced with Xylene Xylene has a refractive index as that of glass and mounting medium It prevents cellular distortion. (6) Mounting - DPX

65. fixation hydration dehydration clearing

66. Factors affecting Pap staining Type of fixatives Regressive or progressive Length of staining time No. Of slides in each dye Age of dyes Moisture and humidity Presence or absence of inflammatory cell changes Quality of cell sample

67. The Papanicolaou Stain

68. ULTRAFAST PAPANICOLAOU STAIN fast as the Diff- Quik stain 90 seconds smeared on a slide allowed to air dry placed in normal saline fixed in a mixture of 4 %formaldehyde and 65% ethanol stained with Richard Allan Hematoxylin 2 and Cytostain

69. May- Grunwald - Giemsa (MGG) Staining method MGG stain is performed in air –dried aspirates or fluids.

70. Immunocytochemistry Detection of surface antigens (markers) on isolated cells The detection is based on specific antigen-antibody binding (immunoreactions). A specific antibody that was produced by single B cell clone identifies an epitope with 8-15 length aminoacid sequence in a protein .

71. ICC in Diagnostic Cytology Applications Tumor Diagnosis/Classification Prognostic/Predictor Markers Target Therapy

72. Immunocytochemistry Fixation Prolonged fixation (wks/months) in formalin may result in antigenic loss Prolonged fixation in alcohol-based fixatives is not a major problem 95% isopropyl alcohol Buffered formalin Formol -acetone Mixture of ethanol & formalin

73. Main methodical steps of immunocytochemistry Cell fixation Antigen unmasking Blocking Selection of appropriate detection signals Parallel detection of more antigens Background staining Controls

74. Frequent enzyme-substrate systems ( 1) Peroxidase (HRPO): Diaminobenzidin (DAB) – brown Aminoethyl-carbasol (AEC)- red True Blue – blue (2) Alkaline phosphatase (ALP): Nitroblue tetrasolium (NBT) – blue Bromo-chloro-indoyl phosphate (BCIP) - blue

75. Commonly Used Markers In Effusions Breast cytology ER Mammaglobin GATA 3 E cadherin P120 catenin

76. Flow cytometry

77. What is flowcytometry Flow means motion Cyto means cell Metry means measure Definition : - An analytical technique in which cell suspension obtained from any unfixed tissue /body fluid, peripheral blood or bone marrow are stained with fluorescently labeled antibody and then subjected to analysis by a instrument called as flow cytometer .

78. Basic components. Fluidics system. Optic system. Electronic system. Associated computer system.

79. before it is subjected to flow cytometer , sample is incubated with the antibody and is washed fixed with 1 % formaldehyde This stabilizes the antigen antibody interaction by creating cross linkages.

80. Florochromes Florescin isothiocyanate (FITC). Phycoerythrin . Texas red. Allophycocyanin . Peridinin chlorophyll. Tandem florochromes .

81. Fluidics Filters Forward scatter and side scatter

82. Flow Cytometry Applications Immunophenotyping . Diagnosis and prognostication of immunodeficiency. To diagnose cause of allograft rejection. Diagnosis of auto antibodies in ITP . To measure nucleic acid content. DNA ploidy study in cancer.

83. Molecular Techniques in Cytopathology Flourescence in situ hybridization (FISH) Polymerase chain reaction (PCR) Microsatellite analysis Laser microdissection Mutation analysis DNA methylation analysis

84. Concluding remarks A specimen must be carefully prepared, well fixed, and stained in its journey to the microscope. Less time is required to prepare staining solution, since ready-made products that produce reliable and consistent staining are available for purchase. Liquid-based and automated systems are entrenched in sophisticated screening programs. Devices such as imaging flow cytometry and 3-dimensional cell scanning promise further advances in analysis capability.

85. thank you

86. References Naylor B, Ramzy I. Cytopathology:the past, the present and the glimpse into the new millenium . In. Gray W, Mckee GT. Diagnostic cytopathology . 2 nd edition. Churchill Livingston.2002. p. 3-13. Bales CE. Laboratory techniques. In. Koss LG. Koss’s diagnostic cytology and its histopathologic bases. 5 th edition. Lippincott Williams and Wilkins. 2005. p. 1570- 1634. Weidmann JE, Keebler CM, Fasik MS. Cytopreparatory techniques. In. Bibbo M, Wilbur DC. Comprehensive cytopatholgy . 3 rd edition. Saunders. 2008. p. 835-58.

87. 4. Cibas ES. Cervical and vaginal cytology. In. Ducatman BS. Cytology: diagnostic principles and clinical correlates.4 th edition. Saunders. 2014. 1-57. 5 . Orell SR, Veilh p. The techniques of FNA cytology. In. Orell SR, Sterret GF. Fine needle aspiration cytology. 5 th edition. Churchill Livingstone. 2011. p. 8-27.