Liquid based cytology ( l b c)

Cervical cytology was introduced by George
Papanicolaou into clinical practice in 1940. In 1945,
the Papanicolaou smear received the endorsement of
the American cancer society as an effective method
for the prevention of cervical cancer .
www.drvikramsaraswat.co.in
www.drsaraswatpathlabs.com

1. LIQUID BASED CYTOLOGY Presented by Dr. Vikram Saraswat Moderator- Dr. Abha Mathur

2. Introduction Cervical cytology was introduced by George Papanicolaou into clinical practice in 1940. In 1945, the Papanicolaou smear received the endorsement of the American cancer society as an effective method for the prevention of cervical cancer . Center of cytology in Vancouver, British Columbia published data which confirmed that cytologic screening leads to a reduction in the rate of invasive cancer of the uterine cervix. It was later established that the sensitivity of the conventional Pap smears for the detection of cervical cancer precursors was less than 50%. Several limitations of conventional smear were identified. Thus Liquid based, thin layer technology was developed to address the limitation of Pap smear.

3. Limitations of conventional pap smear Inadequate samples constitute about 8% of the specimens received. False-negative results as high as 20-30% have been reported, which occurred due to clumping of cells when the cells are not uniformly spread on the glass slide. Sometimes, other contents of the cervical specimen such as blood, mucus, bacteria and yeasts contaminate the sample and prevent the detection of abnormal cells. If exposed to air for too long before being fixed on the slide, cervical cells can become distorted. Human error is probably the primary threat to accurate interpretation. An average Pap smear slide contains 50,000-300,000 cells that must be examined and if the sample contains only a few abnormal cells within a crowded background of healthy cells, the abnormal cells may be missed. Due to limited capacity, long back-logs for reporting cervical cytology were often seen. An automated system was needed to improve screening productivity and reduce manual workload.

4. LIQUID BASED CYTOLOGY ( L B C) It is a method for preparing cytological specimens—in particular from the cervix—for microscopic evaluation in which the patient specimen is suspended in a liquid, which is used to produce a thin layer of cells. Traditional Pap smears are conducted by physicians who use a spatula to collect cervical cells for analysis. A small sample of cells is lightly scraped from the cervix, placed on a microscope slide, and sent to a laboratory for staining and analysis by a cytologist . Unlike a traditional Pap smear, where the cells are placed directly on a microscope slide, in liquid based cytology the head of the spatula is detached and placed into a vial containing a preservative fluid. Most liquid preservatives for liquid based cytology are ethanol-based

5. Liquid based cytology has become increasingly more common in hospitals and clinics since the 1990s. The centrifuging process allows for clearer, more uniform samples that are easier for cytologists to analyze. This has also helped to reduce the number of unreadable or inconclusive tests, requiring fewer women to return to clinics for repeat testing. Liquid samples are also more suitable for human papillomavirus (HPV) testing. Some studies also indicate that liquid based cytology increases the sensitivity and specificity of cytological reports, making the Pap smears more effective in detecting cancers and other diseases

6. Currently, two technical methods, which use automats, were validated by Food and Drug Administration ( FDA,america ) and are used frequently. One is proceeding by fil tration and collection of vacuum-packed cells on a membrane and transferring to the glass slide ( ThinPrep ®, Cytyc ®) . The other is proceeding by centrifugation and sedimentation through a gradient of density ( Surepath ®, Tripath Imaging®, AutoCyte PREP) ® Stands for registered trademark names

7. Methods Not approved by FDA Cytoscreen Turbitec Cellslide ® Papspin ® Centrifugation and sedimentation are performed by manual techniques in these methods, no automate is used.

8. Advantages of LBC Simplifies the collection process for the smear taker Reduces inadequate rate Improves cellular preservation All the collected cells are transferred to the vial Quicker for the laboratory to screen and report as the area to be screened is smaller and circular. Multiple slides can be produced allowing further testing or for teaching and Quality Assurance purposes Residual material in the vial could be made available for associated tests e.g. HPV assay Facilitates Computer Assisted Screening which may also be available in the future Enhances screening processes by improving and standardising the service to the patient

9. Disadvantages of LBC -More expensive than conventional cytology -Cytological interpretation differs slightly from conventional slides with resultant training issues for laboratory staff - Smear taking procedures require a standard approach -Transport of the vials requires a more specialised approach with specialised packing

10. The limitation of conventional pap smear

11. In LBC -

12. With the conventional Pap smear method, cells can be obscured by blood, mucus, and inflammation

13. The LBC method preserves the cells and minimizes cell overlap, blood, mucus, and inflammation.

14. The ThinPrep ® System

15. History of Thin Prep method Cytyc Corporation is a company engaged in the design, development, manufacture, and marketing of clinical products that focus on women’s health since 1987. In order to overcome limitations on conventional pap, the company introduced the ThinPrep Pap Test. In May 1996, the Food and Drug Administration (FDA) in the USA approved the ThinPrep 2000( T 2000 ) as a replacement for the conventional Pap smear method in USA for use in screening for the presence of atypical cells, cervical cancer or its precursor lesions (Low Grade Squamous Intraepithelial Lesions, High Grade Squamous Intraepithelial Lesions), as well as all other cytologic categories

16. method The heart of the ThinPrep ® System is the ThinPrep 2000 Processor, a semi automated slide preparation unit that produces remarkably uniform thin-layer slides, virtually free of obscuring artifacts such as blood, mucous and inflammation. Step 1 A gynecologic sample is collected using a broom-type or cytobrush /spatula cervical sampling device.

17. Step 2 Instead of smearing the cells on a slide, the sampling device is rinsed into a ThinPrep vial containing PreservCyt ® transport medium(buffered methanol soln ). The device is then discarded Step 3 The sample vial is capped, labelled , and sent to the laboratory for slide preparation.

18. Step 4 At the laboratory, the vial is placed into the ThinPrep 2000 Processor. First, a gentle dispersion step breaks up blood, mucous, non-diagnostic debris, and then thoroughly mixes the sample. A negative pressure pulse is generated which draws fluid though a TransCyt ® Filter( a micropore filter) that collects a thin, even layer of diagnostic cellular material. The ThinPrep 2000 Processor constantly monitors the rate of flow through the TransCyt Filter during the collection process to prevent the cellular presentation from being too scant or too dense. The filter is then removed and dabbed onto an electrically charged slide causing cells to transfer onto the glass slide. This is then stained in a separate process using same staining machine as is used for conventional samples.

19. The gentle mixing of the sample before preparing the slides ensures a thorough sampling of the cells present. As part of the validation of the ThinPrep Pap Test, ten or more slides were made from single vials. These studies showed that the first slide prepared from the vial is representative of the remainder of the material . With a conventional Pap smear there is tremendous variability in the number of cells transferred to the slide, from as few as 4,000 to up to 300,000. With the ThinPrep Pap Test, approximately 70,000 diagnostic cells are collected that provide a more representative sampling of the specimen taken from cervix. These are consistently displayed to the cytologist in a high quality preparation Currently a fully automated T3000 system is being used UK and USA, it readily converts vials into slides ready for staining with no intervention.

20. Basic principle of thin prep processor (T3000 fully automated) The ThinPrep Processor makes use of mechanical, pneumatic, and fluidic principles for cell dispersion, collection, and transfer. A rotary drive mechanism gently disperses samples. A pneumatic/fluidic system, controlled by a microprocessor, monitors cell collection. Electrochemical principles, the pneumatic and fluidic systems, the natural binding qualities of cells,and the qualities of the ThinPrep Pap Test Filter are responsible for cell transfer. The ThinPrep Processor slide preparation process can be divided into the following phases Sample preparation/Instrument loading Start of cycle Fluid level detection Dispersion Filter wetting

21. • Cell collection • Waste clearing • Bubble point • Cell transfer • Slide ejection • Completion of cycle Sample Preparation/Instrument Loading In preparation for sample processing, the operator loads four essential items into the ThinPrep Processor: a Sample vial, a ThinPrep Pap Test Filter attached to the filter cap, a ThinPrep Slide and a fixative vial containing a standard laboratory fixative . Start of Cycle When the operator initiates a sequence, the ThinPrep Processor verifies installation Fluid Level Detection The cap seal lowers to seal the filter assembly and the sample vial is raised towards the filter membrane. The sample vial stops when the filter membrane makes contact with the surface of fluid.

22. Dispersion The cap seal lifts and the dispersion system rotates the ThinPrep Pap Test Filter assembly within the cell suspension, creating shear forces in the fluid that are strong enough to separate randomly joined material and disperse mucus. Filter Wetting The head seal lowers to seal the filter assembly. Negative pressure is briefly applied, drawing a small amount of fluid through the ThinPrep Pap Test Filter to wet it. Following wetting, the system gently blows out the liquid in the ThinPrep Pap Test Filter. This clears any cellular material from the filter surface. Cell Collection The filter membrane is biologically neutral and is mounted at one end of the ThinPrep Pap Test Filter cylinder. The membrane is a flat, smooth, porous surface that collects the cellular material on one plane. The pneumatic system applies negative pressure to the filter in a series of pulses. These negative pressure pulses (sips) draw PreservCyt Solution through the filter membrane and collect suspended cellular material onto the outer membrane surface. The collection process ceases when a target filter coverage, predetermined by the processor sequence, is attained. Cell collection is controlled by an embedded microprocessor that monitors the pressure in the ThinPrep Pap Test Filter cylinder. After collection, the cells sit on a single plane over the pores, ready for transfer to the slide.

23. Waste Clearing When collection ends, the ThinPrep Pap Test Filter is withdrawn from the sample vial and the filtrate is aspirated into the waste bottle as the filter is inverted. The collected cells remain on the ThinPrep Pap Test Filter due to the negative holding pressure. Bubble Point Bubble point removes excess fluid from the filter membrane prior to transferring cells onto the slide to enhance cell adhesion to the slide. Bubble point is performed after all of the fluid is evacuated. This is evident by the bubbling activity on the inside of the filter membrane. Cells do not air-dry during bubble point . Cell Transfer When bubble point is complete, the slide handler moves the slide into contact with the inverted ThinPrep Pap Test Filter . The natural adhesion properties of cells and the electrochemical charge of the glass slide are responsible for the transfer of cells from the filter membrane to the slide. The cells have a higher affinity for the glass slide than for the membrane; slight positive air pressure behind the filter membrane enhances cell transfer.

24. Slide Ejection Once cell transfer is complete, the slide is removed from contact with the filter and automatically ejected into the fixative vial. Cycle Completion All the motorized mechanisms return to their initial positions and the display returns to the Main Menu. If the system detects an error during the process, a message will be displayed and an audible alarm will sound

25. The prepared ThinPrep smear is shown in the diagram compared with the conventional Pap smear. The sample prepared by the ThinPrep is evenly distributed on the circle of the slide

26. Microscopically, the uneven distribution of cellular material associated with the Conventional Pap Smear pattern is evident.

27. This slide is the from the same patient as the previous slide. Tissue architecture is maintained. ThinPrep ® rearranges the relationship of cell groups on the glass slide. A group/sheet of endocervical cells present represents this.

28. THIN PREP IMAGING SYSTEM (recent advancement)

29. The ThinPrep Imaging System is the only system that provides Dual Review screening. With this capability, every slide is analyzed by the ThinPrep Imager and screened by an experienced cytotechnologist, thereby combining human expertise with advanced technology to achieve more accurate clinical diagnoses. the ThinPrep Imager scans every cell and cell cluster on the slide, measuring DNA content. The largest and darkest nuclei are identified so that cells can be more accurately assessed for abnormalities by highly skilled cytotechnologists. Cells of interest are highlighted for cytotechnologists' review, helping them to better focus their interpretive skills where it counts most The area of interest is shown in the "crux" of the L-shape when identified by the ThinPrep Imager

30. A comparison of disease detection with the ThinPrep Imaging System vs. manually reviewed ThinPrep Pap Test slides reported a 37% increase in LSIL detection and a 42% increase in HSIL detection with the ThinPrep Imaging System The ThinPrep Imaging System showed a 67% decrease in unsatisfactory and a 15% decrease in ASC-US rates compared to manually reviewed ThinPrep Pap Test slides with 50% reduction in false negative fraction

31. SurePath ™ Liquid-Based Pap Test ( AutoCyte PREP™ System) ( cytorich /prep stain slide processor)

32. Pap Smear Specimen Collection Using SurePath Pap Test Method Option 1: SurePath Sample Collection with Broom-Type Device with Detachable Head 1. Insert the cervix-brush into the endocervical canal. Apply gentle pressure until the bristles form against the cervix. Maintaining gentle pressure, hold the stem between the thumb and forefinger. Rotate the brush five times in a clockwise direction. 2. Place your thumb against the back of the removable collection device tip and disconnect the entire tip from the stem and place in the SurePath preservative vial. 3. The collection device tip should be transferred in the vial. One to three different sampling tips can be left in the SurePath vial. Place the cap on the vial and tighten. 4. Record two patient identifiers, physician name and collection date on the vial. Also record the patient information and medical history on the requisition form. 5. Complete a laboratory requisition form with complete patient information and medical history. 6. Place the vial and requisition in a specimen bag for transport to the laboratory.

33. Option 2: SurePath Sample Collection with Combination Brush/Plastic Spatula Device with Detachable Heads 1. Insert the contoured end of the plastic spatula and rotate 360 degrees around entire exocervix . 2. Snap the device handle and drop the detachable head of the device into the SurePath vial. 3. Insert Cytobrush into the endocervix until only the bottom-most bristles are exposed at the os . Slowly rotate one-fourth to one-half turn in one direction. To reduce unnecessary bleeding, do not over-rotate brush. 4. Snap the device handle and drop the detachable head of the device into the SurePath vial. Place the cap on the vial and tighten. 5. Record two patient identifiers, physician name and collection date on the vial. Also record the patient information and medical history on the requisition form. 6. Complete a laboratory requisition form with complete patient information and medical history. 7. Place the vial and requisition in a specimen bag for transport to the laboratory

34. Collect using either a broom-like device or combination brush/plastic spatula. Drop the detachable head device(s) into the SurePath ™ test vial . Place the cap on the vial and tighten. Send the BD SurePath ™ test vial to the lab for processing

35. Method and principle The Autocyte Prep system/ Sure path method converts liquid suspension of cervical cell sample into a discretely stained homogenous thin layer of cells while maintaining diagnostic cell clusters. The process includes 1) cell preservation (preservative fluid is dil. Soln. of denatured ethanol) 2) randomisation 3)enrichment of diagnostic material 4) automated pipetting 5) sedimentation 6) staining

36. After collection and preservation, the sample is mixed by vortexing ( to re-suspend cells) and dispersed onto a density reagent (contains sodium azide ). An enrichment step consisting of centrifugal sedimentation through density reagent partially removes non diagnostic debris and excess inflammatory cells from sample. After centrifugation, the tube containing the enriched cellular component is placed onto the instrument where the pelleted cells are robotically suspended, mixed and transferred to settling chamber. Here a specialised slide coat is applied to enhance cell adhesion. Cells are sedimented by gravity and then stained using modified papanicolaou staining process. The slide is cleared in xylene and coverslipped .

37. trapped debris SurePath ™ test enriched cell pellet

38. The final product is a well preserved population of stained cells present within 13mm diameter circle on slide. Air drying artefact , obscuring , Overlapping cellular material and debris are largely eliminated. The number of white clood cells is reduced allowing easier visualisation of epithelial cells, diagnostically relevant cells and infectious organisms.

39. AutoCyte PREP System can also be used for preparing non- gynaecological specimens including bronchial and fine-needle aspiration specimens. Stained cytology smears of bronchial specimen (prepared by AutoCyte PREP System)

40. FOCAL POINT SLIDE PROFILER FOCAL POINT GS IMAGE CAPTURER (recent advancement)

41. Focal point slide profiler , an automated screening system, scans slides prepared by SUREPATH method and can also be used on conventional smears. It identifies cells which are potentially abnormal and then ranks the whole sample on its likelihood of being abnormal. This is the only fully automated computerised slide reading application that has been given FDA approval and is in routine use in USA. The other development is the Focal point GS system that captures images,records the location of potentially abnormal cells and then via an automated microscope stage,guides the screener to 10 selected locations. This method has not yet received approval as it has always been recommended that a screener must screen the whole slide.

42. THINPREP v/s SUREPATH

43. THIN PREP v/s SUREPATH sample collection THIN PREP (broom rinsed in the vial) SUREPATH (broom head snapped off And retained in the vial)

44. THIN PREP v/s SUREPATH reception and numbering One of the advantages of the THIN PREP system is that it automatically prints a bar code onto the slide, thus making it a very secure process. With SUREPATH, it must be ensured that samples are labelled correctly throughout the process. Staff receiving specimens must ensure the correct vial and form are matched ( a process analogous to histology specimen reception)

45. Preparation technique thinprep v/s surepath THINPREP PreserveCyt fluid Methanol No vortexing No gradient centrifugation Filter No sedimentation SUREPATH CytoRich fluid Ethanol Vortexing Gradient centrifugation No filter Sedimentation

46. Staining thinprep v/s surepath With Thin prep, slide preparation is automated and yields an unstained slide which can be inserted into a range of automated staining machines. With Surepath , staining is an integral part of the process. The Prep stain processor ( trade name), employed in surepath technique, has higher capacity than Thin prep T3000 ( it produces 48 slides per hour), but it is not fully automated and reuires more skilled manual operation.

47. 13mm 20mm SUREPATH THIN PREP Result -

48. thinprep v/s surepath screening At primary screening, the two methods do have different appearances. The cells in THINPREP are more widely spaced and there is less depth to the field. Glandulare abnormalities show smilar nuclear features but architecture and distribution exhibit significant differences. In THINPREP, the characteristic appearance are of group of cells showing radial alignment of cells with occasional feathering. Palisaded strips with nuclei at multiple levels are seen and occasionally micro acinar structures such as rosettes are visible. Single cells are rarely seen. On SUREPATH, though groups of cells are seen,single abnormal cells are also common. Cells show abnormal nuclei distending the cytoplasm. These cells may be arranged in strips which often show a shared or community border.

49.   Cell block preparation from   SurePath specimen containing glandular epithelium

50. Other LBC methods Though not as popular and effective as the previously mentioned methods, the CYTOSCREEN and CELL SLIDE PROCESSOR are other two notable LBC techniques.

51. CYTOSCREEN method In this method after sample collection,the brush is sunk in a preservation liquid vial. The following stages can be mentioned: – the vial with the removed device is placed in a shaker to take the cells out of the removing device and get an homogenous cell suspension. – automate reading of the cell sample density by using a nephelometer – cells deposition in after centrifugation, resulting a round shaped smear with a diameter of 17 mm.

52. Cytoprep brush and vial with conservation liquid shaker Nephelometer

53. Cell slide processor

54. CELL SLIDE PROCESSOR CellSlide Processor is an automatic computer-controlled instrument, designed to prepare standardized, thin-layer cytological preparations using a filtration process. The processor filters cells from a liquid fixative suspension and transfers them onto a rectangular area of the microscope slide. The instrument operates using membrane filters in a closed circuit system that offers operational security with all types of specimens. All phases of processing, including filtration, residual fluid recovery, membrane transfer and pressure are automatically performed. The instrument is equipped with two filtering towers, which can be operated either independently or in parallel.

55. From a diagnostic standpoint, it is preferable to use a fresh specimen, since cytological samples may lose their integrity if they are processed when a long period of time has elapsed between sample collection and slide processing. However, the CellSlide Pap test solution, a buffered, alcohol-based preservative solution, has been designed to maintain cell integrity from the time of sample collection until the slide is processed (maximum 4 wk) at temperatures between 4 and 30 degree C. Also, a second fixative solution, CellOfix is automatically added to the sample before filtration. This fixative solution contains a polymeric agent with a high viscosity power, dissolved in an alcoholic solution containing colouring agents. The purpose of this agent is to embed the cells,to increase fixative power during filtration and to facilitate the long-term storage of the specimen. The purpose of the colouring agents is to differentiate the processed samples from the non-processed preservative solution. CellOfix is delivered in a concentrated form, which is then diluted in ethyl alcohol or isopropyl alcohol .

56. LBC v/s conventional pap major differences

57. Conventional Pap Smear Liquid Based Cytology Majority of cells not captured. Non-representative transfer of cells. Clumping and overlapping cells. Obscuring material. Virtually all of the sample is collected. Randomized, representative transfer of cells. Even distribution of cells. Minimizing Obscuring material. The difference is obvious

58. LBC v/s CONVENTIONAL PAP screening and morphology Using LBC, modifications must be made to the conventional screening method. The area to be screened is smaller and circular, thus screening must cover 100% of the deposit and should be intensive. There are no breaks in the sample, as there are in conventional smears, meaning that the screener must conentrate throughout the process. The prompt fixation of LBC samples leads to good preservation and this is particularly seen in the clarity of presentation of chromatin. Nuclear membranes are also well visualised . In general, cells appear slightly smaller due to rounding up effect of fixation in a liquid. The clarity of nuclear features is helpful in diagnosing dyskaryosis but training needs to address the tendency to overcall dyskaryosis as subtle variations in chromatin and membrane pattern, invisible on conventional smears, can now be seen .

59. Cells showing Low grade dyskaryosis , esp. those with koilocytosis , are usually easy to find and look identical to similar cells in conventional smear. High grade dyskaryosis (moderate and severe) often appears similar to that seen in conventional smear. In LBC , sometimes severe dyskaryosis may present as dispersed single cells, a recognised but less frequent feature on conventional smear. Hyperchromatic crowded cell groups are a problem both in conventional and LBC cytology, but in latter the size of groups is reduced and in most cases even if grop is too dense to assess nuclear features,the cells on the edge are fully assessable. The improved nuclear clarity also helps in differentiation of benign and abnormal conditions.

60. Future of LBC LBC represents the first major technological change in cervical cytology. Various studies have shown it to be more effective than conventional smear in detecting abnormalities, decreasing inadequacy rates and improving screener productivity. It provides platform for future advances in cervical screening which includes HPV testing and introduction of automated cervical screening system.

61. Thank you