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Histopathological Morphometry of Human Endobronchial ... · PDF file Angenommen vom Fachbereich Medizin der Philipps-Universität Marburg am: 17.08.2009 Gedruckt mit Genehmigung des

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Text of Histopathological Morphometry of Human Endobronchial ... · PDF file Angenommen vom...

  • Aus dem Medizinischen Zentrum für Innere Medizin Aus der klinischen Forschergruppe „Chronische Atemwegserkrankungen“ in der

    Klinik für Pneumologie Geschäftsführender Direktor: Prof. Dr. med. Claus Vogelmeier

    des Fachbereichs Medizin der Philipps-Universität Marburg in Zusammenarbeit mit dem Universitätsklinikum Gießen und Marburg GmbH,

    Standort Marburg

    Histopathological Morphometry of Human Endobronchial Biopsies – a Comparison of Conventional Quantitative Analyses and

    Stereological Designs

    Inaugural-Dissertation zur Erlangung des Doktorgrades der gesamten Humanmedizin

    dem Fachbereich Medizin der Philipps-Universität Marburg

    vorgelegt von

    Vlad Antonio Bratu

    aus Bucureşti, România

    Marburg, 2008

  • Angenommen vom Fachbereich Medizin der Philipps-Universität Marburg am: 17.08.2009

    Gedruckt mit Genehmigung des Fachbereichs.

    Dekan: Prof. Dr. med. Matthias Rothmund

    Referent: Prof. Dr. rer. nat. Heinz Fehrenbach

    Korreferent: Prof. Dr. med. Roland Moll

  • iii

    To my parents, my friends and the family Fehrenbach

  • iv

    To err is human, to forgive is divine –

    but to include errors in your design is

    statistical.

    Leslie Kish

  • Contents

    v

    TABLE OF CONTENTS

    LIST OF TABLES ..........................................................................................................vii

    LIST OF FIGURES...................................................................................................... viii

    LIST OF ABBREVIATIONS AND SYMBOLS ............................................................ ix

    1 Introduction ............................................................................................................. 1

    1.1 Chronic inflammatory airway disorders.................................................................1

    1.2 Bronchial biopsies and airway inflammation..........................................................4

    1.3 What is morphometry?.............................................................................................7

    1.3.1 Classical geometry..................................................................................................7

    1.3.2 Stochastic geometry and probability theory............................................................7

    1.3.3 Unbiased stereology................................................................................................9

    1.4 Principles of unbiased stereology...........................................................................10

    1.4.1 Estimation and bias...............................................................................................10

    1.4.2 Random sampling .................................................................................................12

    1.4.3 Geometrical probes and random geometry ...........................................................12

    1.5 Aim of the study ......................................................................................................18

    2 Material and methods............................................................................................ 19

    2.1 Material ...................................................................................................................19

    2.1.1 Subjects and bioptic material ................................................................................19

    2.1.2 Equipment and software .......................................................................................20

    2.1.3 Specimen collection..............................................................................................21

    2.1.4 Paraffin embedding, deparaffination, hydration, dehydration and mounting ........21

    2.1.5 Histochemical staining procedures .......................................................................22

    2.1.6 Immunohistochemical staining procedures (ABC-Method)..................................22

    2.2 Methods ...................................................................................................................25

    2.2.1 Flexible bronchoscopy and biopsy........................................................................25

    2.2.2 Paraffin-embedding of the biopsies ......................................................................25

    2.2.3 Sectioning and sampling of the paraffin blocks ....................................................26

  • Contents

    vi

    2.2.4 Histochemical staining..........................................................................................28

    2.2.5 Immunohistochemical staining by the indirect Avidin-Biotin-Complex method...29

    2.2.6 Microscopical analyses.........................................................................................31

    2.2.7 Statistical analyses ................................................................................................40

    3 Results .................................................................................................................... 45

    3.1 Biopsy volume .........................................................................................................45

    3.2 Epithelial integrity ..................................................................................................46

    3.3 Thickness of the reticular basement membrane ...................................................51

    3.4 2D and 3D inflammatory cell counts .....................................................................52

    4 Discussion .............................................................................................................. 63

    4.1 Epithelial integrity ..................................................................................................63

    4.2 Thickness of the reticular basement membrane ...................................................67

    4.3 Comparison of 2D and 3D inflammatory cell counts ...........................................70

    4.3.1 Counts of all cell profiles......................................................................................73

    4.3.2 Counts of nuclear profiles.....................................................................................74

    Summary / Zusammenfassung...................................................................................... 80

    Summary ..............................................................................................................................80

    Zusammenfassung ...............................................................................................................82

    Sumar (Romanian) ..............................................................................................................84

    References ...................................................................................................................... 86

    Annexe

  • Contents

    vii

    LIST OF TABLES

    Table 1.1 Stereological terms and their meaning.............................................................11

    Table 1.2 Parameters and probes in stereological designs..............................................13

    Table 2.1 Subject demographics..................................................................................... 20

    Table 3.1 Epithelial morphology by pattern and subject group...................................... 47

    Table 3.2 Quantitative morphological data by group and cell type................................ 53

    Table 3.3 CD68+/CD3+ cell ratios by group and counting design.................................. 56

    Table 3.4 Hypothesis testing and correlation of the CD68+/CD3+ cell ratios between the

    3D and 2D counting designs........................................................................................... 56

  • Contents

    viii

    LIST OF FIGURES

    Figure 2.1 Schematic SURS of the sections of a biopsy ................................................ 27

    Figure 2.2 Assessment of the epithelial integrity by fraction of the RBM area ............. 33

    Figure 2.3 Coherent test system for the estimation of the arithmetic mean thickness of

    the RBM ......................................................................................................................... 36

    Figure 2.4 Physical disector (3D) and profile counting (2D) within a consecutive

    reference and look-up section......................................................................................... 39

    Figure 3.1 Biopsy volumes of the two subject groups ................................................... 45

    Figure 3.2 Heterogeneous morphology of the respiratory epithelium (20x).................. 46

    Figure 3.3 Epithelial integrity by pattern and subject group – comparison with published

    data ................................................................................................................................. 48

    Figure 3.4 Correlation of the epithelial morphology with the biopsy volume ............... 49

    Figure 3.5 Arithmetic mean thickness of the RBM........................................................ 51

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