Manual on Cervical Cytology and Colposcopy Radhika N Joshi
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Cervical CytologyCHAPTER 1

CHAPTER OUTLINE
  • • History
  • • Pap Smear
  • • Papanicolaou's Staining Technique
  • • Interpretation of Smears
  • • Reporting of the Smear
  • • Liquid Base Cytology
  • • Visual Inspection
  • • Management of Smears
  • • Record Sheet
 
HISTORY
Cervical Cytology is a simple, safe, non-invasive method of detecting precancerous changes in cervix. It is accepted as a screening tool for healthy women for evidence of cervical intraepithelial neoplasia (CIN).
  • Dr George Papanicolaou (1883–1962) is considered as the father of cytology.
  • 1928 - Presented a paper on tumor cells, which could be found in vaginal smears in cervix.
  • 1945 - First screening clinic was established in Massachusetts.
  • 1948 - First cytology conference and course in New York, Boston.
  • 1985 - Miller-Population screening is a positive force in control of carcinoma of cervix.
 
Indian History
  • Dr PN Wahi was the Founder of Indian Academy of Cytology in 1970.
  • Dr Hannah Peters, Dr Segulla Aptekar started early detection work in Mumbai.
  • Dr Usha Saraiya founder of Cytology Clinic in Cama and Albless Hospital, Mumbai run by Association of Medical Women of India.
  • 1984 - State cancer control program launched.
 
PAP SMEAR
Important data to be taken in the history:
  1. Age of the Patient.
  2. Date of last menstrual period.
  3. Per speculum findings of the cervix.
  4. Contraceptive history.
  5. Obstetric history
2Apparatus required:
  1. Two clean glass slides labeled.
  2. Cotton swabs, spatulas and cervical brushes.
  3. Jar containing fixative.
  4. Cusco's or Sim's vaginal speculum.
  5. Endometrial aspiration canula and syringes (in case of endometrial cytology).
Fixative percentage types:
  1. Fluid fixative—mixture of equal parts of ether and 95% absolute alcohol.
  2. 95% ethyl alcohol.
  3. 95% isopropyl or 80% isopropyl alcohol.
  4. Absolute methanol.
  5. Cytofix spray.
  6. Spray fixative ordinary hair spray. This is convenient for office gynecology and transportation and for camps.
  7. Polyethylene glycol fixative.
Position of the patient: She is kept in dorsal position. Labia are separated and vaginal speculum is inserted.
Site of smear collection: The most important site for smear collection for cancer detection is squamocolumnar junction and transformation zone.
  1. Upper 1/3rd of lateral vaginal wall for hormonal cytology.
  2. Posterior vaginal pool, ectocervix and endocervix for cancer cytology.
  3. Endometrial aspirate for endometrial cytology. Smears are quickly spread thinly on the slide and immediately immersed in the fixative and period of fixation varies from 30 minutes to 24 hours.
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Figs 1.1A and B: Smear taking: (A) Spatula inserted in os and rotated; (B) Endocervical brush inserted in os and rotated
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Precautions
  1. Presence of water in smears destroys cellular details so the slides, spatulas and cervical brush used must be absolutely dry.
  2. Slides must be fixed immediately. They must not be allowed to dry. With air drying, cells lose their differential staining characteristics.
  3. Smears should be made thin and uniform.
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Figs 1.2A to C: Spreading. Diagrammatic representation of the optimal method of spreading a cervical smear. (A) Spatula spread in flat motion onto slide; (B) Brush rolled onto slide; (C) Evenly spread smear
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Figs 1.3A to C: Fixing: (A) Smear flooded immediately and completely with fixative (ployethylene gycol); should not be allowed to air dry; (B) Slide placed immediately into fixative; should not be allowed to air dry; (C) Smear fixation by spraying
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Fig. 1.4: Superficial cell
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Fig. 1.5: Endocervical cell
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Fig. 1.6: Parabasal cell
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Fig. 1.7: Metaplastic cell
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Fig. 1.8: Intermediate cell
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Fig. 1.9: Cancer cell
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PAPANICOLAOU'S STAINING TECHNIQUE
After fixation in equal parts of 95% alcohol and ether for minimum 1 hour or 95% ethyl alcohol or 95% isopropyl alcohol or 100% methanol. The slides are stained by the following method.
Slides fixed by spray, should be kept overnight in 95% alcohol prior to staining.
1.
95% alcohol
10 dips
2.
50% alcohol
10 dips
3.
Three changes in water
4.
Hematoxylin
1 minute
5.
Three changes in water
6.
Ammonia alcohol
45 seconds.
7.
75% alcohol
10 dips
8.
75% alcohol
10 dps
9.
95% alcohol
10 dips
10.
95% alcohol
10 dips
11.
Orange green
2 minutes
12.
95% alcohol
10 dips
13.
95% alcohol
10 dips
14.
Eosin azure 36(EA 36)
2 and 1/2 minutes
15.
95% alcohol
10 dips
16.
95% alcohol
10 dips
17.
100% absolute alcohol
10 dips
18.
100% absolute alcohol
10 dips
19.
Xylol
15 minutes-overnight
Mount in Canada balsam or DPX (distrene, plasticiser, xylene) mountant
Results: Papanicolaou's stained smear shows
  1. Nucleoli: Red, blue
  2. Nucleus: Bluish purple
  3. Cytoplasm: Superficial cells — pink color.
          Intermediate cells and parabasal cells — green or greenish blue.
          Endocervical, endometrial cells — bluish, green or purple.
          Keratin — orange.
 
Preparation of Stains
1.
Hematoxylin
1 g
Distilled water
200 cc
Absolute alcohol
10 cc
Red mercuric oxide
0.5 g
Ammonium potassium sulphate
20 g
Add 20 g of ammonium potassium sulfate in 200 cc distilled water and boil. Add 1 g hematoxylin, 0.5 g of red mercuric oxide, 10 cc of alcohol, cool and stock in amber colored bottle.
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Figs 1.10A to H: (A) Dip fixed smear for 3 minutes in tap water and blot out excess water from the slide; (B) Dip for 60 sec in RAIPD-PAPTM nuclear stain; (C) Add 3 drops of Scotte's tap water buffer and wash after 10 sec. Blot out excess water form the slide; (D) Dip with two change in RAPID-PAPTM dehydrant for 30 seconds each; (E) Dip for 45 seconds in working cytoplasm stain; (F) Wash in tap water and blot out excess water form the slide; (G) Repeat dehydration in a second bath of RAPID-PAPTM dehydrant for 30 seconds and dry at air; (H) Dip in xylene, dry and mount with cover glass using a drop of DPX.
  1. 7EA36 (Eosin azure 36)
    1. Eosin
    2. Bismarck brown
    3. Light green
      Take : 25 mL of solution I
      5 mL of solution II
      7.5 mL of solution III
    1 g/10 mL distilled water
    1 g/10 mL distilled water
    1 g/10 mL distilled water
    Mix above three solutions and make it upto 1000 mL with 95% alcohol. Add 4 g of phosphotungstic acid and 10 drops of lithium carbonate solution.
  2. Orange G: Orange green powder 5 g/50 mL of distilled water to 50 mL of solution i, add 95% alcohol and make 1000 mL. Add 0.15 g of phosphotungstic acid.
  3. May-Grunwald-Giemsa (MGG) Stain
    May-Grunwald reagent
    Eosin methylene blue
    0.5 g
    Absolute methanol
    100 mL
    Giemsa stain
    Azure 2 eosin
    0.6 g
    Azure 2
    0.16 g
    Glycerine
    50 mL
    Absolute methanol
    100 mL
    Working solution of Giemsa—1.10 dilution with distilled water.
    Staining procedure
    May-Grunwald reagent
    5 minutes
    Running water
    1 minutes
    Working solution of Giemsa
    15 minutes
    Running water
    1–2 minutes
    Air dry and mount with DPX.
    Rapid Pap stain is most useful. When cytology decision is needed urgently or on mass cytology screening graphs. Unlike usual pap stain ‘Rapid Pap’ is user friendly. It is 8 steps method which needs minimum skill.
 
INTERPRETATION OF SMEARS
The smear from a normal cervix may contain:
  • Cells from original squamous epithelium of cervix and vagina
  • Cells from endocervical canal
  • Cells from transformation zone
  • Endometrial cells, leukocytes, red blood cells, contaminants, commensal organisms.
 
Cytological Description of Different Cells
  1. Superficial cells: Large polygonal with transparent cytoplasm and sharp cell borders (45–50 m diameter). Nucleus is pyknotic.
  2. 8Intermediate cells: Slightly smaller than superficial cells (35–40 m diameter) with clearly defined chromatin (vesicular), cynophilic dense cytoplasm, navicular cells (boat shaped) in pregnancy.
  3. Parabasal cells: Small cells (15–30 m diameter) with dense cytoplasm and granular large nucleus. The cells are round or with irregular border because they are fragile.
  4. Columnar cells: These are derived from the endocervical epithelium. They appear in smear as a single cells or a sheet of cells. They are tall columnar with vacuolated cytoplasm and basal nucleus and sometimes a tuft of cilia. The sheet of cells often form honeycomb pattern or palisade of cells.
  5. Endometrial cells: Small round with narrow thin rim of cytoplasm virtually filled with a hyper chromatic granular nucleus.
  6. Doderlin's bacilli: Pale blue thin long rods.
 
Infections and Infestations
  • Trichomonas vaginalis: A flagellated unicellular protozoan pear shaped greyish blue with granules.
  • Gardenella vaginalis: Small gram negative club shaped organisms staining dark blue. The granules accumulate on the surface of squamous cells giving rise to clue cells.
  • Candida albicans: Delicate hyphae and occasional spore forms.
  • Actinomyces: Fluffy ball of bacteria with a size range of 10–30 m. Appears as a grayish blue pear shaped body with single ovoid crescentric nucleus.
  • Herpes simplex virus (HSV): The infected cell often contain several nuclei which mould to one another. Nucleus has a ground glass appearance.
  • Human papilloma virus (HPV): Perinuclear hollows and koilocytes. Enlarged hypochromatic nucleus, additional changes include keratinization, dyskeratosis, parakeratosis, anucleate squamous cells.
  • Metaplastic cells:
    • Immature: Resemble parabasal cells.
    • Mature: Estrogenated squamous epithelial cells.
Cell groups intermingled with exfoliated other cell groups. Cells appear in smear as sheets of cells with dense cytoplasm. Some of the cells show vacuolated cytoplasm. Some have long cytoplasmic processes or intracellular bridges.
 
Atypical Squamous Cells of Undetermined Significance
ASCUS (atypical squamous cells of undetermined significance) is a term coined when reporting of cytological smears started with the Bethesda system. ASCUS means the cytologist cannot determine the significance of the identified abnormal cells, so additional workup for a definitive diagnosis is required.
In the new category of the Bethesda system of reporting ASC (atypical squamous cells) reported as ASCUS which is > 80% and ASC-H which is 10% or less. In ASC-H you cannot exclude high grade squamous intraepithelial lesion (HSIL).
Interpretation of ASC depends on the entire specimen and not just a few cells. The ASC require the cells showing squamous differenciation, increased nucleocytoplasmic ratio and minimal nuclear changes. In ASCUS nuclei are 2.5–3 times (35 m) of normal intermediate cell, increased nucleo–cytoplasmic (N/C) 9ratio, nuclear hyperchomasia and irregularity. ASC-H is in Small cell pattern i.e. high nucleo–cytoplasmic (N/C) ratio, atypical immature metaplasia, chromation irregularity, hyperchromasia, abnormal nuclear shape, favouring HSIL or Crowded sheet pattern i.e polygonal cells, dense cytoplasm, fragments with sharp linear edges.
There are different conditions in which smears cannot be interpreted properly and so they come in ASCUS category like atrophic smear, severe inflammations, necrosis, post-radiation changes, IUCD effect, macrophages, air drying, etc.
 
Low Grade Squamous Intraepithelial Lesion
Squamous cell changes associated with histological cervical intraepithelial neoplasia 1(CIN1) and human papilloma virus (HPV) are combined in low grade squamous intraepithelial lesion (LSIL) according to the Bethesda system. Factors associated with the development of LSIL include young age, presence of high risk HPV and duration of infection. LSIL contains both low risk and high risk types of HPV infections. The cytopathic effect of HPV is characteristic koilocyte with an enlarged irregular nucleus.
Cytological criteria for LSIL are cells occurring singly or in sheets which are mature superficial in type with enlarged nucleus and nuclear atypia. Perinuclear cavitation and keratinisation is often present.
Histology shows loss of normal progressive cellular differentiation. 70–80% of lesion of LSIL are either unchanged or resolve spontaneously.
Colposcopic findings: LSIL lesions are gradual in onset and transient in duration. The lesions are pale, translucent, pink-white or dense snow white in condulomatous change. Low grade lesions are more challenging, less precise and less reproducible.
Low grade squamous intraepithelial lesion (LSIL): Enlargement of cell nucleus. Moderate variation in nuclear size and shape (pleomorphism) and hyperchromatacia. Cytoplasm is frequently pushed to periphery creating the koilocytes.
 
High Grade Squamous Intraepithelial Lesion
High grade squamous intraepithelial lesion (HSIL) are charaterized by progressive dedifferentiation, marked nuclear atypia. Cells are small with decreased cytoplasm. Increased nucleo-cytoplasmic (N/C) ratio. Nuclei are hyperchromatic with chromatin clumping.
 
Microinvasive and Invasive Carcinoma
Cells of abnormal size and shape with hyperchromatic nuclei with irregular coarse chromatin. There can be binucleation, mitosis.
 
Glandular Cells
  • Atypical
  • Adenocarcinoma
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Fig. 1.11: Normal
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Fig. 1.12: NILM
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Fig. 1.13: Superficial cells
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Fig. 1.14: Inetermediate cells
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Fig. 1.15: Parabasal cells
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Figs 1.16A and B: Endometrial cells
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Fig. 1.17: Endocervix: End-on view
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Fig. 1.18: Candida spores and hyphae
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Fig. 1.19: Trichomonas
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Figs 1.20A to C: Metaplastic endocervical cells
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Fig. 1.21: Metaplastic cells
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Figs 1.22A to C: Metaplastic with hyperkeratosis
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Fig. 1.23: Hyperkeratosis
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Fig. 1.24: Hyperkeratinized cells
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Fig. 1.25: Koilocytosis
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Fig. 1.26: LSIL
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Fig. 1.27: ASCUS LSIL
 
ASCUS
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Figs 1.28A to C: (A) Atypical glandular cells; (B) Squamous-cell carcinoma; (C) Squamous-cells keratinizing
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REPORTING OF THE SMEAR
There are many classifications which are used to report the smear. The clinicians must understand these and interpret the report correctly.
 
Papanicolaou's Classification
This is the oldest. The smears are classified from Class I to V. Class I is negative. Class II is negative but atypical cells due to infection are seen. Class III is doubtful. Class IV is positive but isolated atypical cells are seen. Class V is positive with numerous atypical cell groups present.
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Flow chart 1.1: WHO classification of Pap smear
 
WHO Classification
  • Recommendation by International Academy of Cytology.
  • 1970-approved -WHO classification.
  • Dysplasia — term was introduced by Dr. G. Pap in 1949 for describing lesion “less than cancer”.
WHO Classification has effective communication between pathologist and clinician, so it is used in most parts of the world.
 
CIN Classification of Richart
The smears are classified as normal.
  • Cervical intraepithelial neoplasia CIN I
  • Cervical intraepithelial neoplasia CIN II
  • Cervical intraepithelial neoplasia CIN III
  • Invasive carcinoma
  • Neoplesia term was misunderstood and was not accepted by clinicians.
 
The Bethesda System (TBS) 2001
Specimen type: Indicate conventional smear (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, processed and examined, but unsatisfactory for evaluation of epithelial abnormality because of (specify reason).
 
General Categorizatin (Optional)
Negative for intraepithelial lesion or malignancy.
16Epithelial 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.
  • Ancilary testing.
  • Provide a brief description of the test methods and report the result so that it is easily understood by the clinician.
 
Interpretation Result
Negative for intraepithelial lesion or malignacy
(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 hyphae ?
  • Shift in flora suggestive of bacterial vaginosis.
  • Bacteria morphologically consistent with Actinomyces hyphae
  • Cellular changes consistent with herpes simplex virus.
Other Non-neoplasitc 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.
Other
Endometrial cells (in a woman > 40 years of age)
(Specify if ‘negative for squamous intraepithelial 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: moderate and severe dysplasia. CIS/CIN 2 CIN 3
  • High grade squamous intraepithelial lesion (HSIL)
  • Encompassing; moderate and severe dysplasia, CIS/CIN 2 and CIN3
    • With feature suspicious for invasion (if invasion is suspected)
  • Squamous cell carcinoma
17Glandular Cell
  • 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
  • Extrauterine
  • Not otherwise specified (NOS)
 
Other Malignant Neoplasms: (Specify)
Educational notes and suggestions (optional)
Suggestions should be concise and consistent with clinical follow-up guidelines published by professional organizations (references to relevant publications may be included)
 
Classification of Smears
Pap 1954
WHO 1968
CIN 1978
TBS 1988-2001
Class 1
Negative
Negative
WNL*
NILM**
Class 2
Inflammatory, squamous, koilocytotic atypia
Negative
Reactive and reparative changes
ASCUS
Condyloma
Class 3
Dysplasia - Mild
Moderate
Severe
CIN 1
CIN 2
CIN 3
LSIL and Condyloma
HSIL
HSIL
Class 4
Carcinoma in situ
CIN 3
HSIL
Class 5
Invasive carcinoma
Invasive carcinoma
Invasive carcinoma
* WNL = Within normal limits** NILM = Negative for intraepithelial lesion and malignancy
 
LIQUID BASE CYTOLOGY
Cervical Cytology by conventional pap method was introduced be George Papanicolaou in 1940-1945. This method of collection, spreading, fixation, staining and examination under microscope is used even today as standard procedure. But the preparation of conventional slide is variable and poorly controlled. The sensitivity of conventional pap smear for cancer detection is around 50%. This is because of errors in sampling.
The sample is collected with a spatula and /or brush in the same way as for the conventional Pap smear. Instead of smearing the sample on the slide, the specimen is washed directly into a vial containing liquid fixative. Slide preparations are made from the liquid sample. The cells are fixed more uniformly, mucus is dissolved and large cell clusters are removed. This method of specimen collection is more costly and transport is difficult. This method may not be currently available everywhere.
18Liquid based, thin-layer technology is developed recently to overcome major limitation of conventional pap smear.
  1. Failure to capture entire specimen.
  2. Inadequate fixation.
  3. Random distribution of abnormal cells.
  4. Obscuring elements—blood, discharge etc.
  5. Technical variability.
There are less and less ASCUS diagnosis report in liquid base cytology because of better fixation and slide quality.
 
VISUAL INSPECTION
Down staging for cervical cancer means detection of cervical cancer in an earlier stage where it can be cured. Down staging is to be done in asymptomatic women using simple methods. In India patients of cancer cervix approach the clinician at advanced stages. That is why down staging is very important.
(Visual Inspection). 1980 – WHO proposed this method as cervical cytology was not possible for everybody in developing countries. Several pilot studies have showed that upto 50–70% of cases of cervical cancer and CIN are detected by visual inspection.
 
Diagnosis by Visual Inspection
 
Introduction
Considerable research to explore the accuracy and acceptability of visual screening is a process of identifying cervical lesions without pap smear cytology.
Early studies:
  • Visual inspection (VI)
    • Any signs of early cancer (not accurate and useful)
  • Visual inspection with acetic acid (VIA)
    • More promising and useful
    • Acetic acid denatures cellular proteins in nucleus and dehydrates cellular cytoplasm so increased N/C ratio appears white.
 
VIA (Visual Inspection with Acetic Acid) Usefulness
  • Reasonably accurate
  • Sensitivity good in high grade
  • Specificity low
  • Over treatment should be avoided
  • Less effective at age of 50 years because squamocolumnar junction (SCJ) recedes inside
  • Thickness, opacity and border definition of the lesion should be done.
 
VIA Advantages
  • Promising in low resource setting
  • Low cost
  • Simple
  • 19Non physicians can perform with adequate and ongoing training
  • Results available immediately
  • Minimally reliant on infrastructure.
 
Different Procedures
  • VI – Visual inspection
  • VIA – Visual inspection with acetic acid
  • VILI – Visual inspection with Lugol's iodine
  • VIAM – Visual inspection added magnavision
    • VIAM – Magnavision sos.
 
Documentation
  • Odell's diagram
  • Hammond's graph
  • Video recording.
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Fig. 1.29: Magnavision
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Fig. 1.30: Odell's diagram
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Fig. 1.31: Diagrammatic representation of Hammond's graph
 
New Technologies
  • Cervicography
  • Electronic detection: Fluorescence Spectrography
  • Polar probe.
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Visual Inspection with Acetic Acid (VIA): Negative
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Figs 1.32A to C: Normal
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Fig. 1.33: Polyp
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Fig. 1.34: Big ectopy
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Fig. 1.35: Cervicities
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Fig. 1.36: Cut with bleeding
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Fig. 1.37: Unsatisfactory colposcopy
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Fig. 1.38: Cyst
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Fig. 1.39: Ectopy with cyst
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Fig. 1.40: Prolapse
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Figs 1.41A and B: Hyperkerotosis
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Figs 1.42A and B: Warts
 
Visual Inspection with Acetic Acid (VIA): Positive
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Figs 1.43A and B:
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Figs 1.43C to H:
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Figs 1.43A to J: Acetowhite area
 
Visual Inspection with Lugol's Iodine (VILI)
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Figs 1.44A to D: VILI
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MANAGEMENT OF SMEARS
  • Normal-NILM (negative for intraepithelial lesion or malignancy), follow up
  • Infection and organisms: Treated with antimicrobial agent, follow up
  • Endometrial cells in postmenopausal smear—endometrial evaluation
 
Epithelial Cell Abnormalities
 
ASCUS (Atypical squamous cells)
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ASC-H
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LSIL (Low grade squamous intraepithelial lesion)
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HSIL – Leep or Leetz procedures (surgical removals)
Features suspicious for invasion
Squamous cells carcinoma
Adenocarcinoma: Refer to gynecological oncologist
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RECORD SHEET
 
Gynecological Cytology
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