Histopathological morphometry of human endobronchial biopsies [Elektronische Ressource] : a comparison of conventional quantitative analyses and stereological designs / vorgelegt von Vlad Antonio Bratu

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 Bucureti, 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




To my parents, my friends and the family Fehrenbach




iii


















To err is human, to forgive is divine –
but to include errors in your design is
statistical.
Leslie Kish
iv Contents

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
v Contents
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

vi Contents
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

vii Contents
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
Figure 3.6 Anti-CD68 stained sections of endobronchial biopsies (40x)....................... 52