This document discusses liquid based cytology (LBC) and compares it to conventional pap smears. It provides a history of LBC dating back to 1970 and describes two main LBC systems - ThinPrep and SurePath. With ThinPrep, cells are collected in a preservative fluid and then filtered and transferred to a slide. With SurePath, cells are collected in a preservative fluid and separated via density gradient centrifugation before being placed on a slide. Studies have shown LBC reduces inadequate samples and increases specificity compared to conventional pap smears. LBC also better preserves cell morphology. The document focuses on how LBC has advantages over conventional pap smears for detecting cervical intraepithelial neoplasia lesions.

1. Liquid based cytology
By
Dr. Varughese George
Department of Pathology. MGMCRIDR. VARUGHESE GEORGE

2. HISTORY
• Idea of LBC -1970.
• 1987 – NHS CSP (National
Health Service Cervical
Screening Programme) –
successful in decreasing the
incidence and
mortality(80% prevention)
Dr.Georgios Papanikolaou (1920)

3. PAP smear
• The Papanicolau test (also called Pap smear, Pap
test, cervical smear, or smear test) is a
screening test used in gynaecology to detect
premalignant and malignant processes in the
ectocervix.

4. Basic principle :
The technique of removing cells from the surface of the
cervix
scraping with a wooden spatula and
smearing on the glass slide
staining and examining them microscopically
**Ideal focus of sampling to obtain adequacy is Squamo-columnar
Junction

5. Why a PAP?
• The test aims to detect potentially pre-cancerous
changes (cervical intraepithelial neoplasia (CIN) or
cervical dysplasia), which are usually caused by HPV or
any other denovo mutations
• The test may also helps to detect infectious agents
and abnormalities of the cervix

6. Whom to screen? -Indications
• Screening starts at age 2o-25 continues over symptomtic
postmenopausal women.
• screening women with family history of carcinoma cervix or patients
identified to have insitu lesions.
• Screening and follow up on known cases of carcinoma/
radiotherapy/post-hysterectomy status-Vault smear.
• Pap smear screening is still recommended for those who have been
vaccinated against HPV.
• To evaluate hormonal assessment of vaginal smear

7. How to perform?
• Not done during menstruation.
• Insert a speculum into the
patient's vagina, to allow access to
the cervix.
• Samples are collected from the
outer opening or os of the cervix
using an Ayre’s spatula
(conventional PAP),an endocervical
brush, or a plastic-fronded
broom.(LBC)

9. Conventional pap smear slide preparation
Sensitivity-72%
Specificity-94%

10. Reporting according to Bethesda classification[2014]
Specimen type Conventional PAP smear LBC
Adequacy : Epithelial(8000-12,000
cells well preserved and
well visualised.
Glandular
components(10 well
preserved cells in
groups/single)
For LBC -5000 squamous
cells)
Glandular components(10
well preserved cells in
groups/single)
General categorization: Negative for intraepithelial lesion or malignancy[NILM] –
organisms, other non neoplastic findings.
Epithelial cell abnormalities – squamous cells, glandular
cells.

11. Interpretation/result :
• Squamous cell abnormalities (SIL)
– Atypical squamous cells of undetermined significance (ASC-US)
– Atypical squamous cells - cannot exclude HSIL (ASC-H)
– Low-grade squamous intraepithelial lesion (LGSIL or LSIL)
– High-grade squamous intraepithelial lesion (HGSIL or HSIL)
– Squamous cell carcinoma.
• Glandular epithelial cell abnormalities
– Atypical Glandular Cells not otherwise specified (AGC or AGC-NOS)
of endometrial ,endocervical and glandular
– Atypical Glandular Cells favouring neoplastic – endocervical and
glandular
– Endocervical adenocarcinoma insitu
– Adenocarcinoma

12. Interpretation/result :
• Other malignant neoplasms: (specify)
• Adjunctive testing
• Provide a brief description of the test method(s) and report the result so
that it is
• easily understood by the clinician.
• Computer-assisted interpretation of cervical cytology
• If case examined by an automated device, specify device and result.
• Educational notes and comments appended to cytology
• Reports ( optional )
• Suggestions should be concise and consistent with clinical follow-up
guidelines
• published by professional organizations (references to relevant
publications may be
• included).

13. Reporting format
• ** Bethesda system of reporting is followed
in Conventional pap smear as well as LBC

15. Common trouble shootings -CPS
• Using the incorrect sampler or the right sampler
incorrectly & absence of TZ zone
• Cellular transfer onto slide sampling
error –[airdrying artifact]
• Quality of the smear (thickness,
blood, exudate, mucus,poor fixation)
• Missing the abnormal cells.
•
dense inflammation & hemorrhage screening
• Interpreting the abnormal cells.

16. Reasons for Unsatisfactory Smear
The following inhbits intepretation of 75 % of smear : -
• Squamous epithelial component scanty/absent
• Obscuring blood
• Inflammation
• Thick areas
• Mucus
• Poor Fixation
• Air Drying
• Artifact
• Contamination

17. Reasons to improve the PAP smear
• New collection devices (brooms and brushes
rather than spatulas/Q tips, etc)
• Liquid-based Pap Tests rather than smears
• Ancillary tests such as HPV detection
• Computerized screening devices.

18. Liquid based cytology
• Since the mid-1990s, techniques based around placing the
sample into a vial containing a liquid medium that preserves
the cells have been increasingly used.
• The media are primarily ethanol-based.
• Improvised method, can be used as a substitute.
• Works on different principle to ultimately give accurate
results.
• Proper sample acquisition - a cell that is not in the sample
cannot be evaluated.

19. TYPES
• FDA- APPROVED
• 1. Thin –Prep
• 2. Sure Path
• FDA-Not Approved
• Cytoscreen
• Turbitec
• Cellslide
• Papspin

20. Other uses of LBC
• Non-gynaecologic cytopreparation
• 1. thyroid cyst fluid examination
• 2. Oral pathology lesions
• 3. body fluids –pleural effusions, urine
• 4. brushing samples

21. SYSTEMS TO SCREEN
• Two systems:
1. Sure Path (TriPath Imaging): centrifugation and
sedimentation through a pressure gradient
2. Thin Prep(Cytyc Corp): filtration and collection of
Vaccum –packed cells on membrane and
transferring to slide
Sensitivity and specificity has high range of 90%

22. Need for LBC
1. reduces the number of inadequate smears and
need for repeat smears
2. high sensitivity in detecting CIN lesions
3. cellular preservation is preserved
4. preparation as well as storage of in case of fluid
samples

23. THIN PREP
• ThinPrep Imaging System a dual-review computer
system that screens for cancerous and precancerous
cervical cells.
• Dual Review means that your ThinPrep Pap Test gets
screened twice — first by the ThinPrep Imager, and
then by a laboratory professional trained in looking
for abnormal cervical cells.

24. Sample collection
• Obtain cervical specimen prior to manual examination.
• Use an un-lubricated speculum (saline or warm water may be used).
• Vaginal discharge or secretion, when present in large amounts, should be removed
before obtaining the cervical sample so as not to disturb the epithelium
• Use of liquid-based specimen collection minimizes the interference from
factors blood and mucous.
• Different devices :
Cervex-Brush
The Pap Perfect® plastic spatula
Cytobrush® Plus GT
• Both systems require the sample taker to follow the same method of sample collection.

25. Cervical brush used for
LBCs.

26. Thinprep and Surepath
Apply gentle pressure until the
surrounding bristles come into
contact with the ectocervix and splay
out across the cervix. Rotate the
Cervex Broom(TM) 5 times in a
CLOCKWISE direction to obtain the
optimal amount of material.
Note - Do not rotate the broom
anti-clockwise as little or no
sample will be collected rendering
the sample inadequate. The
bristles are 'D' shaped, and will
only collect material if rotated
clockwise.

27. ThinPrep(TM)
Once collected, immediately rinse
the 'broom-head' in the
preservative solution. This is best
achieved by pushing the broom into
the base of the vial 10 times,
forcing the bristles apart. Finally,
swirl the brush vigorously to
release any trapped material and
further break down the previoulsy
released material

28. ThinPrep Pap Test slide preparation

30. THIN PREP
• The vial is received and is processed in two
ways:
– semi-automated method (T2000
processor)
– fully automated method(T3000
processor)
• The vial is agitated and then the fluid is
sucked up through a micropore filter.
• Neutrophils and RBCs are taken up by the
filter but not the epithelial cells – obstruct
the pore.
• There is a pressure gradient developed and
is sensed by the processor.
• This improves the cell yield.

31. • The filter is then removed and dabbed on to a
slide which is electrically charged – permits
the transfer of the cells.
• The slides are then stained as for conventional
methods.
• T3000 processor does not need the cells to be
transferred manually – done automatically.

33. Image analyser for thinprep

34. ThinPrep Pap Test liquid-based slide:
Improvement over the conventional pap
• The ThinPrep Imager scans every cell and cell cluster on the slide,
measuring DNA content. It then identifies the largest and darkest
nuclei, highlighting cells of interest for pathologists' review.
• An Imaged ThinPrep Pap Test slide is placed in the Review Scope,
which takes the pathologist to the appropriate coordinates of the
areas of interest.
• If the pathologist determines the presence of an abnormality, then
he or she will review the entire slide, marking any additional areas
for further review.

39. Imaged slide from the ThinPrep Imaging System:
The area of interest is shown in the
"crux"
of the L-shape when identified by the
ThinPrep Imager
the ThinPrep Imager indicates area of
interest for further human review

40. Conventional Pap Smear ThinPrep Pap Test

42. Photograph showing Trichomonas
Vaginalis by CPS technique
Photograph showing koilocytes
in LSIL by CPS technique

43. Photograph of smear of squamous cell
carcinoma showing tadpole shaped cells
by CPS techniques.
Photograph of smear of squamous cell
carcinoma showing tadpole shaped
cells by LBC techniques.

44. Screening methods
• Area to be screened is smaller and circular.
• Screening must be 100% of the deposit.
• Must be done in 10x. For cellular details,40x may be used.
• Primary screening – both of them have different look.
• Thin prep – more widely spaced than sure path.
• After primary screening , all the negative and inadequate
samples are taken for a second review.
• Must be less than 90seconds.

45. SurePath
Sample Collection:
• Both systems require the sample taker to
follow the same method of sample
collection.
• This test requires a SurePath™ special
collection kit, which includes a
SurePath™ preservative fluid collection
vial and the sampling device(s).

46. SurePath
SurePath liquid-based
collection mediaCervex Brush (Broom)

47. • The device should be twisted slowly.
• Transfer the entire sample by placing your thumb against the back of the
brush pad, and simply disconnect the entire brush from the stem into the
SurePath™ preservative vial.
• For spatula -To obtain an adequate sampling, scrape the ectocervix using
the Pap Perfect® plastic spatula. Disconnect the spatula head into the
SurePath™ Preservative vial.
• For cytobrush - Insert the Cytobrush® Plus GT into the cervix until only the
bottom most fibers are exposed.
• Slowly rotate or turn one half turn in one direction. DO NOT OVER-
ROTATE. Disconnect the brush head and place in the SurePath™
preservative vial.

48. SurePath(TM)
Once collected, transfer the
material to the SurePath(TM)
vial. To do this, place the
thumb against the back of the
'brush-head' and push the
entire 'broom-head' from the
stem into the preservative
solution.

49. • Recap the vial and tighten.
• Record the patient’s first and last name, date
of birth, specimen source and date collected
on the vial. Record the patient’s information
and medical history .
• Place the vial and test request form in a
specimen bag for transport to the laboratory.

50. SURE PATH
• Works on the principle of density gradient.
• Density gradient centrifugation process. (DGC process)
Vials are received
mixed to re-suspend the cells.
A syringe is inserted in to the vial through the cap and the fluid is
dispensed in to a centrifuge tube filled with a density reagent.
First centrifugation @200g for 2 minutes.
Decant
Second centrifugation@800g for 10 minutes
Decant

51. SURE PATH

52. • Transferred to a settling chamber mounted on
the microscope slide.
• Cells settle to form a thin layer and the excess
fluid is discarded.
• Staining is an integral part of the process.
• Prep stain processor.

53. SURE PATH

56. Special Focus on CIN Lesions where
LBC has advantage

57. Special Focus on CIN Lesions where
LBC has advantage

58. Special Focus on CIN Lesions where
LBC has advantage

59. Special Focus on CIN Lesions where
LBC has advantage

60. Normal Smear by both CPS (A) and LBC (B) Methods.

61. Conventional smear V/S LBC
Normal endocervical cells are identified on conventional Pap smear (A)
and liquid-based preparation (B). (Papanicolaou stain, x400)
Normal groups remain intact in the liquid based sample.

62. Conventional smear V/S LBC

63. Conventional smear V/S LBC

64. Conventional smear V/S LBC

65. Conventional smear V/S LBC

66. Conventional smear V/S LBC

67. Conventional smear V/S LBC
Pleomorphic cells from squamous cell cancer are identified in conventional (A)
and liquid-based preparation (B), having similar morphologic features.

68. Conventional smear V/S LBC

69. Conventional smear V/S LBC
Example of Herpes virus present in conventional (A) and liquid-based
preparation (B). (Papanicolaou stain, x400)

70. Conventional smear V/S LBC
Human papillomavirus (HPV) cytopathic effect (LSIL), appears virtually
identical in conventional (A) and liquid-based preparation (B).
(Papanicolaou stain, x400)

71. Pilot studies
• In a UK setting – largest in the number of samples studied.
• It also studied the complete conversion to LBC for the purpose
of screening.
• Two centers used thinprep and the other sure path.
• Included detection of HPV.
• Study showed a decrease in inadequate samples from 9.1% to
1.6%.
• There was increase in the specificity of these tests.

72. Thin prep smear Surepath smear

73. Morphology
• Not much difference from the conventional smears.
• Due to rapid fixation, the cells are well preserved – evidenced
by clarity of nuclear chromatin.
• Nuclear membranes are well visualized.
• Over all appearance of the cell - small – due to “rounding up”
effect of the liquid.
• Cells with low grade dyskaryosis are very easy to find.
• Severe dyskarosis often presents as single and dispersed cells
(a feature less well recognized in the conventional smear)

74. • Hyperchromatic crowded cell groups (HCCG) –
difficult to assess in both LBC and conventional
smears.
• Nuclear clarity and small groups morphology – in
LBC.
• Glandular abnormalities – misdiagnosed as severe
squamous dyskaryosis.
• Thinprep – small groups with radial arrangement of
the cells with occasional feathering.
• Surepath – single abnormal cells are more common.
• Criteria for the no. of abnormal cells in LBC have not
been separately mentioned.

75. Trichomonas vaginalis ( LBP , SurePath ): a 32-year-old woman with
vaginal discharge.The organism’s nucleus, cytoplasmic granules, and fl
agella ( right ) may be better visualized on liquid-based preparations.
Note the kite shape and granules ( bottom right inset )

76. Candida species ( CP ): Torulopsis . Routine screening of a 63-year-old woman. Fungal organisms morphologically consistent with
Candida glabrata (previously known as Torulopsis glabrata ). Note clear halos surrounding the yeast forms ( left ). Bacteria, not
pseudohyphae, are also seen in the background. This organism does not form pseudohyphae and may be pathogenic in
immunocompromised individuals

77. Bacteria – coccobacilli ( LBP , SurePath ). Shift in flora suggestive of bacterial
vaginosis. In a twenty-five-year-old woman.
Note clue cell and the relatively clean background compared to that in CPs below

78. Bacteria morphologically consistent with Actinomyces ( LBP , ThinPrep ). Note that the clumps of protein usually
seen in conventional preparations tend to be washed away in liquid- based preparations leaving only fine thin
bacterial filaments. These are much thinner than the pseudohyphae of Candida spp
Bacteria morphologically consistent with Actinomyces ( CP ). Forty-one-year-oldwoman. Low power shows
“cotton ball” appearance of tangled clumps of filamentous organisms. An acute inflammatory response is
also seen.

79. Cellular changes consistent with herpes simplex virus ( CP ). Note the eosinophilic intranuclear “Cowdry
A-type” inclusions. The “ground-glass” appearance of the nuclei is due to accumulation of viral particles
leading to peripheral margination of chromatin. The inset shows a SurePath liquid-based
preparation with a typical multinucleated herpetic cell showing “ ground- glass” appearance of the nuclei.

80. Appearances considered inadequate for interpretation
Before the lab -Cervix not fully visualized or
sampled
-No brush in vial(sure path)
-Brush left in vial(thin pep)
No endocervical cells Only a cause of inadequacy in women
who were treated or CIN-3 with
endocervical margin involved.
Obscured by blood or polymorphs Extremely rare on LBC
Contamination Use of inappropriate lubricant.

81. ADVANTAGES DISADVANTAGES
More representative sample with random
distribution cells
Preparation is more labor intensive than
conventional
Fewer artifacts in cellular morphology Rounding up of nucleus leading to altered N:C
examples: cytolysis, atrophic changes
Multiple slides and/or ancillary tests possible Requires training for both the screeners and
the smear takers
Cells better preserved and not obscured by
blood, mucus, or inflammatory cells
Screening area reduced & cells in one 10x
focal plane  reduced screening time
Cell nuclei more often take a vesicular delicate
appearance
Both screening and storage can be done More costly than conventional pap smears

82. Future advances
• HPV testing on the same sample.
• Automation – subjective nature of morphological
interpretation – replaced by numerical
quantitative process which was not open to any
ambiguity.
• Computer-assisted Pap test screening detects
cervical cell abnormalities by having a computer
analyze every square millimeter of a Pap test
slide.
• There are currently two such devices that are
FDA-approved and in use in theUnited States.

83. • FocalPoint device (Becton,
Dickinson and Company, Franklin
Lakes, NJ).
• ThinPrep Imaging System (Cytic
Corporation,Boxboro, MA)
• The imaging system scans a slide
and selects 22 areas on the slide
that are the most worrisome for a
squamous intraepithelial lesion
(SIL). And 20 worst cells and 2
worst cell groups are identified.
• Automated stage is used to take
the screener to the identified
feilds, allowing a pathologist to
review those 22 areas and decide
if abnormal cells are present or
not.

84. • The FocalPoint system works in a
different manner and is called a
‘‘primary screening’’ instrument.
• It scans SurePath slides, as well as
conventional slides, but is not
approved for ThinPrep slides.
• At the end of the computer
scanning a given set of slides, it
can declare up to 25% of the
slides in the set to be normal and
need no further review and can
be auto-archived (or filed away).
• The remaining 75% of Pap test
slides in a given set (considered
the ‘‘most abnormal’’) are ranked
into quintiles from 1 to 5 (number
1 quintile being the most
abnormal)

85. • And receive a score related to the percent chance of having an
abnormality compared with the other Pap slides in that set.
• This quintile and percentage information is given to the pathologist who
then completely screens the slide as usual.
• Because of the computer’s prescreening and ranking capabilities, review
of slides by pathologists can pay additional attention to slides considered
at higher risk for abnormalities.
• This allows the reassurance of all Pap tests being reviewed at least twice:
by both a computer and one or two human beings.

86. Summary
• LBC represents the first major technological
change in the cervical cytology.
• It is as effective as conventional cytology for the
detection of high grade abnormalities.
• Offers improvements by decreasing rates of
inadequacy and improving screener productivity.
• Two systems have been approved for use.
• It provides a platform for future advances in
cervical screening.

87. References
Recent advances in
histopathology –Vol 22.
Cytologystuff.com

88. References
• Denton, K.J., 2007. Liquid based cytology for cervical cancer screening. Recent
Advances in Histopathology: 2007(1); pages 127-144
• Simion N, Caruntu I, Avadanei E, Balan R, Amalinei C. Conventional cytology versus
liquid based cytology in cervical pathology: correspondences and inconsistencies
in diagnosis, advantages and limits. Rom J Morphol Embryol. 2014; 55 (4): 1331-
1337
• Mulford DK. Alternatives to the traditional Papanicolaou (Pap) smear - liquid
based cytology (LBC). Lab Med 2003;34:129–131.
• Dhananjaya, C. and Kumari mK, K., Comparison of Manual Liquid Based Cytology
and Conventional Pap Smear in Cervical Cancer Screening. National Journal of
Laboratory Medicine. 2017 Apr, Vol-6(2): pages 32-37
• Denton, K.J., 2007. Liquid based cytology in cervical cancer screening. BMJ: British
Medical Journal, 335(7609), p.1