different methods of preparation of cytological samples for diagnosis .

1. CYTOTECHNIQUES
PRESENTER : DR.
TOUSIF
MODERATOR: DR.
HARISH S PERMI

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

6. CYTOLOGY SPECIMENS
1. peritoneal, pericardial and pleural fluids
2. CSF
3. Nipple discharge
4. Bronchial brushings / washings
5. Sputum
6. Gastric washings
7. Urine sediment
8. Prostatic secretions
9. Cervicovaginal (paps) smear

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

9. BRONCHIAL WASH

10. BRUSHING

11. SCRAPING

12. FINE NEEDLE ASPIRATION CYTOLOGY (FNAC)

13. BODY FLUIDS
Pleural fluid Pericardial fluid

14. BODY FLUIDS
CSF ascitic fluid

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

17. PREFIXATION OF MATERIAL
 Ethyl alcohol 50 %
 Saccomanno’ s fixative
 Shandon mucolexx
 Cytolyt

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 .

20. PRINCIPLE
Hydraulic force
Centrifugal force
•to concentrate cells within a defined area
•a filter card between the chamber sample and the glass
slide resulting in cell to slide adhesion

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.

26. TOUCH IMPRESSION
Impression cytology being
collected From a patient , using a
sterile glass slide with polished
edges.

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

32. FIXATION OF CYTOLOGY SPECIMENS

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

44. LIQUID BASED CYTOLOGY

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

46. LIQUID-BASED CYTOLOGY TECHNIQUES

47. COLLECTION OF SAMPLES
Ayre spatula
Saline moistened cotton tip
applicator

48. Thin prep Path sure

49. THINPREP PROCESSOR
Cell dispersion Cell Collection Cell Transfer

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.

53. PATH SURE
1
4
3
2

54. Conventional smear Thin prep slide

55. Conventional smear Thin prep slide

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

59. STAINING METHODS IN CYTOLOGY
 Papanicolaou Staining Method
 May-Grunwald-Giemsa (MGG) Staining method

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

65. fixation
hydration
dehydration
clearing

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

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

69. MAY-GRUNWALD-GIEMSA (MGG) STAINING METHOD
 MGG stain is performed in air –dried aspirates or
fluids.

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.

72. ICC IN DIAGNOSTIC CYTOLOGY APPLICATIONS
 Tumor Diagnosis/Classification
 Prognostic/Predictor Markers
 Target Therapy

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

77. FLOW CYTOMETRY

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.

79. BASIC COMPONENTS.
 Fluidics system.
 Optic system.
 Electronic system.
 Associated computer system.

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.

81. FLOROCHROMES
 Florescin isothiocyanate (FITC).
 Phycoerythrin.
 Texas red.
 Allophycocyanin.
 Peridinin chlorophyll.
 Tandem florochromes.

82. Fluidics Filters
Forward
scatter and
side scatter

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.

87. thank you

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.