The document discusses Pap smear testing, including:
- The definition and purpose of the Pap smear test in screening for cervical cancer.
- Guidelines for patient preparation, specimen collection using an Ayres spatula and cytobrush, sample preparation including fixation and staining.
- Criteria for evaluating specimen adequacy, including what constitutes an adequate, satisfactory but limited, and unsatisfactory smear.
- What constitutes normal cervical cells and abnormalities that may be detected, following the Bethesda system for reporting cervical cytology.
2. Definition:-
The Babeș-Papanicolaou test (also called Pap
smear, Pap test, cervical smear, or smear test) is
a screening test used to detect potentially
pre-cancerous and cancerous processes in the the
endocervical canal (transformation zone) of the female
reproductive system.
Credit for its development goes to Dr George N.
Papanicolaou.
dr. monika nema
3. PPaattiieenntt pprreeppaarraattiioonn
Women should be tested two weeks
after the first day of their last menstrual
period.(Day 14 of cycle is optimal).
Women should not use any vaginal
medication, contraceptive during the 48
hrs prior to sample collection.
Sexual relationship is not recommended
the night before the test.
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4. SSppeecciimmeenn ccoolllleeccttiioonn
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.
• An optimal sample includes cells from the
ectocervix and endocervix
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6. The location of the squamo-columnar
junction ( 8 mm to 13 mm proximal to
the external cervical OS) in most women
varies with the age and fertility.
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8. AAyyeerrss SSppaattuullaa
Concave end to fit the
cervix
Convex end for vaginal
wall and vaginal pool
scrapings.
One end is longer so
that spatula fits the
external OS.
10. CCyyttoobbrruusshh
Easier to introduce into
narrow cervical canal
because the hairs fold
down along the shaft and
is likely to retain cells.
Insert ~ 2 cm (until
brush is fully inside
canal)
Rotate only 180 degrees
(otherwise will cause
bleeding)
13. FFiixxaattiioonn
Fixative is an agent used to prepare
cytologic specimen for the purpose of
preserving and maintaining the existing
form and structure of all constituent
elements.
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14. 95% Ethanol.
95% Rectified Spirit.
100% Methanol.
80% Isopropanol or Propanol.
Ether/95% Ethanol ( 1: 1).
Spray fixatives contains isopropanol and
propylene glycol.
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16. PPAAPP ssttaaiinn
80% alcohol – 6 to 8 dips.
70% alcohol – 6 to 8 dips.
50% alcohol – 6 to 8 dips.
Distilled water - 6 to 8 dips.
Harris Haematoxylene – 6 minutes.
0.25% HCl- 6 to 8 dips.
Running tap water – 6 minutes.
50% alcohol – 6 to 8 dips.
70% alcohol – 6 to 8 dips.
80% alcohol – 6 to 8 dips.
95% alcohol – 6 to 8 dips.
O.G.G- 2 minutes.
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17. PPAAPP ssttaaiinn
95% alcohol – Rinse in two dishes.
E. A 50 - 2 minutes.
95% alcohol – Rinse in three dishes.
100% alcohol – 6 to 8 dips.
100% alcohol – 6 to 8 dips.
Equal parts of absolute alcohol and xylene– Rinse in two dishes.
Xylene– 6 to 8 dips
Xylene– 6 to 8 dips.
Xylene– 6 to 8 dips
DPX mount
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18. LLiiqquuiidd bbaasseedd ccyyttoollooggyy
The liquid based cytology technique
involves rinsing all the material collected
on the sampling device into a fixative
fluid, creating a cell suspension.
Liquid sample is sent to the laboratory
rather than glass slide preparation.
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20. LIQUID BASED CYTOLOGY
a Different types of brushes alllloowwiinngg ttoo ccoolllleecctt cceellllss
ffrroomm tthhee eeccttoocceerrvviixx aanndd eennddoocceerrvviixx..
Ectocervix and 0.5 cm of
endocervix are sampled
The central bristles of the broom are
inserted into the endocervical canal until
the lateral bristles bend fully against the
ectocervix.
The sampling device is rotated 360 degree
in the clockwise direction twice and then
anticlockwise while maintaining gentle
pressure.
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21. The brush is removed aanndd ddeeppoossiitteedd iinn tthhee mmeetthhaannooll
bbaasseedd lliiqquuiidd mmeeddiiuumm..RRBBCC aarree llyysseedd bbyy ttrraannssppoorrtt
mmeeddiiuumm..
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22. TThhiinn PPrreepp mmeetthhoodd
The entire procedure takes about 70
seconds per slide and results in a thin
deposit of cells in a circle 20 mm in
diameter.
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23. SSuurree ppaatthh
Samples are collected in ethanol based
transport medium.
The tip of the collection device is snipped
off and included in the sample vial.
The equipment to prepare slides includes
a Hettich centrifuge and a PrepStain
robotic sample processer with computer
and monitor.
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24. • 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.
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25. LLIIQQUUIIDD BBAASSEEDD CCYYTTOOLLOOGGYY
• Reduces number of inadequate smears and
need for repeat smears
• Thin-Prep appears to be superior to convention
Pap test in detecting SIL.
• Cellular preservation is enhanced.
• Good fixation results in improved microscopic
details.
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29. What we see under the
microscope. Notice the clean back
ground and how well the cells are
dispersed rendering easier dr. monika to
nema
30. Cervical cytology practice guidelines
Test Requisition
The minimal clinical data:
Age
Date of LMP or onset of menopause
Past or Current history of any abnormalities or treatment
Pregnancy status
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34. IInntteerrmmeeddiiaattee CCeellllss
May be small or large
Round nuclei, nucleus
similar in size as
parabasal cells
Entire cell
approximately twice
the size of parabasal
cells
Cytoplasm becomes
angular, irregular and
folded as cell enlarges dr. monika nema
35. SSuuppeerrffiicciiaall CCeellllss
Largest epithelial cell
As they age and degenerate, the nuclei
becomes small, pyknotic and fades.
Cytoplasm may contain vacuoles with age
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36. SSuuppeerrffiicciiaall CCeellllss CCoonnttiinnuueedd
Cornification is the degeneration process
Superficial cells are commonly called
cornified cells
Once nucleus is lost become Anuclear
cells
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37. BBeetthheessddaa SSyysstteemm 22000011
Specimen type
Indicate conventional smear (Pap smear),
liquid based preparation or other preparation (describe)
(A) Statement of adequacy of the specimen.
Satisfactory
Satisfactory for evaluation but limited by
Unsatisfactory.
(B) General categorization of the diagnosis .
Within normal limits.
Benign cellular changes.
Epithelial cell abnormality.
(C) Descriptive diagnosis
Infections
Reactive changes
Epithelial cell abnormalities
Other malignant neoplasms
Hormonal evaluation.
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38. BBeetthheessddaa SSyysstteemm 22000011
Interpretation/result
Negative for Intraepithelial Lesion or Malignancy (NILM)
Organisms
• Trichomonas vaginalis
• Candida species
• Bacterial vaginosis
• Actinomyces species
• Herpes simplex virus
Other non-neoplastic findings-
Reactive cellular changes associated with
- inflammation (includes typical repair)
- irradiation
- Intrauterine contraceptive device (IUD)
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39. BBeetthheessddaa SSyysstteemm 22000011
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/CIN I
• High grade squamous intraepithelial lesion (HSIL)
- encompassing: moderate and severe dysplasia/CIN2/CIN3/CIS
- with features suspicious for invasion (if invasion suspected)
• Squamous cell carcinoma
GLANDULAR CELL
• Atypical glandular cells of uncertain significance
• Atypical glandular cells- endocervical, endometrial NOS
• AGC, favor neoplastic
• Endocervical Adenocarcinoma in situ
• Adenocarcinoma
- endocervical
- endometrial
- extrauterine
- not otherwise specified (NOS)
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41. AAddeeqquuaattee ssmmeeaarr
An adequate pap smear is one that
includes a sampling of both the exocervix
and endocervix.
An adequate cytologic sample contains
more than 300 squamous cells, including
at least two clusters of 5 cells each of
endocervical and/ or metaplastic cells
with mucus material.
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42. This image was composed to depict the appearance of a 4X field with
approximately 1400 cells. It is to be used as a guide in assessing
squamous cellularity of conventional specimens. An adequate
conventional smear has an estimated minimum of approximately
8,000-12,000 well visualized and preserved squamous cells.
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43. Cytomorphologic Criteria:
Satisfactory squamous cellularity on a ThinPrep slide. Endocervical
cells are also present. An adequate liquid based preparation
should have an estimated minimum of 5,000 well-visualized/
preserved squamous cells
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44. Cytomorphologic Criteria:
Distinct cytoplasmic borders are seen in the cluster of cells on the left,
giving a ?honeycomb? appearance. The cell cluster on the right is seen
from a side view, giving the ?picket fence? appearance. Interpretation is
NILM.
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45. This specimen is unsatisfactory due to scant squamous cellularity seen at10X
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48. ““SSaattiissffaaccoottrryy ffoorr eevvaalluuaattiioonn””
Approriate labelling and identifying
information.
Relevant clinical information.
Adequate numbers of well preserved and
well visualized squamous epithelial cells.
An adequate endocervical transformation
zone component.
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49. SSaattiissffaaccttoorryy ffoorr eevvaalluuaattiioonn bbuutt
lliimmiitteedd bbyy……..
Lack of minimum clinical patient
information.
Partially obscuring
blood,inflammation,thick areas,poor
fixation etc that precludes interpretation
of approximately 50% to 75% of the
epithelial cells.
Lack of endocervical / transformation
zone component.
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50. UUnnssaattiissffaaccttoorryy ffoorr eevvaalluuaattiioonn……
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.
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51. Any epithelial abnormality is of
paramount importance and must be
reported regardless of compromised
specimen adequacy.
If abnormal cells are detected, the
specimen is never categorised as “
UNSATISFACTORY”
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53. LLAACCTTOOBBAACCIILLLLII
Lactobacilli are 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. dr. monika nema
54. BBaacctteerriiaall vvaaggiinnoossiiss
Between puberty and the menopause the presence of
lactobacilli maintains a pH of vagina between 3.8 and
4.2.
Changes in pH over 4.5 leads to non specific vaginitis.
It is an infectious disease classically associated with gray
or white,thin, homogenous discharge that tends to
adhere to vaginal walls.
Exudes a characteristic fishy odour when mixed with
10% KOH.
Gardnerella vaginalis – putative pathogen.
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56. HHeerrppeess ssiimmpplleexx
Cytomorphologic Criteria:
Nuclei showing "ground-glass" appearance. Multinucleation, nuclear
molding, and dense eosinophilic intranuclear inclusions surrounded by a
halo are also seen.
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57. AAccttiinnoommyycceess
Cytomorphologic Criteria:
Tangled clumps of filamentous organisms, often with acute angle
branching, sometimes showing irregular wooly appearance. Swollen
filaments may be seen with clubs at periphery. A cotton ball like acute
inflammatory response is common.
Actinomyces is often associated with intrauterine device (IUD)
usage.
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59. TTrriicchhoommoonnaass vvaaggiinnaalliiss
Cytomorphologic Criteria:
Trichomonas is a pear-shaped, oval to round, cyanophilic organism that
ranges in size from 15-30 microns. The nucleus is pale, vesicular and
centrally located. Eosinophilic granules are often visible in the cytoplasm.
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60. AAccuuttee iinnffllaammmmaattiioonn
Dirty background of the smear.
Desquamation of cells in sheets and aggregates.
Large number of degenerating polymorphs.
Dark pyknotic nuclei in superficial and
intermediate cells.
Perinuclear halos.
Increased number of parabasal cells.
Enlarged endocervical cells with
prominent chromocentres.
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69. IIUUDD EEFFFFEECCTT
Calcified debris
Small cluster of glandular cells with cytoplasmic vacuoles displacing
nuclei.creating a signet-ring appearance
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70. RRAADDIIAATTIIOONN EEFFFFEECCTT
Overall increase in
cell size
Cytoplasmic
vacuolation.
Uneven staining of
cytoplasm.
Nuclear enlargement
with vacuolation.
Multinucleation.
Fragmentation of
nuclei.
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72. EEppiitthheelliiaall cceellll aabbnnoorrmmaalliittyy
The diagnosis of ASCUS ( Atypical
squamous epithelial cells of undetermined
significane) is offered only when the
cytomorphological changes exceed the
parameters related to benign,reactive
processes but fall short of a definite
diagnosis of a squamous intraepithelial
lesion.
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73. AASSCC--UUSS
Cells resemble superficial or
intermediate cells.
Nuclear size is increased 2-3 times.
Nuclear boundraies are regularor with
minimum irregularities.
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74. AASSCC--HH ((ccaannnnoott eexxcclluuddee hhiigghh ggrraaddee
iinnttrraaeeppiitthheelliiaall lleessiioonn))
Cells resemble parabasal or basal in
configuration and size.
Nuclei are hyperchromatic with uneven
chromatin pattern.
Nuclear membrane is thick and uneven.
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81. In CIN I and III , the characteristic
nuclear changes are better observed in
intermediate cells.
As the grades of CIN increase, parabasal
and basal cells start showing nuclear
abnormalities.
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85. HHPPVV iinnffeeccttiioonn
Seen in most of the cases of CIN.
Lesions of CIN I and CIN II are usually
positive for HPV 6,11,31,42.
Lesions of CIN III and invasive cancer are
usually positive for HPV 16,18,33.
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86. KKOOIILLOOCCYYTTEE ((HHPPVV))
Large squamous
cell with
enlarged
hyperchromatic
nucleus & large
sharply
demarcated
perinuclear clear
zone
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87. PPSSEEUUDDOOKKOOIILLOOCCYYTTEESS
Glycogen in squamous
cells can give the
appearance of
"pseudokoilocytosis".
Nuclear abnormalities
required for an
interpretation of ASC-US/
LSIL are absent.
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88. SSqquuaammoouuss cceellll ccaarrcciinnoommaa-- nnoonn
kkeerraattiinniizziinngg
Irregular chromatin,
prominent nucleolus
Dysplastic squamous cells with anisocytosis and anisonucleosis
including keratinization and tadpole cells are diagnostic of invasive
squamous cell carcinoma.
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90. AAttyyppiiccaall eennddoocceerrvviiccaall cceellllss
enlarged round or oval nuclei with prominent
nucleoli.
Mitotic figures
Sheet of cells with enlarged, variably-sized nuclei with some crowding
and overlap of nuclei.
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91. EENNDDOOCCEERRVVIICCAALL AADDEENNOOCCAARRCCIINNOOMMAA
IINN SSIITTUU ((AAIISS))
Atypical columnar endocervical cells, with enlarged, elongated and
hyperchromatic nuclei.
Typical feathering and palisading.
Explanatory Notes:
Pseudostratification, nuclear crowding and feathering are classic features of
AIS dr. monika nema
92. AADDEENNOOCCAARRCCIINNOOMMAA
Cytomorphologic Criteria:
Cluster of cells with enlarged round or oval nuclei, irregular chromatin
distribution and prominent nucleoli.
Explanatory Notes:
Irregular chromatin distribution and prominent or macronucleoli are a
classic findings in invasive endocervical adenocarcinoma.
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93. Invasive adenocarcinoma
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IINNVVAASSIIVVEE AADDEENNOOCCAARRCCIINNOOMMAA::
Cells are round,crowded,
hyperchromatic
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96. HHoorrmmoonnaall ccyyttoollooggyy
Maturation of vaginal squamous cells
form 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.
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97. 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.
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98. CCeelllluullaarr iinnddiicceess ffoorr hhoorrmmoonnaall
aasssseessssmmeenntt
Karyopyknotic index
Eosinophilic index
Folded cell index
Crowded cell index
Maturation index
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99. KKaarryyooppyykknnoottiicc iinnddeexx
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.
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100. EEoossiinnoopphhiilliicc iinnddeexx
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.
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101. FFoollddeedd cceellll iinnddeexx
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.
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102. CCrroowwddeedd cceellll iinnddeexx
Represents the relationship of mature
squamous cells lying in clusters of four or
more cells, to all mature squamous cells.
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103. MMaattuurraattiioonn iinnddeexx
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.
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104. MMaattuurraattiioonn vvaalluuee
Meisels suggested that a value be given to
each category of cells i.e, value of 1.0 to
superficial cells, 0.5 to intermediate cells
and 0.0 to parabasal cells.
This system gives single value ranging
from 0-100 to express hormonal status.
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105. Maturation value
100 – purely superficial cells.
0 – purely parabasal cells.
50-95 – in normal menstruating woman.
< 50 – atrophic squamous epithelium.
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106. DIFFRERENT PHASES OF MENSTRUAL
CYCLE
ESTROGEN PHASE
eosinophilic superficial squamous
cells, clean
background
.
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107. OVULATORY SMEAR
fern-like crystalline structures
eosinophilic superficial
squamous cells
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108. . ( PROGESTERONE PHASE
basophilic squamous cells with folded
cytoplasm (boat cells)
Glycogen
deposits
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109. PROGESTERONE PHASE WITH
ABUNDANT LACTOBACILLI AND
CYTOLYSIS
navicular cells
Naked nuclei
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110. MMeennooppaauussee
Maturation of cervical epithelium is
estrogen dependent
Due to its insufficiency, maturation is
retarded.
Number of mature squamous and
intermediate cells is reduced.
In early menopause intermediate cells
increase in number.
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111. AAttrroopphhyy
Cytomorphologic Criteria:
Parabasal cells and blue blobs.
Explanatory Notes:
Overall cellularity of smear is reduced. Small and large parabasal cells are
seen with almost absence of superficial cells.
Blue blobs are globular collections of basophilic amorphous material
reflecting either degenerated parabasal cells or inspissated mucus
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112. AAmmeerrcciiaann ccaanncceerr ssoocciieettyy
rreeccoommmmeennddaattiioonnss
Screening should begin no later than age 21.
Screening should begin earlier than age 21 if the patient is sexually
active. In this case, it should start 3 years after initiation of vaginal
intercourse.
Once initiated, screening should be performed annually.
After age 30, for women who have had 3 consecutive normal pap
smears, screening frequency may be reduced to every two to
three years.
Women who are HIV positive, immunocompromised should
continue annual screening.
Patient tested positive for HPV, should continue to be screened
indefinitely.
May stop after age 70, if patient is low risk and has had three
normal pap smears over the last 10 years.
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113. In developing countries where there are
no organized screening programme for
pap test due to financial constraints, poor
infrastructure or inadequate human
resources, the WHO recommends five
yearly five pap tests during life span of 35
to 55 years.
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In obtaining the Pap smear, it is important to sample the &quot;Squamo-columnar Junction.&quot; This is the circular area right at the opening of the cervix where the pink, smooth skin of the cervix meets the fiery-red, fragile, mucous-producing lining of the cervical canal. If there is going to be a problem with cancer or precancerous changes, it is this area that is most likely to be effected. This area is also known as the SQJ, or transition zone.
The Ayers spatula is specially designed for obtaining Pap smears. The concave end (curving inward) fits against the cervix, while the convex end (curving outward) is used for scraping vaginal lesions or sampling the &quot;vaginal pool,&quot; the collection of vaginal secretions just below the cervix.
The spatula is made of either wood or plastic. Both give very satisfactory results.
Push the cytobrush into the canal, no deeper than the length of the brush (1.5 cm - 2.0 cm). Rotate the brush 180 degrees (half a circle) and pull the cytobrush straight out. Don&apos;t keep spinning the brush round and round or you will cause bleeding. Even the 180 degree rotation may cause a little bleeding but usually it doesn&apos;t.
Label the slide with pencil on the frosted end.
Two slides may be made, one for the spatula and one for the brush (“two-slide” technique). Alternatively, a single slide may be used (the “one-slide” technique) in which the brush is spread on one half the slide and the spatula is used on the other half. Both techniques give good results.
Conventional Pap Smear (Macroscopic)
•The manual smearing method is readily noted macroscopically. Conventional slide experience is transferable to the ThinPrep process. Cytology does not have to be relearned, base knowledge is merely refined.
CP
•Microscopically, the uneven distribution of cellular material associated with the CP smear pattern is evident.
TP (Macroscopic)
•The key difference in presentation is no longer seeing the smear pattern. ThinPrep takes the same material which is concentrated onto the center of the slide in a thin, uniform layer. Specimen preparation is standardized, eliminating the inconsistency associated with manual preparations.
TP
Same Patient
•Tissue architecture is maintained. TP rearranges the relationship of cell groups on the glass slide. A group/sheet of endocervical cells present represents this.