This document provides an overview of cytological sample preparation and staining techniques. It discusses various cytology specimens including body fluids, exfoliative cytology samples, and fine needle aspiration biopsies. Methods for collecting and preparing samples such as centrifugation and smearing are described. Common staining techniques including Papanicolaou staining, Diff-Quik staining, and May-Grunwald-Giemsa staining are explained. Additional diagnostic tools in cytopathology like immunocytochemistry, flow cytometry, and molecular techniques are also summarized.

1. CYTOLOGICAL SAMPLE
PREPARATION AND STAINING
TECHNIQUES
Yona Mbalibulha

2. INTRODUCTION
 The best morphologic presentation obtained from any
cytologic specimen requires an
Process that it went through during collecting and
preparation. specimen.
understanding of the
Principle of cytotechniques
 To reduce the specimen to a cellular presentation,
can be interpreted and diagnosed.
which

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

4. EXFOLIATIVE CYTOLOGY
 It is the study of cells that have been shed or
removed
organs.
from the epithelial surface of various
smear
wash
scraping brushing

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

6. BRONCHIAL WASH

7. BRUSHING

8. SCRAPING

9. FINE NEEDLE ASPIRATION CYTOLOGY (FNAC)

10. BODY FLUIDS
Pleural fluid Pericardial fluid

11. BODY FLUIDS
CSF ascitic fluid

12. CENTRIFUGATION
 If too much fluid is obtained,.
Centrifuged for 5 mins Sediment
 If little amount of fluid is aspirated (few drops), or if the fluid is
thick, the centrifuge is not required.

13. CYTOCENTRIFUGATION
 It is a special machine that performs a centrifuge and
collection of sediment on the center of the slides.
procedure is useful for hypocellular specimen .
This

14. METHODS OF SMEAR
 streaking
 spreading
 pull apart
PREPARATION:
 touch or impression smear

15. 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

16. SPREADING
- used for thick mucoid secretions
- smears of fresh sputum and bronchial aspirates

17. PULL APART
- for serous fluids, concentrated sputum, and enzymatic
lavage form the GIT
, smears of urinary sediment, vaginal
pool and breast secretions.

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

19. SQUASH SMEAR PREPARATION
 fairly accurate,
 simple and reliable tool for rapid intra-operative
central nervous system lesions.
 Based on two essential factors:
diagnosis of
Availability of very small tissue fragments & good
preservation of fine cellular details.
Not effected by edema, hemorrhage, necrosis &
calcification.
•
•

20. CELL BLOCK
 It is a procedure to convert cell sediment into paraffin
block
 further pathological procedures can be performed like
immunohistochemistry (IHC).

21. Methods of Cell Block Preparation
 Direct processing of tissue fragments present in fluids
 Fixed sediment method
 Bacterial agar method
 Simplified cell block technique
and paraffin
using alcohol, acetone
 Compact cell block technique
 Plasma thrombin method

22. Methods of Cell Block Preparation cont’
 Cell blocks from millipore
 Histogel method
 Gelatin embedding
 Celloidin bag
 Cell block preparation from scraping of cytology smears
 Automated cell block preparation
 Albumin method

23. Example Procedure of cell block prepn
Centrifugation 2400 rpm
Pour off supernatant
Add 5 ml of methanol +formalin (9:1)
Spin at 2400 rpm
methanol+formalin for 30 to 60 min
remove hardened cell button
submit in a cassette

24. 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

25. LIQUID-BASED CYTOLOGYTECHNIQUES

26. Path sure
Thin prep

27. (1)CELL DISPERSION
 Swirling the sampling device in
the preservation solution
 Strong enough to separate
debris and disperse mucus

28. (2) CELL COLLECTION
 A gentle vacuum is created within
the ThinPrep Pap T
est Filter, which
collects cells on the exterior surface
of the membrane.

29. (3) CELL TRANSFER
 the ThinPrep Pap T
est Filter is inverted
and gently pressed against the ThinPrep
Microscope Slide.
 Natural attraction and slight positive air
pressure cause the cells to adhere.

30. PATH SURE
3
4
1 2

31. Conventional smear Thin prep slide

32. Thin prep slide
Conventional smear

33. 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

34. 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

35. 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

36. Staining Methods in cytopathology
• Common staining methods.
– Papanicolaou stain (PAP smear)
– Differential quick method (diff. quick methd)
– Acridine orange technique
– P.A.S
– Mehtylen blue
– Perl’s Prusiian Blue
– Feulgen reaction
– Heamatoxylin and eosin
– Florescent dye techniques
Others.......
Note: Read and make personal notes on these and other
specific cytological methods.

37. General concepts in cell examination
• The overall size and shape of cancer cells are
often abnormal.
• The size and shape of the nucleus of a cancer
cell is often abnormal.
• The nucleus of a cancer cell appears darker
when seen under a microscope after being
stained with certain dyes.

38. Concepts cont......
• Cancer cells do not relate to each other
normally.
• Takes on abnormal functions different from
the parent organ. (form glands, secretions,
hormones )
• cancer cells invade other tissues.
• cancer cells can metastasize

39. Modes of examination.
• Ordinary Microscopy
• Electron Microscopy .
• Flow Cytometry
• Image cytometry
• Molecular Genetic Studies
• Cytogenetics
Note: Read and understand the details of these methods.

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

41. 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.

42. 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

43. 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

44. STEPS OF STAINING PROCEDURE
(1) Fixation
- 95% ethyl alcohol or in other
- minimum of 15 minutes
substitutes
(2) Nuclear staining
 Harris haematoxylin - regressive staining method

45. (3) Cytoplasmic staining
(4) Dehydration
- Rinse the smears in absolute alcohol for two or three
removal of water.
- Alternative to 100% ethanol are
changes for the
- 100% isopropanol and 100% denatured
(5) Clearing
- alcohol is being replaced with Xylene
- Xylene has a refractive index as that of
- It prevents cellular distortion.
(6) Mounting
- DPX
alcohol.
glass and mounting medium

46. clearing
dehydration
hydration
fixation

49. Age of dyes
Type of
fixatives
No. Of slid
es in
dye
each
Moisture a
nd
y
humidit
Regressiv
e or progr
essive
Length of
staining tim
e
Presence or abse
Quality of
ll sample
ce
nce of inflammato
ry cell changes
Factors affecting
Pap staining

50. Diff-Quik Stain:
On-site evaluation of fine-needle aspiration samples requires stain
protocols that are quick and easy to perform and allow for assessment
of adequacy of the sample, i.e., cell types and amounts.
Procedure:
•
•
•
•
•
•
•
Place one drop of aspirate on slide
Allow slide to air dry
and smear
Fix in 95% ethanol - 10 dips
12dips
6-8 dips
- 20 dips
1-
2 -
Place in
Place in
Distilled
solution
solution
water rinse
Review under microscope.
•
purple nucleus
Result:
• The cells stained exhibit a deep
blue for the cytoplasm
and different hues of

52. 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
%formaldehyde and 65% ethanol
4
stained with Richard Allan Hematoxylin 2
Cytostain
and

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

54. 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
protein.
a

55. IHC IN DIAGNOSTIC CYTOLOGY
 Tumor Diagnosis/Classification
APPLICATIONS
 Prognostic/Predictor Markers
 Target Therapy

56. FLOWCYTOMETRY
Definition :
An analytical technique

- in which cell suspension
/body fluid, peripheral
obtained from any unfixed tissue
blood or bone marrow are stained with fluorescently
labeled antibody and then subjected to analysis by a
instrument called as flow cytometer.

57. FLOROCHROMES
 Florescin isothiocyanate (FITC).
 Phycoerythrin.
 T
exas red.
 Allophycocyanin.
 Peridinin chlorophyll.
 T
andem florochromes.

59. FLOW CYTOMETRY APPLICATIONS
 Immunophenotyping.
 Diagnosis and prognostication of
immunodeficiency.
 T
o diagnose cause of allograft rejection.
 Diagnosis of auto antibodies in ITP .
 T
o measure nucleic acid content.
 DNA ploidy study in cancer.

60. MOLECULAR TECHNIQUES IN CYTOPATHOLOGY
 Flourescence in situ hybridization (FISH)
 Polymerase chain reaction (PCR)
 Microsatellite analysis
 Laser microdissection
 Mutation analysis
 DNA methylation analysis

61. 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.
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