Liquid Based Cytology.pptx

LIQUID BASED CYTOLOGY

1. LIQUID BASED CYTOLOGY BY DR. A. B. AJILEYE DEPARTMENT OF BIOMEDICAL LABORATORY SCIENCE, COLLEGE OF MEDICINE, UNIVERSITY OF IBADAN.

2. Introduction Liquid based cytology is a technique that enables cells to be suspended in a monolayer and thus making better morphological assessment possible with improved sensitivity and specificity. The sample is collected, normally by a small brush, in the same way as for a conventional smear test, but rather than the smear being transferred directly to a microscope slide, the sample is deposited into a small bottle of preservative liquid. At the laboratory the liquid is treated to remove other elements such as mucus before a layer of cells is placed on a slide. The technique allows more accurate results . To provide uniformity of the cell population in  each sample.

3. From recent research and development, liquid-based preparations out perform conventional smears because of improved fixation, decreased obscuring factors, and standardization of cell transfer. In direct smears, the cells are not transferred in a representative fashion and that up to 90% of the material scraped from the cervix may be discarded with the sampling device.

4. Conventional Pap Smear method

5. Collection of cervical smears

6. Microscopically, the uneven distribution of cellular material associated with the Conventional Pap Smear pattern is evident. This slide is from the same patient as the Slide by the left hand side. Tissue architecture is maintained . ThinPrep ® rearranges the relationship of cell groups on the glass slide. A group/sheet of endocervical cells present represents this.

7. Reasons for Unsatisfactory Smear (Conventional Pap smear) The following inhibits interpretation of 75 % of smear : - • Squamous epithelial component scanty/absent • Obscuring blood • Inflammation • Thick areas • Mucus • Poor Fixation • Air Drying • Artifact • Contamination

8. Disadvantages of Conventional PAP smears: Despite  the  demonstrated ability of  cervical  cytological  screening  to  reduce  cervical  cancer,  incidence and mortality, the conventional Pap test is less sensitive than is generally believed. The false­  negative rate of the conventional Pap test has been reported to be up to 50%. Approximately  67%  of  false ­negative  results  are  attributable to improper sampling techniques or the  poor  quality  of  slides.  Sampling  errors  may result  when a slide does not contain a representative sample  of the cells from the cervix, or the cells are obscured  by mucous or blood, or are inadequately preserved. Common  findings  on  conventional  Pap  smears  include:  ­thicker and thinner smeared areas, ­air ­drying artifact-variety of artifacts, such as “nuclear feathering

9. Principles of the preparation of Liquid Based Cytology (LBC) slides A sample of cells is collected from the cervix in the same manner as is used in the conventional Pap test, using either a broom type device or a plastic spatula or a cytobrush . Rather than smearing the cytological sample directly onto a microscope slide, the sample cells are transferred into a container of preservative/transport medium. The cell are dispersed in the fluid An aliquot of the suspension  is selected for processing The cells are separated by centrifugation or filtration and deposited on a slide as a thin layer/monolayer by sedimentation or the application of   pressure The slides are stained, mounted and ready for microscopy

10. LBC

11. Methods of carrying out LBC FDA- APPROVED 1. Thin –Prep 2. Sure Path FDA-Not Approved Cytoscreen Turbitec Cell slide Papspin

12. Thin Prep 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.

13. 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.

14. SurePath Sample Collection: This method requires the sample taker to follow the same method of sample collection.

15. 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 ) randomization 3) enrichment of diagnostic material 4 ) automated pipetting 5 ) sedimentation 6 ) staining

16. 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 specialized slide coat is applied to enhance cell adhesion. Cells are sedimented by gravity and then stained using modified papanicolaou staining process.

17. Advantages of LBC Immediate  fixation with enhanced nuclear and cytoplasmic detail All material collected is available for microscopic evaluation A representative sample is prepared for cytological evaluation but multiple samples can be prepared as necessary  Clearer background so that epithelial cells of interest are less likely to be obscured A thin layer of dispersed cells  are spread over a fixed area so that the area to be screened is small  and the preparation takes less time to screen than a conventional smear Unsatisfactory rate decreased LBC samples is suitable for other tests e.g. HPV testing LBC slides are suitable for automated analysis

18. Disadvantages of LBC Smear patterns altered because of randomization of cells Abnormal cells are dispersed Scanty LBC preparations can be difficult to screen and interpret Blood mucous inflammation and malignant diathesis are still present but appear slightly   different Epithelial cells appear mostly as single cells and are slightly smaller than they appear in conventional smears especially endocervical cells and immature metaplastic cells. LBC is more expensive than conventional test

19. Other uses of LBC LBC ThinPrep system has found broad acceptance in non-gynecologic cytopreparation such as: • Thyroid cyst fluid examination. • Oral pathology : diagnostic for various types of oral lesions . body fluids (e.g. urine, pleural effusions), brushing samples (e.g. gastrointestinal tract, lung) fine needle aspiration .