Ex-vivo and In-vivo Optical Molecular Pathology E-Book
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- Herausgeber: Wiley-VCH
- Kategorie: Wissenschaft und neue Technologien
- Sprache: Englisch
The result of a unique collaboration between clinicians, chemists and physicists, this book provides an unparalleled overview of a new generation of diagnostic tools in clinical pathology.
The introductory chapters cover the present status and limitations of currently used methods, followed by an outline of promising novel spectroscopy-based technologies either under development or recently available on the market. The input from both technologists developing these new methods as well as routine clinicians familiar with practical aspects and medical relevance guarantees that this practical work is a valuable asset for a wide audience, including technical personnel and decision makers in treatment centers, experts working in companies developing diagnostic devices, and clinicians specializing in advanced diagnostic methods. Since basic researchers are increasingly adopting novel diagnostic tools developed for human use as well, this will also be of interest for biomedical research institutions with large animal facilities.
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Seitenzahl: 507
Veröffentlichungsjahr: 2014
Ähnliche
Table of Contents
Cover
Related Titles
Title Page
Copyright
List of Contributors
Preface
Chapter 1: Clinical Pathology
1.1 Introduction
1.2 Pathology as a Medical and Research Discipline
1.3 Historical Perspectives
1.4 Specimens
1.5 Conventional Diagnostic Methods in Pathology
1.6 Nonconventional (Ancillary) Diagnostic Methods in Pathology (Molecular Assessment of Tissues)
1.7 Summary of Major Terms in Clinical Pathology
1.8 Limitations of Clinical and Diagnostic Pathology
Further Readings
Chapter 2: Clinical Endoscopy in Gastrointestinal Diseases
2.1 Introduction
2.2 White-Light Endoscopy
2.3 Chromoendoscopy
2.4 Virtual Chromoendoscopy
2.5 Endomicroscopy and Endocytoscopy
2.6 Endoscopic Spectroscopy
2.7 Perspectives and Conclusions
References
Chapter 3: Molecular Pathology via Infrared and Raman Spectral Imaging
3.1 Introduction
3.2 Background
3.3 Methods
3.4 Results and Discussion
3.5 Conclusions
References
Chapter 4: Coherent Raman for Medical Diagnosis
4.1 Introduction
4.2 Raman Contrast for Tissue Imaging
4.3 Coherent Raman Scattering
4.4 Advantage of CRS Imaging
4.5 Applications of CRS in Cellular and Tissue Imaging
4.6 CRS Imaging
In Vivo
4.7 Prospects and Challenges
Funding Source and Acknowledgments
References
Chapter 5: Multimodal Morphochemical Tissue Imaging
5.1 Introduction
5.2 Morphological Techniques
5.3 Functional Techniques
5.4 Morphological Imaging
5.5 Functional Imaging
5.6 Conclusion
Acknowledgments
References
Chapter 6: Molecular Endospectroscopic Approaches
6.1 Introduction
6.2 Endoscopic Imaging Techniques: Sampling Tissue Morphology/Architecture
6.3 Molecular Endospectroscopic Techniques: Probing Native Molecular Signals
6.4 Endoscopic Imaging with Contrast Agents
6.5 Nonlinear Endoscopic Raman Techniques under Development
6.6 Multimodal Endoscopic Detection and Diagnosis
6.7 Conclusions
References
Chapter 7: Image Processing—Chemometric Approaches to Analyze Optical Molecular Images
7.1 Introduction
7.2 Introduction to Statistics
7.3 Pretreatment
7.4 Image Analysis
7.5 Analysis Methods
7.6 Summary
References
Chapter 8: Summary and Conclusions
Index
End User License Agreement
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Guide
Table of Contents
List of Illustrations
Figure 1.1
Figure 1.2
Figure 1.3
Figure 1.4
Figure 1.5
Figure 1.6
Figure 1.7
Figure 1.8
Figure 1.9
Figure 1.10
Figure 1.11
Figure 1.12
Figure 2.1
Figure 2.2
Figure 2.3
Figure 2.4
Figure 2.5
Figure 3.1
Figure 3.2
Figure 3.3
Figure 3.4
Figure 3.5
Figure 3.6
Figure 3.7
Figure 3.8
Figure 3.9
Figure 3.10
Figure 3.11
Figure 3.12
Figure 3.13
Figure 3.14
Figure 3.15
Figure 3.16
Figure 3.17
Figure 3.18
Figure 3.19
Figure 3.20
Figure 3.21
Figure 3.22
Figure 3.23
Figure 3.24
Figure 3.25
Figure 4.1
Figure 4.2
Figure 4.3
Figure 4.4
Figure 4.5
Figure 4.6
Figure 4.7
Figure 4.8
Figure 4.9
Figure 4.10
Figure 4.16
Figure 4.11
Figure 4.12
Figure 4.13
Figure 4.14
Figure 4.15
Figure 4.17
Figure 4.18
Figure 4.19
Figure 4.20
Figure 4.21
Figure 4.22
Figure 4.23
Figure 4.24
Figure 4.25
Figure 4.26
Figure 4.27
Figure 5.1
Figure 5.2
Figure 5.3
Figure 5.4
Figure 5.5
Figure 5.6
Figure 5.7
Figure 6.1
Figure 6.2
Figure 6.3
Figure 6.4
Figure 6.5
Figure 6.6
Figure 6.7
Figure 7.1
Figure 7.2
Figure 7.3
Figure 7.4
Figure 7.5
Figure 7.6
Figure 7.7
Figure 7.8
Figure 7.9
Figure 7.10
Figure 7.11
Figure 7.12
Figure 7.13
Figure 7.14
Figure 7.15
Figure 7.16
Figure 7.17
Figure 7.18
Figure 7.19
Figure 7.20
Figure 7.21
Figure 7.22
Figure 7.23
Figure 7.24
List of Tables
Table 2.1
Table 3.1
Table 4.1
Table 4.2
Table 4.3
Table 6.1
Table 7.1
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Ex-vivo and In-vivo Optical Molecular Pathology
Edited by
Jürgen Popp
The Editor
Prof. Dr. Jürgen Popp
Leibniz Institute of Photonic Technology Jena
Albert-Einstein-Str. 9
D-07745 Jena
Germany
and
Institute of Physical Chemistry & Abbe Center of Photonics
Friedrich-Schiller University Jena
Helmholtzweg 4
D-07743 Jena
Germany
Cover
Background showing tissue imaged with coherent anti-Stokes Raman spectroscopy (CARS, © Leibniz Institute of Photonic Technology Jena). For details of the pictures see figure 3.8.
Limit of Liability/Disclaimer of Warranty: While the publisher and author have used their best efforts in preparing this book, they make no representations or warranties with respect to the accuracy or completeness of the contents of this book and specifically disclaim any implied warranties of merchantability or fitness for a particular purpose. No warranty can be created or extended by sales representatives or written sales materials. The Advice and strategies contained herein may not be suitable for your situation. You should consult with a professional where appropriate. Neither the publisher nor authors shall be liable for any loss of profit or any other commercial damages, including but not limited to special, incidental, consequential, or other damages.
Library of Congress Card No.: applied for
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A catalogue record for this book is available from the British Library.
Bibliographic information published by the Deutsche Nationalbibliothek
The Deutsche Nationalbibliothek lists this publication in the Deutsche Nationalbibliografie; detailed bibliographic data are available on the Internet at <http://dnb.d-nb.de>.
© 2014 Wiley-VCH Verlag & Co. KGaA, Boschstr. 12, 69469 Weinheim, Germany
Wiley-Blackwell is an imprint of John Wiley & Sons, formed by the merger of Wiley's global Scientific, Technical, and Medical business with Blackwell Publishing.
All rights reserved (including those of translation into other languages). No part of this book may be reproduced in any form – by photoprinting, microfilm, or any other means – nor transmitted or translated into a machine language without written permission from the publishers. Registered names, trademarks, etc. used in this book, even when not specifically marked as such, are not to be considered unprotected by law.
Print ISBN: 978-3-527-33513-8
ePDF ISBN: 978-3-527-68190-7
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List of Contributors
Laurence Maximillian Almond
Biophotonics Research Unit
Leadon House
Gloucestershire Royal Hospital
Great Western Road
Gloucester GL1 3NN
UK
Hugh Barr
Biophotonics Research Unit
Leadon House
Gloucestershire Royal Hospital
Great Western Road
Gloucester GL1 3NN
UK
Benjamin Bird
Northeastern University
Laboratory for Spectral Diagnosis (LSpD)
Department of Chemistry and Chemical Biology
Huntington Ave
Boston
Massachusetts 02115
USA
Thomas Bocklitz
Friedrich-Schiller University Jena
Institute of Physical Chemistry
Helmholtzweg 4
D-07743 Jena
Germany
Riccardo Cicchi
National Institute of Optics
National Research Council
Largo E. Fermi 6
Florence
Italy
and
European Laboratory for Non-linear Spectroscopy (LENS)
Via N. Carrara 1
Sesto-Fiorentino
Italy
Max Diem
Northeastern University
Laboratory for Spectral Diagnosis (LSpD)
Department of Chemistry and Chemical Biology
Huntington Ave
Boston
Massachusetts 02115
USA
Joerg Felber
Friedrich-Schiller-Universität Jena
Abteilung für Gastroenterologie
Hepatologie und Infektiologie
Klinik für Innere Medizin II
Erlanger Allee 101
D-07740 Jena
Germany
Jennifer Fore
Northeastern University
Laboratory for Spectral Diagnosis (LSpD)
Department of Chemistry and Chemical Biology
Huntington Ave
Boston
Massachusetts 02115
USA
Martin Goetz
Universitätsklinikum Tübingen
Klinik für Innere Medizin 1
Otfried-Müller-Str 10
D-72076 Tübingen
Germany
Joanne Hutchings
Biophotonics Research Unit
Leadon House
Gloucestershire Royal Hospital
Great Western Road
Gloucester GL1 3NN
UK
Charlotte Kallaway
Biophotonics Research Unit
Leadon House
Gloucestershire Royal Hospital
Great Western Road
Gloucester GL1 3NN
UK
Catherine Kendall
Biophotonics Research Unit
Leadon House
Gloucestershire Royal Hospital
Great Western Road
Gloucester GL1 3NN
UK
Christoph Krafft
Leibniz Institute of Photonic Technology
Jena
Albert-Einstein-Str. 9
D-07745 Jena
Germany
Kathleen Lenau
Northeastern University
Laboratory for Spectral Diagnosis (LSpD)
Department of Chemistry and Chemical Biology
Huntington Ave
Boston
Massachusetts 02115
USA
Christian Matthäus
Friedrich-Schiller University Jena
Institute of Physical Chemistry
Helmholtzweg 4
D-07743 Jena
Germany
Antonella Mazur
Northeastern University
Laboratory for Spectral Diagnosis (LSpD)
Department of Chemistry and Chemical Biology
Huntington Ave
Boston
Massachusetts 02115
USA
Miloš Miljkovi
Northeastern University
Laboratory for Spectral Diagnosis (LSpD)
Department of Chemistry and Chemical Biology
Huntington Ave
Boston
Massachusetts 02115
USA
Masoud Mireskandari
Universitätsklinikum Jena
Institut für Pathologie
Ziegelmühlenweg 1
D-07740 Jena
Germany
Francesco S. Pavone
European Laboratory for Non-linear Spectroscopy (LENS)
Via N. Carrara 1
Sesto-Fiorentino
Italy
and
University of Florence
Department of Physics and Astronomy
Via G. Sansone 1
Sesto Fiorentino
Italy
Iver Petersen
Universitätsklinikum Jena
Institut für Pathologie
Ziegelmühlenweg 1
D-07740 Jena
Germany
Jürgen Popp
Leibniz Institute of Photonic Technology
Jena
Albert-Einstein-Str. 9
D-07745 Jena
Germany
and
Institute of Physical Chemistry & Abbe Center of Photonics
Friedrich-Schiller University Jena
Helmholtzweg 4
D-07743 Jena
Germany
Eric Potma
University of California
School of Physical Sciences
Beckman Laser Institute and Medical Clinic
Health Sciences Road
Irvine
CA 92617
USA
Michael Schmitt
Friedrich-Schiller University Jena
Institute of Physical Chemistry
Helmholtzweg 4
D-07743 Jena
Germany
Jen Schubert
Northeastern University
Laboratory for Spectral Diagnosis (LSpD)
Department of Chemistry and Chemical Biology
Huntington Ave
Boston
Massachusetts 02115
USA
Andreas Stallmach
Friedrich-Schiller-Universität Jena
Abteilung für Gastroenterologie
Hepatologie und Infektiologie
Klinik für Innere Medizin II
Erlanger Allee 101
D-07740 Jena
Germany
Nick Stone
University of Exeter
College of Engineering
Mathematics and Physical Sciences
Streatham Drive
Exeter EX4 4QL
UK
and
Biophotonics Research Unit
Leadon House
Gloucestershire Royal Hospital
Great Western Road
Gloucester GL1 3NN
UK
Jeffrey L. Suhalim
University of California
School of Physical Sciences
Beckman Laser Institute and Medical Clinic
Health Sciences Road
Irvine
CA 92617
USA
James Wood
Biophotonics Research Unit
Leadon House
Gloucestershire Royal Hospital
Great Western Road
Gloucester GL1 3NN
UK
Preface
Spectroscopy, in general, plays an invaluable role in clinical diagnostics. The principle that all of these analytical techniques have in common is the interaction of electromagnetic radiation with the human body or a sample biopsy. All these techniques can be further divided into invasive and noninvasive methods. Long established are colorimetric methods that are based on absorption in the visible region of the spectrum, fluorescence, and optoacoustic techniques. A typical application of fluorescence is, for instance, flow cytometry. Optoacoustic effects are mainly employed for imaging, such as, for instance, the oxygenation level of blood. Examples that utilize light in the near-infrared (NIR) are optical coherence tomography (OCT) and NIR spectroscopy. OCT has become very popular in ophthalmology, dermatology, and cardiology. NIR spectroscopy is used, for instance, to measure blood sugar or to determine the saturation level of hemoglobin.
Visualization techniques commonly used in everyday clinical routines are often based on white light and are essential for the detection and diagnosis of diseases, as well as for the estimation of the risks associated with the disease. Also, for monitoring the development during treatment, these visualization techniques are essential. Endoscopy, for example, can visualize the inside of various organs such as the esophagus, the stomach, or the colon, and is therefore one of the most important techniques in internal medicine. Also very important in pathology are microscopic techniques. Bright-field microscopy is still the most widely used type of microscopy to investigate pathological biopsy samples. It is essential for the evaluation of cancer types, cancer grades, various inflammatory diseases, or changes associated with genetic disorders. There is no doubt that conventional pathology will always be based on these basic visualization techniques. However, by simply looking at structural changes in tissues and cells, there are often diagnostic questions that a pathologist cannot answer with absolute certainty. For instance, it is often very difficult to distinguish between certain cancer types. A better evaluation would often have consequences not only for the diagnosis but also for the treatment of the patient. Another important aspect is the time lag between the inspection of the patient and the preparation of the pathological report. By the application of new technologies, it is possible to have the routinely stained slides of small biopsy samples for microscopic evaluation on the same day of specimen acquisition. In uncomplicated cases, the final diagnosis can be made and the pathology report finalized on the same day. Problems occur if the specimen needs to be evaluated by ancillary methods (immunohistochemistry, molecular analysis) to reach a final and definite diagnosis. These ancillary tests can take a few days to be accomplished. The large resection specimens have to be fixed properly before processing and need to be sectioned cautiously before microscopic evaluations. A time frame of a few days to a week is needed for a complete evaluation of large samples. This unacceptable but legitimate delay contributes sometimes to the discomfort of the patient. Another aspect in pathology is the objectivity of the diagnosis itself. Although nothing can ever replace the trained eye of the pathologist, the evaluation of a pathological sample remains a matter of subjectivity. The same sample given to different pathologists may result in conflicting diagnoses. Interobserver variability is a well-known phenomenon in diagnostic pathology. Although false diagnoses by an experienced pathologist are generally rare, they are not impossible. In general, it would be advantageous if the pathologist could obtain more information about the sample or diseased area he is looking at. There are several imunohistochemical-based protocols, but they are usually limited to the analysis of a single protein. The inspection of multiple biomarkers simultaneously is rare. In addition, it may become expensive and time consuming to investigate a panel of markers. Spectroscopy offers great advantage to actually obtain chemical information about the sample. In other words, qualitative and quantitative biochemical information can be correlated with the pathology. The potential of such a combination is as wide as the field of pathology itself. Knowledge of the chemical composition of the sample would improve the evaluation of the state of the disease: for instance, what grade of malignancy the cancer has reached. It would also be possible to learn more about the origin of a cancer in the case of metastases. A great advantage over histopathology would be a faster pre-evaluation. Spectroscopic screening techniques can be, to a great extent, automated and can be operated by clinical personnel. Therefore, by screening the samples it would be possible to sort the potentially more dangerous cases before evaluation by the pathologist. This could be done on the same day, immediately after the inspection. The time-saving aspect would improve the diagnosis enormously. Additionally, the objective chemical information can be combined with the pathological diagnosis. Another great advantage is that potentially many spectroscopic techniques can be applied in vivo. Therefore, a coupling of spectroscopic fibers with endoscopes is possible. In addition to the visual image, the pathologist obtains valuable information about the chemical composition of the critical area at the same time. The potential to introduce new spectroscopic techniques into the operating theater could have great consequences for improving the decision making of the surgeon.
More recently, molecular spectroscopic techniques that are based on molecular vibrations have been applied to biomedical problems. The concept again is to combine well-established methods from analytical chemistry with optical techniques well established in medicine. The two main vibrational spectroscopy techniques are infrared (IR) spectroscopy, which is based on light absorption within the wavelength range of 2.5–25 μ m, and Raman spectroscopy, which is based on inelastic light scattering in the visible range. The coupling of these techniques to optical instrumentation has led to a tremendous growth within the field of vibrational spectroscopy. For clinical applications, the analytical instrumentation can be combined with either microscopes or endoscopes. The obtained spectral information is in comparison with, for instance, fluorescence very rich and specific. Both IR and Raman microscopy have been successfully employed to study compositional changes associated with various diseases. The two major advantages of both techniques are that they are noninvasive or minimally invasive and can be applied completely label free. Conventional histo- or imunohistochemical pathology and cytology rely on often poorly standardized staining protocols and the trained eye of the pathologist. Conceptually, IR and Raman microscopy can be automated, which would allow faster diagnosis. Because both techniques are noninvasive, the samples can be counterstained after the measurement and compared with standard histopathology. Over the past 10 years, IR and Raman microscopy have been applied to biopsy samples from virtually every organ.
When compared with each other, both techniques have advantages and disadvantages. Absorption measurements in the IR can be obtained relatively fast, which makes it possible to scan whole tissue sections within a reasonable time frame. On the other hand, the penetration depth is relatively small, so that the samples have to be cut into 5–10 μ m thin sections. Also problematic is the high absorption coefficient of water. These facts make IR microscopy very feasible to complement common histopathology and histocytology, but they hinder applications in vivo. Raman measurements can be performed under in vivo conditions, but require longer illumination times.
Other spectroscopic imaging approaches utilize nonlinear optical effects. Under illumination with pulsed laser radiation, molecules show various properties, which are, again, molecule specific and can therefore be used for characterization and identification. Today, most established for biological and medical applications is two-photon excited fluorescence (TPEF) spectroscopy. In TEPF, two photons of relatively long wavelength are used for excitation. Because of the application of the longer wavelength, the method is less invasive and leads to deeper penetration depths. As a consequence, TPEF can be applied in vivo and has been, for instance, used to image neurons. Another two-photon effect is second harmonic generation (SHG), which is ideal for imaging of centrosymmetric molecules such as collagen. Related to vibrational spectroscopy are the nonlinear Raman phenomena of coherent anti-Stokes Raman scattering (CARS) microscopy and stimulated Raman scattering (SRS). CARS microscopy allows rapid imaging of single molecular vibrations over fairly large areas. All these nonlinear imaging techniques can be combined, which is often referred to as multimodal imaging.
This book “Ex Vivo and In Vivo Optical Pathology” aims at introducing all the vibrational spectroscopic and nonlinear techniques in combination with modern pathology and illustrates their enormous potential for clinical applications. In particular, it addresses pathologists and medical doctors, as they are in the key positions to implement any new technical devices associated with the development of new instrumentation. The book should be a motivation to clinicians working in the field of pathology to be open to new technology. As the field of optical pathology is in a state of preclinical development, further collaboration between pathologists, natural scientists, and engineers is essential. It is therefore written in a way to be comprehensible to a very broad audience. Because the communication between experts of the above-mentioned fields is very important, the book addresses current problems from the clinical point of view, as well as technical issues associated with the development. Generally, the contents of the book should be of interest to spectroscopists who work with biological applications in particular. It should furthermore serve as a review and novel update for readers who work in the field of biophotonics, biophysics, and related areas. Last but not least, it should be a motivation for students, especially of the more advanced graduate levels who show an interest in these fields. As biophotonics becomes more and more an independent subject and is meanwhile taught at various universities and institutions, the book may also serve as a reference for instructors.
All contributors to this book are experts in their fields. The book is a consequence of long collaborations between the authors. Because the particular aim of the book is to improve pathology, it is structured in the following way: Chapters 1 and 2 are written by pathologists and clinicians to introduce the “gold standard” methodologies in the clinics and to point out current limitations. Chapter 1 introduces pathology as a diagnostic tool. First, a historical review is given. Standard histopathological routines are then described in detail. The chapter concludes with a list of the most commonly used terminology. Chapter 2 addresses commonly employed endoscopy. It also starts with a historical overview. Common types of clinical endoscopy, such as white-light endoscopy, chromoendoscopy, and endomicroscopy, are explained and compared. This is followed by a brief overview about the spectroscopic methods that have been combined with endoscopy. The chapter concludes with an outlook on future developments. Chapter 3 is a very detailed review of molecular pathology using IR and Raman microscopy. At the beginning, common histopathological techniques are described and compared with regard to their limitations. The spectroscopic basics for IR and Raman spectroscopy are explained, and examples of spectroscopic information and the generation of images are described. In the following, many examples of IR and Raman imaging of pathological tissue and cells are illustrated and how it is possible todistinguish normal from diseased, as for instance, cancer, tissue, and normal from abnormal cells. The novel techniques employed, such as micro-fluidics, are explained. Chapters 4 and 5 introduce nonlinear spectroscopic imaging techniques and their applications. Chapter 4 introduces CARS and SRS imaging and compares both techniques with spontaneous Raman spectroscopy. Examples of cell and tissue imaging in vivo and ex vivo are presented. Finally, current technical aspects and challenges are addressed. Chapter 5 focuses on multimodal imaging and introduces techniques such as TPEF, SHG, fluorescence life time imaging (FLIM), and CARS. Chapter 6 discusses the advantages and challenges of spectroscopy coupled with endoscopy that is referred to as endomicrospectroscopy. Chapter 7 deals with the implementation of modern computational statistics for diagnostic applications, because, for a careful analysis of disease-induced microspectroscopic image alterations (e.g., identification of disease-specific IR or Raman signatures), powerful image processing or chemometric approaches are required. This chapter briefly summarizes the general concepts of chemometric cell/tissue classification procedures or image analysis routines required for a successful realization of optical pathology by the spectroscopic imaging modalities presented in Chapters 3–7.
With this contribution, the authors hope to have accomplished a comprehensive overview of the current status of research in the field of optical pathology and to motivate the readers for further development and clinical applications.
Christian Matthäus, Michael Schmitt, and Jürgen PoppFriedrich-Schiller University JenaInstitute of Physical ChemistryJenaGermany
Iver Petersen and Masoud MireskandariUniversity Hospital JenaInstitute of PathologyJenaGermany
Jürgen PoppLeibniz Institute of Photonic Technology JenaJenaGermany
Chapter 1Clinical Pathology
Masoud Mireskandari and Iver Petersen
1.1 Introduction
Pathologists are referred to sometimes ironically as doctors of dead persons. This has roots in the common idea that pathologists are only responsible for doing autopsies and providing clues for the possible cause(s) of death. What accentuate such misconceptions are the equipment and methods of pathologic examinations. A pathologist never uses the usual tools of ordinary physical examinations such as stethoscopes or sphygmomanometers. He or she has also no direct encounter with patients. There is very little similarity between the image that people have in mind of a physician and of a pathologist.
In this chapter, we try to provide a realistic image about the territories of working of a pathologist. We begin this chapter with a brief definition of the science of pathology and the history of contemporary surgical pathology. Then the reader can find general information about the frequent types of specimens that are handled by pathologists as well as the ordinary diagnostic methods that are applied by them for making an accurate diagnosis. This section is followed by a brief review of the ancillary and more sophisticated diagnostic methods in the field of pathology. In the next section, we will introduce a list of basic definitions that are used frequently by pathologists to describe specifically the different groups of pathologic processes. Finally, we provide some examples of the limitations in the field of diagnostic pathology.
1.2 Pathology as a Medical and Research Discipline
In the study of medicine, pathology functions as a bridge between basic and applied medical sciences and in this way it plays a very substantial role not only in the understanding of the pathophysiologic basis of diseases but also in translating it into the practical management of patients and disease samples.
There are two basic schools of thought about the practice of pathology. In most European countries, a pathologist deals principally with microscopic evaluation of tissue specimens (small biopsy samples as well as large resection specimens) and cytological material. As an adjunct to this histologic and cytological evaluation, a pathologist uses some ancillary methods (such as immunohistochemistry (IHC) or molecular and genetic examinations) for more accurate diagnosis, classification, and prognostication of diseases. In the United States, a pathologist is, in addition, responsible for all laboratory investigations that are elsewhere covered by disciplines such as microbiology and laboratory medicine. These analyses are carried out on body fluids (blood, serous fluids, urine, feces, etc.), secretions of organs (exocrine secretions of pancreas), or other materials that are taken out from or expectorated by a patient (sputum, coverings of skin ulcers, etc.). They cover a broad spectrum of diagnostic methods apart from microscopy, including microbiologic, serologic, biochemical, and microscopic examinations. In this chapter, wherever we use the term pathology, it refers mainly to the macroscopic and microscopic evaluation as well as molecular assessments of tissue samples.
1.3 Historical Perspectives
The microscopic analysis of cells and tissue (e.g., cytology and histology) appeared for the first time in the nineteenth century as an important method for research and diagnosis in the field of medicine. Generally, Xavier Bichat is considered in most publications as the founder of pathology. The branch of histopathology appeared some years later, with Müller publishing a book on the structural characteristics of cancer cells and their growth. Virchow, a student of Müller, introduced the important correlations between cells, which are the smallest units of vital organisms and tissues, disease states, and related disease mechanisms. He became famous worldwide for his cellular pathology studies and his claim that every disease originates from diseased cells or, according to him, “Omnis cellula e cellula.” This statement is valid also today in the era of molecular pathology. The introduction of more innovative techniques, such as the microtome in the year 1839, enabled the pathologist to have better and thinner sections from tissues and had a great influence on the development of pathology. Gradually, the application of frozen section examination found its place in the routine practice of pathology for rapid as well as intraoperative evaluation of the suspected tissues. Another important development was the invention of standard hematoxylin and eosin (H&E) staining in the year 1875. The Carl Zeiss Company developed the first fluorescence microscope in the year 1965 in Göttingen. During the 1980s, the immunohistochemical analysis of tissues developed rapidly, which even today continues to be an invaluable diagnostic tool in pathology laboratories around the world.
Cytopathology is one of the important branches of pathology. By this method, it is possible to analyze all body fluids for the presence of tumor cells and evidences of inflammatory changes. In the middle of the nineteenth century, Virchow introduced the cell as the basic functional element of the body and hence the basic element in the development of diseases. This way he deserves to be considered as the founder of cytopathology. But the development of cytopathology as a diagnostic tool took, in fact, more time. The first important development took place under the influence of the Greek physician Papanicolaou. He introduced in the year 1928 a method of staining cytology smears, which was named after him as the Pap test. His knowledge and works had a great influence on the routine performance of gynecologic cytology and led to a considerable reduction in mortality due to uterine cervical carcinoma by early diagnosis.
1.4 Specimens
1.4.1 Biopsies, Resections, and Cytology
One of the main duties of a pathologist is to provide the clinicians with a precise tissue-based diagnosis, particularly in cases with a complicated disease process or in situations in which there are uncertainties with the clinical diagnosis. In these situations, the pathologists receive a small biopsy sample from a relatively large lesion or organ. Most of the time the questions asked are as follows:
Is there any pathologic change in this specimen?
If yes, is it a preneoplastic, neoplastic, or non-neoplastic lesion?
If it is a (pre)neoplastic lesion, is there any sign of dysplasia or malignancy?
If yes, which type of tumor is it? Is it invasive or noninvasive? What is the grade of the tumor?
If it is a non-neoplastic lesion, which type of disease process can it be? Is it an inflammatory process? Is it an infectious disease? If yes, is there any sign of the responsible infectious agent? If no, which type of inflammatory reaction can it be?
The notable improvement of endoscopic devices and imaging techniques has enabled physicians to gain access to the mucosal coverings of most internal organs and to take samples from them. Accordingly, pathologists encounter these days more frequently small biopsy samples. The most frequent areas of endoscopic samplings are mucosal coverings of the upper and lower intestinal tracts, respiratory tract, acoustic sinuses, female genital tract, urinary tract, and joint spaces.
The same set of questions can be answered by pathologists using other types of specimens that are obtained for cytologic examinations. The fluid accumulations in serosal spaces (pleura and abdominal spaces), secretions of some organs (nipple discharge), expectorated sputum, and voided urine can contain single as well as small aggregates of detached epithelial cells or suspended inflammatory cells, whose morphologic evaluation can serve as a basis for diagnosis. After collection, these fluids are centrifuged. The supernatant fluid, which is usually cell-poor or near completely acellular, can be used for chemical or serologic laboratory examinations. By preparing a direct smear, staining, and microscopically evaluating the cell-rich sediment, a pathologist or cytopathologist can provide an appreciable amount of diagnostic information. It is also possible to prepare a cell block from the sediment and to examine their sections microscopically. Other alternative methods to obtain specimens for cytologic examinations are brushing and washing of the mucosal (respiratory tract, esophagus) and serosal surfaces (washing cytology of Douglas pouch) or extracting fine tissue particles by aspiration using a narrow (fine pore size) needle. Fine needle aspiration (FNA) is a rapid and relatively noninvasive method of sampling, particularly when the target organ is superficial or palpable (thyroid, breast). With the guidance of sonography or computerized tomography (CT), FNA or fine needle biopsy (FNB) can also be used safely to obtain material from more deeply located organs such as pancreas, mediastinal structures, lungs, and liver.
Alternatively, pathologists receive large specimens, for example, resections, which can be different in size and extent from a part of an organ to complete removal of one or many organs together as well as limb amputations. Not infrequently, the reason for such an extensive operation is tissue necrosis and gangrene due to problems of blood supply (ischemia). But most of the time, such a large resection is performed for the complete removal of a malignant tumor as in curative surgery or for the reduction of the size of a tumor as in palliative surgery. Particularly in the case of curative surgeries, a pathologist should thoroughly examine the specimen at both the macroscopic and microscopic levels. The frequently asked questions about such specimens relate to the reconfirmation of diagnosis, grading of the tumor (i.e., degree of malignancy), the extent of tumor infiltration, and the evaluation of resection margins (i.e., if they are tumor-free or affected by the tumor). There are many different recommendations and guidelines for standardization of sampling and for reporting tumor resections (Figure 1.1).
Figure 1.1A radical prostatectomy specimen with both seminal vesicles (a). The outer surface of prostate (resection margin) is marked with blue ink. Such an approach makes the decision about the presence of tumor infiltration at resection margins easier (b). The specimen is completely sectioned in a systematic fashion in small pieces. All tissue fragments are processed and examined microscopically (c).
1.5 Conventional Diagnostic Methods in Pathology
After taking a tissue sample from a patient by any of the above-mentioned methods, it is necessary to fix it. Fixation is a way of treating a tissue using specific kinds of chemicals, usually in the form of fluids. The process of tissue decay and organ destruction begins as soon as the tissue is detached from the body and has lost its source of blood supply. It is a self-destruction and autolytic process that can continue up to the complete destruction of the sample. In the case of inappropriate and untimely fixation, the tissue consistency will be lost and it will not be possible to examine the tissue at both the macroscopic and microscopic levels. In some cases, it is very important that the pathologist provides the clinicians with some information about the characteristics and composition of the constituting cells at the molecular level. Such molecular evaluations are exceedingly difficult if not impossible to carry out on improperly fixed samples. The most universally used fixative solution in most of the pathology laboratories around the world is buffered 4% formalin solution.
There are many other fixatives that can be used in specific situations. Most of them suffer from one or more drawbacks such as high costs, problems with disposal, need for specific methods of tissue processing after fixation, too long a fixation time, and effects on the results of immunohistochemical or molecular examinations.
1.5.1 Cytology
As described above, the specimens that are received for cytologic examinations are usually in the form of an aspirated, expectorated, or washed fluid. One or more smears are usually prepared from the sediment of a centrifuged fluid. Depending on the desired staining method, these smears can be fixed by chemicals or are air-dried. The rest of the sediment can be processed similarly as for a tissue sample by transferring the cells into a network of protein material, for example, protein glycerin or plasma, followed by coagulation with thrombin. The cells are then fixed in formalin followed by paraffin embedding just like a tissue sample. If these cell blocks contain a sufficient number of cells, they serve as a very helpful reserve for further examinations such as immunohistochemical or sometimes molecular genetic tests (Figure 1.2).
Figure 1.2Preparation of cell block from liquid samples. The fluid is centrifuged. A part of the sediment is used for the preparation of cytologic smears by cytocentrifugation (a). The remaining sediment is coagulated by adding plasma and thrombin (b). The coagulated sediment is drained into a plastic basket and processed as a tissue fragment in a tissue processor (c). The paraffin blocks that were prepared from a liquid sample are ready for sectioning by a microtome (d).
1.5.2 Histology
Immediately after submission to a pathology laboratory, every tissue sample is given a numerical code. Different methods of labeling, such as bar coding, can be used for coding the samples.
The process of tissue examination by a pathologist begins by naked eye examination. A lot of information can be obtained after a careful macroscopic tissue examination or grossing. For small biopsy samples, these pieces of information are usually limited to the dimensions, as well as the number, color, and amount of the sample. It provides basic information regarding the adequacy of the specimen for further evaluations. In the case of some specific types of specimens, it is the duty of the pathologist to examine the small specimens by a hand lens or by a low-power microscope before subjecting it to complete formalin fixation and ordinary tissue processing. The best example is the renal needle biopsy. By low-power microscopic examination, the pathologist can tell the clinician whether he or she was successful in obtaining an adequate amount of renal tissue. On the other hand, the pathologist may need to divide the sample appropriately into three portions. Each portion is then handled differently for different methods of examination, that is, fresh tissue for immunofluorescent examination, fixation in glutaraldehyde for electron microscopy, and fixation in buffered formalin for conventional tissue microscopy and specific chemical staining. The last option represents the standard procedure that is applicable in all cases.
The most important role of grossing is in the evaluation of large resection samples. It is evident that microscopic evaluation of a whole resection sample, for example, the complete removal of an organ or extremity, is neither possible nor necessary. There are specific guidelines from which a pathologist can obtain information on how a resection specimen should be sampled and examined for microscopy. In most cases, these resection samples are those that contain a malignant tumor. In this situation, the clinicians might want to know the extent of the tumor and the completeness of its removal. The macroscopic examination defines the exact location, size, shape, and configuration of the tumor, the depth of local invasion (in tumors of luminal structures such as intestinal tract), the relationship with adjacent normal tissue, and the distance from surgical resection margins. It is also necessary to look for lymph nodes to examine them for possible metastatic foci. According to the guidelines, a pathologist takes small tissue fragments from the tumor, resection margins, and lymph nodes, which should not be less than a minimum recommended number. In some types of specimens, for example, radical prostatectomy specimens, it is recommended to completely embed the specimen in thin sections. To maintain the orientation during the microscopic examination, it is sometimes necessary to paint the specific areas such as resection margins by the different colors of specific dyes.
The prepared tissue slices are then placed in a plastic cassette. On this cassette, the code number of the specimen and if necessary the specific code of the area of sampling are written or typed. Now the tissue slices are ready to be processed. Tissue processing is a vital step for preparing the tissue slices for microscopic examinations. This task is performed automatically by a “tissue processor.” The device consists of vessels containing specific chemical compounds (mainly alcohol and xylene) at a previously determined and graded concentration. The processing of tissue is enhanced and accelerated in new-generation tissue processors by the application of microwave energy or vacuum. The tissue processing ends with embedding the tissue in a paraffin block. Now the tissue is ready to be cut to obtain thin slices for microscopic examinations. Using specific sharp blades and a precisely designed device, it is possible to cut the paraffin blocks into very thin sections (preferably 3–5 μm in thickness). The sections are placed on a glass slide, stained, and finally cover by a cover slip. They are now ready for microscopic examination by a pathologist.
1.5.3 Microscopy
A physician collects the necessary information by examining a patient and observing the signs and symptoms of the disease. Then he or she makes a list of differential diagnoses and tries to reduce the size of this table by the application of specific laboratory tests. The final target is to reach an accurate diagnosis. Pathology as a practice has similar components. By careful examination of the microscopic changes on a slide, a pathologist tries to gather specific morphologic signs and symptoms (in this situation the key morphologic findings) in order to have a list of differential diagnoses. The basic forms of pathologic changes (with few exceptions) more and less resemble each other in the different organs and body tissues. For example, an acute or a chronic inflammatory reaction is accompanied almost always by a predominantly neutrophilic or lympho-plasmacytic inflammatory cell infiltration, respectively. The basic microscopic examinations are performed almost always in the first step on H&E stained slides. By using two different acidic (eosin) and basic (hematoxylin) stains, the basophilic components of the cell structure (mainly RNA and DNA) gain a deep blue color and the acidophilic components (cell cytoplasm and interstitial stromal materials) gain a pale to deep pink appearance. Some specific cell components or specific cell types can be amphophilic (neither eosinophilic nor basophilic). Although the basic structure of the tissue and basic forms of pathologic processes are in most cases easily appreciable during this primary microscopic evaluation, it is sometimes necessary to stain new tissue sections to answer specific questions. Some examples of chemical-specific tissue stains are as follows:
Periodic acid-Schiff
(
PAS
): Using this stain, we can see a better reaction of chemicals or structures with a high content of carbohydrates or glycoproteins. It shows better intracytoplasmic or interstitial accumulations of mucinous secretions (for example, in mucin-secreting adenocarcinomas). Some specific forms of microorganisms, for example, fungi, are better recognizable by this type of staining. A pathologist can find more easily the megakaryocytes in a closely packed and hypercellular bone marrow tissue. It is also a very good staining of the basement membrane in different epithelial coverings, and its application plays a crucial role in the microscopic investigation of glomerular diseases in the field of renal pathology (
Figure 1.3
).
Giemsa
: It is one of the basic special stains and is regularly used in the microscopic examination of lymphoid and hematopoietic tissues (lymph node or bone marrow biopsies). In addition to providing better nuclear morphology, application of this staining is a single histologic method for the evaluation of tissue infiltration by mast cells. Most of the microorganisms, particularly bacteria, are better recognizable by this method of staining. A modified Giemsa staining is routinely used in gastric mucosa biopsies for the evaluation of
Helicobacter pylori
infection.
Masson trichrome
: It provides a better evaluation of the extent and severity of tissue fibrosis. In liver pathology, its routine application is one of the bases of diagnosis of advanced liver fibrosis or liver cirrhosis. Its application in medical renal biopsies as adjunct to other specific chemical stains (such as Jones staining) is extremely useful in judging the presence of fibrinoid necrosis, glomerulosclerosis, and abnormal depositions in the mesangial spaces and basement membrane.
Iron staining
: Iron depositions in tissue are recognized in routine H&E staining as coarse dark-brown crystalloid materials. Although an experienced pathologist can recognize iron deposition by noticing the background histologic features and morphologic characteristics, in the liver tissue, for instance, it can be often mistaken for the intracellular bilirubin (a product of hepatocytes) or lipofuscin (a final metabolite of fat in senescent or hypoxic injured cells). In one of the specific iron staining methods (Prussian blue), the iron crystals gain a deep blue stain, while the other two remain unstained. The same staining can help a pathologist to differentiate between iron-laden intra-alveolar macrophages (i.e., heart failure cells) from pigment-laden macrophages with ingested coal particles. The estimation of iron stores in bone marrow specimens is important to differentiate pathologic situations with increased iron stores (for example, myelodysplastic syndromes, sideroblastic anemia, and anemia of chronic disease) from situations with low iron stores (such as iron deficiency or chronic hemorrhagic anemia) (
Figure 1.4
).
Figure 1.3Collection of macrophages with deeply PAS-positive cytoplasm in the lamina propria of a duodenum mucosa biopsy is a characteristic feature of Whipple's disease.
Figure 1.4Iron staining of a lung tissue shows large number of intra-alveolar iron-containing macrophages (blue stained cells). This is an indication of recurrent and chronic intra-alveolar hemorrhage due to blood congestion. Such a situation happens, for example, in patients suffering from heart failure. Accordingly, these cells are called heart failure cells.
1.5.4 Intraoperative Assessment (Frozen Section Examination)
Preparation of formalin-fixed paraffin-embedded (FFPE) tissues is the usual method of tissue processing before microscopic examination, but it is not the only one. In some instances, the pathologists receive the sample in a fresh state (without any fixative or other chemical additives). The interested part(s) of the sample can be embedded in special media and rapidly frozen by immersing the sample in liquid nitrogen. Using a microtome mounted inside a freezer (cryocut microtome), thin tissue sections are prepared and stained. Such type of examination is called frozen section examination and is usually performed for the following purposes:
Intraoperative consultation
: To provide surgeons with an accurate diagnosis or as accurately as possible, the impression about the nature of a pathologic change to avoid a second surgical intervention and reducing the risks of reoperation. In this way, the pathologists are usually asked about the biologic (benign or malignant) behavior of the sampled tissue. This method of diagnosis was more frequent in earlier years. Nowadays, it is performed less frequently because of improvements in preoperative diagnostic methods and nonoperative invasive sampling techniques. Even in cases with confirmed diagnosis of cancer, a pathologist can be asked to determine the extent of a tumor or its grade. These types of information can influence the extent and method of surgery. Another application of frozen section examination during a surgery of a malignant neoplastic process is the evaluation of surgical margins.
Adequacy of sampling
: Even when the pathologist is not specifically asked by a surgeon to provide an accurate intraoperative diagnosis, by using this method the former can assess the adequacy of sampling for further examinations.
Molecular testing
