Histopathology, the study of tissues affected by disease, can be very useful in making a diagnosis and in determining the severity and progression of a disease. Understanding the normal structure and function of different tissues is essential for interpreting the changes that occur during disease. This course introduces the basic principles that apply to the preparation of microscope sections. It also shows how to identify a number of human tissues and interpret the changes that occur in disease.
This OpenLearn course provides a sample of level 1 study in Science
After studying this course, you should be able to:
define all the terms given in bold
outline key features of a number of pathological processes
relate the histological appearance of affected tissues to the underlying pathology
recognise the histological appearance of a number of pathological tissues
understand how sections can be photographed, presented and reported.
Histological examination of tissues can help diagnose disease, because each condition produces a characteristic set of changes in the tissue structure. There are such a wide variety of diseases that histology alone usually cannot produce a diagnosis, although in some cases the histological appearance is definitive. For example, a pathologist might see signs of a viral infection in the brain, because of tissue damage and inflammation, but would be unable to tell what virus is responsible; to identify the virus might require immunohistochemistry (IHC) for the viral protein or more likely, the diagnosis would be confirmed by the symptoms or serology. Conversely, the appearance of 'owl-eye' cells in the brain is diagnostic of a particular type of measles infection (Figure 1). Normally histopathology reports only form one part of the disease picture that the clinician is assembling.
Although diseases are very diverse, the responses made by the body are more limited and fall into specific categories. For example inflammation, may be seen in response to an infection or as a result of physical damage or as part of an autoimmune disease, where the immune system attacks components of the body. The following sections outline some of the more common pathological processes and relate them to examples which can be seen in the virtual microscope.
Infection can affect any tissue of the body, producing cell damage and inflammatory reactions. Viruses are generally too small to be seen in the light microscope, but their presence can often be inferred by the changes they produce in tissue, even if their identity requires confirmation by immunohistochemistry, serology or molecular biology. Bacteria can be seen in the light microscope using high magnification objective lenses; however the numbers of bacteria that are present in a tissue can be highly variable even in one disease. A classic example of this variability is leprosy, where there may be very large numbers of bacteria in the skin (lepromatous leprosy), or very few (tuberculoid leprosy). Distinguishing the type of bacteria in a thin section of a lesion generally requires specialised histological stains, although the morphology of the bacteria may also be informative (Figure 2). As with viral infection, the histological findings are an adjunct to serology and microbiology in producing a diagnosis.
(a) What stain could you use to identify M. tuberculosis in a section of lung? (b) What stain could you use to identify N. gonhorrea in a urethral smear?
(a) Ziehl Nielsen stains mycobacteria red; their identification is aided by the bacterial morphology - mycobacteria are rod-shaped. (b) Gram stain can help distinguish Neisseria, which are gram-negative streptococci from other streptococci and staphylococci which might also be found in the specimen.
Identification of parasites is often difficult by serological methods; however, the appearance of parasite-infected cells (e.g. malaria) or the parasites themselves is absolutely characteristic of the particular infection (Figure 3). Consequently, diagnosis of parasitic infections relies substantially on the initial histological or haematological findings.
Inflammation is a common response to tissue injury or infection. Acute inflammation develops quickly and resolves within days, whereas chronic inflammation can last for months or years, usually because of the persistence of the initiating factor. The histological appearance of acute inflammation is quite different from chronic inflammation and the distinctive features can point to the initiating agent. For example, an infection of the skin with Staphylococcus aureus usually produces an acute inflammatory response, whereas infection with Mycobacterium leprae (leprosy) typically produces persistent infection and chronic inflammation.
There are three main components of inflammation (Figure 4):
All of these processes bring the defence systems of the body to the affected area. The blood contains a number of proteins that stop bleeding, help clear infection and induce repair or regeneration of the tissues. It also contains different types of leukocyte (white blood cells), each of which has evolved to deal with different types of infection. One of the key histological differences between acute and chronic inflammation is seen in the sets of leukocytes that are present in the tissues. In acute inflammation polymorphonuclear neutrophils usually predominate, whereas macrophages and lymphocytes predominate in chronic inflammation. Eosinophils are often prevalent in sites of helminth infections. Hence the characteristics of inflammation are determined both by the tissue in which it occurs and by the initiating agent and its persistence.
Chronic inflammation is seen in diseases where there is persistent infection, usually because the pathogen can resist the body's immune defences. If the infection is cleared, chronic inflammation resolves, but residual damage may still be evident in the tissues. Chronic inflammation also occurs in many autoimmune diseases; in autoimmunity the target of the immune response is one of the body's own proteins or cellular components, and consequently the stimulus for inflammation cannot be cleared, although the condition may improve if the normal controls that prevent autoimmune reactions are restored.
The immune system normally recognises and tolerates all of the body's own tissues. However, in some conditions the immune system reacts against 'self', resulting in autoimmune disease. The targets may be individual molecules found in a specific tissue, or antigens present in many tissues or in the extracellular matrix. Table 1 gives some examples of autoimmune diseases and the target antigens. An example of a tissue-specific autoimmune disease is Hashimoto's thyroiditis, in which lymphocytes recognise thyroglobulin and a thyroid peroxisome antigen (Figure 5).
|Disease||Organ||Target antigens||Histological appearance|
|Destruction of thyroid follicles with severe inflammation|
|Goodpasture's syndrome||Kidney, lung||Basement membranes|
Damage to kidney glomerulus
and/or lung alveolae
|Myasthenia gravis||Skeletal muscle||Acetyl choline receptor||Degeneration of the motor endplate at nerve/ muscle junction|
|Desmosome proteins in keratinocytes||Separation of layers of epithelium|
|Diabetes - type I||Islets of Langerhans|
Pancreatic beta cells
Insulin , GAD (enzyme)
|Selective damage and loss of cells of pancreatic Islets with inflammation|
|Erosion of articular cartilage by fibrous, inflammatory tissue - pannus.|
|Systemic lupus erythematosus||Kidney, skin, CNS||DNA and intracellular antigens||Type-3 hypersensitivity reaction in kidney, damage to glomerulus|
The histological appearance of autoimmune disease depends on the nature of the immune response and the target organ. However a characteristic of many organ-specific diseases is that autoantibodies bind to the antigen within the tissue and recruit inflammatory cells. In this case, direct immunofluorescence microscopy can be used to identify the presence of antibodies, which goes a long way towards providing a diagnosis of the disease (Figure 6). It is also possible to detect autoantibodies in the blood of patients, using the same technique; the patient's serum is first incubated with normal tissue to allow any autoantibodies to bind, and these are then detected, by direct immunofluorescence or immunohistochemistry. Examination of the stained sections can determine not just whether there are autoantibodies in the serum, but also indicate what the target antigen might be, depending on where the autoantibodies are located in the cells.
Hypersensitivity is defined as an immune response, where the reaction is out of proportion to the damage caused by the antigen or pathogen and does more harm than good. Autoimmune diseases are by their very nature a type of hypersensitivity reaction; however, there are many instances where the immune reaction against an antigen or a pathogen is out of proportion to the damage that it causes. A simple example is hay fever or asthma induced by pollen, where the pollen itself is clearly harmless, but the inflammatory reactions, especially in the lung, can be life-threatening. In some infectious diseases, such as M. tuberculosis, a significant component of the pathology is the collateral damage caused to lung tissue by the ongoing immune reaction against the bacteria. Obviously the bacterial infection is itself potentially damaging, but the severity of the disease in different individuals is at least partly due to the variability in their immune responses. Diseases such as multiple sclerosis are even more complex. In this case, it is suspected that there is an autoimmune reaction, although the target antigen is unclear, and there is clearly a hypersensitive response taking place in the brain. The fact that this immune response is particularly damaging is partly related to the nature of the CNS, which is delicate and normally shielded from immune and inflammatory reactions.
Hypersensitivity reactions can be classified into four main types depending on the type of immune response that causes them. Although the causes of hypersensitivity are beyond the scope of this course, the histological appearance of the different types of hypersensitivity reactions is often distinctive and can aid in diagnosis. Referring to the examples given above, hay fever and allergic asthma are examples of type-1 hypersensitivity reactions, which develop rapidly following exposure to antigen. They are characterised by neutrophils and eosinophils in mucosal and submucosal tissues of the respiratory tract; basophils are also common in the bronchial wall in asthma. In contrast, tuberculosis is an example of a type-4 hypersensitivity reaction, which develops slowly, in association with chronic inflammation, and is characterised by macrophages and T-lymphocytes. The other types of hypersensitive reaction are due to antibodies in tissues. For example the autoantibodies seen in pemphigus (Figure 7) are an example of a type-2 reaction, whereas type-3 reactions are caused by the deposition of antigen-antibody complexes from the circulation in organs where filtration occurs, particularly the kidney.
Scarring and fibrosis are seen when the cells of a tissue are damaged or killed and regeneration of the normal tissue architecture cannot take place. For example, in cirrhosis of the liver, the normal hepatocytes are damaged and do not regenerate effectively. The tissue is repaired and replaced by cells such as fibroblasts, which lay down extracellular matrix components including collagen, which can be seen by appropriate histological stains.
How does collagen appear in H&E staining? What stains show collagen more effectively?
In H&E staining collagen is pale pink and often difficult to differentiate from support cells embedded within it. Masson's trichrome stains collagen blue. Van Gieson stains collagen red/pink.
The cells which carry out the repair vary from one tissue to another. For example, following damage to the CNS, a group of glial cells called astrocytes replace damaged neurons, forming a glial scar. Obviously this scar tissue cannot carry out the normal function of nervous tissue, but it also can actively prevent the tissue from regenerating - neurons do not regrow their axons through glial scars. Similarly scar tissue in the skin will usually lack characteristic features of normal skin, such as hair follicles and sweat glands.
Fibrosis also occurs in some infections, particularly if the infectious agent cannot be cleared, fibroblasts lay down areas of extracellular matrix, which walls off the infection. For example, schistosomiasis (a worm infection) in the liver often results in areas of fibrosis surrounding the individual parasites.
Fibrosis and scarring are end-stages of a pathological process in which the body is unable to regenerate normal tissue and does the best it can by patching up the remaining tissue to limit further damage.
In many cases cells can divide and regenerate the tissue, restoring it to virtually normal. For example the basal cells of the skin epidermis can divide to cover a scratch or a graze, provided that it does not extend over too great an area. Epidermal cells from hair follicles can contribute to the regeneration, provided that the damage has not gone too deep. In this case there is a balance between regeneration from the epidermis and repair from the dermal layers, the outcome of which will determine whether a scar is formed or not. The process of normal tissue regeneration can be favoured by closing wounds with stitches, or skin grafts. Conversely, if the damage persists or the area of damage is large, fibrosis and scarring prevail.
The ability to regenerate varies greatly between cell types. For example, neuronal cells have a very limited capacity to regenerate (regrow) their axons if they have been severed, and virtually no capacity to replace themselves by cell division. By contrast, hepatocytes have enormous potential for division, which can be seen following removal of a portion of the liver, following surgery; the remaining cells can divide to fully restore the liver to its original size.
In tissue such as skeletal muscle, regeneration is characterised by an increase in the thickness of myofibres (hypertrophy), but without significant increase in their number. The same effect is seen with adipocytes, which increase or decrease in size (i.e. the volume of the lipid-filled vesicle) in response to fasting or over-eating rather than by changes in cell number. In such tissues, the histological appearance can give an indication of tissue damage that has taken place a long time previously.
Angiogenesis is the process by which new blood vessels grow into tissues, forming capillaries.
Under what circumstances would you expect new vessels to grow into tissues?
An increase in the requirements for oxygen or nutrients stimulate angiogenesis. It may be due to an increased metabolic activity of the organ, e.g. in a muscle following training. Regenerating tissues also require a new blood supply, and angiogenesis is frequently a critical requirement for tumour development.
The process of angiogenesis involves new capillaries sprouting from the side of arterioles and extending as blind-ended tubes in the tissue. Eventually they connect up (form anastemoses) with venules to complete a capillary loop (Figure 8). Regenerating tissue often contains numbers of these developing capillaries; in the skin the base of scars has characteristic pink spots, which are the newly sprouting capillaries.
Cell division is normally a highly regulated process. The numbers of cells in any tissue is usually fairly constant, although some tissues can respond to physiological demand by an increase in cell number.
What process occurs as mountaineers acclimatise to high altitude? Why?
The number of erythrocytes in their blood increases. The fall in the level of oxygen in the air at altitude means that the capacity of the blood to carry oxygen increases in order to compensate. There is a progressive increase in the numbers of erythrocytes over a period of weeks as the bone marrow responds by increasing production.
Other types of cell may increase in numbers in response to appropriate stimuli. For example, in a guitar player, the basal cells of the epidermis in the fingertips can proliferate to produce hard pads of keratin (calluses) caused by repeated contact with the strings. Cell proliferation and the consequent increase in cell numbers seen in these two examples is called hyperplasia. It is a normal physiological response to demand placed on a tissue. The numbers of each cell type are controlled specifically. For example, the numbers of erythrocytes in the blood is controlled by a hormone, erythropoietin; an increase in erythrocyte numbers does not produce any concomitant increase in leukocyte numbers, since leukocyte subsets are each subject to their own controls on cell number.
If cell division becomes poorly regulated, cells may lose some of their morphological characteristics and/or functions. The tissue becomes disordered in appearance, often with an increase in the numbers of immature cells, and greater variability between cells. This appearance is called dysplasia. It should be emphasised that dysplasia does not necessarily show that the cells have become cancerous; however, it does suggest underlying changes in the cells, which may predispose to cancer. In this sense dysplasia may be a stage on the way to cancer development. For example, when histologists screen cervical smears, they are particularly looking for changes in the normal morphology of the cells which indicate pre-cancerous changes.
Neoplasia is the term used to describe the development of tumours or cancerous tissue. The development of a tumour requires a series of changes in the biology of the cell, with progressive loss of the controls that limit cell division. Even a cell which is undergoing uncontrolled proliferation will not necessarily be malignant. Malignancy typically arises when the dividing cells invade the normal tissue and move away from their site of origin. Because of the great variety of different tumours, it is impossible to generalise. Nevertheless it is very important for a pathologist to be able to distinguish between a benign tumour and a malignant cancer, since the treatment required will usually be radically different. Consequently, pathologists often grade tumours according to how malignant/invasive they are. Histologists can get some impression of the rate of cell division within a tissue according to the number of mitotic figures - the number of cells with the nucleus showing the characteristic pattern of separating chromosomes, seen as the cell divides (Figure 9). Invasion of tumour cells within the tissue can be estimated by observing where the cells are in relation to their normal position and in relation to other cells in that tissue, and this forms an important element in the pathological report on a tumour.
Tumours can also move away from their original tissue by invading blood vessels or lymphatic ducts and being carried to distant sites. This process is called metastasis. Tumour cells that are carried through lymphatics will usually metastasize to local lymph nodes - this is the reason that surgeons may remove lymph nodes as well as the original tumour to treat a cancer. Tumours that metastasize via the blood must first invade a blood vessel at the initial tumour site, and then exit the blood vessels in a different organ to establish a new tumour site. Such an event is relatively rare for any individual tumour cell; nevertheless, metastasis accounts for 90% of cancer-related deaths, so identification of metastatic tumours is important both for prognosis and treatment. Pathologists recognise metastatic tumours, because the affected organ contains clumps of cells which are completely uncharacteristic. In some cases the primary tumour-type can be recognised because it has retained some distinctive characteristics of the original cell-type. However, as noted above, the original identify of tumour cells is not always self-evident and this is particularly true of metastatic tumours. Hence, it may be possible to observe a metastatic tumour in a tissue, but be unable to identify the primary cell-type and hence the original site of the tumour, at least by H&E staining. In this case additional staining, particularly immunohistochemistry is valuable to identify the original cell type, because it can provide an important guide for patient-scanning, further surgery, radiotherapy and drug treatment.
When cells die, they do so in two main ways: by apoptosis or necrosis (Figure 10). Apoptosis is programmed cell death; the cell dies as part of its normal programme of development, or it may be lacking in growth factors, or it may be instructed to die by cells of the immune system, because it has become infected. Even pre-cancerous cells may be propelled into apoptosis, by the normal cellular controls that check the development of tumours. In all cases, apoptosis is a highly ordered process. If it occurs as part of a developmental process, it does not induce inflammation - the dead cells are quietly removed by phagocytes within the tissue. Hence, it is often very difficult to identify apoptotic cells within tissues, since they are usually individual cells, with small condensed nuclei and little cytoplasm. Cell death in degenerative conditions (e.g. Alzheimer's disease) appears to occur by apoptosis. Although the loss of individual cells is histologically undramatic, the cumulative loss of cells in such degenerative conditions can cause major loss of function in the affected tissue. Moreover, cell loss may be accompanied by the accumulation of products of tissue breakdown, which are histologically evident.
In contrast necrosis is wholesale unregulated cell death caused by lack of nutrients or infection. For example the failure of the blood supply to an organ due to thrombosis (see below) will cause massive cell death due to lack of oxygen (ischaemia). A large area of cell death caused by ischaemia is called an infarction. Another example of cell necrosis is seen in severe viral infections with cytopathic viruses (e.g. polio). Necrosis is an uncontrolled process and the dying cells release their contents. Areas of necrosis are characterised by infiltration with inflammatory cells; macrophages and neutrophils enter the area over a number of days and weeks in order to clear the dead cells and associated cellular debris. Such large areas of cell loss and inflammation are frequently easily seen in pathological specimens, even without microscopic examination (Figure 11).
Blood clots may form in vessels for a variety of reasons. A blood clot is called a thrombus, and the process by which it forms is thrombosis. Embolism occurs when something is carried through the circulation from one site to another. When a thrombus breaks away and is carried through the circulation, it is referred to as a thromboembolism. Other examples of emboli are tumour cells or air-embolism, where air is accidentally introduced into the circulation by a physician. Thromboembolism can block the downstream blood vessels; emboli formed in veins pass through the heart to block arteries in the other side of the circulation, while thrombi formed in arteries can block vessels in the organ where they form. Exactly which vessels may become blocked also depends on the size of the embolism, and the site determines what damage may follow.
(a) If a thrombus is formed in the veins of the leg, where is it likely to end up? (b) If thrombi form on the tricuspid valves of the heart (leading to the aorta), where might the emboli end up?
(a) Thromboemboli formed in leg veins will usually pass through the heart to end up in the pulmonary arterial circulation, causing damage to the lung. (b) Thrombi formed on the tricuspid valves will pass into the systemic circulation and are particularly damaging if they enter the cerebral or carotid arteries, as they can then damage the brain (stroke).
Cell loss occurs in many tissues with age; the effects are particularly notable in tissues that have a limited capacity for regeneration, such as nerves in the central nervous system, the retina of the eye and the sensory cells of the inner ear. Histologically, it is more difficult to identify something that is not there than a change in the structure of the tissues. In diseases such as Alzheimer's disease there is progressive loss of neurons and shrinkage of the brain, which may be more evident in the gross pathology, although counting the relative numbers of cells within an area can also give some histological indication of the cell loss. For example, the relative numbers of neurons relative to glial cells falls in areas affected by Alzheimer's disease. More evident are characteristic accumulations of proteins. Degenerating neurons leave tangles of fibres (neurofibrillary tangles) produced be degenerating components of the cytoskeleton. In addition there are extracellular accumulations of 'amyloid' within the brain. It is debated whether these deposits are the cause or consequence of the disease, or both.
Amyloid is an extracellular insoluble deposit of protein, and amyloidosis refers to the diseases in which amyloid occurs. In some cases production of amyloid is a primary event, and in others it is secondary to infection or a tumour. The actual protein type varies, depending on the cause of condition. In some cases it affects individual organs, such as the brain in Alzheimer's disease, but in the so-called 'systemic amyloidoses' many organs may be affected, including the lung, kidney, heart and spleen.
What stain can be used to identify amyloid deposits in tissues?
Congo red, as mentioned in Histology (S120_2).
There are a number of hereditary conditions in which the person lacks enzymes that break down particular macromolecules; they are collectively called storage diseases because the components that cannot be degraded within lysosomes accumulate and form insoluble deposits. This is particularly noticeable in the brain, where they are often associated with neurodegenerative diseases (Figure 12).
Digital photography has superseded the use of film for obtaining images of histological sections and many microscopes have a digital camera attached. An image obtained from a slide generally only includes a tiny proportion of the section, however microscope systems are now available that can scan entire slides, providing very large images. Such images can be transmitted electronically, so that a pathologist can 'view' a section from a distant location. Such systems are being increasingly used, although they are still very much the exception to the standard practice where the pathologist observes and records their observations at their own hospital.
Images are not usually obtained for routine work. Since the sections are stored for many years it is always possible to return to them later. However, for presentations, images are essential, and there is some skill in selecting suitable areas of the section to illustrate a point. Journals require a minimum of 300dpi for histological images, usually in jpg or tiff formats. If you are preparing images for publication, it is essential to generate images of acceptable quality and format, by checking the requirements on the journal website beforehand.
When you see micrographs in older text-books, a magnification is usually stated in the legend (e.g. x100). Strictly, this should mean that the magnification of the illustration in the book is 100-fold larger than the original item. However, there is occasionally some ambiguity. For example, the statement can mean that the picture was taken using a microscope set with a 100x magnification (10x objective, 10x eyepiece). Since the light path to the camera is not the same as the light path to the eye (which passes through the eyepieces), these magnifications are not meaningful. Moreover publishers may increase or decrease the size of a micrograph to fit the available space. The stated magnification in the legend should then be corrected, but often it is not.
For these reasons, the use of scale-bars has replaced a statement of magnification. A scale bar, corresponding to a convenient unit of length, is added to the image taken by the camera, and is then an integral part of that image. If the image is increased or reduced in size thereafter, the scale bar changes in proportion, so that it is always possible to see the correct size of the cells or tissue.
Pathological processes leave their imprint on the tissue. Interpreting these changes can give key information about a disease and aid diagnosis. Some of the changes are very subtle, whereas others are easily seen. In all cases it is important to distinguish natural variations from pathological changes, and it requires many years of experience to be able to recognise the different diseases that can occur - even within a single type of tissue. This course should have given you some insight into the subject of histopathology, and the type of work that is done by specialists in this field.
Course image: Larry Darling in Flickr made available under Creative Commons Attribution-NonCommercial-ShareAlike 2.0 Licence.
The material acknowledged below is Proprietary and used under licence (not subject to Creative Commons licence). See terms and conditions.
Grateful acknowledgement is made to the following sources:
Figure 1: Brostoff, J. et al. (1991) Clinical Immunology. Gower Medical Publishing
Figures 2, 3, 5 and 11: David Male
Figure 6: Dr D. Bean
Figure 7: Dr R. Mirakian and Mr P. Collins
Figure 9: Woo, E. K. et al. (2005) 'Myoepithelial carcinoma of the breast: a case report with imaging and pathological findings', British Journal of Radiology, vol. 78, May 2005. The British Institute of Radiology
Figure 12a-e: Claudion, S. (2003) 'Unfolding the role of protein misfolding in neurodegenerative diseases', Nature Reviews Neuroscience, vol. 4, 2003 © Nature Publishing Group
Figure 12f: Riezman, H. (2002) 'The ubiquitin connection', Nature, vol. 416, 28 March 2002. Nature Publishing Group.
Every effort has been made to contact copyright holders. If any have been inadvertently overlooked the publishers will be pleased to make the necessary arrangements at the first opportunity.
Don't miss out:
If reading this text has inspired you to learn more, you may be interested in joining the millions of people who discover our free learning resources and qualifications by visiting The Open University - www.open.edu/ openlearn/ free-courses
Copyright © 2016 The Open University