BASICS OF CYTOLOGY

BMS2125
Objectives:

• Introduction to cytology and difference with tissue biopsy

• The diagnostic role of cytology

• Branches of cytopathology

• Sample collection, fixation and staining techniques

• Diagnostic pitfalls

• Please note: this lecture is mainly adapted from and is a summary of an article by Al-Abbadi M, 2011 for further reading please
refer to Avicenna J Med. 2011 Jul-Sep; 1(1): 18–28.doi: 10.4103/2231-0770.83719: 10.4103/2231-0770.83719
• https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3507055/?report=printable
• Parts of this lecture are also adapted from http://screening.iarc.fr/doc/Cancer_resource_Manual_3_Cytology_New.pdf
• The science of cytology and cytopathology has been
History of Cytopathology
implemented and recognized as early as the 18th and
19th centuries

• However the progress and the standardization of this

branch of pathology were not founded completely until
the late years of the 20th century

• George Papanicolaou, was one of the initial pioneers

who drove the attention to the science of the ability to
make a diagnosis looking at slides with a smear of cells
in the period between 1917 and 1928

• The initial North American scientific papers describing

tumour diagnosis by cytological examination was https://institutions.newscientist.com/article/2202635-georgios-papanikolaou-inventor-
of-the-pap-smear-cervical-cancer-test/
published in 1930 from New York Memorial Hospital by
Drs. Martin and Ellis followed by a publication by Dr.
Stewart in 1933
 TISSUE BIOPSY VERSUS CYTOLOGICAL MATERIAL

• Tissue biopsy offers bigger sample size-

less errors

• Allows for orientation (assess stage of

tumour samples)

• But requires costly and invasive procedure

Uses of Cytopathology:

• To reach a definitive diagnosis that drives treatment decisions

• As a screening tool

• Follow-up of different diseases

• For determination of different prognostic factors

ADVANTAGES OF USING CYTOLOGY

• Safe

• Simple

• Quick

• Cost effective
Branches of Cytopathology

• The science of cytopathology is currently well standardized with two major

branches:

• Exfoliative Cytology

• Aspiration Cytology
Exfoliative cytology:
• The samples represent cells that exfoliate from superficial or deep serosal
or mucosal surfaces
Examples:
• Gynecological samples e.g. cervical smear
• Respiratory/exfoliative cytology:
• which includes bronchial washing, sputum, bronchoalveolar lavage,
and bronchial brushing cytology

• Urinary cytology: Touch preparation cytology technique.

• Urine cytology, bladder washing, and brushing cytology
https://www.semanticscholar.org/paper/Intraoperative-Touch-
Preparation-Cytology-for-in-It-Valdes-
• Body fluid cytology: Boolbol/83428a218a26d36527c13925efa57bd971381366

• Common samples include pleural fluid, pericardial fluid, peritoneal

fluid, and cerebrospinal fluid (CSF) cytology

• Touch preparation:
• Done intra-operatively
• To assess surgical margins
• Gastrointestinal Tract:
• Sampling the mucosa of the gastrointestinal tract is becoming a routine procedure
during endoscopy.
• Brushing samples are used to detect viral and fungal infections, and neoplasia with
its precursor lesions
• Discharge cytology:
• The most common sample is breast nipple discharge that is used as a screening
method for detection of mammary carcinoma

• Scrape cytology:
• Detection of infections and cancer at any surface
• The cut surface of the tissue sample is scraped using a scalpel or the edge of a glass
slide

Procedure of scrape cytology includes scraping from

tumor mass, smearing and fixation in 95% ethyl alcohol
Aspiration Cytology:

• Different names are used including:

• FNA, fine needle aspiration biopsy (FNAB), and needle aspiration biopsy cytology (NABC)
• All of them mean the same thing- aspirating cellular material using a fine needle to make a
diagnosis

• Can be used for:

• Palpable lesions
• Non-palpable lesions
• The non-palpable lesions are usually done with the help of image analysis (CT scan-guided,
ultrasound-guided, fluoroscopy-guided, and recently endoscopic ultrasound-guided fine
needle aspiration)
Fine needle aspiration technique

• There is still no agreed upon standard for the best aspiration technique in cytopathology

• However, all FNA experts agree on one thing, every aspirator have to get comfortable with one
method and modify it as more experience is gained

• The bottom line is to get enough diagnostic cells from the area of interest
FNA techniques:

• Aspiration using a fine needle (gauge range 21-25) without negative pressure or a syringe.
 This technique is also known as “the French technique” and clinicians, radiologists and pathologists
who use this method believe that it is less traumatic than the others and yield enough diagnostic
cells by the mere capillary pressure

• Aspiration using a syringe and needle without negative pressure.

 This method allows the aforementioned capillary pressure to push cells in the hub of the needle
avoiding the trauma of negative pressure.
 It is believed that adding the syringe will allow collection of fluids if the lesion turned to be cystic.

• Aspiration using a syringe and needle with utilization of negative pressure

 The amount of negative pressure varies
 2-3 cm of negative pressure in a 10 ml syringe is commonly used
 https://www.slideshare.net/DRKALPAJYOTI/fine-needle-aspiration-cytology-fnac

Aspiration Biopsy Syringe Gun

https://www.researchgate.net/figure/Procedure-for-fine-needle-aspiration-FNA-guided-by-ultrasound-US-A- http://dxline.info/diseases/fine-needle-aspiration
Needle-inserted_fig2_282394876
Smear Preparation:
Different types of smear preparations are utilized in the cytopathology laboratory, which includes:
• Direct smears:
 The initial smears are usually stained by a quick stain
 Smears are stained following air-drying

• Cytocentrifuge smears:
 which are prepared using the cytocentrifuge method
 This method concentrates the material and is especially advantageous when few cells are
present in a large amount of fluid, such as pleural or peritoneal fluids

• Monolayer liquid-based cytology:

• This technology is now the standard method that is used to prepare Papanicolaou smears

• The Cellblock technique:

• For any remaining material
• Those slides are prepared utilizing formalin-fixed paraffin-embedded technique and are stained
with H&E
Preservation of Smears:
Air-drying:
• followed by hematological stains like May – Grunwald –Giemsa (MGG), Diff Quik, Giemsa
etc.

• In this method, smears are intentionally air dried, but if smears are not correctly made and
dried quickly artifacts will result.

• One advantage is the speed with which smears can be stained especially with use of rapid
stains like Diff Quik (2-3 minutes).

• Rapid stains are particularly useful in preliminary assessment of adequacy of the sample
before the patient is released.

• Colloid, mucin, endocrine cytoplasmic granules etc are better brought out in air-dried
preparations

• Nuclear details are not as clear, cells appear larger than normal

• Unsuitable for Pap staining. Slides could be rehydrated if Pap staining is required
 Fixation of Cytology Specimens

Properties of Cytologic Fixatives:

 Do not excessively shrink or swell cells

 Do not distort or dissolve cellular components

 Inactivate enzymes and preserve nuclear details

 Kill microbes

 Improve optical differentiation and enhance staining properties of the tissues and cell
components
Wet fixation:
 followed by Papanicolaou (pap) or hematoxylin and eosin (H&E) staining

 Rapid fixation in alcohol (wet fixation) is essential for pap staining, which brings out nuclear details
clearly, allowing better identification of malignant cells

 If the smears are not quickly made and fixed, drying artifact can occur in which case, the cytoplasm takes
up more eosin (red color) and nuclear details are less clear

 Hence with pap staining, air-drying is avoided as much as possible by dropping the slides into the fixative
immediately after the smears are made

Time of Fixation:
 Minimum 15 minutes fixation prior to staining is essential.
 Prolonged fixation for several days or even few weeks will not affect the morphology of cells.
 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.
Wet Fixation reagents:
A- Routine fixatives:
• 95% Ethyl Alcohol (Ethanol):
• The ideal fixative
• Ether alcohol mixture:
• consists of equal parts of ether and 95% ethyl alcohol.
• It is an excellent fixative, but ether is not used in most of the laboratories because of its safety
hazards
• 100% Methanol:
• 100% methanol is an acceptable substitute for 95% ethanol.
• Methanol produces less shrinkage than ethanol, but it is more expensive than ethanol
• 80% Propanol and Isopropanol:
• Propanol and Isopropanol cause slightly more cell shrinkage than ether-ethanol or methanol.
• By using lower percentage of these alcohols the shrinkage is balanced by the swelling effect of
water on cells.
• Denatured alcohol:
• It is ethanol that has been changed by the addition of additives in order to render it unsuitable for
human consumption.
• There are many different formulae for denatured alcohol; all of them contain ethanol as the main
ingredient, and hence this can be used at a concentration of 95% or 100%.
• One formula is 90 parts of 95% ethanol + 5 parts of 100% methanol + 5 parts of 100%
isopropanol
B- Coating fixatives:

• Coating fixatives are substitutes for wet fixatives

• They are either aerosols applied by spraying the cellular samples or a liquid base, which is dropped
onto the slide

• They are composed of an alcohol base, which fixes the cells and wax like substance, which forms a
thin protective coating over the cells e.g. Carbowax (Polyethylene Glycol) fixative

• Diaphine fixative Spray coating fixative (Hairspray) with high alcohol content and a minimum of
lanolin or oil is also an effective fixative

• Most of these agents have a dual action in that they fix the cells and, when dry, form a thin
protective coating over the smear

• These fixatives have practical value in situations where smears have to be mailed to a distant
cytology laboratory for evaluation

• The distance from which the slides are sprayed with an aerosol fixative affects the cytology details,
10 to12 inches (25-30 cm) is the optimum distance recommended for aerosol fixative

• Prior to staining, the slides have to be kept overnight in 95% alcohol for removal of the coating
fixative.
C- Special Purpose Fixatives:

• Carnoy’s fixative:
• This is a special purpose fixative for haemorrhagic samples
• The acetic acid in the fixative haemolyses the red blood cells
• It is an excellent nuclear fixative as well as preservative for glycogen but results in
considerable shrinkage of cells and tends to produce over staining in hematoxylin.
• Overfixing in Carnoy’s also results in loss of chromatin material
• Carnoy’s fixative must be prepared fresh when needed and discarded after each use

• AAF Fixative :
• This is the ideal fixative used for cellblock preparation of fluid specimens
• (95% Ethanol 34 ml + formalin 4 ml + Glacial acetic acid 2 ml)
Collection and preservation Pleural, pericardial and peritoneal fluids:

• Can be collected in tubes or syringes that may be either plain or pre- heparinised, to prevent coagulation

• Cells in heparinised fluids do not deteriorate rapidly

• Freshly tapped specimens are preferred for cytology, if facilities for immediate processing are available

• If immediate processing is not possible, it can be preserved in the refrigerator for a period of 24-48 hours

• Preservation of cells by pre-fixation in 50% ethanol is also possible

• Pre-fixation and spray fixatives are recommended when sample has to be sent to a distant laboratory

• 20–30 ml fluid is generally sufficient to get enough cells for cytological evaluation
 Staining of Cytology Specimens
Papanicolaou Staining Method:

• Papanicolaou staining method is the routine

staining procedure used in cytopathology
laboratory

• It is a polychrome staining reaction designed

to display the many variations of cellular
morphology showing degree of cellular
maturity and metabolic activity

• The use of the Papanicolaou stain results in

well stained nuclear chromatin, differential
cytoplasmic counterstaining and cytoplasmic https://en.wikipedia.org/wiki/Papanicolaou_stain

transparency
Nuclei should be crisp, blue to black and the chromatin patterns of the
nucleus should be well defined. Cell cytoplasm stains blue-green
and keratin stains orange in colour
Steps of Papanicolaou Staining Method:
• Fixation:
• in 95% ethyl alcohol or in other substitutes for a minimum of 15 minutes
• Nuclear staining
• It is done by using haematoxylin stain
• Harris haematoxylin or its modified form is used in Papanicolaou staining in regressive method
• This should be followed by differentiation and bluing
• Cytoplasmic staining
• Cytoplasmic stains are OG-6 and EA-36 (Eosin Azure)
• Both are synthetic stains and OG-6 is a monochrome stain while EA-36 is a polychrome stain
• Dehydration
• The smears are rinsed in absolute alcohol for two or three changes for the removal of water
• Clearing
• Cells are not transparent while the smear is in the staining or alcohol solutions
• During clearing, alcohol is being replaced with Xylene, which is also miscible in mounting medium
• Xylene has a refractive index as that of glass and mounting medium and it prevents cellular
distortion
• Mounting of slide
• The mounting media must be miscible with the clearing agent
• Practice is essential to achieve well-mounted slides, free of air bubbles and artifacts
• A minimum of mounting medium should be used
• Haematoxylin and Eosin (H&E) staining method:

• Some laboratories use routine H&E stain for non-

gynecological smears

• H&E stain does not allow clear definition of

nuclear details and differential counterstaining
with cytoplasmic transparency hence
unacceptable for cervical smears

• May-Grunwald-Giemsa (MGG) Staining method:

• Many laboratories use MGG staining method for

cytological diagnosis of non –gynaecological
specimens Peritoneal fluid Cytology, May-Grünwald-Giemsa stain.

• MGG stain is performed in air –dried aspirates or https://www.researchgate.net/figure/Peritoneal-fluid-Cytology-May-Gruenwald-Giemsa-stain-A-An-almost-

pure-population-of_fig2_313495595

fluids
Other cytology stains
Diff-Quik:
• is a commercial Romanowsky stain variant
used to rapidly stain and differentiate a
variety of pathology specimens

• It is most frequently used for blood films and

cytopathological smears, including fine
needle aspirates

• Microbiologic agents, such as bacteria and

fungi appear more easily in Diff-Quik

• The stain can be used to detect Helicobacter

pylori and it may also be used in the
evaluation of sperm morphology

• The Diff-Quick stain consists of a fixative https://en.wikipedia.org/wiki/Diff-Quik

agent (methanol, blue), solution I
(eosinophilic, orange) and solution II
(basophilic, blue). This allows for fixation
and staining.
The Cytopathology Report:
• An informative report should include the following:

• Adequacy
• a statement describing if the material was adequate to make an interpretation

• Diagnosis
• A specific diagnosis is always desired when possible. Sometimes the diagnosis is broad, such as
“positive for malignant cells”, “suspicious for malignancy”

• Descriptive diagnosis (microscopic description)

• Sometimes descriptive diagnosis and microscopic description of the smears

• Comment
• is needed to clarify or add some information of clinical importance

• Recommendations
• call the clinicians and discuss the case either face to face or over the telephone
Cytological features of malignancy:
• There is no single feature that is diagnostic of
malignancy

• It is the group of multiple factors that vary depending

on the tissue aspirated, the collection technique and the
smear preparation method

• The general features of malignancy in cytological

slides are:
• high cellularity
• cellular enlargement
• increased nuclear/cytoplasmic ratio
• nuclear hyperchromasia
• discohesiveness of cells
• prominent and large nucleoli
• abnormal distribution of nuclear chromatin
• increased mitotic activity and specially the
presence of abnormal ones
• nuclear membrane abnormalities . Urothelial cells with dark (jet black) chromatin. Compared with tubular cells that also often have jet black
chromatin, the nuclei of high-grade urothelial carcinoma cells are larger and more irregular in outline, often have
• cellular and nuclear pleomorphism nuclear “points,” and often have a sickle or half-moon shape (A and B: single image; C: composite image;

• background tumour necrosis ( tumour diathesis). Papanicolaou stain, 3 1000). RBC indicates red blood cells.

https://acsjournals.onlinelibrary.wiley.com/doi/pdf/10.1002/cncy.21947
DIAGNOSTIC PITFALLS

• Poor collection technique:

• This can occur when the appropriate slides or containers with appropriate fixatives are not used

• Poor fixation:
• This is sometimes seen when there is no experience with cytopathology material preparation and
collection

• Inflammatory changes:
• extensive inflammation may obscure cellular details and prevent appropriate interpretation. To avoid this
problem, treating the patient and repeating the procedure afterwards is recommended

• Cellular changes related to radiation and/or chemotherapy:

• Certain changes are induced by these treatment modalities. To decrease the pitfalls from these changes,
appropriate and detailed history should be given by clinicians and awareness of the changes by the
pathologist should be taken into consideration

• Atypical cellular changes related to hemorrhage, infarction, or necrosis:

• Awareness of these changes by the cytopathologist is very helpful to prevent both false positive and false
negative diagnosis
False negative results:
• Desmoplasia:
• This is defined as the presence of fibrosis
• Many tumors can cause fibrosis around the malignant cells. The most common are mammary,
pancreatic and billiary tree carcinomas in addition to nodular sclerosing Hodgkin's lymphoma.
• Applying negative pressure and multiple passes during the FNA procedure can help

• Well-differentiated tumor cells:

• Certain tumors are extremely well-differentiated and they resemble their original cells
• For example, well differentiated thyroid follicular carcinoma and well differentiated
hepatocellular carcinoma can be deceiving

• Sampling problems:
• Sometimes the needle is not in the appropriate lesion of interest

• The presence of inflammation, radiation, and chemotherapy:

• changes sometime can be misinterpreted
False Positive Results:

• Pregnancy
• Pregnancy sometimes can increase cell size in Papanicolaou smears.

• Contamination
• Contamination can occur either through the needle tract or during processing.

• Inflammation and inflammatory changes, radiation and chemotherapy

• effects sometimes will lead to false positive diagnosis

• The presence of hemorrhage and infarction

• sometimes induce atypical changes in the cells

• Inexperience by the pathologist

• may induce false positive diagnosis