Liquid Base Cytology

1. Liquid based cytology and
Cytospin
MODERATORS
Dr Kishan
Dr Hemalatha

2. INTRODUCTION :
• Liquid-based cytology is a method of preparing samples for
examination in cytopathology.
• The sample is collected normally by a small brush, in the same way
as for a conventional smear test, but rather the sample is deposited
into a small bottle of preservative liquid, the smear being transferred
directly to a microscope slide.
• For many years, efforts have been made to develop methods that
would enhance the sensitivity and specificity of the Papanicolaou
smear (also called Pap smear).
• Emphasis has been placed on creating automated screening
machines whose success depends on a representative sampling of
cells on standardized slides containing a monolayer of well-stained,
well-preserved cells.
• From this research and development, liquid-based gynecologic
specimen collection has evolved.

3. • The Papanicolaou test (abbreviated as Pap test, also known as Pap
smear (AE) cervical smear , cervical screening, is a method of cervical
screening used to detect potentially precancerous and cancerous processes in
the cervix.
• A Pap smear is performed by opening the vaginal canal with a speculum and
collecting cells at the outer opening of the cervix at the transformation zone (where
the outer squamous cervical cells meet the inner glandular endocervical cells),
using an Ayre spatula.
• The collected cells are examined under a microscope to look for abnormalities.
The test aims to detect potentially precancerous changes.
• The test remains an effective, widely used method for early detection of precancer
and cervical cancer.

5. • There are two types of error in a case of false negative cytology.
• Sampling error: This means that the lesion is present in the body but is
not properly sampled.
• Sampling error is one of the major sources of error.
• Remedies: It is not entirely possible to avoid the sampling error, but with
the help of liquid-based cytology (LBC) we can get the maximum number of
cells on the smear.
• Screening errors: Screening error may be due to failure to find out the
representative cells on the smear or it may be due to interpretation error.
• Remedies: (1) Liquid-based cytology (LBC) may provide a clean
background of the smear which is free of mucus or blood.
• (2) The automated adjunctive technique helps the manual screening by
locating the path of the screening on the computer screen.

6. Liquidbased cytology (LBC)
• The basic principle is to collect cell samples into a liquid fixative
solution and then create a monolayer of cells ready for microscopic
observation after staining.
• It has overcome many limitations of conventional cytology.
• Liquid-based cytology has the added advantage of eliminating human
errors responsible for thick, unsatisfactory smears and poor fixation.
• The presence of the liquid helps collecting cells remaining in the
needle, whereas immediate fixation due to the presence of methanol
or ethanol in the fixative optimizes cell preservation and usually
reduces the bloody background.
• Liquid-based cytology also paved the road to automated cytology and
to the development of automated screening devices.

7. SAMPLE COLLECTION :
• The cervical sample is obtained using a combination of plastic spatula
and cervical broom type device in the same manner of conventional
screening.
• The sample is then rinsed into a vial containing preservative solution.
• The head of the device is kept in the vial containing preservative
solution and the handle is removed.
• The sample is sent to the cytology laboratory for further processing.
• The cells are made discrete and a monolayer preparation is prepared.

9. • Different Company Products :
• two commercially available liquid-based technologies are approved by FDA,
USA:
• ThinPrep Pap test
• SurePath.
• FDA NOT Approved :
• Cytoscreen
• Turbitech
• Cellslide
• Papspin

10. • ThinPrep :
• Cells are collected by a plastic spatula or cytobrush and then transferred to the transport
media provided by the company.
• The vial containing the material along with the filter is introduced into the instrument.
• The filter rotates within the vial which contains the cells in liquid.
• The cells are dispersed mechanically by rotation of the filter.
• The negative pressure is used to draw the fluid through a filter made of neutral
polycarbonate.
• The flow of fluid through the filter is constantly monitored to get an optimal quantity of cells.
• The cells trapped in the filter surface are then automatically transferred to a glass
• slide and fixed immediately.

12. SurePath test :
The cells are collected with the help of plastic device and transferred to a
transport fluid.
In the laboratory, the vial is vortexed and strained to break up mucus and
large cell groups.
The cell suspension is treated through a density gradient centrifugation
process to remove the blood and other insignificant material.
The cell pellet is resuspended and is then allowed to sediment onto a
glass slide

14. SurePathTM slides are prepared and
stained on the BDPrepStainTM using a
gradient density technique and on-
board staining
ThinPrep® slides are prepared using the TP
2000 or TP 5000 Processor® using a
membrne filtration process. Staining is by
conventional Papanicolaou stain or, if
intended for review using the ThinPrep
Imager®, by the ThinPrep Stain®

18. ADVANTAGES :
• The main advantages of LBC are adequate representative material,
complete avoidance of air drying, monolayered preparation and clean
background.
• The background is clean from necrotic debris in LBC smear.
• Infective organisms can also be identified in LBC smear.
• One major advantage of LBC is that the residual sample can be kept
for human papilloma virus (HPV) testing or any other ancillary tests.
• Monolayered cellular smear is of great help in studying individual cells
for automation.
• LBP and cell block material can also be utilized for ancillary studies
such as immunocytochemistry, special stains, and molecular tests.

19. Special attention has been given to the use of residual material in the
vial, which can be used for:
(a) Preparation of additional slides;
(b) Molecular testing of infectious agents;
(c) DNA cytometry.
(d) DNA ploidy analysis.
DISADVANTAGES :
• Liquid-based cytology instrument is very costly.
• The overall processing cost of LBC is also much more than
conventional preparation.
• The laboratory technical staff also needs adequate training for
interpreting LBC smear.

20. Principle advantages and disadvantages

22. Other uses of LBC :
LBC ThinPrep system has found broad acceptance in nongynecologic
cytopreparation such as:
• Thyroid cyst fluid examination.
• Oral pathology: diagnostic for various types of oral lesions.
• Many laboratories have successfully applied this technique to body
fluids (e.g. urine, pleural effusions), brushing samples (e.g.
gastrointestinal tract, lung) and fineneedle aspiration.

23. Gross smears of liquid-based cytology preparation
(SurePath) and
conventional preparation

24. Clean background of liquid-based cytology preparation
(SurePath)
Dirty background and cell clumping of cells in
conventional smear preparation.

25. A conventional cervical smear
containing superfi cial and
intermediate type squamous cells and
a honeycomb sheet of normal
endocervical glandular epithelium.
A liquid-based preparation showing superfi cial and
intermediate type squamous cells and a
honeycomb sheet of normal endocervical
glandular epithelium.

26. Superficial cells
Large polygonal cells, 45-
50 μm in diameter.
Cytoplasm is eosinophilic
and stains pink. Nuclei
are small (2 μm in
diameter), round and
pyknotic.
Intermidiate cells
Polygonal cells, 35-40 μm
in diameter. Slightly
smaller than superficial
cells. Cytoplasm is
cyanophilic and stains
greenish-blue. Nuclei are
vesicular and about 8 μm
in diameter.

27. • Para basal cell
• Round-oval cells, 15-30 μm in
diameter.
• Cytoplasm is cyanophilic and stains
dense greenishblue.
• The nucleus occupies about half of
the cell.

28. Cells of squamous metaplasia
Mature metaplastic cells are almost the
same size as intermediate cells, with
cyanophilic cytoplasm staining blue-green.
The cells are often rounded. Nuclei are
vesicular and may vary in size. They have
finely granular chromatin and nucleoli may
be visible.
Endometrial cells can be found in
loose groups
with intact cytoplasm.

33. Conventional smear showing mature
intermediate type squamous cells containing
large nuclei with coarse chromatin,
consistent
with LSIL or mild dysplasia.
Liquid-based preparations (SP) showing
tissue fragments of intermediate type
squamous cells with enlarged nuclei
consistent with LSIL.
LSIL :

34. Liquid-based preparation showing HSIL,
moderate dysplasia.
The abnormal cells are arranged in a
sheet with well-defined cell borders and
moderate amounts of dense cytoplasm.

37. REPORTING FORMAT
Bethesda system of reporting is followed in both Conventional pap and
LBC.

40. Fibroadenoma of the breast. FNAC. Liquid based preparation.
Papanicolaou stain,

41. CC preparation shows
atypical keratinised cells
entrapped in inflammatory
exudate. (Pap stain x600)
LBC preparation shows atypical
squamous cells with preserved nuclear
and cytoplasmic
details in a cleaner background (Pap
stain x600)
Comparison of CC and LBC in BAL specimen in cases of squamous
cell carcinoma.

42. LBC ON URINE SAMPLE :

44. LBC of pleural fluid showed atypical
mesothelial cells.
The malignant mesothelioma cells are
enormous in size, containing dense,
abundant cytoplasm with fuzzy borders
(arrows).

45. LBC of ascites showed presence of
adenocarcinoma with signet-ring
cells in the fluids.
Gastric brushings showing signet-ring type
adenocarcinoma.
The malignant cells are small with
vacuolated cytoplasm and eccentric nuclei.

46. CYTOSPIN
• Defination : Cytospin technique is a method of performing thin-layer
preparation from fluids through a cytocentrifugation process.
• Principle :
• During the procedure, the material is centrifuged and the cells are
deposited onto a vertical microscope slide. This procedure permits a cell
separation from the fluid medium.
• This technique has a wide variety of applications including cytology,
histology and hematology.

47. Equipment, reagents and cell preparations
•Cytospin™ Centrifuge with cytofunnels and cytoclips (Thermo Scientific).
•Filter cards
•SuperFrost Plus Glass slides
•Cytofunnels
•Fixative: acetone (undiluted), methanol (ice-cold 100% methanol) or
paraformaldehyde (2%) at room temperature.
•Hydrophobic pen
•1% Virkon (Fisher Scientific) or bleach
•Glass pipettes
•Pencil

48. • Procedure :
• To prepare cytocentrifuge smears, a funnel assembly is attached to the front
of a microscope slide.
• The surface of the funnel assembly that is in contact with the slide is lined
with filter paper to absorb excess fluid.
• A few drops of fluid are placed in the funnel.
• The assembly is placed in the cytocentrifuge, which operates at a
low force (600–800 x g) to preserve cellular structure.
• Centrifugal force pushes the fluid through the funnel's opening and
concentrates the cells in a small area of the slide.
• The centrifugation process concentrates cells by about twenty-fold and
creates a one-cell-thick monolayer, allowing for assessment of cellular
morphology.
• The slide can then be fixed and stained.

51. USES :
•Performing differential cell counts on body fluids, such
as serous, synovial and cerebrospinal fluid.
•Cytopathology examination of liquid specimens such as body fluids
and fine needle aspirates.
•Gram staining of fluid specimens for identification of microorganisms.
• Thin watery samples are processed by centrifugation in a cytocentrifuge.

52. • LIMITATIONS :
• The cytocentrifugation process can cause cells to appear distorted.
• Cells located at the centre of the smear may look compressed compared to
cells at the periphery.
• Cell nuclei may develop artifactual clefts, lobes, or holes,and
the cytoplasm may appear vacuolated or develop irregular projections.
• Cytoplasmic granules may be pushed to the periphery of the cell. If the cell
count is high, cells may be distorted due to crowding; therefore, samples with
high cell counts are diluted prior to smear preparation.
• After cytospin, the accessories like the cytofunnel and cytoclip are soak in Cidex OPA
solution (0.55% ortho-phthalaldehyde) at least 10 mins.
• This is to disinfect and remove any precipitate found on the accessories. The filter paper
used is discarded in the biohazard bin.

53. CELL BLOCK TECHNIQUE
• Cell block refers to the examination of sediment, blood clots, or grossly
visible flecks of tissue from cytologic specimens.
• processed by paraffin embedding and staining by Hematoxylin & Eosin.
• Small tissue flecks must be wrapped in tissue paper and then placed in the
cassette.
• Sediments can be embedded in either HistoGel or agar.
• Cell blocks are complementary to the smears and allow extra slides to be
cut for ancillary tests.
• Several methods exist for processing the cell blocks such as agar,
HistoGel, and plasma-thrombin.

54. • Automated Screening
• Automated screening procedure should be designed in such a way
that no dysplastic or malignant cells should be passed as normal or
negative.
• It should not label the normal cell as malignant cells.
• The automated screening technique should be economically cost-
effective and it should shorten the period of screening.
• The two main goals of automated screening are to increase the
accuracy and productivity of the cervical screening.

56. REFERENCES :
• Pranab Dey Diagnostic Cytology
• Sudha R Kini Differential diagnosis in Exfoliative and Aspiration
Cytopathology 2nd edition.
• The Bethesda System and Beyond Atlas for cervical cytology.
• The Bethesda system for Reporting Cervical Cytology.
• Diagnostic Liquid based cytology.
• Research Article of LBC on Urine Samples by using Cytocentrifugation.
• Internet.

57. Thank you