TOPIC: CYTOSCREENING & PAP SMEAR ➢ To review other screeners and biomedical scientist’s ❖ Requires sitting in a confined position
❖ Requires sitting in a confined position carrying out
Specific objectives:
❖ Appreciate the work of a cytoscreener
➢ Not popular in the Philippines, but outside the country,
it is accepted and you earn more.
❖ Discuss the basic procedures involved in Pap Smears
❖ Explain how the Bethesda System 2014 is being reported
Cytoscreener Required in London
Job Purpose
❖ The post holder will be employed as a trainee Cytoscreener,
working towards qualification as a cytology screener
working within National Health Service Cervical Screening
Programme (NHSCSP) guidelines
❖ Providing services to women w/in the screening
programme, diagnosing (-) and unsatisfactory cervical
smears (TAKE NOTE! You’re allowed to diagnose only the
negative and unsatisfactory smears) and referring
abnormalities to senior colleagues
❖ Microscopically recognize the range of normal and
abnormal cellular content, identifying abnormalities thereby
helping to prevent the development of cervical cancer and
reducing the morbidity and prevent the mortality from this
disease.
Cytoscreening / Cytoscreener Main Duties / Responsibilities
❖ Screening and reporting of cervical smear specimens using
knowledge, skills, and competencies attained through the 2-
year NHSCSP training programme
❖ Each specimen requires reviewing microscopically at 100x -
200x magnification and reviewing the patterns of cells and
staining to detect abnormalities
➢ If NO abnormality is detected, to take responsibility for
the diagnostic opinion of negative or unsatisfactory
➢ Where abnormality is detected, to mark the
appropriate cells and refer to a consultant or more
senior member or staff
o Each prep may have up to 300,000 cells
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negative and unsatisfactory preparations as part of the
internal quality control mechanism to ensure that all
non-abnormals are rapidly reviewed by another
member of trained staff
❖ Requires training up to a full working knowledge on the
national standards for reporting cervical smears which
includes knowledge of recall times depending on cytological
assessment, clinical history, and screening history
❖ Requires training up to taking an active part in all QA
measures including rapid review, annual; National
proficiency test and national circulating slides
❖ Must also attend regular update courses to maintain
diagnostic accuracy
Communications and Relationships
❖ Working as part of a team to ensure accurate, timely
reporting of results. Communicating areas of concern
through line manager and working to resolve issues.
❖ The post holder will, when qualified, communicate complex
info directly to other clinicians e.g.
❖ Results of dx in cervical cytology will be discussed with
consultant cytopathologist/senior/chief biomedical
scientist and opinion maybe challenged.
❖ Attending and contributing to department meetings.
Knowledge, Training, and Experience Required to Do the Job
❖ Will gain practical and theoretical knowledge of the
preparation of cervical samples.
❖ A minimum of 2 years in house training, supplemented by
the mandatory courses delivered by the Scottish Training
School and the attainment of the National Certificate of
Cytology.
➢ This certificate examination is a three-part exam; a
prerequisite is the examination of 5,000 slides over a
minimum of 2 years.
❖ When qualified, participation in the National proficiency
testing scheme
❖ When qualified, participation in External and Internal QA
schemes
❖ Update training according to NHSCSP guidelines
❖ Participation in local and national scientific meetings as
appropriate
Demands of the Job
1. Physical Skills
❖ Requires manipulation of cervical smears being viewed
by microscopy for up to 4 hours in a session and on a
daily basis
2) Physical effort (Daily Requirement)
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microscope work e.g. cytology screening and quality
control of cytology slides. (Up to 4 hours/day)
❖ Requires keyboard to be used for up to 2 hours session
3) Mental
❖ Continuous requirement to concentrate for periods of
up to 4 hours/day while screening cervical smears
microscopically.
4) Emotional
❖ Very occasional exposure to situations where the
smear may have been screened and an abnormality
although present not detected and picked up at quality
review
5) Working conditions
❖ Frequent (daily) unpleasant working conditions
handling cervical smear fluids and preparing for
microscopy.
Decisions and Judgements
❖ When trained, will work on own initiative deciding within
competence if a case should be referred to a senior member
of staff for reporting
❖ Rigorous, constant internal QC involves being prepared to
have judgments scrutinized and challenged, which,
although professionally necessary and proper, can be
challenging.
❖ Participation in team discussions on cases where opinions
are challenged and defended.
Most Challenging/Difficult Parts of the Job
2 Cs = Concentration and Confidence
❖ Levels and extent of Concentration required for screening
microscopically over prolonged periods of time.
❖ Maintaining confidence after an abnormality being picked
up at a Quality review which you had not detected.
Cytology
Cyto = cells and logy = logus means study
❖ Study of cells
❖ Most successful application of clinical cytology =
Diagnosing cervical abnormalities before they develop into
invasive cervical carcinoma
➢ Actually guys in the laboratory if you remember your
Clin Mic. you were already doing cytologic examination
there. Imagine the fluids from the peritoneum.
Pericardium, CSF. You are going to examine the cells.
So studying them is already cytology but what makes
it more successful is studying the fluid of the cervix and
identifying cervical cancer, because cervical cancer
can really kill and it is really common among women.
❖ Used for screening and follow-up of cervical carcinoma,
particularly squamous carcinoma
➢ So here, Cytology in cervical cancer is not just to
diagnose but also screen and follow up these
carcinoma particularly squamous cell carcinoma
Nowadays, adenocarcinoma and adeno-squamous
carcinoma are on the rise, so these are studying the
cells in the smears from the cervical and vaginal canal
and these are what makes PAP smear very successful.
❖ Cytology smears are sensitive to abnormalities, although
there is variable interpretation on any particular smear; The
most important factor is to detect an abnormality and to
start an appropriate management plan.
❖ Specimen should be obtained and prepared by trained
individuals.
➢ You interns during the training will prepare the smears
from the obtained from the midwife, nurse,
gynecologist or any practicing physician and you are
going to prepare them for microscopy. If the collector
has inadequate sampling then this cytology will be a
disaster. If you as a cytoscreener will have errors this
will also lead to a disaster.
❖ False (-) tests are often due to poor quality specimens and
inadequate sampling, erroneous interpretation, and error by
screeners.
Pap Smear History
Smear Daddy
PATHOLOGIST George Papanicolaou
❖ exocervix
Discovered in 1928 that uterine cancer could be detected by
❖ ➢
microscopically examining cells from tissue surfaces
scraped off the interior of the vagina.
From the vagina of his wife he was able to take some
➢ Doc doesn’t encourage the use of cotton pledget because
fluid and then examine them
Then identified that there are changes in the cells of the
➢ will absorb the cells. In doing so, the cells may not be
vagina/vaginal canal
❖ It took decades for medical science to recognize the
significance of Papanicolaou’s test, now commonly called a
Thanks to all those who volunteered to help <3 -Bori😿
pap test or pap smear, which provides for quick early 1. Specimens should have a sufficient number of cells from
detection of cervical and uterine cancer. the area in question.
❖ In fact, if he was able to discover it in 1928, it was only in the 2. Smears should contain well preserved cells uniformly
1960s, wherein the American Medical Association (AMA) distributed so that each cell can be individually examined.
began recommending annual pap smears for women in ➢ Medtechs’ job starts here. They prepare the cells in the
1960, and Dr Papanicolaou died only 2 yrs later, before the laboratory.
efficacy of pap smears was widely known 3. Staining procedure should clearly define the details of all
❖ Since his test became routine, the rate of cervical cancer in structures.
North America has dropped by about 70%, and the lives of
millions of women have been extended. Midwives (in the clinics), trained barangay health workers,
➢ This is an example: For the anti-vaxxers, Do we need nurses, post-graduate interns, residents, and gynecologists can
somebody to die before you are going to accept the collect
safety and efficacy of a certain test/vaccine?  2&3 is the job of the medtech.
➢ It happened to Dr. Papanicolaou: 40 yrs before the  1&2 can also be done by the collector because once you
world accepted the safety and efficacy of screening can collect (When the proper collection is being followed)
test, which is simply a PAP Smear then you can provide the sufficient number of cells and
then you can have the cells well-preserved if you can fix
PAP Smear (Test) them right away.
❖ Unlike COVID vaccine which is for treatment, PAP is a
diagnostic tool.
Smear Sampling/Slide Preparation
❖ It can detect cancerous or precancerous conditions of the
(Conventional Cytology)
cervix.
❖ Most invasive cancers of the cervix can be detected early if
women have Pap tests and pelvic examinations.
❖ Screening should start within 3 yrs after first having vaginal
intercourse or after the age of 21.
❖ After the 1st test
➢ There are so many criteria, so many guidelines in the
market. What doc presents here is just the basic since
associations, gynecologists from different countries
have different guidelines
❖ Smearing of the exocervical sample with a wooden spatula
❖ A woman should have a Pap smear every 2-3 years to check
(Ayre’s spatula)
for cervical cancer.
➢ Some may have a longer bifid extremity for a better
➢ Women should have annual pap smears or every 2-3
endocervical sampling
years especially those who are already sexually active.
❖ Smearing of the endocervical sample taken with the thinner
❖ If you are over age 30 or your Pap smears are (-) for 3 yrs in
extremity of the wooden spatula.
a row, the Doctor may tell you that you only need a Pap
❖ This is a spatula. (Used a long time ago but still may be used
smear every 3(5) years.
in barangays and mountainous areas). A piece of wood
➢ Depends on the clinician on how they have assessed
wherein there are 2 tips: one is a bifid (blunt and shaped like
your status.
the end of a bone) and the other one is pointed.
❖ If you or your sexual partner have other new partners, then
➢ The bifid end is used to reach the vagina or the
you should have a pap smear every 2 years.
❖ After age 65-70, most women can stop having pap smears
The pointed end is for the endocervix (Quite painful)
as long as they have had 3 (-) tests within the past 10 years.
▪ Both ends will be used for smearing.
❖ If you have a new sexual partner after age 65, you should
❖ Nowadays, there are midwives that use cotton pledget(?).
begin having Pap smear screening again.
cotton, if wet with the fluid from the cervix and the vagina,
Specimen Collection
❖ In the collection and preparation of smears for cytological
transferred into the smears.
examination, the major objectives are:
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❖ Spray fixation: Immediate, Liquid-Based Preparation vs Conventional Smear
during a few seconds, with a
spray/slide distance around
20 cm.
➢ Done after smearing
➢ Once the gynecologist or
the midwife will get the
spatula/brush/cotton
pledget and make the ❖ Automated machine which
smear. The laboratory forms part of the liquid based
gives 95% alcohol (fixative) to the specimen collector. cytology
After getting the smear, simply drop it in a container ❖ Reduces number of
(e.g. coplin jar) containing the fixative within 1-2 inadequate smears and
seconds. This is because the pap smear is not air dried need for repeat smears
but it is a wet smear (Not like sputum, blood smear) ❖ Thin-Prep appears to be
therefore it should be fixed in the 95% alcohol. superior to convention Pap
➢ Make sure that the distance from the spray to the slide test in detecting SIL
is around 20 cm (7-8 inches). The distance should be ❖ Approved by US Food and
CONVENTIONAL SMEAR
followed because if you are going to spray, the spray Drug Administration in 1997 LIQUID-BASED PREPARATION (LBP)
(PAP SMEAR)
itself gives pressure. If not properly done, this may ❖ Major companies are Cytyc (ThinPath) and TriPath Imaging
❖ put it on the fixative and send
dislodge the cells in the smear. Doc prefers to drop the (SurePath) ❖ get the brush and
it to the lab wherein you will
smear into the fixative and it is very easy. ❖ Can use residual material to prepare cell blocks and for simply place it
perform (...) and compare this
➢ After fixing, the smear will be sent to the lab for immunohistochemistry (immunohistochemical staining onto the smear
using a special machine
examination. and examination afterwards) ❖ directly making
❖ BETTER & MORE EXPENSIVE
➢ These machines are now commonly seen in the smear
because, aside from the
Liquid based Cytology laboratories ❖ all the pus, mucus,
centrifuge, you filter (?) &
blood, and debris
remove all the red cells, pus,
are still present
mucus, & debris

PAPANICOLAOU (PAP) STAIN

❖ alcohol dried; better for nuclear detail (hence, no air drying)

blue- Ribosomes, particularly parabasal cells, mesothelial

green cells, and metaplastic squamous cells

(1) (2) (3) pink Metabolically inactive cells (e.g. superficial cells)
❖ Three examples of liquid-based cytology: cells are
1.) Head of spatula, where cells is lodged, is broken off into small
distributed on a circular surface of variable diameter or
glass vial containing preservative fluid or rinsed directly into orange Keratinized cells or thick specimens (benign or
rectangular.
preservative fluid. malignant)
➢ After putting it in a smear (either rectangle or circle),
❖ Curved part: For the ectocervix then you can produce these slides: ❖ Fix quickly and stain carefully
❖ Tip - Endocervix
❖ Air dried smears are inadequate because it will look like the
➢ As the collector (if you are the physician/ gynecologist/
image
midwife) and into the cervical os(? Choppy part)
below:
➢ The ectocervix of your patient and after that you swirl
this (the spatula) and drop into the fixative or rinse it
directly into the preservative fluid.
2.) Sample is sent to the lab, then spun (centrifuged) and treated
to remove mucus, pus or other obscuring material.
3.) Random sample to remaining cells is taken and deposited
onto a slide.

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 Adequacy Description: Air Drying Artifact SPECIMEN TYPE
❖ Cytomorphologic Criteria: ❖ Indicate:
➢ Enlarged pale nuclei with loss of nuclear detail ➢ Conventional Smear (Pap smear)
➢ Air dried nuclei flatten out (as opposed to well-fixed ➢ Liquid-based preparation (LBP)
cells, which maintain a more 3-dimensional shape), ➢ Other Preparation (describe)
and do not take up stain very well ❖ Diff-Quik stain in Cervical Cytology
➢ an air dried, Giemsa type stain (unlike Pap Smear: which
➢ This leads to enlarged pale appearance
is alcohol dried)
➢ Cytoplasm is also degenerated and evaluation of cells
➢ Better for background material or to assess adequacy
is difficult
of endocervical smears to detect Chlamydia
❖ Air Drying Artifact-Cervical Cytology
trachomatis
➢ Due to delay in immersion in alcohol fixative
➢ Used for fine needle aspirates, not for cervical smears
➢ More common on conventional than liquid based
smears
SPECIMEN ADEQUACY
➢ Specimen is unsatisfactory if more than 75% of cells
❖ Satisfactory for evaluation
show air drying; if less extensive may be mentioned as
➢ Describe presence or absence of
a quality indicator
endocervical/transformation zone component and any
o May cause discrepant diagnosis of LSIL or less for
other quality indicators
HSIL smears
➢ e.g. partially obscuring blood, inflammation, etc.
o Associated with ASCUS in perimenopausal
❖ Unsatisfactory for evaluation (specify reason)
women
➢ Specimen rejected/not processed (specify reason)
o e.g. specimen not labeled, slide broken, etc.
THE BETHESDA SYSTEM 2014
➢ Specimen processed and examined, but unsatisfactory
❖ Specimen Type
for evaluation of epithelial abnormality because of
❖ Specimen Adequacy
(specify reason)
➢ General Categorization (optional)
o e.g. obscuring blood, inflammation, etc.
❖ Interpretation/Result
➢ Adjunctive Testing
➢ Computer-Assisted Interpretation of Cervical Cytology ASSESSMENT OF SPECIMEN ADEQUACY (Satisfactory &
➢ Educational Notes and Comments Appended to Unsatisfactory)
Cytology Reports (optional)
Adequate number of squamous cells
❖ Conventional smear: estimated minimum of approx.
8000-12000 well-preserved and well-visualized
squamous epithelial cells
❖ LBP: minimum limit of 5000 well-preserved and well-
visualized squamous epithelial cells

Representation of microscopic fields with the following

parameters:

OBJECTIVE OCULAR FIELD NUMBER # of “cells”

4x 10x 20 500

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❖ The “cell” size relative to the entire field corresponds
to superficial squamous cells (50 um)
❖ 20 fields would need to be covered at this level of
cellularity to have 10,000 cells
The presence or absence of endocervical cells should be
reported
❖ An adequate number of endocervical cells (at least
10-well preserved endocervical or metaplastic cells)
confirms sampling of transition zone
❖ Presence of endocervical cells indicate that the
upper limit of transformation zone was included, so
collection is adequate
2 “Negative for intraepithelial lesion or malignancy”
❖ Cytology: usually columnar cells (the length is longer
than the width, the nucleus is placed at the base)
with vacuolated or granular cytoplasm, prominent
cell borders, basal nuclei with fine granular
chromatin and occasional nucleoli
❖ Honeycomb appearance en face
❖ Ciliated if tubal metaplasia
Specimen with more than 75% of cells obscured by
inflammation and bacteria is unsatisfactory (however,
should still report presence of abnormal cells)
3
❖ Any specimen with abnormal cells (even with
only one abnormal cell) is by definition
satisfactory by evaluation!
❖ OBSCURING FACTORS:
➢ SATISFACTORY: 50-75% of cells obscured
➢ UNSATISFACTORY: >75% of cells obscured
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❖ SAMPLE REPORTING ORGANISMS
➢ Satisfactory for evaluation. No endocervical/T-zone Trichomonas vaginalis
component is identified. Approximately 60% of the cells
are partially obscured by thick areas
➢ Specimen processed and examined, but unsatisfactory
for evaluation of epithelial abnormality because of
obscuring inflammation.
❖ GENERAL CATEGORIZATION (optional)
➢ Negative for intraepithelial lesion or malignancy (see
Interpretation/Result)
➢ Other: See Interpretation/Result
o e.g. endometrial cells in a woman ≥ 45 years of
age
➢ Epithelial Cell Abnormality: See Interpretation/Result
o specify ‘squamous’ or ‘glandular’ as appropriate
INTERPRETATION/RESULT
❖ COMPUTER-ASSISTED INTERPRETATION OF CERVICAL
CYTOLOGY: if case examined by automated device, specify
device and result
❖ ADJUNCTIVE TESTING: provide a brief description of the test
method(s) and report the result so that it is easily
understood by the clinician
❖ EDUCATIONAL NOTES AND COMMENTS APPENDED TO
CYTOLOGY REPORTS (optional)
❖ Interpretation/Result
➢ When there is no cellular evidence of neoplasia (new
growth), 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
NON-NEOPLASTIC FINDINGS (optional; list is not inclusive)
Non-Neoplastic Cellular Variations
❖ Squamous metaplasia
❖ Keratotic changes
❖ Tubal metaplasia ❖ How to identify T. vaginalis? Look for the thin and elliptical
❖ Atrophy nucleus
❖ Pregnancy-associated changes ➢ Neutrophils: lobulated and segmented nuclei
➢ Squamous epithelial cells: rounded nuclei
Reactive Cellular-Changes Associated With: ❖ Oval or pear-shaped organism
❖ Inflammation (includes typical repair) ❖ 8-30 um
➢ Lymphocytic (follicular) cevicitis) ❖ Trichomonad nucleus is thin and elliptical; must be
❖ Radiation identified to diagnose this infection
❖ Intrauterine Contraceptive Device (IUD) ❖ Flagella are never seen in the Pap Smear
❖ Infection is associated with itching, foul-smelling, yellow-
Glandular Cells Status Post Hysterectomy green discharge, clinically (strawberry cervix: really red)
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Fungal organisms morphologically consistent w/ Candida spp. Bacteria morphologically consistent with Actinomyces spp. Cellular changes consistent with Herpes simplex virus

❖ (referring to picture) we cannot identify the Neisserria or the

rods inside the neutrophil because the neutrophils are small
❖ In PAP smear, we are only the HPO ❖ Cellular changes consistent with Herpes simplex virus
❖ “3 M’s of Herpes”
➢ Margination of nuclei
❖ In PAP Smear, you do not speciate (do not say C. albicans,
➢ Molding - you cannot distinguish a single nucleus
simply say Candida spp.)
anymore
❖ Pseudohyphae (sticks) and yeast (stones)
➢ Multinucleation
❖ Clinical findings include itching and discharge (thin, watery,
❖ Multinucleated cells nuclei have ground glass appearance
or characteristic white cottage cheese-like)
due to accumulation of viral particles, which causes
❖ Candida cannot be speciated based on morphology; a
peripheral margination of chromatin; also nuclear molding
culture needs to be performed
❖ (+) dense, intranuclear Cowdry-type A viral inclusions
❖ Pap smear sensitivity is approximately 80%
❖ High neonatal morbidity & mortality; high risk for CIN/ SIL

Shift in flora suggestive of bacterial vaginosis

Cellular changes consistent with cytomegalovirus
❖ Associated with IUD (intraurine device) use
(one cell with a very big nucleus)
❖ IUD is one way of preventing pregnancy
❖ Variably gram (+), long, thin, filamentous bacteria that are
reddish, branch, are irregularly beaded, and radiate from a
central area
❖ Associated with fuzzy masses of bacteria (“dust bunnies” in
PAP smear)
❖ You have to do a culture in order to speciate (?) this one

❖ Gardnerella vaginalis (clinical condition [you simply say]:

shift in flora suggestive of bacterial vaginosis)
➢ Is a gram (-), comma-shaped coccobacillus
➢ The bacteria tend to agglomerate onto squamous cells
(e.g. clue cells)
➢ Is found in approximately 90% of non-specific bacterial
vaginosis
❖ Clue cells have a velvety surface, obscured cell edges, and ❖ Although rare, CMV may be encountered in PAP smears
are covered by small coccobacilli ❖ The typical cytologic features of cellular enlargement,
❖ Profuse foul-smelling (fishy), yellow-gray discharge, with prominent intranucelar inclusions, and occasional
itching and burning binucleation are present in this specimen.
❖ Bacterial vaginosis is a polymicrobial process ❖ Cytoplasmic inclusions are often indistinct with PAP stains
❖ Cellular change with the virus itself going into the cell

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 ❖ Standard reference or internal control: nuclei of the
intermediate cell
❖ Nuclear area of 35 um2 ot 7 to 8 diameter = rbc
❖ Chromatin pattern
❖ If you are going to identify squamous metaplasia and other
cells which are dysplastic or cancerous, then identify the
nuclei of the intermediate cell.

Intermediate squamous cells - cervical cytology

❖ Predominate in luteal phase
❖ Luteal phase- phase after menstruation where the uterus
will undergo the proliferative phase (ovaries on the other
hand will undergo follicular phase)
❖ Cytology:
❖ Both are STIs (sexually transmitted infection) ➢ Cytoplasm is polygonal, transparent, basophilic,
❖ Gardnerella, bacterial vaginosis, candidiasis are not STIs flat/thin (due to keratin)
➢ You only get Gardnerella and BV is you are doing so ➢ Nucleus is about the size of an rbc, is vesicular, round/
much washing in your vaginal canal oval
➢ Too much washing removes the good bacteria in your ➢ Nuclear texture and size is reference fro dysplasia ❖ The superficial cells have the largest nuclei to cytoplasmic
body ➢ May see cytolysis/ dirty background, and Doderlein ratio
➢ If you are immunocompromised/ diabetic, you could bacilli ❖ More abundant cytoplasm
get Candidiasis but not T. vaginalis or Herpes unless ❖ They are eosinophilic, their nuclei are smaller and pyknotic,
you sexually active and promiscuous dark, and regular in size
❖ They (nuclei) have uniform borders with no discernable
NON-NEOPLASTIC CELLULAR VARATIONS
chromatin pattern
❖ Squamous metaplasia
❖ Keratotic changes
❖ Tubal metaplasia
❖ Atrophy
❖ Pregnancy- associated changes
(Only squamous metaplasia and atrophy will be discussed)

Intermediate Cell Nucleus

❖ This image shows predominantly intermediate

squamous cells
❖ These cells lie beneath the superficial layer
❖ The cytoplasm is generally cyanophilic
❖ The nuclei are round but are not as pyknotic as those of
superficial cells
❖ The chromatin is finely granular and nucleoli are not visible
❖ Cells stained pink: inactive superficial squamous cells
❖ Cells stained purple: intermediate cells (their nuclei is your
reference in evaluating PAP smears)
❖ If you are going to have a PAP smear during your
menstruation, all that will be seen is blood

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 Different Types of Squamous Cells Normal Squamous Parabasal Cells on PAP Smear (High Power)

❖ Squamous metaplasia in a cervical smear

➢ In a squamous metaplastic cell, the nucleus is larger
and the cytoplasm is less abundant compared to
superficial and intermediate cells. The cytoplasm is also
❖ Types of Squamous Cells abnormal and pale-like because they are from
➢ A: Superficial Cells (arrow) ❖ The parabasal cells have a nucleus with finely granular
columnar cells becoming squamous (metaplasia).
➢ B: Intermediate Cells chromatin..
➢ C. Parabasal Cells ❖ The nucleus has a delicate membrane and no visible
➢ D: Metaplastic Cells nucleolus.
❖ The N:C Ratio is much higher than that of the intermediate
cell.
❖ Scattered neutrophils are also seen.
❖ Doc C: “It’s quite difficult if you see both squamous
metaplasia and parabasal cells since they more or less look
the same microscopically. Patient’s age and clinical history
(if the patient is pregnant or undergoing hormonal therapy
to prevent pregnancy) is important to determine parabasal
cells since these are prominent in the aforementioned
cases.”

❖ Squamous Metaplastic Cells (doc is putol-putol in this part -

1:40:00)
➢ “...after pregnancy, they what (?)” :”)

(becomes okay at 1:41:50)

Parabasal Cells (Cervical Cytology)
❖ Associated with atrophy (seen in postpartum women and
❖ Doc C Tips:
menopausal women), postpartum or prolonged use of
➢ Pyknotic nucleus, pink cytoplasm = Superficial
depot-medroxyprogesterone acetate
➢ Vesicular nucleus, bluish cytoplasm = Intermediate
❖ Cytology:
➢ CYTOPLASM: round, dense, basophilic
➢ NUCLEUS: vesicular, central, active, round
➢ May see naked nuclei
➢ Higher N:C Ratio and smaller in size than intermediate
cells ❖ (might wanna watch at 1:43:00 since putol-putol again at
1:43:18-1:44:32)

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 o Dysplastic: At least 3x
Normal Endometrial Glandular Cells • Dysplastic Cell
❖ Clinical History: 60-year old female
❖ Interpretation: NILM: Atrophy with inflammation (atrophic ❖ Under a 40-year old ✓ “Big and dark nuclei”
woman, not menopause. • Grade of Dysplasia
vaginitis)
Presence of endometrial cells. ✓ Amount of cytoplasm
➢ NILM = Negative for Intraepithelial Lesion or
❖ No specific ✓ N:C Ratio
Malignancy
recommendation. (obj. 40x) • Nuclear Membrane Irregularities
❖ Cytomorphologic Criteria: Parabasal cells with mostly
bland nuclei (some showing air drying) ❖ Doc C: “If you see
❖ Some degenerate cells with pyknosis also present. endometrial cells in a younger Doc C: “How are kids produced?
❖ Basophilic granular background with inflammation also patient, you don’t report this ❖ Because of the cervix. It is also called the door.”
present. because there is no specific ❖ In gynecology, this position is called the lethotomy position.
recommendation and since This is undertaken by the patient when trying to get their PAP
RBCs are also abundant.” smear (at the cervix).
General Categorization (Optional)
❖ Negative for Intraepithelial Lesion or Malignancy
❖ Clinical History: The patient is a 52-year old with abnormal
➢ This means there is no cancer and have identified at
uterine bleeding.
least non-neoplastic changes (atrophy and squamous
metaplasia) and able to identify organisms (T.
❖ Interpretation: Endometrial cells are present in a woman
>/=45 years of age. Negative for squamous intraepithelial
vaginalis, C. albicans, G. vaginalis, Actinomyces spp.,
lesions. (See Note)
and cellular changes associated with Herpes and
Cytomegalovirus)
❖ Other: See Interpretation/Result
➢ Example: endometrial cells in a woman >45 years of
age
o Specify if ‘negative for squamous intraepithelial
lesion’)
➢ Endometrial cells (in a woman >45 years of age)
o “Here, it is very important to know that women…
(putol here at 1:45:54 - 1:46:03) ...should be
reported.” :”)
o By the way, you still say that it is “negative for
squamous”, but what we’re talking about here is
not squamous but also endometrial cells.
o Epithelial cell abnormality: See ❖ Note: Endometrial cells after age 45, particularly out of
Interpretation/Result phase or after phase, may be associated with benign
▪ Specify ‘squamous’ or ‘glandular’, as endometrium, hormonal alterations, and less commonly,
appropriate endometrial/uterine abnormalities. Clinical correlation is
recommended.
General Categorization: Other ❖ Doc C: “If you see endometrial cells in a woman >45 years
❖ Endometrial Cells (Cervical Cytology) old and associated with bleeding, then you have to report
➢ Seen during menstrual cycle days 6 to 10 (proliferative because it could be benign, due to hormonal alterations in
phase or exodus phase) the uterus, or cancer. You have to inform the clinician that
❖ Usually on day 6 of menstruation, the uterus will undergo clinical correlation is recommended.”
proliferation, but more will still be shed out from the uterus
and that is called exodus phase (putol at 1:47:16-1:48:45) General Categorization: Epithelial Cell Abnormality
❖ Reports of associated endometrial pathology in post- ❖ Squamous Epithelial Cell Abnormalities
menopausal women with benign endometrial cells at PAP ➢ Recap: For abnormalities, the reference is the nucleus
smear versus no association of intermediate cells.
❖ Try to correlate. This is the cervix (refer to area within broken
❖ Most associated carcinomas are in women aged 45+ years ➢ Nuclear Enlargement
lines in the pic). This is the uterine cavity where the baby will
❖ May also be due to hormone replacement therapy or o Reactive: ½ to 2x
be implanted. From the ovum (ovary) and the sperm here
tamoxifen o ASCUS: 2 ½ to 3x

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 (coming through the vagina), there will be fertilization and
the zygote will be implanted here (uterine cavity).
❖ Your cervix is composed of two parts:
➢ Endocervix
➢ ectocervix/exocervix.
❖ You have a small canal there called the endocervical canal
leading to the uterine cavity.
➢ The bigger canal here is the vagina.
▪ What you see outside, in a pornographic
magazine, is the vulva. You cannot see a vagina.
▪ This vagina is the canal.
▪ It leads to the external os of your ectocervix, goes
down to your endocervix, endocervical canal and
then internal os before opening into the uterine
cavity.
❖ Your collector, the doctor, the gynecologist, the obstetrician,
the midwife, the barangay health worker, the nurse, the
intern, the PGI–good if you have a colposcope, you can view
the cervix.
❖ Look at the shape of the ectocervix leading to the endocervix.
❖ It fits your brush in liquid-based cytology.
❖ From the ectocervix and vagina, you will get the fluid or
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❖ This here, outside, which is part of the skin, is the vulva. It leads
to your vaginal canal. This portion is the cervix. This is the
transformation zone. This is the external os (additional note:
It is the junction between the cervix and the vagina.) This is
the endocervical canal, internal os and uterine cavity.
❖ This is your ectocervix, the door. When talking to your patients,
ang tawag anang cervix is puerta (Spanish for door).
➢ At birth, this is your columnar endocervical epithelial
cells. From the junction, it changes right away to
squamous.
➢ In the young adult, there is a process called ectropion
wherein these endocervical cells will go out and be
exposed to the ectocervix.
➢ However, as adults, this will be restored. The squamo-
columnar junction will be here, and then this one
➢ This is your spatula (the one held by the hand). wherein endocervical cells once were placed in the
▪ Mas painful ang spatula, mas nice ang brush. ectropion is called transformation zone.
➢ With the brush, you just simply twist it and you get part
of the endocervix.
▪ Note that a minimum of 10 endocervical cells
should be present.
scrapings (squamous epithelial cells).
➢ If conventional, minimum of 8000 squamous epithelial
cells.
➢ If liquid-based, minimum of 5000 cells.
❖ In the transformation zone, this is your endocervix (first
picture), see the columnar cells, the glands.
❖ This is your ectocervix (fourth picture), your squamous
epithelial cells.
❖ These are the subcolumnar reserve cells undergoing
columnar differentiation (second picture).
❖ This is your squamous metaplasia (third picture).
➢ This is very important because your human
papillomavirus loves to stay among squamous
metaplastic cells.
Menasalvas, Mueblas, Miranda, Molinas, Manza, Tupaz, Taguba, Damocles, Dignos, Casia, Barrameda, Palang
 ➢ And here, your HPV is the one responsible for more than
90% of your squamous cell carcinoma.
➢ That’s why you need to get these cells in order to
diagnose cancer in your PAP smear.
Here, that is your exocervix and your endocervix. This is your
squamo-columnar junction (arrow in the middle).
Papanicolaou (Pap) stain
❖ After fixation, there are no special handling requirements for
cytological smears.
❖ However, smears which are to be mailed to a laboratory,
should remain in the fixative for about 1 hour.
❖ A second clean glass slide may be placed on each fixed slide
for protection.
❖ In PAP stain, fixative used is 95% alcohol.
❖ The classic form of Pap stain involves 5 dyes in 3 solutions:
1. Haematoxylin - stains cell nuclei
o First OG-6 counterstain
▪ (-6 denotes the used concentration of
phosphotungstic acid; other variants are OG-
5 and OG-8)
2. Orange G - stains keratin
o Second EA (Eosin Azure) counterstain, comprising
three dyes;
▪ The number denotes the proportion of the
dyes, e.g. EA-36, EA-50, EA-65
3. Eosin Y - stains the superficial epithelial squamous cells
(especially the cytoplasm), nucleoli, cilia, and RBCs.
4. Light Green SF yellowish - stains the cytoplasm of all
other cells.
o This dye is now quite expensive and difficult to
obtain.
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o it produces visually different results and is not
considered satisfactory by some.
5. Bismarck brown Y - stains nothing and in
contemporary formulations it is often omitted.
Note: The last 2 stains are not commonly used now which is why
we will stick with the first 3 stains.
Results: Papanicolaou (Pap) stain
❖ Nuclei are stained blue while cytoplasm displays varying
shades of pink, orange, yellow, and green
❖ Stains ribosomes blue-green, particularly in parabasal cells,
mesothelial cells, and metaplastic squamous cells
❖ Stains metabolically inactive cells pink, such as superficial
cells
❖ Stains keratinized cells or thick specimens orange (benign
or malignant)
❖ Pyknotic nucleus, pink cytoplasm = superficial squamous
cell (1)
❖ Standard nucleus (2) is almost as big as an RBC (3)
❖ Intermediate squamous cells (2)
❖ Metaplastic cell cytoplasm (4) looks like a columnar
becoming squamous.
❖ Smaller cells which has metaplastic cells (look at the
cytoplasm, quite irregular) (5)
❖ This one (parabasal cell, 6) is more or less similar to 5,
➢ but 6 has a rounded cytoplasm,
▪ higher N:C ratio because of the nucleus becoming
larger,
▪ cytoplasm is less
➢ also more common in atrophy (post-partum).
➢ If you have a severe infection wherein these ones
(superficial, intermediate and metaplastic cells) will be
removed, you will be exposing the parabasal cells.
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Cervical Cancer
❖ Once cervical cells begin to change, it typically takes 10-15
years before invasive cancer develops.
➢ It takes long to develop unless you have a very high
risky activity, and if you have the high-risk type of virus.
▪ Low-risk types tend to develop slower.
❖ As the cells change, they first become “pre-cancerous”
➢ a condition also known as “dysplasia” or CIN - Cervical
Intraepithelial Neoplasia (newer term).
▪ This means that the cancer is still within the
epithelium.
▪ If detected early, dysplasia can be treated before
the cells become cancerous.
❖ According to a WHO report, cervical cancer claims lives of
around 300,000 women worldwide every year
➢ around 130,000 new cases are reported in India every
year out of which some 8,000 are from West Bengal
alone.
➢ If detected on time, this disease is curable but in most
of the cases, it is detected at an advanced stage,
primarily due to lack of awareness.
Cervical Intraepithelial Neoplasia
❖ The easiest way to differentiate between Normal, CIN I, CIN II,
and CIN III, is to look for the basal cells;
o CIN I- immature cells, occupies ⅓
o CIN II- occupies ⅔
o CIN III- occupies the entire thickness of the epithelium
(Carcinoma In Situ)
 CIN I or LSIL (Low-grade Squamous Intraepithelial Lesion) ❖ Cells have higher N/C ratio, cytoplasm is smaller.
❖ Cytomorphologic Criteria:
➢ Unequivocal nuclear “dysplastic” changes are present
in these relatively small squamous cells.
➢ The nuclear to cytoplasmic ratio suggests moderate
dysplasia (CIN II).

❖ CIN occupying ⅓ of the thickness

❖ Koilocytes (superficial or immature squamous cell)
➢ with large and irregular, well defined perinuclear halos
➢ with cookie cutter border and cytoplasmic thickening,
➢ nuclei are enlarged with undulating (raisin-like)
nuclear membrane
➢ rope-like chromatin; ❖ For mild dysplasia (LSIL), more abundant and mature
➢ often bi- or multinucleation and variation in nuclear cytoplasm and a lower N/C ratio is expected. In this case
size. chromatin changes suggest HSIL; however the N/C ratio is
❖ Nucleus is larger than normal on the low end for HSIL.
❖ Cytoplasm is abundant but with a perinuclear halo due to
CIN III or HSIL (High-grade Squamous Intraepithelial Lesion)
A (Normal); B (CIN I); C (CIN II); D (CIN III) the Human Papillomavirus.

Normal cells CIN II or HSIL (High-grade Squamous Intraepithelial Lesion)

❖ Layers of tissue based on the illustration on the right, from ❖ Occupies the entire thickness of the epithelium with
bottom to top: Basal, Parabasal, Intermediate, Superficial immature cells. Large groups of dark and huge nuclei.
cells. ❖ In papsmear, CIN III is still called HSIL.

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 Other variations:
❖ Atypical Squamous Cells of Undetermined Significance
(ASC-US)

❖ In cases where non-normal CIN I, II, or III are first observed,

the slides should be consulted with and forwarded to the
Pathologist or the Senior MedTech.

➢ Not normal yet is not ILSIL.

➢ Nucleus is large (2x but not 3x). Cytoplasm is still
abundant but absence of halo or irregularities.

❖ Atypical Squamous Cell cannot rule out a High Grade

Squamous Lesion (ASC-H)

➢ Not ILSIL/HSIL.
➢ Irregular nuclear membrane and higher N/C ratio.

Squamous Cell Carcinoma

❖ Occurs when the CIN III cells break the basement
membrane, invading the lamina propria of the cervix.
❖ High N/C ratio, monochromasia, numerous single cells
which are less cohesive, coarse and irregular chromatin,
variable nucleoli among cells, disordered architecture
(Cancer is ugly).

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 Tumor Diathesis-Cervical Cytology
❖ Cytology:
➢ fresh or hemolyzed blood with necrotic cellular debris
in the background.
❖ Associated with invasive carcinoma;
➢ SIL or benign backgrounds are usually “clean”
➢ Often not present if less than 5mm of invasive
squamous cell carcinoma.
❖ For liquid based (Thin-Prep and Sure-Prep), squamous cell
carcinoma may have clinging diathesis.
➢ Necrotic material at the periphery of cell groups or
reduced cellularity.
Questions: green text additional churvanessity nako- Chrispy
1.
❖ Composed of necrotic debris (pinkish granular material),
fibrin (small pinkish-orange threads), inflammatory cells,
and sometimes old blood (smooth, even, orange-pink
material).
➢ Left photo: Squamous cell carcinoma cells with scant
cytoplasm and weird nuclei with irregular, coarse
chromatin.
➢ Right photo: Tumor diathesis. Composed mainly of
lysed blood, granular debris, inflammatory cells and a
stripped nucleus.
❖ Other variations:
➢ Adenocarcinoma in situ of the cervix (in place)
➢ Adenocarcinoma, endocervical (invasive)
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Why can't we just make the preparation cheaper if it's
better? Why do we still have to follow the Conventional
procedure?
a. You need to have the machine. In addition, in Doc’s lab
she made an agreement with the company that all the
patient has to do is purchase the container (cup with
the fixative) and the brush and these already costs Php
800+ . Remember, you have to do the costing: the
machine, the medtech, the electricity… so more or less
it will cost you Php 2k-3k . That is why Liquid-based
cytology is not that common, encouraging to px
especially among Filipinos, especially in this pandemic
where money is really an issue.
b. Whereas, if you have your Conventional Pap smear, the
BHW can do that. Doc discourages the cotton pluget(?)
since the cotton will absorb the cells from the fluid.
They can use the spatula and do scraping and they
can just right away place it on a fixative and send it to
the lab. It is very cheap. They are charging around Php
600 and Php 200 from it will go to the laborator,
compared to your thousands for your Liquid based
Cytology
c. In other countries they have insurance or it’s part of
the insurance that’s why they routinely have the Liquid
based Cytology.
d. We now have a vaccine for HPV: (GARDASIL) sold by
Merck Sharp & Dohme, (and another company doc
forgot, tho from what I’ve researched kani ra na
company ang manufacturer jud).
Menasalvas, Mueblas, Miranda, Molinas, Manza, Tupaz, Taguba, Damocles, Dignos, Casia, Barrameda, Palang
i. There is a type of vaccine that targets the two
(2) high risk viruses (Bivalent)
ii. There is also a type of vaccine that targets four
(4) (Quadrivalent)
1. It targets two (2) low-risk HPV which will
cause warts.
2. The other two is target towards the ones
that can cause cervical cancer
iii. A Nonavalent one also exists that can protect
against nine (9) HP types (this is additional
research not from doc ra)
e. Doc encourages the women to consult a gynecologist
for a vaccine before having sexual intercourse.
However doc is not sure if FDA in the Philippines is
already encouraging HPV vaccines to males also.
Because for males, especially those having MSM (men
having sex with males) there is also not the cervical
cancer but anal cancer which is also Squamous cell
carcinoma. So hopefully, the HPV vaxx can help prevent
this type of carcinoma in men.
2. Why is cytoscreener not a lucrative job?
a) We do not have training here in PH in comparison to
other countries.
b) Doc had a student before who studied in CIM and
underwent training in Pathology, but then she quit here
in PH and went to NZ or Aus and worked as a
cytoscreener and she is actually doing well
there. Sadly, not in PH.
c) Only a very few people or women are representing
themselves in papsmear.
d) Cervical cancer is the #1 killer in women before, but
with papsmear; the mortality/morbidity is low. It’s
cheap and highly accessible.
e) So, doc really encourages us ,especially the females
and those who are sexually active.
THE END
For those that had confusions on what was what in the video and
want to see the video yourselves, I’ll attach the link as well as the
ppt here lng 😊
Video:https://drive.google.com/file/d/1hTFBbiIUU7VkAkFtnEx0RnxEl
s9P3d4C/view
PPT:https://docs.google.com/file/d/1NiCCJNtfu1J7Agdyra2OssGO
h1P-xwZs/edit?filetype=mspresentation
GOOD LUCK TO US!