Cytology

Instructor’s Name (Nathaniel B. Ranon, RMT)

OUTLINE o Direct smears of the material on slides

I. Cytology VII. Color of Stain  Most common and easiest
A. Cytological Stains A. Precautions o Cytospin preparation
B. Sample Collection B. Hematoxylin o Membrane filter preparation
II. Slide Preparation and C. OG and EA Stains o Liquid based preparation
Fixing D. Bluing Solution o Cell blocks
III. Sample Transportation and HCl
IV. Automated Tissue E. Water rinses
SLIDE PREPARATION AND FIXING
Processor F. Alcohol
A. Pap Staining G. Xylene  All preparations are made on clean glass slides
B. Reagents VIII. Maintenance of Stains  Fixative used: ethanol/methanol/spray fixed or air dried
V. Regressive Methods and Solutions depending on the stain to be used
VI. Progressive Methods
 Ethanol Fixation
CYTOLOGY  Wet fixation
 Definition  Immediate immersion in 95% Ethanol placed in couplin jars
 Diagnostic technique used to examine cells from body fluids
and solid tissues to determine the nature of disease.  Air dried smears
 Includes:  Required for stains like MGG
Types of Sampling  Later wet fixed, in Methyl-alcohol for 15-20 minutes during
 Exfoliative staining process
 Abrasive
 Aspirational  Spray fixation
 Exfoliative Cytology  Done using cytosprays
Spontaneously shed cells in body fluids  Employed in many labs as an easier alternative to wet fixation
 Examples:  Smears are air-dried before fixation
 Urine  Cellular characteristics are better preserved if the spray is used
 Sputum as the edges of the smear begins to dry
 CSF  Distance between the nozzle and the smear – 15 to 25 cm
 Effusions (e.g. pleural fluid, pericardial fluid, peritoneal  Fixative bottle held too close – dislodgement of cells from
fluid, etc) the smear
 Abrasive Cytology  Fixative bottle held too far – insufficient amount of fixative
Dislodged cells from body surfaces and mucosal linings  Forceful spraying – creates cellular artifacts by fracturing of
 Examples the cytoplasm
 Bronchiolar wash  Ideal angle - 45°
 Swabs SAMPLE TRANSPORTATION
 Aspirational Cytology  Slide Mailers
Aspirates cells from palpable lump or mass in the body using a  Used for transporting fixed slides
needle with or without suction
AKA: FNAC (Fine Needle Aspiration Cytology)
 Either done directly – palpable lumps
 Under ultrasound/ CT guidance – non-palpable lumps
CYTOPATHOLOGICAL STAINS
 Papanicolaou Stain (PAP)
 Important stain in cytopathology
(Adapted from LabsforLifeProject – Cytology)
 Cervical Cytology
 Used:
 Cervical smears AUTOMATED TISSUE PROCESSOR
 Exfoliative cytology smears  Saves time
 Romanaowski Stain  Produces consistent results
 Used  Overcomes
 FNACs  Laborious steps in processing
 Fluids  Human errors and forgetfulness
 H & E stains
 Used  Display
 Fluids  Processes for start up and shut down of the processor
 FNACs  Details of solution charge
 Used as a Histopathology stain
SAMPLE COLLECTION  Cassettes
 FNAC  Must be cleaned
o Sample is collected by the Pathologists  Place in used saline for one hour
o Activity depends on the site being aspirated
 Pap smears PAP Staining
o Collected by Gynecologists  Polychromatic stain containing multiple dyes to differentially
o Ayer’s spatula stain various components of cells.
 Developed by George Papanicolaou
 Father of Cytopathology
 Principle
 Acid dye stains BASIC components of cells
(Adapted from LabsforLifeProject – Cytology)  Basic dye stains ACID components of cells
 Main Objective of PAP Staining
o Endo-cervical brush  Defining of nuclear details
 Widespread nuclear abnormalities of cancer cells
and their diagnostic significance, good staining of
the nucleus is of primary importance.
 Hematoxylin – stains NUCLEI
 Transparency of cytoplasm
 Important because of varying thickness and
frequent overlapping of cells
 Orange G Stain – Cytoplasmic stain
 Cell Differentiation
(Adapted from LabsforLifeProject – Cytology)  Differences in the staining reaction helps in the
o Sampling of transformation zone is vital to the reporting identification of cell types found in smears.
 Polychromatic solution
 Fluids  Mixture
o Collected by clinicians  Eosin Y
 Preparations from the cellular material can be in the form of  Light green SF
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 Module: Cytology

 Bismarck Brown Y
 Demonstration of differentiation of
squamous cells

REAGENTS
 Harris Hematoxylin
 Nuclear stain – stains cell nuclei blue
 Has chromatin affinity, attaching to sulphate 
groups on the DNA molecule (Adapted from LabsforLifeProject – Cytology)
 Most common – Harris’ hematoxylin
 Others – Gills’ hematoxylin and Hematoxylin S
 Orange Green 6  Resolution:
 1st acidic counterstain (cytoplastic stain)  Take it out from xylene and allow it to dry partially
 Stains matured and keratinized cells  Mount in DPX
 Target structures are stained orange in different  Mounting of Slide
intensities o Mounting media must be MISCIBLE with the CLEARING
 Eosin Azure (EA – 50) AGENT to prevent fading of the stains
 Second counterstain o Practice is essential to achieve well-mounted slides, free of
 Polychrome mixture air bubbles and artifacts
 Eosin Y + Light green SF + Bismarck brown o Minimum of mounting medium should be used
 Eosin Y  Too much mounting medium interferes with
 Gives pink color to microscopic detail, making the cell film appear hazy or
 Cytoplasm of mature squamous cells milky when examined under the HPO
 Nucleoli o Usual size of coverslip
 Cilia  22 x 30 mm
 Red blood cells  If beyond, use another coverslip or add another DPX
 Light green SF
 Stains blue to cytoplasm of cells like COLOR OF STAIN
 Parabasal squamous cells
 Intermediate squamous cells Nuclei Blue
 Columnar cells Acidophilic cells Red
 Bismarck brown Y Basophilic cells Blue green
 Stabilizes the staining Erythrocytes Orange red
Keratin Orange red
Types of Reagents Superficial cells Pink
A. Progressive Intermediate and parabasal Blue green
B. Regressive cells
C. Rapid Eosinophil Orange red
Candida Red
Regressive Methods Trichomonas Grey green
 Nucleus is stained with hematoxylin to an intensity desired
 Intensity of the nuclear staining is controlled by the immersion of Precautions
the slide into a blueing agent 1. Immediate fixation of smears is essential
 Most common blueing agent 2. Solutions and other stains are filtered daily after use to
 Scott’s tap water (pH 8.02) keep them free of sediment
 Nucleus is deliberately over-stained with a non-acidified 3. Alcohols are hygroscopic
hematoxylin 4. If alcohols are left open, the concentration % DECREASES
 Excess stain is removed with dilute HCl solution and appropriate dehydration cannot be achieved
 Decolorizing process is stopped by immersing the slide in a. Keep them covered
running tap water 5. Avoid contamination from one smear to another
 Timing is crucial in the regressive method as destaining may 6. Stains are discarded and replaced as the quality of the
lead to a hyperchromatic nucleus becoming hypochromatic stain deteriorates
7. Place the coverslip on the microslide slowly without
trapping air bubbles
Progressive Methods 8. Stains keep longer if they are stroed in dark colored,
 95% Alcohol Fixation – 15 to 30 minutes stoppered bottles
 Hydration
o 80% Alcohol – 2 minutes
o 60% Alcohol – 2 minutes  POINTS TO REMEMBER
 All stains and alcohol changes will depend on load
o First coplin jar of distilled water – 5 dips
 Any solution which is hazy and turbid should be
o Second coplin jar of distilled water – 6 dips
discarded
 Hematoxylin stain – 3 minutes  All stain changes should be protocoled and logged
 1st nuclear stain
 Wash gently in running water – 30 sec
 Blueing (Scott’s tap water) – 3 minutes Hematoxylin
 Dehydration  Keeps relatively constant staining characteristics.
o Distilled water – 3 minutes  Do not require frequent discarding if small amounts of fresh
o 60% Alcohol - 2 minutes stain are added to replace stain loss due to evaporation
o 80% Alcohol - 2 minutes
o First coplin jar of 95% alcohol – 2 minutes OG and EA Stains
 Orange G stain – 3 minutes  Lose strength more rapidly than hematoxylin
 1st counterstain  Should be replaced each week or as soon as the cells appear
 First coplin jar of 95% alcohol – 2 minutes without crisp staining colors
 Second coplin jar of 95% alcohol – 2 minutes Bluing solution and HCl
 Eosin Azure stain – 3 minutes  Replaced once daily
 2nd counterstain
 First coplin jar of 95% alcohol – 2 minutes
Water rinses
 Second coplin jar of 95% alcohol – 2 minutes  Changed after each use
 First coplin jar of Absolute Alcohol – 2 minutes Alcohol
 Second coplin jar of Absolute Alcohol – 2 minutes  Used for the process of dehydration prior to the cytoplasmic
 Clearing stains is replaced weekly
o First coplin jar of Xylene – 2 minutes  Following the cytoplasmic stains are changed on a rotating basis
o Second coplin jar of Xylene – 2 minutes after each use
o Note:  Immediately following the stain is discarded and the other 2
 If xylene dries completely – CORNFLAKE rinses are moved to the 1st and 2nd position and the fresh unused
ARTIFICATES alcohol is replaced in the 2nd last position
 Ideally this rotation must continue after each staining run
 Absolute alcohols should be changed weekly and is kept water
free by adding silica gel pelletXylene

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 Module: Cytology

 Changed as soon as it becomes tinted with any of the

cytoplasmic stains
 Becomes slightly milky if water is present and clearing process
is disturbed
 Tiny drops of water may be seen microscopically on a plane
above the cell on a slid
 POINTS TO REMEMBER
 Stains may have batch to batch variation in dye
content, requiring change in timing during staining
 Quality of the stained slide is dependent on timing,
solubility and percentage of dye concentration

MAINTENANCE OF STAINS AND SOLUTIONS

 All reagents, stains, and solutions should be stored as
recommended by manufacturer and used within their indicated
expiry dates
 Label should contain information
o Content and quality
o Concentration or titer
o Date received/prepared
o Date of opening
o Storage requirements
o Expiry dates
 Use adequate controls for reagents, stains to check their
performance where a built-in control does not exist
o Ex: buccal mucosal smear for Pap stain
 Reagents and stains should be procured from standard reputed
sources
 Each lot of reagents shall be checked against earlier tested in-
used reagent lots or with suitable reference material before
being placed in service and the results should be recorded

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 Cytology
Instructor’s Name (Nathaniel B. Ranon, RMT)

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