Madhuri ppt path

Exfoliative cytology with pap smear

1. Exfoliative cytology with pap smear Moderator: Dr A Vamseedhar Presenter: Dr Madhuri R eddy

2. DEFINITION :A branch of General Cytology which deals with the microscopic study of cells that have been desquamated from the epithelial surfaces . SPONTANEOUS EXFOLIATION : In this method, cells are collected after they have been spontaneously shed by the body . MECHANICAL EXFOLIATION : Cells are manually scraped/brushed off of a surface in the body . 

3. INDICATIONS 1 . Detection of malignant cells or precancerous lesions in the body 2. Detection of asymptomatic or precancerous cervical lesions in women 3. Assessment of female hormonal status in case of sterility and endocrine disorders 4. Determination of genetic (phenotypic) sex 5. Detection of the presence of infectious microorganisms

4. PRINCIPAL FEATURES OF EXFOLIATIVE CYTOLOGY The technique is applicable to organs with easy clinical access whence the s amples can be obtained. The samples often contain a great variety of cells of various types from many different sources. The cellular constituents are sometimes poorly preserved. The samples may contain inflammatory cells, macrophages, microorganisms, and material of extraneous origin. The signal advantage of exfoliative cytology is the facility with which multiple samples can be obtained.

5. PRINCIPAL TARGETS OF EXFOLIATIVE CYTOLOGY Target Organ techniques Principle lesions to be identified Incidental benefits Female genital tract Smear of material from the vaginal pool obtained by pipette or a dull instrument. Fixation in alcohol or by spray fixative. Precancerous lesions and cancer of the vagina, uterine cervix, endometrium, rarely fallopian tubes, ovaries Identification of infectious agents, such as bacteria, viruses, fungi, or parasites Respiratory tract Sputum: either fresh or collected in fixative (smears and cell blocks) Precancerous states mainly carcinoma in situ and lung cancers Identification of infectious agents, such as bacteria, viruses, fungi, or parasites

6. Continued.. Urinary tract Voided urine; fresh or collected in fixative (smears and cytocentrifuge preparations) Precancerous states, mainly flat carcinoma in situ and high grade cancers Identification of viral infections and effect of drugs Effusions (pleural, peritoneal, or pericardial) Collection of fluid: fresh or in fixative (smears and cell blocks) Metastatic cancer and primary mesotheliomas Other fluids (cerebrospinal fluid, synovial fluid, etc.) Collection in fixative Cytocentrifuge preparations Differential diagnosis between inflammatory processes and metastatic cancer Identification of infectious agents (viruses, fungi)

7. ADVANTAGES Rapid diagnosis - Inexpensive - Simple It is better in evaluating the infectious diseases. Supplement or replace frozen section or biopsy No injury to tissue allowing repeated sampling It is better for hormonal assay Cytopathological smear cover a wider surface than that involved in surgical biopsy.

8. DISADVANTAGES Interpretation of the morphological cellular changes is based only on individual cell observation. Not always fully diagnostic, so it is confirmed by histopathology in some cases. Does n ot determine the size and type of lesion of some cases.

9. FACTORS THAT DETERMINE THE APPEARANCE OF CELLS Type of the technique used. Level of cell maturation at the time of cell collection. Nature of the parent tissue: soft tissue, cyst, or solid organ. Medium of the exfoliated cells. Interval between the stain of the exfoliated cells and collection of samples. Type of fixative, stain, and processing technique used.

10. PAP SMEAR

11. HISTORY The current resurgence of diagnostic cytology is the result of the achievements of Dr. George N. Papanicolaou (1883-1962), an American of Greek descent . He developed a small glass pipette that allowed him to obtain cell samples from the vagina of rodents. In smears, he could determine that, during the menstrual cycle, squamous cells derived from the vaginal epithelium of these animals followed a pattern of maturation and atrophy corresponding to maturation of ova.

12. He made major contributions to the understanding of the hormonal mechanisms of ovulation and menstruation and is considered to be one of the pioneering contributors to reproductive endocrinology. However, his fame is based on an incidental observation of cancer cells in vaginal smears of women whose menstrual cycle he was studying.

13. Dr George N. Papanicolaou

14. ADVANTAGES AS A SCREENING TOOL Relative accessibility of cervix to take the smear Long natural history of cervical carcinogenesis Relative conservative treatment for premalignant lesions Cost effectiveness

15. SIGNIFICANCE Detects precancerous & invasive cancer cervix cases in early stages. Positive screeners can be selected for selective tests and management . With treatment, progression of disease is halted. Thus morbidity associated with advanced cancer decreases. Mortality reduces by 20-60 %. Helps us to study natural history of disease.

16. WHEN TO SCREEN ? Start within 3 years of onset of sexual activity or by age of 21(ACOG) whichever is first . WHO recommends screening after the age of 30. Risk factors for cervical dysplasia Early onset of sexual activity Multiple sexual partners Tobacco Oral contraceptives

17. SCREENING FREQUENCY Yearly ,until three consecutive pap smears are normal and then every three yearly. Annual screening for high-risk women is highly recommended.

18. WHEN TO STOP SCREENING Age 65 and “adequate recent screening” Three consecutive normal pap smears No abnormal pap smears in last 10 years No history of cervical or uterine cancer Hysterectomy for benign disease Hysterectomy for invasive cervical cancer

19. WHAT DOES A PAP TEST INVOLVE? Performing a vaginal speculum exam during which a health care provider takes a sample of cells from a woman’s cervix using a small flat spatula or brush. Smearing and fixing cells onto a glass slide. Sending the slide to a cytology laboratory where it is stained and examined under a microscope to determine cell classification. Transmitting the results back to the provider and then to the woman.

20. PROCEDURE COLLECTION OF SPECIMEN Prerequisites for collection of specimen Women should be tested two weeks after the first day of their last menstrual period .(Day 14 of cycle is optimal ). Proper technique is very important More problems are due to improper sampling than screening Not to be collected during menses Avoid vaginal contraceptives, vaginal medications for at least 48 hrs before taking smear Abstinence from sex for 24 hrs Postpartum smear should be taken only after 6 - 8 weeks of delivery

21. PRECAUTIONS TO BE FOLLOWED • Specimens should be obtained after a nonlubricated speculum (moistened only with warm water if needed) is inserted. • Excess mucus or other discharge should be removed gently with ring forceps holding a folded gauze pad. • The sample should be obtained before the application of acetic acid or Lugol iodine. • An optimal sample includes cells from the ectocervix and endocervix .

22. INFRASTRUCTURE REQUIRED Private exam area Examination table Trained health professionals Sterile vaginal speculum Supplies and equipment for preparing and interpreting the Pap smears (e.g., spatulas, glass slides, fixative, stains, microscopes) Marker/pencil/glass writer/labels Cytology requisition forms Record books or sheets Cytology laboratories with skilled personnel to interpret results Pathologists Transportation of slides to the laboratory and back Information systems to ensure follow-up contact with clients Quality assurance system to maximize accuracy

23. CONVENTIONAL PAP SMEAR Method Patient in dorsal position Good illumination is necessary Cusco’s speculum is inserted to visualise & fix the cervix Inspection of cervix done & findings are noted Ayres spatula is inserted first. It is placed at cervical os so that longer end goes into cervical canal and smaller end rests on ectocervix Spatula is rotated through 360 degrees maintaining contact with ectocervix Do not use too much force [bleeding /pain] Do not use too less force [inadequate sample] Sample is smeared evenly on the slide and fixed immediately Both sides of spatula are to be smeared

24. FIXATION COMMON FIXATIVES 95% Ethanol. 95 % Rectified Spirit. 100 % Methanol. 80 % Isopropanol or Propanol. Ether/95 % Ethanol ( 1: 1). Spray fixatives contains isopropanol and propylene glycol.

25. FIXATION METHODS There are two options for smear fixation. 1.Coating fixatives contain alcohol and polyethylene glycol and are applied by pump sprays, by droppers from dropper bottles, or by pouring from an individual envelope included as part of a slide preparation kit . 2. The smear can be immersed directly into a container filled with 95% ethanol .

26. Slide should be labeled properly with patients name, identification no. and details Detailed history and clinical examination findings are to be mentioned Patient details and clinical findings are to be maintained in a register Advice is given regarding further follow up and treatment

27. STAINING Stains for Pap: 1.Harris Hematoxylin 2.OG 6 Stain Orange Green 6, 0.5 solution in 95% ROH 100 ml Phosphotungstic acid 0.015gm 3. EA 50 light green SF, yellowish 0.1% solution in 95% ROH 45ml Bismarck brown 0.5 in 95% ROH 10 ml Eosin Y, 0.5% in 95% ROH 45 ml Phosphotungstic acid 0.2 gm Lithium Carbonate. Sat. aq. Solution 1 drop

28. PROCEDURE FOR STAININING Fix in ether-ROH and pass thru 80% ROH, 40% ROH and distilled H2O . Stain in Harris Hematoxylin for 4-5 minutes. Wash with H2O. Pass thru 0.25% HCl in 50% ROH. Immerse in 1.5% NH4OH in 70% ROH for 1 minute. Rinse in 70% ROH and pass thru 80% and 95% ROH. Stain with OG 6 for 1.5-2 minutes.

29. Contd.. Pass thru 3 changes of 95% ROH. Stain with EA 65 or EA 50 for 3 minutes. Pass thru 3 changes of 95% ROH. Dehydrate and clear in: a. absolute ROH, b. equal parts of ether and absolute ROH, c. 2 changes of xylol Mount in Canada Balsam.

30. PAP KIT This complete kit contains a Medline speculum, cervical scraper and two 6" polyester-tipped applicators. There's also a pair of latex gloves, lubricating jelly, two frosted end slides, slide holder and mailer.

31. AUTOMATED PAP STAINING

32. 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 transferring to a slide . Sample is processed and resultant thin smear easy to screen.

33. ADVANTAGES 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)

34. DISADVANTAGES More costly than conventional pap smears Preparation is more laborious ,intensive than Conventional Loss of background material Some differences in architecture and morphology Requires training for both the screeners and the smear takers

35. LIQUID BASED CYTOLOGY TECHNIQUES MANUAL METHOD THINPREP PAP TEST SUREPATH PAP TEST MONOPREP PAP TEST

36. MANUAL METHOD LBC FIXATIVE FOR PAP SMEAR Alcohol- 50ml 10% formalin – 50ml Sodium chloride -0.5gm Sodium citrate- 0.5gm

37. POLYMER PREPARATION Agarose- 2gm Polyethylene glycol- 10ml Poly-L-lysine- 2ml Distilled water – 88ml (Filter before use to avoid precipitation) (Solution kept in refrigerator) PHOSPHATE BUFFER PREPARATION Solution A- Sodium hydrogen orthophosphate – 9.45gm Distilled water – 1lt. Solution B- Potassium di hydrogen orthophosphate – 9.8gm Distilled water – 1lt. FOR 100ML BUFFER Solution A- 49.6ml Solution B – 50.4ml (Solution kept in Refrigerator)

38. 1. Receive the sample from the ward uniquely labelled. 2. Mix sample properly before transferring to the test tube. 3. Take clear test tube and transfer the sample to the test tube. 4. Centrifuge at 2000 RPM for 5 minutes 5. Decant supernatant and mix properly. 6. Add1-2ml of polymer reagent to the deposit 7. Centrifuge at 2000 RPM for 5 minutes.

39. 8. Decant supernatant mix deposit properly. 9. Add1-2ml of cold phosphate buffer to the deposit. 10 Centrifuge at 2000 RPM for 5 minutes. 11 Decant supernatant mix properly. 12 Take a clear glass slide. 13. Pipette deposit on the slide in circular 14. Prepared slides can be dried at room temperature, in the hot air over at 40-50’c for 5 to 10minutes. 15. The slide can be further fixed by dipping in 95% alcohol for 2 hours 16. Stained with Pap stain.

40. THIN PREP The practitioner obtains the ThinPrep Pap sample with either a broom-type device or a plastic spatula/ endocervical brush combination . The sampling device is swirled or rinsed in a METHANOL-BASED preservative solution (PreservCyt ) for transport to the cytology laboratory and then discarded . Red blood cells are lysed by the transport medium. The vials are placed one at a time on the ThinPrep 2000 instrument . The entire procedure takes about 70 seconds per slide and results in a thin deposit of cells in a circle 20 mm in diameter (contrast with cytospin diameter=6mm)

41. The sample vial sits on a stage and a hollow plastic cylinder with a 20-mm diameter polycarbonate filter bonded to its lower surface is inserted into the vial. A rotor spins the cylinder for a few seconds, homogeneously dispersing the cells . A vacuum is applied to the cylinder, trapping cells on the filter. The instrument monitors cell density . With continued application of vacuum, the cylinder (with cells attached to the filter) is inverted 180 degrees, and the filter is pressed against a glass slide. The slide is immediately dropped into an alcohol bath .

42. SURE PATH The practitioner snips off the tip of the collection device and includes it in the sample vial . The equipment to prepare slides includes a Hettich centrifuge and a PrepStain robotic sample processer with computer and monitor. The PrepMate ™ is an optional accessory that automates mixing the sample and dispensing it onto the density reagent . Red blood cells and some leukocytes are eliminated by density centrifugation . In addition to preparing an evenly distributed deposit of cells in a circle 13 mm in diameter , the method incorporates a final staining step that discretely stains each individual slide . Media is primarily ETHANOL BASED

43. TThThe sample is quickly vortexed. A proprietary device, the Cyringe ™, disaggregates large clusters by syringing the sample through a small orifice. The sample is poured into a centrifuge tube filled with a density gradient reagent. Sedimentation is performed in a centrifuge. A pellet is obtained and resuspended , and the sedimentation is repeated. The tubes are transferred to the PrepStain instrument, where a robotic arm transfers the fluid into a cylinder. Cells settle by gravity onto a cationic polyelectrolyte-coated slide.The same robotic arm also dispenses sequential stains to individual cylinders.

44. MONOPREP PAP TEST The practitioner obtains the MonoPrep sample with standard collection devices that are swirled or rinsed in a preservative-filled collection vial, after which the sampling device is discarded . Red blood cells are lysed by the transport medium . The vials are delivered to the laboratory where slides are prepared using the MonoPrep Processor, a fully automated, batch processing instrument capable of processing 40 samples per hour, with a throughput capacity of 324 samples per 8-hour run . MonoPrep provides a significant reduction in unsatisfactory slides.

45. An integrated stirrer mixes the specimen briefly to disperse mucus and aggregates . The specimen is aspirated into the hollow stirrer and dual- flotechnology captures a representative sample on a frit-backed filter. The filter is pressed against the slide to transfer the cells onto a 20-mm diameter circular area. After cell transfer, the instrument applies a premeasured amount of alcohol fixative directly onto the slide.

46. THIN PREP SURE PATH

47. CONVENTIONAL PAP VS LBC Conventional Pap Smear Majority of cells not captured Non-representative transfer of cells Clumping and overlapping of cells Obscuring material Liquid-based Cytology Virtually all cells of sample are collected Randomized, representative transfer of cells Even distribution of cells Minimizes obscuring material

48. METHODS OF SCREENING SMEARS

49. CERVICAL HISTOLOGY

50. The uterine cervix protrudes into the upper vagina and contains the endocervical canal, linking the uterine cavity with the vagina. The endocervical canal EC is lined by a single layer of tall columnar mucus-secreting epithelial cells . The ectocervix V is lined by thick stratified squamous epithelium. The junction J between the ecto - and endocervical epithelium is quite abrupt and is normally located at theexternal os , the point at which the endocervical canal opens into the vagina.

51. SQUAMOUS EPITHELIUM The squamous epithelium covering the exocervix is normally noncornified , and it grows, matures, and accumulates glycogen in its upper layers in response to circulating estrogens .

52. ENDOCERVICAL EPITHELIUM The endocervix is lined by a single layer of mucin secreting epithelium composed of cells with small, often basilar , nuclei above which is mucin- filled cytoplasm which imparts a ‘ picket fence’ appearance .

53. TRANSFORMATION ZONE Transformation zone is the zone between the original squamo columnar junction and the new squamo columnar junction .

54. REPORTING SYSTEMS George N Papanicolaou (1954) 5 classifications based on certainty of finding malignant cells Descriptive system – WHO - (1968) based on morphologic criteria – included mild, moderate, severe dysplasia and Ca In Situ Richart – CIN –based on histologic diagnosis Bethesda system – TBS (1988) National cancer institute revised in 1991 , 2001 and 2014

55. NORMAL CELLULAR ELEMENTS A.SQUAMOUS CELLS 1.SUPERFICIAL SQUAMOUS CCELLCELLS The superficial cells have smaller condensed ( pyknotic ) nuclei. Light brown glycogen is present in the cytoplasm. Cells are polygonal in shape with keratohyaline granules in the cytoplasm.

56. 2.INTERMEDIATE CELLS A typical intermediate cell with a polygonal cytoplasmic profile. The nucleus possesses finely granular chromatin with longitudinal groove. The cross-sectional area of the intermediate nucleus is approximately 35 μm 2.

57. 3.PARABASAL CELLS The parabasal cell exhibits typical features with an oval nucleus, fine chromatin, and a cross sectional area of approximately 50 μm 2 . The cytoplasm is dense and heaped up.

58. B.GLANDULAR CELLS 1.ENDOCERVICAL CELLS Endocervical cells when viewed from the side present in a “picket-fence” configuration There is normal nuclear polarity and ample evidence of apical mucin in these columnar cells.

59. 2.ENDOMETRIAL CELLS A tight cluster of endometrial glandular cells with nuclei having cross-sectional areas slightly smaller than the 35 μm 2 of intermediate cells. Endometrial cell nuclear to cytoplasmic ratios are high and the cells tend to form three dimensional groups .

60. The 2014 BETHESDA SYSTEM FOR REPORTING CERVICAL CYTOLOGY SPECIMEN TYPE: Indicate conventional smear (Pap smear) vs. liquid-based preparation vs. other SPECIMEN ADEQUACY • Satisfactory for evaluation ( presence or absence of endocervical /transformation zone component and any other quality indicators, e.g., partially obscuring blood, infl ammation , etc. ) • Unsatisfactory for evaluation – Specimen rejected/not processed – Specimen processed and examined, but unsatisfactory for evaluation of epithelial abnormality(reason)

61. GENERAL CATEGORIZATION • Negative for Intraepithelial Lesion or Malignancy • Others :Endometrial cells ( in a woman ≥45 years of age ) • Epithelial Cell Abnormalities INTERPRETATION/RESULT NEGATIVE FOR INTRAEPITHELIAL LESION OR MALIGNANCY ( When there is no cellular evidence of neoplasia, state this in the GeneralCategorization above and/or in the Interpretation/Result section of the report -whether or not there are organisms or other non-neoplastic findings) OTHER Endometrial cells ( in a woman ≥45 years of age )( Specify if “negative for squamous intraepithelial lesion” ) EPITHELIAL CELL ABNORMALITIES OTHER MALIGNANT NEOPLASMS

62. ADJUNCTIVE TESTING Brief description of the test method(s) and report the result so that it is easily understood by the clinician . COMPUTER-ASSISTED INTERPRETATION OF CERVICAL CYTOLOGY If case examined by an automated device, specify device and result. EDUCATIONAL NOTES AND COMMENTS APPENDED TO CYTOLOGY REPORTS ( optional ) Suggestions should be concise and consistent with clinical follow-up guidelinespublished by professional organizations (references to relevant publications may be included ).

63. ADEQUACY An adequate pap smear is one that includes a sampling of both the exocervix and endocervix . An adequate conventional smear has an estimated minimum of approximately 8,000-12,000 well visualized and preserved squamous cells. An adequate liquid based preparation should have an estimated minimum of 5,000 well visualized/preserved squamous cells

64. “ SATISFACOTRY FOR EVALUATION” Approriate labelling and identifying information . Relevant clinical information. Adequate number of well preserved and well visualized squamous epithelial cells .

65. Satisfactory, but borderline squamous cellularity (LBP, SurePath ). At 40×, there were approximately 11 cells per field when ten microscopic fields along a diameter were evaluated for squamous cellularity; this would give an estimated total cell count between 5,000 and 10,000

66. UNSATISFACTORY FOR EVALUATION Lack of patient identification on specimen. Slide that is broken and cannot be repaired, or cellular material that is inadequately preserved. Scant squamous epithelial component(well preserved and well visualized squamous epithelial cells covering less than 10% of the slide surface) Obscuring that precludes interpretation of approximately 75% or more of epithelial cells .

67. Unsatisfactory due to scant squamous cellularity . Endocervicalcells are seen in a honeycomb arrangement (LBP, ThinPrep at 10× magnification )

68. CONDITIONS WITH ABNORMAL PAP INFECTIONS NON NEOPLASTIC CONDITIONS EPITHELIAL CELL ABNORMALITIES

69. INFECTIONS

70. LACTOBACILLI/DODERLEIN’S BACILLI Lactobacilli are gram-positive facultative anaerobic rod- shaped bacteria observed in about 50% of normal healthy adult female population. These bacilli release enzymes causing extensive cytolysis of glycogen containing cells. Mainly affect intermediate and superficial cells. Parabasal cells are generally spared. Bacteria: lactobacilli and cytolysis ( A:CP ),(B:LBP- Thin Prep ) shows the presence of a cytolytic background with cell debris and numerous stripped nuclei of intermediate cells . A B

71. BACTERIAL VAGINOSIS Bacteria – coccobacilli ( CP :A)(LBP-SUREPATH-B). Shift in flora suggestive of bacterial vaginosis. “Clue cell” and filmy background due to large numbers of coccobacilli. Relatively cleaner background is seen in LBP. A B

72. HERPES SIMPLEX Cellular changes consistent with herpes simplex virus ( CP ). The eosinophilic intranuclear “ Cowdry A-type” inclusions are seen. The “ground-glass” appearance of the nuclei is due to accumulation of viral particles leading to peripheral margination of chromatin. The inset shows a SurePath liquid-based preparation with a typical multinucleated herpetic cell showing “ ground- glass” appearance of the nuclei

73. ACTINOMYCES Actinomyces is often associated with intrauterine device ( IUD) usage. Tangled clumps of filamentous organisms, often with acute angle branching, are recognizable as “cotton ball” clusters on low power . Filaments sometimes have a radial distribution or have an irregular “woolly body”appearance . Masses of leukocytes adherent to microcolonies of the organism with swollen filaments or “clubs” at the periphery may be identified .

74. CANDIDA ALBICANS LBP , ThinPrep : pseudohyphae (left) “spearing” or a “shish kebab” appearance of squamous cells (right). This feature is readily appreciated at low power, even when the pseudohyphae are not prominent.

75. TRICHOMONAS Image A shows CP and B shows LBP ;THIN PREP( polyball appearance of neutrophills ) Trichomonas is a pear-shaped, oval to round, cyanophilic organism that ranges in size from 15-30 microns.The nucleus is pale, vesicular and eccentrically located . Eosinophilic granules are often visible in the cytoplasm . A B

76. CYTOMEGALOVIRUS The cytopathic effect of cytomegalovirus (CMV) affects mostly the endocervical glandular cells but can also be present in stromal cells Cellular and nuclear enlargement. Large eosinophilic intranuclear viral inclusions with a prominent halo. Small cytoplasmic, basophilic inclusions can also be present.

77. NON NEOPLASTIC CONDITIONS

78. Includes the following Squamous metaplasia Keratotic changes Typical parakeratosis Hyperkeratosis Tubal metaplasia Pregnancy related cellular changes Atrophy Reactive /reparative cellular changes Associated with inflammation Lymphocytic cervicitis Associated with radiation Associated with IUD usage

79. SQUAMOUS METAPLASIA ( LBP , SurePath ):A characteristic metaplastic cell is found in the center of the field. The nucleus is round to oval with fi ne, evenly distributed chromatin. The nuclear to cytoplasmic ratio is variable, and in this instance, it approaches one to one. The mean nuclear area is larger than that of the intermediate cell and similar to the parabasal cell at 50 μm 2 .

80. KERATOTIC CHANGES TYPICAL PARAKERATOSIS Both are examples of “typical parakeratosis” showing miniature squamous cells with small bland, pyknotic nuclei. “A” shows the “squamous pearl” formation from a 49-year-old woman being followed up after treatment for SIL. ‘B’ shows small cluster of miniature squamous cells. A B

81. HYPERKERATOSIS A- LBP , ThinPrep shows a group of anucleate squamous at low power . B- LBP , ThinPrep shows anucleate , mature polygonal squamous cells with ghostlike “nuclear holes ” A B

82. TUBAL METAPLASIA CP :Ciliated cells derived from tubal metaplasia . Terminal bar and cilia at left edge ( arrow) LBP , Thin Prep:A linear array of cells showing tubal metaplasia

83. PREGNANCY RELATED CHANGES Hormonal changes Decidual changes Synctiotrophoblastic changes Cytotrophoblastic changes Arias stella reaction

84. HORMONAL CHANGES NAVICULAR CELLS In pregnant women, squamous cells become laden with glycogen, and have a vaguely “ boatlike ” shape referred to as“navicular”cells {A: LBP - ThinPrep , and B : LBP - SurePath } A B

85. ATROPHY Atrophy ( LBP , ThinPrep ): flat , monolayer sheet of parabasal -type cells, with preserved nuclear polarity Atrophy with infl ammation ( CP ):shows classic finding of granular debris in background, degenerating parabasal cells and polymorphonuclear leukocytes.

86. REACTIVES CHANGES ASSOCIATED WITH INFLAMMATION ( CP ) A 26-year-old woman , day 14 of menstrual cycle with mild vaginal discharge. Squamous cells show mild nuclear enlargement with nuclear hypochromasia , perinuclear halos, and cytoplasmic polychromasia resulting in a “moth-eaten” appearance. Trichomonads are seen in the background.

87. LYMPHOCYTIC CERVICITIS ( CP )Abundant lymphoid cells with a tingible body macrophage located centrally LBP-THIN PREP

88. RADIATION EFFECT (CP) A;shows irregularly shaped abundant cytoplasm and the streaming or “windblown” edges of the. Nuclei are typically enlarged and may be pale or become hyperchromatic as nuclear material condenses. Nucleoli are typically seen along with numerous polymorphonuclear leukocytes in the background. B:(LBP , ThinPrep ) radiated cells in liquid-based preparations do not tend to show the streaming and the cytoplasm is typically more dense. Nuclear degeneration and cytoplasmic vacuolization are common in both preparation types A B

89. IUD EFFECT ( CP ) shows small cluster of glandular cells with cytoplasmic vacuoles displacing nuclei. LBP , Thin Prep : cellular groups tend to be tighter.

90. EPITHELIAL CELL ABNORMALITIES

91. ASC-US LBP-SUREPATH Single atypical squamous cell with ill-defined cytoplasmic halo in a background of inflammation. Nuclei are approximately two and one half to three times the area of the nucleus of a normal intermediate squamous cell (approximately 35 mm 2 ) or twice the size of a squamous metaplastic cell nucleus (approximately 50 μm 2 )

92. ASC-US ( CP ): Cells with multinucleation , nuclear enlargement .

93. ASC-H ASC-H ( LBP, SurePath ): Routine cytology for a 30-year-old woman . Rare metaplastic cells with dense cytoplasm and nuclear enlargement with hyperchromasia are present in a background of scattered acute inflammation

94. ATYPICAL GLANDULAR CELLS ATYPICAL ENDOCERVICAL CELLS , (CP): Sheet of glandular cells showing nuclear enlargement, marked variation in nuclear size, prominent nucleoli, and distinct cell borders. ATYPICAL ENDOMETRIAL CELLS ( LBP , ThinPrep ). Three-dimensional grouping of small cells with crowded round or oval nuclei.

95. L-SIL LBP-THIN PREP LBP-THIN PREP:Nuclear enlargement and hyperchromasia of sufficient degree seen for the interpretation of LSIL

96. LBP-THIN PREP LSIL on cytology is characterized by mature squamous cells with enlarged nuclei with variable chromatin and nuclear membranes. Koilocytosis or perinuclear cavitation in the cytoplasm, a characteristic of HPV cytopathic effect is present .

97. Nuclei are generally hyperchromatic but may be normochromatic . Nuclei show variable size ( anisonucleosis ). Chromatin is uniformly distributed and ranges from coarsely granular to smudgy or densely opaque . Contour of nuclear membranes is variable ranging from smooth to very irregular with notches . Binucleation and multinucleation are common . Nucleoli are generally absent or inconspicuous if present. Koilocytosis or perinuclear cavitation consisting of a broad, sharply delineated clear perinuclear zone and a peripheral rim of densely stained cytoplasm is a characteristic viral cytopathic feature but is not required for the interpretation of LSIL Cells may show increased keratinization with dense, eosinophilic cytoplasm with little or no evidence of koilocytosis .

98. MIMICS OF L-SIL PSEUDOKOILOCYTES ( LBP , ThinPrep ): Glycogen in squamous cells can give the appearance of “ pseudokoilocytosis ” . The halos associated with glycogen often have a yellow refractile appearance. HERPES ( LBP , ThinPrep ): A 25-year-old woman. Endocervical cell and intermediate cells showing herpes virus cytopathic effect with clearing of chromatin.

99. H-SIL ( CP ): The dysplastic cells are seen here in a syncytial cluster or hyperchromatic crowded group CP:There is variation in nuclear size and shape, and the cells have delicate cytoplasm

100. The cells of HSIL are smaller and show less cytoplasmic maturity than cells of LSIL. Cells occur singly, in sheets, or in syncytial-like aggregates . Syncytial aggregates of dysplastic cells may result in hyperchromatic crowded groups . Nuclear to cytoplasmic ratio is higher in HSIL compared to LSIL . Contour of the nuclear membrane is quite irregular and frequently demonstrates prominent indentations. Nuclei are generally hyperchromatic but may be normochromatic or even hypochromatic .

101. Chromatin may be fine or coarsely granular and is evenly distributed. Nucleoli are generally absent, but may occasionally be seen, particularly when HSIL extends into endocervical gland spaces or in the background of reactive or reparative change. Appearance of the cytoplasm is variable; it can appear “immature,” lacy, and delicate or densely metaplastic occasionally, the cytoplasm is“mature ” and densely keratinized (keratinizing HSIL)

102. SQUAMOUS CELL CARCINOMA SQUAMOUS CELL CARCINOMA, KERATINIZING ( LBP , SurePath ):The malignant cells in variable shapes and sizes with some keratinized “tadpole cells .” ( CP ): There is markedpleomorphism of cell size and shape, cytoplasmic keratinization, and tumor diathesis in the background

103. Presents predominantly as isolated, single cells and less commonly in cellular aggregates . Marked variation in cellular size and shape is typical, with caudate and spindle cells that frequently contain dense orangeophilic cytoplasm. Nuclei vary markedly in area, nuclear membranes may be irregular, and numerous dense opaque nuclei are often present. Chromatin pattern, when discernible, is coarsely granular and irregularly distributed with chromatin clearing. Macronucleoli may be seen . Associated keratotic changes (hyperkeratosis or parakeratosis) may be present but are not suffi cient for the interpretation of carcinoma in the absence of nuclear abnormalities . A tumor diathesis may be present.

104. ADENOCARCINOMA ADENOCARCINOMA, ENDOCERVICAL ( CP ):Nuclei are enlarged and pleomorphic with irregular chromatin distribution and prominent or macronucleoli . Cytoplasm is finely vacuolated with prominent blood -filled background

105. Abundant abnormal cells, typically with columnar confi guration . Single cells, two-dimensional sheets or three-dimensional clusters, and syncytial aggregates . Enlarged, pleomorphic nuclei demonstrate irregular chromatin distribution, chromatin clearing , and nuclear membrane irregularities . Macronucleoli present Cytoplasm is usually fi nely vacuolated. Necrotic tumor diathesis is common.

106. ADJUNCTIVE TESTING HIGH RISK HPV TESTING There are four hrHPV tests that are FDA approved for performance inassociation with cervical cytology. Three are DNA based and one is RNA based . Triage of an abnormal cytology result by a hrHPV test effectively improves the balance of sensitivity vs. specifi city for colposcopic referral and prevalent disease detection. HPV genotyping refers to the selective reporting of individual HPV types in conjunction(HPV 16 nd 18) with a positive pooled high-risk test IMMUNOCHEMICAL ASSAYS The best-studied biomarkers are p16, ProExC , and Ki67 p16 and ProExC are both markers of an aberrant cell cycle which has been affected by the oncogenic effects of HPV . Ki67 is a marker of cellular proliferation . p16 stains both the nucleus and cytoplasm; ProExC and Ki67 stain the nucleus

107. SAMPLE REPORT SPECIMEN TYPE: Conventional pap smear SPECIMEN ADEQUACY: Satisfactory for evaluation; endocervical /transformation zone present. INTERPRETATION: Negative for intraepithelial lesion or malignancy NOTE High-risk HPV typing is NEGATIVE COMMENT As Per the 2012 ASCCP management guidelines, a negative result for HPV testing when associated with an NILM cytology means the patient has signifi - cantly less than a 1 % chance of HSIL (CIN3) lesion and repeat testing at decreased intervals is not warranted

108. HORMONAL CYTOLOGY Maturation of vaginal squamous cells from one cell to another is hormone dependent . The quantitative ratio between the different cell types can reflect the index of the hormonal status of the female. For hormonal assesment ideal site is lateral vaginal wall. Estrogens have proven action on maturation of squamous epithelium of vagina . Its excess causes enhancement of maturation and the smear contains more of superficial cells, on the other hand its lack causes lower degree of maturation or the atrophy of squamous epithelium,the same effect could be reflected due to antagonistic action of the excess of progesterone .

109. CELLULAR INDICES FOR HORMONAL ASSESSMENT KARYOPYKNOTIC INDEX EOSINOPHILIC INDEX FOLDED CELL INDEX CROWDED CELL INDEX MATURATION INDEX

110. KARYOPYKNOTIC INDEX Ratio between the superficial squamous cells with pyknotic nuclei to all mature squamous cells irrespective of staining character. Peak of KPI usually coincides with the time of ovulation and may reach 50-85 . EOSINOPHILIC INDEX Ratio of mature squamous cells with eosinophilic cytoplasm to all mature squamous cells irrespective of size of nucleus . Peak value is 50-75 during ovulation . MATURATION INDEX It is count of the parabasal cells,intermediate and superficial cells (P : I : S) In a normal menstruating woman during ovulation the menstruation index will be 0/35/65. In postmenopausal it will be 85/15/0.

111. OTHER INDICES FOLDED CELL INDEX Ratio of mature squamous cells with folded margins to all mature squamous cells irrespective of staining chararcter . Folding is usually observed in cells containing glycogen. CROWDED CELL INDEX Represents the relationship of mature squamous cells lying in clusters of four or more cells, to all mature squamous cells .

112. THE MATURATION VALUE Meisels (1967) suggested that a specific numerical value be attached to the three principal subgroups of the squamous cells—a value of 1.0 to superficial squamous cells, a value of 0.5 to intermediate cells, and a value of 0.0 to parabasal cells . The maturation value (MV) in Patient with MI 0:35:65 (normal menstruating woman at time of ovulation ) 0 X 0 = 00 35 X 0.5 = 17.5 65 X 1 = 65.O TOTAL = 82.5 An MV of 100 indicates a pure population of superficial squamous cells, MV of zero indicates a pure population of parabasal cells . For menstruating normal women, the MV is between 50 and 95; for women with varying degrees of atrophy of squamous epithelium, the MV is below 50.

113. TAKE HOME MESSAGE FOR ALL WOMEN: CERVICAL CANCER IS A PREVENTABLE DISEASE "No woman has to die from cervical cancer," says Ault, professor of obstetrics and gynecology at Emory. "It’s almost entirely preventable with recommended Pap guidelines. If everyone got a regular Pap test and the HPV (human papillomavirus) vaccine we could reduce the number of women diagnosed with cervical cancer by more than 75 percent."

114. REFERENCES Koss Diagnostic Cytology and Its Histopathologic Bases, 5th Edition The Bethesda System for Reporting Cervical Cytology Definitions , Criteria, and Explanatory Notes Third Edition CYTOLOGY: DIAGNOSTIC PRINCIPLES AND ISBN: 978-1-4160-5329-3 CLINICAL CORRELATES Copyright © 2009 by Saunders, an imprint of Elsevier Inc . HISTOLOGY FOR PATHOLOGISTS BY STACEY E MILLS. Robbins and Cotran Pathologic Basis of Disease Wheater’s Functional Histology A Text and Colour Atlas

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