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Cytology Processing





Body cavity fluids


Fine needle aspiration biopsy

Full patient name

Unique identifier such as medical record number
Date of birth and age
Date and time of specimen collection
Source or site of the specimen
Type of specimen or examination requested
Collection method
The ordering clinicians full name, and the name and address or other
suitable identifier
Methods of rapid communication such as telephone or FAX numbers of the
ordering clinician
Clinical history, including pertinent physical findings, radiographic findings,
history of environmental exposure, possible exposure to infectious agents,
immunosuppression, chemotherapy, radiation therapy, surgical operations,
history of cancer, previous histological or cytological abnormalities

Methods of Cytologic Processing

Cytologic smear

Cell block



kept refrigerated (4C) up to 72 hours


fixed in 50% ethanol equal to the volume of the

specimen, or in the proprietary transport medium
supplied by the manufacturers of liquid based
systems. Any added fixative should be noted on the


Brushings / Slides

Immersion fixation consists of placing the smear

into 95% alcohol or its equivalent.
If the specimen is immersed in alcohol, it may
remain in the alcohol for transport to the laboratory.
Alternatively, the specimen can be immersed in
alcohol for 20-30 minutes, removed and allowed to
air dry, then placed in a container/mailer to be
transported to the laboratory.

Specimen Processing

Laboratory Sample Processing

Reciept of specimen up to delivery of the stain

slides for microscopy
Identity and integrity of the specimen is
Universal precaution is observed at all times

Laboratory Sample Processing

Reciept and Identification of specimens


pertinent physical findings, radiographic

findings, history of environmental exposure,
possible exposure to infectious agents,
immunosuppression, chemotherapy,
radiation therapy, surgical operations, history
of cancer, previous surgical or cytologic

Laboratory Sample Processing

Specimen condition

Glass slides

Liquid specimens

Brushing specimens


Specimen Processing


Fixed in alcohol Papaniculaou stain

Air -dried Romanowsky

Spray fixative (carbowax) soak in 95% alcohol

Pap stain

Specimen Processing

Liquid specimens

Cell block


Cell Block Procedure

5 mL sample was subjected to fixation for one hour by mixing

with 5 mL of 10% alcohol-formalin (i.e., nine parts of 90%
alcohol and one part of 7.5% formalin).
After one hour, this 10 ml fluid was centrifuged at 2500 rpm for
15 minutes.
The supernatant was discarded and a further 3 mL of fresh
10% alcoholformalin was once again added to the sediment
The sediment containing the cell button of the fluid sample was
scooped out on to the filter paper and this cell button sediment sample
was processed along with other routine histopathological specimens.
The paraffin embedded cell button (cell block) sections of 46
thickness were prepared and stained with the hematoxylin and eosin
stain. Special stains like the periodic acid Schiff (PAS) and
mucicarmine were performed wherever necessary.

Advantage of Cell Block

Recognition of histological patterns of diseases that sometimes

cannot be identified reliably in conventional smears.
Possible to study multiple sections by routine staining, special
staining and immunocytological procedures.
Less cellular dispersal, which permits easier microscopic observation
than do traditional smears.
Less difficulty in spite of background showing excess blood on
microscopic observation.
Possibility of storing slides for retrospective studies. Storage of the
CS is a practical problem.

Papaniculaou stain

Papanicolaou stain uses a standard nuclear

stain, hematoxylin, and two cytoplasmic
counterstains, OG-6 and EA.
Crisp nuclear detail and transparency of the
cytoplasm, which allows the examiner to clearly
visualize cellular morphology.
Either a progressive or regressive technique
may be used for nuclear staining.

Romanowsky stain

Romanowsky stain is recommended for air-dried smears.

Romanowsky stains, mixtures of eosin and methylene blue, are a
family of polychrome stains that produce their effect by the production
of azure dyes as a result of demethylation of thiazines and the acidic
component eosin.
Unlike the Papanicolaou stain they are metachromatic.
Most Romanowsky stains used in cytology are aqueous stains as
opposed to the methyl alcohol based stains of hematology.
Most Romanowsky stains are rapid and are useful in enhancing
pleomorphism, and distinguishing extracellular from intracytoplasmic

Destaining & Restaining Process

Removal of coverslip and mounting medium.

Proceeding backward through the staining

Or soak in acid alcohol until the cells are colorless.

Rinse the slide in water baths.


Reporting of Non Gynecology


Class I Negative for malignant cells.

Class II Atypical cells

Class III- Suspicious for malignancy

Class IV Suggestive of malignancy

Class V Positive for malignant cells

Reporting of Gynecology specimens

Bethesda system

Gynecologic specimens


Exfoliative Cytology
Detection of malignant cells in body fluids,
mainly for staging of cancer.
Detection of precancerous cervical lesions in
women (Cervico-vaginal smear/Pap Smear)
Assessment of female hormonal status in case
of sterility and endocrine disorders.
Maturation index vaginal smears

Determination of genetic sex Barr bodies

Detection of infectious agents.

50% alcohol
Saccomano preservatives (50% alcohol with
Ether alcohol

Best fixatives
Flammable, volatile and fire hazard

Fixation time 20-30 minutes

10 minutes

Precaution During Fixation

Identify the slides before preparing the smears.

Attach a paper clip to the identified end of the slide before

preparing the smear. (Paper clip prevents the slide from
sticking together in the fixative).

Smears should be placed into the fixative container immediately

after preparations.

Place each smear fixative by a single uninterrupted motion to

avoid rippling of the smeared material.

Avoid striking the bottom of the fixing container forcefully to

prevent dislodging the material from the slide.

Gynecology Specimen
1. Conventional
2. Liquid-based
Smear from Transformation zone (T-zone)

Gynecology Specimen

Gynecologic Smears

Endocervical brush
Vaginal scrape
Lateral vaginal scape
4-quadrant vaginal
Vulvar scrape herpes

Cells in cervical vaginal smears

Navicular cells

Non Gynecology Specimens

Respiratory Tract Specimens:
Exclude the possibility of malignancy or infections
(pneumocystis carinii in AIDS)

Type of specimens:
Sputum, BAL/BW and Bronchial Brushing (BB)

Sputum: 3 consecutive morning specimens

Saccomano fluid (50% alcohol and 2% carbowax)

1 air-dried smear stained with Giemsa (or Romanowsky)
2 stained with Pap

Adequacy : alveolar macrophage (<10/smear)

Non Gynecology Specimens

Respiratory Tract Specimens:
Bronchial Brushings 2 labeled slides pull through

Fix spray or 95% alcohol

Bronchial washings ( fluid)

Bronchial Aspirates Suction and place in cap
containing 1-2 ml saline

Non Gynecology Specimens

Gastric secretions and aspirate
Peritoneal, Pleural and Pericardial fluid
Breast secretions

Non Gynecology Specimens

Urinary Tract specimen
Diagnosis of malignancy, usually urothelial in origin

Voided urine
Catheterized specimen
Washings from bladder or renal pelvis