001 Introduction to Cytology
1. Cytology beginning
Dr George Nicolas Papanicolaou
vaginal/cervical smear
detection of hormonal effect
discovery: the ability to detect cancer
beginning of cytology
early detection of cervical cancer
2. Cytopathology
A branch of pathology that studies and diagnoses diseases on the cellular level
It refers to diagnostic techniques that are used to examine cells under the microscopic form various body sites to determine the cause or nature of disease
3. Function of cytopathology lab
Diagnostic test: finds out if a disease is present and, if so, it precisely and accurately classify the disease
Screening test: to find people who might have a certain disease even before they develop symptoms
4. Cervical cancer screening
It is a method of preventing cancer by detecting and treating early abnormalities
HPV infection Low grade cervical dysplasia High grade cervical dysplasia Invasive cancer
HPV infection Pre-cancerous dysplasia Cancer
requires 10~20 years
5. Routine activity in cytology lab
sample receiving
processing
staining
mounting
screening (cytotechnologists)/confirmation (cytopathologist)
report validation
6. Sampling and preparation methods
Pap smear
pap test
collecting from your cervix
procedure to test for cervical cancer in women
can also detect changes in your cervical cells
women >30 years, the pap test may be combined with HPV test
begin at the age of 21
7. Pap smear
proper specimen collections require that specimen integrity is maintained by proper preservative
sample identification and patient identification must be clearly labelled on the specimen container or slide
The sample must be properly transported to the clinical lab in a timely manner
Specimen should be collected preferably 2 weeks after the first day of the last menstrual period and definitely not during menstruation
Patient should be instructed to not use vaginal medication, contraceptives, lubricants or douches 48 hours prior to the test and to refrain from intercourse the night before the examination
8. Patient instructions
2 weeks after the first day of LMP
avoid examination during menses -blood may obscure significant findings
do not use vaginal medication, vaginal contraceptives, or douches for 48 hours before the appointment
intercourse is not recommended the night before the appointment
9. Specimen collection
specimen should be obtained after a non-lubricated speculum (moistened only with warm water) is inserted
excess mucus or other discharge should be removed gently
an optimal sample includes cells from the ectocervix and endocervix
10. Conventional smears
often obtained using a combination of a spatula and brush (spatula is used first)
plastic spatula is recommended because wooden fibers may trap diagnostic materials
the spatula is rotated 360
the brush should be rotated gently only one quarter turn
the sample is smeared on one half of a slide and spray fixed
immediate fixation (within seconds) is critical in order to prevent air-drying artifact (distorts the cells and hinder interpretation)
11. Processing of specimen
Gynaecological specimens
- smeared slide -conventional
- methanol/ethanol based solution -liquid based cytology (ThinPrep, SurePath & etc)
- staining -Papanicolaou stain (Pap stain)
Cytologic smear
- a type of cytologic specimen made by smearing a sample (obtained by a variety of methods from a number of sites), then fixing it and staining it, usually with 95% ethyl alcohol and Pap stain)
Pull push technique
- the choice of technique is determined by the viscosity of the sample
- for mucoid samples (mucus from nasal flushes/bals samples) and viscous fluids (joint)
Squash technique
- slightly force only is applied to the slides as they are placed together and pulled apart parallel
- place a drop of cell deposit near the center of the glass slide
- squash a second slide against the first slide, ensuring slight overlap
- pull the second slide over the first slide, ensuring the two slides maintain contact and remain parallel
- the final preparation is thin and uniform, but should not be so thin as to rapidly air dry
- excessive pressure during smear preparation causes rupturing of cells and yields non-diagnostic smears
- this can be a common problem with lymph node aspirates, (lymphocytes are quite fragile and rupture readily)
- rapidly air-dry the slides
- blowing on the back of the slide with a hair dryer is best
- this is very important because it optimizes cell spreading on the slide, allowing identification of individual cells and detection of small inclusions (bacteria) within the cells
12. Cytology types
Gynae cytology
- includes cervical and vagina
- pap smear
Non gynae cytology
- includes everything except pap smear
- specimens: sputum, fluids, pleural, ascitic, CSF, urine, FNA
- tools of collection: brushing, washing/lavage, Fine Needle Aspiration
13. Exfoliative cytology
Spontaneously shed cells in body fluids
- collected from natural secretions or by paracentesis or lavage, washing or aspirating
- urine, sputum, CSF, effusions in body cavities (pleura, pericardium, peritoneum)
Removed from the epithelial surface of various organs
Brushing
- GIT brushings (esophageal, GI junction, gastric, duodenal, bile duct, other) -using endoscope
- bronchial brushings -using bronchoscope
Washing (lavage) of mucosal or serosal surfaces
- GIT washings (esophageal, gastric, other) -using endoscope
- bronchoalveolar lavage (BAL) -using bronchoscope
Scraping
- oral scraping, skin lesion scraping
Swabbing
- oral swabbing, vaginal swabbing
Serous fluid, urine and cyst fluid
- collected in clean containers
- freshly collected
- gross appearance and volume
- fluid is refrigerated at 4𐩑C (if delay)
- upon preparation, shake gently to disperse the cell
- centrifuge 5~10 mins at 700~1500 rpm
- smear preparation (alcohol fixed and air dried)
- cytospin (alcohol fixed and air dried)
- ThinPrep (alcohol fixed)
- cell block
- stain
- Pap stain (alcohol fixed smear)
- MGG (air dried smear)
- mounting (DPX)
14. Fine Needle Aspiration Cytology (FNA)
obtain material from organs that do not shed cells spontaneously
superficial nodules and organs easily targeted like neck, thyroid or breast
deep organs -guidance of CT, US
breast, thyroid, lymph nodes, liver, lungs, skin, soft tissues and bones
15. ThinPrep
Filtration and collection of vacuum packed cells on a membrane and transferring to glass slide
collection device: broom type device or a plastic spatula or endocervical brush
rinse the sampling devices in a methanol-based preservative solution for transport to the cytology lab
discard all the sampling devices
place the vials one at a time on the ThinPrep 2000 instrument (The entire procedure takes about 70 seconds per slide)
result in an evenly distributed deposit of epithelial cells in a circle 20 mm in diameter (cytospin 6 mm)
this ThinPrep test is significantly more effective than the conventional smear for the detection of LSIL and more severe lesion
the specimen quality was significantly improved over that of the conventional smear
16. SurePath
Centrifugation and sedimentation through a density gradient
in contrast to the ThinPrep method, the clinicians snips off the tip of the collection device and submits it to the lab in the sample vial
it can batch up to 12 specimens
prepare an evenly distributed deposit of cells in a circle 13 mm in diameter
significant increase in the percentage of satisfactory specimens
17. ThinPrep Pap test/Liquid based cytology
Pap smear
- majority of cells not captured
- non-representative transfer of cells
- clumping and overlapping of cells
- obscuring material
ThinPrep
- majority of sample is collected
- randomized representative transfer of cells
- even distribution of cells
- minimizes obscuring material
18. Fixation
Wet fixation
- alcohol-fixed smears
- rapid (before air drying)
- 95% ethanol
- usually used for pap stain
- ideal method for fixing most of the cytological specimens
Dry fixation
- air dried smears
- used for Romanowsky stain
- useful for evaluation of the FNA and effusions (fluids from pleural, pericardial and peritoneal)
19. Staining
Pap stain
- for alcohol-fixed slides
- excellent for nuclear studies
Romanowsky's type stain
- for air-dried slides
- Diff Quik, Wright-Giemsa, MGG
- indicated for cytoplasmic studies (vacuoles, granules, etc)
Additional stains
- special stains
- PAS, GMS, ZN (Ziehl-Neelson stain)
- immunocytochemistry
Hematoxylin: standard nuclear
OG and EA: cytoplasmic stains
4 steps: fixation, nuclear staining, cytoplasmic staining, clearing
20. Pap stain
21. MGG method
May-Grunwald solution : Phosphate buffer (1:1) 10 mins
wash in running tap water
Giemsa stain : Phosphate buffer (1:9) 10 mins
wash in running tap water
air dry
22. Evaluating cytologic sample
Systemic approach
Assessment of
- nucleus, cytoplasm, cell as a whole
- intracellular relationship: cell cohesion, cell formation (acini, ducts, papillae, tissue fragments, epithelial pearls, cell whorls, cell moulding)
- background features: bloody, inflammatory, necrotic
23. Nucleolus
RNA accumulates
prominent nucleolus=metabolically active cells
size, shape, number and variation
24. Nucleus
repository for DNA
benign: uniform size, round/oval contours
malignant: alteration in size and shape, irregular foldings, indentations, irregular membrane, haphazard arrangements
25. Chromatin
DNA responsible for staining properties of nucleus
stain basic dyes proportionately
increase DNA related proteins: hyperchromatic nucleus
malignant nucleus: irregular clumpy chromatin, hyperchromasia
002 The Bethesda System (TBS) of Reporting
1. Specimen type
conventional
liquid-based cytology (high maintenance, expensive)
2. Specimen adequacy
Satisfactory evaluation
cell amount/no
cellularity (LBC 5k, conventional 8~12k well preserved and well visualized squamous epithelial)
5k/area of field or all space have cells (500 x 10)
diameters of SurePath and ThinPrep preparations are 13 and 20 mm
presence or absence of endocervical/TZ component (endocervical/squamous and glandular)
10 well preserved endocervical/squamous metaplastic
singly or in clusters
partially obscuring blood
Unsatisfactory evaluation
rejection
inform the doctor if sample is not enough
specimen not processed or processed and examined but...
not label
broken slide
obscuring blood/thick blood obscuring smear
scanty epithelial cells/lack of cellularity
poor fixation
excessive lubricants (sexual) (jelly)
excessive neutrophils/WBC (purple)=infection
fungal contaminant
3. General categorization
screening normal or abnormal
report higher degree of abnormalities
Negative for Intraepithelial Lesion Malignancy (NILM)
Epithelial cell abnormality
4. Interpretation/Result
NILM/non-neoplastic findings
Atypical cell (abnormal)
Epithelial cell abnormalities
squamous (lighter pink area)
glandular (darker pink area/cervical OS)
5. NILM/non-neoplastic findings
normal cellular elements
infection
non-neoplastic cellular variations
reactive cellular changes associated with
glandular cells status post hysterectomy
6. Normal cellular elements
Squamous
- Basal and Parabasal
- high N:C
- round/oval
- opaque cyanophilic cytoplasm
- vesicular nuclei-dense
- fine chromatin (granules)
- Intermediate
- coarse chromatin
- cytoplasm pale, cyanophilic
- larger, flat
- polyhedral
- Superficial
- small, dark pyknotic nucleus
- thin, semi-translucent cytoplasm, eosinophilic
- orangeophilic with maturation and keratinization
- Endocervical
- eccentric nucleus
- fine chromatin
- abundant, sometimes vacuolated cytoplasm
- inconspicious nucleoli
- in strips: palisading/picket fence
- in sheets: honeycomb
- Endometrial
- similar to cancer cells
- scant cytoplasm
- isolated cells
- balls or small cells
- nuclear fragmentation and molding
- Transitional
- Lower uterine segment cells
7. Non-neoplastic cellular variations
- Squamous metaplasia
- lighter pink
- development, undergo changes or maturation (in between parabasal and intermediate)
- Tubal metaplasia
- glandular
- Atrophy
- parabasal
- flat monolayered sheets
- preserved nuclear polarity
- Atrophy and inflammation
- multinucleated giant cells
- post-menopausal and post-partum
Keratin changes
- Typical parakeratosis (nucleus overproduction)
- small superficial squamous
- orangeophilic, eosinophilic cytoplasm
- single, sheets, whorls
- oval, polyglonal, spindle
- pyknotic nucleus
- Hyperkeratosis
- anucleated
- ghostlike "nuclear halos"
- Pregnancy-related
- high progesterone
- glycogen rich intermediate
- navicular (boat)
8. Reactive cellular changes associated with...
- Inflammation (repair)
- chemotherapy
- radiotherapy
- enlarged nucleus
- bi/multinucleation
- nuclear outline smooth, round, uniform (homogenous) (border)
- fine chromatin
- polychromasia
- cytoplasmic vacuoles
- perinuclear halo
- cytoplasmic processes "spider cells"
- Radiation
- large cell size
- bizarre
- prominent nucleoli
- cytoplasmic vacuoles
- nuclear enlargement
- coarse chromatin
- IntraUterine contraceptive Device
- prevent fertilization
- Y shape device
- irregular cell shapes
- nuclear enlargement
- coarse chromatin
- cytoplasmic vacuoles
- nucleoli
- hyperchromatic nucleus
- lymphocytic (follicular) cervicitis
003 Smear Pattern
1. Hormonal
consistent with patient age, hormonal status, clinical details, H/O
H/O from verbal/request form (hardcopy) for cyto
vaginal epithelium is very sensitive to estrogen and progesterone
2. Hormonal evaluation
Assessment of ovarian function (after hysterectomy, during menstrual cycle, in premature menses)
Assessment of abnormal hormonal production (pregnancy, abortion)
Assessment and guidance of hormonal therapy
3. Key points
Superficial cell influenced by estrogen (ovulation)
Intermediate cell influenced by progesterone
Without estrogen and progesterone, cell remain atrophic (parabasal) during post-menopause/partum
Menstrual (low hormone)--Proliferative--Ovulation (24 h)--Secretory
4. Proliferative phase
mainly influenced by estrogen
promote full maturation of squamous epithelium to level of superficial
5. Secretory phase
primarily influenced by progesterone
inhibit full maturation to level of intermediate
abundant lactobacilli (bare nuclei)
break down cells
reach the glycogen in cytoplasm
folded cells
cells aggregate
6. Menstrual phase
shed endometrial, blood, debris
bloody
neutrophils
7. Atrophic smear pattern
absence of hormonal stimulation
no maturation
Post partum atrophy
may stay atrophic until ovaries begin to cycle hormonally again
common with lactation
Pregnancy atrophy
IntraUterine Fetal Death
Post menopausal atrophy
predominance parabasal
occasionally with intermediate
Peri menopausal cells
hormone level declines during menopause
large intermediate or metaplastic squamous may be seen
8. Reparative changes
spindle, spidery cells
prominent nucleoli
enlarged nucleus
multinucleation
cells in sheets
fibroblasts
9. Radiation
enlarged cells
bizarre
vacuolated
large nucleus with degenerative changes
10. Degeneration
swelling and enlargement
sharply wrinkled nuclear margin
pyknotic nucleus
karyorrhexis
karyolysis
11. Neoplasia
enlarged hyperchromatic irregular nucleus
chromatin clumping
004 Cytology Infection
1. Cytology infection
distinctive cytopathic effect
itch, fever, vaginal discharge (dead WBC/pus/mucus), bad odour
Lactobacilli (NF)
- pH >4.5 acid
- Doderlein bacilli
- late secretory phase
lysed intermediate (clumped)
bare nuclei
2. Gardnerella vaginalis
Cytomorphology:
squamous cells covered by coccobacilli (shaggy)=clue cells
not entire cell covered
sandy
dirty
Description:
women with SIL (HPV)
hormonal replacement therapy post-menopause
Risk factors:
multiple sexual partners
IUCD
prior pregnancy
medication
spermicides
smoking
Signs and symptoms:
yellow, green, mucoid vaginal discharge
asymptomatic
80% sensitive, 87% specific
3. Candida albicans
Cytomorphology:
long, segmented pseudohyphae
protruding form clump of cells
pink
yeast form
budding
Description:
transmit thro fomites, toilet seats
proliferate in changes: vaginal flora or glycogen, diabetes, pregnancy, immunosuppression, antibiotic therapy, oral contraceptive use (if >5 yr, cancer)
Signs and symptoms:
white, curdy discharge
vulvar itching
4. Leptothricia buccalis
Description:
Gram -ve bacilli
thin, segmented hair-like filaments
NF of oral cavity
+TV 75%
5. Trichomonas vaginalis
Cytomorphology:
small pear
pale greyish/blue/green cytoplasm
cannon balls (neutrophils aggregates/grouping)
eccentric nucleus
watery/dirty
Description:
protozoan parasite
attach to cells
associated with vaginitis, cervicitis, urethritis, pelvic inflammatory disease (PID)
HIV
Signs and symptoms:
profuse, malodorous, frothy yellow/green discharge
vulvar itching
burning
postcoital bleeding
5. Human PapillomaVirus
Cytomorphology:
koilocytes (perinuclear halos)
hyperchromatic nucleus
binucleation
Description:
can cause cancer, genital warts
6. Herpes Simplex Virus
Cytomorphology:
cytomegaly
nuclear enlargement
multinucleation
moulding
marked chromatin margination
homogenous ground glass
3D
Description:
immature squamous metaplastic and endocervical
inflammation
7. Actinomyces
Cytomorphology:
dense fuzzy blue/purple masses
long, thin filaments
Decsription:
Gram +ve anaerobic
PID
Signs and symptoms:
malodorous discharge
asymptomatic
8. CytoMegaloVirus
Cytomorphology:
cell and nuclear enlargement
large eosinophilic intranuclear viral inclusions with a prominent halo
small cytoplasmic, basophilic inclusions
Description:
HIV
immunocompromise
005 Pre-malignant
1. Pre-malignant
Low-grade squamous intraepithelial lesion
mild
CIN I
HPV
High-grade squamous intraepithelial lesion
moderate
severe
CIN II, CIN III
2. Epithelial cell abnormalities
benign or malignant
nuclear and cytoplasmic changes
dyskaryosis (mild, moderate, severe)
previously graded as cervical intraepithelial neoplasia (CIN I, II, III)
Bethesda terminology: LSIL and HSIL
HPV infection: redness around cervix
LSIL--HSIL--cancer (bleeding non stop)
3. Features of abnormal nucleus
hyperchromasia, hypochromasia
increased nuclear size/enlargement
abnormal chromatin pattern (coarse/granular)
nucleoli
irregular nuclear membrane
anisonucleosis
bi/multinucleation
pleomorphism
4. Features of cytoplasm
changes in cytoplasmic staining
keratinization
vacuolation
5. Dyskaryosis
abnormal nucleus
grading by assessing nuclear cytoplasmic N/C ratio
higher N/C, more severe
mild: 1/3
moderate: >1/3, <2/3
severe: 2/3
6. LSIL
mildly abnormal + HPV
mild dysplasia, CIN I
nuclear enlargement
hyperchromasia
anisonucleosis
bi/multinucleation
perinuclear halos/koilocytes
dense eosinophilic cytoplasm
increased keratinization
coarse chromatin
inconspicuous or absent nucleoli
irregular contour
singly, clusters, sheets
7. HSIL
higher N:C ratio than LSIL
hyperchromasia
anisonucleosis
coarse chromatin
cyanophilic cytoplasm
smooth wavy contour
inconspicuous or absent nucleoli
8. Dysplasia and HPV
30~70% cases
<20 yr
PCR >90%
serotype 16, 18, 31, 33 most frequently with cancer cervix, vulva and penis
serotype 6, 11 with benign condyloma, rarely malignant lesions
9. Tests
repeat pap test or co-test in 1 or 3 yr for follow-up
HPV test
colposcopy, biopsy, endocervical sampling
endometrial sampling
006 Abnormal Gynae Cytology
1. Squamous Cell Carcinoma (SCC)
2nd most common in Malaysia NCR 2002
35~55 yr
dysplasia--CIS--microinvasive--invasive
43~66% untreated dysplasia progress to microinvasive
duration from CIS prior to invasion: 2~10 yr
range from well-differentiated, keratinizing to poorly differentiated, non-keratinizing tumour
chronic
some cannot be distinguished from HSIL
2. Risk factors of SCC
multiple sexual partners
having more no of children
early marriage
family history/hereditary
smoking
oral contraceptive pills -ovulation still, sperm cannot move to ovum as mucus secretion increase
unprotected sex
early child birth
3. SIC
not metastasize
still confined to the epithelial layer
high-grade dysplasia
begins with mutation
enable it to invade neighboring tissues and shed cells into blood or lymph
4. Microinvasive
neoplasm with 3~5 mm depth of invasion
total or aggregate width <7 mm
infiltration of superficial stroma
diagnosis at 41 yr
most asymptomatic
no lesion visible grossly
colposcopy: abnormal vascular pattern
no lymphatic or vascular invasion
coarse chromatin
nucleoli
similar to HSIL: hyperchromasia, high N:C, bizarre shape, rare with tumour diathesis, syncytial
lysis of RBC/necrosis/WBC
5. Invasive SCC
tumour extends >7 mm into stroma
involve vascular spaces
diagnosis at 51 yr
abnormal vaginal bleeding, poscoital bleeding, intermittent spotting or frank hemorrhage
large nuclei
hyperchromatic
chromatin clumped/coarse
nucleoli
high N:C
tumour diathesis
6. Subtypes
- 75% large cell nonkeratinizing SCC
- high N:C
- hyperchromatic
- coarse chromatin
- scant cytoplasm
- prominent nucleoli
- cyanophilic
- tumour diathesis
- isolated, syncytial, polygonal
- 10~15% keratinizing SCC
- single cells, a few in aggregates
- spindle, caudate, tadpole forms
- squamous pearls
- macronucleoli
- coarse chromatin
- tumour diathesis in 1/2 case
- 5~10% small cell carcinoma
- leopard spot chromatin
- bare nuclei
- no cytoplasm
- scant cytoplasm
- small cells
- round, oval, spindle
- single, clusters, syncytial
- diathesis
007 Glandular Lesions of the Cervix (Essay)
1. Endocervical
eccentrically placed nucleus
palisading
honeycomb
2. Endometrial
balls of small cells
isolated small cells
large nucleus occupied cytoplasm
scant nucleus
dark nucleus
nuclear moulding
nuclear fragmentation
3. Adenocarcinoma -postmenopausal women
involve glandular cells (cervical OS/darker pink area)
uterus, Fallopian tube,
HPV-LSIL-HSIL-Adenocarcinoma In Situ (AIS)-Invasive Adenocarcinoma
endocervical canal
may appear as an ulcer
barrel-shaped cervix
4. AIS
precursor
uncommon lesion
incidence is a mere 0.61
cytomorphology
HSIL
large sheets, overlap
strips, rosettes, syncytial-like sheets
indistinct cell borders
feathery (hairs)
nuclear crowding
nuclei hyperchromatic
nuclei inconspicuous
no tumor diathesis background
5. Endocervical Adenocarcinoma
14~34%
HPV (type 16 and 18)
bleeding or vaginal discharge
asymptomatic
cytomorphology
large sheets
overlap
variation in nuclear size
nuclei large, hyperchromatic
coarse chromatin
macronucleoli
foamy (pale) and abundant cytoplasm
tumor diathesis
6. Endometrial Adenocarcinoma
postmenopausal women
90% vaginal bleeding
prolonged exposure of estrogen
7. Out of phase endometrial cells
a significant finding
abnormal shedding of endometrium
post-menopause or pre-menopause after days 12~15 of cycle -abnormal
risk of its association with malignancy increases
25% 50~59 years
chance of survival is high
small, tight clusters, single cells
scant, cyanophilic, often vacuolated cytoplasm
foamy cytoplasm
enlarged hyperchromatic nuclei
nucleoli
mitotic figures
apoptotic bodies
intracytoplasmic neutrophils
bags of polys
watery or fine granular tumor diathesis
8. Tumor diathesis
cell debris
neutrophils
lysed, old fibrinated blood
fine granular or clear watery transudate
small histiocytes
9. Sources of cells that mimic endometrial adenocarcinoma
10. IUCD
vacuole
nuclear enlargement
nucleoli
HSIL mimickers
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