This document discusses cell blocks, liquid based cytology, and the Bethesda system for reporting cervical cytology. It defines cell blocks and describes their advantages like allowing histological examination and ancillary tests. It outlines the advantages and disadvantages of liquid based cytology compared to conventional pap smears. Finally, it explains the Bethesda system for standardized reporting of cervical cytology results, including categories for negative, epithelial abnormalities, organisms, and other non-neoplastic findings.

2. Points to be discussed
• Cell block- its advantages/disadvantages and
preparation methods and applications.
• Liquid based cytology-its uses/disadvantages.
• Role of liquid based preparation in cervical
cytology
• Bethesda system for reporting cervical cytology

3. What is a cell block????

4. Salvaging of grossly visible flecks of tissue or sediment
Fixation by formalin or any other fixative
Paraffin embedding
Cutting
Staining

5. Cell blocks offer the opportunity to examine the
histological structure and allows the use
of ancillary tests.
Cytology Histopathology
Bridge
Cell blocks

6. Advantages of cell block
Slides are more readily interpretable by histopathologists
Availability of a block facilitates more sections.
Imp residual material is salvaged which is generally not
available in cytology smears .Loose cells, cell aggregates
and microscopic tissue fragments are easily recoverable.
Concentrated in a small area of the slide so examination
less time consuming
Special stains mucicarmine, congo stain, melanin etc

7. Stains for immunocytochemistry
For microorganisms esp fungi and bacteria
Pattern and architectural recognition of tumor possible
CB is simple ,reproducible and readily available in
routine laboratory
No necessity of biopsy
Storage of cell blocks is easy than unstained slides

8. Disadvantages
Compared routine smears takes longer
time
Sparse cellularity
Distortion artifacts

9. Specimen
FNA material
Sputum
Effusion fluids
Urine sediment
Various washings and lavages

10. Material needed

11. Methods of cell block preparation
Direct processing of tissue fragments present in
fluids
Fixed sediment method
Bacterial agar method
Simplified cell block technique using alcohol,
acetone and paraffin
Compact cell block technique
Plasma thrombin method
Cell blocks from millipore

12. Histogel method
Gelatin embedding
Celloidin bag
Cell block preparation from scraping of
cytology smears
Automated cell block preparation
Albumin method

13. .

14. Residual clot /tissue in the hub of the needle is carefully
removed & rinsed in 50% ethanol
Centrifuged at 4000rpm for 6min in a 10ml
disposable to form pellets
Supernatant is discarded & fixative is added to the
deposit
After 1 hr deposit is taken and put into filter paper
& processed as regular paraffin specimen and then
stained routinely by H&E

18. • Cytologic studies of body fluids are done to gain
knowledge about the etiology of effusion.
• Most common reason for evalaution is to
determine whether or not it contains malignant
cells.
• Cytologic evaluation is the best method to detect
malignancy in body cavity fluids
• Diagnostic accuracy for malignancy ranges
from50-80 % with a false negative rate of about
25%, to increase diagnostic accuracy, cell block
preparation is done
Cell blocks in body fluids

19. Few advantages:
Spontaneous formed clot in the body fluid may enmesh
virtually all cells
Induced clot prepared from the sediment obtained by
centrifugation of the fluid specimen would also contain
many cells .Useful in haemorrhagic fluids
Identification of primary site of malignancy can be
enhanced with the use of cell block technique
Histologic patterns of cancer(acinar ,papillary ,duct
like)can be readily identified on cell block.

20.  Psammoma bodies ,granulation tissue ,cholestrol clefts
,microorganisms, fragments of collageous stroma,
hyperpalstic mesothelium,etc can be easily identified
easily.
 Fluid samples or tissue fragments can be preserved in a
refrigerator for 72 hrs before processing in cell blocks.
 IHC on cell blocks are comparable to that of surgical
specimen
 Microarray technique, Molecular tests like FISH ,In situ
PCR.
 Electron microscopy.

21. Role of cell block in non
neoplastic effusion
 Mesothelial cells are often misinterpretated as
metastatic adenocarcinoma
Differentiation between polymorphous lymphocytes of
non neoplastic effusion & monomorphous lymphoid cells
of lymphoproliferative diseases
Rheumatic effusion(elongated & giant cell
multinucleated histiocytes ,granular background ,
cholestrol crystals)
L E cells, granulomas in TB, number of parasitic, fungal
and viral diseases can be picked up

22. Cell blocks in neoplastic effusion
Adenocarcinoma
Signet ring cell carcinoma
Small cell anaplastic carcinoma
Mesotheliomas

28. Cell block is simple, reliable technique suitable for all
types of cytology specimens & does contribute to better
final cytological diagnosis and so should be considered
wherever possible.

29. Liquid Based
Cytology

30. Liquid based cytology
It means cytology (study of cells )through a liquid medium.
Cells are collected from cervix(any other site) are placed
directly into liquid preservative, rather than transferred to
slide.
Sample is processed and resultant thin smear easy to
screen.

31. Samples to Process
• Exfoliative samples
• Urines
• Sputum
• Bronchial washings
• Bronchial brushings
• Body fluids
• Aspiration samples
• FNA Head and neck
• FNA Lymph nodes
• FNA Lung
• FNA Liver
• FNA Breast

32. Advantages
l
Simplifies the collection process for the smear taken
Reduced inadequate rate
 Cells better preserved and not obscured by blood,
mucus, or inflammatory cells
 Infectious organisms retained and better preserved
Quicker to screen and report
Multiple slides can be produced allowing further
testing
Residual material in the vial can be made available
for further tests eg; HPV tests
Facilitates computer assisted screening which can
be available in future(automated cytology)

33.  More costly than conventional pap
smears
 Preparation is more labor intensive than
conventional
 Loss of background material
 Some differences in architecture and
morphology
 Requires training for both the
screeners and the smear takers
Disadvantages

34. Conventional Pap Smear
Overcoming the Inherent Limitations
of the Conventional Pap Smear
 Majority of cells not captured
 Non-representative transfer
of cells
 Clumping and overlapping of
cells
 Obscuring material
 Virtually all cells of sample are
collected
 Randomized, representative
transfer of cells
 Even distribution of cells
 Minimizes obscuring material
Liquid-based Cytology*

35. Conventional smear

36. What we see under the
microscope conventional
smear

37. The prepared slide with the new
ThinPrep

38. What we see under the
microscope. Notice the clean back
ground and how well the cells are
dispersed rendering easier to
interpretation.

39. Liquid based cytology techniques
Thin Prep:
Filtration and collection of vacuum packed
cells on a membrane & transferring to
glass slide
Sure Path:
Centrifugation & sedimentation through a
density gradient

40. Thin prep
Path sure

41. Thin prep

42. 1. Dispersion 2. Cell Collection 3. Cell Transfer
Principle

44. Path sure

45. Morphology
•Clean background
•Cells are shrunken ,more circular
•Cytoplasmic and nuclear features are more easily identified
•Cells with Low grade dyskaryosis ,especially koilocytosis
•Lower nuclear hyperchromasia
•Glandular neoplasms show characteristic picture in thin
film cytology

46. Adequacy
• Conventional pap:
8,000-12,000 squamous cells
• Liquid based cytology:
5000 squamous cells

48. Screening methods
• Area to be sceened is smaller & circular.
Abnormal cells may be located anywhere and it
should cover 100% of deposit & allow for overlap
• Screening is more intensive, 10 x and 40 x
• Screener – must take proper breakes not
exceeding 5 hrs/day.

49. Bethesda System 2001
Nomenclature of cervical cytology on pap
test.

50. Bethesda System 2001
Specimen Type: Indicate conventional Pap smear vs. liquid-based
vs. other
Specimen Adequacy
• Satisfactory for evaluation (describe presence or absence of
endocervical/transformation zone component and any other quality
indicators--e.g., partially obscuring blood, inflammation, etc.)
• Unsatisfactory for evaluation (specify reason)
– Specimen rejected/not processed (specify reason)
– Specimen processed and examined, but unsatisfactory for
evaluation of epithelial abnormality because of (specify reason)

51. General Categorization
• Negative for intraepithelial lesion or malignancy
• Epithelial cell abnormality: See interpretation/result (specify
“squamous” or “glandular” as appropriate)
• Other: See interpretation/result (e.g., endometrial cells in a woman
40 years of age)
Automated Review: If case examined by automated device,
specify device and result
Ancillary Testing: Provide a brief description of the test methods
and report the result so that it is easily understood by the clinician
Bethesda System 2001 (continued)

52. Interpretation/Result
• Negative for Intraepithelial Lesion or Malignancy (when there is no
cellular evidence of neoplasia, state this in the General Categorization
above and/or in the Interpretation/Result section of the report, whether
or not there are organisms or other non-neoplastic findings)
Organisms
– Trichomonas vaginalis
– Fungal organisms morphologically consistent with Candida spp
– Shift in flora suggestive of bacterial vaginosis
– Bacteria morphologically consistent with Actinomyces spp.
– Cellular changes consistent with Herpes simplex virus
Other Non-Neoplastic Findings (optional to report; list not inclusive):
– Reactive cellular changes associated with
• Inflammation (includes typical repair)
• Radiation
• Intrauterine contraceptive device (IUD)
Glandular cells status post hysterectomy
Atrophy
Bethesda System 2001 (continued)

53. Bethesda System 2001 (continued)
• Other (list not inclusive)
– Endometrial cells (in a woman 40 years of age)
(specify if ‘negative for squamous epithelial lesion’)
• Epithelial Cell Abnormalities
– Squamous Cell
• Atypical squamous cells
– Of undetermined significance (ASC-US)
– Cannot exclude HSIL (ASC-H)
• Low-grade squamous intraepithelial lesion (LSIL)
– Encompassing: HPV/mild dysplasia/CIN1
• High-grade squamous intraepithelial lesion (HSIL)
– Encompassing: moderate and severe dysplasia, CIS/CIN2 and
CIN3
– With features suspicious for invasion (if invasion suspected)
• Squamous cell carcinoma

54. Bethesda System 2001 (continued)
– Glandular Cell
• Atypical
– Endocervical cells (NOS* or specify in comments)
– Endometrial cells (NOS or specify in comments)
– Glandular cells (NOS or specify in comments)
• Atypical
– Endocervical cells, favor neoplastic
– Glandular cells, favor neoplastic
• Endocervical adenocarcinoma in situ
• Adenocarcinoma
– Endocervical
– Endometrial
– Extra uterine
– NOS
– Other Malignant Neoplasms: (specify)
* NOS = Not otherwise specified

55. The Bethesda System 2001
• LSIL = HPV / mild dysplasia / CIN1
• HSIL = moderate and severe dysplasia / CIS / CIN2 and
CIN3
• ASCUS = ASC-US (undetermined significance) or
ASC-H (cannot exclude HSIL)

56. Bethesda 2001 Changes
• “SBLB” eliminated
• Unsatisfactory: specimen rejected/not processed; or specimen
processed/examined, but unsatisfactory because of (specify
reason)
• WNL and BCC are now Negative for Intraepithelial Lesions or
Malignancy; includes BCC (e.g., organisms and reactive changes)
as descriptor only
• The multiple subcategories of ASCUS have been reduced to ASC-
US or ASC-H, with no other modifiers
• The subcategories of AGUS (now AGC) have been expanded to
allow for a more descriptive diagnosis of glandular abnormalities;
AIS is now a distinct subcategory

57. Unsatisfactory

58. Satisfactory

59. Actinomyces

67. ASC-US

68. Low grade-sqamous
intraepithelial lesion(LSIL)

69. High grade squamous
intrepithelial lesion(HSIL)

70. Squamous cell carcinoma
(keratinising)

71. Squamous cell carcinoma
(non keratinising)

72. Atypical endocervical cells Atypical endometrial cells

73. Adenocarcinoma
(endocervical)

74. Adenocarcinoma
(endometrial)

75. Adenocarcinoma
(extrauterine)

76. REPORTING FOR CERVICAL CYTOLOGY
Specimen adequacy
Presence or absence of TZ zone
Other quality indicators
Interpretation/Result
• Negative for intraepthelial lesion or malignancy: organisms (T.vaginalis,
Candida ,Actinomyces, Lactobacilli & bacterial vaginosis), other non
neoplastic findings (inflammation ,radiation changes, IUD), atrophy, glandular
cells
• Endometrial cells > 40 yrs of age
• Epithelial abnormalities
 Squamous cell
Atypical squamous cells:
ASC-US and ASC-H
LSIL(low grade squamous intraepithelial lesion)
HSIL(high grade squamous intraepithelial lesion)
SCC(squamous cell carcinoma)
 Glandular cells
Atypical : Endocervical, Endometrial, Glandular
Endocervical adenocarcinoma in situ
Adenocarcinoma: Endocervical, Endometrial ,extrauterine, NOS

78. References
• Bales CE .Cytologic techniques. In Koss LG, editor.Diagnostic
cytopathology and its histopathological bases,4th ed.Philadelphia: J B
Lippincort & company,1992,vol2p1472-74.
• Tapar M, Mishra RK et al. Critical analysis of cell block versus smear
examination in effusions .J Cytol 2009;26:60-64.
• Marluce Bibbo, Comprehensive cytopathology;2nd ed
.Saunders.1997.p.
• Massima Pignatelli, James C. E Underwood. Recent advances in
Histopathology.22.p127-144.
• Diane Solomon, Ritu Nayar. The Bethesda System for reporting
Cervical cytology.2nd ed.Springer.2004.