What difference does a difference make?

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What difference does a difference make?. Elizabeth Little, Ph.D. 26-Oct- 2010. Talk overview. Introduction Tissue thickness variation Using best histological practices Stain intensity variation due to tissue thickness The difference matters Could impact algorithm functionality. - PowerPoint PPT Presentation

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  • What difference does a difference make?Elizabeth Little, Ph.D. 26-Oct- 2010

  • Talk overviewIntroductionTissue thickness variationUsing best histological practices Stain intensity variation due to tissue thicknessThe difference mattersCould impact algorithm functionality

  • Systems integrationsource: www.vagabondish.com

  • The Hematoxylin & Eosin (H&E) slideNumbersIn 2009, 330 million histology slides were produced in the United States83% (274 million) were stained with H&EPathologistPotential first look at the disease stateCostDollars vs. thousands of dollars for more advanced testing

  • Impacts of H&E stain variabilityPathologist workflow is impacted by staining variabilityRepeat slidesImaging workflow is also impacted by staining variabilityAlgorithms can by impacted by stain variability

  • Antecedents that are helpful for H&E slide image analysisControl of the stain variationUnder best practices we can control stain variability to a certain degreeAlgorithms that are robust against stain variation

  • Staining variables we cannot control- tissue type affects stain intensity

  • Intensity LevelPixelcount(N)

  • Staining variables that we have some control over - tissue thickness impacts stain intensity2 micron4 micron

  • Pixelcount(N)

  • Talk overviewIntroductionTissue thickness variationUsing best histological practices Stain intensity variation due to tissue thicknessThe difference mattersCould impact algorithm functionality

  • Possible sources of variations in section thickness in the histology laboratoryFixativeDuration of fixationTissue processingParaffinTissue blockMicrotomeHistologist

  • Objective measure the sectioning process impact on tissue thickness1 tissue block used 1 microtome2 settingsAutomated (32 slides per histologist)Manual (32 slides per histologist)2 histologists22 years of experience vs. 4 years of experience

  • Tissue thickness variability testing outlineSection Tissue was sectioned using a microtome setting of 4 micronsMeasure Section Thickness InterferometryStainH&EMeasure intensityWhole slide imaging

  • Measuring tissue thickness using vertical scanning interferometrysource: cnx.org

  • Tissue thickness using interferometric measurements Glass vs. paraffin Tissue was not measuredInterferometer limitationGlass level variability Measurements taken at 6 locations repeatedly

  • How well are we using the interferometer?

    SourceStandard deviation% ContributionTotal measurement (gage)0.290.80%Repeatability equipment variation0.290.79%Reproducibility operator variation0.030.01%Slide variation3.2099.20%Total variation3.21100.00%

  • How good is our tissue thickness measuring system? - gage R & R Equipment variation 0.79%Operator variation 0.01%Sample variation 99.20%

  • Slice thickness variation by histologist Nominal setting was 4 microns Both Histologists cut significantly thicker than 4 microns Both Histologists cut at significantly different thicknesses from each other

    HistologistNumber of slidesMeasured thickness average S.D. (mm)Combined1284.74 0.161644.65 0.102644.84 0.16

  • Manual vs. automated microtomy impact on tissue thickness Histologist 1 mean thickness was not impacted by microtome setting

    Both histologists had statistically significant more variability using the automated setting as compared to the manual setting

    HistologistMicrotomesettingMeasuredthickness S.D. (mm) 1Automated4.65 0.13Manual4.65 0.082Automated4.91 0.16Manual4.76 0.12

  • Block influences tissue thickness Histologist 1 was the cutter Automated setting used Tissue 3 was cut significantly thinner than tissues 1 & 2

    Tissue blockMeasured thickness average S.D. (um) Tissue one(n=32)4.65 0.13Tissue two(n=16)4.60 0.12Tissue three(n=16)4.36 0.12

  • Summary of tissue thickness measurement results Histology (location within block, slice selection, soaking, etc.)Difference in mean tissue thicknessMicrotome setting automated vs. manualBoth histologists were impacted by setting BlockBlocks 1 and 2 were cut more thickly than block 3

  • Talk overviewIntroductionTissue thickness variationUsing best histological practices Stain intensity variation due to tissue thicknessThe difference mattersCould impact algorithm functionality

  • Stain intensity variation due to tissue thickness - normal breast lymph node study3 micron4 micron

  • Objective measure tissue thickness impacton stain intensityTissue was sectioned and measured for thicknessAll slides were stained using the same methodAll slides were scanned using whole slide imaging and their average intensities were measured

  • Lymph node 1 micron makes a measurable difference

  • Talk overviewIntroductionTissue thickness variationUsing best histological practices Stain intensity variation due to tissue thicknessThe difference mattersCould impact algorithm functionality

  • Grey scale intensity differencesPixel count(N)

  • SummaryExpected vs. measured is differentThe difference is quantifiable Tissue thickness Stain intensityThe difference mattersCould impact algorithm functionality Tissue thickness and stain intensity correlate as expected

  • Further studiesIntensity vs. tissue type Microtome bounceHistology vs. DriftKnifeLocation in blockDegrees of fixation

  • Acknowledgments Cindy Connolly Wendy Lange Allison Cicchini Heather Free Aaron Ewoniuk Jonathan Hall Mike Cohen, Ph.D. David Clark, Ph.D.