Liquid based cytology

Liquid based cytology

1. Liquid based cytology By Dr. Varughese George Department of Pathology. MGMCRI DR. 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)

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

9. 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.

10. 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

11. 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).

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

13. 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.

14. 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

15. 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.

16. 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.

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

18. 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

19. 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%

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

21. 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.

22. 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.

23. Cervical brush used for LBCs.

24. 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.

25. 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

26. ThinPrep Pap Test slide preparation

27. 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.

28. 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.

29. Image analyser for thinprep

30. 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.

31. 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

32. Conventional Pap Smear ThinPrep Pap Test

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

34. 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.

35. 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.

36. 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).

37. SurePath SurePath liquid-based collection media                   Cervex Brush (Broom)

38. 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.

39. 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.

40. 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.

41. 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

42. SURE PATH

43. 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.

44. SURE PATH

45. Special Focus on CIN Lesions where LBC has advantage

46. Special Focus on CIN Lesions where LBC has advantage

47. Special Focus on CIN Lesions where LBC has advantage

48. Special Focus on CIN Lesions where LBC has advantage

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

50. 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.

51. Conventional smear V/S LBC

52. Conventional smear V/S LBC

53. Conventional smear V/S LBC

54. Conventional smear V/S LBC

55. Conventional smear V/S LBC

56. 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.

57. Conventional smear V/S LBC

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

59. 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)

60. 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.

61. Thin prep smear Surepath smear

62. 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)

63. 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.

64. 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 )

65. 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

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

67. 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- old woman . Low power shows “cotton ball” appearance of tangled clumps of filamentous organisms. An acute inflammatory response is also seen.

68. 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.

69. 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.

70. 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

71. 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.

72. 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.

73. 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)

74. 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.

75. 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.

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

77. 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