This document provides information and instructions for various cytological tests including Pap smear, sputum/bronchoalveolar lavage, urine examination, fine needle aspirate collections, and gastric lavage/esophageal brush smears. It describes how specimens should be collected, transported, and prepared as smears or cell blocks. Fixation methods and staining procedures for Papanicolaou, hematoxylin and eosin, and May-Grunwald Giemsa stains are also outlined. The document aims to standardize procedures for cytological analysis across different specimen types.

1. Cytological tests

2. Pap smear:
• PAP smear test is screening test to detect malignant cells of
cervical/ vaginal cancers as well as cells, which represent a
precursor of malignancy through microscopic evaluation of
representative cells.
• Certain infections (candida, trichomonas, etc.) and
inflammatory conditions may also be detected in this
process.
• The smears are scraped from the cervix / vagina and spread
on a slide.
• Specimens are collected by physician at clinic or at nursing
home. Specimens are received either in the form of specific
solution vials or as previously fixed smears (bio-fix spray).
• Smears can also be received in couplin jars containing ether
alcohol as fixative.

3. Sputum/Broncho-Alveolar Lavage/
bronchial Brushing:
• Study fluids such as pleural fluid, peritoneal fluid,
pericardial fluid, synovial fluid, cerebrospinal fluid
etc. are commonly evaluated for presence of
malignant cells.
• Effusions can be neoplastic or non-neoplastic.
• Cells suspension are received from body cavity
fluids. Fluids should be collected in clean, dry
container and should be submitted to the laboratory
as early as possible. In case of delay in transport,
fluid should be preserved at 4◦C.
• Cells can remain viable for 4 days at 4◦C. Heparin
can be used as an anticoagulant to preserve cellular
details.

4. Urine:
• Urine examination in cytology is performed for
detection of inflammatory lesion including specific
infections, diseases related urinary calculi and
neoplasm arising from urinary tract.
• Proper collection of urine is necessary for correct
diagnosis.
• A fully voided fresh specimen of urine is the most
suitable sample for cytological investigations.
• It should not be the first morning sample or a 24
hour collection, since cells deteriorate quickly in
urine

5. Following are the instructions for the
collection urine:
• Allow patient to drink one glass of water every
fifteen minutes for two to three hours.
• At the end of two to thres hours, patient should
collect urine in a clean and dry container( about
50ml capacity).
• Urine sample should be dispatched to the
laboratory without delay.
• Repeat the urine cytological tests for three
successive days.

6. Gastric lavage / Esophageal-Gasstric Brush
Smears/ Scraping from Oral- Laryngeal
Lesions:
• Specimens from gastrointestinal tract are assessed to
rule out malignancy.
• Evolution of flexible fiberoptic endoscopy enables to
view and sample the lesion from GI tract.
• These endoscopes are provided with accessories like
brush, a plastic tubes or forceps for small biopsy.
• For gastric/ esophageal collections, patient is
instructed to have soft meal and water any time one
hour before the procedure.
• After completion of brushing, brush smears are
preoaraed by rotating brush on a glass slide.

7. • The smears are fixed immediately. For collection
of lavage a tube is passed through mouth. 50 to
100ml of normal saline is passed through the tube
and reaspirated back and collected in a tube.
• This aspirate is centrifuged and smears are
prepared from deposit and fixed immediately.
• For collection of colonic lavage, patient is
instructed to have soft meal and laxative at night.
Next day, in the morning, multiple normal saline
enemas are given to the patient.
• Fluid is collected and submitted for cytology
examination. Cytological sample is collected
before the biopsy.

8. Fine Needle Aspirate
Collections(FNAC)
• Fine needle aspirates are usually obtained to diagnose or
rule out malignancy. Infection or inflammatory condition
can also be diagnosed by FNAC.
• It is necessary to indicate, whenever palpable mass or
lesion is visualized within any organ.
• FNAC of deep-seated lesions are perormed under
radiological guidance using a similar technique that is used
for superficial masses.
• FNAC can be performed in out patient department(OPD),
clinics or in specific room in nursing homes. The sample is
received in the form of smears.

9. Following are the requirements for
FNACs:
1. Examination couch with following facilities:
• Pillow
• Disposable bed sheet
• Blanket
• Screen for privacy
2. Solution and equipments:
• Fixative: 8%(v/v) isopropanol
• Formal alcohol
• 10ml syringes
• Needle size 22g and 23g
• Sterile gauge and plaster
• Gloves
• Glass slides with frostedd ends
• Consent form staining solution

10. Procedure
• Palpitation of suspected lump: The lump is localized and
checked by palpating between the tips of the index and middle
fingers. The selected area is clearned with an alcohol swab.
• Aspiration: The needle is inserted through skin rapidly and
then carefully into the lump; plunger is pulled back and needle
is moved in straight line with back and forth motion and
plunger is released before withdrawing needle.
• After withdrawing the needle a gauze piece is applied over the
puncture site with pressure.
• After removing the needle the collected material is placed on
the glass slides.
• Samear preparation: Smears are preparaed by using glass
slides.
• Smears are immediately fixed in 95% alcohol
• Smears are immediately stained with toluidine blue.

13. Fixation of cytological smears
• The specimen should be immediately fixed
while the smears is moist.
• This prevents drying of the smear and the
distortion of the cells.
• Following fixatives are generally used in the
laboratory.

14. Alcohol-Ether Fixative
• Mixed and placed in a jar with a tight stopper.
Fixation is carried out for about 30 minutes,
following by a rinse in alcohol and then the
section is taken to water.
• It is specially used with the Papanicolaou
staininng method.
Absolute ethyl alcohol 50ml
Ether 50ml

15. Schaudinn’s fluid
• Fixation is carried out for about 2 minutes.
• After washing in distilled water, the mercuric
chloride black clumps are removed by addinng a
few drops of saturated alcoholic iodine solution.
• After rinsing in water the smear is taken for
staining.
• This reagent is rapidly in penetrating one and
preserves the smears which are to be stained by
hematoxylin and eosin.

16. Schaudinn’s fluid
Saturated mercuric
chloride solution
60ml
Absolute alcohol 33ml
Glacial acetic acid 1ml

17. Carnoy’s fluid:
• It is prepared as follows:
• The fixation is performed from 30minutes to
3hrs
Absolute alcohol 60ml
chloroform 30ml
Glacial acetic acid 10ml

18. Fixation procedure:
• Fix the pre-labeled smear while moist.
• Fix adequately ( at least for our hour).
• Fixed slides can be kept aside for staining
or can be left in the fixative until ready
for staining .
• Prolonged fixation does not affect the
smear

19. Fixatives used for specimens other
than smears
• 10% neutral buffered formalin
• Bouin’s fluid
• Methanol acetic acid fixative: It is prepared by mixing
1 part of methanol and 19 parts of glacial acetic acid.
This fixative is recommended for cytology and flow
cytometry.
• Ethanol formalin fixative:It is used as mixture of 1
part of 40%(v/v) formaldehyde and 9 partsof absolute
alcohol.
• Formal vapor: These are recommended for
demonstration of lipids.

20. Preparation of smears:
• Smears are prepared according to the type of the
specimen. By using wire loop or an applicator stick,
fixed specimen or sediment is transferred on a
cleanand pre- labeled slide. Following methods can
be used to prepare smears:
• Streaking: This method is used for the mucoid
specimens such as vaginal secretion, sputum or
gastric contents. About 2/3 area of the slide is
covered by spreading the specimen uniformly. The
smears are placed in the fixative immediately.

21. Spreading:
• A moderately thick flim is prepared by this
method which maintains the cellular
interrelationship. This method is used for the
speimens such as sputum and bronchial
aspirates.
• Direct smears: Cervical smears, breast
secretions, FNAC are directly smeared on a
slide. The slides are then paced in a jar
containing fixative or can be coated with a
spray fixative.

22. • Pick and smear method: It is used for mucinous
specimens such as sputum, bronchial lavage and gastric
lavage. Pick and smear method is performed as follows:
• Specimen should be poured in to a petri dish under
aspetic conditions.
• Gross description of the sputum sample is documented.
• The sputum can be teased out using forceps and blood
stained; discoloured and purulent areas can be selected
and placed onto the glass slides.
• About three smears are prepared.
• Once prepared the smear should be immersed in the
fixative-containing jar immediately.
• Smears should be fixed in fixative for at least 30
minutes and then proceed for staining.

24. Saccomanno’s technique
• It is performed for mucinous specimen by using
saccomanno’s fixative. It is prepared as follows:
Carbowax 2g is mixed in 100ml od 50%(v/v) alcohol.
Following procedure is used for fixation.
• Collect specimen in saccomanno’s fixative.
• Pour in a blender (for 5-10sec).
• Pour in the 50ml tube.
• Centerifuge for 10 minutes.
• Decant and agitate well .
• Aspirate sediment and place a drop on a slide and
smear it.

26. Cell block technique
• Cell- block technique is a meethod of preparing
material for microscopic examination using a
paraffin wax embeddig method.
• This supports in providing more definitive
cytopathology diagnosis.
• Cell blocks can be prepared from fluid specimens
of all types, particularly from effusions, edometrial
aspirates and brush samples as well as fine needle
washing.

28. Liquid based cytology(LBC):
• Liquid based cytology technique has increased
sensitivity and specificity of Pap smear technique.
• Sample is collected using a brush and is rinsed in
the fluid.
• This ensures complete transfer of material collected
for the testing.
• The smears preapred using LBC minimizes
obscuring inflammatory cells, red blood cells,
necrosis and displays monolayer of well- preserved
cells.

31. Papanicolaou stain(PAP stain)
• Reagents :
• Harris alum hematoxylin
• 0.5% (v/v) hydrochloric acid in 70% ethanol( acid –
alcohol reagent)
• 0.5%(v/v)hydrochloric acid in distilled water.
• 0.25%(v/v)hydrochloric acid in distilled water
• Orange G6 (OG6) and EA36 and 50 (eosin-azure):
This reagents are avaliable commercially.

32. Procedure :
• Fix the smears in equal parts of 95% ethanol and ether.
• Take the smears to water ( hydration): 6 dips in each of the
following solutions of ethanol
• 80% (v/v)
• 70% (v/v) and
• 50% (v/v).
• Rinse gently in distilled water.
• Stain in diluted Harris’ hematoxylin for 2 to 3 minutes.
• Rinse in distilled water
• Dip in 0.25% (v/v) hydrochloric acid 6 times.( or in 0.5%(v/v)
hydrochloric acid 3 times)
• Place under running tap water for 5 minutes.
• Check up the slides under the microscope to see if the nuclei
are stained properly. (if over stained, decolorize in acid-
alcohol and if pale, return to hematoxylin stain)

33. • Dehydrate through distilled water and alcohol solutions
• 50% (v/v)
• 70% (v/v)
• 80% (v/v)and
• 95% (v/v) by dipping 6 times in each solution.
• Stain in OG6 for 2 minutes
• Rinse in 95% (v/v) alcohol(3 separate changes) and 6
dips each.
• Satin in EA36 (or EA50) for 2 minutes.
• Rinse in 95%(v/v) alcohol (3 separate changes)
• Dehydration in absolute alcohol(2 changes)
• Clear in alcohol xylene (6 dips), following by 3 changes
in xylene (6dips)
• Mount in any satisfactory neutral medium

34. Precautions:
• This slides should be handled carefully during
rinsing and washig. Rough handling may wash
off the specimen.
• Results :
• Nuclei: Blue
• Cytoplasm: Varying shades of pink, blue, yellow
green, grey.

35. The method is highly reproducible.
The method is used for the smears such
as
• Vaginal
• Cervical
• Prostatic
• Urinary as well as for the smears made from
the specimen such as
• CSF
• Pleural fluid and
• Other fluids.

37. Rapid papanicolaou staining:
• Stock reagents:
• Eosin Y 20% aqueous
• Orange G-6 3% aqueous
• Light Green SF 3% aqueous. Working reagent is
prepared as follows.
Eosin 20ml
Orange G-6 6ml
Light green SF 20ml
Isopropyl alcohol 760ml
Methanol 240ml
Glacial acetic acid 10ml
Phosphotungstic 5g

38. • Mix reagents and light green slowly to avoid
precipitation.
• Procedure : Nuclear staining can be performed as
routine PAP procedure followed by using modified
EA regent.
• OG- EA reagent treatment: 2-4min.
• 95% alcohol: 10 dips, with 2 changes
• Xylene: 10 dips with 2 changes
• Blot dry and mount with DPX

39. Hematoxylin and eosin staining
• Reagents:
• Harris’ hematoxylin
• 1.0% (v/v) hydrochloric acid in 70% ethanol(
acid- alcohol reagent)
• Eosin (Y): 1% (w/v) aqueous

40. Procedure:
• Treat with 95% alcohol for 2 minutes.
• Wash in running tap water for 2-3 minutes
• Stain in harris hematoxylin for 5-10 minutes
• Wash in running tap water for 5 minutes.
• Differentiate in acid alcohol solution for 10-15
seconds
• Wash in running tap water for 5-10 minutes
• Treat with eosin solution using 2 dips
• Blot and dehydration in absolute alcohol.
• Clear in xylene and fix coverslip by using a drop of
DPX.

41. May grunwald giemsa stain
• It is useful technique for FNAC smears, and body
fluids.
• May-Grunwald Giemsa stain is Romanowsky staining
technique containing two dyes, methylene blue and
eosin Y.
• During staining procedure, methylene blue on
oxidation produces colored compound known as azure
that has ability to combine with eosin.
• Methylene blue azure is blue violet and as follow
stains mainly acidic cell comonents.
• Eosin is red in color and stains more basic structures.
Combination of acidic and basic dye optimizes
staining reaction.

42. Reagents:
• May-Grunwald stain (stock): It is prepared as
follow:
• Mix and store at room temperature (25◦ C ± 5◦C).
May-Grunwald power 2.5g
Methanol 1000ml

43. May –Grunwald working solution: It is
prepared as follows:
• This working solution should be prepared fresh
everyday.
Stock solution 2ml
Methanol 8ml

44. Giemsa stock solution: It is prepared as
follows:
Giemsa powder 1gm
Glycerin 54ml
Methanol 84ml

45. Stock buffer solution ( pH 6.8): It is
prepared as follows:
• Mix 50ml of K2HPO4 with 236ml of NaOH.
K2HPO4 2.7% (w/v)in
distilled water
NaOH 0.8g% (w/v) in
distilled water

46. • Working buffer solution: It is prepared as follows:
• Mix 40ml of stock buffer solution with 60ml of
distilled water.
• Working Giemsa solution: It is prepared as follows:
It is prepared fresh everyday by mixing 1ml of
Giemsa solution and 9ml working buffer solution.

47. Procedure:
• Fix smears in methanol for 15 minutes.
• Stain the snears with working May-Grunwald
solution for 25 minutes
• Wash in tap water.
• Stain the smears with working Giemsa stain for 20
minutes.
• Wash in running tap water, dry and mount with
DPX.

48. Rapid stains:
• These stain are used mainly to check adequacy of
fine needle aspirates. Rapid stains can also be used
for immediate evaluation to control cross
contamination. The dyes used in rapid staining
procedures are, Methylene blue, Thionin blue, and
Toluidine blue.
• Toluidine bue stain is prepared as follows:
Toluidine blue 0.5g
95% ethyl alcohol 20ml
Distilled water 80ml

49. Toluidine blue staining procedure: It is
performed as follows:
• Fix the smear for 10 to 15 seconds in 95% alcohol
• Blot dry.
• Treat with toluidine blue, use 10 dips.
• Rinse in water to remove excess of stain.
• Put a coverslip on wet smears and submit for
microscopic examination

50. Maintainence of the stains
• Following precautions are taken while using the stains.
• Stain used must be certified by biological stains
commission.
• Dye content on the certified label of dye must be
considered in stain preparation.
• Contamination control: Gynec and Non- gynec slides
are stained separately. Cells are detached from the slides
during staining. These cells can cause cross
contamination which results in female in false
diagnosis.
• Filter stains and reagents daily.
• Non- Gynec specimens have high potential for cross
contminationduring staining procedures
• Daily microscopic checks are documented for the
quality control of stains.

51. Demonstration of Barr body:

52. • The sex chromatin or Barr body is a condensation of
chromatin present at the nucleus of cells in female
individuals.
• Their observation is possible in different cell types
and is used for the rapid diagnosis of biological sex.
• Sample:
• Scrapings from buccal mucosa, or in some women
from the vaginal wall.
• Buccal mucosa is scrapped and smear the slide.
• While the Drumstick is done on the white
cells (WBC) to see the presence of drumstick.

54. Normal
• Male = No barr bodies are seen.
• Female = Barr body is positive.
• Interpretation
• This Barr body test is advised for the screening of
ambiguous or doubtful sex characters.
• Turner’s syndrome female (XO) will have no barr body.
• Male with Klinefelter syndrome (XXY) will have a barr
body.
• Female with XXX chromosome will have two barr
bodies.
– The female cell with four X chromosomes has three barr
bodies.