Conventional Smears and LBC Similarities And Differences

Transcription

1 Conventional Smears and LBC Similarities And Differences Dr. Mina Desai FRCPath; CBE Head of the Service/ Manchester Cytology Centre Director/Northwest Cytology Training Centre Past President/ British Society For Clinical Cytology U.K.

2 Manchester

3 Manchester Cytology Center One of the largest Lab In UK With Affiliated Training School Fully converted to LBC in 2005 Started with 70% TP, 30% SP Hub for processing of both systems of LBC Laboratory workload 300,000/annum for processing Screening workload 225,000/annum. Changed to 70% SP, 30% TP Converted to SP only in October 2010 Workload reduced to 135,000/year Majority of Laboratory staff trained for both systems of LBC Introduced Imager and Focalpoint for automated screening for National Trial

4 Manchester Cytology Lab Experience with Automation Cytyc Imager BD FocalPoint GS Imaging System National HTA funded MAVARIC Trial

5 Manchester Cytology Training Centre Teaching activities NW Regional Training School

6 Conventional and LBC COMPARATIVE FEATURES Conventional Smear Heterogeneous Graphic cell localization k cells/slide Variable fixation Thick uneven groups need frequent focusing Dirty background Variable preservation LBC Slides Homogeneous Random cell presentation k cells/slide Uniform fixation Uniform thin layer Not single cell monolayers Clean background Well preserved cells

7 Liquid Based Cytology Morphological Appearances Comparison of Conventional and LBC Well preserved cells in a liquid medium more rounded single cells. Cell aggregates where present are smaller and evenly spread Blood and polymorphs in the background not obscuring the cells Improved staining more nuclear detail visible Homogeneous cell distribution loss of cell to cell associations (e.g. streaks of histiocytes) Same criteria used for grading dyskaryosis/sil Surepath Conventional Thinprep

8 General appearance of ThinPrep slides 1.9cm diameter circle 70k vs 250k cells per sample Well-demarcated edge no drift Cells evenly distributed Holes in between cells Good Fixation and nuclear detail Cleaner background Traditional smear pattern lost Rounded up, small, single cells and small clusters

9 General appearance of SurePath slides 1.3cm diameter circle of cells Densely cellular More 3D effect seen Need to focus more often than with Thinprep slides Not as many holes as with Thinprep Need to use high power more often Other appearances are similar to Thinprep

10 Advantages LBC Advantages and Disadvantages screening Small No. of cells 70,000 or less (conventional 250,000 cells) Disadvantages Traditional smear pattern absent Mechanical artefact is eliminated Cells have no company Cellular material not pulled out in mucus Cells round up Well preserved single cells, therefore need to go on high power too often Preparation artefact

11 Advantages LBC Advantages and Disadvantages Small area to screen Good fixation Good nuclear detail Disadvantages Nuclear detail too good More borderline/asc reports during learning curve

12 System Specific Advantages and Disadvantages Surepath Good Points Not-so-good Small area to screen In-your-face chromatin Low grade dysk /LSIL easy Cell crowding with 3D effect difficult areas are Small cell and HCCG Atypical glandular can be in single cell Monotone/two-tone staining with loss of orange colour

13 System Specific Advantages and Disadvantages Good Points Thinprep Not-so-good Monolayer effect Flexibility with staining Gaps in-between cells give rest to the eyes Larger area to screen Difficult areas are Metaplastic vs.hsil, pale cell and bland cell Atypical glandular can be without feathering

14 Conventional & Liquid Based Cytology Common Errors Screening Errors Interpretation Errors + Preparation Errors

15 LBC Screening Error Sparse dyskaryosis/sil A feature of conventional and Both systems of LBC Litigation cell

16 LBC SCREENING METHOD COMPARISON CONVENTIONAL mm vertically or horizontally SUREPATH mm diameter circle screen vertically and horizontally during learning period THINPREP mm diameter circle screen one way and around the periphery during learning period With all 3 systems need to screen Systemic, Slowly and with Significant overlap. Focus and use high power more often ( Remember 3S ) Significant overlap = (1/3 to >4/5)

17 Surepath Primary Screening technique Use one of the following screening methods initially during learning period : Screen at half speed of conventional screening i.e. x 10 Screen smears in one direction looking for single abnormal cells and screen second time across at 90º studying sheets/groups i.e. x 10 Screen initially under x 20 until you are familiar with the smear pattern of that particular slide

18 Surepath Screening technique Key to good screening technique is maximum overlapping of fields at x10 and slow speed. Screen Systemic, Slowly and with Significant overlap. Focus and use high power more often ( Remember 3S ) Significant overlap = (1/3 to >4/5)

19 ThinPrep Primary Screening Technique during learning curve Systematic Slow at x10 1/3 to 4/5 Overlap Up/Down and periphery OR Left/Right and periphery Frequent use of High power For Routine use No need to screen periphery separately

20 Interpretation error No difference between conventional and LBC Pattern recognition and experience

21 Liquid Based Cytology Interpretation errors Small cell Pale cell Bland cell Syncitial group Hyperchromatic crowded groups Microbiopsies Small Keratinized cells Scanty Dyskaryosis ( HSIL) Dysk in metaplastic cells Koilocyte or NOT Multinucleate cells Same pitfalls as in conventional smears. Need to change the baseline criteria of abnormality due to in your face chromatin in LBC

22 LBC Normal Cells Classic Criteria is retained. Squamous cells More folded cells v shaped cells Nuclear chromatin granular Often nuclear folding or grooving with notches in the nuclear membrane Reason for increase ASC/Borderline reports during learning curve Metaplastic cells Nuclei appear larger, more granular and hyperchromatic Normal mitoses not uncommon Reason for increase ASC/BNC reports during learning curve Endocervical cells Artefact associated with cervex broom Individual cells, smaller groups or in sheets may be honeycomb Starburst or cartwheel arrangements common Cilia often seen Endometrial cell Tight cell ball (top hat) or Well preserved loose cell groupings with depth of focus and disordered arrangement (3D clusters) Major pitfall of HCCG with false +ve diagnosis of HSIL/ Severe dyskaryosis

23 LBC Normal Cells Classic Criteria is retained. Squamous cells More folded cells v shaped cells Nuclear chromatin granular Often nuclear folding or grooving with notches in the nuclear membrane Reason for increase ASC/Borderline reports during learning curve Metaplastic cells Nuclei appear larger, more granular and hyperchromatic Normal mitoses not uncommon Reason for increase ASC/BNC reports during learning curve

24 LBC Normal Cells Classic criteria are retained Endocervical cells Artefact associated with cervex broom Individual cells, smaller groups or in sheets may be honeycomb Starburst or cartwheel arrangements common Cilia often seen Endometrial cells Tight cell ball (top hat) or Well preserved loose cell groupings with depth of focus and disordered arrangement (3D clusters) Major pitfall of HCCG with false +ve diagnosis of HSIL/ Severe dyskaryosis More LUS cells and combination of EC and EM in same cluster

25 LBC Organisms Thinprep Surepath No change

26 LBC Low Grade Dyskaryosis/LSIL Abnormal cells appear either singly or in sheets Easy to find if Hyperchromatic Pale cell dysk (SIL) easy to miss Low grade dysk (LSIL) in rounded cells in LBCmisinterpreted as HSIL/Mod. Dyskaryosis resulting in Low PPV

27 LBC High Grade Dyskaryosis/HSIL More Isolated cells rather than streaks More three dimensional groups May have smaller nuclei but with higher N/C ratio than found in conventional Small isolated cells stand out due to clean background. Nuclear membrane and chromatin distribution easily identified. Surepath more dysk (SIL) in HCCG Thinprep more Dysk (SIL) in metaplastic

28 LBC Squamous carcinoma Tumour diathesis still present Fibre Cells still present Prominent Nucleoli easily seen

29 HCCG Major Pitfall in Surepath LBC ALL of the following cells are normal!

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31 HCCG Major Pitfall in Surepath LBC ALL of the following cells are abnormal!

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33 Metaplastic Cells Major Pitfall in Thinprep LBC Differential diagnosis is often between normal reactive or moderate (HSIL) dyskaryosis

34 Metaplastic Cells Major Pitfall in Thinprep LBC Spot the Difference

35 Metaplastic Cells Major Pitfall in Thinprep LBC Dyskaryosis/SIL Metaplastic cells

36 LBC Glandular lesions/cgin/ais More single cells and small strips with community borders Psuedostratification and Hyperchromasia Changes more subtle in individual cells due to better fixation Prominent Macro nucleoli, Gland openings and rosettes Feathering present but not as pronounced Vaculation of the cytoplasm Scalloped borders

37 LBC Normal Cells Additional Pitfalls Due To sampling technique Broom artefact distorted tangles of disrupted cells and streaked DNA Endocervical sampling in the atrophic cervix Unexpected May be wrongly interpreted often as high-grade squamous dyskaryosis (HSIL) Lower uterine segment sampling in the post-surgical cervix Interpreted as residual/recurrent high-grade disease

38 LBC Unanswered Questions

39 1.0% Threshold The adequacy dilemma: How many cells are enough? Abnormal detection rate % NB: Hypothetical graph 0 5K 10K 15K 20K Cellularity

40 The adequacy dilemma: where should we set the threshold? 1.0% Threshold Abnormal detection rate % Lower threshold = lower inadequate rates, lower cost but lower detection rates Bethesda Higher threshold = higher detection rates but higher inadequate rates and higher cost NB: Hypothetical graph 0 5K 10K 15K 20K Cellularity

41 The Bethesda System Guidelines Minimum of 10 microscopic fields at 40x should be counted along a diameter that includes the centre of the preparation The average cell number per 40x to achieve 5000 minimum is shown in the table below FN20 eyepiece/ 10X obj. FN20 eyepiece/ 40X obj. FN22 eyepiece/ 10X obj. FN22 eyepiece/ 40X obj. PREP DIAM (mm) AREA FN20 10X Cells/ fields for 5000 Total FN20 40X Cells/ fields for 5000 Total FN22 10X Cells/ field for 5000 Total FN22 40X Cells/ field for 5000 Total

42 SurePath adequacy: macro and micro 27 cells per x40 field = 15K

43 Thinprep Total material must be more than 50% 2/3 of this material should be well preserved and visible squamous cells

44 Definition of adequacy Bethesda minimum 5,000 cells UK uses different definitions for each system and different centres are using different cellular cut off 5,000, 10,000, 15,000 in different centres HTA funded trial in UK will report next year Dr. Desai and Dr. Turnbull are lead investigators

45 Traditional and LBC Conclusions Major difference between conventional and LBC Cytotechnologists do NOT want to go back to conventionals There are major differences between Surepath and Thinprep in sample preparation,cell presentation and screening methods. Finding abnormal small cells in Surepath requires different screening techniques for different slide presentations Generally, SP requires slower search strategy and greater overlap than ThinPrep and conventional smears Only minor differences are present in Interpretation of cells between two methods Be extra vigilant with HCGs in Surepath and Metaplastic cells in Thinprep Modify your existing interpretative skills otherwise more ASC and more HG reports with reduced PPV Be careful with Preperation artifact LBC has reduced inadequate smears but adequacy is not defined LBC has opened the door for HPV and molecular testing

46 Acknowledgement My special thanks to Dr. Persad & Dr. Bijal shah Jean Mather & Andrew Evered For Producing images and drawings and Writing some of the texts I have also used images from Cytyc, Surepath and NHS CSP Atlas

47 The End