2. INTRODUCTION
The best morphologic presentation obtained from any
cytologic specimen requires an understanding of the
factors that went into collecting and preparing the
specimen.
to reduce the specimen to a cellular presentation, which
can be interpreted and diagnosed.
Principle of cytotechnique
3. HISTORY OF CYTODIAGNOSIS
The first era
- 19 century
- exfoliated cancer cells
had been described in
all types of specimen
-1861
-Pharyngeal secretion
-Post mortem
-Keratinizing squamous cell carcinoma
4. HISTORY OF CYTODIAGNOSIS
The second era
- era of development and
expansion
-recognized the importance of
of wet fixation of cytological
specimens
- screening of cervical cancer
Dr. George N Papanicolaou
5. HISTORY OF CYTODIAGNOSIS
The third era
- era of consolidation
- technique of FNAC
- Diagnostic cytology and its
histopathologic basis
the fourth era
- the Bethesda system of
reporting cervical/vaginal
cytology diagnoses
Dr. Leopold G Koss
7. TYPES OF CYTOLOGY SAMPLES
Exfoliative
cytology
Aspiration cytology Fin
e Needle Aspiration Cyto
logy (FNAC)
Body fluids
8. EXFOLIATIVE CYTOLOGY
It is the study of cells that have been shed or
removed from the epithelial surface of various
organs.
wash smear
scraping brushing
15. EVALUATION OF SPECIMEN:
Collection Preparation
•pH
•Protein content
•Enzymatic activity
•Bacteria +/-
Fresh material
Prefixation of m
aterial
??????Prefixatives
16. FRESH MATERIAL
↑↑ mucus
↑↑ protein
Low
mucus or
protein
•Sputum
•Bronchial aspirates
•Mucocoele fluid
•Pleural
•Peritoneal
•pericardial
•Urine
•CSF
12 to 24 hrs if
refrigerated
24 to 48 hrs
Without
refrigeration
1 to 2 hour
delay even if
refrigerated
18. CENTRIFUGATION
If too much fluid is obtained,.
If little amount of fluid is aspirated (few drops), or if the fluid is
thick, the centrifuge doesn’t required.
Centrifuged for 5 mins Sediment
19. CYTOCENTRIFUGATION
It is a special machine that performs a centrifuge and
collection of sediment on the center of the slides. This
procedure is useful for hypocellular specimen .
21. CYTOCENTRIFUGATION
Shandon cytocentrifuge I
Shandon cytocentrifuge II
Shandon cytocentrifuge III
Wescor cytopro
Hettick cytocentrifuge
Leif’s centrifugal cytology buckets
22. METHODS OF SMEAR PREPARATION:
streaking
spreading
pull apart
touch or impression smear
23. STREAKING
- Used for preparing mucoid secretions ,
vaginal secretions, sputum and gastric content
-use a spatula, dissecting needle or applicator stick and
streak in a zigzag fashion
24. SPREADING
- used for thick mucoid secretions
- smears of fresh sputum and bronchial aspirates
25. PULL APART
- for serous fluids, concentrated sputum, and enzymatic
lavage form the GIT, smears of urinary sediment, vaginal
pool and breast secretions.
27. SQUASH SMEAR PREPARATION
fairly accurate,
simple and reliable tool for rapid intra-operative diagnosis of
central nervous system lesions.
Based on two essential factors:
• Availability of very small tissue fragments & good
preservation of fine cellular details.
• Not effected by edema, hemorrhage, necrosis &
calcification.
28. CELL BLOCK
It is a procedure to convert cell sediment in to paraffin
block
further pathological procedures can be performed like
immunohistochemistry (IHC).
29. METHODS OF CELL BLOCK PREPARATION
Direct processing of tissue fragments present in fluids
Fixed sediment method
Bacterial agar method
Simplified cell block technique using alcohol, acetone
and paraffin
Compact cell block technique
Plasma thrombin method
30. METHODS OF CELL BLOCK PREPARATION
Cell blocks from millipore
Histogel method
Gelatin embedding
Celloidin bag
Cell block preparation from scraping of cytology smears
Automated cell block preparation
Albumin method
31. PROCEDURES
Centrifugation 2400 rpm
Pour off supernatant
Add 5 ml of methanol +formalin (9:1)
methanol+formalin for 30 to 60 min
Spin at 2400 rpm
remove hardened cell button
submit in a cassette
33. FIXATION OF CYTOLOGY SPECIMENS
fixation means :
- prevention of degeneration of cells and tissue
- preservation of cells as close as possible to the
living state
specific periods of time
changes the physical and chemical state of the cells
34. AN APPROPRIATE FIXATIVE FOR CYTODIAGNOSTIC
PURPOSES SHOULD PERFORM THE FOLLOWING FUNCTIONS
Penetrate cells rapidly
Minimize cell shrinkage
Maintain morphologic integrity
Deactivate autolytic enzymes
Replace cellular water
Facilitate diffusion of dyes across cell boundaries
Help cells adhere to a glass surface
Provide consistent results over time
35. FIXATION METHODS
Wet Fixation
Wet Fixation with Air Drying
Spray Fixation
Liquid-based Fixation for Papanicolaou Tests
Lysing Fixation for Bloody Samples
Air drying
36. WET FIXATION
95% Ethyl Alcohol (Ethanol) • ideal fixative recommended
• dehydrating agent
• desired amount of cell contraction
• yield optimal chromatin detail
characteristics
100 % ethanol •similar effect on cells
•more expensive
Ether alcohol mixture • ether and 95% ethyl alcohol
• 1 : 1
• excellent fixative, but ether
100% Methanol • produces less shrinkage than
ethanol
• more expensive
80% Propanol and Isopropanol •cause slightly more cell shrinkage
Denatured alcohol • 90 parts of 95% ethanol + 5 parts of 100%
methanol + 5 parts of
100% isopropanol.
37. TIME OF FIXATION
Minimum 15 minutes fixation
Can be Prolonged
several days or even few weeks
If smears are to be preserved over a long period of time in
alcohol, it is better to store them in capped containers in the
refrigerator.
38. COATING FIXATIVES
Aerosols or liquid base
Dual action
Carbowax (Polyethylene Glycol) fixative.
Diaphine fixative Spray coating fixative (Hairspray)
have practical value in situations where smears have to be mailed to a
distant cytology laboratory for evaluation
not recommended for bloody smears
alcohol base - fixes the cells
wax like substance - forms a thin protective coating
39. • THE DISTANCE FROM WHICH THE SLIDES ARE SPRAYED WITH AN
AEROSOL FIXATIVE AFFECTS THE CYTOLOGY DETAILS
-
PRIOR TO STAINING, THE SLIDES HAVE TO BE KEPT OVERNIGHT IN
95% ALCOHOL FOR REMOVAL OF THE COATING FIXATIVE.
40. if the carbowax is not removed completely, Nuclei will then
appear foggy and lack chromatinic detail and the cytoplasm may
exhibit a pale blue color
carbowax not removed
Lack of chromatin details and hazy appearance of cell
HSIL, Pap Hp
41. CARNOY’S FIXATIVE
special purpose fixative for haemorrhagic samples
absolute ethanol, chloroform and glacial acetic acid
6 : 3 : 1
acetic acid in the fixative haemolyses the red blood cells.
Modified Carnoy’s
an excellent nuclear fixative as well as a preservative of
glycogen
95% ethanol Chloroform Glacial acetic acid
7 2.5 0.5
6 3 1
6 1
42. OTHER SOLUTIONS USED TO LYSE RED BLOOD CELLS:
Clarke’s solution: absolute ethanol, glacial acetic acid (3 : 1)
One drop of conc HCl per 500 mL of 95% ethanol
Ten percent glacial acetic acid (this is followed by placing the
slide in 95% ethanol)
Commercially available
fixatives such as CytoRich Red
43. REHYDRATION OF AIR DRIED SMEARS
Unfixed, air-dried gynaecological smears received from
peripheral areas can be used for Papanicolaou staining by
rehydration method
The simplest rehydration technique is to place
air dried cytological specimens
50 % aqueous solution of glycerine
2 rinses in 95% ethyl alcohol
Pap staining
45. LIQUID-BASED CYTOLOGY
cytology (the study of cells) through a liquid medium
Cells are collected from cervix(any other site) are placed
directly into liquid preservative, rather than transferred to
slide.
Sample is processed and resultant thin smear easy to screen
50. (1)CELL DISPERSION
Swirling the sampling device in
the preservation solution
Strong enough to separate
debris and disperse mucus
51. (2) CELL COLLECTION
A gentle vacuum is created within
the ThinPrep Pap Test Filter, which
collects cells on the exterior surface
of the membrane.
52. (3) CELL TRANSFER
the ThinPrep Pap Test Filter is inverted
and gently pressed against the ThinPrep
Microscope Slide.
Natural attraction and slight positive air
pressure cause the cells to adhere.
56. Convetional
papanicolao
u
ThinPrep SurePath
Fixation Ethanol methanol Ethanol
Collection Smear on
slide
Sample rinsed
in vial
Collection
device left in
vial
Cell sample Random
distribution
Uniform
distribution
over 20 mm
of slide
Uniform
distribution
over 13 mm
of slide
57. Collection device EC brush,
spatula, Cervex-
Brush
EC brush,
spatula, Cervex-
Brush rinsed
in vial
Cervex-Brush
most effective,
tip
deposited into
vial
Preservation
artifacts
Air drying,
blood,
inflammation,
irregular
distribution of
cells
All preservation
artifacts greatly
reduced
All preservation
artifacts greatly
reduced
58. Automated
processing
Not applicable Vacuum
pressure through
TransCyte filter
Gravity
sedimentation
process
Imaging
technology
Not applicable Available Available
Ancillary testing Not applicable HPV, Chlamydia,
gonorrhea
HPV, Chlamydia,
gonorrhea
60. PAPANICOLAOU STAINING METHOD
named after Dr. George N. Papanicolaou
polychrome staining reaction
display the many variations of cellular morphology
showing degree of cellular maturity and metabolic
activity.
61. PRINCIPLES
Hydration and dehydration:
– Hydration prepares the cell sample for uptake of the nuclear
dye;
– dehydration prepares the cell sample for uptake of the
counterstains.
Dehydration and clearing solutions result in cellular
transparency and prepare the cell sample for the final steps
62. STAINS
Nuclear staining: Hematoxylin
Two cytoplasmic counter staining:
(1) Orange G - (OG)-6, OG-5 and OG-8 is an acidic dye, stains
keratin a bright, intense orange.
(2) Eosin Azure (EA) - EA-36,
EA-50 and EA-65 including
three stains
–Eosin Y
–Light Green
–Bismarck brown Y
63. STEPS OF STAINING PROCEDURE
(1) Fixation
- 95% ethyl alcohol or in other substitutes
- minimum of 15 minutes
(2) Nuclear staining
Harris haematoxylin - regressive staining method
Gill I, - progressive staining method.
Gill II, - progressive staining method.
Gill III - progressive staining method
64. (3) Cytoplasmic staining
(4) Dehydration
- Rinse the smears in absolute alcohol for two or three changes for the
removal of water.
- Alternative to 100% ethanol are
- 100% isopropanol and 100% denatured alcohol.
(5) Clearing
- alcohol is being replaced with Xylene
- Xylene has a refractive index as that of glass and mounting medium
- It prevents cellular distortion.
(6) Mounting
- DPX
66. Factors affecting
Pap staining
Type o
f fixativ
es
Regressiv
e or progr
essive
Length of
staining tim
e
No. Of slid
es in each
dye
Age of dy
es
Moisture a
nd humidit
y
Presence or abse
nce of inflammato
ry cell changes
Quality of ce
ll sample
67.
68. ULTRAFAST PAPANICOLAOU STAIN
fast as the Diff-Quik stain
90 seconds
smeared on a slide
allowed to air dry
placed in normal saline
fixed in a mixture of
4 %formaldehyde and 65% ethanol
stained with Richard Allan Hematoxylin 2 and
Cytostain
71. IMMUNOCYTOCHEMISTRY
Detection of surface antigens (markers) on isolated cells
The detection is based on specific antigen-antibody binding
(immunoreactions).
A specific antibody that was produced by single B cell clone
identifies an epitope with 8-15 length aminoacid sequence in a
protein.
73. IMMUNOCYTOCHEMISTRY FIXATION
Prolonged fixation (wks/months) in formalin may result in
antigenic loss
Prolonged fixation in alcohol-based fixatives is not a major
problem
•95% isopropyl alcohol
•Buffered formalin
• Formol-acetone
• Mixture of ethanol & formalin
74. MAIN METHODICAL STEPS OF
IMMUNOCYTOCHEMISTRY
Cell fixation
Antigen unmasking
Blocking
Selection of appropriate detection signals
Parallel detection of more antigens
Background staining
Controls
75. FREQUENT ENZYME-SUBSTRATE SYSTEMS
(1) Peroxidase (HRPO):
Diaminobenzidin (DAB) – brown
Aminoethyl-carbasol (AEC)- red
True Blue – blue
(2) Alkaline phosphatase (ALP):
Nitroblue tetrasolium (NBT) – blue
Bromo-chloro-indoyl phosphate (BCIP) - blue
76. COMMONLY USED MARKERS IN EFFUSIONS
Breast cytology
ER
Mammaglobin
GATA 3
E cadherin
P120 catenin
78. WHAT IS FLOWCYTOMETRY
Flow means motion
Cyto means cell
Metry means measure
Definition :
- An analytical technique in which cell suspension
obtained from any unfixed tissue /body fluid, peripheral
blood or bone marrow are stained with fluorescently
labeled antibody and then subjected to analysis by a
instrument called as flow cytometer.
80. BEFORE IT IS SUBJECTED TO FLOW CYTOMETER,
sample is incubated with the antibody and is washed
fixed with 1 % formaldehyde
This stabilizes the antigen antibody interaction by
creating cross linkages.
84. FLOW CYTOMETRY APPLICATIONS
Immunophenotyping.
Diagnosis and prognostication of
immunodeficiency.
To diagnose cause of allograft rejection.
Diagnosis of auto antibodies in ITP .
To measure nucleic acid content.
DNA ploidy study in cancer.
85. MOLECULAR TECHNIQUES IN CYTOPATHOLOGY
Flourescence in situ hybridization (FISH)
Polymerase chain reaction (PCR)
Microsatellite analysis
Laser microdissection
Mutation analysis
DNA methylation analysis
86. CONCLUDING REMARKS
A specimen must be carefully prepared, well fixed, and
stained in its journey to the microscope.
Less time is required to prepare staining solution, since
ready-made products that produce reliable and consistent
staining are available for purchase.
Liquid-based and automated systems are entrenched in
sophisticated screening programs.
Devices such as imaging flow cytometry and 3-dimensional
cell scanning promise further advances in analysis
capability.
88. REFERENCES
1. Naylor B, Ramzy I. Cytopathology:the past, the present and the
glimpse into the new millenium. In. Gray W, Mckee GT. Diagnostic
cytopathology. 2nd edition. Churchill Livingston.2002. p. 3-13.
2. Bales CE. Laboratory techniques. In. Koss LG. Koss’s diagnostic
cytology and its histopathologic bases. 5th edition. Lippincott
Williams and Wilkins. 2005. p. 1570- 1634.
3. Weidmann JE, Keebler CM, Fasik MS. Cytopreparatory techniques.
In. Bibbo M, Wilbur DC. Comprehensive cytopatholgy. 3rd edition.
Saunders. 2008. p. 835-58.
89. 4. Cibas ES. Cervical and vaginal cytology. In. Ducatman BS.
Cytology: diagnostic principles and clinical correlates.4th edition.
Saunders. 2014. 1-57.
5 . Orell SR, Veilh p. The techniques of FNA cytology. In. Orell SR,
Sterret GF. Fine needle aspiration cytology. 5th edition. Churchill
Livingstone. 2011. p. 8-27.