Liquid Based Cytology by Dr Prashant pachuari

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1. Liquid Based Cytology By Dr. Prashant Pachauri Second year resident Under the Guidance of Dr. Arohi Parekh Assistant Professor Department of Pathology GMC, Bhavnagar.

2. Automation Technology that helps the person to have the work without much human assistance. The automation in cytology laboratory includes: 1) Preparation and staining of smear including mounting 2) Scanning the whole slide and give preliminary report 3) Final diagnosis to correlate clinically and other investigations

3. Aims of Automation The primary purpose of automation include: To have well spread , debris-free, monolayer smear preparation To have automated staining and cover slipping Whole –slide scanning To give the task based information

4. Liquid Based Cytology Liquid based cytology technique is an important step in preparing cervical smear and other exfoliative cytology to overcome many limitations of conventional cytology. Conventional preparation of cervical smear was quick, relatively cheap technique. Conventional preparation may often be inadequate, non representative and may be obscured by blood, debris or may show air drying artefact

5. Collection procedure of LBC Sample is collected by cervical brush supplied by company ↓ The brush is gently dipped into vial containing preservative solution ↓ The sample is sent to laboratory for further processing ↓ The cells are made discrete , and a monolayer preparation is prepared

6. Commercially Available Products: FDA approved only two commercially available liquid based technologies: 1) ThinPrep (TP) Pap Test (1996) 2) SurePath (1999) Non FDA approved liquid based technologies: Cyto screen Turbitec Cellslide Papspin

7. ThinPrep Cells are collected by plastic spatula and transferred to transport media Processor- TP 5000 it can automatically process 1-20 samples in a batch within 45 minutes Principle steps of TP processing are: 1) Dispersion 2) Cell collection 3) Cell transfer

8. Dispersion: Sample is collected in 20 ml of PreservCyt sol for fixation ↓ A small hollow cylinder tube covered with a filter at one end is immersed in PreservCyt solution ↓ The tube is rotated rapidly to disperse the cells mechanically

9. Cell Collection: A continuous negative pressure is created through the cylinder to drain the fluid within the vial ↓ The cells are stuck on the surface of the filter ↓ The flow of the fluid through the filter is constantly monitor to get an optimal quantity of cells by adjusting negative pressure with inbuilt software

10. Cell Transfer: The cells are entrapped over the outer surface of the filter. The TP filter now automatically transfers the cells on the circular area of the glass slides. The slide is immediately dipped with fixative.

11. SurePath Test Processor- BD Totalys TM multiprocessors. It can process 96 vials in a batch in a time. The cells are collected with the help of a plastic device and transferred to transport fluid.

12. The basic principles of processing are Vortexing: Helps to disperse a cell mechanically Cell Enrichment: Subsequently PREPSTAIN density reagent is added to the sample and centrifuged. Supernatant is decanted by vaccum suction and the remaining fluid is again centrifuged to get a rich cell pellet. Resuspension: The cell pellet is revortexed to resuspend the cellular material and then transferred to PREPSTAIN slide processor. Density gradient sedimentation: The cells are sedimented to precoated slide with gravitational force in PREPSTAIN Settling chamber.

13. ThinPrep vs SurePath Techniques Features ThinPrep SurePath Collection PreservCyt fluid, methanol based Cytorich fluid, ethanol based Single cell preparation By rotatory filter within in the vial By vortexing Cell concentration By negative suction By density gradient Monolayered cells Present, good No monolayered cells Cell concentration Relatively less Good cell concentration

14. Comparison of LBC and Conventional preparation LBC Conventional PAP Sample collection Liquid preservative On glass slide Air drying No Yes at time Number of cells Much more and adequate Less Screening time Less More Background Clean May be obscured by blood, polymorphs and mucus Sensitivity High Low Further ancillary test Possible for HPV Not possible Cost of equipment High No cost Cost to the patient High Low Training Needs adequate training Routinely trained technician Monolayer Yes in ThinPrep No Automation Easy to implement Difficult

15. Advantages of LBC: Significantly reduces the load of unsatisfactory samples ThinPrep system is significantly more effective then the conventional PAP smear for the detection of LSIL and more severe lesions. HPV DNA testing performed on the residual liquid after liquid based cytology sample is extracted. Disadvantages of LBC: - Instrument is costly - Laboratory technician needs adequate training

16. Other uses of LBC: Non gynecological cytopreparation : Thyroid cyst fluid examination Oral pathology lesion Body fluids – pleural effusions, urine Brushing samples

17. Normal cells in cervical smear Superficial squamous cell Intermediate cell Parabasal and basal cell Endocervical cells Metaplastic squamous cell

18. Features Superficial Intermediate Parabasal Endocervical Cell arrangement Discrete Discrete Discrete Honeycomb shape Cell size Largest 30-40 μ Larger 30-40 μ Smaller 25-30 μ Smallest 20 μ Cell shape Polyhedral Polyhedral Round Round to columnar Nucleus Central pyknotic Vesicular to fine granular chromatin Round: occupies most of the cytoplasm and chromatin is finely granular Round, central to eccentric Cytoplasm Orangeophilic Pink to greenish Dense cytoplasm Vacuolated cytoplasm

19. Minimum Squamous Cellularity Criteria: LBP from a woman with cervix should have estimated of atleast 5000 well visualized / well preserved squamous or squamous metaplasia. This range is applied exclusively to squamous cells . Endocervical cells and completely obscured cells should be excluded. Women who have had chemo- radiation therapy , who are postmenopausal with atrophic changes or who are post hysterectomy may have sample with fewer then 5000 may be considered satisfactory.

20. Patient history must be taken into consideration in such cases. Sample less than 2000 cells however should be considered unsatisfactory in most circumstances. For Conventional smear: 8000-12000 squamous cell