BMS 4004 Advanced Cellular Pathology (January 13, 2023)

Cytology I – Techniques and application

Peter NG
Cyto Lab ic, MT, PYNEH

1
 Definition

Cytology is the microscopic study of cells from

various body sites to determine the cause or nature
of disease.
 CYTOLOGY
 Cytology is the study of cells from different organs of the
body
*
 George N Papanicolou introduced cytology as a tool to
detect cancer and pre-cancer in 1928
 Diagnostic cytology is the process of studying cells to
identify diseases, particular cancer
 These cells may have been shed by itself; aspirated by
needle; or scraped or washed from epithelial surfaces
 The nuclear morphology and cytoplasmic characteristic of
cells are under microscopic evaluation

3
 CYTOLOGY
Diagnostic cytology divides into two major components

1. Exfoliative cytology
2. Aspiration cytology

Classified in the way of cell detach

4
 EXFOLIATIVE CYTOLOGY
 Spontaneous shedding is a function of normal epithelium
 Epithelial surfaces undergo constant growth and continue
to shed worn out cells which are replaced by new ones
 Sputum, urine, body fluids & CSF are the examples
 Artificial exfoliation occurs when any surface is scraped
and viable cells are traumatically exfoliated before their
natural time of shedding
 Forcibly removed from a surface using as sampling
device, e.g. wooden spatula or brush
 Examples of these modalities include pap smears,
brushings, etc.
 Cytology Specimens

Sputum
Fine needle Urine, Body
Gynaecological
fluid
aspiration
specimen
(FNA)
(Gyn)
Non-Gyn

Aspiration Spontaneous & Artificial

artificial exfoliation exfoliation
 FINE NEEDLE ASPIRATION
CYTOLOGY
Fine needle aspiration cytology (FNAC)
 FNAC is a diagnostic procedure where a needle is
inserted into your body, and a small amount of tissue is
sucked out for examination under a microscope.

 Fine needle aspirations are often performed when a

suspicious lump is found, for example a breast lump or
enlarged lymph node, or if an abnormality is detected on
an imaging examination such as x-ray, ultrasound or
mammography.

7
 Brain storm

Fine needle aspiration cytology (FNAC)

Accompany with imaging exam ….

Can FNA work with PET scan or MRI?

PET (Positron emission tomography) scan

PET scan is an imaging test that can help reveal the metabolic or
biochemical function of the tissues and organs

MRI (Magnetic resonance imaging)

MRI is a type of scan that uses strong magnetic fields and
radiowaves to produce detailed images of the inside of the body
8
 Role of Cytology Lab

• Screening (篩查): Symptomatic/ Asymptomatic

. Eg. Cervical screening, screening for anal cancer
• Primary Diagnosis: Symptomatic
• Prognosis: eg. Sm C Ca vs adenocarcinoma of Ca Lung
• Prediction of tumor behavior:
• Secondary/ recurrent diagnoses:
• Staging (tumor): eg. Peritoneal washings => staging gyn
malignancies => ovarian, fallopian tube, endometrial; eg.
LSIL => HSIL => SCC
10
years
of staging
 APPLICATIONS

X
Non-Gynecological / Fine Needle Aspiration Cytology

 The earlier detection of malignant and so increasing the

chance of cure with simpler treatment
 Evaluation of radiotherapy effect in treating cancer
 Follow-up of surgically treated cancer patients
 Recognition of certain infections & allergic reactions

10
 APPLICATIONS

X
Gynecological Cytology

 The detection of premalignant and malignant lesions

 The identification of vaginal infections
 The assessment of hormone function

11
 ADVANTAGES
Cytology is
Histology
 Simple method and is easier to get
 Causes less discomfort to the patient
 Less likely to result in serious complications
 Inexpensive (less cost)
 Reproducible
 Decrease in extent of surgery or radio-therapy
required
 Speedy result

12
 LIMITATIONS
Cytology is
Histology
 Interrelation and arrangement of the cells cannot be
known
 Interpretation of the morphologic cellular changes is
sometimes subjective seach person's interpretation differs
 Location of the lesion cannot be pinpointed
 Screening is time-consuming
 Cytologic diagnosis in NOT always final
t
supportivegnosis

13
 ACCURACY
The accuracy of the cytologic diagnosis depends on
laboratory procedures used to process the specimens. It
include:
 Sample Collection
 Fixation & fixatives
 Preparation
 Staining & mounting
 Microscopic examination (Screening)

Each steps are critical because cells that are inadequate

collected, poorly fixed, prepared, stained and screened
can lead to false positive or negative diagnosis
14
 Sample Collection
Non-Gynecologic
 The site from which the sample is collected dictates the
method of collection.
 The importance of proper specimen collection and
submission is essential.
 The method of collection affects the morphology of the
cellular samples.
 The laboratory must provide instructions for proper
collection of all non-gynecological specimens. (The
instructions must be available to personnel at the location
where the specimens are collected.)
 The laboratory provides feedback on the adequacy of the
sample via individual reports
bronchial:more clamped cells
diff. method diffcell morphology
->
15
 Sample Collection
Non-Gynecologic

Specimen type
 Sputum
 Tracheal aspirate
 Bronchial brush, washings, aspirate & lavage
 Pleural, peritoneal & pericardial fluid
 Urine, ureter brush
 Cerebrospinal fluid
 Bile / Bile duct brush
 Joint fluid

16
 Sample Collection
Sputum
have macrophage, tresputum Fresh sputum is
really
 Morning specimen resulting from overnight better?
accumulation
of secretion yields best results overnight accumulation of more
secretion
cells
W

chigh yield sputum)

 Three to five consecutive days’ sputum samples should
be examined to ensure maximum diagnostic accuracy
 Fresh unfixed specimens are better than prefixed
specimens
 Mucus apparently coats the cells, protecting them against
rapid degeneration
 Refrigeration slows down the bacterial growth, which
causes cellular damage
 The cells in specimens diluted with saliva are not as well
protected and may deteriorate more rapidly
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 skip Sample Collection
Sputum
Collection:  First morning deep cough sputum before
breakfast or after chest physiotherapy.
 Avoid contamination by saliva or food.
 Three consecutive morning sputum are
required to ensure best diagnostic results. (not
3 samples on the same day)
 Collect in black plastic bottle.

Fixation:  Not required

Handling:  Kept refrigerated at 4° C before sending to the

laboratory.
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skip Sample Collection
Bronchial specimen
 Development of the flexible bronchoscope in the late 1960s
 Bronchoscopy is an endoscopic technique of visualizing the
inside of the airways for diagnostic and therapeutic
purposes
 It contains a fiberoptic system that transmits an image from
the tip of the instrument to an eyepiece or video camera at
the opposite end.
 Complications of bronchoscopy are rare

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skip Sample Collection
Bronchial trap, aspirate, washing, and lavage
Collection:  Any volume will do.

Fixation:  Add an equal volume of 50% ethanol to the

sample.

Handling:  The source and site from which the sample

originates must be clearly indicated on the
request.

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skip Sample Collection
Bronchial specimen

21
 Sample Collection
Bronchoscopic Specimen
Bronchial
Bronchial  material obtained by brushing a bronchial lesion under
brushing
aspirate??
direct vision during bronchoscopy is smeared directly
onto glass slide.

Bronchial  from a specific area of the bronchial tree

washing  by instilling 10ml of normal saline in small portions and
re-aspirating the material with suction

Bronchial trap  the mucus traps that are used to suction secretions
during the bronchoscopy procedure

Bronchial lavage  is advanced into a bronchial segment until it gently

occludes the lumen (at the most distal accessible pt)
 aliquots of 20-60 ml of sterile normal saline are infused
and re-aspirated and collected in a suction trap

22
 Sample Collection
Urinary Tract System
 The urinary tract is composed of the kidneys, the ureters,
urinary bladder and the urethra
 They are lined by a highly specialized and unique
epithelium "the urothelium" also known as transitional
epithelium
 Clinical indications for urine cytology
 Hematuria
 Follow-up of patients treated for UC
 High risk of bladder cancer

23
 Sample Collection
Urinary Tract Specimen
Voided urine  Easily obtained
 May be heavily contaminated with vaginal secretions &
squamous cells from perineum in female patients
Catheterized urine  For patients who cannot void urine, or with Foley catheter
inserted
 Often contain urothelial fragments scraped by the
catheter
Cystoscopy urine  Urine collected during cystoscopy

Bladder wash  By irrigation of bladder with normal saline through

cystoscope

Ureteral & renal  By normal saline irrigation of suspected lesion via catheter
pelvic washes whose tip is placed distal to the lesion

Retrograde brushing  By brushing the lesion with a brush inserted into the ureter
via cytoscope
bladder removed
&
Ileal conduit urine  In patients with total cystectomy
use ileum to re-construct bladder (urothelial carcinoma) 24
 Sample Collection
Urine : Voided, catheterized (ureter, bladder)

Collection:  Fresh urine collected at anytime will do, NOT

first morning specimen. overnight (high toxicity, degenerated)
cells

 At least 10-20ml is required for voided sample.

 Any volume for others are acceptable.

Fixation:  Add an equal volume of 50% ethanol to the

sample.

Handling:  Urine is very “toxic” to cells and cause speedy

degeneration.
 All urine sample must be fixed immediately.
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 Cytology of Effusions
Body Cavities
 The Lt & Rt pleural cavities – enclose the lung
 The abdominal (peritoneal) cavity – enclose the intestine
tract
 The pericardial cavity - enclose the heart

26
 Sample Collection
Effusions
 The pleural, pericardial, and peritoneal cavities are lined by
a single layer of flat mesothelial cells called the serosa
 Normally, these cavities are collapsed and contain only a
small amount of fluid, enough to lubricate the adjacent
surfaces as they move over each
 In disease states, a greater amount of fluid accumulates in
body cavities — an effusion
 Inflammation, circulatory disturbances, & malignant tumours
are common causes of effusions
 Serous or “body cavity fluids” are usually collected with
aseptic technique by needle puncture and aspiration of the
body cavity fluid

27
 Sample Collection
Body Cavities - Effusions

Pleural fluid in
pleural cavity

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 Sample Collection
Effusions

 Effusions are classified clinically as transudative or

exudative.
 Transudates result from an imbalance of intravascular
hydrostatic and oncotic pressures
  hydrostatic and  oncotic pressures favour transudates
produced

 hydrostatic pressures => push water and small molecules

out of the blood
 oncotic pressures (osmotic pressure) => induced by the
proteins, notably albumin => pull on fluid back into the
circulatory system
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 Sample Collection
Effusions

 Common causes
 Congestive heart failure (CHF)
 Cirrhosis
 Nephrotic syndrome

 Transudates have a low lactate dehydrogenase (LDH) and

low total protein concentration.
VS

Exudate: LDH ↑ protein

+

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 Sample Collection
Effusions
 Exudates result from injury to the mesothelium, as occurs
with
 Malignancy
 Pneumonia
 Lupus
 Rheumatoid pleuritis
 Pulmonary infarction
 Trauma
 Exudates have a relatively high LDH and total protein
concentration

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 Sample Collection
Effusions
 Malignant tumors are a common cause of exudates because
the serosal surfaces are a frequent site of metastasis for
many tumors
 The only primary tumour of serous membranes is
mesothelioma that is strongly associated with exposure to
asbestos.TEAF

Mesothelioma Ferruginous body

 Sample Collection
Effusions
 Body cavity fluid contain greater concentrations of thrombin
and fibrinogen than blood and will act to form fibrin (i.e. a clot)
 Fibrin, in turn, may entrap potentially diagnostic cells
 Cell block can be made from the clot so that the cells can be
examined
 Cytology is more sensitive than blind biopsy for detecting
serosal malignancy (71% versus 45%), presumably because
fluid provides a more representative sample.
 Specimens are obtained by inserting a needle into the pleural
space (thoracentesis), pericardial space (pericardiocentesis),
and peritoneal cavity (paracentesis).

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skip Sample Collection
Paracentesis

Needle enters the peritoneal cavity and ascitic

fluid will fill the syringe (red arrow)
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skip Sample Collection
Body fluid : Pleural, peritoneal, pericardial, CSF

Collection:  Freshly tapped fluid, NOT from drain bottle.

 Any volume will do.

Fixation:  Add an equal volume of 50% ethanol to the

sample.

Handling:  Kept refrigerated if cannot be sent

immediately.

35
 Sample Collection
Fine Needle Aspiration

 Fine needle aspiration (FNA) is a diagnostic procedure used

to investigate lumps or masses
 A small needle is inserted into the mass for sampling of cells
using negative pressure on the syringe that, after being
stained, will be examined under a microscope

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skip Sample Collection
Fine Needle Aspiration

 Aspiration of cells/ tissue fragments using fine needles (

21- 25 G) ; external diameter 0.6 to 1.0 mm
 1.5 inches long needle ( radiologists use longer needles)
 Standard disposable plastic syringes of 10ml are used
 Reusable syringe holder (piston) is used for the needle
can be precisely controlled and guided with one hand

37
 Sample Collection
Fine Needle Aspiration
Procedure
 Suction is applied after entering the lesion and maintain the suction
 Needle is moved vigorously back and forth in a sawing or cutting
motion too strongnegative pressure, collect bloody cells instead of clean cells
 Changing the direction a few times, ensuring that the needle is
inside the mass throughout
 Before withdrawing the needle, suction is released
 The piston is just allowed to slowly fall back by itself (never push)
 Needle is pulled straight out

38
 Sample Collection
Fine Needle Aspiration

Precautions
 Purpose of suction is to pull the tissue against the cutting
edge of the needle and to pull the dislodged tissue
fragments and cells into the lumen of the needle.
 Material is obtained by cutting motion of the needle and
not by suction.
 Failure to release negative pressure within the lesion will
cause the aspirated material to enter the syringe, which is
difficult to recover

39
 FNA: Advantages
 Simple to perform - safe method
 Cost of health care is reduced - a number of surgery
became unnecessary
 Relatively painless and causes minimal discomfort
 Can be performed on outpatients
 Be readily repeated
 Be used for multiple lesions
 Suitable for debilitated patients
 Greatly reduces the hazard of puncture trauma
 The risk of tumour spread is considered insignificant
 Cheap
 Speedy result

40
 FNA: Complications
 Needle trauma
blood -> metastasis
 Needle track seeding malignant cells from needle will
dislodge & enter

but low chance

 Hematoma
 Pain
 Pneumothorax

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 Sample Collection
Fine Needle Aspiration
 Lesions can be palpable
 Thyroid
 Salivary gland (parotid, submandibular)
 Lymph node
 Breast
 Lesions can be deep-seated - guided by radiologic imaging:
CT (combines a series of x-ray images taken from different
angles), ultrasound (sound waves), fluoroscopy (continuous
x-ray image, real time)
I cannotapply to FNA)

 Lung
Can we use PET scan not
imaging
 Liver (positron emission
I apply FNA)
cannot to

 Pancreas tomography) or MRI

 Adrenal (Magnetic resonance magneticllrpte
 Thyroid
imaging) for FNA? 42
skip Sample Collection
Fine Needle Aspiration
 Fine needle aspiration cytology (FNAC) may be performed
on palpable (superficial) lesions or with radiological,
endoscopic guidance (deep lesions)
 The skin should be cleansed with an alcohol swab prior to
puncture for superficial FNAC
 For percutaneous FNAC of deep lesions, sterile or aseptic
technique is used
 Local anesthesia may be used
 For solid lesions multiple passes with separate needles are
performed

43
Sample Collection
Fine Needle Aspiration

Needle

Adrenal
mass

44
Computerized Tomography Scan
Endoscopic Ultrasound (EUS) FNA
 APPLICATION of FNA
 At on-site evaluation, two direct smears are prepared; and
immediately fixed in alcohol and one is stained with rapid
H&E for on-site examination
 Rapid stain such as a toluidine blue stained wet film, a
Romanowsky can also be used for specimen adequacy
 Criteria for adequacy, which includes
 cellularity
 site-specific architectural features
 cellular elements
 The remaining material is rinsed with preservative solution
(e.g., CytoRich Red) and sent to the laboratory for
processing according to the laboratory’s protocol
 Whenever possible, cell blocks are prepared

47
skip Sample Collection
Fine Needle Aspiration
Smears:  Make 2 direct smears from materials in the
needle.
 Each smear must be fixed immediately
upon preparation while still “wet” to avoid
air-dry artifacts.
 Drop it immediately into 95% ethanol or
spray it with spray fixatives.

Cell block:  Rinse the syringe 2 to 3 times in 50%

ethanol or CytoRich Red solution.
Handling:  Send the direct smears & rinsing fluid to the
laboratory as soon as possible.

48
 Sample Collection
Gynecologic

 Cancer of the uterine cervix is the commonest cancer in

the Female Genital Tract.
 Almost all invasive cancers of the cervix are preceded by
a phase of preinvasive disease
 A continuing spectrum of events progressing from cervical
intraepithelial neoplasia (CIN) grade I to III including

&old
carcinoma in-situ before progressing to squamous cell
carcinoma is demonstrated microscopically
 This progressive course takes about 10 to 20 years
term

Narnames LSIL HSIL -sC

- -

49
skip Sample Collection
Gynecologic
(Conventional Method)

Preparation:  Normally 1 to 2 smear(s) is enough.

Fixation:  Each smear must be fixed immediately

upon preparation while still “wet” to avoid
air-dry artifacts.
 Drop it immediately into 95% ethanol or
spray it with spray fixatives.

Handling:  Send to the laboratory as soon as

possible.

50
 Sample Collection
Gynecologic
(Conventional Method)

Ayre's spatula 51
 Sample Collection
Gynecologic
(Liquid-Based Method)
no loss of cellular material
Collection:  Use preservative solution with cervex
brush.

Fixation:  Put the broom into the collection vial

containing preservatives OR rinse the
device by rotating it in the solution at
least 10 times.
Handling:  Send to the laboratory as soon as
possible.

52
 Sample Collection
Conventional Vs Liquid-Based Method
Conventional Smear LBG Method

Over 80% of collected sample 100% of collected sample are rinsed

discarded into preservative vial
keep 100% preserve

Air-drying artefacts may happen Cells preserved immediately artefact

↓

↓
clumping of colony into a fluid transport medium

Obscuring elements limit accurate Minimizes non-diagnostic debris,

diagnosis obscuring blood, mucus & WBC
(cleaner background)

Uneven distribution of cells onto a slide Cells evenly distributed on a slide in a

uniform layer homogeneity randomization
↑

Prepare 1-2 smears only Multiple preparations from same

sample 53
 Brain Storm
Gynecologic – specimen collection
Transformation Zone (TZ); or
Squamous Columnar Junction (SCJ)

Usually collect cells

from TZ.

WHY?

54
 HPV – Human papilloma virus

Sampling:
TZ/SCJ
squamous
↓
columnar

I
squamous columnarjunction
Inepithelial
protection, HPVattack
↳
malignancy)

When immature basal cells do not have the protective cover of more mature cells,
they are vulnerable to infection. Exposure of the immature basal cells can occur
through metaplastic process if the transition zone of the cervix or through external
insults resulting in mucosal or epithelial injury
skip

Location of TZ:
A) Before menarche;
B) after puberty and
at early reproductive
age; C) in a woman
in her 30s; D) in a
perimenopausal
woman; E) in a
postmenopausal
woman

56
 Fixation
 Cells in fluid without fixation will undergo autolysis because
they contain proteolytic enzymes which destroy their protein
contents when they are sampled
 Good fixation is an essential first step for correct
interpretation of the microscopic features present in the
examined cells
 Maintain as closely as possible the cytomorphologic
characteristic and the cytochemical elements of the cells
 Reduce artificial cell changes while producing an optimal
diagnostic cell sample

57
 Aims of Fixation
 Prevention of autolysis (disintegration of cells caused by
the release of damaging intracellular enzymes)
 Prevention of bacterial attack
 Leaves cells in a condition that allows subsequent staining
techniques
 Cells should remain as close to their living state as
possible
 Do not excessively shrink or swell cells
 Do not distort or dissolve cellular components

58
 Morphology of Irreversible Cell Death
 Pyknosis – Nuclear shrinkage. Condensation of chromatin
 Karyorrhexis – Nuclear fragmentation
 Karyolysis – Nuclear fading. Lysis of chromatin due to action of
endonucleases (loss of nuclear staining, DNA breaking down &
disappearing) Capoptosis)
poorfixation, lysis of nucleus

59
skip Fixation
An ideal fixative
 Penetrate cells rapidly
 Minimize cell shrinkage
 Inactivate autolytic enzyme
 Maintain morphologic integrity
 Replace cellular water
 Allow permeability of dyes
 Permit cell adhesion to a glass surface
 Be bactericidal
 Be reproducible

60
 Fixation
Act of fixation
 By stabilizing cell proteins and inactivating enzymes.
 Rendering proteins insoluble, the whole cell structure is
given mechanical strength
 Change the chemical boding of soluble protein to
structural proteins (may give proteins into 3 dimensional
structure)
 The most common precipitating fixatives are ethanol and
methanol.

61
 Fixation
 The use of prefixatives has become a common practice to
preserve cytology specimens
 Low grade alcohol (lower alcohol content) should be used
to fix the cells away from autolysis or degeneration
 Common prefixatives include 50% ethanol, Saccomanno
fluid (50% ethanol & 2% carbowax) and alcoholic saline
 Thin layer technologies have developed preservative fluid
containing weak alcohol and antimicrobial compounds as
prefixative solutions
*
 The SurePath system (BD) uses a weak ethyl alcohol
*
based solution, while ThinPrep system (Hologic) uses a
weak methyl alcohol based prefixative

62
 Pre-Fixative
50% Ethanol
 Best universal fixative of fluid specimens
 It precipitate large protein molecules and are good for
cytological preservation
 Ethyl alcohol in a concentration higher than 50% should
not be used in collecting fluids rich in protein BECAUSE
the sediment becomes hardened and very difficult to
spread on glass slides
surface hardened X
penetrate
highgrade alcohol
->

63
 Pre-Fixative
Coating fixative
 It is the combination of an alcohol-base, which fixes the cells,
and a wax-like substance (polyethylene glycol ), which forms
a thin protective coating over the cells
 Remove water-soluble coating before staining
 Reason : Hinder the penetration of stains into the cell
 Method : Immerse in tap water or 95% ethanol at least 30’ prior
to staining
X alcohol
use: base
spray

64
 Coating Fixative
 Coating fixative is useful in situations where use of wet
fixatives is impractical
 Relatively expensive
 When using the spray fixative, the slide should not be
closer than 15cm from the can or too far.

65
 Pre-Fixative
Disadvantages in using the pre-fixative
 Coagulates protein which not pass more readily through
membrane filters &
easily dislodge
too thick

 Interferes with the adherence of cells to glass slides

 “Rounds up” cells that make stains difficult to penetrate
cell
 Prefixed specimens may prevent special staining (eg.
ORO, crystals demonstration)
I dissolve crystals

66
 Pre-Fixative
 Some laboratories prefer that all non-gynecologic
specimens are collected fresh, with no fixative or additive,
and sent to the laboratory as soon as possible or kept
refrigerated until sent

67
 Post-Fixative
 All fresh prepared smears should be immediately fixed by
 Alcohol fixative or
 Coating fixative or
 Air drying CFNA:thyroid/lymph node
 Alcohol fixatives are specifically recommended for
cytological preparation

68
 Alcohol Fixatives

95% Ethanol
 Recommended for the fixation of all routinely prepared
smears which give excellent results

69
 Fixation of Cytologic Specimens
Type of Specimen Pre-fixative Post-fixative for
Smears
CSF 50% Ethanol (1:1) 95% Ethanol
Bronchial aspirate
Pleural, peritoneal &
pericardial fluid
Urine
CBD brushing
Rinsing fluid for FNA

Gastric washing 95% Ethanol (1:1)

I
95% Ethanol
need to degenerate enzymes

Joint fluid for crystal Fresh / 100% Ethanol (1:1) 100% Ethanol

Liquid base gynecological CytoRich Preservative Fluid Reagent Graded Alcohol /

specimen / PreserCyt Solution 95% Ethanol

70
 Cytopreparatory Techniques

Brain Storm
Cytology = study of cells by microscope

How????

71
Cytopreparatory Techniques

Centrifugation

Sedimentation
(cell
concentrate)
 Cytopreparatory Techniques
Specimen preparation:

Cytospin

Smearing

SurePath
Centrifugation

ThinPrep

Glass slide
Cell block

73
 Cytopreparatory Techniques
Aim: Adhere the cells onto the slide for microscopic
examination

Cell Cytospin SurePath ThinPrep Smearing Direct Conventional

block smear smear

74
skip Cytopreparatory Techniques

 Aim: Adhere the cells onto the slide for microscopic

examination with good morphologic detail and evenly
thinlayer of cells
 There are different methods are available to prepare cytology
smears
 Certain techniques work best for particular specimen types
 Know each cytopreparatory techniques will help to determine
the best way for individual specimens giving a uniform,
homogenous smear for microscopic examination

75
skip Cytopreparatory Techniques

 Centrifugation (Smearing)
 Cytocentriguation (Cytospin)
 Pick & Smear Method
 Liquid-based Thin-Layer Preparation
 Cell Block / Agar Block / PT Cell Block

76
 Cell Adhesion
Adhesive “Aids”
 Cytologic specimens are spread on slides in an effort to
keep cells from falling off when immersed in alcohol by
using
 Albuminized slides
 Silane-coated frosted slides (aminoalkylsilane)
 Positive charged slides
 Sand-blasted frosted slides
 Provide special surfaces help cells adhere to slides

·Xadd adhesive
·
drop albumin into contrifugation
cell suspension by
 Centrifugation
 Fluid specimens of all types usually require some method
of concentration to facilitate the deposition of the
suspended cells onto the glass slides
 Concentration of fluid specimens has been traditionally
achieved by large-volume centrifugation
 Using a swing out centrifuge instead of fixed angle
centrifuge ensuring the cells are concentrated at the
bottom of the conical tube, rather than along the side

78
 Centrifugation
 Use to separate cells from fluid
 Time & speed: vary from lab. to lab. (personal choice)
 An ideal cell sediment can be obtained using 600xg
(1500rpm) for 10 minutes
 Tissue fragments and large particles with higher density
are usually in the lower level of sediment after
centrifugation
 Higher speed will tightly pack the cells that may produce
an uneven material onto the smear because hard to
disperse the clumpy sediment
 Lower speed do not have enough force to concentrate the
cells to the bottom of the centrifuge tube

79
Centrifugation

80
 Smearing
 Discard the supernatant
 Re-mix the sediment by using a pasteur pipette
 Transfer the cell concentrate onto albuminized glass slides
 Make the smear by covering with another slide and gently
pulling the two aparts
 The smears should then be fixed immediately in 95% ethanol
sample cells spread DATE AFFF!
smearing for
X urine

81
Smearing

82
 Cytocentrifugation

 The fluid samples with low cell content such as CSF and
urine are centrifuged in Cytospin where the cells are
sedimented directly on the glass slides
 Fluid specimen should be pre-centrifuged to obtain
concentrated cell samples
 Specially designed slide centrifuge which in one operation
performs:
a. Centrifugation
b. Deposition
c. Concentration/collection of cells onto a glass slide

83
Cytocentrifugation
1. Glass slide
2. Filter card
3. Sample chamber (Cytofunnel)
4. Slide clip

84
 Cytocentrifugation
 Prepare labelled coated slides
 Load the slide, filter card and sample chamber into the slide
clip
 Pipette the prepared fluid sample into the chamber
 Load the prepared assemblies into the cytospin head
 Run the program

85
 Pick & Smear Method
 Preparation used for sputum sample
 Using wooden applicator sticks and forceps, select any
blood stained or discolored tissue-like material
 Transfer onto the clean side of a slide
 With another clean glass slide, crush the material using a
gentle rotary motion
 Slide them apart to produce two evenly spread smears
(pull apart method)

86
Pick & Smear Method

87
 Sputum Preparation
 Because of the relatively poor diagnostic sensitivity, break
up the mucus (mucolysis) before smear preparation is the
way to maximize detection rate
 Chemical methods achieve the liquefaction of mucus to
release the admixed cells, which can then be concentrated
by centrifugation prior to the preparation of glass slides
alcohol
replaced by
30%

 Dithiothreitol (DTT) is a commonly used substances to

reduce the viscosity of mucus (others including enzymes,
peptides, and dilute mineral acids )
 Using thinlayer methods

88
Liquid-based Thin-Layer Preparation

 Liquid based cytology (LBC) is a method of preparing cells

for examination in a laboratory
 A small sample of cells are placed directly into a vial
containing a preservative fluid
 3 liquid based Papanicolaou (Pap) tests are FDA
approved for use in the United States as replacements for
the conventional Pap smear

89
 Available approved LBC Systems
FDA Approval
 ThinPrep (Cytyc,Hologic, Marlborough, MA) was FDA-
approved on May 1996
 SurePath (BD DiagnosticsTriPath, Burlington, NC)
receiving FDA approval in June 1999
 MonoPrep Pap (MonoGen, Lincolnshire, IL) tests approved
on Mar 2006
 ThinPrep (TP) and SurePath (SP) are most currently used
liquid-based cytology preparations

90
 Liquid-based Thin-Layer Preparation

SurePathTM BD Totalys™ SlidePrep

• Homogenization
• Randomization 91
Liquid-based Thin-Layer Preparation

ThinPrep 2000 ThinPrep 5000

92
 Principles of the preparation of
Liquid Based Cytology (LBC) slides
1
 A sample of cells is collected e.g. from the cervix in the normal
way using a spatula or broom sampling device
2
 The sample is transferred into a container of preservative/
transport medium
3
 The cell are dispersed in the fluid
4
 An aliquot of the suspension is selected for processing
5
 The cells are separated by centrifugation or filtration and
deposited on a slide as a thin layer / monolayer by
sedimentation or the application of pressure
6 7
 The slides are stained , mounted ready for microscopy

93
ThinPrep System

94
 ThinPrep Preparation Process
1. Dispersion
The Filter rotates within the sample vial, separating
debris and dispersing mucus without adversely affecting
the appearance of cells.

2. Cell Collection
A gentle vacuum collects cells on the exterior surface of
the Filter membrane.

3. Cell Transfer
After the cells are collected, the Filter is inverted and
gently pressed against the ThinPrep Microscope Slide.
Surface tension and air pressure cause the cells to
adhere to the Slide, resulting in an even distribution
within a circular area.
95
 ThinPrep

Specimen preparation: ThinPrep (T2000 processor)

Remove
Specime supernatan
n + 50% t + 10%
ethanol Vortex, then
centrifugation Acetic acid
CytoLyt

Vortex, then
centrifugation
 ThinPrep

Specimen preparation: ThinPrep

Filter

Resuspense
sediment
into vial
containing
PreservCyt Cells
SPIN
Negative
pressure
 General Appearance of ThinPrep
Slides
 2.0 cm diameter circle
 70k vs 250k cells per sample
 Well-demarcated edge – no ‘drift’
 Cells evenly distributed
 Holes in between cells
EFETIT
*
 Good Fixation and nuclear detail
 Cleaner background

101
 ThinPrep 5000
Walkaway automation for efficient workflow
• • Continuous, hands-free processing with up to
45 minutes of walkaway time means operators
are free to focus on other tasks
• • Carousels can be loaded at accessioning and
easily transported
• • Load up to 20 vials, slides and filters
• • Processed slides are racked and ready for
staining. Output racks can be loaded directly into
select stainers and coverslippers*
*Adapters may be required for some models.
 ThinPrep 5000
Intuitive design
• • User-friendly touch screen interface for real-time
progress tracking and reporting
Ergonomic design to reduce repetitive strain
• • Automated vial uncapping and re-capping
• • Minimizes risk of repetitive strain injuries for
operators
Chain-of-custody verification for each sample
• • Barcodes on the vial are automatically matched to
the label on the slide, reducing the possibility of errors
 SurePath System
Specimen preparation: Totalys SlidePrep System

Centrifuge PrepMate Vortexer

SlidePrep system
 SurePath System

Specimen preparation: Totalys SlidePrep System (SurePath)

slow speed:RBC, debris ontop
Still remain
Rbc, adequate
inflammatory amount of
cells, debris background
Specimen material
Centrifugation Aspirate the Centrifugation
200rcf, 2 min upper portion 800rcf, 10 min

Density
Gradient
Reagent
heavy:bottom
light:surface
skip SurePath Preparation Process

 Vortex sample => release all cells from the brush

 PrepMate
 agitate sample (further release cells from brush/
mucinous material + randomization)
 Transfer sample into tube (with density reagent)

 1st centrifugation & evacuation => remove

excessive rbc, inflammatory cells, debris etc.
 2nd centrifugation => get cell sediment

108
 SurePath Preparation Process
 The cell enrichment process combines gravity dispersion
and centrifugation to separate obscuring blood, mucus,
inflammation and other debris from diagnostic material,
creating an enriched cellular sample.

109
 SurePath Preparation Process

 Decant supernatant & vortex

 Place sample onto SlidePrep System
 Run the program
 Detach chambers & mounting

110
 SurePath System

Specimen preparation: Totalys SlidePrep System

Settling
Chamber

Specimen
 SurePath System

Specimen preparation: Totalys SlidePrep System

Gravitational
force
 SurePath System
(cell stick onto the slide by gravitational force

 Automated sample handling

 Minimizes debris, RBC’s, mucus, and inflammatory cells
by using Density Reagent
 Consistent staining (programmable) but hrartefact
↑

 Easy to screen 13mm circle

 More 3D effect
I
e.g. glandular cells still 3D-shape
on slides

#
②

113
skip Other preparation system

• AutoCyte Preparation System (APS) => BD SurePath –

PrepStain  Slide processor, US

• ThinPrep® Processor – Hologic, US

• MonoPrep Pap (MonoGen, Lincolnshire, IL) test

• EASYPREP (+REBA HPV-ID), Korea

• HURO PATH Celltrazone Co. Ltd, Korea
• LITUO liquid based cytology slide processor LT-YJ2000,
China
• E-PREP System, India
skip

HURO PATH Celltrazone Co. Ltd, Korea

skip

https://detail.en.china.cn/provide/p129078
809.html
skip

• EASYPREP (+REBA HPV-ID), Korea

https://www.zafire.com.ph/wp- EASYPREP® System is a liquid-based cytology specimen collection,
content/uploads/2018/04/EASYPREP%20B preservation and automated slide preparation system that produces
rochure%20Rev.02(4p)-170403.pdf standardized thinlayer slides for cervical cytology specimens and other non-
gynecologic cytology specimens.
 Liquid-based Preparation:
Advantages
 Easy and uniform sampling
 Ensures 100% of the collected sample is processing without
loss
 Rapid fixation
 Reproducible
 Removal of obscuring elements
 Improving detection & reproducibility
 Reduction in false negative rate

118
 Liquid-based Preparation:
Disadvantages
 More costly than conventional pap smear
 Preparation is more labor intensive than conventional
 Loss of background material
 Some differences in architectures and morphology
 Require training for the screeners

119
 Comparison between Conventional
Method & Liquid Base Method

Conventional Pap Smear Liquid-based Cytology

 Majority of cells not captured
 Non-representative transfer of
cells
 Clumping and overlapping of cells
 Obscuring material
 Air-drying artefacts may happen
 Virtually all cells of samples are
collected
 Randomized, representative
transfer of cells
 Even distribution of cells
 Minimizes obscuring material
Comparison between Conventional
Method & Liquid Base Method
Conventional ThinPrep SurePath
smear

Fixation Ethanol Methanol Ethanol

Collection Smear on slide Sample rinsed in vial Collection device left

in vial

Cell sample Random distribution Uniform distribution Uniform distribution

over 20mm of slide over 13mm of slide

121
randomization)

(brisk of contamination)
It cell debris

3D cellsonslide (alcohol.IIA-A-I..** I
 Cell block

Advantages
 Provide and demonstrate the architecture of histologic
pattern (more informative than the direct smears)
 Enable special staining / immunocytochemistry studies
and helps in differential diagnosis

Disadvantages EAAREFAcell loss

 A delay of approximately 24 hrs for processing
 Increase cost

122
skip Cell block
 Centrifuge the specimen in 7.5% neutral buffered formalin at
1500 rpm for 15 min
 Pour off supernatant and drain well
 Carefully remove the sediment by means of forceps
 Wrap in lens paper
 Place the wrapped sediment into a labelled cassette
 Embed, cut, mount and stain according to standard
histological techniques

123
skip
Cell block

124
less cell loss
Agar block
temp, degenerate cellular material e.g. protein
X meltathigh
 Two drops of melted 1% agar
 Trim the excess agar from the
sediment
 Stain and wrap the agar button
in a lens paper
 Place the wrapped sediment
into a labeled tissue capsule
and process according to
standard histological techniques

125
 X:GX
Pusepatient plasmaltesting
cell-free DNA combined patients'
DNAC
↓ ② have

Plasma-Thrombin Cell Block Method

may

(fibrin) plasma thrombinefibrinogen

For material too sparse to be picked up for Cell Block

Method
1. Centrifuge the specimen at 1500 rpm for 10 minutes
2. Decant the supernatant
3. Add normal saline and well mix
4. Centrifuge at 3000 rpm for 10 minutes. (Turn off the braking mechanism).
5. Decant supernatant. (Using a pipette if the material is not compacted enough.)
6. Add 2-3 drops of de-frost plasma
7. Add one drop of working thrombin
8. Set aside the tube until a gelatinous clot is formed.
9. Carefully remove the clot from the tube by using a wooden applicator or
forceps onto a filter paper
10. Roll the clot with the wooden applicator to squeeze out excess fluid and round
up the clot
11. Dye the clot with Gill’s Hx and wrap in the lens paper
12. Place the wrapped clot into a labeled tissue capsule
13. Process in the tissue processor
14. Embed, cut, mount and stain according to standard histological techniques

126
skip

Plasma-Thrombin Cell Block Method

127
 Staining
 The outcome of an optimal staining is crisp nuclear detail
and transparency of the cytoplasm, which allows the
examiner to clearly visualize cellular morphology.

 Nuclear stain + Counter stain

 H&E & Pap stain are commonly use in cytology

128
 Staining
Purpose
 Stain preparations to
facilitate cell visibility,
detection, and
interpretation
 Definition of nuclear detail
 Cytoplasmic transparency
 Cell differentiation

Bronchial Aspirate (Pap, 400x)

129
Hematoxylein & Eosin (H&E) Stain
 H&E is the most widely used stain
 A combination of a basic stain (hematoxylin) and an acidic
stain (eosin)
 Hematoxylin strongly binds to acidic components of a cell,
most importantly to the phosphate groups of nuclear DNA
 Eosin which react with basic cell components of a cell on
the slide to give a pink colour
 Offers little cytoplasmic differentiation

130
 H&E staining
Gill’s Eosin
Haematoxylin

Nuclear stain Counter stain

• Haematoxylin • Eosin Y (alcohol

• Sodium Iodate & water soluble)
• Aluminum sulphate
• Calcium chloride
• Glacial acetic acid
• Ethylene glycol
Gill's 1:11 have less
hematoxylin

Gill’s I, II & III

(ingredients % & strength)
 H&E - Nuclear stain
extracted from heartwood tree
(Haematoxylon Campechianum) Contains metal ion:
Most commonly
aluminum alum => Al3+
Eg. Sodium iodate Covalent (trivalent metal)
Haematoxylin bond
Oxidation/ Ripen

Anionic, does not have

+ -
much affinity for DNA Haematein Mordant Nuclei
acid

Dye-metal complex Basophilic structures

Basic dye (positive charge/ (anionic), chromatin,
cationic stain) ribosomes

Acidic acid=> to stabilize

Glycerol => to stabilize & reduce evaporation
 Staining Method
Nuclear Stain
Progressive Method
 Stain for the desired color intensity & sets the nuclear dye
in place by blueing agent

Regressive Method
 Over-stain with a hematoxylin, then remove the excess
stain with dilute HCl

133
 H&E – Counter stain
Eosin
• Counter stain,
• Synthetic dye, fluorescent,
xanthene dye Bluing solution
• (to give contrast color) (alkaline)
• Anionic
• -ve charge
• Acidic stain
Nuclei
Bind to and form salts with basic, or eosinophilic,
compounds like proteins containing amino acid
residues such as arginine & lysine
Staining based on the actions of bromine on eosin
Cytoplasm
Pink:
Cytoplasm,
Collagen, muscle fibres
Extracellular matrix
skip Papanicolaou’s Stain

 It was developed by the Greek doctor George Nicholas

Papanicolaou, the father of cytopathology
 First reported that uterine cancer could be diagnosed by
means of a vaginal smear in 1928
 Invention of the Papanicolaou test, commonly known as the
Pap smear or Pap test, which is used worldwide for the
detection and prevention of cervical cancer
 He also developed the particular polychrome staining
reaction designed to demonstrate variations of cellular
maturity and metabolic activity – Papanicolaou’s stain or
Pap stain

135
skil
Papanicolaou Staining Method
 The Papanicolaou stain is a polychrome stain
 The Pap. method involves nuclear stain & cytoplasmic
counterstain
 OG-6 and EA are the two cytoplasmic counterstains
 Modifications of the technique vary from laboratory to
laboratory and it is the change of the length of time in the
hematoxylin and EA dyes

136
skip

Principle of Papanicolaou Stain

Nucleus Staining
 Hematoxylin is a basic dye extracted from the heartwood of
the tree Haematoxylum Campechianum
 Hematoxylin is a very weak stain by itself
 Hematoxylin is converted by an oxidizing agent to hematein,
which upon addition of alum (mordant, e.g. potassium alum)
will form a chelate bond with aluminum, resulting in
aluminum rack
 This is positively charged and has staining ability which
strongly binds to acidic component of a cell, and upon
binding to the negatively charged phosphate group of a
nucleic acid will turn it dark purple

137
 alcohol-based
PAP stain
-
Hx OG EA
Haematoxylin: Orange Eosin Azure (eg.
Nucleus - blue Green EA31, 50, 65)
(eg. OG6) Acidic stain
Acidic Contains
stain • Eosin Y
• Light green SF
• Bismark brown

Counterstain:
• OG: stains the cytoplasm of mature keratinized cell => orange
• Bright and dense orange: hyperkaratinization (eg. Squamous cell
carcinoma)
• Eosin: stains the cytoplasm of mature squamous cells, nucleoli, rbc,
cilia => pink
• Light green: stains the cytoplasm of active cells (eg. Columnar cells,
intermediate, parabasal squamous cells) => blue; blueish green

All OG, Eosin & light green => acidic dye EA & OG => to differential different cell function
 Papanicolaou Staining Method

Cytoplasmic Stain
Orange 6 (OG 6)
 An acidic, monochromatic
dye
 Stains keratin a bright and
intense orange

Sputum (Pap, 200x)

139
 Papanicolaou Staining Method
Cytoplasmic Stain
EA (Eosin Azure)
 A polychrome stain composed of three dyes: light green,
eosin and Bismarck brown
 EA-36, EA-50, EA-65: the number denotes the proportion
of the dyes
 Light green stains the cell that are metabolically active in
blue colour like parabasal squamous cells, intermediate
squamous cells & columnar cells
 Eosin gives a pink colour to cytoplasm of mature
squamous cells, nucleoli, cilia and red blood cells
 Bismarck brown does not add a characteristic color to the
cytoplasm and sometimes it is often omitted

140
 Brain storming

EA & OG are both counter stain

Can we use EA-OG combo

(mixture)?
 PAP stain
Hx EA-OG
combo
EA - OG
What is the difference if
EAOG combo is used?
EAOG
combo

EEFA!
 Staining Procedures
(1009,95%;70%)
Hydration 14 staining:be reminded that his water based

 From fixative (95% alcohol) the cells are hydrated through a

graded series of alcohols to water preparatory to
haematoxylin staining
1709 95%>100%)

Dehydration
 Dehydration prepares the cell sample for uptake of the
cytoplasmic counterstains (alcohol based)

Clearing (xylene)
 Result in cellular transparency (pass through of light)

143
 Causes of Inconsistent Staining
Factors affect the Pap staining reaction:
 Type of fixative used
 Type of hematoxylin formula selected
 Formulas of the counterstains
 Length of staining times
 pH of tap water and staining solutions
 Age of the dyes used
 Insufficient rinsing after acid
 Air drying of slides between solutions
 Improper draining of slides during staining
 Inconsistency of the staining technique

144
 Hints in Staining Slides
 Stains, alcohol & xylene should be filtered before use
 Gentle dipping of slides will avoid cell loss and possible cross
contamination
 Slides should be well drained between each solution
 Discard the water rinse after each use
 Slides should not be permitted to sit in the alcohol solutions
following the eosin, OG and EA dyes to avoid discoloration
 Gentle dipping of slides will avoid cell loss and possible cross
contamination
 The level of solutions should be maintained during the entire
staining process
 The level of the solutions following the OG or EA dye should be
higher in order to achieve complete washing the excessive stain
and dehydration
 Solutions should be kept covered when not in use
145
 Auto-staining Machine

Advantages
 Menu-driven safe and homogeneous slide staining
mechanism
 Fully automatic and programmable reagent management
system
 Flexible transmission mechanism & precise positioning
system ensure stable staining
 LCD display with touch pad key menu / TFT Touch screen
display with touch controls and menu

146
May-Grunwald-Giemsa (MGG) Stain

 MGG stain is a type of Romanowsky-Giemsa stains

 It is useful for studying cell morphology in air-dried smears
 May-Grünwald Giemsa (MGG) staining is a mix of two
neutral stains:
 May-Grünwald stain composed of an acidic stain (eosin) and
a basic stain (methylene blue)
 Giemsa stain composed of eosin and another metachromatic
basic stain: azure of methylene
 Brings out a variety of cytoplasmic granules, inclusions,
vacuoles, basement membrane material
 Chromatin feature is difficult to assess
 Air-drying may induce artifacts
147
 Routine Staining
Specimen Method
Gynecological conventional smear / Papanicolaou stain (EA 50)
Thinprep preparation

AutoCyte gynecological preparation Papanicolaou stain

(EA 50/OG 6 mixture)

Non-gynecological smear Papanicolaou stain (EA 65) / H&E

Aspiration smear Papanicolaou stain (EA 65) / H&E

Air-dried smear MGG / Diff-Quik stain

Cell block section H&E

Smear or Cell block for special Special & Immunocytochemical

purposes stains
148
skip
Special Stains
 The common special
stains used in
histopathology can also
used for cytological
smears without major
modification

Hemosiderin laden macrophages

149
(Bronchial aspirate, Perl’s, 200x)
skip

Immunohistochemical Stains
 Alcohol fixed smears, air-
dried smears or sections of
formalin fixed cell blocks
are suitable for
immunoperoxidase
staining with a variety of
markers
 Detection of surface
antigens (markers) on
isolated cells
 The detection is based on
specific antigen-antibody
binding Metastatic adenocarcinoma
(Pleural fluid, CK20, 200x) 150
skip
Staining Machine
• SAKURA TISSUE-Tek DRS 2000
• Varistain Gemini
• Thermo Scientific Varistain V24-4
• Vector Nate VM100
• EMCO Fastainer
• Trivatron Compass Stainer
• MEDITE TCA 44-720
• ROCHE Ventana HE 600
• AEROSPRAY CYTOLOGY slide stainer/
Cytocentrifuge
skip
Staining Machine
• Dakewe
• Leica HistoCore SPECTRA station
• Leica ST4020 small linear stainer
• Leica ST5020 Multistainer
• Leica ST5010 Autostainer XL

Some staining machine may accompany with coverslipper

(ie. autostainer + auto-coverslipping)
 Staining Machine
My workplace:

Dakewe Leica Fume hood

Autostainer coverslipper I
quality of
check
slide
SCREENING

154
 Diagnosis of Malignancy: Cardinal
Rules

Malignancy determined By nuclear features

Type of malignancy By functional differentiation

determined of cytoplasm

155
*
Cellular Alteration in Malignancy
 Nucleus
 Nuclear-cytoplasmic ratio (N/C ratio)
 Cytoplasm
 Cellular arrangement Lalign / dumping)
 Background Idiathesis:degeneration ofcell and cell debris
release

↑
degeneration of nucleus

156
 Nucleus
 The assessment of a cell’s nucleus is one of the most
important tasks in cytopathology
 Nuclear size, chromatin density, nuclear membrane, and the
presence of nucleoli are important features of nuclear
morphology
 The nucleus contains the genomic DNA, histones, and several
proteins that are responsible for DNA replication, repair, and
transcription of genetic information
 Histones are basic proteins that build a structural unit together
with the DNA, called the nucleosome
 Nucleosomes form the fundamental repeating units of nuclear
chromatin

157
skip
Nucleus
 Chromatin represents the complex structure of proteins and
DNA in the nucleus of non-mitotic cells.
 Two conformations of chromatin are discriminated:
euchromatin and heterochromatin.

158
skip
Nucleus
Nuclear membrane
 Nuclear envelope is composed of outer nuclear membrane,
INM, and nuclear lamina
 Nuclear lamina is made up of lamin
 The qualitative or quantitative changes of nuclear lamin are
responsible for alteration of nuclear margin and shape
 Nuclear margin irregularities are in association with
mutation of lamin
 Other factors: gene silencing, extracellular matrix (ECM),
RET-PTC oncogenic changes, NM protein and nuclear
skeleton altered
 Nuclear margin irregularity may be seen as nuclear
grooving, nuclear molding, and nuclear convolutions

159
skip Nucleus
Chromatin
 Nuclear chromatin pattern alteration is the hall mark of
the malignant nuclei
 Chromatin pattern of nucleus of normal cell is usually fine
 The genes packed in chromatin are in two forms, less
compact active euchromatin and more compact inactive
heterochromatin
 Heterochromatin is situated in the peripheral part of the
nucleus, whereas euchromatin is located in the more central
part of chromosome
 Irregular thickening of nuclear margin in malignant nuclei is
due to the peripheral heterochromatin formation

160
skip Nucleus
Nucleolus
 The changes of nucleolar composition, enlargement, and
pleomorphism are the characteristics of malignant cells
 Nucleolus is the organelle of the interphase nucleus and is
closely related with biogenesis of ribosomes
 The basic function of the nucleolus is ribosome biogenesis
and the rate depends on the metabolic activity of the cell
 The proliferating cells have increased demand for protein
synthesis, which is reflected on the increased rate of
ribosomal biogenesis

161
skip Nucleus
Nucleolus
 As the cancer cell has the higher growth fraction (increased
cell proliferation) shows nucleolar enlargement
 Cell proliferation rate may not be the sole factor of the
nucleolar change, also tumor suppressors and oncoproteins,
such as p53 and pRb, are directly related to the degree of
alteration of the pathway

162
 Nucleus

Cytologic criteria of malignancy

 Variation in size (anisonucleosis)
 Variation in shape (nuclear pleomorphism)
 Nuclear outline
 Irregular nuclear outline
 Irregular thickened nuclear membrane

163
 Nucleus
Cytologic criteria of malignancy
 Chromatin pattern
 Pattern is altered
 Chromatin clumps appear with sharp, well defined borders
 Coarsely granular Igranules nucleus inside

 Hyperchromatism
 Variation in chromatin staining
 Nucleolus (RNA) malignant changes
 Angulated shape
 Massive size
 Increased number
 Mitosis (especially if atypical)
↓ active cells (reactive)

164
 Normal cells: Nucleus

 Uniform nuclear size and

shape
 Fine chromatin and evenly
distributed
 Regular and smooth
nuclear membrane
TI7HT-
 Inconspicuous nucleoli

165
Malignant cells: Nucleus

chromatin
I coarse
&

① nucleolus
↳
malignancy

②
Nucelar size variation

166
Malignant cells: Nucleus
Hyperchromatic nuclei

167
Malignant cells: Nucleus

Coarse
chromatin

168
Malignant cells: Nucleus

Irregular nuclear
membrane

169
Malignant cells: Nucleus

hyperchromasia
Irregular nuclear
membrane

170
 Malignant cells: Nucleus
Thickened
nuclear
membrane

coarse math
Prominent
nucleolus

171
 Malignant cells: Nucleus

Prominent
nucleoli

L
Ya mocav inous
vacuolated
cytoplasm

172
Malignant cells: Nucleus
foamyvacuolated cytoplasm
&

“Angulated”
nucleolus

173
skip

Malignant cells: Nuclear-Cytoplasmic Ratio

 Increased N/C ratio in malignant cells
 Due to nuclear enlargement & decrease in cellular
maturation
 Degree of cellular maturation inversely proportional to
N/C ratio: more immature the cell, higher the N/C ratio &
vice versa

Nucleus

Cytoplasm 174
Malignant cells: Nuclear-Cytoplasmic Ratio

Increased N/C ratio

 Malignant cells: Cytoplasm
cells
 Variation in cell size squamous-likeepithelial
cells
 Bizarre cell shape glandular:columnar shape
 Vacuolization honey
comb

 Keratinization
 Lesser differentiation ChighN:Cratio)

foamy cytoplasm

r
V

cell
squamous
X carcinoma
OGstain)
Ikeratinized:
 Malignant cells: Cytoplasm
glandular epithelial cells nucleol
predominant vacuoles
⑨
> adenocarcinoma

Vacuolated cytoplasm
Malignant cells: Cytoplasm

cell carcinoma
squamous
M
V

Keratinizing cytoplasm
Malignant cells: Cellular Arrangement
of nucleus
overproliferation
V

 Nuclear crowding & overlapping

 Loss of nuclear polarity "honey combarrangement
<- normal:
chromating

chromatin
irregular
abnormal:

 Tumour cell cannibalism paper rock

2

 Decrease cell cohesion

179
Malignant cells: Cellular Arrangement

Nuclear crowding & overlapping

 Malignant cells: Cellular Arrangement

·honeycomre
⑨
W

->
irregular
arrangement

Loss of nuclear polarity

Malignant cells: Cellular Arrangement
chromatin
X
coarse

set
5 predominant

paper rock

Tumor cell cannibalism

Malignant cells: Cellular Arrangement

Decrease cell cohesion

Malignant Lesions: Slide Background

Increased cellularity
Malignant Lesions: Slide Background
hyperchromain
⑨ neon of alls)

<- rupture
o f
sell membrane

Tumor diathesis 185

Malignant Lesions: Mitosis

Abnormal mitotic figures 186

 Benign Cells Cancer Cells

Cell size & shape Regular Variation in size and shape

(not good criterial

Nuclear size Regular Significant variation in nuclear size

(anisonucleosis)

Nuclear shape Generally round, oval or bean Abnormal shape

shaped

Chromatin Finely granular chromatin, evenly Coarse granular chromatin,

structure distributed unevenly distribution

Nucleoli Small , even size , few in number Large, irregular, variable in size &
shape & in number

Hyperchromasia Rarely seen Common reflecting increased

(affected by procedures) chromatin content or rapid cell
turnover or both
Cohesiveness Well formed cell junctions Loss of cohesiveness

Mitoses Occasionally seen Abnormal mitoses frequently found

187
 General Rules
 Cytologic diagnosis of malignancy should not be made on
the basis of a single criteron

 Cytologic diagnosis of malignancy should not be made on

a single cell

 Proper cytologic assessment requires optimal technical

preparations & adequate clinical information

188
 Difficulties in Morphological
Interpretation
 Varies malignant tumors exhibit various cytologic appearances
 Different types of tumors may exhibit similar cytologic
appearances
 Individual tumors of the same type may exhibit different
cytological appearances
 Specimen from the same tumor obtained by different cytologic
methods may exhibit different cytologic appearances
 The same specimen prepared by different cytologic techniques
may exhibit differences in cytomorphology
① ②
 Irritated epithelial cells and reactive cells may meet cytologic
criteria for malignancy ③ predominant nucleolus

189
 Difficulties in Morphological
Interpretation
 Varies malignant tumors exhibit various cytologic
appearances chromatincoarse

/
V

(Xsee nucleoli)
Squamous cell Adenocarcinoma Small cell carcinoma
carcinoma

(non-keratinized
predominant nucleolus
keratinized:X predominant nucleolus
 Difficulties in Morphological
Interpretation
 Individual tumors of the same type may exhibit different
cytological appearances

Adenocarcinoma of cervix Adenocarcinoma of lung

 Difficulties in Morphological
Interpretation
 Irritated epithelial cells and reactive cells may meet
cytologic criteria for malignancy
vacuolated cytoplasm
predominantnucleoli

cell
x ring

Reactive mesothelial cells Adenocarcinoma of stomach

 Cytological Diagnosis:
Cautions
 An adequate clinical history
 Awareness of the advantages and disadvantages
associated with alternative sites & methods of collection
 That the lesion be accessible
 That the lesion exfoliates cells or can be abraded
 That the specimen be suitably fixed & stained
 That the slide be critically examined in its entirety
 The final decision concerning a specimen should only be
made in the light of clinical & pathological confirmation
 Digital pathology Imaging
for QC of
system
purpose instead diagnosis
(to improve sensitivity)
• Automation of cytopathology has been
performed since the 1980s
• BD FocalPointTM GS imaging system and
ThinPrep imaging system are widely used

• FocalPoint => utilizes algorithms for image

recognition;
• ThinPrep imaging sys => based on
densitometric measurement of nuclei
(Al method)
 Digital pathology Imaging
system
***ScanS ->
YRAREA.

• Recent years, artificial intelligence (AI)-based

technologies have been increasingly explored
for cytopathology application
• ‘deep learning’ extracts hierarchies of features
without the need for a human to define them
Examples:
• Roche uPath – Image analysis algorithms
• VENTANA slide scanner with Roche Image
Management software
 HOLOGIC – Genius Digital Diagnostics System

Slide scanner can scan a slide in

different layers and compile shape
images of different layers as one.
You don’t have to focus up and
down
 Digital pathology Imaging
system
Concern:
• Slide scanner: speed, resolution (10x, 40x),
layers of a slide to be scanned
• Learning period for different specimen (Gyn,
sputum, body fluid etc.)
• Learning for different preparation methods
(eg. Fixation, Switch from Thinprep to
SurePath => learn again!)
 Digital pathology Imaging
system
Concern:
• Reporting system => can the machine create
report automatically? (authorization)
• Use for QC only?
• Interference (eg. Bubbles, air dry, EM in urine
etc.)
• Image storage vs Slide storage
• Barcode system (slide and scanner)
 Summary
 Cytology is diagnostic method

 Cytology is quick, inexpensive and accurate method,

with a little risk to patient

 Requires good communication with clinicians and

correlation with other diagnostic methods

 Requires continual learning and education

THANK YOU!

Q&A