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Introduction to histology

Introduction

Histology is the study of tissues and their structure. Disease processes affect tissues in distinctive ways, which depend on the type of tissue, and the disease itself. Hospital laboratories prepare microscope sections which are stained to show key features of the cells and anatomical structures within the tissues.

This OpenLearn course provides a sample of level 1 study in Science

Learning outcomes

After studying this course, you should be able to:

  • define all the terms given in bold

  • outline the services provided by a hospital histology laboratory, and who uses them

  • outline the processes involved in the preparation of tissue sections and explain the purpose of each of these processes

  • identify a number of basic tissue-types from their microscopic appearance

  • understand why histology is essential for accurate diagnosis and monitoring of disease progression.

1 What can histology and histopathology tell us?

Histology is the study of tissues and their structure. Disease processes affect tissues in distinctive ways, which depend on the type of tissue, the disease itself and how it has progressed. Histopathology, the study of tissues affected by disease, can be very useful in making a diagnosis and in determining the severity and progress of a condition. Histopathology units are found in most hospitals and there are also independent private laboratories. The services provided by these laboratories can be accessed by general practitioners (GPs), although the range of tissues that a GP can send for analysis is much more limited than those coming from a hospital surgery or from a mortuary. Because of the great variety of tests that are available and the high level of skill that is needed to carry them out and interpret them, many laboratories specialise in particular tissues or types of diagnosis. For example, a neuropathology laboratory will focus on understanding diseases that affect the nervous system. Units often work in groups across regions to provide as broad a range of services as possible. Even so, only the most diagnostically useful tests are generally available in hospital laboratories; research laboratories use many histological techniques that are limited to specific projects.

1.1 Macro versus micro

The conventional view of histopathology involves someone looking down a microscope. Indeed most histological work does involve preparation of tissues for microscopy, observation of sections and reporting of the findings. However a pathologist can often tell a great deal about a tissue without using a microscope. For example the brain of a person affected by multiple sclerosis has distinct lesions a few millimetres across called plaques in which myelin (the insulating element surrounding nerves) is damaged by inflammation. The plaques can readily be seen with the naked eye (Figure 1). Large specimens that can be examined macroscopically are usually only available post mortem or following surgical removal of tissue; biopsy specimens which consist of a needleful of cells or a flake of tissue can only be examined microscopically.

Figure 1 A post-mortem slice of a brain from a person who had multiple sclerosis. The plaque (arrowed) is located in the white matter of the brain, near a ventricle. (The brain ventricles are fluid-filled cavities that are continuous with the central canal of the spinal cord.)

1.2 Cells and tissues

Cells have distinctive shapes and functions which depend on how they have differentiated, the proteins that they express, and their interactions with other cell types. It is difficult to be precise, but there are probably at least 200 different types of cell within the body. Disease processes affect individual cells in many ways; they may cause them to die, to change their shape, to divide, to move or to invade other tissues. Any of these changes also affect the anatomy of the tissue. Understanding the changes that are characteristic of a disease requires a detailed knowledge of the normal appearance of cells and tissues, and the range of normality. Many tissues change considerably with age, so that something that is normal in an adult would not be normal in a child. For example, the thymus gland gradually decreases in size with age, so a large and hypercellular thymus in an old person could indicate some underlying pathology.

The term tissue is used here to describe any collection of cells; most tissues and organs consist of many different cell types with separate anatomical components, including blood vessels and lymphoid tissues, in addition to the characteristic cells of that tissue.

1.3 Primary and secondary changes

The histological changes seen in a tissue may be primary (a cause of the disease process) or secondary (a consequence of the disease). For example if blood pressure is high due to vascular disease, it may cause an increase in the volume of the heart muscle as the heart finds it more difficult to pump blood into the circulation. A further consequence may be a decrease in the volume of the heart ventricles so that less blood is pumped with each contraction. Interpreting the changes seen histologically, to identify the underlying pathology, requires a sound understanding of the disease process. It takes many years of study and observation to build up a good knowledge of the appearance of normal and diseased tissues, and many pathologists specialise in particular tissues or groups of disease. In this course, we only have space to introduce the subject and outline how it can help with diagnosis of disease.

1.4 Identifying cells and structures

Most cells and cellular elements are virtually transparent, so it is difficult to distinguish individual cells and cellular structures without staining the cells in some way before viewing them. There are two main ways of staining cells. The traditional method involves the use of dyes that selectively bind to different structures within the cell. More recently, techniques using antibodies have been developed which can stain individual molecules.

1.4.1 Histochemistry

Histologists have developed a wide variety of different staining techniques to identify different elements within tissues. This methodology is referred to as histochemistry. The most commonly used stain for medical diagnosis is haematoxylin and eosin (commonly abbreviated H&E). Haematoxylin is an alkaline dye which stains acidic structures a blue/purple colour. Eosin is an acidic dye which stains basic structures a deep pink colour. Cellular and extracellular components that are neutral take up neither stain and appear relatively clear.

SAQ 1

What colour will cell nuclei be stained by H&E? Why?

Answer

Deep purple/blue. The nucleus of a cell contains DNA (an acid) so it binds the basic dye.

Figure 2 shows 'sections' that illustrate the principles of histology and histopathology - the great majority of these sections have been stained with H&E (Figure 2). Some structures, such as the extracellular matrix, do not stain well with H&E, so we have included a few sections with different stains that allow the identification of particular tissue elements. These additional stains include Giemsa, Masson's trichrome (Figure 3), and Van Gieson. The characteristics and use of different stains is explained more fully in the course Introduction to microscopy.

Figure 2 A section of the adrenal cortex, stained with haematoxylin and eosin. Cell nuclei are stained deep blue and the cytoplasm is pink. The collagenous capsule of the organ (longer arrow) is also pink. Red blood cells (erythrocytes) seen within small blood vessels (short arrow) are stained red. Scale bar (bottom right) = 50 μm.
Figure 3 Two sections across the wall of the aorta from a child; (a) is stained conventionally with haematoxylin and eosin; (b) is stained with elastic Van Gieson which identifies the elastin fibres (black) in the media of the artery and the collagen (red) in the adventitia.

1.4.2 Immunohistochemistry

In the last thirty years staining methods using antibodies have been increasingly developed. When applied to staining for the light microscope, these techniques are collectively referred to as immunocytochemistry (ICC) or immunohistochemistry (IHC) (Figure 4). Antibodies specifically bind and detect individual proteins, so they can be selected to identify the presence or location of a single protein, that is diagnostically discriminating. Such characteristic proteins of a particular cell type or disease are called markers. Introduction to microscopy examines markers in greater depth and the ways in which antibodies can be used to stain sections.

Figure 4 Neurons in the cortex of the brain are identified by immunohistochemistry using an antibody directed against neurofilaments (protein filaments in the axons of the neurons). (Courtesy of Dr Eva Del Valle Suarez)

2 Preparing tissue for histopathology

In order to take any tissue, it is first necessary to obtain the informed consent of the patient, i.e. the person must understand why the tissue is taken and what it will be used for. The regulations that govern this area differ from one country to another, but in the UK, the Human Tissue Authority (HTA) oversees the process, which may be regulated by the Human Tissue Act 2004. Tissue which is taken from live individuals for diagnosis or treatment requires consent which is generally taken when the person consents to their treatment, and does not fall within the scope of the Act; tissue taken for essentially any other purpose is regulated by the Act. Since each individual must know at the time they give consent what their tissue may be used for, tissue that is taken for one purpose (e.g. diagnosis or a particular research project) cannot be used for another purpose later.

SAQ 2

A patient arrives in a hospital from a car accident, in which they sustained injuries to the leg. The surgeon has to remove a small piece of skin in order to set the bone. This piece of skin would normally go into clinical waste and be incinerated, but the histology laboratory would like to have a small piece of normal skin as a control in order to validate their immunohistochemistry. Is it permissible to use the clinical waste for this purpose?

Answer

No it is not, because the patient was in no position to give informed consent for the use of their tissue, and it does not aid their own diagnosis or treatment.

2.1 Fixation

It is important that the original structure of the tissue is preserved before it reaches the histopathology laboratory. As they die, cells break down releasing enzymes from their lysosomes and other intracellular organelles, which start to hydrolyse components of the tissue - a process termed autolysis. The degree of breakdown depends on the tissue and what has happened. For example, post mortem tissue is not usually taken until several hours or days after the person has died, and this would have undergone much more autolysis than a surgical specimen. Different components of tissue vary in their sensitivity to enzymatic digestion - RNA is particularly sensitive, most proteins less so and DNA may remain intact for very long periods. Cells and soft tissues tend to be more susceptible to breakdown than bone, cartilage, tendons and proteins of the extracellular matrix. A histologist has to be aware of all these processes and distinguish between changes that are due to a disease and those due to tissue autolysis.

Hydrolysis is the breakdown of proteins and nucleic acids, by reaction with water. These reactions are generally slow, but occur quickly if catalysed by enzymes. Proteins are hydrolysed by proteases; DNA and RNA are hydrolysed by nucleases.

SAQ 3

Why might you expect DNA to be more stable than RNA?

Answer

DNA is the genetic material, which must essentially remain stable for generations. RNA is an intermediate required in the formation of proteins. It is unstable and it breaks down in the cell when synthesis of the proteins is no longer required.

To minimise tissue breakdown, samples are often placed in a solution of fixative. However, since different fixation procedures are appropriate for each staining technique, it is important to know what technique will be applied to a tissue when it is taken. In addition to preventing autolysis, fixation may serve to retain the structure of the tissue and limit microbial growth. The most widely used fixative for light microscopy is 4% formaldehyde (formalin). The aldehyde group reacts covalently with amine groups (NH2) on amino acids such as lysine which blocks the activity of proteins, including lysosomal enzymes. Glutaraldehyde acts in a similar way, but because it has two aldehyde groups it can cross-link proteins and tends to harden tissue and preserve morphology (anatomical structure and cell shape). The two fixatives may be used in combination for particular purposes (Figure 5).

Figure 5 Fixation reaction of glutaraldehyde. The amine groups (-NH2) on amino acid residues in the protein, react with aldehyde groups (-CHO) in the fixative.

Another approach is the use of reagents such as ethanol, methanol or acetone, which disrupt hydrophobic bonds and protein structure and remove water from the tissue. There are critical differences between these two classes of fixative; aldehydes destroy amine groups, but tend to maintain tissue structure well: alcohols usually result in poorer preservation of structure (because they dehydrate cells) but do not destroy amine groups and they can preserve some secondary structure in proteins.

These considerations are important if immunohistochemistry (IHC) is to be used on the tissue. Antibodies bind to groups of amino acids within their specific target (antigen). If the target includes lysine, then the ability of the antibody to bind to the antigen may be destroyed. Histologists try to use antibodies that will work using a variety of fixatives; however, this is not always possible. This is the reason why specific fixation procedures may be recommended or required for particular applications.

Of course, if it is intended to derive live cells from tissue, then it must not be fixed. Freezing damages cells because of the formation of ice crystals, so tissue for cell culture is generally put into a culture medium, at 4ºC, and processed as quickly as possible.

2.2 Embedding and sectioning

Tissue that has been received in the laboratory needs to be prepared for sectioning. A variety of instruments are used to cut the sections and the protocol depends on the application. In most cases the tissue requires embedding in a medium, which allows thin sections to be cut cleanly; most tissues for routine histology are embedded in wax blocks. This requires that water is removed from the tissue and progressively replaced by wax, which can be solidified later to make a tissue block suitable for sectioning. The tissue is progressively dehydrated by immersing it in successively higher concentrations of alcohol (ethanol), before transfer to the organic solvent xylene and finally embedding in wax. Xylene is used at the final stage because wax is soluble in xylene, but not alcohol, so the wax can readily permeate the tissue. In a large pathology laboratory, much of this tissue processing is automated in order to save time and to produce consistent results.

A number of devices are available for cutting sections:

  • Microtome cuts thin sections (1-50µm) from fixed, embedded tissue (1µm =10-6 metres)
  • Vibratome uses a vibrating blade to cut thicker sections from fresh or fixed tissue (up to 200µm).
  • Cryostat cuts sections from deep-frozen blocks of unfixed tissue.

Most sectioning in routine histopathology departments is done with a microtome producing sections of ~3µm thickness, from tissue that has been embedded in wax.

SAQ 4

How thick is a typical cell in relation to the thickness of a 3µm section? How many cells might be seen by focusing up and down through the full depth of a section?

Answer

Most cells are >3µm in thickness, although it depends on the cell type and its shape. Hence a section will be less than one cell thick.

The vibratome and cryostat are often used to cut unfixed sections, and these are often more suitable for antibody staining, but they are not the first choice for routine sectioning (antibodies are expensive and the staining procedure takes several hours. H&E is inexpensive and the staining can be done within minutes). Normally a H&E-stained section is examined first, before deciding whether additional tests or staining procedures are required. Even then, the antibody-staining is normally done on wax-embedded sections, using antibodies that are known to work on fixed sections.

3 How a histopathology department works

Pathology laboratories are very busy places; a laboratory in a medium-sized hospital with five to seven staff could be handling 100 tissue samples each day, producing up to 300 stained slides (Figure 6).

Figure 6 A histopathology department in a busy hospital handles hundreds of tissue samples each week.

Tissue samples are sent to the laboratory from a variety of sources, including operating theatres, surgeries and mortuaries. Each sample is accompanied by a form with basic patient information (full name and date of birth), a note of the tissue-type, and may also include a query as to the possible diagnosis. Each sample will be assigned a laboratory number as soon as it arrives - tracking the tissue accurately through the system is absolutely critical, because any mistake in identifying the tissue could have serious clinical consequences.

The great majority of the samples arrive in formalin. Since it takes some time for the fixative to permeate large tissue specimens, the pathologist will first assess whether the tissue is adequately fixed; if it is, they will then direct the cut-up of the specimen. Within a large piece of tissue, the important area for examination may be quite localised. By observation of the whole piece of tissue and using experience, the pathologist decides exactly which areas should be examined microscopically. In addition, the orientation of the tissue in relation to the plane of the sections is decided. For example, a blood vessel which is cut in a transverse plane (cross-section) will appear quite different and yield different information from a vessel that is sectioned in a longitudinal plane. A single piece of tissue might have several cuts taken from it in order to give the best chance of identifying affected areas. The patient number and information on the size and margin of the cut is noted on the form and on the cassettes that hold the tissue samples (Figure 7).

Figure 7 A cassette-dispensing machine. Tissue is processed and sectioned on plastic cassettes which also carry the information that identifies the patient and the type of tissue. Colour-coding of the cassettes allows the staff to identify particularly urgent specimens (red) or those that require handling in a special way.

For biopsy specimens and small pieces of tissue, a biomedical scientist or a pathologist will assess the tissue and decide how it is to be sectioned. From this point all the tissue is passed over to the laboratory staff, who take it through the processes of embedding and sectioning. Tissue samples are loaded into cassettes which are then processed by a machine which takes the tissue successively through the stages of alcohol dehydration, xylene treatment and permeation with molten wax. The entire process usually runs overnight, at which stage the cassettes are transferred to an embedding machine, which holds the tissue in molten wax until it is ready to put into tissue moulds. The moulds are then filled with additional wax and chilled so that the wax sets, and the entire block containing the tissue and the cassette (with patient information) is then mounted on the chuck of the microtome.

Sectioning of the tissues is skilled and done by hand (Figure 8). A number of slides are prepared from each block, possibly at different levels through the block, depending on the surgical specimen. The section(s) are then transferred on racks into a staining machine which does the basic H&E stain (Figure 9). Finally coverslips are added to the sections, either manually or by another machine (Figure 10).

Figure 8 (a) A medical laboratory scientist examines the tissue block to determine the location of the tissue and then (b) cuts sections from the tissue block on a microtome.
Figure 9 The staining machine has baths containing different stains. It is programmed so that the robotic arm moves racks of sections between the different baths at the correct time, so that each rack remains in each staining solution for the correct amount of time.
Figure 10 A coverslipping machine. The machine takes slides individually, places a streak of mounting medium onto the section and then lowers a coverslip onto the section, before conveying it to a rack, ready for microscopy.

3.1 Quality control and diagnosis

At this point, one of the biomedical scientists will examine the section under the microscope to ensure that it is the correct type of tissue, corresponding with that noted on the form. They will also check that the sectioning and staining has been carried out properly (Figure 11). If it is not, repeating the sectioning and staining will be necessary. The whole of this process, from the time that the tissue arrives in the laboratory until the time that stained slides are ready to be examined takes one day.

Figure 11 The initial checking of the sections is done by a medical laboratory scientist in the laboratory, who ensures that the identity of the tissue and the original tissue blocks correspond, and that the processing and staining has produced good sections.

At this stage a pathologist examines the slides - usually the pathologist who directed the original cut-up of the tissue. They may be examining up to 120 slides in a day, so the amount of time available for each one is quite limited. Here again, experience is very important in rapidly identifying the characteristic appearance of a disease process. The pathologist's report on the slide is dictated to audio, for transcription by medical secretaries, who may add this information to the original pathology request form. In many cases, however, the pathologist's report will be transcribed directly back to the central patient's record, which is held electronically.

Also at this time, the pathologist may identify additional tests that they want done on the tissues; for example, sections from additional areas of the block, different histochemical stains or immunohistochemistry. Perhaps 15 per cent of the blocks will require further examinations. The variety of additional tests is quite large (~40 different stain types, and ~80 different immunohistochemistry stains, although this will depend greatly on the laboratory and its speciality); consequently many of the additional stains are done individually, by hand, by the laboratory staff, although the more commonly used stains may be done by machine. Standard protocols are used, but the large number and variety of tests that are being done means that great accuracy and careful time management is needed to carry out this work; five to ten different staining protocols may be carried out by one person in a single session. In some laboratories immunohistochemistry is partly automated by the use of a machine that takes the sections through the successive antibody treatments, wash steps and final enzymatic staining. The results from the additional assays are again examined by the pathologist and reported back onto the patient record, which is now available to the clinician who requested the histology.

Tissue and sections that are taken for histology are retained in archive for at least twenty-five years, so that they can be reviewed again if necessary. The archives of any pathology laboratory are extensive and the filing of the material is a major activity. Many laboratories store older material off-site, because of space considerations.

Conclusion

The relationship between the function of cells and organs is reflected in the organisation of tissues, visualised under the microscope. Hence histology supports the study of cell biology at all levels. Histology is also very important in diagnosis of disease and hospitals have associated laboratories and systems for examining and reporting on tissue resections and biopsies.

Acknowledgements

Course image: Wikimedia 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:

We are most grateful to Peter Mooney and the staff of the Cellular Pathology Laboratory (MK NHS Trust) for allowing us to photograph their course, and for explaining their work.

Figure 1: Health Sciences Center, University of Utah; Figure 4: Courtesy of Dr Eva Del Valle Suarez.

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.

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